Handbook of Meat Processing

3. Handbook of
Meat Processing

5. Handbook of
Meat Processing
Fidel Toldrá
EDITOR
A John Wiley & Sons, Inc., Publication

6. Edition first published 2010
© 2010 Blackwell Publishing
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Library of Congress Cataloging-in-Publication Data
Handbook 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. Meat
industry and trade—Handbooks, manuals, etc. I. Toldrá, Fidel.
TS1960.H36 2010
664′.9—dc22
2009037503
A 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 Limited
Printed in Singapore
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1 2010

7. Contents
Preface ix
List of Contributors xi
About the Editor xv
PART 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. Nychas
10. Starter Cultures for Meat Fermentation 199
Pier Sandro Cocconcelli and Cecilia Fontana
11. Drying 219
Endre Zukál and Kálmán Incze
v

8. vi Contents
12. Smoking 231
Zdzisław E. Sikorski and Edward Kolakowski
´
13. Meat Packaging 247
Maurice G. O’Sullivan and Joseph P. Kerry
14. Novel Technologies for Microbial Spoilage Prevention 263
Oleksandr Tokarskyy and Douglas L. Marshall
15. Plant Cleaning and Sanitation 287
Stefania Quintavalla
PART II. Products 299
16. Cooked Ham 301
Fidel Toldrá, Leticia Mora, and Mónica Flores
17. Cooked Sausages 313
Eero Puolanne
18. Bacon 327
Peter R. Sheard
19. Canned Products and Pâté 337
Isabel Guerrero Legarreta
20. Dry-Cured Ham 351
Fidel Toldrá and M. Concepción Aristoy
21. Mold-Ripened Sausages 363
Kálmán Incze
22. Semidry and Dry Fermented Sausages 379
Graciela Vignolo, Cecilia Fontana, and Silvina Fadda
23. Restructured Whole-Tissue Meats 399
Mustafa M. Farouk
24. Functional Meat Products 423
Keizo Arihara and Motoko Ohata
PART III. Controls 441
25. Physical Sensors for Quality Control during Processing 443
Marta Castro-Giráldez, Pedro José Fito, Fidel Toldrá, and Pedro Fito
26. Sensory Evaluation of Meat Products 457
Geoffrey R. Nute
27. Detection of Chemical Hazards 469
Milagro Reig and Fidel Toldrá
28. Microbial Hazards in Foods: Food-Borne Infections and Intoxications 481
Daniel Y. C. Fung

9. Contents vii
29. Assessment of Genetically Modified Organisms (GMO) in Meat Products
by PCR 501
Marta Hernández, Alejandro Ferrando, and David Rodríguez-Lázaro
30. HACCP: Hazard Analysis Critical Control Point 519
Maria João Fraqueza and António Salvador Barreto
31. Quality Assurance 547
Friedrich-Karl Lücke
Index 561

11. Preface
For centuries, meat and its derived products worldwide meat products such as cooked
have constituted some of the most important ham and sausages, bacon, canned products
foods 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 quality
meat processing, giving the latest advances of meat and meat products, including physi-
in technologies, manufacturing processes, cal sensors, sensory evaluation, chemical
and 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 fifteen
part deals with the description of meat chem- countries. The editor wishes to thank all the
istry, its quality for further processing, contributors for their hard work and for
and the main technologies used in meat sharing their valuable experience, as well as
processing, 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 kind
smoking, packaging, novel technologies, support and coordination of this book.
and cleaning. The second part describes the
manufacture and main characteristics of Fidel Toldrá
ix

13. Contributors
Irene Allais Susan Brewer
Cemagref, 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.edu
E-mail: irene.allais@cemagref.fr
Janice A. Callahan
Keizo Arihara Food Technology and Safety Laboratory,
Department of Animal Science, Kitasato Bldg 201, BARC-East, Beltsville, Maryland
University, Towada-shi, Aomori 034-8628, 20705, USA.
Japan. E-mail: Janice.callahan@ars.usda.gov
E-mail: arihara@vmas.kitasato-u.ac.jp
Marta Castro-Giráldez
M. Concepción Aristoy Institute of Food Engineering for
Department of Food Science, Instituto de Development, Universidad Politécnica de
Agroquímica 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 Ricerche
António Salvador Barreto Biotecnologiche, Università Cattolica del
Faculdade de Medicina Veterinária, Sacro Cuore, Piacenza-Cremona, Italy.
DPASA, TULisbon, Av. da Universidade E-mail: pier.cocconcelli@unicatt.it
Tecnica, Polo Universitário, Alto da Ajuda,
1300-477 Lisboa, Portugal. Eleftherios H. Drosinos
Laboratory of Food Quality Control and
Jane Ann Boles Hygiene, Department of Food Science and
Animal and Range Sciences, 119 Technology, Agricultural University of
Linfield Hall, Bozeman, Montana Athens, Iera Odos 75, Votanikos, 11855
59717, USA. Athens, Greece.
E-mail: jboles@montana.edu E-mail: ehd@aua.gr
Brian C. Bowker Janet S. Eastridge
Food Technology and Safety Laboratory, Food Technology and Safety Laboratory,
Bldg 201, BARC-East, Beltsville, Bldg 201, BARC-East, Beltsville, Maryland
Maryland 20705, USA. 20705, USA.
E-mail: brian.bowker@ars.usda.gov E-mail: janet.eastridge@ars.usda.gov
xi

