DEVELOPMENTS IN FOOD SCIENCE 37 B
F O O D FLAVORS: GENERATION, ANALYSIS A N D PROCESS INFLUENCE
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DEVELOPMENTS IN FOOD SCIENCE 37 B
F O O D FLAVORS: GENERATION, ANALYSIS A N D PROCESS INFLUENCE PROCEEDINGS OF THE 8TH INTERNATIONAL FLAVOR CONFERENCE, COS, GREECE, 6-8 JULY 1994 Edited by GEORGE CHARALAMBOUS
1995
ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon - Tokyo
ELSEVIER SCIENCE B.V. Molenwerf 1 P.O. Box 211,1000 AE Amsterdam, The Netherlands
ISBN: 0-444-82013-2 © 1995 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521,1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside of the USA, should be referred to the copyright owner, Elsevier Science B.V., unless othenA/ise specified. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in The Netherlands
CONTENTS Forword Introduction List of Contributors Enzyme Reactions in Reverse Micelles A. GUPTE, R. NAGARAJAN and A. KILARA
VII VIII IX 1
Food Applications of Biopolymers - Theory and Practice I.S. CHRONAKIS and S. KASAPIS
75
Sorghum Grain and Quality of its Edible Products Y.G. MOHARRAM and A.M.A. YOUSSEF
Ill
GC-MS Analysis of Artemisia herba alba Asso Essential Oils from Algeria G. VERNIN, O. MERAD, G.M.F. VERNIN, R.M. ZAMKOTSIAN and C. PARKANYI
147
The Volatile Flavor of Fresh Gentiana lutea L. Roots I. ARBERAS, M.J. LEITON, J.B. DOMINGUEZ, J.M. BUENO, A. ARINO, E. de DIEGO, G. RENOBALES and M. de RENOBALES
207
Oriental Natural Flavor: Liquid and Spray-Dried Flavor of "Jeruk Purut" {Citrus hystrix DC) Leaves C. HANNY WIJAYA
235
Simple Analytical Technique for the Determination of Raspberry Flavor in a Complex Gelatin Matrix - Data and Methodology R.K. SALEEB
249
Stir-Fried/Saute Flavors - Recent Developments in the USA A.S. KIRATSOUS and M. ISHIKAWA
265
Formation of Sulfiir-Containing Flavor Compounds from AUylic Alcohol Precursors G.P. RIZZI
289
Changes in Chemical Composition of the Essential Oil of Chios "Mastic Resin" from Pistacia lentiscus var. Chia Tree During Solidification and Storage D. PAPANICOLAOU, M. MELANITOU and K. KATSABOXAKIS A New Method for Harvesting of Chios "Mastic Resin" in a Fluid Form D. PAPANICOLAOU, M. MELANITOU, K. KATSABOXAKIS, D. BOGIS and K. STAMOULA
303 311
VI
The Solubility and the Phase Equilibria of Essential Oil with Carbon Dioxide Calculated Using a Cubic Equation of State F.A. CABRAL and M.A. de A. MEIRELES
331
Authentication of Natural Flavours Using SNIF-NMR - New Developments on Mustard Oil and Safron G. MARTIN, G. REMAUD and GJ. MARTIN
355
Nitrogen-Specific Liquid Chromatography Detections of Nucleotides and Nucleosides by HPLC-CLND E.M. FUJINARI and J.D. MANES
379
Optimization of Virgin Olive Oil Quality in Relation to Fruit Ripening and Storage E. MONTELEONE, G. CAPORALE, L. LENCIONI, F. FAVATI and M. BERTUCCIOLI
397
Evaluation and Quantification of Potent Odorants of Greek Virgin Olive Oils G. BLEKAS and H. GUTH
419
Rapid Extraction and Determination of Phenols in Extra Virgin Olive Oil F. FAVATI, G. CAPORALE, E. MONTELEONE and M. BERTUCCIOLI
429
Effect of y-Radiation on Migration of Dioctyl Adipate Plasticizer from Food Grade PVC Film into Olive Oil A.E. GOULAS and M.G. KONTOMINAS
453
Subjective Odor Evaluation of Toasted Canola Oil D. PARK, J.A. MAGA and D.L. JOHNSON
465
Effect of Natural Antioxidants on the Stability of Canola Oil F. SHAHIDI and U. WANASUNDARA
469
The Aroma Components of Freshly Boiled Potatoes and Freeze-Dried Potatoes M.J. XU
481
Influence of Dips, Modified Atmospheric Packaging, and Storage Time on the Enzymatic Discoloration of Processed Raw Potatoes J.A. MAGA An Application of Deuterated Sex Pheromone Mimics of the American Cockroach {Periplaneta americana, L.), to the Study of Wright's Vibrational Theory of Olfaction B.R. HAVENS and C.E. MELOAN Testing Wright's Theory of Olfaction with Selectively Deuterated (E)-2-Hexen-l-al Compounds D.F. DeCOU (III) and C.E. MELOAN Properties of Extruded Rice Flour and Acorn Squash (Cucubita pepo) Blends G. MORINI and J.A. MAGA
491
497
525 549
Vll
Chestnut (Castanea molissimd) Flour Extrusion G. MORINI and J.A. MAGA
557
Changes in the the Fatty Acid Composition of Roasted and Boiled Chinese (Castanea molissima) and Italian (C. sativd) Chestnuts Grown at the Same Location G. MORINI and J.A. MAGA
563
Roles of Formulation and Extrusion Variables on the Properties of Potato-Based Half Snacks C.H. KIM and J.A. MAGA
569
Cereal Protein and Carbohydrate Digestibility as Affected by Extrusion K. DAHLIN and K. LORENZ Zinc Availability in Low- and High-Phytic Acid Extruded and non-Extruded Rat Diets D.E. BEST and J.A. MAGA
575 . . . . 595
Nutrition in the Critically 111 Infant D.E. WITHINGTON Nutritional Benefit of Edible Oil Processing to Decrease Cardiac Risk Factors: In vivo Studies with Mustard, Rape Seed Oils Low and High in Erucic Acid and Com Oil T. WATKINS, P. LENZ, R. SIDERITS, M. STRUCK and M. BIERENBAUM
625
633
Improving Atherogenic Risk Factors with Flax Seed Bread T. WATKINS, A.C. TOMEO, M.L. STRUCK, L. PALUMBO and M.L. BIERENBAUM
649
Cadmium in Cereal Products - Nutritional Importance K. LORENZ, A. WINATA and L. EOFF
659
The Red Microalga Rhodella reticulata as a Source of a Dietary Q-3 Highly Unsaturated Fatty Acid-Eicosapentaenoic Acid A. YARON, I. DVIR, M. MAISLOS, S. MOKADY and S. (MALIS) ARAD
665
Study of Emulsifying Porperties of Low-in-Cholesterol Egg Yolk Prepared with the Use of Polysorbate-80 A. PARASKEVOPOULOU and V. KIOSSEOGLOU
675
Volatile Nitrosamines in Foods - An Update R.A. SCANLAN
685
A Consumer Survey on Food Additives T. ALTUG and Y. ELMACI
705
Accelaration of Flavour Formation During Cheese Ripening M. EL SODA
721
Soy Bean Cheese L.G. SIAPANTAS
747
VIU
Proteolytic Enzymes of Lactic Acid Bacteria S. TAKAFUJI, T. IWASAKI, M. SASAKI and P.S.T. TAN Chemical and Volatile Organic Compounds Composition of Whey Protein Concentrate I. LAYE, D. KARLESKIND and C.V. MORR
753 . . . 769
Aroma Compounds in Green Coffee W. HOLSCHER and H. STEINHART
785
Formation of Furfuryl Mercaptan in Coffee Model Systems T.H. PARLIMENT and H.D. STAHL
805
Effect of Oxidation Products of Scented Tea Aroma Compounds on Flavor of Tea Infusion J. POKORNY, F. PUDIL, K. ULMANNOVA and E. FIKOVA
815
The Study on Aluminum State in Tea-Water by ^^Al NMR Spectroscopy Method D. QI, J. TONG, Y. SUN, S. CHEN and S. LUO
827
Flavour Composition of Some Lemon-Like Aroma Herbs from Lithuania P.R. VENSKUTONIS, A. DAPKEVICIUS and M. BARANAUSKIEN E
833
GLC Analysis and Comparison of the Flavor of Different Populations of Basil G. PETROPOULOS and A-M. VLACHOU
849
Microwave Extraction of Basil Aroma Compounds L.F. Di CESARE, M. RIVA and A. SCHIRALDI
857
Screening for Antioxidant Activity of Essential Oils Obtained from Spices V. LAGOURI and D. BOSKOU
869
Influence of Sleeted Additives on the Stability of Saffron Pigments in Aqueous Extracts O. ORFANOU and M. TSIMIDOU The Effect of Drying Kinetics on Peppermint Quality H. AKBABA and T. gAKALOZ Contributions of Nonvolatile Flavor Precursors of Garlic to Thermal Flavor Generation C-T. HO, T-H. YU and L-Y. LIN Volatile Compounds from Dried Jimbu {Allium wallichii) A. KATTEL and J.A. MAGA
. . . 881 895 . . . 909 919
Size Exclusion Chromatography with Nitrogen Detection of Peptides and Food Grade Protein Hydrolysates by HPLC-CLND E.M. FUJINARI and J.D. MANES
929
Application of GC-MS Analysis for Studies on Bioshythesis of Lower Terpenes Incorporating Deuterated Precursors in Plant Cultured Cells K. NABETA
951
Gas Chromatographic/Mass Spectrometry Analytical Characterisation of Smoke-Liquid Flavourings to evaluate the Opportunity of use as Antibacterial Agents F. TATEO, G. CASERIO, A. ORLANDI and S. GIOVANDITTO
971
Determination of Low Levels of Aziridine in Food-Simulating Liquids by Capillary Gas Chromatography P.G. DEMERTZIS, R. FRANZ and O. PIRINGER
981
Effect of Heat Treatment on Moisture Sorption Behavior of Wheat Flours Using a Hygrometric Technique 995 K.A. RIGANAKOS and M.G. KONTOMINAS Isolation and Identification of Off-Flavor Components from Soy Milk L. FL\SHIM and H. CHAVERON
1007
Carbohydrate Composition of Raw Vegetable Soybeans Grown at the Same Location O.O. FAPOJUWO and J.A. MAGA
. . . . 1021
Pyrazine Composition as Influenced by the Smoking Conditions of Hickory Sawdust Z. CHEN and J.A. MAGA
. . . . 1025
Functional and Sensory Characteristics of Quinoa in Foods K. LORENZ, L. COULTER and D. JOHNSON
1031
Processing Equipment and Food Quality A.E. KOSTAROPOULOS and G.D. SARAVACOS
1043
Non Enzymatic Browning in Air-Drying of Washed Raisins V.T. KARATHANOS, T. KARANIKOLAS, A.E. KOSTAROPOULOS, and G.D. SARAVACOS
1057
Microwave Heating of Water-Ethanol Mixtures A. PAOLI and A. SCHIRALDI
1065
Changes in Volatile Composition of Kluyveromyces lactis Broth during Fermentation J. JIANG
. . . . 1073
Isolation and Partial Characterization of Oilseed Phenolics and Evaluation of their Antioxidant Activity F. SHAHIDI, U. WANASUNDARA and R. AMAROWICZ The Effect of Polymers on the Vapor Pressure of an 0/W Microemulsion System J.L. CAVALLO and H.L. ROSANO Prediction of Moisture Barrier Requirements for an Effervescent Sinle Serve AspartameSweetened Tablet D. APOSTOLOPOULOS and R. FUSI
1087 1101
1119
Comparing the Rates of Development of Temperature Distributions in Foods Shaped as Spheres, Cylinders and Thick Films A.E. GROSSER
1133
Effect of Oxygen on the Ethyl Acetate Production from Continuous Ethanol Stream by Candida utilis in submerged cultures G. CORZO, S. REVAH and P. CHRISTEN
1141
Changes in Microstructure and Thermal Properties of Thermally Processed Comstarch/Soy Protein Isolate Model Food Systems F.A. NYANZI, J.A. MAGA and C. EVANS
1155
Evaluation of Cookeina sulcipes as an Edible Mushroom: Determination of its Biomass Composition
1165
J.E. S A N C H E Z , A . M . M A R T I N and A.D. SANCHEZ
Interactions Between Polysaccharides and Aroma Compounds S. LANGOURIEUX and J. CROUZET Water and Ethanol Adsorption on Starchy Substrates as Biomass Separation Systems G. VARELI, P.G. DEMERTZIS and K. AKRIDA-DEMERTZI
1173 . . . . 1187
Some Mulitvariate Perspectives on Shelf life Research R.H. ALBERT and C. ZERVOS
1201
Nitrite Alternatives for Processed Meats F. SHAHIDI and R.B. PEGG
1223
Potential for Growth and Inhibition of Listeria monocytogenes in Meat and Poultry Products J.N. SOFOS, W.B. BARBOSA, H.J. WEDERQUIST, G.R. SCHMIDT and G.C. SMITH
1243
Quality of Extrusion-Cooked Poultry Meat Products J.N. SOFOS, A.S. BA-JABER, G.R. SCHMIDT and J.A. MAGA
1265
Use of Starch for Water Binding in Restructured Beef Products J.N. SOFOS, J.A. PEREJDA and G.R. SCHMIDT
1281
Enzyme Generation of Free Amino Acids and its Nutritional Significance in Processed Pork Meats F. TOLDRA, M. FLORES and M-C. ARISTOY
1303
Isolation of Flavor Peptides from Raw Pork Meat and Dry-Cured Ham M-C. ARISTOY and F. TOLDRA
1323
The Effect of Fat Content on the Quality of Ground Beef Patties N.H. WONG and J.A. MAGA
1345
Fractionation and Characterization of Extracts of Chicken Fat Obtained with Supercritical Carbon Dioxide D.L. TAYLOR and D.K. LARICK BMP: A Flavor Enhancing Peptide Found Naturally in Beef. Its Chemical Synthesis, Descriptive Sensory Analysis and Some Factors Affecting its Usefulness A.M. SPANIER, J.M. BLAND, J.A. MILLER, J. GLINKA, W. WASZ and T. DUGGINS
1353
1365
Effects of storage Under Carbon Dioxide Atmosphere on the Volatiles, Phenylalanine Ammonia Lyase Activity and Water Soluble Constituents of Strawberry Fruits 1379 V. DOURTOGLOU, A. GALLY, V. TYCHOPOULOS, N. YANNOVITS, F. BOIS, M. ALEXANDRI, S. MALLIOU, M. RISSAKIS and M. BONY Studies on the Hydrolysis of Fish Protein by Enzymatic Treatment A.M. MARTIN and D. PORTER
1395
Production of Protein Hydrolysate from Lobster (Panulirus spp.) G.H.F. VIEIRA, A.M. MARTIN, S. SAKER-SAMPAIAO, C.A. SOBREIRAROCHA and R.C.F. GONCALVES
1405
Sensory Acceptance and Overall Quality of a Histidine Added Fish Sauce N.G. SANCEDA, T. KURATA and N. ARAKAWA
1417
Extraction of Value-Added Components from Shellfish Processing Discards F. SHAfflDI
1427
Protein Concentratres from Underutilized Aquatic Species F. SHAHIDI
1441
Extending the Shelf-Life of Seafood Using a Multiple Barrier Process S.M. CONSTANTINIDES, S.M. EINARSSON, Y. BENJA-ARPORN and A. PAPPAS
1453
Fresh Orange Juice Flavor: a Quantitative and Qualitative Determination of the Volatile Constituents M.G. MOSHONAS and P.E. SHAW
1479
Effect of Microwave Heating on Roasted Nut Flavor D.E. ZOOK, C. MACKU and D. DEMING
1493
Peanut Flavor Formation During Roasting as Affected by Atmospheric Conditions R.Y.-Y. CHIOU and C-Y. TSENG
1519
Color Sorting of Lightly Roasted and Deskinned Peanut Kernels to Diminish Aflatoxin and Retain the Processing Potency R. Y.-Y. CHIOU, P-Y. WU and Y-H. YEN
1533
Carbohydrate Metabolism in Peanuts During Postharvest Curing and Maturation J.R. VERCELLOTTI, T.H. SANDERS, S.-Y. CHUNG, K.L. BETT and B.T. VINYARD
1547
The Phenolic Composition of Table Grapes E. REVILLA, J.M. ESCALONA, E. ALONSO and V. KOVAC
1579
Selection of Spontaneous Strains of Saccharomyces cerevisiae as Starters in their Viticultural Area A.I. BRIONES, J.F. UBEDA, M.D. CABEZUDO and P. MARTIN-ALVAREZ Hydrolysis of Grape Glycosides by Enological Yeast P-Glucosidases I. ROSI, P. DOMIZIO, M. VINELLA and M. SALICONE Analytical Research to Identify Illegal Modifications of D/H Values in Sugar Mixtures F. TATEO, G. CANTELE, B. DAMIA, G. RUSSO, L. PANZA and E. BOUSQUET Partial Characterization of P-Damascenone Precursors and Toxicity Studies of Free P-Damascenon in Cell Cultures of Vitis x labruscana cv. Concord Grapes K.B. SHURE and T.E. AGREE
1597
1623 . . 1637
1645
Influence of Nitrogen Compounds in Grapes on Aroma Compounds of Wines A. RAPP and G. VERSINI
1659
The Contribution of Oak Lactone to the Aroma of Wood-Aged Wine J.R. PIGGOTT, J.M. CONNER and J.L. MELVIN
1695
Possibilities of Characterizing Wine Varieties by Means of Volatile Flavor Compounds A. RAPP Classification of Italian Wines on a Regional Scale by Means of a Multi-Isotopic Analysis A. MONETTI, G. VERSINI and F. RENIERO
. . 1703
1723
Flavour Development in Whisky Maturation J.R. PIGGOTT, J.M. CONNER and A. PATERSON
1731
Investigation of Flavour Compounds in Whiskey Spent Lees K. MacNAMARA, P. BRUNERIE, F. SQUARCIA and A. ROZENBLUM
1753
An Application of Centrifugal Counter-Current Chromatography on Flavor Chemistry Separation of Aroma Substances in Whisky New Distillates T. TANIGUCHI, N. MIYAJIMA and H. KOMURA Aroma Compounds of Arbutus Distillates G. VERSINI, R. SEEBER, A. DALLA SERRA, G. SFERLAZZO, B. de CARVALHO and F. RENIERO
1767 1779
XIU
Aroma Components of Raki i. YAVA§ and A. RAPP
1791
Recent Evaluation of Malt Quality M. MOLL
1813
Lipolytic Activity of Cheese Related Microorganisms and its Impact on Cheese Flavour M. EL SODA, J. LAW, E. TSAKALIDOU and G. KALANTZOPOULOS Fermentation of Black Olives with Application of Starter Culture and Aeration M. BORCAKLI, G. OZAY and L ALPERDEN Changes in Soluble Sugars in Various Tissues of Cultivated Mushroom, Agaricus bisporus. During Postharves Storage S.O. AJLOUNI, R.B. BEELMAN, D.B. THOMPSON and J-L. MAU Concentration of Heavy Metals and Nutrients in the Leaves of Certain Forest Species Irrigated by Treated Wastewater P. DRAKATOS, G. KALLISTRATOS, I. FANARIOTOU, 1. KALAVROUZIOTIS, D. SKOURAS and M. STOYIANNI
. . 1823 1849
1865
1881
A Rapid Method for Determining the Water Dynamics (Uptake and Loss) of MoistureSensitive Foods S.G. GILBERT, G.W. GREENWAY, J.X. LIU and O. RAMON
1895
The Inhibitory Effect of the Essential Oils from Basil (Ocimum basilicum) and Sage {Salva officinalis) in Broth and in Model Food System Ch.Ch. TASSOU and G.J. E. NYCHAS
1925
Comparison of some Physicochemical Characteristics Between Solid and Fluid Chios Mastic Resin M. MELANITOU, D. PAPANICOLAOU, K. KATSABOXAKIS and K. STAMOULA Food Production in Arid and Desert Areas Using the "KALLIDENDRON" TECHNOLOGY G. KALLISTRATOS, P. DRAKATOS and I.-M. KALLISTRATOS
1937
1947
The Essential Oil of Allium Sativum L., Liliaceae (Garlic) N.A. SHAATH and F.B. FLORES
2025
Microbeam Molecular Spectroscopy of Biological Materials D.L. WETZEL
2039
Analysis of Drinking Water near and Far from Thermal Springs Using Instrumental Neutron Activation Analysis E.T. CONTIS Emulsifying Properties of Lupin Seed Proteins S. ALAMANOU and G, DOXASTAKIS
2109 2129
XIV
Multiresponse Optimization by a Normalized Function Approach J.D. FLOROS and H. LIANG Back Propagation Neural Networks: Theory and Applications for Food Science and Technology V. GNANASEKHARAN and J.D. FLOROS
2139
2151
Gentic Algorithms and Fuzzy Theory for Optimization and Control of Food Processes . . . . 2169 LG. VRADIS and J.D. FLOROS Volatile Compounds in Wheat Cultivars from Several Locations in Kansas L.M. SEITZ
2183
Autolysis of Lactid Acid Bacteria: Impact on Flavor Development in Cheese M. EL SODA, N. FARKYE, J.C. VUILLEMARD, R. SIMARD, N. OLSON, W. EL KHOLY, E. DAKO, E. MEDRANO, M. GABER and L. LIM
2205
SUBJECT INDEX
2225
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1101
The Effect of Polymers on the Vapor Pressure of an OAV Microemulsion System J. L. Cavallo^ and H. L. Rosano^ ^Kraft General Foods, Maxwell House Coffee Company, 555 South Broadway, Tarrytown, NY 10591, U.S.A. ^The City College, City University of New York, Department of Chemistry, 138th Street & Convent Avenue, New York, NY 10031, U.S.A. Abstract The vapor pressure of an oil-in-water microemulsion system consisting of n-octane/20cc of 5% NaCl + 0.01 N NaOH/lg of sodium dodecyl sulfate (SDS)/ 3.7g of dodecyl dimethylamine oxide (DDAO) and n-octane/SDS/water/titrated to clarity with DDAO was determined as a function of the volume of hydrocarbon in the dispersed phase. In the presence of a cationic polymer. Polymer JR, the dispersed droplets are destabihzed due to surfactant depletion from the microemulsion droplet interface. In the presence of an anionic polymer, carboxymethyl cellulose (CMC), the microemulsion droplets appear to be stabilized over larger volume fractions of oil. Introduction Emulsions play a key role in many cosmetics and industrial products in current use. Much has been written over the last three decades on the stability and formation of these oil-in-water (o/w) or water-in-oil (w/o) dispersions (1). Today's industrial formulator is primarily concerned with understanding and developing the most functional o/w or w/o systems possible for a particular appHcation. Stable transparent systems (whether o/w or w/o) called microemulsions can be formulated when the right surface-active ingredients are used (2,3). Possible appHcation for these systems range from products with extended shelf life for the food industry to dehvery systems for active ingredients in pharmaceuticals. More recently, fluorocarbon microemulsions have been developed as media for oxygen transport and as possible synthetic blood substitutes. Mixtures of oil and water in the presence of surface-active agents usually form coarse, optically opaque emulsions that separate on standing. In some
1102 cases, transparent mixtures of oil and water can be prepared when the initial emulsion is titrated to clarity (microemulsion) with a cosurfactant. Relatively small negative free energy changes may explain why the method of preparation plays an important role in microemulsion formation (4). The proper method of adding the ingredients appears to be responsible for producing a transitory zero interfacial tension due to diffusion of cosurfactant across the oil/water interface, during which time both maximum interface generation and spontaneous clearing of the primary emulsion occur (2, 3). The most significant difference between emulsions (opaque) and microemulsions (transparent) lies in the fact that while applying work on an emulsion or increasing the surfactant concentration usually improves stability. This is not the case with microemulsions, which appear to be dependent for their formation on specific interactions among the constituent molecules. If these interactions do not take place, neither applying work nor increasing the surfactant concentration will produce a microemulsion. On the other hand, once the conditions are right, spontaneous formation occurs and little mechanical work is required (5). Terms like microemulsion (6), swollen micellar solution (7), micellar emulsion (8, 9), middle phase (10), unstable microemulsion (11), and spontaneous transparent emulsion (12) have all been used to describe these systems. The broadest definition may simply be "a transparent dispersion of oil, water, and surfactant that forms spontaneously upon addition of a cosurfactant." As originally suggested by Schulman et al. (13, 14), microemulsions form when the surfactant and cosurfactant, in just the right ratio, produce a mixed adsorbed film that reduces the interfacial tension y^ to a value below zero. They concluded that y^ must have a metastable negative value, giving a negative free energy variation -y^ dA (where dA is the change in interfacial area) responsible for spontaneous dispersion. The interfacial tension y^ in the presence of a mixed film is given by Yi = Yo/w - ^i where yo/w is the o/w interfacial tension in the presence of the film and ni is the interfacial surface pressure of the film. At equilibrium y^ becomes zero. If this concept of zero interfacial tension is accepted, stabilization of the microemulsion is implied. This model does not seem to be conceptually valid; however, since a zero y^ would not require the dispersed phase to be distributed in spherical droplets, as is found in the systems under discussion (15). Rosano et al. (11) have considered the dynamic role of the cosurfactant in lowering the interfacial tension during the titration of a coarse emulsion into a transparent dispersion. They pointed out that during the addition of a cosurfactant to an emulsion (either o/w or w/o), excess cosurfactant accumulated at the oil/water interface during transport, reducing the interfacial tension to well
1103 below the positive equilibrium value. The surfactant retards the cosurfactant interfacial transport; a prolonged low interfacial tension helps in the formation of a large increase in the interfacial area. Eventually, YI regains a positive value responsible for the resolution of the system into microemulsion droplets. Emulsion and microemulsion stability appear to be dependent not on the value of the y^ alone but rather, and primarily, on the structure of the film surrounding each droplet (3, 16). For a given oil/surfactant pair, cosurfactant steric requirements determine the volume of the dispersed phase that can be stabilized. These systems apear to be oU and cosurfactant dependent. Surfactant, cosurfactant, the nature of the oil, and the nature of the aqueous phase are four interacting variables that determine the size of the dispersed phase droplet when microemulsions are formed. A wealth of experimental results show that only specific component combinations can produce transparent systems and that the various components must be put together in just the right order to produce a microemulsion. We are thus left with two basic questions: 1. Are these systems kineticaUy stable, since they may show path dependency in their formation? 2. Are they thermodynamicaUy stable, even though their occasional path dependency properties may reflect activation energy barriers that these systems must overcome during their formation? In a previous paper (4) it was shown that o/w emulsions of sodium long-chain sulfate/n-hydrocarbon/5% NaCl can be titrated to clarity with specific long-chain dimethylamine oxides. For the six systems investigated, all were found to have small negative free energy values associated with cosurfactant absorption during microemulsion formation. These small free energy values seem to explain why the manner of combining the various components is important during microemulsion formation. The right order of addition appears to lower activation energy barriers that these systems must overcome during their formation. In addition, microemulsion formation appears to be an entropy-driven process, as shown by titration experiments. It has always been assumed that microemulsions are composed of discrete individual spherical droplets (3). In a previous paper (17), we demonstrated that o/w microemulsions prepared with low volume fraction of hydrocarbon show an overall decreased vapor pressure relative to the vapor pressure of the continuous surfactant phase. This behavior is similar to that of a high molecular weight polymer solution or a regular coUoidal dispersion, where the vapor pressure of the solution is less than that of the continuous phase. It was also demonstrated, based on vapor pressure analysis, that o/w microemulsions clearly exhibit two distinct regions of transparency depending on the volume of hydrocarbon in the dispersed phase. For low volumes of hydrocarbon, encapsulated non-interacting droplets are formed in solution, while for higher volumes of hydrocarbon, a dynamic merging equilibrium exists in which the droplets are continuously breaking and reforming; i.e., percolation (17, 18). Similar phase behavior has been demonstrated by Weatherford (19) using vapor
1104 pressure analysis for w/o diesel fuel microemulsion systems composed of a surfactant mixture of oleyldiethanolamide, diethanolamide, and diethanolanmonium oleate. The results which indicate that two regimes of differing phase behavior exist as the water content is varied, suggest a transition from inverted micellar solutions, at low water concentrations to microemulsions at a higher water content. These results support the fact that the internal droplet structure can vary depending on the internal phase volume, an important physical-chemical property of microemulsion systems of considerable interest to the formulator of consumer goods. Since the dispersed phase volume governs the structure and properties of the droplets formed, microemulsions offer a broad array of applications. For example, microemulsion systems can be used to encapsulate and reduce the rate of oxidation and/or hydrolysis of oils (or oil-soluble ingredients). This effect is due to the fact that, for low volume fractions of oil, microemulsions offer complete encapsulation of the oil by the surfactant sheath surrounding the droplets. A specific example might involve the encapsulation of citrus oil in o/w microemulsions. In addition, since the presence of droplets (low volume fraction of oil) results in a lowering of the vapor pressure, these systems can be used to stabilize and deliver hydrocarbons with high vapor pressure (e.g., isopentane and pentane). This effect cannot be achieved with conventional emulsion systems. Droplet aggregation in w/o microemulsions has also been investigated and discussed by a variety of authors (3, 10, 17). For systems containing low concentrations of water, isolated non-interacting droplets have been found to be the primary droplet structure (18). At higher water concentrations, droplet interactions were found to be either repulsive, with short-lived collisions and no overlap between colliding droplet interfaces, or attractive, with collisions of larger durations and the formation of transient droplet clusters. It has also been shown that the probability of such transient merging in ternary systems is low (~ 10"3 per collision). For quaternary systems the merging probability between droplets is quite high (~ 1), indicating that the interactions between droplet cores play an important role in the droplet structure of these systems. The need to understand factors related to the stability of coUoidal dispersions has long been the central motivating factor in the study and development of surface science. "Stability" in this context is generally understood to mean kinetic stability; i.e., stability imposed by a strong repulsive barrier acting against contact between the suspended particles (20). For emulsion systems, London-van der Waals dispersion forces are at the origin of the tendency of coUoidal systems to coagulate and aggregate. The repulsive forces needed to stabilize the dispersion against these attractive forces are usually of two tj^es: 1. Coulombic repulsion due to electric charges on the particle surface; e.g., electrostatic interactions between the ionic double layers surrounding the particles. 2. Static repulsion introduced by large molecules or polymers adsorbed on the particle surface.
1105 In the case of microemulsions, stability concerns are similar to those encountered for emulsion systems (or colloidal dispersions), with emphasis placed on reducing and/or retarding the rate of droplet coalescence or aggregation. As suggested by Napper (21), the most functional stabilizers are amphipathic molecules that are either block or graft co-polymers. These molecules have been shown to be effective stabilizers when added to colloidal dispersions already formed (or vice versa). The most effective stabilizers consist of both anchor groups and stabilizing moieties. The stabilizing moieties must be soluble in the dispersion medium to be effective, whereas the anchor groups function most efficiently if they are nominally insoluble in the dispersion medium and have an affinity to adsorb at the particle (droplet) interface. This paper will present a basic overview of the physical-chemical properties of microemulsions. Topics highhghted will include microemulsion preparation, vapor pressure measurements, activation energy determination, and the relationship between vapor pressure measurements and microemulsion droplet size. In addition, the effect of dissolved polymers in the continuous phase of o/w microemulsions will be investigated in order to demonstrate the effect these large molecules have on dispersed droplet interactions. Both anionic and cationic polymers will be investigated. It is hypothesized that polymers added to the continuous phase of microemulsion systems may reinforce the encapsulated droplets or to some extent prevent the droplets from merging. If the presence of polymers in the aqueous phase can alter droplet interactions, this effect should be demonstrated by vapor pressure analysis. Experimental Section •
Chemicals
Polymer JR-400, a quaternary water-soluble cationic nitrogen-substituted cellulose ether (Union Carbide Corp.) and sodium carboxymethyl cellulose (CMC), a low viscosity anionic water soluble pol5mier, 99.5% purity (approx. Mol. Wt. 90,000) (Hercules Inc.), were used. N-octane (99% + gold label) and sodium dodecyl sulfate (SDS) were provided by Aldrich Chemical Company. Dodecyldimethylamine oxide (DDAO) 30% active was obtained from Ony^ Chemical Company. Freshly distilled water was used in all solution preparations. •
Preparation and Vapor Pressure Measurements of Polymer Microemulsion Systems
In a water-jacketed beaker maintained at 25°C, initial emulsions were prepared containing 15 cc of water, 1 g SDS, and various volumes of n-octane. These emulsions were titrated to clarity (92% T @ 520 nm.) with DDAO, using the titration technique (2). To these transparent systems 5 cc of a 0.1% JR-400 or 0.1% CMC polymer solution was added. At the concentration of polymer
1106 used, no viscosity changes were observed. In all cases the systems remained transparent upon addition of polymer. The microemulsions were then thoroughly stirred for 15 minutes to ensure complete mixing. For each microemulsion the vapor pressure was determined with an isoteniscope. The microemulsion was placed in the isoteniscope and submerged in a hot water bath, and the system was allowed to boil for 10 minutes to evacuate any excess air present. The bath was then slowly cooled untU an equilibrium between the liquid and vapor was obtained in the isoteniscope, at which the pressure within the system and the temperature of the water bath were recorded. The pressure was then further reduced, and the procedure was repeated. Using the ClausiusClapeyron equation
^ 0
R
IT
(1)
To J
where JO a n d p o ^^® the vapor pressures at T a n d TQ, AH^ is the heat of vaporization, and R is the gas constant, the vapor pressure and heat of vaporization were determined. •
Activation Energy
As shown in Figure 1, the slope of the vapor pressure curve increases with temperature. [This effect was thoroughly discussed in a previous paper (17)]. It has been hypothesized that this increase is due to the breaking and re-forming of droplets in solution (2, 17). As the temperature is increased, the rate of breaking increases, as does the breaking rate constant K = (dP/dV) T • Using an Arrhenius type equation: hi K.
n
I
^1
1.
I
(2)
where K^ and K2, are the slopes corresponding to temperatures T^ and T2 and R is the gas constant, an activation energy E^ of 35.4 kJ/mol was calculated, corresponding to the amount of energy required for hydrocarbon molecules within the droplet core to get into the vapor phase. This value is higher than that for the microemulsion prepared in a water continuous phase where E^ = 31.5 kJ/mol., and higher than the activation energy of pure n-octane, Ea = 8.8 kJ/mol., obtained through viscosity measurements by plotting In r\ vs. I/T- The difference appears to be related to both the strength of the surfactant sheath and the effect of poljmaers at the interface.
1107 Results The vapor pressure for each microemulsion was determined from Eq. 1 by plotting In p vs. 1/T. In each case a straight line was obtained, with excellent correlation (>0.99). From the slope of the line the heat of vaporization was determined. The same procedure was repeated for all systems investigated containing constant amounts of saline, surfactant, and cosurfactant, and various volumes of n-octane.
VOL. OCTANE • !
Figure 1: Vapor pressure diagram for the microemulsions systems: 5% NaCl (pH 12)/sodium dodecyl sulfate/n-octane/dodecyldimethylamine oxide between 15 and 40°C. Plotted as a function of amount of n-octane.
1108 Vapor pressure vs. volume of n-octane was determined for microemulsions between 15 and 40°C. The results, shown in Figure 1, demonstrate that two distinct regions are present, depending on the volume of n-octane used. Microemulsion systems prepared with up to 1.1 cm^ of n-octane show a decreased vapor pressure (e.g., at 30°C, 32.4 mm Hg for the system prepared with no oil to 25.4 mm Hg). Within this region the vapor pressure remains constant. This behavior is similar to that of a high molecular weight polymer solution or a regular colloidal dispersion where the vapor pressure of the solution is less than that of the continuous phase. Assuming that this region is composed of monodispersed noninteracting droplets, and using van't Hoff equation GMW =
RT — T
^^^
where GMW is the average gram molecular weight, R the gas constant, T temperature, C solution concentration (g/1), and n=pQ-p [i.e., the vapor pressure difference between the solution containing no oil (p^ and microemulsions containing various volumes of n-octane (p)], the GMW per droplet was calculated. GMW values were determined for systems containing up to 1.4 cm^ of n-octane. From our experimental results, for volumes of n-octane larger than 1.1 cm^, the increase in the value of the GMW indicates that the droplets start to percolate. It should also be noted that systems prepared containing 0.01-0.1 cm^ of n-octane clearly demonstrate that lamellar structures exist between SDS/ DDAO. The viscosity within this region was found to increase to ca. 100 cp and decrease to ca. 10 cp when the microemulsion contained 0.1 cm^ of n-octane. Although the vapor pressure of these systems was not determined, the addition of hydrocarbon clearly resulted in the resolution of the lamellar structures into microdroplets producing measurable osmotic pressures. Particle Size Measurements. From the average GMW we determined droplet radii by considering that the ratio of GMW to SW (the average weight of the associated surfactant and cosurfactant at the droplet interface) gives the number of surfactant molecules at the droplet surface. For spherical droplets of surface area 4nx^ GMW SW
=
^^ 4T"5"
(4)
where a is the average cross-sectional area per surfactant molecule, droplet radii values were determined, assuming o = 45A2 per molecule (7). These values were found to be in agreement with results obtained by photon correlation spectroscopy (8), as shown in Table 1.
1109 TABLE 1: Average GMW per Droplet and Radii Values Determined by Vapor Pressure Analysis (Assuming a = 45 A^ per molecule) and by Photon Correlation Spectroscopy as a Function of Increasing Volume of n-octane at 30°C Vol. n-octane, cm^ 0.1 0.25 0.5 0.75 1.0 1.1 1.25 1.4
GMW 30°C 4.95 X 105 5.9 X 105 4.99 X 105 6.14x105 6.03 X 105 6.29 X 105 1.77 X 106 1.98 X lO'^
radiuSyp, A 85.4 93.2 85.8 95.1 94.3 96.3 161.5 540.2
radiumpQg, A 80.8 76.8 85.6 80.5 94.4 115.0 198.0 958.0
Beyond 1.1 cm^ of n-octane, the vapor pressure increases, as shown in Figure 1. Within this region droplets appear to merge and re-form continuously. At 30°C, these systems remain transparent (90% transparent @ 520 nm) up to 1.5 cm^ of n-octane. Between 1.5 and 2.5 cm^ of n-octane the systems become cloudy, whUe at 2.5 cm^ of n-octane, separation is clearly visible. At 2.8 cm^ of n-octane, the vapor pressure increases to 52 mm Hg, the sum of the individual components (saline surfactant solution 32.4 mm Hg and 19 mm Hg for n-octane), suggesting an emulsification-failure instability (11). When droplets contain a small volume of n-octane, the hydrocarbon is strongly associated with the tails of the surfactant molecules. This interaction precludes the presence of any n-octane vapor in the vapor phase above the microemulsion system. The effect of increasing the volume of n-octane results in an increase in the total number of droplets and a lowering of the vapor pressure. As the volume of n-octane is further increased, the situation changes: the radius of the droplets increases, more n-octane is present in the droplet core, and the microemulsion shows the characteristics of bulk n-octane. At this point, closed and open microemulsion droplets exist, with a resultant increase in vapor pressure. Increasing the volume of n-octane results in a core showing characteristics of bulk hydrocarbon. The hydrocarbon vapor is free to enter the vapor phase, resulting in an increase in the measured vapor pressure. Similar droplet behavior has been shown to exist for AOT/isooctane/water microemulsions systems as the water content was varied (12). Figure 2 shows the vapor pressure behavior of an o/w microemulsion for various volumes of n-octane in the presence and absence of polymer (anionic and cationic) dissolved in the aqueous phase. Curve A represents the vapor pressure behavior of a microemulsion prepared with 1 g SDS / 20 cc water / 3.7 g DDAO and various volumes of n-octane in the dispersed phase. As indicated by this curve (see discussion of Figure 1) the vapor pressure above the microemulsion
1110 surface decreases when the dispersed phase contains low volume fractions of noctane. The structure of the droplets within this region of low vapor pressure is that of encapsulated droplets surrounded by a surfactant/ cosurfactant sheath. Increasing the volume of n-octane in the dispersed phase results in an increase in the measured vapor pressure above the microemulsion surface. This increase in vapor pressure is due to the existence of a dynamic equilibrium between the merging and re-forming of droplets in solution. The vapor pressure increases until a maximum vapor pressure is reached (-^ 50 mm Hg). Further addition of n-octane results in the separation of oil and water; i.e., a two-phase system is formed.
60
50
tyc
fy^ L*s-*^' 0-0—0—»~
VAPOR PRESSURE
A
B
40
(mm Hg) 30
20
m
10
m
y
i
VOLUME N-OCTANE (ml)
1 FIGURE 2:
1
1
Change in vapor pressure vs. volume of n-octane for a microemulsion prepared with water / 1 g SDS / n-octane and DDAO (curve A) containing Polymer JR (curve B) or CMC (curve C) in the continuous phase.
nil Curve B shows the vapor pressure behavior of a microemulsion prepared with 1 g SDS /15 cc water + 5 cc of 0.1% Polymer JR-400 / 2.4 g DDAO and increasing amounts of n-octane. It is observed that the vapor pressure decreased to approximately 24 mm Hg upon addition of 0.25 cc n-octane and remains low up to the addition of 0.5 cc. When the volume of n-octane exceeds 0.5 cc, a sharp increase in vapor pressure results. For a microemulsion prepared with 0.8 cc n-octane, a maximum vapor pressure of 41 mm Hg is reached. The vapor pressure was found to remain high up to the addition of 1.3 cc, where phase separation occurs. Curve C shows the vapor pressure of a microemulsion prepared with 1 g SDS /15 cc water + 5 cc of 0.1% CMC / 3.7 g DDAO and various volumes of noctane. Vapor pressure results indicate that when CMC is added to the continuous phase of microemulsions containing large volumes of n-octane, the vapor pressure is reduced compared to that for microemulsions prepared in a pure aqueous phase. This result indicates that CMC acts as a stabilizer for the microemulsion by preventing and/or reducing the rate of droplets merging. For 1 cc of n-octane, it can be seen that the vapor pressure is approximately equal to that of a microemulsion prepared in pure water. Increasing the volume of n-octane does not result in a sharp vapor pressure increase. It is also interesting to note that phase separation was observed for microemulsions prepared in pure water containing 2 cc of n-octane in the dispersed phase, but not for microemulsions containing CMC. This fact further supports the hypothesis that the role played by CMC in this particular system is that of a stabilizer. These results clearly demonstrate that the droplet interactions in microemulsion systems can be strongly influenced by the addition of polymers with the end result, in some cases, of stabilization or destabilization of the microemulsion. Discussion and Conclusion The results shown in Figure 2 demonstrate that cationic or anionic poljmaers added to the continuous phase of o/w microemulsion systems can significantly influence droplet-droplet interactions over a wide range of oil volumes. Surface tension measurements on solutions of Polymer JR in water show that the polymer is a weak surface-active agent. At a level of 0.1%, the polymer was found to reduce the surface tension of pure water by ca. 4 dyn/cm (23). This small reduction of the surface tension of water suggests that the polymer is very active in the bulk phase. Using surface tension data, Goddard (23) demonstrated that addition of Polymer JR caused a marked decrease in the surface tension of SDS surfactant solutions at low concentrations. He suggested that, in certain cases, complexes would form between cationic polymers and anionic surfactants. This interaction would modify the pol5nner and render it more surface active by adsorption of surfactant "head-head" onto cationic sites along the polymer chain. Modification
1112 of the polymer was demonstrated by an increase in the overall viscosity of the system. Addition of Polymer JK introduced into the continuous phase of an o/w microemulsion prepared with water / SDS / n-octane and DDAO seems to have a destabilizing effect on the microemulsion droplets. For relatively small volumes of n-octane, the vapor pressure was found to increase sharply, leading to a breakdown of the microemulsion and to eventual phase separation. It was thought that with the addition of a cationic polymer to a microemulsion prepared with an anionic surfactant, the polymer would "wrap around" the microemulsion droplets and enhance their stability over larger volumes of n-octane. Contrary to expectation, however, destabiUzation of the microemulsion occurred. These results are consistent with the formation of complexes between the cationic polymers and SDS. Adsorption of SDS onto the cationic sites along the polymer chain reduces the amount of surfactant at the o/w interface. The complex formed between Polymer JR and SDS reduces droplet stability. Microemulsion systems prepared with CMC in the aqueous phase are shown to exhibit a stabilizing effect when compared to microemulsions prepared in a pure water continuous phase or a continuous phase containing Polymer JR. Figure 2 shows that when CMC is added to the continuous phase of a microemulsion containing large volumes of n-octane, the vapor pressure is lower than for microemulsions prepared in a pure water phase. Since there is no association between the surfactant and the anionic polymer, there is no depletion of the surfactant at the o/w interface. In addition, since both surfactant and polymer are anionic, they produce a repulsive interaction. This interaction aids in keeping the surfactant at the o/w interface while enhancing the droplet stability. The interaction between hydroxyethylceUulose and SDS has been investigated by Goddard (23) using surface tension measurements . These measurements clearly show that there is Uttle or no association between hydroxyethylceUulose and SDS in the bulk phase; when hydroxyethylceUulose is added to an SDS solution, no appreciable surface tension lowering occurs. This suggests that microemulsions containing CMC in the aqueous phase contain only unassociated CMC. Free CMC in the bulk phase should therefore exhibit a repulsive interaction between other CMC molecules and the anionic surfactant heads of the microemulsion droplets. This effect should interfere with microemulsion droplet merging resulting in stable microdroplets over larger volumes of hydrocarbon. Figures 3 and 4 show the interaction between microdroplets in the presence of cationic or anionic polymers.
1113
Figure 3: Microdroplet behavior in solution; (A) water continuous phase; (B) water + Polymer JR.
F i ^ r e 4: Microdroplet behavior in a water + CMC solution.
1114 It has been shown that mixing surfactants and poljmaers in solution leads, under certain circumstances, to complex formation through binding or adsorption of the surfactant molecules either individually or as aggregates onto the macromolecule (24). In the case of a cationic pol5maer and an anionic surfactant, complex formation occurs at very low polymer concentrations (<0.1%), with precipitation of the complex. It has also been suggested, by Goddard et al. (25), that increasing the surfactant concentration can result in a resolubilization of the precipitate as a polyelectrolyte complex with a net charge opposite that of the original poljnner molecule. In reference to Figure 2 (Curve B), it is observed that as the volume of n-octane is increased, the vapor pressure decreases (between 0.2 cc and 0.8 cc n-octane). Beyond 0.8 cc n-octane the vapor pressure increases to 40.8 mm Hg whUe the system remains clear. Beyond 1.0 cc of n-octane, the system begins to cloud until phase separation occurs. A possible explanation for the extensive lowering of the vapor pressure (between 0.2 and 0.8 cc of n-octane) is that the lowering is the result of both the presence of microdroplets in solution and the formation of the surfactant/polymer complex between SDS and Pol5nner JR. Since the reduction in vapor pressure is a colligative property and is related to the concentration of both droplets and macromolecules in solution, increasing the concentration of the surfactant/polymer complex will reduce the vapor pressure above the microemulsion surface, as seen in curve B (Figure 2). Several authors (26, 27) have proposed theoretical accounts of the cooperative linking of surfactants onto polyions based on the Zimm-Bragg Theory of helixcoil transitions in polypeptides (28). These theories are best summed up as describing a nucleation and growth mechanism between the surfactant and the polymer. If CMC added to microemulsions stabilizes the droplets, the activation energy required to break the surfactant sheath surrounding the droplets should be slightly higher than in the absence of CMC. The activation energy for microemulsions prepared with CMC in the dispersed phase demonstrates that microemulsion droplets are significantly stabilized in the presence of CMC, as shown by the vapor pressure curve in Figure 2. The activation energy was found to be 35.4 kJ/mol. This value clearly demonstrates a stabilizing effect over microemulsions prepared in pure water, where the activation energy was found to be 31.5 kJ/mol. Surfactant/polymer association ment that activation energy values for microemulsions prepared in the presence of Polymer JR were not obtained, since these systems rapidly separated. Activation energy barriers to coalescence between two stabilized w/o microemulsion droplets have been determined (3). As two droplets approach each other, the interfacial films surrounding each of the droplets begin to mix. As the monolayers mix, solvent is dispersed from the interfacial layers, causing an increase in free energy as the film becomes locally more concentrated in surfactant and cosurfactant. When such a change in composition increases the free energy, the droplets wiU be stabilized against coalescence. In systems where the dispersed phase volume was increased from 1.0 to 1.44 ml while the
1115 droplet radius was kept at 25 A, a constant amount of surfactant caused an energy change of 2kT. Systems with smaller dispersed phase volume were found to have higher energy barriers against coalescence, since the concentration of surface-active components at the interface is higher. In conclusion, the vapor pressure results discussed in this paper demonstrate that two distinct regions of vapor pressure behavior exist depending on the volume of oil in the dispersed phase. At low volume fractions of oil, homogeneous, non-interacting droplets exist. These droplets are encapsulated by a surfactant/cosurfactant sheath. At high volume fractions of oil, a dynamic equilibrium exists between the breaking and re-forming of the droplets. It has also been shown that the presence of poljnners can stabilize or destabilize the microdroplets present in solution. A microemulsion formulated with an anionic surfactant (e.g., SDS) can be stabilized by an anionic polymer (CMC) present in the continuous phase. The mechanism proposed takes into account the repulsive interactions between the surfactant and the polymer. This interaction is responsible for maintaining the surfactant at the o/w interface and enhanced droplet stability. In addition, the presence of the anionic polymer reduces the rate of droplet merging, another factor that improves droplet stability. Microemulsions formulated with an anionic surfactant (e.g., SDS) and in the presence of a cationic polymer (Polymer JR) result in destabilization of the microdroplets. Droplet destabilization in the presence of a cationic polymer is due to the formation of a polymer/surfactant complex. The formation of this complex results in the depletion of surfactant from the droplet interface. The results highUghted in this article clearly demonstrate that microemulsions can be formulated for many industrial applications. The use of microemulsion systems offers many advantages over conventional emulsions. It is left to the industrial formulator to design these systems for specific product applications.
1116 References 1 H. L. Rosano, J. L. Cavallo, D. L. Chang, J. H. Whittam. J. Soc. Cosmet., Chem. 39 (1988) 201, 2 H. L. Rosano. Journal Soc. Cosmetic Chem. 25 (1974) 609. 3 W. E. F. Gerbacia, H. L. Rosano, H. L. CoUoid and Interface Science. M. Kerker, Academic Press, New York. Vol. II (1976) 245. 4 H. L. Rosano, J. L. Cavallo, G. B. Lyons. In Microemulsion Systems. H. L. Rosano and M. Clausse, editors. Marcel Better, Inc. New York, Basel. (1987) 259-275. 5 T. F. Tadros. Structure/Performance Relationships in Surfactants. M. J. Rosen (ed).. 253rd ACS Symposium Series ACS. Washington, DC. (1984) 154. 6 W. Stoeckenius, J. H. Schulman, L. M. Prince. KoUoidZ. 169 (1960) 170. 7 K. Shinoda, H. Kunieda. KoUoidZ. 42(a) (1973) 381. 8 A. W. Adamson. J. CoUoid and Interface Sci. 39 (2) (1969) 261. 9 W. E. Gerbacia, H. L. Rosano, M. Zajac. Am. Oil Chem. Soc. 53 (1976) 101. 10 M. L. Robbins. in MiceUization, Solubilization and Microemulsions. R. L. Mital, (Ed). Plenum Press. New York, vol.2 (1977) 713-753. L. E. Scriven. in MiceUization, Solubilization and Microemulsions. R. L. Mital, (Ed). Plenum Press. New York. vol. 2 (1977) 877-893. 11 H. L. Rosano, T. Lan, A. Weiss, J. H. Whittam, W. E. Gerbacia. J. Phys. Chem. 85 (1981) 468. 12 T. P. Hoar, J. H. Schulman. Nature. 152 (1943) 102. 13 J. E. Bowcott, J. H. Schulman. Z. Electro-Chem. 59(4) (1955) 283. 14 J. H. Schulman, J. H. Montague. Ann. N.Y. Acad. Sci. 92 (1961) 366. 15 W. E. Gerbacia, H. L. Rosano. Journal Colloid and Interface Sci. 44 (1973) 242. 16 H. L. Rosano, T. Lan, A. Weiss, W. E. F. Gerbacia, J. H. Whittam. Journal CoUoid and Interface Sci. 72a (2) (1979).
1117 17 J. L. Cavallo, H. L. Rosano. Journal Phys. Chem. 90 (1986) 6817. 18 A. M. Cazabat, D. Chatenay, P. Guering, W. Urback, D. Langerin. Meunier J. 19 W. D. Weatherford. Journal Dispersion Sci. Technology. 6 (1985) 467. 20 C. S. Hirtzel, R. Rajagopalan. Chem. Eng. Commun. Science Publishers, Inc. 33 (1985) 301. 21 D. H. Napper. Journal Colloid Interface Sci. 58 (1977) 2. 22 D. L. Chang, H. L. Rosano, A. E. Woodward. Langmuir 1 (1985) 669-672. 23 E. D. Goddard, T. Phillips, and R. B. Hannan. Journal Soc. Cosmet, Chem. 26 (1975) 461. 24 L.Mars. Thesis. City University of New York. 1990. 25 E. D. Goddard and R. B. Hannan. Journal CoUoid Interface Sci. 55 (1976) 73. 26 I. Satake, J. T. Yang. Biopolymers. 15 (1976) 2263. 27 G.Schwartz. Eur. Journal Biochem. 12 (1970) 442. 28 B. H. Zimm, J. K. Bragg. Journal Chem. Phys. 31 (1959) 526.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
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Prediction of moisture barrier requirements for an effervescent single serve aspartame sweetened tablet D. Apostolopoulos ^ and R. Fusi ^ a Kraft General Foods, Technical Center, Glenview, IL 60025, USA b Kraft General Foods, Technical Center, Tarrytown, NY 10591, USA Abstract Competition is forcing food companies to be more aggressive and move new products into the marketplace rapidly. Package developers to meet such time constraints often select packaging for a new product based on best guess choices. The result of such practices is usually over or underpackaging at a substantial cost to the company due to associated product failures, excessive use of packaging materials, and delayed introduction to marketplace. For moisture sensitive foods, the use of a moisture transport model can help package developers to predict the packaging requirements of such foods, avoid problems of the nature described above, and maintain packaging cost at a reasonable level. This paper demonstrates the use of such a model to identify the packaging needs of aspartame sweetened tablets and confirm a 1-year shelf-life prior to product introduction. In our study, the tablets, being an effervescent product, provided a challenge in the effort to generate moisture sorption data needed for the model. Thus, existing experimental procedures were modified and new ones developed to overcome the problems stemming from the effervescent character of the tablets included in this study. 1. INTRODUCTION An effervescent single serve aspartame sweetened tablet was developed in several fruit flavors. Tablets are going to be packed into plastic cylindrical tubes which can be carried around in a pocket or a purse and conveniently used at home, office or wherever an individual needs to prepare and enjoy a glass of refreshing beverage. Two (2) tablets can be mixed with cold water and stirred until dissolved to make up an eight ounce serving of beverage. The dissolution of the tablets in water is facilitated by stirring and some effervescent action resulting from penetration of water into the tablet where it triggers the reaction between citric acid and potassium bicarbonate to form CO2 that upon its rapid release aids in the disintegration and dissolution of the tablets. Effervescent aspartame sweetened tablets are very sensitive to moisture.
1120 Exposure to any relative humidity that may result in moisture sorption can be detrimental to their quality. For instance, moisture sorption resulting in a rise of the moisture content of the tablets above a critical level can affect dissolution, which is very important for the performance of the product during its end use by the consumer. Moisture sorbed on the surface of the tablets starts diffusing into the compressed matrix, slowly dissolving and bringing into intimate contact potassium bicarbonate and citric acid. These ingredients react to form potassium citrate and CO 2 that slowly escapes during storage and before the product has reached the consumer. Premature loss of most CO2 results in loss of the effervescent action needed to aid the disintegration and dissolution of the tablet during the preparation of the beverage, thus, rendering the product unacceptable. Furthermore, moisture sorption followed by a significant increase in the moisture content of the tablets can cause expansion and disintegration of the tablets during distribution resulting in an unacceptable product (Hine, 1987). Thus, it becomes apparent that the beverage tablets storage stability depends on control of moisture pick up from the surroundings atmosphere. The water activity of the tablets is relatively low, about 0.35 and the water vapor pressure in the surroundings atmosphere can be as high as 100%. Such difference in water vapor pressures constitutes a potential which can drive the system to equilibrium by transfer of moisture from the environment to tablets. As a result, changes occur in the moisture content of the tablets which are usually directly proportional to vapor gradient. The rate at which the moisture content of the tablets changes depends on the makeup of the product, i.e. its hygroscopicity, the temperature and the atmospheric pressure (Hall, 1991). Nevertheless, moisture transfer to the tablets without any package protection can occur so fast that the shelf-life of the tablets could turn out to be very short. In packaged tablets the changes can be relatively slower. Packaging opposes this tendency by inserting an effective "moisture barrier" between the tablets and the potential gradient. Therefore, selection and use of any package that does not provide a sufficiently effective moisture barrier would result in inadequate protection of the product and a shelf-life shorter than desired. On the other hand, selection and use of a package that provides a perfect moisture barrier i.e. glass or metal it would completely isolate the tablets from any external environment, but such packaging may be very expensive, and usually unnecessary. Given that packaging may constitute a major component of the production cost, it was thought that the best package for the tablets would be a plasticbased package that is just good enough in moisture barrier properties to retard moisture transfer to such an extent that deterioration does not occur before the product is consumed. Obviously, knowledge of the WVTR or the protection that such a plastic-based package can offer is critical in making the right package selection. Traditionally, the selection of a package is accomplished through actual storage trials. For instance, tablets in various packages under evaluation can be stored either at the expected storage conditions or under "accelerated test" conditions. The package that delivers the shelf-life desired and yet is considered to be the most economic one, obviously, should be the package of choice (Mizrahi et al., 1970).
1121 Using actual storage trials to select the appropriate package for a moisture sensitive product can be very expensive and time consuming especially if a large number of package-product combinations have been included for evaluation. Reduction of the storage trial samples to those combinations of package and product that are likely to give the desired shelf-life would simplify the task of package selection and result in substantial savings. A means for identifying viable storage trial samples is provided through the use of a moisture transport prediction model. The latter is based on equations which describe the mechanism of moisture transport through a packaging barrier and which can be used to predict the packaging protection required to keep the product within certain A^ limits for its shelf-life period. The model described above is applicable for foods deteriorating through reactions sensitive to infiltration of atmospheric moisture, and is basically making use of interrelationships among variables, such as, the slope of the product's moisture sorption isotherm, the initial and maximum allowable water contents, the water activities that correspond to those moisture contents, the surface area of the package and the permeability of the packaging material in use, to obtain the required shelf-life at minimum cost under the storage conditions evaluated (Heiss, 1978; Eichner, 1985). In other words, the moisture transport prediction model relates shelf-life to the quantity of water that has to permeate through package to render the product unacceptable, and ultimately to the permeation properties of the package. It is this link between shelf-life and the permeation properties of the package that could provide the means for estimating the moisture barrier oi' the maximum WVTR of the package to yield the shelf-life desired. This WVTR value can then be compared to literature values of packaging materials to identify candidate materials. The chosen materials are therefore already screened for barrier performance. This offers an improved procedure for new product development because the materials that ultimately are entered into test are already prime candidates. Gross over or under packaging is therefore avoided, thus, resulting in substantial reduction of the product development cost. Savings would result directly through the reduction in trials, the choice of the most cost-effective packaging, as well as, indirectly through the reduced risk of failure for product packaged in the package of the right choice. The moisture transport prediction model can also provide guidelines for product formulation that may be effective in reducing demands on the protection expected of the package and thus reduce packaging costs. As indicated earlier, information required with the moisture transport prediction model includes the moisture sorption isotherm for the product under study at the storage temperature evaluated, as well as, the maximum allowable or critical moisture content and critical relative humidity at which the product is rendered unacceptable. Determination of the moisture sorption isotherm, critical moisture content, and critical relative humidity for m i s t moisture sensitive food products can be easily carried out by using the gravimetric method (Gal, 1981; Troller and Christian, 1978; Labuza, 1983). This method requires that samples of the product are suspended over various saturated salt solutions which at a given temperature provide a wide range of relative humidities.
1122 However, the gravimetric method as described in the literature, was considered unsuitable for determination of the moisture sorption isotherm for effervescent tablets. Such tablets would equilibrate with low relative humidity environments, but at higher relative humidities they would begin to effervesce and release CO 2 that would escape in the environment, thus, interfering with the accuracy of the gravimetric method. Equally challenging, was the determination of the maximum allowable or critical moisture content and critical RH for the tablets. The increased release and loss of CO2 that occurred as the tablets were exposed to higher RH, was found to affect the dissolution of the tablets, judged to be the most critical factor in impacting the product's shelf-life. Thus, samples exposed to various RH had to be monitored for CO2 release and not just visually inspected for caking in order to determine their critical moisture content and critical RH. Thus, the objectives of this study were: i) Overcome the problems stemming from the effervescent nature of tablets and determine accurately the moisture sorption isotherm, critical moisture content, and critical RH, ii) Apply the data above to a moisture transport prediction model for selecting an optimal packaging that would be cost effective, provide adequate protection against moisture, and deliver the shelf-life desired for effervescent tablets. 2. MATERIALS AND METHODS 2.1. Production of Tablets Aspartame sweetened lemon flavored tablets included in this study were produced for experimental purposes at the General Foods technical center in Tarrytown. Production consisted of ingredient mixing, tableting, alcohol glazing, and drying (Smith et al., 1993). Ingredients, such as, citric acid (provides tartness), mannitol (aids dissolving), potassium bicarbonate (aids dissolving), aspartame (sweetener), maltodextrin (from corn), lemon juice solids, natural lemon flavor, ascorbic acid (vitamin C), titanium dioxide (for color), potassium citrate (controls acidity), yellow 5, and BHA (preserves freshness), were preweighed and thoroughly mixed under controlled humidity (<20%). About 1.3 grams of powdered mix were fed manually into a punch tablet press and compressed to about 2000 psi to form a tablet. Tablets injected from the punches were collected on a non woven wire mesh tray and subjected to alcohol glazing for about 30 seconds. Alcohol vapor adsorbed on the surface of tablet partially solubilized aspartame and citric acid which upon drying fused together to form a coating that increased the hardness of the tablet without altering its ability to dissolve. The thickness of the coating was controlled by the concentration of the alcohol in the chamber and residence time of the tablets in the glazer. The concentration of alcohol in the glazer atmosphere was maintained at 81% saturation. This was achieved with direct heating of the air at a dry bulb temperature of 39°C and also by controlling the amount of heat used to vaporize the alcohol into the glazing chamber so the wet bulb temperature was at 35°C. After alcohol glazing, tablets were dried for
1123
15 minutes at 140°F to remove residual alcohol. Drying was carried out by placing the trays containing the tablets in a drying oven equipped with air circulation and exhaust. Following a cooling period glazed tablets were tested for hardness, thickness, diameter, dissolution, taste, and final product cleanliness and then packaged. Final product hardness, dissolution, thickness, and diameter targets were as follows: hardness: 30-40 newtons, dissolution: 30 seconds, thickness: 3 mm, and diameter: 22.41 mm. After testing tablets were foil overwrapped and packed by hand into glass jars to be protected from moisture until used for the study. 2.2. Moisture Sorption Isotherm Determination
MC m^^qWhere:
^^ 100-%/MC ^ 100
CI) ^ ^
= moisture content at a given salt solution relative humidity, in percentage of dry weight. Wi = weight of sample in aluminum weighing dish before exposed to any relative humidity, in grams. W2 = weight of sample in aluminum weighing dish after equilibrated at a given salt relative humidity, in grams. %IMC = initial moisture content of sample in aluminum weighing dish before exposed to any relative humidity, (wet basis). MCeq
Three replicate determinations were made of the moisture content for each mix and relative humidity. Equation 2 was used to calculate the moisture sorption isotherm of the full formula tablet from the moisture sorption data generated experimentally for the two mixes that make up the final product.
loo
^^^ ~ Where:
MCp MCi MC2 X
^^^ = Moisture content of the final product at a given salt solution relative humidity, in percent of dry weight. = Moisture content of the full formula minus the potassium bicarbonate at a given salt solution relative humidity, in percent of dry weight. = Moisture content of the potassium bicarbonate at a given salt solution relative humidity, in percent of dry weight. = percent of potassium bicarbonate in the full formula.
A plot of the equilibrium moisture contents obtained from the above equation versus relative humidities, resulted in construction of the sorption isotherm of
1124 the tablets at 73°F. 2.3. Initial Moisture Content Determination Knowledge of the initial moisture content (IMC) for the full formula tablets, full formula mix less the potassium bicarbonate, and potassium bicarbonate itself was necessary to calculate their moisture sorption isotherms, determine their critical moisture content, and carry out the moisture barrier/packaging requirements prediction process. The IMC of the tablets and the formula mixes mentioned above was determined by using a thermal drying method. Two tablets or about 3 grams of each mix included in this study were placed in pretared aluminum weighing dishes, weighed and placed in a vacuum oven over a drying agent (CaS04). The oven was set at 70°C and 30 in Hg of vacuum. The aluminum weighing dishes containing the samples were weighed until no further weight losses were observed (5-6 hours) and the moisture losses represented by the sample weight decrease were used to calculate the initial moisture content from Equation (A.O.A.C., 1975; TroUer and Christian, 1978). IMC = ^-^ Where:
^ 100 IMC W"! W2
(3) = Initial moisture content on wet basis, in percent. = weight of sample before drying, in grams. = weight of sample after drying, in grams.
2.4. Determination of Mode of Deterioration, Critical Moisture Content, and Critical Relative Humidity. For dry products, a rise in the moisture content or water activity above a critical level can cause microbiological, chemical, as well as physical changes such as agglomeration, caking, sogginess e.t.c, which usually constitute the major modes of deterioration and render the quality of dry foods unacceptable. These levels are defined as the critical moisture content and critical water activity or relative humidity, respectively (Taoukis et al., 1988). The critical moisture level can vary depending upon the different types of deterioration. As a result, the mode of deterioration needs to be systematically investigated in order to achieve the proper clarification of the problem (Heiss and Eichner, 1971). In cases where the mode of deterioration is manifested through physical changes the critical moisture content and critical water activity or relative humidity can be easily defined during the determination of their moisture sorption isotherm obtained by using the gravimetric method. Typically, dry product samples after equilibration over different relative humidities are visually inspected for agglomeration, caking, loss of any functional properties e.g. flowability, scoopability, and textural or color changes. The lowest relative humidity where those changes start taking place is recorded as the critical relative humidity or water activity. The moisture content that corresponds to critical relative humidity and which can obtained from the moisture sorption isotherm is the critical moisture content. However, for a product, such as effervescent tablets, the mode of deterioration determined to be limiting to the product's shelf-life does not involve changes
1125 that can be observed through a visual inspection of product equilibrated to different relative humidities. Preliminary experiments indicated that tablets exposed to higher relative humidities exhibited a CO2 loss that was found to have a significant effect on tablet dissolution. Tablet dissolution times increased with loss of CO2. Thus, formation and loss of CO2 with moisture sorption was considered to be the deterioration mechanism or mode most impacting the product's shelf-life. In order to determine the critical relative humidity, the relative humidity above which the tablets exhibited unacceptable dissolution times, samples exposed to various RH had to be monitored for CO2 release and tested for dissolution. 2.5. Monitoring of Carbon Dioxide Formation Four (4) tablets were placed in aluminum weighing dishes and suspended over salt solutions prepared and held in hermetically sealed half a quart Mason jars which were kept in environmental control chambers set at 73°F. The relative humidities of the salt solutions used ranged from 6% to 80%. Through a sampling port installed on the lids of the Mason jars and using a gas-tight syringe, periodically for about four weeks 1 ml headspace aliquots were withdrawn from the Mason jars and injected into a 3700 Varian gas chromatograph set up for CO2 analysis. The gas chromatograph was equipped with a thermal conductivity detector (TCD), a 6' x 1/4" CTR I chromatographic column, and a data-logger based on an IBM PC used with a Labtech Notebook data acquisition software. The gas chromatograph operation conditions were as follows: column temperature 25°C, injection port temperature: 40°C, detector temperature: 80°C, filament current: 157 ma, and carrier gas-helium-flow rate: 65cc/min. The percentage of CO 2 in the analyzed headspace aliquots was calculated by reference to a calibration curve constructed by injecting standards of known CO2 concentrations. 2.6. Dissolution Test Five (5) tablets were placed in aluminum weighing dishes and suspended over salt solutions held in half a quart Mason jars kept at 73°F. The relative humidities of the salt solutions ranged from 8% to 80%. During the 4 week period of sample equilibration, tablets exposed to various relative humidities were taken out of the Mason jars and tested for dissolution. Each tablet was dropped into a cup containing 120 ml distilled water at room temperature (73°F). Stirring of the water was initiated 10 seconds later and continued until the tablet was completely dissolved. The time required for dissolving the tablet was recordered as dissolution time. 2.7. Prediction of the Moisture Barrier/Package Requirements. As shown by Heiss (1958), Karel (1967), Heiss and Eichner, (1972), prediction of the shelf-life or determination of the moisture barrier/packaging requirements for moisture sensitive foods, such as the tablets, requires accurate knowledge of the rate of transport of water vapor across the packaging barrier. The moisture transport through the packaging barrier is described by a pseudo-steady state equation based on Fick's and Henry's laws.
1126
^
=
k/l^A{P,,,-P,,)
Where:
(4)
- rate of water vapor transport per day = permeance to moisture for packaging (g/day m2 mm Hg) A = package area (m2) Pin, Pout = water vapor partial pressure inside and outside the package, respectively (mm Hg) dw Idt kll
The equation above was converted to Equation 5, which contains expressions that can be obtained experimentally and conviniently applied to determine the shelf-life for a dry product in a package of a given moisture barrier. t=
(w,,-w,,) WVTR{%RH,,,-%RH,,)
w = ^^ cr
Where:
(5)
(6)
100
Wdr=Wi,,-
100
Wi,-%IMC 100
(7)
= the product oikll-A in Equation 4, which is the water vapor transmission rate for the package to deliver the shelf-life desired, grams of water per package and day. t - target shelf-life, in days. Wdr = weight of dry product in the package at the time of packaging, in grams. Wcr = weight of packaged product equilibrated at the critical relative humidity. ^in - weight of packaged product with a moisture content equal to the initial moisture content the product exhibited at the time of packaging, in grams. MCcr = critical moisture content or moisture content of product after equilibrated at the critical relative humidity, in percent. %RHout = storage relative humidity %RHin = relative humidity inside the package right after packaging as a result of the initial moisture content of the product WVTR
Experimental data obtained on the initial moisture content, critical relative humidity, and moisture sorption of the tablets, as well as other information pertinent to the storage conditions provided all the parameters needed to be applied by a computer program to Equation 5 to determine what should be the WVTR of the package that would ensure delivery of the desired shelf-life (Heiss and Eichner, 1971).
1127 Several basic assumptions must be made to predict the packaging requirements of the tablets using Equation 5. The first assumption was that the moisture rapidly equilibrates within the package; this has been found to hold true (Hendel et al., 1958; Heiss, 1958). In other words, it was assumed that the controlling mechanism is the moisture transport through the package, the resistance of moisture vapor diffusion into food being relatively negligible. Other assumptions were that the storage conditions, temperature and % RH remained constant, (which is not necessary correct,) and the moisture sorption isotherm is linear between the initial moisture content and the moisture content at the critical relative humidity (Labuza, 1982). However, what was considered to most affect the accuracy of the outcome resulting from the solution of Equation 5 is the fact that the inside package vapor pressure Pin is not constant but rather increases as the product gains moisture. Thus, there is no constant driving force; rather the driving force decreases with time, slowing down moisture gain. Using a constant-driving force assumption leads to overprediction of moisture exchange and thus to overprotection as well as increased costs (Karel and Labuza, 1969). In order to minimize the error introduced by the decrease of the driving force over time, an analytical solution was applied to Equation 5. The sorption isotherm between the initial moisture content and the critical moisture content was divided graphically into small linear segments. Water vapor partial pressure, (Pin) or relative humidity, (RHin) inside the package corresponding to the start of each linear segment was determined from the moisture sorption isotherm. Then Equation 5 was applied separately for each individual segment to determine the time interval required for the moisture that permeates through the package to be adsorbed by the product and move the moisture content of the product along each individual segment. The time intervals corresponding to those linear sorption isotherm segments were summed up to calculate shelf-life for a given WVTR. Then the shelf-life value obtained was substituted for the target shelf-life value and Equation 5 was solved for WVTR to determine what must be the WVTR for the package that would deliver the target shelf-life. 3. RESULTS AND DISCUSSION The initial moisture content for lemon flavored tablets, mix without potassium bicarbonate, and potassium bicarbonate itself are given in the table below. Table 1 Initial moisture content for lemon flavored tablets and mix variables Product Lemon Flavored Tablets Lemon Flavored Mix Less the Potassium Bicarbonate Potassium Bicarbonate
Initial Moisture Content-Wet Basis, (%) 1.70 ± 0.06 1.41 ± 0.07 0.046 ± 0.009
1128 The moisture sorption isotherms obtained experimentally for the mix less the potassium bicarbonate, as well as the potassium bicarbonate itself, are presented graphically in Figures 1 and 2, respectively. The moisture sorption isotherm as calculated for the tablets is shown in Figure 3.
20 40 60 80 Relative Humidity, (%)
100
Figure 1. Moisture/ sorption isotherm for lemonflaX^oredtablets total mix less the potassium bicarbonate at 73°F.
I"
" r"
r
20 40 60 80 Relative Humidity, (%)
100
Figure 2. Moisture sorption isotherm for potassium bicarbonate at 73°F.
As seen from the graph of Fig. 3, tablets exposed to various RH absorbed very little moisture up to about 45% RH. Moisture sorption started increasing gradually over the 45-65% RH range, and it rose sharply as RH increased above 75%. Figures 4 and 5 show the effect of moisture sorption over different RH on the formation of CO2 and the dissolution time of the tablets. Carbon dioxide started forming in significant amounts at 48.6% RH. The dissolution time was shown to be increasing over the lower RH range, 10-20%, and above 43.9%. The reason that dissolution time increased over the lower RH range is because tablets exposed to such very low RH overdried and hardened, therefore, it was difficult for water to penetrate the matrix. The average of the above two RH (48.6% and 43.9%) was selected as the critical RH or the RH above "1 \ r which the tablets are expected to 100 20 40 60 80 exhibit dissolution times greater Relative Humidity, (%) t h a n 30 seconds, something t h a t perceivably would render the prodFigure 3. Moisture sorption isouct unacceptable by the consumer therm for lemon flavored tablets at (see Table 2). 73'^F.
1129
I - "* r 40 60 Relative Humidity, (%)
r
80
100
Figure 4. Evolution of carbon dioxide with time for lemon flavored tablets over various RH at 73°F. 300250o
-e—Ohrs -B- - 75hrs ^ - - 175 hrs -X- - 530 hrs
200H
150X
lOOH
/
50-
/
0
/
—e—e—o
e-e-n 10
o
\ \ 1 20 30 40 Relative Humidity, (%)
\— 50
60
Figure 5. Dissolution of lemon flavored tablets exposed over time to various RH at 73°F.
1130 This data suggests that packaged tablets should not see RH that exceed 46.25% and moisture content should remain lower than 2.87%, in order for them to exhibit an acceptable dissolution (dissolution time of about 30 seconds) upon use by the consumer. Table 2 Critical relative humidity and critical moisture content for lemon flavored tablets at 73°F. Critical Relative Humidity, (%)
Critical Moisture Content-Dry Basis, (%)
46.25
2.87
Table 3 below provides a summary of the moisture barrier/packaging requirements as predicted for 10 single-serving portions or a number of 20 tables (total product weight: 25.56 grams), a target shelf-life of 1 year, and a range of storage conditions representing a temperate climate (73°F/50%-70% RH). Table 3 Moisture barrier requirements for lemon flavored tablets Target Shelf-Life
Storage Conditions
Package WVTR (grams/package.day)
12 Months
73°F/50%RH 73°F/60%RH 73°F/70%RH
0.016 0.005 0.003
As indicated by data presented in Table 3, the package i.e. a tablet holder tube intended for packaging of tablets should have a WVTR of 0.003 grms/day or lower in order to ensure a 1-year shelf-life under the storage conditions selected. Information such as the above is vital in terms of helping the package developer to select and develop the appropriate package for effervescent tablets. For instance, since such a low WVTR can be provided only by packaging materials with very good moisture barrier characteristics, such as high density polyethylene, (HDPE), which may be the packaging material of choice. Thus, with this in mind the package developer now can proceed with the development, for example, of HDPE tubes (and a suitable plug closure), sufficient in size to accommodate 20 tablets. Developed prototypes can be tested for moisture permeation to determine their W\^Ti?. A comparison of the measured WVTR to WVTR predicted would indicate whether the HDPE tube as designed would provide adequate protection for the tablets or whether adjustments need to be made on the wall thickness to match the protection required.
1131 An optimal wall thickness which can be achieved through downgauging or upgauging of the wall thickness of the prototype tubes would ensure the protection needed at minimal cost. 4. REFERENCES A.O.A.C. 1975. "Methods of Analysis", 11th ed. Assoc. Off. Anal. Cem., Washington, D.C. Bone, D.P. 1987. Practical applications of water activity and moisture relations in foods. In "Water Activity: Theory and Applications to Food", L.B. Rockland and L.R. Beuchat (Ed.), p.369. Marcel Dekker, Inc.New York and Basel. Eihner, K. 1985. The influence of water content and water activity on chemical changes in foods of low moisture content under packaging aspects. In"Food Packaging and Preservation", M. Mathlouthi (Ed.), p.67, Elsevier Applied Science Publishers. Gal, S. 1981. Recent advances in techniques for obtaining complete sorption isotherms. In "Water Activity: Influences on Food Quality", L.B. Rockland and G.F. Stewart (Ed.), p.89. Academic Press. Hall, J. 1991. Food product shelf-life. How long before it's gone? Medallion Laboratories Analytical Progress 8(1): 1. Hendel, C.E., Legault, R., Talburt, N.F., Burr, H.K., and Wilke, C.R. 1958. Water vapor transfer in the in-package desiccation of dehydrated foods. In "Fundamental Aspects of the Dehydration of Foodstuffs", p.89, Soc. Chem. Ind., Metchium & Son, Ltd, London. Heiss, R. 1958. Shelf-life determination. Modern Packaging 31(8):119. Heiss, R. and Eichner, K. 1971. Moisture content and shelf-life; Part 1, Food Manufacture, 46(5):53. Heiss, R. and Eichner, K. 1971. Moisture content and shelf-life; Part 2, Food Manufacture, 46(6):37. Hine, D.J. (1987). Shelf-life prediction. In "Modern Processing, Packaging and Distribution Systems for Food", F.A. Paine (Ed.), p.62, AVI, New York. Karel, M. 1967. Use tests-only real way to determine effect of package on food quality. Food in Canada 27:43. Karel, M. and Labuza, T. P. 1969. Optimization of protective packaging of space foods. U.S. Air Force contract F-43-609-68-C-0015. Aerospace Space Med. School, San Antonio, Texas. Labuza, T. 1982. Moisture gain and loss in packaged foods. Food Tech., April 1982, p.92. Labuza, T. 1983. Standard procedure for isotherm determination, Food Research, 28(4):258. Marsh, k. 1985. Computer-aided shelf-life prediction. In "Mastering Today's Packaging Needs", TAPPI PRESS, p.43. Marsh, K.S., Ambrosio, T. and Morton, D.K. 1988. Stability evaluation of a dynamic system. J. Packaging Tech., 2(6):260.
1132 Mizrahi, S., Labuza, T.P. and Karel, M. 1970. Computer-aided predictions of extent of browning in dehydrated cabbage. J. Food Sci., 35:800. Smith, S.L., Jackson, R.R., Albaum, J.D., Fusi, R.W., and Doherty, S.S. 1993. Process for beverage tablets and products therefrom. United States Patent, Patent Number: 5,254,355. Taoukis, P.S., Meskine, A.EL, and Labuza, T.P. 1988. Moisture transfer and shelf life of packaged foods. In " Food and Packaging Interactions", J.H. Hotchkiss (Ed.), p.243, Amer. Chem. Soc, Washington, DC. TroUer, A. J. and Christian, H.B. J. 1978. Methods. In "Water Activity and Food", J.A. Troller and J.H.B. Christian (Ed.), p.l2, Academic Press.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
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Comparing The Rates Of Development Of Temperature Distributions In Foods Shaped as Spheres, Cylinders and Thick Films. Arthur E. Grosser Department of Chemistry, MoGill University, 801 Sherbrooke St., W., Montreal, Quebec, H3A 2K6, Canada Abstract The rates of development of the thermal distributions that arise in heated foods are given by equations that do not lend themselves to simple computation. Nor do they make evident how the physical and thermal properties of the food control the process. The systems considered are foods of different geometries which are heated from ambient temperature by immersion in a heat bath. The half-heating time is the time necessary for the temperature at a given position in the food to increase by one-half the difference between bath and ambient temperatures. Using this quantity leads to simple relations which aid in estimating these distributions, the rate at which they develop, and how they depend on the properties of the material and the experimental variables. These expressions are compared to codify this method for spheres, cylinders and thick films, and it is found that one equation will describe the behavior of all these systems.
1. INTRODUCTION Foods that are heated from ambient temperature develop thermal gradients that determine the heat transfer through them and drive the rate constants for the chemical transformations. This may also be important for the packaging material if it is sufficiently thick relative to the food and not chemically inert. It is crucial to food chemistry to be able to calculate these gradients and the rates at which they develop. Unfortunately, the heat flow equations are of sufficient
1134 complexity that it is difficult to estimate these rates, even in a semiquantitative manner. Moreover, it is not apparent how the properties of the food (e.g., its size and geometry, heat conductivity and heat capacity) and the experimental variables (the initial ambient temperature of the food and the bath temperature) are related to these rates. Although useful approximations do exist for this problem [1,2], they do not present the food chemist with a transparent view of the process. The half-heating time offers a way to find this information in a relatively simple manner. We will present these results and show that a comparison of the expressions for various geometries leads to a one master equation valid for all cases over a wide range of conditions.
2. MODELS For the purposes of these models the object will be assumed to be of uniform composition with a thermal diffusivity, K, equal to k/pcp, k being the thermal conductivity and pcp the volumetric heat capacity, composed of p, the density, and Cp, the specific heat (or heat capacity per unit mass). The thermal diffusivity is assumed to be constant, and the surface heat transfer coefficient is assumed to be infinite. The food system is initially at a uniform ambient temperature, Ta, and at time, t, equal to zero, is placed in an infinite heat bath of temperature, Tb- The term (Tb- Ta) is denoted by AT. a.
Spherical Foods:
For a sphere of radius, a, the heat flow equations yield [3] the equation for the temperature, T(r,t), at a radial distance, r, and time, t: [T(r,t)-Ta]/AT = 1 + ( 2 a / j c r ) 2 { { - 1 ) " / nHsln(nJtr / a)}exp(-Kn^jt^t / a^) n=i
(1)
1135 b.
Cylindrical Foods:
Similarly, for an infinite cylinder of outer radius a, the heat flow equations [4] yield the equation for the temperature, T{r,t), at the radius, r, and time, t: [T(r,t)-T3]/AT=12 2 {exp(-Kp„2t/a2)}xJ^(p„r/a)/[PnJi(p„)l n=1
(2)
where Jo and J i are the zero-order and first-order Bessel functions, respectively, and Pn is the nth root of the zero-order Bessel functions.
c.
Thick Films:
For an infinite thick film of thickness 2a, the equation [5] for the temperature, T(r, t), at the distance from the center of the film, r, and the time, t, is: I T ( r , t ) - T a ] / AT =1 00
( 4 / Jl)
2 { { ( - 1 ) " / ( 2 n + 1 ) } { e x p [ - ( 2 n + 1 ) ^ r ^ K t / 4a^l}x n=0 {cos[(2n + 1)Jtr/ 2 a l } }
(3)
(For a model heated only on one side, with the other side held at Ta, 2a is replaced by a and the origin of r is at the side held at Ta.) An example of the relative behavior of these three systems is shown in Figure 1 below which displays the temperature rise for food samples of the three geometries with the same values of the physical properties, as given in the Figure caption.. The
half-heating
time,
T i / 2,
is the
time
at
which
temperature difference between the object and its surroundings the initial temperature difference, Tb-Ta-
the
is half
1136 200
That is, at T i / 2 r
sphere
r
20
?/ /
/ ^ /•^/
/
^.^""'"""^cyl
T ( r . T i / 2 ) - T a = 0.5(Tb-Ta)
^ _ ™ " - ™ -
^
-^ ^
1 1
'C^1r" 1 1
v-^"'"^
r
X
^,.- •-" film
In
Figure
1
at
left,
the
vertical arrows indicate the
_^,:^-"~
half-heating
__,„"
times
for
the
three cases.
i^^^
_
^
t, min
L_^
20
Figure 1. Temperature (^C) from exact computation (equations 1, 2 and 3) vs. time (min.) for the three geometries. Ta = 20^0, Tb = 200OC, K= 0.0020 cm^/min., a = 1.0 cm and r = 0.5 cm.
3. HALF-HEATING TIME APPROXIMATIONS The half-heating time equation for spheres is found to be [6] X | / 2(sphere)
=
( a ^ / 3r^K)[ln4 -7?T^ I 6 a ^ l
(4)
Similarly, the half-heating time equation for infinite cylinders is [7]: Xx/ 2 ( c y n n d e r )
=
( a ^ / K)[0.20133 - ( r ^ / 4 a ^ ) l
(5)
and that for thick films is [8]: X,/ 2(film)
=
( 4 a ^ / 3t^K)[ln(8/ n) - j c ^ r ^ / 8a^]
(6)
These equations may be more easily compared by using the
reduced
distance, a, defined as a = r/a.
position
dependence of T1/2
This allows us to plot
on one set of axes, as shown below.
the
1137 20
f
1 1/2,1
min
M
film
V
cyl
1 •"" •^ • ^+ k
The relative behavior of the half-heating times as a function of the reduced distance, a, is shown in Figure 2 at left.
'K
•
sphere
+ •
+
M
•
—i
0
a
t
__*
1
Figure 2. T i /2(nnin.) from equations 4, 5 and 6 vs. o for the models in Figure 1
4. COMPARISON OF HEATING TIMES BY GEOMETRY All the half-heating time equations can be put in the form
^1/2 = {a2/K)[B - Ca2]
(7)
where Table 1 below indicates the values of the B and C terms. geometry
B
sphere cylinder film
0.140461 0.20133 0.378824
c = (In4)/Jt2 = ln(1.223) = 4ln(8/3i) /3i2
1/6 1/4 1/2
Table 1. Values of the B and C terms for the three geometries. The most important consequence of this set of equations is the prediction of a linear dependencies of the half-heating time on o^, on a2, and on 1/K, Figure 3, below, shows a test of the first of these relations.
1138
.film ''1/2,
Figure 3. Ti / 2 (min.) from equations 4,5 and 6 vs. a2 for the same models as the previous Figures.
•s
''++cyl + + — • • sphere , 0 0
min
K
+
+
*
•
J
t It
a
1
As shown in references 6-8, these equations are accurate at all but the highest values of a, that is, at all regions away from the periphery of the food. They may be (conservatively) estimated to be valid when the reduced distance, a, defined as a = r/a, is less than or equal to 0.8. The largest errors will arise from the assumptions that K i s constant and the surface heat transfer coefficient is infinite.
5. DISCUSSION A way of analyzing the meaning of these equations is to define a2/K as the "response time", tr- The response time gives the Inherent time for the food's center to rise in temperature in response to a heat input. The reciprocal of tr is thus the speed with which a food characterized by the thermal diffusivity, K , and the dimension, a, shows such a temperature rise. The half heating time equations can therefore a l l be represented by tl/2
= tr[B - Ca2]
(8)
In this form, T 1 / 2 can be seen to be the manner by which the response time, t r , is modified by two factors.
The geometry of the food
1139 is reflected in the B term, while the C term gives the sensitivity of tr to the position In the food at which the temperature is measured. We may now ask if the quantitative value of these terms are reasonable. Taking the sphere as our reference geometry, we can compare the relatives values of these terms, as shown in Table 2.
B/B(sphere)
C/C(sphere)
1.000 1.436 2.707
1.0 1.5 3.0
geometry sphere cylinder [film
Table 2 Values of the B and C terms for three geometries, divided by the values for B and C for spherical geometry, respectively. It is, of course, clear that the sphere is the quickest, and the film the slowest to be cooked. The regularity in the variation of the C terms is intriguing. zero there. time
T1/2
At the center of the food
Is trB, since a is
As the measurement point moves outwards the
decreases,
but
much
more
sensitively
for
the
half-heating
film
than
cylinder, which In turn is more sensitive than the sphere.
the
But one
should be cautious in trying to rationalize the relative values of the B and C coefficients because the film and cylinder are "infinite" bodies, while the sphere is not. To determine the
regions of
validity
of these
equations
it i s
useful to use the limiting condition that T 1 / 2 must go to zero as a nears
unity:
instantaneously.
the
temperature
at
the
periphery
rises
almost
Our approximations are not valid at such large values
of a, for they predict
X-\ / 2 equal to zero at
B= Co^.
This gives an
upper limit to the range of validity of these expressions, namely, 0 ^ a ^ a c r i t i c a l . where Ocritical ^ critical
are
slightly
0.879, and 0.870 for
different
is given by (BIC)^f^. for
sphere, cylinder
always greater than 0.8.
each
of
the
and film,
The values of
geometries
respectively)
(0.918, but are
1140 Indeed, when we examine the ratios of the B and C constants it is evident that they must run in rough parallel or the a critical values w i l l not lie so close together. This can be seen in Figure 3 which shows the half-heating times going to zero at roughly the same values of a^. In conclusion, one equation serves to give accurate half-heating times for a variety of foods of simple geometry
5. ACKNOWLEDGMENTS The author thanks the Vernon W. Krieble Foundation Department of Chemistry for their generous support.
and
the
6. REFERENCES 1. I. J. Pflug, J. L Blaisdell and J. Kopelman, "Developing TimeTemperature Curves for Objects That Can Be Approximated by a Sphere, Infinite Plate, or Infinite Cylinder", ASHRAE Trans., 71(1), 1965, 238-248. 2. Ramaswamy, H. S. Lo, K.V. and A. Tung, "Simplified Equations f o r Transient Temperatures in Conductive Foods with Convective Heat Transfer at the Surface", J. Food Sci.. 47, 2042-2047. 3. H. S. Carslaw and J. C Jaeger, Conduction of Heat in Solids, 2nd ed., Oxford,London, 1959, 233. 4. ibid., 199. 5. ibid., 100. 6. A. E. Grosser, "Approximation Method for Thermal Gradients in Spherical Objects", J. Thermophyslcs and Heat Transfer, 7, (1993) 536-538. 7. A. E. Grosser, "Approximation Method for Rate of Appearance of Temperature Distributions in Cylindrical Foods", J. Food and Agricultural Chem., 42, (1994) 166-168. 8. A. E. Grosser and Sophie M. K. Brunet, "Approximation Method For Rate Of Appearance Of Temperature Distributions In Thick Films", Food Control (in press, 1994).
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1141
Effect of oxygen on the ethyl acetate production from continuous ethanol stream by Candida utilis in submerged cultures G. Corzo% S. Revah^ and P. Christen^ ^Dpto de Ingenieria de Procesos, Universidad Autonoma Metropolitana Iztapalapa, Apdo Postal 55-534, CP 09340, Mexico D.F., MEXICO. ^ ORSTOM (Institut Frangais de Recherche Scientifique pour le Developpement en Cooperation), Ciceron 609, Col. Los Morales, CP 11530, Mexico D.F., MEXICO. Abstract. The purpose of this work was to compare ethyl acetate production by C. utilis at different oxygen concentrations in an iron free medium. The study was achieved in a batch stirred tank reactor and gaseous ethanol was fed continuously in the air stream. The acetaldehyde production as well as the cell growth were increased when oxygen level was maintained between 5% and 15 % rather than 0% and 2%. It was also found that the ethyl acetate maximum concentration was inferior (5.1 g/l against 8.0 g/1) at higher oxygen levels but with a higher productivity (0.1 g/l.h) in that case. Finally, ethyl acetate and acetaldehyde productions were found to be directly dependent on the growth phase in both cases.
D^RODUCTION Since the end of the seventies, there has been an increased interest in the use of flavors that can be labeled as "natural" in the food industry. This trend has motivated research to look for new sources of these products. Among the alternatives are the production from plant tissue cultures [1], the enzymatic generation of flavor either by adding new enzymes or by using the native system of the substrate [2] or by generating de novo compounds from microorganisms [3]. In Europe, as in the USA, the natural flavoring substances are those obtained by appropriate physical process or enzymatic or microbiological processes from material of vegetable or animal origin [4]. Products, including food ingredients - such as flavor compounds - must obtain the GRAS (generally recognized as safe) status; which is the case for most of the biotechnological products likely to be used in the food industry. The list of microorganisms that can produce flavors or flavor enhancers is broad [3], and there are already a great number of processes successfully applied in the industry [5]. While microorganisms have a definite role in the production of the typical flavors in fermented foods, fewer are the cases where the microorganisms generate these flavors under controlled conditions to optimize the production of the flavoring agent. Nevertheless, some examples can be found for bacteria (e.g. Streptococcus lactis subsp. diacetylactis for diacetyl [6], Zymomonas mobilis for acetaldehyde [7], Pseudomonas fragi for strawberry flavor due to C4-C6 ethyl
1142 esters [8]), yeasts (e.g. Yarrowia lipolytica for 4-decalactone [9]) or molds (e.g. Penicillium roqueforti for ketones [10] or lactones [11], Ceratocystis fimbriata for banana-like aroma [12]). The role of yeasts, such as Saccharomyces cerevisiae, in the formation of secondary aromas in the alcoholic fermentation must be considered: fusel oils [13], acylated amines [14] or esters [15]. Among the diversity of flavoring compounds secreted by yeasts, acetaldehyde and ethyl acetate are of relevance as additives in the food industry. The former delivers a fresh or fruity flavor to food such as fruits, bread, vegetables, dairy products and candies. It is also known to be highly reactive and is used to produce chemicals such as acetic acid, ethyl acetate, butanol,...[16]. The latter, characterized by a fruity note, is present in almost all the fruits and used in ice creams, candies, baked goods or chewing gums [16]. Both are listed as GRAS by the US Food & Drug Administration and their annual consumption as flavoring agent, in the U.S. in 1987, was about 147 and 14 tons, respectively [17]. Although ester production by microorganisms was recognized since the beginning of the century, it was until the fifties that the formation of these compounds by yeasts was studied with special interest in ethyl acetate. Works with Hansenula and Pichia strains [18,19] grown on glucose demonstrated that ethyl acetate formation was due to the aerobic utilization of the ethanol formed. It was also shown that acetaldehyde and acetic acid were also produced and it was suggested t h a t the ester formation had a survival value for the yeasts by preventing the accumulation of toxic amounts of free acetic acid. For the cases of Hansenula mrakii and Geotrichum penicillatum, it was shown that not only ethyl acetate was formed but also other acetate esters, such as isobutyl, isopentyl or 2methylbutyl acetates from the alcohols formed from deamination and decarboxylation of amino acids [3,20]. Nevertheless the mechanism of ethyl acetate formation seems to be different from the other esters because it is produced in the growth phase while the other esters are formed in the stationary phase. In this phase the microorganisms are also able to convert added alcohols and carboxylic acids into esters. The authors pointed out that a clear diauxic effect was observed when glucose was exhausted in H. mrakii cultures [21]. Another yeast, Kluyveromyces fragilis, showed its ability to produce ethyl acetate from whey permeate but only if lactose and ethanol are both present in the broth [22,23]. The levels of dissolved oxygen were shown to have a primordial importance on the orientation of the metabolism [23]. Ester formation (specially ethyl and isoamyl acetates) has been also thoroughly studied in the alcoholic fermentation with Saccharomyces cerevisiae [24,25]. The membrane bound enzyme alcohol acetyl transferase has been suggested to produce the ethyl acetate [26]. S. cerevisiae fermentation produces also acetaldehyde which is very important due to its low sensory threshold value. Saccharomyces rouxii, used in the soy-sauce fermentation, is also known for producing ethyl acetate [27]. In that case, the yeast was found to produce large amounts of the ester from glucose and added ethanol (conversion yield about 16 %) but showed a poor ability to produce it from ethanol alone. An inhibitory effect of sodium chloride was established. The yeast Candida utilis has been shown to produce ethyl acetate and acetaldehyde from ethanol as sole carbon source [28-30]. The production of the volatiles was shown to be dependent on lack of iron in the medium. It has been
1143 suggested that this limitation inhibits the TCA cycle thus causing an accumulation of acetyl-CoA which, through an alcohol acetyl transferase, produces the ethyl acetate. Similar observations were made with C. pseudotropicalis, but inferior ethyl acetate concentrations were reached [31]. The ester is mainly formed when there are low ethanol concentrations in the medium. This has led some authors to propose that this transformation may be used as an alternative method to treat dilute ethanol streams such as those obtained in brewing [29]. When the alcohol concentration was increased in the medium beyond 35 g/1, the metabolism of the yeast shifts to produce acetaldehyde. Rapid accumulation of acetaldehyde may inhibit acetyl-CoA formation thus reducing the production of ethyl acetate. Acetaldehyde can be further oxidized to acetic acid which may in turn reduce the yeast activity by lowering the pH. As any production of microbial metabolites, ethyl acetate and acetaldehyde formation is strongly dependent on the environmental conditions. For <S. cerevisiae ethyl acetate formation is strongly reduced when maltose concentration is high in the wort [24]. Oxygen has shown to be very important since the earlier studies [18,19] which showed that ethyl acetate was only formed when the culture was aerobic. Aeration rate plays a very important role as it is directly linked to oxygen availability on the medium which, in turn, influences growth and ethyl acetate yields in H. anomala [32] and in C. utilis [29]. On the other hand both studies showed t h a t higher aeration rates bring about a reduction in ethyl acetate concentration in the medium, but this effect could be attributed to stripping. The pH is another important parameter for ethyl acetate production hyJL^ anomala [19] where acidic pH was studied and for C utilis where neutral pH was more suitable for acetaldehyde production [28]. For ethyl acetate production, a pH between 5.0 and 7.0 is preferred although values above 6.0 favor acetate formation [29]. The pH has shown to have an influence in the ethanol oxidation by C. utilis yeasts [33] where at a pH of 4.2, maximum respiration rates are observed. Besides the experiments for the production of volatile compounds by viable yeast cells, recent reports have shown other biotechnological possibilities. Ethanol has been converted into acetaldehyde by resting cells of Pichia pastoris [34,35] in a semi-batch fermenter with added ethanol in air. Also, dried Hansenula polymorpha cells have been tried in a gas solid bioreactor to transform gaseous ethanol into acetaldehyde [36]. Continuous conversions were achieved under a reduced water content (about 8%). In previous works, the culture of C utilis on solid supports was successfully achieved with glucose or partially hydrolyzed starch [37] or gaseous ethanol [38] as sole carbon sources. A particular attention was given on the kinetic parameters and a diauxic growth was shown as also observed by other authors for H. anomala. A clear limitation on growth was observed when low nitrogen concentration was used [38]. The aim of the present work was to study the effect on oxygen levels on growth and metabolites accumulation by C. utilis, in submerged cultures, when gaseous ethanol was fed as sole carbon source.
1144 MATERIAL AND METHODS Microorganism and culture medium Candida utilis ATCC 9950 was used throughout this study. It was grown on Potato Dextrose Agar slants for 3 days at 30°C and then maintained at 6°C. To prepare the inoculum, it was grown in 50 ml of dextrose (20 g/1) and malt extract (20 g/1) medium in 150 ml Erlenmeyer flask with continuous shaking at 200 rpm at 30°C. The yeast was then grown on the iron free minimal salts medium previously described by Thomas and Dawson [39] which has been already used in various studies [28,29,37]. Fermentation conditions Fermentation experiments were run in a 3 1 Bioflo III fermenter (New Brunswick, USA). To avoid evaporation of ethanol and other volatile products, a condenser fed continuously with a refrigerant at -10°C was installed at the gas exit of the fermenter. Culture volume was 2.2 1 and temperature was 30°C. The pH was maintained at 6.0 with NaOH 0.5 IM. Inoculum size was 10% to give an initial concentration of 1x10^ cells/1 and initial ethanol concentration was fixed to 3.0 g/1. Air was fed at a rate of 24 1/h (0.2 vvm) and agitation was controlled to keep dissolved oxygen levels in the vessel at levels below 3% for experiment 1 (Exp. 1) and between 5 and 15% for experiment 2 (Exp. 2). Agitation varjdng from 200 to 500 rpm for Exp. 1 and up to 600 rpm for Exp. 2 were required. Ammonium sulfate concentration was 2.5 g/1 in Exp.l and reduced to half in Exp.2 to limit yeast growth in the culture. Once the initial lag phase was finishing, ethanol was fed in the vapor form continuously by bubbling the incoming air through a flask containing liquid ethanol. Aeration was made with an appropriate flow to keep ethanol concentration in the fermenter below 25 g/1. This concentration was selected because it has been reported that ethanol concentrations above 25 g/1 favor acetaldehyde production over ethyl acetate production [29] and a strong decrease of respiratory quotient (q02) was observed for ethanol levels up to 30 g/1 [33]. Added ethanol in both experiments is represented in Figure 1. Analytical methods Total biomass was determined by dry weight method. Results are expressed as g/1. Cell mortality was determined by methylene blue coloring method and cell count in a Neubauer chamber and was expressed as % of total population [37] . Total acid production was related to the added NaOH required to regulate pH to 6. Ethanol, ethyl acetate and acetaldehyde in the broth were determined by gas c h r o m a t o g r a p h y . The analysis was made with a 5890 Hewlett-Packard chromatograph equipped with a flame ionization detector. Nitrogen was used as carrier gas at a rate of 4 ml/mn. Split ratio was 1:50. Temperature were: injector and detector, 180°C; oven, 40°C. Separation was achieved with a Megabore HP-1 column (Length, 5m; Inner diameter, 0.53 mm). Concentrations were reported as g/1. Respirometry (02 and C02 measurements) was realized with a Gow-lMac chromatograph equipped with a thermal conductivity detector and a concentric column CTR-1 (Alltech, USA). Helium was used as carrier gas (flow rate, 60 ml/mn). Carbon dioxide production rate (CDPR), oxygen uptake rate (OUR) were expressed in mmol C02 produced (or 02 consumed) / h. 1 reactor. Respiratory quotient (R.Q.) was calculated as follows : RQ = CDPR/OUR.
1145 Acetic acid and other organic compounds were determined by liquid chromatography using a 1081 Hewlett Packard chromatograph equipped with an UV detector (X= 210 nm). Separation was achieved with an Aminex HPX 87H column (Bio-Rad, USA). Oven temperature was 65 °C and 6 mmolar sulfuric acid was used as eluent at a rate of 0.8 ml/mn.
200 r
• Experiment 1
12
24
36 48 Time (h)
84
Figure 1. Added ethanol profiles for both experiments. RESULTS AND DISCUSSION Figures 2a (Exp. 1) and 2b (Exp. 2) show the evolution of biomass, its mortality and the actual ethanol concentrations in the medium for both experiments. It can be seen that dissolved oxygen has an influence on the duration of the lag phase. The yeast needed only 12 hours in Exp. 2 to begin to grow on ethanol against more than 24 hours in Exp. 1, in this case dissolved oxygen was less than 2% since the 6th. hour. Growth stopped in Exp. 2 at 48 hours as a result of the limitation in the nitrogen source with a maximum value reached of about 10 g/1, this value was attained when ethanol concentration was 15 g/1. Further accumulation in this case was due to the input through the gas phase. In Exp. 1, no limitation was observed up to the final time and the biomass reached more than 18 g/1 under similar ethanol concentrations than Exp. 2. For Exp. 1, yeast mortality was 20% up to the 66th hour and then increased to reach 50% at the end of the fermentation. This augmentation corresponds to the second feed in ethanol (hour 62). Mortality was more important for Exp. 2 where it was 50% until the 50th hour and reached a value of 85% at the end. This was probably due to the increase in ethanol concentration and the accumulation of acidic compounds and acetaldehyde (see Figure 3b) in the medium at this time.
1146 25 r
•-•O"" Mortality
—*—Biomass — -D — Ethanol
48 Time (h) Figure 2a. Evolution of biomass, ethanol and mortality yield for Exp. 1. 25 1
,^
§,_^
—O---- Mortality
— A - - Biomass - - D -- Ethanol
-1
i—1
z?"^
20
80
/,••
//
o
2
15
60 o
c« (/3
a
(5 o
100
/
10 _ [ y5 ~
- - d
%
-
. 40
.••
^JT
1
^ ^
r\m»^r
20
^' y
^ • • ^
0 ^ ()
/
-
p
1
24
•
Time (h)
1
48
n
•
72
Figure 2b. Evolution of biomass, ethanol and mortality yield for Exp. 2. Figure 3a and 3b represents the consumption of ethanol, as obtained from the mass balance, and the evolution of acetaldehyde, acetic acid and ethyl acetate in the medium.
1147
- • — Ethyl acetate - O- - Acetaldehyde T3
a — Acetic acid
>%
1 •i^
••it— Ethanol
73
^ ^ c> 4-J
(D cd ^ TJ
">.
:S PQ
§
48 Time (h) Figure 3a. Evolution of products and total ethanol consumption in Exp. 1.
25 r
>>^^*s 43
3 T^ dj
c> c3
C3
O cd
20 h
- # — Ethyl acetate - o- - Acetaldehyde a — Acetic acid -•^-" Ethanol
70
A
60
H o
50 15 h
40
o
d-o^.
4->
(U
o 10 h
o o 3
-I 30
cd cd T3
A 20
,__, c >. cd ^ W
o
-I 10 24
Time (h)
48
0 72
Figure 3b. Evolution of products and total ethanol consumption in Exp. 2. The ethanol consumption was slower in Exp. 1 which required to stop substrate feeding from hour 40 to hour 60, as seen in figure 1, to keep the concentration below 25 g/1. This problem did not occur in Exp. 2, despite a lower biomass concentration, and ethanol with an average flow rate of 3.7 g/h was fed
1148 without exceeding 22 g/1 in the fermenter. In this experiment, the lag phase was shorter and productivities in ethyl acetate and acetaldehyde were 0.105 and 0.437 g/l.h, respectively. To try to understand the evolution of these products in both experiments, the synthesis pathway of each compound was considered. It has been proposed that, in yeasts such as Hansenula, Kluyveromyces or Candida, ethyl acetate is formed according to the following reactions [32, 29, 22]: Ethanol + 1/2 02 Acetaldehyde + 1/2 02 Acetyl CoA + Ethanol
> Acetaldehyde + H2O > Acetic acid - Fe*++
+ Fe**+
> Acetyl CoA
> TCA Cycle
> Ethyl acetate + H2O
C. utilis was grown in iron limitation which resulted in decreased molar growth yield with respect to carbon substrates and in increased specific rates of oxygen uptakes [39]. Growth under reduced iron concentrations is limited by the available metabolic energy. IMoreover, ethyl acetate production, under these conditions, was favored because acetyl-CoA is diverted to ester formation rather than being oxidized through the tricarboxylic acid (TCA) cycle [39]. In Exp. 1, ethyl acetate production began simultaneously with growth (after 40 h), to reach a concentration of more than 7 g/1 in the bioreactor. Acetaldehyde synthesis started before, as soon as ethanol was fed to the reactor. It did not accumulate and a concentration of 0.8 g/1 was attained. Acetic acid production seemed to be linked to ethyl acetate production and hence to growth. A concentration of 4 g/1 was attained. No inhibition in ethyl acetate synthesis was observed despite ethanol concentrations above 20 g/1. In Exp. 2, the accumulation of the three compounds started at the same time than growth, but with faster production of acetaldehyde than other compounds. In that case, a very high acetaldehyde concentration was observed (more than 22 g/1). This concentration has been reported to be inhibitory for ethyl acetate synthesis by C. utilis probably because it reduces acetyl-CoA formation, an intermediate in the synthesis of the ester [28]. It is also lethal for cells, as can be seen for the cell mortality after 66 hours. It must pointed out that the ethyl acetate concentration reached its maximum (5.2 g/1) rapidly (only 20 hours after the beginning of its production). It appeared that acetic acid production was not inhibited by the high acetaldehyde concentration and this is a confirmation of the inhibition of the esterification step. It can be concluded t h a t oxygen limitation may favor ethyl acetate production while acetaldehyde accumulation needs higher dissolved oxygen levels. This trend was also observed with K fragilis but at higher values (40 % and 70 % of dissolved oxygen for ethyl acetate and acetaldehyde, respectively) [23]. Besides the volatile compounds mentioned above, C. utilis produces other non volatile substances. Figure 4 relates the consumption of NaOH, to maintain a constant pH against the acetic acid produced.
1149 700
— # — Experiment 1 - - O - - Experiment 2
600
0
,^
200 100 150 Acetic acid (meq) Figure 4. Evolution of added sodium hydroxide against acetic acid production for both experiments.
50
It can be seen that the amount of sodium hydroxide needed to neutralize the acids released in the medium is much more important than the amount needed to neutralize the sole acetic acid. The slope of the curves are 3.95 for Exp. 1 and 3.18 for Exp. 2. Although consumption of ammonium sulfate, the nitrogen source, is known to reduce pH, it was only used in low concentrations (max. 2.5 g/1). From analysis of the broth by HPLC with an UV detector, pyruvic and lactic acids were identified by retention time comparison, and quantified at concentrations up to 2 g/1. The production of lactic [40], pyruvic and succinic acids [41] by Candida species were already reported. Such accumulations in both experiments may also be related to the formation of other compounds such as fusel alcohols or glycerol (which was detected in these experiments). Table 1 shows the yields of dry weight, acetic acid, acetaldehyde and ethyl acetate per gram of ethanol which were obtained from the carbon balance of both experiments. Yields (g compound / g ethanol) Biomass Ethyl acetate Acetic acid Acetaldehyde
Exp. 1 0.510 0.123 0.084 0.004
Exp. 2 0.201 0.194 0.088 0.240
Table 1 - Yields of some products during the growth of C utilis. These data show clearly the influence of nitrogen concentration on biomass production as well as the importance of the dissolved oxygen levels on the
1150 metabolites production. Medium used in Exp. 2 contained a concentration twice lower in nitrogen source than medium used in Exp. 1, which explains a lower biomass synthesis. This was already observed with the same media used in solid state cultures of C utilis [38]. The results given in table 1 show an increase of acetaldehyde and a decrease of biomass per gram of substrate on higher oxygen levels. Also, a slight increase on acetaldehyde and acetic acid production was observed in these conditions. So that, formation of end products was favored at high levels of oxygen. Also, higher oxygen levels enhance the formation of acetaldehyde over ethyl acetate. Furthermore, these conditions favored also higher specific productivities (expressed as grams of product per gram of biomass). While different distribution in biomass and products between the experiments was observed, both systems showed similar substrate carbon conversion capacities, (68% and 71% was transformed into biomass and products for Exp. 1 and Exp. 2, respectively). Hernandez and Johnson reported a value of 0.68 g of cells per gram of ethanol growing C. utilis in Erlenmeyer flasks in a non-iron-deficient medium. The iron-limited media decreases the efficiency of energy and growth yields with respect to the carbon sources, but it increases the specific rates of oxygen in microorganisms [42]. Therefore, it was expected to obtain low 3delds in the formation of cellular material, but the ethyl acetate production and acetaldehyde production were increased due to the presence of ethyl acetate substrates such as oxygen, acetic acid and ethanol. Table 2 resumes the results for ethyl acetate production from various studies. Yeast Candida utilis Hansenula anomala Kluyveromyces fragilis Saccharomyces rouxii Candida pseudotropicalis Candida utilis Candida utilis
Particular conditions
Yield (g/gEtOH) Nitrogen free medium+ ethanol (10 g/1) 0.254 Continuous culture (D=0.1 h"i) 0.014 0.300 Lactose + ethanol (10 g/1) 0.308 Glucose + ethanol (46 mg/1) Iron free mediima + ethanol (30 g/1) 0.191 This study, Exp. 1 This study, Exp.2
0.123 0.194
Ref.
29 32 22 27 31 _ -
Table 2 - Yields in ethyl acetate conversion from ethanol for various yeasts. Figures 5a (Exp. 1) and 5b (Exp. 2) show the rates of oxygen uptake, of carbon dioxide production and from the respiratory quotient (R.Q.). Carbon dioxide production followed approximatively growth evolution. Oxygen consumption was much more important than carbon dioxide production and this is reflected in the RQ. In Exp. 1, this coefficient is constant around 0.33 while in Exp. 2, it slowly increased from 0.15 to 0. 4. The values found agree with the carbon mass balance analysis as reported previously [43]. With the reported values in Table 1 mass balance predicts that lower RQ should be expected when increased volatiles and acid production. This is basically due to the oxidation of the ethanol. Moreover, increased biomass yields should also reduce RQ. Respirometry analysis gives a good indication of product and biomass productions.
1151 50 r-
J2
s
i
J, Pi
Q
u oi O 48 Time (h) Figure 5a. Respirometric activity for Exp.l. 35 r [ 30
f --B-R.Q.
^—s^
413
sO
B B
p^
25 20
U
g
10
O
L
5 0
V
U
15 r
-1
yf
r
Q
u
"1 0.6 H
—e—OUR --••-•CDPR
/
/
/
^V^&n \
-/
\
0
O
0.3
7 ^ ' •
• • ' '
i^—r^
0.4 -§ A "j
>v
ET
0.5
1
24
,
Time (h)
1
A
^
I
0.2
CD
3
.
48
72
Figure 5b. Respirometric activity for Exp. 2. Figure 6 shows the formation of cells versus the consumption of oxygen. It is observed that O2 consumption continues after the cells have reached their maximum growth. This is due to the continuous production of organic acids such as acetic which require ethanol oxidation. There is not much information available to compare these results with those reported in the literature. Hernandez and Johnson [42] reported a
1152 yield of 0.61 g of dry cells of C utilis per gram of oxygen when such microorganism was grown in ethanol medium. Moreover, Paca and Votruba [32], observed that different levels of the oxygen affected the growth rate of C. utilis 106, but no specific data were available in such report. Armstrong et al. [29] reported that high levels of aeration increase the ethyl acetate production, but also neither oxygen yields nor respiratory quotients were reported. 30 r c o '^ o
2
OH
c3
s
2
o H 20
120 40 60 80 100 Total oxygen consumption (g) Figure 6. Relationship between biomass production and oxygen consumption in both experiments. CONCLUSION Submerged culture of C utilis on continuously fed ethanol vapor as sole carbon source, was found to be a suitable process for the production of ethyl acetate as well as acetaldehyde by C. utilis. The production was favored by increasing the levels of oxygen in ethanol containing iron-deficient media. These conditions inhibit the formation of energy through the electron transport system. As a consequence of such inhibition, different mechanisms for fueling and biosynthetic pathways are present in the yeast and therefore, other products such as pyruvic and lactic acids, are formed as well. The oxygen supply to this media plays an important role during the growth of the yeast; for example, the specific substrate and end-product yields, and the respiratory quotient are dependent on oxygen concentration in the media. It was also observed that direct measurement of carbon dioxide production was a good indicator of growth. Respiratory quotient was found to reflect oxidized metabolites excretion in the medium. The physiology of C. utilis in these conditions is interesting for potential applications such as assimilation of ethanol in industrial gas streams. This work was achieved under research agreement between ORSTOM (France) and the UAM (Mexico).
1153 REFERENCES 1. R.J. Whitaker and D.A. Evans, Food Technol., 41(9) (1987) 86. 2. P. Christen and A. Lopez-Munguia, Food Biotechnol., Accepted for publication. 1994. 3. L. Janssens, H.L. de Footer, N.M. Schamp and E.J. Vandamme, Proc. Biochem. 27 (1992) 195. 4. P.S.J. Cheetham, The flavour and fragrance industry. In: Biotechnology. The science and the business, V. Moses and R.E. Cape.(eds.), Harwood Acad. London, 26 (1991) 481. 5. P.S.J. Cheetham, Trends Biotechnol., 11 (1993) 478. 6. G.M. Kempler, Adv. Appl. Microbiol., 29 (1983) 29. 7. M.S.A. Wecker and R.R. Zall, Appl. Env. Microbiol., 53(12) (1987) 2815. 8. F. Cormier, Y. Raymond, C.P. Champagne and A. Morin. J. Agric. Food Chem., 39 (1991) 159. 9. M.I. Farbood and B.J. Willis, (1985) US Patent No 4 560 656. 10. C. Creuly, C. Larroche and J.B. Gros, Appl. Microbiol. Biotechnol., 34 (1) (1990) 20. 11. P. Chalier and J. Crouzet, Biotechnol. Lett., 14(4) (1992) 275. 12. P. Christen and M. Raimbault, Biotechnol. Lett., 13(7) (1991) 521. 13. E. Peynaud and G. Guimberteau, Ann. Technol. Agric, 11 (1962) 85. 14. P. Schreier, F. Drawert, A. Junker, H. Barton, and G. Leopold, Z. Lebensm. Unters. Forch., 162 (1976) 279. 15. H. Suomalainen, J. Inst. Brew., 87 (1981) 296. 16. Fenaroli's handbook of flavor ingredients, 2nd Ed., Vol. 2, CRC Press, Boca Raton, Fla. 1975. 17. F. Welsh, W.D. Murray and R.E. WiUiams, Crit. Rev. Biotechnol., 9(2) (1989) 105. 18. R. Davies, E.A. Falkiner, J.F. Wilkinson and J.L. Peel, Biochem. J., 49(1951) 58. 19. J. Tabachnick and M.A. Joslyn, J. Bacteriol., 65 (1953) 1. 20. L. Janssens, H.L. de Footer, E.J. Vandamme and N.M. Schamp, Med. Fac. Landbouww. Rijskuniv. Gent, 52(4) (1987) 1907. 21. L. Janssens, H.L. de Footer, L. Demey, E.J. Vandamme and N.M. Schamp, Med. Fac. Landbouww. Rijskuniv. Gent, 53(4b) (1988) 2071 22. H. Kallel-Mhiri and A. Miclo, FEMS Microbiol. Lett., I l l (1993) 207. 23. H. Kallel-Mhiri, J.M. Engasser and A. Miclo, Appl. Microbiol. Biotechnol., 40 (1993) 201. 24. S. Shindo, J. Murakami and S. Koshino, J. Ferment. Bioeng., 73 (1992) 370.
1154 25. E. Longo, J.B. Velazquez, C. Sieiro, J. Cansado, P. Calo and T.G. Villa, World .J. Microbiol. BiotechnoL, 8 (1992) 539 26. K. Yoshioka and N. Hashimoto, Agric. Biol. Chem., 45(1) (1981) 2183. 27. F.M. Yong, K.H. Lee and H.A. Wong, J. Food TechnoL, 16 (1981) 177. 28. D.W. Armstrong, S.M. Martin and H. Yamazaki, Biotech.Lett., 6(3) (1984) 183. 29. D.W. Armstrong, S.M. Martin and H. Yamazaki, Biotech. Bioeng., 36 (1984) 1038. 30. W.D. Murray, S.J.B. DuflF, P.H. Lanthier, D.W. Armstrong, F.W. Welsh and R.E. Williams, In: Frontiers of Flavor. G. Charalambous (ed.). Proceedings of the 5th. International Flavor Conference, Porto Karras, Chalkidiki, Greece, Elsevier Science Publishers B.V., Amsterdam. (1988) 1. 31. A. Willets, Antonie van Leeuwenhoek, 56 (1989) 175. 32. J. BoL, W. Knol and B. ten Brink, Dechema-Monographs, 105 (1987) 235. 33. J. Paca and J. Votruba, Appl. Microbiol. BiotechnoL, 33, (1990) 438. 34. W.D. Murray, S.J.B. Duff and P.H. Lanthier, Biomass, 23(3) (1990) 229 35. H.K. Chiang, G.L. Foutch and W.W. Fish, Appl. Biochem. Biotech., 28/29 (1991) 513. 36. C.H. Kim and S. KRhee, BiotechnoL Lett., 14(11) (1992) 1059. 37. P. Christen, R. Auria, C. Vega, E. Villegas and S. Revah, BiotechnoL Adv., 11 (1993) 549. 38. P. Christen, R. Auria, R. Marcos, E. Villegas and S. Revah, Adv. Bioprocess Eng., (1994). In press. 39. K.C. Thomas and P.S.S. Dawson, Can. J. Microbiol., 24 (1978) 440. 40. A. Prell, J. Paca and K. Sigler, Appl. Microbiol. BiotechnoL, 36 (1991) 236. 41. A. CoUings, A.R. Holmes and M. G. Shepherd, Biomedical Lett., 46 (1991) 285. 42. E. Hernandez and M.J. Johnson, J. BacterioL, 94(4) (1967) 996. 43. L.E. Erickson, I. G. Minkievich and V.K. Eroshin, Biotech. Bioeng., 20 (1978) 1595.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1155
Changes in microstructure and thermal properties of thermally processed cornstarch/soy protein isolate model food systems F.A. Nyanzi, J.A. Maga and C. Evans Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523, U.S.A.
Abstract
Fluorescent microscopy and differential scanning calorimetry were used to study the influence of thermal processing on the microstructure and thermal properties of cornstarch/soy protein model food systems. Both analytical methods showed that extrusion thermal processing at 50'C affected the physical characteristics of cornstarch/soy protein samples almost to the same extent as conventional heating at 150**C. For example, the enthalpies of transition were 0.8J/g for samples conventionally processed at IBO'C and 0.6J/g for samples extruded at 50'C. The micrographs also showed that the starch granules were not completely disrupted by conventional thermal processing at 150*C but extrusion thermal processing gelatinized the starch granules at 50'C. Overall, the greatest microstructure modifications of starch or protein were clearly shown to be due to extrusion when compared to conventional thermal processing.
1.
INTRODUCTION
Thermal processing of food systems induces changes in microstructure [1] and the thermal properties [2] of food components. Studies of these changes may contribute to the understanding of how food components can influence the functionality of food systems. Electron microscopy has been used by researchers [1,3-6] to study the microstructure of food systems. Other researchers [7-10] have followed changes in thermal properties of thermally processed samples by the use of differential scanning calorimetry. Fluorescent microscopy has received very limited attention in the study of microstructural changes in processed samples. A study by Goossens et al. [11] showed that fluorescent microscopy is a good method for identifying protein and starch in wheat samples. This method has not yet been shown to be applicable to thermally processed samples. Therefore, the purpose of the present study was to use fluorescent microscopy in conjunction with differential scanning calorimetry to determine the changes in microstructure and thermal properties induced by thermal processing method and temperature.
1156 2.
MATERIALS AND METHODS
2.1. Materials The ingredients consisted of commercial cornstarch (Pure-Dent B700: a gift from Grain Processing Corporation, Muscatine, Iowa) and commercial soy protein isolate (Ardex R: a gift from Archer Daniels Midland Company (ADM), Decatur, Illinois). 2.2
Cornstarch preparation for extrusion A 2kg starch sample was mixed with 188 ml of 0.08N sodium hydroxide solution for 10 minutes using a Hobart mixer. Model D-300 (The Hobart Manufacturing Company, Troy, Ohio, USA) set at the lowest speed. The prepared sample had 20% w/w moisture and pH=7. Samples were sealed in plastic bags and left at room temperature for extrusion processing the following day. 2.3
Protein pellet preparation for extrusion Two ml of 0.3N sodium hydroxide was mixed with 2.5 g of soy protein isolate, and a round pellet was manually made. The prepared pellet had a pH of 7. These pellets were stored overnight at room temperature in sealed plastic containers to prevent moisture loss, for extrusion processing the following day. 2.4
Soy protein isolate and/or cornstarch preparation for conventional processing Samples containing lOg of cornstarch or (75:25) cornstarch:soy protein isolate were mixed manually with a glass rod for 1 minute while adding 2 ml of 1.5N sodium hydroxide. Samples were left in sealed containers overnight at room temperature before an aliquot was packed in stainless steel tubes for processing. Stainless tubes 15cm long with an inside diameter of 0.40cm were used in this method. The samples were packed in the tubes and both ends were tightly sealed with metal screw covers. The tubes, with their respective samples, were temperature- and moisture-equilibrated overnight before thermal processing. 2.5
Extrusion processing Extrusion thermal processing was performed in a single screw Brabender Plasticorder Extruder, Model PL-V500 (C.W. Brabender Instruments, Inc., South Hackensack, New Jersey), equipped with a variable D-C drive unit, a tachometer, and a torque meter. The extruder barrel had a diameter of 19.05mm with a 20:1 length:diameter ratio and eight 0.79 X 3.18mm longitudinal grooves. In addition to a tapered screw with a 3:1 compression ratio, a die with a diameter of 4.8mm was used. The temperature of the extruder barrel was controlled by two electrically heated zones. The first heating zone was in the compression section and the second one in the metering section just next to the die. The dough temperature was maintained by compressed-air-cooled barrel collars. The compressed air was controlled by thermostats found inside the barrel wall. The preconditioned starch was fed manually into the feed zone of the extruder while bringing the screw speed to the processing speed of 120 rpm. After a steady state flow, as shown by a torque variation of +2.5 inch-pound, had been maintained and the processing dough temperature just before the die (either 50'C or 150'C) obtained, an experimental protein pellet was introduced in the feed zone. Immediately after a pellet was introduced, the extrudate strand was cut off and discarded. The subsequent exiting extrudate strand, which contained the protein sample, was
1157 cut off and saved. The presence of protein was determined by observing the difference in color of the exiting strand. 2.6
Conventional thermal processing An oil bath at 160'C was used to process the samples. The tubes to be processed, and another tube containing a thermocouple to monitor the temperature, were placed in metal racks, and immersed in the heated oil-bath. When the thermocouple registered the desired processing temperature (either 50'C or 150'C), the processed tubes were removed and immediately immersed into an icecold water-bath to stop the effects of thermal processing. For each set of tubes to be processed, a temperature-control tube containing the same soy protein and/or cornstarch preparation and a thermocouple was used. 2.7
Fluorescent microscopy Fluorescent microscopy was used to determine the microstructure changes in processed cornstarch/soy protein isolate samples. Samples were prepared by embedding 1 cm sections in paraffin blocks. The embedded samples were sectioned into 5 iim slices with a microtome and mounted on a microscope slide. Acridine orange fluorochrome was used in order to differentiate starch from protein structures as per Goossens et al. [11]. A Carl Zeiss fluorescent microscope equipped with 40X objective lens, 47 background filter, BG12 excitation filter, a 200W mercury arc lamp, and a camera body with automatic exposure control, were used to record microstructure of the samples on Ektachrome 100 ASA film. 2.8
Differential scanning calorimetry The method of Kugimiya and Donovan [12], with minor modifications, was employed to analyze thermal characteristics of experimental samples. A 2 to 3 mg sample of thermally processed cornstarch/soy protein isolate was weighed directly into a tared aluminum hermetic pan. A microsyringe was used to deliver lO^L of distilled water into the pan containing the sample and the pan was sealed by a press. The sealed pans were stored overnight at room temperature before thermal analysis. A DuPont 910 cell base and 9900 computer/thermal analyzer were used. The cell was purged with nitrogen gas at a rate of 40 ml per second. The heating rate was 10"C per minute from 20'C to 120'C. Enthalpies of transition ( A H ) , onset temperature (T^), and peak maximum temperature (Tp) were determined by a DuPont computer program General Analysis Utility Version 2.1.
3. RESULTS AND DISCUSSION 3.1
Microstructure determination by fluorescent microscopy Figure 1 shows the microstructure of the starch-protein sample conventionally processed at 50'C. No observable structural changes were detected with the starch granules or the soy proteins. The starch granules and protein particles appear to be intact.
1158
Figure 1 Fluorescent micrograph of cornstarch/soy protein isolate (75:25) sample showing the effect of thermal processing by conventional method (oil-bath heating) at 50'C, 20%w/w feed moisture, on the protein and starch microstructure of the sample.
1159
Figure 2 Fluorescent micrograph of cornstarch/soy protein isolate (75:25) sample showing the effect of thermal processing by extrusion method at 50'C, 20% w/w feed moisture, on the protein and starch microstructure of the sample.
In contrast, a micrograph of an extruded sample at 50'C, shows "melted" starch and a possible aggregation of protein particles (Figure 2). Shear and pressure during extrusion thermal processing, regardless of the temperature levels, appear to drastically alter the microstructure of starch granules.
1160
Figure 3 Fluorescent micrograph of cornstarch/soy protein isolate (75:25) sample showing the effect of thermal processing by conventional method (oil-bath heating) at 150'C, 20% w/w feed moisture, on the protein and starch microstructure.
Increasing thermal processing temperatures to 150'C, for the conventional method, influenced the microstructure for both starch and protein. Figure 3 shows that most of starch granules have been disrupted and "melted" with a few unaffected granules. Protein particles appear to have been unfolded and spread over the "melted" starch mass.
1161
Figure 4 Fluorescent micrograph of cornstarch/soy protein isolate (75:25) sample showing the effect of thermal processing by extrusion method at 150'C, 20% w/w feed moisture, on the protein and starch microstructure.
Extrusion thermal processing at 150T causes protein unfolding and realignment as seen in Figure 4. Protein fibers are observed in the micrograph. The micrograph shows that the starch granules were completely "melted" and that some of the starch is intermeshed with the protein strands. Starch appears to be adsorbed to the protein fibers.
1162
m
3 Q. O fi-
IT
ro 3 3: ro o O
TQmpQraturQ (°C) Figure 5 Differential scanning calorimetry thermograms of cornstarch/soy protein isolate (75:25) samples. Thermograms indicated are for samples: Aunprocessed, B and D - conventional method processed at 50'C and 150'C, respectively; C and E extrusion method processed at 50*'C and 150'C, respectively. All samples had 20% w/w feed moisture before processing.
3.2
Differential scanning calorimetry thermoanalysis Thermoanalysis data (Table 1) and DSC thermograms (Figure 5) show thermal properties of cornstarch-soy protein isolate samples thermally processed by both procedures at 50'C and 150'C, along with a non-heat processed control.
A single enthalpy peak occurred for the unprocessed starch/protein blend as shown in thermogram A. The peak had an enthalpy of transition (AH) of 7.4J/g and peak temperature (Tp) of 74.3*'C. Unprocessed starch alone was determined to have an enthalpy of transition of 16.9 J/g which was within the range of 15.5 J/g to 31.8 J/g as determined by Stevens and Elton [13]. The peak appeared in the same temperature range where cornstarch gelatinization enthalpy normally occurs as
1163 reported by Stevens and Elton [13]. Therefore, the peak was attributed to the cornstarch gelatinization endotherm. The thermogram did not show other peaks generally attributed to the enthalpy of protein denaturation [14]. The reason for the absence of the protein peak might have been due to the nature of protein since commercial soy protein isolate had been shown to produce no detectable peaks with DSC thermoanalysis [10]. The thermogram for a starch-protein blend conventionally processed at 50'C (Figure 5, thermogram B) did not produce detectable thermal property changes in the sample components. The samples had virtually the same enthalpy of transition and T as the unprocessed sample. This may have been the result of a very short duration of heating or that heating the sample to 50'C was not enough to cause any thermal changes in the sample. Parsons and Patterson [15] showed that thermal properties of food systems are influenced by both temperature and duration of heat treatment. Therefore, increasing thermal processing duration might have caused some changes in at least the enthalpy peak of transition. The effect of thermal processing on the thermoprofile of a starch-protein blend was demonstrated when the samples were extruded at 50'C (Figure 5). Table 1 showed that the enthalpy of transition was decreased from 7.4 J/g (unprocessed sample) to 0.6 J/g for the extruded sample but only a slight decrease for a sample conventionally processed at 50"C. This showed that extrusion thermal processing affects the thermal properties of a food system differently than conventional thermal processing. The low temperature when coupled with shear and pressure during extrusion caused changes in the thermal properties of a food system. When the starch-protein sample was conventionally heated to 150'C, the enthalpy peak measured by DSC was virtually the same as that of samples extruded at SO^'C (Figure 5 ) . DSC thermograms showed that there was incomplete starch gelatinization even at a processing temperature of 150'C. This observation is in line with the observation that the amount of water [9,16,17] and the presence of protein [18] in a starch sample influence starch gelatinization, thus influencing the size of the enthalpy peak. In contrast, extrusion thermal processing of samples at 150*C caused the elimination of any detectable DSC enthalpy peak. Table 1 Effect of thermal processing method and processing temperature on onset temperature (To), peak maximum temperature (Tp), and transition enthalpy (AH) of cornstarch/soy protein isolate bend (75:25). All samples were thermally processed at 20% w/w feed moisture. Scanning was performed at temperatures form 20''C to 120^C at a heating rate of lO'^C per minute. Processing method Unprocessed Conventional Extrusion Conventional Extrusion
Processing temp. f'C)
50 50 150 150
TJ'C) 69.0 68.5 70.4 70.5
+ + + +
ND
Tp(^C) 0,2 0.5 0.2 0.2
74.3 74.8 75.0 75.1
+ + + +
ND
AH(J/q) 0.1 0.1 0.2 0.1
7.4 + 0.5 6.9 + 0.3 0.62 + 0.05 0.78 + 0.08
ND
1164 ND:
4. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Not detected
REFERENCES L. Jing-ming and Z. Sen-lin, Starch, 42 (1990) 96. C.G. Biladeris, Food Chem. 10 (1983) 239. M.U. Taranto, G.F. Ceqla and K.C. Rhee, J. Food Sci., 43 (1978) 973. M.J. Gomez and J.M. Aguilera, J. Food Sci., 48 (1983) 378. L.C. Lin and T. Ito, J. Food Technol., 21 (1986) 133. M. Bhattacharya and M.A. Hanna, Lebensm. Wiss. Technol., 20 (1987) 195. J.W. Donovan and R.A. Beardslee, J. Biol. Chem., 250 (1975) 1966. A.M. Hermansson, J. Text. Studies, 9 (1978) 33. C.G. Biliaderis, T.J. Maurice and J.R. Vose, J. Food Sci., 45 (1980) 1669. S.D. Arntfield and F.D. Murray, Can. Inst. Food Sci. Technol. J., 14 (1981) 289. J. Goossens, F. Derez and K.H. Bahr, Starch, 40 (1988) 327. M. Kugimiya and J.W. Donovan, J. Food Sci., 46 (1981) 765. D.J. Stevens and G.A.H. Elton, Starch, 23 (1971) 8. F.W. Sosulski, R. Hoover, R.T. Tyler, R.T. Murray and S.D. Arntfield, Starch, 37 (1985) 257. S.E. Parsons and R.L.S. Patterson, J. Food Technol., 21 (1986) 123. J. W. Donovan, Biopolymers, 18 (1979) 263. D.J. Burt and P.L. Russell, Starch, 35 (1983) 354. R.a. Gryzybowski and B.J. Donnelly, J. Food Sci., 42 (1977) 1304.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
Evaluation of Cookeina sulcipes of its B i o m a s s Composition
1165
as an Edible Mushroom: D e t e r m i n a t i o n
Jose E. Sanchez^, Antonio M. Martin^ and Angel D. Sanchez^ ^ Centro de Investigadones Ecologicas del Sureste, Apdo. Postal 36, Tapachula, Chiapas, Mexico
30700
^ Memorial University of Newfoundland, St. John's, Newfoundland, Canada AlB 3X9 ^ Escuela de Qmmica, Universidad Nacional Autonoma de Chiapas, Km. 2.5 Carr. Antiguo Aeropuerto, 30700 Tapachula, Chiapas, Mexico
Abstract Cookeina sulcipes is a relatively unknown edible tropical mushroom. This mushroom was studied with the aim of evaluating its chemical composition to estimate its potential for commercial production. Determinations were conducted of the following biomass components of the mushroom: amino acids, ash, carbohydrates, carotenoids, fatty acids, fibre, lipids, protein, mineral composition and water content. Among the main results, it was found that C. sulcipes possesses a high protein content, a relatively high content of phosphorus and a low content of lipids. All the essential amino acids were present, albeit the concentration of tryptophan was relatively low. Therefore, it may be concluded that this mushroom biomass could be considered a valuable human food.
1. INTRODUCTION Cookeina sulcipes is an ascomycete from the tropicgJ areas of the earth that grows in mushroom form on dead logs of the cacao plant Theobroma cacao. Few studies have been conducted on this species, and only two references have been found to report that C. sulcipes is eaten in the Mexican regions of Veracruz and
1166 Chiapas (Chac6n, 1988; Sanchez et aL, 1994). In addition to its value as food, this mushroom is attractive due to its pink colour and its shape, and it is frequently used as an ornament. Edible mushrooms are considered to be nutritious foods, due to their relatively high protein contents (15 to 50 % of the biomass, on a dry weight basis). In addition, edible mushrooms generally contain all amino acids essential for human nutrition, being especially rich in lysine and leucine. The scarcity of general scientific information about C sulcipes includes a lack of information about its chemical composition. The study of the composition of exotic edible mushrooms is important before their commercialization is attempted (Martin, 1992). Given the fact that the use of this mushroom as food is widespread among rural populations in the above mentioned Mexican regions, and because there is an absence of information about its nutritional characteristics, the present work was conducted with the objective of determining the chemical composition of C. sulcipes. It is expected that a deeper knowledge of the mushroom's characteristics will awaken interest in its commercial exploitation.
2. MATERIALS AND METHODS 2.1. Mushroom Fresh fi'uiting bodies of C. sulcipes were collected from the region of Soconusco, State of Chiapas, Mexico. To preserve the mushrooms, they were dried to a final moisture content of 10 % (total weight). 2.2. Analytical Methods The analyses of moistiire content, crude fibre and ash were conducted according to the methods of the Association of Official Analytical Chemists (Anon., 1984). The determination of the crude protein of the mushroom was done by finding the total nitrogen content by the Kjeldahl method, and multiplying the resulting value by the factor 4.38. This value was chosen instead of the usual conversion factor of N X 6.25 to accoimt for the non-protein nitrogen associated with the chitin of the mushroom cell wall (Bano and Rajarathnam, 1989). The mushroom biomass was analyzed for amino acids (Blackburn, 1968; Penke et aL, 1974), carbohydrate content (Quarmby and Allen, 1989), carotenoids (An et aL, 1989), and minerals (Anon., 1974). The total lipid content analysis was conducted following the method ofBligh and Dyer (1959). The fatty acid determination involved extraction by the method ofBligh and Dyer (1959), followed by a modified form of the procedure of Thompson (1969). In this procedure, the lipids were dried imder nitrogen, transmethylated (Keoiigh and
1167 Kariel, 1987), extracted into hexane, re-dried under nitrogen, dissolved in CSg, and analyzed using a Perkin Elmer model 8310 gas chromatograph, with a Supelco SP 2330 resin-packed colunm, at 170°C and 3 x 10"^° amp/mv sensitivity.
3. RESULTS AND DISCUSSION The general composition of C. sulcipes is presented in Table 1. The information is presented for both the cap (pileus) and the stem (stipe) of the mushrooms. It can be seen that there are differences between the compositions of the two main parts of the mushroom. The water content is high for both, being higher for the stems, which gives these a more tender consistency. The protein values for both parts compare well with those reported for other edible mushrooms. For example, Bano and Rajarathnam (1989) reported the following protein values, in % dry weight: Pleurotus ostreatus, 10.5 %; Volvariella displasia, 28,5 %; Agaricus bisporus, 26,3%; Pleurotus flabellatus, 21,6%; and Lentinus edodes, 17.5 %, The higher protein content in the caps than in the stems agrees with the findings of Jandaik et al, (1979) in wild Indian mushrooms. It is important to emphasise that some authors utilize the factor N X 6.25, which is generally used for cereals and other foods, for calculating the protein content of mushrooms (Manu-Tawiah and Martin, 1987). Therefore, their final protein content values appear to be higher than those reported in this work, in which the factor N X 4.38 is utilized, as mentioned above. The lipid contents are similar to those reported by Singh and Verma (1991) for Pleurotus djamor (2,75%) and Pleurotus platypus (2.59%). The values found for the lipids fall within the range of 2 to 6 % (dry basis) reported for mushrooms (Li and Chang, 1989). The crude fibre content of C. sulcipes is high, which is common for mushrooms grown on woody materials. Another example of this is Pleurotus limpidUy which attains a crude fibre content of 27.6 % of its total dry weight (Bano and Rajarathnam, 1989). The ash content of C. sulcipes is relatively high, but within the higher limit of ash contents reported for mushrooms (Bano and Rajarathnam, 1989). However, the carbohydrate content is low compared to those reported by Lau (1989). The reddish colour of the caps of C, sulcipes, collected in the soluble lipid fraction of the extract produced by the method of An et al, (1989), indicated the presence of carotenoids in the mushroom. Due to the presence of impurities, it was not possible to make a final analytical determination of the types of carotenoids and their concentrations. Further studies will need to be conducted
1168 in this regard. The presence of carotenoids in C. sulcipes could increase its value as a source of pigments for the food industry. Research is being actively conducted in finding carotenoid-producing microorganisms (Martin et al,, 1993a), and in optimizing the production by microorganisms of such important carotenoids as astaxanthin (An et al., 1989; Martin et al., 19936,c), which is responsible for coloured salmonid flesh. Table 1 Proximate composition of Cookeina sulcipes, % dry weight ^ Component Moisture Dry matter Ash Crude lipids Crude fibre Crude protein (N X 4.38) Soluble Carbohydrates Carotenoids
Cap 79.9 ± 1.5 20.01 ± 1.5 10.4 ± 1.5 ^ 2.95 ± 0.1 " 14.3 ± 0.32 24.79 ± 0.16 6.8 ± 0.15 ' present
Stem 85.4 ± 1.2 14.6 ± 1.23 9.5 ± 1.2 ^ 2.7 ± 0.12 ^ not determined 20.68 ± 0.27 6.2 ± 0.22 ^
Mean of three determinations ± standard deviations. Values in the same column with the same superscript are not statistically different (P < 0.05). Table 2 presents the amino add content of C. sulcipes. With the exception of asparagine, which is a non-essential amino acid, the mushroom contains all the amino adds commonly found. The concentrations of the essential amino adds are adequate, except that the content of trj^tophan is low in comparison with that reported for other mushrooms (Chang and Miles, 1989). The mineral contents are presented in Table 3. Phosphorous appears to be abundant, and the main constituent of the mineral fraction in the mushroom, espedally in the fruiting body (sporophore). The concentrations of Fe, Mg and Ca, among others, are high. However, Mn is present in very low amounts. Although most of the Mg is concentrated in the stem, the general pattern is that the cap has a higher mineral content than the stem. Fatty acid determinations were conducted for the caps (Table 4). The analysis reveals that C. sulcipes contains fatty adds with carbon chain lengths between 14 and 24. The most abundant ones are the so-called essential fatty adds with 18-carbon chain lengths, such as linoleic, oleic and stearic acids, and also palmitic add. They represent 92 % of the total fatty add content of the mushroom. Parent and Thoen (1979), while listing palmitic, palmitoleic, oleic and linoleic acids as the main fatty acids present in mushrooms, indicated that palmitoleic
1169 a d d s were not always present. Manu-Tawiah and Martin (1987) also reported that the fatty acid more abundant in Pleurotus ostreatus was linoleic.
Table 2 Amino a d d composition of Cookeina sulcipes (g / 100 g protein) Amino a d d Alanine Arginine Aspartic a d d Cystine Glutamic a d d Glutamine Glydne Histidine Isoleudne Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Cap 3.72 1.66 5.6 0.60 8.04 3.33 2.27 0.78 1.76 2.24 1.92 0.52 1.80 2.99 2.34 2.36 0.03 1.47 1.88
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Stem 0.3" 0.15" 0.2 ^ 0.1" 0.4 ^ 0.4 ^ 0.5 ^ 0.2 " 0.1 ^ 0.2 " 0.3" 0.1 ^ 0.2 ^ 0.3 ^ 0.4" 0.1 " 0.01 ^ 0.2 ^ 0.3"
2.08 1.26 3.69 1.45 4.11 12.47 1.60 0.70 1.33 1.82 1.83 0.44 1.54 1.62 1.499 2.12 0.03 1.00 1.92
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 ^ 0.3 ^ 0.2 ^ 0.1 ^ 0.3 ^ 0.5 ^ 0.1 ^ 0.1 ^ 0.1 ^ 0.2 ^ 0.2" 0.85 ^ 0.2 ^ 0.2 ^ 0.2" 0.3 ^ 0.02 ^ 0.1 ^ 0.2 ^
Mean of three determinations ± standard deviations. Values in the same column with the same superscript are not statistically different (P < 0.05)
4. CONCLUSIONS The chemical analysis of the mushroom C sulcipes revealed a high protein content (20.6 - 24.8 %), with the presence of all essential amino acids, although with a low tryptophan concentration. The mushroom biomass is abundant in phosphorus and has a low lipid content. Therefore, this mushroom could be considered a nutritious and healthy food.
1170 Table 3 Mineral content of Cookeina sulcipes (ppm) Mineral Calcium 42 Calcium 43 Copper Iron 56 Iron 57 Magnesivmi Manganese Phosphorus Zinc
Cap 313.9 238.5 214.25 1080.2 1146.1 632.1 3.88 12902 803.33
Stem
± 1.0 ± 5.2 ± 1.9 ± 8.5 ± 7.7 ±4.1 ± 0.2 " ± 30.0 ± 8.2
326.6 97 10.46 71.5 71.5 1385.1 3.08 3728 57.68
± 2.5 ± 3.1 ± 0.5 ± 2.3 ± 0.8 ± 10.4 ± 0.4 ' ± 5.9 ±3.1
Mean of three determinations ± standard deviations. Values in the same colimin with the same superscript are not statistically different (P < 0.05)
Table 4 Determination of fatty acids of the cap (pileus) of Cookeina sulcipes Fatty add name (chain length) (14:0) (14:1) Palmitic (16:0) Palmitoleic (16:1) Stearic (18:0) (18:1) Oleic (18:2) Linoleic (18:3N3) Arachidic (20:0) (20:1) (20:2) (20:5N3) Behenic (22:0) (22:1N11) (22:6N3) (24:0) Mjrristic
mg / 100 g dry fvmgal biomass 2.56 0.525 21.24 1.99 23.19 33.8 122.56 0.69 0.34 0.92 1.03 3.29 0.96 1.08 1.99 2.39
Mean of three determinations ± standard deviations.
± 0.48 ± 0.04 ± 0.9 ± 0.028 ± 0.57 ± 0.61 ± 0.03 ± 0.12 ± 0.01 ±0.04 ± 0.10 ± 2.1 ± 0.01 ± 0.01 ± 0.07 ± 0.01
1171 5, ACKNOWLEDGEMENTS The authors would Uke to thank Dr. G. Guzmdn of the mycological herbarium of the Instituto de Ecologia, Mexico, for the identification of the collected samples of C. sulcipes. Also, the authors wish to thank the Campo Experimental Rosario Izapa, Tapachula, Chiapas, Mexico, for the use of their facilities in the realization of part of this work. Thanks are also given to the following members of the Department ofBiochemistry, Memorial University of Newfoundland: Mr. D.Hall and Ms. S. Banfield for the amino acid analysis; Mr. K. Kean for the fatty acid analysis, and Mr. P. Bemister for his assistance with the manuscript.
6. REFERENCES An, G.-H.; Schuman, D.B. and Johnson, E.A. (1989). Isolation of Phaffia rhodozyma mutants with increased astaxanthin content. Appl. Environ. Microbiol., 55(1): 116-124. Anonymous (1974). Reference Analytical Methods Manual. Water Quality Branch, Environment Canada, Ottawa. Anonymous (1984). Official Methods of Analysis, 14*^ edn. (ed. William, S.). Association of Official Analytical Chemists, ArUngton, VA, pp. 38, 56. Bano, Z. and Rajarathnam, S. (1989). Pleurotus mushroom as a nutritious food. In: Tropical Mushrooms. Biological Nature and Cultivation Methods, 3""** edn. (eds. Chang, S.-T. and Quimio, T.H.). The Chinese University Press, Hong Kong, pp. 363-380. Blackburn, S. (1968). Amino Acid Determination: Methods and Techniques. Marcel Dekker, New York, pp. 21-22. Bligh, E.G. and Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 911-917. Chacon, S. (1988). Conocimiento etnomicologico de los hongos en Plan de Palmar, mimicipio de Papantla, Veracruz. Mic. Neotrop. Aplic., 1: 45-54. Chang, S.-T. and Miles, P.G. (1989). Edible Mushrooms and their Cultivation. C.R.C. Press, Boca - Raton, FL. Jandaik, C.L.; Bhandari, A.R.; Arora, C.L. and Rangad, C O . (1979). Chemical composition of some edible fimgi. In: Mushroom Science X (Part II). Proceedings of the Tenth International Congress on the Science and Cultivation of Edible Fungi (ed. Delmas, J.). Tardy Quercy, Bordeaux, France, pp. 685-688. Keough, K.M.W. and Kariel, N. (1987). Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains. Biochim. Biophys. Acta, 902: 11-18.
1172 Lau, O.-W. (1989). Methods of chemical analysis of mushrooms. In: Tropical Mushrooms, Biological Nature and Cultivation Methods, S""^ edn. (eds. Chang, S.-T. and Quimio, T.H.). The Chinese University Press, Hong Kong, pp. 88, 94. Li, G.S.F. and Chang, S.T. (1989). Nutritive value of VbZi;arieZZa i;oZi;accae. In: Tropical Mushrooms. Biological Nature and Cultivation Methods, ^""^ edn. (eds. Chang, S.-T. and Quimio, T.H.). The Chinese University Press, Hong Kong, pp. 202-203. Manu-Tawiah, W. and Martin, A.M. (1987). Chemical composition of Pleurotus ostreatus mycelial biomass. Food Microbiol,, 4: 303-310. Martin, A.M. (1992). Study of the growth and biomass composition of the edible mushroom Pleurotus ostreatus. In: Food Science and Human Nutrition (ed. Charalambous, G.). Elsevier Science Publishers, Amsterdam, pp. 239-248. Martin, A.M.; Lu, C. and Patel, T.R. (1993a). Growth parameters for the yeast Rhodotorula rubra grown in peat extracts. J. Ferment, Bioeng,, 76(4): 321-325. Martin, A.M.; Acheampong, E.; Patel, T.R., and Chornet, E. (19936). Study of growth parameters for Phaffia rhodozyma cultivated in peat hydrolysates. Appl, Biochem, BiotechnoL, 37: 235-241. Martin, A.M.; Acheampong, E. and Patel, T.R. (1993c). Production of astaxanthin by Phaffia rhodozyma using peat hydrolysates as substrate. J. Chem, Tech, BiotechnoL, 58: 223-230. Parent, G. and Thoen, D. (1979). Considerations sur la teneur en proteines et en acides gras de quelques especes de champignons comestibles du Shaba (Zaire). In: Mushroom Science X (Part II), Proceedings of the Tenth International Congress on the Science and Cultivation of Edible Fungi (ed. Delmas, J.). Tardy Quercy, Bordeaux, France, pp. 689-693. Penke, B.; Ferenczi, R. and Kovacs, K. (1974). A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analyt, Biochem., 60: 45-50. Quarmby, C. and Allen, S.E. (1989). Organic constituents. In: Chemical Analysis of Ecological Materials, 2""^ edn. (ed. Allen, S.E.). Blackwell Scientific, Oxford, pp. 160-200. Sanchez, A.D.; Chacon, S. and Sanchez, J.E. (1994). Produccion natural de Cookeina sulcipes (Ascomycotina, Pezizales) en la region de Tapachula, Chiapas (Mexico). Rev. Mex. Mic, 9: 47-56. Singh, S.M. and Verma, R.N. (1991). Nutritional and toxicological evaluation of Pleurotus spp. J, Food Sci, TechnoL, 28(4): 259-260. Thompson, W. (1969). Positional distribution of fatty acids in brain polyphosphoinositides. Biochim. Biophys. Acta, 187: 150-153.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1173
Interactions between polysaccharides and aroma compounds S. Langourieux and J. Crouzet Laboratoire de Genie Biologique et Sciences des Aliments, Unite de Microbiologie et Biochimie Industrielles, Associee a I'lNRA, Universite de Montpellier H F 34095 Montpellier Cedex 05 Abstract The presence of interactions between several polysaccharides : modified and wayy^ starches, dextrin, dextrans, hydroxypropyl celluloses, galactomannans and model aroma compounds : limonene, isoamyl acetate, ethyl hexanoate and fi-ionone, was studied by the use of the exponential dilution technique. The observed decrease of the reduced infinite dilution activity coefficient of these compounds when the concentration of polysaccharides is increased shows that the retention of aroma compoimds occurs for gdl the polysaccharides studied excepted for dextrans. With these polymers, an increase of the reduced dilution activity coefficients of volatile compounds, indicative of a ssdting out effect, was noticed. The determination of aroma compounds solubility in water £ind in aqueous solutions of polysaccharides such as dextrin and fi-cyclodextrin by Dynamic Coupled Column Liquid Chromatography (DCCLC) allows the characterization of the complexes. 1. INTRODUCTION The stimulation of odour receptors by aroma compoimds is strongly dependent on the concentration of these compounds in the vapor phase present in the mouth. This concentration is influenced by the volatility of aroma compounds but also by their release from the foods. These two parameters being dependent of several factors such as the quantity of aroma compounds present, the partition coefficients between the gas and the condensed phases, the temperature, the composition and the viscosity of the food and naturally the interactions of aroma compounds between themselves and the interactions between aroma compounds and food components such as proteins, lipids or carbohydrates. The aroma compounds may be dissolved, absorbed or adsorbed, covalently linked, trapped or encapsulated or limited in their diffusion as a result of the presence of foods components. The relative importance of these phenomena varies according to the chemical properties of aroma compounds and the physiced and chemical properties of the food. For example volatile compounds are disolved by lipidic phases whereas they are probably included in starch when this compound is the most important constituant of a food product. A good knowledge of the interactions between aroma compounds and several major food components is of great interest in order to control the aromatisation process.
1174 More particularly the studies of interactions between aroma compounds were developped in order to overcome t h e difficulties encountered for t h e removal of off-flavours from products such as soy or fish proteins (1,2 ) or in the contrary, related to the flavoring of proteinaceous foods (3). The formation of inclusion complexes during drying of systems containing amidon or cellulose, water and aroma compounds was pointed out ( 4,5). Later the presence of these complexes with dextrins, pectins, agar, methyl cellulose, and foods containing these compounds was established ( 6 ). Interactions between aroma compounds and macromolecules i n aqueous solution are generaly studied using equilibrium methods. Several drawbacks : difficulty to determine the equilibrium, time required, non specific binding and volatilization of aroma compounds are associated to t h e use of these techniques. Morever the static equilibrium observed is quite different of the equilibrium occuring for volatiles released in the mouth. In t h e present work dynamic methods: exponential dilution and dynamic coupled column liquid chromatography (DCCLC) were used for the study of interactions between model aroma compounds and several polysaccharides : modified and waxy starches, dextrin, dextran, hydroxypropyl celluloses, galactomans and fi-cyclodextrin i n aqueous solution. 2, METHODS 2.1. Exponential dilution gas flow rate slow
liiylil
Figure l.Variation of the concentration of the solute in the gas phase versus stripping time When a dilute aqueous solution of a volatile compound contained in an equilibrium cell is stripped by an inert gas the concentration of the solute in
1175 the gas phase vary as indicated (Fig 1) (7-10). In a first step the concentration increase with time, then a plateau is reached with a constant concentration in the gas phase indicative of the equilibrium. This step is followed by a decrease of the concentration according to an exponential law. At hight gas flow rate, 30 to 100 ml.min-i, according to the nature of volatile compound, only the exponential decrease with a short equilibrium phase is observed. The concentration decrease is related to the infinite dilution activity coefficient T^i by the expression : logS = l o g S o . ^ ^ Y r t
(,)
where S is the GLC peak area, So the GLC peak area extrapolated to zero time, D the carrier gas flow rate (ml.min-^), N the moles of solvent, R the gas constant (ml at mg .mol'^.K-^), T the temperature (K), P^ithe vapor pressure of the pure solute (atm) and t the time (min). T^i is calculated from the values of the slope of the straight line obtained by plotting log S versus time. When interactions between volatile compounds and macromolecular systems occurs in aqueous solution the infinite dilution activity coefficient is modified, a decrease is indicative of the fixation of the solute whereas an increase agrees with a salting out effect. A reduced activity coefficient:
»ir
oo
^i
(2)
where T^im is the value of y^i in the presence of the macromolecular system may be introduced. This value gives indications concerning the nature and the intensity of the interactions (10-13). 2.2, Dynamic Coupled Column l i q u i d Chromatography (DCCLC) DCCLC was initially introduced by May et al (14,15) for the determination of aqueous solubility of hydrophobic compounds : polycyclic aromatic hydrocarbons. This method (Fig 2) is based on pumping water through a column, "generator column", containing glass beads coated with the aromatic compounds. The concentration of these compounds is determined by HPLC on a Ci8 reverse phase, "analytical column", either directly or after extraction on
1176 a short Cis column, "extractor column".
Gsneraior column
Figure 2. DCCLC experimental device This method which avoids several problems, such as stability of solutions and losses of compounds to surfaces due to adsorption, allows the determination of the solubility of hydrophobic compounds with a precision and a reproductibility better than ± 3 %. DCCLC was used for the study of cyclodextrin inclusion complexes and more particularly for the determination of the formation constants (16). When aqueous solution of cyclodextrin is used instead of water for the flowing of the generator column an increase of the solubility of PAH as a result of complex formation is noticed. The formation constant for the complex corresponding to the equilibrium:
1177 Cy + P ^
• CyP
[Cy] tSo
(3)
(4)
may be calculated assuming that the cyclodextrin concentration is not depleted to an appreciate extent by complexation (less than 0.1 %) according to : Kf:
[Cy] tSo
(5)
where So is the solubility in water, St the solubility in aqueous solution of cyclodextrin, [Cylt the initial cyclodextrin concentration. 3. CHARACTERIZATION OF INTERACTIONS BY EXPONENTIAL DILUTION 3.1. Interaction with starches Several modified corn starches or waxy corn starches were studied according to the numerous results concerning the interactions between aroma compounds and several starches. The depressive effect of starches is well illustrated by the decrease of the relative volatilety aiw:
Pw (6) in the presence of modified com starches (Fig 3). These results, indicative of the existence of low interactions is in good agreement with results previously reported for potato starch. The association constants obtained for several volatile ligands vary from 10 to 2600 M^^If the effect of the different com starches on the relative volatility of isoamyl acetate is roughly the same, more important interactions, between ethyl hexanoate and modified way corn starches (Clearan CH20® and CI20®) t h a n between this compound and modified corn starches (Clearan MB70® and MH20®). The decrease of volatility expressed by the reduced infinite dilution activity coefficient Y^ir is dependent of the quantity of starch added.
1178
•
00 00
^ 3C00-J
CVi
u
o
::;
CM O <0
2000
>
pi:
1000 H
o ^ ^ i^ ^ r-. S 00 S ?
i
Isoamyl acetate
•
30%
CleargumMB70
•
30%
CiearamMH20
S
30 %
Clearam CH 20
M 30% 00
c
water
Clearam a 20
CO
o o
m I
ethyl hexanoate
limonene
Figure 3. Variations of the reduced volatility at 25° C of aroma compounds in the presence of several modified com and waxy corn starches For modified corn starch Clearam MH 20® (Fig 4) the decrease of isoamyl acetate yooir is weak whereas a rapid decrease from 10 to 15 % of starch followed by a slow decrease was noticed. Simular results have been reported for Cleargun HB70®(17). 1.2
1,0
0.8 H 0.6
0.2-4
0.0
—r~ 10
— I —
20
— I —
30
40
Figure 4. Variations of the reduced activity coefficient of isoamylacetate ( • ) and ethyl hexanoate ( ^ ) as function of modified com starch per cent
1179 When modified waxy corn (Clearam CH 20®) was used (Fig 6) these two phases were not detected, the decrease is approximatively linear with a slope comparable to the one obtained for the second part of the previous plot.
0,1
0,2
0.3
0,4
weight fraction of waxv corn starch
Figure 5.Variations of the reduced activity coefficient of isoamyl acetate( • ) and ethyl hexanoate(A) as a function of modified waxy corn starch weight fraction (17). It may be assumed that the first phase observed for the modified com starch - the com starch is constituted of about 25 % of amylose and 75 % of amylopectin - is representative of the formation of inclusion complexes, with a hight affinity constant with amylose. T h e formation of inclusion complexes between several aroma compounds and the helicoidal conformation of potato starch molecules is well documented (18-20). The main parameter involved in the fixation being the structure of the helice in one hand, the molecular size and the polarity of ligands in the other hand. The second category of interactions with a low affinity constant may be explained by the binding of volatile molecules to the surface of the amylose helice or to the amylopectin chains. The waxy corn starch is constituted by 100% of amylopectin. However it was recently reported that the binding at the surface of amylase is hightly improbable and that a part of amylopectin is able to be imply in the formation of inclusion complexes (20). Unspecific interactions and particularly interactions with lipids present in corn starch may also contribute to the depression of volatile compound concentration in the gas phase.
1180 3 ^ . Interactions with dextrin The dextrin used (Tackidex JO60 K® Roquette freres) is a corn dextrin with a good solubility a n d a low viscosity in aqueous solution. The results concerning the interactions between this dextrin and limonene and ethyl hexanoate are given Figure 6.
0,00
0,01
0,02
0,03
0.04
0,05
0,06
weight fraction of dextrin
Figure 6. Variations of the reduced activity coeficient of ethyl hexanoate (A)and limonene ( • ) as a function of dextrin Tackidex JO60 K® weight fraction (17). For the two volatile compounds a linear decrease of y^ir as a function of increasing quantities of dextrin was observed. This result, in good agreement with previously reported data (22,23), is indicative of the existence of ouly one kind of interactions. Important differences may be noticed according to the nature of the volatile compounds. For a weight fraction of dextrin equal to 0.05 a 25 % decrease of the activity coefficient of ethyl hexanoate was noticed, this decrease is less important that the decrease observed for the same weight fraction of modified com starch. In the contrary the depressive effect on limonene is much more important in the presence of dextrin (50 %) than in the presence of modified corn starch. 3.3. Interactions with galactomannans Galactomannans, the main polysaccharides present in locust and guar flours are constituted by linear chains of fi-D-mannans linked in a(l-4) and irregularly substituted by galactose units Hnked in a(l-6). These compounds are used as stabilizers in foods.
1181 The curves obtained for the variations of yoo^r versus the weight fraction of the two galactomannans are simular for a same volatile compounds. The most important depressive effect was obtained when Umonene is used. 70 % of decrease for a weight fraction of hydrocoUoids less than 0.01. Only 25 to 40 % is reached when the volatile compounds used is ethyl hexanoate in the same conditions, morever the initial decrease is followed by a plateau. However the observed decrease of the volatility may be the result of the viscosity increase of the medium. Several authors (24,25) have established a connection b e t w e e n t h e i n c r e a s e of viscosity in t h e presence of galactomannans and the decrease of the sensory properties of the product. In the contrary according to Pangborn and Szczesmak (26) the decrease of odor and flavor intensities observed in the presence of hydrocoUoids is relatively independent of the viscosity and specific of the combination hydrocoUoidaroma compounds. The plateau obtained when the aroma compound used is ethyl hexanoate may be the result of mass transport limitations when the weight fractions of galactomann is increased. 3.4. Interactions with hydroxypropyl celluloses The general shape of the curves obtained when solutions of hydroxypropyl cellulose are used (Fig 7) is similar to t h a t obtained with galactomannans.
Figure 7. Variations of the reduced activity coeficient of ethyl hexanoate (A)and limonene ( • ) as a function of hydroxy propyl cellulose J K 491 per cent. According to the great variation of the viscosity developped by the two hydroxypropyl celluloses, 3.65 mPa.s for AA 111 and 348 m Pa.s for J K 491 for 2 % solutions it may be assumed that the observed decrease of yooj^ is not the consequence of the increase of viscosity. Moreover Richon et al (27) have report that valid infinite dilution activity coefficient may be obtained for viscous media, up to 1000 mPa.s when a special
1182 equilibrium cell was used as in the present work. In these conditions the depressive effect observed in the presence of hydroxypropyl celluloses are probably the result of interactions between these compounds and volatils compounds. 3.5. Interactions with dextrans Dextrans with molecular weight between 60 000 and 90 000 and 100 000 and 200 000 were used. As previously reported by King (23) and increase of "/^ir, indicative of a saltingout effect, was noticed (Fig 8). 1.8
•
:
1,6 J
•
ft ft
•
I
1,4 J
/iT" U-
1 m) • 1,0 ^HK^ 0,00
I
0,02
«
I
i
I1
'
T "— ,
0,04 0,06 0,08 weight fraction of dextran
J
0,10
,
0,12
Figure 8. Variations of the reduced activity coeficient of ethyl hexanoate ( • ) and limonene ( • ) as a function of dextran (MW 100 000 and 200 000) weight fraction (17).
3.6. Interactions with B-cydodextrins As for modified corn starch two slopes are detected in the curves giving the decrease of yooj^ of limonene and B-ionone as a function of fi-cyclodextrin per cent in the solution (Fig 9).
1183
0,75
0,50
0.25
0.25
Figure 9. Variations of the reduced activity coeficient of fi-ionone ( • )and limonene ( • ) as a function of B-cyclodextrin per cent. The important decrease of aroma compounds volatility detected at low Bcyclodextrin per cent is probably the result of the formation of 1-1 inclusion complexes (25 ). On the other hand the slow decrease may be the consequence of t h e adsorption of aroma compounds at the surface of the 6-cyclodextrin molecule or of the formation of inclusion complexes with a stcechiometry different from 1. 4. QUANTITATIVE STUDY BY DCCLC 4.1. Association constant between aroma compoiuids and dextrin The results obtained with limonene using dextrin solutions from 0.5 to 5 % are given table 1. The fact that no significative differences between the values was noticed shows t h a t the association constant of complexes is independent of the dextrin concentration.
1184 Table 1. Association constants (M"l) for dextrin-limonene complex as a function of dextrin per cent. dextrin per cent
Ka (M"l)
0.5 1.0
15 607 ± 2 094 16122 ± 2 573
2.5
13 316 1713
5.0
11963 ±603
In the case of 13-ionone (table 2) a regular decrease of the association constant when the elution volume is increased. According to Blyshak et al . (16) this phenomena is indicative of an irreversible adsorption of 6-ionone on dextrin molecules. Table 2. Association constants (M"l) for dextrin-B-ionone complex as a function of the elution volume (dextrin per cent: 2.5).
elution volimie (mL)
40 120 200 280
Ka (M"l)
31 3028 2728 2395
4.2. Association constant between aroma compounds and fi-cyclodextrin The determination of the association constants between limonene or fiionone and fi-cyclodextrin was performed for several fi-cyclodextrin percent, 0.01 and 0.5 % in the first case, 0.05 and 0.1 % in the second case. For the two compounds a decrease of the calculated association constant when the elution volume is increased (Fig 10, 11) is noticed. This decrease is associated to a sealing of the generator column probably by the insoluble complexes.
1185 1CCC
Eluticn volume "nir)
Figure 10. Variations of the association constant for 6-cyclodextrin/ fi-ionone complexes as a function of the elution volume for two B-cyclodextrin per cent (•)lg/L,(D)5g/L. bwCC i 5CCC -
N.
k
4CCC -
3CCG -
a •
"..
2CCC -
• \ l
1CCC 10C
2CC
3CC
^OC
5CC
6CC
Elution volume (mT!
Figure 11. Variations of the association constant for B-cyclodextrin/ limonene complexes as a function of the elution volume for two B-cyclodextrin per cent (A)0.5gn.,(B)lg/L. Association constant between B-cyclodextrin and aroma compounds giving soluble complexes such as 2-methoxynaphtalene, ethyl salicylate or 1-menthol were obtained using DCCLC (28).
1186 References 1. J.E. Kinsella and S. Damodaran.in Foods and Beverages. Acad. Press, New York, 1980, p 95. 2. S. Damodaran and J.E. Kinsella, J. Agric. Food Chem., 1983, 31, 856. 3. K.L.F. Franzen and J.E. Kinsella, J. Agric. Food Chem., 1974, ^ , 675. 4. H. Staudinger and M. Staudinger, Makromol. Chem. 1953,^,148. 5. M. Ulmann and F. Schierbaum, KoUoid Z. ,1958,156,156 6. H.G. Maier, Angew. Chem. Interat. , 1970, 9,917 7. J.C. Leroi, J.C. Masson, H. Renon, J.F. Fabries and H. Sannnier. Ind. Eng. Chem. Process. Des. Dev., 1977,16,135. 8. P. Duhem. Contribution a Tetude des melanges **eau-hydrocarburessolvant polaire". Representation des equilibres de phases par le modele NRTL. These Aix-Marseille H, 1979. 9. A. Sadafian and J. Crouzet. Flav. Frag. J., 1987, 2,103. 10. F. Sorrentino, A. Voilley, D. Richon. AIChE, 1986, ^ , 1988. 11. A. Sadafian and J. Crouzet.in Progress in Terpen Chemistry. Ed. Frontieres, Gif sur Yvette, 1986, p 165. 12. A. Lebert and D. Richon. J. Food Sci., 1984, ^ , 1301. 13. A. Lebert and D. Richon. J. Agric. Food Chem., 1984, ^ , 1151. 14. W.E. May, S.R Wasik and D.H. Freeman. Anal. Chem., 1978, 50,175. 15. W.E. May, S.R Wasik and D.H. Freeman. Anal. Chem., 1978, 50,997. 16. L.A. Blishak, K.Y. Dodson, G. Patonay; I.M. Warner and W.E. May Anal. Chem., 1989, 61,955. 17 S. Langourieux and J. Crouzet, Lebens.-Wiss. u Technol.,1994, in press 18. F. Osman-Ismail.and J. Solms, Lebens.-Wiss. u Technol.,1973, 6,147. 19. M.A. Rutschmann ,J. Heiniger,V. Pliska, and J. Solms,. Lebens.-Wiss. u Technol. 1989, ^ , 240. 20. M.A. Rutschmann and J. Solms, Lebens.-Wiss. u Technol.,1990, 23, 70. 21. M.A. Rutschmann and J. Solms, Lebens.-Wiss. u Technol.,1990,^, 83. 22. A. Lebert and D. Richon, J. Agric. Food Chem.,1984, S , 1156. 23. C.J.King,in Physical Properties of Food. AVI Publication Company, Westport CT,1983, p 399). 24. K. Paulus and E.M. Hass, J. Text. Stud., 1970 J , 502. 25. Y. Malkki, R.L. Heino, and K; Autio, Food Hydrocoll., 1993, 6, 525. 26. R. M. Pangbornand A.S. Szczesniak, J. Text Stud., 1974,4,467. 27. D. Richon, F. Sorrentino and A. Voilley, Ind. Eng. Chem. Des. Dev. , 1985,J4,1160 28. S. Langourieux. Interactions Ligand-Recepteur : Cas des Composes d'Arome en Solutions Aqueuses. These Montpellier II, 1993.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
Water and ethanol adsorption biomass separation systems
on
starchy
1187
substrates
as
G. Vareli, P. G. Demertzis and K. Akrida-Demertzi
Laboratory of Food Chemistry, Department of Chemistry, University of loannina, 45110-loannina, Greece
Abstract Adsorption of water and ethanol on corn meal and wheat flour has been studied in the temperature range of 50-90 ^C using inverse gas chromatography (IGC). The results showed that adsorption of water was affected more significantly by the kind of starchy adsorber than the adsorption of ethanol. Values for thermodynamic parameters such as Gibb's free energy (AGs) and enthalpy (heat) (AHs) corresponding to sorption of water and ethanol by the substrates have been calculated using chromatographic retention data. It was found that adsorption was more spontaneous at lower temperatures as expected.
1.
INTRODUCTION
Interest in the production of energy from renewable resources was prompted by the 1973 oil crisis. For automotive transport, liquid fuels are considered essential, and ethanol derived from sugar or starch by hydrolysis and fermentation is one of the most studied renewable alternatives. Although the economics of pure ethanol as a fuel look less attractive at present, there is a growing interest in oxygenated organics as replacers for lead tetra-ethyl in high octane gasoline, and again ethanol is one of the contenders. Alcohols can be produced from either grains or biomass by first converting these materials to fermentable sugars [ 1-4 ]. The sugars are then fermented, typically with yeast, to give a broth containing 6 to 12 persent ethanol along with small amounts of aldehydes, ketones, amyl alcohols (fusel oils), and methanol [ 5 ].
1188 The final step, distillation to water-free alcohol, consumes 50 to 80 percent of the energy used in a typical fermentation ethanol manufacturing process [ 6,7 ], which is a major problem in the use of fermentation ethanol as a liquid fuel [ 6-8 ]. Recovery of ethanol from the fermentation broth is at least a three-step process: (a) distillation of dilute aqueous ethanol to its azeotrope ( 95.57 percent ethanol by weight ), (b) distillation using a third component - either an organic solvent or a strong salt solution- to break up the azeotrope and remove the remaining water, and (c) distillation to separate water from the third component so that it can be recycled. Trace constituents, including pentanol (fusel oil) and methanol, can be removed by additional distillation, but this is not necessary for ethanol to be blended with gasoline. Ladisch and Dyck [ 9 ] reported that most of the energy consumption occurs in the first stage when raising the distillate composition above about 85% ethanol. They and other investigators have proposed adsorption as an alternative to distillation, either by adsorbing alcohol from dilute aqueous solution, or by adsorbing water from a richer alcohol-water mixture. The alternative process, adsorption of water from ethanol, is in large-scale commersial use in the United States, and has been studied in the vapor phase by certain investigators in Purdue University [ 9,10-14]. They proposed a system in which distillation was used to concentrate the fermentation broth to between 75 and 90% ethanol, followed by treatment with a water-selective adsorbent such as CM cellulose, cornstarch, CaO etc. The system seems very promising, and can produce 99% ethanol with an energy consumption of about 3.9 MJ Kg'^-I compared with values of 6-9 MJ Kg'^-I for a distillation process. Although materials such as cornmeal and potato starch have been used as adsorbents [ 15,16 ], no systematic efforts have been made to elucidate the selectivity of these substrates, the effects of its composition and temperature on the relative separation potential of starch based materials. In this paper, an in depth study of the adsorption behavior of water and ethanol on corn meal and wheat flour of greek origin in the range of temperature 5090 oC using inverse gas chromatography (IGC ) is reported.
2. MATERIALS AND METHODS Inverse gas chromatography Inverse gas chromatography Is a well established technique and its main focus is the investigation of the interaction of solutes with a substrate which
1189 comprises the stationary phase of a gas chromatographic column [ 17-20 ]. If very small amounts of a solute are used, then the solute undergoes partitioning while being eluted. Although chromatographic systems are usually not in true equilibrium, use of small amounts of solute and low flow rates allows the approximation of equilibrium contitions. The time which elapses from injection of the sample to the recording of the peak maximum is the retention time. The net retention time is the difference between the retention of a solute and an unadsorbed
(unretained)
indicator. Air was used as the unretained compound in this work. If the net retention times of two solutes are compared, a separation factor can be obtained S= tNl/tN2
(1)
where : s is the separation factor, tNi is the retention time of the first solute, and tN2 is the net retention time of the second solute. It is also possible to obtain thermodynamic parameters using IGC. The volume of carrier gas which is necessary to elute an adsorbed solute from the column under the specific conditions of temperature and pressure of the column is called net retention volume and is calculated from the following equation : VN = i F c ( t R - t A )
(2)
where : Fc is the corrected flow rate at the conditions of the oven : Tov{Po-Pw) Fc=
(3) F
Tf,Po
F is the flow rate measured at the flow meter conditions, Tov is the temperature of the oven, Tfi is the temperature of the flow meter, Po is the outlet pressure of the column, Pw is the vapor pressure of water inside the soap bubble flow meter. Parameter j is the compressibility factor:
1190
j=
3 —
(Pi/Po)2-1
(4)
{Pi/Po)3-1
where : Pi is the column inlet pressure. The specific retention volume Vg° is the net retention volume corrected to standard temperature and pressure and is given by the following equation: VN 273.15 Vg°=
(5)
w^T
where : Ws is the weight of stationary phase and T is the temperature of the column. The partition coefficient Kp is calculated from : VgOpT Kp
(6) 273.15
where p is the density of the packing material. The free energy of adsorption, AGs°, of a solute on the column packing can be estimated using: AGs = RT InKp
(7)
the heat (enthalpy) of adsorption AHs is given by AHs R
d In VgO
(8)
d1/T
Preparation of columns Corn meal and wheat flour of 70% rate extraction were purchased from a local supermarket. Samples were dried in a vacuum oven at 50 ^C for 24h, then dessicated for 1 h. The 60-80 Mesh fractions were obtained and stored in the
1191 dessicator. The samples prepared in this way were diluted with an inert support, Chromosorb WAW, DMCS, 60-80 Mesh, purchased by Serva Germany. The inert support was dried under the same conditions and stored in a dessicator. To make retention time measurements with water, samples were diluted to 10 parts to 90 parts of inert support. The dilution was necessary because of the very long water retention times on starch-based materials which would otherwise make the experiments extremely time consuming. This proved to be a negligible source of error since water retention times on 100% Chromosorb WAW columns ranged from 0.07 min/ g at 90 ^C to 0.08 min/ g at 50C, while water retention times on food substrates ranged from 10.3 min/ g at 90 ^C to 48.5 min/ g at 50 ^C. To make ethanol retention times measurements, 50% starch-based material and 50% inert support columns were constructed. The greater percentage of starch was necessary because of relatively small retention times on both inert support and starchy materials. The retention time per gram for a 100% starchy material column (tNs/g) was estimated from the following equation:
( t N T - tNA/g M A ) tNs/g=
(9) Ms
where, tNj is the net retention time of the solute in the diluted sample column, T N A/g is the net retention time per gram of the solute in the 100% Chromosorb column at the same temperature as the diluted sample column. MA is the mass of the Chromosorb in the diluted sample column, Ms is the mass of the starch in the diluted sample column. The density
of the two starchy
materials was determined with a
stereopycnometer (model SPY-3, Quantachrome,USA ) and the average values of several measurements were 1.462 ml/ g for cornmeal and 1.490 ml/ g for wheat flour. Aluminium tubing was used for the construction of chromatographic columns with a 6.35-mm o.d. and a length of 1m. To begin packing the columns, the tubing was straightened, silane treated glass wool was used to plug one end, and this same end was attached to a vacuum pump. The container holding the stationary phase was weighed. A few grams were put in the column and packing was accomplished with the aid of a mechanical vibrator. The supported stationary phase was continually added and packed until the column was filled, at which time the container was weighed again. The other end of the column was sealed with glass wool; the column was then twisted into spiral form and placed in the chromatograph with only its inlet
1192 port connected. Each analytical column was conditioned at least 12 h by passing helium carrier gas through the column.
GC instrumentation A Shimadzu 8A gas chromatograph was used equipped with a thermal conductivity detector. The reference column was prepared in the same way as the analytical columns . Pressure was regulated with a two-stage regulator and set at 6 Kg / cm'^2. Inlet pressure to the column was determined by connecting a U-type mercury manometer to the inlet part of the oven. The manometer was allowed 5 min to equilibrate. Pressure drops in the column ranged from 0.19 atm to 0.338 atm depending on the flow rate of the carrier gas. The thermal conductivity detector temperature was set at 200 o C. The injection port was also set at the same temperature. The carrier gas velocities ( flow rates ) were measured with a soap bubble flow meter attached to the thermal conductivity detector outlet and adjusted to 35 ml/ min. Flow rates were determined for each column at each temperature after a steady baseline of the recorder indicated equilibration of the column. Injection port septa were conditioned at the temperature of the oven and changed every 40-50 injections, or when leaking was apparent by visual inspection. A 5|il syringe was used to inject 2^1 of the sample into the chromatograph.
3. RESULTS
AND
DISCUSSION
Figures 1 and 2 show the net retention time per gram of material for water and ethanol with both materials studied (corn meal and wheat flour), in the temperature range 50 - 90 ^C. It is observed that the net retention times of water are higher than those of ethanol at all temperatures and for both substrates. A second observation is that net retention times for both water and ethanol decrease as the temperature increases, but for water the decrease is more rapid than for ethanol. For example, the net retention time of water for wheat flour decreases from approx. 48.5 min/ g at 50 ^C to approx. 10.3 min/ g at 90 ^C, while the net retention time of ethanol for the same material decreases from approx. 0.11 min/ g at 50 ^C to approx. 0.05 min/ g at 90 ^c. Another observation is that retention times for water are higher for wheat flour, as compared to the respective for corn meal. For example, at 50 ^C the net retention time for wheat flour is approx. 48.5 min/g, while the respective one for corn meal is approx. 18.5min/g. This could be partially attributed to the higher fat content of corn
1193 50
O -O-
Wheat flour
-ffl—
Corn meal
40 H c
E <
o DC
30
LU Q-
LU
^ H Z O F Iz LU H
O 20
LU
OC h-
LU Z
10
I
40
50
I
60
I
70
I
80
90
100
TEMPERATURE,*'C
Figure 1. Net retention time per gram of material for water on wheat flour and corn meal.
1194 0.12 -H— O
"O-
corn meal wheat flour
0.1
O c E III
0.08 H
H Z O LJ
I-
LU
CC I-
0.06
LU
0.04
0.02
— I —
40
50
I
— I —
60
70
~n— 80
— I —
90
100
TEMPERATURE, ^'C
Figure 2. Net retention time per gram of material for ethanol on wheat flour and corn meal.
1195 500
<>
-m-
corn meal
•-0-
wheat flour
400
b. DC
O
o
h-
o < z g
300
< cc <
o
Q_ LU CO
200
100
40
I
50
—r60
I
I
I
70
80
90
100
TEMPERATURE, "C
Figure 3. Separation factor s for water and ethanol on wheat flour and corn meal
1196 meal. On the other hand, the net retention times of ethanol are also higher in case of wheat flour but the differences are less pronounced. To understand the separation potential of water from ethanol, a comparison of net retention times of water and ethanol by taking the ratio of the net retention time per gram of water to the net retention time per gram of ethanol is necessary (Fig. 3). From this figure it is obvious that this ratio, called separation factor s, becomes greater as the temperature decreases. For example, at 90 o C the separation factor for corn meal is equal to 152, while at 50 ^C it becomes 245. Greater values for separation factor s were obtained for wheat flour, suggesting better separation of the two solutes on this material than on corn meal. More specifically, at 90 ^C the separation factor for wheat flour is 193 and by lowering the temperature to 50^0 it increases to 452. This was expected, because as it was noticed above, the net retention time for water is affected more from the type of material than is the net retention time of ethanol.The above observations lead to the suggestion that wheat flour at 50 ^C, proves to have the higher separation factor. To gain a more detailed understanding of the adsorption process of the two solutes, two thermodynamic parametes were calculated from the specific retention volumes. The first thermodynamic parameter obtained is the free energy of adsorption, AGs°. The values of free energies of adsorption are shown in Table 1. Higher negative values are obtained for water than for ethanol, and this confirms that water is adsorbed more strongly than ethanol in all temperatures and by both materials. Besides, for both the solutes, it is shown that lower temperatures are more favourable for their adsorption, which is stronger for wheat flour than for corn meal. The second thermodynamic parameter is the enthalpy (heat) of adsorption, AHs. In figure 4, InVgO is plotted vs. 1/ T (eq.8), and the slope of the line is equal to AHs/ R. Values of enthalpies of sorption are reported in Table 2. Heats of adsorption of water are - 8.19 Kcal / gmol for corn meal and -9.16 Kcal / gmol for wheat flour. These values show that water is adsorbed more strongly by wheat flour. For ethanol, heats of adsorption are - 5.44 Kcal / gmol for corn meal and 5.81 Kcal / gmol for wheat flour. Although for ethanol the negative value of heat of adsorption is higher for wheat flour, the difference of the enthalpies between the two materials is not so pronounced. This suggests that adsorption of ethanol is not affected significantly by the kind of substrate used. Moreover, the AHs° values obtained are within the range of the average physical adsorption values (3- 9 kcal/gmol), but significantly smaller than chemisorption where typical heats of adsorption are in the range 20-40 kcal/gmol [21].
1197 Table 1 Free energy of adsorption, AGs° ( Kcal / gmol).
50
Temperature (°C) 60 70 80
90
Water Corn meal Wheat flour
-4.33 -4.98
-4.18 - 4.96
4.08 - 4.84
-3.97 -4.72
-3.95 -4.57
Corn meal Wheat flour
-0.80 -1.06
- 0.62 -1.03
Ethanol - 0.47 -0.88
-0.37 -0.78
-0.32 -0.76
Table 2 Enthalpy of adsorption, AHs^,( Kcal /gmol).
corn meal
Water Ethanol
8.19 5.44
wheat flour
-9.16 -5.81
REFERENCES 1. N.P. Cheremisinoff, P.N. Cheremisinoff, and F. Ellerbusch, Biomass. Applications, Technology, and Production, Marcel Dekker, New York (1980). 2. M. R. Ladisch, Process Biochem. 14, 21 (1979). 3. G.T. Tsao, M. Ladisch, T. A. Hsu, B. Dale, T. Chou, Ann. Rep. Ferment. 2,1 (1978). 4. M. R. Ladisch, C. M. Ladisch, G. T. Tsao, Science, 201, 743 (1978). 5. L. F. Hatch, Ethyl Alcohol, Enjay Chemical Company, New York, (1962).
1198
water
• >
BB
com meal
O
wheat flour
2H
ethanol
— I —
2.7
2.8
2.9 1 / T x IC'S,
3
3.1
3.2
K'^( -1 )
Figure 4. In Vg°vs.1/ T* 10''3, for water and ethanol, on wheat flour and corn meal.
1199 6. M. L David, G. S. Hammaker, R. J. Buzenberg, J. P. Wanger, Gasohol Economic Feasibility Study, Development, Planning and Research Associates, Inc., Manhattan, Kan. (1978). 7. T. K. Ghose and R. D. Tyagi, Biotechnol. Bioeng.,21, 1387 (1979). 8. W. 0. Burrows, C. M. Hudson, M. L. Kaesser, N. A. Santer, Changing Portable Energy Sources - An Assessment, John Deere Co, Moline,lll (1977). 9. M. R. Ladisch and K. K. Dyck, Science, 205, 898 (1979). 10. M. R. Ladisch, M. Voloch, J. Hong, P. Bienkowski, and G. T. Tsao, lEC Process Des. Develop., 23, 437 (1984). 11. M. Voloch, J. Hong, avd M. R. Ladisch, Second Chemical Congress of North American Continent, Las Vegas, NV, 1980, paper 43, Microbiol.Biochem. Technol. (1980). 12. J. Hong, M. Voloch, M. R. Ladisch, and G. T. Tsao, Biotechnol. Bioeng., 24, 725 (1982). 13. P. R. Bienkowski, A Barthe, M. Voloch, R. N. Neuman, and M. R. Ladisch, Biotechnol. Bioeng., 27, 960 (1986). 14. R. Neuman, M. Voloch, P. Bienkowski, and M. R. Ladisch, lEC Fundam., 25, 422 (1986). 15. A. A. Hassaballah, and J. H. Hills, Biotechnol. Bioeng. 35, 598 (1990). 16. V. Rebar, E. R. Fischbach, D. Apostolopoulos, and J. L. Kokini, Biotechnol. Bioeng. 26, 513(1984). 17. R. J. Laub, and R. L. Pecsok, Physicochemical Applications of Gas Chromatography, Wiley, New York (1978). 18. R. L. Grob, Modern Practice of Gas Chromatography, Wiley, New York (1977). 19. V. G. Berezkin, V. R. Alishoyev, and I. B. Nemirovskaya, Gas Chromatography of Polymers, Elsevier, New York (1977). 20. V. G. Berezkin, Analytical Reaction Gas Chromatography, Plenum, New York (1982). 21. A.W. Adamson, Physical Chemistry of Surfaces, Wiley, New York (1976).
This Page Intentionally Left Blank
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1201
SOME MULTIVARIATE PERSPECTIVES ON SHELF LIFE RESEARCH 1
2
R.H. Albert, Ph.D., and C. Zervos, Ph.D. ^US FDA/CFSAN, Washington, DC 20204 US FDA/CDER, Washington, DC 20204 (deceased) (Contents do not necessarily reflect policies and decisions of the FDA.) The purpose of statistics is not mere numbers: the purpose of statistics is understanding. As a bonus, insight and inspiration may also be achieved. Multivariate statistics is a key to understanding but it is a set of tools, not an end in itself. These tools can find, and indeed, have found, application in most aspects of shelf life studies. Some specific areas may be cited here:the chemical analyzing of food components, the creating of relevant variables, the processing of foods, and the bringing of insights from different disciplines to bear on the problems of food technology. A. Chemical Analysis A crucial feature in shelf life research is the accurate identification of the chemicals present and the determination of their concentrations. The time course of the decomposition during the shelf life must be monitored by careful analysis of the metabolic products produced, with a view to determining the reaction kinetics of the decomposition. Whether deterioration is due to bacterial decomposition or to protein denaturation or to oxidation of linoleic acid or related unsaturated fatty acids, the researcher needs accurate and precise determination of the chemical intermediates that are involved in the complex process of food spoilage. Even such a routine problem of improving the resolution of the peaks in gas chromatograms has been attacked using multivariate techniques. Morgan and Deming {S.L. Morgan and S. N. Deming, J. Chromatogr. 112, 267-286 (1975)} determined optimal instrument settings, using a multivariate technique called simplex optimization {W. Spendley, G. R. Hext, and F.R. Himsworth, Technometrics, 4, 441-461(1962)}. Youden {W.J. Youden,"Critical Evaluation (Ruggedness) of an Analytical Procedure," in "Encyclopedia of Industrial Chemical Analysis, Wiley-Interscience, New York, 1966, pp. 755-788} has popularized the standard statistical technique known as fractional factorial design, whereby chemical analysis methods are pre-optimized prior to being subjected to interlaboratory validation. Called by Youden "ruggedizing", this multivariate approach is an integral part of the AOAC International [formerly Association of Official Analytical Chemists] protocol for development of chemical analysis methods for regulating foods and drugs. To avoid "statistics shock" that could frighten away chemists , Youden furnished a "template" for optimizing over a
1202 set of up to seven operating variables, provided that the variables could take on only two states. Typical of such "binary" or two state control variables are temperature(high/low), solvent (ethanol/water), flow rate (fast/slow), and pressure(high/low). The variables ("features") need not be such mundane entities: they could be weighted sums of such mundane entities. In this way the researcher might find the fractional factorial exploration might be more effective if, say, principal components were used. The template from Youden consists of a prescribed set of 8 experiments to be run, each experiment having a different combination of binary states for each of the 7 variables. A more complete menu of experimental designs can be found in the text by Haaland {Perry D. Haaland, "Experimental Design in Biotechnology",Marcel Dekker,New York (1989)}. This text includes the "Plackett-Burman designs",which include an 12-experiment design for 8 variables. Even now such pragmatic simplifications as those made by Youden are needed to bring the benefits of multivariate statistics to those involved in food chemistry, and in particular to those involved in investigating shelf life. As Aishima and Nakai {Tetsuo Aishima and Shuryo Nakai, Food Reviews International, 7(1), 33-101(1991)} state, "When a new idea is introduced.., a strong suspicion of its practical value among scientists [develops and].. chemometrics[=multivariate statistics applied to chemical problems]... is still unpopular among flavor researchers." Calibration curves{D.L. Massart, B.G.M. Vandeginste, S.N. Deming, Y. Mischotte, and L. Kaufman, "Chemometrics: a Textbook", Elsevier, Amsterdam (1988)} have the potential of being more effective and less liable to noise and interferences when multivariate techniques are used. Fatty acids may absorb at more than one infrared frequency and by utilizing more than one of the peaks, in particular by using a weighted sum of the absorption peak areas, as the y-variable to plot vs. the true concentration or true concentration added as the x-variable. Obviously, such an approach can be applied as well to other absorption spectroscopies and even to mass spectroscopy. Meglen {Robert R. Meglen, Fresenius J. Anal. Chem, 338, 363367(1990)} has reported on the use of principal component analysis for multivariate quality control of food reference samples. He was able to uncover anomalous samples and to find suggestive groupings of the samples into meaningful clusters. For this particular study, he was concerned with how closely food reference materials match corresponding real foods. In addition, the author offers the tantalizing prospect, with some hypothetical sketches, of how principal components can be used for multicomponent data from foods to quantify the effectiveness of the standard reference materials being used. In the use of such standard reference materials, it is essential that the profile of the reference standard as to analytes and their concentration approximate as closely as possible the actual food matrix. Consider how inefficient and costly it would be to create one single reference material for just one analyte at a
1203 time. By thinking globally, by using a multivariate approach, optimal multipurpose reference standards can be developed. The author's work vividly illustrates how we need multivariate techniques in order to catch up with the large data bases now becoming available from our instruments. A more general discussion by the same author {R. R. Meglen, Chemometrics and Intelligent Laboratory Systems, 3,17-29(1988)} treats of the role of chemometrics in chemical and measurement sciences. Currie {Lloyd A. Currie, "The Importance of Chemometrics in Biomedical Measurements"(chapter 6, pp. 74-100) in "Biological Trace Elements Research: Multidisciplinary Perspectives", edited by K. S. Subramanian, G.V. Iyengar, and K. Okamoto,ACS Symposium Series 445, American Chemical Society, Washington, DC (1991)}, in a review chapter, gives a good example of the use of the Youden ruggedizing approach in an air pollution study. He also gives an example of the simplex technique for optimizing instrument operating conditions and settings. Furthermore, he discusses multivariate quality control. Thus, he touches upon the key points addressed in this chapter. Even the basic processes of designing chemical determinations and evaluating the results can be improved by application of multivariate statistical techniques. Resistance to these techniques will wear away as their utility becomes evident and as the flood of data from our instruments force new perspectives on data processing, to go from raw data to meaningful information. B.DESIGNING RELEVANT VARIABLES Clearly multivariate statistical techniques can facilitate measuring chemical concentration. Now it will be shown that these techniques can be used as well to develop meaningful variables that go beyond mere individual chemical concentrations. Consider for example the problem of measuring rancidity. Oxidative rancidity in fats and oils has been with us since prehistoric times. Surely one of the reasons that the Europeans of Columbus' time were so avid for the spices of the Orient was to cover up the off-flavor and off-smell of meat in those prerefrigerator days. The efficacy of smoking meat, which deposits antioxidative phenols, must have been a serendipitous discovery. Typical rancidity is a result of a complex sequence of oxidations of fats and oils; consequently when a quantitative measure of rancidity is called for, some measure of extent of oxidation is thereby needed. Gray {J.I. Gray, "Simple Chemical and Physical Methods for Measuring Flavor Quality of Fats and Oils", chapter 12, pp. 223239, of "Flavor Chemistry of Fats and Oils, edited by David B. Min and Thomas H. Smouse, American Oil Chemists' Society (1985)} lists 5 analytical chemical procedures for assessing the extent of oxidation of fats and fat-containing foods. These methods are: peroxide value, TBA(thiobarbituric acid), carbonyl value.
1204 anisidine value, and the Kreis test result. Each of these tests actually detects a different aspect of the oxidation process. For example, the much-used TBA essentially just measures the malonaldehyde concentration in the food matrix. The author mentions some objections to this measure of rancidity, among them being the possibility of other materials absorbing at the 532 nm peak, where the TBA-malonaldehyde complex is traditionally measured. The complex also has an absorbance peak at 550 nm. Doesn't this suggest that some combination of the two peaks might be used to determine the concentration of the TBA-malonaldehyde complex? Pohle et al.{W. D. Pohle, R. L. Gregory, and B. Van Giessen, JAOCS 41, 649-?(1964)} did find that the flavor score could be estimated from the TBA value for the fats and oil that they studied. Similarly, the peroxide number could be used to predict what the flavor score would be. All 5 tests simply measure different aspects of oxidative degradation, like the five blind men describing an elephant. The peroxide test actually measures the concentration of the hydroperoxides that are the initial products of lipid oxidations but these are transitory and unstable. If it is assumed that the flavor score has some objectively-verifiable consistent meaning, the problem of combining the five measures to give a better fit to the flavor score would be an interesting research problem. Hopefully some of the weighting factors would be so small that those particular measures could be omitted as being insignificant. Multivariate techniques would also show which of the 5 measures are so highly correlated that some might be redundant and need not be considered. In short it might be fruitful to combine the scores for each of the measures for rancidity to create a new measure of rancidity. To further refine this rancidity assessment, the measures from the "dynamic methods" listed by Gray could be included: Schaal oven test, active oxygen methods, and various oxygen absorption tests. Even the results of standard spectroscopic and polarographic techniques could be incorporated into this suggested multivariate measure of rancidity. How important such a refinement of the rancidity measure might be is seen in the work of Verma et al. (Meat Sci. 14, 91-?,(1985), as reported by Ledward {Dave Ledward,Food Science & Technology Today, 1(3), 153-155(1987)}. The problem addressed was the role of iron-containing proteins (hemoproteins) in lipid oxidation during the spoilage of raw meat. The investigation entailed the study of the catalytic effect of various hemoglobin and myoglobin derivatives on the formation of thiobarbituric acid reactive compounds. Thus, the TBA score, as is usual, was used as a surrogate for the extent of rancidity. Despite the imprecision of such a measure, Verma et al. apparently were able to show that the ferric containing hemoproteins were the major catalysts for oxidative degradation. Duerr and Schobinger{P. Duerr and U. Schobinger, pp.179193,"Flavour '81", 3rd Weurman Symposium, Proceedings of the International Conference, Munich April 28-30,1981, ed. by Peter Schreier, Walter de Gruyter,New York (1981)} claim that about 330
1205 volatiles have been found in orange fruit and juice. Since offflavor in orange juice is indicated by the presence of alphaterpineol, the authors plotted the concentration of this volatile material in orange juice as a function of time, up to 90 days for storage temperatures of 4°, 20°, and 32° Centigrade in cardboard packages and in a glass bottle. Their conclusion was that the increase of alpha-terpineol was linear over 90 days and was strong in the glass bottled juice. As part of their shelf life experiments, they noted that neral and octanal decreased in the course of time over 90 days of storage and that, as expected, the decrease was greater at the higher temperatures. Why not sum up with appropriate coefficients the concentrations of these three well-defined volatiles—alpha-terpineol, neral, and octanal—to obtain a possibly more effective and reliable measure of orange juice deterioration? Such a synthetic variable may or may not have a physical interpretation. As in much of multivariate research, the scientist deals interactively with the statistical results and brings to bear his/her own skill and knowledge. Exploration is often necessary to seek reasonable conclusions. Multivariate statistics does not invariably give the right answer, but it does provide more choices for the statistician. In the case of apple juice, the authors report the work of Koch et al. who found that a simple sum of the 2-hexenal and 2-hexenol concentrations is highly correlated to the intensity of apple juice essence. In other words, the Koch composite variable, that could be used to monitor deterioration in apple juice, is simply : new variable = .5 x hexenal concentration + .5 x hexenol concentration= the simple average of the two concentrations. The researcher is invited to let his/her imagination soar above routine, one-variable-at-a-time computations, one variable at a time. Even in multivariate statistics, the imagination can come into play: an initial transformation of the raw data may provide more tractable variables, even prior to data reduction and manipulation via statistics. For example, a logarithm of a variable may be a better representation of reality than just the simple variable. In his study of off-flavors in canned beef sterilized by heat. Von Sydow{E. Von Sydow, Proc. R. Soc. Lond. B. 191, 145-153(1975)} found a linear relation between the so called "retort off-flavor" and the following variable: the square root of the product of the hydrogen sulfide concentration and the 2-methylthiophene concentration. Another example of the need for a created variable arises from the term "shelf life" itself. People in the field would agree that the word puts too much emphasis on the time aspect and that other factors should be considered as well. Certainly temperature or storage temperature is crucial to how well a stored food or drug retains its desirable features. In fact, reality is a bit more complicated as far as temperature goes: a typical product may undergo several temperature environments as it goes from manufacturer to truck to warehouse to retailer. So temperature history is a factor. In fact, such a time-temperature-history based variable would be pertinent to the cyclic refrigeration
1206 scheme proposed by Scott,Steffe and Heldman {E. P. Scott, J. F. Steffe, and D.R. Heldman, in "Changing Food Technology",pp.189208, edited by M. Kroger and A. Freed, Technomic Publishing Co.,Lancaster, PA (1989)}. Also relevant to quality retention are the conditions under which the product is kept by the consumer after purchase. Certainly, a variable analogous to degree-days would be a less-misleading and a more informative indicator of durability than would a mere declaration of some expiration date or time period. Handling, processing, additives, acidity, and wrapping are among other key factors that need to be considered when trying to quantify the durability of the desirable features of a food or drug product. A composite score might be feasible that would in effect be a linear discriminant function, serving to indicate when the values taken on by the environmental factors/time /processing / etc. cause this function to exceed some critical value. Karel{Marcus Karel, Chapter 17, "Focal Issues in Food Science and Engineering", in "Food Product Development", ed. by Ernst Gaf and Israel Sam Saguy, Van Nostrand Reinhold, New York(1991)} echoes the fact that shelf life is a function not simply of time but of conditions of storage. He alludes to a device that can be scanned at the checkout counter to indicate not only how long the product has been on the shelf but also the temperature conditions the product has been exposed to. The readout consists of the number of "equivalent shelf days"—a parameter that provides a measure of both time-on-shelf and abuse. A critical value for this designed variable can be determined to warn the consumer. Of course, nature does not usually make abrupt jumps: the transition from acceptable quality to unacceptable quality is arbitrary but the line must be drawn somewhere and thinking in terms of a linear discriminant function may help draw that line. Multivariate statistics can certainly help in determining a flavor score, that evanescent quantity often determined by the opinion of expert or not-so-expert panels. D. Thompson {David Thompson,Food Science and Technology Today 3(2),83-88(1989)} has been advocating a special multivariate approach to sensory panel evaluation. This approach, called Generalized Procrustes Analysis (GPA), differs from the usual flavor scoring. In the typical panel set up, a common set of vocabulary terms is established and the panel members are instructed on how to assign a score for each component of a flavor (e.g.,fruitiness, tartness, etc.) in the food items being tested. The GPA approach lets each panelist establish his/her own idiosyncratic set of variables and via a computer-intensive technique called multidimensional scaling the score space of each of the panelists is rotated to maximize the geometric similarity of the different spaces. Once a consensus score space is obtained, then the usual principal component analysis to adjust for correlations among the flavor components is performed. Use of principal components, that is, the use of special linear combinations of the separate flavor component scores may reduce the number of factors that need to be considered and my indicate superfluous variables and might point
1207 to suggestive high correlations among some of the flavor components. A recent book dedicated to application of multivariate statistics to sensory data has been written by Burgard and Kuznicki{David R. Burgard and James T. Kuznicki,"Chemometrics: Chemical and Sensory Data",CRC Press, Boca Raton (1990)}. It strives to bridge the gaps among analytical chemistry, sensory evaluation, and multivariate statistics. Individual chapters are dedicated to correlation and regression, discriminant analysis, and factor analysis. Factor analysis is the multivariate technique that is the most concerned with uncovering new variables but it is not primarily a routine tool. The mathematics can be formidable, even for a statistician, and a great demand is made on the researcher's creativity, insight, and luck. No pat formulas or algorithms exist in factor analysis and a certain ambiguity and arbitrariness exists in the artificial constructs of this technique. For example,an example using orange tea sensory attributes is presented, for which an "orangey" construct and a "spicey" construct are produced. However, Burgard and Kuznicki attractively present factor analysis for those who might want to explore the techniques. They exhaustively work through one particular data set and teach the essential features of this challenging tool in the tool kit of multivariate statistics. We (Zervos and Albert) have written a chapter in "Off-Flavors in Foods and Beverages" {C. Zervos and R. H. Albert, "Chemometries: the Use and Multivariate Methods for the Determination and Characterization of Off-Flavors", in "Off-Flavors in Foods and Beverages", edited by G. Charalambous, Elsevier Science Publishers, Amsterdam (1992)} that gives a sample of the use of multivariate statistics in dealing with off-flavors. In this survey, the work of Bertuccioli et al. {M. Bertuccioli, G. Montedoro and S. Clementi....,(1986)} among many others, was cited as typical of the kind of multivariate applications to flavor research actually being done. In our chapter, we demonstrated how clique analysis, a specialized version of clustering that lets an element belong to more than one group, can be applied to the Bertuccioli data to gain insight into the relationships among the variables used to measure the chemicals evolved during the storage of Provolone cheese. Variables that belonged together were flagged, wherein "belonging together" could mean, under favorable circumstances, that any variable in a clique might be able to act as a surrogate for all the other variables in the same clique. Thus, multivariate statistics can not only create new variables, it can reduce the number of variables that have to be dealt with. Subsequent correlation studies of the simplified variable set with sensory data could thereby be expedited. A much more recent paper on an aging index for cheese has been reported by Banks et al.{J.M. Banks, E. Y. Brechany, W.W. Christie, E. A. Hunter, and D. D. Muir, Food Research International, 25, 365-373(1992)} , who investigated the volatile
1208 components of Cheddar cheese as indicators of cheese maturity, flavor , and odor. Thirty-one gas liquid chromatograph peaks were studied from each of 12 cheese samples. A "partial least squares analysis"(which might have actually be a canonical correlation analysis) was carried out to relate the sensory scores (19 sensory attributes were used) from various panels and the chemical composition (31 peak areas). With only 12 samples, the use of multivariate statistics may be like using a sledgehammer to crack a walnut. Nevertheless, the authors do give for the few data points involved an interesting demonstration of how some multivariate techniques might be used, especially to generate those special linear combinations called principal components. Another dairy product of crucial concern in shelf life studies is milk. Leland {J.V. Leland, G. A. Reineccius, and M. Lahiff, J. Dairy Sci. 70(3), 524-533(1987)} studied 42 samples of milk, oxidized to various degrees using copper wire mesh. The oxidized material served as a surrogate for ordinary spoiled milk, but under controlled conditions. The authors analyzed 22 components via head space gas chromatography and concurrently used a panel of five experts to evaluate the milk for quality. In this study, they employed the special linear combination of variables called a linear discriminant function. Both a principal component and a linear discriminant function are linear combinations of variables, in this case linear combinations of component concentrations. However, the "essences" are different: the principal component is intended to capture as much of the variance structure, being that linear combination with the highest variance, subject to certain restrictions. By contrast, the linear discriminant function(which is just a variable too, of course) is that combination that gives a maximum separation among the centers of gravity of the sets of points corresponding to different groups (low quality, medium quality, high quality). The linear discriminant function often has associated with it a boundary value such that if the numerical value of the function for an unknown milk sample exceeds this critical value, it is assigned to one category, "high quality" for example, and if the value is not exceeded, the sample is assigned to a different category. Principal components are used for purposes of data reduction by capturing the variance structure of the data set by means of a reduced number of variables. Linear discriminant functions serve to categorize and to separate the data into distinct a priori groupings. A good example of a linear discriminant function is to be found in an article by Nishimura and Kato{Toshihide Nishimura and Hiromichi Kato, Food Reviews International, 4(2). 175-194(1988)} on the taste of free amino acids and peptides. The authors observe that proteins without taste, when hydrolyzed by proteases, produce bitter peptides. They studied the reported decomposition during storage of miso as indicated by the change in average peptide lengths with storage time, up to 50 days. In order to predict the bitterness on any given peptide they cite from the literature a table of coefficients for each of 16 key
1209 amino acids. A linear discriminant function is given by the sum of the number of occurrences of each amino acid in a given peptide, weighted by these coefficients divided by the number of amino acid residues in the peptide. If the numerical value of this sum, that is, if the value for this linear discriminant function, exceeds 1400, then the peptide will be expected to elicit a bitter taste. A chemical explanation is available for the relative values of these coefficients or weighting factors: the bitterness of peptides is caused by the hydrophobic property of the amino acid side chain. The authors sought a quantitative description of the frequent observation that the flavors of beef, pork, and chicken are actually improved by storage at low temperatures for certain periods of time. The linear discriminant score at least provides a handle on the problem. C. MULTIVARIATE STATISTICS FOR ASSESSING PROCESSING CONDITIONS Processing conditions can have a profound effect on the quality and stability of food and drug products. A wide range of factors can be involved in carrying out a process with the result that multivariate tools are required to monitor and control them. Alt and Smith{F. B. Alt and N.D. Smith, "Multivariate Process Control,",Ch. 17, in "Quality Control and Reliability", ed. by P. R. Krishnaiah and C. R. Rao, North-Holland Press, Amsterdam (1988)} develop some multivariate process control techniques that rely on the fundamental vector of means for each of the N processing conditions and on the corresponding N x N variancecovariance matrix. A useful way to quantify the effect of the array of processing conditions on the array of product quality indicators is the multivariate technique of canonical correlation. Canonical correlation is especially adept at relating a cluster of variables of one type with a cluster of variables of another type. Typically, one type of variable could be processing or storage conditions and the other type could be the cluster of "flavor notes" associated with the product. Also of potential value in assessing processing conditions are linear discriminant functions. These serve to delimit the values for the acceptable processing conditions, providing a multidimensional boundary between acceptable and unacceptable processing conditions. Here are some examples of multivariate processing studies. As an object lesson in how multivariate statistics might be of use, one can consider the paper by Lima and Cal-Vidal {A. W. O. Lima and J. Cal-Vidal, "Estimation of shelf life of film-packaged freeze-dried banana" in Journal of Stored Products Research 24(2),pp 73-78(1988)}, where the effect of various factors on the shelf life of film-packaged freeze-dried bananas was studied. For polyethylene films of different thicknesses, a plot is made of shelf life vs. water vapor pressure and a plot is made of
1210 shelf life vs the reciprocal temperature; a similar pair of plots is made for polypropylene files of varying thicknesses. Some three-dimensional plots might be useful to demonstrate the interactions between the factors of temperature, film thickness, and water vapor pressure as they combine to determine the shelf life. The actual estimation of the shelf life was based in part on some semi-empirical formulas for moisture transfer rates. Mittal et al.{G.S. Mittal, R. Nadulski, S. Barbut, and S. C. Negi, "textural profile analysis test conditions for meat products",Food Research International 25, 411-417(1992)} studied the effects of test conditions on the "texture profile"of three beef products: wieners, salamis and corned beef. The testing factors (which here may be viewed as processing factors) included diameter to length ratios, % compression, and the speeds of the compressing device; among the texture factors("parameters") were two different types of hardness, cohesiveness, springiness, chewiness and gumminess. Common-sense adjustment of the data, prior to analysis, was made to account for cross section area and for strain—an object lesson in the value of post-editing of raw experimental data to express the results in more meaningful terms. This was obviously a multivariate study that may or may not have benefited from application of canonical analysis. In fact, while multiple regression might have been of value, it could well be that, for a subtle reason, canonical analysis would be INAPPROPRIATE in this particular research. The hindrance is that each operating condition might be able to be assigned a level or value independently of the level or value or any of the other operating conditions. In such circumstances, the variancecovariance matrix for just the operating conditions could be manipulated to contain any values whatsoever. This would be a subtle but real violation of the assumptions underlying canonical correlation. In truth, the non-diagonal elements of the matrix for the processing factors are all zero if indeed the processing factors can be assigned values independently of one another. What the authors did calculate was the correlation of each of the separate operating conditions with each of the texture parameters. No variance-covariance matrix was used or needed by the authors. The authors could draw some valid conclusions as to the optimum testing conditions. Radiation to retard or prevent bacterial spoilage has been a controversial but often successful processing procedure. In a recent article, Narvaiz et al. {P. Narvaiz, G. Lescano, and E. Kairiyama, "Physicochemical and sensory analyses on egg powder irradiated to inactivate Salmonella and reduce microbial load",Journal of Food Safety, 112, 263-282(1992)} reported on the results of a study to identify adequate gamma radiation dose consistent with retaining egg powder quality. Four different radiation intensities were utilized to inactivate Salmonella : 0, 2, 5, and 10 kGy, where Gy represent a gray = 1 joule of absorbed energy per kilogram. These were the 4 processing conditions studies. Among the features of the resulting product measured over a 4 month period were: peroxide number, visible and
1211 ultraviolet absorption peak areas, foam stability and viscosity. Concurrently sensory panelists scored such properties as external appearance, odor, flavor, and acceptability. Microbial concentrations (MPN*s=most probable numbers) were also determined of course. No detailed attempt was made by the authors to apply multivariate techniques: each factor or feature was looked at separately, except to note that when rancidity( as measured by the peroxide number) was high so was the rejection rate by the sensory panelists. For the author's purposes, the nearlyunivariate approach used may be perfectly adequate. Four general types of quality deterioration during production, transportation, and storage are: (1) physical—e.g. drying; (2)chemical—e.g. rancidity; (3)enzymic—e.g., browning; and (4)microbiological—e.g., microorganism growth. In order to process food to ensure the removal or diminishing of this lastmentioned type of spoilage, a realistic predictive mathematical model of microbial growth and survival in foods is needed. This need has been discussed, for example, by G. Gould {Grahame Gould, "Predictive Mathematical Modelling of Microbial Growth and Survival in Foods", in "Food Science and Technology, Vol 3(2),pp 89-92,(1989)} The model must successfully predict the microbial behavior results for the many processing alternatives, including heating, irradiation, decontamination with gases, drying, acidifying, adding preservatives, decontaminating the ingredients. His work is an exemplar of how modern science now is so data-rich: he was able to capture simultaneously the growth patterns within 24 cultures with differing pH and salinity values. With the instrumentation used he could actually have studied up to 200 such cultures and could monitor the timedependence of the bacterial growth via automated optical density measurements. His report is part of a long-range data-base creation project to uncover and record the key determinants of microbial growth for a wide spectrum of microorganisms under a wide range of processing conditions. First comes data, then information, then finally wisdom. "Hurdle technology" in the processing and designing of food for safe storage is a multifactor approach that preserves food by combining methods. The "hurdles" may include such processing conditions as temperature, acidity, reduction-oxidation potential, atmosphere modification, presence of antioxidants, and presence of antimicrobials. "The concept is that for a given food the bacteria should not be able to 'jump over' all the hurdles present and so should be inhibited. If several hurdles are used simultaneously, a gentle preservation could be applied, which nevertheless secures stable and safe foods of high sensory and nutritional properties. This is due to the fact that the different hurdles in a food often have a synergistic (enhancing) effect."{anonymous, "Food Science and Technology Today",6(3), pg. 139(1992)} A good example of the application of hurdle technology is to be found in the paper by Chirife and Favetto {Jorge Chirife and
1212 Guillermo Favetto,"Food Research International" 25, 389396(1992)—Some physico-chemical basis of food preservation by combined methods}. They review such key processing and deterioration factors as water activity (a^), pH, solute effects, and temperature and consider the interaction of these factors. The water activity has historically been a more meaningful indicator than the simple moisture content because the activity includes the effects of the food matrix. Chirife and Favetto explore the combined effects of decreasing water activity and heating. By lowering the water activity, not so much heating is needed to deter bacterial growth. Be aware that the choice of the word "hurdle" to describe such a multivariate situation can mislead the reader, in that the process designer does not envision each of the conditions to be met sequentially, as in a hurdle race; rather, the process variables must be considered simultaneously. Chao and Rizvi {R. R. Chao and S. H. Rizvi, "Maximization of produce shelf life through modified microatmoshpere packaging", from the book "Changing Food Technology 2", pp. 175-178, ed. by Manfred Kroger and Allen Freed, Technomic Publishing Co., Lancaster, PA (1989)} must deal with multivariate processing conditions in their study of designing a modified microatmosphere packaging system to extend the shelf life of apples. Among the parameters to be juggled simultaneously were: storage temperature, time required to reach desired oxygen and carbon dioxide concentration levels, the surface for gas exchange, oxygen and carbon dioxide permeabilities of the wrapping film, and the packaging materials. Part of the strategy to cope with the complex interactions was to use mass balance equations for the gases within the packaging. Mass balance equations simply express the conservation of mass and facilitate the mathematical description of the processes taking place within the film-wrapped product as the product consumes oxygen and generates carbon dioxide, while simultaneously both these gases are permeating through the film. In thinking about such hurdle approaches, we could picture a three-dimensional space, with its three coordinate axes corresponding respectively to water activity, pH, and temperature. Any point in this space corresponds to a set of process conditions. Imagine a three dimensional figure like an icosahedron such that this figure is made up of all those points that correspond to an adequate combination of processing conditions. Any point lying outside this figure would correspond to an unacceptable combination of processing conditions. The shape of this figure—the boundary between acceptable and not acceptable — can be delineated with the help of multivariate techniques. Even if the boundary is more like a crumpled sheet than a polyhedron, application of techniques like piece-wise regression will help trace out this boundary and will help to indicate suitable combinations of operating conditions that will achieve the goal of safe product with less expenditure. As novel food products, such as low-acid foods or low-fat foods, designed
1213 to cater to new fads and fears, penetrate the market place, the application of hurdle technology and its inherent multivariate approach will gain in importance. TRANSCENDING STATISTICS AND PEEKING OVER THE FENCE INTO OTHER FIELDS Man does not live by statistics alone. The food technologist is truly confronted with the need to adopt a multidisciplinary approach to his/her problems. From ANTHROPOLOGY to ZOOLOGY, with important stops at ECONOMICS and PSYCHOLOGY, the cumulated store of many sciences must be tapped by those concerned with food sciences and new contributions to this store often have to be made. For example, the clique analysis referred to above, had its origin in SOCIOLOGY, with the first rigorous formula for complete enumeration of all cliques to be found in the journal Sociometry.IF. Harary and I.e. Ross, "A procedure for clique detection using the group matrix", "Sociometry",20(3),205215(1957)} Mathematics, and in particular multivariate statistics, can provide a unifying approach to assessing the results and estimating the uncertainties. To cite one example of how the scientist might have to adjust to a whole new paradigm: consider a simple sensory panel problem, involving just a paired comparison test. Each panel member is presented with two items and he/she is asked to select which item is "better" on the basis of some pre-agreed-upon protocol for evaluation. If three items are involved—cheese, chicken, and chocolate—• it is not inconceivable that the panelists (a) prefer cheese over chicken, and (b) prefer chicken over chocolate, and yet (c)prefer chocolate over cheese. Note how this set of preferences wreaks havoc on our ordinary quantitative training. Since Euclid, the third century B.C. geometrician and educator from Alexandria, every school person has been taught that if a is greater than b and if b is greater than c, then a is also greater than c. Mathematics label such behavior as "transitive." For example, 5 is greater than 3 and 3 is greater than 1, so 5 is greater than 1. Note that such is not the behavior in the cheesechicken-chocolate dilemma(trilemma?). Numerical instincts fail us. Merely assigning a "satisfaction value" or "util" can't possibly capture this counter-intuitive behavior. In one ECONOMICS textbook, Walsh {Vivian Charles Walsh, "Introduction to Contemporary Microeconomics",McGraw-Hill, New York(1970)} derides the use of utils, calling such a practice a relic from crude unthinking British empiricism of the nineteenth century. Walsh proceeds to explain "indifference curves" —curves that do not rely on exact numerical values, but instead depend on preference rankings. However the cheese-chicken-chocolate problem cannot be solved by this simple ruse of ranking and of "revealed preferences." If one insists on numerical values when transitivity does not hold, the researcher must literally rise above the "x-axis" and
1214 consider an item's utility to be expressed as a vector in multidimensional space; then the non-Euclidean preference ranking might be representable in a geometric fashion. Think higherdimensionally. Some of the more ambitious and daring might even want to explore the use of tensors to express utility. If this much trouble can realistically be expected to happen from time to time from a simple pair comparison, then imagine how creative the researcher needs to be in coping with the "duo-trio" test and the "triangular" test employed in sophisticated sensory panel evaluations.{Roland Harper,"A guide to sensory analysis and its development","Food Science and Technology Today",1(2),pp.7376(1987)} Much of the initial portion of this chapter has dealt with the concept, maligned above, of utility. Used by economists, philosophers, insurance companies, and management scientists (see below), the word "utility" is difficult to define: an adequate definition requires precision-honed mathematical symbolism. "Utility index" as defined by Dyckman et al. {T. R. Dyckman, S. Smidt, and A. K. McAdams,"Management Decision Making Under Uncertainty", Macmillan, London(1969)} is "a real number that gives a preference measure attaching to an outcome or the payoff for an outcome." The authors then go on to define a utility function as a function that assigns a value to a "gamble" such that if gamble g^ is preferred to gamble gg, then the corresponding value assigned by the utility function for the gamble g^ is greater than that assigned to the gamble g2. Note that, by the use of a definition in terms of real numbers, the transitivity condition is assumed to hold. From previous discussions in this chapter, it should be clear that use of principal components might be a fruitful means to composite several variables to create a utility function. Even the linear combination resulting from multivariate regression could be a candidate for utility function. Even adding cross terms in a regression, such as sweetness x density, is not out of the question as part of an exploration to find a suitable utility function. A realistic utility function must combine a widely disparate array of variables, both objective and subjective. Consider how difficult it would be to create a single utility function that mirrors the "healthiness" of food. "Is 5 units of vitamin B^g worth 3 units of vitamin A?" Such questions arise naturally as possible explicit formulas for a utility function are reviewed. Now try to contrive a solution to the harder problem of coming up will a utility function that mirrors the "quality" of food: in addition to "healthiness" variables, such as vitamin and fat content, all the "hedonistic variables, such as texture and taste, are to be considered. It may well be that in such situations the search for a single utility function is like the search for the holy grail: a noble enterprise but doomed to failure. It is perhaps not a matter of time and improved technology until such an explicit all-encompassing utility function can be devised; the goal may be illusory and may never ever be achieved.
1215 Another complication with utility functions as currently envisioned is that the utility indices are often erroneously assumed to be additive. What is after all merely an ordinal ranking is treated as a full-fledged continuous variable (i.e.,as a "ratio" variable). This mistake is particularly insidious when decisions are based on the results of multiplying the utility of each choice by the a priori known probability of some relevant conditions holding true. A utility index of 5 is not five times higher than a utility index of 1.: therefore 1/10 of 5 utils is not equivalent to 1 util in value. In fact, such a multiplication does not make any sense. Ranks don't work that way. This striving after utility functions does force the researcher to examine the actual physical problem closely. Therefore, the misuse of utility functions does not preclude them from being used as a crude guide. They're better than nothing. As discussed in the preliminary portions in this chapter, the transition from individual utility functions to an overall utility function must be made in order to intelligently assess options in foods and in life. Here we transgress into the realms of POLITICS and PHILOSOPHY. A name exists for such an overall merged utility function: it is called, by Nobel-prize-winning Kenneth J. Arrow and by others, the social welfare function. If conceptual and practical difficulties arise for individual utility functions, then no better can be expected from the social or communal welfare function. Arrow's definition, given on page 12 of his book "Social Choice and Individual Values"{Kenneth J. Arrow,"Social Choice and Individual Values",Yale University Press, New Haven (1963)} is as follows: "By a social welfare function will be meant a process or rule which, for each set of individual orderings R^,..., R„ for alternative social states (one ordering for each individual) provides a corresponding social ordering of alternative social states,R." Much of the book is devoted to establishing conditions under which such a function can exist. Clearly "not yet ready for prime time", the problem of integrating individual preferences into communal preferences is of deep, abiding concern that has important impact on how we vote, how we are governed, how we are taxed, and how we live. Associated with the problem of utility is that fact that utility is a function of how people perceive their needs and wants. Thus, the realm of PSYCHOLOGY is important in assessing and interpreting people's preferences. Shelf life is related to the retention of the quality of a food product, but the perception of quality is highly subjective. Even health aspects —matters of life and death— can be viewed differently by different peoples. Consider our attitude toward aflatoxin-contaminated grain versus the attitude toward the same grain of starving Somalis. As the proverb states, "Ones man's meat is other man's poison." ["One man's Mede is another man's Persian" might be an older version of this proverb.] Consider how different tastes are. Pork is anathema to both Jews and Muslims, yet some Polynesian islands are reputed to exist where pork is considered such a supreme
1216 delicacy that women are allowed to consume pork only on special occasions. New Englanders prefer brown eggs. As we mentioned in our chapter on the application of chemometrics to flavor studies {Zervos and Albert, op.cit.}, year-old decayed whale meat is considered a delicacy in Iceland and the off-flavor, as the nonafficionados would dub it, is essential to the enjoyment of this delicacy. As Williams et al.{Anthony P. Williams, Clive de W. Blackburn and Paul Gibbs, "Techniques to improve the safety and extend the shelf life of foods" in "Food Science and Technology Today" 6(3) 148-151(1992)} state, "... the criteria used to describe deterioration should be relevant to the cause for which the food would become unfit." In other words, good quality depends on both subjective and objective factors. Surveys are undertaken periodically to determine just what the salient factors are in determining food preferences. A study of food consumption trends in the United Kingdom has been described by A.M. Rees. {Ann Maree Rees,"Factors influencing consumer choice", in "Journal of the Society of Dairy Technology", pp.112116,vol 45(4),(1992)} She particularly focuses on the influence of such new phenomena as housewives' working, the introduction of microwave ovens, the increase in snacking, and the obsession with "natural" food. In the United States, the results of a survey of factors affecting food consumption has been described by C. I. Waslien.{Carol I. Waslien, "Factors influencing food selection in the American diet", pp. 239-269, in the book "Advances in Food Research Volume 32, edited by C. O. Chichester and B. S. Schweigert,Academic Press, New York(1988)} She describes the effects of age on food preference and food attitudes and on meal patterns. The complex issues of sex differences vis-a-vis senses of taste and smell, food values and attitudes, and actual food selection are also addressed. Moreover, racial and ethnic patterns are discerned. She describes the dramatic alterations in the make-up of U.S. society, in age distribution, and in life styles. In her conclusion, she states that "The marked changes ... demand research methodologies that can consider multiple factors simultaneouslyfitalics ours] and are still rapid enough to make predictions before the population has changed again." She also calls for techniques than can take advantage of computerized statistical procedures on the diverse populations needed to assess the full range of food-choice factors. The "hard sciences", in contradistinction to the "soft sciences" like economics, of course would also be expected to contribute to food technology and the understanding and prolonging of shelf life. CHEMICAL ENGINEERING is replete with multivariable approaches that apply to foods. A paper by Saguy and Karel {I. Saguy and M. Karel, "Modeling of quality deterioration during food processing and storage",Food Technology,34(2),pp.7885(1980)} on modeling quality deterioration during processing and storage exemplifies how chemical engineers approach the complex situations frequently encountered in food technology. A catalog of typical statistical routines is presented, including multiple linear regression and stepwise regression. Emphasis is on the
1217 kinetics of the decomposition of food. Lima and CalVidal{op.cit.}, by their model-based equations, also provide a useful illustration of the chemical engineering approach to coping with all the variables that must be considered in food processing, even though they may fail to go far enough. Even a certain mathematical technique from another of the "hard sciences", specifically from THEORETICAL BIOLOGY, may find application in shelf life work, especially in its chemical analysis and processing aspects. Called the "genetic algorithm", this technique, whose goal is to seek optimum values for instrumental and processing variables, is being actively explored for future routine application. Lucasius and Kateman{C.B. Lucasius and G. Kateman, "genetic algorithms for large-scale optimization in chemometrics: an application",Trends in Analytical Chemistry, 10(8),254-261(1991)}, in order to suggest fruitful application of the genetic algorithm, address the important chemical analysis problem of finding the wavelengths to use to estimate a complex mixture of chemicals, each having a different individual spectrum, i.e., each having a different functional relation between the absorbance, in the example used, of ultraviolet light and the wavelength of the ultraviolet light. The mixture dealt with in this feasibility study consisted of the four RNA nucleotides: adenylic, cytidylic, guanylic, and uridylic. The criterion of "goodness" is the selectivity, which can be calculated via the set of so-called Lambert-Beer equations and which is uniquely and directly calculable for a given choice of wavelengths. In the initial stage of the genetic algorithm, strings of I's and O's were generated at random. Because 36 absorption wavelengths had to be considered, each string was composed of 36 I's and O's, where a 1 at the n* position in a string indicates that the n wavelength is used, while a 0 in that position indicates that the corresponding wavelength is not to be used. Each string is a coding for what wavelengths are to be used concurrently and which are not to be used. For each of the starting set of 36-bit-long strings, a selectivity value — a measure of "goodness"— can be straightforwardly be computed. Since this selectivity is simply a numerical value, and not something so complicated as a preference vector alluded to above, the initial strings can be ranked in order from most selective to least selective. From this initial set, a certain number are selected with a probability proportional to the selectivity and these selected ones are allowed to "breed", creating a new generation. Then the strings in this new generation are evaluated and a sensitivity-based selection is again made to determine who shall reproduce the next generation. The algorithm resorts to this selection procedure, instead of picking say the top 35% of the strings, to ensure that every string has a chance, however small, of contributing to the ultimate goal of finding that string of I's and O's that yields the very highest numerical value for the sensitivity. Also, such a probabilistic approach prevents genetic dead ends, where the same situations keep recurring: in the genetic algorithm.
1218 something new and invigorating can always be introduced by chance alone. In mimicking nature, the genetic algorithm resorts to analogs of the crossovers and mutations found in genes. In crossovers, two strings are split at some random point along their respective lengths, say between position 29 and 30 and a pair of two new strings is generated: (a)the 29-bit portion of the first string is coupled to the 36 minus 29 = 7-bit string of the second and (b)the 7-bit segment of the first string is coupled to the 29-bit segment of the second string. As an analog to mutations, a certain number of bits are reversed at random, I's becoming O's and O's becoming I's. As each generation is created, the strings become associated with higher and higher sensitivity values. However, since the process is random, it is interesting to note that you can get different alleged optima for different random starting sequences of strings. A common-sense solution to this non-reproducibility problem is to resort to this efficient genetic algorithm to zeroin on a good '•fertile region" and to allow some more tedious optimization or "hill-climbing" algorithms slog through the computations to find the unique optimum. The food technologist may wonder why all this bit-playing is necessary, when it is a matter of just trying out all the possibilities and identifying that one set of conditions — t h a t one set of wavelengths— that yields the highest value. For some of the most interesting and important optimization problems, such a complete, exhaustive search may entail more computer time than is left in the life of the planet earth. The genetic algorithm is a powerful general-purpose search strategy that has its greatest strength in those large-scale problems that have no analytical solutions. The genetic algorithm, of course, could effectively be applied to the optimization problems of food processing. As such, it would provide an alternative to the already well-established bag of tricks employed by the discipline known as MANAGEMENT SCIENCE. Under the rubric of "operations research", this collection of procedures, seeks, inter alia, to uncover those values for a set of variables that optimize the value of some function. This function is called the "objective function", equivalent to the sensitivity in the above genetic algorithm. This function provides a measure of goodness. The search process strives to achieve the highest goodness, subject to the constraints imposed by limited resources. A frequent exercise is to calculate the least-cost nutritionally-adequate diet: given the nutrient composition of available foods and the price of these foods and the minimum daily requirements, the question is "What is the lowest cost diet consistent with the constraints of nutritional adequacy?" The well-developed arsenal in operations research {Frederick S.
