1. Food Chemistry

2. Springer-VerlagBerlin Heidelberg GmbH 3. H.-D. Belitz W. Grosch P. SchieberleFood ChemistryTranslation from the Fifth German Editionby M. M. BurghagenThird revised Edition with 472 Figures over 900 Formula and 620 Tables, Springer 4. Professor Dr.-Ing. H.-D. Belitz tProfessor Dr.-Ing. W GroschInstitut fUr Lebensmitte1chemie der Technischen Universitt MUnchenand former Deputy Director of the Deutsche Forschungsanstalt fiir Lebensmitte1chemie,MUnchenLichtenbergstraBe 4D-85748 Garching, FRGProfessor Dr. rer. nat. P. SchieberleInstitut fiir Lebensmitte1chemie der Technischen Universitt Munchenand Director of the Deutsche Forschungsanstalt fUr Lebensmittelchemie, MUnchenLichtenbergstraBe 4D-85748 Garching, FRGTranslators:First edition:Professor Dr. D. HadziyevSecond edition:Peter Hessel (chapters O and 1)Christiane Sprinz (chapter 2)ISBN 978-3-540-40818-5Dr. Sabine Jordan (:chapter 3)Dr. Margaret Burghagen (chapters 4-23)Third edition:Dr. Margaret BurghagenLibrary of Congress Cataloging-in-Publication DataBelitz, H.-D, (Hans-Dieter)[Lehrbuch der Lebensmittelchemie. English]Food chemistry 1 H.-D. Belitz, W. Grosch, P. Schieberle ; translation from the fifthGerman edition by M.M.Burghagen ... [et al.]. - 3rd rev. ed.p.cm.Inc1udes bibliographical references and index.ISBN 978-3-540-40818-5 ISBN 978-3-662-07279-0 (eBook)DOI 10.1007/978-3-662-07279-01. Food-Analysis. 1. Grosch, W. (Wemer) II. Schieberle, Peter. III. Title.TX545.B3513 2004 664'.07-dc22 2004041327This work is subject to copyright. All rights are reserved, whether the whole or par! of the material is concemed,specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproduction on microfilms or in any other ways, and storage in data banks. Duplication of this publicationor parts thereofis only permitted under the provisions ofthe German Copyright Law of September 9, 1965,in its current version, and permission for use must always be obtained from Springer-Verlag Berlin Heidelberg GmbH.Violations are liable for prosecution under the German Copyright Law. Springer-Verlag Berlin Heidelberg 1987, 1999 and 2004Originally published by Springer-Verlag Berlin Heidelberg New York in 2004The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective lawsand regulations and therefore free for general use.Cover design: KiinkelLopka GmbH, HeidelbergTypesetting: Fotosatz-Service Kohler GmbH, 97084 Wiirzburg52/3020xv - 5 4 3 2 1 0- Printed on acid-free paper 5. Preface to the Third English EditionThe third English edition of "Food Chemistry" is a translation of the fifth Germanedition of this textbook. The text has been revised, but most of theproduction data are for the year 1999.We are very grateful to Dr. Margaret Burghagen who translated the manuscript.It was a pleasure to work with her.We would also like to thank Mrs. 1. Jauker for assistance in completing themanuscript.Garching, February 2004 W Grosch, P. SchieberlePreface to the Second English EditionThe second edition of "Food Chemistry" is a translation of the fourth Germanedition of this textbook. The text has been corrected only in a few places, e. g.,most of the production data are presented for the year 1996.The preparation of this edition was greatly delayed due to the deaths of ProfessorDr. H.-D. Belitz in March 1993 and of Professor Dr. D. Hadziyev, who translatedthe first edition, in July 1995. H.-D. Belitz worked on the preparation of thesecond edition.Dr. Margaret Burghagen translated most of the extensive changes incorporatedinto this new edition and revised the entire text. I am greatly indebted to her forher excellent work. It was a pleasure to work with her.I gratefully acknowledge the help of my colleagues who made valuablecriticisms and contributed to the improvement of the text. I particularly thankDr. M. C. Kuhn, Holland.I would also like to thank Mrs. R. Jauker for assistance in completing themanuscript and for proofreading and my son B. Grosch for assistance in preparingthe index.Garching, January 1999 WGrosch 6. Preface to the Fifth German EditionThe continuation ofthis textbook was considerably delayed by the death of Prof.Dr. H.-D. Belitz in March 1993. This new edition follows the time-tested conceptdescribed in the preface to the first German edition. All the chapters havebeen thoroughly revised and updated. For example, the following changes havebeen made:All the data on the composition of foods have been updated.In the chapter on enzymes, the pressure dependence of the activity, the polymerasechain reaction and the detection of food modified with geneticengineering techniques have all been included.An extension of the chapter on lipids was necessary because of the latest researchon enzymes (lipase, lip oxygenase and allene oxide synthase) and antioxidants.The chapter on carbohydrates now includes a completely new presentation ofthe Maillard reaction.The chapter on aroma substances has been revised completely. The sameapplies to the sections on aroma substances in individual foods, where quantitativeresults have been presented for the first time.The data on the occurrence and recommended intake of vitamins andminerals have been updated.In the chapter on additives, the sections on emulsifiers and fat substitutes havebeen extended.The information on the raw materials and production of individual foods hasbeen corrected in accordance with the latest technological advances and newresults pertaining to the constituents have been presented. The main emphasishas been put on milk, meat, fish, cereals, beer, wine and coffee.Some components of foods, which are currently of greater interest, have beenincluded or the text has been extended, e. g., conjugated linoleic acids, allergenicproteins, bifidogenic oligo saccharides and sexual hormones.The literature pertaining to each chapter has been supplemented.We would like to thank a number of colleagues who helped us with their constructivecriticism in the preparation of this manuscript. We are specially gratefulto Prof. Dr. A. Rapp (Chapter 20, Wine), Prof. Dr. H. Scherz (data on thecomposition of foods), Prof. Dr. J. Weder (corrections) and Dr. H. Wiesser (cerealproteins).We also thank Mrs. S. Bijewitz and Mrs. R. Jauker for their support in the preparationof this manuscript, Dr. E. Kirchhoff and Prof. Dr. H. Scherz who helpedwith proofreading and Dipl.-Chem. B. Grosch who cooperated in the preparationof the index.Garching, November 2000 W Grosch, P. Schieberle 7. Preface to the First German EditionThe very rapid development of food chemistry and technology over the last twodecades, which is due to a remarkable increase in the analytical and manufacturingpossibilities, makes the complete lack of a comprehensive, teaching orreference text particularly noticeable. It is hoped that this textbook of foodchemistry will help to fill this gap. In writing this volume we were able to drawon our experience from the lectures which we have given, covering variousscientific subjects, over the past fifteen years at the Technical University ofMunich.Since a separate treatment of the important food constituents (proteins, lipids,carbohydrates, flavor compounds, etc.,) and of the important food groups (milk,meat, eggs, cereals, fruits, vegetables, etc.,) has proved successful in our lectures,the subject matter is also organized in the same way in this book.Compounds which are found only in particular foods are discussed where theyplaya distinctive role while food additives and contaminants are treated in theirown chapters. The physical and chemical properties of the important constituentsof foods are discussed in detail where these form the basis forunderstanding either the reactions which occur, or can be expected to occur, duringthe production, processing, storage and handling of foods or the methodsused in analyzing them. An attempt has also been made to clarify the relationshipbetween the structure and properties at the level of individual food constituentsand at the level of the whole food system.The book focuses on the chemistry of foodstuffs and does not consider nationalor international food regulations. We have also omitted a broader discussion ofaspects related to the nutritional value, the processing and the toxicology offoods. All of these are an essential part of the training of a food chemist but,because of the extent of the subject matter and the consequent specialization,must today be the subject of separate books. Nevertheless, for all importantfoods we have included brief discussions of manufacturing processes and theirparameters since these are closely related to the chemical reactions occurring infoods.Commodity and production data of importance to food chemists are mainly givenin tabular form. Each chapter includes some references which are not intendedto form an exhaustive list. No preference or judgement should be inferredfrom the choice of references; they are given simply to encourage further reading.Additional literature of a more general nature is given at the end of thebook.This book is primarily aimed both at students of food and general chemistry butalso at those students of other disciplines who are required or choose to studyfood chemistry as a supplementary subject. We also hope that this comprehensivetext will prove useful to both food chemists and chemists who have completedtheir formal education.We thank sincerely Mrs. A. ModI (food chemist), Mrs. R. Berger, Mrs. I. Hofmeier,Mrs. E. Hortig, Mrs. F. Lynen and Mrs. K. Wiist for their help during thepreparation of the manuscript and its proofreading. We are very grateful toSpringer Verlag for their consideration of our wishes and for the agreeablecooperation.Garching, July 1982 H.