14. xii Contributors
Silvina Fadda Maria João Fraqueza
Centro de Referencia para Lactobacilos Faculdade de Medicina Veterinária,
(CERELA), CONICET., Chacabuco 145, DPASA, TULisbon, Av. da Universidade
T4000ILC Tucumán, Argentina. Tecnica, Polo Universitário, Alto da Ajuda,
E-mail: fadda@cerela.org.ar 1300-477 Lisboa, Portugal.
E-mail: mjoaofraqueza@fmv.utl.pt
Mustafa M. Farouk
AgResearch MIRINZ, Ruakura Research Daniel Y. C. Fung
Centre, East Street, Private Bag 3123, Department of Animal Sciences and
Hamilton 3240, New Zealand. Industry, 207 Call Hall, Kansas State
E-mail: mustafa.farouk@agresearch.co.nz University, Manhattan, Kansas 66506,
USA.
E-mail: dfung@ksu.edu
Alejandro Ferrando
Departamento de Bioquímica y Biología
Molecular, Facultad de Biología, Isabel Guerrero Legarreta
Universidad 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.mx
Development, Universidad Politécnica de
Valencia, Camino de Vera s/n, 46022
Valencia, Spain. Marta Hernández
E-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, 47071
Institute of Food Engineering for
Valladolid, Spain.
Development, Universidad Politécnica de
Valencia, Camino de Vera s/n, 46022
Valencia, Spain. Karl O. Honikel
E-mail: pjfito@tal.upv.es Max Rubner-Institut, Arbeitsgruppe
Analytik, Kulmbach, Germany.
E-mail: karl-otto.honikel@t-online.de
Mónica Flores
Department of Food Science, Instituto de
Agroquímica y Tecnología de Alimentos Elisabeth Huff-Lonergan
(CSIC), PO Box 73, 46100 Burjassot, Muscle Biology, Department of Animal
Valencia, Spain. Science, Iowa State University, 2275 Kildee
E-mail: mflores@iata.csic.es Hall, Ames, IA 50011 USA. E-mail:
elonerga@iastate.edu
Cecilia Fontana
Centro de Referencia para Lactobacilos Kálmán Incze
(CERELA), CONICET., Chacabuco 145, Hungarian Meat Research Institute, 1097
T4000ILC Tucumán, Argentina. Budapest, Gubacsi út 6/b, Hungary.
E-mail: cecilia.fontana@unicatt.it E-mail: ohki@interware.hu

15. Contributors xiii
Christian James Douglas L. Marshall
Food 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, Colorado
Education(GIFHE), HSI Building, Origin 80639 USA.
Way, Europarc, Grimsby, North East E-mail: douglas.marshall@unco.edu
Lincolnshire, DN37 9TZ UK.
E-mail: JamesC@grimsby.ac.uk Leticia Mora
Department of Food Science, Instituto de
Agroquímica y Tecnología de Alimentos
Stephen J. James
(CSIC), PO Box 73, 46100 Burjassot
Food Refrigeration and Process Engineering
Valencia, Spain.
Research Centre (FRPERC), The Grimsby
E-mail: lemoso@iata.csic.es
Institute of Further and Higher
Education(GIFHE), HSI Building, Origin
Way, Europarc, Grimsby, North East Geoffrey R. Nute
Lincolnshire, DN37 9TZ UK. University of Bristol, School of Clinical
E-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.uk
Department of Food and Nutritional
Sciences, University College Cork, Ireland. George-John E. Nychas
E-mail: Joe.Kerry@ucc.ie Laboratory of Food Microbiology &
Biotechnology, Department of Food
Edward Kołakowski Science & Technology, Agricultural
Department of Food Science and University of Athens, Iera Odos 75, Athens
Technology, Agricultural University of 11855, Greece.
Szczecin, Papie a Pawła VI St. 3, 71-459 E-mail: gjn@aua.gr
Szczecin, 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 and
Microbiological Sciences, North Dakota
Maurice G. O’Sullivan
State University, 1523 Centennial Blvd,
Department of Food and Nutritional
130A Van Es Hall, Fargo, North Dakota
Sciences, University College Cork, Ireland.
58105, USA.
E-mail: maurice.osullivan@ucc.ie
E-mail: Catherine.Logue@ndsu.edu
Spiros Paramithiotis
Friedrich-Karl Lücke Laboratory of Food Quality Control and
Hochschule Fulda (University of Applied Hygiene, Department of Food Science and
Sciences), P.O. Box 2254, 36012 Fulda, Technology, Agricultural University of
Germany. Athens, Iera Odos 75, 11855 Athens,
E-mail: friedrich-karl.luecke@t-online.de Greece.

