Advances in Food and Nutrition Research, Volume 58

Advances in FOOD AND NUTRITION RESEARCH VOLUME 58 ADVISORY BOARDS KEN BUCKLE University of New South Wales, Austral...

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Advances in

FOOD AND NUTRITION RESEARCH VOLUME

58

ADVISORY BOARDS KEN BUCKLE University of New South Wales, Australia

MARY ELLEN CAMIRE University of Maine, USA

ROGER CLEMENS University of Southern California, USA

HILDEGARDE HEYMANN University of California, Davis, USA

ROBERT HUTKINS University of Nebraska, USA

RONALD JACKSON Quebec, Canada

HUUB LELIEVELD Global Harmonization Initiative, The Netherlands

DARYL B. LUND University of Wisconsin, USA

CONNIE WEAVER Purdue University, USA

RONALD WROLSTAD Oregon State University, USA

SERIES EDITORS GEORGE F. STEWART

(1948–1982)

EMIL M. MRAK

(1948–1987)

C. O. CHICHESTER

(1959–1988)

BERNARD S. SCHWEIGERT (1984–1988) JOHN E. KINSELLA

(1989–1993)

STEVE L. TAYLOR

(1995–

)

Advances in

FOOD AND NUTRITION RESEARCH VOLUME

58 Edited by

STEVE L. TAYLOR University of Nebraska, Lincoln

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 32 Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK First edition 2009 Copyright # 2009 Elsevier Inc. 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. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice 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. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. ISBN: 978-0-12-374441-8 ISSN: 1043-4526 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

1. Quinoa (Chenopodium quinoa Willd.): Composition, Chemistry, Nutritional, and Functional Properties

vii

1

Lilian E. Abugoch James Introduction Chemical, Nutritional, and Physical Properties Proteins Carbohydrates Lipids and Lipidic Compound Antioxidant Capacity, Phenolic Compounds, and Flavonoids Saponins Minerals and Vitamins Functional Properties Present and Future Uses of QS References

I. II. III. IV. V. VI. VII. VIII. IX. X.

2. Chemoinformatics—Applications in Food Chemistry

2 4 6 10 15 18 18 19 20 24 25

33

Karina Martinez-Mayorga and Jose L. Medina-Franco I. Introduction

Molecular Descriptors and Physicochemical Properties Molecular Databases and Chemical Space Chemoinformatics in Food Chemistry Examples of Molecular Similarity, Pharmacophore Modeling, Molecular Docking, and QSAR in Food or Food-Related Components VI. Concluding Remarks and Perspectives Acknowledgments References II. III. IV. V.

3. Processing of Food Wastes

34 36 37 40

43 52 53 53

57

Maria R. Kosseva I. Introduction II. Sources and Characterization of Food Wastes III. Recovering of Added-Value Products from FVW (Upgrading Concept)

58 63 69

v

vi

Contents

Multifunctional Food Ingredient Production from FVW Vegetable Residues as Bioadsorbents for Wastewater Treatment Using Eggshell Added-value Products from Whey Food Waste Treatment FCM Aspects Aimed in Sustainable Food System Development Summary and Future Prospects References

IV. V. VI. VII. VIII. IX. X.

4. Technological and Microbiological Aspects of Traditional Balsamic Vinegar and Their Influence on Quality and Sensorial Properties

82 94 98 98 100 116 120 123

137

Paolo Giudici, Maria Gullo, Lisa Solieri, and Pasquale Massimiliano Falcone I. Introduction II. Basic Technology III. Chemical Composition IV. Physical Properties V. Conclusion

References

5. Nanostructured Materials in the Food Industry

138 148 168 176 177 178

183

Mary Ann Augustin and Peerasak Sanguansri Introduction Approaches for Nanoscale Manipulation of Materials Processes for Structuring of Food Materials Nanostructured Materials Functionality and Applications of Nanostrucutured Materials Nanotechnology and Society The Future Acknowledgment References

I. II. III. IV. V. VI. VII.

6. Gossypol-A Polyphenolic Compound from Cotton Plant

184 185 185 192 199 206 206 207 207

215

Xi Wang, Cheryl Page Howell, Feng Chen, Juanjuan Yin, and Yueming Jiang Overview of Cotton and Cottonseed Products Occurrence of Gossypol Physiochemical Properties of Gossypol Gossypol Analyses Agricultural Implication Biological Properties Clinical Implication Conclusions References

I. II. III. IV. V. VI. VII. VIII.

Index

216 218 218 226 228 233 249 251 251 265

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

Lilian E. Abugoch James

Departamento Ciencia de los Alimentos y Tecnologı´a Quı´mica, Facultad de Ciencias Quı´micas y Farmace´uticas, Universidad de Chile, Vicun˜a Mackenna 20, Santiago, Chile (1) Mary Ann Augustin

CSIRO Preventative Health National Flagship, Adelaide, South Australia 5000, Australia; CSIRO Food and Nutritional Sciences, Private Bag 16, Werribee, Victoria 3030, Australia (183) Feng Chen

Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634 (215) Pasquale Massimiliano Falcone

Department of Agricultural and Food Science, Amendola, 2, 42100 Reggio Emilia, Italy (137) Paolo Giudici

Department of Agricultural and Food Science, Amendola, 2, 42100 Reggio Emilia, Italy (137) Maria Gullo

Department of Agricultural and Food Science, Amendola, 2, 42100 Reggio Emilia, Italy (137) Cheryl Page Howell

Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634 (215) Yueming Jiang

South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P. R. China (215) Maria R. Kosseva

UCD School of Chemical & Bioprocess Engineering, College of Engineering, Mathematical & Physical Sciences, University College Dublin, Belfield, Dublin 4, Ireland (57)

vii

viii

Contributors

Karina Martinez-Mayorga

Torrey Pines Institute for Molecular Studies, 11370 SW Village Parkway, Port St. Lucie, Florida 34987 (33) Jose L. Medina-Franco

Torrey Pines Institute for Molecular Studies, 11370 SW Village Parkway, Port St. Lucie, Florida 34987 (33) Peerasak Sanguansri

CSIRO Food and Nutritional Sciences, Private Bag 16, Werribee, Victoria 3030, Australia (183) Lisa Solieri

Department of Agricultural and Food Science, Amendola, 2, 42100 Reggio Emilia, Italy (137) Xi Wang

Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634 (215) Juanjuan Yin

Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634 (215)

CHAPTER

1 Quinoa (Chenopodium quinoa Willd.): Composition, Chemistry, Nutritional, and Functional Properties Lilian E. Abugoch James

Contents

I. Introduction II. Chemical, Nutritional, and Physical Properties III. Proteins A. Chemical and nutritional aspects B. Active biopeptides C. Structural aspects IV. Carbohydrates A. Composition, physical, chemical, and structural properties V. Lipids and Lipidic Compound A. Composition, nutritional properties VI. Antioxidant Capacity, Phenolic Compounds, and Flavonoids VII. Saponins VIII. Minerals and Vitamins IX. Functional Properties A. Functional properties of quinoa flour B. Functional properties of quinoa protein C. Functional properties of quinoa starch X. Present and Future Uses of QS References

2 4 6 6 9 9 10 10 15 15 18 18 19 20 21 23 24 24 25

Departamento Ciencia de los Alimentos y Tecnologı´a Quı´mica, Facultad de Ciencias Quı´micas y Farmace´uticas, Universidad de Chile, Vicun˜a Mackenna 20, Santiago, Chile Advances in Food and Nutrition Research, Volume 58 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)58001-1

#

2009 Elsevier Inc. All rights reserved.

1

2

Abstract

Lilian E. Abugoch James

Quinoa (Chenopodium quinoa Willd.), which is considered a pseudocereal or pseudograin, has been recognized as a complete food due to its protein quality. It has remarkable nutritional properties; not only from its protein content (15%) but also from its great amino acid balance. It is an important source of minerals and vitamins, and has also been found to contain compounds like polyphenols, phytosterols, and flavonoids with possible nutraceutical benefits. It has some functional (technological) properties like solubility, water-holding capacity (WHC), gelation, emulsifying, and foaming that allow diversified uses. Besides, it has been considered an oil crop, with an interesting proportion of omega-6 and a notable vitamin E content. Quinoa starch has physicochemical properties (such as viscosity, freeze stability) which give it functional properties with novel uses. Quinoa has a high nutritional value and has recently been used as a novel functional food because of all these properties; it is a promising alternative cultivar.

I. INTRODUCTION Quinoa is one of the seeds considered as pseudocereals; it is a broadleaf plant that has been used like the cereals. This crop was an important food for the Incas and still remains as an important food crop for the Quechua and Aymara peoples of the rural regions. A native of the Andes, quinoa dates back more than 5000 years. It was called ‘‘the mother grain’’ by the Incas; it sustained the Inca community and was considered sacred. This seed was the major crop of the pre-Columbian cultures in Latin America. After the arrival of the Spaniards, its use, consumption and cultivation was almost eliminated and only remained in the farmers’ traditions. Quinoa grains have an established excellent nutritional food quality, and that is the reason for the great recent interest in it. Botanically, quinoa belongs to the class Dicotyledoneae, family Chenopodiaceae, genus Chenopodium, and species quinoa. The full name Chenopodium quinoa Willd. (Marticorena and Quezada, 1985; Winton and Winton, 1932) includes the author abbreviation corresponding to Carl Ludwig Willdenow. The species Chenopodium quinoa Willd. includes both domesticated and free-living weedy forms (Wilson, 1981, 1988). Chenopodium species are used either as whole plants or parts of the plant. There is great diversity in plants and inflorescences (Mujica and Jacobsen, 2006). The genus Chenopodium includes about 250 species (Bhargava et al., 2005). Quinoa is an annual plant found in the Andean region of South America, between sea level and the heights of the Bolivian Altiplano at around 4000 m above sea level. It produces flat, oval-shaped seeds that are usually pale yellow but can range in color from pink

Quinoa Chemical Nutritional Functional Properties

3

to black. The adaptation of certain quinoa varieties is possible even under marginal environments for the production of seeds with high protein and mineral content (Karyotis et al., 2003). Quinoa’s aptitude to produce highprotein grains under ecologically extreme conditions makes it important for the diversification of agriculture as in high-altitude regions of the Himalayas and North Indian Plains (Bhargava et al., 2005). Quinoa is reported to be one of the few crop plants grown in the salt level of southern Bolivia and northern Chile (Jacobsen et al., 2000; Tagle and Planella, 2002). Salinity influences plant growth, seed yield, and seed quality even of halophytic crops such as quinoa. Plant growth, total seed yield, number of seeds, fresh weight, and dry weight of seeds are reduced in the presence of salinity. Only at high salinity, protein content increases in these seeds, while total carbohydrate content decreases (Koyro and Eisa, 2007). While most quinoa is still grown in South America, it is also cultivated in the USA (Colorado and California), China, Europe, Canada, and India. It is also cultivated experimentally in Finland and the UK. Increasing amounts are being exported to the developed world like Europe and the USA. It is currently produced in Bolivia, Peru, and Ecuador; in Chile almost all quinoa seed (QS) is exported to Europe and the USA. In Europe quinoa was introduced in England in the 1970s, and later research projects focused on its production for humans and/or as a fodder crop under temperate conditions (Jacobsen and Stlen, 1993; Jacobsen et al., 1994). Quinoa production has increased in the last 20 years, especially in Bolivia. The main producing countries are Bolivia, Peru, and Ecuador, which in 2007 produced 61,490 tons, up from 19,000 tons in 1973 (FAOSTAT, 2008). During 2007 quinoa production was 34,000 tons in Peru, 26,800 tons in Bolivia, and 690 tons in Ecuador (FAOSTAT, 2008). Quinoa is a very interesting food due to its complete nutritional characteristics. It is a starchy dicotyledonous seed, and therefore not a cereal, so it is known as a pseudocereal (Ahamed et al., 1998; Ando et al., 2002; Chauhan et al., 1992a,b; Lindeboom, 2005; Oshodi et al., 1999; Ranhotra et al., 1993; USDA, 2005; Wright et al., 2002). This seed has been attracting attention because of the quality and nutritional value of its proteins (Ranhotra et al., 1993). It is rich in the essential amino acid lysine, making it a more complete protein than many vegetables. It does not contain gluten, so it can be eaten by people who have celiac disease as well as by those who are allergic to wheat. The oil fraction of the seeds is of high quality and highly nutritious. It is also rich in iron and magnesium and provides fiber, vitamin E, copper and phosphorus, as well as some B vitamins, potassium, and zinc. Quinoa has an outer seed layer that contains saponins, which are toxic and bitter tasting, making necessary its elimination before eating or processing for the manufacture of food products. The plant’s saponin content is a protective feature.

4

Lilian E. Abugoch James

The seeds are small and have been used as flour, toasted, added to soups, or made into bread. Nowadays new food products featuring ancient grains are appearing in the market worldwide, giving new possibilities for grains like quinoa. With the emerging quinoa market the consumer trend towards ancient grains is expected to keep increasing, with international support from both political and industry organizations in Europe (Tellers, 2008). The first few quinoa products are beginning to appear in the European market. In 2003, the UK-based Anglesey introduced a chilled quinoa meat substitute called Quinova. With increasing interest in grain diversification, the food industry in 2008 can show a change in its tactics leading to new ways of revenue potential from these ancient grains (Launois, 2008; Tellers, 2008). This review presents a summary of the available literature on the composition, chemistry, functional, and nutritional properties of quinoa seed. The focus is on macrocomponents, which are mainly responsible for the functional properties.

II. CHEMICAL, NUTRITIONAL, AND PHYSICAL PROPERTIES QS are a complete food with high-nutritional value due mainly to their high content of good quality protein (Abugoch et al., 2008; Gross et al., 1989; Mahoney et al., 1975; Oshodi et al., 1999; Ranhotra et al., 1993). Besides their protein content, many studies have been made of their lipids (Koziol, 1993; Ruales and Nair, 1993), starch (Atwell et al., 1983; Coulter and Lorenz, 1990), minerals (Oshodi et al., 1999), and saponin (Chauhan et al., 1992a,b; Mastebroek et al., 2000). QS contain minerals and vitamins like vitamin B (Koziol, 1993), vitamin C (Koziol, 1993; Lintschinger et al., 1997), and vitamin E (Coulter and Lorenz, 1990; Ng and Anderson, 2005; Repo-Carrasco et al., 2003; Ruales and Nair, 1993). There is an extensive literature on QS covering different aspects, including the composition of reserves (Ando et al., 2002), and chemical characterization of proteins (Abugoch et al., 2008; Brinegar and Goundan, 1993; Brinegar et al., 1996), fatty acid composition of the oils (Ando et al., 2002; Wood et al., 1993) mineral content (Ando et al., 2002; Koziol, 1993); and functional and nutritional values (Abugoch et al., 2008; Ogungbenle, 2003; Ogungbenle et al., 2009; Ranhotra et al., 1993; Ruales and Nair, 1993). However, it is necessary to consider its saponins, which are present in the pericarp of the seeds and must be removed before their use and consumption. Biopolymers are found in specific parts of the grain (Fig. 1.1) (Prego et al., 1998). For instance, starch grains (Fig. 1.2) occupy the cells of the perisperm, while lipid bodies, protein bodies with globoid crystals of phytin, and proplastids with deposits of phytoferritin are the storage components of the endosperm and embryo tissues (Ando et al., 2002;

Quinoa Chemical Nutritional Functional Properties

5

EN SC PE C C R F

P

SA H

FIGURE 1.1 Medial longitudinal section of quinoa seed showing the pericarp (PE), seed coat (SC), hypocotyl-radical axis (H), cotelydons (C), endosperm (EN) (in the micropylar region only), radicle (R), funicle (F), shoot appendix (SA) and perisperm (P). Bar ¼ 500 mm. (Prego et al., 1998. Reproduced with author’s permission).

FIGURE 1.2 Scanning electron micrographs (10,000 magnification) of quinoa starch (Qian and Kuhn, 1999; Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission).

6

Lilian E. Abugoch James

Prego et al., 1998). The embryo that surrounds the perisperm is dicotyledonous and is part of the bran fraction of the seed; it is high in proteins and lipids, and contains most of the ash, fiber, and saponins (Mastebroek et al., 2000; Varriano-Marston and DeFrancisco, 1984). The shape of QS is similar to a flattened sphere; their mean equivalent diameter varies from 1.4 to 1.6 mm (Chauhan et al., 1992a,b; Vilche et al., 2003). As mentioned previously, carbohydrates, proteins, and lipids are the main component of the seeds, and they are mostly responsible for the functional properties that have made them new ingredients in the development of new products. QS can be very important for improving food supplies (Repo-Carrasco et al., 2003; Tellers, 2008) and as alternative food sources in other regions such as the USA and Europe (Castillo, 1995; Tellers, 2008).

III. PROTEINS A. Chemical and nutritional aspects The mean protein content reported in the literature for QS is 12–23% (Abugoch et al., 2008; Ando et al., 2002; Gonzalez et al., 1989; Karyotis et al., 2003; Koziol, 1992; Ruales and Nair, 1994a,b). Compared to cereal grains, the total protein content of QS (16.3% dry basis (db)) is higher than that of barley (11% db), rice (7.5% db), or corn (13.4% db), and is comparable to that of wheat (15.4% db) (Abugoch et al., 2008; USDA, 2005). QS contain relatively minor proteins compared to legume seeds (Table 1.1). The amino acid composition of QS has been studied (Ranhotra et al., 1993; Repo-Carrasco et al., 2003; Ruales and Nair, 1993; Wright et al., 2002). Relative to cereal grains, quinoa proteins (QPs) are particularly high in lysine, the limiting amino acid in most cereal grains (Table 1.1). Their essential amino acid balance is excellent because of a wider amino acid range than in cereals and legumes (Ruales and Nair, 1993), with higher lysine (5.1–6.4%) and methionine (0.4–1%) contents (Bhargava et al., 2003; National Academy of Sciences, 1975; Prakash and Pal, 1998). QPs have higher histidine content than barley, soy, or wheat proteins, while the methionine þ cystine content of quinoa is adequate for children (2–12 years old) and adults (Table 1.2), it is similar to that of barley and soy, and lower than the amounts in wheat. According to the FAO/WHO suggested requirements (Table 1.2) for a 10-year-old children, QPs have adequate levels of aromatic amino acids (phenylalanine and tyrosine) and similarly in histidine, isoleucine, threonine, phenylalanine, tyrosine, and valine contents. By comparison (Table 1.2), lysine and leucine in QPs are limiting amino acids for 2–5-year-old infants or children, while all the essential amino acids of this protein are sufficient according to FAO/WHO

Quinoa Chemical Nutritional Functional Properties

TABLE 1.1

Amino acids composition of quinoa seed, barley, soybeans, and wheata Quinoa seed

Barley pearled Soybean raw

Amino acid

Arginine Aspartic acid Cystine Glycine Glutamic acid Histidine Isoleucine Leucine Lysine Methyonine Phenylalanine Serine Threonine Tryphtophan Tyrosine Valine Alanine a

7

Wheat durum

mg/g protein

77.3 80.3 14.4 49.2 132.1 28.8 35.7 59.5 54.2 21.8 42 40.2 29.8 11.8 18.9 42.1 41.6

50.1 62.5 22.1 36.2 261.2 22.5 36.5 98.2 37.2 19.2 56.1 42.2 34 16.6 28.7 49 39

69.5 136.3 12.1 38.6 151 26.7 44.5 72 57.8 10.6 49.2 50 38.6 12.2 36.2 47.6 42.2

83.4 94 20.5 45.5 195.1 23.5 43.2 82.8 36.2 23.5 53.5 52.6 35.8 11.5 33.4 61.1 58

USDA (2005).

suggested requirements for 10–12-year-old children. The two quinoa isolates studied in this work showed a good amino acid profile and could be a good source of proteins for feeding infants and children. The nutritional value of a food is determined by its protein quality, which depends mainly on its amino acid content, digestibility, influence of antinutritional factors, and the tryptophan to a large neutral amino acids ratio (Comai et al., 2007). Mahoney et al. (1975) reported the protein efficiency ratio (PER) values for QP, and the protein quality of cooked quinoa was like that of casein. According to these authors, the PER of the cooked quinoa was 30% greater than that of uncooked quinoa. Recently, Ranhotra et al. (1993) also concluded that the quality of protein in quinoa equals that of casein. Gross et al. (1989) reported a high apparent digestibility and a high PER of washed QS; they found that the PER is almost equal to that of casein. Digestibility of the proteins in raw washed quinoa was described by Ruales and Nair (1993), who found 83% (casein, 91%). Both reports (Gross et al., 1989; Ruales and Nair, 1993) showed that it is necessary to remove the saponins to increase digestibility.

TABLE 1.2 Comparison of essential amino acids content of barley, corn and wheat to FAO/WHO suggested requirement Barley Quinoa seeda pearleda Amino acids

Histidine Isoleucine Leucine Lysine Methyonine and Cystine Phenylalanine and Tyrosine Threonine Tryphtophfan Valine a b

Soybeans rawa

Wheat duruma

mg/g protein

FAO/WHO suggested requirementsb 2–5-yearold 10–12-yearold

Adult

28.8 35.7 59.5 54.2 36.2

22.5 36.5 98.2 37.2 41.3

27.6 44.5 72 57.8 28.9

23.5 38.9 68.1 22.1 22.7

19 28 66 58 25

19 28 44 44 22

16 13 19 16 17

60.9

84.7

84.8

85.9

63

22

19

29.8 11.4 42.1

34 16.6 49

38.6 12 57.1

26.7 12.8 41.6

34 11 35

28 9 25

9 5 13

USDA (2005). Friedman and Brandon (2001).

Quinoa Chemical Nutritional Functional Properties

9

Protein digestibility can increase with adequate heat treatment (Ruales and Nair, 1993). Lopez de Romana et al. (1981) found that digestibility of QS is the limiting factor in protein and energy utilization, and that milling improves significantly the digestibility of fat and carbohydrates. Lorenz and Coulter (1991) obtained corn grits with different levels of quinoa and found that quinoa addition produced extruded products which were higher in protein than corn grit products, but had a somewhat lower in vitro digestibility. The importance of the nonprotein tryptophan fraction is due to the fact that it is the only one that can enter the brain and is more easily absorbed, so it guarantees a greater amount available for uptake by the central nervous system. So the tryptophan content of QPs is similar to that of wheat, but higher than that of other cereals (Comai et al., 2007). Free tryptophan in quinoa flour has values similar to those of wheat and oat; lower than those of barley and pearl millet, but higher than that in rice, maize, and rye (Comai et al., 2007).

B. Active biopeptides Aluko and Monu (2003) obtained active biopeptides by enzymatic hydrolysis, and they suggest that short-chain peptides are more active than long-chain peptides. Low-molecular-weight peptides possess higher potential than high-molecular-weight peptides as antihypertensive agents or as compounds that reduce the amount of free radicals.

C. Structural aspects QS, like those of other plants, store proteins in the embryo to provide nutrients for growth and development (Herman and Larkins, 1999). In the food area, proteins stored in seeds are the source of the proteins consumed directly as food by humans (Shewry et al., 1995). Stored proteins provide building blocks for rapid growth upon seed and pollen germination (Herman and Larkins, 1999). Osborne (1924) introduced a classification of plant proteins based on their solubility in a series of solvents, such as albumins in water, and globulins in saline. Albumins and globulins represent the main storage proteins in QS (Brinegar and Goundan, 1993; Brinegar et al., 1996). QS proteins have been characterized electrophoretically by different authors (Abugoch et al., 2008; Brinegar and Goundan, 1993; Brinegar et al., 1996; Fairbanks et al., 1989). Fairbanks et al. (1989) showed that QS polypeptides can be classified as albumin or globulin. Insignificant amounts of protein were present in the prolamin fraction, and all the polypeptides in the glutelin fraction had electrophoretic mobilities identical to those of albumins and globulins (Fairbanks et al., 1989).

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Lilian E. Abugoch James

Brinegar and Goundan (1993) specifically characterized individual seed storage proteins by isolating and characterizing the 11S seed storage protein, which they call chenopodin. The 11S globulin is a hexameric protein consisting of six pairs of acidic and basic subunits, with each subunit pair connected by a disulfide bond; the sequence similarities of six binding regions suggest that the quinoa 11S hexamer has a structure similar to glycinin (Barrett, 2006). Chenopodin, one of the major storage protein fractions (37% of total protein), is an oligomeric protein with a quaternary structure that was purified by gel filtration (320 kDa) (Brinegar and Goundan, 1993). Quinoa globulin is made of monomers or subunits each of which consists of a basic and an acidic polypeptide, with molecular mass of 20–25 and 30–40 kDa, respectively, linked by disulfide bonds (Abugoch et al., 2008; Brinegar and Goundan, 1993). Brinegar and Goundan (1993) determined the amino acid composition of the A and B polypeptides, and compared it with the composition of the native chenopodin. Chenopodin has a high content of glutamine— glutamic acid, asparagines—aspartic acid, arginine, serine, leucine, and glycine. According to the FAO reference protein (FAO, 1973), chenopodin meets the requirements for leucine, isoleucine, and phenylalanine þ tyrosine. The other major protein (35% of total protein) is a 2S-type protein also known as albumin according to Osborne (1924); with a molecular mass of 8–9 kDa. Brinegar et al. (1996) reported for the purified quinoa 2S protein fraction an electrophoretically heterogeneous collection of polypeptides having molecular mass of 8–9 kDa under reducing conditions. The amino acid composition of this protein showed that it is high in cysteine, arginine, and histidine (Brinegar et al., 1996).

IV. CARBOHYDRATES A. Composition, physical, chemical, and structural properties Carbohydrates can be classified according to their degree of polymerization into three principal groups: sugars (monosaccharides, disaccharides, polyols), oligosaccharides, and polysaccharides (starch and nonstarch) (FAO, 1998). Table 1.3 presents the carbohydrate composition of QS, barley, and rice. The carbohydrate (by difference, db) content of QS is comparable to that of barley and rice. Starch is the major component of quinoa carbohydrates, and it is present between 32% and 69.2% (Ahamed et al., 1998; Ando et al., 2002; Chauhan et al., 1992a,b; Lindeboom, 2005; Oshodi et al., 1999; Ranhotra et al., 1993; USDA, 2005; Wright et al., 2002). Besides, total dietary fiber of quinoa is near that of cereals (7–9.7% db), and the soluble fiber content is reported between 1.3% and 6.1% (db)

11

Quinoa Chemical Nutritional Functional Properties

TABLE 1.3

Carbohydrate composition of quinoa seed, rice, and barley (% dry basis)

Carbohydrate by difference Starch Fiber total dietary Insoluble fiber Soluble fiber Sugar a b c d

Quinoa

Ricea

Barleya

73.6a–74b 52.2a–69.2c 7a–9.7d 6.8c–8.4d 6.1c–1.3d 2.9d

79.2

77.7

2.8

15.6

0.8

Data from USDA (2005). Data from Wright et al. (2002). Data from Mundigler (1998). Data from Ranhotra et al. (1993).

(Table 1.3). Finally, there is about 3% of simple sugars (Ranhotra et al., 1993). The individual sugars present in quinoa are mostly maltose, followed by D-galactose and D-ribose, and it also contains fructose and glucose (Oshodi et al., 1999). Carbohydrates play a basic nutritional function and they may have different physiological health effects, such as: provision of energy, effects on satiety/gastric emptying, control of blood glucose and insulin metabolism; protein glycosylation; cholesterol and triglyceride metabolism (FAO, 1998). Carbohydrates from quinoa can be considered a nutraceutical food because they have beneficial hypoglycemic effects and induce lowering of free fatty acids. Studies made in individuals with celiac disease showed that the glycemic index of quinoa was slightly lower than that of gluten-free pasta and bread (Berti et al., 2004). Besides, quinoa induced lower free fatty acid levels than gluten-free pasta and significantly lower triglyceride concentrations compared to gluten-free bread (Berti et al., 2004). Some nutraceutical effects of quinoa have been reported, but that requires further study (Berti et al., 2004). In vitro digestibility (a-amylase) of raw quinoa starch was reported at 22%, while that of autoclaved, cooked, and drum-dried samples was 32%, 45%, and 73%, respectively (Ruales and Nair, 1994a). Saponins did not affect the digestibility of the starch. The total dietary fiber content in quinoa flour is affected by thermal treatment, while the insoluble dietary fiber fraction does not change with heat treatment (Ruales and Nair, 1994b).

1. Structure of quinoa starch Starch is second only to cellulose in natural abundance, and it is the major energy reserve in plants. The most important sources of starch are cereal grains, legumes, and tubers. The glucose polymers that make up starch come in two molecular forms, linear and branched. The former is referred

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Lilian E. Abugoch James

TABLE 1.4 Starch composition of quinoa, rice, barley (% dry basis)

Amylose Amylopectin a b c d e f

Quinoaa,b,c,d

Ricee

Barleyf

3.5–22.5 77.5

7.4–29.8 61

1–45

Tang et al. (2002). Qian and Kuhn (1999). Tari et al. (2003). Lindeboom (2005). Tukomane and Varavinit (2008). Morrison et al. (1986).

to as amylose and the latter as amylopectin. In nature, a-D-glucose is used to form the starch polymers (Murphy, 2000). Quinoa starch consists of two polysaccharides: amylose and amylopectin. In native starches, the amylose content is 20–30% and the amylopectin content is 70–80%. The amylose content (Table 1.4) of quinoa starch varies between 3% and 20% (Inouchi et al., 1999; Lindeboom, 2005; Praznik et al., 1999; Qian and Kuhn, 1999; Tang et al., 2002; Watanabe et al., 2007). The amylose fraction of quinoa starch is low, similar to that of some rice varieties, and higher than that of some barley varieties (Morrison et al., 1986) (Table 1.4). Quinoa starch has an average molar mass of 11.3 106 g/mol, a value lower than that of waxy corn starch (17.4 106 g/mol) or rice starch (0.52–1.96 108 g/mol) (Park et al., 2007; Praznik et al., 1999), and higher than that of wheat starch (5.5 106 g/mol) (Praznik et al., 1999). Quinoa starch is highly branched, with a minimum degree of polymerization of 4600 glucan units, a maximum of 161,000, and a weighted average of 70,000 (Praznik et al., 1999). Chain length can depend on the botanical origin of the starch, but it will be of the order of 500–6000 glucose units. According to Tang et al. (2002) the number-average degree of polymerization of quinoa amylose (900) is lower than that of barley (1,700). Amylose has an average of 11.6 chains per molecule. Amylopectin is one of the largest molecules in nature. Very few results on the molecular weight of cereal amylopectin have been reported because cereal starches are difficult to dissolve in water and may be easily degraded. In the literature, amylose is determined directly, but amylopectin only by difference. In quinoa starch the amylopectin content according to Tari et al. (2003) is 77.5%. The amylopectin fraction is high and comparable to that of some varieties of rice (Tukomane and Varavinit, 2008) (Table 1.4). Quinoa amylopectin has a unique chain length distribution as a waxy amylopectin, with 6700 glucan units for the amylopectin fraction of quinoa starch (Tang et al., 2002). Quinoa amylopectin, like that of amaranth and buckwheat, contains a large number of short chains from 8 to 12 units and a small number of longer chains of 13–20, compared to the endosperm starches of other

Quinoa Chemical Nutritional Functional Properties

TABLE 1.5

Granule size of starches from quinoa, amaranth, rice, barley (mm)

Quinoa a

0.6 –2 a b c d e

13

b,c

Amaranth

1–2

c

Rice

3–8

d

Barley

2–3 and 12–32e

Ruales and Nair (1993). Tang et al. (2002). Qian and Kuhn (1999). Cle´dat et al. (2004). Lindeboom et al. (2004).

cereals (Inouchi et al., 1999). Quinoa glucans were classified by Praznik et al. (1999) as amylopectin-type short-chain branched glucan. Granule size affects the physicochemical characteristics of starch. Granule size and shape are related to the biological source from which the starch is isolated. In general, granule size may vary from less than 1 mm to more than 100 mm according to Lindeboom et al., 2004, who defined the following classes according to size: large (>25 mm), medium (10–25 mm), small (5–10 mm), and very small (<5 mm) granules. Quinoa starch has a very small granule size and has been reported to be 1–2 mm (Ando et al., 2002; Atwell et al., 1983; Chauhan et al., 1992a,b; Lindeboom, 2005; Lorenz, 2006; Qian and Kuhn, 1999; Tang et al., 2002; Tari et al., 2003). Table 1.5 shows granule sizes from different origins, showing that quinoa starch is comparable to that of amaranth and smaller than that of rice or barley. Quinoa starch has small granules and can be used to produce a creamy, smooth texture that exhibits properties similar to fats, or it can be incorporated into biofilms (Lindeboom et al., 2004). Figure 1.2 shows the polygonal shape of quinoa starch by scanning electron microscopy (SEM) (Lindeboom, 2005; Qian and Kuhn, 1999; Wang et al., 2003), similar to that of amaranth and rice starch (Kong et al., 2009; Qian and Kuhn, 1999). According to Ruales and Nair (1994a) the starch in QS also has polygonal granules, and they found that particles can be present singly and as spherical aggregates. The 20–30-mm diameter starch granule aggregates are packed in the quinoa perisperm (Ando et al., 2002). X-ray diffraction studies have been used to explain the structure of whole starch and amylose. Starch granules, depending on their botanical origin, amylose/amylopectin ratio, and amylopectin branch length, show three types of X-ray diffraction patterns, associated with different crystalline polymorphic forms: A-type (cereal), B-type (tubers), and C-type (A and B crystals coexisting in the granule) (Lopez-Rubio et al., 2008; Qian and Kuhn, 1999). Quinoa starch presents the typical A X-ray diffraction pattern reflections at 15.3 , 17 , 18 , 20 , and 23.4 2y angles; characteristic of cereal starches (Lopez-Rubio et al., 2004; Qian and Kuhn, 1999; Watanabe et al., 2007). The degree of relative crystallinity is between 35% and 43% (Qian and Kuhn, 1999; Tang et al., 2002; Watanabe et al., 2007).

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Lilian E. Abugoch James

The relative crystallinity of quinoa starch has been described as higher than that of normal barley starch, lower than that of amaranth starch, and similar to that of waxy barley starch (Qian and Kuhn, 1999; Tang et al., 2002). For each kind of starch granule it is possible to find a characteristic thermogram by differential scanning calorimetry (DSC). Thermograms of quinoa starch show two thermal transitions; one for gelatinization of the starch and another for the amylose–lipid complex (Ruales and Nair, 1994b; Tang et al., 2002). The gelatinization properties of starch are related to a variety of factors including the size, proportion and kind of crystalline organization, and the ultrastructure of the starch granule. Quinoa starch gelatinizes at a relatively low temperature (T0 ¼ 46.1–57.4 C, Tp ¼ 54.2– 61.9 C, Tc ¼ 66.2–68.5 C) (Inouchi et al., 1999). The first thermal transition gives the gelatinization temperature, and it has been reported between 62.6 and 67 C (Qian and Kuhn, 1999; Ruales and Nair, 1994b; Tang et al., 2002). For this transition the enthalpy reported for quinoa starch is between 1.66 and 12.2 J/g (Qian and Kuhn, 1999; Tang et al., 2002). Comparative thermal properties are presented in Table 1.6. It shows that quinoa starch has a similar gelatinization temperature than amaranth starch and higher than rice starch. According to Lindeboom (2005), the gelatinization onset and peak temperatures of quinoa starches ranged from 44.6 to 53.7 C and from 50.5 to 61.7 C, respectively, and the gelatinization enthalpies from 12.8 to 15 J/g of dry starch. The gelatinization temperatures are positively dependent of amylose content (Lindeboom, 2005; Youa and Izydorczyk, 2007). The quinoa starches exhibited lower gelatinization temperatures than waxy barley and amaranth starches (Qian and Kuhn, 1999; Youa and Izydorczyk, 2007). The pasting properties of quinoa starch are reported by Qian and Kuhn (1999) and show a pasting temperature of 66.8 C, comparable to quinoa starch pasting values (63–64 C) reported by Lindeboom (2005). TABLE 1.6 Thermal properties of some starches

Gelatinization enthalphy DH ( J/g) T0 C Tp C Tc C a

Quinoaa,b

Waxy barleyc

Amarantha

1.66–15

14.8

2.58

44.6–59.9 54.5–69.3 71–86.4

66.4

66.3 74.5 86.9

Qian and Kuhn (1999). Lindeboom (2005). c Youa and Izydorczyk (2007). T0: Gelatinization onset temperature ( C).Tp: Gelatinization peak temperature ( C).Tc: Gelatinization conclusion temperature ( C). b

Quinoa Chemical Nutritional Functional Properties

15

Rapid Visco Analysis (RVA) shows the normal pasting feature of cereal and root starches (Qian and Kuhn, 1999). Finally, quinoa starch has excellent stability under freezing and retrogradation processes (Ahamed et al., 1998). Quinoa starch can be affected by heat treatment, showing changes in the degree and extent of degradation (Ruales and Nair, 1994b).

V. LIPIDS AND LIPIDIC COMPOUND A. Composition, nutritional properties QS have been considered an alternative oilseed crop due to their lipidic fraction (Koziol, 1993). Besides the high content and good biological quality of their proteins, QS have an interesting lipid composition of about 1.8–9.5% (Koziol, 1993; Masson and Mella, 1985; Oshodi et al., 1999; Ranhotra et al., 1993; Ryan et al., 2007; USDA, 2005; Wood et al., 1993). Quinoa has an oil content (7% dry basis) higher than corn (4.9% dry basis) and lower than soy (20.9% dry basis) (Koziol, 1993; USDA, 2005). Cytochemical and ultrastructural analyses reported by Prego et al. (1998) show that lipid bodies are the storage components of the cells of the endosperm and embryo tissues (Fig. 1.3). According to Przybylski et al. (1994), QS lipids contain high amounts of neutral lipids in all the seed fractions analyzed. Triglycerides are the major fraction present, accounting for over 50% of the neutral lipids. Diglycerides are present in whole seeds and contribute 20% of the neutral lipid fraction. Lysophosphatidyl ethanolamine and phosphatidyl choline are the most abundant (57%) of the total polar lipids (Przybylski et al., 1994). Some researchers have characterized the fatty acid composition of quinoa lipids (Table 1.7) as follows: total saturated 19–12.3%, mainly palmitic acid; total monounsaturated 25–28.7%, mainly oleic acid, and total polyunsaturated 58.3%—chiefly linoleic acid (about 90%) (Masson and Mella, 1985; Oshodi et al., 1999; Ranhotra et al., 1993; Ryan et al., 2007; USDA, 2005; Wood et al., 1993). Omega-6 and omega-3 fatty acids are essential fatty acids because they cannot be synthesized by humans, who must obtain them from foods. The essential fatty acids are metabolized to longer chain fatty acids of 20 and 22 carbon atoms. Linoleic acid is metabolized to arachidonic acid and linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA and DHA play important roles in prostaglandin metabolism, thrombosis and atherosclerosis, immunology and inflammation, and membrane function (Simopoulos, 1991; Youdim et al., 2000). The fatty acid profile of QS is similar to corn and soybean oil (Koziol, 1992; Oshodi et al., 1999; Youdim et al., 2000). Essential fatty acids are important acids, like linoleic and linolenic acids, that are necessary substrates in animal metabolism. Linoleic acid (C18:2)

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Lilian E. Abugoch James

PB

L

ER

FIGURE 1.3 Transmission electron micrographs of sections of the endosperm. An enlargement section of a cell of the endosperm showing lipid bodies (L) and protein bodies (PB), next to endoplasmic reticulum (Prego et al., 1998. Reproduced with author’s permission).

is one of the most abundant polyunsaturated fatty acids (PUFA) identified in QS; PUFAs have several positive effects on cardiovascular disease (Abeywardena et al., 1991; Keys and Parlin, 1966) and improved insulin sensitivity (Lovejoy, 1999). The oil fraction of QS has high quality and is highly nutritious, based on the fact that it has a high degree of unsaturation, with a polyunsaturation index of 3.9–4.7. In this fraction, not only the fatty acid composition is important. Another important feature is the natural presence of a high amount of vitamin E (a-tocopherol), 0.59–2.6 mg/100 g in the seeds (Coulter and Lorenz, 1990; Ryan et al., 2007; USDA, 2005), which acts as a natural defense against lipid oxidation (Ng et al., 2007). This fact could lead to a very stable oil from QS, with vitamin E acting as a natural antioxidant. The (b þ g)-tocopherol content in quinoa whole flour has been reported as 3.1–5.5 mg/100 g (Ruales and Nair, 1993; Ryan et al., 2007). The chemical stability of the lipids in quinoa flour was studied by Ng et al. (2007), who

Quinoa Chemical Nutritional Functional Properties

TABLE 1.7

a b

17

Fatty acid composition of crude fat from quinoa seed, corn, and soy oil

Fatty acid

Quinoaa

Soyb

Cornb

Saturated Myristic C14:0 Palmitic C16:0 Stearic C18:0

0.1–2.4 9.2–11.1 0.6–1.1

Traces 10.7 3.6

Traces 10.7 2.8

Monounsaturated Myristoleic C14:1 Palmitoleic C16:1 Oleic C18:1

1 0.2–1.2 22.8–29.5

– 0.2 22

– trazas 26.1

Polyunsaturated (PUFA) Linoleic C18:2 (n 6) Linolenic C18:3 (n 3)

48.1–52.3 4.6–8

56 7

57.7 2.2

Masson and Mella (1985). USDA (2005).

found that the lipids were stable during 30 days, and this stability is due to vitamin E present naturally. Squalene and phytosterols are components present in the unsaponifiable lipid fraction of foods (as tocopherols). Squalene is an intermediary in cholesterol biosynthesis, and 33.9–58.4 mg/100 g of it was found in the lipid fraction of quinoa ( Jahaniaval et al., 2000; Ryan et al., 2007); squalene is the biochemical precursor of the whole family of steroids, and besides their effective antioxidant activity, tocotrienols have other important functions, in particular in maintaining a healthy cardiovascular system and a possible role in protection against cancer (Nesaretnam, 2008). Squalene is used as a bactericide and as an intermediate in many pharmaceuticals, organic coloring materials, rubber chemicals, and surfaceactive agents (Ahamed et al., 1998). Phytosterols are natural components of plant cell membranes that are abundant in vegetable oils, seeds, and grains. Phytosterols have different biological effects such as antiinflammatory, antioxidative, and anticarcinogenic activity, and cholesterol-lowering capacity (Moreau et al., 2002). The levels of phytosterols from QS reported by Ryan et al. (2007) were b-sitosterol 63.7 mg/100 g, campesterol 15.6 mg/100 g, and stigmasterols 3.2 mg/100 g, which are the most abundant plant sterols. These levels are higher than in pumpkin seeds, barley, and maize, but lower than in lentils, chick peas, or sesame seeds (Ryan et al., 2007). The recommended doses of free phytosterols are 0.8–1.0 g of equivalents per day, including natural sources, and they are important dietary components for lowering low density lipoprotein (LDL) cholesterol and maintaining good heart health (Berger et al., 2004).

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Lilian E. Abugoch James

VI. ANTIOXIDANT CAPACITY, PHENOLIC COMPOUNDS, AND FLAVONOIDS Zhu et al. (2001) have isolated six flavonol glycosides from QS; these compounds exhibited antioxidant capacity, suggesting that QSs can serve as a good source of free radical scavenging agents. Gorinstein et al. (2008) reported a 0.051% db tannin content for quinoa, a value comparable to that of amaranth. The reported contents (db) were 251.5 mg/g of ferulic acid, 0.8 mg/g of p-coumaric acid, and 6.31 mg/g of caffeic acid (Gorinstein et al., 2008). These authors found antioxidant values expressed as total radical-trapping antioxidative potential (TRAP), ferric ion-reducing antioxidant power (FRAP), cupric-reducing antioxidant capacity (CUPRAC), and nitric oxide (NO). The TRAP value for quinoa was 251 nM mL 1 in acetone extract and 1.686 nM mL 1 in water extract, the FRAP value was 2.3 mM trolox equivalent g 1, a CUPRAC value of 5 mM trolox equivalent g 1; and 32% of NO. Gorinstein et al. (2008) showed that quinoa has higher antioxidant activity than some cereals (rice and buckwheat).

VII. SAPONINS Saponins are a wide group of glycosides found in plants; their name comes from the plant genus Saponaria, whose root was used as soap (sapo, onis ¼ soap) (Sparg et al., 2004); so they are water soluble and form foaming solutions. Saponins are steroid or triterpenoid glycosides, with the latter found more commonly in crops (Francis et al., 2002). These compounds have a bitter taste and are considered toxic in large amounts. They are present in the whole quinoa plant; where their natural function is to defend the plant from the external medium. In general, QSs contain saponins in the seed coat (except sweet varieties, without saponin or containing less than 0.11%). Saponins are the main antinutritional factor present in the seed cover (Ruales and Nair, 1994a,b); studies in rats revealed that animals fed with unwashed quinoa diets showed growth damage and reduced food conversion efficiency (Gee et al., 1996). According to their chemical structure, saponins can be partially removed by washing with water (Chauhan et al., 1999), but even after washing some saponin remains in the seed. Zhu et al. (2002) recommended the use of slightly alkaline water rather than neutral water to debitter QSs. Brady et al. (2007) have reported that the bitter taste imparted by saponins could potentially be reduced by extrusion and roasting processes. Saponins are compounds that contain sugar chains and a triterpenoid aglycone (sapogenin) in their structure (Sparg et al., 2004). They are categorized according to the number of sugar chains in their structure as

Quinoa Chemical Nutritional Functional Properties

19

mono-, di-, or tridesmosidic. Four main structures of sapogenins have been identified in quinoa: doleanolic acid, hederagenin, phytolaccagenic acid, and 30-o-methylspergulagenat (Zhu et al., 2002). The major carbohydrates are glucose, arabinose and galactose. Besides, 20 triterpene saponins have been isolated from different parts of Chenopodium quinoa (flowers, fruits, seed coats, and seeds) (Kuljanabhagavad et al., 2008; Zhu et al., 2002). The sapogenin content in seeds of sweet genotypes varied from 0.02% to 0.04% and in seeds of bitter genotypes from 0.14% to 2.3% (Mastebroek ¨ stu¨ndag˘ and Mazza, 2007). These values are higher et al., 2000; Gu¨c¸lu¨-U than those in soybean and oat, but lower than in green pea and yucca ¨ stu¨ndag˘ and Mazza, 2007). (Gu¨c¸lu¨-U Saponins have been considered toxic for different organisms. Meyer et al. (1990) found toxicity to brine shrimp. Woldemichael and Wink (2001) found monodesmoside saponins hemolytically active. The hemolysis may be produced by the interaction of the saponins with membranes, producing pores that lead to rupture of the (Seeman et al., 1973). Kuljanabhagavad et al. (2008) described mainly saponins with an aldehyde group as cytotoxic in HeLa (cervix adenocarcinoma) cell line. Saponins have shown insecticidal, antibiotic, fungicidal, and pharmacologic activity. Woldemichael and Wink (2001) found five quinoa saponins (glycosides of oleanolic acid and hederagenin) that showed some antifungal activity on Candida albicans; Stuardo and San Martı´n (2008) found higher antifungal activity against Botrytis cinerea with alkali-treated quinoa saponin. Nowadays saponins have been studied because different beneficial properties to health have been described. Saponins possess a broad variety of biological effects: analgesic, antiinflammatory, antimicrobial, antioxidant, antiviral, and cytotoxic activity, effect on the absorption of minerals and vitamins and on animal growth, hemolytic and immunostimulatory effects, increased permeability of the intestinal mucosa neuro¨ stu¨ndag˘ and protective action, and reduction of fat absorption (Gu¨c¸lu¨-U Mazza, 2007). However, the biological properties of quinoa saponins require further study. Finally, saponins have commercial–industrial importance as they are used in the preparation of soaps, detergents, and shampoos.

VIII. MINERALS AND VITAMINS QS are also rich in micronutrients such as minerals and vitamins. Table 1.8 shows the mineral content of QS and quinoa flour. The main minerals are potassium, phosphorus, and magnesium (Table 1.8). According to the National Academy of Sciences (2004) the magnesium, manganese, copper, and iron present in 100 g of QS cover the daily needs of

20

Lilian E. Abugoch James

TABLE 1.8 Mineral composition whole quinoa seed, dehulled quinoa seed, quinoa flour, oat, barley (mg/100 g)

Calcium Phosphorous Potassium Magnesium Iron Manganese Copper Zinc Sodium

Whole QSa Dehulled QSa Quinoa flourb,c Oatd

Barleyd

86.3 411 732 502 15 n.r. n.r. 4 n.r

29 221 280 79 2.5 1.3 0.4 2.1 9

55.1 404.9 656 467.9 14.2 n.r. n.r. 4 n.r.

70–86 22–462 714–855 161–232 2.6–6.3 3.5 0.7–7.6 3.2–3.8 2.7–93

58 734 566 235 5.4 5.6 0.4 3.11 4

a

Konishi et al. (2004). Ranhotra et al. (1993). Oshodi et al. (1999). d USDA (2005). n.r.: not reported. b c

infants and adults, while the phosphorus and zinc content in 100 g is sufficient for children, but covers 40–60% of the daily needs of adults. The potassium content can contribute between 18% and 22% of infant and adult requirements, while the calcium content can contribute 10% of the requirements. However, the mineral content of QS is higher than that of cereals like oat (except phosphorus) or barley, especially that of potassium, magnesium, and calcium (Table 1.8). In their research, Konishi et al. (2004) found that abrasion of QS (for saponin elimination) caused specifically a decrease in calcium content. On the other hand, they found that the distribution of minerals in QS revealed that phosphorus and magnesium were localized in embryonic tissue, while calcium and potassium were present in the pericarp (Table 1.8). The vitamin content (Table 1.9) is also interesting, because QS have high levels of vitamin B6 and total folate, whose amounts in 100 g can cover the requirements of children and adults. The riboflavin content in 100 g contributes 80% of the daily needs of children and 40% of those of adults (National Academy of Sciences, 2004). The niacin content does not cover the daily needs, but is beneficial in the diet. Thiamin values in quinoa are lower than those in oat or barley, but those of niacin, riboflavin, vitamin B6, and total folate are higher (Ranhotra et al., 1993; USDA, 2005).

IX. FUNCTIONAL PROPERTIES The functional properties of food biopolymers are important in food product formulation and manufacture, because their technological properties are dependent on the use of biopolymers. These properties are

21

Quinoa Chemical Nutritional Functional Properties

TABLE 1.9

Vitamin composition quinoa flour, oat, barley (mg/100 g)

Thiamin Riboflavin Niacin B6 Folate total a b

Quinoa floura,b

Oatb

Barleyb

0.29–0.36 0.30–0.32 1.24–1.52 0.487b 0.184b

0.763 0.139 0.961 0.119 0.056

0.191 0.114 4.604 0.260 0.023

Ranhotra et al. (1993). USDA (2005).

related to interaction with water, such as water-holding capacity (WHC), water imbibing capacity (WIC), solubility, viscosity. Another group of functional properties are related to polymer interaction such as gelation, and finally interfacial properties like foaming and emulsifying. The natural polymers like protein or starch have diverse and heterogeneous structures that condition their use in the food industry. The functional properties of food macromolecules are dependent on many factors such as exposure groups, hydrophobic area, water activity, ionic force, pH, temperature, size, charge density, hydrophilic/hydrophobic ratio, and changes in the environment. Some functional properties of quinoa flour and of each component of QS are described below and are shown synoptically in Table 1.10.

A. Functional properties of quinoa flour Some functional properties of quinoa flour have been described, mainly solubility, WHC, gelation, and foaming and emulsifying capacity. Solubility is related to the hydrophilic–hydrophobic balance of the proteins and the thermodynamics of its interaction with the solvent. Protein solubility is pH dependent. Ruales et al. (1993) and Oshodi et al. (1999) described the functional properties of quinoa flour. Ruales et al. (1993) studied the protein solubility of quinoa flour in relation to heat (cooking and autoclaving) and found that solubility is higher in cooked samples with solubility values of 5.4–15.6%. Ogungbenle (2003) and Oshodi et al. (1999) studied solubility related to pH and found solubility values of about 15–52%, corresponding to minimum solubility at pH 6 and maximum at pH 10. The solubility values of quinoa flour in the acid pH region imply that the protein may be useful in the formulation of beverages, dehydrated soups and sauces, and low-acid foods. Another property related to hydration is the WHC, which is expressed as weight increase. Ogungbenle (2003) and Ogungbenle et al. (2009) reported the same value of 147%. The WHC decreased from 147% to

22

Lilian E. Abugoch James

TABLE 1.10 Functional properties of quinoa

a b c d e f g h i j

Flour

Solubilitya,b,c, water holding capacityb,c,d, oil holding capacity, emulsifying and foaming capacityb,c,d, gelationb,c,d

Protein concentrate and protein isolate

Solubilitye,f, water-holding capacitye,f, water imbibing capacity f, emulsifying and foaming capacitye

Starch

Water absorption powerg, solubilityh,i, viscosityh,i, freeze–thaw stabilityh,i, water binding capacityi, Brabender viscographj

Ruales et al. (1993). Oshodi et al. (1999). Ogungbenle (2003). Ogungbenle et al. (2009). Aluko and Monu (2003). Abugoch et al. (2008). Tang et al. (2002). Ahamed et al. (1996). Lindeboom (2005). Praznik et al. (1999).

79.5% in the presence of salts (salt concentration between 0.5% and 10%) (Ogungbenle et al., 2009). The gelation property was determined by the lowest flour concentration required for gelation (Alobo, 2003). According to Ogungbenle (2003) and Oshodi et al. (1999), the lowest gelation concentration of quinoa was 16% (w/v) in distilled water. The addition of salts decreased the lowest gelation concentration of 10–14% (Ogungbenle et al., 2009). Quinoa flour may not be a good gel forming agent. However, it was observed that addition of different salts at low concentration (0.5%) improved the gel forming property of quinoa, and this effect was better with KCl (Ogungbenle et al., 2009). The other functional properties measured in quinoa flour are those related to surface tension, like foaming and emulsifying capacity. The foaming capacity and stability of the flour were low, with volume increase values between 9% and 4% stability (Ogungbenle, 2003). The effect of salts on the foaming capacity was studied by Ogungbenle et al. (2009), who found an increase in foaming capacity and stability with salt addition, especially of Na2SO4, KCl, NaCl, and CH3COONa at high concentrations (10%). Quinoa has a low-foaming capacity and stability, and salt addition may improve this property, but high concentrations around 10% are not useful for human consumption. In this relation, further studies are needed on enzymatic crosslinking of QPs using transglutaminase. Emulsifying capacity and stability were measured by Ogungbenle (2003), Oshodi et al. (1999), and Ogungbenle et al. (2009); they found an emulsifying capacity of 104% with a stability of 45% (according to the methods

Quinoa Chemical Nutritional Functional Properties

23

described by Alobo, 2003), and also a salt dependence. The same authors described 46% as oil absorption capacity for quinoa flour, a property that decreased with salt addition (Ogungbenle et al., 2009). Water and oil absorption are good, enhancing the potential of QS in human food and formulations like beverages, sauces, desserts, and sausages. Park and Morita (2005) studied the possibility of using germinated quinoa flour as a bioactive ingredient for applications in processing food like bread. The physical properties and baking quality of dough made from wheat flour with 10% ungerminated (control), and 24-, 48-, and 72-h-germinated quinoa flours were studied. They obtained bread of good nutritional quality, achieving an increase of the amount of free amino acids and a large loaf volume with the 24-h germinated quinoa flour. These results are useful for practical breadmaking, and the germinated quinoa flour may be applied as a useful food bioingredient. In this aspect, further studies of the nutritional value and sensory evaluation of germinated quinoa flour are needed for industrial applications in food processing (Park and Morita, 2005).

B. Functional properties of quinoa protein Aluko and Monu (2003) studied the use of enzymatic hydrolysis to improve some functional properties of QPs. They found that protein solubility of the hydrolysate was over 80%, a value higher than that of protein concentrate. The protein concentrate (obtained by an alkaline method) had minimum solubility at pH 4–6 ( 5%) and maximum solubility at alkaline pH (70%); Aluko and Monu (2003) also measured foam expansion and stability (expressed as %). Protein concentrate showed the smaller foam expansion (<20%), but protein hydrolysate presented values over 160%. Foam stability was better with protein concentrate. The emulsifying activity index and stability were also measured by Aluko and Monu (2003), who found high stability for the hydrolysate, but a small activity index. The hydrolyzed proteins are not as adequate for food emulsions as the protein concentrate. Abugoch et al. (2008) obtained two quinoa protein isolates (treated at pH 9 and 11) and studied solubility and the influence of pH. The minimum protein solubility was found in the pH 3–4 range. For the isolate (pH 9) about 77% at above pH 5, the other isolate (pH 11) presented 30% as maximum solubility. The WHC reported was similar for both isolates (around 3.5–5 mL water/g protein). Finally, the WIC was higher for the isolate treated at pH 11 (3.5 mL of water/g of isolate). Both isolates can be used as a good source of nutrition for infants and children; protein isolate (pH 9) may be used as an ingredient in nutritive beverages, and the other isolate (pH 11) may be used as an ingredient in sauces, sausages, and soups.

