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Handbook of meat processing

  1. 1. Handbook ofMeat Processing
  2. 2. Handbook of Meat ProcessingFidel ToldráEDITOR A John Wiley & Sons, Inc., Publication
  3. 3. Edition first published 2010© 2010 Blackwell PublishingBlackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishingprogram has been merged with Wiley’s global Scientific, Technical, and Medical business to formWiley-Blackwell.Editorial Office2121 State Avenue, Ames, Iowa 50014-8300, USAFor details of our global editorial offices, for customer services, and for information about how to applyfor permission to reuse the copyright material in this book, please see our website at www.wiley.com/wiley-blackwell.Authorization to photocopy items for internal or personal use, or the internal or personal use of specificclients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the CopyrightClearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have beengranted a photocopy license by CCC, a separate system of payments has been arranged. The fee codesfor users of the Transactional Reporting Service are ISBN-13: 978-0-8138-2182-5/2010.Designations used by companies to distinguish their products are often claimed as trademarks. All brandnames and product names used in this book are trade names, service marks, trademarks or registeredtrademarks of their respective owners. The publisher is not associated with any product or vendor men-tioned in this book. This publication is designed to provide accurate and authoritative information inregard to the subject matter covered. It is sold on the understanding that the publisher is not engaged inrendering professional services. If professional advice or other expert assistance is required, the servicesof a competent professional should be sought.Library of Congress Cataloging-in-Publication DataHandbook of meat processing / edited by Fidel Toldrá. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-2182-5 (hardback : alk. paper) 1. Meat—Handbooks, manuals, etc. 2. Meatindustry and trade—Handbooks, manuals, etc. I. Toldrá, Fidel. TS1960.H36 2010 664′.9—dc22 2009037503A catalog record for this book is available from the U.S. Library of Congress.Set in 10 on 12 pt Times by Toppan Best-set Premedia LimitedPrinted in SingaporeDisclaimerThe publisher and the author make no representations or warranties with respect to the accuracy orcompleteness of the contents of this work and specifically disclaim all warranties, including withoutlimitation warranties of fitness for a particular purpose. No warranty may be created or extended by salesor promotional materials. The advice and strategies contained herein may not be suitable for every situ-ation. This work is sold with the understanding that the publisher is not engaged in rendering legal,accounting, or other professional services. If professional assistance is required, the services of a com-petent professional person should be sought. Neither the publisher nor the author shall be liable fordamages arising herefrom. The fact that an organization or Website is referred to in this work as a cita-tion and/or a potential source of further information does not mean that the author or the publisherendorses the information the organization or Website may provide or recommendations it may make.Further, readers should be aware that Internet Websites listed in this work may have changed or disap-peared between when this work was written and when it is read.1 2010
  4. 4. ContentsPreface ixList of Contributors xiAbout the Editor xvPART I. Technologies 3 1. Chemistry and Biochemistry of Meat 5 Elisabeth Huff-Lonergan 2. Technological Quality of Meat for Processing 25 Susan Brewer 3. Meat Decontamination 43 Panagiotis N. Skandamis, George-John E. Nychas, and John N. Sofos 4. Aging/Tenderization Mechanisms 87 Brian C. Bowker, Janet S. Eastridge, Ernie W. Paroczay, Janice A. Callahan, and Morse B. Solomon 5. Freezing/Thawing 105 Christian James and Stephen J. James 6. Curing 125 Karl O. Honikel 7. Emulsification 143 Irene Allais 8. Thermal Processing 169 Jane Ann Boles 9. Fermentation: Microbiology and Biochemistry 185 Spiros Paramithiotis, Eleftherios H. Drosinos, John N. Sofos, and George-John E. Nychas10. Starter Cultures for Meat Fermentation 199 Pier Sandro Cocconcelli and Cecilia Fontana11. Drying 219 Endre Zukál and Kálmán Incze v
  5. 5. vi Contents12. Smoking 231 Zdzisław E. Sikorski and Edward Kolakowski ´13. Meat Packaging 247 Maurice G. O’Sullivan and Joseph P. Kerry14. Novel Technologies for Microbial Spoilage Prevention 263 Oleksandr Tokarskyy and Douglas L. Marshall15. Plant Cleaning and Sanitation 287 Stefania QuintavallaPART II. Products 29916. Cooked Ham 301 Fidel Toldrá, Leticia Mora, and Mónica Flores17. Cooked Sausages 313 Eero Puolanne18. Bacon 327 Peter R. Sheard19. Canned Products and Pâté 337 Isabel Guerrero Legarreta20. Dry-Cured Ham 351 Fidel Toldrá and M. Concepción Aristoy21. Mold-Ripened Sausages 363 Kálmán Incze22. Semidry and Dry Fermented Sausages 379 Graciela Vignolo, Cecilia Fontana, and Silvina Fadda23. Restructured Whole-Tissue Meats 399 Mustafa M. Farouk24. Functional Meat Products 423 Keizo Arihara and Motoko OhataPART III. Controls 44125. Physical Sensors for Quality Control during Processing 443 Marta Castro-Giráldez, Pedro José Fito, Fidel Toldrá, and Pedro Fito26. Sensory Evaluation of Meat Products 457 Geoffrey R. Nute27. Detection of Chemical Hazards 469 Milagro Reig and Fidel Toldrá28. Microbial Hazards in Foods: Food-Borne Infections and Intoxications 481 Daniel Y. C. Fung
  6. 6. Contents vii29. Assessment of Genetically Modified Organisms (GMO) in Meat Products by PCR 501 Marta Hernández, Alejandro Ferrando, and David Rodríguez-Lázaro30. HACCP: Hazard Analysis Critical Control Point 519 Maria João Fraqueza and António Salvador Barreto31. Quality Assurance 547 Friedrich-Karl LückeIndex 561
  7. 7. PrefaceFor centuries, meat and its derived products worldwide meat products such as cookedhave constituted some of the most important ham and sausages, bacon, canned productsfoods consumed in many countries around and pâté, dry-cured ham, mold-ripened sau-the world. Despite this important role, there sages, semidry and dry fermented sausages,are few books dealing with meat and its restructured meats, and functional meat prod-processing technologies. This book provides ucts. The third part presents efficient strate-the reader with an extensive description of gies to control the sensory and safety qualitymeat processing, giving the latest advances of meat and meat products, including physi-in technologies, manufacturing processes, cal sensors, sensory evaluation, chemicaland tools for the effective control of safety and microbial hazards, detection of GMOs,and quality during processing. HACCP, and quality assurance. To achieve this goal, the book contains 31 The chapters have been written by distin-chapters distributed in three parts. The first guished international experts from fifteenpart deals with the description of meat chem- countries. The editor wishes to thank all theistry, its quality for further processing, contributors for their hard work and forand the main technologies used in meat sharing their valuable experience, as well asprocessing, such as decontamination, aging, to thank the production team at Wiley-freezing, curing, emulsification, thermal pro- Blackwell. I also want to express my appre-cessing, fermentation, starter cultures, drying, ciation to Ms. Susan Engelken for her kindsmoking, packaging, novel technologies, support and coordination of this book.and cleaning. The second part describes themanufacture and main characteristics of Fidel Toldrá ix