16. xiv Contributors
Ernie W. Paroczay Panagiotis N. Skandamis
Food Technology and Safety Laboratory, Laboratory of Food Quality Control and
Bldg 201, BARC-East, Beltsville, Hygiene, Department of Food Science and
Maryland 20705, USA. Technology, Agricultural University of
E-mail: ernie.paroczay@ars.usda.gov Athens, Iera Odos 75, Votanikos, 11855
Athens, Greece.
Eero Puolanne
Department of Food Technology, Viikki John N. Sofos
EE, 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.EDU
Stefania Quintavalla
Department of Microbiology, SSICA, V.le
Tanara 31/A, 43100, Parma, Italy. Morse B. Solomon
E-mail address: stefania.quintavalla@ssica.it Food Technology and Safety Laboratory,
Bldg 201, BARC-East, Beltsville, Maryland
Milagro Reig 20705, USA.
Institute of Food Engineering for E-mail: Morse.Solomon@ARS.USDA.GOV
Development, Universidad Politécnica de
Valencia, Camino de Vera s/n, 46022 Oleksandr Tokarskyy
Valencia, Spain. Department of Food Science, Nutrition, and
E-mail: mareirie@doctor.upv.es Health Promotion, Mississippi State
University, Box 9805, Mississippi State
David Rodríguez-Lázaro University, Mississippi 39762 USA.
Food Safety and Technology Group,
Instituto Tecnológico Agrario de Castilla y Fidel Toldrá
León (ITACyL), Ctra. Burgos km.119, Department of Food Science, Instituto de
Finca Zamadueñas, 47071 Valladolid, Agroquímica y Tecnología de Alimentos
Spain. (CSIC), PO Box 73, 46100 Burjassot,
E-mail: ita-rodlazda@itacyl.es Valencia, Spain.
E-mail: ftoldra@iata.csic.es
Peter R. Sheard
Division of Farm Animal Science, School
of Clinical Veterinary Science, University Graciela Vignolo
of Bristol, Bristol BS40 5DU, Avon, UK. Centro de Referencia para Lactobacilos
E-mail: Peter.Sheard@bristol.ac.uk (CERELA), CONICET., Chacabuco 145,
T4000ILC Tucumán, Argentina.
Zdzisław E. Sikorski E-mail: vignolo@cerela.org.ar
Department of Food Chemistry, Gdansk
´
University of Technology Endre Zukál
E-mail: sikorski@chem.pg.gda.pl OR Hungarian Meat Research Institute,
zdzsikor@pg.gda.pl Budapest 1097, Gubacsi út 6/b, Hungary.

17. About the Editor
Fidel Toldrá, Ph.D., is a research professor at years, including Handbook of Muscle
the Department of Food Science, Instituto de Foods Analysis and Handbook of Processed
Agroquímica y Tecnología de Alimentos Meats and Poultry Analysis (2009), Meat
(CSIC), and serves as European editor of Biotechnology and Safety of Meat and
Trends in Food Science & Technology, editor Processed Meat (2008, 2009), Handbook of
in chief of Current Nutrition & Food Science, Food Product Manufacturing (2007),
and as section editor of the Journal of Muscle Advances in Food Diagnostics, and Handbook
Foods. 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 2002
Journal 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 Secretariat
Food Safety Authority panel on flavorings, and was elected in 2008 as Fellow of the
enzymes, 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 Technologists
associate editor of several books in recent (IFT).
xv

19. Handbook of
Meat Processing

21. Part I
Technologies

23. Chapter 1
Chemistry and Biochemistry of Meat
Elisabeth Huff-Lonergan
Introduction 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 the
nized 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, water
are required for the movement of limbs,
is the primary component of sarcoplasmic
for 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 cellular
such as maintaining balance and coordina-
processes; and for transport of nutrients
tion. Muscle movement and metabolism
within the cell, between cells, and between
are associated with other diverse functions
the muscle and the vascular system.
such as aiding in movement of blood and
The second largest component of muscle
lymph and also in maintaining body tempera-
is protein (U.S. Department of Agriculture
ture. 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 that
cell to maintain energy supplies. Few cells
figure can range from 16 to 22%. Proteins
are required to generate as much force and
serve myriad functions and are the primary
undergo as dramatic shifts in rate of metabo-
solid component in muscle. The functions of
lism as muscle cells. The ability of living
proteins are quite varied. Muscle proteins are
skeletal muscle to undergo relatively large
involved in maintaining the structure and
intracellular changes also influences its
organization of the muscle and muscle cells
response 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 are
and metabolism of the muscle are key to its
associated with the contractile organelles, the
function and to the maintenance of its integ-
myofibril, and are thus termed myofibrillar
rity both during contraction and during the
proteins. In general, the myofibrillar proteins
early postmortem period. Ultimately, these
are not soluble at low ionic strengths found
postmortem changes will influence the suit-
in skeletal muscle (ionic strength ≤0.15), but
ability 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 movement
Muscle 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 also
2008). In living tissue, the average water many soluble proteins (sarcoplasmic pro-
5