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C. Functional properties of quinoa starch Tang et al. (2002) measured the water absorption power of the starch granules of quinoa, and obtained a sigmoid sorption isotherm. Ahamed et al. (1996) and Lindeboom (2005) found lower solubility and viscosity for quinoa starch, and unusual freeze–thaw stability. Water binding capacity was reported by Lindeboom (2005) between 49.5% and 93%, values lower than those of corn starch (117%). Praznik et al. (1999) investigated some technological properties. Dependence of viscosity on temperature was determined for 5% (w/w) quinoa starch suspensions in the 55–95 C range, and quinoa glucans disintegrate at 55 C. The viscosity of 10% aqueous quinoa starch suspensions in Brabender (BU) was 1960 BU at 70 C, comparable to waxy maize (1870 BU, 80 C), and higher than wheat (910 BU, 45 C) or amaranth (580 BU, 74 C) (Praznik et al., 1999). Quinoa starch may be used as a novel food source according to its properties (Watanabe et al., 2007).

X. PRESENT AND FUTURE USES OF QS Quinoa is well adapted to extreme weather conditions, and it is currently produced by Bolivia, Peru, Ecuador, Chile, Argentina, and Colombia. It is basically exported as dry and saponin-free quinoa, with Europe and the USA as the main consumers. Future uses can be wide-ranging, like textured and fermented products. There are many ways in which it can be consumed: cooked, AS flour, extruded. Quinoa meat substitute has been introduced in Europe (Tellers, 2008). There are several developments with quinoa flour at a smaller scale, like bread, cookies, muffins, pasta, snacks, drinks, flakes, breakfast cereals, baby foods, beer, diet supplements, and extrudates (Ahamed et al., 1997; Bhargava et al., 2006; Caperuto et al., 2000; Chauhan et al., 1992a,b; Dogan and Karwe, 2003; Linnemann and Dijkstra, 2002; Morita et al., 2001). Coulter and Lorenz (1991) obtained extruded corn grits–quinoa blends that had high protein quality and solubility and an acceptable sensory evaluation. Caperuto et al. (2000) developed gluten-free quinoa spaghetti and obtained a product without loss of solids and acceptable weight and volume increase upon cooking, while the adhesiveness of the cooked product was not very high. The product was sensorially accepted by the panelists. Quinoa flour does not have good baking properties like wheat gluten proteins. The wheat proteins are able to form a viscoelastic network when flour is mixed with water to form dough, and these viscoelastic properties allow the use of wheat to produce bread and other processed foods (Shewry et al., 2002). Quinoa bread has been made by including 10% of wheat flour (Chauhan et al., 1992a,b). However, the enzyme

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transglutaminase (TGase) is promising for developing a protein structure, as reported by Kova´cs (2003). Use of the enzyme reduced the polypeptides of the low-molecular-weight fractions and the soluble protein fractions when producing pasta. There have been positive reports about TGase that induced crosslinking and polymerization of food proteins, such as milk proteins (Han and Damodaran, 1996), soy proteins (Sakamoto et al., 1994), and fish proteins (Norziah et al., 2008) to improve physicochemical properties. There are some gluten-free products without good baking properties for celiac groups, and quinoa provides an opportunity to develop gluten-free cereal-based products (Gallagher et al., 2004). Dogan and Karwe (2003) showed that quinoa can be used to make novel, healthy, extruded, snack-type food products. They got a good product with maximum expansion, minimum density, high degree of gelatinization, and low water solubility index (16% feed moisture content, 130 C die temperature, and 375 rpm screw speed). Quinoa has shown a high nutritional value and only recently is being used as a novel functional food. However, it is very important to increase and promote QS production, diversify production, and enhance its consumption. An important aspect to consider for promoting quinoa consumption is to inform consumers of the good properties of quinoa and let them incorporate it in their daily diet as a healthy, nutritious, good tasting, and versatile food. Alternatively, it is necessary to develop new functional products that can be available on the market for the ordinary user, and scale them up to industrial level.

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Ryan, E., Galvin, K., O’Connor, T., Maguire, A., and O’Brien, N. (2007). Phytosterol, squalene, tocopherol content and fatty acid profile of selected seeds, grains, and legumes. Plant Foods Hum. Nutr. 62, 85–91. Sakamoto, H., Kumazawa, Y., and Motoki, M. (1994). Strength of protein gels prepared with microbial transglutaminase as related to reaction conditions. J. Food Sci. 59(4), 866–871. Seeman, P., Cheng, D., and Iles, G. (1973). Structure of membrane holes in osmotic and saponin hemolysis. J. Cell Biol. 56, 519–527. Shewry, P., Napier, A., and Tatham, A. (1995). Seed storage proteins: Structures and biosynthesis. Plant Cell 7, 945–956. Shewry, P., Halford, N., Belton, P., and Tatham, A. (2002). The structure and properties of gluten: An elastic protein from wheat grain. Phil. Trans. R. Soc. Lond. B 357, 133–142. Simopoulos, A. (1991). Omega-3 fatty acids in health and disease and in growth and development. Am. J. Clin. Nutr. 54, 438–463. Sparg, S., Light, M., and van Staden, J. (2004). Biological activities and distribution of plant saponins. J. Ethnopharmacol. 94(2–3), 219–243. Stuardo, M. and San Martı´n, R. (2008). Antifungal properties of quinoa (Chenopodium quinoa Willd.) alkali treated previous termsaponinsnext term against Botrytis cinerea. Ind. Crops Prod. 27(3), 296–302. Tagle, M. B., and Planella, M. T. (2002). La quinoa en la zona central de Chile. Supervivencia de una tradicio´n prehispana. (Ed. Iku), Santiago, pp. 25–29. Tang, H., Watanabe, K., and Mitsunaga, T. (2002). Characterization of storage starches from quinoa, barley and adzuki seeds. Carbohydr. Polym. 49(1), 13–22. Tari, T., Annapure, U., Singhal, R., and Kulkarni, P. (2003). Starch-based spherical aggregates: Screening of small granule sized starches for entrapment of a model flavouring compound, vanillin. Carbohydr. Polym. 53, 45–51. Tellers (2008). http://www.frost.com/prod/servlet/market-insight-top.pag?Src¼RSS& docid¼125516124. Tukomane, T., and Varavinit, S. (2008). Classification of rice starch amylose content from rheological changes of starch paste after cold recrystallization. Starch/Sta¨rke, 60, 292–297. USDA U.S. Department of Agriculture, Agricultural Research Service. (2005). USDA National Nutrient Database for Standard Reference, Release 18. Nutrient Data Laboratory Home Page, http://www.nal.usda.gov/fnic/foodcomp. Varriano-Marston, E. and DeFrancisco, A. (1984). Ultrastructure of quinoa fruit (Chenopodium quinoa Willd.). Food Microstruct. 3, 165–173. Vilche, C., Gely, M., and Santalla, E. (2003). Physical properties of quinoa seeds. BioSys. Eng. 86(1), 59–65. Wang, Y., Liu, W., and Sun, Z. (2003). Effects of granule size and shape on morphology and tensile properties of LDPE and starch blends. J. Mater. Sci. Lett. 22, 57–59. Watanabe, K., Peng, L., Tang, H., and Mitsunaga, T. (2007). Molecular structural characteristics of quinoa starch. Food Sci. Technol. Res. 13(1), 73–76. Wilson, H. (1981). Genetic variation among South America populations of tetraploid Chenopodium sect. Chenopodium subsect. Cellulata Syst. Bot. 6, 380–398. Wilson, H. (1988). Quinua biosystematics. I: Domesticated populations. Econ. Bot. 42, 461–477. Winton, A. and Winton, K. (1932). The structure and composition of foods. In ‘‘Vol. 1 Cereals, Starch. Oil Seeds, Nuts, Oils, Forage Plants’’ (John Wiley and Sons, Ed.), pp. 322–325. John Wiley and Sons, London. Woldemichael, G. and Wink, M. (2001). Identification and biological activities of triterpenoid saponins from Chenopodium quinoa. J. Agric. Food Chem. 49, 2327–2332. Wood, S., Lawson, L., Fairbanks, D., Robinson, L., and Andersen, W. (1993). Seed lipid content and fatty acid composition of three quinoa cultivars. J. Food Comp. Anal. 6, 41–44. Wright, K., Pike, O., Fairbanks, D., and Huber, C. (2002). Composition of atriplex hortensis, sweet and bitter Chenopodium quinoa seeds. J. Food Sci. 67(4), 1380–1383.

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Youa, S. and Izydorczyk, M. (2007). Comparison of the physicochemical properties of barley starches after partial a-amylolysis and acid/alcohol hydrolysis. Carbohydr. Polym. 69(3), 489–502. Youdim, K., Martin, A., and Joseph, J. (2000). Essential fatty acids and the brain: Possible health implications. Int. J. Dev. Neurosci. 18(4–5), 383–399. Zhu, N., Sheng, S., Li, D., Lavoie, E., Karwe, M., Rosen, R., and Chi-Tang Hi, C. (2001). Antioxidative flavonoid glycosides from quinoa seeds (Chenopodium Quinoa Willd.). J. Food Lipids 8, 37–44. Zhu, N., Sheng, S., Sang, S., Jhoo, S., Bai, S., Karwe, M., Rosen, R., and Ho, C. (2002). Triterpene saponins from debittered quinoa (Chenopodium quinoa) seeds. J. Agric. Food Chem. 50, 865–867.

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CHAPTER

2 Chemoinformatics— Applications in Food Chemistry Karina Martinez-Mayorga and Jose L. Medina-Franco

Contents

Abstract

I. Introduction II. Molecular Descriptors and Physicochemical Properties III. Molecular Databases and Chemical Space IV. Chemoinformatics in Food Chemistry V. Examples of Molecular Similarity, Pharmacophore Modeling, Molecular Docking, and QSAR in Food or Food-Related Components A. Molecular similarity B. Pharmacophore model C. QSAR and QSPR D. Molecular docking VI. Concluding Remarks and Perspectives Acknowledgments References

34 36 37 40

43 43 47 48 49 52 53 53

The aim of the present chapter is to present the current research and potential applications of chemoinformatics tools in food chemistry. First, the importance and variety of molecular descriptors and physicochemical properties is delineated, and then a survey and chemical space analysis of representative databases with emphasis on food-related ones is presented. A brief description of methods commonly used in molecular design, followed by examples in food chemistry are presented, such methods include similarity searching, pharmacophore modeling, quantitative

Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987 Advances in Food and Nutrition Research, Volume 58 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)58002-3

#

2009 Elsevier Inc. All rights reserved.

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structure–activity relationships (QSAR), and molecular docking. The relatedness to virtual screening is emphasized and the perspectives from this field are presented at the end.

I. INTRODUCTION Chemoinformatics is a relatively new research field which has received several definitions. Among the more often cited are: The mixing of information resources to transform data into information, and information into knowledge, for the intended propose of making better decisions faster in the arena of drug lead identification and optimization. (Brown, 1998) Chem(o)informatics is a generic term that encompasses the design, creation, organization, management, retrieval, analysis, dissemination, visualization and use of chemical information. (Paris, 2000) Accordingly, managing chemical information and computational tools requires knowledge of multiple fields, and in most cases familiarity with a biological system is required in order to generate meaningful models. Chemoinformatics often merits a department of its own in universities and industrial organizations. Very recently the first Master in Science and chemoinformatics courses have been implemented at the University of Sheffield (http://www.shef.ac.uk/is/research/groups/chem/courses. html), the University of Manchester (http://www.informatics.manchester. ac.uk/teaching/), and Indiana University (http://cheminfo.informatics. indiana.edu/). Wild and Wiggings (2006) have described these courses and also the challenges of chemoinformatics education including current trends involving distance education and intensive short courses. Accordingly, specific journals that are related to or include chemoinformatics in their scope are: Journal of Chemical information and Modeling, Journal of Computer Aided Molecular Design, Journal of Chemical Graphics and Modeling, Journal of Molecular Modeling, Current Computer Aided Drug Design, and the most recent Journal of Chemoinformatics which is Open Access and Molecular Informatics. Importantly, while chemoinformatics has been developed with special attention to drug design, the scope is not limited to here and it can be use in other areas like food science, materials science, or polymers. Figure 2.1 shows a short description and methodologies classified into molecular modeling, chemoinformatics, and bioinformatics. The boundaries are soft; however, it is not uncommon to use a combination of methods. For instance, a pharmacophore or docking model is often used for virtual screening. In general, the outcome is structure–property relationships and in the case of virtual screening the prediction ultimately of new molecules having a certain desired property is the final goal.

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Molecular modeling

Chemoinformatics

Bioinformatics

Model or mimic the behavior of molecules and molecular systems considering threedimensional structures

Design, creation, analysis, management, recovery, organization, distribution, visualization and use chemical information Storage and retreival Database mining Molecular descriptors Similarity methods Diversity analysis Library design Virtual screening

Informational techniques applied to solve biological problems usually at the molecular level

Molecular mechanics Molecular dynamics Docking Pharmacophore model Ab initio DFT Semiempirical

35

Biological databases Sequence alignment Gene prediction Molecular phylogenetics Structural bioinformatics Genomics and proteomics

FIGURE 2.1 General classification and definitions of computational methods employed in molecular design in chemistry and biology.

Analysis of chemical information can be performed at different levels depending on the goal and the type and amount of information available. In general, large databases are used for studying the chemical space or as a data source for virtual screening, whereas smaller datasets (<50 compounds) can be used to generate, for instance, pharmacophore or quantitative structure–activity relationships (QSARs) models. In many cases, the system under study involves interactions of small molecules (ligands) with a particular target(s). When the 3D structure of the target is known from X-ray crystallography, NMR, or homology models, the technique of choice is molecular docking. If the 3D structure of the target is unknown, the models are built based on the information of one or more ligands used as reference, such methods being referred to as ligand-based methods, among which, similarity analysis, pharmacophore modeling, and QSAR are the most popular. Figure 2.2 shows some of these methods and how they can be used in combination; it also shows the outline of the following sections in this chapter. First, the concept of chemical space, including a sketch of molecular descriptors, physicochemical properties, and databases is presented. Then, a brief description of molecular similarity, pharmacophore modeling, docking, and QSAR models with the incorporation of examples of food-related components is described. These methods are mainly used to develop and analyze SAR and the resultant models can be used to perform virtual screening. Comprehensive reviews of each of these methods are described elsewhere (Alvarez and Shoichet, 2005; Leach and Willet, 2003; Varnek and Tropsha, 2008).

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Chemical space

Ligand based

Target based

* Similarity searching * Pharmacophore modeling * QSAR

* Docking * De novo design

Virtual screening

FIGURE 2.2 Exploration of the chemical space includes the classification and analysis of databases. To generate structural models several methodologies have been developed, they can be classified into ligand-based and target-based methods. Models derived from these methods are frequently used as a source for virtual screening.

II. MOLECULAR DESCRIPTORS AND PHYSICOCHEMICAL PROPERTIES Chemoinformatics is characterized by the use of large amounts of information. Specific ways to represent the molecules, and to organize and analyze the data have been and continue to be developed. There are different ways to represent the molecules, and they can be classified according to the information that they encode. The most basic level corresponds to representations that depend on or are associated with one dimensional (1D) representation, such as molecular weight. The next level corresponds to the 2D representations associated with the connectivity of the molecules without the consideration of the stereochemistry. In 3D methods the incorporation of stereochemistry conveys not only the specification of the chirality of stereogenic centers but also the possible conformation or conformations. In this regard, the research fields dedicated to conformational analysis have an important impact in reactivity prediction, molecular design, and stability. Examples of 1D, 2D, and

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TABLE 2.1

Molecular descriptors and related properties Descriptor

1D 2D

Simple filter Complex filter Fingerprints

Physicochemical properties

3D

37

Quantum chemical descriptors 3D pharmacophore patterns

SAR patterns Autocorrelation coefficients Virtual screening

Physicochemical and structural properties

Molecular weight Basicity, acidity, etc. Atom types and substructures in a binary representation Lipophilicity, pKa values, ADME, or toxicity parameters Change distribution in the molecules Targets interaction sites (Hbonds, electrostatic, etc.) Different scaffolds can be recognized Pharmacological activity Similarity of shape Pharmacological activity

3D descriptors along with the property represented are summarized in Table 2.1. A number of methods have been developed to store and share information. It is desirable that such information is stored in an ‘‘inexpensive’’ way and, depending on the task, retaining as much structural detail as possible. Based on the molecular representation used, the molecules are stored in suitable formatted files. For instance, for 2D representations, the molecules can be represented by fingerprints, SMILES, or SMARTS strings; such files use a small amount of memory. For 3D representations, typical formats include .sdf, .pdb, .mol, and .mol2; in all these cases the files contain the 3D coordinates of the structures, thus the conformation can be defined.

III. MOLECULAR DATABASES AND CHEMICAL SPACE The advent of experimental techniques such as combinatorial and parallel chemical synthesis, and high-throughput screening has enabled the production of massive amounts of data. These compound databases have played a key role in drug design (Miller, 2002) and other research areas such as Agrochemistry and Food Chemistry. Current computational

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capabilities permit the development of molecular databases, not only to store the information but also to link and mine the data. In addition to proprietary databases used in industry and other research groups, there are extensive collections of commercially available resources. Furthermore, institutional and collaborative efforts have made publicly available web-based databases for research and educational purposes. A review of large compound databases used in drug discovery has been published by Scior et al. (2007). Representative databases with general, food, and pharmaceutical scope are listed in Table 2.2. The Generally Recognized as Safe (GRAS) compounds (Table 2.2) is a collection of molecules used in food and beverage products. A fraction of this collection is available from Flavor-Base as a list of chemical names (http://www.leffingwell.com/flavbase.htm). The chemical names can be automatically converted to chemical structures using available software. GRAS compounds have been compared to drugs, revealing significant differences in molecular size, flexibility, and atom composition (Sprous and Salemme, 2007). The Distributed Structure-Searchable Toxicity (DSSTox) Database Network is a public database containing more than 1000 molecules annotated with toxicity data. The database can be searched online. The chemical structures can also be downloaded from the web site for analysis (vide infra). TABLE 2.2

Examples of large molecular databases used in research

Databases

Web sites

GRAS (Generally Recognized as Safe)

http://www.foodsafety.gov/ dms/eafus.html/ http://www. leffingwell.com/flavbase.htm http://www.epa.gov/ncct/dsstox/

DSSTox (Distributed StructureSearchable Toxicity) SuperScent BIOPEP ZINC MMsINC PubChem Developmental Therapeutics Program, National Cancer Institute DrugBank

http://bioinformatics.charite.de/ superscent/ http://www.uwm.edu.pl/ biochemia http://zinc.docking.org/ http://mms.dsfarm.unipd.it/ MMsINC.html http://pubchem.ncbi.nlm.nih.gov/ http://dtp.nci.nih.gov/ http:// cactus.nci.nih.gov/ http://www.drugbank.ca/

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SuperScent is a database comprising approximately 2000 scents (Dunkel et al., 2008). The database can be browsed online and includes web-based similarity searching. The molecules are linked to PubChem (vide infra). Additional databases in the ‘‘Super’’ collection are SuperToxic, SuperSite, and SuperTarget. The SuperScent web site (Table 2.2) has links to other public databases such as SenseLab, Pherobase, Scentbase, Flavornet, and Leffingwell. The Protein and Bioactive Peptide Sequences (BIOPEP) is a public database (Dziuba et al., 1999) containing 707 proteins, 2123 bioactive peptides, 65 allergenic proteins with their epitopes, and 224 sensory peptides and amino acids. Databases include information such as sequence, number of amino acid residues, molecular weight, activity, and references. BIOPEP can be browsed online and the user can search for a specific sequence. ZINC (Irwin and Shoichet, 2005) is a freely accessible database of increasing use in virtual screening and other computational applications. ZINC contains over 8 million purchasable compounds and it is a very attractive source of compounds to perform chemoinformatic comparisons with other collections. The MMsINC database (Masciocchi et al., 2009) is a public web-based resource with more than 4 million nonredundant compounds for virtual screening and other computational applications. Substructure and similarity searching can be performed. MMsINC is linked to other databases such as FDA, PubChem, Protein Data Bank, and ZINC. Table 2.2 also lists three publicly available databases commonly used in drug research. PubChem is accessed through the National Library of Medicine (Austin et al., 2004) and contains chemical structure information and corresponding activity across a number of biological assays. The system links the compound information with biomedical literature and it is possible to perform web-based similarity searching. PubChem also enables one to download files with structures to perform chemoinformatic analysis off-line. The National Cancer Institute (NCI) database is a freely available collection of more than 200,000 compounds and it is frequently used by research groups to perform virtual screening followed by experimental testing. Table 2.2 contains two links where it is possible to mine the database online and to download structure files for analysis. DrugBank (Wishart et al., 2008) is a public resource containing a collection of nearly 4800 entries including FDA-approved small molecule and protein/peptide drugs, nutraceuticals, and experimental agents. The database has extensive information related to the chemical, pharmacological, and pharmaceutical information of the chemical agent as well as to the drug target. DrugBank can be searched online and the structure files can also be downloaded (vide infra).

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The continued growth in the number of compounds stored in corporate, commercial, and public databases raises questions regarding the relationships of molecules within compound collections and across different databases. Common questions are related to the assessment of structural diversity of molecules, degree of overlap between compound datasets, and analysis and visualization of the chemical space. In fact, the chemical space or chemical universe coverage of compound databases has become a central concept not only in drug discovery but also in other research areas. Analysis and visualization of the chemical space of in-house, commercial, public, and virtual compound databases has a number of applications including diversity analysis, in silico property profiling, data mining, virtual screening, library design, prioritization in screening campaigns, and database acquisition (Medina-Franco et al., 2008). Chemical space has been defined as ‘‘the total descriptor space that encompasses all the small carbon-based molecules that could in principle be created.’’ (Dobson, 2004) A number of methods have been developed to analyze and visualize such descriptor space. Such methods have been applied to compare the chemical space covered by compound collections from different sources including natural products, drugs, metabolites, toxic and nontoxic substances, among other numerous datasets. These comparisons have been reviewed recently by Medina-Franco et al. (2008). It is important to mention here that visualization of the chemical space of a molecular database will depend on the molecular representation of the compounds to define the multidimensional descriptor space and the visualization technique to reduce the multidimensional space into a lower, 2D or 3D plot. In other words, the chemical space of a compound database will not be unique (Medina-Franco et al., 2008). However, using the same descriptors to compare different databases enables the assessment of such collection relative to the descriptors used.

IV. CHEMOINFORMATICS IN FOOD CHEMISTRY An example of a nonpharmaceutical use of chemoinformatics is the work described by Sprous and Salemme (2007) in the flavor portion of the GRAS compounds. Similar analysis could potentially be performed with other databases of, for example, nutraceuticals, flavors, colors, etc. In principle, nutraceuticals exemplify food components with drug-like properties. One of the major challenges in pharmaceutical campaigns is the toxicity of the potential drugs. It should be noted that the toxicity of a given compound is dose-dependent. The National Academy of Sciences Report 2007 (see http://www.nap.edu/catalog.php? record_id¼11970#toc) identified the need of a major shift on the assessment of chemical’s toxicity, and computational methods are

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recognized as key components in such transformation. Comparison of the chemical space of food component databases with those of other databases is expected to inform in both directions, that is, from the food chemistry and the pharmaceutical point of view. How do the molecular properties of the nutraceuticals compare with those of drugs and toxic compounds? Figure 2.3 shows a comparison of the chemical space of the following three databases: DSSTox (1216 compounds), 92 nutraceuticals obtained from ZINC (vide supra) and 1490 drugs obtained from DrugBank. The chemical space was obtained by representing the molecules using six physicochemical properties namely molecular weight (MW), number of rotatable bonds (RB), hydrogen bond acceptors (HBA), hydrogen bond donors (HBD), topological polar surface area (TPSA), and the octanol/ water partition coefficient (SlogP). To obtain the visual representation of

A

8 B

6

6

4

4 PC2

PC2

8

2

2

0

0

−2

−2 0

5

10 PC1

15

20

−4

8 C

8

6

6

4

4

PC2

PC2

−4

2

2

0

0

−2

−2

−4

0

5

10 PC1

15

20

−4

0

5

10 PC1

15

20

0

5

10 PC1

15

20

D

FIGURE 2.3 Chemical space of three databases obtained by principal component analysis of six (scaled) physicochemical properties. The first two principal components (PC1 and PC2) plotted here account for 77.2% of the variance: (A) three databases superimposed; (B) 1216 compounds from the Distributed Structure-Searchable Toxicity (DSSTox ▲); (C) 92 nutraceuticals (○); and (D) 1490 drugs from DrugBank (□).

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the chemical space, a principal component analysis was performed considering the six above-mentioned properties (after scaling). A similar procedure has been reported before by our group to compare a collection of drugs and natural products among other compound collections using the same properties (Singh et al., 2009). Figure 2.3A shows all databases in the same space. Extensive overlap between the nutraceuticals, drugs, and compounds from the DSSTox database is readily observed. However, there are also clear differences such as the large number of DSSTox compounds located in a different region of the chemical space as compared to drugs and nutraceuticals. For clarity, Figure 2.3B–D shows each compound collection separately but within the same coordinates as used in Fig. 2.3A. From Fig. 2.3B and C the large diversity of compounds in the DSSTox and nutraceuticals databases can be inferred. One should keep in mind that the chemical space depicted in Fig. 2.3 is valid within the framework of the six physicochemical properties used. Table 2.3 shows a comparison of the median, mean, and standard deviation of the distribution of the six properties for each of the three databases. From the median and mean values it can be deduced that several properties do not follow a normal distribution. Comparing the statistics for the six properties it can be concluded that a major difference is MW. This property is obviously associated with the size of the molecules. In general, molecules in the DSSTox have larger MW than the compounds in the nutraceuticals and drugs databases. Notably, the standard deviation is quite large in the DSSTox set indicating a large variability in MW (actually the MW ranges between 46 and 1626). The nutraceuticals have, in general, a lower MW than the drugs but with a greater standard deviation. Interestingly, all three databases TABLE 2.3 Summary of the distribution of the six physicochemical properties used to compare DSSTox, nutraceuticals, and drug databases Median/mean (S.D.) Property

Molecular weight SlogP TPSA RB HBA HBD

DSSTox (n ¼ 1216)

Nutraceuticals (n ¼ 92)

Drugs (n ¼ 1490)

312/346 (167)

266/256 (115)

310/310 (91)

1.6/1.3 (2.9) 67.6/82.6 (61.8) 5.0/5.3 (4.0) 2.0/3.1 (2.9) 1.0/1.4 (1.7)

0.8/0.3 (4.2) 69.1/83.9 (54.3)

1.6/1.4 (2.6) 67.1/71.8 (41.5) 5.0/5.1 (3.3) 2.0/2.5 (1.7) 1.0/1.2 (1.2)

5.0/4.9 (3.3) 1.0/1.9 (2.0) 1.0/1.3 (1.3)

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have, on average, a similar number of RB, a property associated with molecular flexibility. However, considering the smaller size of the nutraceuticals as deduced from the MW it can be concluded that these molecules are more flexible than the drugs and compounds in the DSSTox database. These are some examples of the analysis that can be performed for three representative datasets in Table 2.2. Similar analysis using these or other molecular representations can be conducted for other databases. In fact, a comparison of the collection of drugs analyzed here with natural products and compounds obtained from PubChem has been published elsewhere (Singh et al., 2009).

V. EXAMPLES OF MOLECULAR SIMILARITY, PHARMACOPHORE MODELING, MOLECULAR DOCKING, AND QSAR IN FOOD OR FOOD-RELATED COMPONENTS Going deeper into the comparison and classification of chemical structures, a set of computational techniques are available. This section is organized to present a brief description of currently used methodologies, namely; molecular similarity, pharmacophore modeling, molecular docking, and QSAR followed by application of these methods to food-related components. Of particular importance is to keep in mind the in vivo nature of foodrelated studies, where it is expected the simultaneous activation of several biochemical pathways. For that reason, the selection and applicability of the different methods need to be understood. In addition, the source of the data may play a major role, in most cases the information is collected from different laboratories and the variability of the data might be due not only to the chemical structure but also to the different conditions under which the experiments were performed. As an example, problems arising from the collection of data in olfaction studies have been reviewed by Chastrette (1998).

A. Molecular similarity Similarity searches are based on the hypothesis that similar molecules will have similar properties (Johnson and Maggiora, 1990). The similarity then is not a unique value; rather it is always referred to a reference compound(s), and also depends on the representation used to describe the molecules (Johnson and Maggiora, 1990). The dependence on the reference molecule and the similarity measure employed is frequently alleviated by the use of fusion methods, which consist of extracting a new column in the hit list based on the maximum, or median similarity among

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methods or queries (Willett, 2006a,b; Willett et al., 1998). Recently, we have shown the use of both measures (maximum and median) in one plot, called multifusion similarity maps (Medina-Franco et al., 2007). The changes in biological response with the changes in the structure have been rationalized as activity landscapes (Peltason and Bajorath, 2007). Even in the case of the interaction of one ligand with one receptor, the activity or property landscape can have different shapes. Changes in the biological response associated with proportional changes in the structure are in a region of smooth landscape. However, there are situations where small changes in the structure produce large changes in the biological response. Such features on the property landscape have been termed ‘‘activity cliffs’’ (Johnson and Maggiora, 1990). To alleviate the dependence on the molecular representation we have proposed the identification of activity cliffs by using multiple representations and find ‘‘consensus activity cliffs’’(Medina-Franco et al., 2009). Structure–activity relationships have been largely employed for molecular design; these correlations depend on the molecular representation and the activity landscape. The molecular representation depends only on the small molecule, whereas the activity landscape provides information on the ligand–receptor complex, for example, how permissive the binding pocket is. To exemplify the molecular similarity approach, a set of odorants (compared to benzaldehyde) will be presented. The discovery and characterization of the olfactory receptors in the early 1990s by Buck and Axel (1991) facilitated the understanding of the mechanism of olfaction and biochemical principles involved. The olfactory receptors belong to the G-protein coupled receptor (GPCR) superfamily. These proteins are involved in a variety of biological processes like vision and pain and are one of the principal targets with therapeutic importance, that is, 45% of currently marketed drugs target GPCRs. Rhodopsin, responsible for dim light vision in eukaryotes, was the first GPCR for which the X-ray crystal structure was resolved (Palczewski et al., 2000), albeit in the inactive state. In addition to the dark or inactive state of rhodopsin, intermediates to the active state (Nakamichi and Okada, 2006a,b; Ruprecht et al., 2004; Salom et al., 2006; Tikhonova et al., 2008) and the X-ray crystal structure of the b-adrenergic receptor have also been resolved (Bhattacharya et al., 2008; Cherezov et al., 2007; Cong et al., 2001; Rasmussen et al., 2007; Rosenbaum et al., 2007). These advances in the GPCR field have allowed the generation of homology models of related proteins, such as the olfactory receptors. The homology models can then be used to analyze interactions of small molecules in the binding site. It can also be used to dock ligand databases into the receptor and thereby to enrich pharmacophore models. In 2001, Zozulya et al. (2001) reported the identification of 347 olfactory receptors in the human genome. A complication with olfactory receptors is that the

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same ligand can bind to multiple receptors while one receptor is able to host different ligands, this is indicative of the high plasticity of olfactory receptors. Knowledge of the structural requirements for binding to the olfactory receptors will help on the development of structure–odor relationships. A recent review by Zarzo (2007) provides an overview of the different theories that have been proposed on the basis of olfaction. One of the earliest theories is the profile-functional group theory of olfaction, where the association of the functional groups to a particular odor is highlighted. A good example is the thiol (–SH) group, which imparts a rotten eggs or garlic odor to many of the molecules in which it is present. A later theory proposes that the shape of molecules influences their binding to certain nasal receptors. This theory was first proposed by Moncrieff (1967) in 1949 and was latter referred to as ‘‘stereochemical theory’’ by Amoore (1963). As early as 1963, Amoore postulated that the size and shape of the molecule determined its odor. As a means of quantification, Amoore (1963) produced silhouette photographs of a scale model of the benzaldehyde molecule, and compared the silhouette of homologous compounds, taking benzaldehyde as the standard. This early form of molecular similarity leads to a reasonably good odor–structure correlation, and he conducted similar analysis on other molecular sets (Amoore, 1967; Amoore et al., 1967). To exemplify a molecular similarity method, we employed here a 3D shape-based molecular similarity approach using OpenEye scientific software (OpenEye). A set of 27 molecules (Amoore, 1971) were compared to benzaldehyde (query molecule). The representation used here is based on the volume of each molecule. A conformational ensemble is built for the molecules in the database, whereas the conformation of the query remains fixed (the chemical nature of benzaldehyde does not entail different conformers, though in many cases the conformation of the query molecules might be complex and crucial). After the conformers of each molecule in the data set are built, each one of them is compared with the query and a similarity value is computed. For the particular program employed here (ROCS), the similarity is quantified as a score formed by two terms, one takes into account the chemical nature of the molecules while the other relies on molecular shape, such score is referred to as combo score. The maximum similarity value is 2 which can only be obtained from the comparison of a molecule with itself in the exact same conformation (perfect match). The normalized values (from 0 to 1) for the odor and combo score similarities are compared in the graph shown in Fig. 2.4. As can be observed, as the combo score increases, the odor similarity to benzaldehyde also increases. This correlation shows that part of the odor similarity was captured by the molecular

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0.9 0.8

Odor sim (normalized)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.1

0.2

0.3 0.4 0.5 0.6 Combo score (normalized)

0.7

0.8

0.9

FIGURE 2.4 Odor–structure similarity plot. X-axis represents 3D shape similarity; Y-axis shows odor similarity to benzaldehyde. Shape by functional group: squares (CHO) and circles (NO).

similarity employed here. Other examples have been reported recently (Martinez-Mayorga et al., 2008; Yongye et al., 2009). Limitations in the rational design of new odorants have been described (Sell, 2006, 2008). Moreover, minute structural differences resulting on significant odor changes have discouraged attempts at using SAR and rational design. In line with the ‘‘activity cliff’’ concept (Johnson and Maggiora, 1990) (vide supra), there are several examples of what can be called ‘‘odor cliffs’’, a collection of such examples was published by Sell (2006). It is likely that activity landscapes are heterogeneous (Peltason and Bajorath), in other words, for the same system there are regions where smooth SAR can be found, but there are other regions with many activity cliffs. Even for one receptor, the presence of activity cliffs is not uncommon; however, they are rather important because they give information about regions in the receptor-binding pocket that are very selective. When more than one receptor is involved in the biological process corresponding to an expected property (as in the case of olfactory

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receptors), a much more complicated situation arises, where simultaneous activity landscapes are possible. Concerning the selectivity of receptors, when they allow the binding of very different ligands they are regarded as permissive (Peltason and Bajorath, 2008). In those cases, ligands of different sizes, chemical nature, and conformation can be accommodated, showing that the binding site is flexible. When a receptor is able to carry only specific ligands, it is called selective and is expected to have multiple activity cliffs. Thus, smooth landscapes are the best scenario for the development of structure–activity relationships, not only for similarity searches but also for pharmacophore and QSAR models (Maggiora, 2006). In many cases, multiple biochemical pathways are activated by the intake of therapeutical drugs. The activation of side mechanisms coincides with the consequent side effects. This concept has led to research areas like polypharmacology as well as re-purposing studies. In such cases, the possibility of activating of multiple biochemical pathways is employed to explore alternative therapeutic uses of the same compound. In addition to the ligand–receptor process, it is necessary to take into consideration the other mechanistic features that might also be involved and affect the analysis, such as the metabolism, transport based on carry proteins, co-factors, influence of metals, etc.

B. Pharmacophore model In pharmacophore modeling, the aim is to obtain a hypothesis that best describes the chemical features and conformation of molecules responsible for biological activity. The features generally evaluated are hydrogen bond acceptors, hydrogen bond donors, aliphatic hydrophobic groups, negatively ionizable groups, positively ionizable groups, and aromatic hydrophobic groups. In addition to the pharmacophoric features, current programs (Dixon et al., 2006; Kirchmair et al., 2007) allow the generation of excluded volumes that are meant to mimic regions occupied by the receptor. The pharmacophore generated can be then used for virtual screening of a compound collection. A detailed review with practical aspects and successful examples of pharmacophore-based virtual screening in the pharmaceutical field is described by van Drie (2003). An example of a pharmacophore model is presented in Fig. 2.5 which shows several pharmacophoric features for a methanesulfonamide ligand in the binding pocket of a kinase receptor, produced with LigandScout (Kirchmair et al., 2007). In addition to pharmaceutical applications, pharmacophore models have also been generated to analyze structure–odor relationships. In spite of complications arising from the need for more information on the mechanistic basis of olfaction, successful models have been developed

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FIGURE 2.5 Pharmacophore model (produced using LigandScout) showing a methanesulfonamide ligand in the binding pocket of a cyclin-dependent kinase (PDB code: 1KE6). Pharmacophoric features: white arrow, hydrogen bond donor; gray arrow, hydrogen bond acceptor; white spheres, aromatic group; gray spheres, excluded volumes; protein is shown as mash ribbons.

(Kraft et al., 2000). This particular type of pharmacophore has been termed an olpfactophore and a review by Kraft includes the development of olfactophores and SAR for the major odor notes of relevance in perfumery: ‘‘fruity,’’ ‘‘marine,’’ ‘‘green,’’ ‘‘floral,’’ ‘‘spicy,’’ ‘‘woody,’’ ‘‘amber,’’ and ‘‘musky’’ (Kraft et al., 2000). In addition to perfumery, odors are also of great importance in the food industry, for example, in the development of flavors.

C. QSAR and QSPR Among the computational methods available, QSARs, or more general, quantitative structure–property relationships (QSPR) have been widely used not only in drug design and environmental chemistry but also in food-related studies. QSPR studies are grounded in the concept that a property (e.g., biological activity, reactivity, toxicity, volatility, etc.) depends on the molecular structure and that is possible to find a mathematical or quantitative relationship between that property and a suitable molecular representation (e.g., some combination of descriptors). Depending on the molecular representation used, several types of QSAR studies can be carried out, such as 2D- or 3D-QSAR studies,

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where molecules are represented using 2D or 3D descriptors, respectively (vide supra). The first investigations in QSAR aimed to identify linear relationships between the property and molecular descriptors, which were either calculated or measured experimentally. Current QSAR models can be quite complex. One of the goals of QSAR studies is to help explain retrospectively the response or property of a molecule with a rationale based on molecular structure. A second major goal and challenge of QSAR or QSPR studies is to develop models that are able to predict quantitatively the property of new molecules either real or virtual compounds. Thus, successful predictive QSAR models can have a tremendous impact in the design of new molecules. Furthermore, predictive models are useful to perform in silico predictions of the properties of new structures. In virtual screening, those molecules that are predicted to have the desired property according to the QSAR model are selected as best candidates. Reviews, examples, caveats, and modified versions of QSAR are described elsewhere (Kubinyi, 1997a,b; Wermuth, 2008). Some recent examples reported in the food chemistry field are summarized in Table 2.4.

D. Molecular docking Automated molecular docking aims to predict the best conformation of a molecule to fit into the target binding pocket (Kitchen et al., 2004). The Protein Data Bank (Berman et al., 2000) is a widely used source of target crystal structures as well as 3D coordinates of conformational ensembles obtained by NMR. In the absence of crystallographic structures, however, homology models can be used instead. Predicting the preferred conformation of a molecule with a target frequently comprises two major steps namely docking and scoring. Several docking programs are able to dock hundreds or even thousands of molecules relatively quickly so that virtual screening of large compound collections is feasible. Examples of docking programs are Autodock, (Morris et al., 2009) Gold, Glide (Glide, 2008), and Fred (McGann et al., 2003). For virtual screening proposes, Glide, for instance, has implemented a high-throughput docking algorithm that is designed for fast filtering. Then a more refined docking protocol can be used, to follow up. One of the main areas of improvement in docking is the refinement of scoring functions to predict more accurately ligand–target interactions. In some cases biologically-relevant ‘‘poses’’ or docking solutions are found although they are not necessarily scored as the top ranked solutions. Figure 2.6 shows the docking of a small molecule with the enzyme PTP-1B (PDB code 2F71) using the program Autodock. A food-related example of molecular docking was published by Pripp (2007) on angiotensin converting enzyme inhibitory dipeptides. In an

TABLE 2.4

Recent examples of QSAR models in food chemistry

Computational method

Relevant property

Food relatedness

Title

References

Quantitative structure–activity relationships (QSAR)

Carcinogens

Food contact materials

Regul. Toxicol. Phaimacol. 2008, 50, 50–58

Docking and virtual screening

Angiotensin converting enzyme inhibitors Aroma release

Food derived peptides— milk

Structure–activity relationship analysis tools: validation and applicability in predicting carcinogens Docking and virtual screening of ACE inhibitory dipeptides

Quantitative structure–property relationships (QSPR) QSAR

Molecular dynamics (MD), docking, QSAR

Yogurt

Prediction of chemical residues

Tuna

Mutagenic potency

Cooked meat

Effect of thickeners on aroma compound behavior in a model dairy gel A new risk framework for predicting chemical residue(s)—preliminary research for PCBs and PCDD/Fs in farmed Australian Southern Bluefin Tuna (Thunnus maccoyli) Mutagenic potency of foodderived heterocyclic amines

Eur. Food Res. Technol. 2007, 225, 589–592 J. Agric. Food Chem. 2008, 55, 4835–4841 Chem. Eng. Process. 2007, 46, 491–496

Mutation. Res. 2007, 616, 90–94

QSAR

Cognitive and neurological deficiencies

Milk

QSAR

Angiotensin converting enzyme inhibitors

Food derived peptides – Milk

QSAR

Antimicrobial, ACEinhibitory and bitter tasting peptides Antibacterial

Several—review

3D-QSAR (CoMFA)

QSAR

Bioaccumulation potential of organic chemicals in aquatic food webs

Food safety

Food safety

Quantitative structure– activity relationship of prolyl oligopeptidase inhibitory peptides derived from b-casein using simple amino acid descriptors Structural requirements of angiotensin I-converting enzyme inhibitory peptides: QSAR study of di- and tripeptides Quantitative structure– activity relationship modeling of peptides and proteins as a tool in food science 3D-QSAR, synthesis, and antimicrobial activity of 1-alkylpyhdinium compounds as potential agents to improve food safety A generic QSAR for assessing the bioaccumulation potential of organic chemicals in aquatic food webs

J. Agric. Food Chem. 2006, 54, 224–228

J. Agric. Food Chem. 2006, 54, 732–738

Trends Food Sci. Technol. 2005, 16, 484–494

Euro J. Med. Chem. 2005, 40, 840–849

QSAR Comb. Sci. 2003, 22, 337–345

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FIGURE 2.6 code 2F71).

Docking of a small organic molecule into the catalytic site of PTP-1B (PDB

analysis of bioactive peptides derived from food proteins, he found that docking and virtual screening were feasible techniques to identify promising bioactive peptides. As pointed out in his paper, this approach does not fully replace experimental work, though it can contribute to the molecular understanding of bioactivity, and prioritization in virtual screening (Pripp, 2007).

VI. CONCLUDING REMARKS AND PERSPECTIVES The ultimate goals of the methods described in Section IV fall in the understanding of structure–activity relationships and the discovery of new molecules. SAR is an inherent part of the derivation of the models, whereas the virtual screening of databases against the models not only serves as a means of validation but also provides candidates with a better chance (compared to random selection) of having the desired property. The development of new descriptors, modified versions of established methods, or the creation of new approaches is constantly evolving in the scientific community, not only in the molecular modeling field but also in the analysis, mining, and processing of the chemical information. All of the computational methods presented here have been used in connection with food or food-related components, and they can potentially be applied to other areas in food chemistry. The better mechanistic pathways for the property response are understood, then the more suited methods will become in analyzing the very

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rich, diverse, and important body of information that comprises food chemistry.

ACKNOWLEDGMENTS The authors are thankful to Dr. Terry Peppard (Robertet Flavors, Inc.) for valuable discussions and for proof reading this book chapter. This work was supported by the State of Florida, Executive Officer of the Governor’s Office of Tourism Trade and Economic Development. We thank OpenEye Scientific Software for providing OMEGA, ROCS, FRED, and VIDA programs.

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Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Trong, I. L., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745. Paris, G. (2000). ‘‘Balancing the Needs of the Recruiters and the Aims of the Educators’’. http://www.warr.com. Peltason, L. and Bajorath, J. (2007). Molecular similarity analysis uncovers heterogeneous structure-activity relationships and variable activity landscapes. Chem. Biol. 14, 489–497. Peltason, L. and Bajorath, J. (2008). Molecular similarity analysis in virtual screening. In ‘‘Chemoinformatics Approaches to Virtual Screening’’, (A. Varnek and A. Tropsha, eds), pp. 120–147. RSC Publishing, Cambridge, UK. Pripp, A. H. (2007). Docking and virtual screening of ACE inhibitory dipeptides. Eur. Food. Res. Technol. 225, 589–592. Rasmussen, S. G. F., Choi, H.-J., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R. P., Sanishvili, R., Fischetti, R. F., Schertler, G. F. X., Weis, W. I., et al. (2007). Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450, 383–387. ROCS version 2.3.1 OpenEye Scientific Software: Santa Fe, NM, USA, www.eyesopen.com. Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G. F., Thian, F. S., Kobilka, T. S., Choi, H.-J., Yao, X.-J., Weis, W. I., Stevens, R. C., and Kobilka, B. K. (2007). GPCR engineering yields high-resolution structural insights into beta2 adrenergic receptor function. Science 318, 1266–1273. Ruprecht, J. J., Mielke, T., Vogel, R., Villa, C., and Schertler, G. F. X. (2004). Electron crystallography reveals the structure of metarhodopsin I. EMBO J. 23, 3609–3620. Salom, D., Lodowski, D. T., Stenkamp, R. E., Trong, I. L., Golczak, M., Jastrzebska, B., Harris, T., Ballesteros, J. A., and Palczewski, K. (2006). Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proc. Natl. Acad. Sci. USA 103, 16123–16128. Scior, T., Bernard, P., Medina-Franco, J. L., and Maggiora, G. M. (2007). Large compound databases for structure-activity relationships studies in drug discovery. Mini-Rev. Med. Chem. 7, 851–860. Sell, C. S. (2006). On the unpredictability of odor. Angew. Chem. Int. Ed. 45, 6254–6261. Sell, C. S. (2008). Rational odorant design: Fantasy or feasibility? Perfum. Flavor. 33, 48–52. Singh, N., Guha, R., Giulianotti, M. A., Pinilla, C., Houghten, R. A., and Medina-Franco, J. L. (2009). Chemoinformatic analysis of combinatorial libraries, drugs, natural products, and molecular libraries small molecule repository. J. Chem. Inf. Model. 49, 1010–1024. Sprous, D. G. and Salemme, F. R. (2007). A comparison of the chemical properties of drugs and FEMA/FDA notified GRAS chemical compounds used in the food industry. Food Chem. Toxicol. 45, 1419–1427. Tikhonova, I. G., Best, R. B., Engel, S., Gershengorn, M. C., Hummer, G., and Costanzi, S. (2008). Atomistic insights into rhodopsin activation from a dynamic model. J. Am. Chem. Soc. 130, 10141–10149. van Drie, J. H. (2003). Pharmacophore discovery—Lessons learned. Curr. Pharm. Desgn. 9, 1649. Varnek, A. and Tropsha, A. (2008). ‘‘Chemoinformatics Approaches to Virtual Screening’’. RSC Publishing, Cambridge, UK. Wermuth, C. G. (2008). The Practice of Medicinal Chemistry. Elsevier, Academic Press, London, UK. Wild, D. J. and Wiggings, G. D. (2006). Challenges for chemoinformatics education in drug discovery. Drug Discov. Today 11, 436–439. Willett, P. (2006a). Enhancing the effectiveness of ligand-based virtual screening using data fusion. QSAR Comb. Sci. 25, 1143–1152.

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CHAPTER

3 Processing of Food Wastes Maria R. Kosseva1

Contents

I. Introduction A. Food industry wastes: Problems and opportunities B. Development of green production processes II. Sources and Characterization of Food Wastes A. Fruit-and-vegetable wastes B. Olive oil industry C. Fermentation industry D. Dairy industry E. Meat and poultry industry F. Seafood by-products III. Recovering of Added-Value Products from FVW (Upgrading Concept) A. Challenge for the vegetable industry B. SSF of fruit/vegetable by-products IV. Multifunctional Food Ingredient Production from FVW A. Dietary fibers B. Coloring agents and antioxidants C. Gelation properties D. Oil and meal E. Food preservation F. Production of biopolymers, films, food packaging G. Derivatives of meat wastes H. Derivatives of seafood wastes

58 58 60 63 63 64 64 65 67 68 69 69 70 82 83 84 87 88 88 89 89 91

UCD School of Chemical & Bioprocess Engineering, College of Engineering, Mathematical & Physical Sciences, University College Dublin, Belfield, Dublin 4, Ireland 1 Corresponding author: Maria R. Kosseva, e-mail: [email protected] Advances in Food and Nutrition Research, Volume 58 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)58003-5

#

2009 Elsevier Inc. All rights reserved.

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V. Vegetable Residues as Bioadsorbents for Wastewater Treatment A. Biosorption of metal ions B. Adsorption of dyes from wastewater VI. Using Eggshell VII. Added-value Products from Whey VIII. Food Waste Treatment A. Bioprocessing of FVWs B. Biodiesel production C. Anaerobic treatment of dairy wastes D. Aerobic treatment of dairy wastes IX. FCM Aspects Aimed in Sustainable Food System Development A. User-oriented innovation in the food sector B. Market-oriented research C. Integrated product development and sustainability D. The food market focus X. Summary and Future Prospects References

Abstract

94 94 97 98 98 100 100 107 108 111 116 117 117 118 118 120 123

Every year almost 45 billion kg of fresh vegetables, fruits, milk, and grain products is lost to waste in the United States. According to the EPA, the disposal of this costs approximately $1 billion. In the United Kingdom, 20 million ton of food waste is produced annually. Every tonne of food waste means 4.5 ton of CO2 emissions. The food wastes are generated largely by the fruit-and-vegetable/olive oil, fermentation, dairy, meat, and seafood industries. The aim of this chapter is to emphasize existing trends in the food waste processing technologies during the last 15 years. The chapter consists of three major parts, which distinguish recovery of addedvalue products (the upgrading concept), the food waste treatment technologies as well as the food chain management for sustainable food system development. The aim of the final part is to summarize recent research on user-oriented innovation in the food sector, emphasizing on circular structure of a sustainable economy.

I. INTRODUCTION A. Food industry wastes: Problems and opportunities Official surveys indicate that every year more than 160 billion kg of edible food is available for human consumption in the United States. Of that total, nearly 30% (45 billion kg)—including fresh vegetables, fruits, milk, and grain products—is lost to waste by retailers, restaurants, and

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consumers (Rizvi, 2004). It costs the United States around $1 billion every year just to dispose of all its food waste, according to the EPA. Proportionately, the United Kingdom and Japan have traditionally been among the worst offenders worldwide in recent years when it comes to food waste, discarding between 30% and 40% of their food produce annually. The headline figures suggest that 6.7 million ton of household food is wasted each year in the United Kingdom. In total, 20 million ton of food waste is produced annually in the United Kingdom, with the producers, processors, institutions, retailers, and others involved outside the household accounting for 13 million ton. Taking food stuffs specifically 4.4 million apples, 1 million slices of ham, and 440,000 ready meals per day are thrown away. The report claims that this involved 18 million ton of CO2 emissions, because every tonne of food waste means 4.5 ton of CO2 emissions. In the developing world, people spend up to 80% of their income on food: the price of food has risen by over 50% in 2008, on top of a 27% rise in 2007, according to the UN’s Friends of the Earth (FOE). FOE also admits that just 0.36% of the UK’s electricity needs could be met by anaerobic digestion (AD), if 5.5 million ton of food waste was treated by AD, it could only generate enough electricity to power 164,000 houses. In the western world, food is produced, processed, transported, sold, driven home and then, 33% of the time, thrown into the bin for landfill. Then in landfill, methane gas is given off, which is far more destructive than CO2. Fresh fruits, vegetables, and salads make up the largest category of waste, according to the The UK’s Waste & Resources Action Program (WRAP) report, clocking in at 1.4 million ton per year (Moore, 2008). Furthermore, every year the European food-processing industry produces vast volumes of aqueous wastes. These include: fruit and vegetable residues and discarded items, molasses, and bagasse from sugar refining, bones, flesh, and blood from meat and fish processing, stillage and other residues from wineries, distilleries and breweries, dairy wastes such as cheese whey, and wastewaters from washing, blanching and cooling operations (Arora et al., 2002). Many of these contain low levels of suspended solids and low concentrations of dissolved materials. Apart from the environmental challenges posed, such streams represent considerable amounts of potentially reusable materials and energy. Much of the material generated as wastes by the food-processing industries throughout Europe—and about to be generated within bio-fuels programs—contains components that could be utilized as substrates and nutrients in a variety of microbial/enzymic processes, to give rise to added-value products. Varieties of processes exist that do this worldwide, some having operated for many years. Joshi (2002) and Marwaha and Arora’s studies (2000) are two examples of extensive discussions of current industrial exploitation and future possibilities within this area. Added-value products actually

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produced from food industry wastes, or potentially so, include animal feed, single-cell protein (SCP) and other fermented edible products, baker’s yeast, organic acids, amino acids, enzymes (e.g., lipases, amylases, cellulases), flavors and pigments, the bio-preservative bacteriocin (from the culture of Lactococcus lactis on cheese whey) and microbial gums and polysaccharides ( Joshi, 2002). According to the European Landfill Directive, the amount of biodegradable waste sent to landfills in member countries by 2020 must reach 35% of the levels reached in 1995. Therefore, the European food-processing industry operations are having to comply with increasingly more stringent EU environmental regulations related to disposal or utilization of by-products and wastes. These include growing restrictions on land spraying with agro-industrial wastes, and on disposal within landfill operations, and the requirements to produce end products that are stabilized and hygienic. Unless suitable technologies are found for the processing and utilization of waste by-products, large numbers of food-processing operations will be under threat. The aim of this work is to provide a comprehensive literature survey on food waste processing technologies, published during the last 15 years. This chapter consists of three parts: the first describes the upgrading concept, for example, recovery of added-value products; the second summarizes the latest knowledge in the field of treatment technologies and our own investigations; the third part is related to the food chain management (FCM) for sustainable food system development. The aim of the final part is to summarize recent research on user-oriented innovation in the food sector, emphasizing on circular structure of a sustainable economy. When discussing the environmental impact of food production it is important to use a holistic approach, which can integrate the environmental aspects into the product development and food production. As the food supply chain is complex, environmental impacts can occur in different places and different times for a single food product. Life cycle assessment (LCA) provides a way of addressing this problem. LCA gives businesses the opportunity to anticipate environmental issues and integrate the environmental dimension into products and processes. Important issues directly related to food processing are energy and waste management. Food production in general uses significant amounts of energy and produces relatively large amounts of wastes, particularly, packaging wastes.

B. Development of green production processes The waste management hierarchy is one of the guiding principles of the zero waste practice (Fig. 3.1). By analogy with this principle, the development of green production processes can be achieved following the short-, medium-, and long-term goals (Laufenberg et al., 2003).

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61

Most preferable

Avoid Reduce Reuse Recover Treat Dispose

Least preferable

FIGURE 3.1

The food waste management hierarchy.

1. Short-term goals involve waste minimization by reduction and recycling of valuable substances, by-products and residues with reduction of emission and risk as a final outcome. 2. Medium-term goals include development of efficient production process, adding value to by-products. The outcome for the companies is their higher environmental responsibility accompanied by competitive advantages. 3. Long-term goals consist of step-by-step implementation of environmentally friendly manufacturing, developing ‘‘innovative products.’’ The ultimate outcome is design of innovative food products like functional foods, which can open new markets and meet green productivity objectives.

1. Holistic approach in food production When discussing the environmental impact of food production it is important to use a holistic approach. This approach tries to connect different goals, such as highest product quality and safety, highest production efficiency and the integration of environmental aspects into product development and food production. Within the concept every factor and aspect should be taken into account in a coherent manner (Laufenberg et al., 2003). Present R&D in food technology is unthinkable

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without taking environmental aspects into account. A responsible management of inadequate resources is needed especially in view of tighter living spaces. Based on these considerations, the holistic concept of food production shown in Fig. 3.2 has been developed.

2. Green production strategy Green or clean production can be considered so far as a strategic element in manufacturing technology for present and future products in several industrial branches. Demand is focused on the development of costeffective technology, the optimization of processes including separation steps, alternative processes for the reduction of wastes, optimization of the use of resources and improvement in production efficiency (Paul and Ohlrogge, 1998). Hence current industrial waste management techniques can be classified into three options: source reduction via in-plant modification, waste recovery/recycle or waste treatment by detoxifying, neutralizing or destroying the undesirable compounds. The first two options plant modification and waste recovery/recycle represent the most promising waste management strategies. Indeed, waste recovery is a particularly attractive option. Significant environmental and economic benefits can accrue from separating industrial wastes with the objective of recycling/reusing these valuable components and/or the bulk of water.

Product safety, sensorial quality, suitable packaging Product quality

Synthesys

FIGURE 3.2

Production efficiency

Environmental protection

Process know-how Increase in productivity

Waste prevention Added value to co-products Optimised energy consumption Packaging design

The holistic concept of food production (Laufenberg et al., 2003).

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63

Promising concepts include pervaporation in hybrid processes (Hausmanns et al., 1999) or the upgrading of vegetable residues to create a secondary use for the ‘‘waste products’’ (Laufenberg et al., 1999). The goal of green production is to fulfill our need for products in a sustainable way, that is, using renewable, nonhazardous materials and energy efficiently while conserving bio-diversity. Clean production systems are circular and use fewer materials and less water and energy, as a result resources flow through the production–consumption cycle at slower rates (Fig. 3.3).