1219 Hillier and Gerald J. Lieberman, "Operations Research",HoldenDay, San Francisco(1974)}includes the techniques called linear programming, non-linear programming, dynamic programming, integer programming, PERT (Program Evaluation and Review Technique) and CPM(Critical Path Method). Also part of operations research is a classic repertoire of problems addressed by these weapons, among them: the warehouse problem, the knapsack problem, and the optimal-reorder frequency problem. While not strictly in the domain of statistics, many of these areas of operations research rely heavily on statistics to model reality and to establish values for key parameters along with estimates of the uncertainty in these parameters. The following is a sample of some of the shelf life problems where operations research approaches may help provide the important answers: *The simplex method {Lloyd Currie, op.cit.},which was alluded to previously for optimizing experimental operating conditions, is a specialized development for chemists, based on the fundamental linear programming prescriptions of operations research. *The processing of food in preparation for storage should be as rapid as possible. PERT and CPM help identify bottlenecks in production processes and allow the manager to focus on streamlining just those step that make a difference in the overall time required. *The insight into process sequencing provided by PERT and CPM can be applied to the HACCP[Hazard Analysis of Critical Control Points] monitoring of a food production process, such as fish packing. HACCP is a form of quality control that is vital when rare but serious defects are involved. A good example would be contamination by Salmonella; the infestation in a food is unlikely, but when it does occur it can be lethal. Mere statistical sampling will not work well since to detect something that has a probability of .001 of happening will require prohibitively large samples. Instead, HACCP strives to identify those features of a process most liable to cause serious defects. It then focuses on assuring that those features are operating properly. A illustration of HACCP in practice would be the daily verification that the fish packers in an assembly line are wearing intact gloves. *The retail grocer doesn't want to have too much product waiting on the shelves. The Japanese have introduced the concept of "just in time delivery" in shipping parts to an assembly site that basically allows the manufacturer to keep no parts inventory at all. The grocer of course needs some inventory of the food product even though the ideal situation would be for the shipment of a unit of product to arrive just as the customer is about to buy. The strategy that is adopted by the grocer must take into account the fact that perishable products have a utility diminishing with time and have an availability that also diminishes with time as the consumer purchases the product. This type of problem is addressed in operations research under the general heading of inventory models. *The classic warehouse problem can help the food manufacturer decide from which storage facilities to ship to which retail outlets. The formulation of this problem is as follows: a
1220 manufacture has F factories, each producing a given amount of product and S stores that each demand some amount of product. The problem is to assign the product from the F factories to the S stores is such a way as to minimize the costs. For shelf life considerations, the cost might be taken to be transportation time. Here, as in any much of practical research, the problem is may not necessarily be solvable by simply plugging into a formula, even though the problem here is a classic one. Resorting to the tried and true formulation, the assignments will minimize the average transit time. What if, instead, it is more important to minimize the maximum transit or shipping time? That is, suppose it is imperative because of a spoilage threat that no single product shipment take more than 36 hours. No easy answer exists as to how to determine the optimum assignments in this case, but operations research provides at least a starting point for ferreting out a solution. Thus, many fields can contribute to the effective investigation and control of shelf life. IMPLEMENTING THE STATISTICAL PROCEDURES VIA COMPUTER SOFTWARE The value of multivariate statistics and of the multivariate approach has been demonstrated. However, unless the food scientist can actually apply the techniques, he/she is doomed to be a passive on-looker. It is obvious that there is very little that a scientist can do in the realm of multivariate statistics without resorting to a computer. A few simple multivariate approaches that require little if any computer power might be tried during preliminary data exploration: (a) Simply graphing one variable vs. another can suggest trends. (b) When the resulting so-called scatterplot is enclosed in a "convex hull", the limits of the variables are easier to spot. The convex hull of a set of points in a plane is simply the smallest polygon that contains all the points and has all its interior angles less than 180 degrees. If you have N points in your plot you can connect each point with every other point— there are N x (N-1)/2 such lines in all—and then trace out the outermost lines. At each corner of the convex hull is a data point, with any non-corner data points all contained within the convex hull. (c) You can plot one variable vs. another variable for several different categories and the respective convex hulls can provide boundaries to categorize new unknown samples. (d) You can fit a straight line to the scattered points— or you can transform the values—take the logarithm or raise to a power for example—and then fit a straight line, either via conventional regression formulas or by a minimax procedure.{Robert J. Blodgett, U.S. Food and Drug Administration, Washington, DC 20204. Personal communication. This minimax procedure is not generally known but offers an alternative to the ordinary regression procedure, which makes certain demands on the
1221 behavior of the data. The undemanding minimax-fitted straight line provides the minimum maximum "miss" or individual error: for each point in your set of plotted points, note how much this minimax line misses it as measured vertically. You will have N such values or misses. The worst of these N deviations is the maximum miss for the minimax line.. If you draw a straight line other than the minimax straight line, the fit will have at least one point missed by more than this maximum miss. To get the minimax line, draw the convex hull and find which corner is the furthest away from the opposite side of the convex hull. Then draw a line passing half-way between this corner and side, parallel to the side. This line is the minimax line, useful for spotting outlying data points and for indicating possible ranges for trends.} That's pretty much all you can do without computer power but fortunately the requisite computer power—both hardware and software—is available to apply multivariate statistics techniques to data. There is however both good news and bad news. The good news is that there are literally hundreds of software packages that include multivariate subroutines. And these proliferating software packages can be run on a personal computer: mainframe computers are not needed except for truly enormous data sets. That's also the bad news, namely, there are scores of multivariate statistics software packages. The uncontrolled growth of such packages makes selection difficult and some horror stories exist about the quality and validity of some of the programs in some of the packages. See, for example, the article by G.E. Dallal {Gerard E. Dallal, "Statistical microcomputing—like it is.", The American Statistician", 42(3) 212-216(1988)},where 5 different packages are put through the paces and so are found wanting. These were only 5 out of the more than 200 packages that Dallal claims are available just for the IBM PC alone.{Note that the naming of any commercial make of computer or software package is in no way to be construed as an endorsement. No conclusions may be drawn as to the relative merits of similar competing products.} Caution must always be exercised in the blind use of any technique and the very fact that a computer does the computations in no way relieves the scientist from the responsibility of knowing what's going on. Searle{Shayle R. Searle, "Statistical Computing Packages: Some Words of Caution", "The American Statistician", 43(4)189-190(1989)} issues the following warning: "With little or no knowledge of statistics, people with data can so easily have the data processed by a package that does the arithmetic for whatever sophisticated analysis they choose, whether the choice [of type of analysis] is appropriate or not." He recounts some horror stories of computer packages being abused. As far as mainframe packages are concerned, five are discussed in a book by Wolff and Parsons.{ Diane D. Wolff and Michael L. Parsons, "Pattern Recognition Approach to Data
1222 Interpretation",Plenum Press, New York(1983). Of these, at least three (namely SAS [Statistical Analysis System] and SPSS [Statistical Package for Social Sciences]) are available for personal computers and are we11-documented and supported. We ourselves use the software package called Statgraphics and we have seen references in British publications to a package called Genstat. Some shopping may have to be done but having a software package is the sine qua non for doing multivariate statistics. It is necessary but not sufficient however. A willingness to explore and to learn is required and a cheerful attitude in the face of adversity. Goethe, who was born before the golden age of multivariate statistics and computers and who was habitually successful in everything he undertook, always advised any friends fearful of failure in some enterprise:" Vertraue und verhandle!""Have faith and just do it!"
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
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Nitrite alternatives for processed meats F. Shahidi and R,B. Pegg Department of Biochemistry, Memorial University of Newfoundland, St. John's, NF, AlB 3X9, Canada. Abstract Nitrite is an essential ingredient used in the curing of meat products. It is responsible for the characteristic color, flavor and extended shelf-life and microbial stability of cured products. However, it is also responsible for the production of carcinogenic A^-nitrosamines in certain cured products under some processing conditions. Nitrite-free meat products such as wieners and salami have been prepared using curing systems consisting of the preformed cooked cured-meat pigment, CCMP, an antioxidant, a sequestrant and an antimicrobial agent. The color, oxidative stability, and flavor of the cooked treated products, as determined by Hunter L, a, b values, 2thiobarbituric acid (TBA) test, and sensory means, respectively, were similar to those of their nitrite-cured counterparts. Absence of A^-nitrosamines in the nitrite-free cooked products was confirmed using a gas chromatography-thermal energy analyzer (GCTEA) methodology. Similar results were obtained when the above nitrite-free curing mixtures were used in fish-based products, where nitrite curing produced substantial amounts of A^-nitrosodimethylamine. Furthermore, rates of color fading of samples treated with either CCMP or nitrite were similar, thus suggesting that presence of residual nitrite in processed meats may not be necessary for color stability of such products. 1. INTRODUCTION Cured meats represent a large portion of the processed meat products consumed in North America. These processed meats are attractive in their color, texture and flavor and are popular with consumers because they combine this variety with the convenience of extended storage stability. With the advent of modem refrigeration, the importance of nitrite curing of meat for its preservation has declined, but consumers have acquired a taste for certain cured-meats and still demand their preparation.
1224 The origin of meat curing is lost in antiquity [1], but it was not until the turn of this century that nitrite was ascertained to be the fundamental ingredient of the curing process. This ubiquitous compound, whose regulated use has been in effect since 1925 in the USA [2], is responsible for the development of the characteristic pink color [3] and flavor of cooked cured-meats [4]. It also acts as an antioxidative agent, thus, delaying the onset of deterioration of meat flavor, thereby providing an extended shelflife to processed meat products [5-7]. Most importantly, nitrite, in combination with sodium chloride, has bacteriostatic action and inhibits production of Clostridium botulinum neurotoxin [8,9]. Even with all of the benefits conferred by this multifunctional food additive, addition of nitrite to meat and meat products is a source of concern, as it is responsible for the formation of carcinogenic A^-nitrosamines. These carcinogens may be formed by the reaction of nitrite and its dissociation products with secondary amines of muscle tissues during processing, cooking or after ingestion of nitrite-cured meat. A^Nitrosopyrrolidine and //-nitrosodimethylamine, examples of such reaction products, have been detected at low, ppb, levels in fried bacon [10,11]. Because the rate ofNnitrosamine production is proportional to the square of the concentration of residual nitrite in meats [12], a reduction in the level of nitrite added to meats has proven to be an effective measure in reducing the risk of N-nitrosamine formation. Use of A^nitrosamine-blocking agents such as a-tocopherol together with ascorbates has also been employed [13]. Due to the risks of botulinal poisoning, the meat industry is committed to the use of nitrite in cured products because they have no suitable alternative at hand. The necessity for studying the formation and occurrence of A^nitrosamines in cured meats and other food systems stems from the absolute nature of the Food and Drug Regulations in the USA and other regulatory bodies in Canada and Europe which deny the use of any food additive which in itself is carcinogenic or produces carcinogens in foods. Therefore, it is only reasonable that usage of nitrite in cured meats be reduced, or phased-out when effective and safe substitutes are found. Since it is unlikely that a single compound will be found that can perform all of the functions of nitrite, efforts in the past have been concentrated on developing alternatives which performed a selected function of nitrite. For example, a large number of colorants to substitute nitrite has been developed [14], but toxicity problems have prevented their use in meat processing. Sweet [15] was the first researcher to report the development of nitrite-free curing systems to confer the different functions of nitrite. In our laboratory, efforts towards the development of composite nitrite-free curing systems which bestow the characteristic and desirable attributes of cooked cured-meat products without the fear of A^-nitrosamine formation, and which may be employed at the industrial level, have continued. This chapter summarizes the benefits and drawbacks of nitrite in meat curing as well as the development and efficacy of compounds which have been examined and may be used in nitrite-free formulations to reproduce the desirable characteristics of nitrite-cured meats.
1225 2.
NITRITE ALTERNATIVES
2.1 Sensory Attributes 2.1.1 Color Characteristics. Visual appearance is one of the main factors influencing consumers when assessing the quality and palatability of meat products. Many experiments have been performed which support the idea that certain colors do, in fact, influence food acceptance [16]. The color of meat is attributable mainly to the hemoprotein, myoglobin [3]. Addition of nitrite to meat, followed by thermal processing, produces a relatively stable pink-colored pigment. The precise sequence of events resulting in the formation of the cured-meat pigment in meat is not fully understood. As well, the chemical structure of the resultant pigment after thermal processing of nitrite-cured meat has long been a subject of dispute. While Tarladgis [17], Lee and Cassens [18] and Renerre and Rougie [19] have concluded that the cooked cured-meat pigment is a dinitrosyl ferrohemochrome, Bonnett et al. [20] and Killday et al. [21] have shown that a mononitrosyl compound is responsible for the characteristic color of cured meats (Figure 1). Wayland and Olson [22] have also shown that a substituted dinitrosyl protoheme complex may exist under a positive pressure of nitric oxide. Although it is important to find a substitute for nitrite that reproduces the characteristic cured-meat color, the National Academy of Sciences [14] noted that there are few reports of attempts to find compounds or processes that mimic the selective color fixing effect of nitrite in muscle tissue. Toxilogical data on some of these potential colorants for cured meat are limited or non-existent. Sweet [15], in his composite nitrite-free curing system, used erythrosine as the colorant. In 1982, the National Academy of Sciences [14] reported that although a stable uniform cured-meat color can be achieved with as low as 50 ppm sodium nitrite, no suitable means of fixing color in cured meat, other than reduced nitrite levels, have been demonstrated to be effective in products made under commercial conditions. Shahidi and co-workers attempted to solve this problem by using the approach of Sweet, but their colorant of choice was the actual cooked cured-meat pigment (CCMP). The CCMP was preformed outside of the meat matrix and then applied to meat systems. The color characteristics of such nitrite-free systems have successfully duplicated those of nitrite, as will be outlined below. Smith and Burge [23] tried to mimic cured meat color using protoporphyrin-IX, but Hunter L, a, b values and spectra of pigments extracted from protoporphyrin-IX-treated systems were markedly different from those of nitritecured samples. Presence of iron in the protoporphyrin ring is essential for the development of the typical cured color in meats [24]. Preparation of CCMP from the reaction of bovine red blood cells with a nitrosating agent directly or indirectly through a hemin intermediate has been reported [25-28]; a flow diagram of its preparation is presented in Figure 2. Initially, it was proposed that this preformed pigment was a dinitrosyl ferrohemochrome, but citations in literature, as listed above, have suggested that the pigment may indeed be a mononitrosyl heme complex. The issue of the exact chemical nature of the pigment, before and after application to meat, is uncertain, therefore, we have simply referred
1226
HgC =
CH
HgC = CH =
CH
CH«
CH =
CHo
CH =
CHo
[NO], H"^ Reductant
HOOC NO
Nitric Oxide Myoglobin
[NO]
Thermal Processing
NO HgC =
CH
HgC =
CHo
-CH=CH2
H3C -^
/
[NO], H"^ Reductant
4^
H3C
- CHg
HgC -
HOOC
COOH
HOOC
Hemin
CH
CCMP
Figure 1. Formation of the cooked cured-meat pigment from myoglobin and hemin. to the colorant as CCMP. However, recent EPR {i.e., electron paramagnetic resonance) studies in our laboratory have confirmed that the preformed CCMP used in composite nitrite-free curing systems is a mononitrosyl hemochromogen not a dinitrosyl ferrohemochrome. The details of this study will be communicated elsewhere. The stability of the preformed CCMP is limited when light and oxygen are present
1227 [26], but the pigment remains stable for extended periods of time in sealed ampules under a positive pressure of nitric oxide [29]. Entrapment of the pigment in foodgrade wall materials such as cyclodextrins and modified and/or hydrolyzed starches forming a powdered cooked cured-meat pigment (PCCMP) was carried out. The PCCMP prepared from one or a combination of encapsulating agents protected CCMP for at least 18 months as shown by their visible absorption spectra and the Hunter L, a, b and hue angle (tan"^ b/a) values of pigment-treated meats (Table 1). The preformed CCMP, prepared directly or indirectly from bovine red blood cells, had identical spectral characteristics to that of the pigment extracted from a commercial sample of ham (Figure 3). Upon its addition to both comminuted and selected solid cuts of meat, the characteristic color of nitrite-cured meat was reproduced after thermal processing of the meats (Table 1). Since the preformed CCMP is the natural colorant of cured meat, it should be readily acceptable for industrial applications. Pilot-scale experiments for the preparation of wiener and Bovine Red Blood Cells HOAc/NaCl Mixing Protein Separation Formation of Hemin Base/Reductant/ [NO]
Liquids
i
Hemin Crystals
Formation of Pigment Separation Liquids Cooked Cured-Meat Pigment (CCMP) Figure 2. Flow diagram for the preparation of the cooked cured-meat pigment (CCMP) from bovine red blood cells.
1228 Table 1 Hunter L, a, b values of nitrite-cured and pigment-treated cooked pork/ Hunter Values Additives, ppm Control, no additive NaNO^, 150 CCMP, 12 Protoporphyrin-IX, 60 PCCMP, 35
L
a
b
Hue angle
63.3 62.4 60.7 52.1 58.5
4.1
11.6
11.6 11.8 6.8 11.7
9.4 10.1
70.5 39.3 38.5 54.1 40.8
9.5 9.4
^AU pork samples contained 20% (w/w) distilled water and 550 ppm sodium ascorbate. PP-IX - protoporphyrin-IX.
> \ s 0.6
o c
CO
o
0.4
V,,.,
;\
v.
-'^•.....
\ .
•
^\
\
\
< \
0.2
\
\
\
i •
\ \» \ \^ \ \\
Vv^ " - - ^
500
550
600
Wavelength (nm) Figure 3. Absorption spectra of 4:1 (v/v) acetonerwater extracts of: CCMP prepared from bovine red blood cells,--^-^- ; CCMP prepared from hemin,; pigments extracted from nitrite-cured ham — — - ; and pigments extracted from CCMP-treated cooked pork, . Adapted from reference [66]. salami products as well as evaluation of their color quality by subjective or objective methods of analyses have been reported [30-32]. The color intensity of wiener and salami products cured with nitrite or CCMP was practically indistinguishable from one another visually as was reflected in their Hunter L, a, b values (data not shown).
1229 Although pigment-treated samples had slightly lower Hunter L values denoting darker products, they were not visually unattractive and their color was in fact preferred to that of their nitrite-cured counterparts in some cases. Further studies showed that the color intensity of nitrite- or CCMP-cured products depended on the myoglobin content of the original meats. Table 2 shows the dependence of Hunter color parameters on the content of myoglobin in the samples. An increase in the myoglobin content of meat brought about a decrease in Hunter L and hue angle values and a corresponding increase in Hunter a values. Consequently, meats containing a higher myoglobin content when cured with either nitrite or CCMP gave products with a deeper red color. Furthermore, it was revealed that the level of CCMP required to impart a typical cured color to products, equivalent to that of nitrite, depended on the concentration of the native muscle pigments in meat. Muscle tissue richer in myoglobin necessitated addition of higher CCMP levels to attain the characteristic color of cooked cured meat (Table 3). As outlined in Table 3, Shahidi and Pegg [33] applied CCMP to a widerange of muscle foods with myoglobin concentrations ranging from 0.4 to 59 mg/g of wet tissue. Table 2 Dependence of Hunter color values of cooked ground pork systems on their myoglobin content.^ Nitrite-Cured (156 ppm)
Pigment-Treated (12 ppm)
Myoglobin mg/g
L
a
Hue Angle
L
a
Hue Angle
0.76+0.02 1.22±0.06 1.76±0.06
64.2±0.3 57.810.5 56.7+0.7
10.8±0.2 13.4±0.2 14.2+0.4
43.1 ±0.7 34.510.5 33.010.8
63.310.4 57.110.2 55.210.3
11.810.2 13.210.2 14.210.3
39.410.7 34.610.5 33.510.7
^AU samples were prepared with 20% (w/w) distilled water and 550 ppm sodium ascorbate to which either sodium nitrite or the cooked cured-meat pigment was added. Myoglobin (Mb) content was determined according to the procedure of Rickansrud and Henrickson [34], and its content is reported as mg Mb equivalents/g tissue. Table 3 Total hemoprotein pigment content of muscle foods and the amount of preformed cooked cured-meat pigment (CCMP) required to achieve a cured color in products^ Muscle Tissue Total Pigment (mg/g) CCMP (ppm)
Chicken Breast
Pork
Lamb
Beef
Seal
0.4 6
1.2 8
2.1 12
4.5 24
59.0 48
^AU systems contained 20% (w/w) distilled water and 550 ppm sodium ascorbate. Total pigment content determined according to the procedure of Rickansrud and Henrickson [34], and is reported as mg myoglobin equivalents/g tissue.
1230 The color stability of CCMP-treated and nitrite-cured samples was evaluated. Vacuum packaged samples were stored at 4°C underfluorescentlighting. This set-up was designed to simulate the extent of color fading of cured meats in display cases of supermarkets, but intense fluorescent lighting (375 lux) was used to accelerate the color fading process. Both CCMP-treated and nitrite-cured samples faded rapidly during the first 6 h period as reflected by increasing Hunter hue angle values (Figure 4). The rate of color fading of both systems was found to be similar. This trend was surprising because literature suggests that presence of residual nitrite in cured meats serves as a nitrosation source for refixation of disrupted NO molecules from CCMP. It may therefore by concluded that under extreme storage conditions, presence of residual nitrite has little effect on color fixation of cured meats.
Figure 4. Dependence of Hunter hue angle values of meats treated with varying concentrations of cooked cured-meat pigment (CCMP) or sodium nitrite (NaNOj) during exposure to fluorescent lighting over an 18 h period.
1231 2.1.2 Flavor and Oxidative Stability. The role of nitrite in cured meat flavor is complex and the chemical changes that are responsible for this unique flavor brought about in meat are not entirely understood [35]. Cured meat flavor is perhaps a composite sensation arising from the cumulative effect of many compounds. Research into cured meat flavor has been divided into two main areas, namely the sensory evaluation of flavor imparted to meat by nitrite, and the qualitative and quantitative identification of volatile and non-volatile components responsible for it, but caution must be exercised. A compound-by-compound search of meat flavor volatiles may misidentify the true nature of cured meat flavor because a mixture of two or more odors can produce an aroma that is perceived as qualitatively distinct from the odors of their components. Although presence of yet unappreciated substances, in minute quantities, may be responsible for cured flavor, there is no doubt that nitrite influences the flavor of cured meats by virtue of its antioxidative properties and stabilization of microsomal lipids and heme pigments [5,36-38]. The lipid component of freshly cooked meats contributes to their desirable and characteristic flavor, but its oxidation affects palatability, and products so formed may have adverse health effects [39]. Phospholipids are most susceptible to autoxidation and form products such as malonaldehyde, pentanal and hexanal [40,41] which are known to be correlated with off-flavor development in uncured meats. Cross and Ziegler [40] examined the volatile constituents isolated from uncured and cured hams by a GC methodology. Qualitatively, the volatile compounds of cured ham were similar to uncured samples, but were quantitatively different. They reported that hexanal and pentanal were present in appreciable amounts in the volatiles of uncured, but were barely detectable in the volatiles of cured ham. Swain [42] concurred with this finding and reported that nitrite appeared to retard the formation of higher molecular-weight aldehydes (i.e. > C5). Cross and Ziegler [40] also noted that the volatiles, after passage through a solution of 2,4-dinitrophenylhydrazine, had the characteristic cured-ham aroma, regardless of whether cured or uncured hams were used. Cured and uncured chicken and beef volatiles, after stripping their carbonyl compounds by passage through 2,4dinitrophenylhydrazine solutions, also possessed an aroma similar to that of cured ham. These authors concluded that treating meat with nitrite does not seem to contribute any new volatile compounds to the flavor of cooked meats, with the exception of nitrogen oxides that are not present in cooked uncured meat. Therefore, they postulated that cured-ham aroma represents the basic flavor of meat derived from precursors other than triacylglycerols, and that the aromas of various types of cooked meat depend on the spectrum of carbonyl compounds derived by lipid oxidation. Shahidi [35] reported that the elimination of lipid oxidation, either by curing or by stripping of carbonyl compounds from volatiles of untreated cooked meats, caused a major effect on the flavor perception of meats, but this author noted that qualitative differences due to the possible presence of less active flavor components can not be ruled out. Nonetheless, GC analyses of the volatiles of cured meat revealed a much simpler spectrum than their uncured counterparts; the concentration of carbonyl compounds produced from autoxidation of meat lipids was markedly reduced by the presence of nitrite in the system (Table 4). Shahidi [35] proposed that any agent, or
1232 combination of agents that prevents lipid oxidation, with the exception of nitrite precursors, would in principal, duplicate the antioxidant role of nitrite in the curing process, thereby preventing hexanal generation and meat flavor deterioration (MFD). According to Shahidi [43], this is in line with findings of other researchers and its validity was confirmed by preliminary sensory evaluations, but mutton was not included in these studies. A simplistic view, attempting to present a unifying theory of the origin of the basic flavor of meat, species differentiation, and MFD is as follows: when meat is thermally processed, it acquires a characteristic species flavor which results from volatile carbonyl compounds, such as hexanal and pentanal, formed by oxidation of its lipid components (i.e., primarily phospholipids). Further oxidation during storage of cooked meat results in the deterioration of its flavor. Curing with nitrite suppresses the formation of oxidation products. It may be assumed that the flavor of nitrite-cured meats is actually the basic natural flavor of meat from different species without being influenced by overtone carbonyls derived from oxidation of their lipid components. Further support for this view has recently been provided by Ramarathnam et al. [44], but the postulate does not easily explain the fact that the intensity of cured meat flavor is proportional to the logarithm of nitrite concentrations as reported by MacDougall et al. [45], or the apparent persistence of the characteristic "mutton" flavor after nitrite curing of sheep meat [46]. Table 4 Effect of curing with nitrite on the concentration of carbonyl compounds of thermally processed ground pork. Relative Concentration Carbonyl Compound
Hexanal Pentanal Heptanal Octanal Nonanal 2-Octenal 2-Nonenal 2-Decenal 2-Undecenal 2,4-Decadienal
Uncured
Nitrite-Cured'
Nitrite-Free Cured^
100 31.3 3.8 3.6 8.8 2.0 1.0 1.1 1.4 1.1
7.0 0.5 <0.5 <0.5 0.5 ~ — — 0.5 —
6.5 0.5 0.5 0.5 0.7 ~ <0.1 0.5 <0.1
^Sample contained 550 ppm sodium ascorbate. ^Sample contained the preformed cooked cured-meat pigment, 12 ppm; sodium tripolyphosphate, 3000 ppm; sodium ascorbate, 550 ppm; and tert-butylhydroquinone, 30 ppm. Adapted from references [35] and [66].
1233 To reproduce the antioxidative efficacy of nitrite, we have examined a number of antioxidants [47], sequestrants [48] and their combinations [49]. The concentration of carbonyl compounds produced in these systems from the autoxidation of meat lipids was markedly reduced when combinations containing polyphosphates, ascorbates and low levels of an antioxidant were used. The spectrum of notable carbonyl compounds was similar to the nitrite-cured system. Among the antioxidants used, BHA and TBHQ were the most effective, even at 30 ppm, for retarding oxidation during a 5week storage at 4°C, as measured by the 2-thiobarbituric acid test (Table 5). Among the food-grade sequestrants, sodium acid pyrophosphate (SAPP), tetrasodium pyrophosphate (TSPP), sodium tripolyphosphate (STPP) and ethylenediaminetetraacetic acid (EDTA) were most effective. A strong synergistic action was noted when ascorbates were used in combination with polyphosphates (Figure 5). Addition of small quantities (i.e., 30 ppm) of an antioxidant, such as BHA or TBHQ to the above systems, had a minor effect on the TBA values, but conferred a positive influence on their sensory characteristics as noted by untrained panelists [50]. Similar antioxidant effects were observed when plant phenolic compounds were used [51]. Interestingly, the preformed CCMP had a weak, but noticeable antioxidative effect of its own [47]. Good correlations between the TBA values and the sensory data, as well as between the hexanal content of the meats and their sensory acceptability existed [52]. Table 5 TBA numbers of cooked pork treated with different additives after a 5-week storage period at 4°C.^ Experiment Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Additive(s), ppm Control, no additives Sodium nitrite, 150 Butylated hydroxyanisole, 30 tert-Butylhydroquinone, 30 Sodium tripolyphosphate, 3000 Tetrasodium pyrophosphate, 3000 Sodium hexametaphosphate, 3000 (5) + Sodium ascorbate, 550 (6) + Sodium ascorbate, 550 (7) + Sodium ascorbate, 550 (8) + (3) (8) + (4) Cooked cured-meat pigment, 12 (11)+ (13) (12) + (13) (14) + Sodium hypophosphite, 3000 (15) + Sodium hypophosphite, 3000
^Adapted from reference [66].
TBA Number, ppm 15.46 0.63 0.44 0.35 1.86 1.66 7.71 0.27 0.23 0.29 0.20 0.18 9.89 0.34 0.24 0.28 0.21
1234
1.00
0.75 h
0.50 h E c
CM CO
ij in CO
0.15 h
8
C CO jQ
u.
O
0.10
cn
JQ
<
0.05 h
0.00 Storage Period at 4°C (Days) Figure 5. Synergistic effect of polyphosphates with sodium ascorbate in meat systems. Symbols (full) are: • , SHMP; • , STPP; A , TSPP; • , SAPP. Corresponding open symbols are for the same polyphosphates with sodium ascorbate. Adapted from reference [66]. 2.2 Safety Considerations 2.2.1 Antimicrobial Action. Nitrite exerts a concentration-dependent antimicrobial effect in cured meat products, including, but not limited to, inhibition of the outgrowth of spores of putrefactive and pathogenic bacteria such as Clostridium botulinum [53]. The degree of protection provided to cooked meats against microbial contamination
1235 depends on many factors including the concentration of residual nitrite present, duration of temperature abuse and extent of contamination. Nitrite also retards microbial spoilage of cured meats by anaerobic and aerobic spore-forming bacteria. The mechanism(s) by which nitrite inhibits the outgrowth of spores and the growth of vegetative cells and microorganisms is not fully understood, but it appears that a reaction with iron-containing enzymes is involved. A better understanding of the exact mechanism(s) of the antimicrobial role of nitrite is still required. Irrespective of future decisions on the fate of nitrite, its removal or reduction must be counter-balanced by alternatives that will assure the safety from botulinal hazards in abused products [54]. Furthermore, the traditional identity of cured meat products must be retained. According to Sofos and Busta [55], any compound to be considered as an alternative to nitrite should be suitable for use in all cured meat products and should control other microorganisms of public health significance, delay product spoilage, and not interfere with beneficial microorganisms such as lactic acidproducing cultures, necessary for the manufacture of fermented meat products. The compound of choice must also be (a) at least as effective as nitrite itself (b) safe, (c) heat stable, (d) flavorless, and (e) preferably effective at low concentrations. Several alternatives to nitrite for its antimicrobial action have been tested. These include sorbic acid and its potassium salt, sodium hypophosphite, fumarate esters, parabens, and lactic acid-producing bacteria (LAPB). These could be used either alone or together with low concentrations of nitrite [14]. Sorbic acid and its potassium salt have been found to be safe additives and are permitted in a variety of foodstuffs. Sofos et al. [56-58] reported that sorbic acid when used at a level of 2000 ppm delayed Clostridium botulinum toxin production in wieners to an extent similar to that of 156 ppm nitrite; but the effects were pH-dependent and were operative at pH < 6.0. Tanaka et al. [59] demonstrated that potassium sorbate addition to wieners at 2700 ppm provided an anticlostridial action similar to that of 100 ppm nitrite. Sodium hypophosphite is a GRAS (generally recognized as safe) substance and may perhaps be used at 3000 ppm level in meats. Microbiological studies indicated that a total or partial replacement of nitrite with this compound effectively inhibits production of Clostridium botulinum toxins [60]. Fumarate esters are effective at 1250 ppm or higher levels in bacon [61] while parabens had little antibotulinal activity in frankfurters. Wood et al. [62] evaluated the antibotulinal activity of sodium hypophosphite, potassium sorbate and monomethyl fumarate in nitrite-free curing systems. The treatment containing 3000 ppm sodium hypophosphite, together with CCMP, sodium ascorbate, STPP and TBHQ, most closely resembled that of nitrite at 150 ppm in its ability to prevent spore outgrowth and toxin production. Monomethyl fumarate at 1250 ppm was slightly less effective than sodium hypophosphite (Table 6). These additives had no adverse effect on the oxidative stability or the color of formulated pork products in model systems [63]. Various LAPB have demonstrated excellent protection against Clostridium botulinum toxin production in nitrite-free cured bacon. Moreover, some strains of LAPB produce bacteriocins (/.^., proteins with antibacterial activity) which prevent other bacteria such as Clostridium botulinum from flourishing. Research into the use of bacteriocins or bacteriocinogenic LAPB for meat preservation is still in its infancy, and existing difficulties must be overcome before
1236 LAPB can be used commercially to extend the storage life and to enhance the safety of meats [64]. Application of irradiation, at 5 or 10 kGy, to meats treated with CCMP not only ensured the keeping quality of the products, but it did not have any negative effect on their color stability [65]. Table 6 Effect of treatment composition on gas and toxin production by Clostridium botulinum in pork.* Incubation at 27°C (days) Treatment^ 1. 2. 3. 4. 5. 6. 7. 8. 9.
Control NaNO^, 150 (2) + ASC CCMP, 12 (4) + ASC + STPP + TBHQ (4) + SHP (5) + SHP (5) +PS (5) + MMF
1
2
3
34/34+ 0/36- 11/36+ 5/18+ 0/36- 0/32- 15/3017/17+ 12/37+ 25/25+ 1/18- 1/16+ 2/14+ 6/35- 3/27+ 1/240/39- 32/35+ 3/3+ 0/37- 1/33- 17/31+
4
6
8
27
8/13+ 5/14+
2/2+ 4/6+
0/1-
0/1-
4/12+ 6/22+
1/2+ 5/13+
0/11/2+
0/10/1-
9/18+
3/4+
0/1+
0/1-
* Adapted from Wood et al. [62]. Number of packs showing gas production/total number of packs. Toxin present, +; toxin absent, -; ^presence of toxin was not tested. ^Additives were: sodium ascorbate, ASC; cooked cured-meat pigment, CCMP; sodium tripolyphosphate, STPP; tert-butylhydroquinone, TBHQ; sodium hypophosphite, SHP; potassium sorbate, PS; and monomethyl fumarate, MMF. 2.2.2 The A^-Nitrosamines. Nitrite-free ingredients containing the preformed CCMP have been shown to duplicate the color, flavor and bacteriostases of nitrite in model meat systems as reported by Shahidi and co-workers [30,33,47,62,63,66]. Although we have proposed the use of these nitrite-free systems as an alternative to nitrite, the absence of carcinogenic A^-nitrosamines in formulated products must be confirmed. The presence of all possible volatile A^-nitrosamines was tested in various cooked treated muscle food systems, and only N-nitrosodimethylamine (NDMA) was detected, but only in some of the cooked treated systems examined. Table 7 summarizes the content of volatile NDMA in cooked, nitrite-cured and pigment-treated pork, cod, and cod surimi. No measurable amount of NDMA was detected in the control, nitritecured or CCMP-treated pork systems, but trace quantities of NDMA have been reported in nitrite-cured pork-containing products [11]. Additionally, no detectable amount of NDMA was observed in control fish systems. The occurrence of NDMA in nitrite-treated cod was expected because the formation of A^-nitrosamines in cured fish has been shown to occur primarily in salt-water species [67]. Only ca, 1 ppb of NDMA was detected which may reflect the very fresh nature and careful processing
1237 of the fish used. The precursor of NDMA, dimethylamine (DMA), is formed in the muscles of fish as a result of activity of endogenous enzymes on trimethylamine Table 7 Effects of nitrite and CCMP on the formation of A^-nitrosodimethylamine (NDMA) in pork, cod or cod surimi systems.^ Species
Additive, ppm
NDMA, ppb
Pork
No additive NaNOj, 156 CCMP, 12 CCMP, 24
<0.2 <0.2 <0.2 <0.2
Cod
No additive NaNOj, 156 CCMP, 12
<0.2 0.9 <0.2
Cod Surimi
No additive NaNOj, 156 CCMP, 12
<0.2 <0.2 <0.2
^All meat systems were treated with 20% (w/w) distilled water and 550 ppm sodium ascorbate. Detection threshold of NDMA is 0.2 ppb. Adapted from reference [28]. N-oxide. Perhaps only partial degradation of trimethylamine A^-oxide to DMA had occurred in the fish muscle tissue by the time of its use. Although NDMA was detected in nitrite-cured cod, its absence was noted in CCMP-treated samples. This may imply that no disproportionation of CCMP had occurred or that it did not produce a sufficient quantity of nitric oxide to take part in possible transnitrosation reactions. Furthermore, absence of A^-nitrosamines in nitrite-cured cod surimi tends to suggest that washing of cod muscles may be an effective means to remove DMA, trimethylamine, and trimethylamine //-oxide in order to avoid their nitrosation. The effect of sodium nitrite and CCMP at 156 and 12 ppm levels, respectively, on NDMA formation in hybrid meat/fish systems is presented in Table 8. No detectable NDMA was noted in control formulations as expected, but addition of nitrite to hybrid pork systems containing 15 and 50% cod produced 0.3 and 1.0 ppb of NDMA, respectively. The quantities of NDMA in these systems are equal to or less than that found in nitrite-cured cod (Table 7). Again, no measurable amount of NDMA was detected in CCMP-treated hybrid products for the same reasons as stated for pigment-treated systems. Unlike the nitrite-treated cod surimi sample, NDMA was noticed at 0.2 ppb in pork/cod surimi hybrid formulations both at 15 and 50% substitution. Aqueous washing of minced cod flesh did not remove sufficient quantities of trimethylamine, trimethylamine N-oxide or DMA from the fish matrix to prevent nitrosation of DMA even though the 0.2 ppb concentration is at the detection limit of the TEA analyzer. Furthermore, CCMP-treated counterparts were free of detectable NDMA for the same reasons cited above for the pigment-treated cod.
1238 Table 8 Effects of nitrite and CCMP on the formation of N-nitrosodimethylamine (NDMA) in hybrid pork and cod or cod surimi systems.^
Species
Additive, ppm
NDMA, ppb
Pork (85%) + Cod (15%)
No additive NaNO^, 156 CCMP, 12
<0.2 0.3 <0.2
Pork (85%) + Cod Surimi (15%)
No additive NaNOj, 156 CCMP, 12
<0.2 0.2 <0.2
Pork (50%) + Cod (50%)
No additive NaNOj, 156 CCMP, 12
<0.2 1.0 <0.2
Pork (50%) + Cod Surimi (50%)
No additive NaNOj, 156 CCMP, 12
<0.2 0.2 <0.2
^AU meat systems were treated with 20% (w/w) distilled water and 550 ppm sodium ascorbate. Detection threshold of NDMA is 0.2 ppb. Adapted from reference [28]. 3.
SUMMARY
Several nitrite-free combinations consisting of the preformed CCMP, a sequestrant and an antioxidant, possibly together with an antimicrobial agent, have been formulated for meat curing. These mixtures have been found effective in reproducing the color, the oxidative stability and flavor, as well as the antimicrobial effects of nitrite. Use of other antimicrobial agents such as bacteriocins may prove advantageous. Additionally, nitrite-free curing systems containing the preformed CCMP can be successfully employed in the preparation of processed meat products without the fear of A^-nitrosamine formation. It has been demonstrated that nitrite-free curing of fish or fishery by-products in combination with red meats in the production of novel cured products, free of A^-nitrosamines, is now feasible. Hence, this would not only make use of underutilized fish protein, but it also has the potential to increase the nutritional and sensory quality of formulated products.
4.
ACKNOWLEDGEMENTS
We are grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support. The A^-nitrosamine work was carried out by Dr. N.P. Sen of the Food Research Division of the Bureau of Chemical Safety, Health Protection Branch in Ottawa.
1239 5. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
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F. Shahidi, R.B. Pegg and N.P. Sen, In Proceedings of the First International Conference on The Impact of Food Research on New Product Development, R. Ali and PJ. Barlow (eds.), January 24-26, University of Karachi, Pakistan, University of Humberside, UK, pp. 221-245, 1993. R.B. Pegg, Ph.D. thesis, Memorial University of Newfoundland, St. John's, NF, 1993. F. Shahidi and R.B. Pegg, J. Muscle Foods, 2 (1991) 297. D.A. Rickansrud and R.L. Henrickson, J. Food Sci., 32 (1967) 57. F. Shahidi, In Flavor Chemistry. Trends and Developments, R. Teranishi, R.G. Buttery and F. Shahidi (eds.), ACS Symposium Series 388, American Chemical Society, Washington, DC, pp. 188-201, 1989. K. Sato and G.R. Hegarty, J. Food Sci., 36 (1971) 1098. J.O. Igene, J.A. King, A.M. Pearson and J.I. Gray, J. Agric. Food Chem., 27 (1979) 838. J.I. Gray and A.M. Pearson, In Advances in Meat Research. Vol 3: Restructured Meat and Poultry Products, A.M. Pearson and T.R. Dutson (eds.). Van Nostrand Reinhold Co., New York, NY, pp. 221-269, 1987. R.J. Shamberger, T.L. Andreone and C.E. Wills, J. Natl. Cancer Inst., 53 (1974) 1404. C.K. Cross and P. Ziegler, J. Food Sci., 30 (1965) 610. F. Shahidi and R.B. Pegg, J. Food Lipids, 1 (1994) 177. J.W. Swain, Ph.D. thesis. University of Missouri, Columbia, MO, 1972. F. Shahidi, In Lipid Oxidation in Food, A.J. St. Angelo (ed.), ACS Symposium Series 500, American Chemical Society, Washington, DC, pp. 161-182, 1992. N. Ramarathnam, L.J. Rubin and L.L Diosady, J. Agric. Food Chem., 39 (1991) 1839. D.B. MacDougall, D.S. Mottram and D.N. Rhodes, J. Sci. Food Agric, 26 (1975) 1743. D.H. Reid, O.A. Young and T.J. Braggins, Meat Sci., 35 (1993) 171. F. Shahidi, L.J. Rubin and D.F. Wood, J. Food Sci., 52 (1987) 564. F. Shahidi, L.J. Rubin, L.L. Diosady, N. Kassam and J. C. Li Sui Fong, Food Chem., 21 (1986) 145. F. Shahidi, L.J. Rubin and D.F. Wood, Food Chem., 23 (1987) 151. J. Yun, F. Shahidi, L.J. Rubin and L.L. Diosady, Can Inst. Food Sci. Technol. J., 20 (1987) 246. F. Shahidi and P.K.J.P.D. Wanasundara, CRC Crit. Rev. Food Sci. Nutr., 32 (1992) 67. F. Shahidi, J. Yun, L.J. Rubin and D.F. Wood, Can. Inst. Food Sci. Technol. J., 20 (1987) 104. M.D. Pierson and L.A. Smoot, CRC Crit. Rev. Food Sci. Nutr., 17 (1982) 141. F. Shahidi and R.B. Pegg, Can. Chem. News, 43(2) (1991) 12. J.N. Sofos and F.F. Busta, Food Technol., 34(5) (1980) 244. J.N. Sofos, F.F. Busta, K. Bhothipaksa and C.E. Allen, J. Food Sci., 44 (1979) 668.
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J.N. Sofos, F.F. Busta and C.E. Allen, Appl. Environ. Microbiol., 37 (1979) 1103. J.N. Sofos, F.F. Busta, K. Bhothipaksa, C.E. Allen, M.C. Robach and M.W. Paquette, J. Food Sci., 45 (1980) 1285. K. Tanaka, K.C. Chung, H. Hayatsu and T. Kada, Food Cosmet. Toxicol., 16 (1978) 209. R.J. Banner, Food Eng., 53(1) (1981) 130. C.N. Huhtanen, In Developments in Industrial Microbiology, Volume 25, Proceedings of the Fortieth General Meeting of the Society for Industrial Microbiology, August 14-19, 1983, Sarasota, FL, pp. 349-362, 1984. D.S. Wood, D.L. Collins-Thompson, W.R. Usbome and B. Picard, J. Food Prot., 49 (1986) 691. F. Shahidi, L.J. Rubin and D.F. Wood, Meat Sci., 22 (1988) 73. M.E. Stiles and J.W. Hastings, Trends Food Sci. Technol., 2 (1991) 247. F. Shahidi, R.B. Pegg and K. Shamsuzzaman, J. Food Sci., 56 (1991) 1450. F. Shahidi and R.B. Pegg, Food Chem., 43 (1992) 185. Z. Sikorski and S. Kostuch, Food Chem., 9 (1982) 213.
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Potential for growth and inhibition of Listeria monocytogenes in meat and poultry products J.N. Sofos, W.B. Barbosa, H.J. Wederquist, G.R. Schmidt and G.C. Smith Department of Animal Sciences and Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523, U.S.A. Abstract The widespread distribution of Listeria monocytogenes in food plants and products, its potential to multiply at cold temperatures and under vacuum, and the high fatality rate it causes in immunocompromised human populations, make it of importance to study its potential to proliferate or to be inhibited in various foods. Studies in our laboratory have examined the potential for growth and inhibition of this pathogen in meat and poultry products. Growth of the pathogen in vacuum-packaged, fresh ground beef stored at 4°C was slow and depended on pH and bacterial strain. At pH values of approximately 5.5, growth of various strains was either nonexistent or very slow. At pH values above 6.0, growth was more pronounced. In contrast to fresh ground beef, growth of the pathogen was rapid and extensive in cooked bologna-type sausage formulated with mechanically deboned turkey meat and stored under vacuum at 4°C. This extensive growth was significantly inhibited by inclusion of sodium acetate (0.5%), sodium lactate (2.0%) or potassium sorbate (0.26%) in the sausage formulation. 1.
INTRODUCTION
Listeriosis is described as a sporadic bacterial infection of animals resulting in encephalitis or meningoencephalitis in adult ruminants, septicemia or focal hepatic necrosis in young ruminants and monogastric animals, and septicemia with myocardial degeneration or hepatic necrosis in birds (Anonymous, 1986). It is also described as causing abortion or perinatal infection in all susceptible mammals, especially cattle and sheep in cold weather, where death can occur 4 to 48 hours after the appearance of symptoms. The agent of the disease is Listeria monocytogenes, and listeriosis has been characterized as a zoonosis and a potential hazard for owners and veterinarians who handle sick animals. However, L. monocytogenes is also considered a human foodbome pathogen, and the Centers for Disease Control and Prevention (CDC) estimate that approximately 1,850 human cases of listeriosis occur every year in the United States with 425 resulting in death (Schuchat et al., 1992). The clinical signs of the disease in humans are not different than those afflicting other mammals. Human listeriosis can manifest itself as an invasive or noninvasive disease (Schuchat et al., 1991). The invasive form is rare. Factors such as virulence of the
1244 infecting strain, susceptibility of the host and size of the inoculum may determine occurrence of the invasive disease. Listeriosis is usually associated with immunocompromised people, including pregnant women, causing meningitis or meningoencephalitis (Schuchat et al., 1991). The most common immunosuppressive conditions present in non-pregnant adults suffering listeriosis infections are corticosteroid therapy, cancer and AIDS or HIV infection. Many patients, however, had illnesses which are not considered immunosuppressive, such as heart or renal diseases and diabetes (Schuchat et al., 1992). Among pregnant women, abortion, stillbirth, preterm labor or early-onset infection of the neonate are major manifestations. The non-invasive or mild listeriosis syndrome has not been well characterized (Schuchat et al., 1991). Listeria monocytogenes is a Gram-positive, facultative bacterium widely distributed in the natural environment, which over the last several years has become increasingly recognized as an important foodborne pathogen. After the occurrence of several large foodbome outbreaks and associate deaths, L. monocytogenes became the object of considerable public concern (Gellin and Broome, 1989). The food industry, academia, and regulatory and health agencies have focused much attention on gaining an understanding of the nature of the problem, and developing strategies for prevention and control of foodbome listeriosis. Listeria monocytogenes is considered to be a threat to food safety because of its pathogenicity and its ability to proliferate at refrigeration temperatures, since it can grow at 0.4 to 45°C (Ryser and Marth, 1991). The pathogen can also survive freezing and dry conditions, tolerate very high salt concentrations and grow under a wide range of pH levels. Many questions are still to be answered about listeriosis and its transmission with foods. Several kinds of foods are known to carry the pathogen, and it is believed that foodbome transmission may account for a substantial portion of sporadic listeriosis (Pinner et al., 1992; Schuchat et al., 1992). However, very little is known about infective dosage, host susceptibility and strain virulence to establish a direct cause and effect relationship between foods containing L. monocytogenes and the onset of sporadic cases of listeriosis. Epidemiologic studies have reported that, among immunosuppressed patients, eating undercooked chicken increased the risk of listeriosis (Schuchat et al., 1992), but only one documented case of listeriosis in the United States has been directly linked to consumption of poultry products (Anonymous, 1989). Meat and poultry, however, may play a more important role in the epidemiology of listeriosis because the pathogen has been isolated from up to 92% of retail beef mince samples (Lowry and Tiong, 1988), and it is frequently found in slaughterhouses around the world (Genigeorgis et al., 1989, 1990; Gobat and Jenmii, 1990; Pociechaetal., 1991). The behavior of L. monocytogenes in meat or poultry, however, is not completely known; research has produced variable results. Although meat seems to offer all the necessary conditions for growth of the pathogen, studies have shown that the interaction of different factors may reverse this situation. The effect of factors such as L. monocytogenes strains, pH, gas atmosphere composition, spoilage flora, organic salts and temperature on the behavior of the pathogen in meat or meat products needs to be well understood. Strain differences may play a major role in the fate of the pathogen in food products. Listeria monocytogenes includes several strains of different pathogenicity, and perhaps, distinct growth behavior as well. However, most of the research published has tested the behavior of one particular strain (Scott A) in foods (Buchanan and Klawitter, 1991;
1245 Dickson, 1990; Fain et al., 1991; Harrison et al., 1991; Kallander et al., 1991), including meats. Very often, when Scott A is not the strain studied, a pool of different strains is used (Glass and Doyle, 1989; Palumbo and Williams, 1991; Sorrells and Enigl, 1990; Yen et al., 1991, 1992). Listeria monocytogenes is a particularly difficult organism to control in foodprocessing establishments. Refrigerated food plants, in particular, provide conditions which allow for L. monocytogenes survival and growth. Since L. monocytogenes can and will multiply at refrigeration temperatures and in the presence of nitrites and salt, attention is also being directed at investigating this pathogen in ready-to-eat foods, including meat and poultry products. Temperatures of 4 to 10°C are not uncommon in food distribution systems and in retail environments. Home refrigerators have been found to average close to 7°C (Junttila et al., 1988). In a study conducted by Grau and Vanderlinde in 1992, listeriae were detected on 93 of 175 samples of vacuum-packaged processed meats obtained from retail stores, and 5% of the samples contained more than 1000 colony forming units (CFU) per gram. Today, food manufacturers rely on a variety of processing techniques to produce safe and wholesome products with acceptable shelf life. The most common methods of food preservation include heating, cooling, freezing, drying, fermentation and modified atmosphere packaging, few of which can guarantee complete destruction of the hardy L. monocytogenes bacteria. Incorporation of chemical preservatives such as sodium chloride, sodium nitrite, sodium acetate, sodium bicarbonate, sodium lactate or potassium sorbate, may play an important role in the control of L. monocytogenes when used in combination with other processing techniques. Studies in our laboratory have examined the potential for growth and inhibition of L. monocytogenes in fresh and processed meat and poultry products. 1.1
Fate of Listeria monocytogenes in meat and poultry
It is well established that L. monocytogenes is frequently present in meat and poultry products, and that it is difficult to totally eliminate the pathogen from raw meat products. The role of meat and poultry in the transmission of listeriosis to humans, however, is still unknown. In general, meat products frequently carry the pathogen, but not many cases of listeriosis have been associated with consumption of meat or poultry (Barbosa, 1993; Wederquist, 1993). As mentioned above, one case of listeriosis resulting in death has been associated with consumption of turkey frankfurters (Anonymous, 1989). The patient was a woman who had breast cancer. A package of the product found in her refrigerator was contaminated with L. monocytogenes, and so were unopened packages of the same brand in a nearby store. 1.2
Raw meat and poultry
Theoretically, meat and poultry should offer good environments for growth of L. monocytogenes. Studies have shown, however, that this may not always be true, especially in red meats. Inexplicable differences in growth behavior of L. monocytogenes on different meats have been observed (Doyle, 1988), but overall, the number of cells will increase or stay the same after different periods of storage at refrigeration temperature (Sofos, 1992). Significant growth of L. monocytogenes strain Scott A was not detected in hamburger, irradiated hamburger, sausage, or chicken salad stored at 4°C for 7 days, but it was reported
1246 in milk at the same temperature (Buchanan et al., 1987). Survival of L. monocytogenes, but not growth, was observed in ground beef (pH 5.6 to 5.9) stored in oxygen-permeable or vacuum packages held at 4°C for 2 weeks (Johnson et al., 1988). The number of L. monocytogenes remained similar (10^ to 10^ CFU/g) throughout the 14 days of the study, and no differences were observed between the two strains (Scott A and V-7) tested. The authors speculated that meat might be deficient in a nutrient required by the organism to grow. Growth of L. monocytogenes was not observed in ground beef or liver stored at 4 and 25°C (Shelef, 1989), but the potential of meat lacking a necessary ingredient for growth of L. monocytogenes was not confirmed by the author. Three strains of L. monocytogenes (Scott A, Brie-1 and ATCC 35152) were inoculated in ground beef (pH 5.8) and liver (pH 5.6), and incubated at 4°C for 40 days. Despite the differences in composition and pattern of spoilage between the two meats, Listeria counts did not change during the storage period. In the same research study, water, glucose and calcium were added to some of the ground beef samples, but these ingredients did not change the behavior of the microorganism. Growth could only be observed in meat extract, obtained after blending and centrifugation of the meat. It was concluded that all the necessary ingredients for growth of L. monocytogenes should be associated with the water-soluble portion of the meat. According to the author, centrifugation must have either removed possible suppressing compounds associated with the muscle, or released vital nutrients which were not available to the bacteria before (Shelef, 1989). Another paper has endorsed the potential presence of an inhibitory factor against L. monocyro^^«^5 in beef (Buchanan and Klawitter, 1991). Listeria monocytogenes strain Scott A did not grow in either irradiation-sterilized or regular vacuumpackaged raw ground beef stored at 5°C for 800 hours, but growth was observed in cooked meat stored under the same conditions. Whatever was inhibiting L. monocytogenes in the raw meat was lost or was not present in the cooked product. Other studies, however, have contradicted the above findings. Packaging ground beef (pH > 6) under CO2 was more effective in inhibiting growth of L. monocytogenes than vacuum packaging (Gill and Reichel, 1989). Growth occurred in vacuum packages held at 0°C or above, but it was not observed at -2°C. The growth rate of the pathogen was always slower than the growth rate of the natural bacterial flora or other pathogens such as Yersinia enterocolitica and Aeromonas hydrophila. Listeria monocytogenes stopped growing or died in some occasions when the natural flora approached maximum numbers, showing that the native flora can interfere with its behavior in meat. Lag phase became longer as the temperature of storage decreased from 10°C to 5°, 2° or 0°C. Other studies have also speculated about the effect of the natural flora on the inhibition of L. monocytogenes (Buchanan and Klawitter, 1991; Kaya and Schmidt, 1991). Listeria monocytogenes was not able to grow in beef of pH 5.6 stored at 2 or 4°C for 9 weeks. At 7°C, growth occurred just after 3 weeks (pH 5.6). Lactic acid bacteria grew fast and became the dominant flora at 2 and 4°C (pH 5.6), overcoming the number of L. monocytogenes. In high pH beef (pH>6.0), however, growth of L. monocytogenes was observed at 2, 4, 7 and 10°C. At the lowest temperatures, growth started within one week of storage, while at 7 and 10°C, the number of listeriae cells increased more than one power of ten in less than 3 days. Brochothrix thermosphacta grew well in the high pH meat and surpassed L. monocytogenes. Lactic acid bacteria did not grow in the high pH meat, and the authors concluded that growth of listeriae was dependent on the interaction of temperature, pH and competing flora. Growth inhibition of L. monocytogenes in minced beef by lactic acid bacteria has been
1247 demonstrated previously (Gouet et al., 1978). Listeria monocytogenes could not grow in meat (pH 5.8) inoculated with Lactobacillus plantarum, but grew well in the presence of Pseudomonas fluorescens and Escherichia coli. The pH of meat samples inoculated with both L. monocytogenes and L. plantarum remained almost the same during the 17 days of study, but pH increased from 5.8 to 6.5 in 17 days in meat samples inoculated with L. monocytogenes in combination with P. fluorescens and E. coli (Gouet et al., 1978). Another study showed that L. monocytogenes Scott A was able to grow in vacuum packaged lean beef tissue held at 5°C (Dickson, 1990). A four log increase in the number of listeriae present was observed after 21 days of storage, but the microorganism started growing after 7 days. The pH of samples was not reported, however. Listeria monocytogenes, strain Murray B, grew in fat and lean beef striploins of pH between 5.5 to 6.1 packaged under vacuum and stored at 0 and 5.3°C (Grau and Vanderlinde, 1990). Growth was more pronounced in fat than in lean tissue at both 0 and 5.3°C. At 0°C (meat pH 5.5 to 5.7), growth in the lean tissue was severely inhibited until 61 days of storage, and after 76 days, the number of listeriae was just 5 times the initial number. At 5.3°C, growth started after a lag phase of 5 to 6 days. Listeria monocytogenes grew better in lean tissue with pH 6.0 to 6.1 than in the lower pH muscle, but growth in the fat portion was still more accentuated than in the lean. Different results were obtained by Chung et al. (1989), who found that L. monocytogenes strain Scott A grew better in lean than in fat beef tissue incubated at 5°C for a week. The pathogen was able to grow in chicken broth (pH 6.4) incubated at temperatures as low as -0.4°C (Walker et al., 1990). When listeriae inocula were pre-incubated at 30°C instead of 4°C, multiplication was observed above 0.5°C. It was reported that cultures preincubated at 30°C could have had a more intense "cold shock" when transferred to very low temperatures. Because these cultures were grown at optimal temperatures, they were probably more sensitive to extensive reductions in temperature (Walker et al., 1990). Under modified atmosphere packaging (72.5/22.5/5 %, N2/C02/02)L. monocytogenes multiplied in minced chicken incubated at 4, 10 or 27°C (Wimpfheimer et al., 1990). Neither listeriae initial number nor high initial aerobic counts affected changes of the microorganism under these conditions. The modified atmosphere packaging was more effective in controlling the natural flora, showing that growth could occur before spoilage of the meat. The pathogen failed to grow at 4, 10 or 27°C when oxygen was not present in the package (75/25, CO2/N2). Another study reported growth of L. monocytogenes in fresh chicken legs (pH 6.52) held at 6°C for 17 days (Zeitoun and Debevere, 1991). Growth was noticed after 6 days of storage under modified atmosphere packaging (90/10%, CO2/O2) or air. The pathogen was partially inhibited when lactic acid and sodium lactate were sprayed on the samples. Hart et al. (1991) described the behavior of L. monocytogenes in skinless chicken breasts (pH = 5.8) packed under different concentrations of CO2 (air, 30% CO2 + ah-, 30% CO2 + N2 and 100% CO2), and incubated at 1, 6 or 15°C. Counts of L. monocytogenes did not change in samples stored at 1°C for up to 17 days, independent of gaseous environment. Little growth was observed at 6°C when the listeriae inoculum was grown at 37°C, but incubating the inoculum culture at 1°C led to slow growth in the inoculated meat. At 15°C, counts of L. monocytogenes increased about 100-fold in two days when air was present in the package, but less growth occurred under 100% CO2. Although storage temperature was the major limiting factor for growth of the pathogen, modified atmosphere packaging determined the type of the spoilage bacteria present which could have
1248 affected the performance of L. monocytogenes. The pH of the chicken breasts (5.8), which was below the pH usually reported in other experiments (above 6), may be an important factor to be considered when analyzing the results. Listeria monocytogenes grew better in air-packaged than in vacuum-packaged chicken stored at 4°C for 15 days, but it could not be detected in air-packaged irradiated chicken (2.5 kGy) during the same period (Varabioff et al., 1992). Surprisingly, surviving cells were detected in vacuum-packaged irradiated chicken after 7 days of storage, indicating that the irradiation dose may not have been sufficient to eliminate the bacteria in poultry. Despite the differences in results presented by different studies, it seems that some growth of L. monocytogenes is expected to occur in fresh meat and poultry, with growth at refrigerated temperature being more extensive in poultry than in red meats. 1.3
Cooked or processed meat and poultry
The ability of L. monocytogenes to survive or grow in processed meat products will depend on the product itself (Glass and Doyle, 1989), the resistance of the organism to heat, presence of chemical additives, post-processing contamination and their interactions with temperature, pH and gas atmosphere. A composite of five strains of L. monocytogenes, representing two serotypes, was inoculated in several meat products, which were then incubated at 4°C for 12 weeks (Glass and Doyle, 1989). Listeria monocytogenes grew well in some wiener products, ham, bologna and bratwurst, but it grew exceptionally well (within 4 weeks) on sliced chicken and turkey. It survived but did not grow in summer sausage and grew slightly in cooked roast beef. Poultry and other products with pH above 6 offered the best conditions for growth. Listeria monocytogenes strain Murray B was able to proliferate in vacuum-packaged corned beef and ham incubated at different temperatures (Grau and Vanderlinde, 1992). In corned beef, the growth rate was determined by temperature of incubation. At 9 and 15 °C, L. monocytogenes multiplied at the same rate or faster than the natural flora, while at 5°C its growth rate was half of that of the natural flora. In ham, the growth rate of L. monocytogenes was determined by the interaction of temperature and sodium nitrite concentration. As the storage temperature decreased, the pathogen grew slowly in hams with higher levels of residual sodium nitrite. Growth of L. monocytogenes strain Scott A was detected on cooked chicken loaf stored under air and two modified atmospheres at 3, 7 and 11°C for 6 days (Ingham et al., 1990). Modified atmosphere packaging did not stop the growth of L. monocytogenes, but the microorganism grew better in the presence of oxygen. Similar results were observed in precooked chicken nuggets (Marshall et al., 1991). Buncic et al. (1991) reported growth of L. monocytogenes in vacuum packaged frankfurters stored at 4°C. The number of L. monocytogenes increased about 30-fold after 10 days and 420 tunes after 20 days of storage, but it failed to multiply in fermented sausages at room temperature (18 to 22°C) for 20 days. In fact, the number of L. monocytogenes decreased by approximately 2 logs in the first 11 days of storage and remained the same during the following days. The decrease in pH and water activity during the process, as well as the presence of Lactobacillus spp. were probably responsible for the inhibition of L. monocytogenes. Subsequent tests demonstrated that culture filtrates from strams of L. plantarum (pH 3.42 to 4.52) isolated from the sausage possessed inhibitory activity against L. monocytogenes in agar media (zones of inhibition of
1249 4.5 to 11 mm). Zones of inhibition decreased to diameters of 0.5 to 1.2, however, when the pH of the culture filtrates was adjusted to 7.0 1.4
Effects of additives on growth
1.4.a Effect of pH and water activity Traditionally, L. monocytogenes has been considered to be relatively sensitive to acidic environments below pH 5.5 (Gray and Killinger, 1966). However, several newer studies have revealed that in microbiological media, L. monocytogenes was able to grow at pH levels as low as 4.4 (Buchanan and Phillips, 1990; Conner et al., 1986; Sorrells et al., 1989). Consequently, the presumed minimum pH value for growth of the organism has been adjusted downward. The minimum pH at which L. monocytogenes can grow is, of course, dependent upon the incubation temperature, time, and the type of acidifying agent present in the growth substrate (Ryser and Marth, 1991). Sorrells et al. (1989) studied the effect of pH and other factors on the growth and survival of L. monocytogenes and found that inhibition of the pathogen in the presence of an acidic environment appeared to be a function of the synergism between the type of acid and the incubation temperature. At a given pH value, the authors concluded that the antimicrobial activity of the acids followed the sequence: acetic acid > lactic acid > citric acid > malic acid > hydrochloric acid at all incubation times and temperatures. The most inhibition of L. monocytogenes occurred at 35°C, the most growth was observed at 25°C, and the greatest survival was detected at 10°C. The final pH of the culture medium decreased to 3.8 in the presence of hydrochloric acid after 28 days of incubation. A significant observation made in this study was that L. monocytogenes was able to proliferate at the pH value of 4.4, which is much lower than that which had been previously reported (Gray and Killinger, 1966). In addition, the study may have significance in minimally processed and refrigerated acid or acidified foods, due to the observation that L. monocytogenes was able to tolerate a low pH at temperatures of 10°C. In additional work by Sorrells and Enigl (1990), the effect of pH, acidulant, sodium chloride and temperature on the growth of L. monocytogenes was investigated. The authors determined that the minimum pH/salt level for initiation of growth of the organism ranged from pH of 5.0-5.6 when combined with 8-10% sodium chloride (incubated at 25 and 35°C), to pH of 5.6 when combined with 8% sodium chloride and incubated at 10°C. In this study, L. monocytogenes appeared to persist and be tolerant in a system which combined a low pH, high salt and low temperature environment. Listeria monocytogenes, like most other bacterial species, exhibits optimal growth at a water activity (a^) of approximately 0.97 (Petran and Zottola, 1989). Yet, when compared to other microorganisms, L. monocytogenes also has the rather unique ability to proliferate at a^ values as low as 0.92 (Ryser and Marth, 1991). The effect of a^, however, is variable with different solutes. The minimum a^ values for glycerol, sodium chloride and propylene glycol were 0.90, 0.92 and 0.97, respectively (Miller, 1992).
1250 1.4.b Sodium acetate Sodium acetate is a derivative of acetic acid in the form of a sodium salt. According to the United States Code of Federal Regulations (21; 184.1721), sodium acetate is approved as a generally recognized as safe (GRAS) substance for miscellaneous and general purpose usage. It has been used as a pH control agent, flavoring agent and adjuvant, and shows antimicrobial properties (Doores, 1990). Maximum levels of sodium acetate that are recommended for various food product categories range from 0.007% in breakfast cereal to 0.5% in fats and oils. Some derivatives of acetic acid in the form of calcium, sodium or potassium salts can be substituted for acetic acid in certain formulations (Doores, 1990). One of the primary uses of acetic acid in food has been that of an acidulant, which has excellent antimicrobial properties. This activity has been attributed to lowering of the pH below that needed for optimal growth (Doores, 1990). The effect of organic acids, including acetic acid, on reducing microbial loads on poultry and red meat have been investigated. The decimal reduction times for Salmonella newport, Salmonella typhimurium and Campylobacter jejuni in scald tank water used in poultry processing dropped almost one log cycle after exposure to 0.1% acetic acid at 52°C (Okrend et al., 1986), while addition of either 0.5% or 1.0% acetic acid caused either a 99% reduction in plate counts or inmiediate death of the bacteria, respectively (Lillard et al., 1987; Okrend et al., 1986). Although numerous studies have shown that bacterial numbers can be reduced by 90-99% if beef or pork carcasses or primal cuts are washed and sanitized with 3.0% acetic acid (Anderson et al., 1979; BiemuUer et al., 1973, Cacciarelli et al., 1983; Marchall et al., 1977), few studies exist on the antimicrobial, and particularly the antilisterial, effects of its counterpart salt, sodium acetate, in vacuum-packaged or processed meat products. Mendonca et al. (1989) found that unmersing fresh pork in solutions containing 3.0% acetic acid effectively inhibited growth of enterobacteriaceae, lactobacilli, anaerobes and facultative anaerobes during storage in vacuum packages. Pork chops dipped in solutions containing 1.5% acetic acid/1.5% sodium acetate supported microbial growth significantly lower than controls dipped in sterile water. The authors concluded that surface treatment of beef steaks with a potassium sorbate-phosphate-sodium chloride-sodium acetate solution was very effective in extending their microbiological shelf life in vacuum packages during 12 weeks of storage at 2-4°C (Unda et al., 1990). Roskey and Lachica (1992) found that growth of L. monocytogenes in broth with 0.5% sodium acetate was inhibited at pH of 5.6 and temperatures of less than 35°C, but it proceeded at pH of 6.0. A concentration of 1.0% sodium acetate was required to inhibit L. monocytogenes at pH of 6.0, but it was effective only at temperatures below 15°C. The effectiveness of sodium acetate was not diminished in the presence of a high initial inoculum of L. monocytogenes. In presterilized beef, 0.5% sodium acetate inhibited L. monocytogenes growth, but in presterilized chicken, a 1.0% level of the additive was required for inhibition, presumably because of higher pH and fat content. The authors concluded that 0.5% sodium acetate may be a valuable and appropriate preservative for red meat and luncheon meats. These studies indicate that acetic acid is effective for controlling growth of a wide range of microorganisms in meat and other types of food. Sodium acetate may have potential as an antimicrobial additive for meat, since the salts of acetic acid are expected to have the same antimicrobial properties as the acid, when used at comparable pH levels. Research is needed to evaluate the antimicrobial
1251 and antilisteriai effects of sodium acetate, and to determine if the additive would affect physical and organoleptic properties of meat products. 1.4.C Sodium bicarbonate Sodium bicarbonate is a low-cost, nontoxic food additive which has GRAS status, and is widely used in the food industry at levels of up to 2% as a leavening agent, for pH control, and to contribute to flavor and texture development in food products (Lindsay, 1985). Although data on the antimicrobial properties of sodium bicarbonate are quite limited, the compound is used in dental preparations where it inhibits periodontal bacteria (Cerra and Killoy, 1982; Newbrun et al., 1984; Miyasaki et al., 1986). Montville and Goldstein (1987) found that sodium bicarbonate was effective in reducing survival of Aspergillus parasiticus and altering the distribution of aflatoxin in cells of the mold which did survive. Corral et al. (1988) noted that E. coli, L. plantarum, Staphylococcus aureus and Pseudomonas aeruginosa counts were reduced 10,000-fold by 0.12 M (1.00% w/v) of sodium bicarbonate incorporated into the growth media. Moreover, plate counts for yeasts such as Saccharomyces cerevisiae and Hansenula wingei were reduced 100,000-fold by 0.06 M sodium bicarbonate. Bechtel et al. (1985) examined the effect of substituting sodium bicarbonate for sodium chloride in pork and beef frankfurter formulations with either 2% sodium chloride (control), 1% sodium chloride and 1% sodium bicarbonate, or 2% sodium bicarbonate. Frankfurters were stored vacuum-packaged for 30 days at 4°C, and results at 0, 15 or 30 days revealed no significant differences in total plate counts among treatments. The pH of the sodium chloride control was 6.0, while sodium bicarbonate at levels of 1% and 2% elevated the pH of the frankfurters to 7.5 and 8.2, respectively. Curran et al. (1990) reported that dipping cod fillets in 8.0% sodium bicarbonate or 12% ammonium bicarbonate solution resulted in marked reductions of microbial growth compared to untreated samples after storage at 4°C for 8 days. Total plate counts, proteolytic bacteria and hydrogen sulfide-producing bacteria were inhibited by the test solutions. Treated fish exhibited improved texture and moisture retention, yet also had significantly lower aroma and overall acceptability scores. Therefore, it appears that sodium bicarbonate may have some potential as an antimicrobial food additive, which may be useful in inhibiting the growth of acid tolerant microorganisms such as Lactobacillus spp., proteolytic bacteria and certain yeasts and molds. Since sodium bicarbonate, used at levels of 1.0 to 2.0%, may raise the pH of the food one full pH unit or greater, its uses are limited to certain foods in which this may be a desirable attribute. 1.4.d Sodium lactate Sodium lactate has been recognized in recent years as a food ingredient which enhances sensory properties and inhibits microbial growth. The preservative properties of sodium lactate can be attributed to various mechanisms, including feedback inhibition, intracellular acidulation, interference with proton transfer across the cell membrane and lowering of product water activity (Bacus and Bontenbal, 1991). Natural sodium lactate is commercially produced by fermentation; is considered GRAS by the Food and Drug Administration; and is approved by the U.S. Department of Agriculture for use in meat and poultry products. In addition, sodium lactate serves as a firming agent with humectant
1252 properties. Papadopoulos et al. (1991) recommended use of sodium lactate at levels of 2-3 % (of a 100% solution), based on final weight, to enhance flavor and extend shelf life of fresh and cooked meat and poultry products. Sodium lactate exhibits antimicrobial activity against many foodbome pathogens, including Clostridium botulinum. Maas et al. (1989) reported that incorporation of 2.0 to 3.5% (based on final product) sodium lactate into conmiinuted, raw turkey meat delayed toxin production by C. botulinum in cook-in-bag turkey products. Listeria monocytogenes, Salmonella spp., E. coli and S. aureus, were also reportedly inhibited by lactate (Bacus and Bontenbal, 1991). Shelef and Yang (1991) found that L. monocytogenes could also be inhibited by sodium or potassium lactate in broth, chicken and beef. Studies with tryptic soy broth showed that sodium lactate concentrations higher than 5.0% delayed growth of three strains of L. monocytogenes. Experunents in sterile, conmiinuted chicken and beef at 5°, 20° and 35°C demonstrated suppression of growth by 4.0% lactate, which increased with decrease in storage temperature. Listeria monocytogenes consistently exhibited greater sensitivity to lactate in beef than in chicken, displaying an extended lag phase of 1-2 weeks during storage at 5°C. When 4.0% lactate was combined with 3.0% sodium chloride or 140 ppm sodium nitrite, the antimicrobial effect was not enhanced. The authors also noted that incorporation of potassium or sodium lactate did not alter the pH of the chicken or beef, and no significant difference was observed between the effect of the two salts, inferring that the lactate ion may be responsible for the delay in listerial growth (Shelef and Yang, 1991). Unda et al. (1991) concluded that in temperatureabused (10, 25°C) beef roasts, incorporation of lactate into the brine afforded protection against the survival of Clostridia. Listeria survival was reduced by lactate and monolaurin in recooked surface-inoculated roasts. Research reported by Bacus and Bontenbal (1991) indicated that sodium lactate was effective when used to control growth of L. monocytogenes in both cured and uncured meat and poultry products. Frankfurter treatments formulated without sodium lactate allowed growth of 10^ CFU/g during storage for 46 days at 4°C, while L. monocytogenes counts actually decreased in test formulations containing 2 or 4% sodium lactate. Sodium lactate also extended the shelf life of the product by decreasing growth of the microflora normally associated with frankfurters. Sodium lactate used at 2% and 3% also inhibited for 14 or 21 days growth of L. monocytogenes inoculated postprocessing on uncured cooked chicken rolls (Bacus and Bontenbal, 1991). Chen and Shelef (1992) studied the relationship between water activity, salts of lactic acid and growth of L. monocytogenes in a meat model system consisting of cooked strained beef ranging in moisture content from 25 to 85% (wt/wt). Sodium lactate, when used at a level of 4.0% (based on finished product), suppressed listerial growth at moisture levels of greater than 55% (a^ > 0.964), and inhibited growth in meat with moisture levels of 25-55% (a^ < 0.964). The authors reported that lactate concentrations less than 4.0% were not listeriostatic, but combinations of 2.0 or 3.0% lactate with 2.0% sodium chloride in samples with 55 % moisture did inhibit growth. The potassium and calcium salts of lactic acid were as effective as the sodium salt in suppressing water activity of the meat system, and growth of L. monocytogenes. These studies suggest that incorporation of lactate into meat products may extend shelf life, suppress microbial growth, and even enhance flavor in some cases. Additional studies are needed to establish the optimum usage level for meat products, and to further evaluate the effect of sodium lactate on the microorganisms in meat.
1253 1.4.e Potassium sorbate Sorbic acid and potassium sorbate have been used throughout the food industry to extend the shelf life of many foods, including bakery items, cereals, fruits and vegetables, butter, cheese and meat products (Sofos, 1989). According to reviews by Robach and Sofos (1982) and Sofos and Busta (1981), sorbates can delay growth and toxin production by C botulinum, and extend the shelf life and decrease the growth of other pathogenic microorganisms in products such as bacon, fresh poultry and cooked, cured or fermented red meat and poultry products. Research conducted by El-Shenawy and Marth (1988) examined the effect of potassium sorbate against L. monocytogenes in tryptose broth adjusted to a pH of 5.6 or 5.0, and incubated at various temperatures. At pH 5.6 and storage at 4°C, L. monocytogenes was inactivated by 0.25 or 0.3% potassium sorbate after 66 and 60 days, respectively. The organism grew at all potassium sorbate concentrations (0.005-0.30%) when incubated at 13°C and pH 5.6, but maximum populations were directly related to the additive concentration. According to the data collected in this experiment, the ability of potassium sorbate to prevent growth of L. monocytogenes is directly related to temperature and pH. Ryser and Marth (1988) determined that four strains of L. monocytogenes were eliminated faster from cold-pack cheese food with a pH of 5.45, which contained 0.30% potassium sorbate than from cheese food manufactured at pH 5.2 without preservative. In additional studies, El-Shenawy and Marth (1991) reported that incorporation of organic acids such as acetic, tartaric, lactic or citric acid enhanced the antilisterial activity of potassium sorbate. Unda et al. (1991) examined the effect of selected antimicrobials in combinations including potassium sorbate on the survival and inhibition of L. monocytogenes and C. sporogenes in cook-in-bag beef roasts. Acetic acid- and potassium sorbate-containing brines exhibited significant inhibitory properties against anaerobic and facultative anaerobic bacteria in both refrigerated and temperature-abused beef roasts. These studies indicated that sorbate is a valuable food additive and is an effective inhibitor of several types of microorganisms, including bacteria, yeasts and molds, when used at appropriate levels. More research is needed to understand the mechanisms by which sorbate inhibits these microorganisms, and to examine the effect of sorbate on meat pathogens such as L. monocytogenes. 2.
EXPERIMENTAL
As indicated above, several studies have reported conflicting results relative to the potential for growth of L. monocytogenes in fresh meat. In general, it is not clear whether the pathogen is able to proliferate in uncooked, refrigerated beef, and pH value is among the factors which may be responsible for such variability. Studies with ground beef of pH > 6.0 have detected growth of L. monocytogenes at refrigeration temperatures (Gill and Reichel, 1989; Grau and Vanderlinde, 1990; Kaya and Schmidt, 1991), while at pH values <6.0, no growth of the pathogen has been observed in most of the studies with refrigerated ground beef (Buchanan et al., 1987; Johnson et al., 1988; Shelef, 1989; Buchanan and Klawitter, 1991; Kaya and Schmidt, 1991). In addition to pH, physiological differences in strains may be a factor influencing the results of such studies. It is not clear at this time how important differences in strain behavior may be to the fate of L. monocytogenes in uncooked, refrigerated beef of different pH.
1254 In contrast to fresh beef, listeriae were detected regularly in samples of ready-to-eat processed meats obtained from retail stores (Grau and Vanderlinde, 1992; Johnson et al., 1990). Glass and Doyle (1989) reported growth of L. monocytogenes at 4.4°C on ham, sliced chicken and turkey products, wieners and fresh bratwurst. An important observation from this study was that processed meats with pH values of > 6.0 allowed for better growth of L. monocytogenes than meat products with a lower pH. Thus, the high pH (>6.0) of products from poultry meat, such as turkey bologna and frankfurters, may allow for the survival and growth of L. monocytogenes. If that is the case, then there is a need for inhibition of proliferation of L. monocytogenes in processed poultry products. Studies in our laboratory (Barbosa, 1993; Wederquist, 1993) have investigated the potential for growth of L. monocytogenes in refrigerated vacuum-packaged ground beef as affected by differences in strains and pH, and its potential for growth and inhibition with chemical additives in vacuum-packaged refrigerated turkey bologna-type sausage. 2.1
Fresh ground beef
Fresh ground beef samples from top rounds were selected on the basis of their pH, and were divided into those having an average pH of 5.47, which is normal for fresh beef, and others having an elevated average pH of 6.14. The fat content of all treatments was in the range 2.93-3.22%. The ground beef was formed in patties which were inoculated with one of two strains of L. monocytogenes (Na-16/serotype l/2a or Scott A/serotype 4b). The inocula of each strain were prepared in tryptic soy broth (TSB) containing 0.6% yeast extract (Difco Laboratories, Detroit, MI) at 4.0°C. Individual patties were inoculated with sterile distilled water or with the suspension of each strain at 3-4 log colony forming units - CFU/g. Each ground beef patty (approximately 80 g) was inoculated with 0.1 mL of cell suspension or sterile distilled water. Individual patties were vacuum packaged, stored at 4°C and analyzed at weekly intervals during 42 days of storage. During storage, the patties (two per treatment and sampling time) were analyzed for pH, total aerobic plate counts and L. monocytogenes counts. Each experiment was repeated 2-3 times. Total aerobic plate counts were determined on tryptic soy agar (TSA, Difco). Plates were incubated at 35°C for 48 hours. Counts of L. monocytogenes were determined by plating on lithium chloride-phenyethanol-moxalactam-tellurite (LPMT) agar plates incubated at 35°C for 48 hours. The fat content of the ground beef samples was determined by petroleum ether (Mallinckrodt, Paris, KY) extraction of moisture-free samples according to AOAC procedures (Association of Official Analytical Chemists, 1990). Moisture content was determined by drying under vacuum for 12 hours at 60-65 °C. The pH of the samples was determined on blends consisting of one part of meat and three parts of phosphate buffer solution with a Coming flat combination electrode (Coming Glasswork, Medfield, MA). Each experiment was repeated 2-4 times. 2.2
Turkey bologna sausage
A typical turkey bologna sausage product was formulated to contain 7.464 kg of mechanically deboned turkey meat, 0.908 kg of water, 181.6 g of dextrose, 181.6 g of com symp solids, 181.6 g of sodium chloride, 81.7 g of dry mustard, 36.3 g of phosphate, 1.416 g of sodium nitrite, 4.54 g of sodium erythorbate, 22.7 g of paprika, 4.54 g of onion
1255 powder, 4.54 g of garlic powder, 4.54 g of coriander and 4.54 g of white pepper. In addition to the control, experimental treatments were formulated to contain chemical additives, which included 0.5% sodium acetate, 1.0% sodium bicarbonate, 2.0% sodium lactate and 0.26% potassium sorbate. Raw, mechanically deboned turkey meat (MDTM) and the other ingredients were weighed in 9.08 kg batches which were mixed and chopped in a Meissner bowl chopper. The bologna emulsions were extracted into 7.62-cm diameter fibrous cellulose casings (Koch, Kansas City, MO), which were weighed and cooked in a smokehouse to an internal temperature of 70°C. The bologna sausage was then cooled by showering with cold water and stored at 4°C. After it was chilled for 12 hours, the bologna was peeled to remove the casings, and sliced (4-5 mm thick). Two bologna slices (30 g each) at a time were inoculated with 0.1 mL of inoculum dispensed in the center between the slices and spread evenly across the surface with a sterile bent glass rod. The inoculum consisted of a composite of seven strains (four serotype 1/2 and three serotype 4) prepared at 35°C in TSB. The two slices were then wrapped in plastic wrap, vacuum packaged at 120 mmHg and analyzed during storage at 4°C for 98 days. Two samples per treatment and sampling time were analyzed for L. monocytogenes counts by plating on TSA, incubated at 35 °C for 48 hours. The experiment was repeated two times. The pH of the bologna samples was measured in duplicate, at each weekly plating interval, similar to raw fresh beef. Moisture, fat and protein contents were determined according to procedures described above. 3.
RESULTS AND DISCUSSION
3.1
Raw fresh beef
Overall, there was a slight decrease in the pH of normal (5.47-5.48) initial pH ground beef patties during storage at 4°C, but no major differences were detected among treatments inoculated with different strains of L. monocytogenes (Table 1). Ground beef patties of high initial (6.12-6.14) pH showed no major changes in pH during storage at 4°C. Immediately after inoculation, the counts of L. monocytogenes of both strains in patties of normal and high pH were similar (Table 2). With storage in vacuum packages at 4°C, certain changes were detected in counts of L. monocytogenes. The changes, however, depended on L. monocytogenes strain and product pH. In normal pH ground beef, counts of strain Na-16 increased by approximately 1 log CFU/g, while those of strain Scott A decreased slightly. In ground beef patties of high initial pH, L. monocytogenes counts of strain Na-16 increased by more than 2 log CFU/g during the 42 days of storage, while those of strain Scott A increased very little ( < 1 log CFU/g). Total aerobic mesophilic plate counts were similar within each pH group of ground beef patties inoculated with L. monocytogenes (Table 3). In ground beef of normal initial pH, growth was similar (approximately 2 log CFU/g) irrespective of strain of L. monocytogenes inoculated in the ground beef. In high pH ground beef, growth was higher (approximately 4 log CFU/g) compared to normal pH ground beef, but still similar between strains of L. monocytogenes (Barbosa et al., 1994).
1256 Table 1 Changes in pH of ground beef patties of normal and high initial pH inoculated with each of two strains of Listeria monocytogenes (Na-16 and Scott A) during storage in vacuum packages at 4°C Normal initial pH
High initial pH
Days of storage (4°C)
Na-16
Scott A
Na-16
Scott A
0 7 14 21 28 35 42
5.48 5.49 5.58 5.43 5.39 5.36 5.31
5.47 5.54 5.47 5.35 5.47 5.25 5.45
6.14 6.15 6.08 6.16 6.16 6.29 6.20
6.12 6.24 6.13 6.09 6.15 6.26 6.19
Table 2 Counts (log CFU/g) of two Listeria monocytogenes strains (Na-16 and Scott A) inoculated in ground beef patties of normal and high initial pH, and stored vacuum-packaged at 4°C Normal initial pH Days of storage (4°C) 0 7 14 21 28 35 42 CFU: colony formmg umts.
High initial pH
Na-16
Scott A
Na-16
Scott A
4.23 4.26 5.30 5.10 5.48 5.34 4.63
4.00 3.01 3.43 3.37 3.53 2.94 3.67
4.51 4.54 5.61 6.38 6.75 6.80 7.06
3.58 2.60 3.88 4.05 4.30 4.39 3.42
Overall, multiplication of L. monocytogenes in vacuum-packaged ground beef stored at 4°C was very slow, while the spoilage or total aerobic bacterial flora multiplied faster and reached higher numbers than L. monocytogenes in ground beef of either normal or high pH. Growth of both, L. monocytogenes and total aerobic bacteria, however, was different depending on ground beef pH and strain of inoculum. When the pH of ground beef was greater than 6.0, multiplication of bacteria, including L. monocytogenes, was rapid and extensive. At lower pH, growth was less extensive, especially for L. monocytogenes. Actually, in normal pH ground beef, strain Scott A of L. monocytogenes decreased in numbers during storage, while strain Na-16 increased only slightly. Even in high pH ground beef, growth of strain Scott A was limited. Therefore, growth of L. monocytogenes in vacuum-packaged ground beef was influenced by strain of inoculum and pH of meat. Strain Scott A, which is used extensively in research, multiplied very slowly or not at all, while strain Na-16 multiplied faster, especially when the ground beef pH value exceeded 6.0.
1257 Overall, L. monocytogenes either did not multiply or multiplied very slowly in vacuum packaged ground beef of normal initial pH (5.47) at 4°C, but growth was more extensive in ground beef of high initial pH (6.14). Table 3 Total aerobic plate counts (log CFU/g) in ground beef patties of normal and high initial pH inoculated with two strains (Na-16, Scott A) of Listeria monocytogenes, and stored vacuumpackaged at 4°C Normal initial pH Days of storage (4°C) 0 7 14 21 28 35 42 CFU: colony forming units. 3.2
High initial pH
Na-16
Scott A
Na-16
Scott A
4.74 5.34 6.27 5.99 6.60 6.63 6.39
4.21 4.55 5.90 6.05 6.04 6.21 6.60
4.79 7.02 7.49 7.89 7.98 8.56 8.62
4.41 6.05 6.88 7.47 7.43 8.04 8.42
Turkey bologna sausage
The initial pH value of 6.58 for the control turkey bologna product increased with added chemicals, especially with sodium bicarbonate (Table 4). With storage in vacuum packages at 4°C for 98 days, the pH of all treatments decreased gradually but still remained above 6.0. These pH values should not be restrictive to L. monocytogenes growth (Wederquist et al., 1994). Table 4 Changes in pH of turkey bologna sausage formulated with various additives, inoculated with Listeria monocytogenes after processing and stored vacuum packaged at 4°C Days of storage (4°C) 0 14 28 42 56 70 84 98
Control 6.58 6.50 6.53 6.48 6.44 6.26 6.13 6.11
Sodium acetate (0.5%)
Sodium bicarbonate (1.0%)
Sodium lactate (2.0%)
Potassium sorbate (0.26%)
6.63 6.54 6.62 6.49 6.50 6.41 6.25 5.88
7.59 7.46 7.59 7.51 7.43 7.29 7.14 7.07
6.73 6.65 6.70 6.58 6.48 6.52 6.43 6.35
6.59 6.48 6.58 6.49 6.51 6.48 6.39 6.30
1258 Growth (Table 5) of L. monocytogenes in the vacuum-packaged turkey bologna sausage without additives (control) was very rapid, reaching 5.79 log CFU/g by 28 days of storage (4°C). After 42 days of storage, growth had exceeded 7 log CFU/g. Thus, in this product, which was cooked and of high pH, growth of L. monocytogenes inoculated on cooked slices after processing was very rapid and extensive, compared to fresh raw ground beef of lower pH (Table 2). Therefore, there is a need for inhibition of L. monocytogenes growth in cooked sausage products. Table 5 Counts (log CFU/g) of Listeria monocytogenes inoculated after processing in turkey bologna sausage which was formulated with various additives and stored vacuum packaged at 4°C Days of storage (4°C)
Control
0 2.59 14 4.49 28 5.79 42 7.86 56 8.88 70 8.71 84 8.43 98 8.51 CFU: colony formmg umts.
Sodium acetate (0.5%)
Sodium bicarbonate (1.0%)
Sodium lactate (2.0%)
Potassium sorbate (0.26%)
2.47 3.14 3.31 3.14 3.69 3.80 3.19 3.25
2.31 4.19 6.76 7.60 8.23 9.09 8.53 8.62
2.46 2.35 2.09 3.24 3.60 4.36 4.07 4.97
2.18 2.77 3.04 3.14 3.19 3.74 4.25 4.26
Of the four additives and at the concentrations tested, sodium acetate, sodium lactate and potassium sorbate were effective, while sodium bicarbonate, which increased the pH to above 7.0, was ineffective in inhibiting L. monocytogenes inoculated in turkey bologna after heat processing (Table 5). Counts of L. monocytogenes during the 98-day period increased by 1.33, 2.51 and 2.08 log CFU/g in treatments with acetate, lactate and sorbate, respectively. Growth in the control and bicarbonate treatments was 6.29 and 6.78 log CFU/g, respectively. Changes in total aerobic plate counts (Table 6) were similar to those forL. monocytogenes. The results show the potential for extensive growth of L. monocytogenes in turkey bologna sausage contaminated post-processing. The results also indicated that growth of L. monocytogenes in vacuum packaged refrigerated turkey bologna may be significantly reduced through inclusion in the formulation of 0.5% sodium acetate, 2.0% sodium lactate or 0.26% potassium sorbate. 4.
CONCLUSIONS
Growth of L. monocytogenes in ground top rounds of beef stored under vacuum at 4°C depended on strain of the pathogen and initial pH of the meat. In meat of pH less than 6.0, growth was limited, with one strain of L. monocytogenes multiplying slightly and the second decreasing somewhat in counts during storage. In ground beef of pH above 6.0,
1259 growth of the pathogen was more extensive, but still depended on strain. The commonly studied strain Scott A multiplied only slightly, while strain Na-16 increased by more than 2 log CFU/g during the 42 days of storage. Overall, however, growth of L. monocytogenes in refrigerated ground beef is limited, compared to total aerobic mesophilic bacteria. The limited growth of L. monocytogenes, especially of strain Scott A and at the lower pH, may have been due to competing microbial flora producing inhibitors such as bacteriocins. Buchanan and Klawitter (1992a,b) have reported on a strain of Camobacterium piscicola LK5 that was isolated from ground beef and inhibited L. monocytogenes strain Scott A in refrigerated foods. Meat pH values lower than 6.0 select for growth of lactic acid producing bacteria. Table 6 Total aerobic plate counts (log CFU/g) in turkey bologna sausage formulated with various additives and stored vacuum-packaged at 4°C Days of storage (4°C)
Control
0 2.75 14 4.52 28 5.80 42 7.88 8.96 56 70 8.75 84 8.52 98 8.59 CPU: colony forming units.
Sodium acetate (0.5%)
Sodium bicarbonate (1.0%)
Sodium lactate (2.0%)
Potassium sorbate (0.26%)
2.58 3.21 3.71 3.32 3.75 4.05 3.66 4.38
2.35 4.29 6.79 7.74 8.27 9.16 8.74 8.70
2.49 2.43 3.10 3.30 4.04 4.43 4.12 5.08
2.44 2.97 3.16 3.23 3.21 3.86 4.38 4.35
In contrast, growth of L. monocytogenes inoculated in cooked turkey bologna sausage after slicing was very rapid and extensive during storage in vacuum packages at 4°C. However, this growth was greatly inhibited by additives such as sodium acetate, sodium lactate and potassium sorbate. Sodium bicarbonate increased product pH and did not inhibit growth of L. monocytogenes. It should be noted that in addition to being cooked, the turkey bologna was different than ground beef in that its pH exceeded the value of 6.5. 5.
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1260 AOAC. Meat and meat products. In: K. Helrich (Ed.). Official Methods of Analysis of the Association of Food Chemists. Vol. 2, 15th Ed. Assoc, of Offic. Analyt. Chem., Inc., Arlington, VA. p. 931, 1990. Bacus, J. and E. Bontenbal, Meat and Poultry 37(6) (1991) 64-69. Barbosa, W.B. Fate of different species and strains of Listeria in broth and vacuum packaged ground beef. M.S. Thesis, Colorado State University, Fort Collins, CO, 1993. Barbosa, W.B., J.N. Sofos, G.R. Schmidt and G.C. Smith, J. Food Prot. (1994) (in press). Bechtel, P.J., F.K. McKeith, S.E. Martin, E.J. Basgall and J.E. Movakofski, Food Prot. 48 (1985) 861-864. BiemuUer, G.W., J.A. Carpenter and A.E. Reynolds, J. Food Sci., 38 (1973) 261-264. Buchanan, R.L. and J.G. Phillips, J. Food Prot., 53 (1990) 370-376, 381. Buchanan, R.L. and L.A. Klawitter, Int. J. Food Microbiol., 12 (1991) 235-246. Buchanan, R.L. and L.A. Klawitter, J. Food Safety, 12 (1992a) 199-217. Buchanan, R.L. and L.A. Klawitter, J. Food Safety, 12 (1992b) 219-236. Buchanan, R.L., H.G. Stahl and D.L. Archer, Food Microbiol., 4 (1987) 269-275. Buncic, S., L. Paunovic and D. Radisic, J. Food Prot., 54 (1991) 413-417. Cacciarelli, M.A., W.C. Stringer, M.E. Anderson and H.D. Naumann, J. Food Prot., 46 (1983) 231-232. Cerra, M.B. and W.J. Killoy, J. Periodont., 53 (1982) 599. Chen, N. and L.A. Shelef, J. Food Prot., 55 (1992) 574-578. Chung, K-T., J.S. Dickson and J.D. Crouse, J. Food Prot., 52 (1989) 173-177. Conner, D.E., R.E. Brackett and L.R. Beuchat, Appl. Envir. Microbiol., 52 (1986) 59-63. Corral, L.G., L.S. Post and T.J. Montville, J. Food Sci., 53 (1988) 981-982. Curran, D.K., B.J. Tepper and T.J. Montville, J. Food Sci., 55 (1990) 1564-1566. Dickson, J., J. Food Safety, 10 (1990) 165-174.
1261 Doores, S. pH control agents and acidulants. In: Food Additives. A.L. Branen, M. Davidson and S. Salminen (Eds.). Marcel Dekker, Inc. New York, NY pp. 447488, 1990. Doyle, M.P., Food TechnoL, 42(4) (1988) 169-171. El-Shenawy, M.A. and E.H. Marth, J. Food. Prot., 51 (1988) 842-847. El-Shenawy, M.A. and E.H. Marth, J. Food Prot., 54 (1991) 593-597. Fain, A.R. Jr., I.E. Line, A.B. Moran, L.M. Martin, R.V. Lechowich, J.M. Carosella and W.L. Brown, J. Food Prot., 54 (1991) 756-761. Gellin, B.G. and C.V. Broome, J. Am. Med. Assoc, 261 (1989) 1313-1320. Genigeorgis, C.A., D. Dutulescu and J.F. Garayzabal, J. Food Prot., 52 (1989) 618-624. Genigeorgis, C.A., P. Oanca and D. Dutulescu, J. Food Prot., 53 (1990) 282-288. Gill, C O . and M.P. Reichel, Food Microbiol., 6 (1989) 223-230. Glass, K.A. and M.P. Doyle, Appl. Environ. Microbiol., 55 (1989) 1565-1569. Gobat, P-F. and T. Jemmi, Fleischwirtsch., 70 (1990) 1448-1450. Gouet, P.H., J. Labadie and C. Serratore, Zbl. Bakt. Hyg., 166 (1978) 87-94. Grau, E.H. and P.B. Vanderlinde, J. Food Prot., 53 (1990) 739-741. Grau, E.H. and P.B. Vanderlinde, J. Food Prot., 55 (1992) 4-7. Gray, M.L. and A.H. Killinger, Bacteriol. Rev., 30 (1966) 309-382. Harrison, M.A., Y. Huang, C. Chao and T. Shineman, J. Food Prot., 54 (1991) 524-527. Hart, C D . , G.C Mead and A.P. Norris, J. Appl. Bacteriol., 70 (1991) 40-46. Ingham, S.C, J.M. Escude and P. McCown, J. Food Prot., 53 (1990) 289-291. Johnson, J.L., M.P. Doyle and R.G. Cassens, Int. J. Food Microbiol., 6 (1988) 243-247. Johnson, J.L., M.P. Doyle and R.G. Cassens, J. Food Prot., 53 (1990) 81-91. Juntilla, J.R., S.I. Niemela and J. Him, J. Appl. Bacteriol., 65 (1988) 321-327.
1262 Kallander, K.D., A.D. Hitchins, G.A. Lancette, J.A. Schmieg, G.R. Garcia, H.M. Solomon and J.N. Sofos, J. Food Prot., 54 (1991) 302-304. Kaya, M. and U. Schmidt, Fleischwirtsch., 71 (1991) 424-426. Lillard, H.S., L.C. Blankenship, J. A. Dickens, S.E. Craven and A.D. Shackelford, J. Food Prot, 50 (1987) 112-114. Lindsay, R.C. Food Additives. Ch. 10. In: Food Chemistry. O.R. Fennema (Ed.). Marcel Dekker, Inc., New York, NY. pp. 632-675, 1985. Lowry, P.D. and I. Tiong. The incidence of Listeria monocytogenes in meat and meat products: Factors affecting distribution. In: Proc. 34th Int. Congress Meat Sci. Technol., Part B. pp. 528-530, 1988. Maas, M.R., K.A. Glass and M.P. Doyle, Appl. Environ. Microbiol., 55 (1989) 22262229. Marchall, R.T., M.E. Anderson, H.D. Naumann and W.C. Stringer. J. Food Prot., 40 (1977) 246-247. Mendonca, A.F., R.A. Molins, A.A. Kraft and H.W. Walker, J. Food Sci., 54 (1989) 1821. Miller, A.J., J. Food Prot., 55 (1992) 414-418. Miyasaki, K.T., R.J. Genco and M.E. Wilson, J. Dent. Res., 65 (1986) 1142. Montville, T.J. and P.K. Goldstein, Appl. Environ. Microbiol., 53 (1987) 2303. Newbrun, E., C.I. Hoover and M.I. Ryder, J. Periodontol., 55 (1984) 658-659. Okrend, A.J., R.W. Johnston and A.B. Moran, J. Food Prot., 49 (1986) 500-503. Palumbo, S.A. and A.C. Williams, Food Microbiol., 8 (1991) 63-68. Papadopoulos, L.S., R.K. Miller, G.R. Acuff, C. Vanderzant and H.R. Cross, J. Food Sci., 56 (1991) 341-347. Petran, R.L. and E.A. Zottola, J. Food Sci., 54 (1989) 458-460. Pinner, R.W., A. Schuchat, B. Swaminathan, P.S. Hayes, K.A. Deaver, R.E. Weaver, B.D. Plikaytis, M. Reeves, C.V. Broome, J.D. Wenger and the Listeria Study Group, JAMA, 267(15) (1992) 2046-2050.
1263 Pociecha, J.Z., K.R. Smith and G.J. Manderson, Int. J. Food Microbiol., 13 (1991) 321328. Robach, M.C. and J.N. Sofos, J. Food Prot., 45 (1982) 374-383. Roskey, C.T. and R.V. Lachica. Effect of sodium acetate and sodium lactate on the growth of Listeria monocytogenes in broth and in meat. Institute of Food Technologists Annual Meeting, New Orleans, LA. Abstr. No. 658, 1992. Ryser, E.T. and E.H. Marth, J. Food Prot., 51 (1988) 615-621, 625. Ryser, E.T. and E.H. Marth. Listeria, Listeriosis and Food Safety. Marcel Dekker, Inc. New York, NY, 1991. Schuchat, A., B. Swaminathan and C.V. Broome, Clin. Microbiol. Rev., 4(2) (1991) 169183. Schuchat, A., K.A. Deaver, J.D. Wenger, B.D. Plikaytis, L. Mascola, R.W. Pinner, A.L. Reingold, C.V. Broome and the Listeria Study Group, JAMA, 267(15) (1992) 20412045. Shelef, L.A.. J. Food Prot., 52 (1989) 379-383. Shelef, L.A. and Q. Yang, J. Food Prot., 54 (1991) 283-287. Sofos, J.N. Sorbate Food Preservatives. CRC Press, Inc. Boca Raton, FL, 1989. Sofos, J.N. Listeria monocytogenes and its fate in meat products. In: G. Charalambous (Ed.). Food Science and Human Nutrition. Elsevier Science Publishers. New York, NY. pp. 743-760, 1992. Sofos, J.N. and F.F. Busta, J. Food Prot., 44 (1991) 614-622. Sorrels, K.M. and D.C. Enigl, J. Food Safety, 11 (1990) 31-37. Sorrells, K.M., D.C. Enigl and J.R. Hatfield, J. Food Prot., 52 (1989) 571-573. Unda, J.R., R.A. Molins and H.W. Walker, J. Food Sci., 55 (1990) 323-326. Unda, J.R., R.A. Molins and H.W. Walker, J. Food Sci., 56 (1991) 198-205. Varabioff, Y., G.E. Mitchell and S.M. Nottingham, J. Food Prot., 55 (1992) 389-391. Walker, S.J., P. Archer and J.G. Banks, J. Appl. Bacteriol., 68 (1990) 157-162.
1264 Wederquist, H.J. Thermal destruction of Listeria monocytogenes in turkey bologna. M.S. Thesis, Colorado State University, Fort CoUms, CO, 1993. Wederquist, H.J., J.N. Sofos and G.R. Schmidt, J. Food Sci., (in press), 1994. Whnpfheimer, L., N.S. Altmanand J.H. Hotchkiss, Int. J. Food Microbiol., 11 (1990) 205214. Yen, L.C., J.N. Sofos and G.R. Schmidt, J. Food Prot., 54 (1991) 408-412. Yen, L.C., J.N. Sofos and G.R. Schmidt, Lebensm. -Wiss. u. -TechnoL, 25 (1992) 61-65. Zeitoun, A.A.M. and J.M. Debevere, Int. J. Food Microbiol., 14 (1991) 161-170.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1265
Quality of extrusion-cooked poultry meat products J.N. Sofos, A.S. Ba-Jaber, G.R. Schmidt and J.A. Maga Department of Animal Sciences and Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523, U.S.A. Abstract A series of studies in our laboratory have examined the feasibility of extrusion cooking of hand-deboned chicken meat (HDCM) and mechanically-deboned turkey meat (MDTM) combinations in a single screw extruder with various nonmeat binders. The results are useful in the selection of binders, meat combinations and concentrations that have the potential for use in the production of acceptable products. The studies reported here evaluated the twenty most acceptable product formulations, in terms of binding and texture, for their tenderness, juiciness, flavor and overall satisfaction. The eight most acceptable formulations were then evaluated for the effect of boiling on their cooking yield, proximate composition and appearance. Products with 20-60% MDTM (80-40% HDCM), 15-22% soy protein isolate (SPI) and 0.5-1.0% kappa-carrageenan (K-C), were the most acceptable, while those with 80-100% MDTM were the least acceptable. The average fat level of the boiled products was 1.3-10.3% and their protein content 24.5-33.4%. Therefore, it is possible to produce acceptable and nutritious extruded products from low cost meat when combined with appropriate binders. 1.
INTRODUCTION
Extrusion cooking of meat is difficult due to its high moisture content and the presence of fat. These ingredients result in product gushing out of the die in small granules or backflowing of the raw material from the extruder inlet (Megard et al., 1985; Ba-Jaber et al., 1992a; Sofos et al., 1992). These problems are caused because fat and moisture act as lubricants of the extruder die and screw resulting in product slippage or blockage of the die. Therefore, any material that exits the die is of limited cohesion, crumbly and friable. These problems may be avoided by drying or defatting the meat before extrusion, and/or mixing with nonmeat binders and gelling agents, such as soybean proteins, starches, wheat flour, potato products and gums. Use of nonmeat ingredients in extruded meat products eliminates the above technical problems; improves binding and texture; and reduces their total fat, while increasing protein contents. Several studies have indicated that, with various modifications, extrusion cooking of muscle tissues is possible and may be useful in upgrading animal products and by-products (Lawrie and Ledward, 1983; Areas and Lawrie, 1984; Kristensen et al., 1984; Alvarez et al., 1990).
1266 Mechanically deboned meat is an underutilized, economical source of animal protein, which was restructured with a twin screw extruder in the presence of gelling and binding agents such as soy protein isolates, wheat flour, com starch, egg white concentrate, carrageenan, sodium chloride, sodium phosphate, sodium alginate and calcium chloride (Megard et al., 1985). Another study on restructuring mechanically deboned chicken meat with a twin-screw extruder found that soy protein isolate and wheat gluten were less effective than starch for increasing apparent tensile stress and Wamer-Bratzler shear stress of extruded products. These parameters also increased as a function of temperature up to 104°C (Alvarez etal., 1990). Several other studies have been published and deal with development of meat-containing products, including low-fat snacks, processed by extrusion cooking, with nonmeat ingredients such as com products (Clarke et al., 1989; Chung et al, 1989; Tarte et al., 1987), modified potato starch and hydrolyzed vegetable proteins (Thomas et al., 1989), gums, maltodextrins and soybean proteins (Chung et al., 1989). Meats tested include beef, pork and mechanically deboned turkey meat, with nonmeat ingredient levels as high as 50-60% (Clarke et al., 1989) and meat up to 10% in the dehydrated state (Thomas et al., 1989). Other studies have used similar principles to develop extrusion cooked products based on raw materials from fish (Suzuki et al., 1988; Sasamoto et al., 1989; Bhattacharya et al., 1988). Texturized blends of meat proteins and com starch were formed with a twin screw extmder at various feeding rates, temperatures, shear rates and pH values (Chakraborty et al., 1987). The results indicated that lower extrusion temperatures and higher pH values produced extmdates of better quality. Ground meat with 20-40% moisture was extrusion processed to form an expanded meat product (Hale, 1970). Extrusion of meat mixed with plant materials can be used to produce meat-based snacks, chunks or powders for soups or stews, and pasta products fortified with meat. It should be mentioned again that these products are based on lower value raw meat; are of low fat; have no added sodium chloride; are of high protein contents; and if they can be made shelf-stable they should be very useful in development of value-added items which should increase overall consumption of meat by providing customers with nutritious, safe and reasonably priced food items. Both soy protein isolate (SPI) and mechanically-deboned meat have characteristic offflavors which may affect the acceptability of the final product. The off-flavor of the soy protein, which has been described as "beany" or "cereal-like" (Southard, 1985), had a significant effect in reducing the acceptability, especially when used at high levels (25%) in beef patties. Extruded products examined in previous studies in our laboratory contained both mechanically deboned turkey meat (MDTM) and SPI (Ba-Jaber et al., 1992a,b,c; Sofos et al., 1992). These products had no added salt or spices, factors which can significantly improve the acceptability of meat products (Sofos, 1983a,b; Sofos et al., 1977; Southard, 1985). The objective of this study was to evaluate the eating quality of extrusion cooked products formulated with MDTM, hand-deboned chicken meat (HDCM), SPI, kappacarrageenan (K-C) gum and oat fiber. Twenty combinations of these ingredients yielding products of acceptable particle binding and texture were evaluated for their juiciness, tendemess, flavor and overall satisfaction. The effect of adding salt to the eating quality of the products was also evaluated. Then the eight most acceptable formulations were evaluated for the effect of boiling on composition and quality.
1267 2.
EXPERIMENTAL
2.1 Product formulations The formulations found to possess acceptable particle binding and textural properties (Ba Jaber, 1990) and which were evaluated in this study are presented in Table 1. The table also shows the extrusion parameters of temperature and extruder screw speed used. The MDTM and the corresponding HDCM amounts tested were in the range of 0% to 100%. The SPI levels ranged from 14% to 26%, while those of K-C were in the range of 0% to 1.0%, and oat fiber 0% to 2%. The extrusion temperature ranged between 45°C and 105°C, and the extruder screw speed between 60 rpm and 100 rpm. Following this study, the eight most acceptable formations in terms of binding, texture and eating quality, which are shown in Table 2, were evaluated for the effect of boiling on their composition and quality. The MDTM and HDCM levels tested were 0-100%; the SPI levels 15-24%; the K-C 0-1%; and the oat fiber 0-2%. The extrusion temperature was either 95°C or 105°C, and the extrusion screw speed 100 rpm, with one exception, which was 80 rpm. Table 1 The extrusion variables and formulations of the 20 treatments of extruded products prepared for eating quality evaluation
Treatment
MDTM: HDCM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MDTM:HDCM = SPI = Soy protein
SPI (%)
Extrusion temperature (°C)
Extrusion screw speed (rpm)
K-C gum (%)
24 100:0 95 100 0.6 22 95 100 0.6 100:0 22 95 100 0.9 100:0 16 95 80 0.0 10:90 20:80 22 95 60 0.0 22 95 80 0.0 60:40 22 105 60 0.0 60:40 0.0 20:80 21 95 100 0.5 60:40 22 105 100 1.0 60:40 22 95 100 24 0.0 60:40 105 100 24 0.5 60:40 95 100 0.0 22 105 100 80:20 0.5 22 95 100 80:20 24 0.0 95 100 80:20 24 105 1.0 80:20 100 0.5 26 95 100 100:0 0.5 12 95 0:100 100 15 0.0 0:100 95 100 14 0.0 0:100 95 100 Mechanically-deboned turkey meat/hand-deboned chicken meat isolate; K-C gum = Kappa-carrageenan gum.
Oat fiber (%) 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
1268 Table 2 The extrusion variables and formulations of the 8 selected treatments to study the effects of cooking on the quality of the extruded products
Treatment 1 2 3 4 5 6 7 8
MDTMiHDCM
SPI (%)
60:40 20:80 60:40 60:40 80:20 80:20 0:100 100:0
22 20 22 22 22 24 15 22
Extrusion temperature (°C) 95 95 105 95 95 105 95 95
Extrusion screw speed (rpm) 80 100 100 100 100 100 100 100
K-C gum (%)
Oat fiber (%)
0.0 0.0 0.5 1.0 0.5 1.0 0.0 0.6
0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0
MDTM:HDCM = Mechanically-deboned turkey meat/hand-deboned chicken meat. SPI = Soy protein isolate. K-C gum = Kappa-carrageenan gum. The hand-deboned chicken meat used was derived from broilers purchased from a supermarket. The chicken meat was skinned, deboned and cut. Then it was ground through a plate with 0.3-cm diameter holes in a Univex grinder (#PB2, Univex Co., Somerville, NC) and mixed at low speed for one minute in a laboratory mixer (model K45SS, Hobart Mfg. Co., St. Joseph, MO). Next, the nonmeat ingredients were slowly added during low-speed mixing to avoid spattering. The mixing of the meat and nonmeat ingredients was continued at low speed for 5 min, after which the mixing process was stopped. All of the nonmeat ingredients on the sides of the bowl were pushed down with a plastic spatula. Mixing was then resumed for another 8-10 min until the ingredients became uniform and no lumps appeared in the dough. The dough was then removed from the mixer bowl and divided into two portions. Each portion was placed in a plastic bag, labeled A and B, and refrigerated (4°C) until it was extruded later on the same day. Extrusion was performed in a Brabender plasticoder extruder, model PL-V500 (C.W. Brabender Instruments, Inc., South Hackensack, NJ). The diameter of the barrel of the extruder was 19.00 mm, with a 20:1 length-to-diameter ratio and eight 0.79 x 3.18 mm longitudinal grooves. A screw configuration of 1:1 compression ratio was used, and the die plate used was 5.10 cm long with a 0.87-cm diameter opening. The two zones of the extruder were electrically heated and compressed air-cooled collars controlled by thermostats were used to control the temperature of the barrel. Two thermocouples were placed through the barrel wall and indicated the temperature of the dough in the extruder (Likimani et al., 1990, 1991). The extruder was equipped with a variable speed D-C drive unit, a tachometer and a balanced-type torque meter (model 7540, Eaton Corporation, Troy, MI). Ground, moistened com grits were choke-fed into the extruder until a steady state of operation was achieved. Samples of the mixtures from bag A were then fed manually and continuously into the extruder. Ground, moistened com was fed between treatments. After
1269 the A samples were extruded, the B samples were extruded in the same mamier. Each sample was collected from the die outlet in a plastic cup and transferred to a plastic bag. The extruded samples were allowed to cool for approximately 30 min and were stored for one day in the refrigerator (4°C) for subsequent evaluation. Observations on the behavior of the products during extrusion were recorded. 2.2 Sensory evaluation Seven experienced judges from the Department of Food Science and Human Nutrition at Colorado State University were asked to evaluate the product sensory quality parameters of juiciness, tenderness, flavor and overall satisfaction, based on 8-point hedonic scales. For juiciness, the score of 8 represented extremely juicy, and 1 extremely dry; for tenderness, the score of 8 was for products that were extremely tender and 1 for products that were extremely tough; for flavor and overall satisfaction, the score of 8 was like extremely and 1 dislike extremely. The panelists were asked to evaluate the products in two sessions on two consecutive days. The evaluation of the products was done on the second and third day after extrusion. On the day of evaluation, the products were taken from the refrigerator and allowed to reach room temperature (1-1.5 hours). An amount of approximately 1 kg from each treatment was cut into pieces of uniform shape (1.5-2 cm length). Each of the samples was then cooked separately in boiling water for 12 min (treatment 19 was also cooked in 2.5% salted water for 12 min). Each cooked sample was transferred to a plastic cup and again allowed to reach room temperature. The panelists were served in succession a 5- to 10-gram sample from each treatment on a plastic plate. Between each taste test of the samples, the panelists rinsed their mouth with cold water and cleansed their palates with cookies. 2.3 Effect of boiling As indicated, the treatments resulting in the most acceptable extruded products in terms of binding and eating quality from the above study were chosen for further study (Table 2). Samples (10 g) of extruded product from each of the eight treatments (Table 2) were weighed at room temperature and put into boiling water and allowed to cook for 5, 10 or 15 min. After cooking, they were taken out of the boiling water and drained, reweighed while hot, and then allowed to reach room temperature (approximately 10 min). Then the samples were packaged and stored in the refrigerator until the time of proximate analysis. 2.4 Proximate analysis and pH The proximate composition and the pH of the products were determined before and after extrusion. Moisture and fat were determined on a 5-7 gram sample which was weighed inside a fat extraction thimble and heated in a vacuum oven (model 5851, National Appliance Co., Portland, OR) for 24 hours at 65°C. The dry sample was then allowed to cool in a dessicator for 30 min before reweighing. The fat content of the dry sample was then determined by pentane extraction using the Soxtec #1043 extraction unit (Tecator, Inc., Herdon, VA). For protein determination, a 2-gram sample was weighed and analyzed with the Tecator system (Tecator, Inc., Hemdon, VA) for Micro-Kjeldahl nitrogen determination (% protein = N x 6.25). The pH of the products was determined by blending a 10-gram sample with 40 grams of distilled, deionized water for 1 min using an Osterizer Galaxie
1270 blender (Oster Corp., Milwaukee, WI). The pH was then determmed with a Coming pH meter 124 (Coming Glass Works, Medfield, MA). 2.5 Statistical analysis The results were analyzed by analysis of variance (Steel and Torrie, 1980) in order to determine significance of differences among treatments in product juiciness, tendemess, flavor and overall satisfaction. Analysis of variance was also used to determine the effect of salt on the acceptability of the final products. Means were separated with Duncan's procedure. 3. RESULTS AND DISCUSSION The results of the first experiment showed significant (P<0.05) panelist effects on juiciness, tendemess, flavor and overall satisfaction. But among the 20 extruded products evaluated, the treatment differences were not significant (P>0.05) in terms of flavor and overall satisfaction. However, a significant difference (P<0.05) in juiciness and tendemess was noted (Table 3). In general, the results showed that the higher the MDTM and/or SPI level, the less acceptable were the products (Table 4). Table 3 The significance, means and standard errors for juiciness, tendemess, flavor and overall satisfaction scores of 20 treatments of Table 1 (seven panelists) Evaluation description Juiciness Tendemess Flavor Overall satisfaction
Significance among treatments 0.05 0.05 N.S. N.S.
Significance among panelists
Mean
Standard error
0.008 0.005 0.000 0.000
4.68 5.17 4.66 4.59
0.9534 0.7784 0.9194 0.7960
N.S. = Not significant. Treatments number 10 and 12 were the most acceptable, and their corresponding formulations were 60:40 MDTM to HDCM, 22% SPI and 1% K-C, 60:40 MDTM to HDCM, SPI (24%), 24% SPI and 0.5% K-C. In general, the standard errors (Table 3) were very high, indicating large variation in scores. Using salt caused a significant (P<0.05) improvement in all of the four properties of treatment 19 (data not shown). It should be noted that three judges scored the salted product as "like very much" (7 points out of the 8point scale). This showed clearly the importance of the salt in the product. The pH, fat and moisture contents of the twenty products are presented in Table 5. The raw product pH was generally greater than 6.40 and it increased slightly after extrusion. The fat and moisture contents of the products were in the ranges 1.30-10.32% and 43.74-62.13%, respectively. This indicates that the products were not only of low fat, but also of high protein content. Thus, the products are desirable from a nutritional standpoint, but their high pH raises concems about their microbiological stability.
1271 Table 4 Means (seven panelists) and the significant (P<0.05) differences (abed . . .) in juiciness, tenderness, flavor and overall satisfaction scores among the 20 selected extrusion-cooked products of Table 1 Treatment
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47pbc
4.86*'"" 4 43bdac 4 ]^3bdac 4 72bac 4 2Qbdac
The eight most acceptable products (Table 2) were boiled to evaluate the effect of cooking on their stability. Table 6 shows the effect of boiling time on the moisture content of the eight extruded products. In general, boiling the products for up to 10 min resulted in a moisture gain. This increase in moisture was extended in some products by increasing the cooking time to 15 min, whereas some products started to decrease in moisture content when the cooking time was extended from 10 to 15 min. Overall, increasing the boiling time to 15 min increased ( P < 0 . 0 5 ) moisture content of the eight products. Among the eight products, there was a significant (P<0.05) difference in terms of moisture gain. Since the eight products had different nonmeat ingredients and MDTM levels and were treated with different extrusion variables, it is difficult to specify which factor had the greatest effect in causing the difference in the moisture gain among them. A general point which can be made here, however, is that the binding of the products was still acceptable after 15 min of boiling. The water gain of the products when boiled for 10 min was in the range of 3.5 percentage points (product-7) to 15.7 percentage points (product-8). When the products were boiled for 15 min, the water gain was in the range of 2.5 percentage points for product-7 and up to 18.6 percentage points for product-8 (Table 6). Since the levels of the SPI used were similar (22-24%) in most treatments (except for treatment 7), it is difficult to determine its real contribution to water gain, but it should have played an
1272 important role in moisture gain and in cooking yields, as indicated by other studies. The acceptable product binding, even after boiling, may probably be due to the interaction between the meat and the soy proteins and the matrix-forming properties of the soy proteins (Dudonis and Lasztity, 1986). The gelation characteristic of soy proteins is heat inducible and its strength increases to its maximum at boiling point temperatures and above (Schmidt and Morris, 1984; Hermansson, 1986). Table 5 The pH and proximate composition of the 20 products of Table 1 before and after extrusion After extrusion
Before extrusion Treatment
pH
Moisture (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
6.80 6.80 6.81 6.50 6.56 6.63 6.62 6.50 6.72 6.67 6.64 6.70 6.70 6.69 6.70 6.70 6.82 6.45 6.45 6.47
49.09 50.01 51.25 59.65 55.36 52.87 53.59 43.74 52.58 53.82 51.05 52.00 52.68 51.73 51.64 49.28 50.75 62.13 59.57 58.83
Fat (%) 9.51 8.17 9.71 1.98 1.30 7.60 8.19 2.75 7.90 6.79 6.93 6.59 9.64 9.22 9.42 9.90 10.32 2.23 1.77 2.94
pH 6.93 6.87 6.92 6.52 6.63 6.81 6.70 6.54 6.75 6.68 6.76 6.78 6.82 6.82 6.79 6.83 6.88 6.46 6.49 6.53
Apparently, the interaction between SPI and meat proteins resulted in very low losses (0.2-2.5%) of protein from the products when boiled (Table 7). Overall, however, the products were of very high (>20%) protein content. This makes them useftil in feeding programs for people on poor diets. Table 8 shows the effect of cooking time on the fat content of the eight treatments. Overall, the results show that by increasing the cooking time, the amount of fat released from the product increased significantly (P<0.05), and that differences in fat losses among the eight treatments were also significant (P<0.05). As the level of MDTM was increased in the product, more fat was released during boiling. Overall, however, the products were of low (< 12%) fat content, which is also nutritionally important.
1273 Table 6 The average (%) moisture content (± standard deviation) of the eight extruded products (Table 2) cooked in boiling water for various times Boiling time (min) Treatment 1 2 3 4 5 6 7 8
0 53.8 57.2 54.1 53.1 51.5 51.0 61.9 50.2
± ± ± + ± ± ± ±
5 0.4 1.5 1.1 0.9 0.5 0.1 0.6 0.8
60.8 62.4 60.7 61.4 62.9 62.3 64.5 63.3
+ ± + + ± ± + ±
10 0.4 0.3 0.7 0.8 0.2 0.2 0.2 0.2
64.8 65.3 62.5 63.0 65.8 64.4 65.4 65.8
± + ± ± ± ± ± ±
15 0.3 0.5 0.3 0.5 0.2 0.7 0.1 0.1
65.3 64.6 62.9 63.8 66.8 65.8 64.4 68.8
± ± ± ± ± ± ± ±
0.2 0.4 0.2 0.1 0.2 0.1 0.1 0.2
Table 7 The average (%) protein content (+ standard deviation) of the eight extruded products (Table 2) cooked in boiling water for various times (moisture adjusted to 60%) Boiling time (min) Treatment 1 2 3 4 5 6 7 8
0 28.5 34.4 29.8 28.4 28.4 27.9 36.2 26.8
± ± + ± ± + ± ±
10
5 0.3 0.1 0.2 0.2 0.2 0.5 2.0 0.3
28.3 34.2 28.6 28.4 28.5 27.7 33.5 25.5
± + ± ± + ± ± +
0.2 0.2 0.2 0.2 0.3 0.2 0.6 0.3
28.5 33.5 28.5 28.4 28.1 27.7 33.8 25.0
± ± ± ± ± ± ± ±
15 0.3 0.3 0.4 0.2 0.2 0.3 0.3 0.7
28.0 33.4 27.6 28.4 28.4 27.6 33.1 24.5
± ± ± ± ± ± ± +
0.4 0.1 0.8 0.7 0.5 0.2 0.2 0.3
Table 8 The average (%) fat content (± standard deviation) of the eight extruded products (Table 2) cooked in boiling water for various times (moisture adjusted to 60%) Boiling time (min) Treatment 1 2 3 4 5 6 7 8
9.4 3.1 7.5 8.1 9.4 11.1 1.2 11.8
± ± ± ± + ± ± ±
10
5
0 0.4 0.9 0.3 0.7 0.2 0.4 0.2 0.3
7.6 3.1 6.5 6.8 8.2 11.0 1.2 11.5
± 0.3 ± 0.6 ± 0.3 ±0.1 ± 1.2 + 0.5 ± 0.0 + 0.5
7.7 2.9 6.8 6.2 6.8 7.5 1.1 11.5
+ ± + ± + ± ± ±
15 0.1 0.1 0.1 0.1 0.2 0.8 0.0 0.1
6.9 2.8 6.8 5.8 6.3 6.8 1.0 10.4
± ± ± ± ± ± ± ±
0.2 0.5 0.1 0.1 0.0 1.5 0.0 0.1
1274 The addition of oat fiber somehow improved retention of fat in the product when cooked in boiling water, as shown in treatment 8 (Table 8). In general, losses of fat can be explained by the fact that the matrix which forms between the proteins of the product is not strong enough to hold much fat when boiled, so the fat is released. It was evident that when the products were cooked for only 5 min, the emulsion characteristic of the nonmeat ingredients was contributing to the fat-holding property. Boiling of the eight products for 5-15 min resulted in weight gains of 4.46% to 21.70%, depending on formulation and boiling time (Table 9). These gains in water diluted the fat content of the formulations. It is interesting to note that the treatment with oat fiber absorbed more water. Figures 1-3 show various treatments of Table 2 in the unboiled and boiled state, and inmiersed in liquid. It is obvious that extrusion cooking yields some products of acceptable bind and integrity, even when mechanically deboned turkey meat was present in the formulation. 4.
CONCLUSIONS
The results of the first study indicated that: 1. Products containing 20 to 60% MDTM, 80 to 40% HDCM and SPI levels of 15 to 22% with 0.5 to 1 % kappa-carrageenan gum were judged as acceptable; that is, their overall satisfaction scores were in the range of "like slightly" to "like moderately," specifically 5 to 6 points out of an 8-point hedonic scale. However, the products that contained higher MDTM levels, 80 to 100%, and higher SPI, 22 to 24%, were disliked slightly (4 out of 8 points in the hedonic scale). 2. Addition of salt caused significant improvement in product acceptability, from "like slightly" (5 points) to "like very much" (6 points out of the 8-point hedonic scale). The results of the second study indicated that: 1. Protein and fat losses and moisture gains were significantly affected by cooking time (0-15 minutes). 2. There were significant differences among the eight treatments in terms of moisture gain, fat and protein losses. 3. The quality (binding and texture) of the products did not change when boiled for 15 minutes. 4. Under the conditions of this study, oat fiber had a tendency to weaken the matrix which is formed by the interaction of SPI and the meat protein, but it also held fat during cooking. Although there were statistically significant differences among the 8 treatments, from a practical point of view, that of the producer and the consumer, there were no major differences among the treatments in terms of their composition and quality when cooked for 15 minutes in boiling water. In general, they were well-bound before, during and after cooking. Actually, their eating quality was improved by cooking. Furthermore, all treatments were high in protein (in the range of 24 to 28%) and low in fat (an average of about 6%), which is important to consumers. Additional studies need to be done on these products in terms of improving their eating quality by using spices, salts and antioxidants, and in terms of shelf-life.
1275 Table 9 The total weight and weight gain of the eight products (Table 2) before and after boiling for 5 to 15 minutes Boiling (min)
Treatment
Weight before boiling (g)
Weight after boilmg (g)
Difference in weight (g)
% Total gain
5
1 2 3 4 5 6 7 8
6.457 6.055 7.218 6.494 8.234 7.171 4.284 8.007
7.167 6.492 7.830 7.052 8.751 8.071 4.507 8.831
0.710 0.437 0.612 0.558 0.517 0.900 0.223 0.824
11.00 7.21 8.48 8.59 6.28 12.55 5.21 10.29
10
1 2 3 4 5 6 7 8
5.873 5.357 6.092 7.919 7.569 6.329 4.509 9.444
6.660 6.001 6.770 8.852 8.502 7.600 4.710 11.245
0.787 0.644 0.678 0.933 0.933 1.271 0.201 1.801
13.40 12.02 11.13 11.78 12.33 20.08 4.46 19.07
15
1 2 3 4 5 6 7 8
7.752 5.693 5.790 7.899 8.760 5.783 5.080 8.774
8.535 6.295 6.711 8.854 9.714 6.655 5.511 10.678
0.783 0.602 0.921 0.955 0.954 0.872 0.431 1.900
10.10 10.57 15.91 12.09 10.89 15.08 8.48 21.70
1276
BOILED
UNBOILED
MDTM(%): SPI(%): K«C{%): TEMPfX::):
Figure 1.
20 20 0 95
60 22 1 95
80 22 0.5 95
0 15 0.5 95
Unboiled and boiled products of treatments 2, 4, 5 and 7 of Table 2 (MDTM: mechanically deboned turkey meat; SPI: soy protein isolate; K-C: kappacarrageenan gum; TEMP: extrusion temperature).
mnM(%): SFI(%): K>C(%): TEMFfC);
20
W
0 95
60 22 0.5 105
60 22 1.0 95
m
22 0.5 95
m
24 1.0 105
0 15 0 95
M)ILED Figure 2.
Boiled products, shown in liquid, of treatments 2, 3, 4, 5, 6 and 7 of Table 2 (MDTM: mechanically deboned chicken meat; SPI: soy protein isolate; K-C: kappa-carrageenan gum; TEMP: extrusion temperature).
1277
SPI 20%
MDIM
BOILED Figure 3.
TEMP 9$**C
UNBOILED
Unboiled and boiled samples of treatment 2 of Table 2 (MDTM: mechanically deboned turkey meat; SPI: soy protein isolate; K-C: kappacarrageenan gum; TEMP: extrusion temperature).
MDTM 60%
SPI 22%
UNBOILED Figure 4.
K~C 0%
K-C 1%
TEMP 95°C
BOILED
Unboiled and boiled samples of treatment 4 of Table 2 (MDTM: mechanically deboned chicken meat; SPI: soy protein isolate; K-C: kappacarrageenan gum; TEMP: extrusion temperature).
1278
m%
Figure 5.
n%
03%
105X
Sample of treatment 3 (Table 2) stored in liquid after boiling for 10 min (MDTM: mechanically deboned turkey meat; SPI: soy protein isolate; K-C: kappa-carrageenan gum; Temp: extrusion temperature).
5. REFERENCES Alvarez V.B., D.M. Smith, R.G. Morgan and A.M. Booren, J. Food Sci., 55 (1990) 942946. Areas J.A.G. and R.A. Lawrie, Meat Sci., 11 (1984) 275-299. Ba-Jaber, A.S. Restructuring of poultry meat with nonmeat ingredients by extrusion cooking. Ph.D. Dissertation, Colorado State University, Fort Collins, CO, 1990. Ba-Jaber, A.S., J.N. Sofos, G.R. Schmidt and J.A. Maga. Extrusion cooking of chicken meat with various nonmeat ingredients. In: Food Science and Human Nutrition, G. Charalambous (ed.). Elsevier Science Publishers, London, pp. 761-782, 1992a. Ba-Jaber, A.S., J.N. Sofos, G.R. Schmidt and J.A. Maga, Lebensm. Wissensch. und Technologic, 25 (1992b) 135-137. Ba-Jaber, A.S., J.N. Sofos, G.R. Schmidt and J.A. Maga, J. Muscle Foods, 4 (1992c) 2739. Bhattacharya, S., H. Das and A.N. Bose, Food Chem. 28 (1988) 225-231.
1279 Chakraborty, K.C., S. Richardson and R. Villota. Twin-screw extrusion performance of meat proteins and interactions with starch. Ann. Mtg. Inst. Food Technol., Abstr. 18, 1987. Chung, S., P. Bechtel and R. Villota. Production of meat-based intermediate moisture snack foods by twin-screw extrusion of expanded meat-based products. Ann. Meet. Inst. Food Technol., Abstr. 258, 1989. Clarke, A.D., F. Hsieh and S. Mulvaney. Processing of mechanically-deboned turkey and com flour mixtures by twin screw extrusion. IFT Annual Meeting, Abstr. No. 127, 1989. Dudonis, W. and R. Lasztity, Die Nahrung, 30 (1986) 434-436. Hale, D. 1970. Expanded meat product. Canad. Patent 842,723. Hermansson, A.M., American Oil Chem. Soc. J., 63 (1986) 658-666. Kristensen K.H., P. Gry and F. Holm. Extruded protein-rich animal by-products with improved texture. In: Thermal Processing and Quality of Foods. P. Zeuthen, J.C. Cheftel, C. Eriksson, M. Jul, H. Leniger, P. Linko, G. Varela and G. Vos (eds). Elsevier Appl. Science Publish., London, pp. 113-121, 1984. Lawrie R.A. and D.A. Ledward. Texturization of recovered proteins. In: Upgrading Waste for Feeds and Food. D.A. Ledward, A.J. Taylor and R.A. Lawrie (eds.). Butterworths, London, pp. 163-182, 1983. Likimani, T.A., J.N. Sofos, J.A. Maga and J.M. Harper, J. Food Sci., 55 (1990) 13881393. Likimani, T.A., J.N. Sofos, J.A. Maga and J.M. Harper, J. Food Sci., 56 (1991) 99-105, 108. Megard, D., N. Kitabatake and J.G. Cheftel, J. Food Sci., 50 (1985) 1364-1369. Sasamoto, Y., Y. Kammuri, K. Sawa, M. Araki, S. Morimoto, F. Mitsui and N. Miyazaki. 1989. Process for processing and treating raw materials of marine products. U.S. Patent 4,816,278. Schmidt, R. and H. Morris, Food Technol., 38(5) (1984) 85-96. Sofos, J.N., J. Food Sci., 48 (1983a) 1692-1699. Sofos, J.N., J. Food Sci., 48 (1983b) 1684-1691.
1280 Sofos, J.N., A.S. Ba-Jaber and G.R. Schmidt. Extrusion cooking of mechanically deboned turkey meat with soy protein, kappa-carrageenan and oat fiber. Proc. 38th Int. Congr. Meat Sci. and Technol., Clermont-Ferrand, France. Vol. 5, pp. 1125-1128, 1992. Sofos, J.N., I. Noda and C. Allen, J. Food Sci., 42 (1977) 879-884. Southard, C.L. Non-conventional extenders of processed meat products. M.S. Thesis, Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, 1985. Steel, R.G. and J. Torrie. Principles and Procedures of Statistics. 2nd Ed. McGraw-Hill Book Co., New York, NY, 1980. Suzuki, H., B.S. Chung, S. Isobe, S. Hayakawa and S. Wada, J. Food Sci., 53 (1988) 1659-1661. Tarte, R., R.A. Molins and M. Kazemzadeh. Development of a beef/com extruded snack model product. Ann. Mtg. Inst. Food Technol., Chicago, IL. Abstr. 284, 1989. Thomas, L., P. Bechtel and R. Villota. Effects of composition and process parameters on twin-screw extrusion of expanded meat-based products. Ann. Mtg. Inst. Food Technol., Chicago, IL. Abstr. 118, 1989.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1281
Use of starch for water binding in restructured beef products J.N. Sofos, J.A. Perejda and G.R. Schmidt Department of Animal Sciences and Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523, U.S.A. Abstract Sodium chloride (salt), phosphates and mechanical treatment solubilize proteins which increase fat and water binding in meat products. The algin/calcium binding system also enhances meat particle cohesion, while starches may be added to foods to bind water and increase viscosity. Studies in our laboratory examined cooking yields and binding or cohesion in beef and in beef formulated with salt/phosphate (1.5%/0.3%) or the algin/calcium (0.4%/0.075%) binder with and without added starch (2%) and water (10%). The ground beef was mixed with the ingredients, packaged in casings, and cooked in a 70°C water bath for 90 min. The pH of the salt/phosphate products (6.03 + 0.06) was higher (P<0.05) than other treatments (5.81 ± 0.05 and 5.78 + 0.05). Cooking yields increased with added starch in both no-water-added and water-added all-beef and algin/calcium formulations, but not in the salt/phosphate products, which even without added starch had higher cooking yields than the other treatments. Salt/phosphate formulations with or without added starch had higher (P<0.05) binding values (Newtons) than all other formulations, while added starch had only a minor effect on binding. 1.
EVTRODUCTION
Lean mammalian muscle from which meat is derived has a typical composition of 75% water, 19% protein, 2.5% lipid, 1.2% carbohydrate and 2.3% miscellaneous soluble non-protein substances (Lawrie, 1985). The protein component is comprised of myofibrillar proteins (60%), sarcoplasmic proteins (29%) and connective tissue proteins (11%). The myofibrillar fraction which is mostly high-molecular weight, salt-extractable fibrous proteins is largely responsible for three physicochemical events; protein-water interactions, proteinlipid associations, and protein-protein aggregation (Acton et al., 1982). Functionally, these terms describe water-binding capacity, fat-holding capacity, and gelation. Each of these three is important in meat processing and can affect sensory attributes of the final product. The water component in beef interacts with hydrophilic groups of the side chains of the proteins via polar interactions (Shut, 1976). This has been determined to represent only 5-10% of the total water component (Shut, 1976; Acton et al., 1982). The remainder and majority is believed to be immobilized due to membranes and filaments of structural proteins via capillary action (Hamm, 1960; Shut, 1976; Acton et al., 1982).
1282 Protein-lipid interactions are involved in three events: gel strength, lipid particles surrounded by a protein film, and the physical characteristics of the fat, including melting point and degree of fat tissue disruption (Jones, 1984). Proteins act as emulsifiers because their polar side chains orient towards water molecules and nonpolar side chains surround fat particles (Acton and Saffle, 1971). Protein-protein interactions are evident as inter- and intra-molecular bonds that break during heating, and alter the secondary and tertiary structure of the proteins. Gelation occurs during subsequent altered bond reformation (Acton and Dick, 1984), and has been described to involve the orderly interaction of proteins, which may or may not be denatured, and which leads to the formation of a three dimensional well-ordered structural matrix (Hermansson, 1978). Beef processing traditionally involves addition of sodium chloride and alkaline phosphate with and without mechanical treatment to positively affect these three events and sensory attributes of the final product. 1.1
Effects of salt and phosphate
Processors, through the use of salt (sodium chloride), phosphate and mechanical treatment can offer consumers a variety of meat products, including sausages, which are desired for their palatability, shelf-life, cost and convenience. Most cooked sausage products contain approximately 2-3% salt (Offer and Trinick, 1983; Schmidt, 1988). However, salt has also been shown to act as a pro-oxidant accelerating oxidative reactions of unsaturated lipids, thus increasing rancidity (Gray, 1978; Obanu et al., 1980). Protein solubilization through addition of salt increases water-binding capacity of myofibrillar proteins. Salt is believed to produce a shift in the isoelectric point of proteins to a lower pH value (Hamm, 1960). This results in an increased net negative charge at the existing pH from ionizable carboxyl groups on the protein (Shut, 1976; Acton et al., 1982). Subsequent increased repulsion between charged groups produces greater separation between protein chains and creates a matrix capable of entrapping water (Hamm, 1960). Hydrated chloride ions are attracted to positively charged groups and cause inter- and intra-salt bridges to break further increasing water-binding capacity (Shut, 1976). Offer and Trinick (1983) suggested moisture losses/gains in rabbit psoas muscle were due to changes in meat myofibrils and the subsequent myofilament lattice upon treatment with pyrophosphate-saline solutions. Paterson et al. (1988) observed increased fiber swelling and extraction of myofibrillar proteins as salt concentration increased. Phosphates reduce salt concentrations required for maxunum swelling of myofibrils (Offer and Trinick, 1983) and help release proteins for gel formation (Siegel and Schmidt, 1979). Bendall (1954) reported a synergistic increase in water absorption in minced rabbit muscle after addition of salt and pyrophosphate. This was believed to be due to cleavage of actomyosin. Phosphates increase water and meat particle binding in meat products through one or more of the following ways: increasing pH, increase in product ionic strength, dissociating actomyosin into actin and myosin and binding to meat proteins (Hamm, 1970). Product pH, salt concentration and phosphate-type are also involved with phosphate functionality (Trout and Schmidt, 1986). The proposed mechanism suggests that as myosin molecules denature during thermal processing, unprotonated histidine residues are exposed, resulting in elevated pH when solvent protons are donated to negatively charged histidine residues (Goodno and Swenson, 1975).
1283 Effects of salt and phosphate on beef and pork frankfurters were studied by Whiting (1984b). Reducing salt levels from 2.5% to 1.5% increased water and fat exudate, while the addition of 0.12% or 0.25% sodium acid pyrophosphate (SAPP) counteracted these effects. SAPP reduced product pH, and after adjustment to typical pH levels, the exudate decreased. Huffman et al. (1987) reported phosphate additions (0.25%, 0.4%) resulted in lower thiobarbituric acid (TBA) values in structured beef and pork nuggets throughout twenty weeks of storage. Evaluating for off-flavors, a trained sensory panel was unable to detect significant differences (P < 0.05) between treatments with added phosphates and those without until the sixteenth and twentieth week of storage for restructured pork and beef nuggets, respectively. Meat protein solubilization and swelling of myofibrils has been found to be tune dependent and occurs primarily on the meat surface (Voyle et al., 1984). Mechanical treatment in the form of tumbling, massaging and brine injection increases meat surface area for the action of salt and phosphate to occur. The goal of the massaging process is to develop a protein-rich exudate without gross physical damage to the meat (Theno et al., 1978). Advantages include reduced processing time and improved tenderness (Siegel et al., 1978). This is accomplished without extensive physical damage which could compromise desired textural properties. 1.2
Beef consumption and health concerns
Current health concerns associated with food consumption arise from overconsumption of total calories, total fat, saturated fatty acids and cholesterol (National Academy of Sciences/National Research Council, 1988). Between 1976-1986, per capita consumption of beef as a percentage of total meat consumption dropped from 43% to 35%, while chicken consumption increased from 20% to 26% (USDA, 1988b). Data indicate a changing consumer environment that is affecting beef consumption. Consumer surveys in the United States indicate that interest in health and nutrition is at least affecting people's perception of their buying habits. While surveyors are aware that for various reasons what people say and what is actually done may be different, surveys can be accurate in measuring attitudes (Gallup Organization, 1987). Surveys conducted for the National Restaurant Association (NRA) in 1983 and 1986, indicated that consumers had changed food choices both at home and away because of health and nutrition concerns (NRA, 1986). Changes included increased consumption of vegetables, fish and salads, while decreasing salt, fat, meat and fried foods. Four polls conducted since 1982 indicated that 47-55 % of restaurant patrons surveyed would be likely or fairly likely to order low-salt meals (Gallup Organization, 1988). The National Food Consumption Survey, 1977-1979 and Continuing Survey of Food Intake by Individuals, 1985 support results from consumer surveys (USDA, 1985a,b). Another survey for the American Meat Institute and The National Live Stock and Meat Board examined consumer attitudes toward whole muscle cuts of beef (Yankelovich et al., 1985). Relative to 1985, respondents indicated decreased meat consumption for health reasons and were more concerned about salt and cholesterol in the diet. As many as nine out of ten reported exercising care over fat intake and there was a positive responsiveness to the concept of leaner and calorie-reduced meat products. Convenience had become increasingly important.
1284 1.3
Regulatory changes affecting fat levels in processed beef
In response to a petition from the American Meat Institute, the United States Food Safety and Inspection Service (FSIS) responded to the desire for low-fat processed meat products by revising the standard of identity for frankfurters and similar cooked sausages (USDA, 1988a). New regulations allow for a maximum combination of 40% fat and added water in these products while continuing to restrict the maximum fat content to no more than 30% of the cooked product. This allows processors to formulate leaner products replacing fat with water. In the past, added water was limited to 10% of the finished product. In addition, use of the term "lite" in these products requires a 25% reduction in fat content. 1.4
Processed beef products with reduced levels of salt
Sodium chloride (salt) is added to meat for flavoring, as an antimicrobial agent, and for its effect on muscle proteins. Fat is an important contributor to the texture and taste of products. However, consumer interest to limit sodium and fat intake for health reasons has prompted continued research into low-salt and low-fat meat products. Low-salt, 0.5 %-l .5 %, meat emulsions have the potential for decreased emulsion stability as determined by increased cook losses and/or decreased gel strength (Sofos, 1983a,b; Whiting, 1984a; Barbut, 1988). In another study, Sofos (1985) reported that addition of potassium sorbate, 0.26% in low-salt meat batters, increased cook yields. Investigations into the sensory impact of reducing salt levels in beef and pork frankfurters indicated that levels below 2.0% resulted in products having unacceptable flavor, texture, overall acceptability and reduced shelf-life (Sofos, 1983b). Barbut (1988) reported reductions in texture profile analysis as salt levels decreased in mechanically deboned chicken meat. Addition of phosphate (0.4%) counteracted these losses and improved texture analysis scores. Lowering fat content in frankfurters has been reported to increase toughness and alter product quality (Sofos and Allen, 1977; Paul and Foget, 1983). 1.5
Alginate and interactions with protein
Alginates are naturally occurring long chain carbohydrate derivatives from brown seaweed, Pheaophyceae, consisting of uronic acid (D-mannuronic acid and L-guluronic acid residues) joined by 1,4-glycosidic bonds. The uronic acid ratio affects rheological, gelation and ion exchange properties of the alginate (Cottrell and Kovacs, 1977). Homopolymeric blocks of guluronic acid residues are responsible for the cooperative binding of calcium cations leading to gel formation (Smidsrod and Haug, 1972; Grant et al., 1973). Gel strength depends on the nature of the divalent cation with the order: Ba"'>Sr+>Ca"'> >Mg'" (Smidsrod, 1974). Grant et al. (1973) proposed the "eggbox" model to describe gelation. Evidence, however, supports a dimeric association rather than a buckled sheet structure suggested in the original "eggbox" model (Morris et al., 1977). Studies of algin/calcium gels interacting with proteins during gel formation indicate electrostatic forces to be mainly responsible for the formation and stability of proteinpoly saccharide gels (Bemal et al., 1987). Interactions were believed to be due to negatively charged side chains of amino acids in the protein (Imeson et al., 1977; Ledward, 1979). Ensor et al. (1991) reporting on differential scanning calorimetric studies of meat protein-
1285 alginate mixtures found that algin/calcium gels were responsible for thermal destabilization of crude meat protein extracts indicating changes in the physical state of the proteins. This provides evidence that algin/calcium gels interacting with meat protein components which could influence product texture. Stainsby (1980) discussed alginate and meat protein interactions involving lysine amino acids and the mannuronic acid component of alginate to develop thermostable covalent bonds. In addition, it was described that binding and retention of water in complex food gels containing large molecular weight components such as proteins and polysaccharides can show advantages from mutual interactions between the two components. Imeson et al. (1977) reported that electrostatic forces are responsible for interactions between charged polysaccharides such as sodium alginate and proteins leading to decreases in protein denaturation temperatures and development of high molecular weight protein-polysaccharide complexes. 1.6
Restructured meat
Restructuring requires binding between meat pieces while retaining desired textural characteristics associated with intact meat (King and Macfarlane, 1987). Raw materials are sectioned and formed or flaked and formed to produce a value-added meat product of intermediate cost (Berry, 1987). Meat particle size has been found to affect physical and sensory properties of restructured meats (Durland et al., 1982; Berry et al., 1987; Clarke et al., 1988a). Raw steaks made from large meat particles had large particles of fat and were judged to be too fatty in appearance, while those made from smaller particles were more acceptable (Durland et al., 1982). Deformation values, an index of elasticity, increased as flake size decreased (Durland et al., 1982). Berry et al. (1987) reported that as flake size increased, visually detected fibrousness, first bite hardness, cohesiveness and shear force increased. Other variables affecting the finished product include added proteins such as myosin or soy and processing conditions, mixing, cooking time and temperature (Berry, 1987). 1.7
Alg* ^/calcium restructured beef
Means and Schmidt (1986) reported on an algin/calcium meat binding system to produce restructured beef capable of binding in the raw state. Binding of meat particles was achieved by reacting sodium alginate and calcium carbonate to form an insoluble algin/calcium gel. The process was patented (Schmidt and Means, 1986) and approved for commercial use (USDA, 1986). Algin/calcium (AC) restructured beef products had raw bind values that were statistically greater than salt and phosphate added controls (alpha=0.10). Sensory analysis of raw products comparing color and percent discoloration found AC treatments to be statistically similar to pure ground beef products, while salt/phosphate treatments were darker and more discolored (Means and Schmidt, 1986). No differences in cooked aroma were detected. Increasing levels of sodium alginate did produce detectable off-flavors due to sodium alginate that had not yet reacted with calcium ions. Objectionable mouthfeel characteristics described as mealy/slippery were observed with high levels of sodium alginate (1.2%) or low levels of calcium carbonate (0.072%). The average raw pH of AC products was
:s6 significantly higher than no-additive controls. This was noted to be undesirable as high pH meat is known to spoil more quickly (Egan, 1984). Means and Schmidt (1987) included glucono-delta-lactone (GDL), a slow-release acid, in an attempt to alleviate the objectionable niouthfeel observed at higher alginate levels. Raw and cooked bind scores for AC steaks were significantly greater (P < 0.05) than all beef or salt/phosphate treatments, although GDL did not appear to be a factor in this. AC ingredients did not contribute to meat discoloration. Sensory scores for cooked AC steaks had significantly stronger (P < 0.05) off-flavors and less desirable mouthfeel, indicating that GDL was not effective. Shelf-life studies indicated that AC treatments under aerobic conditions were similar to all-beef controls, although in vacuo samples supported microbial growth sooner than controls. Clarke et al. (1988b) reported addition of a slow release acidifier, Capshure® (Balchem Corp., Slate Hill, NY), an encapsulated lactic acid/calcium lactate blend, to effectively reduce product pH at levels of 0.4% sodium alginate, 0.067% calcium carbonate, and 0.10% lactic acid/calcium lactate. Percent cook yield and cook bind scores were greater than all-beef controls (P=0.05) at this and higher binder levels reportedly due to inhibition of moisture migration. Schmidt et al. (1988) illustrated AC technology to be effective and acceptable in restructured beef which has been injected with aqueous solutions of soy protein concentrate. Subsequently, AC technology was shown to be effective in restructured turkey products (Ensor et al., 1989). Optimal raw and cooked product bind was achieved with combinations of 0.4% sodium alginate, 0.075-0.1875% calcium carbonate and 0.6% lactate acid/calcium lactate. Panelists detected no treatment differences (P>0.05) for aroma, flavor or juiciness of the cooked products. Higher levels of acidifier may have been responsible for this. 1.8
Starch
Starch is found in seeds, tubers and stems of plants. Native starch is composed of two carbohydrate polymers of anhydroglucose, amy lose and amylopectin, linked primarily through alpha-D-(l,4) glucosidic bonds. Amylose is a straight chain polymer containing 200-2000 anhydroglucose units, depending on botanical source. Amylopectin is a much larger branched chain molecule with molecular weights in the millions. Branching occurs via alpha-D-(l,6) bonds (Swinkels, 1985; Wurzburg, 1986). Properties of amylose and amylopectin differ significantly in aqueous solutions. Amylose is difficult to dissolve and unstable in aqueous solutions, as the molecules tend to aggregate and precipitate (retrogradation), whereas amylopectin because of its branched structure is more stable and less likely to retrograde (Kennedy et al., 1984). Through chemical treatment, starch can be altered to withstand chemical and physical abuses which would normally cause undesirable gelation or breakdown (Anonymous, 1978; Wurzburg, 1986). Starch manufacturers are able to control viscosity, texture, cold-water swelling and sensitivities to breakdown (McCleary, 1980). Also, gelatinizationbehavior and viscosity can be alTccted by coexistent substances such as sugar or salt (Yamada et al., 1986). CTOSS linking is an important chemical modification which can control swelling and suhscqucnl rupturing ol starch granules during cooking. Introduction of disfunctional agents c;ip;iblc of reacting with hydroxyl groups of two different molecules within the granule act to reinforce natural hydrogen bonding and retard the rate of granule swelling and reduce
1287 chance of rupture (Wurzburg, 1970). Cooked pastes are more viscous, heavily bodied and less likely to break down with extended cooking times, increased acid or severe agitation (Langan, 1986). Introduction of monovalent ions onto hydroxyl groups can increase freeze-thaw stability and lower starch gelatinization temperature (Wurzburg, 1972). Pregelatinization produces starches that are capable of cold-water swelling through simultaneous cooking and drying (Powell, 1967). Rate of rehydration and texture of the finished paste can also be varied through chemistry, processing techniques and physical form (Langan, 1986). 1.9
Processing using starch and other carbohydrates
Starch binders added to fresh and salami sausages served as functional ingredients to maintain juiciness, flavor and acceptability as rated by sensory panels (Sison and Almira, 1974; Sison et al., 1974). Bushway et al. (1982) tested frankfurters containing 3% potato starch or potato flour. Sensory panels rated potato starch and flour treatments to be more tender and juicy than controls containing wheat flour. Starch is often added to surimi to improve textural properties. Wu et al. (1985a) examined rheological and thermodynamic effects of several cook-up and pregelatinized starch types included in a surimi formulation. Textural properties of cooked surimi were dependent on gelatinization characteristics of the starches, such as gelatinization temperature, degree of swelling and water uptake of the starch granules. Starch gelatinization occurred at increased temperatures when included in surimi mixtures and was attributed to the presence of salt and sucrose in surimi (Wu et al., 1985b). Impaired protein gelation was observed when an instant tapioca starch was included. This correlated with shear stress measurements. Comer et al. (1986) reported wheat flour and modified com starch improved stability and textural firmness of a comminuted meat product. Scanning electron micrographs revealed the presence of birefringent starch molecules, indicating incomplete gelatinization. Bonnefin and Baumgartner (1988) examined the effects of seven starch-based binders added to a comminuted meat product at levels of 3 %, 6 % and 9 %. Treatments were cooked to 65, 70 and 75 °C. They found binders in general had favorable effects on reducing expressible moisture, but varying effects on product compression. 2.
EXPERIMENTAL
Heat-induced protein denaturation can cause weight losses in beef products as water and fat migrate to meat surfaces and are expressed as drip and through water vaporization. This is undesirable for processors selling products based on weight and also for consumers who identify tenderness and juiciness as desirable product attributes. Addition of sodium chloride (salt), alkaline phosphate and mechanical treatment solubilize proteins, developing a water and meat binding matrix during cooking. Consumers generally desire the characteristic flavor and texture that is developed. Restructured meats are based on this process resulting in an intermediate priced product providing added value to under-utilized cuts of meat with textural characteristics which resemble those of intact meat (Berry, 1987).
1288 However, there is a growing population who are reducing dietary sodium, fat and total calories for nutrition and health-related reasons. Researchers and processors have shown an active interest in developing low sodium products and replacing fat with water. As indicated, algin/calcium (AC) restructured beef utilizes the reaction between sodium alginate and calcium ions to develop a thermostable gel without application of heat; and is an alternative to the use of salt and phosphate (Means and Schmidt, 1986). The binder has been shown to increase bind of raw and cooked products (Means and Schmidt, 1986; Means et al., 1987; Clarke et al., 1988b; Ensor et al., 1989) and percent cooking yield relative to all-beef control products (Ensor et al., 1991). Preliminary studies in our laboratory comparing AC and salt/phosphate (SP) restructured beef cooked in a closed system showed that SP treatments had greater cooking yield and binding force than the AC counterparts. Starches and gums are added to many foods to bind water, increase viscosity and improve textural characteristics. They are allowed in sausage-type products in the United States in quantities up to 3.5% by weight (Schmidt, 1986). Starch and beef are two different food systems with the potential to bind water. Both develop a structural component which is time- and temperature-dependent. Within a single product, synergistic or cooperative effects would be based on compatibilities between the two systems. This would include processing requirements as well as mutual interactions between the two components. Since functional, nutritional and economic advantages exist for development of beef products that bind water through addition of starch rather than sodium chloride and phosphate, the experimental objectives of studies done in our laboratory were to measure differences in percent cooking yield and cooked product binding strength between three meat systems; all-beef, algin/calcium and salt/phosphate restructured beef; and to examine the ability of starch to compensate for differences in the meat restructuring systems (Perejda, 1991). 2.1
Experimental design
Through a completely randomized block design, changes in two dependent variables (i.e., percent cook yield and cooked particle binding) were measured as a function of addition of starch (2%) and addition of water (10%) to ground beef with no additives (control) or ground beef restructured with salt/phosphate (SP) and algin/calcium (AC). 2.2
Materials
Boneless riblifter beef (78.7% moisture, 4.2% fat and pH of 5.58) was purchased from a local processor and kept frozen (0°C) in vacuum packages until used over a threemonth period. Ingredients in the AC formulation included: 0.4% sodium alginate (Manugel DMB, Kelco, San Diego, CA), 0.075% calcium carbonate (Gamma Sperse 80, Georgia Marble Co., Tate, GA), and 0.6% encapsulated lactic acid/calcium lactate (CapShure® LCL-135-50; 30% lactic acid, 20% calcium lactate, 50% hydrogenated vegetable oil, Balchem Corp., Slate Hill, NY). Salt/phosphate treatments included 1.5% sodium chloride (Morton-Thiokol,
1289 Chicago, IL) and 0.3% sodium tripolyphosphate (FMC Corporation, Philadelphia, PA), both food grade. Starch-added treatments included a modified waxy maize starch designed for use in conmiinuted meat products (LoTemp 452, A.E. Staely Mfg. Co., Decatur, IL) at the 2% level based on the weight of the formulation. This granular starch had a pasting temperature of 36°C; and the peak viscosity of a 5% aqueous solution was 590 Brabender units at 70°C. Distilled and deionized water when added was at the 10% level. 2.3
Product preparation
Beef was thawed for 12-16 hours at 4°C, and then ground twice through a Hobart grinder (Hobart Corp., Troy, OH). First it was ground through a kidney-shaped plate (5 cm X 2 cm), and then through a 4.8-mm round hole plate. Sample aliquots were removed and frozen for later determination of fat and moisture contents. Treatment samples (350 ± 1 g) were portioned, covered with plastic and refrigerated (4°C) until processed 0-6 hours later. The processing room temperature was maintained between 4-9°C. Each treatment was mixed for 2.5 min in a Kitchen-Aid Mixer (model K45SS, Hobart Co., Troy, OH) at speed two with a paddle attachment. Based on dry weight, preweighed ingredients were added at 30-second intervals. Order of addition and ingredient levels are listed in Table 1. Samples were removed for raw product pH determination, while the remaining product was extruded into pre-weighed 6-cm diameter cellulose casings (Hutchinson and Co., Chicago, IL) using a hand operating extruder, they were tensioned and clipped closed. After weighing, the samples in the casings were vacuum packaged (Multivac, Allgau, W. Germany) and refrigerated 20-24 hours (4°C) prior to heat processing. Table 1 Sequence and time table for ingredient addition to treatments and controls Ingredient and time of addition (sec) Treatment
0
30
60
90
150
All-beef
Starch then Mix water
Mix
Mix
Stop mixing
Algin/calcium (AC)
Starch then water
Calcium carbonate (0.075%)
Acidifier^ (0.6%)
Stop mixing
Salt/phosphate (SP)
Starch then Salt (1.5%) and water phosphate (0.3%)
Mix
Mix
Stop mixing
Sodium alginate (0.4%)
Capshure® ~ lactic acid, calcium lactate blend. Packaged products were heated for 90 min in a water bath (70 ± 1°C). The final temperature was attained after approximately 30 min. Thermocouple probes connected to a Speedomax H recorder (Leeds and Northrup Co., North Wales, PA) charted a constant
1290 record of both internal sample temperature and water temperature. After cooking, the products were cooled 15 min in an ice water bath and refrigerated for twelve hours (4°C). 2.4
pH determination
The pH of previously frozen samples of the raw meat, in addition to refrigerated raw and cooked products, was measured within 48 hours after thawing or cooking. The pH was measured by combining 20 ± 0.1 g meat with 80 ± 1 g deionized distilled water and blending one minute (Oster Corp., Milwaukee, WI). A Coming Model 125 pH meter (Coming Glassworks, Medfield, MA), equipped with a Coming combination electrode (Cat. No. 476192) measured pH. 2.5
Percent cook yield determination
Casings as well as excess liquid and solid fat deposits were removed prior to cooked product weighing. Percent cooking yield based on meat and dry ingredients allowed for comparisons across different water levels: Cooking Yield (%) = cooked product weight x (theoretical weight plus water) x 100 raw product weight (theoretical weight less water) 2.6
Cooked product binding determination
Clarke et al. (1988a,b) reported significant (P<0.05) correlations between objective and sensory methods for texture determination of restructured products; indicating that penetration force measurements may be useful in predicting meat particle binding strength. Using an apparatus described by Field et al. (1984), meat particle binding was estimated by the force required for a 1.8-cm spherical brass probe to penetrate a meat sample placed over a 5-cm diameter opening. Meat samples (10 + 1 mm thick) were cut perpendicular to the long axis. The force was measured using a 100 Newton load cell transducer attached to a J.J. Lloyd Tensile Testing matching, type T5002 (Pacific Scientific, Santa Ana, CA) with a crosshead speed of 110 mm/min. Force measurements were recorded (J.J. Lloyd Recorder, model PL3 XY/t, Pacific Scientific, Santa Ana, CA) as peak deflection and intemal calibration standards allowed for length to force conversions. Average values of four to six readings per treatment, tested at room temperature, were taken within 48 hours after cooking. 2.7
Statistical analysis
When statistical analysis of variance of the results (SAS Institute, Inc., 1988) revealed significant (P<0.05) main effects, Duncan's New Multiple Range Test separated significant means (Duncan, 1955).
1291 3.
RESULTS AND DISCUSSION
3.1
Effects on pH
The restructured meat formulation had significant effects (P<0.05) on pH of both raw and cooked products (Table 2). The SP products had higher (P<0.05) pH values than the AC and all-beef treatments. The increase in pH resulted from the addition of phosphate, which has been previously reported (Bendall, 1954; Hamm, 1960; Goodno and Swenson, 1975; Trout and Schmidt, 1983, 1986a,b). The pH values of all-beef and AC products were not significantly (P>0.05) different from each other. The lack of statistical significance (P>0.05) in raw product pH values indicated that the addition of sodium alginate, calcium carbonate and the acidifier did not affect pH. This is in contrast to results of previous experiments with AC beef (Means and Schmidt, 1986; Means et al., 1987; Clarke et al., 1987), where pH values of similar products were reported to be above 5.8. However, pH values above 5.8 may present problems due to limited shelf-life (Egan, 1984). Previous attempts to reduce the pH by addition of 0.2% glucono-delta-lactone (GDL), a slow release acid, were unsuccessful (Means et al., 1987). Addition of CapShure®, an encapsulated lactic acid and calcium lactate blend, was found effective in reducing pH in AC products (Clarke et al., 1988b; Ensor et al., 1989). Therefore, inclusion of this acidifier was responsible for reduced pH of the raw AC treatment in the present study. Table 2 Effects of meat type, starch and water level on the pH of raw and cooked products Product Beef
Starch (%)
Water (%)
0 2 0 2
0 0 10 10
Mean ± SD AC
0 2 0 2
0 0 10 10
Mean ± SD SP
0 2 0 2
0 0 10 10
Raw product pH A B 5.53 5.56 5.52 5.55
5.58 5.60 5.50 5.60
Cooked product pH B A 5.72 5.79 5.75 5.82
5.83 5.88 5.85 5.83
5.56 ± 0.05*^
5.81 ± 0.05''
5.50 5.52 5.54 5.55
5.77 5.78 5.70 5.79
5.56 5.66 5.51 5.51
5.73 5.82 5.81 5.85
5.54 ± 0.04^
5.78 ± 0.05''
5.78 5.85 5.81 5.82
5.98 5.94 5.99 6.03
5.84 5.88 5.84 5.86
6.12 6.06 6.10 6.00
6.03 ± 0.06^ Mean ± SD 5.83 ± 0.02^ A ^ T " Replicates 1 and 2; AC: Algin/calcium restructured beef; SP: Salt/phosphate restructured beef; SD: Standard deviation. ^'^ Means in the same column with different superscripts are significantly different (P < 0.05).
1292 Replicate effects on pH (Table 2) were significant (P < 0.025) and may have been due to variability in the pH of the raw meat (5.54-5.68). These differences could have affected cooking yield (%) and cooked product binding values because small differences in pH potentially lead to larger differences in functional properties (Trout and Schmidt, 1983). 3.2
Percent cooking yield
To emphasize effects on water binding, the percent cooking yield was based on raw meat weight plus added weight of dry ingredients. This permitted percent cooking yield values to be in excess of 100% and was a measure of water that was added to the formulation originally and that was bound during cooking and retained after cooking. Within each water level and without addition of starch, all-beef products were lowest (P<0.05) in percent cooking yield; AC products were intermediate; and SP the highest (Table 3). Table 3 Analysis of variance table for cooking yields Source
DF
Model Error Meat type (MT) Starch level (SL) Water level (WL) Replication MTxSL MTxWL SLxWL
10 13 2 1 1 1 2 2 1
Mean squares 207.441 5.551 502.600 545.688 231.757 0.002 137.631 2.837 10.827
F-value
P-value
37.37
< 0.0001
90.54 98.30 41.75 0.00 24.79 0.51 1.95
< 0.0001 < 0.0001 < 0.0001 >0.9 < 0.0001 >0.6 >0.1
DF = Degrees of freedom. Analysis of variance (Table 3) of the results on cooking yield found significant (P<0.05) main effects due to meat type, starch addition and water addition. There was a significant meat type by starch interaction (P<0.05) indicating variable starch effects on cook yield for the different meat types indicating analysis of main effects could mask significant effects that would be revealed through analysis of individual treatments. Effects due to replication were not significant (P>0.9). Analysis of significant meat type effects averaged over starch and water levels showed that the SP meat products had greater cooking yields than AC products, which had higher yields than the all-beef product (P< 0.005). Salt and phosphate in meats increase pH and ionic strength, solubilize myofibrillar proteins, chelate metal ions and bind phosphate ions to meat products (Bendall, 1954; Voyle et al., 1984; Lewis et al., 1986; Trout and Schmidt, 1986a,b, 1987; Paterson et al., 1988). Concentrations of these two ingredients in SP products approximated an ionic strength of 0.32 based on calculations of Trout and Schmidt (1987). They reported that cooking losses were prevented in frankfurters having a raw pH of 6.0 when ionic strengths were above 0.32. Their use of a meat batter with raw pH of 6.0 would be expected to increase cooking yields compared to the coarsely ground beef and raw
1293 pH of 5.83 used in the present study. Despite these differences, SP samples in the present study were expected to maximize percent cooking yield. Significant differences (P<0.05) in percent cooking yield between AC and all-beef products may have been due to three potential reasons. The first and most simple one involves binding of water by sodium alginate, which is a hydrocolloid (Glicksman, 1982). Secondly, Morris et al. (1977) described the development of a three dimensional algin/calcium structure which may bind water. This is the same structure responsible for the meat particle binding described by Means and Schmidt (1986). Finally, protein-carbohydrate interactions as described by Imeson et al. (1977) and Stainsby (1980) and inferred by Means and Schmidt (1986) may have developed a matrix capable of binding water to increase cooking yields. It is not known whether one of the above potential mechanisms may prevent interactions from another by utilizing the same functional groups. Earlier studies with AC beef (Means et al., 1987) found significant (P<0.05) differences in cooking yield between all-beef, AC and SP meat products. This may have been due to the method of cooking, which was open-hearth grilling in these earlier studies, and would promote moisture loss through evaporation. In contrast, cooking in a closed system such as employed in the present study would promote moisture retention. The concentrations of sodium alginate and calcium carbonate and the acidifiers used were different in the earlier studies (Means et al., 1987). The results indicated that starch contributed (P<0.05) to increased cooking yield. Others have reported similar findings using wheat flour and modified com starch (Comer et al., 1986), potato starch (Comer and Dempster, 1981), various gums (Foegeding and Ramsey, 1987) and various starch binders (Sison and Almira, 1974; Sison et al., 1974; Bonnefin and Baumgartner, 1988). Added water significantly (P<0.05) increased cookings yield of all three types of meat products when starch was present (Table 4). In the absence of starch, only SP samples realized significant (P<0.05) increases in cook yield due to water addition, suggesting that under these processing conditions, starch addition to increase cook yield in meats containing salt and phosphate is unnecessary, but this may not be the case where levels of salt and phosphate are reduced. Cooking yield values of starch-added AC and SP samples at both water levels were not significantly (P>0.05) different (Table 4), which demonstrates the ability of starch to increase percent cooking yield in AC products. Addition of 2% starch significantly (P<0.05) increased percent cooking yield only for all-beef and AC treatments at both water levels (Table 4). This supports the conclusion that starch addition to SP samples was not needed to increase cooking yield. Differences in starch effects between SP and the other two meat types explain the significant (P<0.05) meat type by starch interaction. 3.3
Cooked product bind
Statistical analysis of the cooked product binding value results indicated significant main effects (P<0.05) to include: meat type, water level and interaction of the two (Table 5). Starch and replication effects were not significant (P>0.05).
1294 Table 4 Effects of meat type, starch and added water on the cooking yields and binding force of meat products* Product
Starch (%)
Water (%)
Cooking yield (%)
Beef
0 2 0 2
0 0 10 10
74.8 90.0 78.0 97.5
AC
0 2 0 2
0 0 10 10
87.8 95.5 90.8 04.1 104.1
± + + ± + +
Cooked product bind (N)
0.5^ 1.2"^ 0.3^ 0.3^ 1.9''
12.0 10.0 6.0 6.0
± ± ± +
0.00^ 1.4^ 0.0^ 0.0^
± 4.6^ ± 0.7''^ 0.7^^ + 3.1"* 3.1"* ± 2.7^
14.0 23.0 10.5 11.5
± ± + +
1.4^ 1.4^ 1.0** 0.7^
O.l'' 0 0 96.0 ± O.l*' 50.0 ± 9.4^ 97.9 ± 0.4^^ 2 0 0.4^^ 56.0 ± 1.4^ 04.7 ± 0.5^ 0 10 104.7 35.5 ± 4.9*^ 2 10 104.5 04.5 ± 3.2^ 37.0 + 1.4' * Numbers are means ± standard deviations. AC: Algin/calcium restructured beef; SP: Salt/phosphate restructured beef; N = Newtons. ^^ Means in the same column with different superscripts are significantly different (P < 0.05). SP
Analysis of meat type main effects indicated that the values for SP products were greater (P<0.05) than those for AC products, which were greater than those for all-beef. Water addition decreased (P<0.05) cooked product bind values when averaged over all treatments. Salt and phosphate should have affected cooked product bind of the SP treatment by mechanisms similar to those discussed for percent cook yield, which includes increased pH and ionic strength, phosphate binding to meat proteins, protein solubilization and chelating metal ions (Bendall, 1954; Voyle et al., 1984; Lewis et al., 1986; Trout and Schmidt, 1986a,b, 1987; Paterson et al., 1988). Table 5 Analysis of variance table for cooked product bind Source DF Model 10 Error 13 2 Meat type (MT) Starch level (SL) 1 Water level (WL) 1 Replication 1 MTxSL 2 MTxWL 2 SLxWL 1 DF = Degrees of freedom.
Mean squares 678.641 12.247 2982.125 40.042 570.375 0.042 20.042 76.625 18.375
F-value
P-value
55.41
< 0.0001
243.50 3.27 46.57 0.00 1.64 6.26 1.50
< 0.0001 >0.05 < 0.0001 >0.9 >0.05 <0.01 >0.05
1295 Ten percent water added to SP samples at both starch levels and for AC samples containing added starch significantly (P<0.05) decreased bind, but no effects were observed for all-beef samples (Table 4). Although cook bind for SP products was reduced following water addition, values were three and six times greater than AC and all-beef treatments, respectively. Differences in bind between water-added AC and all-beef treatments were not significant (P>0.05), although values for all-beef were 40-50% less than AC products (Table 4). The slight but nonsignificant increases in cooked bind for AC products without starch addition compared to the all-beef control suggest that the algin/calcium binder may have contributed to binding through a structural component similar to a protein matrix physically binding water or by absorbent water as a hydrocoUoid. Of more interest is the significant (P<0.05) increase in bind when starch was added to AC products, suggesting a synergistic effect between algin/calcium and starch. Starch also affected cook yield, but the increases were observed for both all-beef and AC, which does not imply starch and algin/calcium interactions, although they may exist. Measurements of cook bind in this experiment were described as the force required to fracture the bonds or interactions between adjacent particles, such as protein-protein, carbohydrate-carbohydrate or protein-carbohydrate. Differences between SP and all-beef samples illustrated increased protein-protein interactions followed with subsequent increases in percent cook yield and cook product bind resulting from salt and phosphate addition. Differences between AC and all-beef samples illustrated increased percent cooking yield and was supported by similarities between starch-added AC and SP products at both water levels. Addition of sodium alginate, calcium carbonate and acidifier has not been shown to increase protein extraction and protein-protein interactions, thus it is logical to assume that the increased cook yield was due to protein-carbohydrate or carbohydrate-carbohydrate interactions involving both starch and alginate reacting separately or in combination with beef proteins. Similar comparisons of cook bind show that salt and phosphate developed greater bind than AC at all combinations of starch and water addition. This suggests that there are differences in binding mechanisms between salt and phosphate and those for carbohydrate ingredients, such as alginate. 4.
CONCLUSIONS
The experimental results of the study have shown that percent cooking yield for algin/calcium restructured beef (AC) containing added starch (2%) and water (10%) was not significantly (P>0.05) different than that of salt/phosphate (SP) samples when cooked in a closed system. Addition of starch increased cooked bind of AC products, but cooked bind of AC samples was lower than that of SP treatments. The cooked bind of AC samples was higher, but was not different from that of all-beef samples, and addition of starch and water did not reduce cook bind relative to AC controls without added starch or water. There may be a synergistic effect on cook bind between AC ingredients and starch. Thus, AC beef may provide an alternative to the use of salt and phosphate in restructured meats that does not require the use of salt but maintains functional and economic advantages afforded by use of salt and phosphate. The ability to maximize the potential for this will be related to
1296 maintaining an acceptable level of cook bind. Differences in starch variety as well as modifications during starch processing may affect starch-starch and starch-protein interactions as related to cook bind. Sensory panel evaluations may better determine changes in texture resulting from addition of starch and subsequent increases in cook yield. The results of this study indicate the need for further work designed to investigate potential interactive mechanisms between the functional ingredients, beef proteins, algin/calcium gels and starch. Investigations on microbial growth resulting from starch addition to beef not having the preservative effects of salt would be necessary since combinations of starch and meat may present an environment well suited to undesirable microbial growth. 5.
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1300 Sison, E.G., E.G. Almira and A.B. Naval, Philippine Agriculturalist, 58 (1974) 367-372. Smidsrod, O., Faraday Discuss. Ghem. Soc, 57 (1974) 263-274. Smidsrod, O. and A. Haug, Acta Ghem. Scand., 26 (1972) 2063-2074. Sofos, J.N., J. Food Sci., 48 (1983a) 1684-1691. Sofos, J.N., J. Food Sci., 48 (1983b) 1692-1699. Sofos, J.N., J. Food Sci., 50 (1985) 1571-1575. Sofos, J.N. and G.E. Allen, J. Food Sci., 42 (1977) 875-878. Stainsby, G., Food Ghemistry, 6 (1980) 3-14. Swinkels, J.J.M., Starch., 37 (1985) 1-5. Theno, D.M., D.G. Siegel and G.R. Schmidt, J. Food Sci., 43 (1978) 483-487. Trout, G.R. and G.R. Schmidt. Utilization of phosphates in meat products. Gited in: Proc. of the 36th Annual Reciprocal Meat Gonference. Natl. Livestock and Meat Board, Ghicago, IL, 1983. Trout, G.R. and G.R. Schmidt, J. Food Sci., 511 (1986a) 416-1423. Trout, G.R. and G.R. Schmidt, J. Agric. Food Ghem., 34 (1986b) 41-45. Trout, G.R. and G.R. Schmidt, Meat Sci., 20 (1987) 129-147. United States Department of Agriculture (USDA). Men 19-50 years, 1 day, Nationwide food consumption survey, continuing survey of food intakes by individuals. Report No. 85-3, 1985a. United States Department of Agriculture (USDA). Women 19-50 years and their children 1-5 years, 1 day. Nationwide food consumption survey, continuing survey of food intakes by individuals. Report No. 85-1, 1985b. United States Department of Agriculture (USDA), Federal Register 51(159) (1986) 2945629458. United States Department of Agriculture (USDA), Federal Register 53(50) 1988a 8425-8429. United States Department of Agriculture (USDA). Meats processed under federal inspection. In: Meat Facts, 1988. American Meat Institute, Arlington, VA, 1988b
1301 Voyle, C.A., P.D. Jolly and G.W. Offer, Food Microstructure, 3 (1984) 113-126. Whiting, R.C., J. Food Sci., 49 (1984a) 1350-1354, 1362. Whiting, R.C., J. Food Sci., 49 (1984b) 1355-1357. Wu, M.C., D.D. Hamann and T.C. Lanier, J. Texture Studies, 16 (1985a) 53-74. Wu, M.C., T.C. Lanier and D.D. Hamann, J. Food Sci., 50 (1985b) 14-19, 25. Wurzburg, O.B. Corn starch and modified starch. In: Products in the Wet Milling Industry, Symp. Proc, Com Refiners Assoc, Ch. 3. Washington, D.C., 1970. Wurzburg, O.B. Starch in the food industry. In: Handbook of Food Additives. T.E. Furia (Ed.), pp. 361-395. CRC Press, Boca Raton, FL, 1972. Wurzburg, O.B. Introduction. In: Modified Starches: Properties and Uses. Wurzburg (Ed.), pp. 3-16. CRC Press, Boca Raton, FL, 1986.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1303
Enzyme generation of free amino adds and its nutriti(xial significancje in procjessed pcK-k meats Fidel Toldra, Monica Flores and M-Concepcion Aristoy Institute de Agroquimica y Tecnologia de Alimentos (C.S.I.C.), Jaime Roig 11, 46010 Valencia, Spain Abstract Proteolytic events taking place during pork meat ageing and curing processes are described. Muscle cathepsins and calpains provide suitable substrate for muscle peptidases and aminopeptidases which ultimately generate small peptides and free amino acids. The levels of small peptides and free amino acids in all meats are higher after processing than before as a result of intense action of those proteases. Increases of the free essential amino acids such as valine, methionine, isoleucine, leucine, phenylalanine, tryptophan and lysine are observed along the conditioning of meat and are especially large in the processing of dry-cured ham. Thus, aged meat but very especially dry-cured ham have an important nutritional significance when the energy intake is low or in poor nutritional quality diet. 1. NUTRITIONAL ASPECTS OF MEAT PROTEINS AND AMINO ACIDS Proteins have many functions in our organism. Amino acids are used for protein synthesis but they also participate in numerous biochemical pathways and act as precursors of active metabolites. Some non-essential amino acids may actually become limiting, in certain physiological states, and the ratio between two amino acids may assume importance. Free amino acids and small peptides are absorbed through the brush-border cells of the intestinal mucosa. Several peptides can exhibit physiological effects beneficial for health. This is the case, for instance, of carnosine which is an effective antioxidant. Requirements for proteins vary with age and physiological status. In fact, they are increased during growth and product secretion. Dietary protein may be also used for energy production if the calory intake is not enough. FAGAVHO/UNU proposed in 1985 [1] the following protein allowances for dietary proteins of good nutritional quality : 2.25-1.17 g/Kg/d for infants (0-2 years), 1.13-0.99 g/Kg/d for children (2-10 years), 1.00-0.86 g/Kg/d for adolescents (10-18 years) and 0.75 g/Kg/d for adults (over 18 years). There are different methods, most of them based on measuring the available lysine, for evaluating the quality of a protein [2] : i) The amino acid score, ii) the biological value, iii) the protein efficiency rate and iv) the
1304 net protein utilization. A new quality evaluation system was recently proposed. It was based on the protein digestibility corrected amino acid score which means that amino acid composition is multiplied by a digestibility factor [3]. Eating high quality proteins can satisfy sensory acceptance but also the requirements for growth and maintenance for a better health. Another interesting aspect to consider is in evaluating and comparing the quantity of protein or amino acids per calorie consumed, also known as protein density. This is becoming usual because of the increased number of elderly in our population. In general, the nutrient composition per calorie is an important consideration [3]. Adults have to meet their protein and amino acid requirements from available protein sources which usually are from animal (meat, fish, egg,...) or plant (legumes, cereals, roots,...) origin. Animal proteins are usually of high quality. These proteins contain relatively high percentage of the essential amino acids and their proportion is such that nutritional requirements can easily be met. In fact, they contain sufficient amounts of all the essential amino acids required for growth and maintenance of body tissues. For this reason, these proteins are considered as high biological value (HBV). As an example, the total amino acids content of pork meat (M. Semimembranosus), given in table 1, shows high levels of all the essential amino acids as well as taurine that is becoming important over the last years because of its role in nerve function. The protein density of meat is dependent upon its fat content so that fattier meats have lower protein density than leaner meats. There are also small variations in the amino acid content of different cuts of the same species but they can be large if connective tissue is present. This is due to the amino acid composition of connective tissue protein which is different from that of skeletal muscle. The reason is that collagen, as main component of the connective tissue, contains higher amounts of proline, hydroxyproline and glycine as well as lower levels of tryptophan, sulfur-containing amino acids and tyrosine [3]. The biological utilization of meat proteins is primarily dependent on its digestibility by gastric juice peptidases, pancreatic (trypsin, chymotrypsin, carboxypeptidases A and B and elastase) and intestinal aminopeptidases and dipeptidases as well as its composition in essential amino acids. It also depends on the absorption or transport of amino acids and di- and tripeptides into the blood [4,5]. On the other hand, plant protein sources are known to be limiting in several essential amino acids. For instance, lysine and sometimes threonine and tryptophan are usually limiting in nuts and cereals, sulfur amino acids are limiting in legumes, ....[1,2]. Foods can be supplemented with amino acids resulting in nutritional improvement. So, for instance, hog pancreatic proteases are used to produce small peptides which are more easily absorbed and are less allergenic that the intact protein. This is the reason why oligopeptide mixtures have high potentials for preventive and therapeutic hypoallergenic formulations [1]. In this sense, proteins in vegetable roots are very rich, 50% or even more, in small peptides and free amino acids. Free essential amino acids are of great importance when the energy intake is low or in a poor nutritional quality diet. Thus, the changes in total and free amino acid composition as a result of meat and meat products processing should be taken into
1305 account when designing protein requirements for children, elderly and diseased people. Table 1. Total content of amino acids in raw pork meat (M. Semimembranosus). Sample was hydrolyzed in 6N HCl containing 0.5% phenol at 110*^0 for 22 hours. Results for each amino acid are expressed as both g/lOOg of raw muscle and relative contribution (%). Total content
Amino acids g/lOOg
%
Aspartic acid
0.91
4.45
Glutamic acid
L58
7.72
Serine
L04
5.08
Glycine
0.99
4.84
jS-alanine
0.28
1.37
Taurine
0.02
0.10
Tyrosine
0.76
3.71
Proline
0.95
4.64
Alanine
L17
5.72
Ornithine
0.02
0.10
Arginine
LSI
8.85
Cysteine
0.21
1.03
Threonine
1.28
6.26
Valine
0.99
4.84
Methionine
1.21
5.91
Isoleucine
0.85
4.15
Leucine
1.60
7.82
Phenylalanine
0.91
4.45
Histidine
1.65
8.07
Lysine
2.23
10.90
TOTAL
20.46
100.00
Essential
1306 2. THE MUSCLE ENZYME SYSTEM IN POSTMORTEM PROTEOLYSIS Numerous changes occur in skeletal muscle during postmortem storage. These changes have been mainly studied in relation to the increase of meat tenderness. Even though the process is complex and not fully understood, the biochemical and structural changes taking place in meat can be explained as the result of a synergistic action of calpains and lysosomal cathepsins [6,7]. Some weakening of myofibrils is also attributed to the high ionic strength in postmortem muscle [6,7]. The main changes consist in the fragmentation of myofibrils as a result of Z-disc weakening, the degradation of desmin, titin and nebulin as well as the appearance of a polypeptide with a molecular mass of 95 KDa and another one around 30 KDa associated to troponin T disappearance [8,9]. Sarcoplasmic proteins also experience some proteolytic degradation during postmortem storage although they do not contribute to increased tenderness [10]. The complete muscle enzyme system involved in the proteolytical events taking place in postmortem muscle is schematized in figure 1. The first step consists in the action of muscle proteinases (calpains I and II, cathepsins B, D and L and the multicatalytic proteinase complex). These enzymes, which are naturally located inside the muscle, have been mainly studied because of their contribution to tenderness. 2.1. Calpains The calpain enzyme system (EC 3.4.22.-) has been widely reviewed [9,11,12]. There is strong evidence indicating them as responsible for postmortem tenderization even though they do not degrade myosin, actin or a -actinin [13]. They degrade Z-disc, titin, nebulin, desmin and troponin-T with the appearance of a characteristic 30 KDa fragment associated to troponin-T degradation. This system consists in four proteins [14]: i) ju-calpain or calpain I which requires 5-70 jLiM of Ca^^ for activity, ii) m-calpain or calpain II which requires 100-200 fiM of Ca^^, iii) a third calpain which is not well known yet [15] and iv) calpastatin, a polypeptide acting as specific inhibitor for calpains. These proteinases have optimal activity at neutral pH. 2.2. Lysosomal cathepsins There are several lysosomal cathepsins located in muscle and of significance to muscle protein degradation. The most important are [16]: i) Cathepsin B (EC 3.4.22.1.), a cysteine proteinase with maximal activity at pH 3.5-6.0, which can hydrolyze myosin, actin, tropomyosin, troponin T and collagen, ii) cathepsin L (EC 3.4.22.15), also a cysteine proteinase with optimal activity at pH 3.0-6.0, which in addition can hydrolyze titin, nebulin, a-actinin, desmin and elastin, iii) cathepsin D (EC 3.4.23.5.), an aspartyl proteinase with optimal activity at acid pH, which can hydrolyze titin, myosin, nebulin, actin and tropomyosin and, finally, iv) cathepsin H (EC 3.4.22.-), a cysteine lysosomal enzyme with exo and endopeptidase activities. Its optimal activity is around pH 6.5-6.8 although the exopeptidase activity is limited to unblocked amino terminals and, furthermore, its endo-proteinase activity is weak [16]. As lysosomes exist in muscle, cathepsins may
1307 collaborate with calpains in hydrolyzing muscle proteins, especially myosin, actin and a-actinin which are not degraded by calpains, in long term processes such as dry-cured meats [17,18].
Myofibrillar and sarooplasmic proteins Cathepsins B, D and L Calpains I and II Multy catalytic proteinase complex
Pdypeptides
Dipeptidyl and Tripeptidyl peptidases
Small peptides
Aminopeptidases Di and Tripeptidases
Free amino adds
Figure 1. Pathway of muscle proteins hydrolysis during meat ageing and ripening.
1308 2.3. Multicatalytic proteinase ocmplex This enzyme has received many names in the scientific literature such as proteasome, macropain, multicatalytic complex,... It has a very large complex structure with cylindrical shape [19] and its activity at all sites is optimal at neutral to weakly basic pH [19]. The enzyme has different kind of proteolytic activity [13] such as trypsin-like, with optimal pH 8.5, chymotrypsin-like, with optimal pH 7.0 and peptidyl-glutamyl-peptide hydrolyzing, optimal pH 7.0. There is also activity for cleaving bonds on the carboxyl side of aromatic and branched chain amino acids and also for bonds between small neutral amino acids [20]. The function of the multicatalytic proteinase complex is still not well understood but it is thought to participate possibly in antigen processing and on various aspects of ubiquitin-dependent proteolysis [21]. It does not degrade myofibrils [13]. 2.4. Peptidases These enzymes constitute another step in the proteolytical chain (see figure 1). There are several peptidases known to exist in muscle and with different names as dipeptidyl peptidases and tripeptidyl peptidases because they remove dipeptides and tripeptides, respectively, from the aminus terminus of proteins and polypeptides. The most important are : i) Dipeptidyl peptidase I (EC 3.4.14.1.) or cathepsin C is a cystein protease located in lysosomes and with optimal pH 5.06.0. This enzyme has a molecular mass about 200 KDa and requires the presence of thiol compounds and halide ions. Its activity ceases when either arginine or lysine appears in the N-terminal or a proline residue on the penultimate peptide bond [22], ii) Dipeptidyl peptidase II (EC 3.4.14.2.) is a serin protease also located in lysosomes. It is not a metal-dependent enzyme [23] and differs from dipeptidyl peptidase I in its substrate specificity and lack of sulhydryl and halide requirements. The enzyme has a molecular mass about 130 KDa and its optimal activity is at pH 4.5-5.5. Although it does not need activators, the rate of hydrolysis is increased in the presence of an alanine or proline residue at the penultimate peptide bond at the N-terminal position [24], iii)Dipeptidyl peptidase III (EC 3.4.14.4.) is located in the cytosol and has an optimal pH around 7.0-8.0 This enzyme has a very low activity in muscle [25], iv) Dipeptidyl peptidase IV (EC 3.4.14.5.), also located in the cytosol, has an optimal pH around 8.0 and removes N-terminal dipeptides when the penultimate residue is proline or alanine [26] and v) Tripeptidyl peptidase is a serin protease located in lysosomes. It has a molecular mass about 200 KDa and an optimal pH 4.0. This enzyme releases tripeptides from the N-terminal [22]. Carboxipeptidase A or cathepsin A and carboxypeptidase B or cathepsin B2 are located in lysosomes and hydrolyze the C-terminal residues from N-acetylated polypeptides [26]. They are serin and cystein proteases, respectively and have an optimal pH around 5.0-5.5 [22,27]. 2.5. Aminopeptidases The last step in the proteolysis observed in postmortem muscle, as reflected in figure 1, is the generation of free amino acids. In fact, there are several studies showing an increase in free amino acids during the postmortem ageing/storage of
1309 meat and contributing to the improvement of meat taste [28-30]. This increase has been attributed to the action of muscle aminopeptidases active at neutral pH [3133]. A noticeable increase in free amino acids has been observed in other processed pork products such as dry-cured sausages [34,35] and dry-cured ham [36,37] and has been also related to muscle aminopeptidases [38]. Aminopeptidases are enzymes which hydrolyze peptide bonds near the amino terminus of many proteins and polypeptides. Dipeptidases (prolyl-, proline-, glycylleucine- and glycyl-glycine-) and tripeptidases are a kind of aminopeptidases, located in the lysosomes, which hydrolyze the peptide bond of the amino terminus of dipeptides and tripeptides, respectively [22,26] although these particular enzymes will not be further discussed in this chapter. The role of aminopeptidases in muscle is presumably in the latter stages of protein breakdown. They degrade peptides by removing single amino acid residues sequentially from the N-terminus and can have great importance in peptide turnover [39] as well as in the pathogenesis of many diseases [39,40]. As these enzymes are directly involved in free amino acids generation, they will be widely described. Aminopeptidases may be classified in many different ways although the most usual is according to the relative efficiency with which residues are removed even though they usually present broad specificities. Thus, alanyl aminopeptidase would preferentially hydrolyze peptides with an N-terminal alanine residue while leucyl aminopeptidase would hydrolyze preferentially those peptide bonds adjacent to an N-terminal leucine residue. There are several aminopeptidases known to exist in skeletal muscle [22,39] and their main properties are briefly summarized for a better understanding of their significance in postmortem muscle: 2.5.1. Alanyl amiiK^ieptidase This enzyme (EC 3.4.11.14.) is the most important aminopeptidase in muscle. It has received many different names in the scientific literature. Some of them are thiol-activated or puromycin-sensitive aminopeptidase; major, M-like, cytosol or C aminopeptidase. It accounts for as much as 86% of the total aminopeptidase activity in the cytosolic fraction of skeletal muscle [39,41]. It is also found in other tissues such as brain [42-45], liver [46,47], kidney [48] and lens [49]. Alanyl aminopeptidase has a neutral optimal pH, 6.5-7.0 [43,46,47], and a broad substrate specificity towards aromatic, aliphatic and basic aminoacyl-bonds [41,45,50-52] as shown in table 2. It also has a slight activity towards several dipeptidyl substrates [53] although does not show any endopeptidase activity. This enzyme has a molecular mass about 106 KDa [52] and is considered a metalloenzyme [43] with zinc located near the substrate binding site. Other authors [54] suggested that the enzyme forms a cobalt+metal-enzyme complex. The enzyme is highly stimulated by Ca""^ [22,39,45,50], Co^^ [46,47,51,55] ions and sulfhydryl compounds such as 2-mercaptoethanol and dithiothreitol [46,47,49,50]. There are several effective inhibitors as metal-chelating agents, sulfhydryl reagents, bestatin or puromycin [22,43,46,47]. The enzyme seems to have an essential cysteinyl residue since the activity is enhanced by sulfhydryl compounds and inhibited by sulfhydryl reagents as p-chloromercurybenzoate [44,47,49,56]. It is very stable when stored at 5''C [52] as shown in table 3.
1310 Table 2. Activity of the purified soluble alanyl aminopeptidase and aminopeptidase B on aminoacyl-amidomethylcoumarin derivatives. Both enzymes were purified from porcine skeletal muscle and assayed as previously described [52,62]. Amino acids-AMC
Alanyl Aminopeptidase *
Aminopeptidase B *
Phenylalanine-
210.0
5.9
Lysine-
130.0
47.0
Methionine-
124.0
Alanine-
100.0
2.5
Leucine-
98.0
0.2
Arginine-
64.0
100.0
Tyrosine-
10.7
0.0
Serine-
7.3
0.0
Proline-
5.8
5.1
Glycine-
5.0
0.0
7-Glutamic acid-
0.0
6.0
Pyroglutamic acid-
0.0
0.0
Valine-
3.3
7.4
Gly-Arg-
2.5
Arg-Arg-
4.8
Lys-Ala-
0.7
N-CBZ-Phe-Arg-
0.0
0.0
Z-Arg-Arg-
0.0
0.0
* Activity is expressed as a percentage relative to the alanine and arginine derivatives, respectively. 2.5.2. Aminopeptidase B This enzyme (EC 3.4.11.6.) is located in the cytosol. It represents a 11% of the total aminopeptidase activity in the cytosolic fraction of skeletal muscle. It is also found in other tissues [56-58] and organs as liver [59,60] and brain [61]. Aminopeptidase B purified from porcine skeletal muscle has been characterized as a chloride-activated enzyme hydrolyzing basic termini [62]. This enzyme catalyzes the release of arginine, lysine [39,58,60,61] and, although at a lower
1311 rate, phenylalanine, valine, proline and alanine (see table 2). It has a molecular mass of 76 KDa and an optimal pH around 6.5 in the presence of 0.2 M of NaCl. Inhibition by EDTA and phenanthroline [56,57,59,61,62] suggests that metal ions are essential to the activity although other authors [63] could not demonstrated it by metal analysis. Other inhibitors are bestatin and arphamenine B [64]. Neither amastatin, which is a powerful inhibitor of the leucyl aminopeptidase [22,61,61], nor puromycin, a powerful inhibitor of the alanyl aminopeptidase [50,62,65], inhibit aminopeptidase B. There are other basic termini hydrolyzing enzymes such as cathepsin H and hydrolase H [65] although they are different several properties such as molecular mass and inhibition by E-64 and bestatin, respectively. It is not so stable as alanyl aminopeptidase but its half-life is also significative at the temperatures shown in table 3. Table 3. Half-life, expressed in hours, of the purified soluble alanyl aminopeptidase and aminopeptidase B incubated at pH 7.0 and different temperatures. Activities were assayed as previously described [52,62]. Temperature (°C)
Alanyl Ai
Dpeptidase
Aminopeptidase B
5
982.10
126.800
15
138.10
75.500
25
49.20
68.600
35
26.20
25.700
50
11.90
2.200
65
0.02
0.008
2.5.3. Leucyl aimnopeptidase This aminopeptidase (EC 3.4.11.1.) is a zinc metalloenzyme located in the cytosol and with optimal al]5:aline pH. This enzyme is the most extensively studied aminopeptidase, especially in bovine lens [66], and its structure completely determined [40,67]. The molecular mass is 324 KDa and consists of six subunits of 54 KDa each. There are two zinc ions per subunit. Leucyl aminopeptidase catalyzes the release of leucine and methionine as well as hydrophobic amino acids hke tyrosine and phenylalanine from the N-terminal [68] even though it can also remove other residues but at lower rates [67]. The enzyme is activated by Mn^^ and Mg^2 [g9.Y2] and inhibited by Ni^^ Cu^^ Zn^^ Hg^^ ^^^ Q^^2 [73J Leucyl aminopeptidase is also inhibited by bestatin, amastatin and EDTA [67,74].
1312 2.5.4. I^ro^utamyl peptidase I This is a particular enzyme with omega peptidase activity (EC 3.4.19.3.). It is widely distributed in the cytosol and has an optimal pH around 7.5 and a molecular mass about 24 KDa. This enzyme catalyzes the release of N-terminal pyroglutamyl group from polypeptides. It can also hydrolyze several synthetic peptides with a five-membered ureido ring at the amino terminus but can not appreciably catalyze the hydrolysis of peptides with six-membered ureido rings at this position. So, this enzyme is very sensitive to the ring size of the terminating group but less sensitive to the detailed structure [75]. It is inhibited by phenanthrolin and thiol-blocking reagents [22,39,53]. 2.5.5. Glutamyl amiiK^jeptidase It is also known as a-aminopeptidase (EC 3.4.11.7.) because specifically hydrolyzes aspartyl or glutamyl residues from peptides. It has an optimal pH of 7.5 and is activated by Ca"^^ and Ba"^^ [22,74]. The molecular mass is very high, around 350-400 KDa for the detergent solubilized enzyme and 250-270 KDa for the protease solubilized P-form [22]. Its activity in muscle is very low and, in fact, it only accounts less than 0.2% of the total aminopeptidase activity in the cytosolic fraction [39]. 3. EFFECTS OF PROCESSING ON AMINO ACIDS AVAILABIUTY Processing and storage of meat and meat products might affect possitively or negatively the nutritional properties. Water activity and pH are important factors affecting degradation rates but also the degree of thermal processing as in cooked meats or time and temperature of ripening in the case of fermented sausages or dry-cured ham may contribute to a higher or lower extent of nutritional degradation. Most of the amino acids of meat are resistant to the effects of cooking. A notable exception are reductions in the availability of lysine, methionine and tryptophan. The essential amino acid methionine is limiting in many foods because it is oxidized or its binding to proteins reduce its nutritional availability because of poor digestibility. On the other hand, tryptophan losses during food processing are difficult to be monitored because of the lack of reliable anal5d:ical methods [76]. However, this amino acid is stable in the absence of oxidizing agents even during heat treatments such as industrial or home cooking but can be significantly degraded in severe treatments at high temperatures [76]. There are several reactions involved in the loss of availability of amino acids: The Maillard reaction is the most important carbonyl-amine reaction. Maillard reactions of proteins, peptides and free amino acids with sugars cause deterioration of nutritional contents during meat processing. The rate and extent is affected by water activity, temperature, concentration and type of reactants, pH and is also dependent on the type of carbohydrate present. Lysine, methionine and tryptophan are especially degraded as a result of advanced Maillard reactions [77]. The result is a loss of bioavailability of these residues causing a loss of nutritional
1313 quality by decreasing digestibility or absorption [78-80]. The biological consequences of the Maillard reaction upon lysine has been extensively studied and confirmed to be substantial [81,82] while the loss of tryptophan is only slightly influenced [76]. Amino acids can also be lost through other reactions ocurring when cooking. Amino acids may also react with sugars or their degradation products to yield pyrazines [83] when heating at 70''C and increasing to a maximum at 120''C [84]. Threonine and serine may also form pyrazines just by heating [85]. However, no pyrazines could be found when glycine, alanine, phenylalanine, jS-alanine, leucine, isoleucine, valine, methionine, cystine, tyrosine, histidine, proline, hydroxyproline, tryptophan, lysine, aspartic acid, asparagine, glutamic acid or glutamine were individually subjected to pyrolysis [86]. Other associated reactions which usually occur simultaneously to Maillard reactions are the racemization of the chiral a-carbon in amino acid residues and/or cross-linking of the protein chain [87]. Under these circumstances different reactions might happen. One of the most important is the lysinoalanine formation which consists in a 2 step process: first, the formation of a dehydroalanine intermediate resulting from hydroxide ion-catalyzed elimination reactions of serine, threonine and cystine and, second, that intermediate reacts with the eamino group of lysine to form a lysinoalanine cross-link [80]. This compound causes histological changes in the descending portions of the proximal tubules of rat kidneys [80]. The enzyme transglutaminase catalyzes the formation of 7-glutamyl-£-N-lysyl bonds [88] even though these peptide bonds appear to be as digestible as other peptide bonds and, thus, is totally available as a source of lysine [89]. Other crosslinking reactions resulting from heating of proteins consist in the amidation or transamidation reactions between aspartic acid, glutamic acid, asparagine or glutamine and the s-amine group of lysine [90]. However, the extent of these reactions in processed pork meat products is quite low because heat treatment is far from alkaline conditions which are optimal for maximal reaction rates. Racemization and cross-linking of proteins have a reduction effect on protein digestibility and biological value. Racemization may form D-L, L-D and D-D peptide bonds inaccessible to proteolytic enzymes so that nonmetabolizable or nonutilizable forms of amino acids have been generated [80]. D-amino acids are not utilized by humans with the exception of D-phenylalanine which has ben reported to be partially utilized [87,91].
4. FREE AMINO ACIDS GENERATION DURING MEAT CONDITIONING Changes in the levels of free amino acids during the refrigerated storage (2-4''C) of pork meat for 7 days are shown in table 4. As can be observed, all free amino acids except for jS-alanine, arginine, proline and tryptophan, increased during storage. The larger and significative (P<0.05) increases were for aspartic acid (1.03 mg/100 g), glutamic acid (2.35 mg/100 g), glutamine (4.35 mg/100 g), taurine (3.07
1314 Table 4. Changes in the free amino acids contents in the muscle Longissimus dorsi during the refrigerated storage of pork meat. Results are expressed as means (mg free amino acid/lOOg muscle) of 6 samples and standard deviation (SD). Amino acids
Raw meat
Aged meat
mean
SD
mean
Aspartic acid*
0.39
0.09
1.42
0.23
+1.03
Glutamic acid*
2.03
0.75
4.38
0.09
+2.35
2.02
0.39
2.74
0.44
+0.72
Asparagine
0.91
0.17
1.15
0.17
+0.24
Glycine
6.01
0.42
6.22
0.99
+0.21
38.88
4.96
43.23
2.85
+4.35
/3-Alanine
1.99
0.46
1.85
0.70
-0.14
Taurine*
18.83
2.42
21.90
4.12
+3.07
Tyrosine*
2.11
0.14
2.98
0.31
+0.87
Proline
2.83
0.28
2.70
0.27
-0.13
Alanine
11.29
1.23
11.76
0.96
+0.47
Ornithine
0.83
0.18
0.90
0.36
+0.07
Arginine
5.19
0.51
4.60
0.42
-0.59
Threonine
2.86
0.38
3.08
0.33
+0.22
Valine
2.78
0.22
3.22
0.32
+0.44
Methionine*
0.90
0.12
2.02
0.29
+1.12
Isoleucine*
1.52
0.10
2.27
0.21
+0.75
Leucine*
2.43
0.28
3.45
0.37
+1.02
Phenylalanine*
1.51
0.19
2.30
0.26
+0.79
Tryptophan
0.29
0.11
0.28
0.02
-0.01
Histidine*
2.90
0.24
3.67
0.52
+0.77
Lysine
2.57
0.46
3.24
0.38
+0.67
Serine
Glutamine*
SD
Increment A
Essential
Significantly different at P > 0.05
1315 mg/100 g), histidine (0.77 mg/100 g), tyrosine (0.87 mg/100 g), methionine (1.12 mg/100 g), isoleucine (0.75 mg/100 g), leucine (1.02 mg/100 g) and phenylalanine (0.79 mg/100 g). A greater free amino acids release was observed by other authors [28,29,31] in meat homogenates with significant increases of aspartic acid, glutamic acid, serine, methionine, isoleucine, leucine, tyrosine, phenylalanine, arginine, asparagine and proline but in that case proteolysis was faster because of the previous homogenization and there were more peptides produced by endopeptidases. Similar increases have been observed in beef, chicken [28,29] and rabbit [31]. Muscle aminopeptidases remain active along the complete conditioning (see table 5) and keep most of their original activity so that they have full capability to hydrolyze peptide bonds at the N-terminal position. Table 5. Muscle aminopeptidase activity in raw and aged pork meat (7 days at 2-4''C) and in cooked and dry-cured ham. Activities were assayed in M. Biceps femoris as previously described [52,62] and are expressed as ^mol of hydrolyzed substrate per hour and gram of muscle (U/g). Aminopeptidase
Raw
Aged
Cooked
Dry-cured
Alanyl Ap.
9.10
8.50
0.011
2.79
Ap. B
1.26
1.40
0.002
0.07
Leu Ap.
0.28
0.31
0.000
0.07
Pyroglu Ap.
0.50
0.44
0.002
0.02
Comparing the increase of free amino acids during the meat conditioning with the substrate specificity of muscle aminopeptidases shown in table 2, it would be possible to get an idea of the degree of contribution of these enzymes. So, aminopeptidase B which mainly hydrolyzes basic amino acids such as arginine and lysine would not contribute significantly because the lack of basic amino acids release. On the other hand, alanyl aminopeptidase has a very broad specificity (see table 2) and can hydrolyze phenylalanine, lysine, methionine, leucine, arginine, alanine, tyrosine, serine, proline, valine and glycine which is in great accordance to the free amino acids generated during storage. This enzyme is also dependent on the pork breed type [92]. Glutamyl aminopeptidase can also participate in the generation of glutamic and aspartic acids even though its activity is very low in muscle. Finally, leucyl aminopeptidase could contribute restrictively because the pH of meat, about 5.6-6.3, is far from its optimal pH for maximal activity, 9.0-9.5. These facts indicate that alanyl aminopeptidase would be the major exopeptidase contributing to the release of free amino acids during meat ageing.
1316 Glutamyl and leucyl aminopeptidases would participate in a lower degree. Finally, the contribution of aminopeptidase B seems to be almost negligible. 5. FREEAMNOACIDS GENERATION DUKING CURED MEATPROCESSING Salt, nitrate and/or nitrite constitute the main curing ingredients. There is a wide variety of cured meat products although most of them can be classified as wet- or dry-cured [93]. Cooked and dry-cured ham are good representatives of both kind of respective curing procedures. 5.1. Cod^^ed ham It is one of the most important wet-cured meat products. The curing ingredients are dissolved in water to form a pickle or brine which will penetrate into the meat by injection [93]. Cooking may slightly affect lysine and methionine but, very especially, tryptophan [3]. The generation of free amino acids during the cooked ham processing is shown in table 6. As can be observed, there is no tryptophan at the end of the process confirming its complete degradation. Glutamine also disappears although partially. In general, there is an important increase in the content of free amino acids due to the process. The larger increases correspond to aspartic acid (14.6 mg/100 g), glutamic acid (36.8 mg/100 g), alanine (12.7 mg/100 g), serine (8.0 mg/100 g), arginine (10.4 mg/100 g) and lysine (9.5 mg/100 g). Thermal treatment consists in a heat gradient through the ham until reaching 69''C at the core and then cooling till refrigeration temperatures. The activity of the alanyl aminopeptidase at the end of the process is quite low, as reflected in table 5, while the activity of the rest of aminopeptidase is almost negligible. However, the half-lives of both purified alanyl aminopeptidase and aminopeptidase B were determined in our laboratory and are shown in table 3. Both enzymes have significative half-lives such as 11.9 and 2.2 hours, respectively, when incubated at 50''C. Furthermore, 50''C is the optimal temperature for alanyl aminopeptidase [52]. It means that both but especially alanyl aminopeptidase can be very active during thermal treatment and thus can participate in a very active way in the generation of free amino acids even though the enzyme activity is extremely low at the end of the process as reflected in table 5. Acid and basic amino acids would be generated as a result of glutamyl aminopeptidase and aminopeptidase B, respectively. Furthermore, these enzymes have also showed activity when incubated in the presence of all the curing ingredients in model solutions [94,95]. 5.2. Dry-cured ham This is a traditional meat product where curing ingredients, without any added water, are rubbed into the surface of hams. The origin of the process is lost in the antiquity but it has many variations depending on the area and traditions. In general, the process consists in a salting (about 9-11 days at 2-4''C), post-salting (20-40 days at 2-4T), resting (1-2 months at 6-9^C) and ripening/drying (6-12 months at 12-20''C). The thermal treatment is very mild in opposition to cooked meat products.
1317 Table 6. Changes in the free amino acids contents in the muscle Biceps femoris during the processing of cooked and dry-cured ham. Results are expressed as means of 6 samples (mg free amino acid/lOOg muscle) and net increments (A) in relation to raw meat. Amino acids
Raw Meat
Cooked
ham
Dry-cured
ham
mean
A
14.60
301.07
300.37
42.25
36.85
498.63
493.23
2.38
10.40
8.03
250.30
247.93
Asparagine
1.03
2.25
1.23
27.10
26.08
Glycine
7.30
13.83
6.53
216.10
208.80
25.43
13.20
- 12.23
24.60
-0.83
Tyrosine
2.20
7.60
5.40
171.90
169.70
Proline
3.18
7.28
4.10
288.63
285.46
Alanine
14.50
27.25
12.75
389.33
374.83
Ornithine
1.21
2.50
1.30
56.17
54.96
Arginine
3.88
14.30
10.43
230.80
226.93
Threonine
3.30
9.05
5.76
279.90
276.61
Valine
3.40
7.60
4.20
315.47
312.07
Methionine
1.37
4.40
3.03
133.70
132.33
Isoleucine
1.78
5.90
4.13
218.30
216.53
Leucine
2.82
6.80
3.98
342.67
339.85
Phenylalanine
2.03
6.98
4.95
209.17
207.14
Tryptophan
0.75
0.00
-0.75
31.43
30.68
Histidine
3.20
10.95
7.75
97.97
94.77
Lysine
3.12
12.60
9.48
734.57
731.45
mean
mean
Aspartic acid
0.70
15.30
Glutamic acid
5.40
Serine
Glutamine
A
Essential
The generation of free amino acids in dry-cured ham is incredibly high, as shown in table 6, confirming an intense degree of proteolysis [36,37,96]. There are very large increases of lysine (731.5 mg/100 g), alanine (374.8 mg/100 g), leucine (339.9
1318 mg/100 g), aspartic (300.4 mg/100 g) and glutamic (493.2 mg/100 g) acids, valine (312.1 mg/100 g), proline (285.5 mg/100 g), threonine (276.6 mg/100 g), serine (247.9 mg/100 g), arginine (226.9 mg/100 g), phenylalanine (207.1 mg/100 g) and tyrosine (169.7 mg/100 g). Similar large increases have been observed in CountryStyle [97] and Parma [98] hams. The high content of lysine (734.6 mg/100 g) indicates a high degree of digestibility of ham proteins since this amino acid reflects availability for absorption without need for further digestion [99,100]. A loss in this amino acid would cause a loss in nutritional quality. On the other hand, the amount of taurine is as high as 81.2 mg/lOOg. This residue has increased in importance over the last years [101,102] because it seems to be essential for cats and probably primates including humans [103] and also would play a role in nerve function [104]. The enzymology of dry-curing processes has been recently reviewed [105]. Cathepsins are active through the complete process [17] and a high degree of proteolysis is observed [18,36,37,96] while the activity of calpains is restricted to the initial stage [105]. Aminopeptidases also show some activity at the end of the process, as reflected in table 5, showing a good stability also confirmed in previous assays with the pure enzymes [52,62]. Both groups of enzymes, cathepsins and aminopeptidases, have been assayed in model solutions representing different conditions and stages of the process and have showed significative levels of activity [94,95,106-108]. The major increases in free amino acids during the processing of dry-cured ham is in good accordance with the specificity of the alanyl aminopeptidase and, in fact, this enzyme seems to be the most important. Aminopeptidase B which is activated by the chloride present in the ham in the early stages, would also contribute in generating the basic amino acids while glutamyl aminopeptidase would generate the aspartic and glutamic acids. Finally, leucyl aminopeptidase would have a minor role since its activity is restricted by the pH in ham, 6.0-6.4, which is far from the optimum. In summary, processed pork meats present higher levels of free amino acids than raw postmortem muscle. Cooked ham and very especially dry-cured ham constitute a concentrated source of free essential amino acids which can be an advantage in diets of poor nutritional quality or for those individuals with high protein requirements but small apetites such as children or some convalescents. Acknovdedgements Grant AIR2-CT93-1757 from the EC is acknowledged. 6. REFERENCES 1 2 3
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1323
IsdaticHi d£ flavor peptides from raw pork meat and dry-cured ham M-Concepci6n Aristoy and Fidel Toldra Institute de Agroquimica y Tecnologia de Alimentos (C.S.I.C.), Jaime Roig 11, 46010 Valencia, Spain Abstract Many flavor peptides in raw pork meat and dry-cured ham are generated as a consequence of muscle protein degradation. Low molecular mass peptides (Mr<3000 Da) of the water-soluble extract from both pork meat and dry-cured ham were fractionated by gel filtration chromatography. The fractions were found to have a wide range of flavors as revealed by sensory testing. Bitterness was detected in the earlier-running fractions (molecular mass around 1800 Da) and a slightly acid taste in the latter-running fractions (below 1000 Da). A savoury ham flavor when assaying dry-cured ham extracts and a brothy/umami flavor in the case of meat were detected at the molecular mass range 1500-1700 Da. Peptide profiling of these desirable fractions by reverse-phase HPLC and free solution capillary electrophoresis showed that they were composed largely of compounds thought to be hydrophilic peptides. 1. FLAVOR COMPOUNDS IN MUSCLE FOODS Raw meat has a blood-like flavor, due to the presence of blood salts and products of pyrolysis and saliva [1], with some overtones due to species, food and environment of the animal. Meat acceptance involves all the consumer's senses. Taste and odor sensations are developed in meat exposed to heat and then masticated [1,2]. Taste may be defined as a sensory attribute of soluble substances perceptible via specific molecular receptors located at the tongue, usually the sweet, bitter, sour, salty and umami sensations [1]. Aroma would de defined as the smell of meat before it is put in the mouth and odor as the retronasal smell of meat in the mouth [2]. Flavor represents an interaction of smell, that is detected in the nose, with the five basic tastes [2]. But there are many factors influencing the flavor of meat such as genetic (species, breed and sex) or other environmental (age, nutrition and stress) factors [3]. There is a similarity in the flavor of meat from lean beef, lamb and pork. So, the flavor precursors of lean meats are in the water-soluble-fraction while the fatsoluble substances would contribute to flavor species differences [4]. There are
1324 many compounds acting as aqueous meat flavor precursors [5,6] but can be mainly grouped in three types : Taste compounds, flavor enhancers and aroma precursors. Sugars, organic acids, amino acids, peptides and nucleotides contribute to taste. Monosodium glutamate and 5'-nucleotides, mainly 5'-IMP and 5'-GMP which are generated in aged meat from ATP, contribute to enhance flavor. The ATP concentration declines rapidly with time post mortem, generating AMP which is further deaminated to IMP. The IMP is further degraded to inosine followed by slow base hydrolysis forming hypoxanthine [7]. Finally, the most important watersoluble aroma precursors are vitamins, in particular thiamine or vitamin B l [6,S]. During postmortem ageing/ripening there is an increase of free amino acids and peptides as a result of an intense proteolysis. These compounds, that are generated as a result of the action of muscle aminopeptidases [9-11], contribute to improve the meat taste [12-14]. The increase in the levels of free amino acids and peptides in pork and chicken during meat conditioning is particularly relevant. These results corresponded to a higher brothy taste intensity in pork and chicken after meat storage [12-14]. In the case of beef however there is an alteration in sensory attributes. Desirable flavors such as beefy and brothy diminish while bitter and sour flavors increase [15]. Sulfur components, including thiols and sulfides, which are reported to be in approximately equal concentrations in beef and pork [16] may have a large flavor impact [17]. These free amino acids important for meat flavor are derived from myofibrillar proteins in view of their high content in sulphur amino acids while proteolysis of collagen or sarcoplasmic proteins is less significative for flavor because of their reduced content of sulphur amino acids [18]. Heat-induced changes have been reported for high molecular mass (21-200 KDa) myofibrillar proteins with an associated increase in the proportion of low molecular mass (<16 KDa) peptides and especially between 2.5 and 1.8 KDa [19]. Peptides with molecular mass below 5 KDa can be considered as presumptive flavor principles since they fit the dimensional characteristics and requirements for ligand-receptor interaction [20]. The concentrations of myofibrillar proteins breakdown products may vary depending on the degree of enzyme hydrolysis. In any case, there is a mixture of proteins, peptides, free amino acids, nucleotides, sugars and other low molecular mass water-soluble components that can react with one another when heating [16]. The result consists in the formation of a large number of volatile compounds which are essential for cooked pork meat flavor [21]. On the other hand, the natural peptides carnosine and anserine do not seem to experience relevant changes during meat ageing [12]. Synthetic extracts prepared with free amino acids, IMP and NaCl reproduced quite well the brothy, umami and salty taste of natural soups, but failed in sourness and bitterness concluding that some other components were required even though they were able to reproduce the brothy taste [12,22,23]. There is always a polar function and a hydrophobic group within the molecule of a bitter compound. The polar function probably affects taste quality while the hydrophobic group would affect taste intensity [24]. The same authors observed that the total length along the peptide chain of the molecule would also affect significatively. The bitter taste of some amino acids and peptides may be decreased by an excess
1325 of glutamic acid as in the case of Swiss cheese [25]. On the other hand, the sweetness of the amino acids is greatly increased with an increase of NaCl concentration [26]. 2. FLAVOR PEPHDES IN BAW FORK MEAT 2.1. Gel filtration Raw pork meat was extracted with 0.1 N HCl solution as previously described [27]. Once centrifuged and concentrated, the water-soluble extract was fractionated in a Sephadex G-25 column. The plots of absorbances (214, 254 and 280 nm) vs elution volumes during gel filtration is shown in figure 1. The taste of the pooled fractions from the gel filtration profile (figure 1) is described in table 1. The fractions were tasted by pipetting a fixed volume onto the tongue of staff volunteers. The results show that the first peak (fraction 1), with a bitter/sour taste, corresponds to polypeptides with Mr higher than 1700 Da while the savoury/brothy tastes were found in fractions 2 and 3 corresponding to Mr between 1500 and 1700. It should be taken into account that this kind of matrix may present ionic interactions affecting elution volumes [28,29]. In fact, previous assays (data not shown) revealed the co-elution of salt with several free amino acids as glutamic acid, methionine and phenylalanine at 250-270 ml of elution volume. On the other hand, tryptophan experiences the greatest retardation and elutes in the peak corresponding to fraction 8. The resulting fractions with brothy/savoury/umami tastes (1-4) were hydrolyzed. The amino acids present in each fraction is shown in table 2. Fraction 1 contains a high proportion of lysine, histidine and j3-alanine. It is known that lysine contributes to bitterness [30]. The highest amount of total amino acids (345.83 ^mol) is in fraction 2. It shows high levels of jS-alanine and histidine resulting from carnosine but also of glutamic acid, threonine, alanine, arginine, proline, valine, isoleucine, leucine and lysine. The levels of taurine and ornitine are also high and contribute to saltiness [14]. Fraction 3 has a noticeable brothy/umami/sweet taste even though the amount of total amino acids is quite low (63.93 jamol). However, there is some glutamic acid and methionine and the brothy/umami taste is also reinforced by 5'-IMP that co-elutes in that fraction. Significative levels of glycine, serine and alanine contribute to the sweetness of that fraction [14,30]. The level of total amino acids in fraction 4 is as low as 6.80 ^mol but the brothy/umami taste is still noticed. Although the levels of arginine and phenylalanine are high, this fraction does not show bitter taste. Bitterness is probably masked by the high relative levels of glycine as reported by other authors [30]. 2.2. Reverse-phase HPLC The obtention of peptide mappings through this technique contributes to a deeper understanding of the path of proteolysis in ageing/ripening processes as well as a better study of proteolytic breakdown products [28,31-33]. The reversephase HPLC peptide profiles of fractions 1 and 3-7 are shown in Figure 2.
1326
I 280nm
0
200
AOO
600
VOLUME (ml)
Figure 1. Fractionation of a water-soluble raw pork meat extract in a Sephadex G-25 gel filtration column (2.6 x 55 cm). The column was previously equilibrated with 0.01 N HCl in pure water and calibrated with standards of molecular mass range between 204 and 6500 Da. The flow rate was 18 ml/hour and 6 ml fractions were collected. Sample (50 g) was previously deproteinized at 4*^0 for 24 hours by ethanol addition (1:3), concentrated to dryness, resuspended up to 10 ml and applied to the column. Table 1. Taste of selected gel filtration fractions from raw pork meat extract. Fraction number
Taste description
1
Bitter, sour, slightly brothy
2
Brothy, salty
3
Brothy/umami, sweet
4
Brothy/umami
5
Slightly bitter
6
Sour
7
No noticeable taste
8
No noticeable taste
1327 Table 2. Amino acid analysis of gel filtration fractions from raw pork meat extract. Fractions were hydrolyzed in 6 N HCl containing 0.5 % phenol at llO^'C for 22 hours. Fractions (Mole percentage)
Amino acids 1
2
3
Aspartic acid
1.19
0.03
0.25
2.08
Glutamic acid
1.30
0.54
8.52
2.49
Serine
3.89
0.16
3.34
2.45
Glycine
5.73
0.01
8.70
61.00
29.78
37.82
13.64
2.13
Taurine
0.83
3.11
21.55
1.91
Tyrosine
0.58
0.01
0.88
0.39
Proline
8.63
0.78
1.29
0.42
Alanine
2.39
5.03
19.67
2.43
Ornithine
0.00
0.46
0.36
1.63
Arginine
6.12
0.33
0.66
7.67
Cysteine+cystine
0.00
0.00
0.00
0.00
Threonine
1.43
0.81
2.68
2.99
Valine
1.28
0.85
1.05
0.50
Methionine
0.62
0.04
1.04
1.66
Isoleucine
0.82
0.53
0.84
0.45
Leucine
2.04
0.81
1.36
0.50
Phenylalanine
0.83
0.15
0.50
6.88
Histidine
19.09
46.96
13.33
1.57
Lysine
13.45
1.32
0.34
0.85
7-aminobutyric acid
0.00
0.25
0.00
0.00
TOTAL (^mol)
6.26
345.83
63.93
6.80
B-Alanine
4
1328
TIME(min)
Figure 2. Reverse-phase HPLC separation of gel filtration raw pork meat fractions 1 and 3-7 on a LiChospher 100 RP-18 (5^m, 250x4 mm). The column was eluted with a linear gradient of 0.1% v/v TFA in water:acetonitrile mixture (99:1) to 0.07% v/v TFA in water acetonitrile (40:60) for 30 min.
1329 Carnosine and anserine, natural meat dipeptides, are eluting at 8-8.5 min in fraction 1. However, the brothy/umami taste detected in fraction 3, and in less scale in fraction 4, shows a noticeable amount of early-eluting hydrophylic peptides. This kind of peptides have been reported to be responsible of savoury/brothy flavors in cheese [28]. Fractions 3 and 4 also show peaks of 5'-IMP and phenylalanine eluting at 11.2 and 17.5 min, respectively. Peptides containing high levels of hydrophobic amino acids are known to impart a bitter flavor in cheese and other foods [20,28]. These kind of peptides exhibit long retention time^^. on a reverse-phase column but this is not the case of meat. In any case, fraction 5 is evaluated as slightly bitter (see table 1) and the chromatogram in figure 2 reveals the presence of tyrosine eluting at 14.4 min. This amino acid is known to impart a bitter taste [30]. Furthermore, hypoxanthine, which is a bitter compound [6], and inosine elute at 12.4 min. The levels of peaks in fraction 7 are extremely low and, in fact, no noticeable taste is detected. Finally, fraction 8 only gives one peak corresponding to tryptophan (data not shown). A reverse-phase HPLC chromatogram of the entire raw pork meat extract is shown in figure 3. Free amino acids are distributed according to their respective polarity. So, polar amino acids as glutamic acid, histidine, lysine, glycine and serine, ... elute at the beginning (6-7min) while the aromatic amino acids such as
5
15
25
TIME (min) Figure 3. Reverse-phase HPLC separation of the pork meat extract.
1330
1
3 ijL
L
E
c o o
LU O
5
r
-z. <
J
lij-jjL_JL_
en o
„i
CO
m
6
<
0
•i_ 1
7 1
i
_Aw
1
15
1
1 J
30 0
A 1
15
A ._
x I 1
30
TIME (min) Figure 4. Capillary electrophoretic separations of gel filtration raw pork meat fractions 1 and 3-7. Running buffer: 60 mM sodium phosphate, pH 2.5, with 60 mM ZnS04. T = 35^C. Running voltage: 20 kV.