  8. 8. ContributorsIrene Allais Susan BrewerCemagref, UMR Genial, Equipe Automat Food Science and Human Nutrition,& Qualite Alimentaire, 24 Av Landais, University of Illinois, USA.F-63172 Aubiere 1, France. E-mail: msbrewer@illinois.eduE-mail: irene.allais@cemagref.fr Janice A. CallahanKeizo Arihara Food Technology and Safety Laboratory,Department of Animal Science, Kitasato Bldg 201, BARC-East, Beltsville, MarylandUniversity, Towada-shi, Aomori 034-8628, 20705, USA.Japan. E-mail: Janice.callahan@ars.usda.govE-mail: arihara@vmas.kitasato-u.ac.jp Marta Castro-GiráldezM. Concepción Aristoy Institute of Food Engineering forDepartment of Food Science, Instituto de Development, Universidad Politécnica deAgroquímica y Tecnología de Alimentos Valencia, Camino de Vera s/n, 46022(CSIC), PO Box 73, 46100 Burjassot Valencia, Spain.(Valencia), Spain.E-mail: mcaristoy@iata.csic.es Pier Sandro Cocconcelli Istituto di Microbiologia, Centro RicercheAntónio Salvador Barreto Biotecnologiche, Università Cattolica delFaculdade de Medicina Veterinária, Sacro Cuore, Piacenza-Cremona, Italy.DPASA, TULisbon, Av. da Universidade E-mail: pier.cocconcelli@unicatt.itTecnica, Polo Universitário, Alto da Ajuda,1300-477 Lisboa, Portugal. Eleftherios H. Drosinos Laboratory of Food Quality Control andJane Ann Boles Hygiene, Department of Food Science andAnimal and Range Sciences, 119 Technology, Agricultural University ofLinfield Hall, Bozeman, Montana Athens, Iera Odos 75, Votanikos, 1185559717, USA. Athens, Greece.E-mail: jboles@montana.edu E-mail: ehd@aua.grBrian C. Bowker Janet S. EastridgeFood Technology and Safety Laboratory, Food Technology and Safety Laboratory,Bldg 201, BARC-East, Beltsville, Bldg 201, BARC-East, Beltsville, MarylandMaryland 20705, USA. 20705, USA.E-mail: brian.bowker@ars.usda.gov E-mail: janet.eastridge@ars.usda.gov xi
  9. 9. xii ContributorsSilvina Fadda Maria João FraquezaCentro de Referencia para Lactobacilos Faculdade de Medicina Veterinária,(CERELA), CONICET., Chacabuco 145, DPASA, TULisbon, Av. da UniversidadeT4000ILC Tucumán, Argentina. Tecnica, Polo Universitário, Alto da Ajuda,E-mail: fadda@cerela.org.ar 1300-477 Lisboa, Portugal. E-mail: mjoaofraqueza@fmv.utl.ptMustafa M. FaroukAgResearch MIRINZ, Ruakura Research Daniel Y. C. FungCentre, East Street, Private Bag 3123, Department of Animal Sciences andHamilton 3240, New Zealand. Industry, 207 Call Hall, Kansas StateE-mail: mustafa.farouk@agresearch.co.nz University, Manhattan, Kansas 66506, USA. E-mail: dfung@ksu.eduAlejandro FerrandoDepartamento de Bioquímica y BiologíaMolecular, Facultad de Biología, Isabel Guerrero LegarretaUniversidad de Valencia, Dr Moliner, 50, Departamento de Biotecnología,Burjassot, 46100 Valencia, Spain. Universidad Autónoma, Metropolitana, Unidad Iztapalapa, San Rafael Atlixco 186, Del. Iztapalapa, Apartado Postal 55-535,Pedro Fito C.P. 092340, Mexico City.Institute of Food Engineering for E-mail: meat@xanum.uam.mxDevelopment, Universidad Politécnica deValencia, Camino de Vera s/n, 46022Valencia, Spain. Marta HernándezE-mail: pfito@tal.upv.es Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de Castilla y León (ITACyL), Ctra.Pedro José Fito Burgos km.119, Finca Zamadueñas, 47071Institute of Food Engineering for Valladolid, Spain.Development, Universidad Politécnica deValencia, Camino de Vera s/n, 46022Valencia, Spain. Karl O. HonikelE-mail: pjfito@tal.upv.es Max Rubner-Institut, Arbeitsgruppe Analytik, Kulmbach, Germany. E-mail: karl-otto.honikel@t-online.deMónica FloresDepartment of Food Science, Instituto deAgroquímica y Tecnología de Alimentos Elisabeth Huff-Lonergan(CSIC), PO Box 73, 46100 Burjassot, Muscle Biology, Department of AnimalValencia, Spain. Science, Iowa State University, 2275 KildeeE-mail: mflores@iata.csic.es Hall, Ames, IA 50011 USA. E-mail: elonerga@iastate.eduCecilia FontanaCentro de Referencia para Lactobacilos Kálmán Incze(CERELA), CONICET., Chacabuco 145, Hungarian Meat Research Institute, 1097T4000ILC Tucumán, Argentina. Budapest, Gubacsi út 6/b, Hungary.E-mail: cecilia.fontana@unicatt.it E-mail: ohki@interware.hu
  10. 10. Contributors xiiiChristian James Douglas L. MarshallFood Refrigeration and Process Engineering College of Natural and Health Sciences,Research Centre (FRPERC), The Grimsby University of Northern Colorado,Institute of Further and Higher Campus Box 134, Greeley, ColoradoEducation(GIFHE), HSI Building, Origin 80639 USA.Way, Europarc, Grimsby, North East E-mail: douglas.marshall@unco.eduLincolnshire, DN37 9TZ UK.E-mail: JamesC@grimsby.ac.uk Leticia Mora Department of Food Science, Instituto de Agroquímica y Tecnología de AlimentosStephen J. James (CSIC), PO Box 73, 46100 BurjassotFood Refrigeration and Process Engineering Valencia, Spain.Research Centre (FRPERC), The Grimsby E-mail: lemoso@iata.csic.esInstitute of Further and HigherEducation(GIFHE), HSI Building, OriginWay, Europarc, Grimsby, North East Geoffrey R. NuteLincolnshire, DN37 9TZ UK. University of Bristol, School of ClinicalE-mail: jamess@grimsby.ac.uk Veterinary Science, Division of Farm Animal Science, Bristol BS40 5DU, Avon, England.Joseph P. Kerry E-mail: Geoff.Nute@bristol.ac.ukDepartment of Food and NutritionalSciences, University College Cork, Ireland. George-John E. NychasE-mail: Joe.Kerry@ucc.ie Laboratory of Food Microbiology & Biotechnology, Department of FoodEdward Kołakowski Science & Technology, AgriculturalDepartment of Food Science and University of Athens, Iera Odos 75, AthensTechnology, Agricultural University of 11855, Greece.Szczecin, Papie a Pawła VI St. 3, 71-459 E-mail: gjn@aua.grSzczecin, Poland.E-mail: ekolakowski@tz.ar.szczecin.pl Motoko Ohata Department of Animal Science, Kitasato University, Towada-shi, Aomori 034-8628,Catherine M. Logue Japan.Department of Veterinary andMicrobiological Sciences, North Dakota Maurice G. O’SullivanState University, 1523 Centennial Blvd, Department of Food and Nutritional130A Van Es Hall, Fargo, North Dakota Sciences, University College Cork, Ireland.58105, USA. E-mail: maurice.osullivan@ucc.ieE-mail: Catherine.Logue@ndsu.edu Spiros ParamithiotisFriedrich-Karl Lücke Laboratory of Food Quality Control andHochschule Fulda (University of Applied Hygiene, Department of Food Science andSciences), P.O. Box 2254, 36012 Fulda, Technology, Agricultural University ofGermany. Athens, Iera Odos 75, 11855 Athens,E-mail: friedrich-karl.luecke@t-online.de Greece.
  11. 11. xiv ContributorsErnie W. Paroczay Panagiotis N. SkandamisFood Technology and Safety Laboratory, Laboratory of Food Quality Control andBldg 201, BARC-East, Beltsville, Hygiene, Department of Food Science andMaryland 20705, USA. Technology, Agricultural University ofE-mail: ernie.paroczay@ars.usda.gov Athens, Iera Odos 75, Votanikos, 11855 Athens, Greece.Eero PuolanneDepartment of Food Technology, Viikki John N. SofosEE, P.O. Box 66, 00014 Helsinki, Finland. Colorado State University, Fort Collins,E-mail: Eero.Puolanne@helsinki.fi Colorado 80523, USA. E-mail: John.Sofos@ColoState.EDUStefania QuintavallaDepartment of Microbiology, SSICA, V.leTanara 31/A, 43100, Parma, Italy. Morse B. SolomonE-mail address: stefania.quintavalla@ssica.it Food Technology and Safety Laboratory, Bldg 201, BARC-East, Beltsville, MarylandMilagro Reig 20705, USA.Institute of Food Engineering for E-mail: Morse.Solomon@ARS.USDA.GOVDevelopment, Universidad Politécnica deValencia, Camino de Vera s/n, 46022 Oleksandr TokarskyyValencia, Spain. Department of Food Science, Nutrition, andE-mail: mareirie@doctor.upv.es Health Promotion, Mississippi State University, Box 9805, Mississippi StateDavid Rodríguez-Lázaro University, Mississippi 39762 USA.Food Safety and Technology Group,Instituto Tecnológico Agrario de Castilla y Fidel ToldráLeón (ITACyL), Ctra. Burgos km.119, Department of Food Science, Instituto deFinca Zamadueñas, 47071 Valladolid, Agroquímica y Tecnología de AlimentosSpain. (CSIC), PO Box 73, 46100 Burjassot,E-mail: ita-rodlazda@itacyl.es Valencia, Spain. E-mail: ftoldra@iata.csic.esPeter R. SheardDivision of Farm Animal Science, Schoolof Clinical Veterinary Science, University Graciela Vignoloof Bristol, Bristol BS40 5DU, Avon, UK. Centro de Referencia para LactobacilosE-mail: Peter.Sheard@bristol.ac.uk (CERELA), CONICET., Chacabuco 145, T4000ILC Tucumán, Argentina.Zdzisław E. Sikorski E-mail: vignolo@cerela.org.arDepartment of Food Chemistry, Gdansk ´University of Technology Endre ZukálE-mail: sikorski@chem.pg.gda.pl OR Hungarian Meat Research Institute,zdzsikor@pg.gda.pl Budapest 1097, Gubacsi út 6/b, Hungary.