II. SOURCES AND CHARACTERIZATION OF FOOD WASTES A. Fruit-and-vegetable wastes Fruit-and-vegetable wastes (FVWs) are produced in large quantities in markets and constitute a source of nuisance in municipal landfills because of their high biodegradability (Misi and Forster, 2002). For example, in the central distribution market for food (meat, fish, fruit, and vegetables) Mercabarna (Barcelona), the total amount of wastes coming from fruit and vegetables is around 90 ton per day during 250 days per year (Mata-Alvarez et al., 1992). The whole production of FVW collected from the market of Tunisia has been measured and estimated to be 180 ton per month (Boualagui et al., 2003). In India, FVW constitute about 5.6 million ton annually and currently these wastes are disposed by dumping on the outskirts of cities (Srilatha et al., 1995). The wastes from fruit-and-vegetable processing industries generally contain large amounts of suspended solids (SS) and high values of biological (BOD)

Dy

cle

FIGURE 3.3

2

:w as te

Utilisation

Manufacturing Using clean technology

Durable, repairable and necessary products

wa ste p

Base materials

tion, lo ven re

roducts life p ng

Cycle 1:

Renewable reduced resources

in ycl rec red uctio ste n, non-hazardous wa

g

Circular structure of a sustainable economy (Stahel, 2008).

Non-toxic minimal waste

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Maria R. Kosseva

and chemical oxygen demands (COD). Other parameters of interest are pH, dissolved oxygen tension, concentration of total solids (TSs). Indicative values of BOD, COD, SS, and pH for the processing of some fruit and vegetables are summarized in Table 3.1. According to Verrier et al. (1987) and Ruynal et al. (1998), the total initial solid concentration of FVW is between 8 and 18%, with a total volatile solids (VSs) content of about 87%. The organic fraction includes about 75% sugars and hemicellulose, 9% cellulose and 5% lignin. In general, these wastes consist of hydrocarbons and relatively small amounts of proteins and fat with an acidic pH (Riggle, 1989), and moisture content of 80–90% (Grobe, 1994). The wastewaters contain dissolved compounds, pesticides, herbicides, and cleaning chemicals.

B. Olive oil industry Liquid waste from olive oil industry is a dark-colored juice, which contains organic substances such as sugars, organic acids, poly-alcohols, pectins, colloids, tannins, and lipids (Table 3.2). The difficulty of disposing olive oil mill wastewaters (OMW) is mainly related to its high BOD, COD, and high concentration of organic substances; for example, phenols, which make degradation a difficult and expensive task (Saez et al., 1992).

C. Fermentation industry The fermentation industry is divided into three main categories: brewing, distilling, and wine manufacture. Each of these industries produces liquid waste with many common characteristics, such as high BODs and CODs, but differs in the concentration of the organic compounds such as tannins,

TABLE 3.1 Fruit-and-vegetable waste characteristics (Thassitou and Arvanitoyannis, 2001) Waste characteristics Fruit/vegetable

BOD (mg/L)

COD (mg/L)

SS (mg/L)

pH

Apples Carrots Cherries Corn Grapefruit Green peas Tomatoes

9600 1350 2550 1550 1000 800 1025

18700 2300 2500 2500 1900 1650 1500

450 4120 400 210 250 260 950

5.9 8.7 6.5 6.9 7.4 6.9 7.9

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65

TABLE 3.2 Chemical composition of organic fraction and quality characteristics of liquid olive oil waste (Thassitou and Arvanitoyannis, 2001) Components

Values (%)

Parameters

Values

Lipids Organic acid Pectins, colloids, tannins Sugars Total nitrogen content

1.0–1.5 0.5–1.55 1.0–1.5 2.0–8.0 1.2–1.5

pH SS TS BOD COD

3–5 65,000 mg/L 6.39% 43,000 mg/L 100,000 mg/L

phenols, and organic acid. The difficulty in dealing with fermentation wastewaters is in the flows and loads of the waste.

D. Dairy industry The dairy industry represents a major and important part of the food industry and contributes significant liquid wastes, whose disposal requires a large amount of capital investment. On the basis of cheese consumption and production details (British Cheese Board, 2004), it is estimated that approximately 9 million ton of cheese per annum is produced within the EU, giving rise to an annual whey production figure of the order of 50 million m3. About 50% of total world cheese-whey production is treated and transformed into various food products, of which about 45% is used directly in liquid form, 30% in the form of powered cheese whey, 15% as lactose and de-lactosed by-products, and rest as cheese-whey-protein concentrates. Waste from the dairy industries contributes substantially to the pollution of surface waters and soil. Some of their characteristics can be summarized as follows: high organic load (e.g., fatty acids and lactose), considerable variations in pH (4.2–9.4), relatively large load of suspended solids (0.4–2 g/L), and large variations in waste supply. The dairy wastewater may contain proteins, salts, fatty substances, lactose, and various kinds of cleaning chemicals (Kosseva et al., 2003). The presence of detergents and their additives in dairy wastewater hardly influences the total COD in contrast to milk, cream or whey, the high CODs of which are likely to have a dominating effect (Table 3.3). Detergents may be alkaline or acid, and very often contain additives like phosphates, sequestering agents, surfactants, and so on. EDTA is commonly used as a substitute for polyphosphates. It has a low biodegradability and remains in the wastewater after treatment. Surfactants have been shown to affect strongly the ecosystems of rivers and are toxic to aquatic animals (Thassitou and Arvanitoyannis, 2001).

TABLE 3.3

Characteristics of dairy waste effluents (combined from Wildbrett, 1988; Demirel et al., 2005)

Product/substance

Cream, 30% fat Whole milk, 3.5% fat Skim milk Whey Na-dodecyl benzol sulfonate Na-ethoxy alkyl sulphate Dialkyl dimethyl ammonium chloride (C18–C20) Sodium hydroxide Phosphoric acid Detergent and disinfectant 1 (with QAC) Detergent and disinfectant 2 Detergent and disinfectant 3 (with surfactant)

Concentration (g/L)

COD (g/L)

BOD (g/L)

pH

Suspended solids (g/L)

Volatile solids (g/L)

– – – – 0.1

850–860 760–210 9–100 68–75 0.216

1.20–4.00

8–11 6.92 6.92 3.5–6.5

0.35–1.00 0.34–1.73 0.09–0.45 0.50–2.50

0.33–0.94 0.255–0.83

0.1 0.1

0.178 0.235

10.0 10.0 10.0

0 0 2250

10.0 5.0

0.017 0.147

0.50–1.30

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E. Meat and poultry industry Meat, poultry, and fish industries produce the highest loads of waste within the food industry. The meat industry contains slaughterhouses and processing units where meat is prepared, cut in pieces and is either frozen, cooked, cured, smoked, or made into sausages. Slaughterhouses are more important than the other units in terms of environmental pollution. The wastes coming from these units contain various quantities of blood, fats, residues from the intestine, paunch grass, and manure (Cournoyer, 1996). Slaughterhouse wastewater is typically high in both moisture (90–95%) and nitrogen, has a high BOD, and is odorous. Proper management is a prerequisite to ensure that potentially high levels of pathogens are eliminated. Cooper and Russell (1992) investigated and published a summary of the treatment technologies of 44 meat processing plants in New Zealand. The management of nitrogen in both land application and direct discharge to receiving water was the critical point. The operation of a pilot-scale UASB treating the effluent from a beef slaughtering operation was reported by Torkian et al. (2003a). The researchers were able to obtain steady-state operation of the UASB at organic loading rates (OLRs) of 13–39 kg soluble COD/m3 day and HRTs of 2–7 h under mesophilic conditions. SCOD removals of 75–90% were obtained at these loading rates with influent feed concentrations of 3000–4500 mg soluble COD/L. In a connected study, the effluent from the UASB was processed through a pilot-scale rotating biological contactor (RBC) to obtain additional organic load reduction (Torkian et al., 2003b). At OLRs of 5.3 g soluble BOD/m3 day, soluble BOD removals of 85% were obtained, with a vast majority occurring within the first half of the six-stage reactor. As part of a wastewater treatability study, Del Pozo et al. (2003) noted that the high COD concentrations (7230 mg/L) warranted anaerobic treatment and conducted a series of anaerobic batch tests on a beef slaughterhouse wastewater that yielded COD removals of 80%. Bohdziewicz et al. (2003) were successful in obtaining high contaminant removals from a meat processing wastewater using ultrafiltration followed by reverse osmosis. Total Phosporous and Total Nitrogen were removed at greater than 98% efficiencies, and BOD and COD removals were greater than 99%. Membrane fouling has commonly been cited as the primary impediment to the use of filtration technologies in the meat processing industry. Allie et al. (2003) reported on the use of lipases and proteases to effectively remove fouling proteins and lipids from flat-sheet polysulphone ultrafiltration membranes. Poultry wastes are equally problematic to meat wastes. Starkey (2000) reviewed the considerations for selection of a treatment system for poultry processing wastewater, including land availability, previous site history, publicly owned treatment work discharge, conventional waste

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treatment systems, and land application systems. The performance of anaerobic treatment systems, including lagoons, contact processes, sludge beds, filters, packed beds, and hybrid reactors were outlined (Ross and Valentine, 1992). In another study on combined treatment, anaerobic and aerobic fixed-film reactors in tandem were used for the treatment of poultry processing wastewater (Del Pozo and Diez, 2003). COD removals of 92% were observed with system OLRs of 0.39 kg COD/m3 day and 95% total Kjeldahl nitrogen (TKN) removals for applied N loads of 0.064 kg TKN/m3 day. The authors reported the effects on nutrient and organic removals at varying recycle rates between the two reactors and varying reactor size. Pretreatment is also regarded as necessary for poultry waste to reduce the moisture and increase the porosity with the addition of bulking agents, which also increase the aeration and carbon level in wastewater. Proper treatment is needed to eliminate the pathogens.

F. Seafood by-products One of the most important environmental problems which are characteristic of coastline areas is the large volume of waste generated by fishing, aquaculture, or foodstuff processing industries. Usually, these by-products are dumped into the sea without a previous treatment of depuration neither evidently nor by valorization. Among these food products, the cooked cephalopod (particularly octopus) has higher commercial value and larger production of wastewater, particularly in the NW Spain (Vazquez and Murado, 2008). These massive spills with high protein concentration generate a negative environmental impact on the marine ecosystems. Over the past two decades, the shellfish industry has also experienced a significant expansion, making crustacean wastes materials available concentrated in some areas and in larger quantities. The most commercially harvested crustacean species are crab, shrimp, prawn, Antarctic krill, and cray fish. In the year 2005–2006, 145,180 Gg of frozen shrimps were produced, and it can be estimated that nearly 150,000– 175,000 Gg of shrimp waste per annum to be generated from shrimp processing companies in India (Babu et al., 2008). Use of these crustaceous wastes has been of interest to researchers for two reasons: (1) these wastes are highly perishable and create environmental pollution (Tan and Lee, 2002). (2) They are the rich sources of protein, chitin, and carotenoids. These large quantities of waste materials are useful in production of chitin, which is the second most abundant natural polymer on Earth (Table 3.4). Tuna including yellowfin, skipjack, bluefin, albacore, and bigeye is one of the worldwide favorite fish species (Aewsiri et al., 2008). The total catch of tuna in the world has increased continuously from 0.4 to 3.9

Processing of Food Wastes

TABLE 3.4 2008)

69

Proximate composition of shrimp shell and crab (Santhosh and Mathew,

Parameter

Shrimp shell (%)

Crab (%)

Moisture Ash (dry basis) Protein (dry basis) Chitin (dry basis) Fat (dry basis)

75–80 30–35 35–40 15–20 3–5

70 45–50 30–35 13–15 1.0–1.5

million metric ton from 1950 to 2000 (Miyake et al., 2004). In Thailand, tuna is usually processed as canned products, which are exported to many countries over the world. During the processing, a large amount of wastes involving skin, bone, and fin is generated (Shahidi, 1994). These wastes are commonly utilized as low value fish meal or fertilizer. So far, the utilization of fish processing wastes has been paid increasing attention as the promising means to increase revenue for producer and to decrease the cost for disposal or management of those wastes. Fishery wastes can be used for enzyme recovery (Klomklao et al., 2005), protein hydrolyzate production (Slizyte et al., 2005), collagen extraction (Fernandez-Dıaz et al., 2001; Muyonga et al., 2004a), and gelatin extraction (Choi and Regenstein, 2000; Muyonga et al., 2004b).

III. RECOVERING OF ADDED-VALUE PRODUCTS FROM FVW (UPGRADING CONCEPT) A. Challenge for the vegetable industry Considering the vegetable industry, the green production goals could be fulfilled by the usual approaches such as minimization, disposal, feeding, fertilization/composting, closed loop production, or conversion (Laufenberg, 2003). The upgrading concept tries to add value to the byproducts and residues. This medium-term goal results in the creation of innovative and industrially important metabolites and products like: Flavors produced by bioconversion of waste material or solid-state

fermentation (SSF)

Dietary fibers as matrices for flavors, dyes, or antioxidants Pectin and gelling agents with defined properties using synergetic

effects

Designer dietary fibers for application in bread or beverages

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Effective and low-cost bioadsorbents, which can be easily desorbed or

biodegraded after use

Hybrid processes combining adsorption and membrane processes for

an advanced wastewater treatment and internal process water recycle Thus the introduced concept is a further step toward environmentally gentle manufacturing. The concept does not present any immediate patent solutions or recipes, because industrial food production is an interactive process, which needs to fulfill all three conditions: quality, efficiency, and environmental protection as mentioned above. The result is a stepby-step waste reduction with simultaneously rising productivity, not obtained by restrictions but by opportunities. The advantages for industry and environment are as follows:

Closed loop of valuable constituents Preservation of resources Discovery of niche markets Environmental protection, combined with Reduced waste disposal costs

Important factor for the upgrading process is the development of a procedure using technical standard equipment. Goal of the upgrading is a product with desired, reproducible properties designed under economical and ecological conditions.

B. SSF of fruit/vegetable by-products The exploitation of by-products of fruit and vegetable processing as a source of functional compounds and their application in food is a promising field. The highly valuable composition of apple pomace and possible strategies of utilization by SSF have recently been reviewed by Vendruscolo et al. (2008).

1. Apple pomace Apple pomace and its aqueous extract present a great potential for use as substrates in biotechnological processes. It is a heterogeneous mixture consisting of peel, core, seed, calyx, stem, and soft tissue (GrigelmoMiguel and Martın-Belloso, 1999). It has high water content and is mainly composed of insoluble carbohydrates such as cellulose, hemicellulose, and lignin. Simple sugars, such as glucose, fructose, and sucrose, as well as small amounts of minerals, proteins, and vitamins, are part of apple pomace composition (Jin et al., 2002; Zheng and Shetty, 2000a). The pomace is very inexpensive and is abundantly available during the harvesting season, and several microorganisms can use this apple residue as a substrate. Bacteria, yeast, and fungi have been cultivated on apple

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pomace for different purposes. Filamentous fungi, especially basidiomycetes, are the most suitable microorganisms for growing on fruit processing residues. Table 3.5 presents a list of different biotechnological applications of apple pomace, with the respective microorganism and the fermentation process applied (Vendruscolo et al., 2008) with the following abbreviations: SSF—solid-state fermentation; SmF—submerged fermentation. Several studies and patents have been described regarding the employment of this residue for the production of value-added compounds, such as enzymes, SCP, biopolymers, fatty acids, polysaccharides, and organic acids, among others. Biotechnological applications of the apple pomace are interesting not only from the point of view of low-cost substrate, but also in solving problems related to the disposal of the pomace, a pollution source that has been gaining a lot of attention in apple-producing areas. Several operational variables must be considered and optimized to effectively use the apple pomace in bioprocesses; strain type, reactor design, aeration, pH, moisture, and nutrient supplementation are only a few examples of these fundamental process variables that are crucial for the economic viability of using the apple pomace as a substrate for biotechnological applications.

2. Production of enzymes The most important area of apple pomace utilization is the production of enzymes. Polygalacturonases or hydrolytic depolymerases are enzymes involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing, degumming of plant rough fibers, and treatment of pectic wastewaters. Seyis and Aksoz (2005) investigated the use of apple pomace, orange pomace, orange peel, lemon pomace, lemon peel, pear peel, banana peel, melon peel, and hazelnut shell as substrate for xylanase production using Trichoderma harzianum. The maximum enzyme activity was observed when melon peel was used as the substrate for SSF, followed by the apple pomace and hazelnut shell. Villas-Boas et al. (2002) found a novel lignocellulolytic activity of Candida utilis during SSF on apple pomace. Hydrolytic and oxidative enzymes of C. utilis, excreted to the culture medium during solidsubstrate cultivation, were identified, evaluated, and quantified. The soluble lignin fraction of the apple pomace was consumed at very significant levels (76%), when compared to the nonfermented apple pomace. The enzyme produced by C. utilis with the highest activity was a pectinase (23 U/mL). The yeast showed a significant manganese-dependent peroxidase activity (19.1 U/mL) and low cellulase (3.0 U/mL) and xylanase (1.2 U/mL) activities, suggesting that C. utilis has the ability to use lignocellulose as a substrate.

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TABLE 3.5

Bioprocesses using apple pomace as substrate (Vendruscolo et al., 2008)

Application

Microorganism

Process

References

Aspergillus foetidus

SSF

Lignocellulolytic enzymes Pectin methylesterase

Candida utilis

SSF

Aspergillus niger

Pectinases Pectolytic enzymes

Polyporus squamosus A. niger

SSF/ SmF SmF SSF

Polygalacturonase

Lentinus edodes

SSF

Hang and Woodams (1994) Villas-Boas et al. (2002) Joshi et al. (2006) Pericin et al. (1999) Berovic and Ostroversnik (1997) Zheng and Shetty (2000b)

Enzyme production: b-glucosidase

Aroma compound production: Aroma compounds Rhizopus sp. Rhizopus oryzae Aroma compounds Kluyveromyces marxianus Fruity aroma Ceratocystis fimbriata Phenolic compounds Trichoderma viride Trichoderma harzianum Trichoderma pseudokoningii Nutritional enrichment: Animal feed Gongronella butleri Nutritional Candida utilis enrichment Kloeckera sp. Protein enrichment Rhizopus oligosporus

SSF

Christen et al. (2000)

SSF

Medeiros et al. (2000) Bramorski et al. (1998) Zheng and Shetty (2000a)

SSF SSF

SSF SSF SSF

Vendruscolo (2005) Devrajan et al. (2004) Albuquerque et al. (2006) (continued)

Processing of Food Wastes

TABLE 3.5

73

(continued)

Application

Microorganism

Heteropolysaccharide production: Chitosan G. butleri

Process

References

SSF

Streit et al. (2004) Streit et al. (2004) and Vendruscolo (2005) Jin et al. (2002)

Chitosan

G. butleri

SmF

Heteropolysaccharide

Beijerinckia indica Xanthomonas campestris

SmF

Xanthan

SSF

Stredansky and Conti (1999) Shojaosadati and Babaeipour (2002) Ngadi and Correa (1992a) Stredansky et al. (2000)

Other products: Citric acid

A. niger

SSF

Ethanol

S. cerevisiae

SSF

g -Linolenic acid

Thamnidium elegans Mortierella isabelina Cunninghamella elegans

SSF

Recently, Joshi et al. (2006) reported the production of pectin methylesterase by Aspergillus niger using apple pomace as culture medium comparing the SmF and SSF. The pectin methylesterase activity was 2.3 times higher when produced by SSF than by SmF. This study corroborates the fact that SSF is the more adequate process for apple pomace bioconversion. Dry citrus peels are rich in pectin, cellulose, and hemicellulose and may be used as a fermentation substrate. Production of multienzyme preparations containing pectinolytic, cellulolytic, and xylanolytic enzymes by the mesophilic fungi A. niger BTL, Fusarium oxysporum F3, Neurospora crassa DSM 1129, and Penicillium decumbens under SSF on dry orange peels was enhanced by optimization of initial pH of the culture medium and initial moisture level (Mamma et al., 2008). Under optimal conditions A. niger

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BTL was by far the most potent strain in polygalacturonase and pectate lyase, production followed by F. oxysporum F3, N. crassa DSM 1129, and P. decumbens. N. crassa DSM 1129 produced the highest endoglucanase activity and P. decumbens the lowest one. Comparison of xylanase production revealed that A. niger BTL produced the highest activity followed by N. crassa DSM 1129, P. decumbens and F. oxysporum F3. N. crassa DSM 1129 and P. decumbens did not produce any b-xylosidase activity, while A. niger BTL produced approximately 10 times more b-xylosidase than F. oxysporum F3. The highest invertase activity was produced by A. niger BTL while the lowest ones by F. oxysporum F3 and P. decumbens. After SSF of the four fungi, under optimal conditions, the fermented substrate was either directly exposed to autohydrolysis or new material was added, and the in situ produced multienzyme systems were successfully used for the partial degradation of orange peels polysaccharides and the liberation of fermentable sugars. Feruloyl esterase (FAE) and xylanase activities were detected in culture supernatants from Humicola grisea var. thermoidea and Talaromyces stipitatus grown on brewers’ spent grain (BSG) and wheat bran (WB), two agro-industrial by-products. Maximum activities were detected from cultures of H. grisea grown at 150 rpm, with 16.9 and 9.1 U/ml of xylanase activity on BSG and WB, respectively. Maximum FAE activity was 0.47 and 0.33 U/ml on BSG and WB, respectively. Analysis of residual cell wall material after microbial growth shows the preferential solubilization of arabinoxylan and cellulose, two main polysaccharides present in BSG and WB. The production of low-cost cell-wall-deconstructing enzymes on agro-industrial by-products could lead to the production of low-cost enzymes for use in the valorization of food-processing wastes (Mandalari et al., 2008).

3. Production of aroma compounds The European Community guidelines 88/388/ EWG and 9/71/EWG subdivide flavors/aromas into six categories, the first of which describes regulations for the food labeling as ‘‘natural flavor.’’ Natural flavors are chemical substances with aroma properties that are produced from feedstock of plant or animal origin by means of physical, enzymatic, or microbiological processing. The microbial synthesis of these natural flavors is generally carried out by SmF. Due to the high costs of this currently used technology on an industrial scale, there is a need for developing low-cost processes even for cheaper molecules like benzaldehyde. This could be achieved by exploration of the metabolic pathways and by alternative technology such as SSF (Couto, 2008). The use of biotechnology for the production of natural aroma compounds by fermentation or bioconversion using microorganisms is an economic alternative to the difficult and expensive extraction from raw materials such as

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plants (Daigle et al., 1999). Currently, it is estimated that around 100 different aroma compounds are produced commercially by fermentation (Medeiros et al., 2006). Furthermore, the world market of aroma chemicals, fragrances, and flavors has a growth rate of 4–5% per year. Because of a higher consumer acceptance there is an increasing economic interest in natural flavors (Table 3.6). Bramorski et al. (1998) analyzed the production of aroma compounds by Ceratocystis fimbriata under seven different medium compositions (prepared by mixing cassava bagasse, apple pomace, amaranth, and soya bean). The aroma production was growth dependent, and the maximum aroma intensity was detected in a few hours around the maximum respirometric activity. The medium containing apple pomace produced a strong fruity aroma after 21 h of cultivation. This same medium was used by Christen et al. (2000) for the production of volatile compounds by Rhizopus strains. Authors found that the production of volatile compounds was related mainly to the medium used, and no difference was observed among the strains studied. The odors detected have a slight alcoholic note, and the apple pomace produced intermediate results, compared with the amaranth grain supplied with mineral salt solution. Another source of aroma compounds is grape pomace, the main byproduct of wine production, which consists of skins, seeds, and stalks, reaching an estimated amount of 13% by weight of processed grape (Torres et al., 2002). The chemical composition of grape pomace is rather complex: alcohols, acids, aldehydes, esters, pectins, polyphenols, mineral substances, sugars, and so on are the most represented classes of compounds (Mantell et al., 2003; Murthy et al., 2002). The evaluation of the qualitative aspects of a grape pomace is carried out in view of the production of high-quality grappa; otherwise the grape pomace is used for alcohol distillation, or thrown away. The best grape pomaces are highly rich in vinous liquid with a moisture degree ranging from 55% to 70%, which allows to exploit the raw material better and to extract the organoleptic characteristics of the native vine. The volatile components of a grape pomace were recent studied and reported by Ruberto et al. (2008). Percentages of compounds were determined from their peak areas in the GC–FID profiles, using gas chromatograph with a flame ionization detector and gas chromatography–mass spectrometry.

4. Production of ethanol A solid-state fermentation process for the production of ethanol from apple pomace by Saccharomyces cerevisiae was described by Khosravi and Shojaosadati (2003). A moisture content of 75% (wt/wt), an initial sugar concentration of 26% (wt/wt), and a nitrogen content of 1% (wt/wt) were the conditions used to obtain 2.5% (wt/wt) ethanol without saccharification and 8% (wt/wt) with saccharification. The results indicate that

TABLE 3.6 Flavors and biofine chemicals produced by SSF of vegetable residues (selection from Laufenberg, 2003) Year

Residual matter

Description/conversion principle

Product

2000

Spent malt grains, apple pomace (Stredansky et al., 2000)

2000

Cassava bagasse, apple pomace (Christen et al., 2000) Cassava bagasse, wheat bran and sugarcane bagasse (Bramorski et al., 1998) Citrus, apple, sugar beet pomace (Grohmann and Bothast, 1994) Cranberry pomace (fish offal) (Zheng and Shetty, 1998)

T. elegans CCF 1456 degraded the substrate in a ratio of 3 to 1 (AP to SMG), precursor peanut oil even increased the yield Four strains of Rhizopus, two residues and two precursors, mixed substrate combinations C. fimbriata, ability to generate fruity aromas in dependence on the substrate used

g-Linolenic acid was produced in a yield of 5.17 g per kg dry substrate; with peanut oil precursor 8.75 g/kg DM Volatile carbons as flavors; acetaldehyde, ethanol, propanol, esters Banana flavor and fruity complex flavors

Microbial conversion by enzymatic hydrolysis

Pectin, substrate, liquid biofuel

Trichoderma viride, Rhizopus CaCO3 was added as neutralizer, water for aw adjustment

Polymeric dye decolorizing isolate for wastewater treatment, extracellular enzymes

1997

1994

1998

2001

Linseed cake, castor oil cake, olive press cake, sunflower cake (Laufenberg et al., 2001)

1998

Olive cake, sugarcane bagasse (Cordova et al., 1998) Olive pomace (Haddadin et al., 1999)

1999

1995 1997 1997 1994

Pineapple waste (Tran and Mitchell, 1995) Potato waste (Lucas et al., 1997) Sugar beet pulp, cereal bran (Asther et al., 1997) Tomato pomace (Carvalheiro et al., 1994)

Moniliella suaveolens, Trichoderma harzianum, Pityrosporum ovale, and Ceratocytis moniliformis form decalactones (problems with phenolic components) Lipase degrading fat in olive cake

Four microorganisms, delignification, saccharification with Trichoderma sp., biomass formation with Candida utilis and Saccharomyces cerevisiae A. foetidus produces citric acid 16.1 g/ 100 g DM and 3% methanol Amylases Commensalism of two microorganisms degrading the substrate Co-cultures of Trichoderma reesei and Sporotrichum sp. are degrading cellulose and hemicellulose fraction

Acceptable yields on olive press cake and castor oil cake, d- and c-decalactone are produced

Enzyme product applied in bakery goods, confectionery, pharmaceuticals Crude protein enriched from 5.9% to 40.3%. Source for animal fodder Pharmaceuticals, food industry, preserving agent Bakery goods, breweries, textile industry Flavor vanillin 67% less cellulose, 73% less hemicellulose, enhanced lignin and protein content

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the alcohol fermentation from apple pomace is an efficient method to reduce waste disposal, with the concomitant production of ethanol. Nogueira et al. (2005) evaluated the alcoholic fermentation of the aqueous extract of apple pomace. Apple juice, pomace extract, and pomace extract added with sucrose provided after fermentation 6.90%, 4.30%, and 7.30% ethanol, respectively. A fermentation yield of 60% was obtained when pomace extract was used, showing that it is a suitable substrate for alcohol production. For bioconversion of bean curd refuse, a processing by-product of bean curd, ethanol-producing anaerobic thermophiles were newly isolated. Both of them degraded hemicellulose, but not cellulose at all. Phylogenetically, strains belong to the Clostridium and Thermoanaerobacterium genus. Aerobic thermophiles degrading cellulose were also newly isolated. This strain belongs to the Geobacillus genus phylogenetically. The co-culture also significantly reduced CH3SH production, leading to the prevention of offensive odor (Miyazaki et al., 2008).

5. Production of organic acids Shojaosadati and Babaeipour (2002) used apple pomace as substrate for the production of citric acid using A. niger via SSF in column reactors. They evaluated several cultivation parameters, such as aeration rate (0.8, 1.4, and 2.0 L/min), bed height (4, 7, and 10 cm), particle size (0.6–1.18, 1.18–1.70, and 1.70–2.36 mm), and moisture content (70%, 74%, and 78%). For citric acid yield, the aeration rate and particle size were the most important parameters. Neither the bed height nor the moisture content was found to significantly affect citric acid production. The operating conditions that maximized citric acid production consisted of low aeration rate (0.8 L/min), high bed height (10 cm), large particle size (1.70–2.36 mm), and elevated moisture content (78%). Apple pomace has also been used for fatty acid production. Stredansky et al. (2000) evaluated the g-linolenic acid (GLA) production in Thamnidium elegans by SSF. Apple pomace and spent malt grain were used as the major substrate components for the production of high-value fungal oil containing up to 11.43% biologically active GLA. Apple pomace is a potential substrate for lactic acid production. Lactic acid has a number of applications in food technology (as acidulant, flavor, and preservative), pharmaceuticals and chemicals (Hofvendalh and Hahn-Hagerdal, 2000). The world market for lactic acid is growing every year, and its current production is about 150 million lb per year. The worldwide market growth is expected to be between 10% and 15% per year (Wassewar, 2005). When samples of apple pomace were subjected to enzymatic hydrolysis, the glucose and fructose present in the raw material as free monosaccharides were extracted at the beginning of the process. Using low cellulase and cellobiase charges (8.5 FPU/g-solid

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and 8.5 IU/ g-solid, respectively), 79% of total glucan was saccharified after 12 h, leading to solutions containing up to 43.8 g monosaccharides/L (glucose, 22.8 g/L; fructose, 14.8 g/L; xylose þ mannose þ galactose, 2.5 g/L; arabinose þ rhamnose, 2.8 g/L). These results correspond to a monosaccharide/cellulase ratio of 0.06 g/FPU and to a volumetric productivity of 3.65 g of monosaccharides/L h. Liquors obtained under these conditions were used for fermentative lactic acid production with Lactobacillus rhamnosus CECT-288, leading to media containing up to 32.5 g/L of L-lactic acid after 6 h (volumetric productivity ¼ 5.41 g/L h, product yield ¼ 0.88 g/g) (Gullon et al., 2008). Apple pomace shows several advantages as a raw material for lactic acid manufacture, including: (i) high content of free glucose and fructose, which are excellent carbon sources for lactic acid production (Hofvendalh and Hahn-Hagerdal, 2000); (ii) high content of polysaccharides (cellulose, starch, and hemicelluloses) which can be enzymatically hydrolyzed to give monosaccharides; (iii) presence of other compounds (e.g., monosaccharides other than glucose and fructose, di- and oligo-saccharides, citric acid, and malic acid) which can be metabolized by lactic bacteria (Carr et al., 2002); and (iv) presence of metal ions (Mg, Mn, Fe, etc.) which could limit the cost of nutrient supplementation for fermentation media.

6. Production of polysaccharides

Jin et al. (2002, 2006) examined the potential of three agro-industrial byproducts to be used as substrate for the production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica in SmF under the same cultivation conditions. By-products from apple juice production, for example, soy sauce production, and the manufacturing processes of Sikhye (fermented rice punch), for example, a traditional Korean food, were tested. The apple pomace was found to be the best carbon source for PS-7 production compared to the other by-products, giving a production of 4.09 g/L after 48 h of cultivation. When Sikhye by-product was used as substrate, 3.00 g/L of PS-7 was formed, and using the soy sauce residue, 0.96 g/L of PS-7 was observed. Xanthan gum is the most important microbial polysaccharide from the commercial point of view, with a worldwide production of about 30,000 ton/a. It has widespread commercial applications as a viscosity enhancer and stabilizer in the food, pharmaceutical and petrochemical industries (Papagianni et al., 2001). The rheological behavior of the fermentation broth causes serious problems of mixing, heat transfer, and oxygen supply, thus limiting the maximum gum concentration achievable as well as the product quality (Wecker and Onken, 1991). Several strategies have been developed to overcome these problems including use of two-level impellers. The use of cheap substrates, instead of the commonly used glucose or sucrose, might result in a lower cost of the final product.

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Stredansky and Conti (1999) proposed the use of SSF as an alternative strategy for the production of xanthan by Xanthomonas campestris, since solid substrates reproduce the natural habitat of this phytopathogenic bacterium. This technique allows overcoming problems connected with broth viscosity and, in addition, utilizes cheap substrates. Streit et al. (2004) studied the production of fungal chitosan in SmF and SSF (column reactors) using the watery extract of apple pomace and the pressed apple pomace as substrate, respectively. Among the microorganisms studied, the fungus Gongronella butleri yielded the best results for the production of chitosan in SmF and SSF. Grown on the watery extract of apple pomace, the G. butleri presented the highest productivity (0.091 g/L h) and chitosan content in the biomass (0.1783 g/g of apple pomace) for a medium supplemented with 40 g/L of reducing sugars and 2.5 g/L of sodium nitrate. Vendruscolo (2005) used an external loop airlift bioreactor for chitosan production by G. butleri CCT 4274 on the watery extract of apple pomace. The experiments using higher levels of aeration (0.6 volume of air per volume of liquid per minute) provided greater concentrations of biomass, attaining 8.06 and 9.61 g/L, in the production of 873 and 1062 mg/L of chitosan, respectively. These findings demonstrated the adequacy of the airlift bioreactor for the cultivation of microorganisms with emphasis on the production of chitosan.

7. Production of baker’s yeast Bhushan and Joshi (2006) used apple pomace extract as a carbon source in an aerobic-fed batch culture for the production of baker’s yeast. The fermentable sugar concentration in the bioreactor was regulated at 1–2%, and a biomass yield of 0.48 g/g of sugar was obtained. Interestingly, the dough-raising capacity of the baker’s yeast grown on the apple pomace extract was apparently the same as that of commercial yeast. The use of apple pomace extract as substrate is a useful alternative to molasses, traditionally used as a carbon source for baker’s yeast production.

8. Production of pigments Attri and Joshi (2005) used an apple pomace-based medium to examine the effect of carbon and nitrogen sources on carotenoid production by Micrococcus sp. Using 20 g/L of apple pomace in the basic medium provided the best growth conditions for the microorganism. Maximum biomass (4.13 g/L) and pigment (9.97 mg per 100 g of medium) yields were achieved when the medium was supplemented with 0.2% fructose. Optimal conditions for carotenoid production were 35 C, pH 6.0, and cultivation time of 96 h. The same authors (Attri and Joshi, 2006) studied carotenoid production by Chromobacter sp. Using the same basic medium (20 g/L of apple pomace), they found a high production of biomass

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(6.6 g/L) and carotenoids (46.6 mg per 100 g of medium) and with a shorter incubation period (48 h). These differences showed that the production of carotenoids can be improved by an accurate choice of organism. Skin, rich in lycopene, is an important component of waste originating from tomato paste manufacturing plants (Kaur, et al., 2008). Lycopene is the principal carotenoid, causing the characteristic red hue of tomatoes (Shi and Le Maguer, 2000). Several epidemiological studies reported that lycopene-rich diets have beneficial effects on human health (Arab and Steck, 2000; Sharoni et al., 2000). A possible role has been suggested for tomatoes and tomato products in preventing cardiovascular disease and protecting against some types of cancer (based on lycopene content) (Willcox et al., 2003). Maximum lycopene (1.98 mg/100 g) was extracted when the solvent/meal ratio adjusted to 30:1 v/w, number of extractions— 4, temperature 50 C, particle size—0.15 mm, and extraction time—8 min (Kaur et al., 2008).

9. Feed protein

Song et al. (2005a) developed a method for producing SCP from apple pomace by dual SSF. This method comprises four different steps: (1) preparation of an SSF medium with pulverized apple pomace, (2) inoculating cultured mixed mature strain for SSF, (3) rapidly drying the resulting fermentation product at low temperature, and (4) subjecting the dried product to solid fermentation in another SSF medium to obtain the final product (patent no. CN 1673343). The same group of researchers (Song et al., 2005b) developed another method for producing feed protein by liquid–solid fermentation of apple pomace (patent no. CN 1663421). As compared with solid fermentation, this method has the advantage of reduced consumption of medium for seed culture, reduced cost, and applicability to large-scale production. The nutritional quality of a fibrous by-product or residue from a food manufacturing process was improved by inoculating it with filamentous fungus, and fermenting the fibrous product or residues was proposed by Power and Power (2005).

10. Antibiotics Antibiotics are required in large quantity for the persistent fight against bacterial diseases; and so research has concentrated on producing them at the highest concentration with minimal energy input. Yang (1996) reported the production of oxytetracycline by Strepfomyces rimosus in SSF using corncob, a cellulosic waste, as a substrate. This author found that the oxytetracycline produced by SSF was more stable than that produced by SmF and the energy input was also less (Yang and Ling, 1989). In addition, the product presented the advantage that it could be temporarily stored without losing activity significantly. Adinarayana et al.

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(2003a) tested several substrates (wheat bran, wheat rawa, bombay rawa, barley, and rice bran) to produce cephalosporin C by Acremonium chrysogenum under SSF. Physical and chemical parameters were also optimized. Thus, a maximum productivity of cephalosporin C (22,281 mg/g) was achieved using wheat rawa and soluble starch (1%) and yeast extract (1%) as additives, an incubation period of 5 days, an incubation temperature of 30 C, an inoculum level of 10%, a ratio of salt solution to weight of wheat bran of 1.5:10, a moisture content of solid substrate of 80% and pH 6.5. Adinarayana et al. (2003b) also reported the production of neomycin by Streptomyces marinensis under SSF using wheat rawa as a supportsubstrate. Ellaiah et al. (2004) tested several support-substrates (wheat bran, wheat rawa, rice bran, rice rawa, rice husk, rice straw, maize bran, ragi bran, green gram bran, black gram bran, red gram bran, corn flour, jowar flour, sago, and sugar cane bagasse) for neomycin production by a mutant strain of S. marinensis, under SSF. The accumulation of neomycin by SSF was 1.85 times higher than the SmF. Asagbra et al. (2005) assessed the ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273, S. aureofaciens NRRL B2183, and S. vendagensis ATCC 25507 to produce tetracycline under SSF conditions using peanut shells, corn cob, corn pomace, and cassava peels as substrates. They found that peanut shells were the most effective substrate. Mizumoto et al. (2006) reported the production of the lipopeptide antibiotic iturin A by Bacillus subtilis using soya bean curd residue, okara, a by-product of tofu manufacture in SSF. After 4 days of incubation, iturin A production reached 3300 mg/kg wet solid material (14 g/kg dry solid content material), which was approximately 10-fold higher than that in SmF.

IV. MULTIFUNCTIONAL FOOD INGREDIENT PRODUCTION FROM FVW Several research groups have been working on the development of multifunctional ingredients from vegetable residues and its application in different food products. The crude fiber content combined with at least one other property enables them to fulfill several functions in food as depicted by Laufenberg et al. (2003). Operating areas of multifunctional food ingredients due to food properties and quality: 1. Nutritional and healthy quality, for example, vitamin content, dietary fiber content. 2. Food product structure, for example, porosity, network structure. 3. Sensorial properties, for example, texture/structure, mouth feel, freshness.

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4. Physical properties, for example, density, viscosity. 5. Processing properties, for example, water binding ability, emulsifying properties. A couple of quality determining food properties can be governed by the application of these food ingredients. The raw material mostly used is carrot pomace (Filipini and Hogg, 1997; Lucas et al., 1997), followed by citrus waste (Sreenath et al., 1995; Widmer and Montanari, 1995), grape or apple pomace (Masoodi and Chauhan, 1998), sugar beet pomace (Koksel and Ozboy, 1999), orange, mango, and apple peel (Larrauri et al., 1999). New approaches try to use the dietary fibers as a matrix for the encapsulation of antioxidants (Saura-Calixto, 1998) or flavors (Zeller, 1999), using both the physiological effects and the technological advantages in the form of a controlled release.

A. Dietary fibers A number of researchers have used fruits and vegetable by-products such as apple, pear, orange, peach, blackcurrant, cherry, artichoke, asparagus, onion, carrot pomace (Nawirska and Kwasnievska, 2005; Ng et al., 1999) as sources of dietary fiber supplements in refined food. Dietary fiber concentrates from vegetables showed a high total dietary fiber content and better insoluble/soluble dietary fiber ratios than cereal brans (Grigelmo-Miguel and Martin-Belloso, 1999). The preparation of dietary fibers from food by-products was summarized by Larrauri et al. (1999). Cauliflower has a very high waste index (Kulkarni et al., 2001) and is an excellent source of protein (16.1%), cellulose (16%), and hemicellulose (8%) (Wadhwa et al., 2006). It is considered as a rich source of dietary fiber and it possess both antioxidant and anticarcinogenic properties. Phenolic compounds and vitamin C are the major antioxidants of brassica vegetables, due to their high content and high antioxidant activity (Podsedek, 2007). Lipid-soluble antioxidants (carotenoids and vitamin E) are responsible for up to 20% of the brassica total antioxidant activity. The level of nonstarch polysaccharide (NPS) in the upper cauliflower stem is similar to that of the floret and both are rich in pectic polysaccharides, while the cauliflower lower stem is rich in NPS due mainly to cellulose and xylan deposition (Femenia et al., 1998). Stojceska et al. (2008) studied the incorporation of cauliflower trimmings into ready-to-eat expanded products (snacks) and their effect on the textural and functional properties of extrudates. It was found that addition of cauliflower significantly increased the dietary fiber and levels of proteins. Extrusion cooking significantly (P < 0.0001) increased the level of phenolic compounds and antioxidants but significantly (P < 0.001) decreased protein in vitro digestibility and fiber content in

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the extruded products. The expansion indices, total cell area of the products, wall thickness showed negative correlation to the level of cauliflower. Sensory test panel indicated that cauliflower could be incorporated into ready-to-eat expanded products up to the level of 10%. Content and composition of dietary fibers of some residues have been summarized and listed by Laufenberg (2003). The high crude fiber content of the vegetable pomace (in total 20–65% DM) suggests its utilization as a crude fiber ‘‘bread improver.’’ In bread and bakery goods, as well as in pastry, cereals, and dairy products, the investigated carrot pomace works as a stabilizer. Besides crude fiber it is rich in provitamins, color, and natural acids. It represents several functional properties as above mentioned, additionally substitutes sourdough in bread, carrot pomace is acidifying agent, preservative, or antioxidant in several food products (Filipini and Hogg, 1997; Masoodi and Chauhan, 1998).

B. Coloring agents and antioxidants In beverages, carrot pomace, or citrus waste will stabilize the natural color, improve the vitamin and fiber content, enhance the viscosity (mouthfeel) (Laufenberg et al., 1996), and enrich or adjust the cloudy appearance (Sreenath et al., 1995). The organoleptic and chemical properties offer a widespread use in healthy and functional drinks and selected fruit juices. Olive oil wastewaters are rich in antioxidant compounds, particularly in hydroxytyrosol derivatives (Visioli et al., 1999). Hydroxytyrosol strongly inhibited low-density lipoprotein oxidation stimulated by 2,20 azobis(2-aminopropane) hydrochloride (Aruoma et al., 1998). Further investigations point out that hydroxytyrosol and oleuropein are potent scavengers of superoxide radicals (Visioli et al, 1998). Tyrosol and hydroxytyrosol are dose-dependently absorbed by humans and eliminated as their glucuronide conjugates, indicating a good bioavailability (Visioli et al., 2000).

1. Polyphenolic compounds Saura-Calixto (1998) produced a dietary fiber rich in associated polyphenolic compounds combining in a single material the physiological effects of both dietary fiber and antioxidants. Fiber matrices could act as support for biocolorants made of anthocyanins from olive cake (Clemente et al., 1997), lycopenes from tomato skins (Al-Wandawi et al., 1985) or b-carotene from carrot pomace. Phenolic compounds are powerful antioxidants and may possess potential pharmacological properties, already widely used with green tea catechins (Nwuha et al., 1999) or ferulic acid extracted from sugar beet pulp (Couteau and Mathaly, 1998) which could make them desirable ingredients in the developing market of ‘‘functional foods’’ for

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health. Bioflavonoids like hesperidin, naringin, or rutin are able to normalize capillary permeability and vascular brittleness, therefore they are frequently called vitamin P factors. Hesperidin is applied in vein medication, acts antiviral in flue therapy, and owns artificial sweetener properties; hydrated naringin is 300 times sweeter than saccharose, neohesperidin almost 2000 times. Grape skin extract in powder form is commercially available as a natural food coloring agent. Besides the blue– red color the food will be enriched with ‘‘healthy’’ polyphenols (Anon, 1999). The fermentation of dietary fibers increases digestibility, shelf life and preserves the bioactivity of the components. The food and agricultural products processing industries generate substantial quantities of phenolic-rich by-products, which could be valuable natural sources of antioxidants to be employed as ingredients. For example, more than 450,000 ton of onion wastes is produced annually in the European Union, mainly in the United Kingdom, Holland and Spain (Roldan et al., 2008). Some of these by-products have been the subject of investigations and have proved to be effective sources of phenolic antioxidants (Balasundram et al., 2006). Onion shows a variety of pharmacological effects such as growth inhibition of tumor and microbial cells, reduction of cancer risk, scavenging of free radicals, and protection against cardiovascular disease, which are attributed to specific sulfurcontaining compounds and flavonoids (Ly et al., 2005). In addition, onions have been found to have antioxidant properties in different in vitro models (Kim and Kim, 2006). Recent studies of Roldan et al. (2008) have shown that sulfhydryl (SH or thiol) groups are good inhibitors of the enzyme polyphenol oxidase (PPO) (Ding et al., 2002). Therefore, it is assumed that the thiol compounds contained in onion might be the active components responsible for the PPO inhibitory effect of onion. Onion extracts could be used as natural food ingredients for the prevention of browning caused by PPO (Kim et al., 2005). Onion wastes have been stabilized by thermal treatments—freezing, pasteurization, and sterilization—to evaluate the effect of the processing and stabilization treatment on the bioactive composition, antioxidant activity and PPO enzyme inhibition capacity. Processing of ‘‘Recas’’ onion wastes to obtain a paste (mixture content) and applying a mild pasteurization were the best alternatives to obtain an interesting stabilized onion by-product with good antioxidant properties that made useful its use as functional food ingredient. Grapes are among the world’s largest fruit crop with more than 60 million ton produced annually. About 80% of the total crop is used in wine making (Mazza and Miniati, 1993), and pomace represents approximately 20% of the weight of grapes processed. From these data, it can be calculated that grape pomace amounts to more than 9 million ton per year. A great range of products such as ethanol, tartrates, citric acid, grape seed oil, hydrocolloids, and dietary fiber are recovered from grape

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pomace (Girdhar and Satyanarayana, 2000). Anthocyanins, catechins, flavonol glycosides, phenolic acids and alcohols and stilbenes are the principal phenolic constituents of grape pomace. Anthocyanins have been considered the most valuable components, and methods for their extraction have been reported (Mazza, 1995). In Chardonnay grape pomace, 17 polyphenolic constituents were identified by NMR spectroscopy (Lu & Foo, 1999). Chardonnay pomace was also a source of two unusual dimeric flavanols (Foo et al., 1998). Catechin, epicatechin, epicatechin gallate, and epigallocatechin were the major constitutive units of grape skin tannins (Souquet et al., 1996). A new class of compounds, aminoethylthio- flavan-3-ol conjugates, has been obtained from grape pomace by thiolysis of polymeric proanthocyanidins in the presence of cysteamine (Torres and Bobet, 2001). Grape seeds are rich sources of polyphenolics, especially of procyanidins, which have been shown to act as strong antioxidants and exert health-promoting effects (Jayaprakasha et al., 2001). Addition of supplementary quantities of grape seeds to grape juice increased catechin and procyanidin contents of wines (Kovac et al., 1995).

2. Coloring agents More than 200,000 ton of red beet are produced in Western Europe annually, most of which (90%) is consumed as vegetable. The remainder is processed into juice, coloring foodstuff and food colorant, the latter commonly known as beetroot red (Henry, 1996). Though still rich in betalains, the pomace from the juice industry accounting for 15–30% of the raw material (Schieber et al., 2001) is disposed as feed or manure. The colored portion of the beetroot ranges from 0.4 to 2.0% of the dry matter, depending on intraspecific variability, edaphic factors, and postharvest treatments. Beets are ranked among the 10 most potent vegetables with respect to antioxidant capacity ascribed to a total phenolic content of 50– 60 mmol/g dry weight (Vinson et al., 1998). A more recent investigation showed that total phenolics decreased in the order peel (50%), crown (37%), and flesh (13%). Epidermal and subepidermal tissues, that is, the peel, also carried the main portion of betalains with up to 54%, being lower in crown (32%) and flesh (14%). Whereas the colored fraction consisted of betacyanins and betaxanthins, the phenolic portion of the peel showed ltryptophane, p-coumaric and ferulic acids, as well as cyclodopa glucoside derivatives (Kujala et al., 2001). Toxicological studies revealed that betanin, the major compound from red beet, did not exert allergic potential, nor mutagenic or hepatocarcinogenic effects. High content in folic acid amounting to 15.8 mg/g dry matter is another nutritional feature of beets (Wang and Goldman, 1997).

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C. Gelation properties Pectic substances have an important influence on food texture and are used in products like jams, jellies, dairy products, beverages, pastries, and confectioneries. More and more they are used in pharmaceutics and cosmetics as well. Pectin is located in the cell walls of vegetables and fruits; the use of residual matter as a potential pectin source is commercially and environmentally interesting. Pectin is heterogeneous complex polysaccharide. All pectin molecules contain linear segments of (1 ! 4)linked a-D-galacto-pyranosyluronic acid units with some of the carboxyl groups esterified with methanol. The gelation mechanism of pectins is mainly governed by their degree of esterification (DE). Commonly, two types of pectin gels are distinguished. The first type made from high methoxyl pectins (DE beyond 50%) form gels in an acidic environment and in the presence of sucrose. The second type of pectin gel is composed of low methoxyl pectins (DE below 50%). These pectins form gels in presence of a divalent metal ions, for example, calcium ions. In both cases, gelation and gel properties depend on many factors, including pH, temperature, DE, sugar, Calcium ions, and pectin content. Apple pomace is a natural source of pectic substances, being an important raw material for pectin production throughout the world, it contains 10–15% of pectin on a dry matter basis (Vendruscolo et al., 2008). In apple pomace, the pectin is mainly present as protopectin, an acid-soluble polysaccharide. Canteri-Schemin et al. (2005) studied the effects of particle size, apple variety, and type of acid on the extraction of pectin from apple pomace. The authors found that higher extraction yields (around 14%) were obtained when pomace particles larger than 106 mm and smaller than 250 mm were used. Marcon et al. (2005), using an experimental design, found that the best yield of pectin extraction from apple pomace (16.8% wt/wt) was obtained with higher temperatures (100 C, 80 min). Wang et al. (2007) studied the applicability of microwave-assisted extraction to obtain pectin from apple pomace. They studied the effect of four different factors (extraction time, pH of acid solution, solid:liquid ratio, and microwave power) on the pectin yield. An extraction time of 20.8 min, pH 1.01, solid:liquid ratio of 0.069, and a microwave power of 499.4 W produced the highest extraction yield (0.315 g per 2 g of dried apple pomace). According to the authors, these process conditions allowed an important reduction in the time required for pectin extraction. The presence of up to 30% pectin in a dry residual matter basis like sugar beet pulp, carrot pomace, potato pulp, or lemon peel and its availability in large quantities have made extraction worthwhile.

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D. Oil and meal Goldenberry pomace (seeds and skins) represents the waste obtained during juice processing (around 27.4% of fruit weight). In the present contribution, the potential of goldenberry pomace for use as a substrate for the production of edible oil was evaluated (Ramadan et al., 2008). Toward developing goldenberry as a commercial crop, the results provide important data for the industrial application of goldenberry. Three extraction methods were checked for the best oil yield. The n-hexaneextractable oil (expressed as SE) content of the raw by-products was estimated to be 19.3%. Enzymatic treatment with pectinases and cellulases followed by centrifugation in aqueous system (expressed as EAE enzyme–aqueous–extract) or followed by solvent extraction (expressed as ESE enzyme–solvent–extract) was also investigated for recovery of oil from pomace fruit. Enzymatic hydrolysis of pomace followed by extraction with n-hexane reduced the extraction time and enhanced oil extractability up to a maximum of around 7.60%. Moreover, enzymation followed by solvent extraction increased the levels of protein, carbohydrates, fiber, and ash in the remaining meal. The study covers also the chemical composition and some fractional properties of different pomace extracts (EAE, SE, and ESE). Concerning the oil composition, there were relatively no changes noted in the fatty acid pattern of the oils extracted with different techniques. As a first step toward developing goldenberry as a commercial crop, the data obtained will be useful as an indication of the potentially economical utility of goldenberry pomace as a source of edible oil and functional products. Although goldenberry is a part of a supplemental diet in many parts of the world and its consumption is becoming increasingly popular also in the nonproducer countries, information on the phytochemicals in this fruit is limited. Yet these phytochemicals may bring nutraceutical and functional benefits to food systems. A variety of health-promoting products improved from goldenberry pomace may include ground dried skins and extracts obtained from skins and/or seeds. The levels of polar lipids, unsaponifiables, peroxides, and phenolics in different extracts were associated with oxidative stability and radical scavenging activity.

E. Food preservation Evaluation of olive- and grape-based natural extracts as potential preservatives for food was carried out by Serra et al. (2008). The antimicrobial activities of two waste-derived extracts, from olive oil and wine production, both rich in polyphenols, and three standard well recognized antioxidants (quercetin, hydroxytyrosol, and oleuropein) were investigated against five microbial species (Escherichia coli, Salmonella poona, Bacillus

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cereus, S. cerevisiae, and Candida albicans). The tests were carried out using a microplate photometer assay. The results obtained suggest that the natural extracts may have important applications in the future as natural antimicrobial agents for food industry as well as for medical use. The natural extracts showed more antimicrobial activity than shown by the selected antioxidants alone against all microorganisms.

F. Production of biopolymers, films, food packaging Increasing interest in high-quality food products with increased shelf life and reduced environmental impact has encouraged the study and development of edible and/or biodegradable polymer films and coatings. Edible films provide the opportunity to effectively control mass transfer among different components in a food or between the food and its surrounding environment. Materials used to produce edible films can be divided into four categories: biopolymer hydrocolloids, lipids, resins, and composites. Biopolymer hydrocolloids include proteins such as gelatin, keratin, collagen, casein, soy protein, whey protein, myofibrillar proteins, wheat gluten, and corn zein; and polysaccharides such as starch, starch derivatives, cellulose derivatives, and plant gums. Suitable lipids include waxes, acylglycerols, and fatty acids. Resins include shellac and wood rosin. Composites generally contain both lipid and hydrocolloid components in the form of a bilayer or an emulsion (Perez-Gago and Krochta, 2005). Proteins such as wheat gluten, corn zein, soy protein, myofibrillar proteins, and whey proteins have been successfully formed into films using thermoplastic processes such as compression molding and extrusion. Thermoplastic processing can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used. Addition of water, glycerol, sorbitol, sucrose, and other plasticizers allows the proteins to undergo the glass transition and facilitates deformation and processability without thermal degradation. Target film variables, important in predicting biopackage performance under various conditions, include mechanical, thermal, barrier, and microstructural properties. Film applications include their use as wraps, pouches, bags, casings, and sachets to protect foods, reduce waste, and improve package recyclability (Hernandez-Izquierdo and Krochta, 2008).