1331 tyrosine, phenylalanine and tryptophan elute as late as 14.4, 17.2 and 19.9 min, respectively. Mid-eluting peaks are due to non-polar aliphatic amino acids such as valine, methionine,..., nucleosides and nucleotides (eluting at 11-13 min) and peptides, for instance, natural dipeptides as carnosine and anserine that elute at 8.3 min. Most of the peptides can be considered as hydrophylic because they elute at low ACN percentages in difference to cheese that presents a high proportion of hydrophobic peptides [28]. 2.3. Free Sdiuticm Capillary Electrc^hcxiesis This technique has revolutioned protein research and its use for peptide mapping studies is increasing. Amino acids and peptides are separated as a function of their electronegativity and size so that they elute earlier al lower electronegativities and sizes. Thus, basic amino acids and peptides containing those amino acids, as carnosine and anserine, elute earlier while aromatic amino acids and peptides containing them elute later [34-36]. The addition of Zn"^^ helps to improve the resolution, especially in peptides containing histidine [37]. Fractions 1 and 3-7 from gel filtration were further analyzed by free solution capillary electrophoresis and the electropherograms are shown in figure 4. Nucleosides and nucleotides do not appear under the conditions used in this study. The electropherograms show a different protein/peptide/free amino acid composition for each fraction. So, fraction 1 reveals several peaks corresponding to peptides containing arginine, lysine and histidine such as natural dipeptides carnosine and anserine. Fractions 3 and 4 show a late-eluting peak at 27 min corresponding to phenylalanine. Both fractions differ in the early-eluting peaks. On the other hand, fractions 5 and 6 show a peak due to tyrosine at 28.3 min but fraction 6 presents a late-eluting peak at 24.4 min. This is in accordance to the reverse-phase HPLC chromatograms shown in figure 2. Few peaks appear in fraction 7 while fraction 8 only show one peak corresponding to tryptophan (data not shown). Both fractions have no noticeable taste. 3. DRY-CURED HAM 3.1. Process tedmdogy Dry-cured ham constitutes one of the oldest meat products and, in fact, its origin is lost in antiquity. The process consists in several stages [38]. The raw hams are selected and classified according to pH, color, weight,...and then they are pre-salted with a mixture of curing ingredients consisting in salt, nitrate and/or nitrite and adjuncts (ascorbic acid). These curing ingredients are rubbed onto the lean muscle surface of the ham. Hams are subsequently placed fat side down, completely covered by salt and arranged in single layers, up to six layers, without touching each other. This salting stage usually takes 1-1.5 days per kilogram and allows an initial solubilization of curing ingredients in the moisture present in meat. The temperature is kept at 2-4''C. Once the salting is finished, the excess of cure is washed off and the hams placed for 20 to 50 days in an air-conditioned chamber (2-6^0) with controlled temperature and relative humidity cycles. The
1332 objective of this stage, usually known as post-salting or resting, is to achieve a complete and homogeneous salt distribution or equalization through the entire piece. The hams are then placed in natural or air-conditioned drying chambers with controlled, time, temperature and relative humidity cycles. Temperature is usually held between 14 and 20''C while the relative humidity is decreased from 90 to 70%. This ripening/drying period may be as short as 4 months (rapid process) or extend until 6 to 18 months (slow process). Some of the most well-known dry-cured hams are Spanish Serrano and Iberian hams, Italian Parma ham and French Bayonne ham. In each case, the technology may slightly differ. Other hams such as the American Country-Style and German Westphalia and Northern European hams may be smoked after postsalting and aged for a short period between 1 to 3 months [39]. 3.2. Process biochemistry Many biochemical reactions take place during the entire process. Most of these reactions are of proteolytic or lipolytic nature. Other reactions lead to the oxidation of lipids, protein-lipid interactions or the development of the typical cured meat color due to the formation of nitrosometmyoglobin. 3.2.1. Protedysis The proteolysis is very intense and is extensively reported in the literature as an increase in the non-protein nitrogen [40-42]. There is a change in extractability of several myofibrillar proteins [43] and a marked disappearance of myosin and the simultaneous appearance of a 150, 95 and 16 KDa fragments and numerous polypeptides in the ranges 50-100 KDa and 20-45 KDa [44]. The enzymology of dry-cured ham has been reviewed [38]. There is activity of cathepsins B, H and L along the complete process [45] with significative recoveries even after 15 months except for cathepsin D [44] which is almost negligible after 6 months. An excess of cathepsin activity may result in an excesive softness and tyrosine/peptides precipitation on the cut surface [46,47]. On the other hand, calpains disappear very early in the process proving they are quite unstable enzymes [48,49]. Muscle aminopeptidases are also active along the process and are important since they hydrolyze high amounts of amino acids from proteins and peptides at the Nterminal position [50,51]. In fact, a noticeable increase in the concentration of free amino acids in relation to raw meat is reported for all kind of hams [27,52-54]. 3.2.2. lipolysis Subcutaneous adipose tissue lipolysis is consequence of the action of several endogenous lipases which generate free fatty acids by hydrolysis of the ester bonds with glycerol [55]. These enzymes are the hormone-sensitive lipase and lipoprotein lipase which hydrolyze long chain triacylglycerols at neutral and basic pH, respectively [55]. The monoacylglycerol lipase is responsible for the final production of free fatty acids and glycerol by hydrolyzing mono and diacylglycerols [56]. The intense lipolysis is reported as an increase in the concentration (8-30 times the initial amount) of free fatty acids, especially between 2 and 10 months of process, correlated with a decrease in the concentration of triacylglycerols
1333 [57,58]. Lipoprotein lipase is active up to 5 months of process while the hormonesensitive lipase is active throughout the entire process [57]. Intramuscular lipids also experience an intense lipolysis although mostly in polar lipids [57-60]. There are several muscle lipases involved [55]. The most important are the lysosomal acid lipase and the neutral lipase which hydrolyze triacylglycerols at acid and neutral pH, respectively. Lysosomal acid lipase is active through the complete process even though limited hydrolysis of triacylglycerols has been reported [57,59,61]. There are also several phospholipases (Al and A2) which are capable to hydrolyze phospholipids at the sn-1 and sn-2 acyl esters. Phospholipid content is decreased markedly along the process while free fatty acids increase [57,62]. A good correlation has been reported between both trends confirming phospholipids hydrolysis as the main lipolytic phenomena in muscle [58,61,62]. 3.2.3. Oxidaticm This is a phenomena of auto-oxidation of unsaturated fatty acids that are the substrate for lipid oxidation. In general, it is autocatalytic because the oxidation products themselves catalyze the reaction and accelerate the oxidation rate [63]. There is also enzymic lipid peroxidation associated with muscle microsomes [64,65]. Several long chain polyunsaturated fatty acids decrease after 5 months of process [57]. There are several factors affecting lipid oxidation such as the lipid composition, catalysts (metal cations, heme iron,...) and antioxidants (nitrite in cured meats). NaCl acts as a prooxidant promoting lipid oxidation [66]. Some oxidation products might contribute to the cured meat flavor. So, aldehydes and ketones resulting from lipid oxidation impart different flavors as burnt, sweet, fatty, painty, metallic and rancid [21]. 3.3. FlavtH' development There is little information regarding the origin of cured-meat flavor. Some studies have been carried out trying to relate sensory properties of dry-cured ham to chemical components, either volatile or non-volatile [58,60,67,68]. It is evident that process technology and/or the characteristics of the raw material can seriously affect the biochemical reactions and thus sensory profiles even though the classes of volatile and non-volatile compounds appear to be similar [67]. 3.3.1. Non-volatile oonpounds It is evident that free fatty acids, free amino acids and small peptides contribute to the flavor of many foods [69] and thus they should be also important for dry-cured ham. The intensity of cured meat flavor is greatly influenced by the increase in the levels of salt [70]. It also affects the perception of saltiness and sweetness components in the flavor [71,72]. However the concentration of nitrate and nitrite might be reduced without affecting flavor [70]. In fact, the presence of nitrite has been reported to have no influence on the final flavor [71]. Proteolysis and the total amount of peptides and free amino acids contribute to a strong aged flavor [67]. However, an excess of proteolysis may result in unpleasant off-flavors such as bitter-like or metal aftertaste [46,68]. The same authors reported that an
1334 excess of dry-curing time did not result in a better aged taste. Increased quality of dry-cured ham has been observed in the presence of high levels of tyrosine and lysine while negative effects are attributed to asparagine [67]. Glutamic acid was proved to contribute to saltiness while acid taste was strongly correlated to phenylalanine and isoleucine and negatively correlated to tyrosine [67]. 3.3.2. Vdatile cxmpounds Several studies have been carried out trying to identify the volatile compounds in dry-cured ham. First reports [73] indicated that degradation products of ham lipids, which increase during ham ripening, constituted flavor compounds and also included precursors that are converted to flavor compounds along the process. Other compounds reported to contribute to the flavor were the carbonyl compounds resulting from the autooxidation of the lipids [69]. Their levels were found to increase with time [74]. The compounds in the volatile fraction were tentatively identified [69] and found to be not water-soluble. More than 80 compounds have already been identified in french dry-cured hams [75,76]. These compounds may be originated from the biochemical reactions during the process, from the pig feed or from the technological process [75]. The levels of several ketones and 1-butanol were correlated with the aromas of dry-cured ham. However, rancid aroma was related to aldehydes, ethylacetate, 2,3-pentanedione and nonane [60]. In the case of the Spanish Iberian ham, over 64 volatile compounds have been identified [77]. Italian Parma hams showed the formation of short-chain volatile compounds, 3- and 4-carbon alcohols and esters, with a lateral methyl group contributed possitively to the aroma of ham while long-chain volatiles were negatively correlated [67]. A great decrease in the volatiles concentration has been reported in the cured meats when compared to the uncured [78-80]. The role of nitrite is not clear [71]. In fact, untrained panelists have been unable to differentiate amongst the flavor of nitrite-cured meats from different species such as beef, chicken, mutton and pork. They found that flavor acceptability of nitrite-free and nitrite-cured meat was the same indicating that nitrite is not an essential ingredient [80]. However, it seems that nitrite complexes and stabilizes the membrane lipids [81] so that the addition of nitrite depresses lipid oxidation and reduces the influence due to overtones derived from autooxidation/degradation of their lipid components [80]. 4. FLAVOR PEFnDES IN DRY-CURED HAM 4.1. Gel filtration Dry-cured ham was extracted in the same way as raw pork meat and then fractionated in a Sephadex G-25 column. The chromatograms are shown in figure 5. The taste of the pooled fractions is described in table 3. The results show a bitter/sour peak (fraction 1) that, as in the case of pork meat, corresponds to Mr higher than 1700 Da. In the case of ham, the savoury taste is concentrated in fractions 2 and 3, although the latter combined with saltiness. In fact, there is coelution of salt in fraction 3. The Mr of these fractions are between 1500 and 1700
1335 Da. Fraction 4 has a brothy/umami taste similar to that found in raw pork meat. Fractions 5 and 7 do not have noticeable taste. However, fraction 6 gives a bitter taste which is logical because of the presence of tyrosine and hypoxanthine (data not shown). The resulting fractions (1-4) with savoury/brothy/umami taste were hydrolyzed. The results for amino acids analysis of the fractions show the savoury fractions 2 and 3 to have the highest levels of total amino acids (358.86 and 278.67 ^mol, respectively). They contain the greatest concentrations of glycine, lysine, serine, taurine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine and the lowest levels of cysteine/cystine. A similar study made with the water-soluble fraction of cheese indicated that savoury cheese flavor components were of low Mr (<1000). The fraction richer in methionine and leucine gave the greatest flavor intensity and just in that fraction the NaCl concentration was highest and contributed additionally to the flavor intensity [28]. Savoury fractions in dry-cured ham appear to be richer in methionine and leucine although Mr is slightly higher. Most of the lysine as well as jS-alanine, histidine and cysteine/cystine is in fraction 1 considered as bitter/sour. These data are consistent to other authors [30] who found that the presence of lysine contributes to bitterness. i3-alanine and histidine mainly come from carnosine. On the other hand, fraction 3 contains a high amount of arginine but the levels of glycine and serine are also very high masliing the bitterness due to arginine [30,86], Fraction 4, which gives a brothy/umami taste, has a very high level of phenylalanine. Most of it is in the form of free amino acid. The levels of glutamic acid, serine, glycine, histidine, alanine, methionine and lysine are higher than fraction 4 of raw pork meat, also having a similar taste, but seem to be enough for masking the bitter taste of phenylalanine. 4.2. Reverse-phase HPLC The reverse-phase HPLC peptide profiles of fraction 1-6 are shown in figure 6. Fractions 2 and 3, where a savoury/desirable taste is detected, are relatively rich in hydrophylic nitrogen compounds. This is in accordance to other foods as cheese [28] and fish protein hydrolyzates [82] where desirable flavor was attributed to small peptides with a high content of hydrophylic amino acids, especially glutamyl, aspartyl and seryl residues. Savoury fractions also show a high content of free glutamate and low of aspartate [28]. Fraction 4, which has a brothy/umami taste, shows a lower amount of early-eluting material than fractions 2 and 3. Lateeluting peaks at 14.6 and 17.3 min are due to t5rrosine and phenylalanine, respectively. Other peaks (from approximately 10 min to 13 min) appear in fractions 5 and 6 with absence of hydrophylic peptides. This last fraction, giving a bitter taste, indicates an association between late-eluting material from reversephase columns and bitterness as also observed in cheese [28,83]. Free tryptophan elutes alone in fraction 8 (data not shown) confirming the slightly bitterness of that fraction. Finally, a reverse-phase chromatogram of the complete dry-cured ham extract is shown in figure 7. This is usual in studies following proteolysis profiles along processes [52,84-88]. When comparing this figure with figure 3 of the
1336
LU O
-z. <
o CO <
200
400
600
VOLUME (ml) Figure 5. Fractionation of a water-soluble dry-cured ham extract in a Sephadex G-25 gel filtration column (2.6 x 55 cm). The column was previously equilibrated with 0.01 N HCl in pure water. The flow rate was 18 ml/hour and 6 ml Abactions were collected. Sample (25 g) was previously deproteinized at 4^Q> for 24 hours by ethanol addition (1:3), concentrated to dryness, resuspended up to 10 ml and applied to the column. Table 3. Taste of selected gel filtration fractions from dry-cured ham extract. Fraction number
Taste description
1
Bitter, sour
2
Savoury
3
Savoury, salty
4
Brothy/umami
5
No noticeable taste
6
Bitter
7
No noticeable taste
8
Slightly bitter
1337 Table 4. Amino acid analysis of gel filtration fractions from dry-cured ham extract. Fractions were hydrolyzed in 6 N HCl containing 0.5 % phenol at llO^'C for 22 hours. Fractions (Mole percentage)
Amino acids 1
2
3
Aspartic acid
2.59
1.55
2.19
0.41
Glutamic acid
5.53
3.37
3.43
0.45
Serine
2.18
6.89
11.14
0.87
Glycine
5.61
12.69
15.35
1.63
24.95
1.23
0.70
0.19
Taurine
0.25
3.03
1.61
0.16
Tyrosine
0.04
1.30
0.31
0.00
Proline
4.30
12.64
2.77
0.00
Alanine
2.87
9.90
8.85
0.80
Ornithine
0.54
0.27
0.43
0.23
Arginine
1.12
6.32
16.27
0.00
Cysteine+cystine
5.32
0.40
2.23
0.00
Threonine
2.92
7.60
6.55
0.39
Valine
5.26
7.12
3.30
0.00
Methionine
0.90
5.24
6.51
0.34
Isoleucine
2.00
6.43
3.28
0.05
Leucine
2.52
9.81
4.92
0.08
Phenylalanine
0.27
0.24
5.45
93.58
13.73
2.73
3.70
0.34
Lysine
17.1
1.24
0.45
0.48
7-aminobutyric acid
0.00
0.00
0.56
0.00
195.78
358.86
B-Alanine
Histidine
TOTAL (lumol)
TiB.G?
4
72.23
1338
TIME (mln)
Figure 6. Reverse-phase HPLC separation of gel filtration dry-cured ham fractions 1-6 on a LiChrospher 100 RP-18 (5 fim, 250x4 mm). The column was eluted with a linear gradient of 0.1% TFA in water:acetonitrile mixture (99:1) to 0.07% TFA in water: acetonitrile (40:60) for 30 min.
1339 raw pork meat there is a clear evidence of an intense proteolysis due to the drycuring process. There is a noticeable increase in the levels of all peptides as well as free tyrosine, phenylalanine and tryptophan, eluting at 14.4, 17.2 and 19.9, respectively. On the other hand, it should be mentioned that curing agents also appear in the chromatogram. So, nitrate elutes with the front while ascorbate elutes at around 8 min.
5
15
TIME (min) Figure 7. Reverse-phase HPLC separation of the dry-cured ham extract. 4.3. Free Sdutioti CapUlary Electn^hoaresis Electropherograms of dry-cured ham extracts from gel filtration are shown in figure 8. In general, there are more peaks than in raw pork meat (see figure 4). Fraction 1 shows many peaks distributed along the complete electropherogram. The early-eluting peaks correspond to basic amino acids or peptides containing them. These peaks are present in all fractions except fractions 5 and 6. There is certain similarity between fractions 2 and 3. Both have several early-eluting peaks. The peak due to phenylalanine is higher in fraction 3 than in fraction 2 as
1340
1
2 UUVXSAAAJLJI
e
c o o
CM
n
,3
LU O < GO Q:
oif)
^
ly
kw>y—A>^"
5
DQ <
, 1
0
jU'JLuJ
±
15
jjl i
v*-^
1
1 i
1
6
i
,_ 1
, .___
30 0
TIME (min)
1 - 11 1
15
i_J
30
Figure 8. Capillary electrophoretic separations of gel filtration dry-cured ham fractions 1-6. Running buffer: 60 mM sodium phosphate, pH 2.5, with 60 mM ZnS04. T = 3 5 T . Running voltage : 20 kV.
1341 also observed by reverse-phase HPLC (see figure 6). This is also the case of fraction 4 which confirms the high amount of free phenylalanine as observed in table 4. Fractions 5 and 6 only present a few late-eluting peaks. Acknovdedgements Grant ALI91-0752 from the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT), Spain, is acknowledged.
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1343 52 F. Toldra and M-C. Aristoy, Int. J. Food Sci. Nutr. 44 (1993) 215. 53 M. Bellati, G. Dazzi, R. Chizzolini, F. Palmia and G. Parolari, Ind. Conserve 58 (1983) 143. 54 E.G. Piotrowski, L.L. Zaika and A.E. Wasserman, J. Food Sci. 35 (1970) 321. 55 M-J. Motilva, F. Toldra and J. Flores, Z. Lebensm. Unters. Forsch. 195 (1992) 446. 56 M-J. Motilva, F. Toldra, M-C. Aristoy and J. Flores, J. Food Biochem. 16 (1993) 323. 57 M-J. Motilva, F. Toldra, P. Nieto and J. Flores, Food Chem. 48 (1993) 121. 58 S. Buscailhon and G. Monin, Viand. Prod. Carn. 15 (1994) 23. 59 M-J. Motilva, F. Toldra, M. Nadal and J. Flores, J. Food Sci. 59 (1994), in press. 60 S. Buscailhon, J.L. Berdague, J. Bousset, M. Cornet, G. Gandemer, C. Touraille and G. Monin, Meat Sci. 37 (1994) 229. 61 S. Buscailhon, G. Gandemer and G. Monin, Meat Sci. 37 (1994) 245. 62 J. Flores, P. Nieto, S. Bermell and M-C. Miralles, Rev. Agroquim. Tecnol. Aliment. 25 (1985) 117. 63 D.A. Lillard, In: Lipids as a source of flavor (M.K. Supran ed.) ACS Symp. Series 75, Washington 1978, 68. 64 K.S. Rhee, T.R. Dutson and J.W. Savell, J. Food Biochem. 9 (1985) 27. 65 J. Kanner, M.A. Salan, S. Harel and I. Shegalovich, J. Agric. Food Chem. 39 (1991) 242. 66 K.S. Rhee, Food Technol.42 (1988) 127. 67 M. Careri, A. Mangia, G. Barbieri, L. Bolzoni, R. Virgili and G. parolari, J. Food Sci. 58 (1993) 968. 68 P. Baldini, M. Bellatti, G. Camorali, F. Palmia, G. Parolari, M. Reverberi, G. Pezzani, C. Guerrieri, R. Raczynski and P. Riveldi, Ind. Conserve 67 (1992) 149. 69 D.A. Lillard and J.C. Ayres, Food Technol. 23 (1969) 251. 70 B.D. Eakes and T.N. Blumer, J. Food Sci. 40 (1975) 977. 71 J.I. Gray and A.M. Pearson, Adv. Food Res. 29 (1984) 2. 72 D.A. Froelich, E.A. GuUett and W.R. Usborne, J. Food Sci. 48 (1983) 152. 73 J.D. Kemp, H.C. McCampbellandR.B. Grainger, Food Technol. 11 (1957) 321. 74 H.W. Ockerman, T.N. Blumer and H.B. Craig, J. Food Sci. 29 (1964) 123. 75 J.L. Berdague, C. Denoyer, J-L. Quere and E. Semon, J. Agric. Food Chem. 39 (1991) 1257. 76 S. Buscailhon, J.L. Berdague and G. Monin, J. Sci. Food Agric. 63 (1993) 69. 77 M.O. Lopez, L. Hoz, M.I. Cambero, E. gallardo, G. reglero and J.A. Ordonez, Meat Sci. 30 (1992) 267. 78 N. Ramarathnm, L.J. Rubin and L.D. Diosady, J. Agric. Food Chem. 39 (1991) 344. 79 N. Ramarathnm, L.J. Rubin and L.D. Diosady, J. Agric. Food Chem. 39 (1991) 1839. 80 F. Shahidi, In: Flavor chemistry: Trends and developments (R. Teranishi, R.G. Buttery and F. Shahidi eds.) ACS Symp. Series 388, Washington 1989, 188. 81 A.M. Pearson, J.D. Love and F.B. Shorland, Adv. Food Res. 23 (1977) 1.
1344 82 83 84 85 86 87 88
M. Noguchi, S. Aral, M. Yamashita, H. Kato and M. Fujimaki, J. Agric. Food Chem. 23 (1975) 49. A.J. Cliffe and B.A. Law, Food Chem. 36 (1990) 73. D. Gonzalez, M.C. Polo and M. Ramos, J. Dairy Res. 58 (1991) 363. T.M. Christensen, K.R. Kristiansen and J.S. Madsen, J. Dairy res. 56 (1989) 823. D.W. Stanley, Can. Inst. Food Sci. Technol. J. 14 (1981) 49. M. O'SuUivan and P.F. Fox, J. Dairy Res. 57 (1989) 135. S.H. Mojarro, R. Amado, E. Arrigoni and J. Solms, J. Food Sci. 56 (1991) 943.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1345
The effect of fat content on the quality of ground beef patties N.H. Wong and J.A. Maga Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 80523, U.S.A.
Abstract
Ground beef patties were prepared containing varying fat levels (4,12,20 and 30%). They were cooked under standardized conditions and trained sensory panelists evaluated each treatment for differences in juiciness, tenderness, aroma, beef flavor intensity, and overall eating quality. In addition, cooking loss was determined. In general, increasing fat levels resulted in higher juiciness and tenderness scores (P<.05), and lower cooking yield. However, sensory characteristics such as aroma, beef flavor intensity, and overall eating quality were not statistically affected by fat level (P>.05)
1.
INTRODUCTION
Meat is a highly nutritious food that is an essential part of a healthy diet. Red meat (beef, veal, pork, lamb) is a major component of the American diet supplying approximately 15% of the calories, 27% of the protein, and 28% of the fat consumed in a normal diet [1]. Currently, American per capita consumption of beef is approximately 160 kg yearly. Ground beef, in turn, is one of the least expensive beef products available to consumers, and represents approximately 4% of the American food budget [2]. American food consumption patterns have dramatically changed in the past two decades. Trends indicate a shift in consumption of fats with a decrease in visible, separable fat consumption and an increase in the intake of low-fat animal products such as low-fat milk and fish. Increased consumer concern relative to ground beef with high fat content along with saturated fatty acid and cholesterol consumption has lead to increased consumer emphasis in lowering fat levels in ground beef. Recent research that considered the effects of extremely low levels of fat in ground beef has concentrated mainly on cholesterol and caloric content [3]. The National Restaurant Association conducted a study in 1987 and found that at least half of those interviewed were making a conscious effort to restrict their consumption of fat and cholesterol [4]. The need for further fat reduction in our diets, including ground beef patties, has been further emphasized by the recommendations of the American Cancer Society and the American Heart Association to restrict calories from fat to less than 30% of total caloric intake. Yankelovich [5] found that more than two thirds of consumers surveyed in 1985 had some concerns about health, with one of their major concerns being the amount of fat in their diets. A survey conducted by Burke [6] found that a majority of consumers were limiting the amount of fat, calories, and cholesterol in their
1346 diets. Berry et al. [7] indicated that with exception of younger and lower income consumers (who consider price as most important) leanness is the most important factor in making ground beef purchases. Several studies have shown significantly lower sensory scores for tenderness and juiciness in low-fat ground beef. Kragel et al. [8] found that fat levels above 21% produced greater tenderness and juiciness but did not alter flavor compared to patties formulated to have 9.5% fat. More recently, Huffman et al. [9] reported improvements in juiciness with increases in fat from 5 to 20%, but tenderness and flavor did not change as a result of variations in fat level. Berry and Hasty [7] evaluated the effects of low-fat levels on sensory, shear, cooking, and chemical properties of ground beef patties. Samples were targeted at 0, 4, 8, 12, and 16% fat and were cooked to achieve similar cooking yields. The results illustrated that tenderness, juiciness, and flavor ratings decreased as fat levels decreased. Patties processed with 0% fat were rated lower in juiciness and flavor compared to all other fat levels. Cross et al. [11] evaluated the effects of fat level and source on the chemical, sensory, and cooking properties of ground beef patties. In this experiment, ground beef patties were prepared from varying fat levels to final raw fat contents of 16, 20, 24, and 28%. Sensory characteristics such as tenderness, juiciness, connective tissue amount, and flavor intensity were evaluated by trained sensory panelists. Cooking losses were also evaluated as part of other patty characteristics. The results showed that higher tenderness and juiciness scores resulted from higher fat in formulation. However, cooking losses was not affected by fat level. They concluded that sensory ratings and cooking properties were not significantly affected by fat source. Due to the change of food patterns in the past 20 years, developing a low-fat beef patty to meet consumer demand is of great importance. Meat palatability is dependent upon factors such as flavor, juiciness, tenderness, and appearance. Generally, lean ground beef has a lower palatability linked with the decrease in fat. Therefore, acceptance palatability must remain an important consideration in any effort to reduce fat in mat products. Sensory properties such as tenderness, juiciness, beef flavor intensity, aroma, and overall eating quality are important to test the overall acceptability of the product. Laboratory and consumer studies have shown that tenderness is the most important sensory attribute of beef [12]. It is greatly influenced by the amount of connective tissue and fat in the meat. Juiciness of cooked beef may be separated into two effects: the first is the impression of wetness during the first few chews which is produced by the rapid release of meat fluids, and the second is one of sustained juiciness, largely due to the stimulatory effect of fat on salivation [12]. Tenderness and juiciness are closely related. The more tender the meat, the more juicy it appears. Aroma is the smell or odor that represents the sensory attributes of certain volatile substances perceptibly by the olfactory organ. Beef flavor is difficult to evaluate and describe. Generally, it is due to the aroma or odor developed during cooking. Overall eating quality may be evaluated based on the combination of tenderness, juiciness, and beef flavor intensity. The present study was undertaken to develop an understanding on the effects of fat on the sensory characteristics of ground beef patties and develop an acceptable low-fat all-beef patty.
1347
2. MATERIALS AND METHODS 2.1. Product formulation Extra lean ground beef was purchased from a local grocery store. It was analyzed in triplicate by Soxhlet extraction for six hours using petroleum ether as the extraction solvent. The average fat content was 4.2% Ground beef suet was utilized as the additional fat source. Its fat content was determined to be 94%. Based on their fat contents, approximate amounts of ground lean beef and ground suet were mixed in a Kitchen Aid Model K45SS mixer for two minutes to obtain products containing 4, 12, 20 or 30% fat. Each batch was then processed into 110 g + 5 g patties using a Hollmatic 200 patty machine. Patties were frozen at -28'C for 12 days before evaluation. 2.2. Cooking procedure Frozen patties were first weighed and cooked on a pre-heated 250*0 Presto electric griddle. Each patty was cooked 2 minutes, turned over, cooked for another 2 minutes, turned again, cooked for 1 minute, and turned over again and cooked for 1 minute. The patties were cooked until mid-doneness where the internal temperature of patties was 72''C. After being cooked, the patties were blotted dry with paper towels, and reweighed to determine the percent cooking yield. 2.3. Sensory evaluation Table 1 Sensory score sheet utilized JUICINESS 8 extremely juicy 7 very juicy 6 moderately juicy 5 slightly juicy 4 slightly dry 3 moderately dry 2 very dry 1 extremely dry
TENDERNESS 8 extremely tender 7 very tender 6 moderately tender 5 slightly tender 4 slightly tough 3 moderately tough 2 very tough 1 extremely tough
FLAVOR INTENSITY 8 extremely desirable 7 very desirable 6 moderately desirable 5 slightly desirable 4 slightly undesirable 3 moderately undesirable 2 very undesirable 1 extremely undesirable
OVERALL ACCEPTABILITY 8 extremely acceptable 7 very acceptable 6 moderately acceptabl e 5 slightly acceptable 4 slightly unacceptabl e 3 moderately unacceptable 2 very unacceptable 1 extremely unacceptable
AROMA DESIRABILITY 8 extremely desirable 7 very desirable 6 moderately desirable 5 slightly desirable 4 slightly undesirable 3 moderately undesirable 2 very undesirable 1 extremely undesirable
1348 An eight-member, college-age trained panel (four males and four females) evaluated the ground beef patties for tenderness, juiciness, beef flavor, intensity, aroma, and overall eating quality by means of an eight-point, structured scale (8 = extremely juicy, tender, desirable aroma, desirable flavor intensity, and acceptable overall eating quality; 1 = extremely tough, dry, undesirable aroma, undesirable flavor intensity, and unacceptable overall eating quality (Table 1). Panelists consumed unsalted, warm water between samples. Each formulation was evaluated three times by each of the members for a total of 24 judgments. All evaluations were done in one session of 45 minutes in a specialized sensory room with controlled light intensity. 2.4
Statistical analysis Analysis of variance procedures were used to test the effects of fat level on the sensory characteristics of ground beef patties with an alpha level of .05. 3. RESULTS AND DISCUSSION 3.1
Juiciness
There was a significant increase (P<.05) in juiciness as the fat level of ground beef patties increased based on the sensory scores (Table 2). The results from juiciness revealed that patties formulated to 12 and 20% fat were significantly more juicy than patties containing 4% fat. Similar results were obtained for patties formulated at 30% as compared to patties with 12 and 20% fat. However, there was no significant increase in juiciness on patties formulated at 12 and 20% fat. Table 2 Sensory scores of ground beef patties with different levels of fat Property* Juiciness Texture Aroma Flavor Overall Eatiing Qual ity
4 3.5 3.7 5.5 5.7 5.7
12 4.9 5.1 5.6 5.8 5.8
% Fat 20
4.9 5.1 5.1 5.4 5.8
30
6.9 6.8 5.4 5.5 5.8
PValuBS .0001" .0001"* .4998"^ .7590^^ .9646"^
All values are ratings based on an eight-point scale: 8 = extremely juicy, tender, desirable aroma, desirable flavor intensity, and acceptable overall eating quality; 1 = extremely tough, dry, undesirable aroma, undesirable flavor intensity, and unacceptable overall eating quality. Each value is an average for 24 judgments, one for each of eight panelists in each of three replications. D = significantly different (d<.05) NS = not significantly different (d>.05)
1349 There was a direct relationship between juiciness and the fat content of patties. The result of the study revealed that fat present in meat (sensory score increased as level of fat increased) may act as a means of lubrication and sustained juiciness [13]. Juiciness depends on the amount of liquid released during mastication both from the patties and saliva. Fat may affect saliva production through controlling the ease with which the patty is chewed or by introducing flavor compounds which stimulate saliva flow [13]. In patties containing low levels of fat, i.e. 4%, the absence of fat in the meat which has lost a high proportion of water during cooking will cause it to be registered as "dry" by taste panelists [13]. 3.2
Tenderness
Like juiciness, tenderness increased significantly as the percentage of fat in the patties increased (Table 2 ) . Sensory panel results for tenderness revealed that patties containing 12 and 20% fat were significantly (P<.05) more tender than patties containing 4% fat. Similarly, patties containing 30% fat were more tender than patties formulated at 12 and 20% fat. However, there was no significant increase in juiciness in patties formulated at 12 and 20% fat. There was a significant increase in tenderness as percent fat increased in patties. This may be due to the intramuscular lipids in high-fat patty formulation, i.e. 20 and 30%, may act as a lubricant in mastication, thus improving the apparent tenderness and easing the process of swallowing [14]. Another possible reason for the direct relationship between tenderness and fat may be due to the amount of connective tissue in the patties. Connective tissue is a major component of skeletal muscle and is composed of extracellular fibers of collagen, elastin, etc. and contribute to the "toughness" of meat. Berry et al. [11] found that the amount of connective tissue detected by the panel decreased significantly as the level of fat increased. With high-fat formulation patties, i.e. 30%, there was a lower amount of connective tissue, and hence the patties appeared to be more tender. However, the amount of connective tissue is dependent on the age of the animal [15]. 3.3
Aroma
Aroma was not significantly affected (P>.05) by fat level (Table 2 ) . Even though there was no significant difference, patties formulated at 12% fat were rated highest than other formulations, whereas patties containing 20% fat were the lowest. However, in some of the panelists' comments, they smelled "fatty" in higher fat patty formulations, i.e. 20 and 30% fat. Perhaps, volatile compounds in triglycerides or fatty acids may have evaporated during cooking to give that distinctive smell. 3.4
Flavor intensity
Sensory evaluation (Table 2) showed no effect of added fat on the judges' evaluations of the desirability of the flavor intensity of ground beef patties. Even though with no significant difference, patties with 12% fat received the highest score whereas patties with 20% fat received the lowest score. It appeared that the differences in fat in cooked patties was not a major factor in flavor differences. However, some panelists listed that they experienced a "funny" taste but did not describe exactly what it was in the low-fat formulated
1350 patties, i.e. 4 and 12% fat. The "odd" taste could be "metallic" or "other" flavors as a result of the reduction in ground beef flavors in low-fat level patties [16]. 3.5 Overall eating quality Overall eating quality did not differ significantly among patties containing 4, 12, 20 and 30% fat (Table 2). Patties containing 12, 20 and 30% fat were rated the same score and higher than patties containing 4% fat. As noted earlier, overall eating quality was judged on the overall attributes of juiciness, tenderness, aroma, and beef flavor. The results of this study showed that tenderness and juiciness were not major contributors to the overall acceptability of the product. 3.6 Cooking yield Total cooking yield decreased as the percent fat level of patties increased (Table 3). Patties formulated at 4 and 12% fat had 80% cooking yield, 20% fat had 72% cooking yield, and 30% fat had 60% cooking yield. Most of the weight loss in the low-fat patty was due to water loss during cooking. The patty with more fat only appeared to lose more weight (lower percent cooking yield) during cooking because the liquid fat remained in the pan after cooking; whereas water lost during cooking in the low-fat patty evaporated and the loss was not apparent [10]. Thus, it appeared that the difference between low- and high-fat ground beef patties narrowed considerably during cooking. Table 3 Cooking yield of ground beef patties with different fat levels* Fat level 1%) 4 12 20 30
Cooking yield 1%) 80 80 72 60
*: Each value is an average for six determinations, two for each of three replications.
4.
REFERENCES
1 National Research Council, National Academy Press, Washington, DC, 1988. 2 J.D. Sink, Meat Ind., 26 (1986) 26. 3 L.m. Hoelscher, J.W. Savell, K.S. Rhee and H.R. Cross, J. Food Sci., 52 (1987) 883. 4 National Restaurant Association, Washington, DC 1987. 5 B. Yankelovich, American Meat Institute, Washington, DC 1985. 6 C D . Burke, American Meat Institute, Washington, DC, 1985. 7 B.W. Berry and J.R. Hasty, J. Cons. Stud. Home Econ., 3 (1989) 413. 8 K.K. Kregel, K.J. Prusa and K.V. Hughes, J. Food Sci., 51 (1986) 1162.
1351 9 10 11 12 13 14 15 16
D.L. Huffman, W.R. Egbert, M.A. Browning and S.B. Jungst, J. Food Sci., 55 (1990) 9. B.W. Berry, J. Food Sci., 57 (1992) 537. H.R. Cross, B.W. Berry and L.H. Wells, J. Food Sci., 45 (1980) 791. H.R. Cross and A.J. Overby, World Animal Science, Vol. 2, Elsevier, Amsterdam, 1988. T.N. Blumer, J. An. Sci., 22 (1963) 771. J.D. Wood and A.V. Fisher, Reducing Fat in Meat Animals, Elsevier, Amsterdam, 1990. M.P. Penfield and A.M. Campbell, Experimental Food Science, 3rd Ed., Academic Press, New York, 1990. P.J. Bechtel, Muscle as Food, Academic Press, New York, 1986.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1353
Fractionation and characterization of extracts of chicicen fat obtained with supercritical carbon dioxide D. L. Taylor^'^ and D. K. Larick^ ^Department of Food Science, North Carolina State University, Box 7624, Raleigh, NC, 2 7 6 9 5 - 7 6 2 4 , U.S.A. "^This material is based upon work supported under a National Science Foundation Graduate Research Fellowship.
Abstract Cooked chicken fat was fractionated using supercritical CO2 at 40°C and each of three pressures, 10.3 MPa, 20.7 MPa, and 31.0 MPa. Fatty acid and volatile profiles were obtained via capillary gas chromatography and GC-mass spectrometry. Monounsaturated and polyunsaturated fatty acid concentrations (mg/g extract) increased with increasing extraction pressure. Total volatiles (ppm) increased with decreasing extraction pressure. The concentrations of branched hydrocarbons, enals, ketones, alcohols/phenols, and lactones were influenced by extraction pressure.
1.
INTRODUCTION
The importance of nutritional aspects, especially lowered fat content, in consumer purchasing criteria for foods (National Broiler Council, 1992) and, consequently, in research and development trends (Best, 1992) encourages the study of non-traditional methods of obtaining natural flavor extracts for use in lowfat products which typically have inherent flavor disadvantages. In addition, utilization of excess lipid by-products that result from commercial slaughter as substrates for those extracts could be economically advantageous. The use of supercritical fluid extraction of various food components is receiving more attention due to the inherent advantages of the process including elimination of organic solvents, less destruction of thermally labile constituents as compared to steam distillation, lowered extraction times, and easily manipulated conditions. A supercritical fluid may be considered as a fluid phase above its critical temperature and pressure (Caragay, 1981). Such systems possess properties between those of liquids and gases. Mass transfer properties resemble those of gases while solvation properties more closely resemble liquids; high diffusivity and low surface tension facilitate penetration of solid-like food matrices and extraction of components of interest. Carbon dioxide (CO2) is a popular
1354 supercritical fluid due to its similarity to organic solvents (Hyatt, 1984) and relatively low critical temperature (31.1°C) along with the advantages of being inexpensive, nontoxic, inert, and nonflammable. The solubility of various components in supercritical CO2 is dependent on temperature and pressure (Caragay, 1981). At a given temperature, solvent power of a supercritical fluid increases with density as a function of pressure (Brogle, 1982) while density of the fluid increases with decreasing temperature at constant pressure. Removal of CO2 from extracts is facile via sublimation of the fluid upon depressurization with the accompanying release of now-insoluble extract. Rizvi etal. (1986) reported current commercial applications of the process in the decaffeination of coffee beans and the recovery of hops extract as well as in the recovery of spice essences. Patented supercritical fluid processes include removal of oil from oilseeds, deodorization of fats and oils, and the fractionation of certain fats and oils (Rizvi et a/., 1986). In animal products, supercritical fluid extraction has been used to remove cholesterol and lipids from milk fat, egg yolk, and muscle tissues (Bradley, 1989; Froningefa/., 1990; Chao eta/., 1 9 9 1 ; Kmg eta/., 1989; Hardardottir and Kinsella, 1988). Successful fractionation of lipids in milk fat using supercritical CO2 has been demonstrated, indicating the ability to achieve molecular weight separation (Arul et a/., 1987). Short and medium chain fatty acids decreased in concentration among extracted fractions as the solvent power of the supercritical fluid increased while concentration of long chain fatty acids increased with solvent power. Separations were partially attributed to differences in the molecular weight and volatility of the triglyceride components (Arul et a/., 1987). Also, Merkle and Larick (1994a) fractionated beef fat and selectively extracted saturated and monounsaturated triglycerides based on molecular weight and solvent density. Most efficient separation should occur near the critical point of the supercritical fluid where there is the greatest variation in the dissolving power of the fluid with incremental changes in conditions (Allada, 1984). Flavor compounds have been extracted and concentrated from milk fat with supercritical CO2, indicating the importance of vapor pressure differences between volatile flavor components and nonvolatile triglycerides in selectivity. Among volatile compounds, concentration in the extract decreased with increasing molecular weight due to the vapor pressure effect (de Haan and de Graauw, 1990). Merkle and Larick (1994c) also demonstrated the ability to concentrate beef flavor volatiles with supercritical fluid extraction. Over 300 compounds have been identified in relation to chicken flavor to date. Sulfur compounds, as a class, have been identified as important to overall chicken flavor but largely in relation to "meaty" aroma and flavor notes (Minor et a/., 1965; Chang and Peterson, 1977). Numerous researchers point to the importance of the lipid portion for the distinguishing species aroma and/or flavor notes and the lean portion for the "meaty" sensations (Hornstein and Crowe, 1960; Minor ef a/., 1965; Wasserman and Talley, 1968). Lipid components serve as a source of flavor compounds in several ways: degradation of fatty acids upon heating, oxidation reactions, and the release of fat soluble compounds that were previously stored in the adipose tissue. Specific allusion to the importance of carbonyl compounds for species differences has been made by numerous
1355 researchers (Gray et al., 1 9 8 1 ; Shahidi et al., 1986; Rubin and Shahidi, 1988). Carbonyl spectrum differences are largely due to varying fatty acid profiles in the triglyceride portions (Gray et al., 1 9 8 1 ; Ramarathnam et al., 1991). Indeed, the low threshold values of some carbonyls make them potential flavor sources even in minute amounts (Ramaswamy and Richards, 1982). While no single compound has been proven to be responsible for "chicken" flavor, several compounds have been recognized in significantly higher concentrations in chicken as compared to other meat species. Ramarathnam et al. (1991) identified 2-hexenal, nonanal, decanal, 2-octenal, and 4-ethylbenzaldehyde in disproportionately higher concentrations in chicken than in beef or pork. Gasser and Grosch (1990) noted the importance of 2(E),4(E)-decadienal and gamma-dodecalactone as odorants in chicken broth over broths from cow and ox. Decadienal in chicken meat lipids has been noted to be very odorous (Pippen and Nonaka, 1960). The objective of this research was to investigate the fractionating ability of supercritical CO2 on cooked chicken fat by assessment of fatty acid and volatile profiles. In addition, identification of potential uses for the extracts was another goal of the studies.
2. MATERIALS AND METHODS 2 . 1 . Sample Preparation Chicken fat was collected from a commercial processor and ground once through a .95 cm plate. Portions (500g) were weighed and vacuum packaged in low permeability bags (Cryovac Corp., Duncan, SC) and overwrapped with polyethylene coated freezer paper. Samples were stored at -20°C and thawed at refrigeration temperature prior to use. Prior to extraction, fat was heated in a convection oven to 80°C in 500 mL Pyrex glass beakers to melt the triglyceride portion and create a substrate mimetic of roasted chicken fat. An unextracted portion was placed in a Pyrex tube (16 mm x 125 mm), flushed with nitrogen gas, sealed with a teflon-lined cap, frozen at -10°C and retained as a control. 2.2. Extractions A Superpressure model 46-13421-2 supercritical fluid extractor (Newport Scientific, Jessup, MD) equipped with a 69.0 MPa double end diaphragm compressor was used to fractionate the cooked chicken fat. Aliquots (250 g) were immobilized between t w o plugs of glass wool and loaded into a 0.845 L (internal volume) stainless steel extraction vessel. Vessel temperature was maintained at 40°C via an internal thermocouple and temperature controller with heat tape wrapped around the outside of the vessel. Stainless steel transfer lines were wrapped with insulation to maintain temperature within the system. Continuous extractions were carried out at each of three pressures, 10.3, 20.7, and 31.0 MPa, using supercritical CO2 at a flow rate of 10-15 L per minute to a total flow volume of 500 L of ambient CO2 measured using a flow totalizer. Extracts were collected in a .500 L Pyrex round bottom flask upon fluid depressurization at ambient
1356 temperature. Extracts were transferred to Pyrex tubes (16 mm x 125 mm), flushed with nitrogen gas, sealed with a teflon-lined cap, and frozen at -10°C until analysis. 2.3. Fatty acid analysis Extracts and unextracted control were melted in a 60°C water bath. Fatty acid methyl esters (FAME) were prepared for each treatment using a modified method (Morrison and Smith, 1964). A 0.5 microliter sample was injected directly onto a 30 m DB-23 capillary column (J & W Scientific, Folsom, CA) with internal diameter .25 mm and film thickness .25 micron. An oven temperature program of 150°C to 215°C at 4°C per minute and 215°C to 225°C at 2°C per minute was used. Esters were analyzed using a Hewlett Packard 5890 gas chromatograph equipped with flame ionization detector and maintained with a head pressure of 26 psi, helium carrier gas flow rate of 1.36 mL per minute, and split ratio of 2 6 . 7 : 1 . Data were analyzed using the Maxima 820 Chromatography Workstation (Millipore, Waters Chromatography Division, Milford, MA). An internal standard, heptadecenoic acid (CI7:1), was used for quantitation purposes. Fatty acid identifications were based on retention times of reference standards (Nu Check Prep, Inc., Elysian, MN). Response factors were derived by comparing peak areas of known quantities of reference standards to the peak area of the internal standard.
2.4. Volatile analysis Extracts and unextracted control were melted in a 60°C water bath. Samples (300 mg) were placed into another tube and 1026 ng of internal standard, 2,3,4-trimethylpentane, were added. The tube was sealed and vortexed. Aliquots (100 mg) were placed between t w o plugs of pesticide grade glass wool in a 9 mm X 85 mm glass tube. With the six-port sample valve in the no-flow position, the tube was positioned in an External Closed Inlet Device (Scientific Instrument Service, River Ridge, LA). Volatiles were stripped from the sample via this method of dynamic purge and trap sampling for a total of five minutes. Inlet, valve, and carrier lines were maintained at 1 50°C, 160°C, and 170°C, respectively. Volatiles were flushed onto a 30 m DB-5 capillary column (J & W Scientific, Folsom, CA) with internal diameter .32 mm and film thickness 1.0 micron within a Varian 3700 gas chromatograph (Varian, Palo Alto, CA) equipped with a flame ionization detector and maintained with head pressure of 16 psi, helium carrier gas flow rate of 5.73 mL per minute, and split ratio of 2 0 . 1 2 : 1 . An oven temperature program of -30°C to 290°C at 4°C per minute was used with a one minute hold at -30°C. Data were analyzed via the same software as for fatty acid analyses. Quantitation of volatiles was based on relative peak areas compared to peak area of the internal standard. Volatile identifications were based on GC-mass spectrometry using a Hewlett Packard 5897 gas chromatograph-mass spectrometer with both electrical and chemical ionization methods and comparison to NIH/EPA reference spectra (NIH/EPA, 1978).
1357 2.5. Experimental design Fatty acid and volatile profiles were replicated three times on different batches of fat for the four treatments, three extracts and unextracted control. Fatty acid and volatile concentrations were analyzed via analysis of variance using a randomized complete block design with replicates as blocks. Waller-Duncan kratio t-tests were used to separate means (SAS, 1990).
3. RESULTS AND DISCUSSION 3 . 1 . Fatty acid analysis Nineteen fatty acids were identified. Eight were saturated, ranging from 12 to 22 carbons in chain length; four were monounsaturated, ranging from 14 to 20 carbons; and seven were polyunsaturated, ranging from 18 to 22 carbons (Table 1). Fatty acids identified in the greatest concentrations were octadecenoic (C18:1), hexadecanoic (C16:0), octadecadienoic (C18:2), hexadecenoic (C16:1), and octadecanoic (CI8:0). By class, the monounsaturated fatty acids were predominate in concentration followed by saturated and polyunsaturated fatty acids (Table 2). Extraction pressure influenced concentrations of eight individual identified fatty acids and t w o classes. Among treatments, total fatty acid concentration was lowest for the 10.3 MPa extract. This trend results from the lower density and solvent strength of the supercritical fluid at lower pressure which inhibits the extraction capabilities. The unextracted control yielded the highest concentration of monounsaturated and polyunsaturated fatty acids, and concentration decreased among extracts as extraction pressure decreased (Fig. 1). The enhanced polarity of monounsaturated and polyunsaturated fatty acids required greater solvent strength for extraction. Because solvent strength decreases as a function of density with decreasing extraction pressure, the concentration of these classes of fatty acids in the extracts decreased with decreasing extraction pressure. Similar results were achieved by Merkle and Larick (1994b) with beef fat. No significant differences existed between treatments for concentrations of individual identified fatty acids of chain length less than eighteen carbons indicating that the supercritical fluid possessed sufficient solvent strength at all pressures to extract the shorter chain, lower molecular weight acids. Ratios of saturated to unsaturated (S/U), saturated to monounsaturated (S/M), and saturated to polyunsaturated (S/P) fatty acids indicate the inability of any of the pressure treatments to extract unsaturated fatty acids on a level that equals their natural abundance in the unextracted control. Among extracts, S/U, S/M, and S/P ratios increase with decreasing extraction pressure showing the greater ability of higher pressure treatments to extract the more polar unsaturated fatty acids (Table 3).
1358 Table 1 Fatty acids identified in supercritical fluid extracts of chicken fat Saturated
Monounsaturated
Polyunsaturated
Dodecanoic (12:0)
Tetradecenoic (14:1)
Octadecadienoic (18:2)
Tetradecanoic (14:0)
Hexadecenoic (16:1)
Octadecatrienoic (18:3)
Pentadecanoic (15:0)
Octadecenoic (18:1)
Eicosadienoic (20:2)
Hexadecanoic (16:0)
Eicosenoic (20:1)
Eicosatrienoic (20:3)
Heptadecanoic (17:0)
Eicosatetraenoic (20:4)
Octadecanoic (18:0)
Docosatetraenoic (22:4)
Eicosanoic (20:0)
Docosahexaenoic (22:6)
Docosanoic (22:0)
Table 2 Mean concentrations of predominate fatty acids and categories of fatty acids in supercritical fluid extracts of chicken fat (mg/g extract) Treatment Control
10.3 MPa
20.7 MPa
31.0 MPa
16:0
219.99'
213.58'
240.55'
229.85'
16:1
55.94'
60.51'
66.64'
63.21'
18:0
58.78'
39.32=^
50.69"
51.90"
18:1
360.29'
262.44"
333.17'
343.19'
18:2
165.36'
128.29"
162.59'
166.27'
Saturated
289.65'
266.73'
304.81'
294.58'
Monounsaturated
421.09'
327.15"
404.64'
411.07'
Polyunsaturated
179.05'
138.01"
174.75'
178.42'
Total
988.16'
840.32'
992.60'
986.32'
""means in same row with same letter do not differ significantly (P^O.05).
1359 Figure 1. Concentration of fatty acid classes
Saturated I Control
Polyunsaturated Monounsaturated rai0.3MPa Q20.7MPa HSl.OMPa
Table 3 Ratios of saturated to unsaturated, saturated to monounsaturated, and saturated to polyunsaturated fatty acid concentrations in supercritical fluid extracts of chicken fat Treatnnent Control
10.3 MPa
20.7 MPa
31.0 MPa
Saturated/Unsaturated
0.48
0.57
0.53
0.50
Saturated/Monounsaturated
0.69
0.82
0.75
0.72
Saturated/Polyunsaturated
1.63
1.92
1.75
1.65
3.2. Volatile Analysis Over all treatments, 318 volatiles have been quantified. Of those quantified, 77 have been placed into compound classes (Table 4). Of these, 53 have been identified, excluding isomeric designations. Branched alkanes, aldehydes, and enals appeared in the greatest concentrations (ppm) as groups. The individual compounds present in the greatest concentrations (ppm) were primarily aldehydes, enals, and acids. Treatments influenced volatile concentration (ppm) for 62 compounds, of which 23 were classed volatiles. Total volatiles, branched alkanes, enals, ketones, alcohols and phenols, and lactones were influenced by treatments. (Figures 2 and 3). Total volatiles were concentrated 12 fold for the 10.3 MPa extract versus the control and, among extracts, total volatile concentration decreased with increasing
1360 extraction pressure. The supercritical fluid at lower pressure with lower solvent strength was less able to solubilize less volatile and higher molecular weight fatty acids and triglycerides that would dilute the volatile concentration in the extract. Classes of compounds were concentrated from 5 fold for branched alkanes to 50 fold for alcohols/phenols over the control, depending on extraction pressure, with enals being concentrated 10 fold in the 10.3 MPa extract versus the unextracted control. Individual aldehydes and enals, were concentrated up to 70 fold. Several researchers have indicated the importance of various aldehydes and enals in "chicken" aroma and flavor notes (Gasser and Grosch, 1990; Pippen and Nonaka, 1960; Ramarathnam et al., 1991). Volatile concentration factors of 20-50 using supercritical CO2 were reported by de Haan et al. (1990) with milk fat and concentration factors of up to 1000 were modeled. Merkle and Larick (1994c) demonstrated the ability to concentrate volatiles from beef fat with supercritical fluid extraction most efficiently at lower pressures near the critical pressure of the fluid; however, their results show that enal concentration increased with increasing pressure, likely due to the identification of more higher molecular weight enals in the beef fat. The presence of alkanes, alkenes, carbonyl compounds, alcohols, and acids is attributable to the thermal and oxidative decomposition of the triglyceride and fatty acid components while phenols and aromatic compounds are likely derived from the breakdown of aromatic amino acids found in connective tissue.
Table 4 Classification of volatiles from supercritical fluid extracts of chicken fat Compound class
Number of volatiles quantified
Branched alkanes
30
Enals
9
Aldehydes
8
Alkenes
6
Alcohols/phenols
5
Ketones
4
Other^
15
^includes straight chain and halogenated alkanes, lactones, acids, and aromatics
4. CONCLUSIONS By manipulation of extraction pressure, the fatty acid and volatile profiles of supercritical fluid extracts of chicken fat may be altered. Volatile constituents that contribute to aroma and flavor, especially those reported to be associated with "chicken" notes, may be concentrated with this extraction method. Most efficient
1361 Figure 2. Concentration of total volatiles 150 B a. c
I 100 c
o
c o o
1 ^^ o > c CD
^H I Control
i
^
Total Volatiles
n i 0 . 3 M P a O 2 0 . 7 M P a IZlSl.OMPa
Figure 3. Concentration of volatile classes
Branched alkanes Alcohols/phenols Lactones Enals Ketones • Control n i 0 . 3 M P a O 2 0 . 7 M P a Zl31.0MPa
concentration of volatiles occurred at pressures near the critical pressure. The opposite effect of pressure on concentrations of fatty acids and volatiles demonstrate the ability to fractionate based on molecular weight/volatility and polarity of compounds. There is potential use for the extracts with high volatile concentrations as natural flavorings in low-fat meat products.
1362 5. REFERENCES Allada, S.R. 1984. Solubility parameters of supercritical fluids. Ind. Eng. Chem. Process Des. Dev. 23: 344-348. Arul, J . , Boudreau, A., Makhlouf, J . , Tardif, R., and Sahasrabudhe, M.R. 1987. Fractionation of anhydrous milk fat by supercritical carbon dioxide. J . Food Sci. 52: 1231-1236. Best, D. 1992. R & D horizons: fat and calorie reduction. Prep. Foods. 162: 64. Bradley, R.L. 1989. Removal of cholesterol from milk fat using supercritical carbon dioxide. J . Dairy Sci. 72: 2834-2840. Brogle, H. 1982. CO2 In solvent extraction. Chem. Ind. 19: 385-390. Caragay, A.B. 1 9 8 1 . Supercritical fluids for extraction of flavors and fragrances from natural products. Perf. Flav. 6: 43-55. Chang, S.S. and Peterson, R.J. 1977. Symposium: the basis of quality in muscle foods: recent developments In the flavor of meat. J . Food Sci. 4 2 : 298-305. Chao, R.R., Mulvaney, S.J., Bailey, M.E., and Fernando, L.N. 1 9 9 1 . Supercritical CO2 conditions affecting extraction of lipid and cholesterol from ground beef. J. Food Sci. 56: 183-187. de Haan, A.B. and de Graauw, J . 1990. Extraction of flavors from milk fat with supercritical carbon dioxide. J . Supercrit. Fluids. 3: 15-19. Froning, G.W., Wehling, R.L., Cuppett, S.L., Pierce, M.M., Niemann, L., and Siekman, D.K. 1990. Extraction of cholesterol and other lipids from dried egg yolk using supercritical carbon dioxide. J . Food Sci. 55: 95-98. Gasser, U. and Grosch, W. 1990. Primary odorants of chicken broth. Z. Lebensm. Unters. Forsch. 190: 3-8. Gray, J . I . , MacDonald, B., Pearson, A . M . , and Morton, I.D. 1 9 8 1 . Role of nitrite In cured meat flavor: a review. J . Food Protect. 44: 302-312. Hardardottir, I. and Kinsella, J.E. 1988. Extraction of lipid and cholesterol from fish muscle with supercritical fluids. J . Food Sci. 53: 1 6 5 6 - 1 6 6 1 . Hornstein, I. and Crowe, P.P. 1960. Flavor studies on beef and pork. J . Agric. Food Chem. 8: 494-498. Hyatt, J.A. 1984. Liquid and supercritical carbon dioxide as organic solvents. J . Org. Chem. 49: 5 0 9 7 - 5 1 0 1 . King, J.W., Johnson, J.H., and Friedrich, J.P. 1989. Extraction of fat tissue from meat products with supercritical carbon dioxide. J . Agric. Food Chem. 37:951-954. Merkle, J.A. and Larick, D.K. 1994a. Triglyceride content of supercritical carbon dioxide extracted fractions of beef fat. J . Food Sci. 58: 1237-1240. Merkle, J.A. and Larick, D.K. 1994b. Fatty acid content of supercritical carbon dioxide extracted fractions of beef fat. J . Food Sci. (In Press). Merkle, J.A. and Larick, D.K. 1994c. Conditions for extraction and concentration of beef fat volatiles with supercritical carbon dioxide. J . Food Sci. (In Press). Minor, L.J., Pearson, A . M . , Dawson, L.E., and Schweigert, B.S. 1965. Chicken flavor: the identification of some chemical components and the importance of sulfur compounds in the cooked volatile fraction. J . Food Sci.
30: 686-696.
1363 Morrison, W.R. and Smith, L.M. 1964. Preparation of fatty acid methyl esters and dimethlyacetals from lipids with boron fluoride-methanol. J . Lipid Res. 5: 600-608. National Broiler Council. 1992. 28(22). 1 1 5 5 1 5th Street, N.W., Washington, D.C. NIH/EPA Chemical Information System. 1978. U.S. Government Printing Office. Washington, D.C. Pippen, E.L. and Nonaka, M. 1960. Volatile carbonyl compounds of cooked chicken. II. compounds volatilized with steam during cooking. Food Res. 25: 764-769. Ramarathnam, N., Rubin, L.J., and Diosady, L.L. 1 9 9 1 . Studies on meat flavor. 2. a quantitative investigation of the volatile carbonyls and hydrocarbons in uncured and cured beef and chicken. J . Agric. Food Chem. 39: 1839-1847. Ramaswamy, H.S. and Richards, J.F. 1982. Flavor of poultry meat - a review. Can. Inst. Food Sci. Technol. J . 15: 7-18. Rizvi, S.S.H., Daniels, J.A., Benado, A.L., and Zollweg, J.A. 1986. Supercritical fluid extraction: operating principles and food applications. Food Tech. 4 0 : 57-64. Rubin, L.J. and Shahidi, F. 1988. Lipid oxidation and the flavor of meat products. Proceedings, 34th International Congress of Meat Science Technology, Brisbane, Australia, pp. 2 9 5 - 3 0 1 . SAS Institute, Inc. 1990. SAS/STAT User's Guide, Version 6, Fourth Edition, SAS Institute, Cary, NC. Shahidi, F., Rubin, L.J., and D'Souza, L.A. 1986. Meat flavor volatiles: a review of the composition, techniques of analysis, and sensory evaluation. CRC Crit. Rev. Food Sci. Nutr. 24: 141-243. Wasserman, A.E. and Talley, F. 1968. Organoleptic identification of roasted beef, veal, lamb and pork as affected by fat. J . Food Sci. 33: 219-223.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1365
BMP: a flavor enhancing peptide found naturaUy in beef. Its chemical synthesis, descriptive sensory analysis, and some factors affecting its usefulness. A. M. Spanie^^ J. M. Bland% J. A. Miller^ J. Glinka\ W. Wasz^ and T. Duggins^ ^United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, Louisiana 70124 ^F&C International, Inc., 890 Redna Terrace, Cincinnati, Ohio 45215 Abstract Through the millennia, muscle tissue from a variety of terrestrial, airbome, and aquatic animals, has been one of the most highly prized foods in the human diet. Meat is typically eaten because people like the taste of it. Recently, a small linear peptide was found in meat that elicits a savory taste. The peptide was named BMP because it is found in beef (B), and it enhances the meaty (M) flavor of the beef, and is a peptide (P). Because of the its ability to enhance the meat's flavor, large quantities of the BMP were synthesized to ascertain testing the its flavor enhancing characteristics and to study some of its physicochemical properties. The peptide was found to be similar to monosodium glutamate (MSG) in its ability to enhance the flavor of a beef gravy yet it did not present the salty taste of MSG. BMP was also shown to be resistant to the direct, but not the indirect, effect of lipid oxidation. 1. INTRODUCTION Preparation of a food with a desired taste requires a complete understanding of the factors involved in both the development and the deterioration of flavor in that food. This, of course, involves a thorough understanding of the rules goveming muscle/meat structure and function and the nature of any given reaction in the muscle food. Although we are still a long way from completely unlocking the puzzling relationships between meat structure and flavor, some of the technical achievements over the last few decades have provided the basic machinery to study the complex nature of the problem of food quality in general and muscle food quality in particular [19, 35, 36, 42, 43]. The final flavor of any muscle food is dependent upon many factors. The prime participants in meat flavor development include the composition and relative proportion of the lipids, proteins, and carbohydrates in the meat source [36, 42]. During the postmortem conditioning period and during cooking/storage, muscle foods show a significant alteration in the type and level of chemical components, such as sugars, organic acids, peptides and free amino acids, and products of adenine nucleotide metabolism, for example adenosine triphosphate (ATP). Many of these changes are due to enhanced hydrolytic activity [45]. Regardless of the history of the meat, heating develops both desirable and undesirable flavor from the thermal degradation and the interaction of sugars, amino acids, proteins, nucleotides, Maillard reactions, and lipid oxidation [39, 41, 46]. Thus, the chemical modifications that occur during postmortem agmg and during the subsequent handling of meat serve as a pool of reactive flavor compounds and flavor intermediates that later interact to form additional flavor notes during cooking, e.g., sugars and amino acids react during heating
1366 to form Maillard reaction products [1, 2]. It is apparent, therefore, that the development of flavor in meat is an extremely complicated process that occurs continuously from before slaughter, through cooking and storage, and ending when the food is eaten and the flavor perceived. The final quality of meat, therefore, involves both extemal and intemal factors. These factors, when combined, establish flavor quaUty and influence the purchasing decision of the consumer. In general, the final flavor quality of a muscle food is dependent upon several anteand postmortem factors. These factors include, and are not limited to, the animal's age, breed, sex, and nutritional status, as well as all of the postmortem handling and cooking protocols, including the manner of slaughter [12, 16, 21, 24, 29 - 32, 42, 44, 50]. Because of the importance of muscle foods to human diet, the remainder of this manuscript will focus its attention on a key peptidic flavor component found in beef, BMP. 2. NATURAL PRODUCTION OF BMP 2.1 Initial discovery and characterization In 1978, Yamasaki and Maekawa [52] reported the finding of a delicious tasting, peptide in the aqueous fraction of beef that had been incubated with the plant protease, papain. These investigators isolated and purified the peptide and determined its primary structure by Edman degradation for the N-terminal sequence and the Cpase A method for the C-terminal sequence. The structure was determined to be: H-Lys-Gly-Asp-Glu-Glu-Ser-LeuAla-OH. They named the peptide "delicious peptide" because of its "umami" taste. In English, the Japanese word "umami" translates into "delicious" and "savory." These terms will be used interchangeably in this manuscript, hi subsequent work by these investigators [53] they were able to confirm the primary structure of the delicious peptide by chemically comparing it to a similar peptide that they had synthesized. A decade after the first reported finding of a delicious peptide formed in the papain digests of beef. Dr. Okai and colleagues [48] examined the relationship between the taste and the primary structure of the delicious peptide and its analogues. These and other investigators [25] determined that the delicious peptide had both a "umami" and a sour flavor. The aqueous perception threshold was determined to be 1.41mM. 2.2 Proposed in vivo mechanism of BMP production. Through the millennia muscle tissuefi-oma variety of terrestrial, airbome, and aquatic animals, has been cosidered to be a most highly prized foods in the human diet. While the typical meat scientist will use the word "muscle" and "meat" interchangeably, there are some differences. Muscle or muscle tissue typically describes that organ of the animal that is responsible for locomotion. After the animal's death or during an ischemic episode such as that occurring in the heart during a heart attack [38, 47] the tissue goes through many significant chemical, structural and functional changes (Figure 1). It is only after these changes have occurred that muscle is referred to as meat. To a typical consumer, meat also implies a product that may include some fat and bone. The term "meat" is commonly
1367 reserved for use when referring to the tissue of mammals, while muscle tissue of other species is referred to by names, such as chicken, duck, turkey, catfish, cod, flounder, hake or in more general terms such as poultry or fish [17]. EARLY EVENTS IN MUSCLE ATROPHY, CELLULAR/nSSUE NECROSIS & CELLULAR DEATH.
"THE CONVERSION OF MUSCLE TO MEAT." 1 1
NUMBERS OF LARGER VISIBLE LYSOSOMES
T 1
ACTIVATION OF INTRA-LYSOSOMAL ACID HYDROLASES
^ 1 1
i
METABOUC ENERGV HIODUCTION
H T
T
RELEASE OF SOLUBLE INTRACELLULAR COMPONENTS
T
K L P
1 1 1
1 I I INTRA LmM I CELLULAR
i
LYSOSOMAL MEMBRANE STABILTTY
T
RELEASE OF ACTIVE HYDROLASES
\X Ir
J ! • ^^ I
CYTOSOLIC DEGRADATION AND INACTIVATION OF REGULATORY A N D STRUCTURAL PROTEINS.
INACTIVATION
INHIBITION
DESTABILIZATION AND DISRUPTION OF ORGANELLES A N D MEMBRANES.
J
CELLULAR DEATH
Figure 1. SCHEMATIC OF THE CONVERSION OF MUSCLE TO MEAT
In the living animal, muscle receives energy rich compounds such as the carbohydrates, fats, amino acids, oxygen etc., directly via diffusion and transport from the blood stream. Immediately following death, this flow of blood is curtailed, no new energy rich compounds are brought to the tissue, and tissue metabolites are removed. Therefore, several key changes begin to occur in the muscle, notably, the reduction in the level of the high energy phosphate, adenosine triphosphate (ATP). Other work has demonstrated that the reduction in ATP coincides with the appearance of larger lysosomes. At the same time that ATP levels are dropping, intracellular pH begins to drop making the tissue increasingly more acidic ... from the normal of 7.0 to 5.3 - 5.8 depending upon the species of animal and the preslaughter glycogen content... due to the lactate and proton buildup from glycolytic backup [33]. The latter is due to the cessation of the electron transport system (Krebs cycle) caused by the lack of oxygen availabihty. The reduction in ATP and drop in pH will continue as the tissue tries to maintain its normal metabolic and physiological processes without the benefit of free circulation. These biochemical changes lead to a number of structural changes, notably the decrease in lysosomal membrane stability [38]. The destabilized
1368 lysosomes release their hydrolases into the cytosol where the acidic environment is optimal for their activity, thereby leading to alterations to the structural and regulatory proteins and cellular components (Figure 1). Eventually the muscle is converted into meat during the days or weeks of storage (typically refrigerated) depending upon the animal species and storage conditions [42]. This enhanced hydrolytic activity leads to the production of many flavor precursors that will interact upon cooking to yield additional flavorful components [16, 45]. Several factors suggested that the delicious peptide, reported by Yamasaki and Maekawa [52] in papain digests of beef, might be formed in meat under normal postmortem conditions. First, although water accounts for 70-80% by weight of the bulk of muscle [17], the greatest percentage, i.e. about 16 - 23% by weight, of the lean muscle is protein. This makes this tissue a remarkable reservoir for the production of flavorful amino acids and peptides such as the delicious peptide of Yamasaki and Maekawa [52]. Second, muscle contains thiol dependent proteinases [4 - 6, 37] that are similar in activity to papain [3]. These proteinases include cathepsins B, H, and L. Last, the proteinases have an optimum level of activity in the range of pH found in slaughtered/aged beef. This led to studies that demonstrated the natural occurrence of this peptide in untreated beef extracts [43]. The delicious peptide of Yamasaki and Maekawa [52] was renamed BMP for beefy meaty peptide. BMP is given its acronym because it is found naturally in beef, (B), it enhances beefs meaty (M) flavor, and is of protein (P) origin. The structure of BMP is "LysGly-Asp-Glu-Glu-Ser-Leu-Ala" as seen in the computer generated model below (Figure 2).
Figure 2. THE CHEMICAL STRUCTURE OF BMP.
3. SYNTHESIS OF BMP Routine solid phase methods were employed to synthesize large quantities of BMP for sensory evaluation and for examining some of its physical and chemical properties. A peptide synthesizer (Biosearch/Milligen 9500 made by Millipore Corp.) using Fmoc chemistry with PAC-resin (methylbenzhydrylamine resin with a hydroxymethyl phenoxyacetic acid
1369 linker) was used to synthesize BMP on a scale of 2.3 mmoles. The final yield was approximately 2 grams of BMP. Side chain protecting groups were removed and the peptide cleaved from the resin by treatment with trifluoroacetic acidiwateriethane dithiol (90:5:5, v:v:v). The resin was removed by filtration into cold (-78° C) ethyl ether (70 mL ether / mL of trifluoroacetic acid solution). Pyridine was added to enhance precipitation. The resulting solid material was filtered, washed with ether, and dried over potassium hydroxide in vacuo ovemight. The molecular mass of the BMP was found to be between 847 and 848 as determined by Electrospray mass spectrometry (Figure 3).
Electrospray Mass Spectrum of BMP 800-
• mass = 847 Synthesized BMP
7004-
73 $cans acquired!at a rate of 5.764 seconds/3can Flow = 2.1 jjl/min
600-
SV=2.34KV,SI=150juA, N=235vi REP=15 BLK|=225JH=51JLENS=137 \
500j—
IVIasises acquired as 50-1200.
4004---
30o3— 200q— 100H--
iiini 840
HfinFlnn
850
860
m
T 870
i
Flnnnnnnfiinnll
880
li890nRHFi^Finfnpm900 T"^
Mass Figure 3. MASS SPECTRUM OF BEEFY MEATY PEPTmE (BMP). Conditions of the determination are written in the figure.
Salts and contaminants were removed from the preparation by DEAE-Sephadex A25 ion exchange resin eluted with a pH 8.0 ammonium carbonate buffer. The purity of the material was determined by capillary electrophoresis (Figure 4) of the material both before and after the ion exchange chromatography. The electropherogram indicates the importance of selecting the correct wavelength for analysis of BMP or any other small peptide, since the amino acids composing the analyte may only be measurable at specific wavelengths such as 200 nM but not at others such as 255 nm. This becomes particularly important when looking at peptide-products of protein digestion.
1370
Wavelength 1 • Pure @200
0)
^^Pure
0.020
o c
0.015
o
0.010
<
0.005
@255
Crude @200 Crude @255
0.000 H^ 2
3
4
5
6
Electophoretic
7
8
9
Mobility
10
11
12
(minutes)
Figure 4. CAPILLARY ELECTROPHEROGRAM OF BMP. A BioFocus 3000 capillary electrophoresis instrument (Bio-Rad^ with fast-scan optics was used to examine the level of purification of synthesized BMP. The samples were injected with constant pressure of 40 psi*sec. The separation of the crude (freshly synthesized) and the purified (desalted) BMP was performed using a constant voltage of 8 kV, a current limit of 50.0 //A and a polarity of positive to negative. The capillary column was a 24 cm X 25 /mi coated cartridge (Bio-Rad 148-3031) maintained at 16° C during the entire run. Total running time was 15 minutes. Inlet and outlet buffer contained 0.1 M phosphate buffer, at pH 2.5. Samples were placed in 0.01 M phosphate buffer with the same pH.
4. FLAVOR OF BMP 4.1 Umami/savory of BMP vs. MSG In 1909, Ikeda [18] named the distinctive taste of monosodium glutamate (MSG) and L-glutamate, "umami." Umami is derived from the Japanese word meaning "delicious" or "savory." For the remainder of this discussion, these terms will be used interchangeably. Since then significant interest in umami has been developed by food scientists and may be most clearly seen in the text on Umami edited by Kawamura and Kare [22]. Sensory evaluation was performed by 4-6 trained flavorists using the synthesized BMP both in water and in a beef flavored gravy . The threshold for BMP (MW = 848) is 1.41 mM or 0.16% by weight while that for MSG (MW = 169.1) is 1.56 mM or 0.026% by weight. In an aqueous solution at 5x threshold level (0.8% by weight), BMP presented a more up-front and more pronounced mouthfeel when compared to MSG. At this concentration BMP did not give a salty taste although it did give the impression reminiscent of salt replacers.
1371 The flavor enhancing activity of BMP became more pronounced when compared to MSG in a beef flavored gravy (Figure 5). Both BMP and MSG showed maximal savory development in the natural beef gravy when present at their aqueous threshold level. When the amount of either BMP or MSG was reduced by half only MSG showed a reduction in its intensity of savory development. At half-threshold, all three preparations of BMP still maintained the same intensity and flavor as at threshold. When MSG and BMP levels were again dropped in half to one quarter (25%) of their aqueous threshold concentration, the flavor of the MSG-containing gravy was identical to that of the control gravy. BMP at 25% threshold still maintained its near threshold level of response with some minor variability appearing. At quarter threshold (0.04% BMP by weight), BMP still presented a full-mouth and rounded-out flavor. Since MSG did not affect the flavor of the gravy at 25% of threshold, no further dilutions were made. On the other hand, BMP level was again diluted in half to 12.5% of threshold and tested for its flavor enhancing effect. At this level much of the original savory effect was still observed, but appeared much more variable in the three preparations. Thus, compared to MSG, BMP did not give a mouth watering, salt-like effect, but did give a full or creamy sensation while also enhancing the flavor.
iiiiiiBlBlieiiiiliii^^
BMP-I
-
% Threshold • 12.5%
BMP-II
^;.s; 25%
BMP-Ill |N.D.
• 50% 100%
MSG Control 0
20 40 60 80 100 Relative Savoriness/umami (% Maximum)
Figure 5. A COMPARISON OF THE FLAVOR ENHANCING ABILITY OF BNff AND MSG IN A NATURAL BEEF GRAVY. Three different preparations of BMP (BMP-I, BMP-II, and BMP-Ill; aqueous threshold level 1.41 mM; 0.16% by weight) were added at either threshold or below threshold level to a natural beef gravy and compared to similarly diluted MSG sample (aqueous threshold level 1.56 mM. 0.026% by weight). Their ability to affect the savory flavor of the gravy was determined by a panel of 4-6 trained flavorists at F&C International. Data is presented as relative to the threshold sensation.
The process of tasting involves the temporary adsorption of the taste molecules onto specific sites on the cell surface of the receptor of the taste buds. Taste properties of many foods are determined, in part, by their amino acid content [23]. The experiments of Drs.
1372 Tamura, Okai, and Kuramitsu [25, 48] show that BMP presents a savory/wmami taste only in its native form. Such experiments indicated that the smaller fragments of BMP elicit a response different from that of the parent peptide. For example, a "salty" taste is elicited from the HCl residue of Lys-Gly-, a "sweet" taste from the Lys-Gly-Asp residue, a "sour" taste from the Asp-Glu-Glu residue, and a "bitter" taste from the Ser-Leu-Ala residue [48]. Many theories have been presented on taste reception and several models have been presented for the probable mode of action of flavor enhancers [27, 28]. One of these models suggested and demonstrated that "sweetness" and "bittemess" are recognized at the same receptor [27,28]. Building upon this foundation of models, this author [42] proposed a single receptor model. This receptor would not only recognize sweet and bitter, but also sour and sawoTylumami [42]. Based on this model, the data available to us from our experiments to date, and some of the theories put forward by Dr. Nagodawithana in his article, we have proposed a model for the perception of BMP (Figure 6).
A proposed mechanism for perception of umami (savory, delicious)
SAVORY/M/«a/m + ENHANCED
FOOD
FLAVOR
SAVORY + enhanced umami Hydrophobic group Hydrophobic group or Electropositive group :^cctroneg^tive group
Figure 6. MOLECULAR MODELS FOR THE ACTION OF BMP.
The figure on the upper left is an extension of the proposed mechanism of Spanier and Miller [42]. In this case the lysine of the N-terminal end of BMP binds to that portion of the receptor that binds hydrophobic and/or electropositive groups while the first glutamine binds to the receptor that binds electronegative groups. This gives the distinctive savory flavor. One can get a savory/wmami response by MSG and related compounds when they bind to site B' only. BMP enhances the flavor of a food component when one portion of the food component first binds to its target receptor while the other portion of the food component binds to the ionized
1373 form of the carboxylic group on the second glutamate of BMP. The carboxylic group of the other glutamate molecule ties to the binding site of the receptor (see upper right diagram of Figure 6 and below). MSG works similarly with one carboxylic group binding to the B' site of the receptor and the other ionized carboxylic group to the food component. FOOD COMPONENT NH2-Lys-Gly-Glu-Glu-Ser-Leu-Ala-C00H I I A' B' receptor receptor site site
BMP enhancement of the flavor of a food component,
Another way that BMP might exert its effect in flavor enhancement is for the Lys and the first g/wtamine to be attached to a single receptor (as in top left diagram of Figure 6) and the second g/Mtamine to the B' site of a second receptor (as in the bottom left diagram in Figure 6). This would, speculatively, lead to an enhancement of the intensity of the ssLWory/umami response or a longer-lasting response (see below). receptor site B' NH2-Lys-Gly-Glu-Glu-Ser-Leu-Ala-C00B I I A' B' receptor receptor site site
BMP intensified enhancement of sayory/umami or lengthening of response
Although these mechanisms for taste perception are theoretical, the model, on the basis of physiochemical and biochemical properties, attempts to explain many of the sensory experiences and experimental data that have been generated over the years on studies of flavor potentiation. This is particularly true of savory food systems. For additional timely information on this subject the authors recommend the examination of the article by Dr. Nagodawithana [27]. 4.2 Saltiness of BMP vs. MSG The American consumer is acutely aware of the problems associated with high levels of sodium in the diet. This includes such factors as hypertension, a dilemma to those with cardiovascular problems, and toxemia, a problem to both mother and child in pregnancy. The consumer typically associates high sodium levels with saltiness although it is predominantly the chloride ion that imparts the salty taste. Since the name MSG indicates that it contains sodium as part of its structure, we determined to examine and compare the relative saltiness
1374 of MSG against BMP (Figure 7). It is evident that when MSG is added at its aqueous threshold level to a natural beef gravy, it presents an enhancement of the salty taste. Because many people like the salty taste of some foods, its saltiness (due to the chloride ion) should not be viewed as a negative flavor component. On the other hand, the saltiness perceived with MSG is detrimental to ones health as it is directly equated to an equal elevation in the food's content of sodium. BMP at threshold level did not enhance the saltiness of the gravy above its normal sensory response (Figure 7). This was probably due to our use of the volatile ammonium carbonate buffer in the final desalting step of the synthesis and purification of BMP. When the level of MSG and BMP were dropped to half (50%) of their threshold concentration, the saltiness response of MSG dropped to about half while there was no appreciable change in the saltiness response of BMP in all three preparations examined. When the level of MSG and BMP were again dropped in half to one quarter (25%) of its threshold concentration, the saltiness response of MSG again dropped but was still higher than the gravy without any additional flavor extender. BMP in all three preparations did not elicit a salty response. SALTINESS (% Maximum)
BMP-I
BMP-
Control GROUP
Figure 7. THE SALTINESS OF BMP VS. MSG IN A NATURAL BEEF GRAVY. Threshold level of BMP in aqueous solution is 1.41 mM while that for MSG is 1.56 mM. Samples were added to the gravy either at their full threshold level or at 50% or 25% of die direshold level.
5. FACTORS AFFECTING BMP A major factor involved with the deterioration of flavor in foods is the problem that was once named warmed-over flavor [WOF; 49] and has now been more appropriately named, MFD for meat flavor deterioration [40]. MFD is a continual process occurring in meat both in uncooked and in cooked meat. The basic difference is that MFD occurs more rapidly in meat that has been ground since the surface area exposed to oxygen is greater and the mechanical shearing of grinding has ruptured many cells. It is also because the heating
1375 associated with cooking has denatured many proteins and has changed the water activity such that more surface is again exposed to oxygen. As the meat lipids oxidize, they produce many secondary reaction products, such as alcohols, hydrocarbons, ketones, fatty acids and aldehydes, each capable of supplying a different aroma, and collectively, several different aromas [13 - 15, 26, 51]. During the process of lipid oxidation, there is a significant cascade of free radicals formed [20, 34] that have the potential of affecting the meat proteins and peptides [7-11]. Recent studies in this laboratory have shown how several meat proteins and peptides are affected by the free radicals derived during lipid oxidation [43]. The desired proteinaceous material was entrapped or encapsulated into an artificial membrane system, i.e. multilamellar liposomes, made from endogenous meat lipids. When exposed to a system containing dihydroxyfumarate, ferric iron, and adenosine diphosphate, hydroxyl-radicals were formed causing a cascade of free radical formation in the liposomes. Several enzymes were activated while others were inactivated by the ensuing free radical reactions. BMP, when entrapped alone within the multilamellar membrane system, was shown to be not affected in any manner by the reaction. On the other hand, electrophoretic analysis indicated that when BMP was entrapped as part of a meat extract, its structure was altered [43]. A mechanism was presented in which the free radical mechanism activated a proteinase that then cleaved the BMP into smaller peptide fragments. Thus, BMP appears to be resistant to direct attack and degradation by free radicals [43]. 6. CONCLUSION AND COMMENTS Beefy meaty peptide, BMP, is a flavor peptide found to occur naturally in beef. Its activity appears to be to enhance the flavor of the food it is added to by a receptor mechanism proposed in the text. This flavor enhancing peptide appears to elicit an effect even at concentrations below its aqueous threshold level presumably because of its enhanced interaction with the receptor or the food compound. Unlike monosodium glutamate, MSG, BMP does not exhibit the salty effect and does not contain high levels of sodium. BMP is resistant to direct attack from free radicals. Technology and biotechnology are becoming available for the production or enhancement of higher quality foods and food products. Results from the mechanistic-approach to meat flavor research will permit better predictive, adaptive and/or management methods for enhancing the flavor quality of meat to be developed. Extension of these findings obtained from meat, e.g., the flavor enhancing abilities of BMP, will permit us to utilize meat and meat by-products for the enhancement of the flavor and nutritional quality of other foods. One method aheady initiated by several concems has been the use of biotechnology to mass produce BMP for consumption and utilization in food and food products. However, significant impediments to implementing this knowledge are found both in necessary and required regulatory affairs and in the socioeconomic concems aroused in the pubHc when the food supply is being manipulated by these new techniques. The main thrust of future research should be directed toward the discovery of more natural flavor constituents so that muscle food products of high nutritional and flavor value can meet the concems and demands of tomorrow's consumers regarding how these products are produced.
1376 REFERENCES 1 M. E. Bailey, Food Technol. 42(1988): 123-126. 2 M. E. Bailey, S. Y. Shin-Lee, H. P. Dupuy, A. J. St. Angelo, and J. R. Vercellotti, in: Warmed-over Flavor of Meat. A. J. St. Angelo and M. E. Bailey, eds Orlando, FL: Academic Press, 1987 pp. 137-266. 3 A. J. Barrett, ed. in: Proteinases in mammalian cells and tissues, A. J. Barrett and J. T. Dingle, eds. Oxford: North-Holland Publishing Co., 1977 4 J. W. C. Bird, J. H. Carter, R. E. Triemer, R. M. Brooks and A. M. Spanier, Fed. Proc. 39(1980):20-25. 5 J. W. C. Bird, A. M. Spanier, and W. N. Schwartz, in: Protein Turnover and Lysosome Function. H. L. Segal and D. J. Doyle, eds. New York: Acad. Press, 1978 pp 589604. 6 J. W. C. Bird, W. N. Schwartz, and A. M. Spanier, Acta biol. med. germ. 36(1977): 1587-1604. 7 K. J. A. Davies, J. Biol. Chem. 262(1987): 9895-9901. 8 K. J. A. Davies, M.E. Delsignore, J. Biol. Chem. 262(1987): 9908-9914. 9 K. J. A. Davies, M.E. Delsignore, and S. W. Lin, J. Biol. Chem. 262(1987): 99029907. 10 K. J. A. Davies and A. L. Goldberg, J. Biol. Chem. 262(1987a): 8220-8226. 11 K. J. A. Davies and A. L. Goldberg, J. Biol. Chem. 262(1987b): 8227-8234. 12 M. A. Etherington, J. Taylor and E. Dansfield. Meat Sci. 20(1987): 1-18. 13 D. A. Forss, Lipids 13(1972): 181-258. 14 E. N. Frankel, in: Recent Advances in the Chemistry of Meat. A. J. Bailey, ed. The Royal Society of Chemistry: London, England, 1984 pp. 87-118. 15 U. Gasser, W. Grosch, Z. Lebensm. Unters. Forsch. 86(1988):489-494. 16 K. O. Honikel, in: New Technologies for Meat and Meat Products, F. J. M. Smulders, F. Toldra, J. Flores, and M. Prieto, eds., Ecceamst: Audet Tijdschriften B.V. 1992 pp. 135-160. 17 H. O. Hultin, J. Chem. Education 61(1984):289-298. 18 K. Ikeda, Original Communication of the 8th Intemational Congress of Applied Chemistry. 18(1912):147-149. 19 G. L Lnafidon and A. M. Spanier, Trends in Food Sci. & Techn. 1994 In press 20 J. Kanner, in: Lipid Oxidation in Food. ACS Symposium Series No. 500 A. J. St. Angelo, ed. Washington, D.C.: ACS Books, Inc. 1992 pp. 55-73. 21 H. Kato and T. Nishimura, in: Umami: A Basic Taste, Y. Kawamura and M. R. Kare, eds.. New York: Marcel Dekker 1987 pp. 289-306. 22 Y. Kawamura and M. R. Kare, eds. in: Umami: A Basic Taste. Physiology. Biochemistry. Nutrition. Food Science. New York: Marcel Dekker, Inc. 1987 649 pages. 23 J. Kirimura, A. Shimizu, A. Kimizuka, T. Ninomiya, and N. Katsuya, J. Agric. Food Chem. 17(1969):689-697. 24 M. Koohmaraie, A. S. Babiker, A. L. Schroeder, R. A. Merkel and T. R. Dutson, J. Food Sci. 53(1988): 1638-1641
1377 25 R. Kuramitsu, M. Tamura, M. Nakatani, and H. Okai, in: Food Flavor and Safety. Molecular Analysis and Design, ACS Symposium Series No. 528. A.M. Spanier, H. Okai, and M. Tamura, eds., Washington, D.C.: ACS Books, Inc. 1993 pp. 138-148. 26 G. MacLeod and J.M. Ames, Flavor Fragrance J. 1(1986):91-104. 27 T. Nagodawithana, Food Technology 48(1994):79-85. 28 K. Nakamura and H. Okai, in: Food Flavor and Safety. Molecular Analysis and Design, ACS Symposium Series No. 528. A.M. Spanier, H. Okai, and M. Tamura, eds., Washington, D.C.: ACS Books, Inc. 1993 pp. 28-35. 29 A. Ouali, N. Garrell, A. Obled, C. Deval, C. Valin, and I. F. Penny, Meat Sci., 19(1987):83-100. 30 A. Ouali J. Muscle Foods. 1(1990): 129-165. 31 A. J. St. Angelo, J. R. Vercellotti, M. G. Legendre, C. H. Vinnett, C. James, and H. P. Dupuy. J. Food Sci. 52(1987): 1163-1168. 32 A. J. St. Angelo, J. R. Vercellotti, J. P. Dupuy and A. M. Spanier, Food Technol. 42(1988):133-138. 33 P. Sellier and G. Monin, J. Muscle Foods, 5(1994): 187-219. 34 M. G. Simic, S. V. Jovanovic, and E. Niki, in: Lipid Oxidation in Food. ACS Symposium Series No. 500 A. J. St. Angelo, ed. Washington, D.C.: ACS Books, Inc. 1992 pp. 14-32. 35 F. J. M. Smulders, F. Toldra, J. Flores, and M Prieto, (eds.), in: New Technologies for Meat and Meat Products. Ecceamst; Audet Tijdschriften B.V., 1992 386 pgs. 36 A. M. Spanier, in: Food Science and Human Nutrition, G. Charalambous, ed., Amsterdam: Elsevier Science Publishers B.V., 1992 pp 695-709. 37 A. M. Spanier and J. W. C. Bird, Muscle and Nerve 5(1982):313-320. 38 A. M. Spanier, B. F. Dickens, and W. B. Weglicki, Am. J. Physiol. 249(Heart Circ. Physiol 18; 1985):H20-H28. 39 A. M. Spanier and T. Drumm-Boylston, Food Chem. 50( 1994):251-259. 40 A. M. Spanier, J. V. Edwards, and H. P. Dupuy, Food Tech. 42(1988): 110-118. 41 A. M. Spanier, C. C. Grimm, and J. A. Miller, in: Sulfur Compounds in Foods, ACS Symposium Series No. 564 C. J. Mussinan and M. E. Keelan, eds., Washington, D.C.: ACS Books, Inc. 1994 pp. 49-62. 42 A. M. Spanier and J. A. Miller, in: Food Ravor and Safety. Molecular Analysis and Design, ACS Symposium Series No. 528. A.M. Spanier, H. Okai, and M. Tamura, eds., Washington, D.C.: ACS Books, Inc. 1993 pp. 78-97. 43 A. M. Spanier, J. A. Miller and J. M. Bland, in: Lipid Oxidation in Food. ACS Symposium Series No. 500 A. J. St. Angelo, ed. Washington, D.C.: ACS Books, Inc. 1992 pp. 104-119. 44 A. M. Spanier and P. B. Johnsen, in: Molecular Approaches to Improving Food Quality and Safety, D. Bhatnagar and T. E. Cleveland, eds. New York: avi book. Van Nostrand Reinhold 1992 pp. 229-241. 45 A.M. Spanier, K. W. McMillin, and J. A. Miller J. Food Sci. 55(1990):318-326. 46 A. M. Spanier, A. J. St. Angelo, C. C. Grimm, and J. A. Miller in: Lipids in Food Flavors, ACS Symposium Series No. 558 C-T. Ho and T. G. Hartman, eds., Washington, D.C.: ACS Books, Inc. 1994 pp. 78-97. 47 A. M. Spanier and W. B. Weglicki, Am. J. Physiol. 243(Heart Circ. Physiol 12; 1982):H448-H455.
1378 48 M. Tamura, T. Nakatsuka, M. Tada, Y. Kawasaki, E. Kikuchi, and H. Okai, Agric. Biol. Chem. 53(1989):319-325. 49 M. J. Tims and B. M. Watts, Food Tech. 12(1958):240-243. 50 C. Valin and A. Ouali, in: New Technologies for Meat and Meat Products, F. J. M. Smulders, F. Toldra, J. Flores, and M. Prieto, eds., Ecceamst: Audet Tijdschriften B.V. 1992 pp. 163-178. 51 A. E. Wasserman and F. Talley, J. Food Sci. 33(1968):219-223. 52 Y. Yamasaki and K. Maekawa, Agric. Biol. Chem. 42(1978): 1761-1765. 53 Y. Yamasaki and K. Maekawa, Agric. Biol. Chem. 44(1980):93-97.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1379
Effects of storage under CO2 atmosphere on the volatiles, Phenylalanine Ammonia - Lyase activity and water soluble constituents of strawberry fruits. V. Dourtoglou*, A. Gaily, V. Tychopoulos, N. Yannovits, F. Bois, M. Alexandri, S. Malliou, M. Rissakis, M. Bony Vicryl SA., Viltanioti street, 14564 Kifissia, Athens, Greece
Abstract In this work, the effect of a modified atmosphere with carbon dioxide (CO2) on the biochemical pathways, responsible for the production of the secondary metabolites ; such as aroma, anthocyanins, flavonoids etc... ; was studied. The results of the effect of CO2 treatment can be correlated with biochemical events such as the expression of the enzymes, responsible for the aroma development ; the levels of the endogenous hormones, (e.g. lAA, ethylene), resulted to the further maturation of fruits. The commercial applications of this type of experiments are extensive and are correlated with the production of fruit juices and wines, as well as with the conservation of fresh fruits.
INTRODUCTION Fruit volatiles have been the subject of many research works since they have scientific and commercial importance. Most studies of plant volatiles were undertaken with the aim to identify the substances responsible for the characteristic aroma and flavor of plant material. Recently researchers focused on the biogenesis of volatiles. The enzymic systems responsible for the volatiles formation were studied and the compounds for the typical aroma were related to enzymatic activities. R. Tressl and F. Drawert in Biogenesis of Banana volatiles, [1], showed that the phenylalanine is a common precursor of a number of volatiles through a pathway in which the key enzyme is Phenylalanine Ammonia-Lyase (PAL). Later P.Schreier in the Chromatographic studies of biogenesis of plant volatiles, [2], reported a review of the enzymatic mechanisms which are responsible for the volatiles formation.
1380 The volatiles of cultivated strawberries have been studied extensively, during the last 35 years. Winter and Willhalm [3], identified over 60 compounds in Fragaria ananassa (var. Surprice des Halles). Tressl et al [4], identified about 200 compounds in the berries of the cultivar Revata . According to M. Larsen et al. [5], 2,5dimethyl-4-hydroxy-3-(2H)-furanone, linalool and ethyl hexanoate were found to be important strawberry aroma compounds (wild and cultivar strawberries), whereas ethyl butanoate, methyl butanoate, y-decalactone and 2-heptanone were found to be important cultivarspecific aroma compounds. In addition to 2,5-dimethyl-4-hydroxy-3-(2H)-furanone the methylether 2,5-dimethyl-4-methoxy-3-(2H)-furanone (mesifuran) was found to be a typical compound of strawberry aroma [6], [7]. There are various works reporting the biochemical pathway by which these compounds are synthesized ; but there is no evidence correlating these substances with the CO2 shock treatment. Dourtoglou et al and cited references [8], reported that during carbonic maceration (storage under saturated CO2 atmosphere) in grapes, biochemical changes occur resulting in an intracellular modification of aroma precursors. This leads to a typical aroma of the grape must which persists after the fermentation of the treated grapes. Volatiles and the typical aroma of wines were due to compounds such as vinyl-4-guaiacol, vinyl-4-phenol, eugenol, methyl and ethyl vanillate and ethyl cinnamate, which were obtained through the above process. All these compounds have as common precursor the aromatic amino acid Phenylalanine and are formed through a pathway in which the key enzyme is PAL. Grierson D. et al [28], described PAL in detail. The properties of PAL from higher plants and its position in phenyl propanoid metabolism was briefly reviewd by D.H. Jones [14]. PAL catalyses the elimination of NH3 from Lphenylalanine to give ^rans-cinnamate (Scheme 1) [15]. This deamination is an antiperiplanar reaction leading directly to trans -cinnamic acid [16] [17]. COOH
phenylalanine •
ammonia L-phenylalanine
Scheme 1
^
lyase
trans-cinnamic
acid
1381 Approximately six substituted cinnamates, via cinnamoyl-CoA esters © conjugates ® lignin (D a flavonoid
metabolic
pathways
may
be
distinguished
from
leading to: such as chlorogenic acid compound
via the ortho-hydroxylation of cinnamic acids by a cytochrome P-450 enzyme system leading to : ® coumarins via a coupling reaction leading to : (D 4-phenylcoumarins via chain shortening of the cinnamic acids yields : (D benzoate and substituted benzoates. These different metabolic options imply specialised cells or different location within the cells. However, may imply also a complex regulation of PAL activity. Evidence has accumulated that PAL occurs not only in cytoplasm but also in plastids, mitochondria, and microbodies [18] [19]. A portion of PAL fraction seems to be bound to the membrane. The difference in properties could be correlated with differences in localisation. For example, in leaves and roots of oak two forms of PAL are known. PAL.I is associated with a mitochondria-microbody and with benzoate synthetase activity, whereas PAL.II is associated with the microsomal fraction and the cinnamate-4-hydroxylase activity [20]. Apart from the ripening process, another scientifically and commercially important plant property is defence response to various abiotic and biotic stimuli such as light, wounding, fungal elicitors, artificial elicitors and pathogen infection. Different kinds of stress activate common inducible defence mechanisms which lead to the accumulation of host-synthesized antibiotics, deposition of lignin-like material, accumulation of hydroxyproline-rich glycoproteins and proteinase inhibitors and to an increase in hydrolytic enzyme activities. Cinnamate is a competitive inhibitor of phenylalanine, that means cinnamate could provoke a negative feedback on PAL. But due to the cell compartimentation, it seems that the concentration of cinnamate in the cell is low enough to have a negative effect on PAL. Coumarates are also inhibitors of a form of PAL, like benzoate and salicylate are for other PAL forms ; whereas magnesium, cytokinines act as PAL elicitors and induce a peak of PAL activity in a few days.
1382 The pH optimum for PAL at saturating substrate concentrations is about 8.7. A bell-shaped pH-activity curve has been reported in all cases; for example the potato enzyme is active from 7.25 to 10.25 [21]. PAL is present in cytosol and cytosol pH changes from 7 to 9 when the tissue is exposed in the light. So changes in PAL activity could be expected during a light exposure. After a stimulus and a short latency period PAL activity rises to a maximum (at which point synthesis is equal to breakdown) and then followed by a rapid fall to a lower level than the pre-stimulus. In order to interpret these results hypotheses were proposed. Most of them refer to PAL-IS where IS stands for inactivating system. The main agent is probably a proteolytic enzyme specific toward PAL. PAL activity responds to various external stimuli, such as light, wounding, fungal elicitor, infection, as well as to the developmental stage of plants. [21] In most etiolated plants studied light treatment causes an increase to PAL activity, [22], which is usually transcient and occurs in three phases : a lag phase (about 90 min); a phase of increase in activity(3-20h) and a phase of decrease in activity (3-20h).The photoreceptors involved vary with different plants[23]. M.Zucker [24], found that there is not an absolute light requirement, but exposure to light clearly increases the level of enzyme activity. Several attempts have been made but still the photocontrol of PAL is not clear. The present evidence clearly indicates that at least in some plants the photoregulation of PAL levels may be exerted via relatively specific macromolecular PAL inhibitors and thus through activation of the enzyme [25]. Exposure of fresh fruits and vegetables to O2 levels below or CO2 levels above their tolerance limits, results in various physiological disorders. In a study of CO2 effects on phenolic metabolism in lettuce tissue, found that 15% CO2 induced PAL activity which was correlated well with the development of brown stains. However, the mechanisms by which reduced O2 and/or elevated CO2 induces these physiological disorders are not known [25]. On the other hand it was found that when grapes are held under CO2 atmosphere aromatic compounds are formed in a large scale. PAL is the key enzyme for the formation of all these aromatic compounds. A high concentration of CO2 could represent another form of stress, probably by lowering the pH of plant tissue or by directly inhibiting respiratory enzymes and subsequently inducing higher PAL activity [8]. It has been reported, [22], that wounding increases PAL protein content. The rapidity of increase from very low, nearly undetectebale basal levels suggests that wounding stimulates mRNA transcription rather than its stability [25]. Based on the results previously reported by Dourtoglou et al. [8] an attempt to understand the role of the CO2 atmosphere to the PAL activity and to the way that the storage under CO2 can affects the post-harvesting development of the aroma in strawberry fruits took place. After a period of storage under saturated
1383 C02 atmosphere, strawberries were examined for PAL activity and volatiles. Results indicated that certain volatiles had been formed, derivated from the shikimic acid pathway where PAL is a key enzyme. These results may indicate a correlation between CO2, PAL activity and volatiles. During the determination of aroma compounds it was found that CO2 storage has an effect on indolyl acetic acid ethyl ester, which is an important endogenous hormone and has effects on the mechanism of fruit set, [10], [11].
MATERIALS AND METHODS Plant
material A strawberry (Frasaria ananassa Duch.) cultivar used in this study was the day neutral cv. Brighton. Sixty grams of these strawberries were put in darkglass bottle (2-litre volume). The bottle was filled with CO2. Another sixty grams were put in an open bottle (air sample). Samples were taken and assayed every day. Another experiment was made using a strawberry cultivar obtained from the local market. We did not give much attention to the name of this cultivar because the volatiles formed by this process are independant of the cultivar. Forty kilograms of this cultivar were placed in a heavy duty plastic vessel (170-litre volume) in the dark ; the vessel was closed hermetically and was connected to a carbon dioxide cylinder through a PVC tube(as it is described by Dourtoglou and al.[8]). This cultivar was also examined under air. Samples were taken and assayed every day.
PAL
assay The method used was based on the assay for PAL activity as Dourtoglou et al described [8]. The tissue (Ig of berries) was homogenised directly with 20 mg Phenylalanine ; 3 x weight borate buffer ( 0 Boric acid : 12.37g ; NaOH (IN) 100 ml; the volume was adjusted to 1000 ml with water, ® HCl (O.IN) ; a mixture of 73 ml ® + 27 ml ® was made to pH 8.8. The buffer also contained 5 mM mercaptoethanol). A blank was prepared as above with no substrate (phenylalanine). The homogenate was incubated for 1 hour at 40°C. The reaction was stopped by addition of 3 ml HCl 5N. With enzyme preparation of low specific activity, the acidified reaction mixture was heated in a boiling water-bath for 10 min. After the addition of 0.1 x weight Polyvinyl-pyrrolidone, samples were centrifuged at 12,000 rpm for 20 min at 4°C. The acidified reaction mixture (supernatant) was extracted 3 times (3 x 10 ml) with ether; aliquots of ether phase were removed, and the ether was evaporated under a stream of air at 65^0. The residue that remained was dissolved in 0.05M NaOH and the absorbance at
1384 290nm was determined. One unit of PAL activity was defined as the amount of enzyme that produced one jimole of cinnamic acid in 1 hour under the specified conditions described. Determination ofvolatiles Fifteen grams of each sample of strawberries (CO2 and AIR) were pressed and homogenised in a mortar. The product was centrifuged with 75 ml of distilled FREON 11 (12000 rpm at -4°C for 10 min). The freon extracts were dried by adding 1 gram of Na2S04 ; the freon was evaporated with a Vigreux column at room temperature and the residues were analysed by GC-MS. GC-MS analysis A Hewlett-Packard 5890 chromatograph was coupled with mass spectrometer system (5970) equipped with EI source. Gas Chromatographic conditions were as follows: column, SE-30 fused silica capillary length, 12m x 0.2 mm; film thickness 0.33 |im; injector temperature 200°C; transfer line temperature 230°C; carrier gas Helium; flow rate 0.0160cm3/s; sample volume 0.1 |al; split ratio 1:100. For the analysis the following multistep temperature program was used initial T °C 60
initial time (min) 3.0
rate (°C/min) 3.0 3.0 5.0 10.0
final To C 80 150 200 230
final time (min) 1.00 0.00 0.00 20.00
Scan acquisition : Start time 0.00 25.00 —
low mass 33.0 35.0 50.0
High mass 320.0 450.0 550.0
Scan threshold 1300 1300 1000
samples 3 3 2
scans /sec 0.75 0.52 0.86
1385 HPLC analysis An HPLC instrument Perkin Elmer Series 4 and a Perkin Elmer LC-85 UVA/'IS detector (multiwave length) were used for this work. HPLC conditions : The column used was a Spherisorb Cis ODS-2 (4.6x250 mm). The solvent used was: A: water/CHaCOOH (97.5/2.5) B: Methanol Detection UVA^IS X,28onm, >w5oonm Solvent Gradient: STEP FLOW* (min) (ml/min) 1 0 1 6 17 1 5 1 1 10 *In the set of analysis were different.
A%
B%
93 7 85 15 25 75 0 100 0 100 of Brighton cv. a flow of l,5ml/min was used and the RT
HPLC sample preparation a.Methanol extraction 2.5g of fruit were extracted with a volume of MeOH of 25ml at room temperature. The methanolic extracts were combined , filtrated and the methanol was removed in a rotary evaporator under vacuum (temperature did not exceed 400C).The residue was dissolved in 0.6 ml MeOH and injected to HPLC. b.Methanol/HCL extraction 2.5g of fruits were extracted with 25ml MeOH/HCL (99/l).The samples were left for 24h at O^C, subsequently filtrated and the filtrate was evaporated in the rotary evaporator under vacuum (temperature did not exceed 4 0 ^ 0 . The residue was dissolved in 0.6 ml MeOH and injected to the HPLC.
RESULTS AND DISCUSSION
Recent works described strawberry (Frasaria species) aroma as a mixture of 354 different compounds, [11], [12], [13]. In Table 1 the compounds derived from shikimic acid pathway are underlined.
1386 Table 1 Identified volatiles of strawberry fi*uits [[11],[12] Compound _^cidsEsters ...?^9^P}9.3.9?:^. propanoic acid ....?;.??.?.!'JtlXte?.R.^.^?.i9..?l9.i.4... hexanoic acid ....99.^???.9i9..?9.i4 ....4.?.9.?^.?9l.^9.?9?;^.
dodecanoic acid ....^.9.^.'^?.4^9.^.^9^9..?.9M ....^.9.?^?.^.9.9.?^??l9.i9..?.9A^ ••••^r.^?.?'9.!??.?.?.l?Jl9..^9.!f^.
Alcohols ethanol 3-methyl-2-butanol 3-methyl-l-butanol 1-pentanol 3-penten-l;ol 1-hexanol trans-2-hexen-l^ cis-3-hexen;l-ol ....?.".9.!'.l}y.l:A;A9.^.?i??.9.1 1-heptanol ....?.".^.9P.!'.?^.f?.9.^
1-octanol 2-nonanol 1-decanol 2-undecanol 2-tridecanol ....?'R.9.'^!'.?^.4.99.^.^9.\
linalool a-terpineol ci^tronellol myrtenol
••.•^•9.^.?.Y.1.^.\9.9.^9.\ •.••^•'•P.^.9.?.y.lP.y.9P.^.?.9.1 •••.^r.^?.?'9l?.Q.^.^.Y!.^.\9.9.1l9.l...
nerolidol Carbonyl compounds hexanal 2-hexanal 2-heptanone 2-nonanone 2-undecanone
....?.".!'.VA^^9.^.'^9?.9. ....?.".P.9.'S!'.^^99.?.^9P.9.
acetoin benzaldehyde verbenone
Compound vanillin 2-pentanone butyl formate hexyl formate ethyl acetate butyl acetate 3-methyl-2-butenyl a^^^ hexyl acetate trans-2-hexenyl^a^^^ cis-3-hexenyl acetate octyl acetate decyj acetate ethyl acetoacetate benzyl acetate caryeyl acetate^ methyl butanoate ethyl butanoate hexyl butanoa^^^ decyl butanoate ethyl crotonate methyl hexanoate ethyl hexanoate methyl decanoate methyl dodecanoate methyl salicilate methyl nicotinate methyl cinnama^^^ methyl anthrani^^^^^ methylN-formyla^^ diethyl ether pentane dichlorometh^^ chloroform dodecane tetradecane pentadecane styrene phenylacetylene linalool oxide y-hexalactone y-heptalactone 5-hexalactone y-octalactone 5-octalactone 2,5-dimethyl4-methoxy-3(2H)furanone 2,5-dimethyl-4-hydroxy -3(2H)furanone 2-methoxy-4-yiny^^^ 4-yinylphenol eugenql y-decalactone
1387 As Table 1 shows only a few compounds of strawberry aroma are derived from the Phenylalanine pathway. Except cinnamic acid, which has been identified by many reserchers, other compounds are 2-phenylethanol, methylsalicylate and some other in lower percentages. Generally, it can be said that the derivatives of cinnamic acid do not characterise the typical aroma of strawberry. During these experiments (heavy duty plastic vessel (ITOli.), 40kg of strawberries and CO2 atmosphere, results are shown in Table 2), an increase of the substanses derived from Phenylalanine is observed. Table 2 also shows that other substanses, like mesifuran or ethyl ester of Indolyl Acetic Acid (lAA-Et), are increased under CO2 atmosphere. The results were obtained during five (5) days of storage under CO2 atmosphere. The structure of strawberries and the texture in general had been decayed and they could be only used for the production of fruit juices or strawberry wine. Table 2 GC-MS Analysis of Volatiles of Strawberries samples (%) compounds ethanol;2-ethoxy acetate methyl hexanoate hexanoic acid benzyl alcohol ^ limonene mesifuran phenyl acetic acid i phenyl ethyl alcohol * nonanal + linalool benzyl acetate ^ epoxy linalool ethyl benzoate ^ methyl salicylate i myrtenol acetate...+ decanal gamma octalactone + anisaldehyde ^ linalyl octanoic acid phenyl ethyl acetate ^ bornyl acetate nonanoic acid eugenol ^ octyl-isobutyrate methyl piperonylate decanoic acid dodecanal
composition of volatils 0-day AIR 0,99 0,18 0,13 0,16 trace ^ 1,53 2,49 0,35 0,41 1,09
002
3,87
5,17 2,17 trace 1,15
trace trace 0,32 0,53
trace 0,14 0,35 trace trace
0,47 0,43 0,12 0,48
trace trace
0,71
0,68
trace trace trace trace trace
1388 Table 2 (continued)
compounds ethyl decanoate dimethyl phtalate gamma-decalactone geranyl acetone 2,6-di-ter-butyl quinone cinnamic acid ^ ethyl cinnamate ^ methyl vanillate ^ propyl p hydroxy benzoate ^ p hydroxy phenyl ethyl acetate ^ diethyl phtalate diethyl phtalate + nerolidol lauric acid ethyl laurate gamma-dodecalactone methyl acetyl o-aminobenzoate carotenoid degraded compound benzyl benzoate + hydrocarbure myristic acid ethyl p-hydroxy cinnamate ^ ethyl myristate pentadecanoic acid hexadecanol benzyl salicylate ^ isopropyl myristate indolyl ethyl acetate octadecanal methyl palmitate dichloro benzophenone palmitoleic acid palmitic acid ethyl palmitate isopropylpalmitate oleyl alcohol methyl oleate methyl stearate linoleic acid oleic acid ethyl linoleate + ethyl linolenate stearic acid stearic ac + ethyl oleate ethyl stearate dichloro benzophenone omite (pesticid)
0-day 0,25 0,30 trace 0,20
composition of volatils AIR C02 trace 0,23
3,37
0,51 0,18
3,67 0,77 trace trace trace 2,61
0,84 1,26 0,56
3739
i"76 0,30 0,92 trace trace
5,46
1,47 0,30 trace
0,96
1,60 1,57
0,48 0,74
1,65 0,72 10,18 0,50 0,44 0,81 0,39 1,12 5,07
4,44
15,92
11,66 0,84
4,43 14,51
0,76 2,86 7,74
7,53
1,59 5,68
0,98
6,66 0,49 4,33
trace trace 2,41 trace 0,69 0,56
0,22 trace 3,58
1389 Table 2 (continued) compounds dibutyl phtalate dioctyl phtalate squalene total identified compounds total phenylalanine derivates other unidentified compounds
composition of volatils AIR C02 2,88 2,74 7,08 2,80 0,38 52,93 64,38 74,94 0,16 1,45 7,89 47,07 35,62 25,06
0-day
1 Compounds derivated from phenylalanine 2 trace are not considered as value
In a second series of experiments(dark-glass bottle, 60g strawberry fruits ; results in table 3), the effect of CO2 in some volatiles was studied. According to the Table 3, an increase of the Phenylalanine derivatives was observed. In these experiments and against to other reports mesifuran had a higher concentration under air than under carbon dioxide. As it was discussed at the introduction the increase of cinnamate derivatives can be explained through an activation of enzymic systems of PAL or by a hydrolysis of various Phenylalanine derivatives. The latter assumption seems less possible because, as it is shown in Tables 2 and 3, apart from the free cinnamic acid and the phenyl ethyl alcohol, an increase of compounds that are ethyl and methyl esters of the previous refered, are observed. Also, Table 3 shows that ethyl cinnamate and methyl salicylate are present in a proportion of 0.3-0.5 % after CO2 treatment, while they were not initially in the fresh sample of strawberry. Table 3 Volatiles of Strawberry fruits cv. Brighton (%).
0-day PHE derivatives maximum linalool + nonanal benzyl acetate methyl salicylate cinnamic acid ethyl cinnamate
total mesifuran
storage conditions AIR 2 PAL activity decreasing 0,96 0,16
trace ^ 0,00 0,66
1,12 8,30
C02 = minimum 0,84 0,35 0,23 0,74 0,35 2,51 4,17
10-day : strawberries are put in freezing just after picking 2 AIR, CO2 : strawberries are stored 1 day on the indicated atmosphere ^trace is not considered as value PHE means phenylalanine
1390 Another question raised during this experiment was the involvement of the cultivar variability to volatiles formation when is treated with CO2. Table 4 shows the results obtained when a strawberry cultivar of the local market was examined for 6 days under CO2. The differences observed in compounds derived from Phenylalanine are important and show that they increased after CO2 shock. These results confirm that this response to CO2 shock is independent of plant cultivar. Table 4 Volatiles of Strawberry fruits from local market stored under CO2 atmosphere (%) PHE derivatives ^ benzyl acetate phenyl ethyl acetate p-hydroxy phenyl ethyl acetate methyl salicylate ethyl salicylate benzyl alcohol phenyl ethyl alcohol eugenol ethyl benzoate cinnamic ac + ethyl methoxy benzoate ethyl cinnamate ethyl p-hydroxy cinnamate total other important volatiles isoamyl acetate 2-methyl butyl acetate indolyl ethyl acetate linalool linalool +nonanal methyl thioacetic acide ethyl ester mesifuran 2,5-dimethyl-hydroxy-3(2H)furanone total
fresh
4 days juice fruit trace 0,20 0,47 0,50 0,20 0,25 trace trace
6 days
0,29
4,37
3,22
trace 0,27
0,10 0,31
trace 0,68
10,16 trace 0,16
1 day
trace^
0,66
0,35 0,66
0,91
0,73 5,68
5,35
ripe 1 0,18
0,92 0,87 0,35
0,07 0,97 0,73 14,23
0,18
0,61 0,28 7,19
2,61 0,57
9,92
0,58
0,66
0,37
2,54
0,08 4,27
2,95
7,54
5,85
1,56
3,12
5,29
10,51
10,72
0,69 17
1,56
trace
1 ripe : strawberry fruits ripened in normal conditions (air) 2 traces are not considered as value 3 PHE means phenylalanine
1391 HPLC analysis of water soluble compounds is given in Table 5 and an increase is shown in anthocyans, methyl cinnamate, cinnamic acid and furan derivatives. The HPLC sample preparation did influence the chromatographic profile.The extraction with methanol extracted plant phenolics and other polar compounds. The extraction with MeOH/HCl probably caused a mild hydrolysis of components been in a "bound form" previously. A comparison between the two maners of extraction is possible and the results are semiquantitave (since in the sample preparation used the same amount of plant material and the final dillutions were made with the same volume of solvant and the injections were 6 \)1 each). Differences are observed within the same sample due to the different way of extraction. In Table 5 the values of peak height per peak are given. The main differences were observed in peaks with retention time (RT) 4.60, 5.50, 11.69 (all peaks are unknown except the cinnamic acid and methyl cinnamate content ; samples were spicked with the appropriate standard (the components with RT 1620 are atributed to anthocyanins chromatogram at X.500, shows these peaks). This patern was the same in all samples examined. However differences were observed due to the sample origin and the period of storage under CO2 (for example cinnamic acid in Tables 5 and 6). Using the results shown in Table 5 and comparing separately the MeOH extracts and the MeOH/HCl extracts significant differences can be seen. Table 5 HPLC analysis of strawberry fruits from the local market stored under CO2 atmosphere RT
Dayi 0 1 4 6
4.60 B 100 341 212 NA
A 27 62 24 87 RT11.69
Dayi 0 1 4 6
A 86 101 60 42
RT
B 473 1213 991 NA
Anthocyans A B 186 273 453 223 242 185 86 NA
A 49 35 61 42
5.501
RT8.74
fraision^ B 1898 5704 5300 NA
Cinnamic ac. A B 12 59 68 24 70 22 NA 44
A 23 34 71 12
B 24 49 32 NA
methvl cinnamate A B 97 3 58 24 124 2 NA 32
1392 Table 6 HPLC analysis of strawberry fruits cv. Brighton
fresh day 1 O2 day 1 CO2
Cinnamic acid A 30 53 734
1 Days under CO2 atmosphere ^Fraision : 2,5-dimethyl-hydroxy-3(2H)-furanone A = MeOH extraction B = MeOH / HCl extraction and mild hydrolysis RT means Retention Time Finally it is very interesting that the major volatiles in strawberry (mesifuran and linalool), exist in high concentrations after CO2 treatment. It can not be concluded with the existing results if it is an in vitro synthesis or a release from a pre-existing bound form during the action of glycositic enzymes. According to the Table 5, an increase of the bound forms of the above compounds is observed, because of a mild hydrolysis with MeOH/HCl which results to high peaks. The effect of CO2 is not only shown in Phenylalanine derivatives but also in tryptophan and one of its derivatives, lAA-Et. Table 3 and 4 show that the percentage of lAA-Et is increasing to 2-10 % of the total extracted volatiles. The phenomenon is noteworth and could be connected with the maturation and the senescence in general of the strawberry fruits. Former reports indicate that after an increase in lAA-Et concentration, observed during the tenth day after anthesis, it decreases to zero levels towards the fruit maturation. If CO2 shock induces lAA-Et formation it would mean the action of an enzymatic system and whether is created de novo or is released from a bound form. Another assumption which can not be proved from the existing data is that CO2 shock treatment induces not only PAL, but also various glucosidases, pectinases and pectinasterases, which hydrolyse pectins and glucosidically bound forms of the above compound resulting in an increase of its percentage after the extraction. In order to determine a possible enzymic induction PAL activity was measured and varies as Figure 1 shows. The behaviour of PAL under CO2 was evaluated and measured in many experiments and was found that it does not always follow this model. It was assumed that this was due to experimental errors during the assay. On the other hand previous work in grapes, during two
1393 continuous years and in four different cultivars, showed the same model ; induction of PAL activity was observed after 6 to 10 days under CO2 atmosphere [8]. A similar model was reported by Siriphanich and Kader [27] during their work in lettuce.
Figure 1. PAL activity of strawberry fruits from local market stored under AIR and CO2 atmosphere
CONCLUSION The above results strongly indicate that treatment of strawberry fruits with CO2 saturated atmosphere has a significant effect on the volatiles, PAL activity and water soluble constituents. During this study many questions raised and there is still a lot of research to be done since these results are of great scientific and commercial interest.
REFERENCES 1. 2. 3. 4. 5. 6.
Tressl R., Drawert Fr. ; J. Agr. Food Chem., 21 (1973) 4: 560 Schreier P . ; Chromatographic studies of biogenesis of plant volatiles, Huething Verlag: Heildeberg, (1982): 76 Winter M., Wilhalm B. ; Helv. Chim. Acta, 47(1964): 1215 Tressl. R., Drawert F. , Heimann W. ; Z. Naturforsch. B, 24(1969): 1201 Larsen M., Poll L., Olsen C.E.; Z.Lebensm Unters Forsch, 195(1992):536 Re L., Manrer B. and Ohloff G.; Helv. Chim. Acta, 56(1973):1882
1394 7 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Hirvi T. ; Lebensm. Wiss. TeghnoL, 16(1983): 157 Dourtoglou V., Yannovits N., Tychopoulos V., Vamvakias M. ; J. Agric. Food Chem., 42(1994) 2: 338 Archbold D.D. and Dennis Jr. F.G. ; J. Amer. Soc. Hort. Sci., 109(1984) 3: 330-335 ; 110(1985) 6 : 816 Darnell R.L and Martin G.C. ; J. Amer. Soc. Sci., 112(1987) 5 : 804 Pyysalo T., Honkanen E. and Hirvi T. ; J. Agric. Food Chem., 27(1979) 1 : 19 Larsen M., Poll L., and Olsen C.E. ; Z Lebensm Unters Forsh, 195(1992) :536 Maarse H., Visscher C.A. ; Volatile compounds in food, TNO-CIVO, 1 (1989) : 189-197 Jones D.H. ; Phytochemistry 23(1994) 7: 1349 Koukol J., Conn E.E., J. Biol. Chem., 236(1961): 2692 Hanson H.R., Wightman R.H., Stauton J., Battersby A.R. ; Chem. Commun., (1971) : 185 Ife R., Haslam E. ; J. Chem. Soc. C(1971) : 2818 Hanson H.R., Wightman R.H., Stauton J., Battersby A.R. ; Chem. Commun., (1971) : 185 Alibert G., Ranjeva R., Boudet A.M. ; Physiol, veg., 15(1977) : 279 Mc Clure J., Recent Adv Phytochem, 12(1979): 525 Alibert G., Ranjeva R., Boudet A.M. ; Biochim. Biophys. Acta, 279(1972) :282 Havir E.A., Hanson K.R. ; Biochemistry, 7(1968): 1904 Camm E.L. and Towers G.H.N. ; Progr.Phytochem, 4(1977): 169 Smith H., Billet E.E., Giles A.B. ; edited by Smith H. ; chapter 6 ; Ac. pr.(1977) Zucker M. ; Plant Physiology, 40(Sept.l965) 5: 779 Kader A.A. ; F. Tech., (May 1986): 99 Smith B.G. and Rubery P.H. ; Plant Sci. Letters , 15(1979) : 29 Siriphanich J. and Kader A.A. ; J. Amer. Soc. Hort. Sci. 110(1985a) :249 and 110(1985b) : 333 Given N.K., Venis M.A. and Grierson D., J. Plant Phys., 133(1988): 25 and 133(1988):31
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1395
S t u d i e s o n t h e Hydrolysis of F i s h P r o t e i n by Enzsnnatic T r e a t m e n t Antonio M. Martin and Darren Porter Food Science Program, Department of Biochemistry, Memorial University of Newfoimdland, St. Joha's, Newfoundland, Canada AlB 3X9 Abstract Enzymatic treatment offish biomass with proteolj^ic enzymes in laboratory reactors resulted in the production of protein hydrolysates. Optimization of the main process parameters for the proteolytic reaction was conducted. The parameters optimized were temperature, reaction time, pH, and enz5nne / substrate ratio. After the hydrolytic treatment was concluded, the enzyme was inactivated by heat and the resulting slurry, containing the solubilized fish protein, filtered to separate residual bones and scales. Drying the fish protein solution to a powdered form concluded the process. The powder produced was analyzed for chemical composition. 1.
INTRODUCTION
In general, the main purpose for producing fish protein concentrates (FPC) has been to develop a new food source. Green and Mattick (1979) indicated that its production has been expected to help alleviate some of the problems facing the fishing industry, such as incomplete utilization of the fish catch and perishability of the product. Fish protein, being of animal origin, provides a good pattern of essential amino acids and thus has good nutritive value. An important objective of the projects conducted in the development of FPC has been to use it for the protein enrichment of foods. In addition, Martin and Patel (1991) indicated the importance of the production of FPC for the bioconversion of fisheries biomass from underutilized fish species and, possibly, from seafood processing wastes. Among the methods for the production of FPC are those involving the hydrolysis of the fish proteins to produce fish protein hydrolysates (FPH). Fish hydrolysation implies the liquification of the fish, and includes the use of enzymatic hydrolysis (Hale, 1974). Spinelli et al. (1972) reported the production of FPC with functional properties. A process for the enzymatic conversion of underutilized fish to a liquified product was patented by Keys and Meinke (1966). The term liquified fish protein concentrate (LFPC) has been applied to the product resulting from enzymatic digestion (Watanabe et al., 1974). Capelin, Mallotus villosus, is a fish found seasonally in numerous coastal areas including the Grand Banks of Newfoundland (Ackman et al., 1969). This
1396 species is high in protein and therefore can be converted into FPC, in powdered form, and incorporated into such foods as soups, breads and cereals (Yu and Tan, 1990). Studies have suggested that an increase in hydrolysis time, or in the enzyme / substrate ratio, will result in a decrease in the average chain length of the peptides in the soluble fraction. Prolonged proteolysis may result in highly soluble peptides, and may also promote the generation of bitter flavours in the hydrolysed product (Quaglia and Orban, 1987). The selection of the proteolytic enzyme is a factor of major importance in the process. Mohr (1978) observed that endopeptidases with low specificity, such as bacterial proteases, are more effective for hydrolysis offish protein than highly specific ones, such as trypsin. Liu and Pigott (1981) commented on the high cost of pepsin as an agent for the hydrolysis offish, presenting a method for producing inexpensive crude pepsin. Windsor and Barlow (1981) remarked that the cost of an enzyme rises with its purity, and that the use of a mixture of enzymes could solve the problem of off-flavours that are produced if an enzyme with less purity is used. Bhumiratana et al. (1977) studied the solubilization of FPC by trypsin in batch, semi-batch and continuous-flow membrane reactors. This work was conducted with the objective of developing a soluble fish protein powder from male capelin by enzymatic methods, based on the premise that the availability of inexpensive male capelin, left over from the capelin roe fishery, could make the production of FPH economical.
2.
EXPERIMENTAL
2.1. Materials Whole male frozen capelin were supplied by the Newfoundland and Labrador Institute of Fisheries and Marine Technology, St. John's, NF. Alcalase 2.4L, Neutrase 0.5L, and PTN3.0 type Special were obtained from Novo Nordisk Bioindustrials Inc., Danbury, CT. 2.2. Hydrolysis experiments The three enzymes were each tested at various concentrations (0.05 to 1.50 % w/w, enzyme / H2O). The enzyme and concentration that resulted in the highest percent hydrolysis was then tested at various temperatures. The best results of this work were then used as the basis for testing the effect of pH on percent hydrolysis, and subsequently for the effect of time. In these experiments, 20 g samples of whole capelin were mixed with 20 mL of water and heated to the desired temperature. The pH was adjusted to the desired level and maintained using O.IN NaOH. Each enz3nne was then added in separate experiments and the hydrolysis was allowed to proceed for a given time. At five-minute intervals, the amount of base added was recorded in order to determine the degree of hydrolysis by the pH-stat method (Boyce, 1986). After the completion of the hydrolysis
1397 time, the enzjnne was deactivated by heating the solution to 85°C. The solution was centrifuged and the yield determined. 2.3. Analytical methods Amino acids The samples were hydrolysed with 6N HCl imder vacuum for 24 h at 110°C (Blackburn, 1968). They were then reconstituted with 0.6M lithiimi citrate buffer and analyzed with a Beckman 121 MB amino acid analyzer using a single colimm method (Mondino et al., 1972; Ohara and Ariyoshi, 1979). An analysis for tryptophan was performed by the method of Penke et al. (1974). Ash
The A.O.A.C. method 14.006 (Anon., 1980) was used to find the ash content of the samples.
Moistiu'e The moisture content was determined according to A.O.A.C. method 7.003 (Anon., 1980). Total hpids The total lipids content was determined using the method of Bligh and Dyer (1959). Total nitrogen The A.O.A.C. modified micro-Kjeldahl method 47.021 (Anon., 1980) was employed to find the total nitrogen content. 3.
RESULTS AND DISCUSSION
3.1. Optimization of reaction parameters for the commercial enzymes Initially, the enzymes Neutrase 0.5L, Alcalase 2.4L, and PTN 3.0S Type Special were tested to find which would result in the highest protein solubilization. The enzyme PTN 3.OS Type Special resulted in poor hydrolysis of the substrate and hence low yield, in comparison to Neutrase 0.5L and Alcalase 2.4L, so results were not presented for this enz5rme and no further work was done with it. Both Neutrase 0.5L and Alcalase 2.4L resulted in good hydrolysis and showed varying effects when reaction parameters were changed. Subsequently, more testing was performed using these two enzymes, and it was found that Alcalase 2,4L gave a higher degree of hydrolysis than Neutrase 0.5L, for similar enzyme concentrations, as can be seen in Tables 1 and 2. Indeed, it can be seen that, in comparison to Neutrase 0.5L, using less Alcalase 2.4L resulted in more hydrolysis, so Alcalase 2.4L was judged to be more effective. With regards to the effect of the concentration of enzyme in the reaction solution (the enzyme / H2O
1398 ratio), it was found that percent hydrolysis increases with increasing concentrations of enz5nne, but only up to a certain point. Table 1 Effect of varying concentrations otNeutrase 0.5L on protein hydrolysis ^ % Enzyme (w/w, enz3mae/H20)
^
Degree of Hydrolysis (%) "
0.05 8.72 ± 0.28 0.10 9.46 ± 1.34 0.15 9.44 ± 0.88 0.25 9.61 ± 0.69 0.50 8.87 ± 0.15 1.00 8.20 ± 0.06 1.50 7.00 ± 0.18 Whole male capelin hydrolysed at 60°C and pH 7.8 for 15 minutes. Mean values of three determinations ± standard deviation.
Table 2 Effect of varying concentrations ofAlcalase 2.4L on protein hydrolysis ^ % Enzyme (w/w, enz3nne/H20) 0.05 0.10 0.15 0.20 0.25 0.30 0.45 0.90 1.25 ^
Degree of Hydrolysis (%) " 17.69 17.66 17.70 23.42 20.09 18.72 15.36 13.60 12.15
± ± ± ± ± ± ± ± ±
2.12 0.06 2.47 0.27 1.88 1.72 0.75 0.91 0.01
Whole male capelin hydrolysed at 50°C and pH 8.0 for 15 minutes. Mean values of three determinations ± standard deviation.
Based on preliminary studies, the experiments presented in Tables 1 and 2 were conducted at temperatures of 60°C (or Neutrase 0.5L and 50°C for Alcalase 2AL, However, as Table 3 indicates, the degree of hydrolysis for the latter at 60°C was also higher than for the former at this temperature. Table 3 shows that with increasing temperature the percent hydrolysis increased to a maximimi, and then decreased. Because the difference in percent hydrolysis for Alcalase 2.4L at 50°C and 60°C was not very notable, and considering that a lower processing temperature would result in a better FPH product (also, a lower percent
1399 hydrolysis would give a less bitter product), it was decided that 50°C would be chosen as the hydrolysis temperature for this work. Table 3 Effect of temperature on protein hydrolysis using Alcalase 2.4L ^ Temperature ± 2°C 40 50 60 70 ^
% Hydrolysis ^ 11.76 17.06 20.14 18.61
± ± ± ±
1.24 0.60 1.15 1.93
Whole male capelin hydrolysed at pH 8.0 for 15 minutes, enzyme concentration 0.20 % (w/w, enz5mae/H20). Mean values of three determinations ± standard deviation.
It should be noted that, in this work, the experiments for the optimization of the various parameters in the hydrolysation processes were conducted in blocks, with one block of experiments allocated for each parameter. Therefore, variabilities found among blocks of experiments in the percent hydrolysis values obtained with the optimized parameters were due to expected biological variation, mostly occurring in the substrate or raw material employed. However, the validity of the optimized parameters is verified by the replications of the experiments in each block, conducted imder the same operating conditions. The effects of pH on percent protein hydrolysis can be seen in Table 4, which indicates that at 50°C and 0.20 % Alcalase 2.4Ly a pH of 8.0 was optimum for hydrolysis. The results of the experiments to find the relationship between time of reaction and degree of hydrolysis can be seen in Table 5, which shows a reduction in the rate of hydrolysis after 15 minutes, and that there were no significant differences for the percent hydrolysis at 15 minutes and at longer times up to 30 minutes. In addition, in separate experiments, it was found that the quality of the FPH deteriorated with longer hydrolysis times, with increasing degrees of bitterness. From the point of view of the process, the optimum was identified as the combination of enzyme and process parameters that produced the highest yield of solubilized protein. However, in addition to yield optimization, the best overall process should be determined considering the functional properties and composition of the protein product.
1400 Table 4 Effect of pH on protein hydrolysis using Alcalase 2.4L ^ pH ± 0.05 7.80 7.90 8.00 8.10 ^
% Hydrolysis ^ 18.33 18.68 21.40 19.74
± ± ± ±
1.38 1.31 0.81 1.79
Whole male capelin hydrolysed at 50°C for 15 minutes, concentration 0.20 % (w/w, enzyme/H20). Mean values of three determinations ± standard deviation.
enzyme
Table 5 Effect of time on protein hydrolysis using Alcalase 2.4L ^ Time (minutes) 5 10 15 20 25 30 35 40 45 50 55 60 65 70 ^
% Hydrolysis " 12.07 ± 14.81 ± 16.20 ± 17.03 ± 17.57 ± 18.02 ± 18.31 ± 18.48 ± 18.70 ± 18.85 ± 18.86 ± 19.02 ± 19.06 ± 19.07 ±
2.56 2.45 2.76 2.51 2.57 2.39 2.29 2.24 2.34 2.20 2.22 2.16 2.11 2.11
Whole male capelin hydrolysed at 50°C and pH 8.0, enzyme concentration 0.20 % (w/w, enzyme/HgO). Mean values of three determinations ± standard deviation.
3.2. Composition of the fish protein hydrolysate Table 6 shows the proximate composition, and Table 7 presents the amino acid content of the FPH produced. Its protein content and quality compares well with other similar products, making it potentially useful for protein supplementation of foods and feeds. Work is in progress concerning some of the functional properties of the FPH produced. Preliminary results also indicate that
1401 this product could be incorporated into foodstuflfs that require some of the characteristic properties of protein products. Table 6 Proximate analysis of FPH produced using Alcalase 2AL ^ Component
^ " 4.
%
Ash 14.45 ± 0.23 Lipids 8.55 ± 0.07 Protein ^ 75.78 ± 0.78 Mean values of three determinations ± standard deviations. Nitrogen content X 6.25. CONCLUSIONS
The use of whole male capelin in hydrolysis processes with the proteolytic enz5nne Alcalase 2.4L proved to be successful for the production of FPH in powdered form. The powders were high in protein, low in fat, relatively odourless, and light yellow in colour. 5.
ACKNOWLEDGEMENTS
Funding for this work was provided by the Atlantic Canada Opportunities Agency (A.C.O.A.), the Canadian Centre for Fisheries Innovation (C.C.F.I.), and Seabright Corporation, all of St. John's, Newfoundland, Canada. The authors would like to thank Mr. D. Hall for the amino acid analysis, and Mr. P. Bemister for assisting with the manuscript. Mr. Hall and Mr. Bemister are with the Biochemistry Department, Memorial University of Newfoundland, St. John's, Newfoimdland.
1402 Table 7 Amino acid content of FPH produced using Alcalase 2AL Amino Add or Derivative Alanine Ammonia Arginine Aspartic acid P-Alanine Cystine Ethanolamine Glutamic add Glydne Histidine^ Hydroxylysine Hydroxjrproline Isoleudne^ Leudne^ Lysine^ Methionine^ 1-Methyl Histidine 3-Methyl Histidine Ornithine Phenylalanine^ Phosphoserine Proline Serine Taurine Threonine^ Tyrosine Tryptophan^ Valine^ ^ Essential amino adds.
mg/g protein 45.4 7.7 39.1 63.4 1.1 6.5 1.1 85.7 39.7 14,3 1.1 2.8 31.0 51.2 61.5 23.0 2.2 0.1 3.4 26.1 0.2 40.3 32.3 7.2 34.3 23.3 1.7 39.9
1403 6.
REFERENCES
Ackman, R.G., Ke, P.J., MacCallum, W.A. and Adams, D.R. (1969). Newfoundland capelin lipids: Fatty acid composition and alteration during frozen storage. J. Fish. Res, Board Canada, 26, 2037-2060. Anonymous (1980). Official Methods of Analysis, 13th edition, ed. W. Horwitz. Association of Official Anal3rtical Chemists, Washington, D.C. Bhumiratana, S., Hill, C.G., Jr., and Amundson, C.H. (1977). Enzymatic solubilization offish protein concentrate in membrane reactors. J. Food Sci., 42(4), 1016-1021. Blackburn, S. (1968). Amino Acid Determination: Methods and Techniques. Marcel Dekker Inc., New York, pp. 21-22. Bligh, E.G. and Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911-917. Boyce, C.O.L. (1986). Novo's Handbook of Practical Biotechnology, Novo Industri A/S, Bagsvaerd, Denmark, pp. 125. Green, J.H. and Mattick, J.F. (1979). Fishery waste management. In: Food Processing Waste Management, eds. J.H. Green and A. Kramer. AVI, Westport, CT, pp. 202-227. Hale, M.B. (1974). Using enzymes to make fish protein concentrates. In: Marine Fisheries Review, D83 Technical Information Division, Environmental Science Information Centre, NOAA, Washington, D.C. Paper 1034, vol. 36 (2), pp. 15-18. Keys, C.W. and Meinke, W.W. (1966). Methods of processing fish. U.S. Patent 3,249,442. Liu, L.L. and Pigott, G.M. (1981). Preparation and use of inexpensive crude pepsin for enzyme hydrolysis offish. J. Food Sci., 46, 1569 - 1572. Martin, A.M. and Patel, T.R. (1991). Bioconversion of wastes fi:om marine organisms. In: Bioconversion of Waste Materials to Industrial Products, ed. A.M. Martin. Elsevier Applied Science, London, pp. 417-440. Mohr, V. (1978). Fish protein concentrate production by enzymatic hydrolysis. In: Biochemical Aspects of New Protein Foods, eds. J. AdlerNissen, B.D. Eggimi, L. Munck and H.S. Olsen. Proc. 11th FEBS Meeting, 44, 53-62, Pergamon Press, Oxford. Mondino, A., Bongiovanni, G., Fumero, S. and Rossi, L. (1972). An improved method of plasma deproteination with sulphosalicylic acid for determining amino acids and related compounds. J. Chromatog., 74, 255-263. Ohara, I. and Ariyoshi, S. (1979). Comparison of protein precipitants for the determination of fi:ee amino acids in plasma. Agric. Biol. Chem., 43(7), 1473-1478. Penke, B., Ferencze, R. and Kovacs, K. (1974). A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analyt. Biochem., 60, 45-50.
1404 Quaglia, G.B. and Orban, E. (1987). Enzymic solubilisation of proteins of sardine (Sardina pilchardus) by commercial proteases. J. Sci, Food Agric, 38, 263-269. Spinelli, J., Koury, B. and Miller, R. (1972). Approaches to the utiUzation offish for the preparation of protein isolates. J. Food Sci., 37, 599-603. Watanabe, T., Ebine, H. and Okada, M. (1974). New protein food technologies in Japan. In: New Protein Foods: Technology, ed. A.M. Altschul. Academic Press, New York, pp. 414-453. Windsor, M. and Barlow, S. (1981). Fish protein concentrate. In: Introduction to Fishery By-Products. Fishing News Books, Farnham, Surrey, U.K., pp. 111-123. Yu, S.Y. and Tan, L.K. (1990). Acceptability of crackers with fish protein hydrolysate. Int. J. Food ScL Technol, 25, 204-208.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
Production of Protein Hydrolysate from Lobster (Panulirus
1405
spp.)
Gustavo H.F. Vieira ^, Antonio M. Martin ^, Silvana Saker-Sampaiao ^, Carlos A. Sobreira-Rocha ^, and Raimunda C.F. Goncalves ^ ^ Laboratorio de Ciencias do Mar (LABOMAR) - Universidade Federal do Ceara (U.F.C.), Fortaleza, Ceard, Brasil ^ Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AlB 3X9 Abstract Protein hydrolysates were prepared using waste meat from lobsters (Panulirus spp.) from the northeast coast of Brazil. Solubilization of the protein, measured by the liberation of tyrosine, was conducted with proteolytic enzymes. A statistical analysis of the hydrolysis process results indicated that they were dependent on time, type of enzyme, and the enzyme concentration. The optimum results, including those of total amino adds and free amino acids analyses, were obtained for the products prepared using a fungal protease. A statistical analysis of the results of a taste panel was conducted and the potential of employing these hydrolysates as flavourants is presented. The fimctional properties of the products were reported as good.
INTRODUCTION The objective of recovering protein from wastes from food processing operations has long been of interest in many areas of the world. Wastes from finfish and shellfish fisheries operations are especially attractive, given the quality of the protein from marine organisms. The potential for the production of several types of protein concentrates from seafood processing wastes has been reviewed by Martin and Patel (1991). The term fish protein concentrates has been used to identify the product of several methods of treatment of fisheries wastes for the production of food or feed material with a high protein content. In general, products made by mechanical, chemical and biological methods could be included in this definition. Extraction procedures, mostly based on the use of organic solvents to extract lipids, were among the initial methods studied for the production of fish protein concentrates (Knobl, 1967). Several problems are associated with such methods.
1406 such as potential safety and toxicity hazards, and deficiency of fiinctional properties (Green and Mattick, 1979). Other important methods for the production of protein concentrates involve the hydrolysis of proteins. In the case offish, the product of this is known as fish protein hydrolysates. Fish hydrolysation implies the liquification of the fish. Alkalis or acids have been used in the solubilization of fish protein (Finch, 1970). Studies on the enz3rmatic hydrolysis of animal protein have included the solubilization of protein from fisheries biomass. The works reporting this include those of Tannenbaimi et aL (1970), Rasekh and Metz (1973), Quaglia and Orban (1987), and Vieira et aL (1992). For the production offish protein hydrolysates, the selection of the proteolytic enzyme is a factor of great importance. Animal, plant and microbial proteases are available. Each group of enzjones has its own characteristics, which could have different degrees of applicability to a specific protein hydrolysis. B^rzana and Garcia-Garibay (1994) and Venugopal (1994) presented comprehensive reviews on the production of fish protein concentrates and hydrolysates. In general, biological methods such as the use of proteases for the hydrolysis of proteins have several advantages over other methods practised earlier, such as chemical hydrolysis (Loffler, 1986). Enzjrmes are specific, and their reactions can be conducted at pH and temperature values that could prevent deleterious effects. It has been reported that biological methods of hydrolysis result in a product with better flavour characteristics (Hale, 1972). Two species of spiny lobsters, Panulirus argus and Panulirus laevicauda (Latreille), are fished along the northeast coast of Brazil. Generally, their heads (cephalothorax), which contain about 20 % of the total animal's meat, are discharged as wastes (Vieira et aL, 1992). The purpose of this work was to determine the dependence of the hydrolysis process on several operating parameters, and to analyze the final product for total and free amino adds, and amino sugars contents.
EXPERIMENTAL 2.1. Waste protein raw material After the removal of the gills, viscera, and exoskeletonfi*omheads of Panulirus spp. lobster from the state of Ceard, Brazil, the recovered meat material was ground and cooked for 5 minutes.
1407 2.2. Proteolytic enzymes The proteases, obtained from Sigma Chemical Company (P.O. Box 14508, St. Louis, MO, U.S.A., 63178 - 9916), were papain, pepsin and fungal protease. 2.3. Enzymatic hydrolysis In separate experiments, papain, pepsin and fungal protease were added to partially-cooked, homogenized lobster meat at enzyme concentrations of 0.2, 0.4, and 0.5 % (wet weight basis). Control runs were also conducted without enzymes. The hydrolysation experiments were conducted for five hours at a temperature of 37°C. The pH was adjusted according to the requirements for the specific enzyme used, by adding NaOH or H3PO4. The pH level was maintained at 2.0 for pepsin, and 7.5 for the fungal protease and papain. The tyrosine level in the filtered hydrolysed solution was measured following the methods of Lowry et al, (1951) and Ainouz et al. (1972). When the hydrolysis process was finished, the mixture was heated to deactivate the enzyme and filtered. Finally, the pH was adjusted to neutral, and the hydrolysate preserved by freeze-drying. 2.4. Amino acids and amino sugar analysis The total amino acids contents were determined according to the method of Penke et al. (1974). Cystine and methionine contents were determined according to the method of Blackburn (1968). Free amino acids contents were determined according to the method of Ohara and Arioshi (1979), and the amino sugar contents were determined as by Madden et al. (1982). The amino acid analyses were done using a Beckman Model 121-MB autoanalyzer (Beckman Instruments, Palo Alto, California). 2.5. Statistical analysis The variations of the tyrosine (]ig/g sample) during hydrolysis of the lobster head meat was assessed using the following statistical model:
Yijk = | i + Oj + pj + Yk + (ocp)ij + (pY)jk + ^ij-k where: Yyk
denotes the tyrosine concentration {]ig/g sample), as a function of the i^^ reaction time (1, 2, 3,4, 5 hours), the j**^ enzymatic treatment (papain, pepsin, fimgal protease), and the k**^ enzyme concentration (0.2 %, 0.4 %. 0.5 %);
]l
denotes the overall mean;
OCj
denotes the effect of the i*^ reaction time;
Pj
denotes the effect of the j * ^ treatment;
1408 y^
denotes the effect of the k*^ enzyme concentration;
(aP)^- denotes the interaction effect of the i*^ reaction time against the j * ^ treatment; (PY)jk denotes the interaction effect of the j * ^ enzymatic treatment against the k*^ enzyme concentration, and; 8iji^
denotes the error term; i = 1, 2, 3, 4, 5 (reaction time) j = 1, 2, 3 (treatment) k = 1, 2, 3 (enzjnne concentration).
This factorial model was fitted to the tyrosine, where reaction time, enzymatic treatment (papain, pepsin and fungal protease) and enzymal concentration were taken as factors. The Tukey test was employed to identify the significant differences among means. All statistical analyses were performed using the Statistical Analysis System software package (SAS Inc., 1990, North Carolina, USA).
RESULTS AND DISCUSSION Figure 1 shows the process results of the hydrolysis of Panulirus spp. meat with the enz3maes papain, pepsin and fimgal protease, expressed as the tyrosine levels produced for each enzyme at the three concentrations employed. At approximately five hours, the extent of hydrolysis attained a plateau, therefore no experiments were conducted for longer times. For the enz3nne concentration values tested, the higher the concentration the higher the degree of hydrolysation (Vieira et aL, 1994). The ANOVA (Analysis of Variance) test was conducted to assess the effect of reaction time, enzymatic treatment and enzyme concentration on the t3rrosine concentration (Table 1). From this table, it can be seen that the F values for all the main effects are highly significant. Therefore, the tjrosine concentration was dependant on all those factors. It can also be seen that the interactions are significant as well, particularly for enzymatic treatment and enzyme concentration. Having seen that the F test is statistically significant, the Tukey test showed that, in relation to the reaction time, there was a significant difference in the tyrosine concentration between the 1, 2 and 3-hour reaction times and the 4 and 5-hour reaction times. There was no statistical difference between the 4 and 5-hour reaction times.
1409 1,600 1,400 1,200 1,000 BOO 600 400 200 0
Figure 1.
PAPA IN
PEPSIN
FUNGAL PROTEASE
Hydrolysis of lobster {Panulirus spp.) protein with different concentrations of proteolytic enzymes (wet basis). (From Vieira et al.y 1994). 0.2 %,
0.4 %,
0.5%
Table 1 Analysis of variance for t3rrosine
Source of Variation
df
Time Treatment Concentration Time x Treatment Treatment x Concentration Error
4 1426973.4 356743.3 2 2012959.5 1006479.7 2 12394566.2 6197283.1 8 288737.2 36092.2 4 506240.7 126560.2 114 663566.7 5820.8
SS
MS
F
Pr > F
61.29 172.91 1064.69 6.20 21.74
0.0001 0.0001 0.0001 0.0001 0.0001
The concentration of enzjmcie that caused the highest degree of hydrolysis in each case was 0.5 % (Vieira et al.y 1994). All data presented hereafter represent experiments done with this enzyme concentration.
1410 The free amino acid contents of the hydrolysates produced with each enzyme are presented in Table 2. It shows that they contained high amounts of alanine, arginine, glycine, lysine and taurine. This measurement describes the degree of hydrolysis with each enzjmcie used, suggesting that the stronger the enzyme activity is, the higher the free amino acid content of the hydrolysate will be. It has been observed (Vieira, 1989) that lobster tail defrosting drip contains arginine, glycine, alanine, taurine and histidine as free amino adds in concentrations similar to those observed in the hydrolysates prepared in this work. A comparison of the total amino acids contents of the various hydrolysates presented in Table 3 reveals that this is highest in the hydrolysate prepared using the fungal protease, followed by those prepared with papain and pepsin. In general, the essential amino add patterns of the three hydrolysates were similar. They were also comparable to those reported for fish protein concentrates (Hale, 1972; Rasekh and Metz, 1973; Gildberg et aL, 1989), and fall within the range of the F.A.O. / W.H.O. provisional essential amino adds patterns of ideal protein diets for infants, children, and adults (Anon., 1973). Table 4 presents the contents of the amino sugars galactosamine and glucosamine in the protein hydrolysates. The highest levels of glucosamine and galactosamine were obtained when fimgal protease was used for the hydrolysis, followed by papain and pepsin. The results of the sensorial analyses conducted with four trained taste panel members are presented in Tables 5 and 6. The experiments were conducted according to the method of Moraes (1988). The panel was given samples of hydrolysed lobster meat from all treatments. Control samples, produced without any enzyme, were also given. The panel members tasted each product and attributed a value to it from 1 to 13, answering the question "indicate the intensity of bitter taste in each sample, according to the scale:"
1
2 3 4
Without Bitterness
Very Light
5 6
7
Moderate
8
9
10
Very Bitter
11
12
13
Excessive
The results were subjected to an analysis of variance utilizing a completely randomized design. The samples were found to have no bitter flavour, and no difference in taste was detected between the products tested.
1411 Table 2 Free amino acids of the protein hydrolysate from lobster waste meat (mg amino acid • g"^ of protein) Treatment Amino add Alanine Arginine Asparagine Aspartic acid Citrulline Cysteic acid Cystine Ethanolamine Glutamic add Glutamine Glydne Histidine Hydroxyproline Isoleudne Leudne Lysine Methionine Ornithine Phenylalanine Proline Sarcosine Serine Tavirine Threonine Tryptophan Tyrosine Valine
Papain 6.44 31.58 1.08 1.53 0.82 0.86 0.38 0.11 1.46 1.97 14.91 1.56 0.38 1.39 2.95 5.18 1.79 1.10 2.34 4.10 1.43 1.39 18.08 1.03 3.65 2.65 1.89
Pepsin 5.96 27.33 1.73 1.38 0.14 0.61 0.41 0.12 1.40 0.39 14.36 1.18 0.19 1.06 2.06 3.56 1.61 0.93 1.89 3.49 1.84 1.47 17.78 1.28 1.69 1.91 1.29
Fmigal protease 6.64 33.69 1.70 1.30 0.33 0.45 0.29 0.10 1.47 2.06 13.63 1.78 0.50 2.29 7.96 6.33 2.94 0.99 5.55 4.44 2.12 1.79 18.20 1.91 2.49 2.55 2.62
1412 Table 3 Total amino a d d composition of the protein hydrolysate from lobster waste meat (mg amino acid • g"^ of protein) Treatment Amino A d d Alanine Arginine Aspartic acid Cysteic acid Glutamic acid Glycine Histidine Isoleudne Leudne Lysine Methionine sulfone Phenylaleinine Proline Serine Threonine Tryptophan Tyrosine Valine
4.
Papain 53.2 91.8 103.0 8.1 133.1 51.2 27.3 48.8 80.0 79.5 17.3 47.1 51.5 38.2 47.0 12.9 46.0 52.2
Pepsin 29.9 85.4 100.3 10.6 127.3 50.9 24.3 43.9 72.4 70.0 21.2 42.3 50.0 36.2 41.6 11.1 42.9 46.9
Fungal protease 50.4 89.4 105.3 11.2 128.1 50.7 25.6 49.6 80.5 80.0 24.6 47.4 52.7 36.3 46.2 12.0 46.3 53.6
CONCLUSIONS
The use of hydrolysed lobster head meat as a dietary protein source is promising because, in addition to its protein value, it contains all the essential amino acids in high concentrations. Considering that the test panel results were very promising with regards to the flavour characteristics of the product, further studies will determine the potential of this product as a flavourant for the food industry, and for its possible incorporation into such products as soups, sauces and gravies. This potential is further enhanced by the fact that some functional properties of these hydrolysates, such as solubility, wettability and emulsification, have been found to be good (Vieira et aL, 1994).
1413 Table 4 Amino sugar content of the protein hydrolysate from lobster processing waste (mg • g'^ of hydrolysate) Treatment Compound
Pepsin
Papain
Fungal protease
Galactosamine
0.67
0.45
0.93
Glucosamine
3.23
1.44
3.28
Table 5 Results of the sensorial analysis of protein hydrolysate from lobster waste meat Taster
Values Papain
PepsinL
Control
Fungal protease
1
2
3
1
2
3
1
2
3
1 2
A
2
2
1
1
1
1
2
2
2
1
1
1
B
3
2
4
2
3
2
2
2
3
1
1
1
C
1
4
4
1
1
1
1
1
1
1
1
1
D
2
3
2
1
1
1
1
1
1
1
1
1
3
Table 6 Results of the statistical analysis of the sensorial analysis data for protein hydrolysate from lobster waste meat Variation Source
DF
SQ
MS
Among Treatments
3
11.74
5.58
Residue
8 11
22.51 39.25
2.81 -
1,99 n.s. a = 0.05
1414 5.
ACKNOWLEDGMENTS
This project was funded in part by a Canadian International Development Agency (C.I.D A.) grant. The authors would like to thank Drs. A. Dickinson and R. Vieira, Project Coordinators of M.U.N. - C.I.D.A. - U.F.C. The authors also wish to thank the following members of the Department of Biochemistry, Memorial University of Newfoundland: Mr. S. Omar for his assistance in the experiments, Mr. D. Hall and Ms. S. Banfield for the amino a d d analysis, and Mr. P. Bemister for his assistance with the manuscript.
6,
REFERENCES
Ainouz, I.L.; Xavier-Filho, J. and Gomes-Filho, E. (1972). Atividade proteolitica em sementes de Vigna sinensis seridd. Cien, Cult., 24: 104. Anonymous. (1973). Energy and Protein Requirements, Report of a Joint FAOAVHO ad hoc Expert Committee. WHO Tech. Rep. Ser. 522, World Health Organization, Geneva. Birzana, E. and Garcia-Garibay, M. (1994). Production offish protein concentrates. In: Fisheries Processing, Biotechnological Applications, ed. Martin, A.M. Chapman and Hall Ltd., London (In press). Blackburn, S. (1968). Amino Acid Determination: Methods and Techniques, Marcel Dekker, New York, pp. 21, 128. Finch, R. (1970). Fish protein for himian foods. CRC Critical Revs, in Food TechnoL, 1: 519-580. Gildberg, A.; Batista, I. and Strom, E. (1989). Preparation and characterization of peptones obtained by a two-step enzymatic hydrolysis of whole fish. Biotechnol. Appl. Biochem., 11: 413-423. Green, J.H. and Mattick, J.F. (1979). Fishery waste management. In: Food Processing Waste Management, eds. Green, J.H. and Kramer, A. A.V.I., Westport, CT, pp. 202-227. Hale, M.B. (1972). Making fish protein concentrates by enzymatic hydrolysis. NOAA Technical Report NMFS SSRF-657. U.S. Department of Commerce, Seattle, WA, pp. 1-31. Kirk, R.E. (1968). Experimental Design: Procedures for the Behavioral Sciences. Brooks-Cole, Belmont, CA. Knobl, G.M., Jr. (1967). The fish protein concentrate story. Food TechnoL, 21(8): 56-59. Loffler, A. (1986). Proteolytic enzymes: sources and applications. Food TechnoL, 40(1): 63-70. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randall, R.J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem., 193: 265-275.
1415 Madden, D.E.; Alpenfels, W.F.; Mathews, R.A. and Newson, A.E. (1982). Improved method for the determination of hexosamine using the Beckman 121-M amino acid analyzer. J, Chromatog., 248: 476-482. Martin, A.M. and Patel, T.R. (1991). Bioconversion of wastes from marine organisms. In: Bioconversion of Waste Materials to Industrial Products, ed. Martin, A.M. Elsevier Applied Science, London, pp. 417-440. Moraes, M.A.C. (1988). In: Metodos para Avaliaqao Sensorial dos AlimentoSy 6*^ Edition, University of Campinas, Sao Paulo, Brazil, pp. 1-93. Ohara, I. and Arioshi, S. (1979). Comparison of protein precipitants for the determination of free amino acids in plasma. Agric, Biol. Chem., 43(7): 14731478. Penke, B.; Ferencze, R. and Kovacs, K. (1974). A new acid hydrolysis method for determining trjrptophan in peptides and proteins. Analyt. Biochem., 60: 45-50. Quaglia, G.B. and Orban, E. (1987). Influence of degree of hydrolysis on the solubility of the protein hydrolysates from sardine {Sardina pilchardus). J. Sci. FoodAgric.y 38: 271-276. Rasekh, J. and Metz, A. (1973). Add precipitated fish protein isolate exhibits good fimctional properties. Food Prod. Devel., 7(8): 18-24. Tannenbaima, S.R.; Ahem, M. and Bates, R.P. (1970). Solubilization of protein concentrate. Food Technol., 24: 96-101. Venugopal, V. (1994). Production of fish protein hydrolysates by microorganisms. In: Fisheries Processing. Biotechnological Applications, ed. Martin, A.M. Chapman and Hall, London (In press). Vieira, G.H.F. (1989). Influencia do uso do tripolifosfato de sodio na conservacao de caudas de lagosta por congelamento, DSc tese, Universidade de Sao Paulo (USP), Sao Paulo, Brasil. Vieira, G.H.F.; Saker-Sampaiao, S.; Goncalves, R.C.F. and Reis, S.B. (1992). Preparagao de peptone a partir de subprodutos do pescado. II - Avaliacao biologica. XIII Congresso Brasileiro de Ciencia e Tecnologia de Alimentos, Sao Paulo, SP, Brasil, p. 23. Vieira, G.H.F.; Martin, A.M.; Saker-Sampaiao, S.; Omar, S. and Goncalves, R.C.F. (1994). Manuscript submitted.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1417
SENSORY ACCEPTANCE AND OVERALL QUALITY OF A HISTIDINE ADDED FISH SAUCE. Norlita G. Sanceda^, Tadao Kurata'' and Nobuhiko Arakawa^. ^Nutrition and Food Science Department, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan i n s t i t u t e of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan
Abstract A 4 month incubated histidine added fish sauce wherein the hydrolysis of fish protein was accelerated during the fermentation process, was sensorially evaluated in respect to taste, smell, color and overall quality and compared with the control. Sensory evaluation result showed that the accelerated hydrolysis product fish sauce brought about by the addition of histidine was acceptable in respect to taste, smell, color and overall quality but not the control which was found to be still in the immature stage. A difference between the control and the histidine added sample was confirmed and that the concentrations of volatile acids in the control was higher than the histidine added sample. Panelists who were not familiar with the fish sauce preferred the histidine added sauce over the commercial fish sauces while those who were familiar, preferred the traditionally produced commercial sauce but accepted the histidine added sauce. Addition of histidine during fermentation did not increase the amount of histamine in the fish sauce. Introduction Fish sauce is a clear brown liquid hydrolysis product of salted fish obtained about 1 year after salting and possesses a characteristic smell. The traditional way of producing fish sauce is by mixing fish and salt at the ratio of 2:1 or 3^1, the fish and salt ratio depends on the quality of fish and the kind of salt used, and allowed to ferment for from 9 months to one year. It is a popular condiment in Southeast Asia and in some cases and in certain social classes, it is a major
1418 source of protein in the diets. Lafont [1] reported that fish sauces should not just be considered as condiments. Those with 1% nitrogen or more could be considered a little better than condiments. Fish sauces contain about 20g/L of nitrogen of which 16 g is in the form of amino acids, thus they maybe considered a significant source of protein. The high salt content inhibits large intake thus the nutritive importance is limited but inspite of this, the product continues to gain popularity and become a necessity in the diets [ 2 ] . For example, in the Philippines, records showed that the demand of the sauce far exceeds the supply not only domestically but internationally, which poses a serious problem inspite of the entry of large scale fishermen into the business. The long waiting involved in the manufacture is one major drawback and probably the most important factor limiting the viability of the industry. Several studies on the acceleration of hydrolysis of fish protein during the fermentation process in the manufacture of fish sauce had been reported [ 3 - 5 ] . In our previous work [ 6 ] , addition of histidine to fish mixture accelerated the hydrolysis of fish protein during fermentation process in the manufacture of fish sauce. To determine the usefulness of this work both to the industry and to the researchers in the related field, the product was subjected to a sensory evaluation with the aim to: 1) determine the acceptability in respect to taste, smell, color and overall quality; 2) test whether a difference existed between products; 3) examine the quality and quantity of volatile acids in the products and 4) investigate the possible decarboxylation of histidine to histamine. Materials and Methods Sardines obtained in Japan fish market were used. Histidine of Nacalai Tesque Inc., Kyoto, Japan was also used. Standard volatile acids were puchased from Tokyo Kasei Kogyo Co. Ltd, Tokyo, Japan. Samll Scale Fish Sauce Preparation One kilo of fish Iwashi (Sardinops melanostictus sardine) were sliced into about 4 cm with gut included, mixed with salt at a ratio of 2.5^1, put in layers in a glass beaker and put weights on it. The beaker was covered with a laboratory film (Parafilm) and allow to ferment for about 4 months at 32^C. This mixture was used as control. In another mixture, the same procedure mentioned above was used except that histidine was added in it. After about 4 months, the mixtures were filtered and the liquid was subjected to sensory test. The rest of the liquid was used for the volatile acids analysis. Sensory Evaluation Sensory evaluation based on taste, smell, color and overall quality
1419 including appearance was performed. The tests samples consisted of the control, histidine added sauce incubated for about 4 months and a 9 month incubated sauce, all prepared in the laboratory, and two kinds of commercial fish sauces obtained in bottles from their country of origin. A total of 20 untrained panelists were employed. These panelists consisted of 10 female Japanese, age 20-35, their first time to try the product, and 10 female Filipinos residing in Japan, age range similar with the Japanese panelists, whose daily diets include fish sauce. Acceptability test, ranking degree of preference and paired difference test for taste, smell, color and overall quality were carried out. In all these tests, the coded samples were presented in the same amount, appearance, consistency and temperature. For the assesment of aroma, each fish sauce sample was absorbed to about half a strip of filter paper and panelists sniffed the samples. For the taste tests, about 1 mL of the sample, in teaspoons, were given to the panelists one after the other. Panelists were ask not to swallow the samples and to wash their mouths with water between samples. They were not allowed to talk to each other during the test. Resmelling and retasting were allowed. Along with taste and smell, the color was judged by visual observations. The tests samples in glass beakers on white paper under a white-lighted room in the same quantity, appearance, and temperature were presented to the panelists at a time for evaluation. Previous reports [7-9] illustrated the importance of volatile acids in the aroma of fish sauce. In this work, an analytical study on the volatile acids concentrations of the histidine added sample and the control, both incubated for about 4 months was done. Collection of volatile compounds The 4 month fermented liquid of the histidine added fish mixture and the control added with an internal standard (Dodecane) were steam distilled under reduced pressure for about 4 hours at about 45^C and the distillates were extracted with diethyl ether. Water from the extracts was removed by adding sodium anhydrous sulfate allowing to stand overnight after which the extracts were concentrated in a usual manner and analyzed for the acids volatiles. Gas Chromatography (GC) Gas chromatography was accomplished using a Hitachi 663-30 model gas chromatography equipped with a flame ionization detector. Separation of aroma compounds was done on a 0.25 mm i.d. x 50 m fused silica column coated with PEG 20M. The column temperature was programmed at 60'^C held for 4 min to 180^C at 2^C/min. The injection port was kept at 200^C. The carrier gas was Helium and Nitrogen. Concrete identification of volatile acids was carried out employing co-chromatogaphy using standard acids. Relative concentrations of the volatile acids were calculated using an integrator attached to the gas chromatograph.
1420 Histamine Assay Histamine was assayed by a rapid method using an oxygen electrode sensor with an enzyme amine oxidase [ 1 0 ] . Results and discussion Addition of histidine to fish mixture accelerated the hydrolysis of fish protein during fermentation in the manufacture of fish sauce [ 6 ] . The liquefaction rate in the histidine added was much faster than the control (Table 1 ) . The total protein as well as the amino acids were higher than the control and comparable, if not higher, to the reference (commercial fish sauce). To determine the practicability of this result, a sensory evaluation was carried out. Result showed that the taste, smell, color and overall quality of the histidine added sample was acceptable with an average rating of 4.53, 4.71, 4.01 and 4.10 for taste, smell, color and overall, respectively, on a hedonic scale of 1 = least acceptable and 5 = most acceptable, while the control had an average of 2.51, 1.81 and 1.97 for taste, smell and overall quality, the control was hardly acceptable to either of the groups, however the rating 'for the color was not significantly different for the two samples (Table 2 ) . Table 1 Liquefaction (mL) Incubation period 2 Samples
A
control histidine added
160 180
4 months B
B
A
350 460
2^0 301
412 599
Control : Fish:salt (30%) A) : 500g Fish + salt + 1% histidine B) : lOOOg fish + salt + 2% histidine Table 2
Samples
Means of the acceptability of taste, smell, color and overall quality of histidine added fish sauce and commercial fish sauces Taste
Smell
Color
Overall Quality
Means Control*
2Til
Tsi
Til
1797
1421 Table 2
Means of the acceptability of taste, smell, color and overall quality of histidine added fish sauce and commercial fish sauces 4.10 4.01 Histidine added* 4.53 4.71 4.50 9 months incubation**4.80 4.43 4.51 4.79 4.77 Commercial Patis 4.85 4.80 4.76 4.55 Commercial Namplaa 4,75 4.71 * : Control and Histidine added were incubated for about 4 months ** : incubated in the laboratory *** : Philippine fish sauce **** : Thailand fish sauce Each mean is the average of 60 observations Panelists were 10 Japanese and 10 Filipinos residing in Japan 1 = least acceptable 5 = most acceptable Ratings of the panelists using a paired difference test on the products showed that the control and the histidine added fish sauce were different in respect to smell, color and overall quality but not significantly for the taste (Table 3 ) . Both sauces were so salty that it was difficult to tell the difference. Generally, aroma is used as a gauge to measure the quality of fish sauce since the salt concentration is so high that it tends to overpower other flavor constituents. The smell of the control was described to be very fishy, a sign that the product was still immature or not ripe, and more acidic compared to the histidine added which was more mature or ripe but a little sweet and less acidic. Table 3 Taste
2.01
Rating of the Difference test for the control and the histidine added fish sauce Smell
4.11
Color
Overall Quality
3.21
3.80
The same panelists were employed for the acceptability test and ranking test of preference Rating is the average of 60 observations 1 = very similar 5 = very different When a ranking degree of preference was conducted by the groups of panelists, a very interesting phenomenon was observed. The Japanese panelists who were not familiar with the product preferred the histidine added fish sauce over the traditionally produced commercial fish sauces (Table 4 . 1 ) , however, the Filipino panelists who were familiar with fish sauce, particularly patis, a Philippine fish sauce, preferred the
1422 coimnercial ones over the histidine added sample (Table 4 . 2 ) . The control was hardly preferred by either of the groups. Table 4.1
Ranking Degree of Preference Test
Samples
Taste
Smell
Color
Overall Quality
Control Histidine added 9 Mo incubation Commercial Patis Commercial Namplaa
1.77= 4.66a 3.67b 3.67b 3.63b
1.23= 4.13a 3.27 3.13 3.03
3.06 3.90= 3.67" 3.63b 3.37
1.23= 4.16= 3.23 3.32 3.17
* • Panelists were untrained Japanese not familiar with fish sauce, their first to try it Each mean is the average of 30 observations 1 = least like 5 = most like a * siginificantly different at , 0.01 b * significantly different at ,0/05 Values without any superscripts are not significantly different Significane difference test was based on; A. Kramer, G. Kahan, D. Cooper and A. Papavasiliou. 1974. A non-parametric ranking method for the stat istical sensory data. Chemical Senses and Flavor 1, 121-133. Table 4.2
Ranking Degree of Preference Test
Samples
Taste
Smell
Color
Overall Quality
Control Histidine added 9 Mo incubation Commercial Patis Commercial Namplaa
1.13= 3.43 4.23= 4.66= 3.53
1.20= 4.00= 4.70= 4.66= 3.13
3.66" 2.30 4.03a 4.03= 4.13=
1.53= 3.26 4.20= 4.66= 3.80=
* ' Panelists were untrained Filipinos living in Japan and whose diets usually consist of Patis Each mean is the average of 30 observations 1 = least like 5 = most like a * siginificantly different at , 0.01 b * significantly different at ,0/05 Values without any superscripts are not significantly different Significane difference test was based on; A. Kramer, G. Kahan, D. Cooper and A. Papavasiliou. 1974. A non-parametric ranking method for the statistical sensory data. Chemical Senses and Flavor 1, 121-133.
1423 Analytical study revealed that the volatile acids in the control was much higher than the histidine added fish sauce (Table 5 ) . Sensory evaluation showed that the control was more acidic than the histidine added fish sauce. This phenomenon might be due to the difference in the Quantity of acids in both samples. In our previous work [ 1 1 ] , we reported that the aroma of a lysine added fish sauce was different from that of the control. It seemed that addition of basic amino acids brings changes in the aroma of the sauce. Table 5 Percentage of volatile acids in the acid fraction of fish sauces incubated for about 4 months Acids
Acetic Propionic iso-Butyric n-Butyric iso-Valeric n=valeric iso-Hexanoic n-Hexanoic n-Heptanoic iso-Nonanoic n-Octanoic n- Nonaoic n-Decanoic
Control Mean±S.E. % 12.32±0.22 27.58 + 0.26 0.40 + 0.00 13.06 + 0.12 1.05 + 0.05 1.07±0.03 tr tr tr tr tr tr tr
Histidine added Mean+S.E. % 6.11±0.06a 23.42 + 0.233 tr 11.16±0.02a 0.74 + 0.02b 0.81±0.01b tr tr ND tr ND tr tr
Values are the average of three replicates a : significantly different at <0.001 b : significantly different at <0.01 tr : values with less than 0.01% ND • not detected Histamine is formed in foods largely from the growth of microorganisms that possess the enzyme histidine decaboxylase [12] and this enzyme converts histidine to histamine via an enzymatic decarboxylation reaction [ 1 3 ] . Outbreaks of histamine poisoning have implicated both scombroid (tuna, mackerel) and non-scombroid (mahi mahi, sardines) fish. Histamine causes a rather short, mild illness and variety of symptoms can be encountered including gastrointestinal (nausea, vomiting etc.) cutaneous (rash, urticaria) hemodynamic (hypotension), and neurological (flushing, itching, burning, headaches) symptoms [14,15]. In this study, addition of histidine to fish mixture during fermentation process in the manufacture of fish sauce did not increase the amount of histamine. Histamine was detected in a very low concentration in both the control
1424 and the histidine added sample (Table 6 ) . The amount of histamine detected in the commercial fish sauces analyzed was also very low and is out of the hazardous level set by the U.S [ 1 6 ] . During the sensory evaluation, no symptoms of fish poisoning mentioned above was detected among the panelists although fish containing hazardous levels of histamine are not always detected by organoleptic examination [ 1 3 ] . Looking at the results, it seemed that histidine was not decarboxlyzed into histamine during the fermentation process, probably due to the high salt content of the mixture or the absence of appropriate microbes that could decarboxylyze histidine to histamine since the fish used were in the fresh stage. The formation of histamine and tyramine in miso appears to be greatly inhibited by a high salt concentration [ 1 7 ] . Table 6
Histamine contents in fish samples
Samples
Mean±S.E. (mg/mL)
A) Small scale prepared Control Histidine added
(1%)* 0.12+0.02 0.05+0.00
B)Commercial fish sauces Patis Namplaa Namplaa (made in Japan Shottsuru Anaerobically fermented
0.04+0.01 0.43+0.02 0.37+0.01 nd nd
(2%)* 0.20+0.01 0.15 + 0.01
Small scale prepared fish sauces were done in the laboratory Values are average of two replicates * : Histidine added to the mixture before incubation Histamine was assayed by an Oxygen sensor using fungal amine oxidase All commercial fish sauces were obtained in their country of origin Patis : Philippines; Namplaa - Thailand; Namplaa made in Japan Ganjang : Korea; Shottsuru : Japan; Anaerobically fermented • Japan Conclusion Sensory evaluation result showed that the accelerated hydrolysis product fish sauce brought about by the addition of histidine was acceptable in respect to taste, smell, color and overall quality but not the control which was found to be still in the immature stage. A difference between the control and the histidine added sample was confirmed and that the concentrations of volatile acids in the control was higher than the histidine added sample. Panelists who were not
1425 familiar with the fish sauce preferred the histidine added sauce over the commercial fish sauces while those who were familiar preferred the produced commercial sauce but accepted the histidine added sauce. Addition of histidine during feremntation did not increase the amount of histamine. References 1 R. Lafont, Proc. Indo-Pac. Fish. Coun. 5th Mtg., Bangkok, Sets 11 and 111, (1955) p. 163. 2 Fisheries Products Manual, Indo Pacific Fisheries Council, FAO Regional Office for Asia and the Far East, Bangkok, Thailand, 1961. 3 C.G. Beddows and A.G. Ardeshir, J. Food Technol., 14 (1979) 603 and 613 . 4 S. Murayama, D.C. Calvez and P. Natayachin, Tokyo Regional Fisheries Lab, Contribution A. No. 164, 1961. 5 A. Gilberg, E.J. Hermes and M.F. Orejana, J. Sci. Food Agric. 35 (1984) 1363. 6 N. Sanceda, T. Kurata and N. Arakawa, Abstract. 15th International Congress in Nutrition, Adelaide, Australia, 1993. 7 N. Sanceda, T. Kurata and N. Arakawa, Phil. Agric. 66 (1983) 176. 8 N. Sanceda, T. Kurata and N. Arakawa, Agric. Biol. Chem. 48 (1984) 3047. 9 J. Dougan and G. Howard, J. Sci. Food Afric. 26 (1975) 887. 10 M. Ohashi, M. Suzuki, K. Nomura, M. Otsuka and N. Arakawa, J. Fd. Sci (1993) In Press. 11 N. Sanceda, T. Kurata and N. Arakawa, J. Fd. Sci. 55 (1990) 983. 12 S.H. and W.D. Brown, Adv. Food Research 34 (1978) 113. 13 S.L.Taylor and S. S. Summner, In D.E. Kramer and J. Listen (eds.), Seafood Quality Determination. Elsevier Science Publishers B.V. Amsterdam, 1986. 14 C.K. Murray, G. Hobbs, and R.G. Gilbert, J. Hygiene, 88 (1982) 215. 15 M.H. Merson, W./B. Baine, E.J. Gangarosa and R.C. Swanson, J. Am. Med. A s s o c , 228 (1974) 1268. 16 S.L. Taylor, Monograph, World Health Organization (1985) 1. 17 K.D.H. Chin and P.E. Koehler, J. Food Sci. 49 (1983) 423.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1427
Extraction of value-added components from shellHsh processing discards F. Shahidi Departments of Biochemistry and Chemistry, Memorial University of Newfoundland, St. John's, NF, AlB 3X9, Canada. Abstract There has been a growing interest in natural ingredients such as chitin, carotenoids, flavorants, enzymes and proteins which could be isolated from shellfish processing discards. Proteins from shellfish may be recovered using a base extraction or enzyme hydrolysis process. After demineralization, chitin is produced which could be used directly as a support for enzymes or converted to chitosan and its derivatives to be employed in a variety of food applications. While shellfish flavorants may be recovered, as aqueous extracts, from discards or cook-water, the carotenoid pigments are extracted into oil at 60°C or using an enzyme-assisted process. Subsequently, the extracted pigments are used in preparation of formulated feed for aquaculture of salmonid fish species. Arctic char {Salvelinus alpinus) acquired greater than 4 fig/g carotenoids after a 9 to 15 weeks of feeding on pigmented diets. 1. INTRODUCTION The seafood processing industry produces large amounts of shellwaste which have traditionally been hauled into the ocean or dumped in-land [1]. However, environmental restrictions and a better understanding of potential value of processing discards for a variety of applications has resulted in efforts to find uses for these materials. Coupled with the above problem, there has been a growing interest in natural ingredients which are readily available from shellfish discards. Processing by-products from shellfish are made up primarily of protein residues from body sections such as heads and carapace, as well as minerals and chitin which constitute the exoskeleton of crab, shrimp and lobster [2]. In addition, it is possible to recover minor components such as carotenoid pigments, flavorants and enzymes from the waste. While flavorants can be readily recovered from the cook water or the proteinaceous materials, enzymes such as chitinase, alkaline phosphatase, hyaluronidase and jS-N-acetyl glucosaminidase may be isolated from the thaw water
1428
of frozen raw shrimps [3] and potentially from other shellfish species. The present paper provides information on the characteristics of both the major and minor components of shellfish processing discards and presents examples related to their value-added utilization. 2. SHELLFISH DISCARD COMPONENTS Table 1 summarizes selected compositional characteristics of processing discards from shrimp. The major constituents of the discard are proteins, minerals and chitin. The minor constituents include carotenoids as given in Table 1. Details on the constituents of discards and their value-added utilization are provided below. Table 1 The content of chitin, proteins and carotenoids, on a dry-weight basis, in shrimp and crab processing discards. Species (Segment) Shrimp (Head and Carapace) Crab (Backs) Crab (Claws) Crab (Legs) Crab (Shoulders) Crab (Tips)
Chitin, % 17.0 22.3 23.7 32.3 26.9 27.9
± ± ± ± ± ±
0.3 0.5 0.3 0.1 0.1 0.2
Protein, _% 41.9 18.6 17.2 15.7 24.0 18.0
± ± ± ± ± ±
0.2 0.1 0.2 0.1 0.2 0.1
Total Carotenoids, Mg/g 147.7 119.6 16.4 34.3 26.8 19.8
± 2.5 ± 2.5 ± 0.1 ± 0.2 ± 0.1 ±1.1
2.1 Proteins and Flavorants Proteins in shellwaste may be isolated by extraction into a 5 % (w/v) solution of hot sodium or potassium hydroxide [4]. The resultant proteins generally have an amino acid composition similar to those in the starting material (see Table 2), however, care must be exercised to prevent the formation of lysinoalanine in the resultant product. It is also possible to extract the flavorants from cook water of crab or from processing discards by extraction into hot water prior to deproteinization. The resultant material may then be subjected to ultrafiltration/concentration and dehydration. Enzyme-assisted proteolysis may be used to extract proteins with flavor-enhancing effects from shellfish processing discards. In all cases the products obtained included carotenoid and/or carotenoproteins. Studies on potential use of shellfish protein hydrolyzates in surimi-products as protein fortifying agents and to enhance moisture retention in products and as flavor enhancers as well as in aquaculture feed formulations is being continued in our laboratories.
1429 Table 2 Essential amino acid profile of shrimp and crab shell waste proteins (g/lOOg protein). Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Plienylalanine Threonine Tryptophan Valine
Shrimp 6.13 2.24 5.78 7.01 6.58 2.41 5.13 4.14 1.19 5.95
+ + ± ± ± ± + ± + +
0.07 0.09 0.13 0.02 0.07 0.08 0.07 0.20 0.07 0.06
Crab 6.66 3.58 2.67 5.14 2.51 1.93 5.98 4.74 0.78 7.07
+ + ± ± ± ± ± ± ± ±
0.02 0.01 0.02 0.02 0.07 0.00 0.01 0.02 0.01 0.10
2.2 Enzymes Most of the catch of shrimp (Pandalus borealis) isft-ozenas raw, whole blocks. At the processing plants, the frozen blocks are thawed by circulating spraying water. The resultant waste water contains a number of digestive enzymes. The waste water may be collected and clarified using ferric chloride and concentrated by ultrafiltration employing a molecular weight cut-off of 10,000 Daltons. A simplified scheme for production of enzymesft*omshrimp thaw water is given in Figure 1. The enzymes hyaluronidase, jS-N-acetyl glucosaminidase, chitinase and alkaline phosphatase were recovered in the retentate with a yield of 65-100% and with 17-86% increase in their specific activities [3]. Biotechnological application of enzymes so isolated in targeted applications is of interest 2.3 Chitin and Chitosan Two potentially valuable products from shellfish processing discards are chitin and chitosan. First commercially produced in 1971 chitin and chitosan are the subject of much research worldwide and are currently being produced by up to 15 companies in Japan and several in the United States and elsewhere. Sometimes referred to as crab shell powders, they find application in a wide variety of fields. Chitin is a natural biopolymer with 2-deoxy-2-acetaminoglucose monomers linked together via a i8-l,4 linkage. Chitosan is referred to the deacetylated (to different degrees) form of chitin [5]. Manufacturing of chitosan may be carried out by boiling of chitin in a concentrated sodium hydroxide solution or incubating at 25-30°C for a period of 1-30 days in 30-65% NaOH solutions [6]. The chemical structures of chitin and its monomer N-acetyl-D-glucosamine as well as chitosan are shown in Figure 2. Chemically, chitin is analogous to cellulose except that the 2-hydroxy substituent in the glucose units of cellulose are replaced by a 2acetylamino group. Meanwhile, chitosans possess varying levels of free amino groups which are capable of strong H-bonding and ionic interaction.
1430 THAW WATER FROM FROZEN SHRIMP K2HPO3 Solution
1
pH ADJUSTMENT F e d , Solution
i
CLARIFICATION
CENTRIFUGATION Solids ULTRAFILTRATION
FREEZE DRYING
T
Water
ENZYME SEPARATION
ALKALINE PHOSPHATES HYALURONIDASE N-ACETYLGLUCOSAMINIDASE CHITINASE Figure I. Recovery of enzymes from thaw water of frozen shrimp [2].
The global annual production of crustacean discards, on a dry weight basis, is estimated at 1.44 million metric tons [7]. The content of chitin in shellfish discards is between 20 and 50% of the total dry weight. Thus a minimum of 200,000 metric tons of chitin could potentially become available. Currently only a few thousand tons of chitin are produced annually. Table 1 summarizes the content of chitin in shrimp and different sections of crab processing discards. Other major components of the waste are proteins and minerals. The process by which chitin is produced may be summarized in the flowsheet of Figure 3. Different unit operations involved are grinding of the shells to a uniform reduced size followed by the removal of proteins and minerals. The latter two steps may be easily interchanged [8]. However, in commercial practice, generally proteins
1431 CH^OH
HO
NH
HO
/ \
OH
CO CHo
N-Acetyl-D-glucosamine CH^ CO CH^OH
HO
\
HO
NH
CHjOH
/ CO \
NH
HO
/
CO
CHo
NHo
\
Chitin
NHn
HO
CH^OH
HO
CHoOH
NH
CH^OH
CH3
CH^OH
HO
NHo
Chitosan
Figure 2. Chemical structures of N-acetyl-D-glucosamine, chitin and chitosan.
are first extracted by base. The demineralization is achieved using a dilute hydrochloric acid solution. Although the resultant calcium chloride may be used in the pulp and paper manufacturing, the dehydration process required for its recovery is commercially unattractive. Other potential applications of calcium chloride as a 30% solution is as a dust control agent for spraying on mud roads. It should be noted that minerals and chitin in dehydrated, deproteinized shellwaste are present in nearly equal amounts. The deproteinized and demineralized shells contain mainly chitin with less than 10% moisture and 2% ash. The degree of deacetylation may vary between 10 and
1432 20%. In order to assure high quality of chitin, it is necessary to acquire fresh shells and to adequately control reaction time and temperature as well as the concentration of reagents involved. Highest molecular weight chitin with lowest ash content obtains a better market value since the quality of chitin derivatives such as chitosan is primarily dictated by the quality of the starting material. SHELLFISH DISCARD GRINDING 5% HotNaOII
t DEPROTEINIZATION
WASHING 2% Cold HCl
•
DEMINERALIZATION WASHING DRYING CHITIN 50% Hot NaOH
1 DEACETYLATION
WASHING
DRYING CHITOSAN Figure 3. Simplified flowsheet for manufacturing of chitin and chitosan from shellfish discards.
1433 Chitin is insoluble in almost every common organic solvent and in acidic, basic and neutral aqueous solutions. However, chitosan is soluble in dilute acids in aqueous solutions. Production of chitosan follows simple unit operations of deacetylation, washing and drying (Figure 3). The quality characteristics of chitosans depend, to a large extent, on the degree of deacetylation of the macromolecule. The crude chitosan may be further purified by dissolution in a suitable acid solution followed by filtration and precipitation by pH adjustment. Chitosan derivatives with different functionalities may be prepared in order to address specific needs of the user industries. Table 3 summarizes some of the numerous applications of chitinous materials in different areas of food, agriculture, cosmetics, textile, paper, biomedical, biotechnology, water treatment and removal of radioactive and poisonous metals [9-14]. Although different applications of chitin derivatives have received considerable attention, the market realities with respect to competition from alternative materials and supplies may prove challenging. A novel approach for utilization of chitinous materials was recently examined in our laboratories. N,0-Carboxymethylchitosan (NOCC) was prepared in a laboratory scale or obtainedft*omNovo Chem. Inc. (Halifax, NS). It was noted that NOCC and its lactate, acetate and pyrrolidine carboxylate salts were able to prevent cooked meat flavor deterioration over a 9-day storage period at refrigerated temperatures. The mean inhibitory effect of NOCC and its aforementioned derivatives at 0.05-0.3% addition level in the formation of 2-thiobarbituric acid reactive substances was 46.7, 69.9, 43.4 and 66.3%, respectively. The mechanism by which this inhibition takes place is thought to be related to the chelation of free iron which is released from hemoproteins of meat during heat processing. This would in turn inhibit the catalytic activity of iron ions. Recently, Han and Shahidi [15] reported immobilization of seal gastric proteases (SGP) on glutaraldehyde-treated chitin. The average degree of immobilization was 20% and the immobilized SGP exhibited optimum performance at pH 2.0, and were most stable at pH 4.0. The half-life of the immobilized enzymes with continuous operation for hemoglobin hydrolysis at 22°C was 90h. The immobilized SGP were also used successfully for clotting of milk in cheese-making. However, less conformational flexibility of the immobilized proteases resulted in their lower proteolytic activity towards substrates. 2.4 N-Acetylglucosamine The compound N-acetylglucosamine (NAG) is the monomer of chitin. It has been recognized that NAG has anti-inflammatory effect and may possess interesting characteristics. Although chemical preparation of NAG is feasible, a commercially attractive process for its production would be much desirable. Therefore, consideration of reaction conditions under high pressure may prove beneficial. Use of NAG in different synthetic applications may also be of interest to biochemists. 2.5 Carotenoids and Carotenoproteins A minor, but important class of compounds in crustacean processing discards is carotenoids. Carotenoids in seafoods are oxygenated forms of j8-carotene referred to
1434 Table 3 Some applications of chitin and its derivatives. Area of Application
Examples
Food
Clarification of wine and juice Dietary fibre Protein flocculation Removal of tannins Chromatography
Agriculture
Coating for delayed ripening of fruits Seed coating Nutrient control release Nematode treating Animal feed
Water Treatment
Food processing Potable drinking water Removal of dyes Removal of metals, pesticides and PCB's Sewage treatment
Additive
Inhibition of oxidation Thickener; stabilizer Texture modifier Slow-release additive support
Biomedical
Hypocholesterolemic effect Wound care; eye bandage Drug delivery Biomaterials Dental applications
Cosmetics
Moisturizers Thickener in low pH products Film formers; emulsifiers Antistatic Hair and skin care
Biotechnology
Enzyme immobilization Cell immobilization Encapsulation Filter aid Protein recovery
Textile/Paper
Coatings; fibres Dyeability Wet strength Retention aid
1435 as xanthophylls. They may be extracted from shellfish using appropriate solvents. Carotenoproteins from processing discards of shellfish have also been isolated using enzyme-assisted separation techniques [16]. Figure 4 illustrates the chemical nature of some of the important xanthophylls found in shellfish processing discards. A close scrutiny of the results summarized in Table 4 indicates that Astaxanthin diester is the major carotenoid present in the processing discards of shrimp and crab [5]. Unesterified astaxanthin and astaxanthin monoester were present in smaller amounts, followed by astacene, lutein and zeaxanthin.
OH
OH
Zeaxanthin OH
DH
Figure 4. Chemical structures of major xanthophylls (carotenoids) of shellfish processing discards.
1436
Table 4 Distribution (% of total) of xanthophylls in shrimp and crab processing discards. Shrimp
Carotenoid Astaxanthin Astaxanthin monoester Astaxanthin diester Astacene Lutein Zeaxanthin Unidentified
3.95 ± 19.72 ± 74.29 ± Nil Nil 0.62 ± NU
Crab* 21.16 ± 1.15 5.11 ± 0.23 56.57 ± 1.60 3.26 ± 0.47 8.24 ± 0.30 4.64 ± 0.76 0.22 ± 0.05
0.19 0.19 0.38 0.05
*Soft-shelled crab shells contained 2.66% canthaxanthin. Carotenoids from shellfish discards, mainly astaxanthin and its esters, may be extracted into a vegetable or marine oil at a ratio of 1:2 (v/w) at 60°C for 30 min [8]. The oil may be reused to enrich it with carotenoids. Such oils may be used in the formulation of feed for salmonidfishspecies. Salmonids such as Arctic char, rainbow trout and salmon, like other animals, are unable to perform a de novo synthesis of carotenoids. However, they can acquire adequate pigmentation from the diets containing carotenoids. Yeasts, bacteria, molds and higher plants are capable of synthesizing carotenoids acetate according to the scheme given in Figure 5. Acetate (2-carbons)
"^
Mevalonic acid (6-carbons) CO,
\ Isopentanyl pyrophosphate (5-carbons) Dimethylallyl pyrophosphate
^ pjQ
>- V ^"^^ "^ ^"^^ animals C-20 + C-20
I
Carotenoids
Figure 5. Biosynthesis of carotenoids
>- Steroids
1437 Incorporation of carotenoids in formulated feeds for Arctic char (Salvelinus alpinus) resulted in their assimilation in the flesh and skin tissues of fish. The total content of carotenoids in fish tissues after 5, 9, 12 and 15 weeks of feeding on diets containing 65 ppm astaxanthin are shown in Table 5. It is generally accepted that 3-4 /xg/g carotenoids are required for distinct visual color impression of salmonids [17]. The carotenoid levels observed in flesh (4.10-5.56 ^g/g wet tissue) and skin (24.8541.25 /ig/g wet tissue) after 9-15 weeks of feeding were adequate and similar to that of pigmented rainbow trout, brook trout and coho salmon. Table 5 Total carotenoid content of Arctic char fillet and skin fed 15 weeks on astaxanthin-containing diet and then depigmented on diets without carotenoids. Carotenoid Content, /ig/g wet-tissue Status
Fillet
Skin
PIGMENTATION PERIOD 5 weeks 9 weeks 12 weeks 15 weeks
2.55 4.10 4.69 5.56
± ± ± ±
0.41 0.06 0.31 0.11
14.86 36.01 24.85 41.21
± ± ± +
0.18 1.57 2.51 2.27
DEPIGMENTATION PERIOD 3 weeks 7 weeks
2.38 ± 1.73 1.89 ± 1.60
29.97 ± 0.09 18.32 ± 5.09
Subsequent withdrawal of pigments from feed resulted in a 66% decrease in the pigmentation of Arctic char flesh after a 7-week period. A similar degree of depigmentation was also observed with skin of Arctic char which lost 55% of its initial carotenoid content over the same period. Assuming a growth rate of approximately 100 g/week/fish, a depigmentation of approximately 30-35% might be anticipated. Therefore, the observed values demonstrate that the depigmentation process includes metabolizing of carotenoids by the fish in addition to a simple dilution effect brought about by the growth of Arctic char [18-21]. Further studies on the chemical nature of carotenoids in the flesh and skin of farmed char indicated that dietary carotenoids were assimilated without any chemical changes, except perhaps esterification/de-esterification, in thefishtissues. Thus feeds containing astaxanthin, canthaxanthin and their combination, as well as shrimp meal as such led to the presence of astaxanthin, canthaxanthin, their combination as well as astaxanthin in the flesh and skin of the fish, respectively. In all cases examined, leutein was present, perhaps derived originally from the corn meal used in feed formulations [20].
1438 3. SUMMARY As the supply of raw material from aquatic resources is shrinking and trends towards production of aquacultured species continue, full utilization of marine processing by-products and underutilized species becomes more urgent. Coupled with environmental restrictions, efforts for value-added utilization of natural ingredients from shellfish by-products is expected to proceed at a faster pace in the coming years. 4. ACKNOWLEDGEMENTS Financial support in the form of a subvention grant from the Department of Fisheries and Oceans and Natural Sciences and Engineering Research Council of Canada is acknowledged.
5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
F. Shahidi, In Seafoods: Chemistry, Processing Technology and Quality, F. Shahidi and J.R. Botta (eds), Blackie Academic and Professional, Glasgow, pp. 320-334, 1994. F. Shahidi, In Seafood Proteins, Z.E. Sikorski, B.S. Pan and F. Shahidi (eds.). Chapman & Hall, New York, pp. 171-193, 1994. R. L. Olsen, A. Johansen and B. Mymes, Process Biochem., 25 (1990) 67. F. Shahidi, J. Synowiecki and M. Naczk, In Seafood Science and Technology, E.G. Bligh (ed.). Fishing News Books, Oxford, pp. 300-304, 1992. F. Shahidi and J. Synowiecki, In Advances in Chitin and Chitosan, C.J. Brine, P.A. Sanford and J.P. Zikakis (eds.), Elsevier Applied Science, London and New York, pp. 617-626, 1992. A. Alimuniar and R. Zainuddin, In Advances in Chitin and Chitosan, C.J. Beine, P.A. Sanford and J.P. Zikakis (eds.), Elsevier Applied Science, London and New York, pp. 627-632, 1992. 0 . Skaugrud and G. Sargent, G., In Proceedings of the International By-Products Conference, Anchorage, Alaska, pp. 61-69, 1990. F. Shahidi and J. Synowiecki, J. Agric. Food Chem., 39 (1991) 1527. M.J. Brzeski, INFOFISH International, 5 (1987) 31-33. D. Knorr, Food Technol., 38(1) (1984) 85. D. Knorr, Food Technol., 45(1) (1991) 114. S. Michihiro, S. Watanabe, A. Kishi, M. Izume and A. Ohtakara, Lipids 23 (1988) 187. H.K. No and S.P. Meyers, J. Agric. Food Chem., 37 (1989) 580. Y. Pandya and D. Knorr, Process Biochem., 26 (1991) 25. X.-Q. Han, and F. Shahidi, Food Chem., In press. B.K. Simpson and N.F. Haard, J. Applied Biochem., 7 (1985) 212.
1439 17 O.J. Tonissen, R.W. Hardy and K.D. Shearer, CRC Crit. Rev. Aquatic Sci., 1 (1989) 209. 18 F. Shahidi, J. Synowiecki and R.W. Penney, Meat Focus International, 1 (1992) 319. 19 F. Shahidi, J. Synowiecki and R.W. Penney, J. Aquatic Food Prod. Technol., 2(1) (1993) 99. 20 F. Shahidi, J. Synowiecki and R.W. Penney, Food Chem., In press. 21 J. Synowiecki, F. Shahidi and R.W. Penney, J. Aquatic Food Prod. Technol., 2(3) (1994) 37.
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1441
Protein Concentrates from Underutilized Aquatic Species F. Shahidi Departments of Biochemistry and Chemistry, Memorial University of Newfoundland, St. John's, NF, AlB 3X9, Canada. Abstract Protein concentrates were prepared from a number of underutilized aquatic species namely capelin, herring and mackerel. A simple sequential washing of comminuted meats using water, 0.5% NaCl and 0.5% NaHCOg was employed and the resultant washed meats were suspended in equal amounts of water, in some cases containing a small amount of HO Ac. The protein solution so prepared was thermostable and produced a concentrate upon dehydration. Alternatively, enzyme-assisted hydrolysis of mechanically-deboned fish or seal meats provided soluble, decolorized protein preparations which upon dehydration yielded concentrates with attractive functional properties. The protein preparations so produced may be used in a variety of applications either as such or as a fortifier in food formulations. 1. INTRODUCTION The global annual harvest of pelagic fish species such as capelin, herring, mackerel, among others, exceeds 20 million metric tons [1]. However, due to a high lipid content and rapid development of rancidity, these fish have found limited consumer acceptance. Therefore, production of novel food ingredients from such underutilized aquatic species is desirable. Fish proteins possess excellent amino acid scores and digestibility characteristics and as such may be used to enhance the nutritive value of cereal-based foods. However, lack of solubility of myofibrillar proteins in water as well as their sensitivity to denaturation has hampered efforts towards full utilization of seafood proteins. Production of fish protein concentrates employ processes which may be classified as chemical, biological or physical [2]. In the chemical processes, solvents are used to remove lipids and water fi^om fish and the remaining proteins and minerals are subsequently dried. Biological methods may employ either enzymes or microorganisms to release protein from the oil and water in the tissue. In the physical procedures, mechanical means are generally used to remove oil and water. Lack of solubility of myofibrillar proteins has been related to the tendency of myosin molecules to interact with one another under physiological conditions and in
1442
the presence of enzymes and low-molecular-weight components that adhere to them [3,4]. A significant portion of these adhering compounds may be removed from fish mince upon washing with water. The resultant washed meat has enhanced gelling properties and as such may be used for production of texturized products [3,5,6]. Stanley et ah [7] have recently used a similar aqueous washing process to solubilize up to 36% of the total proteins from various beef and chicken muscles. On the other hand, enzymatic hydrolysis yields shorter chain proteins and polypeptides as well as free amino acids. These latter compounds are generally freely soluble in aqueous solutions. The present study reports the development of a novel process for production of a low-viscosity, thermostable and shelf-stable myofibrillar protein preparation in water. Production of protein hydrolyzates from underutilized aquatic species, such as capelin and harp seal, for food applications is also considered. 2. PROTEIN CONCENTRATES 2.1 Low viscosity, thermostable protein dispersions Freshly landed capelin {Mallotus villosus) were mechanically deboned using a Baader 694 deboning machine with a drum orifice size of 3 mm. For production of dispersions from mackerel {Scomber scombrus) and herring (Clupea harengus), the dark meat was removed from skinned fillets and the resultant meat was comminuted. The preparation of thermostable water dispersions of fish structural proteins, which may then be concentrated by suitable dehydration methods, involves washing of the comminuted muscle tissues to remove soluble components and lipids. Conformational changes in proteins enhances the entrapment of water in the gel matrices [8]. Making use of this property, we have developed water dispersions of myofibrillar proteins from a number of underutilized fish species. Figure 1 outiines the general process for preparation of such thermostable protein dispersions [9]. However, the specifics of the process may vary from one species to another. The mince obtained by mechanical deboning or from manual separation of the meat was soaked in cold water with gentle stirring. The solids were then further washed with dilute saline and sodium bicarbonate solutions followed by a final wash in cold water. The washed samples of meat were then suspended in cold water, at up to 50%, and homogenized. It may be necessary, depending on the species of fish under investigation, to add a few drops of acetic acid to the dispersion to lower the pH to about 3.0-3.5 in order to enhance gelation [10]. Although the added acid brought about an increase in the viscosity of mackerel and herring homogenates, the viscosity of the mixture was lowered by heating the dispersion to 50°C. Acetic acid, when present, may also act as a preservative during the storage of the product prior to its dehydration/concentration.
1443 FISH GUTTING AND FILLETING
COMMINUTING WASHING IN COLD WATER WASHING IN SODIUM BICARBONATE WASHING IN COLD WATER HOMOGENIZATION IN COLD WATER LOWERING OF pH BY ACETIC ACID (if necessary)
HEATING SIEVING SPRAY DRYING
J
FUNCTIONAL PROTEIN POWDER Figure 1. Flowsheet for production of functional fish protein powders.
1444 Table 1 summarizes the proximate composition of unwashed and washed fish meat before the final homogenization step in the production of thermostable protein products from capelin, herring and mackerel [5,11,12]. In all cases, the washed meat had a higher moisture and a lower lipid content as compared with its unwashed counterpart. In all cases, the washing process produced a light colored product which was nearly odorless. The treatment also increased the mass of the sample by about 20% (w/w), presumably due to increased hydration of proteins. Table 1 Proximate composition (weight %) of unwashed and washed fish meat. Species/Component
Unwashed
Washed
CAPELIN Moisture Crude Protein (N x 6.25) Lipid
83.98 ± 0.20 12.70 ± 0.31
93.19 ± 0.10 4.5.1 ± 0.15
1.98 ± 0.01
1.13 ± 0.04
HERRING Moisture Crude Protein (N x 6.25)
72.4 ± 0.10 17.4 ± 1.20
86.7 ± 2.30 8.30 ± 0.20
Lipid
8.10 ± 1.20
4.10 ± 0.10
MACKEREL Moisture Crude Protein (N x 6.25) Lipid
63.6 ± 0.30 19.30 ± 0.20 14.0 ± 0.50
82.20 ± 1.60 9.60 ± 1 . 1 0 5.30 ± 0.20
The aqueous dispersions of washed meats from herring and mackerel were highly viscous and their viscosity was both temperature and concentration dependent. As the temperature of the dispersions increased, their viscosity decreased, however, upon subsequent cooling the apparent viscosity of the dispersion was mostly recovered. In addition, the viscosity of the herring and mackerel dispersions was increased to > 5 Pa-s when 2.4% proteins were present. For capelin dispersions, heating of the samples to 80-100°C resulted in a permanent loss of viscosity (Table 2). However, for herring and mackerel dispersions loss of viscosity was achieved only when acetic acid was added to the washed samples prior to heat treatment (Table 2). The dispersions with reduced apparent viscosity showed remarkable thermal stability. The proteins were stable when heated to 100°C for 30 min and even moderate concentrations of salt in the solution did not influence the solubility characteristics of the dispersions. Stable proteins are of interest to study structurefunction relationships and to examine possibilities of their use in biological applications [13]. Doi [14] reported that under controlled pH, ionic strength and heating one could obtain transparent gels from globular protein with potential applications in food preparations. Use of these dispersions as a protein obtained
1445 Table 2 Effect of Heating Time at 100°C on protein solubility of washed comminuted fish meat. Heating Time, min
Capelin
Herring
100 92.5 88.0 88.0 88.0
100 97.0 98.1 98.0 89.0
0 10 20 25 30
Mackerel 100 85.0 84.0 84.5 83.0
Table 3 Amino acid composition of capelin, herring and mackerel and their thermostable aqueous dispersions (g/100 g protein). Amino Acid Alanine Arginine Aspartic Acid Cysteine Glutamic Acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tryosine Valine
Capelin
Herring
Mackerel
Original Dispersion Meat
Original Dispersion Meat
Original Meat Dispersion
6.08 6.33 10.88 0.93 14.86 5.33 2.53 4.72 9.19 9.54 3.08 4.42 3.63 4.41 4.83 1.08 4.00 5.72
6.11 6.53 10.87 0.90 14.64 5.74 2.18 4.88 9.25 9.47 3.11 4.20 3.93 4.26 4.95 0.99 4.11 5.79
5.97 5.93 9.92 1.73 13.50 4.48 2.71 4.29 8.03 8.98 2.41 4.00 3.47 3.83 4.54 1.21 3.57 6.00
5.90 6.53 10.80 9.98 14.20 4.40 2.17 4.77 8.10 9.45 2.50 4.40 3.80 3.90 5.00 1.24 3.75 5.55
5.87 6.33 10.39 0.81 13.76 4.73 4.82 4.57 8.03 9.28 3.69 4.07 3.50 4.18 5.50 1.33 3.69 5.47
5.91 6.53 10.78 0.64 14.30 4.58 3.07 5.07 8.88 9.31 3.25 4.37 3.92 4.58 5.83 1.10 3.78 5.76
supplement in extrusion cooking of cereal-based products and development of a functional powder by suitable dehydration of the dispersion could be feasible. Venugopal et aL [15] have recently reported the preparation of such functional proteins from shark muscles and their subsequent use in the production of extruded products. The fish protein concentrates (powders) prepared from different species in this study had a protein content of > 85 %. The amino acid composition of products so
1446 was similar to that of the proteins present in the starting muscle tissues (Table 3). However, the content of individual free amino acids in the products was much lower than those present in the starting materials as exemplified for capelin (Table 4). Thus, the dispersions and powders so prepared had the advantage of being bland in taste. In addition, the washing process removed most of the hemoproteins from the original muscle tissues and products so obtained had a milky white color. The Hunter L, a, b values for herring dispersions were 74.7+0.8, -1.9±0.3 and 9.1 ±0.9, respectively. Storage of the preparation at 2°C for up to 3 weeks did not induce any color change in the product. However, storage at ambient temperatures under aerobic conditions led to their slight yellowing after a 1- to 3-week storage which increased their corresponding Hunter b values to 15.4-19.2. Table 4 Free amino acid content of unwashed and washed dispersions of capelin and herring (mg/lOOg protein).' Capelin Amino Acid
Herring
Original Meat
Dispersion
Original Meat
Dispersion
44.8 11.7 22.7 2.3 43.6 22.1 10.0 19.5 37.4 39.3 14.0 17.7 22.6 26.9 129.6 22.3 3.1 16.6 31.1
3.3 3.5 2.3 trace 2.8 1.4 0.8 1.6 3.0 10.2 1.5 2.4 1.3 2.7 4.1 1.6 0.2 2.1 2.3
23.0 10.0 23.0 — 10.0 10.7 16.0 8.5 28.0 71.0 34.0 9.0 5.0 5.0 ND 34.0 5.0 4.6 35.0
3.9 9.0 3.9 — 3.4 1.3 0.8 2.3 1.1 2.6 5.4 5.4 1.4 1.4 ND 2.3 — 4.0 3.2
Alanine Arginine Aspartic Acid Cysteine Glutamic Acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Taurine Threonine Tryptophan Tryosine Valine
•ND, not determined. 2.2 Protein hydrolyzates Freshly landed male capelin {Mallotus villosus) and carcass meat from harp seal (Phoca groenlandica) were mechanically deboned, as before. The comminuted raw materials were then suspended in water and enzyme was added to the slurry. The reaction was allowed to proceed between 2h and 1 week, depending on the activity
1447 of the enzyme employed, process temperature and other factors. After separation of solids, the aqueous layer was clarified, pH was adjusted and dehydrated. The process may include sterilization at different stages, if necessary. Figure 2 outlines the main steps in production of protein hydrolyzate fi-om the raw material [16,17]. Typical protein yield and proximate composition of hydrolyzates from capelin (Biocapelin®) and harp seal (Biosea-L®) are given in Table 5. Although many factors affect the yield of hydrolysis, the type of enzyme employed had a marked effect on this and also on the characteristics of the final product. The enzymes examined in the present investigation included papain, Alcalase, Neutrase and endogenous autolytic enzymes of each species. High yields of protein recovery by Alcalase and its low cost may provide an incentive for its use in commercial operations. CAPELIN/HARP SEAL MECHANICAL DEBONING DISSOLUTION/SUSPENSION pH ADJUSTMENT
HYDROLYSIS ENZYME INACTIVATION SEPARATION & CLARIFICATION DEHYDRATION
T
HYDROLYZATE POWDER Figure 2. Flowsheet for production of hydrolyzates from underutilized species.
1448 Table 5 Typical protein recovery (%) and composition of hydrolyzates from capelin (Biocapelin®) and harp seal (Biosea-L®). Material Capelin Biocapelin Seal Biosea-L
Protein Recovery 51.6-70.6 58.0-72.4
Composition, % Protein
Lipid
Moisture
Ash
13.6-14.1 65.9-73.3 23.0-24.2 64.1-84.9
3.3-3.9 0.2-0.4 3.5-3.9 0.2-2.0
78.0-78.3 5.3-6.3 70.6-70.9 6.1-6.2
2.4-2.5 14.9-20.6 1.8-2.1 17.7-10.9
A close scrutiny of the results presented in Table 5 indicates that hydrolyzates generally possessed a lower lipid content than their original protein source, on dry weight basis. As hydrolysis proceeds, elaborate membrane system of the muscle cells tend to round up and form insoluble vesicles, thus allowing the removal of membrane structural lipids. Consequently, protein hydrolyzates are expected to be more stable towards oxidative deterioration. In addition, hydrolyzates produced from both capelin and seal meat had an ivory-white color. Thus Hunter L, a, b values for Biosea-L® were 74.2-84.6, -1.0-1.1 and 11.0-13.3, respectively, as compared with those of 16.6, 4.3 and 2.1 for the original seal meat. The amino acid composition of Biocapelin® and Biosea-L® produced by Alcalaseassisted hydrolysis were compared with those of their corresponding muscle tissues. Results presented in Table 6 indicate that the amino acid profiles for both species remain generally unchanged. In case of Biocapelin®, however, sensitive amino acids such as methionine and tryptophan were somewhat affected. The hydrolyzates prepared from capelin and seal meat had excellent solubility characteristics at pH values ranging from 2.0 to 10.4. While 90.36-98.57% of nitrogenous compounds of Biocapelin® were soluble, corresponding values for Biosea-L® varied between 93.45 and 98.05% (Table 7). The fat adsorption, moisture retention, emulsification properties and whippability of the hydrolyzates were also excellent [16,17]. In addition, when hydrolyzates were added to meat model systems at 0.5-3.0% levels an increase of up to 4.0% in the cooking yield of processed meats was noticed [17]. Furthermore, Biocapelin® inhibited oxidation of meat lipids by 17.7-60.4% as reflected in the 2-thiobarbituric acid values of the treated meat systems. The mechanism by which this antioxidant effect is exerted may be related to the action of hydrolyzed molecules as chelators of metal ions which may be released during the heat processing of meat. However, a study of the inhibitory effects of Biocapelin® and Biosea-L® against bleaching of jS-carotene in a jS-carotenelinoleate system [18] demonstrated that while the former always exhibited an inhibitory effect, the latter acted as a pro-oxidant when employed at higher concentrations (Table 8). Preliminary chromatographic separation of the hydrolyzates indicated that while antioxidant components were ninhydrin-positive, pro-oxidant activity was due to ninhydrin-negative components. Further studies are required for
1449 better evaluation and structural identification of the biologically active components of hydrolyzates. Table 6 Typical amino acid composition (%) of Biocapelin® and Biosea-L®. Seal
Capeliin Amino Acid Alanine Arginine Aspartic Acid' Cysteine Glutamic acid*" Glycine Histidine Hydroxylysine Hydroxyproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Original Meat
Biocapelin
Original Meat
5.57 5.99 8.88 1.33 13.19 5.32 2.43 0.09 0.42 4.72 8.15 8.47 3.09 3.80 3.70 4.18 4.82 1.07 3.34 5.71
6.00 5.70 9.89 1.34 13.43 5.14 2.09 0.17 0.46 4.25 7.60 8.49 2.05 3.19 3.67 4.24 4.56 0.43 2.47 5.77
5.88 6.21 8.23 0.87 11.46 4.47 5.01 0.04 0.75 4.58 7.44 8.72 3.89 4.57 3.89 3.98 4.53 1.20 2.85 5.80
Biosea-L 5.80 6.02 8.90 1.01 12.53 5.58 5.25 0.10 0.85 3.92 8.50 9.44 3.86 3.96 3.89 3.60 3.82 1.19 2.46 4.62
^Includes asparagine. •^Includes glutamine. Table 7 Effect of pH on the Solubility of Biosea-L®. pH
Solubility
pH
Solubility
2.50 4.00 5.50 6.40
93.45 ±0.55 94.43+0.15 95.96+0.01 96.93±0.00
7.20 7.40 9.00 10.40
97.21+0.11 98.05+0.01 97.91+0.05 98.05±0.12
1450 Table 8 Inhibitory effect (% IE) of Biocapelin® and Biosea-L® against the bleaching of jScarotene in model systems.* Hydrolyzate Concentration, %
Incubation Period, min 30
60
BIOCAPELIN 0.02 0.04 0.10 0.20
11.5 16.7 33.3 38.5
8.5 12.2 19.5 24.4
BIOSEA-L 0.02 0.04 0.10 0.20
9.0 12.8 2.6 -2.6
3.7 7.3 -2.4 -7.3
"Calculated as 100 (B-A)/(C-A), where A = Absorption of the control sample; B = absorption of the sample-containing hydrolyzate; and C = absorption of the control sample prior to incubation. 3.
SUMMARY
The present study demonstrates that underutilized aquatic species may be used to produce useable protein preparations in the form of low viscosity, thermostable dispersions or readily water-soluble hydrolyzates. Potential areas of application of the dispersions include production of functional protein powders and extruded products. Meanwhile, prepared hydrolyzates have numerous application potentials in the food industry for production of adiletic drinks, geriatric foods and for enhancing process yield of fabricated muscle-based foods.
4.
REFERENCES
1
A.L. Fink, L.J. Calciano, Y. Goto and D.R. Palleros, In Current Research in Protein Chemistry: Techniques, Structure and Function, J.J. Villafranca (ed.). Academic Press, New York, pp. 417-424, 1990. B.R. Stillings and G.M. Knobl, Jr., J. Am. Oil Chem. S o c , 48 (1971) 412. T. Suzuki, Fish and Krill Processing Technology, Applied Science Publishers, London, 1981. T. Nakagawa, F. Nagayama, H. Ozaki, S. Watabe and K. Hashimoto, Nippon Suisan Gakkaishi, 55 (1989) 1945. V. Venugopal and F. Shahidi, J. Food Sci., 59 (1994) 265.
2 3 4 5
1451
6 7 8 9 10 11 12 13 14 15 16 17 18
V. Venugopal and F. Shahidi, CRC Crit. Rev. Food Sci. Nutr., In press. D.W. Stanley, A.P.Stone and H.O. Hultin, J. Agric. Food Chem., 42 (1994) 863. G.R. Ziegler and E.A. Foegeding, In Advances in Food Research, Volume 34, I.E. Kinsella (ed.). Academic Press, New York, pp. 203-298, 1990. F. Shahidi and V. Venugopal, Meat Focus International, 2 (1993) 443. K. Freitheim, B. Egelandsdal, O. Harbitz and K. Samejima, Food Chem., 18 (1985) 169. F. Shahidi and A.C. Onodenalore, Food Chem., In press. F. Shahidi and V. Venugopal, J. Agric. Food Chem., In press. Y. Nosoh and T. Sekiguchi, In Protein Stability and Stabilization through Protein Engineering. Ellis Horwood Ltd., Sussex, England, pp. 101-123, 1991. E. Doi, Trends in Food Sci. Technol., 4 (1993) 1. V. Venugopal, S.N. Doke, P.M. Nair and F. Shahidi, Meat Focus International, In press. F. Shahidi, J. Synowiecki and J. Balejko, J. Agric. Food Chem., In press. F. Shahidi, X.-Q. Han and J. Synowiecki, Food Chem., In press. H.E. Miller, J. Am. Oil Chem. Soc, 45 (1971) 91.
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1453
EXTENDING THE SHELF-LIFE OF SEAFOOD USING A MULTIPLE BARRIER PROCESS Spiros M. Constantinides, Sigurdur M. Einarsson, Yenchit Benja-arporn and Apostolos Pappas Department of Food Science and Nutrition, University of Rhode Island, West Kingston, RI., 02892, USA ABSTRACT New processes to extend the shelf-life of seafood have recently become the goals for research and development in the seafood industry. A new approach to extend the shelf-life of fresh seafood, in this case dogfish (Squalus acanthias) , was investigated using a combination of heat (hot corn oil, 175°C) and acidity (acetic acid), followed by vacuum packaging before storing at refrigeration temperature for at least one month. The synergistic effect due to the multiple barriers applied produced an intermediate product, not ready to eat, with extended shelf-life. The product had to be treated as raw fish would before consumption, i.e. cooked in some manner to prepare a meal. After one month of storage at refrigeration temperature, the bacterial count was insignificant. Taste panel tests on the treated samples showed similarity to the fresh untreated dogfish samples. INTRODUCTION The demand in the consumer market for convenience foods in the US was estimated to increase from $30.5 billion in 1985 to about 48.0 billion in 1990 (1). One type of such foods emerging are the minimally processed refrigerated foods (MPRF) which appear to be satisfying the consumer needs for convenience and variety. Shelf-life is prolonged in different ways, such as, an initial heat treatment, vacuum packaging and refrigeration or a combination of different barriers acting in a synergistic manner (2) (3) . The stability depends on several barriers which are not strong enough individually to inactivate undesirable microorganisms but can act together to provide additive synergistic effects. MPRF require a number of such barriers to provide safety. Moberg (2) cites fermented sausage as an example of MPRF. Multiple barriers are used, such as, starter cultures providing the competitive flora which act to increase the acidity, salt helps to decrease water activity, and the addition of nitrite together with low temperature all act to enhance shelf-life and decrease the possibility of food borne disease.
1454 The safety and stability of traditional foods upon storage is based on either low water activity or low pH. Extrinsic factors can be introduced to the food system to contribute further to the prolonged safe storage of the food product. Such factors could be salt, sugar, antimycotic agents, and various additives which are not favored by the consumers in general. Gould (4) has categorized the general procedures which are used to preserve foods. These are: cooling, freezing, drying, curing, vacuum packaging, f e r m e n t a t i o n s , preservatives, pasteurization, sterilization, ionizing radiation, aseptic processing, and heat treatment of food ingredients and packaging materials to reduce microbial contamination. Genigeorgis (5) discussed adequately the effect of low temperature as one of the most important extrinsic factors to control microbial growth in foods. Another factor is vacuum and modified-atmosphere packaging of foods. Microorganisms that need oxidative metabolism to survive and grow are inhibited by such techniques where oxygen is removed. It is the anaerobic microorganisms that will be able to survive and cause subsequent deterioration to food. Acidification is an ancient old method of prolonging the safety of food. The pH level of 4.5 or below is widely regarded as inhibitory to the growth of Clostridium botulinum. Weak acids, which are used as preservatives are incompletely dissociated at pH values of most foods. Their inhibitory activity relies not only on the H"^ concentration, but also on the additional inhibitory effects from the undissociated acid or its anion (6). The undissociated portion being lipophilic can move across the cell membrane causing damage to the metabolism of the microorganism. Heat treatments of different kinds, pasteurization, sterilization, blanching, frying inactivate the vegetative microorganisms and drastic heat treatments would inactivate the spores. It should be noted that any of the procedures mentioned earlier, i.e. acidification or heat treatment can increase the sensitivity of microorganisms especially if those factors are used in a combined manner, requiring less of each treatment to bring about the desired inhibitory effect. Ceilings and Yu (7) attempted to produce an intermediate moisture fish product by combining deep frying of the product with the use of selected solutes in which the fish portion was infused. Although the samples were free of bacteria, they were tough and dry, and the taste panel studies indicated a score of slightly like to dislike. Blumenthal (8) has covered extensively the topic of deep-fat frying. During the frying process there is a mass transfer of water migrating from the interior outward toward the exterior surfaces thus replacing water lost due to dehydration. This "pumping" phenomenon is described by Lydersen (9). Water in the food which is being fried
1455 carries off thermal energy from the hot frying oil surrounding the frying food. This prevents charring or burning caused by excessive dehydration. Although the temperature of the oil may be 180®C, the temperature of the food frying is only about 100®C (8). A new method which in some way utilizes the combined method of processing is the method of food preparation called "sous vide". Complete meals are prepared under extreme sanitary conditions, vacuum packed, and cooked in a pouch or in a rigid container, chilled and stored at refrigeration temperature for about one month depending on the product and the method of cooking. The product is subsequently reheated before it is consumed. Although sous vide processing offers numerous advantages to the consumer it poses a potential public health hazard (10). The psychrotrophic food borne pathogens such as Clostridium botulinum are of concern since vacuum packaging provides suitable environment and the heat treatment is usually not d r a s t i c e n o u g h to p r o d u c e c o m m e r c i a l s t e r i l i t y . Consequently, the products require refrigeration below 3°C to prevent spoilage and ensure product safety (11). Several studies have shown that normal refrigeration alone is not adequate to suppress the growth of type E Clostridium botulinum (11). It has been reported that Clostridium botulinum type E grew and produced toxin in heat-sterilized beef stew substrate at 3.3°C within 31 days. Cann et al. (12) observed growth and toxin production by type E Clostridium botulinum in fresh herring after 15 days storage at 5°C. Kautter (13) observed growth and toxin production in inoculated, vacuum-packaged smoked fish held 5 days at 10®C. Solomon et al. (14) reported toxin production in crabmeat held at 12°C for 14 days. Modified atmosphere (MA) storage together with refrigeration has been shown by Stier et al (15) to significantly increase the shelf life of fresh fish. No toxigenesis was observed in either the air or modified samples stored at 4.4°C, but all inoculated samples held at 22.2°C were toxic within 2-3 days. Tsang et al. (16) found that spores of Clostridium botulinum E were capable of growth and toxin production at 26°C in citric acid acidified systems at pH 4.2. However, growth and toxin production were not detected below pH 5.0 when acetic acid was used. Ikawa and Genigeorgis (17) showed that the probability of toxigenesis by one spore was significantly affected by temperature, and storage time and not by the modified atmosphere. No toxin production was detected at 4°C. At 30®C storage, spoilage of fillets followed toxigenesis. Hanschild (18) discussed the potential risk from extended shelf-life of refrigerated foods by Clostridium botulinum. The nonproteolytic types of Clostridium botulinum have the ability to grow at refrigeration temperature. If given the time and proper medium, these organisms can grow and produce toxin at 40<>F (4.4«C) .
1456 FDA has always been concerned over botulism especially for vacuum packaged foods with a over 0.93, and a hydrogen ion concentration (pH) of over 4V6. Fish and fish products because of their potential of carrying higher levels of Clostridium botulinum, type E spores, and because of the history of fish associated botulism, those products should not be vacuum-packed (19) . Schmidt et al (20) found that growth of psychrotrophic toxigenic Clostridia has not been observed below 3°C. Also that botulism risk is further reduced by taking into account the inactivation of botulinum toxin occurring during the culinary preparation of most of the vacuum-packed refrigerated foods of extended durability. Shelf-life can be defined as the time it takes a given product to deteriorate to an acceptable degree under some specific conditions of processing, packaging and storage. An extended shelf-life refrigerated food can be defined as one in which the refrigerated shelf-life is 50% to 400% longer than that of a comparable product (21). Shelf-life of fresh fish is limited and therefore seafood safety has always been a challenging issue to tackle. Microorganisms and the endogenous enzymatic activities occurring after death of the captured fish are considered to be the primary cause of spoilage. The processes used to extend the shelf-life and control spoilage depend on inhibiting microbial action and in general controlling post harvest biochemical reactions. The common processes used utilize heat such as in canning; low temperature such as in chilling and in freezing; moisture and water activity reduction, such as in drying, salting or even smoking of fish. Such processes can extend the shelf-life anywhere from nine days (chilled fish) to two years or more (frozen fish). Chilled fish in ice does not change significantly the fresh characteristics of the fish. All other processes do alter somewhat the original fresh state characteristics of the freshly caught fish, that is, the original qualities are impaired. Changes in consumer attitude about health and nutrition have increased the worldwide demand and prices of seafood. Eating patterns in the developed countries have emphasized seafood quality and freshness and product diversity. Moreover, new processes to extend shelf-life of seafood have become goals for research and development. The objective of the research conducted was to investigate the efficacy of utilizing a multiple barrier process to prolong the fresh characteristics of fish beyond the nine day limit at refrigeration temperature, and inhibit spoilage, thus producing a safe product for the consumer to use after one month of storage at refrigeration temperature. The final product would be cooked and prepared into a meal in a manner similar to fresh fish. Determining the efficacy of a number of combinations of barriers in inhibiting the growth of microorganisms would be valuable in product development and safety of the food item.
1457 This is of special importance to storage of foods where refrigeration is the primary barrier. Any additional barriers included in the process will add to the safety and prolong the safety of the food product, and may also ensure against problems arising from some temperature abuse during storage or transportation. In this particular study, dogfish (Squalus acanthias) was selected. It is an underutilized species and has not yet penetrated the US market for reasons such as, the intense ammonia build-up from the urea present in the tissues, lack of product development, lack of market analysis and promotion, and in general the consumer being unfamiliar with that species of fish (22) . On the other hand in many European countries it is considered a delicacy and in England the fish and chips market is based partly on dogfish. MATERIALS AND METHODS Preparation of fish All seafood was obtained from local seafood processing plants, Seafresh U.S.A. Inc., Narragansett, RI and Town Dock, Galille, RI. The fish were brought in as fillets or backs. After the fish had been skinned and prepared into backs, fillets or portions, they were washed to remove urea, drained and then either dipped in various concentrations of acetic acid for different periods of time and packed under aerobic conditions in freezer bags or immersed into a deep fryer (Tefal, Model 8219) that contained hot (175°C) Mazola corn oil for different periods of time and then immediately dipped into cold (10°C) acetic acid of different concentrations. The fish were then vacuum packed in standard 4 mil vacuum pouches size 8 X 10" (Market sales Co., Newton, MA) and stored at refrigeration temperature (3-4°C). The following analyses were conducted periodically for up to 4 weeks. Microbiological analysis a) Sample preparation : 11 g of dogfish (Squalus acanthias) were aseptically transferred to a sterile blender jar with 99 ml of 0.1% sterile peptone water and homogenized at a high speed for 1 minute. This gave a lO-i dilution. Serial dilutions of lO"^ , 10"^ , and 10"^ were prepared by using 0.1% peptone water. b) Total Bacterial Count (TBC) : TEC was conducted aerobically by using the plate count agar method according to Post (23) . Plate count agar (Difco) was prepared by homogenizing 23.5 g agar in 1000 ml of distilled water. Duplicate samples of 1 ml and 0.1 of each dilution were pipetted into sterilized and appropriately labeled petri dishes. Twelve to 15 ml of cooled (44-46°C) plate count agar was poured into each plate. The sample and agar medium were immediately mixed and then allowed to solidify. The petri dishes were inverted and incubated aerobically at 37°C
1458 for 48±2 hours. For anaerobic determinations the plates were put in a Gas pak jar and incubated at 37®C for 48±2 hours. The total colony counts were recorded as total bacterial counts or colony forming units per gram (cfu/g). Chemical analysis a) Trimethylamine (TMA) was determined according to Woyewoda et al. (24) . Twenty five g of comminuted fish and 50 ml of 7.5% Trichloroacetic acid (TCA) were added to a blender jar and blended well. The homogenate was filtered through Whatman #2 filter paper and was kept in the freezer (-20°C) before further analysis. Aliquots (1 or 2 ml) of samples and water (2-3 ml) were added to a clean screw-capped tube to bring the total volume to 4.0 ml for both standard and blank. 1.0, 2.0, and 3.0 ml of working standard solutions containing 10.0 /xg TMA/ml were used. The blank contained 4.0 ml of water. One ml of 10% formaldehyde, 10 ml of dried toluene and 3 ml of 25% KOH were added to the tube respectively and the mixture was blended in a vortex mixer for 15 seconds. About 7 ml of toluene (upper layer) was transferred to another tube containing 0.3-0.4 g of anhydrous Na SO and mixed with a vortex mixer again. Four ml of this toluene solution was transferred into a test tube containing 4 ml of 0.2% picric acid in toluene. The absorbance was measured in a Perkin Elmer Lambda 4B Spectrophotometer at 410 nm. The result was expressed in mg TMA/lOOg of fish tissue. b) Thiobarbituric acid (TBA) : TBA values were determined according to Vyneke (25) . Ten g of comminuted fish were mixed with 50 ml of 7.5% TCA, containing 0.1% propylgalate and 0.1% EDTA, for one minute. The homogenate was filtered through Whatman #2. One ml of extract was added to a screw-capped tube containing 5 ml TBA solution (0.2884 g of 2-Thiobarbituric acid in 100 ml of deionized water, boiled with agitation to dissolve the TBA and then cooled) and 4 ml of deionized water. Test tubes were placed in boiling water for about 40 minutes. The tubes were cooled and the absorbance was read at 538 nm in a Perkin Elmer Lambda 4 B spectrophotometer. TBA was expressed in mg malondialdehyde (MDA)/kg sample. Standard curve; 0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 2.0 ml of TEP working solution (1/100,000 dilution of 1,1,3,3-tetraethoxypropane or 9.121 jLtg/ml) were used in determining the standard curve. Both samples and standard solutions were treated in the same way. The c) Acidity was done in accordance to AOAC (2 6 ) . fish sample was comminuted and 5 g were weighed into a screwed-capped bottle and 100 ml of distilled water was added. The sample was blended well in a rotator for 30 minutes and filtered through Whatman paper #4. Ten ml of the mixture was put into a 250 ml Erlenmeyer flask and 100 ml of distilled water was added. The mixture was then swirled to mix. A few drops of phenolphthalein indicator
1459 were added, and the mixture was then titrated with 0.01 N NaOH to a persistent pink color. Distilled water was used as a blank, but to determine the proportion of acetic acid in the treated samples, fresh dogfish was used as a blank. Acidity was expressed as percentage of acetic acid. d) pH determination: The pH of the fish muscle was determined according to Wong et al. (27) by homogenizing 20 g of fish muscle with 40 ml of distilled water. As a general rule the ratio of water to fish muscle should be 2:1. The pH of the homogenate was measured by using the Fisher Accumet Model 910 pH meter and corrected to zero dilution according to the following formula: pH (O) = pH(D)-0.068D^'^ where pH (O)
= pH at zero dilution
pH (D) = pH of homogenate D = dilution ratio = vol. of water added to sample in ml wt. of sample in g e) Urea analysis: The procedure to determine the urea content was based on a modification of the AOAC (26) procedure. One gram of comminuted fillet was put into a 100 volumetric flask, containing 1 g of vegetable charcoal, 5 ml of 22% Zn(0Ac)2 . H2O, 5 ml of 10.6% K4Fe(CN)6 3 H2 0 and about 50 ml of distilled water was added to this mixture and shaken well for about 30 minutes. The flask was made up to volume and the solution was allowed to settle. The mixture was then filtered through Whatman paper #40. Five ml of the clear filtrate was transferred into a tube that contained 5 ml of 1.45% p-dimethylaminobenzaldehyde (Eastman Kodak Co. #95). The mixture was shaken well and allowed to stand for about 10 minutes at room temperature. A reference standard and a blank was included in the group. Samples were read at 42 0 nm on a Perkin Elmer Lambda 4 B Spectrophotometer. A standard curve was prepared using a reagent grade urea. f) Water activity: The water activity of fresh and treated dogfish was measured by using Beckman Humidat-ICI hygrometer. The instrument was calibrated with saturated sodium chloride and saturated potassium chloride. About 5 g of the comminuted sample was transferred into a small plate that was inserted to the cell of the hygrometer. Steady reading (equilibrium) was obtained after approximately 30 minutes. g) Moisture was determined according to the AOAC procedure (26) , and the Soxhlet method was used for fat determination. Physical analysis a) Heat penetration measurements were performed on the fish during the frying process, using Omega RD-TC
1460 thermocouple module for series RD 2000 recorder, Omega Engineering, Inc. Different weights of fish (200 and 400 g of fish), against a set volume of oil (1.9 liters) were tested when the probes were inserted at various depths in the fish (1, 2, 8-10, and 20 m m ) , and in the oil. The temperature changes were recorded until the temperature in the fish reached at least 100°C. b) Color measurements: The color of untreated and treated dogfish was determined by using the MINOLTA Chroma meter CR-200b. Sensory evaluation Sensory evaluation was conducted according to Meilgarrd et al. (28) after verifying the safety of the product by testing it for microbial counts and TMA. The 9-points hedonic test was used (l=dislike extremely, 9=like extremely) to compare between untreated dogfish, untreated cod, and treated dogfish which had been stored for a certain period of time. All samples were deep-fried in hot (175°C) corn oil for up to 10 minutes before they were served to a panel of fish users. Samples were evaluated for taste, texture, odor, appearance and general acceptance using a hedonic scale method. Statistical analysis was carried out using Microsoft Excel version 4.0 program. RESULTS AND DISCUSSION The multibarrier process used in this study to extend the shelf-life of dogfish is shown in Figure 1. The key barriers that have contributed to inhibiting deterioration were the hot oil treatment' and the subsequent dip in cold acetic acid. The hot oil treatment, which did not exceed 4 minutes at 175°C, was not sufficient to cook the fish flesh, but allowed the heat to penetrate and inhibit spoilage. The next barrier, the acetic acid dip, somewhat lowered the pH and also penetrated to some extend the fish flesh tissue, producing an additional inhibiting effect on the microorganisms. The vacuum packaging step, did not allow aerobes to grow and suppressed oxidation. The experimental data to follow show that the combined barriers of heat, acid, vacuum and refrigeration did not permit growth of aerobes or anaerobes over a period of at least one month, the bacterial load remaining at an insignificant or too low a level to count. The initial washing also contributed to lowering the initial population of microorganisms on the fish tissue. It must be borne in mind that the final product is only an intermediate product, which cannot be consumed as such. The product has to be treated as fresh raw fish before consumption, i.e. cooked to prepare a meal by frying, broiling, boiling etc. The last step should also contribute in destroying microorganisms that may have survived, or toxins that could have been produced.