  12. 12. About the EditorFidel Toldrá, Ph.D., is a research professor at years, including Handbook of Musclethe Department of Food Science, Instituto de Foods Analysis and Handbook of ProcessedAgroquímica y Tecnología de Alimentos Meats and Poultry Analysis (2009), Meat(CSIC), and serves as European editor of Biotechnology and Safety of Meat andTrends in Food Science & Technology, editor Processed Meat (2008, 2009), Handbook ofin chief of Current Nutrition & Food Science, Food Product Manufacturing (2007),and as section editor of the Journal of Muscle Advances in Food Diagnostics, and HandbookFoods. He is also serving on the editorial of Fermented Meat and Poultry (2007, 2008).board of the journals Food Chemistry, Meat Professor Toldrá also wrote the book Dry-Science, Open Nutrition Journal, Food Cured Meat Products (2002).Analytical Methods, Open Enzyme Inhibition Professor Toldrá was awarded the 2002Journal and Journal of Food and Nutrition International Prize for meat science and tech-Research. He is a member of the European nology by the International Meat SecretariatFood Safety Authority panel on flavorings, and was elected in 2008 as Fellow of theenzymes, processing aids, and materials in International Academy of Food Science &contact with foods. Technology (IAFOST) and in 2009 as Professor Toldrá has acted as editor or Fellow of the Institute of Food Technologistsassociate editor of several books in recent (IFT). xv
  13. 13. Handbook ofMeat Processing
  14. 14. Part ITechnologies
  15. 15. Chapter 1Chemistry and Biochemistry of MeatElisabeth Huff-LonerganIntroduction content is 75% of the weight of the muscle; however, can vary, particularly in postmor-Muscle cells are among the most highly orga- tem muscle (range of 65–80%). Within thenized cells in the animal body and perform a muscle, it is the primary component of extra-varied array of mechanical functions. They cellular fluid. Within the muscle cell, waterare required for the movement of limbs, is the primary component of sarcoplasmicfor locomotion and other gross movements, (cytoplasmic) fluid. It is important in thermo-and they must also perform finer tasks regulation; as a medium for many cellularsuch as maintaining balance and coordina- processes; and for transport of nutrientstion. Muscle movement and metabolism within the cell, between cells, and betweenare associated with other diverse functions the muscle and the vascular system.such as aiding in movement of blood and The second largest component of musclelymph and also in maintaining body tempera- is protein (U.S. Department of Agricultureture. All of these functions are dependent 2008). Protein makes up an average of 18.5%on cellular metabolism and the ability of the of the weight of the muscle, though thatcell to maintain energy supplies. Few cells figure can range from 16 to 22%. Proteinsare required to generate as much force and serve myriad functions and are the primaryundergo as dramatic shifts in rate of metabo- solid component in muscle. The functions oflism as muscle cells. The ability of living proteins are quite varied. Muscle proteins areskeletal muscle to undergo relatively large involved in maintaining the structure andintracellular changes also influences its organization of the muscle and muscle cellsresponse to the drastic alterations that occur (the role of highly insoluble stromal pro-during the first few hours following exsan- teins). They are also important in the contrac-guination. Thus the organization, structure, tile process. These proteins primarily areand metabolism of the muscle are key to its associated with the contractile organelles, thefunction and to the maintenance of its integ- myofibril, and are thus termed myofibrillarrity both during contraction and during the proteins. In general, the myofibrillar proteinsearly postmortem period. Ultimately, these are not soluble at low ionic strengths foundpostmortem changes will influence the suit- in skeletal muscle (ionic strength ≤0.15), butability of meat for further processing. can be solubilized at higher ionic strengths (≥0.3). This class of proteins includes both the proteins directly involved in movementMuscle Composition (contractile proteins) and proteins that regu-The largest constituent of muscle is water late the interactions between the contractile(Table 1.1; U.S. Department of Agriculture proteins (regulatory proteins). There are also2008). In living tissue, the average water many soluble proteins (sarcoplasmic pro- 5
  16. 16. 6 Chapter 1Table 1.1. Composition of Mammalian Muscle complex lipid found in muscle. In this class Component % of Muscle Weight of lipids, one of the hydroxyl groups of glyc- Water 75% (65–80%) erol is esterified to a phosphate group, while Protein 18.5% (16–22%) the other constituents are fatty acids. The Lipid 3% (1–13%) fatty acids associated with phospholipids are Carbohydrate 1% (0.5–1.5%) Non-Protein Nitrogenous 1.7% (1–2%) typically unsaturated. Phospholipids in skel- Substances etal muscle are commonly associated with Other Non-Protein 0.85% (0.5–1%) membranes. The relative high degree of Substances (minerals, vitamins, etc.) unsaturation of the fatty acids associated with the phospholipids is a contributing factor toNumbers in parentheses indicate the average range ofthat component.(U.S. Department of Agriculture, 2008) the fluidity of the cell membranes. Carbohydrates make up a relatively small percentage of muscle tissue, making up about 1% of the total muscle weight (range of 0.5–teins) that include proteins involved in cel- 1.5%). The carbohydrate that makes up thelular signaling processes and enzymes largest percentage is glycogen. Other carbo-important in metabolism and protein degra- hydrates include glucose, intermediates ofdation/cellular remodeling. glycogen metabolism, and other mono- and The lipid content of the muscle can vary disaccharides. Glycosoaminoglycans are alsogreatly due to many factors, including animal found in muscle and are associated with theage, nutritional level of the animal, and connective tissue.muscle type. It is important to note that the There are numerous non-protein nitroge-lipid content varies inversely with the water nous compounds in skeletal muscle. Theycontent (Callow 1948). Some lipid is stored include substances such as creatine and cre-inside the muscle cell; however, within a atine phosphate, nucleotides (ATP, ADP),muscle, the bulk of the lipid is found between free amino acids, peptides (anserine, carno-muscle bundles (groupings of muscle cells). sine), and other non-protein substances.Average lipid content of skeletal muscle isabout 3% of the muscle weight, but the range Muscle Structurecan be as much as 1–13% (U.S. Departmentof Agriculture 2008). In skeletal muscle, Skeletal muscle has a very complex organi-lipid plays roles in energy storage, membrane zation, in part to allow muscle to efficientlystructure, and in various other processes in transmit force originating in the myofibrils tothe organ, including immune responses and the entire muscle and ultimately, to the limbcellular recognition pathways. or structure that is moved. A relatively thick The two major types of lipid found in sheath of connective tissue, the epimysium,skeletal muscle are triglycerides and phos- encloses the entire muscle. In most muscles,pholipids. Triglycerides make up the greatest the epimysium is continuous, with tendonsproportion of lipid associated with muscle. that link muscles to bones. The muscle isTriglycerides (triacylglycerides) consist of a subdivided into bundles or groupings ofglycerol molecule in which the hydroxyl muscle cells. These bundles (also known asgroups are esterified with three fatty acids. fasciculi) are surrounded by another sheathThe melting point and the iodine number of of connective tissue, the perimysium. A thinlipid that is associated with the muscle is layer of connective tissue, the endomysium,determined by the chain length and the degree surrounds the muscle cells themselves. Theof saturation of the fatty acids. Phospholipids endomysium lies above the muscle cell mem-(phosphoglycerides) are another type of brane (sarcolemma) and consists of a base-
  17. 17. Chemistry and Biochemistry of Meat 7ment membrane that is associated with an basis, they make up approximately 10–12%outer layer (reticular layer) that is surrounded of the total weight of fresh skeletal muscle.by a layer of fine collagen fibrils imbedded Therefore, they are very important in meatin a matrix (Bailey and Light 1989). chemistry and in determining the functional- Skeletal muscles are highly diverse, in ity of meat proteins.part because of the diversity of actions they Myofibrils are the contractile “machinery”are asked to perform. Much of this diversity of the cell and, like the cells where theyoccurs not only at the gross level, but also at reside, are very highly organized. Whenthe muscle cell (fiber) level. First, not only examining a myofibril, one of the first obser-do muscles vary in size, they can also vary vations that can be made is that the cylindri-in the number of cells. For example, the cal organelle is made up of repeating units.muscle that is responsible for adjusting the These repeating units are known as sarco-tension of the eardrum (tensor tympani) meres. Contained in each sarcomere are allhas only a few hundred muscle cells, while the structural elements needed to perform thethe medial gastrocnemius (used in humans physical act of contraction at the molecularfor walking) has over a million muscle cells level. Current proteomic analysis estimates(Feinstein et al. 1955). Not only does the that over 65 proteins make up the structurenumber of cells influence muscle function of the sarcomere (Fraterman et al. 2007).and ultimately, meat quality, but also the Given that the sarcomere is the most basicstructure of the muscle cells themselves unit of the cell and that the number quoted inhas a profound effect on the function of this analysis did not take into account theliving muscle and on the functionality of multiple isoforms of the proteins, this numbermeat. is quite high. Many of the proteins interact Muscle cells are striated, meaning that with each other in a highly coordinatedwhen viewed under a polarized light micro- fashion, and some of the interactions are justscope, distinct banding patterns or striations now being discovered.are observed. This appearance is due to spe- The structure of the sarcomere is respon-cialized organelles, myofibrils, found in sible for the striated appearance of the musclemuscle cells. The myofibrils have a striated, cell. The striations arise from the alternating,or banded, appearance because different protein dense A-bands and less dense I-bandsregions have different refractive properties. within the myofibril. Bisecting the I-bandsThe light bands have a consistent index of are dark lines known as Z-lines. The structurerefraction (isotropic). Therefore, these bands between two Z-lines is the sarcomere. In aare called I-bands in reference to this isotro- relaxed muscle cell, the distance betweenpic property. The dark band appears dark two Z-lines (and thus the length of the sarco-because it is anisotropic and is thus called the mere) is approximately 2.2 μm. A singleA-band. myofibril is made up of a large number of The myofibrils are abundant in skeletal sarcomeres in series. The length of the myo-muscle cells, making up nearly 80–90% of fibril and also the muscle cell is dependentthe volume of the cell. Myofibrillar proteins on the number of sarcomeres. For example,are relatively insoluble at physiological ionic the semitendinosus, a long muscle, has beenstrength, requiring an ionic strength greater estimated to have somewhere in the neigh-than 0.3 to be extracted from muscle. For this borhood of 5.8 × 104 to 6.6 × 104 sarcomeresreason, they are often referred to as “salt- per muscle fiber, while the soleus has beensoluble” proteins. Myofibrillar proteins make estimated to have approximately 1.4 × 104up approximately 50–60% of the total extract- (Wickiewicz et al. 1983). Adjacent myofi-able muscle proteins. On a whole muscle brils are attached to each other at the Z-line