24. 6 Chapter 1
Table 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 to
Numbers in parentheses indicate the average range of
that 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 the
lular signaling processes and enzymes largest percentage is glycogen. Other carbo-
important in metabolism and protein degra- hydrates include glucose, intermediates of
dation/cellular remodeling. glycogen metabolism, and other mono- and
The lipid content of the muscle can vary disaccharides. Glycosoaminoglycans are also
greatly due to many factors, including animal found in muscle and are associated with the
age, 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. They
content (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 is
about 3% of the muscle weight, but the range
Muscle Structure
can be as much as 1–13% (U.S. Department
of 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 efficiently
structure, and in various other processes in transmit force originating in the myofibrils to
the organ, including immune responses and the entire muscle and ultimately, to the limb
cellular 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 tendons
proportion of lipid associated with muscle. that link muscles to bones. The muscle is
Triglycerides (triacylglycerides) consist of a subdivided into bundles or groupings of
glycerol molecule in which the hydroxyl muscle cells. These bundles (also known as
groups are esterified with three fatty acids. fasciculi) are surrounded by another sheath
The melting point and the iodine number of of connective tissue, the perimysium. A thin
lipid 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. The
of 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-

25. Chemistry and Biochemistry of Meat 7
ment 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 meat
in 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 they
occurs not only at the gross level, but also at reside, are very highly organized. When
the 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 all
has only a few hundred muscle cells, while the structural elements needed to perform the
the medial gastrocnemius (used in humans physical act of contraction at the molecular
for 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 structure
number 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 basic
structure of the muscle cells themselves unit of the cell and that the number quoted in
has a profound effect on the function of this analysis did not take into account the
living muscle and on the functionality of multiple isoforms of the proteins, this number
meat. is quite high. Many of the proteins interact
Muscle cells are striated, meaning that with each other in a highly coordinated
when viewed under a polarized light micro- fashion, and some of the interactions are just
scope, 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 muscle
muscle 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-bands
regions have different refractive properties. within the myofibril. Bisecting the I-bands
The light bands have a consistent index of are dark lines known as Z-lines. The structure
refraction (isotropic). Therefore, these bands between two Z-lines is the sarcomere. In a
are called I-bands in reference to this isotro- relaxed muscle cell, the distance between
pic 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 single
A-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 dependent
the 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 been
strength, 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 sarcomeres
reason, they are often referred to as “salt- per muscle fiber, while the soleus has been
soluble” proteins. Myofibrillar proteins make estimated to have approximately 1.4 × 104
up 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

26. 8 Chapter 1
by 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 is
attached to the cell membrane (sarcolemma) required for enzymatic activity, and the other
by intermediate filaments that interact not has regulatory functions.
only with the Z-line, but also with structures Actin is the second-most abundant protein
at 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 approximately
marily of the protein titin (Wang et al. 1979; 400 actin molecules per thin filament. Thus
Wang 1984; Wang et al. 1984; Wang and the molecular weight of each thin filament
Wright 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) are
development in postmortem muscle, it is the 1 μm in length and are anchored in the
interdigitating thick and thin filaments that Z-line.
supply the “machinery” needed for these pro- Two other proteins that are important in
cesses and give skeletal muscle cells their muscle contraction and are associated with
characteristic 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 makes
the 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 polypeptide
1984). The backbone of the thin filaments is chains (alpha and beta) The alpha chain has
made 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 of
the protein myosin. Together, these two pro- approximately 36,000. These two chains
teins make up nearly 70% of the proteins in interact with each other to form a helix. The
the 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 termed
elle. Myosin is a negatively charged protein troponin I (MW 23,000), troponin C (MW
with 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 calcium
daltons) that contains six polypeptides. released from the sarcoplasmic reticulum,
Myosin consists of an alpha helical tail (or troponin I can inhibit the interaction between
rod) region that forms the backbone of the actin and myosin, and troponin T binds very
thick filament and a globular head region that strongly to tropomyosin. The cooperative
extends from the thick filament and interacts action of troponin and tropomyosin in
with 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 actin
which is important in the regulation of con- and myosin and thus is a major regulator of
traction. Each myosin molecule contains two contraction. Calcium that is released from the
heavy chains (approximately 220,000 daltons sarcoplasmic reticulum is bound to the tropo-

27. Chemistry and Biochemistry of Meat 9
nin complex and the resulting conformational Central to the existence of the muscle cell
changes within troponin cause tropomyosin is the production of adenosine triphosphate
to move away from sites on actin to which (ATP), the energy currency of the cell. ATP
myosin binds and allows myosin and actin to consists of adenosine (an adenine ring and a
interact. ribose sugar) and three phosphate groups (tri-
For contraction to occur, the thick and thin phosphate). Cleavage of the bonds between
filaments 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 the
to as actomyosin. In electron micrograph control of the concentrations of key ions (like
images of contracted muscle or of postrigor calcium) in the muscle cell. Cleavage of Pi
muscle, the actomyosin looks very much like from ATP produces adenosine diphosphate
cross-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 ATP
versible and are also known as rigor bonds, is central to survival of the cell, there is a
as 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 and
globular head of myosin also has enzymatic in the very early postmortem period.
activity; it can hydrolyze ATP and liberate Muscular activity is dependent on ample
energy. In living muscle during contraction, supplies of ATP within the muscle. Since it
the ATPase activity of myosin provides is so vital, muscle cells have developed
energy 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 in
the center of the sarcomere. This produces the muscle cell, such as glycogen, lipids, and
contraction by shortening the myofibril, the phosphagens (phosphocreatine, ATP), and it
muscle cell, and eventually, the muscle. The can use energy sources recruited from the
myosin and actin can disassociate when a blood stream (blood glucose and circulating
new molecule of ATP is bound to the myosin lipids). Which of these reserves (intracellular
head (Goll et al. 1984). In postrigor muscle, or circulating) the muscle cell uses depends
the 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, the
permanent. muscle will utilize a higher proportion of
energy sources from the blood stream and
lipid stored in the muscle cell. These will be
Muscle Metabolism
metabolized to produce ATP using aerobic
From a metabolic point of view, energy use pathways. Obviously, ample oxygen is
and production in skeletal muscle is simply required for this process to proceed. During
nothing short of amazing in its range and high intensity activity, during which ATP is
responsiveness. 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 utilized
This can represent an increase in the mus- very quickly and their depletion leads to
cle’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 is
1999). critical; available ATP must remain above

