Advances in Food Research, Volume 23

ADVANCES IN FOOD RESEARCH VOLUME 23 Contributors to This Volume Kan-lchi Hayakawa Rauno A. Lampi Jane D. Love June O...

Author: C O Chichester

23 downloads 1666 Views 21MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

ADVANCES IN FOOD RESEARCH VOLUME 23

Contributors to This Volume

Kan-lchi Hayakawa Rauno A. Lampi Jane D. Love June Olley A. M. Pearson P. Haridas Rao F. B. Shorland S. R. Shurpalekar S. J. Thrower

ADVANCES IN FOOD RESEARCH VOLUME 23

Edited by C. 0. CHICHESTER The Nutrition Foundation, Inc. New York, New York and University of Rhode Island Kingston, Rhode Island

G. F . STEWART University of California Davis, California

E. M. MRAK University of California Davis, California

Editorial Board J. F. KEFFORD S. LEPKOVSKY EDWARD SELTZER W. M. URBAIN

E. C. BATE-SMITH W. H. COOK J. HAWTHORN M. A. JOSLYN

J. R. VICKERY

1977

ACADEMIC PRESS

New York

San Francisco

A Subsidiary of Harcourt Brace Jovanovich, Publishers

London

COPYRIGHT @ 1977, BY ACADLMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PIiOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM T HE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kirr*gdorti Edition priblislred b y ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:48-7808 ISBN 0-12-016423-X PR1NTI.D IN THE UNITED STATES OF AMERICA

CONTENTS Contributors t o Volume 23

......................... . . . . . . . . . . . . . . . . . . . .

vii

"Warmed-Over" Flavor in Meat. Poultry. and Fish

A . M . Pearson. Jane D . Love. and F . B . Shorland

I . Introduction ................................................... I1 . Classification and Significance of Lipids .............................. 111. Structure of Lipids .............................................. 1 v. Composition of Animal Fats ....................................... V . Role of Lipids in Meat Flavor-Desirable and Undesirable . . . . . . . . . . . . . . . . VI . Mechanisms of Lipid Oxidation .................................... VII . Development of WOF ............................................ VIII . Prevention of WOF in Meat. Poultry. and Fish ......................... IX . Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 5 20 37 39 46 57

59 61

Mathematical Methods for Estimating Proper Thermal Processes and Their Computer Implementation

Kan-Ichi Hayakawa 1. I1. 111. IV . V. VI .

Introduction ............................... . . . . . . . . . . . . . . . . . . . . Basic Principles for Determining Proper Heat Processes . . . . . . . . . . . . . . . . . . Published Procedures for Determining Proper Heat Processes . . . . . . . . . . . . . . Computerized Estimation of Heat Processes ........................... ResearchNeeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nomenclature .................................................. Appendix A: Computer Programming Terminology ..................... Appendix B: Computer Programs ................................... References ....................................................

76 76 83 91 104 106 108 110 139

Abalone-An Esoteric Food

June Olley and S . J . Thrower 1. Introduction ................................................... 11. Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. AbaIoneasFood ................................................

144 145 146 V

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

CONTENTS Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catching and Handling of Abalone at Sea ............................. Physiology of Abalone in Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technology of Preserving Abalone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quality Aspects .............................. By-products of Abalone Processing ......................... ... Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147 166 166 168 174 178 179 179

Wheat Germ

S . R . Shurpalckar and P . Haridas Rao 188 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . 190 I I . Structural Components of the Germ ......... . 197 111. Separation of the Germ . . . . . . . . . . . . 204 IV . Chemical Composition of the Germ . . . . . . . . . . ....................... ...................... V . Nutritive Value of the Germ . V1. Storage and Stabilization of th erm ............................... 258 273 VII . Wheat Germ and Bread-Making Quality .............................. 282 VIII . F o o d U s e s o f t h e G e r m . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 1X. ResearchNeeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . ............................................ 289

Flexible Packaging for Thermoprocessed Foods

Rauno A . Lampi I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Early Efforts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .... ....... 111. Materials IV . Package D ................................................ V . Food Product Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....... ................. V1. Production Systems VII . Sealing . . . . . . . . . . ......................................... VIII . Filling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Air Removal ................................................... X. ................ XI . ................ XI1 . Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI11.

SubjectIndex

.......................................................

306 309 312 322 324 346 364 369 375 399 404

429

CONTRIBUTORS TO VOLUME 23 Numbers in parentheses indicate the pages on which the authors' contributions begin.

KAN-ICHI HAYAKAWA, Food Science Department, Cook College, Rutgers, The State University of New Jersey, New Bnmswick, New Jersey (75) RAUNO A. LAMPI, Food Engineering Laboratoty, U. S. Army Natick Research and Development Command, Natick, Massachusetts (305) JANE D. LOVE, Food and Nutrition Department, Iowa State University, Ames, Iowa (1) TUNE OLLEY, CSIRO Division of Food Research, Tasmanian Food Research Unit, Hobart, Tasmania (143)

A. M. PEARSON, Department of Food Science and Human Num'tion, Michigan State University,East Lansing, Michigan (1)

P. HARIDAS RAO, Flour Milling and Baking Technology Discipline, Central Food Technological Research Institute, Mysore, India (187) F. B. SHORLAND, Department of Biochemistty, Victoria University of Wellington, Private Bag, Wellington,New Zealand (1) S. R. SHURF'ALEKAR, Flour Milling and Baking Technology Discipline, Central Food Technological Research Institute, Mysore, India (1 87) S. J. THROWER, CSIRO Division of Food Research, Tasmanian Food Research Unit, Hobart, Tmnania (143)

This Page Intentionally Left Blank

"WARMED-OVER" FLAVOR IN MEAT. POULTRY. AND FISH A . M . PEARSON.* JANE D . LOVE.t AND F . B . SHORLANDS 1. Introduction

.................................................... ...............................

I1. Classification and Significance of Lipids

111. Structure of Lipids ............................................... A. Triglycerides ................................................. B. Phospholipids ................................................ C. Sphingolipids and Glycolipids .................................... D. Nonsaponifiable Matter and Some Related Lipids ..................... E Lipids in Membranes ........................................... 1V. Composition of Animal Fats ........................................ A . Depot Fats .................................................. B . TissueLipids ................................................. V. Role of Lipids in Meat Flavor-Desirable and Undesirable ................. VI . Mechanisms of Lipid Oxidation ..................................... A . Autoxidation ................................................. B Catalysts of Lipid Oxidation ..................................... C. Comparison of Heme and Nonheme Iron as Prooxidants in Muscle Tissue . D. Phospholipid Oxidation ......................................... VI1. Development of WOF ............................................. A . Species Differences in WOF ...................................... B. Influence of Deboned Meat ...................................... C Influence of Heating ........................................... D Influence of Chopping and Emulsifying ........................ E . Effects of Curing .............................................. VIII . Prevention of WOF in Meat. Poultry, and Fish .......................... A . Antioxidants and Cheliting Agents ................................ B. Reducing Conditions ........................................... C Practical Implications .......................................... IX . Research Needs .................................................. References .....................................................

.

.

. .

.

.

2 3 5

5 9 15

18 20 20 20 33 37 39 39 40 45 45 46 46 47 48 53 54 57 57 58 59 59 61

*Department of Food Science and Human Nutrition. Michigan State University. East Lansing. Michigan. t Food and Nutrition Department. Iowa State University. Ames. Iowa. $Department of Biochemistry. Victoria University of Wellington. Private Bag. Wellington. New Zealand . 1

2

A. M. PEARSON, JANE D. LOVE, A N D F. B. SHORLAND

I. INTRODUCTION The term “warmed-over” flavor (WOF) was first used by Tims and Watts (1958) t o describe the rapid development of oxidized flavor in refrigerated cooked meats, in which a rancid or stale flavor usually becomes apparent within 48 hours at 4°C. This is in marked contrast t o the slow onset uf rancidity commonly encountered in raw meats, fatty tissues, rendered fat, or lard, which is normally not apparent until they have been stored for weeks or months. Although WOF has generally been recognized as pertaining only to cooked meat, there is now evidence that it develops just as rapidly in raw meat that has been ground and exposed to the air (Greene, 1969; Sato and Hegarty, 1971). Even though most of the work on WOF has been directed toward the red meats, there is good evidence that it is equally if not even more serious in poultry and fish. Despite the occurrence of WOF in ground, uncooked meat products under certain conditions, it is of much greater importance in cooked meats. Even though WOF was not identified as a problem by food scientists until relatively recently, its existence has no doubt been recognized by consumers for many years as evidenced by their aversion to “warmed-over” roasts, steaks, and other “leftover” meat items. The rapid increase in fast food facilities, such as airlines, food vendors, and franchises, and the development of precooked quick-frozen meals, commonly called TV dinners, has undoubtedly increased the frequency and extent of problems caused by WOF. The continued development and success of both fast food facilities and precooked frozen meals will largely depend on the ability of processors t o circumvent the development of WOF. It is, therefore, clear that an understanding of WOF is important to future developments in the food industry. In the present review, the authors follow Tims and Watts (1958) in considering the rapid development of lipid oxidation as the primary cause of WOF. This view is consistent with the reports of Sato and Hegarty (1971), of Sato et af. (1973), and of Love (1972) showing that a decline in acceptability is associated with a rise in TBA (2-thiobarbituric acid) values. AS with other examples of oxidative rancidity, it is inevitable that the process of lipid oxidation results in the formation of many different compounds. Some of these compounds, such as n-hexanal, contribute t o the undesirable odors and flavors associated with rancidity, whereas other compounds formed are probably desirable or irrelevant. The nature and proportion of such compounds in meat will depend at least in part on the composition of the fat of the animal from which it is derived, which in turn may reflect a variety of factors including the nature of the diet. For the purpose of this review, we have chosen t o confine the discussion to a consideration of the composition of lipids in meat, poultry, and fish and of prooxidants and antioxidants controlling the oxidative processes concerned with the development of WOF in cooked meats.

WOF IN MEAT, POULTRY, AND FISH

3

Since the present discussion is not a review of traditional oxidative rancidity per se, interested readers are referred to an excellent symposium on food lipids and their oxidation, edited by Schultz et al. (1962), which covers all aspects of autoxidation in foods.

I I. CLASS1F ICATl ON AND SIGN I F ICANCE OF LIPIDS Since animal fats represent a variety of lipid components, some understanding of the classification and structure of lipids is essential in explaining their role in the development of WOF. Not only do animal fats and other lipids create problems in meat, poultry, and fish products by virtue of their propensity for undesirable odor and flavor changes, but they are also essential t o the desirable sensory properties of these same products and enhance their flavor and aroma (Hornstein, 1967; Herz and Chang, 1970) and increase their tenderness and juiciness (Blumer, 1963; Pearson, 1966). In Bloor’s (1943) classification of lipids, there are two main subdivisionssimple and compound-which denote, respectively, lipids that contain carbon, hydrogen, and oxygen only, and those that also contain phosphorus, nitrogen, or sulfur. A third subdivision, the “derived” lipids, is also given by Bloor (1943), but, as these are not generally present as such in living tissues, being derived upon hydrolysis (Mitchell, 1946), they will not be considered here. Simple lipids include fats or triglycerides, and waxes. Fats are esters of glycerol and higher fatty acids, whereas waxes are esters of Zllgher fatty alcohols and higher fatty acids. Sterol esters and vitamin A esters may also be included in this category. Compound lipids contain other groups in addition to the alcohols. In the subdivision of compound lipids, there are various schemes and terminologies. Although differentiation of lipids into simple and compound classes gives an overall picture of the lipid categories, it may obscure relationships-for example, between triglycerides and the diacylphospholipids, which have a common biosynthetic origin. The type of classification used in this review is based on the following systems of derivation: (1) esters of glycerol (fats or triglycerides); (2) esters of long-chain fatty alcohols and sterols; (3) esters of glycerophosphoric acid coupled with a nitrogen base and/or carbohydrate (phospholipids); and (4) derivatives of the longchain hydroxyamino alcohol sphingosine (sphingolipids). It is believed that recognition of the biosynthetic routes provides an important means for discerning relationshps between the lipid classes and for assigning structures to these compounds. This point of view has been borne in mind in the treatment of the lipids for the purpose of the present review.

4

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Before consideration of the structure of the lipids, it is relevant to outline the nature of the various lipid components in relation to their significance to WOF. Obviously, fats or triglycerides are the most abundant lipids present in meat. As currently practiced, meat production involves changing a carcass that is almost devoid of fat at birth into one that may contain up to five times as much fat as protein at the time of slaughter (Shorland, 1955). Despite the high content of fat, these lipids are generally less important than the phospholipids. This is especially true for phosphatidyl ethanolamine, in which the highly unsaturated Czo and Czz acids not normally found in meat fats are concentrated. These unsaturated fatty acids are particularly reactive in autoxidation and probably, therefore, relevant to the development of WOF. In addition, phospholipids together with lesser amounts of cholesterol tend to remain at a more or less constant level for a given tissue, constituting 0.7 to 1.0% of lean meat. On the other hand, the level of fat may be altered in a dramatic fashion with age and state of nutrition. For this reason, Terroine (1920) referred to phospholipids and cholesterol as the “element constant,” in contrast to fats which he described as the “element variable.” The various lipid classes are seldom isolated as such by normal procedures of extraction. The concept of a wax, for example, as an ester formed by the interaction of a higher fatty alcohol with a higher fatty acid may be applied t o some extent to the head oil of the sperm whale (Hilditch and h v e r n , 1929). Plant waxes, however, include, besides esters, fatty acids, fatty alcohols, higher ketones, and other high-melting-point components (Morice and Shorland, 1973). Rendered animal fats are composed almost entirely of triglycerides but will include before refining some phospholipids as well as unsaponifiable matter (usually less than 1%) that remains after the refining process (Dugan, 1971). As a broad principle, it does not necessarily follow that the components extracted by lipid solvents are mixed together in the tissues in the same proportions in which they occur after extraction. It does not even necessarily follow that the lipid components appear in the same form in which they are extracted. Although fats generally occur as such in adipose tissue, there is evidence that phospholipids and other complex lipids are mainly, if not wholly, present in combination with or in strong association with proteins. Such lipid-protein combinations, which are called lipoproteins, form an integral part of the cell membranes and are associated with the control of extracellularintracellular ionic gradients, as well as with the operation of enzymic processes such as oxidative phosphorylation (Lehninger, 1970). Proteolipids are involved similarly in the structure of the myelin sheath of the nervous system. This group of lipid-protein combinations includes cerebrosides as well as phospholipids, and is distinguished from the lipoproteins by its solubility in nonpolar solvents (LeBaron, 1969). Soluble lipoproteins, involving triglycerides (Zilversmit, 1969), phospholipids, and cholesterol (Margolis, 1969),

WOF IN MEAT, POULTRY, AND FISH

5

are responsible for the transport of lipids from the gut by way of the lymphatics and the bloodstream to various parts of the body. Likewise, the combination of fatty acids with albumin provides for the transfer in the blood of lipids from the depot fat to other parts of the body (Gurr and James, 1971). Thus, in the blood even triglycerides do not occur entirely as such, but in part are bound as a lipoprotein complex. With these preliminary remarks it is hoped that the lipid entities about to be discussed in detail will be seen more closely within the framework of their actual occurrence in the tissues and that the performance of the lipids after extraction in relation to autoxidation and other reactions is not necessarily indicative of the processes that occur in intact tissues, such as meat.

Ill. STRUCTURE OF LIPIDS A. TRIGLYCERIDES Fats or triglycerides, as already indicated, are the most abundant of the lipids and comprise the fatty acid esters of the 3-carbon sugar alcohol glycerol. The structure and system of numbering of the carbon atoms of the glycerides are given in Fig. lB, where the two end carbons are labeled 1 and 3, respectively, whereas the middle carbon is labeled 2 (Gunstone, 1967). This numbering system is used throughout. The system of nomenclature recommended by the IUPAC-IUB Commission on Biochemical Nomenclature of Lipids (1967) eliminates the confusion of previous names by relating back naturally occurring triglycerides and glycerophospholipids to L-glyceraldehyde (Fig. 1A, B, and C) in much the same way as most naturally occurring amino acids are related to the L series. The abolition of the D and L terminology and substitution of unambiguous numbering of the glycerol carbons in glycerides (Fig. 1B) has been responsible for the clarity of t h s system of nomenclature. On this basis, naturally occurring phosphatidyl choline becomes 1,2-diacyl-sn-glycero-3-phosphorylcholine or the shorter generic name of 3-sn-phosphoryl choline, where sn stands for stereochemical numbering. The sn system thus serves to indicate relationships between naturally occurring triglycerides and phospholipids, but, once these have been clarified, it is convenient to return to the less-cumbersome, commonly used terms, such as phosphatidyl choline and ethanolamine. In addition to the triglycerides may be mentioned the related galactolipids, in which the fatty acid component of carbon 3 is replaced by a sugar molecule as indicated in Fig. 2A, B, and C. The galactolipids occur as the main lipid components of pasture (Weenink, 1959, 1961; Shorland, 1961), and similar compounds involving mannose and other sugars are found in bacteria. Other related compounds include the glyceryl ethers derived from the C16 , C18 -satu-

6

A. M. PEARSON, JANE D. LOVE, AND I'. 5. SHORLAND 0 THO

110

+-

CH:

II

H:

11

'

C 0-{-H

CH,OH

0

711:

II

0 C

R,

( B ) Triglvccwde

0 II

R,

0 C

II ' R 2 C 0-C-H

R,

0

Cfl!

( A ) I.-(;lvceraldeh\de

II

o r

1

0

2

I1

C H 2 0 P OX

3

I

OH

R , , R2.;ind R , rqiresenl alkvl groups corrrponding t o different fattv nrid residues In druclure C . sn stands for stereorhrmiral numheriix and X for une of the following siibslituenls. Phospholipid

Substiluent

H HO CH:

CH:

HO CHI

CH,N+(CH,),

Phosphalidrc acid

NH2

Phuspliatidgl e t h w d i n i i n e Phosphatidyl choline

HO C1I2 C H ' NH: I

Phosphatidyl serinr

COOll

OH

OH

Phosphatidyl inositol ( A )

(A) The inosilul substituent ma! also be n1~uinositul-4-phusphate u r 4.5-diphosphate.

CH, OH CH . OH . C H I . Of1

Ptiosphatidylglycer[il (GPG)

0

CH? . O H -CII

OH . C H ?

I1 I

0-P-o

(xi? - r i i - c t i 2

I

OH O=('

I

I Rt

(: o=c I

('Ardioii;m

I

R>

FIG. 1 . Structure of diacylglycerophospholipids.

rated, and Cle -monounsaturated alcohols known, respectively, as chimyl, batyl, and selachyl alcohols. These compounds, which may be esterified with one or two fatty acids, occur mainly in the liver oil of sharks (Hilditch and Williams, 1964). In recent years, Schogt et al. (1960) have also found glyceryl ethers in animal fats. A detailed review is given by Snyder (1969).

I

WOF IN MEAT, POULTRY, AND FISH

In Nature the formation of simple triglycerides containing only one specific fatty acid is rare. Almost invariably fats consist of mixed triglycerides containing different fatty acids as shown in Fig. 1B. The occurrence of monoglycerides, although not entirely unknown, is rare. Monoglycerides, for example, have been reported in hog pancreas by Jones et af. (1949).

HOQ'o[-H,C

OH CH CH:

OH

(B)1,Z-Diaryl-

0 'COR,

0 COR?

[ ~ - l ~ - ~ n l a c t ~ , p y r a n a s v l - (3)] l ' - -sn-glycerol

,

.OH

O "Q 0

on

O.COR,

,O-COR,

(C)1 ,Z-Diaryl-

[n- D-galartopyranosvl-(

galactopyranosyl-( 1'-3)]

(-&

-sn-glycerol

1

-

6 1-0- D-

HO

0-CH.

no

I .

CHO . C O . R ,

I CH,

'

0 ' CO ' R 3

( D ) 1.2-Diacyl [ 6-sullo-I)-quinovop~r~nos?l11-3)

3 -sn-

glycerol

FIG. 2. Diacylglycerol lipids.

8

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Of the various attempts to describe the pattern of fatty acid distribution in natural fats, the 1,3-random, 2-random distribution proposed by Vander Wal (1960, 1964) has been widely accepted. In a variety of different fats the composition of the trisaturated, disaturated, monounsaturated, monosaturated, diunsaturated, and triunsaturated glycerides found experimentally is in agreement with predictions by the Vander Wal theory (1964). More recently, Brockerhoff (1965, 1967) has shown by stereospecific analysis that positions 1 and 3 of triglycerides are in fact distinguishable. Thus, the Vander Wal theory based on the equivalence of positions 1 and 3 may require some adjustments. The distinction between positions 1, 2 , and 3 of mixed triglycerides is perhaps most readily seen by the work of Lands and Slakey (1966) and Lands et al. (1966). From mixtures of 1,2- and 2,3-diglycerides produced by the action of lipase on triglycerides, Lands and Slakey (1966) found that only the 1,2-diglycerides reacted with the enzyme diglyceride kinase to form 1,2-diacylglycero-3-phosphate, which was attacked specifically by phospholipase A to yield l-acylglycero-3-phosphate, thereby distinguishing between fatty acids in positions 1 and 2. The fatty acids in position 3 were then determined by difference. Natural fats are composed mainly of the straight-chain, even-numbered carbon fatty acids, typically containing sixteen and eighteen carbon atoms (Dugan, 1971). Even in animal fats, which tend to be more uniform in their fatty acid makeup than those of plants, the range of fatty acids encountered is very wide. For example, Hansen er al. (1958) found that in beef tallow, in addition to fatty acids containing sixteen and eighteen carbon atoms, there occurred trace amounts of all the even-numbered carbon fatty acids from Clo to Cz6 and the odd-numbered carbon fatty acids from Cll to Czs. The most abundant and widespread fatty acid is oleic (octadec-cis-9-enoic) acid. Although not so uniformly or prominently distributed, other unsaturated fatty acids, namely linoleic (octadec-cis-9-cis-12-enoic) acid and palmitoleic (hexadec-cis-9-enoic) acid, appear to be nearly as ubiquitous. Of the saturated fatty acids, palmitic (hexadecanoic) acid is the most prominent, and like oleic acid it is seldom if ever absent from any of the natural fats (Hilditch and Williams, 1964). As a guide to the distribution of fatty acids, Boekenoogen (1941) as quoted by Hilditch and Williams (1964) has assessed the percentage distribution of fatty acids in world-wide commercial vegetable fats as oleic, 34; linoleic, 29; palmitic, 11 ; lauric, 7; linolenic, 6; myristic, 3 ; erucic, 3; stearic, 3; and all others, 4. Animals on fat-free diets synthesize fats endogenously from the nonfatty components of the diet. As a rule, endogenous fat contains about 25% palmitic acid, the remaining fatty acids being mainly oleic with minor amounts of stearic, myristic, and palmitoleic (Shorland, 1955). In practice, however, animals have access to dietary fat, the fatty acids of which are reflected in the depot fat, adding variety to the fatty acids present. Invariably, such fat contains linoleic

WOF IN MEAT, POULTRY, AND FISH

9

acid, which is often accompanied by linolenic (octadec-cis-9, cis-12, cis-15trienoic) acid. By chain elongation and desaturation, these acids provide the Czo and CZz polyunsaturated fatty acids of animal phospholipids (Gunstone, 1967). In recent years, it has been established that minor amounts of odd-numbered fatty acids, especially saturated and C1, , as well as pentadeccis-9-enoic and heptadeccis-9-enoic acids, and branched-chain fatty acids occur in animal fats (Hansen et d., 1958). Branched-chain fatty acids have also been isolated from the fats of ruminants (Shorland, 1962). These include iso, CH3-CH-(CH2 ),-COOH

I

CH, acids containing thirteen to eighteen carbon atoms and (t)-anteiso, CH,-CH2-CH-(CH2

),-COOH

acids containing thirteen, fifteen, and seventeen carbon atoms and phytanic (3,7,11,15-tetramethyl hexadecanoic) acid. Other minor fatty acids found in animal fats include 11-cyclohexyl undecanoic (Hansen and Gerson, 1967) and monomethoxy stearic acid isomers (Hansen and Smith, 1966). Hydrogenation by rumen microorganisms results in further diversification of the fatty acid composition of the dietary unsaturated fatty acids. It has been shown that linolenic acid may be saturated to give high levels of stearic acid and of trans and positional isomers of oleic and linoleic acid not found elsewhere in natural fats (Shorland et a/., 1957). B. PHOSPHOLIPIDS Phospholipids include those lipids that contain phosphoric acid as part of their molecular structure. Because of the variety of components that make up lipid molecules, it is readily seen that phospholipids do not necessarily all conform to the same structural pattern. In the present section attention is confined mainly to the 1,2-diacyl-sn-glycero-3-phosphoryl phospholipids, which cover the greater part of the naturally occurring phospholipids. These diacylesters of glycerophosphoric acid have a common biosynthetic origin with the triglycerides, both being derived (as will be discussed later) from 1,2-diglycerides (Shorland, 1962). In the next section concerning the sphingolipids, it will be seen that this group of lipids, based on the long-chain amino diol sphingosine, includes a group of compounds known as sphingomyelins. Because of the attachment of phosphoryl

10

A.

M. PEARSON, JANE I). LOVI:, AND

1,'. B. SHOKLAND

choline t o the hydroxyl of carbon 1 of sphingosine in the sphingomyelins, these compounds are also referred t o as phospholipids even though n o glycerol moiety is present in the molecule. The conformation of this compound with its two long chains consisting of a fatty acid molecule and of sphingosine with a polar head group of phosphoryl choline resembles that of phosphatidyl choline (Lehninger, 1970). Thus, sphingomyelin is closely related t o the diacyl glycerophospholipids despite the absence of the glycerol moiety. In plasmalogens, which are found in minor amounts in animal tissues, a vinyl replaces the fatty acid attached to the hydroxyl ether, -CH2 -0-CH=ClI-R, group of carbon 1 of the glycerol moiety of phosphatidyl ethanolamine, constituting an aldehydogenic chain (Rapport et al., 1957). Plasmalogens also occur with choline attached to the phosphoric acid radical, as in phosphatidyl choline (Klenk and Gehrmann, 1955; Rapport and Alonzo, 1955). Naturally occurring phospholipids, like triglycerides, have a mixed type of fatty acid distribution shown in their glycerol moiety (Fig. 1C). In the phospholipids of most animal tissues, there is a marked tendency for the saturated fatty acids t o occur in position 1 and the unsaturated acids to occupy position 2 (Gurr and James, 1971). In addition t o changes in fatty acid composition and the arrangement of the fatty acids on the glycerol moiety, these 1,2-diacyl-snglycero-3-phosphoryl derivatives (Fig. 1 C ) show variations in the substituents X attached to the phosphoric acid moiety. As shown in Fig. 1 , the phosphoric acid is united through an ester linkage either t o a nitrogenous base, such as choline, ethanolaminc. or serine, t o a cyclic hexose (myoinositol or a phosphate derivative of myoinositol), or to glycerol or a phosphate derivative thereof (Shorland, 1962). The names of the phospholipids corresponding t o the above substituents are also listed in Fig. 1. I n most cases, the glycerol moiety of the phospholipid is fully acylated, but in some glycerophospholipids. such as cardiolipin, the substituent glycerol derivdtive may be partially acylatcd (Macfarlane and Gray, 1957). A broad similarity in the composition of phospholipids in the tissues of a variety of mammals and birds has been established (Body ef al., 1966; Ansell and Hawthorne, 1964). Probably more is known about the pattern of distribution of phospholipids within the tissues o f sheep than in any other species. Body et al. (1966) found in the total tissues of maternal and fetal sheep that the component phospholipids expressed as a percentage of the total phospholipids were approximately as follows: phosphatidyl choline (including minor amounts of lysophosphatidyl choline), 45; phosphatidyl ethanolamine, 2 5 ; sphingomyelin, 1 1 ; phosphatidyl serine, 7; phosphatidyl inositol, 4; and other, 8. The distribution of the component phospholipids has been found to be broadly similar in other tissues of the sheep, including liver (Peters and Smith, 1964); brain, kidney, lung, heart, and skeletal muscle (Dawson, 1960); and rumen and abomasum (Body ef af., 1970). However, small differences in phospholipid composition

WOF IN MEAT, POULTRY, AND FISH

11

between tissues d o exist. Thus, the heart is relatively rich in cardiolipin (Pangborn, 1947). The lung and kidney (Dawson, 1960) and the rumen and abomasum (Body et ul., 1970) contain relatively high levels of sphingomyelin. Furthermore, sphmgolipids, including cerebrosides, are abundant in the membranes of brain and nerve cells, particularly in the myelin sheath (Lehninger, 1970). In contrast to the tissues mentioned, the phospholipids of the red cell membrane were found by De Gier and Van Deenan (1961) t o vary greatly among different species. In order of ranking, the phosphatidyl choline content in rat, rabbit, man, pig, cow, and sheep decreased from 58% to 1% with concomitant increases in the sphingomyelin content. Each type of phospholipid tends to have its characteristic fatty acid composition. In animal tissues, phosphatidyl ethanolamine is notably rich in polyunsaturated Czo and Czz fatty acids, which are derived from dietary linoleic and linolenic acids by the addition of acetate to the carbonyl end followed by desaturation (Gunstone, 1967). Linoleic acid gives rise to fatty acids with the first double bond at carbon 6 from the methyl end group (linoleic or 0 6 type), and linolenic acid forms the linolenic or 03 type with the unsaturation commencing at carbon 3 from the methyl end group (Steinberg et ul., 1956, 1957). As all members of the linoleic and linolenic families are characterized by a system of interrupted methylenic double bonds, it is convenient t o designate the polyunsaturated fatty acids in terms of their chain length, number of double bonds, and origin. Thus, in the linoleic series linoleic acid is denoted as 18:2, w6 and arachidonic (eicosatetraenoic) acid as 20:4, w6. In the linolenic acid series, linolenic acid becomes 18:3, 03; eicosapentaenoic acid, 20:5, 03; docosapentaenoic, 22:5, w3; and docosahexaenoic, 22:6, w3. Under conditions of linoleic acid insufficiency, oleic acid (18: 1, w9) is converted to eicosatrienoic (20:3, w9) acid. Indeed the high ratio of 20:3, 0 9 to 20:4, w 6 found by Body and Shorland (1974) in the phospholipid fractions of fetal sheep has been considered by Noble et al. (1972) to be indicative of a linoleic acid deficiency. The amounts and kinds of polyunsaturated fatty acids recorded for the phosphatidyl ethanolamine fractions may vary with the levels and ratios of linoleic and linolenic acids in the diet as well as with the conditions of analysis (Body and Shorland, 1974). The earlier analysis of Hornstein el ul, (1961) for the phospholipids of pork and beef muscle showed the presence of 20:4, w6 as the only polyunsaturated component besides 18:2, w6. Later, Hornstein et ul. (1967) reported that beef muscle phospholipids included 22:6, w3; 22:3, w6; and 22:4, w6; in addition to 20:4, 06. In more recent studies by Body and Shorland (1974) on the rumen and abomasum tissues of fetal and maternal sheep it was shown that the polyunsaturated fatty acids of the phosphatidyl ethanolamine fraction ranged from 17 to 43% of the total fatty acids. The main polyunsaturated components were 20:4, w6 and 22:5, w3, with lesser amounts of 20:5, w 3 and 22:6, 03, and 18:2, w6 and 18:3, 03. Trace amounts of 20:2, 06; 20:3,

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

12

R, CO . S-CoA

Hl-

+

-

C -OH

HO-4-H

I II H,-C-0-P-OH I OH

R2 CO . S-CoA

I

R,CO

'

+

0 ' GH

R,CO

'

I

CO R ,

I II H,C ' 0-P-OH

I

OH (B) 1,Z-Diacyl-sn-glycero-

(A) sn-3-Glycerophosphoric acid

CH, . O . C O R ,

CH,. 0

~ ~ c o . 0 . ~

phosphoric acid (phosphatidic acid)

GH, . O . C O R ,

. S . CoA

R,CO

, o .CH dH, . OH

CHI . O ,CO R,

(D)Triglyceride

(C) 12-Diglyceride

1

CDP choline

$HI , O . C O R , I RICO.O,CH 0

I

CH,

II

o P . OCH, . C H ~ N + ( C H )3 ~ I

9 (F)Phosphatidyl choline

0 (CH,),N* . CH,

It

'

CH, .O-P-O I

HO

OH

(E) Cytidine diphoaphate choline

FIG. 3. Biosynthesis of phospholipids.

0 6 ; 22:3, 0 6 ; 22:4, 0 6 ; and 22:5, 0 6 were also found. In earlier analysis (Shorland et al., 1966) a similarly complex mixture of polyenoic unsaturated Czo and Czz acids was found for the phosphatidyl ethanolamine fraction isolated from the total tissues of fetal and maternal sheep. At that stage, however, the positions of the double bonds in terms of 0 9 , 06, and 0 3 had not yet been assigned.

WOF IN MEAT, POULTRY, AND FISH

13

Phosphatidyl ethnnolnmine likewise results from 1.2-diglyceride and CDP ethanolarnine. However, phouphatidyl inoaitol and phosphatidyl glycerol are formed from CDP glyceride and not from the CDP base.

$H2 .O . CO . R1 I

R,CO .O .FH

I

CH,

'

OH

CTP

FH, . O . C O . R ,

R2C0.0-cjH I CH,

il , o .P - 0 I

II

(C) 1,2-Diglyceride

OH

OH

0

H

no

(HI CDP diglyceride inositol

6 'ds?

. P . 0-CH I

OH

1

F H 2 0 ' CO ' R,

I

on

,

(I) Phosphatidyl inositol

FIG. 3. (Continued).

In addition to other fatty acids normally associated with phospholipids, the phosphatidyl ethanolamine fraction contained up to 8.5% of cyclopropane fatty acids, including 2,3-methylene hexadecanoic and 2,3-methylene octadecanoic acids not previously recorded by other workers. The extraordinary specificity of phosphatidyl ethanolamine is indicated by the absence of the cyclopropane fatty acids from all other phospholipid fractions. The high content of polyunsaturated fatty acids in the phosphatidyl ethanolamine fraction (17 to 43%) may be compared with the lower values in the phosphatidyl choline (7 to 25%) and sphingomyelin ( 1 to 4%) fractions (Body and Shorland, 1974). Conversely and in agreement with the results of other workers, Body and Shorland (1974) found that the low level of palmitic acid (approximately 8%) in the phosphatidyl ethanolamine fraction increased to 25 to 30% in the phosphatidyl choline fraction and to 29 to 52% in the sphingomyelin fraction. Consistent with the findings of others for mammalian tissues, the sphingomyelin fractions contained a relatively high content (1 6 to 27%) of higher saturated acids including 22:0, 23:0, 24:0, and 25:O as well as a high content (5 to 10%) of the unsaturated acid 24:1, 0 9 (Body and Shorland, 1974). The tendency for each type of phospholipid to be associated with a specific fatty acid makeup is further illustrated by cardiolipin found in heart muscle.

14

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Macfarlane and Gray (1957) reported that the sample isolated from heart muscle showed the following percent composition of fatty acids: 16:0, 0.5; 18:0, 0.8; 16:1, 5.2; 18:1, 11.0; 18:2, 72.0; 18:3, 8.0; and Cz0 polyenoic acid, 1.5. The near absence of saturated fatty acids and the presence of 18:2 as the predominant fatty acid readily distinguishes this phospholipid from all others. The related phosphatidyl glycerol and lyso-bis-phosphatidic acid isolated by Body and Gray (1967a) from pig lung and the semi-lyso-bis-phosphatidicacid found in rabbit lung by Body and Gray (1967b) offer further examples of fatty acid specificity. These trace phospholipid components are composed mainly of palmitic, stearic, and oleic acids, in contrast to cardiolipin in which linoleic acid predominates. The ratios of saturated to unsaturated fatty acids in phosphatidyl glycerol, in lyso-bis-phosphatidic acid, and in semi-lyso-bis-phosphatidic acid were found to be 1.O, 2.0, and 4.0, respectively. As already indicated, the 1,2-diacyl-sn-glycero-3-phosphoryl phospholipids and the triglycerides have a common biosynthetic origin, both being derived from 1,2-diacyl-sn-glycero1. Kornberg and Pricer (1953a,b) found that the sediment of the liver homogenate of the guinea pig possessed an enzyme capable of synthesizing phosphatidic acid (Fig. 3B) from acyl CoA and glycerophosphoric acid (Fig. 3A). This enzyme has a distinct optimum for CI6 and CIS fatty acids. Phosphatidic acids are not found in fresh tissues, probably owing t o their rapid dephosphorylation t o form orthophosphate and 1,2-diglyceride (Fig. 3C). An enzyme concerned with this reaction was shown t o be present in chicken liver by Weiss et al. (1956). The diglyceride thus produced then reacts with acyl CoA to form triglyceride (Fig. 3D) or with cytidine diphosphocholine (Fig. 3E) to form CH,O. CO ' R ,

R,CO

'

I 0-CH

ethanolamine

I

8

CH20. P-0 I

'

CH, . CH2 ' NH2

0

ethanolamine :

Serine

CH,O

I R2CO-0.CH

'

CO ' R ,

I

I

0 (C) Phosphatidyl serine

(A) Phosphatidyl ethanolamine

I

1 CH,O

R,CO

'

0

'

I CH

S-adenosyl methionine

'

CO ' R,

I f l

CHI . O - P - O ' C H , I

II

CH, ' O - P - 0 . C H 2

+

'CH, "(CH,),

0 -

(B)Phosphatidyl choline

FIG. 4. The interconversion of phospholipids.

.CH.COOH I NHi

WOF IN MEAT, POULTRY, AND FISH

15

phosphatidyl choline (Fig. 3F). Phosphatidyl ethanolamine likewise results from 1,2-diglyceride and CDP ethanolamine (Kennedy and Weiss, 1956). However, phosphatidyl inositol (Fig. 31) is considered to result from the reaction of CDP 1,2-diglycerides (Fig. 3H) and the hydroxyl group of myoinositol (Shribney and Kennedy, 1958). The biosynthesis of phosphatidyl glycerol follows a similar route, and in bacteria this route is followed generally in the synthesis of glycerophospholipids (Gurr and James, 1971). The glycerophospholipids exhibit some degree of interchange as outlined by Gurr and James (1971). Phosphatidyl choline (Fig. 4B) may be derived from phosphatidyl ethanolamine (Fig. 4A) by enzymic methylation by way of the active form of methionine (S-adenosyl methionine). In addition, phosphatidyl serine (Fig. 4C) and phosphatidyl ethanolamine (Fig. 4A) undergo a “base exchange” reaction catalyzed by an enzyme from a liver microsomal fraction in the presence of Ca2+ ions. Furthermore, phosphatidyl serine (Fig. 4C) may be decarboxylated to form phosphatidyl ethanolamine (Fig. 4A), but this reaction is not considered important, as the in vivo incorporation of labeled phosphate by phosphatidyl serine is slow compared with that of phosphatidyl ethanolamine. C. SPHINGOLIPIDS AND GLYCOLIPIDS The sphingolipids are derived from the amide diol, sphingosine (~-erythro-2amino-h.ans-4-octadecene-1,3-diol, Fig. 5A). Glycerol is absent from the molecule. The biosynthesis of sphingosine shows that it is derived from palmitoyl CoA and serine (see Fig. 6). The sphingolipids are characterized by the attachment of a fatty acid to the amide group of the sphingosine molecule. In some sphingolipids, known as sphingomyelins, the primary hydroxyl of sphingosine is attached to phosphoryl choline, giving rise to sphingomyelins. In other sphngolipids, called glycolipids, the primary hydroxyl group is attached to a hexose or a polysaccharide. As shown in Fig. 5B, sphingosine is transformed into ceramide by acylation of the amide group. On acylation of the primary hydroxyl group of ceramide with phosphoryl choline, sphingomyelin is formed (Fig. 5C). The resemblance of this compound in structure and conformation to phosphatidyl choline justifies its classification as a phospholipid along with the diacyl glycerophospholipids. In place of phosphoryl choline, the primary hydroxyl group of ceramide may combine with a hexose to form a 0-glycosidic linkage. Such compounds are known as cerebrosides. A typical cerebroside is shown in Fig. 5D. Another group of sphingolipids resembling the cerebrosides are known as gangliosides. According to Lehninger (1970), these glycolipids possess a complex of oligosaccharides forming a polar head of large size in place of the hexose unit of cerebrosides. They are present on the outer surface cell membranes, especially of nerve cells. On hydrolysis, brain gangliosides yield fatty acids, sphingosine, sugars (D-glucose

16

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

AH

NHl

(A) D-Erythro-rronss-l,3-dihydroxy-2-arnino-4octadecene (sphingosine)

Sphingolipids

(B) Ceramide:

N-acyl-D-erthrosphingosine

CH,(CH,),,CH=CH-CH

I OH

- CH

I NH I

. CH,OH

co I

R These are ceramide phosphorylcholines.

(C) Sphingornyelins:

CH~(CH~),,CH=CH-CH-CH 'CH, I I OH NH

B

. o . P .OCH*

I

I

.cH,N+(cH,),

0

co I

R (0) Cerehrosides:

These are rnonohexosides In which the hexose is attached by a P-glycosidic linkage to the primary hydroxyl group of ceramide. Cerasine is 1-O-(p-D -galactopyranosyl)-N-tetracosanoyl~-erythrosphingosine.

CH3(CH, ),,CH=CH

'

CH- CH- CH,O-gal I I OH NH I

co I R

FIG. 5. Structure of sphingolipids.

and D-galactose), and amino sugar derivatives (N-acetyl galactosamine and Nacetyl neuraminic acid). The fatty acids of sphingomyelins from nonneural tissues are characterized by the near absence of polyunsaturated fatty acids. The main component fatty acids are 16:O and 24:1, 0 9 . The 18:0 fatty acid is also prominent together with 20:O to 26:0, inclusive (Body and Shorland, 1974; Svennerholm el a l , 1966). In sphingomyelins from neural sources, such as brain, 18:O tends to predominate, especially in fetal brain tissue. With increasing age the content of 18:O fatty acid falls, while that of 22:O to 26:0, inclusive, increases (Stenhagen and Svennerholm, 1965). The fatty acid composition of cerebrosides is variable. Some cerebrosides contain mainly CI6 to CI8 fatty acids and others C22 to CZ6 fatty acids (Lehninger, 1970). It has been shown by Trams and Lauter (1962) that the fatty acids of brain gangliosides from various species consist mainly (72 to 96%) of

WOF IN MEAT, POULTRY, AND FISH

17

stearic acid. Thus, the sphmgolipids are characterized mainly by having longchain saturated or monounsaturated fatty acids, and the near absence of polyunsaturated fatty acids. The biosynthesis of the sphingolipids and the related cerebrosides is indicated in Fig. 6. According to Stoffel (1971), the long-accepted view that palmitaldehyde and serine condense to form dihydrosphingosine, which is desaturated by an FAD enzyme, must be modified. The reaction actually involves the condensation of palmitoy1 CoA with decarboxylation to 3-ketodihydrosphingosine(Fig. 6A), with pyridoxal phosphate being the necessary coenzyme. The 3-keto group is reduced by a NADPHz -dependent reductase to dihydrosphingosine. Intracerebral injection of labeled dihydrosphingosine into young rats has indicated that cis elimination of hydrogen on carbons 4 and 5 leads to the formation of a trans double bond (Stoffel, 1971). The steps in the formation of ceramide, sphingomyelin, and cerebrosides are summarized by Lehninger (1970) as outlined in Fig. 6B t o E. The great selectivity shown by the different categories of phospholipids for different fatty acids is well known. In particular, the iong-chain highly unsaturated fatty acids are concentrated in the phosphatidyl ethanolamine fraction, whereas the sphingomyelin fraction is characterized by the presence of longchain saturated fatty acids and the absence of polyunsaturated acids. Lands et al. (1966) and Lands and Slakey (1966) have suggested that the enzyme phospholipase A, which is present in the tissues, and hydrolyzes the fatty acids at the 2 position, provides a biosynthetic mechanism for modifying preformed phospholipid molecules. The differences in fatty acid patterns between phospholipids may also arise through transacylation or again through the fatty acid composition of each phospholipid inherent in the diglyceride selected for its formation. The enzyme system available selects diglycerides appropriate in fatty acid composition to the phospholipid to be synthesized. CH:OH

0 (A)

II

R-C-SCoA

+

I

H-C-NH,

COOH Palmitnyl C o A

+

-

0

II

+ CoASH

R--C--CH-CH~OH I

NH:

Serine

;7

+ NADPll

(B)

R-CC-CII-CH~OH

(C1

Sphingosine t

(D)

Ceramide t rytidine diphosphocholine (CDP choline)

(E)

Ceramide t UDP galactose

t

11'

R--CII--CI1--CII,OII

I 011

1 NH:

I

NADP'

D~hydrosphingi~sine

H

'

COSCoA

ceramide

galactocerebroside

sphingomyelin t C M P

+ UDP

FIG. 6. Biosynthesis of sphingolipids.

18

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

D. NONSAPONIFIABLE MATTER AND SOME RELATED LIPIDS In contrast to most other lipids, the nonsaponifiable fraction does not yield fatty acids on hydrolysis (Lehninger, 1970). This dass of lipids is composed of two main groups-namely, the steroids and terpenes-which are closely related by virtue of common 5-carbon building blocks (Fig. 7). The steroids are derivatives of the perhydrocyclopentanophenanthrenenucleus, in which three fused cyclohexane rings occur in the phenanthrene arrangement (Lehninger, 1970). Among the most important naturally occurring steroids are the sex hormones, the adrenocortical hormones, and the sterols, the class of steroids which includes the important metabolic compound, cholesterol (Lehninger, 1970). The sterols contain an alcoholic hydroxyl group at C-3 of the perhydrocyclopentanophenanthrene nucleus and a branched aliphatic chain of eight or more carbons atoms at C-17. They are present as free alcohols or as long-chain fatty acids esterified with the hydroxl group at C-3. Cholesterol (Fig. 7B) is the most abundant sterol in animal tissues, occurring in both the free and combined forms (Lehninger, 1970). It is readily extracted with chloroform, ether, benzene, or hot alcohol, but is insoluble in water. It is mixed with glycerides and phospholipids and endows lipids with lipophilic properties. It is found in appreciable amounts in the membranes of the endoplasmic reticulum and mitochondria. Biosynthesis of cholesterol as outlined by Lehninger (1970) begins with the condensation of three molecules of acetyl CoA t o form mevalonic acid (Fig. 7C), which is converted into squalene-an open-chain dihydrotriterpene compound (Fig. 7D). Squalene, which is found in small amounts in liver and adipose tissue of most higher animals, is then used t o synthesize lanosterol (Fig. 7E). Lanosterol is then converted into cholesterol, which along with phosphoglycerides is an integral part of animal membranes. Another group of compounds that is frequently present in trace amounts in the unsaponifiable fraction from animal fats is the terpenes. They are constructed of multiples of the 5-carbon compound isoprene (2-methyl-l,3butadiene). Terpenes containing two isoprene units are called monoterpenes, those with three isoprene units are called sesquiterpenes, and those containing four, six, and eight isoprenes are named diterpenes, triterpenes, and tetraterpenes (Lehninger, 1970). Thus, squalene, which is an intermediate in the biosynthesis of cholesterol, is classified as a triterpene. Squalene has been shown to be a constituent of the nonsaponifiables from pork adipose tissue (Williams and Pearson, 1965). The unsaponifiable fraction of animal fats also contains the male and female sex hormones and a number of related compounds, among which is 5a-androst16-en-3-one,the compound identified as being responsible for boar odor in pork (Patterson, 1968, 1969). Although the unsaponifiable fraction contains small

(A) Perhydrocyclopentanophenanthrene nucleus showing the numbering system far the four rings. labeled A. B. C. and D. This nucleus is common to all steriad compounds. includisg the sex hormones, adrenocortical hormones, and sterols.

&

FH' H-C-(CH,),-CH

?HI

c\ c/c,p\c

CH, C

-

HO

/C\

c::

I

I

I

:

IC

C-C

1

C '

c,c,'/c,c cl /c-c I l c, c /c* c /c

(B) Cholesterol and its carbon skeleton

(C) Mevalonic acid

'(D)Squalene showing cyclic carbon nucleus (compare with cholesterol above) ,CH, H-C=C

I

YH, 'CH,

8 pH*

H-

HO

H,C

C-CH,

H

CH,

(ElLanosterol an intermediate in cholesterol synthesis

FIG. 7. The basic structure of steroids and some intermediates involved in the biosynthesis af cholesterol.

20

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

amounts or traces of many biologically important compounds, the significance of this fraction to the development of WOF is completely unknown. The presence of some of these compounds in membranes, particularly of cholesterol, could influence the availability of the active lipid components in animal membranes to oxidation, and thus have an influence on WOF development.

E. LIPIDS IN MEMBRANES Cell membranes vary from 60 t o 100 A in thickness and contain about 60% protein and 40% fat (Masaro, 1968), although the percentages vary for different membranes (Korn, 1966; Rothfield and Finkelstein, 1968). Although there are a number of different theories on membrane structure, most state that the membrane consists of a bilayer of mixed polar lipids with their hydrophilic heads oriented outward and their hydrocarbon chains oriented inward to form a continuous hydrocarbon phase. The outer surfaces are coated with a monomolecular layer of protein with the polypeptide chains extended outward. Each type of membrane contains characteristic types of polar lipids in fixed molar ratios. Erythrocyte membranes contain relatively large (40to 55%) and constant amounts of cholesterol, which varies from species to species (Lehninger, 1970). Phosphatidyl ethanolamine also comprises a rather consistent proportion of the erythrocyte lipids, whereas the ratio of phosphatidyl choline t o sphingomyelin varies greatly with the species, the ratio in rats being much higher than in sheep. Most of the polar lipids are located in the membranes. Thus, the membranes contain nearly all the phospholipids, sphingolipids, and glycolipids, as well as cholesterol. The structure of these lipids makes them quite susceptible t o oxidation. Several workers (Tims and Watts, 1958; Sat0 and Hegarty, 1971; Yamauchi, 1972a) have suggested that heating of muscle tissue makes the phospholipids susceptible to oxidation, and thereby accelerates the development of WOF. The structural features of animal membranes proposed by Rothfield and Finkelstein (1968) further suggest that the lipoproteins of membranes could also be involved in the development of WOF. Thus, the review on the structure of lipids given here has concentrated on the lipid components of membranes. More details on membranes and different theories concerning their structure are given by Korn (1966), Van Deenen (1966), and Rothfield and Finkelstein (1968); while the occurrence of phospholipids is covered by Ansell and H a w thorne (1964).

I V . COMPOSITION OF ANIMAL FATS For the purpose of this discussion animal fats will include those from the red meats (beef, lamb, mutton, pork, veal, and large game animals, such as moose,

WOF IN MEAT, POULTRY, AND FISH

21

elk, deer, and others), poultry, and fish. Such fats may vary considerably in composition both within and among species, but particularly among species. The species-characteristic differences in the flavor of meat have been widely accepted as being associated with differences in the composition of the lipids (Hornstein and Crowe, 1960, 1964; Hornstein et al., 1963; Hornstein, 1967). Obviously, such differences may contribute to the susceptibility and severity of problems due to WOF. Lipids in meat, poultry, and fish are commonly classified as depot or adipose tissue and as intramuscular or tissue lipids (Watts, 1962; Love and Pearson, 1971). The depot fats are largely localized as subcutaneous deposits, although appreciable amounts may be present in the thoracic and abdominal cavities and between the muscles as intermuscular deposits. The triglycerides are the principal lipid components of adipose tissue and can be extracted readily with chloroform, carbon tetrachloride, petroleum ether, and other nonpolar solvents (Watts, 1962). Compared with the depot fats, the tissue lipids contain proportionately larger amounts of phospholipids which occur largely, if not entireIy, in association with proteins as lipoproteins and proteolipids (Watts, 1962). Folch et al. (1557) have shown that chloroform-methanol mixtures extract over 99% of the total lipids from animal tissues. Thus, chloroform-methanol mixtures have been widely used for extracting total lipid from animal tissues by a number of investigators (Macfarlane et al., 1960; Marco et al., 196 1; Kono and Colowick, 1961; Hornstein et al., 1961). A. DEPOTFATS Depot fat or adipose tissue consists mainly of triglycerides. The triglycerides are deposited largely as fat globules localized within the individual cells, while tissue lipids are an integral part of various cellular structures, such as the cell wall (Kono and Colowick, 1961), the mitochondria (Holman and Widmer, 1969), the sarcoplasmic reticulum (Newbold et ul., 1973), and the microsomes (Macfarlane et al., 1960). In spite of the deposition of adipose tissue in a fairly consistent pattern, it is influenced by species, diet, environment, sex, and other factors (Deuel, 1955).

I . Species Differences Differences among species in the composition and structure of adipose tissue are well documented. According to Hilditch and Lovern (1936), the ability of animals to alter and deposit dietary fats in a form characteristic of the species is related to its position in the evolutionary scale. Thus, as we proceed from fishes through reptiles to mammals, there is an evolutionary change involving a marked simplification in fatty acid composition characterized by disappearance of CZO

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

22

and CZ2 polyunsaturated fatty acids and to a lesser extent of hexadecenoic acid. These changes coincide with the appearance of increasing amounts of saturated fatty acids in the depot fats of the higher animals. As shown in Table I, fish have lesser amounts of saturated c 1 6 acids and more unsaturated c 1 6 , Czo ,and CZ2 fatty acids than the fowl, pig, or ox, whereas the latter species have larger amounts of saturated CI6 acids and unsaturated C 18 fatty acids. Freshwater fish contain less unsaturated Cz2 fatty acids and greater amounts of unsaturated C I S acids than marine fish, while the whale is intermediate between the marine and freshwater fishes. The domestic fowl is similar to the ox and pig, except for a higher content of CIS polyunsaturated fatty acids and a slightly higher content of C I 6 unsaturated fatty acids. The depot fats of amphibia and reptiles are intermediate in fatty acid composition between those of the aquatic species and the higher land mammals. Both the pig and the domestic fowl have substantial amounts of polyunsaturated fatty acids (more than 5%), compared with less than 5% in the depot fat of the ox. The flesh from both freshwater and marine fishes contains a larger proportion of polyunsaturated fatty acids and is more susceptible to rancidity than poultry and the red meats. Poultry, in turn, is more susceptible to oxidation than pork, which is more susceptible than beef and lamb; lamb or mutton is the least susceptible to development of rancidity and the most saturated (Wilson el al., 1976).

TABLE 1 1;ATTY ACID COMPOSITION 01: DEPOT FATS FROM DIFFERENT SPECIES 01;A N I M A L S EXPRESSED AS WEIGHT PERCENT'

Species

Saturated

Number of carbon atoms.

~-~

Fish, freshwater Fish, marine Whalc brog Tortoise Lizard Domestic fowl Rat Pig

ox

_.

16 13-1 5 12-15 12-1 5 11 14 18 25-26 24-28 25-29 21-29

Unsaturated

~-

16

ca. 20 15-18 15-18 15 9 10 6-7 7-8 2-3 2-3

-

18

~ -- -

4045 27-30 3540 52 65 56 ca. 60 ca. 60 5045 40-50

22

20 ~~

ca. 12 20-25 15-20 15 I 5 0.5-1 .o 0.34.5 0.3-1 .O 0.2-0.5

0-5 8-1 2 5-10

'Data taken from Hilditch and Lovern (1936) and Hilditch and Williams (1964).

WOF IN MEAT, POULTRY,AND FISH

23

Table I1 shows the comparative fatty acid composition of beef, lamb, chicken, pork, and fish fats. Examination of the data shows that sheep fat has a higher percentage of saturated fatty acids than beef fat (55% compared with 44%), with the major differences in the unsaturated fatty acids being in 16:l and 18:1, where sheep fat is, respectively, about 3% and 8% lower than beef fat. Pig fat is less saturated than beef fat containing only 37.5% saturated fatty acids, whereas the major difference in the unsaturated fatty acids is about 8.5% more 18:2 in pig fat. Chicken fat is appreciably lower (7%) in saturated fatty acids than pork TABLE I1 FATTY ACID COMPOSITION O F BEEF, LAMB, CHICKEN, PORK, AND FISH FATS EXPRESSED AS PERCENTAGE O F TOTAL ~~

Fatty acids 12:o 14:O 15:O 16:O 17:O 18:O 20:o 14:1 16:1 18:l 18:2 18:3 18:4 20: 1 20:2 20:3 20:4 20:5 22: 1

225 22:6

~~

Bovine fatsubcutaneous‘ 3.2 0.5 24.8 1.8 13.7 0.9 4.4 46.9 1.9 0.2

Ovine fatsubcutaneousb

Chicken depot fat‘

0 .8f 5.2 0.6 24.6

0.1 0.7 0.2 22.8 0.2 6.5

1.o

22.9 0.8 1.6 38.7g 1.2 0.6 0.2

0.2 5.7 37.0 23.7 1.3 0.2 0.7 0.1 0.2

Pork outer backfatd

Fish herring oile

1.2

6.7

22.7

11.5

13.7

1.4

4.1 47.8 10.5

8.6 14.4 1.2 0.9 1.45 14.3

0.86 5.62 22.00 1.08 3.35

‘Data from Dryden et al. (1973). bData from Cramer er al. (1967). ‘Data from Katz et al. (1966). dData from Koch et al. (1968a). ‘Data adapted from Ackman el (11. (1973). fAlso includes 10:0, 0.4%. In addition, 14-is0, traces; lSbranched, 0.6%; 16-iso, 0.2%; 17-branched, 0.5%; and 18-iso, 0.5% were present. These acids are also known to occur in bovine fats (Hansen et al., 1958). gIncludes 0.8% trans-16-octadecenoic acid.

24

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

fat, and, although it is nearly 12%lower in 18:1, it contains over 12.5%more 18:2 and smaller amounts of Czo polyunsaturated fatty acids. Fish fat contains the least amount of saturated fatty acids (about 19.5%)and is the highest in the Czo unsaturated and polyunsaturated acids. Shorland (1952) questioned the viewpoint advanced by Hilditch and Lovern (1936), which theorized that changes in the fatty acid composition of different animal species were related to their evolutionary development. The fact that the fats of aquatic mammals and fishes have a similar fatty acid composition suggested that environment rather than evolutionary development has been responsible for the formation of an “aquatic type of fat.” Bearing these facts in mind, Shorland (1952) pointed out that the increasing simplicity of composition in animal fats as one proceeds from the lower to the more highly organized forms of life is mainly due to the fortuitous influence of diet, and only in part to the animals themselves. Therefore, Shorland (1952) proposed that animal depot fats can be classified into the following three main categories: (1) those animal fats whose composition is almost entirely dependent on the diet; (2) those animals having depot fats whose composition is influenced by both dietary fat and endogenously altered lipids; and (3) those animals having fats whose composition is not influenced by diet but in which the dietary lipids are altered and deposited in a form characteristic of the species. The fish is an example of the first category, in which the fat deposited is almost completely dependent on the diet and is essentially identical in composition. The second group is typical of most mammals and birds, in which the depot fats are influenced to some extent by diet, but still are deposited in a similar pattern for a given species. Thus, the pig and the chicken will produce a somewhat different type of fat on the same diet, even though the fatty acid pattern within each species can also be altered by changing the composition of the diet. In the third category the ruminant animals are a good example, since diet has little or no influence on the composition of the deposited fats. Certain monogastric marsupials, in which microbial fermentation plays an important role, such as the quokka and wallaby also belong to this group. In this group (Hartman et al., 1955), the depot fats are remarkably similar in composition regardless of diet. Maynard and Loosli (1962) have pointed out that different species lay down depot fats from the same diet in a characteristic manner, but that the location of the fat depot within the body may also influence the composition. The present authors concede that there may be species-characteristic features in relation to the formation of depot fat. For example, a review by Shorland (1962) shows that the internal (perinephric) fats of the beef, sheep, and pig are distinctly richer in stearic acid but lower in oleic acid than the external (subcutaneous) fats. Further, the bone lipids of these same species are markedly lower in stearic and palmitic acid than the subcutaneous fats. In the horse, however, the

WOF IN MEAT, POULTRY, AND FISH

25

differences in the fatty acid composition of the external and internal fats are negligible, although the bone lipids possess less saturated fatty acids than other tissues (Shorland, 1962). Perhaps the most distinctive species differences aside from those found between ruminants and nonruminants are shown by the fats from different parts of the bird. The fatty acid composition of the abdominal, gizzard, neck, and other body fats of birds is not only remarkably similar (Hilditch et al., 1934; Katz et al., 1966), but the lipids from chicken bone are relatively high in palmitic and stearic acid (Moerck and Ball, 1974), suggesting that they do not differ greatly from other depot fats. Although Shorland and Czochanska (1970) found that the depot fat of man, the pig, and the rat on a coconut oil ration failed t o contain lauric and myristic acid in the proportions found in the diet, the depot fat of the chicken closely reflected the composition of the same diet (Table 111). The nonruminant animals generally have the ability to convert lauric and myristic acid to palmitate by addition of acetate, which the chicken is unable t o do (Shorland and Stannard, 1972), thus reflecting a species difference in the pattern of fatty acid deposition. Nevertheless, the influence of dietary fat on the pattern of fatty acid deposition generally seems to be more important than the TABLE I11 FATTY ACID COMPOSITION OF THE DEPOT FATS O F PIGS, RATS, MAN, AND HENS OPI COCONUT MEAL DIETS, EXPRESSED AS PERCENT OF TOTAL FATTY ACIDSa

Hen

Coconut Fatty acid Saturated 8:O lo:o 12:o 14:O 15:o 16:O 18:O 20:o Unsaturated 12: 1 14: 1 16: 1 18:l 18:2 18:3

Embryo

Meat

Pig

Rat

Humanb

Coconut diet

10.5 40.9 15.9 14.3 8.0 -

16.4 43.2 19.7 10.1 2.8 -

0.2 16.3 31.3

-

0.4 0.3 11.5 16.1 0.5 20.4 2.5 0.2

-

-

0.1 31.8 18.1

1.3 55.6 18.1 0.3 10.2 2.8 -

-

1.2 24.0 4.1 -

-

1.1

-

-

-

-

-

3.0 9.6 29.0 3.2 0.1

6.7 42.5 20.8 -

-

-

28.5 4.1

18.6 2.7 0.6

-

-

-

-

4.1

9.6 0.8

6.6 1.2

0.6 1.5 4.1 20.1 1.2

-

-

13.6 1.3

-

_.

9.2 9.2 1.3 -

aData from Shorland and Czochanska (1970). *Also includes 13:0,0.3%; 17:0,0.5%; 17:1,0.6%; 19:1,0.1%;and 20:1,0.6%.

Normal diet

A. M. PEARSON, JANII I>. LOVE, AND I:. B. SHORLAND

26

species effect. In spite of the major role of diet on the composition of the depot fats, species differences still appear to be of major importance in the development of WOF (Wilson er al., 1976).

2. Influence of Diet Although species differences in the composition of depot fats may be related to the composition of the diet (Shorland, 1952), dietary influences within a species are more interesting and are also more controllable. Even though basic work with the rat aids in explaining the exact influence of diet on fat metabolism and deposition (Anderson and Mendell, 1928; Mendell and Anderson, 1930), the pig is more important as a food, and thus serves better for investigating the extent and severity of WOF. Ellis (1933) has reviewed the problem of “soft pork,” which is largely due to allowing growing, fattening pigs free access (“hogging-off ’) to feeds with a high content of unsaturated lipids. Peanuts, soybeans, acorns, and chuffas are the principal feeds contributing to soft pork, although corn also contains relatively unsaturated Lipids. The data taken from Ellis and lsbell(1926a,b) and shown in Table IV clearly dcmonstrate the influence of diet on some measures of carcass firmness and the proportion of different fatty acids in the depot fat of the pig. Not only is the effect of the lipid composition of the diet shown, but also the influence of the amount of lipid as demonstrated by using two levels of corn oil. At the higher level, corn oil produced a much softer fat, as shown by a higher iodine number, a lower melting point, and a lower firmness grade, as well as by a greater proportion of unsaturated fatty acids.

TABLE IV INFLUENCE O F DIETARY OIL SOURCES O N THE FIRMNESS O F THE CARCASS O F THE PIC A N D ITS FATTY ACID COMPOSITION W H E N A D D E D T O A BASAL CORN A N D TANKAGE

RATION^ Fatty acid (%)

011

supplement -~

~

~

Peanut oil,4.1% Cottonseed oil, 4.1% Soybean oil, 4.1% Corn oil, 4.1% ’ Corn oil, 11.5%

birmness gradeb ~

Melting point

Iodine number

34.3 45.3 31.2 36.3 24.5

72.5 64.4

.

~~

MS ti MS MS 0

-

‘Data from lllis and Isbell (1926a,b). bH, hard; MS, medium soft; 0, oily.

75.7

76.3 97.2

Oleic - .-._

47.9 35.9 43.3 45.0 41.4

Total saturated

Linoleic ~

13.8 15.7 18.6 16.8 31.3

~~

32.5 43.0 33.8 33.0 23.1

WOF IN MEAT, POULTRY, A N D FISH

21

I t is well established that soft pork is more susceptible to autoxidation (Ellis, 1933), which presumably occurs at the site of the double bonds in the fatty acids. Thus, it would be expected that diets causing unsaturation of the depot lipids would increase susceptibility to oxidation and rancidity, and thereby might decrease acceptability in meat, poultry, and fish. As already indicated in the discussion on species differences, the composition of the depot fats of nonruminants tends to reflect that of the dietary fat, whereas in ruminants the depot fats are not influenced to any extent by diet (Shorland, 1950). This point can be seen by comparing the fatty acid composition of pasture-fed animals, both ruminants and nonruminants, as shown in Table V. The incorporation of the main fatty acid components of pasture into the depot fats of the rabbit and the horse (nonruminants) and its virtual exclusion from the depot fats of deer, cattle, and sheep (ruminants) are clearly shown in Table V. These results are in agreement with those of Thomas et al. (1935) and of Pearson (1949), who found that the depot fats of ruminants are not greatly altered by the diets. It must be borne in mind, however, that the changes in the composition of depot fats are typically measured by means of fatty acid analysis and measurement of iodine values, which do not take into account mns acids. When truns acids are measured, there is a change in the interpretation of the results given in Table V as shown by Shorland et al. (1955, 1957). The major dietary unsaturated fatty acid of pasture (linolenic acid) is in fact mainly hydrogenated to stearic acid, but much of the remainder appears as trans monoenes, including elaidic acid and vaccenic acid, which in the normal course of analysis would be recorded as oleic acid. Thus, the depot fats of ruminants, which appear to be almost completely resistant to changes in composition by dietary fats (Shorland, 1950), are shown to be responsive to a small degree if these changes are evaluated in terms of trans acid content. The presence of the traits and positional isomers of oleic and linoleic acid is exclusively characteristic of ruminants and other animals with a ruminant-like digestive system (Hartman et al., 1954, 1955). Thus, nonruminants readily incorporate the unsaturated fatty acids of the diet into depot fats, but depot fats of ruminants, although not directly responsive to the unsaturated fatty acids, show a limited response by the deposition of Runs acids derived from the dietary unsaturated acids. For ruminants to become directly responsive to dietary unsaturated fats, it is necessary to bypass the rumen by means of a duodenal fistula (Ogilvie et al., 1961) or to otherwise protect the dietary fat from the action of the rumen microorganisms. Scott et al. (1970, 1971) and Cook et ul. (1970) have developed a procedure for treating the lipids in the diet of ruminants with formaldehyde so that they are protected against saturation by the action of the rumen microorganisms. By using the formaldehyde treatment, it was shown that the proportion of unsaturated fatty

TABLE V COMPARISON O F THE FATTY ACID COMPOSITION O F RYEGRASS AND THE DEPOT FATS O F GRAZING ANIMALS EXPRESSED A S WEIGHT PERCENTa

Saturated

__

Unsaturated ~~

Species

12:O

14:O

16:O

18:O

20:O

14:l

16:l

18:l

18:2

18:3

20-22

Transacids

Ryegrass (Lolium perenne) Nonruminants Wild rabbit (abdominal) Horse (total fatty tissues) Ruminants Deer (perirenal) Ox (caul and kidney) Sheep (total fatty tissues)

0.4

1.4

10.6

1.5

0.4

0.7

4.1

4.6

11.6

62.8

1.9

Not detected

-

1.6 2.4

22.1 29.7

6.4 4.3

0.8 0.2

0.4 1.4

4.4 6.5

12.7 32.5

7.9 3.8

42.4 16.1

1.3 3.1

Not detected Not detected

5.1 2.7 3.5

35.9 27.8 25.0

29.6 21.6 22.2

2.9 0.7

0.2 0.3 0.5

2.2 2.5 1.7

17.0 42.5 44.2

1.2 0.5 Tr

1.0 0.3 TI

4.8 1.8 0.9

3.5 4.8 11.2

‘Data from Shorland (1962).

-

0.1 -

1.3

WOF IN MEAT, POULTRY, AND FISH

29

acids was greatly increased in the meat and milk of cattle and sheep fed the treated diets. The increased proportions of unsaturated fatty acids in milk and meat from animals fed the formaldehyde-treated supplements may be useful in determining the diets of high-risk coronary patients for reduction of blood cholesterol (Scott et al., 1970; Cook et al., 1970). Obviously, the products produced-meat and milk-being more unsaturated, would be more susceptible to the development of oxidative rancidity and related flavor problems. The presence or absence of tocopherols (vitamin E) in animal tissues can also influence rancidity and, presumably, the development of WOF. Lundberg et a f . (1944) demonstrated that the rat could deposit dietary or injected tocopherols into the abdominal fat depots, with the level of deposition being closely related to the levels administered. In many of the earlier investigations, nevertheless, attempts to improve the stability of meat against oxidative rancidity by vitamin E supplementation have been disappointing. Watts et al. (1946) showed that pork fat is unusually low in tocopherols, but that either feeding or injection increased tissue levels. However, the level of increase was relatively small, and the authors concluded that it was not large enough to be of significance in preventing rancidity in pork sausage prepared from the meat of vitamin Esupplemented animals. Although there are considerable data on the effects of vitamin E supplementation on tissue levels in chicken (Bunyan et al., 1967; J . E. Webb et al., 1972, 1974) and turkey (R. W. Webb et al., 1972; J. E. Webb et al., 1973), the improvement in the stability of the fatty tissues to oxidation is not clear-cut. J. E. Webb et al. (1972, 1974) reported that vitamin E supplementation improved the TBA numbers of precooked, frozen turkey and chicken parts, but they were unable to establish a relationship between panel scores and TBA values. In spite of this, Webb et al. (1974) indicated that precooked, frozen, and stored poultry parts had improved stability and flavor, even at relatively low levels of vitamin E and short feeding periods. In keeping with the generally poor absorption of vitamin E, Caravaggi and Wright (1969) found that sheep fed a-tocopherol acetate excreted all of it withn 4 days. In contrast, evidence is accumulating to show that large doses of vitamin E confer stability on meat and depot fats and enhance their acceptability. Thus, Hvidsten and Astrup (1963) showed that the keeping qualities and flavor of pork could be improved by feeding 40 mg of vitamin E per pig per day, which gave a total intake of 2000 to 5000 IU of vitamin E (2000 to 5000 mg of dl-a-tocopherol). Astrup (1973) has confirmed the earlier observations and has indicated that similar improvements were obtained in poultry meat by increased levels of dietary vitamin E in the studies conducted by Mecchi et al. (1956). Merk (1959) has indicated that vitamin E supplementation in the diet of dairy cows improves the stability of butter made from their cream, and Dunkley et al. (1967) have reported improvement in the flavor of the milk. Grau and Fleischmann (1965)

30

A. M. PEARSON, JANE D. LOVE, AND I;. B. SHORLAND

have also claimed that cervelat-type sausage is improved if meat from pigs given dietary vitamin E supplements is used in the formulation. The most spectacular results were recently reported by Ellis et al. (1974). They found that it was possible to produce meat of superior keeping quality from veal calves by feeding vitamin E along with milk from cows fed protected safflower oil and then by weaning the calves and feeding a ration containing protected safflower oil. In comparison with commercial samples, there was an increase (12 to 13%) in the linoleic acid content, while the level of tocopherol in the round fat increased some sevenfold, and the induction period for rancidity increased from 7 t o over 27 days. The fat from the round of calves fed unprotected safflower oil and vitamin E in the milk was lower in linoleic acid and had an increased induction period up to 47 days. Although lowering the levels of polyunsaturated fatty acids in the diet of the pig might be expected to increase the stability of the fat to oxidative rancidity, studies by Dahl(l960) using a fat-free diet supplemented with vitamin E showed that the stability of the fat was no greater than that for fat produced on a diet containing 3 to 5% of oil with 30 to 400/0 linoleic acid. It is possible that the absence of fat in the fat-free diet may have impaired the absorption of vitamin E, thereby overriding any possible effect of vitamin E on stability. We concur with the view of Horn et al. (1974) that dietary requirements for vitamin E are higher for animals fed rations high in polyunsaturates, since polyunsaturated fatty acids promote lipid peroxidation. Thus, animals fed high levels of polyunsaturates, particularly poultry and pigs, would have low levels of natural antioxidants in their tissues, which may make their meat more susceptible to the development of WOF.

3. Environmental Effects The effect of environmental temperature on the firmness of depot fat was first demonstrated by Henriques and Hansen (1901), who showed that covering of pigs with sheepskin coats resulted in a marked decrease in the iodine numbers of the subcutaneous fat layer. Cramer and Marchello (1964) observed marked seasonal variations in the fatty acid composition of subcutaneous fat from lamb, which they attributed to differences in environmental temperatures. Link et al. (1970a,b) found that both beef depot fat and beef intramuscular fat changed in composition at different seasons of the year, apparently a result of changes in the environmental temperature. In a subsequent study, Marchello et al. (1967) demonstrated that colder temperatures resulted in softer body fats with lower melting points and higher iodine numbers. Fats from fishes and aquatic animals living in cold environments, such as the polar regions, are generally more unsaturated and have lower melting points than fats from animals living in warm environments (Lewis, 1967). In birds, where the

WOF IN MEAT, POULTRY, AND FISH

31

skin is well protected from external environmental temperatures, the depot fats are fairly uniform in composition regardless of whether they are from the internal organs or from superficial tissues (Deuel, 1955). The influence of internal environment on the firmness and composition of fat is well recognized, with body fats becoming progressively firmer from surface subcutaneous locations to the deep abdominal fats and kidney fats (Trowbridge and Moulton, 1909). This is borne out by the decrease in iodine number and the higher melting point of kidney fat as compared with subcutaneous fat (Cramer and Marchello, 1964; Marchello ef al., 1967), as well as by the lower content of unsaturated fatty acids of internal fats (Koch ef al., 1968b). The superior firmness and better keeping quality of leaf lard (kidney fat) of the pig and of kidney fat from cattle and sheep in comparison with those of subcutaneous fat are well recognized (American Meat Institute, 1944, 1945). While conceding that environmental temperature may play a role in determining the degree of unsaturation of fat, it must be borne in mind that not all observations support this view. For example, in the horse there is little or no difference in fatty acid composition between the internal perinephric fats and the external surface fats, which are subject to the same temperature variations as the corresponding fats of pigs (Shorland, 1962). Furthermore, Callow (1936), recognizing that there were wide variations of iodine values in the back fat of the pig despite the fact that there was a constant temperature along the back, put forward an alternative theory that the composition of animal fats is influenced by the rate of growth. He explained this view as follows: “It appears probable that the faster the rate of growth at which fat is deposited in fatty tissues the more saturated the fat becomes.” This would be expected in view of the fact that fats in such deposits can either be formed from fats and oils in the diet or be synthesized from carbohydrates. Since the fats and oils in the diet are limited in amount, a considerable proportion of the fat must be synthesized from carbohydrates. This leads t o an increase in saturation of the deposited fat, because fat synthesized from carbohydrates is relatively saturated, with an iodine number of 50 to 60, whereas fat formed from oils in the diet usually have iodine numbers over 100 (Callow, 1936). The difficulties in fully accepting the temperature theory of Henriques and Hansen (1901) or the growth rate theory of Callow (1936) are numerous, as was pointed out by Shorland (1955). Suffice it to say that on fat-free diets the variations in iodine value along the back of the pig still occur, regardless of temperature or growth rate (Shorland et al., 1944). Contrary to expectations based on the growth theory of Callow (1936), the amount of dietary fat deposited as lauric and myristic acid increased for pigs growing more quickly on the same diet (Shorland and de la Mare, 1945). Even though environment may influence the composition of animal depot fats, it is obvious that other factors are at least equally important.

32

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

4. Sex Differences

Generally, the female has a higher fat content than the uncastrated male animal of the same species (Deuel, 1955). On comparing the female with the castrated male, however, the differences are variable, depending on the species. In the pig, the castrated male (barrow) is considerably fatter than the female (gilt) at usual slaughter weights (Bruner et aZ., 1958; Pearson et aZ., 1958a,b; Cox, 1963). With cattle, the heifer (female) fattens at an earlier age and a lighter weight than the steer (castrated male), and when carried to the same weight is considerably fatter (Hedrick et aZ., 1969). However, the composition of the intramuscular lipids was the same for steers and heifers (Link et aZ., 1970b). Similarly, the ewe (female) lamb is fatter than the wether (castrated male) lamb (Cunningham et al., 1967). The female (pullet) chicken prior t o beginning to lay is also fatter than the uncastrated male or rooster (Acosta et al., 1966). Ovariectomy or spaying of the female increases the deposition of fat, just as castration does in the male (Deuel, 1955). However, spaying is relatively uncommon in meat animals and probably has little influence on the lipid composition of meat. Thus, sex-related differences in the amount, distribution, and composition of lipids in meat and poultry appear to be associated with differences between uncastrated males and females and between castrated males and uncastrated males and females. Differences in the degree of unsaturation between the sexes are probably minor, but could occur; they may then influence susceptibility to development of rancidity and could thus affect flavor. 5. Age Effects

In young animals subcutaneous fat constitutes a large proportion of the total fat, usually accounting for 85 to 95% of the stored fat (Deuel, 1955). The quantities of intermuscular, genital, and perirenal fat are low in young animals and increase with age until they reach a maximum and level off (Deuel, 1955). Thus, the relative quantities of fat in meat from young and older animals are relatively different. Callow (1958) and Callow and Searle (1956) have shown that firmness of fat tends to increase with age in both lambs and cattle. As fat content increases, there is a decline in relative water content. Moulton (1923) concluded that on a fat-free basis all mammals show a relative decrease in water content as they mature, accompanied by a corresponding increase in protein and ash content until the proportion of these components becomes constant at chemical maturity. Stated another way, the most striking change in the composition of the body is a rapid increase in fat. Williams et al. (1945) have shown that the increase in fat content of the rat from birth to maturity is due almost entirely to a threefold increase in triglycerides. The age-associated compositional changes in meat animals may be even more marked. For instance, the

WOF IN MEAT, POULTRY, AND FISH

33

newborn Romney lamb, which weighs only 2.6 to 4.4 kg, contains 2.8 to 4.0% total lipid with phospholipids constituting 14.3 to 20.870, whereas the mature ewe, which weighs 35.6 to 44.4 kg, contains 32.0 to 42.5%total lipids including 1.25 to 2.5% phospholipids (Body et al., 1966). The age-associated compositional changes in other mammals appear to be similar, which would be true for the red meat-producing species. The chicken is different from mammals, since it has relatively high blood and liver lipid levels at the time of hatching, but the levels have been shown to decline markedly by 36 days of age (Entenman et al., 1940). Even in broilers, which may be slaughtered as early as 6 weeks of age, the major changes in blood and liver lipid levels would have already occurred, so that the triglycerides constitute the majority of the lipids. However, the more highly unsaturated fatty acid content of the adipose tissues of both fish and poultry generally makes their postmortem tissues more susceptible to oxidative rancidity than is true for the fatty tissues from the red meats.

B. TISSUE LIPIDS Intramuscular or tissue lipids expressed as percentage of raw tissues vary less in amount and in composition than adipose tissue lipids, although there are proportionately large changes as the total fat content varies (Link et al., 1970b). Intramuscular lipids are composed of deposits of triglycerides in fat cells, which is commonly called marbling in the red meats, and of membrane-bound lipids consisting chiefly of the phospholipids and lipoproteins (Love and Pearson, 1971).

1. Marbling Fat Although the amount of tissue lipids in meat is highly variable, ranging from values lower than 2.0% to over 12.0% in beef (Orme etal., 1958), the composition of the intramuscular fat is relatively constant. This is shown by the data from Koch el al. (1968b) on the fatty acid conposition of backfat and of intramuscular fat of pigs after two different periods of time on a fattening ration, as presented in TabIe VI. The data show that the proportion of total saturated fatty acids in backfat increased by a total of 2.2% during fattening, whereas it decreased by 1.3% for intramuscular fat. Thus, the change in the amount of intramuscular fat was not only less, but on the average was in the opposite direction. Terroine (1920) recognized the great variability in the amount of depot fat, which includes the marbling fat, by labeling it the “element variable” in contrast to the essential structural lipids, which he termed the “element constant.” The difference between the total lipid content of a muscle from which all external

34

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

TABLE VI MEAN FATTY ACID COMPOSITION O F BACKFAT AND INTKAMUSCULAR FAT FROM FATTENING PIGS"

Back fatb Fatty acids

Initiald .

14:O (myristic) 16:O (palmitic) 18:O (stearic) Total saturated 16: 1 (palrnitoleic) 18:l (oleic) 18:2 (linoleic)

Finale

lntraniuscular fatC ~nitiaP

binale

0.6 24.2 5.2 30.1

0.6 23.0 5.2 28.8 4.9 52.4 12.5

~~-

0.9 25.1 7.1 33.1 4.9 45.3 15.4

1 .o 25.4 8.9 35.3 4.9 45.8 12.6

5.7

48.4 14.3

"Data from Koch ef al. (1968b). bBackfat values are for inner layer and are averages of three pigs. Clntramuscular fat is for longissirnus muscle and is the average value for three pigs. dInitial values were taken when pigs were allotted at an average of 88 kg live weight. eFinal values were taken after 1 1 weeks on a corn-soybean meal diet.

fat has been removed and the essential structural lipids consists of the marbling fat. According to Dugan (1971), the essential membrane-bound lipids (phospholipids) constitute only 0.5 to 1.0% of the total tissue, whereas Wilson et al. (1975) have reported the phospholipid content to vary from a low of 0.5% for beef red muscle to a high of 1.6% for chicken red muscle (thigh). Campbell and Turkki (1967) reported the phospholipid content of beef muscle to be 0.56 to 0.6 1%; the value for pork muscle was 0.68%. In analyzing for marbling fat, the essential membrane-bound lipids are generally ignored and are included in the total. In terms of percentage this is only a small error, but from the standpoint of the relative role of the two fractions in the development of WOF it could be of major importance. Marbling has been claimed to be a major contributor to the sensory properties of the red meats; it enhances the flavor and aroma (Hornstein, 1967; Herz and Chang, 1970), improves juiciness (Blumer, 1963), and plays some role in tenderness (Pearson, 1966). It has also been demonstrated by Wasserman and Talley (1968) that fat is an important aid in the identification of roasted beef, veal, lamb, and pork. Fat has also been shown to be the major contributor to the aroma of poultry broth (Pippen ef al., 1954). Even though marbling fat is essentially of the same composition as adipose tissue (Table IV), the fat cells are in close proximity to the lean tissues and thus could conceivably be more important to WOF than the other adipose tissue deposits, such as subcutaneous and perirenal fat.

WOF IN MEAT, POULTRY, AND FISH

35

2. Membrane-Bound Lipids The membrane-bound lipids are an essential part of the muscle fiber sarcolemma or muscle cell wall (Kono and Colowick, 1961), the mitochondria (Holman and Widmer, 1969), the niicrosomes (Macfarlane et al., 1960), and the sarcoplasmic reticulum (Newbold et al., 1973). Although membrane-bound lipids (phospholipids) can be altered by diet (Igene, 1976), the changes are relatively small as compared to those of the depot fats. The membrane-bound lipids are composed mainly of phospholipids and lipoproteins (Rothfield and Finkelstein, 1968) and vary from location to location within the carcass (Dugan, 1971). Dugan (1971) reported that phospholipid content varies as a function of total lipid content, with the amount of phospholipid in the total lipid fraction increasing from 10 to 70% as the fat content of muscle decreases from 5 to 1% of the muscle weight. This means that the proportion of phospholipid in a muscle increases as the percentage of fat declines. Thus, the phospholipid content tends to be inversely related to the lipid content of meat, which merely reflects the constancy of the phospholipid fraction even though fat content is highly variable. Nevertheless, there is still considerable variation in the phospholipid content among species (Kaucher et al., 1944) and from location to location within the same species (Gray and Macfarlane, 1961; Dugan, 1971). Poultry meat and fish muscle are known to be higher in phospholipids than the red meats (Watts, 1954, 1963; Younathan and Watts, 1960: Zipser and Watts, 1961a; Zipser et al., 1962; Acosta et al., 1966). Furthermore, there is evidence with rabbits (Bloor, 1943) and poultry (Acosta et al., 1966) that more active muscles contain a greater quantity of phospholipids. However, Katz el al. (1966) have shown that dark meat (legs) from chickens contains only about half as much phospholipids as white meat (breast). Several studies have shown fish (Zipser and Watts, 1961a; Zipser et al., 1962) and poultry (Acosta etal., 1966) to have higher levels of phospholipids and to be more susceptible to oxidative rancidity during refrigerated storage than beef. The speed of oxidation in fatty fish under refrigeration is particularly striking (Zipser and Watts, 1961a; Zipser et al., 1962). Younathan and Watts (1960) have made similar observations on pork, but oxidative rancidity develops somewhat more slowly. As mentioned earlier in the discussion of the structure of phospholipids, the component phospholipids expressed as a percentage of the total phospholipids are somewhat similar in most animal tissues. Thus, the main components consist of approximately 45% phosphatidyl choline, 25% phosphatidyl ethanolarnine, and 10% spldngornyelin, together with lesser amounts of phosphatidyl serine, phosphatidyl inositol, and other minor components (Body et al.. 1966, 1970). Irrespective of species, the phospholipids are characterized by their high levels of polyunsaturated fatty acids. As a consequence, the differences in fatty acid composition of the triglyceride and phospholipid fractions of fish are small

TABLE V11 FATTY ACID COMPOSITION O F THE MAJOR PHOSPHOLIPIDS IN THE R U M E N A N D ABOMASUM 017 THE MATURE

Runlen

SHEEP^.^

Abomasum

MethylestersC

PE

PC

Sph

PE

PC

14:O 15:o 16:O 17:O 18:O 19:Od 20:o 21:o 22:o 23:O 24:O 25:O 15Ve 17V 19v

Tr

0.6 0.7 26.9 1.2 19.4 0.1 0.1

0.2 0.2 35.8 2.3 15.3 0.2 0.9

0.1 0.2 5.1 1.5 18.0

Tr TI

Tr

0.6

10.4 4.9

TI

11.1

X

15:0Br 16:1,w , f 17:1,w R g 18:1, w 9 24:1,w 9 18:2,w 6 20:2,w6 18:3,w , 20:3,w 9 20:3,w6 22:3,w g 22:3,w 6 20:4,w 6 22:4,w 6 20:5,w j 22:5,w 6 22:5,w j 22:6,w j

0.1 6.9 1.8 17.9 Tr TI Tr

0.2 Tr

1.6 2.3 3.3 1.3 TI

2.1 1.5 27.9 4.3 1.1

0.2 0.2

0.5

0.5

0.2 4.2 8.2 0.7 0.1 0.6 0.6

27.6 5.1 1.2 2.9 0.1

TI

TI

0.4 8.4 0.7 4.3

0.8 2.1

TI

TI 1.3

0.5 1.1

0.8

1.9 0.4

0.6 0.6 0.7

0.4

Tr

0.5 Tr

4.2

0.1

8 .O

0.2 1.9 1.5 2.3

Tr

2.5 0.5 0.7

7.1 3.1

0.6

Tr Tr

26.4 1.1 18.5 0.3

0.1 0.1 52.2 2.0 13.3 0.2 1 .o

1.2 2.8 20.1

4.7 1.0 2.3 1.0 1.0 0.3

Tr

3.3 6.0 Tr Tr

TI

3.9 0.1

23.3 7.9 0.9

2.6 0.5 1.0

0.5 0.1 3.7 5.2 0.9 0.3 0.5

0.3 Tr

TI

0.4

11.9 1.3 5.3 TI 11.2 4.6

5.5

1 .o

0.7 3.0

0.7

2.5 0.9

0.6

‘Data from Body and Shorland (1974). bAbbreviations: PE, phosphatidyl ethanolamine; PC, phosphatidyl choline; Sph, sphingomyelin. ‘Designations: chain length, degree of unsaturation, and w position represent the position of the first double bond from the terminal group of the molecule. V indicates cyclopropane structure. X = unidentified. Br = branched chain. TI = trace, less than 0.1%. dContains methyl trans-octadec-16enoate. eContains 16:O Br, and the presence of the cyclopropane derivative is only tentative. fContains 17:OBr. gContains 18:OBr.

WOF IN MEAT, POULTRY, AND FISH

37

(Shorland, 1962). These differences increase as we proceed successively from poultry to pigs through ruminants, in which the polyunsaturated fatty acids are confined largely to the phospholipid fraction (Shorland, 1962). Within the phospholipid fraction, the phospholipid components tend to have a characteristic fatty acid composition. Thus, the sphingomyelin fraction contains little polyunsaturated fatty acid but is composed mainly of higher saturated and monounsaturated fatty acids (Body and Shorland, 1974). The greatest concentration of polyunsaturated fatty acids is found in the phosphatidyt ethanolamine fraction, which is, therefore, of most interest in connection with studies on WOF. The phosphatidyl choline fraction is intermediate in fatty acid composition between sphingomyelin and phosphatidyl ethanolamine (Body and Shorland, 1974). As indicated earlier, the delineation of types of polyunsaturated fatty acids was far from complete in earlier analyses. In recent years, many more polyunsaturated fatty acids have been identified in animal tissues, as shown in Table VII .

V. ROLE OF LIPIDS IN MEAT FLAVORDESIRABLE AND UNDESIRABLE Kramlich and Pearson (1958) first demonstrated that the characteristic flavor of meat was water-soluble. Later Hornstein and Crowe (1960) confirmed the fact that the basic meaty flavor resides in the water-soluble fraction and showed that it was essentially the same for all species, whereas the characteristic species flavor and aroma appeared to arise from the lipids. Hornstein ef al. (1963) then proceeded to demonstrate the similarity in the nature of lean extracts from beef and whale muscle, although there were distinct differences in flavor, due to variation in the lipid fractions. These studies have clearly shown that meat flavor per se resides in the water-soluble extract, while the species-characteristic flavor and aroma originate from the lipid fraction of meat. Several excellent reviews have summarized the evidence (Hornstein and Crowe, 1964; Hornstein, 1967; Herz and Chang, 1970). Further proof for the role of fat in the species differences in flavor was provided by Wasserman and Talley (1968), who showed that fat greatly improved the number of correct identifications for roasted beef, veal, pork, and lamb by taste panel members. Apart from the effects of fat on the characteristic species flavor of meat, there is clear-cut evidence that undesirable flavors can be transferred from the feed to milk and meat. Roberts (1965) has pointed out that a variety of weeds and feeds influence the flavor of milk, and in some cases presumably the flavor of meat. Even inhalation of strong odors may cause off-flavors in milk. Although meat appears to be less susceptible than milk to flavor changes due to the diet of the animal, Kemp and Varney (1955) showed that cattle on pastures containing wild

38

A. M. PEARSON, JANE D. LOVE, A N D F. B. SHORLAND

onions produced meat with an objectionable flavor. The meat from lambs grazed on white clover (Trifoliumrepens) was shown t o have a stronger flavor than that from similar lambs grazed on ryegrass (Lolium perenne) pasture (Cramer et al., 1967; Shorland ei al., 1970), presumably owing to differences in the lipid components. Likewise, strong and unacceptable flavors and/or aromas have been observed in the meat from iambs grazing on rape, vetch, oats, and alfalfa pasture (Park et al., 1972a,b). Although some of the off-flavors transferred to meat by the diet may be due to the lipid components, others may be associated with certain volatile components in the tissues, including disulfides, mercaptans, and organic sulfides, which are associated with other components in the tissues. Lipids can contribute both desirable and undesirable flavors to meats. Among the desirable flavors are the characteristic species-associated flavors and aromas that occur in beef and pork (Hornstein and Crowe, 1960), in beef and whale meat (Hornstein et al., 1963), and in lamb (Hofstrand and Jacobson, 1960; Hornstein and Crowe, 1963). These flavors and aromas are generally accepted as desirable by at least some segment of the consuming public, although others may object to certain of these flavors, as is the case with lamb (Hofstrand and Jacobson, 1960). Although oxidation is usually considered to produce undesirable flavors in meats, a notable exception is observed in dry-cured country hams, which are normally aged to improve their flavor and aroma (Cecil and Woodroof, 1954; Kemp et al., 1957). In fact, the aged flavor does not occur in country cured hams until hydrolysis of some of the fat and a certain amount of oxidation ensues (Blumer, 1954; Kemp et al., 1957). The best indication of aged flavor development has been found to be the amount of free fatty acids (Blumer, 1954; Kemp et al., 1957). It is also possible that oxidative breakdown may have a beneficial effect on the flavor of some of the fermented sausages, in which there is an increase in free fatty acids during ripening. As already indicated, lipids may impart objectionable odors t o meat and meat products. Such odors are most frequently due t o oxidation products (Gaddis et al., 1961; Kesinkel et al., 1964; MacLean and Castell, 1964). The resulting rancid flavors are well known and easily recognized by consumers (Watts, 1962). Gaddis et al. (1961) reported n-hexanal to be a product of autoxidation of Iinoleate, and Evans (1961) proposed a mechanism for its formation. ElGharbawi and Dugan (1965) found that n-hexanal greatly increased during storage of freeze-dried beef, presumably as a consequence of oxidation. Cross and Ziegler (1965) noted that hexanal occurred in greater quantities in uncured than in cured ham, apparently because of more extensive lipid oxidation in the uncured product. Love and Pearson (1976) also observed an increase in the hexanal concentration that was associated with oxidation in a model meat system. The addition of tripolyphosphate caused a 50% decrease in hexanal, whereas 5 ppm of Fe2+to the model system resulted in a twofold increase. These

WOF IN MEAT, POULTRY, AND FISH

39

results support the probable role of oxidation in production of hexanal in meat systems, which may then be involved in WOF. The so-called “sex odor” in pork, which emanates from the heated fat of the uncastrated male (boar) pig, is also another flavor defect in meat that many consumers find objectionable. Craig er al. (1962) showed the responsible component(s) to be localized in the unsaponifiable fraction of the fat, and it was later shown by Patterson (1968, 1969), to be due at least in part t o a steroid compound, a-androst-16-en-3-one. More recently, Thompson e l al. (1972) offered presumptive evidence that at least four steroids may contribute to the undesirable, aroma from boar fat. Thus, it is shown that the undesirable flavors and aromas in meat can arise from either naturally occurring lipids, or more commonly from breakdown products of lipid oxidation. Possibilities also exist for a variety of interactions of lipids and their breakdown products with the other meat constituents, which may produce both desirable and undesirable flavors and aromas in meat. Herz and Chang (1970) have explored some of the possible interactions in an excellent review on the chemistry of meat flavor. Boelens er al. (1974) have also examined some of the organic sulfur compounds formed by reactions with fatty aldehydes, hydrogen sulfide, thiols, and ammonia and their possible contribution to food flavors. They demonstrated that saturated aldehydes react with both gaseous and liquid hydrogen sulfide to produce a number of organic sulfur compounds. Unsaturated aldehydes, which may occur in meat, were shown to react with hydrogen sulfide and thiols to give addition products in which the sulfur becomes linked at the double bond. Saturated aldehydes, hydrogen sulfide, and thiols were also shown to react and produce other organic sulfur compounds that may contribute to the aromas and flavors of meat, poultry, and fish. Since the purpose of this review is to examine WOF and not meat flavor, readers are referred to the review of Herz and Chang (1970) and the recent paper by Boelens e l al. (1974) which summarize current information on meat flavor.

VI. MECHANISMS OF LIPID OXIDATION A. AUTOXIDATION Lundberg (1962) has reviewed the mechanisms involved in autocatalytic autoxidation. It is generally accepted that a free radical chain mechanism, shown in the following simplified scheme, is involved: Initiation (1)

RH+O,---*R’+’OH

40

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Propagarion (2) (3)

R' + 0 , 4 ROO' ROO' + RH +ROOH + R'

The reaction is initiated when a labile hydrogen is abstracted from a site on the lipid (RH), with the production of lipid radicals (R') as shown in step 1. Reaction with oxygen (step 2) which yields peroxyl radicals (ROO') is followed by the abstraction of another hydrogen (step 3). A hydroperoxide (ROOH) and another free radical (R'),which is capable of perpetuating the chain, are formed. Decomposition of the ROOH species forms more free radicals, which participate further in the chain reactions (Dugan, 1961). Lundberg (1962) stated that the hydroperoxides may exist in equilibrium as follows: 2ROOH + (ROOH),

At low total peroxide concentrations, hydroperoxide decomposition is mainly monomolecular (Lundberg, 1962) and may be illustrated by ROOH .+ RO' + 'OH (Dugan, 1961). At high hydroperoxide concentrations a bimolecular decomposition occurs (Lundberg, 1962). The decomposition may be schematically illustrated by 2ROOH + ROO' + RO' + H20 (Dugan, 1961). While hydroperoxides are widely held to be the primary products of the oxidation of unsaturated lipids, the products resulting from hydroperoxide degradation are responsible for the occurrence of off-flavors in oxidized lipids. Keeney (1962) has discussed the complex and dynamic nature of the secondary degradation products arising as a result of the oxidation of unsaturated lipids. While an autocatalytic mechanism may describe the oxidative processes occurring in a highly refined fat or oil, the situation in a food, such as meat, is more complex. The reactivity of food lipids is, of course, influenced by the degree of unsaturation of the constituent fatty acids, as well as by the presence of activating or inhbiting substances. The speed with which undesirable rancid odors and flavors develop in animal tissues indicates the presence of substances promoting the oxidation of the unsaturated lipid components (Dugan, 1961). Hemoprotein muscle pigments and metals, especially iron, have been implicated as prooxidants in meat (Tappel ef al., 1961 ;Tappel, 1962; Watts, 1962; Liu and Watts, 1970; Sat0 and Hegarty, 1971).

B. CATALYSTS OF LIPID OXIDATION

1. Heme Compounds The catalytic effect of iron porphyrins on the oxidative deterioration of polyunsaturated fatty acids was first described by Robinson (1924). The heme-

WOF IN MEAT, POULTRY, AND FISH

41

catalyzed oxidation of unsaturated fatty acids has been extensively studied, and acceleration of lipid oxidation due to a variety of heme compounds is now a generally accepted phenomenon. Tappel (1962) has reviewed some of the voluminous literature dealing with heme-catalyzed lipid oxidation. Catalysis by iron porphyrins is characterized by rapid initiation and propagation of the lipid oxidation chain reaction (Tappel, 1962). According to Tappel (1962), catalytic homolytic cleavage of the COOOHbond of the hydroperoxide is a general property of hematin catalysts. He suggested the mechanism shown here as the most probable one for hematin-catalyzed unsaturated lipid oxidation.

L

\

The hematin compound (a) and lipid peroxide (LOOH) are postulated to form an activated compound (b). Subsequent scission of the peroxide bond occurs, resulting in the production of a lipid radical (LO') and a heme radical (c). Abstraction of a hydrogen atom (H) from a lipid molecule (LH) regenerates the hematin and produces a lipid radical (L). Tappel (1962) also suggested that a direct attack on the lipid by the heme compound could result in generation of lipid radicals according to the following mechanism: LH + hematin - Fe'+ -+ L' + hematin - Fe'+ + HI

Tarladgis (1961) attributed the catalytic activity of ferric hemoproteins to the paramagnetic character of the porphyrin-bound iron. He suggested that the presence of five unpaired electrons in metmyoglobin produces a strong magnetic field that would favor the initiation of free radical formation. Decomposition of hydroperoxides could be mediated through the donation of an electron from the n cloud of the porphyrin ring. While the prooxidant activity of hemes has been known for many years, it has been recognized more recently that heme compounds can also act as antioxidants. Maier and Tappel (1959), using a fixed heme concentration, observed that, when the linoleate concentration dropped below a specified level, lengthy

42

A. M. PEARSON, JANE D. LOVE, A N D I:. B. SHORLAND

induction periods occurred. Banks et al. (1961) found acceleration of fatty acid oxidation with increasing cytochrome c concentrations, up to a maximum; further increases resulted in inhibition. Lewis and Wills (1963) have also reported that the prooxidant or antioxidant activity of a heme compound is determined by the ratio of heme to unsaturated fatty acid. Linoleate-to-heme ratios for maximum catalysis of lipid oxidation were determined by Kendrick and Watts (1969). They reported optimum linoleate-to-heme ratios of 100 for hemin and catalase, 250 for metmyoglobin, 400 for cytochrome c and 500 for methemoglobin. At heme concentrations of two to four times the optimum catalytic amount, they noted that lipid oxidation did not occur. They theorized that a stable lipid hydroperoxide-heme derivative was formed at inhibitory heme concentrations. At lower heme concentrations, it was postulated that the heme may be unable to contain the lipid radicals, and oxidation results, with eventual destruction of the heme. Hirano and Olcott (1971) also reported that high concentrations of heme compounds inhibited lipid oxidation, while heme compounds at lower concentrations accelerated oxygen uptake. These authors pointed out the importance of controlling the initial levels of hydroperoxide present in the lipid used in model system experiments. Nakamura and Nishida (1971) reported that the association of fatty acids with hemoglobin was responsible for the observed dependence of lipid oxidation on hemoglobin concentration. As the hemoglobin concentration in a linoleic acid emulsion increased, they noted that an increasing amount of linoleic acid was associated with the hemoglobin. When more than 77% of the linoleate was bound to the hemoglobin, a lengthy induction period was observed. They further reported that the visible spectra of the hemoglobin indicated that it existed in a low-spin ferric form during the induction period. A carboxylate ion and a cis double bond in the fatty acid structure were also required for the binding of the fatty acid to the hemoglobin. The ratio of hemoprotein to unsaturated fatty acid in muscle tissue could influence the extent of lipid oxidation occurring in the muscle. Myoglobin, the oxygen-binding muscle pigment, and hemoglobin, from blood trapped in muscle tissue, are the major sources of iron in muscle (Bodwell and McClain, 1971). Craig cr al. (1966) reported that the total heme pigment content in the longissinius dorsi muscle of beef animals is 3.79 mg/gm. In beef foreshank, these authors reported the myoglobin and hemoglobin content to be 4.25 mg/gm. Liu and Watts (1970) pointed out that a rough calculation of the ratio of heme to unsaturated fatty acid in muscle indicates that the hemes could exert a prooxidant effect on lipid oxidation. One can only speculate about the actual degree of contact between hemoproteins and unsaturated lipid in muscle. Myoglobin is located in the cytoplasm, whiIe unsaturated lipids are integral parts of cellular structures. Thus, contact between lipid and myoglobin would be limited,

W0.F IN MEAT, POULTRY, AND FISH

43

and myoglobin could be inhibiting in localized areas of the cell and out of contact with unsaturated fatty acids in other areas (Liu and Watts, 1970).

2. Metal Ions Ingold (1962) has summarized the activity of heavy metals in increasing the rate of oxidation of food lipids. He pointed out that metals such as iron, cobalt, and copper, possessing two or more valency states with a suitable oxidationreduction potential between them, are particularly important catalysts. Ingold (1962) also stated that the effect of metals can be reflected in an altered rate of chain initiation, propagation, or termination, as well as by an altered rate of hydroperoxide decomposition. The basic function of the metal catalyst is to increase the rate of formation of free radicals (Ingold, 1962). Heaton and Uri (1961) have shown that metal ions in their higher valency states will react directly with lipid substrates. Ingold (1962) has suggested that, while direct metalsubstrate reactions may be a major source of radicals in the early stages of oxidation, at later stages tlus effect is less important than other possible reactions. The equation for a reaction involving metal catalysis of this type is schematically illustrated here.

Metal ion (M)reacts with lipid (RH) t o yield a lipid radical (R'). Heaton and Uri (1961) have shown that metals in their lower valency states may initiate lipid oxidation chain reactions directly. Uri (1956) and Heaton and Uri (1961) suggested that the first stage in this process may be represented as an activation of dissolved oxygen:

A subsequent reaction with the organic substrate then generated free radicals (Brown e f al., 1963). Uri (1956) has described a commonly accepted mechanism for metal catalysis involving the oxidation of a metal ion with hydroperoxide decomposition resulting as follows:

Ferrous iron has been shown to have greater prooxidant activity then ferric iron in a number of experimental systems (Brown er al., 1963; Wills, 1965; O'Brien, 1969; Sato and Hegarty, 197 1). Several investigators, including Wills

44

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

(1965), Barber (1966), Sato and Hegarty (1971), and Love (1972), have reported that low levels of ascorbic acid increase the efficiency of iron as a catalyst for lipid oxidation, presumably by regenerating the active ferrous ion. Ingold (1962) has pointed out that metals exist as hydrated ions in an aqueous lipid system. He proposed that the water-soluble radicals and products of lipid oxidation enter the aqueous phase as lipid oxidation proceeds. He then suggested that metals may react with the water-soluble species in the aqueous phase or with the substrate, radicals, or hydroperoxides at the oil-water interface. The activity of metal ions in contact with lipid substrates can be either accelerating or inhibiting (Marcuse and Fredriksson, 1971). Marcuse and Fredriksson (1971) examined this conversion phenomenon at low oxygen pressures. They observed that Fe3+ and Cu2+ exhibited prooxidant activity up to a maximum concentration; then a decrease in prooxidant activity was noted. These authors reported that this conversion was more marked at low oxygen pressures; they theorized that, under these conditions, oxygen-independent chain-terminating reactions become dominant. The conversion reportedly did not occur with Fez+ and Co2+. The ratio of concentrations of metal and substrate and the presence of anions may also influence this type of conversion. The influence of metal ions on the oxidation of lipids in a gel form was studied by Ellis ef al. (1970, 1971). Gels were composed of carboxymethyl cellulose, lard, and water in a ratio of 1-2-40 by weight (Ellis el al., 1971). The pH of the gels was adjusted to pH 5.5 t o approximate the pH of meat. Ferrous iron had the greatest prooxidant activity of the metals studied, followed by Fe3+, Cu+, Cuz+ and Ni2+, in decreasing order of activity. In the system studied, MnZ+and SnZ+ exerted an antioxidant effect. The prooxidant activity of low levels of contaminating iron in muscle foods is well known (Moskovits and Kielsmeier, 1960; MacLean and Castell, 1964). Only recently has the nonheme iron endogenous to muscle been attributed a major role as a catalyst of oxidative rancidity. Sato and Hegarty (1971) showed that nonheme iron accelerated the oxidation of lipids in water-extracted cooked meat. They also reported that myoglobin and hemoglobin failed to act as prooxidants in the cooked meat systems studied. Data presented by Love and Pearson (1974) confirm the results of Sato and Hegarty (1971). Most of the iron in animal tissue occurs in hemoproteins; however, a number of substances in animals contain nonheme iron (San Pietro, 1965). Iron-protein complexes (ferritin, hemosiderin, and transferrin) function in the storage and transport of nonheme iron (Mahler and Cordes, 1966). Small amounts of nonheme iron-containing proteins appear to perform key functions in electron transport (San Pietro, 1965; Mahler and Cordes, 1966). Enzymes such as succinic dehydrogenase, DPNH-cytochrome reductase, and xanthine oxidase contain nonheme iron. Wills (1966) has demonstrated that, when nonheme iron is released from ferritin, it becomes an active catalyst of lipid oxidation. Ascorbic acid can function in the release of nonheme iron from iron-containing

WOF IN MEAT, POULTRY, AND FISH

45

proteins (Wills, 1966). This may partially explain why low levels of ascorbic acid enhance lipid oxidation. C. COMPARISON OF HEME AND NONHEME IRON AS PROOXIDANTS IN MUSCLE TISSUE Many studies have focused on the rapid nonenzymic lipid oxidation taking place in tissue homogenates and particulate fractions exposed to atmospheric oxygen. Wills (1966) attempted to assess the relative importance of hemoprotein and nonheme iron catalysts of lipid oxidation in various animal tissues incubated in air. He concluded that both heme and nonheme iron were present in most tissue fractions and were capable of catalyzing the oxidation of unsaturated fatty acids. His results indicated that nonheme iron was a more active prooxidant at acid pH values, whereas hemoproteins were less pH-sensitive. He also reported that nonheme iron was apparently more important than heme proteins in catalyzing the oxidation of the endogenous lipids in tissue homogenates. Iron has been shown to induce oxidation of lipids in mitochondria and microsomes (McKnight and Hunter, 1965; Wills, 1969), in emulsions of fatty acids (Liu, 1970), and in fatty acid hydroperoxides (O’Brien, 1969). Barber (1966) has also suggested that nonheme iron and ascorbic acid constitute the normal prooxidant system in animal tissues. It is clear that either nonheme iron or hemoproteins can function as prooxidants when in contact with purified lipids. The situation in muscle is more complex. Liu and Watts (1970) compared the activity of heme and nonheme iron as catalysts of oxidative rancidity in meats. They concluded that both heme and nonheme iron function as catalysts of lipid oxidation in cooked meat. In contrast, Sat0 and Hegarty (1971) presented evidence that nonheme iron and ascorbic acid catalyzed lipid oxidation in cooked meat. Heme compounds were found t o have little effect on the development of WOF. Love (1972) presented findings that confirmed the observations of Sato and Hegarty (1971). Metmyoglobin did not influence TBA values in cooked meat, which had been water-extracted to remove prooxidants prior t o cooking. No acceleration was observed when metmyoglobin was added at levels of 1 to 10 mg/gm of muscle. Levels of ferrous iron as low as 1 ppm resulted in enhanced lipid oxidation in the samples of water-extracted cooked meat. It is important t o assess the relative contributions of hemoproteins and nonheme iron to the prooxidant activity of muscle, since pH variation and additives may exert different effects on heme and nonheme iron catalysis. D. PHOSPHOLIPID OXIDATION Phospholipids have been shown to be the lipid component most rapidly oxidized in cooked meat (Younathan and Watts, 1960), in lipid fractions

46

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

isolated from beef and pork (Hornstein et al., 1961), in freeze-dried beef (El-Gharbawi and Dugan, 1965), and in cod muscle (Roubal, 1967). Phospholipids offer a more complex oxidation system than neutral lipids. The tendency of phospholipids to oxidize very rapidly is at least partially due to their high content of unsaturated fatty acids (Lea, 1957). The phosphorylated bases may also affect the oxidation of the unsaturated fatty acids in the phospholipid molecule (Corliss, 1968; Lee-Shin and Smith, 197 1). Lee-Shin and Smith (1971) studied the effects of the functional groups of the phosphoryl bases of phosphatidyl ethanolamine and phosphatidyl choline on the oxidation of methyl linoleate in aqueous emulsions. They reported that ethanolamine and o-phosphoethanolamine increased oxygen uptake by methyl linoleate at pH 7.9 but decreased oxygen consumption at pH 10.2. The cholinecontaining groups were reported t o have no effect on the rate of lipid oxidation. Corliss (1968) has reported that the induction period for oxidation of phospholipids is a function of the nitrogen-containing moiety. He found that the ethanolamine moiety of phosphatidyl ethanolamine exerts a greater prooxidant effect than does the choline portion of phosphatidyl choline. He then concluded that the rate of phospholipid oxidation during the steady state was a function of the unsaturation of the fatty acid components of phospholipids. Love (1972) and Love and Pearson (1976) reported on metmyoblobin and ferrous iron as prooxidants in aqueous dispersions of phosphatidyl ethanolamine. Both metmyoglobin and ferrous iron were effective in increasing the oxidation of phosphatidyl ethanolamine at pH 5.5. At pH 7.0, however, neither metmyoglobin nor ferrous iron appeared to exert a prooxidant effect. Heme destruction appeared to result during metmyoglobin-catalyzed oxidation of phosphatidyl ethanolamine at pH 5.5. At pH 7.0, however, the hemoprotein did not appear to be destroyed, even though the lipid was undergoing oxidation.

VII. DEVELOPMENT OF WOF A. SPECIES DIFFERENCES IN WOF Wilson (1974) examined the meat from several species for susceptibility to the development of WOF as measured by TBA analysis. Results indicated that turkey $was most susceptible to WOF, followed by chicken, pork, beef, and mutton in that order. The red muscles consistently suffered more from WOF than the white muscles from the same species. Thus, breast muscle from turkey and chicken or the white portion of the semitendinosus muscle of the pig had lower TBA values after heating followed by refrigerated storage than similarly treated red muscles from the same species (thigh muscle for turkey and chicken and the red portion of the semitendinosus muscle of the pig). Although fish were

WOF IN MEAT, POULTRY, AND FISH

41

not included in this study, the high correlation between TBA numbers and phospholipids (Wilson et a/., 1976) suggests that fish would suffer extensively from WOF, and would be more like poultry than the red meats. However, wide differences among different species of fish would be expected in view of their variability in composition (Lovern, 1956a,b). Studies by Wilson et al. (1976) indicate that phospholipids are major contributors to the development of WOF in turkey, chicken, beef, and lamb, but that total lipids play a more important role than phospholipids in the development of WOF in pork. The role of phospholipids in the development of WOF in fish is not clear, but the relatively high content of unsaturated fatty acids in the triglycerides of fish would suggest that both triglycerides and phospholipids (Lovern, 1956a,b; Zipser and Watts, 1961a) may make the flesh of fish subject to rapid development of oxidative rancidity. Although TBA numbers have been widely used as a m’easure of WOF (Tims and Watts, 1958; Zipser and Watts, 1961b; Sat0 and Hegarty, 1971; Love and Pearson, 1974; Wilson et al., 1976) observations in our laboratory (A. M. Pearson and F. B. Shorland, unpublished data) suggest that TBA numbers are not closely indicative of WOF in different species. Thus, a slightly higher TBA value for one species than for another may not indicate the relative amount of WOF between the two species. In other words, the data are not directly translatable from species to species, especially where differences are relatively small. However, it is believed that large differences in TBA values among species are of general value in assessing the relative severity of WOF. B. INFLUENCE OF DEBONED MEAT Boneless meat is the major ingredient in sausages and related meat products (Kramlich et al., 1973). Development of mechanical deboning machines has made it economically feasible to bone poultry parts and fish, so that large quantities of mechanically deboned meat are now available for processing into sausages and other meat products. Mechanically deboned red meats have not yet been allowed in sausages, although deboned poultry meat has been permitted for several years. Since deboned meat contains large quantities of fat, it is susceptible to oxidative deterioration (Satterlee et al., 1971; Dimick et al., 1972; Field, 1974). It has been shown by Satterlee el al. (1971) that a large proportion of the fat is derived from the skin of poultry, although bone marrow may also contribute a small portion of lipid. The problem of oxidation of the lipids in deboned meat has led to the recommendation that deboned meat should be processed quickly in order to avoid development of rancidity (Dimick et al., 1972; Grunden et al., 1972). Bacterial contamination is also a factor in the stability of deboned meat (Ostovar et al., 1971), which is more serious in poultry because the air sacs are

48

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

connected to the large bones and are thus a source of added contamination. Since the bones of most mammals are not in direct contact with the respiratory system, there is less problem of contamination from the bone marrow (Field, 1974). Nevertheless, the bone marrow in deboned meat and poultry is a source of lipid and may contribute to rancidity (Moerck and Ball, 1974). The bone oils from the ox, sheep, and pig are easily distinguished from subcutaneous fats from the same species by virtue of their markedly lower content of stearic and palmitic acid (Shorland, 1962). There may also be marked variations in the fatty acid composition of bone tissues. For example, it has been shown by Shorland et al. (1962) that the mean iodine value for the fat from the femur of the sheep was 45.1, compared with 54.3 for the tibia tarsus and 79.9 for the metacarpus. Even though the differences in fatty acid composition of external and internal fats of the horse are negligible, the bone fats are distinctive in possessing less saturated fatty acids than other tissues (Shorland, 1962). This clearly shows that bone fats may differ in their susceptibility to oxidative deterioration and may influence the development of WOF in some meat, poultry, and fish products. Mechanically deboned meats and poultry (and presumably fish) have been shown to contain appreciably more calcium than hand-boned meat (Field, 1974). This could materially improve the calcium-to-phosphorus ratio, but it probably has little effect on the stability of either the raw or finished products. Although processing of deboned meats has been shown to improve their oxidative stability (Froning, 1970; Satterlee et al-, 1971; Grunden et al., 1972) and reduce bacteriological problems (Ostovar et al., 1971), a relatively high polyunsaturated fatty acid content could contribute to the development of WOF problems in products containing appreciable quantities of deboned meat. C. INFLUENCE OF HEATING

I . Normal Cooking A number of researchers have noted the accelerating effect of heating on the development of oxidative rancidity in meat and meat products (Younathan and Watts, 1959, 1960; Chang ef al., 1961; Kesinkel et al., 1964; Sat0 and Hegarty, 1971; Keller and Kinsella, 1973). Cooked meat exposed to oxygen can develop off-flavors in a matter of a few hours. The rapid oxidation of lipids in cooked meat has been attributed to the irreversible conversion of iron in the porphyrin ring of myoglobin pigments to the ferric form during heating (Younathan and Watts, 1959). The extent of lipid oxidation in cooked meat appears to be related to the intensity of heat treatment (Yamauchi, 1972a). Yamauchi (1972a) reported the

WOF IN MEAT, POULTRY, AND FISH

49

effects of heating beef, pork, and mutton at various temperatures on the development of rancidity as indicated by TBA values. The development of rancidity was most rapid in meat that was heated at 70°C for one hour. TBA values of cooked meat decreased as the cooking temperature was raised above 80°C. Yamauchi (1972a) also studied lipid oxidation in chicken muscle which had been fractionated prior to heating. Heating mitochondrial, microsomal, and myofibrillar fractions of chicken muscle accelerated the production of TBAreactive material, while heating the sarcoplasmic fraction resulted in only a slight increase in TBA values. Addition of the sarcoplasm to the microsomes, mitochondria, and myofibrils accelerated the lipid oxidation processes occurring in these fractions. Since the microsomes and mitochondria appeared to undergo more lipid oxidation after heating than did the myofibrillar fraction, Yamauchi (1972a) proposed that the destabilization of muscle during heating is related to protein denaturation in lipoproteins. Denaturation of lipoproteins in the microsomes and mitochondria presumably rendered the unsaturated fatty acids in the lipoproteins more susceptible to oxidation. Other researchers have examined the effect of heat on meat lipids. On heating, the neutral lipids are lost from the meat more readily than the phospholipids (Campbell and Turkki, 1967). Campbell and Turkki (1967) have reported that cooking beef or pork by a dry-heat method fails to appreciably change the fatty acid composition of the phospholipids. Giam and Dugan (1965) also observed that there was little difference in the fatty acid content of free or bound lipids in freeze-dried raw or cooked meat. Chang and Watts (1952) had previously noted that the fatty acid composition of ether-extractable lipids of beef and poultry was not changed by cooking. Keller and Kinsella (1973) studied phospholipid changes and lipid oxidation during cooking of ground beef patties. Negligible amounts of phospholipids were lost in the drip from cooked meat. Decreases in phospholipids were observed with some cooking treatments. These authors reported a 25% decrease in the arachidonic acid content of phosphatidyl ethanolamine in ground round cooked on a Teflon skillet. The arachidonic acid content in phosphatidyl ethanolamine decreased from 39.0 to 28.5 mole % during cooking. The value for arachidonic acid in phosphatidyl choline was 6.8 mole %, and this value was not changed by cooking. Keller and Kinsella (1973) also observed increases in TBA values on cooking and further increases when cooked samples were stored for 36 days at -1 8°C. During heating, changes occur in the muscle pigments which may affect their prooxidant activity. Denaturation of hemoproteins has been reported to increase their prooxidant activity (Banks, 1961; Eriksson et al., 1971). Banks (1961) proposed that undenatured cytochrome c may actually prolong the induction period during oxidation of unsaturated fat. He suggested that undenatured

50

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

hemoproteins may break down hydroperoxides so that they are not capable of initiating oxidation. He advanced the hypothesis that a denatured form of the heme compound accelerates oxidation following the induction period. Eriksson et al. (1970) have studied the effect of denaturation on the prooxidant activity of the hemoproteins, catalase and peroxidase. They reported that heat and chemical denaturing agents increase the prooxidant activity of these hemoproteins. According t o these authors, spectral analysis indicates that there is no change in the oxidation state of the iron in the porphyrin ring. They attributed the observed increase in prooxidant activity to an unfolding of the protein to allow greater exposure of heme groups to the substrate. Eriksson and Vallentin (1973) reported that the heat-induced increase in the prooxidant activity of peroxidase is pH-dependent. At pHs below 5.0 they observed no increase in the prooxidant activity of peroxidase on heating; apparently the heme group was masked from contact with the lipid in thermal aggregates formed at low pH values. After heating at pH 7.5, they found that the heme groups were highly exposed to the lipid. Spectral analysis revealed that heme migration and destruction by heat (especially at 140°C) also influenced the prooxidant activity of heated catalase. Ledward (1971) has studied the nature of the cooked meat pigment. His study indicates that myoglobin coprecipitates with other muscle proteins during heating. Increasing temperature was presumed to result in conformational changes in the hematin environment. Ledward (1971) also postulated that denaturated proteins may attack the hematin, resulting in the replacement of apomyoglobin by other proteins. He suggested that the pigments then aggregate and precipitate with other unreacted denatured proteins to form a range of denatured hemoproteins. Ledward (1971) also observed that the ferric hematin in meat possesses some low-spin characteristics. Generally, iron porphyrins that have low-spin states are less effective prooxidants. Koch (1962) cited S. J. Bishov and S. A. Henick (unpublished data) as showing that heat denaturation of heme pigments reduced their prooxidant activity. They observed that cooked dehydrated meat was more stable than raw dehydrated meat. Lipid oxidation rates in model systems containing heated muscle myoglobin extracts were also reduced compared with rates observed when unheated myoglobin was used.