  18. 18. 8 Chapter 1by proteinacious filaments, known as inter- each) and two sets of light chains (14,000–mediate filaments. Outermost myofibrils are 20,000 daltons). One of the light chains isattached to the cell membrane (sarcolemma) required for enzymatic activity, and the otherby intermediate filaments that interact not has regulatory functions.only with the Z-line, but also with structures Actin is the second-most abundant proteinat the sarcolemma known as costameres in the myofibril, accounting for approxi-(Robson et al. 2004). mately 20% of the total protein in the myo- Myofibrils are made up of many myofila- fibril. Actin is a globular protein (G-actin)ments, of which there are two major types, that polymerizes to form filaments (F-actin).classified as thick and thin filaments. There G-actin has a molecular weight of approxi-is also a third filament system composed pri- mately 42,000. There are approximatelymarily of the protein titin (Wang et al. 1979; 400 actin molecules per thin filament. ThusWang 1984; Wang et al. 1984; Wang and the molecular weight of each thin filamentWright 1988; Wang et al. 1991; Ma et al. is approximately 1.7 × 107 (Squire 1981).2006;). With respect to contraction and rigor The thin filaments (F-actin polymers) aredevelopment in postmortem muscle, it is the 1 μm in length and are anchored in theinterdigitating thick and thin filaments that Z-line.supply the “machinery” needed for these pro- Two other proteins that are important incesses and give skeletal muscle cells their muscle contraction and are associated withcharacteristic appearance (Squire 1981). the thin filament are tropomyosin and tropo-Within the myofibril, the less dense I-band is nin. Tropomyosin is the second-most abun-made up primarily of thin filaments, while dant protein in the thin filament and makesthe A-band is made up of thick filaments and up about 7% of the total myofibrillar protein.some overlapping thin filaments (Goll et al. Tropomyosin is made up of two polypeptide1984). The backbone of the thin filaments is chains (alpha and beta) The alpha chain hasmade up primarily of the protein actin, while an approximate molecular weight of 34,000,the largest component of the thick filament is and the beta chain has a molecular weight ofthe protein myosin. Together, these two pro- approximately 36,000. These two chainsteins make up nearly 70% of the proteins in interact with each other to form a helix. Thethe myofibril of the skeletal muscle cell. native tropomyosin molecule interacts with Myosin is the most abundant myofibrillar the troponin molecule to regulate contrac-protein in skeletal muscle, making up approx- tion. Native troponin is a complex that con-imately 50% of the total protein in this organ- sists of three subunits. These are termedelle. Myosin is a negatively charged protein troponin I (MW 23,000), troponin C (MWwith an isoelectric point of 5.3. Myosin is 18,000), and troponin T (MW 37,000).a large protein (approximately 500,000 Troponin C has the ability to bind calciumdaltons) that contains six polypeptides. released from the sarcoplasmic reticulum,Myosin consists of an alpha helical tail (or troponin I can inhibit the interaction betweenrod) region that forms the backbone of the actin and myosin, and troponin T binds verythick filament and a globular head region that strongly to tropomyosin. The cooperativeextends from the thick filament and interacts action of troponin and tropomyosin inwith actin in the thin filament. The head response to calcium increases in the sarco-region of myosin also has ATPase activity, plasm regulates the interaction between actinwhich is important in the regulation of con- and myosin and thus is a major regulator oftraction. Each myosin molecule contains two contraction. Calcium that is released from theheavy chains (approximately 220,000 daltons sarcoplasmic reticulum is bound to the tropo-
  19. 19. Chemistry and Biochemistry of Meat 9nin complex and the resulting conformational Central to the existence of the muscle cellchanges within troponin cause tropomyosin is the production of adenosine triphosphateto move away from sites on actin to which (ATP), the energy currency of the cell. ATPmyosin binds and allows myosin and actin to consists of adenosine (an adenine ring and ainteract. ribose sugar) and three phosphate groups (tri- For contraction to occur, the thick and thin phosphate). Cleavage of the bonds betweenfilaments interact via the head region of the phosphates (Pi) and the rest of the mole-myosin. The complex formed by the interac- cule provides energy for many cellular func-tion of myosin and actin is often referred tions, including muscle contraction and theto as actomyosin. In electron micrograph control of the concentrations of key ions (likeimages of contracted muscle or of postrigor calcium) in the muscle cell. Cleavage of Pimuscle, the actomyosin looks very much like from ATP produces adenosine diphosphatecross-bridges between the thick and thin fila- (ADP), and cleavage of pyorphosphate (PPi)ments; indeed, it is often referred to as such. from ATP produces adenosine monophos-In postmortem muscle, these bonds are irre- phate (AMP). Since the availability of ATPversible and are also known as rigor bonds, is central to survival of the cell, there is aas they are the genesis of the stiffness (rigor) highly coordinated effort by the cell to main-that develops in postmortem muscle. The tain its production in both living tissue andglobular head of myosin also has enzymatic in the very early postmortem period.activity; it can hydrolyze ATP and liberate Muscular activity is dependent on ampleenergy. In living muscle during contraction, supplies of ATP within the muscle. Since itthe ATPase activity of myosin provides is so vital, muscle cells have developedenergy for myosin bound to actin to swivel several ways of producing/regenerating ATP.and ultimately pull the thin filaments toward Muscle can use energy precursors stored inthe center of the sarcomere. This produces the muscle cell, such as glycogen, lipids, andcontraction by shortening the myofibril, the phosphagens (phosphocreatine, ATP), and itmuscle cell, and eventually, the muscle. The can use energy sources recruited from themyosin and actin can disassociate when a blood stream (blood glucose and circulatingnew molecule of ATP is bound to the myosin lipids). Which of these reserves (intracellularhead (Goll et al. 1984). In postrigor muscle, or circulating) the muscle cell uses dependsthe supply of ATP is depleted, resulting in on the activity the muscle is undergoing.the actomyosin bonds becoming essentially When the activity is of lower intensity, thepermanent. muscle will utilize a higher proportion of energy sources from the blood stream and lipid stored in the muscle cell. These will beMuscle Metabolism metabolized to produce ATP using aerobicFrom a metabolic point of view, energy use pathways. Obviously, ample oxygen isand production in skeletal muscle is simply required for this process to proceed. Duringnothing short of amazing in its range and high intensity activity, during which ATP isresponsiveness. In an actively exercising used very rapidly, the muscle uses intracel-animal, muscle can account for as much as lular stores of phosphagens or glycogen.90% of the oxygen consumption in the body. These two sources, however, are utilizedThis can represent an increase in the mus- very quickly and their depletion leads tocle’s metabolic rate of as much as 200% from fatigue. This is not a trivial point.the resting state (Hargreaves and Thompson Concentration of ATP in skeletal muscle is1999). critical; available ATP must remain above
  20. 20. 10 Chapter 1approximately 30% of the resting stores, or with ATP (100 mmol/kg dry muscle weightrelaxation cannot occur. This is because for phosphocreatine compared with 25 mmol/relaxation of contraction is dependent on kg dry muscle weight for ATP) but very lowATP, which is especially important because abundance compared with glycogen (500removal of calcium from the sarcoplasm is mmol/kg dry muscle weight for glycogen).an ATP-dependent process (Hargreaves and Phosphocreatine can easily transfer a phos-Thompson 1999). phate group to ADP in a reaction catalyzed The primary fuels for muscle cells include by creatine kinase. This reaction is easilyphosphocreatine, glycogen, glucose lactate, reversible and phosphocreatine suppliesfree fatty acids, and triglycerides. Glucose can be readily restored when ATP demandand glycogen are the preferred substrates for is low. In living muscle, when activity ismuscle metabolism and can be utilized either intense, this system can be advantageous, asaerobically (oxidative phosphorylation) or it consumes H+ and thus can reduce theanaerobically (anaearobic glycolysis). Lipid muscle cell acidosis that is associated withand lactate utilization require oxygen. Lipids anaerobic glycolysis. Another advantage ofare a very energy-dense storage system and the system is that the catalyzing enzyme isare very efficient with respect to the high located very close to the actomyosin ATPaseamount of ATP that can be generated per unit and also at the sarcoplasmic reticulum (whereof substrate. However, the rate of synthesis calcium is actively taken up from the sarco-of ATP is much slower than when glycogen plasm to regulate contraction) and at the sar-is used (1.5 mmol/kg/sec for free fatty acids colemma. However, this system is not acompared with 3 mmol/kg/sec for glycogen major contributor to postmortem metabo-utilized aerobically and 5 mmol/kg/sec when lism, as the supplies are depleted fairlyglycogen is used in anaerobic glycolysis) rapidly.(Joanisse 2004). In general, glycogen is the preferred Aerobic metabolism, the most efficient substrate for the generation of ATP, eitherenergy system, requires oxygen to operate, through the oxidative phosphorylation orand that oxygen is supplied by the blood through anaerobic glycolysis (Fig. 1.1). Onesupply to the muscle and by the oxygen trans- of the key steps in the fate of glycogen isporter, myoglobin. It has been estimated that whether or not an intermediate to the process,in working muscle, the myoglobin is some- pyruvate, enters the mitochondria to bewhere in the neighborhood of 50% saturated. completely broken down to CO2 and H2OUnder conditions of extreme hypoxia (as (yielding 38 mol of ATP per mole of oxidizedfound in postmortem muscle), oxygen sup- glucose-1-P produced from glycogen orplies are depleted because blood flow is not 36 mol if the initial substrate is glucose),sufficient (or does not exist), and myoglobin or if it ends in lactate via the anaerobic gly-oxygen reserves are depleted if this state con- colysis pathway. The anaerobic pathway,tinues long enough. Prior to exsanguination, while comparatively less efficient (yieldingthe oxidation of glycogen or other substrates 3 mol of ATP per mole of glucose-1-P pro-to form water and carbon dioxide via oxida- duced from glycogen or 2 mol if the initialtive phosphorylation is a very efficient way substrate is glucose), is much better at pro-for the cell to regenerate ATP. However, ducing ATP at a higher rate. Early postmor-after exsanguination, the muscle cell must tem muscle obviously uses the anaerobicturn solely to anaerobic pathways for energy pathway, as oxygen supplies are rapidlyproduction. depleted. This results in the buildup of the Phosphocreatine in living, rested muscle end product, lactate (lactic acid), resulting inis available in moderate abundance compared pH decline.