28. 10 Chapter 1
approximately 30% of the resting stores, or with ATP (100 mmol/kg dry muscle weight
relaxation 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 low
ATP, which is especially important because abundance compared with glycogen (500
removal 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 easily
phosphocreatine, glycogen, glucose lactate, reversible and phosphocreatine supplies
free fatty acids, and triglycerides. Glucose can be readily restored when ATP demand
and glycogen are the preferred substrates for is low. In living muscle, when activity is
muscle metabolism and can be utilized either intense, this system can be advantageous, as
aerobically (oxidative phosphorylation) or it consumes H+ and thus can reduce the
anaerobically (anaearobic glycolysis). Lipid muscle cell acidosis that is associated with
and lactate utilization require oxygen. Lipids anaerobic glycolysis. Another advantage of
are a very energy-dense storage system and the system is that the catalyzing enzyme is
are very efficient with respect to the high located very close to the actomyosin ATPase
amount of ATP that can be generated per unit and also at the sarcoplasmic reticulum (where
of 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 a
compared 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 fairly
glycogen 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, either
energy system, requires oxygen to operate, through the oxidative phosphorylation or
and that oxygen is supplied by the blood through anaerobic glycolysis (Fig. 1.1). One
supply to the muscle and by the oxygen trans- of the key steps in the fate of glycogen is
porter, 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 be
where in the neighborhood of 50% saturated. completely broken down to CO2 and H2O
Under conditions of extreme hypoxia (as (yielding 38 mol of ATP per mole of oxidized
found in postmortem muscle), oxygen sup- glucose-1-P produced from glycogen or
plies 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 (yielding
the 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 initial
tive 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 anaerobic
turn solely to anaerobic pathways for energy pathway, as oxygen supplies are rapidly
production. depleted. This results in the buildup of the
Phosphocreatine in living, rested muscle end product, lactate (lactic acid), resulting in
is available in moderate abundance compared pH decline.

29. Chemistry and Biochemistry of Meat 11
Figure 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 localizing
Tenderization
in the M-line at the center of the sarcomere
During refrigerated storage, it is well known and the N-terminal forming an integral part
that 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 the
occurring in the architecture of the myofibril thick filaments, maintaining the location of
and its associated proteins. There are several the thick filaments between the Z-lines. Titin
key 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 of
Titin
the earliest proteins expressed, and it is
Titin (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 the
addition 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 titin
abundant. A single titin molecule is estimated in living cells, it is quite conceivable that

30. 12 Chapter 1
its degradation in postmortem muscle would extends from the Z-line to the pointed ends
lead to weakening of the longitudinal struc- of the thin filament. The C-terminal end of
ture of the myofibrillar sarcomere and integ- nebulin is embedded into the Z-line. Nebulin
rity of muscle. This weakening, in conjunction is highly nonextensible and has been referred
with 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 the
radation 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 been
Melody 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 anchoring
only slightly faster under SDS-PAGE con- the thin filament to the Z-line (Wang and
ditions than intact titin. This product migrates Wright 1988; Komiyama et al. 1992).
at approximately 2,400 kDa (Kurzban and Degradation of nebulin postmortem could
Wang 1988, 1987; Huff-Lonergan et al. weaken the thin filament linkages at the
1995). Another titin degradation product Z-line, and/or of the thin filaments in the
that 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 to
1995). This latter polypeptide has been be capable of linking actin and myosin (Root
shown 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 regulatory
in the I-band (Kimura et al. 1992), although function in skeletal muscle contraction (Root
the 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 junction
certain. The 1,200-kDa polypeptide has been have the ability to bind to actin, myosin, and
documented to appear earlier postmortem in calmodulin (Root and Wang 2001). More
myofibrils from aged beef that had lower interesting, this portion of nebulin (spanning
shear force (and more desirable tenderness the A-I junction) has been shown to inhibit
scores) 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 region
derness scores (Huff-Lonergan et al. 1995, of nebulin also has been suggested to inhibit
1996a, b). The T2 polypeptide can also be the sliding velocities of actin filaments over
subsequently degraded or altered during myosin. If the latter role is confirmed, then it
normal postmortem aging. Studies that have is also possible that nebulin’s postmortem
used 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 and
periods of postmortem storage or μ-calpain interactions of thick and thin filaments in
digestion (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;