G. Derivatives of meat wastes Collagen, a by-product of meat production, finds only little use in the production of food for its low nutritional value and deficit in essential amino acids (Langmaier et al., 2008). At present, the principal field of its industrial use is in the production of leather—a natural product widely

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used for clothing, footwear, and fancy goods manufacture. An important field also is the manufacture of edible (biodegradable) meat product casings, which, due to the preserved fibrous structure of collagen, display excellent mechanical characteristics (Osburn, 2002). A minor use in terms of volume, but not in significance, is the application in human medicine (tissue bio-engineering—vascular prostheses, membranes, transport systems for antibiotics, steroids and other drugs, implant matrices, haemostatic foams, burn dressings, etc.). A detailed survey of such recent applications was given by Lee et al. (2001). In packaging technology, films or foils obtained from protein hydrogels have the advantage of a high barrier capacity for oxygen (oxidation of packed materials, especially of lipoid character), carbon dioxide (important in packaging and preserving fruit and vegetables) and aromatic substances (spices and other ingredients of semifinished food products). Their hydrophilic character, on the other hand, limits their barrier capacity for humidity (water vapor), mostly accompanied by their solubility in water. Mechanical strength and elongation are somewhat lower when compared with similar packages based on synthetic polymers, whereas, fragility is higher. These characteristics may be controlled by adding suitable plasticizers, which, however, usually further increase the hydrophilic character of plasticized films. The hydrophilic character of proteinic films and foils can be best controlled by increasing the cross-link density attainable through cross-linking reactions by specific enzymes—peroxidase from horseradish, amino-transferases of bacterial origin—Streptoverticilium sp., Streptomyces sp., and others: (Carvalho de and Grosso, 2004; Henning and van Nostrum, 2002), or aldehydes, most often formaldehyde, glyoxal, or glutaraldehyde (Bigi et al., 2001). A suitable degree of cross-linking enables the control of the rate of dissolving of film (foil) and thus also the rate of releasing active component from such packages. This is of significance for maintaining the required concentration of active substance—for example, drugs in the bloodstream, or farming chemicals (fertilizers, insecticides, pesticides, and others) in soil or in other environments. Enzymatic hydrolyzates of waste collagen proteins (H), from current industrial manufacture (leather, edible meat product casings, etc.) of mean molecular mass 20–30 kDa by a reaction with dialdehyde starch (DAS), produces hydrogels applicable as biodegradable (or even edible) packaging materials for food, cosmetic, and pharmaceutical products. Thermo-reversibility of prepared hydrogels is given by concentrations of H and DAS in a reaction mixture. At concentrations of H 25–30% (w/w) and that of DAS 15–20% (related to weight of hydrolyzate), thermo-reversible hydrogels arise, which can be processed into packaging materials by a technique similar to that of soft gelatin capsules (SGC). Exceeding the limit of 20% DAS leads to hydrogels that are thermo-reversible only in part, a further increase in DAS concentration

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then leads to thermo-irreversible gels whose processing into biodegradable packaging materials necessitates employment of other procedures. Thus, better utilization of collagen raw material is achieved, and ecologically unfavorable impacts of the collagen-processing industry are reduced, namely ammonia pollution of wastewaters and the inevitable disposal of solid, unprocessed collagen waste in landfills (Langmaier et al., 2008).

H. Derivatives of seafood wastes 1. Production of carotenoids Enzymatic isolation of carotenoid–protein complex from shrimp head waste was carried out by Babu et al. (2008). The carotenoids extracted gave maximum yield over the traditional, solvent extraction process, and SC–CO2 extraction. Trypsin recovered highest amount of carotenoids from all types of head wastes, but pepsin and papain also showed good recoveries of carotenoids. The percent of recovery varied with the raw materials and the trend was Penaeus indicus > Penaeus monodon (culture) > Metapenaeus monocerous > Penaeus monodon (wild). The loss of carotenoids during processing of frozen carotenoid–protein cake (CPC) to freezedried product was noticed in all trials. Astaxanthin was the main stable pigment and its proportion in total carotenoids increased in freeze-dried product with the loss of minor carotenoids such as b-carotene and their derivatives. The predominance of astaxanthin in the carotenoids indicates that the both frozen CPC and freeze-dried CPC are good source of natural antioxidant and also natural carotenoids.

2. Production of glucosamine and carboxymethylchitin Shrimp shell waste can be economically converted to chitin, a mucopolysaccharide (Santhosh and Mathew, 2008). This marine polysaccharide and its derivatives hold a major part in our lives as medicines, cosmetics, textiles, paper, food, and other branches of industry because of their unique nature in properties such as low toxicity, biocompatibility, hydrophobicity, etc. Hydrolysis of chitin yields a value added product, glucosamine. Carboxymethylchitin is another derivative of chitin, prepared by the carboxymethylation reaction. Glucosamine is the natural component of glycoproteins found in connective tissues and gastrointestinal mucosal membranes. This monosaccharide is involved in the formation of nails, tendons, skin, eyes, bones, ligaments, and heart valves. It also plays a role in the mucus secretions of the respiratory and urinary tracts. It is incorporated in the biosynthesis of glycosaminoglycans and proteoglycans, which are essential for the extracellular matrix of connective tissues. Several clinical studies have been reported that glucosamine works better in reducing the symptoms of

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osteoarthritis. Glucosamine inhibits the cartilage-destructive enzyme collagenase. Glucosamine helps in the synthesis of cartilage by increasing key components of cartilage such as glycosaminoglycans. Various reports confirmed that diabetes patients could also consume glucosamine, which will not increase blood glucose level. Glucosamine appears to undergo a significant first-pass effect in the liver, which metabolizes a significant proportion of the dose to CO2, water, and urea. Carboxymethylchitin is having profound versatile applications. Carboxymethylchitin is popularly used in cosmetic products as smoothener, moisturizer, cleaner for face skin conditioning, and cell activator. Carboxymethylchitin is extensively used in wound dressing. For wound dressing, this polymer must be cross-linked to prolong its dimensional integrity during use. One important characteristic feature of carboxymethylchitin is that it is soluble not only in acid media but at any pH range. This unique property makes carboxymethylchitin different from other derivatives of chitin. Solubility of carboxymethylchitin at any pH makes it advantageous to use in food products and cosmetics. Carboxymethylchitin is used to preserve fruits also.

3. Production of gelatin The amount of gelatin used in the worldwide food industry is increasing annually (Montero and Gomez-Guillen, 2000). The estimated world usage of gelatin is 200,000 MT/year (Badii & Howell, 2005). Generally, gelatin is commercially made from skins and skeletons of bovine and porcine by alkaline or acidic extraction. However, the occurrences of bovine spongiform encephalopathy and foot/mouth diseases have led to the major concern of human health. Thus by-products of mammalians are limited for production of collagen and gelatin as the functional food, cosmetic, and pharmaceutical products (Cho et al., 2005). Studies on extraction and functional properties of gelatin from fish by-products, such as skin and bone, have been reported (Choi and Regenstein, 2000; Fernandez-Dıaz et al., 2001). Gelatin was extracted from precooked tuna caudal fin with the yield of 1.99% (Aewsiri et al., 2008). Tuna fin gelatin (TFG) contained high protein content (89.54%) with hydroxyproline content of 14.12 mg/g. TFG comprised a lower content of high-molecular-weight cross-links and hydroxyproline content than porcine skin gelatin (PSG). However, proline content in TFG was twofold higher than that of PSG. The highest bloom strength and turbidity of TFG were observed at pH 6, while the lowest solubility was noticeable at the same pH. The bloom strength of TFG gel was lower than that of PSG gel at all pHs. TFG exhibited the lower emulsifying activity but greater emulsifying stability than PSG (P < 0.05). TFG showed poor foaming

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properties than PSG. The tensile strength, elongation at break, water vapor permeability of film from PSG was greater than those of TFG (P < 0.05). The study revealed that gelatin of good quality can be prepared from tuna processing discards.

4. Production of marine peptone A diverse group of peptones obtained by enzymatic hydrolysis of wastewater from the industrial processing of octopus showed their effectiveness to promote the growth of lactic acid bacteria (LAB) and the production of bacteriocins. The highest nisin formation by L. lactis was reached using peptones from papain hydrolysis for 24 h (enzyme concentration: 1.25 mg papain/g protein). On the other hand, the highest pediocin production by Pediococcus acidilactici was obtained with peptones derived from 4 h of pepsin digestion (enzyme concentration: 3.75 mg papain/g protein). Thus, these marine peptones are promising alternatives to currently available and expensive commercial medium as well as a possible solution to valorize this problematic wastewater (Vazquez and Murado, 2008; Vazquez et al., 2004). From the viewpoint of their industrial importance, LAB are classified as one of the greatest and most important microbial groups due to their significant role in food fermentation and preservation, as a natural microflora or as an inoculum added under controlled process conditions. Among the molecules produced by these microorganisms which present antimicrobial activity are lactic and acetic acid, ethanol, diacetyl, 2,3butanediol, and bacteriocins. Bacteriocins produced by LAB are peptides with antimicrobial activity and have great importance to the food industry, as they are innocuous, sensitive to digestive proteases of vertebrates, and do not change the organoleptic properties of the food. LAB and, specifically, bacteriocins productions are very fastidious due to the need for rich growth media containing nutrients such as carbohydrates, nucleic acids, minerals, vitamins and, mainly, amino acids, proteins, or protein hydrolyzates. For example, the standard laboratory media (MRS, TGE, APT) solve the problem of protein sources, incorporating products such as bactopeptone, tryptone, meat extract, or yeast extract (sometimes all of these) on formulations which reach expensive costs. The use of low-cost proteins or protein fractions will bring about a reduction in large-scale production costs. Furthermore, when food waste is used to obtain these nutritional sources (e.g., waste generated by industries which process foodstuffs of marine origin), a complete productive cycle is closed: the recycling and valorization of pollutant waste and the obtaining of a product of high added value, used for control and preservation of foodstuffs (LAB and bacteriocins).

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V. VEGETABLE RESIDUES AS BIOADSORBENTS FOR WASTEWATER TREATMENT Conventional methods for treating wastewater containing dyes, aromatic compounds, or heavy metals are coagulation, flocculation, reverse osmosis, nanofiltration and pervaporation (Paul and Ohlrogge, 1998), and activated carbon adsorption, the latter of which is combined with membrane processes like nanofiltration (Eilers and Melin, 1999) or ultrafiltration (Lenggenhager and Lyndon, 1997). Additional efforts are focused on the creation of ‘‘bioadsorbents’’ with improved functionality, using their natural content of adsorptive components or enhancing their adsorption rate by combination of favored raw materials. A number of low-cost adsorbents have been tried for wastewater treatment like wool fibers (Balkose and Baltacioglu, 1992), microbial biosorbents (Xie et al., 1996), pillared clays (Baksh et al., 1992), coir pith untreated (Namasivayam and Kadirvelu, 1996) or activated carbon (Namasivayam and Kadirvelu, 1997), banana pith (Namasivayam and Kanchana, 1992), orange peel (Namasivayam et al., 1996), peanut and walnut shells (Randall et al., 1975), modified onion skin (Bankar and Dara, 1982), corncobs (Tsai et al., 1998), the combination of onion skin with corncobs (Odozi and Emelike, 1985), peanut skin (Randall et al., 1975), palm kernel husk (Omgbu and Iweanya, 1990), pecan (Ahmenda et al., 2000a,b) and almond shells (Toles et al., 2000), or functionalized lignin extracted from sugarcane bagasse (Peternele et al., 1999). Suitable is even black currant and apple dietary fiber because of its binding capacity for cadmium and lead (Borycka and Zuchowski, 1998). The pretreatment methods for these materials differ, reaching from chemical extraction of lignin (Peternele et al., 1999) to adding chemicals and further pyrolysis (Ahmenda et al., 2000a,b; Toles et al., 2000), from polymerization (Bankar and Dara, 1982) to just cutting, drying, and grinding (Namasivayam et al., 1996).

A. Biosorption of metal ions Biosorption can be used as a cost-effective and efficient technique for the removal of toxic heavy metals from wastewater. Toles et al. (2000) investigated the adsorptive properties of air-activated almond shells toward several organics and copper. The almond shell carbon could remove more than 400% of Cu2þ from the solution compared to commercial carbon Norite RO3515. The organic adsorption of almond shell carbon was lower compared to Filtrasorbe 400, ranging between 84% and 92% of the Calgone carbon total adsorption. Convincing as well is the cost estimation: commercial carbons are produced for US $3.30 1/kg, almond shell carbons for US $2.45 1/kg. Johns et al. (1998) compared seven commercial

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granulated activated carbons (GAC) with GACs made of residual matter like almond shells, oil palm shells, sugarcane bagasse, rice straw, soybean hull, peanut, and walnut shells. Both CO2 and steam activated nutshell carbons consistently removed more total organics than the commercial GACs. The soybean hull-based GACs showed three or four times higher copper adsorption compared with all other commercial or coproductbased GACs. Effective adsorption is as well feasible without physical or chemical activation. The raw material has only been cut, dried, and ground before the experiments. Laufenberg et al. (2003) reviewed the most important influencing adsorption parameters, for example, residue combinations and synergies, particle size, adsorbent dosage, removed component and its initial concentration, agitation time and contact time, pH-value, surface area, targeted metabolism, binding mechanisms, bioreactor design used as well as posttreatment procedure. The most appropriate bioreactor design is a packed bed column, as adsorption is much more effective in a packed bed than in a stirred tank bioreactor. A packed bed will permit faster mass transfer and higher conversion, assuming that a large volume of solution is to be fed through a small bed of adsorbing solid. The bed is completely uniformly packed and the flow moving evenly, without dispersion and independent of the bed’s radius. Hence in the packed bed the concentration in the solid is in equilibrium with the high feed concentration. In stirred tank loaded solid reaches equilibrium with depleted solution which is less than with the feed solution. Therefore, yields are much less effective (Cussler, 1997). In recent years, attention has been focused on the utilization of unmodified or modified rice husk as a sorbent for the removal of pollutants. Rice husk is the outer covering of paddy and accounts for 20–25% of its weight. It is removed during rice milling and is used mainly as fuel generating CO2 and other forms of pollution to the environment. The annual generation of rice husk in India is in the range of 18–22 million ton. Unmodified rice husk has been evaluated for their ability to bind metal ions. Various modifications on rice husk have been reported to enhance sorption capacities for metal ions and other pollutants (Kumar and Bandyopadhyay, 2006). Mohan and Sreelakshmi (2008) reported the results of the study on the performance of low-cost adsorbent such as raw rice husk (RRH) and phosphate-treated rice husk (PRH) in removing the heavy metals such as lead, copper, zinc, and manganese. The adsorbent materials adopted were found to be an efficient media for the removal of heavy metals in continuous mode using fixed bed column. The column studies were conducted with 10 mg/L of individual and combined metal solution with a flow rate of 20 ml/min with different bed depths such as 10, 20, and 30 cm. The breakthrough time was also found to increase from 1.3 to 3.5 h for Pb (II), 4.0–9.0 h for Cu(II), 12.5–25.4 h for Zn (II), and 3.0 to 11.3 h for Mn (II) with increase in bed height from 10 to 30 cm for PRH.

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Different column design parameters like depth of exchange zone, adsorption rate, adsorption capacity, and so on were calculated. It was found that the adsorption capacity and adsorption rate constant were increased and the minimum column bed depth required was reduced when the rice husk is treated with phosphate, when compared with that of RRH. Orange waste, produced during juicing has been loaded with zirconium(IV) so as to examine its adsorption behavior for both As (V) and As (III) from an aquatic environment. Immobilization of zirconium onto the orange waste creates a very good adsorbent for arsenic. Adsorption kinetics of As (V) at different concentrations are well described in terms of pseudo-second-order rate equation with respect to adsorption capacity and correlation coefficients. Arsenate was strongly adsorbed in the pH range from 2 to 6, while arsenite was strongly adsorbed between pH 9 and 10. Moreover, equimolar (0.27 mM) addition of other anionic species such as chloride, carbonate, and sulfate had no influence on the adsorption of arsenate and arsenite. The maximum adsorption capacity of the Zr(IV)loaded onto a saponified orange waste (SOW) gel was evaluated as 88 and 130 mg/g for As(V) and As(III), respectively. Column adsorption tests suggested that complete removal of arsenic was achievable at up to 120 Bed Volumes (BV) for As (V) and 80 BV for As (III). Elution of both arsenate and arsenite was accomplished using 1 M NaOH without any leakage of the loaded zirconium. Thus this efficient and abundant bio-waste was successfully employed by Biswas et al. (2008) for the remediation of an aquatic environment polluted with arsenic. Microbial biomass, such as fungi, would be particularly cost-effective as there are many food-processing plants in both Turkey and the United States, and many other countries that could provide wastewater as substrate at a very low cost for the cultivation of these. Dried biomass of Rhizopus oligosporus produced using wet milling corn-processing wastewater as organic substrate was used as an adsorbent for Copper ions in water. The adsorption process was carried out in a batch process and the effects of contact time (1–48 h), initial pH (2.0–6.0), initial metal ion concentration (20–100 mg/L), and temperature (20–38 C) on the adsorption were investigated by Ozsoy et al. (2008). Experimental results showed that the maximum adsorption capacity was achieved at pH 5.0, and adsorbed Cu(II) ion concentration was increased with increasing initial metal concentration and contact time. The isothermal data could be described well by the Langmuir equations and monolayer capacity had a mean value of 79.37 mg/g. A pseudo-second-order reaction model provided the best description of the data with a correlation coefficient 0.99 for different initial metal concentrations. Thermodynamic parameters indicated that biosorption of Cu(II) on R. oligosporus dried biomass was exothermic and spontaneous. The results of FTIR analyses indicated that amide I and hydroxyl groups of adsorbent played important role in binding Cu (II).

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Schiewer and Patil (2008) investigated the removal of cadmium by fruit wastes (derived from several citrus fruits, apples, and grapes). Citrus peels were identified as the most promising biosorbent due to high metal uptake in conjunction with physical stability. Uptake was rapid with equilibrium reached after 30–80 min depending on the particle size (0.18–0.9 mm). Sorption kinetics followed a second-order model. Sorption equilibrium isotherms could be described by the Langmuir model in some cases, whereas in others an S-shaped isotherm was observed, that did not follow the Langmuir isotherm model. The metal uptake increased with pH, with uptake capacities ranging between 0.5 and 0.9 meq/g of dry peel. Due to their low cost, good uptake capacity, and rapid kinetics, citrus peels are a promising biosorbent material warranting further study.

B. Adsorption of dyes from wastewater The wastewaters discharged from dyeing processes exhibit low BOD, high COD, are highly colored, hot and alkaline, containing high amounts of dissolved solids. There is a wide range of pH, making conventional biological and chemical treatment processes difficult (Lee, et al., 1999). The dyes are highly colored polymers and have low biodegradability. The disposal of colored wastes is undesirable because of their toxicity to aquatic life and carcinogenicity. While cassava is an important crop across a wide range of tropical environment, cassava peels are an agricultural waste from the foodprocessing industry. Activated carbon prepared from cassava peel was used as an adsorbent in removal of dyes and metal ions from aqueous solutions. The material impregnated with H3PO4 showed higher efficiency than the heat-treated material (Rajeshvarisivaraj et al., 2001). Tsai et al. (2008) proved feasibility to utilize the food-processing waste for removing dye from the industrial dying wastewater. The beer brewery waste has been shown to be a low-cost adsorbent for the removal of basic dye from the aqueous solution as compared to its precursor (i.e., diatomite) based on its physical and chemical characterizations including surface area, pore volume, scanning electron microscopy, and nonmineral elemental analyses. The pore properties of this waste were significantly larger than those of its raw material, reflecting that the trapped organic matrices contained in the waste probably provided additional adsorption sites and/or adsorption area. The results of preliminary adsorption kinetics showed that the diatomite waste could be directly used as a potential adsorbent for removal of methylene blue on the basis of its adsorption– biosorption mechanisms. The adsorption parameters thus obtained from the pseudo-second-order model were in accordance with their pore properties. From the results of adsorption isotherm at 298 K and the applicability examinations in treating industrial wastewater containing basic

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dye, it was further found that the adsorption capacities of diatomite waste were superior to those of diatomite, which were also in good agreement with their corresponding physical properties.

VI. USING EGGSHELL Large quantities of eggshell waste are discarded in the food-processing industry. Freire et al. (2008) investigated the incorporation of eggshell waste as a raw material into a wall tile body, replacing natural carbonate material by up to 15 wt%. Formulations containing eggshell were uniaxially dry pressed and fired at 1150 C using a fast firing cycle. Physicomechanical properties of the fired tiles (e.g., linear shrinkage, water absorption, apparent density, flexural strength) were then determined. Development of the microstructure was followed by scanning electron microscope (SEM) and X-ray diffraction (XRD) analyses. The results showed that eggshell waste could be used in wall tiles, in the range 5–10 wt%, as a partial replacement for traditional carbonate-based materials with only a slight decrease in the end product properties.

VII. ADDED-VALUE PRODUCTS FROM WHEY Much of the material generated as wastes by the dairy industries throughout Europe contains components that could be utilized as substrates and nutrients in a variety of microbial/enzymic processes, to give rise to added-value products. Varieties of processes exist that do this worldwide, some having operated for many years. Joshi (2002) and Marwaha and Arora (2000) are two examples of extensive discussions of current industrial exploitation and future possibilities within this area. Added-value products, actually produced from dairy industry wastes, include animal feed, single-cell protein and other fermented edible products, baker’s yeast, organic acids, amino acids, enzymes (e.g., lipases, amylases, cellulases), flavors and pigments, the bio-preservative bacteriocin (from the culture of L. lactis on cheese whey) and microbial gums and polysaccharides (Joshi, 2002). A good example of a waste that has received considerable attention as a source of added-value products is cheese whey, which in itself contains many nutrients. Marwaha and Arora (2000) have tracked the products currently produced from whey, and the main destinations of unutilized whey for disposal, summarized in Table 3.7. Recently, demand for whey started to increase with news of the benefits that the high-quality proteins found in whey provide children, adults, and the elderly. Increased pharmaceutical applications of protein fractions for the control of blood pressure and for inducing sleep might

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Whey Utilization or disposal (Marwaha and Arora, 2000)

Processing scheme

Condensed þ Dry whole whey Demineralized whey Refined lactose Ultrafiltration: Whey protein concentrate Ultrafiltration: whey permeate

Added-value product

Pollutant

Human food Animal feed Baby food Edible lactose Animal feed Edible protein Refined lactose

Disposed as waste (see below)

Lactosehydrolyzed products Fermentation products Unutilized whey: for disposal

Disposal on land Into inland surface water Into a common sewer U/F Whey permeate disposed of as waste

further enlarge the market. The World Market for Whey and Lactose Products 2006–2010—From commodities to value-added ingredients clearly demonstrates how whey continues to show significant growth rate both in volume terms and particularly in value terms. There has been a significant increase in consumer products launches containing Whey Protein Concentrate from 2001–2003 to 2004–2006 corresponding to approx. 60%. Peters (2005) evaluated economic consequences of the cheese making process trough several example calculations concerning processing of whey in relation to cheese making throughput and several whey processing alternatives. All value-added enhancements by conversion of whey into whey protein concentrates create a larger stream of an aqueous lactose fraction, with the exception of lactoferrin extraction. This means that the high price which can be obtained for whey protein isolate products has to take into account the large quantity of lactose permeate that will necessarily be created in parallel. The most beneficial step in increasing value for whey products would be to add more value to the lactose

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fraction. The production of galacto-oligosaccharides for the displacement of antibiotics in animal feeding is promising to influence the lactose market. It was calculated that the price of edible lactose has a greater influence on the economics than the price of whey protein. One example of whey utilization technologies is the production of alcohol from cheese whey at the Carbery Milk Products Ltd. factory, Ballineen, Ireland. The Carberry plant produces 2.59% v/v of ethanol from 4.7 w/v of lactose in whey permeate (Barry, 1982). Because the Kluveromyces species, used in anaerobic fermentations, have low ethanol tolerance, preconcentration of the lactose is not possible, so fermentation and distillation costs are considerable. Under Irish conditions potable alcohol is the most profitable outlet but in other countries, anhydrous alcohol for industrial or power uses may be more attractive (Ozmihci and Kargi, 2008).

VIII. FOOD WASTE TREATMENT Technologies for treatment of aqueous food industry waste streams: Reduction of BOD and COD is one of the most pressing tasks for a process treating wastes such as those discussed above. Traditional bioconversion technologies for achieving that aim are essentially those developed for sewage treatment and are used widely. They include: (a) Aerobic processes, such as the activated sludge process (including Deep Shaft) and trickling filters (and other biofilm-based designs). Here, flocs or films of microorganisms act as adsorption points and powerful oxidizing catalysts that convert organic materials essentially to carbon dioxide and more biomass. When operated continuously, a retention time of approximately 15 days is common. (b) Anaerobic processes, such as various designs of the anaerobic digester. In these processes, organic material is converted to methane and carbon dioxide (‘‘biogas’’) and a biomass sludge.

A. Bioprocessing of FVWs 1. Anaerobic digestion Among the several processes that are being used nowadays for treatment of FVW, the ones described are the following: anaerobic digestion, anaerobic co-digestion, and biodiesel production. Anaerobic digestion converts biomass waste to biogas and compost using bacteria in the absence of oxygen. The biogas is mainly a mixture of CO2 and CH4. The biogas is partly utilized to heat the digestion reactors. The rest can be used to generate electricity and/or heat (e.g., with a gas engine) or, after

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treatment, be fed into the natural gas grid. The biomethanation of FVW is accomplished by series of biochemical transformations, which can be roughly separated in four metabolic stages (Bouallagui et al., 2005) (Fig. 3.4). Usually, the choice of a temperature range for anaerobic digestion is strictly dependent on the bioclimatic conditions. In Sweden, for example, research is currently undertaken for a possible anaerobic digestion under low-temperature conditions. In the United States, anaerobic digestion of sludge under thermophilic conditions has been abandoned, although it is well established in Europe, especially for the treatment of the organic fraction of municipal solid waste (OF-MSW) (Ahring et al., 2002). In tropical countries, like in Tunisia, where the ambient temperature is higher than 25 C during a period of more than 8 months in a year, thermophilic anaerobic digestion is readily applicable. Bouallagui et al. (2004) compared the performance of anaerobic digestion of FVW in the thermophilic (55 C) process with those under psychrophilic (20 C) and mesophilic (35 C) conditions in a tubular anaerobic digester on a laboratory scale. The aim of this study was to examine the effect of temperature on the anaerobic digestion of FVWs for several retention times and feed concentrations and to compare the energy balance of the process under

Fruit and vegetable wastes: cellulose, hemicellulose, pectin, fat, protein, lignin, reducing and non-reducing sugars Hydrolysis

One-stage system

Acidogenesis

Intermediary products: VFA Inhibition

Inhibition Acetogenesis

H2

CO2

Acetate

Inhibition

Inhibition

Two-stage system

Amino acids, alcohols, sugars, long chain fatty acids

Methanogenesis H2O + CH 4 + CO2

FIGURE 3.4 Reaction scheme for anaerobic digestion of particulate organic material of FVW (Bouallagui et al., 2005).

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psychrophilic, mesophilic, and thermophilic conditions. The hydraulic retention time (HRT) ranged from 10 to 20 days, and raw FVW was supplied in a semicontinuous mode at various concentrations of TSs (4, 6, 8, and 10% on dry weight). Biogas production from the experimental thermophilic digester was higher on average than from psychrophilic and mesophilic digesters by 144% and 41%, respectively. The net energy production in the thermophilic digester was 195.7 and 49.07 kJ/day higher than that for the psychrophilic and mesophilic digesters, respectively. The relation between the daily production of biogas and the temperature indicates that for the same produced quantity of biogas, the size of the thermophilic digester can be reduced with regard to that of the psychrophilic and the mesophilic digesters. Bouallagui et al. (2005) reviewed the potential of anaerobic digestion for material recovery and energy production from FVW containing 8–18% TSs, with a total VSs content of 86–92%. The organic fraction includes about 75% easy biodegradable matter (sugars and hemicellulose), 9% cellulose, and 5% lignin. Anaerobic digestion of FVW was studied under different operating conditions using different types of bioreactors. It permits the conversion of 70–95% of organic matter to methane, with a volumetric OLR of 1–6.8 g VS/L day. A major limitation of anaerobic digestion of FVW is a rapid acidification of these wastes decreasing the pH in the reactor, and a larger volatile fatty acids (VFAs) production, which stress and inhibit the activity of methanogenic bacteria. Continuous two-phase systems appear as more highly efficient technologies for anaerobic digestion of FVW. Their greatest advantage lies in the buffering of the OLR taking place in the first stage, allowing a more constant feeding rate of the methanogenic second stage. Using a two-stage system involving a thermophilic liquefaction reactor and a mesophilic anaerobic filter, over 95% volatile solids were converted to methane at a volumetric loading rate of 5.65 g VS/L. The average methane production yield was about 420 L/kg added VS. Alvarez et al. (1992) reported that biomethanation of food-market waste resulted in a production of 0.64 m3 biogas/kg TSs added. The biogas yield from canteen wastes, which was a mixture of FVW, when subjected to anaerobic digestion varied from 0.82 to 0.9 m3/kg of VS added (Nand et al., 1991). Viswanath et al. (1992) reported a production of 0.12 m3 biogas/kg TS added with HRT of 16 days and the biogas yield varied between 0.6 and 1.0 m3/kg VS/day from the same type of waste. Biomethanation of banana peel and pineapple wastes studied by Bardiya et al. (1996) at various HRTs showed a higher rate of gas production at lower retention time. The biochemical methane potential of 54 fruits and vegetable wastes samples and eight standard biomass samples were determined by Gunaseelan (2004) to compare the extents and the rates of their conversion

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to methane. The ultimate methane yields (B0) and methane production rate constant of fruit wastes ranged from 0.18 to 0.732 L/g VS added and 0.016 to 0.122 1/d, respectively, and that of vegetable wastes ranged from 0.19 to 0.4 L/g (VS) added and methane production rate ranged from 0.053 to 0.125 1/d, respectively. Temperature had no effect on the B0 of mango peels; however, the conversion kinetics was higher at 35 C than at 28 C. All the samples of fruits and vegetable wastes tested gave monophasic curves of methane production. Substantial differences were observed in the methane yields and kinetics among the varieties in mango, banana, and orange. Different fruit parts within the same variety showed different yields in orange, pomegranate, grape vine, and sapota. The methane yields from the mango peels of some of the varieties, orange wastes, pomegranate rotten seeds, and lemon pressings were significantly (P < 0.05) higher than the cellulose. Methane yields and kinetics of vegetable wastes in different varieties as well as within different plant parts of the same variety differed. Onion peels exhibited yields significantly (P < 0.05) similar to cellulose, while a majority of the vegetable wastes exhibited yields greater than 0.3 L/g VS. Rotten tomato, onion peels, pest infested brinjal, lady’s finger stalk, coriander plant wastes, cabbage leaves, and cauliflower stalk, turnip leaves, radish shoots, and green pea pods exhibited methane yields greater than 0.3 L/g VS added. Methane yields from these wastes varied among various varieties and different plant parts of the same variety. In coriander plant wastes, methane yield for leaves was higher than that of structural roots. These results provide a database on the extent and the rates of conversion of fruits and vegetable solid wastes that significantly contribute to the OF-MSWs. Anaerobic digestion of wastewater from jam industries was studied in a continuous reactor with different OLRs and the optimum OLR was 6.5 kgCOD/m3/day when it was operated with 3 days HRT. The biodegradability of wastewater in batch experiments was about 90%. The removal effciency of total COD and soluble COD were found to 82% and 85%, respectively. The specific methane production was 0.28 m3/kg of COD removed/day (Mohan and Sunny, 2008). Arvanitoyannis and Varzakas (2008) summarized in their recent review the main advantages of the biodegradation waste management as follows: It allows reducing the volume of organic wastes The biological hazard of the wastes can be controlled This system may be compatible with the other biological ELSS

(greenhouses)

The biogas manufactured can be used to produce electricity The water obtained in the biodegradation processes may be used for the

other needs of the space vehicle

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A valuable effluent is also obtained, which eventually can be used as an

excellent soil conditioner after minor treatments (Converti et al., 1999)

High OLRs and low sludge production are among the many advan-

tages anaerobic process exhibit over other biological unit operations (Batstone et al., 2001) Biomethanization of fruit wastes is the best-suited treatment as the process not only adds energy in the form of methane, but also results in a highly stabilized and treated effuent. Compared to the aerobic method, the use of anaerobic digesters in processing these waste streams provides greater economic and environmental benefits and advantages. Besides reducing the amount of green house gases by controlled use of methane from waste, the substitution of oil and coal with bioenergy will result in saving the global environment by reducing the use of fossil fuels. Anaerobic digestion has many environmental benefits including the production of a renewable energy carrier, the possibility of nutrient recycling, and the reduction of waste volumes. Many kinds of organic waste have been digested anaerobically in a successful way, such as sewage sludge, industrial waste, slaughterhouse waste, FVW, manure, and agricultural biomass. The wastes have been treated both separately and in co-digestion processes. In co-digestion, it is important to consider the effects of the different incoming waste streams. Better handling and digestibility can be achieved by mixing solid waste with diluted waste. Furthermore, the successful mixing of different wastes results in a better digestion performance by improving the content of the nutrients and even reduces the negative effect of toxic compounds on the digestion process. Many studies have been carried out both in batch and continuous modes, to determine how co-digestion of different organic solid wastes including FVW with cattle slurry can improve the efficiency of degradation (Callaghan et al., 1999, 2002). The digestion of cattle slurries and of a range of agricultural wastes has been evaluated and has been successful according to Callaghan et al. (2002). Previous batch studies have shown that based on VSs reduction, total methane production and methane yield, co-digestions of cattle slurry (CS) with FVWs and with chicken manure (CM) were among the more promising combinations. A continuously stirred tank reactor (18 L) was used as a mesophilic (35 C) anaerobic reactor to examine the effect of adding the FVW and CM to a system which was digesting CS (Callaghan et al., 2002). The retention time was kept at 21 days and the loading rate maintained in the range 3.19–5.01 kg VS/m.d. Increasing the proportion of FVW from 20% to 50% improved the methane yield from 0.23 to 0.45 m3CH4 /kg VS added, and caused the VS reduction to decrease slightly. Chicken manure was not as successful as a co-digestate. As the amount of CM in the feed and the organic loading was increased, the VS reduction deteriorated and the methane yield decreased. This appeared to be caused by ammonia inhibition.

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Gomez et al. (2006) compared the digestion of primary sludge (PS) against co-digestion of this waste together with the fruit and vegetable fraction of municipal solid wastes (FVF MSW), evaluating the production of gas, the influence of mixing conditions, and the performance of the system under different OLRs. The anaerobic digestion process was evaluated under static conditions and with different mixing conditions, with good results being found for the digesters with limited mixing, this representing an energy saving. The results for co-digestion of mixtures of PSþFVF MSW are better than those obtained from the digestion of PS on its own. Biogas production for co-digestion is much greater thanks to the larger VS content of this feedstock. Nevertheless, biogas yield and specific gas production for the two digestion processes are similar, with values in the range 0.6–0.8 L/g VS destroyed for the first parameter and in the range 0.4–0.6 L/g VS fed for the second. The co-digestion process was also evaluated at different OLRs under low mixing conditions, with stable performance being obtained even when the systems were overloaded. Co-digestion is of considerable technical interest, since it allows the use of existing installations and greatly increases biogas production and the energy produced in cogeneration units. Anaerobic digestion can be carried out using three different systems, first batch systems with the advantage of simple design and process control, robustness toward coarse and heavy contaminants, and lower investment costs. The application of sequencing batch reactor (SBR) technology in anaerobic treatment of FVW is another batch system of interest due to its inherent operational flexibility, characterized by a high degree of process flexibility in terms of cycle time and sequence, no requirement for separate clarifiers, and retention of a higher concentration of slowgrowing anaerobic bacteria within the reactor (Dague et al., 1992) (Fig. 3.5). Hydrogen–methane two-stage fermentation technology was developed by Nishio and Nakashimada (2007), in which the hydrogen produced in the first stage was used for a fuel cell system to generate electricity, and the methane produced in the second stage was used to generate heat energy to heat the two reactors and satisfy heat requirements. The technology proposed is effective for the treatment of sugarrich wastewaters, bread wastes, soybean paste, and brewery wastes. Evaluation of co-digestion with the OF-MSWs has been evaluated by Fernandez et al. (2005) for the treatment of fats of different origin. The process of co-digestion was conducted in a pilot plant working in the semicontinuous regime in the mesophilic range (37 C) and the HRT was 17 days. During the start-up period the digester was fed with increasing quantities of a simulated OF-MSW (diluted dry pet food). When the designed organic loading was reached, a co-digestion process was

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A G D E

G

A E

P G

A

B

R CSTR

A

E FFR E

C

G

G

E

E

A

A

R ASBR

SBH UASB

FFR

FIGURE 3.5 Processes used for FVW anaerobic treatment: (A) continuously stirred tank reactor (CSTR); (B) tubular reactor; (C) two-phase integrated anaerobic solid bed hydrolyser (SBH) and upflow anaerobic sludge blanket (UASB); (D) two-phase integrated anaerobic CSTR and fixed film reactor (FFR); and (E) two-phase integrated anaerobic sequencing batch reactor and FFR (Bouallagui et al., 2005).

initiated. The fat used consisted of animal fat waste from the food industry, with a similar long-chain fatty acid (LCFA) profile to that of the diluted dry pet food. Animal fat was suddenly substituted by vegetable fat (coconut oil) maintaining the organic loading. The LCFA profile for vegetable fat is completely different from that of animal fat and simulated OF-MSW being short-chain-saturated LFCA the most predominant (lauric acid, mystiric acid, and palmitic acid accounting for the 74% of the total LCFA content). No accumulation of LCFA or VFAs was detected in either case. After a short adaptation period, total fat removal throughout the experiment was

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over 88%, whereas biogas and methane yields were very similar to those of simulated OF-MSW. This proved to be an effective method and suitable technology for the treatment of waste through anaerobic co-digestion of OF-MSW and fat wastes to obtain a renewable source of energy from biogas. Briefly, anaerobic digestion leads to the overall gasification of organic wastewaters and wastes, producing methane and carbon dioxide; this gasification contributes to reduction of organic matter and recovery of energy from organic carbon in cost-effective manner.

B. Biodiesel production Biodiesel, as an alternative fuel, has many merits. It is derived from a renewable, domestic resource, thereby relieving reliance on petroleum fuel imports. It is biodegradable and nontoxic. Compared to petroleumbased diesel, biodiesel has a more favorable combustion emission profile, such as low emissions of carbon monoxide, particulate matter, and unburned hydrocarbons. Carbon dioxide produced by combustion of biodiesel can be recycled by photosynthesis, thereby minimizing the impact of biodiesel combustion on the greenhouse effect (Agarwal and Das, 2001). Four different continuous process flow sheets for biodiesel production from virgin vegetable oil or waste cooking oil under alkaline or acidic conditions on a commercial scale were developed by Zhang et al. (2003). Two of them were alkali-catalyzed processes, the former using virgin oil and the latter using waste cooking oil. The remaining two processes were acid-catalyzed processes using waste cooking oil as the raw material. Detailed operating conditions and equipment designs for each process were obtained. Stainless steel was used for the trans-esterification reactor in the designs for the alkali-catalyzed processes in this study. The material of construction of other equipment in the alkali-catalyzed processes was carbon steel. For the acid-catalyzed system, a stainless steel (type 316) reactor was used. A technological assessment of these four processes was carried out to evaluate their technical benefits and limitations. Analysis showed that the alkali-catalyzed process using virgin vegetable oil as the raw material required the fewest and smallest process equipment units but at a higher raw material cost than the other processes. The use of waste cooking oil to produce biodiesel reduced the raw material cost. The acid-catalyzed process using waste cooking oil proved to be technically feasible with less complexity than the alkali-catalyzed process using waste cooking oil, thereby making it a competitive alternative to commercial biodiesel production by the alkali-catalyzed process. The alkali-catalyzed process using virgin oil was the simplest with the least amount of process equipment but had a higher raw material cost than other processes. The method using waste cooking oil was the most complex process

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with the greatest number of equipment pieces due to the addition of a pretreatment unit for free fatty acids removal despite the reduced raw material cost. The acid-catalyzed process using waste cooking oil had less equipment pieces than the previous process, but the large methanol requirement resulted in more and larger trans-esterification reactors, as well as a larger methanol distillation column. Methanol distillation was carried out immediately following trans-esterification to reduce the load in downstream units in this process but more pieces of equipment made from stainless steel material were necessary than the first two processes. In brief, for process simplicity, the alkali-catalyzed process using virgin vegetable oil is recommended. However, if the raw material cost is of concern, the acid-catalyzed process using waste cooking oil is a relatively simple process and proved to be a competitive alternative to the first two processes (Zhang et al., 2003). Tashtoush et al. (2003) investigated the feasibility of utilizing a renewable and low-cost fuel raw material (a waste vegetable oil) as a diesel fuel replacement in small-scale applications such as in residential heating boilers. They examined the aspects of combustion performance and emissions of the ethyl ester of used palm oil (biodiesel) relative to the baseline diesel fuel in a water-cooled furnace. The combustion efficiency, Zc, and exhaust temperature, Texh, as well as the common pollutants and emissions were tested over a wide range of air/fuel ratio ranging from very lean to very rich (10:1–20:1). All tests were conducted at two different energy inputs for both fuels. The findings showed that, at the lower energy rate used, biodiesel burned more efficiently with higher combustion efficiency and exhaust temperature of, respectively, 66% and 600 C compared to 56% and 560 C for the diesel fuel. At the higher energy input, the biodiesel combustion performance deteriorated and was inferior to diesel fuel due to its high viscosity, density and low volatility. As for emissions, biodiesel emitted fewer pollutants at both energy levels over the whole range of A/F ratio considered. World food consumption produces large quantities of waste (used or fryer) vegetable oil, WVO. In many world regions, most WVO produced is disposed of inappropriately. Consequently, the above-mentioned study was initiated to examine the potential of WVO as an alternative source of thermal energy.

C. Anaerobic treatment of dairy wastes Anaerobic treatment applications for dairy industry wastewaters have been evaluated in a number of previous studies (Backman et al., 1985; Barford et al., 1986; Clanton et al., 1985; Hills and Kayhanian, 1985; Lo and Liao, 1986a,b; Lo et al., 1987; Mendez et al., 1989; Samson et al., 1985; Toldra et al., 1987). More recent information about anaerobic treatment practices of dairy waste streams is also presented by Demirel et al. (2005) (Tables 3.8 and 3.9).

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TABLE 3.8

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Typical operating conditions for anaerobic digesters (Wheatley, 1990)

Anaerobic digester configuration

Load (kg COD/ m3day)

Retention

COD removal (%)

CSTR Anaerobic filter UASB Fluidized bed

0.5–2.5 2–10 2–15 2–50

1–5 days 10–50 h 8–50 h 0.5–24 h

80–90 70–80 70–90 70–80

Conventional anaerobic treatment processes are often used for treating dairy wastewaters. Particularly anaerobic filters and UASB reactors are the most common reactor configurations employed. Actually, the UASB reactors are very suitable for treating food industry wastewaters, since they can treat large volumes of wastewaters in a relatively short period of time. More research should be directed toward treatment of dairy wastewaters in pilot and full-scale UASB reactors in near future, to make use of these potential advantages outlined. Lipid degradation and inhibition in single-phase anaerobic systems is frequently discussed in literature, since lipids are potential inhibitors in anaerobic systems, which can often be encountered by environmental engineers and wastewater treatment plant operators. Moreover, high concentrations of suspended solids in dairy waste streams can also affect the performance of conventional anaerobic treatment processes adversely, particularly the most commonly used upflow anaerobic filters. Thus, two-phase anaerobic digestion processes should be considered more often to overcome these problems that may be experienced in conventional single-phase design applications, since twophase anaerobic treatment systems are reported to produce better results with various industrial wastewaters, such as olive oil mill and foodprocessing effluents, which are high in suspended solids and lipids content. When two-phase anaerobic digestion processes are evaluated as a whole, it is clear that the acid phase digestion of dairy wastewaters is actually investigated in various aspects. However, data especially for fullscale two-phase applications for dairy effluents in literature are scarce. Furthermore, in addition to degradation of lipids, protein solubilization should be investigated more comprehensively during acid phase digestion of dairy wastes with relatively high protein content, because there is contradictory information in literature about protein solubilization with different wastewater types during anaerobic acidogenesis. Since high rate anaerobic treatment of dairy wastes (or any industrial wastewater) with a relatively higher content of particulates, fats and proteins can often be problematic, modeling studies simulating biodegradation mechanisms of these components can extensively be explored. Removal of nitrogen and

TABLE 3.9

Anaerobic/aerobic treatment performance levels for dairy wastewaters (Demirel et al., 2005) Application status

Effluent type

System configuration

Removal

Milk bottling plant

DAFþupflow anaerobic filter (UAF) Downflow–upflow hybrid anaerobic reactor (DUHR) þ SBR UASB pond þ aerated pond

38–50% BOD5 (DAF) >90% BOD5 (UAF) >85% COD (UAF) 98% COD (DUHR) >90% COD (SBR)

Pilot scale

Kasapgil et al. (1994)

Laboratory scale

Malaspina et al. (1995)

98% (BOD5) 96% (COD) 98% (TSS) >90% (COD)

Full scale

Monroy et al. (1995)

Laboratory scale

Comeau et al. (1996)

Industrial Laboratory scale

Garrido et al. (2001)

Cheese whey

Cheese wastewater

Synthetic milk powder/ butter factory wastewater Wastewater from an industrial milk analysis laboratory

AAO activated sludge

Anaerobic filter þ SBR

98% (COD), 99% (nitrogen)

References

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phosphorus from dairy wastewaters has recently gained significant attention, due to more strict environmental regulations, so current research efforts clearly seem to focus on this particular topic. Recently, bench–pilot and full-scale applications of combined treatment methods for nutrient removal from dairy waste effluents are frequently encountered. It is obvious that as the regulations for discharge of nutrients become stricter in time, new modifications in existing treatment plants will eventually be incorporated. Finally, since the anaerobic digestion process is an imperative tool for the production of clean energy sources, such as hydrogen and methane, biogas production from high-strength dairy industry wastes will always be of paramount importance, as a valuable renewable energy source, for both developed and developing countries in future. Particularly, production of hydrogen by acidogenesis of high-strength dairy waste effluents is currently worth investigating.

D. Aerobic treatment of dairy wastes Land disposal of whey as a waste product has been practiced not only in Europe but also in both the United States of America and Canada over the past 50 years. Although whey production has increased over the past 28 years by 165% in both countries, the utilization and disposal practices have remained essentially the same. However, because of its high BOD (40,000–60,000 mg/L), whey disrupts the biological process of conventional sewage treatment plants and its disposal into these plants has, therefore, been banned by many municipalities (Singh and Ghaly, 2006). Biodegradability evaluation of dairy effluents was studies by Janczukowicz et al. (2008). The results obtained proved that all dairy production effluents can be treated together, with the exception of whey, whose complex biodegradation demands may cause too much burden to any wastewater treatment technological system and thus should be managed within a separate installation. The pollutants in the cheese and cottage cheese whey proved to be the most resistant to biodegradation. Various methods for dairy waste treatment based on mesophilic aerobic and anaerobic digestions of whey and whey derivatives by yeasts have been reported by Cristiani-Urbina et al. (2000).

1. Thermophilic bioremediation for dairy waste management A dairy farm processing 100 ton of milk per day produces approximately the same quantity of organic products in its effluent as would a town with 55,000 residents. However, legislative regulations for the dumping of whey are forcing industries to come up with alternatives to make this process of elimination environmentally safer. One with attractive potential involves the use of thermophilic microorganisms to produce a

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pasteurized, easily dewatered sludge at temperatures that facilitate enhanced levels of energy recovery. Processing options include the associated production of low COD treated wastewater (Kosseva et al., 2001), or of added-value products such as xanthan gum (Papagianni et al., 2001) and polyhydroxyalkanoates (Pantazaki et al., 2003). Aerobic treatment involving populations of thermophilic bacteria offers a wide spectrum of benefits. One of these is the potential for the biodegradation of organics in high-temperature wastewaters, which eliminates the need for cooling them prior to treatment. Operation under thermophilic conditions gives a high rate of biodegradation, which is 3–10 times higher than with a mesophilic process, and lends itself to high process stability. High temperatures also support the inactivation of the pathogens present in the wastewater (Cheunbarn and Pagilla, 2000; Nakano and Matsamura, 2001), which is one of the main aims of the treatment process. That makes aerobic thermophilic processing suitable for stabilization of the sludge and for rendering it hygienic, so that it can be exploited as a fertilizer. As part of a project funded under the Fifth FRAMEWORK program of the European Commission, we have developed a bioremediation technology for cheese whey, associated with reduction of COD of the treated waste at elevated temperatures. This novel approach is an application of the standards for food industry environmental management systems, notably ISO 14000 (Boudouropoulos and Arvanitoyannis, 2000). Main advantages of thermophilic biological methods are:

Low mass yield Rapid kinetics High-temperature operation Stable process control of aerobic systems Production of pathogen-free products Energy generation

It is known that the composition of whey varies with a season, the acidity of nonpasteurized whey is higher during summer and the lactose concentration is lower than in winter. Kosseva et al. (2003, 2007) developed two strategies (two-stage and one-stage processes) for the bioremediation of blue Stilton whey applicable during whole year. It employed both naturally occurring thermotolerant organisms found in whey (LAB and yeast) and a thermophilic isolate. In 2003, a comparative study of two double-staged strategies was reported, using a thermophilic mixed population of Bacillus sp., isolated from a FVW. The source of these organisms shows robust properties and potential for degrading a broad spectrum of food wastes, for example, potato and grain distillery slops, and potato processing waters. Strategy 1: An anaerobic, mesophilic first stage, followed by an aerobic,

mesophilic second stage (45 C)

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Strategy 2: An anaerobic, mesophilic first stage (45 C), followed by an

aerobic, thermophilic second stage (55–65 C)

Strategy 3: An aerobic thermophilic single stage (55–65 C) was reported

in 2007. In the first stage of the first two strategies, anaerobic mesophilic conditions allow the development of activity of Streptococcus sp. and ‘‘lactic yeast’’ (isolated from blue Stilton whey), which consume lactose and produce lactate, ethanol and carbon dioxide, and further biomass. In the second stage, aerobic conditions are employed which are favorable to the activity of an added mixed population of Bacillus sp., which degrades all available organic acids and ethanol, producing CO2 and further biomass. The following reaction scheme was proposed for the anaerobic mesophilic stage: The homofermentative LAB (identified by Ercolini et al., 2003) produce lactase, which hydrolyzes the lactose found in whey to glucose and galactose: C12 H22 O11 þ H2 O ! C6 H12 O6 þ C6 H12 O6

(1)

Lactic acid is produced via the Emden–Meyerhof–Parnas glycolitic pathway, via pyruvic acid (showing only the main reagents and products): 2 C6 H12 O6 ! 4 C3 H4 O3 þ 8 Hþ ! 4 C3 H6 O3 þ 4 ATP

(2)

The thermotolerant yeast directly utilizes lactose to produce ethanol and carbon dioxide: C12 H22 O11 þ H2 O ! 4C2 H5 OH þ 4CO2

(3)

Anaerobic biomass formation might be described by the following simplified reaction scheme: aCHx Oy þ bHl Om Nn ! CHa Ob Nw þ cH2 O þ dCO2

(4)

where CHxOy is a carbon source, HlOmNn is a nitrogen source, and CHaObNw is biomass formed. During the thermophilic stage, the following reaction scheme, involving the Bacillus sp., was observed in chronological order for all temperatures: Ethanol bio-oxidation to acetic acid: C2 H5 OH þ O2 ! C2 H4 O2 þ H2 O

(5)

Bio-oxidation of acetic acid: C2 H4 O2 þ 2O2 ! 2CO2 þ 2H2 O

(6)

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Bio-oxidation of lactic acid: C3 H6 O3 þ 3O2 ! 3CO2 þ 3H2 O

(7)

Bio-oxidation of citric acid: C6 H8 O7 þ 4:5O2 ! 6CO2 þ 4H2 O

(8)

Aerobic biomass formation: eCHx Oy þ f O2 þ gHl Om Nn ! CHd Oe Nj þ hH2 O þ iCO2

(9)

where CHdOeNj is biomass formed. We propose the following bioremediation pattern for the aerobic single-stage process (Strategy 3) (Kosseva et al., 2007): LAB, Lactococcus sp. available in the Stilton whey, consume lactose, producing lactate: C12 H22 O11 þ H2 O ! 4C3 H6 O3

(10)

Thermotolerant yeasts shift their metabolism to acetic acid and biomass production under aerobic conditions: C12 H22 O11 þ H2 O ! 6C2 H4 O2

(11)

Thermophilic bacteria Bacillus sp. consume lactate and acetate with main products carbon dioxide and biomass. C3 H6 O3 þ 3O2 ! 3CO2 þ 3H2 O

(12)

C2 H4 O2 þ 2O2 ! 2CO2 þ 2H2 O

(13)

Biomass formation occurs simultaneously: eCHx Oy þ f O2 þ gHl Om Nn ! CHd Oe Nj þ hH2 O þ iCO2

(14)

A comparison of the effectiveness between the three strategies: A comparison of the three bioremediation strategies for the management of dairy waste is summarized below: Strategy 1 Strategy 2 Strategy 3 (two-stage) (two-stage) (one-stage) 55, 60, 65 C Second stage Second stage 45 C 55–65 C DOT > 65% DOT < 80% DOT ¼ 20, 40, 60, 80% RQ ¼ 1 RQ ¼ 1 RQ ¼ 1

Processing of Food Wastes

Average velocity of lactate biodegradation: VLA 0.50 g/ (L h)

VLA 0.96 g/ (L h)

Average velocity of COD removal: VCOD 0.74 g/(L h)

VCOD 1.57 g/ (L h)

Total removal of soluble COD ¼ 68%

Total removal of soluble COD ¼ 62.5%

Total reduction of soluble protein ¼ 59%

Total reduction of soluble protein ¼ 47.5%

115

55 C: VLA 0.87 g/(L h) VCOD 1.56 g/(L h) COD removal 80–94% 60 C: VLA 0.80 g/(L h) VCOD 1.40 g/(L h) COD removal 60–65.7% 65 C: VLA 1.01 g/(L h) VCOD 1.35 g/(L h) COD removal 60–77% –

Following the mesophilic–thermophilic strategy, approximately 100% reduction of soluble COD and lactose was recorded accompanied with a 90% decrease in soluble protein in batch cultures. Applying single stage thermophilic strategy, high conversions in the range of 80–100% were obtained at 55 C and DOT ¼ 20, 40, 60, and 80%. Consumption of lactose and organic acids was 90–100%. Biodegradation profiles at 60 C and dissolved oxygen levels of 40% and 80% showed the conversions of lactose and organic acids in the range of 65–74%. At 65 C thermophilic bacteria seem to grow mainly on lactate. Lactate consumption was between 87.5% and 92%. The efficiency of COD removal was approximately 20% lower than that observed at 55 C. The demand for N-source in the course of the biodegradation process was higher under thermophilic than under mesophilic conditions, which also helped nitrogen removal from the whey effluent (Krzywonos et al., 2008). Removal of nitrogen and phosphorus from dairy wastewaters has recently gained significant attention, due to more strict environmental regulations.

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Summarizing, we developed the thermophilic bioremediation technology for treatment of cheese whey. The thermophilic microbial populations Bacillus sp. successfully reduced the polluting load of the whey stream. The process was capable of reducing pollution loads in cheese whey up to 93% at 55 C, and up to 70% at 65 C in a conventional aerated stirred tank bioreactor, in a way that complies with EU guidelines on sanitization of bio-waste. Mass balance based mathematical models have been developed using simplified modifications of the IAWQ Activated Sludge Model’s concepts of ‘‘lumping’’ mixed populations and mixed substrates into a small number of ‘‘clusters’’ of ‘‘equivalent’’ substrate or biomass. Reasonably good fits to process data were obtained using these models over a range of temperatures, including those within the thermophilic region. Values of ‘‘best fit’’ model parameters were generated to predict biomass specific growth rates. The average specific growth rate calculated was 0.097 1/h at 55 C while the experimental one was 0.079 1/h. At 65 C the calculated average specific growth rate was 0.075 1/h while the experimental one was 0.089 1/h. The results obtained suggest that temperature may have exerted a larger influence on the biodegradation process than dissolved oxygen, as the composition of the microbial community changed with temperature over the range 55–65 C. The average biomass yields generated varied from 0.350 (on lactose substrate) to 0.430 g/g (on lactic acid substrate) and were 0.86 g/g on acetic acid substrate, whereas yields calculated using the model varied from 0.325 (on lactose substrate) to 0.410 g/g (on lactic acid substrate), being 1.01 g/g on acetic acid substrate. Our investigations suggest that modeling of complex bioreaction systems via ‘‘lumping’’ of key substrates and microbial species into a limited number of ‘‘equivalent clusters’’ is worthy of consideration as a possible means of facilitating rapid process development and practical process operation. Other members of our consortium (Cibis et al., 2002) have investigated thermophilic aerobic biodegradation of potato slops (distillation residue) from a rural distillery. The COD levels of this fraction ranged from 49 to 104 g/L, and the main contributions to the COD come from organic acids, reducing substances and glycerol. The highest removal efficiency of approximately 77% was achieved at 60 C using a similar mixed population of Bacillus sp., isolated from the same FVW and adapted to the above fraction.

IX. FCM ASPECTS AIMED IN SUSTAINABLE FOOD SYSTEM DEVELOPMENT The current practices of pollution control and waste management cannot completely meet the increasingly strict requirements for the reduction of environmental contamination. A present day challenge for the manufacturer

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is to develop and master technical tools and approaches that will integrate environmental objectives into design decisions. The manufacturing industry has to include the optimization of product-integrated environmental protection into strategic planning, research and development. These challenges cannot be met only by any individual enterprise but require a concentrated effort of specific actions and coordination of initiatives (Fitz and Schiefer, 2008). FCM aims at providing support for the identification and realization of ‘‘best’’ concepts for such actions and coordination needs. This support provides enterprises with the means for improving their own and the sector’s competitiveness, sustainability, and responsibility toward the expectations of its customers and the society (Ondersteijn et al., 2006).