2. Effects of Retorting or Overheating The effects of mild heating on W O F have already been discussed. As indicated earlier, normal cooking appears to disrupt the muscle membranes and contributes to the development of WOF (Sato and Hegarty, 1971). However, overcooking, as is common in canned meats, protects against WOF, apparently

WOI: IN MEAT, POULTRY, AND FISH

51

by producing compounds that possess antioxidant activity (Sato et al., 1973). Zipser and Watts (1961 b) first observed the production of antioxidant activity on heating meat to high temperatures, but did not determine the nature of the protective substances. They did, however, demonstrate that slurries from overcooked meat imparted antioxidant activity to normally roasted sliced beef. Sato et al. (1973) studied the reaction that is responsible for producing antioxidant activity during retorting of meat. They concluded that the substances responsible for producing antioxidative compounds were the result of a heat-catalyzed interaction between amino acids or proteins with carbohydrates. If this is true, one would expect meat in combination with carbohydrate to be more stable against the development of WOF, which does, in fact, occur in meat loaves where carbohydrates are common ingredients (Sato et al., 1973). The compounds produced seem to be products of nonenzymic browning. For example, Sato et al. (1973) found that reductic acid, maltol, and products of the amino sugar reaction were effective inhibitors of development of WOF in cooked ground beef. This supports the earlier findings of Tarr and Cooke (1949) showing that reductic acid retards fat oxidation in frozen minced red salmon and herring tissues. Thus, the evidence shows that overcooking of meat produces antioxidants through the browning reaction. The browning reaction is discussed in greater detail by Hodge (1967) and Reynolds (1963, 1965). Yamauchi (1972b) has proposed a different mechanism to explain the antioxidant activity in overheated meat. He proposed that, with extensive heating, the peroxides of the polyunsaturated fatty acids are created and degrade the myoglobin. The degradation products are presumed to have an antioxidant effect.

3. Oxidation during Storage of Meat Products The rapidity of onset of lipid oxidation in cooked meat products poses particular problems in handling if the development of off-flavors is to be avoided. Tims and Watts (1958) noted that flavor deteriorated rapidly in cooked beef after only a few hours of refrigerated storage. In addition to the rapid lipid oxidation observed in cooked meat, lipid oxidation occurs in raw meat with adverse changes in color and flavor resulting (Greene, 1969). Lower levels of lipid oxidation have been observed in cooked, cured meat than in uncured samples. The pink, ferrous form of the cured meat pigment apparently does not cause rapid lipid oxidation. On storage of cured meat, conversion of the pigment to the brown ferric form can result in increased TBA values for the stored product (Younathan and Watts, 1959). Poultry products are very susceptible to the development of off-flavors due to oxidative rancidity. With the trend toward production of heat-and-serve precooked poultry products, the loss of desirable fresh flavors and the development

52

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

of stale flavors becomes a severe problem. The use of mechanically deboned poultry meat, which is highly susceptible to lipid oxidation, might also increase the tendency of poultry products to oxidize. Antioxidants added to cooking water for turkey increase the stability of frozen meat covered with a sauce or gravy made with the fat (Lineweaver el aL. 1952). Products surrounded by a sauce or gravy that limits contact with air may have a longer storage life. Products such as fried chicken are more difficult to package in a manner that effectively excludes air, and warmed-over flavors develop. Nitrogen packing and avoidance of elevated temperatures during frozen storage are effective in preventing rancidity in frozen fried chicken (Hanson ef al., 1959). Dawson and Sison (1973) investigated the stability and acceptability of phosphate-treated, precooked chicken pieces reheated with microwave energy. Taste panels scored phosphate-treated chicken, that was microwave steam-cooked and browned by pressure frying, higher than chicken prepared by other methods. After storage, flavor scores were significantly lowered, and Lee and Dawson (1973) found that the chicken muscle lipids decreased in unsaturation during storage. They reported that both frozen storage of chicken and the use of reheated corn oil in frying resulted in a tendency to oxidize. The shelf life of stored seafood products is shortened by oxidative rancidity. During frozen storage, fatty fish such as herring, mackerel, and salmon are prone to undergo lipid oxidation even at -20°C (Sweet, 1973). Lean fish such as haddock and cod also oxidize when stored in the frozen state (Sweet, 1973). Fish may be more unstable during frozen storage then beef or chicken, probably owing to the high degree of unsaturation in the fish lipids (Olcott, 1962) and to the high concentrations of metals in seafood (Sweet, 1973). Oxidation of lipids in fish is apparently confined to the outer surfaces of the product (Sweet, 1973; Yu et al., 1973). Yu and co-workers (1973) have studied the effect of packaging as a deterrent to lipid oxidation during frozen storage of salmon steaks. They reported that vacuum packaging improved the sensory scores of salmon stored at -18°C. The importance of maintaining a constant temperature was also emphasized, as temperature fluctuations to above -18°C resulted in the development of more off-flavor, fading of orange-red pigments, and eventual production of a yellow discoloration. Martinez and Labuza (1 968) have also discussed the loss of astacene pigment and the production of brown discoloration in oxidizing freeze-dried salmon. Awad et al. (1969) noted that lipid oxidation and the production of off-flavors appeared to be linked to loss of protein solubility of frozen whitefish muscle. They proposed that the insolubilization of whitefish muscle proteins during frozen storage may at least partially result from the interaction of myofibrillar proteins with products of lipid autoxidation. Similar results have been obtained

WOF IN MEAT, POULTRY, AND FISH

53

for frozen plaice fillets by Dyer and Morton (1956), who demonstrated a relationship between protein denaturation and lipid deterioration during frozen storage. The extractability of frozen stored fish muscle has been observed to be related to muscle tenderness; loss of solubility of muscle protein was accompanied by a decrease in tenderness. D. INFLUENCE OF CHOPPING AND EMULSIFYING Sat0 and Hegarty (1971) reported that WOF develops in raw meat within one hour after grinding and exposure to air at room temperature. The odor and flavor changes were accompanied by a large increase in TBA values. These authors postulated that any process causing disruption of the muscle membrane system, such as grinding or cooking, results in exposure of the labile lipid components to oxygen, and thus accelerates development of oxidative rancidity. They also suggested that any catalysts of lipid oxidation present in the muscle system are brought into contact with the oxidation-susceptible lipids and may also contribute to the rapid development of WOF. Although studies on WOF development in sausage emulsions are lacking, the effects of membrane breakdown appear to be essentially the same as that of grinding. Several investigators (Wasserman and Talley, 1972; Simon et al., 1973; Bailey and Swain, 1973) have shown that nitrite improves the flavor of frankfurters, apparently by inhibiting oxidation. This suggests that addition of nitrite during chopping may play an important role in preventing WOF. Even though the evidence is purely circumstantial, some studies (Wasierman and Talley, 1972; Simon et a/., 1973) imply that emulsion-type products are particularly susceptible to rapid oxidative changes, while nitrite is an effective inhibitor. Thus, it can be assumed that chopping and emulsification are at least as likely to cause warmed-over flavor as grinding or mincing of muscle. If, in fact, the disruption of membranes and bringing of oxidative catalysts into contact with oxidationsusceptible lipids is the cause of WOF (Sato and Hegarty, 1971), it would be expected that emulsions are at least as susceptible to the development of WOF as ground meat, if not more so. Since ferrous iron has been demonstrated to enhance the development of WOF (Sato and Hegarty, 1971; Love, 1972), the use of equipment made of iron would accelerate oxidation. Although most modern choppers and emulsifiers are made of stainless steel, any equipment having iron surfaces that come into contact with meat, poultry, or fish products would catalyze the rapid development of WOF and cause a serious industry problem. Such problems could occur at any stage of processing and may require special trouble shooting to locate their source.

54

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

E. EFFECTS OF CURING Although curing was developed without any concern for WOF, it is interesting to note that phosphates, nitrites, and ascorbates, which are frequently used in meat curing, inhibit the development of WOF (Sato and Hegarty, 1971). 1. Action of Nitrites

Nitrite has been shown to eliminate WOF at a level of 220 ppm and to inhibit development of WOF at 50 ppm (Sato and Hegarty, 1971). Unfortunately, the legal limit of 156 ppm of nitrite allowed in cured meat was not investigated. Bailey and Swain (1973) have also confirmed the effectiveness of nitrite in preventing oxidation of meat (fresh ham) when stored under refrigeration (7.2"C). They further confirmed the effectiveness of nitrite in preventing WOF by subjective panel scores, thus verifying the inhibition of oxidation as shown by low TBA values. Such results offer an explanation for the better flavor of nitrite-cured pork as reported by Cho and Bratzler (1970). Since the muscle membranes appear to be the site of oxidation during the development of WOF (Sato and Hegarty, 1971), the nitrite must either stabilize the lipid components of the membranes or else inhibit the natural prooxidants present in muscle. Zipser et al. (1964) have proposed that nitrite forms a stable complex with iron porphyrins in heat-denatured meat, thereby inhibiting the development of WOF. Since nonheme iron seems to be the major lipid prooxidant in meat systems (Sato and Hegarty, 1971; Love and Pearson, 1974), it seems more probable that nitrite complexes and stabilizes the lipids in the membranes. Further support for this is given by Liu and Watts (1970), who pointed out that myoglobin is in solution in the cytoplasm and thus is separated from phospholipids, which are in the membranes and particulates. However, it may be that, as cooking breaks down the membranes, the lipid constituents and heme components are brought into contact. In this case, the stabilization of myoglobin by nitrite would have the same effects as stabilization of the membranes per se. On the other hand, EDTA has been shown to inhibit oxidation of fresh meat (Sato and Hegarty, 1971), which supports the rote of nonheme iron as a prooxidant and gives further credence to the possible stabilizing influence of nitrites on the membrane components of muscle.

2. Influence of Phosphates Tims and Watts (1958) showed that the addition of phosphates protects cooked meat from autoxidation. This was true for pyro-, tripoly-, and hexametaphosphate, but orthophosphates gave no protection. The effects of these three phosphate compounds in preventing WOF has also been verified by Sat0 and

WOF IN MEAT, POULTRY, AND FISH

55

Hegarty (1971), who have demonstrated that all three compounds markedly lower TBA values in cooked ground beef stored at 2°C. The mechanism by which phosphates prevent autoxidation appears to be related to their ability to sequester metals (Watts, 1950; Timms and Watts, 1958). Thus, phosphates would serve to chelate any metal ions, particularly ferrous iron ions, which are the major prooxidants in meat systems (Love and Pearson, 1974). These facts suggest that phosphates may be as important for preventing rancidity in cured meat products as they are for improving water retention.

3. Role of Ascorbates Ascorbic acid alone at low levels (up to 100 ppm) catalyzes the development of WOF as shown by increased TBA values (Tims and Watts, 1958; Sat0 and Hegarty, 1971). At high levels (1000 ppm and up), however, ascorbic acid retards autoxidation (Sato and Hegarty, 1971), probably by upsetting the balance between ferrous and ferric iron or by acting as an oxygen scavenger. Tims and Watts (1958) demonstrated that ascorbic acid in combination with phosphates acts synergistically to protect against rancidity. Sato and Hegarty (197 1) verified the enhanced antioxidant activity of the ascorbic acid-phosphate combinations and theorized that ascorbic acid functions by keeping a portion of the iron in the ferrous state. These results indicate that ascorbic acid and phosphates in combination have an important synergistic action in preventing the development of oxidation in cured meats (Chang and Watts, 1949). The use of phosphates alone or in combination with ascorbates may offer an explanation for the infrequency of rancidity in cured meats, in addition to the influence of nitrites. Certainly use of these compounds appears to offer real possibilities in preventing WOF in precooked meats, such as TV dinners.

4. Antioxidant Activity of Other Ingredients Younathan and Watts (1960) demonstrated that added antioxidants were particularly effective in reducing rancidity in the phospholipid and proteolipid fractions of pork, which appear to be mainly responsible for WOF. Following up on these studies, Watts (1962) then investigated the effects of a variety of plant extracts on the prevention of rancidity, since plant flavonoids are potent antioxidants. She demonstrated that pepper (pods and seeds), onion extract, and potato peelings are effective antioxidants, and when added to sliced roast beef they materially reduced WOF as shown by panel acceptability and TBA values. It seems likely that other plant ingredients used for seasoning sausages and certain meat products have considerable antioxidant activity. Oleoresins and

56

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

essential oils, which are frequently used in sausages, are low in flavone content (Kramlich et al., 1973) and therefore contain little antioxidant activity. Even though whole spices have antioxidant activity by virtue of their flavonoid activity, they may be contaminated by lipolytic bacteria, which may actually accelerate the development of rancidity (Kramlich et aZ., 1973). Pratt and Watts (1964) demonstrated that extracts from a number of plant sources (green onions, green peppers, potato peelings, and green pepper seeds) are effective antioxidants for meat. Nevertheless, there has been little follow-up on using plant extracts for preventing WOF in cooked meats, particularly in precooked meals where combining vegetable extracts with meat may prove to be an acceptable procedure for retarding the development of rancidity. Smoke is commonly used as a flavoring ingredient for cured meat. Part of its usefulness is known to be related to its antioxidant properties (Kramlich et al., 1973). Erdman et d. (1954) showed that liquid smoke greatly retarded oxidation in fatty fishes. Although ascorbic acid alone was not effective in retarding oxidation, in combination with liquid smoke it appeared to have a synergistic effect in reducing tissue oxidation. Thus, smoking of meat no doubt contributes to preventing the development of WOF.

5. Sodium Chloride The activity of sodium chloride in initiating color and flavor changes in meat lipids is well recognized but poorly understood. Lea (1937) suggested that NaCl influences lipid oxidation by promoting the activity of lipoxidase in meat. Later work by Banks (1961) and Tappel (1952) showed that meat does not contain lipoxidase and indicated that heme compounds are the factor promoting oxidation. Chang and Watts (1950) observed that NaCl showed no greater accelerating effect on rancidity in the presence of hemoglobin or muscle extract than in their absence. These workers reported a direct prooxidant effect by NaCl solutions of 15% or more or by dry salt when contact between the lard and the prooxidant was extensive. Chang and Watts (1950) also observed that the effect of NaCl on fat oxidation depended on the amount of moisture in the system. Moisture level also affects the ability of hemoproteins to act as prooxidants. Pokorny and Janick (1971) have reported that heme derivatives do not accelerate oxidation of lipids mixed with protein in the absence of water. At a level of 60% water, they noted that hemes begin to exert prooxidant activity. Fishwick and Zrnarlicki (1970) reported similar results with freeze-dried meat. Autoxidation of lipids catalyzed by heme pigments was not a major deteriorative process in freeze-dried turkey muscle, according to their report. When freezedried turkey muscle was rehydrated to a water content of not less than 50% of that of fresh muscle, a proportion of the heme pigment was reported to be present in the form of a high-spin complex, metmyoglobin, which catalyzes oxidation of unsaturated lipids in the rehydrated muscle.

WOF IN MEAT, POULTRY, AND FISH

51

Mabrouk and Dugan (1960) observed that autoxidation of aqueous emulsions of methyl linoleate was inhibited as the concentration of dissolved NaCl in the system was increased. These authors postulated that the inhibition might result from decreased solubility of oxygen in the emulsions containing salt. Ellis and co-workers (1968) investigated the mechanisms involved in NaClinduced rancidity in cured pork. They reported than an increase in NaCl concentration accelerated autoxidation but did not affect the decomposition of hydroperoxides to monocarbonyls. High proportions of lean increased autoxidation and the ratio of monocarbonyls to peroxides. Ellis et nl. (1968) suggested that NaCl may activate a component in the lean resulting in a change in oxidation characteristics of pork adipose tissue. Zipser et al. (1964) have reported that heating meat with nitrite converts the pigments to ferrous nitric oxide hemochromogen, which does not possess prooxidant activity. They reported that addition of NaCl resulted in acceleration of oxidation in freezer-stored, cooked cured meat. Olson and Rust (1973) added various salt formulations to dry-cured hams and noted the oxidative rancidity that developed as a result of each treatment. They assessed the extent of rancidity by determining TBA values for the lean and fat portions of the hams. Rancidity was reported to occur in the fat, even though a low-metal salt was used in the curing process. Olson and Rust (1973) noted that TBA values in the fat portion were decreased when a salt containing antioxidant was used in the cure. A taste panel preferred the antioxidant-treated samples; however, the panel did not express a preference for samples cured with lowmetal salt over control hams. At the current time, the processes involved in salt-catalyzed oxidation of triglycerides are not completely understood. Knowledge of the independent oxidative influences of NaCl, trace metal ions, and heme pigments is necessary to clarify the mechanisms involved in this important deteriorative reaction.

VIII. PREVENTION OF WOF IN MEAT, POULTRY, AND FISH A. ANTIOXIDANTS AND CHELATING AGENTS The inhibition of lipid oxidation due to nitrites, phosphates, ascorbates, and other curing ingredients has been discussed in the section on the effects of curing on the development of WOF. Antioxidant treatment of cooked, uncured meat can result in improvements in flavor (Watts, 1962). Polyphosphates provide protection against the development of oxidative rancidity in cooked meat at concentrations as low as 0.01 to 0.05% (Watts, 1962). Greene (1969) has reported that polyphosphates are ineffective as inhibitors of lipid oxidation in raw meat, presumably owing to hydrolysis by muscle phosphatases.

58

A. M. PEARSON, J A N E D. LOVE, A N D F. B. SHORLAND

Antioxidants have been shown to extend the storage life o f fresh fish (Sweet, 1973). Swect (1973) reported that control samples of salmon or trout developed TBA values indicative of significant rancidity in less than 24 hours at 5°C. Antioxidants increased the length of storage until significant TBA values were attained. The most potent inhibitors of oxidation were combinations of either butylated hydroxyanisole (BHA) or tert-butyl-hydroquinone (BTHQ) with EDTA or citric acid. Yu e t al. (1973) evaluated the effect of added antioxidants and vacuum packaging on the shelf life of frozen silver salmon stored at -18°C. Antioxidants did not improve the sensory scores of samples that were vacuum-packed. Peroxide values of fat extracted from frozen fish were reduced by vacuum packaging and further reduced by antioxidant treatment. A promising approach to retarding lipid oxidation in meat products is through the use of naturally occurring antioxidant substances as discussed by Watts (1 962). Flavonoids often have potent antioxidant activity (Watts, 1962). Pratt (1972) has shown that extracts of soybeans and soy products can retard oxidation in slices of cooked beef. Sato et al. (1973) showed that the aqueous extract from retorted meat possessed antioxidant activity and suggested that this was due to reducing compounds produced in browning-type reactions. Bishov and Henick (1972) reported that autolyzed yeast protein and hydrolyzed vegetable protein inhibited lipid oxidation in model systems containing corn oil which was free of antioxidants. These authors also reported that autolyzed yeast protein acted synergistically with BHA or a-tocopherol. B. REDUCING CONDITIONS Greene (1969) has investigated methods of preventing discoloration, due t o metniyoglobin formation, and off-odors, due to lipid oxidation, in raw meat. Wrapping meat in oxygen-impermeable film prevented metmyoglobin formation and lipid oxidation during storage, if sufficient reducing activity was present in the meat. There is a great variability in metmyoglobin reducing activity among different meat samples, and some samples studied did not have sufficient reducing activity to make wrapping in oxygen-impermeable film effective in preventing rancidity. Greene (1969, 1971) investigated the use of antioxidants to retard pigment and lipid oxidation in raw ground beef. Greene (1969) reported that butylated hydroxyanisole and propyl gallate inhibit lipid oxidation in raw ground meat. Grcene (I 971) reported that a combination of propyl gallate or butylated hydroxyanisole and ascorbic acid is effective in retarding lipid and pigment oxidation in ground beef for up to 8 days of refrigerator storage. Sodium tripolyphosphate was found t o be ineffective as an antioxidant unless the meat was first heated to at least 7OoC, presumably since it was hydrolyzed by phosphatases in the raw muscle (Greene, 1969).

WOF IN MEAT, POULTRY, AND FISH

59

C. PRACTICAL IMPLICATIONS The rapidity of onset of lipid oxidation in a variety of meat products poses particular problems in handling if the development of undesirable flavors and colors is to be avoided. Proper handling of meat products, including packaging to exclude air, and storage at a constant, low temperature, can aid in maintenance of quality. The use of natural antioxidants from plant sources or of antioxidants produced during retorting of meat appears to have much promise in a number of meat products. Control of lipid oxidation in raw meat by packaging in oxygenimpermeable wrap is difficult to achieve in some cases, owing to large variations in the reducing activity in meat samples.

IX. RESEARCH NEEDS Although nonheme iron has been shown to be the major prooxidant in cooked meat (Sato and Hegarty, 1971 ; Love and Pearson, 1974), its origin is unknown. It is possible that myoglobin serves as the source of nonheme iron, which could account for the fact that a number of investigators (Lewis and Wills, 1963; Kendrick and Watts, 1969; Hirano and Olcott, 1971; Nakamura and Nishida, 1971) have reported that myoglobin is the major prooxidant in meat. To resolve the question as to whether or not myoglobin may break down and serve as a source of nonheme iron in the meat system, labeling studies using radioactive iron ( 59 Fe) could be utilized. Since 59 Fe is a beta-ray emitter and has a half-life of 46 days, it could be utilized for labeling myoglobin and following the fate of the iron to see if myoglobin does in fact serve as the source of nonheme iron in cooked meat. There are also a number of nonheme iron-containing proteins that could be the source of the nonheme iron in meat (San Pietro, 1965); they too should be labeled with s9 Fe and followed to see if they provide nonheme iron in cooked meat. The proteins containing nonheme iron function in respiratory chain enzymes and are reviewed in detail in a symposium edited by San Pietro (1965). These proteins could serve as the source of nonheme iron and provide the oxidative catalyst for the development of WOF. Thus, labeled nonheme ironcontaining proteins could be followed t o ascertain if the respiratory chain enzymes are the source of prooxidant activity in cooked meat. Labeled iron could also provide information on the mechanism for the development of WOF, especially with reference to the role of cooking and subsequent refrigerated storage. Such studies may be used to elucidate the relative importance of different steps in the initiation of WOF. Although Wilson et al. (1976) observed a relationship between phospholipid levels and the development of WOF in meat from different species, there was also evidence that total lipids are more important than phospholipids in the

60

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

development of WOF in pork. The data for pork plus other observations in our laboratory (A.M. Pearson and F. B. Shorland, unpublished data) suggest that the mechanism for the development of WOF may differ for meat from different species. Thus, research is needed to determine exactly how variations in the composition of the triglycerides and phospholipids influence oxidative changes in meat, poultry, and fish. Special emphasis should be given to the proportion of unsaturated, and especially polyunsaturated, t o saturated fatty acids in both the triglycerides and phospholipids, as well as to differences among individual phospholipids and individual fatty acids with reference to the rate of lipid oxidation. Such variations may help to explain species differences in the development of WOF. Studies on the composition of the triglycerides and the phospholipids in red and white muscles, such as the breast and leg muscles of the chicken and turkey or the red and white portions of the semitendinosus muscle of the pig, could also be helpful in elucidating the factors controlling the development of WOF. Fragmentary evidence suggests that susceptibility to WOF differs for the red meats, poultry, and fish, yet little is known about the amount of variation among and within species. Thus, the entire area of compositional variation associated with a species and among species needs to be thoroughly investigated and clarified in relationship to WOF. Pratt and Watts (1964) reported that plants contain antioxidant activity and protect meat products against rancidity. Pratt (1 972) has recently suggested that water extracts of soybeans contain antioxidants. Various protein additives for meat loaves have been shown to inhibit the development of WOF (Sato et a[., 1973), yet the exact nature of their effects has not been explained. Studies to elucidate the mechanism by which the various protein additives (nonfat dry milk, dried whey, soy proteins, and cottonseed flour) exert their protective action against WOF could be useful, not only in explaining its development, but also in leading t o processing procedures that could circumvent the problem in other meat products. The increased use of different protein additives, especially of the extruded soy proteins or extracted soy flours, would make such studies most timely and important. Related studies using nitrites, phosphates, antioxidants, and other similar WOF inhibitors should also be more fully explored and exploited. Stewart et al. (1965) demonstrated that fresh raw meat reduces metmyoglobin by virtue of the action of indigenous reducing enzymes, which retard tissue oxidation. Although these authors suggested a possible reductive chain in tissues, more evidence and confirmation of the reductive mechanism is needed. Since they observed great variation in the metmyoglobin-reducing activity of meat from different animals and of different muscles from the same animal, the causes of such variation need investigating. It is conceivable that the status of metmyoglobin-reducing activity of the fresh meat could influence the degree of WOF

WOF IN MEAT, POULTRY, AND FISH

61

after cooking, although cooking would largely inactivate the naturally occurring enzymes. Since metmyoglobin-reducing activity increased with pH from 5.1 to 7.1 and with temperature from 3” to 35°C (Stewart et al., 1965), the effects of pH and temperature on development of WOF should be investigated. Stewart et al. (1965) reported that intact cuts of meat maintained their ability to reduce metmyoglobin for several days at refrigerator temperatures, while ground meat rapidly lost its ability to reduce metmyoglobin under the same conditions. Sat0 and Hegarty (1971) have also shown that ground raw beef rapidly becomes oxidized and upon cooking produces WOF. Thus, the significance of the presence or exclusion of oxygen from raw meat prior t o cooking may be a major factor in preventing WOF and should be carefully investigated in conjunction with studies on the role of metmyoglobin-reducing activity in the prevention of WOF in meat, poultry, and fish. Finally, one should bear in mind that the natural level of tocopherols present in the tissues may be sufficient to retard oxidation. Thus, unexplained differences in the prevalence of WOF may be due to differences in the amount of tocopherols in the meat itself and may be worthy of investigation.

REFERENCES Ackman, R. G., Ke, P. J., and Jangaard, P. M. 1973. Fractional vacuum distillation of herring oil methyl esters. J. Am. Oil Chem. Soc. 50, 1. Acosta, S. O., Marion, W. W., and Forsythe, R. H. 1966. Total lipids and phospholipids in turkey tissues. Poult. Sci 45, 169. American Meat Institute. 1944. “Pork Operations,” 5th ed. Institute of Meat Packing, University of Chicago, Chicago, Illinois. American Meat Institute. 1945. “Beef, Veal and Lamb Operations,” 4th ed. Institute of Meat Packing, University of Chicago, Chicago, Illinois. Anderson, W. E., and Mendell, L. B. 1928. The relation of diet to the quality of the fat produced in the animal body. J. BioL Chem. 76,729. Ansell, G. B., and Hawthorne, J. N., eds. 1964. “Phospholipids: Chemistry, Metabolism and Function,;’ BBA Libr., Vol. 3. Elsevier, Amsterdam. Astrup, H. N. 1973. Vitamin E and the quality of pork. Acta Agric. Scand., Suppl. 19, 152. Awad, A., Powrie, W. D., and Fennema, 0. 1969. Deterioration of fresh-water whitefish muscle during frozen storage at -10°C.J. Food Sci 34, 1. Bailey, M. E., and Swain, J. W. 1973. Influence of nitrite on meat flavor. Proc. Meat Ind. Res ConJ, 1973 p. 29. Banks, A. 1961. The role of cytochrome-c in the autoxidation of unsaturated fatty acids. Chem. Id.(London) 2, 4 0 (Abstr. of lecture in Oils and Fat Group, Hull, England, Dec. 5, 1960). Banks, A., Eddie, E., and Smith, J. G. M. 1961. Reactions of cytochrome-c with methyl linoleate hydroperoxide. Nature (London) 190,908. Barber, A. A. 1966. Lipid peroxidation in rat tissue homogenates: Interaction of iron and ascorbic acid as the normal catalytic mechanism. Lipids 1, 146. Bishov, S. J., and Henick, S. A. 1972. Antioxidant effect of protein hydrolyzates in a freeze-dried model system. J. Food Sci 37, 873.

62

A. M. PEARSON, JANE D. LOVE, AND 1;. B. SHORLAND

Moor, W. R. 1943. “Biochemistry of the 1;;itty Acids.” Van Nostrand-Reinhold, Princeton. New Jcrsey. Blumer, T. N. 1954. Chemical compounds associated with aged ham flavor. Ph.D. Thcsis, Michigan State Univcrsity. East Lansing. Blumer, T. N. 1963. Kclationship of marbling to the palatability of beef. J. Ai?i!t?.Sci. 22, 771. Bodwell, C. E., and McClain. P. 1:. 1971. Proteins. 112 “The Science of Meat and Meat Products” ( J . 1,‘. Price and B. S. Schweigert, cds.), 2nd cd., p. 78. Freeman, San Francisco, California. Body, D. R.. and Gray, G . M. 1967a. The isolation and characterization of phosphatidyl glycerol and its structural isomer from pig lung. Chem. Phys. Lipids 1 , 243. Body, D. R., and Gray, G . M. 1967b. Semi-lyso-bis-phosphatidic acid: An unusual phospholipid isolated from rabbit lung. C/?em.Phys Lipids 1 , 4 2 4 . Body, D. It.. and Shorland, F. B. 1974. The fatty acid composition of thc main phospholipid fractions of the rumen and abomasum tissues of foetal and adult sheep. J. Sci Food Agric. 25, 197. Uody, D. R., Shorland, I:. B., and Gass, J . P. 1966. Foetal and maternal lipids of Romney sheep. 1. The composition of the lipids of the total tissues. Biochim. Biophys. Acra 125, 207. Body, D. R., Shorland, 1:. B., and Czochanska, Z. 1970. Changes in the composition of the rumen and abomasum lipids of sheep from birth to maturity. J. Sci Food Agric. 21, 220. Roekenoogen, H. A. 1941. OLiet?, Vetten Oliezadun 26, 143 (quoted by Hilditch and Williams, 1964). Boelens, M., van der Linde, L. M., dcValois, P. J . , van Dort, H. M., and Takken, 11. J. 1974. Organic sulfur compounds from fatty aldehydes, hydrogen sulfide, thiols and ammonia as flavor constituents. J. A@. Food Chem. 22, 1071. Hrockerhoff, H. 1965. Stereospecific analysis of triglycerides: An analysis of human depot fat. Arch. Biochem. Biophys. 110, 586. Brockerhoff, H. 1967. Stereospecific analysis of triglycerides: An alternative method. J. Lipid Res. 8 , 167. Brown, W. D., Harris, L. S., and Olcott, H. S. 1963. Catalysis of unsaturated lipid oxidation by iron protoporphyrin derivatives. Arch. Biochem. Biophys. 101, 14. Bruner, W. H., Cahill, V. R., Robinson, W. L., and Wilson, R. F. 1958. Performance of barrow and gilt littermate pairs at the Ohio swine evaluation station. J. Anim. Sci. 17, 875. Bunyan, J . , Diplock, A. T., and Grccn, J . 1967. Effccts of vitamin E deficiency on total polyunsaturated fatty acids in ratsand chicks. Br. J. Nurr. 21, 217. Callow, E. H. 1936. “Quality in the Pig Carcass,’’ p. 43. Food Invest. Board Great Britain. Callow, E. H. 1958. Comparative studies of meat. VI. Factors affecting the iodinc number o f f a t from the fatty and muscular tissues of 1amb.J. Agric. Sci 51, 361. Callow, I<. H., and Searle, S. R. 1956. Comparative studies of meat. V. Factors affecting the iodine number of the fat from thc fatty and muscular tissues of cattle. J. Agvic. Sci. 48, 61. Campbell, A. M., and Turkki, P. R. 1967. Lipids of raw and cooked ground beef and pork. J. Food Sci. 32, 143. Carravaggi, C., and Wright, E. 1969. The retention and excretion b y sheep of a-tocopherol after oral or intra-muscular administration. N . Z . J. Agric. Res. 12, 655. Cecil, S. R., and Woodroof, J . C . 1954. Effect of storage temperatures on the aging of country-style hams. Food Techrzol. 8 , 216.

WOF IN MEAT, POULTRY, AND FISH

63

Chang, I., and Watts, B. M. 1949. Antioxidants in hemoglobin catalyzed oxidation of unsaturated fats. Food Technol. 3, 332. Chang, I., and Watts, B. M. 1950. Some effects of salt and moisture o n rancidity in fats. FoodRes. 15, 3 1 3 . Chang, I., and Watts, B. M. 1952. The fatty acid content of meat and poultry before and after cooking. J. Am. Oil Chem. SOC.29, 334. Chang, Pi-Yu, Younathan, M. T., and Watts, B. M. 1961. Lipid oxidation of precooked beef preserved by refrigeration, freezing and irradiation. Food Technol. 15, 168. Cho, 1. C., and Bratzler, L. J. 1970. Effect of sodium nitrite o n flavor of cured pork. J. Food Sci 35,668. Cook, L. J., Scott, T. W., Ferguson, K. A., and McDonald, I. W. 1970. Production of polyunsaturated ruminant body fats. Nature (London) 228, 178. Corliss, G. A. 1968. Phospholipid oxidation in emulsions. Ph.D. Thesis, Michigan State University, East Lansing. Cox, D. F. 1963. Breed and sex effects on the relationship between weight and fatness measured at a constant age in swine. J. Anim. Sci 22, 1091. Craig, H. B., Pearson, A. M., and Webb, N. B. 1962. Fractionation of the component(s) responsible for sex odor/flavor in pork. Food Res. 27, 29. Craig, H . B., Blumer, T. N., Smart, W. W. G., Jr., and Wise, M. B. 1966. Evaluation of hemoglobin, myoglobin and blood oxygen content and organoleptic qualities of beef from steers fed grain on pasture o r cut forage and grain in dry lot. L-Anim. Sci 25, 1128. Cramer, D. A., and Marchello, J. A. 1964. Seasonal and sex patterns in fat composition of growing lambs. J. Anim. Sci 26,683. Cramer, D. A., Barton, R. A., Shorland, F. B., and Czochanska, Z. 1967. A comparison of the effects of white clover (Trifolium repens) and of perennial rye grass (Lolium perenne) o n fat composition and flavour of lamb. J. Agric. Sci 69, 367. Cross, C. K., and Ziegler, P. 1965. A comparison of the volatile fractions from cured and uncured meats. J. Food Sci. 30,610. Cunningham, N. L., Carpenter, Z. L., King, G. T., Butler, 0. D., and Shelton, J. M. 1967. Relationship of linear measurements and certain carcass characteristics to retail value, quality and tenderness of ewe, wether and ram lambs. J Anim. Sci 26,683. Dahl, 0 . 1960. Influence of the basal diet o n the quality of pig fat. 11. Feeding a diet very low in fat. Acta Am‘c. Scand. 10, 33. Dawson, L. E., and Sison, E. C. 1973. Stability and acceptability of phosphate-treated and precooked chicken pieces reheated with microwave energy. J. Food Sci 38, 161. Dawson, R. M. C. 1960. A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75,45. De Gier, J., and Van Deenan, L. L. M. 1961. Some lipid characteristics of red cell membranes of various animal species. Biochim. Biophys. Acta 49, 286. Deuel, H. J., Jr. 1955. “The Lipids-Their Chemistry and Biochemistry,” Vol. 11. Wiley (Interscience), New York. Dimick, P. S., MacNeil, J. H., and Grunden, L. P. 1972. Poultry product quality. Carbonyl composition and organoleptic evaluation of mechanically deboned poultry meat. J. Food Sci 37,544. Dryden, F. D., Marchello, J. A., Figroid, W. C., and Hale, W. H. 1973. Composition changes in bovine subcutaneous lipid as influenced by dietary fat. J. Anim. Sci. 36, 19. Dugan, L. R. 1971. Fats. In “The Science of Meat and Meat Products” (J. F. Price and B. S. Schweigert, eds.), 2nd ed., p. 133. Freeman, San Francisco, California.

64

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Dugan, L. R., Jr. 1961. Development and inhibition of oxidative rancidity in foods. Food Technol. 15, 10. Dunkley, W. L., Ronning, M., Frank, A. A., and Robb, J. 1967. Supplementing rations with tocopherol and ethoxyquin to increase stability of milk. J. Dairy Sci 50,492. Dyer, W. J., and Morton, M. L. 1956. Storage of frozen plaice fillets. J. Fish. Res. Board Can. 13, 129. El-Gharbawi, M. I., and Dugan, L. R., Jr. 1965. Stability of nitrogeneous compounds and lipids during storage of freeze-dried raw beef. J. Food Sci 3 0 , 8 17. Ellis, N. R. 1933. Changes in quantity and composition of fat in hogs fed a peanut ration followed by a corn ration. US.,Dep. Agric., Tech. Bull. 368. Ellis, N. R., and Isbell, H. S. 1926a. Soft pork studies. 11. The influence of the character of the ration upon the composition of body fat of hogs. J. Biol. Chem. 69,219. Ellis, N. R., and Isbell, H . S. 1926b. Soft pork studies. 111. The effect of food fat upon body fat, as shown by the separation of the individual fatty acids of the body fat. J. Biol. Chem. 69,239. Ellis, R., Currie, G. T., Thornton, F. E., Bollinger, N. C., and Gaddis, A. M. 1968. Carbonyls in oxidizing fat. 11. The effect of the prooxidant activity of sodium chloride on pork tissue. J. Food Sci. 33, 5 5 5 . Ellis, R., Gaddis, A. M., Currie, G . T., and Thornton, F. E. 1970. Sodium chloride effect on autoxidation of the lard component of a gel. J. FoodSci 35,52. Ellis, R . , Currie, C. T., and Gaddis, A. M. 1971. Metal ion catalysis of autoxidation in a lard gel. J. A m . Oil Chem. SOC.48, 640. Ellis, R., Kimoto, W. I., Bitman, I., and Edmonson, L. F. 1974. Effect of induced high linoleic acid and tocopherol content on the oxidative stability of rendered veal fat. J. Am. Oil Chem. SOC.5 1 , 4 . Entenman, C., Lorenz, F. W., and Chaikoff, I. L. 1940. The lipid content of blood, liver, and yolk sac of the newly hatched chick and changes that occur in these tissues during the first month of life. J. Biol. Chem. 133, 231. Erdman, A. M., Watts, B. M., and Ellias, L. C. 1954. Smoke flavor and ascorbic acid as preservatives for fatty fish. Food Technol. 8 , 320. Eriksson, C. E., and Vallentin, K. 1973. Thermal activation of peroxidase as a lipid oxidation catalyst. J. Am. Oil Chem. SOC.50, 264. Eriksson, C. E., Olsson, P. A., and Svensson, S. G . 1970. Oxidation of fatty acids by heat treated hemoproteins. Lipids 5, 365. Eriksson, C. E., Olsson, P. A., and Svensson, S. G . 1971. Denatured hemoproteins as catalysts in lipid oxidation. J. Am . Oil Chem. SOC.48,442. Evans, C. D. 1961. Chemical changes accompanying flavor deterioration of vegetable oils. In “Proceedings Flavor Chemistry Symposium,” p. 123. Campbell Soup Company, Camden, New Jersey. Field, R. A. 1974. Mechanically deboned meat. Proc. Mear Ind. Rex Conf., 1974 p. 35. Fields, M. D., and Dunker, C. F. 1952. Quality and nutritive properties of different types of commercially cured hams. I. Curing methods and chemical composition. Food Technol. 6, 329. Fishwick, M. J., and Zmarlicki, S. 1970. Freeze-dried turkey muscle. I. Changes in nitrogenous compounds and lipids of dehydrated turkey during storage. J. Sci Food Agric. 21, 155. Folch, J., Lees, M., and Stanley, G . H. S. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 266,497. Froning, G . W. 1970. Poultry meat sources and their emulsifying characteristics as related to processing variables. Poult. Sci 49, 1625. Gaddis, A. M., Ellis, R., and Currie, G. T. 1961. Carbonyls in oxidizing fat. V. The

WOF IN MEAT, POULTRY, AND FISH

65

composition of neutral volatile monocarbonyl compounds from autoxidized oleate, linoleate, linoleate esters and fats. J. Am Oil Chem SOC.38, 371. Giam, I., and Dugan, L. R., Jr. 1965. The fatty acid composition of free and bound lipids in freeze-dried meats. J. Food Sci 30, 262. Grau, R., and Fleischmann, 0. 1965. Ueber den Einfluss der Verfutterung von Vitamin E an Schweine auf die Haltbarkeit des Fettes und fetthaltige Erzeugnisse. Z. Lebensm. Unters. Forsch. 130, 270. Gray, G. M., and Macfarlane, M. G. 196 1. Composition of phospholipids of rabbit, pigeon and trout muscles and various pig tissues. Biochem J, 81,480. Greene, B. E. 1969. Lipid oxidation and pigment changes in raw beef. J. FoodSci 34, 110. Greene, B. E. 1971. Retardation of oxidative color changes in raw ground beef. J. FoodSci. 36,940. Grunden, L. P., MacNeil, J. H., and Dimick, P. S. 1972. Poultry product quality: Chemical and physical characteristics of mechanically deboned poultry meat. J. Food Sci. 37, 24 7. Gunstone, F. D. 1967. “An Introduction t o the Chemistry and Biochemistry of Fatty Acids and their Glycerides,” 2nd ed. Chapman & Hall, London. Gurr, M. I., and James, A. T. 1971. “Lipid Biochemistry: An Introduction.” Cornell Univ. Press, Ithaca, New York. Hansen, R. P., and Gerson, T. 1967. The isolation and identification of ll-cyclohexylundecanoic acid from sheep perinephric fat. J. Sci Food Agric. 18,225. Hansen, R. P., and Smith, 3. 1966. The occurrence of methyl methoxy stearate isomers in the methyl esters prepared from sheep perinephric fat. Lipids 1, 316. Hansen, R. P., Shorland, F. B., and Cooke, N. J. 1958. Trace constituents of hydrogenated ox perinephric fat. J. Sci. FoodAgric. 9 , 391. Hanson, H. L., Fletcher, L. R., and Lineweaver, H. 1959. Time-temperature tolerance of frozen foods. XVII. Frozen fried chicken. Food Technol. 13,221. Hartman, L., Shorland, F. B., and McDonald, I. R. C. 1954. Occurrence of truns acids in animal fats. Nature (London) 174,185. Hartman, L., Shorland, F. B., and McDonald, I. R. C. 1955. The trans-unsaturated acid contents of fats of ruminants and non-ruminants. Biochem. J. 61,603. Heaton, F. W., and Uri, N. 1961. The aerobic oxidation of unsaturated fatty acids and their esters: Cobalt stearate-catalyzed oxidation of linoleic acid. J. Lipid Rex 2, 152. Hedrick, H. B., Thompson, G. B., and Krause, G . F. 1969. Comparison of feedlot performance and carcass characteristics of half-sib bulls, steers and heifers. J. Anim. Sci. 29, 687. Henriques, V., and Hansen, C. 1901. Vergleichende Untersuchungen cber die chemische Zusammensetzung des tierischen Fettes. Skund. Arch. PhysioL 11, 15 1. Herz, K. O., and Chang, S. S. 1970. Meat flavor. Adv. Food Res. 18, 1. Hilditch, T. P., and Lovern, J. A. 1929. Head and blubber oils of the sperm whale. 11. Investigation of some of the component wax esters and the general structure of the oils. J. SOC.Chem. Ind., London48, 359T. Hilditch, T. P., and Lovern, J. A. 1936. The evolution of natural fats. A general survey. Nature (London) 137,478. Hilditch, T. P., and Williams, P. N. 1964. “The Chemical Constitution of Natural Fats,” 4th ed. Chapman ti Hall, London. Hilditch, T. P., Jones, E. C., and Rhead, A. J. 1934. The body fats of the hen. Biochem. J. 28,786. Hirano, Y., and Olcott, H. S. 1971. Effect of heme compounds on lipid oxidation. J. Am. OilChem. SOC. 48,523. Hodge, J. E. 1967. Origin of flavor in foods. Non-enzymatic browning reactions. In

66

A. M . PEARSON, JANE D. LOVE, AND F. B. SHORLAND

“Symposium on Foods: Chemistry and Physiology of Flavors” (H. W. Schultz, E. A. Day, and L. M. Libbcy, eds.), p. 465. Avi Publ. Co., Westport, Connecticut. Hofstrand, J., and Jacobson, M. 1960. The role of fat in the flavor of lamb and mutton as tested with broths and with depot fats. Food Rex 25, 706. Holman, R. T., and Widmer, C. 1969. Polyunsaturated fatty acids in beef heart mitochondria and derived enzymatically active lipoprotein fractions. J. Biol. Chem. 234, 2269. Horn, L. R., Barker, M . O., Reed, G., and Brin, M. 1974. Studies on peroxidative hemolysis and erythrocyte fatty acids in the rabbit: Effect of PUFA and vitamin E. J. Nutr. 104, 192. Hornstein, I. 1967. Flavor of red meats. In “Symposium of Foods: Chemistry and Physiology of Flavors” (H. W. Schultz, E. A. Day, and L. M. Libbey, eds.), p. 228. Avi Publ. Co., Wcstport, Connecticut. Hornstein, I., and Crowe, P. F. 1960. Meat flavor chemistry: Studies on beef and pork. J. Agric. Food Chem 8,494. Hornstein, I., and Crowe, P. F. 1963. Meat flavor: Lamb. J. Agric. Food Chem. 11, 147. Hornstein, l., and Crowe, P. F. 1964. Meat flavor. A Review. J Gas Chromarogr. 2, 128. Hornstein, I., Crowe, P. F., and Heimberg, M. J. 1961. Fatty acid composition of meat tissue lipids. J. Food Sci. 26, 5 8 1. Hornstein, I., Crowe, P. F., and Hiner, R. L. 1967. Composition of lipids in some beef muscles. J. Food Sci. 32, 650. Hornstein, I., Crowe, P. F., and Sulzbacher, W. L. 1963. Flavor of beef and whale meat. Nature (London) 199, 1252. Hvidsten, H., and Astrup, H. M. 1963. The effect of vitamin E on the keeping quality and flavour of pork. Acta Agric. Scand. 13, 259. Igene, J. 0. 1976. Effects of dietary fats and vitamin E upon the stability of meat in frozen storage. M.S. Thesis, Michigan State Univ., East Lansing, Michigan. Ingold, K. V. 1962. Metal catalysis. In “Symposium o n Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 93. Avi Publ. Co., Westport, Connecticut. 1.U.P.A.C.-1.U.B. Comniission in Biochemical Nomenclature. 1967. The nomenclature of lipids. Biochem. J . 105, 897. Jones, M. E., Koch, I:. C., Heath, A. E., and Munson, P. L. 1949. Isolation of a-monopalmitin from hog pancreas. J. Biol. Chem. 181, 755. Katz, M. A., Dugan, L. R., Jr., and Dawson, L. E. 1966. Fatty acids in neutral lipids and phospholipids from chicken tissues. J. Food Sci. 3 1, 7 17. Kaucher, M., Galbraith, H., Button, V., and Williams, H. H. 1944. The distribution of lipids in animal tissues. Arch. Biochem. 3, 203. Keeney, M. 1962. Sccondary degradation products. In “Symposium o n Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 79. Avi Publ. Co., Westport, Connecticut. Keller, J. D., and Kinsella, J. E. 1973. Phospholipid changes and lipid oxidation during cooking and frozen storage of raw ground beef. J. Food Sci. 38, 1200. Kemp, J . D., and Varncy, W. Y. 1955. Onion flavor in beef. Annu. Livestock Field Day Rep. Univ. Ky. p. 4. Kemp, J. D., McCampbcll, H. C., and Grainger, R. B. 1957. Procedure for sampling and laboratory rendering of ham fat and characteristics of hams and fat during aging. Food Technol. 1I , 32 1. Kendrick, J., and Watts, B. M. 1969. Acceleration and inhibition of lipid oxidation by heme compounds. Lipids 4 , 4 5 4 .

WOF IN MEAT, POULTRY, AND FISH

67

Kennedy, E. P., and Weiss, S. B. 1956. The function of cytidine coenzymes in the biosynthesis of phospholipids. J. BioL Chem. 222, 193. Kesinkel, A., Ayres, J. C., and Synder, H. E. 1964. Determination of oxidative changes in raw meats by the 2-thiobarbituric acid method. Food Technol, 18, 223. Klenk, E., and Gehrmann, G. 1955. uber die Glycerinphosphatide des Rinderherzmuskels und das Vorkommen von cholinhaltigen Acetalphosphatiden. Hoppe Seyler’s Z . Physiol. Chem. 292,110. Koch, D. E., Parr, A. F., and Merkel, R. A. 1968a. Fatty acid composition of inner and outler layers of porcine backfat as affected by energy level, sex and sire. J. Food Sci 33, 176. Koch, D. E., Pearson, A. M., Magee, W. T., Hoefer, J. A., and Schweigert, B. S. 1968b. Effect of diet on the fatty acid composition of pork fat. J. Anim. Sci 27, 360. Koch, R. B. 1962. Dehydrated foods and model systems. In “Symposium on Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 122. Avi Publ. Co., Westport, Connecticut. Kono, T., and Colowick, S. P. 1961. Isolation of skeletal muscle cell membrane and some of its properties. Arch. Biochem. Biophys. 93, 520. Korn, E. D. 1966. Structure of biological membranes. Science 153, 1491. Kornberg, A., and Pricer, W. E., Jr. 1953a. Enzymatic synthesis of the coenzyme A derivatives of long-chain fatty acids. J. Biol. Chem. 204, 329. Kornberg, A., and Pricer, W. E., Jr. 1953b. Enzymatic esterification of a-glycerophosphate by long chain fatty acids. J. Biol. Chem. 204, 345. Kramlich, W. E., and Pearson, A. M. 1958. Preliminary studies on meat flavor. Food Res. 23,567. Kramlich, W. E., Pearson, A. M., and Tauber, F. W. 1973. “Processed Meats.” Avi Publ., Westport, Connecticut. Lands, W. E. M., and Slakey, P. M. 1966. A solved problem of triglyceride analysis. Lipids 1, 295. Lands, W. E. M., Pieringer, R. A., Slakey, P. M., and Zschake, A. 1966. A micromethod for the stereospecific determination of triglyceride structure. Lipids 1, 444. Lea, C. H. 1937. The influence of tissue oxidases on rancidity. Oxidation of the fat of bacon. J. Soc. Chem. Ind., London 56,376T. Lea, C. H. 1957. Deteriorative reactions involving phospholipids and lipoproteins. J. Sci. Food Agric. 8, 1. LeBaron, F. N. 1969. The lipid-protein complexes of myelin. In “Structural and Functional Aspects of Lipoproteins in Living Systems” (E. Tria and A. M. Scanu, eds.), p. 201. Academic Press, New York. Ledward, D. A. 1971. On the nature of cooked meat hemoprotein. J. Food Sci. 36,883. Lee, W. T., and Dawson, L. E. 1973. Chicken lipid changes during cooking in fresh and reused cooking oil. J. Food Sci. 38, 1232. Lee-Shin, T., and Smith, L, M. 1971. Role of the bases and phosphoryl bases of phospholipids in the autoxidation of methyl linoleate emulsions. Lipids 6, 196. Lehninger, A. L. 1970. “Biochemistry.” Worth, New York. Lewis, R. W. 1967. The fatty acid composition of some marine animals from various depths. J. Fish. R e s Board Can. 24, 1101. Lewis, S. E., and Wills, E. D. 1963. Inhibition of the autoxidation of unsaturated fatty acids by hematin proteins. Biochim. Biophys Acta 70, 336. Lineweaver, H., Anderson, J. D., and Hanson, H. L. 1952. Effect of antioxidant on rancidity development in frozen creamed turkey. Food Technol. 6, 1. Link, B. A., Bray, R. W.,Cassens, R. G., and Kauffman, R. G. 1970a. Fatty acid

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

68

composition of bovine subcutaneous adipose tissue lipids during growth. J. Anim. Sci.

30, 722. Link, B. A., Bray, R. W., Cassens, R. G., and Kauffman, R. G . 1970b. Fatty acid composition of bovine skeletal muscle lipids during growth. J. Anim. Sci. 30, 726. Liu, H. 1970. Catalysts of lipid peroxidation in meats. I. Linoleate peroxidation catalyzed by MetMb or Fe (11)-EDTA. J. Food Sci 3 5 , 590. Liu, H. P., and Watts, B. M. 1970. Catalysts of lipid peroxidation in meats. 3. Catalysts of oxidative rancidity in meats. J. Food Sci. 35,596. Love, J. D. 1972. A comparison of myoglobin and nonheme iron as prooxidants in cooked meat and dispersions of phospholipid. Ph.D. Thesis, Michigan State University, East Lansing. Love, J. D., and Pearson, A. M. 1971. Lipid oxidation in meat and meat products-a review. J. Am. Oil Chem. SOC.48,547. Love, J. D., and Pearson, A. M. 1974. Metmyoglobin and non-heme iron as prooxidants in cooked meat. J. Agric. Food Chem. 22, 1032. Love, J. D., and Pearson, A. M. 1976. Metmyoglobin and nonheme iron as prooxidants in egg yolk phospholipid dispersions and cooked meat. J. Agric. Food Chem. 24,494. Lovern, J. A. 1956a. The phospholipids of fish. J. Sci. Food Agric. 7, 729. Lovern, J. A. 1956b. The lipids of fish. 8. The triglycerides and cholesterol esters of haddock flesh. Biochem. J. 63,313. Lundberg, W. 0. 1962. Mechanisms. In “Symposium on Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 31. Avi Publ. Co., Westport, Connecticut. Lundberg, W. O., Barnes, R. H., Clausen, M., and Burr, G. 0. 1944. The deposition and storage of a-tocopherol in abdominal fats. J. Biol. Chem. 153, 265. Mabrouk, A. F., and Dugan, L. R., Jr. 1960. A kinetic study of the autoxidation of methyl linoleate and linoleic acid emulsions in the presence of sodium chloride. J. Am. Oil Chem. SOC. 37,486. Macfarlane, M. G., and Gray, G . M. 1957. Composition of cardiolipin. Biochem J. 67, 25P. Macfarlane, M. G., Gray, G. M., and Wheeldon, L. W. 1960. Fatty acids of phospholipids from mitochondria and microsomes of rat liver, Biochem. J. 74,43P. McKnight, R. C., and Hunter, F. E. 1965. Effects of inorganic iron on the thiobarbituric acid method for determination of lipid peroxides. Biochim Biophys. Acta 98, 643. MacLean, J., and Castell, C. H. 1964. Rancidity in lean fish muscle. I. A proposed accelerated cooper-catalyzed method for evaluating the tendency of fish muscle to become rancid. J. Can. Fish. Res. Board 21, 1345. Mahler, H. R., and Cordes, E. H. 1966. “Biological Chemistry,” p. 581. Harper, New York. Maier, V. P., and Tappel, A. L. 1959. Rate studies of unsaturated fatty acid oxidation catalyzed by hematin compounds. J. Am. Oil Chem. SOC.36,8. Marchello, J. A., Cramer, D. A., and Miller, L. J. 1967. Effects of ambient temperature on certain ovine fat characteristics. J. Anim. Sci. 26, 294. Marco, G . J., Machlin, L. J., Emery, E., and Gordon, R. S . 1961. Dietary effects of fats upon fatty acid composition of the mitochondria. Arch. Biochem. Biophys. 94, 115. Marcuse, R., and Fredriksson, P.-0. 1971. Fat oxidation at low oxygen pressure. III. Kinetic studies on linoleic acid oxidation in emulsions in the presence of added metal salts. J. Am. Oil Chem. SOC.48,448. Margolis, S. 1969. Structure of very low and low density ljpoproteins. In “Structural and Functional Aspects of Lipoproteins in Living Systems” (E. Tria and A. M. Scanu, eds.), p. 369. Academic Press, New York.

WOF IN MEAT, POULTRY, AND FISH

69

Martinez, F., and Labuza, T. P. 1968. Rate of deterioration of freeze-dried salmon as a function of relative humidity. J. Food Sci. 33, 241. Masaro, E. J. 1968. “Physiological Chemistry of Lipids in Mammals.” Saunders, Philadelphia, Pennsylvania. Maynard, L. A., and Loosli, J. K. 1962. “Animal Nutrition,” 5th ed. McGraw-HiU, New York. Mecchi, E. P., Poole, M. F., Bechman, G. A., Hamachi, M., and Klose, A. A. 1956. The role of tocopherol content in the comparative stability of chicken and turkey fat. Poult. Sci. 35, 1238. Mendell, L. B., and Anderson, W. E. 1930. Some relations of diet to fat deposition in the body. Yale J. Biol. Med. 3, 107. Merk, W. 1959. “Uber die Wirkung einer Tocopherol-Anreichung im Kraftfutter von Milchkuhen auf Menge und Beschaffenheit der erzeugen Milch,” Prem. Arb. 2891. ETH, Ziirich (quoted by Astrup, 1973). Mitchell, P. H. 1946. “A Textbook of Biochemistry.” McCraw-Hill, New York. Moerck, K. E., and Ball, H. R., Jr. 1974. Lipid oxidation in mechanically deboned chicken meat. J. Food Sci. 39, 876. Morice, I. M., and Shorland, F. B. 1973. The composition of surface waxes of apple fruits and changes during storage. J. Sci Food Agric. 24, 13 3 1, Moskovits, V. G., and Kielsmeier, E. W. 1960. An effect of iron and sodium chloride on flavor in sausage. Proc. Res. Conf: A m Meat Inst. Found. 12th Circ. 61, 121. Moulton, C. R. 1923. Age and chemical development in mammals. J. Biol. Chem. 57, 79. Nakamura, Y., and Nishida, T. 1971. Effect of hemoglobin concentration on the oxidation of linoleic acid. J. Lipid Res. 12, 149. Newbold, R. P., Tume, R. K., and Horgan, D. J. 1973. Effect of feeding a protected safflower oil supplement on the composition and properties of the sarcoplasmic reticulum and on postmortem changes in bovine skeletal muscle. J. Food Sci. 38, 821. Noble, R. C., Steele, W., and Moore, J. J. 1972. The metabolism of linoleic acid by the young lamb. Br. J. Nutr. 27,503. O’Brien, P. J. 1969. Intracellular mechanisms for the decomposition of a lipid peroxide. I. Decomposition of a lipid peroxide by metal ions, heme compounds and nucelophiles. Can. J. Biochem. 47,485. Ogilvie, B. M., McClymont, G. L., and Shorland, F. B. 1961. The effect of duodenal administration of highly unsaturated fatty acids upon the composition of ruminant depot fat. Nature (Londonj 190, 725. Olcott, H. S. 1962. Marine products. In “Symposium on Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 173. Avi Publ. CO., Westport, Connecticut. Olson, D. G., and Rust, R. E. 1973. Oxidative rancidity in dry-cured hams: Effect of low pro-oxidant and antioxidant salt formulations. J. Food Sci 38, 25 1. Orme, L. E., Pearson, A. M., Bratzler, L. J., and Magee, W. T. 1958. Specific gravity as an objective measure of marbling. J. Anim. Sci 17, 693. Ostovar, K., MacNeil, J. H., and O’Donnell, K. 1971. Poultry product quality. 5. Microbiological evaluation of mechanically deboned poultry meat. J. Food Sci 36, 1005. Pangborn, M. C. 1947. The composition of cardiolipin. J. Biol. Chem. 168, 351. Park, R. J., Corbett, J. L., and Furnival, E. P. 1972a. Flavour differences in meat from lambs grazed on lucerne (Medicago sativa) or phalaris (Phalaris tuberosa) pastures. J. Agric. Sci 78,41. Park, R. J., Spurway, R. A., and Wheeler, 3. L. 1972b. Flavour differences in meat from sheep grazed on pasture or winter forage crops. J. Agric. Sci 78, 53.

70

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Patterson, R. L. S. 1968. Sa-androst-16-ene-3-0ne: Compound responsible for taint in boar fat. J. Sci Food Agric. 19, 31. Patterson, R. L. S. 1969. Boar taint: Its chemical nature and estimation. In “Symposium on Meat Production from Entire Male Animals” (D. N. Rhodes, ed.), p. 247. Churchill, London. Pearson, A. M. 1949. The influence of the rate of freezing and length of freezer-storage upon the quality of beef. Ph.D. Thesis, Cornell University, Ithaca, New York. Pearson, A. M. 1966. Desirability of beef-its characteristics and their measurement. J. Anim. Sci 25, 843. Pearson, A. M., Bratzler, L, J,, and Magee, W. T. 1958a. Some simple cut indices for predicting carcass traits of swine. I. Cut-out and loin lean area. J. h i m . Sci. 17, 20. Pearson, A. M., Bratzler, L. J., and Magee, W. T. 1958b. Some simple cut indices for predicting carcass traits of swine. 11. Supplementary measures of leanness. J. Anim. Sci. 17, 27. Peters, J. A., and Smith, L. M. 1964. The composition of the liver lipids of sheep and the effect of early sporidesmin poisoning. Biochem. J. 92, 379. Pippen, E. L., Campbell, A. A,, and Streeter, J. V. 1954. Flavor studies. Origin of chicken flavor. J. Agric. Food Chem. 2, 364. Pokorny, J., and Janick, G. 1971. The effect of haem derivatives on the oxidation of lipids mixed with protein. Nahrung 15, 389. Pratt, D. E. 1972. Water soluble antioxidant activity in soybeans. J. Food Sci. 37, 322. Pratt, D. E., and Watts, B. M. 1964. Antioxidant activity of vegetable extracts. I. Flavone aglycones. J. Food Sci. 29, 27. Rapport, M. M., and Alonzo, N. 1955. Identification of phosphatidal choline as the major constituent of beef heart lecithin. J. Biol. Chem. 217, 199. Rapport, M. M., Lerner, B., Alonzo, N., and Franzel, R. E. 1957. The structure of plasmalogens. 11. Crystalline lysophosphatidal ethanolamine (acetal phospholipide). J. Biol. Chem. 225, 859. Reynolds, T. M. 1963. Chemistry of nonenzymatic browning. I. The reaction between aldoses and amines. Adv. Food Rex 12, 1. Reynolds, T. M. 1965. Chemistry of nonenzymatic browning 11. Adv. Food Rex 14, 167. Roberts, W. M. 1965. Protection of milk quality through management practices. In “Symposium on Food Quality-Effects of Production Practices and Processing” (G. W. Irving, Jr. and S. R. Hoover, eds.), Publ. No. 77, p. 153. Am. Assoc. Adv. Sci., Washington, D. C. Robinson, M. E. 1924. Haemoglobin and methaemoglobin as oxidative catalysts. Biochem. J. 18, 255. Rothfield, L. and Finkelstein, A. 1968. Membrane biochemistry. Annu. Rev. Biochem. 37, 463. Roubal, W. T. 1967. Oxidative deterioration of flesh lipids of pacific cod (Gadus macrocephalus).J. Am. Oil Chem. Soc. 44, 325. San Pietro, A. 1965. “Symposium on Non-Heme Iron Proteins.” Antioch Press, Yellow Springs, Ohio. Sato, K., and Hegarty, G. R. 1971. Warmed-over flavor in cooked meats. J. Food Sci 36, 1098. Sato, K., Hegarty, G. R., and Herring, H. K. 1973. The inhibition of warmed-over flavor in cooked meats. J. Food Sci. 38, 398. Satterlee, L. D., Froning, G. W., and Janky, D. M. 1971. Influence of skin content on the composition of mechanically deboned poultry meat. J. Food Sci. 36, 979.

WOF IN MEAT, POULTRY, AND FISH

71

Schogt, J. C. M., Haverkamp-Begemann, P., and Koster, J. 1960. Nonphosphatide aldehydogenic lipids in milk fat, beef tallow and ox heart. J. Lipid Res. 1, 446. Schultz, H. W., Day, E. A., and Sinnhuber, R. O., eds. 1962. “Symposium on Foods: Lipids and their oxidation.” Avi Publ., Westport, Connecticut. Scott, T. W., Cook, L. J., Ferguson, K. A., McDonald, I. W., Buchanan, R. A., and Hills, G. L. 1970. Production of poly-unsaturated milk fat in domestic ruminants. Aust. J. Sci 32, 291. Scott, T. W., Cook, L. J., and Mills, S. C. 1971. Protection of dietary polyunsaturated fatty acids against microbial hydrogenation in ruminants. J. A m . Oil Chem. SOC.48, 358. Shorland, F. B. 1950. Effect of dietary fat on the composition of the depot fats of animals. Nature (London) 165,766. Shorland, F. B. 1952. Evolution of animal fats. Nature (London) 170, 924. Shorland, F. B. 1955. Formation of animal fats. Prog. Chem. Fats Orher Lipids 275. Shorland, F. B. 1961. Acetone-soluble lipids of grasses and other forage plants. 11. General observations on the properties of the lipids with special reference to the yield of fatty acids. J. Sci. Food Agric. 12, 39. Shorland, F. B. 1962. The comparative aspects of fatty acid occurrence and distribution. Comp. Biochem. 3, 1. Shorland, F. B., and Czochanska, Z. 1970. The fatty acid composition of the depot fats of hens fed on coconut meal: Differences in fat metabolism between birds and mammals. Aust. J. Sci 32, 330. Shorland, F. B., and de la Mare, P. B. D. 1945. Studies on the component fatty acids of the back fat with special reference to carcass quality. The relation between growth rate and chemical composition of pig depot fat. J. Agric. Sci. 35, 39. Shorland, F. B., and Stannard, D. J . 1972. (Stannard, D. J. 1975. Mitochondrial fatty acid synthesis. Unpublished Ph.D. Thesis, Victoria Univ. of Wellington, New Zealand). Shorland, F. B., Hansen, R. P., and Hogan, K. J. 1944. Studies on the fats of the bacon pig with reference to carcass quality. I. Iodine value of the back fat under different conditions of feeding. Emp. J. Exp. Agric. 12, 103. Shorland, F. B., Weenink, R. O., and Johns, A. T. 1955. Effect of the rumen on dietary fat. Nature (London) 175, 1129. Shorland, F. B., Weenink, R. O., Johns, A. T., and McDonald, I. R. C. 1957. The effect of sheep rumen contents on unsaturated fatty acids. Biochem. J. 67, 328. Shorland, F. B., Barton, R. A., and Rae, A. L. 1962. Variations in the iodine value of fats from the fatty, muscular and bone tissues of mature Romney ewes. J. Agric. Sci. 59, 1. Shorland, F. B., Body, D. R., and Gass, J. P. 1966. The foetal and maternal lipids of Romney sheep. 11. The fatty acid composition of the lipids from the total tissues. Biochim. Biophys. Acta 125, 217. Shorland, F. B., Czochanska, Z., Moy, M., Barton, R. A., and Rae, A. L. 1970. Influence of pasture species on the flavour, odour and keeping quality of lamb and mutton. J. Sci. Food Agric. 2 I , 1. Shribney, M., and Kennedy, E. P. 1958. The enzymic synthesis of sphingomyelin. J. Biol. Chem. 233,1315. Simon, S . , Ellis, D. E., McDonald, D. B., Miller, D. G., Waldman, R. C., and Westerberg, D. 1973. Influence of nitrite and nitrate curing ingredients on quality of packaged frankfurters. J. Food Sci 38, 919. Snyder, F. 1969. The biochemistry of lipids containing ether bonds. Prog. Chem Fats Ofher Lipids 10, Part 3, 287. Steinberg, G., Slaton, W. H., Jr., Howton, D. R., and Meade, J. F. 1956. Metabolism of

72

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

essential fatty acids. IV. Incorporation of linoleate into arachidonic acid. J. Biol. Chem. 220, 257. Steinberg, G . , Slaton, W. H., Jr., Howton, D. R., and Meade, I. F. 1957. Metabolism of essential fatty acids. V. Metabolic pathway of linolenic acid. J. Biol. Chem. 224, 841. Stenhagen, S., and Svennerholm, L. 1965. Fatty acid composition of human brain sphingomyelins: Normal variation with age and changes during myelin disorders. Lipid Res. 6, 146. Stewart, M. R., Hutchins, €3. K., Zipser, M. W., and Watts, B. M. 1965. Enzymatic reduction of metmyoglobin by ground beef. J. Food Sci. 30,487. Stoffel, W. 1971. Sphingolipids. Annu. Rev. Biochem. 40, 57. Svennerholm, E., Stallbert-Stenhagen, S., and Svennerholm, L. 1966. Fatty acid composition of sphingomyelins in blood, spleen, placenta, liver, lung and kidney. Biochim. Biophys. Acta 125, 60. Sweet, C. W. 1973. Activity of antioxidants in fresh fish. J. FoodSci. 38, 1260. Tappel, A. L. 1952. Linoleate oxidation catalyzed by hog muscle and adipose tissue extract. Food Res. 17, 550. Tappel, A. L. 1962. Heme compounds and lipoxidase as biocatalysts. In “Symposium o n Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 122. Avi Publ., Westport, Connecticut. Tappel, A. L., Brown, W. D., Zalkin, H., and Maier, V. P. 1961. Unsaturated lipid peroxidation catalyzed by hematin compounds and its inhibition by vitamin E. J. Am. Oil Chem. SOC. 3 8 , 5 . Tarladgis, B. G. 1961. An hypothesis for the mechanism of the heme-catalyzed lipid oxidation in animal tissues. J. Am. Oil Chem. SOC.36, 479. T a n , H. L. A., and Cooke, N. E. 1949. Control of rancidity in fish flesh. 3. Carbonylenediols as antioxidants. J. Fish Res. Board Can. 7, 522. Terroine, E. F. 1920. Contribution h la connaissance de la physiologie des substances grasses e t lipoidiques. Ann. Sci. Nat., 2001.Biol. Anim. [ 101 4 5. Thomas, B. H., Culbertson, C. C., Beard, F., Shulz, J. A., and Lowe, B. 1935. Effect of oils of different iodine numbers upon the quality and palatability of beef. Proc. Am. SOC. Anim.Prod. p. 286. Thompson, R. H., Jr., Pearson, A. M., and Banks, K. A. 1972. Identification of some C,g -A16 steroids contributing to sex odor in p0rk.J. Agric. Food Chem. 20, 185. Tims, M. J., and Watts, B. M. 1958. Protection of cooked meats with phosphates. Food Technol. 12, 240. Trams, E. G., and LPuter, C. J. 1962. On the isolation and characterization of gangliosides. Biochim. Biophys. Acta 60, 350. Trowbridge, P. F., and Moulton, C. R. 1909. Composition of the fat of beef animals on different planes of nutrition. J. Ind. Eng. Chem. 1, 761. Uri, N. 1956. Metal ion catalysis and polarity of environment in the aerobic oxidation of unsaturated fatty acids. Nature (London] 177, 1177. Van Deenen, L. L. M. 1966. Phosphotipids and biomembranes. Prog. Clzem. Fats Other Lipids 8 , 1. Vander Wal, R. I. 1960. Calculation of the distribution of the saturated and unsaturated acyl groups in fats from pancreatic lipase data. J. Am. Oil Chem. Soc. 37, 18. Vander Wal, R. J. 1964. Triglyceride structure. Adu. Lipid Res. 2, 1. Wasserman, A. E., and Talley, F. 1968. Organoleptic identification of roasted beef, veal, lamb and pork as affected by fat. J. Food Sci. 33, 219. Wasserman, A. E., and Talley, F. 1972. The effect of sodium nitrite on the flavor of frankfurters. J. Food Sci. 37, 536.

WOF IN MEAT, POULTRY, AND FISH

73

Watts, B. M. 1950. Polyphosphates as synergistic antioxidants. J. Am, Oil Chem. Soc. 27, 48. Watts, B. M. 1954. Oxidative rancidity and discoloration in meat. Adv. Food Res. 5 , 1. Watts, B. M. 1962. Meat products. In “Symposium on Foods: Lipids and their Oxidation” (H. W. Schultz, E. A. Day, and R. 0. Sinnhuber, eds.), p. 202. Avi Publ. Co., Westport, Connecticut. Watts, B. M. 1963. Biochemical aspects of meat preservation. Proc. Int. Congr. Biochem. Sth, 1961 p. 223. Watts, B. M., Cunha, T. J., and Major, R. 1946. Effect of feeding and injecting hogs with tocopherols on the susceptibility of pork fat to rancidity. Oil Soap 23, 254. Webb, J. E., Bronson, C. C., and Yates, J. D. 1972. Effects of feeding antioxidants on rancidity development in precooked, frozen broiler parts. Poult. Sci. 51, 1601. Webb, J. E., Bronson, C. C., and Yates, J. D. 1973. Effects of feeding fish meal and tocopherol on flavor of precooked, frozen turkey meat. Poult. Sci 52, 1029. Webb, J. E., Bronson, C. C., and Yates, J. D. 1974. Effects of dietary fat and dl-a-tocopheryl on stability characteristics of precooked frozen broiler parts. J. Food Sci. 39, 133. Webb, R. W., Marion, W. W., and Hayse, P. L. 1972. Effects of tocopherol supplementation on the quality of precooked and mechanically deboned turkey meat. J. Food Sci. 37, 853. Weenink, R. 0. 1959. A note o n the acetone-soluble lipids of forage grasses and clovers. N.Z. J. Sci. 2, 273. Weenink, R . 0. 1961. Acetone-soluble lipids of grasses and other pasture species. I. Galactolipids of red clover (Trifolium pratense) leaves. J. Sci. Food Agric. 12, 34. Weiss, S. B., Smith, S. W., and Kennedy, E. P. 1956. Net synthesis of lecithin in an isolated enzyme system. Nature (London) 178, 594. Williams, H. H., Galbraith, H., Kaucher, M., and Macy, I. G. 1945. The influence of age and diet on the lipid composition of the rat. J. Biol. Chem. 161,463. Williams, L. D., and Pearson, A. M. 1965. Unsaponifiable fraction of pork fat as related to boar odor. J. Agric. Food Chem. 13, 573. Wills, E. D. 1965. Mechanism of lipid peroxide formation in tissues. Role of metals and haematin proteins in the catalysis of thc oxidation of unsaturated fatty acids. Biochim. Biophys Acta 98, 238. Wills. E. D. 1966. Mechanism of lipid peroxide formation in animal tissues. Biochem. J. 99, 667. Wills, E. D. 1969. Lipid peroxide formation in microsomes. Biochem. J. 113, 315. Wilson, B. R. 1974. Effect of total lipids and phospholipids o n warmed-over flavor measured by TBA analysis in muscle from several species. M.S. Thesis, Michigan State University, East Lansing. Wilson, B. R., Pearson, A. M., and Shorland, F. B. 1976. Effect of total lipds and phospholipids on warm’ed-over flavor in red and white muscles from several species as measured by TBA analysis. J. Agric. Food Chem. 2 4 . 7 . Yamauchi, K . 1972a. Effect of heat treatment on the development of oxidative rancidity in meat and its isolated tissue fraction. Bull. Fac. Agric., Miyazaki Univ. 19, 147. Yamauchi, K . 1972b. Antioxidant in over-heated meat. Miyuzaki Daiguku Nogakubu, Kenkyu Hokoku 19,402. Younathan, M. T., and Watts, B. M. 1959. Relationship of meat pigments to lipid oxidation. Food Res. 24, 728. Younathan, M. T., and Watts, B. M. 1960. Oxidation of tissue lipids in cooked pork. Food Res. 25,538.

74

A. M. PEARSON, JANE D. LOVE, AND F. B. SHORLAND

Yu, T. C., Sinnhuber, R. O., and Crawford, D. L. 1973. Effect of packaging on shelf life of frozen silver salmon steaks. J. Food Sci. 38, 1197. Zilversmit, D. B. 1969. Chylomicrons. In “Structural and Functional Aspects of Lipoproteins in Living Systems” (E. Tria and A. M. Scanu, eds.), p. 329. Academic Press, New York. Zipser, M. W., and Watts, B. M. 1961a. Oxidative rancidity in cooked mullet. Food Technol.

15,318. Zipser, M. W., and Watts, B. M. 1961b. Lipid oxidation in heat-sterilized beef. Food Technol. 15,445. Zipser, M. W., Dupont, J . , and Watts, B. M. 1962. Extraction of lipids from oxidizing mullet. J. Food Sci. 27, 135. Zipser, M. W., Kwon, T. W., and Watts, B. M. 1964. Oxidative changes in cured and uncured frozen cooked pork. J. Agric. Food Chem. 12, 1U5.

MATH EMATICAL METHODS FOR EST1MATING PROPER THERMAL PROCESSES AND THEIR COMPUTER IMPLEMENTATION* KAN-ICHI HAYAKAWA Food Science Department. Cook College Rutgers. The State University of New Jersey New Brunswick. New Jersey

I . Introduction

.

....................................................

................... ...........................................

11 Basic Principles for Determining Proper Heat Processes

. .

A Basic DataRequired B Basic Principles ............................................... 111 Published Procedures for Determining Proper Heat Processes ............... A . Group I Procedures B Group I1 Procedures ........................................... IV Computerized Estimation of Heat Processes ............................ A Programs for Estimating Parametric Values .......................... B Programs for Estimating Heat Processes without Manual Calculations V . ResearchNeeds .................................................. A . Correction for Sterilizing Effect of Heating during Initial Coming-Up Time . . B Influence of Statistical Variations in Experimental Parameters on Sterilizing Effect ...................................................... C . Heat Processing of Liquid or Semisolid Food ........................ VI . Nomenclature ................................................... Appendix A: Computer Programming Terminology ...................... Appendix B: Computer Programs .................................... References .....................................................

.

. . . . .

............................................

......

76 76 76 82 83 84 88

91 91 97 104 104

105 106 106 108 110 139

*A paper of the Journal Series. New Jersey Agricultural Experiment Station. Rutgers. The State University of New Jersey. Food Science Department. Cook College. New Brunswick. New Jersey .

75

KAN-ICHI HAYAKAWA

16

I. INTRODUCTION Several procedures have been employed or investigated for the sterilization of food products, including chemical treatment, heat, ionization radiation, and microwave sterilizations. Among these procedures, heat sterilization has been widely used because of its reliability and economic feasibility. In the present paper, published mathematical procedures for determining safe heat sterilization are reviewed for their overall characteristics and limitations. Most available procedures require elaborate computations through the use of several experimental parameters. To eliminate or to minimize manual computations for the mathematical determination, several computer programs have been reported in published articles. The computational characteristics of these programs will be examined, together with a program package recently developed by the present author.

II.

BASIC PRINCIPLES FOR DETERMINING PROPER HEAT PROCESSES

Principles for determining safe heat processes have been presented in detail in several textbooks (Ball and Olson, 1957; Charm, 1971; Stumbo, 1973). Basic principles for this determination will be reviewed briefly here in order to facilitate our discussion, since the terminology and symbols used in these textbooks are not uniform.

A. BASIC DATA REQUIRED In order to determine safe heat processes, the following two groups of data are required: (1) Thermal death time characteristics of target microorganisms and/or other target factors in the food to be sterilized. (2) Temperature response characteristics of the food to be sterilized. The microbial and engineering principles which have been used for unifying these data will be discussed first. 1. Thermal Death Time Characteristics Target microorganisms for heat processing are those of one species or one type having all the foIIowing characteristics.

1. They live in the food to be sterilized. 2. They are harmful to humans if surviving microorganisms or their toxin or reactive factors are consumed, or they are harmful to food quality if they are not inactivated or modified.

ESTIMATION OF THERMAL PROCESS

I1

3. They grow or react at the normal storage temperature of heat-processed food. 4. They have the strongest thermal resistance among the microbial species or reactive factors which have all the above characteristics. The conditions for postproduction storage of the food product should be considered as well as the poisonous characteristics of the microorganisms or reactive factors. For example, when food is stored at temperatures below 113"F, there will most likely be no growth of obligate thermophilic bacteria. In this case, it is not required to inactivate these thermophilic spores completely, even though their thermal resistance is frequently strongest among the species in the food. On the other hand, when the food product is stored in tropical zones, it might be subjected to temperatures higher than 113°F. In this case, the thermophilic bacterial spores should be inactivated if they are harmful to man or to food quality. Many factors influence the thermal resistance of microorganisms. Some of them are: age and form of the microorganisms, concentration of the microorganisms, microbial species, chemical composition of the microbial media, pH of the microbial media, heating time and temperature, and water activity of the media. Among these, the pH of the microbial media is one of the most important factors insofar as the heat processing of food is concerned, since the pH of food is not greatly adjustable in most cases. To sterilize food having a pH of about 4.5 and higher, Clostridium botulinum types A and B spores have been widely selected as target microorganisms. Molds, yeast, and bacteria, which tolerate acidity, are target microorganisms for the sterilization of food having a pH value lower than 4.5. For example, Bacillus coagulans spores and Saccharomyces cerevisiae yeasts are target microorganisms for heat processing of tomato and of mandarin orange segments, respectively. Thermal death time characteristics of microorganisms are represented by a survivor curve and a thermal death time (TDT) curve. The survivor curve is obtained by plotting common logarithms of survivor concentrations against heating times at a constant temperature (Fig. 1). The curve is represented by Eq.

Because of the exponential characteristics of a survivor curve, there is no absolutely zero concentration of survivors no matter how long the microbial suspension is heated. Therefore, the following specific concentration of survivors has been selected to define a thermal death time at any constant temperature: *All symbols used are defined in the Nomenclature section.

KAN-ICHI HAYAKAWA

78

I

I

-D+ I

HEATING TIME (MIN )

FIG. 1. Survivor of a thermally vulnerable factor. Reproduced from Food Technology Vol. 23, No. 8, pp. 104-108 (1969). Copyright by the Institute of Food Technologists. The reproduction was approved by this Institute.

the survival concentration at which the probability of survivors in the medium is so low that there is no toxic effect to humans when it is consumed or there is no harmful effect to the quality of the medium. This probability must be extremely small if the microorganisms are highly toxic. Stumbo (1949) proposed that the concentration of surviving Clostridium botulirium spores should be reduced to lo-'* X co. Since the reduction factor, lo-'', is based on the production statistics of canned food in the late 1940's, this factor should be revised if his assumption is valid because the production of canned food has greatly increased since that time. The isothermal heating time, which is required to reduce survivor concentration to 10- X co , may be easily estimated by Eq. (2).

I f R = 1,wehave

Equations ( 2 ) and (2a) are for calculating thermal death time, 7,from a D value. Moats et al. (1971) observed that the survivor curves for each of several bacterial species used for their investigation had complex configurations and

ESTIMATION OF THERMAL PROCESS

19

were not approximated by one initial curvilinear segment and one subsequent straight-line segment as shown in Fig. 1. A close examination of the survivor curves which they presented reveals that each curve could be approximated by one initial curvilinear segment and two or more straight-line segments. The thermal death times of these bacterial species may be easily estimated from the last line segments by calculations similar to those stated above. A thermal death time (TDT) curve for microorganisms is obtained by plotting common logarithms of TDT against temperatures and is mathematically represented by Eq. (3).

This equation is used for calculating TDT's at various temperatures provided two experimental constants are given: TDT, T,, at a reference temperature, T,; and slope index, z , of a TDT curve. For heat processing of low acid or nonacid food, 250" F has been used as the reference temperature, T,.. Several researchers (Deidoerfer and Humphrey, 1959; Simpson and Williams, 1974) have used standard equations for chemical reaction kinetics in their estimation of heat processes. These equations are reaction rate and Arrhenius equations. By applying these equations to data obtained from thermal inactivation tests, two parametric values-reaction rate constants and an activation energy-are determined and are used for the process estimation. The reaction rate constants, k , are obtained from the slope of the isothermal survivor curve, and the activation energy is obtained from the slope of a curve which is determined by plotting the logarithms of k values against the reciprocal of the absolute temperatures. There is the following equivalence between D and z values and the reaction kinetic parameters:

k

=

AE

=

In IOID (In 10) ' R . T,T/z

where T, and Tare expressed as absolute temperatures. It is clear from the above equations that a z value is dependent on temperature if AE is independent of temperature. The use of the parametric values shows an excellent way of applying reaction kinetics to the evaluation of heat processes. However, the parametric values do not have true reaction kinetic significance, since they are usually obtained by assuming that the process of thermal inactivation of microorganisms is a unistep first-order chemical reaction. This assumption is probably not the correct one, since an inactivation process consists of much more complicated processes, as observed by many researchers (Moats er al., 1971). Therefore, the estimated reaction rate constants and the activation energy are considered to be semi-

80

KAN-ICHI HAYAKAWA

empirical parameters. It has been observed that estimated activation energies for the thermal inactivation of microorganisms or nutrients are less dependent on temperature when compared with the temperature dependency of corresponding z values. This dependency is examined mathematically by assuming that changes in the slope of a thermal death time or Arrhenius curve are caused by errors in D or k values. According to this examination, we have Change in slope

change i n slope of

Arrhenius curve where T, and Tare expressed as absolute temperatures. It is clear from the above equation that the slope of an Arrhenius curve is more strongly influenced by errors in k values when it is compared with the slope of a thermal death time curve. Therefore, errors in D values are not responsible for the observed temperature dependency of z values. This temperature dependency might be intrinsically related to the inactivation of thermally vulnerable factors. According to the present author’s calculations, results obtained by using the values of reaction kinetic parameters are almost identical to those obtained by using D and z values. I t is also noted that the application of the parametric values requires more computation than the process of estimation through the use of the D and z values. As stated above, there are initial curvilinear portions in most isothermal inactivation curves (survivor curves). Therefore, the simple application of the reaction kinetic equations does not provide means for accommodating these initial portions in process evaluation. In the following discussion, D and z values are used as parameters for therinal process evaluation, since these empirical constants have been widely and successfully employed by many worlcers.

2. Temperature Response Characteristics of Food Many formulas are available for determining temperature response characteristics of food to be sterilized. These formulas are classified into theoretical formulas (Ball and Olson, 1957; Charm, 1971; Gillespy, 1951; Hayakawa, 1971 ; Hayakawa and Ball, 1968, 1969, 1971; Hicks, 1951; Ikegami, 1974; Stevens ct al., 1973; Stumbo, 1973) and empirical formulas (Ball, 1923; Ball and Olson, 1957; Griffin et aZ., 1971; Hayakawa, 1970,1971; Stumbo, 1973). The theoretical formulas are analytical or numerical solutions of theoretical heat balance equations, and the empirical formulas are obtained by mathematically correlating experimental temperature data. The general characteristics of the theoretical and empirical formulas have been discussed in detail by the present

ESTIMATION OF THERMAL PROCESS

81

author (1972). Among the formulas available, Ball’s empirical formulas (1923) have been widely utilized for determining these characteristics. One heat process is divided into two phases: heating and cooling. Heating curves have been widely used for representing the temperature response characteristics of food in heating phases, and cooling curves for representing those characteristics in cooling phases. In most cases, each of these two curves is approximated by two or more segments. Since Ball’s formula for calculating an initial curvilinear portion of a curve is not generally applicable, the present author (1970, 1971) obtained the following formulas: Curvilinear portion o f heating curve:

(9

(ii)

0.045 < j,, < 0.4 T1 - T = (TI - T,) . 10-(‘/E)”o /3 = (0.3 - log^, jjJ0.3 E = 0.3&(0.3 - logloj h ) 0.4 T1 - T

05

t

5

tl

(4a) (4W (4c)

Ij, < 1 =

(T, - Tfl)COt(B1+~/4)

O I t < I ,

(54

Curvilinear portion of cooling curve: The formulas for the cooling curve can be obtained by replacing the symbols in the above equations as follows:

For example, Eq (5b) is transformed as follows:

Formulas for the linear portion of the heating or cooling curve have been given in published articles (Ball, 1923; Ball and Olson, 1957; Stumbo, 1973). These formulas are given below:

82

KAN-ICHI HAYAKAWA

Linear portion of heating cuwe:

Linear portion of cooling cuwe:

The length of the curvilinear portion, following relationships:

ti,

may be estimated by using the

tl/slope index 'v 0.3 when 0.045 5 intercept coefficient 5 0.4 tl/slope index 'v 0.9 X ( 1 - intercept coefficient) when 0.4 < intercept coefficient < 1 11 = 0 when intercept coefficient = I fllslope index 'v 0.7 X (intercept coefficient - 1) when 1 < intercept coefficient < 3.0

(9a) (9b) (9c) (94

According to the present author's limited examinations, Eqs. (6a), (6b), and (9d) give a reliable estimation of food temperatures when a j h or j , value is as large as 4.0.

B. BASIC PRINCIPLES Estimation of a parametric value called the sterilizing value, F p , is a basis for the mathematical determination of proper heat processes. This value is calculated by Eq. (10):

F,

=

lofe

L dr

(10)

where

In Eq. (loa), food temperatures, T, during a heating process are determined experimentally, empirically, or theoretically. An Fp value represents the duration of a hypothetical heat process applied to food. At the zero time of this hypothetical process, the food temperature

ESTIMATION OF THERMAL PROCESS

83

increases instantaneously to T,"F and is maintained at T,"F during the entire process. The food temperature is then instantaneously lowered t o a sublethal temperature at Fp time units from the beginning of the hypothetical process. It is important to mention that the sterilization effect of the hypothetical process is identical to that of a real process. For the evaluation of a heat process, an Fp value, which is calculated by Eq. (lo), is compared with a TDT of target microorganisms at T,."F. It has been common practice that a microbiologically determined TDT is modified by increasing it by 20% for this comparison in order t o include a safety factor. An Fp value of a proper heat process should be equal t o or slightly greater than this modified TDT. Mathematical problems for estimating heat processes are classified as types A and B. Those of type A are for calculating the sterilizing value of a given process by using the time-temperature relationship of food, which is determined experimentally or predicted mathematically. Problems of type B are for determining a proper heat process, which produces the desired level of sterility.

I l l . PUBLISHED PROCEDURES FOR DETERMINING PROPER HEAT PROCESSES Many procedures are available for the mathematical determination of proper heat processes. These procedures may be classified into two groups: those based on the concentration of survivors at the slowest heating point in the food group, group I (Ball, 1923; Ball and Olson, 1957; Bigelow et al., 1920; Flambert and Deltour, 1972b; Gillespy, 1953; Griffin et al., 1969, 1971; Hayakawa, 1968, 1970, 1973; Herndon, 1971; Hicks, 1958a,b; Jakobsen, 1954; Patashnik, 1953; Pflug, 1968; Schultz and Olson, 1940; Shapton and Lovelock, 1971; Stumbo, 1973), and those based on the mass average concentration of survivors in the food, group I1 (Ball and Olson, 1957; Gillespy, 1951 ;Hayakawa, 1969; Stumbo, 1953, 1973). Group I procedures are based on the evaluation of heat energy applied t o a specific location in the food, and group I1 procedures are based on the evaluation of heat energy applied t o every volumetric element in the food. A criticalpoint sterilizing value, F p , and a mass average sterilizing value, F p , which are used in group I and group I1 procedures, respectively, are defined as follows: (C/CO)critirni

point

('/co)

=

R . 10--FpiDr

(1 1)

=

R . lO-"slUr

(1 la)

In determining microbiologically safe processes, procedures of both groups produce similar results. Since group I procedures require considerably less computa-

84

KAN-ICHI HAYAKAWA

tion than group I1 procedures, however, the former procedures are more convenient for this determination. On the other hand, group I1 procedures are required for estimating the nutritional or organoleptic quality of processed food, since the other procedures d o not give information on this quality for the entire volume of food product. it should be noted that group I and group I1 procedures are not mutually exclusive insofar as the required calculations are concerned. In order to use group I1 procedures, mathematical techniques developed for group 1 procedures are frequently utilized. A. GROUP1 PROCEDURES

Group I procedures are classified further into general methods and formula methods. The general methods d o not provide the means for predicting temperature history curves of food subjected to heat processing. These methods are usually applied to temperature history curves obtained through heat transfer experiments. On the other hand, formula methods provide means for predicting temperature history curves.

1. General Methods General methods are used for estimating F p values through the graphical or numerical estimation of the area under a lethality curve. This curve is obtained by plotting lethal rate value, L , against processing times, t . The general method, which was developed by Bigelow et al. (1920), has historical significance, since it is the first mathematical method successfully applied for the determination of safe heat processes. Since then, several general methods have been reported in published articles. Schultz and Olson (1940) have developed the use of lethal rate paper to eliminate the need for estimating L values. The lethal rate paper is obtained by scaling the y axis of regular graph paper with temperatures that correspond to the L values. For this scaling, Eq. (12) is used.

Lethal rate paper may be used to estimate Fp values for only one z value, which is selected to prepare this paper. The present author (1968) developed a method of applying lethal rate paper to different z values. Patashnik (1953) reported a method of calculating Fp values by numerically integrating through the rectangular approximation of the area under a segment of the lethal rate curve, which corresponds to each of the uniform time intervals. The mathematical error of this approximation is probably greater than the one associated with the estimation of Fp values by Simpson’s rule, which has frequently been used for

ESTIMATION OF THERMAL PROCESS

85

this estimation. The numerical calculations required for process estimation are greatly simplified by the application of a Gaussian integration formula (Hayakawa, 1968). According to numerical experimentations, a sufficiently accurate Fp value was obtained when L values were estimated from only six food temperatures, which corresponded to six properly spaced processing time values. Most numerical tables of L values have been prepared by using temperature expressed in Fahrenheit units. When one needs to estimate Fp values from Celsius temperature data, these data should be converted to Fahrenheit in order to use the numerical tables. Shapton and Lovelock (1971) prepared L-value tables by using temperatures in Celsius units to eliminate this conversion and by assuming that z = 10°C. One of their tables gives L values which are applicable when T,. = 120"C, and another may be used to estimate L values when T, is equal to any one temperature in the range 60" 100°C.

-

2. Formula Methods

One of the most classical formula methods was developed by Ball (1923). Ball's method has historical significance, since it is the first formula method reported in the literature. He derived formulas through the analytical integration of Eq. (1) by using several empirical formulas for predicting food temperatures. The linear portions of the heating and cooling curves are approximated by Eqs. (8a) and (8b), respectively. The curvilinear portion of a cooling curve is approximated by Eq. (13).

T

=

To

+ 0.3m - [0.3m/(f,0.0759)] 2/(0.0759f,.)2 + tC2

0 5 t , 5 0.141fc

(13)

This equation looks different from Ball's equation; however, the equations are mathematically identical to each other, since Eq. (13) was obtained by simplifying Ball's equation. Equation (13) was derived by assuming that tl = O.141fc and thatj, = 1.41. By substituting the empirical formulas for estimating food temperatures into Eq. (lo), Ball derived an analytical expression for calculating the Fp value of a heat process. He then prepared parametric charts and tables for estimating heat processes by means of this equation. Ball's method produces some errors because of the assumption stated above when j c values are considerably different from 1.41 and when the relative sterilizing effect gained during the cooling phase of a heat process, Fpc,is not negligibly small. Ball and Olson (1957) improved Ball's original procedure by the introduction of two new dimensionless parameters. These parameters are related to the sterilizing values of the heating and cooling phases of heat processes. The use of the two parameters greatly simplifies the calculations required for estimation of

86

KAN-1CHI HAYAKAWA

the heat process, especially when there are one or more break points in a heating curve. However, errors associated with the improved procedure are similar t o those of the original procedure, since the assumptions used t o derive Eq. (13) were not modified. Hicks (1958) found several mathematical errors in Ball and Olson's parametric values, which are related t o the sterilizing values of heating phases, Fp,, . He prepared a numerical table of recalculated parametric values. Pflug (1968) compiled abridged tables from Ball and Olson's tables and also from Hick's tables in parametric values and considerably simplified the calculations required for process evaluations. Herndon er al. (1968) reported numerical values of a new dimensionless parameter for estimating Fph values by using a digital computer. There is no significant difference between their parametric values and those given by Ball and Olson insofar as their applications to process evaluations are concerned. Griffin er al. (1971) derived the following equations for calculating food temperatures in the cooling phase of a heat process.

These equations were used to compute parametric values for estimating Fpc values with a computer. According to the present author's examination, the use of these parametric values results in considerably underestimated Fpc values, especially when j c values are greater than unity. Formulas that include hyperbolic functions were used by Jakobsen (1954) to represent heating and cooling curves. From these formulas, he derived analytical expressions for calculating Fp values and prepared parametric charts for this calculation. These charts are applicable to limited cases, since Jakobsen assumed that j , = 2.04. Stumbo and Longley (1966) observed that variable j , values should be considered instead of one futed j, value in order to accurately estimate the sterilizing effect of heat processes. They obtained numerical tables of parametric values, which are related to the Fp values of whole processes. These values were calculated by graphically integrating lethal rate curves, which were obtained from manually drawn heating and cooling curves. There may be some ambiguity in the time-temperature relationships indicated by these curves, since they were drawn with visual judgment. Stumbo (1973) recently recalculated the parametric values by using transientstate temperatures, which were theoretically predicted by means of a formula for heat conduction in a finite cylinder. Cooling curves with different j , values

ESTIMATION OF THERMAL PROCESS

87

were obtained by estimating temperatures at different locations in the cylinder. It is known that theoretical j , values at the center and surface of the cylinders are equal, respectively, to 2.04 and 0.00 and that j , values at intermediate points are within these two limiting values. The general applicability of Stumbo's revised tables to liquid or semisolid food should be carefully examined, since temperature history curves estimated from a heat conduction equation in the solid of a finite cylinder do not necessarily represent those of nonsolid foods. The present author (1970, 1974) prepared parametric tables by using the empirical formulas discussed previously, Eqs. (4a) through (9d). Manual calculations, which are required for estimating heat processes, are greatly simplified by use of these tables because no interpolation of z values is required. Proper heat processes for various z values may be easily estimated from the parametric tables for one fmed z value when the following two theorems are applied. Theorem A. A value for a process parameter, U , for any z value, z b . can be estimated from a U value for one reference z value, z , , by applying Eqs. (I5a) and (1 5b).

Equation (15a) clearly indicates that U values for any z values, z b , can be obtained from a table or chart for U values for one specific z value, z a , by modifying this temperature difference T - T I ,as ( T - T I ) / K s . 77zheorern B. A U value for the cooling phase of the heat process, U,, can be estimated by applying Eq. (16).

In Eq. (16), Ugcsignifies a U value when the reference temperature is Tg instead of T I .To estimate U, values, it is not necessary to prepare tables of U, values for various cooling water temperatures, for various z values, or for various values for T , - Tg. It is sufficient to consider the variation of V, values due to the variations of the values for Tg- T, and j,. Flambert and Deltour (1972b) prepared their parametric tables by using an analytical formula for heat conduction in a finite cylinder. This formula contains an infinite summation series. They used all nonnegligible terms of this series, and

88

KAN-ICHI HAYAKAWA

they did not simplify the formula by imposing restrictive assumptions t o derive a mathematical expression for the estimation of F p values. Deidoerfer and Humphrey (1959) derived a set of analytical formulas for estimating the sterilizing effect when liquid food is processed by various heat exchangers. They assumed uniform temperature distribution throughout the food when it is processed batchwise, and uniform cross-sectional temperature distribution when it is processed continuously. They also assumed that R = 1.0. They used the following parametric value for process evaluation: 0 =

In (co/c)

This value is called the design criterion of sterilization by them and is related to a sterilizing value, F p , as follows:

Fp = Dr(C - In R)/ln 10

(18)

B. GROUP I1 PROCEDURES All procedures of group 11, which are available in published articles, are applicable only to solid food, since analytical or numerical solutions of simple heat conduction equations are used for the development of these procedures. Gillespy (195 1) prepared parametric tables for estimating mass average sterilizing values. For this preparation, he used an analytical formula for heat conduction in a finite cylinder. He approximated an infinite summation series in this formula with its first term for the preparation. He also simplified preparatory calculations by assuming that an Fpc value is equal to an additional sterilizing value gained when the length of the heating phase was increased by 0.3 X jj, minutes. Although this assumption is based on the examination of experimental data, its validity is questionable, since the constant, 0.3, is probably dependent on heating time, the temperature of the heating medium, the f, value, the z value, the j , value, and the temperature of the cooling medium. Stumbo (1953) developed his procedure by using the heat conduction formula, which is similar to the one used by Gillespy. Stumbo estimated the distribution of surviving target microorganisms at the end of a heat process through the combined use of this formula and published parametric charts (Ball, 1923). From this distribution, he then derived a remarkably simple formula for correlating a mass average sterilizing value and a sterilizing value at a critical point in food. As stated previously, Ball's charts were obtained by assuming that j, = 1.41 and jj, = f,. Therefore, the formula was derived by indirectly assuming that a j , value at any location in the food was equal to 1.41. Even though these unreal assumptions were used, the formula has been widely adopted for process evaluation because of its simplicity.

ESTIMATION OF THERMAL PROCESS

89

Timbers and Hayakawa (1967) observed that Stumbo’s formula did not give reliable answers when it was applied to the z and D, values of organoleptic or nutritional factors which are considerably greater than those of microorganisms. Jen et al. (1971) removed this limitation by deriving another formula, which also does not require complicated calculations for evaluating mass average sterilizing values. A mathematical process for this derivation is similar to that employed by Stumbo (1953). The formula derived by Jen eial. is given below.

According to the present author’s dimensional analysis (1969), six dimensionless groups are required for the unique mathematical estimation of a mass average sterilizing value. Neither Stumbo’s formula nor that of Jen et al. includes the required number of dimensionless groups. For example, no parameter representing the relative shape of a can is included. Although the dimensionless groups might not all be required for the fair estimation of mass average sterilizing values of commercial processes, the reliability of the formula derived by Jen el al. should be investigated in the future. Ball and Olson (1957) developed a procedure for process evaluation based essentially on numerical integration of concentrations of a remaining vulnerable factor in eleven pseudo-isothermal Tegions. These concentrations were estimated through the combined use of a simplified heat conduction formula, which is similar to the one used by Gillespy (1951), and parametric tables which they prepared. Through the mathematical analysis of the heat conduction equation, they observed that fh values at any locations in a cylindrical can of solid food were all identical and that j h values changed with location in the food. They also noticed that food temperatures in the linear portion of a heating curve at any fmed processing time could be uniquely estimated when fh and j h values were given, provided the initial temperature distribution was uniform. Therefore, they concluded that food temperatures at any locations whose j h values were identical are redundant to each other during any time of heating. An isothermal plane in the food was then obtained by tracing iso-jh value plane. For convenience, the ratio of a j h value at any location to the one at the central location was used instead of the j h value. An isothermal plan was then represented by the iso-h value plane. Equation (20) was used for estimating the distribution of A values in the food.

We note from Eq. (20) that a h value on the surface of the food is zero and that one at the center is unity.

90

KAN-ICHI HAYAKAWA

Ball and Olson divided the entire volume of the food with eleven iso-h planes. The h values of these planes were 0,0.05, 0.15,0.25,0.35,. . . ,0.85,0.95, and 1. A volumetric fraction, which was enclosed by two successive planes, was assumed to be an isothermal region, and the mean of the h values of these two planes was assigned to this assumed isothermal region except for the central and surface regions. The h values of the central and surface regions were assumed to be unity and zero, respectively. Ball and Olson then estimated the volumetric fraction of each pseudo-isothermal region. The volumetric fractions given by them are questionable values, since the sum of all fractions is not equal to unity but t o 0.9027. The time-temperature relationship of each iso-h region in the linear portion of a heating curve is represented by Eq. (21).

The curvilinear portion of a heating curve was replaced with an extrapolated line segment of a curve obtained by Eq. (21). The parametric tables used for the calculation were prepared by Ball and Olson by assuming that j , = 1.41, as mentioned previously. Therefore, they further assumed that there were sudden temperature drops, d , at the beginning of cooling, whose magnitudes were location-variable as shown in Eq. (22).

No proof is given for verifying the validity of the assumed temperature drops. By using the above described timetemperature relationship, a sterilizing value was obtained for each region. From this value, a survival ratio was estimated from Eq. (23), which is almost identical to Eq. (1 1).

A mass average survival ratio, Z/co, was estimated from a product of each survival ratio and the corresponding volumetric fraction (Eq. 24).

A mass average sterilizing value, Fp,was then finally obtained by using Eq. (25).

ESTIMATION OF THERMAL PROCESS

91

The present author (1969) obtained parametric values for estimating Fp through the mathematical combination of heat conduction formulas and reaction kinetic formulas for the inactivation of a thermally vulnerable factor. An analytical formula used for this estimation is given later in Section IV,A. Thiamine concentrations in several canned foods, which were experimentally processed at the present author’s laboratory, were estimated by using the parametric values; the values agreed fairly well with those determined chemically. Manson et al. (1974) developed a quite interesting method for estimating thermal processes applied to solid food in a pear-shaped can. This can is usually used for packing cured fresh ham. Mass average sterilizing values were estimated through the combined application of the geometric analysis of Smith et al. (1967) and the formula of Jen et al. (1971). These estimated values agree fairly well with those computed numerically by the finite difference approximation of a heat conduction equation.

I V . COMPUTERIZED ESTIMATION OF HEAT PROCESSES With the increased availability of high-speed computers, several research workers reported the computerized estimation of thermal processes based on the use of specially prepared computer programs. These programs may be classified into two groups, depending on their objectives: (1) those for computing parametric values which are used for heat process estimation, and (2) those for directly estimating heat processes without intermediate manual calculation. This classification is used for convenience in the following discussion. A. PROGRAMS FOR ESTIMATING PARAMETRIC VALUES

A group of programs, which will be reviewed first, were prepared to compute parametric values for thermal process evaluation. These parametric values were intended to be used for evaluating safe thermal processes through manual calculations. Therefore, these programs do not provide an economical means for computerized estimation. However, when properly modified, they may be used for directly estimating the sterilizing effect of thermal processes. Theoretical formulas for transient-state heat conduction have been successfully utilized by several workers for evaluating thermal processes applied to cylindrical cans of solid food (Ball, 1923; Flambert and Deltour, 1972a,b; Gillespy, 1951; Hayakawa, 1969; Stumbo, 1973). Ball and Olson (1957) observed that the surface temperature of canned food was almost identical to the heating medium

92

KAN-ICHI HAYAKAWA

temperature during most of the heat processes when the food was processed with steam. Therefore, analytical formulas, derived by assuming fairly large surface conductance, have been commonly employed for thermal process evaluation. Most research workers approximated heat conduction equations by their respective first terms for the evaluation, with limited analysis of errors caused by this approximation. As stated previously, the present author (1969) developed a computer program for estimating the parametric values of a group I1 procedure through the use of all non-negligible terms in heat conduction formulas. A flow diagram of this program is shown in Fig. 2. An analytical formula used in the program is given below. Since the temperature distribution in a cylindrical can of thermally conductive food was symmetrical with respect to the central axis during heat processing, the mass average survival ratio, C/co, of a thermally vulnerable factor in the food at the end of heat processing was estimated by the following equations:

where

and G(B, p , {)

=

8

- 2-

?r k = l n = l

JdPkP) ~

e-.4B

. __.

JI(pk)pk 2n - 1

sin (2n - I ) x {

(26c)

The integrand of Eq. (24) is the local survival ratio, c/co, of a vulnerable factor. Equations (26a) and (26b) estimate the local sterilizing values of heating and cooling phases of a process, respectively. Equation (26c) is a solution of an FIG. 2. Flow diagram of a program for computing parametric values for estimating mass average sterilizing values. Reproduced from Food Technology, Vol. 21, No. 8, pp. 17-24 (1 967). Copyright by the Institute of Food Technologists. The reproduction was approved by this Institute.

0 Start

'

C a l c u l a-t e u

AB (n 5 1, 2 , . . . , N ) v a l u e s a t

.

4

13 l o c a t i o n s i n the food.

Subprogram for c a l c u l a t i n g the temperature distribution

%5-

c a l c u l a t i n g the z e r o o r d e r B e s s e l function

LOG U = ALOG 10 (U) I

A r r a n g e LOG u v a l u e s in the o r d e r of t h e i r n u m e r i c a l m a g n i t u d e

Subprogram for a r r a n g i n g a g r o u p of

i

P l o t the heating c u r v e s a t the 13 l o c a t i o n s

4

Find B v a l u e which

+

Two s u b p r o g r a m s f o r

u s i n g the p r e v i o u s 1

Compute

F v a l u e s of t h e h e a t i n g P

Subprogram for plotting p o i n t s

p h a s e of t h e 13 l o c a t i o n s

t

Compute the m a s s of s u r v i v o r s a t t h e e f o r v a r i o u s up. T,,

[

I

T,, a n d Ch v a l u e s

t

C o m p u t e Fnh v a l u e s of t h e cooling p h a s e

I

Subprogram for a n interpolation formula

1

a t t h e 13 l c c a t i o n s

1.

C o m p u t e t h e mass a v e r a g e c o n c e n t r a t i o n of s u r v i v o r s a t t h e end of t h e cooling p h a s e f o r v a r i o u s Ug. T , , T,, a n d cb values

[

t Compute

F P X Compute /

Fp

P r i n t o u t the G / ( D r * C b )v a l u e s a n d Fp/(D; values a t the center

Cb)

-

c

94

KAN-ICHI HAYAKAWA

equation for thermal conduction in a finite cylinder (Ball and Olson, 1957; Carslaw and Jaeger, 1959). After a number of trial computations, the following relationship was selected for determining the Bf value which represents the length of the cooling phase.

The exponent of the integrand in Eq. (26) was rearranged as follows for easier computation by a digital computer:

where HINT and CINT are defined as follows:

After preliminary computations, Simpson’s formula and a four-point Lobatto formula were selected to calculate the values of HINT and CINT because these formulas produced an accurate estimation of these values. Simpson’s formula was used because we employed short increments in B values for the integration. The domain of B, which corresponds to one log-cycle change in u values, was divided into about thirty equal intervals. The four-point Lobatto formula was utilized because it gave an exact value for integration of any polynomial of any degree less than or equal to 5 (Michels, 1963) and because it was applied t o a narrow range of B values. These two formulas were used to compute one single integration for the following reasons. Temperatures estimated at equal time intervals were used for calculating the integration, and also in most cases the upper limit of the integration was not located at a B value at which a computed temperature was given. Through a careful analysis of Eq. (26), a twelve-point formula (Hammer and Straut, 1958) was selected to calculate the values of the double integration in this equation. This formula was reliable, since it gave exact results on double integration of any polynomial of any degree equal to or less than 7 (Tyler, 1953), and since the results of the trial computations indicate that the distribution of survival ratio (clc,) in the food could be expressed by a polynomial of a degree equal to or less than 7. Finally, the mass average sterilizing value ( F p )is obtained by Eq. (25).

ESTIMATION OF THERMAL PROCESS

95

As stated previously, six dimensionless groups were required for this evaluation. The dimensionless groups selected were:

The values of the dimensionless group, F k , which contained a mass average sterilizing value, were estimated for all possible combinations of the other five dimensionless groups. Flambert and Deltour (1972b) developed their program for estimating parametric values used for evaluating sterilizing values at the centers of cylindrical cans of solid food by means of an analytical formula similar to Eq. (26c). It should be noted that their reaction kinetic parameters, D, and z , , for thermal inactivation of microorganisms are different from those commonly used, as shown below.

Empirical formulas have been frequently used for the computerized estimation of heat processes. Equations (4a) through (9d) were used for estimating values of two dimensionless parameters employed for thermal process evaluation by the present author (1974). These dimensionless groups are Uh/f and U / f . The values of U,, and U represent, respectively. the sterilizing value of the heating phase of a thermal process and of an entire process when the reference temperature in Eq. (loa) is equal to T I .A seven-point Lobatto quadrature formula was chosen for the numerical integration of Eq. (10). According to computational experiments the errors in u h / f and U / f values estimated by this formula were less than 5%. An abridged flow diagram of the programs is shown in Fig. 3. To calculate U,/f and U/f values, the following specific values of experimental parameters were assumed: f = 30 minutes, j h = 1.0, To = 150"F, T I = 250"F, and f h = f, = f. The first value was used, since the values of the two dimensionless groups were practically independent of the f values. The values of the two dimensionless groups were calculated for various g values instead of for various t b values. Thc initial temperatures, T o , of most heat processes are in a range of temperatures whose lethal rates, L , are negligibly small. In this situation, values of the two groups are practically independent of j h and To values. Therefore, the second and third assumed values were included. The fourth value was employed

KAN-ICHI HAYAKAWA

96 M

X

BLOCK DATA

SUBROUTINES

F'IG. 3. 1,'low diagram of program for estimating Uh/f'and U/f values.

because u h and U were independent of the T I value. The combined use of the U,,/f and U / f values enables us to evaluate thermal processes even when h,#fC. Therefore, the last value was chosen to simplify computations. The program prepared consists of one main program, one block data subprogram, and five subroutine subprograms." The names of the five subroutine subprograms are HEAT, COOL, COOLA, RATE, and PLT. Subroutine HEAT is for calculating seven temperatures from a simple heating curve. These seven temperatures were evaluated at the abscissas which are specified by the sevenpoint Lobatto quadrature formula. Subroutines COOL and COOLA are similar to subroutine HEAT and were used to estimate temperatures in the curvilinear and linear portions of a cooling curve. Subroutines HEAT and COOL were prepared by using Eqs. (4a) through (9d). As stated previously these equations are applicable when a j l r or j c value is in the range 0.045 < G h orj,) < 3.00. If any j h or j , value outside this range is used in these subroutines, computation will be forced t o be terminated. A proper message will be printed t o identify the reason for termination. Subroutine RATE is for calculating a sterilizing value from a set of seven temperatures, which are estimated by subroutines HEAT, COOL, or COOLA. Subroutine PLT is for plotting a matrix o f parametric values estimated on log-log paper. By this subroutine, one sheet of 2 X 2 log-log graph paper is produced out of each page of computer printout. and plotting continues until all data points are properly placed on one or more separate sheets of the graph paper. All scales of the graph paper are properly labeled by the same *Some tcchnical terms cmploycd in computer programming are briefly defined in Appendix A for those who arc not familiar with them.

ESTIMATION OF THERMAL PROCESS

97

subroutine. By using subroutine PLT, the values of log,, (Uh/f) or log,, ( U / f ) were plotted against the values of log,, g. The block data subprogram is for defining the following constants: (1) Abscissa (aa) and weight ( h ) of seven-point Lobatto quadrature formula. (2) Data on intercept coefficients of the cooling curve Oc), on temperature differences between the food and the heating medium at the end of the heating phases dp), on the temperature of the cooling medium (T,,,), and on the slope index for the thermal death time curve of the target microorganisms (z). (3) A-FORMAT characters to be used for plotting curves (HMAE and SETSU). (4) A-FORMAT characters for producing variable format statements (FMA). Table I shows variables whose data are stored in labeled common blocks. The program discussed above was obtained through the considerable modification of another program, which was used for developing a method for thermal process evaluation described previously (Hayakawa, 1970). Griffin el al. (1969) and Herndon er al. (1968), prepared computer programs for calculating parametric values similar to Uh/f and U / f by using Eqs. (Sa), (Sb), (15), and (16). Simpson’s integration formula was used to compute these parametric values.