31. Chemistry and Biochemistry of Meat 13
Huff-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-T
Troponin-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 of
ance of polypeptides migrating at approxi- troponin-T and the concomitant production
mately 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 an
et al. 1974; MacBride and Parrish 1977; indicator of overall postmortem proteolysis
Olson 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 integral
nin-T can be degraded by μ-calpain in vitro part of skeletal muscle thin filaments (Greaser
to 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 careful
in 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 and
identified in postmortem muscle. Increasing through its interaction with tropomyosin aids
postmortem time has been shown to be asso- in regulating the thin filament during skeletal
ciated with the appearance of two major muscle contraction (Greaser and Gergely
bands (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 that
28 kDa, which label with monoclonal anti- postmortem degradation of troponin-T and
bodies to troponin-T (Huff-Lonergan et al. disruption of its interactions with other thin
1996a). In addition, the increasing postmor- filament proteins aids in the disruption of the
tem aging time was also associated with a thin filaments in the I-band, possibly leading
loss of troponin-T, as has been reported in to fragmentation of the myofibril and overall
numerous studies (Olson et al. 1977; muscle integrity. During postmortem aging,
Koohmaraie et al. 1984a, b; Ho et al. 1994). the myofibrils in postmortem bovine muscle
It 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-myosin
studies have shown labeling of two very interactions (Greaser and Gergely, 1971;
closely spaced bands corresponding to intact Hitchcock, 1975; McKay et al. 1997; Lehman
troponin-T. This is likely due to isoforms of et al. 2001), it is also conceivable that its
troponin-T that are known to exist in skeletal postmortem degradation may lead to changes
muscle (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 in
Both the appearance of the 30- and 28-kDa the I-band, alters thick and thin filament
bands and the disappearance of the intact interactions, or simply reflects overall protein
troponin-T in the myofibril are very strongly degradation, its degradation and appearance

32. 14 Chapter 1
of 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 enzyme
Koohmaraie et al. 1984a, b; Koohmaraie μ-calpain may be, at least in part, responsible
1992; 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 this
Lonergan 1999). degradation is truly directly linked to tender-
ization or is simply an indicator of overall
postmortem proteolysis remains to be
Desmin
determined.
It has been suggested that desmin, an inter-
mediate filament protein (O’Shea et al. 1979;
Filamin
Robson 1989) localized at the periphery of
the 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 protein
development 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 et
al. 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). The
of myofibrils. They connect adjacent myofi- amount of filamin in skeletal and cardiac
brils at the level of their Z-lines, and the muscle is very low (approximately ≤0.1% of
myofibrils to other cellular structures, includ- the total muscle protein). In skeletal and
ing the sarcolemma (Robson, 1989; Robson cardiac muscle, filamin is localized at the
et al. 1995). Desmin may be important in periphery of the myofibrillar Z-disk, and it
maintaining 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 of
fibrils) 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 in
membrane, could affect the development of lateral register. Degradation of filamin may
tenderness. Desmin is degraded during post- also alter linkages connecting the peripheral
mortem 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 between
and 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 associations
Furthermore, it has been documented that or costameres (Robson et al. 1995). A study
desmin is degraded more rapidly in myofi- using myofibrils from beef showed that some
brils from samples with low shear force filamin was degraded to form an approxi-
and higher water-holding capacity (Huff- mately 240-kDa degradation product that
Lonergan et al. 1996a; Huff-Lonergan and migrated as a doublet in both myofibrils from
Lonergan, 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) has
This degradation product also has been been seen in cultured embryonic skeletal
shown to be present in μ-calpain-digested muscle cells and was attributed to calpain

33. Chemistry and Biochemistry of Meat 15
activity (Robson et al. 1995). Uytterhaegen the total water in muscle cells; depending on
et al. (1994) have shown increased degrada- the measurement system used, approximately
tion 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. Since
stimulate proteolysis and postmortem tender- the total concentration of protein in muscle
ization (Wheeler et al. 1992; Harris et al. is approximately 200 mg/g, this bound water
2001). Compared with other skeletal muscle only makes up less than a tenth of the total
proteins, relatively little has been done to water in muscle. The amount of bound water
fully characterize the role of this protein in changes very little if at all in postrigor muscle
postmortem tenderization of beef. Further (Offer and Knight 1988b).
studies that employ a combination of sen- Another fraction of water that can be
sitive detection methods (e.g., one- and found in muscles and in meat is termed
two-dimensional gels, Western blotting, entrapped (also referred to as immobilized)
immunomicroscopy) are needed to determine water (Fennema 1985). The water molecules
the role of filamin in skeletal muscle systems in this fraction may be held either by steric
and 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, this
Water-Holding Capacity/Drip
water does not flow freely from the tissue, yet
Loss Evolution
it can be removed by drying and can be easily
Lean muscle contains approximately 75% converted to ice during freezing. Entrapped
water. The other main components include or immobilized water is most affected by the
protein (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 can
analyzed as ash, approximately 1%). The also eventually escape as purge (Offer and
majority of water in muscle is held within the Knight 1988b).
structure of the muscle and muscle cells. Free water is water whose flow from the
Specifically, within the muscle cell, water is tissue is unimpeded. Weak surface forces
found 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-rigor
fibrils and the cell membrane (sarcolemma), meat, but can develop as conditions change
between muscle cells, and between muscle that allow the entrapped water to move from
bundles (groups of muscle cells) (Offer and the structures where it is found (Fennema
Cousins 1992). 1985).
Water is a dipolar molecule and as such is The majority of the water that is affected
attracted to charged species like proteins. In by the process of converting muscle to meat
fact, 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 possible
bound water is water that exists in the vicin- in meat is the goal of many processors. Some
ity of nonaqueous constituents (like proteins) of the factors that can influence the retention
and has reduced mobility (i.e., does not easily of entrapped water include manipulation of
move to other compartments). This water is the net charge of myofibrillar proteins and
very 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