1461 MULTEBAREUER PROCESS USED FOR EXTENDING THE SHELFLIFE OF DOGFISH Fresh fish
I
Skiiuiecl Prepared into backs,fillets,orportions Washed in water (Removal of urea) Heat treatment (Hot oil, 175 C)
I
Dipped in cold Acetic acid (5%) Drained
I I
Vacuum-packed
Stored at refrigeration terapei-ature
Dogfish contain which is quickly Figure 1.urea Flow cliart of the multibarrier process. converted to ammonia through microbial action. In order to eliminate that substrate adequate washing would remove about 60% of the urea in about 30 minutes (Table 1). This washing step also helps in reducing the initial microbial population by at least one logarithmic cycle. Table 1 The urea content of fresh dogfish fillets (1.5-2.0cm thick) and the percentage loss of urea from the meat after washing and soaking in cold water Time (min.) 0 15 30 60 90 120
% Urea 1.37±0.10 0.98±0.20 0.56±0.18 0.50±0.15 0.48±0.30 0.48±0.20
% Urea Loss 0 28.5 59.1 63.5 65.0 65.0
Average of two composite samples ± standard deviation. The hot oil treatment step was one of the critical steps in destroying the microorganisms. Figure 2 shows the effect
1462 of hot oil treatments alone on fish. The control sample of fish deteriorated after 6 days, the bacterial population being too high to count. Hot oil treatment of above 2 minutes was sufficient to extend the shelf-life of the fish for at least 25 days in the refrigerator. A 4 minute period (F.) was adopted for additional safety.
CO
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D
Time (days) Figure 2 . CFU/g of dogfish fillets, treated in hot oil for various times (1/4,1/2,1,2, 3, and 4 min.), vacuum packed, and stored at refrigeration temperature
Time (days) Figure 3. TMA-N of dogfish fillets, treated in hot oil for various times(l/4,1/2,1,2,3, and 4 min.), vacuum packed, and stored at refrigeration temperature
An i n d i c a t i o n of q u a l i t y or d e t e r i o r a t i o n i s t r i m e t h y l a m i n e (TMA) formation i n many f i s h s p e c i e s . Trimethylamine oxide (TMAO) i s converted t o TMA through facultative bacterial action. Hoogland (29) demonstrated the p o t e n t i a l use of trimethylamine (TMA) as an o b j e c t i v e quality indicator. For most f i s h t h e u s u a l s e n s o r y borderline of acceptable quality for cod and similar f i s h i s
1463 15 mg TMA-N per 100 g tissue (Dyer and Dyer, 30) . Levels above 15 mg TMA-N (1.08 micro moles TMA) in 100 gr of fish tissue are considered to be a sign of spoilage in fish (31). Figure 3 indicates the corresponding stability of the treated samples, with TMA levels being negligible for the hot oil treated samples. The surface pH (Figure 4) also did not rise as it did in the control and the samples that received a short hot oil treatment such as Fl/4. In this latter case the pH increase is mainly due to the formation of amines and other basic products.
5.
Time (days) Figure 4. Surface pH of dogfish fillets, treated in hot oil for various times (1/4,1/2,1,2,3 and 4 min.), vacuum packed, and stored at refrigeration temperature
The effect of hot oil on microbial growth shown above is due to the heat generated by the corn oil. Figures 5 and 6 show the heat penetration pattern. It takes a little over 4 minutes for heat to penetrate at a depth of one centimeter in the fish fillet producing a temperature of about 70®C. In about eight minutes the maximum temperature is reached, about 100**C, and this continues as long as moisture is lost from the surface as explained earlier in the introduction. The corn oil temperature was around 175*C. The surface layers of the fish flesh reached high temperatures relatively rapidly (Figure 6). Since the treated fish is to be consumed by cooking it further in some manner where heat is involved, it has been found that it takes about 4 minutes for the treated sample, when fried in hot oil to reach about 80®C. Normally a treated fish portion of that size would require a minimum of additional six minutes of frying, that is, the temperature in the middle of the fillet (8-10 mm depth) would reach about 100*>C. This high temperature for that period of heat treatment will destroy the vegetative cells and inactivate the toxins of Clostridium botulinum type E (32).
1464
s
I 4
Time (Min)
6
Figure 5. Heat penetration curve of the fresh washed dogfish heated in corn oil (175 C). Probe in the middle of the fish fillet: 8-10 mm depth.
Depth (mm) Figure 6. Time required for temperature to reach 60 C ,72 C and 100 C at different depths (mm) of dogfish fillets
1465 The next barrier to be studied independently of the other b a r r i e r s w a s t h e a c e t i c acid t r e a t m e n t . Different concentrations of acetic acid (1%, 3%, 5%) were used. The fish fillets were dipped for 2 m i n u t e s , in cold (lO^C) a c e t i c acid v a c u u m - p a c k e d and stored at refrigeration temperature. The concentration of 3% and 5% was efficient in controlling the growth of microorganisms as shown in Figure 7. The TMA-N levels reflected this inhibition of microbial growth (Figure 8) . Furthermore, the surface pH did not rise in the 3% and 5% treated samples, remaining c o n s t a n t between 5.0 to 5.8. However, in the 1% acid treated samples, the pH rose to a high level of above pH 8 (Figure 9) due to the products of deterioration formed.
Time (days)
Figure 7. CFU/g of Dogfish fUlets, dipped in various concentrations of Acetic acid (1%, S%, and 5%) for 2 min., vacuum packed and stored at refrigeration teno^rature. Vl% = Dip in 1% acetic acid for 2 min. V3% = Dip in 3% acetic acid for 2 min. V5% = Dip in 5% acetic acid for 2 min.
1466
Time (days) Figure 8. TMA-N /100 g of Dogfish fillets, dipped in various concentrations of Acetic acid (1%, 3%, and 5%) for 2 min., vacuum packed, and stored at refrigeration temperature Vl% = Dip in 1% acetic acid for 2 min. V3% = Dip in 3 % acetic acid for 2 min. V5% = Dip in 5% acetic acid for 2 min.
9n •
8-
8
I
pHVl%
• V3% • V5%
•
o
76-
bH
»
—•
•
1
—•
1
10
-r-^
1
Time (days) 20
0
30
Figure 9. Surface pH of Dogfish fillets, dipped in various concentrations of Acetic acid (1, 3, and 5%) for 2 min., vacuum packed, and stored in refrigeration temperature Vl% = Dip in 1% acetic acid for 2 min. V3% = Dip in 3% acetic acid for 2 min. V5% = Dip in 5% acetic acid for 2 min.
The dipping time in the acetic acid was deemed to be important. Figure 10 shows that a one minute dip in acetic acid is not adequate in inhibiting subsequent growth during refrigerated storage. Figure 11 also confirms the fact that the acid contributes to maintaining a lower pH for an extended period of time. The control sample of fish with no acid treatment reached a level of pH 7-8 in 10 days (samples spoiled), whereas the treated samples showed a constant level of pH between pH 5.0 and 5.4.
1467
Time (days) Figure 10. CFU/g of Dogfish fillets, under various dipping times (1,2,3, and 4 minutes) in 5% Acetic acid, vacuum packed, and stored at re&igeration temperature VI = Dip in 5% acetic acid for 1 min. V2 = Dip in 5% acetic acid for 2 min. V3 = Dip in 5% acetic acid for 3 min. V4 = Dip in 5% acetic acid for 4 min.
Xfl
TIME (days) Figure 11. Surface pH of dogfish fillets, under various dipping times (1,2,3, and 4 minutes) in 5% Acetic acid, vacuum packed, and stored at refrigeration temperature
1468 The experiments above dealt with fish samples that were treated in acetic acid, vacuum packed and refrigerated. The following set of experiments dealt with samples that were not vacuum-packed but stored under aerobic conditions at refrigeration temperatures. Figure 12 shows the effect of different concentrations of acetic acid. In this case concentrations of above 4% were able to inhibit growth in fish samples stored at refrigeration temperatures. Compared to the vacuum-packed samples (Figure 7) a 3% acetic acid concentration inhibited growth for at least 28 days, whereas under aerobic conditions a 3% acetic acid concentrations failed to inhibit growth (Figure 1 2 ) . Moreover, a 5 minute dip was necessary in this case (aerobic storage) to inhibit g r o w t h in 3 w e e k s s t o r a g e (Figure 1 3 ) . The aerobic microorganisms appear to be able to overcome the inhibitory effect of the acetic acid environment with time.
"S
f
Time (days)
Fissure 12. CFU/g of dogHsh backs (4 inches in length) dipped in different concentrations of acetic acid for 3 min., and stored under aerobic conditions at refrigeration temperature
1469
14 Time (days) Figure 13. CFU/g of dogfish backs (4 inches in length) dipped in 3 acetic acid for 1,3, and 5 min., stored under aerobic conditions at refrigeration temperature VI = Dip in 3% acetic acid for 1 min. V3 = Dip in 3% acetic acid for 3 min. V5 = Dip in 3% acetic acid for 5 min.
The processing parameters were finally optimized and it was found that following the initial washing, a four minute hot oil treatment (175«»C) , followed by a cold (lO^C) acetic acid (5%) dip for 2 minutes gave the best combination of barriers and an extended shelf-life for at least one month at refrigeration temperature of the vacuum-packed dogfish samples. Bacterial counts were too low to count for even up to three months storage at refrigeration temperature and the TMA level was low (1.30 mg TMA-N per 100 gr of fish) after 30 days storage. It is important to note the pH pattern appearing in the fish samples which had been treated (hot oil, 175®C for 4 minutes, and a dip in 5% acetic acid for 2 minutes and subsequently vacuum packed). Table 2 and Table 3 depict the difference in the pH change of the control (untreated) fish samples and the treated samples. The control samples, as expected, rose from pH 6.0 to pH 8.0, whereas the treated samples remained at a constant level of about pH 5.5 for a period of 30 days. A fresh fish sample will spoil in about six to nine days at refrigeration temperature and its pH would also rise to pH 8.0. On the other hand, the acidity concentration expressed as acetic acid in the treated samples remained around the initial level of about 0.35% throughout the storage period.
1470 Table 2 pH change with time at refrigeration temperature of fresh washed dogfish fillets Time (days)
pH
0 3 6 8 10 20
5.9 6.0 6.0 6.0 7.0 8.0
Table 3 pH change with time at refrigeration temperature of treated dogfish fillets (F4V2) Time (days) 0 5 10 14 20 25 30
pH 5.4 5.5 5.5 5.5 5.5 5.4 5.3
F4V2 = Hot oil treatment (175<*C) for 4 min and a dip in 5% acetic acid for 2 min, vacuum packed. Thiobarbituric acid (TBA) studies were conducted to assess the oxidation process that the fish samples had undergone. The untreated control samples which were stored at refrigeration temperature showed a rapid increase in TBA in comparison to the treated samples stored at refrigeration temperature and those untreated samples which were frozen or just vacuum-packed and refrigerated (Figure 14). According to Regenstein and Regenstein (31) a value of around 4 mg MDA/kg is considered low for TBA in fish. The treated samples maintained a value of about 5 mg MDA/kg over a period of 30 days at r e f r i g e r a t i o n t e m p e r a t u r e . Consequently, little, if any, rancidity occurred during the storage period of 30 days at refrigeration temperature. As expected vacuum packaging reduced greatly the oxidative rancidity process.
1471
I
10
20
30
40
TIME(days) Figure 14. TBA change with time (expressed as mg MDAyKg of d o ^ s h ) of various samples. 1. untreated dogfish fillets stored at refi*igeration temperature (4 C) 2. untreated dogfish fillets stored at fi'eezer temperature (-23 C) 3. untreated dogfish fillets vacuum packed stored at refi-igeration temperature (4 C) F4V2. treated dogfish fillets in hot corn oil (175 C) for 4 min then dipped in 5% acetic acid, vacuum packed and stored at refii-igeration temperature (4 C)
The ultimate goal of such a process is to produce a safe product which would have an extended shelf-life at refrigeration temperature and vary little from its original fresh fish characteristics. It should be borne in mind that this product is to be handled as "fresh" fish and cooked into a meal as "fresh" fish. A color study was conducted to see whether there were any significant changes between the untreated (fresh fish) and treated fish samples (F4V2) and also to determine any color changes with time in the treated samples. Whiteness and yellowness was determined. The whiteness value was the same in both type of samples (Figure 15) and remained at the same level during the storage time. However, the treated samples showed a higher yellowness value (Figure 16) , and that value was more or less maintained throughout the storage period. The yellowness was mainly due to the hot oil treatment in the initial steps of the process.
1472 100 80 0)
60 401 85.9
83.4
83.1
83.6
84
84.7
0 5 10 Treated (F4V2)
14
21
25
30
83.4
83.7
20 H
00 Untreated
Time (Days) Figure 15. Color change (L* value -whiteness) with time at refrigiration temperature of untreated and treated (F4V2) dogfish fillets
20
I
10 H 13.5
13.8
15.3
15.1
15.3
5 10 Treated (F4V2)
14
21
25
30
12.8
*
12.8
6.55
0 Untreated
0
Time (Days) Figure 16. Color change (B* value-yellowness) with time at refrigeration temperature of untreated and treated (F4V2) dogfish fillets
1473 T h i s p r o c e s s d i d n o t c a u s e any r e d u c t i o n i n t h e w a t e r a c t i v i t y (aw ) . The aw d i d n o t c h a n g e i n t h e t r e a t e d s a m p l e s (F4V2) i n c o m p a r i s o n t o t h e u n t r e a t e d c o n t r o l s a m p l e s (aw = 0 . 9 8 5 ) . The minimum aw f o r g r o w t h i s a b o u t 0.90 for pathogenic b a c t e r i a (33). The t r e a t e d p r o d u c t r e m a i n e d m o i s t b u t had a lower m o i s t u r e c o n t e n t a s compared to the untreated sample. As w a t e r w a s l o s t , o i l was a b s o r b e d i n t o t h e f i s h t i s s u e s from t h e c o r n o i l medium (Table 4 ) .
Table 4 Moisture and fat content of dogfish fillets Sample Fresh-^ Treated^ Outer^ Inner 1 2 3 4
^2^^ 74.18±0.44 67.26±0.72 65.07±1.65 68.86±0.36
Fat% 6.63±0.63 15.3610.24 17.16±1.61 14.36±1.01
Fresh corresponds to the fresh untreated dogfish. Treated corresponds to the F4V2 process. Outer corresponds to the outer layers of the processed dogfish (F4V2) fillets (1/4 cm). Inner corresponds to the inner layers of the same processed dogfish fillets.
Taste panel tests were conducted on control samples (frozen dogfish, fresh cod, fresh dogfish) and on the treated dogfish (F4V2) which had been stored under refrigeration temperature for 18 days. This product was compared to the other forms of fish above (Figure 17) . There was no significant difference regarding taste among all the four types of seafood. As for texture and odor the panel results did not show any significant difference between the treated dogfish and the fresh dogfish. Cod appeared to be significantly different from all the other samples when appearance was evaluated. As for general acceptability there was no significant difference among all samples. When the baking method of preparation was used, again there was no significant difference among the samples (Figure 18). The acetic acid imparts a slight tart flavor to the product which to most fish users is acceptable and preferred. It can be concluded that treated dogfish, using the above process, can be utilized as fresh fish in preparing various dishes and it is equally acceptable as fresh dogfish. Ongoing research on other fish species (skates, squid) utilizing this process appears to work well, the barrier parameters being different for each species.
1474
H • B 0 E3
Taste Texture Odor Appearance General Accept
H • B 0 •
Taste Texture Odor Appearance General Accept.
Xfl
Frozen dogfish
Fresh dogfish
Treated dogfish
Figure 17. Sensory evaluation of cod & dogfish (fried) after 18 days of storage at refirigeration temperature Treated dogfish = Hot oil (175 C) treatment for 4 min. and dip in 5% Acetic acid for 2 min.
fresh dogfish
Frozen dogfish
Treated dogfish
Figure 18. Sensory evaluation of cod & dogfish (broiled) after 18 days of storage at refrigeration temperature Treated dogfish = Hot oil treatment for 4 min. and dip in 5% Acetic acid for 2 min.
1475 CONCLUSIONS The shelf-life of seafood can be extended to at least one month at refrigeration temperature by utilizing a multibarrier process. The process consisted of a four minute hot oil (175**C) treatment in a deep fryer followed by a two minute dip in a cold (10**C) 5% acetic acid and then vacuum packaging before storage at refrigeration temperature. The bacterial count was too low to count after one month of storage. The TMA levels also remained low, 1.30 mg TMA-N per 100 gr of fish, during storage. The pH did not rise, it remained at about 5.5 throughout. The heat penetration into the fish tissue was rapid. A temperature of 72®C was reached in about 4.5 minutes at a depth of one centimeter of fish tissue, and within eight minutes the temperature was 100®C at that point. Color (whiteness and yellowness) showed little change with time of storage. Taste panel tests revealed that the treated product, after eighteen days of storage at 3°-4°C, was acceptable and compared well with fresh samples of the same species and with other popular fish species such as cod. ACKNOWLEDGMENTS This study was partially funded by the New England Fisheries Development Association through a grant by the Saltonstall-Kennedy Program. Fish were provided by Sea Fresh USA, Inc., Narragansett, RI. , by The Town Dock, Galilee, R.I., and by Family Fisheries Corp., New Bedford, MA.
1476 REFERENCES
10 11
12 13 14
15 16
El-Hag, N, The refrigerated food industry; current status and developing trends. Food Technol., 43 (1989) 96. Moberg, L., Minimally processed refrigerated foods: other methods of preservation, with emphasis on combined methods and synergistic interactions, In: Proc. 1989 IFT Short Course, Minimally Processed Refrigerated Foods, Continuing Education Comm. IFT and Refrigerated and Frozen Foods Division, IFT, Chicago, (1989) p 41. Leistner, L., In: Food Quality and Nutrition, Ed. W.K. Downey, Applied Science Publishers, London (1978) 553. Gould, G.W., In: Mechanisms of action of food preservation procedures, ed. G.W. Gould, Elsevier Applied Science, London, (1989) p 1. Genigeorgis, C , Principles of preservation by refrigeration, modified atmosphere and control of water activity. In: Proc. 1989, IFT Short Course, Minimally Processed Refrigerated Foods, Continuing Eduation Comm. IFT and Refrigerated and Frozen Foods Division, IFT, Chicago, (1989) p 13. Sorvells, K.M., D.C. Enigl and J.R. Hatfield., J. Food Protection, 52 (1989) 571. Collins, J.L. and A.K. Yu, Stability and acceptance of intermediate moisture, deep-fried catfish. J. Food Science, 40 (1975) 858. Blumenthal, M.M., A new look at the chemistry and physics of deep-fat frying. Food Technol., 45 (1991) 68. Lydersen, A.L., In: Mass Transfer in Enginering Practice, (1983) John Wiley and Sons, N.Y. Rhodehamel, E.J., FDA's concerning with sous vide processing. Food Technol., 46 (1992), 73. Conner, D.E., V.N. Scott, D.T. Bernard and D.A. Kautter, Potential Clostridium botulinum hazards associated with extended shelf-life refrigerated foods: A review. J. Food Safety, 10 (1989) 131. Cann, D . C , B.B. Wilson, G. Hobbs and J.M. Shewan, The Growth and toxin production of CI. Botulinum in certain vacuum packed fish. J. Appl Bacterid., 28 (1965) 431. Kautter, D.A., CI. botulinum type E in smoked fish, J. Food Sci., 29 (1964) 843. Solomon, H.M., R.K. Lyne, T. Lilly Jr., and D.A. Kautter, Effect of low temperature on growth of CI. botulinum in meat of the blue crab. J. Food Prot., 40 (1977) 5. Stier, R.F , Bell, L., K.A. Ito, B.D. Shafer, L.A. Brown, M.L Seeger, B.H. Allen, M.N. Porcuna and P. Lerke. J. Food Sci., 46 (1981) 1639. Tsang, N., L.S. Post and M. Solberg, Growth and toxin production by CI botulinum in model acidified systems. J. Food Sci., 50 (1985) 961.
1477 17
18 19 20 21
22 23 24
25
26 27 28 29
30 31 32 33
Ikawa, J.Y., and C. Genigeorgis, Probability of growth and toxin production by non-proteolytic CI. botulinum in rockfish fillets stored under modified atmospheres. Intern. J. of Food Microbiology, 4 (1987) 167. Hauschild A.H.W., CI. botulinum In: Food Borne Bacterial Pathogens, ed. M.P. Doyle. Marcel Dekker, N.Y. (1980) p 111. Schwarz, T.L., FDA position on CAP/MAP production and storage. Presentaton at the Food Safety Pack, 1989, Chicago. Schmidt, C.F., R.V., Lechowick and J.F. Folinazzo, Growth and toxin production by type E Clostridium botulinum below 40°F. J. Food Sci., 26 (1961) 626. Hotchkiss, J.H., Shelf-life of minimally processed refrigerated foods. In: Proc. 1989 IFT Short Course, "Minimally Processed Refrigerated Foods", Continuing Education Comm. IFT and Refrigerated and Frozen Food Division, IFT, Chicago, (1989) p 137. Jhaveri, S.N. and S.M. Constantinides. Chemical composition and shelf-life study of grayfish (Squalus acanthias). J. Food Sci., 47 (1982) 188. Post, F.J., In: A laboratory manual for food microbiology and biotechnology. Star Publishing Co., Belmont, CA, p 7 (1988). Woyewoda, A.D., S.J. Shaw, P.J. Ke, and B.G. Burns, Recommended laboratory methods for assessment of fish quality. In: Canadian Technical Report of Fish and Aquatic Science, No. 1448; (1986) p 35. Vyneke, W., Evaluation of the direct thiobarbituric acid extract method for determining oxidative rancidity in Mackerel (Scomber Scombrus). Sonderdruckand Fiette Siefes Anstrichemittel, 77(1975) 239. AOAC, In: Official Methods of Analysis, 14th Ed. Association of Official Analytical Chemists, Washington, D.C. (1985). Wong, R., G. Fletcher, and J. Ryder, Manual of Analytical Methods for seafood research. In: DSIR Crop Research Seafood Report No. 2 (1991) p 36. Meilgaard, M., V.C. Gail, and B.T. Carr, In: Sensory evaluation techniques, 2nd ed. CRC Press, Inc. (1991). Hoogland, P.L., Grading fish for quality II. Statistical analysis of the results of experiments regarding grades and trimethylamine values. J. Fish Res. Bd. Can. 15 (1953) 717. Dyer, F.E. and W.J. Dyer, Changes in the palatability of cod fillets. J. Fish Res. Bd. Can. 7 (1949) 449. Regenstein, J.M. and C.E. Regenstein. In: Introduction to fish technology, Osprey Book Van Nostrand Reinhold, NY. (1991) p 67. Licciardello, J.J., T.R. Nickerson, C.A. Ribish, and S.A. Goldblith, Thermal inactivation of type E botulinium toxin. Appl. Microbiol. 15 (1967) 249. Russel, N.J. and G.W. Gould, Factors affecting growth and survival. In: Food preservatives. Van Nostrand Reinhold, NY, (1991) p 13.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1479
FRESH ORANGE JUICE FLAVOR: A QUANTITATIVE AND QUALITATIVE DETERMINATION OF THE VOLATILE CONSTITUENTS. MANUEL G. MOSHONAS and PHILIP E. SHAW U.S. Citrus and Subtropical Products Laboratory, 600 Avenue S, N.W. (P.O. Box 1909), Winter Haven, Florida 33883-1909 (USA) SUMMARY The volatile constituents responsible for the highly desirable flavor of fresh orange juice were qualitatively and quantitatively analysed using a dynamic headspace gas chromatographic system. Forty-nine volatile constituents were identifled in thirteen orange juice samples extracted both mechanically and by hand from six different cultivars that included Valencia, Hamlin, Pineapple, navel and Ambersweet. Forty-six of these constituents were quantified. The data generated from these analyses provide the most extensive database available for volatile constituents in fresh orange juice. INTRODUCTION The desirable flavor of fresh orange juice has made it the most popular of all fruit juices. This delicate flavor is widely believed to be due to a complex mbcture of numerous volatile constituents that have an interdependent quantitative relationship. The complexity, physical characteristics and varying concentrations of the volatile flavor and aroma components of fresh orange juice have made it difficult to identify and ^South Atlantic Area, Agricultural Research Service, U.S. Department of Agriculture. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.
1480 quantitate these constituents. However, since this group of interdependent compounds and their quantitative relationships produce the dehcate fresh orange juice flavor, both the identity and the quantity of each constituent must be determined if we hope to unravel nature's formula for this highly desirable juice flavor. Other researchers have reported fresh orange juice volatiles (Schreier et al, 1977, 1979; Schreier, 1981; Sauri et al., 1980; Rodriguez and Culbertson, 1983; Shaw, 1991; Nisperos-Carriedo and Shaw, 1990; Lum et al, 1990; Park and Venables, 1991; Moshonas and Shaw, 1987, 1994). These studies have resulted in the identification of most of the flavor constituents.
Unfortunately the
methodology used to separate and identify these compounds resulted in losses that made it extremely difficult to obtain the quantitative data needed to produce an accurate and complete database of orange juice flavor constituents. In order to evaluate and understand how this complex mixture of flavor volatiles produce fresh orange juiceflavor,accurate quantitative information on these volatiles must be obtained. In accomplishing this objective, consideration must be given to expected quantitative differences among cultivars and between fresh juices being produced both by mechanical and hand extraction. Taking all of these parameters into consideration will lead to data showing a range of quantitative values for each fresh orange juice volatile. This will serve as a database for comparison with similarflavorconstituent databases from processed orange juice products, thus making it possible to determine changes inflavorquality due to processing, storage, the addition of various natural flavor fractions or other factors that might have an effect on flavor. Quantitative analyses of volatile flavor constituents in fresh orange juice were
1481 conducted in our laboratory and others.
However, most of these analyses employed
preparatory steps such as solvent extraction or liquid chromatography followed by removal of solvent by evaporation which caused variable losses in the quantity of each volatile constituent. The analyses were then completed using gas chromatographic (GC) methods. These methods not only incur partial losses of volatile components but are difficult to carry out when large numbers of juice samples must be analysed. Furthermore, in previous studies, quantative data on orange volatiles were reported either from analyses of one to two samples such as that from Schreier et al (1977), or on only a few volatile orange constituents such as the GC analysis reported by Pino (1982) who quantified seven constituents and Rodriguez and Culbertson (1983) who quantified eight constituents , each involving a single sample of fresh orange juice. Recent studies of fresh orange juice volatiles at our laboratory have also yielded quantitative data. Moshonas and Shaw (1987) analysed one sample each of fresh Temple and Valencia orange juice and quantified 24 constituents. In 1990, Nisperos-Carriedo and Shaw analysed fifteen orange juice samples and quantified 20 constituents using static headspace gas chromatography. Lizotte and Shaw (1992) also used static headspace analysis and quantified 22 constituents in several orange juice samples. Moshonas and Shaw (1992) analysed four orange juice samples using both static and dynamic headspace gas chromatography and quantified 16 constituents and Shaw et al (1993) used static headspace analysis to quantify 19 volatile constituents in four fresh orange juice samples. Although the use of gas chromatographs equipped with static headspace injectors alleviated the problem of losing a portion of the volatile constituents experienced in extraction or column methods.
1482 we found that the relatively small sample delivered by the static headspace injector allowed only a limited number of volatile orange juice constituents to be quantified. Moshonas and Shaw (1994), using a GC/dynamic headspace injector system to analyze fresh orange juice samples, identified and quantified 46 volatile flavor constituents, thereby providing the most extensive database yet determined for volatile constituents present in fresh orange juice. The dynamic headspace injector/GC system was an important contributor in allowing a large number of fresh orange juice volatile flavor constituents to be quantified while coincidently eliminating the possibility of component losses during the analysis.
DYNAMIC HEADSPACE INJECTOR/GAS CHROMATOGRAPHY SYSTEM The Chrompack Purge and Trap Injector (Raritan, NJ) used at our laboratory had an advantage over dynamic headspace systems using a packed collection trap when samples such as orange juice which contain a high soluble solids content, were analysed. The Chrompack system uses compact open tubing construction with a section of open fused silica capillary as a trap. The trap can be cooled to any temperature approaching that of liquid nitrogen and leaves no residual sample that can be carried to subsequent analyses after the gas chromatographic run. When a dynamic headspace injector using a packed trap was employed in numerous attempts to analyze orange juice samples, the trapping system was contaminated by juice soluble solids. This caused a carry over of volatile constituents from sample to sample, thus precluding accurate quantitative analysis of juices. There is also a notable advantage in our dynamic headspace method over the static headspace injector system. In the dynamic system, sampling occurs over a time period chosen by the analyst
1483 which allows for the collection of sample adequate for a complete and thorough analysis of a large number of volatile constituents. Typically up to a 10 minute collection period can be employed. The upper time limit is determined by the fact that eventually sufficient water vapor will be collected as ice in the cooled portion of the trap and block gas flow through the system described below. The systems using static headspace injectors, pressurize the sample vial followed by a release of pressure to force a controlled volume of the headspace gases onto the GC column. Heating the juice sample to a higher temperature and increasing vial pressure are the two parameters that can increase sample size during injection, but the heating temperature is limited by the boiling point of the major component, water, and pressurization is limited by the construction of the pressurized vial. This relatively low sample size limits the number of volatile constituents that can be analysed. Orange juice samples analysed using a static headspace injector system had to be heated to 80°C for 15 min equilibration to provide an acceptable but limited sample injection, (Shaw et al, 1991). Lowering the sample temperature limited volatilization and reduced the number of constituents that could be analysed. In our latest investigation of fresh orange juice flavor volatiles, fresh orange juice samples were analysed with a Hewlett-Packard Model 5890 gas chromatograph equipped with a Chrompack Purge and Trap Injector. A 30 m x 0.53 mm i.d. HP-5 capillary column with 2.65 fim film thickness (Hewlett Packard, Wilmington, DE) was employed with both the FID detector and injection port at 250°C. Temperature was held at 40°C for 6 min, then programmed at 6°C min'^ to 200°C final temperature. Column flow rate was 8ml/min. Peak
1484 areas were used for integration of each component. The system for purging and cryofocusing components on the cold capillary trap is shown schematically in Fig. 1. A 5 ml juice sample was placed in sample flask 1 and the flask was equilibrated at 40°C during the trapping sequence. A helium flow purge of 18 ml/min was passed over the sample via a needle (2) for 6 min. The entrained juice volatiles and water vapor passed through condenser 3 cooled to 0°C to remove most of the water in order to avoid blockage of the second cold trap by ice crystals. The flow passed through a glass tube (4), heated at 120°C to prevent component condensation. Volatiles were then cryofocused on the capillary tubing cold trap (5). The temperature was maintained at 130°C by a stream of liquid nitrogen vapors, entering the compartment from twin openings 6. Once the sample was collected, the cold trap was flash heated to 250°C and the sample was injected into the gas chromatograph.
The trap and purge sequences were fully
automated.
QUALITATIVE AND QUANTITATIVE ANALYSIS OF FRESH ORANGE JUICE VOLATILE CONSTITUENTS Volatile flavor constituents found in orange juice originate from three sources. The juice contained in the juice sacs which is released during extraction is the source for the volatile water-soluble compounds. Two types of oil, juice oil and peel oil, contribute the oilsoluble compounds to the flavor of fresh orange juice. Juice oil is present in globular bodies within the juice sacs and it becomes dispersed in the juice during extraction. In 1952, Rice et al demonstrated the presence of about 0.005% juice oil in juice extracted from fruit that
1485 Figure 1. Schematic of dynamic headspace injector
He
1486 had been carefully hand-peeled before extraction to exclude the presence of peel oil. Hand extraction of unpeeled oranges introduced peel oil into the juice, in addition to juice oil, and doubled the amount of total oil present. Commercial orange juice typically contains about 0.015-0.025% total oil (Kimball, 1991), thus making peel oil the major source for oil components in mechanically extracted juices. In a recent investigation at our laboratory (Moshonas and Shaw, 1994) forty-nine volatile flavor constituents were identified in thirteen fresh orange juice samples extracted both mechanically and by hand from six different cultivars that included Valencia, Hamlin, Pineapple, navel and Ambersweet. Forty-six of these constituents were quantified in each juice thus providing the data necessary to determine a range of concentrations for each of these volatile constituents in fresh orange juice. Volatile orange juice flavor constituents were separated and identified by a gas chromatographic/mass spectrometer (GC/MS) system. A Hewlett-Packard Model 5970B MSD, GC-MS, was used with a 0.32 mm x 50 m fused silica column of crossed-linked 5% phenylmethyl silicone. Column oven temperature was held at 55°C for 9 min, raised at 7.5°C/min to 220°C and held there for 30 min. Injection port and ionizing source were kept at 275°C, and the transfer line was kept at 280°C. Mass spectral matches were made by comparison of mass spectra and retention times with those of authentic compounds. Retention times of components were also compared with those of standards prepared above and by enrichment of juice with authentic samples followed by analysis using the headspace GC system described above. Table 1 lists the forty-nine volatile constituents identified in fresh orange juice. All
1487 of the compounds had been identified earUer as constituents of orange juice, peel oil or other natural orange flavor fractions (Maarse and Visscher, 1989). Prior to quantitative analysis of the volatile constituents of freshly squeezed orange juice, a standard peak area calibration curve was determined for all 46 identified volatile constituents that were to be quantified in fresh orange juice. Concentrations for each compound were calculated with regression equations ascertained from headspace analyses data on four different concentrations of each constituent added to a bland orange juice reconstituted to 11.8 °Brix from a concentrated orange juice (pumpout) that contained no flavor constituents (except limonene). Each standard solution was kept for 3 hours at room temperature and then overnight at 5°C to permit equilibration of the hydrocarbon standards between pulp and juice (Shaw et al, 1994b). This equilibration period afforded more precise results for the oil-soluble components than when no equilibration period was used, but had no effect on the precision of the water-soluble components (Shaw et al, 1994a). Quantitative analysis of the fresh juice flavor volatiles was then completed using the dynamic headspace injector/GC system described above. Quantitative values (in ppm) for each component in thirteen fresh orange juice samples are listed in Table 1. Most of the compounds listed in Table 1 are water soluble constituents originating in the aqueous portion of the juice sacs. Comparison of quantities of these constituents found in hand expressed and mechanically expressed Pineapple and Valencia juices shows little difference between amounts present.
Water soluble constituents evaluated and
considered important to orange juice flavor include methyl butanoate, ethyl butanoate, ethyl acetate, ethyl propionate, ethyl 3-hydroxyhexanoate, (Z)-3-hexen-l-ol and ethanol (Shaw,
1488 1991). A comparison of mechanically expressed and hand expressed juices shows the contribution of peel oil from mechanically expressed juices results in a higher concentration of seventeen oil soluble juice constituents in those samples. Nine of these constituents are considered to make important contributions to orange juice flavor (Shaw, 1991). These are limonene, a-pinene, myrcene, neral, geranial, linalool, octanal, nonanal and decanal. However, higher levels of these compounds in orange juice are not necessarily better for good fresh orange flavor. The contribution to juice flavor of the remaining eight oil soluble constituents, octanol, carvone (J^)-linalool oxide, a-phellandrene, 5-3-carene and y-terpinene has not been determined. The information shown in Table 1 provides the most complete database yet determined for quantities of volatile flavor constituents present in fresh orange juice. It shows for each of the 46 components a range of values that might be expected in fresh orange juice. In addition, it indicates the relatively large effect mechanical extraction of juice has on the quantities of oil-soluble volatile components, and shows the relativley minor effect it has on water-soluble juice components. Such quantitative databases on citrus juice volatile components are an essential part of computerized multivariate analysis studies recently being used to evaluate citrus juice products (Shaw et ah, 1993, 1994a, 1994b).
Table 1 .
Volatile Constituents Identified and Quantified (Parts per Million) in Fresh-Squeezed Orange Juice. Valencia mech"
~0mponent' methanole ethanol l-propanol ethyl acetate 2-methyl-3-buten-2-01 2-methylpropanol butanol l-penten-3-01 l-penten-3-one 2-pentanol ethyl propionate methyl butanoate 3-methyl butanol 2-methyl butanol l-pentanol 3-methyl-2-buten-1-01 ethyl butanoates (E)-2-hexenal (Z)-3-hexen-1-01" hexanol heptanal a-p inene sabinene myrcene ethyl hexanoate octanal e-phellandrene b-3-carene
handC
mech A
56 460 0.20 0.15 0.18 0.026 0.026 0.034 0.029 0.11 0.028 0.019 0.12 0.018 0.019 0.034 0.70 0.037 0.15 0.048 0.0006 1.09 0.051 4.1 0.076 0.65 0.029 0.094
Pineapple hand
B 85 730 0.38 0.23 0.32 0.11 0.027 0.15 0.047 0.13 0.016 0.027 0.39 0.067 0.031 0.052 0.89 0.058 0.66 0.29 tr 0.16 0.017 0.58 0.076 0.0025 0.009 0.003
C 51 580 0.25 0.17 0.22 0.024 0.026 0.12 0.018 0.11 0.010 0.011 0.10 0.012 0.026 0.018 0.82 0.032 0.48 0.17 tr 0.13 0.018 0.44 0.070 0.0027 0.008 0.003
navel, handd A B A B 3 31 36 78 240 330 22 890 0.16 0.19 0.050 1.14 0.12 0.13 0.077 0.17 0.047 0.21 0.11 0.13 0.015 0.069 tr' 0.10 0.027 0.024 0.006 0.22 0.004 0.064 0.066 0.021 0.015 tr 0.008 0.023 0.02 0.02 0.14 0.14 0.003 0.0036 0.023 0.028 0.018 0.033 0.0001 0.008 0.074 0.22 0.021 0.15 0,009 0,029 tr 0.025 0,021 tr tr 0.021 0.071 0.048 0.020 tr 0.70 tr 0.43 0.95 0.015 0.010 0.011 0.018 0.16 0.54 0.26 0.30 0.067 0.096 0.005 tr tr tr tr tr 0.25 0.43 0.13 0.13 0.16 0.26 0.015 0.015 0.48 0.49 1.06 1.90 0.0087 0.24 0.086 0.13 0,0041 0,0041 0.056 0.086 0,008 0.008 0.011 0.018 ND' ND 0.019 0.059 Hamlin. hand
Pera, hand 7 760 0.31 0.28 0.41 0.008 0.015 0.11 0.016 0.066 0.017 0.003 0.0004 0.0027 0.023 0.11 1.03 0.018 0.47 0.26 tr 0.29 0.079 1.09 0.14 0.0019 0.014 0.008
Ambersweet mech hand B A 7 76 22 415 0.077 0.16 0.082 0.12 0.057 0.11 0,0010 0.046 0.012 0.031 0.064 0.081 0.11 0.092 0.21 0.24 0.008 0.021 0.006 0.028 0.007 0.33 0.002 0.037 0.031 0.045 tr 0.096 0.85 0.81 0.005 0.012 0.84 0.34 0.039 0.066 0.002 tr 1.03 0.39 0.19 0.023 4.0 1.5 0,049 0.040 0.037 0.0058 0.022 0.012 0.003 ND
Table 1
ContLnued Valencia mechb
componenta 1imonene 13-oc imene 7-terpinene octanol (Z)-linalool oxide (H)-linalool oxide linalool nonanal ethyl-3-hydroxy-hexanoate ethyl octanoate terpinene-4-01 decanal a-terpineol neral carvone geranial perillaldehyde valencene
A 76 0.025 0,010 0.13 0.048 0.081 0.75 0.022 0.27 0.035 0.11 0.16 0.19 0.0005 0.058 0.0005 0.034 2.1
B 134 0.072 0,010 0.37 0.11 0.14 0.92 0.082 0.49 0.023 0.20 0.45 0.91 0.028 0.11 0.035 0.097 4.4
handC C 18 0.018 0.002 0.089
mech
Pineavple hand
Hamlin, hand
navel, hand"
A
B
B
A
B
29 0.023 0.003 0.080
C 24 0.027 0.003 0.086
A
24 0.026 0.004 0.078
24 0.030 0.030 0.073
ND
ND
ND
ND
0.022 0.13 0.001 0.28 0.031
191 0.28 0.013 0.46 0.14 0.30 3.7 0.087 0.39 0.20
ND
ND
0.016 tr tr 0.004 tr 0.012 3.30
0.50 tr 0.0018 0.069 0.0019 0.060 5.1
0.038 0.033 0.003 0.35 0.009 0.18 0.022 tr 0.0004 0.012 0.0003 0.003 3.6
0.033 0.053 0.003 0.32 0.008 0.17 0.022 tr 0.001 0.016 0.0013 0.004 2.4
0.031 0.016 0.004 0.32 0.010 0.17 0.021 tr tr 0.018 0.012 0.004 5.1
0.036 0.013 0.003 0.34 0.008 0.15 0.019 tr 0.0004 0.020 0.0004 0.003 1.6
43 0.020 0.003 0.166 0.012 0.054 0.17 0.007 tr 0.006 0.071 0.057 tr 0.023 tr 0.033 0.034 1.2
62 0.040 0.002 0.23 0.014 0.15 0.47 0.025 tr 0.010 0.14 0.29 0.18 0.023 tr 0.032 0.016 3.7
ND
'Listed in increasing retention order on a nonpolar capillary GC column DMech, mechanically expressed with an FMC in-line extractor. %and, hand expressed with an electric kitchen juicer. 'Sample A, Florida navel; sample B, California navel. 'A minor mount of acetaldehyde coeluted with this component. 'tr, trace, detected but too small to quantify. QA minor mount of hexanal coeluted with this component. hA minor mount of (I?)-2-hexenol coeluted with this component. 'ND = not detected.
Pera, hand 53 0.017 0.004 0.080 0.0078 0.047 0.21 tr 0.33 0.063
ND 0.057 tr 0.007 0.013 0.004 0.069 12.1
Ambersweet mech hand A
167 0.058 0.004 0.083 0.011 0.078 1.02 0.025 0.34 0.010 0.10 0.18 3.7 0.020 0.10 0.022 0.010 6.5
B
65 0.028 0.004 0.075 0.017 0.033 0.39 0.007 0.38 0.015 0.085 0.043 0.13 tr 0.10 tr 0.022 0.83
1491 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Kimball, D. Citrus Processing Quality Control and Technology, Van Nostrand Reinhold, New York, 1991, pp 73-101. Lizotte, P.A.; Shaw, P.E. Flavor volatiles in Valencia orange and their quantitative changes caused by vacuum infiltration of butanal. Lebensm,-Wiss. u-TechnoL 1992, 25, 80-82. Lum, O.L.; Wang, M.K.; Lee, C.K. A simple headspace-gas chromatographic method for quantitative determination of organic volatiles of fresh orange juice. Food Chem. 1990, 37, 313-317. Maarse, H.; Visscher, C.A., Eds., Volatile Compounds in Food, 6th Editioru Qualitative and Quantitative Data, Vol. 1, TNO-CIVO Food Analysis Institute, Zeist, The Netherlands, 1989. Moshonas, M.G.; Shaw, P.E. Quantitative analysis of orange juice flavor volatiles by direct-injection gas chromatography. /. Agric, Food Chem. 1987, 35, 161-165. Moshonas, M.G.; Shaw, P.E. Comparison of static and dynamic headspace gas chromatography for quantitative determination of volatile orange juice constituents. Lebensm.'Wiss. u-Technol 1992, 25, 236-239. Moshonas, M.G.; Shaw, P.E. Quantitative determination of 46 volatile constituents in fresh, unpasteurized orange juice using headspace gas chromatography. /. Agric. Food Chem. 1994, 42, 1525-1528. Nisperos-Carriedo, M.O.; Shaw, P.E. Comparison of volatile flavor components in fresh and processed orange juices. /. Agric. Food Chem. 1990, 38, 1048-1052. Park, J.S.; Venables, A.C. Analysis of packaged orange juice volatiles using headspace gas chromatography. /. Chromatogr. 1991, 540, 456-463. Pino, J. Correlation between sensory and gas-chromatographic measurements on orange volatiles. Acta Aliment, 1982,11, 1-9. Rice, R.G.; Keller, G.J.; Beavens, E.A. Flavor fortification of California frozen orange concentrate. Food Technol. 1952, 6, 35-39. Rodriguez, P.A.; Culbertson, C.R. Quantitative headspace analysis of selected compounds in equilibrium with orange juice. In: Instrumental Analysis of Foods; Charalambous, G., Inglett, G., Eds.; Academic Press; New York, 1983; p. 187. Sauri, E.; Nadal, I.; Alberola, J.; Sendra, J.M.; Izquierdo, L.J. Study on the aromatic evaction of orange juice. II. Comparison among vacuum distillation techniques for the isolation of volatiles. Rev. Agroquim. Tecnol. Aliment. 1980, 20(2), 220-230. Schreier, P. Changes of flavour compounds during processing of fruit juices. Long Ashton Symp. [Proc.]. 1981, 7, 355-371. Schreier, P.; Drawert, F.; Heindze, I. The quantitative composition of natural and technologically modified aromas of plants. VI. Distribution and changes of aroma compounds during thermal concentration of orange juice. Chem. Mikrobiol. Technol. Lebensm. 1979, 6, 71-77.
1492 16.
17. 18. 19. 20.
21.
Schreier, P.; Drawert, F.; Junker, A.; Mick, W.Z. The quantitative composition of natural and technologically changed aromas of plants. 11. Aroma compounds in oranges and their changes during juice processing. Z. Lebensm, Unters. Forsch. 1977, 164, 188-193. Shaw, P.E. Fruits II. In Volatile Compounds in Foods and Beverages', Maarse, H., Ed.; Dekker, New York, 1991; pp 305-328. Shaw, P.E.; Buslig, B.S.; Moshonas, M.G. Classification of commercial orange juice types by pattern recognition involving volatile constituents quantified by gas chromatography. /. Agric. Food Chem. 1993, 41, 809-813. Shaw, P.E.; Buslig, B.S.; Moshonas, M.G. Classification of orange and grapefruit juices by pattern recognition technigues. Fruit Processing 1994a, (2), 45-49. Shaw, P.E.; Moshonas, M.G.; Buslig, B.S. Multivariate analysis for classification of commercial orange juice products by volatile constituents using headspace gas chromatography. In Advances in Fruit Flavors; Leahy, M.; Rouseff, R.L., Eds.; American Chem. Soc; Washington, D.C., 1994b. Shaw, P.E.; Moshonas, M.G.; Pesis, E. Changes during storage of oranges pretreated with nitrogen, carbon dioxide and acetaldehyde in air. /. Food ScL 1991,56, 469-474.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1493
EFFECT OF MICROWAVE HEATING ON ROASTED NUT FLAVOR D. E. Zook^, C. Macku^, and D. Demingb ^Planters Company, Research and Development Department, 200 DeForest Ave., East Hanover, N J 07936, USA. ^Nabisco Foods Group, Fundamental Science Department, 200 DeForest Ave., East Hanover, N J 07936, USA. Abstract The microwave oven has opened up a new dimension in food preparation and management. Wherever it is used, a special set of conditions must be developed for best results in flavor, texture, and quality of a final product. In this study, microwave heating is being used to maximize the perception of "fresh roasted nuts." Headspace analysis was used as a non-invasive sampling method, in combination with gas chromatography/mass spectrometry (SIM) technique, to directly monitor changes in flavor and aroma. This technique has been chosen to select specific volatiles for process control, to monitor the development of off flavors, and to identify and determine the best conditions to microwave roasted nuts. Headspace and gas chromatography (GC) have been used to analyze the volatiles that are produced during microwave heating of roasted nuts. This study will help to identify the amount of volatiles that yield a good peanut flavor, and determine a system that will maximize the characteristics of a microwavable nut product. Introduction Previous studies have reviewed the flavor volatiles of fresh roasted peanuts and other nut types after various roasting processes, including oil roast and dry roast. However, no studies to date have reviewed the flavor volatiles of roasted nuts after microwave heating. The composition and formation of roasted peanut flavor has been studied and reported by numerous authors in the past. One of the first studies on the effect of roasting on peanuts was reported by Pickett and Holly (1,2). They identified carbon dioxide, water, ammonia, hydrogen sulfide, and carbonyl
1494 compounds as the main volatile components of peanuts. Walradt (3) was one of the first to identify and report 187 compounds in roasted peanuts, including 17 pyrazines which had never been previously reported. Van Straten (4) later listed 277 compounds in roasted peanuts. Lee, et al (5,6) reported 67 compounds that were previously unidentified, of which three were pyrazines directly related to roasted peanut flavor. Softly and Templeman (7) reported the relationship between amino acids, reducing sugars, and lipids, in raw peanuts and the desirable and undesirable flavor notes they produce during roasting. Various authors have continued to report their findings on roasted peanut flavor and the volatiles responsible for that flavor. This review monitors the changes of important peanut flavor volatiles during microwave processing and determines a system that will maximize the characteristics of a microwavable nut product. 1. Materials and Methods 1.1. Food Samples for Flavor Analysis The following reviews those peanut samples used for analysis: Sample #1. Blanched, Medium-Virginia peanuts. Industrially-Oil Roasted at 320°F for 5.5 to 6.5 minutes. Glazed and salted. Sample #2. Blanched, Medium-Virginia peanuts. Laboratory-Oil Roasted at 320°F for 6.5 minutes. Glazed Only. Sample #3. Blanched, Medium-Virginia peanuts. Laboratory-Oil Roasted at 320°F for 4 minutes. Unglazed, unsalted. Sample #4. Blanched, Medium-Virginia peanuts. Laboratory-Dry Roasted at 290°F for 25 minutes. Unglazed, unsalted. After roasting, samples were kept in mason jars at 65°F (25°C) and capped after purging their headspace with Argon gas. Nut samples remained in containers for no longer than one week.
1495 1.2. Microwave Processing A uniform layer of two-hundred grams of peanuts was placed in a microwavable, glass dish (16 cm in diameter and 8 cm in height). Peanuts and glass container were placed in a Litton, Generation II microwave (Amana, 10). Peanuts were microwaved on high power (approximately 800 watts) for 0, 1, 2, 2.5, and 3 minutes. 1.3. Food Sample Preparation for Analysis After microwave heating, a sub-sample was immediately set aside. Fifty grams of the microwaved peanuts was ground with a Krups Mini-Pro blender for 3 seconds and the temperature recorded. For the industrially oil roasted samples, a separate 100 grams of the whole peanuts were placed in a mason jar with argon-purged headspace for physiochemical analysis (color and moisture analysis). Seven grams of the ground peanuts were taken from the sub-sample and placed into a 22 ml headspace sampUng vial, capped with teflon Hned septum, and set for analysis. Ten grams of the ground peanuts were taken from the sub-sample and placed on a foil tray, for immediate moisture analysis. 1.4. Headspace Samphng Procedure Headspace sampUng vials were placed in a Tekmar 7000 automatic headspace sampler (Cincinnati, OH) which was operated with Tekmar software, TEKLINK (version 1.01). Headspace volatiles were cryofocused at the head of a GC capillary column with a Tekmar Cryofocusing Module^^^. Table I reviews the parameters that were followed for headspace analysis.
1496 TABLE I. Method Followed Headspace Sampler.
PARAMETER Platen Temp Sample Equil. Time Vial Size Cryo Cooldown Time Cryo Cooldown Temperature Vial Press. Time Pres. Equil. Time Loop Fill Time Loop Equil. Time Inject Time Cryo Inject Time (Cryo warm-up) Cryo Inject Temperature Sample Loop Temperature Line Temperature Cryo Union Temperature Injections per vial
for the Tekmar
7000
Automatic
VALUE
55°C 30 min. 22 mL 4.00 min. -180°C 0.25 min. 0.25 min. 0.10 min. 0.15 min. 1.50 min. 0.60 min. 80°C 105°C 105°C 105°C 2
1.5. Gas Chromatography Procedure Gas chromatography was used for flavor profile analysis. The volatiles were injected into a DB-Wax 60m capillary column (0.25mm in diameter, 0.25um in film thickness) housed in a Hewlett-Packard 5890 Series II gas chromatograph. The GC was interfaced to a Hewlett-Packard 5971 mass selective detector (MSD), and operated under single ion monitoring mode (SIM). HeHum was used as the carrier gas. The GC oven was programmed as follows:
Time 8 minutes 8 °C/min 15 min
Temperature 40°C 40°C to 185° 185°C
1497 1.6. Mass Spectrometry Procedure The following parameters were used for the Hewlett-Packard 5971 MSD:
Interface Vacuum EMV Ionization Mode
250°C 3.0 x lO-^ Atm 2100 V 70eV SIM (see Table II)
Table II shows the method followed for headspace sampUng and analysis. TABLE II. Method Followed for Headspace Sampling / Chromatography / Mass Spectrometry (SIM) Analysis.
GROUP NUMBER
START TIME (min)
Gas
IONS MONITORED
0.00
27.0 45.0 58.0 72.0 82.0
29.0 47.0 62.0 74.0 86.0
31.0 57.0 68.0 78.0 97.0
10.25
56.0 81.0 92.0
79.0 82.0 94.0
80.0 91.0
14.50
60.0 80.0 96.0 108.0
67.0 81.0 97.0 121.0
70.0 95.0 100.0 126.0
1498 1.7. Compounds Monitored by Headspace Sampling / Gas Chromatography/ Mass Spectrometry Technique Peak areas of selected MS ions (Table V) were divided by the peak area of ion 78 produced by benzene (external standard). Benzene (Aldrich Chemical Co, Milwaukee, Wl) was dissolved in peanut oil (Planters Co, Winston-Salem, NC) at a concentration of 5 ppm. Three mLs of the oily solution were placed in a 22 mL Tekmar vial and analyzed as shown in Table 1 and Table 11. The external standard was run before and after analyzing the test samples. 1.8. RGB (Red, Green, Blue) Color Analysis The sum of red, green and blue colors were measured to determine the increase in browning, and color variation throughout the sample after microwave heating. A random sample of 18-19 whole peanuts (approximately 20 grams) was selected and spht by hand. Four of these peanut-halves were evaluated using an Olympus CUE-3 True Color Image Analyzer for camera-based colorimetry (Precision Instrument Division, Cherry Hill, NJ) and the recorded values were means of the inside of these four peanut-halves. The IBM/PC system included a CCD color camera equipped with three separate primary color signals to provide values for red (R), green (G), and blue (B) Hght reflectance from the surface of the sample. Video prints of images of the internal portion of the peanuts were obtained using a SONY Color Video Printer UP-5000. Peanuts were then ground to a uniform particle size using a commercial coffee mill. Grinding was controlled to minimize heat exposure to the sample and to avoid sample pasting. Approximately 10 grams of the ground sample was firmly packed into a small cup (diameter = 33 mm; height = 12 mm). The surface was then flattened and smoothed with the bottom of a glass Petri dish. Color was measured using an the Olympus CUE-3 True Color Image Analyzer. Recorded value is a mean of two measurements. The surface was scraped and fresh sample was flattened and smoothed between measurements.
1499 1.9. Moisture Analysis Ten grams of the ground peanuts were placed on a foil tray and placed in a CompuTrac Moisture Analyzer, by Motorola, Model No. MA-5 (Cherry Hill, NJ). The moisture on the ground sample was measured at a final temperature of 132°C until the sample was equilibrated by the instrument and the moisture of the sample was displayed and recorded. 2. R e s u l t s 2.1. Total Ion Chromatogram Figure 1 shows a Total Ion Chromatogram of the volatile chemicals present after microwave heating of oil roasted nuts for two minutes on high power. 2.2. Compounds Monitored by Headspace Sampling / Gas Chromatography / Mass Spectrometry Technique. Table III shows the 32 compounds that were monitored for, the retention times, and the MSD ion for quantitation, followed by GC/MS (SIM) analysis. (Numbers correspond to peak numbers in Figure 7). Tables IV and V show the 32 compounds and the relative quantities of each compound in oil roast and dry roast microwaved peanuts, respectively.
1500 Figure 1. Total Ion Chromatogram of Oil Roasted Microwaved for Two Minutes.
Peanuts
2000
~ i — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — I — ^ — I — I — I — I — I — I — I — I — I — [ -
Time ->
6.00
8.00
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
1501 TABLE III. List of Compounds Monitored by Headspace Sampling / Gas Chromatography / Mass Spectrometry. Peak#
"12
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Compound ^
Retention Time*^
m/z lon^
5^6 5.7 5.9 5.9 6.1 6.9 7.4 7.7 7.8 8.2 8.3 9.3 11.0 11.9 12.0 13.5 15.4 15.6 16.3 16.7 16.8 18.06 18.10 18.30 18.60 19.2 19.4 19.6 20.5 20.7 21.6 21.6
58 57 27 58 74 72 72 58 58 45 45 58 91 94 56 81 70 80 70 100 67 108 108 108 108 126 121 121 60 95 95 67
Prop anal Octane Isobutanal Dimethylketone Methyl acetate Butanal Methylethylketone 2-Methylbutanal Isopentanal 2-Propanol Ethanol Pentanal Toluene Dimethyldisulfide (DMDS) Hexanal Methylpyrrole 2-Methylbutanol Pyrazine 1-Pentanol 2-Methyl, 3-ketofuran Methylpyrazine 2,5-Dimethylpyrazine 2,6-Diniethylpyrazine 2-Ethylpyrazine 2,3 -Dimethylpyr azine Dimethyltrisulfide (DMTS) 2-Methyl, 5-ethylpyrazine Trimethylpyrazine Acetic Acid Acetylfuran MethyLfurylketone Pyrrole
^ Numbers correspond with peak numbers in Figure 7. •^ Retention Time in minutes. ^ m/z ion followed under SIM mode (Table II).
1502 Table IV. Table of Relative Quantities for Volatiles Isolated From Lab Roasted, Oil Roasted Peanut Samples Peak # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Compound Prop anal Octane Isobutanal Dimethylketone Methyl acetate Butanal Methylethylketone 2-Methylbutanal Isopentanal 2-Propanol Ethanol Pentanal Toluene Dimethyldisulfide (DMDS) Hexanal Methylpyrrole 2-Methylbutanol Pyrazine 1-Pentanol 2-Methyl, 3-ketofuran Methylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine 2 -Ethylpyr azine 2,3-Dimethylpyrazine Dimethyltrisulfide (DMTS) 2-Methyl, 5-ethylpyrazine Trimethylpyr azine Acetic Acid Acetylfuran Methylfurylketone Pyrrole
0 min.
1 min.
2 min.
3 min.
31 41 342 330 58 3 9 360 48 56 53 12 4 53 35 126 3 93 6 2 9 50 2 0.3 0.1 16 6 1 17 3 0.7 2
18 26 218 206 38 8 8 257 59 46 42 9 3 73 23 103 3 77 4 5 7 47 3 0.2 0.06 20 5 1 15 2 0.7 2
n
12 21 163 214 44 10 6 161 122 23 24 11 5 57 22 109 4 81 3 5 24 70 3 1.1 0.2 6 7 6 14 6 1.3 2
18 162 154 29 5 5 213 125 21 26 8 4 64 21 89 6 66 3 5 7 46 2 0.2 0.1 15 5 2 15 3 0.8 5
1503 Table V. Table of Relative Quantities for Volatiles Isolated For Lab Roasted, Dry Roasted Peanut Samples. Peak # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Compound Prop anal Octane Isobutanal Dimethylketone Methyl acetate Butanal Methylethylketone 2-Methylbutanal Isopentanal 2-Propanol Ethanol Pentanal Toluene Dimethyldisulfide (DMDS) Hexanal Methylpyrrole 2-Methylbutanol Pyrazine 1-Pentanol 2-Methyl, 3-ketofuran Methylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine 2-Ethylpyrazine 2,3-Dimethylpyrazine Dimethyltrisulfide (DMTS) 2-Methyl, 5-ethylpyrazine Trimethylpyrazine Acetic Acid Acetylfuran Methylfurylketone Pyrrole
0 min.
1 min.
2 min.
3 min.
12 19 159 162 59 2 6 125 43 61 94 17 4 54 3 108 5 79 10 2 6 37 4 3 3 49 4 2 6 1 0.6 2
7 12 151 123 47 7 5 52 68 50 48 8 4 50 11 99 5 73 3 4 5 29 9 2 1 41 4 2 7 1 0.5 2
6 9 80 126 30 1 4 81 115 35 42 7 4 53 10 85 4 62 3 3 5 30 1 1 1 44 3 4 4 2 0.6 2
5 11 62 127 47 10 8 60 95 25 22 11 5 35 8 113 2 82 2 5 35 41 2 2 0.7 11 5 1 3 5 1.4 8
1504 2.3. RGB Color Analysis The Oil Roasted samples were tested for RGB color after microwaving for 0, 1,2, and 3 minutes. A sum of the RGB values provides a measure of total light reflectance which is known as "paleness," where higher values correspond to a paler product. The paleness/darkness value measure the magnitude of color, as it increases with the extent of the progress of Maillard reactions during roasting. The following table reviews the results of this analysis:
RGB Color Analysis of Microwaved Peanuts (Average values of 3 replicates). Microwave Time (min)
RGB Color
Range
0 1 1 2 2 3 3
167 162 179 170 155 127 151
127 75 97 123 75 178 145
The RGB color remains constant for samples microwaved up to 2 minutes on high power. At three minutes, the number becomes smaller due to "hot spots" where samples tend to burn from excessive microwave heating. The data indicates that the range of RGB numbers is reduced at 1 and/or 2 minutes, which expresses a more homogeneous color. This range increases at 3 minutes, again due to the "hot spots" created during excessive microwave heating. This phenomenon was also demonstrated on studies in 1970 at the USDA, using a 25 KW oven. Results showed that blanched nuts that were placed in the microwave oven for various periods of time prior to cooking, resulted in a more uniform color and less resistance to splitting (17).
1505 2.4. Moisture Analysis The following Table displays the results of this analysis: CompuTrac Moisture Analysis of Microwaved Peanuts (Averages values of 2 replicates).
Microwave Time (min)
Dry Roast
Oil Roast
0 1 2 3
0.68 0.59 0.45 0.40
1.02 0.64 0.52 0.37
3. Discussion The purpose of this study was to better understand the effect of microwave heating on nut products, concentrating mainly on roasted peanuts. Various samples of peanuts were studied in order to better understand which type of roasting method was best suited for microwave exposure. Overall, a microwave heating time of two minutes on high power, for oil roasted and/or dry roasted peanuts, seemed to have the best qualitative and quantitative results. Two minutes of microwave heating allows for a more consistent, even color development. Two minutes of microwave heating on high power also produces a chromatograph with maximized peaks that are believed to represent attributes characteristic of good peanut aroma and flavor. The two minute samples qualitatively had the most peanut flavor and the highest sweetness level while warm. In evaluating the samples, the following sensory attributes were monitored: beany/raw flavor, roasted peanut flavor, dark roasted peanut flavor, sweetness, and bitterness. Textural attributes that were evaluated include: hardness, crispiness, and density (or cohesiveness of mass). The volatiles found in these samples, as will be presented in the results section, clearly shows that the best profile for a potentially good roasted peanut flavor lies in the product that is microwaved on high power for two minutes. This sample expressed a higher concentration of pyrazines, dimethyltrisulfide, and other components that have been estabUshed as important compounds to roasted peanut flavor.
1506 Two minutes also reduces the chances of non-uniform heating which can result in burnt product, that occurs at 2.5 and 3 minutes. Oil roast products have a better profile and final product, quantitatively and quahtatively than dry roast product that is placed in the microwave for any period of time. Finally, it was found that it is quite difficult to obtain a roasted product out of the microwave. When starting with an under-roasted peanut, the temperatures that must be reached for cooking to occur, which would allow for the Maillard reaction and Strecker degradation to be achieved, can only be reached at a point where scorching and non-uniform heating become an issue. An industrially oil roasted sample was chosen in order to observe the differences of industrially produced samples vs. laboratory produced samples (as not all of the samples could be obtained from industry). The comparative sample, made in the laboratory was then tested to better understand the differences that may be seen in additional samples. An under-roasted oil roast sample, made in the laboratory, was then tested to see if roasted peanut flavor could be developed from microwave heating. Finally, a dry roast sample was tested to better understand the effect that microwave heating had on dry roasted product vs. oil roasted product. The important compound classes in raw peanuts relative to the development of peanut flavor in roasted peanuts include amino acids, reducing sugars and lipids. These components are the precursors for pyrazines, carbonyl and sulfur compounds, and the various other important components that make up roasted peanut flavor. These precursors develop into the flavor components that lead to roasted peanut flavor during roasting via the Maillard reaction, and Strecker degredation, for most compounds. The following reviews the major components that are developed during microwave heating that are found to be important to roasted peanut flavor. Figures 2 through 13 show the relative amounts of isobutanal, ethanol, dimethyldisulfide (DMDS), dimethyltrisuffide (DMTS), and acetic acid. These peanut flavor components were monitored by GC/MS headspace analysis as they are representative of trends during microwave heating. Pyrazines are the most significant class of compounds found in roasted peanut flavor that are responsible for the nutty characteristics of peanut flavor. They are formed by the reaction of amino acids and reducing sugars under the Maillard Reaction (3). Ethylpyrazines (Figure 2) as well as propylpyrazines, and methylpyrazine (Figure 3), stay relatively constant throughout microwave heating for all oil roast and dry roast samples. These compounds also show an increase in relative quantity after three minutes of microwave heating. A dramatic increase is noted for methylpyrazine after three minutes of microwave heating for both oil roast and dry roast samples.
1507 The inital quantity of pyrazines is lower in dry roast than in oil roast products. Both products display a sHght decrease in Pyrazine (Figure 4) after one and two minutes of microwave heating, and hke the trend of the previously mentioned pyrazines, express a slight increase after three minutes of microwave heating. Figure 2.
2,5/ 2,6- / 2,3- Dimethytpyrazinesand 2-Ethylpyrazine(RT = 18.06,18.10,18.60, and 18.30 consecutively)
10 +
i 1
2 Micnivuave (minutes)
Figure 3. Methylpyrazine (RT = 1 6 . 8 minutes)
~ Oil Roast "Dry Roast
1508 Figure 4. Pyrazins (RT = 15.6 minutes)
- » - Oil Roast ' ® 'Dry Roast
Microwave (minutes)
Sulfur containing compounds are also a significant contributor to peanut flavor and are also formed by the Maillard reaction of reducing sugars with sulfur containing amino acids; specifically methionine and cystine (7). Sulfur derivatives in large quantity can produce off flavors and burnt notes in peanut flavor. Dimethyl disulfide (DMDS), reaches a minimum at three minutes for both products. The oil roast sample from the lab displays a maximum quantity of DMDS (Figure 5) after one minute of microwave heating, and begins to decrease in quantity thereafter. The level of DMDS in the dry roast sample remains relatively cost ant up to two minutes of microwave heating, but shows a decrease in relative quantity after three minutes of microwave heating. Figure 5. Dimethyldisulfide (RT = 11.90 minutes)
- ^ Oil Roast -®-Dry Roast
1509 Dimethyltrisulfide (DMTS) shows a maximum at one and two minutes for oil roast products, as well as for dry roast products. It is interesting to note that the oil roast product had a better qualitative roasted peanut flavor than the dry roast product overall. Both products showed the best quahtative products at two minutes. Dimethyltrisulfide (Figure 6) in conjunction with methanethiol, dimethylsulfide, and dimethyldisulfide, are important to the flavor of freshly roasted peanuts as previously expressed. The level of DMTS in the dry roast product was much higher initially and at all microwave times, than for oil roast. Both products show a significant decrease in DMTS after three minutes of microwave heating. F i g u r e 6. Dimethyltrisulfide (RT = 19.20 minutes)
- Oil Roast Dry Roast
Microwave (minutes)
Carbonyl compounds are formed via the Maillard reaction and Strecker degredation (9). Isobutanal, Isopentanal, and 2-Methylbutanal (Figure 7) are breakdown products of amino acids. These compounds have been identified as significant to peanut flavor and are found in high concentrations. Carbonyl compounds can be formed by Maillard reaction (10), and contribute to peanut flavor (9). Pentanal and Hexanal are formed by lipid oxidation, and have been identified as negative contributors to peanut flavor. The aldehydes that are formed by these reactions may have a tendency to breakdown or volatize rather than be constructed, as microwave energy may not generate enough heat to induce the formation of these compounds. At a temperature that this reaction is capable of taking place, nonuniform heating has already caused scorching and burning of the product. The burnt, dark roasted notes, then cover up any of the peanut flavor that is characterized by the aldehydes that are then able to emerge. It was found by Mason (18) that roasted peanuts are characterized by very large quantities of 2-methylpropanal, 3-methylbutanal, and 2methylbutanal. Mason suggested that these low molecular weight aldehydes.
1510 Figure 7. 2Methylbutanal (RT = 7.7 minutes)
*
Oil Roast
®
Dry Roast I
!
M i c r o w a v e (minutes)
Pentanal (Figure 8) is a typical carbonyl compound which has been identified in peanuts. Buckholz (20) reported pentanal as organoleptically representing harsh, green, solventy notes. Pentanal exhibited a decrease for both oil roast and dry roast after one and two minutes of microwave heating. There was a slight increase in pentanal for both samples after three minutes of microwave heating. Hexanal has a maximum quantity for oil roast at zero minutes and decreases after one minute of microwave heating. The level then remains constant up to three minutes of microwave heating. Dry roast h a s a minimum level of hexanal at zero minutes and presents an increase in hexanal after one minute of microwave heating and then expresses the same trend as oil roast in that it remains constant up to three minutes of microwave heating. Figure 8. Pentanal (RT = 9 . 3 minutes)
••"Oil Roast - ® - D r y Roast
Microwave (minutssi
1511 Acids and alcohols have been found in high quantities in previous studies (6). They range from C1-C20. C2-C6 have been discovered to have the most impact on peanut flavor and have been found to be sUghtly more soluble than the C8-C20 compounds, which have very Httle if no impact on flavor (6). Acids are formed by the oxidation of higher molecular weight fatty acids. Acetic acid (Figure 9) levels were much higher for oil roast samples initially and throughout microwave heating. The levels of acetic acid remained relatively constant up to three minutes of microwave heating and displayed a slight decrease for the oil roast samples. The level of acetic acid for the dry roast product decreases slightly with increased microwave time. The level for the dry roast sample decreases significantly with increased microwave heating. The destruction of such a polar compound would be expected as these molecules absorb microwave energy easily. Figure 9.
Acetic Acid (RT = 20.50)
18 T 16 14 12 s
10
» Oil Roast ® Dry Roast
1
1.5 Microwave (minutes)
Alcohols are also formed by the oxidation of higher molecular weight fatty acids (11). Ethanol (Figure 10), as well as 2-propanol and isopentanol, decrease during microwave heating for the oil roasted product. The dry roasted product decreases even more significantly during microwave heating.
1512 Figure 10. Ethanol (RT = 8.3 minutes)
' O i l Roast Dry Roast
1.5 Microwave (minutes)
Isobutanal (Figure 11), as well as isopentanal, and 2-methylbutanal are intermediate pyrolysis products of proteins. They display a dramatic decrease after one and two minutes of microwave heating for all oil roast and dry roast products. The oil roasted product had a higher initial and final amount of isobutanal than the dry roast product. During the latter stages of heating, the concentration of isobutanal remains constant. This may be the result of a breakdown of these compounds during microwave heating, in which the compounds are not able to reach temperatures in which they would be formed. Figure 11. Isobutanal (RT = 5.9 minutes)
1513 Acetylfuran (Figure 12) as well as methylfurylketone (Figure 13) and 2methyl, 3-ketofuran, remained the same and increased after two minutes in the microwave for the oil roast samples. Due to their wide distribution and the diversity of their structures, furanoid compounds play an important role in food flavor. They are usually formed by degradation of carbohydrates (19). The oil roast sample showed a very sHght decrease with initial microwave heating. Oil roast product also displayed an increase in acetylfuran after two minutes in the microwave. Acetylfuran remained constant for initial microwave heating for dry roast product, but began to increase after two minutes of microwave heating. Methylfurylketone showed an initial decUne in relative quantity for dry roast during initial heating, but increase after one minute in the microwave. The concentration of Methylfurylketone remained more constant during initial microwave heating for oil roast, but also displayed an increase after one minute of microwave heating. Figure 12. Acetylfuran (RT = 20.7 minutes)
M icrowave (minutes)
Figure 13. Methylfurylketone (RT = 21.6 minutes)
- Oil Roast "TJry Roast
trowave (minute*)
1514 The temperature of the nut samples was taken immediately after microwave heating. F i ^ r e 14 plots the temperatures that were recorded from the ground samples. Figure 14.
Temperature (C) vs. Microwave Time (minutes)
* Oil Roasted, Salted Peanuts Oil Roasted, Unsalted Peanuts
On the average, the stated power output of domestic microwave ovens is between 600-1000 watts (13). Some of the very low-powered ovens or the larger powered ovens are not being included in this discussion in order to avoid confusion. In addition, this is setting aside the questions raised by alternative modes of operation being offered, such as pulsing verses steady application of power and fully variable cooking speed vs. fixed, or merely high and low selections, and does not include modern microwaves having infrared sensors, humidity detectors, and auto cook functions. We have chosen to use high power for all experiments mentioned. The 800 watt level, chosen for consistency in the discussion, will give an even four minutes per pound for many typical foods. When an institutional size oven is considered, the consistent level is high power, approximately 1600 watts (13). When referring to an institutional size oven, a basic time of two minutes per pound should be considered. Exceptions, of course, can be noted for various oven makers. When the power is the controllable and variable factor, an oven that may be set on low power, may only put out 400 watts or less (13). In this case, the microwave times will be double or longer depending on where the manufacturer has set the low power output. When using low power, this would call for longer microwave times, which would be adequate or even preferred for warming, defrosting of slightly frozen products, slow cooking
1515 techniques, etc. Low power was not chosen for this experiment in order to remain constant in recording data and reporting results. It must also be considered that not all microwaves have the same power efficiency, that is, the amount of energy that is actually available from the appUance, for use in cooking. Although improvements of microwaves continue, the percent efficiency that can be expected is in the realm of 6065% (14). The operating power is given by the manufacturer of the microwave, whereas the input wattage can be measured with a simple meter while the oven is in use. The power levels of a microwave oven are controlled in various manners. One method is to vary the Une voltage supphed to the transformer in the oven's power supply to correspondingly vary the ouput of the magnetron. Another method is to use an electronically controlled duty cylce to obtain lower power levels, in which the oven automatically alternates periods of microwave generation with rest periods (Hegenbart, 1994). Either method would have an effect on the power efficiency of an oven at various power levels. The actual cooking efficiency of a food is more dependent on the dipole molecule activity in the foods, as the power of the microwave is converted to heat directly in the food (George, 1993). Foods having higher levels of moisture would have a greater cooking efficiency. Foods high in fats would not have as great a cooking efficiency, but would be able to maintain the heat uptake for a longer period of time. Interesting to note, the moisture for dry roast peanuts was initially signfficantly lower than the moisture for oil roast peanuts. After one minute, the difference in moisture between the two samples is dramatically closer, and continues this trend until they are almost equal at three minutes, where scorching has occurred. In the case of peanuts, it takes approximately 30 minutes at 290°F - 320°F to cook them in a conventional forced air oven. In a microwave, although it is difficult to obtain a good, even roast, the temperature of the nuts can reach cooking temperatures within minutes. It is our experience in this research that preroasted nuts can reach temperatures of 140°F within 2 to 2.5 minutes at high power, enough to invoke the roasting process. At 3 minutes, a temperature of 167°F may be reached, and hot spots and scorching will occur. Figure 14 shows the temperatures recorded at various microwave times. In addition, nonuniform heating, caused by the "overlapping of microwaves in the oven, which in turn yield the two-dimensional model of a waveform that forms a pattern of standing waves that is unique to every microwave oven cavity and will change depending on the temperature of the oven cavity and the load placed within," will vary the actual cooking efficiency of a food in the oven (15).
1516 New developments in microwave technology are addressing issues related to the lack of browning and crisping, lack of uniformity of heating, and lack of flavor development that may occur during microwave heating. In terms of the product itself, it has been found that electrical properties of foods play a very critical role, as described by George, (16). As explained in this review of microwave heating, the addition of ionic compounds such as salts or flavorings can have a detrimental effect on microwave heat penetration. It was concluded that salt or flavorings or other compounds having a high ionic content, may induce a high surface heating effect, with little energy penetrating the interior portion of the product. In the case of using the microwave to warm the nut, having salt on the surface may therefore, actually be beneficial. In one respect, this would be advantageous as the goal is to only warm the nuts, and have little effect on the textural and eating characteristics of the product (no cooking is involved). On the other hand, this would reduce the amount of time the nuts would hold the heat, and the benefit of warming may not be as apparent, ff the product is not eaten immediately after microwaving. Although this study did not evaluate the length of time the heat was held by the peanuts, all samples displayed the same heating trend and experienced nonuniform heating at the same microwave times. The temperatures were taken on ground samples, which would measure the internal temperature of the nut, it is interesting to note that the product that was industrially oil roasted, glazed and salted, displayed a lower temperature trend than the product that was oil roasted in the lab and glazed only. An additional set of data was recorded and plotted in Figure 14, using industrially oil roasted peanuts with salt and unsalted. The results were not as dramatic, however, the trend for higher penetration of heat in the unsalted product is still apparent and significant. The presence of salt does seem to support the findings by George in that a higher ground (internal) temperature was observed on the unsalted product. Today's consumer is consistently demanding products and packaging that allow for rapid and convenient food preparation in the home. Microwave ownership around the world is rapidly increasing. In the United States alone, the ownership of microwaves increased from 81% of U.S. households owning a microwave in 1989 to 93% in 1993 (12). It is apparent that today's food manufacturer must sell foods that are capable of fast and easy preparation. With the increase in microwave oven penetration, the food market has experienced an increase in food products specially made for microwave heating. Microwave cooking departs in many interesting and novel ways from routine cooking and offers variety to those who are prepared to try various cooking devices.
1517 This study has evaluated the effects of microwave heating on roasted nuts, and has concentrated on the effects microwave heating has on those compounds listed in Figure 1, which have been found to be the most important compounds related to peanut flavor. This study found that a microwave time of two minutes yields the best overall peanut aroma and flavor quality for roasted peanuts. References 1. Pickett, T.A., K.F. Holly, Peanut Roasting Studies at GA. Agr. Exp. Station (1943). 2. Pickett, T.A., K.F. Holly, Peanut Roasting Studies at GA. Agr. Exp. Station (1952). 3. Walradt, J.R., A.O. Pittett, T.E. Kinhn, R. Maralinhara, and A. Sanderson, J. Agr. Food. Chem. 19:972 (1971). 4. Van Straten, S., Volatile Compounds in Food. Control Institute for Nutrition and Food Research, Zeist, The Netherlands, (1977). 5. Lee, M-H, Studies on Roasted Peanuts and Peanut Butter Flavor. Ph.D. Thesis. Dept.of Food Science, Rutgers Univ. New Brunswick, N J (1980). 6. Lee, M-H, C.T. Ho, and S.S. Chang, et al, J. Food Sci., 47:127 (1982). 7. Softly, B.J., and G. Templeman, A Critical Review of Natural and Artificial Peanut Flavors. Internal Research Study at Standard Brands, East Hanover, N J (1982). 8. Softly, B.J., J. Sourby, and G. Templeman, 1980 Peanut Flavor Model. Internal Research Study at Standard Brands, East Hanover, N J (1982). 9. Schoenberg, A., R. Moubashev, J. Moustafa, J. Chem. Soc, p. 163 (1948). 10. Wu, C , The Isolation and Fractionation of Volatile Flavor Constituents of Roasted Peanuts, Ph.D. Thesis. Dept. of Food Science, Rutgers Univ., New Brunswick, NJ, (1977). 11. Fennema, O.R., Food Chemistry, edited by S. R. Tannenbaum, and P. Walstra, Marcel Dekker, Inc, New York, 1985, pp. 207-210. 12. Happel, M.E., Microwave World, 13(2), pp. 7-8 (1992).
1518 13. Van Zante, H., The Microwave Oven, Houghton Mifflin Co., Boston, 1973. 14. Copson, D., Microwave Heating, AVI Publishing Co., Westport, 1975. 15. Hegenbart, S., Food Product Design, April, 1994, pp. 23-28. 16. George, R.M., Trends in Food Science and Technology, (4), 190-394 (1993). 17. Sopina, S., (Personal Communication), Research conducted at USDA, California, 1970. 18. Mason, M.E., B. Johnson, and M. Hamming, J. Agr. Food Chem., 14:454 (1966). 19. Ho, C , L. Min-Hsiung, and S. Chang, J. Food Sci., 47:127 (1981). 20. Buckholz, L.L., Jr., H. Daun, E. Stier, R. Trout, J. Food Sci., 45 (3): 547 (1980).
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1519
Peanut flavor formation during roasting as affected by atmospheric conditions Robin Y.-Y. Chiou and Chin-Yin Tseng Department of Food Industry, National Chiayi Institute of Agriculture, Chiayi, Taiwan, Republic of China Abstract Peanut kernels (Tainan 9, a Spanish cv. ) were roasted in a stainless steel vessel at 210 °C under a continuous flow of air, N2, CO2, and O2 at 13 L/min and without aeration as a control, for 0, 10, 18, and 25 min. Hunter L values of the deskinned kernels decreased while a and b values increased with roasting time. The most and the least significant changes in color of peanuts occurred under O2 and CO2 atmosphere, respectively. The best flavor was obtained by roasting peanuts under N2 and CQ2 for 18 min. Decreases of a-amino nitrogen, glucose and sucrose were dependent on the extent of heat treatment (roasting time) rather than on atmospheric gas composition. When raw peanut oil was combined with partially defatted peanut meal at a ratio of 10:1 (v/w) and foasted at 160 ^C for 30 min in a closed chamber under various atmospheric conditions. The addition of 10-20 % moisture to peanut meal was essential for the formation of peanutty flavor in oil. A unique and pleasant roasted peanut flavor was achieved when roasting was carried out under atmospheres of CO2 . Fatty acid composition in oils as affected by atmospheric conditions varied only within a limited range. 1. INTRODUTION When raw peanuts are roasted or oil fried, a pleasant "peanutty" flavor is produced in a rather short of time. A marked flavor difference resulting from roasting or frying is well recognized by the consumer. Fried peanuts were preferred to roasted peanuts. One of the most significant differences between
1520
roasting and deep frying of peanuts is the involvement of air during the process of heat treatment. Considerable variation in flavor and other sensory characteristics of roasted and fried peanuts suggests that flavor formation is undoubtedly affected by the gaseous atmosphere in contact with peanuts during the roasting or frying process. Some conventional roasters are heated via natural gas flames; with the Bauer roaster (Bauer and Bros. Co.) [1], abundant carbon dioxide is produced simultaneously in the atmospheric environment during roasting. Some roasters are designed such that burning fuel does not come in direct contact with peanuts. Others use electricity as the heat source, which does not create a significant alteration in the roasting atmosphere. Depending upon the type of roaster used, unique peanut flavor and other sensory characteristics eventually result. When peanut kernels are spUt, chopped into pieces, ground into meal, and pressed to expel oil before being subjected to roasting, the intensity of the unique roasted flavor in each successive product is diminished. The smaller the particle size, the larger the total surface areas exposed to the roasting atmosphere. The presence of atmospheric O2 during roasting might promote the development of imdesirable flavor or mask the intensity of desired flavors. Therefore, exclusion of O2 in the roasting atmosphere may enchance the production of desired peanuttyflavorcompounds. In this study, the objective was to investigate the effect of various atmospheric gas conditions on roasting characteristics of peanut kernels and peanut oils. Flavor generation resulting from the roasting raw peanut oils together with small amounts of partially (Jefatted peanut meal under various atmospheric environments was investigated. The effects of moisture content of the peanut meal on flavor formation during roasting and sensory evaluations^ and chemical analysis of theflavor-relatedcomponents were conducted. 2. MATERIALS AND METHODS 2.h Peanuts Freshly harvested and dried peanuts (Tainan 9, a Spanish cultivar) were hand-shelled, visually sorted, packaged in polyethylene/nylon laminated bags, and stored at -IS'^C until used. The moisture content of kernels was 7.30± 0.30% (dry basis). Peanuts were removed from the freezer and tempered at room temperature for 24 h before being subjected to various experimental
1521
treatments. 2.2. Peanut roasting and sensory evaluation of the deskinned roasted kernels A high-pressure reactor vessel (600 cm^) (Berghof 575001, Berghof GmbH) equipped with a gas-flushing device and a stirrer was used to roast peanut kernels. The heating mantle temperature wa^ controlled at 210 ""C and tempered 2 h before peanuts were roasted. The atmospheric conditions applied to peanuts consisted of flushing the vessel at a rate of 1.3L/niin (LPM) (flow rate was calibrated and controlled according to the molecular weights of gases) with air, nitrogen, carbon dioxide, and oxygen. Peanuts roasted under an air atmosphere without flushing served as the control. For the entire time of roasting (0, 10, 18, or 25 min), 60 g of ppanut kernels was mixed with the stirrer adjusted at 85 ± 5 rpm. After roasting , the kernels were spread on a tray, cooled at room temperature, deskinned manually, and stored in brown sample vials at -18 "^C until subjected to analyses.*The colors of the deskinned kernels, expressed as Hunter L, a, and b values, were measured [2]. For sensory evaluation, five trained panelists were instructed to use a category hedonic scale (9 to 1) to evaluate flavor notes; 6-9 indicated an increased intensity increment of the peanutty flavor; 5 was the midpoint ; 4 to 1 indicated an increased intensity increment of the unpleasant off-flavor. 2.3. Determination of total a-amino nitrogen, soluble carbohydrate, sucose and glucose contents Unroasted peanut kemds were freeze-dried (Lab Conco Freeze Drier 80), ground with a cyclone mill to prepare peanut meals, and defatted with nhexane. Methanol-chloroform-water (MCW) extraction and determination of total a-amino nitrogen, soluble carbohydrate, sucose and glucose contents were done according to the procedure of Young et al. [3] with modifications by Rodriguez et al. [4] and Chiou et al. [5]. Sucrose contents were determined with a HPLC (Alcott 760 pump) equipped with a refractive index detector (ERC7515A, Erma Co. Inc.). A carbohydrate analysis column (HC-75, Hamilton Co.) was operated at 90 ^C and a flow rate of 1.2 mL/min; water was used as an eluent. The injection volume was 20 |LIL, which was prepared by vacuum drying 0.1 mL of MCW extract followed with rehydration of 2.0 mL of deionized water.
1522
2.4 Raw peanut oil and partially defatted meal preparation The moisture content of the peanut kernels was increased to 9.0% by spraying with a predetermined amount of deionized water. After equilibration at 4 ^^C for 3 days, peanuts were pressed hydraulically (150-200Kg/cm^) to prepare raw peanut oil and partially defatted kernels. The raw oil was passed through filter paper (Advantec No. 2, Toyo, Japan). The partially defatted kernels were deskinned manually and pulverized with a cyclone mill to prepare peanut meal (l-nrni screen) and stored at -25 "^C for fiirther use. Moisture, crude lipid, and protein (N x 5.46) contents of the defatted meals were 9.90, 29.93, and 33.43% (wet basis), respectively. 2.5. Peanut oil roasting and organoleptic evaluation Prior to roasting under various atmospheric conditions, 2 mL of raw oil and 0.2 g of the partially defatted peanut meal were deposited in a digestion tube, flushed with the designed gas for 1 min, and sealed with a Teflon screw stopper. Controls consisted of 2 mL of raw peanut oil under CO2 and ambient atmospheric gas conditions. Oil and meal mixtures were roasted at 160 ^C for 30 min. using a heating module (Pierce Reacti-Therm, Pierce Chemical Co., Rockford, IL). The tubes were cooled at room temperature, and the clarified oil ftactions were deposited in brown glass vials and stored at -25 ^'C before being subjected to sensory evatution us^ng the procedure described as above ?ihd chemical analysis. 2.6. Effect of moisture content of defatted peanut meal on peanutty flavor formation during roasting Partially defatted peanut meal wasfireeze^driedand ground manually to prepare dehydrated peanut powder. Dehydrated peanut powder (0.2 g) was placed in a series of digestion tubes, and 0, 5, 15, 20, 30, 40, 50, and 100 |uL of deionized water was added to result in 0, 2.5, 7.5, 10, 15, 20, 25, and 50% moisture content, respectively. Raw peanut oil (2.0 mL) was added to each tube, which was then flushed with CO2 for 1 min, sealed with a screw stopper, and heated at 160 ^C for 30 min. After cooling to room temperature, the oil was withdrawn from each tube and subjected to sensory evaluation using the procedure described above.
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2.7. Determination of fatty acid composition The fatty acid composition of peanut oils was determined using the methylation procedure reported by Chiou et al. [6] and gas chromatography. A packed column (GP 3% SP 2310/2% SP 2300 on 100/120 Chromosorb W AW, 6 ft X 1/8 in. stainless steel column, Supelco Inc., Bellefonte, PA) was used. The injector and detector temperatures were 230 and 250 °C, respectively, sample size was 0.5|LIL, and carrier gas (He) flow rate was 20mL/min. For each run, the initial colunm temperature held at 190 "^C for 2 min, was programmed to increase to 220 °C at 2 °C/niin, and was then held for 5 min. Data were collected by an integrator/recorder. The relative proportion of each component fatty acid was expressed as a percentage of the total peak areas. 2.8. Statistics Two replicate experiments were conducted. Means of the determinations with standard deviation are reported. Significant differences among samples were analyzed by the statistical t-test. 3. RESULTS AND DISCUSSION Color characteristics of deskinned unroasted and roasted peanut kernels, expressed as Hunter L, a, and b values, are shown in Table 1. In general. Hunter L values decreased while a and b values increased with time of roasting. Decreases in L values resulted in visibly darker color and were significantly affected by the atmospheric gas composition. When peanut kernels were roasted for 25 min, the lowest L value was observed for kernels roasted under an oxygen environment. Effects from other gases, in decreasing order, were roasting without aeration (control) and flushing with nitrogen, air, and carbon dioxide. Undoubtedly, changes in color of peanut kernels during, roasting influenced by the amount of oxygen present in the atmosphere. However, L values of kernels roasted under a nitrogen atmosphere were not significantly different from those of kernels roasted under air, which suggests that nitrogen might be involved in the discoloration process. When kernels were roasted without aeration (control), L values also changed considerably. This might be attributed to a slightly higher temperature which occurred due to slower mass transfer in water evaporation in peanuts not subjected to aeration compared to peanuts roasted under aeration orflushingwith test gases .
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During roasting, a values increased with roasting time and were influenced by atmospheric gas conditions. The most obvious change among samples was observed when peanuts were roasted for 18 min (Table 1). The highest a value was observed for peanuts roasted under an oxygen environment, followed in order by kernels roasted without aeration and under nitrogen, air, and carbon dioxide. When peanut kernels were roasted for up to 18 min, b values steadily increased. After 25 min of roasting, some b values decreased slightly. Atmospheric gas composition had a less marked effect on b values. After 25 min of roasting, the highest and lowest b values were observed for peanuts roasted under carbon dioxide and oxygen, respectively.
Table 1 Color variations of peanut kernels roasted uilder various atmospheric gas environments' gas
color determination^ L at roasting time of
environment
Omin
W/0
a at roasting time of
b at roasting time of
18 min
25 min
69.3±0.1 66.2±0.3 51.8±1.2 43.2±0.1 3.7±0.1
6.5±0.i
15.7±0.4
17.6±0.4 15.4±0.1
19.6±0.2 21.4±0.7
19.0±0.1
air
69.3±0.i 66.7±0.4 56.9±0.4 46.1±0.9 3.7±0.1
5.9±0.1
14.2±0.4
17.8±0.1
15.4±0.1
19.0±0.1 22.5±0.1
20.4±0.3
N2
69.3±0.1 67.2±0.1 54.3±0.3 45.0±1.5 3.7±0.1
5.5±0.6
15.5±0.3
17.5±0.6 15.4±0.1
19.1±0.3 22.2±0.1
20.0tl.0
CO2
69.3±0.1 67.8±0.l
59.9±0.2 48.0±1.2 3.7±0.1
4.5±0.1
12.5±0.I
17.2±0.5 15.4±0.1
17.8±0.2 22.0±0.1
20.9±0.6
O2
69.3±0.1 65.6±0.2 52.0±0.6 40.6±0.3 3.7±0.I
7.2±0.1
16.1±0.1
17.8±0.4 15.4±0.1 20.3±0.1 21.6±0.2
18.0±0.3
18 min
25 min
0 mm
10 min
10 min
Omin
10 min
18 min
25 min
^ Mean of 60 determinations with deviation Reprinted from Ref. 12. with courtesy of Amencan Chemistry' Society
Category hedonic scores indicating the sensory peanutty flavor intensity of peanut kernels roasted under various atmospheric conditions are presented in Figure 1. When peanuts were lightly roasted for 10 min, all sensory scores were less than 5 and were essentially independent of atmospheric conditions. After 18 min of roasting, the highest flavor score was obtained by roasting peanut kernels under carbon dioxide and under an ambient enviromrient. These scores were followed in decreasing order for peanuts roasted under nitrogen, under air, without aeration, and under oxygen in the environment. When peanuts were roasted under oxygen for 18 min, the flavor score was 3.5 and
1525
unpleasant off-flavors were detected. Therefore, elevated temperature in combination with elevated atmospheric oxygen content facilitates the formation of the off-flavors. Most peanuts roasted for 25 min were over-roasted and received lower scores compared to those roasted for 18 min. The lowest score was observed for peanuts roasted under oxygen, followed in order by peanuts roasted under air, without aeration, under carbon dioxide, and under nitrogen. Total a-amino nitrogen contents in peanut kernels subjected to roasting under various conditions of atmospheric gas are presented in Table 2. The fre6 amino acids decreased significantly with time of roasting. Roasting time, i.e., the extent of heat treatment, governed the changp of total a-amino nitrogen. However, the effect of atmospheric composition on the total a-amino nitrogen content was not significant. A correlation in the a-amino nitrogen content and sensory evaluation scores (Figure 1) did not exist. Although amino acids are the precursors for the peanutty flavor formation [4,7,8], their attributes and function related to flavor development during roasting have not been clearly defined. According to the studies of Newell et al. [7], amino acids are classified as typical and atypical peanut flavor precursors. The involvement of atypical flavor precursors does not always contribute positively to peanutty flavor formation. In a previous study [5], changes in specific amino acid content in peanuts during roasting was significantly affected by the nature and extent of heat treatment, internal temperature, and moistue content of kernels. Soluble carbohydrate analyses (Table 2) revealed that sugar concentration in peanuts was higher than that reported by Oupadissakoon et al. [9] and Rodriguez et al. [4] but lower than that reported by Mason et al. [8]. In this study, except for peanuts roasted under oxygen for 25 min, total soluble carbohydrate increased as a result of roasting. Sucrose, except for peanuts roasted under air, increased slightly in the early stage of roasting, i.e., the first 10 min of roasting. Oupadissakoon and Yoimg [10] deep-fiied peanuts at 147 ^C for 9-14 min and reported that sucrose contents in some roasted peanuts were higher than the contents in unroasted peanuts. Glucose content also increased during the first 10 min when peanuts were roasted under gas-flushing conditions. Therefore, changes in carbohydrate composition during peanut roasting were variable. In general, soluble carbohydrate, sucrose, and glucose contents increased considerably in the early stage of roasting and tiien decreased with roasting time. Changes in glucose content were not completely consistent with changes
1526
in total soluble carbohydrate and sucrose contents. When peanuts were roasted for 10 min, the glucose content remained essentially unchanged. The glucose content subsequently decreased with time of roasting. After 25 min, the lowest glucose content was observed in peanuts roasted under nitrogen, followed in order for peanuts roasted imder oxygen, without aeration, under carbon dioxide, and under air. This order does not correlate with the order of color change as influenced by roasting conditions (Table 1), indicating that glucose played only a partial role in browning reactions related to color and flavor development. A considerable amount of soluble carbohydrate, sucrose, and glucose was reduced in peanuts roasted under oxygen for 25 min. At the same time, flavor was judged less desirable (Figure 1). On the other hand, peanuts roasted under carbon dioxide for 18 min had the best organoleptic flavor quality* Soluble carbohydrate, sucrose, and glucose were still present in substantial concentrations in these peanuts. Since the concentrations of soluble carbohydrate, sucrose, and glucose may increase due to hydrolysis of insoluble carbohydrates or otherwise decrease due to involvement in further chemical reactions, an explanation for changes in flavor as influenced by the amount of soluble carbohydrate, sucrose, or glucose in roasted peanuts cannot be offered. Flavor evaluation scores for raw peanut oil roasted with partially defatted peaniit meal are presnted in Figure 2. Peanutty flavor formation during roasting was significantly influenced by the addition of partially defatted peanut meal and varied depending upon the atmospheric gas content. The highest and lowest flavor scores resulted from roasting oil with partially defatted meal under CO2 and O2, respectively. When oil was roasted under He and N2, flavor scores were slightly lower than that for oil and meal roasted under CO2. Satisfactory typical peanutty flavor was not achieved by simply roasting the raw oil (without addition of the peanut meal) under an open atmosphere or under CO2 or by roasting the oil with peanut meal under open, vacuum, and air conditions. Since the best flavor was obtained by roasting peanuts under N2 or CQ2 (Figure 1). The presence of O2 apparently influenced the performance of typical peanutty flavor after roasting by inhibition of the flavor formation or direct destruction of the flavor compounds during roasting. On the basis of the general perceptions and descriptions given by panelists that more oxygen in the roasting envirormient resulted in more unpleasant flavor, it was conjectured that oxidation of peanut components at an elevated temperature resulted in
1527
compounds with off-flavors which may mask or interfere with the normal intensity of typical peanutty flavor.
^ ^ •o
4»
Figure 1. Sensory evaluation scores of peanut kernels roasted under various atmospheric gas environments: (open bar) without aeration (control); (diagonally lined bar) air; (horizontally lined bar) nitrogen; (cross-hatched bar) carbon dioxide;(black bar) oxygen. Reprinted from Ref. 12. with courtesy of American Chemistry Society.
Partially defatted peanut meal contains the precursors of peanutty flavor. When raw peanut oil was roasted in the absence of peanut meal under CO2, maybe due to the absence of these flavor precursors, the peanutty flavor did not develop (Figure 2). Peanutty flavor formation during roasting is a result of reactions between amino acids and reducing sugars, using lipid as the reaction medium [10]. During roasting of whole peanut kernels, exposure of the internal portion of cotyledons to oxygen is limited and release of moisture is slow. The initial moisture content of peanut kernels greatly influences reactions of flavor-related precursors and eventually influences flavor formation during roasting [5]. However, when ground raw peanut meal is conventionally roasted in an oven at an appropriate temperature, due to extensive exposure to O2 from
1528
air and rapid release of moisture by surface evaporation, a pleasant roasted peanut flavor does not develop.
Table 2 Total a-amino nitrogen, soluble carbohydrate, sucrose, glucose, and conarachin contents in peanut kernels roasted under various atmospheric gas environments roasting time, mm 0
a-amino nitrogen," mg of amino acid/g of protein 7.65 ± 0.77
W/0
10 18 25
6.37 ± 0.15 3.21 ± 0.15 1.41 ± 0.10
air
10 18 25
N2
soluble carbohydrate," mg/g of defatted meal 86.24 ± 6.60
sucrose," mg/g of defatted meal 75.30 ± 3.18
glucose," mg/g of defatted meal 0.417 ± 0.026
97.38 ± 0.96 95.63 ± 1.28 94.67 ± 1.43
77.56 ± 7.44 82.64 ± 1.52 68.04 ± 3.04
0.398 ± 0.012 0.323 ± 0.007 0.289 ± 0.019
5.93 ± 0.41 3.89 ± 0.33 1.75 ± 0.13
90.86 ± 2.71 99.61 ± 1.28 90.85 ± 2.07
70.96 ± 0.64 72.48 ± 6.50 68.40 ± 13.32
0.404 db 0.016 0.356 ± 0.006 0.305 ± 0.009
10 18 25
6.21 ± 0.71 3.51 ± 0.21 1.51 ± 0.13
97.30 ± 1.36 100.48 ± 1.03 87.91 ± 1.03
81.50 ± 0.78 81.12 ± 1.30 70.02 ± 1.72
0.406 ± 0.010 0.312 ± 0.009 0.266 ± 0.017
CO2
10 18 25
6.39 ± 0.54 4.27 ± 0.25 1.66 ± 0.16
94.67 ± 5.41 96.90 ± 7.48 91.81 ± 1.75
79.82 ± 3.12 71.04 ± 7.98 67.42 ± 4.58
0.411 ± 0.013 0.320 ± 0.012 0.302 ± 0.006
O2
10 18 25
6.12 ± 0.55 3.23 ± 0.14 1.47 ± 0.06
97.38 ± 0.64 95.23 ± 1.35 83.30 db 1.20
81.68 ± 3.18 80.80 ± 2.46 61.14 ± 6.16
0.408 ± 0.002 0.314 ± 0.001 0.282 ± 0.002
gas environment before roasting
Mean of two determinations from two duplicate experiments. ReprintedfromRef. 12. with courtesy of American Chemistry Society.
On the basis of the observation that typical roasted peanut flavor can be enhanced by roasting raw peanut oil and peanut meal under CO2, the effect of moisture content of the peanut meal on the roasted flavor formation was further studied (Figure 3). Flavor scores for the roasted oils increased significantly with increased moisture content (0-15%) of the peanut meals and decreased when the moisture content was higher than 20%. Chiou et al. [5] roasted peanut kernels containing various initial moisture contents and reported that reactions of flavor-related precursors, i.e., amiiio acids and soluble carbohydrates, were influenced significanfly by the moisture content of the peanut kernels. Since a closed system was used in the present study, the moisture in the roasting chamber may have played a role in facilitating hydrolysis of macromolecules to release flavor precursors and enhance flavor formation.
1529
9.
X
81
1
7 6 o u I-
o
5
1
4
1 X
1I
1
31-
A
U. 2
I/I
;?
Ar
>. ^
^-
4' ^ O ^
^ > /^ ^
::S^
NJ
o
Roasting condition Figure 2. Flavor scores for raw peanut oil and oil roasted with partially defatted peanut meal under various atmospheric conditions. Reprinted from Ref. 11. with courtesy of American Chemistry Society. The fatty acid composition of raw and roasted peanut oils is presented in Table 3. Changes in fatty acid profiles as a result of roasting were limited. Peanut oil roasted with peanut meal under vacuum had the highest 16:0 content and the lowest 18:0, 20:0, 20:1, and 24:0 contents. Peanut oil roasted with peanut meal under CO2 had the lowest levels of 16:0 and the highest levels of linoleic acid (18:2) and 22:0. In addition, peanut oil roasted with peanut meal under O2 had the lowest level of linoleic acid (18:2) and its linoleic acid (18:3) content was not detected.
1530
O
o CI
10
20
30
40
50
Moisture content , Q^^ Figure 3. Flavor scores for raw peanut oil roasted with partially defatted peanut meal having various moisture contents. Reprinted from Ref. 11. with courtesy of American Chemistry Society. Tables Fatty acid composition of raw peanut oil and roasted with peanut meal at 160 ^C for 30 min under various atmospheric conditions atmospheric condition
fattv acid. % 14:0
16:0
18:0
18:3 20:0 20:1 22:0 24:0 40.25±0.06 0.03±0.01 1.45±0.05 0.90±0.02 3.07±0.25 1.08±0.09 39.97±0.06 0.04±0.01 1.36±0.05 0.84±0.02 2.70±0.14 0.98±0.09 40.27±0.06 0.05±0.01 1.47±0.02 0.91 ±0.02 3.12±0.04 1.07±0.03 40.01 ±0.14 0.05±0.0I 1.41 ±0.08 0.90±0.03 2.86±0.14 0.94±0.17 40.14±0.01 0.04±0.01 1.19±0.01 0.75±0.01 2.13±0.04 0.60±0.08 40.12±0.15 0.05±0.01 1.41 ±0.04 0.86±0.02 2.91±0.01 1.02±0.07 1.44±0.02 0.87±0.01 3.08±0.01 1.06±0.01 40.42±0.17 1.33±0.03 0.81 ±0.02 2.72±0.14 0.92±0.02 39.78±0.09 N2C 40.25±0.10 0.03±0.00 1.30±0.03 0.80±0.02 2.59±0.02 0.90±0.03 He'^ 40.03±0.11 0.04±0.01 1.35±0.01 0.83±0.02 2.70±0.19 0.91 ±0.08 ^ Raw oil was roasted without peanut meal at open condition. *' Raw oil was roasted vithout peanut meal under CO?. ^ Raw oil was roasted with defatted paenut meal in the ratio of 10/1 (w/w) under the specilied atmosphenc condition. Repnnted from Ref. 11. with courtesy of American Chcmistrv Socictv unroasted control P control II'' open*^ vacuum'^ air* C02C CHc
0.02±0.00 0.03±O.0P 0.02±0.01 0.03±0.00 0.04±0.01 0.03±0.00 0.03±0.01 0.03±0.0l 0.04+0.01 0.03 ±0.00
12.11±0.28 3.23±0.02 13.11±0.35 3.14±0.00 11.85±0.04 3.29±0.01 12.75±0.53 3.i9±0.04 14.46dt0.09 3.05±0.04 12.81±0.32 3.18±0.01 12.06±0.27 3.22±0.01 13.45±0.00 3.18±0.02 13.34±0.22 3.17±0.03 13.43±0.36 3.13±0.0l
18:1
37.58±0.08 37.54±0.10 37.66±0.03 37.48±0.23 37.25±0.14 37.29±0.08 37.51±0.10 37.54±0.07 37.32±0.01 37.25±0.04
18:2
1531
In conclusion, roasted peanut flavor formation was significantly influenced by atmospheric conditions during roasting. For peanut kernel roasting at 210 ""C, the best flavor was obtained by roasting kemels under N2 and CQ2 for 18 min. The presence of sufficient amount of O2 during roasting played a role to mask the typical flavor performance due to generation of rancid flavor simulaneously. For improvement of peanut oil flavor, the flavor was effectively enhanced by roasting raw peanut oil in combination with partially defatted peanut meal at a ratio of 10:1 (v/w) roasted at 160 ""C for 30 min under CO2 condition. The addition of 10-20 % moisture to peanut meal was essential for the formation of peanuttyflavorin oil. 4. ACKNOWLEDGMENT The financial supported by National Science Council, ROC (NSC 810406-E021-01; NSC 82-0406-E021-01), Valuable advice on manuscript preparation by Dr. L. R. Beuchet, University of Georgia are acknowledged. 5. REFERENCES 1. J. G. Woodroof, Peanuts: production, processing, products; AVI Publishing Co., Westport, CT, 1983. 2. T.-T. Tsai, R. Y.-Y. Chiou and T.-C. Lin, Food Sci., 15 (1988) 211. 3. C. T. Young, R. S. Matiock, M. E. Mason and G. R. Waller, J. Am. Oil Chem. Soc, 51 (1974) 269. 4. M. M. Rodriguez, S. M. Basha and T. H. Sanders, J. Agric. Food Chem., 37 (1989) 760. 5. R. Y.-Y. Chiou, Y.-S. Chang, T.-T. Tsai and S. Ho, J. Agric. Food Chem., 39 (1991) 1155. 6. R. Y.-Y. Chiou, S. Femg and Y.-F. Liu, J. Food Sci., 57 (1992) 998. 7. J. A. Newell, M. E Mason and R. S. Matiock, J. Agric. Food Chem., 15 (1%7) 767. 8. M. E. Mason, J. A. Newell, J. B. Rohnson, P. E. Koehler and G. R. Waller, J. Agric. Food Chem., 17(1969) 728. 9. C. Oupadissakoon , C. T. Young and R.W. Mozingo, Penaut Sci., 7 (1980) 55. 10. C. Oupadissakoon and C. T. Young, Peanut Sci., 11 (1984) 6.
1532
11. R. Y.-Y. Chiou, S.-L. Cheng, C.-Y. Tseng and T.-C. Lin, J. Agric. Food Chem., 41(1993)1110. 12. R. Y.-Y. Chiou, C.-Y. Tseng and S. Ho, J. Agric. Food Chem., 39 (1991) 1852.
G. Chai;alambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1533
Color sorting of lightly roasted and deskinned peanut kernels to diminish aflatoxin and retain the processing potency Robin Y.-Y. Chiou^ Pei-Yin Wu^ and Yue-Homg Yen^ Department of Food Industry, National Chiayi Institute of Agriculture, Chiayi, Taiwan, Republic of China Department of Food Engineering, Da Yeh Institute of Technology, Chunghwa, Taiwan, Republic of China Abstract Moistened peanut kernels were inoculated with conidiospores of Aspergillus parasiticus and incubated at ambient temperature for 35 days. Free fatty acid, free threonine, proline, glycine, alanine, leucine, tyrosine, lysine, phenylalanine, histidine and arginine contents increased while sucrose, free glutamic acid, aspaitic acid and serine contents decreased in the inoculated peanuts. When the incubated kernels were roasted at 160°C, 15 mm was appropriate to result in noticeable discoloration of the infected and deskinned kernels vvhich Vvcre available for color sorting. Average aflatoxin content in the discolored sublets was 18,200 ppb and was not detected in the unblemished sublets. A success in apphcation of this process in the commercial processing line to diminish aflatoxin and subjection of the sorted lots for further processing v/as aciiieved. 1. INTRODUCTION Peanut kernels are a good substrate for mold growth when the moisture content, temperature and time permit. The growth of aflatoxigenic molds and subsequent aflatoxin production in peanut attracts a great deal of public concern. In many cases, mold growth results in discoloration of kernels. Removal of the discolored kernels in lots of raw and unblanched peanut is the m.ost common means to diminish aflatoxin contamination in peanut products.
1534
Dickens and Whitaker [1] reported that an average 72% of the aflatoxin in peanuts was removed by electronic sorting and subsequent hand picking of the discolored kernels. However, the efficacy of aflatoxin removal with electronic sorting was highly variable among peanut kernel lots. Since mold growth on peanut kernels does not always result in significant discoloration, unblemished kernels may be contaminated with aflatoxin at a low level. Mold mycelium and spores on kernel surfaces may be removed by routine handhng, rendering kernels acceptable by color sorting. Wilson and Flowers [2] stated that a low level of aflatoxin contamination in peanuts may be endemic and current sorting procedures may not be effective in removing unblemished and contaminated peanuts. In addition to color sorting of peanuts by hand or an electronic sorting machine, other sorting systems have been developed. Pelletier and Reizuer [3] reported that fluorescence sorting of raw and unblanched peanut kernels was not as effective as an aflatoxin control method. Although electronic color sorting has been shown to be efficient in removing aflatoxin-contaminated peanuts from sound peanuts, hand sorting is more effective. Flotation separation and density characterization of aflatoxin-contaminated and sound peanuts have been extensively investigated [4,5]. Separation of aflatoxin-contaminated kernels from sound kernels by hydrogen peroxide treatment has also been attempted [6]. However, when sorting was conducted with unblanched raw peanuts, complete removal of the aflatoxin-contaminated kernels was not achieved. Growths of molds on peanuts causes a reduction of dry matter and oil content, an increase in free fatty acids and deterioration of seed quality and nutritive value [7]. Hydrolysis of the macromolecules, e.g., proteins [8,9], lipids [10] and polysaccharides, occurs during mold infection, resulting in the release of free amino acids, free fatty acids and simple sugars. These breakdown products contribute to color development in peanut kernels during roasting. IVIore rapid color formation is hkely to occur in infected kernels compared to sound kernels. Color sorting is comparatively more efficient for deskinned kernels than for unblanched kernels, reagardless of the degree of infection. In this study, moistened peanut kernels inoculated with Aspergillus parasiticus were incubated at ambient temperature to promote mold growth.
1535
Infected peanuts were roasted for various times followed by analysis for color. Mold populations and compositional changes in infected peanut kernels were determined. Light roasting and deskinning of peanut kernels was done on a conimercial processing line prior to sorting. Aflatoxin content in unblemished and discolored sublots of kernels was determined. Processing potency of the lightly roasted and color sorted peanut kernels was also evaluated. 2. MATERIALS AND METHODS 2A. Peanuts Freshly harvested, sun dried, shelled and hand sorted peanut kernels (Tainan #9, a Spanish cultivar, 7.5% moisture, dry basis) were used for mold inoculation studies. Prior to use, kernels were packaged in polypropylene bags and stored at -25 ^C. Sealed bags of kernels were removed from the freezer and tempered at ambient temperature (25-30 ^C ) overnight before opening. A collaborative study was conducted in a peanut processing company using commercial lots of peanuts destined for honey-roast processing. Commercially obtained kernels were size graded, electronically sorted and hand picked prior to roasting and deskinning. 2.2. A. parasiticus infection of peanuts and determination of total mold count For each experiment, 200 g of peanut kernels in polypropylene bags were evenly sprayed with 15 g of water to increase the moisture content to approximately 15% (dry basis). Twenty-five kernels were gently rolled on the surface of a 7-day-old culture of A. parasiticus NRRL 2999 grown on yeastmalt agar, added to each 200-g quantity of peanut kernels and thoroughly mixed. The procedure for inoculadon resulted in 9.8 X 10"^ conidia per gram of peanut kernels as determined by plating appropriately diluted suspensions of v/ashed kernels on oxytetracychne glucose yeast extract (OGYE) agar and d for 3 days at amient temperature. Suspensions were prepared by combining 45 ml of phosphate buffer (pH 7.0, 0.05 M and containing 0.01% Tv/een 20) v/ith inoculated kernels and vigoriously shaking for 2 min and subjecting to subsequent dilutions. Bags contairdng inoculated peanuts were punctured with a pin to facilitate
1536 air exchange and incubated at ambient temperature (25-30 ^C)for 35 days. After incubation, total mold populations on kernels were determined as described above . The remaining peanuts were heated at 45 ^C in an oven for 48 h to reduce moisture content to an original level. These peanuts were used in the following experiments. 2.3. Compositional analyses of A. parasiticus infected peanuts A. parasiticus infected peanut kernels were freeze-dried and deskinned. Kernels were then hydrauhcally pressed to prepare oils and paitially defatted press cake. Free fatty acid (FFA) , conjugated diene hydroperoxide (CDHP) contents and fatty acid composition of the oils were determined [11,12]. Partially defatted flour was prepared by grinding the press cake in a cyclone mill and defatting with n-hexane (- 20 ^C). Sucrose, glucose and free amino acid contents were determined [13]. 2.4. Color determination of peanuts during roasting Mold-infected and sound (uninfected) peanut kernels were deskinned, placed on a hot aluminum block and roasted in an oven at 160 ^C. The block v/as removed from the oven at 5-min intervals for the purpose of photographing. The color of the deskinned peanuts in photographs, expressed as L , a and b values, were measured with a color difference meter (Nippon Denshoku Ltd., Osaka, Japan). At each roasting time, color differences between A. parasiticus infected and sound peanuts were dertermined by subtracting L, a and b values measured for each treatment. 2.5. Peanut roasting, color sorting and chemical analyses Preliminary results indicated that mold infected peanut kernels discolored more rapidly than did uninfected kernels during roasting at 160 °C for 15 min. A. parasiticus infected peanut kernels, as well as raw kernels, were roasted in an oven at 160 ^C for 15 min, manually deskinned, hand sorted and divided into unblemished (accepted) and discolored (rejected) sublots. Sound raw kernels and the unblemished kernels were freeze dried and subjected to sucrose, CDHP, FFA and free amino acid analysis following the procedure described previously. On a commercial scale, 200-kg batches of peanut kernels were
1537
roasted at 160 ^C for 15 min in a roller roaster. After roasting, peanuts were desldnned and hand sorted. Weight percentages of unblemished and discolored kernels in sublets were determined and samples from sublots were subjected to aflatoxin analysis. 2.6. Aflatoxin analysis Peanut kernels were ground into meal with a cyclone mill. The procedure reported by Tarter et al. [14] was followed for aflatoxin extraction with minor modification. Meal (10 g) was mixed with 40 ml of methanol and 10 ml of 0.1 N KCl, hom.ogenized for 1 min with a homogenizer (Polytron FT Mr-3000, ICinematica AG, Littau, Switzerland) and then filtered using Advantec #2 filter paper (Toyo, Japan). Twenty miUiUters of the filtrate were mixed with 20 ml of hexane and 20 ml of NaCl (10 %) in a 250-ml flask with a sihcone stopper and shaken at 250 rpm with an orbital shaker for 10 min. Then, 25 ml of the aqueous solution was mixed with 5 mi of dichloromethane and shaken as described above. After the dichloromethane was withdrawn, the remaining solution was extracted twice with 5 ml of dichloromethane. The pooled dichloromethane extract solution v/as placed in a desiccator and evaporated to dryness with an aspirator. Methanol (0.5 ml) was added to dissolve extracted substances; 5 jil of the solution was applied to a TLC plate (DC-Alufolien Kieselgel 60F254, E. Merck, Darmstadt, Germany) which was then developed with chloroform-acetone (9/1, v/v). The quantity of aflatoxins separated on TLC plates was determined using a fluorescence densitometer (Scanner II, Camag, Switzerland ) and an aflatoxin standard containing known quantities of aflatoxin Bi, B2, Gi and G2 spread on the TLC plate concurrently. 2.7. Processing quality evaluation of the lightly roasted and color sorted peanut kernels Sound raw kernels were roasted in an oven at 160 ^C for 45 min to reach a medium extent of roasting. After roasting, kernels were manually deskinned and prepared as a control sample for sensory evaluation. As a preliminary result, based on the color performance during roasting, an additional 50 min of roasting time was required to roast the hghtly roasted, deskinned and color sorted kernels at 160 ^C to reach the same extent of roasting in terms of color formation. Color of both kernels expressed as L , a and b values was
1538
determined as described above. In the meanwhile, the kernels were subjected to sensory evaluation using a triangle test participated by 60 panelists and instructed to differentiate the samples mainly by roasted peanutty flavor and texture. Texture of the kernels expressed as breaking intensity, softy and hardness was also determined with a rheometer [15]. 2.8. Statistics Duphcate experiments were conducted. Means of determinations with standai^d deviations ai*e presented. 3. RESULTS AND DISCUSSION Free fatty acid, conjugated diene hydroperoxide (CDHP) contents and fatty acid composition of peanut oils prepared from Aspergillus parasiticus infected peanut kernels are shown in Table 1. The free fatty acid content in infected peanuts was significantly higher than the control peanuts. This is in agreement with the observations described by Pattee and Sessoms [10] and Diener [8]. Lipolysis .of peanut oils resulted from lypase action of molds. However, extensive Upid peroxidation of oil did not occur. The CDHP content in mold-infected peanuts was not significantly higher than that in control peanuts. This was further supported by the fact that change in fatty acid composition resulting from mold infection was not significant (Table 1). In other words, the increase in free fatty acid content in oils of mold-infected peanuts did not necessarily influence the overall fatty acid composition. The highest mold population was observed in peanuts infected with A. parasiticus. Mold populations were very low in peanuts that were not iHoistened, being similar to populations in peanuts stored frozen during the incubated period. Sucrose, glucose and free amino acid contents in infected peanut kernels are present in Table 2. The difference between two control peanuts was limited. Tlie sucrose content was lower in infected peanuts than in control peanuts while glucose content was shghtly higher in infected peanuts. The total free amino acid content v/as higher in control peanuts than in infected peanuts. In comparison of infected and control II peanuts. Most of amino acids except glutamic acid, aspartic acid and serine increased. Increases in threonine.
1539
proline, glycine, alanine, leucine, tyrosine, lysine, phenylalanine, histidine and arginine contents in infected peanuts were pronounced. Increase of some amino acids undoubtedly affects color and flavor formation during roasting [16]. Table 1 Free fatty acid, conjugated diene hydroperoxide content and fatty acid composition of peanut oils, and mold populations in peanut kernels after A, parasiticus infection Treatments and determinations^ Item A. parasiticus infected Control I*^ Control I f FFA, mg/g oil 0.42±0.01 0.62±0.01 56.79±23.63 CDHP,OD 234nin/mg oil 0.12±0.01 0.13±0.01 0.12±0.01 Fatty acids, % 14:0 0.03±0 0.03±0 0.03±0 16:0 12.80+0.04 12.97±0.02 12.75±0.03 18:0 3.58+0.06 3.57±0.01 3.58+0.01 18:1 38.51±0.05 38.17±0.32 38.23+0.02 18:2 38.40±0.01 38.57±0.05 38.79±0.22 1.60±0 1.55+0.03 20:0 1.60±0.01 20:1 0.81±0 0.83±0 0.83±0.01 22:0 3.03±0.01 3.10±0.01 2.90±0 1.14±0.03 24:0 1.14±0.02 1.06±0.01 Mold count, CFU/g peanut <10^ <102 2.3x10^ a)Mean of determinations with standard deviation b)Controi I : raw kernels stored at •-25°C c)Control II : non-moistened kernels stored at ambient temperature
Color changes occurred in deskinned kernels during roasting at 160 ^C for 30 min, as evidenced by differences of L, a and b values between A. parasitciis infected and sound kernels (Figure 1). After 15 min of roasting, infected kernels underwent considerably more changes in color than did the control (sound) kernels. Rapid discoloration of the mold infected kernels
1540
facilitate sorting by hand or machine. Visually, the extent of color difference between infected and control kernels decreased after 15 min of roasting. Therefore, 15 min of roasting was designated as an appropriate time to enable differentiation of infected and sound kernels. Table 2 Sucrose, glucose and free amino acid contests in peanut kernels after A. parasiticus infection Treaments and determinations^ A. parasiticus Items infected Control IF Control I*^ Sucrose,mg/g defatted meal 85.41±0.25 37.04±7.96 85.40±0.43 Glucose,mg/g defatted meal 1.56±0.02 1.62±0.09 1.72±0.27 Free amino acid, mg/g protein 0.12±0 Asp 0.20±0 0.22±0.01 Thr 0.19±0.01 0.45±0.02 0.38±0.02 Ser 0.54±0.01 2.68±0.09 0.64±0 Glu 2.69±0.05 0.31±0 0.24+0.02 Pro 0.23±0.01 0.11±0.01 0.41±0.01 Gly 0.11±0 1.22±0.06 0.74±0.03 Ala 0.80±0.01 0.25+0 0.26+0.01 Val 0.16±0 Met 0.11±0.02 0.11±0.01 He 0.11±0 0.06±0.01 0.14±0.01 Leu 0.06±0 0.36±0.01 Tyr 0.15±0 0.13±0 0.95±0.05 Phe 0.76±0.06 0.78±0.09 I-iis 0.11+0.01 0.22±0 0.08±0 Lys 0.02±0 0.12±0.01 0.03±0 0.29±0.06 Arg 0.13±0 0.13±0 5.71 Total 6,07 6.02 a)Mean of detenninations with standard deviation b)Control I : raw kernels stored at -25''C c)ControI II : non-moistened kernels stored at ambienit temperature
1541
>
Time, min Figure 1.Color changes expressed as differences of L, a and b values between Asperillus parasiticus infected and sound kernels during roasting at 160 ^C for 30 min. Since peanut roasting characteristics are affected by their initial moisture content [13], moistened and mold infected peanut kernels were dried at 45 °C to their original moisture contents prior to roasting at 160 °C for 15 min. After roasting, peanuts were manually deskinned, sorted into sound (unblemished) and discolored sublots and subjected to weight percentage determination and aflatoxin analysis (Table 3). Weight p.ercentages of the sound sublots in A. parasiticus infected lots was 7.7 %. Aflatoxin was not detected in unblemished sublots. The average aflatoxin content was 18,200 ppb in the A. parasiticus infected and discolored sublots and varied considerably among lots . Sucrose, CDHP, free fatty acid, and free amino acid contents in raw and lightly roasted and color sorted peanuts are shown in Table 4. In general, the difference was very Umited. CDHP content was shghtly higher and serine.
1542
glutamic acid, proline, phenylalanine and histidine contents were slightly lower in the lightly roasted and color sorted peanuts tlian in the raw peanuts.
Weight percentages and aflatoxin content in sound and discolored sublots sorted from A. parasiticus infected and commercial lots of peanut kernels roasting and d(^skinning Sample Sublots Weight Aflatoxin, ppb^ B2 Gi G2 percentage Bi lots Control I^^ Sound 100 nd nd nd nd Discolored 0 100 Control 11^ Sound nd nd nd nd Discolored 0 Aspl-'Asp5 nd nd nd Sound 7.7 nd (5 lots) Discolored 92.3 5411 1200 7045 4511 COMl~COM17 (17 lots)
Sound Discolored
COM18--COM30 (13 lots)
Sound
99.9 0.1 99.9
of peanuts after light
total nd nd nd
±2594
±640
±5614
±4012
18200 ±7270
nd
nd
nd
nd
nd
142.78
122.17
402.08 ±302.99 nd
73.34
68.56
±97.60
±82.28
±104.53 ±149.95
nd
nd
nd
nd
Discolored 0.1 nd nd nd nd a)Mean of determinations with standard deviation b)Controi I : raw kernels stored at -25°C c)Control II : non-moistened kernels stored at ambient temperature Asp : Aspergillus parasiticus infected lots COM : Commercial lots
nd
When Hght roasting, deskinning and color sorting was applied to a commercial scale using 30 lots (200 kg for each), aflatoxin was not detected in sound sublots. Aflatoxin content varied considerably among discolored sublots. Aflatoxin was not detected in 13 lots. In the remaining 17 lots, the average aflatoxin content was 402 ppb and ranged from non-detected to 976 ppb. Since the commercial peanut kernels were size graded, electronically sorted and hand
1543 picked, the weight percentage of discolored peanut kernels after Hght roasting and deskinning was about 0.1 %. Based on the fact that aflatoxin was not detected in the sound sublets, complete removal of aflatoxin by the appUcation of light roatsting, deskinning and color sorting was nearly achieved.
Table 4 Compositional comparison between raw and the light roasted and color sorted Dcanut kernels Peanut and determinations* Item Raw peanuts Lightly roasted and sorted Sucrose, mg/g 81.83±2.80 81.94±2.06 CDHP, OD 234nm/mg oil 0.120±0.01 0.134±0.03 FFA, mg/g oil 0.597±0.02 0.596±0.05 Free amino acids, mg/g protein Asp 0.20±0 0.22±0.01 Ser 0.54±0.01 0.47±0 Glu 2.69+0.05 2.66±0.01 Pro 0.23±0.01 0.19±0 Gly 0.11±0 0.09±0.01 Ala 0.80±0.01 0.78±0.01 Val 0.16±0 0.23±0.02 lie 0.11±0 0.10+0 Tyr 0.15+0 0.15±0 Leu 0.06+0 0.07±0 Phe 0.76±0.06 0.68±0.01 His 0.11 ±0.01 0.08±0 Lys 0.02±0 0.02±0 Arg 0.13±0 0.13±0 Total 6.07 5.87 *Mean of determinations with standard deviation
From the viewpoint of peanut industry, processing potency of the lightly roasted and sorted peanut lots is seriously concerned. When the kernels were subjected to roasting, an additional 50 min of roasting was required to reach the same extent of roasting in comparison to raw kernels subjected to direct
1544
roasting (Table 5). Based on the results obtained in the sensory evaluation, the organoleptic quality of the hghtly roasted and color sorted kernels subjected to further roasting for 50 min was close to that of raw kernels roasted directly at the same temperature for 45 min. Therefore, except the consideration of additional time and fuel was required for the roasting of the lightly roasted and sorted peanuts, the processing potency of the sorted peanuts was retained. Table 5 Color and texture determinations and sensory evaluation of the light roasted and color sorted peanuts subjected to further roasting at 160°C for 50 min (indirect roasting) and raw kernels subjected to the same roasting condition for 45 min (direct roasting) Peanut and determinations* Item Indirect roasting Direct roasting Color 62.14±2.2 61.34±2.88 L 4.85±1.04 5.41±1.06 a 20.68±0.51 b 20.41±0.65 Texture 1223±228 1234±304 BRK Int, mega g/cm 0.09±0.01 0.09±0.01 Softy, cm/dyn 1843±1103 Hardness, mega dyn/cm 2108±1250 Sensory evaluation Triangle test Wrong: 61.67% Right: 38.33% *Mean of determinations with standard deviation In conclusion, A. parasiticus infected peanut kernels underwent color changes more rapidly during roasting than did sound kernels. Fifteen mdn of roasting was sufficient to differentiate and sort the discolored kernels. Deskinning of the kernels before sorting, aflatoxin was not detected in the sound sublots generated from laboratory lots or in commercial lots. Based on the fact that desirable roasted peanut flavor and texture was achieved by subjecting the lightly roasted and sorted peanut to further roasting, the processing potency of the Ughtly roasted and color sorted kernels was accepted by the industry.
1545
4. ACICNOWLEDGEMENT The financial support for this study by National Science Council, R.O.C. (NSC 83-0406-E021-001), kindly supply of peanut processing facility by Everwinning Co. (Chiayi, Taiwan), valuable advice in the manuscript preparation by Dr. L. R. Beuchat, University of Georgia and the helpful assistance in the laboratory by Ms S. H. Lin are acknowledged. 5. REFERENCES 1. J. W. Dickens and T. B. Wliitaker, Peanut Sci., 8 (1975) 11. 2. D. M. Wilson and R. A. Flowers, J. Am. Oil Chem. Soc, 67 (1978) 111 A. 3. M. J. Pelletier and J. R. Reizner, Peanut Sci., 19 (1992) 15. 4. J. C. Henderson, S. H. Kreutzer, A. A. Schmidt and W. R. Hagen, Flotation Sepai-ation of Aflatoxin Contaminated Grain or Nuts, US Patent No. 4 795 651 (1989) 5. V. Gnanasekharan, M. S. Chinnan and J. W. Domer, Peanut Sci., 19 (1992) 24. 6. M. R. S. Clavero, Y.-C. Hung and L. R. Beuchat, J. Food Prot., 56 (1993) 130. 7. U. L. Diener, In Peanuts: Culture and Uses, Am. Peanuts Res. Educ. Assoc, Stillwater, Ok, 1973. 8. J. P. Cherry, L. R. Beuchat and P. E. Koehler, J. Agric. Food Chem., 26 (1978)242. 9. J. P. Cherry, C. T. Young and L. R. Beuchat, Canadian J. Botany, 53 (1975) 2639. 10. H. E. Pattee and S. L. Sessoms, J. Am. Oil Chem. Soc, 44 (1967) 61. 11. S. H. Yoon, S. K. Kim, M. G. Shin and K. H. Kim, J. Am. Oil Chem. Soc, 62 (1985) 1487. 12. R. Y-Y. Chiou, S.-L. Cheng, C.-Y. Tseng, T.-C. Lin, J. Agric. Food Chem., 41 (1993) 1110. 13. R. Y.-Y. Chiou, Y.-S. Chang, T.-T. Tsai and S. Ho, J. Agric. Food Chem.,
1546
39(1991)1155. 14. E. J. Tarter, J. P. Hanchay and P. M. Scott, J. Am. Oil Chem. Soc, 67 (1984) 597. 15. L.-I. Bai, R.Y.-Y. Chiou and C.-W. Chen, Food Sci., 17 (1990) 47. 16. J. A. Newell, M. E. Mason and R. S. Malock, J. Agric. Food Chem., 15 (1967)767.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1547
Carbohydrate metabolism in peanuts during postharvest curing and maturation J.R. Vercellotti% T.H. Sanders^ S-Y. Chung% K.L. Bete, and B.T.Vinyard^ aU.S.D.A.-A.R.S.-Southern Regional Research Center, PO Box 19687, New Orleans, Louisiana 70179-0687, USA ^U.S.D.A.-A.R.S.-Market Quality and Handling Research, Dept. of Food Science, North Carolina State University, PO Box 7624, Raleigh, North CaroUna 27695, USA Abstract Metabolism of low molecular weight peanut (Arachis hypogaea, L.) carbohydrate was studied over three crop years to compare windrow drying (4 days) followed by forced air finishing with more gradual moisture removal in stackpole curing (up to 40 days). Soluble carbohydrates were comprised of reactive hydroxycarbonyls (reductones; ca. 12% of total carbohydrates by weight, consisting of 2 to 5 carbon chain metabolic species) as well as inositol, glucose, fructose, sucrose (>80% total sugars), raffinose, and stachyose. In both windrow and stackpole curing, carbohydrate content of the immature peanuts (yellow/orange hull scrape color) declined from >9% to 8% of seed weight as their seed mass contribution diminished from ca. 40% to <20% of total seed harvested. Mature peanuts (black/brown hull scrape color) maintained a steady carbohydrate content (5 to 6% of seed weight) throughout curing, but increased in total seed mass from ca. 60% to >80% throughout postharvest maturation of the immature seed. This data set supports the hypothesis that seed carbohydrate content is inversely proportional to seed maturity but relatively constant for a maturity class during curing. The concentration of all sugars decreased upon maturation during curing in the order: stachyose>glucose>fructose>sucrose>reductones>raffinose>inositol 1. INTRODUCTION In order to achieve highest grades of peanuts (Arachis hypogaea, L.) with desirable flavor and other physical properties, both optimum harvest date and careful curing through controlled postharvest moisture reduction must be observed [1-4]. Sanders and coworkers published a communication in 1990 on peanut curing
1548 which related continued maturation to slow curing processes [5]. Pattee and coworkers [7-8] studied changes in composition of various parts of the peanut during the maturation process and emphasized the importance of optimization of harvest date for quality product. Maturity, curing, and the interaction between these two variables have previously been shown to affect peanut flavor [1, 9-11]. Curing refers to the process during which the high moisture content of the freshly dug peanuts (often >60%) is reduced to a final stable equilibrium level of the dormant seed [6]. Before approximately 1953 peanuts were stacked around poles to dry for several months. Today 2 to 3 days of windrow drying are followed by mechanized picking and drying with forced hot air for 18 to 24 hours to achieve a final moisture content of 9 to 10%. Little is understood, however, about the simultaneous biochemical processes which occur during the curing process and result in hydrolytic or other bioenergetic reactions. Recent developments in flavor chemistry have focused on the key role of the pyrolytic reactivity of matrix components of cereals, coffee and cocoa beans, chicory, etc., [12] under roasting conditions to generate both olfactory and taste stimuli important to the overall flavor [13]. Similar effects have been found in the matrix components of the peanut [14-19] which undergo thermal transformations during roasting. Peanut proteins, polysaccharides and other complex carbohydrates, and lipids (also involved in pyrolytic reactions of the roasting process) contribute to flavor and physical properties of the roasted peanut [20]. As confirmation of high performance ion chromatographic determination of sugars and highly reactive, electron transfer hydroxycarbonyls (reductones; ca. 12% by weight of 2 to 5 carbon species from the peanut metabolic carbon pool) in complex mixtures of peanut flavor precursors, extracts from defatted samples were derivatized as methylated alditol acetates to identify and semi-quantitate them by gas chromatography-mass spectroscopy-total ion current detection (G.C.-M.S.T.I.C.) [21]. Though documented in some detail in various papers and reviews [20, 22-27], the more reactive hydroxycarbonyl compounds (reductones) have not been qualitatively or quantitatively defined as a metabolically changing set of precursors of roasted peanut flavor, principally for lack of suitable analytical methodology. The complexity of peanut matrix carbohydrate as contributing to its roast and physical colloidal properties was demonstrated recently in a paper by Cardozo and Li [28] in which they determined nonstarch polysaccharide dietary fiber content in peanuts (87 percent) by summing the quantities of component sugars analyzed as alditol acetates by gas chromatography. In Vercellotti et al. [21] ion chromatography/ integrated pulsed amperometric detection data from carbohydrate extracts (80% ethanol) of defatted raw peanuts were compared to G.C.-M.S.-T.LC. chromatograms of sugar derivatives made (after stabilization through reduction) by methylation analysis and acetylation. The technique was first tested on a set of samples from one crop year [21] representing stages of peanut maturity and turnover of metabolites during postharvest maturation and curing. Although there are many interfering substances in the ion chromatography such as amino acids, peptides, tannin, and carboxylic acids, the G.C.-IM.S.-T.LC. total ion
1549 chromatograms of the characteristic methylated derivatives confirm relative quantities as well as identities of the reductone/sugar components separated as ion chromatography peaks. The results of Vercellotti et al. [211 indicated depletion of sugars and reductones from immature peanuts during the curing process and stabilization of mature peanuts at much lower level of reactive reductone precursors. As reported earlier [10-11] peanuts from immature classes developed more fruity fermented off-flavor and less roasted peanutty flavor than mature peanuts when all were cured above ambient temperature. The importance of the relationship between degree of maturity and flavor properties in cured peanuts is apparent from the previous studies on the effects of maturity on roast color and descriptive flavor of peanuts [1]. In the present paper, changes in carbohydrate content during postharvest maturation and curing were tested for three crop years, 1989, 1990, and 1991 in an attempt to establish a quantitative relationship between final dry weight percent of each hull scrape color maturity stage with carbohydrate carbon pool turnover. 2. MATERIALS AND METHODS 2.1. General Methods for Determination of Peanut Matrix Composition Methods of the AOAC [29] and AOCS [30] were consulted for proximate compositional analysis and other special techniques such as total dietary fiber determination [31]. Statistical analysis was performed with SAS/STAT User's Guide and software [32] employing correlation matrix for each maturity level; carbohydrate trends across curing time by maturity scatter and regression equations; maturity analysis of covariance comparing maturity trends for each response; principal component analysis for carbohydrates and maturity classes; and stepwise discriminent analysis. Florunner peanuts were selected from a 1989, 1990, and 1991 crop years curing study with various maturity samples separated by hand into hull-scraped classes [4] of yellow, orange, brown, and black in ascending degree of maturity, respectively [1, 5,10, 33, 48-34]. In each of the crop years samples were taken at time of digging (120 or 135 days after planting, harvest day); after four days windrow drying and final commercial-scale, forced warm air wagon drying for 21 hours (Windrow Day 4). Stackpole-cured peanuts also were harvested at 120 or 135 days of growth with samples taken after 10 to 12 days (Stack 10); after 20 to 23 days (Stack 20); and after 40 to 43 days of slow curing (Stack 40). For windrow and stackpole samples, immature (yellow and orange, hull-scraped maturity class) and mature (brown and black, hull-scraped maturity class) peanuts were hand selected. After defatting with hexane (to include nonpolar triglycerides), some sugars, peptides, amino acids, phospholipids, and glycolipids, as well as conjugated saponins, were extracted into methanol:chloroform (3:2) (Figure 1). From the same meal as previously extracted with methanol:chloroform the soluble reductones and further low molecular weight
1550 Hexane Defatted Dry Peanut Meal, 250 mg Add 3.4 ml methanol/chloroform (60:25) 0.6 ml water
Vortex 3 minutes Centrifuge @ 1000 rcf for 5 min Remove supernatant Repeat extraction Pool extracts
Organic Layer
Meal (damp) Add 2 ml of 80% ethanol
Vortex 3 minutes Centrifuge® 1000 rcf for 5 m i n u t e s Remove supernatant Repeat extraction Pool extracts
i i
Meal Mea
Organic Layer I
I Combine (ca. 12 ml)
I Discard Filter (Millex HV 0.45 pm) into a weighed flask
Remove solvent on rotary evaporator. Dry in vacuum oven at ambient temperature over calcium sulfate. Determine weight of sample. (Sirupy sample can be frozen at -SO'C for storage.;
Add 18 megOhm water by weight to sirup to a concentration of 2.5 mg/g. Swirl to dissolve.
Filter (Millex GV 0.22 pm) into 15 ml tube Keep in ice.
Dispense in 1ml aliquots into cleaned, baked borosilicate snap, crimp vials. Place caps on, but do not crimp. Freeze at -60°C .
When frozen, crimp vials quickly to avoid thawing. Store at -80°C until analyzed.
Figure 1. Extraction of low molecular weight carbon compounds from defatted peanut meal for chromatography.
1551 oligosaccharides were extracted with 80% aqueous ethanol. The combined organic extracts were concentrated by rotary evaporation under mild conditions followed by lyophilization to ensure that no condensation or Maillard-type reactions (Figure 1) occurred. After dissolving in sufficient water to solubilize the foamy solid, the sample was filtered through a Swinney adapter fitted with a 0.45 yxm nylon filter covered with a spun glass filter disk. The concentration of each sample was brought to 2500 parts per million (0.25% weight/weight) by weighing diluting water for use in chromatographic and colorimetric experiments. 2.2. Chromatographic and colorimetric techniques Pronase^^, a-amylase, amyloglucosidase, pectin lyase, and papain were obtained from Sigma Chemical (St. Louis, MO). Fractionation of proteinaceous material by ammonium sulfate gradients was carried out as described by Nasir-ud Din et al. [35]. Protein was determined by the method of Lowry et al. [36], as adapted by Smith et al. [37], using bicinchoninic acid as copper (I) complexing agent (Pierce Chemical, Inc., Rockford, IL, Bulletin 23225). Free amino acids and peptides were estimated by quantitative ninhydrin assay adapted by Yemm and Cocking [26,38]. Uronic acids and pectin were quantitated by the m-hydroxybiphenyl determination of Blumenkrantz and Asboe-Hansen [39]. Total carbohydrate, both free and hydrolyzable to reducing sugar, was determined by the phenol-sulfuric acid method of Dubois et al. [40]. Glycosidic hydrolysis was done by the procedure of Albersheim et al. [41] with trifluoroacetic acid or by the primary and secondary sulfuric acid hydrolyses reported by Englyst and Kingman [42]. Component sugars were determined as alditol acetates, prepared by the procedure of Metz et al. [43] and Li et al. [44] using the gas chromatographic equipment described below for the methylation analyses. Gas chromatography was carried out with a DB-225 capillary column (0.33 mm x 30 m; J & W Scientific, Folsom, CA), instead of a packed OV-225 column, on a Hewlett-Packard Model 5790 gas chromatograph with flame ionization detector, computing integrator, and HP 3359 laboratory automation system. Thin layer chromatography was carried out on the sirupy 80% ethanol extract on Kieselgel 60 F-254 glass plates coated with 0.25 mm thickness of silica gel (Merck, Darmstadt) as reported previously [21]. The plates were developed with ethyl acetate:acetic acid:methanol:water (5:3:3:2) and zones detected with ultraviolet light or by spraying with 5% sulfuric acid in ethanol, followed by heating at 110°C to char the zones. 2.3. Liquid chromatographic analyses of the soluble extract Gel-filtration was effected using a Waters HPLC system with multiple wavelength ultraviolet detector and two Dupont (Wilmington, DE) GF-250 and GF450 columns coupled consecutively (0.2 M, pH 7, potassium phosphate buffer), with molecular weight calibration through appropriate polysaccharide, peptide, and protein standards (Sigma Chemical, St. Louis, MO). Sugars in the combined organic soluble fraction described above (Figure 1) were also determined by ion chromatography with a Dionex BioLC instrument using a Dionex CarboPac PAl
1552 column and integrated pulsed amperometric detection (H.P.L.C.-I.C.-I.P.A.D.) with 2-amino-2-deoxy-D-glucose as internal standard [21]. Each chromatographic sample was run in triplicate, and precision as well as recovery of spiked standard samples had a range of ± 1-3% standard deviation throughout the study. Reductones were estimated from the area of the total complex multiple peaks occurring at retention times less than that of the internal standard (7.2 min) less the area of the inositol peak (2.3 min). 3. RESULTS AND DISCUSSION 3.1. Total dry weight percentage of peanuts across curing treatments In the present paper, in addition to the 1991 crop year peanut curing study considered in Vercellotti et al. [21], the previous two crop years, 1989 and 1990, have now also been analyzed for changes in carbohydrate and total weight percent of hull-scraped maturity stages during curing; results of all three crop years are consolidated in this study of the data. Plant physiological and seed developmental data on the role of maturation in quality of stackpole cured peanuts are currently in preparation and will soon be reported in detail by Sanders and coworkers [45]. The changes in maturity classes as the harvested sample underwent moisture removal are illustrated in Figures 2, 3, and 4. The 4-day windrow reference sample in each year was also compared to the stackpole or slow curing treatment on the same figures. These three figures indicate similar maturity distributions for all three crop years. The principal conclusion is that during the slow curing process (stackpole) significant peanut maturation occurred for all three crop years and it appears that a similar trend occurred in windrow drying. The mature brown and black classes of peanuts increased and plateaued in total weight percentage after 20 to 30 days in the stackpole and after only four days in the windrow followed by forced air dr3dng to 7 to 8% moisture. Concomitantly, the immature orange and yellow maturity classes decreased proportionately. To summarize this observation, as previously described by Sanders et al. [5], during slow curing peanuts continue to be metabolically active and apparently immature peanuts are being converted towards more stable, greater-valued mature peanuts. Thus, proper curing is a process necessary to maximize peanut physical properties and roasted flavor potential, as proven by the large number of papers cited in this report. However, the total mass of peanuts in each maturity class is not identical each crop year. For example, the total yield of dry weight was not as great in the 1990 crop year, probably because it was a much drier year, but the same general trends regarding quantitative relationships between total mass and carbohydrate content hold as in the 1989 and 1991 crops (approximately 25% greater 3deld than in 1990). Similar changes are noted below in the diminished carbohydrate content of the 1990 crop year stackpole and windrow peanuts presumably also due to drought effects.
WEIGHT % -
80 70
-5-\----.-
--
--
Immature
Black
Maturity Stage
80
1 , \
- -
l iane\r 113)
Figure 2. Final shelled ~veightdistributiotl, windrow and stackpolc pcanuts. 1989 crop J. car.
--=---
WEIGHT %
-- ---/--,
. . 7
i !
70 --, . 60 :.i,------! '
80
j
- -
.
,
4-Bindrnrr
Yellnw . .
Maturity Stage
:p-:
- -- -
I Ian crl
----lo
Lp,e
lid!
Figure 3. Final shelled weight distribution, windrow and stackpole peanuts. 1990 crop ycar.
Days Cured
WEIGHT %
4-Hindnrrr
- .-
Maturity Stage
.-
Days Cu
nu!
IInncs~
Figure 4. Final shelled weight distribution, windrow and stackpole peanuts. 1991 crop year.
Pll
1556 3.2. Extraction and analyses of carbohydrates by ion chromatographypulsed amperometric detection Comparison of >500 samples required optimization of extraction with highest recoveries (>95%) obtained using combined residues from both methanol:chloroform (3:2) followed by 80% ethanol (Figure 1). Recoveries of spiked sugar samples in the defatted peanut meal were nearly quantitative as estimated by I.C.-I.P.A.D. (100 ± 3%). Table 1 gives the mean distribution and standard deviations (n = 9) of peanut sugars across maturity stages for the three crop years of the curing study. Table 2 shows the grand mean and standard deviations (n = 9) of total peanut soluble carbohydrate in four maturity stages for three crop years. The % standard deviation values in Tables 1 and 2 represent both variability in the analytical method as well as crop year differences in plant metabolism. The changes in sugar composition with postharvest maturation during curing shown in Tables 1 and 2 are the basis for the curing model described below. In Table 3 the percent mean and standard deviation (n = 9) proximate protein and oil analyses for peanut curing over the three years are an attempt to bracket the regularity of the metabolic process from year to year as well as within maturity stages. Amino acids, peptides, structural and storage polysaccharides such as pectin or starch were quantified and confirmed in these proximate compositional analyses according to the methodology cited in Materials and Methods. Although the mesocarp color is related to maturity and compositional characteristics of peanuts [46-49], the exact biochemical relationships that take place simultaneously in the seed during postharvest maturation with those in the mesocarp, used to identify the maturity stage as pigmentation markers, is unknown. The mixtures of maturity stages and years represented in Table 3 for changes of major metabolites such as lipid accumulation and protein synthesis with maturation are significant, and Table 3 confirms the previous work cited by Sanders et al. [5, 33, 48]. Peptides and amino acids are also extracted into this mixture along with glycosides of tannins or phenolic acids. In the previous paper [21] detailing the identification of sugars and reductones from the 1991 crop year curing study for peanut soluble carbohydrates, only the 80% ethanol extract was pooled for ion chromatographic analysis. Here, the combined organic extracts (Figure 1) used in the present study permitted greater efficiency of recovery from spiked samples. In addition, the other components mentioned above are most probably part of the Maillard browning or caramelization substrates for forming peanut flavor [20, 50-51]. The enhanced sensitivity of the pulsed amperometric detector for the reducing sugars or polyhydroxy carbonyl metabolites permitted quantitation of this extract as the detector discriminated between the carbohydrates and other natural product components such as peptides because the carbohydrates are so much more electroreactive. The low molecular weight, methanol:chloroform (3:2) and 80%-ethanol soluble combined fractions (Figure 1) were found to consist of non-volatile sugars, peptides, amino acids, carboxylic acids, phenolic glycosides, etc. (Table 1). Upon gel
Table 1. Mean distribution and standard deviations of peanut sugar distribution across maturity stages for three crop years. 1989. 1990. and 1991 crop years. Variation wlth maturity. % Average Carbohydrate + / - Standard Deviation (n=9) Maturity stage sugars
Days Cured
Stackpole -
Harvest day
10 Days
Windrow 23 Days
40 Days
-
4 Days
Black fructose glucose inosltol ramnose stachyose reductones sucrose Brown fructose glucose inositol ramnose stachyose reductones sucrose
0.007 0.060 0.075 0.080 0.230 0.728 3.081
t 0.002
Orange fructose glucose lnosltol raffinose stachyose reductones sucrose
0.03 1 0.076 0.1 18 0.147 0.598 1.12 1 3.649
r 0.008 r 0.012
Yellow fructose glucose inositol raffinose stachyose reductones sucrose
0.034 0.080 0.153 0.192 0.730 1.203 4.393
r 0.005
r 0.004 t 0.012
r 0.004 t 0.040
t 0.029 t 0.081
t 0.013
t 0.004 t 0.049
0.074 t 0.366 ?
t 0.008
t 0.007
r 0.012
t 0.092
r 0.100 r 0.121
0.005 0.053 0.069 0.065 0.243 0.738 2.682
t 0.001
0.027 0.061 0.117 0.138 0.622 1.130 3.371
t 0.005
0.029 0.067 0.135 0.159 0.687 1.12 1 4.151
t 0.005
?
t
r t ?
t
0.005 0.004 0.006 0.016 0.039 0.137
0.013 0.023 0.004 0.040 f 0.054 0.289 f t f t
+
t 0.005 t 0.003
0.018 r 0.013 ? 0.097 t 0.127 ?
0.004 0.049 0.065 0.059 0.236 0.744 2.558
t 0.000
0.022 0.059 0.1 13 0.138 0.597 1.060 3.223
t 0.005
0.026 0.063 0.126 0.155 0.676 1.051 3.992
k
t 0.003
t 0.005 t 0.004
r 0.016 t 0.027
r 0.137
t 0.008 t 0.022 t 0.007
r 0.049 t 0.073
t 0.266
0.005
t 0.004
t 0.003
r 0.016 t 0.013
r 0.096 r 0.084
0.004 0.049 0.060 0.053 0.223 0.737 2.404
t 0.000
0.020 0.05 1 0.107 0.136 0.579 1.010 3.173
t 0.004 t 0.008
0.026 0.061 0.122 0.145 0.612 1.040 3.925
r 0.005
r 0.005 t 0.004
r 0.003 r 0.010 t 0.014
r 0.127
r 0.018 f 0.001
r 0.051 t 0.064 t 0.335
t 0.002 t 0.008 r 0.020 ? 0.022 t 0.099 ? 0.088
0.004 0.062 0.079 0.073 0.252 0.785 2.811
f 0.000 t 0.003
0.029 0.068 0.119 0.144 0.638 1.063 3.504
r 0.005
0.027 0.070 0.143 0.171 0.702 1.161 4.348
?
t 0.007 t 0.002 r 0.012 r 0.033 r 0.136
r 0.008 r 0.015 r 0.004 ?
0.044
t 0.083
t 0.315
0.004 t 0.003 t 0.005 r 0.014 r 0.007 r 0.051 ? 0.249
Table 2. Grand means of total peanut soluble carbohydrate in four maturity stages for three crop years Crop years 1989. 1990. and 1991. Average per cent total carbohydrate with standard deviation (n=9). YELLOW DAYS Harvest Day Stackpole- 10 Stackpole-23 Satckpole-40 Windrow-4
% f std. dev.
6.78 6.35 6.09 5.93 6.62
f 0.32 f 0.24 f 0.19 f 0.1 7 f 0.22
MATURITY ORANGE %
5.74 5.47 5.22 5.08 5.33
+
std. dev.
f 0.52 f 0.41 f 0.43 f 0.47 2 0.49
BROWN
BLACK
% f std. dev.
% f std. dev.
4.26 3.85 3.71 3.53 4.07
0.08 f 0.18 f 0.18 f 0.15 f 0.18 f
3.56 3.59 3.54 3.60 3.61
f 0.60 f 0.51 f 0.51 f 0.49 f 0.38
Table 3. Mean proximate protein and oil analyses for peanut curing study over three crop years. Crop years 1989. 1990. and 1991. Average per cent total protein and oil with standard deviations (n=9). Analyses based on full fat cotyledons after drying to equilibrium moisture. YELLOW Maturity stage
% f std. dev.
ORANGE
MATURITY
% f std.dev.
BROWN
BLACK
% f std. dev.
% f std.dev.
Protein (a)
23.4 f 1.4
25.2 f 1.8
29.8 f 2.4
31.3 f 1.6
Oil
38.3 f 2.1
41.6 f 1.4
45.3 f 3.2
49.2 f 2.4
(b)
(a)Kjeldahl nitrogen with factor of 6.23 for estimation of total protein. Corrected for moisture. Calculation based on defatted. extracted meal as residue from treatment in Figure 1.
(b)Corrected for moisture. O i l content based on whole seed extracted with hexane.
L
ul
x! Lhl
1559 filtration according to the method in Section 2.3 above, distribution of size exclusion sample indicated molecular weight <5000 D and a peak with maximum at 1300 D. Results reported in Table 1 and Table 2 for carbohydrate compositions of all the maturity stages of peanuts as means of all three crop years are consistent with previous compositional studies of carbohydrates in peanuts [22-24, 27-28, 52-53] except that no free 2-amino-2-deoxy-D-glucose was found in these samples as was reported by Basha [27]. Upon mild concentration of the organic solution the resulting freeze dried sirup had a pleasant butterscotch aroma with notes of raw beany still present. Thin layer chromatography of the peanut extract sirup separated the major components into zones that were, by visual inspection, similar to the major components of the ion chromatograms. Since each sirup contained amino acids, carboxylic acids, carbohydrates, peptides, glycolipids, saponins, etc., quantitation was only referred to HPLC response factors for external response factors of all the carbohydrate species of components. The quantitations by external standards were spot checked for comparison with the internal standard, 2-amino-2-deoxy-D-glucose, and both methods found to be in good agreement. However, since both amino acids and carboxylic acids gave either much lower responses with the I.P.A.D. or retention times grouped outside of the ranges of the sugars and reductones, assignments of identities of the sugars are on a sound basis (retention time windows of the sugars have <2% relative standard deviation) (c/., [54]). Response factors for each sugar were carefully calibrated on the H.P.L.C.-I.e.-I.P.A.D. with simple regression analysis of precision for triplicate runs (<1 to 3% depending on the sugar). The relative quantities of each sugar component as defined and reported here for the soluble sugars and reductones in the tables and figures are in percent extractable sugar from hexane-defatted meal corrected for initial moisture. 3.3. Carbohydrates as defining changes in carbon pools of peanuts during postharvest curing and maturation In the present report, the methodology was designed to define and characterize changes during curing in carbon pools or carbohydrate precursors of peanut flavor for mature (black hull scrape class) versus immature peanuts (orange hull scrape class) [4, 47, 55]. Simultaneously, with the weight changes shown in Figures 2, 3, and 4 concerning total dry weight seed mass for both stackpole treatment and windrow curing in each maturity class per crop year, the carbohydrate content was extensively tracked as an attempt to understand the nature of the curing metabolism. Comparison of ion chromatography of the low molecular weight sugar alcohols and reductones (Figure 5 for the windrow study; Figures 6, 7, and 8 for the stackpole study) and other sugars (identified by I.C.-I.P.A.D. and G.C.-M.S.-T.I.C. retention times) permitted formulation of a biochemical model of carbohydrate turnover in peanut curing. Total carbohydrates are shown in Figures 9, 10, and 11 for the four maturity stages of the stackpole study in all three crop years. Total carbohydrates are not plotted separately for the windrow references but are similar
--
4 ---
3
*
--
-
.. -
1
*-
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% Carbohydrate
- 4
-
. ---
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2
-
-*7
-
-- ---
- --- P ; % -?
' F
*
InnritoI Glrrc.use
Frrtcrow
Figure 5.
0
-F Rmrrn
Maturity Stage
--
' < ,P
Yellou
Sucrn~e a Red11ctme.v ~tac~ryose Raf/i,ln~e
I
--
-
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@#/
---
-- ---_ -------_ '9
1
?
I ,
t
I
---
prr
Rtack
--
4
--
-.---
---
3 2 1 0
Ilarvest Day &Day4of Crop Year
Windrow study:sugar distribution in peanuts. Variation with maturity. Crop years 1989, 1990, 1991.
% Carbohydrate #
-
Sucrose
Reducrortes Stachyose
Ramnose Inositol
Glucose Fructnse
0
4.5 4
3.5
Bmwn
Maturity Stage
~hsk
HmD.y
Days Cured
Figure 6. Stackpole study: sugar distribution in peanuts. Variation with maturity. 1989 crop year.
% Carbohydrate
Maturity Stage
I lanet thy
Days Cured
Figure 7. Stackpole study: sugar distribution in peanuts. Variation with maturity. 1990 crop year.
% Carbohydrate
Maturity Stage Frrrcrow
Black
Hanat Day
Days Cured
0
Figure 8. stackpole study: sugar distribution in peanuts. Variation with maturity. 1991 crop year.
1564 to the stackpole samples and are shown as bars on the same graphs as the stackpole data (Figures 9,10, and 11). The profiles indicate that these metabolites decrease to a stable concentration by the time curing dehydration reaches equilibrium in both windrow/wagon-dried peanuts as well as in longer term stackpole dr3dng. The following conclusions were drawn about the total carbohydratesfi:*omorganic extracts: - The immature (yellow or orange hull-scraped classes) peanuts have more low molecular weight reducing substances as well as oligosaccharides than the mature (brown or black) peanuts at all postharvest stages of curing. - Carbohydrates and polyhydroxy reductones in immature peanuts essentially remain unchanged harvest day to the final day of drying either as windrow/wagon dried peanuts or in stackpoles, but still have final levels higher than the mature (brown or black). - The carbohydrate component peaks of mature (brown or black) peanuts at the harvest date are not distinguishable by ion chromatography fi-om blacks separated either from windrow or stackpole drying (including final samples), but change only slightly in quantities of most sugars and reductones during either kind of curing treatment. - The windrow or stackpole-cured samples possess an ion chromatogram of sirupy sugar extract identical to the mature black samples for all three crop years. The relative quantities of 2-, 3-, 4-, and 5-carbon pool of reducing or hydroxylated fractions decreased as the immature samples declined in total weight percentage while curing came to completion (Tables 1 and 2), and oil and total protein increased (Table 3). Thin layer chromatograms of the sirupy peanut extract, as described above, indicated densities of zone patterns which followed essentially the same conclusions as the HPLC data, namely, that as curing progressed the composition of the extract from the immature peanuts decreased quantitatively in complexity whereas the extracts of the mature, black peanuts were essentially the same throughout the curing period. Oxidation processes are implicated in some of these changes, and further investigation must be carried out to create a metabolic regulation model of the bioenergetics involved [19, 56-58]. When the three years of windrow and stackpole cured samples in Figures 5 through 8 were compared for differences in carbohydrates across maturity lines, soluble carbohydrates were comprised of reactive hydroxycarbonyls (reductones; ca. 12% of total carbohydrates by weight, consisting of 2 to 5 carbon species from the metabolic carbon pool) as well as inositol, glucose, fructose, sucrose (>80% total sugars), raffinose, and stachyose. In both windrow and stackpole curing carbohydrate content of the immature peanuts (yellow/orange hull scrape color) declined from >9% to 8% of seed weight as their seed mass contribution diminished from 40% to <20% of total seed harvested. Mature peanuts (black/brown hull scrape color) maintained a steady carbohydrate content (5 to 6% of seed weight) throughout curing, but increased in total seed mass from 60% to >80% throughout postharvest maturation of the immature seed.
6
Maturity Stage Figure 9. Total carbohydrate distribution in peanuts. Stackpole and windrow samples. 1989 crop year.
-
Cn
S
Figure 10. Total carbohydrate distribution in peanuts. Stackpole and windrow samples. 1990 crop year.
% Car
Maturity Stage
Black
Harvest Day
Days Cured
Figure 1 1 . Total carbohydrate distribution in peanuts. Stackpole and windrow samples. 1991 crop year.
1568 Mass spectral chromatograms of total ion currents (TIC) for methylated alditol acetates as derivatized extracts of the harvest day immature peanuts were compared with the harvest day mature peanuts for all three crop years [21] as confirmation that the same kinds of processes were occurring throughout maturation. The data are not shown but were similar to that already reported for the 1991 crop year in Vercellotti et al. [21]. The peak areas of all samples measured by H.P.L.C.-I.C.-I.P.A.D. are relatable to the G.C.-JM.S.-T.I.C. responses for the derivatized extract of the florunner windrow references for each crop year. This indicates that sugar and reductone compounds are diagnostic for precursors generated in the process of maturation. On the other hand, using visual pattern recognition, the immature samples have much higher responses for most components in the T.I.C. than the mature samples, indicating that the mature peanut has less of these carbon pool precursors even at harvest day for all three crop years. The harvest day immature peanuts also have a number of additional peaks, which are not present in the matures at any point of the curing treatment. It is interesting, however, that each maturity class possesses essentially the same key carbohydrate-related compounds except that the relative quantities are less in all the mature samples. This pattern of immature peanuts possessing more of the low molecular weight reductones than the mature continues in all the representative mature and immature samples. Without question, biochemical conversion of carbohydrates in the immature peanuts takes place during curing, with the result that immature peanuts, on the basis of carbohydrate content, are being converted to mature peanuts, much as was indicated by the conversion of yellow and orange mesocarp maturity stages to brown and black mesocarps during the same period. 3.4, Correlation of total carbohydrate with final weight yield of peanuts for each maturity stage A correlation was made through principal component analysis [32] for the variables of total weight percentage of harvested peanuts and total carbohydrate in each maturity class combining crop years. The data in Figures 9, 10, and 11 for the total carbohydrates in each maturity class of the stackpole study for each crop year when compared with total weight percentage of these same samples shown in Figures 2, 3, and 4 illustrate their inverse proportionality. The regularity of carbohydrate consumption by peanut metabolism in converting from the less mature to more mature stage is indicative of sharp delineation between maturity classes of these samples in the visually chosen, hand selected maturity stage pods. Simple linear regression analysis of these same data also demonstrated that seed carbohydrate content is inversely proportional to seed maturity. In particular, the divergent concentrations of carbohydrate between the yellow immature peanuts and the black mature sample is diagnostic of wide differences in roast properties such as rate of pigmented color formation or roasted peanut character note. Again, across an individual maturity stage there is little change in the total carbohydrate
1569 content during the curing process just as was noted in Figures 5, 6, 7, and 8 for the individual carbohydrates that compose the total carbohydrate. 3.5. Mass balance of total carbohydrate content normalized over total dry w e i g h t of each maturity class during curing period: A contour model of the dynamic changes of carbohydrate in the maturing seed When normalization of total carbohydrate content of each maturity class was made over total dry weight for the entire period of 3 crop years; 5 treatments (harvest 0 day, 3 stackpole drying times, 4-day windrow); 4 maturity classes; and total carbohydrate in the mass of a maturity class, a contour diagram emerged with a graphic depiction of the dynamic process of carbohydrate changes with maturation during curing. A simple description of this normalization in terms of the distribution of highly reactive carbohydrate at any one time over the curing period is as follows: (Percent total carbohydrate) x (Mass of final dry weight kernels per maturity class) = (Mass balance of total carbohydrate at a discrete interval in the curing period) The results of this carbohydrate distribution, expressed as grams carbohydrate per 100 grams of final cured peanuts are shown in Figures 12, 13, and 14 for all three crop years. Again, the dynamics of the system is indicated, revealing that total carbohydrate is depleted from harvest day to final curing in both the stackpole and windrow samples. Stepwise discriminant analysis [32] also confirms that all of these individual sugars for the data set are effective in discriminating between maturity and immaturity in cured peanuts. Maturity in cured peanuts is related to low soluble carbohydrate content (5 to 6% total) but immature peanuts have as much as >9% total carbohydrate at equilibrium moisture content (Tables 1 and 2). Relative quantities of all sugars were indicative of a difference between mature and immature cured peanuts. Stachyose was the strongest indicator and inositol was the weakest: stachyose>glucose>fructose>sucrose>reductones>raffinose>inositol. According to this model, therefore, total carbohydrate content as well as the concentrations of individual sugars in decreasing order of probability stated in the previous sentence can be used as markers to describe peanut maturation. These results are in substantial agreement with the data of McMeans et al. [24] and Ross and Mixon [52] for changes in carbohydrate distribution with maturation. More substantially, this work extends the principles of the dynamics of peanut maturation reviewed by Sanders et al. [19] and covered in experimental detail by Sanders [49] on maturity distribution in commercially sized florunner peanuts. In addition, these relationships between total reactive carbohydrate, including reductones, in the maturity classes of peanuts at time of harvest and roast quality.
-
W T % CARBOHYDRATE
Maturity Stage
Harvest Day
--='=F+-
Days Cured
Figure 12. Mass balance o f carbohydrates. Curing study distribution over total seed mass and curing time. 1989 crop year.
W T % CARBOHYDRATE d
Maturity Stage
Harvest Day
Days Cured
Figure 13. Mass balance of carbohydrates. Curing study distribution over total seed mass and curing time. 1990 crop year.
WT % CARBOHYDRATE
3
\
-
Maturity Stage
Harvest Day
Days Cured
Figure 14. Mass balance of carbohydrates. Curing study distribution over total seed mass and curing time. 1991 crop year.
1573 as demonstrated in Sanders et al. [1] as well as Vercellotti et al. [14] and Crippen et al. [15], are important keys to shaping total peanut flavor quality of products. 4. SmVCVIARY AND CONCLUSIONS The significance of the above analyses of peanut postharvest curing metabolism lies in the fact that thermal cleavage of sugars and reductones or glycosidic linkages in glycoproteins and cell wall polysaccharides generate sweet aromatic attributes per se, or other reductones [57, 59-61] that are quite reactive in heterocyclic ring closures leading to the myriad roasted peanutty flavor compounds [14-18]. These same reductones, such as the pyranones, hydroxyfuranones, or furaneols, can also contribute to the high impact positive character note, sweet aromatic; to fi:'uity/fermented off-flavors; or to principal characteristic fruit flavors in themselves when in too high concentration (e.g., the hydroxyfuranones are important contributors to pineapple, strawberry, passion fruit, apricot, etc., flavors). Damage to peanut cell walls in pre- or postharvest treatments results in irretrievable losses due to off-flavors such as fruity/fermented that permeate improperly dried or freeze damaged peanuts [1, 11, 58, 62-64]. In addition, many lipid oxidation products are linked glycosidically or through hemiacetal or ketal linkages and are released from polysaccharides during the roasting process. The fuller roasted peanut flavor from mature peanuts, and the poorer potential for production of the delicate balance of high impact flavor component in immature peanuts has been demonstrated by a number of careful studies [1, 15, 58]. Proper harvest and curing techniques will result in fewer immature peanuts of various commercial seed sizes and should result in a higher quality product because the off-flavor and low impact notes from the immature peanuts will be far less concentrated [1, 10, 64]. ACKNOWLEDGEIVIENT Gratitude is expressed by the collaborating authors to Dr. Daniel T. Grimm, USDA-ARS, IVIarket Quality and Handling Research, North Carolina State University, Raleigh, NC, for his generous expert critique and discussion of the peanut physiology and biochemistry aspects of this manuscript. The authors also thank Mr. E. Jay Williams and John A. Lansden of the National Peanut Research Laboratory, USDA-ARS, Dawson, GA, as well as Drs. Terri D. Boylston and Casey Grimm of SRRC, New Orleans, for assistance in sample maturity assessment and valuable discussions about peanut analyses as related to flavor precursor properties. The skilled technical assistance of James F. Hochadel, Eldwin St. Cyr, Larry Greene, Edwina Gill, Jerry Kirksey, Larry Boihem, and Gioconda Lau is gratefully acknowledged. The computer graphics in this paper by Ms. Brenda Lemoine are also gratefully recognized.
1574 5. REFERENCES 1. Sanders, T.H., Vercellotti, J.R., Crippen, K.L., and Civille, G.V. (1989). Effect of maturity on roast color and descriptive flavor of peanuts. J. Food Sci., 54, 475-477. 2. Pattee, H.E., Wynne, J . C , Sanders, T.H., and Schubert, A.M. (1980). Relation of the seed/hull ratio to yield and dollar value in peanut production. Peanut ScL, 7, 74-77. 3. Sanders, T.H., Williams, E.J., Schubert, A.M., and Pattee, H.E. (1980). Peanut maturity method evaluations. I. Southeast. Peanut ScL, 7, 78-82. 4. Williams, E.J., and Drexler, J.S. (1981). A non-destructive method for determining peanut pod maturity. Peanut Science, 8, 134-141. 5. Sanders, T.H., Lansden, J.A., Vercellotti, J.R., and Crippen, K.L. (1990). The role of maturation in quality of stackpole cured peanuts. Proc. Am, Peanut Res. Educ. Soc, 22, 71. 6. Young, J.H., Person, N.K., Donald, J.O., and Mayfield, W.D. (1982). Harvesting, curing and energy utilization. In Peanut Science and Technology, eds. H.E. Pattee & C.T. Young. American Peanut Research and Education Society, Inc., Yoakum, TX, Ch.l2, pp. 458-485. 7. Pattee, H.E., Purcell, A.E., and Johns, E.B. (1969). Changes in carotenoid and oil content during maturation of peanut seeds. J. Am. Oil Chemists Soc, 46, 629-631. 8. Pattee, H.E., Johns, E.B., Singleton, J.A., and Sanders, T.H. (1974). Composition changes of peanut fruit parts during maturation. Peanut Sci., 1, 57-62. 9. Young, C.T. (1973). Influence of drying temperature at harvest on major volatiles released during roasting of peanuts. J. Food Sci., 38, 123-125. 10. Sanders, T.H., Vercellotti, J.R., Blankenship, P.D., Crippen, K.L., and Civille, G.V. (1989). Interaction of maturity and curing temperature on descriptive flavor of peanuts. J. Food Sci., 54, 1066-1069. 11. Pattee, H.E., Yokoyama, W.H., Collins, M.F., and Giesbrecht, F.G. (1990). Interrelationships between headspace volatile concentration, marketing grades, and flavor in runner-type peanuts. J. Agric. Food Chem., 38, 10551060. 12. Pazola, Z., and Cieslak, J. (1979). Changes in carbohydrates during the production of coffee substitute extracts, especially in the roasting process. Food Chemistry, 4, 41-52. 13. Grosch, W., and Schieberle, P. (1991). Bread. In Volatile compounds in foods and beverages, ed. H. Maarse, Marcel Dekker, Inc., New York. pp. 41-77. 14. Vercellotti, J.R., Crippen, K.L., Lovegren, N.V., and Sanders, T.H. (1992). Defining roasted peanut flavor quality. Part 1. Correlation of GC volatiles with roast color as an estimate of quality. In Food Science and Human Nutrition. 29, ed. G. Charalambous. Elsevier Publishing Co., Amsterdam, The Netherlands, pp. 183-209.
1575 15. Crippen, K.L., Vercellotti, J.R., Lovegren, N.V., and Sanders, T.H. (1992). Defining roasted peanut flavor quality. Part 2. Correlation of GC Volatiles and sensory flavor attributes. In Food Science and Human Nutrition.29, ed. G. Charalambous. Elsevier Publishing Co., Amsterdam, The Netherlands, pp. 211-227. 16. Vercellotti, J.R., Mills, O.E., Bett, K.L., and Sullen, D.L. (1992). Gas chromatographic analyses of lipid oxidation volatiles in foods. In Lipid Oxidation in Foods. ACS Symposium Series 500. ed. A.J. St. Angelo. American Chemical Society, Washington, D.C., pp. 232-265. 17. Bett, K.L., and Boylston, T.D. (1992). Effect of storage on roasted peanut quality:Descriptive sensory analysis and gas chromatographic techniques. In Lipid Oxidation in Food, ed. A.J. St. Angelo. ACS S3m[iposium Series 500, American Chemical Society, Washington, DC, pp. 322-343. 18. Vercellotti, J.R., Bett, K.L., and Choi, K.S. (1993). Carbohydrates as sources of roasted peanut flavors and aromas. In 7th International Flavor Symposium, 1992, Samos, Greece, ed. G. Charalambous. Elsevier Publishing Co., Amsterdam, The Netherlands, pp. 232-265. 19. Sanders, T.H., Vercellotti, J.R., and Grimm, D.T. (1993). Shelf life of peanuts and peanut products. Ed. G. Charalambous, Shelf Life Studies of Foods and Beverages. Chemical, Biological, Physical, and Nutritional Aspects. Elsevier Science Publishers, B.V., Amsterdam, pp. 289-309. 20. Ahmed, E.M., and Young, C.T. (1982). Composition, quality, and flavor of peanuts. In Peanut Science and Technology, eds. H.E. Pattee & C.T. Young. American Peanut Research and Education Society, Inc., Yoakum, TX, Ch.l7, pp. 655-688. 21. Vercellotti, J.R., Munchausen, L.L., Sanders, T.H., Garegg, P.J., and Seffers, P. (1994). Confirmation of sugars and reductones in complex peanut flavor precursor extracts by ion chromatography and methylation analysis. Food Chemistry, 50, 221-230. 22. Tharanathan, R.N., Wankhede, D.B., and Raghavendra Rao, M.R. (1975). Carbohydrate composition of groundnuts (Arachis hypogea). J. Sci. Fd. Agric, 26, 749-754. 23. Tharanathan, R.N., Wankhede, D.B., and Raghavendra Rao, M.R. (1976). Mono- and oligosaccharide composition of groundnut (Arachis hypogea). J. Food Sci., 4 1 , 715-716. 24. McMeans, J.L., Sanders, T.H., Wood, B.W., and Blankenship, P.D. (1990). Soil temperature effects on free carbohydrate concentrations in peanut {Arachis hypogaea L.) seed. Peanut Sci., 17, 31-36. 25. Sheppard, A.J., and Rudolf, T.S. (1991). Analysis of peanuts and peanut products for total lipids, fatty acids, and proximates. Peanut Science, 18, 5154. 26. Basha, S.M., Sanders, T.H., Blankenship, P.D., and Vercellotti, J.R. (1991). Effect of curing temperature and seed maturity status on peanut seed and paste composition. J. Food Comp. Anal., 4, 337-345.
1576 27. Basha, S.M. (1992). Soluble sugar composition of peanut seed. J. Agric. Food Chem., 40, 780-783. 28. Cardozo, M.S., and Li., B.W. (1994). Total dietary fiber content of selected nuts by two enz3anatic-gravimetric methods. J. Food Comp. and Anal., 7, 37-43. 29. American Oil Chemists Society (1969). Official and Tentative Methods. 3rd edition. Chicago, IL. 30. Association of Official Analytical Chemists (1990), Official Methods of Analysis, 15th edition. Washington, DC. Vol. 1, Chapters 2 and 3, pp. 9-69. 31. Prosky, L., Asp, N.-G., Furda, I., Devries, J.W., Schweizer, T.F., and Harland, B.F. (1988). Determination of total dietary fiber in foods, food products, and total diets: interlaboratory study. J. Assoc. Off. Analyt. Chem., 67, 1044-1052. 32. SAS. (1988). SAS/STAT User's Guide, Release 6.03 Edn. SAS Institute, Inc., Gary, N.C. 33. Sanders, T.H., Schubert, A.M., and Pattee, H.E. (1982). Maturity methodology and postharvest physiology. In Peanut Science and Technology, eds. H.E. Pattee and C.T. Young. American Peanut Research and Education Society, Inc., Yoakum, TX, Ch.l6, pp. 624-654. 34. Sanders, T.H., Blankenship, P.D., Vercellotti, J.R., and Crippen, K.L. (1990). Interaction of curing temperature and inherent maturity distributions on descriptive flavor of commercial grade sizes of Florunner peanuts. Peanut ScL, 17, 85-89. 35. Nasir-ud Din, Vercellotti, J.R., Jeanloz, R.W., and McArthur, J.W. (1981). Studies on bonnet monkey cervical mucus. The effect of proteases on mucus glycoproteins of Macaca radiata. Biochim. Biophys. Acta, 678, 483-496. 36. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951). Protein measurement with the Folin-Phenol reagent. J. Biol. Chem.,193, 265-275. 37. Smith, P.K., Krohn, R.I., Hermanson, G.T., MaUia, A.K., Gartner, F.H., Provanzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., and D.C. Klenk (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 76-85. 38. Yemm, E.W., and Cocking, E.G. (1955). Determination of amino acids with ninhydrin. Analyst, 80, 209-213. 39. Blumenkrantz, N., and Asboe-Hansen, G. (1973). A new method for quantitative determination of uronic acids. Anal. Biochem., 54, 484-489. 40. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356. 41. Albersheim, P., Nevins, D.J., Enghsh, P.D., and Karr, A. (1967). A method for the analysis of sugars in plant cell-wall polysaccharides by gas chromatography. Carbohydr. Res., 5, 340-345. 42. Englyst, H.N., and Kingman, S.M. (1990). Dietary fiber and resistant starch: Nutritional classification of plant polysaccharides. In Dietary Fiber, Eds. D. Rritchevsl^, C. Bonfield, and J.W. Anderson, Plenum Press, New York. pp. 49-65.
1577 43. Metz, J., Ebert, W., and Weicher, H. (1970). Quantitative determination of neutral and amino sugars by gas-liquid chromatography. Chromatographia, 4, 345-350. 44. Li, B.W., Schuhmann, P.J., and Wolf, W.R. (1985). Chromatographic determinations of sugars and starch in a diet composite reference material. J. Agric. Food. Chem., 33, 531-536. 45. SanderSjT.H., and coworkers. (1994). Unpublished data on stackpole curing of peanuts for the 1989 and 1990 crop years. Manuscript in preparation. 46. Mohapatra, S.C, and Pattee, H.E. (1974). Lipid metabolism in dehydrating peanut kernels. Physiol. Plant, 28, 320-326. 47. Pattee, H.E., Register, E.W., and Giesbrecht, E.G. (1989). Interrelationships between headspace volatile concentration, selected seed size categories, and flavor in large-seeded virginia-type peanuts. Peanut Sci., 16, 38-42. 48. Sanders, T.H., Lansden, J.A., Greene, R.L., Drexler, J.S., and Williams, E.J. (1982). Oil characteristics of peanut fruit separated by a nondestructive maturity classification method. Peanut Sci., 9, 20-23. 49. Sanders, T.H. (1989). Maturity distribution in commercially sized florunner peanuts. Peanut Science, 16, 91-95. 50. Koehler, P.E., Mason, M.E., and Newell, J.A. (1969). Formation of pyrazine compounds in sugar-amino acid model systems. J. Agric. Food Chem., 17, 393-396. 51. Koehler, P.E., and Odell, G.V. (1970). Factors affecting the formation of pyrazine compounds in sugar-amine reactions. J. Agric. Food Chem., 18, 895-898. 52. Ross, L.F., and Mixon, A.C. (1989). Changes in soluble carbohydrates in developing seeds from Florunner peanuts (Arachis hypogaea L.). J. Food Comp. Anal, 2, 157-160. 53. Basha, S.M., and Young, C.T. (1992). Effect of blanching and blanching method on peanut seed composition. Oleagineux, 47, 531-535. 54. LaCourse, W.R., and Johnson, D.C. (1993). Optimization of waveforms for pulsed amperometric detection of carbohydrates based on pulsed voltammetry. Anal. Chem., 65, 50-55. 55. WilHams, E.J., Ware, G.O., Lee, J.Y., and Drexler, J.S. (1987). Effect of pod maturity and plant age on the seed size distribution of Florunner peanuts. Peanut Sci., 14, 79-83. 56. Stadtman, E.R., and Oliver, C.N. (1991). Metal-catalyzed oxidation of proteins. J. Biol. Chem., 266, 2005-2008. 57. Kanner, J. (1992). Mechanism of nonenzymic lipid peroxidation in muscle foods. In Lipid Oxidation in Food, ed. A.J. St. Angelo. ACS Symposium Series 500, American Chemical Society, Washington, DC, pp. 55-73. 58. Ory, R.L., Crippen, K.L., and Lovegren, N.V. (1992). Off-flavors in peanuts and peanut product. In Off-Flavors in Foods and Beverages, ed. G. Charalambous, Elsevier Publishing Co., Amsterdam, The Netherlands, pp. 57-75.
1578 59. Theander, O. (1987). Conversion of carbohydrates to phenols and enones with special reference to humification and to colour formation in pulping and wood processing. In S.S. Stivala, V. Crescenzi, and I.C.M. Dea, (Eds.), Industrial Polysaccharides. The Impact of Biotechnology and Advanced Methodologies. Gordon and Breach Science Publishers, New York, pp. 481-492. 60. Bailey, M.E., and Um, K.W. (1992). Maillard reaction products and Kpid oxidation. In Lipid Oxidation in Food, ed. A.J. St. Angelo. ACS Symposium Series 500, American Chemical Society, Washington, DC, pp. 122-139. 61. Vernin, G., Metzger, J., Obretenov, T., Suon, K-N., and Praise, D. (1988). GC/MS (EI, PCI, SIM)-Data bank analysis of volatile compounds arising from thermal degradation of glucose-valine Amadori intermediates. In Flavors and Fragrances: A World Perspective, eds. B.M. Lawrence, B.D. Mookherjee, and B.J. Willis, Proc. of 10th International Congress of Essential Oils, Fragrances, and Flavors. Elsevier Science Publishers, Amsterdam, pp. 999-1028. 62. Bailey, W.K., Pickett, T.A., and Futral, J.G. (1954). The Peanut Journal and Nut World. June, 1954. pp. 15,37-39. 63. Whitaker, T.B., and Dickens, J.W. (1964). The effects of curing on respiration and off-flavor in peanuts. Proc. Third National Peanut Res. Conf., July 910, 1964. pp. 71-80. 64. Johnson, P.B., Civille, G.V., Vercellotti, J.R., Sanders, T.H., and Dus, C.A. (1988). Development of a lexicon for the description of peanut flavor. J. Sensory Studies, 3, 9-17.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1579
THE PHENOLIC COMPOSITION OF TABLE GRAPES E. Revilla\ J.M. Escalona*, E. Alonso* and V. Kovac'' *Departamento de Quimica Agncola, Geologia y Geoquimica. Universidad Autonoma de Madrid, 28049 Madrid, Spain. '^Tehnoloski Fakultet, BL Cara Lazara 1, 21000 Novi Sad, Vojvodina, Federal Republic of Yugoslavia. Abstract The phenolic composition of more than twenty table grape cultivars has been studied by colorimetric and chromatographic techniques. The experimental data have been analysed by several statistical methods. Results show that it is possible to classify the different cultivars according to their phenolic composition. 1. INTRODUCTION Sensory propierties of fruits and of their processed products are appraised by their esthetic and hedonistic values, and by the identification or determination of their commercial value. Different plant phenolics (anthocyanins, flavonols, catechins, proanthocyanidins, cinnamic acid esters,...) have a decisive role in determining some of these features, Le., color, biterness and astringency. For all these reasons, there is an extensive literature on the phenolic composition of edible fruits, which has been recently reviewed [1]. The phenolic composition of grape cultivars used for winemaking is well known [1-3] because phenolic compounds are typical components of wines which have particular importance in Enology. Two gropus of phenolic compounds present in grapes and wines have been extensively studied in the last 15 years by HPLC techniques: anthocyanins, responsible for the color of red grapes and, hence, for the color of red, rosd and clairet wines [4-7], and catechins and oligomeric proanthocyanidins, responsible for the astringency of grapes and wines [8-11]. In addition, anthocyanins, catechins and proanthocyanidins have shown some interesting pharmacological properties as vascular protectors, which are related to their anti-inflamatory activity [12-13]. Fresh grapes may be considered an important source of vitamins and iron for human nutrition [14]. The fleshy part of a grape berry is relatively low in phenolic compounds, as with most fruits. However, it is common to consume the skins of grape and often the seeds as well, and these tissues are rich in phenolic compounds. Table grapes have a
1580 growing importance in many viticultural countries: in the last twenty years, the world production of table grapes increased from 6. l(f kg to more than 8.10^ kg, specially in Africa and America [15]. In 1991, the production of table grapes represent about 20% of grapes produced for different purposes. Turkey, Italy, USA, the former Soviet Union, Chile, Spain and Greece are the countries with a extremely high production of table grapes (table 1). Table 1 Production of table grapes (10^ kg) in selected countries. OIV data [16]. Country
1971-1975
1976-1980
Italy ex-USSR
963 721 674 64 528 467 228
1125 1012
Turkey Chile
USA Spain Greece
802 167 382 428 257
1981-1985
1986-1990
1991
1992
1480 1012* 802*
1454 1012* 802*
1411 1012*
1765 1012*
167 490 467 288
468 558 465 270
913 668 562 459 307
921 686 572 407 206
*Estiiations
Unfortunately, the phenolic composition of table grape cultivars is not well known, despite their economical and nutritional importance. For these reasons, it has been considered of great interest the study of these components in several table grape cultivars, and specially the content of catechins and proanthocyanidins in their skins and seeds. 2. MATERIALS AND METHODS 2.1. Sampling Samples of white and red table grape cultivars were collected in the vineyards of "El Encin" Experimental Field, Comunidad de Madrid, in 1992 and 1993. Each sample was composed of 10 mature clusters from different vines. Cultivars, sampling dates, weigth of 100 berries and sugar content of must are summarized in tables 2 and 3. 2.2. Preparation of samples Once in the laboratory, berries were separated from clusters and 100 berries were randomly sampled. Seeds, skins and pulp were separated and placed in flasks containing 150 ml methanol. Then, the samples were pulped with a Kinematica PCU-2 mixer, and flasks kept at -24**C for a night Afterwards, the samples were centrifuged at 4000 rpm for 20 min in a Sorvall RC-SB refrigerated centrifuge, and the supernatant liquids were stored. The residues were extracted sequentially with 80 % methanol at room temperature
1581 Table 2 Sampling dates, weight of 100 berries and sugar content in must of white table grape cultivars.
Cultivar
Sampling date
Weight of 100 berries (g)
Albillo Aledo Aledo Real Chasselas Doree Chelva Chelva Dominga Dominga Halvasla Malvasia de Sitges Hantuo Hantiio Moscatel de Malaga Ohanes Ohanes Valenci Blanco
24/09/92 03/10/92 03/10/92 03/09/93 14/09/92 23/09/93 03/10/92 20/10/93 24/09/92 24/09/92 14/09/92 23/09/93 24/09/92 13/10/92 20/10/93 14/09/92
265 407 417 130 296 321 462 617 258 173 255 321 277 355 358 232
Sugar content of must (g/1) 214 186 166 208 175 191 188 156 180 255 152 191 199 189 176 179
Table 3 Sampling dates, weight of 100 berries and sugar content of must in red table grape cultivars.
Cultivar
Sampling date
Weight of 100 berries (g)
Alphonse Lavallee Barlinka Gros Colman Moscatel Tinto Moscatel Tinto Muscat of Hambourg Muscat of Hambourg Muscat of Madersfield Napoleon Napoleon Negra Tardla Planta Mula Valenci Tinto Valenci Tinto
17/09/93 13/10/92 24/09/92 24/09/92 17/09/93 14/09/92 03/09/93 17/09/93 13/10/92 30/09/93 13/10/92 13/10/92 14/09/92 17/09/93
564 538 524 228 163 236 253 265 462 392 493 248 285 296
Sugar content of must (g/1) 176 158 156 181 189 218 210 229 175 152 210 174 202 220
1582 for 4 hours, with 50% methanol at room temperature for 4 hours, with distilled water at -24°C for 15 hours, and with 75% acetone at room temperature for 1 hour. Afterwards, the five extracts obtained for each sample were mixed, the volume raised to 250 ml for seeds and pulp, and to 300 ml for skins, and stored at -24°C prior to analysis. 2.3. Colorimetric analysis Total phenolics in the extracts of seeds, skins and pulp were determined by colorimetry with phosphotungstic-phosphomolibdic acid [17]. Results were expressed as gallic acid equivalents. Total anthocyans were measured in the skin extracts of red cultivars according to Ribereau-Gayon and Stonestreet's method [18]. Results were expressed as malvidin-3-glucoside equivalents. 2.4. Fractionation of phenolic compounds Prior to HPLC analysis of catechins and proanthocyanidins, grape extracts were fractionated by column chromatography. For this purpose, an aliquot (25 ml) of extract was partialUy evaporated under vacuum at 30°C to remove methanol and acetone. Then, pH was adjusted to 7.0, and the volume was restored to 25 ml with distilled water adjusted at pH 7.0. The fractionation was carried out with two Waters Cig SEP-PAK cartridges connected by a rubber tube (preconditioned by secuentially passing 10 ml methanol and 2.5 ml distilled water adjusted to pH 7.0 dropwise) by injecting 1 ml of the alcohol-free extract Then, 10 ml distilled water adjusted to pH 7.0 was passed through the cartridges to elute acidic phenolic compounds. The cartridges were dried under Nj, and 10 ml ethyl acetate passed through them to elute catechins and proanthocyanins. Afterwards, the cartridges were washed sequentially passing 10 ml methanol acidulated to HCl 0.1% and 10 ml methanol through them to elute other phenolics, mainly anthocyans and red and brown polymers. The ethyl acetate fraction was dried under vacuum at 30°C, and the residue dissolved in 1 ml 50% methanol and stored at 0°C prior to analysis. Table 4 Linear gradient used in HPLC analysis of catechins and proanthocyanidins. Solvent B
Tine (Bin)
Flow rate (Bl/iin)
Solvent A
(1)
W
0 47 55 65 70
0.8 0.8 0.8 0.8 0.8
10 82 100 100 10
90 18 0 0 90
2.5. HPLC analysis of catechins and proanthocyanidins A chromatograph equipped with M501 and M510 pumps (Waters Associates), a 720
1583 gradient controler (Waters Associates), a Rheodyne 7125 inyection valve furnished with a 20 /xl sample loop, a Linear UVIS 200 variable wavelength visible-ultraviolet detector set at 280 nm, and a Spectra Phyisics SP4290 integrator were used. The separation of catechins and proanthocyanins was carried out on a reversed phase Nova-Pak Cjg iron cartridge, 3.9 mm i.d. x 150 mm long (Waters Associates), placed in a water bath at 32°C, using a linear gradient of 10% acetic acid (solvent A) in water (solvent B), as shown in table 4. The quantitation of catechins and proanthocyanidins was achieved by an external standard procedure, using multiple-point calibration. Results were expressed as (+)catechin equivalents. A typical chromatogram is shown in figure 1. Retention times for several catechins and procyanidins were as follows: procyanidin B3 (+)-catechin procyanidin B2 procyanidin CI
(peak (peak (peak (peak
1), 9-10 min 3), 15 min 5), 24-25 min 7), 47-48 min
- procyanidin Bl - procyanidin B4 - (-)-epicatechin
(peak 2), 11-12 min (peak 4), 18-19 min (peak 6), 32-33 min
50 ( min) Figure 1. Chromatogram of Alphonse Lavallee seeds extract For key to substances, see text
1584 2.5. Statistical analysis Multivariate statistical analyses (correlation analysis and cluster analysis) were performed by statistical software package STATGRAPHICS [19], in a Tandom TM6105 computer. 3. RESULTS AND DISCUSSION 3.1. Samples harvested in 1992 The amount, on a weight/weight basis, of total phenolics in berries of red and white table grape cultivars harvested in 1992 showed a great variability (tables 5 and 6). Phenolic compounds were accumulated ussually in seeds and, to a lesser extent, in skins. The content of total phenolics in white grapes ranged from 2147 mg/kg grapes (Moscatel de Malaga) to 4779 mg/kg grapes (Malvasia de Sitges). In most cases, cultivars with small-sized berries contained the highest amount of total phenolics on a weight/weight basis. The contribution of seeds to total phenolics was higher in cultivars with small-sized berries than in those with big ones. On the other hand, the contribution of skins to total phenolics was higher in cultivars with big-sized berries (i.e., Dominga) than in those with small ones. The content of total phenolics in pulp was very low (less than 6% of total phenolics), and in some cultivars with small-sized berries (i.e., Malvasia de Sitges) it was as rich as in some cultivars with big-sized berries (Le., Dominga).
Table 5. Content of total phenolics and total catechins and procyanidins (iig/kg grapes), and their distribution (I) in the different parts of the berry in white table grape cultivars. 1992 harvest. Total phenol ics
Catechins and procyanidins
Cultivar
Albillo Aledo Aledo Real Chelva Dominga Malvasia Malvasia de Sitges Mantiio Moscatel de Malaga Ohanes Valenci Blanco
Entire berries
Seeds
3487 2578 2443 3720 2877 3068 4779 3385 2147 3449 3728
89.6 76.7 75.4 80.7 65.0 91.8 85.3 79.4 78.3 76.9 82.4
Skins
Pulp
7.8
2.6 4.3 5.4 3.6 4.7 0.9 4.5 1.7 5.6 3.0 2.3
19.0 19.2 15.7 30.3
7.3 10.2 18.8 16.1 20.0 15.3
Entire berries
460 382 317 954 243 383 480 658 540 229 681
Seeds
Skins
98.0 96.3 95.6 96.9 76.1 98.2 94.2 98.3 94.3 98.3 95.0
2.0 3.7 4.4 3.1 23.9
1.2 5.8 1.7 5.7 1.7 5.0
1585 Table 6 Content of total phenolics, total catechins and procyanidins and total anthocyans (mg/kg grapes), and the distribution (I) of total phenolics and total catechins and procyanidins in the different parts of the berry in red table grape cultivars. 1992 harvest. Catechins and procyanidins
Total phenolics Total Cultivar Entire berries Seeds Skins Pulp
Alphonse Lavallee Barlinka Gros Colian Hoscatel Tinto Muscat of Hambourg Napoleon Negra Tardia Planta Mula Valenci Tinto
4798 4207 2282 4075 3690 3394 4471 3283 3961
69.9 66.7 65.5 83.3 52.8 56.5 43.9 79.7 66.7
25.9 32.2 32.4 14.8 47.2 37.9 48.7 19.0 31.8
4.2 1.1 2.1 1.9 5.2 5.6 7.4 1.3 1.5
Anthocyans
762 266 154 26 387 259 371 88 207
Entire berries Seeds
983 394 746 605 1108
327 819 578 569
93.8 89.3 97.6 97.2 96.9 90.2 94.4 92.4 97.5
Skins
6.2 10.7
2.4 2.8 3.1 9.8 5.6 7.6 2.5
Red grapes contained an amount of total phenolics quite similar to that of white grapes on a weight/weight basis, ranging from 2282 mg/kg grapes (Gros Colman) to 4398 mg/kg grapes (Alphonse Lavallde). Nevertheless, the distribution of total phenolics in seeds and skins of red cultivars was quite different from that observed in white grapes. This fact may be explained by the presence of anthocyans in the skins of red cultivars. The content of these molecules ranged from 26 mg/kg grapes (Moscatel Tinto) to 762 mg/kg grapes (Alphonse Lavallee), see table 6. Hence, skins of red cultivars contained more than 25% of total phenolics, except in those which contained a very low amount of anthocyans (Moscatel Tinto and Planta Mula). As in white cultivars, the content of phenolics in pulp was very low, less than 8 % of total phenolics. Several catechins and procyanidins were measured by HPLC in seeds, skins and pulp extracts of white and red cultivars. Pulp was extremely low in catechins and proanthocyanidins, which were present at trace levels. The total content of catechins and procyanidins in entire berries and their distribution in seeds and skins are given in tables 5 and 6. The amount of these compounds ranged from 243 mg/kg grapes (Dominga) to 954 mg/kg grapes (Chelva) in white cultivars, and from 327 mg/kg grapes (Napoleon) to 1108 mg/kg grapes (Muscat of Hambourg) in red cultivars. These data are quite similar to those reported for grape cultivars used for winemaking [9, 20], and reinforce the finding that white grapes and red grapes contain a similar amount of catechins and procyanidins. Seeds contained more than 89 % of catechins and procyanidins present in grapes, except in Dominga, that contained a remarkable amount of these compounds in skins (23.9%). The relatively high amount of catechins and procyanidins in the skins of Dominga may be related to the high amount of total phenolics observed.
1586 Tables 7 and 8 summarized the total content of catechins and procyanidins in seeds and skins on a weight/weight basis, and the percentages of the different compounds which were determined. Skins present a very low amount of several procyanidins, and only the percentage of total procyanidins is given. Table 7 Total content of catechins and procyanidins neasured by HPLC (ing/kg grapes) and percentage of different catechins and procyanidins in seeds of white table cultivars harvested in 1992. Seeds
Skins
Cultivar
Albino Aledo Aledo Real Chelva Doninga Halvasia Halvasla de Sitges Mantiio Hoscatel de Malaga Ohanes Valencl Blanco
C+P
CAT EPI
451 368 303 925 185 376 452 647 509 225 647
48.3 31.8 44.1 60.0 42.0 54.8 56.4 56.4 59.3 34.7 62.1
31.7 36.1 25.9 18.4 29.2 15.9 19.7 22.6
B3
B4
8.6 3.5 9.2 3.0 10.5 3.2 4.1 4.0 14.9 2.6 3.5 6.9 3.1 4.8 5.6 4.7 4.1 10.2 3.6 3.6 5.9
1.8 7.1 7.7 6.4 5.6 5.3 6.4 1.5 9.0
B2
Bl „
1.4 0.3 1.1 2.9 8.6 6.3 30.2 2.2 " 16.7
01
C+P
CAT
EPI
Pr
6.0
9 14 14 29 58 7 28 9 31 4 34
55.5 71.5 92.9 62.0 39.6 42.9 82.1 72.7 51.6 75.1 67.6
33.3
11.2 28.5
20.7
17.3 58.6 57.1
11.4
8.6 8.4 5.6 19.4
4.6 9.2 7.9 14.7 4.4 6.8 4.9
-
0.8 -
7.1
10.7
7.2
8.1 6.5 8.9
18.2 41.9 24.9 23.5
Table 8 Total content of catechins and procyanidins measured by HPLC (ng/kg grapes) and percentage of different catechins and procyanidins in seeds of red table cultivars harvested in 1992. Seeds
Skins
Cultivar
Alphonse Lavall^e Barlinka Gros Colman Hoscatel Tinto Muscat of Haibourg Napoleon Negra Tardla Planta Mula Valenci Tinto
C+P
CAT
EPI
Bl
B2
922 352 728 588
40.6 39.8 50.4 43.0 49.8 35.9 44.8 40.6 54.8
27.1 26.1 17.0 17.5 24.3 31.2 20.0 21.2 22.5
2.5 3.1 2.2 6.3 3.0 3.7 4.1 1.2 3.2
4.7 6.2 4.0 4.4 4.9 9.2 9.4 3.3 4.0
1074
295 773 570 555
B4
CI
C+P
CAT
9.4 6.1 3.7 8.4 5.4 8.5 4.5 7.2 1.7 7.1 0.9 11.3 1.2 6.3 3.2 4.5
9.6 3.7 6.6
61 42 18 17 34 32 46 8 14
78.7 69.0 77.7 58.8 73.5 43.7 67.4 100.0 71.4
B3
17.3 11.4
14.8
6.3 11.2
9.4 26.1
7.7
EPI 1.7 1.4 5.8 2.9 6.5 13.6
Pr 19.6 28.6 16.6 35.3 20.0 43.7 28.3
4.3 7.2 21.4
1587 (-l-)-catechin predominated in seeds, except in the case of Aledo. The percentages of (-)-epicatechin were higher than those of each procyanidin, except for Malvasia and Planta Mula, that contained a high amount of procyanidin CI (19.4 mg/kg grapes and 26.1 mg/kg grapes respectively). White cultivars comprising a low amount of catechins and procyanidins in seeds (Aledo Real, Ohanes and Dominga) contained a relatively high amount of procyanidin B2. In some cases, the amount of procyanidin Bl was extremely low, specially in white cultivars. In ten cultivars (three white and seven red), the amount of procyanidins was higher than 30% of the total amount of catechins and procyanidins measured by HPLC. These cultivars may be classified taking into the account the main procyanidin present in seeds: procyanidin CI (Aledo, Alphonso Lavallde, Moscatel Tinto, Napoleon and Planta Mula), procyanidin B4 (Moscatel de Malaga, Ohanes and Negra Tardia) or procyanidin B3 (Barlinka and Gros Colman). The amount of procyanidins was less than 30% in ten cultivars (eight white and two red) that may be classified into three groups on the basis of the main procyanidin present in seeds: procyanidin CI (Chelva, Mantiio, Malvasia and Valenci Tinto), procyanidin B4 (Valenci Blanco and Muscat of Hambourg) or procynidin B2 (Albillo, Aledo, Dominga and Malvasia de Sitges). In most cultivars, the content of (+)-catechin in skins ranged from 50% to 80% of total catechins and procyanidins, and there was a relatively high amount of (-)-epicatechin and procyanidins. Three cultivars (Malvasia, Valenci Blanco and Planta Mula) contained a very high amount of (+)-catechin, more than 80%. On the other hand, Malvasia de Sitges, Albillo and Napoledn contained a relatively low amount of (+)-catechin, less than 50%, and a very high amount of procyanidins. The association between pairs of several analytical variables, determined in white and red cultivars sampled in 1992, was measured by correlation analysis, using the following variables: -
total phenolics in seeds (pfts) total phenolics in skins (pfth) total phenolics in pulp (pftp) (+)-catechin in seeds (cats) (-)-epicatechin in seeds (epis) procyanidin B2 in seeds (b2s) procyanidin B3 in seeds (b3s) procyanidin B4 in seeds (b4s) (+)-catechin in skins (cath)
Results, summarized in table 9, show a close association between the contents of total phenolics in skins and pulp, and between the amount of several pairs of catechins and procyanidins in seeds. Nevertheless, the association between the content of total phenolics in seeds and the amount of different catechins and procyanidins in seeds is very loose.
1588 Table 9 Correlation coefficients and their significance level for several analytical variables deteriined in extracts of table grape cultivars saipled in 1992.
pfth pftp cats epis
b2s b4s els cath
pfts
pfth
pftp
cats
epis
b2s
b4s
els
-0.3071 0.0006 0.1782 0.1409 -0.1292 -0.1072 0.1653 0.0463
0.6111* 0.2285 0.4932* 0.6467* 0.5074* 0.0972 0.5884*
0.1853 0.3452 0.8057* 0.5278* -0.0621 0.5957*
0.6788* 0.4188 0.6756* 0.3850 0.3402
0.6379* 0.5379* 0.4233 0.5016*
0.6375* 0.1122 0.5148*
0.4142 0.4545*
0.0390
* significance level less than 0.05
Cluster analysis was performed to group table grape cultivars sampled in 1992 on the basis of their phenolic composition. Two sets of analytical variables (total phenolics in different parts of the grape berry, and several catechins and procyanidins in seeds and skins) were considered independently. Cluster analysis performed with variables corresponding to total phenolics in the different parts of the grape berry allows classifying cultivars into three clusters (figure 2). Most cultivars, red and white, were grouped into cluster 1. Only the cultivars with a very high amount of total phenolics in seeds or in skins were grouped into other clusters. Malvasia de Sitges and Alphonse Lavallee, which contained a high amount of total phenolics in seeds (pftp), were grouped into cluster 2. Negra Tardia, which contained a high amount of total phenolics in skins (pfth), was grouped into cluster 3. Cluster analysis performed with analytical variables coresponding to several catechins and procyanidins in seeds and skins allow us to group cultivars into three clusters (figure 3). Most cultivars, red and white, were grouped into cluster 1. Cluster 2 consisted of two cultivars (Muscat of Hambourg and Chelva) with a high amount of (+)-catechin in seeds (cats), and cluster 3 of two cultivars (Alphonse Lavallee and Negra Tardia) with a high amount of (H-)-catechin in skins (cath). According to the results obtained by cluster analyses, it seems evident that most table grape cultivars sampled in 1992 contained a quite similar amount of total phenolics, catechins and procyanidins in the different parts of the berry, and only cultivars with a relatively high content of phenolics in skins or in seeds may be distinguished from the others.
1589
1400
2400
pfts
3400
4400
Figure 2. Plot of clusters for variables corresponding to total phenolics in seeds, skins and pulp. Key to cultivars: AB, Aledo; AF, Alphonse Lavallde; AL, Aledo; AR, Aledo Real; BA, Barlinka; CH, Chelva; DO, Dominga; GC, Gros Colman; MH, Muscat of Hambourg; MM, Moscatel de Malaga; MN, Mantuo; MS, Malvasfa de Sitges; MT, Moscatel Tinto; MV, Malvasfa; NA, Napoledn; NT, Negra Tardia; OH, Ghanes; PM, Planta Mula; VA, Valencf; VT, Valencf Tinto.
200
400
600
cats Figure 3. Plot of clusters for variables corresponding to catechins and procyanidins in seeds and skins. For key to cultivars, see figure 2.
1590 3.1 Samples harvested in 1993. Five white and six red table cultivars were sampled in 1993. Like in 1992, results showed a great variability (tables 10 and 11). White cultivars, with the exception of Chasselas Doree, contained a lower amount of total phenolics than red cultivars. The contribution of seeds to total phenolics was higher than the contribution of skins, while pulp contained a very low amount of phenolics, less than 5 %. The content of anthocyans in red cultivars ranged from 267 mg/kg grapes (Muscat of Hambourg) to 1291 mg/kg grapes (Alphonse Lavallee). In most cases, the content of anthocyans was reciprocal to the relative amount of total phenolics in skins.
Table 10 Content of total phenolics and total catechins and procyanidins (mg/kg grapes), and their distribution (I) in the different parts of the berry in white grape cultivars. 1993 harvest. Total catechins and procyanidins
Total phenolics Cultivar Entire berries Seeds
Chasselas Doree Chelva Doninga Mantuo Ohanes
7537 3001 3402 2991 2709
80.2 84.0 74.6 68.8 55.0
Skins
Pulp
Entire berries
Seeds
Skins
18.7 15.3 24.1 28.8 41.0
1.1 0.7 1.3 2.6 4.0
393 469 183 391 196
95.4 98.9 86.9 95.1 82.6
4.6 1.1 13.1
4.9 17.4
Table 11 Content of total phenolics, total anthocyans and total catechins and procyanidins (ng/kg grapes), and the distribution (I) of total phenolics and total catechins and procyanidins in the different parts of the berry in red grape cultivars. 1993 harvest. Total phenolics
Total catechins and procyanidins
Cultivar
Total anthocyans Entire berries Seeds Skins
Alphonso Lavallee Moscatel Tinto Muscat of Hambourg Muscat of Madersfield Napoleon Valencl Tinto
5401 6738 7438 6616 3837 3613
53.2 82.0 72.2 65.3 59.3 62.6
Pulp
44.02.8 15.72.3 25.32.5 32.3 2.4 36.1 4.6 34.3 3.1
Entire berries
1291
319 267 446 499 361
511 551 918 665 286 325
Seeds
97.0 98.0 96.0 92.5 89.9 91.3
Skins
3.0 2.0 4.0 7.5 11.1 8.7
1591 The content of total catechins and procyanidins in grape berries and their distribution in seeds and skins are given in tables 10 and 11. The total amount of these compounds ranged from 183 mg/kg grapes (Dominga) to 469 mg/kg grapes (Chelva) in white cultivars, and from 183 mg/kg grapes (Napoleon) to 918 mg/kg grapes (Muscat of Hambourg) in red cultivars. Seeds contained most of catechins and procyanidins present in grapes, and the contribution of skins was less than 10%, except in the cases of Dominga, Ohanes and Napoleon. Tables 12 and 13 summarized the total content of catechins and procyanidins in seeds and skins on a weight/weight basis, and the percentage of the different compounds determined. Skins present a very low amount of several procyanidins, and only the percentage of total procyanidins is given. Table 12 Total content of catechins and procyanidins measured by HPLC (ig/kg grapes) and percentages of different catechins and procyanidins in seeds and skins of white table cultivars harvested in 1993. Skins
Seeds Cultivar
Chasselas Doree Chelva Dominga Hantiio Ohanes
C+P
CAT EPI Bl B2
B3 B4 01
C+P
CAT EPI
Pr
375 464 159 372 164
36.3 61.2 35.2 63.7 50.6
6.9 7.8 5.0 4.8 5.5
. 0.6 0.2 0.8
18 5 24 19 32
50.0 16.6 40.4 20.0 45.8 8.4 36.8 10.5 40.6 12.5
33.4 40.0 45.8 52.7 47.9
40.3 22.6 39.0 23.1 30.5
2.1 0.9 1.3 0.8 0.6
8.5 4.5 11.9 4.3 6.1
5.8 3.0 6.9 3.0 5.9
Table 13 Total content of catechins and procyanidins measured by HPLC (mg/kg grapes) and percentages of different catechins and procyanidins in seeds and skins of red table cultivars harvested in 1993. Skins
Seeds Cultivar
C+P Alphonso Lavallee Hoscatel Tinto Muscat of Madersfield Muscat of Hambourg Napoleon Valencl Tinto
496 540 615 877 257 297
CAT EPI Bl 45.2 56.9 47.8 50.4 41.6 57.9
33.3 30.9 35.6 35.6 41.6 28.0
1.0 0.4 1.5 1.1 1.2 1.0
B2 B3
B4 Cl
4.2 12.5 0.4 3.1 6.70.9 5.4 5.20.5 3.3 7.60.3 5.4 4.30.4 4.4 3.40.3
3.4 1.1 4.1 2.7 5.4 4.0
C+P 15 11 50 41 29 28
CAT EPI
40.0 13.4 54.5 9.1 50.0 6.0 63.4 9.8 48.3 10.4 10.7 7.2
Pr 46.6 36.4 44.0 26.8 41.3 82.1
1592 (+)-catechin predominated in seeds, except in the case of Chasselas Doree, Dominga and Napoleon, which contained a very high amount of (-)-epicatechin. Usually, the amount of procyanidins B2, B3 and CI was quite similar, except in Chelva, Alphonse Lavallde and Napoledn, which showed a relatively high amount of procyanidin B3. The amount of procyanidins Bl and B4 was very low in each cultivar. Skins showed a remarkable proportion of procyanidins compared to catechinsechins, which was higher than in seeds. Nevertheless, (+)-catechin and (-)-epicatechin predominated, except in Valenci Tinto and Mantuo. In these cultivars, procyanidins were 82.1 % and 52.7% of total catechins and procyanidins in skins, respectively. 3.3 Comparision of samples collected in 1992 and 1993 Four white cultivars (Chelva, Dominga, Mantuo and Ohanes) and five red cultivars (Alphonse Lavallee, Moscatel Tinto, Muscat of Hambourg, Napoledn and Valenci Tinto) were sampled in 1992 and 1993. Results are quite different for both years, even in cultivars sampled with a similar degree of maturity, as illustrated in table 14, where can be seen the ratio between the values obtained in 1993 and 1992 for several analytical variables. Anyway, in each case, ratios for total phenolics, catechins and procyanidins and total anthocyans are quite different These differences are probably due to different climatic conditions, specially solar radiation and day/night temperature altemance [1], and are in line with data previously reported for anthocyanins in red cultivars [6, 21, 22], and for catechins and procyanidins in red and white cultivars used in winemaking [23].
Table 14 Ratio of 1993 and 1992 data for some analytical parameters in several grape cultivars. Total phenolics Catechins and procyanidins (entire grapes) (entire grapes)
Cultivar
Sugar content (must)
Chelva Dominga Mantuo Ohanes
1.09 0.83 1.25 0.93
0.81 1.18 0.88 0.78
0.49 0.75 0.59 0.86
Alphonse Lavallee Moscatel Tinto Muscat of Hambourg Napole6n Valenci Tinto
1.04 1.04 0.96 0.87 1.09
1.13 1.65 2.02 1.13 0.91
0.52 0.91 0.83 0.87 0.57
Total anthocyans (skins)
1.69 12.27 0.69 1.93 1.74
1593 8000
6000
4000
2000
Figure 4. Weigth of 100 berries (WB), total phenolics in entire berries (TP), total catechins and procyanidins in entire berries (C+P), and total anthocyans (TA) of Muscat of Hambourg sampled in 1992 (dots) and 1993 (lines). Weigth of 100 berries is given in grams, the other parameters, in mg/kg grapes.
100
^B 80
60
40
20
m
m TP/8eed8
JESi^^aTP/8kln8
TP/pulp
C*P/8eecl8
C*P/8kln8
Figure 5. Distribution of total phenolics (TP) in seeds, skins and pulp, and of total catechins and procyanidins (C+P) in seeds and skins of Muscat of Hambourg sampled in 1992 (dots) and 1993 (lines).
1594 Figures 4 to 6 display data obtained for samples of Muscat of Hambourg harvested in 1992 and 1993, that illustrate the differences in the phenolic composition of grapes with a similar degree of maturity. The content of total phenolics and total catechins and procyanidins in entire berries, and that of total anthocyans in skins changed dramatically from 1992 to 1993 (figure 4). Total phenolics were more abundant in 1993 than in 1992, but the contents of total catechins and procyanidins and total anthocyans were lower in 1993 than in 1992. The distribution of total phenolics in the different parts of berries was different in 1992 and 1993, but that of catechins and procyanidins in seeds and skins was virtually the same (figure 5). Nevertheless, the percentages of (-)-epicatechin and of several procyanidins in seeds changed dramatically year to year (figure 6).
CAT
EPI
B1
B2
B3
84
C1
Figure 6. Distribution of (+)-catechin (CAT), (-)-epicatechin (EPI), procyanidin Bl (Bl), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4) and procyanidin CI (CI) in seeds of Muscat of Hambourg sampled in 1992 (dots) and 1993 (lines). 4. CONCLUSIONS The amount of total phenolics, catechins and procyanidins in berries of table grape cultivars studied is quite similar to that reported for grape cultivars used for winemaking. These substances are accumulated usually in seeds and, to a lesser extent, in skins. Pulp contained a very low amunt of these components. The content of total phenolics in the skins of red cultivars is slightly higher than in the skins of white cultivars, due to the accumulation of anthocyans in red cultivars. In most cultivars, (+)-catechin predominates in seeds and skins, and the amount of procyanidins is lower than 50% of the total
1595 amount of catechins and procyanidins measured by HPLC. Correlation analysis of several analytical variables determined in cultivars sampled in 1992 shows a close association between the contents of total phenolics in skins and pulp, and between the amount of several pairs of catechins and procyanidins in seeds. Cluster analysis allows the classification of cultivars. Most cultivars, red and white, are grouped in the same cluster. Only cultivars with a relatively high content of phenolics in skins or in seeds may be distinguished from the others. Comparision of the results obtained for cultivars sampled in 1992 and 1993 shows that there are important differences from year to year, even in cultivars sampled with a similar degree of maturity, what is probably due to different climatic conditions, specially solar radiation and day/night temperature alternance. 5. ACKNOWLEDGMENTS Dr. J. Borrego (Departamento de Viticultura y Enologia, "El Encin" Experimental Field, Comunidad de Madrid) is gratefully acknowledged for his valuable help. 6. REFERENCES 1 J.J. Macheix, A. Fleuriet and J. Billot, Fruit Phenolics, CRC Press, Boca Raton, 1990. 2 V.L. Singleton and P. Essau, Phenolic Substances in Grapes and Wine, and Their Significance, Academic Press, New York, 1969. 3 P. Ribereau-Gayon, in: P. Markakis (ed.), Anthocyanins as Food Colours, Academic Press, New York, 1982, pp. 209-224. 4 L.W. Wulf and C.W. Nagel, Am. J. Enol. Vitic, 29 (1978) 42. 5 J. Bakker and C.F. Timberlake, J. Sci. Food Agric, 36 (1985) 1325. 6 J.P. Roggero, S. Coen and B. Ragonnet, Am. J. Enol. Vitic, 37 (1986) 77. 7 J. Cacho, P. Fernandez, V. Ferreira and J.A Castells, Am. J. Enol. Vitic, 43 (1992) 244. 8 AG.H. Lea, R Bridle and C.F. Timberlake, Am. J. Enol. Vitic, 30 (1979) 289. 9 M. Bourzeix, D. Weyland and N. Heredia, Bull. OIV, 59 (1986) 1171. 10 C.Y. Lee and AW. Jaworski, Am. J. Enol. Vitic, 40 (1989) 43. 11 V. Kovac, E. Alonso, M. Bourzeix and E. Revilla, J. Agric Food Chem., 40 (1992) 1953. 12 H. Pourrat, P. Bastide, P. Dorier and A. Pourrat, Chim. Ther., 2 (1967) 33. 13 J. Masquelier, In: C.R. Symp. Int "L'Alimentation et la Consommation du Vin", Verona, Italy, 1982, pp. 147-152. 14 OMS/WHO, Los alimentos y la salud, Salvat, Barcelona, 1987. 15 L. Hidalgo, Tratado de Viticultura, Mundi-Prensa, Madrid, 1993. 16 R. Tinlot and M. Rousseau, Bull. OIV, 66 (1993) 861. 17 V.L. Singleton and J. Rossi, Am. J. Enol. Vitic, 16 (1965) 144. 18 P. Ribereau-Gayon and E. Stonestreet, Bull. Soc Chim. Fr., 9 (1965) 2649.
1596 19 STATGRAPHICS User's Guide, Statistical Graphics Corp., Rockville, MD, 1987. 20 V. Kovac, M. Bourzeix, N. Heredia and E. Alonso, Jug. Vinograd. Vinarst, 25 (1991) 10. 21 W.M. Kliewer and H.B. Schultz, Am. J. Enol. Vitic, 24 (1973) 17. 22 W.M. Kliewer, Am. J. Enol. Vitic, 28 (1977) 96. 23 M. Bourzeix, In: ler. Forum Nutrition-Industrie: Raisin, Vin, Nutrition et Sante, Lyon, France, 1990.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1597
SELECTION OF SPONTANEOUS STRAINS OF Saccharomvces cerevisiae AS STARTERS IN THEIR VITICULTURAL AREA A.I. Briones\ J.F. Ubeda^, M.D. Cabezudo^ and P. Martin-Alvarez^ Facultad de Ciencias Quimicas. Universidad de Castilla-La Mancha. Campus Universitario, 13071 Ciudad Real (Spain) 2
. ^ ^ Escuela Universitaria de Ingenieria Tecnica Agricola. Universidad de Castilla-La Mancha. Ronda de Calatrava s/n, 13071 Ciudad Real (Spain) Institute de Fermentaciones Industriales. Consejo Superior de Investigaciones Cientlficas (CSIC) . c/ Juan de la Cierva, 3, 28006 Madrid. (Spain)
SUMMARY The study of the spontaneous flora of each viticultural zone contributed in the past to the detection of many genera, species and strains whose identity was not always distinctly determined and neither was the differences among them evident. Recently, two facts should be pointed out: on the one hand, the classification of yeasts according to the criterion of Barnett et al.(2) which considerably decreased the number of catalogued species, and on the other hand, the assurance that when SO2 is added to the must which is due to undergo fermentation, only S. cerevisiae strains are viable throughout the process. When the selection of the best starters for each varietal wine within each zone is intended it is advisable to take into account the following: a) The selected yeasts should have interesting properties from an enological point of view: good fermentation rate, capacity for completely consuming reducing sugars, resistance towards ethanol, tolerance towards SO2/ and inability to produce SH2 or any other off-flavours, being desirable that they should possess a killer character, or at least be resistant towards the toxin activity. b) Among all the yeasts which possess the above mentioned characteristics, the ones which allow processing of wines with better sensory attributes than the corresponding traditional wines should be selected. The number of yeast isolates obtained in a routine sampling is very large and performing all the tests on the isolates is expensive, tedious and time consuming.
1598 For this reason the present study which involves three different cellars has been carried out and consists of the following steps: a) Observation of the karyotype of 392 isolates by means of the CHEF technique and matching of the different profiles resulting in 174 different profiles, 26 of which are the most abundant in the Spanish zone of "Valdepefias", which in turn are classified in four large karyotype groups which differ in the location of the CHEF bands. Of the four groups, two happen to be the major ones and their most relevant characteristic with regard to the other groups is that they possess two bands of 280 and 455 Kb, respectively, which are not present in the karyotype of the rest of the groups. According to this research only 12% of the tested yeasts show a good fermentation rate, tolerance towards both ethanol and SO2, and inability to produce SH2 or any unwanted off-flavours. The number of strains which addition to this give rise to wines with better sensory characteristics are reduced to 5%. Due to the fact the CHEF technique allows the separation of 13 bands, being 16 the chromosomes corresponding to S. cerevisiae. the information provided by the former technique has not allowed the establishment of a link between the enological, chemical and sensory characteristics within the studied yeasts, on the one hand, and the karyotype profile revealed by CHEF bands, on the other hand.
1. INTRODUCTION When trying to systematise the microbiological techniques used in the identification of yeasts, it is important to take into account the relevant role of yeasts in the fermentation process. It must be also remembered that the authocthonous flora covers the grape skins of vintage and that the possibilities of surviving are limited if these grape skins are not broken. There is also the fact that year after year yeasts are preserved in resistant forms in the cellars themselves. On the other hand, the companies are interested in yeasts that being capable of carrying out the alcoholic fermentation are also performe this process at both maximum speed and yield. It is convenient to differentiate yeasts by their ability to produce a particular compound in the wine, being present in larger amounts if it has a favourable incidence, or null if it confers some kind of non characteristic off-flavour taste. For this purpose identification techniques based on morphological observations, type of reproduction, and physiological characteristics, together with the analysis of both the proteins and fatty acids pool in yeasts (51) can be appealed to. In this way identification of a particular yeast can be achieved in every case. And in those cases where a more precise identification should be required, Biomolecular techniques should be then used. Exhaustive studies have been carried out in traditionally
1599 enological countries with regard to the existing yeast flora in fermenting musts. Results obtained show large differences not only within geographically distant zones but within close zones. Explanation of the former facts has to be ascribed to the identification criterion followed in each case. Thus, for example, some criteria are physiological and morphological, whereas others are based on reproductive characteristics. This may lead to some kind of confusion. Nowadays it is considered that almost all species belonging to Saccharomyces genus are varieties of cerevisiae species (2, 36). The identification of yeasts belonging to Saccharomyces genus as far as the level of strain is of great interest. Several authors (16, 37) suggested that the DNA fingerprint is invaluable for the routine identification and patenting of yeast strains and the monitoring of culture collections. Vezinhet and Lacroix (64) had proposed a genetic key of strains in relation to their resistance towards particular antibiotics. This technique happened to be useful in the investigation of the predominance of starters in wine processing although no information of other existing strains was obtained. Alternative techniques have been used in the identification of yeast strains. In some of them in order to obtain reproducible results the cell development has to be strictly controlled, which implies a disadvantage. Analysis of the monocarboxylic fatty acids freed from the free cellular extracts is a technique which allows differentiation among only some particular strains (49, 55); and by means of the study of the protein profile it has been proved that some Saccharomyces strains can be distinguished by this technique (8, 59). Genetic techniques present several advantages and amongst the more used are the following ones: Genomic DNA hybridization by means of specific probes. It consists in hybridizing a zone of the non codificant genome by using probes and comparing the DNA patterns in order to observe similarities among them. Unless an appropriate probe is employed in this particular technique a good differentiation is not achieved (66). DNAmt restriction analysis. DNAmt of yeasts contains from 65 to 80 Kb and if restriction enzymes are used, the molecule is then cut into smaller fragments of different sizes which can be separated by using electrophoretic techniques. Bands allow to distinguish different strains and it is a simple technique which has proved to be of great efficiency (21, 47, 62). Chromosome electrophoresis in pulsed field. Since the studies carried out by Schwartz and Cantor (52) this technique has been widely used in order to separate and characterize DNA molecules of whole chromosomes in all kinds of cells (bacteriae, vegetal cells, animal cells, etc) . Applied to S. cerevisae it shows a good possibility when differentiating among strains (57, 67). The information given by the chromosomal polymorphism is sufficient to establish the karyotype as an individual characteristic of each strain. This polymorphism is mainly observed in chromosomes of
1600 small size (250-800 Kb) . Several authors have proved differences with regard to electrophoretic karyotype among either yeasts used in enology from different collections or in the study of the spontaneous flora (24, 29, 43, 46, 54, 60, 65). Selection of starters. Traditional fermentation is performed by means of the spontaneous flora existing in both the cellar and the flora which covers the grape. In order to obtain wines provided with the typical characteristics of Appelations d'origine Controlee (AOC) wines some enologists only trust aspects such as climate, grape variety, technological process, etc, but yeasts are also of great importance. The use of one kind of yeast strain on different musts gives rise to different results, although it is intended that a specific yeast should lead to the making of a particular wine in order to obtain in a regular way its typical characteristics (13, 18). Nowadays it is admitted that the use of starters assures a rapid start of the fermentation process, low risk in the development of off-flavours and the opportunity of enhancing special qualities of a certain strain, which in turn endues an increase in the overall quality of the wine (19, 29) . The first two yeasts commercialized as wine starters were the so-called first generation and were recommended in order to prevent fermentative stops, although they were very sensitive towards the activity of the killer toxin. Starters of the second generation appeared next: yeasts which were adapted to musts from different viticultural regions, because they were thought to have a positive influence in the quality of wine. Finally, yeasts of the third generation are strains with an important enzymatic activity, especially with regard to the SD-glucosidase enzyme responsible for the hydrolysis of bonded terpenes, which are strongly related to the must variety (23). Appropriate wine making yeasts can be obtained by selecting the best ones within the natural flora (38) or by means of genetic manipulation of the existing yeasts. The latter is based on the recombinant DNA, which consists in inserting genes originated from other microorganisms and whose expression can improve the strain of the receiving yeast without alteration of its own characteristics (63). Selection usually takes place in specialised laboratories among isolates belonging to S. cerevisiae species, especially due to its high fermentative ability and its high resistance towards SO2. This selection must be carried out according to scientific criteria, after taking into account studies of the microbiological environment of the cellar and the knowledge of the metabolism in different available strains (14). The selection methodology has been experienced by different authors (5, 6, 11, 12, 17, 26) who have performed several tests on different musts which have been sterilised and fermented under standard conditions. The more relevant qualities before definitely selecting a particular strain are summed up as follows: a) Good fermentation rate at any temperature in the range from 18 to 30°C: Control takes place under several variables such as, for
1601 example, CO2 given off, decrease in sugars, increase in the alcoholic content etc., against time, e.i., any property directly or indirectly related to the course of the fermentation process. And an efficient sugar consumption from the medium, since a rapid fermentation does not necessarily involve a complete fermentation. b) Inability to produce SH, that can be proved by the blackening of PbAcO, and the absence of any other off-flavour. c) Tolerance towards ethanol obviously of great importance.
and
SOo.
These
properties
are
d) Killer factor. The enological interest of using killer yeasts as starters is due to the fact that they usually dominate the fermentation process and therefore avoid contamination of containers by sensitive strains (56) . Several authors (3, 7, 5 8 ) , state on the contrary that the use of killer strains is very dangerous since they contaminate both the equipment and the containers in the cellars in such a way that the subsequent use of sensitive strains could give rise to stuck fermentations. Other problems can also arise (high production of volatile acidity and SO2, of acetaldehyde and lactic acid, or low alcoholic content), and therefore suggest the use of neutral strains. e) Sensory evaluation. It is obvious that what has been mentioned up to now is of great importance in order to evaluate the fitness of a S. cerevisiae strain, although tasting provides the definitive test with regard to the selection process of strains. Wines present such a complex chemical composition that even nowadays physico-chemical analysis is insufficient in order to predict their organoleptic quality. To sum up, a yeast strain can present the best qualities with regard to the technological points mentioned above, but should be discarded if it gave rise to poor wines. Therefore each strain must be judged from the sensory profile of the wine. It is likely to consider that in the future, genetic manipulated microorganisms will have application in enology. Nevertheless, at present the enological industry has the challenge to find strains provided with optimum abilities and intimately adapted to ferment the musts of each zone. For this purpose it is important to possess a great number of isolates; to apply classification techniques which clearly inform similarities and differences among isolates and to configure strain groups which differ among them, although the grouped individuals may not be identical. In this way, information becomes easier to handle and a more detailed identification of yeasts is allowed. Other complementary techniques permit to discard the less interesting strains and to select the more interesting ones, according to the technical characteristics and especially to the sensory characteristics of the resulting wines. In order to respond to this ambition of the modern industry there are many techniques and criteria to be applied related to one another, and the approach to the subject in a systematic way becomes therefore necessary.
1602 2. THE SPANISH REGION OF "LA MANCHA" AND ITS VITICULTURAL ZONE "VALDEPENAS" "La Mancha" is located in the geographical centre of the Iberian Peninsula and accounts for 21% (4.2 million Ha) of the total agricultural area of Spain. Its climate can be described as extreme, with cold winters and both hot and dry summers, especially in Albacete and Cuenca. Also belonging to "La Mancha" is "Valdepefias" a viticultural zone to the east of Madrid-Seville route, characterised for its milder winters and summers. Several data corresponding to a period of time close to vintage (which is usually due on the second half of September) are shown in Table 1.
Table 1 Climate in "Valdepefias" (Spain) in 1991 TEMPERATURE
Months JUNE JULY AUGUST SEPTEMBER
Max T (°C)
Min T (°C)
31.7 35.9 36.5 31.1
15.9 20.1 19.5 16.5
RAINFALL Precipitations (mm^) 1.0 6.1 0.3 51.9
Humidity (max/min(%) 78/34 68/35 66/37 85/40
Nowadays, the vine extension of "La Mancha" covers about 584.100 Ha, approximately 43% of which are regarded as four "Appelations d'origine Controlee" (AOC): "Almansa", "La Mancha", "Mentrida" and "Valdepefias" together with other quality distinctions. On the other hand, the "Valdepefias" AOC is well-known all over the world. Only until recently about 20 million HI of new wine and 1.5 million HI of must were produced in "La Mancha" per year, although these figures have decreased due to agreements with the European Union. Nevertheless, "La Mancha" contributes significantly to the Spanish production of table wines: 61% of white, 21.5% of red and 42% of red claret and rosee wines. Besides, 58.5% of the wines used for vinegar production in Spain are originally from "La Mancha". The AOC "Valdepefias" wines involve a production of about 650.000 HI per vintage, and exportation of wines is increasing considerably at present.