B. PROGRAMS FOR ESTIMATING HEAT PROCESSES WITHOUT MANUAL CALCULATIONS Most of the programs described above may be used for the direct evaluation of thermal processes without manual calculations when they are properly modified. However, several programs are available for this evaluation without modification. These programs as well as one recently developed by the present author are reviewed for their computational characteristics. 1. Programs Available in Published Articles

Timbers and Hayakawa (1967) developed two different computer programs for solving type A problems for the estimation of heat processes. These programs TABLE I DATA DEFINED IN LABELED COMMON BLOCKS

Common block names ~

Variables whose values are stored in common blocks

Programs in which values stored are used

~~~~

COMA COMH

COMF HAYA PROC

ab

h FMA HMAE, SETSU to g, T,, z

Subroutines HEAT, COOL, and COOLA Subroutines RATE Subroutines PLT Subroutines PLT Main program

98

KAN-ICHI HAYAKAWA

are for calculating mass average sterilizing values by Ball and Olson’s procedure (1957) and by Stumbo’s procedure (1953). Abridged flow charts of these programs are shown in Fig. 4. The programs are applicable to a cylindrical can of solid food and to a heating curve with one linear line segment. The use of parametric values provided by Ball and Olson (1957) or by Stumbo (1973) is not an efficient method for computerized estimation of thermal processes, since considerable core space is required for storing a large number of the parametric values and since sterilizing values may be easily and quickly computed from heat transfer data through the use of proper numerical integration formulas.

Calculate: Fpo and FpA of entire process.

Fp for each

Calculate:

( /Co)

Calculate: FpA , (C/Co)h, and (C/Co) of th,e cooling phase i

I

Calculate:

I

Calculate: D, and z

I

h

1

(C/C,) ,Fp

Y Print out: tables (c/Co) Fp for each z , D

Ball’s method

Stumbo’s method

FIG. 4. Flow diagrams of programs for solving type A problems by using group I1 procedures. Reproduced from Food Technology, Vol. 21, No. 8, pp. 17-24 (1967). Copyright by the Institute of Food Technologists. The reproduction was approved by this Institute.

ESTIMATION OF THERMAL PROCESS

99

In the program prepared, mass average sterilizing values were directly computed through application of local sterilizing values estimated by substituting Eqs. (8b), (15), (21), and (20) into Eq. (12). For this computation, Eqs. (22), (23), and (24) were employed. The Lobatto numerical integration formula was used to estimate these sterilizing values. The values of the volumetric fractions of pseudo-isothermal regions given by Ball and Olson (1957) were not used because they were questionable, as stated previously. The values of the fractions which were estimated by the present author (1958) were used in the program. The computational process by which these values were estimated is briefly described below because they are the most important parametric values for estimating mass average sterilizing values and because Ball and Olson (1957) did not clearly discuss the process in their textbook. Any iso-X plane is symmetrical with respect to the central axis of a cylindrical can of solid food and is obtained by rotating a proper curvilinear line segment around this axis. Therefore, a volumetric fraction enclosed with an iso-A plane is obtained by applying an integral method for estimating the volume of solid of revolution. We obtained Eq. (29) for estimating thls fraction, Y A ,

where the upper limit of the integration, P A , is the solution of Eq. (29a).

The volumetric fraction enclosed with the iso-X planes were computed by using Gregory’s integration formula (Hilderbrand, 1956) for these A values: 0.05,0.15, 0.25,. . . , 0.85 and 0.95. The fraction for each pseudo-isothermal region considered by Ball and Olson (1957) was then estimated from the computed results (Table 11). To prepare another program, we used a formula for estimating mass average sterilizing values, which was derived by Stumbo (1953). Since Stumbo used Ball’s parametric charts (1923), central and off-central sterilizing values, which were required in this formula, were estimated from the same equations employed in Timber and Hayakawa’s program stated above. The program may be easily modified to use the formula of Jen e l al. (1971) instead of Stumbo’s formula, since these two formulas are similar to each other. The formula of Jen er ai. was not utilized because it was not available when we were developing the program. Manson and Zahradnik (1967) prepared their program for solving type A problems by utilizing Stumbo’s formula. This program employed parametric

100

KAN-ICHI HAYAKAWA

TABLE I1 VOLUMETRIC FRACTIONS O F PSEUDO-ISOTHERMAL REGIONS IN A CYLINDRICAL CAN OF SOLID FOOD

Volumetric fraction

A value of pseudo-isothermal region

_

_

~

~~

~

~

.. .__

0.155 0.245 0.155 0.1 20 0.095 0.070 0.055 0.045 0.033

0 0.1

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 .o

0.020

0.007

tables prepared by Stumbo and Longley (1966), which were stored in a computer. Teixeira et al. (1969) obtained a computer program for estimating the local and mass average survival ratios of a theramlly vulnerable factor in a cylindrical can of solid food. This program may be used for solving type A problems. Transient-state temperature distributions in solid food, which were used for this estimation, were computed by means of a finite difference technique, although several analytical formulas were available for determining the temperature distributions. A program similar to that of Teixeira et al. was reported by Flambert and Deltour (1972a). Manson er al. (1970) obtained their program for solving type A problems by using a group I1 procedure. This program is applicable to a rectangular can of solid food. A finite difference technique was used for computing transientstate temperature distributions in food, although several analytical formulas were available for estimating transient-state temperature distributions in the brick-shaped solid. Sasseen’s program (1969) is for estimating sterilizing values by Ball’s method (1923). This program utilizes parametric values prepared by Ball rather than directly computing sterilizing values from the temperature history of the food. The program is applicable for solving both type A and type B problems by using a group I procedure and allows one break point in a linear line portion of a heating curve.

2. New Program Developed The present author obtained a new program package for estimating thermal processes. Fortran IV computer language was used for the program. The pro

ESTIMATION OF THERMAL PROCESS

101

grams are the most versatile when they are compared with those published and they may be used t o solve most problems discussed in standard textbooks on thermal processing (Ball and Olson, 1957; Stumbo, 1973). Both group I and group I1 procedures are included in the program. The program is applicable t o a heating curve with zero or up to two break points in its linear portion. Equations (4a) through (9d) were used to estimate the temperature history curves of food from which sterilizing values were computed. The formula of Jen el ul. was used as a group I1 procedure for estimating mass average sterilizing values because of its simplicity. An abridged flow diagram is shown in Fig. 5. The programs consist of one main program, seven subroutine subprograms, four function subprograms, and one block data subprogram. The complete listing of the program is given in Appendix B, since Fortran IV language, which was used for preparing the present program, is one of the most widely used ones and since it is compatible with most of the digital computers available. Subroutine SETU is for computing an answer for a given problem on thermal process evaluation and uses all other subprograms. Subroutines RATE, COOL, and COOLA are identical to those shown in Fig. 3. Subroutine HEAT is a modified version of subroutine HEAT shown in Fig. 3. This modified subroutine computes food temperatures on a heating curve at each of equally spaced time points whose number is less than or equal t o 300. The modification was required to facilitate iterative computations for solving type B problems. Subroutine SIMP is for estimating a sterilizing value by using Simpson’s formula. A sterilizing value associated with one narrow time interval of thermal processing is estimated by subroutine FDIF. The formula derived by Jen et al. is included in function FMAS. Functions FTC and FX are used for semilogarithmic and linear interpolations of functional MAIN PROGRAM FUNCTION SUBPROGRAM

SUBROUTINE SUEPROGRAM

(START)

WRITE ANSWER

BLOCK

FIG. 5. Flow diagram of new program for solving types A and B problems by using eithei group I or group I1 procedure.

102

KAN-ICHI HAYAKAWA

values, respectively. Lethal rate values are estimated by function RT, and the length of a curvilinear portion of a heatin or cooling curve is estimated by function TLX. The programs contain all the subprograms required for heat process evaluation, and n o knowledge of computer programming is required when they are properly placed in a computer system. Three data cards are required for one estimation as explained in the listings of the main program and in subroutine SETU. There are eighty columns in one computer card, and any proper character may be key-punched into each of these columns o r a blank space may be assigned to it without key-punching. The first data card should contain codes for identifying a heat process, and the number of characters, which include number of blanks, should be equal to or less than eighty. Typical codes include product name, formulation, and can size. The values of T o , T I , T,, j h , fhl , f h 2 , fh3 , and g b 1 should be punched into the second card in the order given. Ten columns of the card are assigned to each of these values. The first through the tenth columns are for T o , the eleventh through the twentieth are for T I ,and so on. Each value may be key-punched at any location within the ten columns provided a decimal point is properly placed. The third card should contain the values o f g b 2 , j c , fc, D,,z , Tg, T b , and Fp (or Fp). The assignment of columns for these values is similar to that of the second card. The following values should be properly placed in the cards for solving any problem: To, T I , T,, j h , j c , fc, z . Rules for placing other values are given below. 1. Heating curve a. No break point f h l = f h 2 = f h 3 = a given fh value g b l = g b 2 =-1.o b. One break point fhl = a givenfhl value f h 2 = f h 3 = a given f h 2 value gbl = a given g b value gb2 =-1.0 c, Two break points: Given fhl , f h 2 , fh3, placed properly. 2. Group of procedure a. Group I D, = -1.0 b. Group11 D, = a given D, value 3. Type of problems a. Type A problem b' =-1 P

gbl

, and gb2 values should be

ESTIMATION OF THERMAL PROCESS

Tg

103

Tg value ={ a- 1given or , if a value is given a given tb value ={ if aorTgvalue is given tb

tb

-1,

b.

Either a T, or a f b value may be used to define the length of heating phase of thermal process. Type B problem Fp = a given Fp or Fp value T g = t b = -1 .O

As an example, consider the estimation of a mass average sterilizing value of a 211 X 300 can of spinach purCe subjected to the following heat process conditions: To = 180"F, T I = 250"F, T,,, = 55"F, j h = 1.85, fh = 33.0 minutes, j c = 1.56, f, = 45.5 minutes, D, = 130 minutes, z = 49.3"F,and t b = 60 minutes. Sample input data for the above stated estimation are given below.

The first card 21 1 X 300 CAN, SPINACH PUREE, RUN NO. KH-GT-1 The second card 180.0 250.0 55.0 1.85 33.0 33.0 33.0 -1.0 The third card -1.0 1.56 45.5 130.0 49.3 -1.0 60.0 -1.0 If the above problem is for estimating a proper heat process, which produces

Fp

= 40.0 minutes, the third card should be revised as follows:

-1.0

1.56 45.5 130.0 49.3 -1.0 -1.0 40.0

It should be noted that decimal points are properly placed in all numerical data. It was observed that the formula of Jen el al. gave unusual answers because a central sterilizing value for some combination of parametric values became greater than on off-central sterilizing value especially when j,, < 1. Whenever this situation is encountered, the program prints out the message "NO ANSWER IS ESTIMATED SINCE JEN ET AL.'S FORMULA IS NOT USABLE BECAUSE A CENTRAL FP VALUE IS LARGER THAN AN OFFCENTRAL FP VALUE" and proceeds to the next set of processing data t o be evaluated. It should be noted, however, that this unusual situation does not often occur, since the formula should be applied to solid food only and since the j, values of most solid foods are larger than 1.

KAN-ICHI HAYAKAWA

104

As stated previously, the empirical formulas are applicable when 0.045 < ( A or j,) < 3.00. The lower limit of this applicable range is sufficiently low, since most j h or j c values are larger than this limit. In some cases, however,jh and j , values exceed the higher limit of 3.00. Whenever either or both of these parametric values are outside the applicable range, the program prints out “TM & T VALUES ESTIMATED BY SUBROUTINE COOL (or HEAT) ARE QUESTIONABLE SINCE CJ (or HJ) > 3.0 (or < 0.045)’’ After printing this message, the program continues to solve a problem for process evaluation without terminating computations. About one hundred problems for heat process evaluations were collected from published articles or prepared from experimental data obtained at the present author’s laboratory. These problems were then solved by using a digital computer with the application of the programs and also solved manually by applying the procedures used in the programs. There was excellent agreement between computer-produced answers and those obtained by manual calculations. To compile the program 158,000 cores are required, and 78,000 cores are required for computation. Computer time required for solving a type A or B problem is less that 1 second when an IBM370/158 computer is used. As mentioned previously, the empirical formulas of temperature history curves, which were used for preparing the present computer program, estimate most accurately the sterilizing effect of thermal processes when they are compared with those available in published articles (Hayakawa, 1970). Therefore, the present program may be used for the reliable determination of safe thermal processes.

V.

RESEARCH NEEDS

Mathematical procedures for heat process evaluation have been modified and improved by many research workers during the past half-century since the publication of Ball’s pioneering work in 1923. Although most of the major problems were solved by these workers, further research needs to be done in the future are discussed below. A. CORRECTION FOR STERILIZING EFFECT OF HEATING DURING INITIAL COMING-UP TIME The coming-up time is defined as the time required for the temperature of the heating medium around the food to reach a target holding temperature at the beginning of a heat process. Most experimental parameters of heating and cooling curves reported in the literature are usable when there is no or a negligibly short coming-up time, t,, at the beginning of a heat process. It is also observed that recommended heat

ESTIMATION OF THERMAL PROCESS

105

processing times and temperatures are applicable when there is no or a negligibly small t , value. However, some commercial heat processes d o not satisfy this condition. Therefore, to utilize the experimental parameters or the recommended processing data, the sterilizing effect gained during the comingup time heating has been frequently expressed in terms of the processing time at the holding temperature of the heating medium by using a method developed by Ball (1923). Ball observed that heat treatment during coming-up time was equivalent to heating for 0.42 X t, minutes at the target temperature. This finding was based on limited heat transfer experiments conducted with No. 2 cans of cream-style corn or of 4% starch solution. The cans were heated in a water bath whose temperature was changed linearly with time. Alstrand and Benjamin (1949) examined experimentally the validity for the general use of 0.42 as a correction factor for comingup time heating by using No. 2 cans of cream-style corn or of 5% bentonite suspension. Sample cans were processed in a steam retort. To compute the correction factors of coming-up time heating, they used Ball’s parametric chart (1923) for thermal process evaluation. It was found that the factor of 0.42 was satisfactory for correcting their collected data, although there were wide variations in correction factors determined experimentally. According to the present author’s theoretical analysis (Hayakawa and Ball, 1971), the general application of the correction factor of 0.42 is questionable, since it is probably dependent on several experimental parameters. These parameters include fh and j h values, z values, and can shapes. It was also observed that this correction factor, which was based on the use of a mass average sterilization value for thermal process evaluation, was different from the correction factor based on the use of a sterilizing value at the slowest heating point. Ikegami (1974) reported this mathematical analysis of the temperature response of solid food during coming-up heating time. His analysis contained several questionable mathematical treatments. Further investigations are required to verify the correction factor which is currently used in the food industry, since several of the questions stated above have not been answered.

B. INFLUENCE O F STATISTICAL VARIATIONS IN EXPERIMENTAL PARAMETERS ON STERILIZING EFFECT There are inherent statistical variations in all experimental parameters used for the evaluation of heat processes. For example, j values which were determined from the same experimental data by different individuals, vary more than 10% according to the present author’s observation. Hicks (1961) examined qualitatively the influence of these variations on the sterilizing effect of heat processes. Herndon (197 1) analyzed mathematical and

106

KAN-ICHI HAYAKAWA

experimental relationships between variations in fh and j h values and the sterilizing values of heat processes. Several investigators (Manson and Cullen, 1974; Rao and Loncin, 1974a,b; Simpson and Williams, 1974; Veerkamp et al., 1974) analyzed the influence of variable residence times in continuous heat exchangers on sterilizing effect. Investigations are needed to answer questions on the normal magnitudes of statistical variations in all important experimental parameters and on the influence of these variations on the lethal effect of heat processes.

C . HEAT PROCESSING OF LIQUID OR SEMISOLID FOOD The mathematical evaluation of a heat process applied to liquid or semisolid food is commonly based on the use of a temperature history curve, which is applicable to the slowest heating point in the food. However, a food particle or volumetric element does not stay stationary but moves around in a containx during a heat process because of natural or forced convection. A sterilizing value calculated from a temperature history curve applicable to the slowest heating point is probably underestimated, since a food particle or volumetric element located at this point at a given heating processing time moves away from it at another time. Stevens et al. (1973) analyzed mathematically the sterilizing effect of a heat process applied to a can of liquid food subjected to naturally convective movement. There is at least academic interest in further experimental and theoretical examination of this sterilizing effect. Results of these investigations will be particularly useful when liquid or semisolid food is processed for a short time at a high temperature or when temperature distribution in the food is not uniform during processing.

VI. NOMENCLATURE A

= (Pk/S)2

+ (2n - 1)2n2.

Inside radius of can (inches). Abscissas for seven-point Lobatto quadrature formula. ab B = cutf12. = (0.5tb/o,) loVI - Z W z . cb CINT Definite integral defined by Eq. (260. C Volumetric concentration of surviving microorganisms or active factor (concentration of remainder per cubic inch). D Decimal reduction time of a thermally vulnerable factor when it is heated at a constant temperature (minutes). d Assumed temperature drop at beginning of cooling phase. See Eq. ( 2 2 ) (F). a

.

ESTIMATION OF THERMAL PROCESS

E

107

1

Constant used in empirical temperature history curve of food. This constant is estimated by Eq. (4c). = F p / ( D , Cb). Sterilizing value of the thermal process. See Eq. (10). Constant which is related t o a slope of a heating or cooling curve (minutes). Function defined by Eq. (26c). = T I - Tg (FO). Difference between food temperature and heating medium temperature when a break point is ovserved on a heating curve (F"). Weights for seven-point Lobatto integration formula. Definite integral defined by Eq. ( 2 6 0 . Zero-order Bessel function of first kind. First-order Bessel function of first kind. Constant related to an intercept coefficient of a temperature history curve of food. Reaction rate constant Letal rate defined by Eq. (10a). Inside height of can (inches).

m

= Tg - T ,

n Pk R

Dummy integer. kth positive root of Jo ( X ) = 0. Constant related to an intercept coefficient of a survivor curve of a thermally vulnerable factor. When there is no initial curvilinear portion in a survivor curve, R = 1. See Eq. (I). Radical distance measured from a central axis of a cylindrical can (inches). =all. Temperature (OF).

Fk

Fp f

G( ) g gb

h HINT Jo ( ) J1( ) j

k L

r S

T T, T, t tl t,

U U

v

X y z z, zb

(FO).

= (Ti - T,)/z.

- To )/Z. Time (minutes). Length of curvilinear portion of heatin curve (minutes). Coming-up time (minutes). Sterilizing value of food when T, = T1 (O F). = (Tl - T)/(T, - To). Volumetric fraction of space enclosed with iso-h plane. Dummy variable. Axial distance measured from bottom end of a cylindrical can (inches). Constant related to slope for thermal death time curve of thermally vulnerable factor (FO). Fixed z value (F'). Any z value (FO). = (Ti

108

a

P Y AE 1

e*

x

KAN-ICHI HAYAKAWA

Thermal diffusivity of food (square inches per minute). Constant used in empirical formula for temperature history curve of food. This constant is estimated by Eq. (4b), (Sb), or (6b). Reduction index. See Eq. ( 2 ) . Activation energy. =y/l. = (TI - Tg)/(T1 - To). Ratio of j value determined at center of canned food to another j value determined at any location in the same food.

P

=r/a:

7

Thermal death time of thermally vulnerable factor (minutes). Volumetric fraction of pseudo-isothermal region. = h ( c o /c).

w

V

SUBSCRIPTS C

e

f g

h

m 0 r S

s2 W

x 1

2,3

Value related to cooling phase of thermal process. End of a heat process. Value related to end of cooling phase of thermal process. Value related to end of heating phase of thermal process. Value related to heating phase of thermal process. Values used by Flambert and Deltour (1972b). Initial value. Value at reference temperature, T,. T, is usually 2 5 0 " ~ . Value monitored at center of food. Value monitored at an off-center point of food. The value o f g , which is applicable to this point, is g/2. The symbols are used as Fpsz in Eq. (19). Value related to cooling medium. Value monitoried at any iso-X region in food. Constant holding temperature of heating medium, when it is used with T. The first value when it is used with fk or gb. Second and third values, respectively.

A bar (-) over a term indicates mean value.

APPENDIX A: COMPUTER PROGRAMMING TERMINOLOGY A-Formated Character: Characters or symbols which are read from a computer card or magnetic tape by an input device are usually stored in a computer without any modification and used for identifying data points plotted or for

ESTIMATION OF THERMAL PROCESS

109

writing codes with an output device. These characters or symbols are called A-formated characters. Block Data Subprogram: A subprogram for defining a constant commonly used in a main program, function subprogram, or subroutine subprogram. Compiling: Translating a relatively machine-independent program into specific machine-language routines. Core: All operations described in a program are internally converted into a series of binary digits by a computer. During computational work, each digit is stored in one core of a computer. Function Subprogram: A subprogram for defining a mathematical function. One name is assigned to each function subprogram to identify it without any confusion. To actually compute a functional value, the values of the variables should be supplied to a function subprogram through the use of another program. Main Program: A program for completing a set of planned work through the proper use of computational steps. Subprograms are frequently employed for this completion. Subprogram: One subprogram contains a set of computational steps for completing a specific work. Each subprogram can be compiled independently. Subroutine Subprogram: One subprogram for doing planned computational work. The results of this work are not always the numerical values of mathematical functions. One name is assigned t o each subroutine subprogram to avoid confusion, Input data should be properly supplied to a subroutine subprogram for actually completing a planned work through the use of another program.

110

KAN-ICHI HAYAKAWA

APPENDIX B: COMPUTER PROGRAMS c

c

c

oooooo*ooooo*o,ooooooo*ooooooooo*oooooooooooooooooo~*oooooooooooooovoo

oooooooUOouoooooooooUoUooUUOoOooouOooUOUOooUUUUOooOoooUuUOuuOoO~~oO~UOo ooooooooooooooooo.oooo*ooooo*oo*oo*ooooooooooooooooooooo*o*oooo*oooooo

c ooooOUooOuoooooooooooUUo~OUoooo~oUOooUUOUooOUUUooooouoOuOUO~UoO~~oOuO~o C C C M A I N PROGRAM FOR H E A l P'HOttSS E V A L U A T I O N l H A Y A 6 A k A . CMLET I N 6 I

A I C H E 1 Y ? 4 DECtMULH

c

C C C C C C C C C C C C C C C C C C C C C C C C C C C

N 0 M E N C L A 1 IJ y t 0 0 0 ~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 ~ ~ 0 0 C J * 0 2 5 0 , F H l s F h L ' F n d , FPI b t l l r bMLt H J - TG. T M G ~TU, I W ' T I * F SLE S U R H O U l I N t SETU.

00000000000000000

CI

0000~000000000~000000000~00000000000004000000000000000~*00~00000000000

1 p U I 0 A T A QOOOOOOOOOOOOOO~OOOOOOOOOOOOOOO CODE FOR I U t N l l F Y I N b PHOCtSS O A I A . THE NO. OF CHARACTtHS WHICH l F r L L U D t 5 BLANK SPACC5 SHOULU BF LOUAL 10 UP L L 5 5 THAN AU. 2ND CARO TO' T l r l W i HJ' F e l t t H 2 r FHdi bMl FORMAT5 A H t Uk10.0. 3HD CARO GI321 CJI L I O L > O s 2 1 161 IMG* F P FORMAT5 AHL Ub 10.0. REPLAT LARDS 1 , 2 9 AND 3 WHEN I H E R t AHE k O H t THAN ONE 5 t l OF DATA FOR PROCESS E S T I H A I I U N . 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 0 SUMPHOGHAMS R t O U I R E U O O O O ~ O O O O O O O O O O O O O O O O O O Q ~ O O O O O O 1. SUBHOUIINE COOL' COOLA' FCOL, L U I F t HEAT, HATE' 5 t T U s > I M P 2. F U N C T I D N FTG' F X i k T * TLX 3. M O C K UATA SUBPRUGHAH DEFINING D A T A OF AML b n ooooooooooooo~ooooooooooo*ooooooooooooooooooooooooooo~ooooooo~~ooooaoo ~ 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 0 0 Iy

IS1 CARO

...................................................................... ...................................................................... CUMMON/COHA/AMC~Il/COMh/hl7l

D I M E N S I O N C O D E f 6 U ) r S Y H S ( 8 0 ) . SYML(BU1 DATA CODES SYHR' SYHC/ttOO* 1 . U O * ' * ' r LIUO'0'/ 1 0 0 R L P D ( 5 , r 1 , ENO=COUl CUOE 1 FORHAI(UO&ll O R t A D ( 5 r 2 1 1 0 s 1 1 9 T W * HJ, F H l t F H L t F H d r G B l r GI321 C J t Cv UC>U* 1 2 , T b i THGv F P 2 FORMAI(BFIO.OI

5 Y W L 1 CODE 1x9 ~ U A I / / I X I U O A I I W I T E ( 6 r 51 T O P 1 1 ' T * r HJ' F H l r t H L t FH3r GI319 G 8 2 * LJI CI 2 50F0HMAl(lX1///2lXv ' I N P U T DATA USEU FOR t S I I M A T l O N * / / 1 4 X ' '10 = F7.C' 4 x 1 'TI = 1 ' F 1 . 2 1 4 x 1 ' T W = ' 9 F7.2/4&' 2 'HJ ' 9 F7.2' 4x1 ' F H l F 7 . 2 ~ 4 x 9 ' F H 2 i 1 , F1.L. 4x1 3 *FH3 F7.2/4X* 'Ctll ' 9 F7.L. 4 x 1 'btl2 = ' 9 F7.C/4Xr 4 'CJ = ' 9 F7.2, 4 x 9 ' C = F7.L. 4 x 1 'L = ' 9 F7.C) IF(FP.bT.O.1 GO TU 6 Y R I T E ( 6 v 81 8 F O H H A l ( c l X * ' T H I S 1 5 A I Y P E A l"WOt(LtH'1

Y H I T E ( b r 4 1 SYMM.

4 FORMAI(lHl//lX*

UUAI/

'*

**

@

YKITEIbr

1 0 1 TGI

10 FCIRMA114X* 'TG = I f (U25U.GT.O.l GU

*

IMd

**

ti.27

QXr

'TM8

=

1 1

tl.2)

10 I

WhITL(b, Y l Y O F O H H A 7 ( 4 X * 'A C H I l l C A L P O I N I 5 I E R I L I L I N G VALUC IS U5EU AS P C H I T t R lION'/IUX* 'FOR P H U L L S b E S T I M A I I O N ' I W k I T t l 6 t 1 1 1 FPP

ESTIMATION OF THERMAL PROCESS 1 1 F O H M A I l l l X ~ "5iTtklLlZINb VALUt ESIIMATEU

1.1

111

f8.31

60 10 1 0 0

7 WkITEl6. 121 D 2 5 0 1 2 O F 0 H H A 1 1 4 X ~ 'DZ50 ' 9 t7.2/ 4 x 1 ' M A 5 5 AVtHAGL S T E H I L l L l N 6 VALUE I 1s USEU 4 s I CWIltHIUN'/ 1 0 x 1 ' F O R P H U C E 5 S tSTIMATIUN'1 WHITk16, 13) F P P 13 FUt?MA115X* ' M A S S AV. 5IERILILING V A L U E t 5 1 I M A T t O F8.3) G U TO 1 0 0 6 W k I T C l 6 * 14) 14 F U U M A I ( 4 X * ' T H I 5 I5 A IVPE tl PkOBLtM'l Y H I T L l 6 r 15) F P 15 F O R M A l l 4 X * 'FP = ' t t 1 . Z ) IFlU25O.GT.O.) G O 10 16 Wk'ITEI6,

Y)

WHITt16,

20) T A h 5

20 FORMAllllAr 'PROPltH PHIJCESSINCI T I M E t S T I M A I E D GU TO 1 0 0 16 n N I T k i 6 .

121 I X ~ U WRIlkl6r LO) 7ANS GO TO 100 200 S T O P €NU

Fb.3)

KAN-ICHI HAYAKAWA

112

c ooooooooooooooooooooooooooooooo.ooooooooooooooooo*oooo*o*o~.oooooooo*o c uooOouoOoOuOoOoooooOOuOuoOuooooOoOOooOOOOooooOOooooooooooO~oOooooOooLJo c oooooooeoooooooOoooooooooooooooo*ooooooooo~oooooooooooooooooooo*ooooo~ c OooOOuoooOOOoooooOo~OOu~OuO~oOo~oOOooO~OOooOOu~ooOOooooooOOoOooooOoOoo C C C C C C C C C C C

C C C C

C C C C C C C C C C C C

C C C C

C C C C C C C C C C C C

c

C C C C C C C C C

S U B R O U T I N t SUMPROGRAM k O H t S T I H A T I N G I'HOPER H t A T PHOCESSE5 Of C A N N t U FOOD. E M P I H I C A L FORMULA5 OF H E A T I N G AND COOLING CUHVES OF FOOD, WHICH WEHt UEVtLOPEO BY HAYAKAWAIFOOD TtCHNOL.9 2 4 1 1 4 0 7 1 1 9 7 0 ) 1 r WtRE U5EU I N THIS PHOGRAMlPHEPAHtU UY K. HAYAKAWA. OCI.9 1974) N U M t N C L A T IJ H E 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SLOPE I N O L X O r COOLING CUHVE(MIN.) I N l E R C E P T C O E k F I C l t N T Ok COOLINB C U H V t AT C R I T I C A L P O I N T I N CANNED F O O U l U 1 M E N 5 I O N L t S 5 I . 0 2 5 0 L I t C I M A L R E D U C l l U N l l M E OF T A R b t l MICHOORGANISMS A I 250 OtO. F. WHEN A S T E H I L I Z I N b VALUL AT A C H I T I C A L P O I N T I N k O O U IS USED A 5 A C R I T E H I U N FUH PROCESS E 5 I I M A T I O N ~ SET LIZ50 -1.0. YHEN A MASS AVEHPCIt S l t H I L I L I N b V A L U t I 5 USE0 AS T H I S C H I T E R I O N * AN A C T U A L UZ>O VALUL SHOULD n t USEO I MIN. ). FHl SLOPE I N D E X O t H E A I I N G CUHVE. WHtN I H t H E A H t ONE O H MORE tlHEAK P O I N T S I N T H t CUHVEI T H I 5 R E P H k 5 E N I S THE SLOPE I N O t X OF A P O R T I O N OF IHt C W V E B t F O H E T H t 1 S 1 HKEAK P O I N T I M I N . ) . F t i d S I M I L A R TO k h l t X t I ' T T H I S H E P H C 5 t N T S I H E SLOPE INOEX OF A PORl l O N OF A H t A l l N b C U H V t AFTEH THE IS1 BREAK P O l N l WHEN THERE AHL ONE OR H U H t BREAK P O I N T S I N T H t CURVEIMIN.). F H 3 S I M I L A R TO F h l t X t P T THIS H L P H E 5 t N T 5 IHE SLOPE I N O t X OF A PORTION OF A H t A l l N G CURVE A P T t H T H t LND BHEAK P O I N T WHEN l H t R t ARE TkU B H t A K P O l N l S I N A CUHVt(M1N.). FP TAHGET S T E R I L I L I N b V A L U t . THE SUHROUTINE SETU E S T I M A T E S A PROL t S S I N G T I M t wHILH PROOUCtS I H l 5 VALUt. WHEN ONE N t t D S TU E S -1. I I M A T E AN k P P VALUE FOH A G I V t N TG OH TMG VALUE, 5 t T F P IMIN.). F P P S l t R I L I Z I N G VALUE 10 BE E 5 T I M A T t U . T H t SUBROUTINE S t T U CALCUL A T E T H I S VALUL WHEN AN ACTUAL I G OW IMG VALUE 15 61VENIMIN.) GBI TtMPEHATURE O I k F E H t N C E BEIWEEN PO00 AND H E A T I N G MtOIUM AT 1 S T W E A K P O I N l O t H t A T I N G CUHVE. 5 t T btll = -1.0 WHEN THERE IS NO BREAK P O l N l I N A H E A T I N G C U H V E I F . DEG.). bBZ TtMPERATUHE D I k F E H t N C E UEIWEEN FOOD ANO H E A T I N G M t D l U M AT LNO W E A K P O I N T LJk H t A T I N G CUHVE. S t T bl3L = -1.0 WHEN THERE IS ONE OR NO B H t A K I'OINT I N A C U H V t l M l N . ) . HJ INTERCEPT C O t k k I C l t N T OF H t A T I N b C U H V t AT C R I T I C A L P O I N T I N CANNtO F O O L I ( U I M E N S I O N L t S 5 ) . TANS L t N G T H OF H t A l l N G PHASE TO BE E S I I M A T t O . A HEAT PHOCtSS WITH TANS MIN. OF P N O L t S S I N b I I M E PHODUCL5 A TARGET S I E H I L I Z I N G VALUL F P I M I k . ) . TG FOOD T E M P E R A l U H t A 1 E N 0 Ok H E A T I N G PHASE OF THLRMAL PROCE5.S. WHEN A P R O t l L t M I5 FOR t S l l M A l I N G TAN5 OR WHEN AN A C l U A L VALUE Ok TMG IS b I V t N 1 5ET TG = -1.0 IDEb. k ) . TMb L t N G T H OF H t A l l N G PHASE. WHEN A P R O b L t M I S FOR E S I I M A T I N G TANS O Y YHEN AN A L I U A L TG V A L U t I5 GIVEN. SET TMG = -1.OlMIN.). TO I N I T I A L T E M P t K A T U H t OF FOODlDtG. F ) . TW COOLING MEDIUM TEMPERATUHt(DE6. k . 1 . TI HOLDING TEMPEHATUHt OF H E A T I N O M t D I U M l D E G . F ) . Z SLOPE INDEX O t THtHMAL UEATH T I M t CUHVE OF TARGET MICROU H b A N I S H S t k . UEb.1. ooooooe*oooooooooe 1 N p u 7 A A ooooooooooooooo~oo~oooeoooo~oo 1. DATA H t O U I R E O FOh ANY P H O C E 5 S t S T I M A I I O N CI CJ, H J * TO, TM1 1 1 1 L 2. E S T I M A I I O N OF S l t K l L I L l N G V A L U t FOH C I I V t N lHEHMAL P H O L t S S I N G . O L 5 0 ACTUAL V A L U t WHtN A MA5S A V E H A b t S l t H I L I Z l N b V A L U t 15 TO d t t S T I M A T t . Ud>O = -1.0 WHtN A 5 l E H I L I L I N G VALUE A 1 A CHIIILAL P O I h T I5 T H t C H I T L H I O N . F H l r FHZI FH3 F h l = FHZ = k H 3 w H t N T H t H t I5 NO B H t A K P O I N T . k H l i F H 2 = F H 3 WHtN T H t H t IS U N t R H t A K POIhrT

00000000000~000000

C CJ

ESTIMATION OF THERMAL PROCESS C C

C C C C

C C C C C C C

C C C

c

C C C

C C C C

C C C C

c c

113

F H l . FH21 F H 3 WHtN THEHt ARE TWO BWAK POINTS. FP = -1.0 G B l r bud G t l l = GB2 = -1.0 CHEN THtHE IS NO 6KtAK POINT. 6619 6 8 2 = -1.0 WHtN THLHt I 5 ONE HWtAK POINT. b B l r GB2 WHtN THtHE A K t TWO BHtAK POINTS. TG Tb = ACTUAL VALUE WHEN T H I S VALUt I 5 blVEN. TG = -1.0 WHtN AN ACTUAL VALUE OF THG I 5 GIVtN. THG THG = ACTUAL VALUt WHEN T H I S VALUE 1s bIVEN. THb -1.0 WHEN AN ACTUAL T G VALUt IS b I V t N . 3. E S T I H A I I O N OF L t N b l H Uk HEATING PHA5t FHOM bIVEhr S T E H l L l L I N G VALUE DZSO, F H l . F H 2 r Fhdr # b l r GRL Stt AtlOVE. FP ACIUAL S T E R I L I L I N b VALUt bIVEN. TG = -1.0 THb = -1.0 **o~ao*oo****o**** 0 u p u T 0 A 1 A o.ot**e.*o.*QIoooo*aoooo**ao FPP ESIIMATED S l t k ’ l L I L l N G VALUL FOH l Y P t A PROtlLEHf ESIIMATION NO. I ) . OTHEHWl5t FPP = -10. TANS t 5 l I M A T E D L t N b l H lW HEATINb PHASt FOR IYPE b PROBLtH( E S T I M C I l O N NO. 2 ) . OTHtHYISE TANS = -10. OOO~QO*OQ*IQ*O**** SOU~RO~JHAHSHEuUIRtO ~ 0 ~ 1 0 ~ ~ t 0 Q I 0 I ~ 0 Q V 0 ~ 0 . Q Q Q I I I . 1. SUbROUllNE COOL1 COOLA, FCOL. k D I r r HEAT. H A T t r 51HP 2. FUNCTION FTGr F X r R T r TLX 3. BLOCK O A T A PROGHAM DEFINING DATA OF AUC b H

...................................................................... c ...................................................................... ~

o

~

~

o

o

o

o

~

e

~

~

o

~

i

~

o

o

o

o

~

~

~

~

a

o

~

~

o

~

~

o

~

o

C .

~

~

~

o

~

KAN-ICHI HAYAKAWA

OU 1 0 0 1

TI

=

I(I)

= I t 51

ESTIMATION OF THERMAL PROCESS

302 303

LO 50

21

8

23 Y

7

27 28 L')

26

30

304 46

45

32

3 1 NXY = I

115

116

KAN-ICHI HAYAKAWA

TH2(NX 1) = T O 1 (NX N X Y ) 0 L ) € L ( ~ ) TMMZ(Z) 4 8 IF(AU5(OIF).LT.l.t-5) 60 TO 3U5 GH2 0 10. O O ( - O I F / Fn3) TUX = I 1 GO TO 3 0 6 Gn2 3 0 5 TUX = I 1 NX hXY 306 NXZ = 5 1 IF(NXL.LE.1) b0 IU 34 T X Y = (NXZ 1) 0 U t L ( 1 ) CALL I i t A T ( 1 . v F H ~ , r w . 9 TI, -1.. I X Y ~N X L ~OELX. 00 35 I = 1, NXL

-

33 01F

-

--

-

-

TI = 1 1 1 )

11 = NA + NXY 35 T r i 2 ( 1 1 1 = T I

+

1

GO T O 3 b 34 It(NXL.tQ.1) bO 1U 37 It(NIL.LT.0) 60 I U 3 8 60 T O 36 3 7 T H Z ( 5 l ) = TOX W T O 36

36

40

41

25

42

TM.

I )

ESTIMATION OF THERMAL PROCESS

TOX = 11 bll 7 0 JOB

-

117

Gail

19 T H 2 l l U L ) TOX GO TO 5 0 C ALL t L E M E N l 5 I N VECTONS T t i t TH2t b T h M ARE t S l l M A T t D . 60 7U 5 2 2 2 IF lFP.GT.O.1 C THIS IS A l Y P t A PRUtlLtM. TAN5 = -10. Ik(TG.LT.O.1 GO T O 53 Ik(ABS(TG-TI+GRI).LT.I.E-3) GO TO 5 4 CHI) b O I 0 5 5 1FITG.LT.Tl IF(ABS(TG 11 + bUL).LT.l.t-J) GU 10 5 6 GBL) b0 10 5 7 IF(TG.LT.11 THIZUJl)~Ll.l.E-3) GO TO 513 IF(ABS(TG It(T6.LT.TH(203)) b O 10 5 9 GO TO bO 53 IF(TMb.LT.O.1 GO 10 IUL IF(AB5(THG T ~ B ( l ~ ) . L l . l . E - 3 ~ GO TO 5 4 IF(THb.LT.TMB(11) b O 10 6 2 IFIABSITHG THB(C)).LI.l.E-31 GO T O 56 IF(TMb.LT.TMtl(2)) b O 10 6 4 THM(LU3)).LT.l.t-J) GO 10 5 U IFIABSITMG IfITMG.LT.THH(203)) GU TO 66 GO TO 67 5 4 CALL SIMP(THr D t L 1 l ) r 5 1 9 2 1 kPH1 CALL FCOLlFPCt CJI Cv I H ( 5 1 1 ~I W v 2 ) FPP = kPH + FPC I f 1025U.LT.O.l GO 10 bU CALL 51HPlTHZt U t L ( l ) r 519 L t FPHJ L * T H L ( 5 1 ) * l n t 21 CALL FCOL(FPC9 CJ/L.r FPR = f P H + FPC FPP.LT.1.t-4) GO TO 1 0 0 IF(FPK FPP FMAS(FPP9 FP'Ht OL501 GO TO 68 700 Y k I T E l 6 r 7 0 1 ) 7 0 1 0 F O R M A ~ I l H l 'NO ~ PN5WEH IS ESTlMATtO SINCE JEN t T AL"S FORMULA IS lNOT UbABLE*/ 1 x1 'UtCAUSE A CtNTRAL FP VALUE IS LARbkW IHAN AN OFF 2CtNTRAL FP V A L U t ' ) FPP = -10. TANS = -10. GO TO b8 5 5 1 = 1 78 IF(Al35ITHII) TG).LT.I.E-31 GO TO 6 Y IFlTHlIl.LT.T6J GO TO 10 I f (I.bt.2) GO TO 7 1 77 V R I T k ( 6 r 7 2 ) 7 2 F O K M A T l l H l r ' T H t Y t 1 5 NO ANSWtH FOR THIS VHOBLtH S I N C t I G < T O * ) FPP = 0. GO TO b8 / I J = I 7 6 T b H ( 1 ) = THII I1 TbM(2) = T H ( I 1 DU 7 3 11 = 1 9 2 CALL S I M P ( T h t D t L l l l r I + 11 2 1 Zt FPHI C A L L FLOL(FPCt CJI C t I b M ( I I ) * TW* L l 7 3 F P M I I I ) = FPH + FPL FPP F X I F P M ( 1 ) r P V M t L r 9 T G M l l l r IGMI219 I b ) IF(D25O.LT.O.l GO I O 68 I6lJ.tO.21 GO TO 14

- --

-

-

f

-

-

-

I18

KAN-ICHI HAYAKAWA J = J + l F P l = kPP DO 75 I k = 1. 3 0 0 TH1 = I H 2 ( I X ) 75 T H ( I X ) = TH1 C J = L J / 2. T G Z X = TG TG = I 1 (Tl I b 1 / 2. GO TO f b

-

-

1 4 FP2 = kPP IF(FPL kP1.LT.l.t-4) GO T O 1 0 0 FPP = F M A ~ ( F P I . brdt U L ~ O ) CJ = LJ 2.

-

*

Tb

= IbZA

GU T O 68 69 IF(l.tO.1) GO TU / I CALL 5 I M P ( T H t 0 t L I I ) t I t 2 1 FPH) CALL ~ L O L I F P C I L J t Lt I H ( 1 ) t 1 W t L ) FPP = FPH FPC IF(LJZ50.LT.O.) b O 10 b U CALL 5IMP(Th2i U t L ( I ) t I t 2s k P H ) C A L L FCOL(FPC1 CJ/Z.r L I T H L 1 1 ) t I # * Z) FP'H = bPH FPC IF(FPM FPP.LT.1.t-41 bO T O 100 FPP = ~ M A ~ ( F P PkI V H t U C L O ) GO T O 6 h 70 1 = 1 + 1 IF(I.Lt.51) GU TO f t ) GO T O 309 62 1 = 1 6 2 I F ( A ~ S ( T H M ~ I ) - T M b ) . L T . I . E - 3 ) bO T U 6 Y I F (THM(l).LT.TMb) btJ 10 8 0 GO T O 8 1 IF(1.6t.2) GO TO 17 IMG. T H M ( I - l l t 8 1 TG = k I G ( T 1 t T H ( l ) r T t i ( I - 1 ) t 6 0 TO I 1 80 I = l * l IF(I.Lt.51) bO T O a 2 309 CHITL(6t 310)

-

OEL(1))

ESTIMATION OF THERMAL PROCESS

119

TI

86 TkS(I1

CALL S I H P ( T H S I O t L ( 3 ) * 101. 2.9 FPH-i) C A L L FCOL(FPC, L J , L, I H ( Z O J I , T W , L I FPP = F P H l + FPHL FPh3 kPL Ik(D25U.LT.O.) b O I U btl CALL 5 I M P I T h 2 , UtL(I), 51, L I f P H I ) 00 t17 1 = I t 51

T I = lHc(1 + 5 1 ) 87 T H S ( 1 ) = T I CALL 5 I H P ( T H S v O t L ( t ? ) * 51, DU d 8 I = l r 1 0 1

TI = inz(I

4

L,

FPHr'l

102)

88 THS(I) = T I CALL S I M P f T H S , D t L I J t , 1019 2 1 F P H J ) CALL FLOL(FPC, L J / Z . r Lv THLIZ03), T W I FP'P = F P H l FPhZ + F r H 3 t f P L IF(FPK FPP.LT.1.L-41 bO TO 100 FPP t Y A S ( F P P . F P K t UZ50) bU TO b 8

L)

-

57 I = sc

Y1 IF(A8S(TH(Il

-

If(TH(l).bT.TG) I = l + l

16I.LT.l.E-31

bO TO 110

6 0 TO YO

If(I.Lt.102) GO T O Y 1 W l ? I T E ( b i '92) Y 2 0 F O H M A I ( I H 1 . 'NO F P P IS E S T I M A l t O 5INCE I > 1 0 2 AT ST. NO. 9 2 OF SU 1 8 k O U T l N t SETUO) FPP = 1.€+5 GO T O 66 9 0 IF(I.bE.531 60 T O Y 3 h K I T t ( b . 94) 9 4 0 F O H M A l ( l H l . *NO F P P IS E S T I H A I ~ O5 I N C E I < 53 A T ST. NU. 9 0 O t SUkJ 1 H O U T I N t SETU') FPP 1.E*5 GO TO 6 6 9 3 J - I Y 9 CALL S I M P ( T H r D t L I I ) r 5 1 , L, t P H 1 ) oc, r 5 n = 1 , 51

120

117

126

121

122

123

125

I24

11s 131

KAN-ICHI HAYAKAWA

121

ESTIMATION OF THERMAL PROCESS 127 ThSlKl = 1 K CALL 5lHPlTHSr D t L l 2 ) r 517 2' F P H Z ) I X = 1 102 OU 128 K = I r I X TK = I H I K + 1 0 2 ) 128 T h S l K ) = TK C A L L 5 I H P l T H S * U t L I J ) * 1 X r L r FPH3) CALL FCOLIFPC, L J r C s 1 H l I ) r I N * L ) FPP = F P H l + FPHL FPH3 FPL bO 10 6 U IFlD250.LT.O.l 1FlJJ.tQ.Z) GO T O 1 2 9 JJ = JJ + 1 FPli = bPP C J = C J / 2. UU 130 K 1' 3 0 0

-

Tn = I H 2 l K l

130 THIK)

=

Tt.

GO TO 1 3 1

-

129 I F I F P P FPH.LT.1.t-4) 60 TO 1 0 0 FPP = FMAblFPWr k V V 9 UL501 G O 10 6 8 110 IklI.tU.521 60 T O >4 CALL 5IMPITHv D t L l l l r 519 2 , Fb'Hl) 1k = 1 51 UU 1 3 2 K = 1 1 I X TK = I H I K + 5 1 ) 132 TMSIKI = TK CALL SIMPlTHSv U t L I L ) r 1 x 9 L. f P H L I CALL FCOLIFPC' C J I C * I n 1 I ) t I N ' L ) FPP = FPH1 + FPHL FPL b O 10 bU IFlOL>U.LT.O.) CALL SIMPlTH2* D t L I l ) , 519 L P F P H I I DO 133 K = I r I X TK = l H 2 l K + 5 1 ) 1 J 3 THSIKI = T K CALL SIMPITHS, O t L I L l r 1 x 1 L r FPHL) CALL FCOLIFPCs C J / C . r CI T H L 1 l ) r I Y r 21 FPH = F P H l FPHZ + FVC FPP.LT.1.t-41 60 TO 100 IFIFPI( FPP FHA5(FPPr F P M r OL50) G O TO 6 8 66 I = lU3 137 I F l A M 5 I T H H l I ) 1Mbl.Ll.l.t-3) GO TO 115 I F (THHlI).LT.TMG) bO 10 1 3 4 IFll.bt.104) GO I O 135 UCcITEl6r 1361 1 3 6 0 F O R W A l l l H l r 'NO FPP 15 ESTIHAILD SINLE 1 < 1 0 4 A T ST. kO. 1SUBROUIINt SETU') FPP = I.E+5 GO TO 68 134 I = I + 1 IFlI.Lt.203) ti0 1O 131 YHITElb* 138) 138OFORMAll1Hlr 'NO F V P 1s E S T I H A l t D SINLE 1 > 203 A T ST. NO. 1UtrROUllNE S E T U ' ) FPP l.E*5 G O TO btl 1 3 5 TG = F l G I T l r THlIlr THII-111 lHG* 1 H M I I - l ) r O E L l 3 1 ) 60 TO 1 1 7 60 I F I T 1 TG.GT.1.t-3) bU TO 111 IFlAM5lTl TGl.LI.1.t-3) GO 10 3 1 4 UHITtlbr 3 1 2 ) 3 1 2 0 F O R H A l l l H l r 'NO FPP IS L S T l M A l t D MY SUMHOUTINE SETU 5 l k C t l * / l A * 9PWOBAMLLY YOUH t R R O R . t'LEASt CHtCK YOUH O A T A ' I GO TO 68 3 1 4 Y W I T t l C * 315) 3 1 5 0 F O R M A T l l H l i 'NO F V P IS E S T I M A ~ ~ t l YD 5 U W O U l I N E S t T U SINCE

-

-

-

1 3 6 OF

138 OF S

- -

TG > 11.

TG = 11.

122

KAN-ICHI HAYAKAWA

l*/ 1 X t *TRY AGAIN UY LNTERING A COHHtSPUNUING TMG VALUt INSTEAU OF IA.*IHANX FOR 2 * / 1 X t ' T H E TG VALUtr Y H I C M YOU HAVt JUST tNTEHcD.*/ 3YUUH PATIENCE') GO TO 6 8 ALOblUI(Tl-TH(2U31)/ (11 1G)l 3 1 I THD = F H 3 OtLE = TWJ / 50. 1 4 6 TH(204) = T I i ( 2 0 3 ) TM2(C'04) = T I i 2 ( 2 0 3 1 00 1 3 9 K 1 9 50 T1M = DELt * K TM( K + 204) = I 1 (I1 T H ( C 0 4 ) I 0 10. a * (-TIM / F H 3 l 139 TH.?(K 204) = T I ( I 1 -THClLO4)) * 10. ** ( - T I M / F H J I J = l 1 4 5 CALL S l M P ( T M r O t L ( I 1 v 5 1 9 Z r F P t i l l DO 140 K l r 51 TK IM( K 511 1 4 0 T M S I K I = TK CALL SlMP(THSt D E L I L ) t 519 L t FPHC) 00 1 4 1 K = 1 r 1 U I TK = 1 M ( K + 1021 1 4 1 T M S ( K 1 = TI( CALL SlMP(TIiSv O t L ( 3 ) v 1 0 1 1 2 9 FPti3) DO 14L K = 1 r 51

-

--

Th

= IM(K

-

203)

1 4 2 T k S ( K 1 = TK CALL SlMP(TMSt U t L t r 3 1 9 Z r FPH4) CALL FCOL(FPCr C J v C, l H ( 2 5 4 l v TUv Z ) FPP = F P H l FPnL F P M 3 + FPn4 + FPL b0 10 b8 IF(O23U.LT.O.I IF(J.tU.2) GO TO 1 4 3 FPl = rPP J = J + l 00 144 I X I t 300 THI = TH2(I) 144 TM(1XJ = THI C J = C J / 2. GO TO 1 4 5 1 4 3 FP2 = FPP IF(FPC FP1.LT.I.t-4) GO T O 700 FPP = FMAS(FP1v F P L t U d 5 0 ) CJ = CJ 2. GO TO 6 8 6 7 DELE (TMG T H W ~ L 0 3 1 1 / 50. GO T O 1 4 6 C PROGRAMS FOR TYPE B PHOBLtMS BEGIN FROM HERt. bO 10 I02 5 2 IflTANS.LT.0,) FPP = -10.0 CALL SIMP(THv D t L ( l ) r 5 1 9 Z r F P H l ) CALL SlMP(THZr U t L ( 1 ) v 51, Z t FPHA)

-

-

~

I D 0 154 IF(I.Nt.501 GO T O 3 1 6 FPP = U. GO T O 147 316 CALL kCOL(FPCv C J v C t l H ( 5 1 - 1 ) ~ T W t L ) FPP = F P H l FPC GO 10 147 IF(Di?bO.LT.O.) CALL kCOL(FPCv CJ/L.r Ct T H d ( 5 l - I ) t 1Ur 2 ) FPQ = FPHA FPC IF(FPU FPP.LT.1.t-41 GO TO 700 FPP = t M A S ( f P P t FPUI UC'SO) 147 I F ( A B ~ ( F P - F P P 1 . L t . l . t - 3 1 b0 T O 14U IF(FP.LT.FPP) GO I U 1+Y

-

THM(51 1 4 8 TANS GO T O 6 8

-

11

ESTIMATION OF THERMAL PROCESS

123

149 I = I 1 IF(I.Lt.501 GO 10 1 5 1 WHITE(6t 1521 1 5 2 0 F O R M A l ( l H l * 'TANS tSTIMATE0. IS Q U t S I I O N A b L t SINCE I > 5 0 A T 5 1 . NO 1. 149 OF S U H R O U I I N t S t l U ' ) TANS = 0 . GO TO 68 151 CALL F O I F ( O E L F I I l r T H ( 5 l I + 1). I H f 3 l I ) , DELll)r LI FPHl FPHl DtLt If(O2SO.LT.O.l b0 10 153 CALL F U I F ( D E L F t I l r T H L ( 5 1 I 1 1 9 THZOI 11, O E L I I l t 7) FPHA FPHA DtLt 153 FPH = FPP GO T O I 5 4 1 5 0 DO 155 J = 1 , 51 TJ lHlJ 51) 155 T t i S t J ) = T J CALL SIMP(THSv UtL(Zl* 51. L t F P h L ) 00 156 J 1' 51 T J = I H Z ( J + 511 156 THS(J1 = T J CALL S l M P ( T H S t D t L ( L ) * 51, F t FPH81 1 = 0 164 CALL FCOL(FPC9 C J t C t I H ( l 0 2 1 1 9 I W I LI FPP C P H l + FPHZ + FVC GO 10 157 IF(O25U.LT.O.l CALL kCOL(FPC9 C J / L . s C t THt(lO2 1 ) ' I W r 21 FPQ = PPHA + FPhH FPC IFCFPU FPP.LT.1.t-41 GO T O 1 0 0 FPP = FMAS(FPP* FPUs UZ5U) 157 IF(ABS(FP FPP1.Lt.l.t-41 b O TO 158 GO 10 1 > Y IF(FP.LT.FPP1 IF(I.tU.01 GO TO IbO TANS FX(THH(1UZ 111 TrlMllUZ I + 1 1 s FPP' F P R t F P ) GO TO 68 158 TANS = THM(102 11 GO TO 6 8 159 I = I + I IF(I.Lt.50) GO TO 161 U R I T E ( 6 t 162) 1 6 2 0 F O R M A T ( l H l t 'ESTIMATEU TANS I5 Q U t S l l O N A B L t S I N C L I > 5 O A T 51. NO 1.159 Or SUBROUTINt ScIIU'1 TANS THH(521

-

-

-

-

-

-

-

-

-

-

-

-

-

OtLk FPHZ FPH2 IF(O250.LT.O.) bO 10 l b 3 CALL P O I F ( O E L F + 1 1 , T H L ( l O L - I * I l r lHd(lUZ-llr FWe = F P H ~ DtLt F W = kPP GO T O I 6 4 CONTINUt OU 165 J l r 1U1 TJ = I H ( J + 1021 ThS(J1 = T J CALL S l H P ( 1 h S t U t L ( 3 ) r 1 0 l t L t f P H 3 ) 00 1 6 6 J I r 101 T J = lH21J IOCI THS(J1 TJ CALL 5 l M P ( T h S * O t L I j I r 101, 11 FPHC)

-

1b3 160 165

166

I = O

-

1 7 4 CALL CCOL(FPCt C J * C, I H ( t 0 . l I ) * IN* LI FPP = FPHI FPnZ FPHj CPL IFIDZ5O.LT.O.) b0 IO 1 6 7 CALL CCOL(FPCI C J 1 d . r C , THZ(ZU3-l)r T W I L ) FPQ = kPHA + FPHU FVHC + k P C IF(FPO FPP.LT.1.t-6) GO T O 1 0 0

-

UEL(L)* L )

KAN-ICHI HAYAKAWA FIJP = FMAS(FPP. t r u * ULWI 1 6 7 I F ( A M ~ ( F P FPPI. L t . I.€-31 GO TU I68 IF(FP.Lt.FPP1 GO I U 1bY IF(l.tO.0) GO T O 1 1 0 TAN5 F X 1 T H M ( 2 O d - l l i 1 H M ( 2 U . 3 - I + l I * FPP, t P K * t P 1 GO TO 66 11 1 6 8 TANS = THMlZ03 GU TO 6 8 169 I = 1 + I IF(l.Lt.lUO1 GO 1U I 7 1 W H I T E ( 6 9 1721 1 7 2 O F O R M A l ~ l H l t 'TANS t5TIMATED I5 OUtSIIONAULE SINCE I > 1 0 0 P T 51. 10. I 6 Y OF SUtlROUllNt 5tTU'l TANS = THM(1031 GO T O 66 1 7 1 CALL FDIF(DELF9 11, T H ( 2 0 3 - l * l l r I H ( Z 0 3 - 1 1 , D E L ( 3 9 LI FPH3 = FPH3 DtLt I F (DZ50.LT.O.l GO I U 113 CALL FDIF(DCLFv 1 1 , THL(203-1*11 9 THCI203-I1 9 UEL 319 LI FPHC = FPHC DtLt 1 7 3 FPH = FPP GO T O 174 170 TH(Z041 = T H ( 2 O J l T k 2 ( 2 0 4 1 = TH2(CUJI THMIt204l = THH(LUJ1 DtLE (FP FPPI 4 1. / HT(Tlr 21 * 1.b / S O . DO 1 7 5 J = 1 9 5 0 T I M = DELE * J THM(204 Jl THH(LO41 + T I M TH(Z04 + Jl = T I (TI TH(20411 10. ** ( - 1 l h / FHJI 1 7 5 TH2(204 + J ) = T I (11 THZ(Z0411 10. 0 4 ( - T I M / k H 3 1 DO 1 7 6 K = 1, 51 TK = TH(203 + K l 176 T H S f K I TK CALL 5IMP(THS* D E L t r > I * 2 1 FPH41 00 1 7 1 K l r 51

-

-

N

-

-

-

--

TK

--

*

lMZ(L03 + K I

177 T H S ( K 1 =

TK

CALL 5IMP(THS. U t L t i > I t 2 1 FIJHDI 1 - 0 1 8 6 CALL t C U L ( F P C * L J * C , l H ( 2 5 4 - 1 1 ~ 1 W q 21 FPP F P H l + FPHC F r H 3 + tPH4 FIJC If(lJL50.Lt.O.l b O 10 1 1 8 CALL tLOL(FPC, L J / t . v Ct ThC(C54 1 1 r I W . 21 FIJQ = FPHA + FPHD + FIJHZ kPHU flJL It(bPL) FPP.LT.1.t-41 bU TO 1 0 0 FPP f M A b ( F P P * FVU. ULSOI 17M IF(AD5(FP FIJP1.Lt.l.t-31 c10 T O 11Y I t (FP.LT.FPP1 GO I U l 6 U Ik(I.tU.01 GO T O 161 Tnh5 = F X ( T H H ( 2 5 4 - I 1 t l H M ( L 5 4 - 1 * l l i t P P * tIJt3. kP1 6U T O b 8 1 7 9 TAN5 = THM(Z54 11

-

-

-

GO T O 68

-

1 8 1 WN(ITt(6r I 6 Z I lUZOFORMAT(1Hlr 'NO 1AN5 I5 C S T l M A l t D SINCE IANS > T H M ( L 5 4 ) A T S T . NO. 1 I U Z OF SURROUTlhrt 5 t l U ' l GO TO 6 b leu I = I 4 1 IFtI.Lt.SU1 GO 1 0 I U 3 W H I T E ( 6 1 1841 l b 4 0 F U R M A l ( l H l r 'TANS t S T l M A T t 0 I5 QUtSIIONAULt S l N C t I 50 A T S I . NO 1. 180 OF 5UflROUlINt S t l U ' l GC, T O 66

125

ESTIMATION OF THERMAL PROCESS

-

CALL f O I F l D E L F * l l r T H d ( 2 5 4 - I * l l v FPHO = FPrlD DtLt l a 5 Fr'R = k P P GO T O l M 6

702 WRIIL(6.

lHL1254-lJr

21

OELE'

7031

7 0 3 0 F O R M A l l l H l ~ 'NO AN5WEH IS E S I l M A T t O 5 I N L L FPs THG b T b AH€ ALL NEG I A T I V L VALUES.'/ I A , 'I'LtASE CliLCK YOUH INPUT D A T A . ' ) 6 M CONTINUt RLTUHN

END

c ***Uoo**~o*******ooU********o***ooo*********ooU*o**oo****~**o***o***** c oooOOOoOOOOooOOOOOoOOuOOOOOOOOau~~UaaO~OOOa~OOOOo~OOOOoUOOOOOOOOOOOOOO c o*o*.*****o**oooo*ooo**ooooo***~**oo*o*o*oo**oo*o~**ooo**ooooooooo**o* c oooooooooooooooooooo~u~~~~~aao~v~~~oov~~~oo~~v~r~oava C C

C FUMCTION SJBPHOGHAM

bUK L ~ J I I M A T I N MASS b AV.

"

5 l t H I L l Z I N G VALUt BY

C USIhb JEN t T AL'S FOHMULA. C * 0 0 ~ 0 ~ 0 6 I * O Q O * O * O #N 0 M t N C L A 7 H E O ~ O O C DXX DLLIMAL HEDC~CIIUNIIMt A T L50 Dtb. t I M I N I . C FXX CtkTRAL FP V A L U t I M I N l . C FYY OFFCENTHAL PP VALUElHlN.1.

c

~

~

~

O

~

O

*

O

~

......................................................................

O

~

FUNCTIUN FHAS(FXX, t r r a O X X J IF(FYY FXX.LT.l.t-IUJ GO TU 1 0 tWAS = FXX U I X * ALOGIU(1DXX + 1U.Y 0 IFYY FXX 1 ) I / OAXI GO TO d 1 WkITE(6, 31 3 0 F O H M A I I l H I . 'FYAS LUNNOT Mk t b I l H A T L U BY PUNCTION FMA5 SINCk P X X > 1 bYY'1 CALL t a l l 2 HLTUKN END

-

-

~

~

~

O

O

~

KAN-ICHI H A Y A K A W A

126 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

SUBHOUTINt SUBPROGRAM t O H C A L C U L A I I N G A 5 P E C I b I E D NUMBEH Ut FOOD T t M P A H A l U H t S ON A H t A l l N G L U R V t .

......................................................... Fti

SLOPE INDEX OF H t A I I N G CUNVt(M1N.) I N l t X C t P T C O t b b l C I t t v l OF r l t A T l N G C U N V t l D I M E N S I O N L C 5 5 ) NTHM NUMMLH O F FOOU l t M r t H A T U H C 5 T O M t E S I I M A T E D . THI5 NUMMELI M U S T tk GRtATEH l h A N L AND L t S 5 THAN OR t U U A L TO 3 0 0 . ( 1 N l t G E H 1 OIMENSIUNLES5J. T FOUU TEMPEHATUHtS t 5 T I M A l t U ( D E b H t t F.) TG FOOU 7 t M P t H A l L K t A 1 t N U OF H E A l I N b P H A b t l D E G R t E F.1 TM t i t A l I N 6 T I M E 5 A I wtilLH FOOU TEMPtHATUHt5 REACH TO 1 " S I T I M E J TMG L t N b T H OF H E A I I N b t'HASL(T1MLl TO I N I I I A L TEMPtMAIUHL OF f O U U ( D E b H t t F a ) TI TEMPEHATUHE UP H t A l l N G M t D I U M ( U t b H E E F a )

HJ

O

O

O

~

I N P U T DATA: Ftit TO, HJ, t I T H E l 4 Tb OH TMG GRAMS. WHtN A TG -1.0. WHLN A TMG

O

~

~

~

~

~

O

~

O

O

~

~

~

11. lbi TMbr NTHM V A L l r t MAY ME U 5 t U AS D N t O t I N P U T S FOR T H I S SUBPHOV A L l r t 15 USE0 AS AN INPUT, 1HG MUST BE EOUAIEO TU V A L l r t IS USEU, lb MU51 BE -1.0.

0~**0*********0**0*******0**O*0*****0*0***0****0****O**0****0********

OUTPUT DATA: 11 T M 1 DEL

..................................................................... .....................................................................

90

1

O

~

e

~

ESTIMATION OF THERMAL PROCESS ~ ~ F U Q M A I ~ ~' TXMI 6 i s l w t Jn < 0.045'1 2 T L = 0.3 * CH

I

t>llMATtO

R N = AN(HJ) kl = U A I H J t FH. H N ) I f (lb.LT.0.01 GU I U

Y 8

11 10

tl

TtMPL = TD(TLI IC(Tb.Lt.TEMPL) b U TU 9 THH = TID(l61 i n = TG GU 10 10 TMH = T I P ( d t l b r HNJ Tn = TCI 60 TO 1 0 I f (TMb.LT.TL) bU I U I 1 TH = TDlTM61 TMH = Th6 GO TO 1 0 T H = T A t T M G v R - HN) TWH = TMG T(1) = TO TN(1I = 0. D t L = 1MH / N X X T l N T H M J = TH T H ( N T H H 1 = TMH 00 1 0 0 I = 21 NXX TNI = O t L * ( I 11 TH(1) = TMI 1FITMI.Gt.TL) bO 10 102 T ( I ) = T A ( T M I t Uv H N ) GU TO 1 0 0

-

102 T I I ) TDITMII 100 C O N T I F t U t GO

ro

60

-

0.9 * F H * ( 1 . nJ) c) Mb(TL1 bU 10 19 IF(T6.LT.O.Ot TtMPL = TD(TL) IF(TG.LE.TEMPL) b O TU 20 T h H = TIOITG) TH = TG GO TO C 1 20 TMH = T l H ( t 4 v T G ) T h = 16 GO T O C 1 19 I C ( T M b . L T . T L ) G U IU << Tn = I u ( T M 6 ) THH = TMG

3 TL

= =

GU T O z 1 22 TH = l H ( t 3 i TMH

21 T ( 1 )

=

TMG)

TMG

=

TO 0.

TM(1) = T ( N T H M ) = TH T H ( N T H M ) = TMH D t L = IllH / NAX U U 30 I = 21 N X X T M I = IJLL * ( 1 I) TM(1) 5 TMI IC ITMI.6E.TLl b0 10 J Z T(I) = TbIBt I P l ) GO T O JO 32 T ( I ) = TD(THI1 30 C O N I I N U L GO TO 6 0 7 IF ( T 6 . L T . 0 . 0 1 bU I U 34 TMH = T I D I T b )

-

127

tJY 5 U U H O U I I N t H t A T AhL U U t S T I O N A U L k

KAN-ICHI HAYAKAWA

128 Tn = TG GO TO 35 34 Th = T D ( T M G ) THH = TMG

35 ~ ( i )= i n THfl) = 0 .

T(NTHM) = T H TH(NTHM1 TMH D t L = I H H / NXX DO 40 I = 2 r NXX T H I = UEL * ( I 1) TM(I1 = THI = TD(TH1) T(I) 40 cot41 I h U t GO T O 6 0 4 YHITE(6r 4 3 ) 430FOf4MAlllXr * T M 6 I L b l I M A T t U BY 5 U t l H O U I I N t HEAT A H €

-

6

45 44

47 46

57 55 60

UUk5TIONAtILt

ESTIMATION OF THERMAL PROCESS

129

c

ooooooooo.ooooooooaoooooooooooooooooooaoooooaoooooooooooooooooooooooa

c

ooooooaoooooa.ooooooooaoooooooooooaoooooooooo~oaoaoooooooaooooooooaoo oooooOoOOuOoOOoOOoouu~~otiUuO~O~u~OOoouOOuooU~UOOoO~~~oOoO~O~OOOo~Uo~oU

c ooooooooooOooooooOoooOOoOuOooooOouOooUouOoooOOo~ooOOooouoOOooooOoOoooO c C C SUBROUTINt SUBPROGRAM t O H t S T I M A T 1 N G A S l E R l L l L I N G VALUE t H O H TWO FOOO C TEMPEHATUHtS. THESE IWU TLMPERATUHLS A R t MONllURED DEL M I N U l t S C APART. THEY ARE HUNlTOMtD OUHINC, THE H E A I I N G PHASE OF IHCHMAL PKOC CESSING. C 0 ~ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0N M t N 2 L A T u H E OOtOOOOOOOOOOOOOOOOOOOOOO~O C OEL TIME INTEHVAL(t4IN.). C DELF S I t H I L I Z I N G VALUE t S T I M A T t O ( O U 1 P U T . M1N.I. C TH FOOO TEMPkRAIUHt. TH > TL (DEC,. t . 1 . C TL FOOD TEMPtRATUHt. I L < TH (DEb. t . ) . C 11 HOLDING TEMP. OF HCATING HtDIUM(OEG. F . 1 . C 2 SLOPE INDEX OF THtHMAL U E A l H I I M t CUHVEIF. OEG.). c o.oo.oaoooo.aooo.o~ooooooooooooooooaoooooaoaooooooooooooaoaoaoaoooooo C THESE TWO FUNCTION SbdWObHAMS A H t REOUINEO. F I G AND R T .

c c

C C

..................................................................... ..................................................................... SUBROUTINE F D I F ( 0 t L F r 1 1 , THr TLt D t L r L ) TH = t I G ( T 1 r THr !Lr U.5 0 U E L r 0.9 U E L ) DELF = DEL / 6 . 0 0 ( R I ( T L r 2 ) + 4. 0 H T I l M r RLTUHN END

2)

+

RT(THr L ) )

C

c

C C C C C FUNCTION SU8PHOGRAM f U H CALCULATING L t I t i A L H A l t VALUES C C pq t N C L A 1 u W t 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 0 0 0 0 0 0 0 C 00000000000000000 N T F O O D TEMPERATUHt(UtC,. F ) . C C Z SLOPE INDEX U t THtHMAL U t A T H l I M t C U H V t I F . OtG.). C ooooooooooo~ooo.ooooooaoooooooaoooaoooooooooooooooooooooooaoooooooooo C C C C FUhrCTlON t t T ( T t L ) IFIABStT 250.).Ll.l.t-5) GO 10 I TkAT = I T 250.) / Z IF(THAI.LT.-6.0) bU TU 3 R T = 10. 0 0 T H A I GO TO C 3 RT = I.OE-6 CiO TO C 1 H I = 1.0 2 HtTURN ElvO

..................................................................... ..................................................................... --

KAN-ICHI HAYAKAWA

c C FUNCTION SUBPROGRAM FUH L I N E A R I N l t R P U L A I I O N . C T H I S PROGRAM IS FOR t S l l M A l I N G THt V A L U t OF A uEPLNUENT V A H l A B L E i F X i C YHICH CORHtSPONDS TO IHE VALUE OF AN I N U t P E N U t N T V A R I A U L t i T X . C INOEPENUtNT V A H I A V L t UtVtNOENT V A H I A t j L t

...................................................................... .................... -----------------C C C

IU

C

c

FA Ftl FX

1A 1x

...................................................................... ......................................................................

o~oo.*ooOoooooooo.oooooovo.oo*.ooooooooooo~~.ooovooooo*ovooooo*ooovvo

C C

-

-

FUNCTION F X ( F A 9 F U r T A v Tt3. FA = F A + ( T X I A J 0 (FB RETURN END

IIO FA) /

(

18

-

IA

1

C C C

C

C C C FUNCTION 5UUPKOGdAM f U H L U b b H I T H M l L I N l t k ' P O L A I I O N UF T t M P t k A l U k t C H t A 1 1 h G CUHVE C C

C C

C C C

C C C C C C

C C C C

..................................................................... .....................................................................

1 2

3

Ulu

N

m

r

c

m

*

-c

T. .

-

no

-4-

a

-ffiC

1

TIC D - c

To

0

7

+ 7-

D m r -D

.

-

~

.

o 20.2

-

h

-

rlnr

D

-uc

O

I\-

.-

m w ~ n n d - - clioc--l-l i ocnw z r - x ~ a Cr D~D - c xC - c c c+nrr D r r - - n 1 I Z c r r i ~- ~ r r ~C r P K- I x I1 I1 c 0-zco 2 x c c xc.u C U Z C * D C t L D C - I 1 - \ TI+c +on II 11 o n -

W

~

xr-u.

D C D 0

0

r-r-W

e

M O O N +

rnlLn*

a

.

C

O

- ? * -

cr-*

C X O .

~

0

l i a 8

<,-a

(I

F *

0

e

*0

t

f

9

. * 8

-.

*

T

1

u *

*

- c t

D r E

x c t

P

D D U

... ..... .... .. .. .. ..

.. .. .. ..

o c w 0 ..r b d . * .

*

........x z - * .. C * C t C * C *

t

C

C

OCO

O

G

0

C Q C D E O C t

-

L

P

C

C

I

r

r, C c

D

1

c

x

P.

-

z

D

C O G 8

c o t *

c

* G O

c o c *

c*c* c c

c o t t

C E C O

C E C d c o 5 0 C C C L

c

C E C O

c*c*

c o c o c o c +

C E C t

c":c": c o c

C O C 4

c*c* <

c o s a C O - 9 c o o t 2 C

h

c o c o

C * C * C D C O

r

z

--

v

2 * 3 *

= E C O

C 9 C O

C

c*c*

c 4o cc : t

c*c,*

C t C Q

c * c * c c c *

cote

C * C *

c c c u

c

c t c *

C t C t C C C Q

c *c*

-m

D

"I

-3a

-

I

u

(I

-

x

c

7

C I C l

c o o 9

C

t

7

c*ct

C 9 C f

- I , *

C O G *

C L C t coca

C D C t

c o c o C O C O c o c *

'=*co c t c o

C E C 9

cote

c e c a

o*c*

z

c c

Z

c uu

v

.(I

(I2

t r

x +

C C

no

-u zc ca r

4

4

. C

*

I rub-

r r c

.o

c

-c: c0 n c

L

G C -

c c

-c

0C-

c

cor -uc -L c cc

cc-i

00 -0

..r

4-42

I

c c

z

-7

--2
--7

z r r c 0-r c - 4 z I1 c

mz-4c

.. .. .... .. .. .. .. . . - ... . .... .. .. . .-. . ... .. .. .. .. .. .. .. L

4 0

L

.

c

r

2-s cz c r

-r

-0c

DO

I Fc-

-nc r D

0

z -4zn

T

L . 4 4

t

4

I

2

c

vl

z

rr

.. .. .. ... .. ... ..

t

4 4 4 4

i

4 L

:: zI :

C. x, c r

4 c - 4

.. .. .... . .. .. -

a

.. .. ... . .. .. .. .. .. .. .. .. ..

T

rrcc

orz c

IT

- l c o

T Z *

rr-c 4 vlrc 4 v l 4 m 4 . . - c t m - 4

0

- v l 4

r z r

L

4 4 4 c - 7 4

o n n r ) n n n CI P n c,n n n r. n nn

c

N

c

U.

c-

QXJ? 0-1

- * r

C

. D

emc .coil.

.

c N

X

c

w

\

<

D

2

.

N

<

X

.-

t

c f

I

N

-

4

2

+

-

- I -

-X

*

-.

N -

r

-.

- l x

I -

-urn r

c

I1 -I I1

TCT

c-

I

z

I1

X7O-l

W

-I

D

c

A

C

2

F

n

2

c

u)

w c

c-

IF

-c

I1 II F,x

- . .\ - --

-z

1-12

2 - 2 2 Ou)

-4 I1

L

N

c

I-.

W’

zz

C-

0

c

C

0

Q

t

T

-

n

h

.

?

z

?

c

F

<

o w

e r

W-

I <

2 F ENN -v:

0

o c 1 zc 2 2 . 2 2 u.c Ic- c m z c c-

2 I1

-

I-.C

CI-.

-a z g

.. .. ..

t

0.

0

-c

no

0

*t

c

r-c-m-.

rm.

C 4 Z D

OD-

*

KAN-ICHI HAYAKAWA

134 C C C

c

C

c

C SUtlHOUTINt WbPHOCIQAPI FUH L A L C U L P I I N G S t V E h I t M P E H n l U R t 5 Ulu A C U H V I L I C N t A R YOHTIUN UF A CUULlNcr LUHVt. I H t S t I t M P t K A I U * E . S A R t U S t U T O CALCUC L A T t A 5 T t H I L I Z I N b VPLUt UY U S I N b 1HE S t V € N t'U1'VT L O M A T 1 0 UUAUUATUHk C FOWMULI. M t N C L A 1 U H t VVVVVVOOVVVVVOVVVVVVVV C V O O V V O V V V V V V O V V V V V V O V V ,q CJ 1 N l t r ( C t P T C O t t F l C I t N T Uf CUOLINCI L U H V t ( U I k t N S I O N L t 5 5 ) C FC 5LUk't I N O t X U t LUOLING C U H V t ( k 1 N . ) C T 5 t V t h T E M P t R A I U ~ t St S T I M A T t U ( 0 t b K t t b . 1 C TCI FOUU T t k P E A A I b H t A l 8 E G I N h l N G UF L O O L l N 6 P H A S t ( D k 6 H t t F . ) C TL LtNCITH OF L U H V I L I N t A K P01(71ON U t LOOLING CUHVt(M1N.) C Tk CUULINb T I N E 5 A l WHICH S t V t N T t M Y t e A l U H t S A H t E S T I H A l t D ( M I N . ) C TC TtMl'tHATUHk O t L U U L I W MtDIUM(UtCIHEE b e ) C

"

c

vovvvvvvvovvovovvvvvvvvovvvvv~vvvvv~v~vvvvvvovvvvvvvvvvvvvvvvvvvvv~vv

C INPUT DATA: FCI Tbr CJI C C OUTPUT U A I A : TI TLI TM C C

C C C

TW

..................................................................... ..................................................................... S U b H O U I l N t C O U L ( L J , F L * TLI CUHMON/COMA/AbC I II DIMtNSIUN T h ( 1 ) . I ( / ) O T X A ( Y * B Y . Y N ) = IW + (TG 1 0 (l./YN)l) TY) TXB(YI B Y ) = T Y + ( 1 6 T X C ( Y * dY) = TW (Tb TW) T M X ( X 1 T K ) = TI( / Z . + TK

--

OG

I

50

=

=

0.

WKITk(br

12)

TM(I)

50 T ( I ) = U. IF(LJ.Gt.U.045)

10

-

lbr

TWI

rw)

0

00 00

X /

It41

I)

txv(-L..iu25M5

(CUTAN(BY * Y (CUS(UY0Y))

~XP(ALUG(Y/UY)

0.7853Y tl

) )

2.

19 I 6 U 10 11

I Z O F U R M A I ( 1 X ~ ' T M b I VALUES t S l l M A l t U BY SUBROUTINE COUL A Y t (rut511 lONAt4Lt SINCE C J < 0.045') GU T O 13 11 IF(CJ.LE.O.4) GO I U 13 I F ( C J . L E . O . Y ~ ~ ~ Y Y )bO 10 I 4 IF(CJ.Lt.1.00001) bo IU 15 IF(LJ.Lk.J.0) GU 10 16 Y H I T t ( 6 r 17) ~ ~ O F U H M A I ( I X I ' T M b I VALUES t S 1 1 M P l t U BY SUtlROUTINE COOL ARE. O U t b T I 10NAt4Lt SINCE C J > 3 . 0 ' )

13

20

1Y

18

15

ESTIMATION OF THERMAL PROCESS

135

L h

c

c

c

h

0

+

0

c

c

C

o

-c c- - C-N

-

h

- I n

C.

cc

C C C C

- 4

-

c

...

I

x

-

\

X

E*

-n

-47

1.

a t X <

4

a -u

N

z-

-

u

0 * R

w

-nhh

Z

n

D

0

x-x

9Z F

-lo

m.5

\

U P.

i

o

0

1

--

0-0

D - C

\I0 D

.. .. .. ..

N

D

3

-l

r

N

-

L

r

o

4

r

... . .. .. .. .. ...... .. ...... .. .. .. ... . .. .. .. .. ... . .. .. .. .. .. .. ..

.. .. .. .. - .. .. .. ... ..... .. .. .. .. .. .. .. .. .. .. .. ..

I

-l

--

z

C

r

-l

c

1

- C

-

c. - - ----

- \ \ -

I

c

4

-r

v)

I

z

e

--

9

+

- 1 L + U X-l *-I

c.

+

C

c

11

-

-. -

CN

c

Z Z F D-l

i X

-lzn n - r

-

D U - C

-i

a

0

Z

I1 -4

r x

.c\

*

0

11 O c

X

coa-l O

7--

III

1

9

C-

-

0

cc

C

-

c s - 4

-

^ A ^ <

2c .I m

.-D C Z

4C

C Z W

*T V_

DZ

r z r m0

-r

CD

--

X Z

ncc

CD

z-l

C

UT

- 2

C

F, Uc

m X FI

n o vt

c - r r t -2-C x<x- r x m rmr wxxx- CZC c-z w c I1 \ - -I+ no-

G-l-l-l-l

.. t

*t

e

0

t

0

*t 9 *t

t

t

1

t t

4

- 4 - 4

F t

v,

m

o

D

t

4

D I W

- l x a

I X

N-l-l

0

t

t

t

t t t

4

0

* t

1

*tt

8

0

0

0

0

t

t 9

0

t

4

0

*

t

0

4

t

*

t

4

- 0

- a

t

A

I C I 2 I C

I c

I

i

1

~

-n

n

f

+

-a-i-i =roo

4

v

0

I I

I I I I I

D C X F

i'd

I I

I I I

I W

1

Z i L Z I -

0 0 3 0

rrrri-

+-I44

A

L S r

I 7 0

x - x x c r c c

L

nlrl

r

F C

m o

C

~

C D

+

D

W X v F C D

vmz-lz

I O D -

n-IIrr

D

F F o D

v - r c C D Z x x r

XiD

crow

xccc DD Xl +c-lm

F C F . 9

m-l s o cm x Z: V D

4 4 m m

D X 2

-lam 7 m z c r v 0 0 c n r m

zzcc

EIz2ZF

X I T X

c

-Iozuz

D F F X -

x - < v -

F D F - D

TZW

I-

T-lZD

+xxmr rnrrown

c z- c -r

c U C T I u e m C -rz. x

~ r zr c x

X

7ucoc I D O ~ + ~ r r u zlnc-x P.myrzc c o c ScU< 4r ~ T rr c x

0

z--cz v z z - v c c

c r h

0-24

rn-ec

T

+ F r * X W

- 1 -lee

Fl-rVTIr

ESTIMATION OF THERMAL PROCESS 1 TtND

=

TIH((TtlL

+

Irrl / 2.1

7 CWNllNUt

l ( l J = THL T M I l I = TL DU 4 1 = 2.

I

Ik(I.tU.41 60 I U I F (CJ.Lt.0.99999Yl I f (CJ.Lt.1.00001J 11 TMT = IM?.(ABCIIls 6 T M ( I 1 = TMT GO T O 13 I2 TMT IMY(ABC(I1s GO T O 6

13 111)

5

b U I U 11 b U 10 12 ltNUs TLI

IENUI

= TXITHTI

GO TO 4 S IF(CJ.Lt.U.9999YYI b O 10 14 bI) 10 15 IFICJ.Lt.1.000011 14 TMT = I l E N D + 1 L I / 2. GU T O 6 15 T M T = I E N U / 2. GO T O 6 4 C0NTI)UUE GU TO I6

2 TkND = TIH(TLOw1 GO TO I

3 TLND =

GO T O I 16 HtTUHN END

T I M ( T W + U.01

*

(TBL

-

TWIJ

137

KAN-ICHI HAYAKAWA L

vvvvv~vavvvvavvvvvvvvvvvvvvvvvvvvvvavvvavvvvvvvvvvavvvvvvvvvvvvvvavov

C UOUUUUL1~UUUUUU~~UOUO~UUU~OUUU~OU~IJUUO~UUUU~~~UUUO~~~~~UU~UUOUUUUO~

~~a~vvavo~vvvvvvvvv~~vv~v~vv~vvvv~vvvvvvevvvv~ovvvvvvvvvovvvvvvvoe~vv

c uUUuuuuuuuuuouuuuul~uuuuuuuuuuuuuuuuuOuuuuuOuuuuuuuuuuOuuuuuuuuuuuuuuu L

5 U H h O U T I N t SUUPHO6RAC t D H L A L C U L A l l N G S l t R I L A L I N G V A L I J t S UY AF.PLYINLC LUBATTO 5 t V L N P O I h l T U U P U H A I U H t kOHMULA. v o v v a v v v v v v ~ a v a v v v a t v v ,y u M t p, L L A I u H t v v v v v v o v ~ v ~ o v ~ v ~ v C C

L C C

L

c

v e ~ v v

R 5 l t N I L I Z I N G VALUt LPLLULAltb(M1N.) I S t V E N T E M P E H A I U K t b USEL, 1 0 CALCULATL H V A L u t ( U t b H t t k . I T U GIN L U b t W L I M I T 01 A N l t G H A T l O N ( M I N . ) TtNU Ul'l'tk! L I Y I T 0t I N l t b H A T I O N ( M 1 N . I L 5LbPE I N O L X U t T h t H M A L O t A l H I I M t C U K V t OF T A H u k T MILHOUWbANISM5(f. OtbKLC). vvvvvvvvvvvvvvaavvvv*vvvvvQ~vvCvvvv*vQvv~vav**vvvvvuvv~vvvvvvv~vvvvvu

L i t w r C

C C

c L C C

UAIA: 11 L * lnG1N1 OUTPUT O B I & :

TtNU

..................................................................... ..................................................................... N

2 4

5 r(A HA + ti111 1 CUNTINUt

0

I f (TBbIh.bE.l.Ot-J) H = TtND/c'. UP GO TO I 6 H = (ItNl> I

c

-

IU.

**

((Tl1)

-

L5U.I

/

L

I

b U TU b

T U t i l h 1 / L.

HA

HtTlJKN ENU

vvvv*vva*vovvvvvvvvvvevvvvvvvvovvvvvvaovvv*vovvvvvvvvavvvvvo*vvvvvv~v

c

~oo~o~oOuOuOOooOooouO0Ou~UOOoOoOUU0ooOOuOoo~OuOoo~u~UO~oUO~OUoooO0OOuu

c

uoOuouoOUuoOOuooOOouOuOu~uuu~OOuOuD~~uUuU~U~uuuooU~~OoUU~uouuoUl~uuUUuU

C

V ~ O V C V V V C + V V C V V ~ V I ~ V V ~ V V V ~ V ~ V ~ C V V V V ~ a V O e V 4 ~ Q V ~ ~ ~ O O V 4 V ~ V V O V a V O V V V V ~ V

C C

C trLOCK LJATA SUUPHOGRAM k O H O E t I N I N G T H t V A L U t UP PUL AWO H C C BLOCK UATA LUMMUN/LOMA/A~CLI)/COMH/H(7) OOATA ABC/-I.UI -U.UJUc'2J9* -0.SbUU48Ue. 0.0, O.4bbCl4bMr 1 U.8JU22391 l.O/* ii/U.U4IoIYOr U.27bbCbO1 0.4317454, 2 0.48IblLO1 0 . 4 J I /4>49 O.C/b8c' bUi o.U4/61YU/

END

ESTIMATION O F THERMAL PROCESS

139

REFERENCES Alstrand, D. V., and Benjamin, H. A. 1949. Thermal processing of canned foods in tin containers. V. Effect of retorting procedures o n sterilizing values in canned foods. Food Res. 14, 253. Ball, C. 0. 1923. Thermal process time for canned food. Bull. Natl. Res. Counc. (U.S.) 7-1 (37). Ball, C. O., and Olson, F. C. W. 11957. “Sterilization in Food Technology.” McGraw-Hill, New York. Bigelow, W. D., Bohart, G . S., Richardson, A. C., and Ball, C. 0. 1920. “Heat Penetration in Processing Canned Foods,” Bull. No. 16-L. Res. Lab. Natl. Canners Assoc., Washington, D.C. Carslaw, H. S., and Jaeger, J. C. 1959. “Conduction of Heat in Solids.” Oxford Univ. Press, London and New York. Charm, S. E. 1971. “Fundamentals of Food Engineering.” Avi Publ., Westport, Connecticut. Deidoerfer, F. H., and Humphrey, A. C. 1959. Analytical method for calculating heat sterilization times. Appl. Microbiol. 1, 256. Flambert, F., and Deltour, J. 1972a. Localization of the critical area in thermally-processes conduction heated canned food. Lebensm.-Wiss. t Technol. 5 , 7 . Flambert, F., and Deltour, J. 1972b. Exact lethality calculation for sterilizing process. I . Principles of the Method. Lebensm- Wiss. t Technol. 5 , 72. Gillespy, T. G. 1951. Estimation of sterilizing values of processes as applied t o canned foods. 1. Packs heating by conduction. J. Sci. Food Agric. 2, 107. Gillespy, T. G. 1953. 11. Packs heating by conduction: Complex processing conditions and value of coming-up time of retort. J. Sci. Food Agric. 4 , 553. Griffin, R. C., Jr., Herndon, D. H., and Ball, C. 0. 1969. Use of computer derived tables t o calculate sterilizing processes for packaging foods. 2. Application t o broken-line heating curves. Food Technol. 23, 121. Griffin, R. C., Jr., Herndon, D. H., and Ball, C. 0. 1971. 3. Application t o cooling curve. Food Technol. 25, 36. Hammer, P. C., and Straut, A. H. 1958. Numerical evaluation of multiple integrals. 11. Math. Tables Other Aids Comput. 12, 272. Hayakawa, Kan-ichi. 1958. On the theoretical evaluation of heat processes. Part 4. Canners J. (Tokyo) 37, 107. Hayakawa, Kan-ichi, 1968. A procedure for calculating sterilizing value of a thermal process. Food Technol. 22,905. Hayakawa, Kan-ichi. 1969. New parameters for calculating mass average sterilizing value to estimate nutrients in thermally processed food. Can Inst. Food Technol. J. 2, 165. Hayakawa, Kan-ichi. 1970. Experimental formulas for accurate estimate of food temperature and their application t o thermal process evaluation. Food Technol. 24, 1407. Hayakawa, Kan-ichi. 1971. Estimating food temperature during various processing or handling treatments. J. Food Sci 36, 378. Hayakawa, Kan-ichi. 1972. Estimating temperatures of foods during various heating o r cooling treatments. ASHRAE J. 14,65. Hayakawa, Kan-ichi. 1973. Modified lethal rate paper technique for thermal process evaluation. Can. Inst. Food Technol. J. 6 , 295. Hayakawa, Kan-ichi. 1974. Heat sterilizing of biological material. In “The Computer Aids for Chemical Engineering Education” (R. Gordon, ed.), Vol. 4 , pp. 199-243. Aztec Publ. Co., Austin, Texas.

140

KAN-ICHI HAYAKAWA

Hayakawa, Kan-ichi, and Ball, C. 0. 1968. A note on theoretical heating curve of a cylindrical can of thermally conductive food. Can.Insr. Food Technol. J. 1,54. Hayakawa, Kan-ichi, and Ball, C. 0. 1969. A note on theoretical cooling curve of a cylindrical can of thermally conductive food. Can. Inst. Food Technnol. J. 2, 115. Hayakawa, Kan-ichi, and Ball, C. 0. 1971. Theoretical formulas for temperature in cans of solid food and evaluating various heating processes. J. Food Sci 36, 306. Hemdon, D. H. 1971. Population distribution of heat rise curves as a significant variable in heat sterilization process calculation. J. Food Sci. 36, 299. Hemdon, D. H., Griffin, R. C., Jr., and Ball, C. 0. 1968. Use of computer-derived tables t o calculate sterilizing processes for packaged foods. Food Technol. 22,473. Hicks, E. W. 195 I . On the evaluation of canning processes. Food Technof. 5, 134. Hicks, E. W. 1958a. Evaluation of canning processes when g is less than 0.1. Food Technol. 12, 116. Hicks, E. W. 1958b. A revised table of ph function of Ball and Olson. Food Res. 23, 396. Hicks, E. W. 1961. Uncertainties in canning process calculations. Food Rex 26, 218. Hilderbrand, F. B. 1956. “lntroduction to Numerical Analysis,” p. 155. McGraw-Hill, New York. Ikegami, Y . 1974. Effect of “come-up” on processing of canned food with steam. CannersJ. (Tokyo) 53, 79. Jakobsen, F. 1954. A note on process evaluation. Food Rex 19,66. Jen, Y., Manson, J. E., Stumbo, C. R., and Zahradnik, J. W. 1971. A procedure for estimating sterilization and quality factor degradation in thermally processed foods. J. Food Sci. 36,692. Manson, J. E., and Cullen, J. F. 1974. Thermal process simulation for aseptic processing of foods containing discrete particulate matter. J. Food Sci. 39, 1084. Manson, J. E., and Zahradnik, J. W. 1967. Computer thermal process determination for conduction-heated foods. Food Technol 21, 1206. Manson, J. E., Zahradnik, J. W., and Stumbo, C. R. 1970. Evaluation of lethality and nutrient retentions of conduction-heating food in rectangula containers. Food Technol. 24,1297. Manson, J. E., Stumbo, C. R., and Zahradnik, J. W. 1974. Evaluationof thermalprocessesfor conduction heating foods in pear-shaped containers. J. Food ScL 39, 276. Michels, H. H. 1963. Abscissas and weight coefficients for Lobatto quadrature. Math. Comput. 17, 237. Moats, W. A., Dabbah, R., and Edwards, V. M. 1971. Interpretation of nonlogarithmic survivor curves of heated bacteria. J. Food Sci. 36, 523. Patashnik, M. 1953. A simplified procedure for thermal process evaluation. Food Technol. 7, 1. Pflug, 1. J. 1968. Evaluating the lethality of heat processes using a method employing Hick’s table. Food Technol 22, 1153. Rao, M. A., and Loncin, M. 1974a. Residence time distribution and its role in continuous pasteurization. Part I. Food Sci + Technol. 7, 5 . Rao, M. A., and Loncin, M. 1974b. Resident time distribution and its role in continuous pasteurization. Part 11. Food Sci + Technol 7 , 14. Sasseen, D. M. 1969. “Computer Programs for Process Calculation by the Simplified Ball Formula Method.” West. Br. Lab., Natl. Canners Assoc., Berkeley, California. Schultz, 0. T., and Olson, F. C. W. 1940. Thermal processing of canned foods in tin containers. 111. Recent improvements in the general method of thermalprocess calculation-a special coordinate paper and methods of converting initial and retort temperature. Food Res. 5, 399.

ESTIMATION OF THERMAL PROCESS

141

Shapton, D. A., and Lovelock, D. W. 1971. The evaluation of sterilization and pasteurization processes from temperature measurements in degree Celsisus (“C). J. Appl. Bacteriol. 34,49 1. Simpson, S . G., and Williams, M. C. 1974. An analysis of high temperature/short time sterilization during laminar flow. J. Food Sci 39, 1047. Smith, R. E., Nelson, C . L., and Henrickson, R. L. 1967. Application of geometry analysis of anomalous shapes to problems in transient heat transfer. Trans ASAE 10, 236. Stevens, P. M., Zahradnik, J. W., Dixon, J. R., Zinsmeister, G., Stumbo, C. R., and Torrance, K. B. 1973. Convection heating foods: Lethality calculation including product circulation. Presented at 33rd Annu. Meet. Inst. Food TechnoL Stumbo, C . R. 1953. New procedures for evaluating thermal processes for foods in 47.

Stumbo, C. R. 1950. New procedures for evaluating thermal processes for foods in cylindrical containers. Food Technol. 7 , 309. Stumbo, C. R. 1973. “Thermobacteriology in Food Processing,” 2nd ed. Academic Press, New York. Stumbo, C. R., and Longley, R. E. 1966. New parameters for process calculation. Food TechnoL 20,34 1. Teixeira, A., Dixon, J. R., Zahradnik, J. W., and Zinsmeister, C. E. 1969. Computer determination of spore survival distributions in thermally processed conduction-heated foods. Food Technol. 23, 78. Timbers, C. E., and Hayakawa, Kan-ichi. 1967. Mass average sterilizing value for thermal process. I. Comparison of existing procedure for calculating mass average sterilizing value. Food Technol. 21, 1069. Tyler, G. W. 1953. Numerical integration of function of several variables. Can. J. Math. 5 , 393.

Veerkamp, C. H., Romijn, A. J. M., and Pal, J. C. 1974. Influence of varying residence time distribution on inactivation of microorganisms during pasteurization of egg products. Food Sci + TechnoL 7,306.

This Page Intentionally Left Blank

ABALONE-AN ESOTERIC FOOD JUNE OLLEY AND S. J . THROWER CSIRO Division of Food Research Tasmanian Food Research Unit Hobart, Tasmania

I . Introduction

....................................................

............................... 11. Anatomy ................... 111. AbaloneasFood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

144 145

147 IV. Chemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Proximate Analysis . . . . . . . ... ....................... 147 B. Effects of Size and Season . . ................................. 150 C. Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 D. Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ .156 E. Nucleotides and Other UV-Absorbing Extractives . . . . . . . . . . . . . . . . . . . . . 157 F. Lipids . . . . . . . . . . . . . . . . . . ................................. 160 C . Compounds Containing Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 H. Naturally Occurring Pigments . . . . . . ...................... 163 I. Volatile Bases . . . . . . . . . . . . . . . . . . ...................... 164 J. Miscellaneous Compounds ....................................... 165 V. Catching and Handling of Abalone a t Sea . . . . . . . . . V1. Physiology of Abalone in Air . . . . . . . . . . . . . . . . . . VII. Technology 01' Preserving Abalone . . . . . . A. Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 B. Brining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 ............................................ 171 ........................................... 112 ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 VIII. Quality Aspects . . A. Texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 ........................................... 174 .......................................... 177 ...................... 178 1X. By-products of Abalone Processing . . . . . . . . .... . 179 X. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

143

JUNE OLLEY AND S. J . THROWER

144

I. INTRODUCTION Abalone is the American name for large marine gastropods of the family Haliotidae. Some members of this family have been used as food by man from prehistoric times. Aristotle (ca. 347 B.C.) called it Apia lepas, the wild limpet, explaining that others called it 7’haZattion u s , the marine ear. Ebert (1969) lists twenty-five different local names for Haliotidae eaten in different parts of the world; the selection given in Table I ranges from the evocative Orechio de San F’ietro of the Adriatic to the mundane-sounding name mutton fish given by the Australians. The name “abalone” is becoming the one in most common use in the English tongue, displacing the earlier English word “ormer.” Ebert (1969) discusses the geographical distribution of the various species which differ markedly in their maximum size and in their color. Their taking and processing have become industries in several countries, and in recent years Australia has become the world’s largest producer (Montgomery, 1966; Harrison, 1969). The tonnage caught compared with the amount and value of the total fish catch for various countries is shown in Table 11. The biochemistry of the abalone, particularly that of the nucleotides, has been studied most intensively by the Japanese. Much of the published work on the TABLE I COMMON NAMES OF SOME O F THE SPECIES O F ABALONE EATEN IN DIFFERENT PARTS OF T H E WORLD’

Country

Common name

Australia Channel Islands France Japan

Mutton fish or abalone Ormer, Sie-ieu Ormer, Sie-ieu Awabi

Mediterranean Mexico

Orecchiale, Orechio de San Pietro Aulone

New Zealand South Africa United States

Paua Perlemoen Abalone

-.

Species

Notohaliotis ruber Schismotis laevigata Halio tis tub ercu lata Haliotis tuberculata Haliotis gigantea Haliotis sieboldii Haliotis discus , Haliotis kamtschatkana Haliotis lamellosa Haliotis corrugata Haliotis fulgens Haliotis iris Haliotis midae Haliotis rufescens Haliotis corrugata Haliotis cracherodii

aFroni information in Ebert (1969). This is not intended as an exhaustive list.

(black lip) (green lip)

(red) (pink) (black)

ABALONE-AN ESOTERIC FOOD

145

TABLE 11 STATlSTICS OF ABALONE CATCH FOR THE YEARS 1971 AND 1972‘

Country Australia

Year

Nominal catch of abalone (1000 metric tons)

1971 8.0 1972 8.0 Japan 1971 5.7 1972 5.9 Mexico 1971 4.0(FAO 1972 4.0 estimate) United States 1971 1.7 1972 1.4 Newzealand 1971 1.5 1972 0.7 South Africa 1971 1.2 1972 0.9 Korea (Republic) 1971 0.6 1972 1.0 Taiwan 1971 0.1 Canada 1972 0.1 Total 1971 22.8 1972 22.0

Total nominal Abalone catch of (% of total marine species (1000 metric tons) catch) 110.9 113.0 9,894.5 10,247.8 402.5

7.20 7.10 0.06 0.06 0.99

Value of abalone (% of total financial return) 5.08 0.63 2.00

-

-

-

2,766.8 2,649.5 65.8 58.3 1,103.4 1,120.2 1,073.7 1,338.6 650.2 1,073.5 16,067.8 16,600.9

0.06 0.05 2.28 1.20 0.11 0.08 0.06 0.07 0.02 0.01 0.14 0.1 3

-

1.92 1.11 -

0.63 0.58 3.86 0.08 0.01

‘From the Food and Agriculture Organization (1972,1973).

technology of processing stems from the authors’ laboratory, and this work has been done on the local Tasmanian species ~ o t o ~ a l i o truber i s (Haliotis ruber Leach). During the course of this work much unpublished experience has also been gained; the authors make no apology for drawing heavily on this, as there is so little literature on abalone technology apart from work in the Japanese language.

I I . ANATOMY The detailed morphology of the abalone is described in a monograph by Crofts (1929). The muscular foot is used for locomotion and attachment to rocks below water level. It is surrounded by a highly vascularized frill or epipodium, which has a sensory function. The epipodium has a number of horizontal fiber bundles which serve to retract the sensory collarette under the shell. The adductor muscle is attached t o the shell and runs vertically down into the pedal sole, the part of the foot that grips onto a surface. Two pedal arteries and a

146

JUNE OLLEY AND S. J. THROWER

FIG. 1. Two views of shucked abalone. The left-hand specimen shows the dark ventral surface of the pedal sole and its darker pigmented edges. The dark under-surface of the epipodium should also be noted. The specimen on the right shows the top of the white oval central pillar of the adductor muscle where it attaches t o the shell. This muscle can be seen to rise from the dorsal side of the pedal sole. The mouthparts with a radula tooth and the sensory margins of the epipodium are clearly visible.

pedal vein run the length of the pedal sole and can be clearly seen in a dried product of good quality, which should be translucent (see Section VII,D). Dorsal and ventral views of shucked abalone foot are shown in Fig. 1.

1 1 1 . ABALONE AS FOOD Asian peoples, notably the Chinese and Japanese, are the main consumers of abalone, and this shellfish is thought to have aphrodisiac properties. In Japan, the tougher varieties are thinly sliced and served raw (“sashimi”), while the softer varieties with less collagen are considered suitable for boiling or steaming (Takayama el al., 1970). The Japanese cook, dry, and smoke the foot, and also can it (Tanikawa, 1971). They also eat the viscera and roes, the former usually cooked or salted (Hashimoto and Tsutsumi, 1961).

ABALONE-AN ESOTERIC FOOD

147

In the Channel Islands of the English Channel abalone is traditionally stewed. In Australia and the United States the epipodium is often removed and the foot beaten into steaks which are then fried in bread crumbs. This is a wasteful process, and some attempts have been made to develop products from the trimmings. A delicious soup has been made in Australia, but this would require market promotion to become popular. Factory offcuts have also been minced and canned in brine or white wine (Dreosti and Atkinson, 1966), or made into patties which are packaged and frozen (Cox, 1962). Dried abalone is regarded as a great delicacy in Asian countries. The two types of product in commercial use have been described by Young et id. (1973). The first is a semidried product, transshipped chilled as the whole foot and then sold in thin slices packed in polyethylene pouches; the second is a whole, fully dried abalone foot, the moisture content of which is ultimately determined by the relative humidity of its environment. The uses of the dried and semidried products have been described by Young et al. (1973), and a new dish obtained by frying the semidried product is suggested. Although abalone is a gourmet’s food, it is interesting to compare its value with that of other seafoods as a source of high-quality protein, vitamins, and minerals. Matsuno (1970) and Matsuno et al. (1972) have recently compiled tables of the essential amino acids and chemical scores of one hundred and fifty-two Japanese food items. Abalone, number 72 on the list, did not rate very high, having an essential amino acid index of 57 and a chemical scQre limited by either lysine or sulfur amino acids. However, there is a marked seasonal variation in the protein content of abalone and mobilization of the amino acid pools (see Sections IV,A and IV,D). Intengan et al. (1956) and the U.S. Department of Health, Education and Welfare (1972) have reported the vitamin and mineral content of abalone muscle. The riboflavin and niacin content of abalone are low for marine products, while reports of thiamine content range from nil to 0.24 mg per 100 gm of flesh, which is quite high for a seafood. Fisher et al. (1956) found low levels of vitamin A but high levels of P-carotene in intact specimens of Haliotis fulgens. Abalone being a mollusk, the calcium content of the flesh is low. The phosphorus content is normal for muscle tissue, while the iron content can be quite variable even within a species (van der Merwe, 1954).

IV. CHEMICAL COMPOSITION A. PROXIMATE ANALYSIS The main components of abalone muscle as reported by various authors are listed in Table 111. Agricultural products, both animal and vegetable, are har-

TABLE 111 PROXIMATE ANALYSIS OF ABALONE

Water Speciesb Japan H. gigan tea sieboldii H. gigan tea discus H. gigontea (Gmelin) H. gigantea (Gmelin) H. gigantea

H. discus hannai H. japonica Reeve United States H. cracherodii Pacific Coast

(%I 78-83 78-90 I5 82 76

Protein (%)

-

9.4 -

72-18 76

7.5-12.5 10.2

68-12 71

18-23 23

Total N Glycogen 01 6.25(%) carbohydrate (%)

X

12.5-17.0 9.4-1 7.5 22.5 10.4 20.1 12.5-19.0 13.7

-

2.3

0.14.5 7.0 1.5-7 5 -

Fat (%)

MUSCLE^ Ash (%)

Nonnitrogen solids (%)

0.45 0.2 0.4 1.0-1.5 0.3

0.75-3.0 1.6

1.2-2.5 1.35

-

-

References Takayama er al. (1970) Takayama er al. (1970) Simidu et al. (1953) Konosu and Mori (1959) U.S. Department of Health, Education and Welfare (1972) Tanikawa and Yamashita (1961) Suyama and Sekine (1965) Webber (1970) Albrecht (1921)

Australia N.ruber

74-78

-

-

Top of adductor muscle

-

-

16.s19.5

-

-

-

7.0-10.0

Pedal sole

-

-

16.0-17.5

-

-

-

7.0-11.0

Epipodium

-

-

9.0-13.0

-

-

-

3.5-6.5

-

0.4

0.4

2.8

Korea H, giguntea nordntis

76

20

-

J. Olley C. Baldwin L. Barker, and C. Paice (unpublished results) 1969-1973) J. Olley, C. Baldwin, L. Barker, and C. Paice (unpublished results, 1969-1973) J. Olley C. Baldwin L. Barker and C. Paice (unpublished results) 1969-1973) J. Olley, C. Baldwin, L. Barker, and C. Paice (unpublished results, 1969-1973)

Song (1973)

%dwell et UL (1974) have also reported the proximate analysis of abalone from four literature sources. All are listed as H. kumtschutkunu, although only Butler (1958) identifies his sample as such. %here would appear to be some confusion in species classification.

JUNE OLLEY AND S. J. THROWER

150

vested at the optimum stage of growth or in season. Marine products are often gathered the year around, and, as Love (1970) has pointed out, too little attention has been paid to the variations which ensue owing t o size and season. These variations are fully apparent in Table 111. They make the listing of major components to one decimal point (e.g., U.S. Department of Health, Education and Welfare, 1972) completely inappropriate and have many implications relating t o the flavor, texture, and general appearance of products.

B. EFFECTS OF SIZE AND SEASON The relative proportions of edible material of abalone vary with the sexual cycle (Table IV). Webber (1970) found that the depletion of the foot tissue t o build up gonads was real rather than relative by expressing the results as a percentage of the weight of the shell. This dropped from 120% in January t o 82%in August, just prior to spawning. Composition also changes with the size of the animal. In the authors’ laboratory, the solids content of the foot of N. tuber has been directly related to size, and the water content inversely. This correlation remains through various processing treatments (Fig. 2)-for example, in batches of abalone which have been subjected to different brining regimes or different freezing and thawing cycles, the size effect predominating over the treatment effects. The negative correlation of water content with size (p < 0.001) appears to be caused by build-up of nonprotein solids in the pedal sole (J. Olley, C. Baldwin, L. Barker, and C . Paice, unpublished results, 1969-1973). Of these, a high proportion is probably glycogen. The weight of the whole shellfish is some indication of age within a species, although Newman (1969) has found that mean annual temperature has a marked effect on the maximum size attained. It is impossible to predict the water content, the salt uptake, or the moisture loss that will result from a particular TABLE IV SEASONAL VARIATIONS IN THE RELATIVE PROPORTIONS O F ORGANS OF H . crocheroidiia

Gonad __ __ Unripe ____ 4-10% 6-7% 5660% 26-3 1%

~

Organ Gonad Digestive tissue Foot Other tissues

-

condition ~

-

Ripe 18% 7% 45% 30%

~

Spent _ 3% 8% 55% 34%

_

aFrom the data of Webber (1970); results expressed as a percentage of rhe total soft parts of the animal.

_

15 1

ABALONE-AN ESOTERIC FOOD

36-

-

-3 3 2 k

I

i 2 8 # t (L

n 24

20

:

0

D

a0

A&%

2

0

100

200

300

FIG. 2. Relationship of solids content and weight of abalone muscle. Open symbols, foot and epipodium; closed symbols, foot alone 0 , fresh, drained for 0.5 hour after shucking 0 , fresh, drained for 4 hours after shucking. brined in 10% brine, 66 hours. A , brined overnight, boiled for 1 hour. 0, brought t o the boil and canned. Individual slopes from eleven different processing treatments did not differ from each other 0, > 0.75). A common slope of 0.012 was obtained. The common slope was statistically significant (p < 0.01). For clarity only five treatments have been shown.

.,

process without taking the size of the animal into account. Abalone should therefore be carefully randomized between all experimental treatments, and size used as a covariant in all experiments where there is a large range.

C. PROTEINS 1. The Mystey of the Protein Composition The literature on abalone proteins shows many anomolies. These, although not resolved, are best sorted out by considering foot muscle as the three distinct anatomical features-adductor, pedal sole, and epipodium, which contain different mixtures of proteins; by noting the physicochemical properties of the muscle proteins; and by comparing the amino acid composition of the muscle with the amino acid composition of known isolated proteins. Song (1973) has presented evidence that the central oval pillar, the adductor muscle, is composed of paramyosin fibrils in an amorphous background. The electron micrograph shown in Fig. 3a indicates the presence of many actin

152

JUNE OLLEY AND S. J . THROWER

filaments as well as paramyosin. The extracellular space, on the other hand, is seen t o contain collagen fibrils (Fig. 3b), together with a great deal of amorphous material which is in fact the main extracellular component. The proteins of the pedal sole have been examined by Song (1973) with the light microscope and shown to be mostly connective tissue, with “things” that entwine together among them. The high collagen content of the pedal sole and epipodium has been confirmed chemically by Kimura and Kubota (1968) and James and Olley (1971a). However, Tanikawa and Yarnashita (1961) and Takayama et al. (1970) both noted a very marked decrease in collagen in the summer months and a replacement with nonprotein nitrogen. Abalone muscle is extremely tough, and therefore the proportions of the four types of protein-water-soluble (enzymes), salt-soluble (myofibrillar proteins), alkali-soluble (abductin?), and insoluble (connective tissue)-are difficult to determine. Results vary with the extent of comminution and the experimental approach (Migita and Matsumoto, 1959; Tanikawa and Yamashita, 1961; Takayama et al., 1970; Pyeun er aZ., 1973). Pyeun el al. (1973) found a fairly large variation among four individual specimens, but after mincing the central columnar muscle with 1 M KCl and 0.1 NNaOH overnight they found that an average 22% of the protein was water-soluble, 34%was salt-soluble, 20% was alkalisoluble, and the stroma amounted to 24%. Tanikawa and Yamashita (1961) found far less salt-soluble protein when they extracted the whole abalone foot. Pyeun et al. calculated from the ultracentrifugation pattern that paramyosin constituted 65% of the salt-soluble protein. Minor peaks appeared to be actomyosin and myosin. The stroma content of some samples was as high as 30% and, because of its high hydroxyproline content, was equated by Takayama et al. (1970) with collagen. The amino acid composition of known or likely individual proteins of abalone muscle is listed in Table V and compared with data from the literature for protein or total amino acids of whole abalone foot muscle. Some of the individual proteins have not been isolated from abalone itself, so the analyses have been taken from other species for general guidance. Despite the large variations in total amino acid and/or protein content in the three species examined, the amino acid pattern was remarkably similar with two notable exceptions. Haliotis tuberculata, analyzed in Belgium, with the lowest total amino acid content had a high content of free arginine (Fiorkin and FIG. 3. (a) Electron micrograph of fresh abalone adductor muscle showing paramyosin fibrils (pf) together with actin filaments (a). Large glycogen granules are present in the matrix. Magnification 27,OOOX. (b) Extracellular space of fresh abalone adductor muscle. A collagen fibril with characteristic banding is surrounded by amorphous material. Magnification 79,OOOX. (c) Muscle from abalone after drying. Paramyosin is the principal remaining filamentous element, but extensive vacuolation of the muscle has occurred. Magnification 9500X. Courtesy of D. J. Morton.

TABLE V AMINO ACID COMPOSITION O F VARIOUS INDIVIDUAL MUSCLE PROTEINS COMPARED WITH THAT OF WHOLE ABALONE MUSCLE: RESIDUES PER 1000 RESIDUES

Individual muscle proteins

Amino acid Aspartic acid Threonine Serine Glutamicacid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine

Paramyosin, abalone a b

110 35 45 200 19 98 39 12 34 102 10 6 60 7 91

107 35 46 188 0 25 88 30 11 37 100 13 9 62 6 101

Whole abalone muscle

Protein

Total amino acids

Actin, Venusc

Abductin, Pectend

Elastin, oxd

Collagen, abalonee

H . japonicaf (10.2%)'

H. tuberculatag

88 58 62 103 44 66 76 40 26 51 80 36 32 56 20 49

18 11 69 12 13 630 33 6 90 7 2 1 93 8 6 3

4 7 7 13 143 364 194 135 < 1 28 60 8 27 3 1 5

59 17 55 96 102 334 87 18 12 13 28 7

97 54 64 131 58 132 94 47 21 40 78 25 29 56 12 48

66 44

I 8 1 60

(6.25%)'

H. tuberculatag H . gigantea (7.1%)' (20. I%Y 64 41

-

-

115 60 270 103 43

111 55 25 8

-

-

41 64 17 21 58 10 89

40 64 17 21 56 9 31

101

43

102 50 66 133 57 138 96 45 21 38 74 28 28 49 13 49

'Woods and Pont (1971). bF'yeun et al. (1973). 'Kominz (1966). dKelly and Rice (1966). eKimura and Matsuura (1974). Abalone collagen showed the chain composition [a] and, besides the amino acids listed above, contained 13.8 residues of hydroxylysine per 1000,80.4 residues of 4hydroxyproline, and 0.6 residue of 3hydroxyproline. fcalculated from Suyama and Sekine (1965). gCalculated from Florkin and BncteuxCrdgoire (1972). 'Calculated from the U.S. Department of Health, Education and Welfare (1972). i Percentage fresh weight of tissue.

ABALONE-AN ESOTERIC FOOD

155

Bricteux-Gregoire, 1972). The possible significance of this is discussed in Section IV,D. The glycine content of H. tuberculata protein was exceptionally high. When the amino acid contents of the species are converted to grams per 100 gm of flesh, and compared with each other, it is surprising to find that the glycine content of all three species remains approximately the same, while the other amino acids are roughly in proportion to the total proteins or total amino acids. It would seem that the glycine in H. tuberculata is in a form which is not readily mobilized. This amino acid in fact becomes central t o puzzling aspects of the structure of abalone muscle. The amino acid composition of abalone was therefore compared with the composition of isolated proteins to search for a protein of high glycine content. Paramyosin of adductor muscle has only 19 residues per 1000 (Table V) of glycine, and it is not surprising therefore that Tanikawa and Yamashita (1961) detected no glycine spot in their paper chromatograms of the hydrolyzate of the large central muscle. The structure of collagen requires that every third molecule should be glycine (Piez, 1966). The polypeptide backbones of the collagen structure are tightly packed, so there is no room for an amino acid with a side chain. The Belgian analyses would indicate that H. tuberculata was almost 87% collagen, which is highly unlikely, and we therefore searched in the literature for a protein of even higher glycine content. Abductin, the rubberlike protein with elastic properties found in the hinge ligament of the scallop (Pecten), is almost two-thirds glycine (Kelly and Rice, 1966) and is soluble in alkali. Abductin also contains a high proportion of methionine, which was not measured in the Belgian experiments. The Belgian laboratory did not find this remarkable percentage of glycine in the protein of the other mollusks examined. One must either doubt the particular analysis of abalone or invoke an abductin-like protein as a major component of abalone when the total protein content is low.

2. Physicochemical Properties of the Protein The isoelectric point of the watersoluble protein in abalone muscle is pH 4.8 to 5.2, and that of the 0.5 M salt-soluble protein is pH 5.2 (Tanikawa and Yamashita, 1961). These authors distinguish between two distinct types of protein in white muscle, one flocculating at temperatures between 40" and 55°C and the other at temperatures between 60" and 70°C. They have carried out an extensive study on the effects of various cations and anions on the degree of swelling of these muscle proteins.

3. Essential and Nonessential Amino Acids of Total Body Protein Glutamic and aspartic acids, alanine, and leucine were the four predominant amino acids in the whole-body protein of Haliotis rufescens. Studies with

156

JUNE OLLEY AND S. J . THROWER

[U-14C] glucose showed that glutamic and aspartic acid, alanine, half-cystine, glycine, serine, and proline were nonessential, while threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, lysine, histidine, and arginine incorporated n o label and were inferred to be essential (Allen and Kilgore, 1975). These data have relevance to current efforts in California and Japan t o culture abalone (Shepherd, 1976).

D. AMINOACIDS Free amino acids perform a number of functions in shellfish. Some understanding of these functions is crucial to any discussion of the free amino acid pool. The content of free amino acids in abalone tissue has been estimated by Hashimoto (1965) at 2.2% fresh weight and by Florkin and Bricteux-GrCgoire (1972) at 0.8% fresh weight. Seasonal variations might be expected to occur, however. Takayama et al. (1970) reported a 50% depletion of collagen during the summer months with a corresponding increase in the level of nonprotein nitrogen. Such changes could drastically alter the size and nature of the amino acid pool, since the amino acids released would be components of protein. Osmoregulation is a problem faced by many mollusks living in estuarine areas in which changes in salinity subject the animal to osmotic stresses which could lead to a depletion of chemical compounds, especially essential amino acids. Such animals often build up a reserve of nonessential amino acids which can be “sacrificed” to protect essential amino acids. Florkin (1964) listed glutamic acid, glutamine, glycine, proline, alanine, and taurine as osmoregulating substances, and estimated that such substances can account for one-third of the total intracellular modification of osmotic pressure. b n g e (1963) found that taurine concentration in the mussel could range from 0 to 50 mM, depending on the salinity of its environment, and suggested that this acid is responsible for osmoregulation. It should be noted, however, that the habitats of abalone are usually marine (Stephenson, 1924; Crofts, 1929; Shepherd, 1975) and would not be subject to any marked changes in salinity that might require large concentrations of osmoregulators; indeed, Stephenson (1924) quotes experiments done by Beudant in 1816 which showed that abalone were less tolerant to changes in salinity than were other animals living on the seashore. Despite this, the five predominant free amino acids found in abalone muscle by Hashimoto (1965) were taurine (43%), arginine (14%), glycine (8%), glutamic acid (5%), and alanine (4.5%). Florkin and Bricteux-GrCgoire (1972) did not report any taurine in H. tuberculata but listed the predominant amino acids as arginine (68%), glycine (8.3%), and glutamic acid (5.8%). Takayama et d. (1970) estimated that arginine accounted for 62 t o 76%of the free amino nitrogen in abalone meat. Taurine was first isolated from abalone seventy years ago (Mendel,

ABALONE-AN ESOTERIC FOOD

157

1904), and abalone was used by Schmidt and Watson (1918) as a source of taurine for experimental purposes, ample evidence that it can be present in large amounts. Its absence from the Belgian analyses could be due to depletion by osmoregulatory processes, or to the analytical approach. Taurine is not absorbed by sulfonated polystyrene resins when conventional buffers are used in amino acid analysis (Jones, 1955), a fact that was responsible for its omission from the analyses of cod by Shewan (1953). If this was the cause of the discrepancy, however, it is puzzling that Tanikawa and Yamashita (1961) showed neither arginine nor taurine in their paper chromatograms of H. discus but did show a clear spot of aspartic acid, an amino acid which Hashimoto (1965) estimated at only 9 mg per 100 gm of flesh. Schafer (1961) claims that aspartic acid, phenylalanine, and three unidentified compounds staining with ninhydrin are symptomatic of marine pollution with depletion of available oxygen. Phosphoarginine is a hlghenergy intermediate in the synthesis of ATP from ADP; indeed, van Thoai and Roche (1964) list phosphoarginine as the sole phosphagen found in mollusks. Concentrations of free arginine in the tissues of abalone reported by Florkin and Bricteux-Grdgoire (1972) (34.1 pmole/gm) are of the same order of magnitude as that reported by Sidhu ef al. (1974) (26.5 pmole/gm) for phosphoarginine in the muscle of the rock lobster, so it is possible that the free arginine in abalone muscle is the breakdown product of phosphoarginine in the dead animal. E. NUCLEOTIDES AND OTHER UV-ABSORBING EXTRACTIVES Most studies of nucleotides in abalone have been performed by Asian workers (Arai, 1961, 1966a,b; Arai and Saito, 1961; Arai et al., 1968; Nishita et al., 1965,1966;Nishita, 1967; Seki et al., 1967; Seki, 1971; Song, 1973). There are marked differences in the composition of nucleotides in the viscera and in the muscle. Seki ef al. (1967) found that 54%of nucleotides in the hepatopancreas of H. discus were of the “adenylic series,” and 37%were of the “uridylic series.” The nucleotides in abalone muscle are, for the most part based on adenosine, although Nishita (1967) has found traces of uridine diphosphate derivatives in extracts of H. discus hannai. The levels of adenine nucleotides reported in the literature (4 to 5 pmole/gm) are similar to those in surf clam (Arai et al., 1968) and ark shell (Arai, 1961), but half the levels reported in scallop (Arai et aL, 1968) and lobster (Sidhu ef al., 1974). Laboratory studies have shown that ATP is broken down during storage of abalone muscle and AMP accumulates (Arai, 1961, 1966b; Arai et al., 1968), while it is claimed that inosinic acid (IMP) is absent (Fujita and Hashimoto: 1960). The more usual pathway of nucleotide breakdown in marine species is the further deamination of AMP to form IMP which finally breaks down to hypoxanthine. This unusual absence of breakdown of AMP in abalone is

JUNE OLLEY AND S. J . THROWER

158

puzzling, because Seki (1971) found and partially purified an AMP deaminase from H. discus. It is possible that the activity of this enzyme is reduced by inhibitors (Conway and Cooke, 1939). Arai (1966b) found that addition of 5’-nucleotidase to abalone muscle resulted in rapid accumulation of adenosine and stated that “dephosphorylation of AMP was the limiting step in the decomposing pathway of AMP.” In commercial practice it is found that AMP usually accumulates during storage of abalone at chill temperatures, while in the frozen state (Arai, 1966a) or in reconstituted freeze-dried products (Arai et al., 1968) a mixture of ADP and AMP accumulates. There is, however, some confusion about the rate of ATP degradation and AMP accumulation. Song (1973) found that ATP was completely degraded within 8 hours of storage at room temperature, whereas Arai (1961) found that complete ATP degradation took 50 hours under these conditions. It should be noted that both hypoxanthine and adenosine have been detected in abalone (N. ruber) that died during commercial shipment of live animals, and in frozen material rejected by Japanese importers (J. Olley, C. Baldwin, L. Barker and C. Paice, unpublished results, 1969-1973). It is possible that damage to the organs during live transport in bulk released enzymes which produced these compounds, since IMP and hypoxanthine have been found in the viscera of abalone (Seki et al., 1967). Attempts to reproduce these effects on single animals in the authors’ laboratory have always proved unsuccessful. Nucleotides are commonly measured by the time-consuming method of column chromatography. A simpler way of measuring changes in the nucleotides has been devised by Jones and Murray (1964). A neutralized perchloric acid extract of the product is shaken up with an anion-exchange resin; nucleotides are exchanged onto the resin, while the material not exchanged represents a mixture of nucleosides, hypoxanthine, picolinic acid, and other unidentified compounds. The difference between readings of the extract before and after shaking, taken at TABLE VI SPECTRAL CHAKACTEKISTICS O F UV-ABOSRBING COMPOUNDS FOUND IN SOME SHELLFISH Amax

Compound

(nm) ~~

Adenine nucleotides Adenosine Hypoxanthine Homarine Trigonclline

259 259.5 249.5 272 264-265 271 (minor)

250 nm 260 nm 0.78-0.80 0.78 1.32 0.40

280 nm 260 nm

References .~

0.15-0.16 0.14 0.09 1.42

Burton (1969) Burton (1 969) Burton (1969) Tarr and Comer (1965) Hiltt (1970)

159

ABALONE-AN ESTOERIC FOOD

appropriate wavelengths between 210 nm and 350 nm gives an indication of the nucleotide pattern. In this method, compounds are usually identified by spectral characteristics and (250 nm/260 nm) and (280 nm/260 nm) ratios (Table V1). such as h,,,(nm), Typical spectra for N. ruber are shown in Fig. 4. It can be seen that the spectra of the fraction held by the resin approximates to that of adenine nudeotides, while the fraction not so held has a spectrum close to that of homarine, slightly modified by the presence of trigonelline, which was subsequently separated from abalone in the authors’ laboratory by the method of Hiltz (1970). Hirano (1975)

Dead in shell

Fresh

Mexican dried

Dried

091

0.8

1

I

I

‘.\ 200

250

300

200

250

300

200

250

300

350

200

250

300

350

Wovelength Imp1

FIG. 4. Spectral characteristics of perchloric acid extracts of abalone neutralized to pH 6.5: -total extract; -----after shaking with resin; ---- compounds absorbed onto resin, by difference. ,A,

~ _ _ _ _

Fresh, (0-72 hours at 6°C) Dead in shell Dried Mexican dried

of compounds (nm)

Exchanged onto resin

Not exchanged onto resin

259 25 1 259 256, 306, 314

213 2 11 212 210, 322

160

JUNE OLLEY AND S . J. THROWER

has found that there is a seasonal variation in the homarine content of abalone muscle and liver, an approximate doubling of the homarine content of the muscle being accompanied by a halving of that in the liver. A number of other compounds which absorb in the UV have been found in abalone. Nishita et al. (1966) found two unidentified compounds with A, 310 nm and 330 nm by column chromatography of acid-soluble extracts of H. discus. J. Olley, C . Baldwin, L. Barker, and C. Paice (unpublished results, 1969-1973) used the Jones and Murray (1964) method described above t o analyze perchloric acid extracts of dried abalone from Mexico (Fig. 4). They at 314 nm and a minor peak at 310 nm found a large acidic peak (A,,,=) absorbed onto the resin, and a compound with an even larger spectral peak (A, 322 nm) which was not absorbed. These compounds with A, greater than 300 nm were not observed in N. ruber, the Tasmanian abalone. It is possible that the geographical distribution of these species is responsible for the presence or absence of such compounds. Shibata (1969) found a range of such 315 to 323 nm) is algae and corals from the compounds designated S-320 (A, Great Barrier Reef off northern Australia. He suggested that they might be pigment precursors or UV filtering substances in organisms that live in tropical waters.

F. LIPIDS The lipid depots of the abalone are in the organs and vary through the reproductive cycle, building up to high levels in the digestive tissue and then concentrating in the ovaries as the eggs ripen (Webber, 1970). Levels in the foot fluctuate from 5 to 10% dry weight, but these fluctuations show no seasonal pattern. The lipid content of abalone muscle is shown in Table 111, and the more detailed composition in Table VII. Phospholipids, the basis of membrane structure, are present in the foot in about the amounts that might be expected for muscle tissue (J. Olley, unpublished, 1962, quoted by Lovern. 1962). Very low levels of total lipid reported in Table 111 may be due to methods of extraction. The fatty acid composition of abalone (Table VII) is quite similar for different species and is remarkable for the high content of arachidonic acid, unusual in marine species. All species contained 30 to 35% polyunsaturated acid, but only the South African species contained appreciable quantities of C22:6 acid. Shimma and Taguchi (1964) have attributed a high content of C20:4 and C22:5 acids to animals that feed on a seaweed diet; C22:6 acids are attributed t o a plankton diet. The fatty acid composition of the viscera is similar to that of the foot (Table VII). The unsaponifiable lipids of the viscera of H. discus were largely cholesterol and glyceryl ethers (Hayashi and Yamada, 1972; Koga, 1970).

TABLE VII PRINCIPAL COMPONENTS O F T H E LIPIDS O F ABALONE

H. midae,a foot, Fatty acidse

Neutral lipid

Phospholipid

C14:O C16:O C16: 1 C18:O C18: 1 C20:4 C20:5 c22:5 C22:6

6 25 11 8 22 4 6 2 1

5 22 4

Total lipid (% fresh weight) Phospholipid (% total lipid) Sterol (% total lipid)

'De Koning (1966). bBannatyne and Thomas (1969). 'Shimma and Taguchi (1964). dHayashi and Yamada (1972). ePercentages of the total fatty acids. fNot reported.

H. iris,b

H.japonica,'

H.discus,'

foot,

foot,

foot,

H. discus,d viscera,

total lipid

total lipid

total lipid

total lipid

4 21

14 24 6 3 19

5

5

23 2 7 16 13 8 10

20 4 4 17 12 10 8

1 .O

1.o

70 12

N R ~ NR

0.58 NR NR

8 17 13 5 4 9

3 5 16 11 9 7 0.56 NR NR

5

6 1 Trace 6.1 9.9 4.4

162

JUNE OLLEY AND S. J. THROWER

The proportions of choline, ethanolamine, inositol, and serine in the phospholipids of abalone muscle differ little from those of the muscles of other phyla (White, 1973), and the analysis of pink abalone (Haliotis corrugafa) muscle (Simon and Rouser, 1969) is strikingly similar to that for the total soft parts of whole abalone, Haliofis midae (de Koning, 1966). De Koning found that the ethanolamine phospholipids were predominately plasmalogens, and the sphingolipid was unusual, liberating 2-aminoethylphosphonic acid on hydrolysis. Phosphonolipids which seem to be resistant to enzymic attack are present in those phyla that present a living naked membrane to the environment (Rosenberg, 1973). It is predicted that these compounds would be deposited in human tissue after the consumption of edible mollusks. No work has been done on the development of rancidity in frozen-stored or dried abalone. Bannatyne and Thomas (1969) pointed out, however, that the presence of large amounts of polyunsaturated acids with four, five and six double bonds could render shellfish extremely susceptible to oxidative rancidity. Castell and Spears (1968) found that invertebrate muscle, particularly that of lobsters, was strongly protected against rancidity induced by metals, especially Cuz+. Lobsters, like abalone, have large amounts of hemocyanin, a copper protein pigment which functions in oxygen transport (Pilson, 1965), and it is possible that this compound in its reduced form in the flesh could bind with oxygen and so prevent oxidation of the lipids.

G . COMPOUNDS CONTAINING SUGARS The main energy reserve of abalone muscle is glycogen, the amount of which varies markedly with season and species (Table 111). There is a lack of information about the levels of free sugars and related compounds in abalone muscle. The levels of enzymes concerned with carbohydrate metabolism in the muscle were shown by Albrecht (1921) to be undetectable, although, as this author pointed out, in an animal with such a low overall metabolic level “an amount of enzyme too small to be detected is still sufficient to hydrolyze the glycogen stored in the pedal muscle.” As indicated in Section VII,D, very pronounced browning reactions, presumably involving sugars and amino acids, do occur during the drying of abalone, if the material is not thoroughly leached by boiling, and it is highly probable that such compounds occur in the muscle, if only seasonally. The enzyme systems concerned with sugar metabolism in the viscera have been studied in some detail. As befits a herbivorous animal, the digestive juices (Albrecht, 1921) and the hepatopancreas (Bennett e l al., 1971) are rich in glycosidases; the pattern of these enzymes in the hepatopancreas has adapted to break down the type of bonds encountered in the weed and algae on which the abalone feeds.

ABALONE-AN ESOTERIC FOOD

163

The enzymes of the normal glycolysis and pentose phosphate pathways have been shown to operate in the hepatopancreas of H. rufesceris (Bennett and Nakada, 1968). The activities of these enzymes were quite low, however, especially lactic dehydrogenase which was, relatively speaking much lower than a-glycerophosphate dehydrogenase. It has been suggested that abalone metabolism may be modified in a similar way to that of clams, oysters, and mussels (Simpson and Awapara, 1966; Stokes and Awapara, 1968; de Zwaan and Zandee, 1972; de Zwaan and van Marrewijk, 1973) in which alanine and succinate are the main end products of metabolism with small amounts of D-lactate and glutamic acid also formed. No information is available about carbohydrate metabolism in abalone muscle, but Gade and Zebe (1973) have shown that anaerobic pathways in the adductor muscles of six different species of mussels lead to the production of succinate and octopine rather than the normal L-lactate of mammalian systems. However, see Section IV,J. The mucous substances which exude from the pedal sole of abalone muscle have been studied by Tanikawa et al. (1962). Three mucoproteins have been isolated and their constituent carbohydrates characterized. Glucuronic acid, galactose, and N-acetyglucosamine were present in all three compounds, fructose was also present in two, and fucose was detected in one mucoprotein. The collagens of invertebrates are firmly bound to carbohydrates which affect their structure as observed under the electron microscope (Hunt, 1970). Kimura (1972) found that the collagen of H. discus contains 4.2% sugars, mostly glucose and galactose glycosylated to hydroxylysine, 47% of the hydroxylysine in the molecule being glycosylated. H. NATURALLY OCCURRING PIGMENTS The most obvious pigments visible in an intact abalone are located in the epipodium and extend into the edges of the pedal sole; the common names of different species are often derived from the pigments in these parts-for example, green lip (S. luevigata) and black lip (N. ruber). Color drawings of these animals can be seen in Australian Fisheries 32(7). Other pigments occur in the shell and muscle of the abalone. Chew (1973) has examined the pigments of the epidermis and tissue of the pedal sole and epipodial region of H. iris (HI), N. ruber (NR), and S. lueviguta (SL). He distinguished between the black (HI and HR) and iridescent green (SL) pigments in the epidermis of the epipodial region. He also found pigments from green to brown in the epidermis and tissue of the pedal sole, and green (HI) and yellow (NR) chromatophores occurring in tissues below the surface of the pedal sole and epipodium. These green and yellow pigments were not identified, but all pigments gave the infrared spectra of certain melanins, those of the epidermal tissue resembling pigments of Arenicola and Holothunu and those of the muscle

164

J U N E OLLEY AND S. J. THROWER

resembling the melanins of Sepia (Bonner and Duncan, 1962). By inference from the large difference in chromatographic mobility, Chew concluded that the different pigments were in fact different polymers of melanin. The nitrogen content of the isolated pigments varied from 2 to 5%, indicating a mixture of indole and catechol melanins. It should be noted that Hackman and Goldberg (1971) have criticized infrared analysis of melanins as being meaningless, since tannins, lignins, and humic acids give spectra similar t o those of the melanins. &Carotene, as previously mentioned, and unidentified xanthophylls (Fisher ef al., 1956) are also present in extracts of whole abalone. Bilichromes or bile pigments are found in the shell (Goodwin, 1972). The beautiful mother of pearl colors of the abalone shell, which determine its value as jewelery, are to some extent influenced by the seaweeds eaten by the shellfish (Ino, 1953; Leighton, 1961; Poore, 1972; Sakai, 1962). I. VOLATILE BASES Spoilage of foodstuffs produces volatile compounds which are responsible for the putrid odor of "stale" food. One class of such compounds is the volatile bases, usually measured as total volatile bases (TVBs). The volatile bases produced during spoilage of unfrozen material are usually trimethylamine (TMA) and ammonia. The Codex Alimentarius Committee has recommended that the maximum concentration of TMA allowed in fishery products should be 5 to 10 mg of TMA-N per 100 gm. Tozawa ef al. (1971) have pointed out, however, that tests that measure other amines together with TMA are often better indicators of organoleptic quality than those that measure TMA alone. TMA is formed by the breakdown of trimethylamine oxide (TMAO) in the flesh. There are conflicting reports in the literature about the level of TMAO in abalone muscle. Konosu and Maeda (1961) claimed that TMAO was virtually absent from abalone (H. gigantea), while Simidu er al. (1953) found appreciable amounts in flesh of that species. The latter authors showed that the level of TMAO decreased from 60 to 33 mg of N per 100 gm during storage for six days at 8°C to 1O"C, and this decrease was accompanied by a rise of 30mgper lOOgm in TVB-N. Tanikawa er al. (1962) showed that volatile base nitrogen reached the organoleptically unacceptable levels of 15 to 18 mg per 100 gm quite quickly (Table VIII), and this level of TVB was associated with a slight putrefactive odor. It should be noted that, in species where large quantities of arginine are present, Bacterial spoilage produces omithine and ammonia (Sidhu er al., 1974), although the arginine content of abalone is open to question (see Section IV,D), and omithine has not so far been reported in abalone. Mollusks have large reserves of glycogen in the muscle, and after death these are broken down to organic acids by the animal's own enzymes, causing the pH

ABALONE-AN ESOTERIC FOOD

165

TABLE VIII SPOILAGE OF ABALONE MUSCLE'

Holding conditions

Temperature

Eviscerated In shell Eviscerated In shell In shell

5

120

5 20 20

216 19 19

35

17

e C)

Time to reach unacce table levels of volatile base (hr)

E

'From Tanikawa eral. (1962). *15 to 18 mg per 100 gm organoleptically unacceptable.