34. 16 Chapter 1
amount of extracellular space within the relaxation (Millman et al. 1981; Millman
muscle 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 this
in Muscles That Affect
water can be affected by changes in volume
Water-Holding Capacity
as muscle undergoes rigor. As muscle goes
During the conversion of muscle to meat, into rigor, cross-bridges form between the
anaerobic 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 and
builds up in the tissue, leading to a reduction Trinick 1983). It has been shown that as the
in 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 between
charge of the protein is zero, meaning the the thick filaments declines an average of
numbers of positive and negative charges 2.5 nm (Diesbourg et al. 1988). This decline
on the proteins are essentially equal. These in filament spacing may force sarcoplasmic
positive and negative groups within the fluid from between the myofilaments to the
protein are attracted to each other and result extramyofibrillar space. Indeed, it has been
in a reduction in the amount of water that can hypothesized that enough fluid may be lost
be attracted and held by that protein. from the intramyofibrillar space to increase
Additionally, since like charges repel, as the the extramyofibrillar volume by as much as
net charge of the proteins that make up the 1.6 times more than its pre-rigor volume
myofibril approaches zero (diminished net (Bendall and Swatland 1988).
negative or positive charge), repulsion of During the development of rigor, the
structures within the myofibril is reduced, diameter of muscle cells decreases (Hegarty
allowing those structures to pack more 1970; Swatland and Belfry 1985) and is
closely together. The end result of this is a likely the result of transmittal of the lateral
reduction of space within the myofibril. shrinkage of the myofibrils to the entire cell
Partial denaturation of the myosin head at (Diesbourg et al. 1988). Additionally, during
low 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 water
large part of the shrinkage in myofibrillar within the myofibril. In fact, it has been
lattice 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-field
muscle cell. As mentioned previously, much nuclear magnetic resonance (NMR) studies
of the water inside living muscle cells is have been used to gain a more complete
located within the myofibril. In fact, it is esti- understanding of the relationship between
mated that as much as 85% of the water in a muscle cell structure and water distribution
muscle cell is held in the myofibrils. Much (Bertram et al. 2002). These studies have
of that water is held by capillary forces suggested that within the myofibril, a higher
arising from the arrangement of the thick and proportion of water is held in the I-band than
thin filaments within the myofibril. In living in the more protein-dense A-band. This
muscle, it has been shown that sarcomeres observation may help explain why shorter
remain isovolumetric during contraction and sarcomeres (especially in cold-shortened

35. Chemistry and Biochemistry of Meat 17
muscle) 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 framework
would lead to shortening and subsequent responsible for attaching the myofibrils to the
lowering of the volume of the I-band region sarcolemma. Proteins that make up or are
in myofibril. Loss of volume in this myofi- associated with the intermediate filaments
brillar 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 costameric
of water from the myofibrillar structure linkages remain intact during the conversion
into 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 be
fact, 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 ultimately
the gradual mobilization of water from the reduce volume of the muscle cell itself (Offer
intramyofibrillar spaces to the extramyofi- and Knight 1988b; Kristensen and Purslow
brillar spaces may be key in providing a 2001; Melody et al. 2004). Thus, the rigor
source of drip. process could result in mobilization of water
All the previously mentioned processes not only out of the myofibril, but also out of
influence the amount of water in the myofi- the extramyofibril spaces as the overall
bril. 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 has
responsible for the movement of fluid to the been observed in postmortem muscle (Offer
extracellular space and ultimately out of the and Cousins 1992). This water that is expelled
muscle. The myofibrils are linked to each from the myofibril and ultimately the muscle
other and to the cell membrane via proteina- cell eventually collects in the extracellular
cious connections (Wang and Ramirez- space. Several studies have shown that gaps
Mitchell 1983). These connections, if they develop between muscle cells and between
are maintained intact in postmortem muscle, muscle bundles during the postrigor period
would 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 are
et al. 1988; Morrison et al. 1998; Kristensen the primary channels by which purge is
and 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 “drip
constriction of the entire muscle cell, thus channels.”
creating channels between cells and between
bundles of cells that can funnel drip out
Postmortem Changes in Muscle
of the product (Offer and Knight 1988).
That Influence Quality
Extracellular space around muscle fibers con-
tinually increases up to 24 hours postmortem, As muscle is converted to meat, many
but 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 from
postmortem, perhaps due to fluid outflow aerobic to anaerobic metabolism favoring the
from these major channels (Schafer et al. production of lactic acid, resulting in the pH
2002). These linkages between adjacent of the tissue declining from near neutrality to
myofibrils 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