  21. 21. Chemistry and Biochemistry of Meat 11Figure 1.1. ATP production in muscle.Major Postmortem Changes to be between 2 and 2.5 μM in length. In stri-in Muscle ated muscle, titin thus spans fully half of a sarcomere, with its C-terminal end localizingTenderization in the M-line at the center of the sarcomereDuring refrigerated storage, it is well known and the N-terminal forming an integral partthat meat becomes more tender. It is com- of the Z-line. Titin aids in maintaining sarco-monly accepted that the product becomes meric alignment of the myofibril during con-more tender because of proteolytic changes traction. Titin integrates the Z-line and theoccurring in the architecture of the myofibril thick filaments, maintaining the location ofand its associated proteins. There are several the thick filaments between the Z-lines. Titinkey proteins that are degraded during post- is also hypothesized to play a role in generat-mortem aging. ing at least a portion of the passive tension that is present in skeletal muscle cells. During development of the myofibril, titin is one ofTitin the earliest proteins expressed, and it isTitin (aka connectin) is a megaprotein that is thought to act as a “molecular ruler” by pro-approximately 3 megadaltons in size. In viding a scaffolding or template for theaddition to being the largest protein found in developing myofibril (Clark et al. 2002).mammalian tissues, it is also the third-most Due to the aforementioned roles of titinabundant. A single titin molecule is estimated in living cells, it is quite conceivable that
  22. 22. 12 Chapter 1its degradation in postmortem muscle would extends from the Z-line to the pointed endslead to weakening of the longitudinal struc- of the thin filament. The C-terminal end ofture of the myofibrillar sarcomere and integ- nebulin is embedded into the Z-line. Nebulinrity of muscle. This weakening, in conjunction is highly nonextensible and has been referredwith other changes in postmortem muscle, to as a molecular ruler that during develop-could lead to enhanced tenderness. The deg- ment may serve to define the length of theradation of titin has been observed in several thin filaments (Kruger et al. 1991). Nebulin,studies (Lusby et al. 1983; Zeece et al. 1986; via its intimate association with the thin fila-Astier et al. 1993; Huff-Lonergan et al. 1995; ment (Lukoyanova et al. 2002), has beenMelody et al. 2004; Rowe et al. 2004a, b). hypothesized to constitute part of a compos-When titin is degraded, a major degradation ite nebulin/thin filament (Pfuhl et al. 1994;product, termed T2, is observed that migrates Robson et al. 1995) and may aid in anchoringonly slightly faster under SDS-PAGE con- the thin filament to the Z-line (Wang andditions than intact titin. This product migrates Wright 1988; Komiyama et al. 1992).at approximately 2,400 kDa (Kurzban and Degradation of nebulin postmortem couldWang 1988, 1987; Huff-Lonergan et al. weaken the thin filament linkages at the1995). Another titin degradation product Z-line, and/or of the thin filaments in thethat has been observed by SDS-PAGE an- nearby I-band regions (Taylor et al. 1995),alysis migrates at approximately 1,200 kDa and thereby weaken the structure of the(Matsuura et al. 1991; Huff-Lonergan et al. muscle cell. Nebulin has also been shown to1995). This latter polypeptide has been be capable of linking actin and myosin (Rootshown to contain the portion of titin that and Wang 1994a, b). It has been hypothe-extends from the Z-line to near the N2 line sized that nebulin may also have a regulatoryin the I-band (Kimura et al. 1992), although function in skeletal muscle contraction (Rootthe exact position that the 1200 kDa polypep- and Wang 1994a, b; Bang et al. 2006).tide reaches in the sarcomere is still not Portions of nebulin that span the A-I junctioncertain. The 1,200-kDa polypeptide has been have the ability to bind to actin, myosin, anddocumented to appear earlier postmortem in calmodulin (Root and Wang 2001). Moremyofibrils from aged beef that had lower interesting, this portion of nebulin (spanningshear force (and more desirable tenderness the A-I junction) has been shown to inhibitscores) than in samples from product that had actomyosin ATPase activity (Root and Wang,higher shear force and/or less favorable ten- 2001; Lukoyanova et al. 2002). This regionderness scores (Huff-Lonergan et al. 1995, of nebulin also has been suggested to inhibit1996a, b). The T2 polypeptide can also be the sliding velocities of actin filaments oversubsequently degraded or altered during myosin. If the latter role is confirmed, then itnormal postmortem aging. Studies that have is also possible that nebulin’s postmortemused antibodies against titin have been shown degradation may alter actin-myosin interac-to cease to recognize T2 after prolonged tions in such a way that the alignment andperiods of postmortem storage or μ-calpain interactions of thick and thin filaments indigestion (Ho et al. 1994; Huff-Lonergan postmortem muscle is disrupted. This, too,et al. 1996a) could lead to an increase in postmortem ten- derization. Nebulin degradation does seem to be correlated to postmortem tenderization,Nebulin although the exact cause-and-effect relation-Nebulin is another mega-protein (Mr 600– ship remains to be substantiated (Huff-900 kDa) in the sarcomere. This protein Lonergan et al. 1995; Taylor et al. 1995;
  23. 23. Chemistry and Biochemistry of Meat 13Huff-Lonergan et al. 1996a; Melody et al. related to the shear force (Penny 1976; Huff-2004). Lonergan et al. 1996b; Huff-Lonergan and Lonergan, 1999; Lonergan et al. 2001; Rowe et al. 2003; Rowe et al. 2004a). Troponin-TTroponin-T is a substrate for μ-calpain, and it is hypoth-For many years it has been recognized that esized that μ-calpain is at least partly respon-the degradation of troponin-T and the appear- sible for the postmortem degradation ofance of polypeptides migrating at approxi- troponin-T and the concomitant productionmately 30 kDa are strongly related to, or of the 28- and 30-kDa polypeptides.correlated with, the tenderness of beef (Penny Degradation of troponin-T may simply be anet al. 1974; MacBride and Parrish 1977; indicator of overall postmortem proteolysisOlson and Parrish 1977; Olson et al. 1977). (i.e., it occurs as meat becomes more tender).It has been shown that purified bovine tropo- However, because troponin-T is an integralnin-T can be degraded by μ-calpain in vitro part of skeletal muscle thin filaments (Greaserto produce polypeptides in the 30-kDa region and Gergely 1971), its role in postmortem(Olson et al. 1977). In addition, polypeptides tenderization may warrant more carefulin the 30-kDa region found in aged bovine examination as has been suggested (Ho et al.muscle specifically have been shown to be 1994; Uytterhaegen et al. 1994; Taylor et al.products of troponin-T by using Western 1995; Huff-Lonergan et al. 1996b). Indeed,blotting techniques (Ho et al. 1994). Often, the troponin-T subunit makes up the elon-more than one fragment of troponin-T can be gated portion of the troponin molecule andidentified in postmortem muscle. Increasing through its interaction with tropomyosin aidspostmortem time has been shown to be asso- in regulating the thin filament during skeletalciated with the appearance of two major muscle contraction (Greaser and Gergelybands (each is likely a closely spaced doublet 1971; Hitchcock 1975; McKay et al. 1997;of polypeptides) of approximately 30 and Lehman et al. 2001). It is conceivable that28 kDa, which label with monoclonal anti- postmortem degradation of troponin-T andbodies to troponin-T (Huff-Lonergan et al. disruption of its interactions with other thin1996a). In addition, the increasing postmor- filament proteins aids in the disruption of thetem aging time was also associated with a thin filaments in the I-band, possibly leadingloss of troponin-T, as has been reported in to fragmentation of the myofibril and overallnumerous studies (Olson et al. 1977; muscle integrity. During postmortem aging,Koohmaraie et al. 1984a, b; Ho et al. 1994). the myofibrils in postmortem bovine muscleIt has recently been shown that troponin-T is are broken in the I-band region (Taylor et al.cleaved in its glutamic acid-rich amino-ter- 1995). Because troponin-T is part of the reg-minal region (Muroya et al. 2007). Some ulatory complex that mediates actin-myosinstudies have shown labeling of two very interactions (Greaser and Gergely, 1971;closely spaced bands corresponding to intact Hitchcock, 1975; McKay et al. 1997; Lehmantroponin-T. This is likely due to isoforms of et al. 2001), it is also conceivable that itstroponin-T that are known to exist in skeletal postmortem degradation may lead to changesmuscle (Briggs et al. 1990; Malhotra 1994; involving thick and thin filament interac-Muroya et al. 2007), including specifically tions. Regardless of whether or not troponin-bovine skeletal muscle (Muroya et al. 2007). -T aids in disruption of the thin filament inBoth the appearance of the 30- and 28-kDa the I-band, alters thick and thin filamentbands and the disappearance of the intact interactions, or simply reflects overall proteintroponin-T in the myofibril are very strongly degradation, its degradation and appearance