to fall. The volatile bases produced ultimately cause a slight rise in pH, but even in putrid abalone the pH is 6.2 to 6.4, the normal pH of fresh vertebrate fish. Volatile bases are less volatile at this lower pH, and are less apparent as spoilage odors than in spoiled fish, which often has a pH of about 7.2. J. MISCELLANEOUS COMPOUNDS

Albrecht (192 1) asserted that creatine and creatinine are present in abalone muscle, but these compounds have not since been reported. Paper chromatography using suitable indicator sprays or dips is a helpful method of detecting such compounds. Using paper chromatography with either isopropano1:ammonia or ethano1:ammonia as solvent systems and heating the paper with Dragendorff s reagent (Beers, 1967), J. Olley, C. Baldwin, L. Barker, and C. Paice (unpublished results, 1969-1973) detected a compound with R i s identical to that of carnitine. Using the rather crude estimate that the log of the concentration in the spot is proportional to its area, it was found that N. ruber contained 70 to 180 mg per 100 gm of muscle of carnitine. Carnitine plays a general role in the mitochondrial transport of fatty acids. It has not been reported previously in marine gastropods, although both carnitine and atrinine, an isomer of carnitine, were isolated from the adductor muscle of the fan mussel, Atrina pectinatu japonica, by Konosu et al. (1970) using ion-exchange chromatography. The R f of the latter compound in the solvents used by J. Olley, C. Baldwin, L. Barker and C. Paice (unpublished results, 1969-1973) is not known. The quantity of glycine betaine determined by J. Olley, C. Baldwin, L. Barker, and C. Paice (unpublished results, 1969-1973) using the paper chromatographic technique was 550 to 750 mg per 100 gm of muscle, which is a little less than the level (975 mg per 100 gm) reported by Hashimoto (1965). Tanikawa et al. (1962) studied the organic bases produced when abalone putrifies, using paper chromatography and treating the chromatograms with a

166

JUNE OLLEY AND S. J. THROWER

methanol solution saturated with FeC13. Agmatine, cadaverine, putrescine, and histamine were tentatively identified. Octopine as detected by the Sakaguchi reagent has not been found in extracts of abalone muscle (Regnouf and van Thoai, 1970).

V. CATCHING AND HANDLING OF ABALONE A T SEA Methods of gathering abalone vary. In the Channel Islands in the English Channel the public can collect “ormers” off the beaches. Usually abalone are caught by divers using artificial breathing apparatus at depths varying from 5 t o 40 meters. Abalone live on rocks, grazing on the algae on the surface, and on free seaweed (Shepherd, 1975). The cryptic habitat of the young animals tends to protect them (Witherspoon, 1976). The diver locates the abalone and then inserts a flat, spatula-like “abalone iron” under the foot, taking care not to damage the pedal sole, and quickly prizes the animal off the rock. Once taken to the surface, the abalone can be either shucked at sea or transported live to the factory. Distances from the fishng grounds to the processing factory are important. Small “runabout” boats, 3 to 4 meters in length, stay at sea for 10 hours and then land abalone on the beach for truck transport to the factory. Larger, ocean-going heavy displacement vessels up to 24 meters in length can operate up to 500 km from port and stay at sea for periods up to five days. Such vessels are usually equipped with wells filled with recirculating sea water in which the abalone can be stored live. To keep the animals alive for any length of time the water should be well aerated (Stephenson, 1924). The Australian Code of Practice for the handling of abalone (Anonymous, 1972a) advises that shucked abalone shall be held at 6°C and not for more than 72 hours. Storage of abalone in ice results in appreciable increases in weight due to the absorption of fresh water (James and Olley, 1970). Thls water is lost during subsequent processing; therefore processors are opposed to paying for abalone that have been iced.

VI. PHYSIOLOGY OF ABALONE IN AIR Unlike intertidal mollusks, most abalone usually live completely submerged in sea water. The removal of abalone from water into air places them in a completely unnatural environment. The changes that occur have not been studied previously. Since the subject may have important implications for live

ABALONE-AN ESOTERIC FOOD

0

.j

3960 0

*I

E ,14 00

1 f -0

167

1200

0

N

.-C c .-0

1000

$

800

.0)

C

8

600

u .-

8 400 0

.-

5

200

0

10

20

30

40

50

60

70

80

90

100

110

120

Time in air lh)

FIG. 5. Changes in the amino acid concentrations in serial 10-ml fractions of the liquor lost by individual abalone when held in air over a fraction collector at various temperatures. The stippled area shows the range of amino acids in blood as reported by Pilson (1965) and J. L. Smith (unpublished). The progressive increase in amino acid in the fluid indicates bleeding initially, and after death occurs, can also indicate autolysis of the tissues. Kinoshita and Nakagawa (1934) reported 8% loss of body weight from abalone held in air at 20.7"C, 10.6"C, and 5.8"C. Their data are indicated on the figure. The animals are near death when this weight loss occurs. The long periods during which abalone held at 6°C and 12°C lose copious amounts of fluid but relatively little amino acid is worthy of note.

transport of abalone in air and for storage of abalone prior to processing, some observations from the authors' laboratory may be of value. A detailed account of investigations made by J.L. Smith into the physiology of N. ruber in air was given by James and Olley (1974). Factors that influenced the survival rate were size, temperature, and time out of water. It was found that the optimum temperature for survival was 6°C; at temperatures below 6°C the animals suffered from cold shock, and at temperatures above 6°C mortality was inversely related to temperature, and obeyed the Arrhenius law (Olley, 1971; Olley and Ratkowsky, 1973) with an apparent activation energy of 17,000 cal/mole. The higher the temperature is above 6"C, the sooner the animals die and the faster the pH falls.* Respiration of abalone was followed by measuring oxygen consumption and carbon dioxide production in an enclosed space. Small abalone held in air very *These results should be compared with the Q l o of abalone in water (Uki and Kikuchi, 1975).

168

JUNE OLLEY AND S . J. THROWER

quickly switched over to an anaerobic respiration in which C02 was produced but very little O2 consumed.t Larger animals (500 to 700 gm) took longer to become anaerobic. The metabolic pathways used by abalone, which lead to the production of organic acids, are discussed in Section IV,G. The pH of abalone stays normal for a period that varies depending on the size of the anitnal and the ambient temperature-for example, 40 hours a t 12°C (James and Olley, 1974). Abalone held in air lose a liquor that is at first colorless and has a low protein and amino acid content. After a period, the duration of which depends on the ambient temperature, the exudate becomes blue and assumes the composition of blood (Fig. 5). The animal in fact appears to start bleeding. At 12°C this happened between 30 and 40 hours, which was when the pH started to fall (James and Olley, 1974).

VII. TECHNOLOGY OF PRESERVING ABALONE Although a small proportion of the abalone caught may be eaten fresh, most of the catch is processed by freezing, drying, or canning to ensure an adequate shelf life. Details of the Japanese technology are described by Tanikawa (1971). Before abalone can be processed, the shell and viscera are removed. This is done by inserting a flat spatula under the shell and severing the adductor muscle at its point of attachment to the shell. The mouth parts remain with the foot and must be cleanly removed. Blood pours from the severed main artery of a freshly shucked abalone. After an initial rush, the drainage of blood continues until up to 40% of the original muscle weight may be lost. Smaller animals either contain or lose less blood in proportion to their size than do larger ones (James and Olley, 1974). A residual volume of blood, which may account for up to 15% of the weight, often remains in the foot after draining, and is lost in subsequent processing. Postmortem glycolysis produces organic acids (see Section IV,G) which lower the pH of the flesh, making it tough and reducing its water-holding capacity (James and Olley, 1970). This occurs whether the animal is held intact in air or whether only the shucked foot is held. The consequences in relation to processing yields are discussed in Section VII,C. A. FREEZING Abalone is frozen for sale and for prolonged transport prior to further processing (Montgomery, 1966). There is always a residual volume of blood in ?Readers are referred to the excellent review of Hochachka (1975) on mechanisms underlying animal life without oxygen.

ABALONE-AN ESOTERIC FOOD

169

the foot. If this volume is h g h owing to inadequate draining, sheets of "ice" may form in the blocks, and the exporter may be accused of watering the product (James and Olley, 1974). The animals are cleaned, usually mechanically, to remove surface pigments, and trimmed to suit customer requirements. They are then frozen either individually, or in blocks using metal molds; after freezing, the product is wrapped in polyethylene before being packed into cardboard cartons (Anonymous, 1971). It is difficult to generalize on the effects of freezing abalone, as Japanese and Australian experience would indicate that it is easy to freeze abalone before rigor, while Korean work indicates that this is difficult because ATP breaks down rapidly in the muscle (see Section IV,E). Song (1973), finding rapid breakdown in H. giganfeu, investigated the effects of four different rates of freezing, including rapid freezing with liquid nitrogen. The tangy crispness as measured by compression tests was best maintained by nitrogen freezing; although the values fell on storage, they remained higher than values from other methods. Taste panel assessments of texture and taste were also highest for liquid nitrogen-frozen products. James (1974) also found that liquid nitrogen freezing produces a high-quality product withN. ruber, but it could not prevent a rapid contraction of the muscle on cooling, which squeezed out the blood remaining in the foot. Song (1973) showed that with the slowest rate of freezing much less liquor could be expressed from the foot using the Wierbicki and TABLE IX FINAL p~ AND WATER CONTENT OF

ABALONE^-'

Freezing and storage temperature d -7"

c

-1 8" C

Thawing treatment

PH

Water contente (%)

PH

Water contente (%)

Water at 20°C, 2 hours Air at 12"C, overnight Air at 0.5"C, overnight

6.12 6.08 6.25

72.79 73.93 74.50

6.10 6.14 6.33

71.83 72.83 14.30

aAbalone were frozen, stored for 12 days, thawed, and cooked at 90°C for 1 hour. Experiments were carried out with individual abalone. Thawing conditions were similar to the conditions of abalone at the outside of a 5- to 10-pound block, the middle of it, and a point in between. Abalone frozen at -7°C were stored at -7°C; the thermal arrest period was 17 hours. Abalone frozen at -18°C were stored at -18°C; the thermal arrest period was 5 hours. bSize range, shucked, 110 to 275 gm. 'All values in the table are the means of six analyses. dFreezing rate and temperature of storage had no significant effects on the final pH of the cooked abalone. Thawing method had a highly significant effect on the ultimate pH @ < 0.001). eOven&ied for 16 hours at 105°C.

170

JUNE OLLEY AND S. J. THROWER

Deatherage (1958) test, but it is not clear from his paper whether this liquor had already been lost during the slow freezing. When ATP breakdown is a slow process, glycolysis and a consequent fall in pH can occur on thawing. Maintenance of a high pH and moisture content are important if the thawed product is to be subsequently used for canning. Experiments in the authors’ laboratory have shown that the method of thawing is more important in determining the pH and water content in the thawed and cooked product than the rate of freezing or storage temperature over short periods (Table IX). B. BRINING Before abalone are canned or dried, they are usually brined to remove mucus from the surface, and to facilitate removal of the surface pigments, which would otherwise penetrate into the flesh during cooking. In Japan abalone is dry-salted t o 10% salt in the product (Tanikawa, 1971). Australian processors, however, usually steep the shucked abalone in brine solutions. Young and Olley (1974) have investigated the effects of brine concentration on quality and yields of the canned product. They found that size and freshness had a profound effect on yield. With fresh material, the weaker brine solutions (1 to 4%) resulted in an increase in weight, whereas solutions above 4% resulted in loss of weight. This amounted to approximately 18 gm per 100 gm of flesh in 10% brine solution. The weaker the brine used, the higher was the water retention on subsequent cooking and canning. With stale material, weight losses during brining decreased greatly, but the decrease in water-holding capacity of the muscle at the lower pH led to greater losses during cooking. This is analogous to work on the canning of pork (Callow, 1937). Although 10% brine was necessary for adequate cleaning, augmentation of weaker salt solutions with polyphosphates permitted adequate cleaning with weaker brine and thus lower weight losses resulted; 5% potassium chloride with 1% “Mera 67,” a commercial mixed polyphosphate, provided the best compromise between weight loss and effective cleaning. New Zealand abalone (H. iris) is more difficult to clean, and Chew (1973) has suggested immersion in 5% sodium hydrosulfite at 20°C followed by heating to the boiling point to produce a clean product. Sodium sulfite added at 0.25% to can brine prevents reversion of color. When large abalone are brined, the salt does not penetrate into the flesh quickly enough to lower the water activity; brining a 290-gm abalone for 66 hours in 10% brine only lowers the overall water activity to 0.96 which is not low enough to stabilize the product against attack by spoilage microorganisms (Bone, 1973). Water activity at the center would be higher than this, whereas that at the surface would be lower, because the surface is in contact with the 10% brine solution. This salt concentration gradient may explain why brined but

17 1

ABALONE-AN ESOTERIC FOOD

uncooked abalone rotted during drying, releasing a noisome orange liquid which spread from the internal blood vessels into the muscle (Young e? al., 1973).

C. CANNING After brining the abalone are usually rumbled in a rotary washer fitted with warm-water sprays. Some processors then precook because this helps to remove mucin from improperly cleaned material and makes the drained weight easier to predict. Other processors can directly after washing. Abalone are canned either in water or in brine up to 4% strength, depending on the previous brining treatment. The retorting conditions needed t o sterilize the contents are shown in Table X. It is usually accepted that a one-pound can must contain at least 8 ounces of abalone. The lughly priced Mexican and Californian products usually contain one large animal weighing as much as 11 ounces, whereas Japanese abalone, being extremely small, are often packed six or seven to a can. These small abalone are frequently more tender than the Australian and Mexican products, possibly because of the shorter retorting time required; in general they have a higher pH, but i t is not known if this is because they are fresher or because they have been chemically treated. In the canning of abalone the drained weight of the contents is the yield of most importance to the processor. He often has difficulty, therefore, in understanding that a large loss in drained weight after canning may still in fact indicate a high yield in terms of the original weight of raw material. An abalone with a TABLE X RETORT CONDITIONS FOR 301

x

4 1 I CANS OF

ABALONE^'^

Mollusks per can

Initial temperature F)

Time at 240°F (min)

Large Large

2 2

100 160

65

Medium Medium

3 3

100 160

65 5s

Small Small

5

5

100 160

55 50

Mollusk size

e

IS

‘The retort processes were specified to give commercial sterility. Where abalone are of mixed sizes in a production batch, the largest retorting time would have to be used. %oard (1966).

112

JUNE OLLEY AND S. J. THROWER

high pH or one that has been put in weak brine, or not brined at all, contains more water than one of low pH or one that has been put in strong brine before canning. The former abalone lose more water on canning because they have more to lose. The yield from such material in relation to the original weight of raw material is greater, however, than that from the latter material (Young and Olley, 1974). Sometimes a deterioration occurs in the flesh in the center of the abalone, reminiscent of “honey-combing” in tuna that is stale when canned. The center or core of the muscle is oversoft and is like mince in appearance, while the exterior is “blown up” and the epidermis may show splitting. Tanikawa (1971) claims that honey-combing in tuna is caused by gases generated in the center of the muscle escaping through the soft but not yet coagulated collagen in the outer parts. In stale abalone the pedal arteries and vein, which are lined with collagen, are notably dilated, and bacterial action starts in these areas, presumably with production of volatile compounds. Swelling of the foot with splitting of the epidermis has been produced in the authors’ laboratory by brining in 9% salt t 1% sodium carbonate which would release bubbles of C02 in the flesh. Hypochlorite sometimes used by the fishermen at sea as a preservative could also cause release of gas, and similar problems with the core of the foot have been encountered. D. DRYING The uses of dried abalone and a description of the types of product have been given (see Section 111). Traditional methods of drying abalone vary. In Mexico abalone are sun-dried, whereas in Japan a complicated process involving alternate drying and boiling treatments over a period of up t o 1 month has been in use from very early times (Tanikawa, 1971). Doe et al. (1973) have described the optimal conditions that should be used to produce a high-quality product, and these will be summarized briefly. The foot is brined overnight in 10% brine, then precooked by bringing it t o a boil in 15 minutes and boiling gently for a further 5 minutes. This denatures the enzymes and removes the initial bacterial load, particularly in the blood vessels. The foot is then dried mechanically, at a recommended temperature of 30” to 40°C and wind speed of 1.5 t o 2.0 m/sec, care being taken to adjust the humidity so as to keep water activity low enough to prevent microbiological deterioration. If faster rates of drying are used, case hardening may occur; this can be avoided by slightly humidifying the drying airstream. Water diffusivity through the muscle is of the same magnitude as that for beef steak. Doe (1973) has developed a mathematical model for drying with shrinkage for abalone, beef steak, and gelatin.

ABALONE-AN ESOTERIC FOOD

173

The characteristics required in a highquality semidried product for the Hawiian trade have been described by Young et al. (1973). The abalone should have a translucent amber color, clearly showing the dark markings of the pedal arteries, vein, and sinuses (Fig. 6). Darkcolored opaque products are less popular (Tanikawa, 1971). Thin slices of abalone should stretch elastically when pulled from the margins. Flavor, color, and translucence of the product depend on the degree to which browning precursors have been Ieached out during precooking. Strong browning reactions which are known to produce flavorous compounds occur when the abalone is not well cooked before drying. Too long a cooking leaches browning precursors and gives a tasteless product. The product should have a slightly salty flavor and may be lightly smoked to add a characteristic tang. The flavor of the semidried product when cooked in oil is reminiscent of bacon. The succulence of the dried product when cooked in the Chinese manner depends on the ease of reconstitution. The water-holding capacity of rehydrated muscle is less than that of other shellfish, being reported as 44 to 50%moisture by Arai et al. (1968) and as 55 to 57% by Young et al. (1973). A white to yellowish powder consisting of recrystallized bases, amino acids, and salts sometimes appears with storage on the surface of the dried product (Tanikawa, 1971 ; Young et al., 1973). Although these deposits can be prevented (Tanikawa, 1971), the authors have been told that the buyer may expect to see them.

FIG. 6. Photograph of a translucent sample of N. ruber dried under ideal conditions (Young e l a!., 1973). Note two pedal arteries and median pedal vein running the length of the foot, and the numerous blood sinuses.

174

JUNE OLLEY AND S. J. THROWER

V I I I . QUALITY ASPECTS A. TEXTURE The texture of abalone meat is related to the distribution of protein within the foot (see Section IV,C) and the treatments to which the material has been subjected. In raw abalone the epipodium and pedal sole, being rich in collagen, are noticeably tough, whereas the adductor muscle is markedly softer (James and Olley, 1971a). Cooking converts the collagen of abalone to gelatin, the extent of conversion varying with the anatomical origin of the tissue considered (Table XI); overall, after 1 hour’s boiling the conversion is about 40%. On canning, almost all the collagen is gelatinized and the pattern of texture is reversed; the epipodium and pedal sole become soft and succulent, while the adductor muscle becomes tougher, particularly at the base of the pillar where the protein concentration is greatest. James and Olley (1971a) have shown this clearly in axonometric diagrams obtained by measuring the texture over slices of uniform thickness, with a single punch maturometer. The hydration of the protein is controlled by pH, and these authors found that the force required to pierce a slice of abalone could vary sixfold depending on pH, moisture, and protein content of the segment. However, the pattern of relative toughness in relation to anatomy remains the same over a slice (see Fig 3 of James and Olley, 1971a). Love et al. (1974) have pointed out the difficulty in distinguishing between changes in texture of fish caused by different moisture and protein contents and pH values, because the three parameters are so intimately related. James and Olley (1970, 197 l b ) obtained highly significant correlations between pH, moisture content, and texture. Hamm (1966) has described in detail the effects of pH on meat proteins. The variation in the ultimate pH of meat is usually not great, but abalone can be eaten in the pH range 5.8 to 7.0, depending on the freshness. The textural changes are extremely obvious even to an untrained taste panel, tasting abalone for the first time (James and Olley, 1970). The semidried product is “chewy” and elastic. This elasticity is uniform over a slice (Young et al., 1973) and may perhaps be attributed to the paramyosin spindles which are the only filamentous elements that can be clearly seen in the dried product (Fig. 3c). A process for tenderizing abalone steaks with proteolytic enzymes has been patented (Anonymous, 1 9 7 2 ~ ) .

B. FLAVOR The flavor of abalone is very subtle. A number of reports by Japanese workers suggest very strongly that free amino acids are the most important taste con-

TABLE XI COLLAGEN AND GELATIN IN PROCESSED ABALONE'

Process ~

Weight of individual abalone (gm)

Sample

Collagen (% total solids)

Gelatinb (% total solids)

Percent conversion

~

Boiled for 1 hour

180

(a) Top of adductor muscle (b) Pedal sole (c) Epipodium (d) Cooking water Weighted total a 4

Canned, retorted at 116°C for 65 minutes

152

Whole foot

1.42' (2.26) 2.63 (3.32) 26.14 (31.30) -

6.87 (8.50)

1.67' 3.39 8.21 0.04

(2.41) (3.86) (8.27) (0.05)

4.75 (5.20)

54' 56 24

(52) (54) (21)

-

37.9 (40.8)

14.35

'From data supplied by F. Young (unpublished results, 1973). bGelatin was defined as hydroxyproline soluble in 10% trichloroacetic acid (Hughes, 1963). Hydroxyproline was determined by the method of Bergman and Loxley (1969). The spectrum of the chloramine-T oxidation products with Ehrlich's reagent was monitored frequently for peaks other than the hydroxyproline peak of 558 nm. 'The recovery of hydroxyproline added before the addition of chloramine-T varied from 80 to 99% for pedal sole and epipodium and from 60 to 70% for the adductor muscle. Values in parentheses were adjusted for recoveries.

176

JUNE OLLEY AND S. J . THROWER

stituents in abalone meat. Simidu et al. (1953) suggested that the monoamino acids were “the very part of the tasty stuffs” in abalone. Takagi and Simidu (1962a) intimated that the “sweet” taste of shellfish was primarily due to free glycine. In a later paper Takagi and Simidu (1962b) showed that the seasonal depletion of.monoamino acids and consequent lowering of the ratio of monoamino acid amino N to monoamino total N reduces the palatability of oysters during summer spawning. It is possible that there is a relationship between changes in taste and protein breakdown. Hashimoto ( I 965) investigated the taste of abalone by mixing synthetic chemicals in the same proportions as they occur in aqueous extracts of abalone meat, and then removing one or more components from the mixture. He found that glycine and alanine were the most potent taste-producing substances, whereas glycine-betaine, glutamic acid, and AMP contributed to a “meaty” taste. Glycogen gave the test solution “body, harmony and smoothness in taste.” The 50% depletion of collagen levels in the early summer months (see Section IV,D) would presumably release large amounts of free glycine (see collagen composition in Table V) which could account for the improvement in taste in the early part of summer reported by Takayama et al. (1970).

PIG. 7. Ultraviolet spectra between 200 and 400 nm of the dialyzable compounds from the can liquor of N. ruber retorted at 240°F for 6 5 minutes (Young unpublished results, 1973). -Spectra in acid; - - - spectra at pH 7.0. Compounds were separated exactly as described by Christianson et al. (1960). The eluates were neutralized and diluted t o O.S% salt strength. A taste panel of seven members was able t o distinguish fractions from 0.5% salt solution alone. Definite flavors were attributed t o seven fractions: 3, iodine; 6, sweet; 8 , sweet; 9, bitter, acid, or oysters; 12, sweet; 16, bitter with iodine odor; 25, metallic, chemical. Two fractions, 1 and 20, had a negative compensatory action o n the 0.5% salt in the solution (Amerine ef a l , 1965). Fractions 1 1 and 13 had thc characteristic spectra of homarine and trigonelline. The spectra emphasize the complexity of the components present after canning and are a guide t o the elution characteristics of the flavorous compounds, but should not be equated with them. Numbers in parentheses indicate serial volumes of HCI eluate.

ABALONE-AN ESOTERIC FOOD

177

During canning, many new compounds are formed including peptides from the breakdown of proteins. Figure 7 shows the characteristic spectra of twenty-five compounds from the dialyzate of abalone can liquor; many of the fractions had definite flavors, although the tastes were not well defined. Because abalone flavor is so subtle, any chemical used to preserve or bleach the product can be readily detected. Shellfish flavors are sometimes added to the brine in cans to counteract the use of such chemicals (Chew, 1973). C. ODOR AND APPEARANCE The smell of abalone, like the flavor, is delicate, and any chemical preservatives are easily noticed. The raw material may be disfigured by nicks in the pedal sole and tears in the epipodium. Thick mucus collects on the pedal sole of stale raw material. Splits in the circumference of the pedal sole are caused by too rapid temperature changes during cooking. A desirable appearance of the canned product is dependent on the almost complete removal of the melanin-like pigments from the surface of the foot and epipodium (Section VI1,C). Penetration of brine produces color changes in the flesh, (Dreosti and Jantjies, 1966; van der Merwe and le Roux, 1952). The unbrined muscle is flesh colored, changing from pinkish yellow, to pink, to brown with increasing penetration of salt. Desalting after brining tends to produce a bluish tinge. Brining and canning of stale material can produce livid pink patches withm the flesh (J. Olley, C. Baldwin, L. Barker, and C. Paice, unpublished results, 1969-1973). Van der Merwe (1954) noted a deep blue color along the mantle and in the fringes when abalone from the West Coast of Africa are canned. This effect was also noted in Tasmanian abalone during 1971 when seawater temperatures exceeded 18°C (Winstanley, 1972) and in South Africa in 1975 when high water temperatures were experienced ( S . Rudd, personal communication). Blood pigments in invertebrates are more oxygenated when the animals are under stress. Van der Merwe (1954) found more iron in blue areas than in normal flesh, but Olcott and Thrower (1972) found that the epipodium, where bluing is most intense, always contains relatively high concentrations of iron whether bluing has developed or not. They attributed the bluing to the oxidation state of hemocyanin which is colorless when reduced and blue when oxidized, and they were able to reverse the bluing with dithionite or ascorbic acid before canning, but not afterward (James et al., 1973). Van der Merwe (1954) found that 0.5%citric acid (based on the weight of the flesh) eliminated discoloration but gave an acid flavor to the product. He also found that not all abalone of a batch turned blue on canning, thus agreeing with Pilson (1965), who observed remarkable differ-

178

J U N E OLLEY AND S . J. THROWER

ences in the hemocyanin content of the blood of individual abalone. The significance of this appears in conjunction with the fact that the blue discoloration occurs around blood vessels and sinuses. Abalone with uniformly brown flesh are richer in iron and have to be down-graded, because of poorer consumer acceptance. The liquor in the can should be a very light amber color. Mexican abalone has a dense white opalescent can liquor and is highly prized, yet Tanikawa (1971) states that the can liquor should be clear. The discrepancy lies in the fact that when abalone are improperly cleaned they also yield a turbid solution. It is often not pure white, and has particles of melanin floating in it. The white opalescence is glycogen whch would add to the “body” of a soup. James et al. (197 1 ) found a highly significant correlation (r = 0.84, p < 0.001, d.f. 37) between the turbidity of abalone can juice measured in a simple optical turbidity comparator with the glycogen content of the juice which ranged from 0.5 to 13%.

IX. BY-PRODUCTS OF ABALONE PROCESSING Shells with a good nacre are used for jewelry. The viscera may be sold for human consumption (see Section 111) or they may be used as crayfish bait, but in many instances they are discarded. The Scandinavian countries have specialized in the ensilage of all kinds of protein waste by adding a carbohydrate source and malted barley for the breakdown of starch. Olley (1972) described the ease with which abalone viscera are ensiled in this way. The viscera themselves contain a wide range of enzymes (Albrecht, 192 I), and the hepatopancreas contains powerful glycosidases (Bennett et d., 1971) (see Section IV,G). Where a source of free sugars such as apple pomace is readily available, ensiling is very rapid. Typical silages prepared in Tasmania, using 10% carbohydrate, contained 28 to 35% solids: 11 to 13% protein, 2.3% fat, 2% salt, and 0.1 to 0.3% calcium. The quantity in the diet of pigs and poultry must be limited to 40% (0.8% marine fat in the diet), or tainting of the flesh will result. Even at this low level of marine fat, pork patties made from the loin area of the pig had a bland odor and flavor (Anonymous, 1972b; Chapman et al., 1972). This change from a typical odor and flavor of pork was caused by a 0.2% increase in C20:3 or 4, a 0.15% increase in C22:4, and a 0.09% increase in C22:5 polyunsaturated acids in the pork fat. It has been known for some fifteen years that the feeding of fish silage made from fatty fish, particularly herrings, produces a fishy taint and smell in the carcasses of animals (Braude, 1962). The production of pigs and poultry with natural flavor reduced or completely lacking owing t o the laying down of trace quantities of extra polyunsaturated acids in the flesh is, however, just beginning to be recognized (Wessels et al., 1973).

ABALONE-AN ESOTERIC FOOD

179

Animals fed abalone viscera should be kept out of the sunlight, as they might be photosensitized by a photodynamic agent in the abalone liver (Hashlmoto et al., 1960; Hashimoto and Tsutsumi, 1961).

X. RESEARCH NEEDS A knowledge of the basic biochemistry and physiology of an animal is often needed to improve technology, although an empirical approach will sometimes yield solutions. The authors found that as more information was collected and collated most of their initial generalizations were contradicted. This is because most of the references are to work done by biochemists and food scientists rather than by physiologists and anatomists. The exact origin of the tissue sample analyzed is seldom mentioned; neither is the size, season, or stage of the sexual cycle, all of which have marked effects on the composition. Thus, with many aspects of the biochemical composition, one might almost say “start again.” There are, of course, notable exceptions to this criticism. A knowledge of the exact protein components and their distribution in the tissue is an academic challenge which might well lead to technological advances. The complex interaction of factors influencing the amino acid pool offers scope for further study. The factors that determine the rate of nucleotide breakdown are unclear. Neither the intermediates nor the end products of glycogen degradation in the muscle have been studied. Outstanding technological problems which have yet to be satisfactorily solved include improved methods for the removal of melanoi’d pigments from the surface of the foot and epipodium, a method of prevention of seasonal bluing which is acceptable in food legislation, improvements in the reliability of techniques for live transport to ensure a higher survival rate, and the development of aquaculture techniques to protect natural populations while maintaining an adequate supply of this highly priced seafood. The mixture of collagen gelatin and glycogen which often constitutes a large part of abalone flesh would hardly seem to justify the high price paid for this seafood; yet Asian peoples have eaten abalone as an “elixir of life” for centuries. For all we know they may have been intuitively cognizant of the heat-stable, antiviral (Li, 1960), and antibiotic (Prescott and Li, 1960) substances which are now known to be present in abalone muscle. This aspect-abalone as a protective food-opens perhaps the most exciting avenue for future work.

REFERENCES Albrecht, P. G. 1921. Chemical study o f several marine molluscs of the Pacific Coast. J. Biol. Chem. 45. 395.

180

JUNE OLLEY AND S. J. THROWER

Allen. W. V., and Kilgorc J. 1975. The essential amino acid rcquircmcnts of the red abalonc, Haliotis rufiwens. Comp. Biochem. Physiol. A . 50, 77 1. Amerinc, M. A,, Pangborn, R. M., and Roessler, E. B. 1965. “Principles of Sensory Evaluation of Food.” p. 121. Academic Press, New York. Anonymous. 1971. “Survey of Australian Seafood Processing Industry,” p. 90. Aust Govt. Publ. Scrv., Canberra. Anonymous. 1972a. Code for handling and processing abalone. Aust. Fish. 31(5), 9. Anonymous. 1972b. Trcatinp abalonc waste. Rural Res. CSIRO 7 7 , 28. Anonymous. 1972c. Trcating abalone meat. Australian Patent 427 865. Arai, K. 1961. 11. Acid soluble nucleotides in muscle of marine invertebrates. Effects of storing temperature upon thc contcnt of muscular nucleotides of some seashell. Bull. Fac. Fish. Hokkaido Uhiv. 11, 225. Arai, K. 1966a. Nucleotides in the muscle of marine invertebrates. Bull. Jpn. Soc. Sci. Fish. 32, 174. Arai, K. 1966b. V. Acid solublc nucleotidcs in the muscle of marine invertebrates. Degradation of adenylic acid in the muscles of scallop and abalone. Bull. Fac. Fish. Hokkaido Univ. 17, 91. Arai, K., and Saito,T. 1961. Changes in adenine nucleotides in the muscles of some marine invertebrates. Nature (London) 192, 45 1. Arai, K., Kobayashi, K., and Saito, T. 1968. On the acid soluble nucleotides of the freeze-dried muscle of some marine invertebrates. Eiyo To Shokuryo 20,413. Aristotle. ca. 347 B.C. “Historia Animalium,” Vol. 4, p. 4. 529b, 15 Bekker Berol. Bannatyne, W. R., and Thomas, J. 1969. Fatty acid composition of New Zealand shellfish lipids. N. Z. J. Sci. 12, 207. Beers, J. R. 1967. The species distribution of some naturally occurring quaternary ammonium compounds. Comp. Biochem. Physiol. 21, 11. Bennett, R., and Nakada, H. I. 1968. Comparative carbohydrate metabolism of marine mollusks. I. The intermediary metabolism of Mytilus californianus and Haliotis rufescens. Comp. Biochem. Physiol. 24,787. Bennctt, R., Thanassi, N. and Nakada. H. 1. 1971. Hepatopancreas glycosidases of the abalone (Haliotis rufescens). Comp. Biochem. Physiol. B 40, 807. Bergman, I., and Loxley, R. 1969. Lung tissue hydrolysates: Studies of the optimum conditions for the spcctrophotometric determination of hydroxyproline Analyst (London) 94,575. Board, P. W. 1966. “Retort Processes for Canned Abalone.” CSIRO (unpublished report). Bone, D. 1973. Water activity in intermediate moisture foods. Food Technol. (Chicago) 27(4), 71. Bonner, T. G., and Duncan, A. 1962. Infra-red spectra of some melanins. Nature (London) 194, 1078. Braudc, R. 1962. Fish and fishery products in pig nutrition. I n “Fish in Nutrition” (E. Hecn and R. Kreuzer, eds.), p. 332. Fishing News (Books) Ltd., London. Burton, K. 1969. Spectral data and pK values for purines, pyrimidines, nucleosides and nucleotides. In “Data for Biochemical Research” (R. M. C. Dawson et al., eds.), 2nd ed., p. 169. Oxford Univ. Prcss (Clarcndon), London and New York. Butler, C.. 1958. Nutritional value of fish in reference to atherosclerosis and current dictary research. Commer. Fish. Rev. 20(7), 7. Callow, E. H. 1937. The electrical resistance and micro-structure of muscular tissue. D.S.I.R. Rep. Food Invest. Board p. 46. Castell, C. H., and Spears, D. M. 1968. Heavy metal ions and the development of rancidity in blended fish muscle. J. Fish. Res. Board Can. 25, 639.

ABALONE-AN ESOTERIC FOOD

181

Chapman, J. L., Fogerty, A. C., and OUey, J. 1972. Abalone silage. Aust., CSIRO, Div. Food Res., Rep. Rex p. 22. Chew, D. 1973. Pigments of New Zealand abalone (Haliotis iris Martyn). M.Sc. Thesis, University of New South Wales, Sydney, Australia. Christianson, D. D., Wall, J. S., Dimler, R. J., and Senti, F. R. 1960. Separation and determination of quaternary nitrogen compounds and other nitrogenous substances by ion exchange chromatography. Anal. Chem. 32,874. Conway, E. J., and Cooke, R. 1939. The deaminases of adenosine and adenylic acid in blood and tissues. Biochem J. 33,479. Cox, K. W. 1962. California abalones, Family Haliotidae. Fish. Bull. Calif:No. 118. Crofts, D. R. 1929. Haliotis. Liverpool Biol. SOC.Proc. Trans, Mem. No. 29. de Koning, A. J. 1966. Phospholipids of marine origin. IV. The abalone (Haliotis midae).J. Sci Food Agric. 17,460. de Zwaan, A., and van Marrewijk, W. J. 1973. Anaerobic glucose metabolism in the sea mussel Mytilus edulis L. Comp. Biochem. Physiol. B. 44,429. de Zwaan, A., and Zandee, D. 1. 1972. The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Mytilus edulis L. Comp. Biochem Physiol. B 43,47. Doe, P. E. 1973. A mathematical model of the shrinking of solids during drying with experimental data for abalone, beef steak and gelatin slabs. Aust. Conf Heat Mass transfer, 1st. 1973, Sect 4.5, p. 9. Doe, P. E., James, D. G., and Young F. 1973. Studies on the processing of abalone V. Dried abalone: Techniques and characteristics of drying. Food Technol. Aust. 25, 189. Dreosti, G. M., and Atkinson, A. 1966. Canned mince from abalone offcuts. Fishing Ind. Rex Inst. Cape Town, 20th Annu. Rep. p. 52. Dreosti, G . M., and Jantjies, M. 1966 Effect of salting on the colour of abalone. Fish. Ind. Rex Inst. Cape Town, 20th Annu. Rep. p. 24. Ebert, E. E. 1969. Abalone. In “The Encyclopedia of Marine Resources’ (F. E. Firth ed.), p. 2. Van Nostrand-Reinhold Princeton, New Jersey. Food and Agriculture Organization. 1972. Yearb. Fish Statist. 33. Food and Agriculture Organization. 1973. Yearb. Fish Statist. 34. Fisher, L. R., Kon, S. K., and Thompson, S. Y. 1956. Vitamin A and carotenoids in certain invertebrates. IV. Mollusca: Loricata, Lamellibranchiata and Gastropoda. J. Mar. Biol. Assoc. U.K. 35,41. Florkin, M. 1964. Perspectives in comparative biochemistry. In “Taxonomic Biochemistry and Serology” (C. A. Leone, ed.), p. 5 1. Ronald Press, New York. Florkin, M., and BricteuxGrdgoire S. 1972. Nitrogen metabolism in molluscs. Chem 2001. 7, 301. Fujita, T., and Hashimoto Y. 1960. Inosinic acid content of foodstuffs. 111. Marine products. Bull. Jpn. SOC.Sci. Fish. 26, 907. Gade, G., and Zebe, E. 1973. The anaerobic pathway in molluscan muscles. J. Comp Physiol. 85, 29 1. Goodwin, T. 1972. Pigments of mollusca. Chem Zool. 7,187. Hackman, R. H., and Goldberg, M. 1971. Microchemical detection of melanins. Anal. Biochem. 41,279. Hamm, R. 1966. Heating of muscle systems. In “The Physiology and Biochemistry of Muscle as a Food” (E. J. Briskey, R. C. Cassens, and J. C. Trautman, eds.), p. 363. Univ. of Wisconsin Press, Madison. Harrison, A. J. 1969. Australian abalone industry. Aust. Fish. 28(9), 2.

182

JUNE OLLEY AND S. J. THROWER

Hashimoto, Y. 1965. Taste-producing substances in marine products. In “The Technology of Fish Utilization” (R. Kreuzer, cd.), p. 57. Fishing News (Books) Ltd, London. Hashimoto, Y., and Tsutsumi, J . 1961. Isolation of a photodynamic agent from the liver of abalone, Halioris discus hannai. Bull. Jpn. SOC.Sci. Fish. 27, 859. Hashimoto, Y., Naito, K . , and Tsutsumi, J . 1960. Photosensitization of animals by the viscera of abalones, Halioris spp. Bull. Jpn. SOC.Sci. Fish. 26, 1216. Hayashi, K., and Yamada, M. 1972. Studies on the lipids of shell-fish I . On the visceral lipid composition of abalone, Haliotis discus hannai (INO). Bull. Jpn. SOC.Sci. Fish. 38, 255. Hiltz, D. F. 1970. Occurrence of Trigonelline (N-methyl nicotinic acid) in the adductor muscle of a Lamellibranch, the sea scallop (Placopecren magellanicus). J. Fish. Res. Board Can. 27,604. Hirano, T. 1975. On the distribution and seasonal variation of homarinc in some marine invertebrates. Bull. Jpn. SOC.Sci. Fish. 41, 1047. Hochachka, P. W. 1975. An exploration of metabolic and enzyme mechanisms underlying animal life without oxygen. In “Biochemical and Biophysical Perspectives in Marine Biology” (D. C. Malins and J . R. Sargent, eds.), p. 107. Academic Press, New York. Hughes, R. B. 1963. Chemical studies on the herring (Cupea hnrengus). VII. Collagen and cohesiveness in heat-processed herring and observations on a seasonal variation in collagen content. J. Sci. Food Agric. 14,432. Hunt, S. 1970. “Polysaccharide-Protein Complexes in Invertebrates,” p. 245. Academic Press, New York. Ino, T. 1953. Biological studies on the propagation of Japanese abalone (genus ffalioris). Bull. TokaiReg. Fish. Res. Lab. 5, 1. Intengan, C. L., Alejo, L. G., Concepcion, I., Corpus, V. A., Salud R. D., del Rosario, 1.. Gomez, R., and Henson, J . 1956. Composition of Philippine foods. V. Philipp J . Sci. 85, 203. James, D. G. 1974. Abalone. Aust.. CSIRO, Div. Food Res., Rep. Res. p. 31. James, D. G., and Olley, J. 1970. Moisture and pH changes as criteria of frcrhness in abalone and their relationship t o texture of the canned product. Food Technol. Attsr. 22, 350. Jamcs, D. G., and Olley, J. 1971a. Studies on the processing of abalone. 11. The maturometer as a guide t o canned abalone texture. Food Tcchnol. Aust. 23, 394. James, D. G., and Olley. J. 1971b. Studies on the processing of abalone. 111. The cftect of processing variables on abalone texture with special reference to brining. Food Technol. Aust. 23,444. James, D. G., and Olley, J. 1974. The abalone industry in Australia. In “Fishery Products” (R. Kreuzer, cd.), p. 238. Fishing News (Books) Ltd., London. James, D. G., Olley, J., and Smith, J . L. 1971. Abalone. Aust., CSIRO, Div. Food Res., Rep. Res. p. 22. James, D. G., Olcott, H. S., Olley, J., and Young, F. 1973. Abalone, cleaning and discoloration. Aust., CSIRO, Div. Food Res., Rep. Res. p, 38. Jones, N. R. 1955. The free amino-acids of fish. I . Taurine in the skeletal muscle of codling (Gadus callarius). J. Sci. Food Agric. 6, 3. Jones, N. R., and Murray, J. 1964. Rapid measures of nucleotide dephosphorylation in iced fish muscle. Their value as indices of frcshness and/or inosine 5’-monophosphate concentration. J. Sci. Food Agric. 15, 684. Kelly, R. E., and Rice, R. V. 1966. Abductin: A rubber-like protein from the internal triangular hinge ligamcnts of Pecren. Science 155, 208. Kimura, S. 1972. Studies on marine invertebrate collagens. V. The neutral sugar composition and glycosylated hydroxylysine contents of several collagens. Bull. Jpn. SOC. Sci. Fish. 38. 1153.

ABALONE-AN ESOTERIC FOOD

183

Kimura, S., and Kubota, M. 1968. Some properties of collagen from the abalone. Bull. Jpn. SOC.Sci. Fish. 34, 925. Kimura, S . , and Matsuura, F. 1974. The chain compositions of several invertebrate collagens. J. Biochem. (Tokyo) 75, 123 1 . Kinoshita, T., and Nakagawa, A. 1934. Temperature and vitality of abalone in air. Hokkaido Fish. Exp. Stn. Rep. No. 258, p. 573. Koga, Y. 1970. Studies o n cholesterol in foods. 111. Cholesterol crude protein index (CCPI). J. Jpn. SOC. Food Nutr. 2 3 , 4 12. Kominz, D. R. 1966. ‘*Phylogeny of Immunity,” p. 49. Univ. of Florida Press, Gainesville. Konosu, S., and Maeda, Y. 1961. Muscle extracts of aquatic animals. IV. Distribution of nitrogenous constituents in the muscle extracts of an abalone, Haliotis gigantea discus Reeve. Bull. Jpn. SOC.Sci. Fish. 27, 251. Konosu, S., and Mori, T. 1959. Amino acid composition of shellfish proteins. Bull. Jpn. SOC. Sci. Fish. 25, 153. Konosu, S., Chen, Y.,and Watanabe, K. 1970. Atrinine, a new betaine isolated from the Sci. Fish. 36, 941. adductor muscle of fan-mussel. Bull. Jpn. SOC. Lange, R. 1963. The osmotic function of amino acids and taurine in the mussel, Mytilus edulis. Comp. Biochem. Physiol. 10, 173. Leighton, D. 1961. Observations of the effects of diet on shell colouration in the red abalone Haliotis rufescens Swainson. Veliger 4, 29. Li, C. P. 1960. Antimicrobial effect of abalone juice. Proc. SOC.Exp. Biol. Med. 103, 522. Love, R. M. 1970. “The Chemical Biology of Fishes with a Key t o the Literature,” Part I. Academic Press, New York. Love. R. M., Robertson, I . , Smith, G. L., and Whittle, K. J. 1974. The texture of cod muscle. J. Texture Stud. 5 , 201. Lovern, J. A. 1962. In “Fish in Nutrition” (E. Heen and R. Kreuzer, eds.), p. 332. Fishing News (Books) Ltd., London. Matsuno, N. 1970. Essential amino acid index (EAA-index) of foods in Japan. Eiyoguku Zasshi 28(1), 35. Matsuno, N., Fukami, H., and Linuma, M. 1972. Essential amino acid EAA-index and A/T chemical score of foods in Japan. Eiyoguku Zasshi 30(1), 35. Mendel, L. B. 1904. Uber das vorkommen von taurin in den muskeln von weichtieren. Beitr. Chem. Physiol. Pathol. 5, 582. Migita, M., and Matsumoto, J . J. 1959. A comparative study on the extractability of muscle proteins of some animals. Bull. Jpn. SOC.Sci. Fish. 24, 75 1 . Montgomery, W. A. 1966. Processing and canning abalone. Aust. Fish. Newsl. 25, 23. Newman, G. G. 1969. Distribution of the abalone (Haliotis midae) and the effect of temperature on productivity. Repub. S. Afr, Div. Sea Fish. Invest., Rep. No. 74. Nishita, K. 1967. Studies on the organic phosphates in the muscles of aquatic animals. XIX. Isolation and identification of UDP-derivatives in the muscle of abalone and squid. Bull. Fac. Fish. Hokkaido Univ. 17, 193. Nishita, K., Arai, K., and Saito, T. 1965. Occurrence of homarine in the muscles of some marine invertebrates. Bull. Fuc. Fish. Hokkaido Univ. 16, 114. Nishita, K., Arai, K., and Saito, T. 1966. Studies on the organic phosphates in muscle of aquatic animals. XVIII. Acid-soluble nucleotides in the muscle of abalone. Bull. Fac. Fish. Hokkaido Univ. 17, 139. Olcott, H. S., and Thrower, S. J. 1972. Abalone; discoloration. Aust., CSIRO, Div. Food Res., Rep. Res. p. 22. Olley, J. 1971. Handling of abalone. In “Report on Quality in Fish Products,” Semin. No. 3. p. 89. Fishing Ind. Board, Wellington, New Zealand.

184

JUNE OLLEY AND S. J. THROWER

Olley, J . 1972. Unconventional sources of fish protein. CSIRO Food Res. Q. 32, 27. Olley, J . , and Ratkowsky. D. A. 1973. Temperature function integration and its importance in the storage and distribution of flesh foods above the freezing point. Food Technol. Aust. 25, 66. Piez, K. A. 1966. Collagen. (I? “The Physiology and Biochemistry of Muscle as a I:ood” (F,. J. Briskey, R. G. Cassens, and J . C. Trautman, eds.), p. 135. Univ. of Wisconsin Press, Madison. Pikkarainen, J . . Kantanen, J . , Vastaniliki, M.. Lampiaho, K., Kari, A., and Kulonen, E. 1968. On collagens of invertebrates with special reference to Mvfilus edulis. Bur. J. Biochem. 4,555. Pilson, M. E. Q. 1965. Variation of haemocyanin concentration in the blood of four species of Haliotis. Biol. Bull. 128, 459. Poore. C;. C. D. 1972. lkology o f New Zcaland abalone. Huliotis species (Mollusca : Gastropoda). I. Feeding. N.Z. J. Mar. Freshwater Rcs. 6 , 11. Prescott, B., and Li, C. P. 1960. Abalone juice. fractionation and antibacterial spectrum. Proc. Soc. Bxp. Biol. Med. 105,498. Pyeun. J . H . , Flashimoto. K.. and Matsuura. I:. 1973. Isolation and characterisation of abalone paramyosin. Bull. Jpn. Soc. Sci. Fish. 39, 395. Regnout. I;., and van Thoai, N . 1970. Octopinc and lactate dehydrogenase in mollusc muscles. Comp. Biochem. Phjasiol. 3 2 , 4 1 I . Rosenberg, H. 1973. Phosphonolipids. I n “l:orm and Function of Phospholipids” (G. B. Ansell, J . N . Hawthorne, and R. M. C. Dawson, eds.), BBA Libr., Vol. 3, p. 333. Elsevier, Amsterdam. Sakai, S. 1962. Ecological studies on the abalone. Haliotis discus haiwai. Experimental studies on the food habit. Bull. Jpn. SOC.Sci. Fish. 28, 766. Schafcr. R. D. 1961. Effects of pollution on the free amino acid content of two marine invertebrates. PUC.Sci. 15,49. Schmidt, C. L. A., and Watson. T. 1918. A method for the preparation of taurin in large quantities. J. Biol. Cbern. 33,499. Seki, N . 197 1 . AMP dcaminase from abalone muscle. Bull. Jpn. SOC.Sci. Fish. 37, 87 1. Seki, N., Arai, K., and Saito, T. 1967. I. Studies on the organic phosphates in viscera of aquatic animals. Acid-soluble nuclcotidcs in the hepatopancreas of abalone. Bull. Fat. Fish. Hokkaido Univ. 17, 184. Shepherd, S. A. 1975. Distribution, habitat and feeding habitats of abalone. Ausf. Fish. 34(1), 12. Shepherd, S. A. 1976. Breeding, larval development and culture of abalone. Ausf. Fish. 3544). I. Shewan, J . M. 1953. Thc nitrogenous extractives from fresh fish muscle. 11. Comparison of several Gadoid and Elasmobranch species. J. Sci. Food Agric. 4, 565. Shibata, K. 1969. Pigments and a W-absorbing substance in corals and a blue-green alga living in the Great Harrier Reef. Pfant Cell Physiol. 10, 325. Shimma, Y . , and Taguchi, H . 1964. A comparative study o n fatty acid composition of shellfish. Bull. J p . Soc. Sci. Fish. 30, 153. Sidhu, G., Montgomery, W., and Brown, M. 1974. Post mortem changes and spoilage in rock lobster muscle. I . Biochemical changes and rigor mortis in Jusus novae-hollundiuc. J. Food Technol. 9, 357. Sidwell. V. D., Foncannon, P. R . . Moore, N. S., and Bonnet, J. C. 1974. Composition of the edible portion of raw (fresh or frozen) crustaceans, finfish, and mollusks. I . Protein, fat moisture, ash, carbohydrate, energy value, and cholesterol. Mar. Fish. Rev. 36, 21. Simidu, W., Hibiki, S., Sibata, S., and Takeda, K. 1953. Studies on muscle of aquatic

ABALONE-AN ESOTERIC FOOD

185

animals. XVI. Distribution of extractive nitrogens in muscles of several kinds of gastropod. Bull. Jpn SOC.Sci. Fish. 19, 871. Simon, G., and Rouser, G. 1969. Species variations in phospholipid class distribution of organs. 11. Heart and skeletal muscle. Lipids 4,607. Simpson, J. W., and Awapara, T. 1966. The pathway of glucose degradation in some invertebrates. Comp. Biochem. Physiol. 18, 1537. Song, D. J. 1973. Effects of freezing on the quality of abalone. Reito 48, 5. Stephenson, T. A. 1924. Notes on Haliotis tuberculata I. J. Mar. Biol. Assoc. U.K. 13,480. Stokes, T. M., and Awapara, J. 1968. Alanine and succinate as end products of glucose degradation in the clam Rangia cuneata. Comp. Biochem. Physiol. 25, 883. Suyama, M., and Sekine, Y. 1965. Studies on the amino acid composition of shell-fish proteins. BUN.Jpn. Soc. Sci. Fish. 31,634. Takagi, I., and Simidu, W. 1962a. Studies on muscle of aquatic animals. XXXIV. Constituents and extractive nitrogens in a few species of shellfish. Bull. Jpn. SOC.Sci. Fish. 28, 1192. Takagi, I., and Simidu, W. 1962b. Studies on the muscle of aquatic animals. XXXV. Seasonal variation of chemical constituents and extractive nitrogens in some species of shellfish. Bull. Jpn. SOC.Sci. Fish. 29, 66. Takayama, N., Yamamoto, Y., Kadowaki, Y., and Endo, K. 1970. Chemical components of abalone meat. Kaseigaku Zasshi 21, 239. Tanikawa, E. 1971. “Marine Products in Japan,” pp. 188, 241, and 290. KoseishaKoseikaku Co., Tokyo. Tanikawa, E., and Yamashita, J . 1961. Chemical studies on the meat of abalone (Haliotis discus hannai INO). BUN. Fac. Fish. Hokkaido Univ. 12, 2 10. Tanikawa, E., Akiba, M., and Yamashita, J. 1962. Chemical studies on the meat of abalone (Haliotis discus hannai INO). Bull. Fac. Fish. Hokkaido Univ. 12, 293. Tarr, H. L. A,, and Comer, A. G. 1965. Nucleotides and related compounds, sugars, and homarine in shrimp. J. Fish. Res. Board Can. 22, 307. Tozawa, H., Enokihara, K., and Amano, K. 1971. Proposed modification of Dyer’s Method for trimethylamine determination in cod fish. In “Fish Inspection and Quality Control” (R. Kreuzer, ed.), p. 187. Fishery News (Books) Ltd. London. Uki, N., and Kikuchi, S. 1975. Oxygen consumption of the abalone Haliotis discus Hannai in relation to body size and temperature. Bull. Tohuku Reg. Fish Res. Lab 35, 73. U.S. Department of Health, Education and Welfare. 1972. “Food Composition Table for Use in East Asia,” DHEW Publ. No. (NIH) 73465. USDHEW, Washington, D.C. van der Merwe, R. P. 1954. Canned abalone: Blue discoloration. Fishing Ind. Res. Inst. Cape Town, 8th Annu. Rep. p. 23. van der Merwe, R. P., and le Roux, G. J. 1952. Abalone desalting before canning; colour vs salt content. FishingInd. Res. Inst. Cape Town, 5th Annu. Rep. p. 15. van Thoai, N., and Roche, J. 1964. Diversity of phosphagens. In “Taxonomic Biochemistry and Serology” (C. A. Leone, ed.), p. 347. Ronald Press, New York. Webber, H. H. 1970. Changes in metabolite composition during the reproductive cycle of the abalone Haliotis cracheroidii (Gastropoda : Prosobranchiata). Physiol. Zool. 43, 21 3. Wessels, J . P. H., Atkinson, A., van der Merwe, R. P., and de Jongh, J. H. 1973. Flavour studies with fish meals and with fish oil fractions in broiler diets. J. Sci. Food Agric. 24, 451. White, D. A. 1973. The phospholipid composition of mammalian tissues. I n “Form and Function of Phospholipids” ( G . B. Ansell, J. N. Hawthorne, and R. M. C. Dawson, eds.), BBA Libr., Vol. 3, p. 441. Elsevier, Amsterdam.

186

JUNE OLLEY AND S. J. THROWER

Wierbicki, F., and Deatherage, F. E. 1958. Determination of water-holding capacity of fresh meats. J. Agric. Food Chem. 6 , 387. Winstanley, R. H. 1972. Abalone and rock lobster mortality and abnormal water temperatures. Tus. Fish. Res. 6(1), 21. Witherspoon, N. B. 1976. Sizes in the Tasmanian commercial catch of black-lip abalone (Huliotis rubel.)-possible effects of changing habitat during growth. Tus. Fish. Res. 9(1), 15. Woods, E. F., and Pont, M. J . 1971. Characterization of some invertebrate tropomyosins. Biochemistry 10, 270. Young, F., and Olley, J. 1974. Studies o n the processing of abalone. VI. The effect of brine composition on the quality and yields of canned abalone. Food Technol. Aust. 26,96. Young, F., James, D. C . , Olley, J., and Doe, P. E. 1973. Studies on the processing of abalone. IV. Dried abalone; products, quality and marketing. Food Technol. Aust 25, 142.

WHEAT GERM S . R . SHURPALEKAR AND P . HARIDAS RAO Flour Milling and Baking Technology Discipline Central Food Technological Research Institute Mysore. India

I . Introduction .................................................... 188 I1. Structural Components of the Germ . ............................. 190 A. Structure of the Germ .......................................... 190 B. Components of the Germ ....................................... 194 C. Methods for Separation and Determination of StructuralComponents . . . . . 195 D . Germ Content and Composition of Its Structural Components . . . . . . . . . . . 196 Ill . Separation of the Germ ........................................... 197 A . Separation of Whole Germ ...................................... 198 B. Separation of Flaked Germ ....................................... 199 C. Physical Characteristics of Mill Germ .............................. 200 D. Air Classification of the Germ .................................... 201 IV. Chemical Composition of the Germ .................................. 204 A . DissectedGerm ............................................... 205 B . Mill Germ ................................................... 211 C. Summary .................................................... 241 V . Nutritive Value of the Germ ........................................ 242 A . Nutritional Evaluation by Chemical Methods ........................ 242 B . Nutritional Evaluation by Biological Methods ........................ 244 C. Supplementary Value of Wheat Germ .............................. 246 D . Effect of Processing on the Nutritive Value of the Germ ................ 249 E. Effect of Supplementing the Germ with Amino Acids 253 F . Toxic Factors in the Germ ...................................... 255 G . Summary .................................................... 258 VI . Storage and Stabilization of the Germ ................................ 258 A . Storage Studies ............................................... 259 B Methods of Stabilization of Wheat Germ ............................ 263 C. Effect of Storage and Stabilization on the Nutrients ................... 270 D. Summary .................................................... 273 VII Wheat Germ and Bread-Making Quality ............................... 273 A . Earlier Studies ................................................ 273

.................

.

.

187

188

S. R. SHURPALEKAR AND P. HARIDAS RAO

B. Recentstudies ............................................... 277 C. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 VIII. Food Uses of the Germ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 A. Bakery and Pastry Products . . . . . . . . . .......................... 282 284 B. Supplement for Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Germ Oil ................................. . . . . . . . . D. Fermented Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Vitamin Concentrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 F. Animal Feeds .................................. G. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Research Needs .................................... . . . . . . . . 287 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

I. INTRODUCTION Wheat is the major staple food in the dietary of people in Europe, Australia, North and South America, and some parts of Asia. It accounts for 20 to 80% of the total food consumption in various regions of the world. Wheat grain consists of the endosperm, the bran (layers of seed coat), and the germ, which account for 81 to 84%, 14 to 16%, and 2 to 3% of the grain, respectively. Most of the wheat produced in the world is processed in commercial roller flour mills into white flour or semolina, for use in the manufacture of bread, cookies, crackers, and macaroni products. In developing countries, it is processed into whole-wheat flour or semolina in hammer or disc mills of widely varying capacities. These products are used in preparing sweet and savory items for breakfast or other meals, snacks, chaparis (unleavened pancakes), wholewheat bread or rolls, etc. Commercial milling of wheat into flour aims at the maximum extraction of the endosperm with the minimum possible contamination by bran and the germ, which form the by-products of the flour milling industry and are generally used in animal feed formulations. Depending on the extraction rate, varying proportions of the germ also finds their way into the flour during milling. This is not desirable, as the presence of the germ affects the storage stability as well as the baking quality of the flour. Wheat germ is a unique source of highly concentrated nutrients. It offers three times as much protein of high biological value, seven times as much fat, fifteen times as much sugar, and six times as much mineral content when compared with flour from the endosperm. In addition, wheat germ is the richest known source of tocopherols (vitamin E) of plant origin and also a rich source of thiamine, riboflavin, and niacin. The presence of large amounts of fats and sugars makes wheat germ highly palatable. Toasting is reported to improve its flavor. Early investigators reported that, qualitatively as well as quantitatively, cereals are poor sources of protein as indicated by their low biological value or protein

189

WHEAT GERM

efficiency ratio. Interestingly enough, wheat germ proteins have been classed with superior animal proteins. In view of its high nutritive value and palatability, wheat germ offers an excellent source of proteins and vitamins for fortification of food products. Its use in bakery products, especially bread and biscuits, has received special attention. In many of the western countries, it is also used as a breakfast cereal. The only drawback in the extensive utilization of wheat germ has been its poor storage stability, owing to the presence of large amounts of fats and of oxidative as well as hydrolytic enzymes, which render the product highly susceptible to rancidity (Kuhl, 1941). Many processing methods have been reported to improve the stability and hence the shelf life of wheat germ (Rothe, 1963). Some of the heat processing techniques are also reported to improve its nutritive value by the destruction of the antinutritional factors present in the germ. The wide prevalence of protein and calorie malnutrition among the vulnerable segments of the population in many developing countries has been reported by TABLE I WORLD PRODUCTION O F

Regionlcountry

WHEAT^

Production (million metric tons)

Economically developed North America United States Canada Western Europe France Germany 0c ean ia Other developed

56.56 42.04 14.51 56.1 1 18.12 6.61 7.04 2.29 Total 122.00

Economically developing Africa Latin America Argentina Near East Far East India

6.16 12.45 8.10 26.34 33.93 26.48 Total 78.87

Economically centrally planned Asia China Europe, USSR

34.86 34.50 1 1 1.88 Total 146.73 WORLD TOTAL

'FA0 (1972).

347.60

190

S . R. SHURPALEKAR AND P. HARIDAS RAO

several nutrition workers (Scrimshaw and Behar, 1959; Bressani and Elias, 1968; Narayana Rao and Swaminathan, 1970). Consequently, a worldwide search has been in progress during the last few decades t o discover hitherto unknown or unutilized resources of food nutrients to meet the challenge of hunger. In addition t o oilseed meals, wheat germ, being a by-product of the flour milling industry, forms one of the most potential sources of much-needed proteins, calories, and vitamins a t a relatively low cost. Based on a world figure of about 3 5 0 million metric tons of wheat per annum (breakdown given in Table I), about 7 million metric tons of wheat germ providing nearly 2 million metric tons of protein and approximately 25 billion calories are potentially available. Thus, a tremendous scope exists for harnessing wheat germ t o improve the nutritional status of the needy millions. Even though a considerable amount of research has been carried out by several workers, no comprehensive review on wheat germ covering various aspects relating t o processing, nutritive value, and utilization has been published so far. The available literature on the structure, separation, composition, nutritive value, stabilization, and food uses of wheat germ is reviewed in this chapter.

II. STRUCTURAL COMPONENTS OF THE GERM Several workers (Vogl, 1899; Tschirch and Oesterle, 1900; Percival, 1921; Winton and Winton, 1932; Hector, 1936; Hayward, 1938; Fairclough, 1947; Gassner, 1951) have investigated the structure of various parts of the wheat kernel including that of the germ. Knowledge of the structure of the germ and the tissues surrounding it was found t o be important for its efficient separation during the milling process and for its utilization. The related studies were restricted to some aspects of its structure and its relation t o water penetration into the starchy endosperm of the wheat kernel during the conditioning operations of tlie milling process. Milling efficiency was found t o be largely dependent on water penetration during conditioning. Knowledge of the microscopic structure of the germ has also been useful in tlie work of quality control laboratories of wheat-based industries as well as in quality evaluation of animal feed formulations.

A. STRUCTURE OF THE GERM The germ is a separate and distinct part of the wheat kernel. Partly embedded in the endosperm, it is located at the base of the wheat kernel. There are natural FIG. 1 . Wheat kernel (Pawnee variety) bisected longitudinally through the crease (20X). This is a composite p h o t o p p h tliat givcs an idealized view of the cut surface at the right and of onc flank of the crease at the left (by courtesy, Northern Utilization Research Branch, U.S. Dept. Agr., Peoria, Illinois).

BRUSH

, OUTER PERICARP ALEURONE LAYER STARCHY ENDOSPERM NUCELLAR PROJECTION PIGMENT STRAND VASCULAR BUNDLE PERICARP I N CREASE REGIC

A

ENDOSPERM CAVITY

B C SCUTELLUM COL EOPTILE

D

PLUMULE

EPIBLAST PRIMARY ROOT COLEORHIZA SEED C O A T

FIG. 2. Transections of wheat kernel (Pawnee variety) at planes A, B, C, and D indicated

in Fig. 1 (by courtesy Northern Utilization Research Branch, U.S.Dept. Agr., Peoria, Illinois).

WHEAT GERM

193

lines of separation of the germ from the endosperm and the bran. Thus, it would appear that the germ could be easily separated even from the dry kernel. Actually, the tissues of the germ and the endosperm are in intimate contact with each other, probably through a cementing layer in between. Moistening the wheat may help in weakening the cementing layer between the two structural components (Bradbury el al., 1956d), as in corn (Wolf e l a t , 1952), and ease their separation. Structurally, the wheat kernel may be divided into three main parts (Fig. 1): (1) the germ (the embryo), which produces the new plant; (2) the endosperm, which provides the food for the new plant when the embryo starts to grow; and (3) the various outer coverings, collectively called bran, which protects the

FIG. 3. Longitudinal section of germ cut parallel to the crease (44X). E. endosperm: VS, ventral scale; Cp, coleoptile; Sc, scutellum;,El, epithelium; FL, foliage leaves; P1, plumule; SA, stem apex; B, bran; ScN, scutellar node; Eb, epiblast; PR, primary root; Co, cortex; VC, vascular cylinder; RC, root cap; Cr, coleorhiza; SC, seed coat.

194

S. R. SHURPALEKAR AND P. HARIDAS RAO

kernel. Bradbury et al. (1956a-d) have described exhaustively the structure of the wheat kernel and its various parts, including the germ, with photomicrographs. B. COMPONENTS OF THE GERM Wheat germ is composed of two major parts-the embryonic axis and the scutellum-and also the epiblast, which is of minor importance (Figs. 1, 2, and 3). On germination, the embryonic axis develops into a seedling, and the scutellum nourishes it. 1. Embryonic Axis

The embryonic axis is composed of a shoot (plumule) pointing toward the brush end of the grain and a primary root pointing toward the base. The shoot consists of a stem apex, several embryonic foliage leaves, and a protective sheath, called the coleoptile. The coleoptile is a cone-shaped sheath covered by a very delicate cuticle. Near its tip is a small pore through which foliage leaves emerge during germination. The primary root and two hairs of secondary lateral roots are protected by a sheath known as the coleorhiza. The coleorhiza is composed of parenchyma cells, covered by inner and outer epidermal layers. The walls of these cells are slightly thicker in the tip region of the coleorhiza.

2. Scutellum The part of the germ that is attached to the side of the embryonic axis nearest to the endosperm is called the scutellum. Its convex face is embedded in the endosperm, and its slightly concave surface partly encloses the embryonic axis. The slight projection near the tip of the scutellum is called the ventral scale. During germination, the scutellum supplies food, which has been stored in the endosperm during maturation, and becomes a digesting and absorping organ for the transfer of food from the endosperm to the growing part of the embryonic axis (Percival, 1921). The epidermis of the scutellum, adjacent to the endosperm, is modified to form a layer of secreting cells called the epithelium. The surface of the scutellum of bread wheats is free from invaginations or “glands,” unlike corn germ. Only a few “glands” sometimes appear near the tip of the scutellum. The provascular bundle of the scutellum consists of many protoxylem and protophloem cells which have a smaller diameter than the parenchyma cells. This bundle extends from the scutellum node into the upper part of the scutellum. Near the tip of the scutellum it divides into many small branches which extend to a considerable distance. The largest proportion of the scutellum consists of unspecialized cells

WHEAT GERM

195

Wheat kernel I

I

Pericorp

Seed 1

I -Seed

coat

1 Germ (embryo)

Endosperm I

I

I

I Epiblast

I

+I Scutellum

Epitheleum

Parenchyma

Embryonic axis

Provascular

Plumule,

tissues

including

Primary root covered by

Secondary lateral

coleoptile

coleorhiza

rootlets

-

Bran

(parenchyma), which form the body of the scutellum. In addition to the nucleus and the cytoplasm, these parenchyma cells contain a considerable amount of protein and fat and very little starch.

3. Epiblast The epiblast, a scale-like structure with little morphological significance (Avery, 1930), is present opposite the scutellum, on the other side of the embryonic axis. Different parts of the germ are clearly shown in Figs. 2 and 3. The relationship of the different parts of the wheat kernel with special reference to the germ is given in Fig. 4.

C. METHODS FOR SEPARATION AND DETERMINATION OF STRUCTURAL COMPONENTS The relative merits of different methods for separation of the structural components of the germ have been described by MacMasters et al. (1971). 1. Methods of Separation

a. Hand-Dissection of the Untreated Kernel. For separation of major components like the scutellum and the embryonic axis, hand-dissection is accurate,

196

S. R . SHURPALEKAR AND P. HARIDAS RAO

but it is laborious and time-consuming. Also, the separation of all the constituent parts of these major components cannot be achieved by this method. b. Hand-Dissection of the Kernel after Soaking It in Water. Soaking the kernel in water prior to dissection results in greater accuracy. However, soaking may alter the chemical composition of different parts, owing to the movement and redistribution of soluble matter. This method is also time-consuming. c. Maceration of the Kernel by Mechanical Treatment in Chemical Reagents. This method is comparatively quick, and many samples can be dissected in a short time. The possibility of change in the chemical constituents due to their movements or due to absorption by the reagent is the main limitation of this method. d. Separation of the Components in Organic Liquids after Mechanical Treatment of Mill Products. This method is also very rapid, but complete separation of all structural components is not possible.

2. Quantitative Determination of Structural Components Most of the quantitative data are obtained by separating the kernel components by the above-mentioned methods and immediately weighing the products. In some cases, the quantitative data are calculated by comparing the data on the chemical composition of different components of the kernel under study with those of well-defined pure components. The reliability of this method depends on the correctness of the data on the chemical composition of the pure structural components, which are generally hand-picked from mill products. Although this method is not as accurate as the hand-dissection and weighing method, larger numbers of samples can be handled.

D. GERM CONTENT AND COMPOSITION OF ITS STRUCTURAL COMPONENTS According to Bailey (1938), durum wheat has a higher germ content, 2.9476, followed by soft wheat, 2.6676, and spring wheat, 2.23%. Recently, Matveef (1965) observed that hard wheats contain a higher proportion of the germ (2.4 to 3.9%) than soft wheats (2.1 to 3.2%). Also, smaller kernels of any one variety have a smaller proportion of the germ than large kernels. The data given in Table 11 on the composition of the germ and its component parts indicate that the weight of the germ in relation to kernel weight as well as the proportion of the embryonic axis and the scutellum varies widely. These variations have been attributed to varietal differences, kernel size, and method of dissection. In general, the proportion of the scutellum was found to be higher than that of the embryonic axis.

WHEAT GERM

197

TABLE I1 COMPOSITION' OF WHEAT GERM A N D ITS COMPONENTS

Number of samples Total germ 4 (France) 4 20 (American) 5 (Australia) 8 (Russia) -

156 3 11 -

1.2 -1.5 2.5 -3.6 2.64 3.10 2.56-3.25 2.8 -3.5 2.1 -3.9 2.0 -3.1 3.4 -3.8 2.64 3.2 -3.8 -

Embryonic axis Scutellum

References

-___-

-

1.0 -1.6 1.25 1.40 -

-

-

1.4 -2.0 1.39 1.70 -

-

-

-

1.25

1.39

-

0.90-1.5 1.37

-

1.30-1.80 1.09

Girard and Fleurent (1899) Hinton (1947, 1959) Bailey (1938) Hinton (1962) Grisclienko (1 935) Percival (1921) Matveef (1 965) Mambish (1953) Kazakov (1947) Dubois er al. (1960) Morris et al. (1945) Hinton (1944) Blain and Todd (1955)

uPercent of wheat kernel, on a moisture-free basis.

111.

SEPARATION OF THE GERM

Wheat germ is a by-product of the roller flour milling industry. Its separation from other milled products is important for the following reasons: (1) It adversely affects the keeping quality of flour and other mill feeds. Stevens (1959) reported that 23 to 34% of the oil in flour originated from the germ, owing to expression of the oil during rolling. The presence of highly unsaturated germ oil in flour decreases its storage life because of oxidative rancidity (Kuhl, 1941). (2) The presence of the germ in flour was reported to affect the baking quality and color of the flour (Bull, 1937; Pomeranz et al., 1970b). ( 3 ) Wheat germ is a rich source of protein of high nutritive value, B-group vitamins, and tocopherols, and a potential nutritious food supplement. It thus has great commercial value, fetching more than double the price of other mill feeds, and can be used with advantage in the preparation of speciality breads and also as a vitamin E concentrate. Even though wheat contains 2 to 3.5% of germ, only about 1% is recovered during normal milling operations for the following reasons: (1) Most of the scutellum portion goes into the flour stream, as it is more friable, like the endosperm. The embryonic axis gets flattened between the rollers and can be recovered by proper sieving techniques. (2) Breakage of some of the germ portions into small pieces occurs in between the rollers. The broken portions escape into the floury portion and are not recovered. ( 3 ) Also, some of the germ

198

S. R. SHURPALEKAR AND P. HARIDAS RAO

portion is lost during screen-room operations such as handling, scouring, washing, and brushing. There are two ways by which the germ can be separated in the mill. One method is to separate it in the break system itself, in the form of whole germ, by using a “germ separator” as ancillary equipment. In the other method the middlings containing the germ particles are passed through the reduction rolls, where the germ gets flattened and is removed by sieving.

A. SEPARATION OF WHOLE GERM

I . Use of the Germ Separator A good account of the separation of the germ was given by Lockwood (1952). The germ must be separated before the particles of the endosperm, germ, and bran portions become very small in the break rolls. The particle size should be such that they will not pass through a 28-mesh sieve. The scutellum was present in the stocks released from the second break onward. After the second break it was released mainly in the particle size of coarse semolina, and further grindings produced an increased proportion of fine particles. The first three breaks cut up very little germ and left most of it substantially intact. However, in the later rolls most of the germ was either cut up or overtailed the scalpers, and escaped with the bran. Thus it is imperative that the germ be separated before the middlings enter the fourth break. Kent et al. (1944) found that the release of the scutellum in the break system was earlier in soft wheat than in hard wheat. Further, Kent et al. (1949) reported that the release of the scutellum and the embryo portions depended on the moisture content of the wheat. The higher the moisture content, the higher will be the amount of the scutellum that can be removed, as it is more friable at a low moisture content. Separation of the germ from the other stock was achieved by using a Simon germ separator, which was introduced on the feed to the fourth-break fine rolls. Here much of the germ could be recovered intact. The separator was fed by a stock which was sifted through a No. I6 and over a No. 28 fine wire cover. The separation of the germ from the stock was carried out by using an aspiration technique of increasing intensity. The feed contained 80 to 95% of light particles of the endosperm and bran, and the remaining heavy particles were the germ. To prevent entanglement of the germ with other light material, the stock was subjected to a large volume of air moving at 380 to 440 cfm in successive controlled aspirations of increasing intensity. Uniform stock was fed to each aspiration channel by a patented device consisting of oscillating platforms which push the material under spring-controlled feed gates. A controlled rate of airflow was obtained by careful design of

WHEAT GERM

199

the air channels and the number of curved vanes. The velocity of the first aspiration was adjusted so that only about 30% of the lighter particles was lifted out. The second aspiration was stronger and lifted some more of the lighter stock. The third aspiration lifted the remaining lighter particles except the germ. The aspirated stocks were fed to the fourth-break rolls. The separated whole germ was fed in between the smooth rollers to get the germ flaked. Rolling was done on a conventional 40-inch roller 10 inches in diameter. The speed of the rollers was adjusted in a 5:4 ratio. Purification of the germ from the bran particles could be carried out by using a plansifter, fitted with 18W and 24W sieves. The overtails form the finished pure germ product. The cut-up germ from the first three break rollers finds its way to the reduction rollers along with coarse semolina through the purifier. It gets flattened in the reduction rollers. The advantages in using a germ separator are higher yields of germ and a good-quality flour free of germ contamination.

2. Use of Prebreak Unit The method of separation of the germ by using a prebreak unit was described by Jagbir Singh (1973). The machine manufactured by Sturtevant Mill Co., Boston, called the No. 6 Simpactor, was used as a prebreak unit. This machine, placed before the first break of the roller mill, breaks the wheat kernel, so as to release more free germ. The equipment, consisting of devices such as entoleters, sifters, aspirators, and rollers, separates the germ before sending the broken kernels to the first break rolls. B. SEPARATION OF FLAKED GERM

I . Laboratory Mill Using laboratory mill LM-400, Dimitrov et al. (1972) have reported better yields as well as better quality of germ as compared to Bulgarian standard BDS 5279-70. Later, Dimitrov (1975) also studied the effect of loading germ rolls and plansifter on the yield and oil content of wheat germ: he observed that the optimum loading was 130-150 kg per cenitmeter per 24 hours and 3500 kg per square meter per 24 hours, respectively. Free bran particles to the extent of 7.2% could be removed from the embryo fraction by air separation.

2. Commercial Mill, National Joint Industrial Council of the United Kingdom The National Joint Industrial Council for The Flour Milling Industry (1966) has published a detailed account of separation of the germ.

200

S. R. SHURPALEKAR AND P. HARIDAS RAO

Separation of the germ in the normal milling system is based on its tendency to flake in between the rollers owing to the high moisture content and fat present in the germ. Some of the germ will be cut up in the early break rolls, but most of it gets ruptured in the fourth break. This ruptured germ will have a particle size similar t o that of the middlings. The middlings containing the germ are sent to the purifiers, where most of the bran will be removed. In the purifier, the germ gets mixed up with the scratch stocks, as it is coarser than the semolina. Purified semolina or scratch stocks are sent to the reduction rolls, where the semolina gets further reduced in size, while most of the germ gets flattened and is separated by sieving. Some portion of the germ, however, gets pulverized and is difficult to separate. This method does not require any additional equipment such as a germ separator. The main disadvantage is that the flour obtained is not of good quality, as some of the germ ruptured in the break rolls goes into the flour portion. This imparts a brown color to the flour. The keeping quality of the flour will also be affected, as during flaking in between the reduction rolls the oil of the germ gets extruded into the flour. The mill germ thus obtained, however, contains bran and endosperm as impurities. The major contaminant of the germ is bran. Various methods have been suggested to remove the bran from the germ. Scott (1951) described a method for separation of the bran from the germ, by impinging the mill germ on a slightly moistened smooth surface. The bran particles fall off the surface, but the germ, which adheres more firmly, can be brushed off and collected separately. C. PHYSICAL CHARACTERISTICS OF MILL GERM a. Purify. Grewe and LeClerc (1943) determined the purity of nineteen samples of mill germ, of which only three samples were found to be pure. The remaining samples were contaminated with varying amounts of bran and endosperm. They reported that the carbohydrate content of mill germ was a good index of the purity of the germ (Section IV,B,6). Tkachuk and Irvine (1969) reported more than 20%of bran in mill germ. b. Yield of G e m . The yield of germ depends on the method followed for separation of the germ, the moisture level of the wheat during milling, and the variety of wheat milled. It has been reported that the yield varied from 0.2% to a little more than 1.0% (Farrel er al., 1967; Calhoun et al., 1960). Farrel et al. (1967) have reported yields varying from 0.58 to 1.06% for germ samples obtained from nine varieties of wheat. c. Density of G e m . Farrel er al. (1967) reported that the density of mill germ ranged from 18.5 to 22.6 Ib/ft3. Grewe and LeClerc (1943) found a higher

WHEAT GERM

20 1

TABLE I11 S I E V E ANALYSIS O F M I L L GERM“ ~

Overtailings Tyler sieves

Averageb (%)

Range (%)

+l ow +12w +14W +20w +28W Pan

0.80 11.85 21.30 50.00 9.40 5.60

0.40- 1.62 0.40-28.00 14.06-32.40 36.40-61.20 2.40-14.86 0.80-20.48

“Farrel ef al. (1967). bFor germ samples from nine varieties of wheat.

density for whole germ compared with flaked germ. The density of granular germ ranged between 117 and 143 gm per half-pint, whereas that of flaked germ ranged between 65 and 98 gm. d. Sieve Analysis. Sieve analysis of mill germ was carried out by Farrel et al. (1967) for germ samples from nine varieties of wheat; the average values reported are presented in Table 111. Most of the germ was found in +14W and t20W Tyler sieves.

D. AIR CLASSIFICATIONO F THE GERM Pomeranz et al. (1 970a) have carried out extensive studies on air classification of pin-milled untreated and defatted germ using a Pillsbury Laboratory Model No. 1 classifier. The flow diagram of the germ air classification procedure given in Fig. 5 indicates four fine fractions-B, C, D, and E-and a coarse fraction, EE. Further work on the composition of air-classified germ was reported by Garcia et al. (1972b). They followed the method of Stringfellow and Peplinski (1966) and obtained five air-classified fractions and two coarse fractions, as indicated in Fig. 6. 1. Effect on Proximate Composition

The results of air classification studies as reported by Pomeranz et al. (1970a) are given in Table IV. They observed that air classification of original untreated germ was fast and economical, but the stability was very poor. Fractionation of defatted germ gave a more stable product. The protein increase in the major fraction was 0.9 to 1.6% in untreated germ; in defatted germ it was higher, 2.3 to 3.8%.

202

S. R. SHURPALEKAR A N D P. HARIDAS RAO O R I G I N A L FLOUR-A

100 POUNDS/HOUR

100 POUNDSIHOUR

e0eq

6 DECKS FORWARD (0"LOUVER CURTAIN

CCIARSF

5800 RPM

FRACTION

loo LOUVER

5 0 POUNDS/HOUR

cc

CURTAIN

IS / HOUR

FRACTION

I

10- LOUVER CURTAIN

I

2 DECKS FORWARD 35" LOUVER CURTAIN

FRACTION

IE

EE

FRACTION

FRACTION

FIG. 5. Flow diagram of the germ air classification procedure (Pomeranz ei al., 1970a).

Dry-Milled Wheat Germ

4

Defatted

@

11001

Pin hilled ! X at 9,000 r p m

t

Screen

-60,Mesh ' I

- 6 0 Mesh

c 5 0 Mesh @ -50+50 Mesh

@ 114(1

I

1

-60 Mesh Pin milled 3 X at 14.000 r p m

r

A;

t

Cla$,sificat;cn

0 0 0 0

FIG. 6. Flow diagram for processing of wheat germ. Encircled numbers or letters identify samples used in the study. Numbers in boxes indicate percentage yield.

203

WHEAT GERM TABLE IV ANALYSIS OF AIR-CLASSIFIED FRACTIONS OF WHEAT

Source and fraction Untreated germ A B C D E EE Bag Defatted germ (petroleum ether-extracted) A B C D E EE Bag

GERM@^^

(%I

Average particle size (microns)

Ash (%)

100.0 1.8 11.8 10.4 19.4 56.3 0.3

6.2 6.7 6.8 6.6 7.1 7.1 9.6

10.1 3.9 6.1 8.4 10.0 15.8 2.4

4.7 5.0 5.2 5.1 4.9 4.5 6.3

29.1 28.5 30.0 30.7 27.6 28.1 31.4

100.0 11.9 22.0 24.7 14.3 26.1 1.0

7.2 7.5 7.2 7.5 7.7 7.7 8.9

5.2 2.6 4.7 8.4 10.9 19.8 1.8

5.4 7.6 6.0 5.5 4.6 3.7 7.3

33.0 35.2 36.8 35.3 30.0 26.4 31.2

Yield

Moisture

(%)

Protein (N X 5.45)

(%I

"Pomeranz et al. (1970a). bExpressed on as-is basis.

2. Effect on Roteins and Amino Acid Composition Pomeranz et al. (1970a) also studied the amino acid composition and starch gel electrophoretic patterns in the air-classified fractions of high as well as low protein content. The starch gel electrophoretic patterns showed that the most prominent band found in the sodium chloride extract of the germ was much smaller in the high-protein fraction of defatted germ than in the corresponding low-protein fraction. The amino acids lysine, serine, proline, alanine, leucine, and tyrosine, and the sulfur-containing amino acids were less in the high-protein fractions than in the lowprotein fractions (Pomeranz et aZ., 1970a). Garcia et al. (1972b) did not find any considerable effect of air classification on the amino acid levels of different fractions except the fifth one (Fig. 6). This fifth fraction had the lowest values of lysine and arginine and the highest values of leucine, alanine, and phenylalanine.

S. R. SHURPALEKAR AND P. HARIDAS RAO

204

3. Effect on Mineral Content Garcia et al. (1972b) analyzed the three major and five minor mineral elements in the different air-classified fractions of the germ and found that fraction 1 and possibly fraction 2 had higher mineral contents compared with the original samples (Table V).

4. Effect on Carbohydrates The effects of air classification on the starch, pentosans, and sugar fractions of carbohydrates were studied by Garcia el al. (1972b). Air classification of wheat germ increased the starch content of fractions 3 and 4. Most of the pentosans were removed during screening operations, as indicated by higher values in coarse fractions A and B. Air classification had only a minor effect on modifying the pattern of sugars.

IV. CHEMICAL COMPOSITION OF THE GERM Exhaustive literature is available on the chemical composition of mill germ. In contrast, only limited information is available on dissected germ. This may be TABLE V DISTRIBUTION O F M I N E R A L S ~I N AIR-CLASSIFIED WHEAT G E R M FRACTION$

Minor elements (mg%)

Major elements (%) Defatted germ fractions Original Coarse fraction A Coarse fraction B Air-classified fractions 1 2 3 4 5 Feed to classifierC

Phosphorus Potassium Magnesium Sodium Calcium Iron Zinc Copper

51

13 17 11

20 16 26

1.8 1.6 1.1

77 56 32 30 31 46

16 13 9 9 9 II

21 23 21 17 18 2

1.3 1.1 0.7

1.69 1.77 1.60

1.36 1.69 1.41

0.43 0.63 0.40

18 14 17

58 109

1.84 1.62 1.29 1.26 1.22 1.46

1.55

1.41 1.12 1.10 1.11 1.26

0.5 1 0.44 0.32 0.30 0.28 0.38

21 27 28 22 26 25

‘Moisture-free basis. bGarcia et a l . (1972b). ‘Calculated value based on yield of air-classified fractions 1 through 5.

0.1

0.8 0.9

WHEAT GERM

205

attributed to the ready availability of commercial samples of mill germ, whereas separation of the germ by dissection is quite cumbersome. A. DISSECTED GERM When compared with mill germ, which is admixed with bran and endosperm portions during milling, dissected germ represents the purest form of germ studied. There is more disagreement than agreement in the scanty literature on values reported for dissected germ. This may be due to the differences in the methods followed for dissection, the purity of the dissected parts, and the variety of wheat used for dissection.

1. Proteins Morris er al. (1946) reported germ protein values for three principal classes of American wheat: 25.5%for Thorne variety (soft red winter), 24.3%for Tenmark variety (hard red winter), and 26.8% for Thatcher variety (hard red spring). Protein contents of 28.7% in the scutellum and 33.4% in the embryonic axis were reported by Hinton (1944) for eleven varieties of English wheats. Girard and Fleurent (1 899), however, found that the embryonic axis of germ from a French variety of wheat contained as much as 44%protein. It may be inferred that the embryonic axis contains a higher amount of protein than the scutellum.

2. Sugars The sugar contents of dissected germ and its structural parts were first studied by Dubois er al. (1960) in a soft white wheat variety from Holland. The results TABLE VI CARBOHYDRATES O F THE EMBRYONIC AXIS A N D SCUTELLUM

FRACTION I N HAND-DISSECTED

GERM^

Sugars

Embryonic axis

Scutellum

Total germb

Total sugars‘ Raffinosed Sucrosed

22.0 45.3 54.7

18.4 38.0 62.0

20.1 41.5 58.5

‘Dubois et al. (1960). bCalculated on the basis of data collected for the embryonic axis and the scutellum. ‘Expressed as percent of defatted germ on a dry-weight basis. dpercent of total sugars.

S. R . SHURPALEKAR AND P. HARIDAS RAO

206

TABLE VII EFFECT OF M O I S T U R E CONTENT OF WHEAT O N SUGARS' IN WHEAT

Moisture 9.2% Sugar

Moisture 12.3%

Scutelfum Embryonic axis Scutellum Embryonic axis

GERM^

Moisture 12.9% Scutellum

_ _ _ _ _ . ~ _ _ _

Total sugars (%) Sucrose (% of total sugars) Raffinose (% of total sugars)

13.6

18.4 62.0

22.0

11.8

54.7

57.0

17.9 56.0

51.0

38.0

45.3

43.0

44.0

49.0

'Expressed on a dry-weight basis in defatted germ. bDubois et al. (1960).

of analysis carried out according to the method of Dubois e l al. (1951) indicated that the germ contained mainly sucrose and raffinose and only traces of glucose (Table VI). Sucrose content was higher than raffinose content in the germ and its parts. They further reported that total sugar level was dependent on the moisture level of the grain (Table VII). As the moisture increased in the wheat grain, the total sugar level decreased in the scutellum as well as in the embryonic axis fractions of the germ. This was probably due to the increase in the metabolic activity of the germ with an increase in moisture. During short storage periods, the ratio of sucrose to raffinose remained constant in the embryonic axis but decreased in the scutellum of wheat containing 12 to 13%moisture.

3. Lipids Wide variations in the lipid content of the germ and its structural parts have been reported in the literature. This may be due to the difference in the solvent used for extraction of the lipids. When cold benzene was used for extraction, a lipid content of 12.9% in the scutellum was first reported by Girard and Fleurent (1899). Using petroleum ether extraction, Hinton (1944) found 30.3% of lipids in the scutellum, which was double that found in the embryonic aixs (15.4%). Unlike Hinton (1944), Dubois et al. (1960) found no significant difference in the lipid contents of the scutellum (1 2.6%) and the embryonic axis (1 5.3%) of germ from soft white wheats from Holland. They used diethyl ether for extracting the lipids. Pomeranz and Chung (1965) determined the different fractions of lipids by thin-layer chromatography. The identified fractions and their quantities are given in Table VIII. Triglycerides were the main fraction in nonpolar lipids, whereas glycolipids and phospholipids were the major fractions in polar lipids. The characteristics reported by Hinton (1944) for lipids extracted from the scutellum and the embryonic axis were: acid number, 5.3 and 3.7; iodine

WHEAT GERM

207

TABLE VlIl LIPID FRACTIONS O F DISSECTED GERM‘

Quantity (%)

Lipids Nonpolar lipids Triglycerides Other nonpolar lipids Polar lipids Unidentified Monogalactosyl glyceride Digalactosyl glyceride Phosphatidyl choline

7.06 1.77 0.437 0.559 0.725 1.702

‘Pomeranz and Chung (1965).

number, 116 and 133; and protein content, 0.02%and 0.03%,respectively. The higher acid number for the lipids of the scutellum was explained by its fourfold content of lipase compared with that of the embryonic axis (Pett, 1935). Other lipid characteristics were almost the same for lipids extracted from both the scutellum and the embryonic axis. 4. Minerals Good agreement in the values (range 4.30 to 6.72%) for the ash content of hand-dissected germ was reported by Kazakov (1947), Morris et al. (1946), Mambish (1953), and Hinton (1959). Grischenko (1935), however, reported slightly lower values of 3.70 to 4.35% by using an indirect method of dry separation and calculation. Hinton (1959, 1962) found that the ash in the germ accounted for 8.3 to 14.5% of the total ash in Thatcher, Vilmoria, Argentinian, Egyptian, and Australian (soft white) varieties of wheat. Hinton (1959, 1962) found a 40 to 7% greater concentration of ash in the scutellum than in the embryonic axis of five varieties of wheat. The ash values ranged between 3.51 and 5.36% for the scutellum and between 5.87 and 8.20% for the embryonic axis. Girard and Fleurent (1899) reported 5.45%of ash in the scutellum. The literature on the mineral constituents of dissected germ is very scanty, and values are available only for phosphorus and manganese. Hinton (1944) reported 1.9%total phosphorus in the scutellum, of which 1.3%was phytic phosphorus, whereas the embryonic axis contained 1.16%total phosphorus, of which only 0.39% was phytic phosphorus. Thus the scutellum contained more than three times as much phytic phosphorus as did the embryonic axis. Pringle (1952) reported similar values for total phosphorus and phytic phosphorus in the scutellum as well as in the embryonic axis. The scutellum and the embryonic

208

S . R. SHURPALEKAR AND P. HARIDAS RAO

axis have been reported to contain 178 and 134 ppm of manganese, respectively (Hinton, 1944).

5. Vitamins Proportions of certain B-group vitamins determincd by Hinton et al. (1953) in germ fractions are given in Table IX. a. Thiamine. Ward (1 943) and Fournier (1 946) reported values of 2.03 and 1.35 mg% of thiamine in the embryonic axis and 15.7 and 17.4 mg% in the scutellum, respectively. Hinton (1944) reported similar values of 18 mg% of thiamine in the scutellum and 1.24 mg% in the embryonic axis of germ dissected from eleven varieties of wheat. No significant difference was observed in the thiamine content of the plumule (0.9 mg%) and the radicle (0.72 mg%). A high concentration of thiamine has been reported in the scutellum of durum wheat as compared with the lowest concentration in soft English wheat. It is evident that the thiamine of wheat is mostly concentrated in the scutellum. Although the scutellum constituted only 1.5% of the kernel weight, it contributed about 60% of the total thiamine in wheat, while the embryonic axis contributed only 3%. Hinton (1944) also observed no appreciable change in the thiamine content of the scutellum during germination (Table X) or soaking (Table XI) of the wheat kernel. During soaking, however, movement of thiamine to other parts of the kernel was observed only when its cells were made permeable by the action of acid or chloroform. b. Riboflavin. The germ accounted for about 26% of the riboflavin of the whole wheat kernel. For the Thatcher variety, the embryonic axis of the germ contained 13.8 pglgm, while the scutellum had 12.7 gg/gm (Hinton et al., 1953). c. Niacin. The niacin contents of the scutellum and the embryonic axis accounted for only 1% of the total niacin content of the Thatcher and English varieties (Heathcote et al., 1952). The values for these varieties as determined by the microbiological method were 38.5 and 5 2 &gm of the scutellum and 38.2 and 38.0 pg/gm of the embryonic axis, respectively. TABLE IX DISTRIBUTION OF B-GROUP VITAMINSIN WHEAT GERM'

As % o f that in whole grain Germ fraction Embryonic axis Scutellum

Thiamine

Riboflavin

Niacin

Pyridoxine

Pantothenic acid

2.0 62.5

12.0 14.0

1.0 1.3

8.6 11.6

3.5 4.0

'Hinton ef a[. ( 1 953).

209

WHEAT GERM TABLE X EFFECT O F GERMINATION PERIOD ON THIAMINE CONTENT O F

sc u T E L L u M‘ Germination period (days)

Th iami ne Condition of seed

(Iu/gm)’

First root just visible First root 1 cm long First root 3-5 cm long

53 50 45 25

‘Hinton (1944). ’On a dry-weight basis.

d. Pyridoxine. Clegg and Hinton (1958) determined the pyridoxine content in the structural parts of Thatcher wheat by a microbiological method using Saccharomyces carlsbergensis 4228 as the test organism. They reported 2 1.1 pg/gm in the embryonic axis and 23.2 pg/gm in the scutellum. The aleurone layer, the scutellum, and the embryonic axis contained higher concentrations of pyridoxine compared with other structural parts of wheat grain. e. Pantofhenic Acid. The contribution of pantothenic acid in the germ was only about 7.5% of the total in the kernel. Pantothenic acid contents as reported by Hinton ef al. (1953) were 14.1 pg/gm of the scutellum and 17.1 pg/gm of the embryonic axis. TABLE XI EFFECT O F SOAKING ON THIAMINE CONTENT O F SCUTELLUM

Time of soaking (hours)

Thiamine (Iu/gm)’

0 1 3 6 12

54 53 41 54 54

‘Hinton (1944). ’On a dry-weight basis.

6. Enzymes

Being embryonic in nature, wheat germ contains several enzymes. However, very few data are available on the enzymes of the dissected germ and its structural parts.

210

S. R. SHURPALEKAR AND P. HARIDAS RAO

a. Dipeptidase, Proteinase, arid Lipase. Pett (1935) determined activities of dipeptidase, proteinase, and lipase in various structural parts of Manitoba hard red spring wheat. The values given in Table XI1 indicate that the scutellum contains higher dipeptidase and proteinase activity than the embryonic axis. The lipase activity was considerably higher in the scutellum, which could be explained by its high f a t content. In contrast to these observations, Engel (1945) found no significant difference in the values for lipase, proteinase, and dipeptidase in both the embryonic axis and the scutellum. This variation was attributed to the difference in the techniques adopted for preparation of the germ components. Engel and Heins (1947) reported 0.8 to 1.3 units of proteinase activity in the germ, and observed that the germ was particularly rich in dipep tidase. Pett (1935) also observed that both dipeptidase and proteinase activity in the scutellum and the embryonic axis greatly increased after a germination period of 12 hours. This increase was somewhat greater in the cotyledon than in the radicle portion. The activity of enzymes in the embryonic portion decreased rapidly after germination for 12 hours. In the scutellum, however, i t continued to increase only up to 36 hours of germination. The increase in the activity was reported to be due to the change in the germ from a dormant to an active condition. Unlike dipeptidase and proteinase, the lipase activity of the scutellum dropped suddenly during germination. In contrast, the activity in the cotyledon portion increased during the 12-hour germination period and dropped suddenly thereafter. The activity in the radicle, however, did not show any appreciable change during the 36-hour germination period. b. Lipoxidase, Amylase, Phosphornonoesteruse, Phytase, arid Dehydroascorbic Acid Reductase. By the carotene oxidation method, Blain and Todd (1955) reported almost equal lipoxidase activities of 64 and 6 2 units (defined as the amount required to destroy 0.01 5 mg of carotene in 5 minutes at 20°C) in TABLE XI1 DII’EPTIDASE,PKOTEINASE, A N D LIPASE ACTIVITIES‘ O F WHEAT GERM C O M P O N E N T S ~

Embryonic axis Enzyme

Scutellum

Cotyledon

Radicle

Dipeptidase activity Proteinase activity Lipase activity

17.1 17.8 7.3

11.3 15.8 2.0

20.7 11.8 0.5

‘Expressed as microliters of 0.05 N HCl per milligram of dry material at 40°C. bPett (1935).

WHEAT GERM

21 1

the scutellum and embryonic portions of the germ, respectively. According to Engel (1945), the scutellum had 88 units (milligrams of maltose per hour per cubic millimeter of tissue at 40°C) of amylase activity, whereas the embryonic axis had no activity. Hinton (1 944) found a higher phosphomonoesterase activity in the scutellum (58 King Armstrong units) than in the embryonic axis (36 units) of germ from eleven varieties of wheat. The scutellum of soft wheat (Cuppell deprez) had a high phytase activity of 31.8 units (micrograms of phosphorus per hour per milligram), whereas the embryonic axis had an activity of only 9.0 units (Peers, 1953). However, both the scutellum and the embryonic axis had the same dehydroascorbic acid reductase activity of about 7 0 units (micrograms of ascorbic acid formed per milligram during 10 minutes at 25"C), as reported by Carter and Pace (1964). It may therefore be concluded that the enzyme activities are more concentrated in the scutellum than in the embryonic axis.

B. MILLGERM Unlike dissected germ, the composition of mill germ has been studied in great detail by several workers. This may be partly attributed to the ready availability of mill germ from the commercial roller flour mills. Most of the commercial germ contains some bran and endosperm as impurities. It has also been reported that mill germ lacks some of the scutellum components. Thus, mill germ is probably less representative of a single structural part than other mill products like bran and flour. However, some of the studies carried out have been on the composition of the germ in more or less purified form.

I . Proximate Composition The considerable variation observed in the proximate composition of mill germ is due mainly to different degrees of contamination with bran as well as endosperm. The normal ranges of the proximate composition of commercial samples of mill germ, as reported by Richardson (1884), Grewe and LeClerc (1943), Kent-Jones and Amos (1967), and Farrel et ul. (1967), are given in Table XIII. Grewe and LeClerc (1943) observed that the moisture content of about 9% for the germ was lower than that (13%) of the flour and enhanced the keeping quality of the germ. Lower values (5.2%) of fat indicated contamination of the germ with flour. The germs from hard red spring and durum wheat had appreciably higher fat and protein contents than did the germ from soft wheats. Carbohydrates present in commercial germ could be the best index of purity for most of the germ samples. The carbohydrates in germ fractions from hard red spring and durum wheats were the lowest (24.3%), compared with 30.4% for hard red winter, 36% for white wheat, and 39% for soft red winter wheat. These

S. R . SHURPALEKAR AND P. HARIDAS RAO

212

TABLE XI11 PROXIMATE COMPOSITION O F COMMERCIAL GERM

. ~ and Amos Grewe and LeClercb Richardson Farrel er ~ 1 Kent-Jones (1943) (1884) (1967) (1967) ~

Constituent

Range

(%I

(%) . _-_

. -

Moisture Protein ( N X 6.25) Ether extractives Mineral matter (total ash) Cellulose (fiber) Carbohydrate (by difference) Starch

11.3-12.9 21 .I-24.5 6.5-10.6 3.5- 4.3 2.4- 4.0 ~

14.Ck23.9

Range (70)

Range (%)

Average (%)

Average (%)

9.0-13.0 22.0-32.0 6.0-11.0 4.0- 5.0 1.8- 2.5 35.045.0

7.4-11.5 18.3-35.3 5.2-15.0 3.1- 4.9

9.2 28.9 9.7 4.1

8.4 30.1 12.5 4.6

-

-

-

19.2-53.0

30.4

44.4

-

-

-

~

uValues for germ obtained from nine varieties of wheat. b a l u e s for nineteen samples of commercial germ representing five classes of American wheat.

observations indicated that the flour portion of the germ processed from hard wheats could be easily separated owing to its granular nature as compared with that of soft wheat germ. Fraser and Holmes (1 959) determined the comparative proximate composition (Table XIV) of commercial samples of flour, germ, and bran. The data indicated that the germ contains three times as much protein, seven times as much fat, six times as much ash, and about fifteen times as much sugar as does endosperm flour. Unlike others, these workers have reported the carbohydrate values for different fractions individually and not by difference. It is interesting to note that, compared with other mill products, sugars form the major fraction of the germ carbohydrates. Booth et al. ( 1 941) reported somewhat similar values for all three fractions obtained during the actual milling process. However, the protein content of the germ was only twice that of the endosperm. This variation could be attributed to the purity of the germ and the variety of wheat from which the germ was obtained. In recent years, there have been vast innovations in the flour milling techniques involving heavy feeding of the rolls, reduction in the roller surface area, a shortened milling system, etc. (Jones, 1964). Kent-Jones and Amos (1 967) analyzed the different fractions milled in the improvised flour mill (Table XIV). To summarize, there is considerable variation in the literature values reported for the proximate composition of the germ and its fractions. However, the values reported recently by Farrel et al. (1967) for germ milled from nine varieties of wheat can be taken as the best representative values for normal samples (Table XIII).

WHEAT GERM

213

TABLE XIV PROXIMATE COMPOSITION O F COMMERCIAL M I L L P R O D U C T S ~

Composition (%) Endosperm Constituents

Ka

Fb

Germ Ka

Bran Fb

Ka

Fb ~~

Moisture Protein (N X 5.7) Fat Ash Carbohydrate (by difference) Starch Hemicellulose Sugar Cellulose Total carbohydrate Recovery of fraction

14.7 11.3 0.8 0.4 72.8

14.0 9.6 1.4 0.7 74.3 71.0 1.8 1.1 0.2 74.1 99.8

12.6 29.8 10.6 4.7 41.1 -

11.7 28.5 10.4 4.5 44.9 14.0 6.8 16.2 7.5 44.5 99.0

12.6 13.6 2.8 5.9 66.3 -

-

-

~~

13.2 14.4 4.7 6.3 61.4 8.6 26.2 4.6 21.4 60.8 99.4

‘Kent-Jones and Amos (1967). bFraser and Holmes (1959).

2. Protein a. Conversion Factor for Germ Protein. A factor of 6.25 is being used normally to convert the total nitrogen of the germ into protein even though it was considered high by Jones (1931) and Tkachuk (1969). Jones obtained a conversion factor of 5.8 by taking into consideration the fact that different plant proteins contained various amounts of nitrogen. Later, Tkachuk (1969) reported a conversion factor of 5.45 based on the amino acid content of the germ. This low value reflected the substantial amount of nonprotein nitrogen present in the germ. b. Protein Content. The protein content (N X 6.25) of mill germ has been reported to vary from 18.3 to 36.7% (Richardson, 1884; Jacobs and Rask, 1920; Sullivan and Bailey, 1936a; Booth et al., 1941; Grewe and LeClerc, 1943; Fraser and Holmes, 1959; Kent-Jones and Amos, 1967; Waggle etal., 1967). This wide variation probably reflects on the amount of contamination of the germ as well on as the variety of wheat milled. c. Protein Fractions. Types of protein in wheat germ fractionated according to their solubility characteristics were entirely different from those of flour proteins. There is no literature available t o prove the presence of a gluten type of protein in the germ as exists in flour. Protein fractions of mill germ were first reported as percentages of the total protein by Osborne (1907): albumin, 30.2; globulin, 18.9; gliadin, 14.0; glutenin, 0.3 to 0.37; and insoluble, 30.2. Danielson

S. R. SHURPALEKAR AND P. HARIDAS RAO

214

TABLE XV RELATIVE FRACTIONS OF TOTAL NITROGEN IN WHEAT GERM SAMPLES O F HIGH A N D LOW C A R B O H Y D R A T E C O N T E N T S ~ -~

Relative fraction of total N

3% 5% N not alcohol- NaCI- K, SO,- Cu(OH), precipitated by Total soluble soluble soluble precipitated phosphotungstic Glutenin N N N N N acid NtJ 70%

Sample

-~

Germ high in 4.3 carbohydrates (40.8%)' Germ low in 57 carbohydrates (24.4%)' Individual resultsd 5.1 Wheat flour 2.3 ( f o r comparison)e

~~

.~

~~

15.4

63.8

55.9

92.0

13.6

28.7

12.6

65.7

58.2

88.8

14.0

29.2

13.9 52.6

65.1 24.1

57.0 14.7

90.2 95.7

13.7 3.5

29.1 32.2

'Grewe and LeClerc ( 1 943). bTotal N - (5% K, SO, -soluble N + 70%alcohol-soluble N). 'Average for eight samples. dAverage for nineteen samples. eAvcrage lor four samples.

( 1 949) reported two distinct fractions-alpha and gamma globulins-in wheat germ on the basis of their sedimentation behavior in the ultracentrifuge. However, Pence and Elder (1953) later showed the presence of a third componentdelta globulin-in defatted wheat germ. The nitrogen fractions of nineteen samples of germ determined by Grewe and LRClerc (1943) showed the presence of a high proportion of salt-soluble proteins-albumin and globulin-which accounted for nearly 60% of the total nitrogen (Table XV); in flour they accounted for only about 15%. However, germ protein contained only about 15% of alcohol (70%)-soluble nitrogen, which in the case of flour protein accounted for more than half of the total protein. Pomeranz ef al. (1970a) found that the solubility of germ proteins depended on the drying temperature and concentration of the salt solution used for extraction. The extraction of protein in 0.02 N acetic acid decreased from 32.6% to 1.1% when the drying temperature was increased from 70°C to 130°C. A 3% sodium chloride or calcium chloride solution yielded as high as 83% protein in the extract on a dry basis. The starch gel electrophoresis pattern of gluten and germ proteins showed that the gluten protein was completely absent in the germ extract (Pomeranz e l al.,

WHEAT GERM

215

1970a). The germ protein contained a wide spectrum of fast-moving, salt-soluble proteins, separated into several bands. The most prominent band was somewhat reduced in the sodium chloride extract. This band was much smaller in the high-protein air-classified fraction than in the low-protein one (Fig. 7). From wheat germ Johns and Butler (1962) obtained histones which, unlike the histones of animal tissues, had a higher total lysine content. d. Nonprotein Nitrogen. Wheat germ contained high amounts of nonprotein nitrogen ranging from 11.3 to 15.3% (Osborne, 1907; Grewe and LeClerc, 1943). According to Bailey (1 944) this nitrogen included mainly asparagine,

1

2

3

4

5

6

Water and salt-soluble proteins.

FIG. 7. Starch-gel electrophoretic patterns (migration from slots on top of figure) Of proteins in (1) whole germ, (2) wheat flour gluten, ( 3 ) germ air-fractionated low-proteh fraction, (4) germ air-fractionated high-protein fraction, (5) NaCl extract of g e m proteins, and (6) whole germ.

S. R . SHURPALEKAR AND P. HARIDAS RAO

216

TABLE XVI NONPROTEIN NITROGEN COMPOUNDS O F GERM ~

~~

~

~~

‘

Nitrogen compound

Quantity

References

Asparagine (as total amide N) (lo) Allantoin (as total amidc N) (lo) Betdine (%) Choline (mg/grn)

0.53

Teller and Teller (1932) Frankfurt (1 896) Waggle et al. (1967) Waggle ef al. ( 1 967) Engel (1943) Glick (1945) Geoffroy (1934) Albizatti (1937) Sullivan et al. ( 1 9 3 6 ~ ) Sullivan ; and Howe (1937)

Lecithin (70) Glutathionc (5%)

0.70

0.306-0.604’ 2.59 -3.306 3.00 -2.90 3.04 1.25 0.35 0.46

‘On a 14% moisture basis. ’Range of values for nine samples of gcrm.

betaine, choline, lecithin, allantoin, glutathione, and arginine. Engel (1943) reported higher values of choline in defatted germ than in raw wheat germ. This finding was later discredited by Glick (1945), since practically most of the choline exists in the form of lecithin. Sullivan et al. ( 1 9 3 6 ~ )reported 0.46% of glutathione (Table XVI) in freshly prepared germ, as determined by Weller’s method (1935). Later Howe et al. (1937) obtained 0.1 to 0.2 gm of almost pure glutathione from 2 kg of germ. Wasserman and Burris (1965) isolated hemoprotein from the germ and resolved a major component, WCHP550 (wheat germ hemoprotein SSO), by chromatography. Uroma and Louhivuori (1 954) found that the HC1-acetone-extracted protein fraction, when injected, decreased the eosinophile count in the peripheral blood of rats.

3. Amino Acids The literature values for the amino acid composition of the germ and germ proteins show wide variation. Such variation is attributed mainly to the methodology followed for estimation, the purity of the germ, and the variety of wheat used. Most of the published literature values have been obtained by microbiological methods. In recent years, the automatic ion-exchange chromatographic method has been used because of its simplicity and rapidity as compared with the microbiological method. The main difficulties encountered in different methods of estimation occurred during hydrolysis with 6 N HCI at 1 10°C for 24 hours (Hires et al., 1954), since the rate of stability and the rate of release varied widely for different amino acids. The amino acids serine, threonine, cystine, and methionine were found to be unstable, while valine and isoleucine were released slowly during hydrolysis (Rees, 1946; Tkachuk and Irvine, 1969). This problem

TABLE XVII: AMINO ACID COMPOSITION^ OF MILL GERM PROTEIN

Amino acid Alanine Arginine Aspartic acid Cystine Clutamic acid Clycine Histidine Isoleucine Leucine Lysine Methionine Phenyldanine Proline Serine Threonine Tyrosine Tryptophan Valine Total nitrogen (dry basis) Recovery (%)

Barton-Wright and Moran (1946)b

Block and Mitchell (1946)‘

Horn (1949, 1950)b

-

1.37

6.0 0.8

6.9 -

-

-

3.03 5.23 7.33 5.44 1.28 2.47 6.28

2.5

6.20 -

-

0.90 4.20 5.22 -

“Grams per 16 gm of nitrogen. bMicrobiological method. ‘Chemical method. dDefatted.

-

-

2.1 3.6 6.0 6.2 1.3 3.5 -

-

-

-

6.7 5.5 -

4.2

3.8 3.8 1.0 -

4.0 0.7 4.8 -

-

-

Dunn (1950)b.d -

Block and Dunn Bollingf (1950)b*e (1951)

5.4 -

5.4 13.0 5.7 2.1 3.7 6.0 5.1 1.2 2.5

16.0 5.2 2.3 4.1 5.9 4.3 1.3 3.0

-

-

-

-

-

-

6.0 1.1 2.4 3.9 6.7 5.5 1.4 3.1 5.0 3.8

4.4 -

4.8 -

4.8 -

-

-

-

eDefatted and toasted. fPaper chromatographic method. gIon-exchange chromatographic method.

1.0

Hepburn et al. (1960)b Lyman et al. (1956)b

7.41 -

2.28 3.45 5.95 6.55 1.64 3.90 -

3.95 2.95 1.07 5.02 4.51

Mill A

Mill B

5.23 6.88 7.48 1.04 14.00 5.22 2.26 3.48 5.75 5.28 1.91 3.38 5.03 4.60 3.42 -

5.08 7.04 7.19 1.19 17.30 4.94 2.08 3.28 5.72 4.78 1.73 3.68 6.09 4.60 3.44 -

-

4.63

-

4.40

Kohler Tkachuk and and Palter lrvine (1967)g (1969)g

Miladi et al. (1972)g

5.68 7.36 7.67 1.66 15.30 5.47 2.38 3.58 6.12 5.25 2.26 3.67 4.71 4.24 3.74 2.72 5.25 4.52

5.7 7.5 8.1 1.4 13.3 5.4 2.3 3.1 5.6 6.2 1.6 3.1 3.7 4.3 3.8 2.4 I .4 5.5 6.25

5.21 6.93 7.30 1.88 17.13 5.39 2.38 3.68 6.04

88.00

83.10

89.20

4.92

1.89 3.78 5.23 4.22 3.52 2.58 1.28 5.22 4.45

218

S. R. SHURPALEKAR AND P. HARIDAS RAO

has been overcome by applying suitable correction factors or by converting the unstable amino acids (cystine and methionine) to a more stable form (Schram el GI., 1954). Tkachuk and Irvine (1969) obtained the following correction factors: threonine, 1.07; serine, 1.06; valine, 1.01 ; and isoleucine, I .05. The amino acids of germ proteins and the germ are given in Tables XVII and XVIII, respectively. The values reported by Block and Mitchell (1946) by chemical methods were somewhat different from those determined by the niicrobiological method. This may partly bc due to varietal differences of wheat. Isoleucine and leucine were difficult to estimate by chemical methods, as n o satisfactory method of separation was available. Barton-Wright and Moran (1946) observed that the sum of the isoleucine and leucine values was almost the same by both chemical and microbiological methods. Most of the amino acid values reported by Tkachuk and lrvine (1969) are higher than other reported values, as they were determined in pure commercial germ free from bran. TABLE XVIII A M I N O AC I I ) COMPOSITION O F GERM'

Andrews and l e l t (1941) Barton-Wright Whggle et al. cited in MacMasters et al. and Moran (1967)b (1971)' Amino acid (1946)' __ _ _ _ - -~ . -_ _ ~ 1.34-1.7 1 1.30 Alanine 1.77-2.09 1.71 Argininc 1.92-2.2s 1.86 Aspartic acid 0.50 0.43-0.61 0.26 Cystine 3.654.59 3.48 Glutamic acid 1.32-1.58 1.30 Glycinc 0.70 0.59-0.82 0.56 Histidine 1.91 0.77-0.94 0.84 I soleuc ine 2.66 1.43 Leucine 1.SO-1.75 1.99 1.31 1.30-1.77 Lysinc 0.46 0.47 Methionine 0.39-0.58 0.84 Phen ylalamine 0.86-1 .0 I 0.90 I . 13-1 .52 1.25 Prolinc 1.05-1.28 1.11 Scrine 0.89-1.09 0.85 Threoninc 2.29 0.31 2.44 Tryptophan 0.65-0.78 0.71 Tyrosinc 1.01-1.37 1.23 Valinc 1.52 ~

Horn ef al.' (1946, 1947a,b,c, 1948a.b. 1949a.b)

~~~~

'Crams per 100 gm of germ on a 14% moisture basis. bChromatograpliic determination, range of nine wheats. 'Microbiological determination.