A. User-oriented innovation in the food sector According to CIAA (2007), one of the main issues the food sector needs to deal with is to focus on changing consumer needs. User-oriented innovation is not new to the sector (Grunert et al., 2008). The fork-to-farm approach to food chains—meaning that all participants in the food chain should maximize value creation for the end user—has been promoted in various guises. But some recent developments make user-oriented innovativeness of the food chain more important, for example, public demands with respect to sustainable resource utilization, considerations concerning ethics and the environment and improvement of the work environment. The term user-oriented innovation has been defined as a process toward the development of a new product or service in which an integrated analysis and understanding of the users’ wants, needs, and preference formation play a key role (Grunert et al., 2008). There are three main streams of the user-oriented innovative research in the food sector: (1) understanding user preferences, (2) innovation management, and (3) interactive innovation. Sndergaard and Harmsen (2007) have suggested a new product development model that takes an understanding of consumer quality perception as its point of departure. The basic message of the model is that the quality to be perceived by consumers is to be taken as a starting point, and that the concrete attributes to be built into the product, just as the concrete product attributes to be communicated to the prospective buyer, should be derived from this, and not the other way round. This approach is applicable to all three types of innovations, but is more straightforward in a type I innovation.

B. Market-oriented research Several studies have concluded that market orientation is important for the successful outcome of innovation and this has also been documented specifically for the food industry (Kristensen et al., 1998). Market

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orientation is often defined as a three-step process of collecting, disseminating, and responding to market information. Generating and disseminating information on user and market needs and incorporating these into product development is a prerequisite for user-oriented innovation, because it is essential to gain an understanding of user needs and then to incorporate this knowledge into product development.

C. Integrated product development and sustainability New product development is an interdisciplinary activity requiring contributions from nearly all functions in a company or cross-functional cooperation and representation of user knowledge (Grunert et al., 2008). Among the factors, that impact the success of new products, the use of cross-functional teams in product development is a key success factor (Cooper and Kleinschmidt, 1996). Obtaining successful collaboration can be a challenge. This is usually attributed to differences in orientations, goals, departmental cultures as well as languages that functional representatives bring to the team. Especially, integration between marketing and R&D has been the focus of research indicating that disharmony between marketing and R&D is the rule rather than the exception (Moenaert and Souder, 1990). The interaction between development and use may vary along the ‘‘life’’ of a product. Synthesis oriented approaches for product development suggest a range of methods to be applied along a product’s life cycle from conceptual idea and product design to manufacturing, distribution, sales and scrapping, recycling, and so on. As the food supply chain is complex, environmental impacts can occur in different places and different times for a single food product. LCA provides a way of addressing this problem. LCA gives businesses the opportunity to anticipate environmental issues and integrate the environmental dimension into products and processes. Important issues directly related to food processing are energy and waste management. Food production in general uses significant amounts of energy and produces relatively large amounts of wastes, particularly, packaging wastes (Mattsson and Sonesson, 2003).

D. The food market focus The food sector faces three strategic developments regarding its production: (a) increasing demand for bioenergy, (b) limits in the availability of water, and (c) diminishing production resources (e.g., land for agricultural use). Furthermore, food production will be affected by pressure from a growing world population and the desire for an increased consumption of meat (Pingali, 2007). Possible changes in climate might magnify the consequences. Without innovations, consumers’ need for

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affordable food without compromises in quality, and which continues to retain their trust, cannot be served in the long run. Consumers’ perception of food quality is a dynamic variable. It might focus on products, processes, process management, or management issues such as fairness in trade, working conditions, environmental awareness, or the origin of products. Its understanding depends on lifestyles, cultures, and so on (Grunert and Wills, 2007; Lobb et al., 2007). New types of efficient and responsive coordinated production, distribution, and communication networks must emerge that can support these changing demands, taking into account varying quality parameters, organizational conditions, and different requirements of market segments (Lindgreen, 2003; Taylor & Fearne, 2006). This may include, for example, new organizational structures for flexible chain-encompassing distribution and logistics systems that utilize advanced technologies for communication, control, or tracking and tracing, developments in quality preservation, new packaging and processing technologies, or organizational innovations such as parallel chains that could provide opportunities to better serve the needs of consumers. Finally, consumers want to get the best quality at the lowest prices— but finding out what the best quality is may not always be a straightforward task. Even providing consumers with more information may not solve the problem, as the information may be ignored or misinterpreted. Public policy is often based on the assumption that more information is better, both to improve daily decision-making and in situations of crisis, but the research summarized by Grunert (2005) implies that more information may not only be without effect, but may in some cases increase confusion and consumer concerns. What is needed instead, is information of educational type, which adds value in both promotional but also knowledge-enhancing way. Much food product differentiation has traditionally been dealt with at the processing level. However, there has also been a trend toward increasing differentiation already at the farm level. There are a number of reasons for this. Consumers demand some kinds of product differentiation that by their nature have to be dealt with at the farm level, such as increased animal welfare or organic production. Advances in biotechnology open up new possibilities for differentiation of both animal and plant production. Product differentiation at the processing level involves replication delays for competitors that are usually short, whereas differentiation that goes back to primary production gives better protection against competitive moves. The same conclusion may be valid to the waste product differentiation and reuse. The outlined concept can be naturally transferred to several areas of industrial food production. The intentions of this research area are located at the development of techniques, which fulfill the conditions of environmental protection with costs to a minimum.

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X. SUMMARY AND FUTURE PROSPECTS 1. Large quantities of food wastes are generated all over the world. The environmental pollution problems associated with conventional disposal methods have been an impulse for the search for alternative, environment-friendly methods of handling food wastes. These biodegradable wastes can be used as support-substrates in SSF to produce industrially relevant metabolites, such as enzymes, organic acids, flavor and aroma compounds, and polysaccharides, with a great economical advantage. Thus, cultivation of microorganisms on these wastes may be a value-added process capable of converting these materials into valuable products. However, much remains to be done in this area to develop commercial processes with technoeconomical feasibility. It is envisaged that in the near future we should be able to develop industrial bioprocesses based on SSF for the production of industrially relevant products utilizing food wastes (Couto, 2008). Filamentous fungi are metabolically versatile organisms that are exploited commercially as cell factories for the production of enzymes and a wide variety of metabolites. It was possible to control simultaneous production of pectinolytic, cellulolytic, and xylanolytic enzymes by fungal strains of the genera Aspergillus, Fusarium, Neurospora, and Penicillium and generate multienzyme activities using a simple growth medium consisting of a solid by-product of the citrus processing industry (orange peels) and a mineral medium. Furthermore, the two-stage process proposed which includes coupling enzymatic treatment and solidstate fermentation, resulted in the production of fermentable sugars which could be converted to bioethanol (Mamma et al., 2008). The ability to determine the flux of carbon, for example, into the desired product, and to identify and overcome bottlenecks in its production, will require a combination of bioreactor technology and global methods of analyses that are only now becoming possible. 2. The green production concept shows a good utilization potential for solid vegetable waste. It could achieve a reduction of investment and raw material costs and can contribute to a waste minimized food production. The development of bioadsorbents is a promising area to add value to vegetable residues. They will appear as a cheap and environmentally safe alternative to commercial ion-exchange resins (Laufenberg, 2003). 3. The exploitation of by-products of fruit and vegetable processing as a source of functional compounds and their application in food is a promising field which requires more interdisciplinary research in the following aspects:

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Food-processing technology should be optimized to minimize the

amounts of waste arising

Methods for complete utilization of by-products resulting from food

processing on a large scale and at affordable levels should be developed. Active participation of the food and allied industries with respect to sustainable production and waste management is required Natural toxins such as solanin, patulin, ochratoxin, dioxins, and polycyclic aromatic hydrocarbons need to be excluded by efficient quality control systems including specific microanalytical methods for the characterization and quantification of organic compounds The bioactivity, bioavailability, and toxicology of phytochemicals need to be carefully assessed by in vitro and in vivo studies (Schieber et al., 2001) Functional foods represent an important, innovative, and rapidly growing part of the overall food market. However, their design, that is, their complex matrix and their composition of bioactive principles, requires careful assessment of potential risks, which might arise from isolated compounds recovered from by-products. Furthermore, investigations on stability and interactions of phytochemicals with other food ingredients during processing and storage need to be initiated. Since functional foods are on the boundary between foods and drugs, their regulation still proves difficult. In any case, consumer protection must have priority over economic interests, and health claims need to be substantiated by standardized, scientifically sound and reliable studies. 4. The ready availability of starch-based industrial wastes and their renewable nature merit their use as substrates for poly-betahydroxybutyrate (PHB) production from activated sludge. This would not only utilize the excess sludge generated and reduce the load on landfills, but would also contribute to reduction in the cost of PHB production by avoiding sterile conditions and pure carbon sources for maintenance and growth of pure cultures. PHB content is the most important factor affecting the production cost of PHB due to its effect on PHB yield and recovery efficiency, followed by cultural conditions and carbon substrates used (Khardenavis, 2007). 5. The comparative presentation of the various vegetable waste treatment methodologies showed that though bioremediation stands for the most environmentally friendly technique, its required longer treatment time in conjunction with its weakness to deal with elemental contaminants makes imperative the employment of a second alternative technique which could either be a membrane process (low energy cost, reliability, reduced capital cost) or a coagulation/flocculation method because of its low cost and high effectiveness. Biogas production appears to be another promising and energy effective waste treatment method (Arvanitoyannis and Varzakas, 2008).

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6. Anaerobic digestion represents a commercially viable process to convert FVW to methane gas, a useful energy source. The overall results of anaerobic digestion of FVW suggest that the two-stage system is a promising process to treat these wastes with high efficiency in term of degradation yield and biogas productivity. This efficiency is possible by the adaptation of each ecosystem to its own substrate. The biochemical reactions involved in anaerobic digestion of FVW are taken subsequently under conditions similar to those of the rumen. It is appropriate to view the gastrointestinal tract as an ecological system and that by applying ecological principles, a better understanding of distribution and interaction of organisms can be achieved, and then it could help to design and construct a suitable bioreactor for FVW anaerobic treatment (Bouallagui et al., 2005). 7. Conventional anaerobic treatment processes are often used for treating dairy wastewaters (Demirel et al., 2005). Particularly anaerobic filters and UASB reactors are the most common reactor configurations employed. In fact, the UASB reactors are very suitable for treating food industry wastewaters, since they can treat large volumes of wastewaters in a relatively short period of time. More research should be directed toward treatment of dairy wastewaters in pilot and full-scale UASB reactors in near future, to make use of these potential advantages outlined. Lipid degradation and inhibition in single-phase anaerobic systems can often be encountered by environmental engineers and wastewater treatment plant operators. Moreover, high concentrations of suspended solids in dairy waste streams can also affect the performance of conventional anaerobic treatment processes adversely. Since the anaerobic digestion process is an imperative tool for the production of clean energy sources, such as hydrogen and methane, biogas production from high-strength dairy industry wastes will always be of paramount importance, as a valuable renewable energy source, for both developed and developing countries in future. Particularly, production of hydrogen by acidogenesis of high-strength dairy waste effluents is currently worth investigating. 8. The thermophilic bioremediation technology for treatment of highstrength organic wastewaters appears to combine the advantages of low biomass yields and rapid kinetics associated with high-temperature operation and stable process control of aerobic systems. It also has the potential of both producing pathogen-free products and the generation of energy out of the process. Furthermore, the average velocity of the thermophilic aerobic bioremediation was almost twice as high as that under mesophilic conditions and compared to the fact that COD and soluble protein levels were reduced during the thermophilic process compared to the mesophilic one, calls for further investigation of the opportunities of this particular promising technology

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(Kosseva et al., 2001, 2003, 2007). The aerobic technologies adapted by many dairy industries for processing of their wastewaters are usually, highly energy intensive and may lead to uncertainty regarding a stabilized performance, due to factors such as overloading and bulking sludge. On the contrary, anaerobic technologies are simpler, require a lower budget to operate, and have the potential of producing energy out of the utilization of the main process product, biogas with a high content in methane (Arvanitoyannis and Giakoundis, 2006). 9. Finally, the food industry uses the LCAs to identify the steps in the food chain that have the largest impact on the environment in order to target the improvement efforts. It is then used to choose among alternatives in the selection of raw materials, packaging material, and other inputs as well as waste management strategies. A new trend in society is when food is considered as the ethical and moral values, this will influence LCA. Combining LCA and social values, such as working environment and animal welfare, is the next step in development of food waste technologies.

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CHAPTER

4 Technological and Microbiological Aspects of Traditional Balsamic Vinegar and Their Influence on Quality and Sensorial Properties Paolo Giudici, Maria Gullo, Lisa Solieri, and Pasquale Massimiliano Falcone

Contents

I. Introduction A. The ‘‘balsamic family’’ B. Historical note C. ‘‘Balsamic’’: From Semitic languages to Italian legislation D. Legal aspects E. Sensorial aspects II. Basic Technology A. Raw material B. Cooking technology C. Fermentation D. The barrel set III. Chemical Composition A. Major compounds B. Minor compounds C. Melanoidins and other biopolymers D. TBV composition during the last three decades

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2009 Elsevier Inc. All rights reserved.

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IV. Physical Properties A. Rheological properties B. Color and spectrum absorbance V. Conclusion References

Abstract

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The term ‘‘balsamic’’ is widespread and popular all over the world of vinegar and fancy foods; it is used generally to refer to vinegars and sauces with a sweet and sour taste. However, the original is the European Protected Denomination, registered as ‘‘Aceto Balsamico Tradizionale of Modena, or of Reggio Emilia’’ that should not be confused with the ‘‘Aceto Balsamico di Modena’’ very similar in the name, but completely different for technology, raw material, quality, and sensorial properties. Traditional balsamic vinegar is made by a peculiar procedure, that starts with a thermal concentration of freshly squeezed grape juice, followed by alcoholic and acetic fermentations and, finally, long aging in a wooden barrel set, by a procedure which requires a partial transfer of vinegar from cask to cask with the consequential blending of vinegars of different ages. In addition, water transfer occurs across the wood of the barrels, the result being an increase of solute concentration of the vinegar. The chemical and physical transformations of the vinegar are mainly directed by the low water activity of the vinegar. Highmolecular polymeric compounds are the main and characteristic constituents of original and old traditional balsamic vinegar, and the major cause of its rheological and sensorial properties.

I. INTRODUCTION ‘‘Traditional balsamic vinegar of Modena’’ and ‘‘traditional balsamic vinegar of Reggio Emilia’’ (here collectively abbreviated as TBV) are two similar types of vinegar, both characterized by a strong local identity as well as chemical–physical and sensory properties, defined by Italian and European legislation. One of the main features of TBV is its aging period, fixed at a minimum of 12 years. During this time, chemical–physical changes take place and give to the vinegar its characteristic sensorial properties. In short, time plays the central role in the overall production process. Control over TBV and its reputation is protected by the constitution of local associations of producers and experts in the field, usually called Consortia. The aim of these Consortia is to promote the culture of TBV and to survey their production and distribution. There are now four active Consortia in both of the interested cities—Modena and Reggio Emilia— with the same purpose and functions. For the TBV of Modena, these are Consorzio Produttori Aceto Balsamico Tradizionale di Modena, Consorzio

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Tutela Aceto Balsamico Tradizionale di Modena, and Consorteria dell’Aceto Balsamico Tradizionale di Modena; for the TBV of Reggio Emilia, they are Consorzio fra Produttori di Aceto Balsamico Tradizionale di Reggio Emilia, Confraternita dell’Aceto Balsamico Tradizionale di Reggio Emilia, and Sindacato Produttori Aceto Balsamico Tradizionale di Reggio Emilia.

A. The ‘‘balsamic family’’ TBV belongs to the wider group of vinegars made from grapes, known worldwide under the generic and legally dubious appellation: ‘‘balsamic vinegars.’’ The market for balsamic vinegars and related products has developed very quickly in a short space of time. It is, nowadays, composed of a wide range of products that at first glance can seem quite similar not only in appearance but also from the sensory point of view; however, all these products can actually be very different in respect of their ingredients, market claims, price, and legal status. It can be very difficult to understand the true differences among this heterogeneous group of products. For this reason, we attempt to present here a summary of what can be called the ‘‘balsamic family.’’ A first clarification can be achieved through observing the legal definitions, as a base on which we can identify three sets, and related protection levels: 1. Condiments: This set is composed of products that cannot be defined as vinegars because of their composition, low acidity level or intended use. There are no limitations as to their composition: they can contain thickeners, preservatives, colors, flavors, and any kind of additives, both artificial and natural. They can be liquid, solid, or semisolid. Sometimes they can resemble vinegars, even balsamic vinegars, in many aspects. Their price and their qualitative level are widely variable and depend on the cost of the raw materials. Balsamic sauces, glazes, jellies, flavored vinegars, dual oil and vinegar compositions, various fruit and vinegar compositions, vinaigrettes are members of this set. General food laws, according to national and international regulations, cover these products. The reasons for the production of condiments are manifold: (i) offering variants of extant products, with altered properties, for example, lower acidity, food colouring, flavor, higher viscosity, etc.; (ii) selling good quality products at a low price, for example, by avoiding the aging time; (iii) developing vinegar- or balsamic-like products containing new raw materials, for example, fruits, vegetables, honey, etc. 2. Vinegars: These are a subset of the larger condiments group. According to a generally accepted definition, vinegars are liquids obtained by the acetic fermentation of any suitable foodstuff. Vinegars are legally defined in many states of the world and sometimes are subject to specific legislation. They usually have a minimum acidity level. To cite just a few

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examples in Europe, vinegars must have a minimum of between 5 and 12 g of acetic acid per 100 ml of product, except wine vinegar which has a minimum of 6 g; however, in the United States, the minimum is 4 g/100 ml (FDA ORA quality manual) and in Australia, it is 4 g/100 g (FSANZ Standard 2.10.1). Vinegars are usually cheap everyday condiments, but their price can rise if they require complex production steps, long aging or highly priced raw materials (e.g., PGI wines). This group includes vinegars such as wine vinegar, rice vinegar, apple cider vinegar, malt vinegar, and honey vinegar that are usually obtained from a single foodstuff through alcoholic and subsequent acetic fermentation. For some countries, flavored vinegars are included in this category. 3. Specialty vinegars: This subset includes vinegars that are legally recognized as peculiar and different from the products of the ‘‘normal’’ vinegar group, for historical, cultural, or other plausible reasons. These types of vinegar are subject to special and dedicated regulations or under special protection such as PGI or PDO. The vinegars belonging to this group are often expensive and produced on a reduced or small scale, like TBV. However, they can sometimes reach huge production numbers, like the Jerez Vinegar or the balsamic vinegar of Modena (not to be confused with TBV), which are actually industrial products consumed worldwide like wine vinegar. Balsamic family products can belong either to the condiments set or to the specialty vinegars set. Figure 4.1 illustrates the three legal levels of vinegar and the balsamic family, while Table 4.1 shows a summary of the balsamic family features. The technological and microbiological aspects described in this review are not specifically mentioned, except in regard to TBV.

B. Historical note TBV is generally described as vinegar of ancient origin, possibly dating back to the Middle Ages and deeply embedded in the gastronomic history of the Italian Provinces of Modena and Reggio Emilia. TBV has achieved worldwide fame in recent times, boosted by several marketing promotions. Notwithstanding their famed time-honored traditions, the culture, the history, and the complexity behind this product are far from being correctly reconstructed and properly understood. It seems that serious comprehensive research into the original production process, the aging system, the sensory profile, and the analytical parameters that properly describe TBV has not yet been accomplished. The piecing together of reliable information from various sources, and the consequent reconstruction of the true history of the TBV of Modena or Reggio Emilia, is a very challenging task because documents and testimonies about them are few

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General food regulations (national or international)

Vinegar regulations (national or international)

Condiments

Vinegars

Special protections (PDO, PGI, national or regional regulations)

Specialty vinegars Balsamic vinegars

FIGURE 4.1 The three legal levels of the balsamic family: condiments, vinegars, and specialty protected vinegars.

and often confusing. Secondly, very few of the publications on TBV and related products can be considered as a valuable source of information, because producers or commercial associates to create a cultural background to use for marketing and promotional purposes. The claimed antiquity of this product and its culture is affected by too many expectations, and derives more from ex nihilo publications than from serious research, whether historical or scientific. However, we can report that according to popular folklore, the world famous condiment now known as TBV originated in the Italian region of Emilia. As far as we can deduce from recent research, it has been steadily produced in its present form in the provinces of Modena and Reggio Emilia; however, little is known about the original recipes and related production practices (Benedetti, 2004; Giudici et al., 2008). As an example of a common misunderstanding of the early documents on vinegar, one of the first generally accepted written testimonies about balsamic vinegar is the allusion to a precious and highly prized condiment made in the province of Reggio Emilia in the poem Acta Comitissae Mathildis, also known as Vita Mathildis or De Principibus Canusinis, written in the 12th century by the monk Donizo of Canossa. In the first book of

TABLE 4.1

The main features of vinegars and ‘‘balsamic family’’

Product type

Vinegar

Vinegar (wine, apple, honey, malt, whey, . . .) Special vinegars Jerez vinegar (Spain) Orle´an vinegar (France)

Yes

Yes

Balsamic vinegars Balsamic Yes vinegar Balsamic vinegar of Modena Traditional balsamic vinegar of Modena Traditional balsamic vinegar of Reggio Emilia

Yes > 10%

Yes (should NOT be used) Yes (should NOT be used)

Grape must (%)

Added sugars

Caramel color (E150a–d)

Thickeners

Flavors

Aging

No

No

No

No

Natural flavors only

Optional not mandatory

0–50% (also used as color) No

No

No

No

No

No

No

No

5–60%

No

Yes

5–60% (should be >20%) 50–100 %

No

50–100%

Market protections

Legal acidity limit

State

No

EU 5 –12% USA > 4% AUS > 4%

Liquid

Yes

PGI

>7%

Natural flavors only

Yes

PGI

>5%

Light to highbodied liquid Liquid

No

–

Usually no

No

–

Yes 2% max

No

No

PGI

>6%

No

No

No

No

60 days min (usually not observed) 12 years min (not declarable)

PDO

>4.5%

No

No

No

No

12 years min (not declarable)

PDO

>5%

Light to highbodied liquid Light to highbodied liquid High-bodied liquid

High-bodied liquid

Condiments Liquid condiments not qualifiable as ‘‘vinegar’’ nor ‘‘balsamic’’ Balsamic sauces, glazes or jellies (condiments)

Depends on recipe

Depends on recipe

Permitted—depends on recipe

Permitted

Depends on recipes

Permitted (usually not used)

Permitted

No

No

No

Liquid

Depends on recipe

Modified or

Permitted

No

No

No

Thick liquid— semisolid

native starch (maize, potato, wheat, . . .) Glucose/ fructose syrup (fluid or dehydrated) Pectins Dextrose Gums (xanthan, guar, . . .) Carob seed flour Lactose

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the poem, there is an account of a luxurious gift given to the Emperor Henry III of Franconia by the Marquis Bonifacio, Lord of Canossa (Reggio Emilia), who in order to display his wealth and power, gave as a present a silver barrel filled with the famous, precious, exquisite vinegar made in the Castle of Canossa (Donizo, Acta Comitissae Mathildis). An important detail not mentioned by previous authors is that in the Italian poem the adjective ‘‘balsamic’’ is never used, even though the words ‘‘balsam’’ and ‘‘balsamic’’ were already present in Italian dialects. What this ancient document tells us is only that a famous vinegar was made in this area; nothing is said about its properties and composition. Similarly, other early references to vinegar are always cited in order to demonstrate the ancient tradition of ‘‘balsamic’’ vinegar making; however, the documents from the area of Modena and Reggio Emilia always cite only the word ‘‘vinegar.’’ It seems that the first use of the adjective balsamic, referring to a particular kind of vinegar, is not documented before the 18th century, where it is found mainly in records of donations or nuptial gifts. From the 19th century, however, we can see a number of testimonies clearly speaking of ‘‘balsamic vinegar’’ and the first recipes or suggestions on how to produce it from the grape must (Benedetti, 2004). Even if the conclusion that the balsamic vinegar cited in those records is directly related to the one we know now is in some way inviting, it should be kept in mind that we have no further evidence to back up this statement. According to other authors, it is also possible that the adjective ‘‘balsamic’’ has been used since those early years to designate any kind of generically aromatic vinegar or mixed vinegar—not just the product obtained from the fermentation of grape must alone (Saccani and Ferrari Amorotti, 1999), as it now must be according to the legal definitions of ‘‘traditional balsamic vinegar of Modena’’ and ‘‘traditional balsamic vinegar of Reggio Emilia.’’ These considerations go to show that we cannot be sure when in history the TBV of Modena and Reggio Emilia began to be produced in the way we are accustomed to today. In addition, no conclusive research has been done to understand historical aspects of the production such as the types of grapes (e.g., by pollen analysis), cooking and fermentation methods, and possible aging systems. Regarding the aging practice, we have to note that the technique used today and which is expressly prescribed by law is the so-called rincalzo (‘‘refilling’’), which is carried out by fractional blending in a barrel set, so that the finished product is a mixture of ages, with the average age gradually increasing as the process continues over many years. The vinegar refines in a barrel set of at least five casks of different sizes and woods. Every year, a small quantity of the aged vinegar is withdrawn from the smallest barrel. This barrel is then refilled from the contents of the preceding barrel and this operation is repeated, up to the first and largest cask, which receives the new cooked grape must (Giudici and Rinaldi, 2007). This method of aging is not exclusive to TBV, even

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though it is an essential part of the TBV culture and mandatory in production regulations. It is also possible that the rincalzo method did not originate in Emilia, nor even in Italy: it is clearly a system invented to save time from year to year in the aging of each barrel (see ‘‘Refilling procedure’’ chapter) and is virtually identical to the Spanish solera system. According to extant documents, the solera system was used in Spain after the Napoleonic Wars and began to spread abroad after the second half of the 19th century (Simpson, 2003). It is thus possible that this aging practice spread from Spain to Italy during the Cisalpine Republic (a French client republic founded by Napoleon Bonaparte in Northern Italy that lasted from 1797 to 1802) mediated by French oenological culture. Further historical research in this direction would be highly useful to understand whether the rincalzo and solera systems are in some way related, or developed independently in Spain and Italy.

C. ‘‘Balsamic’’: From Semitic languages to Italian legislation The word balsamic in the English language is attested as having been in use since the Middle Ages as an adjective from the noun balsam, meaning (i) any agency that soothes, restores, or comforts or (ii) certain officinal plants and/or products thereof. The words balsamic and balsam have gone through different language families, over a very long period of time. In the modern languages of Europe and the Americas, the linguistic form of the name and derived adjectives are still well preserved, because of the common Latin and Greek origin, as we can see from Table 4.2. The various forms of the word balsam all come from Latin Balsamum, which itself came from Ancient Greek Ba´lsamon and has been recorded since the 4th century BC. The form Ba´lsamon has actually been borrowed from the Hebrew bas´am, which is related to Aramaic busma and the Arabic bas´am, meaning ‘‘balsam, spice, perfume, incense.’’ The root on which the words balsam/ balsamic has formed is thus clearly of Semitic origin (Table 4.3) and is usually represented as bs´m (Ko¨bler, 2006; Murtonen 1986; Nielsen, 1986; The American Heritage Dictionary of the English Language, 2004; Vocabolario degli Accademici della Crusca, 2008). Regarding the ancestral origin of this ancient root, very little is known and we can only hypothesize that the Semitic bs´m may be akin to an even older root recognizable in a larger number of languages. So, the adjective ‘‘balsamic’’ is associated not only with TBV but also with an entire class of products far less expensive and ‘‘traditional,’’ well represented in the vinegar and condiment market, with much higher selling volumes than TBV (as shown below). This alternative class of products belongs to the ‘‘balsamic vinegar of Modena’’ (BVM) group and has been legally recognized since 1965. Whereas TBV production is small scale and expensive, BVM is cheaper

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TABLE 4.2 Comparison of the word ‘‘balsam’’ in different European languages Language

Form

Attestation time

Modern English Old Spanish (Old Castilian) Middle English

Balsam, balm Balsamo (?) Balsamum/balsaum Basme Basme/balsme/ balme Balsam Balsam/balsame Balsamo

After 1500 AD 900–1500 AD 1175–1225 AD 1220 AD 1000–1300 AD

Old French Old Norse Old English Old High German Vulgar Latin (Toscano Italian)

Balsamo/balsimo

1000–1300 AD Before 1000 AD Around 1000 AD Since 900 AD

The American Heritage Dictionary of the English Language 2004; The Merriam-Webster Online Dictionary; Ko¨bler, 2006; Vocabolario degli Accademici della Crusca (2008).

TABLE 4.3 The borrowing of the balsam forms by Latin and Ancient Greece from Semitic languages Language

Form

Attestation time

Latin Ancient Greek Hebrew Aramaic Arabic

Balsamum Ba´lsamon Bos´em/bas´am Besma/busma Bas´am

4th century BC 4th century BC

The Merriam-Webster Online Dictionary (2004); The American Heritage Dictionary of the English Language (2004).

and intended for a very wide market; thus it is a fully industrial product. To distinguish these two classes of balsamic vinegar, the ‘‘traditional’’ and the ‘‘industrial,’’ in 1983, a group of producers proposed and obtained legal recognition of the denomination ‘‘traditional balsamic vinegar of Modena,’’ which still exists today in the PDO regime granted by the European Union. The difference between the two denominations relies solely on the adjective ‘‘traditional’’; this is misleading, as it means that the cheaper product receives a sort of marketing benefit from TBV, just through the presence of the adjective ‘‘balsamic’’ in its commercial name. Here it is worth mentioning that, through the centuries, the names associated with balsamic vinegar have never been clear to the general public, and that the existence of these two differing ‘‘classes’’ of balsamic

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vinegars has been recorded at least since the 19th century (Saccani and Ferrari Amorotti, 1999), more than 100 years before true industrialization began. This situation is further confused by the fact that before the legal distinction was made between TBV and BVM, balsamic vinegars were recorded in historical documents under a variety of names such as aceto alla modenese (‘‘Modena-style vinegar’’), aceto del duca (Duke’s vinegar), and aceto balsamico naturale (natural balsamic vinegar).

D. Legal aspects TBV was granted PDO status by the European Community on April 17, 2000 by EC Council Regulation No. 813/2000, in which the Production Regulations proposed by the Consortia were accepted, both for Modena and for Reggio Emilia TBV; they were published on May 15, 2000. The two products must be retailed in a distinctive bottle, one for each TBV, with a legally defined shape and design, sealed and numbered. The grape used for the TBV must come from the Provinces of Modena or Reggio Emilia, following the specified local tradition, and without additives. Each TBV has its specific Production Regulation, though there are no substantial differences between the two recipes. Basically, there are only two significant aspects: the minimum total acidity, which is 4.5% for the TBV of Modena and 5% for the TBV of Reggio Emilia; and the minimum density which is 20 C: 1.24 g/ml for the TBV of Modena and 1.20 g/ml for the TBV of Reggio Emilia. The reason for these discriminations is possibly due to a desire to impose a parametric differentiation between the two types of TBV, which would otherwise remain indistinguishable. According to product regulations in force, TBV is made exclusively from a single ingredient: grape must, cooked in open vats, and matured with a long acetification process through natural fermentation, followed by progressive aging in a series of casks made of different woods (oak, chestnut, mulberry, cherry, and juniper being the most common) even though it is still unclear whether the type of wood has any real influence on the final product (Giudici et al., 2008). Concerning the single-ingredient composition, it is necessary to state that, in spite of such strict legal statements, basically derived from historical practice, many producers, from the second half of the 20th century onward, have begun to produce TBV by mixing cooked grape must and wine vinegar, thereby avoiding the difficult fermentation phase. This ‘‘mixture preparation’’ for TBV has probably been borrowed from industrial balsamic vinegar production, which eschews fermentation after blending, thereby gaining faster results, and more control of acidity and viscosity. However, over a long time scale, this system can easily produce negative effects, mostly in the sensorial features, because it does not produce the overall complexity of the fermented vinegar, causing in many

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cases excessive bitterness, loss of heady flavors, flat aromas, and other sensory degradations. Many chemical–physical defects are also associated with the two-ingredient system, such as liquid-to-solid transitions and the progressive loss of total acidity (Giudici et al., 2008).

E. Sensorial aspects A commission of expert tasters trained by the Consortia performs the sensory analysis of TBV. According to production regulations and published PDOs, the sensory profile of TBV should be evaluated by hedonic judgment expressed in all cases through a numeric score by a panel that evaluates the following sensory attributes: (1) visual-related aspects such as free flowing, color and clearness; (2) olfactory-related aspects such as flavors (fragrance) and aroma in terms of their intensity, persistence, and pungency; and (3) taste-related aspects of vinegar body in terms of its intensity, harmony, and acidity. Ideally, the vinegar color is defined as dark brown, nearly black, but full of ‘‘warm light’’; good texture requires the vinegar to be dense, with a fluid- and syrup-like consistency; good fragrance requires the vinegar to be sharp and unmistakably but pleasantly acid; good aroma requires the vinegar to show the traditional, inimitable sweetness, and sourness in perfect proportion: vivid, full-bodied, velvety, intense, and lingering. The score achieved is used by the Consortia to certify the two possible levels of aging that according to product regulations should be specified as (1) ‘‘Affinato’’ (‘‘fine’’) or (2) ‘‘Extravecchio’’ (‘‘fine old’’). No reference to the year of production or the presumed age of the product may be stated on packaging; ‘‘Extravecchio’’ is the only age-related statement permitted on packaging for this aging level. It is remarkable that, in existing regulations, no methods are specified to evaluate in an objective way the effective aging of the products; it is evaluated only through a panel-tasting test, whose effectiveness for this purpose is clearly inadequate. Among the attempts that have been made for age evaluation, a simple calculation model has been proposed recently to estimate the TBV age (see TBV age paragraph) but, at present, no official Consortia in Modena or in Reggio Emilia have adopted it, or any analogous procedure, as a control system.

II. BASIC TECHNOLOGY The process of TBV production can be divided into four main steps as shown in Fig. 4.2: (i) cooking of grape juice; (ii) cooked must fermentation; (iii) acetic oxidation; and (iv) slow aging of vinegar. The cooking of the grape juice takes between 12 and 24 h and produces chemical and physical

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Basic steps

MUST COOKING

ALCOHOLIC FERMENTATION

ACETIC OXIDATION

AGING

Biological transformation

Physical and chemical transformation

FIGURE 4.2

Basic technology of the traditional balsamic vinegar.

modifications that strongly affect the successive stages of TBV production. After cooking, the must undergoes alcoholic fermentation of sugars by yeasts, followed by acetic oxidation of the ethanol by acetic acid bacteria (AAB), both biological processes taking place in a cask, the ‘‘badessa.’’ These two biological processes take more or less 1 year to be fully completed. The alcoholic conversion is easier to control than that of the acetic acid, which is a serious problem for TBV production because incomplete oxidation of the ethanol produces vinegars with low titratable acidity, affecting negatively the sensory perception of the end quality. Finally, such vinegar undergoes slow aging in the barrel set to concentrate flavors. Aromatic compounds accumulate and intensify over decades, with the vinegar kept in fine wooden casks becoming sweet, brown, viscous, and concentrated. The aging of vinegar is the longest step and it occurs inside a set of barrels of different volumes, made of different types of wood.

A. Raw material 1. The grape In oenology, it is a general opinion that the quality of wines is born in the vineyard. It is widely accepted that a large variability in grape composition is expected as a function of the grape cultivar, climate, and agricultural conditions (Ribe´reau-Gayon et al., 1980). Grape variety and cultivar, agronomical operations, climate, and degree of grape ripeness exert a strong influence on wine composition and sensorial properties. The literature on these topics is extensive, consistent, and widely accepted. However, to our knowledge, there are no significant research data related to grape

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composition and TBV quality. Literature on this topic is poor and vague, suggesting simply that the grape needs to be ‘‘well-ripened.’’ This is a really weak point, because TBV is a sweet/sour vinegar and the balance between acidity and sweetness has been proved to be a very important trait, one which, as everyone concerned realizes, may also be influenced by the berry composition. The major grape constituents are sugars and organic acids, the former consisting mainly of glucose and fructose in a ratio close to 1:1, their precise quantity being strongly related to grape variety, climate, and time of harvest. The other constituents, organic acids, are present in salt form: among them are tartrate, malate and, in smaller amounts, citrate. The balance between sugars and acids changes with grape ripening: in particular, the sugar increases with time whereas malate decreases. Grapes permitted in TBV production come from vineyards of the Emilia Romagna region and have a minimum sugar content of 15% (w/w). According to DOP rules, numerous grape varieties are allowed, with white or red berries; among the most important are Lambrusco (including all varieties and clones), Ancellotta, Trebbiano (all varieties and clones), Sauvignon, Sgavetta, Berzemino, and Occhio di Gatta. However, although the DOP rules on grape varieties and their sugar content are very stringent, to date, there has been no scientific study of the quantitative influence of grape composition on TBV quality. In addition, the length of aging and the refilling procedure required in making TBV complicate the picture. Actually, the balance between sugars and acids is easily modified by collecting grapes with different degrees of ripening. Our recent data show that the ratio between sugars (expressed in g/l) and organic acids (expressed as g/l of tartaric acid equivalents) ranges from 13 to 30, when the sugar content of the berries is at least 16% (w/v). But the proper sugar/acid balance for TBV is still an intriguing unsolved question. For example, in the past, it was usual practice to add common ash obtained from burned woods to decrease the acidity of the grape must before cooking (Sacchetti, 1970). Nowadays, this practice is in total disuse, and a contrary approach is standard: grape musts with high-fixed acidity are required to increase the acidity of TBV. In wine science, other grape constituents such as anthocyanins, polyphenols, and tannins have inspired scientific studies due to their central role in sensorial properties, shelf-life, and wine stability (Ribe´reau-Gayon et al., 1980). During the cooking of grape juice in an open pan, a portion of these minor compounds separate from liquid bulk due to their interaction with proteins and other colloidal material; while, another portion can be involved in polymerization reactions with sugars during the aging process (see Section II.D.3). The grape berry composition is per se not uniform: the pulp is richer in sugar and organic acids; the skin in anthocyanin (red grape) and flavonol (white grape); the seed in polyphenols and tannins (Ribe´reau-Gayon et al., 1980). The grapes are crushed with specialized wine making equipment,

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the juice being separated from the pomace by soft pressure for up to a yield of 70%. Juice composition is expected to be strongly influenced by the pressing conditions of grapes: the earlier juice is rich in sugars and acids, the latter in tannins and salts.

B. Cooking technology Fresh grape juice is boiled down to approximately 30% of the original volume to produce a concentrated must. Cooking is carried out in batchtype stainless steel pans operating at atmospheric pressure, heated by a direct flame burner for 12/24 h at a temperature close to boiling point. The heating causes the formation of foams and natural colloids, mainly coagulated proteins, all needing to be removed mechanically from the surface of the must. Cooking is presumed to exert a key role in the TBV quality. The transfer of mass and energy takes place, and many chemical reactions and physical transformations are activated. Cooking induces the formation of compounds that will act as precursors in the formation of particular sensory-related constituents of TBV; at the same time, it induces the formation of compounds potentially toxic for microbial activity and/or human consumption. This is a long-term practice for some traditional Italian foods and beverages, including traditional balsamic vinegars, and for some special liquor known as ‘‘vino cotto’’ (‘‘cooked wine’’). Cooked grape must is also produced in Spain for sweet wines (Riviero-Pe´rez et al., 2002). The general use of cooked must in European countries is regulated by International laws (Regulation CE 1493/99, 1999).

1. Heat-induced changes during cooking a. Solute concentration The most evident effects of cooking are water vaporization and color change. The process of vaporization involves simultaneous heat and mass transfer, and depends on the heat supply (Fig. 4.3). The extent of vaporization is strictly related to the effectiveness of both the mass and energy transfer. Both, in turn, depend on the rheological properties of grape juice as well as on the evaporator size and on fouling resistances increasing during cooking. Mass and energy fluxes are coded and presented in Fig. 4.3. In a simplified model where the juice is brought instantaneously to the boiling point (Tb), the quantitative analysis of fluxes is based on the conservative mass balance equation: G 2 ¼ Li L f while, for water it is

(1)

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Mechanical stirrer

Water vapor

Hot air

Grape must Stainless steel walls

Fire Vapor (G2, Tb)

Cooked must (Lf, wf, Tb)

Grape must (Li, wi, Ti)

Hot air (H2, T2)

Fire (G1, T1)

FIGURE 4.3 Schematic representation of process streams during must cooking (upper side). Symbols indicate the steady-state condition (bottom side): Li (kg/batch) is the fresh grape must entering the open pan; G2 (kg/batch) is the water vapor leaving the open pan; G1 and H2 (kg/batch) are the hot-dried air streams; wi (kg/kg) is the solid concentration in Li; T is temperature ( C).

Li ð1 wi Þ ¼ G2 þ Lf ð1 wf Þ

(2)

Therefore, the amount of water lost by vaporization at boiling temperature per batch can be calculated by wi G 2 ¼ Li 1 (3) wf A mass balance can be analyzed for each solute of interest: in the case where it takes part in a chemical reaction, so that its specific extensive properties (such as mass or moles) can be measured over time, the generation term must be accounted for in the balance equation: inflow outflow þ

ds ¼0 dt

(4)

where ‘‘s’’ is the solute of interest, which can act as reactant, intermediate or product of a reaction.

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The size of the pan is equally important and deserves consideration: it determines the rate of juice-heating. The heat transfer rate, expressed as kg/batch, can be calculated by q ¼ UAðTb TiÞ

(5)

where U is overall heat transfer coefficient (W/m2/ C), A is the area (m2) of the pan walls, and q is the heat flux (kJ/s). It should be noted that the coefficient U is a function of the sum of the resistances to the heat transfer. The most important resistances are the increasing viscosity of the juice and the semisolid layers on juice surfaces.

b. Chemical changes It is well documented that heat up-take induces some complex transformations in sugar-rich foods. Some of them lead to the formation of brown compounds, the so-called ‘‘melanoidins.’’ These compounds are expected to have significant effects on the end quality and in consumer acceptance of widely consumed dietary goods (e.g., coffee, cocoa, bread, malt, and honey) thanks to their antioxidant properties (Delgado-Andrade and Morales, 2005; Rufian-Henares and Morales, 2007; Verzelloni et al., 2007), antimicrobial activity (Rufian-Henares and Morales, 2007), antihypertensive properties (Rufian-Henares and Morales, 2007), prebiotic activities (Borrelli and Fogliano, 2005), browning properties (Gogus et al., 2007; Hofmann, 1998), and foam stability (D’Agostina et al., 2004). Sometimes, melanoidins are considered to be potentially undesirable compounds playing a strong role in the binding of nutritionally important metals (O’Brian and Morrisey, 1989) and flavored compounds (Hofmann et al., 2001). All of these functionalities are presumably derived from the fact that the melanoidin structures are sufficiently diverse to have complex functional behavior. The heat supply leads to the formation of some potential toxicants: methylglyoxal, furfuryl, and furan derivates including 5-hydroxymethylfurfural (HMF). HMF is a cytotoxic, genotoxic, and tumorigenic agent (Janzowski et al., 2000; Zhang et al., 1993). However, HMF and methylglyoxal act as intermediate chemicals and their concentration and lifetime are related to the initial reducing sugar concentration and to the extent of sugar degradation. In model systems, the accumulation of 5-hydroxymethyl furfural is a function of the kind of sugar and amino acids (Gogus et al., 2007); in grape juice, fructose is more reactive than glucose. Masino et al. (2005) observed that cooking promotes the formation of some furanic congeners in grape must: at the end of cooking they found from about 3.4 to 6.8 ppm of HMF; from 3.8 to 2.3 g/kg of furoic acid; and from 7.8 to 4.8 ppm of furfural as a function of the starting grape must. Water activity plays a central role on the kinetics of HMF formation upon grape must cooking (Muratore et al., 2006).

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Sugars undergo other degradation reactions leading to the formation of acetic and lactic acids, the last one in both the D and L isomers (Fig. 4.4). In particular, the extent of these degradations is strictly related to the grape juice composition. The time-dependent accumulation of solutes follows different kinetics leading to high-chemical potentials that are responsible for the accumulation of newly formed compounds with high-molecular size, over 500 kDa (Fig. 4.10).

c. Physical changes During cooking, the grape juice undergoes profound changes related to the solute interactions which generally affect juice density, refraction index, viscosity, boiling point, specific heat, and coefficient of thermal expansion (Rao et al., 1984; Saravacos, 1970; Schwartz and Costell, 1986). Cooking stops all enzymatic browning reactions that rapidly occur inside fresh grape musts by polyphenol oxidase, and progressively promotes grape must discoloration (due to the heat-induced deactivation of proteins including browning enzymes). In addition, it promotes nonenzymatic browning reactions. The most evident effect of water vaporization is the increase of solute concentration and viscosity; clarified juice concentrates show Newtonian behavior (Ibarz and Ortiz, 1993; Rao et al., 1984; Saenz and Costell, 1986; Saravacos, 1970) although some authors have found a small pseudoplasticity in the flow of grape juice for concentrations above 55 Bx (Brix degree). However, other authors have observed that juice concentrates behave as Newtonian fluids even at high-soluble solid concentrations of 60–70 Bx (Rao et al., 1984; Schwartz and Costell, 1986). It has been postulated that pectins and tartrates affect the rheology of grape juice in a significant way during cooking (Moressi and Spinosi, 1984; Saravacos, 1970), but others assert that high-molecular size biopolymers induced by thermal treatment play the most important role (Falcone and Giudici, 2008).

C. Fermentation 1. The scalar fermentation Fermentation is the name conventionally attributed to any industrial transformation that involves microorganisms. In TBV production, there are two distinct fermentations: alcoholic and acetic; the first is carried out by yeasts of different genera and species, the second by AAB. Usually, the two fermentations occur in the same vessel (badessa) and are performed by wild strains without any control. The results are unpredictable: sometimes the alcoholic fermentation is inhibited by acetic acid produced by AAB; often the acetic acid fermentation, for various reasons, does not occur. To solve the problem, a two-stage fermentation procedure has been suggested: the oxidation of ethanol should always follow alcoholic

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1600

5

1400

D-glucose

1200

4

D-fructose D-acetic acid

800

C(t)/C0

3

600 400

2

HMF(t)/HMF(0)

1000

200 1

D-malic acid L-lactic acid D-lactic acid pH Density aw

0

HMF

−200

Titrat. Ac

−400

0 0

5

10

15

20

25

5

4900 4400

4

3900

2900 2400

2

1900

HMF(t)/HMF(0)

C(t)/C0

3400 3

1400 900

1

400 −100

0 0

5

10

15

25

20

Cooking time (h)

HMF aw pH Density D-fructose D-glucose L-tartaric acid L-malic acid Acetic acid L-lactic acid D-lactic acid

0.00

16.00

FIGURE 4.4 Effect of the cooking time on the composition of grape juice from the Trebbiano grape variety. ‘‘A-type’’ grape must was harvested in mid-October 2009; while ‘‘B-type’’ grape must was harvested 2 weeks later. All investigated properties have been normalized in respect to their initial value to the fresh juice; corresponding differences before and after 16 h of heating are reported at the bottom of the table.

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fermentation, in a scalar way and in two different vessels (Giudici et al., 1992). In the past, it was accepted that fermentation of cooked must was a commensalistic interaction between yeasts and AAB (Sacchetti, 1932, 1970). The idea of commensalisms has not completely gone away; and some practices, such as the use of one single vessel for both fermentations, are still in use.

2. Yeast and alcoholic fermentation a. The yeast involved The alcoholic fermentation of grape juice has been an ecologically well-studied process since the time of Pasteur. During the last 100 years, papers have focused on the dynamics of yeasts during wine fermentation, elucidating the role of Saccharomyces and nonSaccharomyces yeasts (Amerine and Kunkee, 1968; Davenport, 1974; Fleet and Heard, 1993; Kunkee and Amerine, 1977; Kunkee and Bisson, 1993). Only recently has the importance of yeasts for TBV production and quality become clear that their metabolism is responsible for major physical–chemical changes of cooked must (Landi et al., 2005; Solieri et al., 2006, 2007). The first study of TBV yeast dates back to the 1930s. In a paper published by Sacchetti (1932), later summarized in a book (Sacchetti, 1932, 1970), the author recognized strains belonging to the genus Zygosaccharomyces (very similar to those recognized as Z. rouxii, according to the latest nomenclature) as the predominant TBV yeasts, and proposed the idea of a commensalistic interaction between yeasts and AAB. In the 1980s, Turtura and coworkers investigated the main TBV-related species (Turtura, 1984, 1986; Turtura and Benfenati, 1988). They reported the presence of Z. bailii and Z. rouxii, identified on the basis of morphophysiological features, such as the ability to grow at 1% acetic acid concentration. Afterward, the occurrence of Saccharomycodes ludwigii strains, together with Z. rouxii and Z. bailii, was demonstrated. Recently, a complex yeast microflora, including Z. bailii, Z. rouxii, and S. ludwigii, Z. mellis, Z. pseudorouxii, Z. bisporus, and Z. lentus, two species belonging to Hanseniaspora genus (H. osmophila and H. valbyensis), two Candida species (C. stellata and C. lactis-condensi) and an S. cerevisiae species have been found by Solieri et al. (2005, 2006). All Zygosaccharomyces species recovered from TBV are osmophilic yeasts growing in media with a high sugar concentration (50–60%) and are responsible for the spoilage of sugary beverages and food (Fleet, 1992; Loureiro and Malfeito-Ferreira, 2003; Pitt, 1975). Among TBV yeasts, it is remarkable that many strains belong to Z. lentus, a new osmotolerant species first described by Steels et al. (1998, 1999) in spoiled beverages, such as orange juice and tomato ketchup. TBV has also been the isolation source of a new putative species, provisionally named Z. pseudorouxii (Solieri et al., 2006).

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Some other TBV-associated species, mainly Candida and Hanseniaspora spp., are rarely detected in spoiled foods and beverages, but are prevalently associated with the early stages of wine fermentation, even though they can also occur during middle and late phases. In spite of being a nonosmophilic species, S. cerevisiae has been frequently detected in the lowest sugary, cooked musts (Solieri et al., 2006); this agrees with Deak and Beuchat (1996) who found that some strains of S. cerevisiae are able to grow in food or beverages with high sugar content.

b. The product of fermentation The influence of S. cerevisiae and nonSaccharomyces yeasts on the flavor of wine and wine vinegar is well characterized (Ciani, 1998; Fleet, 2003). C. stellata strains have been found to produce high glycerol, succinic acid, ethyl acetate, and acetoin concentrations that influence positively the aromatic profile of wine vinegar (Ciani, 1998). Two other TBV-associated yeasts, H. osmophila and S. ludwigii, produce high amounts of ethyl acetate, acetoin, acetic acid, and acetaldehyde and are considered detrimental yeasts in wine fermentation (Ciani and Maccarelli, 1998; Granchi et al., 2002). S. ludwigii has been proposed for continuous production of vinegar by Saeki (1990). The role of yeast secondary metabolites in TBV sensorial quality has not yet been studied; deeper knowledge of this topic is required.

3. Acetic acid bacteria and oxidation The generic name ‘‘acetic acid bacteria’’ indicates a heterogeneous group of strictly aerobic bacteria. Nowadays, the AAB group includes spoilage bacteria of fermented beverages, bacteria exploited in biosynthesis with highly economically relevant molecules, vinegar bacteria, and recently pathogenic bacteria. However, historically, AAB were recognized as ‘‘vinegar bacteria,’’ showing the important traits for bioconversion of ethanol into acetic acid (Gullo and Giudici, 2008). The most relevant phenotypic feature of AAB is related to their ability to carry incomplete oxidation of broad ranges of carbohydrates (aldehydes, ketones, and organic acids) that are secreted almost completely into the medium. In TBV, the oxidative fermentation is carried out by spontaneous acetification due to the natural occurrence of AAB in the environment. Recently, the application of selected AAB strains in TBV production has been proposed, and a procedure has been developed for the scale-up of the fermentation process at the vinegar factory scale (Fig. 4.5) (Gullo et al., 2009). However, the actual basic technology used for cooked must fermentation is without any physical–chemical control; therefore, fermentation breakdowns cannot be predicted. Ecological studies on AAB of TBV were first conducted by culture dependent methods. The oldest studies date back to 1970 and 1988 (Sacchetti, 1970; Turtura and Benfenati, 1988) and deal with the ecological aspects of AAB in TBV.

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Strain selection

Stage 1 (Laboratory) Refillings (701) Factory transfer Tank acetification Stage 2 (Scale-up) Refillings (9001)

Stage 3

Analytical and molecular monitoring

Liquid GY culture (5 ml)

Barrel acetification

FIGURE 4.5 Critical steps in the scale-up of the fermentation by selected starters from laboratory to industrial scale (adapted from Gullo et al., 2009).

More recently, intermediate products of TBV have been investigated using both culture-dependent and -independent methods. In particular, strains belonging to the following species were detected: Gluconacetobacter europaeus, Gluconacetobacter hansenii, Gluconacetobacter xylinus, Acetobacter pasteurianus, Acetobacter aceti, and Acetobacter malorum. All of these species were previously detected in different kinds of vinegars, except for A. malorum (De Vero et al., 2006; Gullo and Giudici, 2006, 2008). Among the species recovered until now, the Ga. europaeus strains seem to be the most widespread.

a. The products of oxidation Many studies have been carried out on AAB metabolism, mainly focusing on the physiological behavior of strains in defined media as well as an understanding of the enzymatic system of AAB. The majority of studies focused primarily on the Gluconobacter oxydans species because of its relevance in biotechnological applications, such as the synthesis of Vitamin C, gluconic acid, and several biopolymers (Adachi et al., 2003; De Vero et al., 2006; Deppenmeir and Ehrenreich, 2009; Macauley et al., 2001). In general, sugars, alcohols, and polyols are oxidized via two alternative pathways by two types of enzyme

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systems, differing in location and function within the cell, that are capable of dehydrogenation reactions. However, the chemical composition of TBV, relating to the AAB oxidation products, is highly variable and depends on several factors such as the type of grape must, cooking modality, oxidation temperature, and others. A lack of information about the compounds originating from AAB is due to the fact that importance has been given to substances detected in the final product while the dynamics of these compounds over the course of the production time has been ignored. Some volatile compounds (acetoin, diacetyl and ethyl acetate) have proved to be useful in characterizing the bacterial strains involved in the acetification process (Gerbi et al., 1995). Interest has been shown in TBV as well as in other vinegars produced by traditional technology, with the aim of differentiating them from vinegars produced by quick processes (Tesfaye et al., 2002). Among organic acids, acetic acid is the most studied compound, due to the fact that it is strictly related to both the safety and the sensorial quality of TBV. High amounts of this organic acid can mask other sensorial properties and potentially adulterate vinegars. Among sugar acids, several authors have studied gluconic acid. The availability of glucose together with very low nitrogen and phosphorus sources are the optimal conditions for the gluconic acid production in vinegar: it has been found at up to 3.0% in TBV (Giudici, 1993; Plessi et al., 1989) and at lower concentrations (0.37% and 0.28%) in wine vinegar, cider vinegar, and balsamic vinegar (the last obtained with a short-time acetification of a blend of cooked grape must and wine vinegar). The occurrence of gluconic acid has been proposed as an indicator of TBV quality, a way of differentiating it from other balsamic vinegars (Giudici and Masini, 1995).

D. The barrel set The TBV is aged in a barrel set usually composed of at least five casks of different sizes and woods, mainly chestnut, acacia, cherry, oak, mulberry, ash, and, in the past, juniper. It is generally agreed that the type of wood used plays an important role in the aging process and sensorial properties of the TBV. The casks are arranged in decreasing scalar volume, generally from 75 to 16 l (Fig. 4.6). The smallest one contains the oldest vinegar of the set and is conventionally numbered ‘‘1.’’ The barrel set behaves essentially as a device for vinegar concentration. Two types of process streams take place: (i) the mass transfer from vinegar bulk toward the ambient throughout the wooden casks (water lost by evaporation, vinegar leakage throughout the staves, and solutes lost by precipitation) and (ii) the mass transfer from cask to cask spanning the barrel set (see ‘‘refilling’’ paragraph). Evaporation varies as a function

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Alcoholic fermentation and acetic oxidation

Badessa 120L

The refilling keeps constant all casks volume

Barrel n. 5 75L

Barrel n. 4 50L

Barrel n. 3 33L

Barrel n. 2 24L

Barrel n. 1 16L

Oak

Mulberry

VTBV= 5L Oak/chestnut

Cherry/oak

Juniper

Chemical and physical aging

FIGURE 4.6 A possible configuration of the barrel set. The withdrawn of TBV is made once per year. The refilling procedure is aimed at keeping constant the volume of vinegar in all the casks. The casks have a decreasing volume. Conventionally the smallest cask is referred to as ‘‘barrel no. 1.’’

of cask features and ambient conditions. Leakage may occur in the presence of holes and/or defects in the wood, often caused by rapid changes of hygroscopic conditions, and may result in buckling and cracking of the stave. Solute precipitation may occur depending on the precise amount of insoluble matter and their degree of solubility. In particular, the wooden cask works as a molecular separation device: low-steric dimension molecules pass selectively throughout the wood pores and the higher ones are retained (Siau, 1984), resulting in a general increase of the solute inside the vinegar bulk. The residence time (RT) of the vinegar inside the barrel set depends on both the rate of water lost by evaporation and the mass flux to volume ratio. The rate of water transfer through the wooden casks is influenced by several factors including the hygroscopicity (water activity) of the vinegar, the relative humidity of the surrounding air, the type of wood, its thickness, and the size and shape of the cask. Another factor is the way the barrels are closed on the topside: historically, each cask had either a large rectangular opening, covered by a cloth, or a round hole covered by a round rock from local rivers.