-D. Belitz, W. Grosch 8. IntroductionFoods are materials which, in their naturally occurring, processed or cookedforms, are consumed by humans as nourishment and for enjoyment.The terms "nourishment" and "enjoyment" introduce two important propertiesof foods: the nutritional value and the hedonic value. The former is relativelyeasy to quantify since all the important nutrients are known and their effects aredefined. Furthermore, there are only a limited number of nutrients. Defining thehedonic value of a food is more difficult because such a definition must take intoaccount all those properties of a food, such as visual appeal, smell, taste andtexture, which interact with the senses. These properties can be influencedby a large number of compounds which in part have not even been identified.Besides their nutritional and hedonic values, foods are increasingly beingjudged according to properties which determine their handling. Thus, the term"convenience foods". An obvious additional requirement of a food is that it befree from toxic materials.Food chemistry is involved not only in elucidating the composition of the rawmaterials and end-products, but also with the changes which occur in food duringits production, processing, storage and cooking. The highly complex natureof food results in a multitude of desired and undesired reactions which are controlledby a variety of parameters. To gain a meaningful insight into these reactions,it is necessary to break up the food into model systems. Thus, starting fromcompositional analyses (detection, isolation and structural characterization offood constituents), the reactions of a single constituent or of a simple mixturecan be followed. Subsequently, an investigation of a food in which an individualreaction dominates can be made. Inherently, such a study starts with a givencompound and is thus not restricted to anyone food or group offoods. Such generalstudies of reactions involving food constituents are supplemented by specialinvestigations which focus on chemical processes in individual foods. Researchof this kind is from the very beginning closely associated with economic andtechnological aspects and contributes, by understanding the basics ofthe chemicalprocesses occurring in foods, both to resolving specific technical problemsand to process optimization.A comprehensive evaluation of foods requires that analytical techniques keeppace with the available technology. As a result a major objective in foodchemistry is concerned with the application and continual development ofanalytical methods. This aspect is particularly important when followingpossible contamination of foods with substances which may involve a healthrisk. Thus, there are close links with environmental problems.Food chemistry research is aimed at establishing objective standards by whichthe criteria mentioned above - nutritional value, hedonic value, absence oftoxic compounds and convenience - can be evaluated. These are a prerequisitefor the industrial production of high quality food in bulk amounts.This brief outline thus indicates that food chemistry, unlike other branches ofchemistry which are concerned either with particular classes of compounds orwith particular methods, is a subject which, both in terms of the actual chemistryand the methods involved, has a very broad field to cover. 9. Table of Contentso VVater0.1 Foreword0.20.2.10.2.20.30.3.10.3.20.3.30.3.40.3.50.411.11.21.2.11.2.21.2.2.11.2.2.21.2.31.2.3.11.2.3.21.2.3.31.2.3.41.2.41.2.4.11.2.4.21.2.4.2.11.2.4.2.21.2.4.2.31.2.4.2.41.2.4.31.2.4.3.11.2.4.3.21.2.4.3.31.2.4.3.41.2.4.3.51.2.4.3.61.2.4.3.71.2.4.41.2.51.2.5.1StructureWater MoleculeLiquid Water and IceEffect on Storage LifeWater Activity ...Water Activity as an IndicatorPhase Transition of Foods Containing WaterWLF EquationConclusionLiteratureAmino Acids, Peptides, ProteinsForewordAmino AcidsGeneral RemarksClassification, Discovery and OccurrenceClassification ...... .Discovery and OccurrencePhysical PropertiesDissociation . . . . . . . .Configuration and Optical ActivitySolubilityUV Absorption ..... ...Chemical Reactions . . . . . . . .Esterification of Carboxyl GroupsReactions of Amino GroupsAcylationAlkylation and Arylation ..Carbamoyl and Thiocarbamoyl DerivativesReactions with Carbonyl Compounds .Reactions Involving Other Functional GroupsLysineArginine ...Aspartic and Glutamic AcidsSerine and ThreonineCysteine and CystineMethionineTyrosineReactions of Amino Acids at Higher TemperaturesSynthetic Amino Acids Utilized for to Increasing the BiologicalValue of Food (Food Fortification)Glutamic Acid . .112335567788999991212131515161616161820212222232323232424242931 10. XII Table of Contents1.2.5.21.2.5.31.2.5.41.2.5.51.2.5.61.2.5.71.2.61.31.3.11.3.21.3.2.11.3.31.3.41.3.4.11.3.4.21.3.4.31.3.4.41.3.4.51.41.4.11.4.1.11.4.1.21.4.1.31.4.1.41.4.1.51.4.21.4.2.11.4.2.21.4.2.2.11.4.2.2.21.4.2.2.31.4.2.2.41.4.2.31.4.2.3.11.4.2.3.21.4.2.3.31.4.2.41.4.31.4.3.11.4.3.21.4.3.31.4.3.41.4.3.51.4.3.61.4.41.4.4.11.4.4.1.11.4.4.1.21.4.4.1.31.4.4.1.41.4.4.21.4.4.31.4.4.41.4.4.51.4.4.6Aspartic AcidLysine ....MethioninePhenylalanineThreonine ..TryptophanSensory PropertiesPeptides ..... .General Remarks, NomenclaturePhysical PropertiesDissociation . . . .Sensory PropertiesIndividual PeptidesGlutathioneCarnosine, Anserine and BalenineNisin ..... .Lysine Pep tidesOther PeptidesProteins ....Amino Acid SequenceAmino Acid Composition, SubunitsTerminal GroupsPartial Hydrolysis . . . . . . . . . .Sequence Analysis . . . . . . . . . .Derivation of Amino Acid Sequence from the NucleotideSequence of the Coding GeneConformation ................... .Extended Peptide Chains ............. .Secondary Structure (Regular Structural Elements)~-SheetHelical Structures . . . . .Reverse Turns ...... .Super-Secondary StructuresTertiary and Quaternary StructuresFibrous Proteins . . .Globular Proteins ..Quaternary StructuresDenaturation . . . .Physical PropertiesDissociation . . . .Optical Activity ..Solubility, Hydration and Swelling PowerFoam Formation and Foam StabilizationGel Formation . . .Emulsifying EffectChemical ReactionsLysine ResidueReactions Which Retain the Positive ChargeReactions Resulting in a Loss of Positive ChargeReactions Resulting in a Negative ChargeReversible Reactions ....... .Arginine Residue ......... .Glutamic and Aspartic Acid ResiduesCystine Residue . .Cysteine ResidueMethionine Residue31323233333333343435353537373838393939414141424345484849505152535353535656585860606162636364646465656666676869 11. 1.4.4.71.4.4.81.4.4.91.4.4.101.4.4.111.4.51.4.5.11.4.5.21.4.5.2.11.4.5.2.21.4.5.2.31.4.5.2.41.4.61.4.6.11.4.6.21.4.6.2.11.4.6.2.21.4.6.2.31.4.6.31.4.6.3.11.4.6.3.21.4.6.3.31.4.71.4.7.11.4.7.21.4.7.31.4.7.3.11.4.7.3.21.522.12.22.2.12.2.22.2.2.12.2.2.22.2.~2.2.42.2.52.2.62.2.72.32.3.l2.3.1.12.3.1.22.3.22.3.2.l2.3.2.22.3.2.32.3.32.3.3.12.3.3.2Table of Contents XIIIHistidine Residue .Tryptophan ResidueTyrosine ResidueBifunctional ReagentsReactions Involved in Food ProcessingEnzyme-Catalyzed ReactionsForeword ...... .Proteolytic EnzymesSerine EndopeptidasesCysteine EndopeptidasesMetalo Peptidases . . . .Aspartic EndopeptidasesChemical and Enzymatic Reactions of Interest to Food ProcessingForeword ...... .Chemical ModificationAcylation ...... .Alkylation . . . . . . .Redox Reactions Involving Cysteine and CystineEnzymatic ModificationDephosphorylationPlastein Reaction .Cross-Linking ...Texturized ProteinsForewordStarting MaterialTexturizationSpin Process . . .Extrusion ProcessLiterature ....EnzymesForewordGeneral Remarks, Isolation and NomenclatureCatalysis ...... .Specificity . . . . . .Substrate SpecificityReaction SpecificityStructure ...... .Isolation and PurificationMultiple Forms of EnzymesNomenclature ..ActivityUnits . .Enzyme CofactorsCosubstrates . . .Nicotinamide Adenine DinucleotideAdenosine TriphosphateProsthetic GroupsFlavins ...... .Hemin ...... .Pyridoxal PhosphateMetal Ions .....Magnesium, Calcium and ZincIron, Copper and Molybdenum696969707074747475757678797979798282828283868787878788888892929292939394949496969797100100100101101102102103103104 12. XIV Table of Contents2.42.4.12.4.1.12.4.1.22.4.1.2.12.4.1.2.22.4.1.2.32.4.22.4.2.12.4.2.22.4.2.32.4.2.42.4.2.52.4.32.52.5.12.5.1.12.5.1.1.12.5.1.1.22.5.1.22.5.1.2.12.5.1.2.22.5.1.32.5.22.5.2.12.5.2.22.5.2.2.12.5.2.2.22.5.2.2.32.5.32.5.42.5.4.12.5.4.22.5.4.32.5.4.42.5.52.5.62.62.6.12.6.1.12.6.1.22.6.1.32.6.22.6.32.6.42.6.4.12.6.4.22.6.4.2.12.6.4.2.22.6.4.2.32.6.4.2.42.72.7.12.7.1.12.7.1.2Theory of Enzyme CatalysisActive Site . . . . . . .Active Site LocalizationSubstrate Binding . . .Stereospecificity . . . ."Lock and Key" Hypothesis.Induced-fit Model ..... .Reasons for Catalytic ActivitySteric Effects - Orientation EffectsStructural Complementarity to Transition State .Entropy Effect . . . . . . . .General Acid-Base CatalysisCovalent Catalysis . . . . . .Closing Remarks ..... .Kinetics of Enzyme-Catalyzed ReactionsEffect of Substrate ConcentrationSingle-Substrate ReactionsMichaelis-Menten EquationDetermination ofK", and VTwo-Substrate ReactionsOrder of Substrate BindingRate Equations for a Two-Substrate ReactionAllosteric EnzymesEffect ofInhibitorsIrreversible InhibitionReversible InhibitionCompetitive InhibitionNon-Competitive InhibitionUncompetitive InhibitionEffect of pH on Enzyme ActivityInfluence of Temperature . . . .Time Dependence of EffectsTemperature Dependence of EffectsTemperature OptimumThermal Stability ..Influence of PressureInfluence of Water . .Enzymatic Analysis .Substrate DeterminationPrinciples ....... .End-Point Method ... .Kinetic Method .... .Determination of Enzyme ActivityEnzyme ImmunoassayPolymerase Chain ReactionPrinciple ofPCRExamples ........ .Addition of Soybeans .. .Genetically Modified SoybeansGenetically Modified TomatoesSpecies Differentiation ....Enzyme Utilization in the Food IndustryTechnical Enzyme PreparationsProduction . . . . . . .Immobilized Enzymes105106106107107108109110110111111112114117117117117117120121121122124125126126126127127128130131131133134136137137137137139139140141142143144144144144144144146146147 13. 2.7.1.2.12.7.1.2.22.7.1.2.32.7.1.2.42.7.22.7.2.12.7.2.1.12.7.2.1.22.7.2.1.32.7.2.1.42.7.2.1.52.7.2.22.7.2.2.12.7.2.2.22.7.2.2.32.7.2.2.42.7.2.2.52.7.2.2.62.7.2.2.72.7.2.2.82.7.2.2.92.7.2.2.102.7.2.2.112.7.2.2.122.7.2.2.132.7.2.2.142.7.2.2.152.7.2.2.162.7.2.32.7.2.42.833.13.23.2.13.2.1.13.2.1.23.2.1.33.2.23.2.2.13.2.2.23.2.2.33.2.2.43.2.2.53.2.33.2.3.13.2.3.23.2.3.2.13.2.3.2.23.2.3.2.33.2.3.2.43.2.4Table of Contents XVBound Enzymes . . . . 147Enzyme Entrapment . . 147Cross-Linked Enzymes 147Properties ..... 147Individual Enzymes 148Oxidoreductases . 