-

0.72 1.27 2.10 2.17 0.44 -

1.40 -

1.68

21 9

WHEAT GERM TABLE XIX

AMINO ACID COMPOSITION" OF WHEAT FLOUR, GLUTEN, AND DEFATTED GERM

O R GERM P R O D U C T S ~ ~ _ _ _ _ _ _ _

~~~

Air-classified germ Amino acid

Flour

Gluten

Whole germ

Lysine Histidine Ammonia Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Me thionine Isoleucine Leucine Tyrosine Phenylalanine

1.78 1.82 3.01 3.23 3.81 2.31 4.43 37.19 11.55 3.37 2.87 1.44 3.99 1.45 3.80 6.64 2.15 5.16

1.53 1.77 3.33 3.14 3.05 2.36 4.18 39.30 11.49 3.03 2.37 1.61 3.58 1.45 3.5 1 6.31 3.30 4.68

7.76 2.65 1.71 8.86 10.21 4.82 4.62 15.45 4.37 6.54 7.00 0.66 5.65 1.88 3.91 6.79 3.12 4.07

NaCl extract of germ

High-protein fraction

Low-protein fraction

8.33 2.58 1.54 9.85 8.81 4.92 4.64 15.94 4.04 6.95 6.89 0.88

6.41 3.35 2.26 10.60 9.67 4.27 4.10 16.47 4.22 6.58 6.40 0.75 5.80 1.77 3.64 6.53 3.02 4.20

7.30 2.40 3.15 7.27 9.58 4.40 4.60 16.76 5.07 6.34 6.70 1.09 5.06 1.89 3.61 7.45 3.34 4.02

5.5 1

2.11 3.59 6.37 3.26 3.86

'Crams per 100 gm of amino acids. bpomeranz et UZ. (1970a).

The amino acid contents of various mill fractions were reported by several workers and in recent years by Hepburn et al. (1960), Waggle et al. (1967), and Kohler and Rhoda (1967). It was found that the germ contained the highest amount of essential amino acids as compared with other milled products. The germ was also rich in the essential amino acids lysine, methionine, and threonine, in which many of the cereals are deficient. Pomeranz et al. (1970a) determined the amino acid content of defatted germ, flour, gluten, salt extract of germ, and air-classified fractions of germ containing high- and low-protein fractions by the ion-exchange chromatographic method. The values reported are given in Table XIX. The amino acid composition of germ protein, which agreed well with the literature values, differed significantly from that of flour proteins or gluten. However, the amino acid composition of both defatted germ protein and the sodium chloride-extracted protein did not show any significant difference. The values for the amino acids lysine, serine, proline, alanine, leucine, and tyrosine, and the sulfur-containing amino acids

S. R. SHURPALEKAR AND P. HARIDAS RAO

220

were higher in the low-protein air-classified fraction than in the high-protein fraction.

4. Nucleic Acid The values reported by Osborne and Harris (1902) and by Javillier and Colin (1933) for the ribonucleic acid (RNA) content of the germ were 3.5% and 4.2%, respectively. The purity and yield of RNA was low when either of the methods described by Osborne and Harris (1902) or by Clark and Schryver (1917) were used. The composition of RNA as reported by Osborne (1907) and Osborne and Hey1 (1908) was similar to that of RNA present in yeast (Calvery and Remson, 1927; Jones and Perkins, 1925; Levene and Bass, 1931; Read and Tottingham, 1917). Lusena (1951) described a method for purification of RNA and isolated 0.8% of RNA of 99% purity from wheat germ. Lipshitz and Chargaff (1956) isolated, purified, and analyzed deoxyribonucleoprotein and deoxyribonucleic acid from wheat germ.

5. Lipids a. variation in Lipid Content. Wheat germ contains a high percentage of fat compared with other mill products like bran, patent flour, and shorts. The values reported for lipid contents of mill germ are given in Table XX. The wide variation in these values is attributed to the following reasons: Method of solvent extraction. Herd and Amos (1 930) observed a higher vahe for lipids obtained by hydrolysis as compared with direct extraction with petroleum ether (Table XX). Higher values were attributed to the extraction of bound lipids, whereas ether extracted more or less free lipids. TABLE XX LIPID CONTENT O F MILL GERM

Quantity (70) Lipid

Range

Average

References

Ether extract

5.05-18.90

9.45

Alcohol extract

7.70-14.14

10.75

Acid hydrolysis

-

Ball (1926); Barton-Wright (1938); Grewe and LcClerc (1943);Herd and Amos (1930); Sullivan and Near (1928, 1933); Sullivan and Bailey (1936a) Barton-Wright (1938); Channon-Foster ( 1 9 3 4 ) ; Herd and Amos (1930); Sullivan and Near (1928, 1933); Sullivan and Bailey (1936a) Herd and Amos (1930)

8.27

22 1

WHEAT GERM

Purity of germ lipids. Herd and Amos (1 930) and Sullivan and Near (1 933) estimated the nitrogen and phosphorus contents in the lipid material obtained by different extraction procedures. The data (Table XXI) indicated that direct extraction and acid hydrolysis gave a product more or less free from nitrogen and phosphorus compared with that obtained by alcohol hydrolysis methods. The high values for nitrogen and phosphorus in lipids obtained by alcohol hydrolysis were attributed to the presence of other lipoid materials such as sterols and pigments. Similarly, Sullivan and Near (1 933) found minimum and maximum contamination with nitrogen and phosphorus in ether and alcoholether extractives, respectively. Variety of wheat. Using the same solvent and extraction procedure, Grewe and LeClerc (1943) reported a range of 5.0 to 15.0% for ether extractives in nineteen samples of commercial germ milled from five varieties of American wheats. The average maximum and minimum lipid contents were found in the germs obtained from durum wheat and soft wheat, respectively. The purity of the germ also contributed considerably to the variation in the lipid contents. b. Composition of Lipids. The composition of germ lipids was extensively studies by Nelson er QZ. (1963a, b), Moruzzi er al. (1969), and Pomeranz er aL (1970a). Different constituents of germ lipids reported by Moruzzi et al. (1969) are given in Tables XXlI and XXIII. Nelson er al. (1963a) separated and identified four lipid fractions from germ, bran, and endosperm by column chromatography (Table XXIV) and showed that more than 74% of lipids of bran and germ were nonpolar (tri-, di-, and monoglycerides, fatty acids, sterols) and less than 25% consisted of polar lipids (phospho- and glycolipids). In contrast, endosperm lipids contained more than 50% of polar lipids and 47% of nonpolar lipids. However, Moruzzi et al. (1969), by extracting the germ lipids according to TABLE XXl LIPID CONTENT O F GERM A N D ITS PURITY AS AFFECTED BY V A R I O U S METHODS O F EXTRACTION'

Methods

-

Lipid content (%)

Soxhlet extraction Petroleum ether Ethyl ether Hydrolysis Alkali Acid Alcohol 'Herd and Amos (1930).

Nitrogen (%.)

Phosphorus (%o)

7.82 8.26

-

-

0.08

0.28

8.80 9.62 10.31

0.16 0.16 0.43

0.53 0.15 0.48

TABLE X X l I LII'II) CONSTITUENTS O F WHEAT GERM'

Mg %. of

Lipid constituent

germ

(9) Total lipids Phospholipidsb Glycolipidsc Neutral fat" Protein Amino nitrogen Total I- rcc

100.00 1.38 0.75 96.21 1.66

9380 130 71 9024 155

0.10 0.01

13

0.99

'Moruzzi et n l . ( 1 969). bMilligrams (if lipid phospliorus X 25. 'Galactose x 4.55. dTotal lipid - (phospholipids + plycolipids + protein). TABLE XXIIl L I P I DCONSTITUENTSor: GEKM'

Pcrcent of germ

Lipid cons t it ucnt ~~

Neutral fat Sterol esters Triglyceridcs and free fatty acids Mono- and diglycerides Polar lipids containing phosphorus Phosphatidyl cholineb Noncholine nonethanolamincb Phosphatidyl etli;inolamineb ~ ~ i o s p ~ i a t serincb idy~ Phosphntidic acid, etc. Polar lipids containing galactosc Protcins of proteolipids 'Moruzzi et al. (1969).

9.02 0.56 6.10 2.40 0.130 0.052 0.0091 0.0 I29 0.0032 0.053 0.071 0.1558 bl:scluding phosphatidic acid.

TABLE XXIV L I P I D F R A C T I O N S O F W A N , G E R M , A N D ENDOSPEKM'

Lipid fraction _ _ ..... Hydrocarbon and stearyl esters Triglyceridcs I"atty acids, sterols, mono- and diglycerides Phospholipids and glycolipids Total 'Nelson ct al. (1963a).

Bran (%)

Germ

0.5 56. I 25.1 22.5 104.2

3.7 57.0 17.8 16.5 95.0

(%I

Endosperm (%) Traces

29.9 17.1 52.4 98.9

WHEAT GERM

223

TABLE XXV PHOSPHOLIPIDSA N D G A L A C T O L I P I D SI N POLAR L I P I D FRACTION O F GERM'

Percent of fraction Fractions

Base found

Phospholipids

Galactoliuids

Phosphatidyl choline (lecithin) Choline and amino-free phospholipids Phosphatidyl ethanolamine Phosphatidyl scrine

Choline

53.32 29.25 13.01 4.40

23.78 64.73 11.49

-

Et hanolamine Serine

-

'Moruzzi ef 01. (1969).

the method of Brady (1964), found as high as 97% of germ lipids to be nonpolar. This variation may be attributed to the difference in the method followed for extracting lipids from the germ. Further separation of polar lipids into four fractions (Table XXV) showed that lecithin was abundant in the iolar lipid fraction. The different fractions identified in nonpolar lipids are indicated in Fig. 8. Fractionation of polar and nonpolar lipids by thin-layer chromatography (Pomeranz e l al., 1970a) showed a virtual absence of polar lipids in free lipids of petroleum ether extractives (Fig. 9). However, bound lipids contained small amounts of polar lipids, presumably consisting of phospholipids and some glycolipids. They reported that free and bound lipids were present in the ratio of 12:2.

1

2

3

4

5

6

FIG. 8. Chromatography o n silicic acid-impregnated paper of different nonpolar lipid fractions from wheat germ. y = yellow; B = blue fluorescence after staining with Rhodamine 6-G and examination under an ultraviolet lamp. 1 = nonpolar lipids of egg yolk: ( l a ) monoglycerides, ( l b ) diglycerides, ( l c ) triglycerides, ( I d ) cholesterol esters; 2 = nonpolar lipids of wheat germ: (2a) monoglycerides, (2b) diglycerides, (2c) triglycerides, (2d) sterol esters + tocopherols; 3 = tocopherol standard; 4 = sterol esters fraction of wheat germ; 5 = triglycerides fraction of wheat germ; 6 = mono- and diglycerides fraction of wheat germ: (6a) monoglycerides, (6b) diglycerides, (6c) tocopherols.

224

S. R. SHURPALEKAR AND P. HARIDAS RAO

c. Phospholipids. The germ contains a high percentage of phospholipids when compared with other mill fractions (Barton-Wright, 1938). The literature values (Barton-Wright, 1938; Channon and Foster, 1934; Sullivan and Near, 1928, 1933) for phospholipids in the germ range from 0.65 to 4.17%. The analysis of phospholipids reported by Channon and Foster (1934) is given in Table XXVI. Fractionation of phospholipids showed the presence of phosphatidic acid (as salts of calcium, magnesium, and potassium), lecithin, and cephalin in the proportion of 4:4:1 on the basis of their phosphorus content. About 42% of the total phosphatide was phosphatidic acid. d. Unsaponi@ble Fraction. The unsaponifiable fraction of mill germ which contained sterols, tocopherols, and pigments ranged from 0.32 to 0.80%, with an average of 0.45% (Ball, 1926; Barton-Wright, 1938; Channon and Foster, 1934; Sullivan and Bailey, 1936b). Sullivan and Bailey (1936b) found 4% of germ fat to be unsaponifiable. Of this, 70% was a mixture of sterols, and the remaining 30% consisted of yellow viscous oil, the composition of which was not de-

1

Nonpolar 2 3

Polar

4

5

6

1

8

FIG. 9. Thin-layer chromatography of lipids in wheat flour and wheat germ. Samples 1 to 4 fractionated with chloroform, 5 to 8 with chloroform-methanol-water (65:25:4). Samples 1 and 5 free lipids (petroleum ether extract) of flour; 2 and 6, bound lipids (water-saturated butanol extract following petroleum ether) of flour; 3 and 7, free lipids of germ; 4 and 8, bound lipids of germ. Tentatively identified as (A) hydrocarbons and steryl esters, (B) triglycerides, (C) diglycerides, (D)free fatty acids, (E) unresolved polar lipids, (F) unresolved nonpolar lipids, (GI digalactosyldiglycerides,and (H)phosphatidyl choline.

WHEAT GERM

225

TABLE XXVI ANALYSIS OF ACETONE-INSOLUBLE FRACTION O F WHEAT GERM OIL'

Characteristics

Average value for seven samples

Yield (as % o f extract) Yield (as "/o of germ) Iodine value Fatty acids (70) Iodine value of fatty acids Nitrogen (70) Phosphorus (%) Ash (%)

12.8 1.1 77.0 56.2 123.0 1.60 2.65 2.70

'Channon and Foster (1934).

termined. However, preliminary work indicated the presence of polyene hydrocarbons and alcohol. About 56% of the sterol was in the free state; the remaining was in the bound form. Ellis (1918) reported 0.5% of sterol in the germ. Later, sterols were shown t o be a mixture of hydrositosterol and four isomeric sitosterols: alpha, beta, gamma (Anderson et aZ., 1926; Ichiba, 1935b), and delta (Ichiba, 1935a). The presence of ergosterol (12 ppm on germ basis) and dihydroergosterol was reported by Drummond et al. (1 935). Fernholz and MacPhillamy (1941) isolated a new phytosterol called campesterol from wheat germ. e. Fatty Acid Composition. Jamieson and Baughman (1932) and Sullivan and Bailey (1936a) reported that linoleic acid accounted for nearly half of the TABLE XXVII FATTY ACID COMPOSITION O F LIPIDS O F WHEAT GERM

Nelson et al. ( I 963b) Fatty acid (methyl esters) Myristate (C-14:O) 16:0) Palmitate (CPalmitoleate (C-16: I) Stearate ((2-18: 0) Oleate (C-18:l) Linolea te (C-18: 2) Linolenate (C-18:3) Arachidate (C-2O:O) Others

Triglycerides (%)

Trace

Trace

18.5 0.7 0.4 17.3 57.0 5.2

19.4 0.8

Trace

0.8

Moruzzi ef al. (1969)

Total

Total lipids (%)

0.5

19.6 52.5 4.5 0.5 2.4

lipids (%)

20.03 0.30 0.30 16.68 56.00 5.05 2.06

Nonpolar lipids

(%I 19.77 0.58 16.50 58.40 4.61 -

Polar lipids (%)

18.39 1.74 1.08 19.44 43.49 12.12 4.09

TABLE XXVIII PHYSICAL A N D CHEMICAL CHARACTERISTICS OF WHEAT GERM LIPIDS

Barton-Wright (1938) Characteristics Specific gravity Refractive index Acid value Saponification value Iodine number Thiocyanogen number Hexabromide number Acetyl value Reichert-Meissel value Polenske number Hehner number Soluble acids as butyric (70) Ester number Unsaponifiable matter (70) Iodine number of unsaponifiable matter Thiocyanogen number of unsaponifiable matter

Petroleum ether extract

Acetone-soluble fraction

-

-

-

-

24.57 184.00 127.40 73.48 2.96

24.40 186.20 130.90 73.52 2.96

-

-

-

-

-

-

-

-

-

-

-

-

4.71 -

5.21 -

-

-

Sullivan and Bailey (1936a)

Channon and Foster (1934)

Jamieson and Baughman (1932)

( 1 926)

Alcohol-ether extract

Alcohol-ether extract

Ether extract

Ether extract

0.9326 (26"/26") 1.4800 (20") 6.95 17.6 184.00 184.0 125.0 (Rosenmund) 111.0 84.7 2.28 16.7 0.77 0.44 89.00 1.44 177.05 4.00 7.03

Ball

0.9268 1.4686 (17'/1°) 1.4762 (25'/25") 0.9249 (25"/1") 21.48 7.6 (20") 186.5 184.13 123.64 (Wijs) 125.6 (Hanus) 79.7 Trace 9.9 0.475 0.2 0.25 0.35 93.70 -

-

~

4.70 97.30

162.65 3.59

62.30

-

-

16.0

16.00

-

17.87

Saturated acids (Twitchell) (%) Saturated acids (Bertram) (%) Saturated acids (lead salt-ether) (%)

16.0

Unsaturated acids (%)

84.0'

84.0'

84.00'

-

134.10

132.90

129.90

120.00

80.87

79.64

79.30

-

160.10

158.40

153.00

-

278.00

278.00

278.00

284.00

212.72

266.00

266.00

262.20

-

245.08

309.00

307.00

281.00

Iodine number of total fatty acids Thiocyanogen number of total fatty acids Iodine number of unsaturated fatty acids Mean molecular weight uf total fatty acids Mean molecular weight of saturated fatty acids Mean molecular weight of unsaturated fatty acids

'Percent of total fatty acid determined. 'Percent of total fatty acid calculated.

-

13.30 (COI.)

15.83

75.30 (corr.) -

84.17' 128.11

160.70

145.97

276.85

228

S. R. SHURPALEKAR AND P. HARIDAS RAO

total fatty acids. The unsaturated fatty acids reported by Sullivan and Bailey (1936a) are: Total unsaturated fatty acids a-Linolenic acid 0-Linolenic acid a-Linoleic acid p-Linoleic acid Oleic acid (by difference)

84.00% 1.83% 1.72% 22.32% 29.99% 28.14%

Palmitic acid, the principal fatty acid, formed 73.5% of the total saturated fatty acids. Jamieson and Baughman (1932), however, reported a higher value of 91%.The remaining 9%was mostly stearic and lignoceric acids. Studies by Nelson et al. (1963b) and Moruzzi et al. (1969), using gas-liquid chromatography of methyl esters of fatty acids, presented similar figures with slightly high values for linoleic and low values for oleic acid (Table XXVII). In addition, some new fatty acids were detected in traces. No significant difference was observed in the fatty acid composition of total lipids and triglycerides except for the higher values of linoleate in the total lipids. Myristate and arachidate were the new fatty acids found in traces in the germ. The fatty acid composition of nonpolar lipids was somewhat different, when compared with that of polar lipids. Nonpolar lipids contained more of linoleate and less of linolinate and oleate. f: Constants of Wheat G e m Oil. The physical and chemical constants of wheat germ oil were reported in the early 1930’s by several workers (Table XXVIII). Many of the constants reported were somewhat similar except for slight variations caused by differences in the me thodology followed for determination. The specific gravity of germ oil was quite normal. The saponification value was comparable to the normal value for vegetable oils. Because of the high iodine value, germ oil was classified among the semidrying oils (Lewkowitsch, 1915). The germ oil had a higher saponification value as well as iodine number, a lower specific gravity, and contained more of the unsaponifiable fraction when compared with flour oil. Ball (1926) found very little difference in the specific gravity of germ oil obtained by ether extraction and extraction under pressure. The iodine value, however, was slightly higher in pressure-extracted oil. The characteristics of Bulgarian wheat germ oil as compared with three commercial samples of imported germ oil have been reviewed by Ivanova et al. ( 1974). Ivanova and Popov (1 974) have compared the characteristics of laboratory-extracted oil from fresh wheat (variety Bezostaia) germ with germ oils extracted (i) commercially, (ii) by pressure, and (iii) by extraction.

WHEAT GERM

229

TABLE XXIX EFFECT OF D R Y I N G ON WEIGHT OF GERM LIPIDS‘ (ON D R Y BASIS)

Conditions of drying 48 h r i n desiccator (gm)

2 hrat 98°C

Solvent used

24 h r i n desiccator (gm)

Ethyl ether Petroleum ether

8.40 7.87

8.40 7.85

15 h r a t

(gm)

98°C (sm)

21 h r a t 98°C (gm)

8.26 7.82

8.26 7.75

8.26 7.75

‘Herd and Amos (1930).

The effects of drying under different conditions on the weight and physical constants of germ oil were studied by Herd and Amos (1930). They reported that the weight of ether-extracted germ lipids was constant even after 24 hours of drying in a vacuum desiccator. Very little loss in weight was observed during the first 2 hours of heating at 98°C. Thereafter, the weight remained constant even after heating for 21 hours (Table XXIX). Even though the weight was constant after 2 hours of drying, the physical constants changed slowly, even beyond the 2-hour period (Table XXX). Bromine values and iodine values decreased as the time of heating increased, and specific gravity increased only slightly. No appreciable difference was observed in the constants of germ oil obtained by using different solvents. g. Stability of Germ Oil. During 30 days of storage of laboratory extracted oil in the dark, Ivanova and Popov (1974) observed that the acidity index of the TABLE XXX EFFECT O F DRYING ON THE PHYSICAL CONSTANTS O F GERM OIL‘

Solvent used Petroleum ether

Ethyl ether

Bromine value

Specific gravity

Air drying 24 hr in desiccator 48 hr in desiccator 12 hr at 98°C 18 hr at 98°C 36 hr at 98°C

1.470 1.479 1.479

-

-

-

-

81.8 81.3

129.9 129.1

81.5

-

-

-

1.480 1.479 1.485

1.480

73.3

116.4

‘Herd and Amos (1930).

(NDzo )

Bromine Iodine value value

Specific gravity (ND”)

Condition of drying

-

-

-

1.488

61.5

230

S. R. SHURPALEKAR AND P. HARIDAS RAO

oil increased from 5.27 t o 17.82 and the peroxide index from 0 to 0.08 a t ambient temperature, but remained unchanged at 4°C. Further, they observed that resistance of germ oil to autooxidation was double (12.7 hours) that of sunflower oil (6.2 hours) in a modified Schaal oven test at 100°C. 6. Carbohydratrs

Generally, the carbohydrate content of any food is calculated by difference. Thc carbohydrate content of mill germ varies widely (19.2 to 53.0%; see Table XIII), depending o n the contamination with the endosperm and bran portions (Grewe and LeClerc, 1943). It is, therefore, the best index of purity of mill germ. a. Carbohydrate. Composition. Fraser and Holmes (1 959) found hemicellulose, cellulose, starch, and sugars as the carbohydrate fractions of mill germ (Table XXXI). Sugars formed the major component of the germ, while the endosperm was rich in starch, and the bran was rich in cellulose and hemicellulose. The starch in the germ was contributed entirely by endosperm impurity, while the cellulose and heniicellulose came from the bran present in the germ. According t o Fraser and Holmes (1957), the germ hemicellulose was made up of 52.1% xylose, 40.9% arabinose, and 7.0% uronic acid. They also found that the germ contained 3.7% pentosans. b. Sugars. Using 80% hot alcohol for extraction, Richardson and Crampton (1886) reported 15 to 18% of total sugars in defatted germ. Of this, 80 t o 90% was sucrose. The rest, consisting of nonfermentable, nonreducing sugars (before hydrolysis), was strongly dextrorotatory and was hydrolyzed by yeast invertase t o reducing substances. These characteristics resembled those of raffinose. Frankfurt ( 1 896) reported 24.34% of total soluble carbohydrates. Of these, 6.89% was raffinose, and the remaining 17.4% was presumed t o be sucrose. TABLE XXXI C ARBOHYDRAT E COMPOSITION O F COMMERCIAL

M I L L PRODUCTS'

As 9,of total carbohydrate Nature of carbohydrate Hemicellulose Cellulose Starch Sugars Total

Endosperm

Germ

Bran

2.4 0.3 95.8

15.3 16.9 31.5 36.3 44.5

43. I 35.2

1.5 74.1

14.2

7.5 60.8

'Calculated from the values reported by 1-raser and Holmes (1959).

23 1

WHEAT GERM

Schulze and Frankfurt (1 894, 1895) reported the presence of both sucrose and raffinose in wheat germ and isolated them in crystalline form. The presence of sucrose and raffinose in commercial wheat germ was confirmed by Power (1913). He isolated and characterized these sugars by their melting points and specific rotations and found that the embryonic axis ’contained a relatively large proportion of glucose, since aqueous extracts of the germ readily yielded glucose phenylosazone. Later, Colin and Belval (1933, 1935) reported a low value of 9.4% for total sugars consisting of 5.2% sucrose, 4.0% raffinose, and 0.2% reducing sugars. Fraser and Holmes (1 959), Dubois et al. (1 960), Linko et al. (1960), and Garcia et al. (1 972b) separated and identified the individual sugars of the germ by chromatographic techniques. The component sugars as reported by these workers are given in Table XXXII. The total sugar content was 16.2% in mill germ and 20.1% in dissected germ, as reported by Fraser and Holmes (1959). This suggested that the contamination of mill germ with endosperm and bran portions decreased the total sugar content; the value reported for dissected germ appeared to be more reliable in view of the purity of the germ samples. The total sugar content of about 16% agreed more closely with the value reported earlier by Schulze (1910) than with that of Colin and Belval(l933). Fraser and Holmes (1959) reported only sucrose and raffinose in dissected germ. On the other hand, they also found, besides sucrose and raffinose, small quantities of glucose and fructose in mill germ. The presence of glucose and fructose was attributed probably to partial hydrolysis of sucrose and raffinose. The percentages of component sugars reported by various workers were comparable (Table XXXII). Linko et al. (1 960) found a sugar, melibiose, which was TABLE XXXII COMPONENT SUGARS OF WHEAT GERM DETERMINED BY CHROMATOGRAPHIC TECHNIQUES

Component sugar (as % of total) Sucrose Raffinose Fructose Glucose Melibiose Unidentified Total sugars

Fraser and Holmes

Dubois ef al.

Linko er nl.

Garcia et al.

(1959)

(1960)

(1960)

(1972b)

62.3 37.1

57.6 31.6 4.8

55.9 38.1 2.8 2.1

64.0 32.0 1.0 2.0a

1.1 Trace

2.0

-

-

Trace -

16.2’

16.8‘

-

“Include maltose. bOn a 11.7%moisture basis. =On a defatted-germ basis. a moisture-free basis.

28.6d

232

S. R. SHURPALEKAR A N D P. HARIDAS RAO

Moisture (70)

Moisture (%)

FIG. 10. The effect of moisture content at 35°C and 50°C on the concentration of soluble sugars.

not reported by earlier workers. Also, they suspected the presence of lactose and galactose in the germ samples. c. Effect of Storage on Sugar Content of the Germ. Linko et al. (1960) determined the sugar content of germ stored for 8 days at different temperature and moisture levels. No change in the sugar content was observed when germ containing 8.9% moisture was stored even at 50°C (Fig. 10). However, changes became more pronounced with increases in moisture content of the germ during storage. Depending on the moisture and temperature of the germ, nonreducing sugars were found to decrease, whereas reducing sugars increased during storage. The decrease in nonreducing sugars was followed by an increase in browning as measured by the fluorescence value. This was due to the reaction of reducing sugars with free amino acids to form an intermediate of nonenzymic browning.

7. Minerals a. Earlier Work. The work up to 1943 was excellently reviewed by Bailey (1944) in his monograph. The methods followed by earlier workers were mostly colorimetric or titrimetric, and some of the analyses were carried out by methods that are presently regarded as of doubtful accuracy. In some cases, precautions were not taken to avoid the interference of other metals in the estimation. Good agreement was observed in many of the values for different minerals (Table XXXIII). Phosphorus, potassium, and magnesium were the major constituents of germ ash. In addition to the values for the mineral constituents given in Table XXXIII, the following values (in parts per million) for individual elements have been

WHEAT GERM

233

TABLE XXXlll MINERAL CONSTITUENTS IN WHEAT GERM

Mineral Phosphorus Potassium Magnesium Calcium Sodium Iron Zinc Copper Manganese Aluminum Sulfur Silicon Chlorine Total ash

McHargue (1925)'

-

270 160 460 150 -

Sullivan and Near (1927)' 12,533 5,542 3,081 692 68 420 9 67 25 240 -

50,410

Howe and Sullivan (1936)a

Zunini (1935)a-b 10,900 1,600 430 100

-

Booth et al. (1941)'

Grewe and LeClerc (1943)' 10,550 9,300 3,220 550 58

18 208

-

2,400 90

2,350 820 42,600

'As parts per million on a dry basis. bAlso detected Zn, Mn, Fe, N, Al, Cu, CO, and B. 'Mean values (on a 14% moisture basis) for nineteen samples covering five varieties of wheats.

reported by various workers: copper, 48 (Guerithault, 1927), 12.7 (Lindow et al., 1929), and 9.0 (Elvehjem and Hart, 1929); aluminum, 143 (Bertrand and Levy, 1931); manganese, 225 (Javillier and Imas, 1926) and 190 (Bruere, 1934); zinc, 199 (Javillier and Imas, 1926); lead, tin, and selenium, 1.5, 0.4, and 0.8, respectively (Kent, 1942); and iron, 416 (Andrews and Felt, 1941). The aluminum and manganese contents in the germ were nearly ten to thirty times as high as in other mill products. b. Phosphonts and Phosphonts Compounds. Phosphorus was the main constituent of germ ash, ranging from 1.04 to 1.41% as reported by several workers. The distribution of phosphorus given in Table XXXIV shows that about 50% of the total phosphorus is phytic phosphorus (Pringle and Moran, 1942; Andrews and Bailey, 1932). Sullivan and Near (1927) reported that the ratio of lipid phosphorus to total phosphorus for patent flour was 0.128, as compared with 0.04 for the germ. Bailey (1944) reported that the inorganic phosphorus content of germ was 0.04%, and it was present mainly as pyrophosphate together with metaphosphate (Sullivan and Near, 1927). c. Recent Work. Czerniejewski et al. (1964) analyzed various minerals by colorimetric, flame photometric, and titrimetric procedures after separating the

234

S. R. SHURPALEKAR AND P. HARIDAS RAO TABLE XXXlV DISTRIBUTION O F PHOSPHORUS IN WHEAT GERM

As 7% of total P

As % of germ

J avillier and Colins (1933)

Phosphorus compound Total Extract by 2% HCl Phytin Lipoid Nuclein Inorganic Others

Andrews and Bailey (1932) 1.244 1.004 0.597 0.071

Zunini (1935)

1.355

~

-

~

Pringle and Moran (1942)

Andrews and Bailey (1932)

1.413 0.674 -

80.7 48.0 5.7

0.567 0.120 0.379 0.289

0.340 0.100 0.368

-

-

-

-

-

-

-

0.576

-

~

46.3

TABLE XXXV RECENT DATA O N M I N E R A L CONSTITUENTSOF WHEAT

GERM^

Garcia et al. (1972a)b Mineral

A

Waggle et al. (1967)'

B

Czerniejewski et al. (1964)

Percent Phosphorus Potassium Magnesium Calcium Sodium

1.08 0.95 0.299 0.044 0.01 0

1.04 0.95 0.319 0.048 0.027

1.01 1.14

0.27 0.05 8 0.024

0.925 0.889 0.268 0.048 0.0232

Parts per million Iron Zinc Mangdnesc Copper MoIybdenum

98.0 143.2 148.4 10.3

102.5 138.7 163.7 9.5

53.6 134.7 135.5 10.2

66.6 100.8 137.4 7.4 0.67

aOn a full fat and dry basis. bMean of triplicate samples. 'Mean value of nine different samples; other minor mineral elements (as parts per million) are: Ba 8.5, A1 5, Sr 2.36, B 5.7, and Se 0.372.

WHEAT GERM

235

elements interfering in the estimation. Later, Waggle et al. (1967) found that germ samples from nine different wheat mixes contained relatively high amounts of mineral constituents compared with samples from whole wheat and flour. Results were obtained by a computer-programmed emission spectrometer without any mention of sample decomposition or ashing procedures. More recently, Garcia et af. (1972a) used a rapid wet-ashing procedure coupled with atomic absorption spectroscopy techniques. The different mineral values (summarized in Table XXXV) are generally in good agreement except for the iron content. The differences in the values for iron, zinc, and copper were attributed to the differences in the ashing techniques. The data reported by different workers indicate that the germ contained higher concentrations of nutritionally important trace elements like zinc, manganese, copper, cobalt, iron, and selenium. 8. Vitamins

Most of the work on the vitamin contents of the germ has been reported for hand-dissected germ and its structural parts. This has already been discussed (Section IV,A,5). Comparatively, very few data are available on the vitamin contents of mill germ. Bailey (1944) had reviewed the literature on vitamins of milled products. Values reported earlier than 1958 varied widely, as there were no standard methods for estimation. Different biological, chemical, and microbiological methods were adopted by various workers. Only relatively recently, Calhoun et al. (1 958) standardized the procedures and the hydrolysis conditions to be followed for wheat and wheat products for estimating vitamins. a. B-Group Vitamins. Jackson et al. (1943) determined the vitamin content of mill products and reported values of 21.3 pg of thiamine and 4.53 pg of riboflavin per gram of germ. The germ contained five times as much thiamine as did whole wheat and about twenty-five times as much as did patent flour. Similarly, values for riboflavin in the germ were about four and seven times as high as those in wheat kernel and patent flour, respectively. They found only 42 pg of niacin per gram of purified germ, as compared with 6 8 pg per gram of mill germ. The higher value in mill germ was due to contamination of the germ with niacin-rich bran portions. Andrews (1 942) and Barton-Wright (1944) reported comparable values for pure and commercial samples of germ. Teply et QI. (1 942) reported the following values (in micrograms per gram) for germ: niacin, 34; pantothenic acid, 15.3; and pyridoxine, 9.6. Among the mill fractions analyzed, the germ contained the highest amounts of pantothenic acid and pyridoxine. Moran and Drummond (1945) reported a comparatively low value of 8.5 pg of pantothenic acid per gram of germ. The pyridoxine contents in the germ, as reported by Siegal et al. (1943) and by Moran and Drummond (1945), were 10.6 and 14.0 pglgm, respectively, as compared with 4.2 pg/gm for whole wheat.

S. R. SHURPALEKAR AND P. HARIDAS RAO

236

TABLE XXXVI VITAMIN CONTENT O F COMMERCIALLY MILLED WHEAT P R O D U C T S ~

Waggle et al. (1967)b

Calhoun et al. (1960) Vitamins

Whole wheat

Patent flours

Germ

-

Germ

Micrograms per gram Thiamine‘ Riboflavin Niacin Pantothenic acidd Folic acid Biotin p-Aminobenzoic acid

3.93 1.07 54.5 10.9 0.50 0.1 14 3.83

0.76 0.32 10.1 4.83 0.11 0.014 0.33

13.5 4.87 45.3 10.4 2.05 0.174 3.7

21.84 5.83 7.5 21.8 2.06 -

Milligrams per gram Cholinee Inositol Pyridoxine Betaine

1.63 3.15

1.61 0.33

2.65 8.52

-

0.01 16 4.83

‘On a 14%moisture basis. bAverage for nine samples. ‘As thiamine hydrochloride. dAs calcium pantothenate. eAs choline chloride.

In later studies, Calhoun et al. (1960) and Waggle et al. (1967) analyzed mill fractions for their vitamin contents by standard methods. The values reported by them (Table XXXVI) compared well and showed that thiamine, riboflavin, folic acid, pyridoxine, betaine, and choline were the highest in the germ. b. Tocopherols. Wheat germ contained more tocopherol (vitamin E) than any other cereal germ (Green et al., 1955). Mill germ contained 256 to 500 ppm of tocopherols with an average of 332 ppm (Eggitt and Norris, 1955; Eggitt and Ward, 1955; Engel, 1942, 1949; LeCoq, 1944b; Moran et QL., 1954). Eggitt and Norris (1955) reported 3.0 and 2.0 mg of tocopherols in 100 gm of oil extracted from commercial and purified germ, respectively. They separated the individual tocopherols by chromatography and reported 49 to 59% a-tocopherol, 28 to 29% 0-tocopherol, and 1.5 to 9% €-tocopherol. Pure mill germ contained less E-tocopherol compared with commercial germ because of the contamination with a bran portion rich in E-tocopherol. The biological activity of each fraction in terms of a-tocopherol was: a-tocopherol, 100; 0-tocopherol, 30; and E-tocopherol, 7.5 (Ward, 1958). Waggle et al. (1967) reported 3 I to 202 ppm of a-tocopherol in nine samples of the germ.

WHEAT GERM

237

9. Pigments

Apart from traces of carotenes, von Euler and Malmberg (1936) found xanthophylls as the main pigments of the germ. Flavone-type pigments were reported to be present in mill germ in greater proportions than in the other mill fractions (Simpson, 1935). About 0.2 to 0.3% of glycoflavones were isolated by King (1962) from wheat germ. 10. Other Organic Compounds

King (1 962) detected phenolic compounds such as phenolic, ferulic, and vanillic acids in commercial germ. Daniels and Martin (1967) reported antioxidant properties of wheat germ oil and attributed it to the presence of caffeine and ferulic acid compounds, analogous to the series present in oat kernels. Daniels (1959) detected the presence of methoxyhydroquinone glycosides in the germ and found it to be mostly concentrated in the germ. Andrews and Viser (1951) found 0.1% of oxalic acid in the germ. Swatditat (1974) has reported a thioctic acid content of 36.59 to 41.04 pmoles per gram in wheat germ as compared to 1.37-2.85 pmoles per gram of flour. 11. Enzymes

Being embryonic in nature, wheat germ contains innumerable types of enzymes, as mentioned earlier (Section IV,A,6). Only a few have been reported for dissected germ. Most of the enzyme studies were carried out on mill germ. Some of the important enzymes in the germ were estimated quantitatively, some were crystallized, and others were only detected. Even some of the values for enzymes that were quantitatively analyzed could not be compared with other literature values, as the methods followed and the units of activity expressed were quite different. a. Lipases. Among the different enzymes present in the germ, lipase is probably the most important enzyme studied. This may be attributed to the major role played by lipase in the development of hydrolytic rancidity during storage of the germ or .products containing germ. Inactivation of lipase assumes paramount importance for improving the shelf life of the germ and germ products. This aspect will be discussed separately in Section VI. Several workers have estimated lipase activity using different substrates, with the exception of Sullivan and Howe (1933). All the workers have reported maximum lipase activity in the germ as compared with other fractions. Lipase activity in the gem. Pett (1935) reported maximum lipase activity in dissected parts of the germ, concentrated in the scutellum. Using monometric techniques and mono-n-butyrin as a substrate, Miller and Kummerow (1948)

238

S. R. SHURPALEKAK A N D P. HARIDAS RAO

reported lipase activity of 132.4 pl of COz for defatted germ, compared with 1.4

PI for patent flour and 3.9 pI for low-grade flour. It is interesting to note that the lipase activity of defatted germ was nearly one hundred times that of patent flour. This lipase activity of the germ agrees with that reported by Engel (1947). Koch et al. (1954) reported five times as much activity in the germ as in patent flour, using butter fat emulsion as a substrate. Luchsinger et al. (1955) deveIoped a sensitive method based on the estimation of glycerol released from a monoolein emulsion at pH 7.4 and 30°C for measuring the low levels of lipase activity. No agreement was found between the relative activities estimated by the methods of Luchsinger et al. (1955) and Koch et al. (1954). This was probably due to the differences in pH, substrate, and buffer concentration used in the two methods. However, the lipase activity in the germ was many times that of the endosperm or patent flour, as observed by Engel (1947) and Miller and Kummerow ( 1 948). With monoolein as substrate, Ferrigan and Geddes (1958) measured the lipase activity of twenty-seven flour streams and five mill feeds from hard red spring wheat and found that the distribution of ash and lipase was similar in the mill streams. Feed streams representing 26.5% of the wheat contained 78.7% of total ash and lipasc. The two germ fractions-fine and coarse-had the highest lipolytic activities of over 2000 units (micrograms of glycerol liberated from monoolein per gram of sample) as compared with 434 units for whole wheat and 12 to 333 units in different flour streams. Properties of lipase. The properties of wheat germ lipase were exhaustively studied by Singer and Hofstee (1948a, b), Mounter and Mounter (1962), Fink and Hay (1969), Stauffer and Glass (1966), Brouillard and Ouellet (1965), and Dirks et al. (1955). Wheat germ lipase had an optimum temperature of 36 to 40°C (Luchsinger et al., 1955) and an optimum pH of 7.2 to 7.9 for esters of low- as well as high-molecular-weight acids like butter fat and monobutyrin (Singer and Hofstee, 1948a; Koch et af., 1954). The lipase of wheat germ was concentrated to eleven times the activity of the original extract by Singer and I-lofstee (1948a). They found that only a single enzymc was responsible for the lipolytic activity, which was inhibited by fluoride, p-amino-phenylarsine, and o-iodosobenzoate. Mounter and Mounter ( I 962) suggested that wheat germ lipase functioned more like an esterase than a lipasc. Lipase fractions. Singer and Hofstce (1948b) found no evidence to prove the presence of more than one enzyme. Stauffer and Glass (1966) separated three enzyme fractions from defatted wheat germ: esterase, with an optimum pH of 7.2 to 7.3; tributyrinase, with an optimum pH of 6.6 to 6.8; and lipase, with an optimum pH of 8.0. Brouillard and Ouellet ( 1 965) chromatographed commercial wheat germ lipase on DEAE cellulose and obtained four fractions which exhibited both esterase and acid phosphatase activity. Fink and Hay (1969) studied

WHEAT GERM

239

germ lipase by means of disc electrophoresis and chromatography on Sephadex columns and obtained three different esterase fractions, which differed in their substrate specificity and pH optima, b. Amylases. Oparin and Kaden (1945) reported that the amylases were not concentrated in the germ. Later, Scholander and Myrback (195 1) investigated the amylase activity of 159 units (milligrams of maltose per minute per gram) in the germ, 192 in bran, and 280 in the flour portion, indicating thereby a relatively low concentration of amylase in the germ. They also reported 5.7 units (relative effect on viscosity) of a-amylase in the germ, which was less than that found in other mill fractions. Proskuryakov and Rodionova (1960) determined the amylase activity in the water-soluble protein fraction of commercial germ. c. Proteuses. As reported by Pett (1935), protease and dipeptidase activities were concentrated more in the dissected germ. Using edestin as a substrate, Engel and Heins (1947) found 0.8 to 1.3 units of proteolytic activity in the germ, compared with 0.1 unit for the endosperm. Balls and Hale (1936) expressed proteolytic activity as increased titer value against 0.1 N KOH after enzyme hydrolysis on a portion equivalent to 2 gm of flour. They reported a highest activity of 2.5 ml as compared with 0.45 ml for white flour and 1.6 ml for whole wheat. In contrast, Howe and Click (1946), with casein as substrate, found the proteolytic activity of the germ to be less than that of whole wheat, bran, and shorts. This was probably due to the fact that Balls and Hale (1936) may have used fractions milled from different wheats. d. Phosphutases. The presence of phytase was reported by Proskuryakov and Rodionova (1960) in commercial wheat germ and by Peers (1953) in dissected germ. Verjee (1969) and Brouillard and Ouellet (1965) separated acid phosphatase from wheat germ by ion-exchange chromatography. The latter workers showed that the wheat germ phosphatase existed in four different active and easily separable molecular forms, which hydrolyzed the esterase substrates. They confirmed the presence of iron in the enzyme. e. Oxiduses. Lipoxiduse. The presence of lipoxidase in commercial germ was first shown by Sumner (1943), who reported 810 units of activity, defined as activity in the presence of 5 mg of linoleic acid catalyzing the reaction of 1 pg of O2 per minute at 25°C and pH 7.0 by using the thiocyanate method. This activity was 2.5% of the activity in soybean meal. Later, using the 0-carotene oxidation method, Miller and Kummerow (1948) reported maximum lipoxidase activity in the germ as compared with other mill fractions. The values for destruction of carotene in the germ and commercial flour were 32% and 1.6%, respectively. By the same method, for seven varieties of wheat, Blain and Todd (1955) also reported 58.5 units of lipoxidase activity for the germ as compared with 1.8 units for the endosperm. Chtuluse. Hawthorn and Todd (1955) reported the possibility of catalase playing a part in the unsaturated fat oxidase system. They observed higher

24 0

S. R. SHURPALEKAR AND P. HARIDAS RAO

activity in purified mill germ as compared with flour and commercial germ, which contained impurities like bran and endosperm having low activity. The relative catalase activity of commercial germ was 83, compared with 1 for the endosperm and 4.4 for bran. Peroxidase. Hagihara et ~ l (1958) . isolated, purified, and crystallized enzymes peroxidase 556 and peroxidase 566 from commercial germ and found that the yield of peroxidase 566 was very low. By using mild conditions, Tagawa and Shin (1959) and Tagawa et ul. (1959) obtained crystalline peroxidase in larger amounts. Later, Shm and Nakamura (1961) improved the method for the extraction and purification of peroxidases which had distinctly different properties. Further work on the isolation and purification step was reported by Sequi et al. (1 968). Other Enzymes. Haghara et al. (1958) crystallized cytochrome c from wheat germ. a-Carboxylase from wheat germ was purified 2700-fold by Singer and Pensky (1952). Heinstein and Stumpf (1969) studied the properties of acetyl coenzyme A and purified it more than 1000-fold. Various glycolytic enzymes of wheat germ and their fractionation were reported by Proskuryakov and Loseva (1962). They reported the presence of aldolase, phosphoglycomutase, apyrase, and hexose-phosphate isomerase. In addition, the following enzymes were reported to be present in mill germ; they are classified according t o the recommendation of the International Union of Biochemistry. Oxidoreductases Dehydrases (Kretovitch, 1945) Dehydrogenase dihydroorotic NAD (Kapoor and Waygood, 1965) TPNH oxidase (Conn e f al., 1952) 6-Phosphogluconate isocitric dehydrogenase and glucose 6phosphate dehydrogenase (Barnett et al., 1953) Alcohol dehydrogenase (Stafford and Vennesland, 1953) Isocitric dehydrogenase, glutamic dehydrogenase, malic dehydrogenase, and succinic dehydrogenase (Sisakyan and Vasil’eva, 1954) TPNH diphorase (Clum and Nason, 1958) Cystine reductase (Proskuryakov and Rodionova, 1960) Peroxidase isoenzymes (Lanzani et a/., 1967) Transferases Glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase (Priest, 1959) Hexokinase (Inoue and Ito, 1959) Glutamic acid decarboxylase (Cheng et al., 1960) Acetyl coenzyme A, transcarboxylase, acyl coenzyme A, and malonyl coenzyme A (Hatch and Stumpf, 1961) Methyl transferase Sadenosyl methionine-methionine (Karr ef al., 1967) Orotidine-5-phosphate pyrophosphorylase (Kapoor and Waygood, 1965) D-Xylulokinase (Zahnley and Axelord, 1965) Uridine diphosphate-D-glucuronic acid decarboxylase (Ankel and Feingold, 1965 ; Castaner and Hassid, 1965)

WHEAT GERM

24 1

Hydrolases Acetylesterase (Jansen et al., 1948) Ribonuclease (Proskuryakov and Nuzhdina, 1960; Lanzani and Lanzani, 1967) Ascorbic acid oxidase, glycerophosphatase, deoxyribonuclease (Proskuryakov and Nuzhdina, 1960) Gentobioside (Yamaha and Cardini, 1960) Aldolase, apyrase, and phosphoglucomutase (Proskuryakov and Loseva, 1962) Phosphoglycerol phosphatase (Rao and Vaidyanathan, 1966) Acidic peptidase (Prentice e l aL, 1967) Ligases (Sy n thetases) Oxalacetic carboxylase (Kraemer et al., 195 1 ) Synthetase, glucosides, and synthetase gentobioside (Yamaha and Cardini, 1960) Synthetase, adenyl succinate (Hatch, 1966) Synthetase oxalyl coenzyme A (Giovanelli, 1966) Uroporphyrinogen 111 cosynthatase (Stevens and Frydman, 1968)

C. SUMMARY The exhaustive studies reported on the chemical composition of the germ cover mainly commercial mill germ which is admixed with impurities like bran and endosperm. Separation of dissected germ of high purity is feasible only on a laboratory scale. Information on its composition is scanty and can be considered to be only of academic interest. Dissected germ naturally contains comparatively larger amounts of proteins, sugars, lipids, thiamine, and tocopherols, and lesser amounts of carbohydrates and phytic phosphorus, compared with mill germ. Compared with wheat flour, the germ is unique in that it provides three times as much protein, seven times as much fat, and about fifteen times as much sugar. In addition, the high sugar content makes it one of the most acceptable foods in its natural form. It is interesting to note that, unlike flour protein, wheat germ lacks in gluten and is rich in salt-soluble proteins. In spite of the high nonprotein nitrogen content of about IS%, the conversion factor used by most of the workers for determining protein remains 6.25. Among vegetable proteins, mill germ has probably the best essential amino acid make-up, which compares well with that of egg protein. It is a rich source of lysine, unlike other cereal proteins. Mill germ has also proved to be an excellent source of B-group vitamins and tocopherols, which enhances the value of the germ as a food supplement. The similarity between the physical properties of germ oil and edible vegetable oils makes it suitable for food uses. Fatty acids of germ lipids are predominantly unsaturated, and over 70% of these are essential ones. However, the high lipase and lipoxidase activities coupled with unsaturated fat pose problems regarding an adequate shelf life of the germ. The abundant data available on commercial mill germ have clearly shown its great potential as an ideal food by itself or as an effective supplement packed with all the desired nutrients.

24 2

S. R . SHURPALEKAR AND P. HARIDAS RAO

V. NUTRITIVE VALUE OF THE GERM In human nutrition, cereals, including wheat, have been reported to be poor sources of quality protein. Wheat germ, however, is unique in containing high proportions of protein, edible oil, sugars, certain B-group vitamins, and tocopherols. The biological value of wheat germ proteins has been reported to equal that of highly rated animal proteins. For the nutritional evaluation of different foods, several workers have reported different methods (McLaughlan and Campbell, 1969), including chemical methods, rat and chick growth methods, and supplementary value studies. They have also studied the toxic or inhibitory factors present and the effect of processing on the nutritive value of wheat germ. A. NUTRITIONAL EVALUATlON BY CHEMICAL METHODS

The amino acid composition of the germ has already been discussed (Section IV,A,2). It has been reported that the amino acid pattern of germ protein compares favorably with that of whole egg protein, indicating that wheat germ proteins have a well-balanced amino acid make-up (Barton-Wright and Moran, 1946). The lysine content of germ ranged between 5.5 and 6.5% and was twice that in whole wheat, three times that in white flour, and comparable to that of egg proteins (7.2%). Germ protein was found to be even superior to other first-class proteins with respect to some amino acids like arginine, cystine, and methionine (Barton-Wright and Moran, 1946). The only limiting amino acid of TABLE XXXVIi CHEMICAL SCORE 01’ ESSENTIAL AMINO ACIDS OF WHEAT

MILL P R O D U C T S ~ ~ ~

Amino acid

Germ

Patent flour

Whole wheat

Isoleucine Leucine Lysine Methionine Phcnylalanine Threonine Tryptophan Valine

59c 69 71 5 3d 66 69 86 77

68 77 28d 56 89 5 2‘ 74 67

63 74 35d

53‘ 83

55 86 71

‘Miladi e l QI. (1972). bValues represent amount of amino acid as a percentage of the amount in egg protein. ‘Second most limiting. dMost limiting.

24 3

WHEAT GERM

the germ was reported to be isoleucine (Mitchell and Block, 1946), the percentage deficit being 62%compared with the isoleucine content of egg protein. The chemical score, defined as the amount of amino acids present as a percentage of the amount in egg protein, was reported for mill products (Table XXXVII) by Miladi et aI. (1972). A majority of the amino acids had a higher chemical score in the germ than in other milled products. Their observation that methionine was the most deficient amino acid in the germ contradicted earlier findings (Osborne, 1907; Moran et al., 1970) that isoleucine was the most deficient amino acid, and methionine the second limiting one. The difference was attributed to the variation in the composition of the sample tested or to differences in the amino acid requirements of experimental animals and their species. Block and Mitchell (1946) reported an inverse relationship between the chemical score (defined as 100 - % deficit in the most limiting amino acid compared with egg protein) and the biological value as shown by rat growth experiments. Interestingly enough, wheat germ was rated much lower according to its chemical rating compared with its actual biological performance. This was attributed to the higher proportion of nonprotein nitrogen present in the germ, which probably contributed to the biological value. Using different parameters, Kasarda et al. (1970) evaluated the amino acids of mill products by determining (1) the ratio of the essential to the total amino TABLE XXXVIII NUTRITIVE VALUE OF MILL PRODUCTS OF WHEAT AND WHOLE EGG PROTEIN"

Essential amino acid

E / T valuesb A / E valuesC Total aromatic amino acids Total sulfur amino acids Threonine Tryptophan Valine Isoleucine Leucine Lysine

Egg reference protein Whole wheat

3.22 195 107 99 31 141 129 172 125

1.99

Flour

2.01

24 3 239 196 115 93d(94)e 83d(84)e 41 37 150 138 122d(95)e 120d(93)e 213 210 82d(66)e 59d(47)e

Wheat germ

2.26 192 109 104 36 145 99d(77)e 170 145

'Kasarda et al. (1970). bEssential amino acids (gm)+ total amino acids (gm). cSpecific amino acid (mg) + total essential amino acids (gm). dValues that are at least 5% lower than the reference pattern. eValues A / E for specific amino acid i A / E for reference pattern X 100. The lowest value shows the first limiting amino acid.

244

S. R. SHURPALEKAR AND P. HARIDAS RAO

acids (E/Z");(2) the patterns of essential amino acids compared with those of a reference whole egg protein; and (3) the ratio of specific essential amino acid to the sum of the essential amino acids (A/E). The nutritive values of proteins of different mill products including the reference whole egg protein are given in Table XXXVIII. Even though the EIT value for egg protein was much higher than that for germ protein, such a high E / T ratio was not necessary for the most efficient use of its amino acids. Only 50% of the essential amino acids of egg protein have been reported to be utilized by human adults for maintaining nitrogen balance (FAO/WHO Joint Expert Group, 1965). It was further confirmed from the A / E values that wheat germ showed much better balance and isoleucine was found to be the only limiting amino acid. B. NUTRITIONAL EVALUATION BY BIOLOGICAL METHODS 1. Rat Growth Experiments

Studies carried out by Boas-Fixsen and Jackson (1932) and by Chick et al. (1935) clearly indicated that the biological value of wheat germ was dependent on the level of protein in the diet. They reported biological values of 69 and 90 at 6.8%and 3.6%levels of protein, respectively, in the diet. Hove and Harrel (1943) found that the quality of wheat germ protein was at least as good as that of animal proteins when the diet contained 5% protein. They further determined the protein efficiency ratio (PER) of wheat germ at a 10% level of protein and observed that a slight variation in the level of the dietary protein affected the PER. The PER decreased from 2.87 to 2.41 when the protein content in the diet was increased from 9.3% to 11.7%. The PER of animal proteins included for comparison and fed at the 10%level were: casein, 2.3; dry skim milk, 2.85; and boiled dry egg white, 2.58. A similar inverse relationship between dietary protein and the PER was also observed by Sure (1957). According to him, 15%protein was the most efficient level of protein intake. A higher protein intake resulted in wastage during metabolism. Jones and Widness (1946) compared the nutritive value of wheat germ with that of corn germ, soybean, peanut and cottonseed flours, dried whole egg powder, dried skim milk powder, and casein. The different diets were adjusted so that they were isocaloric and nutritionally adequate with respect to dietary factors other than the protein. The PER of wheat germ was much higher than that of the oilseed flours and was equal to that of the skim milk powder. Also, wheat germ was found to be nutritionally (PER) better than corn germ at any protein level.

245

WHEAT GERM TABLE XXXIX PROTEIN QUALITY OF M I L L PRODUCTS OF WHEAT'

Mean weight gain (sm)

Mean protein consumed

Product

Protein level in diet (%)

Whole wheat Wheat germ Wheat bran Patent flour Nonfat dry milk solids'

10.2 10.1 9.9 9.8 9.6

41.9 131.8 101.5 19.2 125.0

30.0 45.9 47.3 22.9 44.1

(gm)

Mean protein efficiency ratiob

~

~

1.40 2.81 2.15 0.84 2.84

'Hove et al. (1945). 'Ten rats per group fed for a period of 6 weeks. 'Values from a separate experiment given for comparison.

The nutritional superiority of wheat germ over other mill products was reported by Osborne and Mendel (1919), Hove et al. (1945), and recently by Miladi et al. (1972). Wheat germ had a higher PER (Table XXXIX) than any of the other mill fractions (Hove el al., 1945), and the PER compared well with that of nonfat dry milk solids as reported earlier by Hove and Harrel (1943). Recently, the relative nutritive value (RNV) (defined as the slope of the doseresponse curve obtained with the protein under test, divided by the slope of the response curve obtained with standard protein lactalbumin) and in vitro digestibility of wheat proteins from different mill fractions were determined by Miladi et al. (1972). The gain in body weight and body water after a 3-week experimental period have been used as a measure of the response. The RNV of the patent flour was the lowest (25%), and wheat germ had the highest RNV of 80% (Fig. l l ) , which was even higher than the 74% for casein reported by Hegsted and Chang (1965).

80.

01

shorts, .:germ flour ,.,

1' 1'

I

I

I

I

I

1

2

3

4

5

Total lysine (grnA6 grn N)

FIG. 11. The relative nutritive value (RNV) of different mill products.

24 6

S. R. SHURPALEKAR AND P. HARIDAS RAO TABLE XL EVALUATION O F PROTEIN QUALITY O F WHEAT GERM'

Protein source

Wciglit gain (smIb

Growth index

Isolatcd soy protein (ncgative standard) Viobin fish flour (positive standard) Soy flour Solvent defatted wheat germ Isolated wheat germ protein Least significant range (P= 5%)

290 393 368 396 369 16

100 135 127 136 127

'Rand and Collins (1958). bEqualized pain over protcin-frec diet.

2. Chick Feeding Trials Using growing chicks, Rand and Collins (1958) evaluated the nutritional quality of wheat germ along with that of other animal and oilseed proteins. The results, presented in Table XL, indicated that the nutritive value of defatted wheat germ equaled that of Viobin fish flour used as positive control. Hinners (1958) has reported that high-quality fish meal was even superior to egg white as a sole source of protein. It may thereby be inferred that the wheat germ protein was also superior to the egg white proteins. These results of chick feeding trials have confirmed the earlier finding with rat growth trials. I t is, however, interesting to note that the protein isolated from wheat germ had a lower protein quality index than the parent material. On the basis of net protein utilization (NPU) values, Moran et al. (1970) reported that wheat germ compared favorably with soy flour. Cave et al. (1965), while evaluating the proteins of a series of wheat by-products, found that the growth of chicks was relatively rapid when they were fed on wheat shorts or wheat germ meal diluted with 50% of a corn-soybean ration. Using the growing chicks, Summers et al. (1968) reported a PER of 3.06 and an NPU of 58.8 for nine samples of germ. They further evaluated the various wheat fractions obtained from wheat samples for metabolizable energy (ME), metabolizable dry matter (MDM), and NPU. Wheat germ had a higher ME (2.50 kcal/gm) and a higher MDM (57.5%) than other mill fractions such as shorts and bran.

C. SUPPLEMENTARY VALUE OF WHEAT GERM Generally, protein-rich foods are used as supplements to improve the nutritive value of different staple diets based on cereals and millets. The supplementary

24 7

WHEAT GERM

value of wheat germ to wheat flour, gluten, barley, corn, rice, etc., has been studied by several workers.

1. Germ as a Food Supplement Hove and Harrel (1943) reported that the biological value of poor-quality vegetable protein could be improved to the same extent by supplementing either with wheat germ or with casein. The PER of gluten increased from 0.43 to 2.12 and 2.38, when 25% of the gluten was replaced by wheat germ and casein, respectively. Because of good-quality protein and high levels of thiamine, niacin, and many essential minor elements such as copper, iron, and zinc, these workers found that the addition of wheat germ to the American dietary would not only improve the nutritional quality of the diet but could also replace some of the animal proteins in the diet. Supplementation of patent flour with various levels of low-fat wheat germ markedly improved the PER (Hove er al., 1945). Supplementation with even 3% germ improved the PER of patent flour from 0.84 to 1.19. Beeson er al. (1947) found that supplementing the peas with 25% or 50% of germ brought about a TABLE XLI IMPROVEMENT IN THE NUTRITIVE VALUE O F CEREALS WITH DEFATTED WHEAT GERM FLOUR'

Level o f germ added

I 0%

Diet 1

2 3 4 5 6 7 8

9 10

11

15%

Ad libitum gainb

Increase

Protein source

(pm)

(%)

Wheat flour 1 +DWGF~ Rice 3 + DWGF Barley 5 + DWGF Oat 7 iDWG?: Cereal mixture 9 + DWGF DWGF

107 157 138 185 159 202 221 248 128 151 396

'Rand and Collins (1958). bFeeding period: 7 days. 'Calculated for the same feed intake. dDWGF-defatted wheat germ flour.

46 34

27 13

18

Equalized gain'

Increase

(gm)

(%I

120 202 139 236 161 252 28 1 369 463

69 69 56 31 -

S. R. SHURPALEKAR AND P. HARIDAS RAO

24 8

significant improvement in the growth-promoting value. Westerman e f al. (1952) reported that the addition of defatted germ to enriched flour at 4% and 6% levels increased the growth rate of rats, but 2% of germ had n o effect. However, addition of even 2% of defatted germ to flour that had not been vitamin-enriched resulted in increased growth rate, better reproduction and lactation performance, and increased storage of some vitamins, particularly pantothenic acid in the livers of rats. Crampton and Ashton (1943) observed that supplementing the endosperm of corn, barley, or wheat with wheat germ significantly improved the growth of hogs. Using chicks, Rand and Collins (1958) determined the supplementary value of the germ t o wheat flour, rice, barley, oats, and mixtures of equal parts of these cereals by adding 10% and 15% of defatted and light-toasted wheat germ. They observed a striking improvement in chick weight at the 10%level of the germ supplement. The maximum improvement was observed for rice, and the least for oats. At the 15% level, the weight gain was more significant in all the cereals (Table XLI).

2. Germ Compared with Other Food Supplements Hove et af. (1945) observed from their rat growth experiments that the supplementary value of wheat germ to patent flour was the same as that of nonfat dry milk solids and corn germ meal. However, wheat germ was superior to corn germ (Table XLII) as a supplement to wheat flour (Stare and Hegsted, 1944) and peas (Beeson et al., 1947). Similarly, at the 3% level, Westerman et al. (1954) observed that the supplementary effects of soy flour and wheat germ to nonenriched wheat flour were comparable. TABLE XLII SUPPLEMENTARY VALUE O F WHEAT AND CORN GERM PROTEINS TO

PATENT FLOUR'

Protein supplement

Total protein from supplement (%)

Protein efficiency ratiob

None Dry skim milk Defatted wheat germ Defatted corn germ Soybean oilmeal

0 20 20 20 20

0.78 1.45 1.52 1.13 1.23

'Stare and Hegsted (1944). bDetermined for 3-week period.

Total protein from supplement

(%I -

50 50 50 50

Prdtein efficiency ratiob

2.26 2.26 1.71 1.77

WHEAT GERM

24 9

3. Other Beneficial Effects In addition to improved growth observed as a result of feeding germ supplements, other beneficial effects have been reported. Crampton and Ashton (1943) found that wheat germ had a tendency to stimulate or facilitate synthesis and deposition of body fat from dietary carbohydrates. LeCoq (1944a) observed that the edema and neuromuscular troubles developed by rats fed low-protein diets containing theobromine or caffeine could be prevented by the addition of 40% wheat germ meal to the protein-poor diets. Morgulus and Spencer (1936) reported that muscular dystrophy of rabbits could be prevented by the addition of wheat germ.

D. EFFECT OF PROCESSING ON THE NUTRITIVE VALUE OF THE GERM Raw wheat germ was found to have a “feedy” taste and smell and poor stability owing to hydrolytic and oxidative changes in the germ lipids (Sherwood et al., 1933). These defects have been overcome by heat processing of the germ (discussed in Section VI,B). Hove and Harrel (1943) have found that heat processing of wheat germ, to make it suitable for human consumption and to improve its keeping quality, had no effect on the nutritive value of protein as determined by the PER. The effect of several processing methods on the nutritive value of defatted germ was studied by Rand and Collins (1958) by the chick growth method. The processing of the germ was carried out either by light toasting of the material, TABLE XLlII EFFECT O F MILD HEATING ON PROTEIN QUALITY O F

DEFATTED WHEAT GERM‘

Weight gain Protein source

(sm)

DWGC (untreated) DWC laboratory steamed DWG steamed in pilot plant DWG light toasted Isolated soy protein Viobin fish flour

186 209 202 214 142 204

‘Rand and Collins (1958). bFor 1-week period. ‘DWG-defatted wheat germ.

Protein efficiency ratiob 4.1 4.8 4.8 4.8 3.4 5.0

S. R. SHURPALEKAR AND P. HARIDAS RAO

25 0

until it attained the light-brown color desired, or by steaming the germ at atmospheric pressure for 20 minutes. Light toasting or mild steaming improved the protein quality of wheat germ as shown by the weight gains (Table XLIII). This confirmed the earlier findings that plant proteins improved in digestibility and biological value as a result of heat treatment (Gray et al., 1957). However, severe heat treatment of the germ had an adverse effect on its nutritive value, as shown by the reduction in the weight gain of chicks from 428 t o 248 gm, when they were fed pressed, defatted germ. Rand and Collins (1958) suspected this low value to be due to the severe heat development during expeller-pressing of the germ. The effect of heat treatment on the quality and utilization of germ proteins was studied by Olsen (1967) by feeding weanling rats, The heat treatments consisted in toasting germ in a rotary drum dryer at a product temperature of 121°C for 45 minutes and autoclaving at 15 pounds of pressure (121°C) for 20, 45, and 9 0 minutes. Soybean meal was included for comparison. The results of the studies, reported in Table XLIV, confirmed the excellent nutritional qualities of wheat germ. However, heat processing of the germ decreased the nutritive value as shown by the lower PER of the processed germ, depending on the severity of processing. Olsen (1 967) also found that only arginine and lysine contents were affected to a significant extent by the heat treatment. The arginine content decreased by 7% in toasted samples as well as in samples autoclaved for 20 minutes, and by 12% and 27% in the samples autoclaved for 45 and 90 minutes, respectively. The loss of lysine was even greater than that of arginine-12% for toasted' samples, and 16%, 25%, and 42% for samples autoclaved for 20, 45, and 90 minutes, TABLE XLIV EFFECT 01: HEAT TREATMENT ON THE NUTRITIVE VALUE OF WHEAT GERM

MEAL P R O T E I ~ ~ . ~

Diet Soy bean meal W G M , ~raw WGM, toasted WGM, autoclaved, 20 minutes WGM. autoclaved, 45 minutes WGM, autoclaved, 90 minutes

Weight gain (gm)

37.1 + 36.1 k 28.2 + 30.9 +

1.08' 0.82' 1.25' 1.12' 18.0k 0.52' 7 . 3 ? 1.16'

Feed consumed km)

Protein efficiency ratio

85

3.40 3.50 2.84 3.12 2.31 1.00

86 80 84 69 60

'Olsen (1 967). %slues are means for six rats per group; duration of experiment 7 days. 'Standard error of mean. dWGM-wheat germ meal.

25 1

WHEAT GERM

respectively. The losses of other amino acids were negligible, as seen in Table XLV. Toasting had the least effect on the percentage of absorption of amino acids and nitrogen; autoclaving decreased the same values by 2 to 4%. However, compared with values for raw germ, the absorption values of amino acid and nitrogen decreased to 77% and 63% in samples autoclaved for 45 and 90 minutes, respectively. Some of the amino acids were affected to a greater extent on autoclaving of the germ for 90 minutes. The decreased values for absorption were 42% for lysine, 54% for isoleucine, 56% for vdine, and 58% for leucine. The destruction in the amino acids and the decrease in absorption were reflected by lower weight gain and protein utilization by rats, as shown in Table XLVI. TABLE XLV AMINO ACID COMPOSITION' O F HEATED WHEAT GERM M E A L S ~ ~ ~

Wheat germ meal Autoclaved Amino acid Essential Arginine Histidine Isoleucine Leucine Lysine Methionine Phen ylalanine Threonine Tryptophanf Valine Nonessential Alanine Aspartic acid Glutamic acid Glycine Proline Serine Tyrosine

20

45

90

1.01 2.22 3.15 6.01 5.69 1.29 3.28 3.05 0.96 5.05

1.02 2.28 3.21 6.15 5.45 1.22 3.30 2.12 0.96 5.29

6.65 2.22 3.22 6.11 4.85 1.22 3.34 2.92 0.96 5.08

5.54 2.16 3.34 6.05 3.79 1.21 3.31 2.93 0.96 5.10

5.69 1.65 13.10 5.66 4.18 3.09 2.22

5.11 1.53 14.20 5.80 4.29 2.44 2.18

5.92 1.75 14.63 5.59 4.15 2.83 2.19

5.96 1.20 14.49 5.64 4.30 2.87 2.23

Raw

Toastedd

1.58 2.42 3.46 6.24 6.50 1.27 3.53 2.68 0.98 5.39 6.24 8.43 14.38 6.24 4.05 2.19

2.22

'Grams per 16 gm of nitrogen. bOlsen (1961). CMicrobiological method on acid hydrolyzate (except for tryptophan). dAt a product temperature of 121°C for 45 minutes. eAt 15 psig. fMicrobiologica1 method on alkaline hydrolyzate.

25 2

S. R. SHURPALEKAR AND P. HARIDAS RAO TABLE XLVI

GROWTH, FEED EFFICIENCY, A N D NET PROTEIN U T I L I Z A T I O N BY CHICKS FED W I T H HEAT-PROCESSED WHEAT GERM'

Processing treatment Nitrogen-free diet Wheat germ Raw Toasted Autoclaved at 15 psig for 20 minutes 45 minutes 90 minutes Isolated soybean protein

Weight at 3 weeks (gm)b

Weight gain/ feed consumed

Net protein utilization'

I35 154

0.35 0.38

47.7 53.0

127 118 83 159

0.33 0.29 0.13 0.42

48.4 43.9 32.9 59.4

47

'Moran et al. ( 1 968). bAverage I-week starting weight was 67 gm for each treatment. The 3-week weight represents an average of four pens o f chicks. Each pen had ten chicks. 'Feeding period 2 weeks.

Unlike Olsen (1967), who reported detrimental effects of heat treatment, Moran et al. (1968), in their experiment with growing chicks fed on heatprocessed wheat germ meal, found an improvement in the protein quality after toasting. However, autoclaving for more than 20 minutes at 15 pounds of pressure reduced the weight gains and the NPU values below those observed for raw germ. The NPU values reported at 15% dietary protein level are given in Table XLVI. The observed difference in the quality of toasted or autoclaved germ was reported to be due to moisture content, which could affect both the degree of denaturation and the rate of browning, resulting in the unavailability of certain amino acids. The superiority of toasted germ over germ autoclaved for more than 20 minutes was attributed to the low moisture content of the toasted germ as well as to the removal of moisture during the toasting process. The denaturation occurring during toasting appeared to be sufficient to improve digestibility of proteins but not adequate to destroy the limiting amino acids. The low nutritive value of the autoclaved samples was reported to be due to greater destruction of lysine and arginine in the presence of moisture. Moisture probably facilitated the browning reaction of amino acids with free sugars. The effect of heat treatment on the metabolizable energy (ME) of wheat germ meal was studied by Bayley er al. (1968). They found that the processing effect on the ME varied for different classes of birds-chicks, roosters, and turkeys (Table XLVII). The ME values of the diet containing raw germ for both chicks and roosters were similar. Toasting the germ had no effect on the ME values for chicks, but caused an increase in roosters. Autoclaving resulted in increased ME

WHEAT GERM

25 3

TABLE XLVIl EFFECT OF HEAT PROCESSING O F G E R M ON THEIR METABOLIZABLE ENERGY VALUESa

Metabolizable energyb (kcal/gm) Processing treatment

Gross energy (kcal/gm)

Young

chicks

Roosters

Turkey tom

4.48

2.85 2.85

2.91 3.19

3.26 3.28

2.69 2.77 2.75

3.53 3.19 3.31

3.20 3.08 2.68

-

Nil (raw) Toasted Autoclaved at 15 psig for: 20 minutes 45 minutes 90 minutes

-

-

‘Bayley et al. ( 1 968). bOn a 90%dry-matter basis.

values for roosters, while values for chicks decreased. The ME values of the raw and toasted wheat germ meals for turkey were found to be similar t o those of the toasted wheat germ meals for roosters. However, autoclaving the germ for a longer time reduced its utilization by turkeys by about 18%. E. EFFECT OF SUPPLEMENTING THE GERM WITH AMINO ACIDS

1. R a w G e m Olsen (1967) reported that raw wheat germ supplied less of the essential amino acids than the requirement of rats based on the calculation of the available amino acids (the amino acid content of the germ X percentage of absorption) in the germ. He found that it was necessary to supplement the germ with methionine, threonine, phenylalanine, lysine, isoleucine, valine, leucine, and tryptophan to meet the NRC (National Research Council, United States) requirements for the rat. The results of growth experiments with rats fed on raw wheat germ meal supplemented with only methionine at three different levels and also with the above-mentioned amino acids are given in Table XLVIII. The data indicated that improvements in the nutritive value of wheat germ supplemented either with methionine alone or with the amino acid mixture (calculated to be deficient) were comparable. Although the calculated requirement was 0.25% of methionine, maximum growth was noticed in the diet containing only 0.20% of methionine. It may therefore be inferred either that methionine was underestimated in the protein hydrolyzates or that cystine met more than one-third of the sulfur-amino acid requirement.

254

S. R. SHURPALEKAR A N D P. HARIDAS RAO TABLE XLVIll

EFFECT O F AMINO ACID SUPPLEMENTATION ON NUTRITIVE VALUL O F WHEAT G E R M MEAL I'ROTEINS'

Diet

Weight pain Feed consumed

(gm)

(gm)

Protein efficiency ratiob

Soybcan mcal + 0.20%mcthionine Wheat gcrni meal (WGM) WGM + plutamic acid + ammonium citratc WGM + 0.1 5'X Ill-methionine WGM + 0.20%.DL-methionine WGM + 0.25'2 DL-methionine WGM + amino acid mixture I" WGM + amino acid mixture I l d

68.0 f 1.95 59.8 r 2.81 58.4 f 2.50 66.0 + 2.70 72.1 f 1.83 65.1 + 2.26 67.0 f 2.1 3 68.1 r 0.97

178 r 2.93 175 f 5.59 178 f 4.43 1 7 1 ? 4.29 1 7 6 f 4.34 167 f 3.47 167 f 3.26 167 r 2.56

2.8 1 2.53 2.43 2.77 2.97 2.8 1 2.99 2.87

'Olscn ( I 967). bIktermined for a 2-week period. 'Contained (as percent of the diet) DL-methionine, 0.20; L-threonine, 0.19; L-phenylalanine, 0.18; L-lysine, 0.1 2; L-isoleueine, 0.10; L-valine, 0.06; L-lcucine, 0.04; and L-tryptophan, 0.03. dSamc as aniino acid mixture I except for only 0.09% L-phenylalanine.

Miladi et al. (1972) did not find any improvement in the relative nutritive value (RNV) of the germ when it was supplemented with lysine.

2. Processed Germ In order to overcome the deleterious effect of processing on the nutritive value, Moran et al. (1968) carried out an experiment by feeding chicks with diets containing differently processed wheat germs, which were also supplemented with deficient amino acids-namely, methionine, glycine, cystine, phenylalanine, and tryptophan. Moran et al. (1968) found that supplementing differently processed germ with glycine had no effect on the growth of chicks or on the utilization of feed. However, supplementation with methionine significantly improved the growth of chicks fed with all diets containing processed germ except the diet based on germ autoclaved for 90 minutes. In addition to glycine and methionine, supplementation with tryptophan and phenylalanine did not improve the nutritive value any further. By supplementing the processed germ with an amino acid mixture containing methionine, cystine, lysine, and arginine, Moran et al. (1968) reported an improvement in the feed efficiency and the growth of chicks. However, by eliminating each amino acid from the amino acid mixture, they observed that deficiencies in lysine and arginine were not encountered until the meal was autoclaved for 90 minutes.

WHEAT GERM

255

F. TOXIC FACTORS IN THE GERM Bakke et al. (1930) observed a color change from black to silver gray in the coats of rats receiving a germ diet, whereas a whole wheat diet did not have any effect. However, if the germ diet was continued for long, the coat color returned to normal. They inferred that wheat germ contained a toxic element which was neutralized by feeding the whole grain. They also assumed the development of immunity to the toxicity after long-term feeding with the germ. Famiani (1932) reported that the germ did not make a complete food for rats, as the sexual functions did not develop adequately when only the germ was fed. Creek (1955) first observed that substitution of 15% and 30% of wheat germ for corn meal significantly depressed the growth of chicks, which could not be accounted for by the difference in the calories, minerals, bulk, or amino acids. He suspected that the poor growth was due to some toxic factors present in the germ. Later, Creek et al. (1961) replaced 30% and 55.5% of corn flour in the diets with raw and autoclaved (30 minutes at 15 pounds of pressure) germ and fed young chicks these diets for 10 to 13 days. The data on growth and feed conversion of chicks given in Table XLIX confirmed the earlier finding that raw wheat germ depressed growth and feed efficiency, depending on the amount incorporated in the diet. Improvement in both growth and feed conversion in rats fed an autoclaved germ diet indicated the presence of a thermolabile toxic factor which impaired digestion. The behavior of the raw wheat germ was TABLE XLIX EFFECT O F RAW AND AUTOCLAVED WHEAT GERM MEAL ON GROWTH AND FEED CONVERSION BY CHICKS'

Trial

Group

I

1 2 3 1 2 3

I1

Weight gain

Feed/ weight gain

162 136 150 89 69 91

1.54 1.59 1.58 1.64 2.00 1.58

Diet Control (C)

c + 30% WGM' C + 30% autoclaved WGMd Control (C) C + 55.5% WGM C + 55.5% autoclaved WGMd

aCreek etal. (1961). bAverage values for 10 days of feeding. Significantly different at 1%level: 1 versus 2 and 2 versus 3 in trial I; and 2 versus 1 or 3 in trial 11. Significantly different at 5% level: 1 versus 3 in trial I. 'WGM-wheat germ meal. dAt 15 psig for 30 minutes.

256

S. R. SHURPALEKAR AND P. HARIDAS RAO

reported to be similar to that of raw soybean containing trypsin inhibitor (Brambila et aL, 1961). Determination of the weight of the liver and pancreas of chicks fed the diet containing 55.5% germ showed significant hypertrophy of the liver in birds receiving raw germ. Brambila el al. (1961) suspected that enlargement of the livers was probably due to some compensatory mechanism, similar to that existing in thyroid function. Creek and Vasaitis (1962) further isolated from raw wheat germ a water-soluble factor which inhibited the enzymic digestion of protein. It was thermolabile and very much like the antitrypsin factor found in raw soybean. By analyzing the feces of chicks fed diets containing raw and autoclaved wheat germ, as a sole source of protein, they found that utilization of protein in chicks fed the diets containing raw or autocfaved germ was nearly the same. On the contrary, rats receiving the raw germ excreted about six times as much fat as those receiving the autoclaved germ (Table L). Creek and Vasaitis (1962) inferred that some toxic factors present were blocking the utilization of fat. Using ether-extracted germ, these workers later confirmed that some factor was acting on the fat added to the diet and not on the germ fat. However, they found that nitrogen retention was also improved by the autoclaving treatment, although not to the same extent as fat utilization. Depressed growth as well as fat utilization of raw germ observed by Creek et al. (1962) was further confirmed by Parrish and Bolt (1963). On close observation, however, they found that the diet containing raw wheat germ formed a paste on the beaks in combination with salivary mucus and water. Consequently, these birds consumed correspondingly less of the diet and hence did not grow well. They also observed that the birds receiving the raw germ diet were cleaning their beaks on the wire floors, thus adding sufficient feed fat directly to the excreta. Use of autoclaved germ in the diet altered its consistency so that it did not form a sticky paste and the birds could eat it without any difficulty. Therefore these workers did not believe in the presence of a toxic factor. TABLE L EFFECT OF FEEDING CHICKS WITH RAW OR AUTOCLAVED

WHEAT GERM M E A L AS SOLE SOURCE O F

Weight gain (gm/20 days) Percent nitrogen excreted Percent fat excreted OCreek and Vasaitis (1962). bWGM-wheat germ meal. CAt 15 psig for 30 minutes.

PROTEIN^

Raw WGMb

Autoclaved WGMC

141 41.8 40.8

21 2.0 36.6 6.8

WHEAT GERM

257

Like Parrish and Bolt (1963), Attia and Creek (1965) also observed the formation of paste on the beaks for some of the raw germ samples, but they felt that it was not the sole reason for the poor growth of the chicks. Consequently, to prevent the formation of paste with saliva, they conducted experiments with young chicks by feeding an ultra-high-fat diet. Care was also taken to avoid the possibility of the feed fat’s entering directly into the excreta on the wire floor. The results, given in Table LI, confirm their earlier findings that raw germ depressed growth and interfered with fat absorption. They inferred that the problem of low nutritive value of raw germ was very similar to that of raw soybean which contained hemagglutinin and trypsin inhibitor and reported 2500 units of hemagglutinin in rabbit blood and 5000 units in chick blood per gram of germ. They also observed the pancreatic hypertrophy of the liver. However, autoclaving overcame all detrimental properties of wheat germ. Cave et al. (1965) fed raw wheat germ meal to young chicks at a level of 50% added to a corn-soybean meal diet and observed no adverse effect on growth or on carcass protein. Rehfeld (1967) and Olsen (1967) found no toxic components in raw wheat germ and reported that high nutritional value was reduced by heat treatment. The presence of hemagglutinin and antitrypsin activity in raw germ was further proved by Moran et al. (1968), who estimated their activity by following the method of Liener and Hill (1953) modified by Attia and Creek (1965). Toasting almost completely destroyed hemagglutinin and antitrypsin activity. Autoclaving reduced the toxic factors to zero values (Table LII). Trypsin inhibitor from wheat germ with a molecular weight of 17,000 was isolated and purified later by Karl et al. (1969). TABLE LI EFFECT O F FEEDING RAW AND AUTOCLAVED WHEAT GERM ON GROWTH, PANCREAS SIZE, FAT ABSORPTION, A N D FEED RETENTION I N CHICKS‘

Body weight (gm) after: Trial I

I1 111

Diet

2 weeks

3 weeks

215 236 174 201 146 205

-

Raw germ Autoclaved germb Rawgerm Autoclaved germb Rawgerm Autoclaved germb

‘Parrish and Bolt (1963). bAt 15 psig for 30 minutes.

-

283 364

Pancreas size (mg/lOO sm) after: 2 weeks 462 413 -

3 weeks 33.3 13.6 -

343.0 264.0

Consumed fat (%) excreted in: 2 weeks

3 weeks

-

-

-

24.03 12.6 26.5 11.6

-

25 8

S. R. SHURPALEKAR AND P. HARIDAS RAO TABLE LII EFFECT O F PKOCESSING W H E A T GERM ON HEMAGGLUTININ A N D ANTITKY PSIN A C T I V I T Y ~ , ~

Hemagglu tinin activity‘ Heat treatment of germ

Regular

Papain

Antitrypsin activity

Nil (raw) Toasted Autoclavcd at 15 psig for: 20 minutes 4 5 minutes 90 minutes

2560 Nil

5120 20

Very high Trace

Nil Nil Nil

320 160 160

Nil Nil Nil

‘Moran et al. (1968). bAll data have been expressed on a 14% moisture basis. ‘Expressed as dilution necessary to attain zero activity.

G . SUMMARY It can be inferred from different studies carried out by several workers that wheat germ proteins have a very well-balanced amino acid make-up. The amino acid composition is quite similar to that of egg proteins as indicated by its chemical score. When cereals in general are deficient in lysine, wheat germ is a rich source of lysine. The only limiting amino acid reported was isoleucine. Nutritionally, wheat germ is much superior to other milled products of wheat. Feeding trials with rats, chicks, and other animals have shown conclusively that the nutritive value of the germ is equal to that of any animal protein such as egg, casein, skim milk powder, or fish flour and is much superior to that of protein-rich oilseed flours. Similarly, wheat germ has excellent supplementary value when added to other cereal proteins and compare favorably in this respect with nonfat dry milk solids. Mild heat processing or light toasting of germ improves its nutritive value, probably owing to an improvement in the digestibility of the proteins or to destruction of antinutritional factors like hemagglutinin or trypsin inhibitors, whose presence in the germ is well established. Severe heat processing lowers the nutritive value considerably, depending on the extent of the heat treatment.

VI. STORAGE AND STABILIZATION OF THE GERM The usefulness of wheat germ as a rich source of vitamins and proteins is so well recognized that it is used in its natural form as a component of several

WHEAT GERM

25 9

speciality foods. Extracts of the germ are also used in certain vitamin concentrates. As wheat germ contains a high amount of fat, predominantly unsaturated, protein, and several hydrolytic and oxidative enzymes, it is natural to expect changes in the fat or protein, resulting in the development of an objectionable odor and taste on storage. The demand for wheat germ as a special food has consequently attracted several workers to study its keeping quality and methods to improve this quality. A. STORAGE STUDIES Only a few researchers have systematically studied the shelf life of differently packed wheat germ as influenced by various conditions such as moisture and temperature. The spoilage of the germ during storage has been attributed to different enzymes. Various methods have been suggested to evaluate the freshness or spoilage of stored germ.

1. Effect of Packing under Vacuum or Inert Gases Sherwood et al. (1933) studied the shelf life of whole wheat germ packed in vacuum or with inert gases like nitrogen or carbon dioxide at various temperatures. Alcoholic acidity was used as a measure of the freshness or the spoilage of the germ during storage. Even when samples were stored under the same condition, the acidity changes of vacuum-packed germ were dependent on the purity of the germ. Considering acidity changes as well as organoleptic quality, vacuum-packed germ was found to be better than that packed under inert atmosphere. Pearce (1943) also observed that packing under nitrogen or pelleting of the germ was desirable for a longer shelf life.

2. Effect of Temperature Sherwood et al. (1933) observed that the increase in germ acidity was a function of temperature. Acidity increased eight times as fast at 29°C as at -10°C. In the majority of cases, no unpleasant flavor was observed until the acidity exceeded 0.25%. The germ packed either under vacuum or under nitrogen kept well for over 338 days at -10°C and only for 90 days at 29°C. From their studies on the effect of storage temperature on acidity changes in vacuumpacked germ (Table LIII), they concluded that the germ could be stored under vacuum for a minimum period of 6 months, with the temperature ranging between -10°C and 7"C, without affecting the organoleptic quality. Pearce (1943) reported similar findings from his storage studies covering a temperature range of -40" to 24°C.

S. R. SHURPALEKAR AND P. HARIDAS RAO

260

TABLE LIII CHANGES IN ACIDITY O F VACUUM-PACKED GERM STORED AT DIFFERENT TEMPERATURES~ Aridity (as % H, SO,) at: Storage period (days)

0 31 61 99 126 162 196

0" c

I" c

22°C

29°C

35°C

0.126 0.157 0.160 0.140 0.183 0.249 0.213 0.00044b

0.126 0.165 0.208 0.150 0.193 0.269 0.244 0.00060b

0.126 0.203 0.257 0.270 0.333 0.462 0.500 0.00 191

0.126 0.229 0.330 0.360 0.445 0.546 0.638 0.00261b

0.126 0.3 25 0.462 0.590 0.770 0.808 0.993 0.00442b

aSherwood et al. (1933). bAverage daily increase in acidity.

Fitfield and Bailey (1929) observed a similar acidity-temperature relationship for stored flours of different extraction rates. When the observations made by Sherwood et al. (1933) are compared with those of Fitfield and Bailey (1929) and Markley and Bailey (1931), it is seen that the daily increase in acidity under similar storage conditions was four times as fast in wheat germ as in patent flour.

3. Effect of Moisture Using different solvents for the extraction of lipid, Sullivan and Near (1933) estimated the acidity and the nitrogen and phosphorus contents of lipids in samples stored in glass containers for 6 months. The acidity of the stored germ depended on its moisture level (Table LIV). The acidity (1.27% on H2SO4) of the germ stored at 12.3% moisture was higher than that of the germ stored at 4.3% moisture. On storing the germ in airtight glass containers and cotton bags, they found that the sealed glass container retained the original moisture content, while the germ stored in bags lost considerable moisture (5.5%) through evaporation. Pearce (1943) studied the stability of the germ at different moisture levels ranging from 8.0 t o 26.5% and observed an increased shelf life for the germ when it was stored at a low moisture level. Cuendet et al. (1954) found that the germ developed an undesirable odor within 6 weeks even when it was stored at a moisture level of 3% in a closed glass jar, They also reported that the development of acidity in the various mill fractions was directly proportional to their lipid contents. After a one-year storage period, the acidity of the germ containing 12.9% lipids was about 2.0%, whereas that of patent flour with 1.3% lipids was as low as 0.03%.

26 1

WHEAT GERM TABLE LIV EFFECT O F STORAGE O F GERM AT DIFFERENT MOISTURE LEVELS ON

L I P I D CONSTITUENTS'

6 months of storage at: ~~

12.3% moisture

Initial

~

4.3% moisture

Eb

Pc

Nd

Eb

f

Nd

Eb

Solvent used

(%)

(%)

(%I

(70)

(%)

(5%)

(70)

p' (70)

(%)

Alcohol-ether Ether Acetone

15.62 11.81 12.58

0.50 0.14 0.13

0.41 0.12 0.34

12.81 13.00 14.60

0.30 Trace 0.04

Trace 0.04 0.05

13.68 11.54 12.23

0.560 0.085 0.094

0.35 0.04 0.23

Nd

'Sullivan and Near (1933). bExtractives. 'Phosphorus. dNi trogen.

Sullivan and Near (1933) found that germ samples stored at a higher moisture content showed a marked decrease in the alcohol-ether extractives, and an increase in the ether or acetone extracts. This was due to the solubility differences of lecithin and its split products-that is, fatty acids, choline, and glycerophosphoric acid. The nitrogen and phosphorus contents also decreased in all the extractives. Storage at low moisture levels showed relatively slight changes in germ lipids. The changes observed were explained as being due to enzyme hydrolyses and were correlated with increasing moisture and acidity of the samples. 4. Causes of Spoilage

Rancidity and peroxide values in germ oil increased concurrently during storage, whereas in whole germ the rancidity was detectable even when the peroxide values were relatively low (Pearce, 1943). As such, the peroxide value was found to be a better index of spoilage for the oil than for the germ. It was therefore suspected that protein may be mainly responsible for spoilage of the germ, rather than lipids as reported by Sherwood er al. (1933). Pearce (1943) confirmed this by estimating the fluorescence of potassium chloride extract of defatted wheat germ (Table LV). The fluorescence was attributed t o the products of protein hydrolysis. The results of both Sherwood er al. (1933) and Pearce (1943) were confirmed by Lusena and McFarlane (1945). Yakovenko (1961) reported that microorganisms did not play a decisive role in the development of rancidity in the germ. He observed in stored germ an increased lipoxidase activity which was dependent on the temperature of storage. Maximum lipoxidase activity was observed during 6 months of storage at

S. R. SHURPALEKAR AND P. HARIDAS RAO

26 2

TABLE LV FLUORESCENCE O F 10% POTASSIUM CHLORIDE EXTRACT O F

D E FATTE D

Condition of germ

w H EAT GERM' Fluorescence reading in photofluoronieter

Fresh Storcd for 6 months at:

11.6 11.8

-4O.O"C -17.8"C -

9.4"C

-

1.1"C

11.0

12.4 12.2 14.0

15.6"C 'Pearce (1943).

16" to 21°C. The results indicated a correlation between lipoxidase activity and the development of off-flavors in the germ. According to Rothe and Stoeckel (1962) and Rothe (1963), both lipase and lipoxidase activities in the germ were responsible for the development of offflavors during storage. Stability of the germ was increased by reducing the lipase and lipoxidase activities. Rothe (1963) found that lipase activity decreased rapidly as germ moisture was reduced to 4.5%. Below this moisture level, n o lipase activity was detected. Further, he observed a considerable increase in the fatty acid content of four germ samples stored over a period of 5 weeks at room temperature. From the initial values ranging from 50 to 90 mg, the fatty acid values increased to a range of 185 to 280 mg. This increase resulted in products of varying degrees of bitterness. Later, Rothe er al. (1967) reported that oxidation of fat was the main reason for the development of off-flavors in the germ. According to them, fat is converted to hydroperoxides either by autoxidation or by an enzymic reaction mainly by lipoxidase. Normally the lipoxidase reaction was faster than autoxidation. The hydroperoxides could be further oxidized to polymerized compounds, which impart a bitter taste, or t o carbonyl compounds, which give a rancid taste to stored germ. 5. Evaluation of Freshness

Sherwood er ul. (1933) and Sullivan and Near (1933) used alcoholic acidity as the criterion for assessing the spoilage of the germ during storage. Estimation of fluorescence resulting from products of proteolytic activity was used by Pearce (1943). Yakovenko (1961) found lipoxidase activity to be a measure of freshness. In the studies of Rothe (1963), free fatty acid formed the basis of germ

WHEAT GERM

263

TABLE LVI RELATION BETWEEN DECANAL VALUE AND RANCIDITY

IN GERM‘

Sample

1 2 3 4 5 6 7 8 9 10 11

Organolep tic qualityb

Decanal value (mg%)

Peroxide valueC

Slightly bitter Bitter Bitter

1.4 0.8 1.4 2.1 3.8 4.2 4.2 5.1 6.6 7.4 17.0

0 3 1 1 4 10 4 11 11 22 7

+ + ++ ++

+++ +++

+++

‘Rothe et a!. ( 1 967). b- satisfactory, + rancid, ++ highly rancid, +++ unacceptable. ‘Determined according to the method of Franzke (1956).

stability. Lusena and McFarlane (1945) found a peroxide value of 20 meq/kg of the germ as the threshold value, beyond which the rancidity could be detected by smell and taste. They, however, did not find any relation between lipoxidase activity and subsequent peroxide formation during storage. According to Rothe el al. (1967), the “decanal value” is a good measure of germ freshness. The method for determination of the decanal value consisted in steam distillation of germ fat, formation and separation (by thin-layer chromatography) of hydrazones, and measurement of absorbance of aldehydes of higher chain length. From the results presented in Table LVI, it was inferred that the decanal value was better correlated to rancidity than the peroxide value. The critical decanal value without detectable rancidity was found to be 3 mg per 100 gm of germ. Further, there was a steady increase in the decanal value which could be better correlated with the organoleptic acceptability of stored germ. On the contrary, peroxide values showed an irregular increase or decrease during the storage period. B. METHODS OF STABILIZATION OF WHEAT GERM It is evident from the work of Sherwood et al. (1933) and of Pearce (1943) that raw germ on storage develops a rancid flavor and bitter taste in a short time. Rothe (1963) has observed that, because of the high enzyme activity and

264

S. R . SHURPALEKAR A N D P. HARIDAS RAO

unsaturated fat content in fresh germ, the organoleptic acceptability is affected adversely within a few days. The poor stability of raw germ has restricted its food uses; this problem could be overcome either by inactivating the enzymes or by creating conditions unfavorable for the enzymic activity by suitable means. Some of the important methods for stabilization of the germ and the effect of different treatments on the enzyme activities are discussed below:

1. Heat Processing Light toasting of the germ at 120" to 130"C, until it attained a light brown color, improved its keeping quality as well as its palatability (Hertwig, 1931). The germ thus heated and stored in a glass jar at 50°C remained fresh even after 25 days of storage. When stored in paper cereal cartons at room temperature, the toasted germ was in good condition even after 10 months of storage. Lusena and McFarlane (1945) studied the effect of heating the germ, at various moisture levels for different periods in a hot air oven, on the enzyme activities and its stability during storage at 37°C (Table LVII) in laminated metal foil. They found that all the heat treatments completely destroyed lipoxidase activity; the proteolytic activities were reduced considerably, depending on the moisture level. The enzyme-active raw germ samples showed an unexpectedly smaller increase in peroxides compared with the heat-treated samples. All the samples developed strong off-flavors during 5 weeks of storage. As the germ with low moisture content showed less tendency to develop offflavors in storage, Lusena and McFarlane (1945) subsequently treated raw germ with moist heat followed by dry heat. The treated samples packed in cellophane envelopes were in good condition even after a month's storage at 55°C. It is interesting to note that the peroxide values of both the control and the heat-treated samples were comparable after a month of storage. The other method of stabilization developed by them was direct steaming of the germ in a chamber at 110°C for 30 minutes and then drying it at 100°C under nitrogen. Wierszbowski et al. (1966) stabilized the germ by heat treatment at 240°C for 2 minutes. They observed a decrease in the amylolytic and proteolytic activities as a result of a reduction in the number of -SH groups. Due t o heat treatment, reduction in -SH groups as determined by amperometry has also been reported by Swatditat (1974). Rothe (1963) carried out investigations on the possible ways of stabilizing wheat germ, either by inactivation of the enzyme by heat treatment or by a drying technique t o create conditions unfavorable for enzyme activity. Moisture and dry heat were utilized to improve the keeping quality of the germ by inactivating the lipolytic enzymes responsible for the bitter taste,

TABLE LVII EFFECT O F VARIOUS HEAT TREATMENTS AND SUBSEQUENT STORAGE CONDITIONS ON ENZYME ACTIVITY AND PEROXIDE FORMAT ION^

Lipoxidase Proteolytic activity activity

Heat treatmentb Sample

Peroxides formedC

Duration

System

Storage atmosphere

Moisture (%)

(units/kg)

(units/kg)

Initial

7 days

14 days

1.5 hours 1.5 hours 1.5 hours 1.5 hours 12 hours 12 hours Control-untreated Control-untreated

A.C.d A.C. N.C.e N.C. A.0.f A.O.

Air Nitrogen Air Nitrogen Air Nitrogen Air Nitrogen

10.3 9.6

Nil Nil Nil Nil Nil Nil 537 537

Nil 20 23 16 35 35 42 42

Nil Nil Nil Nil 1.0 1.0 Nil Nil

0.2 0.1 Nil Nil 20.0 10.1 Nil Nil

1.2 1.5

35 days

~

8.6 9.7 0.9 0.9 11.1 11.1

'Lusena and McFarlane (1945), bAt 100°C. 'Expressed as milliequivalents per kilogram of oil during storage at 37°C. dAir-covered. 'Ni trogen-covered. fAir open.

0.9 0.9 39.0 54.7 0.4 0.3

~

20.3 23.4 10.0 5.3 43.0 60.0 16.0 14.3

S. R. SHURPALEKAR AND P. HARIDAS RAO

266

Interesting data regarding the effect of moisture content and temperatures of heat treatment on the lipase activity are presented in Table LVIII. The lipase of the germ containing 12% moisture was found to be quite stable when the germ was heat-treated at 70°C for 24 hours. It could be concluded from the data that, as the moisture content increased at a given temperature of heat treatment, the extent of inactivation also increased. Further, at the same moisture content, the degree of inactivation increased with the increase in the temperature of the heat treatment. Rothe and Stoeckel (1967) studied the effect of a 20-hour heat treatment on lipoxidase and peroxidase activities of wheat germ containing 6.3% moisture. Based on the data collected, a linear equation to correlate the inactivation temperature of the enzyme and the moisture content of the germ has been worked out, and correlation coefficients of 0.990 for peroxidase and 0.998 for lipoxidase have been obtained. Rothe and Stockel (1967) further reported that, on a dry-weight basis, the inactivation temperatures of peroxidase and lipoxidase were 108°C and 67"C, respectively. According to a U.S. patent (1974), the stabilization of wheat germ could be carried out by (i) grinding in an impact mill, (ii) suspending the ground germ in air and heating to about 93°C to lower the moisture content to less than 6%, and (iii) collecting in a cyclone and sifting to remove bran particles. Ivanova et ul. (1975) conducted extensive studies on the heat stabilization of wheat germ by using hot air-stream at 80-1 10°C or steam heating at 0.5-1.75 atmospheres followed by hot air stream drying at 100"-120°C. Drying in a hot air stream at 100°C was found to be a most promising treatment. Heating at TABLE LVIII EFFECT OF MOISTURE CONTENT ON HEAT SENSITIVITY'

OF

WHEAT G E R M L I P A S E ~ . ~

Loss in enzyme activity (%) at: Moisture content (%)

100°C

90°C

80°C

70°C

4

1

9 12 16 19 25

55 100 100 100 100

0 21

0 0 15 53 60 100

0 0 0

15 100

100 100

'Expressed as loss in enzyme activity (%). bEstimated according to Rothe (1961). 'Rothe (1963).

10

28 75

WHEAT GERM

267

100°C for 8 minutes resulted in reduction of moisture from 12.67 to 2.90%. Such a sample could be stored in good condition for about 90 days without any change in its vitamin E content. Only small increases in acid and peroxide values were observed. Heating at 100°C for 3-4 hours facilitated preseving wheat germ without any change in acid value. A moisture content of 3.0-3.5% was considered to be critical for wheat germ stabilization.

2. Ethylene Dichloride Treatment “Tonic” wheat germ, which is a fat-free residue obtained by extraction with ethylene dichloride, has been reported to have good keeping quality (Lusena and McFarlane, 1945). Even though the germ treated with ethylene dichloride stored well for one and one-half months at 55”C, removal of traces of the solvent was a problem. Lusena and McFarlane (1945) found that steaming of the treated germ removed all the residual solvent. Consequently, a process combining both the ethylene dichloride and the steam treatments has been developed by them. A product thus treated had 4.7% moisture and was free of lipolytic or proteolytic activity. Further, the product was quite palatable and retained its freshness for 8 months when stored in cellophane envelopes at 37°C. Untreated germ, however, developed an off-flavor at the end of one month’s storage.

3. Infrared Heat Maes and Bauwen (1951) reported that stability of the germ could be increased by subjecting it to infrared radiation. The stability was dependent on the radiation period, the intensity of the radiation, and the thickness of the germ layer exposed. The acidity of the germ, which reflected the freshness, was much higher in stored raw germ than in treated germ.

4. Treatment with Epoxy Compounds Gaver (1962) patented a process for improving the shelf life of wheat germ by treating it with epoxy compounds. In addition to enhancing its stability against fermentation, rancidity, and discoloration, such treatment also resulted in a germ of improved flavor. The method involved exposing fresh wheat germ in an air-free anhydrous container to a mixture of epoxy compounds consisting of ethylene oxide and propylene oxide until the resulting wheat germ contained about 2 to 5% by weight of reacted epoxy compounds. The treated germ was quite stable when stored for 5 months at room temperature or at 100°F.

26 8

S. R. SHURPALEKAR AND P. HARIDAS RAO

5. Deoiling, Alkali Treatment, and Roller Drying

Grandel (1959) in his patent has described a method for stabilizing and debittering the germ. The steps involved were: (1) deoiling in a hydraulic press until the germ contains less than 4% oil; (2) grinding the deoiled germ in a roller mill, so that the degree of comminution does not exceed 50% (this avoids contamination with bran, containing bitter compounds); (3) treating the fine flour with water containing the requisite amount of sodium carbonate or bicarbonate, corresponding to the acidity of the germ flour; (4) and flaking the germ slurry on a roller dryer at 130" to 140°C.

6. Treatment with Antioxidant Barnes (1948) reported that addition of 5-pentadecylresorcinol at a level of 0.01 to 0.5% to wheat germ enhanced its shelf life by inhibiting the oxidation of germ fat. The germ treated at a 0.2% level did not show any rancidity even after 75 days of storage, whereas raw germ developed a rancid taste in only 17 days. 7. Lowering the Moisture Content By a simple drying technique, Rothe (1963) has reported that the lipase activity of 100% observed in germ samples containing 26.5% moisture decreased steadily to complete inactivation, when the moisture content was reduced to about 4%. The data on the relationship between the lipase activity of the wheat germ and its dependence on moisture content are presented in Fig. 12. A drying technique has been developed by Rothe (1963) for stabilizing the germ by reducing its moisture content to 5%. This is presented schematically in Fig. 13.

Moisture content during enzyme reaction (%)

FIG. 12. The relationship between inactivation of germ lipase and its moisture content.

WHEAT GERM

269 Inlet

Inlet

germ

Air outla

r inlet

watad duct

Outlet

FIG.13. Schematic presentation of apparatus for drying of wheat germ.

Hot air is allowed to enter the chamber from the bottom, and it leaves at the top after carrying away the moisture from the germ sample falling through metallic wire mesh from top to bottom. He has also worked out a process for drying wheat germ to a 5% moisture level by using predried air at temperatures below 35°C. In this way the germ sample can be stabilized without affecting the nutritive components like vitamin E and thiamine and some of the desirable enzymes as well as the fresh taste. Wheat germ thus treated and packed in moisture-proof containers showed n o undesirable changes even after two and one-half years of storage. The keeping quality of the germ in relation to its moisture content, when stored at room temperature in diffused daylight, has been studied by Rothe (1963). The data presented in Table LIX highlight the good keeping quality of the product stored for more than 600 days at a moisture content of 5%. In contrast, in germ samples containing 13% moisture, the onset of bitterness or stale taste was observed in a short period of 3 days. TABLE LIX EFFECT OF MOISTURE CONTENT ON KEEPING QUALITY OF WHEAT

Moisture content (%) 13 9

6 5

GERM'^^

Storage period until onset of astringent or bitter taste (days) 3

21 60 6 00

'Rothe (1963). bStored at room temperature in diffused light.

270

S. R. SHURPALEKAR AND P. HARIDAS RAO

8. Miscellaneous Treatments Donk and MacDonald (1935, 1937) patented a method of stabilizing wheat germ against rancidity, Finely ground germ flour was mixed with sodium chloride to absorb moisture. It was then blended with nonfatty, starchy materials like potato flour or rice flour and dried under a current of nitrogen or carbon dioxide. Musher (1940) stabilized the germ against oxidative deterioration by dispersing it with aqueous skimmed milk and heating it to at least 170°F prior to drying.

C. EFFECT OF STORAGE AND STABILIZATION ON THE NUTRIENTS Hove and Harrel (1943) reported that toasting wheat germ to make it suitable for human consumption improved its keeping quality without affecting its nutritive value. According to Pearce (1943), the thiamine content of the germ, when stored for 6 months at 15.6"C in sealed containers, remained unaffected. Wierszbowski et al. (1966) observed a loss of tocopherols during storage of the germ. On the contrary, lusena and McFarlane (1945) had found earlier that both thiamine and tocopherols in germ samples packed in cellophane bags were quite stable during the stabilization process as well as after 8 months of storage

TABLE LX GLUTATHIONE CONTENT O F RAW A N D T R E A T E D WHEAT

GERM^ Glutathione content ( m d 1 0 0 gm) Oxidized Sample

Total

Reduced

(by difference)

Raw germ Treated germ Steamed at 105-110°C for 30 minutes; finally air-dried at 100°C for 1 hour Exposed to ethylene dichloride vapor;b finally air-dried at 100°C for 1 hour

147.4

102.6

44.8

79.8

80.5

Ni 1

76.7

77.2

Nil

'Lusena and McFarlane (1945). bAt 95" to 100°C for 15 minutes followed b y 15 minutes of steaming at 105" to 110°C.

21 1

WHEAT GERM

at 37°C. They also reported that different treatments for stabilizing the germ destroyed 50% of the total glutathione (Table LX). The reduced glutathione decreased by 25%, while the oxidized form was completely destroyed. Cuendet et al. (1954) observed that the moisture content at which wheat germ was stored had a profound effect on the loss of thiamine (Table LXI), which was greater in the samples containing higher moisture levels. During 3 weeks of storage, little loss of thiamine was found, whereas after 52 weeks, 80% of the original thiamine in the germ was lost. The thiamine content of the germ having 3% moisture remained unchanged during storage. Iwata ef al. (1955a) inferred that the loss of thiamine in raw germ during storage was proportional to the degree of germ damage. The loss of riboflavin in the germ, when stored either at 37°C or at room temperature, was relatively stable, while loss of pyridoxine was observed mainly at higher temperatures of storage. Rothe (1963) studied the effect of storage of differently processed germ products from some European countries on the taste, fat acidity, vitamins, and linoleic acid contents. The data presented in Table LXII highlight clearly the effects of dry and moist heat. Different methods of processing were found to preserve the organoleptic quality and nutrients of the germ samples to different degrees. It is evident from the data that the thiamine and vitamin E contents of product 1 stabilized by dry heating and were significantly lower than the contents of products 2 to 5 processed by moist heat treatments. However, fat acidity was relatively lower for product 1, as compared with products 2 to 5 . The linoleic acid contents of different products were comparable within a range of 43 to 50% on the basis of fat. Organoleptically, dried germ was more acceptable over a longer period of 33 months, as compared with products 1 to 5, which showed different degrees of staleness, bitterness, etc., during 8 months of storage. TABLE LXI EFFECT O F MOISTURE ON THIAMINE CONTENT O F GERM DURING STORAGE AT 37.8"Ca

Thiamine content (rng/lb)b after storage for: Moisture content

(%I

0 week

3 weeks

38 weeks

52 weeks

3 6 10 14

10.1 11.2 10.8

11.3

8.8

11.2 10.4

10.1 9.4 8.0

-

10.5

8.8 8.5 4.8

'Cuendet ef al. (1954). bOn a dry basis.

2.1

TABLE LXII QUALITY PARAMETERS O F STORED WHEAT GERM P R O D U C T S ~

Products Fresh wheat germ Dry wheat gerp Product 1 (GDR)C Product 2 (GDR)‘ Product 3 (FRQd Product 4 (Austria) Product 5 (Switzerland)

Method of Storage period stabilization (months) -

Drying Dry heat Moist heat Moist heat Moist heat Moist heat

‘Rothe (1963). bper 100 gm of dry matter. ‘German Democratic Republic. dFederal Republic of Germany.

0 33 8 8 8 8

8

Taste Fresh Fresh Slightly stale, somewhat bitter Somewhat bitter Rusty, somewhat stale Somewhat stale, bitter Irritating, bitter

Fat acidityb Thiamineb Vitamin Eb Linoleic acid (mgKOH) (vg) (mg) (%in fat)

4Cb100 120 153 216 180 196 290

1500-2500 1780 970 1640 1870 2100 1670

15-30 19 13 18 21 22 20

4 2-5 2 43 48 45 46 44 50

WHEAT GERM

27 3

D. SUMMARY Freshly milled raw germ is highly unstable and deteriorates rapidly within a few days, as it is rich in enzymes, lipase, lipoxidase, and protease. Different studies have indicated that the keeping quality of the germ is dependent on moisture content, temperature and period of storage, mode of packing (inert gases or vacuum), processing treatment, etc. Storing at low moisture of about 5% and low temperatures (less than 10°C) or packing under inert gases or vacuum enhances the shelf life of the germ significantly. Oxidation of fat was one of the main factors contributing to spoilage of the germ during storage. Among different indices such as alcoholic acidity, free fatty acids, peroxide value, and the decanal value used for evaluating the quality of the germ during storage, the decanal value has been claimed to be the best. Many methods such as dry or wet heat processing, or treatment with ethylene dichloride, epoxy compound, infrared heat, antioxidants, sodium chloride or skim milk powder, alkali, etc., or lowering the moisture content have been employed by several workers for stabilization of the germ. The principle governing the stabilization of the germ for improving its shelf life is the inactivation of enzymes. Of these methods, only heat processing appears to be practically feasible on a commercial scale. The stability of different nutrients, mainly vitamins, during storage is dependent on the processing methods employed for stabilization and the storage period.

VII. WHEAT GERM AND BREAD-MAKING QUALITY Exhaustive literature is available on the effect of the use of wheat germ in bread making. Several workers have drawn different conclusions about the beneficial as well as the adverse effects of the germ and its components on dough characteristics and bread-baking quality. Various methods to overcome the deleterious effects on baking quality have been suggested. A. EARLIER STUDIES The constituents in the germ responsible for deleterious or beneficial effects on bread-making quality have been studied by several workers. Consequently, different theories have been put forth to clarify the roles played by these constituents. Various treatments have been suggested to counteract or overcome the adverse effects. Some of the factors-germ constituents or processing conditions-possibly influencing baking quality are discussed below.

214

S. R. SHURPALEKAR AND P. HARIDAS RAO

1. Phosphatides

Geddes (1 930) reported that germ added to fifth-middlings flour significantly reduced its baking quality, as reflected by (1) the poor handling property of the dough, (2) poor loaf volume, and (3) poor crumb characteristics. Addition of bromate to the dough or heating the germ before blending i t with the flour reduced the deleterious effect of the germ. He suspected that oxidation of phosphatides present in the germ was the primary cause of such improvement. However, this was disproved later by Rich (1934), who reported that some germ constituents other than phosphatides were responsible for the adverse effect on baking quality.

2. Glutathione From farinograph data and baking trials using fresh as well as stored germ, Sullivan et al. (1936a,b) observed that some beneficial changes took place in the harmful constituents during storage of the germ (Table LXIII), They also found that the harmful constituent was water-soluble. Later, Sullivan el al. ( 1 9 3 6 ~ ) suggested that glutathione was probably the harmful constituent of the germ. By using the Sullivan test (Sullivan and Hess, 1931), they detected glutathione in the water extract of the germ. This was confirmed by their findings that incorporation of 60 mg of pure isolated glutathione or water extract from 10 gm of germ in the bread recipe had a similar adverse effect on the balung quality (Table LXIV). Bull (1 937) reported that baking quality was not impaired when the coagulable fractions were removed from the water extract of the germ by heating or dialyzing. According to Sullivan et al. (1 937), glutathione activated the proteolytic enzymes, which in turn influenced the gluten quality adversely. The beneficial effects of heat treatment of the germ or addition of oxidizing agents such as TABLE LXIII EFFECT OF GERM INCORPORATION ON BAKING QUALITY O F PATENT FLOUR"

Sample

Dough quality

Loaf volurneb

Patcnt flour (PF) PF + 10%fresh germ (untreated) PF + 10%fresh germ (ether-extracted) PF + 10%stored germC

Strong, elastic Very poor, soft, short, sticky Very poor, soft, short, sticky Poor, soft

100 61 62 66

'Sullivan el al. (1936b). bCalculated on the basis of 100 for bread loaf from 100 gm of patent flour containing 13% protein. CFor 10 months at 13% moisture.

215

WHEAT GERM TABLE LXIV

EFFECT OF GERM GLUTATHIONE ON BAKING QUALITY OF PATENT FLOUR"

Form of glutathione added

Dough quality

Loaf volumeb

Patent flour (PF) PF + water extract from 10%fresh wheat germ PF + 60 rng glutathione from germ PF + 60 mg glutathione from yeast PF + 10%fresh germ PF + 10%fresh germ + 60 mg potassium bromate

Strong and elastic Very poor, very soft Poor, soft, sticky Poor, soft and sticky Poor, soft, dead Fair plus

100 15 19 15 12

89

"Sullivan et al. (1936~). bCalculated on the basis of 100 for bread loaf from 100 gm of patent flour.

potassium bromate (Sullivan et ul., 1936c) or lactic acid (Zav'yalov, 1939, 1940) were found to overcome the adverse effects of the germ on baking quality. This was attributed to the oxidation of reduced glutathione to an oxidized form.

3. Fermented Germ Hullett and Stern (1941) found to their surprise that the harmful effects of the germ on baking quality were no longer observed when a pre-ferment consisting of wheat germ, water, and yeast was used in bread preparation. The reduced glutathione was not detected in the pre-ferment, as indicated by the sodium nitroprusside reaction. However, when boiled germ was used in the pre-ferment, reduced glutathione could be detected. These observations indicated that destruction of glutathione in the germ was connected with an enzyme mechanism effective in the raw germ and not in the boiled germ. Therefore, they did not believe that reduced glutathione (-SH) was converted to the oxidized form ( S : S ) during heating, as suggested by Sullivan et ul. (1937). Further, a minimum of 6 hours of pre-fermentation was required to overcome completely the deleterious effect of the germ on the baking quality. An increase in the temperature (up to 35'C) and in the quantity of yeast and a decrease in the initial pH to 5.0 to 5.5 shortened the pre-fermentation time required for destroying glutathione. Using blends of varying percentages of wheat germ and hard red spring wheat flour, Smith and Geddes (1942) made an interesting observation that the dough-handling qualities and loaf characteristics improved as the fermentation time increased from 1.5 to 4.5 hours. The inclusion of potassium bromate in the bread formulation brought about a spectacular improvement in both the doughhandling properties and the loaf characteristics. These workers also found an improvement in the baking quality when the germ was allowed to stand in an aqueous suspension containing 80 mg of potassium bromate per 100 gm of germ. On the other hand, pre-fermentation of the germ with yeast progressively

276

S. R. SHURPALEKAR AND P. HARIDAS RAO

decreased the harmful effect, depending on the time of fermentation up to 4.5 hours. Inclusion of potassium bromate in this pre-ferment further improved the baking behavior. However, the improvement was more prominent when bromate was added at the dough stage. Under optimum conditions of fermentation time and bromate level, bread baked from patent flour containing the germ was comparable to the quality of control bread based on patent flour alone. The higher efficiency of the bromate, when added to fermenting germ-flour dough rather than to the pre-ferment, indicated that the potassium bromate exerted a direct action on the gluten protein and not on the glutathione, as suggested by Sullivan et al. (1937). In contrast, Elion (1943) observed from his experiments that the beneficial effect of the oxidizing agents was due mainly to the oxidation of glutathione and not to their action on gluten. However, he also found an improvement in the baking quality of bread containing the germ with an increase in fermentation time. He confirmed the earlier findings (Smith and Geddes, 1942) regarding the deleterious effect of wheat germ which was attributed mainly to the activating effect of reduced glutathione on flour proteinases. Stern (1944) did not find any evidence to prove that the deleterious effect of germ glutathione on bread quality was due t o the activation of proteolytic enzymes in the flour. He suspected direct action of the glutathione on the gluten itself. The better characteristics of the dough made with fermented germ were explained by the absence of -SH groups. According to him, the enzyme responsible for the elimination of the -SH groups in fermenting dough was a dehydrogenase of the germ, the presence of which was demonstrated by them. The adverse effect of fermented wheat germ on bread quality, when used at hgh levels, was also reported by Greer et al. (1953). They observed the formation of an undesirable pink or reddish brown discoloration, both in the germ ferment and in the bread crumb, whenever fermentation of the germ was unduly prolonged. From fermented germ, they isolated methoxy-y-benzoquinone, which acted as a bread improver. They suspected that the improving action of the fermented germ was due to the benzoquinone derivative and not to the oxidation of glutathione, as earlier workers suggested (Hullett and Stern, 1941; Smith and Geddes, 1942). 4. Steeping of the G e m According to Grewe and LeClerc (1943), steeping the germ considerably improved its bread-malung properties. The beneficial effect of steeping increased progressively with time up to 8 hours. The addition of potassium bromate during steeping of the germ resulted in further improvement in bread quality. These findings were similar to the earlier observations of Hullett and Stern (1941). However, they inferred that steeping in water itself counteracted the adverse

WHEAT GERM

211

effect of glutathione, and the presence of yeast in the pre-ferment was not essential, as observed by Hullett and Stern (1941). Interestingly enough, Grewe and LeClerc (1943) found that addition of steeped germ at a level of 2.5% or 5% to flour produced bread that was even better than the control bread based on flour alone. Further, steeped germ up to 10% could be added to the flour dough, without any detrimental effect on the loaf quality. Use of even 15 to 20% levels of steeped germ produced acceptable bread, which was only slightly inferior to the control bread. Addition of salt (in amounts normally used in bread making) to the germ during steeping brought about an improvement in the handling property of the dough. This improvement was due to the change in the colloidal properties of the germ as measured by its viscosity. B. RECENT STUDIES In recent years only a few workers have studied the roles played by germ constituents, by the processing of the germ, and by additives in bread quality.

1. Effect of Processed Germ Pomeranz et al. (1970b) and Giacanelli (1973) have demonstrated qualitatively and quantitatively the effect of the inclusion of wheat germ components on bread quality. According to them, the unfavorable influence of certain components on the baking process could be eliminated by heat treatment. a. Processing of the Germ. Processing of the germ as carried out by Pomeranz et al. (1970b) consisted in the following operations: (1) heating the germ having 14.9% moisture for 8 hours at 80°C; (2) subsequent drying t o about 4% moisture; (3) extracting the lipids with petroleum ether; and (4) grinding the extracted germ in a Hobart mill and re-extracting with petroleum ether. b. Inactivation of Glutathione. The inactivation of glutathione (Pomeranz el al., 1970b) during heat treatment was found to be dependent on the temperature and moisture content of the germ (Fig. 14). For the germ containing 14.9% moisture, the inactivation time at 60°C was nearly four times that of the germ containing 23.7% moisture. However, this difference was less pronounced at higher temperatures of heat treatment. c. Effect on Dough Characteristics and Loaf Volume. The effect of the addition of raw and heat-treated germ on loaf volume (Pomeranz el al., 1970b) is shown in Fig. 15. The loaf volume decreased with the increase in levels of raw germ. The volume of the bread made by incorporating heat-treated germ was consistently higher than that of the bread containing raw germ. It was interesting to note that no decrease in loaf volume was observed even in bread containing

27 8

S. R. SHURPALEKAR AND P. HARIDAS RAO

c

900

950 -

--

E

850-

H E Y TREATED

4:-

5

CONTROL

z

60

80

0

100

5

-0

\

10

15

Germ level (%)

Temperature ("C)

FIG. 14 (left). The effect of temperature and moisture on inactivation time ofglutathione in the germ. FIG. 15 (right). The effect of the addition of raw and heat-treated germ on loaf volume.

15% of heat-treated germ. The addition of heat-treated germ to the flour also increased farinograph water absorption by approximately 1% per gram of germ. Further, mixing time decreased in germ-enriched doughs, depending on the level of germ addition. d, Effect of Bromate. In contrast to the requirement of only 10 ppm of bromate for the control bread, a very high level of 70 ppm was required in doughs containing 15% of heat-treated germ. The bromate requirement was even higher when raw germ was used. However, the addition of bromate did not eliminate completely the deleterious effect of raw germ. TABLE LXV EFFECT O F HEAT-TREATED GERM AND POLAR LIPIDS O F

WHEAT FLOUR O N LOAF VOLUME OF

BREAD^

~

Heat-treated germ (%o)

0

5 5 10

10 15 15 15 15 -~

~

'Pomeranz et al. (1970b).

Polar lipids (%)

Loaf volume (ml)

0 0 0.2 0 0.4 0 0.8 1.0 2.0

864 890 900 895 930 885 925 945 95 5

27 9

WHEAT GERM TABLE LXVI EFFECT OF LECITHIN ON LOAF VOLUME OF BREAD BAKED WITH HEAT-TREATED GERM'

Loaf volume (ml) of bread baked with lecithin levels of: Germ added (%)

0.0%

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%

4.0%

0 5 10 15 20 30

864 890 895 885 865 818

855 925 940 920

878 945 943 918 915 845

870 935 1010 940

865

855

865 985

-

-

-

-

915 -

900 855

820

-

-

-

-

-

973 975 920 840

-

940 -

6.0%

-

-

-

-

aPomeranz et al. (1970b).

e. Effect of Polar Lipids. The addition of free polar flour lipids was found to improve the loaf volume of bread containing the germ (Pomeranz et a l , 1970b), as seen in Table LXV. The increase in the loaf volume of bread containing 15% of heat-treated germ was dependent on the level of polar lipids added. On an equiprotein basis, the volume of bread baked with a sodium chloride extract of germ was higher than that of bread baked with heat-treated germ. A similar beneficial effect of polar lipids on the baking quality of wheat flour with or without the germ has been confirmed by Daftary et al. (1968) and by Bolling et al. (1973). As polar lipids contain both phospholipids (lecithin) and glycolipids, Pomeranz et al. (1970b) also studied the effect of incorporation of only phospholipids isolated from soybean on the loaf volume of germ bread. The addition of soya

750

9 I

1

0

1

Lecithin

2

3

(%I

FIG. 16. The effect of lecithin addition on loaf volume of bread baked with 0%, 3%, 6%, and 9%of a sodium chloride extract of germ products.

280

S. R. SHURPALEKAR AND P. HARIDAS RAO

TABLE LXVII EFFECT O F VARIOUS PHOSPHOLIPIDS O N LOAF VOLUME OF

BREAD‘

Loaf volume (ml) of bread bakedb with phospholipid levels of: Lipid

0%

None (control) Polar, weight flour Soya lecithin DL-cY-Lecithin D L-ol-Cephalin Phosphatidyl ethanolamine Phosphatidyl serine Inositol phosphatide

71

1%

1.5%

2%

81 72 83 71 81 74 86

78 75 86 70 84 72 86

79 75 88 72 86 75 90

‘Pomeranz et al. (1970b). bFrom 1 0 gm of flour and 1 gm of germ.

lecithin significantly improved the loaf volume of bread baked with up to 30% of heat-treated germ (Table LXVI). The optimum level of lecithin increased with an increase in the levels of germ added. In bread containing 10 to 30% of wheat germ, the loaf volume was the highest when the lecithin-to-germ ratio was between 1 : l O and 1.5:lO.O. The addition of a small amount of sucrose monomyristate was found to counteract the defect of coarse crumb structure, observed as a result of using lecithin in germ-enriched bread. The effect of lecithin levels on the loaf volume of bread baked with various levels of sodium chloride extract of germ is given in Fig. 16. Bread baked without any germ extract did not show any improvement on addition of lecithin. The effect of various types of synthetic phospholipids on the loaf volume of bread baked from 10 gm of flour and 1 gm of heat-treated germ is given in Table LXVII. DL-ar-Lecithin was found to be a better improver than soya lecithin. Synthetic DLff-cephalin (a phosphatidyl ethanolamine) had n o improving effect. In contrast, phosphatidyl ethanolamine of soybean was an excellent improver. Phosphatidyl serine had very little effect on the loaf volume of germ bread, whereas inositol phosphatide was found to be an excellent improver. These variations in the beneficial effect of phospholipids were suspected to be due to the difference in the fatty acid composition of various phospholipids.