1603 MATERIALS 3.1. Grapes and sample preparation Five subzones of "Valdepefias" AOC region the so-called "Moral", "Vega", "Sierra", "Abertura" and "Pozo de la Serna" were considered for the present study. Clusters from both the upper part of the grapevine and the ground level were selected. Samples were aseptically transported to the laboratory where stems were removed and manual crushing of the grapes took place in order to obtain the corresponding musts. 3.2. Fermenting musts obtained from three cellars Samples from tanks belonging to three different cellars were taken at different fermentation stages during two successive years in order to study the existing yeast flora. Starters were not used in these cellars. Sterilised vessels were inserted up to the geometrical centre of the containers and filled up with 250 mL of must for sampling purposes.
METHODS 4.1. Microbiological counts in both recently obtained and fermenting musts Progressive dilutions were prepared for each must and were then grown on the following culture media: Malt Extract agar pH 3.5, Wallenstein, differential Wallenstein, and on Lysine agar, and incubated at 30°C for 48-72 hours in order to establish which isolates belonged to Saccharomyces genus. Yeast colonies selected at random were purified on malt extract agar for subsequent identification purposes. 4.2. Microbiological, biochemical and genetic study of yeasts The assignment of isolated yeasts to their respective genus was carried out according to the morphological and physological characteristics based on the identification criteria of Barnett et al, (2). The morphological characteristics of the studied yeasts were: appearance of growth in liquid media (ring, film, or sediment development, turbidness, etc), sexual and asexual gemmation). Both reproduction (formation of spores and physiological and biochemical behaviour were established on the basis of carbon, nitrogen and vitamin requirements, cycloheximide resistance, urease synthesis capacity, and response to the diazonium blue to achieve the phylogenetic links between yeasts and basidiomycetes, etc. In order to obtain the S. cerevisiae strains identity an analysis of the electrophoretic karyotypes was carried out. For DNA extraction the method of Schawrtz and Cantor was used (52) with certain modifications. The S. cerevisiae isolates were grown on YM or WL media and after 48-72 hours of incubation the DNA was
1604 then extracted. For this purpose a suspension of approximately 10^ cells/mL in EDTA O.OSM pH 8 was prepared, to which was added a solution of liticase (1 mg/mL) in NaHP04 with 50% glycerol. After 15 minutes at 37°C, a solution of agarose (1.6% in EDTA 0.125M pH 7.5) was added and it was quickly transferred to suitable moulds were it was allowed to gel. The blocks thus obtained were submerged in LET buffer (EDTA 0 . 9M pH 8, tris HCl 0.02iyi pH 7 .5 and g-mercaptoethanol 7.5%) for a period of 4-12 hours at 37°C. After three washes with EDTA 0.05iyi pH 8, the blocks were covered with NDS buffer (EDTA 0. 9M pH 8, tris HCl 0.021X1 pH 7.5, 1% laurylsarcosine, 1 mg/mL proteinase K) and incubated at 50°C for 5-15 hours. After three new washes with EDTA 0.05M pH 8, the blocks were loaded onto wells in a 1% agarose gel. Once the electrophoretic run stopped, the gel was coloured with ethidium bromide at 1% for thirty minutes and was decoloured with the migration buffer for 1-2 hours. It was then photographed with a Polaroid MP4 camera, using Polaroid 667 film. The chomosomal DNA of haploid strain S228C, was used as reference in all electrophoretic runs. For the electrophoretic process, a Bio Rad Contour Homogeneous Electric Field equipment ( CHEF-DR II) was used as follows: migration buffer TBE 0. 5x at 14°C. Pulse time =60 sec/15 h and 90 sec/8 h; 200V. Identification of the physiological race was based on the criterion of Kreger Van Rij (36) by using different sugars as substratum according to the method of microtitulation plate (28). 4.3. Enological characteristics of S. cerevisiae strains In order to study both the fermentation kinetics and the possible SH2 production, a certain volume of inocule from each of the studied strains was added to 100 mL of sterilised media, and a cellular population of 10^ cells/mL was then achieved. Fermentation kinetics were followed by loss of weight in the glass flasks. Rate expressed as the loss of CO2 (g/L) , within 24 and 72 h, against time was used as parameter. The test was carried out in duplicate and PbAcO was used in order to perform a qualitative control on the possible SH^ production by some particular strains. In order to test the strain tolerance towards both SO2 and ethanol a modified methodology (45) was followed. The obtained CO2 was collected under Durham bells: the test was considered positive if within a maximum of a three day incubation period at 30°C the bell was filled up to one third of its capacity. The isolates were subjected to different EtOH and SO2 concentrations being 8°, 10° and 12° for EtOH and 25, 50 and 100 ppm for SO2, respectively. When a test happened to be positive the different isolates were then separated from the media by centrifugation, washed twice and tested for the subsequent concentration in order to work in progressive adaptation conditions of the yeasts. The ability to produce different volatile phenols in different strains was established (27) : each strain was inoculated in an added culture media of p-cumaric acid (0.5 mM) or ferulic acid in the same concentration. Both 4-vinyl phenol and 4-vinyl
1605 guayacol production was determined after 48 h incubation at 30°C by olfactory and spectrophotometric analysis, respectively. The killer toxin was also detected (53). 4.4. Wine making on a small scale Wine micro-processing was carried out by using a model solution. On the other hand Macabeo musts were subjected to light sulphitation (50 ppm of SO2) in the corresponding cellars and once in the laboratory were then decantated after 24 h refrigeration at 4°C. The different substrata were inoculated with the testing yeasts until counts of approximately 10^ cells/mL were obtained. Several fermentations were carried out in duplicate in flasks under controlled temperature, at 25°C. Daily control was performed by weight. The end of the fermentation process was established by the almost loss of sugar. Fining of the wines was performed by using a 5% bentonite aqueous solution and wines were then refrigerated for 24 h and subsequently decanted and tangentially filtrated through 0.45^^ pore membranes. Wines were preserved closed by using crown stoppers and refrigerated until analysis took place. 4.5. Chemical analysis of wines by GC Major volatile components were analysed by GC: Injector and FID detector temperature: 230°C; nitrogen was used as carrier gas. Program temperature:40°C-6°C/min-160°C. Minor volatiles were also analysed by GC, prior to continous extraction of 250 mL of fermented sample with 90 mL of a mixture of pentane and methylene chloride (2:1) for 10 hours. Injector and FID detector temperature: 250°C, carrier gas nitrogen, temperature program: 55°C-2.5°C/min-185°C (61). 4.6. Sensory analysis Sensory analysis was carried out by means of a taster panel from "La Mancha" University in a tasting room adequately equipped according to the ISO 8589-88 standard (32) . Evaluation was focussed on possible defects (stale taste, sulphur compounds, boiling taste...), routinely observation of acidity, astringency, body and after-taste together with the detailed consideration of the following attributes: freshness odour, resin or pine-tree odour, fruit odour and odour intensity. Defective samples were immediately discarded and those which had succeeded this first part of the evaluation were subjected to an overall assesment according the average value corresponding to the scores obtained for each individual attribute. Attributes were judged from "unnoticed" to "clearly perceptible" up to a maximum score of 10 points. 4.7. Statistical methods Multivariate analysis of Principal Component Analysis (PCA)
1606 and Stepwise Discriminant Analysis (SDA) have been applied by means of the statistical package BMDP (20).
SPONTANEOUS FLORA OF GRAPES DURING VINTAGE TIME The existing speculation about uniformity of spontaneous flora for each viticultural zone within years has given rise to an exhaustive study of the authocthonous flora of the different Spanish zones. The former studies were initiated in Spain for "La Mancha" zone in 1957 (9) and subsequently were extended to other viticultural areas (1, 15, 35, 39, 44). Reviewed literature shows the presence of oxidative yeasts in most of the Spanish zones followed in less proportion by the Hanseniaspora, Pichia, Candida and Debaryomyces genera, and with the absence of S. cerevisae in the clusters (22, 31, 40, 41) . Experience reveals that when a defective grape skin disgregation takes place, a significant number of S, cerevisiae isolates are not then achieved. Generally speaking, yeasts belonging to oxidative species such as Sporobolomyces roseus, Rhodotorula crlutinis, Cryptococcus laurentii and Cryptococcus albidus together with smaller proportions of fermentation species such as: Pichia guillermondii, Candida tennuis and Debaryomyces hansenii are abundant in "Valdepefias" vines. The two former species have an insignificant role in the fermentation process of musts since they disappear once the process has started. Sampling of the spontaneous flora of grape clusters belonging to different vines from Valdepenas proved several matters: a) in general, but not always, clusters located near the ground level are favoured by a flora consisting of a larger number of yeast species and b) the number of species vary considerably from one vineyard to another. When the subzones are clx)se differences based on climate are too small to justify differences detected in the flora. Therefore variations should be attributed to possible differences within the ground structure and fertility degree. The establishment of a relationship between the number of colonies isolated for each zone within the five studied, on the one hand, and ground characteristics, on the other hand, has been attempted (33) and further results are given in Table 2. According to the number of colonies, clusters belonging to both subzones "Pozo de la Serna" and "Vega" involve a larger number of yeasts than the rest. These areas posses similar grounds in terms of N and K content. Besides, they present a high lime content (about 18-20%) sustained in a 58-62% of sand. On the other hand, clay content within all subzones is 19-23%. When comparing values expressed for both "Pozo de la Serna" and "Vega" subzones, on the one hand, and "Abertura", "Sierra" and "Moral" subzones, one the other hand, it is noticed that the poorer the corresponding grounds happen to be (in terms of N,K and lime contents and high content in sand) the less abundant is the spontaneous flora.
1607 Table 2 Ground characteristics of the vineyards from five subzones of "Valdepehas" and yeast colonies isolated from the spontaneous flora of the clusters. Ground characteristics Valdepefias (Spain) Subzones:
K N i (meq/lOOg) (Tieq/lOOg)
Spontaneous flora
Lime (%)
Sand (%)
yeasts/mL
Pozo Serna Vega
0.121 0.144
1.64 1.05
18.56 19.86
58.88 61.58
445 448
Abertura Sierra Moral
0.077 0.070 0.033
0.60 0.78 0.64
6.56 6.56 1.32
78.88 74.88 90.88
25 60 212
meq: milliequivalents
6. CHARACTERISTIC YEASTS OF THE CELLARS In each cellar authocthonous species proliferate at a disadvantage of others, being the so-called predominant species the ones which year after year confer their characteristic properties to the wines of each brand. These properties in turn distinguish them from other cellars. Nevertheless, the use of SO2 gives rise to a reduction in the number of species capable of must fermentation according to its SO2 tolerance, ranging from S. cerevisiae (more tolerant) to Hanseniaspora (more sensitive). Experience points out that in cellars where sulphitation takes place S. cerevisiae strains are abundant, and this profile remains year after year (1, 30, 35) . A study including two subsequent vintages was performed in order to observe if predominance of some yeast species over others took place and which were predominant according to the cellar (34). This study involved four cellars from "Valdepehas" OAC and three fermentation tanks for each one (in all 12 tanks) corresponding to vintages 1992 and 1993, respectively. A moderate sulphitation took place in all cellars thus a large population of S. cerevisiae was expected in every case. Data base consisted of 180 isolates for the first year (1992) and 135 for the second year (1993) . All colonies from both vintages (315 colonies) were grown on the proper media in order to establish their belonging to Saccharomyces genus (10) . The obtained results are given in Table 3 where predominance of S. cerevisiae is stated in all cases. Within the two years considered yeasts belonging to other genera were also isolated, especially several Hanseniaspora, Candida and Pichia species, but in a scarce
1608 proportion.
Table 3 Classification of yeast isolates corresponding to fermenting tanks from four cellars of the "Valdepenas" zone (Spain). Number of isolates (genera) Vintages
Saccharomyces
Hanseniaspora
Candida
Pichia
Total
1992
148
28
1
3
180
1993
123
4
6
2
135
It is frequent to find apiculate yeasts belonging to Hanseniaspora genus in warm regions (15) , in our particular case H. occidentalis and H. uvarum were detected. The identified yeasts belonging to Candida genus were C. apicola and C. stellata and belonging to Pichia genus was P. cruillermondii. These yeasts have been also described in white musts from other viticultural regions in the first stages of the fermentation process (1, 41, 42).
7. KARYOTYPE OF THE S. cerevisiae STRAINS ISOLATED IN FERMENTING MUSTS
In another experience similar to the previous one, 392 colonies belonging to S. cerevisiae were obtained from three cellars from which again must fermenting samples were taken from 4, 6 and 4 tanks respectively. The chromosomal profile of the 392 isolates was obtained by means of Contour Homogeneous Electric Field (CHEF). To sum up the results referring to all of the vats in three cellars, the 392 isolates gave rise to 174 patterns with different karyotypes. A screening was performed and the most abundant karyotype patterns in each vat were selected. The 174 different karyotypes gave rise to 26 predominant patterns. On the other hand, after applying Cluster Analysis to the data referred to the 174 karyotypes of the different S. cerevisiae strains (4) the existence of four large groups with different characteristics was revealed. The 2 6 predominant strains belonged to a greater extent to group number 1 (11 strains) and group number 2 (6 strains). Example of these karyotypes are shown in Figure 1. The most outstanding differences in the karyotype of the
1609 four groups are shown in Table 4 (4)
o n OJ
CD
(D 00 (\i CJ
CO CO
a' u
Figure 1. CHEF profile of the different S. cerevisiae strains abundant in "Valdepehas". Main karyotype groups: 1 (strains A83, B156, B159, B230), 2 (strains A3, A6, A26, C286), 3 (strains A78, B162) and 4 (strains B217, C293).
Table 4 Differential bands of the most abundant S. cerevisiae strains in the cellars; 4 groups possessing different karyotype patterns Main strains groups with a different karyotype
CHEF Bands Kb
240
280
355
455
590
675 785 830 X
X
X X X
X X
1610 8. E N O L O G I C A L CHARACTERISTICS DIFFERENT KARYOTYPE
OF
S.
cerevisiae
STRAINS
OF
W h e n the 174 strains of S. cerevisiae of different karyotype w e r e g r o w n on a m o d e l solution in order to ferment, 60 of them h a p p e n e d to p r o d u c e SH2 w h i c h was noticed b y the b l a c k e n i n g of PbAcO. T h e rest e i t h e r p r e s e n t e d slow fermentation rates or w e r e u n a b l e to consume completely the existing sugar of the c o r r e s p o n d i n g m u s t , except for 36 w h o s e fermentation rates ranged b e t w e e n 0.2 and 0.4 g of CO2 g i v e n off p e r litre and h o u r . These 36 strains c h o s e n due to their fermentation rate w e r e subjected to d i f f e r e n t SO2 and ethanol amounts respectively in o r d e r to study their t o l e r a n c e . Only 2 0 easily overcame the m a x i m u m extreme c o n d i t i o n s imposed. They w e r e once a g a i n tested in o r d e r to study if they w e r e capable of p r o d u c i n g the k i l l e r toxin, t o g e t h e r w i t h the resistance or sensitivity towards its activity: 8 h a p p e n e d to b e Killer^ and Resistant^, w h e r e a s the rest, 1 2 , did not p r o d u c e the toxin but w e r e resistant to its activity (K",R^) . The p o s s i b i l i t i e s K^R" and K",R" w e r e not detected (Table 5) . T h e fact that g r o w t h of w i l d strains is not affected by the k i l l e r toxin is a convenient characteristic for practical p u r p o s e s ( 3 ) . In r e l a t i o n to the d e c a r b o x y l a t i o n p r o p e r t y of p h e n o l i c (Pof), 18 strains p r o v e d to be capable of p r o d u c i n g acids v o l a t i l e p h e n o l s as shown in Table 6. This characteristic is common to the m a j o r i t y of the wild S. cerevisiae strains ( 2 7 ) . By a s s o c i a t i n g b o t h the k i l l e r and the Pof character, the results shown in T a b l e 7 w e r e then obtained, w h e r e the independence b e t w e e n the two characters is stated.
Table 5 K i l l e r c h a r a c t e r of the selected S. cerevisiae strains. K i l l e r fenotype K-R+
N^ of
isolates 12
K+R-' K-'RK-R-
8 0 0
9. C H E M I C A L C O M P O S I T I O N OF THE FERMENTED M E D I A
which
The S. cerevisiae strains selected w e r e 20 (numbers 1 - 2 0 ) , p o s s e s s the following c h a r a c t e r i s t i c s : a) they do not
1611 produce SH^, b) their specific fermentation rate ranges from 0.2 to 0.4 g/L/h CO2/ c) they tolerate alcohol in large amounts, d) they tolerate SO2 in amounts of at least 100 ppm and e) most of them are K" R+ and Pof^. On the other hand, the 20 strains belong to the four main groups of different karyotypes (Table 4) found in the "Valdepehas" zone as reported in Table 8.
Table 6 Volatile phenol production by the selected Pof fenotype
S. cerevisiae strains N^ of isolates
Pof(+)
18
Pof(-)
2
Table 7 Killer and Pof character of the
S. cerevisiae selected strains
K-'R-^
K-R+
Pof(+)
8
10
Pof(-)
0
2
Table 8 Ascription of S. cerevisiae selected strains to the main different karyotype groups from the " Valdepehas " zone Main
S. cerevisiae strains
karyotipe profiles
Group 1
Group 2
6*,7,8,12 14,18,20
1,2,3,5 15,16,17
(*)Identification code
Group 3 9,13,19
Group 4 4,10,11
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1613 At this stage of the study, the matter of knowing if all strains gave rise to the same wines in terms of composition was questioned. For this purpose fermentations on a small scale were performed in duplicate, and analysis of the most characteristic volatiles in wines was carried out. The substrata subjected to fermentation were two, I: a model solution (Williams medium) which contains the necessary sources in C and N, minerals and vitamins together with grape fixed acids, and II: a white grape must of Macabeo variety. Several volatiles happened to be present at concentrations lower than 0.5 mg/L, such as: isobutyl-acetate, isoamyl-acetate, 2-phenylethanol-acetate, ethyl-butyrate, diethyl-succinate, butyric acid and 9-decenoic acid. Concentrations corresponding to the rest of the variables are shown in Table 9. The following compounds showed statistical significant differences: ethyl caprylate, 4-ethylhydroxybutyrate, caproic acid and capric acid, depending on the substratum to be fermented (synthetic medium or must) and large differences were also observed in the amounts corresponding to other volatiles. Thus, for example, higher values were observed in both 1-propanol and etoxypropanol and lower in 3-methyl-1-butanol respectively for the synthetic medium. This is probably due to the fact that in the synthetic medium the nitrogen source is provided by ammonium salts whereas aminoacids and several nitrogen compounds are available in the must (25) . When applying Principal Component Analysis to the wines obtained with the 2 0 S. cerevisiae strains on the Macabeo must, it was noticed that the most correlated variables with each Principal Component are the ones reported in Table 10 and when plotting the wines on both axes Figure 2 were then obtained: There are two strains (2 and 6) which are separated from the rest due to the higher values within the variables which are involved in the Principal Component 1. According to the lineal function representing Principal Component 2, there are other strains which differ from the central group due to either their lower values (strains 7, 10 and 11) or their higher values (strains 8, 9 and 18) . Differences of the wines obtained by the strains different from the general set, are shown in Tables 11, 12 and 13. Strains 2 and 6 are responsible for the volatiles in Table 11 in slightly higher amounts than the set. On the contrary, strains 7, 10 and 11 produce in less amounts most of the volatiles in Table 12, except for isobutanol, and the opposite effect is observed in the strains and variables corresponding to Table 13. Results reveal both the important differentiating role of the acids developed by these strains and the remarkable differences observed in relation to 1-propanol and isobutanol (48, 50) . As Stated above, the 20 selected strains presented similar characteristics, although small differences within the volatiles were observed. In order to prove if these differences could be due to a certain karyotype profile of the four found in this study (Table 4) Stepwise Discriminant Analysis (SDA) was applied to the data corresponding to volatiles. A first test showed that
1614 fermented media obtained by using strains of groups 1 and 2 are almost indistinguishable. Whereas fermented media by using groups 3 and 4 differ from the others, as shown in Figure 3. Variables which allow separation within groups are shown in Table 14, and it can be stated that differences are not large. Consequently, there is no evidence (with regard to volatiles) that differences in the karyotype should necessarily lead to different fenotypic behaviour.
Table 10 Principal Component Analysis applied to Macabeo wines obtained by means of the 20 selected strains Principal Component
Explained variance(%)
Cumulative proportion(%)
Variables most correlated
22,10
22,10
ethoxypropanol 1- propanol isobutyric acid isovaleric acid propanediol acetate
17,90
40,00
capric acid isobutanol ethyl caprate caprylic acid
Table 11 Concentration (mg/L) of the variables which more differentiate wines obtained by strains 2 and 6, from the total set 1- propanol isobutyric isovaleric propanediol acid acid acetate X
ethoxy propanol
22,9
1,4
0,6
2,1
0,2
strain 2 wine
48,7
3,4
1,4
6,3
1,1
Strain 6 wine
70,2
7,2
2,0
6,6
1,9
X: mean values corresponding to all the wines
1615 PRINCIPAL
CO M P O N E N T 2
2
-0.5 PRINCIPAL
1 2.5 COMPONENT 1
Figure 2. Application of Principal Component Analysis to the volatiles corresponding to wines obtained by means of several S. cerevisiae strains. Plotting of the 20 selected strains.
Table 12 Concentration (mg/L) of the variables which more differentiate wines obtained by strains 7, 10 and 11, from the total set. capric acid
isobutanol
ethyl caprate
caprylic acid
X
0,8
25,0
0,07
5,0
strain 7 wine
0,3
35,9
0,03
3,4
Strain 10 wine
0,3
38,8
0,03
2,5
Strain 11 wine
0,5
38,9
0,03
3,7
X: mean value corresponding all the wines
1616 Tabla 13 Concentration (mg/L) of the variables which more differentiate wines obtained by strains 8, 9 and 10, from the total set capric acid
isobutanol
ethyl caprate
caprylic acid
0,8
25,0
0,07
5,0
strain 8 wine
1,2
19,5
0,1
6,5
Strain 9 wine
1,2
14,6
0,1
5,8
Strain 18 wine
1,3
12,0
0,1
7,1
_ X
X: mean value corresponding to all the wines
Table 14 Average concentration (mg/L) of the volatiles in the fermented media by the 2 0 selected yeast strains grouped according to the main karyotype groups mean •values (mg/L) Volatiles
groups 1 +2(*)
group 3
group 4
isobutanol
25,1
32,2
28,2
2 methyl-1-butanol
14,1
12,0
18,6
ethyl caprate
0,04
0,06
0,03
butyric acid
0,5
0,5
0,3
capric acid
1,2
1,1
0,42
(*) In table 8 are reported the identification codes for the strains involved in each group
1617 CANO N I C A L
VARUeiE
2
CANONICAL
-1 VARI ABLE 1
Figure 3 . Canonical plot obtained by applying SDA to the volatiles corresponding to synthetic medium by using selected yeasts. Numbers 1, 2, 3 and 4 correspond to the numbers assigned to the 4 different karyotype groups founds in the "Valdepenas" zone.
10.
CHEMICAL COMPOSITION AND SENSORY PROPERTIES OF WINES
It is extremely stimulating to relate the chemical information of wines to sensory information in order to obtain possible relationships. In the present work there are 20 wines of Macabeo variety, obtained each one by using a unique selected strain of S. cerevisiae. whose volatile composition is known. On the other hand, the 20 strains belong to 4 groups of different karyotype profile. Application of different tests of Sensory Analysis resulted in the obtention of high scores for 9 of the 20 wines due to their remarkable attributes and the absence of defects. The rest of the wines proved to be deficient. The relationship between sensory characteristics of wines and the karyotype profile group of the S. cerevisiae which have been involved is reported in Table 15. It can not be stated that either the most valuable wines or the defective wines are linked to a certain karyotype profile. Therefore it does not seem that the karyotype profile of the starters allows the prediction of the sensory quality of wines.
1618 Table 15 Classification of wines according to their sensory properties and to the originating S, cerevisiae strain Karyotype profile Macabeo wines
group 1
group 2
group 3
group 4
wines fermented by With interesting atributes
7*,14,18
1,15,16, 17
With Sensory defects
6,8,12,20
2,3,5
19
9,13
10,11
(*) Identification code
However, it must be pointed out that wines obtained by strains 2, 6, 8, 9, 10 and 11 happened to be outlayers when applying PCA, and have proved to be defective from a sensory point of view. This result shows some kind of relationship between concentration of the most relevant fermentation volatiles and the sensory quality of wines. Nevertheless, it is not sufficient in order to obtain general conclusions.
11. IDENTIFICATION cerevisiae STRAINS
OF THE PHYSIOLOGICAL
RACE
IN SELECTED
The nine S. cerevisiae strains, which gave rise to wines with favourable sensory characteristics, were subjected to several fermentation tests by using different sugars in order to assign their physiological race. Seven strains happened to belong to cerevisiae variety (1, 7, 14, 15, 16, 17 and 18), being the rest (strains 4 and 19) of bayanus variety.
ACKNOWLEDGEMENTS This work was made possibile by financial assistance from the Spanish Interministerial Commission for Science and Technology, Project ALI91-0701. The assistance of M.S. Perez Coello in data handling is
1619 highly appreciate and encouragement of Dr G. Versini is gratefullyacknowledged.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1623
Hydrolysis of grape glycosides by enological yeast p-glucosidases I. Rosl, P. Domizio, M. Vinella and M. Salicone Dipartimento di Biologia, Difesa, Biotecnologie Agro-Forestali, Universita della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy
Abstract Three enological yeast strains, belonging to the species Debaryomyces hansenii, Debaryomyces polymorphus, and Saccharomyces cerevisiae, characterized by an exocellular p-glucosidase activity, were examined for their ability to hydrolize a glycosidic extract from grape juice. The enzymatic preparations (culture supernant fluid) of the different yeasts released different amounts of terpenols such as linalol, a-terpineol, geraniol, nerol, citronellol and benzyl and 2phenylethyl alcohol. The extent of release of the flavour compounds was related to yeast species. When an enzymatic preparation (concentrate culture supernatant) of Debaryomyces hansenii was incubated with a wine containing glycosidic precursors, significant production of monoterpenols and benzyl and 2-phenylethyl alcohol was observed.
1.
INTRODUCTION
Terpenes are a class of compounds responsible for the varietal aroma of many grapes, wines, and other fruits. Among the terpenes, the monoterpenols (linalol, nerol, geraniol, a-terpineol and citronellol) are the most active, from an olfactory point of view, due to their low sensory threshold. For example, linalol has a sensory threshold of 100 |Lig/l, while that of nerol and a-terpineol is three to four times higher (1). However, most of the terpenols in grapes are found in glycosidically bound forms which are almost odourless. Studies conducted to identify the glycosidic part of these flavour precursors, have shown that it is mostly formed by 6-0-a-arabinofuranosyl-p-D-glucopyranosides, 6-0-a-L-
1624 rhamnopyranosyl-p-D-glucopyranosides (rutinosides) (2, 3), 6-0-p-apiofuranosylP-D-glucopyranosides (4) and, to a much lesser degree, p-D-glucopyranosides (2, 3). The aglyconic part is primarily made up of monoterpenols and benzyl and 2phenylethyl alcohol. To release this potential reserve of aroma, studies have been conducted using enzymes with glycosidase activity . Earlier studies showed that hydrolysis of glycosidically bound terpenes occurs in two sequential steps (5). In the first, an a-L-arabinofuranosldase, a-L-rhamnopyranosidase and p-D-apiosidase must break the bond between the glucose and the terminal sugar (rhamnopyranose, arabinofuranose and apiofuranose). In the second step, a p-glucosidase must release the volatile aglycon from the glucose. Enzymatic preparations from plants (grape and sweet almond) and from microorganisms (moulds and yeasts) were studied to evaluate their capacity to hydrolyze the aromatic precursors found in grape and other fruits. Plant-produced p-glucosidases were characterized by a restricted specificity with respect to aglycon, were not very active between pH 3 and 4 and were inhibited by a glucose concentration over 1% (6, 7) .The p-glucosidase of fungal origin reacted strongly to a glucose concentration above 1-1.5 % (8). The P-glucosidases produced by the yeasts {Candida molischiana, C. wickeramii, Saccharomyces cerevisiae) were less sensitive to glucose and had a wider specificity for aglycon (9, 10). In a recent study (11) conducted to show the p-glucosidase activity in yeasts of enological origin, we found a strain of Debaryomyces hansenii which could produce an exocellular p-glucosidase whose activity was not inhibited by high ethanol and glucose concentrations and was not greatly influenced by acidic pH and low temperatures. Currently, we are studying the environmental conditions needed to increase the production of exocellular p-glucosidase by this strain of yeast as well as a strain of Debaryomyces polymorphus and Saccaromyces cerevisiae, which in a previous study, exhibited exocellular p-glucosldase activity. The aim of this work is to verify the capacity of exocellular p-glucosidase, produced by three different yeasts, to hydrolyze the glycosidically bound aroma compounds , in light of their use in juice processing and winemaking.
1625 2. MATERIALS AND METHODS 2.1 Yeasts The strains used were Debaryomyces hansenii 4025, Deb. polymorphus 3631 and Saccharomyces cerevisiae 1014. All strains were obtained from the Industrial Yeast Collection of Dipartimento di Biologia Vegetale, deH'Universita di Perugia (DBVPG). The strains were maintained at 4° C on slopes of yeast malt agar (YM). 2.2 Medium and culture conditions The basal culture medium was Yeast Nitrogen Base (YNB, Difco) : 0.67%, buffered with phosphate tartrate (100 mM, pH 5.0); the carbon source was arbutin (0.5%) for Saccharomyces cerevisiae 1014 and Debaryomyces polymorpiius 3631 and glucose (0.5%) for Debaryomyces iiansenii 4025. Aerobic culture was performed in Erienmeyer flasks filled to one tenth of their volume and shaken at 150 rev min""" on a giratory shaker for 24 h at 25 °C. 2.3 Enzymes After centrifugation (4000 rev min""", 10 min, 4 °C), the culture supernatant fluid of Debaryomyces Iiansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014 and the culture supernatant of Debaryomyces hansenii 4025 concentrated 50-fold by ultrafiltration in an Amicon cell (PM 10 filter) were used for hydrolysis assay. AR 2000 (Gist-Brocade, DAL GIN, Milano, Italy), a commercial preparation of pectolytic enzyme, which is a soluble powder, was also used. 2.4 Enzyme assay p-glucopyranosidase, a-rhamnopyranosidase and a-arabinofuranosidase activities were assayed by measuring the amount of p-nitrophenol (pNP) liberated from the substrates p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-Lrhamnopyranoside or p-nitrophenyl-a-L-arabinofuranoside. According to the previously described method (11), 0.2 ml of each enzymatic preparation was mixed with 0.2 ml of a 5 mM solution of the appropriate glycoside in 100 mM citratephosphate buffer (pH 5.0). The reaction mixture was incubated at 30 °C for 1 h and subsequently 1.2 ml of carbonate buffer (0.2 mM, pH 10.2) were added to stop the reaction. The pNP liberated was measured by spectophotometry at 400 nm in a LBK Ultraspec 4050 spectophotometer. All assay were performed in duplicate and
1626 averaged. One unit of enzymatic activity (U) was defined as jimol of p-nitrophenol released min"'', under the above conditions. 2.5 Protein assay The protein concentration of samples was determined with the Bio-Rad protein reagent with bovine serum albumine as standard (Bio-Rad Laboratories, Richmond, Ca, USA) 2.6 Experiments with grape glycosides a) Isolation of the glycosides 200 ml aliquots of a mixture of Traminer grape juice and water (1:1) orTraminer wine were passed through a reverse-phase CI8 adsorbent column (5g) (Mega Bond Elut, VARIAN, Harbor City, CA, USA) that was activated by flushing with 40 ml of methanol and 40 ml of water. The column was washed with methylene chloride (100 ml) in order to remove free terpenols. Glycosides were then recovered by elution with methanol (100 ml) and stored at -18 °C. Before using, this fraction was concentrated to dryness under vacuum at 30 °C . b)Enzymatic hydrolysis of the glycosides The glycosidic sample obtained from 200 ml of grape juice was dissolved in 50 ml of culture supernatant fluid (pH 4) of Debaryomyces hansenii 4025, Deb. polymorphus 363^ and Saccharomyces cerevisiae 1014. The glycosidic sample obtained from 200 ml of Traminer wine was suspended in 50 ml of lOOmM citratephosphate buffer (pH 5.0) and hydrolyzed with the concentrated supernatant of Debaryomyces hansenii 4025 and with the commercial pectolytic enzymatic preparation (AR 2000). The two enzymatic preparations, having the same [3glucosidase activity (0.7 U), were also used to hydrolyze the terpene glycosides found in 50 ml of Traminer wine (pH 3.1) in which free terpenols were measured. All samples were incubated at 25 °C for 48 h. Controls were run as above but without enzymatic preparation. All assays were performed in triplicate. 2.7 Analysis Liberated monoterpenols were: a) isolated by dynamic headspace analyzer, b) separated by gas chromatography and c) detected and evaluated by ion trap detector.
1627 a) Dynamic headspace sampling of monoterpenols (12) The volatile monoterpenols in the headspace were isolated by dynamic headspace analyzer LSC 2000TM (TEKMAR Inc., Cincinnati, OH, USA). The sample vessel, after conditioning at 25°C for 10 minutes, was purged with helium at 40 ml min-"" for 30 min to isolate headspace monoterpenols. The compounds isolated by purging were then trapped in a TenaxTM , 12x1/8" column. The trapped volatile monoterpenols, after a 10 min dry purge, were desorbed at 220 °C for 20 min, using helium gas at 1 ml min"'', and cryogenically focused at the first 10 cm capillary column which was cooled down to -100 °C by liquid nitrogen. The volatile compounds, condensed in the first 10 cm of the capillary column, were rapidly vaporized at 220 °C, and transfered to the capillary column for the analysis.
b) Gas chromatographic analysis A gas chromatograph (VARIAN 3300, Palo Alto, CA, USA) with an Ion trap detector (Finningan Mat ITD 700) was used to analyze of volatile monoterpenols. A fused-silica capillary column, Supelcowax 10, 60 m x 0.32mm, 0.25 mm film thickness, was used. The helium gas flowrate was 1ml min-""- The initial column temperature of 60 °C was maintained for 10 min after which the temperature was programmed to increase 3°C min-i to the final temperature of 195 °C which was maintained for 10 min. The peak areas of monoterpenols were calculated by peak integration module in the ion trap detector. Figure 1 shows the separation of some volatile monoterpenols and two cyclic alcohols, released by the culture supernatant fluid of Debaryomyces hansenii 4025 from glycoside extract of Traminer grape juice, after 48 h at 25 °C. Figure 2 shows the volatile compounds present in glycoside extract of Traminer grape juice after 48 h at 25 °C, without yeast enzymatic preparation. The identifications in the legend were determined by comparing the results with mass spectra of reference substances. c) Ion trap detection (ITD) Electron impact mass spectra of monoterpenols enzymatically hydrolyzed from the glycosides were recordered by coupling the Supelcowax 10 fused-silica capillary column to ITD. The transfer line was mainatained at 220 °C. The source temperature was 230 °C. Mass spectra were scanned between 50 and 80 eV in the 50-250 m/z range at 2-s intervals. The electron multiplier voltage was 1350 V.
Figure 1. Total ion chromatogram of the headspace compounds from grape glycoside extract after hydrolysis (48h, 25°C) by yeast enzymatic preparation (culture supernatant fluid of Debaryomyces hansenii 4025) l=Linalol, 2=u-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol
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.
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1800 30: 01
'
~
'
2400 40: 01
"
~
'
3000 50:01
_. l
'
~
'
3600 60: 01
Figure 2. (Control) Total ion chromatogram of the headspace cotnpounds from grape glycoside extract after 48 h at 25°C without yeast enzymatic preparation. l=Linalol, 3=Citronellol, 4=Nerol, 5=Geraniol.
-
O\
\D N
l
'
1630 E3 Saccharomyces cerevisiae 400
CO 1^
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Debaryomyces polymorphus Debaryomyces hansenii
z o
3
4
5
COMPOUNDS
Figure 3.
Monoterpenols and aromatic alcohols released from a grape glycoside extract added to the culture supernatant fluid of three yeasts 1=Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1%levels)
1631 E3i Sacch. cerevisiae B Deb. polymorphus H Deb. hansenii
a) Culture supernatant fluid
o
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m AR2000 B Deb. hansenii 147***
D)
b) Wine
3 O
20001
216*
^ •
AR2000 Deb. hansenii
c) Buffer
o
1000
Figure 4
Total amount of monoterpenols (1) and aromatic alcohols (2) released by: a) culture supernatant fluid of three yeasts from grape glycoside extract b) Deb. hanseneii and AR2000 enzymatic preparations from wine (pH 3.1); c) Deb. hanseneii and AR2000 enzymatic preparations from wine glycoside extract in citrate-phosphate buffer (pH 5.0) (LSD value,*, **, *** significant at 5, 1 and 0.1%levels)
1632 ^
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AR2000
EB Deb. hansenii
3 4 5 COMPOUNDS ^
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163***
b) Buffer
3 4 5 COMPOUNDS Figure 5
Monoterpenols and aromatic alcohols released by Deb. hanseneii and AR2000 enzymatic preparations from: a) wine (pH 3.1); b) wine glycoside extract in citratephosphate buffer (pH 5.0) 1 =Linalool, 2=a-Terpineol, 3=Citronellol, 4=Nerol, 5=Geraniol, 6=Benzyl alcohol, 7=2-Phenyl ethyl alcohol. (LSD value, **, *** significant at 1 and 0.1 % levels)
1633 2.8 Chemicals p-nitrophenyl-p-D-glucopyranoside, p-nitrophenyl-a-L-rhamopyranoside, pnitrophenyl-a-L-arabinofuranoside were obtained from Sigma (St. Louis, MO, USA). All other chemicals used were of reagent grade. 3. RESULTS AND
DISCUSSION
The production and properties of yeast-produced p-glucosidases is of interest because of the potential to increase the availability of specific enzymatic preparations which could be used in winemaking to increase the varietal aroma of the wine. There is also interest in the possibilty of arranging selected cultures of yeasts characterised by the presence of active p-glucosidase during and/or after alcoholic fermentation. The hydrolysis values of the terpene glycosides of Traminer grape juice, added to the culture supernatant containing the exocellular p-glucosidase produced by Saccharomyces cerevisiae 1024, Debaryomyces polymorphus 3631 and Debaryomyces hansenii 4025, are reported in Figure 3. More linalol and a terpineol were released by the enzyme present in the supernatant of Deb. polymorphus 3631, while more 2-phenylethyl alcohol was released by the enzyme of Deb. hansenii. The enzymatic preparations of Deb. polymorphus 3631 and Sacch. cerevisiae 1014 released the most monoterpenols (Figure 4, a). The smaller quantity of monoterpenols released by the exocellular enzyme produced by Deb. hanseniii 4025 could be due to the lack of a-L-arabinofuranosidase and a - L rhamnopyranosidase enzymatic activity as reported in Table 1. These results confirm previous findings, i.e. that most of the monoterpenols are found as diglycosides (2, 3). Therefore, the enzymatic preparations of Deb. polymorphus 363^ and Sacch. cerevisiae 1014, which have a-L-arabinofurosidase and a-L-rhamnosidase activity, can release more monoterpenols into the medium. Furthermore, the enzymes produced by these yeasts showed a rather wide specificity. They could attack the glycosides with tertiary alcohols as aglycon, as well as, those with primary alcohols. This behaviour has rarely been found in the enzymatic preparations of either plant or microbial origin studied to date. Having shown that p-glucosidase, produced by Deb. hansenii 4025, had some interesting properties that could be used in winemaking (11), we carried out trials to analyze the hydrolytic behaviour of the culture supernatant fluid, concentrated 50 times, and compared the results to those from the commercial
1634 pectolytic enzyme preparation (AR2000). Recent studies have shown that this enzyme, produced by Aspergillus niger, efficiently releases the monoterpenols from the terpenic glycosides present in white and red aromatic grape must (13). Figure 5 reports the amounts of monoterpenols and benzyl and 2-phenylethyl alcohol released by the two enzymatic preparations added to Traminer wine (pH 3.1) and to a citrate-phosphate buffer (pH 5.0) containing glycosides extracted from the Traminer wine. The enzymatic preparation of Deb. hansenii 4025 released more linalol, a-terpineol, benzyl and 2-phenylethyl alcohol, while the commercial enzyme released more citronellol, nerol and geraniol. This behaviour was noted in both wine and buffer. In the wine, however, the total amount of alcohols (monoterpenols, benzyl and 2-phenylethyl alcohol) released by the two enzymatic preparations was less than in the buffer (Figure 4, b and c); this demonstrates that pH is an important factor for enzymatic activity. Nevertheless, the enzymatic preparation of Deb. hansenih 4025 appears to be less affected by a low pH in the wine because it released more alcohols (Figure 4, b). Table 1 Glycosidase activities in culture supernatant fluid of Debaryomyces hansenii 4025, Debaryomyces polymorphus 363^ and Saccharomyces cerevisiae 1024
Enzyme
Deb. hansenii Deb. polymorphus Sacch. cerevisiae
Substrate pNPp-D-Glucopyranoside 0.50^-51 b 0.60a-33b 1.10a.-5lb
pNPa-L-Arabino-
pNPa-L-Rhamno-
furanoside
pyranoside
0.005^-0.27^ 0.010^-0.05^
0.03^-0.15b 0.03^-0.17b
^Activity expressed as units (U) b Activity expressed as specific activity (U mg-"' protein) 4.
CONCLUSIONS
The data presented here confirm the interest in utilizing yeasts with P-glucosidase activity for enriching the aroma of grape juice and wines by the
1635 hydrolysis of glycosidic precursors. Further study is in progress to purify the pglucosidases of these yeasts and to study the biochemical properties and the possibilities to utilize these enzymes or the yeasts themselves under juice processing and winemaking conditions.
5. REFERENCES 1 2 3 4 5 6 7 8 9 I 0 II 12 13
P. Ribereau-Gayon, J.N. Boidron and A. Terrier, J. Agr. Food Chem. 23 (1975) 1042. P.J. Williams, C.R. Strauss, B. Wilson and R.A. Massy-Westropp, Phytochemestry 21 (1982) 2013. P.J. Williams, C.R. Strauss, Phytochemestry 22 (1983) 2039. S.G. Voirin, R.L Baumes, S.M. Bitteur, Z.Y. Gunata and C.L Bayonove, J. Agr. Food Chem. 38 (1990) 1373. Z.Y. Gunata, S.M. Bitteur, J.M. Brillouet, C.L Bayonove and R.E. Cordonnier, Carbohydr. Res. 184 (1988) 139. C.L Bayonove, Y.Z. Gunata and R.E. Cordonnier, Bulletin de I' O.I.V. 643 (1984)741. A.P. Aryan, B.Wilson, C.R.Strauss, and P.J. Williams, Am. J. Enol. Vltic. 38 (1987) 182. R.E. Cordonnier, Y.Z. Gunata, R.L Baumes and C.L Bayonove, Conn. Vigne Vin23 (1989)7. Y.Z. Gunata, C.L Bayonove, A. Arnaud, and P. Galzy, J. Sci.Food Agric. 50 (1990)499. P.H. Darriet, J.-N. Boidron and D. Dubourdieu, Conn. Vigne Vin 22 (1988) 189. L Rosi, M. Vinella and P. Domizio, J. Appl. Bacteriol. (1994) in press. M. Bertuccioli, in preparation C. Bayonove, Y.Z. Gunata, J.C. Sapis, R.L Baumes, I. Dugelay and C. Grassin, VigneVini 9 (1993) 33.
Research supported by National Research Council of Italy, Special project RAISA, Sub-project No. 4, Paper N. 1491
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1637
Analytical research to identify illegal modifications of D/H values in sugar mixtures F. Tateo^ G. Cantelea B. Damia^ G. Russo^, L. Panza^ and E. Bousquet^ ^Dipartimento di Fisiologia delle Piante Coltivate e Chimica Agraria, Universita degli Studi di Milano, Via Celoria 2, 20133 Milan; Dipartimento di Chimica Organica e Industriale, Universita degli Studi, Via Venezian 21, 20133 Milan; and ^Istituto Chimica Farmaceutica, Universita di Catania, 95125 Catania, Italy Abstract Gas chromatographic/mass spectrometric (GC/MS) and nuclear magnetic resonance (NMR) investigations were carried out to verify the possibility of identifying the presence of perdeuterated ethyl alcohol added to beet sugar to increase the alcoholic grade of wine must. Data were obtained on the limits of significance of the EEC analytical method (based on measurement of the D/H ratio and of 5 C%o) used to reveal such sophistication. The results demonstrated that the percentages of beet saccharose in the order of 20-25% (calculated on the total sugars), appropriately corrected for perdeuterated ethyl alcohol, cannot be revealed with the SNIF-NMR method as published in its present form. The minimum limit of detection of the addition of perdeuterated ethyl alcohol for GC/MS is instead about four times greater than that revealed by the SNIF-NMR method. 1. mXRODUCTION With the approval of regulation no. 2676/90 of the European Economic Community (EEC) [1, 2], the analytical method SNIF-NMR {site-specific natural isotope fractionationnuclear magnetic resonance) for the determination of D/H values in alcohol derived by fermentation was included in the "Official Methods of Analysis of Musts and Wines, Vinegars, and Wine-Making By-products" (Ministerial Decree of 12 March 1986 and successive modifications). The SIRA/IRMS {stable isotope ratio analysis/isotopic ratio mass spectrometry) method for the determination of the C/^^C isotope ratio of alcohol, integrated with NMR, was included in the official analytical methods of the Ministerial Decree of 16 February 1993 [3]. The two analytical methods are targeted to identify sugars foreign to the nature of grape sugars that have been added to concentrated musts. The same methods are used to identify the presence in wines of ethyl alcohol derived from sugars foreign to the nature of the grape. As expressly stated in the Ministerial Decree of 16 February 1993, "the determination of the site-specific content of deuterium in ethyl alcohol according to the method prospected by EEC regulation no. 2676/90 is not probative for sugar or alcohol mixtures derived fi'om plants
1638 of cycle C3 (Calvin's cycle) and C4 (Hatch-Slack's cycle)." In fact, the addition of appropriate mixtures of sugars from beet (cycle C3) and from cane or com (cycle C4) to grape sugars is not unequivocally revealed by the SNIF-NMR method. Instead, the isotopic C/ C ratio in alcohol derived from fermentation shows distinct and different variability intervals for the two plant categories that synthesize sugars according to C3 and C4 cycles. However, the complementary application of the H-NMR method [4] with the isotopic C/ C ratio does not always reveal the addition to musts of sugars other than grape sugar. The reason for this is that NMR and IRMS values may be brought to normal by adding only beet sugar to the grape musts and using perdeuterated ethyl alcohol to correct the D/H ratio. In fact, grapes and beets are both plants of the C3 cycle with similar CI C variability intervals: the relative abundance values (5 C%o) with respect to the international standard of Pee Dee Belemnite are around -24 to -25%o, values that should be better defined. It should also be noted that the variability of normal (D/H)i values for grape alcohol cannot be neglected in the light of various experiments, and the minimum value of such an index has not yet been officially codified by means of strict legal practices. In this report we propose analytical criteria useful to evaluate the significance of the aforementioned official integrated method. Moreover, analytical data are given that can characterize some wine products that derive from the addition of beet sugar syrups, with a "corrected" (D/H)i ratio, to musts as such or to rectified concentrated musts (RCM). Perdeuterated ethyl alcohol has been considered for its availability as the product to increase the (D/H)i ratio. Therefore, in the experimental part that follows, some mixtures of alcohol, resulting from the addition to pure RCM of beet sugar corrected with aliquots of perdeuterated ethyl alcohol, are analyzed by electronic impact (EI) mass spectrometry and by ^H-NMR. 2. EXPERIMENTAL The data refer to two different series of experiments. 1) Measurement of the values of some intensity ratios between ions resulting from EI mass spectrometry, in standard samples constituted of alcohol from beet molasses to which perdeuterated ethyl alcohol (25 to 300 ppm) has been added; 2) %-NMR measurement of (D/H)i values obtained by the SNIF-NMR method [1] on the following samples: a) RCM from grapes, b) beet saccharose syrup corrected for the addition of perdeuterated ethyl alcohol, c) mixtures of a) and b). 2.1. GC/MS measurements Figure 1 shows the comparison between the mass spectrum of a sample of ethyl alcohol from beet molasses and that of perdeuterated ethyl alcohol (ethyl-dsalcohol-d, anhydrous, 99 + atom % D, Aldrich). Comparison shows the possibility to calculate the ratios between the intensities of some fragments, which vary linearly as a function of the composition of the mixture. In particular, the ratios between the peaks m/e 49/46, 49/42 and 51/47 can be considered useful to construct curves representative of the behavior of each of the ratios as a function of the concentration in ppm of the perdeuterated ethyl alcohol added to beet ethyl alcohol. The values of some of the ratios are shown in Table 1.
1639 100
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31 ' 2?
/ 13
15
17
32
24
38
/
42 \
47
\
-20
49 -40 -B0
CD^CD^OD 30 15
20
25
33
30
35
40
45
50
Mass/Charge
Figure I Comparison of the mass spectra of CHSCH2OH and CDSCD2OD. Table I Variations in 49/46, 49/42 and 51/47 ratios as a function of the concentration of perdeuterated ethyl alcohol added to beet molasses ethyl alcohol C2D5OD added (ppm)
49/46
Ratios 49/42
0 25 51 135 254
0.57x10" 0.60x10" 0.64 X 10" 0.77x10" 0.97x10"
2.82x10"^ 2.96x10 3.17x10 3.80x10"^ 4.77x10"^
51/47 2.75 x 10"^ 2.97x10'^ 3.26x10'^ 4.20x10"^ 5.52x10"^
Figure 2 is representative, for individual ratios, of the data given in Table 1. The measurements were made with a Hewlett Packard 5890 gas chromatograph and Hewlett Packard 5971A mass spectrometer with an SPB-TM5 column, operating in SIM and evaluating the fragments at m/e = 41, 42, 46, 47, 49 and 51. Evaluation of these curves showed that the values of the ratios 49/46, 49/42 and 51/47 (obtained for the mixture
1640 "corrected" with CD3CD2OD) were significantly modified only after the addition of about 20 ppmofCD3CD20D.
c)
100
200
3(
^
II 4.5
/• 3.5
^ 2.5
300
0
100 200 C9D5OD ADDED (ppm)
300
Figure 2. MS/SIM abundance ratios 49/46 (I), 49/42 (II) and 51/47 (III) for a sample of ethyl alcoholfrom beet molasses as a function of added perdeuterated ethyl alcohol
1641 2.2. H -NMR measurements Table 2 summarizes the composition of the samples subjected to SNIF-NMR analysis and the corresponding (D/H)i data. Two beet saccharose syrups, A and B, were produced and the D/H ratio corrected with different quantities of perdeuterated ethyl alcohol. The other samples reported in the table were composed of mixtures of A and B with an RCM of known origin. The ^H-NMR spectra were recorded with a Brucker NMR AC 300. Figure 3 shows the ^H-NMR peaks for CH3CHDOH and CH2DCH2OH of the alcohol derived from the RCM/A mixture (70:30). An additional peak attributable to the CD3 group of the added perdeuterated ethyl alcohol is evident, but in a barely analytically probative manner. Instead, there is no anomaly in the CH3CHDOH peak. The RCM/A (50:50) sample showed a distinct CD3 peak (Figure 4). The CH3CHDOH peak did not undergo significant modifications even in this case. A shoulder of the CH3CHDOH peak, in addition to the CD3 peak, attributable to the CD2 of the perdeuterated ethyl alcohol was evident only in the trace relative to the RCM/B (50:50) mixture (Figure 5).
CH3 CHDOH
168
166
164
X Scale: Hertz
CH2D CH2OH
55 53 J X Scale: Hertz
Figure 3. SNIF-NMR method applied to a mixture of RCM/A (70:30, wt/wt) sugars, defined as: 700 g 70 ""Bx RCM + 300 g 60 ""BX beet saccharose syrup ^4.6mg CD3CD2OR
1642
X Scale: Hertz
X Scale: Hertz
Figure 4. SNIF-NMR method applied to a mixture ofRCM/A (50:50, wt/wt) sugars, defined as 500 g 70 ""Bx RCM + 500 g 60 ""Bx beet saccharose syrup + 7.6mg CDSCD2OD.
X Scale: Hertz
X Scale: Hertz
Figure 5. SNIF-NMR method applied to a mixture ofRCM/B (50:50, wt/wt) sugars, defined as: 500 g 70 °Bx RCM + 500 g 60 ""BX beet saccharose syrup + 15.3 mg CD3CD2OD.
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1643
1644 3. CONCLUSIONS The results of our experience allow us to conclude the following. Evaluation by mass spectrometry of some ratios between intensity of characteristic ions can reveal the addition of deuterated ethyl alcohol to mixtures of alcohol from grape and beet sugar (or the addition of deuterated alcohol to mixtures of RCM and beet sugar) only at the level of 20 ppm and above, although a value of 50 ppm is a better confidence level (values expressed in ppm on total alcohol). In other words, mass spectrometry can reveal additions of beet sugar only beyond 40% (calculated on total sugars). NMR can identify the addition of perdeuterated alcohol at concentrations of about 13 ppm (value calculated on total alcohol). This means that it is possible to identify the adulteration only starting from additions of beet saccharose in the order of about 25% (calculated on total sugars). The data are apparent from a careful evaluation of Figure 2 and Table 2. The (D/H)i ratios therefore will not be abnormal when the addition of beet sugar is limited to such values, appropriately using perdeuterated ethyl alcohol as the corrector. The current official methods used to detect alcohol of non-wine origin should thus be considered as insufficient, at least in their present form. 4. REFERENCES 1 2 3 4
Gazzetta Ufficiale della Comunita Europee no. L272, 3/10/1990, progetto di regolamento GEE no. 2676/90 della Commissione del 17/9/1990. Gazzetta Ufficiale della Repubblica Italiana, II Serie Speciale del 19/11/1990, progetto di regolamento (GEE) no. 2676/90 della Gommissione del 17/9/1990. Gazzetta Ufficiale della Repubblica Italiana no. 95, 24/4/1993, Decreto Ministeriale 16/2/1993. G. Martin, S. Brun, Bulletin de I'O.I.V., 671,672 (1987), 131-145.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1645
Partial characterization of |3-damascenone precursors and toxicity studies of free p-damascenone in cell cultures of Vitis x labruscana cv. Concord grapes K.B. Shure and T.E. Acree Food Science and Technology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY Abstract Cell cultures of 'Concord' grapes producing acid hydrolyzable precursors to pdamascenone were analyzed to partially characterize the bound forms of this important aroma compound in callus tissue initiated from immature fruit and petioles. Exogenous free p-damascenone was added to the medium of cell suspensions to determine toxic levels of this compound to 'Concord' fruit cells. Multiple forms of the acid hydrolyzable precursors to p-damascenone were present in the callus tissue and levels of free p-damascenone over 1 mg/l were toxic to the cell suspensions. Introduction Plant cells cultured in vitro should be very useful for the study of both primary and secondary metabolism in plants. Furthermore, the importance of secondary plant metabolites in the development of pharmaceuticals, natural pigments, and odorants makes their production in vitro an attractive area for research. The use of compounds extracted from plants for medicines and perfumery goes back centuries. Herbal remedies and perfumes were made from extracts of fat used to adsorb volatiles from flowers by a process called enfleurage. Today the list of chemical compounds isolated from plants is large but only a small subset of these have been studied using modern techniques of plant cell culture. Examples of work using plant cell cultures to study biologically active compounds including mevalonates (terpenoids and steroids), phenylpropanoids, and alkaloids have been recently reviewed (Stafford and Warren 1991). Examples for the production and accumulation of organic compounds or their biotransformation focused mainly on pharmaceutically active compounds. However, there has been a recent interest in research devoted to chemical compounds responsible for the sensory aspects of foods, generally pigments and odorants (Bell and White 1989; Shuleretal. 1991).
1646 There have been a number of cell culture systems that have been developed to study the flavor compounds produced by plants and fruits. Included in this are in vitro cultures of vanilla plants (Funk and Brodelius 1992), onion (Selby et al. 1980) and garlic (Malpathak and David 1986; Madhavi et al. 1991), strawberries (Hong et al. 1990), guava (Prabha et al. 1990), raspberries (Borejsza-Wysocki and Hrazdina 1994), and grapes (Ambid et al. 1982; Paisarnrat and Ambid 1985). The use of various methods that attempt to increase levels of flavor compounds and other types of secondary metabolites include change in media composition, addition of metabolic precursors to the compound(s) of interest, addition of bio-organic extracts as elicitors, immobilization of cells and in situ adsorption using a solid phase adsorbent (Buitelaar and Tramper 1992). We have recently reported the production of acid hydrolyzable precursors to p-damascenone (bound p-damascenone), an aroma compound important to all species of grapes, in callus and cell suspensions of Vitis x labmscana cv. Concord (Shure and Acree, 1994). Here we report of further work done on these cultures including attempts to increase production in the cell suspensions and partial characterization of the precursors found in callus initiated from both immature fruit and petiole material. Materials and Methods Tissue Culture - Callus of petiole and fruit cells from Vitis x labnjscanacy. Concord were maintained in the dark at 24°C on Gamborgs 85 media supplemented with 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l benzylaminopurine (BA), 0.4% agar, pH 5.7. Cell suspensions were initiated from the fruit callus in a liquid medium of the same composition as the callus medium without agar and shaken in the dark at 24''C at 120 rpm. Details of culture initiation, maintenance, further sample processing and analysis have been described previously (Shure and Acree, 1994). Sample Preparation Callus tissue was collected and homogenized in an equal volume of water followed by addition of CaCl2 as a saturated solution to a concentration of 0.7 M to inhibit oxidative enzyme activity. Homogenates were sonicated in a bath sonlcator for 30 minutes and then centrifuged at 10,000 rpm for 15 minutes at 4°C and the supernatant saved for analysis. Cell suspensions were treated similarly except that water (c.a. 10-20 ml per flask) was only added in the later stages of the growth curve when the cultures became more viscous. Conditions tested for increased production of bound p-damascenone in cell suspensions included increasing sucrose levels to 6%, a treatment shown to have positive
1647 effects on p-damascenone precursor levels in petiole callus (Shure and Acree, 1994), Immobilizing the cells in alginate and the use of XAD-2 as an in situ adsorbent. Isolation of Bound p-damascenone The samples were placed on a glass column (40 cm x 3.6 cm i.d.) containing 25 grams of C-18 reversed phase absorbent, washed with several column volumes of water and the bound forms of p-damascenone eluted in the first two of five column volumes of methanol. The methanol/water extract was then evaporated to dryness at 28°C, 60 mm Hg and resuspended in approximately 1 ml of water. The resuspended sample was hydrolyzed by adding 0.1 M citric acid (pH 2) at a 3:1 ratio and heating the mixture at 90°C for 20 minutes. After rapid cooling in an ice bath, samples were extracted with 2 ml Freon 113™. Partial Characterization of Bound forms of p-damascenone One kg each of petiole and fruit callus were processed as above to the point of resuspension in 1 ml of water after evaporation. To partially characterize the precursors present in these samples a method similar to that described by Roberts et al. (1994) that analyzed bound p-damascenone in apples was used. HPLC utilized a Varian (Sugar Land TX) liquid chromatograph equipped with a Star 9010 solvent delivery system, a Star 9095 autosampler, and a Star 9096 polychrome diode array detector operated in the range of 190-367 nm. The semipreparative (10 mm i.d. x 25 cm) and guard (10 mm i.d. x 5 cm) columns were packed with Microsorb 5 um C-18 (Rainin, Woburn MA). The mobile phase gradient was from 10 to 100% methanol in water at a rate of 5 ml/min for 35 minutes. Five hundred |il of the sample from each of the callus tissues were Injected and 5 ml fractions were collected with a Gilson FC80 Micro Fractionator (Middleton Wl). Two ml of each fraction were combined with 2 ml of 0.1 M citric acid (pH 2) and heated, cooled and extracted under the same conditions as noted above. Immobilization of Cell Suspensions A method similar to that described by Ziyad-Mohamed and Scragg (1990) was used for immobilization of fruit cells in suspension. Fifty grams of cells were filtered from 7 day old cell suspension cultures using Whatman number one filter paper (Whatman Paper Limited, England). The cells were suspended in 200 ml of liquid media containing 4% Na-Alginate (Sigma, St. Louis, MO) and a peristaltic pump (Isco, Inc., Lincoln, NB) was used to pump the mixture via a 1 mm (i.d.) tube into a 0.1 M solution of CaCl2. The beads formed were
1648 approximately 4 mm in diameter and after washing twice with sterile water were transferred to 250 ml of liquid media in a one liter flask and shaken in the dark at 110 rpm for nine days. The beads were then collected in a strainer and placed in 250 ml of 0.1 M trisodium citrate and stirred for four hours to dissolve the calcium alginate beads. The resulting mixture was centrifuged at 5,000 rpm for 10 minutes at 4°C. The pellet was then resuspended in 250 ml of 0.1 M trisodium citrate buffer and stirred for another hour to dissolve any remaining beads. After a final centrifugation the buffer was decanted and the cell mass was weighed. After re-addition of the buffer, the cells were sonicated, recentrifuged at 10,000 rpm for 15 minutes and the supernatant was then analyzed for bound pdamascenone. The liquid media strained from the beads was also tested for the presence of bound p-damascenone to see if any product excretion from the beads occurred. In situ Adsorption by XAD-2 Resin In a method similar to Asada and Shuler (1989) one gram lots of XAD-2 nonionic polymeric adsorbent (Aldrich Chemical Company, Inc., Milwaukee, Wl) were weighed out and wrapped in Miracloth (Calbiochem, La Jolla, CA) and tied with string. This resin has shown to be effective in trapping glycosidlc precursors to p-damascenone (Zhou et al. 1993; Roberts et al. 1994). One resin bag was placed in each of twenty flasks of fruit cell suspension on day five of incubation. At day 10 of incubation the resin bags were removed from the flasks, washed in distilled water, divided into two replicates each consisting of ten bags and placed in 250 ml Erienmeyer flasks. One hundred ml of methanol was added and the flasks were placed on a shaker overnight at 120 rpm. The methanol extract was then removed and evaporated in vacuo, resuspended in one ml of distilled water and assayed for bound p-damascenone as described above. Toxicity of p-damascenone to Cell Suspensions The toxicity of p-damascenone to the suspended cells was tested. A similar study was done previously with cell cultures of Pelargonium fragrans that tested the toxicity of monoterpenes to the cells (Brown et al. 1987) 20% pdamascenone solutions in DMSO, ethanol and propylene glycol were prepared and ten fold serial dilutions were made. Fifty ^il of these solutions were added to 100 ml of liquid media for final concentrations of p-damascenone of 100 mg/l, 10 mg/l, 1 mg/l, and 0.1 mg/l. Controls were also prepared that contained 0.05% solvent. The media solutions were filter sterilized using Corning (Corning NY) 200 ml filter systems equipped with 0.22 |im nylon, sterilizing, low extractable membranes. After inoculation the flasks were measured for initial fresh weight using Cell Volume after Sedimentation (CVS) as described by Blom et al (1992). On day six of incubation, the fresh weights were determined again and a growth
1649 index calculated. Degree of browning was also determined by removing 1 ml of media, diluting four fold and reading the absorbance at 420 nm (Prabha and Patwardhan 1980) on a Gary 219 Spectrophotometer (Varian, Sugar Land TX). Results and Discussion Partial Characterization of Acid Hydrolyzable Precursors to |3damascenone Produced !n vitro Bound forms of p-damascenone from the petiole callus began eluting from the HPLC column with approximately 36% methanol (Figure 1). The most abundant activity was seen at 41% and 46% methanol with smaller levels eluting throughout the methanol gradient to 74% methanol. In the fruit callus damascenone generating activity also began eluting at about 36% methanol with the majority eluting at 41% and 43% (Figure 2). Smaller levels of bound p3)2401 S200i 0)
g 160 o>
8l20i E CO
7 80H io 40i 10
20
h lUiUilU U L L
30 40 50 60 70 % Methanol in Water
80
90
Figure 1. Distribution of bound p-damascenone in petiole callus across C18 HPLC methanol gradient.
damascenone from the fruit callus were also seen to elute throughout the methanol gradient to 56%. This datum indicates that multiple acid hydrolyzable precursors to p-damascenone exist in the callus with the majority of activity being manifested in one or two molecules, p-damascenone and other nor-isoprenoids
1650 are known to be generated from multiple bound forms in the intact 'Concord' grape tissue (Braell et al. 1986), as well as in grape cultivars from the vinifera species (Winterhalter et al. 1990) and in apples (Zhou et al. 1993; Roberts et al. 1994). Utilizing the information from the methanol gradients -;100 D) S 80H o 0)
o
60 H
(0 CO
E 40 H (0
20 H o
ffi
10
20
30 40 50 60 70 % Methanol in Water
80
90
Figure 2. Distribution of bound p-damascenone in fruit callus across C-18 HPLC methanol gradient. in Figures 1 and 2 and comparing to extensive characterization and identification of acid hydrolyzable precursors from apple fruit eluting along the same gradient (Roberts et al. 1994), the compounds of most abundance in the callus tissue may be di- or tri-glycosides of polyhydroxylated compounds known to form p-damascenone under acid hydrolysis conditions such as the allenic triol or the acetylenic diol (Skouroumounis et al. 1992; Winterhalter 1992; Williams 1993). The remaining p-damascenone generating activity could be assigned to the non-glycosylated polyhydroxylated compounds. Immobilization The cells were easily immobilized in the calcium alginate beads and the media and cells soon turned the color (beige to light brown) of the callus tissue used to initiate the suspensions. A measure of the fresh weight of the isolated cell mass after 9 days in a state of immobilization revealed only a 20% increase in biomass, a value more comparable to the callus tissue rather than what is
1651 seen in the freely suspended cultures where a 3-4 fold increase in nnass is noted by that time (Shure and Acree, 1994). Measurement of bound p-damascenone found 0.62 ng/g indicating that in the immobilized state, p-damascenone production increased back up to the levels seen in the callus tissue from values approximately one fifth of that found in non-manipulated cell suspensions (Shure and Acree, 1994). No excretion of the product out of the beads into the media took place as no bound pdamascenone was detected in the medium. This effect of immobilization supports the idea that this technique can be viewed as a primitive attempt to induce biochemical differentiation in fine cell suspensions (Payne et al. 1992). The change in the cells in their color, growth pattern and bound p-damascenone production levels back to what was seen in the callus indicates that increasing the association between cells had a positive effect on the secondary metabolism of bound p-damascenone in this instance. It should be noted however that immobilization proved to be an unreliable method of increasing bound pdamascenone levels in the cell suspensions. Later attempts with this technique resulted in a lack of color change in the media and beads and levels of bound pdamascenone similar to what was usually found in the non-immobilized cell suspensions. //7S/fi/adsorption Twenty four hours after addition of XAD-2 resin bags to the fruit cell suspensions, browning of the cells was noted. On day 10 when the resin bags were harvested, the fresh cell weight of the cultures were less than half than that which is normally seen in these cell suspensions. The browning and decreased growth of the cells indicated that addition of the resins had a negative effect on the suspensions, probably due to an adsorption of media components such as vitamins and hormones as has been seen by other investigators (Robins and Rhodes 1986; Asada and Shuler 1989). Acid hydrolysis of the resuspended methanol extracts used to collect any bound p-damascenone trapped In the XAD2 resin bags did not result in the formation of p-damascenone indicating that no leakage of the bound form into the media from the cells was occurring or being enhanced by in situ adsorption. The results of the p-damascenone precursor content for the various treatments tested are shown in Figure 3. None of the treatments, including increased sucrose levels, had an effect with the exception of immobilization. This treatment showed an increase in levels of bound p-damascenone, however later attempts at immobilization yielded precursor amounts similar to the control. In any case, secretion of the precursor into the media did not occur resulting in a
1652 need to destroy the biomass in order to isolate the bound forms of pdamascenone. ^
0.8
Treatment
Figure 3. Bound p-damascenone levels found In cell suspensions under the following conditions: A) Control, B) Immobilized in calcium alginate beads, C) 6% Sucrose, D) In situ adsorption by XAD-2 resin added to the medium (Bound |3-damascenone levels tested were those extracted directly from the resin). All measurements In duplicate except B. Error bars are standard error for this and all following graphs. Toxicity of |3-damascenone to cells It was necessary to find a suitable solvent carrier for dispersing pdamascenone in the medium for these experiments. Both DMSO and ethanol at low (0.05%) concentrations caused intense browning and inhibition of growth indicating that these organic solvents were highly toxic to the cell suspensions. Organic solvents such as DMSO have typically been used to permeabalize cells in order to allow for excretion of intracellular chemicals of interest (Knorr and Berlin 1987; Brodelius 1988). Many of these experiments have resulted in cell death at solvent concentrations necessary for release of the secondary metabolites. This is presumably due to the loss of compartmentation within the cells allowing for the release of proteases and other destnjctlve enzymes as well
1653 as toxic compounds that had been enclosed within vacuoles (Buitelaar and Tramper1992). Using propylene glycol did allow for cell growth to occur in the control and in the lowest two concentrations of exogenous p-damascenone while the highest two concentrations showed no growth (Figure 4). The highest two concentrations of added p-damascenone resulted in intense browning of the cultures while the control and lower two concentrations showed little or no browning (Figure 5).
0.1 1.0 10 p-damascenone mg/ L
Figure 4. Growth index of cell cultures treated with p-damascenone in propylene glycol. All measurements done in triplicate.
These results indicate the connpatibllity of propylene glycol with the cell cultures when used as a solvent and that free p-damascenone becomes toxic to the cell suspensions above 1 mg/l. The goal of this experiment was to test the toxicity of p-damascenone as had been done previously with monoterpenes in cell cultures of Pelargonium fragrans where concentrations of 500 mg/l proved toxic for all of the monoterpenes tested (Brown et al. 1987). These researchers also showed that many monoterpenes were tolerated by these cells at
1654 concentrations of 100 mg/l, one hundred times the level of the highest tolerated P-damascenone concentrations seen here. 0.5
E 0.4 CM
I 0.3 o 0.2 0)
0.1
0.0 0
0.1 1.0 10 |3-damascenone mg/ L
100
Figure 5. Browning as measured by absorbance at 420 nm of the four fold diluted supernatant of a cell suspension treated with |3-damascenone in propylene glycol. All measurements done in triplicate.
In conclusion, multiple acid hydrolyzable precursors to p-damascenone exist in undifferentiated callus tissue initiated from 'Concord' fruit and petioles. The bound forms of p-damascenone that yield the highest levels of this compound chromatograph similarly to di- and tri-glycoside p-damascenone precursors in apples (Roberts et al. 1994). Immobilization of cell suspensions in alginate beads was capable of elevating the level of p-damascenone precursors in the cells, however the technique was not consistent in accomplishing this, pdamascenone added to the medium was toxic to the cells at levels higher than 1 mg/l. This tolerance threshold was significantly lower than what has been seen previously for many monoterpenes in cell cultures of another species.
1655 References Ambid, C , Moisseeff, M. and Fallot, J. Biogenesis of monoterpenes: Bioconversion of citral by a cell suspension culture of muscat grapes. Plant Cell Reports 1982,1,91-93. Asada, M. and Shuler, M.L Stimulation of ajmalicine production and excretion from Catharanthus-roseus : effects of adsorption in situ, elicitors and alginate immobilization. Appl. Microbiol. Biotechnol. 1989, 30(5), 475-481. Bell, E.R.J, and White, E.B. The potential of biotechnology for the production of flavours and colours for the food industry. International Industrial Biotechinology 1989, 9(3), 20-26. Blom, T.J.M., Kreis, W., van Iren, F. and Libbenba, K.R. A non-Invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures. Plant Cell Reports 1992,11,146-149. Borejsza-Wysocki, W. and Hrazdina, G. Biosynthesis of p-hydroxyphenylbutan-2one in raspberry fruits and tissue cultures. Phytochemistry 1994, 35(3), 623-628. Braell, P.A., Acree, T.E., Butts, R.M. and Zhou, P.G. Isolation of Nonvolatile Precursors of p-Damascenone from Grapes Using Charm Analysis, in Biogeneration of Aromas. T.H. Parliment and R. Croteau eds. Washington D.C., American Chemical Society. 1986; pg. 75-84. Brodelius, P.E. Permeabilization of plant cells for release of intracellularly stored products: viability studies. Appl. Micorbiol. Biotechnol. 1988, 27, 561-566. Brown, J.T., Hegarty, P.K. and Charlwood, B.V. The toxicity of monoterpenes to plant cell cultures. Plant Science 1987, 48,195-201. Buitelaar, R.M. and Tramper, J. Strategies to improve the production of secondary metabolites with plant cell cultures: a literature review. Journal of Biotechnology 1992, 23,111 -141. Funk, C. and Brodelius, P.E. Phenylpropanoid metabolism in suspensoin cultures of Vanilla planifolia Andr. IV. Induction of vanillic acid formation. Plant Physiol. 1992,99,256-262.
1656 Hong, Y., Huang, L, Reineccius, G.A., Harlander, S.K. and Labuza, T.P. Production of aroma compounds from strawberry cell suspension cultures by addition of precursors. Plant Cell, Tissue and Organ Culture 1990, 21, 245-251. Knorr, D. and Berlin, J. Effects of immobilization and permeabilization procedures on growth of Chenopodium rubrum ceWs and amaranthin concentration. Journal of Food Science 1987, 52(5), 1397-1400. Madhavi, D.L, Prabha, T.N., Singh, N.S. and Patwardhan, M.V. Biochemical studies with garlic Allium sativum cell cultures showing different flavor levels. J. Sci. FoodAgric. 1991, 56(1), 15-24. Malpathak, N.P. and David, S.B. Flavor formation in tissue cultures of garlic Alium sativum . Plant Cell Reports ^9BS, 5(6), 446-447. Paisarnrat, S. and Ambid, C. Bioconversion of Geraniol by a Cell Suspension Culture of Muscat Grapes: Development and Metabolic Pathways, in Topics in Flavour Research. R.G. Berger, S. NItz and P. Schreier eds. Marzling Hangenham, H. Eichorn. 1985; pg. 321-333. Payne, G.F., Bringi, V., Prince, C. and Shuler, M.. Plant Cell and Tissue Culture in Liquid Systems. New York, Hansen 1992. Prabha, T.N., Narayanan, M.S. and Patwardhan, M.V. Flavour formation in callus cultures of guava (Psidiumguajava) fruit. J. Sci. FoodAgric. 1990, 50(1), 105110. Prabha, T.N. and Patwardhan, M.V. Polyphenols of avocado and their endogenous oxidation. J. Food Sci. Technol. 1980,17, 215-217. Roberts, D.D., Mordehai, A.P. and Acree, T.E. Detection and partial characterization of eight p-damascenone precursors in apples (Malus domestica Borkh. Cv. Empire). J. Agric. FoodChem. 1994, 42(2), 345-349. Robins, R.J. and Rhodes, M.J. The stimulation of anthraqulnone production by Cinchona-ledgeriana cuWures with polymeric adsorbents. Appl. Microbiol. Biotechnol. 1986, 24(1), 35-41. Selby, C , Turnbull, A. and Collin, H.A. Comparison of the onion plant (Allium cepa) and onion tissue culture. II. Stimulation of flavour precursor synthesis in onion tissue cultures. NewPhytol. 1980, 84, 307-312.
1657 Shuler, M.L, Hirasuna, T.J., Prince, C.L and Bringi, V. Bioreactor Considerations for Producing Flavors and Pigments from Plant Tissue Culture, in Biotechnology and Food Process Engineering. H.G. Schwartzberg and M.A. Rao eds. New York, Basel, Marcel Dekker. 1991; pg. 45-66. Shure, K.B. and Acree, T.E. Production of p-damascenone precursors in cell cultures of Vitis labruscana cv Concord grapes. Plant Cell Reports ^994, in press. Skouroumounis, G.K., Massy, W.R.A., Sefton, M.A. and Williams, P.J. Precursors of damascenone in fruit juices. Tetrahedron Letters 1992, 33, 35333536. Stafford, A. and Warren, G. Biotransformation by Plant Cell Cultures, in Plant Cell and Tissue Culture. A. Stafford and G. Warren eds. Buckingham, Open University Press. 1991; pg. 163-204. Williams, P.J. Hydrolytic Flavor Release in Fruit and Wines through Hydrolysis of Nonvolatile Precursors, in Flavor Science. T.E. Acree and R. Teranishi eds. Washington D.C., ACS. 1993; pg. 287-308. Winterhalter, P. Oxygenated C-13 Norisoprenoids. in Flavor Precursors. R. Teranishi, G.R. Takeoka and M. Guntert eds. Washington D.C., ACS. 1992; pg. 98-115. Winterhalter, P., Sefton, M.A. and Williams, P.J. Volatile CI3-norisoprenold compounds in Riesling wine are generated from multiple precursors. American Journal ofEnology and Viticulture 1990, 41 (4), 277-283. Zhou, P.G., Cox, J.A., Roberts, D.D. and Acree, T.E. p-damascenone precursors in apples, in Progress in Flavour Precursor Studies. P. Schreier and P. Winterhalter eds. Carol Stream IL, Allured Publishing Corp. 1993; pg. 275-278. Ziyad-Mohamed, M.T. and Scragg, A.H. Plant Cell Immobilization in Alginate and Polyurethane Foam, in Methods in Molecular Biology, vol. 6, Plant Cell and Tissue Culture. J.W. Pollard and J.M. Walker eds. Clifton, NJ, The Humana Press. 1990; pg. 525-536.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1659
INFLUENCE OF NITROGEN COMPOUNDS IN GRAPES ON AROMA COMPOUNDS OF WINES A. Rapp and G. Versini Bundesanstalt fur Ziichtungsforschung an Kulturpflanzen, Institut fiir Rebenziichtung Geilweilerhof, 76833 Siebeldingen, BRD Istituto Agrario, 1-38010 San Michele all'Adige, Italia
INTRODUCTION Aroma compounds, as a result of their pronounced effect on our sensory organs, play a definitive role in the quality of our food and luxury products. As in the case with most food products, the aroma or "bouquet" of a wine is influenced by the action of several hundred different compounds (1,2,3,4,5). When dealing with wine aroma, a distinction is made among: - primary or grape aroma: aroma compounds as they occur in the imdamaged plant cells of the grape - secondary grape aroma: aroma compounds formed during the processing of the grapes (crushing, pressing, skin contact) and by chemical, enzymatic-chemical, and thermal reactions in grape must, - fermentation bouquet: aroma compounds formed during the alcoholic fermentation - maturation bouquet: caused by chemical reactions during maturation of the wine. Numerous studies (1,2,3) have shown that the monoterpene compounds form the axis for the sensory expression of the wine bouquet which is typical of its variety (primary or grape aroma) and that they can, therefore, be used analytically for varietal characterization. At present, more than 50 monoterpene compounds are known. Based on the quantitative determination of only 12 monoterpene substances the German white wines can be classified into three groups: "Riesling-type", "Muscat-type" and "Silvaner-WeiBburgunder-type". Such "terpene profiles" are also useful for separating true Riesling (White Riesling) wines from others called Riesling (e.g., Welschriesling,
1660 Kap Riesling, Emerald Riesling) but not produced from grapes of the variety Riesling (2,3,6). By including further components and by means of statistical methods, such as for example linear discriminant analysis, even the different varieties within the mentioned groups (for instance in the "Riesling-group": Riesling, MuUer-Thurgau, Bacchus, Kemer, Scheurebe, Ehrenfelser; in the "Silvaner-group": Silvaner, Weifiburgunder, Rulander) can be distinguished (3,7,94,95).
AROMA COMPOUNDS FORMED DURING FERMENTATION (FERMENTATION BOUQUET)
The essential part of the wine flavour is formed during the alcoholic fermentation (Fig. 1) (2,3,8). The grape odor of the juice is superimposed by the fermentation products which form the vinous flavor of the product. Apart from ethanol and glycerine as well as diols and higher alcohols (2-Methyl-l-propanol, 3-Methyl-1-butanol, 2-Methyl-1-butanol) numerous other wine constituents are formed by yeast metabolism (especially acids, esters, aldehydes, ketones, and S-compounds). Most of the aldehydes present in grape juice are only detectable in wines in the initial phase of fermentation (1). They probably are reduced to alcohols. Except for acetaldehyde, the aldehydes in wine arise from carbohydrate degradation (furfural, 5hydroxymethyl-furfural), originating from lignins (vanillin, cinnamaldehyde), or are formed during wine aging. Most of the ketones reported in grapes are also found in wines, though in small amounts. The sensory impact on wine aroma of ketones, a-diketones and a-hydroxyketones that arise during yeast fermentation (e.g. acetone, acetoin, 2,3-pentanedione) seems to be very low, except for diacetyl which is reported to possess a buttery odor and can increase significantly sometimes after malolactic fermentation. A similar production profile as that for acetoin, diacetyl, and 2,3-pentandion is also applied to formation of acetales from acetaldehyde. At the beginning of the fermentation (high acetaldehyde production-phase), the levels of the corresponding acetales are also at their highest levels. These decreases during the fermentation progress are in line with decreasing acetaldehyde level. The highest levels of acetaldehyde acetales are referring to those, of the cyclic acetales formed from threo- and meso-2,3-butanediol and
1661 glycerine, followed by 1,1-diethoxyetliane and the mixed acetales formed from ethanol, higher alcohols and acetaldehyde. Morio-Muskat Must
iJ Ma7
bk 160156 53 <^ tS 61 S8 Si. U
8S
W337 33 30 32
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272523 1 9 n 13 121110 8 2 26 2<»2118 15 9 1
Wine
LA_4^H!^ 90 M 89 87
86B(> 85
lifii.
80
79'77 75\72 70 67 6563605754^9 US W2 39 35 32 28 62 56 5 l 1.7 U 38 34
iU
m
11 8 S 1 26 22 18 14 25 21 H 1312 W 7 3
Fig. 1: Aromagrams of grape must (above) and the corresponding wine (below)', cultivar Morio-Muskat
The formation of higher alcohols - including 2-phenylethanol but 1-propanol excepted - during yeast fermentation takes place parallel to the ethanol formation. 1propanol increases only until about the half of the fermentation (83). The production of higher alcohols during fermentation results from decarboxylation and reduction of the corresponding a-ketoacids, which are themselves produced by either transamination of amino acids (9,10) following the Ehrlich mechanism, or carbohydrate degradation
1662 (11,12). The contribution from the Ehrlich mechanism is much lower than that from carbohydrate degradation (13). The higher alcohols are present in concentrations above their organoleptic perception threshold. At concentrations below 300 mg/1, they certainly contribute to the desirable complexity of wine. When their concentrations exceed 400 mg/1, the fusel alcohols are regarded as a negative quality factor. Lee et al. (19) foimd a higher content of the total fusel alcohols in red wine (256 mg/1 in Leon Millot wine) than in white-wine (149 mg/1 in Riesling); being, in any case higher in hybrids (e.g. 507 mg/1 in Concord wine). The fatty-acids are formed earlier during the alcoholic fermentation and in higher concentration than their corresponding fatty acid ethyl esters. Relatively few organic acids in wine are volatile enough to contribute to its odor. Odorous acids are acetic acid (vinegary), butanoic acid (spoiled butter) lactic acid and caprylic acid (goaty). Goaty flavour is typical for caprylic acid as found in beer (85). With the exception of acetic acid, their amounts in wines are usually below perception threshold. Medium contents found in a Chardonnay wine for octanoic acid are 7.9 mg/1; std. dev. 4.4 mg/1 and for hexanoic acid 5.8 mg/1; std. dev. 2.9 mg/1 (84). The contents of all fatty acids (C-4 to C-10) increase during fermentation, whereas those of C-16 to C-18-acids decrease (20). Ethyl esters of straight-chain fatty acids and acetates of higher alcohols are the dominanting esters in wine, and they are formed during the alcoholic fermentation (Fig. 2). The yeasts synthesize the acetates in the same way as the fatty acids; water hydrolysis of the acetyl-CoA-derivative gives the fatty acids, and similar, ethanol hydrolysis gives the acetates. Volatile organic sulphur compounds produced by alcohoUc fermentation are of outstanding importance for aromas because, for the most part, they have extremely low perception thresholds. The most important S-containing compound in wine is hydrogen sulphide, which has a recognition threshold below 1 ^g/1. It appears predominantly in high amounts during the fermentation of grapes with free elemental sulphur (21) and causes the "rotten egg" odor. Other odor-intensive sulphur compounds in wine are thioethers (dimethylsulphide, diethylsulphide), thiols (ethanethiol, 4-methylthio-lbutanol, 3-methylthio-l-propanol), thiolanes (2-methylthiolane-3-one, 2-methylthiolane-3-ols), and esters of sulphur-containing acids (methyl and ethyl-3-methylthiopro-
1663 pionate) (14,5,15,86). The odor of trans-2-methylthylthiolane-3-ol is onionlike, that of ethanethiol is fecal.
Vol •/« 12
phenylethylacetate o
propylacetate
2 f
15 (days)
Fig. 2: Formation of acetates ("fermentation compounds") during fermentation (Rapp et al.)
The most volatile phenols which are found in wines are not present in grapes, except acetovanillone (5). They result either from yeast or bacterial metabolism, of corresponding benzoic acid and cinnamic acid or from hydrolysis of higher phenols and phenol glycosides (87). For example, the volatile phenols 4-vinyl-guajacol and 4vinyl-phenol can originate from the cinnamic acids p-cumaric acid and ferulic acid by enzymic or thermal decarboxylation (1). These phenols can influence wine odor. Their content in Moscato rosa-wines ranged from 2 to 8 ^g/1, in wine of white varieties (e.g. Traminer) levels were found of about 1 mg/1 (17,88,89). The process of volatile nitrogen compounds are also determined by wine production. Among the most aboundent substances, N-(2-phenylethyl)-acetamide ranges in Moscato Rosa-wines between 1 to 23 mg/1 while N-(3-methylbutyl)-acetamide ranges between 15 to 72 mg/1. Many remarkablejactones from wines thus far known seem to arise during fermentation through reactions involving 4-oxobutyric acid and, to a lesser extent, 2-oxo-glutaric acid (18). Among the many volatile constituents of wine, the lactones, particularly gamma lactones, occupy a place of prominence not only in terms of their contri-
1664 bution to the total vinous aroma and bouquet, but also because of their physiological properties. Other 5- and y-alkyllactones, the so called "peach lactones", can be present in wines but they could be considered as worthless contributors to the total aroma as shown by Etievant et al. (90). Terpene compounds, as a group, form an important part of the grape bouquet; they belong to the secondary plant constituents, of which the biosynthesis begins with acetyl-CoA. Microorganisms are also able to synthesize terpene compounds (16), but the formation of terpenes by Saccharomyces cerevisiae has not yet been observed. These compounds are not changed by yeast metabolism during fermentation (Fig. 3) (2,3,8) except for geraniol (91) and nerol (92). The monoterpene compounds are, therefore, suitable for the varietal characterization of wines made from different grape varieties (1,3).
Vol. %
12
cis rose oxide trans linalool oxide (f) trans linalool oxide (p) cis linalool oxide (f)
Fig. 3: Behaviour of monoterpene compounds ("grape aroma compounds") during fermentation (Rapp et al.)
1665 INFLUENCE OF VARIOUS FACTORS ON THE PRODUCTION OF FERMENTATION COMPOUNDS
It is fairly well accepted that several factors can change the "fermentation bouquet" of wines. The type of yeast has a great influence on the production of fermentation compounds, but even more important are the clarification and fermentation conditions, including the treatments and the composition of the musts (pH, nitrogen compounds, nitrogen contents). Yeast strains selected for alcoholic fermentation of grape juice have been found to cause appreciable variation in the quality of the wines and in their contents of volatile substances (22). In fact some compounds (e.g. hydrogen sulfide, aldehydes and acetic acid) were present at levels sufficiently high to influence the quality of some wines negatively, whereas in most wines of higher quality, the fatty acid esters were present in concentrations and ratios which are typical of better quality wines. Saccharomyces cerevisiae strains, in general, produce higher quality wines than those of Saccharomyces uvarum, Saccharomyces bayanus and Saccharomyces chevalieri. The total higher alcohol production of 24 selected strains of these subspecies did not vary considerably (22). The same is true for individual higher alcohol contents, except for the 2phenylethanol levels produced by three Saccharomyces uvarum strains. The highly significant negative correlation coefficient between wine quality and 2-phenyIethanol content may suggest that, at the possible extraordinary high levels as found, this alcohol could make a negative contribution to the quality of the wines (22). Also Cavazza et al. (23) found no significant differences in the levels produced of higher alcohols except for 2-phenylethanol and acetamides by different yeast strains. Two of the three 2-phenylethanol high producing strains were classified as Saccharomyces uvarum, the other one as Saccharomyces cerevisiae. The production of higher alcohols depends on the degree of juice turbidity and fermentation temperature. In clearfilteredjuice, however, the production of higher alcohols is independent of fermentation temperature (24). The results of numerous experiments withfilteredjuice and strictly anaerobic fermentation indicated that the concentrations of the 3 main higher alcohols (2-methyl-propanol-l, 3-methyl-butanol-l, 2phenylethanol) amoimted to a rather constant total of 90 mg/1 at 13 °C and 15 °C, as well as at 25 °C. On the other hand, in winefi-omsettled juice, fermented at 13 °C and 25 °C, the total higher alcohol concentration amounted to 170 and 375 mg/1, respectively.
1666 r.g/l
-, MIJLLER THURGAU
00
o
^
lO
"7 CD
o ro m
cr o r- H
H 2: Z) U
Fig. 4: Formation of isoamylacetate during fermentation at different musts and yeasts (Cavazza et al.; 23)
The major factors affecting ester content of wine are medium composition (Fig. 4) and fermentation procedure. Variations in media include carbon source, nitrogen supply, and pH. Important fermentation procedure factors include fermentation temperature, carbon dioxide concentration, oxygenation of the media, and yeast strain selection. Differences in ester production due to yeast species or strains have been reported (Table 1) (22,23,28,29). It has been shown in several studies (25,22) that the contents of individual fatty acid esters such as isoamyl acetate, ethylhexanoate, ethyloctanoate are normally higher in wines with higher quality ratings. The positive contributions of some esters to wine bouquet have, in fact, been demonstrated conclusively (25,22). The fruity esters (isoamyl acetate, isobutyl acetate, ethyl butyrate, hexyl acetate) are produced and retained at the lower fermentation temperature (10 °C). The higher-boiling, more aromatic or "heady" esters (ethyl octanoate, 2-phenylethyl acetate) are produced and retained in the wine in greater amounts at higher fermentation temperatures
1667 Table 1: Influence of yeast strains on ester content of wine (Zeemann et al.; 22)
yeast species
ethyl-
i-amyl
2-phenyl-
ethyl-
ethyl-
and strain
acetate
acetate
ethylacetate
hexanoate
octanoate
56 60 56
12 7 7 14
S. cerevisiae
1
S. cerevisiae
14
S. cerevisiae 372 S. bayanus
390
S. uvarum
355
62 36 67
S. uvarum
402
51
10 13 4
S. uvarum
400
53
1
S. cerevisiae 373
0.22
1.7
0.10
1.1 1.3 2.7 1.4
0.21 0.38 0.36
2.2 4.6 2.1
3.2 0.5 0.2
1.3 1.1 1.1 1.5 0.9 1.1 1.0 0.2
(15 to 20 °C) (26). The aroma of a wine produced by low fermentation temperatures is distinctively characterized by a fruity and soap like odor typical for some esters (27). Levels of fatty acids and their ethyl esters depend on the skin contact time (30), in some cases they could be higher in wines from pressing fractions so as it is usually for the acetates (93). During the second fermentation (sparkling wine production), the decrease of the fatty acid esters depends on the yeast level (31). Cavazza et al. (23) could separate some yeast strains with the aid of discriminant analysis based on the concentrations of esters and acids (7 variables). Nevertheless, Marchetti et al. (32), investigating 28 wines of different varieties, showed that the influence of the must was greater then any of 16 different yeast strains on the production of fermentation aroma compounds (Fig. 5).
AMINO ACIDS OF MUST The characterization of amino acid composition in grape musts and wines is of great interest because such compounds represent an important source of directly assimilable nitrogen and are also precursors in the synthesis of some volatile compounds (e.g. fusel alcohols) in alcoholic fermentation.
1668
8 9
^
60 -
2
r~
1 70 -
*§)
1
2
80 -
i
90
-
a.
100 .
n1
__E
r^ r^ r^
111111 o
u
o
0
rn i
C
a) >oucfi es
C
c c
z
i
g
2 5
7
8
6
i
i
2
1
b| mouts
Fig. 5: Cluster analysis by means of higher alcohols (8 different musts; 16 different yeast strains; Marchetti et al.; 32)
In the 1930s, amino acids were found in musts for the first time. Since then, many authors have dealt with the amino acid composition in grape berries and musts (33,34, 35,36,37,38,39,40,41,42). According to Khewer (35) 60 to 90% of nitrogen content in grape juices is present in form of amino acids. Grape varieties differ from each other merely for the amount of certain common amino acids. Cantagrel et al. (50) compared the amino acid contents of several varieties and found higher levels in Pinot and Syrah. On the other hand, the socalled "Mediterranean" varieties (Carignan, Cinsault, Grenache) showed a lower content, particularly Grenache. The most abundant amino acids are generally arginine, glutamine, proline, alanine, glutamic acid, threonine, serine and y-amino-butyric acid. They represent 80 to 90% of the total amino acid content, and in certain varieties, proline predominates up to 80 to 90% of that level (43). The variations of amino acid profiles in musts depend upon variety, crop level, density of plantation, fertilization and composition of soil, grape ripening and degree of Botrytis cinerea infection, as well as upon harvesting procedure and climatic characteristics. The amino acid content of grape berries increases with the advancement of ripening, with a very different extent (Table 2) (33,35,39,44,45). The decrease in yield/plant brought about an increase in total nitrogen content and in free amino acids level, particularly of that of proline (34). An important increase in amino acid concen-
1669 tration, was observed by increasing the density of plantation (1989). Kliewer (34) proved that the ratio of arginine/proline content in grape juice increases with the crop level at ripeness.
Table 2: Amino acids in grapes during ripening (Riesling 1976; mg/1; Rapp; 57)
9.8.76
23.8.76
8.9.76
22.9.76
60 50 70 106 630 46 60 7 5 13 17
75 109 126 140
128 155 160
1000
1030
165 100 18 11 18 21
278 145 57 20 71
Pheal
48 23 35 69 240 8 10 9 3 8 14
Lys Arg
3 150
5 255
5 490
10 660
778
1490
2540
3320
amino acid
Asp Thre
Ser Glu Glu-NH^
Pro Ala Val Met iLeu
total amino acid
74
73
Nitrogen fertilization influences the amino acids in must (68): Significant differences were found mainly between zero and the 112 kg/ha treatment (Table 3). Nitrogen fertilization above this level did not seem to affect the nitrogen composition of the fruit much, even though nitrate petiole values increased consistently with the amount of nitrogen applied to soil. Botrytis cinerea infection of grapes deeply influences the amino acid content of berries and/or juice (Table 4). The decrease of total amino acid amount can reach up to 80%, though it could differ for each amino acid (39, 40). Such variation of amino acid profile can influence the production of some fermentation components.
1670 Table 3: Effects of nitrogen fertilization of grapevines on amino acid in grape juice (Ough et al.; 68)
nitrogen fertilization (kg/ha)
amino acid
Ala
0
112
448
61
165
159
Val
15
32
28
Leu
25
47
51
iLeu
13
23
28
Ser
26
64
63
Threo
19
68
50
Asp
42
84
70
Glu
219
437
400
Pro
311
627
542
Arg
314
1068
1083
329
701
737
Total N
The amino acid content increases by increasing pressure in the pressing of grapes (46) and, to a smaller extent, by heating crushed grapes (47). Mechanical harvesting brings about an increase of amino acids in musts if compared with manual harvesting, as found by Cantagrel et al. (50): For Grenache the increase ranges between 60 to 70%, for Carignan between 60 to 80%. In that respect, all amino acids show the same tendency. By comparing the influence of climate in the same area (Beaujolais) throughout two vintage years (1977 and 1978) on a specific variety (Gamay), Cantagrel et al. (50) foimd lower amino acid levels in musts of 1978 than in those of 1977. According to Flanzy et al. (48) and Schrader et al. (49), higher levels of amino acids can be found in musts of cooler years than in those of warmer and surmy years. Apparently in cooler years a smaller amount of proteins is synthesized in the not-sufficiently ripening berries.
1671 Table 4: Effects of Botrytis cinera on amino acids (mg/1) in grape juice Rapp et al.; 39)
Bacchus
Castor amino acid
control
attacked by
control
attacked by Botr. cinerea
Botr. cinerea
His
23
14
59
-
Lys
-
10
9
66
Arg
870
78
1100
391
Asp
53
24
24
32
Threo
126
10
80
30
Ser
357
35
248
154
Glu
112
74
47
179
Pro
46
18
82
-
Ala
658
45
125
116
Val
23
5
19
10
iLeu
30
3
42
12
Pheala
30
10
72
25
2461
348
2080
1089
total amino acid
Seeber, Versini et al. (51) investigated the variation of amino acid composition in Chardonnay musts of different vintage years (1986, 1987, 1988) from 31 growing areas in a relatively small geographical region (Trentino, Italy) (Table 5). The musts were all obtained exactly with the same technology. By means of cluster-analysis, certain amino acids were grouped after similar content variation, notwithstanding a different biological origin, in some cases: threonine/serine; isoleucine/leucine/valine; methionine/phenylalanine; proline/y-amino butyric acid. Good linear correlations were found in musts between the content of threonine related to that of serine, as well as to that of tyrosine; arginine related to asparagine and to histidine, proline related to yamino butyric acid and to the sum of amino acids. No correlations were found between sugar contents or crop load/stock (under limited variations) and the sum of amino acids (63). With the aid of a proper stepwise feature selection procedure a pretreatment of data devoted to identifying the variables with highest discriminant ability, six variables we-
1672 re selected, according to the following order: glutamic acid, aspartic acid, proline, leucine, alanine und serine. The discriminant analyses based only on the content of the
Table 5: Amino acids of Chardonnay musts and volatile compounds of the wines (73 samples of 31 regions; 3 vintages: Seeber et al.; 51)
amino acid
1986
1987
1988
mean (mg/1)
mean (mg/1)
mean (mg/1)
aspartic acid
67.9
47.6
threonine
73.4
73.2
77.8
114.6
103.5
106.3
serine
44.8
asparagine
24.7
29.3
26.4
glutamic acid
91.2
135.4
130.9
glutamine
323.7
288.8
227.6
proline
693.1
823.0
614.3
valine
24.7
24.6
29.1
iso-leucine
12.3
11.5
16.9
phenylalanine
25.2
21.7
29.8
243.5
249.6
278.8
arginine
compound
1986
1987
1988
mean (mg/1)
mean (mg/1)
mean (mg/1)
1-propanol
26.0
19.7
19.4
2-methyl-1-propanol
41.3
40.3
46.7
2-methyl-l-butanol
24.9
20.9
28.7
3-methyl-l-butanol
124.9
110.0
137.6
1-hexanol
1.3
1.8
2.0
benzyl alcohol
0.02
0.04
0.03
2-phenylethanol
15.5
18.5
20.3
six amino acids showed a significant differentation between the vintages 1986, 1987, and 1988, but no differentiation among the growing regions was possible (Fig. 6). No significant overlap between 1986 and 1988 vintages was evident. While 1987 exhibited a slightly lower correct predication rate, its characteristics were intermediate between those of 1986 and those of 1988 (51). No discriminations among vintage years were found on the basis of amino acid content in the wines, these values being widely
1673
7
,7
7
7 7
7
6
6
6
Discriminant score 1
Fig. 6: Discriminant Analysis of 73 Chardonnay wines of 31 regions (amino acids) (Seeber, Versini et al; 51) dependent upon yeast metabolism. On the other hand, such discrimination was possible by using other variables (e.g. certain metals and volatiles)
VARIATION OF AMINO ACID LEVEL IN RELATION TO THE ALCOHOLIC FERMENTATION AND AMINO ACID COMPOSITION OF WINES.
Many facts influence the amino acid levels in wine: climate, soil, variety and winemaking technology. The variety seems to have the greatest influence (50). Among the 75 variables considered by Symonds and Cantagrel (65), proline is one of the most si-
1674 gnificant parameter together with shikimic acid, some esters, and magnesium for the statistical discrimination of wines from five varieties (Syrah, Gamay, Pinot noir, Malbec, Carignan). 42 wines of 8 different varieties from Portugal were separated by Vasconcelos et al. (66), with similar statistical procedures, using only the amino acids as variables. Also Etievant et al. (67) considered some amino acid levels as usefijl variables for the varietal and geographical classifications of some French red wines. Peynaud and Lafon-Lafourcade (62) explained the amino acid metabolism through: - direct assimilation - deamination, decarboxylation, and production of ammonia, the preferential nitrogen source for yeasts - transamination. During the alcoholic fermentation, a large variation of nitrogen content in must takes place. The growing yeasts assimilate a part of many N-compounds, so that the decrease of soluble nitrogen can range from 30 to 80%. Some of the free amino acids can decrease even to a larger extent (75 to 90%). At the beginning of fermentation, the contents of many amino acids are strongly reduced, whereas at the end of the process some increase considerably because of being desorbed from yeasts or because of autolysis of the cells (41, 43, 44, 50, 52, 53, 54, 55, 56, 57, 58, 59). Cantagrel et al. (50) assessed the amino acid content of 70 musts and of the corresponding wines. In two cases, the amino acid content in the wines was higher than that in the musts, whereas in seven wines there was a decrease varying between 0 to 30% and in 29 wines even higher than 60%. By fermenting the same musts with the same yeasts in different wineries, a variable decrease of amino acid content was observed. This was explained (69) by the influence of different winemaking processes (e.g. fermentation temperature, and speed). Not all amino acids concentrations decrease during fermentation, most wines have a higher content of lysine, glycine and cysteine than the corresponding musts (41,50,52, 57,58). The proline level in must may either increase or decrease - sometimes considerably - or keep constant because of the fermentation metabolism of Saccharomyces cerevisiae, as Rapp et al. (55,57,58) and other researchers (36,44,50,51,60) found, and that is in relation to the total free amino acid content, less or higher than approximately 700 to 1000 mg/1 (Table 6). The fermentative variation of proline seems to be considerably influenced also by the variety and the climatic characteristics of the vintage year (50): Syrah musts had in
1675 1978 similar average level of this amino acid in Rhone Valley so as in Tarn Valley (273 mg/1 and 292 mg/1), but proline respective contents in corresponding wines were very different: 433 mg/1 for the first area and 1944 mg/1 for the second one. Analogous behavior tendencies were observed in Beaujolais region, if taking into account the subsequent vintage years 1977 and 1988, with respective mean values in the musts of 196 mg/1 and 141 mg/1 and of 205 mg/1 and 436 mg/1 in the corresponding wines.
Table 6: Changes in content of amino acids during fermentation (Rapp, 57)
Riesl ing amino acid
must
SiIvaner
wine
must
wine
His
34
2
59
11
Lys
2
13
27
60
Arg
605
76
1130
707
Asp
75
9
94
13
Thr
80
3
136
26
Ser
188
11
392
132
Glu
106
8
77
47
Pro
122
53
115
308
Ala
56
17
155
25
Glyc
+
8
8
38
Val
24
3
76
26
Met
13
8
8
10
iLeu
46
5
70
16
Leu
55
19
56
15
Tyr
9
12
76
56
61
10
112
36
1340
640
2645
1730
Pheal Total amino acid
In the experimental fermentations with different musts we found (55,57,58) that each amino acid is metabolized by yeasts with a different intensity: glutamine, asparagine, serine, glutamic acid, aspartic acid and arginine are the favorite sources for yeast growth compared to the others such as cysteine, methionine, phenylalanine, glycine, and tryptophane. Similar results were published by Amerine and Joslyn (61). The yeast
1676 metabolism of nitrogen sources is also dependent upon the pressure in the fermentation vessel; less ammonia, leucine, and histidine are consumed in such a situation (64).
INFLUENCE OF AMINO ACID AMOUNT IN MUSTS ON THE FERMENTATIVE PRODUCTION OF VOLATILE AROMA COMPOUNDS IN WINES Higher alcohols As demonstrated in many research works (33,50,51,52,55,57,58,59,70), the fusel alcohol concentration in wine can be determined by the nitrogen content in must, so explaining the importance of amino acid composition in this connection. 1-propanol Seeber, Versini et al. (51) found (Fig. 7) in Chardonnay musts and corresponding wines of three consecutive vintage years, a general positive linear correlation (significance > 99%) between 1-propanol concentration, in the wine and the total free amino acid content in must, the latter varying from ca. 1000 mg/1 to ca. 3000 mg/1. Similar results were also found by Ough et al. (68). They showed that nitrogen fertilization of wines has a determining effect on higher alcohol level and, among other things, increases 1-propanol concentration. Also, addition of (NH4)2HP04 to the must increased the content of 1-propanol, as shown by Arapaa et al. (70) and confirmed by Margheri, Versini et al. (71). By comparing 70 musts and the corresponding wines, Cantagrel et al. (50) found a non linear correlation between threonine concentration in must and 1propanol level in wine, although it significantly increased up to amino acid concentrations of approximately 150 mg/1. The lower quantity of 1-propanol in wines of Grenache, Cinsault and Carignan - the above-mentioned "Mediterranean" varieties could be explained by the likewise lower levels of amino acid nitrogen in their musts. This fact is confirmed by the results for other varieties, e.g. Gamay and Pinot noir, which show opposite composite situations (56). The different volatile compositions, particularly of 1-propanol in wine obtained from mechanically harvested grapes in comparison with those produced from handpicked grapes (50) can be explained by the significantly higher level of amino acids from mechanically harvested grapes, probably the result of longer contact with air and leaves, which increases the possibility of chemical and enzymatic reactions, in the must (50) e.g.
1677 • that of the phenoloxidase, which can oxidize phenolic compounds to quinones, whiche in turn can oxidize amino acids to corresponding keto acids; • an increased proteolitic grape activity because of mechanical processes, which increases the enzymatic transformation of soluble nitrogen (72).
1000
1500
MUST
2000
2500
3000
500
3500
WOO
1500
MUST;
TOTAL AMINO A C m i m n / I )
2000
AMINO ACID
2500
3000
3500
(TOTAL) ( m g / l )
IOOT
225
90-
~ 200 I 175 o 150
A
r--~~- *
>*
~—- _ ^ * ^
=:
I 70 1 * ^
* ^
^
60
" 100
<
^
t
I
-
80-
X
75
/ 30 20 L--'—^""^
tH 50
s:
A A
10 0
1000
500
2000
2500
3000
0
3506
500
MUST: TOTAL AMINO ACID ( m g / l 1
1500
2000
2500
1500
2000
MUST: TOTAL AMINO ACID
^
1000
1000
3000
2500 (mg/l)
3.5
3500
MUST: AMINO ACID (TOTAL) (mg/l 1
Fig. 7: Influence of total amino acids content in must and fermentation compounds in wine (Seeber, Versini et al., 51)
Such reaction mechanisms have opposite effects on the amino acid compositions of musts (50): - decreases in amino acid levels because of their oxidation through quinones; - increases in amino acid levels from increased protease activity.
1678 2-methyl-1 -propanol No significative correlation was found between 2-methyl-1-propanol levels in wines and valine concentration in related musts (50) or even lower correlations were noted in connection with the total amino acid level in musts (68) or valine concentrations in wines (51). 3-methyl-1-butanol and 2-methyl-1-butanol Negative correlations were found between both of these alcohols and total free amino acid content in musts (51,68) (Fig. 7). A linearity with a significance > 95% was assessed only for 3-methyl-1-butanol and for each considered vintage. Such tendencies were also found in connection with structurally higher alcohol corresponding amino acids (leucine and isoleucine) in musts as well as in wines (50,51). This kind of correlation was already assessed by Margheri, Versini et al. (71) by adding different quantities of (NH4)2HP04 to the must, and so decreasing the amylalcohol level in relation to the untreated sample. Cantagrel et al. (50) found that an increase of leucine and isoleucine contents in must up to 50 mg/1 had no influence on the corresponding higher alcohol concentrations, and this confirmed our resuhs with model solutions (55,57,58). 2-phenylethanol By increasing the free amino acid concentration in must, an important decrease of 2phenylethanol (Fig. 7) content in wine was found (51) with a correlation at a significance level > 95%. Such an inverse correlation was also noticed when supplying assimilable N-sources, such as (NH4)2HP04, to the must (71). Houtman et al. (24) obtained similar results. Cantagrel et al. (50) also proved a marked decrease in this alcohol as the phenylalanine content in the must increased up to approximately 50 mg/1, but then remained constant for higher values, even up to 140 mg/1.
Other volatiles (fermentation compounds) Ethyl acetate Seeber, Versini et al. (51) found a positive linear correlation with a significance >95%, existing in every year, and also for the three considered vintage years, between total free amino acid content in musts and that of ethylacetate in wines (Fig. 7). A similar correlation was found by Bell et al. (74) between such esters and N-levels in must.
1679 3-methyl-1-butyl and 2-methyl-l-butyl acetate (isoamyl acetate) The same kind of positive linear correlation as for ethyl acetate, was assessed between the i-amyl-esters and the total free amino acid levels (51) (significance > 95%), but only within each considered vintage year. In fact, there is a different slope and range variation in relation to the vintage year. Ough et al. (73) found a similar correlation by increasing the total nitrogen content from ca. 200 to 1000 mg/1 in Thompson Seedless musts through several different levels of fertilizer application on wines in carefully replicated blocks. The fitness of such a correlation decreased when increasing the molecular weight of the esters. The addition of (NH4)2HP04 to the must seems to give only a limited increase of isoamyl acetate in wine. 2-phenylethyl acetate According to Ough et al. (73), the content of this acetate is negatively correlated with the total nitrogen amount of musts, and from 800 mg/1 of total nitrogen upwards, no more variation was recorded. By adding (NH4)2HP04 to the must (up to 200 mg/1 as N), we observed a constancy in the concentration of this ester as opposed to the marked decrease of the corresponding alcohol, 2-phenylethanol: Seemingly this could result from the opposing influences on the higher alcohol relative to that on the acetate synthesis. Cantagrel et al. (50) did not find a direct relation between free amino acid level in must and that of higher alcohol acetates, thus deducing a greater influence of wine making conditions (e.g.pH and temperature), as compared to the effect of N content. 3 -methylthio propan-1 -ol Its concentration in wine is inversely correlated to the total free amino acid (Fig. 7) amount in must (51), as it is with added (NH4)2HP04 (71). Limited additons to the must of possible methionine biosynthetic precursors like aspartic acid and (NH4)2S04 don't increase significantly methionol production (86).
PRECURSORS OF THE FERMENTATION COMPOUNDS
In earlier years it was believed that the principal higher alcohols were produced from the corresponding amino acids. However, this does not correlate with the fact that these amino acids in the must are at levels 3 to 6 times lower than the corresponding higher alcohols in wine (52). Thoukis (76) and Ingraham et al. (77) showed that the yeast can also produce higher alcohols from sugar. Guyman (78) first demonstrated the
1680 link between keto acids and production of higher alcohols: 1-propanol is yeast produced from a-keto butyric acid. This agrees with increased production of 1-propanol on addition of a-aminobutyric acid. According to Ribereau-Gayon et al. (75) production of higher alcohols is as follows: - 10%fromthe corresponding amino acids, through transamination, decarboxylation and hydrogenation (Ehrlich mechanism), the higher alcohols are produced via the corresponding keto acids - 25%fromthe sugar carbon skeleton - 65%fromother amino acids Thus, must amino acids are not the only source for higher alcohol formation in wine. Yeast can produce higher alcohols from a medium with low amino acids contents. Yeasts can also produce higher alcohols from one single nitrogen source, with levels depending upon the ratio of the assimilable nitrogen content to the sugar content.
EXPERIMENTS WITH MODEL SOLUTIONS TO DETERMINE THE INFLUENCE OF THE NITROGEN SOURCE ON THE FORMATION OF HIGHER ALCOHOLS
In model solutions using only one nitrogen source (amino acids, ammonixmi salt), Rapp et al. showed (55,57,58) that the yeast differently assimilates the nitrogen source and gives different levels of higher alcohols. The influence of nitrogen source can be traced using the ratio of i-amylalcohol to i-butanol formed (Table 7). When the ratio is greater than 1.5, a tendency to i-amylalcohol production is indicated, and when lower than 1.5 this indicates increased production of i-butanol. In addition to i-leucine, production of i-amylalcohol is also significantly influenced by proline, glutamic acid and ammonium sulfate (55,57). Investigations show that this influence of nitrogen source is more significant than that of the yeast strain. In model solutions using different concentrations of the same nitrogen source Rapp et al. found (55,57) that increasing levels of a standard amino acid mixture decreased the level of 3-methyl-l-butanol (Fig. 8). This effect continued up to a level of about 1400 ppm amino acids (250 ppm amino acid-nitrogen); and above this level no further amylalcohol decrease is observed. Arapaa (70) found similar results and established a level of 300 ppm amino acid nitrogen for nofiirtheralcohol decrease.
1681 Table 7: Influence of nitrogen source on production of higher alcohols (mg/1) during fermentation (Rapp Diss. 1965)
nitrogen source
2-methyl-
3-methyl +
propanol-1
2-methyl -
V^B
butanol-1
51
52
1.0
260
77
0.3
methionine
70
128
1.8
tryptophane
58
89
1.5
aspartic acid valine
NH^N03
41
61
1.5
glutamic acid
20
68
3.4
leucine
17
430
25.3
m^)^so^
10
23
2.3
435
4.7
93
i-leucine
'56g/i sucrose (= 70°0e) 200g/i sucrose (= 88°0e)
J^ ia
263g/i sucrose (= 110°0e) 3i7g/i sucrose (= 130°0e)
100
500
900
1300
1700
3700 ng/l AS
Fig. 8: Influence of aminoacid- and sucrose concentrations in must on the production of 3-methyl-l-butanol during fermentation (Rapp 57,55)
1682 Table 8: Distribution (%) of l^C-activity in model fermentation medium (grape juice by adding ^^C-compounds; Rapp et al. 55,57)
1 4 c . compounds
Fraction glucose
glutamic acid
aspartic acid
malic acid
yeast
1.3
22.2
24.9
2.6
CO2
28.9
8.3
27.9
0.1
non volatile
5.5
69.0
29.3
94.6
total volatile
64.3
0.5
17.9
2.6
1 fraction
Table 9: Distribution (%) of ^^C-activity in volatile fraction after fermentation (fermentation of I'^C-compounds with Saccharomyces cerevisiae; Rapp et al. 55,57)
14c _ compounds
Fermentation compounds
glucose
glutamic acid
aspartic acid
malic acid
dest.-fraction total 95
53
60
34
5
47
40
66
ethylacetate
0.5
0.2
0.6
0.1
i-amylacetate
0.1
1.4
0.1
0.4
propanol-1
0.1
1.5
3.8
1.3
2-methyl-propanol-1
1.5
0.9
19.6
31.7
butanol-1
0.1
0.2
0.4
0.5
2.3
40.0
14.8
30.3
0.1
0.4
0.1
ethanol other volatiles other volatile
3-methyl- + 2-methyl-butanol-1 1 pentanol-1
< 0.1 1
In experiments with I'^C-compounds (amino acids, acids, sugar), we investigated (55,57) the relative contribution to the different fermentation substances of the carbon
1683 skeleton of these marker compounds. For example, 17.9% of the ^^C content of aspartic acid was found in the volatile fraction, and 60% in ethanol (Table 8). Corresponding level for glutamic acid were 0.5% in the volatile fraction. Using radiogaschromatography we found that glutamic acid provided the carbon of i-amylalcohol and ibutanol in a ratio of 40 : 1. While when using aspartic acid the ratio was 19.6 : 14.8 (Table 9). Production of higher alcohols is influenced by many factors. The principle factor is the amino acid source for the yeast, and this factor is more important than the actual yeast strain. A strong correlation exists between the amino acid spectrum in must and the absolute and relative levels of the higher alcohols in wine.
INFLUENCE OF THE AMINO ACIDS IN MUST AND THEIR CORRESPONDING FERMENTATION COMPOUNDS ON WINE QUALITY
According to Cantagrel et al. (50), the higher alcohols have a negative influence on wine quality, but Bidan (52) stated only that this could be a possibility and did not accept definite link. I50T
, Y-64.95 + O.I44(X)-0.00006(X^)
O
139
I28|
z (0
<
106
95 200
320
440
560
680
800
TOTAL NITROGEN (mg./l)
Fig. 9: Relationship of juice total nitrogen to wine taste intensity ratings (Sinton, Ough et al. 1978)
1684 Sinton et al. (79) studied the relationship between crop level, juice and wine composition, wine sensory ratings and scores (Fig. 9). Good wine-aroma intensity, flavour intensity, and wine balance were rated on a scale of 1 to 10. Both the individual good wine-aroma ratings and intensity-of-flavour ratings show a significant negative correlation to cropping level. The taste intensity-not specifically good or bad tastes - has a positive correlation with the total nitrogen of must. The same correlation is found between the hexyl acetate /hexanol values in the wine and the good wine - aroma intensity ratings. With the increase of nitrogen levels in must there is also an increase of ester formation during fermentation. Wagner et al. (80) showed that the acetate esters contributed to wine quality. Bell et al. (81) found that wines from unfertilized grapes scored lower in aroma intensity flavour, and overall wine quality than wines madefi'omfertilized grapes. From the combined tasting data and the analytical data, it appears that the greatest influence of nitrogen fertilization on wine quality would be in applications of 0 to 112 kg N/ha. This verifies that the rates of nitrogen fertilization of 56 to 84 kg N/ha recommended in California are in the proper range.
Fig. 10: Relationship between FAN of Musts and Aroma Quality of Wines (Vos, 1981) FAN = free assimilable nitrogen
1685 The initial assimilable nitrogen content (FAN: free amino acids, ammoniacal nitrogen, lower peptides) of must greatly affects the rate of fermentation, the end-product formation and ultimately, the fmal organoleptic quality of the wine (Fig. 10) (82). The correlations between FAN content in must and the formation of 1-propanol acetates and ethyl esters are significant and positive; those of 2-methyl-1-propanol, 3-methyl-lbutanol and hexanol are negative. The inverse relationship between FAN and H2Sproduction implies that when the assimilable nitrogen content of must is insufficient for optimum metabolism, the proteolytic activities of yeast are stimulated. Proteins and higher peptides are hence degraded to assimilable forms in an effort to supplement such deficiency; during this process, the sulphur derivatives of must proteins are liberated as H2S. The amelioration of musts with (NH4)2HP04 resulted in highly significant increases in the esters content of wine as well as significant decrease of higher alcohols. The results confirm the judicious use of (NH4)2HP04 to raise the inital FAN content of musts (when required) and enhances the quality of wines; sensory preference rankings established for overall taste and aroma quality (82).
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1688 28. R.M. Soles, C.S. Ough, R.E. Kunkee Ester concentration differences in wine fermented by various species and strains of yeasts Amer. J. Enol. Vitic. 33, 94-98 (1982) 29. E. Souffleros, A. Bertrand Role de la "soudre de levure" dans la production des substances volatiles au cours de la fermentation du jus de raisin Conn. Vigne vin 13, 181-198 (1979) 30. M. Castino, A. Bosso, G. Marlescalco Elaboratione di vini bianchi con macerazione a freddo in presenza di enzimi pectolitici Vinid'Italia 7-20 (1983) 31. G. Margheri, G. Versini, L. Gianotti Vini Spumanti di Qualita Metodo champenois Vinid'Italia 51-59 (1984) 32. R. Marchetti, M E . Guerzoni Effects de I'interaction souche de levure composition du mout sur la production, au cours de la fermentation, de quelques metabolites volatils Conn. Vigne Vin 21, 113-125 (1987) 33. S. Lafon-Lafourcade, G. Guimberteau Evolution des aminoacides on cours de la maturation des raisins Vitis 3, 130-135(1962) 34. W.M. Kliewer Free amino acids and other nitrogenous fractions in wine grapes J. Food Sci. 35, 17(1970) 35. W.M. Kliewer Changes in the concentration of free amino acids in grape berries during maturation Amer. J. Enol. Vitic. 19, 166-174 (1968) 36. C.S. Ough, R.M. Stashak Further studies on Proline concentrations in grapes and wine Amer. J. Enol. Vitic. 25, 7-12 (1974) 37. M.C. Polo, C. Llaguno Evolution des acides amines libres dans le mout de raisin sous Taction des levures de fleur Connais. Vigne Vin 8, 81-90 (1974) 38. C. Poux Les acides amines dans les mouts et les vins Rev. franc. Oenol. 11, 5-19 (1970)
1689 39. A. Rapp, K.H. Reuther Der Gehalt an freien Aminosauren in Traubenmosten von gesunden und edelfaulen Beeren verschiedener Rebsorten Vitis 10, 51-58(1971) 40. H.H. Dittrich, W.R. Sponholz Die Aminosaureabnahme in Botrytis-infizierten Traubenbeeren und die Bildung hoherer Alkohole in diesen Moten bei ihrer Garung Wein-Wissensch. 30, 188-210 (1975) 41. J.G.B. Castor The free amino acids of must and wines. I. Microbiological estimation of fourteen amino acids in California grape musts Food Research 18, 139-145 (1953) * J.G.B. Castor The free amino acids of must and wines. II. The fate amino acids during alcoholic fermenfermentation Food Research 18, 146-151 (1953) 42. M. Feuillat, J. Bergeret Influence des traitements thermiques de la vendange sur I'extraction des constituants azotes C.R. Acad. Sc. Paris Serie D, 264, 2520-2523 (1967) * M. Feuillat, J. Bergeret Identification et dosage des aminoacides dans les mouts et les vins de Bourgogne C.R. Acad. Sci. Paris Serie D. 264, 1757-1759 (1967) 43. C.S. Ough Proline content of grapes and wines Vitis 7, 321-323(1968) 44. R.M. Kluba, L.R. Mattick, L.R. Hackler Changes in concentration of free and total amino acids of several native american grape cultivars during fermentation Am. J. Enol. Vitic. 29, 181-186 (1978) 45. J.S. Hawker Changes in the activities of malic enzyme malate dehydrogenase, phosphopyruvate carboxylase and pyruvate decarboxylase during the development of a nonclimateric fruit (The grape) Phytochemistry 8, 19-23 (1969) 46. KG. Bergner, H.E. Haller Das Verhalten der freien Aminosauren von WeiBwein im Verlauf der Garung, bei Ausbau, Lagerung und Umgarung Mitt. Klostemeuburg 19, 264-288 (1969)
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1691 57. A. Rapp Uber die Inhaltsstoffe von Traubenmosten und Weinen unter besonderer Beriicksichtigung der fluchtigen Verbindungen und des stofflichen Geschehens wahrend der Hefegarung Diss. Univ. Mainz, 1965 58. A. Rapp, H. StefFan, H. Hastrich, H. Ullemeyer Zur Kenntnis der Biosynthese des Garungsamylalkohols Mitt. Fachgr. Lebensm. und gerichtl. Chemie 29, 33-38 (1975) 59. F. Drawert, A. Rapp Uber Inhaltsstoffe von Mosten und Weinen. VII. Gaschromatographische Untersuchung der Aromastoffe des Weines und ihrer Biogense Vitis 5, 351-376(1966) 60. C. Poux, A. Ouraac Acides Amines libres et polypeptides du vin Ann. Technol. Agric. 19, 217-237 (1970) 61. M.A. Amerine. M.A. Joslym Table wines. The technology of their production University of California Press Berkeley (1970) 62. E. Peynaud, S. Lafon-Lafourcade Sur la nutrition azotee des levures de vin Rev. Ferm. Industr. Alim. 17, 11 (1962) 63. G. Versini, A. Delia Serra, A. Monetti, M. Falcetti, D. Tonon, M. Bertamini Considerazione sulla cariabilita compositiva di alcuni para metri analitici di mosti e vini. Base Spumante Chardonnay del Trentino in Funzione della zona e dell'annata Proceedings 4. Mostra Nationale Spumante Classico, 34-44 (1989) 64. G. Margheri, L. Gianotti, R. Pelligrini, C. Mattarei Vini Spumanti di Qualita metodo champenois Vini d'ltalia 21-26 (1984) 65. P. Symonds, R. Cantagrel Communication presentee a la reunion regionale de la societe des experts chimistes. Toulouse juin 1980; 66. A.M.P. Vasconcelos, H.J. Chaves das Neves HRGC of wine free amino acids as a tool for elementary wine characterization J.H. Res. Chromat. 13, 494-498 (1990) 67. P. Etievant, P. Schlich, J.C. Bouvier, P. Symonds, A. Bertrand Varietal and geographic classification of French Red Wines in terms of Elements, amino acids and aromatic alcohols J. Sci. Food Agric 45, 25-41 (1988)
1692 68. C.S. Ough, A.A. Bell Effects of Nitrogen fertilization on grapevines on amino acids metabolism and higher alcohol formation during grape juice fermentation Am. J. Enol. Vitic. 31, 122-123 (1980) 69. G. Margheri, G. Versini, R. Pelligrini, D. Tanon L'azoto assimilabile e la tiamina in fermentazione, loro importanza qualifattori di qualita 'dei vini Vinid'Italia 71-86 (1986) 70. T. Ayrapaa Biosynthetic formation of higher alcohols by yeasts. Dependance on the nitrogenous nutrient level of the medium J. Inst. Brew. 77, 266-275 (1971) 71. G. Margheri, G. Versini, L. Gianotti, R. Pellegrini Fattori di qualita dei vini bianchi giovanni: influenza dell' azota assimilabile dei mosti e dei componenti aromatici dei vini La Rivista Soc. Ital. LaRivista Soc. Ital. Alimentazione 13, 401-412 (1984) 72. M. Feuillat, G. Brillant, J. Rochard Mise en evidence d'une production de proteases exocellulaires par les levures au cours de la fermentation alcoolique du mout de raisin Conn. Vigne Vin 14, 37-52 (1980) 73. C.S. Ough, T.H. Lee Effect of vineyard nitrogen fertilization level on the formation of some fermentation esters Am. J. Enol. Vitic. 32, 125-127 (1981) 74. A.A. Bell, C.S. Ough, W.M. Kliewer Effects on must and wine composition, rates of fermentation and wine quality of nitrogen fertilization of V. vinifera Var. Thompson seedless grapevines Am. J. Enol. Vitic. 30, 124-129 (1979) 75. J. Ribereau-Gayon, E. Peynaud, P. Ribereau-Gayon, P. Sudraud Traite d'Oenologie Dunod, Paris 1972 76. G. Thoukis Amer. J. Enol. Vitic. 9, 161-163 (1958) 77. J.L. Ingraham, J.F. Guymon The formation of higher alcohols by mutant strains of Saccharomyces cerevisiae Arch. Biochem. Biophys. 88, 157-161 (1960) 78. J.F. Guymon Developments in Industrial Microbiology. Chap II. 88-96, Am. Inst. Biol. Sci. Publ. Washington D C . (1966)
1693 79. T.H. Sinton, C.S. Ough, JJ. Kissler, A.N. Kasimatis Grape Juice indicators for prediction on potential wine quality. I. Relationship between croplevel, juice and wine composition, and wine sensory ratings and Scores Am. J. Enol. Vitic. 29, 267-271 (1978) 80. W.D. Wagner, W.G. Wagner The influence of ester and fusel alcohol content on the quality of dry white wine. S. Afr. J. Agric. Sci 11, 469-476 (1968) 81. A.A. Bell, C.S. Ough, W.M. Kliewer Effect on must and wine composition, rates of fermentation and wine quality of nitrogen fertilization of V. vinifera var. Thompson Seedless grapevines Am. J. Enol. Vitic. 30, 124-128 (1979) 82. P. Voss Assimilable Nitrogen - A factor influencing quality of wines Proceedings, Oenological Symposium, Mainz, Germany, 163-181 (1981) 83. L. Usseglio-Tomasset L'acetato di etile e gli alcooli superori nei vini. Riv. Vitic. Enol. XXIV, 276-286 (1971) 84. G. Versini, G. Margheri Evoluzione di componenti volatili nel corso della elaborizione dei vini spumanti del Trentino. Produceed. Symp. Intern, on sparkling wines, Salice Terme, 10/11. June 1981, Chirotti, Torino, 1981, pagg. 148-158 85. J.F. Clapperton, D.G.W. Brown Caprylic flavour as a feature of beer flavour J. Inst. Brew. 84-90 (1978) 86. V. Lavigne, J.N. Boidron, D. Dubourdieu Formation des composes soufres lourds an cours de la vinification des vins blancs sees. J. Int. Sc. Vigne Vin 26/2, 75-85 (1992) 87. P. Winterhalter, M.A. Shefton, P.J. Williams Two-dimensional GC-DCCC analysis of the glycoconjugates of monoterpenes, norisoprenoids and shikimate-derived metabolites from Riesling wine. J. Agr. Food CHem. 38, 1041-1048 (1990) 88. G. Versini, A. Scienza, A. Dalla Serra, M. Dell'Eva, C. Martin Role du clone et de I'epoque de recolte sur I'arome du Chardonnay: aspects analytiques et sensoriels In: "Actualites oenologiques 89", IV Symp. Intern. Oenologie, Bordeaux, 15.-17. juin 1989, P. Ribereau-Gayon and A. Lonvand eds. Dunod, Paris, 1990, pagg 69-74
1694 89. G. Versini SuU'aroma del vino Traminer Aromatico o Gewurztraminer. Vignevini XII, 57-65(1985) 90. P.X. Etievant, S.N. Issanchon, C.L. Bayonove The flavour of Muscat wine: the sensory contribution of some volatile compounds J. Sci. Food Agric. 34, 497-504 (1983) 91. P. Gramatica, P. Manitto, B.M. Ranzi, A. Delbianco, M. Francavilla Stereospecific reduction of geraniol to (R)-(+)-citronellol by Saccharomyces cerevisiae Experientia 38, 775-776 (1982) 92. R. di Stefano, G. Maggiorotti, S. Gianotti Trasformazioni di nerolo e geraniolo indotte dai lieviti Riv. Vitic. Enol. 1, 43-49 (1992) 93. G. Kinzer, P. Schreier Influence of different pressing systems on the composition of volatile constituents in unfermented grape musts and wines Am. J. Enol. Vitic. 31/1, 7-13 (1980) 94. A. Rapp, C. Volkmann, H. Niebergall Untersuchung fluchtiger Inhaltsstoffe des Weinaromas: Beitrag zur Sortencharakterisierung von Riesling und Neuziichtungen mit Riesling-Abstammung Vitis 32, 171-178(1993) 95. A. Rapp, I. Suckrau, G. Versini Untersuchungen des Trauben- und Weinaromas. Beitrag zur Sortenchrakterisierung neutraler Rebsorten (Silvaner, WeiBburgunder, Rulander) Z. Lebensm. Untersuchung und Forschung 197, 249-254 (1993) 96. D.R. Webster, C.G. Edwards, S.E. Sprayd, J.C.Pterson, B.J. Seymour Influence of vineyard Nitrogen Fertilization on the Concentration of Monoterpenes, Higher Alcohols and Esters in Aged Riesling Wines Amer. J. Enol. Vitic. 44, 275-284 (1993)
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1695
The contribution of oak lactone to the aroma of wood-aged wine J.R. Piggott, J.M. Conner and J.L. Melvin Department of Bioscience and Biotechnology, University of Strathclyde, 131 Albion Street, Glasgow Gl ISD, Scotland Abstract Lactones are important flavour compounds in many foods and beverages because of their potency and varied sensory properties. However, while their importance in the flavours of fruits and dairy products is moderately well understood, the flavour effects of lactones in alcoholic beverages are not clear. Oak lactone has been identified in many wood-aged wines and distilled alcohoHc beverages at around its threshold concentration, but it is uncertain whether this has a significant effect on the flavour. Australian white wines were found to contain between 57 and 142 jig V\ and reds between 13 and 433 \ig 1 "^ Spanish white wines contained from 42 to 538 jig 1 , and reds from 208 to 479 jig 1'^ The odour threshold in 12% ethanol was estimated to be 75 jig 1'^ Oak lactone therefore probably contributes significantly to the aromas of some barrel-aged red and white wines. 1. INTRODUCTION The flavours of wines are due to a composite of compounds derived from various sources, including volatiles present in the grapes, those formed during processing and fermentation, and others resulting from subsequent aging (Schreier, 1979). In the case of wood-aged wines, oak wood can have significant effects on the sensory properties of the product through extraction of flavour compounds or their precursors. Due to the limited life of oak casks, danger of contamination, difficult temperature control and higher costs, maturation in stainless steel has gradually become more popular. However, oak maturation is still crucial to the character of many of the world's fine wines (Chatonnet, 1991). A better knowledge of the physico-chemical changes involved would allow both control and improvement of the quality of such wines. The cis and trans forms of B-methyl- y -octalactone have woody and coconut odours and, along with eugenol and vanillin, are among the major aroma compounds of uncharred oak wood (Masuda and Nishimura, 1971; Chatonnet, 1991). This lactone has been identified in aged alcoholic beverages such as brandy, whisky and wine (hence the common names, "whisky", "quercus" or "oak" lactone). The species of oak, method of drying, level of charring and history of the cask all affect the levels of oak lactones in the wood and in the beverages (Guymon and Crowell, 1972; Otsuka et ai, 1974; Marsal and Sarre, 1987; Boidron et a/., 1988; Maga, 1989; Chatonnet, 1991). Relatively few studies have examined the effect of oak maturation on the aroma composition of wood-aged wines (Simpson and Miller, 1984; Boidron etai, 1988; Chatonnet, 1991). On reviewing the role of oak lactone in wine aroma, Etievant (1991) concluded that the more potent isomer could make a contribution but that there was still a lack of quantitative data for oak lactones in commercial wines. Most Australian wines and wines from the Rioja region of Spain are commonly fermented and/or matured in oak, and are known for their oaJky character. Hence, the aim of this work was to quantify 6-methyl- y octalactone in a selection of wood-aged wines, and to estimate its importance in wine aroma
1696 by determining its odour threshold. 2. EXPERIMENTAL 2.1. Materials Wines (Table 1) were obtained from a retail outlet in Scotland. Reagents for synthesis of lactone were ethyl crotonate, pentanal, benzoyl peroxide (70%) and sodium borohydride (Aldrich Chemical Co. Inc., Gillingham, Dorset, England). Other reagents were pentane and diethyl ether (Glass distilled grade; Rathburn Chemicals Ltd., Walkerburn, Scotland), dichloromethane (HPLC; Rathburn Chemicals Ltd.) and n-hexadecane (BDH Chemicals Ltd., Poole, England). 2.2. Synthesis of B-methyl- y -octalactone Racemic 6-methyl- y -octalactone (3-methyl-4-octanolide) was synthesised by the method of Gunther and Mosandl (1986). Ethyl-3-methyl-4-oxooctanoate was synthesised by reaction of pentanal with ethyl crotonate. At the end of the reaction excess pentanal was removed by distillation under nitrogen. Ethyl-3-methyl-4-oxooctanoate was purified by liquid chromatography on a column of silica (silica gel 60,40-63 |Lmi, 230-400 mesh ASTM; Merck, D-6100 Darmstadt, Germany). The eluent was pentane/diethyl ether (95:5). Reduction of ethyl-3-methyl-4-oxooctanoate with NaBH4 and cyclisation gave racemic 3-methyl-4octanolide. 2.3. Purity of synthesised oak lactone The initial purity of the product of synthesis was about 94% (by GC-MS). The components were separated on a Carbowax BP 20 column (as for wine analysis) and odour evaluation carried out using a Carlo Erba 5300 H.R.G.C. operating in splitless mode and with a sniff port (ODO-1, SGE UK Ltd., Milton Keynes, England). Descriptions and intensities of the odours sniffed were recorded with a tape recorder; starting time and duration of odours were recorded with a stop-watch. The GC profile of the sample was recorded under the same chromatographic conditions. Two peaks (unidentified) with a fruity odour of relatively low intensity eluted before the oak lactone isomers which both had a strong coconut odour. The sample was distilled at 50°C on a rotary evaporator until examination by GC-MS indicated 96% purity. Further purification steps were not used to avoid a reduction in the yield of lactone. The lactone preparation was diluted in pentane until a concentration judged to have a pure odour by sniffing test was determined. The compound (undiluted) was stored in a screw-capped glass vial at -10°C. Analysis for oak lactone was carried out using a Finnegan MAT ITS40 integrated benchtop GC-MS and data analyser. The ionisation mode was electron impact and the ionisation potential 70 eV. Compounds were separated by injecting 1 jil extract onto a 30 m x 0.25 mm DB5 column (J&W Scientific; Jones Chromatography, Hengoed CF8 8AU, Wales), with an initial temperature of 60°C for 3 min, increasing to 250°C at 10°C min'^ with a final 3 min hold at 250°C. Injection port and detector temperatures were 230°C and 150°C, respectively. Carrier gas was helium at 1.8 ml min'\ Identification of cis- and trans-i^-mQthy\- y octalactone was confirmed by comparison of EI mass spectra with published spectra. Purity of the synthetic lactone was estimated from percentage peak areas measured with a computing integrator. 2.4. Odour detection threshold determination The panel (untrained and not selected) was composed of 12 assessors (students and members of staff) of both sexes, between the ages of 20 and 50 years. The odour detection thresholds of 6-methyl- y -octalactone were determined in 12% ethanol, white wine (BV, Table 3) and red wine (CO, Table 3) using the synthesised racemic mixture of cis/trans isomers (96% pure). Test liquids (20 ml) were served in tulip-shaped
1697 Table 1 Wine sample details Principle grape variety Vintage
Winery
Origin
Oak-wood maturation
White wines Australia Chardonnay
1991
Orlando
South East
French & American oak
Chardonnay
1991
Killawarra
South East
Not described
Spain Viura
1991
Castillo de Olite
Navarra(DO)
Not described
Viura
1991
Gran Vendema
Rioja Alta (DO)
Not described
Viura
1991
Bodegas Montecillo (Vina Cumbrero)
Rioja (DO)
Not described
1990
Cosme Palacio y Hermanos
Rioja (DO)
Crianza
Min. of 6mths. Limousin oak
1987
Jose Bezares
Rioja (DO)
12 mths. in oak
1990
Barossa Valley Estates
South East
Not described
Shiraz/ Cabernet Sauvignon
1990
Seaview
South
Seasoned American oak
Shiraz/ Cabernet Sauvignon
1990
Wolf Blass 'Red Label'
South
Not described
1989
Cosme Palacio y Hermanos
Rioja (DO)
Crianza
Min. of 1 year French oak
Viura Viura
Crianza
Red wines Australia Shiraz/ Cabernet Sauvignon
Spain Tempranillo Not detailed Garnacha/ Carifiena Tempranillo
1989
Bodega Farina (Gran Colegiata)
Toro, Zamora
Not described
Crianza
Penedes (DO)
6 mths. new American oak
Rioja Alta (DO)
>2 years in oak
1987
Miguel Torres
Reserva
(Gran Sangredetoro)
1985
Campo Viejo
Reserva
1698 nosing glasses covered with watchglasses. Each panellist sniffed a series of six triangular tests (Meilgaard, 1975a), each containing two controls and one test sample. The position of the 'odd' sample in each triangle was balanced. Test samples increased in concentration by a constant factor C^VlO or ^VlO). Assessors were asked to identify the 'odd' sample in each triangle and to describe the odour difference. 2.5. Analysis for oak lactone Triplicate 30 ml samples of wines were extracted with dichloromethane (3 x 10 ml with agitation on a shaker for one hour). The combined extracts were dried over anhydrous sodium sulphate, filtered and 40 [ig of internal standard (hexadecane) added. The extracts were concentrated to a final volume of 1 ml on a rotary evaporator and then stored in screwcapped glass vials at -10°C until analysis by gas chromatography. Concentrated wine extracts were analysed for oak lactone on a 25 m x 0.32 mm Carbowax BP 20 column (SGE UK Ltd.) using a Carlo Erba 5300 gas chromatograph fitted with a flame ionisation detector. The injection port temperature was 230°C and the detector temperature 250°C. The column temperature was held at 60°C for 2 min, then programmed to 200°C at TC min ^ then increased to 240 °C at 20°C min"^ with a final 2 min hold at 240°C. Helium carrier gas was maintained at 1.8 ml min'^ Injections of 2 jul were made with a split ratio of 20:1. Peaks were identified by comparing retention times with data for the synthetic compound. Concentrations of oak lactones in wine were calculated from the standardised peak area and from the corresponding calibration curve. Solutions of synthetic lactone in 12% aqueous ethanol, ranging from 10 |Lig 1'^ to 1000 |Lig \'\ were extracted under conditions identical to those for wine samples. Calibration curves were prepared for each isomer by plotting area ratios of lactone/hexadecane versus concentration of lactone. 3. RESULTS AND DISCUSSION 3.1. Determination of odour thresholds The estimated odour thresholds are shown in Table 2. The percentage of correct answers for each concentration was corrected for chance and transformed to probits (assuming the distribution of scores followed a cumulative normal probability function). Regression lines between probits and log concentrations were calculated and odour detection thresholds defined as the concentration at 5.00 probits (50% above chance score). The estimated detection threshold in 12% ethanol in water was 75 jiig 1'^ Using a similar method, Boidron et al. (1988) obtained a threshold of 15 |Lig 1'^ for a 1:1 mixture of the two oak lactone isomers in a model wine solution of 12% ethanol. However, the latter study used a trained panel. The numerical value of a threshold varies with the test used to determine it, the assessors and the purity of the stimulus. Also, a flavour compound may be chemically very pure (99% +) and yet organoleptically very impure (Meilgaard, 1975b). Although the impurities present in the synthesised lactone used in this study (96% pure) were judged to be below detection level at the concentrations assessed in the threshold test, the estimated threshold for the lactone might have been affected. To obtain a better indication of the role of oak lactone in the aroma of the wines analysed, the thresholds were also determined in a red and a white wine. Both wines were similar in style to those analysed but were chosen for their lack of contact with wood. These thresholds do not reflect the great variation in composition among wines but the values are valuable because they indicate the concentration above which the lactone would dominate the overall wine aroma. However, analysis of these wines by GC-MS showed the presence of woodderived compounds including the oak lactones at less than 25 \ig 1 "^ The thresholds in wine reported in Table 2 are therefore difference thresholds i.e. the increase which can be smelled. Use of difference thresholds tends to lead to underestimation of the sensory contribution of
1699 Table 2 Odour thresholds and sensory descriptions of B-methyl- y -octalactone Detection threshold Medium Odour (cis + trans 1:1) comments 75 241 (difference) 853 (difference)
Coconut, woody, musty, less harsh
Model soln. (12% Ethanol)
15 ' {cis+trans 1:1) 25 ^ {cis) no*' {trans)
Coconut, oaky
White wine Red wine
120^ 125^
Ethanol 12% vol. White wine Red wine Literature values
' Boidron et aL, 1988 ^ Chatonnet, 1991 Table 3 Estimated concentrations of B-methyl- y -octalactone in wines and odour unit values Ratio Odour units Mean concentration Wine cis: trans sample^ {cis + trans) (ngi-') (n=3) trans c^y cis+trans White KC OC CO VC GV CPB JB Red
13 31 9 14 21 119 475
45 111 15 29 168 231 63
57 142 24 42 188 350 538
3.5 3.6 1.7 2.1 8.1 1.9 7.6
0.8^ 1.9 0.3 0.6 2.5 4.7 7.2
1.9' 4.7 0.7 1.3 6.9 10.3 19.6
BV SS WB CV GC GS CPT
4 56 72 44 146 39 202
9 121 361 164 189 254 277
13 177 433 208 336 293 479
2.6 2.2 5.0 3.8 1.3 6.6 1.4
0.2 2.4 5.8 2.8 4.5 3.9 6.4
0.4 5.4 15.1 7.0 8.9 10.5 12.9
' KC=Killawarra Chardonnay; OC=Orlando Chardonnay; CO=Castillo de Olite; VC=Vina Cumbrero; GV=Gran Vendema; CPB=Cosme Palacio Blanco; JB=Jose Bezares; BV=Barossa Valley; SS=Seaview Shiraz; WB=Wolf Blass; CV=Campo Viejo; GC=Gran Colegiata; GS=Gran Sangredetoro; CPT=Cosme Palacio Tinto Pooled standard deviation between replicate extractions = ^17; ^"34; ^^38 ^ Based on the estimated threshold of 75 ug 1'* determined for cis/trans 1:1 in 12% ethanol ' Based on thresholds of 110 and 25 |Lig 1 for trans and cis respectively (Chatonnet, 1991).
1700 compounds present at close to their threshold (Meilgaard, 1975a) so these values are less adapted to estimation of the importance of the oak lactone. The thresholds in wine were higher than those in 12% ethanol, probably because of effects due to the presence of so many other flavour volatiles (possibly with similar aromas to oak lactone) and interactions with non-volatile components of wine. Again, Boidron et al. (1988) reported thresholds in French wines which were much lower than the thresholds in the present study. Threshold values are very dependent on the medium and because of their inherent variability caution must be exercised in interpreting such values and relating them to concentrations of compounds in wine. In the course of the threshold tests, several judges commented that the wines to which oak lactones had been added at near threshold concentrations had an odour which was less harsh than the control wine. In agreement with other authors (Boidron et al, 1988; Chatonnet, 1991; Etievant, 1991), the odour of the mixture of isomers was described as coconut at higher concentrations and, at near threshold levels, woody and musty. 3.2. Determination of oak lactone in wines The estimated extraction yields were poor (<80%). However, since the calibration curves had high correlation coefficients (0.997 and 0.998 for trans and cis isomers respectively) and since wine samples and standards were extracted under identical conditions, the concentrations reported in Table 3 may be considered as reasonable estimates of the concentrations in the wines studied. The estimated concentrations of cis- and rran^-B-methyl- y -octalactone in the wines analysed are shown in Table 3. Since the two isomers of the synthetic lactone were not purified, the isomer eluting first in GLC on Carbowax BP 20 was designated trans and the second peak, cis, based on the assignments by Masuda and Nishimura (1981) and Chatonnet (1991). The opposite assignment has been assumed by several other authors (Kepner et al, 1972; Otsuka et al, 1974; Marsal and Sarre, 1987; Boidron et al, 1988; Maga, 1989). This has resulted in conflicting reports of which isomer is more potent (Otsuka et al, 1974; Chatonnet, 1991). The pooled standard deviations of triplicate extracts were estimated as 17 and 34 \x.g 1"^ for trans and cis lactones respectively. The concentration of the cis lactone was greater than that of the trans isomer in all of the wines studied. The total concentration of oak lactones ranged from 24 to 538 |ig 1"^ in white wines and the red wines contained from 13 to 479 \ig \'^. Among the white wines, two Riojas contained significantly higher levels of both oak lactones: Jose Bezares (p<0.001) and Cosme Palacio Blanco (p<0.005). Jose Bezares was matured for 12 months in oak casks while the maturation period in Limousin oak for Cosme Palacio Blanco is not known. It is interesting that the ratio of cis:trans in Jose Bezares and Gran Vendema wines was much higher than in all other wines. The ratio of cis:trans in wines depends on the history and treatment of the barrel but a conversion of trans to the apparently more stable and more potent cis form has been observed during subsequent bottle maturation (Chatonnet, 1991). The two AustraUan Chardonnays contained 57 and 142 |Lig 1'^ of total oak lactones with cis:trans ratios of about 3.5. Simpson and Miller (1984) reported levels of only 27 to 81 |Lig 1'^ {cis:trans, 0.8 to 2) for six vintages of Chardonnay aged in new French oak for 2-3 months. The higher concentrations found in Orlando Chardonnay in the present study could reflect its maturation in American as well as French oak since American oak (predominantly Quercus alba) is reported to contain more oak lactone than French Limousin oak (Guymon and Crowell, 1972; Otsuka, 1974). However, more data are required to confirm the higher concentrations in this Chardonnay. In the red wines studied, the total levels of oak lactones were highest in the Cosme Palacio Tinto Rioja (p<0.05) and Wolf Blass Shiraz/Cabernet Sauvignon. Compared with levels in the Gran Sangredetoro and Gran Colegiata Riojas, the high total oak lactone concentration in Wolf Blass was not significantly different but the level of cis lactone was higher (p<0.025). The other two Australian Shiraz Cabernets contained the lowest concentrations. The maturation of the Rioja reserva, Campo Viejo was labelled as more than 2 years in oak. It
1701 was therefore surprising that this wine contained less oak lactone than the other Spanish red wines. The higher concentration (p<0.05) in Cosme Palacio Tinto compared with the white wine from the same winery (Cosme Palacio Blanco) could reflect the maturation of the red in Quercus sessilis which generally contains more oak lactone than the Limousin oak (Quercus pedunculata) used for maturation of the white, as observed in French wines by Boidron^f ^/. (1988). Inferences about the effect of different types of cask, length of aging etc. on levels of oak lactones in wines cannot be made from the limited information available on the vinification and maturation of the wines investigated. 3.3. Estimation of the contribution of oak lactone to wine aroma The contribution of oak lactone to the aroma of the wines analysed is expressed in Table 3 as an "Odour Unit" (Keith and Powers, 1968; Meilgaard, 1975a) i.e. the concentration present in the wine divided by its odour threshold value. This estimate does not take account of synergistic and/or antagonistic effects resulting from interactions which might occur between volatile and non-volatile compounds of the wine. However, the odour unit (OU) does indicate the potential contribution to the overall aroma. Although the ratio of cis:trans isomers varied from 1.3 to 8.1 in the wines, the threshold of 75 |ag 1 in 12% ethanol for a racemic mixture was applied. Chatonnet (1991) found that the threshold for the cis lactone was only about 5 (ig 1'^ less than that for a 1:1 mixture. Thus, the estimated threshold for synthetic oak lactone should only slightly underestimate the sensory importance of this compound in the wines. The concentrations of oak lactones in the base wines used for the threshold tests were below odour threshold levels (0U<1). Oak lactone, and especially the cis isomer, would appear to make a considerable contribution to the aroma of all the other red wines and all the other white wines (estimated OU > 5 for several samples) except for Killawarra Chardonnay and the Viiia Cumbrero Rioja, where levels were close to threshold. Moreover, in two white Riojas, Jose Bezares and Cosme Palacio Blanco, concentrations exceed the estimated difference threshold in white wine (241 |ag 1'^). To estimate the relative contribution of the oak lactones to the overall aromas of these wines, the levels and thresholds of other woodderived aroma components such as eugenol and vanillin should also be determined. 4. CONCLUSIONS B-Methyl- y -octalactone was present at near or above threshold level in all of the wines analysed and the woody aroma of this molecule probably contributes significantly to the overall aroma of some of the white Rioja and certain red wines. The Australian wines could not be distinguished from the Spanish wines studied on the basis of the concentrations of oak lactone. 5. REFERENCES Boidron, J.N., Chatonnet, P. and Pons, M. (1988). Effect of wood on aroma compounds of wine, Connaiss. Vigne Vin, 22(4), 275-294. Chatonnet, P. (1991). Incidences du bois de chene sur la composition chimique et les qualites organoleptiques des vins. Thesis for the Diplome d'etudes et de recherches de I'Universite de Bordeaux, U.F.R. Institut d'Oenologie. Etievant, P.X. (1991). Wine, Food Science and Technology, 44, 483-546. Giinther, C. and Mosandl, A. (1986). Stereoisomeric flavor substances, XII- 3-methyl-4octanolide - "Quercus lactone, Whisky lactone" - structure and properties of the stereoisomers, Liebigs Ann. Chem., 2112-2122. Gyumon, J.F. and Crowell, E.A. (1972). GC-separated brandy components derived from
1702 French and American oaks, Amer. J. Enol. Vitic, 23, 114-120. Keith, E.S. and Powers, JJ. (1968). Determination of flavor threshold levels and subthreshold, additive, and concentration effects, J. Food Science, 33, 213-218. Kepner, R.E., Webb, A.D. and MuUer, C.J. (1972). Identification of 4-hydroxy-3methyloctanoic acid gamma-lactone (5-butyl-4-methyldihydro-2-(3H)-furanone) as a volatile component of oak-wood-aged wines of Vitis vinifera var. Cabernet Sauvignon, Amer. J. Enol. Vitic, 23, 103-105. Maga, J.A. (1989). Formation and extraction of cis- and trans-Q-mQthy\- y -octalactone from Quercus alba. In: Distilled Beverage Flavour (edited by J.R. Piggott and A. Paterson). Pp. 171-176. Chichester: Ellis Horwo9d. Marsal, F. and Sarre, Ch. (1987). Etude par chromatographic en phase gazeuse de substances volatiles issues du bois de chene, Connaiss. Vigne Vin, 21 (1), 71-80. Masuda, M. and Nishimura, K. (1971). Branched nonalactones from some Quercus species, Phytochemistry, 10, 1401-1402. Masuda, M. and Nishimura, K. (1981). Absolute configurations of Quercus lactones (3S, 4R)and (3S, 4S) -3-methyl-4-octanolide, from oak wood and chiroptical properties of monocyclic Y -lactones. Chemistry Letters, 1333-1336. Meilgaard, M.C. (1975a). Flavor chemistry of beer Part 1: Flavor interaction between principal volatiles, MBAA Technical Quarterly, 12(2), 107-117. Meilgaard, M.C. (1975b). Flavor chemistry of beer Part II: Flavor and threshold of 239 aroma volatiles, MBAA Technical Quarterly, 12(3), 151-168. Otsuka, K., Zenibayashi, Y., Itoh, M. and Totsuka, A. (1974). Presence and significance of two diastereomers of B-methyl- y -octalactone in aged distilled liquors, Agr. Biol. Chem., 38(3), 485-490. Schreier, P. (1979). Flavor composition of wines: a review, CRC Critical Reviews in Food Science and Nutrition, 12(1), 59-111. Simpson, R.F. and Miller, G.C. (1984). Aroma composition of Chardonnay wine, Vitis, 23, 143-158. ACKNOWLEDGEMENTS We gratefully acknowledge technical assistance and financial support from Chivas Brothers and the UK Agricultural and Food Research Council.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1703
POSSIBILITIES OF CHARACTERIZING WINE VARIETIES BY MEANS OF VOLATILE FLAVOR COMPOUNDS A. Rapp
Bundesanstalt fur Zuchtimgsforschung an Kulturpflanzen, Institut fur Rebenzuchtung, Geilweilerhof 76833 Siebeldingen, BRD Abstract The aroma of wine consists of 600 to 800 aroma compoimds from which especially those, typical for the variety, are already present in the grapes. There are significant varietal differences between the aromagrams ("fingerprint pattern"). Thus the amount of some flavour compounds ("key substances") shows typical dependence on the variety. Especially monoterpene compounds play an important role in the differentiation of wine varieties. The German white wines can be differentiated into three groups only by quantitative determination of 12 monoterpenes ("terpene profile"). These groups are: "Riesling type", "Muscat type" and "Silvaner-WeiUburgunder type". Such "terpene profiles" are also usefiil for the separation of real Riesling wines from others called Riesling (e.g. Welschriesling, Kap Riesling, Emerald Riesling) but not produced from grapes of the variety Riesling. Including fiirther components and by means of statistical methods (discriminant analysis) even the different varieties within the mentioned groups for instance the "Riesling" - group (e.g. Riesling, Kemer, Ehrenfelser, Bacchus, MiillerThurgau) can be separated from each other. An analytical characterization of the neutral ("Silvaner-Type") grape varieties Silvaner, Rulander (Pinot gris), WeiBburgunder (Pinot blanc) is also possible with about 20 compounds (e.g. monoterpenes, alcohols). Computing at the same time free and
1704 glycosidically bound aroma components (monoterpenes, alcohols, norisoprenes) in discriminant analysis the characterization of the neutral grape varieties can be considerably improved.
INTRODUCTION
Aroma compounds, as a result of their pronounced effect on our sensory organs, play a definitive role in the quality of our food and luxury products. As in the case with most food products, the aroma or "bouquet" of a wine is influenced by the action of several hundred different compounds on the sensory organs. The total content of aroma compounds in wine amounts to approximately 0.8 - 1.2 g/1, which is equivalent to about 1% of the ethanol concentration. The fusel oils, formed during fermentation, are responsible for about 50% of this content. The concentration of the remaining aroma compoimds range from 10""* to 10"^^ g/1 (1). The human sensory organs display somewhat sensitive and variable reactions to these amounts of aroma compounds. Thus threshold values differ considerably and could vary between 10"^ and lO'^^ g/1 (2,3,4). Early investigations into volatile constituents of wine date back to the year 1942, refering to the research of Hennig and Villforth. Bayer et al (5) instituted gas chromatography for the first time towards the end of the 1950's for the determination of wine aroma compoimds. In the following years, several research institutions used this method. Constantly improving physiochemical methods of analysis (gas chromatography), combined with the various possibilities of spectroscopic structure identification (gaschromatography-mass spectrometry), especially the introduction of highly efficient capillary columns and detectors (FID, NPD, FDP, SIM), led to an ever-improving insight into the complex constitution of wine aroma. In various synoptic reviews (1,6,7,8,9,20,38) the aroma compounds identified by many authors up to the present in grape must and wine have been listed. The requirements for an analytical characterization of wine varieties are: - an extensive and artifact-free enrichment of the aroma compounds
1705 - an effective separation by means of gas chromatography - and to find out those compounds which are qualified for a characterization of the varieties and the wine quality. With the aid of suitable extraction methods, for example liquid-liquid extraction with trichlorofluoromethane, the aroma sub- stances can be isolated and enriched fi-om wine as well asfiromgrapes without formation of artifacts (Rapp et al, 15). These aroma concentrations can be separated in many hundred single compounds by means of gas chromatography, using capillary columns.
CHARACTERISTIC AROMA COMPOUNDS OF DIFFERENT GRAPE VARIETIES
Significant differences exist amongst the individual grape varieties pertaining to the composition of the aroma compoimds. In the aromagrams ("finger print patterns") it is clear that the components ("key substances") vary greatly from one another in quantity (Fig. 1). Between the varieties Morio-Muskat (Fig. 1 below) and Riesling (Fig. 1 above), obvious differences in the quantity of several components can be recognized (Fig. 1, marked with arrows).
Riesling
Morio-Muskat
M
I tttt
fttt
Fig. 1: "Fingerprint patterns" of the cultivars Riesling and Morio-Muskat ( t key substances)
1706 It has emerged from numerous studies that the terpenoid compounds form the axis for the sensory expression of the wine bouquet which is typical of its variety and that they can therefore be used analytically for varietal characterization. Apart from the hitherto known compounds (terpene ethers, monoterpene alcohols) Rapp et al (10,11,12,13), Williams et al (14) and Versini, Rapp et al (44,45,46) identified numerous new monoterpene compounds, in particular monoterpene diols in grape must and wine. At present about 70 monoterpene compounds are known. The most prominent terpene alcohols, generally occurring in muscat and related grape aromas, are linalool, geraniol, nerol, citronellol, 3,6-dimethyl-l,5-octadien-l,7-diol and a-terpineol. Terpene compounds may also contribute to the aroma of other non-muscat cultivars.
INFLUENCE BY VARIOUS FACTORS ON THE AROMA COMPOUNDS OF WINE The amoimts of wine aroma components can be influenced by various factors, amongst others the environment (climate, soil), grape variety, the degree of ripeness, fermentation conditions (pH, temperature, yeast flora), wine production (oenological methods, treatment substances) and aging (bottle maturation) of the wine (Fig. 2).
PrimarV' o r G r a p e A r o m a
• SeCOndarV' G r a p e A r o m a
vanety. vmtaue, region, matunty
crushmc. pressmc, skm comaci enzMnatic reactions
Fermentation Bouquet yeast. pH. temperature ammo acids, suizar .
Maturation Bouquet
chemical reactions dunng maturation; pH. temperature, wood-compounds
Winearoma "Wine Bouquet"
Fig. 2: Origin of the Aroma compounds of Wine
1707 The characteristic varietal composition of the monoterpene compounds in the various grape varieties (monoterpene patterns) is only marginally influenced by the growing area. The "fingerprint patterns" of Morio-Muskat samples of different growing areas in Germany show a strong similarity in prominent characteristic monoterpene components (16, 17,9). By comparing the same Riesling-clone from a cooler (Germany) and a warmer (South Africa) area the "monoterpene profiles" are very similar, but the intensities of the monoterpene compounds are lower in the warmer climate and consequently the sensorial taste is different, too (Marais, Rapp et al. 42). The fungus Botrytis cinerea is responsible for the rotting of grapes. Under special climatic conditions it can cause noble rot, which is a prerequisite for the production of botrytized wines having a distinct aroma. Boidron (18) observed decreases in monoterpenes of Botrytis cinerea-infected Muscat varieties. Shimizu et al (19) report that B. cinerea does not produce terpenoids in grapes without terpenes. Rapp and Mandery (21) found that the monoterpene alcohols are changed by Botrytis cinerea. The content of monoterpene alcohols decrease while the content of monoterpenediols increase significantly. In the case of increased skin contact, the content of caproic, caprylic, and caprinic acid, their ethyl esters, as well as the acetic acid esters of the higher alcohols (acetates) decreased (22). On the other hand, skin contact time did not significantly change the contents of monoterpene compounds, which are partly glycosidically bound in the grape berries (23,24,25,26). The release of the glycosidically bound terpenes by means of natural 6glucosidases in the berry, as can be observed in the case of a pH change in the must (to pH 5.0), does not occur in the mash. The essential part of the wine flavour is formed during the alcoholic fermentation. Apart from ethanol and glycerine, as well as diols and higher alcohols, numerous other wine constituents are formed by yeast metabolism (especially acids, esters, aldehydes, ketones and S-compounds). Ethyl esters of straight-chain fatty acids and acetates of higher alcohols are the dominating esters in wine and they are formed during the alcoholic fermentation. These compounds can contribute to the evaluation of optimal wine technology, but are, however, not suitable for a varietal characterization. Microorganisms are also able to synthesize terpene compounds (28), but the formation of terpenes by Saccharomyces cerevisiae has not yet been observed (9,27). The
1708 monoterpene compounds are typical primary aroma components and are therefore suitable for the varietal characterization of wines made from different grape varieties. During the maturation or aging of wine (bottle maturation), various processes influence the volatile substances and thereby also the bouquet of the wine. Rapp et al (30,31) noted that numerous changes take place in the content of single aroma substances during bottle maturation of wine and that can be essentially divided into four aspects: - changes in ester content decrease in acetates increase in mono- and dicarboxylic acid ethyl esters - formation of substances from carotene breakdown - formation of substances from carbohydrate breakdown - acid-catalyzed reactions of monoterpene compounds The fruity acetates are mostly synthesized enzymatically through Saccharomyces cerevisiae during the fermentation. Table 1 shows that the acetates decrease significantly during storage in bottles (30,31). This decrease is recognizable clearly in the comparison of the frozen variant with the cellar-stored variant (Table 1). The contents of the acetates of the alcohols, appearing in the young wine in higher concentrations, decrease and plateau at a level determined by the chemical equilibrium between alcohol, acetic acid and acetate. These equilibrium concentrations of the acetates concerned are achieved in about 6 years. The decrease of acetates accounts the loss of freshness and fiuitiness of wines during bottle aging.
Table 1: Changes in the content of acetates during aging (Riesling) 1976 - 1983 Vintage
1982 1980 1978 1975 1973 1964
-30°C
stored 1
i-amyl-acetate
107
120
58
8
5
10
243
27
38
42
25
4
2
5
27
3
1 2-phenylethylaceteate
"frozen" cellar-
The ethylester of the fatty acids andfiirthermonocarboxylic acids increase significantly during aging. The concentration increase linearly over several years (30,31).
1709 The content of various monoterpene components changes during bottle-storage or rather during the maturation of wine by means of acid-catalyzed reactions (30,31). In the case of several components (e.g. linalool, geraniol, hotrienol and isomers of linalool oxide), an obvious decrease in concentration can be ascertained during the course of aging. In addition to this, compounds are formed which are not present in young wines: amongst others the formation of cis- and trans-l,8-terpine (Table 2). These reactions, influenced by the storage temperature (Table 2), affect the varietal character of a wine during bottle-aging.
Table 2: Changes in monoterpene compounds during bottle aging (Riesling)
Vintage linalool cis-l,8-terpin trans(f)-linalooloxide cis(f)-linalooloxide [_a-terpineol
1982 1980 1974 1964 17 1.5 0.5
2 1.8 0.8 3
1 1 12 4
6 14 6 8
1976 - 1983 n cellar "frozen" stored -30°C 19 3 2 9 2 0.9 3 17 11
The biogenesis of the isomers of vitispirane, l,l,6-trimethyl-l,2-dihydronaphthalene (TDN), damascenone and dihydroactinidiolide is generally due to carotene metabolism during the aging of wine. The formation of TDN is clearly correlated to the organoleptic change in wine during aging and is responsible for a pronounced kerosene or petrol note (30,31,37,42,43). This aroma may become detrimetal to wine quality when present at too high intensities and is generally more prominent in wines (especially Riesling) from hot wine-producing regions (e.g. South Africa, Australia) than in wines from cooler European countries.
1710 ANALYTICAL CHARACTERIZATION OF WINE VARIETIES
Utilizing only 12 monoterpene compounds (e.g. linalool, trans-linalooloxide (f), cislinalooloxide (p), nerol, geraniol, 3,7-dimethyl-octa-l,5-dien-3,7-diol), Rapp et al (33, 13,27,21,32,9, 1,38) succeded in classifying the grape varieties into various aroma types (Fig. 3). In the terpene patterns ("terpene profiles"), clear differences exist between the grape varieties with a muscat-related aroma ("Muscat-type", Fig. 3 above: e.g. Muskateller, Morio-Muskat, Schonburger, Wiirzer, Ga-47-42), and varieties with
!1 9 7 3 3 1
Muskateller
19
7 5 3 1
Huxel
"19 7 5 3 1
•: 9 7 5 3 :
Morto-MusKat
Siegerreoe
1 I 9 7 S 3 1
Kerner
1 9 7 5 3 1
iMuUer-Thurgau
1 9
7 5 3 1
Schonburger
1 9 7 5 3 1
Riesling
1 9 - 5
Wijrzer
1 9 7 5 3 1
Scheureoe
Fig. 3 : "Terpene profiles" of Muscat type (A) and Riesling type (B) varieties. 1 = trans-linalool oxide(f) 2 = cis-Iinalool oxide(f) 3 = linalool 4 = hotrienol 5 = trans-linalool oxide(p) 6 = cis-linalool oxide(p) 7 = 3,7-dimethylocta-l,5-diene-3,7-diol 8 = nerol oxide 9 = citronellol 10 = nerol 11= geraniol 12 = (E)-geranoic acid
1711 a fruity Riesling-related aroma ("Riesling-type"; Fig. 3 below e.g. Riesling, xMiillerThurgau, Kemer, Scheurebe, Orion, Phonix) and the grape varieties with a neutral bouquet ("Silvaner" or "Pinot blanc-type", not shown in figure). An accurate classification of wines into distinct sensory detectable wine types can therefore be effected analytically, only by the content of 12 components (monoterpene compounds). From these results, Rapp et al (17,34,38) in further research were able to attain a pronounced analytical differentation between wines made from the grape variety Riesling and wines just bearing the name Riesling but not made from this variety. All analysed wines from the grape variety Riesling (WeiBer Riesling) possess a similar terpene pattern ("terpene profile") which is displayed by the monoterpene content with normal degrees of variation (caused by varying maturation and aging), but in a composition which is typical of this variety. With the aid of these terpene profiles, a significant analytical differentation could be ascertained between Riesling and Welsch Riesling from different growing regions (Austria, Italy and Yugoslavia) (Fig. 4). In all the Riesling wines, the selected monoterpene compounds (e.g. linalool, translinalooloxide (f), a-terpineol) were present in a 10 to 50-fold higher concentration than in the wines made from Welsch Riesling. Using an example of South Africa wines, it was also demonstrated that a distinct differentation between various Riesling wines is possible using the terpene profiles typical of the variety: e.g. WeiBer Riesling (WR) and Cape Riesling (also sometimes classified as Riesling or Paarl Riesling (R)) (Fig. 5). The terpene profiles of Cape Riesling wine (R), made from the variety with the synonym Riesling vert or Cruchen blanc, is very similar to that of wines made from the grape variety Chenin blanc (St) (17). Based on the gaschromatographic investigations of volatile aroma compounds of 86 **Riesiing type*' wines Rapp et al (51) searched for a way to differ between Riesling and several new varieties which are descended from Riesling (Miiller-Thurgau, Scheurebe, Bacchus, Ehrenfelser, Kemer). Comparing the aromagrams ("finger print patterns") of the examined varieties significant quantitative differences in the wine composition were found. Therefore apart from monoterpene compounds additional components like lactones, esters, acetamides, volatile phenols, alcohols, and sulfur compounds were determined. At first 76 compounds of the aromagrams were identified and their relative amounts were determined. The differences between the varieties were only in the quantitative distribution of the selected aroma compounds.
1712
en c
en c
C7> C
O
OJ
X
cc
Q:
0
-
en c
1 1
1
L.
1
CD
£ u
1 1
•O
a;
1
H
X
1
111,1
.1I1.1.1
C71 C \n
c
iy
OJ
ex
1 ..l.i.l.l.
III!
1231567891011
lljjjl
NORD-JUGOSLAWIEN
NORD-ITALIEN
oSTERREICH
TJ
1 = trans-Linox (f)
5 = Hotrienol
10 = Terpendiol I
2 = cis-Linox (f)
6 = '^-Terpineol
11 = Hydroxyl inalool
3 = Nero 1 ox id
8 = trans-Linox (p)
Fig. 4: "Terpene profiles" of the varieties Riesling and Welschriesling
rel. piak height l-fpans-Linox(f) 2-cis-Linox(f) 3-Neroloxid 4-Linalool 5-Hotrienol 6-a-Terpineoi 7-frans-Linox(p| 8-cis-Linox(p) 9-Terpendiol-I 10-Hydroxylinalool
1234S«7I*10
W.R.
(SA)
123*iS*7l«K
R
ISA)
1 234S
St
47I910
ISA)
Fig. 5: "Terpene profiles" of the cultivars Riesling (W.R.), Paarl Riesling (R) and Chenin blanc
1713 The highest level of hexanol was established for Muller-Thurgau. Scheurebe and Bacchus achieved the highest content of trans-p-linalool oxide. The Ehrenfelser wines contained the highest quantities of linalool of all investigated varieties. Statistical computer programs are very useful for characterizing grape varieties. The first step to recognize the significance of a variable is to plot its amount against the group membership. The correlation coefficient describes the relationship between two variables in correlation analysis. Some analytical data are listed in table 3. The different significance of the single monoterpene compounds for the characterization of the
Table 3: Correlation coefficient of monoterpene compounds for the characterization of the grape varieties
1 Nr.
Verbindung
Korrelationskoefifizient
Trennung
V30
Linalool
0.94 0.91 0.70
Riesling - Ehrenfelser Scheurebe - Ehrenfelser Riesling - Muller-Thurgau
V41
cis-p-Linalooloxid
0.61 0.90
Muller-Thurgau - Scheurebe Muller-Thurgau - Ehrenfelser |
varieties investigated is clearly shown by these results. For the separations of Riesling - Ehrenfelser (correlation coefficient 0.94) and Scheurebe - Ehrenfelser (correlation coefficient 0.91) linalool achieved an essential higher significance than for the separation of Riesling - Miiller-Thurgau (correlation coefficient 0.70). Among all examined monoterpene compounds linalool (V 30), trans-p-linalool oxide (V 39) and 3,7-dimethyl-l,5-octadien-7-ethoxy-3-ol (V 71) are the most significant variables for the separation of the Riesling related new varieties MixUer-Thurgau, Bacchus, Ehrenfelser and Scheurebe (51). For the reduction of the great number of compounds which are first selected for the differentiation of grape varieties, without loss of informations, the regression analysis with the method of backward elimination is choosen. From the original model with numerous variables the variable with least information (F-value) is removed stepwise.
1714 This process of extracting variable is repeated until all variables are removed. After each step the new F-values and R^ (multiple correlation coefficient or multiple coefficient of determination) are calculated. R2 lies clearly between 0 and 1. Values of R^ close to 1 (or 100%) indicate a high probability. Therefore R^ is a good help for the decision how much and which variables will be taken into linear combinations for separating the grape varieties. If there is no substantial increase in R2 from one step to the other then there is no significant influence in the goodness of the separation by adding another variable into linear combination. The regression analysis with the selection method of backward elimination is necessary for the differentiation of the varieties Riesling, Miiller-Thurgau, Kemer, Scheurebe, Bacchus and Ehrenfelser at the same time. This method makes it possible to find
Table 4: Differentiation of grape varieties within the regression analysis (R2 = degree of accuracy) Trennung Riesling - Ehrenfelser Scheurebe - Ehrenfelser Kemer - Miiller-Thurgau Kemer - Bacchus Muller-Thurgau - Bacchus I Bacchus - Ehrenfelser
Trennmodell
R2
V30, V24, V3, V41, V37 V30, V39, V71, V l l VI1, V14, V53, V62, VIO, V48 V71,V14,V11, V51, V48 V39, V51, V10,V14,V48 V30, V39, V54, V71
0.92 0.96 0.94 0.94 0.95 0.95
the right variables and to derive the separating linear combinations. The necessary variables for the separation of every two grape varieties are represented in table 4 in connection with R2 (51). These results show that only 4 to 6 variables are needed to achieve a high significant separation (R^ > 0.9). By means of stepwise discriminant analysis it was possible to ascertain 15 variables for the significant differentiation between Riesling and the new varieties descended from Riesling. The data of the 15 components were taken to calculate three canonical functions (CAN 1, CAN 2, CAN 3). The plot of these functions and the group-centers of the single grape varieties for the separation of Riesling, Muller-Thurgau, Kemer, Scheurebe, Bacchus and Ehrenfelser are shown in Fig. 6 (51). In spite of the great range of the investigated samples (e.g. different growing regions, vintages, not the
1715 same degree of ripeness, various wine technologies) an unequivocal characterization of these grape varieties is possible. In agreement with sensorial analysis in ana-
CflN1
9. 3
1 . 90
- ^ . 53 - 5. 39 2. 20 -10. 96 5. 90
0. 99 2. 69
_0, 52 CflN2
-3. 73
^. 17
o=Riesiing JOf=Ehrenfelser o=Kerner
0=Mueller—Thurgau <^~Scheurebe 0:=Bacchus
Fig. 6: Discriminant analysis of different grape varieties (Riesling and new varieties descended ifrom Riesling) based on 15 compounds lytical characterization the grape varieties Ehrenfelser, Scheurebe and Bacchus can be separated very clearly from Riesling while also in analytical differentiation the two varieties Miiller-Thurgau and Kemer show a great similarity to Riesling. In further investigations the differentiation within the wines of the neutral grape varieties of the "Silvaner or WeiBburgunder type" was attempted (Rapp et al., 52). The discriminant analysis of 92 examined wines is possbile with 23 variables ascertained (Fig. 7). The highly significant components (e.g. 10 monoterpene compounds and
1716 3 C^-alcohols) are found by the selcetion method of backward elimination. The four investigated varieties (Riesling, Silvaner, Rulander, WeiBburgunder) can be separated distinctly, however the two related Pinot-varieties (WeiBburgunder, Rulander) are very closely situated one by the other. This results agree with the sensory evaluation (52).
Fig. 7: Discriminant analysis of different grape varieties (Riesling and the neutral varieties WeiBburgunder, Rulander, Silvaner) based on 23 compounds
In addition to free volatile aroma compounds various components (e.g. monoterpenes, C6-alcohols, aromatic alcohols, norisoprenoids) are glycosidically bound in the grape berries. These glycosidic derivatives can be liberated enzymatically by B-glucosidases and then enrich the flavor of wine (35,36). Such substances could be of interest for the characterization of neutral non-aromatic varieties ("Silvaner-type") because these varieties have only low concentration of free monoterpenes. In Fig. 8 are the concentrations (relative peakheight) of some liberated monoterpens of the examined varieties shown. The linalool oxides of the neutral varieties (Silvaner,
1717 Rulander, WeiBburgunder) show nearly the same distribution but in comparison to Riesling they differ significantly. Comparing the liberated amounts of nerol, a-terpi-
WeiObureunder
Rulander
Silvaner
• trans< fVLinaiooioxia acis(0-l.inalooioxid
WeiBburgunder
Rulander
INeroi
aa-Teroincoi
Riesiinu Scisioi-Linalooioxia
DTercenaiol 1 •Geraniol
Fig. 8: Relative concentrations of some liberated monoterpene compounds (Vintage 1990)
neol, terpendiol-1 and geraniol it can be recognized that substantial differences exist between both the neutral varieties and Riesling. The highest level of terpendiol-1 it liberated from Riesling while Silvaner and Rulander obtain the highest concentrations of bound geraniol (Fig. 8). Distinct differences in the amoimts of the liberated norisoprenoids are found. Rulander achieves the highest level of 3-oxo-a-ionol. Riesling, Silvaner and WeiBburgunder follow with decreasing concentrations. Looking at the bound aroma substances the close relationship between the neutral varieties (Silvaner, Rulander, WeiBburgunder) is clearly shown in spite of the quantitative differences. The non-aromatic varieties contrast distinctly with Riesling in their aroma composition.
1718 Using 18 glycosidically bound aroma substances it could be shown that these components can also contribute significantly to the characterization of grape varieties where monoterpenes and norisoprenoids play an important role. Computing at the same time free and glycosidically bound aroma components in discriminant analysis the characterization of neutral grape varieties (Silvaner, Rulander, WeiBburgimder) can be considerable improved. Further studies should clarify if the methods of analytical differentiation of grape varieties are suited for an early diagnosis for the selection of fungus resistant new varieties. Based on gaschromatographic examinations of numerous wines of Vitis vinifera grape varieties (group E) and of fungus resistant new varieties originating from Vitis vinifera x Villard blanc (group V) and Vitis vinifera x Vi 5861 (Oberlin 595) (group C) it was possible to separate the three groups significantly by selecting 17 aroma
^
Ex A ait (Vi 5861)
^^
EtA ncu iVillara)
3
V. vintfara
1.26
LflE
- 3 . 19 - 0 . I'f
Fig. 9: Discriminant analysis of different groups of wine: E = V. vinifera; V = fungus resistant grape varieties from V. vinifera x Villard blanc; C = fungus resistant grape varieties from V. vinifera x Vi 5861 (Oberlin 595)
1719 compounds (41). The separation of the groups show that fungus resistant new varieties descended from Vitis vinifera x Villard blanc (group V) are very similar to the investigated Vitis vinifera varieties and new varieties descended from Vitis vinifera x Vitis vinifera. However, group C (Vitis vinifera x Vi 5861) is distinctly separated from the Vitis vinifera varieties (Fig. 9). These results are supported by sensoric evaluations. The fungus resistant varieties of group V (e.g. Orion, Phoenix, Sirius, Ga-54-14) are in their sensoric taste similar to the Vitis vinifera varieties while the resistant varieties of group C (e.g. Castor, Pollux, A-100-3) show a significant distance to the wished taste of Vitis vinifera varieties.
References 1. Rapp, A., Mandery, H. (1986) New progress in vine and wine research: wine aroma. Experentia 42, 857-966 2. Boeckh, J. (1972) Die chemischen Sinne - Geruch und Geschmack. In: Gauer, O.H., Cramer, K., June (eds), Handbuch Physiologic des Menschen, Urban und Schwarzenberg, Munchen 3. Guadagni, D.G, Buttery, R.G, Okano, S. (1963) Odour threshold of some organic compounds associated with food flavours. J. Sci. Food Agric. 14, 761-765 4. Demole, E., Engist, P., OhlofF, G. (1982) l-p-Menthen-8-thiol: powerful flavour impact constituent of grape fruit juice (Citrus paradisi) Helv. Chim. Acta. 65, 1785-1794 5. Bayer, E. (1958) Anwendung chromatographischer Methoden zur Qualitatsbeurteilung von Weinen und Mosten. Vitis 1,298-312 6. Drawert, F., Rapp, A. (1968) Gaschromatographische Untersuchung pflanzlicher Aromen. 1. Anreicherung, Trennung und Identifizierung von fliichtigen AromastofFen in Traubenmosten und Weinen. Chromatographia 1, 446-457 7. Schreier, P. (1979) Flavor compositions of wines: a review. CRC Crit. Rev. Food Sci. Nutr. 12, 59-111 8. van Straten, S., Maarse, H. (1983) Volatile compounds in Food qualitative data. Div. for nutrition and food research TNO 9. Rapp, A (1988) Wine Aroma Substances from Gas Chromatographic Analysis. In: Linskens, H.F. and Jackson, J.F.: Wine Analysis, Springer Verlag, Berlin 29-66 10. Rapp, A, Knipser, W., Engel, L. (1980) Identifizierung von 3,7-Dimethyl-okta-l,7dien-3,6-diol in Trauben- und Weinaroma von Muskatsorten. Vitis 19, 226-229
1720 11. Rapp, A., Knipser, W. (1979) 3,7-Dimethyl-okta-l,5-dien- ,7-diol. Eine neue terpenoide Verbindung des Trauben- und Weinaromas. Vitis 18, 229-233 12. Rapp, A., Mandery, H., Ullemeyer, H. (1984) Neue Monoterpendiole in Traubenmosten und Weinen und ihre Bedeutung fiir die Genese cyciischer Monoterpenather. Vitis 23, 84-92 13. Rapp, A., Mandery, H., Giintert, M. (1984) Terpene compounds in wine. In: Nykanen, L., Lehtonen, P. (eds) Flavour research of alcoholic beverages. Instrumental and sensory analysis. Kauppakirjapino Oy Helsinki 255-274 14. Williams, P.J., Strauss, C.R., Wilson, B. (1980) Hydroxylated linalool derivatives as precursors of volatile monoterpenes of muscat grapes. J. Agric. Food Chem. 28, 766-771 15. Rapp, A., Hastrich, H., Engel, L. (1976) Gaschromatographische Untersuchungen uber die Aromastoffe von Weinbeeren. I. Anreicherung und kapillarchromatographische Auftrennung. Vitis 15, 29-36 16. Rapp, A., Hastrich, H. (1978) Gaschromatographische Untersuchungen liber die AromastofFzusanmiensetzung der Rebsorte Rjesling. Vitis 17, 288-298 17. Rapp, A., Guntert, M., Heimann, W. (1985) Beitrag zur Sortencharakterisierung der Rebsorte WeiBer Riesling. I. Untersuchung der Aromastofifzusammensetzung von auslandischen WeiBweinen, die als Sortenbezeichnung des Begriff Riesling tragen. Z. Lebensm. Unters. Forsch. 181, 357-361 18. Boidron, J.N. (1978) Relation entre les substances terpeniques et la qualite du raisin (Role du Botrytis cinerea). Ann. Technol. Agric. 27, 141-145 19. Shimizu, J., Nokora, M., Watanabe, M. (1982) Transformation of terpenoids in grape and must by Botrytis cinerea. Agric. Biol. Chem. 46, 1339-1344 20. Rapp, A. (1992) Aromastoffe des Weines Chemie in unserer Zeit 26, 273-284 21. Rapp, A., Mandery, H., Niebergall, H. (1986) Neue Monoterpendiole in Traubenmost und Wein sowie Kulturen von Botrytis cinerea. Vitis 25, 79-84 22. Rapp, A., Guntert, M., Rieth, W. (1985) EinfluB der Maischestandzeit auf die Aromastoffzusammensetzung des Traubenmostes und Weines. Dt. Lebensmittel-Rundschau 81, 69-72 23. Bayonove, C , Gunata, Y.Z., Cordonnier, R. (1984) Mise en evidence de I'intervention des enzymes dans le developpement de I'arome du jus de muscat avant fermentation: la production des terpenols. Bull. OIV 57, 741-758
1721 24. Gunata, Y.Z. (1984) Recherches sur la fraction liee de nature glycosidique de Tarome du raisin. These Univ. des Sciences et Techniques du Languedoc/France 25. Williams, P.J., Strauss, C.R., Wilson, B., Massy-Westropp, P.A. (1982) Novel monoterpene disaccharide glycosides of Vitis vinifera grapes and wines. Phytochemistry 21, 2013-2020 26. Williams, P.J., Strauss, C.R., Wilson, B., Massy-Westropp, P.A. (1982) Use of C18-reversed-phase liquid chromatography for the isolation of monoterpene glycosides and norisoprenoid precursors from grape and wines. J. Chromatogr. 235, 471-480 27. Rapp, A., Mandery, H., Ullemeyer, H. (1984) Neuere Ergebnisse uber die Aromastoffe des Weines. Oenolog. Symposium Rom, 157-196 28. Hock, R., Benda, J., Schreier, P. (1984) Formation of terpenes by yeasts during alcoholic fermentation. Z. Lebensm. Unters. Forsch. 179, 450-452 29. Rapp, A., Marais, J. (1993) The Shelf Life of Wine: Changes in Aroma Substances during Storage and Ageing of White Wines In: G. Charalambous: "Shelf Life Studies of Foods and Beverages", Elsevier, Amsterdam-London-New York, pp. 891-921 30. Rapp, A., Guntert, M., Ullemeyer, H. (1985) Uber Veranderung der Aromastoffe wahrend der Flaschenlagerung von WeiBweinen der Rebsorte Riesling. Z. Lebensm. Unters. Forsch. 180, 109-116 31. Rapp, A., Guntert, M. (1986) Changes in aroma substances during the storage of white wines in bottles. In: Charalalambous, G. (ed) The shelf life of foods and beverages. Proceedings of Flavour Conference, Rhodos. Elsevier Science BV, Amsterdam 141-167 32. Rapp, A., Knipser, W., Hastrich, H., Engel. L. (1982) Possibilities of characterizing wine quality and wine varieties by means of capillary chromatography. In: Webb, A.D. (ed) Symposium Proceedings Univ. of California, Grape and Wine Centennial (1980) Davis, 304-316 33. Rapp, A., Knipser, W., Engel, L., Hastrich, H. (1983) Capillary-chromatographic Investigations on various grape varieties. In: Charalambous, G. Inglett, G. (eds) Instrumental analysis of foods. Academic Press, New York, 435-454 34. Rapp, A., Guntert, M. (1985) Beitrag zur Sortencharakterisierung der Rebsorte WeiBer Riesling. II. Untersuchung der Aromastoffzusammensetzung in inlandischen WeiBweinen der Rebsorten WeiBer Riesling, Muller-Thurgau und Silvaner. Vitis 24, 139-150 35. GroBmann, M., Rapp, A. (1988) Steigergung des sortentypischen Weinbuketts nach Enzymbehandlung Dt. Lebensmittel-Rundschau 84, 35-37
1722 36. GroBmann, M., Suckrau, I., Rapp, A. (1990) EinfluB kellertechnischer MaBnahmen auf das Sortenbukett Weinwirtschaft-Technik 8, 13-16 37. Marais, J., van, Wyk, C.J., Rapp, A. (1992) Effect of Storage Time, Temperature and Region on the Levels of l,l,6-Trimethyi-l,2-dihydro-naphthaiene and other Volatiles and on Quality of WeiBer Riesling Wines. S. Afr. J. Enol. Vitic. 13, 33-44 38. Rapp, A. (1990) Natural flavours of wine: correlation between instrumental analysis and sensory perception. J. Anal. Chem. 337, 777-785 41. Rapp, A., Ringlage, S. (1989) Vergleichende gaschromatographische Untersuchungen uber die Aromastoffzusammensetzung der Weine verschiedener Vitis-vinifera- und pilzresistenter Rebsorten Vitis 28, 21-29 42. Marais, J., Versini, G., van Wyk, C.J Rapp, A. (1992) Effect of Region on Free and Bound Monoterpene and C^^-Norisoprenoid Concentrations in Weisser Riesling Wines S. Afr. J. Enol. Vitic. 13/2, 71-77 43. Marais, J., van Wyk, C.J., Rapp, A. (1992) Effect of Sunlight and Shade on Norisoprenoid Levels in Maturing WeiBer Riesling and Chenin blanc Grapes and WeiBer Riesling Wines. S. Afr. J. Enol. Vitic. 13, 23-32 44. Versini, G., Dalla Serra, A., Del^va, M., Scienza, A., Rapp, A. (1987) In: Schreier, P. ed. Bioflavour'87, Analysis Biochemistry Biotechnology. Proc. Int. Conf Wurzburg, Germany 1987,16145. Versini, G., Rapp, A., Dalla Serra, A. (1992) Considerations about the Presence of free and bound p-menth-1 enediols in grape Products In: Schreier, P. ed. Bioflavour'92 de Gruyter, Berlin 1992, 243-249 46. Versini, G., Rapp, A., Reniero, F., Mandery, H. (1991) Structural identification and presence of some p-menth-1-enediols in grape products. Vitis 30, 143-149 51. Rapp, A., Volkmann, C , Niebergall, H. (1993) Untersuchung fluchtiger Inhaltsstoffe des Weinaromas: Beitrag zur Sortencharakterisierung von Riesling und Neuziichtungen mit Riesling-Abstammung. Vitis 32, 171-178 52. Rapp, A., Suckrau, I,, Versini, G. (1993) Untersuchungen des Trauben- und Weinaromas. Beitrag zur Sortencharakterisierung neutraler Rebsorten (Silvaner, WeiBburgunder, Rulander) Z. Lebensm. Unters. Forsch. 197, 249-254
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1723
Classification of Italian wines on a regional scale by means of a multi-isotopic analysis A. Monetti, G. Versini and F. Reniero Istituto Agrario di S.Michele all'Adige, Via Mach 1, 38010 S.Michele all'Adige, Italy Abstract Stable isotope analysis in foods has been usefully employed to relate a product with its original matter by examining specific fractions or molecules as well as to find out some kinds of adulterations. For wines, using a wide databank of genuine samples as reference, interesting applications have emerged also in order to identify their geographical origin. In this respect, parameters referred to the sitespecific natural isotope fractionation of deuterium in ethanol determined by NMR spectroscopy, and EEC reference method to identify the botanic origin of ethanol itself - are not sufficient. In the present work on wines, the study has been extended for the parameters ^^C/^^C of ethanol and ^^0/*^0 of water, both determined by isotope ratio mass spectrometry. With this multi-isotopic analysis it has been possible to reduce in a significant way the uncertainty in the determination and validation of the exact origin among different wine-producing Italian regions. 1. INTRODUCTION Stable isotope analysis can be successfully used to control the authenticity of many food products, particularly to detect some adulterations, mostly the addition of foreign sugars, as widely reported in the literature [1,2]. At present, even more attention is paid to this kind of analysis in order to relate a product not only with its original matter - as, e.g., to discriminate among certain C3 plants or between C3 and C4 plants, and the relevant mixtures, through the complementary techniques of sitespecific natural isotopic fractionation (SNIF)-^H NMR and isotopic ratio mass spectrometry (IRMS) on carbon - but also with its geographic origin. In fact, recent studies confirm an interesting tendency of deuterium content, mostly in the methyl position of fermentative alcohol [3,4,5], as well as of '^C/^^C ratio of the alcohol molecule as a whole [6,7,8], but expecially of *^0/^^0 ratio of vegetal water [8,9,10,11], to variate in function of the latitude and other geoclimatic parameters. A recent investigation about this topic, on the basis of SNIF-NMR deuterium content of alcohol of genuine products referred to the six years' Italian data-bank including over 3000 samples of wines, confirmed the dependence of D/Hi on latitude, but not of D/Hu, and therefore the parameter R, derived from both of them did not add any further information. Such parameters were not different enough for each region to allow a univocal characterization when a statistical procedure was applied to validate a sample: except for some very far apart regions, the others are still very overlapped and confused [12]
1724 This research is aiming at improving this goal by considering also the data of 5^^C of alcohol and 5^^0 of wine water. 2. MATERIALS AND METHODS 445 samples of wines and relevant distillates were provided by area offices of the Central Control Service against Adulterations in Foods of the Italian Ministry of Agriculture. The samples belonged to the national databank available at our Institute and were collected in the 1992 vintage. They are referred to grape samples picked in every region at technological ripeness, which were crushed, fermented and distilled in standard conditions at least at 93° proof. Wine samples from the Lombardy region were not provided, so the corresponding 5^^0 values were missing. The sitespecific quantitative analysis of deuterium in ethanol was carried out with a AMX 400 Bruker NMR instrument after the EEC method [13] and with a line broadening of 0.5 Hz as previously detailed [12]. 5^^C content of alcohol and 5^^0 of wine water were measured with a SIRA II VG mass spectrometer after the Italian official method [14] and the literature references [15,16,17], respectively. In order to study the possibility of differentiating between the Italian regions, the data were analysed by means of multivariate analysis of variance (MANOVA) on the parameters D/Hj, D/Hn, 5'^C and 6^^0. The ratio R, as derived from the D/Hi and D/Hn values, was not considered. Multiple comparisons were used (Tukey test) to investigate the nature of the differences among regions. To maximize the discrimination, the linear discriminant analysis (LDA) was applied, using the same variables as in MANOVA. The generalized Mahalanobis distances were computed and their significance tested to quantify the dissimilarity between groups and to derive a classification rule based on the distances between the centroids of each region and the sample dispersion around them [18]. At a general level, the performance of the results has been evaluated examining the validity of the classification criteria on sample resubstitution taking into account the geographical proximities between regions. The analyses were performed using SAS 6.09 [19] on a computer DEC VAX 6000-410 AXP with o.s. OpenVMS 1.5. 3. RESULTS AND DISCUSSION Means and standard deviations of the parameters D/Hi, D/H„, 6''C and 6^^0 are presented in Table 1. In this case, it easy to confirm the tendency of D/Hj and 5'^0 to being correlated with the latitude [ 1,4,6,12]. In order to verify the existence of differences between regions, these were studied by means of MANOVA and the resuhs proved highly significant. The Tukey test was employed to analyse the behaviour of each variable for every region to detect which region could have been responsible for the significant differences. For the D/Hj variable it is confirmed the presence of a gradient North-South (Table 2; regions with the same letter are not different), with the lowest values in alpine regions, inland and at higher latitude. The D/Hn does not present any clear trend (Table 3). b''C (Table 4) and 6''0 (Table 5), with some exceptions, distinguish between northern and southern Italian regions. It is possible, for every variable, to identify different groups, but it is not possible to identify individual regions because they overlap.
1725 Table 1 Means (first row) and standard deviations (second row) of the isotopic parameters Region Abruzzo Apulia Calabria Campania Emilia-Romagna Friuli-Venezia Giulia Latium Liguria Marche Molise Piedmont Sardinia Sicily Tuscany Trentino-Alto Adige Umbria Valle d'Aosta Veneto average
D/H, ppm 101.33 0.88 103.89 1.27 104.04 0.91 102.90 0.89 100.39 1.13 101.41 1.03 102.89 1.13 102.26 0.70 102.04 1.34 102.59 0.37 100.01 1.23 103.01 1.40 104.50 1.39 101.65 1.42 99.43 0.93 101.43 1.22 100.03 0.72 101.28 1.38 101.97 1.88
D/H„ ppm 126.47 2.30 130.35 3.48 134.26 2.74 132.56 0.93 129.67 2.16 128.45 2.15 133.09 2.03 132.09 3.48 129.60 2.08 127.87 1.14 128.75 2.15 131.28 1.36 132.86 2.77 131.88 1.98 126.28 1.54 132.95 1.70 125.62 0.46 128.88 2.80 130.42 3.07
6^'C %o vsPDB -25.06 0.94 -24.64 0.37 -25.43 0.67 -25.68 1.10 -26.55 0.87 -25.65 0.59 -25.52 0.61 -26.80 1.03 -25.86 1.10 -25.47 0.90 -26.45 0.98 -24.87 1.07 -25.07 0.83 -26.17 1.21 -26.97 0.94 -26.23 0.93 -26.31 0.82 -25.88 0.89 -25.80 1.16
5'^0 %o vs SMOW
"~"
3lT
1.32 6.46 1.78 5.22 1.04 2.41 1.07 1.11 2.21 2.64 2.05 5.33 1.13 3.02 1.76 1.38 0.97 4.02 1.04 -0.39 2.84 5.14 1.60 6.12 0.95 3.82 1.63 -0.23 2.52 3.53 1.93 -2.71 0.71 1.76 2.32
105" 2.88
1726 Table 3 Tukey's test for D/H„
Table 2 Tukey's test for D/H,
E E E E E E E E E E
B B B B B B G G G G G G G G
D D D D D D D D D D
A A A A A A F F F F F F F
C C
c c c c
H H H H
Sicily Calabria Apulia Sardinia Campania Latium Molise Liguria Marches Tuscany Umbria Friuli-V.G. Abruzzo Veneto Emilia-R. Valle d'A. Piedmont Trentino-A.A.
E E E E E E E E
D D D D D D D
A A A A A A F F F F F F F F I I I
H H H H H H
D D D D D D D J J J J J J
A A A A A A A A G G G G G G G
C C C C C C C
F F F F F F F
Calabria Latium Umbria Sicily Campania Liguria Tuscany Sardinia Apulia Emilia-R. Marches Veneto Piedmont Friuli-V.G. Molise Abruzzo Trent.-A.A. Valle d'A.
Table 5 Tukey's test for 5''C
Table 4 Tukey's test for 6''0 B B B B B B G G G G G G G G
E E E E E E E
B B B B B B B B
C C C C C C C C H H H H H
Apulia Sicily Latium Calabria Sardinia Molise Tuscany Umbria Abruzzo Liguria Friuli-V.G. Campania Veneto Marches Emilia-R. Trentino-A.A. Piedmont Valled'A.
E E E E E E E E E E E E
B B B B B B B B B B B
D D D D D D D D D D D D
A A A A A A A A A A A
C C C C C C C C C C C C
F F F F F F F F F F F
Apulia Sardinia Abruzzo Sicily Calabria Molise Latium Friuli-V.G. Campania Marches Veneto Tuscany Umbria Valled'A. Piedmont Emilia-R. Liguria Trent.-A.A.
Before performing the linear discriminant analysis (LDA), the samples of Valle d'Aosta were merged with those of Piedmont and the same was done for the samples of Molise and Abruzzi. The LDA was used to maximize the differences between regions by computing new
1727 functions, which aimed at obtaining the best discrimination. The first 3 functions explain 98.2% of the total variability. The first function explains 72.6% of the total variability and is mainly loaded with D/Hi and 5^*0, their standardised coefficients being 0.93 and 0.94, respectively. The second one shows the coefficients 0.23,1.28, -0.43 and -0.74 for D/Hi, D/Hn, 5^^C and 5^*0, so it mainly depends on D/Hn and 8"C. The third is similar to the first function but with an opposite sign for D/Hi (-1.07) and 5^^0 (1.24). These results confirm the role of D/Hi in determining the geographical origin of wines, and highlight the importance of 6**0 in this connection. The generalised Mahalanobis distances between the centroids of each configuration have been calculated in order to obtain a classification rule for assigning an unknown sample to the population whose centroid is closer to the values of the sample. The overlap among regions could be appreciated in Figure 1, where at 90% confidence regions are drawn [18].
-2 0 2 First canonical variate
Figure 1. Centroids of the ItaUan regions (letters) and confidence regions of the most discriminated regions. In the figure the centroids of each region are identified by its initials. It is interesting to see that the first axis separates not only between northern (Trentino-Alto Adige and Piedmont) and southern (Calabria, Apulia, Sicily) regions, but also between Adriatic (underlined lowercase) and Tyrrhenian (italic lowercase) regions; Umbria region is inland and its centroid is intermediate. In Figures 2 and 3 the values of the centroids (x) for each region are presented to show the discriminating capability of the computed functions.
1728
FRIULIVENEZIA GIULIA
VALLE D'AOSTA + PIEDMONT
lit) CALABRIA
Figure 2. Mean value of the first canonical variate for the Italian regions.
TRENTINOALTO ADIGE ^ ^ ^ ^ H l FRIULI^ J l ^ ^ y VENEZIA GIULIA
VALLE D'AOSTA + PIEDMONT 1
x<-1
^^^HkvENETO ^^MM ^^IGURl^
-1<x<0
EMILIA-ROMAGNA
^ ^ ^ t t ^ t e . MARCHES Adriatic Sea
TUSCAN1
9 UMBRIA
SARDINIA
0<x<1 r^zH^wT^^^ x>1
- ^ ^ ^ f e ^ i ^ ^ . ABRUZZI + ^ ^ ^ ^ ^ ^ ^ ^ ^ MOLISE
>-ATIUM^^B|R|^^APULIA CAMPANIA ^ f K Tyrrhenian Sea
SICILY
^ ^ ' ^ ^ •.'-. ;^lTf\
5|fcALABRIA
Figure 3. Mean value of the second canonical variate for the Italian regions.
1729 Employing four isotopic parameters, instead of the SNIF-NMR parameters only, the discrimination among regions has been greatly improved but for certain regions it is still difficult to obtain a clear distinction. The error rates of the LDA are presented in table 6, where the misclassifications between quite far apart regions are bolded. The error rate when considering only this type of misclassifications is about 25%. This enlarged - and for some respect arbitrary - grouping of regions was adopted not so much for reducing the error rate but rather for keeping into consideration the geoclimatic continuum existing between bordering regions and needs surely further reviewing. Table 6 Cross validation summary of discriminant analysis (regions far apart each other are bolded italic) To region: Ab. Cal. Cam. ER
F- La. Li. ]Ma. Pi. Ap. Sa. Si. Tu. T-Um. Ve. VG AA
From region: Abruzzo Calabria Campania Emilia-R. Friuli-V.G. Latium Liguria Marches Piedmont Apuha Sardinia Sicily Tuscany Trentino-A.A. Umbria Veneto
11 0 0 / 3 0 0 1 0 2 0 0 0 0 0 3
0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0
0 0 5 0 0 0 0 2 0 2 0 0 0 0 0 0
"~o" 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0
4 0 0 0 5 0 0 0 1 0 0 0 1 1 0 2
"T""~o""T"~T ""o"~o"~o'~r 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 4 1 5 0 0 5 0 0 0 1 0 32 1 2 0 0 0 2 0 0 2 0 0 0 6 0 0 3
0 0 0 0 6 0 0 3 0 2 0 1 7 0 33 0 0 27 0 9 0 8 2 3 6 0 2 0 5 1
5 0 2 0 0 0 0 0 0 3 0 0 2 1 0 0 9 1 2 0 2 27 1 4 0 0 0 2 0 /
1 4 5 7 4 7 5 9 3 7 3 30 0 16 8
0 0 0 1 0 0 0 0 0 0 0 0 0 16 0 1
0 ~ 0 0 0 2 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 10 1 0 0 10 0 0 1
4. CONCLUSIONS This research can be considered a preliminary study to check the possibility of identifying the geographical origin of Italian wines by using other isotopic variables in addition to those from the SNIF-NMR ethanol analysis. Actually, this possibility was proved, being the single regions more effectively differentiated. In conlusion, if taking into account at the same tune the parameters D/H„ D/Hj,, 6^^C and 6**0 it becomes possible to discrimate at least among geographically very different regions, as e.g. northern and southern Italian regions. A fine discrimination among closer or bordering regions is still very problematic and for this it will surely be necessary to consider also the general climatic characteristics of each area and the weather conditions peculiar for each year.
1730 5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
J. Bricout in: H.-L. Schmidt, H. Forstel and K. Heinzinger (eds.), Stable Isotopes, Elsevier, Amsterdam (1982) 483. B.N. Smith and S. Epstein, Plant Physiol., 47 (1971) 380. G.J. Martin, B.L. Zhang, N. Naulet and M.L. Martin, J. Am. Chem. Soc., 108 (1986) 5116. G.J. Martin, C. Guillou, M.L. Martin, M.T. Cabanis, Y. Tep and J. Aemy, J. Agric. Food Chem., 36(1988)316. F. Reniero, A. Monetti, A. Scienza and M. Simoni, Boll. CIDEAO, 11 (1991) 49. M. Ramponi, Thesis in Chemistry, University of Padua, 1991. G. Versini in: E. Lemperle (ed.). Proceed. 10th Intern. Oenol. Symp., Intern. Ass. Winery Technol. Manag., Breisach (1993) 440. J. Dunbar in: H.-L. Schmidt, H. Forstel and K. Hemzinger (eds.), Stable Isotopes, Elsevier, Amsterdam (1982) 495. J. Bricout, J.Ch. Pontes and L. Merlivat, Conn. Vigne Vin, 2 (1974) 161. H. Forstel and H. Hiitzen, Weinwirtsch.-Technik, 3 (1984) 71. B. Holbach, H. Forstel, H. Otteneder and H. Hiitzen, Z. Lebensm. Unters.- Forsch., 198 (1994) 223. A. Monetti, F. Reniero and G. Versini, Z. Lebensm. Unters.-Forsch., in press. EEC, Gazzetta uficiale delle Comunita europee, 272 (1990) 64. Gazzetta ufficiale della Repubblica italiana, 95, Decreto 16 febbraio 1993. M. Cohn and H.C. Urey, J. Am. Chem. Soc, 60 (1938) 679. S. Epstein and T. Mayeda, Geochim. Cosmochim. Acta, 4 (1953) 213. Office mtemational de la vigne et du vin, FV N°919, 1955/220792. W.J. Krzanowsky, Principles of multivariate analysis, Claredon press, Oxford, 1988. SAS/STAT* User's guide. Version 6, SAS Institute Inc., Gary, NC, 1989.
Acknowledgments This research was developed in collaboration with the Central Control Service against adulterations in food of the Italian Ministry of Agriculture.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1731
Flavour development in whisky maturation J.R. Piggott, J.M. Conner and A. Paterson Department of Bioscience and Biotechnology, University of Strathclyde, 131 Albion Street, Glasgow Gl ISD, Scotland Abstract Whisky and other distilled beverages undergo major changes in flavour during maturation. These include direct flavour effects of materials extracted from the cask-wood, and of products of reactions within and between wood and distillate components. Wood-derived compounds also modify the headspace concentrations of volatile congeners. Dynamic light scattering indicated the presence of structures (agglomerates or micelles), with a mean diameter of 550 to 600 nm in unaged distillates, and 330 to 400 nm in matured distillates. The agglomerates were formed by medium- and long-chain ethyl esters and alcohols, which have limited solubilities in aqueous ethanol and are present at concentrations exceeding this limit in many malt whiskies. Agglomerates reduced the volatility of other congeners, with a proportion of these compounds being partitioned into the agglomerate. The reduction in agglomerate size during maturation, caused by the dissolution of wood components, was accompanied by changes in the solubilisation of distillate components which altered their partitioning between spirit and headspace. 1. INTRODUCTION Freshly distilled whiskies and brandies generally have unacceptable sensory characteristics and are traditionally matured in oak casks for several years to produce a premium product (Philp, 1989). Maturation reactions are complex but involve extraction of wood components, evaporation of low boiling point solutes from the distillate and interactions of wood and distillate components (Nishimura and Matsuyama, 1989). Dissolution of wood components is thought to be of prime importance. It is also known that the character of a matured Scotch whisky is closely related to contents of non-volatile compounds (Piggott et a/., 1992; Clyne et al., 1993) and it has been possible to predict sensory scores for mature characteristics from quantifications of non-volatile compounds (Piggott et al, 1993). Maturation also involves a reduction in the perception of undesirable, or immature characteristics. However, losses of ethanol and water through the cask wood increase the spirit concentrations of many volatile components during maturation (Reazin, 1983; Nishimura et al, 1983). Ethyl esters, particularly acetate, hexanoate, octanoate, decanoate and dodecanoate (Salo et al., 1972), make a major contribution to whisky odour. Both addition or depletion of such esters may have negative effects on aroma intensity (Jounela-Eriksson, 1981). In studies with a redistilled brandy it was observed that tannic acid and oak extract significantly reduced the activity of ethyl esters, with reductions in extraction reaching a maximum for 12 - 14 carbons (Piggott et al., 1992). Dilution of malt spirit for sensory analysis, normally at 22 or 23% ethanol by volume with water (Hardy and Brown, 1989; Perry, 1989), frequently results in a cloudy solution. Dilution greatly affects the solubility of compounds such as these ethyl esters, which are less soluble in water than in ethanol. Ethyl esters are amphiphilic with a polar head group and a hydrocarbon chain tail (Tanford, 1980) and may thus form the visible agglomerates or micelles in the diluted solution. These agglomerates or micelles may
1732 solubilise other spirit congeners, suggesting a mechanism by which esters might affect the perception of many spirit components. The relationship between solution concentration and headspace concentration may be investigated by determination of solute activities. The activity of solutes may be determined directly by measuring the vapour pressure for the compound of interest. However chromatographic quantifications of headspace concentrations are more readily obtained (Grant and Higuchi, 1990). These can be compared with data from headspace concentrations above the pure solute (unit activity). Activity can then be related to the solution concentration by the equation: a = fX, where a is the activity, f the activity coefficient and X^ the concentration of the solute expressed as mole fractions. For an ideal solution f = 1. As a first step to understanding how solute interactions, particularly those between volatile and non-volatile constituents, influence the sensory properties of distilled beverages, we have investigated: 1. The agglomerate particle size and sensory properties at 23% ethanol of the same malt distillate matured in 5 types of oak casks for up to 4 years. Particle sizes were compared with a model solution containing increasing concentrations of the 3 most abundant ethyl esters in the malt distillate (ethyl decanoate, dodecanoate and hexadecanoate). 2. The activity of ethyl esters, in 23% ethanol solutions, in the presence and absence of wood components. 3. The distribution of esters, aliphatic alcohols and aldehydes between solution and ester phases, using a model spirit solution containing ethyl decanoate, dodecanoate and hexadecanoate in the presence and absence of wood extract components. 2. EXPERIMENTAL 2.1. Materials Ethanol was HPLC grade (Rathbum Ltd, Walkerbum, Scotland) and water was distilled and filtered using a Millipore-Q system. Ethyl octanoate, ethyl decanoate, ethyl dodecanoate, ethyl tetradecanoate, ethyl hexadecanoate, dodecanol and tetradecanol were > 97% pure (Sigma Chemical Company, Poole, Dorset, England). Hexanol, octanol and decanol were >98% pure (BDH Ltd, Poole, Dorset, England). Hexanal, octanal, decanal and dodecanal were > 97% pure (Aldrich Chemical Co. Ltd, Gillingham, Dorset, England). Limousin oak extract was supplied by International Flavours and Fragrances (Haverhill, Suffolk, England) and redissolved overnight in 65% v/v ethanol and filtered to give a solution with a pH of 5.5 (Table 1). Whisky samples were provided by Chivas Brothers Ltd, Keith, Scotland. A single batch of new malt distillate was diluted to 63.4% and 67.5% ethanol and filled into five types of casks: new charred American white oak (type 1); new plain American white oak (type 2); used bourbon (type 3); used bourbon used for at least one maturation of Scotch (type 4); and a used bourbon reused repeatedly until judged unable to mature new distillate (type 5). Casks were stored in a bonded warehouse in Scotland and pooled spirit from each cask type was analysed after 3 or 4 years ageing. 2.2. Particle size measurement Whisky samples and model systems were diluted to 23% v/v ethanol and examined in triplicate by dynamic light scattering. Light from a He-Ne laser was scattered from the sample in a 1 cm^ fluorimeter cell positioned in the PCSIOOSM goniometer unit of a PCS system (Malvern Instruments Ltd, Malvern, England) at 20 ± 0.1 °C. Light scattered at 90° was analysed by a Malvern 7032 correlator. The correlator functions were analysed by the method
1733 of cumulants (Koppel, 1972) to give a z-average diffusion coefficient for the scattering particles in the solution. From this the hydrodynamic diameter of the particles was calculated by the Stokes-Einstein relation (Brooksbank et al, 1993). 2.3. Sensory analysis A panel of experienced assessors described the odour of the samples, using a vocabulary of 24 terms (Piggott and Canaway, 1981) shown in Table 2, on an intensity scale from 0-5. The samples were presented to the panel at 23% v/v ethanol concentration in tulip shaped nosing glasses similar to standard wine tasting glasses (BS5586:1978) but of approximately 150 ml capacity, covered with watchglasses and assessed in individual sensory booths under red lighting to minimise colour differences. Each sample was assessed in duplicate and eight samples were assessed per session. Data were collected using the PSA-System (Oliemans, Punter & Partners, PO Box 14167, 3508 SG Utrecht, The Netherlands). 2.4. Gas chromatography Glass vials (20 ml) were fitted with PTFE Uned silicone rubber septa in plastic screw caps. Standard concentrations of esters, alcohols, aldehydes and acids were prepared in 65% ethanol and 3.5 ml placed in a vial and diluted with 6.5 ml of water to a final ethanol concentration of 23 % v/v (pH 6.6). Standard concentrations were also prepared for solutions containing wood extract (2.5 g 1'^ at 65% ethanol) and ethyl esters plus wood extract. Vials containing pure solute (10 ml) were also prepared. Samples were equilibrated in a water bath at 30 °C for at least 30 minutes and a 2.5 ml sample of headspace withdrawn using a 5 ml gas tight syringe, heated to 50 °C. Only one headspace injection was made per vial and samples were analysed in quadruplicate using a Carlo Erba HRGC 5300 Mega series gas chromatograph (Fisons Instruments Ltd, Crawley, England) with an FID linked to TRIO computing integrator. A 0.5 mm x 12 m BPl column (df = 1) (SGE UK Ltd, Milton Keynes, England) was used with a helium carrier gas flow rate of 5 ml min ^ Column was at 60 °C for 1 minute after injection, increasing to 240 °C at 18 °C min"^ The detector temperature was 250 °C. The injector was a cold-on column injector fitted with an external gas tight septum. Ethanol headspace injections followed the same procedure but using a 100 |LI1 syringe and an oven temperature of 40 °C. The glass surface in syringes can adsorb higher boiling compounds that would result in the retention of a proportion of the sample within the syringe after injection. The extent of this adsorption for C8 to C14 compounds was calculated from 3 subsequent blank injections after sampling of the headspace above the pure solute. The results, expressed as cumulative percentages, are given in Table 2. Although the proportion retained by the syringe was high for C12 and C14 compounds, it was constant, irrespective of the amount of analyte present, and so did not result in significant error in the calculation of activity. Flushing syringes with air 3 times prior to sampling and 3 times after injection greatly reduced carry over between injections (<5% for tetradecanol) and no consistent increase was observed over 4 consecutive replicates. Activities were calculated from headspace concentrations, with unit activity taken as the headspace concentration above the pure solute (Grant and Higuchi, 1990). At least 5 points on the linear portion of the plot of activity against concentration, expressed as mole fractions, were used to calculate the activity coefficient (Denbigh, 1981). 2.5. Data analysis Data matrices were analysed by principal components analysis (PCA; Piggott and Sharman, 1986). PCA calculates components, defined as linear combinations of variables (e.g. chemical compounds or sensory descriptors), describing as much of the variance of the original data as possible; the resulting components may be rotated graphically or to a preset mathematical criterion to aid interpretation. This allows the original multi-dimensional matrix to be simplified without substantial loss of information, and so eases interpretation of complex data matrices. The results of PCA can be displayed as two sets of quantities. The first is the
1734 Table 1 Composition of the commercial Limousin oak extract (after dilution to 23% ethanol). Analytical methods from Conner et al. (1992) PH total phenols (mg gallic acid equivalents ml ) gallic acid (mg 1"^) vanillic acid (mg 1"^) vanillin (mg 1'^) syringic acid (mg 1'^) syringaldehyde (mg 1'^)
5.5 0.7 0.4 0.1 0.2 0.2 0.5
Table 2 Cumulative percentage of compounds detected in 3 subsequent blank injections after sampling headspace above pure solute. Blank 3 taken as 100% Compound
Analyte
Blank 1
Blank 2
octanol decanol dodecanol tetradecanol
95.6 89.3 69.7 62.4
98.4 97.7 88.8 85.4
99.4 99.3 96.0 95.0
octanal decanal dodecanal
94.2 87.8 65.3
97.9 94.2 86.4
99.2 95.6 94.6
Table 3 Relationship between previous history of whisky cask, agglomerate average diameter and size range, and sensory score of aged distillates Cask type
Filling strength (%v/v)
z-Average diameter (nm)
Polydispersity
PCI score
1 1 2 2 3 3 4 4 5 5
63.4 67.5 63.4 67.5 63.4 67.5 63.4 67.5 63.4 67.5
348 343 465 500 600 561 560 566 603 581
0.174 0.188 0.137 0.116 0.060 0.105 0.152 0.142 0.060 0.134
1.33 1.14 0.84 0.08 -0.02 -0.17 -1.09 -0.96 -0.71 -0.43
1735 correlations or loadings of the original measured variables with successive components which aid interpretation of the components; the second is the sample scores which show relationships between samples. 3. RESULTS 3.1. Light scattering Light scattering data (Table 3) showed that spirits from the type 1 cask (new charred) contained particles (agglomerates) with diameters in the range 340 - 350 nm and high values for polydispersity, correlated with a high concentration of non-volatile wood-derived material. In spirit matured in type 2 (new plain) casks, z-average diameters were higher at approximately 500 nm, and polydispersity reduced. Spirits matured in the used casks (types 3 - 5 ) contained particles with similar z-average values in the range 550 - 600 nm. Model systems containing 30 - 80 mg 1'^ of an equimolar mixture of ethyl decanoate, dodecanoate and hexadecanoate, and wood extract at 0, 2.5 gl'^ and 5 gl'^ were similarly analysed, immediately after preparation and after a further 7 days (Figure 1). z-Average agglomerate diameters were significantly reduced by wood extract in both cases. After 7 days agglomerate diameters had increased in the absence of wood extract, but not in its presence. Agglomerate diameter was significantly affected by ester concentration in the absence of wood extract and in the solutions containing 2.5 g 1'^ wood extract. 3.2. Sensory analysis Loadings of descriptors on the first principal component of principal components analysis of panel mean sensory data are listed in Table 4. Samples from type 1 casks had higher component scores, related to the descriptors with large positive loadings on the component: sweet, vanilla, woody and smooth; whereas spirits from type 4 and 5 casks had lower component scores, related to those descriptors with negative loadings: grainy, solvent, sour, soapy and grassy. 3.3. Single esters Initial studies were determinations of activity coefficients and the concentrations at which agglomeration occurred for each ester. For even-numbered carbon esters from ethyl decanoate to hexadecanoate, plots of activity against solution concentration followed the general form shown in Figure 2. In each, there was an initial linear relationship between the activity and the solution concentration delineated by the origin (A) and a transition point (B). This was followed by a second linear increase to a maximum (C), followed by decline to a third transition point (D) and a plateau (terminating at E). With ethyl decanoate activity reached a maximum of 0.7 whereas for the longer chain esters plateaux were between 0.9 and 1.0. For each ester, five concentrations within the initial linear portion (AB) were selected. Plotting of activity against concentration provided an activity coefficient (f) for each ester. The concentrations at which agglomeration occurred were determined from the average of the activities in each plateau (DE) and the activity coefficient. The distribution of esters between the solution and associated phases (Figure 3) was calculated from the activity coefficient. Ethanol activity in solutions containing esters at high and low concentrations (regions D to E and A to B in Figure 2, respectively) was determined, but showed no significant differences. Plotting the natural logarithm of the agglomeration concentration against the number of carbons in the ester (Figure 4) produced a gradient indicating a change in the free energy of association of -2724 J mole"^ CH2 (standard deviation (SD) = 193 J mole'^ CH2). For homologous series of ionic amphiphiles at constant ionic strength and most nonionic amphiphiles the free energy of micelle formation changes by about -2930 J mole"^ CH2. The corresponding change in free energy on removing an alkyl chain from water to hydrocarbon for a homologous series of n-alkanes is -3430 J mole"^ CH2 (Tanford, 1980).
1736
S
r,
650
T
600
t
550
t
500
t
»< 450 <
400
t
350
t
300 20
40 60 Esters concentration (mg/1)
80
Plus 2.5 g/1 wood extract —a Plus 5.0 g/1 wood extract Ester mixtureFigure 1. z-Average diameter of agglomerates formed by an equi-molar mixture of three ethyl esters in 23% v/v ethanol with varying levels of wood extract. Open symbols are after 7 days. 0.8 0.7 0.6 ^ 0.5 > 0.4 0.3
t
0.2
t
0.1
0
2 4 6 8 Concentration (mole fraction X 10-6) Figure 2. Typical plot of activity vs total concentration. For each ester, concentrations were selected to give at least five points on AB to calculate the activity coefficient. The average activity of the plateau region was used to estimate the agglomeration concentration (CAC).
1737 Table 4 Loadings of descriptors on first component from principal components analysis of descriptive sensory data Descriptor
Loading
Sweet Vanilla Malty Spicy Woody Fruity (other) Smooth Fruity (estery) Floral Buttery Nutty Phenolic Mouldy Fishy Pungent Catty Meaty Oily Sulphury Soapy Grassy Sour Solvent Grainy
0.42 0.37 0.35 0.34 0.29 0.25 0.21 0.20 0.15 0.11 0.10 0.03 0.01 -0.01 -0.02 -0.04 -0.07 -0.09 -0.11 -0.13 -0.14 -0.15 -0.19 -0.24
Table 5 Critical micelle concentrations for hexa-oxyethylene alkyl ethers (CjE6) at 20 or 25 °C (Shinoda, 1978) and agglomeration concentrations for ethyl esters (EtCj) at 30 °C i
8 10 12 14 16
CiE6
240^ 21*^ 2.V 0.024^
' at 25 °C ^ at 20 °C
EtCj
3.2 0.67 0.042 0.0059
1738
• Solution - Agglomerate
2 4 6 Concentration (mole fraction X 10'^) Figure 3. Plot of concentration of free ester in solution and amount forming agglomerates against overall concentration -11
t
-13
f
-15
t
-17
t
-19 10
12
14 No of carbons
16
18
Figure 4. Plot of the natural logarithm of the agglomeration concentration (In CAC) against the number of carbons in the ethyl ester (gradient = -1.08, sd = 0.08, R^ = 0.990). Agglomeration concentrations are in mole fractions.
1739 Plotting the natural logarithm of the activity coefficient against number of carbons in the ester (Figure 5) indicated an increase in the excess chemical potential of solution (Denbigh, 1981) of 2675 mole^ CHj (SD = 118 J mole^ CH2). Table 5 compares the agglomeration concentrations for the ethyl esters in 23% ethanol with critical micelle concentrations for hexa-oxyethylene alkyl ethers in water (Shinoda, 1978). In each case the ethyl esters gave lower results, despite the higher temperatures used in this experiment. 3.4. Ester mixtures In the second set of experiments ester mixtures were prepared to examine the relationship between agglomerates and the activity of a second ester. It was apparent that the activity coefficient for ethyl decanoate was significantly decreased (p<0.01) by addition of a second ester at 20 mg 1 (Table 6). There were also significant (p<0.05) differences between the effects of octanoate, dodecanoate and hexadecanoate. Table 7 shows the reduction in the activity of ethyl decanoate at 20 mg ml'^ in the presence of other ethyl esters at increasing concentrations. With ethyl octanoate no significant decrease in activity was observed at < 20 mg 1'^ where the sum of solute activities < 0.70. Thereafter decreases in ethyl decanoate activity were balanced by increases in ethyl octanoate activity (f= 0.025 (SD = 0.006), R^ = 0.940). For tetradecanoate and hexadecanoate an activity minimum of 0.20 was reached for ethyl decanoate. For 20 mg 1'^ solutions of esters the weight percentage forming agglomerates ranges from 0 % for octanoate, to 99 % for hexadecanoate. This indicates that the presence of any second ester has a large effect. The structure and distribution of the second ester was of lesser importance. Table 6 also gives the activity coefficients of ethyl dodecanoate in the presence of a second ester at 20 mg 1 ^ These data also support the assertion that it is the presence, rather than structure, of the second ester that is of primary importance. Moreover it is clear that the linearity of activity extended well beyond the agglomeration concentration for ethyl dodecanoate. Figure 6 shows the effect of increasing concentrations of ethyl decanoate on a solution of ethyl dodecanoate and hexadecanoate, each at 20 mg 1'^ The linear increase in the activity of ethyl decanoate is balanced by decreases in the activities of ethyl dodecanoate and hexadecanoate, such that there was no significant change in the sum of the activities of the three esters. A similar experiment (Figure 7) was performed in which ethyl octanoate was added to a solution of three esters; ethyl decanoate, dodecanoate and hexadecanoate, each at 20 mg 1'^ A decrease in hexadecanoate activity was observed but neither decanoate nor dodecanoate activities were affected. 3.5. Effect of wood extract on solutions of single esters Activity coefficients and agglomeration concentrations for ethyl decanoate and dodecanoate were determined in the presence of oak wood extract at a range of concentrations (Table 8). Wood extract at concentrations up to 5 g 1'^ significantly increased the activity coefficient of ethyl decanoate but decreased the agglomeration concentration. At higher concentrations of wood extract the activity coefficient was decreased, but the intercept shifted significantly from the origin. Similar results were observed with ethyl dodecanoate, although these effects were observed at lower concentrations of wood extract. There was also a marked decrease in the activity at the agglomeration concentration. 3.6. Effect of wood extract on solutions of more than one ester The effect on activity coefficients of both ethyl decanoate and dodecanoate in the presence of a second ester and wood extract at 2.5 g 1'^ was also investigated (Table 9). As previously (Table 6), addition of a second ester resulted in significant decreases in the activity coefficient with the nature of the second ester being of lesser importance. Again the linearity of the relationship extended well beyond the agglomeration concentration The effect of wood extract on a distribution of esters in a mixture of decanoate.
1740
fl
20
T
18
f
(L)
o
1^
16
O
o
>^ 14 > C) a 12 10
t —I
1-
12 14 No of carbons
10
16
18
Figure 5. Plot of In (activity coefficient) against the number of carbons in the ester (gradient = 1.05, sd = 0.02, R ' = 0.999). — decanoate
0.8
^• dodecanoate
0.7
I— hexadecanoate
0.6 0.5 *>
1 0.4
0.3 0.2 0.1
0
1
2
3
4
5
Concentration (mole fraction X 10"^) Figure 6. The effect of increasing concentrations of ethyl decanoate on a solution of ethyl dodecanoate and ethyl hexadecanoate (20 mg 1'^ each).
1741 Table 6 The effect of a second ester (20 mg 1'^) on the activity coefficients of ethyl decanoate and dodecanoate Added ester
Activity Coefficient^ Mean (SD)
Intercept
0.233 (0.016) 0.140(0.006) 0.108 (0.007) 0.091 (0.010) 0.080 (0.050)
0.02 0.10 0.06 0.04 0.05
(0.02) (0.01) (0.02) (0.03) (0.02)
0.984 0.998 0.988 0.948 0.987
1.60 0.31 0.23 0.21
0.07 0.02 0.03 0.09
(0.06) (0.05) (0.05) (0.05)
0.902 0.969 0.971 0.973
R^
Mean (SD)
decanoate octanoate dodecanoate tetradecanoate hexadecanoate dodecanoate decanoate tetradecanoate hexadecanoate
(0.37) (0.06) (0.06) (0.04)
^xlO^ (SD) = Standard deviation Table 7 The effect of increasing concentrations of a second ester on the activity of ethyl decanoate (20 mg 1-^) Added ester
Rate of decrease^ Mean (SD)
Intercept Mean (SD)
R^
octanoate (1) dodecanoate (2) tetradecanoate (3) hexadecanoate (4)
-0.024 -0.062 -0.083 -0.162
0.484 0.384 0.350 0.353
0.903 0.800 0.999 1.000
(0.008) (0.018) (0.002) (0.001)
(0.035) (0.026) (0.002) (0.001)
^ Rate of decrease of activity per micro mole fraction added ester (1) concentration range 20 to 50 mg 1 "^ no significant decrease up to 20 mg 1"^ (2) concentration range 5 to 30 mg 1'^ (3) concentration range 5 to 20 mg 1'^ no further decrease at higher concentrations (4) concentration range 5 to 15 mg r \ no further decrease at higher concentrations
1742
•^
0.4
^
0
1
2
3
4
5
6
Concentration (mole fraction X 10'^) Figure 7. The effect of increasing concentrations of ethyl octanoate on a solution of three esters (ethyl decanoate, dodecanoate and hexadecanoate, 20 mg 1'^ each). 1.6 1.5 o ^ 1.4
• ^ VO a 1
§2
1.3
go xd
1.2
oa; '-do
1 1
S c^ -2 S 0.9 <: 0.8
decanoate - dodecanoate • hexadecanoate
0.7 0.6
H-
-h
-+-
2 4 6 Wood extract concentration (g 1-^) Figure 8. Changes in the agglomerate concentrations of ethyl decanoate, dodecanoate and hexadecanoate (20 mg 1"^ each) in the presence of increasing concentrations of wood extract.