148Glucose Oxidase 148Catalase ..... 149Lipoxygenase .. 149Aldehyde Dehydrogenase 149Butanediol Dehydrogenase 149Hydrolases . . . . . . . . . 149Peptidases . . . . . . . . . 149a- and p-Amylases 150Glucan-l,4-a-n-Glucosidase (Glucoamylase) 151Pullulanase (Isoamylase) .. 151Endo-I,3(4)-P-n-Glucanase . . . . 151a-n-Galactosidase . . . . . . . . . 151p-n-Galactosidase (Lactase) 152p-n-Fructofuranosidase (Invertase) 152a-L-Rhamnosidase ...... 152Cellulases and HemiceIlulases 152Lysozyme ..... 152Thioglucosidase . . 152Pectolytic Enzymes 152Lipases 153Tannases . . 153Glutaminase 153Isomerases . 153Transferases 154Literature . 154Lipids .. 157Foreword 157Fatty Acids 158Nomenclature and Classification 158Saturated Fatty Acids . 158Unsaturated Fatty Acids 159Substituted Fatty Acids 163Physical Properties 163Carboxyl Group . . . . 163Crystalline Structure, Melting Points 164Urea Adducts . 165Solubility ..... 165UV-Absorption .. 166Chemical Properties 166Methylation of Carboxyl Groups 166Reactions of Unsaturated Fatty Acids 166Halogen Addition Reactions 167Transformation of Isolene-Type Fatty Acids to Conjugated Fatty Acids 167Formation of a It-Complex with Ag+ Ions 167Hydrogenation. . . . . . . . . . . . . . 167Biosynthesis of Unsaturated Fatty Acids . 168 14. XVI Table of Contents3.33.3.13.3.1.13.3.1.23.3.1.33.3.1.43.3.1.53.3.23.3.2.13.3.2.23.43.4.13.4.1.13.4.1.23.4.1.33.4.23.4.2.13.4.2.23.4.2.33.53.5.13.5.1.13.5.1.23.5.23.63.6.13.6.23.6.2.13.6.2.23.6.33.73.7.13.7.1.13.7.1.23.7.1.2.13.7.1.2.23.7.23.7.2.13.7.2.1.13.7.2.1.23.7.2.1.33.7.2.1.43.7.2.1.53.7.2.1.63.7.2.1.73.7.2.1.83.7.2.1.93.7.2.23.7.2.33.7.2.43.7.2.4.13.7.2.4.23.7.2.4.33.7.2.4.4Acylglycerols ................ .Triacylglycerols (TG) . . . . . . . . . . . . .Nomenclature, Classification, Calorific ValueMelting Properties . . . .Chemical Properties . . .Structural DeterminationBiosynthesis . . . . . . .Mono- and Diacylglycerols (MG, DG)Occurrence, Production .Physical PropertiesPhospho- and GlycolipidsClasses ......... .Phosphatidyl DerivativesGlycerolglycolipidsSphingolipids ..... .Analysis ........ .Extraction, Removal of Non lipidsSeparation and Identification of Classes of ComponentsAnalysis of Lipid ComponentsLipoproteins, MembranesLipoproteins .Definition ....... .Classification ..... .Involvement of Lipids in the Formation of Biological MembranesDiol Lipids, Higher Alcohols, Waxes and CutinDiol Lipids .......... .Higher Alcohols and DerivativesWaxes ....Alkoxy Lipids . . . . . . . . .Cutin ............. .Changes in Acyl Lipids of FoodEnzymatic Hydrolysis . . . . .Triacylglycerol Hydrolases (Lipases)Polar-Lipid HydrolasesPhospholipases .......... .Glycolipid Hydrolases . . . . . . . .Peroxidation of Unsaturated Acyl LipidsAutoxidation . . . . . . . . . . . .Fundamental Steps of AutoxidationMonohydroperoxides ...... .Hydroperoxide-Epidioxides . . . .Initiation of a Radical Chain ReactionPhotooxidationHeavy Metal Ions .Heme(in) CatalysisActivated Oxygen .Secondary ProductsLipoxygenase: Occurrence and PropertiesEnzymatic Degradation of HydroperoxidesHydroperoxide-Protein Interactions ..Products Formed from HydroperoxidesLipid-Protein Complexes ...Protein Changes . . . . . . . .Decomposition of Amino Acids169169169170171172175176176177177177177179179181181181181182182182183184184184185185185186186186186188188189189190190191194195195198199200201205206209209209211212 15. 3.7.33.7.3.13.7.3.23.7.3.2.13.7.3.2.23.7.3.2.33.7.43.7.4.13.7.4.23.7.53.7.63.83.8.13.8.23.8.2.13.8.2.23.8.2.2.13.8.2.2.23.8.2.33.8.2.3.13.8.2.3.23.8.2.43.8.33.8.3.13.8.3.23.8.43.8.4.13.8.4.1.13.8.4.1.23.8.4.23.8.4.33.8.4.43.8.4.53.8.4.5.13.8.4.5.23.8.4.63.944.14.24.2.14.2.1.14.2.1.24.2.1.34.2.24.2.2.14.2.2.24.2.34.2.44.2.4.14.2.4.2Table of Contents XVIIInhibition of Lipid Peroxidation .Antioxidant Activity . .Antioxidants in Food .Natural AntioxidantsSynthetic AntioxidantsSynergists .. . . . . .Fat or Oil Heating (Deep Frying)Autoxidation of Saturated Acyl LipidsPolymerization ........... .Radiolysis . . . . . . . . . . . . . . .Microbial Degradation of Acyl Lipids to Methyl KetonesUnsaponifiable ConstituentsHydrocarbons . . . . . .Steroids ........ .Structure, NOTI1enclatureSteroids of AniTI1al FoodCholesterol ...... .VitaTI1in D . . . . . . . .Plant Steroids (Phytosterols)DesTI1ethylsterols ..... .Methyl- and DiTI1ethylsterolsAnalysis .......... .Tocopherols and TocotrienolsStructure, ITI1portance . . . .Analysis .......... .Carotenoids ........ .CheTI1ical Structure, OccurrenceCarotenes .....XanthophyllsPhysical PropertiesCheTI1ical PropertiesPrecursors of AroTI1a COTI1poundsUse ofCarotenoids in Food ProcessingPlant Extracts .....Individual COTI1poundsAnalysis .LiteratureCarbohydratesForewordMonosaccharidesStructure and NOTI1enclatureNOTI1enclatureConfiguration .. .ConfofTI1ation .. .Physical PropertiesHygroscopicity and SolubilityOptical Rotation, MutarotationSensory Properties ..... .CheTI1ical Reactions and DerivativesReduction to Sugar Alcohols ....Oxidation to Aldonic, Dicarboxylic and Uronic Acids212213213213216217218219221222223224224224224225225226226227229230231231231232233233235237238238241241241241242245245245245245247251254254255255258258259 16. XVIII4.2.4.34.2.4.3.14.2.4.3.24.2.4.3.34.2.4.44.2.4.4.14.2.4.4.24.2.4.4.34.2.4.4.44.2.4.4.54.2.4.4.64.2.4.4.74.2.4.4.84.2.4.4.94.2.4.4.104.2.4.54.2.4.64.2.4.74.2.4.84.2.4.94.34.3.14.3.24.44.4.14.4.24.4.2.14.4.2.24.4.2.34.4.2.44.4.2.54.4.2.64.4.34.4.3.14.4.3.24.4.3.34.4.3.44.4.3.54.4.3.64.4.3.74.4.3.7.14.4.3.7.24.4.44.4.4.14.4.4.1.14.4.4.1.24.4.4.1.34.4.4.24.4.4.2.14.4.4.2.24.4.4.2.34.4.4.2.44.4.4.34.4.4.3.14.4.4.3.24.4.4.3.3Table of ContentsReactions in the Presence of Acids and AlkalisReactions in Strongly Acidic MediaReactions in Strongly Alkaline SolutionCaramelization ............ .Reactions with Amino Compounds (Maillard Reaction)Initial Phase of the Maillard ReactionFormation of Deoxyosones ..... .Secondary Products of 3-DeoxyosonesSecondary Products of I-DeoxyosonesSecondary Products of 4-DeoxyosonesRedox Reactions ........ .Strecker Reaction ........ .Formation of Colored CompoundsProtein Modifications . . . . . . .Inhibition of the Maillard ReactionReactions with Hydroxy Compounds (O-Glycosides)Esters ........ .Ethers ........ .Halodeoxy DerivativesCleavage of Glycols . .Oligo saccharidesStructure and NomenclatureProperties and ReactionsPolysaccharides ....Classification, StructureConformation ..... .Extended or Stretched, Ribbon-Type ConformationHollow Helix-Type ConformationCrumpled-Type ConformationLoosely-Jointed ConformationConformations of HeteroglycansInterchain InteractionsProperties ........... .General Remarks ....... .Perfectly Linear PolysaccharidesBranched Polysaccharides . . . .Linearly Branched PolysaccharidesPolysaccharides with Carboxyl GroupsPolysaccharides with Strongly Acidic GroupsModified Polysaccharides . . . . . . . .Derivatization with Neutral SubstituentsDerivatization with Acidic SubstituentsIndividual PolysaccharidesAgar ........ .Occurrence, IsolationStructure, PropertiesUtilization . . . . . .Alginates ..... .Occurrence, IsolationStructure, PropertiesDerivativesUtilization . . . . .CarrageenansOccurrence, IsolationStructure, PropertiesUtilization . . . . . .260260263267268269270272274279280281282284288288289290291293294294295298298298298299300300300300301301302302304304304304304304304304304305305305305305306306307307307309 17. 4.4.4.44.4.4.4.14.4.4.4.24.4.4.4.34.4.4.54.4.4.5.14.4.4.5.24.4.4.5.34.4.4.64.4.4.6.14.4.4.6.24.4.4.6.34.4.4.74.4.4.7.14.4.4.7.24.4.4.7.34.4.4.84.4.4.8.14.4.4.8.24.4.4.8.34.4.4.94.4.4.9.14.4.4.9.24.4.4.9.34.4.4.104.4.4.10.14.4.4.10.24.4.4.10.34.4.4.114.4.4.11.14.4.4.11.24.4.4.11.34.4.4.124.4.4.12.14.4.4.12.24.4.4.12.34.4.4.134.4.4.13.14.4.4.13.24.4.4.13.34.4.4.144.4.4.14.14.4.4.14.24.4.4.14.34.4.4.14.44.4.4.14.54.4.4.14.64.4.4.154.4.4.15.14.4.4.15.24.4.4.15.34.4.4.15.44.4.4.15.54.4.4.15.64.4.4.15.74.4.4.15.84.4.4.15.9FurcellaranOccurrence, IsolationStructure, PropertiesUtilization . . . . . .Gum Arabic . . . . .Occurrence, IsolationStructure, PropertiesUtilizationGum Ghatti ....OccurrenceStructure, PropertiesUtilization . . .Gum Tragacanth . .OccurrenceStructure, PropertiesUtilizationKarayaGum ....OccurrenceStructure, PropertiesUtilization . . . . .Guaran Gum . . . .Occurrence, IsolationStructure, PropertiesUtilization ..... .Locust Bean Gum . .Occurrence, IsolationStructure, PropertiesUtilization . . . . . .Tamarind Flour ...Occurrence, IsolationStructure, Properties .Utilization . . . . . .Arabinogalactan from LarchOccurrence, IsolationStructure, PropertiesUtilization . . . . . .PectinOccurrence, IsolationStructure, PropertiesUtilization . . . . . .Starch ....... .Occurrence, IsolationStructure and Properties of Starch GranulesStructure and Properties of Amylose ..Structure and Properties of AmylopectinUtilization . . . .Resistant Starches . . . . . . . .Modified Starches . . . . . . . .Mechanically Damaged StarchesExtruded Starches . .Dextrins ...... .Pregelatinized StarchThin-Boiling StarchStarch EthersStarch Esters . . . . .Cross-Linked StarchesOxidized Starches . . .Table of Contents XIX309309309309309309310311311311311311312312312312312312312313313313314314314314314314315315315315315315315316316316316317317317318323325326327327327327327327327328328329329 18. xx Table of Contents4.4.4.164.4.4.16.14.4.4.16.24.4.4.16.34.4.4.174.4.4.17.14.4.4.17.24.4.4.184.4.4.194.4.4.19.14.4.4.19.24.4.4.19.34.4.4.204.4.4.20.14.4.4.20.24.4.4.20.34.4.4.214.4.4.21.14.4.4.21.24.4.4.21.34.4.4.224.4.4.22.14.4.4.22.24.4.4.22.34.4.4.234.4.4.23.14.4.4.23.24.4.54.4.5.14.4.5.1.14.4.5.1.24.4.5.1.34.4.5.1.44.4.5.24.4.5.34.4.5.44.4.5.54.4.64.4.6.14.4.6.24.555.15.1.15.1.25.1.35.1.45.1.55.25.2.15.2.1.15.2.1.25.2.1.3CelluloseOccurrence, IsolationStructure, Properties .Utilization . . . . . .Cellulose DerivativesAlkyl Cellulose, Hydroxyalkyl CelluloseCarboxymethyl CelluloseHemicellulosesXanthan Gum .. . .Occurrence, IsolationStructure, PropertiesUtilization . . . . . .Sc1eroglucan . . . . .Occurrence, IsolationStructure, Properties .UtilizationDextran ......OccurrenceStructure, PropertiesUtilization . . . . .Inulin and OligofiuctoseOccurrenceStructure ........ .Utilization . . . . . . . .Polyvinyl Pyrrolidone (PVP)Structure, Properties . . . . .Utilization ......... .Enzymatic Degradation of PolysaccharidesAmylasesa-Amylase ................ .~-Amylase ................ .Glucan-l,4-a-D-glucosidase (glucoamylase)a-Dextrin Endo-I ,6-a-glucosidase (pullulanase)Pectinolytic Enzymes ..Cellulases ....... .Endo-l ,3( 4)-~-glucanaseHemicellulasesAnalysis of PolysaccharidesThickening AgentsDietary FibersLiterature .....Aroma SubstancesForewordConcept DelineationImpact Compounds of Natural AromasThreshold Value . . . .Aroma Value . . . . . .Off-Flavors, Food TaintsAroma Analysis . . . .Aroma Isolation . . . .Distillation, ExtractionGas Extraction . . .Headspace Analysis . .329329330330330331331332333333333333333333333334334334334334334334334334334334335335335335335335335336336337337337337339339342342342342342344345347347348350350 19. 5.2.25.2.2.15.2.2.25.2.35.2.45.2.55.2.65.2.6.15.2.6.25.2.75.35.3.15.3.1.15.3.1.25.3.1.35.3.1.45.3.1.55.3.1.65.3.1.75.3.1.85.3.25.3.2.15.3.2.25.3.2.35.3.2.45.3.2.55.3.2.65.3.2.75.45.4.15.4.25.55.5.15.5.1.15.5.1.25.5.1.35.5.1.45.5.1.55.5.1.65.5.25.5.35.5.45.5.55.65.6.15.6.25.6.35.766.16.26.2.1Sensory Relevance ........... .Aroma Extract Dilution Analysis (AEDA)Headspace GC OlfactometryEnrichment ...... .Chemical Structure .... .Enantioselective Analysis . .Quantitative Analysis, Aroma ValuesIsotopic Dilution analysis (IDA)Aroma Values (AV) ........ .Aroma Model, Omission ExperimentsIndividual Aroma CompoundsNonenzymatic ReactionsCarbonyl Compounds . . . . .Pyranones .......... .Furanones .......... .Thiols, Thioethers, Di- and TrisulfidesThiazolesPyTroles,PyridinesPyrazinesPhenols ..... .Enzymatic ReactionsCarbonyl Compounds, AlcoholsHydrocarbons, Esters .. .Lactones ......... .Terpenes ......... .Volatile Sulfur CompoundsPyrazines ........ .Skatole, p-Cresol .... .Interactions with Other Food ConstituentsLipids ............. .Proteins, Polysaccharides ... .Natural and Synthetic FlavoringsRaw Materials for EssencesEssential Oils ..Extracts, Absolues . . . . .Distillates ........ .Microbial Aromas . . . . .Synthetic Natural Aroma CompoundsSynthetic Aroma CompoundsEssences ........ .Aromas from PrecursorsStability of Aromas ...Encapsulation of AromasRelationships Between Structure and OdorGeneral Aspects . . .Carbonyl CompoundsAlkylpyrazinesLiteratureVitaminsForewordFat-Soluble VitaminsRetinol (Vitamin A)Table of Contents XXI351351353354355355358358359359361362362362363365369371374376376378380382384391393393394396396398399399399400400400401401401401403404404404406406409409409. 409 20. XXII Table of Contents6.2.1.16.2.1.26.2.1.36.2.26.2.2.16.2.2.26.2.2.36.2.36.2.3.16.2.3.26.2.3.36.2.46.2.4.16.2.4.26.2.4.36.36.3.16.3.1.16.3.1.26.3.1.36.3.26.3.2.16.3.2.26.3.2.36.3.36.3.3.16.3.3.26.3.3.36.3.46.3.4.16.3.4.26.3.4.36.3.56.3.5.16.3.5.26.3.5.36.3.66.3.6.16.3.6.26.3.6.36.3.76.3.7.16.3.7.26.3.7.36.3.86.3.8.16.3.8.26.3.8.36.3.96.3.9.16.3.9.26.3.9.36.4Biological Role .Requirenlent, ()ccurrenceStability, Degradation . .Calciferol (Vitanlin D)Biological Role .....Requirenlent, ()ccurrenceStability, Degradation . .a-Tocopherol (Vitanlin E)Biological Role .....Requirenlent, ()ccurrenceStability, Degradation . .Phytonlenadione (Vitanlin KI , Phylloquinone)Biological Role .....Requirenlent, ()ccurrenceStability, DegradationWater-Soluble VitanlinsThianline (Vitanlin BI )Biological Role ....Requirenlent, ()ccurrenceStability, Degradation . .Riboflavin (Vitanlin B2)Biological Role .....Requirenlent, ()ccurrenceStability, Degradation . .Pyridoxine (Pyridoxal, Vitanlin B6)Biological Role .....Requirenlent, ()ccurrenceStability, DegradationNicotinanlide (Niacin)Biological Role ....Requirenlent, ()ccurrenceStability, Degradation . .Pantothenic AcidBiological Role .....Requirenlent, ()ccurrenceStability, Degradation . .Biotin ......... .Biological Role .... .Requirenlent, ()ccurrenceStability, Degradation . .FolicAcid ....... .Biological Role .... .Requirenlent, ()ccurrenceStability, Degradation . .Cyanocobalanlin (Vitanlin B12)Biological Role .....Requirenlent, ()ccurrence . .Stability, Degradation . . . .L-Ascorbic Acid (Vitanlin C)Biological Role .....Requirenlent, ()ccurrenceStability, DegradationLiterature ....... .409410412412412412413413413413414414414414415415415415418418419419419419420420420420420420420421421421421421421421421421422422422422422422423423423423423424426 21. 77.17.27.2.17.2.27.2.37.2.47.2.57.2.67.37.3.17.3.27.3.2.17.3.2.27.3.2.37.3.2.47.3.2.57.3.2.67.3.2.77.3.2.87.3.2.97.3.2.107.3.2.117.3.37.3.3.17.3.3.27.3.3.37.3.3.47.3.3.57.47.588.18.28.38.48.58.68.6.18.6.28.6.38.6.48.78.88.8.18.8.1.18.8.1.28.8.2MineralsForewordMain ElementsSodiumPotassiumMagnesiumCalcium ..Chloride ..PhosphorusTrace ElementsGeneral RemarksIndividual Trace ElementsIronCopper .. .Zinc ... .ManganeseCobalt ..ChromiumSeleniumMolybdenumNickelFluorine .. .Iodine ... .Ultra-trace ElementsTin .....AluminiumBoronSilicon ...ArsenicMinerals in Food ProcessingLiterature ......... .Food AdditivesForewordVitaminsAmino AcidsMinerals ...Aroma SubstancesFlavor EnhancersMonosodium Glutamate (MSG)5' -Nucleotides . .Maltol ..... .Other CompoundsSugar SubstitutesSweetenersTable of ContentsSweet Taste: Structural RequirementsStructure-Activity Relationships in Sweet CompoundsSynergismSaccharin ...................... .XXIII427427427427429429429429429430430430430430431431431431431431431432432432432432433433433433433434434434435435435435435436436436437437437437438438 22. XXIV8.8.38.8.48.8.58.8.68.8.78.8.88.8.98.8.108.8.118.8.128.8.138.8.148.8.14.18.8.14.28.8.14.38.8.158.8.168.8.178.8.17.18.8.17.28.8.17.38.8.188.8.198.98.108.10.18.10.28.10.38.10.48.10.58.10.68.10.78.10.88.10.98.10.108.10.118.10.128.118.128.12.18.12.28.12.38.12.48.12.58.12.68.12.78.12.88.12.98.12.108.12.118.12.128.12.138.138.14Table of ContentsCyclamate .... .Monellin ..... .Thaumatins ... .Curculin and MiraculinGymnema silvestre ExtractSteviosideOsladinPhyllodulcinGlycyrrhizinNitroanilinesDihydrochalconesUreas and GuanidinesDulcin .. .Suosan .. .GuanidinesOximesOxathiazinone DioxidesDipeptide Esters and AmidesAspartame ...SuperaspartameAlitame ....HernandulcinHalodeoxy SugarsFood Colors . . .Acids ..... .Acetic Acid and Other Fatty AcidsSuccinic Acid ..... .Succinic Acid AnhydrideAdipic AcidFumaric AcidLactic AcidMalicAcid ..Tartaric Acid .Citric Acid . .Phosphoric AcidHydrochloric and Sulfuric AcidsGluconic Acid and Glucono-. 'i0i i " .. c..: ;, ....I .c: I-"-40teins have been elucidated and recorded inseveral data bases.Glycoproteins, such as x-casein (cf. 10.1.2.1.1),various components of egg white (cf. 11.2.3.1)and egg yolk (cf. 11.2.4.1.2), collagen fromconnective tissue (cf. 12.3.2.3.1) and serumproteins of some species of fish ( cf.13.1.4.2.4), contain one or more monosaccharideor oligosaccharide units bound O-glycosidicallyto serine, threonine or 6-hydroxylysineor N-glycosidically to asparagine (Formula1.82). In glycoproteins, the primary structureof the protein is defined genetically. The carbohydratecomponents, however, are enzymaticallycoupled to the protein in a co- or posttranscriptionalstep. Therefore, the carbohydratecomposition of glycoproteins is inhomogeneous(microheterogeneity)."0Q) '0c:'" .= t:l Q) Q) 0 "0 Q) c:.~Q) >-a 'u . '0" ~ c: a>Q) t:l a> " a.a> c: u If) .c III 1,0 " c: 'u 'E I- ~'u ::Ea>"0 Q) '" >- t:l !! c: " ,'V-Diagram (Ramachandran plot). Allowedconformations for amino acids with a C~atomobtained by using normal (-) and lower limit(- - -) contact distances for non-bonded atoms, fromTable 1.20. ~-Sheet structures: antiparallel (l); parallel(2), twisted (3). Helices: n-, left-handed (4),310(5), n, right-handed (6), n (7) 92. 50 1 Amino Acids, Peptides, Proteins~ : " " . .. ;., ::.. ., :;: ' .' . ... . ,. ...-1 80. ::', ': L...J'-."'"'---'----L ___ L-~....:....:'___ _ __ __J-1 80 o4> -180Fig. 1.16. jJ n" db r e(0) CO) (A) (A)~-Pleated sheet, parallel -119 +113 2.0 3.2 1.1~-Pleated sheet, antiparallel -139 +135 2.0 3.4 0.93w-Helix - 49 - 26 2.3 2.0 1.9a-Helix, left-handed coiling + 57 + 47 3.6 1.5 2.3a-Helix, right-handed coiling - 57 - 47 3.6 1.5 2.3n-HelixPolyglycine II- 57 - 70 4.4- 80 +150 3.01.15 2.83.1Polyglycine II,left-handed coilingPoly-L-proline IPolY-L-proline II+ 80 -150 3.0 3.1- 83 + 158 3.3 1.9- 78 + 149 3.0 3.1" Amino acid residues per turn.b The rise along the axis direction, per residue.e The radius of the helix.CommentsOccurs occasionally in neighbouringchain sectors of globular proteinsCommon in proteins and syntheticpolypeptidesObserved at the ends of a-helixesCommon in globular proteins, as a"coiled coil" in fibrous proteinsPolY-D-amino acids poly-(~-benzyl)L-aspartateHypotheticalSimilar to antiparallel ~-pleatedsheetfonnationSynthetic polyglycine is a mixture ofright- and left-handed helices; insome silk fibroins, the left-handedhelix occursSynthetic polY-L-proline, onlycis-peptide bondsAs left-handed polyglycine II, astriple helix in collagen 93. zxFig. 1.17. A pleated sheet structure of a peptidechainSubsequently, adjacent chains interact alongthe x-axis by hydrogen bonding, thus providingthe cross-linking required for stability.When adjacent chains run in the same direction,the peptide chains are parallel. This providesa stabilized, planar, parallel sheet structure.When the chains run in opposite directions,a planar, antiparallel sheet structure isstabilized (Fig. 1.18). The lower free energy,Fig. 1.18. Diagrammatic presentation of antiparalleI(a) and parallel (b) peptide chain arrangements1.4 Proteins 51Fig. 1.19. Diagrammatic presentation of a twistedsheet structure of parallel peptide chains (accordingto Schulz and Schirmer, 1979)twisted sheet structures, in which the mainaxes of the neighboring chains are arranged atan angle of 25 (Fig. 1.19), are more commonthan planar sheet structures.The p structures can also be regarded as specialhelix with a continuation of 2 residuesper turn. With proline, the formation of a pstructure is not possible.1.4.2.2.2 Helical StructuresThere are three regular structural elements inthe range of I/> = - 60 and jJ = - 60 (cf. Fig.1.15) in which the peptide chains are coiledlike a threaded screw. These structures are stabilizedby intrachain hydrogen bridges whichextend almost parallel to the helix axis, crosslinkingthe CO and NH groups, i. e., theCO group of amino acid residue i with theNH group of residue i + 3 (3w-helix), I + 4(a-helix) or i + 5 (n-helix).The most common structure is the a-helix andfor polypeptides from L-amino acids, exclusivelythe right-handed a-helix (Fig. 1.20). Theleft-handed a-helix is energetically unfavourablefor L-amino acids, since the side chainshere are in close contact with the backbone.No a-helix is possible with proline. The3w-he1ix was observed only at the ends ofa-helices but not as an independent regularstructure. The n-helix is hypothetical. Twohelical conformations are known of polyproline(I and II). Polyproline I contains only cispeptidebonds and is right-handed, while polyprolineII contains trans-peptide bonds and isleft-handed. The stability of the two conformationsdepends on the solvent and otherfactors. In water, polyproline II predominates.Polyglycine can also occur in two conforma- 94. 