2. Effect of Germ Lipids Bolling et ul. (1973) carried out experiments to investigate the contradictory conclusions regarding positive (Rohrlich and Schoenmann, 1962) and

WHEAT GERM

28 1

TABLE LXVIII E F F E C T O F WHEAT GERM A N D WHEAT GERM O I L O N L O A F VOLUME OF BREAD BY RAPID MIX T E S f

Level of addition Germ

Breadloafvolume(%)

Germ oil

Controlb

1%

2%

3%

4%

1%

2%

3%

4%

100

95

90

87

85

105

110

115

125

‘Bolling et al. (1973). bBread made of wheat flour without any added germ or germ oil.

negative (Chiu et al., 1968) roles played by the fat in the flour and by fat additives like phosphatides, galactolipids, and mono- and diglycerides in influencing the baking quality of wheat varieties. In order to study the changes brought about in the baking quality of wheat flour by the addition of wheat germ containing 9.1% fat, 21.8% protein, 40.0% starch, and 4.1% ash, the germ was added at 1%, 2%, 3%, and 4% levels. By the rapid mix test, Bolling et al. (1973) demonstrated that the increasing levels of wheat germ added brought about a decrease in the loaf volume, and addition of germ oil at comparable levels improved the loaf volume (Table LXVIII). They concluded, therefore, that the wheat germ oil played a positive role in improving the baking quality of wheat flour. However, the negative effect of nonfat components of wheat germ was higher than the positive effect of wheat germ oil. C. SUMMARY The studies of different workers have clearly shown that inclusion of raw wheat germ in the bread recipe has an adverse effect on the baking quality. Many workers have attributed this adverse effect to the germ constituent, glutathione. The effect of glutathione has been explained in two ways. Glutathione activates proteolytic enzymes, which in turn affect the gluten as well as the bread-making quality. Alternatively, glutathione weakens the gluten by reducing disulfide linkages to sulfhydryl groups. Addition of polar lipids or oxidizing agents like bromate, pre-fermentation or steeping of the germ, and a longer fermentation time brought an improvement in the quality of germ bread. Many workers have concluded that this beneficial effect is due to the oxidation of reduced glutathione (present in raw germ) to oxidized form. It has also been inferred that the improvement is due to the benzoquinone derivatives formed during fermentation. Heat treatment of the germ resulted in a similar improvement, which was attributed to the inactivation of the glutathione.

282

S. R. SHURPALEKAR AND P. HARIDAS RAO

VIII.

FOOD USES OF THE GERM

Until recently, wheat germ was almost wholly disposed of as an animal feed. However, after it was realized that wheat germ contains a significant quantity of proteins of superior nutritive value and vitamins, several workers investigated various aspects of its utilization as a human food. The unique characteristics of palatability and hgh nutritive value place wheat germ on a par with other foods based on animal proteins. Cirilli er al. (1971), from his analytical data on fifty different samples of germ, suggested that such a concentrated source of energy and nutrients as the germ should not go unutilized for human consumption. The addition of the germ to wheat flour would considerably improve its nutritive value. Later, during their studies on durum wheats, Cirilli et al. (1972) observed that durum semolina had a significantly lesser nutritive value, as a result of loss of the germ during milling and purification. They inferred that the nutritive value of milled products for human consumption would certainly increase if the germ could be retained during the milling operation. The poor stability of the germ had restricted its wide usage as a human food, however. But today, various methods are available for stabilization of the germ to prevent its deterioration during storage. Some of the important uses of the germ are discussed below. A. BAKERY AND PASTRY PRODUCTS Among bakery products, germ bread has been studied extensively. Information on other products is scanty.

1. G e m Bread

a. Formulation. A process for the preparation of bread containing 10% of germ has been described by Hullett and Stern (1941). Commercial germ samples from soft red winter or durum wheats were found to be better than samples from hard spring varieties (Grewe and LeClerc, 1943). Bruno (1935) suggested that, when flour is to be used within a short time after milling, the germ could be retained in the flour. If the flour is to be stored for longer duration, it is desirable to add freshly extracted germ, just before using the flour to make bread. Larousse and Blanchet (1971) demonstrated that bread containing 1.5% of stabilized and lyophilized germ is better than control bread. b. Nutritive Value, Bruno (1935) reported that bread containing the germ had better odor, flavor, digestibility, and biological value. After studying growth in rats, Cerquiglini (1938a,b) demonstrated that bread containing 2 to 5% of wheat germ was nutritionally superior to that made from the same flour without any germ. He further observed that, when fasting rats were fed, the gain in

WHEAT GERM

283

weight was 5% greater with germ bread than with the same quantity of control bread. Feeding tests carried out by Bachmann and Leusden (1939) on bread baked from a dough containing 22% of germ showed that as much as 95% of the protein, fat, and carbohydrate of the germ was utilized by the animals. Mice receiving this bread gained in weight and showed a lower incidence of disease and a much longer life span than those receiving bread without the germ. Only a slight, negative nitrogen balance was observed in human subjects fed on a diet consisting of germ bread during a 4-day test period. Gontzea el al. (1970) reported that bread containing wheat germ from which the fat was extracted was definitely better than bread from the same wheat flour enriched with gluten. According to them, the gain in weight, the consumption index, the protein efficiency ratio, the ratio of total nitrogen to creatine nitrogen, the amount of xanthine oxidase of the liver, and the formation of new liver proteins were significantly higher in rats fed on germ bread. Steiger (1973) has reported that wheat germ bread represents special bread enriched with important vitamins. Especially vitamins A and E and the B-group vitamins are present in wheat germ in a well-balanced form, and their beneficial effects have been demonstrated by him. Zaitsev el al. (1974) have observed that in bread made from first grade flour enriched with 5% wheat germ the amounts of protein, lysine and essential amino acids were higher by 4, 17, and 6%, respectively, as compared to the control. In addition, the enriched bread had better taste with pronounced flavor. Use of 1.5% freeze-dried germ has been reported to bring about considerable improvement in flavor as well as yield of bread (Larouse and Blanchat, 1971). c. Speciality Breads. Kent-Jones and Mitchell (1962) suggested that, for a bread to be labeled germ bread, it must contain at least 10%(calculated on a dry-weight basis) of processed wheat germ. Rohrlich and Bruckner (1967) TABLE LXIX CHARACTERISTICS OF COMMERCIALGERM BREADS"

Ingredients Germ meal (Ib) Water (qt) Yeast (02) Salt (02) Process conditions Fermentation period (hr) Proofing time (min) 'Bennion (1967).

Hovis

Daren

Vitbe

14 4 4

14 4 4.5 -

14 4 4.5 -

14

0.5 -

Nil -

2.25 45

-

Nil 30

Turog

4 2 3

284

S. R. SHURPALEKAR AND P. HARIDAS RAO

reported that at least 8 to 10% of germ was required to effectively improve the nutritional value of bread. The best-known types of commercially produced germ bread are Hovis, Daren, Vitbe, and Turog. These breads are prepared by using different germ meal formulations, and they contain the germ cooked with known amounts of salt, soy flour, and wheat flour. Only Turog bread is quite different. It contains smaller percentages of the germ together with caramel, which imparts a characteristic color to the bread. The ingredients for preparation of these bread are summarized in Table LXIX.

2. Biscuits and Cakes Hertwig (1931) reported that biscuits and cakes made from self-rising flour containing toasted germ were highly acceptable and were comparable to products containing no germ. The incorporation of 12 to 15% of toasted germ in the self-rising flour was found to be optimum. The recipe proposed for self-rising flour was: patent flour, 850 gm; toasted germ, 125 to 150 gm; baking soda, 15 gm; sodium acid pyrophosphate, 16.8 gm; calcium acid phosphate, 8.1 gm; common salt, 16 to 18 gm; and dextrose, 15 gm. The keeping quality of this mix was found to be excellent, as no off-flavor developed during storage at 120" to 130°F for 25 days in a closed jar. The authors have also found that up to 15% of toasted germ could be used in biscuits without any adverse effect on its quality. Larousse and Blanchet (1971) reported an improvement in the quality of biscuits when only 0.25% of stabilized and lyophilized wheat germ was included in the biscuit recipe. According to Araki (1971), deterioration of sponge cake can be prevented by incorporation of wheat germ.

3. Pastry Products Wheat germ has aiso been used as one of the optional ingredients for the enrichment of noodles or macaroni products (Federal Register, 1946). In addition to improving the nutritive value, the addition of wheat germ to the pastry products was reported to reduce the cost of the product. B. SUPPLEMENT FOR CEREALS Because of its composition, wheat germ can be used as an effective supplement for improving the nutritive value of cereals, which are known to be nutritionally inferior. The beneficial effect of supplementing with wheat germ on the nutritive value of wheat flour has been already reported. Supplementing cereals like wheat, rice, barley, and oats with 15% of defatted germ significantly improved their nutritive value by 31 to 69%, as shown by rat growth experiment (Rand

WHEAT GERM

285

and Collins, 1958). It has also been suggested that these enriched cereals can be used for the preparation of breakfast foods. C. GERMOIL Wheat germ is a good source of edible salad oil which is classified as a semidrying oil (Lewkowitsch, 1915). In view of the shortage and high cost of edible oils in many developing regions, the extraction of oil from wheat germ will go a long way in augmenting the supplies of edible oils. In addition to its use in the preparation of food products and vitamin concentrates, germ oil is also used in cosmetics (Jagbir Singh, 1973). Wheat germ oil has the potential for use in the preparation of margarine (Netherlands Patent Application, 1970). DeJonge and Erkelens (1969) described a method of preparing edible oil from wheat germ by selective hydrogenation. This oil has a better keeping quality than unhydrogenated wheat germ oil. The hydrogenated oil was found to be suitable for use in bakery products. D. FERMENTED FOODS lwata et al. (1952, 1953) showed that wheat germ can be used in the preparation of miso and koji. Germ miso was prepared by replacing portions of rice and soybean with germ. The chemical characteristics as well as the organoleptic qualities of germ miso were similar to those of miso prepared from soybeans. Koji was prepared from wheat germ by inoculating with Aspergillus oryzae. E. VITAMIN CONCENTRATES Different methods have been described for separating vitamin E or vitamin B complex concentrates from the germ. The presence of high amount of vitamins in the germ, particularly vitamin E, thiamine, riboflavin, and pyridoxine, makes it a very suitable raw material for the enrichment or preparation of vitamin concentrates. The multiple uses of wheat germ suggested by Devyatnin (1944) included the preparation of (1) pyridoxine concentrate, (2) concentrate of the B-group vitamins, and (3) thiamine concentrate from the water-soluble fractions; and the preparation of (4) vitamin E concentrate, (5) sitosterol concentrate, (6) food fat, and (7) raw fat from the fat-soluble fraction.

1. Vitamin E Concentrate The method as described by Evans et al. (1935) involved the extraction of oil with methanol and the separation of the unsaponifiable matter containing

286

S. R. SHURPALEKAR AND P. HARIDAS RAO

vitamin E by using ethyl ether. Borodina (1959) has suggested a method for the purification of vitamin E. Niewiadomski et al. (1961) reported that extraction of vitamin E from germ oil with acetone containing 2%, 5%, and 10%water yielded concentrates containing 0.520%, 0.642%, and 0.7 12% of vitamin E, respectively. Increasing the ratio of acetone to oil from 1 : l to 5:l yielded a concentrate containing about 1% of tocopherols. Popova and Kirova (1964) used alcohol extraction to recover a concentrate containing vitamin E and sugars which has been used in pharmaceutical as well as in food industries. 2. Concentrates of B and E Vitamins Several products like vitamin E-enriched oil, sterols, lecithin, B vitamins, and sugars have been prepared by McFarlane (1950) from alcohol extracts of wheat germ according to a patented process. The residue meal, after separation of the vitamins, served as a protein concentrate for food uses. The preparation, composition, and processing of polyvitamin concentrate from wheat germ has also been reported by Neumann (1952). Ciupercescu (1958) has described methods for the preparation of concentrates of vitamin E and the B vitamins by ethanol extraction of the germ.

F. ANIMAL FEEDS The by-products of the roller flour milling industry in general form very useful and popular constituents of animal feeds. Wheat germ is mostly used as a feed for pigs, poultry, and cattle, since it is rich in protein and thiamine. Lewis and Weisberg (1952) developed a well-balanced ration for poultry using whey solids, buttermilk, and wheat germ. Iwata et al. (1955b) showed that, when compared with wheat and rice bran, wheat germ was a superior cattle feed in increasing the milk yield. The butter of these cows had a better storage life, possibly owing t o the antioxidant effect of tocopherols. Soloveichik (1946) patented a process for the preparation of a feed concentrate containing the B-group vitamins. The mixture of wheat germ, rice bran, and rice hulls was extracted with acidulated water containing alcohol and chloroform. The extract was neutralized with chalk and filtered. The filtrate was then concentrated to a syrupy consistency.

G. SUMMARY From the studies reviewed, it is evident that utilization of wheat germ for food uses has yet to find commercial application even in technologically advanced countries. Most of the work reported has been primarily of laboratory interest. However, wheat germ, which can be recovered as a by-product of the

WHEAT GERM

287

flour milling industry, has great potential as a food supplement and can be used to enhance the nutritive value of many products. In many developing regions there is considerable scope for using wheat germ in commercially produced bakery products, mainly bread and biscuits. Preparation of vitamin E concentrate has probably had a wider application in the pharmaceutical industry. In view of its unique taste as well as make-up of nutrients like protein, amino acids, fats and vitamins, utilization of the germ in feeding the human population deserves more attention, especially in the developing countries.

IX. RESEARCH NEEDS The foregoing account describes studies already carried out by several workers doing research on various aspects relating to the structure, separation, stabilization, chemical composition, and nutritive value of wheat germ with special reference to its effect on the baking quality of wheat flour and food uses. However, certain other aspects of these studies have yet t o receive adequate attention. 1. The recovery of less than 1% germ during milling as compared to 2.5 to 3.0% present in the wheat grain, is rather low. Such a situation may be attributed to the fact that in technologically advanced countries better and cheaper sources of proteins and fat are available. This means that, even today, the major portion of the germ utilized as animal feed becomes unavailable for the population of developing regions, where protein and calorie malnutrition is widely prevalent. It has probably not been considered necessary to extend milling research on recovering the remaining 1.5 to 2.0% of highly nutritive wheat germ. It must be recognized, however, that in developing countries, in spite of the ever-increasing need for proteins and edible oils due to the explosive rise in the population, the required technological base and facilities for improving milling efficiency are lacking. As a result, the developing regions, accounting for nearly one-third of the world’s wheat production, are unable to utilize 2.8 million tons of potentially available proteins and fat-rich material of high nutritive value for human consumption. Further research for improving the milling efficiency should logically come from the technologically advanced regions, which can more readily afford the required inputs and infrastructure in this direction. 2. No milling techniques have been developed to manufacture degermed wheat. This aspect has considerable relevance for wheat varieties that are not used for bread making. Also, a significant portion of the total world wheat produced is not milled in roller flour mills. It is milled only in small units, such as hammer mills or motor-driven mills using grinding stones. The brown flour milled therefrom at very high extraction rate (about 90% or more) is utilized in

288

S. R. SHURPALEKAR A N D P. HARIDAS RAO

traditional foods like chapatti, roti, etc., especially in India and Pakistan. Such a situation calls for urgent action in developing suitable degerming devices which can be attached as accessories to local grinding mills having small capacities of 100 kg per hour. Separation of the germ prior to grinding or during grinding will be helpful in avoiding the loss of the precious nutrients entering the bran portion of whole wheat flour, which is often sieved off before the flour is used in traditional food preparations like chapatis. 3. In spite of the exhaustive literature available on the chemical composition of the germ, very few data have been reported on the chemical composition of the germ from several varieties of wheats grown under different agroclimatic conditions. Such information is likely to be useful for separation of the germ on a commercial scale. In the last decade or so, more and more hybrid varieties of wheat have been cultivated in many regions of the world. However, very few studies have been directed toward an understanding of the effect of hybridization on the structure, chemical composition, and nutritive value of wheat germ and its effect on baking quality. This area of research should be covered more adequately in the coming years. 4. Although the majority of the studies carried out reveal the beneficial effect of heat processing on the nutritive value of the germ, the deleterious effect of heat processing is still being demonstrated by some workers. It is therefore desirable to come to a definite conclusion on the basis of which suitable processing techniques can be decisively worked out to improve the quality of the germ. 5. Most of the processes commercially feasible for the stabilization of wheat germ are patented ones. It may be necessary t o develop efficient processing techniques and suitable equipment of different capacities at moderate cost. This will be especially helpful in processing wheat germ in roller flour mills in whatever quantities it becomes available. An alternative solution would be to transport the wheat germ efficiently from individual flour mills to a central place where it could be conveniently processed for stabilization on a large scale. 6. The adverse effects of the addition of germ on the bread-making quality of wheat flour have been demonstrated clearly by several workers. However, it is interesting to note that the studies on different causes for these adverse effects do not appear t o be conclusive. Further, the remedial measures suggested for correcting these defects and improving the quality of germ bread appear to be inadequate, uneconomical, and to some extent not practicable. Thus, there is still considerable scope for working out a practical and feasible methodology to overcome these defects in germ bread, 7. Most of the work on utilization of the germ has been in the area of bread making. These studies should be extended to other food products as well as to

WHEAT GERM

289

traditional food items, which account for a significant portion of the total wheat consumption. 8. Today, triticale, the first man-made cereal, seems to be one of the promising food crops, especially in the developing regions with limited supplies of irrigation, power, fertilizers, agricultural machineries, etc. Although some work on triticale has been discussed at recent symposia (Tsen, 1974; MacIntyre and Campbell, 1974), no specific investigations have so far been reported on the structure, chemical composition, nutritive value, and separation of triticale germ. Also, its adaptability to processing and usage in food products will have to be studied in the context of different dietary patterns prevailing in various regions of the world.

REFERENCES Albizatti, C. M. 1937. La presencia del glutatione en el germen del trigo y su influencia en las harinas. An. SOC.Cient. Argent. 124, 194. Anderson, R. J., Shriner, R. L., and Burr, G. 0. 1926. The phytosterols of wheat germ oil. J. Am. Chem. SOC.48, 2987. Andrews, J. S., and Bailey, C. H. 1932. Distribution of organic phosphorus in wheat. Ind. Eng. Chem. 24, 80. Andrews, J. S., and Felt, C. 1941. The iron content of cereals. Cereal Chem 18, 819. Andrews, J. S., and Viser, E. T. 1951. The oxalic acid content of some common foods. Food Res. 16, 306. Andrews, S. 1942. Address before the Minnesota Institute of Cereal Chemistry (cited in Bailey, 1944). Ankel, H., and Feingold, D. S. 1965. Biosynthesis of uridine diphosphate D-xylose 1-uridine diphosphate glucuronate carboxylyase of wheat germ. Biochemistry 4, 2468. Araki, J. 1971. Cake taste improvement. Japanese Patent 25,705 /71. Attia, F., and Creek, R. D. 1965. Studies on raw and heated wheat germ for young chicks. Cereal Chem. 42, 494. Avery, G . S., Jr. 1930. Comparative anatomy and morphology of embryos and seedling of maize, oats, and wheat. Bot. Gaz. (Chicago) 89, 1. Bachmann, W., and Leusden, F. P. 1939. Bread containing wheat germ, its composition and biological value. Z. hyg. Infektionskr. 121,506, Chem Ahstr. 34, 5120 (1940). Bailey, C H. 1938. Germ content of American wheats. Cereal Chem. IS, 102. Bailey, C. H. 1944. “The Constituents of Wheat and Wheat Products.” Van Nostrand-Reinhold, Princeton, New Jersey. Bakke, A., Aschehoug, M. V., and Zbinden, C. 1930. A new factor in nutrition. C R. Hehd. Seances Acad. Sci. 191, 1157;Chem Ahstr. 25,989 (1931). Ball, C . D., Jr. 1926. A study of wheat oil. Cereal Chem. 3, 19. Balls, A. K., and Hale, W. S. 1936. Proteolytic enzymes of flour. Cereal Chem 13,54. Barnes, H. M. 1948. 5-Pentadecylresorcinol as an oxidation inhibitor of glycerides. U.S. Patent 2,448,207; Chem Abstr. 43, 884 (1949). Barnett, R. C., Stafford, W. A., Conn, E. E., and Vennesland, B. 1953. Phosphogluconic dehydrogenase in higher plants. Plant Physiol. 28, 115.

290

S. R. SHURPALEKAR AND P. HARIDAS RAO

Barton-Wright, E. C. 1938. Observations of the nature of the lipids of wheat flour, germ and bran. Cereal Chem. 15, 723. Barton-Wright, E. C. 1944. The microbiological assay of nicotinic acid in cereals and other products. Biochem J. 38, 314. Barton-Wright, E. C., and Moran, T. 1946. Microbiological assay of amino acids. 11. The distribution of amino acids in the wheat grain. Analyst (London) 71, 278. Bayley, H. S., Summers, J. D., and Slinger, S. J. 1968. Effect of heat treatment on the metabolizable energy value of wheat germ meal and other wheat milling byproducts. Cereal Chem. 45, 557. Beeson, W. M., Lehrer, W. P., Jr., and Woods, E. 1947. Peas supplemented with wheat germ as a source of protein for growth. J. Nutr. 34,587. Bennion, E. B. 1967. “Breadmaking, its Principles and Practice,” 4th ed. Oxford uliv. Press, London and New York. Bertrand, G., and Levy, G. 1931. Recherches sur la teneur des plantes, et notamment des plantes alimentaires, en aluminum. Bull. SOC.Chim 49, 1417. Blain, J. A., and Todd, J. P. 1955. The lipoxidase activity of wheat. J. Sci. Food Agric. 6 , 471. Block, R. J., and Bolling, D. 1951. Amino acid composition of proteins and foods. “Analytical Method and Results,” 2nd ed. Charles C Thomas, Springfield, Illinois. Block, R. J., and Mitchell, H. H. 1946. The correlation of the amino acid composition of proteins with their nutritive value. Nutr. Abstr. Rev. 16, 249. Boas-Fixsen, M. A., and Jackson, H. M. 1932. Biological value of proeins of wheat corn and milk. Biochem. J. 26, 1923. Bolling, H., El Baya, A. W., and Zwingelberg, H. 1973. Germ fat and baking quality of wheat. Getreide, Mehl Brot. 27, 92. Booth, R. G., Carter, R. H., Jones, C. R., and Moran, T. 1941. The nation’s food. 2. Cereals as food. Chemistry of wheat and wheal products. Chem. Ind. (London) 60, 903. Borodina, Z. V., 1959. Recovery of natural antioxidants from the wastes of the food industry. Sb. Nauchn. Rub., Leningr. Inst. Sov. Torg. No. 15, pp. 33-46. Chem Abstr. 55,202411 (1961). Bradbury, D., Cull, I. M., and MacMasters, M. M. 1956a. Structure of mature wheat kernel. I. Gross anatomy and relationship of parts. Cereal Chem. 33, 329. Bradbury, D., MacMasters, M. M., and Cull, I. M. 1956b. Structure of mature wheat kernel. 11. Microscopic structure of pericarp seed coat and other coverings of the endosperm and germ of hard red winter wheat. Cereal Chem 33, 342. Bradbury, D., MacMasters, M. M., and Cull, 1. M. 1956b. Structure of mature wheat kernael. 111. Microscopic structure of endosperm of hard red winter wheat. Cereal Chem. 33, 361. Bradbury, D., MacMasters, M. M., and Cull, I. M. 1956d. Structure of mature wheat kernal. IV. Microscopic structure of germ of hard red winter wheat. @real Chem. 33, 373. Brady, C. J. 1964. Lipid bound acids in bread bean leaves. Biochem J. 91, 105. Brambila, S., Nesheim, M. C., and Hill, F. W. 1961. Effect of trypsin supplementations on utilization of chick of diet containing raw soyabean oil meal. J. Nurr. 75,12. Bressani, R., and Elias, L. G. 1968. Processed vegetable protein mixtures for human conbumption in developing countries. Adv. Food Res. 16, 1. Brouillard, J., and Ouellet, L. 1965. Acid phosphatases of wheat germ; chromatographic analysis. Can. J. Biochem. 43, 1899. Bruere, P. 1934. Taux et repartition du manganese dans le grain de ble. C. R. Hebd. Seances Acad. Sci. 198, 504.

WHEAT GERM

29 1

Bruno, A. 1935. Wheat germ and quality of the flour. C. R. Hebd. Seances Acad. Agric. Fr. 21,281-2; Chem. Abstr. 29,4840G (1935). Bull, J. E. 1937. An investigation into the cause of the degrading effect of wheat germ on the baking quality of flour. Cereal Chem 14, 244. Calhoun, W. K., Bechtel, W. G., and Bradley, W. B. 1958. The vitamin content of wheat, flour and bread. Cereal Chem. 35, 350. Calhoun, W. K., Hepburn, F. N., and Bradley, W. R. 1960. The distribution of vitamins of wheat in commercial mill products. Cereal Chem 37, 755. Calvery, H. O., and Remson, D. B. 1927. The nucleotide of triticonucleic acid. J. Biol. Chem 73,593-597. Carter, J. E., and Pace, J. 1964. Distribution of dehydroascorbic acid reductase in the wheat grain. Nature (London) 201, 503. Castaner, A. E. G., and Hassid, W. Z. 1965. Properties of uridine diphosphate Dglucuronic acid decarboxylase from wheat germ. Arch. Eiochem Eiophys. 110,462. Cave, N. A. G., Summers, J. D., Slinger, S. J., and Ashton, G . C. 1965. The nutritional value of wheat milling byproducts for the growing chicks. 11. Evaluation of protein. Cereal Chem. 42,533. Cerquiglini, S. 1938a. The nutritive value of bread which contains definite amounts of the wheat germ. Quad. Nutr. 5 , 1-5; Chem Abstr. 34,5127 (1940). Cerquiglini, S. 1938b. The nutritive value (for growth) of bread containing the wheat germ. Quad. Nutr. 5,355; Chem Abstr. 34,7363 (1940). Channon, H. J., and Forster, C. A. M. 1934. The phosphatides of the wheat germ. Biochem J. 28,853. Cheng, Y. Y., Linko, P., and Milner, M. 1960. Glutamic acid decarboxylase in wheat embryos. Plant Physiol. 35,68. Chick, H., Boas-Fixsen, M. A,, Hutchinson, J. C., and Jackson, H. M. 1935. The biological value of proteins. VI. Biochem J. 29, 1712. Chiu, C. M., Pomeranz, Y.,Shogren, M.,and Finney, K. F. 1968. Lipid binding in wheat flours varying in bread making potential. Food Technol. 22, 1157. Cirilli, G., Ghedini, G., and Rocchi, R. 1971. Chemical and nutritional characteristics of soft wheat. Miillerei 24, 574. Cirilli, G., Rocchi, R., and Ghedini, C. 1972. Chemical and nutritional characteristics of durum wheat. Miillerei 25, 278. Ciupercescu, V. 1958. Vitamin concentrates from wheat germ. Lucrar. Inst. Cercet. Aliment. 2,121: Chem Abstr. 52,166311 (1958). Clark, E. P., and Schryver, S. B. 1917. The preparation of plant nucleic acids. Biochem. J. 11, 319. Clegg, K. M., and Hinton, J. J. C. 1958. The microbiological determination of vitamin B6 in wheaten flour, and in fractions of the wheat grain. J. Sci. Food Agric. 9, 717. Clum, H. H., and Nason, A. 1958. Triphosphopyridine nucleotide diaphorase from wheat germ. Plant Physiol. 33, 354. Colin, H., and Belval, H. 1933. La raffinose dans les c6rdales. C.R. Hebd. Seances Acad. Sci. 196, 1825. Colin, H., and Belval, H. 1935. Les glucides de la farine et de la p8te. C. R . Hebd. Seances Acad. Sci. 200, 2032. Conn, E. E., Kraemer, L. M., Liu, P. N., and Vennesland, B. 1952. The aerobic oxidation of reduced triphosphopyridine nucleotide by a wheat germ enzyme system. J. Eiol. Chem. 194, 143. Crampton, E. W., and Ashton, G. C. 1943. The role of germ in the nutritive properties of the cereal grains. Sci Agric. 23,445; Chem Abstr. 37,4442 (1943).

292

S. R. SHURPALEKAR AND P. HARIDAS RAO

Creek, R. D. 1955. Manganese in chickcn nutrition. Ph.D. Thesis, Purdue University, Lafayette, Indiana. Creek, R. D., and Vasaitis, V. 1962. Detection of an antiproteolytic substance in raw wheat germ. Poult. Sci 41, 1351. Creek, R. D., Vasaits, V., and George S. 1961. The improvement of the nutritive value of raw wheat germ meal by autoclaving. Poult. Sci. 40, 822. Creek, R. D., Vasaitis, V., Pollard, W. O., and Schumaier, G . 1962. Evidence for the presence of a thermolabile growth inhibitor in raw wheat germ. Poult. Sci. 41, 901. Cuendet, L. S., Elof, L., Norris, C. G., and Geddes, W. F. 1954. The influence of moisture content and other factor on the stability of wheat flours at 37.8"C. Cereal Chem. 31, 362. Czerniejewski, C. P., Shank, C. W., Bechtel, W. G., and Bradley, W. B. 1964. The minerals of wheat flour and bread. Cereal Chem. 41, 65. Daftary, R. D., Pomeranz, Y., Shogren, M., and Finney, K. F. 1968. Functional breadmaking properties of wheat flour lipids. 2. The role of flour lipid fractions in breadmaking. Food. Technol. 22, 79. Daniels, D. G. H. 1959. The estimation and location of methoxyhydroquinone glycosides in the wheat grain. Cereal Chem. 36, 32. Daniels, D. G. H., and Martin, H. F. 1967. Antioxidants in oats. Monoesters of caffeic and ferulic acids. J. Sci. Food Agric. 18, 589. Danielson, C. E. 1949. Seed globulins of the gramineae and leguminosae. Biochem J. 44, 387. DeJonge, A., and Erkelens, J. 1969. Methods for selective hydrogenation of oils containing linoleic acid and higher unsaturated acids. West German Patent Application 1,467,539. Devyatnin, V. A. 1944. The utilization of wheat embryos. Pishch. Prorn-st.SSSR NO. 9, p. 8. Dimitrov, N. 1975. Effect of some milling machinery characteristics on quality and quantity of wheat embryo fraction. Khranit. Prom. 24,6. Dimitrov, N., Oreskov, I., and Tenovaki, 0. 1972. Development procedure for manufacture of wheat germ. Khran. Prom 21, 20. Dirks, B. M., Boyer, P. D., and Geddes, W. F. 1955. Some properties of fungal lipases and their significance in stored grain. Cereal Chem. 32, 356. Donk, P. J., and MacDonald, A. R. 1935. Preserving wheat germ. British Patent 436,765; Chem Absrr. 30, 1890 (1936). Donk, P. J., and MacDonald, A. R. 1937. Stabilized wheat germ product for use in the manufacture of bakery products. U.S. Patent 2,085,421; Chem. Absrr. 31, 5894 (1937). Drummond, J. C., Singer, E., and MacWalter, R. S. 1935. A study of the unsaponifiable fraction of wheat germ oil with special reference to vitamin E. Biochem. J. 29, 456. Dubois, M., Gilles, K., Hamilton, J. K., Rebers, P. A., and Smith, F. 1951. A colorimetric method for the determination of sugars. Nature (London) 168, 167. Dubois, M., Geddes, W. F., and Smith, F. 1960. The carbohydrates of the Cramineae. X. A quantitative study of the carbohydrates of wheat germ. Cereal Chem 37, 557. Dunn, M. S., and McClure, L. E. 1950. The response of Lactobacillus casei to partial hydrolysate of protein. J. Biol. Chem 184, 223. Eggitt, P. W. R., and Norris, F. W. 1955. The chemical estimation of vitamin-E activity in cereal products. 111. The application of partition chromatography t o the isolation of €-tocopherol from bran and to the determination of the individual tocopherols of cereals. J. Sci Food Agric. 6, 689. Eggitt, P. W. R., and Ward, L. W. 1955. The chemical estimation of vitamin E activity in

WHEAT GERM

29 3

cereal products. 11. Millers’ offals and compound animal foods. J. Sci. Food Agric. 6, 329. Elion, E. 1943. Action of glutathione and wheat germ on dough in relation to proteolytic enzymes in wheat flour. Cereal Chem 20,234. Ellis, M. T. 1918. Contributions to our knowledge of the plant sterols. Part 1. The sterol content of wheat (Triticum sativum). Biochem. J. 12, 160. Elvehjem, C. A., and Hart, E. B. 1929. The copper content of feeding stuffs. J. Biol. Chem 82, 473. Engel, C. 1942. The tocopherol (vitamin E) content of milling products from wheat, rye and barley and the influence of bleaching. Z. Vitaminforsch. 12, 220. Engel, C. 1945. Distribution of amylase, proteinase and esterase in resting cereals. Recl. Trav. Chim Pays-Bas 64, 318. Engel, C. 1947. The distribution of the enzymes in resting cereals. 111. The distribution of esterase in wheat, rye and barley. Biochim. Biophys. Acta 1,278. Engel, C. 1949. Vitamin E in human nutrition. Ann. N. Y. Acad. Sci. 52, 292. Engel, C., and Heins, J. 1947. The distribution of the enzymes in resting cereals. 11. The distribution of the proteolytic enzymes in wheat, rye and barley. Biochim Biophys. Acta 1, 190. Engel, R. W. 1943. The choline content of animal and plant products. J. Nutr. 25,441. Evans, H. M., Murphy, E. A., Archibald, R. C., and Cornish, R. E. 1935. Preparation and properties of vitamin E concentrates. J. Biol. Chern 108, 515. Fairclough, B. 1947. “A Microscopical Examination of the Wheat Grain,’’ Tech. Educ. Ser., Pamphl. No. 14, 2nd ed. National Joint Industrial Council for the Milling Industry, London. Famiani, V. 1932. The nutritive value of wheat germ. Atti Accad. Naz. Lincei, Cl. Sci. Fis.. Mat. Natl., Rend. [6] 16,275; Chem Abstr. 21, 2178 (1933). FAO. 1972. “Production Year Book,” Vol. 26, p. 51. FAO, Rome. FAO/WHO Joint Expert Group. 1965. “Protein Requirements,” F A 0 Nutr. Meet. Rep. Ser. No. 37 and WHO Tech. Rep. Ser. No. 301. FAO, Rome. Farrel, E. P., Ward, A. B., Miller, G. D., and Lovett, L. A. 1967. Extensive analyses of flours and millfeeds made from nine different wheat mixes. 1. Amounts and analyses. Cereal Chem 44.39. Federal Register. (1946). Alimentery pastes. Fed. Regist. 11, 7520; Chem Abstr. 40, 7435 (1946). Fernholz, E., and MacPhillamy, H. B. 1941. Isolation of a new phytosterol: Campesterol. J. Am. Chem. Soc. 63, 1155. Fcrrigan, M., and Geddes, W. F. 1958. The distribution of lipase in the commercial mill products from hard red spring wheat. Cereal Chem 35, 422. Fink, A. L., and Hay, G. W. 1969. Enzymic deacylation of esterified mono- and disac charides. I. Isolation and pruification of an esterase from wheat germ lipase. Can. J. Biochem 47, 135. Fitfield, C. C., and Bailey, C. H. 1929. The march of acidity in stored flours. Cereal Chem 6, 530. Fournier, P. 1946. La repartition de la vitamine B1 dans le grain de bld. Bull. Soc. Sci. Hyg. Aliment. Aliment. Ration. [N. S.] 34, 155. Frankfurt, S. 1896. Zur Kenntnis der chemischen Zusammensetzung des ruhenden Keims von Mticum vulgare. Londwirtsch. Vers.-Stn. 41, 449. Franzke, CI. 1956. Iodometric method for determining the peroxide value of food fats. Z. Lebensm-Untersfersch. 103, 108.

294

S. R. SHURPALEKAR AND P. HARIDAS RAO

Fraser, J . R., and Holmes, D. C. 1957. Proximate analysis of wheat flour carbohydrates. 111. Estimation of the hemicellulose fraction. J. Sci. Food A@. 8, 715. Fraser, J. R., and Holmes, D. C. 1959. Proxiniate analysis of wheat flour carbohydrates. IV. Analysis of wholemeal flour and some of its fractions. J. Sci. Food Agric. 10, 506. Galle, K. L. 1974. Stabilized wheat germ. U.S. Patent 3,783,164. Garcia, W. J., Blessin, C. W., and Inglett, G. F.. 1972a. Mineral constituents in corn and wheat germ by atomic absorption spectroscopy. Cereal Chrin. 49, 158. Garcia, W. J., Gardner, 11. W., Cavius, J. F., Stringfellow, A. C., Blessin, C . W., and Inglitt, G. F. 1972b. Composition of air classified defatted corn and wheat germ flours. Cereal Chem. 49, 499. Gassner, G. 195 1. “Mikroscopische Untersuchung pflanzlicher Nahrungs und Genussmittel,” 2nd ed. Fischer, Jena. Gaver, K. M. 1962. Wheat germ processes. US. Patent 3,046,142. Geddes, W. F. 1930. Chemical and physicwchemical changes in wheat products induced by elevated temperatures. 111. The influence of germ constituents on baking quality and their relation to improvement in flour induced by heat and chemical improvers. Can. J. Res. 2, 195. Geoffroy, R. 1934. Etudes des lipides de quelques farines industrielles Graminees et de Legumineuses. Relations des lipides du froment avee la valeur boulanghe. Bull. SOC. Bor. Fr. 81, 17. Giacanelli, E. 1973. Pane a1 germe (Weizenkeimbrot). Tech. Molit. 24, 8-95. Giovanelli, J. 1966. Oxalyl coenzyme A synthetase from pea seeds. Biochim Biophys. Acta 118, 124. Girard, A., and Fleurent, E. 1899. La composition des blds tendres franpis et dtrangers. Bull. Minist. Agric.. Paris 6, 1032. Click, D. 1945. The choline content of pure varities of wheat, oats, barley, flax, soybeans and milled fractions of wheat. Cereal Chein. 22, 95. Gontzea, I., Sutzesco, P., and Popesco, F. 1970. L’influence du taux d‘extraction sur la valeur nutritive des farines de bld et la possibilitd de son anidlioration par I’incorporation de germes ddlipides. Vitalst. Zivilisationskr. 15, 63-66. Grandel, F. 1959. Debittering of cereal seed germs. U.S. Patent 2,879,167; Chem Abstr. 53,9514b (1959). Gray, J. A., Hill, D. C., and Branion, H. D. 1957. Effect of heat on lysine content of rapeseed. Poult. Sci. 36, 1 1 93. Green, J., Marcinkiewicz, S., and Walt, P. R. 1955. Determination of tocopherols by paper chromatography. J. Sci. Food Agric. 6 , 274. Greer, E. N., Halton, P., Hutchinson, J . B., and Moran, T. 1953. Effects of methoxy-p. benzoquinone and some related compounds on bread quality. J. Sci. Food Agric. 4, 34. Grewe, E., and LeClerc, J. A. 1943. Commercial wheat germ, its composition. Cereal Chem 20, 423. Grischenko, A. P. 1935. Das Verhaltnis der Bestandteile des Weizenkornes und ihr Aschegehalt. Muehlenlaboratorium 5 , 182. Guerithault, B. 1927. Investigation into the presence of copper in plants and in the human organism in a normal and pathological (cancerous) state. Bull. SOC.Sci. f f y g . Aliment. Aliment. Ration. [N.S.] 15, 386; Chem. Abstr. 22, 809 (1928). Hagihara, B., Tagawa, K., Morikawa, I., Shin, M., and Okunuki, K. 1958. Crystallization of cytochrome c from wheat perm. Nature (London) 181, 1590. Hatch, M. D. 1966. Adenylsuccinate synthetase and adenylsuecinate lyase from plant tissues. Biochem J. 98, 198. Hatch, M. D., and Stumpf, P. K . 1961. Fat metabolism in higher plants. XVI. Acetyl

WHEAT GERM

295

co-enzyme A carboxylase and acyl co-enzyme A-malonyl co-enzyme A transcarboxylase from wheat germ. J. Biol. Chem. 236, 2879. Hawthorn, J., and Todd, J. P. 1955. Catalase in relation to the unsaturated-fat oxidase activity of wheat flour. Chem. Ind. (London) p. 446. Hayward, H. E. 1938. “The Structure of Economic Plants.” Macmillan, New York. Heathcote, J. G., Hinton, J. J. C., and Shaw, B. 1952. The distribution of nicotinic acid in wheat and maize. Proc. R. SOC.London, Ser. B 139, 276. Hector, S . M. 1936. “Introduction to the Botany of Field Crops,” Vol. 1. Central News Agency Ltd., Johannesburg, South Africa. Hegsted, D. M., and Chang, Y . 1965. Protein utilization in growing rats. 1. Relative growth index as a bioassay procedure. J. Nutr. 85, 159. Heinstein, P. F., and Stumpf, P. K. 1969. Fat metabolism in higher plants. XXXVIII. Properties of wheat germ acetyl coenzyme A carboxylase. J. Biol. Chem. 244, 5374; Chem. Abstr. 71, 98448 (1969). Hepburn, F. N., Calhoun, W. K., and Bradley, W. B. 1960. The distribution of the amino acids in wheat in commercial mill products. Cereal Chem 37, 749. Herd, C. W., and Amos, A. J. 1930. Fat: Its estimation in wheaten products. Cereal Chem. 7, 251. Hertwig, R. 1931. Mildly toasted wheat germ-a possible merchandisable table cereal. Cereal Chem 8,226. Hinners, S . W. 1958. Bioassay for determining the nutritional adequacy of protein supplements for chick growth (Univ. of Illinois, Urbana). Univ. Microfilms (Ann. Arbor, Mich.), L.C. Card. No. Mic. 58,1704. Diss. Abstr. 18, 1567; Chem. Abstr. 5 2 , 13907 (1958). Hinton, J. J. C. 1944. The chemistry of wheat germ with particular reference to the scutellum. Biochem. J. 38,214. Hinton, J. J. C. 1947. The distribution of vitamin B, and nitrogen in the wheat grain. Proc. R. SOC.London, Ser. B 134,418. Hinton, J. J. C. 1959. The distribution of ash in the wheat kernel. Cereal Chem. 36, 19. Hinton, J. J. C. 1962. Note on the distribution of ash in an Australian wheat. Cereal Chem 39, 341. Hinton, J. J. C., Peers, F. G., and Shaw, B. 1953. The B vitamins in wheat: The unique aleurone layer. Nature (London) 172, 993. Hires, C. H. W., Steen, W. H., and Moore, S. 1954. The amino acid composition of ribonuclease. J. Biol. Chem 211, 941. Horn, M. J., Jones, D. B., and Blum, A. E. 1946. Colorimetric determination of methionine in proteins and foods. J. Biol. Chem. 166,313. Horn, M. J., Jones, D. B., and Blum, A. E. 1947a. Microbiological determination of lysine in proteins and foods. J. Biol. Chem 169,71. Horn, M. J., Jones, D. B., and Blum, A. E. 1947b. Microbiological determination of threonine in proteins and foods. J. Biol. Chem. 169,739. Horn, M. J., Jones, D. B., and Blum, A. E. 1 9 4 7 ~Microbiological . determination of valine in proteins and foods. J. Biol. Chem 170,719. Horn, M. J., Jones, D. B., and Blum, A. E. 1948a. Microbiological determination of histidine in proteins and foods. J. Biol. Chem. 172, 149. Horn, M. J., Jones, D. B., and Blum, A. E. 1948b. Microbiological determination of phenylalanine in proteins and foods. J. Biol. Chem. 176,679. Horn, M. J., Jones, D. B., and Blum, A. E. 1949a. Microbiological determination of leucine in proteins and foods. J. Biol. Chem. 177,697.

296

S. R. SHURPALEKAR AND P. HARIDAS RAO

Horn, M. J., Jones, D. B., and Blum, A. E. 1949b. Microbiological determination of isoleucine in proteins and foods. J. Biol. Chem. 180, 695. Horn, M. J., Jones, D. B., and Blum, A. E. 1950. Methods for microbiological and chemical determination of essential amino acids in proteins and foods. US.,Depr. Agric., Misc. Publ. 696. Hove, E. L., and Harrel, C. G . 1943. The nutritive value of wheat germ protein. Cereal Chem 20, 142. Hove, E. L., Carpenter, L. E., and Harrel, C. G. 1945. The nutritive value of some plant proteins and the supplemented effect of some protein concentration on patent flour and whole wheat. Cereal Chem. 22, 287. Howe, M., and Glick, D. 1946. The proteolytic enzyme activity of milled fractions of wheat and the effect of bromate in flour. Cereal Chem. 23, 360. Howe, M., and Sullivan, B. 1936. Minerals of wheat. 11. The determination of sodium and potassium. Cereal Chem. 13, 61. Howe, M., Sullivan, B., and Frederick, S. 1937. An explanation of the effect of heat treatment on wheat germ. Cereal Chem. 14,489. Hullett, E. W., and Stern, R. 1941. Biological elimination of glutathione from wheat germ and flours used in bread making. Cereal Chem. 18,561. Ichiba, A. 1935a. Phytosterol of wheat germ oil. Sci. Pap. Inst. Phys. L'hem. Res. (Jpn.) 28, 122; Chem. Absrr. 30, 1063 (1936). Ichiba, A. 1935b. A phytosterol in wheat germ. Sci. Pap. Inst. Phys. Chem. Res. (Jpn.) 28, 124;Chem. Absrr. 30,1063 (1936). Inoue, Y., and Ito, T. 1959. Preparation of wheat germ hexokinase. ~ i p p o n ~ o g e i K f f g f f k u Kaishi 33, 659; Chem. Abstr. 62, 6727g (1965). Ivanova, B., and Popov, A. 1974. Bulgarian wheat germ oil. Riv. Ital. Sostanze Grasse 51, 425. Ivanova, B., Dimitrov, N., and Popov, A. 1974. Wheat germ as raw material for nutritional and industrial purposes. Khran. Prom. 23, 26. Ivanova, B., Dimitrov, N., Stefanov, G., and Popov, A. 1975. Stabilization of wheat germ. Khran. Prom 24, 30. Iwata, H., Maesawa, G., and Mohachiro, N. 1952. Wheat germ. VI. Preparation of miso from wheat germ. Eiyo To Shokuryo 5,107; Chem. Abstr. 51, 183628 (1957). Iwata, H., Maesawa, G., and Ito, T. 1953. Wheat germ. VII. Preparation of koji. Eiyo To Shokuryo 6 , 223; Chem. Abstr. 51, 18362h (1957). Iwata, H., Suetsugu, S., Nakaya, T., Hayashida, T., and Kariazona, A. 1955a. Effect of Storage on thiamine, riboflavin and pyridoxine contents of cereal germs. Eiyo To Shokuryo 7, 152; Chem. Abstr. 54, 12410h (1960). Iwata, H., and Arima, K., and Sato, Y. 1955b. Wheat germ. VIII. Effect of wheat germ on milk production. Eiyo To Shokuryo 7,224; Chem Abstr. 54,12410g (1960). Jackson, S. H., Doherty, A., and Malone, V. 1943. The recovery of the B vitamins in the milling of wheat. Cereal Chem. 20,551. Jacobs, B. R., and Rask, 0. S. 1920. Laboratory control of wheat flour milling. fnd. Eng. Chem 12,899. Jagbir Singh. 1973. Wheat germ-separation and utilization. Indian Miller 3 , 19. Jamieson, G . S., and Baughman, W. F. 1932. Wheat germ oil. Oil Soap (Chicago) 9, 136. Jansen, E. F., Fellows-Nutting, M. D., and Balls, A. K. 1948. The reversible inhibition of acetylesterase by di-isopropyl fluorophosphate and tetraethyl pyrophosphate. J. Biol. Chem 175,975. Javillier, M.,and Colin, Y. 1933. Etudes sur le dosage des principes immkdiats phosphores des vdgdtause en w e de recherches de physiologie vdgdtale. Bull. SOC.Chim Biol. 15, 1552.

WHEAT GERM

297

Javillier, M., and Imas, S. 1926. Le manganese dans le grain de bld et dans les produits de mouture. C. R. Seances Acad. Agric. Fr. 12,721. Johns, E. W., and Butler, J. A. V. 1962. Studies on histone: The histone of wheat germ. Biochem J. 84,436. Jones, C. R. 1964. Recent European developments in flour milling technology. Milling 142, 324. Jones, D. B. 1931. Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins. US., Dep. Agric., Circ. 183. Jones, D. B., and Widness, K. D. 1946. The comparative growth promoting value of the processing of wheat germ, corn germ and some other protein foods of plant and animal origin. J. Nutr. 31, 675. Jones, W., and Perkins, M. E. 1925. The nitrogenous groups of plant nucleic acid. J. Biol. Chem 62,557. Kapoor, M., and Waygood, E. R. 1965. Orotidine 5-phosphate pyrophosphatase and dihydroorotic-NAD dehydrogenase in wheat embryos. Can. J. Biochem. 43, 143. Karl, H., Eugen, W., Sibylle, S., and Renate, S. 1969. Plant protease inhibitors. 111. Purification of trypsin inhibitors from wheat and rye seed embryos and location of the active centers. Hoppe-Seyler’s Z. Physiol. Chem. 350, 249. Karr, D., John, T., and Peter, A. 1967. S-Adenosyl methionine: Methionine methyl transferase from wheat germ. Arch. Biochem Biophys. 121, 732. Kasarda, D. D., Nimmo, C. C., and Kohler, G. 0. 1970. Proteins and the amino acid composition of wheat fractions. In “Wheat Chemistry and Technology” (Y.Pomeranz, ed.), 2nd ed., Monogr. Ser., Vol. 111, Revised. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Kazakov, E. D. 1947. Quantitative relationships and ash content of components of wheat kernels. Dok. Akad. Nauk SSSR 58,1081. Kent, N. L. 1942. The occurrence of lead, tin and silver in wheat and its milling products. J. SOC.Chem. Ind., London 61, 183. Kent, N. L., Simpson, A. G., Jones, C. R., and Moran, T. 1944. ‘‘High Vitamin Flour.” Ministry of Food, Cereals Research Station, St. Albans. Kent, N. L., Thomlinson, J., and Jones, C. R. 1949. Scutellum and its separation in milling. Milling 113, 46. Kent-Jones, D. W., and Amos, A. J. 1967. “Modern Cereal Chemistry,” 6th ed. Food Trade Press Ltd., London. Kent-Jones, D. W., and Mitchell, E. F. 1962. “The Practice and Science of Breadmaking.” Northern Publ. Co., Liverpool. King, H. G. C. 1962. Phenolic compounds of commercial wheat germ. J. Food Sci. 27,446. Koch, R. B., Felsher, A. R., Burton, T. H., and Larsen, R. A. 1954. A rapid method for the determination of cereal lipase activity. Cereal Chem. 31, 113. Kohler, G. O., and Palter, R. 1967. Studies on methods for amino acid analysis of wheat products. Cereal Chem 44, 512. Kraemer, L. M., Conn, E. E., and Vennesland, B. 1951. The p-carboxylases of plants. 111. Oxalacetic carboxylase of wheat germ. J. Biol. Chem. 188, 583. Kretovitch, V. L. 1945. “Physiologico-biochemical Bases for the Storage of Grain,’’ (transl. by K. Starr Chester). Akad. Nauk SSSR, Inst. Biokhim., Moscow. Kuhl, H. 1941. The action of cereal fat and free fat acids on the baking capacity of flour with special reference to storage. Mehl Brot 41, 297. Lanzani, G. A., and Lanzani, M. M. 1967. A ribonuclease from the microsomes of wheat germs. Enzymologia 32, 236. Lanzani, G. A., Marchesini, A., Galante, E., Manzocchi, L. A., and Sequi, P. 1967. Peroxidase isoenzymes in wheat germ. Enzymologia 33, 361.

298

S. R. SHURPALEKAR AND P. HARIDAS RAO

Larousse, J., and Blanchet, L. 1971. Studies on the effect of addition of stabilised wheat germ in production of bread and biscuits. Bull. A m Eleves Ec. Fr. Meun. 246, 250. Larousse, J., and Blanchet, L. 1975. Effect of addition of stabilized wheat germ in bakery products. Bull. Anc. Eleves Ec. Meun. 246, 255. LeCoq, R. 1944a. Comparative action of casein and wheat germ meal on experimental production of edema, by dietary i n enbalance and on the alternation of neuro-muscular exitability accompanying syndrome. C. R. Seances SOC.Biol. Ses Fil. 138, 736. LeCoq, R. 1944b. La teneur en vitamine E. de I’huile de germe de blt. C R. SeancesSoc. Biol. Ses Fil. 138, 836. Levcne, P. A., and Bass, L. W. 1931. “Nucleic Acids.” Chem. Catalog Co. (Tudor), New York. Lewis, D., and Weisberg, S. M. 1952. Nutriment for poultry. U.S. Patent 2,620,274; Chem. Abstr. 41,2908f (1953). Lewkowitsch, J. 1915. “Chemical Technology and Analysis of Oils, Fats and Waxes,” 5 t h ed. London. Liener, 1. E., and Hill, F. G. 1953. The effect of heat treatment on the nutritive value and hemagglutinating activity of soyabean oilmeal. J. Nutr. 49,609. Lindow, C. W., Elvehjem, C. A., and Peterson, W. H. 1929. The copper content of plant and animal foods. J. Biol. Chem. 82,465. Linko, P., Cheng, Y., and Milner, M. 1960. Changes in the soluble carbohydrates during browning of wheat embryos. Cereal Chem. 37,548. Lipshitz, R., and Chargaff, E. 1956. Studies on nucleoproteins: Preparation of the Deoxyribonucleoprotein and fractionation of the Deoxyribonucleic acid of wheat germ. Biochim. Biophys. Acta 19, 256. Lockwood, J. 1952. “Flour Milling,” 4th ed. Northern Publ. Co., Liverpool. Luchsinger, W. W., Cuendet, L. S., Boyer, P. D., and Geddes, W. F. 1955. A sensitive method for measuring lipase activity and its application t o wheat products. Cereal Chem. 32, 395. Lusena, C. V. 1951. Preparation of ribonucloprotein and ribonucleic acid from wheat germ. Cereal Chem 28,400. Lusena, C. V., and McFarlane, W. D. 1945. Studies on the processing of wheat germ. Can. J. Biochem. 23, 201. Lyman, C. M., Kuiken, K. A., and Hale, F. 1956. Essential amino acid content of wheat in commercial products. Cereal Chem. 37, 749. McFarlane, W. D. 1950. Vitamin E-enriched oil. U.S. Patent 2,497,317. McHargue, J. S. 1925. The occurrence of copper, manganese, zinc, nickel and cobalt in soils, plants and animals and their possible function as vital factors. J. Agric. Res. 30, 193. MacIntyre, R., and Campbell, M. 1974. In “Triticale.” Proceedings of an International Symposium, Int. Dev. Res. Cent., Ottawa, Canada. McLaughlan, J. M., and Campbell, J. H. 1969. In “Mammalian Protein Metabolism” (H. N. Monro and J. B. Allison, eds.), Vol. 3, p. 39. Academic Press, New York. MacMasters, M. M., Hinton, J. J. C., and Bradbury, D. 1971. Microscopic structure and composition of the wheat kernel. I n “Wheat Chemistry and Technology” (Y. Pomeranz, ed.), 2nd ed. Monogr. Ser., Vol. 111, Revised. Am. Assoc. Cereal Chem., St. Paul, Minnesota. Maes, E., and Bauwen, M. 1951. Influence of infrared radiation in the keeping qualities of wheat germs. Bull. Ec. Meun. Belge 13, 126; Chem. Abstr. 46, 1662 (1952). Mambish, I. E. 1953. The weight relation and ash content of the component parts of the wheat kernel. Tr., Vses. Nauchno-Issled. Inst. Zerna Prod. Ego Pererab. 25, 139. Markley, M. C., and Bailey, C. H. 1931. The march of acidity in stored flour. 11. Cereal Chem. 8. 29.

WHEAT GERM

299

Matveef, M. 1965. Recherche d’une mdthode nouvelle de dosage des enveloppes et du germe du grain de bld. Bull. Anc. Eleves Ec. Fr. Meun. 206,67. Miladi, S., Hegsted, D. M., Saunders, R. M., and Kohler, G. 0. 1972. The relative nutritive value, amino acid content, and digestibility of the proteins of wheat mill fractions. Cereal Chem. 49, 119. MilIer, B. S., and Kummerow, F. A. 1948. The disposition of lipase and ljpoxidase in baking and the effect of their reaction products on consumer acceptability. Cereal Chem. 25, 391. Mitchell, H. H., and Block, R. J. 1946. Some relationships between the amino acid contents of proteins and their nutritive values for the rat. J. B i d . Chem. 58, 163. Moran, E. T., Jr., Summers, J. D., and Bass, E. J. 1968. Heat processing of wheat germ meal and its effect on utilization and protein quality for the growing chick: Toasting and autoclaving. Cereal Chem. 45, 304. Moran, E. T., JL, Summers, J. D., and Pepper, W. F. 1970. Nutritional evaluation of selected milling fractions from wheats of different type and geographical area of production: First three limiting amino acids for the chick and performance under dietary conditions calculated adequate. Poult. Sci. 69, 371. Moran, T., and Drummond, J. 1945. Scientific basis of 80%extraction flour. Lpncet 253,698. Moran, T., Pace, J., and McDermott, E. E. 1954. The lipids in flour-oxidative changes induced by storage and improper treatment. Nature (London) 174,449. Morgulus, S., and Spencer, H. C. 1936. Study of the dietary factors concerned in nutritional muscular dystrophy. J. Nutr. 11,573. Morris, V. H., Alexander, T. L., and Pascoe, E. D. 1945. Studies of the composition of the wheat kernel. I. Distribution of ash and protein in centre sections. Cereal Chem 22, 351. Morris, V. H., Alexander, T. L., and Pascoe, E. D. 1946. Studies of the cornposition of the wheat kernel. 111. Distribution of ash and protein in central and peripheral zones of whole kernels. Cereal Chem. 23,540. Moruzzi, G., Viviani, R., Sechi, A. M., and Lenaz, G. 1969. Studies on compounds and individual lipids of wheat germ. J. Food Sci. 34,581. Mounter, L. A., and Mounter, M. E. 1962. Specificity and properties ofwheat germ esterase. Biochem. f. 85,576. Musher, S. 1940. Stabilized wheat germ food material, etc. U.S. Patent 2,198,218; Chem Abstr. 34,5559 (1940). Narayana Rao, M., and Swaminathan, M. 1970. Plant proteins in the amelioration of protein deficiency states. World Rev. Nutr. Diet. 2, 106. National Joint Industrial Council for the Flour Milling Industry. 1966. “The Practice of Flour Milling.” Northern Publ. Co., Liverpool. Nelson, J. H., Glass, R. L., and Geddes, W. F. 1963a. Silicic acid chromatography of wheat lipids. Cereal Chem. 40, 337. Nelson, J. H., Glass, R. L., and Geddes, W. F. 1963b. The triglycerides and fatty acids of wheat. Cereal Chem 40, 343. Netherlands Patent Application. 1970. Method of production of edible fat products. Patent Appl. No. 6,908,382. Neumann, H. 1952. Preparation, composition and processing of poly-vitamin concentrate from wheat gem. Seifen, Oele, Fette, Wachse 78,291. Niewiadomski, H., Wilczpolski, M., and Ploszynski, M. 1961. Preparation of tocopherol concentrates from wheat germ. Tluszcze Srodki Piorace 5 , 273; Chem. Abstr. 59, 13767f (1963). Olsen, E. M. 1967. Effect of heat treatment on the quality and utilization by the rat of protein in wheat germ meal. Can. J. Biochem. 45, 1673.

300

S. R. SHURPALEKAR AND P. HARIDAS RAO

Oparin, A. l., and Kaden, S. B. 1945. Changes of beta-amylase in wheat seedlings. Biokkimiya 10, 25. Osborne, T. B. 1907. The Proteins of the Wheat Kernel.” Carnegie Institute of Washington, Washington, D.C. Osborne, T. B., and Harris, T. F. 1902. Die Nucleinsaure des Weizenembryos. HoppeSeyler’s Z. Physiol. Chem. 36, 85. Osborne, T. B., and Heyl, F. W. 1908. The prymidine derivatives in nucleic acid. Am. J. Physiol. 21, 157. Osborne, T. B., and Mendel, L. B. 1919. The nutritive value of the wheat kernel and its milling products. J. Biol. Chem. 37, 557. Parrish, J. A., and Bolt, R. J. 1963. Effect of raw wheat germ on nutrition of the chicken. Nature (London) 199, 398. Pearce, J. A. 1943. Effect of storage on thiamine content and on development of rancidity in wheat germ. &n. J. Res., Sect. C 21,57. Peers, F. G. 1953. The phytase of wheat. Biochem. J. 53, 102. Pence, J. W., and Elder, A. H. 1953. The albumin and globulin proteins of wheat. Cereal Chem. 30, 275. Percival, J. 1921. “The Wheat Plant.” Duckworth, London. Pett, L. B. 1935. Studies on the distribution of enzymes in dormant and germinating wheat seeds. I. Dipeptidase and protease. 11. Lipase. Biochem. J. 29, 1898. Pomeranz, Y., and Chung, 0. 1965. The lipid composition of a single wheat kernel and its structural parts. J. Chromatogr. 19, 540. Pomeranz, Y., Carvajal, M. J., Shogren, M. D., Homey, R. C., and Ward, A. B. 1970a. Wheat germ in bread making. I. Composition of germ lipids and germ protein fraction. Cereal Chem. 41, 373. Pomeranz, Y., Carvajal, M. J., Shogren, M. D., Hoseney, R. C., and Finney, K. F. 1970b. Wheat germ in breadmaking. 11. Improving breadmaking properties by physical and chemical methods. Cereal Chem. 47,429, Popova, Y. G., and Kirova, L. A. 1964. Wheat germ-raw material for the preparation of vitamin E concentrates. Nauchni Tr., Vissh Inst. Khranit. Vkusova Prom-st., Plovdiv 11, 139. Power, F. B. 1913. Chemical examination of wheat germ. Pharm. J. 37, 117. Prentice, N., Burger, W. C., Kastenschmidt, I., and Huddle, J. D. 1967. The distribution of acidic and neutral peptidases in barley and wheat kernels. Physiol. Plant. 20, 361. Priest, M. 1959. Distribution of transaminases in the wheat grain. Chem. Ind. (London) 1318. Pringle, W. J. S. 1952. Mineral constituents of wheat and flour. “Food Science.” Cambridge Univ. Press, London and New York. Pringle, W. J. S., and Moran, T. 1942. Phytic acid and its destruction in baking. J. Soc. Chem. Ind., London 61, 108. Proskuryakov, N. I., and Loseva, L. P. 1962. Glycolytic enzymes of wheat grain embryo and their fractionation. Nauchn. Dokl. Vyssh. Shk., Biol. Nauki 3, 1 5 7 ; Chem. Abstr. 60, 8275d (1964). Proskuryakov, N. I., and Nuzhdina, T. N. 1960. Proteins of wheat germ and their enzymic activity. Nauchn. Dokl. Vyssh. Shk., Biol. Nauki 2, 148; Chem. Abstr. 54, 25080e (1960). Proskuryakov, N. I., and Rodionova, I. V. 1960. Enzymic activity of the water soluble proteins of the wheat germ. Biokhim Zerna. 5,109; Chem. Abstr. 54, 228661 (1960). Rand, N. T., and Collins, V. K. 1958. Improving cereals with defatted wheat germ. Food Technol. 12,585.

WHEAT GERM

30 1

Rao, N. A., and Vaidyanathan, C. W. 1966. 2-Phospho-glycerol phosphatase. In “Methods in Enzymology” (W. A. Wood, ed.), Vol. 9, p. 642. Academic Press, New York. Read, B. E., and Tottingham, W. E. 1917. Tritico nucleic acid. J. Biol. Chem. 31, 295. Rees, M. W. 1946. The estimation of threonine and serine in proteins. Biochem. J. 40, 632. Rehfeld, G. 1967. Zur Rage der Toxicitat von Weizenkeimen. Ernaehrungsforfchung 12, 299. Rich, C. E. 1934. The relation of germ content to flour maturation. Cereal Chem. 11, 220. Richardson, C. 1884. The composition of American wheat and born. Burr. Chem. Bull. 4. Richardson, C . , and Crampton, C. A. 1886. Vorlaufige Mitteilung iiber die zusammensetzung des Weizen Keimes und iiber die Anwesenheit von eiher neun Zuekeratrt und von Allantoin. Ber. Dtsch. Chem. Ges. 19, 1180. Rohrlich, M., and Bruckner, G. 1967. “Das Getreide und seine Verarbeitung,” Vol. 4, Part I. Parey, Berlin. Rohrlich, M., and Schoenmann, 1. 1962. Muehle 99,162. Rothe, M. 1961. Acetylesterase in cereals. Getreide Mehl 11, 73. Rothe, M. 1963. Ueber ein neues Stabilisierungsverfahren fur Weizenkeime.Nuhrung 7,579. Rothe, M., and Stoeckel, J. 1962. I. Zur Qualitatsbeurteilung von Weizenkeimen. 11. Haltbarkeit. Getreide Mehl6, 310. Rothe, M., and Stoeckel, J. 1967. Activity changes in enzymes caused by heat (I) lipase acetyl esterase peroxidase and lipoxidase of cereal products. Nahrung 11, 74 1; Chem. Abstr. 68, 11340911(1968). Rothe, M., Woelm, G., and Voigt, 1. 1967. Fat changes in cereal products and their analytical demonstration. 1. Rancidity. Nahrung 11, 149; Chem. Abstr. 67, 42683p (1967). Scholander, H., and Myrback, K. 1951. Amylases in milling products. The use of hydroxyethyl starch for the determination of alpha-amylase. Sven. Kern Tidskr. 63, 250. Schram, E., Moore, S., and Bigwood, E. J. 1954. Chromatographic determination of cystine as cysteic acid. Biochem. J. 57, 33. Schulze, E. 1910. Uber die chemische Zusammeusetzung der Samen unserer Kulturpflanzen. Landwirtsch. Vers.-Stn. 73, 35. Schulze, E., and Frankfurt, S. 1894. Uber das Vorkommen von Raffinose in Keim des Weizenkorn. Ber. Dtsch. Chem. Ges. 27,64. Schulze, E., and Frankfurt, S. 1895. Uber die Verbreitung des Rohrzuchers in den Pflanzen, iiber seine physiologische Rolle und iiber losliche Kohlenhydrate, die ihn begleiten. Hoppe-Seylers’ Z. Physiol. Chem. 20, 5 11. Scott, J. H. 1951. “Flour Milling Processes,” 2nd ed. Chapman & Hall, London. Scrimshaw, N. S., and Behar, M. 1959. World-wide occurrence of protein malnutrition. Fed. Proc., Fed. Am. SOC.Exp. Biol. 18, Suppl. 3,82. Sequi, P., Marchesini, A., and Lanzani, G. A. 1968. Isolation and purification of wheat germ isoperoxidases. Atti Simp. Int. Agrochim. 7, 197. Sherwood, R. C., Andrews, J. S., Wade, W. B., and Bailey, C. H. 1933. The march of acidity in wheat germ during storage. Ind. Eng. Chem. 25,437. Shin, M., and Nakamura, W. 1961. Hemoproteins of wheat germ. Ill. Further studies on purification of peroxidases and some of their properties. J. Biochem. (Tokyo) 50,500. Siegal, L., Melnick, D., and Oser, B. L. 1943. J. Biochem. (Tokyo) 49, 391. Simpson, A. G. 1935. A simple method for determining the “yellowness” and grade of wheat flours. Cereal Chem. 12,569. Singer, T. P., and Hofstee, B. H. J. 1948a. Studies on wheat germ lipase. I. Methods of estimation, purification, and general properties of the enzyme. Arch. Biochem. 18, 229.

302

S. R. SHURPALEKAR AND P. HARIDAS RAO

Singer, T. P., and Hofstee, B. H. J. 1948b. Studies on wheat germ lipase. 11. Kinetics. Arch. Biochem. 18, 245. Singer, T. P., and Pensky, J. 1952. Isolation and properties of the a-carboxylase of wheat germ. J. Biol. Chem. 196, 375. Sisakyan, N. M., and Vasil’eva, N. A. 1954. Oxidation-Reduction processes in hard and soft wheat. Biokhimiya 19, 730; Chem. Abstr. 49, 6389e (1955). Smith, D. E., and Geddes, W. F. 1942. Yeast fermentation and potassium bromate as factors influencing the harmful effects of wheat germ on baking quality. Cereal Chem. 19, 785. Soloveichik, I. Ya. 1946. Food concentrate containing vitamin B-complex. U.S.S.R. Patent 65,874; Chem. Abstr. 41, 5233 (1947). Stafford, H. A., and Vennesland, B. 1953. Alcohol dehydrogenase of wheat germ. Arch. Biochem. Biophys. 44,404. Stare, F. J., and Hegsted, D. M. 1944. The nutritive value of wheat germ, corn germ, and oat proteins. Fed. Proc., Fed. Am. Soc. Exp. Biol. 3, 120. Stauffer, C. E., and Glass, R. L. 1966. The glycerol ester hydrolases of wheat germ. Cereal Chem. 43,644. Steiger, R. 1973. Wheat germ special breads as natural sources of vitamins in nutrition. Osterr. Backer-Ztg. 68, 5. Stern, R. 1944. Effects of flour doughs of extracts from unfermented and fermented wheat germ. Cereal Chem. 21, 81. Stevens, D. J. 1959. The contribution of the germ to the oil content of white flour. Cereal Chem. 36,452. Stevens, E., and Frydnian, B. 1968. Isolation and properties of wheat germ uroporphyrinogen. 111. Cosynthetase. Biochim. Biophys. Acfu 151, 429. Stringfellow, A. C., and Peplinski, A. J. 1966. Air classification of sorghum flours from varieties representing different hardnesses. Cereal Sci. Today 11,438. Sullivan, B., and Bailey, C. H. 1936a. The lipids of the wheat embryo. I . The fatty acids. J. Am. Chern. Soc. 58, 383. Sullivan, B., and Bailey, C. €1. 1936b. The lipids of the wheat embryo. 11. The unsaponifiable fraction. J. Am. Chem. Soc. 58, 390. Sullivan, B., and Howe, M. 1933. Lipases of wheat. J. Am. Chem. Soc. 55, 320. Sullivan, B., and Howe, M. 1937. The isolation of glutathione from wheat germ. J. Am. Chern. Soe. 59, 2742. Sullivan, B., and Near, C. 1927. The ash of hard spring wheat and its products. Ind. Eng. Chem. 19, 498. Sullivan, B., and Near, C. 1928. Lipoid phosphorus of wheat and its distribution. Cereal Cliern. 5, 163. Sullivan, B., and Near, C. 1933. Change of crude lipids of wheat on storage. Ind. Eng. Chem. 25, 100. Sullivan, B., Near, C., and Foley, G . H. 193th. The role of the lipids in relation to flour quality. Cereal Clzem. 13, 318. Sullivan, B., Near, C., and Foley, G. H. 1936b. The harmful action of wheat germ on the baking quality of flour and the constituents responsible for this effect. Cereal Chem. 13, 453. Sullivan, B., Howe, M., and Schmalz, F. D. 1936c. On the presence of glutathione in wheat germ. Cereal Chem. 13,665. Sullivan, B., Howe, M., and Schmalz, F. 1937. An explanation of theeffect of heat treatment on wheat germ. Cereal Chem. 14, 489. Sullivan, M . , and Hess, W. C. 1931. Estimation of cysteine in the presence of glutathione. Public Health Rep. 46, 390.

WHEAT GERM

303

Summers, J. D., Slinger, S. J., Pepper, W. F., and Moran, E. T. 1968. Biological evaluation of selected wheat fractions from nine different wheat samples for energy and protein quality. Poult. Sci. 47, 1753. Sumner, R. J. 1943. Lipoid oxidase studies. A method for the determination of lipoxidase activity. Ind. Eng. Chem., Anal. Ed. 15, 14. Sure, B. 1957. Relative nutritive values of proteins in various foods at increasingly high levels of protein intake. J. Agric. Food Chem. 5,463. Swatditat, A. 1974.Diss.Abstr. Int. B 33(7), 3129. Tagawa, K., and Shin, M. 1959. Hemoproteins of wheat germ. 1. Crystallization and properties of peroxidase from wheat germ. J. Jpn. Biochem. SOC.46, 865. Tagawa, K., Shin, M., and Okunuki, K. 1959. Peroxidases from wheat germ. Nature (LondonJ 183, 111. Teller, G. L., and Teller, W. K. 1932. A study of proteins of wheat bran. Cereal Chem. 9,

560. Teply, L. J., Strong, F. M., and Elvehjem, C. A. 1942.Nicotonic acid, pantothenic acid and pyriodoxine in wheat and wheat products. J. Nutr. 24, 167. Tkachuk, R. 1969. Nitrogen to protein conversion factors for cereals and oilseed meals. Cereal Chem 46,419. Tkachuk, R., and Irvine, G. N. 1969. Amino acid compositions of cereals and oilseed meals. Cereal Chem. 46, 206. Tschirch, A., and Oesterle, 0. 1900. “Anatomischer Atlas der Pharmakognosie und Nahrungsmittelkunde,” 2nd Sect. Tauchnitz Leipzig. Tsen, C. C. 1974. “Triticale: First Man Made Cereal.” Assoc. Cereal Chem., St. Paul, Minnesota. Uroma, E., and Louhivuori, A. 1954. Biologically active proteins from wheat germs. 1. An anti-eosinophilic fraction (A.E.F.). Ann. Med. Exp. Biol. Fenn. 32,42. Verjee, Z. H. M. 1969. Isolation of three acid phosphatases from wheat germ. Eur. J. Biochem. 9,439. Vogl, A. E. 1899.“Die vegetabilischen Nahrungs- und Genussmittel.” Urban & Schwarzenberg, Berlin. von Euler, H., and Mahiberg, M. 1936.Vitamins and catalysts in wheat embryo. Ark. Kemi, Mineral. Geol. 12B,6;Chem Abstr. 30,3860 (1936). Waggle, D. H., Lambert, M. A., Miller, G. D., Farrell, E. P., and Deyoe, C. W. 1967. Extensive analyses of flours and millfeeds made from nine different wheat mixes. 11. Amino acids, minerals, vitamins, and gross energy. Cereal Chem. 44,48. Ward, A. H. 1943.Location of vitamin B, in wheat. Chem Ind. (London) 62, 11. Ward, R. J. 1958. The biological activity of E - and 6-tocopherols. Br. J. Nutr. 12,226. Wasserman, A. R., and Burris, R. N. 1965.Hemoprotein from wheat germ. Phytochemistry

4,413. Weller, G. 1935. Sur le dosage du glutathione dans la levure. Ann. Ferment. [N.S.] 1, No.

27, 108. Westerman, B. D.,Roach, F., and Stone, M. 1952.Improving the nutritive value of flour. V. The effect of the use of defatted wheat germ. J. Nutr. 47, 147. Westerman, B. D.,Oliver, B., and May, E. 1954. Improving the nutritive value of flour. VI. A comparison of the use of soya flour and wheat germ. J. Nutr. 54, 225. Wierzbowski, J., Trojan, M., and Gogolevski, M. 1966. Effect of selected technological measures for stabilizing wheat germs on some of their biochemical properties. Przegl. Zbozowo-Mlyn. 10,292;Chem Abstr. 66,543251(1967). Winton, A. L., and Winton, K. B. 1932.“The Structure and Composition of Foods,” Vol. 1. Wiley, New York.

304

S. R. SHURPALEKAR AND P. HARIDAS RAO

Wolf, M. J., Buzan, C. L., MacMasters, M. M., and Rist, C. E. 1952. Structure of the mature corn kernel. IV. Microscopic structure of the germ of dent corn. Cereal Chem 29, 362. Yakovenko, A. V. 1961. Activity of lipoxidase during rancidity development of germ flakes. Tr. Odessk. Tekhnol. Insf. 11, 31; Chem Absfr. 59, 10689e (1963). Yamaha, T., and Cardini, C. E. 1960. Biosynthesis of plant glycosides. 1 . Monoglucosides. Arch. Biochem. Biophys. 86,127. Zahnley, J. C., and Axelrod, B. 1965. D-Xylulokinase and D-ribulokinase in higher plants. Plant Physiol. 40, 312. Zaitsev, V. I., Shaskina, N. Z., and Tyuterov, S. L. 1974. Improvement of nutritive value of bread by adding wheat germ. Khlibopekarnaya i Kondif. Prom 7, I. Zav’yalov, A. A. 1939. Enriching bread and bread products with vitamins. Khlebopek. Prom-st. No. 3, pp. 9-11. Zav’yalov, A. A. 1940. Enriching bread and bread products with vitamins. Khim. Ref. Zh. No. 1, p. 138; Chem Absfr. 36, 1102 (1942). Zunini, E. 1935. La “spuntatura” di frumento: Componenti inorganici e fosfo-organici. Sua importanza per la reintegrazione degli elementi embrionali nei prodotti della panificazione. Chim. Ind., Argic., Biol. Realiz. Corp. 1 1 , 339.

FLEXIBLE PACKAGING FOR THE RMOPROCESSED FOODS RAUNO A . LAMP1 Food Engineering Laboratory

U.S. Army Natick Research and Development Command Natick. Massachusetts

I. Introduction

....................................................

.................................................... I11. Materials ....................................................... A . General Aspects ............................................... 11. Early Efforts

B . Extractives .................................................. C. Bacterial Penetration ........................................... D. Retortability ................................................. E . CurrentFilms ................................................ 1 v. PackageDesigns ................................................. V. Food Product Development ........................................ A. TypicalProducts .............................................. B. High.Temperature, Short-Time Products ............................ c. Utility ...................................................... D. Quality and Stability ........................................... VI . Production Systems .............................................. A . Overview: A Systems Approach .................................. B. Current Systems and Components ................................. C. Production Reliability .......................................... VII . Sealing ........................................................ A. General Aspects ............................................... B . Seal Definition and Requirements ................................. C. Effect of Retorting and Storage on Heat Seals ....................... D. Significance of Interlamina Bonds in the Seal Area .................... E. Effects of Occluded Particles in Closure Seals ........................ F. Methods for Sealing Retort Pouches ............................... G . SealWrinkles ................................................. H . Seal Contamination ............................................ VIII . Filling ........................................................ A. Definition and Requirements .................................... B. Equipment ..................................................

306 309 312 312 313 315 317 317 322 3 24 325 327 327 328 333 333 336 344 346 346 347 352 353 354 355 359 360 364 364 365 305

306

RAUNO A. LAMPI

.................................................... 369 369 A. General Aspects ............................................... .................................... 371 B. Specific Techniques . . . . . ual Gas Levels ........................ 374 C. Methods for Determining R Retorting ...................................................... 375 A. Consideration of Prepackaging versus Postpackaging Sterilization . . . . . . . . . 375 376 B. Preliminary Heat Transfer Considerations ........................... C. Process Determination .......................................... 380 D. Retorting Techniques and Equipment .............................. 387 Package Durability ............................................... 399 399 A. Performance Tests ............................................. 403 B. Shipping Tests and Experience ................................... Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 A. General Aspects .......................................... 405 B. System Specifications and Sampling Protocol ........................ C. Specific Tests ........................................... Research and Development Needs .............................. A. Product Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 B. Flexible Film Technology ............................. . . . . . 418 C. Equipment Development ........................................ 418 419 Acknowledgment ................................................ 420 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IX. Air Removal

X.

XI.

XII.

XIII.

I. INTRODUCTION The use of a flexible polymeric-foil laminate pouch to contain and maintain the wholesomeness of thermoprocessed foods has been proved to be technically and commercially feasible. This achievement, dating from exploratory studies in the mid to late 1950’s (Hu et ~ l . ,1955; Keller, 1969a; Proctor and Nickerson, 1958; Could et al., 1962; Wallenberg and Jarnhall, 1957), has resulted in the evolution of a new food preservation concept with its own distinct, singular technology. Flexibly packaged thermoprocessed food items (or retort pouches), as typified by Fig. 1, may be compared, with some logic, with items packed in the common three-piece steel plate or drawn aluminum sanitary can. In both instances, heat sterilization and the air tightness of the container assure sterility up to the point of end use. However, the outward appearance of the flat pouch and the convenience of reheating by immersion of the unopened pouch in hot water invite, with equal logic, a comparison with frozen “boil-in-bag” entrke items. True, the evolution of the retort pouch has drawn very heavily on past experiences in many disciplines available from the canning, packaging, and frozen food industries, but there are still a number of specific technical problems and solutions, production-related considerations, and performance requirements and characteristics that differ enough from established methods of preservation and packaging to justify the development of a new, separate expertise.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

307

FIG. 1. Typical retort pouch items: thermoprocessed foods.

As an introduction to this chapter, a brief description of the more important of these technological differences will provide a contextual framework for the detailed technical discussions to follow. The first pacing problem was packaging materials. Unsupported and laminated films were available that exhibited excellent barrier properties, were heatsealable, protected the food products against shipping and handling abuse, and had satisfactory machineability characteristics. In addition to possessing these essential properties, new films were developed that withstood, in final package form, steam or water thermoprocessing at 250°F for 20 to 40 minutes without loss of heat seal or laminate bond and without contamination of the product. Overall package strength, integrity, and resistance t o abuse had to equal those of the metal cans. To achieve these properties, advances were made in devising blends of polymers for films to be concomitantly inert, heat-sealable, dimensionally stable, and inherently resistant to heat at 250°F for typical process times. New adhesive systems were formulated to resist thermoprocessing temperatures, high moisture, and fatty product attack, and to meet existing Food and Drug Administration (FDA) requirements on extractives. The laminating process required unusually close quality surveillance. The packaging materialmachinery interface was most intimately exemplified in heat sealing, where wrinkle-free seal areas were necessary; better control over seal bar pressure,

308

RAUNO A. LAMPI

temperature set point, and temperature distribution had to be exercised; and new fusion, burst, and tensile test criteria had to be met. The final package (end item) itself required considerations and developments different from those of past experience. Generally, food formulations were similar to those for conventional metal cans, with modifications geared to optimize flavor in view of the shorter process times and to facilitate accurate, clean filling. Packaging of some previously heat-sensitive products (meatballs, ham, seafoods) now become feasible. Relative t o sterilization, aseptic techniques were considered as an alternative to postpackaging sterilization, primarily to void the heat-resistance requirements of the materials, but these methods presented formidable additional technical barriers and offered no apparent advantages. The flat, rectangular shape of the pouch and the resultant shorter process times caused new concerns, even with conventional equipment. The approach to high-temperature, short-time (HT/ST) conditions necessitated assurance that enzymes as well as bacteria were inactivated, that Fo and integrated sterilization value concepts were compatible and applicable, that access of the heating medium to each container was carefully and uniformly controlled, and that package shape was physically uniform and individually restricted. The vacuum concept used with metal cans, in terms of negative pressure (mm Hg), no longer applied. Since the package was flexible and the contents were nonporous and nonrigid, the package would collapse or expand until internal and external pressures were equal. This fact, coupled with lowering of the tensile strength of the seal at high temperatures, required the use of hyperbaric retort pressures to prevent pouch rupture during the 250°F processes, but lessened concern over postprocess infection, since no “sucking-in’’ pressure differences existed, especially during cooling. The transition of the retort pouch from the laboratory and pilot plant to a production environment is where the use of standard concepts engineered to perform at new higher levels in terms of inherent performance (such as seal tensile strengths) and reliability (low incidence of faulty closure seals) is best exemplified. The difference here becomes primarily one of magnitude instead of direction. Whether made prior to production or made in-plant on a form-fillseal machine, pouch seals had to meet high strength and defect-free standards. These requirements meant that improvements were necessary in bar design, temperature control, seal bar support and action systems, bar application pressure control, and seal-area alignment systems. Changes in the chassis design of flexible packaging equipment were necessary to facilitate wash-down and to meet U. S. Department of Agriculture (USDA) meat plant sanitation requirements. The wet and splash-prone nature of the foods necessitated improvements and in some instances new methods for the close control and smooth transition of the pouch, especially until the closure seal was made, from one production line function to another. Filling without seal-area contamination required not only a good match of a pump-nozzle combination to the needs of each specific

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

309

product, but frequently redesign to eliminate product accumulation and drip. New nozzle designs have also emerged. The retort pouch differs from dehydrated or frozen food packages, in terms of the definition, measurement, and assurance of overall reliability, in that a greater performance burden is put on the package itself (fewer manufacturing defects, greater durability), and it differs from the metal containers used for thermoprocessed foods in that reliability had to be proved to gain regulatory agency approval and consumer acceptance prior to implementation, rather than be accepted on the basis of empirical data gathered over a century of commercial experience. This latter aspect has contributed to the delay in commercialization of the retort pouch in the United States and to the cautious expansion of its use in Europe and Asia. If the published figure of 0.1% failure for the metal can is accepted as normal, indications (Lampi, 1973; Burke and Schulz, 1972; Tsutsumi, 1974, 1975a; Nughes, 1973) are that the reliability of the retort pouch has been established and that it is at least as durable and safe as the established (proved) metal can. This chapter will cover the use of flexible packaging for thermoprocessed foods in depth, considering all efforts up to 1960 as historical, and all subsequent developments according to specific technical areas. Included will be future research and development needs. Excluded will be any detailed discussion of aseptic packaging (either full or partial), w h c h has separate technical and developmental concerns (Brody, 1973; Kelsey, 1974a,b), or of drawn semirigid containers (Lane and Widner, 1969), which also have their own technology and application. An attempt will be made t o cover the subject internationally, but with recognition that discussion of some developments outside the United States may not be totally comprehensive or current. Proprietary efforts are, of course, excluded. Also, as with any viable area of development, changes occur rapidly, and today’s state of the art is soon passC. Brody (1971) has presented an earlier review of the status of the retort pouch.

II. EARLY EFFORTS The earliest recorded studies, at least in the United States, using flexible packaging materials for thermoprocessed foods are those reported by Hu et al. (1955) and by Nelson et al. (1956) at the University of Illinois, although Gould et al. (1962) stated that the idea was proposed as early as 1940. The studies at the University of Illinois typified those carried out during the period up to 1961, concentrating on establishing basic feasibility, defining areas where improvements in films and techniques were required, and determining tentative material and food product requirements. The University of Illinois researchers systematically screened candidate films for permeability to water vapor and oxygen, for resistance to boiling water and

310

RAUNO A. LAMP1

steam at 250”F, for inertness using packaged water as a flavor and appearance vehicle, and for heat sealability . The surviving materials, polyester (Mylar) and Trithene, were used to package a high-acid and a low-acid product. A water processing technique using super-imposed air to prevent bursting was devised. These studies pointed out the need for stronger, heat-sealable materials with low oxygen and water vapor permeability rates. Wallenberg and Jarnhall (1 957), denoting European interest, also surveyed a variety of films; they concluded that the concept was feasible and cautioned that air must be evacuated prior to sterilization to preclude bursting of the pouch. The U. S. Military (Quartermaster Food and Container Institute for the Armed Forces) saw the potential of retort pouches from a functional aspect for combat rations. The flat shape fitted conveniently into field clothing pockets and load-carrying equipment without restricting physical movement. The softness, to quote Rubinate (1960), eliminated personal injury if the soldier had to “hit the dirt” during combat. These incentives spurred most of the exploratory effort over the period from 1957 to 1960. Keller (1959a,b) reported on the screening of several candidate films, concluding that laminations containing aluminum foil showed the greatest promise. The first successful such material was 75-micron vinyl/9-micron foil/25-micron polyester used for test packs of sliced peaches and 5.5-ounce beefsteaks. McGregor (1959) reported additional successful test packs of diced beef and applesauce; he also illustrated a proposed combat ration emphasizing flexible packaging. Keller (1959a), in addition to the film evaluation and test product efforts, investigated retort processes and recommended that, although a straight steam cook will work if enough residual gases are removed from the pouch prior to sealing, a water cook with superimposed air pressure is preferred. He also reported that the use of a paperboard folder improved performance during rough-handling tests. An alternative to the folder was a die-cut frame. Progress continued to the point where trial “commercial” production runs of peaches, cranberry sauce, blueberries, pears, beefsteaks, chicken a la king, and barbecued beef were made (Tripp, 1961). These test packages were used in field tests under both normal and accelerated wear conditions. As reported by Tripp (1961), when these packages were carried by troops over a 4 d a y period in quantities of three, six, or nine per man, failures were 1.5% at the end of the first day and continued at a negligible rate thereafter. The accelerated use tests involved sixteen traversals of a clothing-wear course by men carrying nine retort pouches in field jacket pockets. No failures occurred over the first two traversals. After sixteen traversals, the overall failure rate was 6.1%. Although exact correlation of these test results with actual field experience could be only approximate, they did clearly show that the retort pouch was durable, at least to the degree that further improvement of the concept was warranted. During this early period, in addition to carrying out internal studies, the Quartermaster supported exploratory contract work to get answers to funda-

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

311

mental questions. Proctor and Nickerson (1958) studied a variety of materials including the Mylar (polyester)/aluminum foil/vinyl material preferred at that time for retort pouches for resistance to bacterial penetration. Results of these screening tests showed that penetration through single and laminated films was infrequent, and, where it did occur, dye penetration tests confirmed the existence of a flaw in the material. Creasing of many films was found to increase the frequency of penetrations, but heat sealing had n o effect. Beadle (1959) reported on a program t o develop new and improved seals for flexible packages, emphasizing those made with foil-containing laminates. His significant conclusion was that a round-jaw sealer is a simpler yet better way than flat bars in terms of both the inherent seal strength and the amount of material used in making the seal. Comprehensive summaries of this early military-oriented effort are given by Rubinate (1960, 1961). There was also some academic and commercial interest in retort pouches. Commercial firms had responded and supplied film materials and packaged foods for the military field tests. Could et al. (1962), reporting on studies started in 1960, confirmed the finding that retort processing was feasible using a water cook with superimposed air pressure. Sterilization was obtained in all cases where the National Canners’ Association recommendations for metal cans were followed. A more uniform rate of heat penetration was obtained when a rack was used to support the pouches during retorting. In addition to Quartermaster-supported efforts during this early period, The Massachusetts Institute of Technology researchers contributed to the state of the art. Wornick et al. (1960) established that flexible films offered little resistance to heat conductivity into the packages and that variances among the materials tested were nil. Davis et al. (1960a) explored volume expansion and generation of internal pressure during processing, showing that pressurevolume relationships changed according t o theory and cautioning that the most critical pouch differential pressure situation occurred at the start of the cool cycle. The use of superimposed air pressure prevented bursting. Davis et al. (1960b) reported that heat processing had little effect on film permeability. Tsutsumi (1972) indicated that the advent of pasteurized (up to 100°C) fish, sausage, ham, and other entrees in Japan after World War I1 provided a background that facilitated the development of retort-sterilized items in laminated flexible materials and the marketing of these items. Felmingham (1964) implied that the Metal Box Company in England, while investigating the use of polypropylene for rigid containers, tested Nylon I1 and polypropylene in flexible forms with unsatisfactory results. Their efforts then turned to a Melinex/foil/polyvinyl chloride (PVC) material with whxh they successfully paralleled results obtained in the United States. Nughes et al. (1973) revealed that Star di Agrate Brianza in Italy initiated limited production in 1960 and has enlarged ever since. The Mylar/foil/PVC combination previously cited was the first relatively successful laminate made. Occasional problems were encountered with delamina-

312

RAUNO A. LAMPI

tion and efforts to employ materials that were odor-free. Horst (1961) was optimistic that better films were in the offing. Long (1962) discussed polyester/ foil/vinyl, polyester/foil/polypropylene, polyester, polypropylene, and polyester/foil/polyethylene, hinting that the last combination had a better performance than the others. Shortly thereafter, Continental Can Company presented a successful combination of 12-micron polyester/9-micron foil/75-micron proprietarily modified polyolefin (called C-79). In such a multifaceted technology there is obviously no distinct dividing line between historical developments and more current techniques. An attempt has been made to cover the basic early work on feasibility of the method and definition of problems. Generally, the developments after 1961 have been refinements, assurances of capabilities, and transitions to commercial production.

I l l . MATERIALS A. GENERAL ASPECTS Without proper materials, there could be no flexible packaging of thermoprocessed foods. In addition to the normal requirements of resistance to abuse desired from any flexible material, the materials for retort pouches are differentiated by the need to withstand thermoprocessing temperatures in final package form. Furthermore, because of the desire for a reasonably long shelf life, laminates containing aluminum foil became prime candidates. The quest consequently narrowed down to a search for single resins, copolymers, or polymer blends to be laminated to protect and supplement the barrier properties of aluminum foil, both internally and externally. This, in turn, entailed the selection or development of appropriate adhesive systems. As previously mentioned, the first such laminate was made of polyester (Mylar), foil, and polyvinyl chloride. This was soon superseded by polyester/ foil/modified polyolefin (high-density polyethylene blended with polyisobutylene), polyester/foil/high-density polyethylene, polyester/foil/nylon, and polyester/foil/cast polypropylene. Long (1962) listed fourteen requirements for retort pouch fdms, as follows: 1. 2. 3. 4. 5. 6. 7. 8.

Low gas (oxygen) permeability-less than 1 cc/100 in? /24 hr/atm. Low water vapor transmission rate-less than 0.05 g m / l O O in.'/24 hr. Resistance to temperatures from below 32'F to at least 250'F. Low hydrophilic properties. Low cost of material and package fabrication. Heat sealability over a wide temperature range. Suitability for food use within FDA regulations. Resistance to penetration of fat, oil, or other food components.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

313

9. Dimensional stability and chemical inertness, with no tendency to impair objectionable odor or flavor to foods. 10. Physical strength to resist any handling abuse. 11. Consumer appeal: transparency (or opaqueness, depending on product), gloss, agreeable feel. 12. Capability of being handled on automatic fabricating and filling equipment. 13. Good aging properties. 14. Good printability.

This formidable list, with some refinements and special requirements (Heidelbaugh and Karel, 1970; Nieboer, 1970; Rubinate, 1964; Szczeblowski, 1971; Schulz, 1973; Tsutsumi, 1972; Nughes, 1971a), has remained valid as a guide for film developments. Where applications were such that shelf life could be shortened and the aluminum foil omitted from the laminate, foil-free laminates have been used and are available (Table I). Requirements were relaxed to a degree where gas TABLE I TYPICAL CURRENT RETORT POUCH MATERIALS

9- to 25-micron polyester/9- to 12-micron foil/-75-micron modified polyolefin (C-79) 12- to 25-micron polyester/9- to 25-micron foil/dO- to 85-micron ethylene-propylene copolymers and/or blends 15- to 30-micron polyamide/50- to 75-micron cast polypropylene 15-micron polyamide/70-micron modified polyolefin (C-76) 30- to 40-micron polyamide/50- to 75-micron mediumdensity polyethylene 30- to SO-micron polyamide/50- to 75-micron polypropylene copolymer and/or blend 12-micron polyester/l2-micron foil/l2-micron polyester/75- to 85-micron ethylene-propylene copolymers and/or blends 12-micron polyester/9-micron foil/lS-micron oriented polyamide (Nylon 6)/50-micron polypropylene

(oxygen) permeabilities can be in the neighborhood of 5 to 6 cc/lOO in?/24 hr/atm and moisture vapor transmission rates of 2 to 3 gm/lOO in?/24 hr are acceptable. Other requirements remain strict.

B. EXTRACTIVES The requirement for resistance to thermal processing temperatures of 250" to 265°F necessitated development of new polymer blends and new adhesives systems that could be exposed to a new high-temperature extractives environ-

314

RAUNO A. LAMPI

ment. Szczeblowski and Rubinate (1965), identifying the films studied by Karel and Wogan (1963), reported that three polyolefin/foil materials met the requirements specified in Section 121.2514, Resinous and Polymeric Coatings, of the U. S. Federal Food, Drug and Cosmetic Act. The foil-bearing materials were tested for resistance to food-simulating solvents such as water and acetic acid solutions, under thermal processing temperature conditions (250°F) and n-heptane at 150°F. The extractives were composed primarily either of low molecular weight fractions of the base polymer or of plasticizers contained in the materials. Ferm (1966) continued studies on extractives, delving primarily into the relationship between n-heptane at 150°F and oils or fats at higher temperatures. The regulations stated that “heptane extractivity results must be divided by a factor of five in arriving at the extractivity for a food product.” Ferm confirmed Karel and Wogan’s observation that the 5-1 ratio did not relate to temperature in the 250°F range, varying widely from film to film and with temperature. Ferm postulated that film extraction is a function more of temperature than of the extracting solvent. In spite of such vagaries, the total extractives for candidate films were below limits and met appropriate regulations; two structures were approved by the USDA for limited use with meats and for general use with poultry. These films were used by the U. S. Army Natick Research and Development Command (NARADCOM) for their field feeding tests during 1965 and 1966, and one was used for their production reliability program (1969 to 1973). In late 1974 and early 1975, approvals by the USDA and the FDA for more comprehensive use-it was hoped for full-scale commercialization-were sought by film suppliers. The petitioning data revealed that traces (0.3 to 3.0 parts per billion) of toluene diisocyanate (TDI), one of the reactants necessary for the adhesive, were being extracted by the food-simulating solvents. Since TDI had not previously been regulated, new petitioning and submission of data were deemed necessary, basically to cover the TDI question but also to reaffirm that other extraction components were identified and within acceptable tolerances. As described by Goldfarb (1973), a polyesterisocyanate adhesive is used. The polyester polymer is first formed by the reaction of a dihydric alcohol with a dibasic acid. Then the polymer is mixed with a coreactant diisocyanate (TDI in this instance), sometimes called a catalyst, and heated. The condensation product is an adhesive. This adhesive system is especially applicable to retort pouches, since it has a strong affinity for foil and the isocyanate component reacts with surface hydroxyl and carboxyl groups of the polyester and available carboxyl groups of corona-treated polyolefins. A typical adhesive system, as described in one petition (Anonymous, 1975b), consists of polyethylene phthalate polymers (polyester), 4’4sopropylidene diphenolepichlorohydrin resins (epoxy), and the reaction reaction products of TDI and trimethylolpropane or its chloroformate (polyurethane). Maximum chemical and thermal properties develop within 7 to 14 days (Morton Chemical Company, 1975).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

315

Toluene diisocyanate, although an irritant, is quite nontoxic (LDS0 of 5.8 mg/kg body weight). On review of the extraction data representing the effects of 250°F processing temperatures (following procotols for can-coating extractives) and equilibrium studies (accelerated by using a 120°F storage temperature), toxicity data, manufacturing quality assurances, and environmental impact statements, it is anticipated that a regulation permitting the use of current films in the United States can be written. As of July 1976, however, initial petitions had been rejected on the basis of inadequate data on which to support the safety of the polyester and epoxy migrants, each of which was present in the extracting medium at a level of 2 parts per million. It should be noted that food-simulating extractants and conditions of test vary widely among international regulatory bodies, making direct comparisons difficult and tenuous. Beyond the inherent low extractives property of retortable films, suppliers and researchers (Duxbury et aL, 1970; Thorpe and Atherton, 1972; Nieboer, 1973) recognized from a practical production aspect that quality assurance surveillance is mandatory on roll stock and for preformed pouches to guarantee both freedom from odor and removal of solvent. Both qualitative (sense of smell) and quantitative (chromatographic) test procedures have been proposed to ascertain that the proper steps were taken during lamination and curing to prevent off-odors, flavors, or “taint” in the final food products. C. BACTERIAL PENETRATION Currently, the resistance of retortable films to attack or penetration by bacteria through means other than actual fractures in the film is taken for

FIG. 2. Bacterial penetration apparatus for flat sheets.

316

RAUNO A. LAMPI

FIG. 3. Pouch biotester.

granted. This confidence stems largely from studies first by Proctor and Nickerson (1958) and then by Hartman et at. (1963), Ronsivalli et at. (1963), Lampi (1967), Griffin et al. (1967), and Payne et al. (1969). Materials were studied in flat sheet and final pouch form, in single-ply and laminate structures, and with and without physical manipulation of package or chemical treatment of penetration media to increase the chances of penetration. Apparatus (Fig. 2) was designed to permit the study of flat sheets (Lampi, 1967) and the “biotesting” of pouches (Maunder et at., 1968). The latter apparatus (shown in Fig. 3) flexes pouches and, apparently through the creation of a momentary pressure difference between the exterior and the interior of each pouch, improves the ability of bacteria (Aerobacter aerogenes) to penetrate through known and readily visible punctures (Szczeblowski and Rubinate, 1965) from a previous 50% to 9 0 to 100% (Lampi, 1967). The biotester has proved to be an important laboratory tool, but it is not appropriate for on-line use. These studies revealed that single plies could contain pinholes permitting bacterial penetrations; three-ply foilcontaining laminates inherently did not show penetrations or pinholes; penetration occurred only where a physical break was also detected; creasing (criss-cross folds) increased the incidence of penetration; direct contact with a hot surface increased the penetration sites for single films only; and heat sealing and retorting per se had no effect on the integrity of the material.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

317

Szczeblowski (1971) reported that pouches either buried in soil or left hanging in air at tropical room conditions-85" to 95"F, relative humidity 95%-considered t o be an extreme measure of resistance, remained resistant to attack and penetration by the indigenous bacteria over an eight-month exposure period. D. RETORTABILITY Long's (1962) third requirement as previously listed for retort pouch films was resistance to temperatures of at least 250"F, which, when transposed to the production situation, means retortability in steam, steam-air, or hyperbaric water cooks. Initial evaluations as cited by Rubinate (1964) consisted of simply exposing sheets of films to the intended heating media. Experience soon taught that films that passed the sheet test failed when formed into pouches and filled with product. Consequently, screening tests for materials now include testing for retortability using actual product [or a synthetic mixture simulating the lipid, acid, and flavoring (straight-chain hydrocarbon) composition of candidate products] packaged as desired in a pouch containing no more residual gases than would be permitted under actual production conditions (usually less than 10 cc). Experience also revealed that films passing retortability tests carried out in home-style pressure cookers or small laboratory retorts failed in production-sized retorts. Further refinements were made in retortability evaluations to impose a controlled fluctuation in the counterpressure during laboratory retorting to simulate the controller response cycle typical of much production equipment. The U. S . Army Natick Research and Development Command has found that a 20-psi set point pressure with fluctuations of +2 psi every 2 minutes over a 30-minute process time relates satisfactorily to the necessary film properties. Film specifications of current suppliers (Duxbury et al., 1970; Nieboer, 1973) include retortability or sterilization resistance requirements, which rely on the use of pouches containing water followed by retorting and visual examination for package change or failure. Undoubtedly such procedures are sufficient for most products, materials, and processes for quality assurance and material acceptance testing, but their adequacy should be definitely confirmed. Other requirements for materials, such as food stability (related to barrier properties and material inertness), physical strength, and heat sealability, will be discussed in succeeding sections. These properties relate more appropriately to the production environment and to the final package. E. CURRENT FILMS Table I lists the material constructions currently in favor. It is acknowledged that such a listing constantly changes and increases. It can be safely postulated that nearly any supplier could offer a material to meet specific needs or

318

RAUNO A. LAMPI

customer preferences. The film and adhesives technology for retort pouch materials has become widespread and well established. During the decade of film development from 1963 to 1973, some trends and some changes of interest, which led to better performance or lower costs, can be delineated. In the United States, where there is no current commercial usage, modified polyolefin (Rubinate, 1960), high-density polyethylene, and polyamide (Nylon 11) (Goldfarb, 1970) were used successfully in the development of military rations and some industry products. Of these materials, the polyamide, because of its cost, and the high-density polyethylene, because of converting difficulties and lack of commercial interest, are no longer widely used. The modified polyolefin continues to be an acceptable inner lamina; however, resin blending, extrusion, and lamination to foil require close surveillance for adherence to exacting procedures. This material dominates Japanese usage (Tsutsumi, 1972) and has been successfully employed in Europe. Polypropylene had early proponents (Felmingham, 1964; Nieboer, 1973; Nughes, 1973) in Europe. Thorpe and Atherton (1972) excluded Nylon 11, high-density polyethylene (HDPE), and rubber-modified HDPE in favor of cast polypropylene for their comprehensive retort pouch studies. They also successfully included modified polypropylene copolymers. STAR (Nughes, 1973) in Italy started with cast polypropylene, but now utilize a polypropylene copolymer. The U. S. Army Natick Research and Development Command, gearing their evaluations to rigorous operational ration requirements, rejected early cast polypropylene and polypropylene blends as the sealant layer because of embrittlement following retorting and lack of adequate resistance to abuse at low temperatures. Recently (Lampi, 1973), newer polypropylene copolymers have been evaluated with results easily equal to those achieved with the former standard, modified polyolefin (HDPE-polyisobutylene). Nieboer (1973) indicates that polypropylene copolymer is now replacing cast polypropylene. Currently, the two prevailing three-ply laminates are polyester/foil/modified polyolefin and polyester/foil/polypropylene-ethylene copolymer. It would appear from the foregoing discussion, and as Kelsey ( 1 9 7 4 ~ has ) also observed, that each film has its proponents. For nonfoil applications, such as vegetables with short shelf lives, cast polypropylene reportedly (Nieboer , 1973) remains in favor as the sealant layer, although medium-density polyethylene has been used for potatoes. Killoran (1974) has reported that bond strengths between the inner ply and the aluminum foil and seal strengths of retortable materials could be significantly increased by gamma radiation at 6 megarads (Mrads) without adverse effects on retortability or heat seal strengths. At 1 8 Mrads, sealability was reduced. Another trend noted has been the tailoring of components to meet specific

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

319

product and performance requirements. Some packs initially had thicker aluminum foil layers in the 12- to 18-micron range. Currently, a 9-micron aluminum thickness predominates, with evidence of better performance (Turtle and Alderson, 1968; Nughes, 1973). The thicker foils tended to result in more flexure failures and, because of their greater rigidity, formed more permanent creases or peaks that acted as loci for abrasion failures. For some high-lipid-content products or highly essential oil-flavored products, an additional ply of polyester has been used between the inner sealant layer and the foil to form an additional barrier and reduce tendencies for delamination. It has been observed that the thickness of the inner ply differs among specific products from Japanese packers, apparently on the basis of proprietary studies showing satisfactory performance with less than a 75-micron thickness for certain items. In Japan (Tsutsumi, 1975) products are being commercially packed in a film described as 12-micro polyester/9-micron aluminum foil/l5-micron oriented Nylon 6/50-micron polypropylene and processed at temperatures up to 275°F for times ranging from 2.7 to 9 minutes. In addition to the development of heat-resistant films, the lamination or converting process has been most critical, but one where much of the technology is proprietary and much of the processing is art. Most failures in materials during retorting have been at the adhesive locale, often aided by dimensional instability (shrinking) of one of the polymer plies or inconsistent control of the retort counterpressure. The adhesive system cited by Goldfarb (1970), a two-part catalytically cured, thermosetting polyurethane type, appears to prevail. Martin (1966) reported on the availability of catalyzed polyester, alkyd, urea, and melamine formaldehyde types of adhesive systems with extremely high heat resistance, but none of these appear to be used. Nieboer (1970), in tracing the development of suitable lamination techniques, lists with equal emphasis not only the curing adhesive system itself, but also pretreatment of films t o promote adhesion (corona discharge, use of primer); process conditions on the laminating machinery; application methods and coating weight applied; and curing-off techniques. To this list should be added the necessity of obtaining properly blended or prepared individual component films. Minor variations in melt index and thickness, for example, have caused difficulties. Since thermoprocessing requires a measurably better performance from laminates that are often exposed to conditions that test their physical capabilities to their limits, quality assurance procedures must be strict and religiously followed. Printing is another problem; the ink itself must be stable, and its presence should not (since it is located between the outer ply and the adhesive) interfere with the integrity of the laminate bond. Judging from the performance and appearance of current packages, these requirements have been met. Attractively printed packages are commercially available in Europe and Japan. To a degree,

320

RAUNO A. LAMPI

the sales appeal function of the printed matter and graphics can be transferred t o the outer carton. Table I1 reflects the ranges of values for the major performance-related specifications for suitable three-ply retort pouch films. Nonfoil films differ in that oxygen permeabilities of 5.5 to 6.0 cm3 /m2 /24 hr prevail, and the shelf life of the product is accordingly limited to 1 to 6 months. Other specifications required by some suppliers include visual examination for delamination or blisters, odor by subjective smelling, elongation, tensile strength, Mullen burst, tear resistance, and block temperature, but those listed in Table I1 are most directly related to performance. All components and the final structure obviously have to meet the safety and extractives criteria of the FDA or equivalent regulatory agency. Table I1 presents specifications on major performance variables that are appropriate for monitoring films during regular procurements. However, full qualification of any candidate material should be based on formation of a pouch, use of TABLE I1 RANGE OF CURRENT PERFORMANCE REQUIREMENTS FOR STERILIZABLE MATERIALS Criteria Requirement Sterilization temperature Oxygen permeability Moisture vapor transmission rate Seal strength (tensile)b Bond strengthb Heat seal range Thickness tolerances

English

Metric

240" -300" F

116"-145"C

= O cc/100 in.'/24

=O cc/m'/24 hr/atm

hr/atm' -0 gm/100 in.'/24 h P 11-20 lb/in. 0.8-2.8 lb/in. 320"-500"F +-0.0001in. 10% of value iO.5 gm/lOO in.'

Burst test

28 lb/in. seal (method l)c 35 psi for 30 secd

Residual solvent (taint)

2 mg/100 in.' (method 4)c 0.5 ppm as tohold

-0 gm/mz /24 hr 2-3.5 kg/lO mm 150-500 gm/lO mm 160"-260" C *2 microns (inner ply only) 10%of value k7.0 gm/m2 (inner ply only) 7.5 kg/lS-mm seal (method 1) 17.2 X lo4 Pascals for 30 sec 30 mg/m' (method 4)

0.5 ppm as toluol

aAssuming shelf life of 6 months or more is desired; zero based on sensitivity of prevailing test procedures. k o v e r s both machine and transverse directions. CDRG methods. dReference: Duxbury et al. (1970).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

321

actual food, and exposure to periodic pressure fluctuations of 2 psi at the desired process temperature. The usual storage tests should be applied to confirm achievable shelf life. Above all, shipping or simulated handling tests, geared to the degree of rough handling expected, should be performed. The NARADCOM testing of retort pouches for operational military rations involves screening candidate materials in their final form (retort pouch of food enclosed in a 16-point Kraftboard folder or carton) by subjecting them to the following sequential test procedures: (1) Cartons (shipping cases of seventy-two foldered or cartoned pouches) vibrated at 1 G for 1 hour. (2) Cartons dropped per ASTM, D-775-68, Objective B (ten random drops from a height of 18 inches). Following the abuse cycles, pouches are visually inspected for failure, incubated for 14 days at 100"F, and then biotested. Based on the performance of cans subjected to the same abuse, a tentative criterion for satisfactory performance of 2%or less failures has been set. The Animal Products Health Inspection Service (APHIS) of the U.S. Department of Agriculture (Sloan, 1973; Kelsey, 1974c) prepared similar package performance test procedures and criteria adjusted to the lesser abuse faced in strictly commercial channels. Their approach included individual pouch drops. Analogous tests, consisting in actual shipping of case lots, followed by unloading, storage, and return shipping, have been reported (Nughes, 1973). Toyo Seikan Kaisha, Ltd. (1973a), in discussing outer packaging, presents vibration test procedures that could be used similarly to test performance. Performance testing will be discussed in greater detail later under Package Durability. Emphasis is placed on relating package performance, even for the criteria listed in Table 11, to actual or simulated handling tests, since, in reality and in comparison, all else is relative. The supporting physical measurements are essential to monitor and rapidly assess expected performance, but, improperly interpreted, they could be unduly restrictive or, conversely, meaningless. Schulz (1973) showed the effect of retorting on the bond strength of one material-a drop from 600 gm/cm to 200 gm/cm. Other materials exhibit a reverse trend-an increase on retorting. Similar differences between two totally satisfactory films (in terms of handling performance) occur in bond strengths over storage periods. Indications are that, as long as laminate bonds in seal areas remain high, bonds in the body area may be very low (tack) without any adverse effects on overall package performance. The performance characteristics of the foil-containing materials listed in Table I are covered by the ranges of performance levels in Table 11. Undoubtedly, the variability-for example, bond strengths from 150 to 500 gm per 10 mmreflects the particular characteristics of each supplier's material as matched to satisfactory final performance. These variabilities among materials and suppliers must be recognized; low values do not necessarily indicate low performance per se.

322

RAUNO A. LAMPI

To summarize, suitable materials have been developed for packaging thermoprocessed foods and even refined in structure to meet specific product requirements. Technology is progressing to create relevant test procedures and performance criteria.

IV. P A C K A G E DESIGNS The predominant design of the retort pouch has been governed by the anticipated marketing or utilitarian advantages: A flat shape (cross section) to obtain high quality through sterility with minimum overcooking of peripheral volumes, and to gain convenience through fast reheating for consumption. A ready-to-eat “wet” food requiring a high degree of package integrity, especially from the heat-sealing aspects. Dimensions that permit efficient usage of shelf and display volumes; for the military, compatibility with field clothing without restriction of physical movement.

The four-seal flat design (Fig. 1) has been universal from the beginning. Fin seal designs and gusset features create multiple seal junctions where leakage is more apt to occur and defects are difficult to detect. Volume and dimension (length and width) relationships are approximately as follows: Four-ounce package-4% X 6 inches outside dimensions with 3/s-inch seals Eight-ounce package-5% X 7 inches outside dimensions with 3/s -inch seals Sixteen-ounce package-6% X 8%inches outside dimensions with 3/8 -inch seals Thirty-two-ounce package-8 X 10 inches outside dimensions with 3/8 -inch seals inch for the 4-ounce size to 15/16 The nominal thickness would range from inches for the 32-ounce size. Laboratory tests have indicated that packages over 30 ounces in net weight require special materials, cartoning, and handling. Within the basic four-seal design parameters, variations and features include: Tear notches for easy opening. Some “unused” package volume in keeping with filling specifications, such as no filling within 1% inches of the open top of the package (Duxbury et al., 1970) to minimize splash or other product contamination of the opposing seal areas. This top area can be folded over to reduce the outer carton size. Secondary top seals with or without trimming to eliminate an unsealed flap or lip (Nieboer, 1970).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

323

Rounded internal and/or external pouch corners to improve appearance and performance and to reduce incidence of interpouch punctures. Combinations of clear and opaque films, featuring one type for each pouch surface. Variations in graphics from complete printing on the pouch, to simple coding on the pouch with extensive printing on the outer carton. A feature of the four-seal design which would also be characteristic of other relatively flat designs, but not to the same degree with designs that are rounder or more block-shaped, is the ease with which swelling due to gas-producing spoilage is observed. C. 0. Payne, Jr. (private communication, 1964) and G. L. Schulz (private communication, 1973) have both photographically recorded that pouches d o swell and that swollen pouches are easy to detect. Schulz established that for identical inoculum and net weights a retort pouch could easily be identified as a swell in 48 hours, whereas a can, where the concave lid required a pressure buildup to flip, took 72 hours for a tenuous identification as a swell. There have been a few instances, such as vegetables in foil-free pouches as described by Nieboer (1970), where individual pouches were not supplied with an outer carton or wrap for graphics, display, and added protection. However, most retort pouches are enclosed in some type of outer box or tray. NARADCOM, for their developmental operational ration items (Szczeblowski, 1971), consider a 16-point Kraftboard folder into which the pouch is glued as an integral part of the total package. A lock-closure feature permits nondestructive visual examination and reclosure. Rubinate (1964) reported that the performance of pouches glued to the folder was four times as good as that of unglued pouches in tests simulating military handling. High-speed photography has shown that the converging ends of the folder restrained the pouch contents and appeared to reduce immediate stress on seal areas on impact in drop tests. To prevent dirt or other foreign matter from getting into the folder and causing abrasion damage, plans include use of a modified glued folder or a flat, glued carton. Commercially, rectangular flat cartons predominate-some with perforated tear lines on the large flat surface to facilitate opening and removal of the pouch from the carton. Spot gluing of pouches to carton wails has been used, although a great many “glued” pouches have worked themselves free during shipment. Goldfarb (1971a) and Thorpe and Atherton (1972), the latter as the result of a direct comparison, concluded that, at least for commercial distribution, gluing of pouches to cartons made no difference and was not necessary, Automatic cartoning has been reported. Toyo Seikan Kaisha, Ltd. (1973a) recommends an outer carton of 300 gm/m2 base weight with glued flaps. The dimensions are specified on the basis of drop and vibration tests and should be 0 to 10 mm shorter than the pouch dimensions

324

RAUNO A. LAMP1

for the length and width. However, the thickness should be 6 t o 10 mm more than that of the pouch. Horizontal packing for shipment is recommended. A relatively recent innovation to extend the shelf life of products in foil-free films and also to make the total package more attractive has been the use of polymer trays with heat-sealed film lids to contain one or two retort pouches. In the United Kingdom, polystyrene trays are lidded with 450 MXXT cellulose film. In France, thermoformed 500-micron PVC trays with polyester-PVdC lids contain clear film pouches. The air in this last tray-pack example is replaced with nitrogen gas to gain a guaranteed shelf life of 6 months for carrots and potatoes. Laminated pouches have been formed from a single web with a bottom gusset which, when extended laterally, provides a base for a free-standing pouch (Anonymous, 1974b). These pouches can be formed on a relatively standard horizontal form-fill-seal type of equipment by the inclusion of a second plow to give a bottom W in lieu of a V, the use of dual bottom sealers, and special side seal formation. Three-ply heat-sterilizable films can be formed into pouches of this free-standing design. Initial applications appear to be juices and drinks.

V. FOOD PRODUCT DEVELOPMENT The flat shape of the package and the resulting shorter process time make retort pouches applicable and advantageous for a wide variety of foods, including fruits, meats, and vegetables along with formulated mixtures thereof. Experience has proved that most particulate items do not lose their integrity during handling in spite of the flexible nature of the packaging material; the packaging of relatively delicate items, therefore, is possible. A variety of bakery items are suitable. Exceptions appear to be foods with bones (although coq au vin is packed), shells, or other sharp, protruding components. Because thermoprocessing is involved, there is a tendency, from the technology aspect, in determining the potential for a candidate product to make a comparison with more conventionally canned items. In a few instances, a comparison is made with boil-in-bag frozen items, representing the end use of both. It would appear more appropriate and objective if the product potential for, or selection of, candidate foods were made on the unique merits of retort pouches. In comparison with some canned items, the retort pouch, with its high-temperature, short-time approach, may result in better quality. In competition with frozen items, convenience and refrigeration-free storage become greater advantages. Judgments should be made on an individual basis. Thorpe and Atherton (1972) agree that retort pouches require a separate technology and choice of products. Over the decade that retort pouches have been under development in the United States and have progressed to commercialization in Japan (Tsutsumi,

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

325

1972), Scandinavia, Italy, West Germany, France, and Great Britain (Nieboer, 1973), and Finland and Israel (Dibbern, 1973), the predominant products have been fabricated meats (sausages) and solid meats in sauce (from stews to slices with gravy). Ethnic flavor inflections prevail. Vegetables in foil-free pouches are also offered.

A. TYPICAL PRODUCTS A detailed review of product listings such as those given by Toyo Seikan Kaisha, Ltd. (1973a), Nieboer (1973), Thorpe and Atherton (1972), and Lampi (1973) and by various trade journals and papers revealed a minimum of eighty brand-name products. Thorpe and Atherton categorized products according to heat-penetration characteristics into those without covering liquor and those with a covering liquor of low viscosity (convection heating). Lampi (1973) categorized by filling modes: extrudable, pumpable: placeable, and dual (pumpable and placeable). Products are available in each of the above groupings, indicating a wide versatility for retort-pouched foods. An arbitrary classification of more than eighty products, given in Table 111, covers the following types: 1 . Sauces or gravies containing pieces or slices of meats, poultry, seafood, and/or vegetables. The sauces include curries, tomato sauces, cream sauces, stroganoff, goulash, and brown gravies with and without wines and specified flavorings to meet national flavor, ethnic, or gourmet images. Many of these items most accurately fit into Nieboer’s terminology as “ready meals.” Most meat pieces fall in the typical stew category; however, many slices, meatballs, and sausages are also offered with a sauce. 2. Straight sauces. These items, such as tomato, cream, and meat sauces, are intended as covering for items such as rice or pasta, which have been offered in dry or precooked form as a companion package to the retort-pouched sauce. As was pointed out by Rees (1973), cream sauces are susceptible to thermal degradation and therefore become a prime candidate for retort pouches. 3. Meats with minimum fluid. Items in this category include sliced luncheon meats, meat loaves, sausages, beefsteaks, and ham slices. Formulations are modified, and precooking, searing, or frying may also be used to render off unsightly fat and extraneous free fluid. Pet foods are included in this category. 4. Vegetables. Vegetables are offered generally in foil-free laminates with a shorter shelf life, ranging from a few weeks to 5 months, depending on package variations. Some are provided as ingredients in gourmet sauces. The marketing intent of these items is the replacement of fresh or frozen items at competitive prices and for convenience. In Canda, Europe, and temporarily in the United States (stopped because of FDA review of the films), peeled and cut potatoes in both foil (6 to 9 months shelf life) and foil-free (3 to 6 months shelf life)

326

RAUNO A. LAMP1 TABLE I l l COMMERCIALLY FEASIBLE RETORT-POUCHED PRODUCTS ~~

Type of product Sauces with meat, poultry, seafood and/or vegetables (ready meals)

Sauces

Meats (minimum liquid)

Vegetables