1743 Table 8 Effect of wood extract on solution thermodynamics of ethyl decanoate and dodecanoate Wood cone. gi-'
Activity Coefficient^
Intercept
CMC*'
R^
3.28 2.30 1.17 <0.09
0.984 0.955 0.989 0.909
0.67 0.43
0.983 0.974 0.999 0.968 0.904 0.990 0.955 0.985
decanoate 0 2.5 5.0 7.5
0.23 0.41 0.49 0.10
(0.02) (0.09) (0.05) (0.02)
1.60 2.13 0.52 2.41 0.73 1.75 0.61 0.74
(0.07) (0.04) (0.02) (0.31) (0.17) (0.12) (0.06) (0.05)
0.02 (0.02) 0.04(0.11) 0.04 (0.06) 0.13 (0.04)
dodecanoate 0 1.2 1.2 2.5 2.5 5.0 5.0 7.5
(AB) (BC) (AB) (BC) (AB) (BC)
0.07 0.00 0.37 -0.01 0.30 -0.01 0.20 0.11
(0.07) (0.08) (0.12) (0.04) (0.12) (0.02) (0.06) (0.02)
0.14 0.09 <0.01
" xlO^ ^ mole fraction xlO"^ AB/BC refer to linear portions of graph in Figure 2 Table 9 Effect of ester and wood extract (2.5 g 1'*) on activity coefficients of ethyl decanoate and dodecanoate Added ester (20 mg 1-^)
Activity Coefficienf
Intercept
R^
0.06 (0.01) 0.07 (0.04) 0.04 (0.01)
0.999 0.932 0.993
0.12 (0.03) 0.15 (0.05) 0.05 (0.04)
0.975 0.983 0.964
decanoate (CIO) dodecanoate (C12) 0.081 (0.003) hexadecanoate (CI6) 0.080 (0.015) C12 + C16 0.062 (0.003) dodecanoate decanoate hexadecanoate C10 + C16 xlO'
0.194 (0.022) 0.188 (0.025) 0.105 (0.014)
1744 dodecanoate and hexadecanoate, each at 20 mg 1'^ was also studied (Figure 8). It was apparent that there was a displacement of ester from solution to agglomerates that reached a maximum at 2.5 g \'\ At concentrations above 3.75 g 1"^ the concentration of esters in solution was increased. For wood extract the effect was greatest for ethyl decanoate and least for ethyl hexadecanoate, in contrast to that observed with addition of ethyl octanoate. 3.7. Distribution of esters in matured spirit samples The calculated agglomeration concentrations for ethyl dodecanoate, tetradecanoate and hexadecanoate (Table 5) were related to the concentrations observed in a malt distillate aged for three years in either glass, new charred (type 1), used Bourbon (type 3), and used (type 4) and exhausted (type 5) Scotch casks (Table 10). From these results the percentage of each of the three esters in the associated phase could be estimated. As predicted, ethyl dodecanoate, tetradecanoate and hexadecanoate were primarily in agglomerates. However ethyl decanoate was more abundant free in solution in glass-aged distillate and in the three casks that would be used for maturation of Scotch malt distillate. In the new charred wood, as used for Bourbon maturations, decanoate was primarily in the associated phase. Table 10 Fatty acid ethyl ester concentrations (C) and percentage forming agglomerates (%A) in a malt distillate aged for 3 years in four types of cask. Concentrations are given as mg 1"^ at nosing strength (23% v/v ethanol) Ester Dist C(%A) CIO C12 C14 C16
19 (42) 16 (87) 3 (92) 11 (99)
Total 49
Type 1 C(%A)
Type 3 C(%A)
Type 4 C(%A)
Type 5 C(%A)
42 (68) 17 (85) 3(99) 10 (99)
22 (37) 17 (86) 3(97) 11 (99)
23 (36) 19 (86) 3(98) 11 (99)
19 (28) 17 (80) 3 (97) 10 (99)
72
53
56
49
CIO = ethyl decanoate; C12 = ethyl dodecanoate; C14 = ethyl tetradecanoate; C16 = ethyl hexadecanoate Dist. = Distillate stored in glass for 3 years; Type 1 = New, charred cask; Type 3 = Used bourbon cask. Type 4 = Used Scotch cask; Type 5 = Exhausted cask 3.8. Alcohols Activity coefficients for alcohols are listed in Table 11. In 23% v/v ethanol the linear portions of the activity curves extrapolated close to the origin. This did not indicate an ideal solution, as activity coefficients were greater than 1. For dodecanol and tetradecanol, activity reached a plateau at approximately 0.7, at solution concentrations of 11 and 1.4 mg T , respectively. For the homologous series of alcohols in 23% ethanol, the logarithm of the activity coefficient and the number of carbons in the alcohol gave a linear relationship with gradient =1.31 (standard deviation (SD) = 0.04), intercept = -2.66 (SD = 0.47) and R^ = 0.997 (Figure 2). This gave an increase in the excess chemical potential of solution (Denbigh, 1981) of 3298 J mole'^ CH2. Similar results were obtained for the homologous series of ethyl esters, where the plateau region was found to be due to the formation of agglomerates. The point where the linear portion of the activity plot and the plateau cross represents the
1745 saturation concentration of singly dispersed molecules (Shinoda, 1978). Table 11 Activity coefficients for homologous series of alcohols in solutions at 23% v/v ethanol at 30 °C, with intercept and R^ for linear portion of plot
Alcohol
Intercept
Activity Coefficient^ Mean (SD)
Mean (SD)
R^
0.18 (0.01) 2.2 (0.2) 37 (4.9) 650 (41) 5100 (670)
0.00 0.00 -0.02 -0.31 0.06
(0.00) (0.00) (0.02) (0.06) (0.04)
0.998 0.991 0.966 0.997 0.966
0.00 0.00 0.01 -0.01 0.15
(0.00) (0.00) (0.02) (0.05) (0.08)
0.996 0.997 0.997 0.979 0.974
0.00 (0.00) 0.00 (0.00) -0.05 (0.02) 0.02 (0.01) 0.22(0.11)
0.996 0.997 0.997 0.999 0.994
ethanol solution hexanol octanol decanol dodecanol tetradecanol
ethanol solution with ethyl esters hexanol octanol decanol dodecanol tetradecanol
0.17 (0.01) 2.0 (0.1) 35 (3.8) 160 (23) 680 (72)
ethanol solution with wood extract hexanol octanol decanol dodecanol tetradecanol
0.20 (0.02) 2.0 (0.1) 66 (3.8) 370 (8) 2800 (190)
ethanol solution with ethyl esters and wood extract hexanol octanol decanol dodecanol tetradecanol
0.21 (0.02) 1.7 (0.11) 53 (8.9) 200 (6.4) 2200 (200)
0.00 0.00 -0.04 -0.04 0.15
(0.00) (0.00) (0.04) (0.01) (0.07)
0.994 0.996 0.973 0.999 0.985
xlO' In the presence of ethyl ester solution but absence of wood extract, activity coefficients for dodecanol and tetradecanol were significantly lower (p<0.01) than in 23% ethanol alone. For the homologous series, the increase in the excess chemical potential of solution was 2644 J mole"^ CH2, significantly lower (p<0.05) than for 23% ethanol. This indicated that a portion of the alcohol was incorporated into the ester agglomerate phase.
1746 For decanol, in 23% v/v ethanol in the absence of esters but presence of wood extract, the activity coefficient was significantly higher (p<0.05) than in 23% ethanol alone. For dodecanol and tetradecanol however, activity coefficients were significantly lower (p<0.05). This is similar to the effect observed for esters. For C12 and C14 esters, low concentrations of wood extract increased activity coefficients and decreased the concentrations at which agglomerates were formed. Either increasing the concentration of wood extract, or the ester chain length, substantially decreased the concentrations at which agglomerates were formed. This suggested that the concentrations of dodecanol and tetradecanol used in activity coefficient determinations were above those required for the development of the agglomerate phase in the presence of wood extract. No significant reductions in the activity coefficients of alcohols were observed through the addition of wood extract to ethyl ester solutions. However, in the presence of hexanol and octanol, addition of wood extract significantly decreased the sum of ester activities (p<0.01). In the presence of dodecanol and tetradecanol, wood extract significantly increased the sum of ester activities (p<0.05) but had no effect in the presence of decanol. Addition of hexanol, octanol and decanol to the ethyl ester solutions had little effect on the activities of ethyl esters independent of the wood extract. Dodecanol and tetradecanol displaced esters from solution to the agglomerate phase, decreasing the sum of ester activities at a rate equal to the increase in alcohol activity. This resulted in no overall change to the sum of solute activities. 3.9. Aldehydes Activity coefficients for aldehydes are listed in Table 12. Again, the linear portions of the activity curves extrapolated close to the origin, but activity coefficients were greater than 1. For the homologous series of aldehydes, the logarithm of the activity coefficient and the number of carbons in the alcohol gave a linear relationship with gradient = 1.40 (SD = 0.04), intercept = -4.94 (SD = 0.20) and R^ = 0.9997 (Figure 2). This corresponded to an increase in the excess chemical potential of solution of 3525 J mole'^ CH2. In the ethyl ester solutions in the absence of wood extract, the activity coefficient for dodecanal was significantly lower (p<0.1) than in 23% ethanol alone. The increase in excess chemical potential per mole CH2 in ethyl ester solution without wood extract, was not significantly different from 23% ethanol. The lower activity coefficients suggested that aldehydes were more soluble in 23% ethanol than either alcohols or esters and, except for dodecanal, were not incorporated into ester agglomerates. The lower activity coefficients for aldehydes were most probably the result of the formation of 1,1-diols, hemiacetals and acetals in solution. In 20% ethanol at 25 °C, hemiacetal and 1,1-diol account for over 60% of the total aldehyde in solution (Perry, 1986). For hexanal, octanal and dodecanal, addition of wood extract to the ethyl ester solution significantly increased (p<0.05) activity coefficients. Changes in the activity coefficients due to the presence of wood extract may have been caused by the decrease in pH affecting the equilibria between aldehyde, 1,1-diol and hemiacetal. For hexanal in ethyl ester solution, the presence of wood extract significantly lowered (p<0.05) the activity coefficient. For octanal, decanal and dodecanal, however this was not observed. Addition of hexanal and octanal to ethyl ester solutions, both in the presence or absence of wood extract, decreased ester activities (Figure 9). The decrease was significantly greater (p<0.01) than the increase in aldehyde activity. Addition of decanal decreased the sum of ester activities at a greater rate than the increase in aldehyde activity except in the presence of wood extract. Addition of dodecanal decreased the sum of ester activities at a similar rate to that of the increase in aldehyde activity, whether wood extract was present or absent. However, in the presence of hexanal and octanal, addition of wood extract decreased the sum of ester activities, with combined reductions reaching a minimum at activity of 0.4. With decanal no change in ester activities was observed, whereas with dodecanal, addition of wood extract yielded an increase.
1747
• decanoate • dodecanoate • hexadecanoate S
20
30
Added hexanal (mg 1"0 Figure 9. Ethyl ester activities, calculated from headspace concentrations above 23% v/v ethanol at 30 °C, as functions of hexanal concentration. Filled symbols represent activities in solutions containing 2.5 g 1'^ wood extract. This suggested that the solubiUty behaviour of aldehydes has two components: a hydrophobic component due to the hydrocarbon chain and common to esters, alcohols and aldehydes; and an effect due to the aldehyde group. In a spirit with an agglomerate phase formed from esters or alcohols, shorter chain aldehydes reduce the total activity or chemical potential of the solution. It is possible, therefore, that the effect of wood extract may be due to the presence of aromatic aldehydes such as vanillin and syringaldehyde. 4. DISCUSSION For solutions of individual esters, plotting activity against total solute concentration yielded a plateau as activity approached unity. There are two alternate hypotheses that may explain this observation. The first is that a surface film forms as the ester is excluded from solution, or alternatively excess ester forms agglomerates or micelles. However, measurements of ethanol activity showed no significant change with increasing ester concentration, which would be expected if a surface film were formed. Consequently it was concluded that the esters were forming agglomerates or micelles. For the homologous series of esters the increase in free energy of association depended on the increase in length of the hydrocarbon chain. This behaviour is an important characteristic of the hydrophobic effect which is mainly responsible for the formation of micelles. In other studies on solutions of amphiphiles, the formation of micellar aggregates accounted for all or the major part of nonideality in these solutions (Tanford, 1980). For this series of ethyl esters, activity coefficients were always much greater than 1 indicating non-ideal solutions. For all ethyl esters the plot of activity against concentration gave a straight line through the origin.
1748 so the choice of whether solutions are ideal or not depends on the convention used for unit activity; either the headspace over the pure solute or an infinitely dilute solution (Denbigh, 1981). Table 12 Activity coefficients for homologous series of aldehydes in solutions at 23% v/v ethanol at 30 °C, with intercept and R^ for linear portion of plot
Aldehyde
Activity Coefficient Mean (SD)
Mean (SD)
R'
31 (1) 596 (15) 8500 (820) 150000 (10400)
0.0 0.1 0.0 0.2
(0.0) (0.1) (0.0) (0.3)
0.999 0.999 0.982 0.991
0.0 0.2 0.0 0.4
(0.0) (0.1) (0.1) (0.6)
0.999 0.998 0.987 0.992
0.0 0.0 0.0 0.1
(0.0) (0.0) (0.0) (0.3)
0.999 1.000 0.979 0.986
Intercept
ethanol solution hexanal octanal decanal dodecanal
ethanol solution with ethyl esters hexanal octanal decanal dodecanal
31 510 6500 98000
(0.0) (16) (700) (7500)
ethanol solution with wood extract hexanal octanal decanal dodecanal
35 (0.0) 770 (0.1) 9500 (725) 170000 (10200)
ethanol solution with ethyl esters and wood extract hexanal octanal decanal dodecanal
28 (0.3) 550 (60) 6900 (840) 120000 (9500)
0.0 0.1 0.0 0.4
(0.0) (0.3) (0.0) (0.2)
0.987 0.977 0.959 0.989
The increase in free energy of association for the homologous series of esters is close to that reported for the formation of micelles. However, the results from dynamic light scattering indicated that "micellar" diameters for ethyl esters in 23% ethanol were extremely large at approximately 500 nm and give a cloudy solution. The agglomeration concentration may therefore represent the cloud point concentration for that ester at 30 °C, and DE (Figure 2) represents the saturation concentration of singly dispersed molecules (Shinoda, 1978). Comparison with hexa-oxyethylene alkyl ethers indicates that the ethyl esters have much lower solubilities as the extent of H-bonding to water is considerably reduced. For ethyl esters, the increase in activity coefficient with hydrocarbon chain length gave an
1749 increase in the excess chemical potential of solution of 2675 J mole'^ CH2. Using the reasoning for changes in the free energy of association, then changes in the excess chemical potential also provided a measure of hydrophobic interaction for that solute. A further consequence is that for increasing ester chain lengths, decreases in the agglomeration concentration are balanced by increases in ester activity coefficients and this, for most esters results in agglomeration when solute activity approaches unity. Consequently, each of the homologous series of esters displays the same maximum activity, or chemical potential in 23% ethanol, above which agglomeration occurs. This limit is exceeded by many malt whiskies. For mixtures of ethyl octanoate and decanoate, no change in the activity coefficients of either ester was observed at activities less than 0.7. Thus the activity coefficients calculated for single esters in solution can be used to calculate the free solution concentration of each ester in a mixture. At values greater than this, increases in the activity of one ester were balanced by decreases in that of the second. In solutions in which a portion of ester has formed agglomerates, observed reductions in activity coefficients must relate to ester being transferred to these agglomerates. Although there are significant differences between esters, such effects are less marked than those arising from the presence of any ester. It can thus be concluded that the presence of a saturated solution is more important than either the structure or quantity of ester present. Increasing the weight percentage of ester in agglomerates resulted in a decrease in the activity of a shorter chain ester maintained at constant concentration. Such changes may be the result of the decreases in the relative mole fraction of the shorter chain ester. The rate of decrease in ethyl decanoate activity increases with the chain length of a second ester. As the minimum activity observed was similar for each ester, changes in rate may relate solely to the activity coefficient of the second ester. In mixtures of three and four esters, increasing the concentration of the ester with the shortest hydrocarbon chain resulted in decreases in the activity of the other esters. The ester with the largest hydrocarbon portion is then displaced preferentially. Thus it can be concluded that the maximum activity calculated for single esters in solution is closely related to the sum of ester activities in a mixture. Previous studies have reported interactions between many classes of volatile flavour compounds with purines (King and Solms, 1982; Seshadri and Dhanraj, 1987). These interactions were attributed to plane-to-plane stacking resulting from hydrophobic and 7C-electron interactions. Compounds lacking an extended 7C-electron system were unable to interact. In our studies on esters the driving force for interaction appears to be the hydrophobic effect, suggesting that other amphiphilic compounds may also be involved. Further the presence of agglomerates may result in the solubilisation of hydrophobic compounds in spirit solutions. Addition of Limousin wood extract was observed to change the solubility of esters in 23% ethanol. Activity coefficients were increased, but both the concentration and the activity at which agglomeration occurred decreased. Reductions in activity coefficients were observed for ethyl tetradecanoate and hexadecanoate with wood extract at any concentration and with ethyl decanoate and dodecanoate at 2.5 g 1'^ As the activity at the agglomeration concentration decreased (Table 8), a further linear increase, with a significantly lower gradient, was observed. This relates to the transition in the plot of activity against concentration in single ester solutions (point B in Figure 2). The dynamic light scattering results indicated that dissolution of wood extract results in a decrease in the agglomerate diameter for ethyl esters in 23% ethanol and it can be postulated that the expansion of BC in Figure 2 is accompanied by the formation of smaller and stabler structures. In mixtures of two esters, despite the increased activity coefficients of the individual esters, addition of wood extract resulted in decreases in the activity coefficient. This suggested that the wood extract enhances the partitioning of the second ester into the agglomerates. The effect is a reduction in ester in the solution concentration in the presence of the wood extract. When mixtures of three esters were analysed, addition of wood extract was found to decrease the ester in solution to a minimum with the greatest effect on the ester with the smallest
1750 hydrocarbon group. Estimating distribution of ester between solution and agglomerate phases in matured distillate samples indicated that dodecanoate, tetradecanoate and hexadecanoate were primarily in the agglomerate phase. Little difference was observed between cask types, though for ethyl decanoate a significantly higher weight percentage was in the agglomerates for the new charred casks. However, values in Table 10 were calculated from the activity coefficients of esters in 23 % ethanol. Variation in concentrations of wood components arising from the different cask types were not included in the equation. From the effects of wood extract described above this is clearly not the case and the results suggest that activity coefficients would be highest in the new charred cask and decrease with increasing cask use. This would result in a highest concentration of ester in the agglomerate for the new charred cask. For the matured distillate samples, scores on the first principal component, from analysis of the matrix of panel mean sensory profiles, were correlated with z-average agglomerate diameter. Spirits with lower agglomerate diameters had higher component scores, related to the descriptors with large positive loadings on the component: sweet, vanilla, woody and smooth; whereas spirits with larger agglomerate diameters had lower component scores, related to those descriptors with negative loadings: grainy, solvent, sour, soapy and grassy. Model solutions indicated that the agglomerates were formed by the ethyl esters in the spirit and the dissolution of wood extractives resulted in the formation of smaller agglomerates. The effect of wood components on the amphiphilic compounds both singly and collectively suggest a change to the physico-chemical properties of the ethanol water solvent. This change appears to reduce the solubility of hydrophobic compounds, which in the presence of esters agglomerates results in increased incorporation of the compound into the agglomerate. Further, from these studies it is clear that an agglomerate phase was present in model solutions studied previously (Piggott et al., 1992). A plausible explanation for differences in extraction resulting from the presence of tannic acid and wood extract may lie in the stability of the esters in the agglomerate phase. It is also possible then, that in matured whisky samples, the stability of agglomerates is related to the concentrations of wood-derived, non-volatile compounds. The influence of this increased stability on the release of aroma compounds into the beverage headspace or retronasal cavity of the human taster is also under investigation. 5. CONCLUSIONS Two important implications for understanding the basis of alcoholic beverage aroma and flavour emerge from this study. First, that for hydrophobic aroma compounds in 23% ethanol, activity and hence headspace concentration, is not solely determined by concentration but by the presence of other hydrophobic compounds in the spirit. Secondly, dissolution of wood extract during maturation may alter the relative activities, and hence headspace concentrations, of certain solutes, with an effect also dependent on spirit composition. 6. REFERENCES Brooksbank, D.V., Davidson, CM., Home, D.S. and Leaver, J. (1993). Influence of electrostatic interactions on beta-casein layers adsorbed on polystyrene latices, J. Chem. Soc. Faraday Trans., 89, 3419-3425. Clyne, J., Conner, J.M., Paterson, A. & Piggott, J.R. (1993). The effect of cask charring on Scotch whisky maturation. Int. J. Food Science Technol., 28, 69-81. Conner, J.M., Paterson, A. and Piggott, J.R. (1992). Analysis of lignin from oak casks used for the maturation of Scotch whisky, J. Sci. Food Agric, 60, 349-353. Denbigh, K. (1981). The Principles of Chemical Equilibrium. Cambridge: Cambridge
1751 University Press. Grant, D.R. and Higuchi, T. (1990). The Solubility of Organic Compounds. New York: J. Wiley & Sons. Hardy, P.J. and Brown, J.H. (1989). Process Control. In: The Science and Technology of Whiskies (edited by J.R. Piggott, R. Sharp and R.E.B. Duncan). Pp 235-263. London: Longman. Jounela-Erickson, P. (1981). Predicative value of sensory and analytical data for distilled beverages. In: Flavour '81 (edited by P. Schreier). Pp. 145-164. Berlin: Walter de Gruyter. King, B.M. and Solms, J. (1982). Interactions of volatile flavour compounds with propyl gallate and other phenols as compared with caffeine, J. Agric. Food Chem., 30, 838-840. Koppel, D.E. (1972). J. Chem. Phys. 57, 4814-4818. Nishimura, K. and Matsuyama, R. (1989). Maturation and maturation chemistry. In: The Science and Technology of Whiskies (edited by J.R. Piggott, R. Sharp and R.E.B. Duncan). Pp. 235-263. London: Longman. Nishimura, K., Ohnishi, M., Masuda, M., Koga, K. and Matsuyama, R. (1983). Reactions of wood components during maturation. In: Flavour of Distilled Beverages: Origin and Development (edited by J.R. Piggott). Pp. 241-255. Chichester: ElUs Horwood. Perry, D.R. (1986). Whisky maturation mechanisms. In: Proceedings of the Second Aviemore Conference on Malting, Brewing and Distilling (edited by I. Campbell and E.G. Priest). Pp. 409-412. London: Institute of Brewing. Perry, D.R. (1989). Odour intensities of whisky compounds. In: Distilled Beverage Flavour: Recent Developments (edited by J.R. Piggott and A. Paterson). Pp. 200-207. Chichester: EUis Horwood. Philp, J. (1989). Cask quality and warehouse conditions. In: The Science and Technology of Whiskies (edited by J.R. Piggott, R. Sharp and R.E.B. Duncan). Pp. 264-294. London: Longman. Piggott, J.R., Conner, J.M., Clyne, J. and Paterson, A. (1993). Effects on Scotch whisky composition and flavour of maturation in oak casks with varying histories. Int. J. Food Sci. Technol., 28, 303-318. Piggott, J.R. and Sharman, K. (1986). Methods for multivariate dimensionality reduction. In: Statistical Procedures in Food Research (edited by J.R. Piggott). Pp. 181-232. London: Elsevier Applied Science. Piggott, J.R., Conner, J.M., Clyne, J. and Paterson, A. (1992). The influence of non-volatile constituents on the extraction of ethyl esters from brandies, J. Sci. Food Agric, 59, 477-482. Piggott, J.R. and Canaway, P.R. (1981). Finding the word for it: methods and uses of descriptive sensory analysis. In: Flavour '81 (edited by P. Schreier). Pp. 33-46. Berlin: Walter de Gruyter. Reazin, G.H. (1983). Chemical analysis of whisky maturation. In: Flavour of Distilled Beverages: Origin and Development (edited by J.R. Piggott). Chichester: Ellis Horwood. Pp. 225-240. Salo, P., Nykanen, L. and Soumalainen, H. (1972). Odour thresholds and relative intensities of volatile aroma components in an artificial beverage imitating whisky. J. Food Sci., 37 394-398. Seshadri, R. and Dhanraj, N. (1987). Flavour interactions in tea. In: Frontiers of Flavour (edited by G. Charalambous). Pp. 169-180. Amsterdam: Elsevier Science Publishers BV. Shinoda, K. (1978). Principles of Solution and Solubility. New York: Marcel Dekker. Tanford, C. (1980). The Hydrophobic Effect: Formation of Micelles and Biological Membranes. New York: J. Wiley & Sons. ACKNOWLEDGEMENTS This research is supported by the UK Agricultural and Food Research Council and Chivas Brothers Ltd, Keith, Scotland. Particle size analyses were performed by David Home at the Hannah Research Institute, Ayr, Scotland.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1753
INVESTIGATION OF FLAVOUR COMPOUNDS IN WHISKEY SPENT LEES. K.MacNAMARAl, P.BRUNERIE^, F.SQUARCIA^, A.ROZENBLUM^. 1. Irish Distillers Group, Bow St. Smithfield, Dublin 7, (Irish Republic) 2. Pemod-Ricard, 120 Av. du Marechal Foch, 94051 Creteil, (France) 3. San Giorgio Flavours, 114 Via Fossata, 10147 Torino, (Italy)
SUMMARY Chromatographic procedures, incorporating some of the latest technology advances, are described for investigation of flavour compounds in whiskey spent lees. A flavour concentrate produced from spent lees, and with an interesting malt-like aroma, is fractionated by preparative GC to isolate and enrich the trace compounds. The interesting fraction is then processed by 2-dimensional GC with simultaneous mass, sulfur and nitrogen selective detection after elution from the second column. In this way several channels of information are generated for each heart cut to allow a comprehensive profiling of the flavour compounds.
INTRODUCTION The whiskey distillation process consists of a series of consecutive rectification steps involving progressive refining of the spirit by selective removal of both volatile and high boiling components. (1) The higher boiling fraction constitutes an undistilled residue and is termed spent lees. The composition of spent lees is determined by the objectives of the various distillation stages, which are essentially maximum ethanol recovery for wash distillation and a specified final gravity for spirit distillation. These requirements in turn restrict the compounds in spent lees to a combination of both non-azeotropic and higher boiling azeotropic-forming congeners. Spent lees congeners are also present to some extent in the actual distilled product, since the above requirements will also represent a distribution or partition of these compounds between the whiskey and its spent lees. Therefore investigation of compounds in spent lees constitutes a convenient approach to possible identification of similar compounds at lower levels in whiskey.
1754 EXPERIMENTAL PREPARATION OF SPENT LEES EXTRACT. 10 liters of fresh spent lees was continuously extracted into a 2:1 mixture of pentane and ether for 22 hours. The extract was then dried over NA2SO4 and carefully concentrated by distillation to give 1ml of a complex extract.
ANALYTICAL GC-MS, NITROGEN AND SULFUR DETECTION A Hewlett-Packard 5890A GC (Hewlett-Packard, Avondale, USA) was configured with a temperature programmable cold injection system (CIS-3, GERSTEL Gmbh, Mulheim an der Ruhr, Germany), an FID, a Nitrogen Thermionic Detector (TID-2, Detector Engineering and Technology, USA) and a Mass Selective Detector (HP 5971A, Hewlett Packard, Avondale, USA) An additional Chemiluminescence Sulfur Detector (Sievers Instruments, Colorado, USA) could also be employed since this system uses an existing FID for flame conversion of organic sulfur to sulfur monoxide in a ceramic probe positioned in the flame. The sulfur monoxide is transported at sonic speed to a reaction cell where its chemiluminescent reaction with ozone is monitored as signal output. A key parameter for maximum sensitivity is proper positioning of the ceramic probe in the FID flame and for this work the manufacturer's standard probe assembly was replaced with a better technically engineered version (Gerstel Gmbh, Mulheim an der Ruhr, Germany) incorporating individual adjustments in any of three dimensions. Use of this unit not only allows rapid and optimum probe positioning, but also locks the probe into this spatial geometry for reproducible nm-to-run performance. Analysis Conditions Column:
50MFFAP
Pneumatics :
od=0.25mm
df=0.25|am
Helium carrier gas at 24psi FID H2,30 mls/min Air, 30mls/min N2, 30mls/min FID/SCD H2, 300mls/min Air, 200mls/min N2, lOmls/min TID-2 H2,5mls/min Air, 60mls/min
1755 Temperatures :
CIS 60° C to 280° C at 12° C /sec Oven 50°C to 220°C at 2°C/sec FID 250°C FID/SCD 280°C TID-2 300°C MSD 280°C
Mass Selective Detector
scan range 25 to 400 amu electron volts 1800
These same detector settings and temperatures were used for subsequent work where this GC becomes the satellite oven in a double oven 2-dimensional configuration.
PREPARATIVE GAS CHROMATOGRAPHY Fig 1 shows a schematic of this system (Gerstel Gmbh, Mulheim an der Ruhr, Germany) which is built into the 5890GC and equiped with a temperature programmable injector. A preparative wide-bore column leads from the injector to an effluent splitter (ES). This latter is a micro-coupling device which fits into the base of the FID and essentially distributes the column effluent with about 1% going to the FID through an 0.05mm fused silica line in order to produce a monitor FID signal. The majority of the sample eluting from the column passes through a heated fused silica transfer capillary (TFC) into the preparative switching device (PSD) which is housed in the preparative unit, The switching device is software controlled and can distribute compounds into any of 6 cooled traps based on the elution pattern indicated by the monitor FID trace.This is possible because lengths and diameters of capillaries from the effluent splitter are chosen so that the appearance of a compound or fraction of compounds on the monitor FID trace is concurrent with the arrival of the corresponding bulk of that compound or fraction of compounds at the preparative switching device. The sample to be fractionated is first chromatographed to generate a monitor FID signal only with the rest of the sample allowed to waste (zero trap). Based on this monitor signal times are suitably programmed to divert compounds or fractions of interest to selected traps. During subsequent "live" runs the preparative switching device diverts the choosen compound or fraction to one of six selected traps through another short capillary which leads from the switching device to the top of the trap. These traps can be cooled with LN2 to any selected temperature down to -196°C to suit the volatility of the compounds. All capillary connections at the effluent splitter and the preparative switching device are sealed with the proven Gerstel Graphpack design.
1756
C2 D2
TFC
(11^=^ TV1-B
B - Trap
sample trap 1 - 6
Figure 1 Principle of preparative device Analysis conditions Column Pneumatics
15M Supelcowax 10
od=0.53mm
df=1.0|im
Helium atSpsi
Temperatures : Cis 60° C to 270° C at 12° C/sec Oven 60° C to 150° C at 3° C/min then 30° C to 270° C and hold 16mins Transfer capillary 270° C Preparative switching device 270° C Traps at ambient temperature
1757 Injection :
Hewlett-Packard 7673A autosampler 2}il per injection. Splitless mode for 2 minutes. 150 injections with 50min cycle time per run.
2-DIMENSIONAL ANALYTICAL GC-MS, NITROGEN AND SULFUR DETECTION. For this approach the previous GC-MS system with its selective detectors was now mstalled as the main or satellite oven in a 2-dimensional configuration. This system has been previously described (2,3) and also incoporates an additional crosspiece after the second column so that the transferred cut from the main column is ftirther distributed simultaneously to the mass spectrometer and selective nitrogen and sulfiir detectors. In 2-dimensional heart cutting each one dimensional run on the precolumn has the potential to provide numerous sub-runs on the main column, and the present system for simultaneous detection of each of these considerably lessens the workload. The system is outlined schematically in Fig 2.
7 8 1 r~^
9
r v^"^
±h^ 5
\
3
U ^—^~X
2
Figure 2 Schematic diagram of the applied system which consists of a temperature programmable cold injection system with a septumless sampling head (1), a GC (2) configured with a monitor FID, column switching device (3) and pneumatics, connected via a heated transfer line incorporating a cryotrap (4) to a second GC (5) which has a micro crosspiece (6) installed after the main column with short colunm segments to nitrogen (7), sulfiir (8) and msd (9) detectors. Analysis conditions Columns
15M Supelcowax 10 od=0.25mm df=0.25^m Precolumn in GC 1 Maincolumn in GC 2 60M 5% Phenyl od=0.25mm df=0.25^m
1758 Column segments to selective detectors
5M 5% Phenyl od=0.25nim df=0.25|im
Column segment to MSD
15M 5% PHENYL od=0.25mm df=0.25|im
Temperatures
Oven 1 Oven 2
60° C to 130° C at 10° C/min, then 2° C/min to 270° C. 40° C to 200° C at 20° C/min, then 3° C/min to 300° C.
For each cut there will be an initial time on the second oven corresponding to the time required to transfer the cut through the heated transfer line into the second column.
RESULTS AND DISCUSSION The investigation into the composition of the spent lees extract consisted of a systematic approach based on the experimental techniques previously described, i.e. one-dimensional capillary analysis with MS and selective detection. preparative cutting and enrichment of a fraction of interest from the extract. further one-dimensional capillary analysis of this fraction with MS and selective detection to allow identification of additional compounds. 2-dimensional analytical capillary GC with simultaneous MS, sulfur and nitrogen detection for identification of interesting sulfur compounds indicated in the enriched cut. Fig 3 shows FID, sulfur and nitrogen traces of the spent lees extract. 2-phenylethanol and fatty acids are the dominant compounds and among a rich collection of sulfiir compounds methionol [3-(methylthio)-l-propanol] is the most abundant. The almost absence of quenching by predominant non-sulfur species on the chemiluminescent detector (4,5 ) is a major advantage for this type of sample, and this is evidenced by the very clear profile of lateeluting sulfur compounds. The nitrogen detector on the other hand is more selective than specific and some response is to be expected from abundant non-nitrogen compounds. A major peak is seen in the nitrogen trace at the elution time of 2-phenylethanol and is probably due to a combination of this compound and a coeluting nitrogen compound. In the splitless traces some peak asymmetry is evident in the region before 2-phenylethanol and this is most likely due to the high boiling point of this compound which is essentially the sample matrix. Since this compound does not elute until well into the run a good focusing effect is not available for those early eluting compounds. Table 1 details the principal compounds identified in the extract by GC-MS and the identifications were made by
Figure 3 FID (A), sulfur (B) and nitrogen (C) traces of the spent lees extract
1760 TABLE 1 Compounds from GC analysis of spent lees extract \
ScaaBttrober 98 205 657 739 786 907 940 1009 1037 1066 1075 1083 1096 1166 1183 1218 1238 1246 1250 1279 1341 1367 1375 1436 1487 1523 1549 1616 1699 1832 1881 1903 1967 2057 2072 2102 2142 2191 2238 2330 2342 2357 2408 2422 2436 2492 2511 2543 2706
ldBn.tiika.^pn: acetaldehyde 6thanol 3 -hydroxy-1 -propanol ethyl lactate 3-ethoxy-1-propanol * acetic acid furfural * ethyl 3-hydroxybutanoate * 2,3-butanediol (isomer 1) * ethyl 4-oxobutanoate * 2,3-butanediol monoacetate 2-methyl propanoic acid * 2,3-butanediol (isomer 2) ethyl levulinate butanoic acid * ^ -butyrolactone ** 1,3-propanediol diacetate furfuryl alcool 3-methyl butanoic acid ** 2(3H)-furanone,5-ethenyldihydro-5-methyl 3(methylthio) propanol pentanoic acid unknown ** 2,7-dimethyl-4,5-octanediol * ethyl 4-hydroxybutanoate * ethyl (iso) nicotinate hexanoic acid * benzyl alcool phenyl ethanol phenol jf-nonalactone octanoic acid 2,6-dimethyl 3,7-octadien-2-ol unknown unknown 4-ethyl phenol * 2-methoxy-4-vinyl phenol unknown decanoic acid 2-phenyl ethyl lactate * unknown unknown * monoethyl succinate unknown * a) 4-hydroxycinnamic acid * benzoic acid unknown dodecanoic acid vanilline unknown
1 2977 a) thermal decomposition product of 4-hydroxycinnamic acid
* : Compounds already reported in foods but not in whiskey (TNO) ** : Compounds not reported previously in foods (TNO)
R^fention md&i \ 851 957 1308 1362 1393 1473 1495 1539 1557 1575 1581 1586 1594 1638 1648 1669 1681 1686 1688 1706 1743 1758 1763 1799 1829 1851 1866 1906 1955 2036 2066 2079 2119 2177 2186 2206 2232 2263 2295 2357 2365 2375 2409 2419 2429 2467 2480 2502 2612 2741
1761
V
. ^ ^
\J u Figure 4 Monitor FID traces of: Spent lees extract (top) Fraction 1 (middle) Fraction 2 (bottom)
%
UJ
1762 comparasion with authentic standards either commercially available or synthesised in the laboratory. In general the elution region after 2-phenylethanol seems to be the most complex and interesting and it was therfore decided tofractionatethe sample by preparative GC to give two fractions, i.e. Fraction 1 : up to and including the 2-phenyl ethanol Fraction 2 : all compounds after 2-phenylethanol. A widebore polar column was used, as a gum phase was considered unsuitable in view of possible disadvantageous interactions between a non-polar phase and a high boiling polar matrix. For this application Supelcowax 10 proved to be a very usefiil carbowax phase as it has a stable upper temperature of 270°C. Fig 4 shows the preparative monitor FID trace of the original extract and the resulting fractions 1 and 2 taken from the traps after 150 autosampler injections. Fraction 2 is practically depleted of 2-phenyl alcohol and the desired enrichment of late-eluting compounds has been achieved. In addition off-line sensory examination indicated that the desirable maltlike aroma in the original extract was almost totally in fraction 2. This fraction (Fig 5) was again chromatographed similar to the original extract and Table 2 outlines some additional compounds found by GC-MS. These compounds are essentially esters of 2-phenylethanol and the fatty acids and their structures are shown in Fig 6.
161B95
2127
1977 1370 133J
1149
2210
715
1224 H * 10941 ^l^lll JlAJJuALWnAO
'Ab\66
WM
>>liJUjW
W:b6
V-yJI
^66' ""'8b':6(i
Figure 5 TIC trace of fraction 2 Since the preparative cutting to give fraction 2 also enriched the late-eluting sulfiir compounds it was decided to investigate these compounds and in particular the possible
1763 TABLE 2 Additional compound s after preparative cutting (fraction 2)
1
ScaB ftuaEnber 114 348 654 716 988 999 1020 1025 1052 1056 1066 1078 1093 1102 1112 1159 1162 1182 1222 1234 1260 1267 1304 1344 1355 1378 1403 1412 1425 1434 1502 1529 1535 1560 1578 1597 1629 1639 1662 1674 1724 1799 1817 1827 1832 1903 1940 1993 2025 2048 2065 2084 2128 2172 2179 2193 2207 2248 2284 2319 2419 2558 2600
Ideotl^Scatiiraf
** *
* * ** * * * * * * * * *
** * ** ** ** ** * * *
** * ** * *
** * * * *
**
3-methyl-l -butanol 1,3-oxathiane methyl decanoate ethyl decanoate unknown 2-phenylethyl acetate unknown ethyl dodecanoate 3-methylbutyl decanoate (1 ou 2)-methyl naphtalene unknown unknown 2-phenylethyl 2-methyIpropanoate 2-phenylethyl propanoate (1 ou 2)-methyl naphtalene -octalactone actinidol (isomer 1) actinidol (isomer 2) 2-phenylethyl butanoate 2-phenylethyl 2-methylbutanoate 2-phenylethyl 3-methylbutanoate diphenyle diphenyle methane ethyl tetradecanoate 1,1-diphenyle ethane 2-phenylethyl pentanoate 2-nonen-4-olide 1,2-diphenyle propane 3,8-terpineol hydrate 1,2-diphenyle ethane -decalactone nonanoic acid 2-phenylethyl hexanoate + unknown unknown 5-decalactone diethyl nonanedioatc unknown 3,4-diethyl biphenyle ethyl hexadecanoate nonenoic acid (unknown structure) 1,1-bis (p-ethylphenyl) ethane famesol unknown 1-hexadecanol 2-phenylethyl octanoate 8-dodecalactone unknown phenyl benzoate ethyl linoleate unknown unknown 2-phenylacetic acid 2-phenylethyl decanoate benzyl benzoate ethyl vanillate 4-hydroxy 3-methoxyacetophenone ethyl 2-phenylethyl succinate tetradecanoic acid unknown p-tert-butyl benzoic acid 2-phenylethyl dodecanoate unknown hexadecanoic acid
*^ ** : See TABLE 1 RiStentioa: index J 1223 1387 1605 1648 1828 1835 1849 1853 1872 1874 1881 1889 1899 1904 1911 1940 1942 1954 1978 1985 2001 2005 2029 2055 2062 2077 2093 2099 2108 2114 2161 2180 2184 2200 2213 2226 2249 2256 2271 2280 2314 2367 2381 2388 2391 2443 2471 2513 2538 2555 2568 2582 2615 2648 2653 2664 2674 2705 2731 2761 2826 2905 2926
1764 presence of similar esters of methionol and fatty acids. Fraction 2 in splitless mode gives a highly complex FID trace and even though the sulfur compounds can be located easily because of the specificity and sensitivity of the chemiluminescence detector, it is almost impossible to obtain clean mass spectra with normal 1-dimensional chromatography. Therefore 2-dimensional capillary polar to non-polar heart cutting was used and the vastly increased resolution due to chromatography on two phases of opposing polarity allows the required mass spectral data to be obtained. Simultaneous sulfiir detection of the cut after the second column also greatly facilitates location of the desired compounds in the MS trace. A similar approach and instrumental configuration was used to profile medium-boiling sulfur compounds in whiskey (6).
1
R: -C2H5
2-phenyl ethyl propanoate
2
R: -CH(CH3)2
2-phenyl ethyl 2-methylpropanoate
1
R: -C3H7
2-phenyl ethyl butanoate
4
R: .CH(CH2)C2H5
2-phenyl ethyl 2-methyl butanoate
5L
R: -CH2CH(CH3)2
2-phenyl ethyl 3-methyl butanoate
6
R: -C4H9
2-phenyl ethyl pentanoate
2
R: -C7H15
2-phenyl ethyl octanoate
£
R: -CH(0H)CH3
2-phenyl ethyl lactate
9.
R: -(CH2)2 COOC2H;5 ethyl 2-phenyl ethyl succinate
Figure 6 New phenyl ethyl esters found in spent lees and not reported yet as occuring in whiskies Fig 7 shows the simultaneous TIC and sulfur trace resulting from one such cut. Ten cuts in total were processed in this fashion and mass spectra were recorded for many new compounds. Two esters of methionol were suggested from mass spectral data and confirmed by the following synthesis of the compounds. 0.1 gr of methionol was mixed with an equimolar amount of the acid and a catalytic amount or p-toluene-sulfonic in ether and left in a sealed ampoule at 35°C for 24 hours. The mixture was then injected into the GC for retention and mass spectral data on the sulfur ester. The structures and mass spectra of the two sulfur esters are given in Fig 8
1765 We are now proceeding to examine the significance of possible trace of these compounds in actual whiskey.
Figure 7 Simultaneous TIC and sulfur traces of a 2-dimensional cut from fraction 2
1766 188
100%
-V
i£
0
161
il lylii,JIMA[
1155
p60 iii|iui|iSminniii»i""i""l""i
100%
11
-V"^ 0
106
• >'l'in.,'|lWi'|
u UlU
»n^ ;i.|'». ^nHlfu-Mylly.',^
t^tn!•m^ll'lll•l|rlll|
Figure 8 Mass spectra of new sulfur compounds found in spent lees 3-(methylthio)propyl decanoate IQ 3-(methylthio)propyl dodecanoate H
REFERENCES 1. Rose, A.H. (1977) Economic Microbiology, Vol 1, Alcoholic Beverages, Academic Press, London. 2. K Mac Namara, P Brunerie, S Keck, A Hoffinann. Food Science and Human Nutrition. (1992). G. Charlambous (Ed). Elsevier Science Publishers. 3. K Mac Namara, A Hoffmann. Fifteenth International Symposium on Capillary Chromatography, Riva del Garda. (1993). 4. N Johansen, J Birks. American Laboratory. (1991). 5. A Hov^ard, L Taylor. Journal of High Resolution Chromatography, Vol 14, December (1991). 6. K Mac Namara. Premier Symposium Scientifique International de Cognac. (1992). R Cantagral (Ed). Lavoiser, Paris. ACKNOWLEDGEMENT Many thanks to Beatrice DECARPENTRIE for substantial technical assistance.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence B.V. rights reserved (p\ 1995 iQQ';Elsevier T7lcm7i*»rScience SiriRnrp R All V All riffhts reserved
1767
An Application of Centrifugal Counter-Current Chromatography on Flavor Chemistry — Separation of Aroma substances in Whisky New Distillates — Takayuki Taniguchi, Norifumi Miyajima and Hajime Komura Suntory Ltd., Research Laboratories of Distilled Spirits and Liqueurs, Suntory Techno-Developement Center, Yamazaki 1023, Shimamoto-cho, Mishima-gun, Osaka 618, JAPAN. Abstract Aroma compounds in whisky new distillates were separated by centrifugal counter-current chromatography. More polar compounds, such as alcohols and aldehydes, were easily separated from less polar esters and acetals as were expected. Moreover, less polar compounds were further separated from each other based on their small difierence of partition coefficients, indices of polarity. Sulfur containing compounds were separated from corresponding non-sulfurous compounds of similar nature because of the higher polarity of sulfur atom(s). In fractions purified from whisky new distillates by this method, three compounds with common 3,4-dithiapentyl residue, namely an alcohol, its ethyl ether and acetate, were identified. INTRODUCTION For the identification of component(s) responsible for any activity, purification techniques sometimes become crucial factor even sophisticated and efficient analytical equipments have been developed. For example, gas chromatography established its position as one of the most efficient and popular analytical methods for aroma substances with rather smaller molecular weights by the introduction of high performance fused silica capillary columns and sensitive and spjeciric detectors. However, the presence of so many aroma components in the mixture requires us to perform preparative separation in order to achieve the determination of the structures of key components. Many different purification techniques such as fractional distillation, liquid chromatography or gas chromatography have been introduced for organic compounds. Counter-current chromatography (CCC) is a liquid-liquid partition chromatography between two immiscible solvent layers. This method has no solicl supports which may give irreversible
1768 adsorption, thus enables us to recover all the components put on separation. CCC is also characteristic with its high volume ratio of the stationary phase to the total column volume, that is, a large capacity for the size of the stationary phase, to permit us preparative scale separation with high resolution. Most of the CCC systems, though they are called "counter-current", have a current of one solvent flow, a mobile phase flow, through another solvent layer which should be kept in the separation column(s), thus called stationary phase. In such system, stationary phase has to be retained in the separation column against the mobile phase flow. Many types of CCC have been developed, but only three of them. Droplet Counter-Current Chromatography(DCCC)[l], Rotation Locular Counter-Current Chromatography(RLCC)[2] and Centrifugal Partition Chromatography(CPC)[3], are commercially available. In general, CCC is most applicable for the separation of rather polar substances such as glycosides and saponines[4]. In the field of flavor science, CCC has been used for the separation of flavor precursors in wines, grapes, and other fruits[5,6]. Only a few applications, however, can be seen for the separation of less polar compounds such as terpenoids[7]. In order to establish the wide applicability of CCC for the separation of less polar compounds, we have separated aroma compounds in whisky new distillates by a commercial Centrifugal Partition Chromatography (CPC), a kind of the droplet counter-current chromatography. In contrast to DCCC, which is performed in the gravity field, CPC is operated in the centrifugal field to retain the stationary phase solvent more firmly in the separation columns. Centrifugal force can also shorten the operation time with high efficiencies by making the mobile phase move much faster through the stationary phase. Centrifugal force can be adjusted by the rotor speed suitable for any solvent systems. In this report, a separation result thus obtained on CPC will be discussed, together with the identification of three sulfur-containing compounds with common 3,4-dithiapentyl group. METHODS Preparation
of Aroma Concentrate from
whisky new
distillates
Whisky new distillate (300 mL) was diluted with 1,500 mL of distilled water and was saturated with NaCl. The aqueous ethanol solution was then extracted with 1,500 mL of distilled dichloromethane for three times. The extracts were combined, dried over anhydrous Na2S04 and concentrated to ca. 3 mL using Snyder column at atmospheric pressure in a water bath (60 °C). Centrifugal
Partition
Chromatography (CPC)
Centrifugal Counter-Current Chromatograph Model CPC-B92 (Sanki Engineering Ltd., Kyoto, Japan) with 12 sets of separation column cartridges Model 250W which can hold 250 mL of solvent was used in this study (Figures 1, 2). Solvent system employed was pentane : 40 %
1769
(a) centrifuge Pwn^P
valve unit
Figure 1. Sanki centrifugal counter-current chromatograph (photographs from Sanki Engineering Ltd.) (a) Total view (b) Separation column cartridges in the rotor
1770 aqueous ethanol : dichloromethane = 6 : 6 : 1 (v/v), and the upper layer (less polar organic layer) was used for the mobile phase while the lower layer (more polar aqueous layer) for the stationary phase. Rotating unit where separation column cartridges sit was rotatecl at 500 r.p.m.(ca. 120 g) throughout the operation. After filling the separation columns with the stationary phase solvent, 1 mL of aroma concentrate was charged through sample inlet loop, then the separation was started by pumping the mobile phase solvent at 3 - 4 mL/min. Under this conmtion, the pressure at pump head should not exceed 50 Kg/cm^. The eluate was collected by 100 drops/fraction with a fraction collector (ISCO Model 1200). After collecting 150 fractions, stationary phase was recovered by reversing the flow direction and pumping ca. 300 mL of the mobile phase solvent. Fractions were combmedf into 17 groups according to aroma characters of each fraction, which was sniffed directly at the mouth of fraction tube. The groups of fractions and the residue fraction recovered from the stationary phase were subjected for gas chromatographic analysis and sensory evaluation.
centrifugal force -^^fliiiiiiiiiiiiii
/ mobile phase flow
Figure 2. Schematic illustration of centrifugal partition chromatography (in ascending mode) 1: rotatory seal joint. 2: connecting tube. 3: separation columns 4: mobile phase solvent. 5: sample inlet loop. 6: pumping unit 7: fraction tubes. Gas
Chromatography
Analysis of principal volatile components in CPC fraction groups and the original whisky new distillates were operated with Hewlett Packard gas chromatograph Model 5890 series II equipped with Flame Ionization Detector (FID). A fused silica mega-bore column coated with
1771 5 % phenylmethylsilicone (J&W DB-5, 30 m, 0.53 mm I.D., 0.15 mm film thickness) was used for all the analyses of the principal components at following temperature condition. Column temperature was raised to 230 °C at 5 ''C/min after holding at 42 °C for 8 minutes and finally held at 230 °C for 33 minutes. Sulfur containing cornpounds were analyzed with Shimadzu gas chromatograph Model GC 14A equipped with Flame Photometric Detector (FFD). A fused silica capillary column coated with polyethylene glycol (J&W DB-WAX, 60 m, 0.32 mm I.D., 0.25 mm film thickness) was employed. Column temperature was programed as follows: Initial temperature was set at 60 °C and raised to 110 °C at 4 °C/min without initial holding time. At 110 °C, the temperature gradient rate was changed to 3 °C/min and was held at 220 °C for 30 minutes. Gas
Chromatography-Mass
Spectrometry
Gas chromatography-mass spectrometry was performed with Hewlett Packard gas chromatograph Model 5890 series II equipped with MSD Model 5971A with interface and ion source temperatures at 280 and 190 °C, respectively. Gas chromatographic condition was the same as those of the principal components except for column; Hewlett Packard, Ultra 2, 50 m, 0.32 mm I.D., 0.52 mm film thickness. The mass spectra were obtained by scanning from m/z 40 to 350 in electron impact mode at 70 eV for EM voltage. Identification of the GC peak components of the principal volatiles as well as sulfur containing compounds were achieved by comparing the retention times of each GC peaks with those of authentic compounds and/or by analyses of their mass spectral data. Sensory Evaluation of each Fraction Group Sensory evaluation of each fraction group was performed as follows: Aliquots of 20 % (v/v) aqueous ethanol (10 mL) were poured into test glasses and a few drops of concentrated CPC fraction groups, of which solvent had been converted to ethanol, were added. Free comments on aroma characters of each fraction group were collected from five well-trained panels. RESULTS AND DISCUSSION Separation of whisky aroma compounds on CPC In counter-current chromatography, separation of compounds in the mixture should be depending solely on the partition coefficients of each components between the stationary phase and the mobile phase. The artition coefficients are determined by the balance between the ydrophobicity and the hydrophilicity, roughly speaking, by the Eolarities of each compounds. Therefore, less polar compounds should e eluted faster because less polar solvent was used for the mobile phase in this study.
P
1772 For CCC, any immiscible solvent sets can be used for the base solvents in combination with intermediate solvent(s) which is miscible with both of the base solvents. In the same time, the base solvents have to have high ability to dissolve compounds in question. However, considering the nature of aroma substances, which exist only in small quantities and tend to vaporize easily azeotropically or by themselves, organic solvents with low boiling points should be employed. The solvent system, pentane-dichloromethane-aqueous ethanol, is one of the combination fulfills the requirements because pentanedichloromethane mixture and/or water can dissolve organic compounds in wide polarity range. In the case of pentane-dichloromethane/water combination with some ethanol as the intermediate solvent, dichloromethane can behave also as the intermediate solvent. The ratio of each solvents, pentane : dichloromethane : 40 % aqueous ethanol 6 : 1 : 6, was obtained empirically by performing preliminary examination. Aroma concentrates obtained from whisky new distillates were separated into 17 fraction groups and the residual stationary phase using CPC. Interesting comments were obtained by sensory evaluation of each fraction groups (Table 1). Particularly in the earlier fraction group, aroma characters were more unique and distinctive than later fraction groups which were less pronounced. No distinctive odor were recognized in fraction group #16 and #17. Table 1 Odor characters of CPC fraction groups Fraction groups 1 2 3 4-5 6-7 8-10 11 12-14 15 16-17 SP
Odor characters
Estery, Fatty, Yeasty Hay-like, Leafy, Husky, Fruity, Refreshing Sweet, Estery, Cooked mash. Hay-like Coffee, Cooked, Toasted, Green tea Cooked, Fatty acid, Amine Aldehydic Metalic, Aldehydic Rosy, Yeasty, Fatty acid. Sweet Rosy, Solvent Ethanol Sweet, Chocolate
SP : Recovery from stationary phase The separation profile of the principal components on CPC is summarized in Figure 3. Macroscopically speaking, polar compounds such as ISO- and active-amyl alcohols and 2-phenethyl alcohol were retained longer in the separation column and were eluted later in the range of fraction groups #11 to #17. More polar compounds, such as ethyl lactate, were still remained in the stationary phase even after collecting 150 fractions (ca. 300 mL), thus these compounds were
1773
I
I
i
TT
n
^6
CHO
s
A ^S^CHO
t£
^ ^^"^^y
rw
S
^^
OH
iRecovery from 10 11 i 12 13 14 15 ;16 17 istationary phase Fraction groups
2 i3
Figure 3. Compounds eluted in eachfractiongroup
-N^. ^
o
-
-H^o'
.
.
^o-H:
A.^A
(4)
-Nf. ^
^
o
o
Fr. 36
^
^
s
s
Fr.37 -FrGrl-
o'^
(1)
(2)
^
^
A
.
^
Ethyl esteijs Acetates
^
(3)
-N/-.
K Fr. 38
^4^=
Fatty Acids
«v—^ ElO
EtO
Fr.40 -FrGr 2-
Jl
Fr.44 FrGr 4-
Figure 4. Compounds eluted in earlierfractionsby CPC (FrGr : Fraction group )
Acetals
Fr.47 -FrGr 5-
1774 recovered only from the stationary phase. On the other hand, less polar compounds such as acetates, fatty acids and their ethyl esters were eluted in the fraction group #1 to #5 as were expected. Microscopically observing (Figure 4), the compounds in the earlier fraction groups were further separated from each other by the small difference of their partition coefficients. In the first fraction, the ethyl esters of C6 to Cl 8 fatty acids and the acetates of C6 to Cl4 alcohols were found. Fatty acids and acetals were eluted a little later than them. The elution tendency of flatty acids was in order that the longer the carbon chains are, the faster they are eluted. Sulfur-containing compounds should play very important roles to the over-all aroma characters of whisky because of th eir unique aroma characters as well as of their low threshold levels. They were separated effectively from corresponding non-sulfurous compounds of similar nature. This achievement is due to the higher polarity of sulfur atom(s). For example, ethyl 3-(methylthio)propionate (1) was separated from ethyl hexanotate (2), and 3-(methylthio)propyl acetate (3) from hexyl acetate (4) as shown in Figure 4. These sulfurous compounds eluted earlier by CPC seem to be responsible for the distinctive aroma characters of the earlier fraction groups (See Table 1). In the case of complex mixture, fractionation is indispensable to correlate the components that may contribute to odor characters of the whole mixture witn their individual odor characters. In the special case such as "rosy" character existing from fraction groups #12 through #15, it is not so difficult to assign this odor to 2-phenethyl alcohol. Iiowever, in general, after only one step separation, it is almost impossible to clarify just by gas chromatographic analysis which compound is responsiole for particular odor characters picked up by the sensory evaluation, because even in only one fraction group, so many of the odorants exist. Therefore, in order to pin-point the key aroma compounds among all volatiles, further fractionation based on different separation mode and/or GC/olfactometry (sniffing) should be mandatory. 2. Identification
of three
sulfur
containing
compounds[8]
Sulfur containing compounds exist at so low level[9] that we have to analyze them sensitively and selectively by GC using Flame Photometric Detector (FPD). However, the identification of the peak component on GC/FPD is difficult in most cases not only due to their very small contents but also because of the simultaneous elution of the interfering non-sulfurous compounds existing by several thousand times (Figure 5). Therefore, we have tried to eliminate the interfering compounds from target peak components by using CPC to make the identification easier. Figure 6 shows GC/FPD chromatograms of original aroma concentrate and three of the CPC fraction groups. Among GC peaks, three compounds A, B and C with peak Nos. 12, 28 and 37 on GC/FPD are recognized as unknown components which were chosen for the identification target in this study. After CPC separation, two compounds A and B were found in fraction group #2 and C in fraction group #17. This means A and B are less polar and C is polar, because the former pair were eluted together with ethyl esters while the latter with alcohols. Furthermore, the fact that acetylation (with acetic anhydride/pyridine mixture) of all the
1775
GC/FID
lU
ul y
LA]lJ^^Jw.A-<-^Wl
GC/FPD
_JJ
VjuOi
LJWV_.J«1
y Ul IAJUI
lLi_L_A_.
_.vA
.^
A-
/v..
Figure 5. GC/FID and GC/FPD chromatograms of aroma concentrate Column : DB-WAX 60 m, 0.32 mm I. D., 0.25 |im film thickness Column temperature : 60-110 °C (4 °C/min), 110-220 °C (3 °C/min), 220 °C (30 min)
(1)
LJI
LMJJ
(2)
(3)
(4)
Figure 6. GC/FPD chromatograms of aroma concentrate (1) and the CPC fraction groups #2(2), #11(3) and #17(4) (1) Aroma concentrate (2) Fraction group #2 (3) Fraction group #11 (4) Fraction group #17
1776 components in fraction group #17 including C shifted the peak position of Cf to peak No. 28 corresponding to B, allowed us to confirm that B at peak No. 28 is an acetate of C. Thus, the presence of OH group in C was expected. GC/FID analysis of CPC fraction groups enabled us to confirm that most of the interfering compounds, which were invisible on FPD chromatograms, were eliminated after the CPC separation, but still it is not pure enough to obtain their mass spectra for the identification. However, after a silica gel chromatography and/or an octadecyl silica chromatography of these fraction groups allowed us to obtain semi-puriiied fraction with which mass spectra of the components were 152 104
59
2"o'
' ' 4*0
79
6'0
413
20
' ' i46
' i^d ' ' 1^6
87
106
60 .
' i66 ' ' lid
80
' ' ' ' I ''""' 1 I ' I ' I 1 I
40
60
I 1 I
80
1 I I I I
lOO
31
64
120
140
160
l60
124
.81
15
B
166
' 1 '' '' I ' ' ' ' I ' ' ' 'I '
93
-]—I—r-'T—I—I—i"'r I—I—\—I—I—I—r-^—I—I—r*—1—I—r—I—i—i—i—r""!—i—i—i—i—i—i—i—r~T—i—i—i
20
40
60
l6o
80
120
140
160
i•
l60
Figure 7. Mass spectra of compounds A, B and C
M^^^S^y^^y
Me
peak No. 12 (A)
Me>.,
J peak No. 28 (B)
Me
Me^,
*0H
peak No. 37 (C)
Scheme 1. Structures of compounds A, B and C
1777 recorded by gas chromatography-mass spectrometry. From the mass spectra of compound A shown in Figure 7, the presence of ethoxy {miz 45) and methyldithio {mIz 79 and/or 80) functional groups were established as well as the molecular formula, C 5 H 1 2 O S 2 . unly two structures were possible for A ; one with two functional groups on the same carbon and the other on different carbons. It should be noted that A gave the superimposable mass spectrum reported by MacNamara [10], but the structure had not been assigned. As mentioned before, B is the acetate of C . Mass spectral study of B also allowed us to evidence the presence of methyldithio group as well as the acetoxy group {mIz 43) in B . Thus, the presence of a common structural fragment, 3,4-dithiapentyl residue, was expected for all three compounds. A , B and C . By comparing the mass spectra and the retention times with those of the synthetic compounds aerived from 2-mercaptoethanol, we could confirm the structure of these compounds being j,4-dithiapentyl ethyl ether (A), 3,4-dithiapentyl acetate (B), and 3,4-dithiapentanol ( C ) as shown in Scheme 1. All these compounds have very similar mushroom-like odor at lower concentration presumably due to their common 3,4-dithiapentyl skeleton.
CONCLUSION Using commercially available counter-current chromatography, aroma substances in whisky new distillates were separated within j to 4 hours without any loss of the components, as: 1. Polar compounds can be separated from less polar compounds. 2. Less polar compounds can be further separated from each other. Particularly, sulfur containing compounds are separated effectively from corresponding non-sulfurous compounds of similar nature because of the higher polarity of sulfur atom(s). Thus, it was shown that counter-current chromatography is effective tool also for the separation of less polar substances. From the CPC fractions separated from whisky new distillates, three sulfurous compounds with a common 3,4-dithiapentyl substructure were identified. There are still several more sulfurous compounds in whisky new distillates, which should be identified though it is known that some sulfurous compounds would be disappearing during maturation [9,11].
REFERENCES AND NOTES 1. 2. 3.
T. Tanimura, J. J. Pisano, Y. Ito, R. L. Bowman, Science, 169, (1970) 54 Y. Ito, R. L. Bowman, J. Chromatogr. Sci., 8, (1970) 315 W. Murayama, T. Kobayashi, Y. Kosuge, H. Yano, Y. Nunogaki, K. Nunogaki, J. Chromatogr., 239, (1982) 643
1778 4. For general concept, see: K. Hostettmann, M. Hostettmann, A. Marston, Preparative Chromatography Techniques, SpringerVerlag, Berlin and Heidelberg, 1986, pp 80 - 126 5. P. J. Williams, in: T. E. Acree, R. Teranishi (eds.), Flavor Science, American Chemical Society, Washington, 1993, pp 287 - 308 6. A. Lutz, P. Winterhalter, J. Agric. Food Chem., 40, (1992) 1116 7. For example: H. Becker, W. C. Hsieh, C. D. Verelis, G. I. T. Fachz. Lab., Supplement Chromatographic, 34, (1981) and H. Becker, J. Reichling, J. Chromatogr., 237, (1982) 307 8. Details of the structural determination should be published elsewhere. 9. M. Masuda, K. Nishimura, J. Food Sci., 47, (1982) 101 10. K.MacNamara, in: R. Cantagrel (ed.). Elaboration et connaissance des spiritueux, Lavoisier-Tec & Doc, Paris, 1993 11. M. Kurokawa, T. Fujii, M. Saita, in: G. Charalambous (ed.). Shelf Life Studies of Foods and Beverages, Elsevier Science Publishers B. V., 1993, pp 991 - 1002
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1779
Aroma compounds of arbutus distillates G. Versinf, R. Seeber^, A. Dalla Se^^a^ G. Sferlazzo^, B. de Carvalho'' and F. Reniero^ ^Laboratorio di Analisi e Ricerca, Istituto Agrario, Via Mach 1, 38010 San Michele all'Adige, Italy ^'Dipartimento di Chimica Industriale, Universita di Bologna, Viale Risorgimento 4, 40136 Bologna, Italy ^Direcgao Regional de Agricultura do Algarve, 8000 Faro, Portugal Abstract The composition of the arbutus distillate, a typical product of Southern Portugal, is studied also in order to give suggestions for improving its quality. 45 samples from 25 distilleries have been submitted to GC and, in part, to GC-MS analyses. A wide number of compounds, mostly of fermentative origin, have been identified. They are often in different quantities and ratios with respect to other fruit distillates. Several compounds of primary origin, of possible sensorial contribution, are identified. The frequent presence of a high level of total acidity, as well as of ethyl acetate, constitue at the moment a sensorial and technological problem to solve adequately. Chemometric techniques based on multivariate analysis have been used to study the relationships among the variables considered, and to grouping the various distillates. By using only two variables of technological and microbiological significance, seven outliers are evidenced, the other products constituting quite a homogeneous kernel. 1. INTRODUCTION The arbutus distillate is a typical product of southern regions of Portugal, obtained with traditional pot stills from fermented fruits of Arbutus unedo L., a wild bush-like tree. In 1990 European Community reserved it two different geographical denominations, medronheira do Algarve and do Bugaco, respectively. The aim of an EC Leader Project for revitalizing the traditional economy of the Serra do Calderao (Alentejo-Algarve) is to support the rural income also through the valorization of this product. In the context of the present project, this research is devoted to determine the composition of the distillate, as well as to advance suggestions for improving the quality level of the production. The work is based on the analysis of a wide series of distillates from different farms.
1780 2. MATERIALS AND METHODS 45 raw distillates of 1992 have been considered. 40 of them refer to double sampling from 20 distilleries, i.e. relative to distillations performed either with or without head cutting. All but one were produced with traditional pot still equipment heated by direct fire (Figure 1).
Figure 1. Drawing of a traditional equipment used for the production of arbutus distillates. The refrigeration coil is usually located outside the distillation room. In the last single case, a different modem equipment (arraste do vapor) was employed, that uses steam blowing through the mash, as it is usual for grape-marc distillation in the Vinho Verde region. Furthermore, in the case of head cutting, which is unusual in the traditional process, the first 10 cm of the mash at the top of concrete tank were discarded, with the aim of even more lowering a possible acetic off-flavour of the distillate. Rectification column was absent and the distillation was performed in a single step.
1781 2.1. GC analytical methods The most aboundant compounds were analysed through direct injection of the distillate in packed and capillary columns, as previously reported [1]. Components present at a level lower than 0.1 mg/L, or corresponding to poorly resolved chromatographic peaks, were enriched by extracting 10 ml of distillate, previously diluted (1:10) with water and added with 2-octanol as internal standard, with pentane/dichloromethane, 2:1, v/v (3 x 20 ml). The extract, after anhydrification with NaS04, was concentrated to about 0.5 ml before GC-MS analysis. GC-EIMS (70 eV) analyses were performed on a HP 5890 gas chromatograph, coupled with a HP 5979 Mass Detector connected with a HP 59943B Wiley Database, and equipped with an apolar PS-264 fused silica capillary column (Mega, Milan; 25 m x 0.25 mm i.d.; df=0.15 jLtm). Experimental conditions were as follows: injector temperature: 220 °C; splitless injection; carrier gas: He; programmed temperature: 1 min at 40 °C; 10 °C/min up to 60 °C; 2.5 ''C/min up to 190 °C; 30 min at 190 °C. The quantification was performed by relating the TIC area of each compound to that of 2-octanol, considering RF=1 for all compounds. Differences with respect to the results obtained by FID-GC analysis with direct injection of the distillate are possible. 2.2. Statistical data treatment Data evaluation was carried out by using PARVUS package [2] on a PC and SAS 6.06 package [3] on a DEC VAX 8250 computer, under VMS 5 - 5 . 1 operating system. 3. RESULTS AND DISCUSSION 3.1. Parameters typifying the arbutus distillates with respect to other fruit distillates Table 1 reports mean values and variability of some parameters (alcoholic proof, pH and total acidity) and of the concentration of compounds - as mg%ml p. A. (pure alcohol) - from GC analysis with direct injection of the distillates. The following remarks are possible: - the alcoholic proof ranges from about 44 to 57 %Vol., except for two samples with markedly lower and higher values, respectively: the range is typical for the distillation technique used; - methanol content (0.90 ± 0.078 g%ml p.A with a maximum at 1.01 g%ml p.A.), is well lower than the upper limit of 1.5 g%ml p.A. allowed by EEC Reg.n.1014/90 and n.3458/92; - the content of fermentation higher alcohols (193.1 ± 45 mg%ml p.A.) is roughly one half with respect to that of other fruit distillates, that of 1-propanol (14.1 ± 2.5 mg%ml p.A.) being even lower. The ratio between 3-methyl- and 2-methyl-l-butanol (3.06 ± 0.24) as well as that between the sum of isoamyl alcohols and 2-methyl-1-propanol (2.92 ± 0.36) are similar to those found for most fruit distillates [4]. 1-Butanol content (0.31 ± 0.23 mg%ml p.A.) is very low, as in the case of cherry brandies [4]. Traces of 2-butanol (1.2 ± 0.45 mg%ml p.A. with a maximum at 2.5 mg%ml p.A.) suggest, in accord with the low content of ethyl lactate (3.4 ± 4.5 mg%ml p.A. with a maximum at 20 mg%ml p.A.), the lack of significant lactic bacteria spoilage;
1782 Table 1 Content and variability of the principal volatile compounds and other parameters characterizing the 45 samples. N COMPOUND
Mean value*
Standard deviation*
Minimum value*
Maximum value*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
51.40 898.3 14.12 1.23 45.48 0.31 32.77 99.78 193.10 0.550 0.670 0.053 0.163 0.076 0.070 0.105 1.94 494.0 1.65 0.249 0.080 0.595 1.47 1.46 0.734 0.352 1.24 0.709 0.149 3.45 0.495 57.32 45.74 3.48 0.327 0.049 1.05 0.188 170.1 4.31 3.06 2.92
4.130 77.90 2.470 0.451 10.720 0.231 9.290 25.567 44.960 0.4190 0.3130 0.3159 0.0914 0.0300 0.0456 0.1023 0.503 563.21 1.760 0.1029 0.0655 0.5893 1.004 0.765 0.431 0.290 0.906 0.795 0.152 4.520 0.318 44.544 23.450 1.594 0.1516 0.0234 0.5633 0.0685 171.03 0.365 0.244 0.357
36.15 689 9.8 1.0 29.8 0.10 17.2 51.0 109.2 0.19 0.23 0.02 0.06 0.03 0.02 0.02 1.05 82.8 0.42 0.10 0.02 0.14 0.56 0.52 0.21 0.06 0.07 0.01 0.01 0.35 0.07 24.3 22.5 1.3 0.12 0.02 0.24 0.07 27.1 3.4 2.6 2.3
64.0 1014 23.1 2.8 85.8 1.3 68.4 180.8 343 2.4 1.8 0.20 0.50 0.20 0.26 0.62 3.1 2807 8.9 0.64 0.26 2.8 5.75 5.0 2.5 1.6 5.3 3.85 0.68 20.0 1.9 319 135 9.3 0.79 0.12 3.6 0.37 889 5.6 3.9 3.8
Ethanol methanol 1-propanol 2-butanol 2-methy 1-1 -propanol 1-butanol 2-methyl-l-butanol 3-methyl-1-butanol higher alcohols 1-hexanol CIS 3-hexen-l-ol 1-heptanol 1-octanol 1-nonanol 1-decanol benzyl alcohol phenethyl alcohol ethyl acetate isoamyl acetate hexyl acetate phenethyl acetate ethyl caproate ethyl caprylate ethyl caprate ethyl laurate ethyl miristate ethyl palmitate ethyl linoleate ethyl linolenate ethyl lactate diethyl succinate acetaldehyde acetal furfural nonanal + linalool trans furan linalool oxide a - terpineol vitispiranes total acidity'' pH 8/7 (7 + 8) / 7
% Vol. for 1, mg%ml p.A. for 2 to 39; ^ as acetic acid
1783 - a limited level of acetaldehyde plus acetal (103 ± 64 mg%ml p.A.) was mostly found, even in the presence of relatively remarkable values for acidity indexes (pH and total acidity) and for ethyl acetate content, which is typical for acetic metabolism of ethanol; - the sum of apple, banana-like fruity scenting acetates of higher alcohols (isoamyl alcohols, 1-hexanol and phenethyl alcohol) is usually not higher than 2 mg%ml p.A., but increases up to about 10 mg%ml p.A. in the correspondence with the highest levels of ethyl acetate; - the contents of low to middle boiling ethyl Cg ^ Cij-esters, with a tropical fruit scent, as well as those of high boiling esters, are also similar to those of other fruit distillates [4]. In particular, ethyl caprylate is as the average at the same level of ethyl caprate, while the unsaturated Cjg-esters in some cases are and in other cases are not present at a significant level; - quite an anomalous situation, probably related with a peculiar catabolism of unsaturated Cjg - acids in such substrate, is found as to C^-alcohol concentrations: a rather low content of 1hexanol (0.55 ± 0.42 mg%ml p.A.) results, strangely lower than that of the cis 3-hexen-l-ol (0.67 ± 0.31 mg%ml p.A.), the trans 3- and 2-hexen-l-ols being only present in traces lower than 10 /xg%ml p.A.. Among the fruit distillates, only the cherry brandy shows a mean value of 1-hexanol at 0.8 mg%ml p.A. [4]. However, the level of C7 -j- Cio-aliphatic alcohols, all coconut-like scenting, is similar to that of most fruit distillates, the highest values resulting in the correspondence to those of Q- and benzyl alcohols, similarly to what has been found in distillates produced by direct steam distillation; - the content of phenethyl alcohol and of furfural varies should vary manly in function of the extent of tail cut. That of furfural (3.5 ± 1 . 6 mg%ml p.A.) shows different values in some distilleries, probably in relation with the kind of mash heating process. Among the varietal compounds which could give a peculiar aroma contribution to this kind of distillates, we count several monoterpenols, like trans and cis furan linalool oxides, a- and jS-terpineol, linalool, geraniol, bomeol and carveol; citronellol, nerol and geraniol only result to be present in traces. Norisoprenoids as vitispiranes, the so-called Riesling acetal (2,2,6,8tetramethyl-7,ll-dioxatricyclo[6.2.1.0*'^]undec-4-ene; [5]) and TDN (l,l,3-trimethyl-l,2dihydronaphthalene) were also detected. Some of these compounds were quantified with respect to 2-octanol in 10 randomized samples by GC-MS analysis. The results are given in Table 2; some reference compounds already quoted in Table 1 are also reported, a-terpineol, the most important monoterpenol, is present at the remarkable level of 1.05 ± 0.56 mg%ml p.A. and likely contributes to a pine-like aroma (threshold level in wine at 0.4 mg/L [6]). The presence of /3-terpineol, already found in Cognac [7], at a level lower than one tenth of a-terpineol can be accounted for by hydrolytic degradation of monoterpene glycosides during the distillation [8]. Linalool, with an average value of 0.1 mg%ml p.A. (threshold level in wine at 0.1 mg/L [6]) could also be flavour-active with a floral muscat-like scent. We recall that bomeol, which was already found in wine distillates [7], and carveol, a typical compound of caraway and dill, both present in a quantity close to 10-20 ii%%m\ p.A., have a camphoraceous, earthy-peppery [9] and a resinous-spicy scent, respectively. An unidentified monoterpenol (unident. 1), with RT on PS-264 apolar column close to that of nerol and characterized by the following MS spectral data, was evidenced: 31(20); 41(100); 43(31); 53(25); 55(19); 67(38); 69(71); 77(21); 79(14); 81(20); 91(20); 93(42); 107(8); 108(9); 109(8); 121(24); 136(5). As for the noisoprenoids found, the vitispiranes are the most aboundant with 0.19 ± 0.068 mg%ml p.A. and roughly equivalent concentrations for the two diastereomers; a
1784 camphoraceous, eucalyptus-like odour contribution from these compounds seems possible, considering the threshold level in wine of the isomer mixture at about 0.4 mg/L [10]. Table 2 Compounds quantified by GC-MS in 10 randomized samples after enrichment with organic solvent. COMPOUND
Mean value*
Standard deviation*
Minimum value *
Maximum value*
MONOTERPENOLS trans furan linalool oxide cis furan linalool oxide linalool a -terpineol /3 -terpineol bomeol carveol unidentified 1
134.7 81.3 92.1 439 29.8 18.0 15.0 37.8
46.4 18.7 36.0 114 8.15 16.3 2.9 13.4
79 53 63 299 20 4 12 19
234 105 178 699 43 62 22 64
NORISOPRENOIDS vitispiranes TDN Riesling acetal
217.6 13.6 132.5
77.8 7.7 47.1
119 6 84
340 26 205
OTHERS styrene ethyl cinnamate ethyl benzoate phenylacetaldehyde ethyl 2-furoate 4-ethyl-2-methoxyphenol nonanal l-octen-3-ol unidentified 2
101.3 27.6 75.7 18.4 65.2 36.7 79.3 10.7 199.8
118.5 13.0 52.5 8.1 22.8 35.2 43.1 9.1 92.5
27 12 21 4 40 7 42 4.5 43
428 55 167 30 HI 104 188 36 327
* as /xg of 2-octanol %ml p.A. The Riesling acetal, already evidenced in quince brandy [11] and mostly also camphoraceous scenting, is present in arbutus distillates at about one half the concentration of vitispiranes, while TDN (14 ± 8 jLtg%ml p.A.) - resembling kerosene flavour at a level of about 20 ptg/L in wine [12], but at 0.6 ppm in wine distillates [13] - is approximately one tenth with respect to Riesling acetal content. It is worth mentioning that Riesling acetal and TDN can be likely derived from glycosides of 3,6-dihydroxy-megastigm-4-en-9-one,
1785 while vitispiranes can derive from those of the corresponding reduced form, the 3,6,9trihydroxy-megastigm-4-ene; both pathways have been proposed and interconnected by Waldmann and Winterhalter [14]. Other peculiar compounds are, among the aldehydes, nonanal, whose approximate average content can be calculated by difference between the sum of nonanal and linalool contents, and single linalool content, as computed by GC-MS. Phenylacetaldehyde is also present in smaller quantity. Some aryl compounds, like ethyl cinnamate, styrene, ethyl benzoate and 4-ethylguaiacol, have also been found. While 4-ethylguaiacol is usually considered a bacterial derivative of ferulic acid, the raspberry, plum-like scenting ethyl cinnamate (28 ± 13 /xg%ml p.A.; threshold level at 10-20 jLtg/L in wine [15]) should be originated, like styrene, by the anaerobic metabolism of intact fruit cells during the silage period, as it is supposed to happen for grape-marcs [16]. A reduction of styrene content could be therefore advisable. This goal could be achieved through a better mashing of fruits at the ensilage. Among other compounds found, we recall traces of l-octen-3-ol, with an intense mushroom odor and a threshold level in wine of 20 fig/L according to Amon et al. [17], likely originated from metabolism of mould. Other compounds identified, but not quantified here, are: 1,1-diethoxy-isopropane, -isobutane, -pentane and -hexane; benzaldehyde and its 1,1-diethoxyderivate; 2-acetylfurane; 5-methyl-2-furfural; diethylmalonate; ethyl heptanoate and nonanoate; C6-C12 fatty acids and relevant methyl and isoamyl esters; ethyl 3phenylpropionate; maltol; C12-C16 aliphatic primary alcohols and diethylfumarate. Finally, the MS-spectra of another unidentified compound (unident. 2), which is present in quite a high content and with RT close to that of phenethyl acetate, is reported: 39(58); 41(21); 51(39); 53(20); 63(17); 65(27); 77(100); 79(73); 91(27); 93(21); 107(25); 108(44); 121(46); 135(1); 150(27). Histograms of data in Table 1 allow the recognition of a rougly normal distribution for most of them, except for 2-butanol and ethyl linoleate and linolenate, that exhibit a dichotomic-like distribution. 3.2. Multivariate data treatments 3.2.1 Cluster analysis on the variables A hierarchic cluster analysis on the variables was performed in order to group them and to draw out information from possible similarities. Among the groupings, we put in evidence those basically justified by similar distillatory behaviour, as already verified in other cases of pot still distillation [1]. Thus, isobutyl and both isoamyl alcohols and the ethyl esters from caproic to palmitic acids are put together; ethyl and isoamyl acetates, acetaldehyde, acetal and 2-butanol as well as all the homologous alcohols from 1-hexanol to 1-decanol are also collected in two different groups. Methanol is grouped together with furfural and alcoholic proof with pH, both these groupings being markedly influenced by tail cut extent. The singling out of the primary compounds, as well as of 1-propanol, could suggest that their variability is conditioned more by casual phenomena or by different substrate characteristics than by differences in distillation procedures.
1786 • W 7.5
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0° 4.4
r ' = 0.8221 5.5 6.6 ETHYL ACETATE
7.7
Figure 2. Correlations between some variables (in mg%ml p.A.); values under logarithmic form; symbol • indicates the objects corresponding to outliers in Figures 3 and 4. From linear correlations between two variables, some exemples of which are reported in Figure 2, possible outliers can be evidenced: some are the same as those resulting from Principal Component Analysis (PCA) data treatment, which is discussed later. 3.2.2 Possible discrimination of the objects on the basis of a distillation variant As for a possible discrimination between the objects of the 20 pairs of products distilled with or without head cutting, respectively, no single variable shows clear difference between the two groups. In order to apply the Linear Discrimination Analysis (LDA) to the two categories, a proper stepwise feature selection procedure has been applied as a pretreatment of data devoted to pick up the variables with highest discriminant ability. If considering only ethyl caproate, the most discriminating variable, a 65.5% classification and 65.6% prediction ability result. The latter datum is worse (61.3%) when adding four more variables, the classification capability improving only a little bit. Acetic spoilage indexes do not result to be useful to this discrimination, probably because such a modification of distillation technology, even if influencing a head-hybrid component as the ethyl caproate - and therefore the similarly distilling compounds - is not sufficient for overcoming some uncontrolled markedly different substrate situations and therefore, in practice, for eliminating a possible acetic scent. An explanation for this could be found in the unusually long time - up to two
1787 or three months -spent in fruit picking, that leads to frequent additions of fresh product to the open tank, covering previous layers already fermenting and possibly also spoiling. 3.2.3 Investigation of possible groupings of objects Since from the above investigation no significant discrimination of the distillates on the basis of the mentioned distillatory variations can be evidenced, each single sample of each distillery can be reasonably considered as a randomized sampling. A first rough comparison among the distillate aroma profiles evidences that both the products from the producer that follows a non-traditional distillation method {arraste por vapor), are characterized by notably higher quantities of several compounds, like not only ethyl and higher alcohols acetates, but also 1-butanol, 1-hexanol, cis 3-hexen-l-ol, a-terpineol and furfurol. At the same time, vitispiranes, methanol and some other aliphatic alcohols are present at the highest levels.
»
- 6 - 3
0
3
6
EIGENVECTOR 2 (16.6% OF VARIANCE) Figure 3. Biplot for the data of distillates. Scores of the objects and loadings of the variables in the plane defined by the first two eigenvectors. Significance of the labels are included in Table 1; symbol • indicates the same outliers. PCA with 34 variables. In order to carry out an unsupervised description of all objects and to reduce the dimensionality of the system described by 34 variables with acceptable normal distribution, PCA was applied to autoscaled data. The compounds 2-butanol, and ethyl linoleate and linolenate were excluded because being nearly dichotomic variables.
1788 The loadings of some variables representative of quoted groupings from cluster analysis, are drawn in the biplot referred to the plane originated by the first two eigenvectors (Figure 3). By taking into account the discriminant scores resulting from the first three eigenvectors explaining 66.7% of the total system variability, at least 8 outliers, only two of which referred to one single producer - the same above mentioned - can be observed. By removing these outliers and applying the same statistical treatment, two other odd samples result as further outliers. No other grouping can be singled out and this could indicate that most distillates represent an interesting quite homogeneous group of products. As a further step, a reduction of variable number was achieved by selecting, via multiregression analysis, those with the highest weight on the dependent variables represented by the eigenvectors of PCA with 34 variables. This regression was calculated five times, corresponding to the component number evidencing the 8 outliers. If the presence of higher loadings for each eigenvector, that corresponding to the variable with lower analytical error and giving diff-^rent distillatory information with respect to the other variables chosen, was selected. The selected variables were ethyl laurate, total acidity, acetaldehyde, phenethyl alcohol, and ethyl acetate.
EIGENVECTOR 2 (23.7% OF VARIANCE) Figure 4. See Figure 3. PCA with 2 variables. By using these data, the PCA data set scores on the plane determined by the first two eigenvectors, put in evidence the same outliers as in the case of 34 variables, one outlier being evidenced on another plane. 7 of the 8 outliers were however evidenced by considering only two variables, the total acidity and ethyl acetate (Figure 4). The outliers are therefore mainly justified by distillatory (head and tail cuts) and/or microbiological (acetic spoilage)
1789 grounds. Most varietal compounds with sensorial influence, which were singled out in the cluster analysis of variables, do not seem to present any classificatory significance. 4. CONCLUSIONS Arbutus distillates produced with traditional equipment present quite homogeneous volatile profiles with interesting peculiarities of sensorial relevance and some off-flavour, which suggests improvements of mashing, silage and storage techniques. The introduction in the distillation of head cutting, as well as of discarding the upper part of siled matter, is not enough for obtaining a significant reduction of a possible acetic scent. As for peculiar flavouring compounds, it is worth to emphasize the presence, above all, of Qf-terpineol and linalool among the monoterpenols, and of vitispiranes, Riesling acetal and TDN among the norisoprenoids. Statistical data treatments using 34 variables show a limited number of outliers in the presence of most products as an undiscriminable kernel. This was confirmed by the results obtained using only two variables, but was not related with primary fruit peculiarities. Acknowledgement This research has been carried out in the frame of an EC Leader Project co-ordinated by Associagao "IN LOCO", 8000 Faro, Portugal. We wish to thank L. Galego, A. Martins, P. Soares and J. Varela for the on farm experimental part and samples collecting, as well as S. Inama, M. Ramponi and L. Ziller for the analytical support. 5. REFERENCES 1 2 3 4 5 6 7 8
G. Versini, A. Monetti, A. Dalla Serra and S. Inama, A. Bertrand (ed.), Les eaux-de vie traditionelles d'origine viticole, Lavoisier - TEC & DOC, Paris (1990) 137. M. Forina, R. Leardi, C. Armanino, S. Lanteri, P. Conti and P. Princi, PARVUS: An extendable package of programs for data exploration, classification and correlation, Elsevier, Amsterdam, 1988. SAS/STAT* User's guide. Version 6. Cary, NC: SAS Institute Inc., 1989. W. Postel, F. Drawert and L. Adam, F. Drawert (ed.), Geruch- und Geschmackstoffe. H. Carl, Numberg (1975) 99. P. Winterhalter, M.A. Sefton and P.J. Williams, Chem. Ind. (London) (1990) 463. A. Terrier, J.N. Boidron and P. Ribereau-Gayon, C.R. Acad. Sc. Paris Ser. D 275 (1972)941. R. ter Heide, P.J. de Valois, J. Visser, P.P. Jaegers and R. Timmer, G. Charalambous (ed.). Analysis of Food and Beverages, Academic Press, New York (1978) 229. P.J. Williams, C.R. Strauss, B. Wilson and R.A. Massy-Westropp, J. Agric. Food Chem., 30(1982) 1219.
1790 9 10 11 12 13 14 15 16 17
K. Bauer, D. Garbe and H. Surburg, Common fragrance and flavor materials, VCH, Weinheim, 1990. R.F. Simpson, C.R. Strauss and PJ. Williams, Chem. Ind. (London), 66 (1977) 663. R. Naf, A. Velluz, R. Decorzant and F. Naf, Tetrahedron Lett., 32 (1991) 753. R.F. Simpson, Chem. Ind. (London) (1978) 37. J.P. Vidal, R. Cantagrel, G. Mazerolles, L. Lurton and J. Gaschet, A. Bertrand (ed.), Les eaux-de-vie traditionelles d'origine viticole, Lavoisier - TEC & DOC, Paris (1990) 165. D. Waldmann and P. Winterhalter, Vitis, 31 (1992) 169. G. Versini, A. Dalla Serra, L Orriols, S. Inama and M. Marchio, Vitis, in press. G. Versini and T. Tomasi, L'Enotecnico, XIX (1983) 595. J.M. Amon, J.M. Vandepeer and R.F. Simpson, Wine Industry Journal, 4 (1989) 62.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1791
AROMA COMPONENTS OF RAKI i. Yava$ a) and A. Rapp ^) ^) Ankara University Agricultural Faculty, Food Engineering Department, TR-06110 Ankara /TURKEY t>) Karlsruhe University, Institute for Food Chemistry, D-76128 Karlsruhe /GERMANY
ABSTRACT Raki is the national alcoholic drink which is produced and consumed in Turkey. Took place 49,5 % in total alcoholic drinks. In experiments using direct injection methods with capillary columns the higher alcohols and 12 further aroma compounds could be separated and determined quantitatively in 35 minutes. After liquid-liquid extraction 110 different aroma compounds are detectable. The aromagrams show significant differences between the different Raki types. With the aid of GC/MS method many compoimds could be identified. By means of regression analysis with the selection method of backward elimination of factors it was possible to find out the right compounds for the differentiation of different Raki types. INTRODUCTION Aniseed aromaticed alcoholic beverages (aromatic high alcohol drinks) are produced by distillation of a mixture of agricultural alcohol and aniseed. These products consumed with different names which are "Raki" in Turkey, "Ouzo" and "Mastika" in Greece and "Pastis" in France, respectively. In Turkey raki consumption take first place concerning alcoholic drinks [1] (Table 1).
1792 Turkish Raki is produced by using high concentrated alcohol (93,5-94% vol) from grape spirit (Suma). Suma is distilled from fermentated raisins with special retorts by using special methods. The distilled alcohol is diluted to 45 % vol, mixed with aniseed (6 hrs steeped in water) and distilled a second time. Thefractionatedalcohol is used for alcoholic drinks which named Raki.
Table 1: Alcoholic Drinks Production in Turkey in 1993
Alcohol (Vol) Product Name
alcohol degree)
(%) Raki Wine Beer Vodka Cin
Turkish Cognac Liqueurs (average) Whiskv 1 Ihlara Brandy Total
45 US 4 40 47 41 30 43 35
-
Liter 67 330 000 27 886 000 552 310 000 7 682 000 3444 000 I 149 »XX) I 640 000 106 000 240 000
'
1 Part of
Production (1) (100'^c ethyl alcohol)
Production (With own
1
Liter 30 298 500 3 206 890 22 092 400 3 072 800 1 618 680 471 090 492 000 45 580 84 000 61 381 940
Part of
Total Distilled Alcohol 1 (•%)
Total Alcohol Drinks C^o)
84.0
49.4 5.2 36.0 5.0 2.6 0.8 0.8 0.1
S5 4J 1.3 1.4 0.1 0.2 1
lOO.O
j
0.1
1)
100.0
1
Reprinted from: Anonymous, Tekel 1993 Yiii Faaliyet Raporu, Alkollii Iqkiler Sanayii Muessesesi Mudurlugii, Istanbul, 1994.
Some countries also try to produce similar alcoholic beverages as "Raki". These are not Raki, because Turkish Raki has some special characteristics which are mentioned below: - Raki should be produced in Turkey - In Raki production raisin based alcohol or other agricultural based alcohol should be distilled two times by using special retort with aniseed {Pimpinella anisum) - Raki should be contain at least lOg sugar and 0.8 - 2.2g anethol per liter - Raisin alcohol should take place at least 40% in total Raki alcohol - Raki should be distilled up to 94,5% alcohol for preventing aromatic compound losses In Turkey distilled alcohol production is made under licence of Turkish Monopoly (Tekel) Organisation by custom laws. For producing 100 liter Raki, 150 kg raisin is
1793 needed for "suma" production[2]. Turkey has 576 000 ha vineyard area and grape production is 3 450 000 metric tons. Nearly 10% grape production is used for Raki production whereas only 1% is used for wine production.
RAKI PRODUCTION In Raki production raisins, aniseed and sugar are used as main ingredients. Some factories use fresh grape in Turkish Monopoly Organisation. In Turkey three different types of Raki are produced which are named Yeni (New) Raki, Kuliip (Clup) Raki and Altmba§ (Golden Head) Raki. Aniseed concentration is 80 g/1 in Yeni Raki, 100 g/1 in Kulup Raki and 120 g/1 in Altinba§ Raki. For Raki production (Figure 1) raisins mashed with a grinder. Before entering grinder tap water is added to the raisins for receive the ready Baume degree in the raisin juice. Baume degree should be 8-10 (d = 1.060 - 1.074). Mixture is heated 60°C for 20-30 minutes and then cooled to 22-25°C degree. After this step mixture is taken in a fermentation tank and then inoculated by yeast {Saccharomyces cerevisiae Rasse M and XII). After 2 - 2.5 fermentation days alcohol content is increased up to 8-10 % vol in mash. This mash is distilled in a continious distillation column to approximately 93.5 - 94% alcohol. This product is named "suma". Suma is stored in tank. Aniseed extract is prepared in a seperate tank. For extraction aniseed is steeped for 6 hours in water. This extract is added 6-12 % to suma which alcohol concentration is diluted with water to 45% vol. Generally distillation period is 46 hours. In fractionated distillation head (first), medium and last product is separated. Medium part is important and used for Raki production which alcohol concentration is 80% vol. First (head) part is 10-11% whereas medium part is 64% [3]. Alcohol concentrations of medium part should be diluted for different Raki types with good quality water. For instance Kulup and Altinba§ Raki contain 50% vol alcohol whereas Yeni Raki contain 45% vol alcohol.
1794
STARTER YEAST
Mash (H-10%, alc(jhoi)
Cuntinious distillation SUMA (93.5-94 %. Alcohol)
Dilution (45 %. Alcohol) ANISEED (6-12 %) •
Fractionated Distillation
M E D I U M PRODUCT
(8U %. Alcohol)
Dilution (45-50. Alcohol) SUGAR (4-6 g/1) Aging (Oak barrel)
YENi RAKI 45 % vol.
KULUP RAKISI 50 <7c vol.
I
II ALTINBA§ RAKISI 50 % vol.
Figure 1: Raki production pathway
At the end of the Raki production 6g sugar should be added to Kuliip and Altinba$ Raki and 4g sugar for Yeni Raki. Then Raki is stored in oak (Teuricum chamaedrys) barrels for a ripening process. This storage period should be minimum 20 days usually 30 days for Yeni Raki 60 - 90 days for Kuliip and Altmbas Raki. During this period airing is needed for 2-3 times.
1795 QUANTITATIVE DETERMINATION OF VOLATILE AROMA COMPONENTS
Generaly, chemical and organoleptic analyses of distilled alcoholic beverages are performing by using gaschromatography. In quantitative analysis not only odor and taste characteristic carried out, also quality and purity are determined. For this reason in distilled alcoholic beverages GC analyses are going to be more safe and important assay. In previous researches, gaschromatographic analyses with various separation columns were revealing in details [4-20]. Drawert and Rapp [4], Rapp et al. [5] examined volatile compounds by direct injection of wine, cognac etc. with different (Carbowax 1550, Diethylhexysebazat + Diethyl tartrat) separation columns. Reinhard [8-10] used Diethyl trimethylpropantripelargonat, 1,2,3-Tris (2-cyanoethoxy)-propan + Diglycerol and Polyethylenglykol coliunns for carrying out volatile compounds of whisky suma and other distilled alcohols. Postel et al. [13-16], determined various aroma compounds by using FFAP, Igepal, Ucon LP 550, Carbowax 400 and Triethanolamin in packed columns. Postel et al. [13], used various columns for determination 65 compounds in whisky quantitatively with direct injection methods. Rapp [19] used different capillary glass columns (90m, Carbowax 400; 0.4 mm i 0) for the determination of volatile compounds in various alcoholic beverages. With this method, using 0.5 - 1.0 ^il direct injection, it is possible to separate 3-Methyl-butanol-l and 2-Methyl-butanol-l and to determine 30 compounds in wines and brandies. Postel et al. [17] determined higher esters (C6-Ci6-ethylester and Cg-C9-methylester) from wine, distilled alcohol by using 50m-Fused silica column (crosslinked 5% Methylsilicone). Aktan and Rapp [21], determined by direct injection on packed columns the higher alcohols in various Raki samples. Yava§ and Rapp [22] determined volatile compoimds (especially anethol) in various Raki samples by direct injection, using appropriate capillary column. In this research Raki samples are obtained from local markets (Table 2).
1796 Table 2: Raki samples
Code
Product Name
A
Altinba§ ralci
50
Kuiup raki
50
-
Yeni raki
45
•
D
Lowcnmiich (Lion milk) Asian siitij
41
Like raki. appetizer produced with aniseed Made in Germany
E
Pernod
43
Oistiled alcohol with aniseed. Made in France
F
Ouzo
42
Greece appetizer, fresh grape, raisin, Macedonia aniseed. Coriander and 9 different spices an aromatic plant
E. Tsantalis, Chaikidiki, Greece
G*
Kulup raki
50
Produced grape and aniseed Made in Turkey
Tekel. Istanbul (Turkish Monopoly) |{
It B
I ^
1 "• 1 1 *
Yeni
Alcohol C^c. Vol)
raki
|
45
repetition of B and C
Type
Produced grape and aniseed 1 Made in Turkey
1
Firm. Company
Tekel. Istanbul (^Turkish Monopoly) ]
Teta AG. Miinchen
Pernod Fils.
Pans
1
1 1 II
I
G and H samples with a long bottle aging
Reprinted from: I. Yava§ and A. Rapp, Zur quantitativen Bestimmung von Anethol und fluchtigenAromakomponenteninverschiedenenRaki-Proben,Dtsch.Lebensm.-Rundsch, 81 (1985) 317-321. GC studies with direct injection methods 25 ml sample and 2 ^1 Pentanol-1 (standard) are mixed and from this mixture 1 jil is injected to glass capillary column directly. GC: Siemens L 350 Glass capillay column: 60m Durabond 5, diameter 0.32 mm, film thickness 1 ^m Split: 1:25 Carrier gas: Hydrogen Temperature programme: 50-160°C, 5°C/min
1797 In this research 12 different volatile compounds of Raki samples were determined quantitatively by using SE 54 (Durabond 5) capillary column (Figure 2,3; Table 3).For the determination of aroma compounds the standard coefficient were 2.0% for methanol, 8.3 % for capric- and caprylic acid ethylester and 5.1 % for anethol respectively.
L. 13 X2X112
n
io
^3
M,__u T.T X7X,12
11
10
TTin
976 5 32 1
•^—9-1?—Tin
Figure 2.
ABCD '
Figure 3.
Altmbaf Rakisi Kulup Rakisi Ycni Raki Asian SutU
TCE" F-
Test xokent (Blank) Ycni Raki Pernod Ou/o
Determined aroma components and peak number (22) -
Methanol
» » » -
Propanol-1 Accijcacjd cihylcsier i-Ouianol ButanoM I,l-Diciho)c>c(h3n
7 - 3-Meihylbui3nol-l 8 - 2-Mcthylbuianol-l 91011 12-
Pcntantil-I (-Standard) Hcxanol-I Caprnicacid ethylcsicr Capiylicacid elhylcMcr
13X| • X,X,Xj-
Anethol Unknoxn Unknown Unknown Unknown
1798 Concerning methanol concentration, minimum methanol were carried out in Pernod (12 mg/100 ml p.a.) whereas maximum in Asian siitu (Lowenmilch = Lion milk, 134 mg/100 ml p.a.). In Turkish Raki propanol-1 differed between 30 to 45 mg/100 ml p.a. whereas very low propanol-1 level were determined in Pernod and Ouzo. Propanol-1 could not be found in Asian siitu (Lowenmilch).
Table 3: Determination of aroma components with direct injection on capillary column in Raki samples (mg/100 ml p.a.)
Peak II Number
1
I 2 3 4 5 6 7 8 10 11 12
1 13
1
Aroma components
Rei.-Time (min)
3,3 4.7 5.6 5,8 6,5 8.3 8,4 8.6 12,9 17,9 25.3 31.0
{
Methanol Propanol-1 Acetic acid ethylcster {•Butanoi Butanol-1 Acetal (1.1-Diathoxyethan) 3-Methyi-butanoi-l 2-Methyt-butanoi-l Hexanol-1 Caproic acid ethylestcr Capiyiic acid ethylester Anethol
Altmba§ A
Kulup B
Yeni Raki C
60 30 11 57 1.2 0.90 143 39 0.20 0.24 0.28 330
78 45 1.4 85 0.06 78 6J 3.7 0.02 0.08
125 39 6.4 105 0J4 2.6 43 15
-
343
-
0.10
-
288
Asian Siitu D 134
-
1.7 0.02
-
0.26 0.09
-
0.18
-
260
'
Pemod E
Ouzo
12 2.0 l.l
48 0.30 0.94 0.05 OJl 0.05 0.20 0.05
-
0.16 0.04
-
0.20 480
i
F
•
0.14 •
92
Reprime d from: L Yava§ and A. Rapo^ i^ur quaiititativei iBesti mmun g von Anethol unc fliichtigen Aromakomponenten in verschiedenen raki-Proben, Dtsch-Lebensm.-Rundsch. 81(1985)317-321.
Acetic acid ethylester level is maximum in Altmba§ Raki (11 mg/100 ml p.a.), Yeni Raki follow with 6.4 mg/100 ml p.a., other samples contain very low amount of acetic acid ethyl ester. Concerning i-butanol in Turkish Raki samples show similar features. But other countries raki samples contain very low concentrations of i-butanol. In the content of amy1 alcohol (3-Methyl-butanol-l and 2-Methyl-butanol-l), develop from the fermentation, there is no significant difference in the Turkish Raki. In opposite Raki of other countries contain a very low content of iso-amylalcohol (<1 mg/ 100 ml p.a.). Anethol is the main aroma component of aniseed products. Anethol content in Turkish Rakis differed between 288 and 343 mg/100 ml p.a. Pemod contains a higher
1799 amount of anethol (480 mg/100 ml p.a.) whereas Ouzo has a minimum level of anethol (92 mg/100 ml p.a.).
GC-Mass-spectrometric analyses of Raki aroma compounds An investigation is performed in 1991 [23], Yeni, Kulup and Altmba$ raki samples produced from Turkish Monopoly (Tekel) Organisation were examined. Extraction of aroma components Raki samples are diluted 1:1 with distilled water according to methods of Rapp et al. [24,25]. Mixture is extracted with Trichlorfluormethan for 20 hours. Before extraction 1 nl 1% Decanol-3 (prepared in ethanol) is used, as standard, per 100 ml sample. Aroma extract is stored in deep freeze (-25°C) until required for GC-analysis. Determination of aroma compounds Before GC analysis 10 ml aroma extract is evaporated in a glass flash in 30°C water bath up to 100 ^1 volume using Vigreux column. This aroma concentrate is stored at -25°C. For GC-analysis, 1 jil aroma concentrate is inject on capillary column with a cooled microliter syringe. Separation columns: - 50 m DB-Wax (Fused silica): 50-180°C, 1.5°C/min - 50 m DB-5 (Fused silica): 50-150°C, 5°C/min Aroma compounds of Raki extract (liquid-liquid-extraction with Trichlorfluormemethan) were determined using polar DB-Wax (50 m) and apolar DB-5 (50 m) capillary columns (Figure 4). Yeni, Altinba§ and Kulup Rakis show similar aromagrams but some of the components showed quantitative differences in the various Raki samples. GC and GC/MS were helpfull for the separation and identification of many components (Table 4). Some of these components show typical characteristics of fresh grapes and fermented raisins. Fermentation aroma components are higher alcohols (Table 4, No. 18-20), fatty acid ethylester (Table 4, No. 7,11,13,22,24) and acetals (Table 4,
1800 Table 4. Volatile aroma components of Turkish rakis Ret. Peak Number mm
Rel. Ret. (DB-Wax 2-Phenylethanol » 1000 38 40 45 53 61 70 75 76 81 101 112 117 123 141 143 171 182 189 193 206 214 240 256 270 272 290 305 310 312 343 362 378 385 387 407 442 450 468 501 513 520 530 542 560 585 590 620 635 700 760
Amount
+ +
I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18a 19 19a 19b 20 21 22 23 24 24a 25 26 27 28 29 30 31 32 32a 33 34 35 36 37 38 39 40 4i 42 43 44 45 46 46a 46b 47 48
5.6 5.9 6.0 6.4 6.8 7,4 7.6 7.7 7.9 8.6 9.6 lO.O 10.2 11.2 11,3 12.9 13,4 13.8 14,0 15,0 15,4 16.7 18.0 18.6 18.8 19,6 20.1 20,6 20.9 22.2 23.2 24.1 24.4 24.6 25.7 27.6 28,1 29.0 30,8 32,1 32.2 32.4 33,7 34.7 35,6 36,1 37.9 38.6 42.1 45.6 47.1 47.9 48,9 49,8 50.3
815 830 850 855
++
48a 49 50
51.5 51.8 52,3
870 880 895
+ + +
m)
-f-
++ + ++ ++ +• •+•
+ + ++ (+) + ++ +
+ (+) + + + + + ++++ + ++
+ + +
+ + -f-4++ (+) ••-
+ +• + (+) + ++ (+) ++ + + + + + ++ + + +++ + +
+ + +
[ -f) a (indctermmaiion timiu)
MS-a in 70 cV m/z Order after Intensity X « Oasispeak
Componcnis
Acetic acid methytester Acetic acid ethylcster 1-Ethoxy-l-mcthoxycthan 1,1-Dicthoxyethan 3-Methyi-butan-l-al 2.4,5-Trimethyl-l .3-dioxolan Propionic acid ethylcster 1,1-Diethoxypropan 2.3-Butandion a-Pinen Dutinc acid ethylcster 2-Butcnal 3- Methylbutinc acid et.ivlester Hexanal 1. l-Dicthoxy-3-Mcthyibuty n 3-Meihylbutylacctat Vaicriansaurecthyiestcr Butanol-l 1.1-Diethoxypentan 2- Butcnic acid ethvlester Amyiacetat Limonen 3-Methylbutanol-l 1.1-Diethoxyhexan Caproic acid ethylester Pentanol-1 2-Ethoxyethanoiacetat Octanon-2 p-Cymol (p-Cymen) 4-Methylpentanol-l 3-Methylpentanol-l Onanthic acid ethylester 6-Methyl-5-hepten-2-on Hexanol-1 ds-3-Hexenol-l Nonanai ds-2-Hexenol-l Capcylic acid ethylester l-Octen-3-ol Hcpianol-l 2-Furancarb«)xaidchyd (Furfural) 2-Ethyihcxanol Benzaldchyd Linalooi Octanol-1 Sesquiterpen: Ccdren Eiitragol (4-Allylanisol) Sesquiterpen Sesquiterpen Sesquiterpen Sesquiterpen cis-Anethol (l-Mothoxy-4-(I-propenyl)-benzol| ScNquitcrpcn: Curcumen Sesquiterpen: Zingibcren Sesquiterpen: Bisabolcn + - little
43,74(M),44,59,42.29 43,45,70.61,73.88(M) 59,45.73.61,89(M-I5) 45.73.103(M-15).75,89.117fM-l) JW,41.43.58.71.57.86(M) 43.44.10I(M-15),55.73.1I5(M-1) 57,73.45.29.75.102(.M).87 59,47.87.75.103(M-29) 43.44.56,86(.Vn 93.79.80.53.121.136(M) 43,71.88.60.101.116fM) 41.70rM)39.69.55 57,102.74,85.115.130fM) 44.56.41.57.72.67. lOO(M) 103.47.75.71,59.87.115(M-45) 43J5,70,73.87(M-J3) 88,85,57,60,73.101,115(M-15) 56.41,43.55,73fM-l) 103.69.47.75. H5(M-45) 69.99,41.86.114(M) 43,70,6l,42,55.87(M-43) 68,93.79,53,107.121,136(M) 55,42,41,43,70,87(M-1) 103,75,47,55,83,129,173(M-1) 88,43,60,73,42,115,129,144<M) 42^5,41.70.87(M-1) 43,72,59,55,73,45,87( M^5) 43,58,71,85,113, i:8(M) 119.91,105,134<M).77,65,51 53,81.50,82,39.96.66.68 56,43.41.69,87.10l(M-l),84 56,69.55,42.57,84.101 (M-1) ^,60.73,70, ll3(M-45), 115 43,108.69,111,126(M),93 56,43,42,55.69,84< M-H^O) 67,55.82fM-H2O),69,57,100(M) 57,4I,82,96,98,142(M) 57,82(M-H20).71.58,67,69 88,101.60,70.127,143,172(M) 57.72,99.81.127(M-1) 70J6,55,41.69.43.83,98(M-H:0) 95,96(M),39.67 93.121,136.79.105.91,77,107 57.43,70,84.55.112( M-H,0) 11 J5,69.43.57.84.96.122 77.106(M). 105,51.50.78 21.93,55,41.43.80.121,136(M) 41,56,55,70.69.84.83.112(M-H20) 161.105,69,91,79.119,133,189,204(M) 14«(M). 121.77.91,105,117,133,51 93.41,79,105,133,161,204(M). 119.77 91,I05,ll9,16l,79,77.133,204(M) 69.41,93,79,109,133,161,204(M) 93,119,91,105,41.133,161,204(M) 14«(M), 147.117.77.121,105.133
119,132,41.91,105.145.202(M) 93,119.69.56.91.133.161.204(M) 69.41.93,67.79.109.119.161.189.204(M) •¥• -^ -t- -^m veiy much •!-+,•»•-»•+- much
Reprinted from: I. Yava§ and A. Rapp, Gaschromatographisch-massenspektrometrische Untersuchungen der Aromastoffe von Raki, Dtsch. Lebensm.-Rundsch. 87 (1991) 41-45.
|
1801 Table 4. Volatile aroma components of Turkish rakis (Cont.) Ret. Peak Number mm
51 52 53 54 55 56 57 58 59 fH) 61 62 62u 62b 63 63a 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 87a 87b 88 88a 89 89a 90 91 92 93 94 95
Rcl. Ret. (DB-Wax) 2-rhenylethanoi - 1000
55,4
945
57,6 58.8 59.3 59.9 61.0 61.7 62.4 63.1 64.4 65.1 65.5 66.1 67.2 68.6 69.3 69.7 70.4 71.6 70.6 71.1 71,4 72.0 72,4 73.8 74.2 74,8 76,1 76,4 78,0 80,0 82.0 83.6 84,0 89,0 90,5 91.8 92.7 94.9 96.3 98.0 98,6 100,2 117.1 118,5 121,7 128.0 150.0 165,0 178,5 187,0 215,0
990 1014 1023 1025 1032 1067 1081 1090 1100 1120 1135 1145 1165 1194 1205 1214 1225 1220 1231 1238 1240 1245 1250 1268 1297 1308 1330 1339 1367 1405 1420 1469 1477 1570 1590 1622 1635 1655 1700 1730 1750 1775 2075 21(X) 2180 2235 2630 2900 3141 3290 3795
Amount
++++
MS-EI in 70 eV m/z Order after lalensity X » Oasispeak
_
CJomnnnrnic
trans-Anethol [l-Methoxy-4-(l-propenyi)-bcnzol|
148(M).77,9l,105,117,147,133
-t+ + + ++ (+) + -h + + +++ + + (+) (+)
43,105,91.79,120.145,160.93 157,143.185.200,170,94,111 2-Phenylethanol 91,92,65,122(M).l2l l.l.6-Trimcthyl-1.2-<Jihydronaphthalin 157(M-15).142,141.115,128 147,135.148,91,77.103.115.163.192 135.123,107.69.79.148.55.164 Eugenolmethylether 178(M).107.91,135.147.163,77 161.91.77.79,55.119,133,147 isom. Eugenolmethylether i78(M).147.107.103,91.77.147,163 Anisaldehyd 135.136(M),77,92.107.63 ij>om. Eugenolmethylether 178(M),135,163,105,147.103.91,77 2-Methoxyphenylaceton 91,121,164(M).65,43,135 cis-Ancthoicpoxid 135.121,105.77.91,164(M) Anisic acid meihylester 135.166<M). 107,92.77.63 -¥• trans-Anetholepoxid 103,121.135,165,164(M) + 119,109.93,161.189.77.204(M) + 105,91.43,93.79,120,131,160,178 ++ 159,43.41.202,187.131,119.91 + 202.187,159.121.77,85.79.147 ++ Anisic acid ethytester 135,152,180(M).77.107.92 ++ 4-Methoxyacetophenon 135,77.150(M),92,107.64,119 isom. Eugenolmethylether 178(M),135,105.163,91,77,103 + +++ 4-Methoxy-l-(2-propanon)beiuol 121,77.91,51,106,164(M).133 110,121.43,77.69.91.135.149 + + 165,137,109.77.83,178 J5 + Eugenol I64(M).121.149,77.91.137.133 + 4-Meihoxy-l-( l-propanon)-benzol 135.107.92.77.63.164(M) + Scsquitcrpcn 119,43,95,67,109,134.161.204(M) +++ 137,165.109.77,94 + 119,43.91,79,159,162,187.220 + -•Anisalkohoi 137.138(M).109.77.43.94,121 +++ + 165.137.109.77.94.149 ++ isom. 4-Methoxypropanonbenzol 135.148,163(M-I),ia3.91,77 ++ isom. 4-Methoxypropanonbenzol 135.77.164(M).92.107,103.149 ++ Sesquitcrpen: Farnesol 69,41,55,118.81.93.121.136 + 109,77,121.135,55.94,164,163 + o-Methoxyzimtaldehyd 161,162(M).131.91,65,77.44.134,119 4-Methoxyphenol + 109.124(M).81.53,62 137.45.109.94.73.138,121 + ++ 137.207.165.109,77.91.121 ++ 137,45,77,94,109.121.138 isom. A net hoi ++ 148(M).147.117,115,77.133 4-Methoxyzimtaldehyd -f- + 162(M).131.161,91.119 i-Eugenol 164(M).149,91,105,103,133,77 ++ Vanillin + 151,152(M).109,81.65,53.137 isom. 4-Methoxypropananbenzol ++ 135.77.107.92.163,164(M) + 165,137,45.77,109.121.148 4-Mcthoxy-zimtalkohol ++ 121.108,9I,77,164(M).55.65,133.115 ++ 43,69,109,93,95.135.151,177 -H 45.44,137.109,77,59 J{9 + 137.109,77,94,89.121
(-»-)» (inde termination limits)
-•• • little
+ •••,-•- + + •
Tiuch
+ -f -f -f« very macb
Reprinted from: I. Yava§ and A. Rapp, Gaschromatographisch-massenspektrometrische Untersuchungen der Aromastoffe von Raki, Dtsdi. Lebensm.-Rundsch. 87(1991) 41-45.
1802 No. 3,8,15,21). Trans-4-methoxy(l-propenyl)-benzol (= trans-anethol; Table 4, No. 51), 4-allylanisol (= estragol; Table 4, No. 48) and 4-Methoxy-l-(2-propanon)-benzol (Table 4, No. 71) are the main characteristic aroma components [26-28, 32] of aniseed products which are related with the quality. These give the characteristic taste and odor of Rakis.
(U ^60 [34 54 '43 22I4I 51 61 45 42 30
16li3l'l23!7| I2 ^fW'^^
Yeni raki
-19—^-
^
^AW 51
45
L..i...>x\J i_J..J_jijJ>' 4139 • I 32 30 27 23 47 34 -^TT
4443-
20
1311
Figure 4: Aromagrams of Yeni Raki [23] (Peak number declared in Table 4) a) 50m DB-5 (apolar), b) 50m DB-Wax (polar)
In Rakis, trans-anethol content differed between 1500 to 1800 mg/1. The amounts of cis-anethol (0.3% of trans-anethol content), estragol, anisaldehyde (2% of transanethol content) are lower. Some results were observed in Greece Ouzo by Kontominas [29] and Soufleros et al. [30]. Liddle et al. [31] determined 4-Methoxy-l-(2-propanon)-benzol (anisketon) which is reaction product of anethol (trans-anetholepoxid). On the other hand further components of aniseed were determinedfromPimpinella anisum: 4-Methoxyphenol (Table 4, No. 86) Eugenol (Table 4, No. 74), Eugenolmethylether (Table 4, No. 58), Acethylanisol (Table 4, No. 69), Linalool (Table 4, No. 42), Anisic acid ethylester (Table 4, No. 68) and a few hydrocarbon compounds. The determined hydrocarbon compounds are: a-pinen, p-cymen, bisabolen (Table 4, No. 50) and zingiberen (Table 4, No. 49) and sesquiterpenes.
1803 In this research for the first time 4-Methoxy-Zimtaldehyd (Table 4, No. 88a) and 4Methoxy-Zimtalcohol (Table 4, No. 92) (typical aroma compounds of Ocimum basilicum and Cinnamon) were determined [23]. Safi-ol [l,2-Benzoldioxol-4-(2-Propenyl)] aroma compound of lUicium anisatum [28] and Myristicin [l,2-Benzoldioxol-6-methoxy-4-(2-propenyl)] were not determined in real Raki samples. GC Analyses of different Raki In this investigation [33] 3 Turkish Raki (Yeni, Kuliip and Altinba§ rakis) which are produced by Tekel (Turkish Monopoly) and Lowenmilch (Asian stitu)fi'omthe German market were examined. Extraktion Raki samples are diluted with water (1:1) [24,25] and extracted with Trichlorfluormethan for 20 hours or with 1,1,2-Trichlor-trifluorethan [34]. Standard solution preparation procedure is the same as previous research [24,25,34]. Separation identification and quantitative analysis of aroma compounds Aroma compound separation and preparation of Trichlorfluormethan extracts are similar with previous research [23]. 1,1,2-Trichlor-trifluorethan extracts were injected directly without any working. Identification of aroma compounds is made by using mass spectrometry and retention time of reference substances. Separation columns: - 50 m DB-Wax (Fused silica): 50-180°C, 1.5°C/min - 50 m DB-5 (Fused silica): 50-180°C, 5.0°C/min Carrier gas: Hydrogen or Helium (GC-MS) In these research the main aim is the comparison of results under equal conditions. For statistical comparison 1,1,2-Trichlor-trifluorethan extracts were used which are injected to DB-5 column directly. For the separation of different Raki types. Statistical Analysis System ready programs are used for correlation analyses [35-37].
1804 Results Aroma compounds extracted with liquid-liquid extraction method from different Raki types showed different features in the GC analysis (Figure 5). Differences occur due to various aroma compounds. For our purposes modified method of Rapp et al. [34] were used. From 10 ml sample a simple and rapid determination for many aroma compounds is possible, without difficult preparation procedure. In Figure 6, chromatograms of 4 different Raki are shown in comparison. Altmbas and Kulup Raki samples are very similar with each other, whereas Yeni Raki and Lowenmilch are different. This simple and rapid method may help to determine 50 different aroma compounds without any difficulty. Statistical computer programs are very useful for characterizing different Rakitypes. The correlation coefficient describes the relationship between two variables in correlation analysis [38]. Some analytical data are listed in Table 5. By means of stepwise discriminant analysis it was possible to ascertain variables for a significant differentiation between the examinated Raki-types. Yeni, Kuliip, Altmbas are significant separated (Fig. 7). The discriminant analysis (Fig. 8) shows also, that Lowenmilch is very different in opposite to the true Turkish Raki (Yeni, Altmba§, Kulup). As shown in Figure 6, aroma compounds of different Yeni and Altinba$ Raki show similar characteristics. For the analytical separation aroma compounds were determined as follows: 3-Methylbutiricacid ethylester, Caproicacid ethylester, Caprylicacid ethylester, Linalool, Estragol, cis-Anethol, Anisaldehyd, Anisicacid ethylester, Eugenolmethylether, i-Eugenol, 4-Methoxy-l-(2-propanon)-benzol and a few sesquiterpenes. Trans-Anethol the main aroma compound of Raki should be used in calculation, too. Lowenmilch shows very different characteristics comparing with Turkish Rakis (Yeni, Altmba$ and Kuliip). Lowenmilch contains very low amount of 3-Methyl-butanol-1, 3-Methylbutiricacid ethylester, Caproicacid ethylester which occur in the fermentation and also Linalool and other compounds. Peak 38 and 39 which are compoimds originate from aniseed (Table 6).
1805
Figure 5: Chromatograms of different Raki (trichlorofluoromethane extracts) (DB-5) [33] 2 = Acetic acid ethylester 7 = Propionic acid ethylester 13 = 3-Methyl butiric acid ethylester 18 = Butanol-l 22 = Caproic acid ethylester 30 = 6-Methyl-5-hepten-2-on 32 = cis-3-Hexenol-l 42 = Linalool 44 = Sesquiteqjen (Cedren) 51 = trans-Anethol 58 = Eugenolmethylether 61 = Anisaldehyd 79 = Anisalkohol
5 = 3-Methyl-butan-l-al 11 = Butiric acid ethylester 16 = 3-Methylbutylacetat 20 = 3-Methyl-butanol-l 23 = Pentanol-1 31 =Hexanol-l 34 = Caprylic acid ethylester 43 = Octanol-1 45 = Estragol (4-Allylanisol) 54 = 2-Phenylethanol 60 = isom. Eugenolmethylether 69 = 4-Methoxyacetophenon
1806
Figure 6: Chromatograms of different Raki (1,1,2-Trichlorotrifluoroethane extracts) (DB-5) [33] 1 » l.l-Diethoxyethan 3 = 2-Mcihyl-buianol-l 8 = l.l-Dicthoxy-2-methyl-propan 11 = l.l-Diethoxy-3-mcthyl-buian 17 » Dicihoxyhexan 25 = Caprylic acid cthylesier 28 a cis-Anethol X » trans-Anethol {4-Methoxy-4(l-propcnyl)-ben2ol] 3 1 a Capric acid ethylester 36 a Eugenoimethylether 38 = Sesquiterpen (Caryophyllcn) 41 = Sesquiterpen (Farnesol) 43 a Sesquiterpen (Bisabolen) 47 = i-Eugenol (2-Methoxy-4(l-propenyl)-phenoi]
2 = 3-Methyl-butanol-I 7 = 3-Methylbutiric acid ethylester 9 = Hexanol-I 14 ' Caproic acid ethylester 20 > Linaiuol 26 • •• Estragol (4-Allyianisol) 29 =' Anisaidehyd (4-Methoxybenzaidehyd) 30 =• Terpen 33 =' Sesquiterpen (Elemen ?) 37 ••' Anisic acid ethylester 40 - 4-Methoxy-l-(2-propanon)-ben2ol 42 = Sesquiterpen (Zingiberen) 45 = Unknown ( M . 137. 135. 109, 77. 94)
1807
O = Yeni
0 = Altinba§
O = Kuliip
Figure 7: Separation of Raki types with 3 parameters by using analytical discrimination [33]
1808
V
0
y^
V 24 V 38
O = Yeni 0 = Altinba§
• —
^
O = Kulup D = Lowenmilch
Figure 8: Separation of Raki types with 3 parameters by using analytical discrimination [33]
Table 5: Components for the separation of different Raki types (correlation coefficients; >0.9 shows > 80% security limits)
Separation Altmba$ from Kulup, Yeni Kulup from Altmba§, Yeni Yeni from Altmba§, Kulup [ Lowenmilch from Altmba$, Yeni, Kuliip
Components (Correlation coefficients) 26 (0.92), 32 (0.87) 46 (0.94), 40 (0.94), 39 (0.94) 2 (0.94) 10 (0.99), 21 (0.99), 17 (0.99) 4 (0.99), 22 (0.99) 38 (0.99), 37 (0.98), 24 (0.97), 39 (0.96), 19 (0.93)
Reprinted from: I. Yava$, A. Rapp and R. Rupprecht; Vergleichende gaschromatographische Untersuchungen von tiirkischen Anis-Spirituosen (Raki), Dtsch. Lebensmittel-Rundschau 87, 242-245 (1991)
1809 Table 6: Aroma components of different Raki (Trichlorofluoromethan-extracts) (content in percent of trans-anethol)
Components
Kulup
Linalool Estragol cis-Anethol Anisaldehyd Anisic acid ethylester 4-Methoxy-l-(2-propanon)benzol Anisalkohol 165. 137, 109, 77...
1.5 16.6 10.5 7.4 0.3 0.6
1.1 14.5 6.2 5.5 0.2 0.6
1.1 1.8 5.8 0.02 0.8
0.05 4.6
0.06 3.1
0.01 1.8
-
0.2
1 Safrol
j
-
AJtinba§
L6 wenmilch
0.03
1
1
Reprinted from: I. Yava§, A. Rapp and R. Rupprecht, Vergieichende gaschromatographische Untersuchungen von tlirkischen Anis-Spirituosen (Raki), Dtscli. Lebensm.Rundsch. 87 (1991) 242-245.
Lowenmilch, Kulup and Altmba^ Raki show slight different characteristics concerning amount of Anisaldehyd, 4-Methoxy-l-(2-propanon)-benzol, Anisalkohol. On the other hand Altinba$ and Kulup Raki show significant differences in the content of Estragol and cis-Anethol. Further in Lowenmilch Safrol (4-Allyl-l,2-methylendioxybenzol) is found with mass spectrometric analysis, in a concentration of 0.2% of transAnethol content [33].
1810 REFEREiNCES 1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21
Anonymous, Tekel 1993 Yili Faaliyet Raporu, Alkollu tqkiler Sanayii iMiiessesesi Mudurlugii, Istanbul, 1994. M. titer, Rakinin Tarihi, Tekel Ekonomik Ara§tirmalar Grubu, Yayin No. Tekel 261, EAG/DKY 93, Istanbul, 1984. I. Fidan and i. §ahin, Alkol ve Alkollu tqkiler Teknolojisi, A.U.Ziraat Fakiiltesi Yayinlan: 1295, Ankara, 1993. F. Drawert and A. Rapp, Gaschromatographische Beurteilung der Qualitiit von Branntweinen, Z. Lebensm. Unters. Forsch. 126 (1965) 105-109. A. Rapp and H. Franck, Uber die Bildung von Aethanol und einiger Aromastoffe bei iModellgarversuchen in Abhangigkeit von der Aminosaurenkonzentration, Vitis 9(1971)299-311. A. Rapp, Les aromes des vins et des eaux-de-vie, Bull. OIV 45 (1972) 151-166. F. Drawert, W. Heimann and G. Tsantalis, Gaschromatographischer Vergleich verschiedener Branntweine, Z. Lebensm. Unters. Forsch. 228 (1967) 170-180. C. Reinhard, Uber gaschromatographische Untersuchungen an Weinbranden, Dtsch. Lebensm,-Rundsch. 67 (1971) 349-352 C. Reinhard, Uber gaschromatographische Untersuchungen in alkoholischen Erzeugnissen, Weinwirtsch. 112 (1976) 172-174. C. Reinhard, Zur Untersuchung und Beurteilung von Whisky, Dtsch. Lebensm.-Rundsch. 73 (1977) 124-129. H. Woidich and W. Pfannhauser, Zur gaschromatographischen Analyse von Branntweinen: quantitative Bestimmung von Acetaldehyd, Essigsaureethylester, Methanol, Propanol-1, Butanol-1, i-Butanol, i-Amylalkohol und Hexanol, Mitt. Klostemeuburg 24 (1974) 155-166. O. Endres, D. Hess, E. Hieke, M.D. Olschimke, A. Rapp and C. Reinhard, Einfluss verschiedener Destiilationsverfahren auf die Zusammensetzung fliichtiger Weininhaltsstoffe in den Destillaten, Dtsch. Lebensm.-Rundsch. 72 (1976) 233-236. W. Postel and L. Adam, Gaschromatographische Charakterisierung von Whisky, Branntweinwirtsch. 116 (1976) 249-254. W. Postel and L. Adam, Gaschromatographische Charakterisierung von Weinbrand, Cognac und Armagnac, Branntweinwirtsch. 119 (1979) 404-409. W. Postel and L Adam, Gaschromatographische Charakterisierung von Weinbrand, Cognac und Armagnac, Branntweinwirtsch. 120 (1980) 154-163. W. Postel and L. Adam, Gaschromatographische Bestimmung der fliichtigen Inhaltsstoffe in extrakthaltigen Spirituosen, Branntweinwirtsch. 121 (1981) 146. W. Postel and L. Adam, Hohere Ester in Wein, Brennwein und Weindestillaten, Dtsch. Lebensm.-Rundsch. 80 (1984) 1-5. S. Nosko, Zur Beurteilung von Williams-Christ-Branntweinen, Dtsch. Lebensm.Rundsch. 70 (1974) 442-447. A. Rapp, Analysis of grapes, Wines and Brandies. In: W.G. Jennings, Applications of glas Capillary Gas Chromatography, Marcel Dekker New York and Basel, 1981. S. Nosko, Zur Beurteilung von Zwetschgenwassern, Branntweinwirtsch. 112 (1972) 133-140. N. Aktan, Y. Sekin and A. Rapp, Raki ve Aporaklarin Ugucu Unsurlari Ozerinde Bir Ara§tirma, E.U.Z.F. Dergisi 17 (1980) 17-22.
1811 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
38
t. Yava§ and A. Rapp, Zur quantitativen Bestimmung von Anethol und fliichtigen Aromakomponenten in verschiedenen Raki-Proben, Dtsch, Lebensm.-Rundsch. 81 (1985)317-321. i. Yava§ and A. Rapp, Gaschromatographisch-massenspektrometrische Untersuchungen der Aromastoffe von Raki, Dtsch. Lebensm.-Rundsch. 87 (1991) 4145. A. Rapp, H. Hastrich and L. Engel, Gaschromatographische Untersuchungen iiber die Aromastoffe von Weinbeeren. 1. Anreicherung und kapillarchromatographische Auftrennung, Vitis 15 (1976) 29-36. A. Rapp, H. iMandery and H. Ullemeyer, Neue Monoterpendiole in Traubenmosten und Weinen und ihre Bedeutung fiir die Genese cyciischer Monoterpenether, Vitis 23 (1984) 84-92. E. Gildemeister and F. Hoffmann, Die atherischen Ole, Band VI, Akademie-Verlag, Berlin, 1961. H. Becker, Vergleichende Untersuchungen iiber die Zusammensetzung der atherischen Ole verschiedener Handelsfriichte von Pimpinelia anisum L., Dtsch. Apotheker-Zeitung 111 (1971) 41-43. S. Van Straten and H. iMaarse, Volatile compounds in Food, Central Inst, for Nutrition and Food Research TNO, Zeist, 1983. M.G. Kontominas, Volatile constituents of Greek Ouzo, J. Agric.Food Chem. 34(1986) 847-849. E. Soufleros and A. Bertrand, Etude sur le Tsipouro", eau de vie de marc traditioneile de Grece, precurseur de TOuzo, Conn. Vigne Vin 21 (1987) 93-111. P.A.P. Liddle and A. Bossard, The Analysis of Anethole and Anethole-FIavoured Beverages by Gas Chromatography, In: L Nykanen and P. Lehtonen "Flavour Research of Alcoholic Beverages", Helsinki Proceedings, 1984. K.H. Kubeczka and I. Bohn, New Constituents from the Essential Oils of Pimpinelia species. In: EJ. Brunke "Progress in essential oil research". Proceedings De Gruyter, Berlin, New York, 1986. i. Yava§, A. Rapp and R. Rupprecht, Vergleichende gaschromatographische Untersuchungen von tlirkischen Anis-Spirituosen (Raki), Dtsch. Lebensm.-Rundsch. 87 (1991) 242-245. A. Rapp, I. Yavas und H. Hastrich, Einfache und schnelle Anreicherung ("Kaltronmethode") von Aromastoffen des Weines und deren quantitative Bestimmung mittels Kapillargaschromatographie. Dtsch. Lebensm.-Rundschau 90 (1994) 171-174. S.D. Schlotzhauer and R.C. Littel, SAS-System for Elementary Statistics, SASInstitute Inc. Cary USA. R. Rupprecht, Using the annotate facility to represent multivariate data by ChernoffFaces, In: Seugi'86, Proceedings of the SAS European Users Group International Conference, SAS-Institute GmbH, 1986. A. Rapp, S. Ringlage, C. Volkmann and R. Rupprecht, Varietal Classification of Wines using statistical methods and the graphical Presentation of the Results, In: P. Ribereau-Gayon and A. Lonvaud "Actualites Oenologiques 89", Dunod, GanthierVillars Paris, 1990. A. Rapp and S. Ringlage, Vergleichende gaschromatographische Untersuchungen uber die Aromastoffzusammensetzung der Weine verschiedener Vitis-vinifera- und pilzresistenter Rebsorten, Vitis 28 (1989) 21-29.
This Page Intentionally Left Blank
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1813
Recent evaluation of malt quality M. Moll Cervac-Est, 1 Allee Chaptal, F-54630 Richardmenil, France
Abstract Evaluation of malt quality has much improved in recent years. There is still a lack of understanding in the malt extract determination where false results are obtained with old obsolete methods. A method with a precise mass balance is available, which is also applicable to industrial brewhouses. Modification and homogeneity of malt deliveries are important and a new automatic instrument is presented. Near infrared reflectance spectroscopy is a powerful tool and many correlations with malt analysis parameters are described. The varietal purity of malt samples can be determined with sufficient accuracy. 1. INTRODUCTION The objective of the evaluation of malt quality is to provide sufficient information to the brewer for it to be possible to predict, at reasonable cost, the behaviour of this raw material during the brewing process. Standard methods are generally applied to regulate the interaction between the malting and the brewing industries (1-4). There are many new methods available today and the weak and strong points of a limited number of methods of analysis will be discussed. Reviews of evaluations of malt quality have been presented by several authors (5-12). 2. EXTRACT DETERMINATION Small scale solid-liquid extraction and solid-liquid separation have been carried out on the laboratory scale (50 g of malt grist and 200-300 ml of water) for more than 100 years (12). Today two main extract determinations are applied in the brewing industry all over the world in the malting-brewing trade:
1814 - the conventional extract determination described in Analytica EBC (1), first standardized in 1898 with a solid-liquid extraction at 45°C and 70°C and solidliquid separation at room temperature, is still applied, - the Institute of Brewing method of mashing using a solid-liquid extraction at 65°C for 1 hour (hot water extract), simulating the infusion mash, has been adopted in 1993 by the EBC Analysis Committee. The weak point is the solidliquid separation at room temperature similar to the previous method. In spite of numerous scientifically founded proposals of changes and introduction of new methods of extract determinations, none of them has been considered seriously for nearly 100 years. Knowledge of biochemistry, enzymology and process-engineering have allowed higher extracts to be obtained in the industrial brewhouse compared to the theoretical laboratory malt extract, which is based essentially on volumetric measurements (12). Malt extract represents all the water soluble substances present in ground malt and comprises : - carbohydrates preformed in the malt or degraded by enzymes during mashing, the glucans and the pentosans, approx. 90-93% - protein components, approx. 4-5% - minerals - lipids approx. 2-3% - polyphenols and others The only way to determine the precise extract of malt is a solid-liquid extraction at 50°C-63°C-75°C with water to malt ratio similar to that used in the brewery, and a solid-liquid separation taking into account the temperature at 75°C with sparging of the spent grains (as shown in Figure 1). A mass balance calculation of this laboratory malt extract determination can also be applied to an industrial brewhouse. This type of approach has been used for many years in the chemical industry, but all attemps in the brewing industry have failed. Only volumetric yield determinations are carried out in breweries worldwide (12,13). 3. MALT MODIFICATION AND HOMOGENEITY Several indirect methods have been applied for the determination of malt modification and homogeneity listed in Table 1.
1815
TEPRAL •
MALT 57 g.
m
WKTER 2 0 0 ml.
g ^ FIITRATIOfc • ADDITION OF 2 x 1 0 0 ml. OF WATER AT 80 X
RATIO WATER/MAU
Fig. 1. TEPRAL mashing andfiltrationconditions.
Figure 1. Mashing and Filtration diagram (Reproduced with the permission of the Journal of the Institute of Brewing).
The majority of these methods need much skill, require a considerable experience and are often time consuming. One of the best approaches in the recent years, for the simultaneous determination of modification and homogeneity in malt, was the Calcofluor. Fast Green staining, developed by Carlsberg (1). This technique of analysis has been abandoned by many maltsters and brewers because of the need to learn special skills to use it, and the long time it takes. Recently, an automatic apparatus for the Calcofluor method has been developed, giving much better results for repeatability (16, 17). Figure 2 illustrates a sheet of results for malt modification and homogeneity.
1816 Table 1 Methods of analysis for the indirect determination of malt modification and homogeneity Method of analysis
Modification
Homogeneity
Fine-coarse extract difference (1)
+
-
Wort viscosity (1)
+
-
Hartong index (6, 7)
+
-
Kolbach Index (4)
+
-
Tepral Extract (12)
+
-
Cold water Extract (3)
+
-
Milling Energy (12)
+
-
NIRS (12)
+
-
Wort Fermentability (4)
+
-
Hagberg falling number (12)
+
-
Hot water extraction 65°C (6, 7)
+
-
fi-glucans (12)
+
-
Zeleny sedimentation (14)
+
-
Sequential pearling (15)
+
-
Brabender hardness (4)
•f
+
Sclerometer (4)
+
+
Murbimeter (4)
+
+
Friabilimeter (1-3)
+
+
Vitreosity (4, 6, 7)
+
+
Methylene blue coloration (12)
+
+
Calcofluor Fast Green (1)
+
+
1817
N 100i U M B E 754 R 0 F
M =
85.9X
H =
59.5X
y.
504
K E R N 254 E L S
0
w '4 V V"
JI
I
I
I
I" I
!
I
I
11 I i j
t< r
f' V 'I " t " f' 1 I
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 100X M O D I F I C A T I O N
Figure 2. Malt modification % and homogeneity %.
4. NIRS (NEAR INFRARED SPECTROSCOPY) Two main measurement principles are used : near infrared transreflectance (NRS) and the near infrared transmission (NRT). Results are calculated from the spectra obtained using several mathematical methods such as analysis of principal components , multivariate regression etc. This elegant method of analysis, which uses ground samples or whole malt kernels, has a high investment cost and is mainly used by laboratories requiring to examine a great number of samples. Several authors applied this method to find correlations with classical malt analysis parameters which are sumarized in Table 2. It is essential to calibrate the mill and the NIRS instruments every
1818 Table 2 Parameters of malt and wort analyses determined with NIRS Analytical parameters
References
Moisture
18,22,24,27,28,30,31,35,36,39,41,47,49, 52,53
Nitrogen / protein
18,19,22, 24, 26-28,30,31,35-40,47-49,52,53
LO.B. Hot Water Extract
21, 29,33,34,36-38,40,41,43,46,47,52,53
EBC Extract
24,25,28,30,39,49
Fine-coarse extract difference
24,46,47,49
Modification (e.g. Calcofluor)
23, 24, 27,31
C-glucan
20,22,32,42,44,47
Starch
24
Hartong 45°C
24,30,31, 39,49
Free amino nitrogen
33-36,52,53
Fermentability
33,34,36,41,52,53
Total carbohydrates
33, 34,36, 52
Total soluble nitrogen
30,39,49,50,51,52,53
Fermentable sugars
33,34,36,50,51,52
Milling energy
37,40
Friability
36
S-methyl-methionine
45
year for each variety of barley malt. The important advantage of the NIRS is simplicity and rapidity : each analysis takes approximately 5 minutes.
1819 5. VARIETAL PURITY OF BARLEY MALT It is for the brewer, and not for the maltster, to mix malts prior to brewing. Two main methods have been developed for the varietal identification of barley malt: - Polyacrylamide gel electrophoresis (Hordein B, C fraction) - Botanical identification on malt samples. This type of evaluation needs much experience and can be only carried out in specialised laboratories such as : SECOBRA, VLB, TNO etc...
6. CONCLUSION Evaluation of malt quality should be mainly located with the eventual user of this raw material, the brewer. The size of the brewery, from micro to multinational industry, is the chief determining factor for malt specification and its control. At the reception of a malt delivery in a brewery, the speed of analysis plays an important role. Rapid methods, such as NIRS and Friabilimeter, allow good evaluation of several essential parameters. For the future, it seems important that the maltings make an effective malt analysis for each delivery, including the main parameters of the signed specifications.
7. REFERENCES 1 2 3
4 5
6
Analytica- EBC, 4th edition, Brauerei-und Getranke-Rundschau, CH8047- Zurich (1987), E 57- E 59. Methods of Analysis of the ASBC, 8 * edition, ASBC, 3340 Pilot Knob Road, St. Paul, Minnesota, 55121-2097, U.S.A. (1992), Malt 1-12. The Institute of Brewing, Recommended Methods of Analysis, The Institute of Brewing, 33 Clarges Street, London, WIY 8EE, GB (1991) 2.12.17. Brautechnische Analysenmethoden, Vol. 1, MEBAK, D- 85350, Freising-Weihenstephan (1978) 153-200. G.H. Palmer and G.N. Bathgate in Y. Pomeranz (ed.) Advances in Cereal Science and Technology, AACC, Inc. St.Paul, Minnesota, U.S.A., 1976,237-324. T. Wainwright and G.K. Bucklee, J. Inst. Brew., 83 (1977) 325-347.
1820 7 8 9 10 11
12
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
M. Moll in J.R.A. Pollock (ed.) Brewing Science, Vol.l, Academic Press, London, 1979,1-143. G.C.J. Muts in H.F. Linskens and J.F. Jackson (eds.) Beer Analysis, Springer Verlag, Berlin, 1988, 3-21. G.H. Palmer (ed.) Cereal Science and Technology, Aberdeen University Press, Aberdeen, 1989. M. Moll (ed.) Bi^res et Coolers, Tec & Doc, Lavoisier, 75384 Paris Cedex 08,1991,20-53. C.W. Bamforth and A.H.P. Barclay in A.W. Mac Gregor and R.S. Bhatty (eds.) Barley : Chemistry and Technology, AACC St. Paul, Minnesota, U.S.A., 1993, 297-354. M. Moll, Evaluation de la qualite brassicole du malt: Application et signification des techniques d'analyses. Thesis, University of Nancy, 1994. M. Moll, D. Leclerc, J.A.Dodds and G. Baluais, J. ASBC, 51 (1993) 45-48 and 49-51. S.G. Reeves, E.D. Baxter, H.L. Martin and T. Wainwright, J. Inst. Brew., 85 (1979) 141-143. M.R. Glennie Holmes, J. Inst. Brew., 96 (1990) 311-321 M. Moll, in Malting and Brewing Technology, 100 Years Institute Meurice, 1993, see also Brauerei-Rundschau, 104 (1993) 217-222. K. Wackerbauer, M. Carnielo and R. Hardt, E.B.C. Proc. 2 4 * Congress, Oslo, 1993,479-486. M. Moll, R. Flayeux and J.M. Lehuede, Bios, 7 (1976) 11, 3-6. Y. Pomeranz, KB. Moore and S. Lai, J. ASBC, 35 (1977) 86-93. M.J. Allison, LA. Cowe and R. Mc Hale, J. Inst. Brew., 84 (1978) 153-155. A.G. Morgan and P.G. Gothard, J. Inst. Brew., 85 (1979) 339-341. A. De Groen, Monatsschrift fiir Brauerei, 33 (1980) 131-135. M. Moll, R. Flayeux and M. Carnielo, J. ASBC, 40 (1982) 155-158. J. Grandclerc, B. Lebordais, M. Carnielo and M. Moll, BrauereiRundschau, 93 (1982) 177-179. C.F. Mc Guire, Cereal Chemistry, 59 (1982) 510-511. W.J.W. Lloyd, in E.G. Priest and I. Campbell (eds.) Proc. 1^^ Aviemore Conference, 1982,157-169. M. Carnielo, J. Grandclerc and M. Moll, E.B.C. Proc, 1 9 * Congress, London, 1983, 619-626. S. Donhauser, E. Geiger, O. Linsenmann and E. Faltemeier, Monatsschrift fur Brauwissenschaft, 36 (1983) 474-477.
1821 29 30 31
32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53
D. Smith, The Brewer, 20 (1984) 246-248. R. Roth, Cerevisia, 9 (1984) 2, 59-62, 65, 66. M. Carnielo, J. Grandclerc, P. Muller, M. Moll, P. Grandvoinnet, M. Berger, J.P. Simiand, M. Lemaitre, D. Mary, F. L'Homme, J.P. Leboeuf, M. Rouiller and M. Mabille, J. Inst. Brew., 91 (1985) 174-179. R.J. Henry, Carbohydr. Chem., 141 (1985) 13-19. S.A. Halsey and O.K. Buckee, E.B.C. Proc, 2 0 * Congress, Helsinki, 1985, 523-530. S.A. Halsey, J. Inst. Brew., 92 (1986) 387-393. D.R. Thronton and C. Williamson, in I. Campbell and F.G. Priest (eds.), Proc. 2^^ Aviemore Conference, 1986, 328-335. S.A. Halsey, J. Inst. Brew., 93 (1987) 407-412. M.J. Allison, A.T. Brown and P.L.Freeman, E.B.C. Proc, 22^^^ Congress, Zurich, 1989, 229-234. M.J. Allison, J. Inst. Brew., 95 (1989) 283-286. T. Zahn, Brauwelt, 139 (1989) 1666,1677,1678. M.J. Allison , in I. Campbell (ed.), Proc. 3^^ Aviemore Conference, 1990, 155-160. T. Zahn, Ibid., 360-362. J.L.M. Lebouille and W.C. Drost, E.B.C. Proc, 23^^ Congress, Lisbon, 1991,125-132. M.J. Allison and A.P. Maule, Ibid.,133-138. T. Zahn, Ibid., 169-175. S.H. Van, E. Bodart and J.P. Dufour, Ibid., 489-496. M.R. Glennie Holmes, J. Inst. Brew., 97 (1991) 283-289. M.R. Glennie Holmes, Ibid., 381-387. J.L.M. Labouille and W.C. Drost, E.B.C. Monograph XX, 1992,14-22. T. Zahn, Ibid., 23-31. K. Sjoholm, J. Tenhunen, S. Home and K. Pietila, E.B.C. Proc, 24^^ Congress, Oslo, 1993, 517-524. J. Tenhunen, K.Sjoholm, K. Pietila and S. Home, J. Inst. Brew., 100 (1994) 11-15. S. Halsey, Ferment, 1 (1988) 2, 48-50. M.O. Proudlove, Ferment, 5(1992) 4, 287-292
This Page Intentionally Left Blank
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1823
Lipol3^ic activity of cheese related microorganismsm and its impact on cheese flavour. M. El Soda^, Jean Law^, Effie Tsakalidou c> and G. Kalantzopoulos^ ^ Department of Dairy Technology, Faculty of Agrigulture Alexandria University, Egj^t. ^ Department of Food Microbiology and National Food Biotechnology centre, University College, Cork, Ireland. c Laboratory of Dairy Research, Agricultural University of Athens, Greece.
Abstract Fat hydrolysis is one of the major biochemical events that takes place during cheese ripening. The action of carboxylesterases on the esters in the aqueous phase of the cheese probably also contributes to the formation of flavour compounds in cheese. The role of lactic acid bacteria and other cheese related microorganisms in fat degradation, the characterization and specificities of their lipases and esterases, as well as the influence of the growth conditions of the cells on enzyme production are discussed. Trends in future research have also been considered.
1. THE IMPACT OF LIPOLYSIS ON CHEESE FLAVOUR Glycolysis, proteolysis, and lipolysis are the principle events occuring during cheese ripening. The extent and impact of each varying, depending on the cheese variety. Lipolysis involves the release of free fatty acid side chains from the triglyceride molecule. These fatty acids, whose carbon chain length can vary considerably depending on the type of milk fat and the hydrolytic agent present can be converted to methylketones and thioesters which contribute, in
1824 addition to the free fatty acids, to the flavour of the ripened product p-keto acids, d-hydroxy acids, and lactones, all of which are breakdown products of triglycerides are also implicated as cheese flavour compounds (Figure 1). Figure 1. Triglyceride breakdown products identified as flavour components in ripened cheese.
Triglycerides n- Fatty Acids B-oxidaUon 7
Methyl ketones Thioesters
"7
Esters Lactones
i
Alcohols A delicate balance exists between the amount of free fatty acids generated which are necessary to give a desirable flavour in cheese, and the development of a rancid taste due to excessive lipolysis. For example, it is generally agreed that the volatile short-chain free fatty acids released by the action of lipase on milk glycerides are responsible for the rancid flavour of milk (1) and indeed ruminant milk fat is unique with respect to the high content of short chain fatty acids bound in the glycerides (2). It must also be noted that fat, and not only fat breakdown products, is important in flavour perception because it is commonly observed that cheese made from skimmed milk does not develop an aroma (3) which suggests that the importance of fat in flavour development extends to its ability to dissolve and retain the flavour components (4). The degree of lipolysis in most varieties of cheese except Blue, Feta, and hard Italian cheeses is limited. Lipolysis occurs most extensively in Blue mould ripened cheeses where up to 25% of the total fatty acids may be liberated (5).
1825 Lipolysis in these cheeses does not result, however, in rancidity, possibly due to neutralization of the pH during ripening. The lipolytic agents in mould ripened cheeses are principally the lipases produced from Penicillium species. P. roquefortii produces two lipases, one of whose optimal activity is in the alkali pH range and one of which is in the acid pH range (6, 7, 8), but both of which apparently contribute to the flavour of Roquefort cheese where the extent of lipolysis is only 8-10% of the total fatty acids (9) (Table 1). Table 1. Concentration of total firee fatty acids i n selected c h e e s e varieties.
Variety
FFA, mg / kg
Cheddar
1028
Roquefort
32,453
Camembert Provolone
681 2,118
It has been documented that P. camemberti produces one lipase which has an optimum pH of 9.0 and temperature of 35°C (10,11) which results in less lipolysis in Camembert than in Roquefort (about 10%) (12). Free fatty acids are converted in Blue cheeses into methylketones via the b-oxidation pathway (Figure 2). The methyl ketones of intermediate chain length (C5-C13) could be the important flavour compounds of mould ripened cheeses (13). The predominant methylketones whose presence is proportional to the level of lipolysis in cheese are 2-heptanone and 2-nonanone which contribute to the flavour and aroma of Blue mould ripened cheeses (14). Since many microorganisms can produce esterases, it is quite likely that aliphatic and aromatic esters w ^ ! A are important cheese flavour compounds are produced as a result of some s t a r t t . activity. Italian cheeses, Provolone in particular, rely for their characteristic taste on quite extensive lipolysis mediated by pre-gastric esterase (PGE) which is added to the cheese milk either in partially purified form or as a component of rennet paste. The specificity of PGE is towards the ester bond at the sn-3 carbon of the triglyceride molecule to jdeld relatively high proportions of butyric acid and other short chain FFA's. The short chain acids in milk fat are predominantly esterified at the sn-3 position and hence the action of PGE results in the release of high concentrations of short and medium chain acids which are responsible, in part, for the characteristic flavour of hard Italian cheeses (15). Surface smear cheeses are flavoured by lipolytic activity from enzymes of Brevibacterium Zinens, yeasts, and in some cases from Geotrichum candidum. Hovewer,in Cheddar, Dutch, and Swiss cheeses, where lipolysis is a
1826 much more subtle event, the sole lipolytic enzjnnes are those from the starter bacteria and any residual milk lipase. Figure 2. Methyl Ketone Formation Via the b-Oxidation P a t h w a y (adapted from Kinsella and Hwang, 1976). O
II R-CH2-CH2-CH2-C-S-CoA Acyl CoA Oxidation HO I II R-CH2-C=C-C-S-CoA I H Enoyl CoA
t
Hydration
OH HO I I II R-CH2-C-C-C-S-CoA I I H H L-Hydroxyacyl CoA
I
Oxidation
o
H-S-CoA
' o
II II R-CH2-C-CH2-C-S-CoA B-KetoacylCoA
i ^
y
I' •
-^
o
Thiolysis II ^ - R-CH2-C-S-CoA Acyl CoA
Deacylation
O
O
O
R-CH2-C-CH2-C-OH B-Ketoacid
+
H-S-CoA
I
Decarboxylation
O II R-CH2-C-CH3 Methyl ketone
^
^Q2
CH3-C-S-CoA Acetyl CoA
1827 1.1. The lipolytic agents i n cheese The principal lipolytic agents in cheese include the native milk lipase, lipases/esterases produced by the starter and non-starter bacteria, lipases from psychrotrophic bacteria and, depending on the cheese variety, enzyme preparations added during manufacturing. The native milk lipase causes significant lipolysis in raw milk cheese and is inactivated at TS^C for 10 seconds, so can thus contribute to lipolysis in cheese made from pasteurized milk (16). Milk lipase is highly selective for fatty acids on the sn-3 position of the triglyceride and, since most of the butyric acid in milk fat is esterified at the sn-3 position, this specificity probably explains t h e disproportionate concentration of free butyric acid in cheese (17). Storage of milk at low temperatures can lead to a significant increase in psychrotrophic bacteria in the cheese milk. Psychrotrophic bacteria are noted for the production of heat stable lipases which are not destroyed by subsequent heat treatment of the milk and which can lead to the production of rancid offflavours in the ripened product. This property of psychrotrophic lipases has prompted studies such as t h a t by Tan and Miller (18) where a lipase gene (1,428 nucleotides) was cloned from Pseudomonas fluorescens B52, a psychrotrophic spoilage bacterium isolated from refrigerated raw milk. The predicted enzjone was found to contain an amino acid sequence highly homologous to the putative substrate-binding domain present within all lipases examined to date (18). In addition to the milk lipase and psychrotrophic lipases, varying lipolytic agents are incorporated into the milk to produce specific cheese varieties. For example, Italian cheese production involves the use of PGE which is present in the r e n n e t paste, and in some other cheese-tj^es, esterases p r e p a r e d commercially ft'om animal or microbial origin are used. With the exception of mould and surface ripened cheeses, the extent of lipolysis is limited but is believed to play an important role in the formation of the characteristic flavour of most other cheeses. Lactic acid bacteria and other cheese related microorganisms such as Propionibacterium, Micrococcus and Bifidobacterium are believed to be major contributors to fat degradation in these cheeses, however, the lipolj^iic activity of these microorganisms has not been extensively reviewed. The present review is, therefore, devoted to the description of the different lipase and esterase systems present in cheese related microorganisms and the possible role of such enzymes in the development of flavour in cheese. Attention will also be given to the areas where more work needs to be accomplished.
2. LACTOCOCCUS AND LACTOBACILLUS In addition to their active role in protein and peptide degradation during cheese ripening, it is also thought that Lactococcus and Lactobacillus play a role in fat hydrolysis during ripening. Interest in their lipolytic activity was shown from as early as 1930. Long and Hammer (19) and Wolf (20) suggested t h a t
1828 certain lactobacilli and lactococci showed weak lipolysis after prolonged incubation of several months. Peterson et al, (21) detected lipase activity in extracts prepared from Cheddar cheese which were shown to hydrolyze tributyrin. Then, in a more extensive study, Peterson and Johnson (22) demonstrated that lactic acid bacteria possess an intracellular lipase which is thought to be liberated after cell autolysis and is able to hydrolyze milk fat. Early evidence for the presence of lipolytic activity was also reported on several other occasions (23, 24, 25, 26). Experiments accomplished using cheese made aseptically from pasteurized, drawn milk (27) also showed that the addition of various starters to the cheese milk lead to the production of appreciable amounts of free fatty acids from milk fat. The most complete and conclusive work accomplished in a cheese system on the lipolytic activity of the lactococci was reported by Stadhouders and Veringa (28). The authors made a Gouda-type cheese using aseptically drawn milk. Cheese was also manufactured under aseptic conditions and 1% of the cheese starter under investigation was added. The results led the authors to reach the following conclusions: 1.) Fatty acids, and more specifically butyric acid, are produced in cheese ripened with Lac^ococci/5 strains. However, some of the higher chain fatty acids (i.e., isovaleric) may also be formed from carbohydrate or protein degradation; 2.) Lactococcus are able to produce free fatty acids from partially hydrolyzed fat at a faster rate when compared to triglycerides, which means that lipolysis in cheese is due to the combined action of the starter and other microorganisms, most likely the gram negative rods, which may provide the lactococci with mono and diglycerides. 2.1 Detection of lipases and esterases in Lactococcus and Lactobacillus strains. Several workers have undertaken comparative studies to describe the lipolytic activity of Loc^ococci^s and Lactobacillus sp. Sato e^aZ. (29) determined the lipolj^ic activity of 12 strains of dairy lactic acid bacteria qualitatively on agar plates in which pigment-stained fats had been suspended. Lb. acidophilus^ Lb. caseij Lb. helveticuSy Lb. lactis, and Leuconostoc citrovorum only slightly changed the colour of fats stained with Nile blue, but when tributyrin was suspended in the agar, clear rings appeared around the colonies. When washed cells of these cultures were suspended in a buffer containing 2% substrate, much more acid was produced from tributyrin than from a butter fat or an olive oil emulsion after 27h at ST^C. Using a more sensitive technique, where a 100-fold concentration of cells was used. Fryer et al. (30) demonstrated t h a t 25 strains of lactococci; 7 Lb caseiy 10 Lb. plantarum^ and 8 Lb. brevis had lipolytic activity to varjdng extents on tributyrin. The lipase and esterase activity of 17 strains of lactic acid bacteria was assayed by Oterholm et al. (31) using a pH stat procedure. All cultures showed detectable activity and hydrolyzed tributyrin more rapidly than tricaproin. In a comparative study where 12 strains of lactic acid bacteria were involved, Searles et al. (32) demonstrated that lactococci and Leuconostoc cultures are significantly more lipolytic than lactobacilli. The specific lipase
1829 activity is expressed as micromoles of free fatty acids per mg of cellular DNA produced in a 10% tributyrin emulsion per hour at 37^C. The values obtained were below 1.0 for the lactobacilli, while they ranged from 2.3 to 33.0 for the lactococci and Leuconostoc cultures. Similar observations were also made by Brandl and Pfleger (33) and Piatkiewicz (34) when they compared the lipolytic and esteroljiiic activities of Lccctococcus dLiidLactobacillus. More recently, El Abboudi et al (35) showed t h a t Lb. casei subsp. rhamnosus exhibited the highest esterase activity when compared to Lb. casei subsp. pseudoplantarum and Lb. casei subsp. casei (Table 2). The increase in activity varied 5- to 10-fold according to the substrate tested. Attention was also given to the quantitative determination of the esterolytic activities of lactococci and lactobacilli. Hosono et al, (36) measured esterase production of 3 lactococci and 2 lactobacilli. Ethyl but3n:*ate production was higher when compared to ethyl caproate production by all the strains tested. Enzjnne production was reduced by incubation under nitrogen and markedly affected by pH. El Soda et al. (37) also demonstrated that, with the exception of Lb. plantaruiUy all Lactobacillus sp. tested hydrolyzed nitrophenyl esters of short chain fatty acids at a higher rate and, as a general rule, the rate of hydrolysis increased with the increase in the number of carbon atoms in the substrate. Table 2 Esterolytic activities of Lactobacillus
casei subspecies
Substrates
Subspecies o{Lactobacilli casei Specific activities! pseudoplantarum rhamnosus casei
p-acetate2
1.03 0.42 1.79 0.85 2.26 0.44 2.03 0.41 3.2
2.54 1.98 4.76 2.86 6.35 2.06 5.56 1.75 3.97
1.5 ...
1.59 0.09 0.16
8.03 3.67 0.24 0.24
0;04
0.18
o-acetate p-butjrate o-butyrate p-caproate o-caproate p-caprylate o-caprylate p-caprate ocaprate p-laurate p-myristate o-myristate
— ...
2 06 1.15 6.88 3.36 11.47 4.59 11.47 4.01
^ These resiilts are means of three replicate analyses made on cell-free extract. 2 All substrates are nitrophenyl derivatives.
1830 The authors also showed that Lb. casei and Lb. plantarum exhibited higher esterolj^ic activities when compared to Lb. brevis or Lb. fermentum, Esterolytic activity in the lactococci and lactobacilli seems to be dependent on the configuration of the substrate used. In fact, El Soda et al. (37, 38), Kamaly et al, (39), and Khalid et aL (40) clearly demonstrated that pnitrophenyl derivatives are hydrolyzed at a significantly higher rate when compared to the o-nitrophenyl derivatives of the corresponding fatty acids. Polyacrylamide gel electrophoresis (PAGE) has been widely used to study the esteroljiiic activity of lactic acid bacteria. Electrophoresis of the crude cell-free extract is usually carried out at pH 8.0 in tris buffer according to the procedure described by Davis (41). Esterase activity is then detected directly on the gels using a- or p-naphthyl derivatives of fatty acids in the presence of a chromogenic reagent. Using this technique, Morichi et aL (42) obtained the electrophoretic patterns of 113 strains of lactic acid bacteria. The esterase patterns of the streptococci were generally species specific. Lb. casei was distinguished from the other lactobacilli by showing a very consistent esterase pattern. Among the lactobacilli, species of the thermobacterium showed significantly lower intensity esterase bands when compared to Lb. casei or Lb. plantarum. PAGE of the esterase patterns of five Lactococcus strains was also considered by Harper et aL (43). An esterase with an RF value of 0.6 was common to the five strains tested and was the only band for the L. lactis subsp. cremoris strains. L. lactis subsp. lactis strains (5lO, C2, and the commercial strains showed a second esterase band with Rf values of 0.67, 0.72, and 0.33, respectively. This study also revealed that L. lactis subsp. cremoris was significantly more active than L. lactis subsp. lactis strains. Moreover, addition of glutathione to the growth medium increased esterase activity. This was later confirmed by Kamaly (44) who found that addition of 0.1% glutathione to the culture medium of the Lactococcus strains led to a 20 - 30% increase in their intracellular lipase activity. The esterase bands of several Lactobacillus sp. were described by El Soda et al. (37, 38). The study revealed the presence of a major esterase band showing an Rf value of 0.48 which hydrolyzed a-naphthyl acetate, propionate, butjn^ate, and valerate in the five strains of Lfe. casei tested. The b-naphthyl derivatives of the previously mentioned fatty acids were also hydrolyzed. This enzyme activity band was also revealed in all the Lb. plantarum strains tested, but showed a narrower specificity. It was also of interest to note that this activity band was also present in all the strains of L6. brevis and Lb. fermentum tested. In the thermobacterium group of the lactobacilli, the esterase bands were distributed from an RF value of 0.58 to 0.14, but no major band common to all the strains reported could be identified. The number of esterase bands detected was strain specific. The PAGE of the esterases was also used by Kamaly et al. (39) to differentiate between (Lac") (Prt") mutants of lactic streptococci. Khalid et al. (40) attempted this technique to distinguish between strains revealing different flavour patterns in cheese making. The PAGE results obtained, however, are not conclusive.
1831 The PAGE technique is also used as a criterion during the selection of native Ldctococcus and Lactobacillus strains isolated from traditional cheeses in Greece (45) and in Egypt (46). 2.2 Localization of the esterase and lipase system of lactic acid bacteria Only limited information is available on the cellular location of the lipase and esterase systems of the lactic acid bacteria, probably due to the absence of a sensitive enough method for the detection of low lipolj^ic activity. The first attempts were accomplished by Oterholm et aL (31). They concluded t h a t the esterolytic activity seems to be more strongly associated with cell particulated matter t h a n lipase. Brown (47) using a cytochemical electron microscopic technique showed that the esterase activity of L6. casei was primarily localized at the plasma membrane and the outer surface of the cell wall. This was recently confirmed by Ezzat et aL (48) who detected cell-wall-associated esterases in several cheese related microorganisms. As for the proteinase activity of lactic bacteria, no extracellular lipolj^ic activity was detected (31, 32, 33) except for the work of Singh et aL (49) and Yu et aL (50) where no evidence for intracellular release was reported. 2.3 Effect of growth conditions and mutation on lipase production Lipol5^ic activity of the dairy streptococci and lactobacilli seems to be influenced by the growth conditions of the cells. El Soda et aL (37, 38) demonstrated that esterase production by several lactobacilli was maximum at the early stationary phase followed by a decrease in enzyme production in the late stationary phase. The decrease in enzymatic activity at the end of the stationary phase was reported to be due to further exposure of the cells to a lower pH. This hypothesis was then confirmed by Khalid et aL (40) when they observed no decrease in the esterase activity of Lfe helveticus cells when grown under pH controlled conditions. A similar trend was also reported by Kamaly et aL (51) for the lipase system of the lactococci. In fact, it was shown that the activity was greater in extracts from logarithmic phase cells when compared to those obtained from stationary phase cells. Higher lipase production from logarithmic phase cells was also observed by Piatkiewicz (34) and Papon and Talon (52). An increase in lipase production by Lactococcus strains was possible after the addition of glutathione to the growth media (43, 51). The reason for this increase could be due to the fact that glutathione protected sulfliydryl groups in the enzymefi:'omoxidizing metabolites in the cell (51). UV irradiation of the cells was also reported to increase fatty acid liberation (53) and carbonyl compound formation from milk fat (54). These results, however, are in contradiction to the work of Piatkiewicz (34) who noticed a decrease in activity of 40 to 60% due to mutation with UV irradiation. Freezing L6. helveticus cells in order to use them in an attenuated form to accelerate cheese ripening led to a decrease in esterolytic activity of 24 to 36% (40). The authors also showed that when Lb, helveticus cells were grown in a
1832
whey based medium a slight increase in the esterase activity could be measured when compared to cells obtained from MRS broth, indicating the possibilities of using a whey based medium for the industrial scale production of Lb. helveticus cells to be used for accelerated cheese ripening. 2.4. Characterization and specificity of the lipase and esterase systems of Lactococcus and Lactobacillus sp. The influence of physical and chemical factors on the esterolytic and lipolytic activities of different species of lactobacilli and lactococci were reported on a few occasions and are summarized in Table 3. Table 3 Characterization of the lipases and esterases of lactic acid bacteria Microorganism
Type of activity
Lactobacillus
Opt. Temp (?C)
Inhibitors
Ref
Ethyl esterase
6.5
32
Co, Mn, Cu
36
Lactococcus sp.
Lipase
7.0
37
20% NaCl
51
Lb. plantarum
Acetyl esterase
6.7
40
Cn, N3
55
Lb. brevis
Lipase
6.5
30
Ag,Hg
56
Lb. casei
Lipase
7.0
37
Ag,Hg
57
Lb. casei, Lb. plantarum. Lb. helveticus
Lipase
37
not reported
59
sp.
Opt. pH
i-8
Attempts were made to purify their esterases and lipases. Oterholm et al. (55) reported the purification of an acetyl ester esterase from Lb plantarum. The enzyme was purified from cell-free extract by ammonium sulfate precipitation, heat treatment, acetone fractionation, and ion-exchange chromatography. Maximum enzyme activity was at pH 6.7 and 40°C. The activity of acetylesterase was not aff^ected by low concentrations of heavy metals such as mercury, or by respiratory poisons such as cyanide and azide. The enzyme had a strong preference for substrate in solution rather than in emulsion and preferentially hydrolyzed substrates containing acetylesters. The partial purification of an intracellular lipase from Lb. brevis was carried out by Chander et al. (56). The enzjnne showed maximum activity at 30^0 after 3.5h incubation at pH 6.5. Salts of Mn, Mg, Na, and Ca stimulated lipase activity while those of Ag, Hg, and Zn were inhibitory. The enzyme was completely inactivated at 62.8oC after 30 min and at 71.7^C after 16 seconds. In their extensive work on the characterization of the lipase enzjmae of Lb. casei subsp. pseudoplantarum, Lee and Lee (57) purified the enzyme 54-fold using several purification steps, including ion exchange chromatography and gel
1833 filtration in an FPLC system. The enzjone was obtained as a single protein band as verified by PAGE and Sodium dodecyl-sulfate PAGE. Optimum conditions for lipolytic activity were observed at pH 7.2 and 320C. Silver and mercury were strong inhibitors of the enzyme activity while magnesium and calcium salts stimulated lipolytic activity. The stability of the esterase system of L6. casei under cheese making conditions and their stability in a cheese system was recently considered (35). The esterolytic activity of the cell-free extract of several Lb. casei strains were measured at pH 5.2. The results were strain specific. Strain UL21 showed 9% more activity at pH 5.0 compared to pH 7.0, while more than a 50% reduction in esterase activity could be measured at pH 5.2 in strain LCIO. Sodium chloride at concentrations up to 8% seems to activate the esterase system of Lb. casei LCIO (Fig. 3). On the other hand, the other Lb. casei strains were inhibited to different extents due to the presence of NaCl. Figure 3. Effect of sodium chloride concentration on the esterolytic activity of Lactobacillus casei. 250-
2% NaCI
4% NaCI
8% NaCI
LC10
-j
n
UL21
1
r
UL 26
UL137
Strain #
Results are expressed as % of the activity measured in the absence of sodium chloride. LCIO: Lactobacillus casei UL21: Lactobacillus casei subsp. casei UL26: Lactobacillus casei subsp. rhamnosus UL137: Lactobacillus casei subsp. pseudoplantarum
1834 An experiment was then conducted where the lyophiHzed extract of Lb. casei was mixed with salt and added to a cheddar cheese curd during the milling process. Esterase activity was then measured in the cheese extract and compared to the values obtained from an untreated cheese. Figure 4 clearly indicates that the intracellular esterases from cheese related microorganisms are active under cheesemaking and ripening conditions for up to 7 weeks and could, therefore, actively contribute to the ripening process. Figure 4. Esterase stability in cheese manufactured with cell-free extract of Lactobacillus casei snhsp. pseudoplantarum.* 1.6-
2
3 4 5 TIME/WEEKS
6
* The control cheese did not exhibit any esterolytic activity Some interest was also given to the specificity of the lipase system. The general conclusion reached by most authors was t h a t lactic acid bacteria hydrolyze triglycerides composed of short chain fatty acids more rapidly than those composed of higher chain fatty acids. Singh et al. (49) found t h a t the intracellular lipase from L. lactis hydrolyzed tributyrin, whereas tripalmitin and triolein hydrolysis was very limited. These observations were also confirmed when cell suspensions of lactic acid bacteria (29) or the crude extracts of Lactobacillus and Lactococcus strains were used (58). In both cases, tribut3a'in emulsions were hydrolyzed more rapidly than butter fat and olive oil. A similar trend can also be found in the work of Chander et ah (56), El Soda et al. (59), and Carini et al. (60). Stadhouders and Veringa (28) examined the rate of lipolysis of partially hydrolyzed fat by the lactic streptococci. It was found that more fatty acids
1835 are formed from mixtures of tri, di, and monoglycerides than from the pure triglycerides which led the authors to conclude that di and monoglycerides are better substrates for starter bacteria. In addition to the Lactococcus and the Lactobacillus, attention was also given to other cheese related microorganisms and will be discussed below.
3. ENTEROCOCCUS Lund (61) studied the soluble cell constituents of Enterococcus faecalis, E. faecium, and E. durans by polyacrylamide gel electrophoresis followed by staining to reveal esterase enzymes. TTie patterns found inE. faecalis differed from those on£^. faecium and E. durans. Esterase activity of E. faecium and E. durans was weaker than that of E. faecalis and in most strains only faint esterase bands were detected. Dovat et aL (62) examined the lipolytic activity of 16 enterococci strains and other lactic streptococci when grown in skim milk, cream, and skim milk containing tributyrin. Using the Nile blue sulphate agar technique, all strains were able to hydrolyze tripropionin. The clear zones around the enterococci colonies were larger than those obtained for other lactic streptococci after prolonged incubation. Tributyrin was less susceptible to hydrolysis but, again, the enterococci were more active than the lactic streptococci. Tricaproin and tricaprylin were less frequently hydrolyzed and triolein was not attacked at all. Chromatographic determination of the volatile fatty acids showed that most enterococci produced more acetic acid than the lactic streptococci. Strain differences within species were apparent as 50% of the Enterococccus durans strains frequently produced as much as ten times more acetic acid than the others. Jensen et al. (63) used 2 strains each ofE. faecalis and E. durans as supplement starters in Cheddar cheese manufacturing. The authors claimed that over the whole ripening period the experimental cheeses, particularly those made with E. durans, contained more free fatty acids than did controls manufactured without enterococci. Martinez-Moreno (64) isolated enterococci from raw ewes' milk and Machengo cheese. The majority of the strains belonged to the E. durans species; E. faecium and E. faecalis were present only in a limited ratio. None of the strains showed lipol3^ic activity towards milk lipids but they were active towards tribut3n:in. This activity was not reduced at pH 5.0. Chander et al. (65) studied the role of some fatty acids on the growth and lipase production of E. faecalis. Short chain fatty acids such as propionic, butyric, caproic and capric stimulated lipase production, while long chain fatty acids such as lauric, myristic, palmitic, stearic and oleic inhibited lipase production. The authors claimed that stimulation of lipase production by short chain fatty acids may be of value in enhancing flavour production hyE. faecalis in foods and dairy products. The same authors (66) described the purification and characterization of a glycerol ester hydrolase (lipase) from E. faecalis. The
1836 molecular weight was 20,900 and its isoelectric point 3.6. The enz3ane had optimum activity at 40°C and pH 7.5; it was stable for 1 month at -IS^C and completely inactivated in 10 min at 90°C. The lipase hydrolyzed tributyrin more easily than tricaproin, tricaprylin and triolein. The relative specificity of the purified lipase for natural triglycerides was in the following order: butter oil > olive oil > linseed oil > coconut oil. Carrasco de Mendozae^ al. (67) evaluated the lipolytic activity of various Enterococcus strains in milk. This was variable from one strain to another. Most bacteria exhibited low activity and only a few, belonging to E. faecalis species, could be classified as highly lipolj^ic. The highest activity usually occured after a 5-day incubation, both for E. faecalis and for E. faecium. Tsakalidou et aL (68) reported the photometric and electrophoretic detection of esterolj^ic activities ofE. durans and E. faecium strains. Spectrophotometric detection was based on the hydrolysis of p-nitrophenyl esters of acetate, butyrate, caproate, caprylate, caprate, laurate, myristate, palmitate, and stearate. For the electrophoretic detection, the a-naphthylamide derivatives of the above listed fatty acids were used as substrates. The E. durans strains were active up to C8, while the E. faecium strains showed activity up to CIS by the photometric method. Using a one-gel electrophoretic system (pH 7.2) both species gave one esterase band (Rf = 0.34) for C2 up to C8. On the contrary, using a two-gel electrophoretic system (pH 8.3) no band was detected for the E. faecium,, but active esterase band(s) were observed for the E. durans strains. Finally, Tsakalidou et aL (69) reported that E. faecium were more active on rnitrophenyl valerate (C5) than E. durans. Both species showed higher esterolytic activity compared to other lactic acid bacteria examined under the same conditions. It appears that enterococci possess active lipase and esterase systems which can be cloned in Lactococcus or Lactobacillus strains in order to improve their lipolytic capabilties.
4. LEUCONOSTOC Fryer et al. (30) studied the lipolytic activity of whole cells of three Leuconostoc mesenteroides strains, using an agar-plate assay at 30°C and pH 7.0, with 0.1% emulsified tributyrin as a substrate. All strains proved positive to varying degrees towards tributyrin. The increase in the area of lipolysis was linear from zero time, no lag in hydrolysis being apparent, indicating that the lipase is close to the cell surface (exoenzyme) and readily released. Morichi et aL (42) tried to differentiate various lactic acid bacteria using the electrophoretic patterns of their soluble proteins and their esterases. The authors determined the esterase patterns of various Leuconostoc strains. All Leu. cremoris strains gave a weak and diffuse esterase band. Often strains of Leu. mesenteroides, 4 strains belonged to the Garvie's physiological group III showing highly active esterase bands. The remaining 6 strains gave variable
1837 esterase p a t t e r n s . Finally, the esterase patterns of Leu. lactis and Leu. dextranicum differed from tfie oiher Leuconostoc species. Oterholm et al, (31) assayed whole cells of a Leu. citrovorum and a Leu. mesenteroides strain for their glycerol ester hydrolase activity by using an improved agar-well technique. In the case of Leu. citrovorum, the activity was also determined in the cell-free extract using a pH-stat procedure. Concerning lipase activity (esterolytic activity directed toward a substrate in emulsion), the whole cells of both strains hydrolyzed tributyrin more actively than they did tricaproin. When other triglycerides in the same homologous series (C8 through C16) or milk fat were used as substrates, complete clearing of the emulsion did not take place. In the cell-free extract of Leu. citrovorum, high lipase (on emulsified tributjn-in) as well as high esterase activity (on aqueous triacetin) were detected. These activities were several-fold greater t h a n those of six species of the genus Lactobacillus tested under the same conditions. Finally, the authors claimed that the lipase of these organisms is an endoenzyme, while the esterase appeared to be strongly associated with the cell particulate material. Chandan et al. (70) studied the lipase activity of 12 lactic cultures. Activity was assayed by the silica gel method, using 10% tributyrin emulsion as a substrate. Leu. dextranicum appeared to be highly lipolytic. Addition of 1% cream or 0.2% casein to the growth medium resulted in a slight increase in the cellular lipase of Leu. citrovorum. Angeles and Marth (71) confirmed the Table 3. Specific activity* of the e s t e r a s e s from Leuconostoc P-C4 0-C4 P-C2 0-C2 P-C6 Leuconostoc sp.
species 0-C6
1.20
0.13
0.13
0.00
0.53
0.13
0.53 0.06
0.09 0.04
0.73 2.44
0.00 0.09
1.11 0.21
0.58 0.00
L. mesenteroidei• 1019 1022 1024
21.10 0.03 0.05
0.76 0.01 0.05
19.60 0.09 0.15
3.20 0.07 0.05
6.00 1.16 0.47
0.13 0.00 0.38
L.
7.20 0.14 0.29
0.88 0.02 0.00
0.51 0.23 0.79
0.19 0.08 0.04
1.55 1.44 3.23
0.48 0.07 0.12
0.18 0.00 0.29
0.03 0.00 0.08
0.35 2.62 0.51
0.35 0.00 0.05
1.79 0.45 1.47
0.84 0.64 0.00
L. mesenteroides suhsp.cremoris
H16 773 347
dextranicum 1025 1027 1028
L. lactis
774 775 776
p : para-nitrophenyl; o : ortho-nitrophenyl; C2: acetate; C4: butyrate; C6: caproate. ""Specific activity was defined as the number of units per mg protein. A unit of enzyme activity was defined as the variation of 0.1 unit of absorbance at 410 nm in 1 min.
1838 lipolysis of tributjTin by Leu. mesenteroides using the agar-well assay technique. Less activity was detected for triolein and none on soybean oil. El Shafei (72) reported the detection of the intracellular esterolytic activities of Leu, mesenteroides. Leu. lactiSyLeu. cremoris and Leu. dextranicum. In the photometrical determination, the rate of hydrolysis of the p-nitrophenyl esters of the fatty acids was higher than that of the o-nitrophenyl derivatives. All strains showed activity on acetate, butjrate and caproate, but none on caprate, laurate and stearate (Table 3). The PAGE pattern of the esterases revealed four activity bands (Rf = 0.29, 0.38, 0.42 and 0.47). The major band (Rf = 0.42) was detected in 75% of the strains. Finally, Tsakalidou et al. (69) examined the esterolytic activity of three Leu. mesenteroides and two Leu. pseudomesenteroides strains using p-nitrophenyl esters of valerate, laurate, and palmitate. All strains tested showed very limited activity in the case of valerate; no hydrolysis was observed for the other two substrates.
5. PEDIOCOCCUS Pediococci were also shown to hydrolyze tributyrin (30). It was also demonstrated that the rate of tributjnin hydrolysis is higher than the rate of hydrolysis of triglycerides containing long chain fatty acids (31, 71). The electrophoretic patterns of their esterase activity were also considered (73) for six strains of Pediococcus pentosaceus and two strains of P. acidilactici. Esterase bands hydrolyzing the a-naphthyl derivative of acetate, propionate, and butyrate were only detected in P. pentosaceus.
6. BIFIDOBACTERIA Bifidobacteria is the group of lactic acid bacteria which has been studied only in the last few years. The reseach work has mainly focussed on the taxonomy, isolation, growth aspects, and probiotic effect, while limited information is available on their biochemical activities. Concerning their lipolytic system, Desjardins et aL (74) determined the enzymatic profiles of 22 isolates of Bifidobacteria using the API ZYM system. Most strains showed low (1 to 2 in the scale of the test system) esterase (C4), esterase-lipase (C8) and lipase (C14) activities. Finally, Ezzat et aL (48) detected cell-wall associated esterases of B. infantis, using chromogenic substrates.
1839
7. PROPIONIBACTERIA Propionibacteria, which are used as starters in Swiss cheese manufacturing, exhibit lipolytic activity (32,75). Attention has also been recently given to their esterase system (76, 77). They seem to differ from previously described microorganisms in this respect since they hydrolyzed the napthyl derivative of lauric, myristic, palmitic, and stearic acids.
8. OTHER CHEESE RELATED MICROORGANISMS Evidence was also reported for the presence of lipase activity in dairy related micrococci (78). The lipase of Brevibacterium linens was also described by Sorhaug and Ordal (79). Detection of esterolytic activity in B. linens was also reported (80).
9. MOLECULAR BIOLOGY OF LIPASE/ESTERASE PRODUCTION IN LACTIC ACID BACTERIA Lipolysis in milk fermentation systems plays a significant role in end product flavour development and, since the starter cultures are also lipolytic, there is growing interest now to understand wholly the role of starter cultures in lipolysis. Several lipases and/or esterases from lactic acid bacteria have been isolated and biochemically characterized. The evolving body of evidence that lipolysis of these bacteria may play a role in the flavour of fermented milk products has prompted interest in understanding the genetic systems underlying lipase and esterase production by these bacteria. Determination of the primary structure of lipases and esterases is necessary to understand the molecular mechanism of the catalytic reaction, substrate recognition, processing, activation, and structure of the enzymes. Determination of the molecular biology of lipase and esterase production will be of utmost value to improve and manipulate starter strains and enhance flavour production in ripened products. The genetics of lipase and esterase production in lactic acid bacteria is in its infancy. It is only in relatively recent times that transformation techniques and gene cloning strategies have been optimized for lactococci, which represent the most explored strain of lactic acid bacteria in genetic terms (for a review see Leenhouts and Venema) (81). Guidelines to understanding the genetics of lipolysis in lactic acid bacteria may be found from information obtained from other systems. The genes for several microbial lipases have been cloned and among those for which nucleotide squences are now available are Bacillus
1840 subtilis (82), Staphylococcus aureus (83), S. hyicus (84), Moraxella TA144 (85), Bacillus stearothermophilus (86), Pseudomonas aeruginosa TE3285, Ps. fluorescens B52, Ps, fluorescens SIKWl, Ps. cepacia, andPs. subsp. M-12-33 (87, 18, 88, 89. respectively), Geotrichum candidum (90), Mucor miehei (91), Staphylococcus epidermidis (92), and Penicillium camembertii U-150 (93). Due to the highly lipolytic nature of Pseudomonas species and the possibility of commercially applying these enzjmaes, a considerable body of research has resulted in understanding pseudomonal lipol5^ic systems fh)m a genetic point of view. A genomic librovy of Pseudomonas aeruginosa TE3285 was constructed in XEMBL3. An oligo-probe synthesized to the partially purified enzyme from this strain localized the lipase gene to a 2.7 kb fragment. A second gene lipB was identified 50 kp downstream from lipK, the sequence of which was highly homologous to that of putative modulators of the production of active lipases in other Pseudomonas species. LipA was expressed in Escherichia coli 1100 only in the presence of the complete lipB (87). Two genes lipA and lipX from Pseudomonas M-12-33 have been cloned and sequenced and shown to be essential for lipase production. LipA encodes a 364 amino acid pre-pro-protein which is then processed to a mature lipase protein of 320 amino acids while lipX encodes a 344 amino acid protein which is involved in the regulation of lipase production (94). The lipase gene from Pseudomonas fragi IFO-12049 was cloned on a 3.3 kb SauSA chromosomal fragment inE. coli and sequenced. The lipase (EC 3.1.1.3) had a molecular mass of 29,966 Da as determined by SDSPAGE which agreed with the predicted size estimated from the sequence (95). Lipase enzymes from Staphylococcus species have also received much attention in the last ten years. The S. epidermidis 9 lipase gene (gehC) was cloned in E. coli and localized on a 2.1 kb sequence by subcloning and transposon mutagenesis. The 2064 nucleotide sequence ofgehC was predicted to encode a lipase of 77 kDa. A 97 kDa lipase was detected in extracts ofE. coli harbouring gehC and it was therefore predicted that a pro-lipase is proteolytically processed in cultures of S. epidermidis during growth (92). The lipase gene from S. hyicus was cloned in S. carnosus and E. coli. The open reading frame comprises 1923 nucleotides which encodes a pre-protein of 641 amino acids with a predicted molecular weight of 71,382. A predicted signal sequence is present at the N-terminus. At the N-terminus the first 16 amino acids are predominantly hydrophilic, followed by a sequence of hydrophobic amino acids (17 - 34) and the sequence Ala-Glu-Ala (36-38), the expected cleavage site for a signal peptidase (84). The S. aureus lipase gene encodes a 76 kDa protein. The extracellular lipase purified from culture supematants is only 45 - 46 kDa. The lipase was shown to be secreted in vivo as an 82 kDa protein with full enzyme activity. It is then processed both in cultures and in cell free supematants to a mature 45-46 kDa protein. Protein sequences demonstrate that the N-terminus region of the 82 kDa prolipase comprising 295 amino acids is cleaved from the central and C-terminal moieties which contain the active site. A metallocysteine protease is probably responsible for initiating the processing (96). Common features emerging from these and other studies which should assist in understanding the genetics of the lipol3^ic systems in lactic acid
1841 bacterial starter cultures indicate that, in general, a conserved pentapeptide Ala-X-Ser-X-Gly may function as the catalytic site. Lipases, like lactococcal serine proteinases, are transcribed as pre-pro-proteins. The pre-protein contains a signal peptide typical of secreted proteins. Cleavage of the signal peptide results in a pro-protein which is modified to the active protein conformation (97) and lipases in general have a catalytic triad Ser/Asp/His typical of the serine proteinases (98). Work on the genetics of lipase/esterase activity in lactic acid bacteria has begun with the focus on L. lactis strains. Esterase activity in L. lactis subsp. cremoris E8 was relatively high compared to other strains of Ixzctococcus which prompted its purification and characterization. N-terminal sequence analysis of the enzyme will prove a valuable tool in localizing and cloning the genes encoding the enzyme (99). The lipase gene from S. subtilis 168 has been cloned on a lactococcal expression vector and expressed in Lactococcus, It is now of interest to assess the affect of expression of this heterologous lipase gene in Lactococcus (100). The availability of genetic information on lipase/esterase activity in Lactococcus and other lactic acid bacteria will be vital for the manipulation of their lipolytic system, by not only over producing and altering the lipase activity but the availability now chromosome integration vectors will allow mutations to be made in genes of interest generating lipase negative strains Such strains will facilitate the conclusive determination of the role of lipolysis by starter cultures in ripening and flavour development in fermented products.
10. CONCLUSION The work discussed in this review clearly indicates the potential of cheese related bacteria to produce lipases and esterases. However, vital information remains to be determined: 1) The precise cellular location of the enzjones and whether some of these microorganisms can secrete their enzymes into the surrounding environment; 2) More enzymes need to be purified to homogeneity and characterized; 3) Although some information is available concerning the specificity of the lipases, more work is needed on partially hydrolyzed fat; 4) Whereas the overall role of the lipase enzymes during ripening is partially understood, the information for the esterases is limited and the nature of the substrate that they hydrolyze in cheese is still unknown. We believe that a great part of the missing information is due to the lack of an accurate and simple method for measuring lipolytic activity. Most of the data discussed in this review were obtained using enzjmae assays based on the measurement of hydrolysis zones in agar plates by determining the liberated fatty acids with pH stat. Both of these methods have their limitations in measuring weak lipolytic activity (for more details, see Jensen, [81]). In the case of esterases, the autohydrolysis of the nitrophenyl derivatives of the various fatty acids at pH values in the alkaline range is a serious limitation to
1842 the method, especially because most of these enzymes show optimum pH in the alkaline range. The development of a reliable method for measuring relatively low lipase and esterase activity is, therefore, imperative for a better understanding of the role of these microorganisms during cheese ripening. Lipase and/or esterase negative mutants obtained through genetic manipulation techniques could lead to an explanation of the role of these enzymes during ripening. The availability of highly lipolj^c cheese related microorganisms through strain selection or genetic engineering would also offer new tools to accelerate cheese ripening research.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1849
Fermentation of black olives with application of starter culture and aeration M. Borcakli, G. Ozay and I. Alperden ^TUBITAK, Marmara Research Center, Department of Food and Refrigeration Technology, P.O. Box. 21, 41470. Gebze, Kocaeli/Tiirkiye.
Abstract The possibility of the application of the selected starter culture to black olives, in batches with and without air injection were investigated in the laboratory. Effect of the pretreatment application on the microbial development, the inoculum level of the starter culture and the composition of the contaminating flora to the fermentation of the olives were examined. Experiments were pursued in regard with microbial and chemical composition, during 160 days of fermentation, including the pretreatment period. Air supply enhanced the growth of microorganisms, particularly allowed an increase in the lactic acid bacteria population within 73 days after inoculation of Lactobacillus plantarum. Starter culture utilization stimulated the reducing sugar consumption between the 50th and the 80th days. Inoculation of starter culture did not inhibit the yeast and the contaminating flora development. The contaminating flora was represented by Lactobacillus delbreuckii and Leuconostoc mesenteroides. In batches inoculated with starter culture the final microorganism populations were consisted of lactic acid bacteria and yeast by half. Besides the polyphenols and reducings sugars, free, volatile acidity and salt content in brine, moisture, ash, protein content and a^ values were determined in the final products. Sensory analyses conducted on the batches revealed higher points for the olives which processed aerobically. The effect of starter culture utilization to the organoleptic properties of the olives in aerated brines needs further experiments.
1. INTRODUCTION In the context of the studies to improve the process of the traditional black olive fermentation, our studies aim to lower the salt content of brine and shorten the fermentation time which takes approximately 10 months and to obtain olives with desired organoleptic properties. Starter culture apphcation to olives is one of the means of providing products with characteristic taste and flavour. Cultures added to brine
1850 accelerate the sugar utilization by reducing the sugar content of final product leading to a long shelf life. In most of the pickling products lactic acid bacteria are the microorganisms used as starter culture which converts sugars present in the product mainly to lactic acid involving a decrease in pH. Occurrence of gas pockets in olive flesh is a frequent defect caused by CO2 produced by microorganisms such as heterofermentative lactic acid bacteria and yeasts. For this reason application of Lactobacillus plantarum which is a most common species found in pickling vegetables and fruits, is preferred as starter culture because of the homofermentative metabolism and diacetyl-acetoin producing ability (1). One of the most effective ways of shortening the processing time is to apply the aeration into olive basins for accelerating the interaction between olive and brine (2, 3). Circulation created by air injection favourite the liberation of CO2 accumulated in brine and the leaching out of the water soluble substances from olives. As the fact that the phenolic compounds has an inhibitory effect on lactic acid bacteria is mentioned by different authors (4, 5, 6) in the actual study besides the starter culture and aeration application, pretreatment was performed as well to remove phenoHc substances from brine and subsequently the establishment of the high and persistent lactic acid bacteria population was induced during fermentation. The main objectives of this study were to determine the effect of starter culture applied at two different concentrations and times on the microbial and chemical composition of olives, towards the pretreatment and air injection.
2. MATERIAL AND METHODS 2.1. Material Fully ripe, raw olives of Gemlik variety with average size of (318 fruits/kg) from the 1991 crop, supplied by Marmarabirlik Union of the Olive Producers Cooperatives (in Bursa) were used in our experiments. 2.2. Microbiological methods a. Isolation and identification of microorganisms Enumeration of microorganisms were carried out on solidified media after preparing homogenized dilutions of 20 ml brine samples in physiological saline solution with Tween 80 at a rate of 1%. Appropriate dilutions were plated on Rogosa Agar (7) and Modified Chalmer's Agar (8) for the lactic acid bacteria, Plate Count Agar plus penicillin for Gram-negative bacteria, Modified Clostridium Medium for anaerobic, sulphite reducing bacteria, Baird-Parker Agar for staphylococci and Oxytetracycline Glucose Yeast Agar for yeasts (9). The Most Probable Number (MPN) Method was used for the enumeration of the coliform bacteria (10). Lactic acid bacteria were isolated and identified by classical methods (7, 10, 11).
1851 b. Origin and selection of starter culture The strain of Lactobacillus plantamm used as starter culture was isolated on Rogosa Agar in our laboratory from spontaneous fermentation of olives in vessels. It was chosen by considering its ability of growing at large range of pH (4-9.6) temperature (10-45° C) and its capacity of rapid fermenting sugars. The production of diacetyl-acetoin was verified by Voges Proskauer method (7). c. Preparation of inoculum Starter cultures were prepared by two successive transfer in 150 ml MRS broth medium with incubation at 30° C for 24 h. Cultures were enumerated prior to inoculation then poured into vessels. d. Olive pretreatment and fermentation conditions Olives were washed and divided into 3 batches and immersed into the water with pH adjusted to 4.2 with acetic acid (glacial). Conditions of fermentations are given in Table 1. Olives in batches 1 and 2 were supplied with air, through a perforated plate placed to the bottom of the vessel and hold in tap water during 24 and 34 days, respectively. Air injected with the flux, ranging between 0.3-0.5 1 h" per one litre of capacity was applied during 8 h per day throughout the fermentation. After pretreatments, before starter culture addition all batches of olives were drained, washed and placed in brine, adjusted to pH 4.2, containing 6% NaCl. Following the pretreatment batch 1, was inoculated with starter culture of about 9.10 cfu ml' after 27 days and starter culture of about 2.10 cfu ml" was added to batch 3, after 17 days. There was no starter culture inoculation in batch 2. Table 1 Conditions of the fermentation processes Batches
Pretreatment (days)^
Aeration (0.5 1 h'M)
1 2 3
24 34 34
+
Starter addition time (days)
Inoculation (cfu ml"^)
27
9.10l«
17
2.10^^
^: Tap water with pH adjusted to 4.2 was used for pretreatments. : Starter cultures were added after brining.
1852 Experiments were carried out in vessels with 15 1 capacity at laboratory temperatures and the ratio of olives to brine was 3 to 2 (w/v). All samples were taken from brine for microbiological analyses and from olives and brine for chemical analyses. Experiments were followed during 160 days of fermentation. 2.3. Chemical methods Titratable acidity, volatile acidity and salt content of olive and brine samples were determined by the titrimetric methods of lOOC (1990). Spectrophotometric determination of polyphenols and analyses of moisture and ash were realized according to the methods of Fernandez-Diez et al, 1985 (12). Protein content (Nx6.25) of the fruits was determined with Kjeltech Auto 1030 Apparatus (Tecator). In reducing sugar analyses, the sugar were extracted according to Richmond et al, 1981 (13) and determined quantitatively, using HPLC (Beckman, USA) system. The HPLC system was consisted of M 421 System Controller, M 112 Solvent Delivery Module, M 210 Injection Part, U-Spherogel Carbohydrate Column (7.5 mm x 30 cm), M 156 RI Detector. Mobile phase was HPLC Grade Water and during analysis, column and mobile phase were heated to 85"* C. Water activity (a^) of the fruits was measured with EEJA-3 electronic a^ measurement device, Novasina, at 25° C. pH values was measured by pH meter (Metrohm).
3. RESULTS AND DISCUSSION 3.1. Composition of raw olives The poor load or absence of lactic acid bacteria in raw olives was indicated by several authors (14, 15). The microbial flora of raw olives used in these experiments was exclusively composed of yeasts, containing 10 cfu g" . Gram-negative, lactic acid, anaerobic sulphite reducing, coliform bacteria and staphylococci were not present in samples. Microbiological and chemical characteristics of raw and ripe olives are given in Table 2. Gemlik variety of olive has high fat and low moisture content in relation to other Turkish olive varieties (20). 3.2. Fermentation of olives In traditional method, the use of high salt concentration (14-16%) and the polyphenol content of olives are two inhibitory factors effecting the growth of lactic acid bacteria. For olives, fermented in brine with low salt concentration, the quantity of phenolic substances contained in olives being variable, depending on the environmental conditions of fruit three growth or annul climatic conditions, is the most important factor which plays the inhibitory effect on lactic acid bacteria growth (16). Prior to fermentation all the batches were maintained in water to reduce the polyphenol content of olives and to
1853 allow the survival of the inoculum. Table 2 Microbiological and physico-chemical composition of raw olives (Gemlik variety) Microorganisms (cfu ml" ) Gram-negative bacteria Lactic acid bacteria Anaerobic sulphite reducing bacteria Coliform bacteria^ Staphylococci Yeasts
Chemical composition (g 100 g" , fruit flesh) <10 <10 <10 <3 <10 10^
4.62 2.57 35.58 42.63 1.60 2.38 0.968
Reducing sugar Polyphenols Moisture Fat^ Ash Protein ^w w
^: Enumerated by MPN method. : Expressed in tannic acid. ^: Expressed in dry basis. In the experiments realized, the polyphenols were diffused from the fruit into the water during pretreatments and then eliminated in part from the fermentation media. Thus, polyphenols in water between 0.76% and 0.78% were discarded by means of the pretreatment water. In this way, at the moment of starter culture addition polyphenol content present in brine were about 0.07% in all batches. A great difference among batches were not observed in the diffusion of polyphenolic substances into the brines during the pretreatments in regard with the presence of air application and two different lengths of pretreatment (the 24th and the 34th days). At the end of the fermentation, polyphenol concentrations in brines were higher (0.15%) in batches 1 and 2, than that (0.125%) of batch 3 (Figure 1) and were between 0.34% and 0.36% (Figure 2) in all the batches. 8
1
In batch 1 the initial lactic acid bacteria population (6.10 cfu ml" ) was increased after inoculation to 10 cfu ml" within 73 days, then decreased to 2.10 cfu ml"^ within 119 days (Figure 3). Whereas in batch 3 the initial concentration of 2.10 cfu ml" was decreased to the same concentration (2.10 cfu ml" ) more rapidly, within 73 days of fermentation without an increment, in spite of higher inoculation (Figure 5). The final concentration of lactic acid bacteria (3.10 cfu ml" ) in batch 3, was lower than that of olives fermented with air supply in batch 1 (10 cfu ml" ) on the 160th day. The higher inoculum concentration (10 cfu ml" ) in batch 3 was not effected the growth of lactic acid bacteria and yeast population of the final product. While in batch 1 the air supply seemed to enhance the growth of lactic acid bacteria and yeasts with
1854 0.3•? 0.25 a c c o
0.2
5^ 0.15i J)
o c
0.1
Brine placing starter addition
a "o 0.05 0
20
AO
60 80 100 120 lAO 160 Fermentation period (days)
180 200
Figure 1. Changes in the polyphenol content in brines, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation. 1.2•2 o
1
Brine placing
1 0.8
Starter addition
1.0.6 "o
S 0.A
JZ Q.
?0.2 20
To
50 80 100 120 1A0 160 Fermentation period (days)
1^0 200
Figure 2. Changes in the polyphenol content in fruits, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
1855
"40
60 80 100 120 UO Fermentation period (days)
160
180
200
Figure 3. Changes in ( • ) lactic acid bacteria and (+) yeast populations during pretreatment and fermentation in batch 1.
(J
o
80 100 120 UO Fermentation period (days)
160
180
200
Figure 4. Changes (+) yeast populations during pretreatment and fermentation in batch 2.
1856
£
"5 U
O
20
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60 80 l o o ilo iZo Fermentation period (days)
i?0
i"§0
200
Figure 5. Changes in ( • ) lactic acid bacteria and (+) yeast populations during pretreatment and fermentation in batch 3. final concentration of 3.10^ cfu ml"^ and 4.10^ cfu ml"^ respectively, on the 160th day. In batches 1 and 2 supplied with air (Figures 3 and 4), yeast population growths were similar and higher than that of batch 3. Starter culture inoculation in batch 1 did not inhibit the yeast growth. The yeast growths were increased in batches 1 and 2 to maximum values of 9.10^ cfu ml"^ and 5.10^ cfu ml'^ respectively, due to the air supply. Similar researches carried on Spanish olive varieties which were fermented aerobically in 1001 fermenters, the maximum levels of yeast concentrations were reached about 10 cfu ml" in studies undertaken by Brenes Barbuena et al., 1986 (17) and Duran Quintana et al., 1991 (18). Maximum yeast growth in batch 3 without air supply was 3.10 cfu ml" . In batches inoculated with starter culture, the final microorganism populations were consisted of lactic acid bacteria and yeast by half. In batch 1, the competing lactic acid bacteria were mainly composed of Lactobacillus delbrueckii along with starter culture. In batch 3 in addition to L. delbrueckii, Leuconostoc mesenteroides species were present as the contaminating flora. There was no lactic acid bacteria growth and contaminating bacteria in batch 2, during fermentation in brine. Changes in salt contents in fruit flesh and brine are given in Figures 6 and 7, respectively. The applied salt concentration (6%) was suitable for lactic acid bacteria growth in olive fermentation. As it is known, high salt concentrations over this level inhibit lactic acid bacteria growth. Penetration of salt into the olives was slightly increased by air injection to reach at the final concentrations between 3% and 3.3% and
1857 the salt concentrations in brine was stable at about 4% NaCl in all batches after the 50th day. During pretreatments, some of the reducing sugars also pass to the water and discarded by brine placing. In fact, 0.29% (batch 1), 0.35% (batch 2) and 0.25% (batch 3) of the sugar contents in the waters were discarded from the fermentation media (Figure 8). Others were consumed by microorganisms present in the media. Leaching out of reducing sugars was evidently more accelerated by means of the aeration in batches 1 and 2 and consumed more rapidly due to the higher levels of microorganisms within 50th and 80th days, compared with batch 3. Thus, at the stage of starter culture inoculation reducing sugar concentrations in brines were 0.25%, 0.26% and 0.1% for batches 1, 2 and 3, respectively. At this moment, reducing sugar contents in olive flesh for the batches 1, 2 and 3 were 1.01%, 1.08% and 1.12%, respectively (Figure 9). As fermentation proceeds, reducing sugars were consumed by microorganisms and consequently converted to lactic acid by homofermentative metabolism of Lactobacillus plantarum and Lac. delbrueckii and to acetic acid CO2 , ethanol etc. in addition to lactic acid, by heterofermentative metabolism of Leuconostoc mesenteroides and yeast strains. The most rapid consumption periods in reducing sugar, correspond to the starter inoculation times in batches 1 and 2. Then reducing sugar utilizations in brines were similar and stable at about value of 0.04% in all the batches during the rest of the fermentation period. In the above mentioned study undertaken by Duran Quintana et al., 1991 (18) the sugar utilization, in the fermentation of olives from the variety of Gordal was similar particularly after the 80th day of fermentation. Reducing sugars in olives from batches 1 and 2 were decreased to the values below 0.1% after the 150th day of fermentation, whilst that of olives from batch 3, was 0.5% on the same day. Table 3 Some characteristics of the final product^ Batches 1 2 3
Moisture
Ash
Protein
a^
49.55 50.30 51.88
3.20 2.49 2.08
1.79 1.91 1.63
0 965 0.966 0.967
\ g 100 g"% fruit flesh.
1858
0
20
40
60 80 100 120 1/0 Fermentation period (days)
160
180
200
Figure 6. Changes in the salt content in fruits, ( • ) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
51
^ 4o
»
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~
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z 3• - 2-
pretrcatmenri
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1
20
1
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1
1
1
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60 80 100 120 140 Fermentation period (days)
1
160
1
180
200
Figure 7. Changes in the salt content in brines, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
1859 0.5'3 0^5 (/) o
Brine placing
5 0.35 t 0.3H
Starter addition
lo.25^ g^ 0.2^ •|0.15H Q::
0.1 0.05 0
AO
60 80 100 120 140 160 Fermentation period (days)
180 200
Figure 8. Changes in the reducing sugar content in brines, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
A.5H O
A-l
u 2 en 3.5
a 2 5-j Brine
/ placing
•D
c
/
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2^
u 1.5 D
cr
1-1
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0.5^ 0
0
20
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60 80 100 120 lAO Fermentation period (days)
160
180
200
Figure 9. Changes in the reducing sugar content in fruits, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
1860 0.7-
8 0.6H a 0.5 Starter addition
O-AH o 0.3 H a 2 0.2
pretrcatments»
-^
'
0.1 20
40
60 80 100 T^O UO 160 Fermentation period (days)
180
200
Figure 10. Changes in the free acidity content in brines, (•) batch 1, brine placed on the 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation. 0.6 0.5 0.A 0.3 0.2
^
pjetreatmenbl
o.H 20
— I —
AO
60 80 100 120 UO Fermentation period (days)
160
180
200
Figure 11. Changes in the content of volatile acidity in brines, (•) batch 1, brine placed on 24th day of fermentation, (+) batch 2 and (o) batch 3, brine placed on the 34th day of fermentation.
1861 As an important fermentation product, free acidity values formed in brines during fermentation processes are given in Figure 10. It can be seen that the free acidity formation has been higher in batches 1 and 3 with maximum values of 0.44% and 0.64%, respectively. The free acidity in batch 2 with the maximum value of 0.27% was attained at the end of the fermentation, on ground of the absence of the lactic acid bacteria growth. Volatile acidity was relatively lower in batches 1 and 2 than batch 3 due to the aeration applied (Figure 11). The moisture, ash and protein contents and a^ values of the final products from the three batches are depicted in Table 3. The moisture content of raw olives was incraesed in final product from value of 35.58%, to 49.55%, 50.30% and 51.88% for batches 1, 2 and 3, respectively. The ash contents were increased in all the batches, but the values from batch 1 were higher than those of batches 2 and 3. The protein content in raw olives was decreased to 1.79%, 1.91% and 1.63% in batches 1, 2 and 3, respectively. The salt contents in the final brines and fruits were between 3.5% and 4% NaCl which correspond approximately to 0.96, a^ values which allow the lactic acid bacteria growth (19). In the sensory analyses conducted with 10 panelists by using 1-9 score scale, properties of olives from the batches with air injection revealed the higher scores than that of the batch without air application. The points obtained were 7.6 and 6.9, for color and apperance, 7.6 and 7, for texture and 7.5 and 6.7 for taste, in the batches with and without air injection, respectively. Further studies must be performed on the olives processed aerobically, to differentiate the organoleptic properties between those fermented with and without starter culture addition. As it is mentioned in our prior studies (20) starter culture utilization in black olives fermentation was considered inapplicable to the olives processed by traditional method, because of the high salt and polyphenol content. From the results of our experiments we conclude that the establishment of lactic acid bacteria population is possible by the application of a pretreatment with pH adjusted water and low salt content brine. It was observed that, keeping olives in water (with 4.2 initial pH value) during 34 days did not cause a detrimental effect on the organoleptic properties of the final product. Aeration accelerated the leaching out of polyphenolic substances and stimulated the reducing sugar consumption by enhancing the microbial growth. Therefore, our results suggest that the combined effects of pretreatment, aeration and starter culture application lead to a rapidly fermented (approximately within 5 months) product with low polyphenol content and desired fermented taste, colour and texture. Thus it appears that the extention of pretreatment within a period of 24 days and application time of the starter culture remain to study in further experiments.
1862 Acknowledgments This work was financially supported by NATO-SFS Programme within the scope of the NATO-TU-FERMENTECH Project. We would like to thank to Prof. Dr. M. Pala Head of our Department for supporting the project studies, to General Directory of Marmarabirlik, Union of the Olive Producers Cooperatives, for their collaboration, to Miss. E. Goziim and Mr. B. Qrak for their technical assistance in laboratory studies.
4. REFERENCES 1 T.J., Montville, M.E., Meyer and A.H.M., Hsu, J. Food Prot. 50 (1) (1987) 42-46. 2 A., Garrido-Fernandez, M.C., Duran Quintana, and P., Garcia Garcia, Grasas y Aceites, 38 (1) (1987) 27-32. 3 M., Borcakh, G., Ozay and I., Alperden, In: G. Charalambous (ed.). Food Flavors, Ingredients and Composition, Elsevier Science Pubhshers, Amsterdam, (1993) 265277. 4 J.L., Ruiz-Barba, R.M., Rios-Sanches, C, Fedriani-Iriso, J.M., Olias, J.L., Rios and R., Jimenez-Diaz, Syst. Appl. Microbiol., 13 (1990) 199-205. 5 J.L., Ruiz-Barba, A., Garrido-Fernandez and R., Diaz-Jimenez, Lett, in Appl. Microbiol, 12 (1991) 65-68. 6 G.J.E., Nychas, C.S., Tassou and R.G., Board, Lett, in Appl. Microbiol., 10 (1990) 217-220. 7 W.F., Harrigan and M.E., McCance (eds.). Laboratory Methods in Food and Dairy Microbiology, Academic Press, London, (1976) 452. 8 v., Vanos and L., Cox, Food Microbiol., 3 (1986) 223-234. 9 Merck, Culture Media Handbook, Darmstadt, (1987) 232. 10 C.H., Collins and P.M., Lyne (eds.). Microbiological Methods. Butter Co. Publishers. London, (1987) 136-141. 11 M.E., Sharp, In: P.M., Starr, H., Stolp, H.G., Triiper, A., Ballows and H.G., Schlegel, (eds.). The Procaryotes, Springer-Verlag, Berlin, 2 (1981) 1653-1679. 12 M.J., Fernandez Diez, R.C., Ramos, A., Garrido-Fernandez, F.G., Cancho, P.G., Pelhso, M.N., Vega, A.H., Moreno, I.M., Mosquera, L.R., Navarro, M.C., Duran Quintana, F.S., Roldan, P.G., Garcia, A.C., Gomez-Millan (eds.), Biotecnologia de la Aceituna de Mesa. Consejo Superior de Investigaciones Cientificas. Instituto de la Grasa. Sevilla, (1985) 475. 13 M.L.,Richmond, S.C., Brandao, J.I., Gray, P., Markakis and CM., Stine, J. Agric. Food Chem., 29 (1981) 4-7. 14 H.P., Fleming, In: A.H., Rose, (ed.) Economic Microbiology. Fermented Foods. Academic Press. London, 7 (1982) 227-258.
1863 15 A., Garrido-Fernandez, M.C., Duran Quintana, F., Gonzales Cancho and M.J., Fernandez Diez, Grasas y Aceites, 23 (1) (1972) 22-31. 16 M.J., Fernandez Diez, H.J., Rehm and G.Reed (eds.), In: Biotechnology, Verlag Chemie. Weinheim, 5 (1983) 380-397. 17 M., Brenes Balbuena, P., Garcia Garcia, M.C., Duran Quintana and A , GarridoFernandez, Grasas y Aceites, 37 (3) (1986) 123-128. 18 M.C., Duran Quintana, M., Brenes Balbuena, P., Garcia Garcia, M.J., Fernandez Gonzalez and A., Garrido Fernandez, Grasas y Aceites, 42 (2) (1991) 106-113. 19 J.A., Troller and J.V., Stinson, Appl. and Environ. Microbiol. 42 (4) (1981) 682-687. 20 M., Borcakli, G., Ozay and I., Alperden, Grasas y Aceites, 44 (4-5) (1993) 253-258.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1865
Changes in Soluble Sugars in Various Tissues of Cultivated Mushrooms, Agaricus bisporus. During Postharvest Storage*
Said O. Ajlouni**, Robert B. Beelman, Donald B. Thompson, and Jeng-Leun Mau Department of Food Science, The Pennsylvania State University, University Park, PA 16802 Keywords: Agaricus bisporus. mannitol, trehalose, ribose Abbreviated title: Mushroom Sugars * Presented at the Annual Meeting of the Institute of Food Technologists, Anaheim, CA, June 16-20, 1990. Abs. No. 239. ** Present address: Atomic Energy Commission, Damascus, Syria.
ABSTRACT Soluble sugar contents in various tissues of two hybrid strains (Ul and U3) of cultivated mushrooms, Agaricus bisporus. were determined during 10 days of storage at 12°C. No significant differences in mannitol, trehalose and ribose contents were observed between these two hybrid strains at harvest. Mannitol content decreased in the cap and lower stipe throughout the storage period, while it remained steady in gills and increased slightly in upper stipes. No significant changes in trehalose contents were observed. Ribose was continuously accumulated in all tissues of the two hybrid strains during postharvest storage.
1866
INTRODUCTION Fresh mushrooms are highly perishable, and their quality declines rapidly after harvest. Quality changes in mushrooms include browning (surface discoloration), dehydration, maturation and senescence. Harvested mushrooms undergo a course of postharvest development similar to that for those allowed to remain growing on the bed. The morphological changes (maturation and senescence) during postharvest storage, especially stipe elongation, gill exposure and sporulation, are supported by water and nutrients that are present in the mushrooms at harvest (Hammond and Nichols, 1975). The mushroom sporophore has a high soluble sugar content, and this appears to be the obvious source of respiratory substrate. Hammond (1977) reported that mannitol, trehalose and a glycogenlike polysaccharide were non-structural carbohydrate present in sufficient quantity to act as substrate in flush development. In Agaricus bisporus. mannitol is the major constituent of the soluble carbohydrate fraction, accounting for up to 40% of the sporophore dry weight (Rast, 1965). The loss of a significant amount of mannitol from the sporophore during postharvest storage indicated that it is the major respiratory substrate in harvest mushrooms (Hammond and Nichols, 1975).
Goody (1974)
suggested that postharvest quality changes in cultivated mushrooms are related to the dynamic redistribution of carbohydrate components. Soluble sugars, which may be utilized in postharvest sporophore development are considered to be important factors in determining the rate of maturation. Elucidation of soluble sugar distribution among various tissues of harvested mushrooms during storage represents an important step towards an understanding of the basic processes related to mushroom quality after harvest. The research reported herein was designed to examine changes in soluble sugars in the sporophore, cap, gills, upper and lower stipes during storage at 12°C.
1867
MATERIALS AND METHDOS Hybrid off white and white strains (Ul and U3, respectively) of Agaricus bisporus were grown on traditional straw-bedded horse manure compost at the Mushroom Test and Demonstration Facility (MTDF) on the Pennsylvania State University campus. Using standard cropping procedure and spawn from different manufacturers, three crops of each hybrid were cultivated at six different times under their optimal growing conditions. Mushrooms harvested on the peak day of the second flush were used in all experiments. Freshly harvested mushrooms were transported within about 1 hr to a cold room (4°C) for about 2 hr before sampling. Mushrooms were sorted on the basis of size and appearance. Diseased, damaged, misshapen, open-veiled and extremely large or small mushrooms (>40 or <25 mm in cap diameter) were discarded. Mushrooms of uniform size (25-40 mm) and maturity of the button stage (stage 1, veil intact tightly) (Schmidt, 1977) were used in this study. To avoid the effects of variations in size and maturity, mushrooms of similar size and maturity from all Ul and U3 crops were selected as the samples. Stipes were trimmed to a mean lower stipe length of 15±2 mm. All acceptable mushrooms were pooled, and then approximately 100-g samples were placed into 227-g linear polystyrene trays and overwrapped with polyvinyl chloride (PVC) stretch film (60 gauge PWMF Vitafilm, Goodyear Tire & Rubber, Akron, OH). To simulate the ideal retail packaging of mushrooms, the packages were sealed by application of mild heat from a hot plate to the tray bottom, and ventilated through two 3-mm
1868 holes punched through self-adhesive labels placed equidistantly on the top of the package. After packaging, three packages of mushrooms from each crop were randomly selected for day 0 analyses. To simulate retail market storage, the rest of packages were then stored in a 12°C incubator (Freas Model 815, Lunair Environmental, WiUiamsport, PA). On days 2, 4, 6, 8 and 10, three packages of mushrooms were randomly selected for the study. All the mushrooms in a package were cut into quarters.
One quarter of each sporophore in a package was used to
represent whole sporophore tissue, while the remaining sporophore quarters were further dissected into cap, gill, upper and lower stipes. Similar tissues from the same package were combined and freeze-dried using a VirTis 15 SRC-X freeze dryer (VirTis, Gardiner, NY). Freezedried tissues were then ground to powder using a Micro Mill (Chemical Rubber, Cleveland, OH) and stored in a desiccator until carbohydrate analysis. A precise quantity (300±0.1 mg) of freeze-dried tissue was extracted with 50 ml 80% aqueous ethanol. A precise quantity (50±0.1 mg) of xylose (Sigma Chemical, St. Louis, MO) was added as an intemal standard. This suspension was shaken for 45 min at room temperature using a Platform Rotator (Haydon Mfg, Torrigton, CT) and filtered through Whatman #4 filter paper. The residue was washed 5 times with additional 5-ml portions of 80% ethanol. The combined filtrate was then rotary evaporated and redissolved in deionized water to a final volume of 50.0 ml. An aliquot of the aqueous extract was passed through a SEP PAK Cig Cartridge (Waters Associates, Milford, MA), and filtered using 0.45 |Lim
1869
Acrodisc-CR Filter (Gelman Science, Ann Arbor, MI) prior to injection on to HPLC. The HPLC system consisted of a Rainin HP/HPX pump, a Rheodyne 7125 injector, a 20 |i.l-sample loop, a Waters 401 differential refractometer, and a Shimadzu C-R3A integrator. A 30 cm x 7.8 mm i.d. Aminex HPX-87C column (Biorad, Rockville Center, NY) was connected with a 3 cm X 4.6 mm i.d. micro guard cartridge. The mobile phase was deionized water at a flow rate of 0.6 ml/min. The column temperature was maintained at 85°C. Each sugar was quantified by comparing the peak area of the sugar to that of the internal standard. The amount of sugar was expressed as the percent of the dry weight of stored mushrooms. A split-plot experimental design was used in this study. Each hybrid strain was considered as a plot and within each plot there were 3 crops. A single crop of one hybrid strain was considered as a replicate (sub-plot). Harvested mushrooms were examined every 2 days during storage for 10 days at 12°C. The experimental data were subjected to the analysis of variance using the General Linear Model Procedures developed by Statistical Analysis System (Helwing and Council, 1985) to determine the least significant difference (LSD) among means. The level of significance used in this research was 0.05.
1870
RESULTS AND DISCUSSION Soluble sugar contents in various tissues of Ul and U3 hybrid strains of Agaricus bisporus on the day of harvest are presented in Table L Mannitol was found to be the major soluble sugar in freshly harvested mushroom sporophores (27.0% of dry weight for Ul hybrids and 25.5% for U3 hybrids). Trehalose contents were similar to those reported by Rast (1965), who found approximately 1% dry weight in the fruit bodies. Ribose was detected in mushroom sporophores for the first time. Except in the trehalose content of upper stipes no significant differences were observed in each sugar content of whole sporophores or various tissues between these two hybrid strains. In both hybrid strains, mannitol was distributed unevenly among various tissues of sporophores. Caps and lower stipes had the highest amount of mannitol, while gill tissues had the lowest. Soluble sugar contents in whole sporophores during storage are shown in Fig. 1. The decrease in mannitol contents of both hybrid strains during storage was similar. However, starting from day 8 on, significant differences in mannitol contents between the two hybrid strains were observed. Trehalose contents of two hybrid strains varied during storage, while ribose contents generally increased. The ribose content of U3 hybrids decreased after 8 days of storage, and differed from that of Ul hybrids at the end of storage period (10 days). Mannitol content decreased steadily in caps and lower stipes, while it remained steady in gills and increased slightly in upper stipes with both hybrid strains (Fig. 2). No differences were found in mannitol contents of
1871
Table 1. Soluble sugar contents (% dry weight) in various tissues of Ul and U3 hybrid strains of Agaricus bisporus on the day of harvest.
Hybrid
Soluble sugar content (%)
Tissue Mannitol
Trehalose
Ribose
Ul
Sporophore Cap Gills Upper stipe Lower stipe
26.97 A 31.45 A 11.43 A 21.25 A 27.83 A
1.87 A 1.85 A 1.66 A 3.14 A 2.03 A
0.37 A 0.59 A 0.25 A 0.58 A 0.15 A
U3
Sporophore Cap Gills Upper stipe Lower stipe
25.51 A 29.86 A 9.81 A 19.22 A 27.66 A
1.01 A 2.95 A 2.60 A 0.73 B 1.03 A
0.58 A 0.53 A 0.43 A 0.33 A 0.29 A
*Values with the same letter for each sugar, are not significantly different (p = 0.05). Comparisons were made within the same tissue for the two hybrids.
0
2
4
6
8
1
0
I
Trehalose
5~
0
2
4
6
8 1 Storage Time (Days)
0
Ribose
0
2
4
Fig. 1. Soluble sugar contents in the sporophore of U1 and U3 hybrid strains of Aearicus bisporus during storage at 12°C. Values with the same letter within a day are not significantly different (p = 0.05).
6
8 1 0
Stipe n
Gills
I
Storage Time (Days) Fig. 2. Mannitol contents in various tissues of U1 and U3 hybrid strains of Agaricus bisponls during storage at 12°C. Values with the same letter within a day are not significantly different
(p = 0.05).
1874
caps, gills and lower stipes between two hybrid strains. After 6 days of storage, upper stipes showed the differences in mannitol contents between two hybrid strains, partially resulting from differences in mannitol contents of whole sporophores after 8 days of storage (Fig. 1). Ba sed on the original mannitol amount at harvest, mannitol losses after 10 days of storage were 7.0% and 10.8% from caps and lower stipes in Ul hybrids, respectively, while in U3 hybrids the losses were 8.5% from caps and 15.1% from lower stipes. The losses in mannitol contents of caps and lower stipes with two hybrid strains were consistent with the decrease in dry weights of caps and lower stipes (Ajlouni, 1991). Moreover, the increase in mannitol contents of gills and upper stipes with two hybrid strains was concurrent with the increase in dry weights of gills and upper stipes (Ajlouni, 1991). These observations are in general agreement with the suggestion of Schmidt (1977) that translocation of mannitol occurred between various tissues of golden-white and off-white strains during postharvest storage. Based on the mass flow hypothesis in plants, as described by Devlin (1975), Schmidt (1977) proposed that the diffusion of mannitol from one tissue to another was driven by a turgor pressure gradient. Pressure would be relieved by the growth of tissues as assimilation of metabolites and water occurred, and the direction of translocation would also be from a region of higher concentration to a region of lower concentration. Since his and our results showed that caps and upper stipes were the most massive tissues and had the greatest concentration of mannitol and moisture, the diffusion of moisture and mannitol would be directed to gills and upper
1875
Stipes, particularly when these tissues have a higher turgor pressure due to their fast growth and development. The cap and lower stipe tissues appear to be the major sources of substrate (mannitol) that supports respiration and development of mushrooms after harvest.
The decrease in mannitol contents of
sporophores is sufficient to account for up to 64% of the total CO2 produced by respiration after 2 days of storage, and for about 45% of the total CO2 produced after 10 days of storage at 12°C (Ajlouni, 1991). Those values were similar to those reported by Hammond and Nichols (1975), who indicated that the decline in mannitol during storage at 18°C was large enough to account for up to 50% of the total CO2 produced. The observation that mannitol contents of upper stipes initially increased and peaked on day 6 with two hybrids strains (Fig. 2) was consistent with the fast elongation of upper and lower stipes during storage up to day 6 (Ajlouni, 1991). This observation was in general agreement with the suggestion that translocation of mannitol and water from lower to upper stipes can cause or (support) growth and cell enlargement of upper stipes (Ajlouni, 1991). Ajlouni et al. (1992) found that the removal of lower stipes immediately after harvest resulted in extended shelf life of harvested mushrooms. That finding might be due to the reduction of substrate available for respiration or translocation from lower to upper stipes, thus preventing growth of upper stipes, which promotes cap opening and thus indirectly extends the shelf life. Variation in the trehalose content of caps was observed within each hybrid strain during the storage period, while no significant differences were found between these two hybrid strains (Fig. 3). The trehalose
4
V)
a
'1
Gills
4
Stipe
Lower Stipe
Storage Time (Days) Fig. 3. Trehalose contents in various tissues of U1 and U3 hybrid strains of Agaricus bisporus during storage at 12°C. Values with the same letter within a day are not significantly different
(p = 0.05).
6'
Gills
A
Lower Stipe
Storage Time (Days) Fig. 4. Ribose contents in various tissues of U 1 and US hybrid strains of Agaricus bisuorus during storage at 12°C. Values with the same letter within a day are not significantly different (p =
1878
contents of gills were higher with Ul hybrids than with U3 hybrids on days 2, 4, 6 and 10. Similariy, the trehalose contents of upper and lower stipes seemed to be higher with Ul hybrids than with U3 hybrids. No significant changes were observed in trehalose contents of whole sporophores (Fig. 1) or various tissues during postharvest storage with two hybrid strains (Fig. 3).
Trehalose levels during growth of sporophores was previously
observed to be similar in all tissues (Hammond and Nichols, 1976), and during postharvest development trehalose levels decreased from approximately 5% dry weight at stage 2 to 1-2% at stage 7 (Hammond and Nichols, 1975). Ribose content of each mushroom tissue increased continuously during postharvest storage for both hybrid strains (Fig. 4). Obviously, most of the difference in the rate of ribose accumulation between sporophores (Fig. 1) of two hybrid strains was attributed to that in the cap tissues (Fig. 4). The increase in ribose content during storage suggests that the pentose phosphate pathway, though which ribose is formed, may be activated during storage, possibly in order to provide enough NADPH necessary for the activity of mannitol dehydrogenase.
The pentose
phosphate pathway begins with the dehydrogenation of glucose 6phosphate, which is catalyzed by the enzyme glucose 6-phosphate dehydrogenase (G6PD). Hammond's (1981) demonstration of the presence of this enzyme in a large quantity in sporophores is consistent with the possibihty of ribose accumulation in mushrooms during postharvest storage through the pentose phosphate pathway. In our study, significant losses of mannitol (Fig. 1 and 2) and increases of ribose (Fig. 1 and 4) during postharvest storage were
1879
observed. The pathways that are involved in these processes produce reducing equivalents (NADH and NADPH, respectively). It is plausible that mushrooms metabolize these reducing equivalents through electron transport and oxidative phosphorylation systems as in the higher plants. The produced ATPs would be used to support the growth of upper stipe and gill tissues and the formation of the spores. Mushrooms may also use the NADPH against oxidizing agents. This might be accomplished in a way similar to that in erythrocytes (Newsholme and Leech, 1983), and in plants (Salisbury and Ross, 1985), where NADPH reduces the oxidized form of glutathione, and the reduced glutathione then reacts with hydrogen peroxide and organic hydroperoxides. The presence of glutathione in mushrooms has not been studied.
REFERENCES Ajlouni, S. O. 1991. Quahty characteristics of two type hybrids of the cultivated mushrooms (Agaricus bisporus) and the improvement of their shelf Hfe using stipe trimming and gamma irradiation. Ph.D. Thesis, The Pennsylvania State University, University Park, PA. Ajlouni, S. O., Beelman, R. B., Thompson, D. B., and J.-L. Mau. 1992. Stipe trimming at harvest increases shelf life of fresh mushrooms (Agaricus bisporus). J. Food Sci. 57: (in press). Devlin, R. M. 1975. "Plant Physiology." Van Norstrand, New York. Gooday, G. W. 1974. Control of development of excised fruit bodies and stipes of Coprinus cinereus. Trans. Br. Mycol. Soc. 62: 391.
1880
Hammond, J. B. W. 1977. Carbohydrate metabolism in Agaricus bisporus: Oxidative pathway in mycelium and sporophore. J. Gen. Microbiol. 102: 245. Hammond, J. B. W. 1981. Variations in enzyme activity during periodic fruiting of Agaricus bisporus. New Phytologist 89: 419. Hammond, J. B. W. and Nichols, R. 1975. Changes in respiration and soluble carbohydrates during the postharvest storage of mushroom (Agaricus bisporus). J. Sci. Food Agric. 26: 835. Hammond, J. B. W. and Nichols, R. 1976. Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: Changes in soluble carbohydrates during growth of mycelium and sporophore. J. Gen. Microbiol 93: 309. Helwing, J. T. and Council, K. A. 1985. SAS User's Guide. SAS Institute, Gary, NC. Newsholme, E. A. and Leech, A. R. 1983. "Biochemistry for the Medical Science." John Wiley, New York. Rast, D. 1965. Zur stoff wechsel physiolgischen bedeutung von mannitol and trehalose in Agaricus bisporus. (eine gaschromatographische studie) (Enghsh Abstract). Planta 64: 81. Salisbury, B. F. and Ross, W. C. 1985. Hormones and growth regulator: Cytokinins, ethylene, abscisic Acid, and other compounds. In "Plant Physiology." Wadsworth, Belmont, CA Schmidt, C. E. 1977. Postharvest quality changes in two off-white strains of cultivated mushrooms Agaricus bisporus. M.S. Thesis, The Pennsylvania State University, University Park, PA.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1881
CONCENTRATION OF HEAVY METALS AND NUTRIENTS IN THE LEAVES OE CERTAIN EOREST SPECIES IRRIGATED BY TREATED WASTEWATER P. Drakatos^, G-, i^allistratos*^j^ I. Panariotou"^, -^ I. Kalavroiiziotis''^ P. Skouras' and M. Stoyianni ^Department of Mechanical Engineering, University of Patras, Greece ^Department of Experimental Physiology, Faculty of Medicine, University of loannina, Greece ^Department of Economics, University of Patras, Greece Abstract In this paper, the ability of certain forest species to absorb heavy metals contained in treated wastev/ater is examined. Forest species adapted to the Greek enviroriirient, specifically 01 ea europea. Geranium spp,, i^ereum oleander ^^^ Myoporum spp. were used. The concentration of heavy mietals was measured in the irrigation water and the leaves and tissues of the different forest species following standard chemical techniques. The results indicate an increased concentration of heavy metals for Kereum oleander, Kyoporum spp. and Geranium, spp., and only moderate concentration for Olea europea. Introduction Land treatm^ent of v/astewater, although with a long international history, is relatively new to Greece. Land treatm.ent has been used in reforestations with very good results. One of tPxe main obstacles that need to be considered ie the tolerance of certain forest species to the high concentrations of heavy mietals present in the treated wastewater. In this work, wastewater from the Wastewater Treatm.ent Plan (WWTP) of the University of Patras was used to irrigc?.te certain forest specieso Olea europea. Geranium species, hereum oleander and Myoporum species were specifically selected for this study because of their adaptability to the Greek environment and their extensive use in floriculture and in landscape architecture.
1882 The main objective of this research was to investigate the tolerance of the above mentioned forest species to the increased concentration of heavy metals contained in the treated wastewater used for irrigation.
Materials and Methods, The chemical quality of the wastewater was periodically monitored in order to determine the concentration in heavy metals at the entrance and exit points of the VAVT?. The experiment involved the use of 200 plants for each of the forest species selected. These plants were one year old at the beginning of the experiment.The plants of each species were divided into two groups: the test treatment group (irrigation with treated wastewater) and the control group (irrigation with ordiuary water). Samples of leaves v/ere taken several times t •roi;ghout a period of one year from both tlie test and the control groups of each species under consideration. These samples were analysed to measure the concentration in neavy metals as well as certain ions, following the standard methoas of the S o M Science Society of America (Page £t al., 1982). The plants v/ere irrigated up to soil water saturation tv;ice every v/eeh. Results and Discussion. Results regarding the mean concentration of different heavy metals in the treated wastewater and the ordinary irrigation water are given in Table 1. All concentrations reported are withi/i the limits set for use in agriculture (Pettygrove and Asano, 1985). Mean concentration of the different heavy metals and nutrients found in the leaves of the species under consideration is shown in Table 2 and in figures 1 to 9. 01 ea europa shov/s a lower concentration in most of the ITeavy m.etals in question - with the exception of copper and boron. However, the concentration of boron, specifically for olive oil trees is desirable since most of the Greek soils cf olive oil tree plantations are deficient in boron.