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Recent evidence shows that the use of hermetically sealed barrels improves the TBV quality because it serves to preserve the vinegar’s aromatic compounds.

1. Yield

The yield can be calculated easily as the mass fraction ratio between the cooked must and TBV withdrawn from the smallest cask of the barrel set: yield ¼

mTBV 100 mFCM

(6)

Yield indicates the ability of a barrel set to concentrate cooked must at given operating conditions. It is worthwhile noting that neither the volume of casks nor their number affects yield, which is affected only by the solid concentration in the cooked must coming from the ‘‘badessa.’’ For example, a cooked must containing 35–40% (in weight) of glucose and fructose is fermented by yeasts to have ethanol at around 5% and residual sugars between 20% and 25%, and at the end of aging between 40% and 50% in weight. Using Eq. (6), it is easy to calculate that a yield of TBV ranging between 40% and 65%—in other words, 3 kg of cooked and acidified must—can provide between 1.2 and 1.8 kg of TBV. The yield of TBV production does not depend on the size of the barrel set; rather, it depends on the flux of masses across the barrel set.

2. Refilling procedure a. Process streams in wooden barrels The barrel set is managed with a

traditional procedure that resembles the Solera method used for making Sherry wine. In particular, a coded procedure is followed for the annual cask refilling, consisting of withdrawing only a part of the vinegar from the smallest cask, which is then refilled with the vinegar coming from the next barrel, and so on. The biggest one receives new cooked and oxidized must (see Fig. 4.6). The purpose of refilling is to keep the volume of the vinegar constant inside every cask of the barrel set, reintegrating the product withdrawn and/or lost by evaporation and/or transferred from cask to cask. The mass transfer of vinegar throughout the wooden staves involves the following aspects: (i) a transition time is required to reach the outside of the stave; (ii) the loss by evaporation consists mainly of water; (iii) the mass flux throughout the staves is a function of wood type; (iv) the mass flux is more pronounced when the wood is in direct contact with the vinegar; and (v) the mass flux changes with ambient conditions. The most common model used to describe the mass transfer across the wooden barrels assumes three mechanisms in series: absorption, diffusion, and evaporation. A Fickian model for calculating water losses from oak casks depending on conditions in aging facilities has been recently proposed by

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Ruiz de Adanaa et al. (2005). These authors proved that the pressure potential drives the water flow across the staves within free domains according to Darcy’s law; while, the diffusion mechanism drives the water transfer within hygroscopic domains inside the staves according to the Fick’s law. The lower the relative humidity, the higher the transfer of water. However, mass diffusion through the wooden staves represents a rate-limiting stage of the vinegar transfer throughout a barrel, and Fick’s second law describes quantitatively the mass transfer under isothermal conditions: @M @ @M ¼ D (7) @t @x @x where x is the thickness of the stave, and D the diffusion coefficient (m2/s) of the wood. In a simplified model, diffusion occurs under a gradient of moisture in the wood, and the mass flux is proportional to the driving force; that is, the difference between the average water content within the wood and the water content at the saturation equilibrium point of wood. Conversely, water may be transferred also from the ambient into the wood. Of course, mass transfer occurs if driving forces can prevail over the opposing resistances such as the concentration of the hygroscopic solutes in vinegar and the thickness of the stave. The aging causes the water activity of vinegar to decrease due to the increase of solute interaction and their ability to link water molecules so that the water activity will become progressively the limiting driving factor for the evaporation. It is useful to define an overall mass transfer coefficient as a function of the ambient conditions so that a surface emission coefficient gradient can be defined by dS ¼

@S @S @S dT þ dRH þ dV @T @RH @V

(8)

where temperature (T), relative humidity (RH), and air velocity (V) contribute differently to the mass transfer. In general, higher temperatures and lower relative humidity make vinegar concentration faster.

b. Vectorial concentration model There is experimental evidence that the concentration of solutes increases from left to right along the barrel set (Fig. 4.6) (Sanarico et al., 2002). This vectorial product concentration (VPC) is due to two additive factors: the annual refilling of casks and the water lost by evaporation. In particular, the refilling procedure is responsible for solute dislocation: the same amount of solutes (dry matter) is transferred from cask to cask (Table 4.4) but in a decreasing volume, as required by the water evaporation. The theoretical basis of the VPC model is explained with the analysis of the degree of freedom for the process

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TABLE 4.4 Analysis of the degree of freedom (DOF) of the process streams across a barrel set consisting of five of casks, see Fig. 4.9 Set of equations (E)

Technological constraints Physical constraints Overall volume transferred across the barrel set Dry matter transferred from cask to cask Mass of water leaving the barrel set Mass of water transferred from cask to cask

Viþ1 ¼ kVi si þ wi ¼ 1 X5 ðWi Þ RTBV ¼ RFCM i¼1 mFCM sFCM ¼ mi si ¼ mTBV sTBV X5 i¼1

ðmw Þi ¼ ½mFCM wFCM ½mTBV wTBV

mFCM ð1 sFCM Þ ¼ mi ð1 si Þ ¼ mTBV ð1 sTBV Þ

Assuming null all vinegar lost (Li), we have 17 unknown variables (i.e., six streams with two components, that is, water and solutes; five streams with one component, i.e., water), 12 mass balance equations, and constraints: DOF ¼ 22-E-11-5. The mass transfer becomes a solvable problem if we have 11 independent equation (E). wi and si are the mass fraction of water and dry solutes within the streams, respectively; Riþ 1,i refers to the vinegar volume withdrawn from the barrel i þ 1 and used to refill the barrel i; Wi are the water volume lost by evaporation; mi are the mass fluxes; i is the number of the barrel (with 1 i 5 and ‘‘1’’ is the smaller barrel).

streams involved in any cask as reported in Fig. 4.7. All sets of equations necessary to evaluate mass transfer are reported in Table 4.4. A practical way to save time in TBV production is to extend the vectorial concentration in the barrel set for example by increasing the number of casks.

3. The aging a. Definition of the age and its descriptors The meaning of the age as related to a special food product such as TBV calls for two distinct concepts. Firstly, irrespective of how much or how little the vinegar properties change, we can define the TBV age as the time that it spends in the barrel set. In this case, the TBV age corresponds to the turnover time; that is, the time it takes to completely replace the product in the cask (RT). Secondly, accounting for time-dependent changes in chemical, physical, and sensorial properties occurring at a given RT, we can define an appropriate physical age of TBV. In both cases, the variable chosen to describe the TBV age must have additive property. Consequently, as is true for every extensive property, the descriptors of TBV age must depend on the size of the barrel set and will accumulate during the storage time.

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W5 (m5)

W4 (m4)

W3 (m3)

W2 (m2) W1 (m1)

R4-3 (m4, s4)

R5-4 (m5, s5)

R3-2 (m3, s3)

R2-1 (m2, s2)

FCM (mFCM,sFCM)

TBV (mTBV, sTBV)

L5 (m5, s5)

L4 (m4, s4)

L3 (m3, s3)

L2 (m2, s2)

L1 (m1, s1)

FIGURE 4.7 Vinegar streams across the barrel set during aging. FCM refers to the new cooked must from the ‘‘badessa’’; TBV refers to the vinegar volume ready to be bottled; Wi refers to the water volume lost in evaporation; Ri þ 1,i refers to the vinegar volume withdrawn from the barrel i þ 1 and used to refill the barrel i; Li refers to the vinegar volume lost from staves; mi are the mass fluxes; si is the mass fraction of dry solutes within vinegar streams; i is the number of the barrel (with 1 i 4 and ‘‘1’’ is the smaller barrel). The vinegar volume inside the casks (i) is kept constant by the annual refilling: the solute translocation caused by refilling practice and water lost for evaporation determines the vectorial product concentration across the barrel set.

b. Residence time Due to the refilling procedure, each barrel contains a blend of vinegars of different composition and age: intuitively, one would expect the age to increase according to the VPC model and, therefore, from the largest barrel to the smallest. What complicates the assessment of RT is the discontinuous refilling practice: each barrel, every year, contains a new blend of vinegars of different ages. Casks behave as continuous evaporators and as splitter devices at the end of each year during refilling (Fig. 4.8). Giudici and Rinaldi (2007) proposed a simple model to calculate the age of the vinegar blend as the pondered value of the RT of vinegar inside the cask (i) before refilling plus the RT that the vinegar used for refilling has spent inside the previous cask (i þ 1): RTvinegar blend n ¼ Vi RTi n þ Ri RTiþ1 n

(9)

where n is the number of years (and the number of refillings performed). In particular, the authors formulated RT(n) as a way of defining the following extensive quantity that accumulates over the years: RTi n ¼ RTi ðn 1Þ þ 1

(10)

Traditional Balsamic Vinegar and Related Products

Residence time (RT) RT(n) = [(n−1) + 1]

Wi (mw)n+1

RT = n

Cask i

Ci (mc, sc) RTc = n + 1

Splitting Ri-1 (mRi-1,sc) RTRi + 1=n + 1

Cask i

Ev aporation

Bi+1 (mBi+1, sRi+1)RT = n

Vi+1 (mVi,sc) RTVi = n+1

[Vinegar blend]n RTBi+1(n) = Vi+1,RTi+1(n)+Ri+1,RTBi+1(n)

Ri+1 (mRi+1, sRi+1) Rti+1 = n

165

RT = n + 1 RT = n + 1 Cask i

Cask i + 1

Ri (mRi,sRi) RTRi = n + 1

Wi+1 (mwi+1)n+1

Re

filling

Bi(mBi,sBi) [Vinegar blend]n+1 RTB3(n) = Vi,RTV3(n+1)+Ri,RTBi(n+1)

FIGURE 4.8 Virtual splitting of the vinegar streams involved in the cask (i) of the barrel set during aging. Casks behave as continuous evaporators and, as splitter devices at the end of each year, during refilling. The age of the vinegar in the cask (i) is the pondered value between the RT of the vinegar coming from the cask (i þ 1) and that of the vinegar present into the cask (i).

This definition of the age is valid for 1 i 4 because the age of the cooked and acidified must is assumed to be nil. The study carried out by Giudici and Rinaldi (2007) demonstrated that: (i) the RT of vinegar in a barrel set reaches an upper finite limit at the steady-state; (ii) limit increases from the biggest to the smallest cask according to the ratio between refilled volume and volume of cask; and (iii) the upper aging limit is a decreasing function of the volume leaving the barrel set, that is, the quantity of TBV withdrawn. It is worth noting that the above-mentioned limits are reached with n; that is, the number of years theoretically goes to infinity, but in reality, each RT limit is more or less reached after very few years of storage (Fig. 4.9A and B). The mathematical model can be used to find how much TBV can be withdrawn to satisfy the legal limit of 12 years or, conversely, to verify whether the minimum legal age required has been reached at a given productivity. According to the proposed model, an increase in the production rate leads to a decrease of RT: in fact, the solids concentration inside the process streams decreases, even though more feed (cooked

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A

Barrel no.1

RT(n)-years

20

Barrel no.2

15

Barrel no.3

10

Barrel no.4

5

Barrel no.5

0 20

40

60

80 n (years)

100

120

B

140

vP = 21 25 vP = 31

RT(n)-years

20

vP = 41

15 vP = 9.0131 10 5 0 20

40

60

80

100

120

140

n (years)

FIGURE 4.9 Numerical simulation of the effects of the refilling on the upper limit of the RT for each barrel of the set with a withdrawal of 3 l (Vp), and on the hypothesis of constant water evaporation (5% of the volume per year) (A); upper limit of the RT for the smallest cask with different volumes of vinegar withdrawn (B). The increase of the vinegar withdrawn leads the vinegar age to decrease (adapted from Giudici and Rinaldi, 2007).

must from badessa) is being processed, because the time of residence is reduced.

c. Physical ripening time At a given RT, each vinegar is characterized by thermodynamic properties that are the results of specific microstates, the actual interaction among all the constituents. Refilling practices, of course, periodically perturb all chemical potentials among vinegar constituents as

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4

Abs(420 nm)

3E

+0

4

2E

+0

3

+0

5

.5 .5

6 .5

min

)

.5

e(

.5

tim

1

27

tion

24

Elu

1 11 4 .5

9

15

21

. 12

18

6E

Cm

rs)

3.5

e

V TB

ag

a (ye

FIGURE 4.10 Distribution of the brown-labeled chromophores of melanoidins accumulated in the vinegar during aging. The vinegar age was calculated as RT; elution time is the HPLC-column elution time; and Cm represent the cooked must for which RT is considered to be nil. Signal is the 420 nm-radiation absorption of melanoidin (adapted from Falcone and Giudici, 2008).

well as the balance of their interactions. The thermodynamic state of TBV is described by intensive and extensive quantities. The former (temperature, volume, pressure, internal energy, chemical potential) are independent of the amount of material in the vinegar at a given time and are therefore not sufficient to provide unbiased information on TBV constituents’ microstate upon ripening. On the other hand, the extensive quantities (entropy, total volume, total surface area, mole of solutes, mass of solutes, number of charges or electrons) may be easily linked to the vinegar age. Vinegar behaved as an ‘‘out of equilibrium’’ system in the early years, with the extensive properties continuously changing towards a ‘‘partial equilibrium’’ after a long RT (Falcone and Giudici, 2008). The products of polymerization reactions are good descriptors of TBV aging since they are related both to RT and PRT. The evolution of the molecular size as a function of RT of TBV is reported in Fig. 4.10. These polymers possess a distribution character in a variety of physical properties simultaneously, such as molar mass, density, chemical composition, viscosity, and thermal properties. The distribution characteristics are good descriptors of the physical age (PRT, Fig. 4.11).

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A MWD parameters

6.0

V1

4.0 1

2

2.0

GMp GMn GMw GMz GMz1 GMv

1 3

4

5

2.0

6

C 1.0

P1

0.8

0.6

0.6

0.4

0.4

0.2

0.2 0

2

4

6

8 10 12 14 16

Age of TBV (years)

2

3

4

5

0.0 D 1.0

0.8

0.0

V2

4.0

0.0

MWD parameters

B 6.0

0.0

6

P7 GMp GMn GMw GMz GMz1 GMv

0

2

4 6

8 10 12 14 16

Age of TBV (years)

FIGURE 4.11 Biopolymers’ absorbance at 280 nm (A, B) and 420 nm (C, D); GMp, GMn; GMw, GMz1, and GMv are the characteristics of the molecular size distribution, having physical meaning as described by Falcone and Giudici (2008), all describing quantitatively the physical age of the traditional balsamic vinegar. All values have been normalized with respect to their initial value in the fresh cooked must. The vinegar age corresponds to the RT (adapted from Falcone and Giudici, 2008).

Attempts to identify descriptors of TBV age have been made by other authors (Cocchi et al., 2002, 2006; Consonni et al., 2008; Masino et al., 2005) but with unsatisfactory results, mainly due to the uncertainty of the RT of the samples investigated.

III. CHEMICAL COMPOSITION The full picture of TBV composition is actually difficult to complete for at least four reasons: (i) the empirical approach that is used to manage the barrel sets; (ii) the technology for TBV production is roughly the same everywhere but there are differences among the producers; (iii) the analytical techniques used to determine vinegar composition are frequently transferred directly from wine science without any specific validation for TBV; and (iv) the data available in the literature are mainly focused on the macro constituents (i.e., all those present with a concentration of g/l of TBV such as reducing sugars and organic acids): little attention has been

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paid to minor but not less important constituents including polyphenols, volatile compounds, and the end-products of nonenzymatic sugar degradation reactions.

A. Major compounds 1. Sugars The first studies on monosaccharides in TBV were based on enzymatic methods (Plessi et al., 1987): the authors proposed glucose, fructose, together with glycerol, ethanol, and xylitol as vinegar quality criteria. D-Glucose and D-fructose have been studied in TBV in relation to its degree of crystallization (Giudici et al., 2004). Other sugars such as xylose, ribose, rhamnose, galactose, arabinose, mannose, and sucrose were detected in very small amounts (g/kg) (Cocchi et al., 2006). Glucose and fructose are the main components of TBV, their average mass fraction being 23.60 ( 3.45) and 21.14 ( 3.57), respectively; the average and standard deviations (SDs) were calculated on the data of 100 samples randomly chosen during the TBV exhibition held in Modena, 2005 (Table 4.5). It is consolidated practice to use the Bx to indicate the overall content of sugars in TBV, but this assumption is conceptually wrong and creates confusion as to the real solute composition. In addition, sugar and Bx are uncorrelated (Fig. 4.12), which makes it impossible to compare old data with new. Each soluble solid affects TBV’s optical properties and the Bx value is the additive and/or synergic result of the contribution of the individual solute to the overall refractive response of TABLE 4.5 Chemical characteristics of traditional balsamic vinegar, expressed as mean of 104 samples (adapted from Falcone et al., 2008)

a

Parameter

Meana

Standard deviation (SD)

Soluble solids Titratable acidity ‘‘R’’ ratio Glucose Fructose Tartaric acid Succinic acid Acetic acid Malic acid Gluconic acid Lactic acid

73.86 6.67 11.27 23.60 21.14 0.78 0.50 1.88 1.04 1.87 0.12

1.73 0.88 1.53 3.45 3.37 0.25 0.70 0.45 0.32 1.27 1.07

Amount expressed in g/100 g of TBV, as mean value of 104 samples presented at the annual competition held at Modena in 2005; titratable acidity is expressed as gram of acetic acid per 100 g of TBV.

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Brix degree

78

72

66 28

31

34

FIGURE 4.12

37 40 43 46 49 D-glucose + D-fructose (g/L)

52

55

58

Relationship between sugars and Brix.

vinegar. Moreover, the solute-specific refractive index depends on several factors including temperature and concentration, as well as molecular weight and polydispersity of the solute. It is easy to infer that the higher the vinegar concentration, the higher the overestimation of sugar content: the solute contribution to the refractive response of the vinegar has been proved to be higher than that of the cooked must, most probably because of the accumulation in vinegar of high-molecular-weight biopolymers (Falcone and Giudici, 2008). The sugars in solution are present in different isoforms, each of them having different solubility and reactivity, which is an important concern for TBV’s physical stability. Both a and b pyranosidic and furanosidic forms are present in sugar solution, whereas, in TBV, glucose is present in the pyranosidic forms and fructose in both pyranosidic and furanosidic forms (Consonni et al., 2008).

2. Organic acids

The ratio between Bx and titratable acidity (‘‘R’’) was first described by Gambigliani Zoccoli (unpublished data) and it is still used as a quality criterion for TBV (Giudici et al., 2006). Tritatable acidity is usually expressed as grams of acetic acid per 100 g of vinegar, but this does not describe either the type or the amount of the individual organic acids. In particular, the sum of the individual acids, expressed as equivalents, is always less than the corresponding titratable acidity. The degree of vinegar sourness is related to the titratable acidity, the pH, the relative amount of dissociated and undissociated acid, buffer capacity and the relative quantities of individual acids. The acids are all more or less sour and some

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have a characteristic flavor (Ough and Amerine 1988): succinic acid is bitter, acetic acid is pungent, and so on. Recently, the concentration of single organic acid has been determined by selective methods such as enzymatic-based techniques (Giudici et al., 2004, Plessi et al., 1987), chromatographic techniques (Cocchi et al., 2002, 2006), and NMR (Consonni et al., 2008). Unfortunately, the data obtained by different methods cannot be compared, due to the different sensitivity and specificity involved. We believe that the most reliable composition data are obtained by enzymatic methods because they allow the separation of D/L isomers. Table 4.5 shows the average and SD of the main individual organic acids and titratable acidity of a large number of TBV samples. The samples have roughly the same titratable acidity as each other, but they show different compositions for the individual acids (with the exception of tartaric acid because the main part of it precipitates as potassium and calcium salts). In general, TBV contains, in descending order: acetic, gluconic, tartaric, succinic, malic, and citric acids, plus a small amount of lactic acid. The origin of the individual acids varies: some arise from grapes, some from yeasts and bacteria metabolism, and others have more than one origin (Table 4.6). Acetic acid is the result of ethanol oxidation by AAB: a very small amount is formed during must cooking due to sugar degradation. Tartaric acid comes only from grapes. Gluconic acid is produced by AAB but can also be a natural constituent of grapes undergoing mold deterioration, that is, by Botrytis cinerea. It has been proposed as an indicator of TBV authenticity (Giudici, 1993). Succinic acid comes from yeast metabolism and D-/L-lactic acid, which is generally present in a low concentration, comes mainly from AAB and the thermal treatment of grape must (Fig. 4.4).

B. Minor compounds 1. Volatile compounds The volatile and aromatic fraction profile of TBV varies strongly in relation to the method used in their determination. Recent studies have shown the presence of several aldehydes, ketones, alcohols, and esters in TBV (Del Signore, 2001; Natera et al., 2003; Zeppa et al., 2002). Quantitative determinations of volatile compounds have been used to group and discriminate vinegars from different origins. Among alcohols, ethanol and 2-propanol are present in a relatively high concentration in common vinegars; while 1-propanol, isobutyl alcohol, isoamyl alcohol, and 1-hexanol are present in BV. Acetaldehyde is present in a relatively high concentration of common vinegars; while diacetyl, hexanal, and heptanal were three and five times higher in TBV, compared to BV and common vinegars. Esters were found in BV and common vinegars in concentrations higher than that found in TBVs, apart from 1,3-butanediol diacetate (Del Signore, 2001).

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TABLE 4.6 Organic acids: origin and microorganism involved in their formation Organic acid

Origin

Metabolism

Microorganism

Tartaric acid

Grapes

Degradation

Lactic acid bacteria Acetic acid bacteria

Acetic acida

Succinic acid

Acetic acid bacteria Yeasts Lactic acid bacteria Yeasts

Citric acid

Grapes

Gluconic acid

Botrytized grapes Acetic acid bacteria Grapes

Malic acid

Yeasts L-Lactic

acid and D-Lactic acida Piruvic acid a

Lactic acid bacteria yeasts Yeasts and bacteria

Oxidation with high oxygen concentration Carbon dioxide and water

Oxidation with high oxygen concentration Acetic acid

Acetic acid bacteria

Acetic acid bacteria Lactic acid bacteria Schyzosacchaomyces

L(þ)-lactic

acid þ carbon dioxide Ethanol þ carbon dioxide Oxidation with high oxygen concentration

Acetic acid bacteria Acetic acid bacteria

Oxidation with high oxygen concentration

Acetic acid bacteria

Small amounts can be produced by sugars thermal degradation.

2. Phenolic compounds Phenols include several hundred compounds that are broadly grouped as flavonoids and nonflavonoids. The former include anthocyanins, catechins, and tannins; the latter include stilbenes and other compounds derived from benzoic, caffeic, and cinnamic acids. In red wine, up to 90% of the phenolic content consists of flavonoids. These compounds derive in a decreasing order from grape stems, seeds,

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and skins. Tannins refer to a wide range of high-molecular-weight compounds including the two classes of compounds generally known as ‘‘natural tannins’’ and ‘‘pigmented tannins.’’ The natural tannins (proanthocyanins) derive, in descending order of volume, from grape skins, stems, and seeds. The reaction between natural tannins and/or anthocyanins with other phenols, mainly catechins, results in the formation of pigmented tannins. Finally, the barrel wood may itself contribute to the formation of the pigmented tannins, in which case they are known as ‘‘hydrolyzable tannins’’ since they contain ellagic and gallic acids that are usually determined by chemical hydrolysis. However, it is reasonable to suppose that the older barrels contribute to the tannin formation less than the new ones. To date, both qualitative and quantitative judgments of the individual phenolic compounds in TBV are still unsatisfactory. The phenols in TBV were first studied in terms of their overall content by colorimetric methods and GC–MS techniques by Plessi et al. (2006); and by means of colorimetry alone by Verzelloni et al. (2007). The latter showed that the overall amount of the phenolic compounds is strictly related to the antioxidant properties of TBV. Phenols take part in the polymerization reactions during the TBV aging (Tagliazucchi et al., 2008; Verzelloni et al., 2007) and probably the reactions start during the cooking of the must. Discrimination of TBV based on phenolic compositions, aroma compounds, and organic acids has been investigated by Natera (2003): data from 83 vinegars of different origins and raw materials were studied by linear discriminating analysis which allowed differentiation between the 88% of samples investigated, according to their raw materials, and the 100% investigated according to the presence or absence of the aging period in wood.

3. Furanic compounds The most studied product of sugar thermal degradation is HMF, but other furanic congeners (furoic acid, formaldehyde, and acetoxymethylfurfural) were quantified in a barrel set (Chinnici et al., 2003; Masino et al., 2005). HMF is a molecule of public concern for its potential toxicological activity; several vinegars were grouped on the basis of the content of this compound. In particular, BVM showed a concentration ranging between 300 and 3300 mg/l, and higher concentrations—up to 5500 mg/kg—were found in TBV samples (Theobald et al., 1998). Other authors reported similar results for BVM (Bononi and Tateo, 2009; Giordano et al., 2003). It is a general opinion that HMF and related congeners are formed during the cooking of the must, and that these compounds are a highly reactive intermediate of sugar degradation, including Maillard reactions (Berg and Van Boekel, 1994). HMF formation and Maillard reactions occur also at room temperature, especially in sugary food with low water activity. TBV is

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a typical beverage in which these reactions occur, because of its peculiar chemical composition, low water activity, and long aging time.

C. Melanoidins and other biopolymers Melanoidins are high-molecular biopolymers, which arise from sugar degradation reactions. They are important for the rheological properties of different vinegars including TBV. Other functionalities arise from their complex molecular structure and size, depending on the extent of the polymerization reactions leading to their formation. Falcone and Giudici (2008) have recently used size-exclusion chromatography with a dual detection system (UV–Vis and DRI) to characterize the distribution properties of the molecular size of these biopolymers; the data obtained suggest that TBV behaves as a heterogeneous mixture of four classes of copolymers at least, all ranging from 0.2 kDa to beyond 2000 kDa, and highly polydispersed in respect to their molecular size and composition. All classes of melanoidins of TBV are labeled by ultraviolet- and/or visible-radiation absorbing chromophores. Since the relative concentration of melanoidins evolves according to their initial value into the cooked must, the size-exclusion chromatographic profiles can be safely used as a fingerprint of the overall composition of the TBV throughout the manufacturing process.

D. TBV composition during the last three decades TBV composition and characteristics have changed over the years. In particular, during the last three decades, the most evident changes have related to the Bx and the titratable acidity levels: from 62 to 73 Bx, and from 9% (sometimes even as low as 11%) to 6%. Nowadays, it is unusual to find a TBV with a titratable acidity greater than 6% (w/w) (Table 4.7). The composition and sensorial properties of TBV are now very different from those of the past: in general, more acid, more pungent, and less sweet. The new taste seems to meet consumer preferences as well as those of the professional panel testers, who assign higher sensory scores to the sweeter vinegars. The relationship between composition and sensorial properties in TBV has been investigated by several authors (Chinnici et al., 2003; Cocchi et al., 2002, 2006; Giordano et al., 2003; Plessi et al., 2006; Sanarico et al., 2002; Zeppa et al., 2002). Unfortunately, different and often opposite conclusions have also been reported. This is due to the lack of preliminary validation studies with reliable procedures aiming to investigate the TBV sample with respect to the age claimed by producers.

TABLE 4.7 Composition related-changes of the traditional baslamic vinegar during the latest three decades (adapted from Giudici et al. 2008) 1982

1996

Sample

1 2 3 4 5 6 7 8 9 10 11 12 Mean

61.00 61.50 64.40 61.40 63.60 63.60 65.70 70.00 63.60 57.30 61.00 59.20 62.69

Br

TA

‘‘R’’

11.40 7.26 9.72 9.90 8.76 7.92 9.12 8.70 8.40 9.00 8.70 11.58 9.21

5.35 8.47 6.62 6.21 7.26 8.03 7.20 8.04 7.57 6.36 7.01 5.11 6.93

73.80 75.80 76.30 70.80 73.80 73.80 71.30 75.80 71.80 72.30 71.80 73.80 73.43

‘‘R’’ is the ratio between Brix and titratable acidity.

Br

2004 TA

‘‘R’’

7.32 8.37 8.99 8.25 7.32 7.69 9.30 8.68 8.31 6.82 6.20 8.80 9.18

10.08 10.06 8.49 8.58 10.08 9.60 7.67 8.73 8.64 10.60 11.58 8.39 9.18

74.50 77.00 71.50 72.50 72.25 74.50 75.00 73.00 73.00 74.00 74.00 74.00 73.77

Br

TA

‘‘R’’

6.52 6.93 6.99 7.17 8.87 7.18 6.31 5.91 7.06 6.49 6.25 8.18 6.99

11.43 11.11 10.23 10.11 8.15 10.38 11.89 12.35 10.34 11.40 11.84 9.05 10.69

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IV. PHYSICAL PROPERTIES A. Rheological properties Aqueous solutions containing glucose, fructose, and acetic acid in a range of concentrations simulating a typical TBV exhibit some Newtonian viscosity; while samples of genuine TBV exhibit Newtonian viscosity of two or three times greater (Falcone et al., 2006). Glucose and fructose act as structure-promoting compounds, but other constituents exert a greater structuring ability (Falcone et al., 2006). In particular, it was recognized that such biopolymers are responsible for the high viscosity level in TBV. As mentioned, these compounds are formed during the cooking of grape juice and accumulate and evolve during aging (Falcone and Giudici, 2008). These polymers contribute to other physical properties including colligative ones, the refractive index, density, specific heat capacity melt, and viscoelastic properties.

B. Color and spectrum absorbance The color of vinegar during aging changes from yellow/brown to brown/ black, due to the accumulation of chromophore-labeled melanoidins (Falcone and Giudici, 2008). At least four classes of melanoidins contribute to this coloration. Falcone and Giudici (2008) propose the ratio between the absorbance at 420 nm of TBV and the absorbance of the cooked must (brown index, BI) as a descriptor of the vinegar color and physical age (PRT): ARTV BI ¼ ACM 420 nm where ARTV is the average residence time of the vinegar (V) calculated according to the Giudici and Rinaldi (2007) model. The BI follows a two-rate kinetic with the second stage starting at about the 6th year of aging with a high rate; the relative melanoidins content follows a similar trend (Fig. 4.13). The UV–Vis spectrum (from 200 to 700 nm) of TBV samples during aging exhibits a well-defined absorption peak at 280 nm and featureless absorptions as the wavelength increases. Spectra of this kind were observed for other naturally occurring melanoidins (Hofmann, 1998; Riviero-Pe´rez et al., 2002; Rizzi, 1993) and in synthetic solution, where the interaction between lysine, glucose, and fructose results in chromophore and molecular size distributions resembling those which occur in the cooked must (Falcone and Giudici, 2008).

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9 G(V4)

8 15

G(V2)

7

G(V1)

6

G(BI)

5 10 4 3 5

2

Browning index, BI (420 nm)

Normalized parameters, G(pi)

G(V3)

1 0

0,00

1,22

3,63

6,21

8,82

11,67

13,94

0

Age of TBV (RTAV), years

FIGURE 4.13 Time evolution vinegar color during aging. G(BI) is the brown index as defined in the text; G(V1), G(V2), G(V3), and G(V4) are the relative concentration of high-molecular size in vinegar melanoidins having brown-labeled (420 nm-radiation absorbing) chromophores. All data are normalized with respect to their initial value (in the cooked must). The age of the vinegar was assessed according to the Giudici and Rinaldi (2006) model (adapted from Falcone and Giudici, 2008).

V. CONCLUSION Several different products are known worldwide with the generic and legally dubious appellation ‘‘balsamic vinegars.’’ It is very difficult to understand the true differences among these products, which include condiments, vinegars, and specialty vinegars legally recognized and protected by special regulations such as PGI and PDO. Most ‘‘balsamic products’’ are simply a blend of ingredients, in different amounts and ratios, such as vinegar, concentrated must, sugars, food colouring, and thickeners. Also among the balsamic vinegars produced in Italy, wide differences exist between the PDOs traditional balsamic vinegar of Modena and the PGI balsamic vinegar of Modena, the first is the only one produced by fermentation, acetification, and aging as described in this review; the second is a blend of wine vinegar and cooked must with the addition of caramel to improve the color. From a scientific point of view, TBV is an important model to understand as it answers important questions in food sciences: fermentation of selective media, sugar degradation, Maillard reactions, mass transfer, physical aging, polymerization reaction

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in complex and long preserved media, and the role of high-molecularweight compounds on rheological properties.

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Steels, H., James, S. A., Roberts, I. N., and Stratford, M. (1998). Zygosaccharomyces lentus: A significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature. J. Appl. Microbiol. 87, 520–527. Steels, H., Bond, C. J., Collins, M. D., Roberts, I. N., Stratford, M., and James, S. A. (1999). Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker. Int. J. Syst. Bacteriol. 49, 319–327. Tagliazucchi, D., Verzelloni, E., and Conte, A. (2008). Antioxidant properties of traditional balsamic vinegar and boiled must model systems. Eur. Food Res.Technol. 227, 835–843. Tesfaye, W., Morales, M. L., Garca-Parrilla, M. C., and Troncoso, A. M. (2002). Wine vinegar: Technology, authenticity and quality evaluation. Trend Food Sci. Technol. 13, 12–21. The American HeritageÒ Dictionary of the English Language, fourth edn. Houghton Mifflin Company (2004). Retrieved October 18 2008 from http://dictionary.reference.com/ browse/balsam and http://dictionary.reference.com/browse/balm. The Merriam-Webster Online Dictionary. Retrieved October 18, 2008 from http://www. merriam-webster.com/dictionary/balsam. Theobald, A., Mller, A., and Anklam, E. (1998). Determination of 5-hydroxymethylfurfural in vinegar samples by HPLC. J. Agric. Food Chem. 46, 1850–1854. Turtura, G. C. (1984). La microflora dell’Aceto Balsamico Naturale. Industrie delle Bevande 4, 100–111. Turtura, G. C. (1986). Microbiologia e chimica dell’Aceto Balsamico Naturale. In ‘‘l’Aceto Balsamico’’ (B. Benedetti, ed.), p. 256. Consorteria dell’Aceto Balsamico di Spilamberto, Modena. Turtura, G. C. and Benfenati, L. (1988). Caratteristiche microbiologiche e chimiche dell’Aceto Balsamico Naturale. Studio del prodotto. Annali di Microbiologia 38, 51–74. Verzelloni, E., Tagliazucchi, D., and Conte, A. (2007). Relationship between the antioxidant properties and the phenolic and flavonoid content in traditional balsamic vinegar. Food Chem. 105, 564–571. Vocabolario degli Accademici della Crusca in rete. Retrieved October 23, 2008 from http:// vocabolario.biblio.cribecu.sns.it/Vocabolario/html/_s_index2.html. Zeppa, G., Giordano, M., Gerbi, V., and Meglioli, G. (2002). Characterisation of volatile compounds in three acetification batteries used for the production of Aceto Balsamico Tradizionale di Reggio Emilia. Ital. J. Food Sci. 14, 247–266. Zhang, X.-M., Chan, C.-C., Stamp, D., Minchin, S., Archer, M. C., and Bruce, W. R. (1993). Initiation and promotion of colonic aberrant crypt foci in rats by 5-hydroxymethyl-2furaldehyde in thermolyzed sucrose. Carcinogenesis 14(4), 773–775.

CHAPTER

5 Nanostructured Materials in the Food Industry Mary Ann Augustin*,† and Peerasak Sanguansri†

Contents

184

I. Introduction II. Approaches for Nanoscale Manipulation of Materials III. Processes for Structuring of Food Materials A. Milling B. Homogenization C. Microfluidization D. Ultrasound E. Electrospraying F. Rapid expansion of supercritical solution process IV. Nanostructured Materials A. Biopolymeric nanostructured particles B. Lipid nanoparticles C. Nanostructured emulsions D. Nanocomposites V. Functionality and Applications of Nanostrucutured Materials A. Nanosensors and nanotracers B. Food packaging and edible coatings C. Encapsulated food components VI. Nanotechnology and Society VII. The Future Acknowledgment References

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* CSIRO Preventative Health National Flagship, Adelaide, South Australia 5000, Australia {

CSIRO Food and Nutritional Sciences, Private Bag 16, Werribee, Victoria 3030, Australia

Advances in Food and Nutrition Research, Volume 58 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)58005-9

#

2009 Elsevier Inc. All rights reserved.

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Abstract

Mary Ann Augustin and Peerasak Sanguansri

Nanotechnology involves the application, production, and processing of materials at the nanometer scale. Biological- and physicalinspired approaches, using both conventional and innovative food processing technologies to manipulate matter at this scale, provide the food industry with materials with new functionalities. Understanding the assembly behavior of native and modified food components is essential in developing nanostructured materials. Functionalized nanostructured materials are finding applications in many sectors of the food industry, including novel nanosensors, new packaging materials with improved mechanical and barrier properties, and efficient and targeted nutrient delivery systems. An improved understanding of the benefits and the risks of the technology based on sound scientific data will help gain the acceptance of nanotechnology by the food industry. New horizons for nanotechnology in food science may be achieved by further research on nanoscale structures and methods to control interactions between single molecules.

I. INTRODUCTION Nanotechnology involves the application, production, and processing of materials at the nanometer scale. It is an enabling technology that can be used to create novel materials, devices, and systems based on manipulation of matter at the nanometer scale. Thus, while the strict definition of nanoparticles refers to entities of less than 100 nm, it is the manipulation of matter at this scale that leads to the development of new materials which may have the same gross composition but widely varying properties. New exciting materials created at the atomic, molecular, and supramolecular scale are proving that nanotechnology will continue to have a significant impact on society. Nanotechnology enables the development of a radically new generation of existing products and processes for diverse industries including manufacturing, electronics, engineering, telecommunications, medicine, agriculture, cosmetics, and food (Chaudhry et al., 2008; Farhang, 2007; Roco, 2003; Sanguansri and Augustin, 2006). In the food industry, the benefits of nanotechnology hold promise for the development of new functional materials, micro- and nanoscale processing, new product development, and design of methods and instrumentation for food safety and biosecurity. Examples of some developments include packaging materials with improved barrier properties and increased resistance to high temperature and mechanical stresses; nutrient delivery systems that enable targeted delivery; nanosensors for detection of pathogens, chemicals, and contaminants; and nanotubes and functionalized membranes for efficient processing and delivery (Moraru et al., 2003; Morrison and Robinson, 2009; Sozer and Kokini, 2009). These

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applications exemplify the use of nanotechnology to achieve products with improved control, selectivity, security, functionality, bioavailability, and product targeting.

II. APPROACHES FOR NANOSCALE MANIPULATION OF MATERIALS Principles and techniques in nanotechnology using biological- and physical-inspired approaches are applied to create and modify materials at the nanoscale level. In the bottom-up approach, materials are constructed by self-assembly. Molecular recognition is a defining feature in developing nanostructures. By self-organization, individual molecules are built-up and integrated into larger units and hierarchical structures with unique functionalities. This process and control of nanostructures requires having compatible building blocks and an understanding of how to control the self-assembly processes (Forster and Konrad, 2003; Seeman and Belcher, 2002). The forces of attraction and repulsion that govern the self-assembly of molecules is influenced by many factors including pH, temperature, concentration, and ionic strength. In addition, the conformation of molecules may be altered by various stresses including mechanical forces (e.g., pressure, extension, ultrasound, shear), electric and magnetic fields. All these factors can change the way molecules assemble, resulting in a variety of structures that can be formed from the same molecular building blocks. The top-down approach looks at shaping the structure of the material to the desired specification and generally involves size reduction. The top-down approach is a process that has been conventionally used by the food industry, but nanotechnological developments have introduced more precise tools that allow better control and finer dispersions to be made. A smaller size leads to a bigger surface area and this alters the functionality of materials. Food nanotechnology exploits both the top-down and bottom-up approaches for the development of new materials. The challenge for the food scientist of the future is to harness the natural self-assembling character of a food ingredient (protein, lipid, or polysaccharide) and design nanostructures for particular applications (Dickinson, 2003).

III. PROCESSES FOR STRUCTURING OF FOOD MATERIALS Various processes may be used to alter the size and structure of food materials at various length scales and this has the potential to modulate the self-assembly behavior of food components and alter functionality.

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Selected technologies that have established use or the potential for structuring food materials in the food industry are discussed briefly.

A. Milling Milling results in particle size reduction. Milling techniques have long been used for size reduction of pharmaceutical powders to improve body absorption (Bentham et al., 2004). An increased surface area of food materials will increase the rate of water absorption of materials, improve solubility of dry products, and increase accessibility of sites for chemical reactions (e.g., oxidation, digestion, flavor release, catalyst, and enzyme activity). The structure of food is also important as it dictates how, when, and where food nutrients and flavors may be released. The effectiveness of nutrient bioavailability in food is in part related to its size although other factors such as interactions of the component with a matrix also influence how the component is released. Ball milling was a more efficient method for extracting arabinoxylan from psyllium seed husk, compared to jet milling when particles were milled to the same size. Ball milling reduced the molecular weight of the arabinoxylan and its viscosity was lowered whereas jet milling did not reduce its molecular weight (van Craeyveld et al., 2003). Examples of other effects of milling on functionality of materials are given in Table 5.1.

B. Homogenization In homogenization, a liquid product or slurry is subjected to a high shear stress. Homogenizers have traditionally been used for reducing the size of fat globules to improve the stability of emulsions. High-pressure homogenizers are capable of producing finer milk emulsions than conventional homogenizers (Thiebaud et al., 2003). Membrane emulsification is an alternative process that may be used to form emulsions with narrow particle size distribution and it requires relatively less surfactant than other high-energy processes (Joscelyne and Tra¨ga˚rdh, 1999). Monodisperse emulsions are formed using cross-flow emulsification when the dispersed phase volume is low but the method is not ready for largescale production of emulsions (Kim and Schroe¨n, 2008). High-pressure homogenization/micronization have been exploited to improve the functionality of a variety of plant materials. High-pressuremicronized particles from starfruit pomace, carrot, and orange (insoluble fiber-rich fraction) had the highest oil- and water-holding capacity, swelling capacity, cation-exchange capacity, glucose adsorption capacity, alpha-amylase inhibitory activity, and pancreatic lipase inhibitory activity compared to that obtained by ball milling or jet milling

TABLE 5.1

Modification of plant-based material properties and functionalities by milling

Process

Product

Functionality

References

Ball milling

Various starches

Fragmented starch particles had improved cold water binding properties and freeze–thaw stability Microfine particles (300–1000 nm) were produced which had enhanced antioxidant activity compared to middle grade tea (>1000 nm) Native particles (657 mm) were reduced to 24 mm. Micronized particles had reduced crystallinity and antiobesity effects in vivo when tested in rat models Micronized starch particles (8–24 mm) had reduced gelatinization temperature Starch granules were partially fragmented; water absorption index and water solubility was improved; crystallinity was destroyed; moisture content during milling had an influence on functional properties of the milled starch

Niemann and Meuser (1996)

Green tea

Konjac flour

Vacuum ball milling High-energy ball milling

Cassava starch Jicama starch and cassava starch

Shibata (2002)

Bin et al. (2005)

Che et al. (2007) Martinez-Bustos et al. (2007)

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(Chau et al., 2006a,b, 2007a). Huang et al. (2008) found that high-pressurehomogenized-micronized fibers (9.7–14.3 mm) of starfruit (insoluble fiber and cellulose) improved bowel health, as demonstrated by decreased caecal ammonia concentration, increased fecal output and lowered colonic bacterial enzyme activity in feces of hamsters. Jet milling of the same material was not as effective. The beneficial effects of size reduction were dependent on the process and on the reduction in particle size achieved (Huang et al., 2008). High-pressure homogenization is also capable of disrupting macromolecules. The structure of biopolymers such as proteins and polysaccharides can be altered, protein/polysaccharide complexes may be formed and in addition aggregates of protein or protein/polysaccharide complexes can be microparticulated by high-pressure homogenizers (Paquin, 1999). Examples of the effects of high-pressure homogenization on macromolecules are given in Table 5.2. Recent work has shown that ultra-high-pressure homogenization can alter the structure of proteins adsorbed at the interface. This will influence the interaction between protein-coated oil globules and consequently its stability (Lee et al., 2009).

C. Microfluidization Microfluidization is a form of homogenization that uses interaction and auxiliary chambers with microchannel architecture designed to provide optimum cavitations (formation and collapse of vapor cavities in flowing liquid), shear (pulling apart), and impact (shattering effect) forces for either size reduction, dispersion, or emulsion formation (Kasaai et al., 2003). One of the benefits of microfluidization over conventional homogenization is the formation of a narrower size distribution of oil droplets when microfluidization is used to prepare emulsions. However, at very high pressures (>40–60 MPa), there is the risk of reagglomeration of droplets (Jafari et al., 2007). Microfluidization can also disrupt aggregates (Table 5.3) and depolymerize macromolecules with subsequent alterations to their functionality (Table 5.4).

D. Ultrasound High-power ultrasound has been used to disrupt cells, disperse aggregates, and modify food texture and crystallization (Knorr et al., 2004). The ultrasonic wave causes intense localized heating and this generates gas bubbles which cavitate and result in intense pressure and shear (Povey and Mason, 1998). It is the high pressure and shear which cause physical disruption of food components and materials and can change the rate of chemical reactions. Kentish et al. (2008) used a flow-through power ultrasound systems at 20–24 kHz to produce an oil-in-water emulsion with

TABLE 5.2

Effects of high-pressure homogenization technologies on macromolecules

Process

Material

Functionality

References

Ultra-high-pressure homogenization

Methylcellulose

Floury et al. (2002)

Dynamic pulsed pressure

Modified corn starch

High-pressure valve homogenization

High methoxy pectin

Ultra-high-pressure homogenization (>100 MPa) degraded methylcellulose; a decrease in intrinsic viscosity was obtained which was correlated to the decrease in molecular weight Processing of modified corn starch at 414 or 620 MPa at 70 C decreased melting temperature but viscosity was not affected Molecular weight of pectin was reduced by homogenization at 124 MPa; of the three pectins that had been depolymerized, only one of these had altered flow behavior

Onwulata and Elchediak (2000)

Corredig and Wicker (2001)

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TABLE 5.3

Microfluidization for disruption of aggregates

Product

Comments

References

Low-protein rice starch

Microfluidization disrupted protein– starch aggregates enabling improved separation by subsequent density-based separation Microfluidization disintegrated aggregated denatured whey proteins formed as a result of heating; the disrupted aggregates were stable to sedimentation but not to reheating

Guraya and James (2002)

Denatured whey protein

Iordache and Jelen (2003)

TABLE 5.4 Effects of microfluidization on macromolecular properties Material

Properties and functionality

References

Xanthan gum

Microfluidization caused depolymerization with molecular weight being lowered from 25 106 to 4 106; the microfluidized product had reduced thickening and stabilizing properties Chitosan was depolymerized but degree of acetylation was not changed Microfluidization of heated starch suspensions reduced molecular weight and increased viscosity

Lagoueyte and Paquin (1998)

Chitosan

Resistant starch

Kasaai et al. (2003) Augustin et al. (2008)

particle size of 135 nm. Tsai et al. (2008) manipulated the particle size of ionotropically gelled chitosan-tripolyphosphate nanoparticles by the use of ultrasound in combination with mechanical shearing. The degradation by ultrasound is due to cavitation effects while degradation by mechanical action is by tearing and stretching effects.

E. Electrospraying Electrospraying is a method to electrostatically manipulate droplet formation. The mechanism involves applying an electric field to pull at the surface of the liquid to form droplets by reducing the surface

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tension force. Nanometer size droplets containing electric charge may be formed using this technique. The introduction of electrical charge to each droplet during its formation allows electrostatic stabilization to take place and prevents coalescence of droplets. Electrostatic manipulation enables control the formation of structures and how they may be deposited or released. An electrified coaxial liquid jet was used to encapsulate inner liquid with an outer liquid surround (Loscertales et al., 2002). The action of the electrohydrodynamic (EHD) forces surrounding the compound jet enable the continuous formation of this core shell structure with a diameter range of tens of nanometers to tens of micrometers. The control of deposition and release is also possible by means of charge control, where liquid can be charged to be attracted to oppositely charged surfaces. The release may be also controlled by selecting a shell material that will disintegrate to release the core when a trigger point is reached. The electrospray process has been successfully applied to produce cocoa butter microcapsules through nano-micrometric coaxial jets caused by EHD forces (Bocanegra et al., 2005). A variation of this method is to spray a conducting liquid inside insulator baths. In this case, the spray dynamic is also dependent on the conducting liquid. This may be advantageous in some controlled applications where liquid–liquid emulsion uniformity is important (Barrero et al., 2004). Electrospraying application in micro/nanoencapsulation and electroemulsification has been reviewed by Jaworek (2008).

F. Rapid expansion of supercritical solution process The rapid expansion of supercritical solution (RESS) process consists of dissolving the product in a supercritical fluid (usually carbon dioxide) and then rapidly depressurizing the solution through a spray nozzle thus causing extremely rapid nucleation of the product into a highly dispersed material. Various technologies based on supercritical fluids are given in Table 5.5. The most important limitation of RESS is the low solubility of compounds in supercritical fluids and the use of co-solvent to improve solubility is usually costly and not economically feasible. As an alternative a supercritical fluid anti-solvent (SAS) process was introduced where a supercritical fluid is used to cause substrate precipitation or recrystallization from a polar liquid solvent (Subramaniam et al., 1999). Zhong et al. (2008) successfully used SAS to produce alcohol soluble zein micro- and nanoparticles. A number of other technologies based on manipulating supercritical fluids have been successfully used to produce nanoparticles (Della Porta and Reverchon, 2008; Matsuyama et al., 2003; Meziani and Sun, 2003; Shariati and Peters, 2003; Subramaniam et al., 1999).

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TABLE 5.5

Rapid expansion supercritical fluid-based technologiesa

Acronym

Description

RESS

Rapid expansion of supercritical solutions through a nozzle causing rapid nucleation of product into highly dispersed nanoparticles Rapid expansion of supercritical solutions into a liquid solvent chamber that can contain surfactant that act to impede particle growth Gas or supercritical fluid antisolvent introduces gas or supercritical fluid to decrease the solvent power of a polar liquid solvent in which a substrate is dissolved causing substrate precipitation or recrystallization Aerosol solvent extraction system involves spraying of a polar liquid with a substrate as fine droplets into an atmosphere of compressed carbon dioxide causing precipitation of fine nanoparticles Solution-enhanced dispersion by supercritical fluid to achieve small droplet size and intense mixing of supercritical fluid and solution for increased transfer rates Particles from gas-saturated solutions or suspensions achieved by dissolving a supercritical fluid into a liquid substrate, or a solution of the substrates in a solvent, or a suspension of the substrate(s) in a solvent followed by rapid depressurization of this mixture through a nozzle causing the formation of solid particles or liquid droplets

RESOLVE

GAS/SAS

ASES

SEDS

PGSS

a

Based on Jung and Perrut (2001) and Meziani et al. (2004).

IV. NANOSTRUCTURED MATERIALS The processes described in Section III have been used to influence the assembly of food components into nano- and micron-sized structures, which are the basis for the hierarchical architectures of food materials on a macroscopic scale. The processing treatments and the order in which they are applied can give rise to food materials with the same gross composition but varying functional properties. The aggregation of materials can be driven by self-assembly of molecules to achieve a state of minimum free energy via noncovalent interactions (hydrophobic interactions, hydrogen bonding, electrostatic interactions, and Van der Waals interactions). An example of self-assembly involving noncovalent

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interactions is the spontaneous formation of monoglycerides dispersed in water. In certain types of aggregation (e.g., heat-induced aggregation of whey proteins), covalent as well as noncovalent interactions can be involved. For the purpose of this chapter, nanostructured materials are divided into biopolymeric nanostructured particles, lipid nanoparticles, emulsions, and composites. In this section, the principles underlying the process for manufacturing these nanoparticles are discussed with limited examples. Further examples of the potential use of nanostructured particles are given in Section V.

A. Biopolymeric nanostructured particles These particles can form as a result of the self-assembly of like biomolecules or complexes of different biomolecules (e.g., proteins, polysaccharides).

1. Whole proteins Self-assembly of proteins under different conditions can lead to the development of different structures depending on the pH of the system and the type of protein. The casein micelle is an example of a naturally occurring nanoparticle formed when the different types of caseins (as1, as2, b, and k) self-assemble around amorphous calcium phosphate. This allows it to be a natural carrier for calcium. The casein micelle also serves as a carrier for hydrophobic bioactives (Livney and Dalgleish, 2007). Treatments such as ultrahigh pressure have been reported to alter the structural characteristics of the casein micelle by partially removing parts of the surface of the casein (Sandra and Dalgleish, 2005). Altering the surface properties of these nanoparticles is expected to alter their functional properties. Proteins (e.g., b-lactoglobulin, bovine serum, albumin, hen egg white ovalbumin, egg white) form particulate gels at their isoelectric point (pI). At a pH that is greater than or less than that of the pI of the protein, fine-stranded gels are formed from denatured globular proteins as a result of aggregation of protein strands. The conditions for formation of protein gels can be manipulated to obtain gels of different textures (Foegeding, 2006). In contrast to the formation of gels with single proteins, gels can be prepared by self-assembly of oppositely charged globular proteins. Nanogels (hydrodynamic radius 100 nm) are formed from mixtures of ovalbumin (pI 4.8) and lysozyme (pI 11). These proteins are mixed at pH 5.3, adjusted to pH 10.3, stirred, and heated. The pH of formation of the gels is between the pI of the two proteins and in this pH region the proteins carry opposite charge and are electrostatically attracted to each other. The gels have a core and shell structure (Yu et al., 2006).

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2. Hydrolyzed protein A unique protein nanotube has been obtained by the self-assembly of partially hydrolyzed a-lactalbumin (a milk protein). This is the only nanotube that is derived from food proteins to date (Graveland-Bikker and de Kruif, 2006). Hydrolysis is carried out using a protease from Bacillus licheniformis under specific conditions. The products of hydrolysis are the building blocks for the formation of the protein-based nanotubes but the hydrolyzates need to be in the presence of a suitable ion for the formation of tubular structures. The a-lactalbumin nanotubes have potential for use in a range of applications. The high aspect ratio and stiffness of the tubes make them effective viscosity modifying agents. The protein nanotubes produce stronger gels than other proteins at equivalent concentrations. The 8 nm cavity of the nanotubes and the ability to control their disassembly make it possible for the tubes to carry and deliver valuable nutrients in food vehicles (Graveland-Bikker and de Kruif, 2006).

3. Polysaccharides Starch and gums are commonly used texturing agents in food. The way in which they assemble dictates their functional properties on a macroscopic scale. Traditionally, the conditions of solutions (e.g., pH, presence of ions) have been altered to obtain various functionalities of these macromolecules. For example, altering the potassium ion concentration of k-carrageenan solution influences the temperature at which this polymer gels. Low methoxy pectins and alginates form gels in the presence of calcium ions. Some examples of high shear processes that can be used to alter the nanostructure of polysaccharides have been provided before, with different shear processes having different effects (Tables 5.2–5.4). In addition, the mode of heating can also influence the nanostructure of these components. Starch gels are traditionally formed by cooling convection-heated aqueous dispersions of starch. Starch gels are also formed using microwave-assisted heating but the gels formed in this way undergo incomplete gelatinization, as evidenced using atomic force microscopy. The extent of changes to the starch nanostructure by microwave heating was dependent on the type of starch used (An et al., 2008). Understanding the changes in the structure of starch at the nanometer scale can guide the selection of suitable starches for microwave processing.

4. Protein–polysaccharide mixtures When proteins and polysaccharides carry an opposite charge, complex formation is driven by the attractive electrostatic interactions between the two biopolymers. Soluble and insoluble complexes may be formed depending on the strength of the interactions, the balance of negative

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and positive charges, and their distribution on the biopolymers, molecular weight, and biopolymer flexibility under the conditions used (e.g., pH, ionic strength, concentrations of biopolymers). At or near charge neutrality, the complexes usually aggregate and dense phases predominate. When the overall charges of the biopolymers are not neutralized, the complexes are soluble. The strongest attraction between proteins and anionic polysaccharides (e.g., carrageenens, pectins) occurs at a pH below the pI of the protein when the protein carries a positive charge. Complexes can also be formed between anionic polysaccharides (e.g., chitosan) and proteins (e.g., whey protein isolate). In the latter case, the interactions are strongest at pH above the pI of the protein where the protein possesses a net negative charge. Recent reviews on protein– polysaccharide interactions discuss the principles and applications of these systems (de Kruif et al., 2004; Turgeon et al., 2007). Interactions between proteins and polysaccharides give rise to various textures in food. Protein-stabilized emulsions can be made more stable by the addition of a polysaccharide. A complex of whey protein isolate and carboxymethylcellulose was found to possess superior emulsifying properties compared to those of the protein alone (Girard et al., 2002). The structure of emulsion interfaces formed by complexes of proteins and carbohydrates can be manipulated by the conditions of the preparation. The sequence of the addition of the biopolymers can alter the interfacial composition of emulsions. The ability to alter interfacial structure of emulsions is a lever which can be used to tailor the delivery of food components and nutrients (Dickinson, 2008). Polysaccharides can be used to control protein adsorption at an air–water interface (Ganzevles et al., 2006). The interface of simultaneously adsorbed films (from mixtures of proteins and polysaccharides) and sequentially adsorbed films (where the protein layer is adsorbed prior to addition of the polysaccharide) are different. The presence of the polysaccharide at the start of the adsorption process hinders the formation of a dense primary interfacial layer (Ganzelves et al., 2008). These observations demonstrate how the order of addition of components can influence interfacial structure. This has implications for foaming and emulsifying applications.