Fruits

soups Bakery items

Typical products Beef stew Stewed veal with peas Hungarian goulash Rice with seafood Assorted meat curries Chicken B la king Sloppy Joe Bar-B-Q chicken Duckling with chestnuts Prepared tomato sauces Rice sauce Cream sauces (with vegetables) Ham Meatballs Chicken hamburger Frankfurters Beef loaf Pet foods Potatoes Sauerkraut Beetroots Beans in tomato sauce Crushed pineapple Apples Applesauce Variety Fruitcake Spice cake Pound cake Rhum baba

Beef stroganoff Sweet and sour pork Sukiyaki Beef bordelaise Bamboo shoots in sauce Beef slices in barbecue sauce Meatballs in barbecue sauce Roasted chicken in sauce Rabbit with rice Bean-cured seasoning sauce Meat sauce (with and without mushrooms) Pork luncheon fingers Sliced pork luncheon meat Beefsteak Ham and chicken loaf Tuna meat (in oil) Carrots (some in sauces) Mushrooms (some in sauces) Celery in cheese sauce Fried rice Rhubarb Prunes Shark’s fin Cherry, maple, chocolate, orange and pineapple nut cakes

laminate pouches have been marketed to the institutional trade. Net weights have ranged from 1.5 to 6 kg; a typical pack in the United States has been 2.25 kg (5 pounds). 5. Fruits. Table 111 indicates those fruits currently on the market or intended for United States military rations. There appears to be little limit on fruits if antibrowning precautions are taken, in lieu of dependence on exposure to the reducing action of the tin plate of cans. Some development efforts have centered on a “dry” pack in which dry sugar and fruit are packaged. The process releases some juices to dissolve the predetermined quantities of sugar.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

327

6 . Soups. Soups differ from the first grouping only in viscosity of the fluid component. There appear to be few restrictions on the variety of soups that can be flexibly packaged. 7. Bakery items. Aside from confections such as rice cakes and rhum baba, the bakery products listed are those developed by NARADCOM for operational rations. The preparation and processing systems that result in a uniform, porous, leavened structure are described under Retorting. Bread, requiring a yeast-based leavening system and flavor note, has been prepared but not with adequate overall hedonic acceptability. Schotte (1974) has, however, reported the successful sterilization of bread by hot-air oven procedures. Temperatures range between 221" and 266"F, and the sterilization times are 1 to 4 hours.

B. HIGH-TEMPERATURE, SHORT-TIME PRODUCTS The foods discussed thus far have been retorted at temperatures in the 240" to 250°F range. There are recent film developments (Komatsu and Yamaguchi, 1975) that permit processing in the 275" to 293°F range. Studies report that, at least for Japanese criteria, beef, pork, mutton, and chicken are prime candidates for this HT/ST approach. Vegetables have been shown to retain a favorable color, and seafoods, such as crab, shrimp, and whitefish, are good candidates. Vegetable protein and starch-based products have shown doubtful results. The significant retention of quality attributes and nutrients for representative Japanese products processed at various temperatures to equal lethality levels (Fo of 3.3 to 3.5) was reported by Komatsu and Yamaguchj (1975). Storage stability data were not reported.

C. UTILITY The retort pouch has been used predominantly for family or home use for entree items and selected vegetables. There are additional potential commercial (ready-to-use ingredients such as fruits for baking), institutional (hospitals, restaurants, schools), and special uses (meals in space, campers' meals, emergency foods for forest firefighters, mine caches, etc). Regardless of the specific end use, much of the utility of the retort pouch is related to its rapid preparation heating characteristics. Costanza (1971) ran immersion heating trials starting with 80°F water and boiling water. His results indicated that when both water and product were initially at 80°F the center temperature of a sauce-type product (Sloppy Joe) lagged behind the water temperature during heating by 24°F. By the time the water boiled, the product temperature was 188"F, too hot for immediate consumption. With a solid item such as frankfurters, the center temperature of the product lagged by 28OF. If the water is brought to

328

RAUNO A. LAMPI

boiling before immersion of the packaged product, the heating time for a sauce to reach 180'F is 4 minutes, and for a solid it is 6 minutes. Costanza (1971) and Bows (1973) determined that retort pouches used as rations during military operations could be heated by holding them over an open flame (with movement 2 inches above the flame) or by using the engine heat of a motor vehicle (intake and exhaust manifolds).

D. QUALITY AND STABILITY The specific quality attributes of retort pouches are related to their thin cross section and the optimization of thermoprocessing without peripheral overcooking that this thnness provides. Quantification of quality is relative and nebulous at best; therefore, perhaps the best evidence of achievable quality is the number of products, including many in the gourmet category, that are successfully being marketed. In many regions, conventional preservation methods for frozen and canned foods have not been as well established as in, for example, the United States, but all methods are growing, and according to reported volume and growth rates (Tsutsumi, 1972; Nieboer, 1973) the retort pouch is gaining acceptance. The U.S. Army, in its last field test (Lampi, 1974) before accepting the retort pouch as a replacement for cans in its operational rations, did gather comparative hedonic rating data. As shown in Fig. 4, main entrke items in the retort pouch rated higher than those in cans in both the Temperate and Arctic zone tests. Stability cannot be divorced from quality. For commercial success, quality must be initially high and must remain that way. An initially poor, but stable,

RATING

MEAL-READY-TO-EAT (FLEXIBLE PACKAGES)

LIKE EXTREMELY

9

LIKE VERY MUCH

a

LIKE MODERATELY

7

LIKE SLIGHTLY

6

NEITHER LIKE NOR OlSLlKE

5

DISLIKE SLIGHTLY

4

DISLIKE MODERATELY

3

DISLIKE VERY MUCH

2

MSLIKE EXTREMELY

1 -

i 7.8

Ly

I-

MEAL, CDMBAT~ND. (CANS)

7.7 7.0

+ 4 W

P Ly P

Bc

1 5.9

-

FIG. 4. Generalized acceptance of thermoprocessed items; Development test 11, meat entrees (hot).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

329

TABLE IV APPROXIMATE SHELF LIFE OF RETORT-POUCHED FOODS

Pouch constructionu 30-micron nylon/60-micron cast polypropylene 40-micron nylon/l2-micron PVdC/S0-micron polypropylene 30-micron nylon/60-micron cast polypropylene 12-micron polyester/9-micron foil/75 -micron polypropylene or polyethylene blend

Approximate shelf life

Outer package

Products

Paperboard carton

Vegetables

Paperboard carton

Meat entrees 5 months at 20°C Vegetables

Formed polymer tray flushed with nitrogen Paperboard carton

Vegetables

3 months at 20°C

Entrees Meats

2 years at 20°C commercially 8 years at 20°C for military rations

1 month at 20°C

uTypical constructions; materials and thicknesses will vary to some degree among packages and specific products.

product is useless. Table IV, without specific product enumeration, gives the approximate shelf life expected with various packaging variables.

I. Foil-Free Laminations The purpose of using transparent, foil-free packages for vegetables and some fruits has been to compete with fresh and frozen produce where a short shelf life is acceptable. Thorpe and Atherton (1972) found that fruits and vegetables in foil-free pouches varied to some extent in their flavor, texture, and color retention compared with canned controls, but that generally a 2- to 4-month shelf life could be expected. Storage away from light and at 37'F usually helped flavor and color stability, but adversely affected texture in some instances (potatoes, cherry pie filling). Changes in chemical components confirm the above subjective findings on stability. Komatsu et al. (1970), using model systems of ascorbic acid, dehydroascorbic acid, glycine, and glucose in various combinations, indicated that significant darkening of the substrate and losses of ascorbic acid occurred within 60 days with a relatively high-barrier, yet foil-free film of 12-micron coated polyester/70-micron polyethylene (oxygen transmission rate of 36 cc/m2124 hr/atm at 30°C,40% R.H.). Films with greater oxygen permeabilities manifested more rapid changes correlated to their permeabilities. Yamaguchi el al. (1971) using cottonseed oil as a model product showed that the peroxide value increased dramatically from an initial 5.0 to 120.0 after 2 months of storage at room temperature in a foil-free two-ply laminate pouch. Heidelbaugh and Karel

330

RAUNO A. LAMPI

(1970) found similar results with two actual food products-cranberry sauce, and vegetables and bacon. Changes in hedonic ratings, browning (optical density at 400 mp), and ascorbic acid for the cranberry sauce, and in hedonic ratings, thiobarbituric acid values, peroxide numbers, and ascorbic acid levels for the vegetables and bacon, after 5 weeks at 27°C (80% R.H.), indicated limitations in the foil-free materials.

2. Foil Laminates The inclusion of a thin aluminum foil layer (9 microns) dramatically improves the shelf life of retort pouches. A shelf life of over 2 years is easily assured. In all the studies reported above on foil-free films the controls were conventional cans (except for the work of Komatsu et al., 1970), and another variable, foilcontaining films, was included. In all instances, chemical changes were negligible with foil laminates and were generally no greater than those observed with cans. In some instances (Yamaguchi et al., 1971), the pouches offered greater protection than the cans. The peroxide value of 3% soybean oil in water in the foil pouch was initially lower than that in the can and remained that way over a 60-day storage period at 30°C. The retention of 0-carotene followed a similar pattern. Frequently, hedonic ratings for retort pouches exceeded those for cans. Heidelbaugh and Karel(l970) indicated such a situation for cranberry sauce and confirmed the earlier findings of Rubinate (1964). Not only did the hedonic rating remain high, but the pouch integrity outlasted that of the can. Thorpe and Atherton (1972) reported similar findings with rhubarb in syrup. Thorpe and Atherton (1972) reported that storage studies of formulated foods in foil laminate pouches revealed a shelf life of 18 months or longer was possible; that the pouches generally exhibited marked advantages in quality over canned controls; and that, specifically, the delicate flavors of one or more of the individual constituents of the foods were retained better in the pouches than in the cans. Table V (Szczeblowski, 1971) shows that candidate items for military rations are stable, exhibiting, with very few exceptions, no significant changes in hedonic ratings over a 2-year period at 70°F or even at 100°F for 12 months. If storage stability beyond 2 years is required, apparently such requirements can be met. Dymit (1973) reported that after 8 years shrimp in a foil laminate was superior in flavor and color to the canned item. NARADCOM has stored available retort-pouched items for 6 to 7 years at room temperature, as shown in Table VI, with some lowering of hedonic ratings but none below the minimally acceptable (neither like nor dislike) level of 5.0. These studies are continuing until confident extrapolation to determine an end point is possible.

331

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS TABLE V

HEDONIC RATINGS OF FLEXIBLY PACKAGED, HEAT-PROCESSED FOODS BEFORE AND AFTER STORAGE

Item

Initial rating

Rating after 18 months at 70°F

Rating after 12 months at 100°F

Ham and chicken Meat loaf Beef steak Chicken loaf Pork sausage links Frankfurters Sliced beef with barbecue sauce Pickle-flavored sauce with ground beef Chicken A la king Beef stew Pineapple Beans and tomato sauce Corn

7.4 6.4 7.1 7.4 6.3 7.2 7.0 6.4 7.4 7.2 8.0 6.8 6.0

7.0 6.9 6.8 7.0 7.1 6.9 6.9 6.5 7.1 7.1 7.7 7.4 6.0

7.1 6.6 6.0 7.0 6.8 5.9 6.7 6.2 7.1 6.6 6.5 7.3 5.4

For the most part, shelf life is governed by the barrier properties of the film. Since it is desirable to keep residual gases (headspace) as low as feasible to avoid pouch failure during retorting, the effects of these residual gases on stability have not been widely researched. Komatsu et al. (1970) have published data showing the effects of initial headspace air contents on a food-simulating system containing 100 mg% ascorbic acid. Measurements taken immediately after retorting at 240'F for 35 minutes showed that, with no gases, 95.5% of the ascorbic acid remained; with 5 cc, 92.3%;with 10 cc, 83.7%; and with 20 cc, 75.7%. The effects of subsequent storage, if any, were not given. NARADCOM TABLE V1 LONG-TERM STORAGE STABILITY O F RETORT-POUCHED FOODS'

Products

Stored 3 years

Frankfurters Beef stew Sloppy Joe Fruitcake Pork sausage Chicken loaf Beef slices in barbecue sauce

6.3 5.9 6.0 6.7 5.5 5.8 6.8

'Values represent hedonic ratings as defined in Fig. 4.

Stored 6 years

Stored 7 years

5.7 5.7 5.9 5.9 5-4 5.3 5.5

332

RAUNO A. LAMPI

has found that certain sensitive items such as frankfurters require very low residual gas levels for adequate stability. Thomas and Sherman (1969) subjected thirteen menus of a military operational ration to one, three, and six freeze-thaw cycles (22°C to -54°C and return). Individual components were blended into a composite sample for each menu; therefore, the stability characteristics of the thermoprocessed meat and bakery items, freeze-dried fruits, and spreads were averaged in the study. Results varied among vitamins and the number of freeze-thaw cycles experienced. In summary, vitamin losses were similar to those occurring during other storage conditions, leaving one to conclude that freeze-thaw cycles per se, although statistically having a significant effect, are not important in terms of product

FILLING NO. 2

CONTINUOUS RETORTING

#

I I

TACK SEAL

I 1

I

RETORTING

1

[

SHAPING

!

RETORT UNLOADING~

I

INSPECTION POINT

CARTONING

FIG. 5. Block diagram of major retort pouch production operations. Not all unit operations are required for specific products. The sequences may also vary.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

333

stability. McNutt and Lee (1974) reported that six freeze-thaw cycles had little effect on the hedonic acceptance ratings of eighteen retort-pouched military ration items in comparison with controls held at 40°F.

VI. PRODUCTION SYSTEMS A. OVERVIEW: A SYSTEMS APPROACH There is wide recognition that the transition of the retort pouch concept from the laboratory to a viable commercial entity requires a comprehensive systems engineering approach. The products are “wet” (high water activity), and violation of package integrity by improper or inadequate production methodology, quality assurance, or post-plant performance can lead to serious product deficiencies and therefore economic losses. The total system, wluch consists of the production operations listed in Fig. 5, has to be, minimally, as reliable (in terms of low numbers of failures) as conventional metal can packing lines. Additionally, there were no century of experience and no recognized performance specifications available as there are for the conventional metal can. Therefore, specific definitions, criteria, and tolerances loose enough to permit establishment of realistic requirements for manufacturing equipment, and yet tight enough to assure package integrity and durability during distribution and consumer usage, were not known. Furthermore, the packer, the film supplier, and the equipment manufacturer were traditionally not used to a comprehensive, cooperative approach. Proprietary interests predominated. In the current industrial environment, nevertheless, a systems approach was recognized as the best. Another early acknowledgment relative to equipment and production techniques was that the basic technology was known, and standard equipment concepts could be used, but they had to perform at higher levels. This was

FIG. 6. Intermittent-motion form-fill-seal packaging machine. Courtesy o f the Packaging Machinery Division, The Rexham Corporation, Rockford, Illinois.

334

RAUNO A. LAMPI

confirmed by the Natick kboratories/Swift & Co. program (Lampi and Rubinate, 1973) in which standard equipment was modified to perform, generally, one magnitude better than previously required. Supportive evidence for this thesis is manifested by Nughes (1971a) in a U.S. Patent on a method for WEB EDGE SENSORS FOR AUTOMATIC WEBCENTERING

fi

,POUCH-FORMING

PLOW

BOTTOM HEAT SEAL COOL BOTTOM SEAL SIDE HEAT SEALCOOL SIDE SEAL PUNCH TEAR NOTCH PHOTO SENSE

CUT-OFF Poucn PICKUPSTATION AIRJET OPENING STATION POUCH-FORMING STATION OPEN STATION FILL STATION NO I

FILL STATION NO 2 OPEN STATION TOP SEAL STATION (OPTIONAL) OPEN STATION

FIG. 7. Schematic drawing of package-forming (top) and package-filling (bottom) stations.

TABLE VII RETORT POUCH PRODUCTION SYSTEMS

Semi-automatic (manual transfer from filler to sealer)

Unit operation

Pouch formation Performed with Thurlings or other pouch presenters Filling-fluids Positive displacement pump (Leonhardt, Bock, or others) Filling-solids Volumetric or manual

Tack or partial seal Air removal

Frequently used

Reynolds-Larsen Bartelt IM pouch former

Natick-Swift Bartlet IM pouch former

Positive displacement Positive displacement Pump Pump Volumetric

Volumetric with plunger

No

No

Modified vacuum canclosing maching using pouch carrier Bartelt IM Modified vacuum canSwissvac, Multivac, etc. machine closing machine (impulse) plus band (hot bar) sealer Carriers on conveyor Accumulated pouch Wire, trays, perforated chains carriers in trays sheet-metal trays (vertical) Optional (steam, steamHydrolok Batch horizontal air, or water), horizontal (steam-air) (water cook) or vertical Swissvac, Multivac B-6, Swissvac Duo Mark I1

Closure seal

Retort packinf

Retort

~

~

~~

Tapered tube (snorkel)

~~

‘Manual placement in carriers, trays, or racks.

Japan (Toyo Seikan) Preformed pouches

Positive displacement pump on Yokohama Y-11-A Volumetric or manual on Yokohama Y-11-A or TOYOTT-6 No

Italy (STAR) ACMA pouch former

Volumetric on ACMA Yes

Tensioning of pouch to form squeezing action on Y-11-A “Triple” seal on rotary filler sealer (hot bar) on Y-71-A Horizontal on perforated trays

Goglio GL-25 compartment

Batch horizontal (steam-air)

Batch horizontal (steam-air)

Goglio GL-25 (impulse) (Unknown)

336

RAUNO A. LAMPI

packaging food products in flexible containers, where not only are film structures described, but all line functions to interface with available equipment are covered. Tsutsumi (1972) describes an identical approach in Japan. In Asia, Europe, and North America, film suppliers offer total processing system support to assure proper use of films. Not only are there interrelationships between operations, such as the effect of the cleanliness of the filling process on the adequacy of the closure seal, or the effect of the efficiency of air removal on the required retort counterpressure, but many of these functions are carried out integrally on one piece of machinery-that is, on a single chassis-and require synchronization. An example of such multifunctional equipment as is used for retort pouches is the intermittent-motion form-fillseal machine, one style of which is shown in Fig. 6 (Rexham Corporation, 1974). The functions it normally performs are shown in Fig. 7. There are versions of such equipment (ACMA S.p.A., 1973; Hamac-Holler Bosch Group, 1974) in use for retort pouches where, after the pouch cutoffstep, the remainder of the operations are performed on the periphery of a rotary table or drive mechanism rather than in a straight-line mode. Although there are feasible, low-production-rate lines that consist mainly of single-function pieces of equipment, several of the multifunctional machines have already been combined to form complete production systems. Before delving more deeply into the major individual production operations of sealing, filling, air removal, and retorting, a brief description of the currently identifiable production systems tabulated in Table VII and a general assessment of their performance characteristics will be given. There are, and will continue to be, variations and improvements. Plant layouts have been published, and it is safe to state that any film supplier and many equipment manufacturers can offer recommendations and layouts for specific products and physical plants. The intent is to identify some prevailing systems and multifunctional equipment. Detailed discussions of key operations will be covered in subsequent sections. B. CURRENT SYSTEMS AND COMPONENTS

1. Semiautomatic Systems Semiautomatic systems differ from the more automated systems primarily in that filling is apt to include manual operations, and the transfer of the filled pouch to the air removal-closure sealing operation is manual. This type of system has been used by Normeat in Denmark, by Lawsons of Dyce in Scotland, by Cranberry Products in Wisconsin, by Brooke Farms in the United Kingdom, and by Swan Valley Ltd. in British Columbia. It is the type suggested for market test and initial commercial production, where production rates in the neighborhood of thirty to forty pouches per minute are adequate.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

331

FIG. 8. Swissvac CVEP vacuum sealing machine. (Courtesy of TransvaeMachinen AG., Luzern, Switzerland.)

Specific systems vary among packers in such areas as the selection of pouchpresenting aids; the type of positive displacement pump used for filling fluids; and the make and style of vacuum-closing equipment. All reported systems use a vacuum chamber or hood system which can vacuumize and seal several pouches at one time. Impulse closure sealing prevails. The Swissvac CVE(P) (Fig. 8) (Transvac-Maschinen AG, 1972) has been widely used, being superseded in some applications by the Swissvac Duo Mark I1 (Transvac-Maschinen AG, 1975; Anonymous, 1975a). The Multivac B-6 series has also been reported in use for this application (Multivac Export AG, 1973). There is no reason why any wellengineered sealer could not be used. Care must be exercised to ensure contamination-free seal areas and wrinkle-free closure seals. Retorting is by batches, and the reported systems use all variations-vertical and horizontal retorts, and water and steamair cooks. 2. Reynolds Metals

Goldfarb (1970, 1971a) described a production system which featured the operations from pouch fabrication through closure sealing on an in-line intermittent-motion, horizontal form-fillseal machine such as that shown in Fig. 6 . The

338

RAUNO A. LAMPI

system was used for packing vegetables in brine. The brine was introduced by means of a positive displacement pump, while the product was fed through a volumetric reciprocating chamber at the bottom of a feed hopper. The pouch opening was controlled by a duckbill. Air was removed by a tapered tube (snorkel) entering and withdrawing from the pouch just prior to closure sealing. The hot-bar closure-sealing operation was followed by a chill-bar station to assure wrinkle-free and bubble-free seals. A continuous Hydrolok retort, described later under Retorting, was used feature steam-air processing. The use of the continuous retort permitted hot-filling of the product, and processes were therefore based on an initial product temperature of 150°F.

3. Natick-Swift Line

In support of the U.S. Military’s desires to use retort pouches for combat rations, an extensive development program emphasizing production reliability was carried out by a consortium of food and equipment firms at the Swift & Co. Research and Development facilities (Duxbury et aZ., 1970; Lampi and Rubinate, 1973). This production system, shown schematically in Fig. 9, also featured a horizontal pouch-forming and filling machine; but instead of using a snorkel approach for air removal, a vacuum closing machine designed to vacuumINSPECTION

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

339

ize and close rectangular cans was modified to accept the taller and thinner pouch carriers. T h s system is illustrated in Fig. 10. The can-seaming operation was replaced by a hot-bar sealing mechanism. The key to the operation of this system, which was designed to package a wide variety of products, was the carrier concept. The carrier concept is not equipment per se, but, as described by Duxbury et al. (1970), Corning (1973), and Lampi and Rubinate (1973), is a technique used in conjunction with a Bartelt intermittent-motion package former and filler, a Continential Can Company vacuum can sealer, and horizontal retorts to gain close control of the pouch throughout several functions. The carrier is an anodized, Teflon-coated, cast aluminum container, as shown in Fig. 11, sized to accept and control the thickness of the filled pouch on the production line from a point after filling to the feed of the dryer or, if n o dryer is needed, to the overwrap operation. The functions performed by the carrier are as follows:

1. It permits accurate control and positioning of the pouch through the vacuumizing process and closure sealing. 2. As a whole and in trays, it becomes a component of the retort rack, providing uniform package thickness and, because of its raised surface ridges, assuring water circulation. VACUUM I

POUCH IN CARRIER

DISCHARGE STATION

FIG. 10. Modified vacuum closing machine.

340

RAUNO A. LAMP1

FIG. 1 1 . Retort pouch carrier.

3. It eliminates the handling of the pouch between unit operations without the need for special conveyors. Standard conveyors for rigid containers may be used . 4. It permits the use of a standard vacuum can-closing machine, modified to accept the carrier and incorporating a closure-heatseal mechanism. The transfer of the pouches to the carriers occurs at the end of the forming and filling machine.

Modifications (Duxbury et al., 1970; Lampi and Rubinate, 1973) were necessary to make the horizontal packaging machine suitable for use in USDAinspected facilities. These included (1) use of square tubing in place of angle or channel construction, (2) smooth welded joints, (3) sealant-sealed mating surfaces, (4) raised rather than recessed alignment keyway, (5) sanitary feet, (6) stainless-steel surfaces above open pouch edges, and (7) USDA-approved paint for surfaces where paint was permitted. The multiple stations permit several-stage filling, the use of tack seals, and shaping of the product by formed flatteners. Retorting was carried out in horizontal batch retorts using a water cook. The specific requirements of the bakery products necessitated close control over the rate of temperature rise during come-up.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

34 1

4. Japanese System Since retort pouches are so widely marketed in Japan, there are undoubtedly modifications and equipment features that are especially designed for many items. However, the basic Japanese production line (Tsutsumi, 1972, 1975) is built around a six- or eight-station rotary fillersealer as illustrated in Fig. 12. This machine functions with premade pouches which go through an opening operation, one or two filling stations, and closure sealing. With fluid-containing products, which dominate the Japanese market, air is removed from the pouches by physical flattening of the pouch and outward tensioning from the two side seals. Another feature of this rotary machine is the triple seal which has a second seal bar at a slightly lower temperature than the first, sealing over the location of the first, and a third, unheated flattening bar.

5. STAR (Italy) System STAR (Star di Agrate Brianza) has evolved a relatively sophisticated production line. The operation starts with roll stock formed into pouches on an in-line section of a form-fillseal machine. After the pouches have been cut into individual units, they are transferred to a rotary filling and sealing section somewhat analogous to the apparatus in Fig. 12, where filling can be performed

FIG. 12. Rotary fiiler-sealer. Courtesy of Toyo Seikan Kaisha, Ltd., Tokyo, Japan.

342

RAUNO A. LAMP1

in single or multiple steps. After formation of a partial closure seal on this apparatus, the pouch is discharged to a pocket conveyor which elevates the pouch to the feed leve1 of a top-loading, rotary-pocket, vacuum-sealing unit. Several pouches can be vacuumized and sealed in each compartment or pocket. In some European systems, the rotary vacuum sealer has reportedly been replaced with the Swissvac Duo Mark 11; sealing is by impulse. Retorting is reportedly in batch horizontal retorts using a steam-air mixture.

6. Hydropac System The Hydropac system (Heid, 1970; Mencacci, 1973) is a proprietary development of the FMC Corporation. The system, as illustrated in Fig. 13, is unique in that exhausting of residual gases and thermoprocessing occur simultaneously and before closure sealing. Filled pouches are held in carriers (inset in Fig. 13) with the top seal surfaces kept stretched to a closed position by means of clips. The pouches in carriers are introduced and passed through a steam or water section where, apparently by means of a combination of heat and pressure, residual gases are thoroughly exhausted. The taut seal surfaces act as a one-way valve. In addition to thorough exhausting, some flushing away of contamination occurs, and the seal areas become preheated so that, when the closure seal is made, less heat is required from the seal bars themselves. The holding time and thermal conditions during the “exhausting” period are chosen to effect a thermoprocess. Heid (1970) illustrated the concept adapted to a hydrostatic cooker as a means of achieving temperatures higher than 100°C. Although there are no technical barriers to assuring that the top lip of the filled but unsealed pouch is kept above the water line, some reluctance should be expected on the part of commercial packers to believe in the efficiency of stretched surfaces to act solely as one-way valves.

7. Circle Design The Circle Design and Manufacturing Corporation (1973) proposed the application of a vertical form-fillseal machine for retort pouches. This equipment features rotary sealing and achieves air removal by means of a steam flush through tubes mounted parallel to the feed tube prior to closure sealing. An apparent limitation of this system would be an inability to package solid items or those requiring a two-step filling operation.

8. Drawn Pouch System Where a short shelf life can be tolerated-for example, for institutional packs of potatoes (2.5 to 5 kg)-a drawn pouch system like that used for frankfurters

INLET LOCK

m

EXIT LOCK

\

THERMAL PROCESSING LINE FOR FLEXIBLE POUCHES (SCHEMATIC)

FIG. 13. Hydropac process system for pouches. Courtesy of FMC Corporation, Santa Clara, California.

344

RAUNO A. LAMP1

and luncheon meats, utilizing foil-free formable film such as polyamide/mediumdensity polyethylene or polyamide/polypropylene, has found application. In this system (Cryovac Bvision of W.R. Grace and Co., 1974), a lower forming web of film is drawn flat and horizontally over forming dies, heated, and formed with the aid of vacuum into tray or pocket configurations. The product is then placed into the tray or pocket, a second polymeric web is drawn over the top of the lower web, and a perimeter heat seal of the two webs around each pocket is made in a vacuumized section of the chassis. The success of this approach depends on the prerequisite that the material be thermoformable. Claimed advantages are that the large, open trays are easier to fill and that the web can be several pockets wide, speeding up production. C. PRODUCTION RELIABILITY Bohrer (1 963), in discussing microbial spoilage of canned foods, delineated two aspects-the efficiency of the heat treatment to sterilize the food, and the ability of the hermetic container to prevent recontamination after processing. The latter requirement can be further broken down into two phases-the ability of the manufacturing operation itself to produce defect-free containers, and the durability of the container during subsequent handling. Rubinate (1 973), reporting on the U.S. Military field testing in 1966 to 1967 of combat rations featuring retort pouches, noted that the defects discovered at the point of end use were related to manufacturing deficiencies-dirty seal areas, body cuts from poor handling, etc. Nieboer (1970) and Nughes (1973) reported on manufacturingrelated problems that were methodically faced and solved. Tsutsumi’s (1975) report on Japanese practices reflected a combination of similar experiences. Sterilization procedures, once established, have not been a cause of spoilage. The criteria for production reliability of retort pouches have been based on the record of the conventional can. Actual numbers may have less significance from a public health aspect than from an economic one because of the multiple screening points of distribution systems and the detectable manifestations of container failure-leakage, swelling, and malodor. The significance of any numerical assessment is further complicated by the practice of classifying defects into assignable (traceable and correctable) and nonassignable (random) causes. Mounce (1966) reported an improvement in guarantees against spoilage with cans, in terms of a decrease in failure rates, from 0.3% in 1931 to 0.004% in 1966. In 1967, a published, acceptable swell rate was given as 0.1%, not necessarily the result of container failure (Anonymous, 1971). California laws require segregation of lots of cans when evidence of spoilage exceeds 0.5% (California Administrative Code, 1954). Using such documentation plus experience and judgment as background, Lampi and Rubinate (1973) reported that a goal of 0.01% or less failures had

345

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

been set for the Natick-Swift Reliability Program to select, test, modify, innovate, and use equipment for the defect-free manufacture of retort pouches. The results of t h s extensive program, covering the preparation of six diverse foods on the system shown in Fig. 9, are given in Table VIII. In addition to the selection and design of proper equipment, key factors to achieving such a low defect rate were the implementation of a sound quality assurance protocol to keep equipment performance in control, and two 100%visual inspections of the pouches during manufacturing-one before retoring and one after. Tsutsumi (1975) reported that Japanese practice is the same-two 100%visual inspections, one before and one after retorting. Although not specifying the outgoing failure rate, Tsutsumi did state that faulty packages from the filling operation ran to 0.2% of each lot run, and those from retorting, 0.02%. Although numerical reports are not readily available elsewhere, the success of the retort pouch in the marketplace attests that manufacturing failure rates are in control and that any of the tested systems can be made to perform reliably. Relative to postthermal process spoilage, the retort pouch has one inherent advantage over the conventional can. Thorpe and Everton (1968) cited work reporting that 2 to 3% of cans with good commercial seams have temporary leaks induced during cooling and for a short time after. The pouch-if the seal TABLE VIII PACKAGE DEFECTS AFTER INCUBATION (PROCESS-ORIENTEDCRITICAL DEFECTS)~

Percent failure by category

Product Pumpable Beef stew Pineapple Placeable Beefsteak Franks Extrudable Fruitcake Ham and chicken loaf Total Average outgoing quality

Number of pouches Closure Gripper Stress Side cuts riser seal incubated seal

48,738 48.965

0.045' -

0.002

-

50,314 50,484

0.012 0.008

0.060 0.067

-

49,189 51,281 298,971

0.004 0.029

-

0.006

0.016

0.022

-

-

0.002

Percent failure Excluding Total grippercutsb 0.047 0.002

0.045 0.002

-

0.072 0.075

0.012 0.008

-

0.010 0.029

0.010 0.029

-

0.001 0.0003 0.0393

%S. Army Contract. DAAG 17-69-C-0160 (Duxberry et nl., 1970). bGripper cuts are a correctable cause of failure. CDoesnot include twenty leakers caused by foaming of gravy (on Jan. 5, 1973).

0.0173

346

RAUNO A. LAMP1

criteria of fusion, burst resistance, and freedom from contamination and severe wrinkles are met-has no such weakness. The fusion seal is a total weld and barrier to bacterial penetration. Production reliability has been and can be attained, and contributing factors can be listed. Including many that are obvious for any manufacturing operation, these factors are: 1. A systems approach-that is, a firm conviction and recognition that film, product, and equipment requirements are interrelated and that each unit operation in the production line interfaces with those before and those after. Cooperative development effort among the various suppliers is necessary. 2. Selection of proper equipment and modification of this equipment to perform at the necessarily high standards. 3. Control of the transfer of pouches between unit operations. 4. Establishment of relevant on-line tests of the empty, filled, and retorted package and implementation of a statistically valid sampling plan to keep the operations within tolerances. As nondestructive test methods become established, they can replace some sampling. 5. Thorough maintenance of equipment. 6. Definition of critical defects and the use of these definitions in two 100% visual inspections for package defects, especially in the seal area. It has been the experience and conclusion of several of those associated with retort pouch manufacture (Mapp, 1971; Tsutsumi, 1975; Lampi, 1973; Nughes er al., 1973) that, if the preceding guidelines are followed, retort pouches can be reliably and efficiently made with no more hazards than are presented by the more conventional three-piece can. Experience has indicated that, if film, product, and production are closely monitored and controlled, there is no need, as stated by Szczeblowski (1971), for 100% incubation or mechanical leak testing of the outgoing product beyond standard sampling for confirmation of sterility.

V I I . SEALING A. GENERAL ASPECTS

Methods for forming and sealing flexible polymeric film pouches have been reviewed thoroughly (W. E. Young, 1967; W. E. Young, 1975; Brown and Keegan, 1973). The equipment aspects have also been covered (McCillan and Neacy, 1964; McCloskey, 1971). This general technology for heat sealing is applicable to the retort pouch system, but refinements t o achieve better and more uniform performance have been necessary.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

347

For the purposes of this review, sealing refers to the forming of pouches from roll stock, regardless of whether the food packing facility uses preformed pouches or roll stock with on-site pouch fabrication. It also includes forming of the two side seals and a single bottom seal, and the final sealing of the closure after filling and air removal. The reliability of the closure seal is directly affected by the ability of the filler operation (s) to leave the opposing seal surfaces free of product contamination, and frequently, but not in this review, filling and sealing are discussed as one operation. Also, it is not uncommon to have side and bottom seals made by hot-bar (resistance-heated) methods and closure seals by impulse methods. Miller (1973) has shown that the folding of the three-ply laminate over a plow in preparation for the formation of side and bottom seals does no harm. Over two 10-foot lengths of 14%-inch-wide material, only eleven breaks were detected, and these were in the foil ply only at the location of the bottom fold.

B. SEAL DEFINITION AND REQUIREMENTS 1. Criteria Primarily because seals have not received attention from a critical performance aspect and because seal failures have occurred within tolerable percentages for less-perishable foods (for foods not requiring a hermetic package), the definition of a good seal has been subjective and based on nebulous criteria. Each authority has had, to some extent, its own vocabulary and indicators of performance (or lack thereof). The requirements for retort pouches-a leaker rate as low as that of the metal can; seals that can stand thermoprocessing temperatures of 250°F or higher; packages that will be durable through the entire distribution system; and shelf-life requirements beyond those of frozen and some dehydrated foodshave necessitated a greater appreciation for the significance of seals and an upgrading and objectivity in definitions. As stated in the section on Materials, the ultimate seal criterion is performance, and the supporting tests, although varying among materials, should relate to the performance standard. For the retort pouch, this standard has been the sanitary can and its historically documented satisfactory performance, Burke and Schulz (1972) compared the resistance of metal caris and retort pouches to a rough-handling cycle representative of the military distribution systems. Briefly (since a more detailed description will be given later under Durability), the cycle consisted of vibration, a series of ten drops from 18 inches (ASTM D-775-68, Objective B), visual examination, and biotesting (Maunder e l al., 1968). The results, shown in Table IX, although not restricted to seal failures alone, present a tentative performance criterion, suitable for military needs, of 2% or less leakers after biotesting. If a finer or more specific breakdown is desired, twenty

348

RAUNO A. LAMP1 TABLE IX COMPARATIVE PERFORMANCE TEST RESULTS ~~~~

~~

~~

Pumpable product (chicken d la king) Material Metal cansa Flexible materialb #I Flexible materialC #2

Semisolid product (beefsteak)

Number tested

Number failed

Percent failure

Number tested

Number failed

Percent failure

1440 1440

32 30

2.22 2.08

720 720

4 2

0.56 0.28

720

5

0.7

7 20

4

0.56

‘Pumpable product in the cans was chicken and noodles. bFlexible material #I = 0.076-mm (0.003-inch) blend of high-density polyethylene and polyisobutylene/0.0089-mm (0.00035-inch) 1145-0 aluminum foil alloy/0.0127-mm (0.0005-inch) polyethylene terephthalate. ‘Flexible material #2 = 0.076-mm (0.003-inch) high density polyethylene/0.0089-mm (0.00035-inch) 1145-0 aluminum foil alloy/-0.0127-mm (0.0005-inch) polyethylene terephthalate.

of the thirty failures for the pumpable product with material # I , featuring a modified polyethylene sealant layer, occurred in the seals or seal junctions for a I .4% failure rate. None of these failures for material #2 (HDPE inner layer) were seal-related. Failures with tfie semisoIid item, beefsteak, were not seal-oriented. Similar criteria could be established for commercial distribution requirements. The flexible packages used in the comparative tests were representative of those prepared in a production environment. A comprehensive development program (Duxbury et al., 1970; Lampi and Rubinate, 1973) had been carried out by a consortium of five suppliers of food, equipment, and materials under contract to NARADCOM to establish that, through proper selection and engineering of equipment, retort pouches could be reliably formed, filled, sealed, handled, and processed. By drawing on these development experiences and on supporting laboratory effort, such as that reported by Shulz (1973), the requirements for a good flexible package seal can be delineated.

2. Fusion Fusion is a requirement that is met when the opposing seal surfaces form a total weld. Such a weld is characterized by the inability to distinguish visually either opposing seal surface at the inner seal junction or after seal tensioning beyond the point of failure. On tensile failure (this can be by manual pulling), fusion exists when fracture of one inner ply at the seal junction occurs and there is delamination of one lamina (Fig. 14). Brown and Keegan (1973) offer further discussion, and Young (1975) shows fusion to be an essential characteristic of a

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

349

good seal. If the seal peels or fails so that the inner seal surfaces are identifiable, the seal should be rejected. The significance of fusion relates directly to performance. Seals examined at the time of creation can meet tensile and burst test criteria without fusion, yet after a short (24 hours plus) storage period (Schulz, 1975) such seals fail when subjected t o simulated handling tests such as vibration and drop cycles. As stated above, measurement is visual, and the results are in terms of attributes-fusion either does or does not exist.

3. Internal Burst Test The internal burst test for seal integrity has been propounded by Schulz (1973) as a good overall measure of the ability of a package to withstand transportation and handling. Duxbury et al, (1970) included internal burst as a necessary criterion. With the apparatus designed by Continental Can and used in the NARADCOM studies cited above, an unsealed or cut and emptied pouch is placed over an air source, the jaws are clamped, and the internal pressure is increased to a predetermined level. Either the pressure to burst, the time to burst at a constant pressure, or adherence to a preset pressure-time cycle is recorded. A heavy metal plate restrains the pouch thickness to l / ~ inch. The seal junction yield should be n o more than l / ~inch; a greater yield generally indicates lack of fusion or material inadequacies.

4

4 SAMPLE UNDER TENSION

I

-

SEAL INTERFACE

TYPICAL FUSION SEAL FAILURE

TYPICAL NONFUSION SEAL FAILURE

PEEL N SEA AREA

I

I

FIG. 14. Fusion versus nonfusion seals.

350

RAUNO A. LAMPI

Retorting and storage (time following sealing) affect achievable burst levels. The following relationships are typical of a polyester/foil/modifed polyolefin laminate : Immediately after sealing: passes 2.4 X 10’ Pa ( 3 5 psig)-30-second hold. Twenty-four hours after sealing: passes 2.1 X lo5 Pa (25 to 30 psig)30-second hold. Retorting and indefinite storage: passes 1.4 X lo5 Pa (20 psig)-30-second hold. Materials and pouches meeting the above criteria have shown a correlation with acceptable tensile strengths and have endured military-level rough handling. An acceptable commercial internal burst criterion is 15 psig for 30 seconds (Shenkenberg, 1975). Other films meeting the post-retort criteria can have varying on-line and pre-retort burst test levels. The prime advantage of the internal burst test is its ability to detect or measure the weakest part of the total seal. A version of the burst test in which the totally sealed, restrained pouch is punctured with a needle through a cured silicone sealant septum or other form of gasket appears feasible and perhaps equally suitable for production line surveillance. At least one European supplier (RWP Flexible Packaging, 1974) specifies internal pressure tests of the restrained pouch for package seal integrity. They also offer a procedure and equipment for a compression strength test where a filled (with water t o approximate product volume) and sealed pouch is compressed between plates that are connected directly to an indicating hydraulic load cell. A static loading is applied across the faces of the pouch. Pouches, per referenced specifications, should withstand a force of 7.5 kg per 15 mm of internal seal length applied for 15 seconds. Y. Tsutsumi (private communication, 1975) has reported a static load test requirement of resistance of a 115 x 155-mm pouch filled with 180 ml of water to 50 kg for 1 minute. Nughes e f al. (1 973) describe an internal-pressure test procedure where the pouch is pressurized to 5 psi and then held submerged in water. Criteria are retention of pressure for 60 seconds and absence of visually detectable leaks.

4. Tensile Test The tensile strength of seals is currently measured dynamically on Instron or similar equipment. RWP Flexible Packaging (1974; since then renamed DRG Flexible Packaging) proposed that satisfactory readings can be obtained by a simple device constructed from a constant-speed motor, clamps, and a spring balance. Sample widths vary among investigators, as do crosshead-to-jaw separation speeds. Regardless of the specifics of the technique, it should be recognized

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

35 1

that the tensile test’s best application is for surveillance on the sealability of materials and as a spot check on sealing conditions and equipment operation. By definition, the tensile test measures the total force or weight required to cause failure over the total width of each sample strip. The detection of any channels or stress points and the effect of occluded particles or other small, weak areas within this dimension are obscured by the adjoining high-value areas. Tensile tests should therefore be supplemented by burst tests. Tensile tests are useful as a quality assurance tool for assessing the inherent sealing qualities of retort pouch films, and they should be mandatory. Duxbury el al. (1970) reported no problems in achieving a tensile strength of 16 lb/in. with 12-micron Mylar/9-micron foil/75-micron modified polyolefin. Neither retorting nor 3 months of storage had any important effect on that tensile strength. Similar results with other films have been noted at NARADCOM. Tensile strength specifications for materials range (laboratory sealing) from 11 to 19 lb/in., yet all packages will perform adequately. These same levels should be achieved by the production equipment, and all specific values and criteria for each film should be related to performance standards. Pflug and Long (1966) studied tensile strengths under retort conditions and concluded that the behavior of a seal under such conditions cannot be completely predicted by tensile tests performed at room temperature. They affirmed the value of fusion as a criterion above tensile strength. Their material was polyester/foil/vinyl; however, similar relationships are likely k i t h other films.

5. Visual Examination Until the use of nondestructive instrumented techniques merit their expense, visual examination beyond that required to establish the presence or absence of TABLE X SUMMARY OF CRITERIA TO DEFINE A HIGH-PERFORMANCE SEAL FOR RETORTED POUCHES

Variable

Criteria English

Fusion Internal pressure

Tensile Visual

Must exist Level Hold time Maximum seal yield Restrained pouch thickness Level Crosshead speed Sample width No visible aberrations

Metric

-

-

20 psig 30 sec 1/16 inch 1/2 inch 12 lb/in. 10 in./min 1/2 in.

1.38 X lo5 Pa 30 sec 1.6 mm 13 mm 2.1 kg/lO mm 25.4 cm/min 13 mm

35 2

RAUNO A. LAMP1

fusion is necessary to assure the absence of heat creep, significant wrinkles (over one-half the seal width), surface irregularities, or occluded matter in the seal area. The criteria that define a good seal are summarized in Table X. This definition is, of course, for one material and is presented as an example of the detailing that is required for retort pouch applications. C. EFFECT OF RETORTING AND STORAGE ON HEAT SEALS Much of the preceding discussion has been concerned with fabricating good, reliable heat seals. Since storage life is of paramount importance, especially for military rations, the ability of seals to remain intact and strong throughout the life of an item is equally important. To determine the effect of storage time and temperature on the strength of heat seals, NARADCOM has conducted storage studies with materials containing modified high-density polyethylene and polypropylene as the heat-sealable inner lamina. Packages for these studies were filled with either ground beef in barbecue sauce, pork sausage, or ham and chicken loaf; retorted; and stored at 100°F or 72°F. Seal strength measurements were made prior to retorting, immediately after retorting, and at 3-month intervals for a total period of 27 months. The upper two curves of Fig. 15 show the average seal strength values for packages with modified high-density polyethylene as the inner ply, containing 3000 SEAL STRENQTH

I161 114)' 1121L

110)-

a3

c

E

1600

\

16)

-

BOND, SEAL AREA

c u) 0

5 2

loo0 141-

0

I

0

3 RETORT t

I

6

1

1

I

I

I

I

1

V

12

16

18

21

24

21

TIME (months)

FIG. 15. Effects of storage on seal strength and seal-area bond; polyester/foil/modified high-density polyethylene. Product was ground beef in barbecue sauce. 100°F storage (solid line); 72°F storage (dotted line).

35 3

FLEXIBLE PACKAGlNG FOR THERMOPROCESSED FOODS 3000 SEAL STRENGTH

500

0

---

12) 1

.--I

-.I

I

-.-.I

I

I

I

I

I

ground beef in barbecue sauce, over a 27-month storage period. The fluctuations are typical for this material and occurred, to some degree, even in pouches that contained only water. With this material, there were no significant differences in the seal strengths of packages stored at 100°F or 72°F. Figure 16 shows (again with the upper two curves) average seal strength values for retort pouches constructed of polyester/aluminum foil/ethylenepolypropylene copolymers. Regardless of the food product, materials made from this structure showed less variation from sample to sample, and both seal and seal area bond strength remained very uniform throughout the test. All materials tested maintained seal strengths near their original values throughout the 27-month storage period. D. SIGNIFICANCE OF INTERLAMINA BONDS IN THE SEAL AREA Complete fusion of the seal interface surfaces has been discussed, and its importance in producing a good heat seal has been emphasized. As indicated in Fig. 14, a good fusion heat seal, when tested, will usually fail through delamination, ideally with approximately half of the sealant material remaining on each of the substrate pieces. Since, with the three-ply structures (polyester/aluminum foil/polyolefin) currently suitable for retort food applications, the inner ply has considerably greater elastic properties than the other layers, the bond between the inner layer and the aluminum substrate in the seal area plays an important part in the seal strength.

354

RAUNO A. LAMPI

Shown in Fig. 15, in addition to the seal strengths already discussed, are bond strength values in the heat seal areas for a typical polyester/aluminum foil/ modified high-density polyethylene laminate. It is apparent that similar patterns are followed by the two properties. Materials with polypropylene as the inner ply, as shown in Fig. 16, showed less fluctuation in bond strength in the seal areas, as well as in seal strength. On the basis of these studies and similar studies conducted with other materials, good seals can be made, in accordance with the definitions presented earlier, that will withstand extended storage without deterioration.

E. EFFECTS O F OCCLUDED PARTICLES IN CLOSURE SEALS To determine the effects of occluded particles in the closure seals of flexible retort pouches, a series of tests were conducted at NARADCOM on pouches with pieces of rubber, 0.16 X 0.16 X 0.08 cm, entrapped in the seals. These tests included two seal widths, 0.32 cm and 0.64 cm, both made with hot-bar sealers. The performances of contaminated and clean seals were compared on the basis of internal burst tests, failures during retorting, and failures during rough handling and storage. There were no significant differences between the two seal widths in the number of pouches passing burst levels when no contamination was present. There also were no significant differences between the burst levels of the two seal widths when contaminated with a piece of rubber as described above. Despite the similarity in resistance to internal pressure at standard conditions, the packages with 0.32-cm-wide seals with particles in the closure seals showed a failure rate of 11% during retorting and a failure rate of 8.3% during rough handling (cycle used by Burke and Schulz, 1972). The pouches with 0.64-cmwide seals had no failures during retorting and rough handling. Samples of the pouches with occluded particles were placed in storage and examined after 6 and 12 months of alternating 3-month storage at 38°C and 85% R.H., and 21°C at 50% R.H., to determine if any changes had occurred. Table XI shows the changes in internal bursting strength that occurred at these intervals. All seals with defects showed a decrease in internal bursting strength after storage. In all instances the greatest decrease occurred during the first 6 months and included the initial strength loss which occurred after retorting. It should be noted that the decrease in the 0.32-cm-wide seals was more pronounced than that in the 0.64-cm-wide seals. On the basis primarily of failure rates noted after retorting and during rough handling, it was concluded that the minimum seal width should be 0.64 cm and that occluded particles in such a seal cannot be tolerated.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

355

TABLE XI EFFECTS OF STORAGE ON BURST LEVELS O F SEALS WITH OCCLUDED PARTICLES"

0.32 cmb

0.64 cm

Control

Defect

Control

Defect

Before retort

40.0

39.5

49.5

45.0

After retort

28.3

29.8

25.6

29.8

6 months

23.4

12.8

25.6

22.0

12 months

21.0

13.0

26.0

21.0

"Values in pounds per square inch, gage (psig). bSeal width.

F. METHODS FOR SEALING RETORT POUCHES The two most common heat-sealing techniques for retort pouches have been the hot-bar (or hot-jaw) sealer and the impulse sealer. Variations in terms of bar configurations, supports, etc., are available. Both techniques form fully reliable seals. The use of an ultrasonic sealer has been reported (Anonymous, 1972).

1. Hot-Bar Sealing The hot-bar technique, which is simply a constant-temperature, resistanceheated metal bar sealing against a rubber-faced anvil, predominates as the method for forming side and bottom pouch seals, and shares popularity with the impulse technique for closure seals. a. Pouch-Forming Seals. Whether made on the pouch-forming section of horizontal form-fillseal equipment (Fig. 7) or on bag-making equipment (for systems where preformed packages are introduced into production), the pouchforming steps consist in forming a bottom seal, cooling that seal by means of separate chilled seal-type bars, forming a double-width vertical seal, chilling that seal, punching a diamond-shaped (or oval) hole in the upper center line of the vertical seal for a tear notch, and finally cutting the vertical (side) seal along its center line to form individual pouches. For some applications, a double-cooling operation is required for the side seal to achieve clean-cut edges on the tear notch; the tear notch may be more appropriate near the bottom of the formed

356

RAUNO A. LAMPI

pouch; and photoelectric sensing for accurate indexing of pouches may be required. Coding of pouches is feasible at the roll stock feed section. Although the bottom of the pouch is a fold, better performance has been obtained when an actual heat seal is superimposed at that location; this bottom seal is, however, frequently narrower than the side or top seal. Sharp 90-degree pouch corners have caused punctures in pouches during production handling where random contact between pouches was permitted. This has been overcome by rounding the external corners (Tsutsumi, 1972). Rounding of internal corners through the use of formed heat bars is optional, since no failures predominantly in pouch corners have been reported. Pouch forming and side and bottom sealing on a commercial horizontal form-fillseal machine under the Natick/Swift Reliability Program have been summarized by Miller (1973) and Lampi and Rubinate (1973). During the feasibility phase of this program, which required that a single production line produce six diversified products as reliably as three-piece metal-can systems did, it became evident that performance levels had to be improved and maintained at high levels. Use of earlier equipment simply had not demanded the degree of performance and control necessary, although, as proved by Miller (1973), the basic techniques and equipment designs were adequate. The prime difficulty was maintaining temperature uniformity over the length of each seal bar of no more than k10"F. The design features and modifications successfully implemented to achieve such performances were: Redesign of the seal bar configuration to position additional seal bar material adjacent to the sealing surface. Selection of optimum bar materials. Relocation and closer sizing (smaller tolerance between the heater and the cartridge hole). Relocation of the thermocouple. Use of high-performance temperature controllers. Isolation of the seal bars from the supporting rocker arms to minimize conduction heat loss. An anticipatory control system, in which full voltage to the bars is supplied for a preset time, eliminated temporary seal bar temperature drops on start-up. On shut-down, radiant heat from the seal bars would ruin the sixth and seventh pouches before the cutoff station; therefore, these pouches were automatically removed by an air jet activated by a counter. The counter could be overridden manually to remove formed pouches at will for quality assurance testing. A more detailed description is given by Duxbury et al. (1 970).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

351

The Circle Design and Manufacturing Corporation (1973) proposed a vertical system in which the side seals are made by constant-resistance heated rotary sealers. Cross seals are made by timed rotation of a cross bar having surfacemounted seal bars. Vacuum is by steam purge. Cooling is accomplished by rotary wheels or chilled cross bars. It is apparent that there is currently available equipment that, suitably engineered, can very satisfactorily form retort pouches. b. Closure Seals. As reported by Mayer and Robe (1963), a snorkel vacuumization technique can be used on horizontal equipment with fluid foods prior to closure sealing on the same chassis. Tsutsumi (1974, 1975), describing Toyo Seikan experience, illustrated equipment (Fig. 12) where hot-bar closure seals are made after residual air has been adequately removed physically by flatteners and after tensioning of the pouch from each side. With each of these systems, the package, especially the seal areas, does not experience immediate post-seal stressing, and seal areas are allowed to cool without undue disturbance. The Toyo Seikan technique appears to be quite successful; seal wrinkles have been eliminated. However, the physical squeezing of the pouch to remove air is limited to fluid foods. The snorkel technique will result in satisfactory seals if no seal contamination from the snorkel tube occurs; such a guarantee is not possible. Since solids (frankfurters and beefsteaks) as well as fluid foods were included, a broader approach has involved the use of the previously described carrier concept to contain and position pouches for vacuumization and closure sealing through a modified metal-can vacuum closing machine (Corning, 1973) (Fig. 10). The can-seamer mechanism is the vacuum closing machine was replaced by a dual-action, “ice-tong” style, hot-bar sealing mechanism. Both the seal bar and the anvil bar were heated. Dwell times were approximately 0.4 second. Earlier studies in the same program had shown the adequacy of both the hot-bar and the impulse techniques; however, the short total retention time allowed for sealing because of the one pouch-sealing station eliminated consideration of impulse sealing. Side seal grippers and surface flattening bars were used to assure flat seal surfaces and elimination of wrinkles. The pocket spacing was reduced from 45 degrees to 22.5 degrees to provide a longer cooling period after seal formation; rapid release of packages to the atmosphere with uncooled seals resulted in stressing and minor seal area wrinkles. Side seal bars are universally characterized by flat surfaces. Surfaces are smooth except for the fine, knurled imprint of Teflon-coated fiberglass cloth. Closure seals have mostly been formed by flat-surfaced bars, a few with fine surface knurling, and a few by bars with a slightly transverse-radiused cross section. Schulz and Mansur (1969), encouraged by the preliminary findings of Beadle (1959), optimized the design of a curved bar (Yi-inch side bar with

358

RAUNO A. LAMP1

-inch transverse-radiused seal surface) and the selection of anvil material [silicone rubber with durometer (Shore A) of 701. Tests, with a flat bar as a control, showed that, in regard to tensile strength, internal burst pressures, and total closure seal failures, highly satisfactory seals could be made with the curved bar even through gross grease and water contamination of opposing seal surfaces. Wilson (1974) provided a variation of the curved-bar approach, using, first, a massive hot bar heated to 10°F below the film sealing temperature to vaporize, and then an impulse sealer for ultimate bonding. Under the Natick/Swift Reliability Program (Duxbury et al., 1970), curved and formed seal bars were evaluated. Although the seal strengths with curved bars were very close to those achieved with flat bars, the decision was made to remain with flat bars because filling was to be contamination-free, and, since minimally acceptable seal strength levels were not known at that time, it was safer to remain with the highest. Subsequent review of the performance of those pouches indicates that the curved bars would have worked to complete satisfaction. In the FMC Hydropac system (Wilson, 1971, 1972b; Heid, 1970) unsealed pouches are held for 12 to 15 minutes in a hot-water or steam atmosphere at 250°F. Sealing is by hot bar, using a curved jaw against an anvil. The curved feature reportedly permits high-quality seals through moisture, and the presealing high-temperature exposure conditions the seal areas so that less heat is necessary during the actual sealing operation. y4

2. Thermal Impulse Sealing Thermal impulse sealing consists of, in rapid succession, clamping the facing seal areas together by a pair of jaws, heating to fusion temperature by means of a short, powerful electrical impulse, and cooling while still under pressure. Impulse sealing generally results in a narrower seal, '/s inch as opposed to the t/4 inch plus that characterizes a hot bar, but it is felt (Duxbury et al., 1970; Nieboer, 1970) that better seals can be achieved through limited or minor contamination. To accommodate varying pouch constructions (mostly thicknesses), either the impulse voltage and/or the impulse duration is controlled. A disadvantage is that, because the cooling cycle is part of the total dwell time, the total sealing time may be too long for economically feasible production rates with specific systems (8 to 15 seconds). This is generally overcome through the use of multiple-head or multiple-pocket equipment where three to ten or more pouches are sealed at one time. Impulse sealing relative to retort pouch applications has centered on the closure seal. Some packers have used both hot-bar and impulse methods; therefore, apparently, their performance is considered to be equivalent. The impulse sealing equipment, known to be used for retort pouches, whether single or multiple

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

359

head, consists of a “pocket” or station for holding the pouch in position between the sealing jaws. An enclosing “clamshell” lid or hood comes down to form a small vacuum chamber around the pouch. Multiple chamber units (Fig. 8) (Transvac-Maschinen AG, 1972) have a belt section for loading which is automatically indexed to the vacuumization and impulse sealing area. Transfer of pouches from the discharge of the filling operation onto the feed-belt pockets of impulse sealing equipment has generally been manual. Consequently, control over the position of the pouch itself and the alignment of seal areas to preclude severe wrinkling is somewhat lacking. Goglio Luigi (1968) constructs a sealer, used by STAR S.p.A., Milan, Italy, which automatically receives tack or partially sealed pouches from a vertical form-and-fill operation, and transfers them to a series of individual vacuum sealing pockets that open at the top and bottom. Because of the generally narrower width of impulse heat seals and the less uniform placement of packages within the sealer, seals are generally positioned a secure distance from the top lip of the pouch, leaving an exposed unsealed area. Nieboer (1973) recommended applying a second seal over the first, extending to the edge of the pouch, and trimming off the excess (by means of a band sealer). 3. Ultrasonic Sealing The principles of ultrasonic sealing have been reviewed by Young (1971). Schulz and Mansur (1969) did not find that ultrasonic techniques had sufficient performance nor that the equipment was suitable for continuous production in 1968. More recently, however, Stevens-Lefield, Ltd., a Scottish company, reported (Anonymous, 1972) sealing filled pouches by ultrasonic welding, using a modified basic welding unit. G. SEAL WRINKLES Regardless of the sealing technique, seal wrinkles can occur, and they are difficult to define and to assess in terms of adverse effects on package performance. Frequently, and perhaps predominantly, irregularities in seal surfaces, not caused by occluded matter, are not channels or leaks and do not constitute a hazard. But, until experience permits otherwise, wrinkles are to be avoided, and seals containing such wrinkles should be rejected. The matter is one of economics and practicality in establishing inspection criteria. There are, as implied above, both true wrinkles and artificial or minor wrinkles. One true wrinkle can be defined as a material fold on one seal surface (see Fig. 17), caused when one seal surface is longer than the other-at least in a localized or narrow area at the moment of seal fusion. This definition would also include a severe foldover of both seal surfaces at the time of sealing (Fig. 17). Minor convolutions evident on both sides-indented on one and raised on the

360

RAUNO A. LAMPI

other-perhaps caused by minor irregularities in the seal bar or anvil surfaces, should not be cause for rejection unless they are large enough to be confused with contamination. Neither should a minor wrinkle emanating from the inner seal junction out into the seal width, but generally not over more than half the seal width, be cause for rejection. These minor wrinkles occasionally occur with hot-bar sealing methods and are usually caused by the collapse of a package around its contents on transfer from vacuum to ambient pressures before the polymeric material in the seal area has adequately cooled. Wrinkles do not occur when the opposing seal surfaces are flat and totally parallel. Some design or, if semiautomatic sealing equipment is used, manipulative guidance to achieve flat surfaces can be given. These recommendations apply to closure seals, the ones where severe wrinkles are more likely to occur. Specification of no filling within 1% inches of the top of the pouch. Appropriate but not necessarily ultrataut tensioning by means of clamps or grippers, spring-loaded tensioning devices, or other mechanical means. Formation of a partial cylindrical shape (or round-cornered partial fold) across the width of the pouch at or immediately adjacent to the location of seal. Cooling, which could be accomplished by a time lag, before release of the pouch from vacuum to atmosphere. A combination of any two or all of the above. By following the last guideline, the incidence of closure seal wrinkles on the “production reliability” project described by Lampi (1973) was reduced to 1 in 500 pouches.

H. SEAL CONTAMINATION Contamination of the closure seal by the product is a more ubiquitous problem, since it can be caused by several packaging operations: filling deficiencies, incorrect vacuumization procedures, or improper handling of the pouch prior to sealing. Solutions to assure reliable final seals in the face of occasional contamination have taken three avenues: eliminating contamination, ignoring it, or detecting packages so affected. 1. Elimination of Contaminarion

Since each product has its own flow characteristics and particle size distribution, detailed filler specifications to eliminate seal area contamination are rather impossible to determine or define. Experience with retort pouch applications has

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

361

revealed steps that, if followed, will minimize seal area contamination resulting from the filling function:

1 . A correct match of filler to product characteristics, preferably established through actual filling tests. For example, although cake dough may resemble chicken loaf mix in apparent consistency, comparative tests (Duxbury et al., 1970) showed that an auger filler (Bartelt Model D) with a sliding tube (essentially a wide-mouthed plug valve) nozzle worked better for the chicken loaf, while a gear pump (Creamery Package stuffer) with a rotary valve nozzle performed better for cake doughs. 2. Nozzle design features such as circumferentially located suction holes on nozzle tips to suck back dripping product, external suction rings, or sheet-metal synchronized guards to physically prevent drippings from contaminating seal surfaces. 3 . Specification of bottom-to-top filling and n o filling within a specified distance of the top of the pouch-for example, within 11/2 inches of the top. 4. Assurance of close control over the size and shape of the pouch opening by means of conveyor clamps on both leading and rear pouch edges, air-jet assistance t o initiate opening, and use of suction cups and/or spoonbill internal forming devices. 5. Use of winged or formed guards t o swing down into the package opening at the moment of filling to physically protect the inner seal surfaces. Irregular or uncontrolled handling during and between unit operations can result in splashing of product up into the seal area. This potential problem in one system (Corning, 1973) was remedied through the use of the carrier previously described. The use of a tack or partial seal below the location of the closure seal has been reported as a step to prevent splash of product into the seal area. Removal of residual package air can also result in splash, especially with viscous products (sugar syrup, gravy) prone to air occlusion. Precautionary measures include the obvious care to avoid occluded air, control over the programming of the rate of air removal, and control over product fill temperature to prevent flashing.

2. Sealing in Spite of Contamination Once seals do become contaminated with product, presumably as a rare occasion, the pouch is not necessarily lost. The FMC Hydropac processing system was described earlier, in which unsealed pouches, with seal areas held taut by tensioning grippers, are immersed to the seal height in a hot-water bath

36 2

RAUNO A. LAMPI

to remove residual gases and clean the seal surfaces of product by a reflux action. SchuIz and Mansur (1969) indicated the feasibility of using a steam flush not only to clean the seal surfaces, but to remove residual air from the pouch prior to sealing. The key to their success was the use of a curved (transverseradiused) sealing bar against a silcone rubber anvil as mentioned earlier to seal through the residual condensed steam. Tsutsumi (1974, 1975) reported a unique, superimposed triple hot-bar approach. The initial hot bar (on apparatus typified by Fig. 12) effects a seal; a second bar, at a slightly lower temperature, flattens any blister that was caused by vaporization of contamination during the initial sealing; and a third, ambienttemperature bar performs a final flattening action on the still-heated polymer. Judging from the results-a reduction in contaminated closure seals from 10% to 0.2%-this technique is effective.

3. Detection of Contamination

In addition to eliminating the causes of defective seals and obtaining sound seals in spite of contamination, efforts should be made to detect defective seals. There are no subjective or objective nondestructive methods currently available for assessing seals for fusion, tensile, or burst strength. One must rely on periodic sampling during production to keep those particular variables in control. However, physical aberrations and contamination can be detected. Visual methods have their disadvantages-mainly subjectivity and human error. A rule of thumb often cited is that visual inspections under ideal conditions are 75% effective. Nevertheless, experiences from three independent sources reveal that, for retort pouches, defect rates can be kept at low levels by visual, on-line, 100% inspection. Nughes (1974) reported on success in Italy; Tsutsumi (1974) from Japan cities the use of visual inspection and final pouch defect rates of 0.02%. Lampi (1973), covering the Natick/Swift production experiences, indicated outgoing seal defect rates of 0.017% on the basic reliability project and a 0.01 1% overall defect rate for a follow-on production effort. If one accepts the published normal failure rate of 0.1% for the common can, the pouch is easily competitive. Figure 17 shows typical defective and acceptable seals and covers those deficiencies of concern to regulatory agencies. The representations in the illustration were obtained by rubbing a lead pencil lightly over actual acceptable and defective seal areas. A comparison of photographs, sketches, and actual defective seals for possible use as visual aids has indicated that actual typical seal defects are the most appropriate and the easiest to use as training aids. The rubbing technique of Fig. 1 7 was the second most illustrative. Nondestructive test apparatus for seal defects is discussed later under Quality Assurance.

FIG. 17. Vtsual tnspcctlon crlteria for closure real

364

RAUNO A. LAMPI

VIII. FILLING A. DEFINITION AND REQUIREMENTS The filling of retort pouches has received considerable engineering attention, since not only does the product require accurate measurement and dosing into the pouch to meet net weight requirements, but the opposing seal surfaces must not be contaminated with food or moisture during any of the mechanical movements of the apparatus. With metal cans, spillage of the product onto the rim of the metal container can usually be “sealed over” without causing visually evident leakage; there is no similar assurance with flexible packages. Although curved-bar sealers (Schulz and Mansur, 1969) can create good-quality seals through fluid contamination, steam flushing and refluxing (Schulz and Mansur, 1969; Wilson, 1974) can remove gross contamination, and preheating of seal areas, such as in the Hydropac system (Heid, 1970), can permit hermetic sealing around or through some contamination, the major burden of effecting clean seals remains with the filling operation. A significant percentage of the processrelated failures (Lampi and Rubinate, 1973) have been traced to contamination of seal areas during the filling operation, although air removal (vacuumization) has been an occasional contributing factor. Thorpe and Atherton (1972) present a pragmatic viewpoint, stating that the design of filling equipment capable of operating at acceptable speeds and of ensuring freedom from seal-area contamination will not be practicable for the complete range of products that might be packed in retort pouches; they suggest that an alternative approach would be to design a method of sealing that could cope with contamination. It would appear appropriate to emphasize both approaches. The filling operation can be subdivided into three functional areas:

1. Presentation or movement of the product by means of pumps, conveyors, augers, etc., to the proximity of the pouch opening. Volumetric or gravimetric control of dosage may be wholly or partially accomplished by this action. 2. Precise positioning and feeding of the product into the pouch itself, generally through a nozzle. The nozzle augments any metering or net weight control function of the pumping means through further quantification by volume control (positive displacement) or by precisely timed cutoff of product. More important, nozzles must be designed t o eliminate or drastically minimize the incidence of seal contamination because of drip or accumulation of the product on the nozzle itself. Beyond proper basic designs and dimensioning t o get tight or sharp cutoff of product, ancillary features, as discussed under elimination of contamination for sealing, such as suck-back, blow-off nozzles

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

365

and/or shields, can alone or in combinations preclude seal-area contamination. Duxbury et al. (1970) reported a constant internal water flush to remove any product build-up within the nozzle mechanism itself. Use of the bottom-to-top filling motion reduces the chance of splash and minimizes air occlusion, the latter of which can be troublesome on subsequent vacuumization. Duxbury el al. (1970) recommended specifying no filling within 11/2 inches of the top of the pouch, and experience proved the recommendation sound. Nughes (197 l b ) recommends leaving a wide space representing even as much as one-third of the pouch volume free for the same reason. 3. Presentation of the opened pouch to the filling station and holding the pouch in position during the filling step itself. This step includes first opening the pouch top by means of splitter bars with help from air jets and sometimes vacuum cups, and then control of the shape of the pouch opening by insertable spoonbill pouch formers; controlled movement of the clamps (both the leading and trailing pouch-holding clamps) toward each other to permit seal surface separation; and the use of formed shields or the similar insertion of metal fingers as reported by Nieboer (1973). On rotary equipment, a similar motion can be cam-activated. Above all, successful filling is assured by the proper match of the filling system to the characteristics of the product. In addition, clean filling can be aided in many instances by control of product consistency, fill temperatures, and product formulation. Formulation procedures or prefill vacuum treatment should be considered to minimize occluded air which could contribute to splash or cavitation, especially on vacuumization.

B. EQUIPMENT Filling systems used or production-tested for retort pouches range from manual to fully automatic with variations and combination of both. Those reported here represent known systems; their delineation is intended to represent typical workable systems, and not to exclude other suitable methods. The most extensive documented filling tests, covering a variety of products, to select, modify, and/or design fillers and nozzles have been those conducted by Duxbury et al. (1970) and by Miller (1973). Their requirements (Lampi and Rubinate, 1973) were to establish filling methods for seventeen diverse military ration items [4.5 to 5.5 ounces of product in a 43/4 X 7l/4-inch (outside dimensions) pouch], using the pouch opening and filling stations of a Bartelt horizontal intermittent-motion P9-11 machine at thirty to sixty pouches per minute. Two hundred and twenty-one separate filling tests were run, and, on the basis of these tests, four fillers and four nozzles, along with matching products

366

RAUNO A. LAMP1

(beef stew consisted of beef slices and stew gravy and vegetables as a two-step operation), were recommended for the system (Table XII). From these seventeen items, six (shown as asterisked components) were selected to represent the combinations of fillers and nozzles suggested for all seventeen items and to represent anticipated problems such as uniform distribution of suspended or solid ingredients (fruitcake and stew), foaming or splash (pineapple), unique cases (four frankfurters), and a two-stage requirement (beef stew) in more extensive production-scale tests. The fillers and dispensing nozzles used to test the items were as described below.

I . GearPump A Creamery Package St. Regis stuffer-filler (sanitary gear pump) was used for the viscous cake doughs. To achieve a positive cutoff of product at the pouch, a rotary valve nozzle was used (schematically illustrated in Fig. 18). The rotating spool valve at the tip of the nozzle was activated by a ball chain drive enclosed in a housing running the length of the nozzle. Bottom-to-top filling action was used. TABLE XI1 FILLER-DISPENSING NOZZLE COMBINATIONS-RETORT POUCHES

Filler Bock piston

Dispensing nozzle 1-inch-diameter plug 5/8-inch-diameter Plug

Creamery package stuffer

Rotary valve

Bartelt model D

Sliding tube

Placeable

None

Productslingredients Beans in tomato sauce Beef stew, vegetables and gravy4 Chicken A la king Ground beef with pickle-flavored sauce Pineapple in syrupa Barbecue sauce Chocolate nut cake Fruit cakea Orange nut cake Pound cake Beef loaf Chicken loaf Ham and chicken lo& Pork sausages Frankfurters4 Beef steaka Beef slices Beef dices"

'Selected for confirmatory testing (beef dices and beef stew, vegetables and gravey, are components of one product).

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS ROTARY CUTOFF VALVE BAKERY PRODUCTS-I 314.X I'

PLUG NOZZLE

361

SLIDING TUBE FILLER

PINEAPPLE- 5/8' I. D. HAM 8 CHICKEN LOAF- I 5/8' I. D. STEW 8 GRAVY- I' I. D.

POSITION ON PRODUCT DELIVERY

POSITION ON END O F DELIVERY FIG. 18. Nozzles for filling flexible pouches.

2. Piston Pump A modified Bock piston filler (featuring a pneumatically activated 90-degree ball valve for controlling product intake into and discharge out of the main pump body) was used for pumpable products such as crushed pineapple and the stew vegetables and gravy. Two plug-type nozzles differing basically in plunger diameter ("8 inch for pineapple and 1 inch for stew gravy component), operating as illustrated in the center of Fig. 18, were used for controlling product discharge in the pouch itself. The plunger was hollow to permit both a suction and blow-off, to be used if needed to prevent product build-up on the end of the nozzle. A unique constant water flush between the internal moving parts prevented any product build-up. In Europe, a widely used piston filler, the Leonhardt dosing pump (Maschinenfabrik Leonhardt OHG, 1974), features a rotating-face plate disk in place of the 90-degree ball valve. The disk has a single opening which, when the disk is rotated, is alternately positioned to permit inflow of product during the suction phase of the pump cycle and, following a returning rotating motion, discharge through a second tube.

3. Auger Filler

A Bartelt Model D conical auger filler was used with highly viscous products such as ham and chicken loaf. A larger-diameter plug nozzle, called a sliding tube

368

RAUNO A. LAMP1

nozzle, shown on the right in Fig. 18, was used for metering and cutoff. Suck-back and water-flush ring features were included. The product was fed as a round slug, later flattened to permit entry of the pouch into the carriers.

4. Placeable Filler The placeable product filler consisted of an intermittently indexed horizontal carousel containing cylindrical openings for beef dices or slots for beef steaks and frankfurters. The loading of the slots can be manual or automated. As the slot was indexed over the open pouch, a piston assist was used to assure dropping of the slot contents into the pouch. No cutoff nozzle was required, but a shaped tube and shields were used to protect the sides of the pouch. A spring steel tensioning device was used to prevent the two center frankfurters from dropping before the two end ones. In each instance, appropriately shaped metal shields were used as a further precaution against seal-area contamination. For all six diverse products, contamination was reduced in continuous production runs to less than one in fifty pouches, and net weights were within tolerances. In the studies reported above, commercial volumetric and net weight fillers were also investigated. For the specific particulate matter used, these fillers were found to be less applicable than the custom-designed rotating carousel ffler. However, Nieboer (1970) reported that a vibratory weigher feeder has been in commercial use with vegetables. The weighed product is discharged by the operator into pouches. Goldfarb (1971a) similarly detailed the feeding of vegetables into pouches on a Bartelt intermittent-motion packaging machine on a volumetric basis. The preheated product was delivered through holes in a reciprocating plastic block at the base of a stainless-steel hopper. The volumes, controlled by hole size, were fed by the reciprocating action of the block to a duckbill which was lowered into the pouch, opened to discharge its contents, and withdrawn in preparation for the next cycle. A vacuum line connected to the base of the hopper prevented liquid accumulation and drippage. In each of the preceding vegetable filling applications, brine was added by positive displacement pumps. While fluid products are generally poured into pouches automatically, solid pieces such as sausages have been successfully placed into pouches by hand with the aid of funnels and similar devices to guide the product and to protect the seal areas. Since commercial acceptance of retort pouches has been assured, these filling operations are reportedly becoming more automated. To summarize, clean filling can be accomplished by automatic and manual methods and is best assured by the proper match of filler to product needs, by the use of properly designed apparatus (close tolerances, good cutoff actions,

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

369

proper sequencing of movements), and by inclusion of ancillary features such as nozzle-end suction devices, pouch formers, and metal seal-area shields.

IX. AIR REMOVAL A. GENERAL ASPECTS The term “air removal” to describe the removal of noncondensible gases from retort pouches prior to closure sealing is a more accurate description than “vacuumization.” Vacuumization implies a pressure-reducing activity and a resulting pressure difference between the outside and the inside of the package; neither of these is necessarily true with the use of flexible packaging for a fluid, nonporous product. That retort pouches should contain as low a volume of residual gas as possible is axiomatic. Many of the supporting reasons for low residual gas levels have been discussed in preceding sections, and others will be discussed later in support of retorting. Some of these reasons are as follows: Product stability. Preclusion of pouch bursting during retorting without resorting to high t? 10 psi) counterpressures. Assurance of uniform and predictable heat transfer during retorting to guarantee sterility. Easier detection of spoilage (swelling). Easier cartoning and casing. There is no equipment solely for air removal; the function is carried out on equipment that also performs the filling and/or, much more frequently, the closure-sealing operation. (See Section VI, Production Systems, for a discussion on multifunctional equipment, and Section VII, Sealing, for a more detailed look at relevant specific equipment.) Table XI11 lists the known techniques for air removal, whether the techniques are at the feasibility stage of development or in full-scale production use. The enumeration of the residual gas levels in Table XI11 is presented strictly as an indication of capability and not as a standard or inviolate measure of what can be or should be expected from each system. Fundamentally, all the production-scale techniques are adequate to remove enough residual air for commercial and military shelf lives and for confidence in thermoprocessing procedures. The exact levels for each technique will be governed by factors such as air occluded in the product prior to or during filling,

TABLE XII: AIR REMOVAL CAPABILITIES OF CANDIDATE AND KNOWN PRODUCTION TECHNIQUES Residual gas Type of equipment

Product types

Source

Net weight

Average

Range

Comments

250 gm

3.0 cc

2.3- 3 . 6 ~ ~Natick analysis

140 gm

1.1 cc

0.9- 5 . 2 ~ ~Natick analysis

Clam-shell chamber

Sausage, frankfurters

Clam-shell chamber (single package)

Meat loaf items, stew, chicken A la king Stews, meats with sauce Stew, curries

Italy (retail samples)

140 gm 400 gm

2.0 cc 15.4 cc

6.3-24.6 cc

Natick analysis

Japan (retail samples)

180 gm

7.9 cc

5.1-10.7 cc

Natick analysis

Vegetables in brine

Reynold's metals

225 gm

3.0 cc

<5 cc

Fruit

Test samples

150 gm

1.7 cc

0.9- 2.5 cc

Ground beef in barbecue sauce Beef stew, pineapple, beefsteak, frankfurters ham and chicken loaf Fruits, peas in water

Test samples

120 gm

2.0 cc

0.4- 4.8 cc

140 gm 125 gm 125 gm 100 gm 140 gm 150 gm Unknown

5.3 cc 6.5 cc 5.1 cc 6.0 cc 4.4 cc 2.3 cc 39.0 cc 10.0 cc 00.4 cc 0.01 cc

Multipackage chamber; pocket conveyor feed Rotary fillersealer; pouch sides stretched before sealing Snorkel (tapered tube through seal area) Rotary turret (FMC) Steam flush Modified can vacuum closing machine with pouch carrier

Water bath, seal surfaces held taut (Hydropac)

Denmark (retail samples Production test

Natick-Swift contract

Test samples, U.S. patent data #3,625,712

2.65.24.44.63.00.7-

8.2 9.0 6.2 8.5 5.9 5.6

cc cc cc

cc cc cc

Production for Market test Prototype tests; Natick analysis Feasibility tests Production runs of 50,000 each; analysis is of 700 packages each Natick analysis of prototypes 0 min, 120°C, H, 0 5 min, 120"C, H, 0 10 min, 120°C, H, 0 20 min, 120"C, H, 0

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

371

retention time under vacuum or steam flush, velocity of vacuumization or steam flush, degree of vacuum desired, and overall production rate.

B. SPECIFIC TECHNIQUES 1. Snorkel

The snorkel technique, consisting of a tube entering the pouch through a short section of unsealed, top-seal area followed by suction, withdrawal of the tube, and sealing of the remaining seal area, was used in early trials (Mayer and Robe, 1963). However, the tube frequently gets contaminated with product and then contaminates the seal area.

2. Vacuum Chambers Where products consist of large or small solids with little or no free fluid, or where a variety of products are to be run on the same production line, the use of vacuum-chamber systems dominates. Many of these systems have been used for vacuum packaging of luncheon meats, and the transition to the use of supported films has required little if any modification. A common machine configuration is typified by the Hamac-Hansella Swissvac CVE(P) (Fig. 8), (Trans-Maschinen AG, 1972) where filled pouches are placed manually onto positions on an intermittent-motion horizontal conveyor in blocks of four or eight. As the conveyor indexes under a hood, the hood closes to form a vacuum chamber. The pouches are then sealed by the impulse mode and released to atmosphere when the hood rises. Typical cycles take 8 to 15 seconds, and reasonable production rates are made possible by the ability to vacumize and seal several pouches at one time. A variation of this type of equipment has a vertically moving conveyor belt (Multivac BG-6 Special, Multivac Export AG, 1973). The hood size for any system can be varied for efficiency in vacuum drawdown. A newer conveying system consists of a long, horizontal, intermittently rotated drum with full-length horizontal support panels to hold pouches during their rotation through a vacuumization and sealing area (SwissVac Duo Mark I or 11) (TransvacMaschinen AG, 1975). Goglio Luigi (1968) offers a system in which the vacuum-sealing chambers (sized to take several bags each) are rotated beneath the discharge ports of a pocket conveyor. Sealing again is by impulse. Rotating turrets with chambers for individual pouches also appear feasible. Woods (1967) has proposed a turret of pockets with individual sealers. Feed is through a top opening, and discharge is from the bottom. An obvious hazard is the possibility of contamination of the seal area during the drop into the sealing chamber. T h s problem can be lessened through the use of tack seals.

372

RAUNO A. LAMPI

As reported more extensively in Section VII, a standard vacuum closing machine for three-piece rectangular cans was modified to accept metal carriers holding individual pouches. In this particular instance, three stages of vacuum, each infinitely variable, were used to control the rate of air removal. Typical gage settings were: first stage-9 inches Hg; second stage-18 inches Hg; third stage-20 inches Hg; and main stage-27 inches Hg.

3. Counterpressure Where the products are predominantly fluids, air can be adequately removed by external pressure or pouch manipulation. Tsutsumi (1972) stated that by stretching the seal area taut, causing the liquid contents to rise just up to the seal area, and then sealing, internal air volumes can be kept as low as 2 cc. This method is applicable to products containing particles up to '12 inch in size, suspended in sauce. Other systems are offered in which the seal area is stretched taut, but further assurance of air removal is obtained by the squeezing action of two opposing plates coming together to push the product up in the pouch (Hamac-Hollar Bosch Group, 1974). A further sophistication is offered by ACMA S.p.A. (1973) (Model 712), which features a double squeezing system. An initial squeezing action by narrow bars partially closes the pouch above thc product, preventing the product from being pushed up into the seal area. A second squeezing action by plates on the body of the pouch then removes entrapped air. Heid (1970) described physical exclusion of residual gases by the use of waterhead pressure (FMC Hydropac system, Fig. 13). Again, seal areas are held taut to form a one-way valve, but instead of plates or pressure pads, the Hydropac system holds unsealed pouches submerged in water to the level of the seal area until gases are exhausted. This system is quite effective, challenging the sensitivity of gas measurement methods. To date, this system has been only pilot-tested. Long el al. (1966) proposed a tortuous path in a one-way valve approach for air removal during microwave sterilization. Conceivably, such an approach could be used in the submerged water-bath approach. Using the Hydropac technique as a necessary basis, Wilson (1971, 1972b) described formation of hydrogen gas in the pouch headspace through the reaction of water permeating the inner sealant layer with the aluminum foil. Concomitantly, a layer of aluminum oxide is formed on the foil surface. The claim is that the hydrogen assists in air removal prior to sealing, and the aluminum oxide improves the barrier characteristics of the pouch. If the pouches are to be sealed prior to heat treatment (sterilization), Wilson (1972a) proposes a preliminary hot-water soak to cause aluminum oxide to form in a layer substantial enough to reduce further hydrogen formation. The practical significance of these results, other than to support the use of the Hydropac technique, has not

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

313

been established at the present time. The conventional sealing operation occurs too rapidly to rely on hydrogen formation to displace air. Katz (1967), in an internal NARADCOM report, showed that the headspace of retort-pouched corn stored at 37°C for 24 months was 60% hydrogen, supporting Wilson’s contention that hydrogen is formed. However, the total gas volume was still a low 2.45 cc, which would have no adverse effect on pouch performance. The state of the art relative to retort pouches has not progressed yet to the point where the exact composition of headspace gas is of interest, since its total volume relative to most canned items is low.

4. Steam Flush Steam has been successfully used over the years to remove air from can headspaces where a high vacuum is required or where the headspace volume is inordinately large (such as canned emergency water). After Schulz and Mansur (1969) had refined their curved-bar sealing technique so that grease and water were no longer a deterrent to high seal strengths, the authors established that steam flushing could also be used to reduce headspace gas levels to 2 cc in 43/4 X 7I/~inchflexible pouches containing 4.5 ounces of product. The success of the technique, of course, hinged on the use of the curved-bar sealing technique to seal through the condensed moisture in the seal area. The FMC Corporation (1973) currently manufactures a Vaporpac laboratory simulator which permits close control of the variables governing the efficiency of steam flushing prior to sealing. Indications are that the technique is very effective. In tests using a 6 X 7-inch pouch filled with varying amounts of salt solution, air removal efficiencies ranged from 93% for a 70-cc presteam air volume to 97 to 98% for a presteam air volume of 10 cc. The FMC standard Vaporpac simulator features impulse sealing. It would appear easily feasible to incorporate steam flushing station(s) onto straight-line fill-and-seal equipment such as the Bartelt IM machine or rotary equipment such as the Yokohama T66F, ACMA 712, or the Bosch MU. The Circle Design and Manufacturing Corporation (1973) described the use of steam nozzles flanking the product feed tube to purge air from the pouch as the pouch was being formed on their continuous vertical forming equipment. Most of the steam gets displaced by product; the remainder condenses.

5. Drawn Pouch Systems Where aluminum foil is not a necessity, horizontal vacuum-forming equipment such as the Hooper 1000, Cryovac Division of W. R. Grace and Co. (1974), can be used. In these systems, air removal is accomplished in a vacuum-chamber section where the top web is circumferentially sealed to each pocket before

374

RAUNO A. LAMP1

cutting and release to atmosphere. Currently, such an approach is used primarily for institutional-size packs, and air removal efficiency is excellent.

C. METHODS FOR DETERMINING RESIDUAL GAS LEVELS I . Destructive Approach Shappee and Werkowski (1972) compared a destructive and a nondestructive method for measuring the residual gas volume of a retort pouch. The destructive method consisted in capturing escaping pouch gas (air) in a graduate cylinder inverted with its open end held under water. The pouch is opened under water, and the escaping air is steered into the cylinder through a funnel. The volume is corrected to atmospheric pressure as follows (Boyle’s law):

where Va = volume of air at atmospheric pressure (ml). Pa = atmospheric pressure (inches Hg). W, = pressure of water level in graduate (inches Hg). V,,, = volume of measured air (ml).

2. Neutral Buoyancy Technique In the neutral buoyancy technique, the test package is initially weighed in water with the pouch held just below water level. Then the pouch is placed into a transparent vacuum chamber in which the pressure is reduced until the pouch just floats. Gas volume is calculated as follows:

where V1 = volume of air (gas) in package at pressure P1 (ml). P1 = atmospheric pressure at time of test (inches Hg). P2 = pressure at time package is in a state of neutral buoyancy in water (inches Hg). D = weight of package in water at pressure P1 (gm). Shappee and Werkowski concluded that the nondestructive method compared very favorably with the standard destructive technique. Correlation coefficients were 0.994 to 0.998 for gas levels in the 0- to 50-ml range for three pouch sizes (284, 568, and 850 gm net weights). The authors presented regression equations for each package size and corrections for waterhead pressure when very accurate determinations are required. Generally, the calculated values (neutral buoyancy)

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

315

were lower than the actual volumes (destructive). Equation derivations are also given. Yamaguchi er al. (1972) similarly found a close correlation between the neutral buoyancy and destructive methods when the weight of the pouches in water ranged from 2 to 10 gm.

3. Pressure Buoyancy Wilson (private communication, 1968) has proposed a nondestructive method based on pressure increase in a chamber as opposed to reduction. In his method, the pouch to be tested for headspace volume is placed inside a pressure chamber which contains water and a beam balance. The pouch is submerged in the water, and the balance is zeroed. Then a known load is added to the beam of the balance. The chamber is closed and the pressure is increased until the balance is rezeroed. Rezeroing can be determined by the closing of an electrical contact. Gas volume is determined by the following equation:

V=

L 1 - (14.7/P)

where V = volume of gas (cc). L = added beam load (gm). P = absolute pressure at rezero (psia). Each method has advantages over the other, depending on the concerns of the tester and the accuracy required. It would appear that both nondestructive techniques are accurate enough for purposes of quality assurance and specification compliance. In fact, a simple method of flotation or sinking in a brine of known density (such as is used for pea grading) may find application.

X. RETORTING A. CONSIDERATION OF PREPACKAGING VERSUS POSTPACKAGING STERILIZATION Recognizing that the resistance of flexible packages to thermoprocessing conditions was a formidable requirement to meet and on the fringes of the prevailing state of the art, early developers (at least NARADCOM) considered the applicability of aseptic procedures (Szczeblowski, 1965) whereby product and package are sterilized separately and brought together in a sterile atmosphere at ambient or near-ambient conditions. This approach would have alleviated stresses on the package (Rubinate, 1964). Aseptic methods had been used up to 1960, albeit in a limited way, for the conventional three-piece metal can.

316

RAUNO A. LAMP1

The decision against aseptic procedures for retort pouches was based on several considerations: 1. None of the sterilizing apparatus for metal containers and none of the heat-dependent sterilizing techniques were suitable for retort packages. An aseptic retort-packaging system had to be totally devised and proved. Since the inception of retort pouch development programs, however, such systems have become available, but only for fluid products. 2. Little was known of the efficacy of film-sterilization procedures, and, apparently (Brody, 1973) further work remains to be done in this area. 3. High-temperature, short-time (HT/ST) heat exchangers were suitable only for fluid products; however, particulate and solid products were of major interest. Currently, development of particulate heaters-coolers is progressing. 4. The thin cross section of the prepared retort pouch (3h inch t o inches) permits rapid heating and cooling, thus providing the quality benefits of HT/ST principles where such is desirable, especially now that films withstanding temperatures of 275°F are available. Where the cook factor, C, (Mansfield, 1965), predominates, such as with many meat products, a lower temperature of 240°F has proved more desirable (Duxbury el al., 1970; Thorpe and Atherton, 1972). Here, the rapid heating characteristics simply minimize peripheral overcooking. 5 . Thermoprocess-resistant films, although not in wide commercial use, were considered feasible and within current state of the converting art. Aseptic procedures would only have eased the burden on adhesive systems, since two- or three-ply materials would have still been necessary for adequate strength, durability, and barrier properties. B. PRELIMINARY HEAT TRANSFER CONSIDERATIONS Heat transfer and related considerations have centered on determining the effects of pouch characteristics on heating and heat measurement and adapting techniques for metal cans to the specific characteristics of flexible packages including the heating mode of a slab as opposed to a cylinder.

1. Material mema1 Resistance As wqs pointed out by Chapman and McKernan (1963), published values for the thermal conductivity of steel (representative of tinplate containers) and of plastics, such as highdensity polyethylene, difference significantly enough to possibly require an increase in process time with plastics. Their work indicated that such could be the case if high-density polyethylene in thicknesses of 200

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

311

and 500 microns were used. Wornick et al. (1960), however, had investigated films in the thinner retort pouch range of 25 to 75 microns and had concluded that the thermal resistance of 75-micron high-density polyethylene and of thinner gages of polyester, highdensity polyethylene, and polypropylene was negligible. The heating lag of water in pouches was no greater than that for water alone. The effect of surface films, the conductivity of the food, and the geometric shape of the container were more important. In view of findings that process-determination methods for conventional cans apply to retort pouches without consideration of material resistance, no further detailed effort on this point has been reported. 2. Residual GaseslCounterpressureRelationship

With the exception of bakery products where the dominant residual component is C 0 2 from the leavening reaction, it is generally recognized that residual gases in retort pouches should be kept as low as possible to preclude chances of rupture during a retort cook cycle. Davis et a1 (1960a) devised apparatus and studied the pouch/retort pressure relationships during processing. They enumerated four causes of internal pressure increase as (1) increase in the vapor pressure of the water in the processed food with increasing temperature; (2) increase in the pressure of air in the headspace with increasing temperature; (3) release of additional air from the product, due to a decrease in gas solubility with increasing temperature; and (4) thermal expansion of the food product itself. They concluded that internal pressures of possible concern were related to the amount of material and enclosed air in the packages and graphically demonstrated that this pressure could be problematical in terms of pressure differential during the cool cycle. Cooling under superimposed air avoided these high-pressure differentials. Wallenberg and Jarnhall (1957) presented a table enumerating ratios of enclosed air volumes to package surfaces that should not be exceeded if pouch bursting is to be prevented. Yamano and Komatsu (1969) detailed the relationships among internal pressures, residual gas volumes, process temperatures, headspace expansion ratios, and steam-air ratios. Their protocol permitted calculation of required counterpressures to prevent failure. Rubinate (1964) reported that conventional steam processing could be used if no more than 10 cc of residual gas were contained in a 4y2 X 7 X %,-inch package containing 4.5 to 5.0 ounces. However, he mentioned what is perhaps the best reason for imposing an air pressure of 3 to 10 psi above the equilibrium pressure for water or steam at the process temperature over the total period of the cook; that reason is pouch restraint to prevent agitation and movement that could rupture the seals. Much pouch agitation is undoubtedly the result of temperature and/or pressure-control characteristics where sharp variations of k2

318

RAUNO A. LAMP1

psi are not uncommon. Whitaker (1971), in describing how to calculate pouch pressures from different causes, pointed out that, when pouch contents are at or near retort temperatures, a sudden reduction in retort temperature or pressure can cause a momentary pressure difference adequate to cause vaporization of pouch contents and an increase in internal pressure. Empirical support for this postulation comes from laboratory tests where a controlled pressure fluctuation of ?2 psi resulted in the failure of filled pouches of marginal seal quality, while closer, more uniform control did not. Aside from assurance of package integrity, superimposed air pressures, according to Keller (1959a) and Nelson and Steinberg (1956), should be maintained to improve heat transfer. The reasoning was that gas and vapor bubbles were kept from expanding and reducing heat transfer by impedance t o heat conduction and convection. Japanese investigators (Toyo Seikan Kaisha, Ltd., 1973a) support these contentions relative to residual gases and offer data to indicate that the headspace gas can also interfere with sterilization. Increasing headspace gas (occluded air) volumes from 0 to 15 cc resulted in changes in fh values from 6 to 7.2 and in sterilization times at 250°F from 6.7 to 7.1 minutes; however, 20 cc of occluded air increased thefh to 9.4 and the sterilization time to 12.5 minutes. In consensus, thermoprocess practice has been the use of superimposed air pressures or appropriate steam-air mixtures to prevent pouch expansion or movement from a preboiling point of the come-up cycle through cooling to again below boiling. With steam-air cooks, additional precautions during the initial introduction of cooling water are necessary to prevent an undesirable pressure upset.

3. Temperature Measurement Temperature measurements for determining heat penetration rates have generally been by conventional copper-constan tan thermocouples for preliminary heat-penetration experiments and batch-production systems. Continuous retorts and microwave techniques required different approaches. Where product characteristics, pouch-holding techniques, and pouch placement within the retort permitted, metal packing glands such as those used for conventional cans (Pflug et al., 1963) have been successfully employed to introduce wire and thermocouples into the pouch. Where greater flexibility of the pouch was to be retained or where holding methods within the process chamber dictated, thermocouple wires have been individually introduced either through seal areas or through small holes in the pouch walls. Pflug et al. (1963) described a procedure whereby 30-gage thermocouple lead wire was introduced through the sea area. The section of wire that passed through the seal area was stripped of all insulation, cleaned with solvent, and coated with a lacquer that

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

379

heal-sealed to the inside laminate of the pouch. Individual leads were separated by at least 0.25 inch to ensure a continuous seal rather than risk bridging across the wires. Rha and Karel (1968) reported success with sealing thermocouples between polyester tapes which were then capable of becoming a part of the pouch heat seal. For added assurance against leakage, they used pieces of rubber on each side of the seal, held in place by spring clips. Thorpe and Atherton (1972) introduced thin, lacquered copper and constantan wires through small holes made in the laminate about y4 inch above the bottom seal and sealed with a small amount of adhesive. A short length of 3M retort tape was used to attach each wire to the outer surface of the pouch to prevent movement of the wire through the adhesive. Once the lead wire has been introduced into the package, positioning can be controlled by several means. Pflug et al. (1963) used a plastic saddle with the metal packing gland to orient the thermocouple tip to the geometric center of the pouch if liquid or semiliquid foods were under investigation. Pflug (1964) also reported using a 1/16 X 1/4 X 5-inch piece of wood placed diagonally across a 4$!! X 7-inch (outside dimensions) pouch with the thermocouple stapled onto the center. With wire introduced through the seal area, Pflug et al. (1963)used an internal gusset fabricated to be part of the pouch. The thermocouple was attached to the apex of the gusset, and because of the V shape of the gusset, remained centered regardless of actual pouch thickness. Thorpe and Atherton (1972) used a rigid nylon spacer. With recipe packs, such as sweet and sour pork and beef strogonoff, the tip of the spacer-positioned thermocouple was secured in the center of a piece of meat of known size. Dewey Redesign (1975) described a disposable thermocouple/pouch arrangement where the lead wire enters through the pouch wall through an epoxy sealant. The thermocouple tip is suspended in the center of a conical coil spring. Continuous retorts present a different problem in temperature measurement. It is not practical to string thermocouple wire through such apparatus to follow a pouch. The direct count reduction method of process determination (Yawger, 1965) would appear applicable if retort thermal conditions are controlled and the repeatability of the cycles is established. Goldfarb (1970, 1971a) described and illustrated in detail a temperature telemetry system in which a thermistor sensor is inserted into the pouch through a packing gland similar to those used for conventional wire systems. The thermistor lead is connected to a battery-powered transmitter. The pouch-transmitter combination, as it passes through the continuous retort, sends radio-frequency signals that are indicated as temperatures on a digital telemetry receiver. This system (Aerotherm Corporation, 1970) has been successfully used for temperature measurements on vegetables packaged in retort pouches using a continuous retort.

380

RAUNO A. LAMP1

With microwave heating methods, nonmetallic sensors must be used. Kenyon et al. (1971) reported that chemically treated paper strips that can be calibrated to measure in 10°F increments alone or sealed into small glass tubing have been utilized for initial heating studies. A controlled browning reaction capable of indicating total heat treatment related to lethality has been developed. It would appear that with microwave systems, extensive inoculated pack studies in lieu of temperature-based calculations and methods are essential to prove effectiveness. C. PROCESS DETERMINATION Thermoprocessing conditions-cook time and temperature-have been generally determined for pouches by methods proved satisfactory over decades of use for metal cans. Even early researchers, such as Keller (1959a) and Could et al. 1962), used procedures established by Ball and subsequent thermoprocess investigators. The following concepts, related to microbial survival, have been fully accepted for direct applicability to retort pouches, since the microbial history and growth environments (foods) were identical: Fa-lethality in terms of minutes at 250°F required to destroy the specified spoilage organism in a specific medium. A “z” of 18 is assumed. D-time in minutes to accomplish a 90% reduction in number of organisms (or spores). z-shape (slope) of the thermal death-time curve in degrees Fahrenheit required for the curve to traverse one log cycle (Dversus OF). Production-scale experience such as that reported by Duxbury er al. (1970) and Lampi (1973) and a review of commercial cook times and temperatures (Tsutsumi, 1972; Goldfarb, 1970) indicate that Fo.values suitable for commercially canned products are generally adequate for retort pouches. Modifications for specific products might be required to adjust for the following factors: 1. Spore distribution versus total heat treatment required for peripheral volumes, based on Stumbo’s (1965) concepts. Roberts (1975) has shown, however, that the F-value distribution for three center-tosurface volume increments based on thickness increments of 1/32 inch for a 3.5 X 6-inch slab were, for his example, 2.310 (center), 2.404, and 2.425 (nearest to surface). 2. The partial choking of convectionheating modes as reported by Pflug (1964). 3. The degree of control exercised over point-to-point temperature variations in the retort (admittedly this is related to a safety factor for retort operation

381

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

rather than strictly to product requirement, but implementation is by increase in Fo if the variation is significant). However, from the nonmicrobial aspects of heating, instead of heat penetration into a finite cylinder, heat transfer with the retort pouch is into a thin slab; this difference has been most thoroughly investigated. Thermal-process parameters, as described by Ball and Olsen, can be established and should be confirmed by directly inoculated packs, but calculation techniques reduce time and permit translation of results from one set of conditions to another. They also provide rationale to support observed changes or events. These methods basically have consisted of integrating the desired lethality characteristics into the heat-penetration curves of cans. As shown in Fig. 19 in two comparisons (same retort load), the centers of retort pouches heat significantly faster than 300 X 200 metal cans of equal net weight. Similar comparisons have been cited by Tsutsumi (1972) and by Pflug et al. (1963). Heat penetration curves for cylindrical cans have been calculated and plotted on semilog paper, forming single or dual (broken) straight lines. The slope of such a curve (the plot of retort temperature minus initial temperature versus time) is termed fh. A thermal lag correction factor, j (designation of the intersection of the extended, straight-line portion of the semilog heating curve with the vertical line representing the beginning of the process), is also used in formulas calculated mathematically. Tsutsumi (1972) indicated that the faster

W

a L W c

I

0

100-

so

CAN, 35 MINUTES POUCH, 10 MINUTES

/’

1

1

1

1

1

1

382

RAUNO A. LAMP1

heating rate of the retort pouch results in an f h (steam-air cook) of 8.8, as opposed to 28.0 for a No. 6 can. Assummg conduction heating, Yamano et al. (1969a) gave the following theoretical values offh for cylinders and slabs: 2.303

Cylindrical shape: f -

1,

- 5 . 7 8 ( 1 , / ~ ) ~t 9.87

Flat slab:

fh=

2.303 -x-

9.87

lf k

Where k = thermal conductivity of contents. 1, = height of cylindrical can. a = radius of cylindrical can. If = height (thickness of flat can). From the above relationships, Yamano et al. (1969a) concluded that, with 25% bentonite, the fh for cylinders should be 3.5 times that of a slab for their specific container sizes. Experimentally, the ratio was 3.2, quite close, which they interpreted to indicate that heating was most influenced by container shape, with little effect from surface heat transfer coefficients. Pflug et al. (1963) also calculated an fh value for a 0.75-inch slab and found that it agreed closely with experimental heating data, indicating that a calculated fh can be useful in checking on experimental data for pouches. Thorpe and Atherton (1972) substituted replotted f h values into equations for determining thermal diffusivities (k). The k values agreed very closely with each other for various shapes, including slabs, and with k values calculated for conduction heating of cans. They concluded that the agreement of k values and j values between pouches and cans indicated that the pouch material tested did not affect the rate of heat transfer. They further cautioned that, with thicknesses of more than 1 y2 inches, the package should be treated as a parallelopiped. Schott et el. (1974) discussed the heat transfer characteristics of retort pouches and added a cautionary note relative to the fat content of meat products. Unless the percentage of fat is relatively closely controlled, it may cause enough variations in the heating parameters, especially the thermal diffusivity, to create problems in sterilization. For example, the thermal diffusivity of m2 /hr for 35% fat. meat with 20% fat is 6 X m2/hr, versus 4.5 X Pflug (1964) and Pflug and Borrero (1967), in their extensive studies to compare various heating media for applicability to commercial retorts and to define specific heating characteristics of each medium, used f h ,j , andF, values as criteria for their comparisons. They concluded that usually fh and j were adequate parameters to describe heat transfer, and this contention appeared to

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

383

be adequate for laboratory heating media and for confirming inoculated-pack studies. In their studies with commercially sized retorts, however, although they found that the three heating media (100% steam, water, steam-air) gave predictable and reproducible results, they noted that some of the semilogarithmic heating plots were straight lines, some broke one time, and some actually were curves. This was especially true of water cooks, which exhibited a slow retort come-up time, with product temperature lagging 20" to 30°F behind water temperatures. For those nonlinear heating situations, they recommended evaluation and design of thermal processes based on the general method. Herndon (1971) reported studies showing that predicted sterilization values for inoculated test packs would be closer to actual test values if the population distribution of the slope indices from a sample of heat-rise curves were used instead of the traditional slowest or mean single value of slope index in the sterilization calculations. To confirm his postulation, the accuracy of the proposed method was checked with 2 4 0 - p retort pouches of whole kernel corn (180 gm of corn and 60 gm of brine), first in a batch laboratory retort and then in a production prototype continuous retort (Hydrolok) using steam-air as the heating medium. These techniques were the basis of the process determinations in the packaging system studies documented by Goldfarb (1970). Yamano er al. (1975), reporting on studies using steam-air mixtures, found no appreciable differences among the heating rate parameters cf) for two types of film (12-micron polyester/50-micron polyethylene and 12-micron polyester/9-micron foil/ 50-micron polyethylene) and among processing temperatures from 105" to 120°C. They presented data showing the effects of varying side lengths with constant thicknesses, and of varying thicknesses with constant side lengths, on f values at 110°C. These could be helpful in determining some final pouch configurations; for example, such studies plus product browning effects appear to be the basis for Komatsu and Yamaguchi's (1975) coefficient of flatness ( K F ) limitation of 0.25, where KF = maximum thickness/maximum distance across the surface. As a whole, the reported experience indicated that heat penetration into retort pouches could usually be described by fh and j , and standard mathematical formulas for process calculations could be used. Frequent exceptions can occur (for example, the heat penetration curve shows n o straight section), and then the general method becomes applicable. All researchers cautioned, and the author agrees, that calculations should be used only as means for assisting process evaluation studies and not as a substitute for full process determination. The significance of excessive residual gas, as mentioned earlier, should not be discounted. Such dual approaches to process determination have been documented by Yamano et al. (1969a) with Chinese meat dumplings and soft spaghetti, and for a

W

m

P

TABLE XIV THERMOPROCESSES FOR REPRESENTATIVE RETORT-POUCHED FOODS: COMMERCIAL-SIZED EQUIPMENT

Package

Product

Retort and media

Net weight

Process

(02)

Length width Thickness (in.) (in.) IT CF) fh

X

Process Cook Total temperature time pressure CF) (min) (psi)

Beef stew Beef curry

Horizontal steam-air (80%vapor)

7.5

6.7 X 5

0.5

70

6

250

30

21

Borscht Sukiyaki

Horizontal steam-air (80%vapor)

7.5

6.7 X 5

0.5

70

6

240

40

21

Macaroni au gratin Chicken and rice

Horizontal steamair (80%vapor)

7.5

6.7 X 5

0.5

70

6

25 0

25

21

Meatballs and spaghetti

Horizontal steam-air (80%vapor)

7.5

6.7 X 5

0.5

70

6

245

35

21

Reference ToyoSeikan Kaisha, Ltd. ( 1973a) Toyo Seikan Kaisha, Ltd. (1973a) Toyo Seikan Kaisha, Ltd. (197 3a) Toyo Seikan Kaisha, Ltd. (1973a)

Horizontal water cook (retort IT of 70'F) Horizontal water cook (retort IT of 70'F) Continuous steam-air (Hydrolok) Vertical water cook Special horizontal steam-air

5.0

7.25 X 4.75

0.75

70

-

240

45

28

Duxbury et al.

5.0

1.25 X 4.15

0.15

70

-

250

20

N/A

Duxbury et al.

8.3

7 X 5.5

Frankfurters

Special horizontal steam-air

4.8

Pork and vegetables

Special horizontal steam-air

6.6

Beef stew Beefsteak Frankfurters Cakes