52 Amino Acids, Peptides, ProteinsFig. 1.20. Right-handed a-helixtions. Polyglycine I is a ~-structure, whilepolyglcine II corresponds largely to the polyprolineII -helix. A helix is characterized by theangles ~ and "', or by the parameters derivedfrom these angles: n, the number of amino acidresidues per turn; d, the rise along the mainaxis per amino acid residue; and r, the radiusof the helix. Thus, the equation for the pitch, p,is p = n . d. The parameters n and d are presentedwithin a ~, '" plot in Fig. 1.21.II-180. '--_....u..._-1---"-'------"--"--'--'L..l..~~_____J _ ___>l-180 D 180ciI-Fig.1.21. cp,ljI-Diagram with marked helix parametersn (- - -) and d (-). (according to Schulz andSchirmer, 1979)7.4.2.2.3 Reverse TurnsAn important conformational feature of globularproteins are the reverse turns ~-turnsand ~-bends. They occur at "hairpin" comers,where the peptide chain changes directionabruptly. Such comers involve four amino acidresidues often including proline and glycine.Several types of turns are known; of greatestimportance are type I (42% of 421 examinedturns), type II (15%) and type III (18%); seeFig. 1.22.In type I, all amino acid residues are allowed,with the exception of proline in position 3.In type II, glycine is required in position 3. Intype III, which corresponds to a 31O-helix, allamino acids are allowed. The sequences of the~-bends oflysozyme are listed in Table 1.22 asan example.Fig. 1.22. Turns of the peptide chains (~-turns), types I-III. 0 = carbon, @ = nitrogen, - = oxygen. Thea-C atoms of the amino acid residues are marked 1-4. X = no side chain allowed 95. Table 1.22. ~-Turns in the peptide chain of eggwhite lysozymeResidue Number Sequence20- 23 Y R G Y36- 39 S N F N39- 42 N T Q A47- 50 T D G S54- 57 G I L E60- 63 S R W W66- 69 D G R T69- 72 T P G S74- 77 N L C N85- 88 S S D I100-103 S D G D103-106 D G M N7.4.2.2.4 Super-Secondary StructuresAnalysis of known protein structures hasdemonstrated that regular elements can existin combined forms. Examples are the coiledcoila-helix (Fig. 1.23, a), chain segments withantiparallel p-structures (p-meander structure;Fig. 1.23, b) and combinations of a-helix andP-structure (e.g., papap; Fig. 1.23 c).1.4.2.3 Tertiary and Quaternary StructuresProteins can be divided into two large groupson the basis of conformation: (a) fibrillar(fibrous) or scleroproteins, and (b) folded orglobular proteins.7.4.2.3.7 Fibrous ProteinsThe entire peptide chain is packed or arrangedwithin a single regular structure for a varietyof fibrous proteins. Examples are wool keratin(a-helix), silk fibroin (p-sheef structure) andcollagen (a triple helix). Stabilization ofthesestructures is achieved by intermolecular bonding(electrostatic interaction and disulfidelinkages, but primarily hydrogen bonds andhydrophobic interactions).7.4.2.3.2 Globular ProteinsRegular structural elements are mixed withrandomly extended chain segments (randomlycoiled structures) in globular proteins. Theproportion of regular structural elements is1.4 Proteins 53bFig. 1.23. Superhelix secondary structure (accordingto Schulz and Schirmer, 1979). a coiled-coila-helix, b ~-meander, c ~a~a~-structurehighly variable: 20-30% in casein, 45% inlysozyme and 75 % in myoglobin (Table 1.23).Five structural subgroups are known in thisgroup of proteins: (1) a-helices occur only; (2)p-structures occur only; (3) a-helical and pstructuralportions occur in separate segmentson the peptide chain; (4) a-helix and p-structuresalternate along the peptide chain; and (5)a-helix and p-structures do not exist.The process of peptide chain folding is not yetfully understood. It begins spontaneously, probablyarising from one center or from severalcenters of high stability in larger proteins. Thetendency to form regular structural elementsshows a very different development in thevarious amino acid residues. Table 1.24 listsdata which were derived from the analysis ofglobular proteins of known conformation. Thedata indicate, for example, that Met, Glu, Leuand Ala are strongly helix-forming. Gly andPro on the other hand show a strong helixbreakingtendency. Val, lIe and Leu promotethe formation of pleated-sheet structures, whileAsp, Glu and Pro prevent them. Pro and Glyare important building blocks of turns. Bymeans of such data it is possible to forecast theexpected conformations for a given aminoacid sequence. 96. 54 1 Amino Acids, Peptides, ProteinsTable 1.23. Proportion of "regular structural elements"present in various globular proteinsProtein a-Helix ~-Struc- nGtureMyoglobin 3- 16"20- 3435- 4150- 5658- 7785- 9399-116123-145Lysozyme 5 - 15~-Casein24- 3480- 8588- 9697-101109-12515141-54129199209n %1415772091823173 75111114695763 49ca. 30ca. 20" Position number of the amino acid residue in thesequence.~: Total number of amino acid residues.n: Amino acid residues within the regular structure.%: Percentage of the amino acid residues present inregular structure.Folding of the peptide chain packs it denselyby formation of a large number of intermolecularnoncovalent bonds. Data on thenature of the bonds involved are provided inTable 1.25.The H-bonds formed between main chains,main and side chains and side-side chains areof particular importance for folding. The portionof polar groups involved in H-bond buildupin proteins of Mr> 8.9 kdal appears to befairly constant at about 50%.The hydrophobic interaction of the nonpolarregions of the peptide chains also plays animportant role in protein folding. These interactionsare responsible for the fact that nonpolargroups are folded to a great extent towardsTable 1.24. Normalized frequencies" of amino acidresidues in the regular structural elements of globularproteinsAmino acid a-Helix(Pa )Ala 1.29Cys 1.11Leu 1.30Met 1.47Glu 1.44Gin 1.27His 1.22Lys 1.23Val 0.91Ile 0.97Phe 1.07Tyr 0.72Trp 0.99Thr 0.82Gly 0.56Ser 0.82Asp 1.04Asn 0.90Pro 0.52Arg 0.96Pleated sheet ~-Turn(P~) (Pt )0.90 0.780.74 0.801.02 0.590.97 0.390.75 1.000.80 0.971.08 0.690.77 0.961.49 0.471.45 0.511.32 0.581.25 1.051.14 0.751.21 1.030.92 1.640.95 1.330.72 1.410.76 1.280.64 1.910.99 0.88" Shown is the fraction of an amino acid in a regularstructural element, related to the fraction of allamino acids of the same structural element. P = 1means random distribution; P > 1 means enrichment,P < 1 means depletion. The data are basedon an analysis of 66 protein structures.the interior of the protein globule. The surfaceareas accessible to water molecules have beencalculated for both unfolded and native foldedforms for a number of monomeric proteinswith known conformations. The proportion ofthe accessible surface in the stretched state,which tends to be burried in the interior of theglobule as a result of folding, is a simple linearfunction of the molecular weight (M). Thegain in free energy for the folded surface is10 kJ nm- 2. Therefore, the total hydrophobiccontribution to free energy due to folding is:(1.94)This relation is valid for a range of 6.1 08 ~ M~ 34.409, but appears to be also valid for largermolecules since they often consist of 97. Table 1.25. Bond-types in proteinsType Examples Bondstrength(kJ/mole)Covalentbonds-s-s- ca. -230Electrostatic -COO-H3N+- -21bonds >c=o O=C< +1.3Hydrogen-O-H ... 0N-H ... O=C 95C), theaggregation to which the lower activationenergy corresponds predominates.The values in Table 1.27 determined foractivation entropy also support the above mentionedattribution. In the temperature range of70-90C, LlS# is always positive, which indi-Table 1.27. Denaturation of p-lactoglobulins A andB (P-LG-A, p-LG-B) and of a-lactalbumin (a-LA)Protein n {} E. In (k.,) ~S*COC) (kJ mol-I) (8-1) (kJ mol-1 K-l)~-LG-A 1.5 70- 90 265.21 84.16 0.44595-150 54.07 14.41 -0.136~-LG-B 1.5 70- 90 279.96 89.43 0.48795-150 47.75 12.66 -0.150a-LA 1.0 70- 80 268.56 84.92 0.45285-150 69.01 16.95 -0.115n: reaction order, 6: temperature, E.: activationenergy, k,,: reaction rate constant, ~S*: activationentropy.200 Na+ > Cs+ > Li+ > NH/;S042 - > citrate 2- > tartrate 2- > acetate>Cl- > N03- > Be > J- > CNS-.(1.99)Multivalent anions are more effective thanmonovalent anions, while divalent cations areless effective than monovalent cations.Since proteins are polar substances, they arehydrated in water. The degree of hydration(g water of hydrationlg protein) is variable. Itis 0.22 for ovalbumin (in ammonium sulfate),0.06 for edestin (in ammonium sulfate), 0.8for p-lactoglobulin and 0.3 for hemoglobin.Approximately 300 water molecules are sufficientto cover the surface of lysozyme (about6000 A2), that is one water molecule per20N.The swelling of insoluble proteins correspondsto the hydration of soluble proteins inthat insertion of water between the peptidechains results in an increase in volume andother changes in the physical properties of theprotein. For example, the diameter of myofibrils(cf. 12.2.1) increases to 2.5 times the originalvalue during rinsing with 1.0 mollLNaCl, which corresponds to a six-fold volumeincrease (cf. 12.5). The amount of water takenup by swelling can amount to a multiple of theprotein dry weight. For example, muscle tissuecontains 3.5-3.6 g water per g protein drymatter.The water retention capacity of protein can beestimated with the following formula:(1.100)(a: g waterig protein; fe' fp, fn : fraction ofcharged, polar, neutral amino acid residues).1.4.3.4 Foam Formation and Foam StabilizationIn several foods, proteins function as foamformingand foam-stabilizing components,for example in baked goods, sweets, dessertsand beer. This varies from one protein to 104. 