1883 The above results are reported for the first time in Greece under a "real conditions" experimental approach. Although indicative of what may he expected, additional research is required. This should be extended to more species over longer tiiDe periods, using wastewater of different qualitative parameters.. In addition, such research should be accompanied by relevant soil analyses for examining the long teriri pollution of soil and water resources. Bibliography E.P.A., 1980. Outside V/astewater Treatment and Disposal SysteraSo Lesign ••anual. U.S.A. E.P.A., 1981. Land Treatment for Municipal V/astewater. Process Design I-anual. Cincinnati, OK, U.S.A. Page, A.L., r'iller, R.H. and heeney, D.R., 1982. liethods of Soil Analysis, Part ". Chemical and I'licrobiological Properties, Second Edition, American Society of Agronomy, Inc., Soil Sciences Society of America, Inc. Kadison, WI, U.S.A. Pettygrove G. and Asano, T., 1985* Irrigation with re 1 aimed ruunicipal wastev/ater. A guidance manual.
TABLE I. Comparison in the coucentration of main nu,trients in treated wastewater and irrigation water.
PH Irrigation water
7,47
Wastewater
7,72
Electrical Conductivity
Ca
Mq
K
Fe
Zn
1.339
143,l
14,7
3,9
0
0,25
1.478
135
17
Cu
Mn
cz
cd
P
Na
0,025
0
0
2
56.25
7,75.0,001 0,105 0,006 0,042 0,039
0
0
4
0.001
pb
0
121
SAR 1,24
2,56
TABLE 2 % Change of the concentration of certain nutrients in plant leats irrigated by treated wastewater and irrigation water.
Irrigation with treated wastewater ~rrigation Water
-Fe(ppm)
Mg(ppm)
Ca
m(ppm)
~ ( p p m ) Cu(ppm1
Zn(ppm)
144,2
204,5
90,12
68
16
51,5 121
85
80
78,6 173,6 157,l
1. OLEA EUROPA
100
57,6
2. NEREUM OLEANDER
100
517,6
97
144,4
51,l
3. GERANIUM S.P.P.
100
110,5
160
90,6
184,6
80,2
40
59
4. MYOPORUM S.P.P.
100
143
115,6
92,7
56,l
81,8
71,7
69,l
125
217,6 137 91,4 223,5
1886 120 T
S
a a a a
I o
Olea
Nereum
Geranium
Myoponim
Figure 1: Concentration of Mn (ppm) in leafs, (white columns are controls and black are treatments).
1887
a
a-
Olea
Nereum
Figure 2: Concentration of B (ppm).
Geranium
Myoporum
1888 30 -r
25 + 0
20 4-
Olea
Nereum
Figure 3: Concentration of Cu (ppm).
Geranium
Myoponim
1889 120 T
S
1
a
105.5 100
80 +
60 -f
40 +
20 +
Olea
Nereum
Figure 4: Concentration of Zn (ppm).
Geranium
Myoponim
1890 137.5
140 T
o o
f
a
Olea
Nereum
Geranium
Figure 5: Concentration of Fe (% of dried substance)
Myoporum
1891 0.6 -r
0.5
o o o
0.4 4-
0.3 4-
I 0.2 +
0.1 4-
Olea
Nereum
Geranium
Figure 6: Concentration of Mg (% of dried substance).
Myoponim
1892 3 r
3
I
"S
o U
Olea
Nereum
Geranium
Figure 7: Concentration of Ca (% of dried substance).
Myoponim
1893 2 -r
a
Olea
Neremn
Geranium
Figure 8: Concentration of K (% of dried substance).
Myoporum
1894 0.4 T
e o o
0.38
0.3 +
B
i
0.2
0.1 +
Olea
Nereum
Geranium
Figure 9: Concentration of P (% of dried substance).
Myoporum
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1895
A Rapid Method for Determining the Water Dynamics (Uptake and Loss) of Moisture Sensitive Foods
S.G.Gilbert*, G.W. Greenway, J.X. Liu and O. Ramon
•address correspondence to: S.G. Gilbert, Center for Packaging Science and Engineering, Rutgers University, Building 3529,
Piscataway, NJ 08855.
ABSTRACT: Modified Frontal Inverse Gas Chromatography (MIGC) was used to determine the sorption behaviour of food materials. The method is dynamic and has the ability to demonstrate and delineate differences in the water exchange mode and kinetics of food systems. Selection of the proper contact area and operating variables provide sorption rates and generate sorption isotherms in several hours, in good agreement with data obtained by using static gravimetric methods which may require many weeks. The differences in sorption kinetics, as related to structure created by various dehydration methods of instant coffee are shown dinstinctly by the isotherms obtained by the MIGC method.
1896 INTRODUCTION The food industry manufactures and distributes many moisture sensitive foods. Any technique which can reduce the time required for the development of a new product will result in both economic and competetive advantages in the marketplace. Slade and Levine(1991), stated that there is a need of alternative experimental approaches and interpretations for prediction of stability, biological behaviour and quality of food systems. The approach is based on the kinetics of water exchange, where neither the water content nor the water activity can adequately describe the kinetic behaviour of real food systems. When the latter are not in equilibrium and therefore have limited shelflife a kinetic approach should be applied. Sorption Isotherms of foods or biological components are conventionally determined gravimetrically using prolonged exposure of the food to a specific relative humidity (expressed as Relative Vapour Pressure, RVP) measuring water uptake or loss. Weeks may be required for the water exchange to reach an equilibrium between the chemical potential of the water in the food product and the water vapor phase surrounding it. A method developed by Gilbert et al.,(1988,ibid,1989) can provide an equivalent water uptake/relative vapour pressure relationship in hours. This is modified inverse gas chromatographic method (MIGC) as used previously by Apostolopoulos and Gilbert (1988) and extended by Ramon et al.( 1990) and Liu et al.(1993). Test
1897 materials are food components or biological materials as a solid, fixed bed, stationary phase whose chemical and physical properties are investigated in relation to variables in the mobile phase such as partial water vapor pressure in the carrier gas. Factors such as temperature and gas flow rate can be varied to measure thermodynamic properties (Apostopoulus and Gilbert, 1990) and the kinetic of the water excange and to relate it to the concentration of water in the mobile and the stationary phases. The main feature of the method, in the revised form, is the capability of characterizing and evaluating the water exchange of metastable foods in a dynamic system. The present study was aimed to show: (a) That structural properties of soluble coffee, produced by different dehydration methods, can be characterized by their sorption behaviour measured with MIGC. (b) That by careful selection of the food-water exchange conditions, controlling the gas chromatograph column design and geometry, and by varying the operating variables, it is possible to obtain in some food systems, in a relative short time, sorption isotherms data similar to those obtained by time averaging methods.
The development of MIGC equations Ferng (1987) and Gilbert (1989) showed that since the product of flow rate, time and concentration equal the input mass, a
1898 constant input concentration provides the calculation of mass from time to any retention volume. The unsorbed mass is directly proportional to the area of the chromatogram at that time with the mass difference giving the mass retained. In the sorbent, the partial water pressure at any time is proportional to the response voltage which is detected by a sensitive thermal conductivity detector TCD. A typical chromatogram of a sorption/desorption cycle is shown in Fig. 1.
Determination of calibration constants Based on the preceeding work, it was shown that empty columns provided for calibration of the TCD for water mass concentration. Empty columns provide basically an essentially constant ratio of input mass to time, at constant flow rate, where the concentration of the water vapor is determined by the temperature of the carrier gas saturated with water vapour. Since there is no sorption, the area of the resulting peak is also proportional to the mass input at any retention time. Thus two constants may be derived:
mass of water injected K, =
g «
time
(1) sec
1899 mass of water injected K, =
g «
chromatographic response area
(2) V x sec
where:
Kt - time constant for the mass entering per unit of time
K^ - area constant that reflects the detector sensitivity
V - response voltage of the detector
The time constant, K^, depends on flow rate and concentration. Since the latter is saturated water vapor pressure, it is directly related to temperature. Liu et al.(1991) observed that the water mass injection holding time in an IGC column for foods which have strong diffusion dependent kinetics the temperature and flow rate conditions may be longer than the holding time of the same water mass injection in an empty column at the same operating conditions. Using a corrected time constant based on the actual holding time (tjj^^x ^^ Fig 1.) they obtained accurate sorption isotherms of flour. For strongly hydrophilic systems such as dextrins as in soluble coffee, the original assumptions are still valid. The area
Fig. 1
Chromatogram (response vs. time) of a typical IGC sorption experiment. The area of the
chromatogram in Fig.1 represents the out going mass of water during sorption or desorption.
5
10
TIME (SEC)
1901 constant, K^, is proportional to the mass per unit area
which is
the product of the pressure and time. Since higher flow rates decrease the time for a given mass input and higher temperatures increase concentration with proportionate decrease in time, the area constant is independent of temperature. The product of flow rate and time is volume, K^ the area constant is directly proportional to flow rate. The linear relation of detector response to mass at constant detector volume and pressure provides the concentration factor so that the mass change is proportional to the response change at constant flow rate. In addition to the constants Kt and K^, the maximum attainable detector response (voltage) is obtained from the empty columns
Determination of the water uptake during the adsorption state in ideal food systems. The water uptake represents the amount of water sorbed during the sorption process per sample dry mass evaluation, and is based on mass balances at sequential water content and RVP-the relative vapor pressure. Assuming constant temperature conditions in the column the mass retained is the difference between the mass in and the mass out and expressed in the following way:
mass retained == mass in - mass out
(3)
1902 The water uptake is defined as:
mass in - mass out Water uptake »
(4) sample dry mass
and is described by the equation:
K^tj - K ^ i
(5) m, where:
W^ - the mass of water uptake by the adsorbent
(g H20/g
dry Material) ti - time elapsed from injection of the probe Ati - the area of the chromatogram
(sec.)
(Volts x Sec.) at t^
m, - the mass of the dry sorbent (g)
The relative vapor pressure is evaluated from the following expression:
Pi RVP «
V,, =
(6)
1903 where:
Pi - the partial water vapor pressure at time t^ P^ - vapor pressure of pure water at temperature T - (the temperature of the experimental measurement) V^i - response voltage of the chromatogram (Fig 1) at t^ V,^ -plateau saturation response voltage of an empty column at the same temperature. The partial water vapor pressure is proportional to the response voltage of the TCD detector at that selected time. The maximum peak height is proportional to the partial vapor pressure of pure water at that column temperature. Thus a complete sorption isotherm can be obtained from a single frontal chromatogram.
MATERIALS AND METHODS
Locally purchased samples of agglomerated spray-dried soluble coffee and of freeze-dried soluble coffee, were used as test materials. Both samples were passed through a 200 mesh screen. The soluble coffees were also subjected to reverse processing. The spray-dried coffee was solublized in distilled water and freeze dried in the Labconco laboratory freeze dryer operating at 25 Torr (condenser set point of -75°C) and passed through a 200 mesh screen. Similarly, the freeze-dried coffee was solubilized
1904 in distilled water and dried on a glass tray which was placed into an air circulation oven for 6 hr. at 70°C. The dried film was scraped off with a razor blade and passed through a 200 mesh screen. A sample of Avicel PH-lOl was obtained from Dr. Wolf at the Federal Research Centre for Nutrition, D-75 Karlsruhe 1, Germany. This material was from the same lot used in a round robin standardized laboratory static sorption isotherm determination program reported by Wolf et al (1984).
COLUMN PREPARATION Very short, 5/32" diameter* 5/8" long (4/16 mm), stainless steel columns were used. The columns were prepared by placing a silanized glass wool plug into a Swagelok (TM) reducer. The reducer was connected to a vacuum hose and vibrated while about 20-100 mg coffee powder was transferred through a funnel into the column. The columns was closed with another small plug of silanized glass wool. The weight of the sample packing was determined gravimetrically. The columns were dried either offcolumn or on-column for 24 hr. at 70°C with an He flow of 40 cc/min.
APPARATUS The apparatus is depicted schematically in Fig. 2. It s operation
1905
HELILS1 TPtf€< &
L^JAL INjeCTlON J ORIS
FLOy CONTROLLER
MERCURY MANOMETER H E A T E D !. COOLED WATER BATH
SOAP BUBBLE FLOW METER
cHEEH
HEATED UATER BATH
THERMAL CONDUCTIVITY DETECTOR
1 HARD CARD
Fig. 2
A/D
PERSONAL COMPUTER
Scheme of the apparatus and the recording system.
1906 was as follows: Pressure regulated Hellium is delivered to the dual flow controller and then to the two injection ports. A selected amount of water equal to about 30% of the mass of the column packing mass/ was injected into either one of the injection ports which are maintained above 100°C. The resultant steam condensed on the interior surface of either the reference column or the pre-column section of the variable column; the precolumn was long enough prevent condensation on the interior of the variable column. The carrier gas was saturated at the operating temperature of the column bath. The column become a device which was similar to a reactor wherein the water vapor sorption took place. The emerging water vapor was quantified by the thermal conductivity detector. The analog chromatographic response voltage was amplified electronically/ digitized by the A/D converter and stored on the hard disk of an IBM-compatible personal computer. The data logging operation was handled by Labtech Notebook software with follow on analysis by Lotus 123. Th follow-on analysis chore had been greatly simplified by a macro program.
OPERATING CONDITIONS The IGC sorption behaviour of coffee solubles and Avicel PH-101 were determined at the operating conditions of: carrier gas(He) flow rate: 40 cc/min column temperature:
20 C or 30 C
1907 Injection port:
150 C
TCD temperature:
140 C
TCD filament current:
143 mA
RESULTS AND DISCUSSION
Figures 3a and 3b show the time and the area calibration constants, as determined in empty columns at 20°C with a 40 ml/min. flow rate. These data clearly show that there was a linear dependence (R = 0.99-1) between the injection mass and the residence time or the area of the chromatogram at that residence time. The peak voltage
was proportional to the partial pressure
of the water at the test temperature. In order to achieve this relation the column must be held at a constant temperature during the data acquisition of the chromatogram. This was achieved, as shown in Fig. 2, by using a carefully controlled water-bath. Fig. 4 shows MIGC determined sorption isotherms of freeze dried and spray dried soluble coffee, measured under the previously mentioned operating dynamic conditions. The dynamic nature of the MIGC, enabled to delineate the differences in the physical structure of the tested samples which were created due to the processing method. The freeze dried sample sorption curves demonstrate clearly differences in the amorphous or glass like solid state of the coffee. On the other hand, the spray dried coffee was closer to a crystalline state. Similar results were
a)
b) Calculation of the time constant from the
Calculation of the area constant from the slope of the probe-mass
slope of the probe-mass vs. area, at constant
vs. area, at constant temperature
temperature and flow rate (20°C, 40 cc/min),
and flow rate (20°C, 40 cc/min)
from empty column
from empty column.
0
1000
2000
3000
4000
0
TIME(SEC)
4
0
12
18
AREA* 10-3 (VOLTS*SEC) Fig. 3
20
-
\D 0 UO
1909
0.20
Q
0,16
O
CNJ X
UJ !!£ < H OZD
a:
LLJ
I—
0,12
0.08
0,04
<
0,00 0,0
0,2
0.4
0,6
0,8
1.0
RELATIVE VAPOR PRESURRE
Fig. 4
IGC sorption isotherms of freeze-dried and spray-dried soluble coffees at 30°C and 40 cc/min carrier gas flow rate. •
- IGC regular freeze-dried coffee
O
- IGC regular spray-dried coffee.
1910 shown by Nickerson (1974) and Saltmarch and Labuza (1980). Cluster analysis. The cluster theory was proposed by Zimm and Lundberg (1956) in order to explain sorption of water in high molecular weight polymers. The cluster function measure the tenndency of adsorbed water molecules to cluster. Zimm (1953) derived the following relationship between the activity coefficient (Yi=a„/<|)i) and the cluster integral Gn. This relation, namely the cluster function has the following form:
where: Vi
-
2 -
The partial molar volume of water The volume fraction of water (1) and sorbent(2), and (t)2 = l-(t>i. Iri terms of molar concentration ©component 1/ (CI = ^i/Vi),
the cluster function may be
expressed as:(8) The mean number of molecules in a cluster is: CiGj^-i-l. Clfunction values more negative than -1, means that the solvent molecules
*i^q-Gn
(8)
1911 are widely
dispersed and attached to specific separate sites on
the biopolymer. If the cluster function is positive for instance CiGii=3/ the interpretation is that a cluster of four molecules is present^ namely three additional molecules in excess of the original one, are forming a cluster. The tendency for water cluster formation is related to the hydrophobicity of the substrate, favouring water/water intereaction and cluster formation. Table 1 show the results of cluster analysis of freeze dried and spray dried isotherms (Fig.4). These data enable to plot Fig.5/ as CiGu versus p, the water vapor pressure. The present results are in good agreement with those obtained by Helen and Gilbert (1985), Demertzis et al.(1989)and Riganakos et al.(1993). It is apparent that the crystallinity of the spray dried coffee sample creates an environment in which the water cluster are formed. The cluster formation data of Fig.5 shows that the freeze-dried(amorphous) dextrinsarehighly hydrophilic, absorbing water molecules primarily as one per dextrin active sorption site until a high RVP is present. Themore crystalline spray-driedmaterial has fewer sitesavailable for sorption. Thus water clusters at the few sites at the surface and by swelling from the energy of cluster formation exposed more sites by disruption of the crystalline intramolecular dextrin bonds. The lower enthalpy of water/water bonds allows for greater fugacity per mole of water content and thus a greater RVP permole
1912
*
10
20 p (mm Hg)
Fig. 5
Water cluster function (CiGn) versus water vapor pressure (p) at 30°C for: •
- IGC regular freeze-dried coffee
O
- IGC regular spray-dried coffee.
1913 of water in the spray-dried coffee dextrins. Fig.6 shows sorption isotherms of different coffee samples processed so that the spray dried coffee became a freeze dried while the feeze-dried coffee was redried on a tray in an circulating oven. The freezing stage^ of the freeze drying reprocess was fast at -70°C (dry ice and ethanol) . Fig. 6 reflects that by freeze-drying a spray dried coffee, a different physical structure was created. This structure showed a similar sorption behaviour to that measured previously on the freeze-dried coffee. Fig.4 representing the amorphous state of the coffee samples. On the other hand, the reprocessing of the freeze dried coffee by atmospheric tray-drying, created a different physical structure in the coffee whith a higher degree of crystallinity than in the freeze dried samples, but lower than in the spray-dried coffee. Thus physical structure and its relation to the water exchange phenomena may be defined by MIGC as shown by Greenway (1988) and Apostolopolus and Gilbert (1989). Fig.7 shows the water isotherms of freeze-dried and spray-dried coffee, obtained by dynamic (MIGC) and by static (gravimetric) methods. The static, time averaging, method cannot reveal differences in the physical structure as does MIGC due to the prolonged time necessary in order to achieve equilibrium. Fig.7 points out that the static isotherm of the freeze-dried coffee at RVP = 0.5 showed a change from an amorphous to a more crystalline state. This recrystallization phenomenon is both RVP and temperature
1914 0.20
Q
0.16 H
cn
\ O
CN X
0.12 H
UJ
<
a.
0.08 H
Z)
cm 0.04 H
0.00 0.0
0.2
0.4
0.6
0.8
1.0
RELATIVE VAPOR PRESURRE Fig. 6
The influence of the "Reverse processing" on the soluble coffee sorption behavior as measured by IGC at 30°C and 40 cc/min. The carrier gas flow rate curve legend is: O
- Regular Spray-dried soluble coffee
V
- Reverse process freeze-dried-to-tray-dried
Y
- Reverse Process spray-dried-to-freeze-dried
1915 0.20
Q
0.16
\ O
CN
0.12 H LI
LLJ
< CL
LU
0.08
0.04
0.00
# 1.0
RELATIVE VAPOR PRESURRE Fig. 7
Comparison between IGC sorption isotherms of soluble coffees obtained at 30®C and 40 cc/min carrier gas flow rates vs. published static data on the same material from (Hayakawa et al./ 1978). The curve legend is: •
- Static Freeze-dried soluble coffee
—
- Static Spray-dried soluble coffee
^^ O
- IGC Freeze-dried soluble coffee - IGC Spray-dried soluble coffee.
1916 dependent (30*^0 as pointed out also by Mackower and Dye (1956), Saltmarch and Labuza (1980). The static isotherms of both spray dried and freeze dried samples measured by Hayakawa et al.(1978) are in good agreement with the IGC sorption isotherm for the freeze-dried coffee. Measurements of the water exchange by a dynamic method like IGC do not create conditions where recrystallization can occur. On the other hand, by selecting appropriate contact efficiency factors and operating conditions it is possible to achieve quasi-equilibrium conditions during the IGC experiments, as shown in Fig.8 for a crystalline material, Avicel Ph-101.
CONCLUSIONS The modified frontal IGC method, enabled to study, sorption behaviour of coffee samples, in a relatively short time. Due to its dynamic nature, it yielded results which indicated differences in the rate of water vapor exchange of moisture sensitive foods, including soluble coffees, due to various physical structure created by different processing methods. The water state as related to differences in structure and morphology was well characterized by the Zimm - Lundberg cluster function. Reverse processing of spray-dried and freeze-dried coffees revealed that physical state and properties and not chemical differences were responsible for differences in sorption behaviour.
1917 0.20
0.00 0.0
0.2
0.4
0.6
0.8
1.0
RELATIVE VAPOR PRESURRE Fig. 8
Comparison between dynamic sorption isotherms (IGC) at various contact efficiency conditions (residence time of the probe as related to the carrier gas flow rate) to static data on Avicel PH-101 from (Wolf et al. 1984). •
- 2.5 cc/min carrier gas flow rate, T = 25°C
O
- Static (Wolf et al. 1984), T = 25°C
—
- 40 cc/min carrier gas flow rate, T = 30°C.
1918 These data indicated that the major differences in water sorption of the two types of coffee was in the kinetics of the diffusion of water into their particles. The reversal experiments showed that the dynamics of the water exchange exert a great influence on the physical structure of dehydrated foods. The IGC method measured these kinetic factors while the static method obscured them. By an appropriate selection of contact efficiency factors, the MIGC sorption isotherm may be in good agreement with the static methods or a different set of factors may be used to produce an isotherm which reveals important kinetic and thermodynamic differences.
1919 REFERENCES Apostolopoulos, D. and Gilbert, S.G. 1984. Water sorption of coffee solubles by inverse gas chromatography. In "Instrumental Analysis of Foods". G. Charalombous and G.Inglett. (Eds.), Academic Press, Inc., New York.
Apostolopoulus, D. and Gilbert, S.G.
1988.
Frontal inverse gas
chromatography as used in studying water sorption of cofee solubles.J.Food Sci.53:882.
Demertzis, P.G., Riganakos, A. and Kontominas, M.G. 1989. Water sorption isotherms of crystalline raffinose by inverse gas crromatography.Int.J.of Food Sci. and Techn. 24:629.
Ferng, A.L. 1987. A study of water sorption of corn starch from various genotypes by gravimetric and inverse gas chromatographic method, PhD. Thesis, Rutgers University, New Brunswick, N.J.
Gilbert, S.G. 1984. Inverse gas chromatography. In "Advances in Chromatography" 23:(Eds.) J.C. Giddings, E. Grushka, J. Gazes, P.R. Brown, Marcel Dekker, Inc., New York.
1920 Gilbert, S.G.
1988. Applications of IGC for research on kinetics
and thermodynamic problems in food science. Symposium on inverse gas chromatography in polymer characterization, TVmerican Chemical Society, Toronto, Ontario, Canada, June, 5-11.
Gilbert, S.G.
1989. Modified frontal chromatographic method for
water sorpyion isotherms of biological macromolecules. In "Inverse Gas Chromatography" p.309, Lloyd, D.R., Ward,T.C. and Schreiber, H.P.(Eds.), ACS Symposium Series 391 Amer. Chem.Soc. publishers, Washington,D.C.
Greenway, G.W. 1988. Water vapor sorption of soluble coffees, corn starches and microcrystalline cellulose by frontal analysis using pulse inverse gas chromatography, Ph.D. Thesis, Rutgers University, New Brunswick, N.J.
Hayakawa, K.I., Mata, J. and Hwang, M.P. 1978. Moisture sorption isotherms of coffee products, J. Food Sci. 43: 1026.
Helen, H. and Gilbert, S.G.
1985. Moisture sorption of dry bakery
products by inverse gas chromatography.J.Food Sci.50:454.
Liu, J.X., Gilbert, S.G., Li, L.Y. and Ramon. 0. 1991. Water relations in food systems, determined by modified frontal inverse chromatography (MIGC) In " Proceedings of the first int. conf. on
1921 food sci. and techn. "p.146. Zeidler, G., Whitaker, J.R., Haard, N. and Luh, B.S.(Eds.) Wuxi, China.
Mackower, B. and Dye, W.B. 1956. Equilibrium moisture content and crystallization of amorphous sucrose and glucose. J. Ag. and Fd. Chem. 4(1):72.
Nickerson, T.A. 1974 Chemistry"
"Lactose in Fundamentals of Diary
Ed. Webb, B.H., Johnson, A.H., and Alfred, J,A.
Avi
Pub. Co. Westport, Conn.
Ramon, 0., Daun, H., Frenkel, C. and Gilbert, S.G. 1990. Caracterization of hydrophilic water related hysteresis in food systems by inverse gas chromatography. In:Flavors and offflavors. Developments in food science:vol. 24:4184 38.Charolombous, G.(Ed.)Elsevier Amsterdam, Neth.
Riganakos, K.A.,Demertzis, P.G. and Kontomlnas, M. 1992. Effect of crystalline sucrose on the water sorption behaviour of wheat flour as studied by inverser gas chromatography. Lebensm.Wiss.u.-Techn.25:389.
Saltmarch, M. and Labuza, T.B. 1980. Influence of relative humidity on the physicochemical state of lactose in spray-dried sweet whey powders. J. Food Sci. 45:1231.
1922 Slade, L. and Levine, H. 1991. Beyond water activity: Recent advances based on alternative to the asssesment of food quality and safety. Critical Reviews in Food Sci. and Nutrition.30,23:115.
Wolf/ W. 1984. The water vapor sorption isotherms of microcrystalline cellulose (mcc) and of purified potato starch. Results of a collaborative study. J. Food Eng. 3:51.
Zimm, B.H. 1953. Simplified relation between thermodynamics and molecular distribution functions for a mixture. J. of Chem. Physics. 21:934.
Zimm, B.H. and Lundberg, J.L. 1956. Sorption of vapors by vhigh polymers. J.of Phys. Chem. 60:425.
1923 ACKNOWLEDGEMENT Journal Series Paper D 10535-13-88, New Jersey Agricultural Experiment Station, Cook College, New Brunswick, NJ 08903. This work was supported in part by state funds and the Center for Advanced Food Technology, a New Jersey Commission on Science and Technology Center.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1925
The Inhibitory Effect of the Essential Oils from Basil (Ocimum basilicum) and sage {Salvia officinalis) in Broth and in Model Food System Tassou Ch.Ch. and Nychas, GJ.E National Agricultural Research Foundation Institute of Technology of Agricultural Products 1, S. Venizelou str., Lycovrisi 14123
INTRODUCTION Food preservation has become a complex problem. New food products are frequently being introduced onto the market. Generally these require a longer self life and greater assurance of freedom from foodbome pathogenic organisms. The search for new substances to be used in food preservation is hampered by regulatory restrictions. Consequently a great deal of time and money may well be required to develop a new chemical preservative and to get it approved especially in view of the public pressure against chemical additives in general. Such obstacles provide new opportunities for those seeking alternative routes in the search for new food preservatives. The excessive use of chemical preservatives, many of which are suspect because of their potential carcinogenic and teratogenic attributes or residual toxicity, has resulted in increasing pressure on food manufacturers to either completely remove chemical preservatives from their food products or to adopt more "natural'' alternatives for the maintenance or extension of a product's self life. There is considerable interest in the possible use of the latter as alternative food additives either to prevent the growth of foodbome pathogens or to delay the onset of food spoilage. For this reason the effect of naturally-occurring compounds, from basil and sage, on Gram negative and Gram positive bacteria have been considered in this context. Indeed many spices and herbs and extracts thereof possess antimicrobial activity. It is almost invariably due to the essential oil fraction (Deans & Ritchie 1987). Thus the essential oils of citrus fruits exhibit antibacterial activity on foodbome bacteria (Dabbah et aL 1970) and moulds (Akgul & Kivanc, 1989) so too have the essential oils of many other plants such as oregano, thyme (Salmeron et aL 1990; Paster et al, 1990), sage, rosemary, clove, coriander etc. (Farag et aL 1989; Aureli et aL 1992; Stecchini et aL 1993). The antibacterial and antimycotic effects of garlic and onion have been well documented also (Mantis et aL 1978; Sharma et aL 1979; Saleem & Al-Delaimy 1982; Conner & Beuchat 1984a,b). The objectives of this work were 1. to establish the antimicrobial effect of naturally occurring essential from sage and basil on food spoilage (Pseudomonas spp.) and foodbome bacteria (Staphylococcus aureus. Salmonella spp.) in broths and the
1926 interactive effect between inoculum size, glucose, and the concentration of essential oil, and 2. to examine the effect of these essential oils (basil and sage) on the above mentioned bacteria in Model Food Systems (such as meat, mayonnaise, meat gravy sauce)
MATERIALS AND METHODS Bacterial strains Gram negatives: Pseudomonasfragi,Salmonella enteritidis, Salmonella typhimurium. Gram positives: Staphylococcus aureus S-6. All the microrganisms were maintained in Nutrient broth at -80° C. A 24h culture of each organism was used in every experiment. Media 1. Nutrient broth (Oxoid Ltd.) 2. SPYE broth (Malthus, code 490-001) 3. Coliform broth (Malthus, code 490-003) 4. XLD (Xylose Lysine Desoxycholate) for the enumeration of salmonellas. 5. PC A (plate count agar, Oxoid) for Total Viable Counts 6. CFC (Oxoid) for pseudomonads counts 7. MRS (Oxoid) for Lactic Acid Bacteria 8. Meat gravy sause containing (% w/v): 1.8g Trypticase Peptone, 1.2g Beef Extract, 0.6g Yeast Extract, 0.2g Carragenan Type II and 2g Starch. All the above media were sterilised by autoclaving at 121° C for 15min. 9. Mayonnaise was made with 300ml virgin olive oil, two whole eggs and 17ml vinegar to drop the pH to 4.5 or 5.2 Additives The essential oils of basil (Ocimum basilicum) and sage {Salvia officinalis) obtained by steam distillation. They were used in quantities of 0, 0.4 and 0.8% (v/v), in broths and m the meat gravy sauce, 0.5% v/w m the case of mayonnaise and almost 0.6% (v/w) in the case of meat. Glucose was added in some cases in quantities of 0, o.l % and 1% (w/v) Experimental procedure When the Malthus Instrument was used, SPYE broth was used for Ps, fragi and coliform broth for Salmonella spp.. In all cases the total volume of the medium, the additives (essential oil of basil and glucose, in the concentrations referred above) and the inoculum in the Malthus cells was 2.5ml. The meat gravy sauce was dispensed in sterile Malthus cells. Essential oil was added in quantities of 0, 0.4 and 0.8% (v/v) and finally 0.1ml of a 24h culture of each of the above microorganisms was added to a final volume of 2.5ml. The cells were incubated
1927 in the Malthus waterbath at 30"" C for a period of 48h. The mayonnaise separated in 25gr portions and placed in sterile plastic bags. In some of these an inoculum (ca. W cells/g) of Salm. enteritidis was added and essential oil (0.5% v/w) of either basil or sage and the bags stored at 20° C. At 24, 48 and 72 hours a sample was taken out of the incubator, 225ml of sterile Ringer's solution was added and decimal dilutions followed. The colonies of Salm, enteritidis were counted on XLD (Xylose Lysine Desoxycholate) Petri dishes after 24h of incubation at 37° C. A piece of lean beef (1cm x 1cm diam) was cut in three, approximately equal, pieces (15g each). One of these was the control while on the two others 1ml of essential oil was spread on their surface. When Salm. enteritidis was used, all the samples immerged in a solution of a 24h culture of the microorganism (ca. lO^ml). In this case one uninoculated sample was immerged in sterile water. The meat pieces were placed in aluminum trays, packaged under vacuum and stored at 10° C. The day of the analysis the samples were taken out and Ig was weighted in 99ml Ringer's solution while the rest meat was packaged again under vacuum and put for storage.
RESULTS a. In broths The essential oil of basil retarded the growth of Ps. fragi in SPYE broth increasing the detection time (Fig 1) and minimizing the final growth (Fig 2). The addition of glucose up to 1 % (w/v) had no effect on the detection time (Fig. la) as well as on the final growth (Fig. 2a). The linear surface analysis revealed that the inhibition of basil essentail oil depended upon the size of the initial inoculum (Fig. 2b).The inhibition of Salmonella enteritidis with basil oil in Coliform broth are shown in three dimensional predicted response surfaces (Figs 3, 4). High concentrations of basil essential oil gave higher detection times for this bacterium and decreased the final growth (Figs. 4b) while the addition of glucose had no effect (Fig. 3b). High inoculum levels reduced the lag phase (Fig. 3a), and slightly increased the final conductance values (Fig. 4a).
1928
a b Fig. 1 The three-dimensional predicted Detection Time (hours) response surface (linear) of Pseudomonas fragi in SPYE broth at 25° C as determined with a Malthus Instrument as a function of (a) basil essential oil (0, 0.4 and 0.8 v/v)%) versus glucose (0, 0.1 and 1 % w/v) and (b) basil essential oil (0, 0.4 and 0.8 v/v)%) versus inoculum size (l:logio 5.8 cfu/ml; lOilogio 6.8 cfu/ml; 100: logio 7.8 cfu/ml)
a b Fig. 2 The three-dimensional predicted Final Growth (microsiemens) response surface (linear) of Pseudomonas fragi in SPYE broth at 25° C as determined with a Malthus Instrument as a function of (a) basil essential oil (0, 0.4 and 0.8 v/v)%) versus glucose (0, 0.1 and 1 % w/v) and (b) versus Inoculum Size (l:logio 5.8 cfu/ml; 10:logio 6.8
1929 cfu/ml; 100: logio 7.8 cfu/ml)
a b Fig. 3 The three-dimensional predicted Detection Time (hours) response surface (quadratic) of Salmonella enteritidis in Coliform Broth media at 37° C as determined with a Malthus Instrument as a function of (a)essential oil (0, 0.4 and 0.8 v/v)%) versus Inoculum Size (l:logio 5.3 cfu/ml; lOilogio 6.3 cfu/ml; 100: logio 7.3 cfu/ml) and (b) basil essential oil (0, 0.4 and 0.8 v/v)%) versus glucose (0, 0.1 and 1 % w/v)
a b Fig. 4 The three-dimensional predicted Final Growth (microsiemesn) response surface (quadratic) of Salmonella enteritidis in Coliform Broth media at 37° C as determined with a Malthus Instrument as a function of (a) basil essential oil (0, 0.4 and 0.8 v/v) %) versus glucose (0, 0.1 and 1 % w/v) and (b) of basil essential oil (0, 0.4 and 0.8 v/v)%) versus Inoculum Size (l:logio 5.3 cfu/ml; 10:logio 6.3 cfu/ml; 100: logio 7.3 cfu/ml)
1930 b. In Model Food Systems 1. In meat gravy sauce The essenti2il oil of basil at concentrations of 0.4 and 0.8% (v/v) inhibited completely the growth of St, aureus (Fig. 5a). It retarded the detection time but allowed growth to occur later for Ps, fragi (Fig. 5b).
a b Fig. 5 The effect of basil essential oil on the growth of (a) Staphylococcus aureus and (b) Pseudomonas fragi in a meat gravy sauce at 30° C as determined with a Malthus Instrument (circle: control; triangle: 0.4% v/v; square: 0.8% v/v)
At both concentrations inhibited completely the growth of 5. enteritidis (Fig. 6a) and at 0.8% S. typhimurium, while at concentration of 0.4%, growth started after several hours (Fig. 6a)
1931
Fig. 6 The effect of basil essential oil on the growth of (a) Salmonella enteritidis and (b) Salmonella tymphimurium in a meat gravy sauce at 30° C as determined with a Malthus Instrument (circle: control; triangle: 0.4% v/v; square: 0.8% v/v) The essential oil of sage inhibited significantly the growth of St aureus (Fig. 7a). It slightly mcreased the detection time of Ps, fragi (Fig. 7b).
6
10
16
a b Fig. 7 The effect of sage essential oil on the growth of (a) Staphylococcus aureus and (b) Pseudomonas fragi in a meat gravy sauce at 30''C as determined with a Malthus Instrument (circle: control; triangle: 0.4% v/v; square: 0.8% v/v) Similar results were obtained with S, enteritidis (Fig. 8a) and 5. typhimurium (Fig. 8b).
1932
a b Fig. 8 The effect of basil essential oil on the growth of (a) Salmonella enteritidis and (b) Salmonella tymphimurium in a meat gravy sauce at 30° C as determined with a Malthus Instrument (circle: control; triangle: 0.4% v/v; square: 0.8% v/v) 2. In meat and mayonnaise The essential oil of sage was more effective than that of basil in preventing the increase of the Total Viable Counts of meat, for the first three days of storage under vacuum at 10° C (Fig. 9a). Similar results were obtained for pseudomonads (Fig. 9b).
a b Fig. 9 The effect of basil and sage essential oils (0.6% v/w) on the (a) Total Viable Count and (b) on pseudomonads of meat stored under vacuum packaging at 10° C (circle: control; triangle: sage; square: basil)
1933 Both the essential oils had almost no effect on lactic acid bacteria of meat (Fig. 10a), but affected appreciably the survival of S, enteritidis (Fig. 10b).
a b Fig. 10 The effect of basil and sage essential oils (0.6% v/w) on the (a) Lactic Acid Bacteria and (b) Salmonella enteritidis of meat stored under vacuum packaging at 10° C (circle: control; triangle: sage; square: basil) Both the essential oils had no effect on the same microorganism in mayonnaise of pH either 4.57 or 5.2 (Figs. 11a & lib).
Time (boura)
a b Fig. 11 The effect of basil and sage essential oils (0.5% v/w) on the survival of Salmonella enteritidis (initial inoculum logio 7.23 cfu/g) on maynonaise with (a) pH 4.57 or (b) pH 5.2 and stored at 20° C (1st row bars: control; 2nd row bars: sage; 3rd row bars: basil)
1934
DISCUSSION The antimicrobial compounds in plant materials are conmionly in the essential oil fraction. These compounds are mainly responsible for the characteristic aroma and flavour. They are recovered from plant materials primarly by steam distillation, although some are expressed cold, by dry or vacuum distillation (Farrel 1985). According to Hargreaves et al. (1975) essential oils are defined as being a group of odorous principles, soluble in alchohol and to a limited extent in water, constisting of a mixture of esters, aldehydes, ketones and terpenes In general Gram positive are more sensitive than Gram negative bacteria to the antimicrobial compounds in the essential oil from spices (Nychas 1995). Variation in the rate or extent of inhibition was also evident among the Gram-negative bacteria. For example Esch, coli was less resistant than Pseudomonas fluorescens or Serratia marcescens when tested with essential oils from sage, rosemary, cumin, caraway, clove and thyme oils (Nychas 1995). Inhibition of growth ranged from 88% With Aerobacter aerogenes to 100% with Alcaligenes faecalis as test organisms. Salmonella enteritidis and S. typhymurium were less sensitive than Pseudomonasfragito various essential oils (Nychas 1995). For example Salmonella typhimurium was found to be more sensitive than Pseudomonas aeruginosa to essential oil from oregano and thyme (Nychas 1995). Deans and Ritchie (1987), who studied the effect of 50 plant essential oils against 25 genera of bacteria, concluded that Gram positive and Gram negative organisms were both susceptible to the essential oils and there was no evidence that the degree of sensitivity to the oils was reflected in the Gram reaction of the organism. The growth of Salmonella spp. was inhibited by essential oils of linden flower, thyme, oregano, orange, lemon, grapefruit, mandarine almond, bay, clove, coriander, cinnamon and pepper (Dabbah et al 1970; Aktug & Karapinar 1987; Deans & Ritchie 1987; Paster et al. 1990) in broths. In this study strong inhibition was observed in most cases in various broths tested. When these two essential oils tested in Model Food systems it was found that their inhibitory action was reduced dramatically. It is worthy of note that many groups of workers concluded that the effectiveness of essential oils decreased when the experiments were conducted "in vivo". This could be due to high protein and fat content of meat which can mask the antimicrobial effect of essential oils (Shelef 1983). It is worth mentioning that, if the exploitation of essential oils is going to be increased, certain parameters and characteristics should be taken into account. For example the extraction methodology, the variation of composition in the same species which may be influenced by geographical location, climatic condition etc. For this reason the active constituent present in ihnibitory oils should be chemically separated and identified. Moreover the mode of action of these compounds should be also examined in more detail.
1935 REFERENCES Akgul,A- and Kivanc, M. J. Sci. Food Agric 1989; 47:129-132 Aktug,S.E and Karapinar,M, Inter. J. Food Microbiol 1987; 4:161-166. Aureli,P., Constantini,A-, and Zolea,S. J. Food Protect 1992 55:344-348 Conner,D.E and Beuchat,L.R Appl. Environ. Microbiol. 1984a; 47:229-233 Conner,D.E and Beuchat,L.R J. Food Sci. 1984b; 49:429 - 434 Deans, S.G and Ritchie,G. Int. J. Food Microbiol. 1987; 5:165-180 Dabbah,R., Edwards,V.M., and Moats,W.A Appl.Microbiol.1970; 19:27-31 Farag,R.S., Daw,Z.Y, Hewedi,F.M., and El-Baroty,G.S.A J. Food Protect. 1989;52: 665-667 Farell,K.T (1985) Spices, condiments and seasonings The AVI, Inc. Westport, Conn., Hargreaves,L.L.,Jarvis,B., Rawlinson,A.P. and Wood,J.M The British Food Manufacturing Industries Research Association Scientific and Technical Surveys 1975 No. 88 Mantis,A.J., Karaioannoglou,P.G., Spanos,G.P., and Panetsos,A.G Lebensm.-Wiss.u.Technol. 1978;11:26-29 Nychas G.J.E New & Emerging Technologies for Food Preservation Blackie Acad. & Prof. 1995 (m press) Paster,N. Juven,B.J.,Shaaya,E., Menasherov,M., NItzan,R., Weisslowicz,H., and Ravid,U. Ltr Appl. Microbiol. 1990;11:33-37 Saleem,Z.M. and Al-Delaimy,K.S J. Food Protect. 1982;45:1007-1010 Sahneron,J. Jordano,R., and Pozo,R. J. Food Protect. 1990;53:697-700 Sharma,A.A., Tewari,G.M., Shrikhande,A.J. Padwal-Desai,S.R. and Bandyopadhyay, C. J.Food Sci. 1979;44:545-1547. Shelef, L.A. J.Food Safety 1983; 6:29-44 Stecchini,M.L., Sarais,I., and Giavedoni,P. J. Food Protect. 1993;56:406-409
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1937
Comparison of some physicochemical characteristics between solid and fluid Chios mastic resin. M. Melanitou \ D. Papanicolaou \ K. Katsaboxakis ^ and K. Stamoula^ ^ National Agricultural Research Foundation (N.AG.RE.F), Institute of Technology of Agricultural Products (I.T.A.P.), 1, S.Venizelou, 141 23, Lycovrissi Attikis, Greece. ^ Mastic tree Growers Union, Chios, Greece
Abstract A connparative study was done on sonne physicochemical characteristics of solid and fluid nnastic resin, harvested by the traditional and a new method. The parameters measured were: solubility, colour, grain size, specific gravity, hardness, viscosity, essential oil content, ash and fatty acids. Colour, hardness and viscosity were the main characteristics which differ significantly between the t w o types of resin and therefore can be used to distinguish the different qualities of mastic resin. Minor differences on the other characteristics were also observed.
1. INTRODUCTION Chios mastic resin is an exclusive and characteristic product of Chios island. There are a f e w studies ( 1 , 2, 4 , 7) about the physicochemical characteristics of this product, all of them dealing with Chios solid mastic resin which has been harvested by the traditional way. According to that method, the resin which secrets from 150-200 small incisions made on the trunk of the tree, Pistacia lentiscus var chia, in the form of tear drops, is left on the trunk or on the ground below the tree for 20-30 days in order to be solidified and can be harvested by hand. In this case, a great amount of its essential oil has been lost due to evaporation. Recently, a new method of harvesting Chios mastic resin has been developed (6), according to which, the resin is harvested In a fluid form by means of special harvesting devices into plastic bags by using the stimulating substance "Ethrel" for increased resin secretion. There is a lack of information about the physicochemical characteristics of the product, solid and fluid, which are necessary for its identification and quality control. Moreover, the physicochemical characteristics of mastic resin are essential
1938 during its processing and subsequent storage. The purpose of this project was a comparative study on sonne physicochemical characteristics of solid and fluid mastic resin, harvested by the traditional and new method.
2. MATERIALS AND METHODS The samples of mastic resin tested were from different commercial types: I) "Hondri" No 1 : solid mastic free from foreign substances, in irregular grains of ca 20 mm diameter, of new harvest. II) The same as (I) but of old harvest (3 years old). III) "Psili" No 1: solid mastic free from foreign substances, in irregular grains of ca 7mm diameter, of new harvest. IV) "Psili" No 1 as (III) but of old harvest. V) Fluid mastic resin: free from foreign substances, harvested by the new method. The physicochemical analysis included the following parameters: - Colour measurement (L, a, b), by a Minolta colorimeter having the capacity of measuring small samples. - Specific gravity measurement based on the anosis phenomenon. - Grain size measurement by means of sieves of 1.41, 4.75 and 7.93 mm diameter. - Ash measurement by burning down at 400° C. - Acidity Index, Saponification Index, Ester number and Iodine number according to Hanus. All the above parameters concerning mastic resin were determined according to the methods described by Galanos and Voudouris (4). - Essential oil determination. The Clevenger apparatus was used for the determination of the essential oil content of the resin by steam distillation. - Solubility of mastic resin. It was estimated macroscopically the fraction of 1 gr of sample that had been dissolved after 24 h at 30 ml of the following solvents: acetone, ethanol, methanol, dioxan, hexan, n-heptane, diethylether, n-butyl-ether, toluene, xylene and chloroform. - Hardness measurement: The tenderometer of the Food Technology Corporation equipped with a special constructed cylinder rod of 4.5 mm diameter was used. The sample grains of solid resin was put in a test cell or a 10mm thick piece of fluid mastic resin and the force required to compress and shear each sample was recorded. - Viscosity measurement by a Brookfield HV-2 viscometer. The sample was placed in a glass tube into a water bath of 95° C. The probe No 4 was used and measurements were taken at 3 different speeds. - Minerals: The N was determined by the Kjeldahl method . The P was determined colorimetrically by the method of ammonium vanadate, K with the flame photometer and Ca, Mg, Mn and Zn by atomic absorption (3).
1939 3. RESULTS AND DISCUSSION 3.1 Solubility of mastic resin The solubility of "psili" mastic resin into the various solvents was found to be: Chloroform > n-butyl-ether> acetone > heptan> methanol > toluene > xylene > ethanol> dioxan> hexane> diethylether. 3.2 Colour of mastic As shown in table 1 which includes the values of L, a, b, colour parameters, a significant difference was found between the commercial types of solid resin and the fluid resin,. The solid resin ("Psili" and "Hondri") of old harvesting gave a higher ( + ) b value which means a higher yellow colouration than the solid resin of new harvesting. The higher ( + ) b value was observed in "Psili" of old harvesting. This could easily be explained by the fact that the relationship surface/volume is higher in that type of resin and therefore a greater surface is exposed to the atmospheric oxygen which accelerates the oxidation reactions.
Table 1: L, a, b parameters of colour for the various commercial types of mastic resin. L
Type of mastic resin
a
b
I. "Hondri" old harvested
61T2
3,2
19,3
II. "Hondri" new harvested
70,1
1,0
9,7
III."Psili" old harvested
57,1
3,7
24,9
IV. "Psili" new harvested
69,6
3,1
18,1
V. Fluid mastic
78,3
0,6
7,7
"Psili" mastic resin of old harvesting gave also a high ( + ) a value. On the contrary, fluid resin gave lower (-I-) a value and higher L value. The differences in colour can be better observed in Figure 2, where the ratios L/b and b/a are presented for the different commercial types of resins. It can be concluded, that colour determination is a quick simple and non destructive method for the characterisation of the types and oldness of mastic resin as well as the discrimination between fluid and solid mastic resin.
1940
RATIOS L / b AND b / a
iL/b
^b/a
(I):"HONDRI"(OLD) (II):"HONDRI" (NEW) (III):'TSILr' (OLD) (IV):'TSILr' (NEW) (V):FLUID MASTIC RESIN Fig.1 : L/a and b/a ratios of the colour parameter of different types of solid resin stored for different times and of fluid resin. 3.3 Grain measurement As it can been seen in fig.2, which includes the results of the mastic resin grain measurements, "psili" mastic is more homogenous than "hondri", as the 8 5 % of its grains belonged at the same fraction (4.75 mm diameter).
7.93 m m
1 mm 1.41 m.m "HONDRI" MASTIC RESIN
TSILI" MASTIC RESIN
Fig. 2: Diagrammatic distribution of grains of solid mastic
("Psili","Hondri").
The type "Hondri" on the other hand, did not seemed to be so homogenous as its grains were distributed at 3 fractions with 12, 11 and 77 % respectively.
1941 3.4. Specific gravity The specific gravity of the different types of mastic resin is shown in Table 2. Table 2: Specific gravity of different types of mastic resin.
Type of resin
specific gravity
"Psili"
1.0782
"Hondri"
0.9889
"Fluid"
0.9637
It can be observed that the lowest value is that of fluid resin. It should be noted however that the specific gravity of mastic grains showed a fluctuation due to foreign materials contained or to air bubbles inside mastic resin mass. 3.5. Hardness and viscosity of mastic The hardness of solid resin grains is shown in Fig. 3. A greater dissimilarity in hardness was observed in grains of "Psili" and "Hondri" type of mastic resin in comparison with fluid resin. In several times, specially with "Psili" mastic grains, the product was so hard that it was impossible to measure its hardness, because sample was broken in pieces, something that was undesirable. HARDNESS
UNITS
3 0 0 O 2 5 0 O 2 0 0 0 1
5 0 0
i
OOO
^•i mm m
5 0 0 O
'PSILI'
'HONDRI'
3^==z±=zt: FLUID
^
Fig.3: Measurements of hardness for different types of solid resin and of fluid resin. As it can be observed in the same figure, the values for hardness of fluid resin were too low, due to the fact that immediately after harvest, the mastic was in a semi-liquid condition. The great similarity in hardness of fluid resin, is very important in the product standardization.
1942 It should be pointed out here that during the period when the mastic was still on the tree and also when it was stored at roonn temperature into plastic bags not sealed hermetically, some Important changes in its rheological properties occurred, due to polymerization and/or oxidation. The main change was the Increase of viscosity. The results of viscosity measurements on solid and fluid mastic resin at 95° C, temperature where mastic was In fluid state, are presented in Fig.4. It can be observed that the viscosity of fluid resin is significantly lower than any other type measured. Also "Psili" type of mastic had a higher viscosity than "Hondri". It seems also that storage time contributes to the increase of viscosity, as the solid resin of old harvesting gave higher values than those recently harvested.
VISCOSITY CPI X 1 . 0 0 0 1 00
DIFFERENT TYPES OF MASTIC RESINS I "HONDRI" (OLD) I "PSILI" (NEW)
I "HONDRI" (NEW) H "PSILI" (OLD) i FLUID MASTIC RESIN
Fig. 4 : Viscosity measurements of different types of mastic resin at 95° C, taken with Brookfield viscometer.
It can be concluded therefore, that viscosity measurements are very useful from technological point of view, but also much slower and more difficult than colour measurements and afterwards sample can not be used for further analysis.
3.6 Essential oil content of mastic. The results of the essential oil determination of different types of mastic resin are included In Fig. 5.
1943 % ESSENTIAL
OIL
CONTENT 14.8
16 14 12 lO
7:i
8 6
3JB.
2.1
4.
4.1 2.59
S O
I
II
DIFFERENT
III
TYPES
IV
V
OF M A S T I C
(I):"H:ONDRI" ( O L D ) (III):"PSILI" (OLD) ( V ) : F L U I D 6 M O N T H S STORAGE
VI
RESINS
( l l ) : " H O N D R I " (NEW) (IV):"PSILI" (NEW) ( V I ) : F L U I D MASTIC R E S I N
Fig 5: Essential oil content of different commercial types of solid resin in comparison with fluid resin and of samples stored for 6 months at room temperature. It can be observed that the essential oil content of fluid mastic immediately after harvest is up to 1 4 . 8 % compared to 3.8% of solid "Hondri" type recently harvested and 4.1 of solid "Psili" type. Howeverjt should be mentioned that a great loss up to 7.7 %, of the essential oil was observed in the fluid resin stored for six months at room temperature into the collecting plastic bags not hermetically closed. This loss could be prevented by freezing the product immediately after harvest . 3.7 Determination of ash The ash content of different types of mastic resin is shown at Fig.6.
ASH
CONTENT %
DIFFERENT TYPES 'HONDRI"(OLD) 'PSILI"(NEW)
OF
MASTIC
1 "HONDRI"(NEW) 1 FLUID MASTIC R E S I N
l"PSILI"(OLD)
Fig.6: Ash content of different types of solid and fluid resin.
1944 The lowest ash content value, far from the others, was observed in fluid resin and the highest value in "Hondri" solid resin. The above results were due to the fact that solid resin ("Psili" or "Hondri") inevitably contains a higher amount of foreign materials (soil, leaves, insects etc) which can not be totally removed. On the contrary, fluid resin harvested into plastics bags and was free from foreign materials.
3.8 Parameters concerning mastic resin Acidity index, saponification index, ester number and iodine number according to Hanus were determined and the results for the different types of resin are shown in Fig.7.
'HONDRr'
"PSILI"
"FLUID"
lESTER NUMBER ^ A C I D I T Y INDEX iSAPONIFICATION INDEX ^ I O D I N E ACC.HANUS Fig.7: Acidity index, saponification index, ester number and iodine number according to Hanus, for different types of mastic resin.
It can be observed that fluid resin differs from solid types, having higher ester and iodine number as well as saponification index and lower acidity index. These results were expected because fluid resin, was less exposed to the atmospheric oxygen, contained more saponified and unsaturated fatty acids and less free acids than solid resin in which more acids were saturated and hyperoxides were produced.
1945 3.9 Mineral content of mastic resin The mineral content of fluid and solid resin is shown in Table 2. Table 2: Mineral content of fluid and solid mastic resin. Minerals
Type of resin
Zn ppm
Ca ppm
Mg ppm
P ppm
Mn ppm
K ppm
Solid resin
1.2
300
20
10
-
-
Fluid resin
0.6
200
30
50
-
The above results are in accordance with those reported by other researchers (7). It is noteworthy though that no nitrogen nor potassium was found, elements which are highly absorbed by plants. It is very interesting to search which is the role of those elements In resin biosynthesis as well as the influence of soil fertilizing in the qualitative and quantitative yield of mastic resin. 4. ACKNOWLEDGMENTS Thanks are due to the General Secretariate of Research and Technology of Greece and to the Mastic Growers Union who funded this project. Also to Mrs Nicolaou J. researcher of Soil Science Institute of N.AG.RE.F for the analysis of minerals. 5. REFERENCES 1. BARTON D.H.R., SEOANE E. (1956): Triterpenoids Part XXII. The constitution and stereochemistry of Mastica dienoic acid. J . Chem. Soc. p.4140-4157. 2. BOAR R.B, COUCHMAN L.A., JAQUES A . J . , PERKINS M.J. (1984): Isolation from Pistacia Resins of a Bicyclic Triterpenoid Representing an Apparent Trapped Intermediate of Squalene 2,3 - Epoxide cylization. J.Am. Chem. Soc. 106, p. 2476-2477. 3. CHAPMAN H.D., PRATT P.F., (1961): Methods of Analysis for soils, plants and water. Univ. of California, Riverside. USA. 4. GALANOS D., VOUDOURIS E. (1983): Univ. Athens.
Introduction at the food inspection.
5.KEHAY0GL0U A., DOXASTAKIS G., KIOSSEOGLOU V., MIKEDIS M., (1993): Compressional Properties of Chios mastiche ( Pistacia lentiscus var.chia) In: Food Flavors Ingredients a< Composition. Ed.Q, Oharalajnbous,ELSEVIER Publishers.p. 429-436. 6. PAPANICOLAOU D., MELANITOU M., KATSABOXAKIS K., BOGIS D. and STAMOULA K. (1994): A new method for harvesting of Chios "Mastic resin" in a fluid form. 8th International Flavor Conference, July 6-8 1994, Cos Island, Greece. 7. PERRIKOS G. (1988): Mastic. The daughter of Chios. Kallimasia, Chios.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
1947
FOOD PRODUCTION IN ARID AND DESERT AREAS USING THE "KALLIDENDRON TECHNOLOGY" G.Kallistratos'', P.Drakatos^ and l.-M.Kallistratos'' ''Department of Experimental Physiology , Faculty of Medicine , University of Joannina , GR-45110 Joannina, Greece ^Department of Mechanical Engineering , Polytechnical School, University of Patras , GR-26000 Patras, Greece ABSTRACT Development and Environmental Protection are usually conflicting situations. But still in many cases, they can be coordinated in such a way that they could function not only in peaceful coexistance, but evenmore, as a complement to each other. Such a case is the so called "Kallidendron Technology" which is a simple, water saving and economic method for planting trees in arid and desert areas. The "Kallidendron Technology" exploits a part of the end products of our consuming society, transforming them into suitable soil for planting trees and simultaneously decreasing the environmental pollution. The application of the "Kallidendron Technology" aims: 1. to achieve a local production of food in dry and desert areas of the Third World Countries, so the poor humans living under famine conditions, can become self-sufficient individuals. 2. to stop the spread of the desert by planting fruit and forest trees with a minimum of water supply. 3. to decrease the "Greenhouse Effect" by planting billions of fruit and forest trees, which could regenerate oxygen from carbon dioxide by means of the process of photosynthesis. 4. in some special cases such as contaminated areas with radioactive materials, for example, in the late Soviet Union (Russia, Ukraina, Bielorussia, Sibiria) and elsewhere i.e. after atomic bomb tests in Pacific Islands etc. to plant forest trees for the production of oxygen, thus transforming them from problematic areas to useful "oxygen lungs". The cost for the concentrates and other materials necessary for the application of the "Kallidendron Technology" are very low compared with those calculated for conventional methods to plant trees. Also the amount of the water needed for irrigation of the trees is about 5 times less than the classical watering systems. The "Kallidendron Technology" can help the poor countries in their economic and social development and, therefore, can be considered as a great contribution to the "Second Green Revolution".
1948 INTRODUCTION The development of the /C4LL/DEA/DR0A/Technology was first conceived after an observation made while visiting the old castle of loannina in northwestern Greece, where a tree was growing in the stone walls of the castle. This was really a surprise, because usually stones are very poor in fertilizers and also they can not retain much moisture in order to deliver to the tree an adequate amount of water. Practically speaking this means that many trees can also grow with small amounts of both, water and fertilizers. In order to prove this observation experimentally, a number of Greek fruit varieties suitable for planting trees were selected during the winter time. They were introduced into a plastic bag of 50 I in volume, containing in a nutrient pocket the necessary ingredients for their physiological growth. These are fertilizers, trace elements, and hydrogels, which could retain water up to 500 times their volume. Six months later, the fruit trees became leaves and flowers and within 8 months from the time of their plantation, they became fruits. The practical consequences of this experiment were: 1. Since the fruit trees were growing in the roof of our Laboratory, which is composed of cement, it became clear, that fruit and forest trees can grow everywhere, in arid and desert areas, stony and sandy places, providing that the bag contains all essential compounds for their growth, which have already been mentioned. 2. Since fruit, from the point of view of nutrition, are ideal victuals because they can deliver energy, vitamins, electrolytes, trace elements and water, indispensable for the physiological functions of human beings, they could be cultivated in arid areas and deserts, in order to improve the situation of the population living under conditions of famine. 3. The KALLIDENDRON TECHNOLOGY could be applied everywhere to dissolve problems related to negative interference with the environment.
MATERIALS AND METHODS In order to plant fruit or a forest tree with the KALLIDENDRON TECHNOLOGY, only 60-150 g of the essential ingredients, the so-called Concentrates, and a stable bag of an adequate volume, usually 15 I for grapes, flowers and bushes, or 50 I is required for most to the trees. The concentrates are composed of:
1949 a. Fertilizers, NPK, containing nitrogen in the fornn of inorganic salts, like amnnoniunn sulphate, ammonium phosphate, ammonium nitrate, etc. As well as organic compounds, mostly urea and Its derivatives. Phosphates, also as salts, and potassium, in several combinations, depending upon the needs of each plant, for its physiological development. NPK fertilizers are enriched in magnesium, manganese, iron, in the form of chelate, molybdenum, etc. b. Trace elements 20-50 g containing cobalt, zinc, selenium, boron, rare earth elements, thorium, uranium, etc and c. Hydrogel 20-50 gr. such as Aquastore, Evergreen-500 (polyacryl amide), or other suitable water absorbing compounds.
THE PRINCIPLE OF THE KALUDENDRON
TECHNOLOGY
The KALUDENDRON system Is composed of three parts 1. THE NUTRIENT POCKET. It is prepared with a mixture of fertilizers (20-50 g) 20-50 g of trace elements, 20-50 g of an hydrogel and 10-20 I of suitable soil. For practical reasons, since the farmers in the less developed countries do not posses the necessary equipment, they can use either one or two soup-spoons full of each compound, or small plastic cups for measuring the corresponding amounts of compounds suitable. 2. TREES. The selection of the trees must be made according to their significance/usefulness for human nutrition, for the economic development of each country and also for Its general uses, which means that it should be able to contain large amounts of water, in order not to be too inflammable thus decreasing the danger of forest fire. The leaves of the tree should occupy a large surface for Intensive production of oxygen through carbon dioxide, by means of the process of Photosynthesis, contributing simultaneously to the reduction of the Green House effect. To have the advantages to be used either as fruit tree, with could cover the calories demand of the humans, and furthermore, their needs of vitamins, electrolytes, trace elements, and as a water supply source, especially to the inhabitants of these deserts. In addition, could be used for feeding domestic animal. To exploit it as wood for the manufactures of furniture, construction of houses, as fuel material for food preparation, and also the selected high quality of trees, for export, contributing to the economical and social development of the country. 3. THE APPROPRIATE SOIL. For planting trees with the KALUDENDRON system it
1950 is prepared with local materials such as solid wastes, from plant and animal origin (free from glass, plastic materials, heavy metals, poisons or other toxic compounds). Furthermore, animal manure from cows, pigs, goats, sheep, camels, poultry, etc. Dry leaves and other plant remains, sludge residues and other appropriate organic sediments, which are mixed with raw materials, such as local earth, or even sand, in the case of deserts, which are available locally and in abundance.
DETAILED DESCRIPTION OF THE KALUDENDRON
METHOD
a. In a bag of appropriate volume, ten litres of the suitable soil for planting trees, are mixed well with 60-120 g of the concentrates, i.e. fertilizers, trace elements and hydrogel. Since the specific gravity of different soils varies considerable according to their origin, the heaviest being the sand of the deserts, it is important to advise to the farmers to use as a measure the volume of a suitable container and not the weight of the material. This part of the mixture is introduced into the bottom of the bag and represents the so called nutrient pocket (see schematic demonstration fig. 5). For most of the trees, the 50 I volume bags are sufficient. For smaller trees, viticulture, bushes, flowers, etc, the 12-15 1 bags are preferable for reasons of economy. For bigger trees, like Sequoia, date trees, etc, 80 I bags or even larger are used if available. For practical reasons, we use at present plastic bags which are cheaper. In order to find some more biodegradable materials to replace the environmentally polluting plastic bags with paper bags in the near future, the corresponding investigations are running now in our laboratory. b. The selected fruit or forest tree, according to the conditions mentioned above for its use as nutritive or social point of view, is introduced into the bag and the roots, together with the lower part of the trunk are covered with 20-30 I of the prepared local soil (Figs. 8,9). c. The lower part of the bag is then perforated with a knife, about 20 cross perforations in the bottom and around the lower part of the bag, which serves i. to enable the exit of the roots from the bag and disperse them in the earth. ii. to eliminate the excess of water, which without the perforations could damage and decay the roots and consequently destroy the tree. ill. the cross shape of the perforations serve as a technical valve to control the pressure of the irrigating water Inside the bag. By irrigating the tree periodically, the water
1951 pressure inside the bag increases spontaneously, and the valves regulate the pressure inside the bag. As soon as the pressure inside and outside the bag is equalized the perforating holes are closed automatically, thus reducing the exit of the irrigating water. iv. The irrigating water passes automatically, due to gravity, through the nutrient pocket, and dissolves small amounts of fertilizers and trace elements which are transported to the roots already outside the bag. Due to the surface tension of the roots, the irrigating water plus fertilizers and trace elements follow the roots also horizontally, thus supplying them with the necessary nutrients. At the same time, desert sand which is composed mainly of silicon oxide, enriched with sodium chloride, due to its origin from the seas, the salt can be washed away, thus eliminating the danger of poisoning the roots with sodium chloride salt. d. The bag with the tree is then buried into the ground up to its neck. e. Finally 10 I of water is added to each tree, which through gravity precipitates to the bottom of the bag and by reaching the nutrient pocket is retained by the hydrogel. The great economy of water compared with the conventional methods for planting trees is obvious. f. The next water supply after 1-4 hydrometer.
weeks depends upon the indications of the
For verandas, roofs, or balconies, a 50 I plastic flowerpot is used containing a mixture of peat and perlite, in order to reduce the weight of the KALLIDENDRON system
MODIFICATION
OF
THE
KALLIDENDRON
TECHNOLOGY
FOR
THE
PRODUCTION OF VEGETABLES AND OTHER ANNUAL FRUITS LIKE MELONS AND WATERMELONS With a slight modification of the KALLIDENDRON technology. It is possible to produce vegetables like tomatoes, eggplant, cucumbers, peppers, etc, as well as melons and watermelons. This modification consists in the following two variations: 1. Instead of burying the bag vertically In the original soil. It Is placed horizontally and 2. The content of the bag is composed entirely of the nutrient pocket. The roots of the plants, after selection, are introduced into the bag through the corresponding holes in
1952 the upper part of the bag. Because of this modification of the KALLIDENDRON method it became possible, by using suitable hybrids, to produce vegetables, as well as melons and water melons, every two to three months, i.e., four to six han/est per year.
PROBLEMS RELATED TO OUR CIVILIZATION AND OUR HIGH STANDARD OF LIVING One of the serious problems of our civilisation is the increased production of solid, liquid and gas waste. It was our endeavour through the application of the KALLIDENDRON technology to find solutions which could reduce these problems if possible to a minimum. This is achieved by exploiting a part of the solid end products of our consumer society for the manufacture of a suitable soil which is used for planting trees. The liquid phase, i.e. sewage waste after a 2nd degree of purification and oxygenation has been used for planting trees. It is obvious that the trees planted with such the KALLIDENDRON system, using as soil garbage of plant and animal origin, animal manure, sewage sludge, mud from lakes, when the latter are used as recipients of household waste, and irrigated with household water, from purification units, are able to regenerate oxygen from carbon dioxide through photosynthesis, thus contributing to the reduction of the environmental pollution from solid, liquid and gas origin. In addition when the purified waste is used for irrigation of the trees has a double function and advantage. It reduces the problem of the lack of water, and increases the quality of coastal waters
PROBLEMS RELEVANT TO THIRD WORLD COUNTRIES IN ARID AND DESERT AREAS The most serious problems which hinder the growth of fruit and forest trees, as well as vegetables in arid areas and deserts, thus contributing to poor per capita food production for their inhabitants are: 1. Infertile soil 2. Water shortage 3. Lack of fertilizers, or
1953 4. Excess of fertilizers and insecticides in order to increase yield, resulting to environnnental pollution 5. Formation of a salt layer, especially in hot climates, through excessive irrigation followed by Intensive evaporation due to high surface temperature 6. Financial difficulties due to the poverty of these countries. However, taking into consideration the basic low of the nature, that trees and vegetables can grow everywhere, even on balconies, roofs, rocks and deserts, provided that the plants can find the suitable conditions for their physiological growth, the KALLIDENDRON technology was developed with the aim to minimize the problems above mentioned through the following manner. 1. Infertile soil. Deserts and arid lands occupy millions of square miles in Africa, Asia, Australia and America North as well as South, with a constant tendency of spreading. Even in Europe, especially a part of the Mediterranean regions are composed of infertile rocks and arid areas. Through the application of the KALLIDENDRON technology in one of these areas near Athens, it became possible to grow fruit trees. 2. Water shortage .It is obvious that a tree, especially during the summer in dry and hot climates, needs large quantities of water and frequent irrigation. Otherwise, through heat and Intensive evaporation of water, dehydration occurs, and the plant dies. According to our personal experience in Saudi Arabia, we know that the trees planted along the streets of Riyadh, capital of the Kingdom, need between 50-100 I of water, 3 times per week. That means, 600-1.200 I per month or 7.200-14.000 I per year per tree. Tacking into account the number of trees existing only in one city and the quantity of water required, it is obvious that the water amount needed yearly for watering the trees just in Riyadh is enormous. Part of the fresh water needed for the excessive irrigation of the trees is produced by desalination of sea water, a very costly procedure. Supplementary water is secured from drilling new wells. Since the yearly rainfall in this country is low, it is evident that the water reserves of the wells will diminish in the future until they dry up completely. Consequently, methods are investigated with the following targets: a. To create a water reserve in the area where the trees grow, and b. To restrict the intensive evaporation of the irrigating water. These two targets can be achieved by mixing a hydrogel with the content of the nutrient pocket. The polymer EVERGREEN-500 and other hydrogels have the property
1954 to bind an annount of water in excess of 500 times their volume, i.e., 20 g of the hydrogel can bind approximately 10 I of water, a quantity which can supply a tree even under extremely dry climatic conditions for many weeks, depending on the species. Of course bananas need more water than olive trees or grapes. With this KALLIDENDRON technology the water amount needed is 10 I, 1-4 times a month, instead of 600-1.200 per month. With the KALLIDENDRON system more than 80% of water is saved. 3. Lack of fertilizers. Occasionally, plants need supplementary fertilizers for their physiological development. This is an additional problem for poor countries, because if they plan to increase food production, they must import most of the fertilizers. The imported fertilizers, besides being expensive, must be paid in foreign currencies, mainly U.S. Dollars, which are not available in the quantities needed. This serious handicap can also be reduced by applying the KALLIDENDRON technology, since only the absolutely necessary amount of fertilizers, typically 20 g, which a tree actually needs, is added to the nutrient pocket. This amount represents 5-10% of the quantity of fertilizers used in the conventional method of plantation, which is the distribution around the tree of 1 to 3 kg per tree. With the KALLIDENDRON technology, only the absolute necessary amount of the fertilizer is introduced into the bag, and this quantity represents the real fertilizer need of the tree. It is evident that with the scarcity of fertilizers in the less developed countries, at least 10 times more trees can be supplied with the same amounts, when the KALLIDENDRON technology is applied. 4. Excess of fertilizers. Excess fertilizers when through rain or wind are transported to the seas, rivers or lakes they can transform them to eutrophic regions, contributing to the environmental pollution. In addition, the cost of food production is increased considerably, because of the wastefulness of these materials. 5. Formation of salt layer, especially in hot climates, through continuous evaporation of the irrigating water. Through the excessive irrigation in hot climates and subsequent evaporation due to the high surface temperature of the soil, cultivated fertile land became arid after centuries, because of the formation of a salt layer which destroys the cultivated plants. Such known examples are the deserts of Mesopotamia, which during the ancient time they were very fertile and could nourish millions of their inhabitants. Further examples are the Sahel zone and others deserts. According to experts' calculations, until the end of our century about 65% of the cultivated land in hot climates will be lost to agriculture. In addition the construction of dams can aggravate the problem. An example of this is the Assuan dam in Egypt, which increased the water capacity of a country but at the same time hindered the flood of the Nile river, which during the past washed off the salt formation of the cultivated land every year. The same problem of salt formation appeared after the construction of other dams in many tropical
1955 hot regions, for example, the Euphrat dam. The same was true in other continents like America, where the salting process of the irrigated land took enormous dimensions. These few examples demonstrate the significance of the KALLIDENDRON technology to postpone the formation of a salt layer and consequently the desertification for many decades of the cultivated land. It is evident that another advantage of this method is that it restricts to a minimum the evaporation of the irrigating water from one square meter and even more down to a few square centimetres, which is the surface of the plastic bag. Furthermore, because the water inside the bag is found deep in the nutrient pocket, far from the evaporation surface and in addition, bound with the water adsorbing hydrogel. These factors are responsible for the very low evaporation of the irrigating water Inside the bag. Measurements performed in the area of El-Harg in Saudi Arabia by the NAFA Enterprises where the KALLIDENDRON technology has been tested since 1984, have revealed that even 28 days after the last watering the plastic bag with a tomato tree contained more than 60% moisture. Considering the hot climate and the dryness of the area, these results become more impressive. Contrary to this, with the conventional irrigating systems, there is around the tree a large evaporating surface. This is necessary in order to supply the tree through the surrounding soil with sufficient water. Under these conditions, with each watering 50 - 80% of the irrigating water is lost by evaporation. Even freshwater contains small amounts of sodium chloride salt, which after every evaporation forms a thin layer around the tree. By repeated irrigations followed by continuously every year and more intensively in hot climates resulting in the permanent loss of cultivating surface and the expansion of the deserts. The extent of these disasters were confirmed by satellite earth scan photographs, where about half of the cultivated land, i.e. 120 million of hectares are already damaged through salt formation in the irrigated areas. It is evident that with the application of the KALLIDENDRON technology, the evaporation of the irrigating water is greatly restricted because of the above mentioned reasons. Consequently,
the lost of cultivated land is not only
stopped, but on the contrary, lost land can be reclaimed for re-cultivations. 6. Financial difficulties due to the poverty in general of the 3rd World Countries. The poor countries of the 3rd World need financial support in order to organise
an
infrastructure,
indispensable
for
their
agricultural
development.
Unfortunately, their external debt, which was estimated a few years ago to be more than 1.300 billion U.S. Dollars, with a continuous tendency to increase, stops every effort for their economic and social development. Despite the up to date success of the KALLIDENDRON technology in several areas of the five continents, one of the greatest handicaps for its propagation and expansion is, besides the lack of foreign currency, their obligations for the increased external debt. This situation will become worse in the future, because even now these countries with great difficulties can pay only a part of
1956 their debt's interest. That means, even without receiving a single Dollar for their developnnent, as further external aid from the rich countries of the North, they are obliged to pay about 130 billion U.S. Dollars every year for accumulated interest to the World Bank and other Bank Consortiums of the rich North. According to objective reports (North south a common future) for each currency unit which the European Nations, except Greece, with the KALLIDENDRON technology, sent to the 3rd World Countries, the so called donors will generally collect directly or indirectly 9 fold in return. The USA will receive 25 fold, including the weapons and other war materials, and Japan 21 fold. It must be emphasized, that the rich Industrialized Northern Nations generally follow a traditional policy of dependency towards the poor South. This constitutes one of the main reasons of the North-South Conflict. On the contrary, the Hellenic Technical Assistance to the 3rd World Countries with the KALLIDENDRON Technology alone has an efficiency index over 10 fold, i.e. the profit for the poor countries is multiplied at least 10 times. This is because i. it saves hard currency for the import from abroad of the commercial soil for planting trees, since this earth is prepared with cheap local materials, such as solid waste, animal manure, dry leaves, and other raw material found locally in abundance, li. it exploits a part of the end products of the human society, which by accumulation provoke serious problems, such as environmental polluting agents, by changing them to advantages as soils suitable for trees, ill. the KALLIDENDRON technology contributes to the economic development of the poor countries, by offering work to the local inhabitants, iv. poor inhabitants of the less developed countries suffering from malnutrition can improve their situation through local food production. They can also become by Increasing their yields, self-sufficient in the future. V. Arid areas, as well as cultivated land already lost can be reclaimed.
1957
ILLUSTRATIONS
1958
Figure 1. The tree growing in tiie walls of the castle of loannina with very little humidity and fertilizers which could be supplied by stones, stimulated the development of the KALLIDENDRON technology.
Figure 2. Selected Greek fruit trees for the Investigation and confirmation of the observation.
1959
Figure 3. In six months, the Greek fruit varieties developed leaves and flowers.
Figure 5. Schematic demonstration KALUDENDRON technology.
Figure 4. Fruit formation (peaches) after 8 months.
of the
1960
Figure 6. Three local farmers In Senegal using cups to measure the quantity of three concentrates.
Figure 7. The use of local material such as litter of plant and animal origin, dry leaves, animal manure etc. for the production of local soil.
1961
Figure 8. Senegal. The prepared soil is transported to the place where the nutrient pocket (rear) and the rest of the ingredients for the KALLIDENDRON technology are prepared.
Figure 9. In case no suitable soil is available, desert sand mixed with animal manure is used (Jilantai desert, Inner Mongolia).
1962
'k^r^
Figure 10. Sewage sludge free of heavy metals can also be used as suitable soil (Patras). The household water after a second degree of purification and oxygenation can also be applied for watering the trees.
Figure 11. The household water from Fig. 10 is an ideal liquid for the growth of the trees, due to its content in substances necessary for their accelerating growth. The eucalyptus tree reaches a height of 5 m after three years.
1963
Figure 12. Perforation of the KALLIDENDRON technology bag (Capo Verde Islands).
Figure 13. The use of sand as soil in the deserts of Inner Mongolia, Jilantai desert.
1964
Figure 14. The use of sand as soil In the deserts of Egypt (desert of Marsa Matruh).
Figure 15. A 16 litre volume plastic bag is use for planting sun flower (Senegal).
1965
Figure 16. A 16 litre volume plastic bag is use for planting grapes (400 km west of Beijing, P.R. China).
^\ ^'^^Mt-
Figure 17. A date tree planted with the KALLIDENDRON technology.
Figure 18. Also Sequoia, the biggesi tree of the earth, can be planted by applying the KALLIDENDRON technology.
1966
Figure 19. Preparation of the soil for tiie modified KALLIDENDRON method (Senegal).
1967
Figure 20. Classification of the bags In lines (Senegal)
,#**.
^mm^ Figure 21. The horizontal bags are covered with sand and only the roots are inside the bag. The rest of the plant is outside.
1968
Figure 22. Fruit formation (watermelon) three months after planting (Senegal).
Figure 23. Rubbish which can be used for the KALLIDENDRON technology. (Rubbish dump of the City of loannina).
1969
Figure 24. Since these end products are not used for the production of suitable soil, they are only covered with sand (loannina, Greece).
Figure 25. The next step is planting the area with forest trees (University staff working on reforestation of the campus).
1970
Figure 26. Sewage household water after a second degree of purification, followed by oxygenation, is ideal for Irrigation of the trees, since it contains materials indispensable to their growth.
1971
Figure 27. A negative example for tine uncontrolled flow of sewage in the gulf of Patras, Greece, the cause of many health and social problems.
Figure 28. Desert is composed of infertile soil which, together with the lack of fresh water reserves, precludes the planting of trees. A general view of the Jilantai desert in Inner Mongolia, where with the application of the KALLIDENDRON technology, a 30% success was achieved in this afforestation endeavour.
1972
^^ji^ Figure 29. A well in the desert near Marsa Matruh, Egypt, which supplied water for planting fruit trees by means of the KALLIDENDRON technology.
1973
Figure 30. The formation of a salt layer due to the intensive evaporation of the irrigating water causes many problems, as this example in the Jilantai desert, Inner Mongolia, illustrates.
Figure 31. A typical arid area in northwestern Greece, where the KALLIDENDRON technology found a successful application.
1974
Figure 32. An arid area near Athens, Greece, which now produces many fruits by means of the KALLIDENDRON technology.
Figure 33. Wells can pump water from a depth of 60 meters in the Jilantai desert of Inner Mongolia and supply the planted trees with the need amount of water. The bags of the KALLIDENDRON technology with their trees are visible in the front of the photograph.
1975
Figure 34. Bananas need much more water compared to other fruit trees, which show a greater resistance to dryness. Bananas planted with the KALLIDENDRON method in Senegal.
^^S^^
/Pi^
©^
'<. ^M'
Figure 35. Minimization of the evaporating surface of the irrigating water through the /CALL/DEA/DROA/technology. Mass production of KALLIDENDRON trees in Senegal. Notice the reduction of the evaporating surface which is restricted only to the diameter of the plastic bag.
1976
Figure 36. Preparation of tiie soil to plant trees with the KALLIDENDRON technology, using local materials in Ethiopia.
Figure 37. Preparation of a roof in loannina, Greece for the application of the KALLIDENDRON technology.
1977
Figure 38. General view of tine roof, two years later.
Wi M
Figure 39. The olive tree of Fig. 38. The bag on the surface is also visible. Notice the narrow width of the soil were the tree is growing.
1978
Figure 40. Apple harvest from the roof (loannina, Greece).
Figure 41. Flowers and grass can be planted on the roof (loannina, Greece).
1979
Figure 42. Rocky mountains and arid areas, like tiiis landscape in Inner Mongolia are suitable places for the application of the KALLIDENDRON technology.
Figure 43. Avocado trees planted in Senegal.
1980
Figure 44. A jojoba planted with the KALLIDENDRON system in Senegal.
1981
Figure 45. A pine tree planted with the KALLIDENDRON technology in a stony and arid areas of the polar zone of Finland.
1982
Figure 46. Professor Drakatos, Polytechnic School, University of Patras, demonstrating a series of forest trees planted through the KALLIDENDRON method on the University campus.
1983
Figure 47. Harvest of peaches, planted through the KALLIDENDRON technology, at the University yard of loannina
Figure 48. Production of an abundance of plumps from the first tree planted in 1979 with the KALLIDENDRON method.
1984
Figure 49. Fruits of a tomato tree (Tomarillos) planted witii tiie KALLIDENDRON system in loannina.
Figure 50. A variety of Greek fruit trees planted with the KALLIDENDRON method in an arid area near Athens.
1985
Figure 51. The use of sewage sludge from a leather factory, which contains enormous amounts of chromium, for the preparation of special soil.
1986
Figure 52. The soil in this photograph is destined for recycling purposes of the heavy metals and can be used to plant under control forest trees like here, plane trees.
1987
Figure 53. Planting of olives, apple trees and other fruit trees in the western Egyptian desert.
Figure 54. Plantation of a Papaya in Djibouti with the KALLIDENDRON technology.
1988
^^*llBf ::*
Figure 55. Introduction of tine KALLIDENDRON technology in the Capo Verde Islands.
Figure 56. An olive tree, just arrived from Greece, is planted in a Hotel at Capo Verde.
1989
Figure 57. Gratis distribution of KALLIDENDRON trees to tine farmers of Capo Verde.
1990
Figure 58. Senegal. Transportation of the prepared KALLIDENDRON trees to their final destination.
Figure 59. Prof. Kallistratos during the presentation of his nnethod in the Faculty of Agriculture in Indonesia.
1991
Figure 60. Prof. Kallistratos demonstrating to Chinese and Mongolian colleagues the principles of the KALLIDENDRON technology.
Figure 61. Discussion with specialists concerning the areas where the KALLIDENDRON method will be applied In Inner Mongolia.
1992
Figure 62. Women preparing tiie nutrient pockets with materials partly sent from Greece and the rest from P.R. China.
Figure 63. Chinese and Mongolian people planting trees with the KALLIDENDRON method in Jilantai desert.
1993
Figure 64. Visit of Prof. Kallistratos, forest station of Hohhot, capital of Inner Mongolia, to discuss with forest specialist the continuation of the desert progrannmes.
Figure 65. Vines, which during the winter (during six months) are covered with sand for protection against the hard low temperature, have in a very short time sprang leaves (Inner Mongolia).
1994
Figure 66. Formation of grapes two years after tiney have been planted. By considering the hard climatic conditions of Inner Mongolia the fruit formation in such a short time is surprisingly good.
Figure 67. plantation of vines in an arid area of Peru.
1995
Figure 68. The situation after two years. The vines are doing very well.
Figure 69. Women from African countries participating in a seminar about the KALLIDENDRON technology, organized by the Greek ministry of Agriculture in the island of Greta.
1996
Figure 70. After the theoretical seminar women have to gain practical experience by preparing the materials for the KALLIDENDRON technology in the Island of Crete.
Figure 71. African style transportation of the 10 litres of water for the Irrigation of the KALLIDENDRON trees already planted.
1997
Figure 72. Prof. Kallistratos and a group of women working for the KALLIDENDRON project in Inner IViongolia.
Figure 73. School children from the Inner Mongolia Autonomous Region help during the afforestation of their area with the KALLIDENDRON system.
1998
Figure 74. The first KALLIDENDRON tree is already prepared with the supervision of their teacher and the children are looking proud their endeavour.
Figure 75. Greek soldiers in the Island of Crete participating in the afforestation efforts by planting the KALLIDENDRON trees.
1999
Figure 76. Chinese soldiers participating in the afforestation of their country.
Figure 77. Monks of the holy mountain of Athos, planting olive trees with the KALLIDENDRON technology.
2000
Figure 78. Monks of a convent in Peloponissos, Greece, receiving a donation of KALLIDENDRON trees to plant in tiieir yard.
Figure 79. Apple trees planted with the KALLIDENDRON method, 400 km west from Beijing.
2001
-.^^^i^^: Figure 80. Distribution of KALLIDENDRON fruit trees to priest-farmers in tiie Island of Crete.
Figure 81. A coconut tree planted In the shores of Senegal, irrigated with brackish water (winter 1987).
2002
Figure 82. The tree in the previous Figure, one year later. Despite its irrigation of brackish water, its growth seems to be normal.
2003
Figure 83. Irrigation of KALLIDENDRON trees planted in the western desert of Egypt with water transported by barrels from the desert wells.
Figure 84. Irrigation of the 5.500 fruit and forest trees planted in Senegal with the KALLIDENDRON technology. The water tank is filled with water with the help of a pump.
2004
Figure 85. By means of a system of underground pipelines, the water from the tank is propagated to the barrels in the proximity of the trees through the physical principle of communicating vessels.
-^^^^^S
Figure 86. The farmer who irrigates the trees has only to walk each time about 50-100 meters to the next barrel, which supplies the water. Othen/vise, he could be obliged, if the pipeline system did not exist, to walk at least a kilometre to bring each time 20 litres of water for irrigating two trees.
2005
Figure 87. Eucalyptus trees planted in three lines to brake the hot wind from Sahara (Senegal, winter 1987).
Figure 88. The Eucalyptus trees in the previous Figure, one year later, showing a very satisfactory growth (Senegal winter, 1988).
2006
Figure 89. The Yellow river in China passes through the desert. In case our project will be realized, it can supply the desert with sufficient amount of water for our KALLIDENDRON programme.
Figure 90. The river Awash in Ethiopia, which vanishes near the Ogaden desert, and could be detected by modern technological methods. If this happens, a part of the desert will be changed to a green belt.
2007
Figure 91. The action is called "Fruit trees against hunger and for the self-sufficient of the people living in problematic areas".
Figure 92. The struggle against desertification.
2008 RESULTS The KALLIDENDRON technology is a product of practical experience based on observations which the nature reveals: how under extreme climatic conditions In conjunction with very little fertilizers and moisture, several plants can sun/ive. With modern technology it is possible to achieve such conditions, which also allow, under these hard climatic conditions, to cultivate selected plants, important to human nutrition. The condition necessary for this is the coincidence of three main factors: a. the seed of the tree b. scarcity of moisture, and c. scarcity of fertilizers. It is clear that many tree varieties need for their growth very little water. Sometimes they can even survive without any drop of water for many years, like the Cactus, Jojoba, etc. In contrast other trees need a lot of water for their growth and especially in order to compensate for the loss through the leaves and other evaporation processes. It is obvious that between the two extremes - a lot or a little water - there are trees which can sun/ive, in dry and hot climatic conditions, with an intermediate amount of water. The practical consequences of this obsen/ation, is to select suitable seeds and trees, which could grow with a small amount of water, e.g. 5-10 I, 1-4 times per month and about 20-50 g of fertilizers and trace elements. The experience gained with the KALLIDENDRON technology during the last ten years, revealed that most of the trees tested can grow with this system, provided that they are protected from the wind by natural or artificial fences, as well as from herbivorous animals, because in arid areas those trees will probably be their most important source of food. Numerous trees and vegetables have been tested with regard to their suitability to the KALLIDENDRON technology. These are contained in Table 1. Other tree varieties, especially tropical as well as of economic value for the social development of the 3rd World countries, are also under investigation.
PRACTICAL APPLICATIONS OF THE KALLIDENDRON
TECHNOLOGY IN
AGRICULTURE. According to the description of the KALLIDENDRON technology, this method has the following fields of application in Agriculture and Environment in general.
2009 1. Plantation of streets, avenues, parks, balconies, roofs, arid areas and rocky mountains with trees, and the formation of oxygen lungs in polluted cities, as well as new forest in problematic areas. 2.
Application In greenhouses for the production of vegetables.
3.
Growing of fruit trees in arid areas and deserts. Most of the trees planted with the
KALLIDENDRON technology in Greece, Africa or in Asia were fruit trees. 4.
Tropical and subtropical trees like Avocado, Battel trees, Palm trees, tomato trees,
Gawavas, Jojoba, Sequoia dendron giganteum and Sequoia sempervlrens, etc. The plantation of arid areas and deserts with edible fruit trees, especially in 3rd World countries could contribute to the struggle against starvation, because of the consumption shown In Tables 2-5. Besides the fruit, which can be eaten by the local population, fruit and forest trees deliver the leaves and other parts of the so called plant blomass, which can be used to feed lambs, goats, cattle, pigs, camel and poultry, in order to complete their nutrition with animal proteins, such as milk and its products as well as, meat, produced by the above mentioned animals. Even at present, the combination of three types of fruit trees, mainly avocados, dates and nuts, may constitute an emergency measure for the survival of starving populations. This combination is demonstrated in Table 5. The minimum energy demand for an adult person In order to cover his basic metabolism is 1.400 kcal or 5.850 kjoule. That means, with the above food ratio of 1.980 kcal has also a reserve for his muscular activity, that is why it is Important in the future to determine the standard of life of the poor countries in kcal or kjoules necessary to Table 1. Successfully tested plants for the KALLIDENDRON technology (Reprinted from G. Kallistratos and U. Kallistratos, J. Orient. African Studies, 1991, p.197). 1. Almond trees 2. Pear 3. Apricot 4. Berry 5. Plum 6. Olive 7. Cherry 8. Lemon 9. Clementine 10. Orange 11. Citron 12. Grapefruit 1 13. Quince
14. Chestnut 15. Walnut 16. Pistachio nut 17. Hazelnut 18. Lotus 19. Apple 20. Peach 21. Nectarine 22. Fig 23. Carob 24. Vine 25. Kiwi 26. Avocado
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
Mango Gawava Macadamia Coconut Coffee Date Tomato tree Jojoba Bamboo Sequoia Banana Sunflower Diverse vegetables
[
2010 Table 2. List of common fruits and their energy value (Reprinted from G. Kallistratos and U. Kallistratos, J. Orient African Studies, 1989, p.141). Fruit
Amount (ing)
Energy delivered (in kcal)
1 Olives
100
150
1 Chestnuts
100
210
1 Apricots
300
160
1 Grapes
300
210
1 Apples
300
160
1 Peaches
300
160
1 Pears
300
160
1 Cherries
300
210
1 Plums
300
200
1 Oranges
two
110
1 Tangerines
two
rio
1
survive by reaching a minimum of 1.400 kcal or 5.850 kjoule daily, instead to calculate their standard of life in U.S. Dollars. The reason is that if it is calculated in U.S. Dollars per capita and year of the poor inhabitants of the earth, this calculation possesses only a relative value and undergoes many variations from country to country, depending upon their wealth or poverty. In addition, the tendency of local inflation must be considered. It is clear that the U.S. Dollar has another value in the rich gulf countries, where it is not enough for a tip, and in Bangladesh and other poor countries, where a whole family can purchase a meal. Furthermore, the value of the U.S. Dollar was Table 3. List of dry nuts (particularly rich energy sources) and their energy value (Reprinted from G. Kallistratos and U. Kallistratos, J. Orient African Studies, 1989, p. 141). Fruit
Amount (ing)
Energy delivered (in kcal) |
1 Almonds
100
650
1 Hazelnuts
100
695
1 Walnuts
300
700
1 Pistachio nuts
300
600
1 Coconuts
300
600
1
2011 Table 4. Energy content of sonne tropical and subtropical fruits (Reprinted fronn G. Kallistratos, and U. Kallistratos, J. Orient African Studies, 1989, p. 141). Fruit
Amount (ing)
Energy delivered (in kcal) |
1 Dates
200
600
1 Mango
300
200
1 Gawava
300
180
1 Papaya
300
120
1 Avocado
One, medium
480
different 50 years ago, and will be changing in the future because of the tendency of inflation. In contrast to the political value for the determination of victuals of the poor populations, the physical units in kcal and recently in kjoule is scientifically more correct. The reason is that it was always constant in the past and will also remain stable in the future. In addition, it does not undergo any variations, or is influenced by economical crises or inflation. Concerning the Greek aid for less developed countries, this is divided in three steps.
1. First aid to produce vegetables like tomatoes, cucumbers, aubergines, etc., melons and watermelons which could be harvest within 3 to 6 months. 2. Short term aid, with plants which can give fruits within 1-2 years, like Bananas, Papayas, Sunflower, etc. Table 5. A simple daily food ration which can be applied to cover the basic energy demands in famine areas (Reprinted from G. Kallistratos etal., Nutr. Diet, 1990, p. 26). Amount (ing)
Energy delivered (in kcal/kJ)
1 Avocado
300
480/2.000
1 Dates
300
900/3.765
Nuts
100
600/2.150
1 Total
700
1.980/8.275
Fruit
11
2012 3. Long term aid, with fruit trees which need several years for fruit production, like Avocado, Apple trees, Lemons, Oranges, Date trees, and especially the rich in calories nut trees. Meanwhile, the afforestation of selected areas of the Countries with forest trees for multiple use and for economic development. The KALLIDENDRON technology is presently applied in a belt around the World within a width extending from the polar region of Finland to South Africa and South America till Central Argentina (Mentosa). The up to date results in all five continents are the following: Europe Finland: Few hundred trees, mainly forest. In South, Middle and North Finland, up to the stony areas of the polar zone. Germany: In spite of the fact that Germany has a fertile soil and regular rain, there have been problematic areas selected, where the plantation of trees was difficult because of the existence of unsuitable soil and for demonstration purposes (200 trees). France: Especially in Midi and other arid places (150 trees). Italy: Spain:
In South Italy and Sicily (100 trees) Especially in the Islands Mallorca and other Canary Islands (see also
references, 300 trees). Portugal: 100 trees. Hellas:
Patras 6.000 trees, loannina 3.500 trees, Athos mountain 1.000 trees, Athens
2.000 Islands of Aegean See 2.500. Total 15.000, mainly fruit trees. Russia: 100 trees, half fruit and half forest. Malta: 50 trees. Total in Europe: 16.200 trees.
Africa Egypt:
1.100 trees, most of them in the Western desert near Marsa Matruh
2013 Libya: 8.000 trees. Sudan: 200 trees. Ethiopia: 300 trees. Djibouti: 100 trees. Senegal: 5.500 trees. Capo Verde Islands:
1.400 trees.
In addition, nnaterials for planting 100 trees were sent to Nigeria, Niger, Ghana, Ivory Coast and Burkina Faso, where we intend to plant 5.000 trees as soon as financial problems are dissolved. In the future Mauritania, Mali, Zambia, Zimbabwe, Tanzania, South Africa and the Island of Mauritius will be considered. Total In Africa: 17.800 trees. Asia Arabic desert between Dubai and Abu Dahbi 2.700 mainly fruit trees Saudi Arabia: 200 trees. Qatar: India:
100 trees. In three experimental stations, in Bengal, Kerala and Hyderabat, 300 trees.
Indonesia: In two experimental stations. In Jakarta, 100 trees and Semarang ,100 trees. Inner Mongolia: 42.000 fruit and forest trees, among them 2.500 Dazi plums, 500 Lanzhou Bajie Apricots, 500 grape-vine trees, 7.000 Apple trees. The average survival rate In the villages Dayang and Haishengbula was 89,91 % after 3 years, in comparison to the conventional method which yielded 60,83 %. The growth of the KALLIDENDRON trees was 43,73 cm/year compared to that of the trees planted with the conventional method, which was 25,57 cm/year. Following the successful results obtained with the first trials, the Science and Technology Commission of the Inner Mongolia Autonomous Region, decided to plant 500.000 more fruit trees in an area between the Yellow River and Hohhot with the KALLIDENDRON Method. In addition, the Multi-trade Station of
2014 Inner Mongolia, proposed a second program for planting 2.000.000 trees. The problem is that for the realization of both programs, a minimum request for five million U.S. Dollars is needed. However, the environmental contribution and the enhancement of the welfare of the farmers in these areas. Is worth the financial burden. To this purpose the Inner Mongolia Science and Technology Commission, opened an account with the Bank of China in Hohhot, for local currency - and U.S. Dollars for foreigners who are interested in supporting this endeavour. P.R.China: In cooperation with the Chinese Academy of Science, Department of Botany, in a sandy area of the Yellow River, 400 kilometres south of Beijing, 1.100 trees. Asia total: 46.600 trees
Oceania Australia:
In New South Wales, 100 trees.
America North (USA): 100 trees. Guatemala: 100 trees. Brazil:
100 trees.
Chile:
100 trees.
Peru: 300 trees. Argentina: 900 trees. Total in North and South America: 1.600 trees To date 82.250 fruit and forest trees have been planted with the Kallldendron technology in all five continents. There are some organisations all over the world who are already familiar and enthusiastic for the introduction and application of the KALLIDENDRON technology in
2015 their countries. These are: 1. Women Organisations especially in Africa and Asia, but also in the Americas. 2. School Children in Greece and P.R. of China (Inner Mongolia). 3. The armed forces in Greece and Inner Mongolia.
In some special cases also the Monks of the Monasteries.
DISCUSSION Problems such as low food production caused by droughts, permanent loss of cultivated land, due to constant spreading of the deserts, in conjunction with the uncontrolled growth of the earth's population, cause regional famine and many diseases resulting from malnutrition. According to experts' calculations, about two thirds of our planet's population is victimized more or less through our active or passive selfdestructive behaviour. Furthermore, by interfering with our sensitive ecological balance which sustains life, our eco-system Is at present in great danger, especially through due to human interference, which causes irreversible environmental pollution. Every day, thousands of humans, and mostly children die of hunger and diseases, mainly in the less developed countries. During the last decades a new form of perilous disease appeared, AIDS, especially in Africa and South America. Among the reasons which decrease the efficiency of foreign aid and turns it to a less effective and more expensive operation, are: 1. High transportation cost from Europe and North America. 2. Shortage of local facilities for the transport of the food to their final destination in the famine areas, due to the absence of infrastructure and lack of advanced technology in most of the less developed countries. 3. Local bureaucracy discouraging and hindering farmers' productivity 4. Poor harvest yields of cultivated areas, due to the lack of suitable hybrids, which could Increase food production (Green Revolution). Furthermore, shortage of foreign currency, a permanent situation of the poor countries, thus inhibiting them to buy from the International Free Markets the amounts of necessary food, fertilizers, insecticides, etc.
2016 5. Civil wars between tribes. 6. Accelerating growth of the population, due to uncontrolled rising of birth rate. As a result of this situation, the per capita food production and consumption of the population living in these areas is very low, reaching the borders of stan/ation and subsequent death. Even in the rich countries, the per capita standard of living of the poor people is also very low due to the high percentage of unemployment. As a supplementary factor, this situation will become worse in the future, because measures taken in the past were also ineffective to stop the accelerating destruction of our planet and consequently the danger to perish will not be limited to the inhabitants of the less developed countries, but will expand all over the world. Generally speaking, the rich countries were unable to solve several critical problems, without at the same time provoking new ones. It is clear that without a practical system which tends to exploit a part of the end products of our consumer society, like the KALLIDENDRON technology it will be very difficult to master the tendency of the gradually increasing destruction of the earth. The KALLIDENDRON technology combines the up to date progress of various scientific fields of basic and applied research. The target of this innovative procedure will be primarily to stop the spread of the deserts and also reclaim land gone already arid. Furthermore, to produce locally cheap agricultural products, which could cover at first partially and eventually total the necessary victuals for the daily energy demands, as well as vitamins, electrolytes and trace elements for the population living in arid and desert areas, within a self-supporting system. It is also important that this innovative technology should avoid repeating the same mistakes made by the developed countries, which are to a great extent responsible for the grave environmental pollution we are facing today. In contrast, they must aim to protect, preserve, or even Improve the world around us. Most of the difficulties mentioned above can be restricted or even avoided through the introduction of the KALLIDENDRON system to the 3rd world countries. The philosophy of this system is an initial aid for self-help, i.e. the encouragement of the population living in famine areas to produce food locally and consequently to increase its food intake. Gradually these areas will become self-sufficient. The supplementary calories delivered by this innovative technology, should be an alternative way against world famine. This solution is independent from the social, economic or political situation of the less developed countries. Three are the main problems in arid and desert areas which are discussed in this chapter:
2017 1. Infertile soil. A part of this problem has been discussed in the previous chapters. Supplementary, the classical fertilizers NPK can be cheaper in the future due to a strong competition between the Chemical Multies (big international companies) and the small Fertilizer companies. It must be pointed out, that all materials used for the KALLIDENDRON technology are available in the open market, either in Greece, or in other European Countries. 2. Scarcity of water. This problem can be bypassed by using water reserves as effectively as possible. That means that with the KALLIDENDRON technology the consumption of water is restricted to a minimum of 10-40 I per month, per tree. Further, economy of water can be achieved by: I. By using brackish water available through the wells. Most of the plants cannot be irrigated with brackish water. Unfortunately, most of the wells in the Sahel Zones and across the shores of the Atlantic and Indian Ocean, deliver slightly saline water. Such a water quality is suitable for planting coconut trees, some varieties of date trees and also tomatoes, which could tolerate brackish water. One example of coconut tree planted in the shore of the Atlantic Ocean in Senegal and irrigated with a slightly saline water pumped from a well is shown in Figs. 81 and 82. ii. Limiting fresh water consumption, in cases it is available in limited amounts. In these cases the number of fruit trees which can be planted with the KALLIDENDRON technology and irrigated with the available fresh water reserves can be increased at least up to fivefold, in comparison with the conventional methods of cultivation, because of the advantages of the technology, especially concerning its water saving properties. The possibility to increase the number of fruit trees growing with the available fresh water supply, contributes positively to the struggle against hunger for the people living in these areas, because fruits can supply everything which is indispensable for the physiological function of the human organism. iii. Planting along the river sides. Two projects, currently under investigation as future endeavours to assure water in the arid regions and deserts of the earth. The systematic plantation of fruit and forest trees in lines at intervals of 5-10 meters, depending upon the variety of the tree, is the target of our investigation. The initial plantation line can be at a distance of 10 meters from the river bank. Starting this program with fruit trees, which could occupy a belt of a width up to 1 kilometre on both sides of the river, planting 100 lines in each side, results in 200 trees. In a sector of 100 kilometres, 2 x 1.000.000 trees could be planted. In order to reduce the cost of energy and the manual work involved in the irrigation of the trees, water pumps working with solar energy can be used. Solar energy is
2018 abundant in these areas. For the propulsion of the pumped water from the river shore to the proximity of the trees, a pipe line system is being tested in Senegal as a model propelling the water by means of the physical principle of communicating vessels, with very good results. The pump system and the resulting distribution of water, is now applied in Senegal to the irrigation of 5.500 trees with very good results, especially with regard to the immense economy of water. The irrigating water enters into the bag in precise amounts and, by careful manipulation, a single drop is not lost. Underground pipeline systems have the advantage that the water which circulates inside the pipelines cannot be evaporated. In contrast, with the open air irrigating canals constructed by the engineers of the former Soviet Union to change the direction of the rivers from the North Arctic Sea and lead them to the deserts of the Central Asian Soviet Republics, the loss of water through evaporation in the warm deserts along the long route of the canals, was very intensive, wasting large amounts of the valuable water through evaporation along the distance from Northern Siberia to the central Asian desert areas. iv. Detection of the underground course of rivers which disappear in the desert. By using modern technology, there is no problem to discover the course of a river which disappears in the desert and continues its underground way to the ocean. Two examples are the Okawango River in Botswana which disappears in the Kalahari desert and also the river Awash in Ethiopia, which vanishes near the Ogaden desert. By determining the underground river bed by means of air photographs, it will be possible to drill numerous wells in the deserts along their subterranean passages, which could deliver the necessary amount of water to plant millions of fruit and forest trees in the actual deserts, and transform them to orchards. 3. Financial difficulties. This is not only a problem of the poor countries, but also a problem of the rich countries, in connection with the environmental pollution which is severe in the industrialized nations. Carbon dioxide is one of the main contributors to the green house effect. In a pilot plan, the possibility of applying the KALLIDENDRON technology for the plantation of roofs, balconies, bare hills surrounding the cities, and other available places with trees, bushes and grass, in order to reduce the smog of polluted cities like Athens, and the possibility to regenerate oxygen from carbon dioxide was investigated. A number of roofs and verandas have already been planted in the cities of Athens and loannina as models to demonstrate how simple and economic is the reduction of smog when the Green Roofs Programmes are realized in cities polluted with exhaust gasses and fumes derived mainly from cars, Industry and household heating installations. It Is estimated that each middle size tree, at the beginning of its plantation, could produce 2-10 I of oxygen daily, from 2-10 I of carbon dioxide. This amount of oxygen production, will gradually increase due to the growth of the tree. In case that each member of a family could plant in the future in their balconies, roofs and
2019 other places between 5-10 mainly fruit trees, the four million inhabitants of Athens, who live at present in the most smoggy city in Europe, can plant 20.000.000 trees. These trees can produce every day about 200.000.000 I of oxygen from 200.000.000 I of carbon dioxide, thus contributing to the reduction of smog, which to a great extent is composed of carbon dioxide. The advantages of this so called Green Roofs Programme are: i. The reduction of the smog in Athens. ii. Low cost for the Hellenic Government, because the plantation of the balconies roofs, etc. with fruit trees, will be a private obligation for the citizens of Athens. iii. The produced fruits can be selected and eaten from the owners of the plants. It is also advisable to plant such fruit trees in Athens, which during the winter time they keep their leaves, like the olive trees, lemons, orange, Clementine, grapefruit etc., and occasionally other trees which lose their leaves in winter such as apple trees, plum, almond, pear, apricot, peaches, fig and other Greek fruit varieties. In order to reduce the Green House Effect and to avoid the increase of Earths' temperature, which threatens to cause a climatic collapse, specialists estimate that it will be indispensable to plant in the near future at least 3 billion trees. The expenses for this life-saving action are estimated at 450 billion U.S. Dollars, i.e. 150 U.S. Dollars per tree. Unfortunately, besides the financial problems, a second question arises about where and how soon the tremendous quantities of water needed to irrigate the 3 billion trees could be available. It is known that most of the areas to be selected are arid regions and deserts which possesses very little water or they are dry. For the three billion trees, the quantity of water needed according to the experience in Saudi Arabia, is calculated between a minimum of 21,5 m^ up to 43 billion m^ water every year. These calculation make the target to reduce the Green House Effect in the near future unrealistic. The KALLIDENDRON technology offers an alternative solution, concerning expenses and irrigation of the tree billion trees. The cost for planting 3 billion trees can be reduce to 4,5 - 9 billion U.S. Dollars and the quantity of water required for irrigation, to 1 - 3 billion m^, an alternative which appears reasonable and realistic, by considering the four possibilities of watering trees in arid areas and also the advantages of the KALLIDENDRON technology which are: 1. Water saving of more than 80 %.
2020 2. Savings up to 95 % in fertilizers. 3. Easy application everywhere, e.g on roofs, balconies, arid areas, deserts, bare hills, etc. 4. Reduction of environmental pollution. 5. Low cost and therefore applicable to conditions in poor countries. 6. Possibility of afforestation during all 12 nnonths of the year. 7. Delayed or postponed fornnation of salt layer. 8. Reduction of the manual work by 50 % or more. Thus, the importance of the
KALLIDENDRON technology for the sun/ival of
starving humans through the action "Fruit trees against hunger" completed with the sentence "and forest trees for environmental improvement' is obvious and very relevant. The KALLIDENDRON technology can be easily and widely applied in poor countries. Furthermore, the significance of the technology to reclaim land already gone arid and to reduce the Green House Effect, is emphasized. Consequently, the rich Industrialized nations and the international organisations, have a moral obligation to support the KALLIDENDRON technology as a part of the 2nd Green Revolution, if they intend to establish a peaceful co-existence between the rich northern and the poor southern nations. That means that the relations between the rich North and the poor South must cease to be a conflict situation and to become a collaboration for a common wealthy and peaceful future. In conclusion, hunger, external Debt, poverty and other unacceptable living conditions in the poor 3rd World countries may cause bloody revolutions, disease and other calamities with many deaths, while the KALLIDENDRON technology could contribute to a peaceful 2nd Green Revolution.
REFERENCES References and articles on the KALLIDENDRON technology 1.
Anonymous, Trees in a bag, hope for Agriculture in dry climates, Greece Today,
9.4.1988, p. 14
2021 2.
P. Drakatos, R. Lyon, G. Chryssolouris and G. Kallistratos, Reliability and
Exploitation of waste-water treatment plants, using diagnostic methods. International Journal of Environmental studies 40 (1992 ) 267 3.
G. Kallistratos et al. Reports of the HYDROBIOLOGICAL RESEARCH CENTRE of
the UNIVERSITY of lOANNINA, 1981 4.
G. Kallistratos and V. Kalfakakou-Vadalouka, Introduction of new varieties of trees
in Greece. Modern Agriculture Technology 9 (1982) 37 5.
G. Kallistratos, A. Evangelou, A. Donos and E. Fasske. Nutrition and
Carcinogenesis. Materia Medica Greca 12 (1984) 213 6.
G. Kallistratos and U. Kallistratos. The up to date experience with the Kallidendron
system for the growth of fruit trees in arid and desert areas. Modern Agriculture Technology (Gr), 26 (1985) 45 7.
G. Kallistratos and U. Kallistratos. The application of innovative methods for food
production in arid and desert areas. XXVI. Intern. Congress of Military Medicine, Marrakesh, Maroc March 23-28, 1986, Published in International Review of the Armed Forces Medical Service 59 (1986) 159. 8.
G.
Kallistratos,
Kallidendron-Verfahren.
Obstbaueme
gegen
Hunger,
Naturwissenschaftliche Rundschau 40(1987)300. 9.
G. Kallistratos, Erfahrung bei der Anwendung von Wasserabsorbens (HYDROGEL)
fuer die Aufforstung Arider Regionen und Wuestengebieten. Arab Technology 6 (1987) 59 10. G. Kallistratos, New developed methods for food production in arid and desert areas. Arab Technology 7 (1988) 20 11. G. Kallistratos and U. Kallistratos, Les Arbres Fruitiers centre la Fain. ATHENA (1988) 64 12. G. Kallistratos and l.-M. Kallistratos. Wachstumspolitik. Die Loesung gegen das Vordringen der Wuesten, Innovatio (1988)46. 13. G. Kallistratos and U. Kallistratos. Fruit trees against Hunger, Journal of Oriental and African Studies, 1 (1989) 134 14. G. Kallistratos and A. Papadopoulos. The Xeroponic (Dryponic) Kallidendron method as an alternative solution for growing trees and vegetables in dry and arid regions of the World. 7th International Congress on soilless culture. Frevohof-Holland 13-21 Mai, 1988. Proceedings p. 243 15.
G. Kallistratos, A. Evangelou, A. Donos and V. Kalfakakou. Nutrition and Cancer.
SEAMEO TROMPED 2nd Seminar on Nutrition. Jakarta/Indonesia, 20-21 March 1989. Proceedings pages 176-191 16.
G. Kallistratos. Plantation of roofs and other arid places with fruit trees by applying
the Kallidendron method, as a contribution to reduce the smog of the City of Athens. Modern Agriculture Technology 4 (1990) 97 17. G. Kallistratos, A. Evangelou and D. Karabetzos, Food Product! ^n in arid areas.
2022 (Gr) Nutrition and Diet 2 (1990) 20 18. G. Kallistratos and P. Drakatos. Reduction of the Environmental Pollution through the Exploitation of the solid and liquid waste in the Kallidendron technology, in Environnnental Contamination, 4th International Conference. Barcelona-Spain, October 1990, Congress Report pp. 398 19. G. Kallistratos and l.-M. Kallistratos, The Kallidendron method as a contribution to the 2nd Green Revolution. Journal of Oriental and African Studies, 3 (1991) 194 20. G. Kallistratos. Hao Bing ZhI Shu Fa (Chinese) Journal of Inner Mongolia Forestry College 14 (1992) 93 21. M. Levidi, North-South, a common future. Hellinas (1988) 4 22. H.F. Liskens. Plastic zak voor tropische plantenteelt. Biovisie (Vakblad voor Biologen) (1988) 68 23. G. Merlkas. Problematic Abandoned areas in Greece and possibilities for their development. Reports Athens Academy of Science 58 (1983) 192 24. J. Papadakis. An interesting method for planting trees. Report Athens Academy of Science 62 (1987) 555 25. C. Tsolodimos. Ein Wissenschaftler ueberlistet mit einfachen Mittein die Natur. Volksblatt Berlin 8. December 1985 26. J. Antonopoulos. Trees in bags for the desserts (Gr) Kyriakatiki Eleftherotypla 29 June 1986 27. Papa Mor Sylla, le "Kallidendron" reduit sensiblement la consommation en eau des plantes. "Le soleil", Dakar SENEGAL 22.8.1986. 28. Anonlmous. Enfin une decouverte interessante. "La Nation" Djibouti 16. 4.1987 29. A. Weig, Ein griechischer Professor will die Wueste fruchtbar machen. Frankfurter Allgemeine Zeltung, 13. October 1987 30. H. Schwabe. Der Mann, der gruenes Leben in die Wuesten der Welt bringt. Hamburger Abendblatt 13 July 1987 31. Personalien, Georgios Kallistratos. "Natur" November 1987 32. C. Velazquez-Padron. El "Sistema Kallidendron". Diario de las Palmas, 10 de Junio 1988 33. Matthaeus Gemeinde Hamburg, Baeume gegen den Hunger 10(1988) 34. C. Michels. An/ores fruiferas a fome. Correis Ris. 19.10.1988 35. C. Velazquez-Padron. Alternativas para cultivar en zonas secas. "Canarias" 13.11.1988 36. M. Katsanopoulou. Athens can be transformed to a wood (Gr). Vima 3.12.1989 37. Anonimous, Hoffnungsbaeume fuer unfruchtbare Oedgebiete, "Heimat-Echo" 31.10.1990 38. G. Koehler. Mit Klaerschlamm und dem Kallidendron-Verfahren gegen die Ausbreitung der Wuesten und gegen den Hunger in der Dritten Welt. Holz-Zentralblatt Nr. 128, 24 October 1990
2023 39. M. Grohe. Eine Birke auf einem Schornstein, Beweis fuer den geringen Wasserbedarf von Baeumen. Holz-Zentralblatt, Nr.130, 29.10.1990 40. M. Tsintilla. The Moses of the 2.000 is the father of the "Kallidendron method". Eleftheros Typos (Gr) 2 July 1991 41. The Science and Technology Commision of Inner Mongolia. Kallidendron Method In Inner Mongolia, an Afforestation method invented by greek prof. Kallistratos, Report December 1992 42. S. Leyva Chinchay. Riego, Nuevo sistema para el desierto, Reyista Agroconsultas No 165, Setiembre 1993 Lima Peru.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2025
THE ESSENTIAL OIL OF ALLIUM SATIVUM L. , LILIACEAE (GARLIC) Nadim A. Shaath*, Ph.D. and Frederick B. Flores Research and Development Laboratory, KATO Worldwide, Ltd., Mount Vernon, New York, 10553 USA and Mohamed Ositian and Mohamed Abd~El Aal Research and Development Department, KATO Aromatic, Harraneya, Cairo, Egypt Abstract The essential oil of Allium Sativum L, ^ Liliaceae (Garlic) obtained by steam distillation from Egypt was analyzed and identified by Capillary Gas Chromatography-Flame Ionization Detection (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) . Over 40 constituents were detected and more than 95% were identified as sulfur containing compounds. Diallyl trisulfide, diallyl disulfide, methyl allyl trisulfide, methyl allyl disulfide and diallyl sulfide were identified as the main constituents. Commercial, technical and statistical data are also presented. Introduction For centuries garlic has been used as a medicine, a condiment and dietary staple, and in several cultures against the evil eye.-*- Egyptian papyrus dating earlier than 1500 BC listed garlic as a cure for many ailments and, in fact, six garlic bulbs were found in King Tut's tomb.^ In ancient Greece, and on the same island, Cos, that the 8th International Flavor Conference was being held, Hippocrates had recommended garlic prescriptions for a number of ailments, as did Aristotle. The Roman naturalist, Pliny, listed many uses for garlic, including driving away scorpions, disinfecting dog bites, curing leprosy, asthma and epilepsy.-^ According to the Talmud, the eating of garlic, "satiated hunger, brightened up the face, improved circulation, killed parasites, kept the body warm and enhanced conjugal love"."^
2026 The Irish, Danes and Russians used garlic for centuries as a treatment for coughs and colds. Louis Pasteur first documented in 1858 that garlic kills bacteria. During World War I, the British Army used garlic to control infections in wounds and doctors in Russia during the war used garlic when they ran out of penicillin and sulfa drugs.^ Recent studies have indicated that garlic can reduce cholesterol, inhibit blood clots, ease asthma, act as an antioxidant^''^ and help prevent heart attacks and strokes.^ Compounds in both onion and garlic had a significant hypolipemic effect on albino rats, reversing a Vitamin D2 induced hyperlipemia.^^ Cancer researchers have found that compounds in garlic can inhibit the growth of tumors in mice and rats. See Table I. (Ref. 8-11). Table I Various Uses of Garlic Cosmetics Clears complexion Culinary Condiments Flavor Enhancer Meat seasoning Antioxidant Non-toxic Repellent Repels birds Some animals & Insects Also Vampires 1
Medicinal Anemia Antibacterial & antifungal Antibiotic properties Anticancer Antitumor Diabetes & hypoglycemia Headaches Heart disorder Hypolipemic Intestinal disorder (Ulcer) Rheumatic diseases Wounds & bites
Garlic pills already have a major following in Europe and more recently in the USA. They achieved their popularity in Europe in part due to clinical studies reporting that the pills can help reduce cholesterol levels. In Germany, claims are made that garlic pills are their top-selling tablets, outselling even aspirin.^^ A number of patents and procedures have been reported on the removal of the non-desirable pungent odor of garlic, providing better consumer acceptability. •'•^'•'•^ Garlic has, however, been affectionately called the "stinking rose" 16 It reeks with healing properties!
2027 Physical Description Garlic belongs to the Lily family which also includes onions, chives, leeks and shallots. ^^ It is a small, hardy perennial bulbous plant of the Amaryllidacae family, allied to the common onion. The strong odor, pungent tasting bulbs is composed of several bulblets, enclosed within a tough outer, whitish skin. See Figure 1.
Figure 1. Garlic - a perennial herb with a bulb divided into segments or cloves. Derived from the Anglo-Saxon word "Garleac", from Gar (which means - a spear) & Leac (-a leek), supposedly meaning "A leek with cloves-like spearheads,"^^ It is known that the major volatile constituents of Garlic are not originally present in the intact cloves. It was found that a colorless, odorless, and water-soluble flavor precursor Alliin - the main active constituent - is present in the intact cells of Garlic. On injury to the cells, the enzyme allinase reacts immediately with alliin to produce the sulfur-containing compound allicin. Allicin is very unstable and is very dependent upon temperature and pH (inactive at pH lower than 3.5). Allicin is further broken down into the strong smelling components, most notably diallyl and allyl diand tri-sulfides, which possess the characteristic odor of garlic. ^"^ See Figure 2.
2028
0 II
0 II
NH2 I [allinase]
CH2=CH-CH2~S-CH2-CH-COOH
>
CH2=CH-CH2-S-S-CH2-CH=CH2
alliin
allicin
i allyl disulfides + allyl trisulfides Figure 2. Enzymatic components.^^
breakdown
of
alliin
to
its
odorous
Garlic oil, allium sativum, is obtained by the steam distillation of the crushed bulbs or cloves of the garlic plant. It is a clear, pale yellow to reddish brown liquid bearing the characteristic pungent, acrid, aromatic odor of garlic.2^ The yields vary between 0.09 to 0.12%, depending upon the cultivation periods and other factors. The imports of garlic and onion oil in 1992 by country of origin is listed below as obtained from the FAS/US Department of Agriculture publication.22 Table I I United Sates: I m p o r t s of G a r l i c and Onion O i l by C o u n t r y of o r i g i n (1992) ORIGIN
i n Kgs
China Colombia Egypt France Hong Kong Italy Mexico Netherlands
261.00 104.30 300.80 23.90 55.40 459.10 850.80 30.50 TOTAL
Source:
US Department of A g r i c u l t u r e ^ ^
2085.80
2029 Studies on garlic oil began as early as 1844 when Wertheim reported that it consisted chiefly of diallyl disulf ide .^-^ In 1945, Cavalito and his co-workers clearly demonstrated that allicin, the principal component, and the volatile sulfur compounds of garlic, where actually secondary compounds formed by the enzymatic action on precursors in the intact bulb.^^ For an excellent review on the sulfur chemistry of garlic and its biosynthesis, the reader is refered to the publication by Professor Eric Block and the references cited therein.^^ Composition of Egyptian Garlic Oil Egyptian garlic oil was analyzed by Capillary GC, using an The instrumental conditions used in FID detector and GC/MS. the analysis are given in Table III. Table III The GC and GC/MS Instrumental Conditions GC CONDITIONS Sample size: O.SOjil Oven Temp.: 65°C to 250°C Program Rate: 4°C/minute Injection Port Temp.: 210°C Detector: FID Carrier Flow: Iml/minute Split Ratio: 150:1 Attenuation: 32 Threshold: 1
GC COLUMN 50m X 0.20mm x 0.33|im (Ultra 1) Hewlett Packard Cross-linked Methyl Silicone Gum Phase Column & a 60m X 0.25mm x 0.25|im (CBX20) Supelcowax 10 INSTRUMENT Perkin-Elmer Autosystem GC II and Hewlett Packard 5840 GC
GC/MS CONDITIONS GC/MS COLUMN Sample size: 0.05fil 50m X 0.20mm x 0.33|im (Ultra 1) Hewlett Packard Cross-linked Oven Temp.: 65°C to 250°C Methyl Silicone Gum Phase Column Program Rate: 4°C/minute Injection Port Temp.: 210°C Detector: TIC INSTRUMENT Carrier Flow: Iml/minute Combined Perkin-Elmer 8500GC Split Ratio: 100:1 Finnigan Ion Trap Detector and Attenuation: 64 Hewlett Packard 5870 MSD Threshold: 1 MS Ionization Voltage: 70eV Scan Time: 67 minutes Start-stop Masses: 35-400 Electron Multiplier Voltage: 2000eV
2030 The main components found in Egyptian garlic oil are diallyl trisulfide, diallyl disulfide, methyl allyl trisulfide, methyl allyl disulfide and diallyl sulfide. See Table IV and Figures 3-5 for the mass spectrum of the top three chemical components of the oil. In addition to the major constituents listed, the following were also found in trace amounts: aniline, 1,2 epithiopropane, 2,4-dimethylfuran, 1-hexanol, methyl thio sulfide, 2,5~dimethylthiophene, 2,5dimethyl tetrahydrothiophene, dipropyl sulfide, 3-methyl-2cyclopenten-1-thione,1,3-dithiane, 1,2-dimercaptocyclopentane, 4-methyl-5-vinyl thiazole, 2-methyl benzaldehyde, 3,5-diethyl1,2,4-trithiolane, allyl-1-propenyltetrasulfide, dipropyl tetrasulfide, myristic acid, pentadecanone, palmitic acid, linoleic acid.^^'^'^ Table IV The Main Chemical Constituents of Egyptian Garlic Oil CHEMICAL CONSTITUENT 1-Propene Methanethiol Allyl thiol Methyl allyl sulfide Dimethyl disulfide Hexanal Diallyl sulfide Methyl allyl disulfide cis-Methyl propenyl disulfide trans-Methyl propenyl disulfide Dimethyl trisulfide 2,4-Heptadien-l-al Diallyl disulfide Allyl propyl disulfide ^6^10^2 (Tent ID) Methyl a l l y l t r i s u l f i d e 3-Vinyl-4H-l,2-dithiin 2-Vinyl-4H-l,3-dithiin Diallyl trisulfide I s o b u t y l i s o t h i o c y a n a t e ( T e n t ID) D i a l l y l t e t r a s u l f i d e (Tent ID)
AREA% 0.02 0.01 0.09 1.07 0.24 0.03 3.01 3.81 0.06 0.15 0.80 0.01 23.95 0.48 1.22
- 0.04 - 0.03 - 0.41 - 1.84 - 0.71 - 0.05 - 5.89 - 6.83 - 0.08 - 0.19 - 1.39 - 0.03 - 34.25 - 1.26 - 3.41
11.02 0.15 0.14 29.22 0.12 0.48
- 17.68 - 0.20 - 0.16 - 39.77 - 0.16 - 0.82
Minimum and maximum values a r e r e p o r t e d for a r e p r e s e n t a t i v e sample of 10 l o t s from October '93 t o A p r i l ' 9 4 , run through a Gas Chromatograph using a non-polar c a p i l l a r y column d e t e c t e d by FID.
Gas Chromatographic a n a l y s i s of g a r l i c o i l may vary whether an FID or an EC d e t e c t o r i s used. FID i s known t o be l e s s
2031 In an sensitive in its response to di- and tri-sulf ides. experiment by Oaks et al. where both detectors were used with a dual-channel system splitting the effluents equally to both detectors, response ratios of di- and tri-sulfides were greater than unity.^^ In addition, gas chromatographic analysis in general tend to distort the actual percentages and components found in products, due to the thermal decomposition encountered in the injection ports and high temperatures in the gas chromatograph.^^
Diallyl disulfide
mv,
113
CeH-,oS2
MW=146
INT
71 79
137
59 1,1 I 85
. il I. 1
T' I ' I ' '
40
Figure 3.
60
l| I 'l,.,"l' I ' I ' I 108
80
179
' I ' I -'I ' I ' I ' I ' 1 ' I
120
140
180
U0
The mass s p e c t r a of d i a l l y l
disulfide:
Diallyl trisulfide 100X
87
^^r^,^^ SSS \ ^ ^ . x ^
C6H>,oS3
MW=178
INT
HI
59 64 7 7 I ' I' I
40 Figure 4.
60
119
13?
153
I'I ' I ''' I ' I ' I ' ' . I''
80
100
120
t
•
I
140
1
*
1
—
160
The mass spectra of diallyl trisulfide
2032
Methyl allyl trisulfide 188*/
41
C4H8S3 113
IKI
105
62
146
f ' I ' V''."i"l r ' I •' T " ' I ' ' I " '
40
Figure 5.
60
80
100
I
I ' I ' I '" I ' I
120
140
160
The mass s p e c t r a of methyl a l l y l t r i s i i l f i d e .
9
UiUl
MW=152
R
2
— I — I — 1 — I — I — I
I—I
1
I
I
I
I
I
I,
I
Figure 6. Capillary Gas Chromatogram of Egyptian Garlic Oil using a non-polar capillary column and detected by Flame Ionization Detector.
2033 We have observed changes in both the profile of the gas chromatographed oil and in the actual percentages of the components present in garlic oil. High Performance Liquid (HPLC)^^'-^^ is a recommended additional Chromatography technique for the thorough analysis of components of garlic oils. Due to the lower temperature requirements of this technique, fewer degradations occur in the particularly vulnerable and labile sulfurous components found in garlic oils. From a quality control perspective, GC analysis is suitable and convenient, however, it is crucial that all parameters of this analytical technique be standardized and constantly monitored and optimized. It is worth noting that garlic oil is also produced in China and Mexico. The Mexican variety we analyzed was of questionable origin and that of China possesses different physico-chemical properties and generally has inferior organoleptic qualities. Table V lists the comparative analysis of Egyptian, Chinese and Mexican garlic oil. Values given are averages of several representative lots from 1993 to 1994. The geographical origin of the oil can be characterized by examining the contents of diallyl trisulfide, diallyl disulfide, methyl allyl disulfide, methyl allyl trisulfide and diallyl sulfide. Table V Comparative Chemical Composition of Random Garlic Oil Samples of Various Geographical Origins CHEMICAL CONSTITUENT Allyl thiol Methyl allyl sulfide Dimethyl disulfide Diallyl sulfide Methyl allyl disulfide Dimethyl trisulfide Diallyl disulfide Allyl propyl disulfide ^6^10^2 (Tent ID) Methyl allyl trisulfide Diallyl trisulfide Diallyl tetrasulfide(Tent ID)
Chinese
Egyptian
Mexican
0.15 0.64 0.23 4.25 2.23 0.18 28.60 0.57 0.91 6.77 50.92
0.21 1.21 0.41 3.69 5.37 1.30 27.45 0.74 1.92
0.14 3.15 0.88 6.96 15.25 1.44 42.46 0.28 0.03
16.82 35.30
10.36 12.52
0.74
0.70
1.09
2034 Egyptian Production at KATO-Egypt Since June 1993 and until now, KATO has produced large quantities of garlic oil with varying yields as shown below: Months Yields
May '93 0.09%
Aug '93 0.12%
Dec '93 0.11%
Jan-Mar '94 0.09%
The data listed above corresponds to the period when the distillation of the garlic itself occured not when it was cultivated. Garlic is generally cultivated in Egypt in April and May. The variation in the yields shown above are a function of three parameters: 1. 2. 3.
The moisture content of the bulbs The cultivation time The period of storage of the bulbs
Given the high levels of moisture in the bulbs when harvested, the yield in May is generally low. As the humidity decreases during storage of the garlic bulbs, the yield is increased as shown in August through December. Later on in the season the yields also decrease again due to the evaporation of some of the volatile constituents of the oil. KATO maintains extensive storage facilities for the different blends and productions of garlic oil to maintain a consistent quality and purity for its customers. The quantitative GCanalysis of Garlic oil production from June 1993 to March 1994 are given in Table VI. Table VI Analysis of Garlic oil production at Kato-Egypt from June'93 to March'94 CHEMICAL CONSTITUENT
Jun.
Jul.
Aug.
Sept.
Oct.
Nov.
Dec.
Feb.
Mar.
Methyl allyl sulfide
0.96
0.82
0.98
1.06
1.12
1.11
1.18
1.12
1.22
Dimethyl disulfide
0.21
0.22
0.27
0.28
0.30
0.31
0.34
0.32
0.35
Diallyl sulfide
3.78
2.98
3.12
3.18
3.55
3.35
3.45
3.29
3.36
Methyl allyl disulf.
5.23
4.93
5.22
5.65
5.88
5.87
6.45
6.65
7.00
0.83
1.07
1.22
1.38
1.36
1.41
1.38
1.37
1.25
33.73
28.36
27.47
28.40
29.94
29.42
30.77
32.05
33.12
Methyl allyl trisulf 13.90
16.16
17.07
17.60
17.15
17.63
16.37
16.15
15.38
37.29
41.96
39.51
37.97
35.62
35.79
33.06
30.55
30.00
Dimethyl trisulfide Diallyl disulfide
Diallyl trisulfide
The data was gathered from a minimum of 2 lots for each month analyzed, run through a polar (carbowax 20M) column and detected by FID.
2035 The physico-chemical porperties of the Egyptian Garlic oil produced conform to the Food Chemical Codex specifications listed below.^2 Refractive Index at 25°C Specific Gravity at 20°C
1.574-1.579 1.076-1.092
1.559-1.579 1.040-1.090
Effects of storage on the quality of Garlic Oil In order to follow changes occurring in the constituents of garlic oil during storage, KATO analyzed a sample of garlic oil by GC at three months intervals. The ratio between sulfur compounds in garlic oil was found to undergo significant and rapid changes during its storage when exposed to light and in half-filled containers. Results are given in Table VII below: Table VII Effects of storage on Garlic Oil CHEMICAL CONSTITUENT
Dec. 1993
Mar. 1994
Methyl allyl sulfide Dimethyl disulfide Diallyl sulfide Methyl allyl disulfide Dimethyl trisulfide Diallyl disulfide Methyl allyl trisulfide Diallyl trisulfide
1.18 0.34 3.45 6.45 1.37 30.77 16.37 33.02
0.95 0.41 3.58 8.07 0.96 37.36 13.16 27.10
Total: Total mono- sulfides: Total di- sulfides: Total tri- sulfides:
92.95 4.63 37.56 50.76
91.59 4.53 45.84 41.22
Total sulfide contents are generally conserved throughout the storage period. Di-sulfides increase during storage while the levels of tri-sulfides decrease. Monosulfides levels remain relatively constant. Therefore, it is possible that an oxidation of the tri-sulfides takes place as shown below:
RSSSR
+
O2
->
RSSR
+
SO2
2036 The proposed mechanism is the following:
RSSSR 4- O2
0 II > RSSSR II 0
=
RSSR + SO2
Little is known about actual processes which cause changes of an essential oil. Usually it is attributed to such general reactions as oxidation, resinification, saponification, polymerization or hydrolysis. These processes are activated by heat, presence of air, moisture and is catalyzed by exposure to light.^-^ Acknowledgments The authors would like to acknowledge the support of the members of both Instrumentation and Quality Control Departments at KATO Worldwide. References * Author to whom all correspondence should be addressed. 1. Studies show garlic reeks with healing properties. By Mary Esch, Daily Bulletin, Mar. 2, 1994, p A12. 2. The Cultivated Gardener; What vegetable besides garlic has a fan club? By Anne Raver, The New York Times, 1991, p 68. 3. M. Esch, Ibid, p A12. 4. P. Airola, Ph.D., The Miracle of Garlic, Health Plus Publishers, 1978, p 15. 5. Ibid. 6. H. Michasu, Jpn. Kokai Tokkyo Koho JP 05,268,906 [93,268,906] (CI. A23L1/30), Oct. 19, 1993. 7. N. Fu, L. Huang, L. Quan, and L. Yan, Zhongguo Yixue Kexueyuan Xuebao, 1993, 15(4), pp 295-301. 8. M. Esch, Ibid, p A12. 9. J. Valnet, M.D., The Practice of Aromatheraphy: Holistic health and the essential oils of flowers and herbs. Destiny Books, New York, 1980, pp 128-132. 10. Garlic: Infection Fighter, by R. McCaleb, il v55. Better Nutrition for Today's Living, August 1993, p 46(3).
2037 11. Big whiffs of garlic repulse insects, too. (nontoxic insect repellents) by R. Tomsho. The Wall Street Journal, August <6, 1991 pp Bl (E&W) . 12. M.H. Abo-doma, M.M. Said, N.E. Shahat, R.F. Riad, and A.R. Nassar, J. Drug Res., 1991, 20(1-2), pp 1-11. 13. After Granola, What? Lichtwer's Little Garlic Pills? by Eben Shapiro, The New York Times, Apr. 5, 1992, p 8. 14. H. Nishimura, Gekkan Fudo Kemikaru, 1993, 9(12), 73-6. 15. J. Morinaga, Jpn. Kokai Tokkyo Koho JP 05,304,924 [93,304,924] (CI. A23L1/212). 19 Nov. 1993. 16. A. Raver, Ibid, p 68. 17. Flavor makeup of onion, garlic revised (Thiosulfinates provide onion and garlic flavors) V143 Science News, Feb. 13 1993, p 110(1) . 18. The Illustrated Encyclopedia of Herbs. Their Medicinal and Culinary Uses, Ed. by Sarah Bunney, Dorset Press, 1992, p 60. 19. L. Lawson and B. Hughes, Planta Med., 1992, 58, pp 345350. 20. E. Gunther, The Essential Oils, Vol III, D. Van Nostrand Co., 1948, pp 67-69. 21. U.S. Essential Oil Trade, May 1993, Foreign Agricultural Service, United States Department of Agriculture, Washington, D.C. 22. Fenaroli's Handbook of Flavor Ingredients, 2nd edition, ed. T.E. Furia and N. Bellanca, CRC Press, Inc. 1975, Vol I, p 359-360. 23. T. Wertheim, Ann. Chem. Pharm., 1844, 22, 193-200. 24. C.J. Cavallito ,J.H. Bailey and J.S. Buck, J. Amer. Chem. S o c , 1945, 67, 1032-1033. 25. E. Block, Angew. Chem. Int. Ed. Engl., 1992, 31, 1135-1178. 26. L.Jirovetz, W. Jaeger, H.P. Koch, and G. Remberg, Z. Lebensm.-Unters. Forsch., 1992, 194(4), pp 363-365. 27. G. Mazza, S. Ciaravalo, G. Chiricosta, S. Celli, Flavour Fragrance J., 1992, 7(3), pp 111-116. 28. D.M. Oaks, H. Hartmann and K.P. Dimick, Analysis of sulfur compounds with electron capture hydrogen flame dual channel chromatography. Anal. Chem., 1964, 36, p 1560. 29. N. Shaath, P. Griffin and G. Andemicael, Sunscreens: Development, Evaluation, and Regulatory Aspects (eds. by N. Lowe and N. Shaath), Marcel Dekker, New York, 1990, pp 509-510. 30. L. Snyder and J. Kirkland, J. Introduction to Modern Liquid Chromatography, 2nd Ed., Wiley, New York, 1979. 31. C. Horvath, High Performance Liquid Chromatography, Advances and Perspectives, Academic Press, New York, 1980. 32. Food Chemical Codex, 3rd Ed.; Food and Nutrition Board, National Research Council, National Academy Press, Washington, DC, 1981, p 132. 33. S.I. Balbaa, Medicinal Plants Constituents, Egyptian Dar El Kotob, Cairo, 1976.
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G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2039
MICROBEAM MOLECULAR SPECTROSCOPY OF BIOLOGICAL MATERIALS
David L. Wetzel, Kansas State University, Microbeam Molecular Spectroscopy Laboratory, Shellenberger Hall, Manhattan, KS 66506. SUMMARY: Molecular vibrations of microscopic areas of biological tissue specimens are observed and measured to provide in situ, highly localized, chemical information. Fourier transform infrared (FT-IR) microspectroscopic interrogation of even single cells is done. Spectra at multiple points within single cells provide images for select wavelengths representing different chemical (functional group) distributions. Spatially resolved microspectroscopy allows study of heterogeneous materials and superimposition of chemical microstructure onto morphology. Recent ultra-spatial resolution has been achieved using a bright infrared source from a synchrotron (National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY) coupled with the Spectra-Tech, Inc. IRjis^"^ scanning infrared microspectrometer. The combination of these powerful tools results in working at the diffraction limit with high signal/noise to obtain good spectra using 6 x 6 micrometer double aperturing and coadding only 16 scans. Individual adjacent cells can be readily interrogated one at a time to seek out chemical differences. INTRODUCTION In biological specimens, the chemistry of single cells can be studied in situ using an infrared spectroscopic probe of cellular and subcellular dimensions. Whereas conventional microscopy has been a most useful tool to establish physical microstructure, microbeam molecular spectrometry offers the means of providing chemical microstructure to complement the former and assist in characterization of the tissue. New technologies have made it possible to combine chemical imaging with structural information. In light microscopy, electronic spectroscopy has been useful where natural pigments exist or where contrast could be produced by the application of stains or fluorescent materials. In vibrational spectroscopy, in particular the mid-infrared region of the spectrum, the use of chemical reagents or stains is not necessary. In 1994, an optically efficient, integrated, infrared microspectrometer was first coupled to a very bright, synchrotron infrared radiation source. This recent development has allowed the use of smaller apertures (6 x 6jLim) with high performance and reduced scanning time. This could be the Utopian goal for which practicing microspectroscopists have hoped.
2040 Spatial resolution of individual cells or other morphological parts of a tissue thin section in the field of an infrared microscope is possible with state-of-the-art equipment. In later generation IR microspectrometers, when the tissue of interest is located by observation with white light, the spectrometer is put into action and mirrors divert the white light and allow infrared radiation to go through an apertured portion of the tissue and collect the infrared spectrum of that particular tissue. This is done in situ without any prior physical or chemical separation. The area of tissue to be scanned is restricted by apertures positioned before both the front surface optical objective and condenser of the infrared microspectrometer. Chemical analysis and microscopy coincide one point at a time in the microscope field. The information from these analyses then can be pooled to get the profile of the entire specimen. Scanning a programmed sequence of spots in a distinct pattern can be used to obtain, unattended, the spectrum at each spot. As information is pulled from the grid for a particular wavelength, a topographical (contour) map is produced for the entire specimen with respect to its localized absorbance at that wavelength. The result is a reconstructed microscopic image that refiects the spectroscopic response for a particular absorption band in the infrared spectrum. Thus, we obtain chemical information for a specific microstructural portion of the specimen. Organic compounds produce infrared fingerprints, making infrared spectroscopy an ideal tool for qualitative analysis. A class of compounds containing certain fiinctional groups is identified by characteristic absorption bands that occur in a particular region of the spectrum. Thus, chemical differences between subsamples within the microscopic field can be illuminated. Relative magnitude of absorption due to a particular APERTURE CASSEGRAINIAN OBJECTIVE (ON AXIS)
functional
quantization, in the experiments that are described. For more than forty years, since World War II, the field of infrared spectroscopy has progressed. During
STAGE
group is used, at least for semi-
much
of that
time,
the
desire
for
microsampling and attempts to obtain infrared microscopic data have been severely frustrated. Experimental limitations have been mostly from
CASSEGRAINIAN CONDENSER (ON AXIS) APERTURE
lack of sufficient infrared energy going through the microscope optics to be sorted out by a conventional dispersive in
infrared
infrared
(interferometric
spectrometer.
spectroscopic optics
and
Advances
instrumentation fast
Fourier
transformation data treatment) have made it possible Fig. 1. Cassegranian objective and condenser optical diagram showing projected aperture before the objective and after the condenser.
to have a high energy throughput spectrometer. It , , , ,, , , i i- i ^as also been possible to condense that high throughput SO that it can traverse a specimen of
2041
Fig. 2. Photograph of the Fourier transform infrared microspectrometer (IR|as™ ) as configured at the Microbeam Molecular Spectroscopy Laboratory of Kansas State University: the IR microscope/IR spectrometer is in the center, programmable microscope stage controller (left), video camera (top) transmits video to the monitor closest to the IRjus™ showing an image of the whole microscope field with the projected aperture (bright spot) superimposed, master computer (right), computer screen (farthest from IRjis™) with spectra displayed, and plotter. Slave computer (not shown) controls the microscope/interferometer/stage and data acquisition. A service module with power supplies and IR source cooling unit comes with the above system but is not shown.
microscopic dimensions. The slit required in dispersive infrared instruments previously limited the energy or allowed passage of high energy only at the expense of spectroscopic resolution. With an interferometer-based spectrometer and Fourier-transform (FT) calculations with rapid low-cost computers, it is possible to access the valuable technique of infrared microscopy conveniently and cost effectively. In the past decade, front-surface-optics (Figure 1) infrared microscopes have been produced as standard FT-IR sampling accessories available for use on all FT-IR instruments. Their use, however, is enhanced if a small detector area (approx. 0.25 mm) is used to match the beam dimensions used in microspectroscopy. This author previously reported on the use of such an accessory to examine wheat crossections and milling fractions (1) in addition to thin
2042
sections of various foodstuffs (2). More recently, a moving stage made it possible to have programmed acquisition from different spots throughout a particular tissue. Still more recently, an integrated system, the IRjis™ molecular microspectrometer, was produced commercially by Spectra-Tech, Inc. (Shelton, CT). We subsequently reported on the integrated instrument and its use in the mapping of wheat and com thin sections in a transmission mode (3). The integrated design is optically more efficient than previous accessory infrared microscopes attached by way of an optical interface to a conventional FT-IR spectrometer. It combines an IR microscope and an FT-IR spectrometer, optimizing the optics. Control of the sample positioning and data acquisition has been improved with the use of separate computers for the microscope and spectrometer. Details of this state-of-the-art equipment (including diagrams) appear in a later section on instrumentation. Now we can have both spatial resolution and spectroscopic resolution and retain excellent signal-to-noise characteristics. This specialized instrument is designed to allow a microscopist to collect spectroscopic data. In general, microscope accessories for FT-IR spectrometers are designed from the spectroscopist's point of view. The IRjis™ is designed as a spectrometer for use in microscopy. Control software is of the point and click menu-driven format. The optics of the molecular microscopy system incorporate an infrared microscope as an integral part of the interferometer bench of an FT-IR spectrometer. In the IRjis™ system, the interferometer bench of a Nicolet (Madison, WI) FT-IR spectrometer has been placed in a vertical position, and the microscope-retaining parts are structurally integrated with the housing of the interferometer. Obtaining a good clear infrared spectrum of a small spot in the field of the microscope provides fundamental chemical information concerning that target tissue. Infrared spectroscopy involves fundamental vibrational frequencies, which are determined by the masses of the atoms vibrating and the restoring force, i.e., chemical bond, between those atoms. Infrared measurements are not dependent on the chemical or absorptive interaction of a stain, the production of fluorescence, or the absence of background fluorescence. All organic compounds produce an infi-ared spectrum. Thus, a comparison of spectra obtained from adjacent tissues within the plant material provides the investigator with information regarding the chemical similarity or dissimilarity between those tissues. In our early studies (2) reported in 1989, we routinely sampled multiple cells, granules, or crystals in a lOO-jim-diameter circular aperture and occasionally used rectangular apertures, typically 36 \xm in length. In our subsequent studies (3) on a similar subject, reported in 1994, we routinely did tissue mapping experiments with 24 x 24 jam apertures before and after the specimen. However, spectra were sometimes painstakingly obtained with apertures as small as 3 X 6 |im customized to the target of interest. A single cell map was also obtained using 7 |Lim apertures. In this report of 1994 experimentation, we describe the use of a synchrotronpowered IRjas™ to overcome signal-limited cases and to operate at the diffraction limit to
2043
attain ultra-spatial resolution. We review the progression of instrumentation to achieve the present commercial and research equipment state-of-the-art. We present results of experiments with synchrotron radiation infrared microspectroscopy (SR-IR|j,s) in the areas of single cell interrogation, high density (100%) small aperture tissue maps to reveal localized chemical heterogeneity, and high speed large area mapping of interesting biological specimens. Infrared Spectroscopy The infrared spectrum involving the fundamental vibration and fingerprint region from 4,000-750 cm'^ has long been a useful tool for describing the molecular structure of compounds synthesized in a chemical processor isolated from natural products. In the past, in order to obtain infrared spectra from biological tissue, either extraction, preparation of a homogenate, or both were required to collect enough sample for conventional infrared techniques. From the infrared spectrum, the presence or absence of various organic functional groups is readily observed. The length of hydrocarbon chains is observed by the relative prominence of CH3 and CH2. Also, the aromatic character or the relative amounts of unsaturation may be detected by C=C or C=C-H vibrational bands. Subtle shifts in the frequency of bands give a clue to the chemical environment of the infrared active group being observed. Infrared spectra of isolated separation fractions and subtle differences between them have been observed by chemists in the past four decades. During this time, the infrared spectrum has become more common in the organic chemistry laboratory than the melting point determination. With the use of dispersive infrared A DIRECTION OF MOTION spectroscopy, macrosamples have been routinely observed between alkali halide salt optical windows. In general, samples subjected to infrared spectroscopy were purified prior to scanning in order to allow interpretation. Dispersive Infrared. Energy throughput of DETECTOR dispersive spectrometers is severely limited by the slits of a monochromator equipped with either a prism or diffraction grating. Infrared microsampling required elaborate beam condensers to squeeze the whole beam, one frequency at a time, through a sample of less than 1 mm in diameter. Appropriate referencing (double beam operation) I SOURCE to account for source intensity and detector responses at each frequency was also necessary. Fig. 3. Diagram of a typical Michelson InterferoWith the light-starved optics of a dispersive meter.
v
2044
instrument, it is mutually exclusive to use an aperture small enough to exclude radiation not passing through the sample and have intensity necessary for a favorable signal-to-noise ratio. Dispersive microspectrometry then could be either not very small or not very good spectroscopically. Thus, in the past, the utility of this analytically valuable part of the spectrum has been restricted by practical functional considerations. In spite of this dravs^back, painstaking pioneering work was accomplished, leading the way to our modem efforts in the field. Interferometric Infrared. In the past decade, interferometers have largely replaced dispersive spectrometers (4). In FT-IR spectroscopy, the interferometer used allows all frequencies to pass through the sample simultaneously (Fellget or multiplex advantage), and the absence of slits provides greatly enhanced optical throughput (Jacquinot advantage). Figure 3 shows the generalized diagram of a Michelson Interferometer. The radiation is divided by a half-silvered mirror into two pathways and ultimately reunited after traversing the alternate pathways. In the one case, the length of the pathway is fixed by a permanently positioned mirror and, in the other case, a moveable mirror adjusts, as a function of time, the path of radiation. When a particular frequency traversing the moveable path adjusts so that the the right distance is reunited with the same frequency of the fixed path, a constructive interference occurs, and a maximum response is produced at that frequency. Thus, as a function of time of the oscillation cycle, a cosine function of path length differences is related to optical frequency, and an interferogram is produced. Multiple oscillations of the mirror generate a summation interferogram to produce suitable signal/noise. In this way, the raw data are accumulated from which a spectrum may be produced using mathematical operations and appropriate ratioing of sample to reference. The design and manufacture of FT-IR spectrometers have progressed to the point that they are used not only by researchers needing a high resolution system, but they are also available to routine users in industry and education. FT-IR spectrometers for obtaining spectra of moving samples on-the-fly are sold as accessory detectors for gas chromatographs. All of this progress has been made possible by a number of technical advances. These include optical verification of the mechanical motion (assuring absolute frequency reproduction) by interference fringes of a laser parallel to but offset from the infrared beam of the interferometer. Another advance is the use of a solid state mercury cadmium telluride (MCT) detector cooled with liquid nitrogen to greatly enhance the sensitivity and, thus, improve the signal-to-noise ratio. Bringing FT-IR into routine usage, formerly occupied by dispersive IR spectrometers, required the development of low cost digital computers to perform the fast Fourier transformation. Microscopy with FT-IR. Early models of infrared microscopes were reviewed by Coates, one of the pioneers in the field, at a meeting of the Federation of Analytical Chemistry and Spectroscopy Societies (FACSS) in Detroit (5). One monograph has already appeared on the subject of FT-IR microscopy (6), and numerous workshops are being conducted on the subject by instrument companies and by Miami University, Ohio. At least one instrument
2045
company holds annual users' group workshops at national spectroscopy meetings. National meetings of the Microscopy Society of America (MSA) and the Microbeam Analytical Society (MAS) now include symposia and sessions on molecular microspectroscopy. IR microspectroscopy papers appear in Spectroscopy meetings such as FACSS and The Pittsburgh Conference on Analytical Chemsitry and Applied Spectroscopy (PITTCON) in the United States, as well as at the International Conference on Fourier Transform Spectroscopy. Current activity in the field has been made possible by the interfacing of infrared microscopes to existing commercial FT-IR spectrometers. Before the front surface optic microscope accessories were commercially available, it was difficult to obtain a good injfrared spectrum of a sample less than 500 |im in diameter. This was formerly accomplished by masking the sample, so that only radiation from a small area could reach the detector. Reffner (7) discussed the historical development of infrared microscopy. The precommercial experimental work that he cites begins with an article appearing in Nature in 1949 entitled "Infrared Spectroscopy with the Reflecting Microscope in Physics, Chemistry, and Biology" (8). This study was done by Barer, Cole, and Thompson at Oxford University using a reflecting microscope developed by Burch (9). It was mounted on a Perkin-Elmer recording IR spectrometer and used on particles, crystals, and fibers of approximately 20-60 |Lim diameter. Also in 1949, Gore established the feasibility of combining light microscopy and infrared spectroscopy and predicted "increased application in the biological and crystal structure fields" (10). Commercial Infrared Microscope Accessories. Coates, Offner, and Siegler of the Perkin-Elmer Company (Norwalk, CT) designed the first commercial infrared microscopy attachment for IR in 1953 (11). This instrument, after improvement, became the Perkin-Elmer Model 85. John Reffner of Spectra-Tech stated that "while the early researchers demonstrated its principles, feasibility and promise, the development of IR microspectroscopy was halted until computers became available." The NanoSpec2 IR (Nanometrics) single beam instrument (1978) recorded and stored the reference and sample spectra in the computer and generated a ratioed absorption spectrum. This eliminated the atmospheric and instrumental background and recorded spectra in only 2 minutes. From that point in history, in the opinion of Reffner, IR microspectroscopy became a practical analytical technology. FT-IR microspectroscopy and its practical advantage was demonstrated by Muggli (12) in 1982, when he successfully attached a Perkin-Elmer Model 85 microscope to a Digilab FTS-20 spectrometer. Subsequently, in 1983, Analect (Irvine, CA) and Digilab (Cambridge, MA) microscope accessories were introduced, and by 1984, Spectra-Tech (Shelton, CT) and Bruker (Billerica, MA) were involved also. During the 1980's, microscopes became available as accessories for nearly all FT-IR spectrometers.
2046 Optical Considerations. Severe diffraction effects at infrared wavelengths require special consideration, discussed by Messerschmidt (13). A microscope designed specifically for FT-IR focuses radiation on the plane of the sample. The radiation exiting the sample is re-imaged at an adjustable aperture. In this way, it is possible to carefully control the area of the sample observed by the detector. When the dimensions of the aperture approach the wavelength being used, one must be concerned about the effects of diffraction. When performing spectroscopy at a wavelength of 5 jim, one could not expect to use an aperture of 5|im. For any given wavelength, a certain minimum, practical, aperture size exists. In cases where the specimen to be observed is too narrow and does not fill the field of view, stray light may be a problem. Diffraction-induced, unwanted sampling of tissue adjacent to the target may be minimized by the design of the infrared microscope. The work reported here incorporates the principle of dual remote image masking (Redundant Aperturing®). Apertures are located both above and below the sample. The price for having two apertures is a slight reduction in the energy transmitted compared to using one aperture. The reduction in stray light is considered worth the price. Synchrotron Radiation Infrared Microspectrometer. The integrated infrared microspectrometer (IR|Lis™) previously described is optically, relatively efficient. In addition to the mirrors of the microscope and the interferometer, it has incorporated a sensitive detector with dimensions appropriate for the microbeam. However, it is equipped with a conventional globar as a source of infrared radiation. Synchrotron radiation is 100-1000 times brighter than the globar and is also more stable. The synchrotron radiation source coupled with the IR|j,s™ exhibits a high signal-to-noise ratio. Synchrotron radiation has a small viewing angle expressed in milliradians and is typically pulsed at 10"*^ seconds. With this bright beam, the problem of insufficient energy to traverse a sample is eliminated, and because the synchrotron radiation is concentrated into a narrow beam, a much smaller percentage of it falls outside of an aperture. Thus, smaller spot sizes can be readily sampled, and even at these dimensions the signal-to-noise characteristics are such that data acquisition times can be reduced because co-adding fewer scans will produce excellent spectra. Characteristics of sychrotron operation do impose experimental or operational inconveniences, and optical nuances of the synchrotron must be dealt with. SpectrQgCQpic Fgatureg in Biological Material? Infrared absorption bands have been catalogued extensively (14-16). Spectra of biological materials or constituents isolated from them are included to illustrate how differences in chemical composition result in substantially different infrared spectroscopic features. Figures 4-7 are spectra of components isolated from grain. Figures 8-11 (17) are obtained from different parts of plant materials (crossections of grain). Figures 12-13 illustrate chemical differences between different parts of a crossection of a plant leaf Figures 14-15
2047
illustrate differences in the spectra obtained from the same mammalian tissue representing lean and fatty portions. Figures 4-7 (l)(representing a single starch granule, single cellulose crystal, a single gluten particle, and lipid extracted from wheat, respectively) show most of the absorption bands that appear in subsequent spectra. Beginning at the higher frequency we observe the 0-H stretch vibrational bands for both the starch and cellulose ca. 3300 cm'^ The gluten, representing protein has an NH stretch vibration which produces a sharper peak slightly shifted from the ca. 3300 cm"' absorption band of OH. Because lipid contains neither OH nor NH, this spectral feature is absent in the lipid spectrum (Figure 7). CH vibrational bands in the 2927 to 2850 cm"' region are present in all four spectra, but they are particularly predominant in the lipid spectrum in the absence of OH or NH. In the region of 1800-1500 cm"' (from high to low) at 1740 cm', a prominent carbonyl appears in the lipid spectrum (Figure 7). A small peak occurs at the same place in the gluten spectrum indicating that the protein specimen used was not completely purified. The next absorption bands of interest appear in the gluten spectrum, amide I and II, respectively, at approximately 1650-1550 cm"'. Other weaker amide bands are present in the gluten spectrum below 1400 cm"'. An HOH deformation band occurs at ca. 1650 cm"'. This can be seen in both of the carbohydrates. In the portion of the spectrum from 1550-800 cm", strong carbohydrate bands are present m both the starch and cellulose (Figures 4 and 5)
^ y^ |s s « ^
'^ a" i •
|i"
pjg 4 spectmm of wheat starch single granule (i). ^^g- 5- Spectrum of cellulose single crystal (i).
Fig. 6. Spectrum of wheat gluten particle (1). particularly in the region o f 1100-1025 cm"'. Fig. 7. Spectrum oflipid (film) extracted from wheat (l).
2048
Fig. 8. Wheat kernel central endosperm, 20 X 60 mm area, 2 minute scan. Ref.(17).
Fig. 9. Wheat kernel subaleurone endosperm, 6 x 12 mm area, 2 minute scan. Ref. (17).
ir (CM-i)
A major difference between these two carbohydrates is the presence of bands of moderate intensity at approximately 1420, 1370, and 1335 cm'^ in the spectrum of cellulose. In the strong lipid spectrum at 1469 cm"^ we observe CH2 stretch at 2927 cm"^. By being acquainted with the spectral characteristics of starch, cellulose, protein, and lipid, you can look for their presence in mixtures or portions of tissue from plant material. This knowledge now can be applied to spectra (Figures 8-15) of different parts of grain, leaf, and mammalian tissue. The spectrum of wheat endosperm. Figure 8, shows the typical 0-H stretch band at 3300 cm'^ as well as the HOH deformation at approximately 1650 cm"^ The most prominent band is the complex carbohydrate band in the region of 1100 to 1000 cm"^ In this case, the peak is at a maximum at approximately 1025 cm'^ Note also that, in the central wheat endosperm, a small amide II band occurs at 1550 cm"^ The subaleurone region of the wheat kernel is located in between the central endosperm and the aleurone cells. In the
2049
Fig. 10. Scutellum portion of wheat germ. Ref. (17).
'
Fig. 11. Embryo portion of wheat germ. Ref. (17).
i-oo-
(CM-l)'
spectrum (Figure 9) of the wheat subaleurone endosperm, note that in comparison to Figure 8, there is an increase in the amide II band at 1550 cm'^ and a decrease in the carbohydrate band at 1025 cm"^ In the spectrum of the scutellum (Figure 10), which is the oil-bearing tissue of the wheat, the lipid bands are very prominent in terms of the strong carbonyl at 1740 cm"\ and the long chain CH2 has greater detail in the region of 2900 to 2800 cm"^ Unsaturation shows at 3005 cm-' as the stretch of the CH attached to the C=C bond. In the embryo portion of the wheat germ (Figure 11), there is a marked decrease in the lipid content compared to the scutellum, whereas the prominence of the amide bands has increased, as one would anticipate. This is shown in both the amide I band at 1650 and the amide II band at 1550 cm'^ In the leaf tissue, a very active portion of the spectrum occurs at frequencies below 1800 cm^ The two spectra in Figures 12 and 13 are from two subsamplings of the same vascular bundle of a single leaf. They were obtained by microspectroscopically interrogating
2050
Fig. 12. Spectrum of leaf tissue with a strong 1509 cm' band.
Fig. 13. Spectrumof leaf tissue with a weak absorption at 1509 cm'.
(cm-l)
spots only 20 jim apart. The spectrum in Figure 12 is from the bundle sheath, which is known to be highly lignified. This structural material in the leaf is obviously chemically different from nearby tissue, as seen by comparison to the spectrum in Figure 13. One band of particular interest is at 1509 cm"^ more prevalent in Figure 12 than in Figure 13. This band cannot be found in spectra of grain sections except in the pigment strand (which appears later in this chapter). Figure 13 also has a high lipid content, based on the carbonyl band at 1740 cm'^ In animal tissue (Figures 14 and 15), the fatty portion is distinctly characterized by the following bands. Carbonyl at 1740 cm'^ and to some degree CH2 stretch at 2927 cm"^ and CH2 bend at 1469 cm'^ are indicative of the lipids that constitute the fatty tissue. This particular tissue is known to contain phospholipids and a conjugate form of the carbohydrate galactose. These appear at 1235 cm"^ and 1085 cm'. Note that the amide II bands are similar in spectra from both tissues.
2051
Fig. 14. Spectrum of lean mammalian tissue.
assotM
0.0500(H
Fig. 15. Spectrum of fatty mammalian tissue.
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—I
1500
INSTRUMENTATION Contemporary Integrated Infrared Microspectroscopy Figure 16 shows the optical diagram of the molecular microspectroscopy system. Note that from the infrared source on the right, the radiation goes from the beamsplitter of the interferometer to the top of the figure, where it is directed down through the microscope optics and focused on the sample stage. It is subsequently condensed and carried back to the interferometer bench and to the MCT detector. From the targeting aperture (shown enlarged in Figure 1) a Cassegrainian objective, focuses radiation onto the sample stage. From this, a Cassegrainian condenser collects the radiation to exit through a second aperture and be directed ultimately to the detector. Optical optimization of the integrated system combining the microscope, computer, and FT-IR spectrometer is evident by comparison of the molecular microspectrometer system (Figure 16) with the FT-IR microscope (Spectra-Tech, Inc.) and
2052 PROJECTED IMAGE OF APERTURED AREA SUPERIMPOSED ON FULL FIELD OF VIEW
NFRARED SOURCE
Fig. 16. IR^is™ instrument crossectional view optical diagram.
typical interface shown in Figure 17 that was used in the work previously reported by this author (2). Note particularly the numerous mirrors in the accessory IR microscope and interface that have been eliminated in the integrated system. In addition to the optical advantage that leads to potentially improved spatial resolution, there is built-in convenience in selecting the apertured area projected against the image of the entire field of view. Actually, the FT-IR microscope was considered to be the first general purpose microscope to combine the high quality visual imaging of a research light microscope with the reflecting optics for infrared microspectroscopy. This microscope has both transmission and reflection illumination. Infrared spectra can be collected by either transmission through the sample or reflection from the surface of the sample. The visible light path is coaxial with the spectrometer's infrared radiation path, thus allowing precise selection of the area to be analyzed. Unlike prior infrared microscopes that functioned primarily as microsampling accessories for infrared spectroscopy, the IR-PLAN™ also provided for high quality imaging, photometric accuracy, and photography. In the later generation instrument, the advantageous optical features of the research model FT-IR microscope were retained, but in addition, integration of the microscope, spectrometer, and computer has optical and functional advantages. Specifically, improved
2053
FROM . 1 - ^ 3 BEAMSPLITTER
8
10 TO / ^ ^ DETECTOR
FROM MICROSCOPE
Fig. 17. Microsampling accessory IR-PLAN™ and optical interface used between the IRPLAN™ and host FT-IR spectrometer (note mirrors 1-10).
throughput allows aperturing down, while retaining acceptable signal-to-noise and allowing good spatial resolution. Mapping experiments are enhanced by this optical advantage and point-to-point data acquisition using the motorized stage. The minimum step size of the stage, as well as the stage position, is controlled through the system's integrated software. Figure 18 shows a block diagram of the entire microbeam molecular spectroscopy system. The integrated microscope/interferometer bench is the heart of the system. It is supported by a utility module containing the power supplies and the circulator for cooling the source. The instrument is operated by a slave microcomputer and is controlled by the master computer, which is equipped with a monitor and keyboard. An auxiliary computer workstation is used to manipulate data from an earlier experiment, while new data are being accumulated. Peripherals include a plotter and a printer. The microscope is equipped with a video camera, which conducts a signal to an electronic video processor and printer and subsequently to a monitor for observation of the optical image. An air cleaning system (Balston, Inc., Lexington, MA) is used to take compressed, predried, house air furnished at 100 psi into the system and remove the CO2 and water vapor, providing a metered flow at reduced pressure to the FT-IR spectrometer. Figure 2 shows a photograph of the system with the image of the microscope field displayed on the video monitor and a spectrum on the computer screen.
VIDEO PRINTER & PROCESSOR
0
NITROGEN PURGE SOURCE
.
SLAVE CPU
VIDEO CAMERA
I
/
VIDEO MONITOR
COMPUTER MONITOR
MICROSPECTROMETER
MICROSCOPE OPTICS
1
FT-IR BENCH
w
-
MASTER CPU
Y
L
L
STAGE CONTROLLER
I COMPUTER MONITOR
PRINTER UTILIW MODULE
PLOTIER
WORKSTATION CPU F i g .18. Confiation of I R p andperipherals in the KSU Microbeam Molecular SpectroscopyLaboratory showingthe microspectrometerin the center,
alsoclockwise: purge source,slave CPU,master CPU,printer, plotter, utilitymodule,workstation,stage controller,video (image) monitor,video printed processor, andvideo camera.
2055
Fig. 19. Diagram of the National Synchrotron Light Source experimental area showing electron source (A), linear accelerator (B), booster accelerator ring (C), and the vacuum ultraviolet (VUV) storage ring with associated beam lines. The x-ray storage ring with its associated beam lines is also shown.
Synchrotron Radiation Coupled to an Integrated IR Microspectrometer. An IR)is™ microspectrometer was set up at the National Synchrotron Light Source at Brookhaven National Laboratory, Upton, New York, by John Reffner of Spectra-Tech, Inc., in cooperation with Gwyn Williams of NSLS, and Larry Carr of the Grumman Corporation, a user of the beamline U2b. Figure 19 shows the overall view of the synchrotron experimental area at the NSLS. In the synchrotron, electrons from an electron source are accelerated with a linear accelerator (LINAC) shown at point B on Figure 19 to an energy of ca. 75 million electron volts (meV). Electrons from the LINAC enter a booster ring, where they are further accelerated and from which they can enter either the x-ray storage ring or the vacuum ultraviolet (VUV) storage ring. Electrons entering the VUV storage ring are at an energy of approximately 750 meV. Figure 20 shows a pictorial view of the VUV storage ring equipped with eight bending magnets. Seventeen radiation extraction ports are at the bends of the ring. Synchrotron light consists of a continuous spectrum of electromagnetic radiation ranging from x-ray to infrared (see Figure 21). At the National Synchrotron Light Source, light is produced by accelerating bunches of electrons in either of the two closed orbit storage rings. When
2056
Fig. 20. Pictorial view of VUV storage ring showing 8 bending magnets, associated vacuum equipment, and the radiation extraction ports located at the bend of each magnet. Ref (18).
2057 1 —
I
V
I
1
1
1
r
UNIVERSAL SYNCHROTRON RADIATION SPECTRUM
\0
10^
10^
10^
10^
10^
10^
electron orbit
Fig. 21. Spectrum of radiationfromsynchrotron including the infrared region. Ref. (18).
electrons are accelerated, they radiate energy in the form of electromagnetic waves (Figure 22A). This has been known for over a century; however, Einstein, as a result of his theory of relativity, showed that the radiation pattern becomes highly forward-directed when the source of radiation is moving close to the speed of light. Figure 22B is a pictorial view of the relativistic concept of a beam emerging from the storage ring. Note that the angle of observation is expressed in milliradians and the pulse duration is 10^^^ seconds. Figure 23 from Williams Fig. 22. Nonrelativistic (A) and relati(18) shows the relative brightness of the NSLS and vistic (B) emission of radiation from radially accelerated electrons. Ref (18). globar sources on a logarithmic scale. Note that the globar is nondirectional, similar to the radiation produced by the filament of a light bulb. The synchrotron radiation, on the other hand, is not only directional but concentrated within a narrow angle into a beam of high flux. As this beam goes from the storage ring and is transferred by various optical members of the system, losses of intensity are minimal. This is also true for the radiation entering the interferometer and infrared microscope optics. Because a large percentage of the beam is concentrated into a small crossectional area, aperturing of the field of the microscope does not discard 1 10 100 1000 as large a percentage of the beam entering the Wavelength [microns] microscope as it would for a more divergent source. Fig. 23. Relative intensities of synchrotron and glowbar sources. Ref (18).
2058
1t5Cr
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1
V
0.1
^^ o
v o
— 1000 fim — 300 fim 100 fim 50 fim
Synchrotron
2000
4000
6000
Frequency [cm ] Frequency [cm ] Fig. 24. Effect of aperturing on signal intensity from synchrotron radiation beam (A) and glowbar (B) source using the same detector. Ref (19). Figures 24A and 24B show, respectively, the effects of aperture size when using the synchrotron and globar sources with the microspectrometer. Because the synchrotron is not a thermal source it does not exhibit the usual thermal noise characteristics of a hot filament. Figures 25 A and 25B at 100% transmission show the relative noise characteristics of the synchrotron and globar, respectively. These data were reported by Larry Carr (19) from measurements at Beamline U2b at the NSLS in the first workshop on Applications of Synchrotron Radiation to Infrared Microspectroscopy at the NSLS in 1994. Radiation extracted from the VUV ring at beamline U2b contains soft x-rays and vacuum ultraviolet radiation in addition to that in the infrared region. Figure 26 shows the system used to extract the infrared beam from the beam line port and direct it to the microspectrometer. The optical arrangement shown in Figure 26 was detailed by Reffner et al. in a recent publication (20). The first mirror Ml is a standard, plane, copper, laser mirror. This mirror is water cooled and absorbs the x-ray and VUV flux but reflects the infrared beam at right angles from the incident radiation to provide for Bremsstrahlung shielding. Other 150
150 Synchrotron
o c o
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100
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2000
4000
6000
2000
4000
6000
Frequency [cm" ] Frequency [cm"^] Fig. 25. Comparison of noise as a function of optical frequency for synchrotron equipped spectrometer (A) and a globar equipped spectrometer (B) when the same detector and optics were used. Ref (19).
2059
TO IR MICROSPECTROMETER
Fig. 26. System used between the synchrotron beam port and the IR microspectrometer. Synchrotron is 3.5 meters upstream from M2. Ref. (20).
,^m^
mirrors throughout the scheme direct radiation to the infrared microspectrometer. The storage ring operates at a high vacuum. The high vacuum is maintained throughout the optical arrangement shown on Figure 26 from the gate valve (left) through the window valve (right) to the KBr window.
The mirror chamber downbeam from the KBr window is purged
continually with boil-off nitrogen. Our first experiments in early 1994 were performed by placing the IRfas™ approximately 2 meters from the mirror chamber and purging a PVC pipe connecting the mirror chamber to the instrument. These microspectroscopy experiments were conducted out in the open on the floor of the experimental facility of the VUV ring at beamline U2b.
In this arrangement, the beamline dedicated to another spectrometer was temporarily
interrupted to perform the first feasibility experiments. The current configuration involves placement of the spectrometer an additional 10 meters downbeam in a controlled atmosphere room more acceptable for microscopy. Figure 27 shows the optical arrangement used to go from the mirror chamber at the beamport to the microspectrometer. At the microspectrometer end is a chamber attached to the spectrometer containing mirrors to steer the beam from the elevation of entry to one suitable for sit down operation of the IR|is™ specially mounted to an
Nitrogen Purge KBr Vindov FroiU2B Beailine" Vacuw
VdCUUA
Nitrogen Purge
ciMiir 10 Meter Pipe Kbr Hindo» Bulkhead
/
H:^! - iRuS I
K6r Vindow
Fig. 27. Optical arrangement used to conduct the synchrotron beam from the mirror chamber at the beamport to the microspectrometer located in an isolated room.
2060 optical table. This table is equipped with three sturdy legs bolted to the reinforced concrete floor. The floor of the VUV storage ring and its beamlines is designed as one large optical bench. The 10 m long PVC pipe closed at both ends with infrared transmitting optical windows (KBr) is evacuated. The mirror chamber of the microspectrometer is purged with boil-off nitrogen and pneumatically isolated from the IRfis™. The entry port, a 1.5 in. hole Fig. 28. Synchrotron beam entering the beamsplitter of the cut in the back plate o f the IR|Lis™ is IR)Lis™ microspectrometer. . i • t T^T^ • t ^
equipped with a KBr window. One mirror was removed from the IRJLIS™ to allow projection of the synchrotron beam onto the beam splitter of the interferometer in place of the internal source. Figure 28 shows introduction of the synchrotron beam on the interferometer compared to the earlier diagram of the IR|as™ shown in Figure 17. The modified infrared microspectrometer is operated as previously described. In the initial tests of the installed instrument, Reffner et. al. found that they could get most of the flux from the synchrotron infrared beam through a 10.5 jim diameter aperture. This was reported as being consistent with what would be expected for a 1 mm beam size demagnified by a factor of 100 ongoing from f/100 optics to f/1 optics. Preparations of Specimens and Routine Procedure Thin sections of specimens were prepared by cryostatic sectioning on a microtome. In preparation for thin sectioning, wheat kernels were soaked in distilled water overnight under refrigeration. In work previously reported by Wetzel, Messerschmidt, and Fulcher (1), approximately 8 |Lim space thicknesses were selected from numerous sample preparation attempts with thicknesses of 5-20 |am. Grain slices that were too thick and dense produced a greater than optimimi absorbance. Attempts to mount the specimen in a polymeric medium resulted in loss of spectral purity. Only Tissue Tek™ (Miles Laboratory, Elkhart, IN) was utilized to surround the specimens. Tissue Tek has prominent absorption maxima that were not detected within the section itself or in crevices of open space in the center of the tissue. After focusing on the subject to be scanned, with the compensator adjusted to the refractive index of a 2 mm thickness of BaF2, the condenser was focused to give a sharp image of the postcondenser aperture. The purpose of the compensator was to correct for the spherical aberration imposed by the use of a mounting medium, in this case BaF2, which has an index of refraction considerably different than that of air. The specimen was positioned, and the aperture adjusted to frame the desired portion for scanning and to exclude unwanted tissue.
2061 After adjusting the pre-objective aperture to coincide with the stage position and the postcondenser aperture, the stage was moved to illuminate an open portion of the BaF2 disk to obtain a reference spectrum. Once the reference spectrum was accumulated, none of the adjustable aperture settings were changed without repeating the reference scan. The transmission mode of spectrum display was selected for each portion, and 250 scans were routinely accumulated to produce the spectrum in the 4000 to 800 cm'^ range. A logarithmic transformation was performed, and the absorbance spectra were stored. In obtaining a series of automatically accumulated spectra, the stage positions corresponding to first and last areas of the specimen to be scanned are marked using the programmable stage controller (Figure 29). With the control software in the "experiment" mode, the number of points Fig. 29. Photomicrograph showing
along the X axis and the number of columns on the Y axis are starting and ending positions 1 and 2 defined. Also, the spectral range, the number of scans to be corresponding to an automated co-added, and the nominal resolution (usually between 8 cm'^ and 2 cm"^) are selected. After the aperture is adjusted and before the automatic data acquisition is initiated, a reference is scanned using an open portion of the BaF2 disk on which the specimen is mounted. While the automated "experiment" is in progress, absorption spectra appear on the monitor in a stacked sequence (Figure 30). Examination of successive spectra readily indicate when changes in the chemistry of the specimen are encountered as it is being traversed in a stage moving procedure. In obtaining data of good spectroscopic merit with adequate spatial resolution, there are some practical tradeoffs. Apertures with dimensions very near to those of the wavelength will introduce interference, and the optics are said to be diffraction limited. Also, an aperture that is too small reduces the total throughput and adversely threatens signal-to-noise. Some practical aperture must be chosen for large cells or densely packed groups of the same type of tissue. In most cases, 250 scans produced useful spectra even in the presence of adverse optical sample defects. When mapping a large section of tissue using a fairly small aperture and collecting 200 or more data points, 8 cm'* resolution was used with 50 scans co-added in order to save computer storage capacity and time. For a given resolution, aperture, and acquisition time, it is advantageous to be concerned with the optics of the sample. In most cases, tissue samples were used uncovered, and not all parts of the material in the field of use were in focus at the same time. It was not possible to immerse the samples in a fluid of approximately matching refractive index. Thus, for plant tissue slices, the scattering effects
2062
1s5o
~3o5o
'
255b
2o5o
WAVENUMBERS Fig. 30. Successive spectra (154) for a 24x24 ^m area acquired during a (14x11 matrix) mapping experiment of a 650x550 |im rectangle. Note 11 cycles. Ref. (3).
were highly significant. These effects are minimized in routine Hght microscopy with a cover slip and a drop of glycerin. In infrared microspectroscopy, this is not possible. The uniformity of focus across a tissue to be mapped with a moving stage experiment requires a compromise in the focus from one portion of the specimen to another or may require rejecting the specimen altogether and preparing another. As an alternative to the uncovered tissue samples in certain instances, the specimen was sandwiched between two 2 mm BaF2 disks with just sufficient compression to produce optical contact in a special squeeze cell. For each of the spectra obtained, once the aperture size and shape were established, a reference spectrum was collected at the same resolution setting and for the same range as the scan of the particular area of tissue chosen. Reference scans were collected through the sandwiched BaF2 specimen disks, but in an open region of the disk. For the BaF2 sandwich configuration, the reference was collected through a crystal of KI placed near the tissue sample (crushed) between the two BaF2 disks.
2063 CONVENTIONAL (THERMAL) SOURCE INFRARED MICROSPECTROSCOPY Although this report features some of our very latest experimentation that utilizes SRIR|Lis, we have collected considerable data with the laboratory version on a regular basis at the Microbeam Molecular Spectroscopy Laboratory at Kansas State University, since the work reported at the 1992 conference, documented in an earlier volume of the Elsevier series. We previously concentrated on collecting infrared data on both sides of a boundary between different botanical parts in order to spectroscopically illucidate the chemical distinction associated with the morphopology of the tissue. Different botanical parts in a crossection of a seed having different chemical compositions are expected to show significant spectroscopic differences. This we observed in the past going from the pericarp at the outer part of the grain crossection through the outer cell wall of the aleurone, the inner aleurone cell wall, and finally the endosperm. In the germ portion of the wheat, a different spectrum reflecting the importance of lipids appears, and the pigment strand in the crease of the wheat kernel has a spectrum all its own. In addition to these anticipated spectroscopic differences based on different known chemical compositions, early on (2) we were pleased to note that, even within botanical parts such as the endosperm and the germ, it was possible to observe significant distinctions. In the subaleurone endosperm compared to the central endosperm, set apart by microscopic dimensions, the amide II band at 1550 cm"^ intensity relative to the carbohydrate band at 1235 cm"^ makes the compositional differences between the hard high protein subaleurone endosperm and the starchy central endosperm quite clear. In addition, within the general area known as the germ, the embryonic axis and the scutellum differ significantly based on the carbonyl band intensity at 1740 cm"^ indicative of larger amounts of lipid in the scutellum. Differences between the primary root and coleorhiza also have been noted. In Figures 31-33, electronic photomicrographs onto which the square aperture is projected and their corresponding spectra illustrate what is meant by spatial resolution and in situ interrogation of select botanical parts within a biological specimen. In Figures 31-33 we observe typical spectra that correspond to different parts of a wheat kernel (17). We have also observed similar and corresponding parts of other grains, particularly com (3). As previsously reported, we successfiilly produced a rectangular map of a single cell shown in Figures 34 and 35. The figure (34B) in which the cell appears as a pyramid is based on its higher concentration of protein compared to the surrounding cell wall, which appears as a trough at the protein wavelength, but appears in Figure 35B as a ridge, indicative of higher carbohydrate content than the cell. In Figure 35B the pit represents the location of the cell, which is lower in carbohydrates than the surrounding cell wall. Although these data have been reported before (3), they are included in this chapter because, in our own work, they represent a breakthrough on the way to in situ single-cell interrogation. A 180 x 180 |im rectangular region of a wheat kernel was mapped (21). Data were taken from the inner edge of the pericarp
2064
Fig. 31. Spectrum of wheat pericarp (A) and the apertured area from which it was obtained (B). Ref (17). ABS
A
Fig. 32. Spectrum of wheat aleurone cell wall (A) and the apertured area from which it was obtained (B).
Fig. 33. Spectrum of a wheat kernel aleurone cell (A) and the apertured area from which it was obtained (B).
2065
Fig. 34. Labeled (A) and stacked (B) contour maps of the baseline corrected 1650 cm' peak area for a single aleurone cell. Note that the stacked contour map is rotated 180°, with the endosperm at the front. Ref (21). eakareal246)
--T'a
xArO
Fig. 35. Labeled (A) and stacked (B) contour maps of the baseline corrected 1250 cm"' peak area for a single aleurone cell, showmg maxima for the cell walls. Ref (21).
(0 on X-axis) through the aleurone layer (90 on x-axis) and into the subaleurone endosperm. Spectra were obtained from a 7 x 7 jam area, and the data from selected absorbance peaks were used to generate the following figures. The aleurone cell wall area with its high lipid content is shown in the C=0 (1740 cm'^) contour map (Figure 36A). The protein distribution from the amide II 1550 cm"^ peak area (Figure 36B) shows a maximum in the subaleurone endosperm in comparison with the aleruone cell area and the central endosperm region shown in the lower right hand comer of the contour plot. Data used for the contour plots in Figure 36 were replotted as 3-D stacked contours (Figure 37). Figure 37A shows the decline in C=0 moving from the aleurone layer into the subaleurone endosperm. In Figure 37B, the location of two
2066
Fig. 36. Baseline-corrected peak contour map of C=0 (lipid) band at 1740 cm'^ (A) and amide II (protein) band at 1550 cm' (B) for a transition from the pericarp to just inside the "*®5 J subaleurone region of a wheat kernel. Units of X-and y-axes are in micrometers. Ref (21). «. ^
aleurone cells is evident based upon localized protein concentrations at the **^ ridge that appears in the 3-D plot between the pericarp and the sub- ^•40 aleurone endosperm (approximately 80 on the X-axis). Figure 37C is a 180° rotation of Figure 37A. The upperlevel contours are the location of the Fig- ^^- Baseline-corrected peak three-dimensional stacked .
„
J 1 •
1-
contour map of C=0 (lipid) band at 1740 c m ' ( A ) , amide II
two aleurone cells and their adjacent ^^^^^^^^^ ^^^ ^^ 1550 ^^., (3^^ ^^^ ^=0 (lipid) band at 1740 cell walls bordered on one side by cm-^ (reverse side) (C). Ref (21). pericarp.
2067
Fig. 38. Sketch of area mapped in com aleurone to Fig. 40. Surface map of baseline-corrected 1025 cm"' endosperm experiment: P = pericarp, A = aleurone, absorbance peak areas for wheat subaleurone to central SAE = subaleurone endosperm, CE = central endo- endosperm transition. Ref. (3). sperm. Ref. (3).
Fig. 39. Surface map of baseline-corrected 1650 cm"' Fig. 41. Surface map of baseline-corrected 1650 cm"' absorbance peak areas for wheat subaleurone to central absorbance peak areas for wheat subaleurone to central endosperm transition. Ref. (3). endosperm transition (rotated 180°). Ref. (3).
A portion of com was mapped corresponding to a square of 200 x 200 |Lim shown on the sketch in Figure 38 representing the transition from subaleurone endosperm to central endosperm. In Figure 39, it is obvious that the amide I band (1650 cm"^), representing protein, diminishes as the infrared beam moves away from the subaleurone endosperm (left) into the central endosperm (right), whereas the carbohydrate band (1025 cm"') increases (Figure 40) in the same direction. Figure 41 is a result of rotating Figure 39 by 180°, thus viewing the plot from central endosperm (left) to subaleurone endosperm (right). Within the germ of com there are significant differences between the scutellum and the embryonic axis. A 300 x 300 jiim region of the com from the scutellum into the central root (Figure 42) was mapped to show these differences. Examining baseline corrected peak areas, the root is very prominently outlined by the absence of carbonyl (lipid), the higher concentration of protein (amide II), and
2068
saoo
—0OS
Fig. 42. Sketch of area mapped in com scutellum to Fig. 44. Surface map of baseline-corrected 1550 cm"' embryo experiment: scutellum (left), root (right). peak areas for com scutellum to embryo transition. Ref. (3).
«'«.68 — e o s
Fig. 43. Surface map of baseline-corrected 1740 cm'* Fig. 45. Surface map of baseline-corrected 1100 cm"* peak areas for com scutellum to embryo transition, peak areas for com scutellum to embryo transition. Ref. (3). Ref. (3).
the absence of carbohydrate. From the 3-D plot (Figure 43), the sharp decline of lipid C=0 (1740 cm-^ from scutellum (left) to root (right) is obvious. The corresponding increase in amide II (protein) at 1550 cm"^ (Figure 44) at the interface is also graphically apparent as is the 1100 cm-^ (Figure 45) band increase well into the central root. The last two series of fimctional group maps clearly showed chemical distinctions within botanical parts. Within the endosperm, a dramatic concentration gradient appeared between the subaleurone and central endosperm. Within the germ, concentration differences were apparent between the scutellum and the embryonic axis.
2069
bg
wm
>> - 2 2 8 8
3818
4010
4201
4393
X
4585
4776
4968
Fig. 46. Contour map of locally baseline-corrected peak area for the peak at 2927 cm' (interpolated from a 13 X 16 spectrum matrix); bg = basal ganglia, wm = white matter, and gm = grey matter. Ref. (22,23).
-3128 3818
4010
Fig. 47. Contour map of locally baseline-corrected peak area for the peak at 1550 cm' (interpolated from a 13 x 16 spectrum matrix); bg = basal ganglia, wm = white matter, and gm = grey matter. Ref. (22,23).
Mapping of animal tissue, a mouse brain (22,23), in Figures 46, and 47, shows a clear distinction between the fatty portion (white matter in the center) and the lean portion (grey matter to the right). A third part of the tissue lying to the left of the fatty ridge contains both fat and lean in a heterogenous mixture. Figure 46 involves a wavelength selective for lipids (2927 cm-^), resulting in a vivid outline of the lipid-bearing tissue. A contour map with respect to the peak area at 1550 cm"^ (Figure 47) indicates that protein concentration shows no distinct pattern to distinguish the three portions of tissue from the same specimen. Other investigations with brain tissue include the observation of demylination in mutant animals (24) and damage to the myelin at a lesion site formed by extravasated blood (25,26). In such cases, the spectrum of the white matter nearly looses its identity and becomes more like that of the grey matter. Recently an animal model of a human disorder that is known to cause a buildup of a particular chemical (psychosine) lead to a microspectroscopic search for localized deposition of this chemical (27). A spectrum of the pure chemical showed the CH2 stretch vibration occurs at 2919.6 cm ^ This absorption maximum is different than the corresponding vibration in the brain tissue at 2925-2924 cm ^ Searching for the effect of a part of the molecular population on the spectrum of the whole is like looking for a needle in a haystack. Nevertheless this piece of circumstantial evidence reveals the presence of psychosine build-up. When spectral subtraction was used by subtracting the spectrum of normal brain tissue from that of the tissue where pathological changes were expected the resultant CH2 band was at 2919.6 cm'^ and thus
2070
2940
2930
2920
2910 2900 Wavcnumber (cm" )
Fig. 48. Spectrum of white matterfromthe hindbfain of a normal mouse (left) compared to the difference between the spectrum of the white matter from the hindbrain of a twitcher mouse and that from a normal mouse (right). The resultant peak is shifted to the right indicating that the peak from the twitcher mouse is broadened to the right, correlating to the psychosine absorption peak at 2919 cm-^ Ref (27).
2945 293b 292b Wavenumber (CM-1)
Fig. 49. The second derivative of three spectra from the hindbrain white matter from normal mice (*) and of three spectra from the hindbrain white matter from twitcher mice (^), revealing a frequency shift in the vicinity of the 2919 cm'^ psychosine absorption peak, Ref (27).
coincident with the corresponding band in psychosine. This is shown in Figure 48. Also, the second derivative of three spectra from the hindbrain of normal mice agreed with each other but were n o t coincident with second derivative of three spectra from the hindbrain of the diseased mice (Figure 49). A mathamatical function describing the shape and position of the band was calculated for 20 spectra from normal brain and 44 spectra for diseased mice. T h e results of these data supported the spectral subtraction and second derivative plot results. This worked because nature had locally concentrated the psychosine. The mapping capabilites of the *Bs microbeam spectroscopy laboratory have been used to study the migration of water in the process of tempering grain (28). Grain that is stored at moisture levels below 15% is "tempered" to a higher moisture level by addition of water prior to milling. To simulate the tempering process, wheat kernels were I soaked in D2O for specified periods of time, Fig. 50. Comparison oftheTsoo'cm-^ region spectrum frozen, and mounted for cryosectioning. After of D2O (A) to spectra of the subaleurone endosperm of sectioning, a specimen was thaw-mounted
2071 covered with a second disc. The resulting sandwich was mounted into a microsample press and placed on the motorized microscope stage. A linear mapping experiment was initiated in which approximately 25 points were taken from just inside the edge of the kernel into the interior portion of the section. The OD stretching vibration occurs at approximately 2500 cm"^ At this frequency, nothing else in the specimen absorbs, and the added heavy water (D2O) is distingusiable from the H2O in the original grain that occurs at 3300 cm'^ In the process of this experimentation, we noted that the OD stretch of the water bound to the grain was shifted somewhat from the OD vibration of true D2O (Figure 50). Figure 51 shows the migration of water throughout the kernel during a period of 4.5 hours. From the dimensions, it appears that a high D2O concentration went no further than the pigment strand. Figure 5IB shows the distribution of the OD after 6.5 hours, when it has been dispersed throughout the kernel. Figure 51C, after an extended period, shows a redistribution of the OD in
an
200 300 DISTANCE (microns)
300 DISTANCE (microns)
300 DISTANCE (microna)
equilibrium state, but not ., . ^, ^ ^ , -,. Fig. 51. Comparison of D2O distributions at various stages necessarily m the form of water. The of tempering: A) 4.5 hrs., B) 6.5 hrs., and C) 9.0 hrs. Ref technique developed is now in routine use (28). for studies of water migration in grain.
2072
SYNCHROTRON RADIATION INFRARED MICROSPECTROSCOPY The purpose of performing Synchrotron Radiation Microspectroscopy experiments was to determine how well we could do without the usual energy-limited conditions at small apertures and work at the diffraction limit. With these basehne data, we then could make comparisons of future, conventional-source microspectroscopy results. Also with SR-IR^jiS, we have unique opportunities: • Small dual apertures (12 ^im x 12 jjim or 6 ixm x 6 y^m) may be used routinely in a short scanning time because of a high signal/noise ratio. By interrogating cells one at a time, highly localized heterogeneity may be revealed. This is possible because the technique is less intrusive than those requiring sample preparation such as homogenization or extraction. • Even when cell boundaries cannot be visualized, mapping with apertures of cellular dimensions may be used to reveal chemical heterogeneities previously unobserved. • Another utility of the high signal/noise ratio and, thus, short acquisition time is the abihty to obtain scans at a large number of spots in a tissue. This allows mapping of large microscopic sections by interpolating spectroscopic data from a grid of points in the field, where each point is of cellular dimensions. What follows are two series of spectra of individual cells. These spectra are followed by a series of spectra of cellular dimensions collected in succession across a specimen. These will illustrate the abruptness of change observable with good spatial resolution. Small aperture, high density mapping of areas 60 x 60 ^.m or 100 x 100 ^jim illustrates localization on a small scale. Maps of parts of tissue further illustrate this capabiHty, and finally, the speed of data collection when using a very bright but low noise (synchrotron) IR source allows scanning of many spots, necessary to generate large maps that provide perspective and aid in communication of the capabiUty of the microbeam molecular spectroscopy technique. Spectra of Individual Cells Various individual cells were scanned (29) by locating them in the field of view of the microscope using either the lOx or 32x Cassegrainian objective. The motorized stage was used to center the cell of interest in the crosshairs of the eyepiece. Coincidence with the
Wovenumber ( e m - l )
Fig. 53. Spectrum of barley pigment strand, 12x9 jjim aperture. Ref. (29).
Fig. 54. Photomicrograph of barley pigment strand showing 12x9 jjim projected aperture. Ref. (29).
2073
Fig.55. Coleorhiza near wheat primary root. Ref. (29).
Fig.56. Epidermis cell (wheat primary root). Ref. (29).
aperture was verified by projection of the°«' upper aperture onto the specimen prior to^^ replacing the bottom aperture and initiatingo.4< the scan. Figure 53 shows the spectrum of parto.3, of the pigment strand obtained from a 12 x 9^^ pjn rectangular spot shown in the electron photomicrograph (Figure 54) of the pigment strand of an 8 jjon thick section of barley. ArHn wheat, a cultivar of the hard Fig.57. Outer row cell (wheat primary root). Ref. (29). white winter (HWW) class, developed and released by the Kansas Agricultural Experi-" ment Station (KAES), was used for the next" few spectra. Individual cell spectra from a 6 x** 6 [Jim aperture recorded at 8 cm" resolution «^ are shown in Figures 55-59. Figure 55 is fromo^ the coleorhiza (outside of the primary root)o. containing lipid. Figure 56 is the epidermis of ^^ the primary root. Figures 57, 58, and 59 are from cells at the outside (cortex), mid circle of Fig.58. Middle row cell (wSprhnary root). Ref. (29).
cells, and inner cells. These spectra were trun-^^ cated because the frequencies at which dif^
0.J
fraction robbed our intensity resulted in ex-
0.4
cessive noise at the lower wavenumbers. Spectra in Figures 60 and 61 are from'' two different spots in a single tissue (8 jim"*' thick) specimen of the retina of a mouse."' They were obtained by coadding 16 scans and"' using 6 X 6 ixm double aperturing at 8 cm"-^ resolution.
3O0O
2500
Wo*enumber (cm-1)
Scattering is not a problem in Fig.59.ImierrowceU(wheatprimaryroot).Ref.(29).
2074
Fig.60. Spectrum of retina tissue, lipid rich. Ref. (30).
Fig. 63. Photomicrograph of a section of Arlm wheat prepared for microspectroscopic examination. Fig.61. Spectrum of a different spot in the same retina as Figure60, This tissue is protein rich. Ref. (30).
Fig62.Fhotonucrographofprimaryrootofhardwheatshowingthesurroundingcolorhiza,theepidern^^ ot the cortex, cells of the central vascular cylinder, and large cell at the core.
2075
most mammalian tissue. The excellent quality of these spectra (30) from relatively few scans demonstrates the capability of using a bright source with a small angle of divergence. Examination of these spectra shows that we are not intensity limited but that below ca, 1100 cm"^ we are, in fact, diffraction limited. Comparison of these spectra shows drastically different chemical composition between the lipid-rich spot Figure 60 (note bands at 1740,2927,2850, and 1469 cm"^) and the protein-rich portion in Figure 61 (note bands 3280,1650, and 1550 cm'^). The primary root of the cereal grain wheat is a good example of cell-to-cell complexity found in a microscopic specimen. The electronic photomicrograph (Figure 62) of a hard wheat primary root shows several different types of cells. The cortex composed of rows of large, thin walled cells is bounded by epidermis at the outer edge to endodermis at the cortex inner boundary just before the central vascular cylinder. This cylinder contains different types of cells, and at its core is one large cell. A detailed treatment of microspectroscopy, chemistry, and morphology of the primary root is beyond the scope of this writing. Extensive microspectroscopy of the roots of several hard wheat specimens will be reported in the primary literature in the future. From that work, we have excerpted examples to show what can be observed with good spatial resolution. The next group of spectra is from individual cell interrogation using 12 x 12 pjn apertures. In each case, the cell targeted was larger than the aperture size and, after selection, was centered in the crosshairs of the eyepiece and in the projected aperture. The focus was checked, lower aperture repositioned, and scanning initiated. Objectives used were either lOx or 32x, and a resolution setting of 8 cm" was used. With a factor of four in the area of the 12 X 12 Jim aperture, compared to the 6 x 6 jim aperture of the previous series, the signal is greater. Also, because the dimensions of the aperture were doubled, the diffraction losses do not require truncation of data at such low wavenumbers. The cells scanned were from sections of a different kernel of Arlin wheat from the same lot (a typical section shown in Figure 63). Thus, this primary root series involves replicates of those in the 6 jim x 6 ^im individual cell series. Figure 64 is the spectrum from a cell in the coleorhiza. The peaks in the CH stretch region show unsaturation at 3015 cm"^ in addition to the usual CH2 and CH3 bands.
Fig.64. Spectrum ofsingle coleorhiza cell. Ref. (29).
Fig.65. Spectrum ofprimary root epidermis. Ref. (29).
2076
Wowwiumb^ (em-1)
Wovenumbw (cm-1)
Fig. 66... Spectnmi of single outer row root cell. Ref. (29). Fig. 70. Spectrum of central core cell. Ref. (29).
2200
2000
1500
(em-1)
Fig. 67. Spectrum of cell from middle row. Ref. (29).
Fig. 68. Spectrum ofcell from the inner root. Ref. (29).
r,' ^nc, .
f n
.1
T> r/onx
Fig. 69. Spectrum ofcell near central core. Ref. (29).
Also, at 1469 cm"-^, the CH2 bend is in evidence, but the strong carbonyl at 1740 cm"^ is the most prevalent feature. It is convenient to compare the peak heights of 2927 cm""*^ (lipid) with NH stretch at 3280 cm"^ (protein) and to compare 1740 cm' (lipid) to amide II at 1550 cm"^ (protein). Figure 65 is from the epidermis (outermost) part of the primary root. Figure 66 from the outer primary root and Figure 67 were obtained for a cell one row to the outside of the midpoint between the epidermis and the central core. In contrast to Figures 66 and 67, the trend is reversed in Figure 68 from the inner root to show a greater contribution from Upid compared to protein again. Figure 69 from a cell one row out from the central core exhibits a similar spectrum, and in Figure 70, the optical difficulty encountered is in evidence in the spectrum of the central core. Linear Sampling Sequence. A data acquisition sequence was taken across a whole wheat section to include germ. The line was chosen to go from outside the germ through the center cell of the primary root and continue through the seed, passing into the crease and subsequently through the cheek exiting the side of the kernel opposite the germ. Approximately 1700 ixm of tissue was Sampled lu r
j
»*
f
2077
Fig. 71. Spectra from the edge of the periciirp into the pericarp and entering into the seed coat.
Fig. 72. Spectra ofthecolorhiza at ad jacent sampUng positions (outer to inner).
3000
1 2500
1
1 2000
Wovtnumbwr (cm-1)
•
1 1500
Spectrum
160 spectra apertured to 6 ^iin x 6 ^m. Figure 71 includes spectra from the edge of the pericarp into the pericarp (spectrum 3) and entering into the seed coat. In the three center spectra, the amide bands are prominent. The carbohydrate band in the 1025-1100 cm^ region does not show in the second spectrum but increases in prominance by the fourth and fifth spectra. Steps in Figure 72 within the coleorhiza show similar but not necessarily identical responses. The first (outermost) spectrum has the lowest and the fifth (innermost) has the highest carbonyl response at 1740 cm"^. Figure 73 shows the transition of coleorhiza into the primary root. The outermost part of the primary root is the epidermis. This could account for some of the irregularity among the first three spectra at the boundary. Spectra four and five are presumed to be from the primary root outer row of cells. In the first three spectra of Figure 74, the lipid content is strongly evident from the 1740 cm' carbonyl band. It is useful to compare the 1740 cm" to the amide bands at 1650 cm and 1550 cm to get a sense of the lipid to protein ratio. The transition from the outer layers of cells into the more
2078 Fig. 73. Transition of coleorhiza into the primary root.
Fig. 74. Transition from outer layers of cells in the primary root into the vascular cylinder.
Fig. 75. Spectra from adjacent points showing the transition from the edge of the central core, through the center, to the opposite edge.
r(cin-1)
2079 Fig. 76. Transitionfromthe vascular q^linder into the outer layer of primary root cells.
Fig. 77. Spectra within the cortex (outer rows) of the primary root.
tightly packed vascular cylinder shows up with spectra four and five when compared to the first three. Because the spectra were obtained from 6 jxm wide spots spaced on 10 jon centers, this means that the transition between the morphological parts is pinpointed by spectroscopic response to within 10 jjim. This illustrates the power of this technique when spatial resolution is quite good. Figure 75 shows only a portion of the fingerprint region of five spectra spanning the single large cell in the center core of the primary root. Spectra two, three, and four are of the one cell. Spectra one and five (before and after) are included to illustrate the symmetry of the tissue features. In Figure 76, the transition from the vascular cylinder (spectra one and two) into the outer layer of cells is apparent from the Upid features including 1740 cm" vs amide bands at 1650 cm" and 1550 cm" . Also compare the intensity of CH bands in the 2927-2850 cm"^ to NH at 3280 cm""^. Because with this aperture size we are diffraction-limited, noise appears at wavenumbers below 1100 cm" . In Figure 77 are several spectra taken within the cortex where a lesser amount of sample was in the beam for
2080
Fig. 78. Transitionfromthe epidermis of the primary root into the coleorhiza.
;^o
Fig. 79. Spectra of seven adjacent points showing a transitionfromthe coleorhiza into the endosperm.
2500 «tovtnumb«r ( e m - 1 )
Sptctnim
thefirsttwo. In Figure 78, a transition occurring from the epidermis (outermost part) of the primary root into the coleorizha is in evidence on the fourth and fifth spectra. Comparing the 1740 cm" to 1550 cm" (amide 11) of the first three spectra with the corresponding pairs of bands in the fourth and fifth spectra shows a dramatic difference. The first spectrum in Figure 79 was obtained several steps away from the fifth spectrum of Figure 78. The trend continues for the pairs of wavelengths cited for the fourth and fifth spectra of Figure 78. All seven of the spectra in Figure 79 arefi"omsuccessive 10 ^im steps of the programmable microscope stage. These seven spectra represent a transition from the coleorhiza of the germ across the depleted layer (spectra four, five, and six) into the endosperm (spectrum seven). Comparison of the lipid-amide pairs of the seventh and first spectra shows a difference. Omitting two data points between the seventh spectrum of Figure 79 and the first spectrum of Figure 80, the next 2-5 spectra of the endosperm are taken in succession. There appear to be only slight if any differences in composition between these data points. Lower absor-
2081 Fig. 80. Continuation of spectra shown in Figure 79.
^6.0
Fig. 81. Spectra of the transition from the endosperm into the outer tissues of the kernel inside of the crease.
r (cm-1)
bance for the second and third spectra implies a separation or thin spot in the tissue section. In Figure 81, the first spectrum shown is comparable to the last spectrum of the previous figures, although four data points were omitted between them. The third, fourth, and fifth spectra are from data points adjacent to each other, and the prominant peaks in the sixth at 1420 cm" and 1370 cm" suggest we are sampling the hemicellulose of the lacy pericarp tissue in the crease. The observation of a carbohydrate band at ca. 1100 cm" is consistent with the above suggestion, as is the rounding of the band at 3300 cm""^ caused by OH in comparison to the sharper peak of NH observed on spectra from Figure 80. The first two spectra in Figure 82 representing the lacy pericarp have a pair of unresolved peaks at 1544 and 1520 cm"^ They also have moderate carbonyl absorptions at ca, 1740 cm"\ and the amide I band at 1650 cm" has a normal position and shape. The third and fourth spectra show that the 1517 cm" peak predominates. Perhaps that is a summation of the 1509 cm" band of pigment strand and amide II of adjacent tissue. Both the fourth and fifth spectra have more lipid, based on
2082
Fig. 82. Transitionfromthe lacy pericarp within the crease into part of the pigment strand.
Fig. 83. These spectra taken in a hnefromthose of previous figures in this series go from the void of the crease into the outer layers (pericarp and aleurone) of the cheek.
Fig. 84. Spectra within the cheek proceedingfromthe endosperm (spectra 2 and 3) to the layers at the outside of the kernel (spectrum?).
2000
1800
1400
1600 (cm-1)
1200
Spectrum
2083
large 1740 cm"^ bands, and the amide 11 band at ecu 1550 cm'^ has a new shape. Because of a void in the tissue at the crease in the wheat kernel, more than 30 data points were omitted before the cheek of the kernel was traversed. Figures 83 and 84 show partial spectra from successive data points (thefifthspectrum in Figure 83 and the first in 84 are the same). Because of a combination of light scattering and diffraction-induced noise, these spectra are truncated at 1200 cm"^ to allow their display. The first spectrum (Figure 83) and last spectrum in Figure 84 probably represent pericarp and aleurone primarily. These have the maximum response at 1740 cm"^ and exhibit the 1420 and 1370 cm"^ bands. The second and third spectra in Figure 84 possibly are sampling some endosperm along with part of the aleurone layer. The scans shown in this sampling of 6 x 6 pinpoints across the germ and other parts of a wheat kernel illustrate the localized chemical differences that may be studied by the spatial resolution achieved with synchrotron-powered infrared microspectroscopy. Mapping of Selected Regions of Biological Tissues With the synchrotron powered infrared microspectrometer we were able to interrogate individual cells by locating them with the microscope and using dual apertures to isolate the infrared beam to a portion of the cell or the whole cell. A second case for using good spatial resolution was in situations where it was difficult to discern with the eye through a light microscope the actual boundaries between cells. Then a procedure was used whereby apertures of cellular dimensions were employed to map a limited area of tissue. An example of this is an area 60 jjjnx 60 ^JLm in which ten steps are taken along the x-axis and a second row of steps offset 6 jim in the y-direction would take place until a 10 x 10 matrix of 6 x 6 jjjn spots had been interrogated. With this procedure it could not be assured that each spectrum was
X
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X
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Fig.85. Contour map of2927cm"^ peak areas from a70 Fig.86. Contour map of 1550 cm"^ peak areas from a 70 x70 jjumarea of mouse brain white matter. Ref. (30). x70 [imarea of mouse brain white matter. Ref. (30).
2084
Fig.87.SpectrumfromliighpointinFig.85. Ref.(30).
Fig. 88. Spectrum fromlowpoint in Fig. 85.Ref. (30).
of only a single cell because the aperture could encompass parts of more than one. However, by using apertures of cellular dimensions it is still possible to get localization of chemical differences in cellular dimensions.__ Brain Tissue. High density mapping of tissue was done where visual distinctions of cell boundaries are not possible but heterogeneities are expected. With the synchrotronpowered SR-IRJJLS, many 60 x 60 jjim regions (or 100 X100 jon rectangles) of various tissues were mapped in at least 10 x 10 matrices. A map of mouse brain white matter is shown as an example of how the use of cellular dimensions and high density (100% sampling) coverage can reveal highly localized heterogeneities. Figure 85 shows the lipid distribution based on 2927 cm'^. Figure 86 also shows the distribution of protein at 1550 cm" . Spectra in Figures 87 and 88 represent lipid high and low points in the map of Figure 85. Spectra in Figure 89 and 90 represent protein
Fig. 89. Spectrum fromhighpoint in Fig. 86. Ref. (30).
(om-l)
Fig. 90. Spectrum fromlowpoint in Fig. 86. Ref. (30).
Fig. 91. Contour map of 1st derivative values calculated for the region of 1550 cm'^ localizing differences in shape and position of this protein band. Ref. (30).
2085 high and low spots in map of Figure 86. In the past, we have observed qualitative differences in lipid by ratioing the areas of CH2 to C = O bands tofindwhere a lesion in a tissue results in breakdown of long chain fatty acids into shorter ones. Here in Figure 91, we see localized qualitative differences in the shape and position of the amide II band at 1550 cm' that may indicate localized differences in the protein. This is one good reason to interrogate individual cells m^/m using spatial resolution. Fig.92. Photomicrographofwheatrootoutercells. Wheat Primary Root Cells. One localized mapping exercise involved a group of cells at the outer part of a primary root in hard wheat. The photomicrograph in Figure 92 shows the segment of the specimen that was interrogated. This was highlighted by projecting a rectangle onto the field of the microscope. Note that at the top of the photograph is observed the outer row of root cells at an angle from the edge of the rectangle. In the upper right comer of the rectangle is material outside the row of cells. This material is actually part of the coleorhiza. In the subsequent plots of data, each at a different wavelength, it is possible to find the coleorhiza and the first 2-3 rows of cells inward from the edge of the root. The contour map in Figure 93 of 3300 cm'^peak areas reflects the NH stretching vibration of protein and the OH stretching vibration of carbohydrate. From thisfigureit is apparent that the greatest density of the chemical compounds exists in the upper right comer where the coleorhiza is located. However, within the cell grouping inside of the primary root there is also considerable amount of heterogeneity represented by thisfigure.The contour map of Figure 94
-lOB - 9 a
-88
-79
-89
-89
-49
-89
-89
-80
-10
Fig. 93. Contour map of 3300 cm" peak areas from wheat root outer cells and adjacent tissue.
Fig. 94. Contour map of 1550 cm'^ peak ; wheat root outer cells and adjacent tissue.
2086
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-88
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-69
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Fig. 95.1420 cm peak area map of wheat root cells.
-108 -06
-66
-79
-69
-69
-49
-99
-89
-80
-10
Fig. 97.1469 cm peak area map of wheat root cells.
-108
-96
-86
-79
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Fig. 96.2927 cm peak area map of wheat root cells.
0
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Fig. 98.1740 cm peak area map of wheat root cells.
representing 1550 cm', indicative of the amide II band, shows a greater amount of protein in the cells of the primary root than at the edge of it or in the adjacent coleorhiza. High spots in the amide II band density within the primary root are considered to be likely positions for individual cells. The contour plot (Figure 95) at 1420 cm', indicative of structural carbohydrate such as the hemicellulose that one would expect to find in cell walls, shows also a heterogeneity possibly representing cell walls among the cells of the primary root. Other chemicals present in this area include lipids. Where lipids are present the concentration of the hydrocarbon chains are reflected by CH2 stretch at 2927 cm' and CH2 bend at 1469 cm . Figures 96 and 97 show the distributions of the CH2 stretch and CH2 bend respectively. Although it is difficult to pick out individual characteristics, it is obvious that there is con-
2087
—XX —XX
Fig.99. Rootcellstackedcontoiirmap(3300cm'^).
—XX —XX
Fig. 101. Root cell stacked contour map (2927 cm"^).
—XX —XX
Fig.100. Root cell stacked contour map (1550 cm"^).
Fig. 102. Root cell stacked contour map (1469 cm"^).
-1
siderably localized heterogeneity of the CH2. The carbonyl group absorbing at 1740 cm" has a distribution shown in Figure 98. Three dimensional stacked contour plots of this same region of the primary root illustrate perhaps better the chemical heterogeneity involved. Figure 99 of the 3300 cm" band shows a general decline in peak area from the coleorhiza into the region of the group of cells. The localization of protein within individual cells is in evidence by the stacked contour plot (Figure 100) of the amide 11 band at 1550 cm' . The distributions of CH2 stretch and CH2bend at 2927 cm"^ and 1469 cm" respectively are shown in Figures 101 and 102. The distribution of the carbonyl band at 1740 cm" shown in Figure 103 is nearly coincident with that of the CH2 vibrations but the geometry is not quite as well defined. The distribution of the band at 1235 cm"^ (Figure 104), usually considered to be that of the F = 0 of the phospholipid, is interesting to note since it shows numerous highs and
2088
—ii
~xx
Fig. 103. Root cell stacked contour map (1740 cm"^).
Fig. 104. Root cell stacked contour map (1235 cm"^).
Fig. 105. Spectrum of high point in map (Fig. 100).
Fig. 106. Spectrum of cell wall from map (Fig. 95).
lows localized within a fairly small area. All of these chemical differences within the tissue have been observed in a 90 x 90 jim rectangle interrogated as an 11 x 11 point matrix. The apertures used were 6 jjon x 6 ^jim. A spectrum from a high point of the 1550 cm" maps (Figures 94 and 100), indicative of one of the cells, is shown inFigure 105. This spectrumrepresents one of the 121 obtained in the high density mapping experiment. Figure 106 is from cell wall tissue between the high spots. Primary and Lateral Root. A 400 x 400 pim portion of the primary and lateral roots shown on the electronic photomicrograph (Figure 107) was mapped by obtaining data with dual (12 X12 pm) apertures (31). On the diagonal is the coleorhiza in between the primary root and adjacent lateral root. This specimen was produced by sectioning the lower part of the wheat kernel containing the germ. The contour maps of 1550 cm" 1740 cm" , 2927 cm" , 1469 cm"\ and 3300 cm" , Figures 108-112, respectively, show a greater amount of density in the specimen for the roots compared to adjacent tissue. Corresponding stacked contour plots of all (Figures 113-117) show a valley on the diagonal in between the two high regions. It would appear in the case of the adjacent primary and lateral roots that the density of these
2089
Kii.*
Fig. 107. Photomicrograph ofprimary and lateral root.
0
37
74
111
148
165
222
258
286
383
370
407
Fig.109.1740 cm'^ map of primary root. Ref.(31).
37
74
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140
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222
208
206
333
370
Fig. 111. 1469 cm' map of primary root. Ref. (31).
57
74
111
148
laS
222' 258
286
333
370
407
37
74
111
148
185
222
20S
333
370
407
Fig. 108.1550 cm map of primaryroot. Ref. (31).
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258
Fig.110. 2927 cm"^ map of primary root. Ref.(31).
407
37
74
llli
148
lOS
222
2S8
288
333
370
Fig!112. 3300 cm'^ map of primary root. Ref. (31).
2090
Fig.113.1550 cm"^ 3-D map of primary root. Ref.(31).
Fig.114.1740 cm"^ 3-D map of primary root. Ref.(31).
Fig. 115. 2927cm"^3-Dmapofprimaryroot. Ref.(31).
Fig.116.1469 cm'^ 3-D map of primary root. Ref.(31).
root tissues is so much greater than the tissue in between them that this density difference is the overpowering issue obscuring chemical differences. This situation would account for the similarity of the stacked contour plots that are observed. The protein reflected by the amide II band at 1550 cm' definitely shows a lesser amount of protein in the tissue between I'^he primary and lateral roots. The spectrum in Figure 118 was extracted from a single point in the mapping experiment chosen in the root where there was a high response for Fig.117.3300cm-^3-Dmapofprimaryroot. Ref(3l). the 1550 cm"^ amide II band. The y-axis is
2091
Fig. 118. Spectnim from high point in map (Figure 113). Fig. 119. Expanded spectrmn from coleorhiza region.
from 0.0 - 0.7 absorbance units. Figure 119, showing a spectrum of the coleorhiza between the root and shoot required a 3.8x scale expansion (0.0 - 0.2 absorbance units) in order to compare chemical differences. Comparing the two spectra shows that in Figure 119 the lipid to protein ratio is clearly greater in the coleorhiza than in the root (Figure 118). The absolute quantity (absorbance) is low based on density and this fact is reflected in the previous figures. As one would expect the three plots showing lipid at 2927 cm"\ 1469 cm"^, and 1740 cm" are all similar in their reflection of amount of those materials in the primary and lateral roots. Only at 1235 cm" , the P = O vibration, does there appear to be a significant population at the tissue in between the roots. These figures are presented to demonstrate the power of the technique at finding localized differences in two different dimensions within the germ of a wheat kernel. Spectra obtained from individual cells in the wheat germ further demonstrate localized differences. These will be discussed in a different section. One of the pieces of tissue surveyed was a segment of a section of oat. The particular oat cultivar chosen is one known to be very high in beta glucan. The beta glucan is typically located just inside the aleurone layer with the subaleurone endosperm. The electronic photomicrograph in Figure 120 shows the outer portion of the oat section from which the maps were taken. No unique band occurs in the spectrum of beta glucan; however, in the region of the spectrum where we normally look for structural carbohydrates such as cellulose or hemicellulose, a band occurs at 1420 cm"^ and another at 1370 cm"^. We chose to use the peak area at 1420 cm"^ to look for large and irregular depositions of this particular carbohydrate. The contour map of the 1420 cm""^ peak area (Figure 121) shows con^ siderable heterogeneity in the left one-third x,. .^^ m. . • i. c . _.• c . ^
''
Fig. 120. Photomicrograph of outer portion of oattsecs
of the graph. These could conceivably repre- tion.
2092
60
Fig. 121. Oatsubaleurone (1420cm"^).Ref (31).
60
70
80
9(
Fig. 122. Oat subaleurone (1550 cm"^). Ref. (31).
w (em-1)
mmm»ttm (em-1)
Fig. 123. Cell wall spectrum from point in oat. Ref. (31). Fig. 124. Oat subaleurone single pointfrommap (31).
sent greater amounts of this particular carbohydrate in this region of the oat tissue. The range of peak area calculations varied by a factor of six from high to low. In Figure 122, show1
1
ing the peak area of the amide II band at 1550 cm", high points in the tissue at 1420 cm" are shown as being the lower points of the 1550 cm"^ plots. This observation may indicate that, in the tissue regions where beta glucan has been deposited, it has crowded out some of the subaleurone endosperm protein that would normally be in these regions. The spectrum in Figure 123 was from a single point in the mapping experiment chosen to show a portion of the cell wall. In this spectrum the 1420-1335 cm" region shows significant absorption, as does the carbohydrate band 1100-1025 cm" . In contrast to the cell wall spectrum, presumed to include beta glucan, in Figure 124, from another point in the same experiment when the protein presence is dominant at 1650 cm"^, 1550 cm"\ and the sharp band at 3280 cm" . In this case the high protein subaleurone endosperm amide II band at 1550 cm" overshadows the 1100-1025 cm" carbohydrate. Because oat endosperm is known to contain lipid, the bands at 2927,1740, and 1469 cm"^ simply verify this fact. Stacked contour maps show a distinct heterogeneity. Figure 125, showing a stacked contour of the peak area at 1420 cm" , in-
2093
Fig. 125. Oat subaleurone stacked contour map (1420 cm-^). Ref. (31).
Fig. 126. Oat subaleurone stacked contour map (2927 cm'^). Ref. (31).
dicates two substantial ridges. Specifically defined ridges of lipid are shown on the 2927 cm" plot in Figure 126. The plot of the' protein in Figure 127 at 1550 cm" indicates ^^ that the protein location is definitely not coincident to that of the structural carbohydrate (1420 cm" ) shown previously in Figure 125. This was a high density map in which a high percentage of the area was sampled. The overall rectangle pattern was 90 x 90 fjon. The area sampled o n each of the 10 X10 points was Fig. 127. Oat subaleurone stacked contour map (1550
6 jjim X 6 pm. This search by spatially resolved ^°^ ^* ^ * ^ ^' microspectroscopy for a particular material within a tissue surrounded by other materials represents a speculative attempt. It is not always possible to spectroscopically define a particular material by using data from just one wavelength. It is possible with statistical data treatment to produce a better multiwavelength definition of the material that we are looking for. One way this could be handled is by using multivariate techniques to define principal components. Maps of various principal components would represent input from multiple wavelengths. In this way, perhaps a more unique definition would exist for the chemistry of the component being sought. Again, this would require development of some robust software to handle the principal component on a routine basis where a large number of spectra are involved. Upon examination of a section of a leaf, in this case the leaf of grass, our attention was directed to a vascular bundle in the center of the leaf (32). This vascular bundle consists of a cluster of conduits through which nutrientsflow.These conduits are surrounded by a protective piece of tissue called the bundle sheath. The bundle sheath in the shape of a horseshoe, in turn, is attached to some other tissue (Figure 128). Some of this tissue is of a lacy nature.
2094
Fig. 128. Photomicrographof leaf vascular bundle.
Fig. 129. Leafvascular bundle (1509 cm'^ peak area map).Ref.(32).
Fig. 130. Leafvascular bundle (1550 cm'^ peak area
having a large number of holes in it. Inside of the sheath but exclusive of the bundle of conduits are characteristic geometric patterns, which upon sectioning, appear as three large holes. In the crossection of the leaf of grass, one would expect a variation in its chemistry compared to that of seeds or of animal tissue. In our previous work (2), we observed a band at 1509 cm" , which we considered may be indicative of the aromatic character of the lignin. With forage crops such as grass, it is of concern in animal feeding that the maximum digestibility of the grass is obtained. This is usually done by animal experiments, where a ratio of weight utilized versus weight consumed is established. In the past, tissue sections such as this have been suspended in rumen fluid and subsequently interrogated with electron microscopy to determine which parts of a stem or leaf section have been consumed through the action of the enzyme. In experimental work by Danny Aiken, USDA, Athens, Georgia, the most resistant tissue was the bundle sheath. Figure 129 for the band at 1509 cm" shows the horseshoe-shaped structure of the bundle sheath. A spectrum for a high point in the sheath map is shown in Figure 12 and a reference point outside of the sheath appears in Figure 13. In contrast, the plot of the 1550 cm"^ in Figure 130 shows the that the protein maximum is outside of the bundle sheath. Stacked contour plots. Figures 131 and 132, show similar examples of the ability of this technique to localize various portions of the tissue with wavelength-specific plots. The locaUzation of absorption at 1469 cm" related to lipid (Figure 133) is ex^ actly the opposite of the protein in Figure 132. We are in hopes that continuation of the work
2095 begun, of which this is a sample, could lead to a method of evaluation of breeders' stock at early generations when only a few leaves are available. At present it is necessary to grow enough material to do animal studies in order to predict the digestibility of future grass cultivars and other forage materials produced as ^ part of a long-term breeding program. Mapping Large Microscopic Specimens We have shown the utility of the synchrotron source system to interrogate single ^^^^ Leafvascular"b„ndle (1509 cm"! 3-D peak cells and to perform high density mapping of areamap). Ref. (32). limited areas with apertures of cellular dimensions. A third way in which the synchrotron powered infrared microspectrometer was ^-lo used to obtain a large number of data points in a small period of time. This enabled us to ^-^ . produce functional group maps of relatively^ large microscopic specimens within a reasonable period of time. The time limita-.. tions using the synchrotron involve approximately four hours between the time that the beam current stabilizes after injection of electrons and the time that it degrades to ap-^. ,^^ , . , u ^ ^ r / . c ^ -i i x
° ^ Fig. 132. Leaf vascular bundle (1550 cm peak area) proximately 1/3 of the original beam current showingproteinmaMmatotheleftandright. Ref.(32).
prior to the next injection. The relatively high signal-to-noise ratio obtained even at small apertures allowed coadding of 16 (in some **^ cases as few as 8) scans before stepping to the i.^^ next point. Actually, the time limitations were required mainly for I/O, data storage, '^ and treatment rather than data acquisition o.^^ (scan time). **-0s Cerebellum. Aportion of the cerebellum of a mouse brain was an interesting subject for a large map, because it is considered to be a layered tissue. From the field of microscope with a 15x objective, a relatively large Fig-133. Leaflipid vascular (1469 cm'^Ref. peak area map) showing at thebundle top and bottom. (32). segment 1600 x 1600 iim was chosen to at-
2096 tempt acquiring data for a wavelength-selective image. Spectra were obtained at 625 points in a 25 X 25 matrix configuration. In this case, a 12 X 12 jxm aperture was used, 8 cm" resolution was selected, and 8 scans were coadded for each spectrum. An electronic photomicrograph (not shown) included a pattern that resembled the boot of Italy. The wavelength-selective image that appeared (Figure 134) was coincident with the pattern observed under the microscope with visible light. Images constructed from these spectra 657 -34a -139 Fig. 134. Cerebellum 2927 cm"^ contour map. Ref.(30). from peak areas at other wavelengths (Figures 135 and 136) did not clearly reveal these features. Thus, the chemistry of the features in the specimen provided the selectivity to enable contrast and produce a meaningful image.. Rye Kernel. The dimensions of the section of rye required mapping of an area of approximately 3,800 p,m x 2,700 jon (648 spectra collected over a 27 x 24 point matrix). Fart of the rye section is shown in the electronic photomicrograph (Figure 137). The germ portion is not shown. A germ portion is -1500 -13BI -1153 -»74 -755 Fig. 135. Cerebellum 1650 cm"^ contour map. Ref.(30). shown in Figure 138. The section has tears in it, however, the outUne remains intact. In this case, it is impossible to obtain data from 100% of the tissue and, thus, representative points are scanned and datafi*omthese points are interpolated so that the plotting routine shows continuity between the points. The individual points from which data were acquired were 12 jjon x 12 iim. In this case, the restriction was done with only a single aperture prior to the specimen. The outline of the seed section is apparent from Figure 139, a contour map of the peak area of the 1025 cm" -1500 -iSOi -IISS absorption band. Similarly, in Figure 140, the Fig. 136. Cerebellum 3300 cm"^ contour map. Ref.(30).
2097
Fig. 140. Ryekemel (3300 cm"^ peak area). Ref.(29). Fig. 137. Photomicrograph of partial rye kernel.
Fig. 138. Photomicrograph of rye kernel germ.
Fig.141. Ryekernel(2927cm"^peakarea). Ref.(29). f^^^
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Fig.139. Rye kernel (1025 cm-^ peak area). Ref.(29).
Fig.142. Rye kernel (1469 cm"^ peak area). Ref.(29).
outline is shown from the large absorption at nearly all portions of the specimen for the 3300 cm' reflecting both NH and OH stretch vibrations. Figures 141 and 142, showing contour maps of CH2 bands at 2927 cm" and 1469 cm" , are indicative of the overall lipid concentration and, in general, of the concentration of organic matter in the beam. Figure 143 of the peak area at 1740 cm" is more selective for actual lipids, and it is no surprise that their con-
2098
Fig.143. Ryekernel(1740cm'^peakarea). Ref.(29).
Fig.144. Ryekemel (1550 cm"^ peak area). Ref.(29).
centration is greatest in the region of the germ. The amount of protein in the germ area is shown in Figure 144 for the peak area of the amide II band at 1550 cm" . When data from the rye were subjected to mapping at 1509 cm , deposition of all of the absorbing material at this wavelength occurred at the region of the pigment strand (Figure 145). We recall that this was the only place in ker«»^'»nels of grain where this band, which we preFig. 145. Rye kernel (1509 cm'^ peak area). Ref.(29) viously found in the vascular bundle of leaves and stems, exists. Stacked contour maps present an interesting look at the data. The area of the 3300 cm" band, shown in Figure 146, is indicative of practically everything in the rye kernel. The shape of the cheeks of the kernel is readily apparent; however, opposite the cheeks, there does not seem to be a very large absorption for the germ. More contrast between the germ and the endosperm-containing portion of the kernel is shown on Figures 147 and 148 involving the CH2 at 2927 cm'"^ and at 1469 cm"^. Note that the germ — 8 0 7 —9
2099
Fig. 147. Rye kernel stacked contour (2927 cm'^ peak area). Ref. (32).
—aor—3*« Fig. 148. Rye kernel stacked contour (1469 cm"^ peak area). Ref. (32).
area at 1740 cm" (Figure 149), which shows great contrast of the germ portion of the kernel in comparison to the rest. However, ••Sj noticeable carbonyl absorption occurs around the edges of the kernel in the region of ^.^^ the pericarp and the aleurone cell walls. This is precisely what we would expect. l.>^ The use of this technique allows representative sampling of a larger portion of tis- **5.j« sue. The fact that each individual area sampled is of cellular dimensions also simultaneously allows one to obtain spectra with high spatial resolutions. In most of our large Fig. 149. Rye kernel stacked contour (1740 cm"^) showmapping experiments, we chose to COadd 16 inglipidatedgeofkernelandingemi.Ref.(32).
spectra; in some cases where time did not allow, it was necessary to coadd only eight. Typically, this required 15-20 seconds. If software is written to store the interferogram without doing the fast Fourier transformation and to minimize the I/O delay of the computer, then perhaps single scans could be run and data could be accumulated for many more positions during a continuous 4-hour block of available synchrotron beamtime. At the present time, data acquisition and storage software do not allow for this rapid operation. Lobe. Microbeam molecular spectroscopy of animal tissue specimens has been particuarly fruitful and interesting. Section preparation has a higher yield than when dealing with seeds and other plant materials. Once the section has been prepared it tends to be optically "user friendly" with the result that scattering, sample thickness, and sample discontinuity are not a problem. Under the section of conventional source IR microspectroscopy
2100 Fig. 150. Contourmap of brain lobe (2927cm-' peak area) showingthe white mater as continuousdark area. Fig. 151. Similar to Fig. 150.White matter localized by continuous darkarea. Ventricle is foundas triangle abovewhite matter. Fig. 152. contour map of 1740cm-' repeats lipid localizationbut with less contour. Fig. 153.~ a p p i ofn 1550 ~ cm"defining onlythe location of the ventricleshown as triangle. Ref. (30).
2101 mapping of normal brain tissue was shown (Figures 46,47) and band assignments were proposed for all frequencies that were characteristic of white matter (22). Once these were established, this information was used in subsequent studies involving mouse brain tissue. With synchrotron-powered microspectroscopy, detailed high density mapping of a 60 x 60 jjim rectangle revealed heterogeneity to within 6 |xm. Subsequently, a 625 point map of a 1600 X 1450 cerebellum showed that a Fig. 154. 2927 cm"^ peak area map. Curvature of outer wavelength selective contour map of this tis- top lobe (front), ridge of white matter in center with greymatter in foreground. Ref. (30). sue produced an image coincident with the viewin the field of the microscope. It has been a long standing goal since our original brain mapping to produce an image of a whole lobe of the brain using the combination of parts into a mosaic if necessary. With the shorter acquisition time of the synchrotron-powered IR^-s, most of one lobe (4100 X 3400 [ixn) was mapped in a single session. In the contour maps that follow the lower left corner shows the curvature of the outer top part of the lobe and the upper left Fig. 155. 2927 cm"^ peak area map, rotated to show shows lobe curvature at the bottom. Figure basal ganglia in foregroimd and top lobe curvature 150 produced from the areas of peaks at 2927 (right). Ref. (30). cm" clearly outlines the strip of white matter. This is repeated in the 1469 cm" contour map in Figure 151 which also shows the location of the ventricle as a heavily contoured (shaded) triangle just above the WM at the right of the figure. The locus of the WM is crudely defined from the 1740 cm" plot (Figure 152) and undefined in Figure 153 from the 1550 cm" plot. Wavelength dependance of selective functional group mapping is coincident Fig. 156. 1235 cm"^ peak area stacked contour map with the microscope view. Three dimensional selective to white matter localization (similar to Fig. maps of the lobe (Figures 154-158) provide an 154). Ref. (30)
2102
Fig. 157. Stacked contour map of 1085 cm" peak area. Lobe localization ofwhite matter similar to Fig. 154. Ref.(30).