Whole kernel corn Beans in brine Cured meat sausage Shrimp SOUP

(1970) (1970) 1.25

150

6

255

13

24

Goldfarb

-

240 275

15 3

-

37

Normeat ToyoSeikan Kaisha, Ltd.

(1973) 7.5 7.0

-

0.5 0.635

185

-

-

0.75

-

-

275

9

37

ToyoSeikan Kaisha, Ltd.

-

0.625

-

-

275

4

37

ToyoSeikan Kaisha, Ltd.

6.7 X 5

(1975) (1975) (1975)

w m VI

386

RAUNO A. LAMPI

continuous steam-air retort (Hydrolok) by Goldfarb (1970, 1973). In the latter study, peas, whole-kernel corn, cut green beans, and mixed vegetables were processed at 255"F, 260"F, and 270°F. A broken heating curve used to calculate corn processes is given. Schmidt and Robertson (1 970) reported inoculated-pack and heat penetration studies with vegetable products. They concluded that heat penetration studies are a valid means for establishing processes, and that F values at the cold spot equal to those of cans would be sufficient to assure Commercial sterility, The results of the inoculated-pack studies were evaluated from the knowledge of the D 250 of the spores (PA 3679 and FS 1518), the total inoculum, and the number of packages spoiled. From these values, the integrated sterilizing value (ISV) was calculated as follows:

where M

= total spore load (number of spores per package times the

number of packages). S = number of packages spoiled. DzsO= resistance of the spores in the specific product. This is the microbiological estimate of the sterilizing value of a given set of processing conditions, and in their studies the agreement between Fo and ISV was rather good, verifying the validity of the heat penetration data. An Fo of 12 was found to be adequate. The ISV recognizes Stumbo's (1965) appreciation that spore distribution throughout a container may be significant, and such recognition may have more significance with 3/h-inch slabs than with 3-inch-diameter cans. Table XIV lists processes for known commercial or near-commercial products in retort pouches. The listing of process parameters for all products would be space-consuming and not overly useful; however, a tabulation of representative commercial processes is given as a guide to typical results. The F , values of commercial processes for cylindrical cans may vary widely (Alstrand and Ecklund, 1952), depending on the specific container size and the product. The processes shown for retort pouches vary similarly, representing Fo values from 2.5 for cured meats to 11 to 12 for some vegetable-in-sauce items. Another comparison of interest is that of process times of retort pouches with those of cans of equivalent product characteristics and net weights. Figure 19 shows heating curves on linear ordinates for chicken A la king in equal weights in cans and pouches. In a water cook, a 35-minute process for the can results in an Fo of 5.2, while only 10 minutes is required for the pouch. Thorpe and Atherton (1972) reported that, at 250"F, 454 gm (I pound) of beef and vegetable curry in a 1 6 2 can required a 70-minute process, while the same net weight in a 0.75-inch-thick pouch required only 2 0 minutes.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

387

D. RETORTING TECHNIQUES AND EQUIPMENT 1. General Aspects In view of the axiom that counterpressure above the process equilibrium is necessary in a commercial retorting cycle from the time the pouch temperatures exceed 212°F during come-up to a return to below 212°F during the cool cycle, the retorting techniques boil down to: (1) a water cook with superimposed air pressure similar to that used for glass containers and (2) mixed steam-air procedures. Possible exceptions are the developmental Hydropac system, where pouch closure seals are made after sterilization, and recently reported HT/ST systems. Batch operations currently prevail in the industry; however, test programs, reports of impending uses in Japan, and a pharmaceutical application in the United States indicate that, when volume merits, continuous retort systems can be used. H u g (1964) and Pflug and Borcero (1967), using both laboratory and commercial batch retorts, made a comparative study of steam, steam-air mixtures, and water as processing media. All, with certain constraints, could sterilize cans and retort pouches. Specific findings included the following: 1. Steam-air mixtures of 90% steam/lO% air and 7.5%steam/25% air at 240°F were similar in temperature distribution characteristics to a water cook at 240°F; 100%steam had less variation than either o f the other media. 2. Heat transfer rates of 100% steam, 90% steam/lO% air, 7.5% steam/25% air, and water are uniform and predictable. In comparing steam-air with water, if the steam percentage is more than 85%, steam-air is the better heating medium; if the steam percentage falls below 8.5%,water is better. 3. The type of heating medium did not measurably affect the quality of the product (shoulder muscle of beef3 for equal processes. It was established, however, that the 0.75-inch-thick pouch produced a significantly better product than the 300 X 201 can. The order of heat transfer efficiency, in general, was: 100% steam best, 90% steam, 75% steam, and water, with the last two about equal. However, a biological study (inoculated packs) to evaluate the different media revealed that, conversely, a water cook was best, steam-air cooks intermediate, and 100% steam, least lethal. Reasons for this reversal were not evident; in general, though, the results paralleled the pressure pattern where the highest pressure showed the least required lethality. 4. When commercial-size vertical or horizontal retorts were used, results confirmed that water and steam-air cooks could be effectively used for retort pouches, but the pragmatic considerations of the complexity of control systems,

388

RAUNO A. LAMP1

the greater need for induced circulation of the heating media, and sensitivity to operator error must be recognized. Recommendations and precautions were enumerated. The choice of water or steam-air as the processing medium becomes a matter of individual preference, and each has its commercial proponents. One basic advantage claimed for steam-air is the rapid come-up rate; however, as proposed by Thorpe and Atherton (1972) and currently offered in commercial systems by FMC and by several companies in Japan, process water can be preheated t o process temperatures if a separate closed-tank system is used. Microwave must be currently considered in the developmental stage.

2. Retort Racks or Trays A universal requirement, independent of the specific heating medium, is control of each pouch during a thermoprocess to assure a repeatable dimension of known thickness (or at least to preclude swelling) and to guarantee equal exposure of each pouch to the heating medium. Indiscriminate piling in a retort basket is hazardous. With bakery products, total shape is governed by control of pouch configuration in the retort. Pflug et al. (1963) used slotted racks of rectangular sheet-metal plates separated by rod-mounted spacers. Through the use of alternating 0.75-inch-wide and 0.375-inch-wide slots, they could control the thickness of the pouch to either of those two dimensions and, by leaving every second slot empty, assure circulation of water (or other heating medium) around each pouch. Circular retort baskets fitted with divider plates can be used with large vertical retorts. The carrier concept (Duxbury et al., 1970; Corning, 1973) is a variation of the vertical rack when carriers are grouped on trays, and it has the further advantage of adaptability to continuous retort systems. Continuous systems such as the Hydrolok (Rexham Corporation, 1973) and the Hydropac (Heid, 1970; Mencacci, 1973) use carriers attached to and moved by conveyor chains through locks into and out of the processing systems. Horizontal trays offer the advantage, in interface with batch systems, of a simpler loading operation. Although trays can be designed (Long, 1971) to permit easy loading and to contain pouches in a vertical position, most are designed for horizontal applications. Figure 20 shows a design featuring slots for better circulation of the heat medium and edges designed to permit stacking. Tray designs feature various types of perforations to assure thorough exposure to the heating medium and circulation. Yamano et al. (1969b) compared horizontal and vertical retort racks using steam-air mixtures as the heating medium. At 220"F, the temperature rise with the vertical type was slightly faster, while at 250"F, the temperature rise was

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS PERFORATIONS

7

389

f OPEN'

END VIEW

FIG. 20. Retort tray.

faster with the horizontal type. They concluded that there was almost no difference between the two orientations. Rectangular slots in the center areas of the trays were intended to improve heat transfer, but their effectiveness could not be determined. It is interesting to note that tray space was twice the normal pouch thickness to allow for horizontal movement of heating media. Retort trays or racks for horizontal retorts are collected on cars (Gee, 1973; Toyo Seikan Kaisha, Ltd., 1973b) consisting essentially of an angle-bar or channel-bar, open-framework base on casters. These are loaded into retorts by means of laid-in tracks, transfer cars, or fork-lift vehicles. There has been discussion but no convincing arguments over horizontal versus vertical pouch attitude during retorting. Long (1971) states that, by experimental evaluation, it is highly desirable to process the packages vertically rather than horizontally in retorts in order to establish reliable heat penetration with vertical flow of the heating medium in support. Thorpe and Atherton (1972) counter by proposing a horizontal position to preclude particle collection at the bottom and residual gases at the top. They claim that heat penetration tests have confirmed that for many products optimum processing conditions are attained when pouches are held in a horizontal plane throughout the processing cycle. Both studies used water cooks. Since successful production has been attained with pouches in the vertical position by the NARADCOM-Swift line (Duxbury et al., 1970) in the United States and by Normeat in Denmark, and in the horizontal

390

RAUNO A. LAMP1

position by STAR in Italy (Nughes, 1973) and the Toyo Seikan system in Japan (Tsutsumi, 1972), the question of pouch attitude appears academic as long as adequate circulation of the heating medium is assured.

3. Water-CookSystem Standard vertical and horizontal retort systems, using the standard water-cook temperature and counterpressure control systems employed for glass jars, are basically suitable for retort pouches. Pflug and Borrero (1967) cautioned that temperature stratification could take place unless water circulation was assured, preferably by mechanical pumping. They recommended the addition of air through steam headers and proposed that, as a design basis, point-to-point temperature variation be no more than 1°F after the retort reaches the process temperature. Robertson (1970), in testing the performance of water-cook retorts 5 feet in diameter and 14 feet horizontally, with a 400-gpm water circulation system (contents circulated once each 5 minutes), was able to meet a criterion of k2"F maximum point-to-point variation with no problems. Thorpe and Atherton (1972) imply a 21°C uniformity in specifying this accuracy for thermometers and calling for equal temperature uniformity. They also concur with a circulation rate of contents once every 5 minutes. Bulletins of the National Canners Association provide additional design guidance, as do the reports by Duxbury et al. (1970) and Thorpe and Atherton (1972). The appearance of a water-cook retort for pouches would be n o different from the basic systems for cans, nor would many control systems. Sams (1973), however, describes experiments with formulations and internal pressure relationships with bakery products that led to a versatile retort-control system. Bakery (cake) product formulations, primarily the leavening systems, were modified to withstand variable external waterhead pressures resulting from specific bottom or top retort locations in a water cook. Related laboratory retorting experiments established the following findings: 1. Leavening, structure setting, and sterilization occurred under different temperature and time conditions. A linear come-up rate of 8°F per minute t o 250°F resulted in good, sterile products (pound cake to fruitcake). 2. Lot-to-lot variations in dough characteristics could not be neglected. 3. An internal pouch pressure, 2 psi higher than the external (retort) pressure, resulted in good uniform porosity, typical of proper baking. Later, during confirmatory production runs (Duxbury et al., 1975), successful prepressurization of the system to 4 to 6 psi was necessary to avoid boiling during retort come-up.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

39 1

4. Pressure within the bakery product pouches was the cumulated effect of leavening (CO, ), occluded air, and product (water vapor); this total pressure exceeded that related to the process temperature over the total cycle period. Since this excess was more than 2 psi over the equilibrium for water at any point in the cycle, the postulation was that pouch pressures could be used to control retort pressure without the danger of causing boiling of the water. On the basis of the preceding relationships and requirements, a retort pressurecontrol system, schematically shown in Fig. 21, was devised (Katz et d.,1973). A measured sample of dough from each individual product lot is placed into a pressure can designed and proved to simulate conditions within a pouch during processing. The pressure within this can is constantly compared with the pressure in the retort chamber by means of a differential pressure (d/P) cell, and when a deviation from the desired differential is sensed, the control system automatically initiates a corrective valve action. With this particular control system, the counterpressure for nonbakery products was obtained by simple prepressurizing of the pressure can. Gee (1973) incorporated the pressure-control system described by Sams (1973) into an automatic-control system where, additionally, time cycles were controlled by a

r---I

1 I

I

I

I I

0l

I I

I

k--

I

Q

11) PRESSURE-TIGHT CONTROL CAN (21 PRESSURE VESSEL I R E T O R T I 13) CONTROL CAN PRESSURE-SENSING CAPILLARY 14) PRESSURE VESSEL PRESSURE-SENSING CAPILLARY

1s) D I F F E R E N T I A L PRESSURE TRANSMITTER (6) A U X I L I A R Y PRESSURE RECORDER I R E T O R T I (7) TEMPERATURE/PRESSURE TRANSMITTER WITH PRESSURE CONTROL RANGE OF 0 TO 50 PSI

181 AIR PRESSURE CONTROL V A L V E NORMALLY CLOSED, OPERATES FROM 9 TO 15 PSI SIGNAL 191 AIR EXHAUST CONTROL V A L V E NORMALLY OPEN, OPERATES FROM 3 TO 9 PSI SIGNAL.

FIG. 21. Bakery product retort control system.

392

RAUNO A. LAMPI

digital set programmer; rates of temperature come-up and cool by steam and water flows, respectively; and temperatures of the cook period by a pneumatic temperature recorder-controller. In Food Engineering (Anonymous, 1966) a simple position transmitter is described that senses the expansion of a representative bakery item pouch during the process and initiates corrective control action. The control system described by Sams and Gee was versatile for a variety of water-cook situations. For nonbakery products, a disadvantage was the slow [low retort initial temperature (IT)] come-up rate. Thorpe and Atherton (1972) reported the use of preheated water to reduce come-up time. The Instruction Manual for Toppan Auto Cooker H, Model 111(Toppan Printing Co., Ltd., 1975), describes a water-cook system using superheated water (250" to 300°F) from a separate tank to fill and sterilize pouches in the main retort chamber. It claims that, compared with the usual steam-sterilization equipment, sterilization times are one-half to one-third for the same sterilization value. Uniform temperature distribution is assured by a water-circulating pump. Steam is introduced into the main retort chamber to maintain temperature control during the actual process. At the end of the sterilization cycle, the hot water is pumped to the storage tank, air is used to maintain total pressure in the retort, and the cooling water is introduced. Toppan recommends this retort system for HTlST (275" to 300°F) processes, in contrast to Toyo Seikan, which avoid water cooks for those conditions. The FMC Corporation (Havighorst, 1975) has designed and constructed a similar system. In addition to the preheated sterilization media feature, the FMC system features an internal rectangular tunnel that holds the special retort carts and is mounted on circular end plates. These end plates are furnished with rollers mounted on their circumference; b y driving the tunnel section on these rollers, a tumbling type of agitation can be obtained if needed. Circulation is provided by water movement horizontally in channels between trays through the tunnel section, with return between the tunnel and retort shell wall.

4. Steam-Air Cooks Pflug and Borrero (1967) found that, if precautions are taken to assure constant flow-through of the heating medium to preclude dead spots, steamair processes could be used with commercial-sized retorts. Yamano and Komatsu (1969) and Yamano et aZ. 1969a,b) extensively studied the use of steamair heating media. Their comprehensive and successful studies have apparently formed the basis for steam-air processes, as they dominate in Japan (Tsutsumi, 1972; Toyo Seikan Kaisha, Ltd., 1973b). Aside from detailing operational parameters and generally reporting results paralleling those of Pflug (1964) on the nature of steam-air as a heating

393

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

tan(#ablr&(

-owr-pmsun d i FJly aU(0mtlc ntort).

Symbol Air- actuated control valve Air valve

(Piping

-

Water SUPP~Y

i

position

I V.

I

I h

1

Water pump

2r-=

Y

Y

1

II h\

T;mperature sensor

Steam

Reducing valve

I

w I

I

-

1 1I

I

Temperature-detecting

..

,.>

E / j I

Pressure introducer

*c,w” -

. . ACZOOV

. -. - -1, ~1

tt.

tieatingSterilizationWater supply CoolingDrainExhaustEnd-

Pr

Outdoor ditch

Temperature controller recorder Control panel

IEnd El I

El

1

Process Cooling timer timer I

FIG. 22. Steam-air retort. Courtesy of Toyo Seikan Kaisha, Ltd., Tokyo, Japan.

394

RAUNO A. LAMPI

medium, Yamano et al. concluded, significantly, that retorts presently used for canning in Japan were suitable, with piping modifications, for steam-air processing of retort pouches. Such a retort with its control system and air supply tank is shown schematically in Fig. 22. In operation, steam is introduced premixed with air. Temperature and pressure are controlled by independent systems. On the basis of considerations of better heat transfer with higher vapor-to-air ratios and the relationship between vapor-to-air ratios and pouch breakage because of occluded air, Toyo Seikan recommends retort operation in their optimum condition ranges as illustrated in Fig. 23. For example, with a retort temperature of 250°F (120"C), and retort gage pressures in the range of 1.4 to 1.8 kg/cm*, vapor ratios would be 85% to 70%, and the preferred pressure at 250°F (120°C) would be 1.5 kg/cm2. Point-to-point retort temperatures during processing are specified to agree within k2"C. On the start of the cool cycle, rapid condensation of steam could cause severe pressure upsets. Methods and rates of adding cooling water should be compatible with pressure-control recovery rates. With the advent of films capable of withstanding retort temperatures of 275" to 300°F, retorts have had to be modified to assure a fast come-up time. Toyo Seikan Kaisha, Ltd. (1975), in its literature on high-temperature films, describes a two-car retort for steamair processing with a separate steam reservoir and an air pressure tank. Steam is injected into the retort to achieve a come-up time of

105' C-

0 0

0.5

1.0

1.5

2 .a

RETORT GAGE PRESSURE (KGICM')

FIG. 23. Optimum operating range for steam-air retort for pouches. Courtesy of Toyo Seikan Kaisha, Ltd., Tokyo, Japan.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

395

1 minute to 275°F. The system permits four types of temperature-pressure profiles to be programmed. For 275°F processes, a straight steam cook with pressure cool or a cycle with superimposed air during most of the cook cycle and all of the cool cycle is proposed. They propose avoiding hot-water circulation systems for these HT/ST applications. Davis et al. (1 972) described successful procedures for retort-pouched, thermoprocessed cakes using steam-air techniques. 5. Continuous Retorts

Continuous retorts are suitable for retort pouches and are beginning to find their way into commercial production systems. One system that has been quite extensively tested is the Hydrolok sterilizer, shown in Fig. 24, and reportedly first used for pouches by Lawler (1967). Goldfarb (1970, 1971a) described in detail its use for test packs of vegetables; procedures for temperature measurement have been discussed earlier in this chapter. More recently, a detailed description of its capabilities, including its usage for pouches, has been presented by the Rexham Corporation (Rexham Corporation, 1973). As described by Goldfarb (1971a), pouches were placed manually into sixsectioned (one pouch per section) carriers which were then fed through a rotary-paddle wheel waterlock into the main retort chamber. Product filling and water-leg temperatures were maintained at 150°F (the IT for process calculations), and processing was accomplished by steam-air mixtures at temperatures as high as 270°F. Water level and water temperature were maintained by a continuous-running centrifugal circulating pump, feeding from an overflow reservoir to the chamber. A controller-actuated valve in a fresh-water make-up line controlled the level. Process steamair uniformity was assured by a circulating fan. Tests indicated that temperatures were controlled within +0.5"F at all points throughout the processing chamber, and pressures within k0.25 psi. A 7-psi air-overriding pressure, determined as the appropriate level by Whitaker's formula (Goldfarb, 1973), was successfully used with pouches. The pressurized, exit water leg was separated from the process section by an insulating baffle plate. Cooling in this exit leg was to 150"F, considered adequate to protect product quality. Removal from the carriers was fully automatic, followed by further cooling by a series of water sprays to a final temperature of 90°F. The Hydrolok has found significant application in Europe for sterilization of pouched milk, and an initial application for retort pouches in the United States for a pharmaceutical product (Anonymous, 197%). Current designs are capable of speeds to 550 pouches per minute and sterilization temperatures to 289°F. In 1975, it was reported that a hydrostatic, continuous sterilizer was designed specifically for retort pouches and shipped for use in Japan. The hydrostatic leg heights were such that air pressure could be used to supplement saturated steam

396

RAUNO A. LAMP1

Minuter

FIG. 24. Hydrolok sterilizer. Courtesy of Packaging Machinery Division, The Rexham Corporation, Rockford, Illinois.

pressures in the sterilization section. The exact amount of overpressure, according to Conley and Kornmann (1975), was based on sterilization temperature, residual gas levels, filling temperature, product characteristics, and pouch material. Pouches were conveyed through the system in a horizontal position in individual pockets. After each pocket or holder was loaded, a visor closed to retain each pouch in its individual compartment. This hydrostatic cooker was designed to receive the output of several filling and sealing lines and to process 480 pouches per minute. The advantages or incentives for its consideration were based on gentleness: no physical abuse during entry into and discharge from the

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

397

sterilization section, and no exposure to abrupt temperature or pressure changes. There have been no reports on its actual usage. A Carvallo hydrostatic sterilizer (Anonymous, 1975), featuring eleven columns for sequential preheating, sterilization, and cooling of retort pouches, has also been proposed. Heid (1970), Wilson (1972a), and Mencacci (1973), all describing the FMC Corporations’s developmental Hydropac (Fig. 13) and Vaporpac systems and the versatility permissible with those techniques, included hydrostatic designs as well as horizontal water-bath concepts. Steam or water has been proposed as the sterilizing medium, since the pouch is unsealed during the cook cycle and residual gases can escape through the tightly stretched “one-way valve”sea1 areas. Hydrostatic cookers involve massive structures and large investments, and they are fundamentally not adaptable to high-temperature/-short-time processes. If, therefore, the use of films capable of withstanding 275OF process temperatures find significant utility (process times of 2 to 9 minutes), the hydrostatic cookers may have limitations.

6. Microwave Processing J m d y (1965) and Long et al. (1966) devised, respectively, techniques for batch and continuous sterilization of foods in flexible containers using microwave energy. Long’s procedure included venting of residual gases through a tortuous patch through the seal area, thereby limiting its use to sterilization temperatures of 212°F. Both procedures were limited to packages formed of nonreflective materials-that is, no aluminum foil. Kenyon (1970) and Kenyon VIEW PORTS

ALUMINUM END BOXES

RECEIVER CHAMBER

ICROWAVE CAVITY

MICROWAVE OVEN-

MICROWAVE ANTENNAE

ENTRANCE VALVE SVSTEM ENTRANCE VALVE SVSTEM

FIBERQLASS EPOXY TUBE

~

ENTRANCE VALVE SYSTEM M I N U M END BOXES VIEW

bolls

FIG. 25. Schematic drawing of microwave processing system.

398

RAUNO A. LAMPI

er al. (1971) advanced the state of the art by devising a continuous pressurized system for sterilization and proposing “clean” or aseptic overpackaging with foil-containing laminates to achieve a reasonable shelf life. Counterpressures were achieved by means of a %foot, cylindrical, fiberglass-reinforced epoxy tube. Figure 25 illustrates the overall appearance of the apparatus. The conveyor belt carrying pouches through the tube was of neoprene-chlorinated sulfonated polyethylene construction. Four water-cooled, modular magnetron generators (Litton Industries, 1.25 kW each at 2450 MHz) provided power. Another pressure lock was used on the discharge end t o remove pouches batchwise after water or air cooling to below 2 12°F. Techniques for temperature measurement have been described. Preliminary runs were made with chicken a la king and frankfurters to demonstrate feasibility. Economics were discussed briefly, based on available data (6 to 8 ounces of product), but without sizing up experience (microwave heating rates are mass-dependent) and consideration of the effect of the more complex and material-consuming double-pouching operation, these could only be rough comparative estimates. For pouches measuring l,$ inch to 1I$! inches thick, where heat penetration rates v h ) in steam-air are one-third of those experienced with equivalent-sized cans and where 275°F processes are becoming commercial, there would appear to be minimal advantage to the use of microwave energy t o further reduce product heating times. No biological studies were reported, but based on “commercial practice,” a process time of 3 to 4 minutes (Fo required assumed to be 3) was cited to give practical sterilization. As a basis for comparison, Goldfarb (1971a) reported a 14-minute cook for mixed vegetables in a continuous steamair retort on the basis of inoculated pack studies. The use of high-temperature films and processes (275°F) cuts process times to 3 to 9 minutes with significant improvements in color, nutrient retention, and texture. Iipoma (1971, 1972) described what appears to be a like if not identical approach in United States patents. Stenstrom (1971a,b,c) similarly covered variations in the specific steps and manipulations of thermoprocessing with microwaves. Omeara er al. (1976) describe a technique for using microwave energy to raise the temperature of foods in foil-free’pouchesfrom 200’F to 250” to 260°F in 1 minute with 2450-MHz microwaves. The pouches are immediately placed in water at 250’F to give the desired F o . Products were found to have superior color and flavor, compared with pouches more conventionally retorted at 240°F for 40 minutes. No comparisons were made with steamair or water processes where the medium was preheated or where temperatures were in the 275°F range. A limitation has been that added salt interferes with the uniformity of microwave heating and apparently cannot be tolerated in the products. Schotte (1974) notes that microwaves are most practical for the pasteurization of packaged sliced bread and pie.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

399

XI. PACKAGE DURABILITY The durability of the retort pouch, defined as the ability of the package to withstand the rigors and physical abuse of normal distribution without excess failures or safety hazards, was among the first of the performance requirements that had to be faced. Since consumers (as well as many packers) would initially be skeptical that a heat-sealed, polymeric pouch could withstand the transportation and handling abuse that conventional three-piece cans could, confidence in the durability had to be firmly established. The durability or transportability of retort pouches has been assessed and established both by laboratory performance tests and by actual shipping tests and experience. A. PERFORMANCE TESTS The general intent of laboratory performance tests is to simulate, by means of controlled drops and vibration, the abuse of shipping and handling. In some instances, consumer handling, such as individual package drops and puncture resistance, are included. These tests are also sometimes supplemented by actual shipment of product. Such tests, on a comparative basis, merely permit judgments or rankings to be made on the adequacy of the basic phckage design-that is, its inherent properties. They do not guarantee the adequacy of daily manufacturing operations, nor d o they safeguard the consumer from occasional defective pouches. In a development program, difficulties frequently arise in that durability judgments have to be made on prototype containers when large-scale production characteristics are not known and could differ. A further difficulty is that results of laboratory abuse tests have a tendency not to correlate well with actual shipping experience. The conclusion-that laboratory vibration tests can be used to select the more abuse-resistant flexible packaging materials but not to predict the exact amount of damage occurring to packages during actual shipping tests-states the best perspective for such testing.

1. STAR Two reports (Nughes, 1973; Nughes et al., 1973) cover measurements of the performance characteristics of retort pouches as manufactured by Star di Agrate Briango, Milan, Italy. a. Immediate Containers. Tests on the immediate container (pouch and carton) consisted in guided drops followed by internal pressurization to 0.35 1 atmosphere (5 psig) for 60 seconds. The guided-drop apparatus assured guiding a package to hit a solid base at an angle of 15 degrees from the horizontal. The

400

RAUNO A. LAMPI

apparatus has a quick-release mechanism adjustable for height. The height of the free fall was adjustable so that the package struck with a force of 20 inchpounds. The height was calculated from the formula H = F/W, where H is the height in inches, W is the gross weight of the package in pounds, and F is 20 inch-pounds. After visual examination, pouches were pressurized with compressed air to 0.351 atmosphere by means of a hypodermic needle. Pressurization was done at the center of the pouch through a leak-proof seal. The pressurized pouch was then submerged in water and could not show leakage over a 60-second period. A normal sampling lot was given as twenty packages, and all were required to pass. b. Shipping Cases. Five shipping cases of product were conditioned at 23" f 2"C, 50% R.H., for 16 hours and then subjected to (1) vibration at 190 to 195 cycles per minute for 30 minutes (maximum case height permitted insertion of '/16 -inch flat board between the case and platform), followed by ( 2 ) five drops from 12 inches per ASTM D-775-6 1. The performance criterion given by Nughes et al. (1973) in their detailing of the test procedures was no visual damage. Nughes (1973) reported no evident failures or breakage and 13.5% minor damage (less evident failure defined as not visible to the naked eye). He further reported that pouches were submitted t o a biotest without spoilage and withstood squeezing in a press at a pressure of 700 kgSupplementary testing consisting in truck shipment of ten cases for a minimum of 500 miles was carried out. On return, the cases were incubated for 15 days at 34°C and examined. The failure rate was not to exceed 0.25%. c. Atncture Tests. One of the more controversial performance tests has been the puncture test. The correlation of its results with the mode and frequency of actual distribution-system failures has not been established; its main value appears to be as a measure of toughness. Based on performance experience in Japan where its use has not been reported, and no puncture-related problems reported, its value is marginal. A significant factor negating its current value is that an overwrap folder or carton is mandatory. If, as reported by Nughes in 1974, films are being developed (for example, incorporating a fabric in the laminate) to eliminate the need for a carton, then the utility of the puncture tests will require reassessment. In the procedure reported by STAR, a weighted plummet having a 1-mm point was dropped,from a height of 25 to 35 mrn onto a test pouch surface. No definition or criteria of failure were given. 2. NARADCOM Experience In the United States, considerable discussion has centered on using tests fundamentally identical to those reported by Nughes et al. (1973), but no

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

40 1

standard procedure currently exists. Undoubtedly, relevancy to actual shipping experience over such a diversified area as North America and equity in performance standards for the entire scope of hermetic containers used have been deterring factors. NARADCOM, designing to rugged military standards which include handling abuses such as obstacle course traversals in field clothing pockets, storage outdoors anywhere in the world, and 75%survival of free-fall air drops, has put candidate retort pouches through a series of severe performance tests. Among the earliest of these were those reported by Rubinate (1964, 1973), which resulted in the recognition that the folder into which the pouch was glued was necessary. Resistance to handling abuse was four times as good with the folder. This requirement was reaffirmed by Fiori and Fishman (1967) and by Lechtur (1967), whose abuse tests included retorting with periodic pressure modulations, horizontal vibration, slow rotation of the pouch on a central axis to cause the product t o flow back and forth, and revolving hexagonal drum drops. The last procedure, with a drop height of 20 inches, clearly indicated the advantage of the folder. Without a folder, retorted pouches could not withstand more than 3600 drops (10 out of 10 failed), whereas pouches in folders showed no failures after 7920 drops (0 out of 5 failed). The foldered pouches were next tested (Burt, 1960, 1963; Brugh, 1964), not in a laboratory, but outdoors under controlled conditions using an obstacle course of sixteen hazards designed to cause accelerated wear on clothing. Pouches were carried in field jacket pockets, and each, excepting failures, was subjected to ten traversals. Failures were higher than those experienced in actual use; for example, chicken 4 la king failed at a 5.5% rate, and green beans at 7.4%. As summed up by Lampi (1968), the significance of these failure rates is a matter of judgment, and the validity of this judgment will be strengthened by experience and comparison with actual use. One summation of the obstacle course tests has been that any package that passes even one traversal is a good one, but conversely, nothing derogatory could be said about the failures. The 5% to 7%failures were judged to be low.

3. Continental a n Company A more formalized performance test procedure, again somewhat analogous to but harsher than the STAR procedure, was developed by Continental Can Company under contract to NARADCOM (Payne et al., 1969). This effort also included development of the biotester previously described under Materials. Although primarily intended for evaluating candidate packages for irradiationsterilized foods, retort pouches (pouch and folder) were run for experience and comparison. The intent was comparison; and the abuse sequence was selected to cause failure (as opposed to a minimum performance standard), which it did.

402

RAUNO A. LAMPI

Their “tentative” performance cycle, after conditioning of shipping cases at 70°F and 50% R.H. for 48 hours, consisted in (1) shipping cases (54 jacketed pouches per case) vibrated at 1 G for 1 hour; (2) shipping cases dropped from 30 inches for ten random drops (ASTM D-1776-61, Objective B); (3) individual jacketed pouches dropped in a random manner ten times from 3 feet; (4) individual pouches subjected to a static load of 200 pounds for 3 minutes; and (5) biotesting of unjacketed pouches for thirty cycles in inoculated water. The results, based on exposure of 418 packages to the abuse cycle, were 5% failures. The investigation concluded that the abuse agreed with the obstacle course simulation of combat abuse, but that, with reference to experience to that time, the cycle was too severe.

4. Direct Comparison of Pouches and Cans The preceding performance tests were conducted with retort pouches alone. General judgments on failure rates, without benefit of experience or comparisons with other containers, were that the retort pouches performed adequately. Burke and Schulz (1972), backing off slightly from the number of treatments used by Payne et al. (1969), made a direct comparison of the resistance of cans and retort pouches to simulated handling abuse. The pouches were 4% X 7 X 3/4 inch glued into folders. Cans were 300 X 200 sanitary cans conforming to the requirements of Federal Specification PPP-(2-29 and represented a recent, 100% inspected procurement lot. Test variables included two three-ply films and two product types-a semisolid item (beefsteak) and a more fluid pumpable product (chicken A la king). Each test package was packed 72 to a 200-pound-test corrugated fiberboard case. The rough handling in cases consisted in ( 1 ) vibration at 1 G for 1 hour; (2) drop tests of ten drops from 18 inches in accordance with ASTM-D-775-68, Objective B, in a prescribed sequence, and (3) biotesting of individual pouches and cans. The biotester was again used. For cans, a desiccator jar fitted with an air pressure system was used. Timer-controlled solenoid valves operated to fluctuate the vacuum (17 to 22 in. Hg) in the jar to cause flexing of the can ends and to copy the flexing action of the pouch biotester. All nonswollen containers were inoculated with bacteria following the post-biotest incubation to confirm the absence of large defects through which gas could have escaped. There were none. The results are shown in Table IX. Although there were differences between the films, the investigators concluded that the two flexible materials tested were capable of withstanding the abuse cycle at least as well as the metal cans. On the strength of permitting a direct comparison with the performance of an accepted, proved safe container and being relatively straightforward to carry out, the abuse cycle used by Burke and Schulz shows promise for application as a performance screening test for containers.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

403

5. Toyo Seikan Kaisha, Ltd.

Toyo Seikan Kaisha, Ltd. ( I 973a) in their brochure discussed the necessity to carefully design individual cartons in terms of product viscosity and resistance to drop and vibration, but gave no standard or sequential performance test procedure. B. SHIPPING TESTS AND EXPERIENCE Shipping tests frequently supplement laboratory performance tests but are also carried out in lieu of them and to gain direct practical experience. The total shipping experience has been favorable, or else the concept would not be as viable as it is currently in Japan and Europe. Goldfarb (1971 a) reported that palletized shipments of retort-pouched vegetables, with canned vegetables as a control, can be successfully made by both rail and truck for distances up to 1000 miles and probably farther. The pouches used for these tests were made with 25-micron Nylon 11 as the heat-seal film. In addition to the basic comparison with cans and the evaluation of truck versus rail modes, variables included banded versus unbanded pallets and gluing versus nongluing of pouches into cartons. Relative to these last two variables, it was found that banding was needed only if the method of shipment allowed load shifting and that there was no need to glue pouches into cartons. Goldfarb suggested that vertical placement of cartons into cases be evaluated to improve compression strength for case stacking. This would be in contradiction to the preferences of Toyo Seikan Kaisha, Ltd. (1973a), but if folders are used it would be compatible with the findings of Burke and Schulz (1972), who found that package orientation made no difference. As mentioned briefly by Lampi (1968) in relation to performance tests and summarized more completely by Szczeblowski (197 1), NARADCOM evaluated shipping and handling early in its development cycle. For an integrated engineering and service test (ET/ST) for an operational ration featuring retort pouches (Paschal1 et al., 1967), approximately 4000 cases of twelve meals per case were shipped from Kansas City, Missouri, to test sites in Virginia, Georgia, North Carolina, Louisiana, and Panama. The report on this field-use test concluded that the item was “excellent” for all means of transportation. A failure rate of 0.3% during the ET/ST attributable to manufacturing deficiencies necessitated a hiatus to establish manufacturing reliability and then, in 1973 to 1974, a repeat of field testing of operational rations featuring retort pouches. The transportation and handling trail consisted of air, rail, commercial motor, military truck, tracked vehicles, sled, air drop, and man carry. Obviously, commercial shipping would not include tracked vehicles across country, trucks on secondary roads, or air drops; however, if the results are favorable with this

404

RAUNO A. LAMP1 TABLE XV DURABILITY O F THERMOPROCESSED FOODS IN FLEXIBLE P A C K A G E S ~

100%inspection site

Number examined

Percent failures

Ration assembly Field test site Ft. Greeley, Alaska Ft. Benning, Georgia, Camp Lejeune, North Carolina Consumption

57,000 54,000 approx.

0.01 1 0.004

30,000 approx.

Ob

aDevelopment test 11, meal ready to eat. *Does not include air-drop tests.

greater level of handling, then a degree of confidence for commercial use is a logical conclusion. The results were favorable, as shown in Table XV. Of 57,000 packages, 100% inspected, at the ration assembly point, only 6, or 0.01 1%, were failures. It was not possible to distinguish any process-oriented failures from transportationcaused ones. On receipt at test sites, only two retort pouches had failed; one side seal leaked, and another had burst on one end. There were no further reports, written or verbal, or other evidence of failure at the time of consumption. The difference in the number of packages examined at the test site and those consumed represents those used for air drop, including free-fall, where the criteria were 100% recovery for parachuted loads and 75% for free-fall. The meals passed the air drop criterion. Those items not needed for the field tests were packed, without folders, on edge in open fiberboard trays, palletized with a shrink film overwrap, and shipped by commercial truck from Chicago to Natick, Massachusetts. There, the 2 10,000 pouches were visually examined and showed no evidence of failures or spoilage. Judging from the documented performance and actual shipping tests, and the success of the concept in Japan and Europe, retort pouches are rugged and can readily withstand the experiences of commercial and military distribution.

XII. QUALITY ASSURANCE A. GENERAL ASPECTS Quality assurance, per Military Standard 109A, comprises a planned and systematic pattern of all actions necessary to provide adequate confidence that a product will perform satisfactorily in service. The key concept or philosophy

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

405

promulgated in that definition is that raw materials, in-process or intermediate product characteristics, and the processing system are all kept performing within tolerances with the assumption that the final product will meet end-item definition and performance requirements. Obviously, these in-process measures are supplemented and tolerances are refined by end-item testing, timely feedback of data, and experience. Relative to retort pouches, this approach to quality assurance is especially pertinent, since from a positive aspect (Lampi, 1973; Tsutsumi, 1975; Nughes, 1974) it has proved successful, and from a negative viewpoint comprehensive leak-testing on a production scale is not yet feasible.

B. SYSTEM SPECIFICATIONS AND SAMPLING PROTOCOL Undoubtedly, individual suppliers and food packers have selected tests and have developed and refined test protocols, sampling levels, and criteria to meet individual proprietary needs. Duxbury et al. (1970), reporting on NARADCOM’s program to establish production reliability capabilities, documented “tentative specification guidelines” for incoming materials and for each unit operation as listed in Fig. 5. These specifications were the standards to which the equipment was designed and the criteria to which each had to perform. These standards were not unlike those listed by DRG (1975), Toyo Seikan Kaisha, Ltd. (1973a), and other film and equipment suppliers and can be presented as an example of a successfully implemented approach. Obviously, fundamental design features and the use of automatic controllers kept many line functions in control, but sampling and individual pouch testing were necessary at key locations. Table XVI shows, for a typical product, beef stew, the sample removal points, tests, sampling levels, and rejection criteria. This plan assumes prior testing of roll stock film (or of preformed pouches, if such is the case) to meet standards. The tests shown were selected for their relevancy to key variables such as seal strength and integrity, and the ease and rapidity with which the tests could be performed. Timely feedback for prompt corrective action was considered vital. Accept/reject criteria were based on the criticality of the variable to overall package performance and the statistical guidance of Military Standard 105D. The objective of the program was assessment of production capability where no firmly established prior art existed. Therefore, it may find its best application immediately on start-up of a new line or product. A reduction in sampling levels seems possible as confidence permits. Shenkenberg (1975) offered further criteria for container defects. In establishing these standards and tests, two cautions soon evolved. First, there must be assurance that laboratory tests and criteria are compatible with on-line machine capabilities; to wit, strengths of seals made on a Sentinel sealer in the laboratory as an assessment of materials’ properties and standard for

TABLE XVI INSPECTION PLAN-BEEF STEW PRODUCTION' .~

Sampling site In process-after pouch formation In process-after closure seal

Final packageafter retorting

~

~~

.-

-

Test

Lot

Sample size

Air burst Pouch dimension Meat weight

1800 pouches (30 minutes of production) 1800 pouches

6 each 30 minutes 6 each 30 minutes 6 each 30 minutes

Meat count

(30 minutes of production)

6 each 30 minutes

Reject criterion 1 1

Corrective action, no rejection per se Corrective action, no rejection per se Corrective action, no rejection per se Corrective action, no rejection per se

Vegetable weight

6 each 30 minutes

Vegetable count

6 each 30 minutes

Top seal air burst Residual gas Visual for defects Net weight Cooked meat weight Vegetable weight Organoleptic acceptability ResiduaJ gas Air burst Visual for defects Bacterial count

1 6 each 30 minutes 2 each 30 minutes 1 100% All defective packages removed 13 Accept 3, reject 4 13 Accept 3, reject 4 13 Accept 3, reject 4 3 Corrective action 2 1 13 (6 bottom and side; 7 top seal) 1 100% All defective packages removed 6 1

'U.S. Army Contract DAAG 17-69€-0160.

2688 pouches (retort load)

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

407

performance should be capable of being duplicated by production pouchforming equipment. This is not always as easy as initially supposed. Second, there should not be conflict between definitive and performance requirements. For example, if a film construction is closely defined and the definition is followed, but delamination or seal failure occurs during production, what corrective action can be taken, and by whom? Preference has been performance. C. SPECIFIC TESTS The specific tests and performance criteria for the quality assurance program illustrated have been discussed individually in preceding sections on materials, sealing, and air removal. The adequacy of retorting can be assessed by using incubation procedures established for cans. It should be noted that two key defects-pouch leakers, and seal area wrinkles and contamination-have been detected by visual examination and removed. Adequate visual guides (Fig. 14 and 17) exist to facilitate seal-area evaluation, and experience has indicated that production leakers are large enough for visual detection. There has been an effort to determine if nondestructive testing methods could be applied or devised to replace the subjective visual examinations. For seal defects (which may or may not be leakers at the time of examination), candidate methods exist; for leakers, there has been n o economically acceptable method yet devised. In addition, the ability to produce retort pouches with a reliability equal to that of the conventional can has negated the essentiality of such tests. 1. Seal Defects

During the interim period of retort pouch development when seal-area contamination was recognized as a defect, but before there was any assurance that defect-free seals could be made with the required regularity under production conditions, NARADCOM investigated several approaches toward a nondestructive test procedure. Under consideration were such techniques as ultrasonics, low-current x-rays, eddy currents, feeler brushes, liquid crystals, polarography, and infrared thermography. Low-current x-rays using a videoscope output did permit detection of ail but grease- and moisture-related contamination, but had two serious disadvantages: (1) maximum scan rate of only eighteen 4l/~-inchseal areas (detection relied on movement of the scan area, and experiments revealed detection problems at higher scan rates), and ( 2 ) the need for human interpretation of the results. a. Infrered Scanning. Studies on infrared thermography (Lampi et al., 1969) led to a single-line scan approach of the closure seal with the output initially an X-Y plot of a signature curve and, eventually, a signal processed to give an

RAUNO A. LAMP1

408

accept/reject decision. The operating technique is illustrated in Fig. 26. A moving carriage holds and passes the seal area first across a heat source and then, very slightly later, in front of an infrared radiometric microscope which measures the temperature of the opposite seal surface. Any impedance to the flux of heat through the seal thickness is detected as a momentary temperature drop. In the basic feasibility studies, the infrared rddiomcter was a Barnes Engineering Company Model RM-2B, and the heat source a Henes Model FT-200 flarneless torch with an 0.125-inch-diameter nozzle. Airflow and temperature were adjustable. Wmi the design and performance criteria were converted into a prototype machine to align, scan, and accept or reject the seal (Ordway and Schulz, 1972). the detector was a modified Thermal Master, Model IT4 (Barnes E.ngineering Company), and the heat source, a Sylvania tungsten halogen projector lamp. The heat from the lamp was concentrated and directed by an elliptical mirror to form a 0.2 X I-inch line image on the scanned surface. The long dimension of the heat image was perpendicular to the direction of seal travel. The transport mechanism for the prototype apparatus consisted of a main conveyor which first aligned the pouches on their long dimension. Then, as the pouches were moved over a transverse conveyor-belt section, this transverse belt positioned the pouches between the jaws of clamps that are integral components of each drive conveyor segment. A linear cam caused closing of the clamp as the

h TRAN SVERS I N G

HEAT SOURCE

CARRIAGE M E C H A N I S M

DIRECTION OF POUCH MOVEMENT-

INFRARED *MICROSCOPE

P U R I N G SCAN

ELECTRONIC x-Y

___

RECORD -€ -

-CONTROL UNIT

FIG. 26. Schematic of infrared wanner for seal defects.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

409

conveyor neared the scanning operation and presented a 1/4 -inch-wide band at a constant speed and controlled plane to the scanner. Lampi et al. (1973) reported on basic acceptance tests and selected optimization studies. The equipment acceptance tests were successfully run with minimum optimization. Simulated package defects consisted primarily of 0.5 and 1.O mg of freeze-dried pork fibers, granulated sugar crystals, dehydrated potato powder, 8-mil-diameter thread, and voids left on withdrawing 26-mil threads. Moisture, grease, and wrinkles were also used. Optimization studies showed that defect detectability is relatively insensitive to heat-lamp voltage and lead distance (distance between heat source and detector) settings, or fluctuations. Signal processing variables can also be set within a range as opposed to optimum specific settings without greatly affecting the detectability of defects. Experiments also indicated that similar packaging materials can manifest different thermal-response characteristics, but that machine adjustments to optimize detectability are possible. The infrared scanner is technically applicable for detecting seal defects at speeds at least up to 6 inches of seal per second. Its pragmatic applicability hinges on three points: (1) effectiveness of measures to eliminate concern over seal contamination (such as clean filling, steam-flushing, or refluxing to remove contamination, curved-bar sealing, long-term documentation to reveal low incidence of contamination), (2) definition of defects and establishment of acceptance/rejection criteria, and (3) justification of its relatively high cost. b. Calipers. An alternate system of detecting defects (Lampi et aL, 1975) was inspired by the principles of operation of a paper sheet weight profiler (Foxboro Company, 1966). This technique relies on the use of calipers t o measure the transverse thickness of paper and, therefore, possibly of seal areas and seal-area irregularities. Preliminary trials indicated that occluded particles and fold-type seal wrinkles could be detected; but sizes, in general, had t o be larger than those detected by the infrared scanner. Alignment problems were also noted. The caliper technique is being reevaluated, since it is less costly than the infrared system, and, since curved-bar sealing methods could remove concern over grease and moisture contamination (which calipers had trouble detecting), only relatively large amounts of occluded matter may need to be detected.

2. Leak Detection Because of the high water activity (a,,,) of most of the products packed, the incidence of leakers in retort pouches could result in economic loss through bacterial recontamination. Furthermore, on the basis of experiences with flexible packaging where higher failure rates, although not condoned, prevailed and

410

RAUNO A. LAMPI

were tolerated, people contemplating using retort pouches for thermoprocessed foods were wary of high leaker rates and voiced a strong desire for on-line detection for 100% inspection. Proctor and Nickerson (1958), Hartman et al. (1963), and Griffin er al. (1967) had shown that single plies of laminate materials for retort pouches contained pinholes that could permit passage of bacteria. They also reported that three-ply laminates were unlikely to contain inherent passageways for bacteria. Tsutsumi (1975a) echoes these latter findings, observing that it would be very rare for pinholes on each film of the three-layer structure to coincide with one another. Therefore, apprehension relative to inherent laminate pinholes or breaks was relieved. Furthermore, promising methodology, as described in the preceding section, existed for seal defects, some of which could be leaks. However, before adequate development and production experience existed to indicate that the leaker rate could be kept low and that leaks were visually detectable (Tsutsumi, 1975a), and before enough data accumulated to indicate that pouches swelled more easily than cans and were equally as identifiable [48 hours to definite visual detection with pouches, as opposed t o 72 to 120 hours for a can with equal inocula of Aerobacrer aerogenes (Schulz, private communication, 1973)], studies were carried out on the definition of leaks in terms of bacterial penetrability and on concepts of leak detection to determine a feasible system. u. Definition of Leaks. As a prerequisite for the selection, development, or even consideration of leak-testing methods and hardware, criteria have been established. Leak test apparatus, based on a pressure-drop principle, is available for empty cans (Borden Company, 1972), with a sensitivity of gas leaks of 0.2 cc of flow per second at a 10-psi pressure difference. Bee er al. (1972) described a microleak detector for metal and glass containers, but gave no quantitative estimate of sensitivity. For flexible films, Japanese studies showed that 50 X SO-micron holes in vinylidene chloride and 80 X 80-micron holes in rubber hydrochloride sausage wraps were safe relative to bacterial spoilage, while 70 X 70-micron and 100 X 100-micron holes in the respective films were dangerous. Payne el al. (1969), in describing the development of the biotester, reported that 11% of 386 pouches of chicken A la king punctured with a 0.032-inch needle did not become contaminated during flexing. Hole sizes for these were reported to average 102 microns in diameter with a range from 33 to 160 microns. It became evident (Ciavarini and Lampi, 1976) that there was inadequate background on which to establish practical leak standards for retort pouches. An additional complicating factor for retort pouches was that fluid can effectively plug small holes and severely reduce the sensitivity of common techniques for detecting gas-flow leaks. Detection of leaks smaller than 1 X lo5 atm cclsec by halogen gas is not possible if moisture is present; otherwise a leak of 1 X atm cclsec can be detected. The common Mead test, which involves

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

41 1

pulling a vacuum on a submerged pouch and inspecting for escaping bubbles, was tried on a 4 j / 4 X 7'/z-inch retort pouch sealed first with air only and then filled with distilled water. A 5-micron-diameter hole was easily detected in the dry pouch body, but even a 68-micron hole was not detectable in the body of a pouch containing distilled water. With the preceding information as background, NARADCOM (Ciavarini and Lampi, 1976) went through a series of experiments in the sequence listed to: 1. Initially determine the smallest defect in a flexible package of wet product, regardless of how created, that bacteria could pass through. Although later findings could make this aspect rather academic, the size limit would establish the ultimate sensitivity that could be required from any inferential, commercially applicable, leak-test method and would act as an initial criterion for subsequent laboratory test methods. 2 . Determine whether gas-flow measurement could be used to define leak size in lieu of a physical dimension, since defects were found to be very irregular in shape, and visual sizing was inadequate. An analysis to establish the type of flow would permit translation of results to other test conditions. 3. Progress to controlled laboratory creation of defects by flexing, puncture, and abrasion to determine the likeliest cause of the smallest defects in terms of bacterial penetration. 4. Finally, assess the size and characteristics of defects, if any, caused by abuse tests simulating the transportation experience of the packages. Pragmatically, the collective results of these studies would permit: 1. Establishment of realistic leak detection criteria. If, for example, abuse tests created only large defects, there would be little advantage in searching for methods to detect significantly smaller ones. Conversely, if small defects are encountered, the data on penetration versus size could then be used to establish a limit. 2 . Judgments relative to the applicability of or even the need for leak detection methods.

Certain initial assumptions and constraints were made and followed: 1. The biotester (Maunder et at., 1968) was assumed t o simulate extreme yet realistic conditions for exposure of packages to viable bacteria; if penetration were to occur, it was assumed it would be in the biotester. 2. The packaging material was limited to a three-ply laminate of 12-micron Mylar/9-micron aluminum foil/75-micron modified polyolefin, or high-density

412

RAUNO A. LAMP1

polyethylene. For simulating transportation and handling abuse, the pouch would be adhered t o and inclosed in a 16-point Kraftboard folder. 3 . The causes of failure, beyond the initial fundamental studies, would be created realistically in mode and magnitude but would be carried out to the point of positive failure (lengthening of time, increasing number of cycles). It was not the basic intent to evaluate or imply performance per se but to gain data on size when failure does occur. The initial task turned out to be extremely difficult. Laser drilling was selected for fabrication of small holes in the three-ply retortable laminate; however, control over exact hole size was not possible, since even minor variations in material thickness affected the outcome. Another problem was the observation of a “flap” of material apparently formed at the moment the laser burning action reached the farthest surface; such defects were not used because of the possibility of the flap’s acting as a valve and interfering with the biotest action or gas-flow measurement. Out of one hundred laser-drilled defects, only twelve were in the size range of interest and clean enough to give reliable results. The results of biotesting pouches of semisolid agar containing dextrose are shown in Fig. 27. The small number of replicates voided statistical analyses or the drawing of very finite conclusions. It would appear that there is little likelihood of penetration through a defect smaller than 11 microns in diameter. If it is desirable to establish a criterion in terms of gas flow, a sensitivity of 5 x lo-’ atm cclsec, using a pressure drop across the film of I92 mm Hg, would be tentatively suggested. The second phase of NARADCOM’s studies involved laboratory abuse to the point of failure simulating the following potential causes of package failure: 1. Flexing. A folded sample in the MIT folding endurance tester was flexed to obtain a point-type failure. One thousand flexes were required for a Mylar/foil/ high-density polyethylene material. 2. Abrasion. An S&S Corrugated Paper Machinery Company scuff tester with a relatively smooth fiberboard as the abrading surface was employed. The location and dimension of the high point of the package-surface crease receiving the abrasive treatment were established by the use of crossed fine wires on the surface of a plastic block which was then vacuum-sealed inside the pouch. The length of treatment was selected to result in perforation of 50% of the samples. 3 . Puncture. Punctures were made by a finely drawn tungsten needle fired to a long, thin point. The results of the laboratory defect-sizing studies were as follows: 1. Flexing. Flexing caused a general area of porosity where microscopically aided visual sizing is not possible. There was no single hole or physical dimension

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

413

.26

P

Q,I

0

PENETRATION 0 N O PENETRATION 0

.2

E E

Q

8

CRITICAL DIAMETER

0 0 4

.16

P 0

Y

0

u)

\

0 0

.1

:

Y

9Y .os .oa

I

I

I

I

I

5

10

15

20

25

HOLE DIAMETER (MICRONS)

FIG. 27. Laser-drilled defects; measured size versus flow rate with notations on bacterial penetration.

to measure. Based on empirical gas-flow sizing (dried nitrogen gas at 192 mm Hg pressure drop), bacterial penetration in one sample out of ninety-nine was noted at 2 X cclsec. The next largest gas-flow rate for a penetrated pouch was 1.1 X cclsec (eight negative replicates in between the two positives), and penetration predominated at all other higher flow rates. Flexing can cause a very small defect (equivalent in gas flow to a 1 1-micron-diameter hole), but, because of the high resistance of the materials to this type of abuse (1000 to 5000 flexes), it is unlikely to be a serious performance factor or to warrant a size criterion based on its characteristics. 2. Abrasion. Forty replicates were abraded for each of three times. No perforations were noted after 1 minutes, and only one-an ellipse equivalent in area to a 124-micron-diameter round hole-after 2 minutes. After 2'14 minutes, one-half of the samples were perforated. The smallest defect was equivalent in area to a 24.4-micron-diameter round hole, while seventeen out of the twenty were equal to or larger than an equivalent 128-micron-diameter round hole. If abrasion is established as a cause of leakage, an initial criterion could be sensitivity to detect a 24-micron-diameter hole.

414

RAUNO A. LAMP1

3. Puncture. It became evident that, as the diameter of the probe was reduced to 100 microns, puncturing the three-ply laminate became very difficult. The best tough probe found was a thin tungsten wire fired to a long, fine point. A rubber stopper enclosing all but the point was used to control the depth of penetration, but, even then, the defects created were relatively large (>lo0 microns) and detectable by visual examination. The laboratory tests indicated that none of the three possible causes of abusive pinholing created leaks smaller than the basic sensitivity criterion of a 1 1-micron-diameter hole or a gas-flow rate of lo-* atm cclsec. They also revealed that (1) the films were inherently resistant to flexing, voiding concern over its being a major cause of pinholing and that (2) abrasion and puncture caused relatively large pinholes. The laboratory tests were followed by a survey of leaks caused by simulated shipping tests. Burke and Schulz (1972), as described earlier, ran comparative performance tests with retort pouches and 200 X 300 three-piece cans. In case lots, each package was put through a vibration and drop test sequence that resulted in a total of forty-one retort pouch failures out of a total of 3600 tested. Table XVII presents the size distribution of these failures. The smallest defects were found in the body areas of two beefsteak packages and, on the basis of subjective, comparative, microscopic examination by experienced personnel, were typical of leaks caused by a combination of abrasion and flexing. These were small in size and could not be detected visually. However, they constituted only 6.3%of the defects (or 0.05% overall failure rate) resulting from an abuse cycle that was unusually severe. If the field tests described by Lampi (1974) represent a truer picture of pouch durability and if the same distribution of leak sizes were applied to the overall TABLE XVII SIZE DISTRIBUTION OF DEFECTS CAUSED BY SIMULATED HANDLING ABUSE CYCLE' Number per range 1 1

1 2 3 24 9

Size

20 X 15 microns 20 X 4 0 microns 100 X 180 microns 200 - 299 microns 300 - 999 microns 1000 microns or greater Undefined, undetectable, or destroyed during pouch opening

Product Beefsteak Beefsteak Chicken A la king Chicken A la king Chicken A la king 3 beefsteak 21 chicken A la king

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

415

leak rate (at ration assembly and in the field) of 0.015%, then one package in 100,000 could have a leaker below 100 microns in size. It would be difficult to justify any significant development of instrumentation for such a small economic loss. No comprehensive biotesting or defect sizing studies were performed on the retort pouches prepared under the Natick-Swift Reliability Program. The causes of failure were contaminated seal areas and body cuts, the latter caused by seal-area tensioning grippers or inadvertent puncture of one pouch by the sharp corner of another. On the basis of the NARADCOM laboratory studies described above and the absence of any swellers in samples from the program after more than three years’ storage, it would appear that visual detection is adequately sensitive and that process-related defects are large. b. Leak Detection Mefhodology. Leak testing methodology has been developed to a sophisticated state, but predominantly for dry systems and low production rate applications. Marr (1968) presents a comprehensive review of various techniques. Marr (1965) discussed the problems and precautions in establishing leakage specifications, including the effects of fluid blockage. In addition to automated leak test apparatus for unfilled cans (an exception to the low production rate applications) mentioned earlier, portable helium leak detection equipment requiring no liquid nitrogen (Varian Associates, 1975) and a broader technique used an infrared sensor to measure the applicable trace gas (suggested as COz) (Modern Controls, Inc., 1974) are currently available for various packing testing situations. Spencer and Bodman (1970) reported on a survey of leak detection techniques and on experiments to define the sensitivity of the most promising approaches specifically for retort pouches. Their initial criteria were a hole 10 microns or larger in diameter in the body of the pouch, or a 30-micron-channel through a 3/8 -inch-wide seal area. The package material was 12-micron polyester/9-micron foil/75-micron polyolefin; the package was 4 l / 2 X 7 inches; and the contents were 4.5 to 5.5 ounces of thermoprocessed foods, both fluid and drier solid or baked items. Further requirements included rapidity (= thirty packages per minute), reasonable cost, and acceptability under Food and Drug Administration regulations. Table XVIII lists, in brief descriptive title form, the various techniques screened. Spencer and Bodman (1970) presented a more detailed description of the principles involved, techniques proposed for actual use, characteristics, and reasons for rejection. Two techniques, although not meeting the 10-micron-hole criterion, were considered most sensitive and were selected for further experimentation. For fluid foods, changes in the conductivity of deionized water caused by product pushed through the leaks by external pressure on the pouch were measured. The apparatus consisted of an inert silicone rubber bag holding

416

RAUNO A. LAMP1 TABLE XVllI

NONDESTRUCTIVE TEST PROCEDURES SURVEYED FOR APPLICABILITY FOR

LEAK TESTING OF FLEXIBLE

Infrared thermography Photoelastici ty Capacitance Radioactive tracer gas Pressure drop Soap bubble Microwave scattering Internal-external gas-producing chemical reaction visible or fluorescent dyes Electrified calcium carbonate particles Helium mass spectrometerb Ultrasonic translation of Schlieren patterns

PACKS^

Vapor pressure changes Infrared spectroscopy Gas chromatography Immersion bubble test Corona effect Beta-particle back scattering Electrical conductivity of a carrier gas Fluorescence quenching Electrical conductivity of carrier fluidC Halogen leak detector Eddy currents Feeler brushes

%pencer and Bodman (1970). bSelected for futther experimentation-“drier” foods. %elected for further experimentation-“wetter” foods.

deionized water. The test pouch was immersed in the water, and pressure was applied externally to both the water bag and the test pouch. Initial tests were made with salt-water solutions and confirmed with actual food products (chicken a la king, beef slices in barbecue sauce, and pickle-flavored sauce with ground beef). With laser-drilled defect holes, tests showed conclusively that the technique could not be depended upon to detect holes 100 microns in diameter or smaller. For products with very low water activity, such as cakes, a helium tracer gas method that uses a sniffer probe to capture the escaping gas in a covering polyethylene envelope was shown to successfully detect holes as small as 30 microns. The duration of the test was 30 seconds. Helium tracer gas techniques were also found to be more applicable to “wet” foods than initially anticipated. By subjecting packages to a vacuum of 1 torr, as under a bell jar, defects as small as 70 microns and possibly as small as 55 microns can be detected. The package must be turned over to allow testing of each side. The time required to test one side of a package in a well-engineered system was estimated t o be less than 15 seconds. Reduction of the test data to equipment designs was not a requirement of the leak detection survey. However, rough calculations indicated that when all the sequential handling, vacuumization, and analytical steps were considered, as well as the times required for the performance of each of these, and the need to test at least thirty, and preferably sixty, packages per minute,

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

417

the equipment would be extremely bulky, complex, and expensive. Furthermore, it would be necessary to add a tracer gas accurately and reliably, in amounts small enough not to contribute to package failure in the retort or to erroneously indicate package swelling. Since the leaker rate has been proved to be low and visual inspection adequate, there would appear to be no mandatory need for 100%leak detection. If rapid and economical techniques can be devised, their use could then be considered.

XIII. RESEARCH AND DEVELOPMENT NEEDS The retort pouch is a reality, and processing technology exists. Much of it has been borrowed from the technologies and systems devised for conventionally canned and frozen food. Some has been developed specifically for the retort pouch (curved-bar sealing, bakery product formulations, and retort control systems, for example). Other than the Food and Drug Administration assessment of the significance of adhesive migrants, there are no deterrents to commercialization in the United States. Progress outside the United States has had no such constraints. Research and development needs are, therefore, related mainly to improving products, methods, and packaging materials to meet the specific product, cost, and marketing requirements of the pouch. There is always room for totally new ideas and ways for accomplishing a task, but the absence of such developments is in no way critical to greater commercialization and utility of the retort pouch. Some areas where improvements and innovations would be welcomed are discussed below. A. PRODUCT RESEARCH Mencacci (1975), summing up the status of the retort pouch and the equipment available, stated that, although some product data are available from canning and freezing techniques, specific formulas and heat transfer data must be developed for each product. Undoubtedly he recognized that the lesser heat exposure of the products can result in different viscosities, textures, and flavors. This lesser heat exposure has already resulted in the use of pouches for some products not totally suitable for cans, such as meatballs in barbecue sauce, fully sterile ham slices, and fish fillet-based items. There is room for the development of additional products that, because of fragility or flavor sensitivity, are currently not suitable for conventional canning. Some products, especially those high in reducing sugars, are prone to darken in the absence of the reducing action of the tinplate surface of conventional cans.

418

RAUNO A. LAMP1

Others rely on the same chemical action for specific flavor characteristics. Studies should be carried out to devise methods to guarantee the applicability of pouches to such items. Bakery product formulations reportedly vary enough from lot to lot that complex retort control systems have been developed to accommodate these variations. Additional experience and studies could result in improvements. Also, although sterilization procedures have been reported for some bread products (as opposed to cake), additional studies appear warranted. Generally, the assumption has been made that the lesser total heat exposure of pouched foods results in higher nutrient levels, as well as improvements in flavor. Relatively few studies have been made, however, to substantiate such a premise. Obviously, many products are processed primarily for optimum acceptability beyond microbial inactivation, and the nutritional aspects in terms of specific vitamins are legitimate secondary considerations. However, especially with the advent of high-temperature films, nutritional studies could be used, as they have in Japan, to further promote the use of retort pouches.

B. FLEXIBLE FILM TECHNOLOGY The ideal film is cheap, totally machineable, light in weight, inert, tough, heat-sealable over a wide temperature range, resistant to exposure to temperatures from -30° to 300°F, and impermeable t o oxygen and water vapor. To date, no single film that is relatively economical and commercially available can even partially fulfill those requirements. Therefore, three-ply laminates have resulted where a shelf life longer than a few weeks or months is desired. Although one should seek the ideal, lesser advances can be highly important. The current apparent concern of the U.S. Food and Drug Administration is adhesive migration. Therefore, any alleviation of this problem-be it elimination of the adhesive for bonding the plies, establishment of an adhesive system that results in no migration, or the development of an acceptable food-contacting polymeric f i m that is impermeable to the adhesive-would be widely welcomed. Any other advances in devising polymeric films, copolymers, or blends that would simplify or make less critical the problem of control over the converting process would aid the acceptance of the retort pouch. In addition, any converting innovations to assure uniform, high-performance laminating of the three plies will find a market.

C. EQUIPMENT DEVELOPMENT The area of processing equipment offers the best potential for improvements. The common complaint about production systems has been the slow rate of 30 to 60 pouches per minute, when production lines for cans run more than 200

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

419

per minute for many competitive items. The desire has been to achieve filling and sealing rates of 120 per minute or more. This should be possible for the following reasons:

1. The high can-filling rates are achieved by using multiple filling heads; that is, a rate of 360 per minute may be attained by a 36-head filer for an individual head rate of 10 cans per minute. For the pouch, individual filing head rates of 30 per minute are already possible, signifying that the pouch is not really very difficult to fill. 2. Continuous-motion form-fill-seal equipment for pouches already exists and has been used for many nonretortable products. 3. Steam flushing for cleansing of the seal area and removal of residual air coupled with curved-bar sealing techniques to seal through any condensed moisture is feasible and should provide a basis for continuous-motion machine concepts. The pouch carrier concept should also form the basis for continuous handling procedures. Filling pumps, nozzles, and associated mechanical manipulations to speed quantitative, clean filling can be improved, especially to further minimize sealarea contamination. Batch, hydrostatic, and some continuous retorts have been adapted successfully for the retort pouch, but improvements in pouch handling and control of media mix, temperature and pressure can be made. It would appear that, because of the rapid heating characteristics of the pouch and the advent of steam flushing for air removal (vacuumization could cause flashing), hot filling of products followed by continuous retorting would be attractive. This is especially true of products to be sterilized at 275°F and higher. The natural technical inclination is to think in terms of larger and more sophisticated equipment. The inherent simplicity of the pouch, however, makes it a strong candidate for food preservation for ,the lesser developed countries. Efforts should be made to devise ethnically acceptable formulations; to ease concern over understerilization (for example, through acidification or control of water activity); to design simple retorting methods, even the use of Fa's significantly above the minimum; to design simple filling and seaming methods using preformed pouches; and to establish rudimentary procedures for assessment of pouch strength.

ACKNOWLEDGMENT I am deeply grateful for the inspiration, guidance, and support of Mr. Frank J. Rubinate before and during the preparation of this chapter. Mr. Rubinate, in the mid to late 1950’s,

420

RAUNO A. LAMPI

foresaw the feasibility and applicability of flexible packaging to thermoprocessed foods; and without his energetic management and backing, the development of the pouch for U.S. Military rations would not have been realized.

REFERENCES ACMA S.p.A. 1973. “ACMA Type 712 Pouchforming Filling and Sealing Machine,” Brochure. ACMA, Bologna, Italy. Aerotherm Corporation. 1970. “Temperature Telemetry Systems for Food Processing,” Unnumbered brochure. Aerotherm Corp., Mountain View, California. Alstrand, D. V., and Ecklund, 0. F. 1952. The mechanics and interpretation of heat penetration tests in canned food. Food Technol. 6(5), 185-189. Anonymous. 1966. $3 for hardware solves control problem. Food Eng. 38(10), 79-81. Anonymous. 1971. “Soup Tests Negative,” Associated Press Article (9/11 issue). Worcester Telegram, Worcester, Massachusetts. Anonymous. 1972. Sterilizable packs for gourmet foods. Packaging 43(506), 25. Anonymous. 1974a. Lemonade in mini-pouch Canada’s latest doy-n-pack. Puckug. Dig. 11 (8),6-7. Anonymous. 1974b. Pouch cookers: Operation of commercially available equipment. Food Eng. 46(12), 70-71. Anonymous. 1975a. Prototype line for the flxible, retortable pouch. Can Puckug. 28(6), 30-33. Anonymous, 1975b. Reynolds Metals Co. Filing of petition for food additive. Fed. Regisf. October.) Anonymous. 1975c. Flexibles: A solution for hospital solutions. Mod. Puckug. 48(9), 6. Beadle, B. W. 1959. Development of new methods of sealing containers. Contract DA19-129-QM-923. Rep. 12. U.S. Army Natick Research and Development Command, Natick, Massachusetts. Bee, G. R., Decamp, R. A., and Denny, C. B. 1972. “Construction and Use of a Vacuum Micro-Leak Detector for Metal and Glass Containers.” Research Laboratories, National Canners Association, Washington, D.C. Bohrer, C. W. 1963. Microbial spoilage of canned foods. “Microbiological Quality of Foods,” pp. 198-204. Academic Press, New York. Borden Company 1972. “Borden Container Leak Tester. Model 500,” Brochure. Bows, P. A. 1973. Practical evaluation of reheating flexible package (Flex-pac) meal components. Memorandum Rep. U.S. Army Natick Research Development Command, Natick, Massachusetts. Brody, A. L. 1971. Food canning in rigid and flexible packages. Crir. Rev. Food Technol. 2, 187-244. Brody, A. L. 1973. Current issues in aseptic packaging. Mod. Puckug. 45(12), 47-57. Brown, F., and Keegan, P. B. 1973. Heat sealing methods. Mod. Puckug. 46(12), 314-317 and 321-327. Brugh, J. F. 1964. “Final Report of Engineer Design Test of Flexible Packages for Heat Processed Foods-Beans, Green; Corn, Whole Kernel; Chicken la King,” USA TECOM Proj. No. 7-3-017343K. U.S. Army General Equipment Test Activity, Ft. Lee, Virginia. Burke, P. T., and Schulz, G. L. 1972. “The Comparative Performance of Flexible Packages

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

421

and Metal Cans,” Tech Rep. 73-62-GP. U.S. Army Natick Laboratories, Natick, Massachusetts. Burt, T. B. 1960. “Engineer Test of Flexible Packages for Heat Processed Foods-Peaches,’’ Tech. Rep. T-163. FEA 60001. Quartermaster Field Evaluation Agency, Ft. Lee, Virginia. Burt, T. B. 1963. “Engineering Test of Packaging, Flexible for Heat-Processed Beefsteak” Pro$ No. 7K-3173-01. U.S. Army Quartermaster Research and Engineering Field Evaluation Agency, Ft. Lee, Virginia. California Administrative Code. 1954. “Spoilage in Canned Food Products,” Title 17, Chapter 5, Subchapter 2, Article 8. Chapman, S., and McKernan, B. J. 1963. Heat conduction into plastic food containers. Food Technol. 17(9). 79-82. Ciavarini, T., and Lampi, T. A. 1976. “Studies on the Nature of Leaks in Flexible Packages for Thermoprocessed Foods,” Tech. Rep. U.S. Natick Research and Development Command, Natick, Massachusetts (in preparation). Circle Design and Manufacturing Corporation. 1973. “The C Circular Kick the Can,” Brochure. Saddle Brook, New Jersey. Conley, W., and Kornmann, W. 1975. Advances in non-rigid container processing. Canner/ Packer 144,60-61. Corning, A. 0. 1973. “Reliability of Packaging Materials and Closure,” Proceedings of the Symposium on Flexible Packaging for Heat-Processed Foods, pp. 3 H 8 . Nat. Acad. Sci.-Nat. Res. Coun., Washington, D.C. Costanza, F. A. 1971. “Report on Heat-up of Flexible Package Using Engine Heat,” Memo. Rep. No. 84. G.E.P.L. U.S. Army Natick Laboratories, Natick, Massachusetts. Cryovac Division of W. R. Grace and Co. 1974. “The Hooper 1000,” Brochure. Duncan, South Carolina. Davis, E. G., Karel, M., and Proctor, B. E. 1960a. The pressure-volume relation in film packages during heat-processing. Food Technol. 14(3), 165-169. Davis, E. G., Karel, M., and Proctor, B. E. 1960b. Heat processing vs permeability. Mod. Packag. 33(7), 208-211,283, and 284. Davis, J. W., Hulsey, R. G., Umina, A. P., and Rahman, A. R. 1972. “Development of Flexibly Packaged Cakes,” Tech. Rep. 73-8-FL. US. Army Natick Laboratories, Natick, Massachusetts. Dewey Redesign. 1975. “Retort Pouch TIC,” Dwg. No. DR-186, Dewey Redesign, Eagle River, Wisconsin. Dibbern, V. 1973. “Food Technology of Military Significance-Foreign,” Rep. No. 3, Flexible Packaging for Thermoprocessed Foods. No. FSTCCW-01-01-74. U.S. Army Foreign Science and Technology Center. Charlottsville, Virginia. DRG Flexible Packaging. 1975. “Sterilite,” Tech. Inf. Note. Duxbury, D. D., Sams, P. F., Howeler, W. F., Gee, J. H., and Miller, W. N. 1970. “Reliability of Flexible Packaging for Thermoprocessed Foods Under Production Conditions. Phase I: Feasibility,” Tech. Rep. 72-77GP. US. Army Natick Laboratories, Natick, Massachusetts. Duxbury, D. D., Sams, P. F., Maloney, J. L..Gee, J. H., and Miller, W. N. 1975. “Reliability of Flexible Packaging for Thermoprocessed Foods under Production Conditions. Phase 11,” Final Rep., Contract DAAG17-69-C-0160. U.S.Army Research and Development Command, Natick, Massachusetts (in preparation). Dymit, J. M. 1973. Today’s food service systems Act. Rep. Res. Dev. Assoc. Mil. Food Packag. Syst. 25(2), 56-63.

422

RAUNO A. LAMP1

Felmingham, J. D. 1964. Plastic and flexible containers for heat sterilized foods. Proc. Int. Food Ind. Cong., I964 pp. 177-182. Ferm, R. L. 1966. “Migration of Flexible Packaging Components into Foods,” Tech. Report 66-56CD. U.S. Army Natick Laboratories, Natick, Massachusetts. Fiori, F., and Fishman, S. 1967. “Abuse Tests on Flexible Packages of Thermoprocessed Foods,” Intern. Rep. U.S. Army Natick Laboratories, Natick, Massachusetts. FMC Corporation. 1973. “Laboratory Process Simulator for Convenience Foods,” Unnumbered brochure. Santa Clara, Calfironia. FMC Corporation. 1975. “FMC Convenience Food Sterilizer,” Unnumbered brochure. Santa Clara, California. Foxboro Company. 1966. “Foxboro General Catalog of Industrial Instrumentation,” Bull. 565, p. A54. Foxboro Co., Foxboro, Massachusetts. Gee, J. H. 1973. “Feasibility of Retorting Foods in Flexible Packages,” Proceedings of the Symposium on Flexible Packaging for Heat-Processed Foods, pp. 5 7-70. Sponsored by U.S. Army Natick Laboratories and National Research Council. Natick, Massachusetts. Goglio Luigi. 1968. “Fres Co. System. Vacuum Packaging Machine Unit GL-25 ,” Brochure. Goldfarb, P. L. 1970. Pouch for low acid foods. I. Mod Packag. 43(12), 70-76. Goldfarb, P. L. 1971a. Pouch for low-acid foods. 11.Mod. Packag. 44(1), 70-76. Goldfarb, P. L. 1971b. Measures temperatures by telemetry. Food Eng. 43(10), 61-63. Goldfarb, P. L. 1973. “Flexible Packaging for Heat Processed Foods-Package, Process, and Product Evaluation.” Packaging Institute, St. Louis, Missouri. Gould, W. A., Geisman, J. R., Weiser, H. H., Bash, W. D., Moore, W. J., Salzer, R. H., and Long, F. E. 1962. Heat processing of vegetables in flexible films. Ohio,Agric. Exp. Stn., Res. Bull. 905. Griffin, R. G., Nosrati, M. H., Lampi, R. A., and Szczeblowski, J. W. 1967. Bacteria1 resistance of films. Mod.Packag. 40(14), 164-167. Hamac-Holler Bosch Group. 1974. “BM-BMK-BMX Flat Bag Form, Fill and Seal Machines,” Brochure. Bergisch-Gladbach, West Germany. Hartman, N. W., Powers, J. J., and Pratt, D. E. 1963. Bacterial permeability of selected food-packaging films. Food Technol. 17(9), 92-98. Havighorst, C. R. 1975. New pouch foods sterilizer offers improved heat transfer. Food Eng. 47(12), 72. Heid, J. L. 1970. Retort process minimizes residual headspace gasses with “breathable” pouch. Food Prod. Dev. 4(6), 82,84, and 86. Heidelbaugh, N. D., and Karel, M. 1970. Changes in pouched heat-processed foods. Mod. Packag. 43(11), 80-90. Hemdon, D. H. 1971. Population distribution of heat rise curves as a significant variable in heat sterilization process calculations. J. Food Sci. 36,299-305. Horst, H. D. 1961. Films and laminates available for flexible packaging of freeze-dried and Thermally processed foods. Act. Rep., Res. Dev. Assoc. Mil. Food Packag. Syst. 13(4), 228-230. Hu, K. H., Nelson, A. I., Legault, R. R., and Steinberg, M. P. 1955. Feasibility of using pastic fhpacages for heat-processed foods. Food Technol. 19(9), 236-240. Karel, M., and Wogan, G. 1963. “Migration of Subsistence from Flexible Containers for Heat-Processed Foods,” Final Rep., Contract DA19-129-QM-2080. U.S. Army Natick Laboratories, Natick, Massachusetts. Katz, M. H., Sams, P. H., and Riley, D. M. 1973. Method for heat processing food products packaged in flexible containers. U.S. Patent 3,769,028. Katz, P. H. 1967. “Controlled Temperature Storage Study of Whole Kernel Corn in Flexible

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

423

Packages and Cans,” Intern. Rep. Packaging Division, U.S. Army Natick Laboratories, Natick, Massachusetts. Keller, R. G. 1959a. “Flexible Packages for Processed Foods,” QMFCIAF Rep. No. 31-59. Quartermaster Food and Container lnstitute for the Armed Forces. Chicago, Illinois. KeUer, R. G. 1959b. Flexible packages for processed foods. Mod. Packag. 33(1), 145-149 and 235. Kelsey, R. J. 1974a. Aseptic-packaging machine design. I. Mod. Packug. 47(2), 37-40. Kelsey, R. J. 1974b. Aseptic packaging machine design. 11. Mod. Packag. 47(8), 37-38. Kelsey, R. J. 1974c. “Flexible cans” for food-this could be the year. Food Drug Packag. 30(3), 1 and 27. Kenyon, E. M. 1970. “The Feasibility of Continuous Heat Sterilization of Food Products Using Microwave Power,” Tech. Rep. 71-8-FL. U.S. Army Natick Laboratories, Natick, Massachusetts. Kenyon, E. M., Westcott, D. E., LaCasse, P., and Gould, J. W. 1971. A system for continuous thermal processing of food pouches using microwave energy. J. Food Sci. 36,289-293. Killoran, J. J. 1974. Irradiation of multilayered materials for packaging thermoprocessed foods. Adv. Chem. Ser. 135, 87-94. Komatsu, Y., and Yamaguchi, K. 1975. Process for the preparation of high-temperature short-time sterilized packaged articles. U.S. Patent 3,892,058. Komatsu, Y., Yamaguchi, K., and Kishimoto, A. 1970. Effect of oxygen transmission rate of retort pouches on the browning of food-simulated systems during the heat-processing and storage. Pap., 17th Annu. Meet. Jpn. SOC,Food Sci. Technol. Lampi, R. A. 1967. Microbial recontamination in flexible films.Act. Rep. Res. Dev. Assoc. Mil. Food Packag. Syst. 19(1), 5 1-58. Lampi, R. A. 1968. The reliability of flexible packages. Act. Rep. Res. Dev. Assoc. Mil. Food Packag. Syst. 20(2), 140-147. Lampi, R. A. 1973. “The Reliabuty Program,” Rep. T-7310. Packaging Institute, St. Louis, Missouri. Lampi, R. A. 1974. “The Current Prognosis for Flexible Packaging of Military Rations,” Presented at Annual Packaging Institute Forum, Chicago, Illinois. Lampi, R. A., and Rubinate, F. J. 1973. Thermoprocessed foods in flexible packages: Transition to production. Package Dev. 3.12-18. Lampi, R . A., Fiori, F., and Hu, K. H. 1969. Finding flexible packaging flaws via infrared. Mod. Packag. 42,197-1 11. Lampi, R. A., Roberts, N. D., and Costanza, F. A. 1973. Automatic infrared radiometric scanning to detect fexible package seal defects. IEEE Trans Ind Electron Control Instrum. 20,205-212. Lampi, R. A., Schulz, G. L., Ciavarini, T., and Burke, P. T. 1976. Performance and integrity of retort pouch seals. Food Technol. 30(2), 38-48. Landy, J. J. 1965. Method of sterilizing food in sealed containers. U.S. Patent 3,215,539. Lane, J. H., and Widmer, K. 1969. “Sterilizable Semi-Rigid Aluminum Food Containers,” Presented at the Preservation and Packaging of Food Congress. Institute of Packaging, Harrogate. Lawler, F. K. 1967. New sterilizer made in France. Food Eng. 39(7), 73-75. Lechtur, C. P. 1967. “Review of Defect Creation Techniques on Flexible Packages of Thermally Processed Foods,” Intern. Rep. U.S. Army Natick Laboratories, Natick, Massachusetts. Lipoma, S. P. 197 1. Apparatus for continuous electromagnetic sterilization. U.S. Patent 3,7 18,082.

424

RAUNO A. LAMP1

Lipoma, S. P. 1972. Method for continuous electromagnetic sterilization of food in a pressure zone. U.S. Patent 3,889,009. Long, F. E. 1962. Flexible packages now withstand heat-processing temperatures of foods. Package Eng. 7(3), 63-80. Long, P. E. 1971. Automatic retort rack system for flexible packages. U.S. Patent

3,622,356. Long, F. E., Shaw, F. B., and Lisle, H. C. 1966. Microwave sterilization and vacuumking of products in flexible packages and apparatus therefor. U.S. Patent 3,261,140. McClosky, R. B. 1971. “Packaging Liquids in Pouches.” Modern Packaging Encyclopedia, McGraw-Hill, New York. MGillan, D., and Neacy, T. 1964.Choosing machinery for flexible packaging. Package Eng.

9(3), 74-79. McGregor, W. S. 1959. Flexible packages for heat processed foods. Act. Rep., Rex. Dev. Assoc. Mill. Food Packag. Syst. 11(2), 89-93. McNutt, J. W., and Lee, F. H. 1974. “Effect of Freeze-Thaw Cycle on Meal, Ready-to-Eat, Individual 1966 Prototype,” Tech. Rep. 74-24-FL.U.S. Army Natick Laboratories, Natick, Massachusetts. Mansfield, T. 1965. High-temperature, short-time sterilization. Food Sci. Technol., Proc. Int. Cong., Ist, 1962 Vol. 4,pp. 311-316. Mapp, A. 1971. “Heat Processed Food in Flexible Packages. A Technical Perspective,” Brochure. RWP Flexible Packaging Ltd. Marr, J. W. 1965. ‘‘Derivation of Leakage Specifications from the Fundamentals of Leakage Phenomena,” Rep. No. 65GL37. Advanced Technology Laboratories, General Electric Co., Schenectady, New York. Marr, J. W. 1968. “Leakage Testing Handbook,” NASA Contract No. NAS7-396.General Electric Co., Schenectady, New York. Martin, E. L. 1966. “New Laminations and their Uses,” Proc. 28th Annu. Packag. Forum. Vol. IV, pp. 266-276. Packaging Institute, St. Louis, Missouri. Maschinenfabrik Leonhardt OHG. 1974. “Leonhardt Spezialmaschinen fur die Nahrung Smittelindustrie. Abfullmaschinen Baureihe A DPZII,” Brochure. Sprendlingen, West Germany. Maunder, D. T., Folinazzo, J. F., and Killoran, J . J. 1968.Bio-test method for determining integrity of flexible packages of shelf-stable foods. Food Technol. 22(5), 81-84. Mayer, P. C., and Robe, K. 1963. Canning without cans. Food Process. 24(1 l), 75. Mencacci, S. A. 1973. “Convenience Foods: A Way of Life.” Packaging Institute, St. Louis, Missouri. Mencacci, S. A. 1975. Equipment systems for sterilizable pouches - USA, Europe, Japan. Act. Rep. Res. Dev. Assoc. Mil.FoodPachg. Syst. 27,161-175. Miller, W. M. 1973. “Reliability of Package Forming and Filling,” Proceedings of the Symposium on Flexible Packaging for Heat-Processed Foods, pp. 4Cb56. Nat. Acad. Sci.-Natl. Res. Counc., Washington, D.C. Modern Controls, Inc. 1974. “The Pac Guard 300,” Brochure. Minneapolis, Minnesota. Morton Chemical Company. 1975. “Adcote Brand Products for the Packaging Industry,” Tech. Inf., Adcote 506.Chicago, Illinois. Mounce, D. M. 1966. Standards for safety for foods in relation to public health. Am. J. Public Health 56,952-956. Multivac Export AG. 1973.“Multivac Brochure.” Koch Equipment Co., Kansas City, Missouri. Nelson, A. I., and Steinberg, M. P. 1956. Retorting foods in plastic bags. Food Eng. 28(1),

92-93.

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

425

Nelson, A. I., Hu, K. H., and Steinberg, M. P. 1956. Heat processible food fdms. Mod. Packag. 20(10), 173-179. Nieboer, S.F.T. 1970. Flexible vacuum packs for processed vegetables. Food Manuf: 45(2), 6M4. Nieboer, S., 1973. “Market Perspectives and Estimates on Retortable Pouch Packaging in Europe,” Rep. T-7312. Packaging Institute, St. Louis, Missouri. Nughes, G. 1971a. Flexible package for foodstuff. U.S. Patent 3,556,816. Nughes, G. 1971b. Method for packaging food products in flexible containers. U.S.Patent 3,s 97,237. Nughes, F. 1973. “Perspective Report on Retortable Pouch Packaging in Europe,” Rep. No. T-7313. Packaging Institute, St. Louis, Missouri. Nughes, F. 1974. “European Developments in Retortable Pouch Packaging,” Presented at Annual Packaging Institute Forum, Chicago, Illinois. Nughes, F., Mantovani, E., and Merloni, R. 1973. Nuova serie di controlli di imballaggi per cibi pronti. Imballaggio 24(209), 14-15. O’Meara, J. P., Wadsworth, C. K., and Farkas, D. F. 1976. “Microwave Heat Sterilization of Foods in Flexible Pouches.” U.S. Army Natick Research and Development Command, Natick, Massachusetts (in preparation). Ordway, G. B., Schulz, G. L. 1972. Inspects package seals. Food Eng. 44(2), 64-65. Payne, G. O., Jr. 1964. Private Communication. Paschall, H. H., Cantrell, J. H., and Ezzard, R. D. 1967. “Integrated Engineering and Service Test of Meal, Ready-to-Eat, Individual (Intermediate Conditions),” USATECOM Proj. No. 8-3-7400-06/07/08. U.S. Army General Equipment Test Activity, Ft. Lee, Virginia. Payne, G. O., Jr., Spiegl, C. J., and Long. F. E. 1969. “Study of Extractable Substance and Microbial Penetration of Polymeric Packaging Materials to Develop Flexible Plastic Containers for Irradiation Sterilized Foods,” Tech. Rep. 69-57-FL. U.S. Army Natick Laboratories, Natick Massachusetts. Pflug, I. J. 1964. “Evaluation of Heating Media for Producing Shelf Stable Food in Flexible Packages. Phase I,” Final Rep., Contract DA19-AMC-145 (N). U.S. Army Natick Laboratories, Natick, Massachusetts. Pflug, I. J., and Borrero, C. 1967. “Heating Media for Processing Foods in Flexible Packages. Phase 11,” Tech. Rep. 6 7 4 7 6 P . U.S. Army Natick Laboratories, Natick, Massachusetts. Pflug, I. J., and Long, F. E. 1966. Static load tests evaluate flexible packaging materials at elevated temperatures. Packge Eng. 11(5), 91-95. Pflug, I. J., Bock, J. H., and Long, F. E. 1963. Sterilization of food in flexible packages. Food Technol. 17(9), 87-92. Proctor, B. E., and Nickerson, J. T. R. 1958. “Investigation of Bacterial Resistance of Packages,” Final Rep., Contract DA19-129-QM-758. U.S. Army Natick Laboratories, Natick, Massachusetts. Rees, J. A. G. 1973. Processing heat-sterilizable flexible packs. Packaging 44(525), 17-18. Rexham Corporation. 1973. “Hydrolock Continuous Cooker/Cooler.” Bull. No. 502. Rockford, Illinois. Rexham Corporation. 1974. “Intermittent Motion Flexible Pouch Packagers,” Bull. No. 101B. Rockford, Illinois. Rha, C., and Karel, M. 1968. “Heat Transfer to Flexibly Packaged Foods. I. Comparison of Theoretical and Experimental Heat Transfer Parameters for Ground Beef,” Contract DAAG 19-68606(N). U.S. Army Natick Research and Development Command, Natick, Massachusetts. Roberts, N. D. 1975. U.S. Army Natick Research and Development Command (private communication).

426

RAUNO A. LAMP1

Robertson, W. F. 1970. “Test Plan Retort Check-Out,” Contract DAAG19-C-69-0160. U.S. Army Natick Laboratories, Natick, Massachusetts. Ronsivalli, L. J., Bemsteins, J. B., and Tinker, B. L. 1963. Method for determining the bacterial permeability of plastic films.Food Technol. 20(8), 1074-1075. Rubinate, F. J. 1960. “Flexible Containers for Heat-PIocessed Foods,” Proceedings of the Conference on Flexible Packaging for Military Food items, pp. 21-28. National Academy of SciencesfQuartermaster Food and Container Institute for the Armed Forces. Rubinate, F. J. 1964. Army’s “obstacle course” yields a new look in food packaging. Food Technof. 18(11), 71-74. Rubinate, F. J. 1973. “Flexible Packaging for Heat-Processed Foods.” Packaging Institute, St. Louis, Missouri. RWP Flexible Packaging. 1974. “Outline Specification for Sterilite MFEP.” Bristol, England. Sams, P. F. 1973. “Bakery Items in Flexible Packages,” Proceedings of the Symposium on Flexible Packaging for Heat Processed Foods, pp. 29-38. U.S. Army Natick Laboratories and National Research Council, Natick, Massachusetts. Schmidt, C. F., and Robertson, W. F. 1970. “Inoculated Pack in 6 X 8 X y.,” Flexible Pouches of Peas with Brine and Peas with Butter Sauce,” Intern. Rep. Packaging Service, Metal Division Research and Development, Continental Can Company. Chicago, Illinois. Schott, O., Pfeiffer, H. J., and Torber, D. 1974. The heat sterilization of menu components in flexible films. Verpack. Folien-Papiere pp. 1-7. Schotte, K. 1974. Sterilized foilds for preserved foods. Verpack. Mag. pp. 1-7. Schulz, G. L. 1973. Private Communication. Schulz, G. L. 1973. “Test Procedures and Performance Values Required to Assure Reliability,’’ Proceedings of the Symposium on Flexible Packaging for Heat-Processed Foods, pp. 71-82. Nat. Acad. Sci.-Natl. Res. Counc., Washington, D.C. Schulz, G. L. 1975. U.S. Army Natick Development Center, Natick, Massachusetts (private communication). Schulz, G. L., and Mansur, R. T. 1969. “Sealing Through Contaminated Pouch Surfaces,” Tech. Rep. 69-76GP. US. Army Natick Laboratories, Natick, Massachusetts. Shappee, J., and Werkowski, S. J. 1972. “Study of A Nondestructive Test for Determining the Volume of Air in Flexible Food Packages,” Tech. Rep. 734-GP. U.S. Army Natick Laboratories, Natick, Massachusetts. Shenkenberg, D. 1975. USDA status on flexible retortable pouches for meat products. Act. Rep., Res. Dev. Assoc. Mil. Food Packag. Syst. 27(1), 157-160. Sloan, J. W. 1973. “USDA Status on Acceptance of Flexible Packaging for Heat Processed Foods.” Packaging Institute, St. Louis, Missouri. Spencer, W. T., and Bodman, H. A. 1970. “Nondestructive Testing of Packages of Thermoprocessed Foods,” Final Rep., Phase 11 (Draft), Contract DAAG1769-C-0013. U.S. Army Natick Laboratories, Natick, Massachusetts. Stenstrom, L. A. 1971a. Method and apparatus for treating heat-sensitive products. U.S. Patent 3,809,844. Stenstrom, L. A. 1971b. Heating of products in electromagnetic field. U.S. Patent 3,809,845. Stenstrom, L. A. 1971c. Heat eeatment of heat-sensitive products. U.S. Patent 3,814,889. Stumbo, C. R. 1965. “Thermobacteriology in Food Processing,” 1st ed. Academic Press, New York. Szczeblowski, J. W. 1965. “Aseptic Packaging,” Intern. Rep. Packaging Division, U.S. Army Natick Laboratories, Natick, Massachusetts. Szczeblowski, J. W. 1971. “An Assessment of the Flexible Packaging System for Heat-

FLEXIBLE PACKAGING FOR THERMOPROCESSED FOODS

427

processed Foods,” Tech. Rep. 71-57GP. U.S. Army Natick Laboratories, Natick, Massachusetts. Szczeblowski, 1. W., and Rubinate, F. J. 1965. Integrity of food packages. Mod. Packag. 38(10), 131-135. Thomas, M. H., and Sherman, D. E. 1969. “Effect of Freeze-Thaw Cycling on the Vitamin Content of the Meal, Ready-to-Eat, Individual,” Tech. Rep. 69-86-FL. U.S. Army Natick Laboratories, Natick, Massachusetts. Thorpe, R. H., and Atherton, D. 1972. “Sterilized Foods in Flexible Packages,” Tech. Bull. No. 21. Fruit and Vegetable Preservation Research Association, Chipping Campden, Gloucestershire, England. Thorpe, R. H., and Everton, J. R. 1968. “Post-Process Sanitation in Canneries,” Tech. Manual No. 1. Fruit and Vegetable Preservation Research Association, Chipping Campden, Gloucestershire, England. Toppan Printing Co., Ltd. 1975. “Instruction Manual for Full-Automatic Over-Heated Water System Retort Sterilization Equipment (Toppan Auto Cooker H, Model 111.” Tokyo, Japan. Toyo Seikan Kaisha, Ltd. 1973a. Brochure on RP-F. Tokyo, Japan. Toyo Seikan Kaisha, Ltd. 1973b. “Fully Automatic Overpressure Retort for Retortable Pouches,” Brochure. Tokyo, Japan. Toyo Seikan Kaisha, Ltd. 1975. Package for Hi-Retort food for more delicate items through hi-RP-F. Brochure, Tokyo, Japan. Transvac-Maschinen AG. 1972. “Swissvac (CVE(P),” Brochure. Lucerne, Switzerland. Transvac-Maschinen AG. 1975. “Swissvac Duo Mark I, Mark 11,” Brochure. Lucerne, Switzerland. Tripp, G. E. 1961. Conventional retorting of flexibly package products. Act. Rep., Res. Dev, Assoc. Mil, Food Packag. Syst. 13, 222-228. Tsutswni, Y. 1972. “Retort pouch”-its development and application to foodstuffs in Japan. J. Plast. 6 , 2 6 3 0 . Tsutsumi, Y. 1974. “The Growth of Food Packed in Retortable Pouches in Japan,” Presented at Annual Packaging Institute Forum, Chicago, Illinois. Tsutsumi, Y. 1975a. The growth of food packed in retortable pouches in Japan. Act. Rep., Res. Dev. Assoc. Mil. Food Packag, Syst. 27(1), 149-153. Tsutsumi, Y. 1975b. Private communication. Turtle, B. I., and Alderson, M.G. 1968. “Sterilizable Aluminum Foil Food Packs,” Pap. No. 13. Symp. Aluminum Foil in Packaging. London, England. Varian Associates. 1975. “Porta-Test Portable Helium Leak Detector,” Brochure. Lexington, Massachusetts. Wallenberg, E., and Jarnhell, B. 1957. Heat sterilization in plastics. Mod. Packag. 31(1), 165-167. Wilson, D . C. 1968. Private communcation. Witaker, W. C. 1971. Processing flexible pouches. Mod. Packag. 44(2), 83-85, and 88. Wilson, D. C. 1971. Control of diffusion of gases in pouches. U.S. Patent 3,625,712. Wilson, D. C. 1972a. Control of hydrogen formation in pouches during heat treatment. U.S. Patent 3,634,099. Wilson, D. C. 1972b. Method of purging air from containers. U.S. Patent 3,658,562. Wilson, D. C. 1974. Package sealing in steam atmosphere. U.S. Patent 3,845,606. Woods, Q. T. 1967. The quest for better vacuum pouch equipment. Package Eng. 12(15), 116-1 2 1. Wornick, R., Karel, M., and Proctor, B. E. 1960. Heat penetration into plastic packages for heat-processed foods. Packag. Eng. 5(7), 33-35.

428

RAUNO A. LAMP1

Yamaguchi, K., Namiki, T., Komatsu, Y., and Kishimoto, A. 1971. Sterilization of foods in flexible packages. Part VII. Storage stability of food-simulated systems in retort pouch-0 carotene. 18th Annu. Meet. Jpn. Soc. Food Sci. Technol. Yamaguchi, K., Komatsu, Y., and Kishimoto, A. 1972. Sterilization of foods in flexible packages. Part VI. Nondestructive method for determining residual air in pouches. J. Food Sci. Technol. 19(7), 316-320. Yamano, Y., and Komatsu, Y. 1969. Sterilization of foods in flexible packages. Part I. Heat characteristics of a pilot retort for sterilization of flexible packages. J. Food Sci. Technol. 16(3), 113-118. Yamano, Y., Komatsu, Y., and Ikegami, Y. 1969a. Sterilization of foods in flexible packages. Part 11. Thermal characteristics and storage stability of various film-wrapped foodstuffs. J. Food Sci. Technol. 16(3), 119-123. Yamano, Y., Yamaguchi, K., Nagata, H., Komatsu, Y., and Kishimoto, A. 1969b. Sterilization of foods in flexible packages. Part 111. Temperature distribution and heat characteristics of an industrial scale retort. J. Food Sci. Technol. 16(3), 124-129. Yamano, Y., Ejiri, K., Endo, T., and Senda, M. 1975. Heat penetration into flexible packages with food-simulated materials. J. Food Sci. Technol. 22(5), 199-204. Yawger, E. S. 1965 Mechanical and bacteriological determinants of process lethality in continuous pressure Cookers. Food Sci. Technol., Proc. Int. Cong., l s t , 1962 Vol. 4, pp. 227-233. Young, W. 1967. “Heat and Sonic Sealing.” Modern Packaging Encyclopedia, McGrawHill, New York. Young, W. 1971. “Heat and Sonic Sealing.” Modern Packaging Encyclopedia, McGrawHill, New York. Young, W. E. 1975. “Sealing Packaging Materials.’’ Modern Packaging Encyclopedia, McGraw-Hill, New York.

SUBJECT INDEX A Abalone, 143-186 anatomy of, 145-146 antiviral and antibiotic activity of, 179 brining of, 170-171 by-products of, 178-179 canning of, 171-172 can liquor in, 178 catching and handling of, 166 chemical composition of, 147-166 effects of size and season on, 150-151 drying of, 172-173 flavor of, 174,176-177 as food, 146-147 freezing of, 168-1 70 gelatin and collagen in, 175,179 lipids of, 160-162 names for, 144 nucleotides of, 157-160 odor and appearance of, 177-178 physiology of, in air, 166-168 pigments in, 163-164 preservation of, 168-173 proteins of, 151-156 amino acids of, 154, 155-157 properties, 155 proximate analysis of, 147-150 quality aspects of, 174-178 research needs on, 179 shell of, 164, 178 spoilage of, 164-165 statistics of catch of, 145 sugar-containing compounds in, 162-163 texture of, 174 UV-absorbing compounds of, 158 viscera of, 178-179 volatile bases in, 164-165

Abomasum, phospholipids in, 36 Abrasion, in flexible-package leak studies, 413 Acetyl coenzyme A, in wheat germ, 240 Acetylesterase, in wheat germ, 241 Acidic peptidase in wheat germ, 241 Acyl coenzyme A, in wheat germ, 240 Adipose tissue, see Depot fats Age, effects on fat composition, 32-33 Agmatine, in putrid abalone, 166 Air, removal of, from thermoprocessed-food packaging, 369-375 Air classification, of wheat germ, 201-204 Alcohol dehydrogenase, in whkat germ, 240 Aldolase, in wheat germ, 241 Amino acids, in wheat germ, 203,216-220 Amino acid composition, of abalone, 154-157 AMP deaminase, in abalone, 158 Amylases, in wheat germ, 210-21 1,239 Animal fats, composition of, 20-37 Antibiotic activity, in abalone, 179 Antioxidants, as preventatives of “warmedover” flavor, 57-58 Antiviral activity, of abalone, 179 Apyrase, in wheat germ, 240, 241 Ascorbates, effect on “warmed-over” flavor, 55 Ascorbic acid oxidase, in wheat germ, 241 ATP, in abalone, 157-158 Auger filler, for thermoprocessed food, 367-368

B Bacillus coagulans, as thermal processing target, 77 429

430

SUBJECT INDEX

Bacteria, penetration into flexible packaging, 315-3 17 Bakery products flexible packaging for, 327 retorting system for, 391 wheat germ use in, 273-281 Beef, fatty acids in fat of, 23 Blichromes, in abalone shell, 164 Biotester, for flexible packaging, 316,411 Biscuits, wheat germ use in, 284 Bran, of wheat kernel, 193-194 Bromate, effects on wheat germ, 278

C Cadaverine, in putrid abalone, 166 Cakes, wheat germ use in, 284 Calipers, use in flexible-package seal defect detection, 409 Canning, of abalone, 171-172 Cans, comparison with flexible packages, 402 Carbohydrates, in wheat germ, 204, 230-232 Cardiolipin, structure of, 6 Carnitine, in abalone, 165 p-Carotene, in abalone, 165 Catalase, in wheat germ, 239-240 Catalysts, for lipid oxidation, 40-45 Ceramide biosynthesis of, 17 structure of, 16 Cereals, wheat germ supplementation of, 284-285 Cerebrosides, structure of, 1 6 , 1 7 Chelating agents, as preventatives of “warmed-over” flavor, 57-58 Chicken, fatty acids in fat of, 23 Cholesterol biosynthesis of, 18 structure of, 18, 19 Chopping, of meat, effect on “warmedover” flavor, 5 3 Circle design, for retort packaging, 342 Clostridiam botulinum, as thermal processing target, 77 Clover, effect on meat flavor, 38 Coconut meal, diets containing, effect on animal depot fats, 25

Collagen, in abalone, 175, 179 Computer use in thermal process estimation, 91-97 programs for, 110-138 Continental Can Company, flexible packaging by durability of, 4 0 1 4 0 2 Cooling curve, curvilinear portion of, 81 Counterpressure, for package air removal, 372-373 Creatine and creatinine, in abalone, 165 Cystine reductase, in wheat germ, 240 Cytochrome c, in wheat germ, 240

D Deboned meat, effect on “warmed-over flavor ,” 4 7 4 8 Dehydrases, in wheat germ, 240 Dehydroascorbic acid reductase, in wheat germ, 210-211 Dehydrogenase dihydroorotic NAD, in wheat germ, 240 Depot fats age effects on, 32-33 composition of, 21-33 diet effects on, 25-30 environmental effects on, 30-31 sex factors affecting, 32 species differences in, 21-26 Dexyribonuclease, in wheat germ, 241 Diacylglycerol lipids, structure of, 7 Diacylglycerophospholipids, structure of, 6 Diet, effects on animal depot fats, 25-30 Dipeptidase, in wheat germ, 210 Drawn pouch system for package air removal, 373-374 for retort packaging, 342, 344 Drying, of abalone, 172-173

E Embryonic axis, of wheat germ, 194 Endosperm, of wheat kernel, 193 Environment, effects on depot fats, 3&32 Enzymes, in wheat germ, 209-21 1 Epiblast, of wheat germ, 195 Epoxy compounds, wheat germ treatment by, 267 Extractives, for flexible packaging, 313-315

43 1

SUBJECT INDEX

F Fatty acids in abalone, 161 in depot fats, 22,23 in wheat germ, 225-228 Feeds and feedstuffs effect on milk and meat flavor, 37-38 wheat germ use in, 286 Fermented foods, wheat germ use in, 285 Ferulic acid, in wheat germ, 237 Films, for flexible packaging, 317-322 Fish fatty acids in fat of, 23 “warmed-over” flavor in, 1-74 Flavor, of abalone, 174,176-177 Flexible packaging, 3 0 5 4 2 8 air removal from, 369-375 bacterial penetration into, 315-317 cans compared to, 402 current fiims for, 317-322 designs for, 322-324 durability of, 399-404 early studies on, 309-3 12 equipment development for, 418-419 extractives for, 313-315 filling of, 364-369 flexible film technology in, 418 food product development in, 324-335 heat-seal quality, retorting effects on, 352-353 for HT/ST products, 327 interlamina bonds in, 353-354 leak detection in, 4 0 9 4 1 7 methodology, 415-417 materials for, 312-322 occluded particles in seals of, 354-355 product research in, 4 1 7 4 1 8 production reliability in, 344-346 production systems for, 333-346 current types, 336-344 quality assurance in, 404-417 quality and stability of, 328-333 research and development needs in, 4 17-41 8 residual gas levels in, 374-375 retortability of, 317 retorting of, 375-399 techniques and equipment for, 387-398 seal contamination in, 360-363

seal defects in, tests for, 407-409 seal wrinkles in, 359-360 sealing in, 346-363 for retort pouches, 355-359 shipping tests on, 403-404 systems approach to, 333-336 for thermo processed foods, 3 0 5 4 2 8 utility of, 327-328 Flexing, in flexible-package leak studies, 412-413 Foil-free laminations, for thermoprocessed foods, 329-330 Foil laminates, for thermoprocessed foods, 330-333 Food, temperature response characteristics of, 80-82 Freezing, of abalone, 168-170 Fruits, flexible packaging for, 326 Fusion, in flexible-packaging sealing, 348-349 G

Galactolipids, in wheat germ, 223 Gases, residual, in flexibly packaged foods, 376-380 Gear pump, for thermoprocessed food Filing, 366-367 Gelatin, in abalone, 175,179 Germ breads, preparation and nutritive value of, 282-284 Gentiobioside, in wheat germ, 241 Glucose 6-phosphate dehydrogenase, in wheat germ, 240 Glutamic acid decarboxylase, in wheat germ, 240 Glutamic dehydrogenase, in wheat germ, 240 Glutathione in wheat germ, 270 effect on baking quality, 274-275 inactivation. 277 Glycerophosphatase, in wheat germ, 24 1 Glycogen in abalone, 162,176,179 in can liquor, 178 Glycolipids, structure and biosynthesis of, 15-17 Glycolytic enzymes, in wheat germ, 240

SUBJECT INDEX

432

H Haliotis sp., see Abalone Heat, effects on “warmed-over” flavor, 48-5 3 Heat processes, see Thermal processes Heat transfer, in thermal processing of foods, 376-380 Heating curve, curvilinear portion of, 81 Heme compounds as catalysts of lipid oxidation, 40-43, 59-60 as prooxidants, 45 Hemocyanin, in abalone, 162 n-Hexanal, in rancidity, 2, 38 Hexokinase, in wheat germ, 240 Hexose-phosphate isomerase in wheat germ, 240 High-temperature/short-time products, packaging for, 327 Histamine, in putrid abalone, 166 Hot-bar sealing, of retort pouches, 355-359 Hydrolases, in wheat germ, 241 Hydrolok continuous retort system, 395-397 Hydropac system for retort packaging, 342 diagram, 343

I Infrared scanning, for flexible-package seal defects, 407-409 Internal burst test, for seal integrity in flexible packaging, 349-350 Iron, as catalyst of lipid oxidation, 43-44, 59 Isocitric dehydrogenase, in wheat germ, 240 Italy, retort packaging (STAR) system for, 341-342

L Lamb, fatty acids in fat of, 23 Lanosterol, structure of, 18, 19 Leak test apparatus, 410 Leaks, detection of, in flexible packages, 409-4 17 Ligases, in wheat germ, 241 Linoleic acid, in natural fats, 8 Linolenic acid, in natural fats, 9 Lipases, in wheat germ, 210,237-239 Lipids in abalone, 160-162 autoxidation of, 39-40 classification and significance of, 3-5 depot fats, see Depot fats meat flavor and, 37-39 oxidation mechanisms in, 39-48 catalysts of, 40-45 polar, effect on wheat germ, 279-280 structure of, 5-20 in membrane’s, 20, 35-37 nonsaponifiable lipids, 18-20 phospholipids, 9-15 sphingolipids and glycolipids, 15-17 triglycerides, 5-9 in tissues, composition of, 33-37 of wheat germ, 22C230 Lipoxidase, in wheat germ, 210-211,239 Liquid food, heat processing of, 106 Lobatto formula, 94

M

Malic dehydrogenase, in wheat germ, 240 Malonyl coenzyme A, in wheat germ, 240 Marbling fat, composition of, 33-34 Meat flavor of, lipid effects on, 37-39 flexible packaging for, 325 storage of, oxidation during, 51-53 “warmed-over” flavor in, 1-74 J Melanins, in abalone, 163-164 Membrane-bound lipids, composition of, 20, Japan, retort packaging for processed foods 35-37 in, 341 Membranes, lipids in, 20 Metal irons, as catalysts of lipid oxidation, 4 3-45 K Methoxyliydroquinone glycosides, in wheat Koji, wheat germ use in, 285 germ, 237

SUBJECT INDEX

433

Phosphates, effect on “warmed-over” flavor, 5 4-5 5 Phosphatides structure of, 6 in wheat germ, effect on baking quality, 274 Phosphoarginine, in abalone, 157 Phosphoglucomutase, in wheat germ, 240, 24 1 6-Phosphogluconate isocitric dehydrogenase, in wheat germ, 240 Phosphoglycerol phosphatase, in wheat germ, 241 Phospholipids in abalone, 162 N biosynthesis of, 12-1 3 NARADCOM system, package durability in, effect on wheat germ, 280 400401 interconversion of, 14 Natick-Swift retort packaging line, oxidation of, 45-46 description of, 338-340 structure of, 9-15 Natural fats, structure of, 8 in wheat germ, 223,224 Niacin, in wheat germ, 208 Phosphomonoesterase, in wheat germ, Nitrites, effect on “warmed-over” flavor, 54 210-211 Nucleic acids, in wheat germ, 220 Phosphorus and phosphorus compounds, in Nucleotides, of abalone, 157-160 wheat germ, 233,234 Phytase, in wheat germ, 210-211 Pigments 0 in abalone, 163-164 Oleic acid, in natural fats, 8 in wheat germ, 237 Ormer, see Abalone Piston pump, for thermoprocessed food Orotidine-5-phosphate pyrophosphorylase, filling, 367 in wheat germ, 240 Placeable filler, for thermoprocessed food, Oxalacetic carboxylase, in wheat germ, 241 368-369 Oxalic acid, in wheat germ, 237 Plant flavonoids, effect on “warmedaver” Oxidases, in wheat germ, 239,240 flavor, 55-56 Oxidation Plasmalogens, structure of, 10 during meat storage, 51-53 Pork of lipids, 39-48 fatty acids in fat of, 23 of phospholipids, 45-46 “sex odor” in, 39 role in meat off-flavors. 38 Poultry, “warmed-over” flavor in, 1-74 Preservation, of abalone, 168-173 Prooxidants, heme compoundgas, 45 P Proteinase, in wheat germ, 210, 239 Proteins Palmitic acid, in natural fats, 8 of abalone, 151-156 Palmitoleic acid, in natural fats, 8 in wheat germ, 203,205,213-216 Pantothenic acid, in wheat germ, 209 Puncture, in flexible-package leak studies, Pastry, wheat germ use in, 284 414 Peroxidases, in wheat germ, 240 Putrescine, in putrid abalone, 166 Phenolic compounds, in wheat germ, 237 Pyridoxine, in wheat germ, 209 Phosphatases, in wheat germ, 239

Methyl transferase S-adenosyl methionine, in wheat germ, 240 Mevalonic acid, structure of, 19 Microorganisms, as target for thermal processes, 76-77 Microwave processing, of flexibly packaged foods, 397-398 Minerals, in wheat germ, 204,207-208, 232-235 Miso, wheat germ use in, 285 Muscle of abalone, 152 amino acids of. 154

434

SUBJECT INDEX

Q Quality, assurance of, in flexibly packaged foods, 404-417

R Racks, for retorting, 388-390 Retort pouches materials for, 313, 326 production systems for, 335 Retorting of flexible-packaged foods, 375-399 Reynolds metals’ system, for flexible packaging, 337-338 Ribonuclease, in wheat germ, 241 Rotary filer-sealer machine, 341 Rumen, phospholipids in, 36 Ruminants, fatty acids in fats of, 2 5 , 2 8 Ryegrass, fatty acids in, comparison with depot fats of grazing animals, 28, 38

Squalene, structure of, 1 8 , 1 9 STAR (Italian) system for retort packaging of processed foods, 341-342 durability of, 399-400 Steam-air cooks, for retorting, 392-395 Steam flush, for package air removal, 373 Steeping, of wheat germ, 276-277 Sterilizing value calculation of, 88 computer program, 93 Steroids, structure of, 18, 19 Succinic dehydrogenase, in wheat germ, 240 Sugars in abalone, 162-163 in wheat germ, 205-206, 230,231 Synthetases, in wheat germ, 241

T

Taurine, in abalone, 156-157 Tensile test, for seal integrity in flexible packaging, 350-351 S Texture, of abalone, 174 Thermal death time (TDT) curve, Salt preservation, of abalone, 170-171 “Sashimi,” abalone as, 146 calculation of, 77, 79 Sauces, flexible packaging for, 325 Thermal impulse sealing of retort pouches, Scutellum, of wheat germ, 194-195 358-359 Sealing Thermal processes in flexible packaging, 346-363 basic principles of, 76-83 of retort pouches, 355-359 computerized estimation of, 91-97 Semisolid food, heat processing of, 106 without manual calculations, 97-104 Sex factors, effect on composition of depot parametric values for, 91-97 fat, 32 programs for 110-138 “Sex odor,” in pork, 39 terminology for, 108-109 Shell of abalone, 164, 178 data required for, 76-82 Shipping tests, of flexible packages, mathematical estimation of, 75-141 403404 research needs for, 104-106 Sieve analysis, of wheat germ, 201 procedures for determination of, 83-91 Simpson’s formula, 94 thermal death time in, 76-80 Snorkel technique, for package air removal, Thermoprocessed foods 371 filling of 364-369 Sodium choloride, effects on “warmedflexible packaging for, 305-428 over” flavor, 56-57 microwave processing of, 397-398 Soups, flexible packaging for, 327 process determination for, 380-386 Sphingolipids, structure and biosynthesis of, Thiamine, in wheat germ, 208,209 15-17 Thioctic acid, in wheat germ, 237 Sphingomyelins, structure of, 16 Tocopherols, in wheat germ, 236 Sphingosine Toxic factors, in wheat germ, 255-258 biosynthesis of, 17 Toyo Seikan Kaisha, Ltd, flexible-package structure of, 16 durability of, 403

SUBJECT INDEX

435

TPNH diphorase, in wheat germ, 240 Wheat, world production of, 189 TPNH oxidase, in wheat germ, 240 Wheat germ, 187-304 T r a n ~ a m i ~ sin e ~wheat , germ, 240 air classification of, 201-204 Transcarboxylase, in wheat germ, 240 amino acid supplementation of, 253-254 Transferases, in wheat germ, 240 amino acids in Trays, for retorting, 388-390 of mill germ, 216-220 Trigylcerides, structure of, 5-9, 10 processing effects on, 203,251 Trimethylamine, from abalone spoilage, 164 nutritive aspects, 242-243 in animal feeds, 286 antioxidants for, 268 U in bakery and pastry products, 282-284 UDP-Dglucuronic acid decarboxylase, in in bread making, 273-281 wheat germ, 240 carbohydrates in Ultrasonic sealing, of retort pouches, 359 of mill germ, 230-232 Uroporphyrinogen 111 cosynthetase, in processing effects on, 204 wheat germ, 241 as cereal supplement, 284-285 chemical composition of, 204-241 of dissected germ, 205-21 1 V of mill germ, 21 1-241 Vacuum chambers, for package air removal, definition of, 193 371-372 deoiling of, 268 Vanillic acid, in wheat germ, 237 drying Of, 268-269 Vegetables, flexible packaging for, 325-326 embryonic axis of, 194 Visual examination, for seal integrity in enzymes in flexible packaging 351-352 of dissected germ, 209-211 Vitamin concentrates from wheat germ, of mill germ, 237-241 epiblast of, 195 285-286 epoxy compound treatment of, 267 Vitamin E, from wheat germ, 285-286 Vitamins, in wheat germ, 208-209, ethylene dichloride treatment of, 267 235-236.271 fatty acids in, 225-228 fermented, 275-276 in fermented foods, 285 W flaked, separation of, 199-200 food uses of, 282-287 “Warmed-over” flavor (WOF), 1-74 freshness evaluation in, 262-263 antioxidant effect dn, 57-58 heat processing of, 264-261 chelating effects on, 57-58 infrared radiation of, 267 chopping and emulsifying effects on, 53 lipids in, 220-230 curing effects on, 54-57 characteristics, 227-228 deboned meat effects on, 47-48 composition, 22 1-22 3 development of, 46-57 heating effects on, 48-53 of dissected germ, 206-207 occurrence of, 2 effect on baking quality, 280-281 variety effects, 221 practical aspects of, 59 minerals in prevention of, 57-59 reducing conditions and, 58 of dissected germ, 207-208 research needs for, 59-61 of mill germ, 232-235 retorting or overheating effects on 50-51 processing effects on, 204 species differences in, 46-47 moisture effects on, 260-261 Water-cook system, for retorting, 390-392 nucleic acids of, 220 Weeds, effect on milk and meat flavor, nutritive value of, 242-258 37-38 by biological method, 244-246

436

SUBJECT INDEX

Wheat germ, continued by chemical methods, 242-244 processing effects on, 249-253 storage effects on, 270-272 supplementary value, 246-249 physical characteristics, 200-201 pigments in, 237 processed, 277-280 proteins in of dissected germ, 205 of mill germ, 213-216 processing effects on, 203 research needs for, 287-289 scutellum of, 194-195 separation of, 197-204 sieve analysis of, 201 spoilage causes in, 261-262 steeping of, 276-277 storage and stabilization of, 258-273 effect on nutrients, 270-272 structural components of, 190 composition, 197 diagram, 195 quantitative determination, 196 separation methods, 195-196

sugars in of dissected germ, 205-206 of mill germ, 230,231 storage effects on, 232 temperature effects on, 259-260 tocopherols in, 237 toxic factors in, 255-258 vacuum packaging of, 259 vitamin concentrates from, 285-286 vitamins in of dissected germ, 208-209 of mill germ, 235-236 storage effects on, 271 Wheat germ oil analysis of, 225 constants of, 228-229 stability of, 229-230 uses of, 285 Wheat kernel structural diagram of, 191 transections of, 192

X D-Xylulokinase, in wheat germ, 240

A B c D E F

7 8 9 O l

2

G 3 H 4 1 5 J 6