B. Lipid nanoparticles Lipid nanoparticles are comprised a lipid core that is surrounded and stabilized by a surface-active material. A single surfactant or a mixture of surfactants such as lecithins and polysorbates may be used to stabilize the lipid. Co-surfactants (e.g., bile salts, butanol) may be incorporated into the formulation. The formulation is homogenized and then cooled, resulting in the formation of the lipid nanoparticle (Weiss et al., 2008). When choosing a fat, it should be recognized that the melting point of

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triglycerides within lipid nanoparticles is lower than that of the bulk fat and, furthermore, that the rate of polymorphic transformations in emulsion is increased compared to that in the bulk lipid phase (Higami et al., 2003). The emulsified lipid carriers are destabilized when recrystallization of the lipids occurs. The nature of the lipid influences the stability of the nanoparticle and their carrier properties. The type of emulsifier used has also an important role in influencing the crystallization process (Westesen et al., 1997). Solid lipid nanoparticles have received attention in the food industries. An advantage of the solid lipid particle over liquid lipids is their improved stability and finer control over the release of components entrapped within the lipid matrix. However, a potential limitation with solid lipid nanoparticles is their susceptibility to aggregation, due to the polymorphic transitions which occur during processing and storage. Strategies that could be used to alleviate this aggregation include an increase in the level of surfactant, choosing a surfactant that can modify polymorphic transitions, or selecting a lipid in which a to b polymorphic transformation is slow (Helgason et al., 2008). Solid lipid nanoparticles have found their place as carriers of a range of lipophilic bioactive compounds (Weiss et al., 2008).

C. Nanostructured emulsions There are many types of emulsions—simple oil-in-water emulsions, double emulsions, multilayered emulsions (formed by layer-by-layer deposition of oppositely charged surfactants), and microemulsions. Various types of oils and surfactants including low-molecular-weight surfactants (e.g., monoglycerides, diglycerides, polysorbates, Tweens) and highmolecular-weight surface-active components (e.g., proteins, gum arabic) may be used in the formulation. Emulsion-based systems of various structures have been developed and except for microemulsions, which are formed spontaneously, most emulsions are formed by homogenization of a dispersion of the oil, water, and surface-active components. The oil–water interface of an emulsion has to be stabilized by a surface-active agent. The formulation and the conditions used for preparation of the emulsion system influence the structure of the emulsion and the architecture of the interface. Proteins form thicker layers at an interface compared to low-molecular-weight surfactants. However, protein-based films are prone to displacement by the more surface-active low-molecular-weight emulsifiers depending on the nature of the interactions between surfactant and protein at the interface and in the continuous phase (Dickinson and Tanai, 1992; Diftis and Kiosseoglou, 2004). In addition, there can be migration of oil between droplets in a mixed oil emulsion. This occurs over time

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in the absence of re-homogenization. If there is re-homogenization, there is instantaneous exchange of oil (Elwell et al., 2004). These results demonstrate the potential for redistribution of the components of the emulsion. However, the challenge is to structure the emulsions so that they have improved kinetic stability and interfaces that display novel functionality (Leal-Calderon et al., 2007).

1. Simple oil-in-water emulsions Most of the conventional triglyceride-based simple emulsions are in the >100 nm to mm range. These emulsions are opaque because at dimensions >100 nm, light is scattered. Recently, Wooster et al. (2008) used the microfluidizer to form transparent long chain triglyceride oil nanoemulsions with average particle size <40 nm that do not undergo Oswald ripening for up to 3 months.

2. Double emulsions Double emulsions (water-in-oil-in-water, W/O/W or oil-in-water-in-oil, O/W/O) have the advantage that they can carry both water and oilsoluble components. These emulsions are normally made using a twostage process. For W/O/W emulsions, a primary water-in-oil emulsion is made and this is followed by emulsification of the primary emulsion in water. One of the drawbacks of double emulsions is that they are prone to destabilization. Increasing the viscosity of the phases is a strategy which may be used to stabilize these emulsions. The use of biopolymer hybrids or conjugates of a protein and polysaccharide as the external emulsifier also helps stabilize double emulsions (Benichou et al., 2004). Most double emulsions are in the micron range and use a mix of surfactants in their formulations. However, recent work has shown that it is possible to form stable double emulsions with droplets under 100 nm (Hanson et al., 2008). These authors proposed the use of racemic, disordered, hydrophobic polypeptide segments which interact via hydrogen bonding to stabilize nanoscale double emulsions.

3. Microemulsions These are transparent isotropic structured fluids composed of two immiscible phases that are stabilized by surfactants. Often a co-surfactant and a co-solvent are present in the formulation. Microemulsions form spontaneously and are thermodynamically stable. Their transparency is due to the small droplet size (<100 nm) in microemulsions (Flanagan and Singh, 2006; Garti and Aserin, 2007).

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4. Structuring emulsions for functionality A growing use of nanostructured emulsions is the delivery of bioactive lyophilized components (McClements et al., 2007). The size of the emulsion droplets and the nature of the interface have a significant impact on the properties of the emulsions. For example, one would expect the rate of reactions that occur at an interface such as oxidation and lipolysis to increase with decreasing droplet size. However, there are conflicting reports in the literature on the effects of droplet size on these reactions. For example, there was no effect of droplet size on oxidation of structured lipid emulsions (Osborn and Akoh, 2004), no clear effects of heat treatment and droplet size on oxidation (Kiokias et al., 2007), and a slower oxidation rate in methyl linoleate emulsions with smaller droplet size (Imai et al., 2008). This shows that the effects of droplet size on the properties of emulsions cannot be considered in isolation from the nature of the interface. There are similar issues when considering lipolysis of emulsions. Armand et al. (1992) found that pancreatic lipase activity was increased with decreasing emulsion size. However, modification of the interface of emulsions by heat treatment of the encapsulant (a mixture of caseinate and modified starch) prior to emulsion formation altered the rate of lipolysis of emulsions in model systems (Chung et al., 2008). The structuring of interfaces for the target delivery of oils and oil-soluble bioactives is currently an active field of research (Singh et al., 2009).

D. Nanocomposites Nanocomposites are typically hybrids of materials. Traditional nanocomposites are made with synthetic polymers and inorganic solids. Nanocomposites may be formed by assembling biopolymers with inorganic nanometer-sized solids. These are known as bio-nanocomposites. Bio-nanocomposites can be engineered to form materials with enhanced mechanical, thermal, and barrier properties. Often the development of bio-nanomaterials is based on bio-inspired approaches and an understanding of bio-mineralization processes (Darder et al., 2007). Compared to conventional nanocomposites, these bio-nanocomposites have the advantage of being more biocompatible and biodegradable. An example of a bio-nanocomposites is the hybrid formed between gelatine and silicates. The formation of this hybrid organic–inorganic composition is due to the strong interaction between the two components. This results in gelatine–silicate composite gels which are weaker than the protein gel under the conditions of the experiment (Coradin et al., 2004). However, these authors suggest that it is possible to manage the self-assembly of the particles by controlling the conditions of the reaction.

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An interesting nanocomposite was formed from natural cellulose fibers incorporated with sucrose (Fahmy et al., 2006). These authors used cotton linters and a key finding was that sucrose could be incorporated into the micropores of mercerized fibers that had not been previously dried. This allowed stronger fibers to be prepared. Although this example is not from the food industry, the finding that sucrose incorporation in nondried fiber preserves the nanostructure may have implications for improving the properties of cellulose used in food applications.

V. FUNCTIONALITY AND APPLICATIONS OF NANOSTRUCUTURED MATERIALS Selected applications of nanotechnology in the food industry are discussed below. These include advances in (a) nanosensors and nanotracers, (b) food packaging and edible coatings, and (c) encapsulated food components. There are also other applications and further information is available in a number of reviews (Chaudhry et al., 2008; Moraru et al., 2003; Sanguansri and Augustin, 2006; Weiss et al., 2006).

A. Nanosensors and nanotracers The introduction of nanotechnology has given sensor specialists the tools required to design and manufacture nanosensors that are capable of rapid measurements on a very small scale. New nanostructures such as carbon nanotubes are used as a component in the construction of nanosensors while processes like lithography and inkjet printing may be used to directly construct tiny electronic circuits and sample delivery channels for nanosensors. The understanding of how biological systems work in the real world is important in identifying how to detect target organic molecules or even microorganisms. A typical nano-biosensor device normally consists of a molecular recognition element which is derived from an understanding of biological systems and a transducer which can be made up using nanotechnological processes or new nanostructures (Vo-Dinh et al., 2001). Nakamura and Karube (2003) have recently reviewed research in biosensors. Table 5.6 shows a number of molecular recognition elements which can be monitored using detectors in transducers to measure specific parameters. The electrical signal may be displayed in a number of different ways, depending on the application requirement. Nanosensors may be used as rapid detectors to quickly identify threats in the case of suspected food poisoning, or integrated into packaging. Rapid detection of toxins and microorganisms is critical for the survival of

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TABLE 5.6 Nanosensor devices based on molecular recognitiona Molecular recognition

Transducer parameters

Detector

Result display

Antibodies Receptors Enzyme

Weight Light Chemical substance Electrical signals Sound

Thermistor Photomultiplier Semiconductor electrodes Quartz crystal microbalance Photodiode

Color changes Light Buzzer

Heat

Sound detector

Animal and plant cells Animal and plant tissues Organelles and microorganism a

Liquid crystal display

Based on Nakamura and Karube (2003).

sufferers in such outbreaks. The use of nanosensors in food packaging to detect growth of microorganisms and change of color when a threshold level is reached is useful for preventing food poisoning. Micromechanical oscillators were developed for rapid detection of active growth of Escherichia coli (Gfeller et al., 2005). The sensor design is based on a nanomechanical cantilever (springboard) microfabricated using standard silicon technology. Its basic principle is to use tiny plates or leaf springs as cantilevers that may be as small as 20 mm wide 100 mm long 0.2 mm thick and attaching a molecular recognition element at the end (Fritz, 2008; Fro´meta, 2006; Ziegler, 2004). The presence of a specific toxin or microbial organism will cause changes in the cantilever which may be measured in resonance frequency and converted to an electrical signal that can be interpreted. Other changes that can be detected by the cantilever include superficial tension, temperature, and mass. Due to its small size this type of sensor can also be designed to detect multiple toxins and microorganisms by putting a series of cantilevers with different molecular recognition elements together in an array mounted on a single chip (Lange et al., 2002; Mabeck and Malliaras, 2006). The use of molecular recognition is based on understanding biological systems and micromechanical devices. This type of sensor is sometimes referred to as bio-micromechanical systems or BioMEMS (Bhattacharya et al., 2007). In some cases, molecular recognition elements such as protein antibodies have stability issues that limit signal transduction. Synthetic materials that can mimic the function of bioreceptors are sometimes used as molecular recognition elements to overcome this

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problem. These synthetic materials are created using molecularly imprinted polymer (MIP) techniques. The synthetic polymer gives high sensitivity and selectivity while maintaining excellent stability (Jenkins et al., 2001). There are a number of nanosensors being developed that have applications in food, either for rapid detection or as nanotracers to show the history of the food product and whether it is of acceptable quality at any given time. More examples of nanosensor development for food applications are shown in Table 5.7.

B. Food packaging and edible coatings Nanomaterial developments are influencing how food is packaged. Bio-nanocomposites with improved thermal, barrier, and mechanical properties are becoming available. Examples of the use of nanostructured materials for packaging applications have been given in Chaudhry et al. (2008) and references therein. One of the first market entries into the food packaging arena was polymer composites containing clay nanoparticles (montmorillonite). The natural nanolayer structure of the clay particles impart improved barrier properties to the clay–polymer composite material. Some of the polymers which have been used in these composites for production of packaging bottles and films include polyamides, polyethylene vinyl acetate, epoxy resins, nylons, and polyethylene terephthalate. Edible coatings have been used in the food industry for extending the shelf-life of food products. Traditionally, the food to be coated is dipped in a polysaccharide or protein-based solution or emulsion and a thin layer of the coating material is formed around the surface of the food product. Developments in nanotechnology with the potential to impact on the edible coating industry have been discussed (Vargas et al., 2008). Multilayered coatings may be obtained using layer-by-layer electrodeposition (Weiss et al., 2006). Sequenced immersion of the product in solutions of oppositely charged polyelectrolytes produces the multilayered coating. This allows the assembly of oppositely charged polyelectrolytes in succession and may, in principle, be applied to the edible coating of fruits. Coatings may be used as carriers of functional ingredients (e.g., antimicrobial agents) by using microencapsulation or nanoencapsulation techniques (Vargas et al., 2008). Edible films based on chitosan, with improved barrier and mechanical properties, may be obtained by incorporating nanoparticles made from unmodified or organically modified montmorillonites, nanosilver, or silver zeolite. The rate of improvement depended on the type of nanoparticle incorporated (Rhim et al., 2006).

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TABLE 5.7 Nanosensors for food application Application

Description

References

Pesticide and insecticide in water

MIPs for sequestering of nonhydrolyzed organophosphate coated on optical fiber Detection of seven different pesticides in water using multiwalled carbon nanotubebased sensor Copper nanoparticle and carbon nanotube-based sensor Multiwalled carbon nanotubebased biosensor with high stability of 86.7% after 4 months MIPs using amorphous, crystalline, and solubilized trypsin as template with 100 ng/ml detection limit A chip-based nanofluidic device that amplifies catechol electrochemical signal; capable of detection of a few hundred molecules Nanoparticle printed onto carbonpaste electrode capable of 8.3 mM detection limit Metal oxide semiconductor thin-film sensors for red wine characterization Multicomponent carbon nanotube composite sensor array for bitter, sweet, salty, umami, and sour detection

Jenkins et al. (2001)

Glucose and other saccharides

Trypsin

Catechol

Ascorbic acid

Electronic nose

Electronic tongue

AsensioRamos et al. (2008) Male et al. (2004) Wang et al. (2003) Hayden et al. (2006)

Wolfrum et al. (2008)

Ambrosi et al. (2008) Garcı´a et al. (2006) Pioggia et al. (2008)

C. Encapsulated food components Nanostructured biopolymers and emulsions lend themselves to encapsulation of food ingredients that need to be protected from their environment until they are released at a specific time or site. Controlled breakdown of the matrix material and release of the encapsulated core

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can be achieved by selection of appropriate encapsulant materials and processing techniques. A recent review of nanostructured assemblies for the delivery of food ingredients provides more detail (Augustin and Hemar, 2009). Selected examples of encapsulated food components are given in Table 5.8. The ability of proteins to form gels and stable emulsions makes them suitable encapsulant materials for delivery of a range of food components. By controlling the microstructure of the gel network, the release of the entrapped food component (e.g., bioactive) can be manipulated (Chen et al., 2006). The nanocomplexes formed between proteins and polysaccharides, and emulsion-based systems have been examined for their ability to protect and deliver a variety of food components. Cross-linking biopolymers has been used to improve microcapsule stability. Glutaraldehyde is an effective cross-linking agent but is not allowed in food. An alternative to glutaraldehyde that has been examined is tannin. An interesting example which capitalizes on the ability of the hydrophobic cavity of b-lactoglobulin to carry a hydrophobic molecules and complexation has been recently reported (Zimet and Livney, 2009). It was found that b-lactoglobulin binds docosahexaenoic acid (DHA) and further that DHA-loaded b-lactoglobulin can form nanocomplexes ( 100 nm) with low methoxy pectin at a pH
TABLE 5.8 Nanostructured microencapsulated food components Encapsulant system

Biopolymeric nanoparticles Gelatin-based simple coacervate Gelatin–gum acacia complex coacervate Carboxymethylated (CM) chitosan–alginate

Core

Comments

References

Capsaicin

Coacervate cross-linked by glutaraldehdye; thermal stability was enhanced Coacervate cross-linked with tannins

Wang et al. (2008)

Capsicin Bovine serum albumin (BSA)

Whey protein isolate–low methoxy pectin

Thiamine

Whey protein isolate– alginate

Riboflavin

BSA-loaded microspheres prepared from carboxy methylated chitosan and alginate with emulsion phase separation and cross-linked with Ca ions Thiamine entrapped in whey protein isolate– pectin complex; entrapment efficiency was highest at pH 3–3.5 Riboflavin-loaded microspheres formed; riboflavin release slowed in simulated gastric fluid with complete release of the bioactive in simulated intestinal fluid

Xing et al. (2004) Zhang et al. (2004)

Be´die´ et al. (2008)

Chen and Subirade (2006)

Emulsions Sunflower oil and surfactants Nonionic food grade emuslifiers/water/ limonene/ethanol/ propyleneglycol Molecular inclusion complexes Starch

b-Carotene

Lycopene

Stearic acid

Particles (400 nm) were formed; the colloidal particles were stable and water-dispersible; carotene was protected against degradation Enhanced solubilization of lycopene; water dilutable food grade microemulsions

Hentschel et al. (2008)

V-type starch inclusion complex formed using continuous dual feed homogenization; potential use as delivery vehicle

Lesmes et al. (2008)

Spernath et al. (2002)

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VI. NANOTECHNOLOGY AND SOCIETY Although there are many potential benefits of nanotechnology, the public perception of the technology will determine how rapidly it will be adopted in the market place. Regulations for the use of nanotechnology need to be developed. Among some of the issues that need to be considered include definition of the particle size range and how it can be measured, the physicochemical properties of the particles and the processes used to produce them and overcoming safety concerns (Chau et al., 2007b). There has to be transparent processes to deal with the safety aspects of the technology (Powell and Colin, 2008). As with any new technology, the potential risks need to be evaluated in a balanced fashion. Stern and McNeil (2008) have recently reviewed the published data on safety concerns surrounding the exposure of humans and the environment to nanomaterials. These authors considered the design of experiments and methodologies used to assess the risks in their analysis of the literature. Taking into consideration where there is agreement or points of difference between the published studies, these authors’ opinion was that published data suggest that the human organs (i.e., lung, gastrointestinal tract, and skin) can serve as significant barriers to many nanomaterials. However, they also advocate a cautious approach to application of nanotechnology until unknown facts such as degree of and hazards of exposure and life cycle analysis of these materials are better defined and understood.

VII. THE FUTURE The ramifications of nanotechnology in the food arena have yet to be fully realized. This requires further research into biopolymer assembly behavior and applications of nanomaterials in the food industry. Researchers should keep abreast of the development of research tools and what is being done to push resolution limits for techniques such as atomic force spectroscopy or the synchrotron coupled to various spectroscopic techniques and higher resolution microscopy. New techniques should be exploited and the knowledge gained used to understand the dynamics and interactions of food materials at the single-molecule level and to describe assembly behavior in quantitative thermodynamic terms. There are questions about the interactions of nanoparticles with the food matrix and within the human body. These questions need to be addressed by future research (Simon and Joner, 2008; Sletmoen et al., 2008). Further research is required on nanoscale structures and methods that can be used to control the interactions between molecules to enable

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nanotechnology to have an impact on food and health. The future challenge would be to decide on what application of nanotechnology in food would be of most benefit to the consumer, environment, and industry. It is important to educate the consumer about the implications of applying nanotechnology in food and for regulatory bodies to take an active role in approvals of new products made as a result of nanotechnology. This would include evidence that food product made as a result of nanotechnology is safe and have benefits that would otherwise not be possible using current practices.

ACKNOWLEDGMENT We gratefully acknowledge the assistance of Christine Margetts for sourcing literature information.

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CHAPTER

6 Gossypol-A Polyphenolic Compound from Cotton Plant Xi Wang,* Cheryl Page Howell,* Feng Chen,† Juanjuan Yin,† and Yueming Jiang‡

Contents

I. II. III. IV. V.

Overview of Cotton and Cottonseed Products Occurrence of Gossypol Physiochemical Properties of Gossypol Gossypol Analyses Agricultural Implication A. Insecticidal activity B. Antifeeding activity C. Toxicity D. Detoxification VI. Biological Properties A. Antioxidant property B. Antifertility activity C. Anticancer activity D. Antivirus activity E. Antiparasitic protozoan activities F. Antimicrobial activity G. Lowering plasma cholesterol levels VII. Clinical Implication VIII. Conclusions References

216 218 218 226 228 228 228 229 232 233 234 235 237 242 244 247 248 249 251 251

* Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634 { {

Department of Food Science and Human Nutrition, Clemson University, Clemson, South Carolina 29634 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P. R. China

Advances in Food and Nutrition Research, Volume 58 ISSN 1043-4526, DOI: 10.1016/S1043-4526(09)58006-0

#

2009 Elsevier Inc. All rights reserved.

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Gossypol (C30H30O8) is a polyphenolic compound derived from the cotton plant (genus Gossypium, family Malvaceae). The presence of six phenolic hydroxyl groups and two aldehydic groups makes gossypol chemically reactive. Gossypol can undergo Schiff base formation, ozonolysis, oxidation, and methylation to form gossypol derivatives. Gossypol and its derivatives have been the target of much research due to their multifaceted biological activities including antifertility, antivirus, anticancer, antioxidant, antitrypanosomal, antimicrobial, and antimalarial activities. Because of restricted rotation of the internaphthyl bond, gossypol is a chiral compound, which has two atropisomers (i.e., (þ)- and ()-gossypol) that exhibit different levels of biological activities. This chapter covers the physiochemical properties, analyses, biological properties, and agricultural and clinical implications of gossypol.

I. OVERVIEW OF COTTON AND COTTONSEED PRODUCTS Cotton has long been known as nature’s unique food and fiber plant. It is produced worldwide in tropical and subtropical regions. During the 2003–2004 marketing year, the world production of cotton was approximately 88 million bales, for which the People’s Republic of China was the largest producer, followed by the United States, India, and Pakistan, which accounted for approximately three-quarters of the world output (USDA, 2003/2004) (Fig. 6.1). If Brazil and Turkey were added, six countries would account for 83% of world cotton production. Regardless of a continuous declining trend of the share of cotton fibers compared to that of the chemical textile fibers since 1970s, world demand and consumption of cotton fiber has been steadily growing along with the worldwide economic growth. However, much of the growth of cotton production since the end of the Second World War was due to improved yield (output from 0.2 t/ha in 1945/1946 to 0.8 t/ha in 2006/2007 according to the International Cotton Advisory Committee—ICAC), rather than to expanded area (cultivated land increased by only 35% over the 1945/ 1946–2006/2007 period, expanding from 22.3 to 34.8 million hectares). Meanwhile, the cottonseed production has increased in the same trend. Still, cottonseed ranks third behind soybean and rapeseed in terms of world’s oilseed production (USDA, 2007) (Table 6.1). The composition of cottonseed, which includes oil, protein, carbohydrates, phosphorous compounds, and minerals, varies considerably depending on plant species, variety, and plantation environment. Besides the cotton fiber, the cottonseed oil, and cottonseed protein are other two major products of cotton plants. The former is embedded as droplets (oil bodies) in the tissue of cottonseed (Markman, 1968), which consists

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217

Others, 17.60% Turkey, 4.80%

China, 29%

Brazil, 5% Parkistan, 9.50% India, 14.20%

FIGURE 6.1 TABLE 6.1

United states, 19.90%

Cotton production by main countries.

World oilseed production 2007

Soybeans Rapeseed Cottonseed Peanut Sunflower seed Palm kernel Copra Total

Million short tons

Million metric tons

242.3 52.5 50 36.5 30.5 12.2 5.9 430.8

219.9 47.6 45.4 33.1 27.7 11.1 5.4 390

of about 45% hull and linters, and 55% kernel in average. The cottonseed kernel is a pointed ovoid body approximately 8–12 mm in length, in which there are innumerable dark spots. These dark spots are pigment glands and unique to the cottonseed. The major component of the pigment gland is free gossypol (FG). Gossypol is a polyphenolic chemical once having a notorious reputation but now considered a promising biological phytochemical. The amount of FG in the cottonseed oil can be significantly affected by the oilseed processing, which can also remarkably influence the protein value of the cottonseed meal (CSM). For example, in screw-pressing (also known as expeller- or cold-pressing), most of the FG binds to amino acids of proteins, which lowers the nutritional value of the protein and the CSM. Although prepress solvent extraction and direct solvent extraction can produce the CSM with highquality protein, they considerably increase the content of FG in the oil.

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Since gossypol has a certain degree of toxicity, its presence will significantly influence and limit the applications of the cottonseed oil and CSM as food and animal feed, respectively. Therefore, the US Food and Drug Administration (FDA, 1974) set up mandatory regulations that any cottonseed protein products intended for human use must contain no more than 450 ppm FG. Likewise, the Protein Advisory Group of the United Nations Food and Agriculture Organization and World Health Organization (FAO/WHO) has set limits of 600 ppm of FG and 12,000 ppm total gossypol (TG) for human consumption. Moreover, some additional caution was suggested concerning the presence of gossypol in both CSM and whole cottonseed when used in dairy rations (Poore and Rogers, 1998).

II. OCCURRENCE OF GOSSYPOL Gossypol is mainly embedded in the cottonseed pigment glands. It constitutes 20–40% of the gland weight and accounts for 0.4–1.7% of the whole kernel. Besides the cottonseed, gossypol glands are also found in some other parts of the cotton plant, such as the bark of plant roots, leaves, seed hulls, and flowers. On the other hand, gossypol content varies largely, depending on the species and variety of cotton plant, climatic and soil conditions of the region, water supply, agrotechnical treatment, and in particular, on the amount and composition of fertilizers used (Markman, 1968; Stansbury et al., 1954). Changes of gossypol content throughout different stages of the cotton maturity have also been reported (Caskey and Gallup, 1931; Gallup, 1927, 1928). Though gossypol only accounts for 0.4–1.7% of whole cottonseed kernel, its production could exceed 40,000 tons annually in the United States alone. Therefore, the gossypol content of cotton plants has become a big issue, both scientifically and commercially, due to its unique biological characteristics (such as antiproliferative activity, antivirus activity, etc.), and influence of its toxicity on use of some commercial cottonseed products such as the CSM as an animal feed, the cottonseed oil in food products, and the cottonseed flour for human consumption. In this context, it is of great significance to review the physiochemical and biological properties of gossypol and its derivatives in the following sections.

III. PHYSIOCHEMICAL PROPERTIES OF GOSSYPOL Gossypol was first discovered and isolated as a crude pigment by Longmore (1886) from cottonseed oil foot, a mixture of precipitated soaps and gums produced in the refining of crude cottonseed oil with

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sodium hydroxide. Forty one years later, its chemical formula C30H30O8 was established by Clark (1927). After another 30 years, its complete structure was verified as 1,10 , 6,60 , 7,70 -hexahydroxy-3,30 -dimethyl-5,50 diisopropyl-2,20 -binaphthyl-8,80 -dialdehyde (Fig. 6.2) by Edwards (1958) when gossypol was completely synthesized. As shown in Fig. 6.2, gossypol contains polar groups (six hydroxyl and two aldehydic groups) making it soluble in most organic solvents, such as methanol, ethanol, isopropanol, butanol, ethylene glycol, dioxane, diethyl ether, acetone, ethyl acetate, chloroform, carbon tetrachloride, ethylene dichloride, phenol, pyridine, melted naphthalene, and heated vegetable oil. It is less soluble in glycerine, cyclohexane, benzene, gasoline, and petroleum ether. However, the presence of two heavy dialkylnaphthalene groups makes it insoluble in water (Markman, 1968). Using diethyl ether, chloroform, and ligroin, Campbell et al. (1937) obtained three gossypol crystals with different melting points of 184, 199, and 214 C, respectively. Although Adams et al. (1960) suggested that these samples were various polymorphic forms of gossypol, it was demonstrated by Ibragimov et al. (1995) that only the last sample with the melting point at 214 C was a nonsolvated compound, while the other two crystals with the melting points of 184 and 199 C, were really the complexes with the solvents of diethyl ether and chloroform, respectively. A comprehensive review of the discovery, determination of structure, and chemistry of gossypol was published by Adams et al. (1960) and Markman (1968).

A

OHC HO 8⬘

7⬘ HO

HO

CHO 1⬘

6⬘ 5⬘

4⬘

OH

1

8

OH

CHO OH

6 OH

2⬘ 2

3⬘

HO

7

HO

OHC

OH

HO

OH

5 3

4

(+)-Gossypol

(−)-Gossypol

B

Gossypol

Bisabolane

FIGURE 6.2 gossypol.

Cadinane

(A) Atropisomers of gossypol and (B) formation of naphthalene ring in

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Gossypol exists in two atropisomers due to restricted rotation of its internaphthyl bond (Fig. 6.2). The naphthalene rings of gossypol are derived from sesquiterpenes of the cadinane family, while the cadinanes are formed in the biogenetic cascade from the bisabolane intermediate by a series of putative 1, 2-shifts and cyclization (Fig. 6.2B). An enantiomeric excess of (þ)-gossypol was usually found in plant species of Gossypium arboreum, G. herbaceum, G. hirsutum, G. aboreum, G. mustelimum, and Thespesia populnea, while ()-gossypol was found in excess in G. barbadens (Cass et al., 1991; Zhou and Lin, 1988). Adams et al. (1960) proposed that gossypol existed in three tautomeric forms: (a) aldehyde, (b) ketol, and (c) hemiacetal (Fig. 6.3) in different solvents. Use of nuclear magnetic resonance (NMR), mass spectral analysis, and UV spectrometry has confirmed its structural changes in various solutions. In basic solvent systems, gossypol existed mainly as ketol, but in ordinary inert solvents, such as chloroform, benzene, acetone, or dioxane, and in acidic conditions, gossypol existed mainly in the aldehyde form (Stipanovic et al., 1973). While in polar solvents such as dimethyl sulfoxide (DMSO) with alkali condition, the hemiacetal form occurred in dynamic equilibrium with the aldehyde form as well as the ketol form (Abdullaev et al., 1990). Since gossypol is commonly dissolved in DMSO as a stock solution for biological studies, several tautomeric forms of gossypol may simultaneously contribute their biological activities. Gossypol is chemically reactive due to the reactivity of carbonyl and phenolic hydroxyl groups as well as its bulky binaphthalene structure. Gossypol can react with other compounds to form bound gossypol (BG). In order to better describe the chemical status (forms) of gossypol in cottonseed products, three terms (i.e., FG, BG, and TG) are frequently used. Based on the AOCS (American Oil Chemists Society) official

A

CHO

OH

HO HO

2

B

C CHOH OH

HOHC HO

O HO

O

2

HO

2

FIGURE 6.3 Tautomeric forms of gossypol: (A) aldehyde, (B) ketol, and (C) hemiacetal.

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definition, FG is defined as gossypol and gossypol derivatives in cottonseed products that can be extracted by 70% aqueous acetone (AOCS, Ba 7-58). BG, formed during cottonseed processing by reaction of gossypol with other compounds, is not soluble in aqueous acetone. TG is defined as gossypol and gossypol derivatives, both FG and BG, which can react with 3-amino-1-propanol in dimethylformamide solution (AOCS, Ba 8-78). However, since the AOCS method (AOCS, Ba 8-78) only measures gossypol, gossypol analogs, and gossypol derivatives that have an available aldehyde moiety for the derivatization reaction, a fraction of gossypol derivatives that are unable to undergo this reaction will be excluded from the TG value. Regardless of the aforementioned shortcomings, FG and TG are still determined empirically, and BG is determined mathematically by the equation BG ¼ TG FG. Some evidence indicates that the majority of gossypol exists as Schiff bases formed by the condensation between the aldehydic groups of gossypol and amino groups of proteins during cottonseed processing (Cater, 1968; Lyman et al., 1959). However, this chemical complex alone cannot fully account for gossypol’s complex behavior. In fact, gossypol could be chelated by iron in cottonseed products to form insoluble metal complexes (Muzaffaruddin and Saxena, 1966), subject to be oxidized (Haas and Shirley, 1965; Scheiffele and Shirley, 1964), or may form gossypol polymers (Anderson et al., 1984). Additionally, its phenolic groups may react to form esters and ethers with other carboxylic compounds and phenols in cottonseed plants. In the past several decades, much work has been done on the Schiff base reaction between gossypol and amino groups (Fig. 6.4). One investigation (Lyman et al., 1959) on gossypol–protein complexes revealed that e-amino group of lysine in crystalline BSA (bovine serum albumin) or cotton protein was involved in the gossypol–protein interaction. Furthermore, it was shown that the molar ratio of free e-amino groups to gossypol varied according to the experimental conditions, but averaged about 1.5 mol of lysine bound per mol of gossypol. At low concentrations of gossypol, the molar ratio of lysine to gossypol was 2:1, indicating that both of the reactive aldehyde groups of gossypol were linked to lysine. On the other hand, sedimentation velocity studies of these gossypol– protein complexes suggested the presence of one to four different

NR OHC HO

OH

OH CHO OH

HC NH2R

OH

HO Gossypol

NR OH

OH CH

HO

OH NaBH CN HO 3

HO

OH

NHR OH CH2

HO

Gossypol schiff base

FIGURE 6.4

NHR H2C OH

Gossypol Schiff base formation.

OH OH

Secondary amine

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compounds, thus indicating that gossypol probably formed a cross-link between two or more protein molecules. Besides, involvement of arginine in the reactions has also been reported (Baliga, 1956; Martinez et al., 1961). Damaty and Hudson (1979) concluded that lysine, serine, methionine, along with some other hydrophobic amino acids in cottonseed protein, were easily able to participate in the formation of insoluble material after their study on the chemical nature of the interactions between gossypol and proteins using selective proteolysis, gel filtration, and amino acid analysis. Reddy and Rao (1987) studied the interactions between gossypol and proteins (i.e., gossypin, congossypin, and glycinin) using a difference spectral method. They found that the interactions were entirely reversible, and suggested that both hydrophobic and ionic interactions were involved in the reaction of gossypol with proteins. Later, Strm-Hansen et al. (1989) studied the interaction of gossypol with amino acids and peptides using circular dichroism (CD) and NMR, which gave evidence that hydrophobic interaction might be responsible for a significant proportion of the interaction between gossypol and proteins. This opinion was also supported by the finding that gossypol was bound competitively at the bilirubin-binding site on albumin (Royer and Vander Jagt, 1983) because this site was known to be linked with many hydrophobic residues, with only one or two positively charged amino acids being present. Later, more model complexes of gossypol with amino acids (lysine, asparagine, glutamine, and glycine), peptides (hippuryl-L-lysine, L-alanyl-L-lysine, glycyl-L-lysine, L-histidyl-L-lysine), and purified proteins (glandless cottonseed protein, cottonseed globulin, insulin) were investigated and partially characterized (Cater, 1968). It was concluded that the rate of reaction of gossypol with amino acids increased with an increase of pH value (5.7–7.5), and was shown to be related to the distance of the amino group from the carboxyl group within the molecule. After using CD to study the reaction of (þ)-gossypol with BSA, human serum albumin, lactate dehydrogenase (LDH), malate dehydrogenase, alkaline phosphatase, lysozyme, protamine, and poly-L-lysine, Whaley et al. (1984a,b) found that (þ)-gossypol bound to albumin with the same affinity as (þ/)-gossypol. A Schiff base is a relatively labile bond that is readily reversible by hydrolysis in aqueous solution and can be chemically stabilized by reduction. The formation of a Schiff base is enhanced at alkaline pH values, but is still not entirely stable unless reduced to a secondary or tertiary amine linkage (Hermanson, 1995). The addition of sodium borohydride or sodium cyanoborohydride will result in reduction of the Schiff base intermediate into a relatively stable secondary amine. Both borohydride and cyanoborohydride have been used for reductive amination purposes, but borohydride will simultaneously reduce the reactive aldehyde groups to hydroxyls and convert Schiff bases present to

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secondary amines. Cyanoborohydride, by contrast, is a milder reducing agent that is at least five times milder than borohydride in reductive amination processes. Cyanoborohydride does not reduce aldehydes, but it is very effective for Schiff base reduction (Hermanson, 1995; Lane, 1975). Thus, higher yields of conjugate formation can be obtained using cyanoborohydride instead of borohydride for the stabilization of Schiff base products. Gossypol is unstable and can be readily oxidized under various reaction conditions into different products with slight structural differences (Fig. 6.5). Oxidation of gossypol usually proceeds in alkaline solution. Ismailov et al. (1994) reported the preparation of gossindane through gossypol oxidation by pure oxygen in alkaline medium. Later, Talipov and Ibragimov (1999) determined the single crystal structure of gossindane through X-ray diffraction analysis and confirmed that the chemical contained an indane nucleus instead of the gossypol naphthalene nucleus, for which the chemical gossindane was named. In addition, gossypolo-binaphthoquinone was prepared in an early stage of the reaction between gossypol and oxygen via a Dakin-type reaction (Scheiffele and Shirley, 1964). Besides o-binaphthoquinone, p-binaphthoquinone, also called gossypolone, can be obtained from the reaction of ferric chloride hexahydrate and gossypol in acetic acid or acetone, which displays an orange color (Shirley and Sheehan, 1955). During the oxidation of gossypol by potassium permanganate in NaOH medium, some decomposition compounds (e.g., formic acid, acetic acid, n-butyric acid, iso-butyric acid) were found by Clark (1928). Gossypol can also be ozonized in acetic acid solution resulting in gossypolic acid in a very low yield, along with large amounts of oxalic acid (Fig. 6.6) (Karrer and Tobler, 1932).

OH

O

OH

O

HO

OH

Gossindane

Pure oxygen in alkaline solution O O HO

OH

OH O O (1)O2,OH−

OHC HO

OH

OH CHO OH

FeCl3

K2MnO4 NaOH⬚C

O

O

O

Gossypolone

CO2

HO HO

O

OH HOAc/acetone HO

+ OH (2) H3O HO

Gossypol-o-binaphthoquinone

OHC HO

O

O O

OH

O OH

FIGURE 6.5 Oxidation of gossypol.

CHO O H O H

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OH CHO OH

CHOOH

HO

OH O3

OH

HO

HO

OH CHO OH

HO

OH

H3COHC H3CO

O

O

COOH

CHOCH3 OCH3

HO

or

OH

Gossypol tetramethyl ether

(CH3)2SO4 O

COCH(CH3)2

COOH

(CH 3 )2 S 04

CH2N2 or CH3I

O

+

Ozonolysis of gossypol.

Gossypol

H3COHC H3CO

COOH

(H3C)2HCOC

FIGURE 6.6 CHOOH

OH

HOOC

CHOCH3 OCH3

H3CO

H (C

04 ) 2S 3

OCH3 Gossypol hexamethyl ether H2SO4

HOHC HO

O

O

CHOH OH

H3CO

OCH3 Gossypol dimethyl ether

FIGURE 6.7

Methylation of gossypol.

Unlike gossypol, gossypol ethers (Fig. 6.7) are much more stable. Gossypol hexamethyl ethers have been synthesized using three different methylation agents: diazomethane in ether solution, methyl iodide as well as dimethyl sulfate; gossypol offered no resistance to methylation when using less drastic methylation agents such as methyl iodide or diazomethane (Haar and Pominski, 1952). Gossypol hexamethyl ether and gossypol tetramethyl ether were formed at the same time during the reaction between gossypol and dimethyl sulfate in methanolic potassium hydroxide. Gossypol tetramethyl ether could be further methylated into gossypol hexamethyl ether. Moreover, gossypol dimethyl ether can be prepared from demethylation of gossypol hexamethyl ether by treatment with a solution of concentrated sulfuric acid in acetic acid (Fig. 6.7).

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With concentrated sulfuric acid the hexamethyl ether of gossypol gives an orange color characteristic in contrast to the tetramethyl ether in a scarlet color (Adams and Geissman, 1938). Additionally, apogossypol is another important functionalized gossypol derivative (Fig. 6.8). It is a dealdehyde product from the reaction between gossypol and 40% aqueous sodium hydroxide at 85 C under a nitrogen atmosphere for 1.75 h (Meltzer et al., 1985). Apogossypol is sensitive and subject to change from its original yellow color into brown within a short time of exposure to the air, therefore, it should be freshly prepared whenever needed. By using dimethyl sulfate, apogossypol can be converted to apogossypol hexamethyl ether, which is much more stable than apogossypol. Apogossypol hexamethyl ether can then be treated with concentrated sulfuric acid to obtain didesisopropyl apogossypol hexamethyl ether. Gossypol can also undergo reduction with LiAlH4, NaBH3CN, or H2 (Pt as catalyst). During the reduction, the aldehydic moieties are reduced to the methyl group (Shirley and Sheehan, 1955; Shirley et al., 1957) or the methanol group (Dao et al., 2000). Due to gossypol’s multifaceted reactivity and potential wide applications in biological aspects, we also strived to combine gossypol and fullerene in an effort to make a nanoparticle-based therapeutic gossypol– fullerene hybrid through a certain synthetic pathway. Gossypol decomposed and formed several unexpected fulleropyrrolidines including (Fig. 6.9) N-methyl, 2-phenylfulleropyrrolidine (1), N-methylfulleropyrrolidine (2), and 1, 2, 2-trimethylfulleropyrrolidine (3), which were chemically and physically characterized via the 1D and 2D NMR, IR, MS as well as X-ray methods.

HO

OHC

OH

OH CHO OH

HO

OH

OH 40% NaOH 850 C

OH

HO

OH

Gossypol

Apogossypol (CH

OCH3

O4 ) 2S

3

OCH3

H3CO

OCH3

H3CO

OCH3

Apogossypol hexamethyl ether

FIGURE 6.8

OH

HO

OCH3

OCH3

Concentrated

H3CO

OCH3

H2SO4

H3CO

OCH3

Didesisopropyl apogossypol hexamethyl ether

Formation of apogossypol and its derivatives.

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N CH3

N CH3 C60 sarcosine

3

PhCl 120 ⬚C

Gossypol

C60 sarcosine

1

PhCH3 110 ⬚C

N CH3

N CH3

2

2

FIGURE 6.9

Syntheses of gossypol–fullerene hybrid.

IV. GOSSYPOL ANALYSES Methods for the quantitative determination of gossypol in cottonseed, oil, press-cake, and meal have been reviewed by Markman (1968). These methods include gravimetric, volumetric, colorimetric, spectrophotometric, polarographic, and luminescent means. Gossypol can be derivatized to form a trimethylsilyl (TMS) derivative subject to the gas chromatography (GC) analysis (Raju and Cater, 1967). Near-infrared reflectance, which has been used in many applications for composition analysis (Norries et al., 1976), also offers a possibility for the measurement of gossypol content (Birth and Ramey, 1982). Furthermore, the structural analyses of gossypol, gossypolone, and correlated derivatives are facilitated by using electron impact-mass spectrometer (EI-MS) and infrared spectrometer, which provide ‘‘fingerprint’’ mass spectra and IR spectra, respectively (Phillip and Hedin, 1990). Compared with GC, high-performance liquid chromatography (HPLC) is more frequently used for the quantitative and qualitative determination of gossypol and gossypol derivatives due to the existence of chromophoric groups that have strong absorption in the UV range. HPLC has been used to determine gossypol and its derivatives in cottonseed, leaves and flower buds, processed oils, and meals after derivatization (Chamkasem, 1988; Hron et al., 1990; Nomeir and Abou-Donia, 1982; Stipanovic et al., 1988). Cai et al. (2004) further demonstrated a simplified HPLC method without gossypol derivatization to analyze gossypol content in various genetic types of cotton varieties on a C18 column with methanol–acetic acid aqueous solution (90:10, v/v) as the mobile phase. Similarly, Aoyama (2008) used a mobile phase of methanol:water (9:1) adjusted to pH 2.6 with phosphoric acid to determine gossypol in feed with UV detector at 254 nm. With the advent of new technology, such as the combination of

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HPLC in tandem of mass spectrometer (LC-MS), determination of gossypol has become more sensitive, accurate, and efficient. For example, Orth et al. (2007) analyzed BG in insects through forming a Schiff base with aniline by using high-pressure liquid chromatography coupled with a triple quadrupole mass spectrometer. Besides the HPLC methods, AOCS official methods (AOCS, Ba 7-58 and Ba 8-78) are also recommended to be used for routine gossypol analyses. However, it is found that the total amount of gossypol determined by HPLC is not always correlated with the results from the AOCS official methods. This phenomenon is ascribed to shortcomings of the AOCS official methods that depend on the aldehydic groups of all the gossypol and gossypol derivatives, which can lead to false readings resulting from the presence of nongossypol aldehydic-containing compounds that can also react with the dye reagent (Stipanovic et al., 1984). Additionally, the AOCS methods are often criticized for their laborious and time-consuming procedures as well as lack of analytical sensitivity and accuracy. To overcome these obstacles in the quantitative and qualitative analyses of gossypol and gossypol derivatives, polyclonal and monoclonal antibodies to gossypol and relevant corresponding immunochemical methods, or the enzyme-linked immunosorbent assays (ELISA), have been developed to simplify and improve the current gossypol analyses (Wang and Plhak, 2000, 2004; Wang et al., 2004, 2005). Based on the principle of antibody–antigen specific interaction, ELISA tests are generally highly sensitive and specific, and favorably comparable with other methods such as the AOCS methods aforementioned. Besides, this method allows for easy visualization of results and can be completed within a relatively shorter time, and suitable for high throughput screening (analysis) of gossypol and its derivatives. This method has been demonstrated to be able to analyze crude extracts, possibly both soluble and insoluble analytes such as matrix-BG in various complex samples. Moreover, another advantage of the ELISA lies in its lower detection limit that enables analysis of samples in small volume or samples containing low gossypol content. It was a big challenge to separate gossypol enantiomers, though they are able to be quantified by HPLC after conversion to the Schiff base diastereoisomers (Cass et al., 1991; Stipanovic et al., 2005, 2006b; Zhou and Lin, 1988). Cass et al. (1999) developed a new method using a chiral HPLC to separate racemic gossypol without the need of derivatization. Dowd (2003) reported a promising method to obtain large enantiomeric crystals of gossypol–acetone (1:3) from acetone solutions of racemic gossypol–acetic acid (1:1) at low temperature. Later, Dowd and Pelitire (2008) isolated the chiral forms of gossypol, 6-methoxy gossypol and 6,60 -dimethoxy gossypol from the root bark of St. Vincent Island cotton

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and quantified the chemicals after the derivatization with R-()-2-amino1-propanol. By combining HPLC-PDA-MS-SPE-NMR with CD, Sprogoe et al. (2008) were able to detect the ()-gossypol in crude extracts of T. danis.

V. AGRICULTURAL IMPLICATION A. Insecticidal activity Gossypol is a secondary metabolite that is present in the pigment glands of cotton plants. It plays an important role in protecting cotton plants from insects (Liu et al., 2008). One study on larvae (El-Sebae et al., 1981) showed that gossypol at a dosage of 1.5% concentration in the feeding diet could significantly reduce the larval weight, and increase the duration of each larval stage. Also, the compound inhibited protease and lipid peroxidase activities as well as ATPase activity in larvae at higher concentrations. Gossypol showed stronger inhibition of mitochondrial ATPase (IC50, 1.7 10 4 M) than fenvalerate (insecticide, IC50, 7.0 10 4 M). These insecticidal activities could result in slower insect hatchability. The study on Helicoverpa zea larvae showed that a diet containing 0.16% racemic gossypol was the most effective on reducing the pest survival compared to (þ)- and ()-gossypol at the same concentration (Stipanovic et al., 2006a). Recent studies demonstrate that the toxicity of gossypol lies in the two aldehydic groups present in the molecule (Przybylski et al., 2008a,b,c), which can lead to changes of the metabolism of sterols, steroids, and fatty acids, and the production of toxic chemicals. Concerning the acute and long-term toxicities of gossypol and some of its derivatives, two methods have been employed to reduce the toxicity of gossypol in an effort to expand its agricultural usage. The first commonly used method is to block its two aldehyde groups by the formation of aza-derivatives and another one is to make a gossypol complex with metal cations. These kinds of chemical treatments can reduce the gossypol toxicity because they are no longer able to covalently bind proteins.

B. Antifeeding activity Gossypol is chemically reactive due to the reactivity of its carbonyl and phenolic hydroxyl groups as well as its bulky binaphthalene structure. Therefore, it has been the compound of greatest concern in cottonseed. During the cottonseed oil processing when the oil is removed from the cottonseed, some gossypol will also be extracted and remain in the oil, but it can be removed in the subsequent refining process, although some oil is lost. Meanwhile, some gossypol can react with other compounds in the

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cottonseed to form BG. This results in an undesirable color and a lowvalue protein in CSM (Jones, 1979; Pons et al., 1951). Some evidence indicates that the majority of gossypol exists in the Schiff bases from the condensation between the aldehydic groups of gossypol and amino groups of proteins during cottonseed processing (Cater, 1968; Lyman et al., 1959). Baliga (1956) found that the e-amino groups of lysine accounted for most of the free amino groups of the cottonseed protein for the reaction in an experimental model when the purified protein was allowed to react with gossypol. This resulted in a decreased availability of lysine to about one-half of its original value (82.9–48.7%). Additionally, involvement of arginine in the reaction has been reported (Martinez et al., 1961). Although the Schiff base reaction is the most well-known reaction of gossypol, it only represents a part of the known chemical activities of gossypol. In fact, gossypol can form other complexes via chelating with metal ions (Muzaffaruddin and Saxena, 1966), or forming gossypol polymers (Anderson et al., 1984), or being oxidized (Scheiffele and Shirley, 1964). Moreover, not only can gossypol bind with proteins in the CSM to render the adjacent peptides unavailable to the proteolytic action but also directly inhibit the activity of certain enzymes such as pepsinogen, pepsin, and trypsin present in the gastrointestinal tract by binding with their free e-amino groups of lysine (Sharma et al., 1978), lowering the digestibility. Thus, the presence of gossypol in cottonseed products generally will lower the nutritional values of the CSM.

C. Toxicity A number of investigators have indicated that gossypol is toxic to monogastric animals as well as young ruminants. Particularly, FG is more toxic to monogastric animals than BG. The most common toxic effect of gossypol was shown in the cardiac irregularity which caused the death of animals due to the prevention of the liberation of oxygen from oxyhemoglobin (Menaul, 1923). Generally, the toxicological doses of gossypol are classified into three levels, which are (1) acute doses causing circulatory failure, (2) subchronic doses causing pulmonary edema, and (3) chronic doses causing symptoms of ill health and malnutrition (Abou-Donia, 1976). However, the toxic doses could not be specifically defined because they depend on different animal models. Since gossypol possesses dose-dependent toxicity, its content in the CSM is limited if the CSM is used as a livestock feed. An early study on male rats (Eagle and Davies, 1958) showed that the oral LD50 values of gossypol ranged from 1061 to 2170 mg/kg, or 2200 to 2600 mg/kg when cottonseed pigment glands (containing 27.0–37.8% of gossypol), were orally administrated, respectively. The cottonseed pigment glands seemed more toxic than pure gossypol for rats in this study. El-Nockerashy

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et al. (1963) obtained similar findings in studying the acute oral toxicity by feeding cottonseed pigment glands, gossypol, diaminogossypol, and gossypurpurin for rats with LD50 values of 1120, 2570, 3270, and 6680 mg/kg body weight, respectively. Calhoun et al. (1990b) compared the toxicity of gossypol acetic acid and FG in CSM and Pima cottonseed by feeding lambs, and found that oral administration of gossypol acetic acid was much more toxic to young lambs than feeding equivalent amounts of FG in CSM or cottonseed. It was hypothesized that gossypol in different chemical states might have different stabilities in the digestive tracts between ruminants and nonruminants, so gossypol administrated in different forms to different animal species exhibited different toxic behaviors. In the study of gossypol’s toxicity on rainbow trout (Herman, 1970), it was found that 95 ppm of FG in the diet could cause histological changes in the fish’s liver and kidney while 100 ppm could cause pathological changes. When the gossypol concentration was raised to 290 ppm, growth suppression was observed. At the 531 ppm level, the fish lost weight for a period of time and suffered severe reduction of hematocrit, hemoglobin, and plasma proteins. Another study on channel catfish (8 weeks old) showed that diets containing approximately 0.14% of FG or added gossypol acetate in CSM could depress catfish growth, but a level of 0.09% or less FG in the diet seemed to be safe if all essential amino acids were in balance (Dorsa and Robinnette, 1982). Trischitta and Faggio (2008) investigated the effect of gossypol on transepithelial ion transport of the intestine of Anguilla anguilla, an European seawater-adapted eel. They found the addition of gossypol resulted in reduced short-circuit current and increased tissue conductance. However, such effects diminished when the eel was pretreated with a calmodulin inhibitor, suggesting that gossypol operated through the Ca2þ calmodulin pathway. Another interesting phenomenon was observed for yolk discoloration when hens were fed with the gossypol-containing feed, due to chemical combination of gossypol with ferric Fe released from yolk (Kemmerer et al., 1966; Schaible et al., 1934). Lordelo et al. (2007) found that the ingestion of (þ)-gossypol in the diet for 18 days reduced egg production in laying and broiler hens, and had a greater effect on egg yolk discoloration than ()-gossypol. Both ()- and (þ)-gossypol can bind with Fe, so the greater potency of (þ)-gossypol to trigger egg yolk discoloration indicates that the mechanisms by which gossypol causes egg yolk discoloration are more complicated than previous hypothesis (Kemmerer et al., 1966). In addition, gossypol caused lower diet consumption in laying hens compared with others fed by alternative dietary treatments. A study on calves (Zelski et al., 1995) showed that a diet containing 33% CSM, in which the concentration of FG was 100–220 ppm, resulted in the death of 24 out of 57 calves, each between 7 and 15 weeks of age. It was believed the gossypol in CSM caused large volumes of serous fluid in the

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body cavities, hard livers of ‘‘nutmeg’’ appearance, and pulmonary congestion. Several calves showed hemosiderosis and fibrosis in some pulmonary vessels of the lungs, in addition to the livers which exhibited periacinar necrosis in acute cases and periacinar fibrosis in chronic cases. Studies on the effects of different processing methods on the availability of gossypol through feeding lambs (Calhoun et al., 1990a,b) and cattle (Calhoun, 1996) demonstrated that gossypol toxicity was influenced by the methods of cottonseed processing, dietary concentrations of iron and calcium, the presence of other toxic terpenoids and chemicals found in cottonseed, as well as the amount and duration of gossypol consumption. Lindsey et al. (1980) studied the physiological responses of lactating cows to gossypol after the animals were fed with CSM containing high FG, and suggested that the rumen microorganisms were responsible for the detoxification that occurred in mature ruminants. It was also proposed that gossypol formed a gossypol–microbial protein complex with the soluble protein in the rumen liquor. This complex was very stable against the enzymatic hydrolysis, and not absorbed in the digestive tract (Reiser and Fu, 1962). It is believed the higher microbial yield characteristic of the rumen is important in supplying enough protein to capture the gossypol, although the microbial population in the rumen is regulated by the ecological balance of conditions that tend to prevail (Van Soest, 1982). This may explain why cattle were found to be more tolerant to gossypol than young calves due to the action of the rumen (Morgan, 1989). However, if the gossypol content is too high in the diet, it will still surpass the rumen’s ability to detoxify. For example, it was found that 348–414 mg of FG per day would cause congestive heart failure in adult goats (East et al., 1994). Similarly, 8 mg of FG and 222 mg of TG (in extracted CSM)/kg body weight per day could cause physical and hematological changes at 2 weeks for cows (Lindsey et al., 1980). Also, the intake of 36.8 mg of FG per kg of body weight per day could have detrimental effects on embryo development in pregnant heifers, and contribute to fertility problems in dairy cows (Villasenor et al., 2008). Besides, gossypol was toxic to swine when the animal was fed by 200–400 ppm of FG. Thus, it was suggested that FG should be less than 100 ppm for growing and fattening swine (Haschek et al., 1989). It is arguable whether FG content is a good predictor of the toxicity of the cottonseed products (Calhoun, 1996; Calhoun et al., 1990a; Eagle and Davies, 1958; Eagle et al., 1956) because measurements of BG, FG, and TG are insufficient. For example, the BG fraction is not always entirely inactive depending on the mechanism(s) of gossypol binding that occurs during the cottonseed processing, the type of animals consuming the gossypol, and the physical form of the feed. Moreover, different gossypol derivatives also yield different physiological activities, and the (þ/)two gossypol enantiomers, possess different biological activities. Study of

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the gossypol enantiomers revealed that only ()-gossypol was toxic to humans, but different ratios of (þ)- and ()-gossypol enantiomers in cotton plants did not affect the toxicity toward insects and pathogens (Liu et al., 2008). Calhoun (1996) suggested that different forms of BG might have different stabilities in vitro and in vivo, possibly because different cottonseed processing conditions favor various forms of BG. The animals selected for bioactivity experiments would also affect the results because of differing digestive environments. Therefore, current results related to the relationship between the available gossypol and its toxicity must be evaluated and interpreted carefully. Also, it should take into account of other factors such as dose and route of administration, the possibility of other toxic materials, and the degree of inactivation of administered gossypol before and/or after administration by reaction with components of the diet (Yu, 1987).