  24. 24. 14 Chapter 1of polypeptides in the 30-kDa region seem to myofibrils (Huff-Lonergan et al. 1996a;be a valuable indicator of beef tenderness Huff-Lonergan and Lonergan, 1999; Carlin(Olson et al. 1977; Olson and Parrish, 1977; et al. 2006). Thus, the proteolytic enzymeKoohmaraie et al. 1984a, b; Koohmaraie μ-calpain may be, at least in part, responsible1992; Huff-Lonergan et al. 1995; Huff- for desmin degradation under normal post-Lonergan et al. 1996a; Huff-Lonergan and mortem aging conditions. Whether or not thisLonergan 1999). degradation is truly directly linked to tender- ization or is simply an indicator of overall postmortem proteolysis remains to beDesmin determined.It has been suggested that desmin, an inter-mediate filament protein (O’Shea et al. 1979; FilaminRobson 1989) localized at the periphery ofthe myofibrillar Z-disk in skeletal muscle Filamin is a large (Mr = 245,000 in skeletal(Richardson et al. 1981), plays a role in the and cardiac muscle) actin-binding proteindevelopment of tenderness (Taylor et al. that exists in numerous cell types (Loo et al.1995; Huff-Lonergan et al. 1996a; Boehm et 1998; Thompson et al. 2000; van der Flier etal. 1998; Melody et al. 2004). The desmin al. 2002). There are several different iso-intermediate filaments surround the Z-lines forms of filamin (Hock et al. 1990). Theof myofibrils. They connect adjacent myofi- amount of filamin in skeletal and cardiacbrils at the level of their Z-lines, and the muscle is very low (approximately ≤0.1% ofmyofibrils to other cellular structures, includ- the total muscle protein). In skeletal anding the sarcolemma (Robson, 1989; Robson cardiac muscle, filamin is localized at theet al. 1995). Desmin may be important in periphery of the myofibrillar Z-disk, and itmaintaining the structural integrity of muscle may be associated with intermediate fila-cells (Robson et al. 1981, 1991). It is possible ments in these regions (Loo et al. 1998;that degradation of structural elements that Thompson et al. 2000; van der Flier et al.connect the major components (i.e., the myo- 2002). Thus, postmortem degradation offibrils) of a muscle cell together, as well as filamin conceivably could disrupt key link-the peripheral layer of myofibrils to the cell ages that serve to help hold myofibrils inmembrane, could affect the development of lateral register. Degradation of filamin maytenderness. Desmin is degraded during post- also alter linkages connecting the peripheralmortem storage (Hwan and Bandman 1989; layer of myofibrils in muscle cells to the sar-Huff-Lonergan et al. 1996a; Huff-Lonergan colemma by weakening interactions betweenand Lonergan, 1999; Melody et al. 2004; peripheral myofibrillar Z-disks and the sarco-Rowe et al. 2004b; Zhang et al. 2006). lemma via intermediate filament associationsFurthermore, it has been documented that or costameres (Robson et al. 1995). A studydesmin is degraded more rapidly in myofi- using myofibrils from beef showed that somebrils from samples with low shear force filamin was degraded to form an approxi-and higher water-holding capacity (Huff- mately 240-kDa degradation product thatLonergan et al. 1996a; Huff-Lonergan and migrated as a doublet in both myofibrils fromLonergan, 1999; Melody et al. 2004; Rowe naturally aged muscle and in μ-calpain-et al. 2004b; Zhang et al. 2006). A major digested myofibrils (Huff-Lonergan et al.degradation product that is often seen in beef 1996a). This same doublet formation (com-is a polypeptide of approximately 38 kDa. posed of intact and degraded filamin) hasThis degradation product also has been been seen in cultured embryonic skeletalshown to be present in μ-calpain-digested muscle cells and was attributed to calpain
  25. 25. Chemistry and Biochemistry of Meat 15activity (Robson et al. 1995). Uytterhaegen the total water in muscle cells; depending onet al. (1994) have shown increased degrada- the measurement system used, approximatelytion of filamin in muscle samples injected 0.5 g of water per gram of protein is esti-with CaCl2, a process that has been shown to mated to be tightly bound to proteins. Sincestimulate proteolysis and postmortem tender- the total concentration of protein in muscleization (Wheeler et al. 1992; Harris et al. is approximately 200 mg/g, this bound water2001). Compared with other skeletal muscle only makes up less than a tenth of the totalproteins, relatively little has been done to water in muscle. The amount of bound waterfully characterize the role of this protein in changes very little if at all in postrigor musclepostmortem tenderization of beef. Further (Offer and Knight 1988b).studies that employ a combination of sen- Another fraction of water that can besitive detection methods (e.g., one- and found in muscles and in meat is termedtwo-dimensional gels, Western blotting, entrapped (also referred to as immobilized)immunomicroscopy) are needed to determine water (Fennema 1985). The water moleculesthe role of filamin in skeletal muscle systems in this fraction may be held either by stericand postmortem tenderization. (space) effects and/or by attraction to the bound water. This water is held within the structure of the muscle but is not bound per se to protein. In early postmortem tissue, thisWater-Holding Capacity/Drip water does not flow freely from the tissue, yetLoss Evolution it can be removed by drying and can be easilyLean muscle contains approximately 75% converted to ice during freezing. Entrappedwater. The other main components include or immobilized water is most affected by theprotein (approximately 18.5%), lipids or fat rigor process and the conversion of muscle(approximately 3%), carbohydrates (approxi- to meat. Upon alteration of muscle cell struc-mately 1%), and vitamins and minerals (often ture and lowering of the pH, this water cananalyzed as ash, approximately 1%). The also eventually escape as purge (Offer andmajority of water in muscle is held within the Knight 1988b).structure of the muscle and muscle cells. Free water is water whose flow from theSpecifically, within the muscle cell, water is tissue is unimpeded. Weak surface forcesfound within the myofibrils, between the mainly hold this fraction of water in meat.myofibrils themselves and between the myo- Free water is not readily seen in pre-rigorfibrils and the cell membrane (sarcolemma), meat, but can develop as conditions changebetween muscle cells, and between muscle that allow the entrapped water to move frombundles (groups of muscle cells) (Offer and the structures where it is found (FennemaCousins 1992). 1985). Water is a dipolar molecule and as such is The majority of the water that is affectedattracted to charged species like proteins. In by the process of converting muscle to meatfact, some of the water in muscle cells is very is the entrapped (immobilized) water.closely bound to protein. By definition, Maintaining as much of this water as possiblebound water is water that exists in the vicin- in meat is the goal of many processors. Someity of nonaqueous constituents (like proteins) of the factors that can influence the retentionand has reduced mobility (i.e., does not easily of entrapped water include manipulation ofmove to other compartments). This water is the net charge of myofibrillar proteins andvery resistant to freezing and to being driven the structure of the muscle cell and its com-off by conventional heating (Fennema 1985). ponents (myofibrils, cytoskeletal linkages,True bound water is a very small fraction of and membrane permeability), as well as the
  26. 26. 16 Chapter 1amount of extracellular space within the relaxation (Millman et al. 1981; Millmanmuscle itself. et al. 1983). This would indicate that in living muscle the amount of water within the fila- mentous structure of the cell would not nec-Physical/Biochemical Factors essarily change. However, the location of thisin Muscles That Affect water can be affected by changes in volumeWater-Holding Capacity as muscle undergoes rigor. As muscle goesDuring the conversion of muscle to meat, into rigor, cross-bridges form between theanaerobic glycolysis is the primary source of thick and thin filaments, thus reducing avail-ATP production. As a result, lactic acid able space for water to reside (Offer andbuilds up in the tissue, leading to a reduction Trinick 1983). It has been shown that as thein pH of the meat. Once the pH has reached pH of porcine muscle is reduced from physi-the isoelectric point (pI) of the major pro- ological values to 5.2–5.6 (near the isoelec-teins, especially myosin (pI = 5.3), the net tric point of myosin), the distance betweencharge of the protein is zero, meaning the the thick filaments declines an average ofnumbers of positive and negative charges 2.5 nm (Diesbourg et al. 1988). This declineon the proteins are essentially equal. These in filament spacing may force sarcoplasmicpositive and negative groups within the fluid from between the myofilaments to theprotein are attracted to each other and result extramyofibrillar space. Indeed, it has beenin a reduction in the amount of water that can hypothesized that enough fluid may be lostbe attracted and held by that protein. from the intramyofibrillar space to increaseAdditionally, since like charges repel, as the the extramyofibrillar volume by as much asnet charge of the proteins that make up the 1.6 times more than