62 1 Amino Acids, Peptides, Proteinsanother. Serum albumin foams very well,while egg albumin does not. Protein mixturessuch as egg white can be particularlywell suited (cf. 11.4.2.2). In that case, theglobulins facilitate foam formation. Ovomucinstabilizes the foam, egg albumin andconalbumin allow its fixation through thermalcoagulation.Foams are dispersions of gases in liquids. Proteinsstabilize by forming flexible, cohesivefilms around the gas bubbles. During impact,the protein is adsorbed at the interface viahydrophobic areas; this is followed by partialunfolding (surface denaturation). The reductionof surface tension caused by proteinadsorption facilitates the formation of newinterfaces and further gas bubbles. The partiallyunfolded proteins associate while formingstabilizing films.The more quickly a protein molecule diffusesinto interfaces and the more easily it is denaturedthere, the more it is able to foam. Thesevalues in turn depend on the molecular mass,the surface hydrophobicity, and the stability ofthe conformation.Foams collapse because large gas bubblesgrow at the expense of smaller bubbles(disproportionation). The protein films counteractthis disproportionation. That is why thestability of a foam depends on the strength ofthe protein film and its permeability for gases.Film strength depends on the adsorbed amountof protein and the ability of the adsorbedmolecules to associate. Surface denaturationgenerally releases additional amino acid sidechains which can enter into intermolecularinteractions. The stronger the cross-linkage,the more stable the film. Since the smallestpossible net charge promotes association, thepH of the system should lie in the range of theisoelectric points of the proteins that participatein film formation.In summary, the ideal foam-forming and foamstabilizingprotein is characterized by a lowmolecular weight, high surface hydrophobicity,good solubility, a small net charge in terms ofthe pH of the food, and easy denaturability.Foams are destroyed by lipids and organic solventssuch as higher alcohols, which due totheir hydrophobicity displace proteins fromthe gas bubble surface without being able toform stable films themselves. Even a low con-centrationof egg yolk, for example, preventsthe bursting of egg white. This is attributed toa disturbance of protein association by thelecithins.The foam-forming and foam-stabilizing characteristicsof proteins can be improved bychemical and physical modification. Thus apartial enzymatic hydrolysis leads to smaller,more quickly diffusing molecules, better solubility,and the release of hydrophobic groups.Disadvantages are the generally lower film stabilityand the loss of thermal coagulability.The characteristics can also be improved byintroducing charged or neutral groups ( cf.1.4.6.2) and by partial thermal denaturation(e.g. of whey proteins). Recently, the additionof strongly alkaline proteins (e. g. clupeines) isbeing tested, which apparently increases theassociation of protein in the films and allowsthe foaming of fatty systems.1.4.3.5 Gel FormationGels are disperse systems of at least two componentsin which the disperse phase in thedispersant forms a cohesive network. They arecharacterized by the lack of fluidity and elasticdeformability. Gels are placed between solutions,in which repulsive forces between moleculesand the disperse phase predominate, andprecipitates, where strong intermolecularinteractions predominate. We differentiate betweentwo types of gel, the polymeric networksand the aggregated dispersions, althoughintermediate forms are found as well.Examples of polymeric networks are the gelsformed by gelatin (cf. 12.3.2.3.1) and polysaccharidessuch as agarose (cf.4.4.4.1.2) andcarrageenan (4.4.4.3.2). Formation of a threedimensionalnetwork takes place through theaggregation of unordered fibrous moleculesvia partly ordered structures, e. g. while doublehelices are formed (cf.4.4.4.3.2, Fig. 4.14,Fig. 12.21). Characteristic for gels ofthis typeis the low polymer concentration (-1 %) aswell as transparency and fine texture. Gel formationis caused by setting a certain pH, byadding certain ions, or by heating/cooling.Since aggregation takes place mostly viaintermolecular hydrogen bonds which easilybreak when heated, polymeric networks arethermo-reversible, i. e. the gels areformed 105. when a solution cools, and they melt againwhen it is heated.Examples of aggregated dispersions are thegels formed by globular proteins after heatingand denaturation. The thermal unfolding of theprotein leads to the release of amino acid sidechains which may enter into intermolecularinteractions. The subsequent association occurswhile small spherical aggregates formwhich combine into linear strands whose interactionestablishes the gel network. Before gelcan be formed in the unordered type of aggregation,a relatively high protein concentration(5 -1 0 %) is necessary. The aggregation rateshould also be slower than the unfolding rate,since otherwise coarse and fairly unstructuredgels are formed, Juch as in the area of the isoelectricpoint. The degree of denaturationnecessary to start aggregation seems todepend on the protein. Since partial denaturationreleases primarily hydrophobic groups,intermolecular hydrophobic bonds generallypredominate, which results in the thermoplastic(thermo-irreversible) character of this geltype, in contrast to the thermoreversible geltype stabilized by hydrogen bonds. Thermoplasticgels do not liquefy when heated, butthey can soften or shrink. In addition tohydrophobic bonds, disulfide bonds formedfrom released thiol groups can also contributeto cross-linkage, as can intermolecular ionicbonds between proteins with different isoelectricpoints in heterogeneous systems (e. g. eggwhite).Gel formation can be improved by adding salt.The moderate increase in ionic strengthincreases interaction between charged macromoleculesor molecule aggregates throughcharge shielding without precipitation occurring.An example is the heat coagulation ofsoybean curd (tofu, cf. 16.3.l.2.3) which ispromoted by calcium ions.1.4.3.6 Emulsifying EffectEmulsions are disperse systems of one or moreimmiscible liquids. They are stabilized byemulsifiers - compounds which form interfacefilms and thus prevent the disperse phasesfrom flowing together (cf. 8.15). Due to theiramphipathic nature, proteins can stabilize o/wemulsions such as milk (cf. 10.l.2.3). This1.4 Proteins 63property is made use of on a large scale in theproduction of food preparations.The adsorption of a protein at the interface ofan oil droplet is thermodynamically favoredbecause the hydrophobic amino acid residuescan then escape the hydrogen bridge networkof the surrounding water molecules. In addition,contact of the protein with the oil dropletresults in the displacement of water moleculesfrom the hydrophobic regions of the oil-waterboundary layer. Therefore, the suitability of aprotein as an emulsifier depends on the rate atwhich it diffuses into the interface and on thedeformability of its conformation under theinfluence of interfacial tension (surface denaturation).The diffusion rate depends on thetemperature and the molecular weight, whichin tum can be influenced by the pH and theionic strength. The adsorbability depends onthe exposure of hydrophilic and hydrophobicgroups and thus on the amino acid profile, aswell as on the pH, the ion strength and the temperature.The conformative stability dependsin the amino acid composition, the molecularweight and the intramolecular disulfide bonds.Therefore, a protein with ideal qualities as anemulsifier for an oil-in-water emulsion wouldhave a relatively low molecular weight, abalanced amino acid composition in terms ofcharged, polar and nonpolar residues, goodwater solubility, well-developed surfacehydrophobicity, and a relatively stable conformation.The J3-casein molecule meets theserequirements because of less pronouncedsecondary structures and no crosslinks due tothe lack of SH groups (cf. 10.l.2.1.1). Theapolar ''tail'' ofthis flexible molecule is adsorbedby the oil phase of the boundary layer andthe polar "head", which projects into theaqueous medium, prevents coalescence.The solubility and emulsifying capacity ofsome proteins can be improved by limitedenzymatic hydrolysis.1.4.4 Chemical ReactionsThe chemical modification of protein is ofimportance for a number of reasons. It providesderivatives suitable for sequence analysis,identifies the reactive groups in catalyticallyactive sites of an enzyme, enables thebinding of protein to a carrier (protein immo- 106. 64 1 Amino Acids, Peptides, Proteinsbilization) and provides changes in proteinproperties which are important in food processing.In contrast to free amino acids andexcept for the relatively small number offunctionalgroups on the terminal amino acids,only the functional groups on protein sidechains are available for chemical reactions.1.4.4.1 Lysine ResidueReactions involving the lysine residue can bedivided into several groups: (a) reactionsleading to a positively charged derivative; (b)reactions eliminating the positive charge; (c)derivatizations introducing a negative charge;and (d) reversible reactions. The latter are ofparticular importance.1.4.4.1.1 Readions Which Retain the PositiveChargeAlkylation of the free amino group of lysinewith aldehydes and ketones is possible, with asimultaneous reduction step:Prot-NH2 + R-CO-R'NaBH" pH 9. 0 ac. 30 min Prot-NH-CHR/R'(R= R' = CH3; R = H. CH3, R' = H)(1.101)A dimethyl derivative [Prot-N(CH3)2J can beobtained with formaldehyde (R=Rj=H) (cf.1.2.4.2.2).Guanidination can be accomplished by usingO-methylisourea as a reactant. a-Aminogroups react at a much slower rate than -aminogroups:NHProt-NH2 + H3C-o-C.r'NH2NH$pH 10.6, 4 ac, 4 days II 2-------.... Prot-NH-C-NH2(1.102)This reaction is used analytically to assess theamount of biologically available -aminogroups and for measuring protein digestibility.Derivatization with imido esters is also possible.The reactant is readily accessible fromthe corresponding nitriles:NH$ 1/ 2Prot-NH2 ------.