D. Detoxification Due to the toxicity of gossypol to animals, a glandless variety of cotton was developed in the early 1960s. It was believed that the value of cottonseed oil and meal would be improved if gossypol were not present. However, this glandless strain was easily susceptible to insect infestation, and attracted field mice and other rodents (Lusas and Jividen, 1987; Stipanovic et al., 1986). Dilday (1986) developed a cotton plant through an interspecific cross of tetraploid (2N ¼ 52) G. hirsutum L. diploid (2N ¼ 26) G. sturtianum Willis. This plant showed gossypol glands in vegetative foliar and fruiting tissues but not in the seed. However, this approach needs more investigation to prove the stability and yield of this plant. Another approach for gossypol removal is by a flotation process based on the specific gravity difference or by centrifugal force, such as the liquid cyclone process which is a physical separation of intact pigment glands (Ridlehuber and Gardner, 1974). Nevertheless, solvent extraction is more popular to remove gossypol because it is widely soluble in various organic solvents like ethyl alcohol, hexane, aqueous acetone, acidic butanol, methylene chloride, aniline, and aliphatic amines, which allows an extraction process with more choices in regard to the safety of solvent, volatility, cost, etc. Among the various extraction methods, extraction with 95% ethanol is more promising for a reduction in TG content up to about 70% in CSM (Hron et al., 1994). Aqueous acetone is also a desirable choice to reduce the FG in CSM (Damaty and Hudson, 1975; Pons and Eaves, 1971). A mixture of ethanol and hexane also reduce both FG and BG in flakes and oil tremendously (Liu et al., 1981). A two-step extraction technique utilizing aqueous and anhydrous acetone, successfully reduced the FG content in gossypol protein concentrate to below FDA standards for human consumption without compromising organoleptic characteristics

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(Gerasimidis et al., 2007). Although solvents have generally been successful in reducing the gossypol content of cottonseed products to quite low levels, how to completely remove the potentially harmful solvent residual is another challenging subject. Chemical methods have been involved in gossypol detoxification. Bressani et al. (1964) reported that an alkaline pH of cooking, associated with calcium ions, was effective in reducing FG and TG in cottonseed flour used for human foods. The addition of calcium increased the effectiveness of the gossypol–iron complex formation, resulting in full protection from gossypol toxicity. Nagalakshmi et al. (2002, 2003) also confirmed the effectiveness of calcium hydroxide for the gossypol detoxification. Iron sulfate is an inexpensive source of Fe that could also reduce gossypol intoxication in nonruminant animals (Ullrey, 1966). Kemmerer et al. (1966) reported that the addition of iron sulfate to feed could prevent yolk discoloration caused by gossypol. Muzaffaruddin and Saxena (1966) showed that a 1:1 molar ratio of ferric iron to gossypol formed an iron– gossypol complex, in which the two perihydroxyl groups of gossypol were the most plausible sites for the ferric irons to be chelated. Barraza et al. (1991) investigated the efficacy of iron sulfate and feed pelleting to detoxify FG in cottonseed diets for dairy calves. Ferrous sulfate added to diets for swine contained 244 and 400 ppm FG, a molar ratio of 0.5:1 for iron to gossypol resulted in partial detoxification and, furthermore, a 1:1 ratio gave complete detoxification. The addition of phospholipids to CSM or CSM coupled with cooking could eliminate some FG and improve the protein quality (Yannai and Bensal, 1983). Microbial fermentation of CSM, which was proposed to detoxify FG in the CSM (Zhang et al., 2006a,b), seems promising because some exoenzymes such as cellulolytic enzymes, amylase, protease, and lipolytic enzymes that are secreted by certain microorganisms, and some vitamins, as well as some unknown active substances are produced in the fermented CSM (Brock et al., 1994), which adds nutritional value of the fermented CSM. Recently, Qian et al. (2008) reported that in situ alkaline-catalyzed transesterification could produce a CSM with FG and TG contents below the FAO standard. However, the requirement for a high amount of methanol usage in the in situ transesterification and the potential energy consumption to remove the methanol in the meal may be an obstacle for its practical application.

VI. BIOLOGICAL PROPERTIES Gossypol is a reactive compound due to the presence of six phenolic hydroxyl groups and two aldehyde groups. This reactivity also contributed to its biological activities, which will be discussed in the following sections.

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A. Antioxidant property Gossypol is a polyphenolic compound from the viewpoint of its chemical structure. Like many other phenolic chemicals, such as butylated hydroxytoluene (BHT), coumaric acid, gallic acid, quercetin, myricetin, catechin, gallocatechin, etc., gossypol is an effective and potent natural antioxidant. For example, gossypol was found to be able to protect carotene in vitro against preformed fat peroxides many decades ago (Hove and Hove, 1944). Hove (1944) confirmed that cottonseed products containing gossypol could inhibit carotene destruction and rancidity development in vitro, and gossypol could act as a carotene-protecting antioxidant in vivo. Gossypol has shown potential in inhibiting rat liver microsomal peroxidation, which is caused by an incubation with ferric/ ascorbate (IC50 < 0.1 mM) (Laughton et al., 1989). Gossypol also exhibited a significant positive effect on oil and biodiesel stability. With a concentration of 0.1% gossypol, the oxidative stability indices (OSI) of cottonseed oil biodiesel could increase to 17.2 h from 4.15 h at 110 C (Fan et al., 2008). In some cases, the modification of the functional groups on gossypol may not affect its original chemical and biological activities. For instance, the modification of aldehydic groups on gossypol to form dianilinogossypol, of which the free carbonyl groups were tied up by the anilido complex, did not decrease the antioxidant activity of the free compound (Bickford et al., 1954; Hove, 1944). Bickford et al. (1954) also found the other Schiff base-formed gossypol derivatives, gossypol-urea, gossypolaminobenzene-thiol, and gossypol-glycineindicates, have roughly equivalent antioxidative ability to gossypol on a molar basis. Gossypol bis (piperinoethylimine) and bis(morpholinoethylimine) also demonstrate potent antioxidant action in human blood serum and rat brain synaptosomes. At equal concentrations, these substances suppressed the peroxidation of lipids in enzymatic and nonenzymatic systems regarding the oxidation of rat liver microsomes (Dalimov et al., 1989). On the contrary, in many other cases, the modification of phenolic hydroxyl groups on gossypol could significantly decrease the ‘‘chemical’’ antioxidative abilities regarding free radical scavenging activity, reducing power, and DNA damage prevention activity (Wang et al., 2008), demonstrating that the hydroxyl groups are critical for the antioxidative activities. For example, 6-methoxy gossypol exhibited a similar free radical scavenging activity as 6,60 -dimethoxy gossypol, while gossypol possessed a stronger radical scavenging activity than either of the methylated derivatives. The concentrations of 6-methoxy gossypol and 6,60 -dimethoxy gossypol needed to scavenge 50% of the free radicals in the test system were twofold higher than that of gossypol (about 16 ppm vs. 8 ppm). Although gossypol, 6-methoxy gossypol, and 6,60 -dimethoxy gossypol all reduced ferric ions to ferrous ions in a dose-dependent manner, gossypol

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235

again showed greater reducing power and higher efficiency than 6-methoxy gossypol or 6,60 -dimethoxy gossypol. However, all three test compounds showed much stronger reducing power than BHT; 6,60 -dimethoxy gossypol at a concentration of 10 ppm exhibited the same reducing power as BHT at a 100-ppm concentration. The relative capability of gossypol and its methylated derivatives to prevent DNA damage caused by ultraviolet light and hydrogen peroxide was consistent with the compounds’ antioxidant effects. This suggests that gossypol’s protection of DNA may occur partially by quenching free radicals, therefore alleviating oxidative stress. A previous study (Li et al., 2000) also found that gossypol demonstrated the ability, in a dose-dependent manner, to protect supercoiled plasmid DNA from damage caused by exposure to Fe3þ/ascorbate.

B. Antifertility activity Gossypol has been studied extensively and intensively as a potential contraceptive agent through in vivo models including rats (Hadley et al., 1981; Lin et al., 1980, 1985), mice (Coulson et al., 1980), monkeys (Shandilya et al., 1982), hamsters (Matlin et al., 1987; Waller et al., 1984), rabbits (Chang et al., 1980), bulls (Arshami and Ruttle, 1988), and male humans (National Coordinating Group on Male Antifertility Agents, 1978). The following section is going to cover some research findings on antifertility activity of gossypol. Oral administration of gossypol acetic acid at a dose of 5 or 10 mg/kg body weight/day for 12 weeks induced sterility in male hamsters and rats. Treatment in male rabbits at doses ranging from 1.25 to 10 mg/kg body weight/day for 5–14 weeks decreased the sperm mortality but did not affect the average number of sperm per ejaculate (Chang et al., 1980). In another study reported by Lin et al. (1980), gossypol treatment at a dose of 30 mg/kg body weight/day was found to markedly diminish sperm production in rats. Hoffer (1982) observed no differences in the morphology of the testis or epididymal sperm in male rats at a low dosage of 7.5 mg/kg body weight/day for 7 weeks. At the higher dosages (20 and 30 mg/kg body weight/day), deleterious changes in epididymal sperm and a limited extent of testicular damage were observed. In a study through oral administration of gossypol at a level of gossyspol acetic acid of 5 or 10 mg/kg body weight/day for 6 months, it was found that adult male cynomoglus monkeys showed a significant decrease in sperm concentration and motility, even though there was neither a significant decrease in circulating levels of testosterone nor a significant difference in plasma testosterone levels (Shandilya et al., 1982). Similarly, daily feeding of gossypol acetic acid (40 mg/kg body weight/ day) to chickens for 18 days resulted in a decrease in semen volume and

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sperm concentration. The treatment also decreased the activities of acrosin, hyaluronidase, and angiotensin converting enzyme, and fertility dropped to zero at the end of the treatment period. Meanwhile, other obvious side effects including a loss of appetite, loss of body weight, and morphological abnormalities in spermatozoa were observed in the treated cocks (Mohan et al., 1989). It was also found that ()-gossypol is more active in its antifertility function than (þ)-gossypol (Matlin et al., 1987). (þ)-Gossypol at the dosage of 30 mg/kg orally for 14 days has neither antifertility effects nor toxicity in male rats, but slight damage was found in the germinal epithelium of the testis in animals dosed for 4 weeks. ()-Gossypol at 30 mg/kg orally for 7 days showed an antifertility effect in male rats (Wang et al., 1987). Gossypolone, an oxidized metabolite of gossypol, displayed less spermicidal activity than gossypol isomers on spermatozoa from human, monkey, rabbit, mouse, rat, and hamster subjects (Kim et al., 1984). The concurrent treatment of gossypol and steroid hormones showed potential in contraceptive activity. For example, the combination of gossypol (12 mg/kg body weight/day) with steroid hormones methyltestosterone at 20 mg/kg body weight/day and ethinyl estradiol at 100 mg/kg body weight/day for 6 weeks, along with a low-dose gossypol alone for 12 weeks as a maintenance dose could damage the epididymal sperms in male rats (Xue, 2000). A similar study on adult Wistar rats under the treatment with a combination of a low dose of gossypol and steroid hormone (methyltestosterone and ethinyl estradiol) via gastric intubation also caused a similar decrease of epididymal sperm motility and epididymal sperm deterioration (Wang et al., 2000). The mechanism illustrating how gossypol exerts contraceptive activity has been exploited by some researchers. Spermatogenesis is a process involving a specific ratio of cells with unique DNA ploidy (1C, 2C, and 4C). Treatment of 20 mg/kg body weight/day of gossypol in rats revealed a significant shift from the 1C stage to the 2C and 4C stages, indicating less sperm available for female fertilization (Ojha et al., 2008). In another study, Bai and Shi (2002) found that the concentration 5 mM gossypol could completely block the T-type Ca2þ currents in mouse spermatogenic cells, and the gossypol-induced inhibition of T-type Ca2þ currents could be responsible for the antifertility activity of the compound. A decreased seminal plasmid lipid concentration contributed to the reduction in sperm counts in rabbits (Shaaban et al., 2008) and bulls (Kelso et al., 1997). Studies on the activity of rabbit sperm acrosomal enzymes have indicated that gossypol at 12–76 mM could significantly inactivate azocoll proteinase, acrosin, neuraminidase, and arylsulfatase. Hyaluronidase, b-glucuronidase, and acid phosphatase were also inhibited at a higher concentration of gossypol (380 mM) (Yuan et al., 1995). Since acrosomal enzymes play important roles in the fertilization process, the inhibition of

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237

these enzymes would alter the acrosome reaction, and interrupt the fertilization process. ATPases are a type of enzyme that can catalyze the decomposition of adenosine triphosphate (ATP) and provide energy for cell growth. Gossypol was found to inhibit the human spermatozoa ATPase activity, lowering the energy available for cell motility (Wu et al., 1998). Taitzoglou et al. (1999) found that plasminogen activator activity from man and ram extracts can be completely inhibited by 350 and 300 mM of gossypol, respectively. Also, a low concentration of gossypol (2.5–40 mM) was found to be able to significantly inhibit plasmin activity in a dose-dependent manner. Since the plasminogen activator/plasmin system plays a role in the entire process of ovum fertilization, the inhibition of both acrosomal plasminogen activator and plasmin activity is a possible mechanism by which gossypol exerts its antifertility effect. In addition, the inhibition of LDH also contributes to the antifertility. Selective inhibitors of human LDH (LDH-C4, -B4, and -C4) targeted to the dinucleotide fold hold promise as male antifertility drugs. Gossypol is a nonselective competitive inhibitor of NADH binding to LDH, with Ki values of 1.9, 1.4, and 4.2 mM for LDH-A4, -B4, and -C4, respectively (Yu et al., 2001). However, gossypol’s antifertility activity may be influenced by many endogenous effectors. Javed and Khan (1999) found that histidine, cysteine, and glycine could block the effect of gossypol acetic acid on the inhibition of purified LDH-X, while arginine, glutamic acid, phenylalanine, and valine were ineffective against the inhibitory action of gossypol acetic acid. In humans, 5-alpha-reductase activity is critical for certain aspects of male sexual differentiation and may be involved in the development of benign prostatic hyperplasia, alopecia, hirsutism, and prostate cancer. Gossypol is a selective noncompetitive inhibitor of the type 1 steroid 5-alpha-reductase isoenzyme and, therefore, may have potency for the prevention or treatment of androgen-dependent disorders. This inhibition seems to require the presence of catechol moieties on the gossypol molecule (Hiipakka et al., 2002).

C. Anticancer activity Gossypol is capable of inhibiting the growth of a variety of cell lines including breast, colon, prostate, and leukemia cells (Balci et al., 1999; Benz et al., 1990; Huang et al., 2006; Zhang et al., 2003). Table 6.2 summarizes the antitumor activities of gossypol against several cancer human cell lines in vitro. These disruptions include inhibition of cytoplasmic and mitochondrial enzymes involved in energy production (Ueno et al., 1988) and uncoupling of oxidative phosphorylation (Abou-Donia and Dieckert, 1974; Flack et al., 1993). In addition, depletion of cellular ATP has been demonstrated in cultured tumor cells (Keniry et al., 1989). Gossypol also

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TABLE 6.2 Antitumor activity of gossypol against several human cancer cell lines in vitro Human cancer cell lines

Ovarian OVCA-420 OVCA-429 OVCA-433 OVCA-432 OVCAR-3

Gossypol activity IC50 (mM)

(þ)/()3.8 2.4 2.5 2.4 1.5

()1.6 0.9 1.2 1.1 0.6

(þ)11.7 11.3 8.5 10.2 5.8

References

Band et al. (1989) Band et al. (1989) Band et al. (1989) Band et al. (1989) Band et al. (1989) Band et al. (1989)

Prostate PC-3 Breast MCF-7 MCF-7 T47-D MCF-7 WT

10

Zhang et al. (2007b)

5 24 5 3.4

MCF-7 ADR

4.3

SKOV-3

5.7

Coyle et al. (1994) Zhang et al. (2007a) Benz et al. (1990) Jaroszewski et al. (1990) Jaroszewski et al. (1990) Le Blanc et al. (2002)

3

20

a

Endomentrial RL95-2 Carvix KB-3

3.4

Le Blanc et al. (2002)

3.8

KB-A1

2.9

KB-V1

4

Hela SiHa SiHa

4 47 14

Jaroszewski et al. (1990) Jaroszewski et al. (1990) Jaroszewski et al. (1990) Coyle et al. (1994) Shelley et al. (2000) Zhang et al. (2007a)

Adrenocortical H295R SW-13

2.9 1.3

Le Blanc et al. (2002) Le Blanc et al. (2002)

Medullary thyroid TT

18.9

Le Blanc et al. (2002)

30

>50

(continued)

Gossypol

TABLE 6.2

239

(continued)

Human cancer cell lines

Gossypol activity IC50 (mM)

Lung A549 H69

0.5 30

Colon Caco 2 Colo201 SW407

17 4 8.2

SW1084

6.5

SW1116

7

HCT-8 HT-29

5

Leukemia JURKAT T HL-60 HL-60 HL-60 HL-60 K562 U937 THP-1

20

5

50

50

20 50 8.3 20

>50

Pancreatic MiaPaCa

3

20

Head and neck UM-SCC

2.5–10

15 15 15 15

References

Chang et al. (2004) Shelley et al. (2000) Zhang et al. (2007a) Coyle et al. (1994) Tuszynski and Cossu (1984) Tuszynski and Cossu (1984) Tuszynski and Cossu (1984) Benz et al. (1990) Zhang et al. (2003) Oliver et al. (2005) Jarvis et al. (1994) Hou et al. (2004) Shelley et al. (2000) Moon et al. (2008b) Moon et al. (2008b) Moon et al. (2008b) Moon et al. (2008b) Benz et al. (1990) Oliver et al. (2004)

Melanoma SK-MEL-19

25

20

>30

SK-MEL-28

23

16.5

>30

WM9 WM56

6.2 6

3.1

14.3

WM164

5.5

Blackstaffe et al. (1997) Blackstaffe et al. (1997) Band et al. (1989) Tuszynski and Cossu (1984) Tuszynski and Cossu (1984) (continued)

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TABLE 6.2

(continued)

Human cancer cell lines

a

Gossypol activity IC50 (mM)

FEMX

2.4

FEMX4AP

5

SK-EML-3

4

References

Jaroszewski et al. (1990) Jaroszewski et al. (1990) Coyle et al. (1994)

No data provided; IC50: median inhibitory concentration. Adapted from Dodou et al. (2005).

inhibits key nuclear enzymes responsible for DNA replication and repair, including DNA polymerase a (Rosenberg et al., 1986) and topoisomerase II, and blocks DNA synthesis in HeLa cells (Wang and Rao, 1984). Hou et al. (2004) found that gossypol at 50 mM for 6 h could induce apoptosis in human promyelocytic leukemia cells (HL-60) (DNA fragmentation, poly (ADP) ribose polymerase cleavage), and also induce the truncation of Bid protein, the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into cytosol, and activation of caspases-3, -8, and -9. At a low dose of 5 mM, gossypol also could cause a significant elevation of caspases-3, -8, and -9, which resulted in cell apoptosis of human colon cancer cell line HCT 116 (Zhang et al., 2007b). Recent studies on human leukemia U937 cells showed gossypol at >10 mM resulted in significant cell cytotoxicity and DNA fragmentation, induced caspase-3 activation and poly(ADP) ribose polymerase cleavage. These properties make gossypol a potential antineoplastic agent. It is reported that inhibition of DNA synthesis can be achieved with 10 mM gossypol by blocking the G1/S checkpoint in MCF-7 cells at 24 h of incubation (Ligueros et al., 1997). Gossypol might regulate cell cycles by modulating the expression of cell-cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. A similar conclusion was obtained from Jiang et al. (2004) that inhibitory effects of gossypol on the proliferation of human prostate cancer PC3 cells were associated with induction of TGF-b1, which in turn influenced the expression of the cellcycle regulatory protein, cyclin D1. In human alveolar lung cancer cells, gossypol induces Fas/Fas ligand-mediated apoptosis (Moon et al., 2008a). Also, gossypol induces transcriptional downregulation and posttranslational modification of hTERT in human leukemia cells, causing inactivation of c-Myc and Akt, respectively. Both c-Myc and Akt are able to regulate various Bcl-2 proteins, the proapoptosis protein family members. Treatment with gossypol also downregulated the expression of NF-kappa B-regulated gene products, including inhibitor of apoptosis protein (IAP)-1, IAP-2, and X-linked IAP. These results suggest that

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241

gossypol-induced apoptosis partially involves suppression of NF-kappa B activity (Moon et al., 2008b). Treatment of Ramos cells with gossypol not only induced cell arrest on the G0/G1 phase but also increased apoptosis and growth inhibition induced by etoposide (VP-16), doxorubicin hydrochloride (ADM), vincristine (VCR), and paclitaxel (taxol) (Li et al., 2008). Liu et al. (2002) found that ()-gossypol was more active in inhibiting breast cancerous epithelial cells (cEC) and cancerous stromal cells (cSC). Meanwhile, the inhibitory activity of ()-gossypol was related to the reduction of the cell-cycle regulator, cyclin D1, and the induction of the cell proliferation inhibitor, TGF-b. In the study of human prostate cancer cells, it was found that ()-gossypol-induced apoptosis was mediated by the regulation of Bcl-2 and caspase families (Huang et al., 2006). Another in vitro study (Mohammad et al., 2005) demonstrated ()-gossypol had significant inhibitive effects against the growth of lymphoma cell line WSU-DLCL2 and fresh cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. ()-Gossypol also induced complete cytochrome c release from mitochondria, increased caspases-3 and -9 activity, and caused apoptotic death without affecting protein levels of Bcl-2, Bcl-X(L), Bax, and Bak. Recent research has revealed that ()-gossypol acts as a BH3 mimetic, binding to the BH3-binding domain in various proapoptotic proteins of the Bcl-2 family, displacing prodeath partners to induce apoptosis (Balakrishnan et al., 2008; Meng et al., 2008). Sikora et al. (2008) found that the combination of gossypol with the antioxidant N-acetyl-cysteine (NAC) to block reactive oxygen species (ROS) would increase the ()-gossypol-induced cytotoxicity in tumor cells, but not normal cells, indicating that concurrent treatment with antioxidants to block ROS prevents oxidative inactivation of ()-gossypol and limits off-target toxicity allowing more potent ()-gossypol-induced antitumor activity. An in vivo study also showed that ()-gossypol significantly enhanced the antitumor activity of X-ray irradiation, leading to tumor regression in the combination therapy by inhibiting both antiapoptotic proteins Bcl-2 and/or Bcl-xL (Xu et al., 2005). A combination of docetaxel and ()-gossypol synergistically enhanced the antitumor activity of docetaxel both in vitro and in vivo in the human prostate cancer PC-3 xenograft model in nude mice. ()-Gossypol exerts its antitumor activity through inhibition of the antiapoptotic protein Bcl-xL accompanied by an increase of proapoptotic Noxa and Puma (Meng et al., 2008). One study on gossypol derivatives (Arnold et al., 2008) showed that apogossypolone could inhibit the growth of the lymphoma cell line WSU-FSCCL with an IC50 of 109 nM, and the activation of caspases-9, -3, and -8 was observed. Hu et al. (2008) found that apogossypol selectively inhibited proliferation of three NPC cell lines (C666-1, CNE-1, and CNE-2) that highly expressed the antiapoptotic Bcl-2 proteins with release of cytochrome c, activation of

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caspases-9 and -3, and apoptosis of sensitive NPC cells (Hu et al., 2008). The toxicity and efficacy study on mice (Kitada et al., 2008) showed that mice tolerate doses of apogossypol two- to four-times higher than gossypol. Apogossypol displayed superior activity to gossypol in terms of reducing splenomegaly and reducing B-cell counts in the spleens of Bcl-2-transgenic mice, indicating the potential of gossypol derivatives for cancer therapy. Gossypolone was less potent than gossypol in inhibiting human breast cancer cells (Gilbert et al., 1995). The reduced effectiveness of gossypolone compared to gossypol in breast cancer cells agrees with the antifertility effects (Kim et al., 1984), but is in contrast to the antisteroidogenic and antireproductive effects of gossypolone, which have shown similar potency as gossypol (Gu and Lin, 1991). Methylated gossypol, 6-methoxy gossypol, and 6,60 -dimethoxy gossypol, compared with the parent compound, showed superior anticancer activity against cervical (SiHa), breast (MCF-7), and colon (Caco-2) cancer cells (Wang et al., 2008). In summary, gossypol is believed to arrest cell growth at the G0/G1 phase and induce cell apoptosis, in cancer cells, by regulating the cell cycle, enzymes, antiapoptosis, and proapoptosis proteins.

D. Antivirus activity Lin et al. (1989, 1993) reported that gossypol inhibited the replication of human immunodeficiency virus type 1 (HIV-1) and found ()-gossypol to be more inhibitory (IC50 ¼ 5.2 mM) compared to the (þ)-gossypol (IC50 ¼ 50.7 mM). Besides HIV-1, gossypol also showed antiviral activity in multiple enveloped viruses including herpes simplex virus type II (HSV-II), influenza virus, and parainfluenza virus (Vander Jagt et al., 2000). Gossypol and a series of periacylated gossylic nitriles (Fig. 6.10) were compared for their antiviral activities against HSV-II and for their toxicities to the host Vero cells. All of the periacylated gossylic nitriles exhibited lower cytotoxicities to the host cell than did the parent compound gossypol. Both gossypol and the series of derivatives exhibited antiviral activities against HSV-II when the virus was treated with concentrations as low as 5 10 7 M. Two of the derivatives, gossylic nitrile-1,10 -diacetate and gossylic nitrile-1,10 -divalerate, were capable of inhibiting viral multiplication in Vero cells that were infected with virus before administration of the drug. Radloff et al. (1986) concluded that modification of gossypol’s aldehydic groups lowered its toxicity to the host Vero cells but did not abolish the compound’s antiviral (HSV-II) activity. Derivatives of gossypol may be useful as antiviral agents. Later, Royer et al. (1991) found that gossypol and its derivatives, gossylic nitrile-1,10 -diacetate, gossylic iminolactone, and gossylic lactone (Fig. 6.10), inhibited the replication of HIV-1 in vitro. Gossylic iminolactone displayed the greatest inhibition, followed by gossypol, gossylic

Gossypol

N C

OR

243

N OR C OMe OMe

MeO MeO

R=O

a Gossylic nitrile 1,l⬘-diacetate

O b Gossylic nitrile 1,l⬘-dipropionate R=O

HN

c Gossylic nitrile 1,l⬘-dibutyrate

R=O

d Gossylic nitrile 1,l⬘-divalerate

R=O

O

HO HO

O

OH OH Gossylic iminolactone, GIL CHO

HO HO

CHO OH OH

1,l⬘-dideoxygossypol (DDG) COOH HO HO

O

NH

COOH OH OH

1,l⬘-dideoxygossylic acid (DDGA)

FIGURE 6.10

O O O

O

O

HO HO

O OH OH

Gossylic lactone CHO HO HO 8-deoxyhemigossypol (DHG) COOH HO HO 8-deoxyhemigossylic acid (DHGA)

Gossypol derivatives.

nitrile-1,10 -diacetate, and gossylic lactone, indicating that derivatives of gossypol can retain antiviral activities. Then, Royer et al. (1995a,b)) tested several other gossypol derivatives for inhibition of HIV 1,10 -dideoxygossypol (DDG), 1,10 -dideoxygossylic acid (DDGA), 8-deoxyhemigossypol (DHG), and 8-deoxyhemigossylic acid (DHGA) (Fig. 6.10). The result showed that DDGA was the most effective in inhibiting the replication of HIV in vitro with EC50 < 1 mM. Meanwhile, DDG was less effective than DDGA. DHG showed some anti-HIV activity, and DHGA was ineffective against HIV. Since all four gossypol derivatives were found to have much lower affinities for albumin than the parent compound gossypol, this would possibly enhance the antivirus activity of the gossypol derivatives in vivo with less interference from in vivo proteins.

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E. Antiparasitic protozoan activities Malaria is a vector-borne infectious disease caused by protozoan parasites. Human malaria is usually caused by the infection of Plasmodium falciparum, P. malariae, P. ovale, and P. vivax (Mendis et al., 2001). It is widespread in tropical and subtropical regions, including Asia, Africa, and parts of the Americas. Each year there are about 350–500 million cases of malaria, and more than 1 million people die (CDC, 2009). A series of gossypol derivatives with modified aldehydic groups and hydroxyl groups (Figs. 6.10 and 6.11) have been shown to inhibit the growth of P. falciparum (Razakantoanina et al., 2000; Royer et al., 1986). Table 6.3 R

R

N H OH

H

H C OH

N H

O

O

HO

OH

Derivatives

R

Methyl gossypol

−CH3

Ethyl gossypol

−CH2CH3

Propyl gossypol

−CH2CH2CH3

Isopropyl gossypol

−CH(CH3)2

Butyl gossypol

−CH2CH2CH2CH3

s-butyl gossypol

−CH2 CH(CH3)−CH3

t-butyl gossypol

−C(CH3)3

Pentyl gossypol

−CH2CH2CH2CH2CH3

Hexyl gossypol

−CH2CH2CH2CH2CH2CH3

Heptyl gossypol

−CH2CH2CH2 CH2 CH2CH2CH3

Dodecyl gossypol

−CH2 (CH2)11CH3

Mepheneta gossypol

−CH2(CH3)CH−C6H5

Phemetb gossypol

−CH(COOCH3)−CH2−C6H5

a: mephenet: -methyl phenylalanine ethyl b: phemet: phenylalanine methyl ester

FIGURE 6.11

Chemical formula for gossypol Schiff bases.

TABLE 6.3

Antimalarial activity of gossypol and its derivatives against P. falciparum in vitro IC50 (mM)a

IC50 (mM)a

Strain

a b c

Strain

Drug

PFB

b

FCB1

Gossypol Methyl gossypol Ethyl gossypol Propyl gossypol Isopropyl gossypol Butyl gossypol s-Butyl gossypol t-Butyl gossypol Pentyl gossypol Hexyl ossypol Heptyl gossypol Dodecyl gossypol Mephenet gossypol Phemet gossypol

15.3 ND 22 16 16.6 42.4 37.8 39.5 ND 67.2 ND 43.2 47 83

28.8 66.2 22.5 20.8 17.6 ND 54 40 ND ND 33.2 37 56 70.3

b

Drug

FCB/NC-1

CDC/I/HB-3c

Gossypol Gossylic nitrile 1,10 -diacetate Gossylic nitrile 1,10 -dipropionate Gossylic nitrile 1,10 -dibutyrate Gossylic nitrile 1,10 -divalerate

13 76 69 26 16

7 36 46 21 12

c

IC50 represents the drug concentration producing 50% inhibition of the growth of P. falciparum in drug free control wells. Chloroquine-resistant strains of P. falciparum. Strains FCB/NC-1 and CDC/I/HB-3 are the chloroquine-resistant and chloroquine-sensitive strains of parasite, respectively. Adapted from Royer et al. (1986) and Razakantoanina et al. (2000).

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summarizes the antimalarial activity of gossypol and its derivatives in vitro. The IC50 values are between 13 and 83 mM for gossypol and gossypol derivatives against P. falciparum. The derivatives with ethyl, propyl, or isopropyl side chains as well as gossylic nitrile 1,10 -divalerate with IC50 values close to gossypol (IC50 ¼ 16 mM) showed stronger inhibition than other gossypol derivatives against the growth of P. falciparum. Royer et al. (1986) proposed that the antimalarial activity of gossypol and gossypol derivatives was through the inhibition of LDH. LDH is the most active and essential enzyme for anaerobic life cycle of P. falciparum. Any compound showing inhibition of this enzyme also kills the parasites (Razakantoanina et al., 2000; Royer et al., 1986). A study on Toxoplasma gondii, a protozoan parasite causing toxoplasmosis, also demonstrated that the inhibition of T. gondii LDH activity is correlated with the inhibition of T. gondii growth in cultures (Dando et al., 2001). In the study on Entamoeba histolytica (Gonzalez-Garza et al., 1993a,b), gossypol also showed the inhibition to alcohol dehydrogenase and malic enzymes, and ()-gossypol was found more active than racemic gossypol and (þ)-gossypol. The ()-gossypol was 3.6 and 13 times more potent than (þ/)- and (þ)-gossypol, respectively, in inhibiting the malic enzyme, and 1.9 and 2.9 times more potent than (þ/)- and (þ)-gossypol, respectively, against the alcohol dehydrogenase. Trypanosomes, protozoan parasites belonging to the subphylum Mastigophora, can cause a chronic infection called sleeping sickness. It has seriously affected the health of people in western and central African countries, and exerted significant mortality in man and livestock. Over 60 million people living in 36 sub-Saharan countries are threatened by sleeping sickness (WHO, 2001) and 48,000 deaths were reported in 2002 (WHO, 2004). In addition, 46 million cattle are exposed to the risk of the sleeping disease. The disease costs an estimated 1340 million USD per year (Kristjanson et al., 1999). However, few drugs are available for the treatment of trypanosomal infections that cause significant mortality in man and livestock in Africa. Gossypol was reported to be able to inhibit trypanosomes (Blanco et al., 1983; Kaminsky and Zweygarth, 1989; Montamat et al., 1982). Montamat et al. (1982) reported that a 5-min exposure to 100 mM gossypol ( 50 ppm) immobilizes cultures of Trypanosoma cruzi. Blanco et al. (1983) reported that a 30-min exposure to 25 mM gossypol ( 12 ppm) immobilizes and alters the cell morphology of T. cruzi. Later, Kaminsky and Zweygarth (1989) reported that, for three separate T. brucei strains (including one drug-resistant strain), the IC50 value for a 24-h gossypol exposure was >10 ppm. Our study showed a similar level of gossypol’s antitrypanosomal activity with IC50 value of 7.8 ppm after 24-h exposure. Moreover, methylated gossypol, both 6-methoxy gossypol (IC50 value, 3.98 ppm) and 6,60 -dimethoxy gossypol (IC50 value, 3.21 ppm) showed more effective inhibition of growth than

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gossypol. In T. cruzi, gossypol has been reported (Gerez de Burgos et al., 1984; Montamat et al., 1982) to inhibit some oxidoreductases, such as, alpha-hydroxyacid and malate dehydrogenases, NAD-linked enzymes, and glutamate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase, NADP-dependent enzymes. Gossypol also inhibits the MDH enzyme of T. cruzi (Gerez de Burgos et al., 1984). Accordingly, the possible mechanism of the antiparasitic effect of gossypol and gossypol derivatives could be the selective inhibition of vital enzymes in the parasites.

F. Antimicrobial activity The antimicrobial properties of gossypol have been reported by several research groups. Gossypol has general antifungal activities with LD50 values from 20 to 100 ppm of pure gossypol (Bell, 1967), and has an inhibitory effect on microorganisms including aerobic sporeformers and lactobacilli and some yeasts (Table 6.4) (Margalith, 1967). Gossypol showed strong antibiotic activity against aerobic sporeformers and lactobacilli, and displayed antagonistic property to some of the more oxidative yeasts. Later, Vadehra et al. (1985) investigated the effects of gossypol on the growth of a variety of bacteria and on spore formation and germination in Bacillus cereus. It has been found that gossypol has more potent TABLE 6.4 Inhibitory effect of gossypol on microorganisms effect of gossypol on microorganisms Organism: minimal inhibitory concentration

a b

Bacteria

mg/ml

Yeasts

mg/ml

Staphylococcus aureus Sarcina lutea Bacillus polymyxa B. megaterium B. licheniformis B. cereus B. thermoacidurans Leuconostoc mesenteroides Lactobacillus delbruckii Escherichia coli Proteus mirabilis Pseudomonas aeruginosa

10 25 50 50 25 50 50 10 20 >200

Saccharomyces cerevisiae S. carlsbergensis Zygosaccharomyces mellis Hansenula anomala Hanseniaspora sp. Candida utilis Debaryomyces nicotianae Pichia membranefaciens Cryptococcus neoformans Rhodotorula mucilaginosa

>200 >200 >200 200a 200a >200 100 25b 25 >200 >200 >200

Caused slight inhibition. Caused complete suppression of film growth. Adapted from Margalith (1967).

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antibacterial properties against Gram-positive organisms (i.e., Streptococcus spp., Bacillus spp., Staphylococcus aureus) than Gram-negative bacteria such as Pseudomonas aeruginosa, Salmonella spp., Klebsiella pneumoniae, Shigella spp., Proteus spp., and Escherichia coli. All of the Gram-positive organisms tested were completely inhibited at a concentration of 100 ppm. None of the tested Gram-negative strains was inhibited at 100 ppm of gossypol, and only one-third of the tested strains were inhibited at 200 ppm of gossypol. The authors proposed that the antibacterial activity of gossypol was related to the Gram character of the organisms. Besides, the chemical and quantitative differences of the cell wall and cell membrane of the Gram-positive and -negative groups may influence the transport of gossypol to its target site (i.e., Gram-positive organisms have high amount of peptidoglycan in the cell wall, and lack the outer membrane found in Gram-negative bacteria). The same research group also found that yeasts, such as Saccharomyces cereviseae, S. uvarum, S. diasticu, were sensitive to gossypol, and the growth were completely inhibited at 50 ppm of gossypol. Subsequent research (Poprawski and Jones, 2001) found that fungi Paecilomyces fumosoroseus (associated with cutaneous and disseminated infections in dogs and cats) were highly tolerant to gossypol even at 500 ppm, but could be strongly inhibited at 1000 ppm of gossypol.

G. Lowering plasma cholesterol levels Cholesterol is a fat-soluble compound found in the body. Having high ‘‘bad’’ cholesterol means you have too much low-density lipoprotein (LDL) in your blood, which is linked to serious problems, such as atherosclerosis and coronary heart attack or stroke. A study on adult male cynomolgus monkeys (Shandilya and Clarkson, 1982) found that gossypol administered orally at 10 mg/kg/day for 6 months could cause a significant decrease in total plasma cholesterol (TPC) and LDL without any significant decrease in plasma high-density lipoprotein (HDL) cholesterol levels. It was proposed that this cholesterol lowering activity may be attributed to (a) gossypol might possibly reduce the intestinal absorption of dietary cholesterol and (b) gossypol may reduce the hepatic synthesis of LDL. Studies with rabbits also showed that dietary cottonseed protein effectively lowers the concentration of plasma cholesterol when compared to the animal protein casein (Beynen and Liepa, 1987), which may be attributed to the present of gossypol in the cottonseed protein. Thrice weekly subcutaneous injection doses of 20 mg/kg body weight to rats for 4 weeks also resulted in lower serum cholesterol (Akingbemi et al., 1995). Another study on rats (Achedume et al., 1994) demonstrated that gossypol consumption had a significant effect on alcohol dehydrogenase and had a profound influence on the regulation of cholesterol level in the liver. A subsequent study on rats (Nwoha and Aire, 1995) showed that

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the administration of gossypol at 20 mg/kg body weight/rat/day for 8 weeks could significantly decrease the serum level of cholesterol in both low- and normal-protein-fed male Wistar rats. The combined administration of gossypol and chloroquine (chloroquine, a 4-aminoquinoline, used for treatment of malaria) to the protein-malnourished rats had more profound effects in decreasing the levels of serum cholesterol and triglycerides compared to normal-protein-fed rats, indicating the implication of the treatment and dietary effect on the level of serum cholesterol. However, the mechanism by which gossypol lowers the serum cholesterol still needs further investigation.

VII. CLINICAL IMPLICATION Gossypol initially spurred a lot of interest due to its contraceptive activity. A large-scale clinical trial that involved 10,000 healthy volunteers was conducted in China in 1978. A dose of 20 mg/day by mouth for 75 days (loading dose), and then 50 mg/week (maintenance dose) were administered to the volunteers. A small portion of the volunteers (0.75%) developed severe hypokalemia and 10% of the men taking gossypol for >1 year acquired irreversible aspermatogenesis (National Coordinating Group on Male Antifertility Agents, 1978). Another international study on 151 men from Brazil, Nigeria, Kenya, and China found that 15 mg/day of gossypol for 12 or 16 weeks, followed by either 7.5 or 10 mg/day for 40 weeks did not cause hypokalemia, and spermatogenesis was recovered after gossypol discontinuation (Coutinho, 2002). A study from Xue (2000) on male volunteers found that taking low doses of gossypol (15 mg/day) could induce antifertility within 12 weeks. Furthermore, all of the volunteers remained infertile without developing hypokalemia and irreversible azoospermia after a low-maintenance dose of gossypol (10 mg/day) for 44 weeks. In contrast, the fertility, induction of abnormal histone-to-protamine replacement reaction, as well as alteration of nuclear basic proteins could be recovered 10 weeks after the withdrawal of drug treatment (Xue, 2000). Two key side effects of high-dose gossypol treatment include irreversible infertility and hypokalemia. The inhibition of gossypol on 11-bhydroxysteroid dehydrogenase (11-b-OHSD) results in hypokalemia. The enzyme, 11-b-OHSD, is present near mineralocorticoid receptors. It oxidizes hydrocortisone to inactive cortisone in the kidney and is an important regulator of renal Kþ clearance. Inhibition of 11-b-OHSD leads to the production of mineralocorticoid in excess, hypokalemia, and hypertension (Reidenberg, 2000). Gossypol inhibits purified 11-b-OHSD from rat liver and human kidney microsomes in a competitive manner. The degree of physiological symptoms due to potassium excretion is correlated to the initial serum potassium level of the individual and can be

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changed by dietary 11-b-OHSD inhibitors, such as polyphenols from tea, naringerin from grapefruit juice, and glycyrrhizic acid from licorice in a synergistic manner (Reidenberg, 2000; Song et al., 1992). The hypokalemia of concern may have been caused by an improper diet of the test subjects (Coutinho, 2002). Hypokalemia is a common occurrence in Chinese men and Chinese people frequently consume tea, providing a possible explanation for the 10% of patients who developed hypokalemia during gossypol trials. Also, permanent infertility could potentially be a manageable side effect by limiting the use of gossypol to patients who are ready to accept the consequences or have already established families as an alternative to a vasectomy. The anticancer activity of gossypol has also gained a big interest in the past several decades. A preliminary study investigated the effects of an increasing dosage of gossypol on 34 patients with advanced cancer (cancer that has spread to other places in the body and usually cannot be cured or controlled with treatment). The resulting emesis was the doselimiting adverse effect in most patients (Stein et al., 1992). A clinical trial conducted on 21 patients with metastatic adrenal cancer revealed that oral gossypol given in doses of 30–70 mg/day was able to induce a tumor response (induce tumor size). All of these patients had little or no response to previous treatments. Three of the eighteen patients who finished the study over the course of 6 weeks showed a 50% decrease in tumor volume (Flack et al., 1993). Patients’ side effects included xerostomia, transient transaminitis, dry skin, fatigue, intermittent nausea, vomiting, transient ileus, and minor hair thinning, yet none of the 18 patients had to withdraw due to these side effects. There was no mortality observed due to administration of gossypol. Also, the highest dosage of gossypol that patients could tolerate was found to be 0.8 mg/kg body weight/day. Another group of 27 patients with progressive or concurrent glial tumors that had already undergone radiation therapy were administered 10 mg gossypol orally twice a day. Two patients exhibited a partial response, one of which lasted 78 weeks. Development of mild thrombocytopenia, hypokalemia, grade 2 hepatic toxicity, and peripheral edema occurred (Bushunow et al., 1999). Twenty women with refractory metastatic breast cancer were involved in a phase I/II study in which each was given a dose of 30–50 mg/day oral gossypol. A minor response was observed in one patient and two patients exhibited >50% reduction in serum tumor markers. Adverse effects included nausea, fatigue, emesis, altered taste sensation, and diarrhea. Dermatologic toxicity limited the dosage, and the maximum tolerable dosage was established as 40 mg/day (Van Poznak et al., 2001). Table 6.5 lists the percentage of patients with the most noted side effects in gossypol clinical trials. A study conducted on one patient with chronic lymphocytic leukemia, in which malignant immunologically incompetent lymphocytes

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Percentage of patients with most noted side effects in gossypol clinical

Side effect

Xerostemia Transient transaminitis Fatigue Nausea Emesis Transient ileus Hypokalemia Thrombocytopenia Hepatic toxicity

Adrenal cancer patients (%) (Flack et al., 1993)

Glioma patients (%) (Bushunow et al., 1999)

Breast cancer patients (%) (Van Poznak et al., 2001)

93 93

3.7 —

— —

64 36 21 21 — — —

— — — — 33.3 7.4 33.3

15 35 20 — — 5 —

accumulate, proposed that the detoxified gossypol found in fresh bovine milk decreased the lymphocyte count over a period of 5 years (Politzer, 2008). Derivates, such as apogossypol, have been shown to have similar antitumor activity with less toxicity (Hu et al., 2008). Perhaps future clinical trials may utilize gossypol derivates that have comparable antitumor activity with less toxicity.

VIII. CONCLUSIONS Gossypol is a polyphenolic aldehydic compound, and it has been studied for its versatile biological activities. Gossypol’s biological activities are based on direct chemical reactions, the inhibition of enzymes, and the regulation of signal transduction pathways. However, due to its toxicity, the application of gossypol is sometimes limited.

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INDEX A American Oil Chemists Society (AOCS), 220, 221 Amylopectin, 12–13 Amylose, 12 Anaerobic co-digestion, FVW, 104–106 Anaerobic digestion, FVW biodegradation waste management, 103–104 choice of temperature, 101 potential for material recovery, 102 reaction scheme, 101 Apogossypol, 225 Apple pomace for fatty acid production, 78 for heteropolysaccharide-7 production, 79 for lactic acid production, 78–79 Aspergillus niger, 73–74 B Bacillus licheniformis, 194 Balsamic vinegar. See Traditional balsamic vinegar (TBV) Barrel cask, TBV production process aging definition, 163 physical ripening time, 166–168 residence time, 164–166 configuration, 160 process streams types, 159 refilling procedure degree of freedom (DOF) analysis, 163 Fickian model, 161–162 mass transfer, 161 vectorial concentration model, 162–163 residence time (RT), 160–161 yield, 161 Beer brewery waste, 97 Beetroot, coloring agents, 86 Bioadsorbents for wastewater treatment dyes adsorption, 97–98 metal ions biosorption, 94–97 Biodiesel production, 107–108

Biopolymeric nanostructured particles hydrolyzed protein, 194 polysaccharides, 194 protein–polysaccharide mixtures, 194–195 whole proteins, 193 Borohydride, 222–223 Bound gossypol (BG), 220, 221, 229, 231, 232 C Candida utilis, 71 Carboxymethylchitin, 91, 92 Carotenoids, seafood wastes, 91 Cauliflower, FVW, 83–84 Ceratocystis fimbriata, 75 Chemoinformatics chemical space exploration, 35–36 computational methods, classification and definition, 35 definitions, 34 in food chemistry chemical space comparison, 41 physicochemical properties distribution, comparison, 42 toxicity, 40 food-related components molecular docking, 49–52 molecular similarity, 43–47 pharmacophore model, 47–48 QSAR and QSPR, 48–49 molecular databases and chemical space Distributed Structure-Searchable Toxicity (DSSTox), 38 DrugBank, 39 generally recognized as safe (GRAS) compounds, 38 MMsINC database, 39 National Cancer Institute (NCI) database, 39 Protein and Bioactive Peptide Sequences (BIOPEP), 39 SuperScent, 39 molecular descriptors and physicochemical properties, 36–37

265

266

Index

Chenopodium quinoa, 19 Cotton and cottonseed products, 216–218 Cyanoborohydride, 223 D Dairy industry wastes, 65–66 Dairy wastes aerobic treatment, 111–116 anaerobic treatment, 108–111 Differential scanning calorimetry (DSC), 14 Docosahexaenoic acid (DHA), 15 E Eicosapentaenoic acid (EPA), 15 European Landfill Directive, 60 F Fermentation industry wastes, 64–65 Fermentation, TBV production process acetic acid bacteria ecological studies, 157–158 oxidation products, 158–159 scalar fermentation, 154–156 yeast and alcoholic fermentation product, 157 zygosaccharomyces, 156 Food chain management (FCM) for sustainable food system development, 116 food market focus, 118–119 integrated product development and sustainability, 118 market-oriented research, 117–118 user-oriented innovation in food sector, 117 Food materials structuring process electrospraying, 190–191 homogenization, 186–188 microfluidization, 188 milling, 186 rapid expansion of supercritical solution (RESS), 191–192 ultrasound, 188–190 Food-related components, chemoinformatics molecular docking Autodock, 49, 52 improvement areas, 49 Protein Data Bank, 49 QSAR models, 50–51 molecular similarity

fusion methods, 43 G-protein coupled receptor (GPCR), 44 odor-structure relationships, 45–46 OpenEye scientific software (OpenEye), 45 stereochemical theory, 45 pharmacophore model, 47–48 QSAR and QSPR, 48–49 Food waste processing FCM, sustainable food system development, 116 food market focus, 118–119 integrated product development and sustainability, 118 market-oriented research, 117–118 user-oriented innovation in food sector, 117 fruit-and-vegetable wastes (FVWs), 63–64 multifunctional food ingredient production, 82–93 recovering added-value products, 69–82 green production processes, development food production, holistic approach, 61–62 green production strategy, 62–63 waste management hierarchy, 60–61 problems and opportunities, 58–60 anaerobic digestion (AD), 59 CO2 emissions, 59 low levels of suspended solids and dissolved materials, 59 restrictions on waste, 60 sources and characterization dairy industry, 65–66 fermentation industry, 64–65 fruit-and-vegetable wastes (FVWs), 63–64 meat and poultry industry, 67–68 olive oil industry, 64 seafood by-products, 68–69 using eggshell, 98 vegetable residues for wastewater treatment dyes adsorption, 97–98 metal ions biosorption, 94–97 waste treatment aerobic treatment of dairy wastes, 111–116 anaerobic treatment of dairy wastes, 108–111

Index

of aqueous food industry waste streams, 100 biodiesel production, 107–108 bioprocessing of FVWs, 100–107 whey, added-value products, 98–100 Free gossypol (FG), 221, 229, 231, 233 Fruit-and-vegetable wastes (FVWs), 63–64 bioprocessing, 100–107 multifunctional food ingredient production, 82 coloring agents and antioxidants, 84–86 dietary fibers, 83–84 food preservation, 88–89 gelation properties, 87 meat waste derivatives, 89–91 oil and meal, 88 production of biopolymers, films, food packaging, 89 seafood waste derivatives, 91–93 recovering added-value products SSF of fruit/vegetable by-products, 70–82 vegetable industry challenges, 69–70 Fusarium oxysporum, 73, 74 G Gelatin, 92–93 Glucosamine, 91–92 Goldenberry pomace, 88 Gongronella butleri, 80 Gossypol, cotton plant agricultural implication antifeeding activity, 228–229 detoxification, 232–233 insecticidal activity, 228 toxicity, 229–232 analyses, 225 AOCS methods, 227 enzyme-linked immunosorbent assays (ELISA), 227 high-performance liquid chromatography (HPLC) method, 226–227 near-infrared reflectance, 226 biological properties anticancer activity, 237–242 antifertility activity, 235–237 antimicrobial activity, 247–248 antioxidant property, 234–235 antiparasitic protozoan activities, 244–247 antivirus activity, 242–243

267

plasma cholesterol levels, 248–249 clinical implication, 249–251 cotton and cottonseed products, overview, 216–218 occurrence, 218 physiochemical properties, 218 apogossypol, 225 chemical formula, 219 methylation, 224 naphthalene rings, 220 oxidation, 223 ozonolysis, 223–224 Schiff base reaction, 221, 222 structure, 219 tautomeric forms, 220 G-protein coupled receptor (GPCR), 44 Grape must production, TBV chemical changes, 153–154 cooking time effect, 155 physical changes, 154 solute concentration, 151–153 Grape pomace, 75 Grapes, 85–86 H Hydrogen–methane two-stage fermentation, 105 5-Hydroxymethyl furfural (HMF), 153 Hydroxytyrosol, 84 Hypokalemia, 249–250 L Lactic acid bacteria (LAB), 93 Lipids and lipidic compound, Quinoa docosahexaenoic acid (DHA), 15 eicosapentaenoic acid (EPA), 15 fatty acid composition, 15, 17 polyunsaturated fatty acids (PUFA), 16 squalene and phytosterols, 17 Liposomes, 203 Lycopene, 81 M Meat and poultry industry wastes, 67–68 Melanoidins, 153, 174 Molecular databases and chemical space, chemoinformatics Distributed Structure-Searchable Toxicity (DSSTox), 38 DrugBank, 39

268

Index

Molecular databases and chemical space, chemoinformatics (cont.) generally recognized as safe (GRAS) compounds, 38 MMsINC database, 39 National Cancer Institute (NCI) database, 39 protein and bioactive peptide sequences (BIOPEP), 39 SuperScent, 39 Molecular docking, 49–52 Molecularly imprinted polymer (MIP) techniques, 201 Molecular similarity fusion methods, 43 G-protein coupled receptor (GPCR), 44 odor-structure relationships, 45–46 OpenEye scientific software (OpenEye), 45 stereochemical theory, 45 N Nanosensors and nanotracers Escherichia coli, 200 molecularly imprinted polymer (MIP) techniques, 201 molecular recognition, 199 Nanostructured materials aggregates disruption, 188–189 biopolymeric nanostructured particles hydrolyzed protein, 194 polysaccharides, 194 protein–polysaccharide mixtures, 194–195 whole proteins, 193 food materials structuring process electrospraying, 190–191 homogenization, 186–188 microfluidization, 188 milling, 186 rapid expansion of supercritical solution (RESS), 191–192 ultrasound, 188–190 functionality and applications encapsulated food components, 202–205 food packaging and edible coatings, 201–202 nanosensors and nanotracers, 199–201 future of, 206–207 high-pressure homogenization effects, 188–189

lipid nanoparticles, 195–196 microencapsulated food components, 204–205 microfluidization effects, 188–189 nanocomposites, 198–199 nanoscale manipulation, 185 nanostructured emulsions double emulsions, 197 microemulsions, 197 simple oil-in-water emulsions, 197 structuring emulsions, functionality, 198 nanotechnology and society, 206 Neurospora crassa, 73, 74 O Oleuropein, 84 Onion wastes, 85 OpenEye scientific software (OpenEye), 45 P Pectin, 87 Pectin methylesterase, 73 Penicillium decumbens, 73, 74 Pharmacophore model, 47–48 Phytosterols, 17 Polyunsaturated fatty acids (PUFA), 16 Q QPs. See Quinoa proteins Quantitative structure–activity relationships (QSARs) models, 35, 48–49 Quantitative structure–property relationships (QSPRs), 48–49 Quinoa antioxidant capacity, phenolic compounds, and flavonoids, 18 carbohydrates amylopectin, 12–13 amylose, 12 differential scanning calorimetry (DSC), 14 gelatinization properties, 14 glucose polymers, 11–12 granule size, 13 polysaccharides, 12 thermal properties, 14 chemical, nutritional, and physical properties, 4–6 chenopodium species, 2–3 functional properties

Index

quinoa flour, 21–23 quinoa protein, 23 quinoa starch, 24 water-holding capacity (WHC), 21 water imbibing capacity (WIC), 21 lipids and lipidic compound docosahexaenoic acid (DHA), 15 eicosapentaenoic acid (EPA), 15 fatty acid composition, 15, 17 polyunsaturated fatty acids (PUFA), 16 squalene and phytosterols, 17 minerals and vitamins, 19–20 proteins active biopeptides, 9 chemical and nutritional aspects, 6–9 structural aspects, 9–10 pseudocereal, 3 saponins, 18–19 uses of, 24–25 Quinoa flour emulsifying capacity and stability, 22–23 functional properties, 22 solubility, 21 Quinoa proteins (QPs) active biopeptides, 9 chemical and nutritional aspects amino acids composition, 6–9 protein efficiency ratio (PER), 7 structural aspects, 9–10 Quinoa starch functional properties, 24 structure, 11–15 R Rapid expansion of supercritical solution (RESS), 191–192 Residence time (RT), 160–161, 164–166 Rhizopus oligosporus, 96 Rhodopsin, 44 S Saponins, 18–19 Seafood by-products wastes, 68–69 Seafood wastes, derivatives carotenoids production, 91 gelatin production, 92–93 glucosamine and carboxymethylchitin production, 91–92 marine peptone production, 93 Solid-state fermentation (SSF), fruit/vegetable by-products

269

antibiotics, 81–82 apple pomace, 70–71 aroma compounds production, 74–75 baker’s yeast production, 80 enzymes production, 71–74 ethanol production, 75–78 feed protein, 81 organic acids production, 78–79 pigments production, 80–81 polysaccharides production, 79–80 Squalene, 17 Supercritical fluid anti-solvent (SAS) process, 191 T Thermophilic bioremediation technology, 111–116 Total gossypol (TG), 221, 233 Traditional balsamic vinegar (TBV) chemical composition characteristics, 169 composition, 174–175 furanic compounds, 173–174 melanoidins, 174 minor compounds, 171–174 organic acids, 170–171 phenolic compounds, 172–173 sugars, 169–170 volatile compounds, 171 condiments, 139, 141 conservative mass balance equation, 151–153 consortia, 138–139 features, 142–143 historical note comprehensive research, 140–141 production aspects, 144–145 testimonies, 141–144 5-hydroxymethyl furfural, 153 legal aspects, 147–148 physical properties color and spectrum absorbance, 176–177 rheological properties, 176 production process barrel set, 159–168 cooking technology, 151–154 cooking time effect, 155 fermentation, 154–159 raw material, 149–151 semitic languages and italian legislation in European languages, 146

270

Index

Traditional balsamic vinegar (TBV) (cont.) traditional vs. industrial, 146–147 various forms, 145 sensorial aspects, 148 vinegars, 139–141 Tuna fin gelatin (TFG), 92–93 U UK’s Waste and Resources Action Program (WRAP), 59 User-oriented innovation in food sector, 117 V Vegetable residues for wastewater treatment dyes adsorption, 97–98

metal ions biosorption, 94–97 Vinegar. See Traditional balsamic vinegar (TBV) W Waste management strategies, 62–63 Waste recovery, 62 Whey utilization and disposal, 99–100 X Xanthan gum, 79 Z Zirconium, 96 Zygosaccharomyces, 156