Fig. 158. Stacked contour map of 1550 cm"^ peak area. Lobe protein does not show localization of white matter.Ref.(30).
excellent perspective. In Figure 154, representing the area of the 2927 cm" peak, the corner to the front corresponds to the outer top of the lobe. The white matter appears as the prominant ridge with the grey matter to the front and the basal ganglia observed by the white matter. Figure 155 has been rotated 90° from Figure 154 to show the basal ganglia. Images from other wavelengths characteristic of white matter 1235 cm' (Figure 156), 1085 cm" 1
1
(Figure 157), also 1469 cm" and 1740 cm" (not shown) to the same degree exhibited a ridge of white matter as anticipated from earlier studies (22), similar to that of Figure 154. The 1
1
1550 cm" map (Figure 158) and the 3300 cm" map (not shown) had no features coincident with the view in the microscope. COMMENTARY Working with the synchrotron as a source enables us to throw off the limitations imposed by low signal. The diffraction limit is still very real as Figure 60 and 61 so readily indicated. With special techniques working at the diffraction limits has been demonstrated. Access to a synchrotron beamline adapted to accept a microspectrometer is not necessarilly possible. When using the synchrotron-powered instrument at the NSLS, the schedule of work on the instrument is dictated by available beamtime. Except for scheduled maintainance and test runs it operates around the clock seven days per week. Beamtime is in 4-5 hour blocks of time between fills of the storage ring. Experiments requiring programmed data acquisition after each stage position change must fit into these time blocks. The synchrotron beam profile is not subject to decay at low wavenumbers as characteristic of a blackbody source which is good, but spikes (transients) sometimes appear. These can be shifted out of the way of the region of interest by control room adjustment of the ring control parameters. The experiment and experimentor are subject to the beam. Of course this is the case with all beamline activity.
2103 Diffraction is a factor to be considered. The effect of diffraction is particularly severe for radiation of longer wavelengths encountered in the infrared region of the spectrum. Diffraction effects make spatial resolution difficult in two ways. Light loss sets practical limits on minimum aperture size and diffraction-induced sampUng outside of the aperture area diminishes spectral purity. Optimization of instrument design and successful use of infrared microscopes requires that attention be paid to the diffraction effect and its impact on the data obtained. "Photometric Considerations in the Design and Use of Infrared Microscope Accessories" by Messerschmidt (13) emphasizes this fact previously discussed by Coates (11) and tells how contributions from unwanted material outside of the projected aperture can be minimized by instrument design characteristics. This included the use of a projected image plane mask to aperture the collected rays that emerged from the sample, as well as use of a similar device before the objective to limit the area of the field sampled. The integrated instrument (IRpiS ™) used in this work has these features incorporated into its design. Instrument optimization allows only the potential for achieving idealized spatial resolution. Use of the instrument may involve several optical compromises and, thus, compromise spatial resolution. This, in turn, affects spectral purity. The work that we have done, particularly with the conventional globar source, is no exception. To allow only narrow aleurone cell walls, as viewed with the IR|JLS™ instrument, to be sampled by the infrared beam, required a nominal aperture of 3 x 6 jjim. As anticipated, the results degraded at lower wavenumbers but useful data were obtained for a reasonable portion of the spectrum. Surprisingly, the optical characteristics of the cell walls allowed transmission of sufficient energy to get enough radiation to the detector to provide a signal/noise acceptable for practical accumulation of data in a reasonable period of time. With the synchrotron radiation source, adequate brightness produces noise-free spectra with small dual apertures and minimal scanning time (Figures 60,61), except below the diffraction limiting wavelength. In this case we anticipate that spectral purity is conserved by the resulting spatial resolution. Optical characteristics of the sample from a select area of the specimen on the microscope stage may Umit success with even the most ideally-optimized instrumentation. Sample preparation is highly important. In transmission work, the effects of sample thickness are obvious. The effects of sample scattering, lensing, or otherwise de-focusing part of the radiation are less obvious. Effects of the sample on band shape and relative intensity have been illustrated and discussed (33). Attenuation of the beam by the sample for any of these reasons compounds the problem of achieving spatially resolved spectra of good quality in a reasonable time. In attempting to record the spectrum of a small area surrounded by chemically dissimilar material, users of even a well designed instrument are forced to compromise the spectral purity of a small target area when a Ught starved condition occurs from attenuation caused by the sample. In single cell interrogation, spectral purity may degrade in practice for cases where the apertures must be small but the sample is optically non-ideal. The nature of the sample may require enlargement of the post-condenser aperture to a point
2104 where it was undoubtedly less effective at clipping off the unwanted, diffracted, stray light, thereby contributing unwanted spectral characteristics of adjacent tissue to our target spectrum. Aperturing down to this size to examine a single cell requires sacrificing all data at lower wavenumbers. Enlargement of the second aperture allows a small percentage of the total radiation to influence the spectrum that we observed. This seemingly non-ideal compromise is usually considered better than the alternatives of no sampling at all, enlarging the aperture (deliberately sampling adjacent material), or usually producing unacceptably noisy spectra requiring co-addition of many scans over an extensive period of time. Use of the synchrotron source in our recent work meant less compromising. Ordinarily, data appearing in most in situ sampling of small areas in a heterogeneous field by contemporary future infrared microscope users must be considered in the light of the compromises made, but not necessarily revealed, by the experimenter. Applications of sampling heterogeneous biological materials at the Microbeam Molecular Spectroscopy Laboratory since its origination in niid-1991 and our recent experience at the National Synchrotron Light Source are exemplified by the data presented in this chapter. For in situ sampling of any single tissue it would be presumptive to regard these first reports to be typical. This most recent consolidation of data, interpretation, and maps will serve as a reference for continued work in our laboratory, and we believe it maybe useful to others working with similar biological materials. CONCLUSION Not only is spatial resolution possible with conventional (globar) source modem Fourier-transform infrared microspectroscopy, but it is enhanced when the bright narrowly divergent synchrotron radiation is used. This latter development will allow future specialized experimentation and presently has provided baseline data for comparison of past and future experiments when spatial resolution and spectral purity are the issue. We have had to wait 30 years for FT-IR spectroscopy to achieve its present popular status. From Michelson's paper of a century ago and recognition of the multiplex and optical throughput advantage to the remarkable achievements of the last 30 years involving the development of rapid-scanning interferometers, introduction of the fast Fourier transform, and commercial introduction of relatively inexpensive digital microcomputers, the present state of infrared microspectroscopy has arrived. As a research analytical chemist, Ifindthe combination of molecular vibrational spectroscopy and microscopy exciting. We can now assist researchers concerned with biological specimens by achieving a molecular chemical dimension to enhance their understanding of the systems where highly localized compositional as well as microstructural information may be of value. Biological applications of spatially resolved infrared microspectroscopy have been reported, but as yet only infrequently. Several are tabulated in a recent review (22) including (34,35). Presumably the availabiUty of molecular
2105 microspectrometers to more practicing microscopists should lead to research applications not yet explored. In his recent guest editorial in Applied Spectroscopy (36), Professor Peter R. Griffiths, speaking as an FT-IR spectroscopist, considers all "accessories" for IR too large to fit in a sample compartment as peripherals. He stated that "far and away the most important peripheral for FT-IR spectrometers has been the microscope. The development of infrared microscopes that has taken place over the last decade has greatly increased the number of samples that can be characterized by FT-IR spectroscopy"... "microscopes are available that with theflickof a switch could be used for either transmission or reflective measurements" ... "objectives have become available for FT-IR microscopes by which ATR (attenuated total reflectance) or grazing incidence R-A spectra of samples smaller than 50 pin on the side can be measured. With computer controlled translation stages and programmedtemperature hot plates, the range of applications that can be attacked by FT-IR microscopy is truly mind boggling." Spectroscopists have recognized the value of microscopy. Now that the sampling techniques and dedicated instrumentation is available, the day may not be far removed when more scientists working in the biological arena may consider molecular microspectroscopy as a highly valued tool. This is an exciting time to be alive and active in the field now that the state-of-the-art includes integration of the disciplines of microscopy, FT-IR spectroscopy, and computer science. Come on in, the water is fine! COWORKERS Without the leadership and inspiration of John Reffner, Gwyn Williams, and Larry Carr, the synchrotron radiation infrared microspectroscopy would never have been possible. I appreciate the opportunity to be involved in the early stages of feasibility studies and system debugging as well as use of the SR-IRftS at the NSLS. Arnold Eilert took time away from AOTF near-IR instrument building and experimentation to visit the NSLS facility for data acquisition and to manage the data handling at the Microbeam Molecular Spectroscopy Laboratory. He also was responsible for the electronic assembly of this manuscript. Liling Cho, Mark Esfeld, Don DeCou, and Ming Zhao of Kansas State University did the voluminous data conversions and plotting. Most plant materials were sectioned by Lukasz Pietrzak and S. Shea Miller (plant scientists) of Agriculture Canada (Ottawa, Ontario). Sectioned grass specimens were courtesy of Danny Aiken, USDA, Athens, Georgia, and all animal tissues were furnished by Steven LeVine, research physiologist/pathologist at the University of Kansas Medical Center, Kansas City, Kansas. Ongoing collaborative work at Kansas State University and at Brookhaven National Laboratory with visiting senior scientists Miller and Pietrzak will be published in the primary literature, and I appreciate not only their contributions to plant microscopy but also the opportunity to use excerpts of microspectroscopic data that they have been involved in obtaining on the IRjiS™ and the SR-IRjiS. Professor Clifton
2106 Meloan of the KSU Chemistry Department and the author estabUshed the Microbeam Molecular Spectroscopy Laboratory. Previous collaboration with Bob Messerschmidt, Gary Fulcher, Elizabeth Aradt, Steve Walchle, Connie Wetzel, Jeff Wilson, and Jason Jarrett contributed indirectly to processes on which this chapter is based. The support of the National Science Foundation EPSCoR:OSR-9255223 and the Kansas Agricultural Experiment Station is greatly appreciated. Contribution no. 95-070 Kansas Agricultural Experiment Station, Manhattan. REFERENCES 1. Wetzel, D. L., Messerschmidt, R. G., and Fulcher, R. G., Chemical Mapping of Wheat Kernels by FTIR Microspectroscopy, Federation of Analytical Chemistry and Spectroscopy Societies, 14th Annual Meeting, Detroit, Oct., 1987. 2. Wetzel, D. L., ^ d Fulcher, R. G., Fourier transform infrared microspectroscopy of food ingredients, in Flavors and Off Flavors, Charalambous, G., Ed., Elsevier: Amsterdam, 1990, pp. 485-510. 3. Wetzel, D. L., Molecular Mapping of Grain with aDedicated Integrated Fourier Transform Microspectrometer, in Food Flavors, Ingredients, and Components, Charalambous, G., Ed., Elsevier: Amsterdam, 1993, pp. 1-50. 4. Griffiths, P. R., and de Haseth, J. A., Fourier Transform Infrared Spectroscopy, John Wiley and Sons: New York, 1986. 5. Coates, V. J., Evolution of Light, Infrared, and Fluorescence Microspectrometry, Federation of Analytical Chemistry and Spectroscopy Societies, 14th Annual Meeting, Detroit, Oct., 1987. 6. Messerschmidt, R. G. and Harthcock, M., Eds., Infrared Microscopy: Theory and Applications, Marcel Dekkar: New York, 1988. 7. Reffner, J. A., Molecular Microspectral Mapping with the FT-IR Microscope, (EMAGMICRO 89, London, Sept., 1989), Inst. Phys. Conf. Ser. No. 98, Chapter 13, lOP Publishing: London, 1990, pp. 559-569. 8. Barer, R., Cole, A. R. H., and Thomas, H. W., Infrared Spectroscopy with the Reflecting Microscope in Physics, Chemistry, and Biology, iNtowre, 163,198(1949). 9. Burch, C. R., Semi-aplanat Reflecting Microscopes, Proc, Phys, Soa, 59,47 (1947). 10. Gore, R. C, Infrared Spectroscopy of Small Samples with the Reflecting Microscope, Science,n0,710(1949). 11. Coates, V. J., Offner, A., and Siegler Jr., E. H., Design and performance of an infrared microscope attachment,/. Opt Soc,Am.,43,984 (1953). 12. MuggU, R. Z., FT-IR Through a Microscope, Inter/Micro-82, Chicago, IL (unpublished).
2107 13. Messerschmidt, R. G., Photometric Considerations in the Design and Use of Infrared Microscopy Accessories, in The Design, Sample Handling, and Applications of Infrared Microscopes, Roush, P. B., Ed., ASTM STP 949, American Society for Testing Materials, Philadelphia, 1987, pp. 12-26. 14. Lin-Vien, D., Colthup, N. B., Fateley, W. G., and Grasselli, J. G., Infrared and Raman Characteristic Frequencies of Organic Molecules, Academic Press: San Diego, 1991. 15. Colthup, N. B., Daly, L. H., and Wiberley, S.E., Introduction to Infrared and Raman Spectroscopy, 3rd Edition, Academic Press: San Diego, 1990. 16. Bellamy, L. J., The Infrared Spectra of Complex Molecules, Wiley: New York, 1975. 17. Wetzel, D. L. and Reffner, J. A , Composition profile in grain thin sections by Fourier transform infrared microspectrometry, in Proceedings of the International Cereal Chemistry Symposium, Vienna, Austria, Lasztity, R., Ed., ICC and Technical University of Budapest, Budapest, Hungary, 1991, pp. 47-52. 18. WiUiams, G. P., What is Synchrotron Radiation - An Introduction, in Proceedings of the First Workshop on Applications of Synchrotron Radiation to Infrared Microspectroscopy, Reffner, J. A. and Williams, G. P., Eds., National Synchrotron Light Source, Brookhaven National Laboratories, Upton, NY, Feb. 3,1994, pp. 2-8. 19. Carr, G. L., Interfacing the Synchrotron to IR Spectrometers, in Proceedings of the First Workshop on Applications of Synchrotron Radiation to Infrared Microspectroscopy", Reffner, J. A. and Williams, G. P., Eds., National Synchrotron Light Source, Brookhaven National Laboratories, Upton, NY, Feb. 3,1994, pp. 9. 20. Reffner, J. A , Carr, G. L., Sutton, S., Hemley, R. J., and WiUiams, G. P., Infrared Microspectroscopy at the NSLS, Synchrotron Radiation News, 7 (2), 30-37 (1994). 21. Wetzel, D. L., and Reffner, J. A , Spatially Resolved FT-IR Microbeam Spectroscopy to Chemically Examine Microstructure of Wheat Kernels, CerealFoods World, 38 (1993). 22. Wetzel, D. L. and Le Vine, S. M., /n situ Fourier Transform Infrared Microspectroscopic Mapping of NormalBrain Tissue, S/7ec^ro5C(9/7y, 8(4), 40-45 (1993). 23. Wetzel, D. L. and LeVine, S. M., Brain Mapping by FT-IR Microspectroscopy, Proc. SPIE-Int Soc. Opt. Eng,, 1575,435-436 (1992). 24. LeVine, S. M. and Wetzel, D. L., Analysis of Brain Tissue by Fourier Transform Infrared Microspectroscopy,^/?;?/. Spectrosc. Rev., 28 (4), 385-412 (1993). 25. Wetzel, D. L. and LeVine, S. M., In situ FT-IR Microspectroscopy of Extravasated Blood Damaged Brain Tissue,Proc. SPIE-Int. Soc. Opt. Eng.,20S9,340-341 (1994). 26. LeVine, S. M. and Wetzel, D. L., In situ Chemical Analysis from Frozen Tissue Sections by Fourier Transform Infrared Microspectroscopy: Examination of White Matter Extravasated Blood in the Rat BTam,Amer. J. Path., (1994), in press. 27. LeVine, S. M., Wetzel, D. L., and Eilert, A. J., Neuropathology of Twitcher Mice: Examination by Histochemistry, Immunohistochemistry, Lectin Histochemistry, and Fourier Transform Infrared Microspectroscopy, Int. J. Devi. Neuroscience, 12 (4), 275288(1994).
2108 28. Wetzel, D. L. and Eilert, A. J., Fourier Transform Infrared Microspectroscopic Observation of the Migration of Water in Individual Wheat Kernels with Tempering Using T>20,Proc, SPIE-Int Soc. Opt Eng., 2089,464-465 (1994). 29. Wetzel, D. L., Eilert, A. J., Pietrzak, L. N., Williams,G.P., and Reffner,J.A., UltraSpatially Resolved FT-IR Microspectroscopy with a SynchrotronBeam Light Source, Meeting of the American Association of Cereal Chemists, Nashville, Oct. 1994. 30. Wetzel, D. L. and LeVine, S. M., FT-IR Microspectroscopy: Applications to Neuropathology, Joint Meeting of The Microscopy Society of America and The Microbeam Analysis Society, New Orleans, Aug. 1994. 31. Wetzel, D. L., Eilert, A. J., Reffner, J. A , Willams, G. P., and Pietrzak, L. N., UltraSpatially Resolved Single Cell FT-IR Microspectroscopy of Biological Tissues, Federation of Analytical Chemistry and Spectroscopy Societies, 21st Annual Meeting St. Louis, Oct. 1994. 32. Wetzel, D. L., Ultra-Spatially Resolved FT-IR Microspectroscopy with a Synchrotron Light Source, 7th International Diffuse Reflectance Spectroscopy Conference, Chambersburg,PA, Aug. 1994, paper no. 18. 33. Bartick, E.G., Considerations for Fiber Sampling with Infrared Microspectroscopy, inTheDesign, Sample Handhng, and AppUcations of Infrared Microscopes, Roush, P.B., Ed., ASTM STP 949, American Society for Testing Materials, Philadelphia, 1987, pp. 64-73. 34. KodaU, D.R., Small, D.M., Powell, J., and Krishnan,K., Infrared Micro-Imaging of Atherosclerotic Arteries,-^;?;?/. Spectrosc,,AS(9>\ 1310(1991). 35. Dong, A., Messerschmidt, R.G., Reffner, J.A., and Caughey, W.S., Infrared Spectroscopy of aSingleCell-TheHumanErythrocyte,5/ocftem.B/op/iy5.i^e5. Commun., 156(2), 752 (1988). 36. Griffiths,P.R.,UpdateonFT-IR,^;7p/.5pec^ro5C.,46(ll), 14A(1992).
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2109
Analysis of Drinking Water Near and Far from Thermal Springs Using Instrumental Neutron Activation Analysis Ellene Tratras Contis Department of Chemistry, Eastern Michigan University, Ypsilanti Mi 48197 USA.
Abstract This project investigated what effect, if any, there was on drinking water near and far from radioactive thermal springs. The focus was to analyze several drinking water samples for trace elements, especially those of nutritional and toxic value. Elements of interest included selenium (Se), vanadium (V), arsenic (As), mercury (Hg), cadmium (Cd). Samples from nine different springs were collected from an island in the eastern Aegean Sea in Greece, Ikaria, which is known for its radioactive therapeutic baths. The liquid samples were irradiated and counted after 3 days and 15 days using the instrumental neutron activation analysis technique. Analysis of the samples revealed typical elements found in natural water: sodium (Na), bromine (Br), potassium (K). In addition, some of the samples indicated ultra traces of uranium (U). These amounts, were attributed to natural radiation found in typical geological formations. Results indicated that there were no statistically significant traces of the elements of interest in the water samples.
introduction There is increasing environmental concern worldwide for the consumption of non contaminated foodstuffs and for a clean supply of drinking water. Many studies have reported what methods can be used to study the contaminants in food and water analytically, especially in trace and ultra trace amounts [1]. These studies and others [2] have set levels for the number and types of toxic elements. Because of this concern, this project investigated what effect, if any, there is on drinking water near and far from springs that are used as therapeutic radioactive mineral baths. The project was implemented to measure any contamination of drinking water supplies with these special mineral springs. The focus of the project was to analyze several drinking water samples for trace elements, especially those of nutritional and toxic value. These elements of interest included selenium (Se), vanadium (V), arsenic (As), mercury (Hg), cadmium (Cd).
2110 Background Radioactive spas or therapy centers are found primarily in Europe, and can be seen from Britain west and south to Greece and Eastern Europe. These centers typically use natural sources of mineral water with enhanced radon-222 (222Rn) content. Natural geologic formations contain naturally decaying uranium-238 (238u). There are various methods of therapy. They include bath procedures, drinking of the water, inhalation therapies, or a mixture of several techniques. Traditions originating from ancient times, like the old Roman baths, recommended the use of these baths for alleviating symptoms of such ailments as joint diseases, like arthritis. To this day people travel from all over the world to participate in arthritis therapies at these spas. Those who use and are prescribed these baths by physicians claim they work. The radioactive baths contain a rich supply of minerals, like sodium (Na), calcium (Ca), magnesium (Mg). The water ranges from warm to hot due to the natural decay process of the uranium. Figure 1 shows a map of Greece in which three major areas of therapeutic baths are found. There are several others, but their dose rates in terms of radioactivity and mineral richness are magnitudes lower than the ones pictured. Geological and mythological history indicates that in the region of the present Aegean Sea, Santorini (also known as Thera) was the indicated center of a large volcanic eruption. From this may have come the generation of many of the Aegean islands. As a result, three major areas in Greece were left with fissures of places where the activity from this eruption would bubble to the surface. These three major area are Edypsos and Kamena Vouria, with four radioactive springs studied; Loutraki, with five springs measured; and Ikaria, with its nine radioactive springs investigated [3, 4]. As Figure 1 shows, the therapeutic spas of Edypsos and Kamena Vouria are found on the mainland region of Attika. The spas of Loutraki are located near the Isthmus of Corinth. The springs and baths on the island of Ikaria are located in the Eastern Aegean Sea.
2111
/
v>
B U L G A R I A
r Y U G O S L A Y I A
J/
^J^-^**'
V--
/
EDYPSOS 5 units 222Rn:
^ .
investigated 1-200 kBq»m-3
226Ra: 1.1-4.8 kBq.ni-3
AEGEAN SEA
KAMENA VOURLA 4 units
^^^!? ^•' ^^ IKARIA 9 sources investigated 222Rn: 160-5700 kBq-ra-3
investigated
226Ra: 0 . 2 - 4 . 7
222Rn: 850 kBq-m-3 226Ra: 1.5 kBq-m-3
P ^ LOUTRAKI
^
n .^
I?;
y\-
5 units investigate!! -Rn: 90-450 kfiq-m" -0.1 kBq-m-
fJ^; i; E D I T E R R A K E A f! SEA
Figure 1. Map of Greece showing three radon spas (Reference 3).
i
kBq-m-3
2112 The most radioactive of the three areas in Greece is on the island of Ikaria. The value found in Figure 1, for Ikaria, the highest value is 160-5700 kBq pfT^. This value can be compared to typical values for 222Rn jp drinking water as follows: an average value of 2-5 Bq m"^ found in open reservoirs of water; values increasing up to 10 Bq m"^ for deep well water sources. In comparison radon decay products in the air equal 2-5 Bq m-3 outdoors, and 5-50 Bq m-3 indoors. The SI unit for radioactivity is the becquerel (Bq). It is defined as one disintegration per second (dps). In Ikaria the nine radioactive springs come from beneath the sea and bubble up through the sea water. The Ikarian Pelagos, the part of the Aegean Sea that borders the southern coast of Ikaria is quite deep, with depths right at the shoreline beginning at 20-30 feet and extending to hundreds of feet within a mile off shore. The southern coast of the island has no beaches. The shoreline is rocky and quite steep, as if part of the island fell into the sea. Consequently the spa centers here are located immediately next to the shoreline. Several studies have investigated thermal radioactive spas in Greece. These baths were topics of investigation in the last century, as well as in the earlier parts of this century [5-9], when the therapeutic spas were more popular. The three major radioactive spas in Greece were most recently studied during the years 1984-87, excluding the year 1986 during which the Chernobyl accident occurred in the former USSR. The study was done to determine the concentrations of radiologically significant natural radionuclides in water [3]. The study also determined the concentrations of indoor air (the air inside the bath houses), and to estimate the annual doses to personnel and patients using these baths. The results obtained were well within the limits set by international agencies. The purpose of this study then was to determine if the natural drinking water on the island of Ikaria (which shows the highest radon levels) was affected by these radioactive springs, both near and far from some of the radioactive sources on the island.
2113 Experimental Setup To see if the radioactive springs were related to the drinking water springs that the island uses, samples were collected from the non radioactive springs. The sample springs from which the drinking water was collected were chosen to be near and far from some of the radioactive thermal springs. Nine drinking water samples were collected. The representative sample collections were taken at sites around the southern, western, and northern coast towns and villages of the island. Table 1 lists the collection sites. Sample #101 came from the capitol city of the island. Agios Kyrikos. Here one of the therapeutic baths is located. The water is pumped from the spring called "Moustafa", which is located along the deep water right off the furthest eastern part of the city. Agios Kyrikos is located on the southern coast of the island, about one-third of the way from the eastern tip. The sample was collected from a public town faucet. This faucet provides water from a mixture of sources, nearby drinking water wells, and from wells to the west near Lefkada, another area where radioactive springs are located. Sample #202 came from the southern village of Manganiti. This village is a one-hour caique (small covered motor boat) ride west of the capitol, Agios Kyrikos. Sample #202 was collected from a public faucet near the harbor. The water comes from a mountain spring above the village. Figure 2 shows the faucet near the boat. Karkinagri, a small village of about 190 summer and winter homes and almost at the western tip of the island, is a two-hour caique ride west of the capitol city. Several samples were collected here. Sample #303, Raxouni, was collected directly from a mountain spring about a half-hour climb from the harbor, and on the face of the mountain known as "Anginofa". Figure 3 shows this source. The Tratradon sample #404, was collected below the mountain spring, directly from a running stream, which Is fed by mountain springs higher on the mountain range. The sample, called Maggouraton #505, was collected at the furthest western edge of the village from a well that feeds a private home water supply used for irrigation. This sample contained debris and particles
2114 Table t Representative Drinking Water Collection Sites on Ikaria, Greece Agios KyrikQS (#101) - Capitol city of island, where one of the therapeutic baths is located. - City is located on southern coast of island. - Sample was collected from town faucet.
Manganiti (#gQ2)
- Southern village, 1-hour caique ride from capitol. - Sample was collected from town faucet.
Raxouni f#303) - Small mountain spring on western side of island. - Sample was collected about one-half hour climb from sea level. Tratradon (#404) - Sample was collected from running mountain stream below mountain spring of #303 above.
Maqgouraton (#505)
- Sample was collected from well in western part of village of Kari
Karkinaori (#606) - Small southwestern village, 2-hour caique ride from capitol. - Sample was collected from kitchen faucet. Stou KalQU (#707) - Remote areas on western part of island. - Sample was collected from running mountain spring at a climb of about one-half hour from sea level.
Kotsagrgij (#8Q8)
- Sample collected from Karkinagri from black rubber hose used to bring irrigation water to one farmer in the village.
EvdilQS (#9Q9) - City on northern coast of the island. - Sample collected from a town faucet. Blank (#1010) - Deionized water from laboratory acidified to a pH of 2.0.
2115
Figure 2. Sample Collection Site in Manganiti (#202).
2116
Figure 3. Sample Collection Site in Karkinagri at Raxouni (#303). from a nearby tree and other foliage. Sample #606, Karkinagri, was obtained from the kitchen faucet of one of the village homes in the northern edge of the village. This water comes from a village reservoir which is filled from several mountain spring sources around the village. Stou Kalou, sample #707, came from a remote area on the western part of the island. The sample was collected from a running mountain spring at a climb of about one-half hour. This water sample was obtained from the highest mountain source, which comes from a height of about 2,000 feet. Figure 4 shows the place on the mountain where the
2117 water runs. This spring feeds into the water supply of two villages, Karkinagri and Trapalou. The Kotsagreli sample, #808, was collected from the black rubber hose used to bring irrigation water to the village.
Figure 4. Sample Collection Site #707 at mountain spring, Stou Kalou.
2118 On the northern edge of the island came the last sample. Sample #909, from the city of Evdilos, was collected from a public town faucet, which is also near the harbor. Figure 5 shows this faucet. Evdilos is one of the largest cities on the island and is located on the northern coast about midway between the two ends of the island. Sample #1010 was a blank solution, made of acidified deionized water. This was done to match the acid conditions under which the representative samples were stored.
Figure 5. Sample Collection Site in Evdilos (#909), town faucet.
2119
Sample Preparation The bottles used for sample collection were made of highest quality polyethylene. They were cleaned with cleaning solution and ultra pure 14 M nitric acid (HNO3) and double distilled water from the laboratory. This was done to prevent any leaching of impurities left from the walls of the bottles to the sample. One-liter bottles were used to collect the samples. The bottle was filled twice and rinsed with the sample to be collected. The third filling was used as the sample. As the samples were collected, the pH of each sample was taken with pH paper as an initial range reading and with a calibrated pocket pH meter. Table 2 lists the pH values of each of the samples. Then each sample was acidified to a pH of about 2, using ultra pure HNO3. The samples were kept refrigerated until used, in order to prevent any leaching of elements from the sample into the walls of the bottle, and to prevent bacterial growth.
Table 2 pH Measurements of Samples Collected #
Sample
pH
Comments
101 202 303 404 505 606 707 808 909
Agios Kyrikos Manganiti Raxouni Tratradon Maggouraton Karkinagri Stou Kalou Kotsagreli Evdilos
7.0 7.6 7.3 7.3 6.8 7.6 7.4 8.0 8.4
Town faucet Town faucet Mountain spring Running stream Well Kitchen faucet Mountain spring Irrigation hose Town faucet
2.0
Distilled water
1010 Blank (with HNO3)
2120 Aliquots of 0.5 mL were used in the analysis [10]. These aliquots were pipetted into 3-mL polyethylene vials and sealed by melting the cap onto the body of the vial with a hot soldering iron. Each sample was double sealed by placing the 3-mL vial into a 5-mL cryogenic screw top vial. Five samples at a
Figure 6. Aluminum sample holder used in the reactor.
2121 time were placed into a round aluminum holder and lowered into the correct position near the reactor core. The holder was rotated constantly to ensure even irradiation. Figure 6 shows the aluminum holder. The "Demokritos" reactor at the National Centre for Scientific Research (NCSR) in Aghia Paraskevi, near Athens, was used for the irradiations. The reactor is a 5 Mw reactor, and was used at full power. Irradiation was done for 20 minutes. The samples were collected in the reactor pool, away from the core, for three days. The aluminum holder was too "hot" to remove from the pool, as were some of the isotopes, which were too "hot" to measure. Health physics personnel were in charge of checking to be sure that the samples were within appropriate radiation guidelines before the samples were removed from the reactor pool.
Experimental Method The method of instrumental neutron activation analysis (INAA) was used to analyze the samples for trace and ultra trace elements [11]. Gamma ray spectroscopy was used to interpret the data. Figure 7 shows a block diagram of the INAA technique. A high purity germanium (HpGe) detector was used. The sample is placed directly next to the detector face for counting. Lead shielding was built around the detector as indicated in Figure 7. Gamma radiation from the sample strikes the detector face and the Ge. This gamma radiation excites the electrons in the Ge into its conduction band causing an electronic signal to be generated. The signal is enhanced by the preamplifier and amplifier to become an analog pulse. The pulse is then digitized and passed to the multichannel analyzer which deposits the signal (count) into the appropriate energy "bin". Since radiation events are not continuous, radiation striking the Ge detector is generally of different energies at different times (fractions of a second). Therefore the multichannel analyzer (MCA) must be able to separate, sort, and store these digitized events quickly. A gamma-ray spectrum thus "grows" over a
2122
o
^1
E
< Q.
E
<
H-
Figure 7. Diagram of Instrumental Neutron Activation Method (Reference 11).
2123 counting time. Peaks at various energies form depending on the type of nuclide, and how much of that nuclide is present. For example, the 24fsia peak will be large and its area may hide other smaller peaks. Therefore, it is necessary to wait until the ^^Na decays (its half-life is 15 hours) and its area decreases. Fortunately, this occurs faster than other longer-lived nuclides (halflives of many hours or days), and more information can be obtained on the samples. The only other way to deal with the large 24Na peak area is to chemically or physically remove the sodium from the sample. However, it can introduce contamination and result in incorrect results. This is the advantage of INAA. There is no need to chemically or physically alter the sample. This will result in more accurate information and less contamination of the sample, a must when determining trace and ultra trace concentrations. Samples were counted for one hour each using the Canberra Multichannel Analyzer Series 35+ System. Figure 8 shows the counting setup in the INAA laboratory. Each sample was counted at an interval of 3 days and at 15 days. This second round of counting was done to see if any longer-lived isotopes of interest would be found hiding under the larger peaks of the shorterlived isotopes. The MCA records and stored the information from the counting interval. Canberra'a Spectra Scan software was used to do the peak searches. This program searches for energy peaks and calculates peak areas. Since all nuclides have unique patterns of energies for their series of gamma-ray radiations, gamma-ray spectra are fingerprints of the various kinds of elements found in a sample [12]. The peak areas give a quantitative evaluation of each element. Measurements into the parts per million and parts per billion range are thus routine for the INAA technique.
2124
Figure 8. Canberra Multichannel Analyzer Series 35+ setup.
RgsMlt^ Hand analysis of the peak searches found the following peaks In each sample spectrum: sodium-24 (24Na), bromine-82 (^^Br), gold-198 O^^Au). Uranium decay products of radon-226 (226Rn), lead-204m (204mpb), and neptunium-239 (239Np) were measured in some of the samples from the mountain springs. This is due to natural radioactivity found in the geologic
2125 formations. The amounts could not be substantiated by the methods used and were not statistically valid (greater than 30% error). This could also mean that the samples became contaminated. Quantitative analysis could not be verified, because the counting statistics were in most cases much greater than 10% error, typically 20-30%. A better technique for these drinking water samples would be to use freeze drying techniques. It would alleviate gas buildup in the sample vials during irradiation, causing leakage of the vial. The gas build up occurs from the radiolytic reactions of water in a radiation field. The gases formed expand, pressure in the vials increase, the vials rupture. Freeze drying techniques concentrate the sample into solid form, which is easier to handle in a reactor core. It would permit greater irradiation times and would therefore Improve counting statistics.
QgnclM^ign^ In summary, the drinking water sources tested on the island of Ikaria at the sample locations are pure. They contain very few elements, an indication of soft water. Results indicate that there were no statistically significant traces of the elements of interest, either of nutritional or toxic value in the drinking water samples. Some of the samples indicated ultra trace amounts of uranium, but since these results were not statistically valid, the results could not be verified if the uranium came from the water samples or were from some sort of contamination of the samples after collection. The pH measurements indicated that the water varied from a pH of 6.8 (sample #505 from a well) to 8.4 (sample #909) from the city public faucet on the northern coast of the island. Environmentally, this indicates that the water samples have little buffering ability, if the water were to become polluted. It also indicates that drinking water sources, are for the most part, from mountain springs, and not from deep wells. The drinking water sources are different from the radioactive mineral thermal springs, which come from deep in the sea and not from the mountain springs.
2126 Acknowledgments The project was funded by a Sabbatical Leave from the Chemistry Department at Eastern Michigan University. It was also supported by a Visiting Scientist position at NCSR "Demokritos" in Greece. The author thanks George N. Contis who assisted in collecting the drinking water samples. Dr. loannis Papazoglou, Director of the Institute for Radiation Technology and Protection, offered office and computer facilities for the project. Figure 9 shows the entrance to the Institute at NCSR. The author also expresses much appreciation to Kostas Papastergiou, Manager of the "Demokritos" reactor laboratory, who provided technical personnel, reactor time, and facilities; and to loannis Anousis, Department Head of the Neutron Activation Analysis Section, who provided exceptional assistance in parameter and analysis setup.
Figure 9. Entrance to the Institute for Radiation Technology and Protection.
2127 References 1
E.T. Contis, "An Overview of Radiochemical Methods to Determine Trace Elements in Food", chapter in Food Flavors, Ingredients and Composition, G. Charalambous (Ed.), Elsevier Publishing, Amsterdam, 1993.
2
Food Flavors, Ingredients and Composition, G. Charalambous (Ed.), Elsevier Publishing, Amsterdam, 1993.
3
P. Kritidis and M. Probonas, "The Greek Radon Spas: Hot Spots of Natural Radioactivity in the Mediterranean Area", Report from the Environmental Radioactivity Laboratory, National Centre of Scientific Research (NCSR) "Demokritos", Aghia Paraskevi, Greece, 1988.
4
Private communication with Dr. A.P. Grimanis' group. Analytical Chemistry Division, NCSR "Demokritos", 1992.
5
M. Pertesis, "The Greek Mineral Springs", Geological Service of Greece, Report #24, Athens, Greece, 1937.
6
M. Pertesis, "About the Radioactive Hot Springs of Ikaria Island", Proceedings of the Academy of Athens, 14,1939.
7
E.K. Platakis, The Radioactive Thermal Mineral Springs of Ikaria (Greek), Athens, 1959.
8
N.G. Leousis, Radioactive Springs of Hellas (Greek), Athens, 1969.
9
K.G. Makris et al. New Research In the Thermal Mineral Radioactive Springs on the Island of Ikaria (Greek), Thessaloniki, Greece, 1965.
10
Private communication with loannis Anousis, Department Head, Neutron Activation Analysis Laboratory, NCSR "Demokritos", Aghia Paraskevi, Greece, 1992.
11
Activation Analysis, volume 2, Z.B. Alfassi (Ed.), CRC Press, Boca Raton, Florida, 1991.
12
S.I. Najafi, "Computerized Analysis of Gamma-Ray Spectra", chapter 2, volume I, Activation Analysis, Z.B. Alfassi (Ed.), CRC Press, Boca Raton, Florida, 1991.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2129
Emulsifying Properties of Lupin Seed Proteins (Lupinus albus, ssp. Graecus).
S. Alamanou and G. Doxastakis. Laboratory of Food Chemistry and Technology, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54006, G R E E C E .
ABSTRACT
The emulsifying properties of lupin seed protein isolates prepared by different methods have been studied. The resulting isolates were used for the preparation of o/w emulsions in order to evaluate their emulsifying properties. It was observed that the isolate prepared by the isoelectric precipitation method is an efficient emulsifier and exhibits satisfactory emulsion-stabilizing ability when compared to the other isolates. 1. INTRODUCTION Legumes are an important source of plant proteins for human consumption. Although legume proteins are low in some essential amino acids, they are the main protein intake in parts of the world where animal protein is limited (1,2). Lupin has a good potential to be a valuable crop both for its protein (35-45%) and oil (11%) content (3). Lupin oil is an excellent source of unsaturated fatty acids (78%), out of which 25-35% are polyunsaturated (4,5).
2130 The polysaccharide lupin fractions are typically nonstarch and likewise similar are the oligosaccharide fractions. In common, lupin seeds contain significant amounts of the oligosaccharides of the raffinose family (6). The increasing interest in protein rich plant seeds, such as lupins, for use in human and animal nutrition also focuses attention on the substances known as antinutritional factors (ANFs). The most important ANFs in legume seeds are protease inhibitors, lectins, tannins, saponins and phytic acid (phytates). Alkaloids are of particular concern in the lupin seeds, which otherwise offer promises as a rich source of protein. Many of the ANFs can be eliminated or inactivated to a large degree by appropiate heating and processing during food preparation. Treatments may include dehulling, presoaking and diffusion, sterilization, steaming and cooking. Wet milling and processing techniques employed during protein concentration and isolation are known to be effective in the detoxification of seed materials (8). The use of lupin products as a source of protein for humans will depend not only upon its nutritional quality but also on its ability to be used as, or incorporated into, foods which will be readily consumed. So, the functional and physical properties rather than the nutritional value of proteins will largely determine their acceptability as ingredients in prepared foods (9,10). One of their most important funtion is their ability to adsorb at the o/w interfaces and stabilize the oil droplets by forming cohesive and mechanically strong interfacial films which may exhibit viscoelasticity (11-14). Difficulties in studying the functional properties of vegetable proteins arise from the complexity and variability of the system. In fact, composition, conformation and structural rigidity of proteins
2131 vary depending on the operating conditions of the process. On top of that, other constituents, such as carbohydrates, phytin, etc. interact with protein during the isolation process and give to their products various functional properties (15-17). This is due to the protein-polysaccharide complexes which exist in lupin seed protein isolates (LSPI) and alter their functional properties. The present work aims to investigate the influence of the extraction methods used for LSPI isolation in relation to the emulsifying properties. This may help to understand better the role of extracton methods to the functional properties of legume proteins. 2.EXPERIMENTAL 2.1. Materials Lupin seeds (Lupinus albus, ssp. Graecus) were provided by a Greek grower (Lasithi, Crete). All the pH adjustments were made with IN NaOH and IN HCl solutions. 2.2.
Preparation of isolates. The dry seeds were ground in a Tomas-Wiley Mill, model ED-5, USA to pass a 100 mesh screen; in this way it is possible during the solubilisation by alkali the protein extraction out of the flour up to 96^ (18). The flour was then defatted with n-hexane (l:3w/v) by Soxhlet method during 8 hrs at around 40*^C until fat content was determined to be 5.0% (19). Solvent residues were removed by drying the flour at room temperature. The lupin flour was diluted in 10 times distilled water and the pH of the solution was increased to 9.0 with continious stirring at room temperature. The slurry was kept at the above pH with periodical stirring for 30 min during which it was found that the maximum amount of protein extracted from the ground flour was achieved. For further increase of this amount the solution was centrifuged at 5000g,
2132 the supernatant was collected and the residual was redissolved with distilled water 1:5 w/v. After a second centrifugation both supernatant solutions were joined. This processing was not continued any more because protein percentage which is extracted after the second residual washing is very small and also the resulting solution volume increases very much causing problems in handling. In this way the protein extract was received and was separated from the insoluble polyssaccharides and crude fiber. The pH of the protein solution was then adjusted to 7.0 so that neutral protein could be finally received by isoelectric precipitation, dialysis and polyacrylamide gel methods as described elsewere (20). 2.3
Analytical determination Nitrogen content was determined by using the Kjeldahl method and N was multiplied by 5.6 when protein content was refered to the intermediate solutions and by 5.7 when it was refered to the final isolate (21). Protein content was expressed on a dry basis which was determined by drying the samples at 105 ^C to constant weight . All the determinations as fat, moisture, ash and nitrogen were performed according to the official methods (22). 2.4
Preparation and stability of o/w emulsions The o/w emulsions were prepared by adding 50ml of corn oil into 50ml of LSPI solution containing 0.5% protein and having a pH of 3.5, 5.5, 6.0 and 6.5 while mixing with the aid of a mechanical stirrer. The crude emulsion, following mixing for 3 min, was then homogenized with an Ultra-Turrax T-25 homogenizer (IKA Instruments, Germany) equipped with a S25 KG-25F dispersing tool, at a speed of 9,500 r.p.m. for 1 min. Emulsification conditions were chosen to result in oil droplets larger than Ipm. A small amount of Penicillin was added to the water phase as a preservative.
2133 The stability against coalescence of the o/w emuslions was studied by storing at 5** C and then by following their oil droplet size distribution pattern changes with ageing time. The droplet size distribution patterns were determined with the light microscope method (23). The distribution patterns were used to calculate the mean volume diameter (Dv) according to equation: SniDi' (1) 2ni The rate of droplet coalesence (K) was calculated from the equation: Nt-No exp[-Kt] (2) where Nt and No are the numbers of droplets per ml of emulsion at times t and t=0, respectively, and: Nt =
10 '^
(3)
nDv where 0
is the oil phase volume.
3. RESULTS AND DISCUSSION Table 1 shows the proximate analysis of LSPI extracted by isoelectric precipitation, dialysis and polyacrylamide gel methods. The lower protein content for LSPI by gel may be due to the low polymer efficiency. Tables 2-4 present the oil droplet size data and the rates of drop coalesence of emulsions stabilised with LSPI prepared by the three different preparation processes. Moreover, the effect of the pH value of the aqueous phase on the emulsion stability was studied.
2134 Table 1. Proximate analysis of LSPI prepared by Results expressed as g/lOOg d.w.
differnt
% constituent
LSPI*
LSPf
Protein (Nx5.7) Oil Ash Moisture
92.1 0.5 0.1 7.3
82.7 0.5 0.2 6.1
methods.
LSPf 41.4 0.7 0.3 7.6
1. LSPI by Isoelectric precipitation 2. LSPI by dialysis 3. LSPI by polyacrylamide gel
Table 2. Effect of LSPI prepared by isoelectric precipitation on the stability against oil droplet coalesence of o/w emulsions at various pH.
Ageing time (hr)
Emulsionl Emulsion2 Dv (pm) 29.6 37.0 38.1 39.3 40.2
3 24 72 144 240 -1)
KxlO^ ( sec
2.8
28.5 37.2 38.9 39.3 40.6 2.3
Emulsions
31.1 33.3 34.3 36.8 37.9 5.9
Emulsion4
33.5 34.1 34.9 36.3 38.2 5.4
Emulsion 1: Prepared with aqueous phase pH at 3.5, Emulsion 2: Prepared with pH at 5.5, Emulsion 3: Prepared with pH at 6.0, Emulsion 4: Prepared with pH at 6.5.
2135 Table 3. Effect of LSPI prepared by dialysis on the stability against oil droplet coalesence of o/w emulsions at various pH. Ageing time (hr)
Emulsionl
Emulsion2
Emulsion3
Emulsion4
Dv(|jm)
3 24 72 144 240
KxlO (sec )
12.7 16.3 17.7 18.8 21.0
18.2 19.9 22.2 23.3 25.1
20.9 23.7 26.8 28.7 30,1
31.1 33.2 37.5 39.2 42.7
5.8
6.6
6.8
5.8
Emulsion 1: Prepared with aqueous phase pH at 3.5, Emulsion 2: Prepared with pH at 5.5, Emulsion 3: Prepared with pH at 6.0, Emulsion 4: Prepared with pH at 6.5. Table 4. Effect of LSPI prepared by polyacrylamide gel on the stability against oil droplet coalesence of o/w emulsions at various pH. Ageing time (hr)
3 24 72 144 240 KxlO^(sec*)
Emulsionl
Emulsion2 Dv(Mm)
Emulsion3
Emulsion2
24.2 24.5 29.7 30.5 31.6
36.9 51.8 53.1 53.9 55.2
34.0 41.6 44.7 46.3 47.5
35.2 39.9 42.7 44.0 46,3
3.1
3.0
4.2
4.5
Emulsion 1: Prepared with aqueous phase pH at 3.5, Emulsion 2: Prepared with pH at 5.5, Emulsion 3: Prepared with pH at 6.0, Emulsion 4: Prepared with pH at 6.5.
2136 The difference in emulsifying and emulsion-stabilizing ability among the LSPI prepared by the three different methods may be attributed either to varieties in composition, since the isolates are expected to contain different amounts of soluble protein fractions (e.g albumins) and carbohydrates or to additional denaturation resulting during the isolation process (Table 1). In order to test this remarque, emulsions were prepared with all LSPI at pH 3.5, 5.5, 6.0 and 6.5. The increase with time of the oil droplet size of the emulsions is given in Tables 2-4. The initial oil droplet sizes (Table 2) are lower near the isoelectric point (e.g. 3.5 and 5.5). This becomes more obvious when comparing the rates of drop coalesence. At the isoelectric region, the LSPI molecules are in a more compact form than at other pH values. They are adsorbed at the oil-water interface in this configuration and so should provide a higher concentration of protein molecules per unit area of interface and consequently, a larger number of interlinkages per unit area, than at other pH values (25). Tables 3 and 4 show that the oil droplet sizes increase faster with ageing time than those of the Table 2, which leads to the conclusion that the LSPI prepared by dialysis and polymer gel are less effective in emulsifying and stabilizing emulsions. This could be due to the less degree of denaturation and unfolding and in the presence of higher amounts of carbohydrates and other constituents.
4. ACKNOWLEDGEMENTS S. Alamanou wishes to thank the Scholarships for financial support.
Foundation
of
State
2137 REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11. 12. 13.
14. 15. 16. 17.
A.A. Seyam, J.O Banasik and D.M. Breen, J. Agric. Food Chem., 31 (1983) 499. J.R. Evans and L.S. Bandemer, J. Agric. Food Chem., 15 (1967) 439. P. Cerletti and M. Duranti, J. Amer. Oil Chem. S o c , 56 (1979) 460. G.D. Hill, Nutr. Abst. Rev., 47 (1977) 511. W. Watkin, Euphyt., 28 (1979) 481. R. Macrae and A. Zand-Moghadddam, J. Sci. Food Agric, 29 (1978) 1083. Y. Birk, In: Y. Birk, A. Dovrat, M. Waldman, C. Uzureau, (Eds). Proceedings of the joint CEC-NCRD workshop, Belgium, (1990) 79 K. Elkowicz and F.W. Sosulski, J. Food Sci., 47 (1982) 1301. W.D. Johuson, J. Amer. Oil Chem Soc., 47 (1970) 402. C.W. Hutton, A.M. Campbell, J. Food Sci., 42 (1977) 457. E.D. Graham and M.C. Phillips, J. Colloid Interf. Sci., 76 (1980) 240. V. Kiosseoglou, K. Theodorakis and G. Doxastakis, Colloid Polym. Sci., 267 (1989) 834. O.D. Velev, A.D. Nikolov, N.D. Denkov, G. Doxastakis, V. Kiosseoglou and G. Stalidis, Food Hydrocoll., 7 (1993) 55. B.E. Elizalde, R.J. De Kanterewicz, A.M.R. Pilosof and G.B. Bartholomai, J. Food Sci., 53 (1988) 845. H.D. Chou, and V.C. Morr, J. Amer. oil Chem. S o c , 56 (1979) 53A. B.V. Tolstoguzon, Ya, V Grinberg, N.A. Gurov, J. Agric. Food Chem., 33 (1985) 151. V. Kiosseoglou, G. Doxastakis, Lebensm.-Wiss. u. Technol., 21 (1988) 33.
2138 18. P. Leonard, Jr. Ruiz and E.L. Hove, J. Sci. Food Agric, 27 (1976) 667. 19. W.W. Christie, Lipid Analysis (1982) 22. 20. S. Alamanou and G. Doxastakis, Food Hydrocoll., (1995, in press). 21. G. Malgarini and B.J.F Hudson, Riv. Ital. Sostanze Grasse, (1980) 378. 22. AOAC. Assoc. Official Analytical Chemists, Washignton, D.C. (1975) 23. T. Mita, E. Iguchi, K. Yamada, S. Matsumoto and D. Yonezawa, J. Texture Studies, 5(1974) 89. 24. P. Sherman, In Emulsion Science, P. Sherman (ed.) Academic Press, London, 1986. 25. G. Doxastakis and P. Sherman, Colloid Polym. Sci. 264 (1986) 254.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2139
MULTIRESPONSE OPTIMIZATION BY A NORMALIZED FUNCTION APPROACH John D. Floros and Hanhua Liang 1160 Smith Hall, Department of Food Science, Purdue University, West Lafayette, IN 47907-1160, USA ABSTRACT A simple new method, called Normalized Function Approach (NFA), was developed for simultaneously optimizing systems defined by several output variables (response functions) and a common set of input variables (factors). For each original response function the difference between the estimated response and its individual optimum was evaluated and normalized over the experimental space. The individually normalized functions were then weighted for their importance and combined into an overall function using a "sum of squares" approach. Simultaneous optimization was completed by minimizing the overall function. Some numerical examples were considered to introduce the new method and several reported applications in the food area were used for verification and comparison with other multiresponse optimization methods. INTRODUCTION Localization of the "best" operating conditions has attracted considerable interest by the food industry during the last few years. For systems with more than one response, searching for the "best" (optimum) conditions becomes a multiresponse optimization (MRO) problem. Many MRO methods have been proposed. The most commonly used methods are: (a) graphical approaches (GA) (Lind et al., 1966; Floros and Chinnan, 1987; Floros and Chinnan, 1988; Floros, 1992); (b) desirability function approach (DFA) (Derringer and Suich, 1980); and (c) generalized distance approach (GDA) (Khuri and Conlon, 1981). All these methods have some advantages and disadvantages. For example, graphical approaches are simple and straightforward methods that usually provide reliable solutions. However, they are time consuming, need advanced computer graphics capabilities, and become complicated when the system under consideration has more than three factors. DFA is a relatively simple and efficient method, but relies heavily on subjective user input to determine critical values. The GDA method does not need any subjective input, it requires only short computer time to obtain a solution, but it is complicated in formulation, can utilize only polynomial functions of the same order, and it may produce nonfeasible solutions. It is evident therefore, that a simple, time efficient and more reliable MRO method is needed. By investigating all the existing MRO methods, three common key steps are apparent and can be stated as follows: I. Normalization. Most methods standardize and evaluate all responses on the same scale. For example, graphical methods project all responses onto the same x plane; the DFA method transforms each response into an individual desirability function.
2140 2. Combination. All existing methods combine all responses into one formulation so that an evaluation can be done simultaneously. For example, graphical methods superimpose all contour plots onto the same figure; the DFA method combines all individual desirabilities into an overall desirability function; and the GDA method introduces the socalled distance function. 3. Optimization. All methods optimize the fmal formulation obtained from step (2) by a univariate optimization method. Based on the above three key steps a new MRO method, called Normalized Function Approach (NFA), is proposed. The new method is simpler, easier to apply, and does not rely on subjective user's input. In this paper, the basic theory of the method is presented, three numerical examples are used to demonstrate how the method works, and finally, three application examples from food processing are employed to show the applicability of the method and to compare its results to those obtained form the GA, DFA and GDA methods. THEORY For a system with m responses and p factors, there must exist a set of expressions which relate each response to the factors
ytj-fMk)
•'^ij
i=l, 2, ...m y=i,2,...« k=l, 2, .../?
[1]
where, yy are responses, x,, are factors, n is the number of investigations for the zth response, and fijj is the error term associated with the /th response and the yth investigation. By assuming that e^ is a randomly distributed error and Efej) = 0, the expected iih response can be expressed as [2]
yi=A(h)
After the functions/ have been found and tested for statistical adequacy, the maximum (yimax) ^rid minimum {y'\,,J values for each response must be located over the experimental space. This can be accomplished through a very simple program written in any computer language. A desirable optimum value (yiopt) must then be chosen for each response in the system. This optimum (yiopi) may take any value in the interval yi^^^x ^ yiopt ^ yiminThe second step involves the transformation of each response into a normalized response, yNi, by
max
min
2141 so that -1 < y^i < 1. All the normalized responses are then combined into an overall function, F, by summing their individual square terms as m ^i yNi
[4]
F =^ m i=l
where, a^ is a weight factor, which allows the user to account for the importance of each response. Values of aj = 1 should be used when all responses have equal (similar) importance. The overall function F has a range of 0 < F < 1. The final step is to find the optimum conditions by minimizing F. Since Eq. [4] is a continuously inivariate function of x^, any single response optimization method can be used. NUMERICAL EXAMPLES Three numerical examples were formulated in order to show how the method works and to illustrate in detail the procedures of each step described above. All examples are multiresponse problems with a single factor, so that the results can be easily plotted and visualized. The general formulation for all examples is: y. = Po + PjX + p^ x^
[5]
The polynomial form is used here simply because it is easy to obtain illustrative curves. The numerical values of parameters BQ, fii and B2 for all three examples are listed in Table 1. Table 1. Regression parameters of Eq. [5] for the three numerical examples Numerical Example
Parameter
Response 60
B.
y2
10.00 2.00
0.00 0.00
20.00 -20.00
2
yx y2
10.00 10.00
20.00 -20.00
20.00 20.00
3
y\
22.20 12.17 1.75
-24.00 16.67 5.00
20.00 16.67 -25.00
1
yi
y^
B2
2142 The first example was formulated so that the two responses (yi and yj) attain their individual optima at the same x value (x=0), which should also be the value of the simultaneous optimum. Fig. lA shows the values of ji and y2 vs. x, while the normalized responses y^^i and yj^2 were calculated according to Eq. [3] and plotted in Fig. IB. The normalized function F was calculated by Eq. [4] using ai=a2 = l and is shown in Fig. IC. The minimum value of function F was found by an unconstrained univariate optimization subroutine in IMSL software library (IMSL, 1987). Individual and simultaneous optimum values for x and y^ are listed in Table 2 along with results obtained by the GDA method. It can be seen (Table 2) that NFA produced the expected results. Table 2. Individual and simultaneous optimum solutions for the three numerical examples as calculated by NFA and GDA methods Numerical Example
1
2
3
Response
yiopt
Individual optima
yi mm y2 max
10.0 2.0
0.0 0.0
Simultaneous optimum (NFA)
yi min y2 max
10.0 2.0
0.0
Simultaneous optimum (GDA)
yi min y2 max
10.0 2.0
0.0
Individual optima
yi min yi niin
5.0 5.0
-0.5 0.5
Simultaneous optimum (NFA)
yj min y2 min
10.0 10.0
0.0
Simultaneous optimum (GDA)
yi min y2 min
10.0 10.0
0.0
Individual optima
yi min y2 ^iri y3 max
15.0 8.0 2.0
0.6 -0.5 0.1
Simultaneous optimum (NFA)
yi min y2 ^ i ^ ys max
21.6 12.6 1.9
0.03
Simultaneous optimum (GDA)
yi min y2 rnin y3 max
20.9 13.2 2.0
0.06
^Opt
The second numerical example (Fig. 2 and Table 2) illustrates a specific case in which the two responses are symmetrical (mirror image) around a vertical line. This obviously indicates that the simultaneous optimum should be located at the intersection of the two responses (at j = 0) when ai=oi2 = l. Again, NFA produced the expected solution. The
2143
Ym 0.0 f
-1.0
-0.5
0.0
0.5
1.0
X
Figure 1: Graphical representation of numerical example 1 showing (A) true responses, (B) normalized responses, and (C) overall normalized function.
2144
-1.0
-0.5
0.0 X
0.5
1.0
Figure 2: Graphical representation of numerical example 2 showing (A) true responses, (B) normalized responses, and (C) overall normalized function.
2145 third example is a more general case, with the three responses attaining their individual optima at different x values. The results (Fig. 3 and Table 2) indicate that the proposed NFA method worked well, and that the solutions obtained were similar to those obtained by the GDA method. APPLICATION EXAMPLES In practice, the word "best" is a relative term. Finding the "best" is eventually replaced by fmding the "better". For this reason, three real optimization problems, dealing with food systems and having known optimum solutions found by other MRO methods and verified by experiments, were used to test the applicability of the NFA method. The first application example was the optimization of a cucumber pickling process (Guillou, 1991). The objective was to locate optimum NaCl, CaCk, and Potassium. Sorbate concentrations that result in good fermentation and produce sound quality pickles (maximum texture (y,), maximum Hydrogen ion concentration (yj), and minimum yeast (log of) contamination (yg)). The regression models for this system can be expressed as Ji = A "-^l^l ^fil^l ^filh t^l \^\ -^filial -"A^^S t^i2^1^21/^13^1^3 ty^23^2^3
t^l
where JCj is the coded NaCl concentration, X2 is the coded CaCl2 concentration, and X3 is the coded Porassium Sorbate concentration. The estimated coefficients of the models and the coefficients of determination were obtained by the PROC RSREG procedure (SAS, 1990) and they are shown in Table 3. The models were statistically acceptable without any significant lack of fit. They adequately represented the system and were used for further investigation. Table 3. Regression coefficients for the three application examples
Bo 15, B, B3
6„ (522 1533
15,2 15,3 (523
R2
Application Example Pepper lye-peeling
Pickle processing
Coefficient in Eq. [6] yx 56.49 8.21 6.27 12.63 -7.61 -4.79 -10.99 -7.35 -7.55 -9.82 0.917
)'2
39.67 -7.11 4.54 5.94 -18.26 -12.07 -13.83 -5.15 -3.97 1.11 0.982
y2
}'3
0.73 -0.38 -0.40 -2.32 1.75 1.55 2.14 -0.57 0.05 -0.09 0.968
39.85 11.78 5.27 17.29 -7.47 1.67 -8.74 -2.41 3.67 -1.46 0.981
-2.55 -2.39 -2.11 -3.74 0.70 0.12 -0.25 0.69 -0.87 -0.90 0.978
Tomato (ye-ireeling yx 92.64 -3.25 -6.89 -4.57 2.10 -1.91 2.88 -1.14 -3.96 -2.69 0.946
yi
3.06 -0.92 -4.06 -2.53 1.83 -0.14 0.58 1.63 -3.92 -1.17 0.897
2146
0
60 40
h
^"^N^^^
20 0 -20 -40
1
•
1.0
.-i
0
I
0.5
i j
yN2
y;v7 J
P'--"^
YNi 0.0 k -0.5 hr
-1.0
•
U-] I
^-±
F 0.4
-1.0
-0.5
0.0
0.5
1.0
X
Figure 3: Graphical representation of numerical example 3 showing (A) true responses, (B) normalized responses, and (C) overall normalized function.
2147 The individual optima {%p^ for all responses were computed and listed in Table 4. The normalizing step was done by using Eq. [3]. The simultaneous optimum found from Eq. [4] by using ai = 1 for all responses was located at Xi = -0.07, X2 = 0.17, X3 = 0.36. The respective response values were y^ = 59.6, y2 = 41.1, yj = 0.7. These results are listed in Table 4 along with the results found from other MRO methods. This solution was within an experimentally verified optimum region and closely agreed with results derived by other optimization methods (GA, DFA, and GDA). The plot ofy^ vs. X3 (whileXi and X2 were held at their simultaneous optimum values) is shown in Fig. 4A. The y^-, vs. X3 is plotted in Fig. 4B, and the combined normalized function F vs. x^ is presented in Fig. 4C. The second application example was the optimization of a pepper lye-peeling process (Floros and Chinnan, 1987). The purpose was to determine a set of optimum processing conditions (lye concentration, x^ temperature, X2, and processing time, X3) suitable for minimizing peeling loss (yO while removing all the skin (y2) from the pepper. The same regression models used for pickle processing (Eq. [6]) were also applied in this case. The estimated coefficients are presented in Table 3. The individual optima for all responses are listed in Table 4. The simultaneous optimum found from Eq. [4] using cej = 1 was located at-^i = 1.00,^2 = -0.12,^3 = -0.88. The respective response values were: y^ = 18.6, and y2 = 1-0. The results are listed in Table 4 along with the results found by other MRO methods. The third application example was the optimization of a tomato lye-peeling process (Floros, 1994). The purpose was to determine a set of optimum processing conditions, (lye concentration, ^i; temperature, X2\ and processing time, x^) suitable for maximizing the yield of the process (y^) while removing all the skin (^2) from the tomato. Table 3 gives the estimated coefficients of the regression models. The simultaneous optimum found from Eq. [4] using a; = 1 was located atx, = -1.00, X2 = 1.00, X3 = -1.00. The respective response values were: yj = 96.5, and y2 = 1.4. The results are listed in Table 4 along with the results found by other MRO methods. CONCLUSIONS All the results obtained by the Normalized Function Approach (NFA) method using numerical examples (Table 2) and application cases (Table 4) were acceptable and compared favorably to those obtained by other MRO methods. Therefore, it can be concluded that NFA is a reliable, simple, easy to apply, and computationally efficient multiresponse optimization method. Furthermore, the NFA does not only give the location of an optimum, but it can be used to plot all normalized responses onto the same scale, allowing the user to see the behavior of each response and how it reaches its optimum.
2148 Table 4. Individual and simultaneous optimum solutions for the three application examples as calculated by NFA and other MRO methods. Application Example
Optimum Factor Values Response
yilOpt
^lopt
^2opt
yi max % max y3 min
60.8 41.9 0.1
0.35 -0.26 0.08
-0.15 0.26 0.13
0.52 0.26 0.54
simultaneous optimum (NFA)
yi max y2 max
59.6 41.1 0.2
-0.07
0.17
0.36
Pickle simultaneous processing optimum (GA)
yi max y2 max y3 min
58.0 40.8 0.5
0+0.4
0+0.7
simultaneous optimum (DFA)
y, max % max y3 min
59.4 38.5 0.7
0.03
0.59
0.16
simultaneous optimum (GDA)
yi max y2 rnax y3 min
59.5 41.4 0.2
-0.13
0.22
0.38
individual optima
y, mm y2 min
0.0 0.0
-1.00 1.00
0.00 1.00
-0.95 1.00
simult. opt. (NFA)
y, mm y2 min
18.6 1.0
1.00
-0.12
-0.88
simult. opt. (GA)
yi min y2 min
19.4 1.0
1.00
0+0.4
simult. opt. (GDA)
yi min y2 min
21.6 1.7
1.00
1.00
0.97
individual optima
y, max y2 min
100.0 0.0
-1.00 0.90
-1.00 1.00
1.00 1.00
simult. opt. (NFA)
y, max y2 min
96.5 1.4
-1.00
1.00
-1.00
simult. opt. (GA)
y, max y2 min
95.9 9.2
-0.7+0.2
simult. opt. (DFA)
yi max y2 min
98.7 7.2
-1.00
0.65
-1.00
simult. opt. (GDA)
y, max y2 min
96.5 2.4
-0.92
0.60
-1.00
individual optima
Pepper lyepeeling
Tomato lyepeeling
0.6+0.2
"^Sopt
0.2+0.35
-0.85+0.1
-0.7+0.1
2149 70
[
60
0
.
1
50 40
yTH
30 20 10
y a x i o l
0 '
•
'
-0.5
0.0
'
^—^
ym
0.2
F 0.1
0.0
-1.0
0.5
1.0
Figure 4: Graphical representation of application example 1 showing (A) true responses, (B) normalized responses, and (C) overall normalized function.
2150 REFERENCES Derringer, G., and R. Suich, 1980, Simultaneous optimization of several response variables, J. Qual. Tech., 12:214-219. Floros, J.D., 1992, Optimization methods in food processing and engineering. In Encyclopedia of Food Science & Technology, Hui, Y.H. (Ed.), pp. 1952-1965, Wiley, NY. Floros, J.D., 1994, Ongoing research data Floros, J.D., and M.S. Chinnan, 1987, Optimization of pimiento pepper lye-peeling process using response surface methodology, Trans. ASAE, 30(2):560-565. Floros, J.D., and M.S. Chinnan, 1988, Computer graphics-assisted optimization for product and process development, Food Tech., 42(2):72-78, 84. Guillou, A.A., 1991, Minimization of the amount of NaCl used during natural cucumber fermentation and storage through multiresponse optimization methods, M.S. Thesis, Purdue University, West Lafayette, IN. IMSL, 1987, "IMSL Math/Library User's Manual", Version 1.0, IMSL, Inc., Houston, TX. Khuri, A.I., and M. Conlon, 1981, Simultaneous optimization of multiple responses represented by polynomial regression functions, Technometrics 23(4):363-375. Lind, E.E., J. Goldin, and J.B. Hickman, 1960, Fitting yield and cost response surfaces, Chem. Eng. Prog., 56:62-68. SAS, 1990, "SAS/STAT User's Guide", Version 6.03, SAS Inst. Inc., Gary, NC.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2151
BACK PROPAGATION NEURAL NETWORKS: THEORY AND APPLICATIONS FOR FOOD SCBENCE AND TECHNOLOGY Vivek Gnanasekharan and John D. Floros Department of Food Science, 1160 Smith Hall, Purdue University, West Lafayette, IN 47907-1160, USA Address inquires to author Floros ABSTRACT Neural networks (NNs), a class of powerful information processing techniques, may be used to characterize complex nonlinear relationships such as those found in food processing operations. A brief overview of the various NNs is presented. In depth discussion of back propagation networks (BPNs), a simple technique with wide range of applicability, follows. The mechanics of constructing and using BPNs including their characteristics, definition, training and testing are covered. The limitations of BPNs and practical guidelines for their implementation are also discussed. Current and potential applications of BPNs in food science and technology are presented with examples in process modeling, optimization and control, food and sensory analysis, and product formulation.
INTRODUCTION The role of food processing is to convert raw food materials into forms of greater utility, stability, nutritive value, convenience and appeal. In contrast to the raw materials used by other industries, raw food materials vary with production season and time, cultivation conditions and location, and post harvest treatment. Thus, the intrinsic variability of food materials is a recurrent and inseparable component of food processing operations. Furthermore, processing of these heterogeneous materials generally involves complex chemical and/or biochemical reactions that may not be amenable to complete characterization. Quality is another significant difference between food and other manufacturing operations. Food quality depends on several synergistic and sometimes subjective factors such as sensory attributes, chemical composition, physical properties, microbiology, toxicology, packaging, shelf life and convenience. Given these complexities, mathematical characterization of food processing operations often results in
2152 nonlinear, time dependent and coupled differential equations. Modeling of such processes in the presence of noisy data (process/product variation) can be counterproductive from a practical standpoint (time, resources and expense). However, conventional process control and/or optimization strategies require representative mathematical models. Logical progression of this discussion points towards the need for a technique capable of abstracting and representing complicated relationships in the presence of imprecise and variable data. In other words, effective optimization and control of food processes would benefit from a mathematically non rigorous pattern recognition technique. Neural networks (NNs), a rapidly maturing information processing paradigm, are best known for their ability to model complex nonlinear relationships in the presence of imprecise data (Hecht-Nielsen, 1988; Bhagat, 1990). Neural networks are known as universal approximators, because they learn to approximate any continuous function or relationship (linear or nonlinear) given sufficient sample data. Furthermore, NNs do not require theoretical or mechanistic information about the process being modeled. Although NNs are heuristic, they are similar to certain parametric and nonparametric statistical techniques such as regression, principal component analysis and cluster analysis (White, 1989). Thus, problems that lend themselves to such techniques can also be solved by NNs. Based on the characteristics of NNs and the needs of food processing, it is apparent that NNs constitute a potentially attractive solution to currently intractable problems in the food industry. While NNs appear to have great potential, they also have several problems. The heuristic nature of NNs makes it difficult to extract information about the process being modeled. NNs are by definition parallel processes and their implementation on serial computers requires extended training time, which limits the complexity of problems to be solved (Nelson and lUingworth, 1991). Nevertheless, NNs have been successfully used to solve several real life problems such as time series forecasting, credit evaluation, recognition of handwriting, sensor data interpretation, and process modeling and diagnosis. Advances in computer technology such as the development of parallel and optical computers will probably make NN applications more powerful in the near future. The objectives of this paper are to: (a) provide an overview of neural networks; (b) briefly discuss the most common types of neural networks; (c) discuss in detail the mechanics of constructing and using back propagation networks (BPN); and (d) summarize NN applications relevant to food science and technology.
NEURAL NETWORKS Biological and Mathematical Models Theoretical explanations of cognitive processes in mammalian brains date back to the work of ancient Greek philosophers Plato and Aristoteles (Kohonen, 1988). However, the explosive growth of NN research and applications was catalyzed by the classical work of McCulloch and Pitts (1943). Their results convinced the scientific community that biological
2153 neural processes can be mathematically approximated for practical and useful purposes. Neural networks are based on a biological model derived from the mammalian cerebral cortex, which is essentially a system of connected components consisting of neurons and synaptic junctions. A synapse is composed of an axon (transmitter) and a dendrite (receiver) (Fig. 1). Signals transmitted across this junction are modified by the strength (or weight) of the junction. Repeated incoming signals act to continuously modify junction strengths, and hence, increase output accuracy. In general, memory corresponds to the strength of a synaptic junction, while experience is manifested as a change in synaptic junction strength. Recall or recognition improves as the difference between synaptic and signal (input stimulus) strengths decreases. The role of a neuron is to sum all weighted inputs from the synapses attached to it, and produce a collective output (Kohonen, 1988; Willard, 1990).
SYNAPTIC JUNCTION
AXON
NEURON
Figure 1. Biological model of a neuron and synapse.
2154
INPUTS SYNAPTIC WEIGHTS SUMMATION FUNCTION
THRESHOLD FUNCTION Yi^yi-yn
OUTPUT
Figure 2. Mathematical model of a processing element. The NN equivalent of a neuron and synapse is called ?i processing element (PE) or node (Fig. 2). The primary distinction between biological (Fig. 1) and mathematical (Fig. 2) models is the presence of a threshold function in the latter. Key activities that occur in a typical PE are: (a) summation of the products of inputs and corresponding weights; and (b) comparison of the sum with a threshold value to determine the output. If the sum of weighted inputs is greater than the threshold value, the PE generates a signal or fires. The threshold function is generally a continuous and bounded nonlinear differentiable function, with sigmoidal functions being the most popular choice (Nelson and Illingworth, 1991). A NN is created by combining several PEs in parallel to form a layer, and then connecting several layers to form a multilayer net (Fig. 3). The input layer serves only to distribute the inputs, while all computations are confined to other layers. All layers between input and output are called hidden layers, and they may or may not be present depending on the application. The various PEs in a NN communicate via connections called interconnects. Feedforward nets are constrained to a strictly forward flow of outputs. Feed back nets can direct PE outputs back (as inputs) to other PEs on the same or preceding layers. Finally, difiilly connected net has all its PE outputs from one layer passing along to all the PEs in the next layer. Under these hierarchical network rules, the NN shown in Fig. 3 is a fiilly connected feed forward net. Specification of a NN architecture or topology involves specification of the: (a) number of PEs; (b) number of layers; and (c) connectivity options (Nelson and Illingworth, 1991). Learning is the process by which a PE uses locally stored information from previous computations to update or change the weights attached to its interconnects, and hence modify its output in response to its inputs. The algorithm defining how the weights are changed is called
2155 INPUTS INPUT LAYER
fflDDEN LAYER
OUTPUT LAYER
OUTPUTS
Figure 3. Schematic of a fully connected NN with one hidden layer.
the learning law. It allows a NN to adapt in response to inputs and organize information within itself. Knowledge in a NN is a function of its topology and the weights associated with its interconnects (Bhagat, 1990). A NN learns to process information during training with supervised or unsupervised trials. Supervised training consists of repeated presentations of inputs and desired (or experimentally measured) outputs until the prediction error is sufficiently reduced. Unsupervised training consists of presenting inputs and allowing the NN to generate its own internal pattern groupings (Nelson and Illingworth, 1991). In either case, the NN is said to have converged when the prediction errors have been reduced to some preset tolerance. At that point the learning phase is terminated. Major Types of Neural Networks Complete specification or NN design requires specification of topology and learning law. While the possible combinations of these parameters are numerous, practical considerations have limited the options to about 50. From those, only 13 types of NNs are used often (Table 1). The characteristics of the various NNs (Table 1) are sufficiently different, and therefore, one NN
Table 1 Characteristics of major neural network models1. -
NN Name
Primary applications
Strengths
Weaknesses
Comments
Adaptive Resonance
Pattern recognition
Resolves complicated patterns
Sensitive to translation, distortion and change of scale
Sophisticated and unexplored
Avalanche
Speech recognition, Robots control
Real time pattern analysis, playback of motor sequences
Difficult to interpolate movements and alter speed
A class of networks
Back Propagation
Wide range including Simple construction and speech synthesis, loan training evaluation, process modeling
Long training time, large training data
Most popular NN, requires supervised training
Bidirectional Associative Memory
Content addressable associative memory
Easy to train, associates fragmented objects
Requires large storage capacity
Data needs to be coded properly
Simple NN, resolves complicated patterns
Long training time
Uses noise functions to find the global minimum
Boltzmann Machine Pattern recognition Brain state in a box
Knowledge extraction Associates fragmented objects
Lack of iterative reasoning
Cerebellatron
Robot control
Requires complex inputs
Playback of motor sequences
Capable of interpolating control motion and speed
Table 1 (Cont.) Characteristics of major neural network modelsl.
Name
Primary applications
Strengths
Weaknesses
Comments
Counterpropagation
Image compression, Statistical analysis
Simple multi-layer NN
Requires a large number of processing elements
Self programming look up table
Hopfield
Image analysis
Madaline
Adaptive signal filtering
Large scale implementation Powerful learning law
Cannot learn, weights must be preset Cannot represent nonlinear relationships
Similar to BPN but less powerful Used commercially for more than 20 years
Neocognitron
Recognition of handwriting
Identification of complex patterns
Requires a large number of processing elements
Sophisticated and complex NN, insensitive to changes in scale, translation and rotation
Perceptron
Typed character recognition
Oldest NN, Implemented in hardware
Cannot recognize complex characters
Not used much at the present
Self-organizing map
Mapping geometrical regions
Superior to many algorithms for numerical calculations
Requires extensive training
Popular for aerodynamic flow computations
1 Based on information presented by Hecht-Nielsen (1988)
2158 type cannot be used for all applications. However, specific characteristics of the back propagation network (BPN) have made it a popular choice for many applications. All NNs consist of simple processing elements interconnected in some fashion. Primary differences exist in their topology, type of learning law utilized, and method of training. For example, the self organizing map is an unsupervised (self training) network, while a BPN requires supervised training. At the other end of the spectrum is the Hopfield network, which requires pre specification of weights because it is not capable of learning. The type of input is yet another point of difference among NNs. Some NNs can utilize only continuous inputs, while others may accept binary data. Furthermore, binary inputs may range from 0 to 1 or from -0.5 to +0.5. Finally, various activation or threshold functions can be used for different applications. Details on the mechanics of different NNs can be obtained from the literature (Kohonen, 1988; Treleaven and Vellasco, 1989; Nelson and Illingworth, 1991). In summary, selection of a NN is primarily based on the application and its constraints.
BACK PROPAGATION NETWORKS Ease of learning, simplicity and other characteristics have made BPNs the most widely used NNs (Hecht-Nielsen, 1988; Bhagat, 1990; Nelson and Illingworth, 1991). BPNs are multilayer feed forward nets characterized by forward flow of information in the prediction mode and backward flow of error corrections in the learning mode (Rumelhart et al., 1986). They are essentially mapping networks that correlate a set of input to a set of output vectors, and can predict inputs from outputs. A BPN may have zero, one or more than one hidden layers depending on the application. Training is accomplished by presenting the BPN with a set of input and output vectors, and by defining an algorithm that iteratively adjusts the relative strengths of interconnects to progressively reduce the difference between predicted and desired outputs (Wasserman, 1989). As the name suggests, BPNs employ a learning rule known as back propagation, a brief description of which follows (Rumelhart et al., 1986). Theoretical Considerations The total input xj to a node j is a linear function of the output yi of all the nodes connected to nodey and the weights Wji of the interconnects between these nodes
^rY^yi^ij
0)
where / is the number of nodes connected to node j . The output yj of a node is a nonlinear (usually sigmoidal) function of its total input
2159
yj = r ^
(2)
The sigmoidal activation function (Eqn. 2) can be replaced by other nonlinear functions as long as they have bounded derivatives. The nonlinear nature of the activation function is critical, because it allows a BPN to solve nonlinear problems. For a fixed finite set of inputoutput pairs, the total error for a particular set of weights can be obtained by comparing the predicted and desired outputs of the entire set of input-output pairs. This total error E is defined as
^ = Z I.{yj,k-djJ
(3)
where, k is the number of input-output pairs, j is the number of output nodes, y is the predicted output and d is the desired output. The total error E is minimized using the gradient descent method, which involves computation of the partial derivatives of E with respect to each weight in the network, and is equivalent to the sum of partial derivatives for all input-output pairs. The partial derivatives are computed in two passes, forward and backward. The forward pass consists of computing the state of each node using Eqns. (1) and (2). The backward pass propagates derivatives from the output to the input layer and starts with computation of DE/dy for each of the output nodes. Differentiation of Eqn. (3) yields
f-yj-i
(4)
The chain rule is then used to compute dE/dxi as
(5)
d^^d^^ dxj
dyj dxj
Differentiation of Eqn. (2) to get dyj/dxj and substitution into Eqn. (5) results in
dE
dE (^
—-—yM-yj) dxj dyj
\ (6)
2160 The partial derivative with respect to a weight Wjj is given by dE _ dE dxj _ BE dWij
dXjdWij
dXj '
and for the fi^ output, the effect of / onj will result in a component dE dXj __ dE dXj dyi
^ii
(8)
dXj
Consideration of all the connections of node / results in
dE dyj
^dE ydxj
Eqns (3) through (9) can be used to compute dE/dy for any node in the layer preceding the last layer. The procedure is then repeated to compute dE/dy and dE/dw for successively lower layers. The weights in the network are progressively updated using the following function
Aw{t) = -ri
+ aAw{t-i)
(10)
dWf where, t is incremented by 1 for each pass (epoch) through all the input-output pairs, r| is the learning rate and a is the momentum coefficient. The learning rate is a multiplier that serves to adjust the size of average weight change. The momentum coefficient determines the relative contribution of current and earlier gradients to weight change. These two parameters and the number and size of hidden layers determine the performance of BPNs. Construction of BPNs The initial task in constructing a BPN is to design its architecture or topology. Definition of topology involves specification of the number of nodes, number of layers and the connectivity options. The number of input and output nodes is determined by the number of inputs and outputs of the target application. For example, a BPN constructed to model a cucumber fermentation process with three processing variables (inputs) and five process responses (outputs) had three nodes in the input layer and five nodes in the output layer (Gnanasekharan and Floros, 1994). The type of threshold function must also be specified. As stated earlier any continuously differentiable and bounded function can be employed. Some examples of possible threshold functions are: sigmoidal, hard limiter (Heaviside), sine, cosine and pseudo-linear (Treleaven and Vellasco, 1989). Quantitative
2161 evidence suggests that the sigmoidal function is superior to other functions because of its ability to discriminate and handle nonlinear relationships between inputs and outputs (Sontag, 1989). Specification of the number of hidden layers and the number of nodes in each hidden layer is not simple. Solution of problems that require nonlinear partition(s) of the inputoutput space requires at least one hidden layer (Wasserman, 1989). The appropriate size (number of nodes) of the hidden layer is problem specific and is typically determined by trial and error. Sub-optimal number of hidden layer nodes may result in loss of discriminating ability, while an excessive number may lead to lack of generalization and extended training time (Fogel, 1991). Increasing the number of hidden layer nodes translates to an increased number of interconnects, and hence, more freedom for the BPN to adjust its weights and achieve a lower prediction error. This is analogous to regression analysis where increasing the number of model parameters results in a better fit. Extending the same concept, BPNs with too many hidden layer nodes effectively memorize the training data patterns, but lose their ability to predict and generalize to new data (Karnin, 1990). Some attempts have been made to develop quantitative criteria for selecting optimal hidden layer size, but none of the techniques can be used with all types of BPNs. Fogel (1991) summarized some of these approaches and developed one technique based on statistical theory. However, for most applications, selecting the number of hidden layer nodes must still be done by a trial and error procedure. Connectivity options (type and extent) constitute the next set of variables. Connections between nodes may be either feed forward or a combination of feed forward and feed back. Classical BPNs are limited to feed forward connections, and therefore, the learning law must be modified to allow for feed back. The extent of connections refers to whether a BPN is fully or partially connected. A fully connected net has more connections than a partially connected one, and requires longer training time. Pruning is a technique that can be used to examine the sensitivity of the net to each connection, and selectively remove redundant connections (Karnin, 1990). As the size of a BPN increases, the number of possible interconnects also increases, and pruning becomes very time consuming. Thus, most existing BPN applications use fully connected nets. The next decision step concerns the choice of appropriate values for momentum coefficient (a) and learning rate (r\). The momentum coefficient is a multiplier that takes values from 0 to 1, and makes weight adjustments proportional to previous weight changes. The choice of this parameter is problem specific, and it is usually selected by a trial and error approach. Large values of a cause the network to closely follow the error surface rather than oscillating from side to side (Wasserman, 1989). Therefore, appropriate choice of a can help in avoiding local minima, and many times it is set to 0.9. The learning rate adjusts the magnitude of average weight changes, and takes values from 0 to 1. Increasing r\ speeds up learning, but it may also cause network instability. In extreme cases, high r| values may even cause network paralysis (failure to learn). The best choice of r[ is also problem specific, and values of 0.6 to 0.9 are often used. For the cucumber fermentation example mentioned earlier (Gnanasekharan and Floros, 1994), values of 0.8 were found to be the best choices for both a and r].
2162 BPN Training and Testing Training is accomplished by presenting the net with several input-output data sets, and allowing weight changes to occur until the difference between desired and predicted outputs is sufficiently reduced (net convergence). The training data must be scaled from 0 to 1 to satisfy the range requirements of the activation function. Before initiating the training phase, all weights should be initialized to small random numbers to prevent failure of learning (Wasserman, 1989). Training could be a time consuming process, because thousands of iterations are generally required for convergence. One complete pass through all the input-output pairs of the training data set is called an epoch. Weight changes and error calculations are normally made at the end of each epoch. If a BPN failed to converge, training must be halted and restarted after changing some parameters (i.e. weights, topology or values of a and r\). Occasionally, convergence to a preset tolerance may not be possible, although prediction errors have been reduced. In such cases, continued training will not result in any further learning, and the options available are to either accept the performance or change some parameters and retrain the net. The size and type of training data can significantly affect the performance of BPNs. It is generally acknowledged that performance of BPNs is directly proportional to training data size. Excessively large amounts of training data may extend training time significantly, while training data of very small size may prevent learning. Boundaries on the number of samples required for successful learning of BPNs with two hidden layers have been established (Mehrotra, 1991). Similar approaches may be possible for other topologies. In practice, the size of training data is generally determined by trial and error. The type of training data is also important, because it captures the characteristics of the problem being solved. Data that are not representative of the true relationship between inputs and outputs would result in poor performance. Successful learning requires that the order of data presentation during training be randomized. Random data presentation ensures that the BPN "remembers" all possible variations in the input-output space. Once the error has reached acceptable limits, BPN training is terminated. Its performance is then evaluated using a test data set, that must be different from the training data set. This process is called the recall phase. During recall, weights are frozen to remain unchanged, and errors are not propagated backwards through the net. Correct predictions (within tolerance) of all outputs during recall is indicative of the ability to generalize. Poor performance would require a change in parameters followed by retraining. A common cause of poor performance during recall is an excessive hidden layer size, which results in memorization of training patterns and poor generalization to test patterns. Limitations and Modifications The primary limitation of BPNs is extended training time, which can be as long as several days or even weeks for complex problems (Wasserman, 1989). Another major limitation is the susceptibility of gradient decent methods to local minima. A BPN can get "stuck" in a local minimum especially when the error surface is highly irregular containing peaks, valleys and folds. Several researchers have developed variations and enhancements of the classical back propagation technique to decrease training time and provide stability. Network paralysis is another problem, which can be prevented by appropriate choice of
2163 learning rates. Temporal instability or a chance of "forgetting" less frequently seen training patterns could also be a problem. This is commonly avoided by randomizing the data presentation sequence and by batching. Batching is a process by which weights are changed after completion of a full epoch and not after presentation of each pattern. Change of the input range from 0 - 1 to ± 0.5 and inclusion of a bias term to the threshold function has been shown to reduce convergence time by upto 50% in certain cases (Wasserman, 1989). Back propagation has been applied to recurrent networks (outputs feedback to inputs) resulting in decreased learning time and enhanced stability (Pineda, 1988). Probabilistic NNs (PNN) have also demonstrated significant decreases in training time when compared to classical back propagation (Specht, 1990). A PNN is similar in structure to a BPN, but differs in that the sigmoidal activation function is replaced by a statistically derived relationship. Specht (1990) reported that PNNs could speed up training time by upto 200,000 times relative to a standard BPN. The gradient descent technique, which is employed in standard BPNs to locate the minimum of the error function, becomes progressively slower as the minimum is approached (Watrous, 1987). Consequently, several approaches to decrease training time revolve around substitution of the gradient descent method with superior techniques such as Quasi-Newton methods (Parker, 1987), and conjugate gradient methods. These techniques are usually computationally more intensive and may not be practical for very large problems. However, they can provide significant speed advantages during training. The conjugate gradient technique may decrease training time by a factor of two. It assures convergence, but has a greater susceptibility to local minima than gradient descent (Leonard and Kramer, 1990). Further discussion of all the modifications developed to increase training speed and stability of BPNs can be found in the literature (Wasserman, 1989; Nelson and Illingworth, 1991).
FOOD RELATED APPLICATIONS OF BPNs Although NNs have been applied for many years in other fields, they have not been used much in food related problems. Recently, with the on-set of process automation in the food industry, several NN applications have been reported. The majority of reported applications employ BPNs for process modeling and control, and data analysis. In general, all documented applications (existing and projected) exhibit some combination of the following features: large data sets, nonlinear relationships, noisy and imprecise data, non normal or serially correlated data, and lack of mechanistic models. Food process control is a natural match for NNs, because many processes are too complex to be modeled mathematically. The approximation capabilities of NNs makes them an ideal choice for generating models needed during process control implementation. Another reason for the predominance of process control applications is the complexity of data produced by modern sensors. NNs have been shown to be very effective in compressing and reducing data produced by different types of sensors or sensor arrays (Fildes and Cinar, 1992).
2164 A BPN was used for real-time estimation and multi-step ahead prediction of enzyme activity and biomass level during glucoamylase fermentation (Linko and Zhu, 1992). The inputs were pH, agitation rate, oxygen consumption rate, carbon dioxide evolution rate, and the accumulated volumes of carbon dioxide released and nitrogen utilized. The outputs were enzyme activity and biomass level. The BPN used for multi-step ahead prediction had two additional nodes for each output to allow for two future sampling times. Different topologies were evaluated and one hidden layer with 10 nodes provided the best results. Good results were obtained compared to off-line analysis. BPNs have also been used to successfully model and control wine fermentation (Cleran et al., 1991), sake brewing (Oishi et al, 1992), saccharomyces cerevisiae fermentation (Shi and Shimizu, 1992), beer brewing (Simutis et al., 1993), cucumber fermentation (Gnanasekharan and Floros, 1994), flat bread extrusion (Erikainen et al., 1994) and on-line package inspection (Harvey, 1992). Other potential BPN applications in food processing operations include adaptive process control and fault diagnosis (Ungar et al., 1990; Ydstie, 1990), and dynamic process modeling (Bhat and McAvoy, 1990). Furthermore, neural networks are being investigated for optimizing sensor data interpretation and achieving real time adaptive control of food processing operations (Willard, 1990; Fildes and Cinar, 1992; Whittaker et al., 1992; Zang and Litchfield, 1992). Borggaard and Thodberg (1992) used a BPN to analyze Near Infra Red (NIR) spectra of ground pork meat. The inputs were the spectral data, while the outputs were experimental measurements of fat, water and protein contents. Principal Component Analysis (PCA) was used to preprocess the spectral data prior to BPN input. Results were superior to those obtained using standard chemometric techniques. BPNs have also been combined with PCA for quantitative determination of apple quality using NIR spectroscopy (Bochereau et al., 1992). Another application of BPNs for spectral data analysis is detection of adulteration of virgin olive oil with other edible oils based on data from pyrolysis mass spectrometry (Goodacre et al., 1992 & 1993). In a different study, a BPN functioned as an electronic nose to classify various alcohols based on data from a computerized sensor array (Gardner et al., 1990). This application is similar to odor discrimination (sensory analysis) and head space gas chromatography. BPN inputs consisted of 12 experimental gas sensor measurements, and BPN outputs were the presence or absence of an alcohol. Five different alcohols were used and correct classifications were achieved in all cases. Data from machine vision was analyzed by BPNs and apples were classified into various quality groups according to their surface characteristics (Yang, 1993). A classification accuracy of 97 % was achieved. A BPN was used to predict protein secondary structure from amino acid sequences (Holley and Karplus, 1989). An average prediction accuracy of 63 % was achieved for proteins with helical, sheet and coil conformations. Prediction accuracy was increased to 79 % by deleting outliers. Prediction of protein functionality (foam capacity, foam stability, and emulsion activity index) based on the physicochemical properties of 11 food proteins was studied by both BPNs and PCA (Arteaga and Nakai, 1993). The predictive ability of a BPN was superior to that of PCA. A modular network consisting of two BPNs coupled together optimized the selection of starches for inclusion in food products (Huang et al., 1993). Each BPN was trained to recognize eight types of starches based on eighteen variables including preferred source, cost, product storage conditions, desired shelf life and texture, and other physicochemical properties. This application is similar to nonlinear
2165 optimization and the authors reported satisfactory results. Finally, BPNs have been used to optimize product formulations (Gill and Shutt, 1992). Although this report describes formulation of adhesives and coatings, similar techniques could be applied to food formulation.
SUMMARY & CONCLUSIONS Neural networks are powerful information processing techniques. Their key characteristics are useful in solving food related problems, and include: (a) insensitivity to noisy and variable data; (b) ability to represent complex nonlinear relationships in multidimensional spaces; (c) ability to handle large data sets; and (e) lack of dependence on theoretical information. Several NN models exist, but each is suited for specific applications. One type of NN, the BPN, is remarkably versatile, simple to construct, and easy to train. However, BPNs may require extensive training time and they are susceptible to local minima. Several food related problems have been successfully resolved through the use of BPNs. Such problems include process modeling, process control, complex data reduction and analysis, and sensor data interpretation. Furthermore, NNs may also be useful in sensory analysis and product formulation.
REFERENCES
Arteaga, G. E. and Nakai, S. 1993. Predicting protein functionality with artificial neural networks: Foaming and emulsifying properties. J. Food Sci. 58(5):1152-1156. Bhagat, P. 1990. An introduction to neural nets. Chem. Eng. Prog. 86(8):55-60 Bhat, N. and McAvoy, T. J. 1990. Use of neural nets for dynamic modeling and control of chemical process systems. Comput. Chem. Eng. 14(4/5):573-583. Bochereau, L., Bourgine, P. and Palagos, B. 1992. A method for prediction by combining data analysis and neural networks: Application to prediction of apple quality using near infra-red spectra. J. Agric. Eng. Res. 51(3):207-216. Borggaard, C. and Thodberg, H. H. 1992. Optimal minimal neural interpretation of spectra. Anal. Chem. 64(5):545-551. Cleran, Y., Thibault, J., Cheruy, A. and Corrieu, G. 1991. Comparison of prediction performances between models obtained by the group method of data handling and neural networks for the alcoholic fermentation rate in enology. J. Ferment, and Bioeng. 71(5):356-362.
2166 Erikainen, T., Zhu, Y. H. and Linko, P. 1994. Neural networks in extrusion process identification and control. Food Control 5(2): 111-119. Fildes, J. M. and Cinar, A. 1992. Sensor fusion and intelligent control for food processing. Proc. Food Processing and Automation Conf, Lexington, KY, May 4-6, ASAE Publ. 02-92, pp. 65-72, ASAE, 2950 Niles Rd., St. Joseph, Michigan. Fogel, D. B. 1991. An information criterion for optimal neural network selection. IEEE Transac. on Neural Networks 2(5):490-497. Gardner, J. W., Hines, E. L. and Wilkinson, M. 1990. Application of artificial neural networks to an electronic olfactory system. Measurement Sci. Technol. 1(5):446-451. Gill, T. and Shutt, J. 1992. Optimizing product formulations using neural networks. Sci. Comput. Autom. 8(10): 19-26 Gnanasekharan, V. and Floros, J. D. 1994. Comparison of back propagation network (BPN) performance and response surface methodology (RSM) for modeling food processes. Proc. of the Conference on Computer Integrated Manufacturing in the Process Industries, pp. 748-763. Goodacre, R., Kell, D. B. and Bianchi, G. 1993. Rapid assessment of the adulteration of virgin olive oils by other seed oils using pyrolysis mass spectrometry and artificial neural networks. J. Sci. Food and Agric. 63(3):297-307. Goodacre, R, Kell, D. B. and Bianchi, G. 1992. Neural networks and olive oil. Nature 359(6396):549. Harvey, R. E. 1992. Plastic containers frustrate inspection. Food Eng. 64(6): 125-128. Hecht-Nielsen, R. 1988. Neurocomputing: picking the human brain. IEEE Spectrum 25(3):36-41. Holley, L. H. and Karplus, M. 1989. Protein secondary structure prediction with a neural network. Proc. Natl. Acad. Sci. 86(1): 152-156. Huang, Y. W., Mithani, R, Takahashi, K., Fan, L. T. and Seib, P. A. 1993. Modular neural networks for identification of starches in manufacturing food products. Biotech. Prog. 9(4):401-410. Kamin, E. D. 1990. A simple procedure for pruning back-propagation trained neural networks. DEEE Transac. on Neural Networks l(2):239-242. Kohonen, T. 1988. An introduction to neural computing. Neural Networks 1(1):3-16. Leonard, J. and Kramer, M. A. 1990. Improvement of the backpropagation algorithm for training neural networks. Computers Chem. Eng. 14(3):337-341. Linko, P. and Zhu, Y. H. 1992. Neural network modelling for real-time variable estimation and prediction in the control of glucoamylase fermentation. Process Biochem. 27(5):275-283. McCulloch, W. S. and Pitts, W. A. 1943. A logical calculus of the ideas immanent in nervous activity. Bull. Math. Biophysics 5:115-133.
2167 Mehrotra, K. G., Mohan, C. K. and Ranka, S. 1991. Bounds on the number of samples needed for neural learning. EEEE Transac. on Neural Networks 2(6):548-558. Nelson, M. M. and Illingworth, W. T. 1991. A Practical Guide to Neural Nets. AddisonWesley Publ. Co. Inc., New York, NY. Oishi, K., Tominaga, M., Kawato, A. and Imayasu, S. 1992. Analysis of the state characteristics of sake brewing with a neural network. J. Ferment, and Bioeng. 73(2):153-158. Parker, D. B. 1987. Optimal algorithms for adaptive networks: Second order back propagation, second order direct propagation, and second order hebbian learning. Proc. IEEE Intl. Conf on Neural Networks, Vol. 2, pp. 593-600. Pineda, F. J. 1988. Generalization of backpropagation to recurrent and higher order networks. In "Neural Information Processing Systems", D. A. Anderson (Ed), pp. 602-611, Amer. Inst. Physics, New York, NY. Rumelhart, D. E., Hinton, G. E. and Williams, R. J. 1986. Learning representations by backpropagating errors. Nature 323(6088):533-536. Shi, Z. and Shimizu, K. 1992. Neuro-fuzzy control of bioreactor systems with pattern recognition. J. Ferment, and Bioeng. 74(l):39-45. Simutis, R., Havlik, I. and Luebbert, A. 1993. Fuzzy-aided neural network for real-time state estimation and process prediction in the alcohol formation step of production scale beer brewing. J. Biotechnol. 27(2):203-215. Sontag, E. D. 1989. Sigmoids distinguish more efficiently than heavisides. Neural Computation l(4):470-472. Specht, D. F. 1990. Probabilistic neural networks. Neural Networks 3(1): 109-118. Treleaven, P. and Vellasco, M. 1989. Neural computing overview. Computer Physics Communic. 57(3):543-559. Ungar, L. H., Powell, B. A. and Kamens, S. N. 1990. Adaptive networks for fault diagnosis and process control. Comput. Chem. Eng. 14(4/5):561-572. Wasserman, P. D. 1989. Neural Computing Theory and Practice. Van Nostrand Reinhold, New York, NY. Watrous, R. L. 1987. Learning algorithms for connectionist networks: Applied gradient methods of nonlinear optimization. Proc. IEEE Intl. Conf on Neural Networks, Vol. 2, pp. 619-627. White, H. 1989. Neural network learning and statistics. AI Expert 4(12):48-52. Whittaker, A. D., Huang, Y. and Cook D. F. 1992. Neural network based control of serial food processes. Proc. Food Processing and Automation Conf, Lexington, KY, May 4-6, ASAE Publ. 02-92, pp. 348-353, ASAE, 2950 Niles Rd., St. Joseph, Michigan.
2168 Willard, J. P. 1990. Neural networks for computer control. Proc. Food Processing and Automation Conf., Lexington, KY, May 6-8, ASAE Publ. 02-90, pp. 167-172, ASAE, 2950 Niles Rd., St. Joseph, Michigan. Yang, Q. 1993. Classification of apple surface features using machine vision and neural networks. Computers and Electronics in Agric. 9(1): 1-12. Ydstie, B. E. 1990. Forecasting and control using adaptive connectionist networks. Comput. Chem. Eng. 14(4/5):583-599. Zhang, Q. and Litchfield, J. B. 1992. Advanced process controls: Applications of adaptive, fuzzy and neural control to the food industry. Proc. Food Processing and Automation Conf, Lexington, KY, May 4-6, ASAE Publ. 02-92, pp. 169-176, ASAE, 2950 Niles Rd., St. Joseph, Michigan.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2169
GENETIC ALGORITHMS AND FUZZY THEORY FOR OPTIMIZATION AND CONTROL OF FOOD PROCESSES loannis G. Vradis and John D. Floros Department of Food Science, Purdue University, 1160 Smith Hall, West Lafayette IN 47907-1160, U.S.A. Address inquires to author Floros ABSTRACT Genetic Algorithms are search algorithms seeking improved (optimized) performance by mimicking the mechanics of natural selection and genetics. Fuzzy set theory imitates the human way of judging by using linguistic descriptions and criteria. A combination of the two methods may assist the optimization and on-line control of food processes. The structure of Genetic Algorithms and the way optimization is implemented through them are presented. Emphasis is placed on classifier systems. Furthermore, fuzzy set theory and its applications in fuzzy control are discussed. The important aspects of combining the two methods (Genetic Algorithms and Fuzzy Control) for simultaneous optimization and control of food processes are investigated. Some potential applications in the food industry are briefly discussed. INTRODUCTION As in all industrial processes, efficiency of food processing operations largely depends on the ability to (a) determine optimum operating conditions, and (b) maintain operation at these optimum conditions. To satisfy these two requirements, it is necessary to properly employ appropriate optimization and process control techniques. In optimization problems the system under consideration is expressed by the socalled objective function. The input to the system is represented by a vector of independent variables, which are either controllable (factors) x^, x^ . . ., x^ or uncontrollable (noise) n^, n^ . . ., n^. The output is represented by a vector of dependent variables (responses) y^, y^ . . ., y^. A common optimization technique is Response Surface Methodology (RSM) in which the method of least squares is used to fit an empirical function to experimental data. The empirical function is then used to estimate the optimum conditions of the respective process.
2170 Automatic control methods aim to achieve and/or maintain the state of a system or process by monitoring certain variables and taking appropriate control actions. The basis of automatic controllers is a precise mathematical model that describes the system under consideration. Complexity and raw material variation make food processes difficult to optimize and control. Often such processes are not fully understood and it is difficult to build adequate mathematical models to describe them. Even if a mathematical model describing a process has been developed and the optimum operating conditions of the process have been estimated, it may still be difficult to run the process efficiently because the properties of the raw material vary. In such cases the optimum conditions and possibly the control parameters may change according to the properties of the raw material. Optimization techniques may be combined with appropriate control methods to improve the performance and efficiency of food processes. For these reasons an on-line optimization and control system capable of adopting easily to input material variation would be beneficial. Genetic Algorithms (GA) and fuzzy set theory have the ability to adopt to process variations and produce desired final products. This ability indicates a great potential for their application in food processing. The objectives of this work are to discuss: (a) the important aspects of coupling genetic algorithms and fuzzy logic for process optimization and control; and (b) potential applications of these methods in food and biochemical processes. GENETIC ALGORITHMS Terninology. Genetic Algorithms (GAs) are a group of optimization techniques. They are search algorithms seeking improved performance by sampling areas of factor space most likely to give good results. GAs are based on the mechanics of natural selection and genetics (Holland, 1975), and examine and manipulate sets of possible solutions. Each solution is represented by a string (A), which consists of characters Uj from an alphabet (V), has finite length ^, carries coded information about inputs, and can be represented symbolically as follows:
^ = «i«2^3- ••««
(1)
Each aj represents an alphabetic character called detector, which in a binary mode (i.e. V={0, 1}) takes the value 0 or 1. The natural analog of a detector is the gene and that of a string is the chromosome. Genetic algorithm optimization techniques deal with populations of strings called structures. Each structure is a set of values of input variables. The biological analog of a structure is the genotype. String populations (structures) are decoded to produce a group of responses, called parameter sets, solution alternatives or points in the solution space; these are analogous to the phenotype in natural systems.
2171 Function, A population of strings evolves in a prespecified number of generations under application of rules such as genetic operators and reproductive plans (Androulakis and Venkatasubramanian, 1990). Similar to events in the biological world, GAs operate by reproduction, crossover and mutation, and they function by manipulating strings (Goldberg, 1989). Reproduction makes exact copies of existing strings (Table 1). The probability of a string to participate in a new generation (to make copies of itself) is proportional to the string's fitness and ability to produce a desired solution. The number of copies of each string is determined by the reproductive plan and depends on its fitness. A string with high merit has a high probability of making more copies of itself These copies create a new population and form a mating pool of individuals with high fitness. Thus, reproduction leads the search towards the optimum. Table 1. An example of string operations using binary alphabet. Original String Reproduction Crossover^ Mutation^ 1100101 1100101 1100|011 1100101 0110011 0110011 01101101 0110001 ^ Vertical lines indicate the position where the original strings were crossed over. 2 Bold face character indicates the detector that randomly changed its original value. Crossover is responsible for the exchange of parts (groups of detectors) between two strings (Table 1). It is the most important operator, because it controls exchange of information. Strings in the mating pool are crossed over to create a new population. Members of this new population combine high or low fitness detectors, and have high or low merit, respectively. The new population is then subjected to reproduction to create copies for a new mating pool. The probability of mutation for any string or detector is the same. Mutation creates new strings of either higher or lower merrit and introduces new knowledge to the populationWhile reproduction produces an accurate copy of the original string, crossover and mutation are responsible for changing the values of detectors in strings. Mutation occurs randomly within a predetermined number of operations (Table 1). GAs are different from other optimization techniques because they: (a) use coding of strings, not parameters; (b) search for populations (regions), not individual points; (c) use the objective function for evaluation, not derivatives or other auxiliary knowledge; and (d) use probabilistic, not deterministic rules. Classifier systems Terminology, GAs are used to develop Genetic Based Machine Learning Systems (GBML). The most common GBML architecture is the classifier system (CS), which learns simple string rules (classifiers) during operation (Goldberg, 1989). A classifier consists of two main parts, the condition and the message. It resemples the rule: //
2172 then <message>, and may have the form (:<message>), where condition and message are strings. Three different levels of activity can be distinguished in a classifier system: (a) performance; (h) simple learning; and (c) discovering (Booker et al., 1987). A classifier system consists of three main components: (a) rule and message system; (b) apportionment of credit system; and (c) genetic algorithm (Goldberg, 1989). Function, The rule and message system is located at the performance level and is a computational scheme that uses rules as its only algorithmic device; these rules are contained within the classifiers. When information from the environment is received, it is coded by the detectors and a string representing a message is formed. This string is posted in the message list and stored along with other messages. From there it might activate classifiers stored in the classifier store. In turn, these classifiers fire messages to be stored in the message list, and might activate either other classifiers or the effectors. Finally, the effectors decode the messages into actions and produce the output of the classifier system to the environment. Not all matching classifiers will post their messages in the message list. The selection is based on individual strength, which is determined by the apportionment of credit system. This system is located at the simple learning level and ranks classifiers according to assinged awards due to their performance (similar to the strength of strings). The more awards obtained by a classifier, the higher the probability of surviving and reproducing. The most prevalent way of ranking classifiers is the bucket brigade algorithm (Holland et al, 1989). This algorithm contains two main operations; (a) auction; and (b) clearing house. At the beginning of any process each classifier present in the classifier store has an initial net worth. When the condition of a classifier is satisfied, the classifier participates in an auction where it is evaluated according to its strength. Each time a classifier is selected to post its message, it pays a fee, through the clearing house to the source of the message that activated it. The source might be either the environment (input), or another classifier. The fee is called a bid and is proportional to the classifier's strength. The bucket brigade selects the best rules, but in order to introduce new ones into the system, genetic algorithms are employed (discovery level). The three operators (reproduction, crossover, mutation) create new rules, which are introduced in the classifier list. Strengths and weaknesses of GAs and CSs, Genetic algorithms are advantageous for use in process control because they are: (a) insensitive to noise (GAs adopt a population rather than a point approach, which resuhs in averaging over all the population and reduction of noise); (b) indifferent to problem structure; and (c) parallel, therefore fast in computation (Davis, 1988; Goldberg, 1988). Genetic Algorithms also have another promissing feature, their extensibility. GAs behave very well with optimization problems that have one optimum. In the case of multimodal problems, the stochastic error associated with the genetic operators makes the population to convergence to only one peak. This problem is known as genetic drift (De Jong, 1975; Deb and Goldberg, 1988; Goldberg and Segrest, 1987) and can be avoided by employing sharing functions (Goldberg and Richardson, 1987). Sharing functions rate the fitness of individuals within a niche (subdomain of a function) by counting the number of
2173 individuals in that niche, and by supporting the formation of niches close to several optima. Sharing functions allocate a certain number of individuals to every optimum pick. Higher picks receive a larger number of individuals. Niche formation accomodates the distribution of individuals in different regions of space, but for different points in time, the operators of dominance and the structures of diploidy are employed. Each information is coded in one pair, exactly like the chromosomes, and through a dominance relation it is decoded to a an expressed string. A primary weakness associated with this technique is its difficulty to generate and maintain long bucket brigade chains (Wilson and Goldberg, 1989). Long bucket brigade chains take a long time to be reinforced (Wilson, 1987; Riolo, 1987). Such approach seems to be practical only for programs that can be divided into a number of smaller steps. AUTOMATIC CONTROL AND FUZZY LOGIC Proportional integral derivative (PDD) controllers require a mathematical model that describes the process to be controlled. Many complex processes cannot be modelled, because of nonlinearities or absence of measuring accuracy. Experienced human operators sometimes achieve better control of such processes. Humans use imprecise linguistic terms to express the state of several variables such as: the temperature is very high, the flow rate is very low, open the valve slightly. Fuzzy control theory tries to imitate the human way of judging and uses linguistic description. Fuzzy controllers are based on thQ fuzzy set theory. Fuzzy set theory. In the classical set theory an item is either a member of a set {crisp set) or not. The process by which members of the universal set X {x ^X) are determined to be either members or nonmembers of a set A can be defined by a characteristic function. This function assigns a value \x (x) to every x G X such that:
..
[l if and only if X G^
Therefore, the characteristic function maps the members of the universal set X to a set^ as follows: H,:X^{0,l}
(3)
In fuzzy set theory, an item may be only partially member of the set (fuzzy set), and its membership is expressed by a membership function maped over the interval [0,1] (Klir and Folger, 1988). The membership function defines the degree of membership of an element x G X to the fuzzy set A. This function maps elements of the universal set over the interval [0, 1]:
2174
1]
ii/.X^[0,
(4)
For example, if the temperature of a room is 30^ C, then it might be high with a membership function value of 0.8 and medium with a membership function value of 0.3, high and medium being fuzzy sets. The theory of fuzzy sets is formulated in terms of the following three operators: complement operator
\ij{x):=\-\i^(x)
union operator
^^^u5W = inax[|^^(x),
intersection operator
\i^^^{x)-mvc^\i^{x),
where.
(5)
|IB(X)]
(6)
M-BW]
(7)
AyjA = X
(8)
These are called standard operations of fuzzy set theory and fmd many applications in fuzzy control. Fuzzy control Fuzzy control processes consist mainly of the steps shown in Fig.l. Process inputs include the state variables that are monitored during the process. These variables are measured and the obtained values are translated by the condition interface into fuzzy linguistic terms (fuzzification). At this stage values are assigned to the membership functions of the linguistic terms. In the fuzzy controller, fuzzy control rules are applied and a fuzzy set is defined over the universe of possible control actions. This fuzzy set is received by the action interface and is translated into a crisp control action (defuzzification) (Efstathiou, 1987).
inputs action interface
process
•
condition interface
outputs Figure 1. Fuzajy CO ntroller.
fuzzy
•
controller
fuzzy control rules
1
fuzzy set definitions
1
1
|
2175 For a fuzzy controller to function, the following must be defined: (a) fuzzy sets for input and output variables; (b) fuzzy control rules; and (c) the method of choosing an output action based on fuzzy results and rules (Kandel, 1992). Fuzzy controllers use rules such as 7/ then . The condition as well as the action are not variables themselves, but fuzzy sets. For example, // then . A set of such fuzzy rules constitute the fuzzy rule base. These rules are evaluated by the compositional rules of inference. The fuzzy rule ''if A then 5" (where A and B are fuzzy sets defined over universal sets X and Y of inputs and outputs, respectively) can be captured by a fuzzy relation R defined on XxY. For each fuzzy rule there are statements T represented by the relation:
|i7,(x, y)= mm[\i^(x\
\is{y)\
(9)
where, x GX and;/ G 7 . A number of these relations form the rule base relation R after setting the membership of (x, y) in R as the maximum of all its membership grades in any of the fuzzy relations r(Pedrycz, 1989). For a given input fuzzy set A' the membership function of the output>- in the set B' is defined by:
\^B'iy) = max{min[|a^,(x), \i^(x, y)]]
(10)
The rules used in fuzzy controllers are usually based on human experience, but they can also be developed using genetic algorithms (Thrift, 1991; Karr, 1991a; Karr, 1991b). It has been reported that a ^wzzy classifier system, which employs a genetic algorithm to evolve fuzzy rules, was proven successfuU in applications of simple static systems (Valenzuela-Rendon, 1991). The value of the output membership function is converted by the action interface into an action command. There are several ways to implement this: (a) maximum criterion method, (b) minimum of maximum method (MOM), (c) center of area or center of gravity method (COA) (Lee, 1990b). There have been several applications of fuzzy controllers in areas such as traffic control (Self, 1990; Nakatsuyama et al., 1990), wastewater treatment (Tong et al., 1980), cement grinding (Gilbert et al., 1989), and medicine (Lim and Teo, 1991). COMBmrnC FUZZY CONTROL AND GENETIC ALGORITHMS Fuzzy controllers do not require the use of a mathematical models that describe a process Thus, it is not necessary to fully understand the mechanisms of the process. Therefore, fuzzy controllers can be used instead of conventional PID controllers in food processes. Furthermore, the fuzzy rules can be created using a classifier system. This
2176 Genetic Based Machine Learning System (GBML) will search for the best "fitted" rules to be used by the fuzzy controller. Description of the controller The heart of any fuzzy controller is a computer program composed of the following parts: (a) condition interface (fuzzifier)', (b) detectors; (c) classifier system (classifier store, message list); (d) effectors^ (e) inference machine-action interface (defuzzifier); (f) bucket brigade; and (g) GA machine (Fig. 2). A traditional fuzzy controller includes only parts (a) through (e), whereas a GA-based fuzzy-classifier is composed of all seven parts.
input
r~i
fuzzifier
bucket brigade
output
classifier store
I
T~r
detectors
message list
_L
defuzzifier
T
GA machine
effectors
Figure 2. GA-based fuzzy controller. a. Condition interface (fuzzifier). Several fuzzy sets can be used to represent input variables. A seven set example with uhra low (UL), very low (VL), low (L), medium (M), high (H), very high (VH), and ultra high (UH) is shown in Fig. 3. If two actual variables, say a and /, where considered, the input to the controller would be the two variables a and /, and their rate of change over time, d and /, respectively.
Figure 3. Membership functions of a and / variables (for every a or I value one membership grade corresponds to each fuzzy set).
2177 In order to reduce the number of input variables, the values of a and / can be combined with their rates of change to form two new variables:
a -a + K-a
(11)
t = i+x-i
(12)
where, K and X are constants with dimensions of time. These constants represent a value of importance assigned to the time derivatives of the variables. Their magnitude depends on how fast the controller and the process react and can be determined experimentally. The a and / values are entered into the fuzzification unit, where a membership grade (|a) is assigned to each fuzzy set depending on its membership function (Fig. 3). The membership grade varies from zero to one, and the value of the variables from minimum to maximum attainable during the experiment. Initial membership functions are given in Fig. 3. b. Detectors. Each fuzzy set is coded in a binary system and forms a message string of the form: ### ###. The first three bits correspond to variable a and the last three to / . c. Classifier system. This consists of the classifier store and the message list. The first contains rules in the form of classifiers: ### ### : ### ### ### . The first two groups of bits are the condition, the last three the action. The groups of the action represent the input variables (three in this case). Fuzzy sets corresponding to the output variables are coded as in Fig. 4. When a message enters the classifier system it is posted in the message list. Then, the classifier store releases a classifier whose condition is satisfied by the message. This classifier is posted in the message list and all previous messages are erased. Each classifier carries the membership grades that correspond to the fuzzy sets of the two input variables. 1 1
UL 000
VL 001
L 010
M Oil
H 100
VH 101
UH 111
1 1
Figure 4. Coding of fuzzy sets in a binary system. d. Effectors. The rules fired by the classifiers are decoded to membership functions. e. Inference machine-action interface (defuzzifier). The rules released are processed by the minimum operation rule given by Eqn. 10 (Lee, 1990a and b). For a given set of input variables (a or / ) and a given rule (classifier), there is a membership grade |^ (A) and \x(L) that corresponds to each fuzzy set. The minimum of these two membership grades is chosen and superimposed on the membership functions of the
2178 output variables (Fig. 5). The area enclosed by the membership functions of the output variables and the chosen membership grade is estimated for every output variable. The union of areas of the same output variable, but from different rules, is then estimated. The analogue value of the output variable can be the gravity center of this union of areas. For example, it can be written:
e =J^
(13)
where, 0: an output variable, |LApg(0.): membership grade for a particular 0 value, FS: fuzzy set of the universe of discourse 0 (UL, VL, L, M, H, VH, UH), n: number of quantization levels.
mmimum VH
VL
VL
1st rule |Li M
UH
VH
VL
2nd rule |J-
— union
output e
p
Figure 5. Minimum operation rule. Equations similar to Eqn. 13 can be used to estimate all variables. An example with two input variables (a , / ), three output variables (0, 7C, p) and for two rules is given in Fig. 5. On the left side are the conditions, and on the right the actions. The control output (combination of the two rules) is given in the lower right side of the figure. The first rule is: if a is L and I is VH then 0 is UH and % is VL and p is VL. The second rule is: if a is M and I is UH then 0 is VH and % is VL and p is I.. The
2179 graphs on the left hand side represent the membership functions of a* and /* for the two rules. One value of \i corresponds to a and another to / . As these values pass through the minimum filter (dotted line) the lowest value within each rule is selected. This minimum value is used as the upper bound to defme areas under the lines of the membership functions of the action fuzzy sets. For each fuzzy set there are two defined areas, one for each rule. These are combined in the graphs on the lower right side of the figure, and a new area is formed for each fuzzy set by superimposition (union). The gravity center of each new area projected on the x axis gives the corresponding value for each fuzzy set variable. f. Bucket brigade. The strength (S) of each rule is evaluated and a credit is assigned by the following equation:
l-[(a
a
) + ( T •/ )]
(14)
where, a and T are constants, whose values are determined according to the importance of a and / responses. Each rule participates in the calculation of output variables depending upon its membership grade. The actual strength of each rule S furnished to the GA machine is:
S = S-\x^^
(15)
where, pi . is the minimum of the membership grades of all fuzzy sets participating in the rule. g. GA machine. Classifiers reproduce and mate according to their strength. The probability/?! of a classifier to reproduce itself is:
P=-^
(15)
and the number of copies v/ it will make is: v.^ri'p.
(16)
where, n is the number of classifiers in the store. The copies participate in the mating pool where they randomly mate. The new classifiers substitude the old ones in the
2180 classifier store. Mutation participates with a probability /?^. The number of mutated classifiers n^yi in each generation is:
^m = n'^-Pm
(17)
where, ^ is the number of bits of the classifier. The position of mutation is randomly selected. The new classifiers are stored in the classifier store and evaluated. In every cycle the weakest classifiers are deleted. POTENTIAL APPLICATIONS From the above discussion it becomes clear that there are two main requirements for application of Genetic Algorithms/Fuzzy Control in food processes. The first is the ability to collect information about the properties of raw materials and final products online. These measurements must be introduced into the Optimizer/Controller, which in turn produces the orders to change the process parameters. The second requirement is related to: (a) the time necessary for sensors to collect and process information and send their measurements to the optimizer/controller; and (b) the time that elapses before the process responds to the changes dictated by the controller. Both times should be as short as possible in order to make the whole system respond and achieve stability quickly. Quick responses result in the development of optimum fuzzy rules in a short time. Slow responses will result in the use of non-optimum fuzzy rules for a long time. Examples of potential applications of Genetic Algorithms/Fuzzy Logic in food processing include oil refining, liquid food pasteurization/sterilization, extrusion processes, some fermentations, extraction and distillation processes. In oil refining it is possible to make fast on-line measurements of acidity and loss of oil in the byproduct sream by using colorimetric techniques and sensitive flowmeters. The response of some refining processes to changes of operating conditions can be very fast. Therefore, oil refining seems to satisfy both requirements set forth for application of Genetic Algorithms and Fuzzy Logic. Similarly, many other food processes fuUfil the requirements for application of Genetic Algorithms and Fuzzy Control. CONCLUSIONS Many food and biochemical processes are complex and not easily modelled. Sometimes input and/or output parameters are difficult to measure. Therefore, such processes are often controlled and adjusted by human experts. Furthermore, the quality of incoming raw material sometimes varies significantly resulting in increased difficulty for process optimization. The use of fuzzy controllers that imitate the human way of thinking, and genetic algorithms with their ability to on-line optimize a process may result in more efficient operations. New processing plants that use GA-based fuzzy-
2(81 classifier control and optimization may become fully automated and time efficient. Old plants may improve their efficiency not by purchasing new equipment, but rather by improving the performance of existing ones. REFERENCES Androulakis, I. P. and Venkatasubramanian, V. 1991. A genetic algorithm framework for process design and optimization. Computers Chem. Engng. 15 (4):217-228. Booker, L. B., Goldberg, D. E., Holland, J., H. 1987. Classifier Systems and Genetic Algorithms, The University of Michigan, Technical Report, No. 8. Davis, L. 1988. Genetic algorithms and simulated annealing. Pitman, London. De Jong, K. A. 1975. An analysis of the behaviour of a class of genetic adaptive systems. (Doctoral dissertation. Department of Computer and Communication Sciences, University of Michigan, Ann Arbor). Dissertation Abstracts International. 36(10), 5HOB. (University microfilms No. 76-9381). Deb, K. and Goldberg, D. E. 1988. An Investigation of niche and species formation in genetic function optimization. Proceedings of the 19th annual Pittsburgh conference. Modelling and simulation. Vogt, W. G, Mickle, M. H. (Eds.), vol. 19, part 5, control, robotics. Efstathiou, J. 1987. Rule-based process control using fuzzy logic. In "Approximate reasoning in intelligent systems, decision and control", Sanchez, E. and Zadeh, L. A. (Eds.), Pergamon Press, Oxford. Gilbert, S., Coromina, J., Gomez, J., Recio, G and Pedersen, M. 1989. Fuzzy logic optimizes cement grinding at the Sanson cement plant, Spain. Zement-Kalk-Gips, 42 (6):286-287. Goldberg, D. E. 1989. Genetic algorithms in search, optimization, and machine learning, Addison-Wesley, Reading, MA. Goldberg, D., E. 1988. Genetic algorithms in adoptive control: Why bother? Workshop on adaptive control strategies for industrial use. Lodge Kananaskis, Alberta, Canada, June 20-22 (preprints). Goldberg, D. E. and Richardson, J. 1987. Genetic algorithms with sharing for multimodal function optimization. In "Genetic algorithms and their applications, Grefenstette, J. J. (Ed.), Lawrence Erlbaum Associates, Publishers, Hillsdale, NJ. Proceedings of the second international conference on genetic algorithms. MIT Cambridge, MA, July 28-31. Goldberg, D. E. and Segrest, P. 1987. Finite Markov chain analysis of genetic algorithms. In "Genetic algorithms and their applications", Grefenstette, J. J. (Ed.), Lawrence Erlbaum Associates Publishers, Hillsdale, NJ. Proceedings of the second international conference on genetic algorithms. MIT Cambridge, MA, July 28-31. Holland, J. H., Holyoak, K. J., Nisbett, R E. and Thagard, P. R. 1989. Induction. Processes of inference, learning, and discovery, MIT Press, Cambridge, MA. Holland, J. H. 1975. Adaptation in natural and artificial systems. The University of Michigan Press, Ann Arbor. Kandel, A. 1992. Fuzzy expert systems, CRC Press, Boca Raton Fl.
2182 Karr, C. 1991a. Genetic algorithms for fuzzy controllers. AI Expert, February:26-33. Karr, C. 1991b. Applying genetics to fuzzy logic. AI Expert, March:38-43. Klir, G. J. and Folger, T. A. 1988. Fuzzy sets, uncertainty, and information. Prentice Hall, Englewood Cliffs, NJ. Lee, C. C. 1990a. Fuzzy logic in control systems: fuzzy logic controller-part I. IEEE Transactions on systems, man, and cybernetics, 20 (2):404-418. Lee, C. C. 1990b. Fuzzy logic in control systems: fuzzy logic controller-part IL IEEE Transactions on systems, man, and cybernetics, 20 (2):419-435. Lim, C. C. and Teo, K. L. 1991. Optimal insulin infusion control via a mathematical blood glucoregulatory model with fuzzy parameters. Cybernetics and Systems 22:1-16. Nakatsuyama, M., Nagahashi, H., Nishizuka, N., and Watanabe, K. 1990. Matrix representation for fuzzy program and its application to traffic control. Automatic Control World Congress. Proceedings of the 11th Triennial World Congress of the International Federation of Automatic Control. Tallinn, Estonia, August 1317, Jaaksoo, U. and Utkin, V. I. (Eds) volume IV, Pergamon Press, Oxford. Pedrycz, W. 1989. Fuzzy control and fuzzy systems. Research Studies Press, Taunton, Somerset, England. Riolo, R. L. 1987. Bucket brigade performance:I Long sequences of classifiers. Genetic algorithms and their applications: Proceedings of the second international conference on "Genetic algorithms", Lawrence Erlbaum Assoc, Hillsdale, New Jersey: Self, K. 1990. Designing with fuzzy logic. IEEE Spectrum, November:42-105. Thrift, P. 1991. Fuzzy logic synthesis with genetic algorithms. Proceedings of the Fourth International Conference on Genetic Algorithms, University of California San Diego, CA July 13-16, (eds.) Belew, R. K. and Booker, L. B. (Eds), Morgan Kaufmann Publishers, San Mateo, California. Tong, R. M., Beck, M. B. and Latten, A. 1980. Fuzzy control of the activated sludge wastewater treatment process. Automatica, 16:659-701. Valenzuela-Rendon, M. 1991. The fuzzy clasifier system: A classifier system for continuously varying variables. Proceedings of the fourth international conference on "Genetic algorithms". University of California San Diego, CA July 13-16, Belew, R. K. and Booker, L. B. (Eds), Morgan Kaufmann Publishers, San Mateo, California. Wilson, S. W. and Goldberg, D. E. 1989. A critical review of classifier systems. Proceedings of the third international conference on "Genetic algorithms", Schaffer, J. D. (Ed.), George Mason University, San Mateo, CA, June 4-7, Morgan Kaufmann. Wilson, S. W. 1987. Hierarchical credit allocation in a classifier system. Proceedings of the tenth international joint conference on "Artificial intelligence", Los Altos, CA, Morgan Kaufmann.
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2183
VOLATILE COMPOUNDS IN WHEAT CULTIVARS FROM SEVERAL LOCATIONS IN KANSAS. Larry M. Seitz U. S. Grain Marketing Research Laboratory, Agricultural Research Service, U. S. Departnnent of Agriculture, 1515 College Ave., Manhattan, KS 66502. Abstract Five cultivars of wheat grain harvested at six Agricultural Experiment Station plots across Kansas in 1992 and 1993 were analyzed for volatiles by using a purge and trap instrument interfaced to a gas chromatograph equipped with infrared and mass spectrometric detectors. Even though odors of most of the samples were normal, more than 100 compounds were observed. Alcohols were most abundant, followed in order by aldehydes, alkanes, alkylbenzenes, ketones, methyl esters, naphthalenes, terpenes, and other miscellaneous compounds. Amounts of some volatiles differed among locations and cultivars, but none of the differences appeared to be associated with intrinsic properties of the cultivars themselves. Evidence for slight insect infestation was found in the 1992 samples. 1. INTRODUCTION Although wheat is an important component of the human diet, there have been only a few reports concerning volatile compounds in raw wheat grain (1-8) or flour (9,10). Other investigations of wheat volatiles have involved analyses of leaves and stems (11-13). In an early study of chemotaxonomic classification of seed, Hougen et al. (3) found mostly slight differences in a relatively small number of volatiles from five cultivars (varieties) of hard red spring wheat. This is the only previous reference to volatiles in specific cultivars of wheats. Development of off-odor detection and classification methodology for possible use in grain grading has been a major stimulus for research on volatile components of wheat and other grains (5-8). Recent research in our laboratory has shown that some specific compounds cause or are associated with off-odors in grains (7,8). However, there was need to know more about 1) how the total volatile profile might vary among good-quality samples with generally normal odor and 2) whether
2184 conditions during grain nnaturation or inherent properties of the cultivars themselves mi-jht influence the connposition of volatiles evolving from the grain. 2. EXPERIMENTAL 2.1 Samples Wheat samples were combine-harvested In 1992 and 1993 from performance test plots managed by the Kansas Agricultural Experiment Station (Kansas State University). Assays were performed on samples from six locations in 1993 [Labette (LB), Franklin (FR), Reno (RN), Ellis (EL), Finney (FN), and Thomas (TH) counties] and three locations in 1992 (FR, RN, and TH). Five cultivars with different parentages were collected from each plot as listed in Tables 1 and 2. As soon as possible after harvest, the samples were stored at about 4°C until analyses were conducted. A few researchers at our laboratory had access to the samples for other tests, so the samples were occasionally removed from cold storage for brief periods oi time. Bushel weights, heading dates, harvest dates, and other data concerning the cultivars were obtained from Reports of Progress published by the Agricultural Experiment Station (14,15). Weather data for each location was supplied by Mary Knapp, Kansas State University Extension Weather Data Library. Just before analysis for volatiles, each sample was smelled by two people with experience in classifing odors in grains. Except for a few 1992 samples with some slight off-odors as discussed below, the odors of all samples were found to be normal. 2.2 Dynamic Headspace Sampling Ninety grams of each whole wheat sample was placed in a glass sample tube of special design to accommodate grain samples. Details of the tube design will be published elsewhere. This special tube was mounted like a regular Tekmar sparger tube on a Tekmar purge and trap Instrument (model LSC 2000) equipped with a sample heater (model 211005) and a capillary interface module (model 2530) (Tekmar Co., Cincinnati, OH). Each grain sample was preheated without gas flow at 60°C for 2 min, and then the volatiles from the heated sample were purged with helium onto a Tenax trap (.29 g, 60-80 mesh, Tekmar Co., Cincinnati, OH). After an 8-min sample-purge time, an 8-min dry purge was performed to remove excess moisture from the Tenax trap, and the collected volatiles were preheated at 175°C and desorbed at 180°C for 4 min. With the capillary interface module, the desorbed
2185 Table 1 Wheat samples harvested from KAES test plots in 1992; County (Abbrv) [region] Franklin (FR) [Eastcentral]
Reno (RN) [Southcentral]
Thomas (TH) [Northwest]
Cultivar
Bu. Wt. (lbs)
Karl
55
TAM-107
49
Harvest Date
7/01
Mat. to Harvest (days)
Rain (days)
Total Rain (cm)
5/04
33
14
15
5/06
31
13
14
5/12
25
9
8
Heading Date
Tomahawk
50
Newton
48
5/12
25
9
8
2163
52
5/08
29
11
10
Karl
60
5/01
27
16
15
TAM-107
53
5/01
27
16
15
5/06
22
11
14
6/22
Tomahawk
56
Newton
53
5/08
20
9
10
2163
55
5/03
25
14
14
Karl
44
5/07
36
12
9
TAM-107
50
5/06
37
13
9
5/11
34
10
8
7/07
TAM-200
55
Newton
49
5/11
32
10
8
2163
57
5/10
33
10
8
Ave. RH
(%)
Ave. Temps Min, Max
(^c)
84
15,27
75
13,24
78
13', 27
^Additional information about column headings: "Bu. Wt. (lbs)" indicates bushel weight (test weight) in pounds, a unit comonly used in the United States. For these cultivars, 60 Ibs/bu or more is considered good quality. "Mat. to Harvest" is the number of days from physiological maturity to harvest. For this calculation, heading to physiological maturity was estimated to be 26 days. All of the weather data applies to the period from physiological maturity to harvest: "Rain" is the number of days with rain, and "Total Rain" is the amount of rain (inches) that fell during that period. Relative humidity (RH) and temperatures were approximately the same for all cultivars within a location. ^A damaging freeze occurred on May 26, 1992, at this location.
2186 Table 2. Wheat samples harvested from KAES test plots In 1993/ County (Abbrv) [region] Labette (LB) [Southeast]
Franklin (FR) [Eastcentral]
Reno (RN) [Southcentral]
Ellis (EL) [Central]
Thomas (TH) [Northwest]
Finney (FN) [Southwest]
Bu. Wt. Cultivar Karl
56
TAM-107
53
Tomahawk
47
Harvest Date
6/28
Mat. to Harvest (days)
Rain (days)
5/07
27
12
13
5/06
28
12
13
5/14
20
8
10
Heading Date
Total Rain (cm)
Newton
52
5/13
21
9
11
2163
50
5/11
23
9
11
Karl
56
5/17
35
13
13
TAM-107
53
5/15
37
15
13
5/22
30
13
13
7/16
Tomahawk
53
Newton
54
5/20
32
13
13
2163
55
5/21
31
13
13
Karl
58
5/17
18
7
6
TAM-107
53
5/17
18
7
6
5/23
12
6
5
6/29
Tomahawk
53
Newton
54
5/22
13
7
6
2163
56
5/21
14
7
6
Karl
58
5/22
19
12
17
TAM-107
58
5/21
20
12
17
5/23
18
11
13
Tomahawk
57
7/05
Newton
55
5/24
17
10
8
2163
55
5/23
18
11
13
Karl
58
5/22
30
12
13
TAM-107
56
5/21
31
12
13
5/22
30
12
13
7/16
Tomahawk
57
Newton
57
5/22
30
12
13
2163
55
5/25
27
12
12
Karl
61
5/20
15
4
7
TAM-107
57
5/20
15
4
7
5/22
13
4
7
6/29
Tomahawk
59
Newton
59
5/22
13
4
7
2163
58
5/24
11
2
5
^See Table 1 for additional information about column headings.
Ave. RH
Ave.Temps Min, Max
83
19,28
82
19,31
75
18,31
73
17,32
71
15,31
65
13,27
(%)
2187 volatiles were cryofocused at -140°C by liquid nitrogen at the top of the gas chromatograph (GC) column. Rapid heating of the cryofocused zone at 180°C for 0.75 min released the components in a narrow band for separation on a GC column. 2.3 Gas Chromatography-Fourier Transform Infrared Spectroscopy-Mass Spectrometry (GC-IR-MS) A model 5890 Series II GC coupled with a model 5965B IR detector (IRD) and a model 5970 mass selective detector (MSD), all from Hewlett Packard Co. (Palo Alto, CA), were used to analyze the grain volatiles. A BPX5 column (50 m x 0.32 mm i.d. X 0.25 um film thickness) from Scientific Glass Engineering lnc.(Austin, TX) was used for separation. Column head pressure was 89.5 kPa (13.0 psi) at 50°C, which gave a helium flow rate of about 1.7 ml/min. Then the flow rate was held constant by automatically increasing pressure as oven temperature increased. Oven temperature was held at 50°C initially for 2 min and then was increased to 140°C at a rate of 7°C/min, and to 230°C at a rate of 17.5°C/min. The temperature of the GC injector zone under the capillary interface module was maintained at 200°C. Effluent from the column first passed through the FTIRD and then into the MSD. IRD conditions included transfer line and flow cell temperatures maintained at 200°C, a liquid nitrogen-cooled Hg-Cd-Te detector (750-4000 cm''), and a spectral resolution of 16 cm"^ at a scan rate of 0.9 spectra/sec. MSD conditions were as follows: direct transfer line temperature, 230°C; ion source temperature, 250°C; ionization voltage, 70 eV; mass range, mass/charge 33-230 amu; scan rate, 1.57 scans/sec; electron multiplier voltage, 2600 V. 2.4 Compound Identification and Quantification Compounds were identified by computer matching of experimental infrared spectra and mass spectra of compounds with standard spectra in two IR vapor phase libraries (HP 59963A EPA and HP 59964A Flavors and Fragrances) and in the HP 59943B Wiley PBM MS database, respectively. All databases were from Hewlett Packard Co. (Palo Alto, CA). Two uL of a standard solution in methanol representing 20 ng each of 1bromo-4-fluorobenzene (BFB) and naphthalene-dg (NAPHD8) was injected at the top of the sample just before starting each analysis. The standards were eluted at 9.22 and 15.00 min, respectively. The NAPHD8 was used primarily as a monitor of column performance and retention time. The BFB was used to normalize day-to-day
2188 instrument response. An authentic standard was not readily available for every compound detected in this study. Methanol solutions of compounds that were available, as marked by an "s" in Table 3, were prepared at concentrations of 10 and 14 ng/uL. For calibration purposes, 2 uL of each standard mixture along with 2 uL of the BFBNAPHD8 standard was injected into an empty sample tube, and then purged as above for grain samples. Extracted ion chromatograms were used for specific detection and quantification of compounds. With each compound for which a standard was available, a response factor was determined, i.e ng per unit of extracted ion area, and then used to calculate the number of ng of compound purged from the 90-gram sample. With each compound for which a standard was not available, an estimated response factor was used based on the values of response factors for similar compounds within the same compound class. 3. RESULTS AND DISCUSSION During a single 8-mln purge with helium at 60°C, only a portion of the total volatiles present in a whole wheat sample was removed. This was demonstrated by the nearly uniform total-volatile results from five consecutive purges of a sample as shown in Figure 1. The increase in amount of total volatiles from the first to the second purge was probably due to more thorough and uniform heating of the sample. Composition of the volatiles changed slightly from the first to the fifth purge, with the first containing highest amounts of the low molecular weight compounds, as expected. Other results presented In this study represent only one purge of each sample. Because it took 30 to 40 min to analyze each sample, replication was limited to only a few samples. Two samples were analyzed in triplicate and two others in duplicate. Overall coefficient of variation averaged 17.1% with a range of 13.0 to 20.5%.
2189 _1.6 CD
P1.4 CL
2 1 +-|o.8+^ 0.6 + '•*—•
o 0.4 > S 0.2 o ^ 0
3 Purge Number
4
Figure 1. Total volatiles (sum of TIC peak areas) from five consecutive purges of a whole wheat sample. Atypical total ion chromatogram (TIC) from the MSD is shown in Figure 2. In addition to TIC's, numerous extracted ion chromatograms for specific ions or ion ranges were obtained to help identify and quantify specific compounds. Infrared data, which was recorded simultaneously with the mass data, especially aided the identification of compounds with relatively strong characteristic absorbances. Figure 3 shows an extracted wavenumber chromatogram (2700 to 3100 cm"^) corresponding to the TIC shown in Figure 2. Other wavenumber ranges were used for improved detection of specific types of compounds such as aldehydes, ketones, etc. Also, the infrared detector provided a means of monitoring the elution of compounds with molecular weight below the range set in the MSD, such as water, methanol, and carbon dioxide. To obtain good chromatograms, it was necessary to avoid excessive amounts of these compounds, especially water and carbon dioxide. Some methanol was present because it was the solvent for the internal standard injected into the sample tube at the start of each analysis. Compounds detected in the whole wheat samples are listed in Table 3 in order of abundance within each class for the 1993 samples. In general, relative abundance of compounds in the 1992 samples were similar to those for the 1993 samples. Some
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Figure 2. An example total ion chromatogram showing MS detection of volatiles purged from whole wheat at 60 "C. Numbered peaks correspond to compounds listed in Table 3. The wheat was Tomahawk cultivar grown in Finney (FN) County in 1993.
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Figure 3. Extracted wavenumber chromatogram (2700 to 3100 cm-') showing infrared detection of volatiles purged from whole wheat at 60 "C. Infrared data and the tic shown in Figure 2 were obtained simultaneously. Other wavenumber ranges were used for improved detection of specific types of compounds such as aldehydes, ketones, etc. Peak numbers correspond to compounds listed in Table 3.
2192 Table 3. Volatile compounds detected in whole wheat harvested at KAES test plots in Kansas in 1992 and 1993, listed in order of abundance within each compound class for 1993 samples. Cpd. No.
Compound Class and Name
Ret. Time "
Average^ ng/sample 1993
Identification Std'
spectra^
330 210 88 52 450 221 206 214 64 125 17 12 2 35 30 0.1
8
MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR ms/ir MS/IR MS/IR MS/IR MS/IR
54 59 24 14 25 24 6 4 1 0.1
s s s s s s s s
MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR ms/ir
63 55 50 26 39 24 10 7 8 5 5 5 3
s
MS/IR ms/ir MS/IR MS/IR ms/ir ms/ir MS/IR ms/ir ms/ir ms ms/ir MS/IR ms/ir
1992
alcohols 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2-propanol 1-hexanol 1-pentanol 1 -propanol 2-methyl-1-propanol 3-methyl-1-butanol 2-methyl-1-butanol 2-heptanol 2-butanol 2-ethyl-1-hexanol 1 -octanol 3-methyl-2-buten-1-ol 1-heptanol ethanol 2-pentanol 1 -octen-3-ol
3.43 7.76 5.82 3.68 4.11 5.32 5.38 8.43 3.89 11.28 12.19 4.00 9.98 3.32 4.80 10.21
280 250 140 77 45 24 23 16 15 14 8 6 5 4 3 0.5
6.41 4.43 4.35 4.80 3.62 12.98 10.72 8.50 10.11 7.56
180 73 36 27 26 17 13 8 5 1
10.49 10.23 6.24 14.78 9.54 13.82 10.32 14.62 9.43 17.25 13.55 12.67 14.16
65 58 41 35 33 18 10 8 8 6 5 4 4
s s s s s s s s s s s s s
aldehydes 17 18 19 20 21 22 23 24 25 26
hexanal 2-methyl-1-butanal 3-methyl-1-butanal pentanal 2-methylpropanal nonanai octanal heptanal benzaldehyde an enai''
alkanes 27 28 29 30 31 32 33 34 35 36 37 38 39
decane butylcyclohexane octane dodecane 5-methylnonane 5-methylundecane 1 -decene cyclododecane or 1-dodecene 4-ethyloctane an alkane an alkane undecane 2-methylundecane
s
2193 Table 3. (Continued) Compound Class and Name
Cpd. No. 40~ 41 42 43 44 45 46
tetradecane nonane an alkane 3-mGthyltridecane an alkane an alkane tridecane
Ret. Time "
Average^ ng/sample 1993
1992
Identification Std^
Spectra^
s
MS/ir MS/ir ms ms ms ms MS/IR
17.86 8.26 13.68 17.52 17.01 17.42 16.50
3 2 2 2 1 0.3 0.3
a3~ 3 3 0.1 0.4 0.0 34
7.84 8.36 5.86 10.65 9.69 9.87 7.66 9.92 10.26 11.23 10.03 12.61 11.26 11.84 12.47 13.41 11.96 12.40 12.18 13.32 11.77
61 45 22 14 9 8 6 4 3 3 3 3 3 2 2 2 2 2 1 1 1
35 27 10 13 8 7 3 3 3 3 3 2 2 2 2 1 2 1 1 1 1
s s s s
3.34 3.84 8.21 10.33 4.64 9.63 5.52 11.56 10.32 6.20
22 19 10 5 4 3 3 2 2 2
18 41 54 5 23 3 2 1 7 5
s
15.34 3.46 8.91 13.29
8 8 6 3
3 18 10 2
alkylbenzenes 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
1,3- & 1,4-dimethylbenzene 1,2-dimethylbenzene toluene 1,2.4-trimethylbenzene propylbenzene ethylmethylbenzene ethylbenzene ethylmethylbenzene ethylmethylbenzene 1,2,3-trlmethylbenzene 1,3,5-trlmethylbenzene ethyl-dimethylbenzene or methyl-(methylethyl)bz ethyl-dimethylbenzene or methyl-(methylethyl)bz diethylbenzene or methyl-propylbenzene methyl-(methylethyl)benzene tetramethylbenzene or ethyl-dimethylbenzene diethylbenzene or ethyl-dimethylbenzene methyl-(methylethyl)benzene methyl-propylbenzene or diethylbenzene ethyl-dimethylbenzene diethylbenzene
68 69 70 71 72 73 74 75 76 77
2-propanone 2-butanone 2-heptanone 6-methyl-5-hepten-2-one 2-pentanone 6-methyl-2-heptanone 2-methyl-3-pentanone 3-octen-2-one 3-octanone 2-hexanone
78 79 80 81
methyl methyl methyl methyl
s s
MS/IR MS/IR MS/IR MS/IR MS ms ms ms ms MS/IR MS/IR ms ms ms ms ms ms ms ms ms ms
ketones s s
s s
MS/IR MS/IR MS/IR MS/IR MS/IR ms/ir ms/ir ms/ir MS/IR MS/IR
methyl esters nonanoate acetate hexanoate octanoate
s s
MS/IR MS/IR MS/IR MS/IR
2194 Table 3. (Continued) Cpd. No. 82~ 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105
Compound Class and Name methyl decanoate methyl pentanoate methyl heptanoate
furans
2-pentylfuran 2-ethylfuran 2-methylfuran 3-methylfuran
naphthalenes
naphthalene 2-methylnaphthalene 1-methylnaphthalene
terpenes
limonene a monoterpene a monoterpene (beta-ocimene ? ) a monoterpene (alpha-terplnene ?)
miscellaneous
2,3-dihydro-1 H-indene a dihydro-methyl-(1H)-indene + tetramethylbz a dihydro-methyl-(1H)-Jndene a dihydro-methyl-(1H)-Jndene or me-propenylbz acetic acid 2(3H)-dihydrofuranone (gamma-butyrolactone) a lactone a lactone a lactone unknown
Ret. Time ~
Average^ ng/sample
Identification
1993
1992
16.95 6.80 11.12
1 0.5
2 0.4
10.41 4.78 3.86 3.93
7 5 4 2
8 6 6 7
15.06 16.96 17.18
13 2 1
12 1 0.4
S
11.35 10.88 10.62 14.66
7 5 4 0.4
5 4 10 0.0
S
MS/IR ms ms ms
11.63 14.11 12.72 13.91 4.32 9.15 9.94 11.74 12.12 11.37
1 1 0.5 0.4
1 0.5 0.5 0.3
S
ms ms ms ms MS/IR MS/IR ir ir ir ms/ir
i
-------
oT"
-------
Std^ s
s 8
S
Spectra^ MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR MS/IR ms ms
^The average represents five cultivars from six locations in 1993, and five cultivars fronn three locations in 1992. Sample size was 90 grams. ^An "S" indicates that an authentic standard was available. ^Capitol letters indicate that spectra from samples clearly matched spectra from the database libraries and/or from the standards. Lower case letters mean that spectra from samples, after considering suggested matches from database libraries, were consistent with the compounds listed. '^Trace amounts of other enals were detected at 5.63, 9.74,11.98, and 14.16 min with IR by using extracted wavenumber chromatograms representing 1714 cm'^
2195 exceptions to that pattern existed among the alcohols, alkanes, and ketones as discussed below. Also listed in Table 3 are times at which compounds were eluted from the GC column and information regarding identification of compounds from MS and/or IR spectra. Identifications were definite with compounds for which an authentic standard was available and/or where spectral identifications are indicated by capltol letters for both MS and IR spectra. Other compound identifications should be considered tentative, especially in cases where spectra indications in Table 3 are represented by lower case letters. Even though exact compound identifications could not be determined for some compounds, there was little or no doubt for most compounds about correct assignment to class due to the availability of both IR and MS spectra. In cases where only MS spectra were available, i.e. for many alkylbenzenes and alkanes, it was clear from the mass spectral patterns what class of compound was being detected. Complete identification of all compounds was beyond the scope of this project. The compounds reported in Table 3 were found consistently in all of the samples, especially if the average abundance was greater than about 2 ng/sample. Because of this consistency, it appeared that none of the samples were contaminated with substances foreign to wheat. Many of the compounds listed here were reported by Wasowicz et al.(4), although they had considered some compounds such as alkanes, methylalkanes, benzenes, xylenes (dimethylbenzenes), and 2-ethyl1-hexanol as possible contaminants. They also detected low concentrations of three terpenes, but llmonene was the only one they reported that could be positively Identified in this work. In common with compounds reported here, Buttery et al.(9) found hexanal, nonanal, hexanol, octanol, 1-octen-3-ol, several enals, naphthalene, and 2-methylnaphthalene in wheat flour. Variation in total volatiles (as measured by summing TIC peak areas for each sample) among locations and cultivars was mostly slight within samples from 1993 and in comparison of samples from FR and RN counties in 1992 with most of the samples from 1993 (Figure 4). There was no consistent pattern in total volatile variations among cultivars that could be linked to inherent differences in the cultivars, i.e. no cultivar consistently produced more or less volatiles than other cultivars at all locations. Conditions before and after harvest that may have affected amounts of volatiles are discussed below. How compounds within a class varied among locations is shown in Figure 5, with each bar representing a ng/sample result averaged over five cultivars as listed
2196
250
1992
Tomahawk TAM-107
RN EL LOCATION Figure 4. Variation in total volatiies (sum of TIC peak areas) among locations and cultivars from whole wheat grown in 1992 and 1993. Locations are identified in Tables 1 and 2.
2197
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Figure 5. Main classes of volatile compounds in wheat grown at three locations in 1992 and six locations in 1993. Locations are identified in Tables 1 and 2.
2198 in Tables 1 and 2. It is clear that alcohols were predominant in wheat grown both years, but especially so in 1992. Aldehydes, alkanes alkylbenzenes, and ketones were .generally more abundant than methyl esters, furans, naphthalenes and monoterpenes. Within each class, some compound amounts varied extensively among locations. The differences are not readily explainable from consideration of weather, days from maturity to harvest, and bushel weights listed in Tables 1 and 2. Evidence presented below suggest that storage conditions may have been a factor in alcohol differences in the 1992 grain. Variations in amounts of compounds among five cultivars for nine classes of compounds in whole wheat grown at six locations in 1993 and three locations in 1992 are shown in Figures 6 and 7, respectively. In some cases, samples representing the five different cultivars gave quite similar results, i.e. aldehydes, furans, methyl esters, and naphthalenes in samples from FR county in 1993. In other cases, variation among cultivars was considerable, i.e. for alkanes in samples from FR county in 1993, and furans and aldehydes in samples from FN county in 1993. Reasons for differences such as these were not apparent from the data concerning bushel weights, maturity, or weather conditions shown in Tables 1 and 2. Within each compound class there was no consistent pattern from one location to the next for either 1993 or 1992 (Figures 6 and 7). In 1993, for example, cultivars Tomahawk and TAM-107 had high levels of aldehydes compared to the other three cultivars in samples from FN county, but that was not consistent for other locations. In both years, terpene contents varied substantially among cultivars with no consistent pattern being evident. There were three notable differences between 1992 and 1993 samples which may be related to adverse moisture or temperature conditions that existed in the grain either before or after harvest. First, elevated amounts of 2-pentanol (Figure 8) and 2-pentanone found in the 1992 samples indicated slight damage from lesser grain borer which probably occurred sometime during the storage and handling of the samples (7). Slight insect damage (mostly holes bored into kernels) was visible in the 1992 samples with the damage being greatest in the wheat from TH county, in accordance with indications from the 2-pentanol and 2-pentanone results. No evidence of insect infestation was found in the 1993 samples. Second, the 1992 samples contained noticeable amounts of tridecane (Table 3), especially in the samples from TH county. Tridecane has been reported as a metabolite of mites (16), which suggests that the grain may have had enough moisture for a brief period to
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H i 2163
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LU 150 -f
<
CO DC LU
100 +
DL
CD
LB
FR
RN
EL
TH FN LOCATION
2-Methyl-1-propanol - e - 3-Methyl-1-butanol
FR
RN
TH
- • - 2-Methyl-1-butanol
Figure 9. Contents of 2-methyl-1-propanol, 3-methyl-1-butanol, and 2-nnethyl1-butanol in whole wheat grown at six locations in 1993 and three locations in 1992. Each point represents an average of results from five cultivars.
2202 support a mite population . Third, enhanced levels of 2-nnethyl-1-propanol, 2-methyl1 -butanol, and 3-methyl-1 -butanol, in the 1992 grain (Figure 9) signify that the 1992 grain nnay have been exposed to some dampness (7,17,18). However, the concentration of 1-octen-3-ol, which is produced by molds and causes a musty odor (4,7,19), was not elevated in the 1992 grain. The odors of the 1992 samples, especially those from TH location, were slightly sour compared to the definite normal wheat odor associated with the 1993 samples. A freeze on May 26,1992, at the TH location caused poor quality grain with low bushel weight and wide variation in seed size, but the relative significance of this factor in causing differences in the amounts of volatiles was unclear because some of the grain from FR county in 1992 was also low in bushel weight (Table 1). Some scab (Fusarium head blight) was present in the grain from FR county both years, but this did not seem to have any particular affect on the volatiles detected. In summary, results from the 1993 wheat samples show that amounts of compounds purged from the grain can vary considerably even though the odors were normal. The only compounds detected (Table 3) that previous research In our laboratory had shown to be associated with off-odors (7,8) included 1-octen-3-ol, 2pentanol and possibly 3-methyl-1-butanol. The latter two were implicated in reduced quality in the 1992 grain as discussed above, whereas the concentration of 1-octen3-ol was low in the grain from both years. Finally, the results presented here do not indicate that inherent properties of a cultivar control or strongly influence relative amounts of volatiles that evolve from the grain.
4. REFERENCES 1. J. Maga, J. Agric. Food Chem. 26 (1978) 175-178. 2. M. McWilliams and A. C. Mackey, J. Food. Sci. 34 (1969) 493-496. 3. F. W. Hougen, M. A. Qullliam, and W. A. Curran, J. Agric. Food Chem. 19 (1971)182-183. 4. E. Wasowicz, E. Kaminski, H. Kollmannsberger, S. Nitz, R. G. Berger, and F. Drawert, Chem. Mikrobiol. Technol. Lebensm. 11 (1988) 161-168. 5. M.C. Ponder, and D.S. Weinberg, Development of an effective method of detecting and identifying foreign odors in grain samples - literature and equipment survey. Southern Research Institute, Birmingham, AL, Contract 53-6395-5-59, U.S. Dept. of Agric, APHIS, 100 North Sixth Street, Minneapolis, MN 55403, 1985.
2203 6. D.S. Weinberg, Development of an effective method of detecting and identifying foreign odors in grain samples. Final Report, U.S. Dept. of Agriculture, Project 5803,VII/F, 1986. 7. L. M. Seitz, and D. B. Sauer, Off-Odors in Grains. In: G. Charalambous (Ed.) Off-Flavors in Foods and Beverages, Elsevier Science Publishers B.V., Amsterdam (1992) 17-35. . 8. L. M. Seitz, and D. B. Sauer, Cereal Foods World (1994) In Press. 9. R. G. Buttery, C. J. Xu, and L. C. Ling, J. Agric. Food Chem. 33 (1985) 115-117. 10. K. Lorenz, and J. Maga, J. Agric. Food Chem. 20 (1972) 769-772 . 11. R. G. Buttery, L. C. Ling, and M. M. Bean, J. Agric. Food Chem. 26 (1978) 179-180. 12. T. R. Hamilton-Kemp, R. A. Andersen, D. F. Hilderbrand, J. H. Loughrin, and P. D. Fleming, Phytochemistry. 26 (1987) 1273-1277. 13. T. R. Hamilton-Kemp, and R. A. Andersen, Phytochemistry. 25 (1986) 241-243. 14. Kansas Agricultural Experiment Station. Kansas performance tests with winter wheat varieties. Report of Progress 665, 1992. 15. Kansas Agricultural Experiment Station. Kansas performance tests with winter wheat varieties. Report of Progress 691 1993. 16. D. Tuma, R. N. Sinha, W. E. Muir, and D. Abramson, J. Chem. Ecol. 16 (1990)713-724. 17. D. Tuma, R. N. Sinha, W. E. Muir, and D. Abramson, Internat. J. Food Microbio. 8 (1989) 103-119. 18. R. N. Sinha, D. Tuma, D. Abramson, and W. E. Muir, Mycopathologia. 10(1988) 153-60. 19. E. Kaminski, S. Stawicki, and E. Wasowicz, Acta. Aliment Pol. 1 (1975) 153 . 5. Acknowledgement I thank Harold Mohr for laboratory assistance and Dr. D. B. Sauer for helpful discussions. U. S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Reference to a company or product does not imply approval or recommendation of the product by the USDA to the exclusion of others that may be suitable.
This Page Intentionally Left Blank
G. Charalambous (Ed.), Food Flavors: Generation, Analysis and Process Influence © 1995 Elsevier Science B.V. All rights reserved
2205
Autolysis of lactic acid bacteria: Impact on flavour development in cheese M. El Sodaa, N. Farkye^, J.C. Vuillemardc, R.E. SimardS N.F. Olsond, W. El Kholy^, E. Dako^, E. Medrano^, M. Gaber^ and L. Lim^ ^Department of Dairy Technology, Faculty of Agriculture, Alexandria University, Egypt l^Dairy Products and Technology Center, Cal. Poly State University, San Luis Obispo, California, USA ^Centre de Recherche STELA, Universite Laval, Quebec, Canada ^Department of Food Science, University of Wisconsin, Madison, Wisconsin, USA
Abstract
Early autolysis of cheese related microorganisms in cheese should potentially release the intracellular enzymes into the cheese matrix. Such an event should lead to a more rapid development of flavour in cheese. The autolytic properties of several cheese related bacteria as well as the different factors affecting the process are discussed. Enzyme release during cell autolysis is also considered.
1. I N T R O D U C T I O N Cheese ripening involves several dynamic and complex biochemical processes including proteolysis, lipolysis, and glycolysis. All three processes act in concert to give each cheese its unique flavor. An imbalance between reactions of the biochemical processes results in undesirable flavors. The biochemical processes are due to activities of residual rennet and, plasmin (proteolysis), lipases and esterases (lipolysis) and enzymes from starter and non-starter lactic acid bacteria (proteolysis, lipolysis and glycolysis). This suggests the importance of lactic acid bacteria in cheese ripening.
2206 Lactic acid bacteria used as starter cultures for cheese manufacture include Lactococcus lactis subsp. cremoris or Lc. lactis subsp. lactis (for Cheddar or Dutch-type cheeses), Streptococcus thermophilus or Lactobacillus delbrueckii subsp. bulgaricus (for Emmental and Italian-t3T)e cheeses). Others include Leuconostoc sp., Lc. lactis subsp. lactis biovar. diacetylactisy and Lactobacillus helveticuSy which are used in Dutch- and Emmental-type cheeses. Also of significance to cheese ripening are non-starter lactic acid bacteria that are indigenous to milk. These include Lactobacillus casei, Lb. plantarum. Lb. brevis, Pediococcus pentosaceus ^ and Micrococcus sp. Interest was also recently given to the use o{Bifidobacterium in cheesemaking. The primary role of starter bacteria in cheese manufacture is the fermentation of lactose to lactic acid. This glycolytic rolb continues during early stages of ripening until lactose is depleted. Production of lactic acid reduces the pH of cheese, thereby creating favorable conditions for other enzymatic reactions. Other glycol5^ic reactions of importance are catabolism of lactate and citrate to volatile organic compounds. For example, in Emmental-type cheese, lactate is metabolised into propionate, acetate, and CO2 by propionic acid bacteria The role of starter and non-starter lactic acid bacteria in lipolysis of cheese is limited. For example, free fatty acid levels in Cheddar cheese after 12 months of ripening is <0.2%. However, because lactic acid bacteria contain intracellular lipase and esterase activities, it is conceivable that these enzjmaes are released into the cheese matrix after cell lysis, thereby contributing to lipolysis. In varieties such as blue-veined cheeses, and Italian cheeses like Romano, Parmesan and Provolone, lipolysis is extensive, but it is due to activities of blue mold, or exogenous lipases or esterases added during manufacture. Of the biochemical processes that occur during ripening, proteolysis is most important. Rennet and plasmin primarily hydrolyse casein into large- and smallchain peptides that are further hydrolysed by proteolytic enzymes of starter and non-starter lactic acid bacteria into amino acids. Several studies have shown a strong positive correlation between the concentration of free amino acids and the intensity cheese flavor, suggesting the importance of lactic acid bacteria in flavor development. Lactic acid bacteria (LAB) have two classes of proteolytic enzymes; proteinases and peptidases. In general, proteinases of most LAB are bound to the cell wall, although some intracellular proteinase activity may be present in some organisms. Peptidases of LAB, however, are intracellular. In actively growing cells, small peptides taken into the cell are hydrolysed by the peptidases into free amino acids that may be released into the growth medium. In cheese, growth of LAB is limited, especially for the lactococci used for manufacture. Thus, increase in levels of free amino acids in cheese during ripening is partly due to activities of peptidases released after lysis of LAB. Thus, there are increasing interests to study the autolysis of LAB cells with the aim of selecting highly autoljiic strains to accelerate cheese ripening. r This paper reviews the literature on autolysis of lactic acid bacteria, and reports authors' preliminary results on autolytic properties of various LAB and their impact on cheese ripening.
2207
2. AUTOLYTIC PROPERTIES OF CHEESE RELATED MICROORGANISMS Autolysis could be defined as the spontaneous disintegration of the bacterial cell as the result of age or unfavourable physiological conditions which activate autolysin(s), enzymes found in the cell and capable of hydrolysing the cell wall peptidoglycan structure. The physiological functions of these enzymes is not fully understood, they probably play a role during cell division, wall growth and wall turnover. This process is of great importance during cheese ripening because it leads to the release of the intracellular enzymes that are now known to play a key role in protein and fat hydrolysis. Studies on tiie autolytic properties of lactic acid bacteria started when Hansen [1] demonstrated in the early fourtys that the growth of Lb. casei in cheese is stimulated in the presence of autolysed cells or cell extracts obtained from Lc. lactis subsp. lactis or Lc. lactis subsp. cremoris. The author also showed that the stimulation was significantly reduced in the presence of suspension of killed cells of the same microorganisms. Almost 30 years later, Ohmiya and Sato [2-3] as part of their extensive work on the proteolytic action of dairy lactic acid bacteria studied the autolysis of these microorganisms in an aseptic rennet curd system. Aseptic cheese was made from milk heated and then treated with hydrogen peroxide. The cells of Lc. lactis subsp. cremoris and Lb. helveticus were then sandwiched between the curds. Aseptic accessories and additives were used during the manufacturing process. A remarkable increase of cell free DNA could be measured in the cheese containing the sandwiched cell. The increase was higher in the curd incubated at 35oC when compared to the curd incubated at lO^C. The authors also reported that the amount of cell free DNA liberated from a cell suspension of the same microorganisms incubated in a conical flask was lower than the values obtained in the cheese system. This was attributed to a possible role of rennet in the promotion of cell autolysis. Evidence for the autolysis of lactic streptococci was reported by McDonald [4], the author's results indicate that slime forming strains exhibit lower autolysin activity when compared to non slime forming strains. Autolysis of lactic streptococci was then confirmed by Langsrud et al. [5]. The authors screened 45 strains of lactococci for their autolytic properties in a buffered growth medium. The obtained results showed wide intra-species variation. The autolysis of Lc. lactis subsp. cremoris ranged between 16 and 79% while the range was 24 to 87% for Lc. lactis subsp. lactis strains. In addition to lactococci, the rate of autolysis of other cheese related cocci was also measured. The reported values for 3 strains of Streptococcus thermophilus were 11, 17 and 24%, for Enterococcus faecium it was 65, 83 and 59%. In the case of Ec. faecalis values of 45, 36 and 45% were calculated for the 3 strains tested. The autol5d;ic properties of several cheese related microorganisms was studied at the University of Alexandria, Egypt. In order to compare the rate of autolysis, bacterial cells were grown in MRS medium, they were then harvested by centrifugation during the early stationary phase and resuspended in phosphate buffer O.IM, pH 5.5 containing IM NaCl and incubated at lO^C. The decrease in
2208 optical density was then measured at 650 nm. The obtained results indicated a low rate of autolysis varying from 2 to 5% after 48 hrs of incubation in the case of Pediococcus pentosaceus, Leuconostoc mesenteroides suhsp, dextranicum^ Bifidobacterium bifidurriy Bifidobacterium infantis, Propionibacterium freudenreichii and Propionibacterium acidopropionici. The rate of autolysis was however significantly increased if lysozjnne was added at a concentration of 10 mg/ml of the cell suspension. The increase in autolysis reached 60 to 80% for all the strains tested after 48 hrs of incubation. A similar work was then accomplished on the following Lactobacillus species: Lb. casei. Lb. plantarum. Lb. helveticus, Lb delbruckii subsp. lactis, Lb. delbruckii subsp. bulgaricus. Lb. fermentumj Lb. brevis and Lb. curvatus [7]. It was of interest to notice t h a t the rate of autolysis of the lactobacilli was higher t h a n the previously described species, the obtained results indicate autolysis rate varying from 30 to 80% according to the species tested. Addition of lysozj^me led to an increase in autolysis which was higher in the case of L6. delbruckii subsp. bulgaricus strains when compared to the other lactobacilli. The rate of autolysis of Lactococcus and Lactobacillus species was compared at different occasions, [8-10]. The general conclusion reached by the different authors indicate a higher rate of autolysis in the case of the Lactobacillus species as illustrated in Figure 1. 2.1. Influence of the physiological age of the cells on autolysis The physiological age of the cells seems to be one of the most important factors for cell autolysis and was investigated by several authors: Coyette and Ghuysen [11] detected autolysin activity in both log and stationary phase cells of Lb. acidophilus but the action of the enzyme on stationary phase cells was inhibited. Similar observations were also reported forL6. fermentum [11] where the autolytic system exhibited greatest activity during the exponential phase of growth and was hardly detected during the stationary phase. Higher rates of autolysis during exponential phase were also described inL6. helveticus [13]. Mou et aZ.[14] harvested cells of Lc. lactis subsp. cremoris during growth of the organism in broth. The autolytic activity was t h e n measured after resuspension of the cells in phosphate buffer. An increase in autolysis was observed during exponential growth reaching maximum activity before transition to the stationary phase. This was then followed by a marked decrease in the autolytic activity. A few years later, Niskasaari [15] confirmed these findings on her work on the characteristics of the autolysis of slime forming Lc. lactis subsp. cremoris strains. Cell autolysis as a function of the physiological age of the cells using the approach described by Mou et al. [14] was investigated on a wide range of species including L6. cosei. Lb. helveticus, Lb. delbruckii subsp. bulgaricus, Lb. fermentum, Lb. brevis, Leuconostoc mesenteroides subsp. dextranicum, Pediococcus pentosaceus, Pediococcus sp. Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris, Propionibacterium freudenreichii, Propionibacterium acidipropionici and Micrococcus sp. At least three strains of each specie were evaluated [6-9]. Figure 2 which illustrates the rate of autolysis ofL6. casei harvested at different phases of growth and subjected to autolysis in a buffer system clearly confirms liie findings of the previous authors. Similar results were also found in all the other species tested but to a different extent.
2209
100 90 80 <2 70 >- 60 50 40 < 30 20 10 0
II.
II l.ill III
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
STRAIN #
Figure 1. Autolysis of different cheese related microorganisms in a buffer system. Micrococcus sp (1^2); Bifidobacterium infantis (3); Bifidobacterium bifidum (4); Propionibacterium shermanii (5,6); Pediococcus pentosaceus (7,8); Leuconostoc mesenteroides subsp. cremoris (9,10); Lactococcus lactis subsp. cremoris (11^12,13,14,15); Lactobacillus casei (16,17,18,19,20).
Log. phase cells Early stationary phase cells Late stationary phase cells
3
4
5
Time / hrs.
n
6
1
24
r
48
Figure 2. Autolysis ofL. casei cells harvested at different stages of growth.
2210 2.2. Influence of physical treatments on the rate of autolysis Some interest was directed to the influence of temperature, freezing and heat shocking on the rate of autolysis of different cheese related microorganisms and will be discussed in this section. 2.2.1. Temperature Bie and Sj5strom [16] evaluated the influence of temperature on the rate of autolysis of a mixed culture from Flora Danica. The culture contained several lactococci and a Leuconostoc. The following temperatures were evaluated 6, 10, 14 and 190c. No autolysis could be measured at G^C during the first 5 days of the experiment, this was then followed by a gradual increase that reached 29% after 33 days of incubation. The autolysis rate increased with the storage temperature, 57% autolysis was obtained after 33 days at 14^0 while 51% autolysis could be measured at 19^0 after 9 days of incubation. When single cultures of Lc. loxitis subsp. cremoris were evaluated [14], it was shown that for strain MLl, there was a similar increase in autolysis at the different temperatures tested (25, 30, 35, 40 and 45^0) during the first 2 hrs of incubation. On the other hand, for strain HP the rate of autolysis increased with temperature however, the rate of autolysis at 40 and 45^0 began to decrease after 3 hrs of incubation. A complete loss of autolysis was then observed at 45^0 aftier 4 hrs of incubation. In a set of experiments where 14 strains of lactic streptococci were preincubated at 30^0, and then stored at 5, 10 and 20^0 [17]. Five Lc. lactis subsp. cremoris strains showed maximum autolysis at 5^0 and minimum autolysis at 30^C while six strains revealed an opposite behaviour (maximum autolysis at 30^0 and very little at 5^0). The remaining strains autolysed at a faster rate at lO^C. In the case of Streptococcus thermophilus, maximal lysis of strains 140/76 and I I F occurred at 45^0 [18]. At 34 and 37^0 a considerable lag period before lysis could be observed while no or very slight lysis of the cells occurred at 30^0. When higher temperatures were evaluated, the authors could not detect any lysis at neither 50 or 55^0. For Lb. helveticuSy the optimal temperature for cell autolysis was found to be in the 40-45^0 range. A total inactivation of the autolytic system was observed after 30 minutes at 60^0 [19]. The influence of temperature on cell autolysis was also considered by El Soda et al. [6,7] on a wide range of cheese related microorganisms including the following genera: Leuconostoc, Pediococcus, Lactococcus, Lactobacillus, Propionibacterium and Bifidobacterium. Two sets of experiments were carried out: in the first set, the rate of autolysis of strains from the species of the previously listed genera was measured at 10, 20, 30, 40 and 50^0. The obtained results indicated t h a t maximum autolysis occurred at temperatures similar or closed to the optimum growth temperature of the organism (Table 1). In the second set of experiments the cell suspensions from the different species were incubated at temperatures used in the ripening or storage of cheeses (4, 8 and 1 2 ^ 0 . The obtained values for all the species tested indicated a higher rate of autolysis at 120c.
2211 Table 1 Optimum conditions for the autolysis of different cheese related microorganisms Optimum temperature
Optimum pH
Optimum NaCl concentration
Lb. casei
30
5.5
0.5M
Lb.
40
5.5
0.5M
Lb. helveticus
50
5.5
0.5M
Lb. delbruckii bulgaricus
50
5.5
0.5M
Lb. delbruckii lactis
50
7.5
0.5M
Lb. brevis
30
7.5
0.5M
Lb. fermentum
30
5.5
0.5M
Pediococcus sp.
30
7.5
l.OM
Pediococcus pentosaceus
40
7.5
0.5M
Leuconostoc mesenteroides citrovorum
30
7.5
l.OM
Leuconostoc mesenteroides dextranicum
30
7.5
O.IM
Bifidobacterium infantis
40
7.5
l.OM
Bifidobacterium bifidum
40
7.5
l.OM
Propionibacterium acidilactici
40
7.5
l.OM
Propionibacterium freudenrechii
40
7.5
l.OM
Microorganism
plantarum
2212 2.2.2. Freeze shocking Several authors studied the effect of freeze shocking on the rate of cell autolysis due to the fact that freezing a bacterial cell at sub optimal temperature can lead to cell wall and cell membrane injury which can then lead to cell lysis. The first attempt was accomplished by Bie and Sjostrom [16] who subjected Lb. helveticus CNRZ 32 to 3 cycles of freezing at -20^0 followed by thawing at 20OC. Release of DNA was then taken as an index for cell lysis. The obtained results indicated a 100% increase in the amount of DNA from the cells that were subjected to the freezing and thawing cycles. It should however be noted t h a t these results were presented as preliminary experiments and were not confirmed in later publications. The previously described results [15] were to some extent confirmed by Ohmiya and Sato [20] who demonstrated t h a t the rate of autolysis of Lb. acidophilus cells stored at -2(PC overnight and then thawed was 45% higher when compared to cells stored at 3^0 for the same period of time. It was also indicated t h a t the storage time at -20^0 had a significant effect on the rate of autolysis. The % autolysis of cells stored at -20^0 for 10 hrs was 50%, the value was 70% if the cells were stored for 2 days at the same temperature. However, a storage time of seven days led to a slight decrease in the rate of autolysis. In an extensive study were the following species were involved Lb. casei, Lb. helveticus, Lb. delbruckii subsp. bulgaricus andL6. delbruckii subsp. lactisy Lb. brevis, Pediococcus pentosaceus, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. cremoris, Propionibacterium freudenrechii, Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris and Micrococcus subsp.. El Soda et al. [6-8] evaluated the effect of freezing at -20OC and then thawing the cells 3 times on the rate of cell lysis. The rate of autolysis which was measured after each cycle indicated that the autolysis was higher in the case of the frozen cells. The cells that were subjected to 2 cycles of freezing and thawing autolysed faster than the cells which were subjected to 1 or 3 cycles of ft*eezing and thawing. The decrease in the rate of autolysis after the third cycle of freezing and thawing may be due to a partial inhibition of the autolysins due to the physical treatment. 2.2.3. H e a t shocking The rate of autolysis of cells that were subjected to heat shocking was considered in several occasions, the available data are however contradictory. The results of Bie and Sjostrom [16] indicate an increase in the rate of autolysis if Lb. helveticus CNRZ 32 is subjected to the following subsequent heat treatments 54, 59 or 6 4 ^ 0 for 20 sec. when compared to non heated cells. In their experiments, the authors did not follow a decrease in cell turbidity and used DNA release as an index for cell autolysis. It was also noticeable t h a t a 52% DNA release could already be measured from the cells subjected to the higher lethal treatment at zero time while a 32% DNA release was recorded for the unheated cells. The release of DNA then reached 88% and 68% after a storage time of 60 days for the cells heated at 64^0/20 sec. and the unheated cells, respectively. However, when cells of the following species: Lb. casei, Lb. plantarum, Lb. helveticus, Lc. lactis subsp. lactis, Lc. lactis subsp. cremoris, Propionibacterium freudenrichii, Leuconostoc mesenteroides subsp. cremoris. Bifidobacterium bifidum
2213 and Bifodobacterium infantis were subjected to a heat treatment of 650C/16 seconds a different trend was noticed. The rate of autolysis of the previously mentioned species was monitored by measuring the decrease in optical density of a cell suspension. The results were also compared to a cell suspension that was frozen to -20^0 for two days and then thawed at 30^C and to a cell suspension of unheated cells. The general trend of the obtained results in the case of the heat shocked cells indicated a lag phase where no autolysis occurred during the first three hours of incubation, followed by little autolysis varjdng from 2 to 10% during the 120 hrs incubation time of the experiment. At similar experimental condition a much higher rate of autolysis varjdng from 30 to 80% according to the specie and/or strain tested could be measured for the freeze shocked cells. Untreated cells revealed autolysis t h a t were in most of the cases higher than the heat shocked cells and lower t h a n the fi^eeze shocked cells (Figure 3). The rather low rate of autolysis of the heat shocked cells was attributed to a partial denaturation of the autolytic system of the cells because of the heat treatment.
Untreated cells Freezing / Thawing 1 cycle Freezing / Thawing 2 cycles Heat shocking
3
-|
1 1 1 r 4 5 6 24 48 120 Time / hrs.
Figure 3. Effect of heat shocking and freezing and thawing cycles on the autolysis of L6. casei. 2.3. Influence of chemical treatment and pH on the rate of autolysis Autolysis of cheese related microorganisms was considered in the presence of several chemical compounds, it was also considered as a function of pH. 2.3.1. Influence of pH on the rate of autolysis In their pioneer work on the autolysis of lactic acid bacteria, Bie and Sjostrom [16] followed the autolysis rate of Lb. helveticus at pH values
2214 encoxintered during cheesemaking (4.9, 5.2, 5.7 and 6.8). The results obtained indicated t h a t the rate of autolysis is pH dependent and increased by increasing the pH. At pH 4.9 and 5.2 no autolysis could be measured during the first 15 days of the experiment. The autolysis of L6. acidophilus as a function of pH was considered by Ohmiya and Sato [20]. The authors used a wide range of pH values (5.5 to 8.0), and report an optimum pH for cell autolysis within a pH range of 6.5 to 7.0 where 20% of the initial turbidity could be measured. Lactobacillus helveticus showed a wider pH optimum ranging from 6.5 to 8.0 [19], the authors however report t h a t their result were not reproducible and differed according to the time of incubation used. A broad pH optimum was also obtained in the case of Lc. lactis subsp. cremoris [14], the maxima differed however according to the strain tested, the obtained values for strain MLl ranged from 6.0 and 6.5 while the corresponding values for strain HP were pH 6.5 and 7.0. Similar results were also obtained by Niskasaara [15] for slime forming strains of Lc. lactis subsp. cremoris. El Soda et al. [6,8,9] reports an optimimi pH of 7.0 and 7.5 for Pediococcus pentosaceus, Pediococcus sp., Leuconostoc mesenteroides subsp. cremoris, Propionibacterium shermanii, Propionibacterium acidilactici, Bifidobacterium bifidum. Bifidobacterium infantis, L. delbruckii subsp. lox^tis and L. delbruckii subsp. bulgaricuSy L. brevis and L. fermentum (Table 1). For L. casei, Lb. plantarum and Lb. helveticus strains, maximum autolysis occurred at pH values ranging from 5.0 to 5.5 [7]. Optimum pH of 5.0 and 5.5 were also reported for 12 strains of L6. casei [21] (Table 1). 2.3.2. Influence of salts on the rate of autolysis Although an enhancement of the rate of autolysis of L6. acidophilus could be measured by Ohmiya and Sato [20] due to the addition of 15% CaCl2 and MgCl2 to the autolysing buffer. An opposite situation was noticed in the case of Lb. helveticus in the presence of MgCl2 [19]. The role of Ca++ or Mg++ on the rate of autolysis is not quite clear and was not discussed by the authors. The influence of sodium chloride was also investigated and was found to increase the rate of autolysis at concentrations that could be found in a cheese system [6-7]. The positive effect of sodium chloride on the rate of autolysis was also confirmed for L6. casei [10]. The increased autolysis in the presence of sodium chloride was explained by Metcalf and Diebel [22]. The authors noticed that suspensions of Be. faecium resuspended in water lysed slowly if sodium chloride was added prior to lysozyme. However, when brief incubation with lysozyme was followed by addition of sodium chloride lysis occured immediately and extensively. This was explained by the fact that in the absence of competing anions, lysozyme, effect a rapid hydrolysis of the cell wall material but coat the negatively charged cell and prevent an over l5^ic reaction. The subsequent introduction of anions disrupt the protective layer and lysis occurs. On the other hand, if sodium chloride is present in the cell suspension and lysozyme is subsequently added a different situation is encountered. The positively charged ions of the salt compete with lysozyme for the negative charges on the cell surface and the negatively charged ions from the salt compete with the negative charges
2215 on the cell for lysozyme. As a result, the rate of lysoz3ane lysis is reduced. Confirmation of these findings with autolysins is still to be demonstrated. 2.3.3. Miscellaneous compounds Ethylene diamine tetraacetic acid (EDTA), trypsin and pepsin inhibitors seem to increase cell lysis [19]. This could be due to the inhibition of the cell proteolytic system t h a t may degrade the autolytic enzymes in the living cells. EDTA chelating action may also eliminate inhibiting cations from the autolytic enzymes environment. Contradictory results were however obtained by El Soda et al. [9] who showed a decrease in cell autolysis in the presence of IxlO"^ M of EDTA or phenjonethylsufonyl fluoride which may suggest the importance of metals and/or serine residues for the catalytic action of the enzymes. The same authors [9] also reported that while a slight increase in the rate of autolysis could be measured in the presence of 1% a-lactalbumin, sodium caseinate did not have any influence on cell ly^is. The carbon source in the growth medium seems to influence the action of autolysins. Rapid autolysis of Lc. lactis subsp. lactis, L. lactis subsp. cremoris, L. lactis subsp. lactis biovar. diacetylactis and Ec. faecalis was observed in a complex medium and in a partially defined casein hydrolysate medium [23]. Several experiments were then run by the same authors [24] on the biovariety diacetylactis for a better understanding of the above described observations. The results could be simimarized as follows: If the organism is grown at 320C in media containing glucose as the energy source, instant cell lysis began immediately after maximal growth or if the growth was stopped with chloramphenicol. The osmotic fragility of the cells was referred to their inability to use glucose as an adequate precursor of galactosamine. On the other hand, growing the organism in media containing galactose, lactose, maltose or glucose as energy source at IT^C resulted in cells that were resistant to lysis. The role of autolysins is not discussed. More recently, Vegarud et al. [17] also demonstrated t h a t glucose grown cells autolysed more rapidly than lactose grown cells. The influence of the energy source on cell autolysis was also described in Streptococcus thermophilus [18], substitution of lactose in M17 broth for sucrose, glucose or galactose resulted in a remarkable change in growth and autolysis. The organism became resistant to lysis in the presence of sucrose. However, when either glucose or galactose were used cells became more susceptible to lysis. This observation was not discussed by the authors and, the possible induction of a lysogenic phage was not verifyed. 2.4. Release of intracellular materials during autolysis For researchers in the cheese area, the release of intracellular enzymes and more particularly proteases, peptidases, esterases and lipases from the autolysing cells is of great importance. In fact, an early release of such enzymes should provide a more rapid development of cheese flavour and thus reduce the ripening time. Evidence for the release of DNA or soluble nitrogeneous components was reported [2-3,16], but will not be considered here. We will however discuss the release of enzymes that could be involved in the ripening process.
2216 Release of intracellular post-proline-dipeptidyl-aminopeptidase (PPDP) from Lc, lax^tis subsp. cremoris was first demonstrated by Wilkinson et al. [25] in a cheese system. Monitoring of PPDP release over a period of 120 days was then accomplished [26]. Very little enzyme release could be detected during the first 20 days of cheese ripening. This is then followed by a gradual increase in PPDP activity which then reaches a peak after 60 days. A decline in activity was then noticed and no PPDP activity was measurable after 120 days of ripening. Increased salt in moisture level in the range 0.4-5% were accompanied by a decrease of PPDP activity. PPDP activity was also lower in cheeses ripened at A^C when compared to cheese ripened at lO^C. Intracellular enzyme release was also studied in two Lac- Prt+ mutant strains Lc. lactis subsp. cremoris andL. Icu^tis subsp. lactis which autolyse rapidly at cheese ripening temperatures and at ionic strengths found in cheese [27]. DNA as well as LDH and proline aminopeptidase activities were found to be higher in the experimental cheese when compared to the control where only the starter organism was present. Proteolysis assessment during ripening using either amino nitrogen determination or FPLC of the peptide fractions also revealed higher values of amino nitrogen in the experimental cheeses. In a rather elegant publication Chapot-Chartier et al. [28] monitored the autolysis of two lactococcal strains in Saint-Paulin type cheese. Hie authors also determined the viability of the organisms in cheese, the morphological changes in the bacteria observed by electron microscopy as well as the release of intracellular peptidases. For one of the strains (Lc. lactis subsp. cremoris AM2) lysis occured from the first week of ripening leading to a decrease of cell viability which was also confirmed by electron microscopy observation. The authors also demonstrated that the aminopeptidase activity was measurable from the first day of ripening and remained active during the 60 days of the experiment. A 30% decrease in activity could however be measured at the end of the ripening period. Maximum aminopeptidase activity was detected after 20 days of ripening. The levels of measurable PPDP activities in the cheese seems to be lower than the aminopeptidases and, no PPDP activity could be measured after 40 days of ripening. In the cheese made with strain Lc. lactis subsp. lactis NCDO 763 an opposite situation could be observed. High cell viability, and no release of enzymes could be monitored during the first 3 weeks of ripening. This work that highlighted the differences in the autolytic properties of the lactococci was confirmed [29] for 3 L. lactis subsp. cremoris strains. These findings also confirm the work on Lb. casei [21], where the release of aminopeptidase activity from a highly autolytic strain as compared to a poorly autolytic strain was monitored in a buffer system. Figure 4 which illustrates these findings clearly demonstrate the higher amounts of aminopeptidase activity released fix)m the highly autolytic strain.
2217
Aminopeptidase from Lb. casei AU 1 Aminopeptidase from Lb. casei AU 2 Autolysis of Lb. casei AUl Autolysis of Lb. casei AU2
"I
1
10 20 30 Time /hrs.
1
40
r
50
Figure 4. Release of aminopeptidase activity from L6. casei cells during autolysis. Aminopeptidase activity was then monitored in Cheddar type cheese where both strains were evaluated separetely. Acidification was accomplished using gluconodeltalactone to eliminate interference from the starter peptidases. The enzyme could be detected in the cheese manufactured with the highly autolytic s t r a i n after 48 h r s of ripening while a week was necessary to detect aminopeptidase activity in the cheese made with the strain showing little autolysis. No aminopeptidase activity was measurable in a control cheese made under the same experimental conditions. Experiments accomplished in a buffer system [10] confirm the results described in cheese systems. Protein, aminopeptidase and PPDP release from Lb. casei were monitored at T^C in a buffer system. As far as enzjone release was concerned (Figure 5) little activity could be measured during the first 3 hrs of incubation, this was then followed by a gradual increase during the first 9 hrs after which both activities reached a plateau. On the other hand, protein release seems to be increasing during the 48 hrs of the experiment.
2218 120• ^"
Intracellular protein release Autolysis
" • h Aminopeptidase release ^*-
PPDA release
Figure 5. Release of aminopeptidase, dipeptidylaminopeptidase and protein from Lb, casei cells during autolysis. Lopez-Fandino and Ardo [30] studied the effect of heat shocking at 67 or 68^C for 15.5-16 seconds on the rate of enzyme release fromL. delbruckii subsp. bulgaricus. Their findings indicate a stimulatory effect of the heat treatment on the leakage of the aminopeptidase. Such results show beyond doubts that the pre-selection of strains based on their autolytic properties is becoming an important criteria which starter manufacturers should consider for their so-called ripening strains. 2.5. Proi)erties of partially purified preparations of autolysins For a better understanding and a possible control of bacterial autolysis, it is essential to isolate and characterize the enz3mies involved in the process. In the case of cheese related microorganisms very little information is available on such enzymes. However, some work was accomplished on closely related microorganisms such as enterococci and Lb, acidophilus and will be described in the review. The autolysin of Lb. acidophilus was first described by Coyette and Ghuysen [11]. The enzyme has the specificity of an endo-N-acetylmuramidase, it hydrolysed both N-acetylmuramic acid and N,0-diacetyl muramic acid linkages. Although, stationary phase cells were very poorly autolytic, the corresponding walls underwent rapid solubilization. The authors reported t h a t mechanical disruption of the cells may have liberated the autolysin which was somehow prevented from reaching the cells.
2219 The characterization of the N-acetylmuramidase was then described [31]. The optimum pH for lysing both intact cells or isolated cell walls was found to be pH 5 and 6. It was however noticed that the buffer used influenced the rate of cell autolysis. In fact, cell lysis is two to four times more rapid in citrate than in acetate buffer. Sulfliydryl groups seem to be required for cell autolysis because the activity is strongly inhibited in the presence of heavy metals and P-chloromercuribenzoate. Autolysin activity was successfully released after extraction of log walls at O^C in the presence of 100 jig/ml of bovine serum albumin. Alkaline extraction with sodium bicarbonate lead to a 90% loss of the enzymatic activity. The solubilized enzyme could then be rebind to sodium dodecylsulfate treated walls. Attention was also given to the autoljdiic system from Lb. fermentum where the autolytic system seems to be more complex when compared to Lb. acidophilus. Neujahr and Logardt [12] indicated that the autolysing system is composed of several enzjnoaes including an endo-N-acetylmuramidase, amidases and possibly an N-acetylhexosamine diacetylase. The autolj^ic activity is inhibited after a heat treatment of 60 to TO^C for 15 minutes or in the presence of 2% sodium dodecylsulfate. The initial rate of autolysis was found to be higher at
450c.
A great deal of attention was given to the autolytic system of Steptococcus faecium ATCC 9790 (Ec. hirae). Most of the work was accomplished by Shockman's group in the USA. The microorganism was shown to possess an autolytic enzjmae showing a single specificity [32-33]. It was also demonstrated that the B-1,4 acetylmuramylhydrolase activity had a very high affinity towards the cell wall of the specie and could only associate to the insoluble cell wall fraction of disrupted cells. The autolytic system is believed to be present in a latent as well as an active form. Activation of the latent form could be successfully achieved by proteinase treatment [34]. Release of the enzymatic activity from cell walls was obtained either by complete autolytic hydrolysis of the cell walls [32] by high salt concentration or dilute alkali treatment [35]. Bacteriolysis was shown to be induced in the presence of the non ionic surfactant triton X-100 which seems to be due to neutralization of one or more inhibitors of the active autoljiiic enzyme. Attention was then given to the purification of the autolytic system. Kawamura and Shockman [36] first described the purification and characterization of the latent form (muramidase-1). A near homogeneity preparation of the enz5ane was obtained after affinity chromatography on concanavalin A-Sepharose 4B. The purified latent form could be activated in the presence of trypsin and showed a molecular weight of 130,000. The molecular weight of the trypsin activated form was 87,000. The enzjone is also thought to be a glycoenzyme. Specificity studies revealed that the enzyme can hydrolyze the peptidoglycans from several bacterial species inchiding Bacillus cereus, Micrococcus luteus, Streptococcus mutants and Lb. acidophilus. However, little activity could be measured on the corresponding intact cells except for Lb. acidophilus. The same authors [37] successfully purified a second enzyme (muramidase-2), the enzyme was removed from the culture supemate by binding to SDS walls of Micrococcus luteus^ it was then eluted with 0.005 ]^I NaOH. Difference in the composition and substrates specificity of the enzyme were clearly demonstrated. It was however suggested that despite differences in
2220
specificity and functions of the two enzymes, defect in one of the enzyme may be at least partially compensated by the presence of the other. Also, a possible role in post-incorporation modifications and remodeling of the wall was given to the 2nd enzyme. More recently, Dolinger et al. [38] demonstrated that muramidase-2 contains two polypeptides, both possessing muramidase activity that could work in concert with muramidase-1. Attention was also given to the autolysins of starter bacteria. Mou et al. [14] suggested that the autolysin of Lc. cremoris subsp. cremoris had the specificity of an endo-N-acetylmuramidase and was localized in the cell wall fraction. The autolytic system of two slime forming and a non slime forming strain of Lc. lactis subsp. cremoris were also investigated. The optimal conditions for autolysis were reported to be a phosphate concentration of 0.01 M, pH 7.0 and 3 7 0 c . The specificity of the enzyme indicate that it is an endo-Nacetylmuramidase showing neither amidase nor endopeptidase activity. A heat treatment of SS^C for 20 min led to a total inhibition of the autolytic activity. Lipoteichoic acid as well as cardiolipin are effective inhibitors of the enzyme.
3. AUTOLYSIS RESEARCH: FUTURE AND PERPSECTIVES Interest in the autolytic properties of lactic acid bacteria and other cheese related microorganisms started in the early fourtys. The publications in this area from 1940 to 1970 represent 15% of a total of 50 publications which to the authors knowledge are the most relevant articles published on the subject. In the time period 1970 to 1979 and 1979 to 1989, 28 and 32% respectively of the articles were published while the remaining 25% appeared after 1989 which indicates the growing interest in the area and we believe that by the beginning of the 21st century much more publications will be available. One of the major problems in autolysis research is probably related to the methods used to follow autolysis in buffer or in a cheese system. The work of Bie and Sjostrom [39] contributed in the advancement of research in this area. The more recent work of Brikeland et al. [27] and Wilkinson et al. [26] as well as the approach of Chapot-Chartier et al. [28] and El Soda et al. [21] made it to some extent possible to study autolysis in cheese. The efforts of Lammers and Noomen [40-41] as well as the development of the model cheese system by Youssef [42] should also contribute to a better understanding of the process. More research is however needed for a better quantification of autolysis in cheese, more sensitive techniques using intracellular markers are needed since the available methods are still not satisfactory and lead to unreproducible results. Very little information on the autolysins of cheese related microorganism was made available. Future research in this area will probably lead to the purification and characterization of the autolytic systems of these technologically important microorganisms. Cloning of the enzyme and the availability of overproducing strains is also the aim of several laboratories. The availability of such information should then lead to the construction of strains that could be inducible to lysis during an appropriate stage of the
2221 manufacturing or the ripening process as previously suggested [43-44], Positive results in this area of research should benefit to the cheese industry and the consumer since at almost no additional costs, accelerated ripening will be provided with the highly autoljdiic strains. The availability of such strains should also contribute to more flavour development in low fat cheese where an overall lack of flavour was often reported.
4. REFERENCES 1
P. A. Hansen, J. Dairy Sci., 24 (1941) 969.
2
K. Ohmiya and Y. Sato, Agr. Biol. Chem., 33 (1969) 1628.
3
K. Ohmiya and Y. Sato, Agr. Biol. Chem., 34 (1970) 457.
4
J. McDonald, Can. J. Microbiol., 17 (1971) 897.
5
T. Langsrud, A. Landaas, and H. B. Castberg, Milchwissenschaft 42 (1987) 556.
6
M. El Soda, W. El Kholy, N. Ezzat and H. El Shafei, Unpublished results (1994).
7
M. El Soda, M. Gaber, N. Ezzat and H. El Shafei, Unpublished results (1994).
8
M. El Soda, M. Gaber and N. Power, Unpublished results (1994).
9
M. El Soda, E. Medrano and N. Farkye, Unpublished results (1994).
10
E. Dako, J. C. Vxiillemard, R. E. Simard and M. El Soda, UnpubUshed results (1994).
11
J. Coyette and J. -M. Ghuysen, Biochem., 9 (1970) 2952.
12
H. Y. Neujahr and I. -M. Logardt, Biochem., 12 (1973) 2578.
13
S. Lortal, M. Rousseau, P. Boyaval, and J. Van Heijenoort, J. Gen. Microbiol., 137 (1991) 549.
14
L. Mou, J. J. Sullivan and G. R. Jago, J. Dairy Res. 43 (1976) 275.
15
K. Niskasaari, J. Dairy Res., 56 (1989) 639.
16
R. Bie, and G. Sjostrom, Milchwissenschaft, 30 (1975) 739.
2222
17
G. Vegarud, H. B. Castberg, and T. Langsrud, J. Dairy Sci., 66 (1983) 2294.
18
E. Sandholm and S. S. Sarimo, FEMS Microbiol. Letters 11 (1981) 125.
19
S. Lortal, P. Boyaval, and J. Van Heijenoort, Le Lait, 69 (1989) 223.
20
K. Ohmiya and Y. Sato, Agr. Biol. Chem., 39 (1975) 585.
21
M. El Soda, L. Lim and N. F. Olson, J. Dairy Sci.,76 (1993) Supplement Page 130 (Abstract).
22
R. H. Metcalf and R. H. Deibel, J. BacterioL, 99 (1969) 674.
23
H. H. Moustafa and E. B. Collins, J. BacterioL, 96 (1968) 117.
24
H. H. Moustafa and E. B. Ck)llins, J. BacterioL, 95 (1968) 592.
25
M. G. Wilkinson, A. M. O'Keeffe and P. F. Fox, Irish J. Food Sd.and TechnoL, 13 (1989) 158.
26
M. G. Wilkinson, T. P. Guinee, and P. F. Fox, Int. Dairy J., 4 (1994) 141.
27
S. E. Birkeland, R.K. Abrahamsen and T. Langsrud, J. Dairy Res. 59 (1992) 389.
28
M. -P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal and J.-C. Gripon, Int. Dairy J., 4 (1994) 251.
29
M. G. Wilkinson, T. P. Guinee, D.M. O'Callaghan, and P. F. Fox, J. Dairy Res., 61 (1994) 249.
30
R. L6pez-Fandifto and Y. Ardo, J. Dairy Res., 58 (1991) 469.
31
J. Coyette and G. D. Shockman, J. of BacterioL, 114 (1973) 34.
32
G. D. Shockman, J .S. Thomson and M. J. Conover, Biochem., 6 (1967) 1054.
33
G. D. Shockman and M. C. Cheney, J. BacterioL, 98 (1969) 1199.
34
G. D. Shockman, H. M. Pooley, and J. S. Thompson, J. BacterioL, 94 (1967) 1525.
35
H. M. Pooley, J. M. Porres-Juan and G. D. Shockman, Biochem. Biophys. Res. Commun, 38 (1970) 1134.
36
T. Kawamura and G. D. Shockman, J. Biol. Chem., 258 (1983) 9514.
2223
37
T. Kawamura and G. D. Shockman, FEMS Microbiol, letters., 19 (1983) 65.
38
D. L. Dolinger, L. Daneo-Moore and G. D. Shockman, J. Bacteriol., 171 (1989) 4355.
39
R. Bie, and G. Sjostrom, Milchwissenschaft, 30 (1975) 653.
40
W. L. Lammers and A. Noomen, FEMS Microbiol. Reviews, Special Issue, 87 (1990) 114 (Abstract).
41
W. L. Lammers and A. Noomen, Proceedings, Cheese Ripening SeminareLund, Sweden 1992, P. 36 (Abstract).
42
Y. B. Youssef, Factors affecting proteolytic action ofLactococcus lactis in cheese, Ph. D. thesis Agricultural University, Wageningen, The Netherlands (1993).
43
J .M. Freitag and L. McKay, J. Dairy Sd., 70 (1987) 1773.
44
J .M. Freitag and L. McKay, J. Dairy Sci., 70 (1987) 1779.
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2225
INDEX
2,4 N-Acetylglucosamine, 1433 Additives food consumer survey, 705-719 saffron pigments stability, 881-849 Adsorption, starchy substances, water/ethanol, 1187-1199 Adulteration, SNIF-NMR detection, 369-373 Aflatoxin, decrease in peanuts, 1533-1546 Alcalase, 1397 et seq. Algin/calcium restructured beef, 1285 Algorithms, genetic, in food processes, 21692182 Alliin, 909, 2027-2028 Allium sativum composition, 2029-2033 storage, 2034-2036 wallichii composition proximate, 920 sulfur compounds, 927 volatiles, 923-925 Allyl isothiocyanate, 362-373 Allylic alcohol precursors, sulfiir-containing flavor compounds, 289-302 mechanism, 291-292 Aminoacids enzyme-generated, pork meat, 1303-1322 raw meat extract, 1327 free, generated during cured meat processing, 1316 meat contitioning, 1313-1316 Aminopeptidases, 1309-1312 Anitbrowning treatments, 491-495 Anti-Listeria activity, 972 Antioxidant activity coefficients, herbs and spices, 873874 influence on saffron pigments stability, 881-894 Antioxidants natural, canola oil, 46-479 oilseed phenolics, 1087-1099 spices, screening, 869-879 A. parasiticus, 1535 et seq.
Arbutus distillates, 1779-1790 Aroma basil, compounds in, 861, 862 C. hystrix DC compounds in, 242 detected odors in, 241-243 flavor powders, 244-245 canola oil, detected odors, 465 G. lutea, compounds in, 212-214 green coffee, compounds in, 788-794, 795802 herbs, lemon-like, compounds in, 833-847 olive oil compounds in, 424-426 detected odors, 423 potatoes, boiled vs. freeze-dried, compounds in, 486-489 raki, 1791-1811 scented tea compounds in, 818 soy milks, 1017 whisky distillates, 1767-1778 wines, 1659-1694 Artemisia herba alba Asso, essential oils, 147-205 collection sites, 162 composition, 186-190, 199 quantitative, 194-197 sensory properties, 200 et seq. Arid and desert areas, food production, 19472023 Ascorbic acid, 886, 887 Aspartame-sweetened tablet, packaging needs, 1119-1132 Atherogenic risk factors, 649-658 Aziridine, determination by capillary GC, 981-993 B BMP factors affecting, 1374-1375 flavor, 1370-1374 muscle conversion to meat, 1367 structure, chemical, 1368 synthesis, 1368-1370 Back propagation neural networks,
2226
food science, 2158-2168 Barrier process, seafood shelflife extension, 1453-1477 Basil aroma compounds, microwave extraction, 857-868 essential oils, inhibitory effect, 1925-1935 flavor comparison, different populations, 849-855 Beany off-flavor, soy milk products, 1007, 1008, 1010 Beef products, restructured, 1281-1301 patties, quality/fat content, 1345-1351 sensory evaluation, 1347-1350 peptide, flavor-enhancing, in, 1365-1378 Beer, origin identification by SNIF-NMR, 359 B-ionone, raspberry, 249-259 Biocatalysis, reverse micelles microbial cells for, 63-65 non-aqueous media, 9-11, 65 organic media, nev^ trends in, 44-63 Bioflavors, 1073; see also K. lactis, 1141 and C utilis 1141-1154 Biological materials, microbeam molecular spectroscopy, 2039-2108 Biopolymers, food applications, 75-109 Biosynthetic pathways, bisabolanes and cadalanes, 952 Bisabolanes, 963-964 Biosynthesis enzymatic vs. classic organic, 2 lower terpenes, 951-970 Biomass C sulpices, determination, 1165-1172 C. w^/7/5, fermentation, 1146-1149, 1152 K. lactis, fermentation 1075 et seq. separation systmes, 1187-1199 Bread flax seed, 649-658 quinoa, 1033 sorghum, 124-130 Browning enzymatic, potatoes, 491-495 non-enzymatic, raisins, 1057-1064
Cadmium, nutritional importance, 659-664 Cakes, sorghum, 130-133 Calpains, 1306 Camphene, 168 Camphor, 161 Candida utilis, submerged cultures, 11411154 Canola oil phenolics as antioxidants, 1087, 1089 stability, effect of natural antioxidants, 469479 toasted, odor evaluation, 465-468 Capillary electropherogram, BMP, 1370 Carbohydrate composition, soybeans, 1023 digestibility, extrusion effect, 575-594 Carbon dioxide essential oils extraction, 331-334 supercritical, in biocatalytic reactions, 5-6 N,0-Carboxymethylchitosan, 1433 Cardiac risk factors, decrease, 633-647 Cardiomyopathy, 633, 646 Carotenoids as food colorants, 882 from crustaceans, 1433-1437 from saffron, 883 CARSO methodology, 406, 412 Carveol, trans, 182 Carvone, 172 Casein bitter score, tryptic digest, 762 hydrolysate molecular weight distribution by SEC, 946 size exclusion chromatogram, 945 scheme for breakdown by lactococci, 754 Castanea molissima, 557, 563 sativa, 563 Catnip {Nepeta cataria L.) composition aroma, 844-845 flavor, 839-840 Cathepsins, 1306 Cereal products, cadmium in, 659-664 protein, extrusion effect on, 575-594 Cheese making, 722-723 products, generation, 747-749
2227
related microorganisms, 1823, 2205 ripening, primary and secondary changes, 724-725 Chestnut fatty acid composition, 563-568 flour extrusion, 557-562 Chicken fat, supercritical carbon dioxide extraction, 1353-1363 fatty acid analysis, 1356-1359 volatiles analysis, 1356, 1359-1360 Chios mastic resin essential oil, 303 harvesting, 311 physicochemical characteristics, 1937 Chitin from shellwaste, 1429-1433 applications, 1433, 1434 manufacturing flowsheet, 1432 Chromatography, centrifugal cunter-current, 1767-1778 Chrysanthenol, 179 Chrysanthenone, 178 Chrysanthenyl acetate, 180 Chylothorax, 629 Cineoles, 169, 170 Citrus hystrix DC, 235-248 extraction, 240 GC, volatile fraction, 241 Clostridium botulinum in meats, 1236, 1252 Coffee green, aroma compounds, 785-803 model systems, flirfuryl mercaptan formation, 805-813 solubles, IGC sorption behavior, 1906-1918 Cola drinks, diet vs. regular, consumer survey, 717 Consumer survey, food additives, 705-719 Conditioning meat, free amino acid generation, 1313-1316 Contour maps, 2065 et seq. Cooked cured-meat pigment absorption spectra, 1228 effect on NDMA formation, 1238 formation, 1226 preparation, flow diagram, 1227 Cookeina sulpices, evaluation as edible mushroom, 166-1170 composition, 1168-1170 Cooking, effect on peanuts, 1268 Com oil, 633-647
meal, adsorption behavior, 1188 Cucubita pepo, 549 squash content and extrudate properties, 553 Cultivars table grape, phenolic composition, 15791596 wheat, volatiles composition, 2192-2197 Cuparene TIC-GC profile, 957 MS spectra, 961, 962 Curcumene, 966 D U-Damascenon precursors, in grapes, 16451657 Davanafurans, 159 Davanone, 159, 184 Debittering activity comparison, lactobacillus spp., 759-762 Delicious, see Umami D/H, see isotopic ration, 360 et seq., 1637 et seq. Deuterated compounds, study/test of Wright's theory of olfaction, 497-524, 525-548 precursors, plant cultured cells, 951-870 Deuterium NMR, 364-365 Dietary unsaturated fatty acid, 665-674 Differential scanning calorimetry thermoanalysis, 1162-1163 Distillates, aroma compounds in arbutus, 1779-1790 equipment, 1780 volatiles, 1782 whisky, 1767-1778 equipment, 1769 separation, sulfur-containing from nonsulfur containing compounds, 1771 et seq. Dogfish fillets CFU/g., 1462, 1464-1469 color change, 1472 content moisture and fat, 1473 urea, 1461 pH, 1463, 1466, 1467, 1470 sensory evaluation, 1474 temperature requirements, 1464 TMA-N, 1462, 1466, 1467
2228
T B A change, 1471 Dragonhead {Dracocephalum moldavica) aroma composition, 844-845 flavor composition, 841-842 Drying process, 896-897 curves, peppermint, 900, 903, 905 Dynamic coupled column LC, 1175 et seq. E EDTA, 886, 887 EEC directive No. 28/388 of 22/6/88, 971 Egg yolk, low-in-cholesterol, 675-684 emulsifying properties, 679-681 Eicosapentaneoic acid, 665-674 Electrospray mass spectrum, BMP (q.v.) 1369 Emulsifying properties, lupin seed proteins, 2129-2138 Enterococcus, 1836-1837 Enzymes proteolytic, lactic acid bacteria, 753-767 reverse micelles catalytic properties, 25-31 gel-entraped, 55-56 PEG modified, 56 polymeric, nanogranule-entrapped, 57-58 solubilization aspects of, 11-21 conformational changes upon, 21-25 Enzymology, reverse micellar, applications in chemical and energy industry, 44 food industry, 37-40 new trends, 44-64 pharmaceutical industry 41-43 Essential oil of A. herba Alba, Algerian, 186-190 B. nigra and B. Juncaea, 361 basil and sage, inhibitory effect, 1925-1935 garlic, 2025-2037 lemon-like, Lithuanian herbs, 835-846 mastic resin, 303-310, 1943 spices, antioxidant activity screening, 869879 Essential oils solubility and phase equilibria with carbon dioxide, 331-354 critical properties, 336-337 equations of state, 333-337 phase equilibria prediction, 338-339
system solute/solute, 34-347 essential oil/carbon dioxide, 347348 Erucic acid, 633 Ethyl acetate production, 1141-1154 Ethyleneimine, see Aziridine Ethrel, 311, 1937 Exponential dilution, 1174-1177 Extruded cereal protein, 575-594 chestnut flour, 557-562 potato-based half-snacks, 569-574 poultry meat products, 1265-1280 quinoa products, 1038, 1039 rat diets, 595-623 rice flour/acomsquash blends, 549-555 sorghum products, 136-142 soybean products, 595 Extrusion processing of foods, 595-604
Fat content beef patties, 1345-1351 chicken, 1353-1363 Fatty acid composition, C. molissima, 561, 566 Fish protein, enzymatic hydrolysis, 1395-1404 sauce, histidine-added, 1417-1425 sensory evaluation, 1418, 1421-1422 volatiles composition, 1423 Flavor BMP, 1370-1375 cheese, 725-726, 1824, 2205 coffee, furfiiryl mercaptan, 805-813 compounds in muscle foods, 1323-1325 raspberry gelatin matrix, 253-260 key aroma, 258-259 sulfur-containing, from allylic alcohol precursors, 289-302 formation acceleration during cheese ripening, 721746 peanuts during roasting, 1519-1532 generation, thermal, in garlic, 909-918 intensity, effect of thickness on, 104-107
2229
natural, authentication by SNIF-NMR, 355378 orange juice, fresh, 1323-1325 roasted peanuts, microwaved, 1493-1518 stir fried - saute, 265-288 whey products, dried, 769-770 whisky, 1731-1751, 1753-1766 wines, 1703-1722 Flavonoids, in canola oil tabilization, 472-478 Flatulence producers, 1023 Flax seed bread, 649-658 Flour quinona/wheat blends, 1035, 1036 wheat adsorption behavior, 1188 moisture sorption behavior, 995-1005 Fluorescent micrographs, microstructure determination, 1158-1161 Food colors, consumer opinion, 716 enzymology in, 37-40 industry, reverse micellar applications in, 37-40 processes, optimization and control, 21692182 production, arid and desert areas, 19472023 photographic exposition, 1927-2023 Formulae, parenteral or enteral, 626 Function approach, normalized for optimizaiton, 2139-3150 Furfriryl alcohols, 294-299 mercaptan, 805-813 Fuzzy theory, food processes, 2169-2182
T radiation, effect on plasticizer migration, 453-463 Garlic essential oil, Egyptian, 2025-2037 constituents, 2033 storage effects, 2034-2037 non-volatile flavor precursors, 909-918 volatiles from non-volatile precursors by thermal degradation, 913 interactions of lipids and non-volatile precursors, 914-915, 916-917 sugars and non-volatile precursors, 914,
916 Gas chromatograms, volatile fractions of aziridine, 986 A. wallichii, 922 A. sativum, 2032 basil oil, 852, 853, 854 bergamot oil, 817 boiled potatoes, 488 C. hystrix, 241 freeze-dried potatoes, 489 G. lutea, 211, 215-219 ginger, natural, 286 green coffee, 800, 801 H. planus, cultured cells, 925 linalool and limonene, 819 soy milk, deodorized vs. non-deodorized, 1012 Gel formation and network properties, 81-83 Gels as enzyme hosts in biocatalysis gelatin, 45-48 lecithin, 48-49 micro-emulsion-based, 44-45 "weak", 89-90 biopolymers, phase-separated, 90-101 Genetic algorithms and frizzy control, 21752180 Gentiana lutea L. Roots, 207-234 aroma/detected odors, 231 compounds identified, 212-214 GC volatile fraction, 211 Glucopyranosyl sinapate, 1098 GPNA, 26 Glucose syrup from sorghum grain, 142 B Glucosidases, enological yeast, 1623-1635 Grains, unprocessed/extruded composition, 582 Grapes glucosides in, hydrolysis, 1623-1635 6-damascenone in, 1645-1647 nitrogen compounds in, 1659-1694 table, phenolic composition, 1579-1596 H Half-heating time approximations, 1136-1137 Half snacks, potato-based, extruded, 569-574 Ham cooked, 1316 dry-cured, 1316-1318, 1331-1341
2230
non-volatiles, 1333-1334 process technology, 1331-1332 volatiles, 1334 Health concerns, beef consumption, 1283 Heating times, comparison by geometry, 1137-1138 Hexanal in soy milk, 1011-1014 (E)-2-Hexen-l-al compounds, deuterated, 525-548 Hickory sawdust smoking, 1025-1029 Hypolipidemic agent, 673 HPLC-CLND, 379-396, 929-949 HPLC chromatogram, olive oil polyphenol extracts, 444
Ikaria, Greece, drinking water collection sites, 2114 Infant, critically ill, nutrition, 625-631 Inverse GC, 1188, 1895 Ion chromatogram microwaved roasted peanuts, 1500 wheat volatiles, 2190-2191 Immunoblotting, cell-free extract, 764-767 Ionizing radiation, effect on additives migration, 454-455 IRs™, 2041, 2052 g/5e^. UR-PLAN™, 2053 Isotherms coffee, freeze-dried vs. spray-dried, 1909 dynamic sorption, 1917 wheat flour, moisture sorption, 999, 1001 Isotopic analysis in food products, 358 carbon, 13, 372, 374 nitrogen, 15, 373 Isotopic ration (D/H), 360-361, 376 D/H values in sugar mixtures, 1637-1644
Jeruk purut, 235-248; see also C. hystrix DC Jimbu, see Allium wallichii and garlic K Kallidendron technology, food production in arid and desert areas, 1247-2023 Kernels, peanuts, 1519-1532, 1533-1545 Ketone, raspberry, 257-261
Kinetics aziridine decomposition, 991 drying, effect on peppermint quality, 895907 enzymatic reactions, reverse micelles, 32-37 non-enzymatic browning, 1063 Kluyveromyces fragilis, ethyl acetate production, 1142 lactis broth fermentatin 1073-1086 changes in major volatiles, 1080-1086 fermentation compounds, 1078-1079
Lactic acid bacteria, proteolytic enzymes, 753-767, 1828-1836 applications, 755 proteolytic pathway, 753-754 raw olives, 1852 Lactobacillus strains comparison, 756-763 Lactone, oak, effect on wine aroma, 16951702 Lemon balm (Melissa officinalis) aroma composition, 844-845 flavor composition, 837-838 Lemon catmint, see Catnip Lemon-like aroma, monoterpenes contribution, 844-845 aroma herbs, 833-848 Leuconostoc, 1837-1839, 1849 LCS, 971 etseq. Lard, autoxidation rate, BHT and spice antioxidants effect, 876 Limonene basil, 861, 862, 864 com/waxy com starches, 1178 GLC analysis, 819 lemon-like herbs, 837-844 odor-sensory profile, 821 orange juice, fresh, 1490 strawberry, 1387 Lipase, C cylindracea, 61 Lipid oxidation, BMP, 1375 Lipolysis, impact on cheese flavor, 18241826 Liposome technology in dairy industry, 736738 Liquid crystal mesophases, lyotropic, 52-55 Lobster, protein hydrolysate production, 1405-1415
2231
free aminoacid content, 1411 sensorial analysis, 1413 Lupin seed proteins, 2129-2138 M Maillar reactions, 273, 280, 781, 794, 812 Malt quality evaluation, 1813-1821 Mannitol, Agaricus bisporus, 1873 Mastic resin essential oil, chemical composition changes during solidification and storage, 303-310 new harvesting method, 311-329 physicochemical characteristics solid and fluid, 1937-1945 Meat curing, nitrite in, 1223-1240 Meat and poultry products, L. monocytogenes growth and inhibition, 1243-1263 Mesifiiran, 1387, 1389, 1390, 1392 Metals, heavy, in forest species leaves, 1886, 1994 Methyl ketone formation, 1826 Micelles, reverse, enzyme reactions in, 1-74 Microalga, source of highly unsaturated fatty acid, 665-674 Microbial cells, whole, for biocatalysis, 63-65 Microdroplet behavior in solution, water, 1113 Microbeam molecular spectroscopy, 20392108 Microemulsions bicontinuous, 61-62 detergentless, 59-61 gelatin gels, 45-48 lecithin gels, 48-49 media for enzymatic reactions, 61-62 organogels, 44-45 polymers, effect on vapor pressure of, 1101-1117 Microspectroscopy, infrared synchrotron radiation, 2072-2104 thermal source, 2063-2071 Microwave extraction, basil aroma compounds, 857-868 heating effect on roasted nut flavor, 1493-1518 water-alcohol mixtures, 1065-1072 Migration of PVC DOA into olive oil, 457462 Model systems, coffee, ftirfiiryl mercaptan
formation, 805-813 Moisture sorption, wheat flours, on heat treatment, 995-1005 Molecular weight and distribution by CLND, 929-949 Monoterpenes, irregular, in A. herha alba, 153 Monoterpenes, aroma contributions to lemonlike aroma, 844, 845 MPRF, 1453 et seq. MSG consumer opinion of, 715 perception mechanism, 1370-1373 Microstructure changes, comstarch/soy protein systems, 1155-1165 Moisture barrier requirements, tablets 11191132 Moisture sorption, aspartame-sweetened tablets, 1123 et seq. M semimembranosus, aminoacid content, 1305 Multivariate perspectives, shelflife research, 1201-1222 Multiresponse optimization methods, 21392150 Multibarrier process, 1453-1478 Multiisotopic analysis, 1723-1730 Muscle protein hydrolysis, pathway, 1307 Mushrooms Agaricus bisporus, 1873 cultivated, soluble sugar changes 1865-1880 Mustard oil, 361-373, 633-647 N Natural (bio-) antioxidants, 1087 et seq. flavors, 1141 et seq., 1073 et seq. Neural networks, food science back propagation, 2158-2163 characteristics, 2156-2157 food-related applications, 2163-3165 Neutrase 0.5 L, 1397 et seq. Neutron activation analysis of drinking water, 2109-2127 pH measurements, 2119 method diagram, 2122 sample collection sites, 2115-2118 Nitrite, alternatives for processed meats, 1223-1241
2232
Nitrosamines, volatile, in foods, 685-704 analytical methodology, 689-690 formation and occurrence barley meat, 694-695 cured meats, 691-694, 1224, 1236-1238 dried foods, 694 dry milk, nonfat, 696-697 migration, 697-698 Nucleotides and nucleosides, detection by HPLC-CLND, 379-396 calibration curves, 388-390 response factors, 391, 394 simultaneous detection, HPLC/UV, 395 traces of standard mixture, 386-387, 393 Nut flavor, roasted, 1493 et seq. Nutrients in forest species leaves, 1884-1885 Nutrition, critically ill infant, 625-631 Nutritional importance cadmium, 659-664 zinc, 595-623, 659 Normalized function approach method, 21392150 O Ocimum basilicum L. essential oil inhibitory effect, 1925-1935 flavor comparison, different populations, 849-855 microwave aroma extraction, 857-868 Odors basil, 861, 862 canola oil, 465 coffee, 797, 799, 801 C. hystrix, 241-243 G. lutea, 231 gentian, 231 herbs, lemon-like, 833-847 olive oil, 423, 424, 426, 864-866 potatoes, boiled vs. freeze dried, 483-487 tea, 822 Odorants, green coffee, derived from: lipid oxidation, 798-799 Strecker degradation, 799-800 sulfur compounds, 799 Off-flavor compounds green coffee, 795, 801 roasted peanuts, 1527 protein isolate, 1266 soy milk, 1007-1019
Oilseed phenolics antioxidant activity fractions canola, 1094, 1095 flaxseed, 1095, 1096 mustard, 1094, 1095 extrants content, 1094 separation, 1093 Oligosaccharide levels, soybeans, flatulence, 1023 Olfaction, Wright's theory of, study with deuterated sex pheromone mimics, 494-524 test with deutereated compounds, 525-548 Olive oil basil-aromatized, 864-866 virgin aziridine in, 989 odorants evaluation, 419-427 phenols determination, 429-452 plasticizer migration, 453-463 quality optimization, 397-418 sensory analysis, 403-411, 438 treatment of grapes, 1058 Olives, black, fermentaion, 1849-1863 Orange juice, fresh, flavor, 1479-1492 volatile constituents analysis, 1484-1488 identified and quantified, 1489-1490 Oregano taxa, carvacrol and thymol content, 875
PAL, USO et seq. Pandalus borealis, 4129 Panulirus spp., 1405-1415 Pasteurization, cold, 455, 461 Pastry, sorghum, 133-136 Peanuts aflatoxin decrease, 1533-1546 carbohydrate metabolism, 1547 flavor formation during roasting, 1519-1532 microwaved, analyses of, 1504, 1505 oil roast vs. dry roast, 1508-1514 Peasy off-flavor, coffee, 801 Pediococcus, 1839 Peppermint quality, drying curves, 900, 903, 905 kinetics, 895-907 Peptides and protein hydrolysates separation by HPLC-CLND, 929-949
2233
Periplaneta americana L., 497-524, 525-548 pH dependent behavior of enzymatic activity, 27-28 memory, 4 Phase equilibria, essential oil/carbon dioxide prediction, 338-340 solubility, 232-233 system: essential oil/C02, 347 solute/solute, 340-343 solute/solvent, 344-347 Phenols liquid condensed smoke, 971-979 oilseeds/antioxidants, 1087-1099 olive oil, extraction/determination, 429-452 Photographic exposition, food production in arid and desert areas, 1957-2023 Phytase activity, 599-600 Phytate, mineral bioavailability, 600 Phytic acid, extruded vs. non-extruded rat diets, 595-623 Piperitol, cis-, 183 Pistacia lentiscus var. Chia chemical composition changes during solidification and storage, 303-310 new harvesting method, 311-329 physicochemical characteristics, 1937-1945 Plasmin, 731 PLS relationship, 447-450 Polyphenol antioxidants, canola oil stabilization, 469479 content, olives, 1854 extraction, olive oil, methods, 432-435, 441, 442, 444 Polysorbate 80, preparation of low-cholesterol egg yolk, 675-684 Pork cooked. Hunter color values, 1228 meats, enzyme-generated free aminoacids, 13031322 flavor peptides in, 1325-1331 Potatoes boiled, aroma components, 481-490 in half snacks, 569-574 processed, enzymatic discoloration, 491-495 Poultry meat products, extrusion-cooked, 1265-1280 Polysaccharides and aroma compounds,
interactions 6-cyclodextrins, 1182-1183 dextrans, 1182 dextrin, 1180 galactomannans, 1180-1181 hydroxypropyl celluloses, 1181-1182 starches, 1177-1179 Porridges, sorghum, 123-124 PR-EOS, 332-335, 344-349 Precursors allylic alcohol, 289-302 garlic, 909-918 intracellular modificaiton during carbonic maceration, 1380 plant cultured cells, deuterated, 951-970 Prenyl alcohol, 298 Processing equipment and food quality, 10431055 distillation, 1050-1051 heat transfer, 1048-1050 mechanical, 1046-1047 sanitation, 1051-1052 Processing effects on aminacid availiability in meats, 1312-1313 Proteolytic activity comparison-lactobacillus and strains, 757-759, 760-762 Protein concentrate from aquatic species, 14411451 composition, 1444, 1445-1446 hydrolysate, 1446-1450 production flowsheet, 1443 fish, enzymatic hydrolysis hydrolysate composition, 1400-1401 reaction parameters optimization 13971400 lobster, enzymatic hydrolysis, 1407 hydrolysate composition, 1411-1413 shellwaste, 1428-1429 Proteins and aminoacids, nutritional aspects in meat, 1303-1305 Proteolysis, postmortem, muscle enzyme system, 1306-1312, 1368 PTN3.05, U91 etseq. PVC plasticizer migration into olive oil, 453463 Pyrazines gentians, 232 green coffee, 800-801 hickory smoke generation, 1027
2234
peanuts oil-roasted, 1502-1503 microwaved, 1507-1508 whey protein concentrate products, 781
Quinoa in foods, 1031-1042 characteristics functional, 1033, 1034 et seq. sensory, 1033, 1036, 1039 R Radon spas, Greece, 2111 Raki, aroma compounds, 1791-1811 Rapeseed oil, low erucic acid, see canola oil, also 633-647 Rates of development, temperature distributions in foods, 133-1140 Raspberry flavor, analytical technique, 249264 Raisins, non-enzymatic browining parameters during air drying air-drying temperature, 1061 color, 1060 moisture control, 1062 Restructured meat, 1285-1286 Reverse micelles, enzyme reactions in, 1-74 enzymatic reactions in, 32-37 enzyme catalysis in, 37-44 enzymes, catalytic properties, 25-31 enzyme solubilization in, 11-21 conformational changes on, 21-25 multienzyme system in, 41 reaction media macroheterogeneous, 3-8 microheterogeneous, 8-9 schematic representation, 12 Reverse processing, coffee, 1903, 1914 Rhodella reticulata, 665-674 Ribose, Agaricus bisporus, 1877 Rice flour/acorn squah blends, 549-555 Rio-flavor, 794-795 Ripening, accelerated, cheese, different methods, 727-742 future developments, 742-743 Risk factors improvement with edible oil processing, 633-647 flax seed bread, 649-658
Saffron, 373-377, 881-894 Saffranal, 374, 376 Sage essential oils, inhibitoryy effect, 19251935 Saltiness, BMP vs. MSG 1374 Salvia officinalis, see Sage Saponins, 1032 Sardines, 1418 et seq. S. cerevisiae strains as starters, 1597-1622 Selenium, dietary, 633 et seq. Sensory analysis, soy milk, 1010-1011, 1014-1015 BMP flavor, 1370-1375 differences, olive oil samples, 406-412 evaluatin beef patties, 1347-1350 dogfish, 1460, 1474 roasted peanuts, 1529-1530 Sesamol, 465 Sesquiterpenes, labelling patterns, 961 Sex pheromone mimics, American cockroach, 497-524 Shelflife extension of seafood, 1453-1477 research, multivariate perspectives, 12011222 Shellfish processing discards, 1427-1439 Shrimp, frozen, thaw water, 1430 Size exclusion chromatography, separation of peptides and protein hydrolysates, 929-949 calibration curves, 937-940 chromatograms, 935, 942-945 schematic, 933 Smoke generation, hickory sawdust, resulting pyrazines - role of air, 1027 moisture, 1028 temperature, 1027 Smoke-liquid flavorings as anti-bacterial agents, 971-979 Sodium benzoate, 889, 890 Solvent systems, enzymtic reactions, water organic single-phase, 6 two-phase, 6-8 Sorghum grain, quality of its edible products, 111146
2235
chemical composition, 114-118 nutritional quality, 118-119 physical properties, 112 ultrastructure, 112-114 milling, 119-123 products, 123-128 Soy bean cheese, 747-752 milk off-flavors, 1007-1019 protein hydrolysate molecular weight distribution by SECCLND, 946 size exclusion chromatography, 942 protein isolate, off-flavor, 1266 Soybeans, carbohydrate composition, 10211024 Spent lees, whisky flavor compounds, 17531756 Spices, antioxidant screening, 869-879 Squalus acanthus, 1457 Starch protein model, food systems, 1155-1164 water binding, restructured beef, 1281-1301 Starter culture, black olives fermentation, 1849-1863 Stir-frying basic techniques, 266-271 flavors, 271-276 contributing technologies, 276-284 Storage studies C. hystrixDC, 238 garlic oil, 2034-2036 mastic resin essential oil, 303-310 mushrooms, cultivated, postharvest, 18651880 olive oil, 412-415 potatoes, 491-495 raspberry, 257-258 saffron pigments, 890, 891 strawberry fruits, under CO2 atmosphere, 1379-1394 tea, 823-824 whey protein concentrate, 777-782 Strawberry fruits, effect of storage under CO2 atmosphere, 1379-1394 Sugar mixtures, D/H values, 1637-1644 Sulfuring, raisins, 1059 Sulfur containing aroma compounds from allylic alcohol precursors, 289-301 Sulfur compounds in Jimbu, 927
SNIF-NMR in authentication of natural flavors, 355-378 applications, 358-360 Superactivity, enzyme, 2 Supercritical fluids carbon dioxide, in biocatalytic reactions, 5-6 chickenfat extraction, 1353-1363 herbs and spices extraction, 279, 331-354
TBA number, cooked pork, 1233 TEARS, 471, 474-475, 640, 642, 645, 653, 1088, 1089, 1091, Tea bergamot, 824 Earl Grey, 823 sensory profiles, fresh vs. stored, 823-824 Tea water, aluminum in, by ^^Al NMR, 827832 Temperature air-drying effect on raisin color, 1061 distributions in foods, 1133-1140 Terpenes, GC-MS studies on biosynthesis of, 951-970 a-Terpineol, 173,231 Texture profile analysis, 100-101 Thickness, effect on flavor/taste intensity, 104-107 Thermal processing, properties of food systems, 1155-1164 Thujone a, 176 6, 177 TLC chromatograms, basil aroma compounds, 863-865 Trehalose, Agaricus bisporus, 1876 Tricyclene, 167 TOTOX, 471, 476-478 Tyrosol, 441 U Umami, 1366, 1370-1374 Underutilized pelagic fish species, protein applications, 1450 concentrates, 1442-1446 hydrolysates, 1446-1450
2236
Value-added components from shellfish discards carotenoids, 1433-1438 chitin and chitosan, 1429-1433 enzymes, 1429 proteins and flavorants, 1428-1429 Vapor pressure measurements, polymer microemulsion systems, 1105-1111 Variables, formulation and extrusion, roles on potatoe-based half snack properties, 539-574 Vegetable protein hydrolysate molecular weight distribution by SECCQLND, 946 size exclusion chromatogram, 943 Viscosity concentration and shear-rate dependence of, 84-88 objective and perceived "thickness", 102103 intrinsic and molecular size, 83-84 Volatiles, see Flavor, also individual materials W Wastewater irrigation 1881-1894 Water analysis by neutron activation, 2109-2127 binding, by starch, in beef products, 12811301 dynamics, moisture-sensitive foods, 18951923 ethanol mixtures, microwave heating, 10651072 Wheat cultivars, volatile compounds 2183-2203 flour, moisture sorption behavior, 995-1005 spectra, 2047 et seq. Whey protein concentrate commercial products flow chart, 772 composition, 775, 776 organic compounds, 115-111 volatile compounds, IIS-ISO Whey protein hydrolysate molecular weight distribution by SEC, 946 size exclusion chromatogram, 944 Whisky distillates, aroma substances separation.
1767-1778 maturation, flavor development in, 17311766 Wines aroma compounds, effect on of grapenitrogen compounds, 1659-1694 characterizaiton by volatile flavor compounds, 1703-1722 chemical composition and sensory properties, 1617-1618 classification by multi-isotopic analysis, 1723-1730 isotopic fingerprint by SNIF-NMR, 358 must, alcoholic grade increase, 1637-1644 phenylalanine, wine aroma precursor, 1380 wood-aged aroma, effect of oak lactone, 1695-1702 Winsor III systems, 62-63 WOF/MFD, 1374
Xanthophylls from shellfish discards biosynthesis, 1436 chemical structures, 1435 distribution, 1436, 1437
Yeast 6-glucosidases, 1623-1635 Yeasts, selection as starters, 1597-1622
Zinc availability, rat diets, 595, 623 Zymogram staining, 763-764