-.pH 9.2, 0 ac, 20 hProt-NH-CR-CNNH$R'OH II 2--------.-. R-CH$ OR'R(1.103)Proteins can be cross-linked with the use of abifunctional imido ester (cf. 1.4.4.10).Treatment of the amino acid residue withamino acid carboxyanhydrides yields a polycondensationreaction product:oProtein-NH2 + O~RyNHo (1.104)Protein-N H-[CO-CH R-NH]n-CO-CH R-NH2The value n depends on reaction conditions.The carboxyanhydrides are readily accessiblethrough interaction of the amino acid withphosgene:R-CH-COOHICOCI2 R-?H-COOHNH2 NH-Co-CI- HCI(1.105)1.4.4.1.2 Reactions Resulting in a Lossof Positive ChargeAcetic anhydride reacts with lysine, cysteine,histidine, serine, threonine and tyrosine residues.Subsequent treatment of the protein withhydroxylamine (1 M, 2 h, pH 9, ODC) leavesonly the acetylated amino groups intact:Prot-NH2(CH3CO),OpH 7-9.5, oac Prot-NH-CO-CH3(1.106)Carbamoylation with cyanate attacks a- and aminogroups as well as cysteine and tyrosineresidues. However, their derivatization isreversible under alkaline conditions: 107. fH2 KOCNProt-NH2 --------+. Prot-NH-C~o pHS, 37C, 12-24h(Ll07)Arylation with l-fluoro-2,4-dinitrobenzene(Sanger s reagent; FDNB) and trinitrobenzenesulfonic acid was outlined in Section 1.2.4.2.2.FDNB also reacts with cysteine, histidine andtyrosine.4-Fluoro-3-nitrobenzene sulfonic acid, a reactantwhich has good solubility in water, is alsoof interest for derivatization of proteins:Prot-NH2 + :{}SO'" (1.1 08)02NRO pH 6-9, 25C -------+. prot-NH-y-SO~Deamination can be accomplished with nitrousacid:HN02Prot-NH2 -----+.pH 4.35, DOCProt-OH + N2(1.109)This reaction involves (X- and E-amino groupsas well as tryptophan, tyrosine, cysteine andmethionine residues.1.4.4.1.3 Reactions Resulting in a NegativeChargeAcylation with dicarboxylic acid anhydrides,e. g. succinic acid anhydride, introduces a carboxylgroup into the protein:~OHNH2SH o ~o - co - (CH2)2 -COOHoNH - co - (CH2)2-COOHpH 8-9 S - co - (CH2)2 -COOH~OHNH - co - (CH2)2 - COOHSH (1.110)1.4 Proteins 65Introduction of a fluorescent acid group is possibleby interaction of the protein with pyridoxalphosphate followed by reduction of theintermediary Schiff base:(1.111)NaSH.pH 6, 25C1.4.4.1.4 Reversible ReactionsN -Maleyl derivatives of proteins are obtainedat alkaline pH by reaction with maleic acidanhydride. The pH < 5, regenerating 09 acylated product is cleaved atthe protein:pH>7Prot-NH2 + 0 h 1-opH arsenite andphosphite> alkanethiol > aminoalkanethiol >thiophenol and cyanide> sulfite> OH- > pnitrophenol> thiosulfate > thiocyanate.Cleavage with sodium borohydride and withthiols was covered in Section 1.2.4.3.5. Completecleavage with sulfite requires that oxidativeagents (e. g. Cu2+) be present and that thepH be higher than 7:RSSR + so~e __ RSSO~ + RSeRSSR (1.123)The resultant S-sulfo derivative is quite stablein neutral and acidic media and is fairlysoluble in water. The S-sulfo group can beeliminated with an excess ofthiol reagent.Cleavage of cystine residues with cyanides(nitriles) is of interest since the thiocyanateformed in the reaction is cyclized to a2-iminothiazolidine derivative with cleavageof the N-acyl bond:1.4 Proteins 67R-CO-NH-~H-Co-NH-R'CH2R"-S-S"""R-Co-NH-CH-Co-NH-R'/bH2 + R"-S9-- NC-S R-co-7--~H-Co-NH-R'C CH's""" -- HN-7 2 HN--~b H-Co-NH-R'R-COOH + CHHN-7 's""" 2 (1.124)This reaction can be utilized for the selectivecleavage of peptide chains. Initially, all thedisulfide bridges are reduced with dithiothreitol,and then are converted to mixed disulfidesthrough reaction with 5,5' -dithio-bis-(2-nitrobenzoicacid). These mixed disulfides are thencleaved by cyanide at pH 7.Electrophilic cleavage occurs with Ag+ andHg+ or Hg2+ as follows:2 Ag$ + 2 RSSR ---+ 2 RSAg + 2 RS$2 RS$ + 20H9 ---+ 2 RSOH ---+ RS02H + RSHRSH + Ag$ ---+ RSAg + H3Ag$ +2RSSR+20H9 ---+ 3 RSAg + RSo,H+H$(1.125)Electrophilic cleavage with H+ is possible onlyin strong acids (e.g. 10 mol/L HCI). The sulfeniumcation which is formed can catalyze adisulfide exchange reaction:RS + R'SSR' ---+ RSSR' + R'S(i>(1.126)In neutral and alkaline solutions a disulfideexchange reaction is catalyzed by the thiolateanion:RSSR R-SOH + RS9R'SSR' + RS9 R'SSR + R'Se(1.127) 110. 68 1 Amino Acids, Peptides, Proteins1.4.4.5 Cysteine Residue (d. also Section1.2.4.3.5)A number of alkylating agents yield derivativeswhich are stable under the conditions foracidic hydrolysis of proteins. The reaction withethylene imine giving an S-aminoethyl derivativeand, hence, an additional linkage positionin the protein for hydrolysis by trypsin, wasmentioned in Section 1.4.1.3. Iodoacetic acid,depending on the pH, can react with cysteine,methionine, lysine and histidine residues:Protein-SHICH,COOH----+. Protein-S-CH,-COOH(1.128)The introduction of methyl groups is possiblewith methyl iodide or methyl isourea, and theintroduction of methylthio groups withmethylthiosulfonylmethane:Protem. -SH --C--H--3-I'- -+ Pr ote. m-S-CH3/NH2Protein-SH + CH:r-O--C~~NH?(1.129)(1.131)--+. Protein-8-S-CH3 + CH3S03HMaleic acid anhydride and methyl-p-nitrobenzenesulfonate are also alkylating agents:Protein-S-~H-COOHCH2-COOH(1.132)Protein-SH + CH30S02-V-N02---+ Protein-S-CH3 (1.133)A number of reagents make it possible tomeasure the thiol group content spectrophotometrically.The molar absorption coefficient, E,for the derivative of azobenzene-2-sulfenylbromide,E353 , is 16,700 M-i cm-i at pH 1:' .... ;~SH + ~=N-O~'.w;~6-~lO(1.134)5,5' -Dithiobis-(2-nitrobenzoic acid) has asomewhat lower E412 of 13,600 at pH 8 for itsproduct, a thionitrobenzoate anion:o,-HOOC o-.+ONO,COOH;~SH ' .... + COOH COOH- , .... ;~S-S-oNo' + H~No,(1.135)The derivative of p-hydroxymercuribenzoatehas an E250 of 7,500 at pH 7, while the derivativeofN-ethylmaleic imide has an E300 of 620at pH 7:Protein-SH + X-H~COOH---+ protein-S-H~COOHo, .... ;'-SH + qN-C,H,oo-+ protein-s'QN-C2Hs~o(1.136)(1.137) 111. Especially suitable for the specific isolation ofcysteine-containing peptides of great sensitivityis N-dimethylaminoazobenzenemaleicacid imide (DABMA).oProtein - SH + 90 %,e. g. a soya protein isolate) is suspended inwater and solubilized by the addition of alkali.The 20 % solution is then aged at pH 11 withconstant stirring. The viscosity rises duringthis time as the protein unfolds. The solution isthen pressed through the orifices of a die(5,000-15,000 orifices, each with a diameterof 0.01-0.08 mm) into a coagulating bath atpH 2-3. This bath contains an acid (citric,acetic, phosphoric, lactic or hydrochloric)and, usually, 10% NaCl. Spinning solutions ofprotein and acidic polysaccharide mixturesalso contain earth alkali salts. The proteinfibers are extended further (to about 2- to 4-times the original length) in a "winding up"step and are bundled into thicker fibers withdiameters of 10-20 mm. The molecular interactionsare enhanced during stretching of thefiber, thus increasing the mechanical strengthof the fiber bundles.The adherent solvent is then removed by pressingthe fibers between rollers, then placingthem in a neutralizing bath (NaHC03+NaCI)of pH 5.5-6 and, occasionally, also in a hardeningbath (conc. NaCl).The fiber bundles may be combined into largeraggregates with diameters of 7 -1 0 cm.Additional treatment involves passage of thebundles through a bath containing a binder andother additives (a protein which coagulateswhen heated, such as egg protein; modifiedstarch or other polysaccharides; aroma compounds;lipids). This treatment producesbundles with improved thermal stability andaroma. A typical bath for fibers which are to beprocessed into a meat analogue might consistof 51 % water, 15% ovalbumin, 10% wheatgluten, 8 % soya flour, 7 % onion powder, 2 %protein hydrolysate, 1 % NaCl, 0.15% monosodiumglutamate and 0.5 % pigments.Finally, the soaked fiber bundles are heatedand chopped.7.4.7.3.2 Extrusion ProcessThe moisture content of the starting material(protein content about 50%, e. g., soya flour) isadjusted to 30-40 % and additives (NaCl, buffers,aroma compounds, pigments) are incorporated.Aroma compounds are added in fat asa carrier, when necessary, after the extrusionstep to compensate for aroma losses. The proteinmixture is fed into the extruder (a thermostaticallycontrolled cylinder or conicalbody which contains a polished, rotating screwwith a gradually decreasing pitch) which isheated to 120-180C and develops a pressureof30-40 bar. Under these conditions the mixtureis transformed into a plastic, viscous statein which solids are dispersed in the moltenprotein. Hydration of the protein takes placeafter partial unfolding of the globular moleculesand stretching and rearrangement of theprotein strands along the direction of masstransfer.The process is affected by the rotation rate andshape ofthe screw and by the heat transfer andviscosity of the extruded material and its residencetime in the extruder.As the molten material exits from the extruder,the water vaporizes, leaving behind vacuolesin the ramified protein strands.The extrusion process is more economicalthan the spin process. However, it yields fiberlikeparticles rather than well-defined fibers. Agreat number and variety of extruders are nowin operation. As with other food processes,there is a trend toward developing and utilizinghigh-temperature/short-time extrusion cooking.1.S LiteratureAeschbach, R., Amado, R., Neukom, H.: Formationof dityrosine cross-links in proteins by oxidationof tyrosine residues. Biochim. Biophys. Acta 439,292 (1976) 131. 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BioI. 105,571 (1978)Bosin, T.R, Krogh, S., Mais, D.: Identification andquantitation of 1 ,2,3,4-Tetrahydro-~-carboline-3-carboxylic acid and I-methyl-1,2,3,4-tetrahydro~-carboline-3-carboxylic acid in beer and wine. 1.Agric. Food Chern. 