36. 18 Chapter 1
calcium, sodium, and potassium pumps to that is involved in increasing the tenderness
function; and (4) an increasing inability of of fresh meat and in influencing fresh meat
the cell to maintain reducing conditions. All water-holding capacity (Huff-Lonergan and
these changes can have a profound effect on Lonergan 2005). Because μ-calpain and
numerous proteins in the muscle cell. The m-calpain enzymes contain both histidine
role of energy depletion and pH change have and SH-containing cysteine residues at their
been covered in this chapter and in other active sites, they are particularly susceptible
reviews (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 in
oughly considered is the impact of other postmortem muscle lead to inactivation or
changes 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 autolysis
Another change that occurs in postmortem (Guttmann et al. 1997; Guttmann and Johnson
muscle during aging of whole muscle prod- 1998; Maddock et al. 2006). In postmortem
ucts is increased oxidation of myofibrillar muscle, there are differences between
and sarcoplasmic proteins (Martinaud et al. muscles in the rate that postmortem oxidation
1997; Rowe et al. 2004a, b). This results in processes occur (Martinaud et al. 1997). It
the conversion of some amino acid residues, has been noted that differences in the rate of
including histidine, to carbonyl derivatives oxidation in muscle tissue are seen when
(Levine et al. 1994; Martinaud et al. 1997) comparing the same muscles between animals
and 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 in
these changes reduce the functionality of pro- diet, breed, antemortem stress, postmortem
teins in postmortem muscle (Xiong and handling of carcasses, etc. In fact, there have
Decker 1995). In living muscle, the redox been reports of differences between animals
state of muscle can influence carbohydrate and between muscles in the activity of some
metabolism by directly affecting enzymes in enzymes involved in the oxidative defense
the glycolytic pathway. Oxidizing agents can system of muscle (Daun et al. 2001).
also influence glucose transport. Hydrogen Therefore, there may be genetic differences
peroxide (H2O2) can mimic insulin and stim- in susceptibility to oxidation that could be
ulate glucose transport in exercising muscle. capitalized on to improve meat quality. It is
H2O2 is increased after exercise, and thus oxi- reasonable to hypothesize that differences in
dation systems may play a role in signaling the antioxidant defense system between
in skeletal muscle (Balon and Yerneni 2001). animals and/or muscles would influence
Alterations in glucose metabolism in the calpain activity, proteolysis, and thus
ante- 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 inhibits
important 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 the
meat quality. The proteolytic enzymes, the substrate it was shown that the presence of
calpains, are implicated in the proteolysis oxidizing species does significantly impede

37. Chemistry and Biochemistry of Meat 19
the ability of calpains to degrade their sub- (NOS). There are three major isoforms of
strates. 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 thought
Leu-Val-Tyr-AMC, regardless of the pH or to be the predominant isoform (Kaminski and
ionic 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 following
strate. This inhibition was reversible, as reaction: L-arginine+NADPH+O2 forming
addition of reducing agent (DTT) to the oxi- L-citrulline+•NO+NADPH+. NO is important
dized samples restored activity. Oxidation in biological systems, particularly because of
also 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 other
et 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 to
et 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. Proteins
increase 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 be
that very low levels of oxidation can influ- inactivated by peroxynitrite. Among these is
ence proteolysis and that increasing the level the sarcoplasmic reticulum Ca2+-ATPase
of antioxidants in meat may have merit in (Klebl et al. 1998). One indirect effect of
improving 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 and
of 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 the
observed in meat. activity of the protein. Because of this, it
has been suggested that S-nitrosylation may
function as a post-translational modification
Nitric 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 ryanodine
term that includes NO and reactive nitrogen receptor and the cysteine protease caspase-
species (RNS), like S-nitrosothyols, per- 3, have been shown to be endogenously
oxynitrate, and metal NO complexes. In nitrosylated, further supporting the sugges-
living tissue, NO is involved in arteriole dila- tion that formation of nitrosothiols may be
tion 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 also
and oxygen to the muscle (Kobzik et al. a cysteine protease that could be influenced
1994; Stamler et al. 2001). NO species are by S-nitrosylation. Small thiol peptides
also implicated in glucose homeostasis and like glutathione can be impacted by nitro-
excitation-contraction coupling. The gas NO sative stress to form compounds like
is 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

38. 20 Chapter 1
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Bendall, J. R., and H. J. Swatland. 1988. A review of the
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Relationship between meat structure, water moblity,
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Boehm, M. L., T. L. Kendall, V. F. Thompson, and D.
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Briggs, M. M., H. D. Mcginnis, and F. Schachat. 1990.
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Developmental Biology 140:253–260.
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X-ray-diffraction measurements of postmortem
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