its pre-rigor volumemyofibril approaches zero (diminished net (Bendall and Swatland 1988).negative or positive charge), repulsion of During the development of rigor, thestructures within the myofibril is reduced, diameter of muscle cells decreases (Hegartyallowing those structures to pack more 1970; Swatland and Belfry 1985) and isclosely together. The end result of this is a likely the result of transmittal of the lateralreduction of space within the myofibril. shrinkage of the myofibrils to the entire cellPartial denaturation of the myosin head at (Diesbourg et al. 1988). Additionally, duringlow pH (especially if the temperature is still rigor development, sarcomeres can shorten;high) is also thought to be responsible for a this also reduces the space available for waterlarge part of the shrinkage in myofibrillar within the myofibril. In fact, it has beenlattice spacing (Offer 1991). shown that drip loss can increase linearly Myofibrils make up a large proportion of with a decrease in the length of the sarco-the muscle cell. These organelles constitute meres in muscle cells (Honikel et al. 1986).as much as 80–90% of the volume of the More recently, highly sensitive low-fieldmuscle cell. As mentioned previously, much nuclear magnetic resonance (NMR) studiesof the water inside living muscle cells is have been used to gain a more completelocated within the myofibril. In fact, it is esti- understanding of the relationship betweenmated that as much as 85% of the water in a muscle cell structure and water distributionmuscle cell is held in the myofibrils. Much (Bertram et al. 2002). These studies haveof that water is held by capillary forces suggested that within the myofibril, a higherarising from the arrangement of the thick and proportion of water is held in the I-band thanthin filaments within the myofibril. In living in the more protein-dense A-band. Thismuscle, it has been shown that sarcomeres observation may help explain why shorterremain isovolumetric during contraction and sarcomeres (especially in cold-shortened
  27. 27. Chemistry and Biochemistry of Meat 17muscle) are often associated with increased associated with intermediate filament struc-drip losses. As the myofibril shortens and tures and structures known as costameres.rigor sets in, the shortening of the sarcomere Costameres provide the structural frameworkwould lead to shortening and subsequent responsible for attaching the myofibrils to thelowering of the volume of the I-band region sarcolemma. Proteins that make up or arein myofibril. Loss of volume in this myofi- associated with the intermediate filamentsbrillar region (where much water may reside), and costameres include (among others)combined with the pH-induced lateral shrink- desmin, filamin, synemin, dystrophin, talin,age of the myofibril, could lead to expulsion and vinculin (Greaser 1991). If costamericof water from the myofibrillar structure linkages remain intact during the conversioninto the extramyofibrillar spaces within the of muscle to meat, shrinkage of the myofi-muscle cell (Bendall and Swatland 1988). In brils as the muscle goes into rigor would befact, recent NMR studies support this hypoth- transmitted to the entire cell via these pro-esis (Bertram et al. 2002). It is thus likely that teinacious linkages and would ultimatelythe gradual mobilization of water from the reduce volume of the muscle cell itself (Offerintramyofibrillar spaces to the extramyofi- and Knight 1988b; Kristensen and Purslowbrillar spaces may be key in providing a 2001; Melody et al. 2004). Thus, the rigorsource of drip. process could result in mobilization of water All the previously mentioned processes not only out of the myofibril, but also out ofinfluence the amount of water in the myofi- the extramyofibril spaces as the overallbril. It is important to note that shrinkage of volume of the cell is constricted. In fact,the myofibrillar lattice alone could not be reduction in the diameter of muscle cells hasresponsible for the movement of fluid to the been observed in postmortem muscle (Offerextracellular space and ultimately out of the and Cousins 1992). This water that is expelledmuscle. The myofibrils are linked to each from the myofibril and ultimately the muscleother and to the cell membrane via proteina- cell eventually collects in the extracellularcious connections (Wang and Ramirez- space. Several studies have shown that gapsMitchell 1983). These connections, if they develop between muscle cells and betweenare maintained intact in postmortem muscle, muscle bundles during the postrigor periodwould transfer the reduction in diameter of (Offer et al. 1989; Offer and Cousins 1992).the myofibrils to the muscle cell (Diesbourg These gaps between muscle bundles areet al. 1988; Morrison et al. 1998; Kristensen the primary channels by which purge isand Purslow 2001; Melody et al. 2004). allowed to flow from the meat; some inves-Myofibril shrinkage can be translated into tigators have actually termed them “dripconstriction of the entire muscle cell, thus channels.”creating channels between cells and betweenbundles of cells that can funnel drip out Postmortem Changes in Muscleof the product (Offer and Knight 1988). That Influence QualityExtracellular space around muscle fibers con-tinually increases up to 24 hours postmortem, As muscle is converted to meat, manybut gaps between muscle fiber bundles changes occur, including: (1) a gradual deple-decrease slightly between nine and 24 hours tion of available energy; (2) a shift frompostmortem, perhaps due to fluid outflow aerobic to anaerobic metabolism favoring thefrom these major channels (Schafer et al. production of lactic acid, resulting in the pH2002). These linkages between adjacent of the tissue declining from near neutrality tomyofibrils and myofibrils and the cell mem- 5.4–5.8; (3) a rise in ionic strength, in part,brane are made up of several proteins that are because of the inability of ATP-dependent
  28. 28. 18 Chapter 1calcium, sodium, and potassium pumps to that is involved in increasing the tendernessfunction; and (4) an increasing inability of of fresh meat and in influencing fresh meatthe cell to maintain reducing conditions. All water-holding capacity (Huff-Lonergan andthese changes can have a profound effect on Lonergan 2005). Because μ-calpain andnumerous proteins in the muscle cell. The m-calpain enzymes contain both histidinerole of energy depletion and pH change have and SH-containing cysteine residues at theirbeen covered in this chapter and in other active sites, they are particularly susceptiblereviews (Offer and Trinick 1983; Offer and to inactivation by oxidation (Lametsch et al.Knight 1988a). What has not been as thor- 2008). Therefore, oxidizing conditions inoughly considered is the impact of other postmortem muscle lead to inactivation orchanges on muscle proteins, such as oxida- modification of calpain activity (Harris et al.tion and nitration. 2001; Rowe et al. 2004a, b; Maddock et al. 2006). In fact, evidence suggests oxidizing conditions inhibit proteolysis by μ-calpain,Protein Oxidation but might not completely inhibit autolysisAnother change that occurs in postmortem (Guttmann et al. 1997; Guttmann and Johnsonmuscle during aging of whole muscle prod- 1998; Maddock et al. 2006). In postmortemucts is increased oxidation of myofibrillar muscle, there are differences betweenand sarcoplasmic proteins (Martinaud et al. muscles in the rate that postmortem oxidation1997; Rowe et al. 2004a, b). This results in processes occur (Martinaud et al. 1997). Itthe conversion of some amino acid residues, has been noted that differences in the rate ofincluding histidine, to carbonyl derivatives oxidation in muscle tissue are seen when(Levine et al. 1994; Martinaud et al. 1997) comparing the same muscles between animalsand can cause the formation of intra- and/or and/or carcasses that have been handled dif-inter-protein disulfide cross-links (Stadtman ferently (Juncher et al. 2001). These differ-1990; Martinaud et al. 1997). In general, both ences may arise because of differences inthese changes reduce the functionality of pro- diet, breed, antemortem stress, postmortemteins in postmortem muscle (Xiong and handling of carcasses, etc. In fact, there haveDecker 1995). In living muscle, the redox been reports of differences between animalsstate of muscle can influence carbohydrate and between muscles in the activity of somemetabolism by directly affecting enzymes in enzymes involved in the oxidative defensethe glycolytic pathway. Oxidizing agents can system of muscle (Daun et al. 2001).also influence glucose transport. Hydrogen Therefore, there may be genetic differencesperoxide (H2O2) can mimic insulin and stim- in susceptibility to oxidation that could beulate glucose transport in exercising muscle. capitalized on to improve meat quality. It isH2O2 is increased after exercise, and thus oxi- reasonable to hypothesize that differences indation systems may play a role in signaling the antioxidant defense system betweenin skeletal muscle (Balon and Yerneni 2001). animals and/or muscles would influenceAlterations in glucose metabolism in the calpain activity, proteolysis, and thusante- and perimortem time period do have the tenderization.potential to cause changes in postmortem Exposure to oxidizing conditions (H2O2)muscle metabolism and thus represent an under postmortem-like conditions inhibitsimportant avenue of future research. calpain activity (Carlin et al. 2006). In a In postmortem muscle, these redox series of in vitro assays using either a fluo-systems may also play a role in influencing rescent peptide or purified myofibrils as themeat quality. The proteolytic enzymes, the substrate it was shown that the presence ofcalpains, are implicated in the proteolysis oxidizing species does significantly impede