34, 843 (1986)Bott, RR, Davies, D.R.: Pepstatin binding to Rhizopuschinensis aspartyl proteinase. In: Peptides:Structure and function (Eds.: Hruby, v.1., Rich,D.H.), p. 531. Pierce Chemical Co.: Rockford, III.1983Brussel, L.B.P., Peer, H.G., van der Heijden, A.:Structure-taste relationship of some sweet-tastingdipeptide esters. Z. Lebensm. Unters. Forsch.159,337 (1975)Chen, C., Pearson, A.M., Gray, 1.1.: Meat Mutagens.Adv. Food Nutr. Res. 34, 387, (1990)Cherry, 1.P. (Ed.): Protein functionality in foods.ACS Symposium Series 147, American ChemicalSociety: Washington, D.C. 1981Creighton, T.E.: Proteins: structures and molecularproperties. WHo Freeman and Co.: New York.1983Croft, L.R: Introduction to protein sequence analysis,2nd edn., John Wiley and Sons, Inc.: Chichester.1980Dagleish, D.G.: Adsorptions of protein and the stabilityof emulsions. Trends Food Sci. Technol. 8, 1(1997)Dickinson, E.: Towards more natural emulsifiers.Trends Food Sci. Technol. 4, 330 (1993)Einsele, A.: Biomass from higher n-alkanes. In: Biotechnology(Eds.: Rehm, H.-1., Reed, G.), Vol. 3,p. 43, Verlag Chemie: Weinheim. 1983Faust, u., Prave. P.: Biomass from methane and methanol.In: Biotechnology (Eds.: Rehm, H.-1.,Reed, G.), Vol. 3, p. 83, Verlag Chemie: Weinheim.19831.5 Literature 89Felton, 1.S., Pais, P., Salmon, C.P., Knize, M.G.:Chemical analysis and significance of heterocyclicaromatic amines. Z. Lebensm. Unters. Forsch.A 207,434 (1998)Finot, P.-A., Mottu, E, Bujard, E., Mauron, 1.:N-substituted lysines as sources of lysine innutrition. Adv. Exp. Med. 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Applied Science Publ.: London.1982Ikenaka, T., Odani, S., Koide, T.: Chemical structureand inhibitory activities of soybean proteinaseinhibitors. Bayer-Symposium V "Proteinase inhibitors",p. 325, Springer-Verlag: Berlin-Heidelberg.1974.Jagerstad, M., Skog, K., Arvidson, P., Solyakov, A.:Chemistry, formation and occurrence of genotoxicheterocyclic arnines identified in modelsystems and cooked foods. Z. Lebensm. Unters.ForschA 207, 419 (1998)Kasai, R., Yamaizumi, Z., Shiomi, T., Yokoyama, S.,Miyazawa, T., Wakabayashi, K., Nagao, M., Sugimura,T., Nishimura, S.: Structure of a potentmutagen isolated from fried beef. Chern. Lett.1981,485Kasai, H., Yamaizumi, Z., Wakabayashi, K., Nagao,M., Sugimura, T., Yokoyama, S., Miyazawa, T.,Spingarn, N.E., Weisberger, 1.H., Nishimura, S.:Potent novel mutagens produced by broiling fishunder normal conditions. Proc. Jpn. Acad. Ser.B56, 278 (1980)Kessler, H.G.: Lebensmittel- und Bioverfahrenstechnik;Molkereitechnologie. 3rd edn., Verlag A.Kessler: Freising, 1988Kinsella, J. E., Shetty, K. 1.: Yeast proteins: Recoveryand functional properties. Adv. Exp. Med. BioI.105,797 (1978) 132. 90 1 Amino Acids, Peptides, ProteinsKinsella, 1 E.: Functional properties of proteins infoods: A survey. Crit. Rev. Food Sci. Nutr. 7,219(1976)Kinsella, 1 E.: Texturized proteins: Fabrication, flavoring,and nutrition. Crit. Rev. Food Sci. Nutr.10, 147 (1978)Kleemann, A., Leuchtenberger, W., Hoppe, B., Tanner,H.: Amino acids. In: Ullmann's encyclopediaof industrial chemistry, 5th Edition, Volume A2, p.57, Verlag VCA, Weinheim, 1986Klostermeier, H., et al.: Proteins. In: Ullmann'sencyclopedia of industrial chemistry, 5th Edition,Volume A22, p. 289, Verlag VCH, Weinheim,1993Kostka, V. 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Chern. 43, 1065(1979)Yamashita, M., Arai, S., Tsai, S.-1., Fujimaki, M.:Plastein reaction as a method for enhancingthe sulfur-containing amino acid level ofsoybean protein. 1. Agric. Food Chern. 19, 1151(1971 )Yamashita, M., Arai, S., Kokubo, S., Aso, K., Fujimaki,M.: Synthesis and characterization of aglutamic acid enriched plastein with greater solubility.1. Agric. Food Chern. 23, 27 (1975)Yoshikawa, M., Tamaki, M., Sugimoto, E., Chiba,H.: Effect of dephosphorylation on the self-associationand the precipitation of ~-Casein. Agric.BioI. Chern. 38, 2051 (1974) 134. 2 Enzymes2.1 ForewordEnzymes are proteins with powerful catalyticactivity. They are synthesized by biologicalcells and in all organisms, they are involvedin chemical reactions related to metabolism.Therefore, enzyme-catalyzed reactions alsoproceed in many foods and thus enhance ordeteriorate food quality. Relevant to thisphenomenon are the ripening of fruits andvegetables, the aging of meat and dairy products,and the processing steps involved in themaking of dough from wheat or rye flours andthe production of alcoholic beverages byfermentation technology.Enzyme inactivation or changes in the distributionpatterns of enzymes in subcellular particlesof a tissue can occur during storage orthermal treatment of food. Since such changesare readily detected by analytical means, enzymesoften serve as suitable indicators forrevealing such treatment of food. Examplesare the detection of pasteurization of milk,beer or honey, and differentiation betweenfresh and deep frozen meat or fish.Enzyme properties are of interest to the foodchemist since enzymes are available in increasingnumbers for enzymatic food analysisor for utilization in industrial food processing.Examples of both aspects of their use are providedin this chapter in section 2.6.4 on foodanalysis and in section 2.7, which covers foodprocessing.Details of enzymes which playa role in foodscience are restricted in this chapter to onlythose enzyme properties which are able to providean insight into the build-up or functionalityof enzymes or can contribute to the understandingof enzyme utilization in food analysisor food processing and storage.2.2 General Remarks, Isolationand Nomenclature2.2.1 CatalysisLet us consider the catalysis of an exergonicreaction:(2.1)with a most frequently occurring case in whichthe reaction does not proceed spontaneously.Reactant A is metastable, since the activationenergy, EA , required to reach the activatedtransition state in which chemical bonds areformed or cleaved in order to yield product P,is exceptionally high (Fig. 2.1).The reaction is accelerated by the addition of asuitable catalyst. It transforms reactant A intointermediary products (EA and EP in Fig.2.1), the transition states of which are at alower energy level than the transition state ofa noncatalyzed reaction (A' in Fig. 2.1). Themolecules of the species A contain enough1 >OlWcllJpReaction coordinateFig. 2.1. Energy profile of an exergonic reactionA ~ P; - without and --- with catalyst E 135. 2.2 General Remarks, Isolation and Nomenclature 93Table 2.1. Examples of catalyst activityReaction Catalyst Activation k,." (25C)energyI. H,O, ~ H,O + 112 0, AbsentIe2. Casein + n H,O ~(n + 1) Peptides3. Ethylbutyrate+ H,O ~ butyric acid+ ethanol4. Saccharose + H,O ~Glucose + Fructose5. Linoleic acid+ 0, ~ Linoleic acidhydroperoxideCatalaseHTrypsinHLipaseHInvertaseAbsentCu'+Lipoxy-genase(kJ'mol-')7556.526.886505517.610746150--27030-5016.71.0-2.1,103- 3.5 . 1081.0- 2.1 . 1061.0- 4.2' 1061.0- 5.6' 10101.0- 10'- 107energy. to combine with the catalyst and, thus,to attam the "activated state" and to form orb~eak the. covalen~ bond that is necessary togIve the mtermedlary product which is thenreleased as product P along with free, unchangedcatalyst. The reaction rate constants , k +1and k_1 , are therefore increased in the presenceof a catalyst. However, the equilibirum con~tant of the reaction, i.e. the ratio kl+Ik_1 = K,IS not altered.Activation energy levels for several reactionsand the corresponding decreases of these energylevels in the presence of chemical or enzymaticcatalysts are provided in Table 2.1.Changes in their reaction rates are also given.In ~ontrast to reactions 1 and 5 (Table 2.1)WhICh proceed at measurable rates even in theabsence of catalysts, hydrolysis reactions 2, 3and 4 occur only in the presence of protons ascatalysts. However, all reaction rates observedin the case of inorganic catalysts are increasedby a f~ctor of at least several orders of magnitudem the presence of suitable enzymes.Because of the powerful activity of enzymestheir presence at levels of 10-8 to 10-6 molll i~sufficient for in vitro experiments. However,the enzyme concentrations found in livingcells are often substantially higher.2.2.2 SpecificityIn addition to an enzyme's ability to substantiallyincrease reaction rates, there is a uniqueenzyme property related to its high specificityfor both the compound to be converted (substratespecificity) and for the type of reactionto be catalysed (reaction specificity).The activities of allosteric enzymes ( cf.2.5.1.3) are affected by specific regulators oreffectors. Th~~, the activities of such enzymesshow an additIOnal regulatory specificity.2.2.2.1 Substrate SpecificityThe substrate specificity of enzymes showsthe following differences. The occurrence of adistinct functional group in the substrate is theonly prerequisite for a few enzymes, such assom~ hY. H-C-OH3 Lactate racemase IU+I - Lactic acid Ox CH3~tate L-0olotp DI-I-Lacticacid;,~:~~te-rn~ transhYdrnalote CH,COCOOHrogenaseFig. 2.2. Examples of reaction specificity of someenzymesOf the four enzymes considered, only the lactateracemase reacts with either of the enantiomersoflactic acid, yielding a racemic mixture.Therefore, enzyme reaction specificity ratherthan substrate specificity is considered as abasis for enzyme classification and nomenclature(cf. 2.2.6).2.2.3 StructureEnzymes are globular proteins with greatlydiffering particle sizes (cf. Table 1.26). As outlinedin section 1.4.2, the protein structure isdetermined by its amino acid sequences andby its conformation, both secondary and tertiary,derived from this sequence. Larger enzymemolecules often consist of two or morepeptide chains (subunits or protomers, cf.Table 1.26) arranged into a specified quaternarystructure (cf. 1.4.2.3). Section 2.4.1 willshow that the three dimensional shape of theenzyme molecule is actually responsible for itsspecificity and its effective role as a catalyst.On the other hand, the protein nature of theenzyme restricts its activity to a relatively narrowpH range (for pH optima, cf. 2.5.3) andheat treatment leads readily to loss of activityby denaturation (cf. 1.4.2.4 and 2.5.4.4).Some enzymes are complexes consisting of aprotein moiety bound firmly to a nonproteincomponent which is involved in catalysis, e. g.a "prosthetic" group (cf. 2.3.2). The activitiesof other enzymes require the presence of acosubstrate which is reversibly bou