  29. 29. Chemistry and Biochemistry of Meat 19the ability of calpains to degrade their sub- (NOS). There are three major isoforms ofstrates. Oxidation with H2O2 significantly NOS: neural, inducible, and endothelial.limits proteolytic activity of μ- and m-calpain Skeletal muscle expresses all three isoforms;against the fluorescent peptide Suc-Leu- however, the neural form, nNOS, is thoughtLeu-Val-Tyr-AMC, regardless of the pH or to be the predominant isoform (Kaminski andionic strength. Similar results were seen Andrade 2001). These enzymes utilize argi-when using purified myofibrils as the sub- nine as a substrate and catalyze the followingstrate. This inhibition was reversible, as reaction: L-arginine+NADPH+O2 formingaddition of reducing agent (DTT) to the oxi- L-citrulline+•NO+NADPH+. NO is importantdized samples restored activity. Oxidation in biological systems, particularly because ofalso has been shown to slow the rate of μ- its role as a second messenger. However,calpain autolysis and could be part of the while NO rapidly diffuses through tissues,mechanism underlying some of the retarda- NO itself is a relatively short-lived species.tion of activity (Guttmann et al. 1997; Carlin It does have the ability to combine with otheret al. 2006). biomolecules that also have physiological Oxidation does occur early in postmortem importance.meat, and it does influence proteolysis (Harris One example of this is its ability toet al. 2001; Rowe et al. 2004b). Rowe et al. combine with superoxide to form the highly(2004) showed that there was a significant oxidizing molecule peroxynitrite. Proteinsincrease in proteolysis of troponin-T in steaks are important biological targets of peroxyni-from alpha-tocopherol-fed steers after 2 days trite, particularly proteins containing cyste-of postmortem aging compared with steers ine, motioning, and/or tryptophan (Radi et al.fed a conventional feedlot diet. This indicates 2000). Several enzymes are known to bethat very low levels of oxidation can influ- inactivated by peroxynitrite. Among these isence proteolysis and that increasing the level the sarcoplasmic reticulum Ca2+-ATPaseof antioxidants in meat may have merit in (Klebl et al. 1998). One indirect effect ofimproving tenderness in future studies. In NO is S-nitrosylation. In most cases, S-fact, low levels of oxidation may be the cause nitrosylation events involve amines andof some heretofore-unexplained variations in thiols. Nitric oxide can interact with cyste-proteolysis and tenderness that have been ines to form nitrosothiols that can alter theobserved in meat. activity of the protein. Because of this, it has been suggested that S-nitrosylation may function as a post-translational modificationNitric Oxide and S-Nitrosylation much like phosphorylation (Jaffrey et al.Nitric oxide (NO) is often used as a general 2001). Some proteins, such as the ryanodineterm that includes NO and reactive nitrogen receptor and the cysteine protease caspase-species (RNS), like S-nitrosothyols, per- 3, have been shown to be endogenouslyoxynitrate, and metal NO complexes. In nitrosylated, further supporting the sugges-living tissue, NO is involved in arteriole dila- tion that formation of nitrosothiols may betion that increases blood flow to muscles, an important regulatory step (Hess et al.resulting in increased delivery of nutrients 2001; Hess et al. 2005). μ-Calpain is alsoand oxygen to the muscle (Kobzik et al. a cysteine protease that could be influenced1994; Stamler et al. 2001). NO species are by S-nitrosylation. Small thiol peptidesalso implicated in glucose homeostasis and like glutathione can be impacted by nitro-excitation-contraction coupling. The gas NO sative stress to form compounds likeis produced in biological systems by a family S-nitrosoglutathione (GSNO). These com-of enzymes known as nitric oxide synthases pounds can, in turn, influence other proteins
  30. 30. 20 Chapter 1by transnitrosating other reduced thiols Bang, M.-L., X. Li, R. Littlefield, S. Bremner, A. Thor, K. U. Knowlton, R. L. Lieber, and J. Chen. 2006.(Miranda et al. 2000). Nebulin-deficient mice exhibit shorter thin filament Aspects of skeletal muscle function that lengths and reduced contractile function in skeletalcan be affected by increased NO production muscle. Journal of Cell Biology 173:905–916. Bendall, J. R., and H. J. Swatland. 1988. A review of theinclude inhibition of excitation-contraction relationships of ph with physical aspects of porkcoupling, increased glucose uptake, decreased quality. Meat Science 24:85–126.mitochondrial respiration, and decreased Bertram, H. C., P. P. Purslow, and H. J. Andersen. 2002. Relationship between meat structure, water moblity,force production. The decrease in force is and distribution: A low-field nuclear magnetic reso-apparently because of an inhibitory effect nance study. Journal of Agricultural and Foodthat NO has on actomyosin ATPase activity, Chemistry 50:824–829. Boehm, M. L., T. L. Kendall, V. F. Thompson, and D.which leads to less cross-bridge cycling. E. Goll. 1998. Changes in the calpains and calpastatinS-nitroslyation of the ryanodine receptor during postmortem storage of bovine muscle. Journal(calcium release channel in the sarcoplasmic of Animal Science 76:2415–2434. Briggs, M. M., H. D. Mcginnis, and F. Schachat. 1990.reticulum) may also play a role on modulat- Transitions from fetal to fast troponin-t isoforms areing contraction. This protein is responsible coordinated with changes in tropomyosin and alpha-for releasing calcium from the sarcoplasmic actinin isoforms in developing rabbit skeletal-muscle. Developmental Biology 140:253–260.reticulum into the sarcoplasm. S-nitrosylation Callow, E. H. 1948. Comparative studies of meat. II.of a cysteine in the ryanodine receptor will Changes in the carcass during growth and fatteningincrease its activity. This effect is reversible and their relation to the chemical composition of the fatty and muscular tissues. Journal of Agricultural(Kobzik et al. 1994). Because muscle con- Science 38:174.tains all the compounds needed to form these Carlin, K. R., E. Huff-Lonergan, L. J. Rowe, and S. M.intermediates, it stands to reason that they Lonergan. 2006. Effect of oxidation, ph, and ionic strength on calpastatin inhibition of μ- and m-calpain.could be important in the conversion of Journal of Animal Science 84:925–937.muscle to meat. Clark, K. A., A. S. McElhinny, M. C. Beckerle, and C. It is clear that the composition, structure, C. Gregorio. 2002. Striated muscle cytoarchitecture: An intricate web of form and function. Annual Reviewand metabolic properties of skeletal muscle of Cell and Developmental Biology 18:637–706.have enormous impacts on the quality of Daun, C., M. Johansson, G. Onning, and B. Akesson.fresh meat and, in turn, its suitability as a 2001. Glutathione peroxidase activity, tissue and soluble selenium content in beef and pork in relationraw material for further processed meat. to meat ageing and pig rn phenotype. Food ChemistryContinued attention to factors that regulate 73:313–319.changes in early postmortem muscle will Diesbourg, L., H. J. Swatland, and B. M. Millman. 1988. X-ray-diffraction measurements of postmortemimprove the quality and consistency of fresh changes in the myofilament lattice of pork. Journal ofmeat. This, in turn, will improve the consis- Animal Science 66:1048–1054.tency of the quality of further processed Feinstein, B., B. Lindegard, E. Nyman, and G. Wohlfart. 1955. Morphologic studies of motor units in normalproducts. human muscles. Acta Anatomica 23:127–142. Fennema, O. R. 1985. Water and ice. In Food Chemistry, O. R. Fennema (ed.). New York: Marcel Dekker.References Fraterman, S., U. Zeiger, T. S. Khurana, M. Wilm, and N. A. Rubinstein. 2007. Quantitative proteomics pro-Astier, C., J. P. Labbe, C. Roustan, and Y. Benyamin. filing of sarcomere associated proteins in limb and 1993. Effects of different enzymatic treatments on the extraocular muscle allotypes. Molecular Cell release of titin fragments from rabbit skeletal myofi- Proteomics 6:728–737. brils—Purification of an 800 kda titin polypeptide. Goll, D. E., R. M. Robson, and M. H. Stromer. 1984. Biochemical Journal 290:731–734. Skeletal muscle, nervous system, temperature regula-Bailey, A. J., and N. D. Light. 1989. Connective Tissue tion, and special senses. In Duke’s Physiology of in Meat and Meat Products. Barking, UK: Elsevier Domestic Animals, M. J. Swensen (ed.), pp. 548–580. Applied Science. Ithaca, N.Y.: Cornell University Press.Balon, T. W., and K. K. Yerneni. 2001. Redox regula- Greaser, M. L. 1991. An overview of the muscle tion of skeletal muscle glucose transport. Medicine cell cytoskeleton. Reciprocal Meats Conference and Science in Sports and Exercise 33:382–385. Proceedings 1–5.