Retrospective Theses and Dissertations Iowa State University Capstones, Theses andDissertations

1975

Postmortem physical and chemical events relatedto bovine skeletal muscle tenderizationDennis Gene OlsonIowa State University

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OLSON, Dennis Gene, 1947-POSTMOKTEM PHYSICAL AND CHEMICAL EVENTS RELATED TO BOVINE SKELETAL MUSCLE TENDERIZATION.

Iowa State University, Ph.D., 1975 Food Technology

Xerox University Microfilms, Ann Arbor, Michigan 48106

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

Postmortem physical and chemical events related

to bovine skeletal muscle tenderization

by

Dennis Gene Olson

â Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Bequirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Animal Science

fiajor; Heat Science

Approved;

arg

For the Hajor Department

For &h^#aduate College

Iowa state Oniversity AmeSf Iowa

/

1975

Signature was redacted for privacy.

Signature was redacted for privacy.

Signature was redacted for privacy.

il

TABLE OF CONTENTS PAGE

INTRODUCTION 1

Abbreviations 3

LITERATOHE REVIS» 5

Muscle Biology 5

Postiùortea Huscle 19

Influences oa Postmortem Muscle Tenderness 45

METHODS AND MATERIALS 67

Source of Muscle Tissue 67

Myofibril Preparation 69

Myofibril Fragmentation Determination 70

Light Microscop y 70

Troponin Preparation 71

Calcium-Activated-Factor (CAF) Preparation and Assays 72

Sodium Dodecyl Sulfate (SDS) Polyacrylamide G®1 Electrophoresis 74

Muscle Fiber Tensile Strength Measurement 76

Sarner-Bratzler {i-Bj Shear-Force and Sensory Panel Determinations 76

Data Analysis 78

RESULTS 79

DISCUSSION 171

SUMMARY 192

CONCLUSIONS 197

BIBLIOGRAPHY 201

ACKNOULEDGEHENTS

1

INTRODUCTION

Shortly after the essaagiiination of an animal, a series

of chemical and physical events are initiated in the first

24-48 hours post-mortem. Daring this time skeletal muscle

transforms from a physical state of being elastic and pliable

to a state of being stiff and rigid, termed rigor mortis, to

one of being pliable again. Energy stores in muscle are de­

pleted and muscle pH falls due to anaerobic glycolysis during

rigor mortis. The significance of tbese chemical aad physi­

cal changes is that almost all meat quality attributes are

established at this time. Rigor mortis and its associated

events and relationships to meat tenderness have intrigued

meats researchers for many years. However, most researchers

,have been unsuccessful in elucidating and relating these

changes to tenderness.

Tenderness is one of the meat quality factors that is

considered to be very important in meat palatability. It is

eell known that a number of variables affect tenderness, but

the one that is still not understood is the large increase

that occurs in tenderness diiring pûstâOEtes storage. For

example, it has been shown that muscles left attached to ths

carcass skeleton are least tender immediately after death and

become progressively more tender with increasing time post­

mortem. The causes for the increase in meat tenderness dur­

ing storage have remained obscure even though many postmortem

2

biochemical and structural muscle changes have been well

characterized.

It is known, for example, that myofibril structure

weakens during postmortem storage, but it is unknown what

causes this structural weakening or whether it is related to

meat tenderness. It is also known that postmortem muscle

tenderization is influenced primarily by alterations in myo­

fibrillar proteins and not sarcoplasmic or stroma proteins,

but it is unknown what myofibrillar protein alterations di­

rectly influence meat tenderness or what causes those altera­

tions. It has been hypothesized that postmortem muscle ten­

derization results from proteolysis by enzymes which have

been isolated from muscle, but it has not been shown that

muscle proteases directly affect muscle tenderness, While

many postmortem muscle characteristics have been thoroughly

investigated and well established, the associatiou ox these

characteristics to postmortem muscle tenderization has not

been clearly made.

Z-line degradation in myofibrils has been shown to occar

during postmortem muscle storage which causes myofibrils to

fragment into small pieces. Z-line degradation clearly has

an influence on the strength of the muscle fiber and hence

should be related to meat tenderness. Also a

calciuE-activatad-sarcoplasmic protease (CAF) has been shows

to selectively degrade Z-lines in myofibrils. It seems

3

probable that CàF may cause a weakening in ths myofibril

structure daring postmortem storage which would be reflected

in an increase in meat tenderness.

The objectives of this study were to 1) develop a quan­

titative method to determine the extent of Z-liae degradation

by measuring myofibril fragmentation, 2) characterize specif­

ic changes in myofibrillar proteins during postmortem storage

since sarcoplasmic and stroma proteins have little influence

on postmortem tenderness changes, 3) determine relationships

of CAF activity to postmortem myofibrillar protein changes

and myofibril fragmentation and 4) correlate postmortem chan­

ges in myofibrillar proteins and myofibril fragmentation to

postmortem changes in meat tenderness.

abbreviations

h angstroms

ATP adenosine triphosphate

âTPase adenosine triphosphatase

C Celsius

Ca2+ calcium

Câ? calcium-activated-factor

cm centimeter

CP creatine phosphate

EDTA ethylenediaminetetraacetic acid

g gravity

gm grams

4

hr hour

K potassium

kg kilogram

H molar

MCE mercaptoethanol

mg milligram

Hg2+ magnésium

ml milliliter

mM millimolar

P probability

SDS sodium dodecyl sulfate

SR sarcoplasmic reticulum

V volume

w Height

W-B Warner-Bratzler

ug microgram

us sicrosetsr

y.S.D.a. Dnited States Departseat of Agriculture

5

LITERATURE REVIEW

Muscle Biology

Skeletal muscle is a highly specialized tissue used foe

skeletal support and locomotion. A discussion ox the intri­

cacies of muscle and its relationship to meat requires a re­

view of some of the fundamental structural features of skele­

tal muscle- The following brief discussion of skeletal mus­

cle structure is based on reviews of the microstructure of

muscle and muscle eoaponeats by Brisksy and Fskazasa (1971)#

Gould (1973) and Huxley (1972). From a gross anatomical

viewpoint, skeletal muscles are usually conical in shape, ta­

pering at both ends where they are attached by tendons to the

skeleton. Hicrostructurally a connective tissue sheath sur­

rounds an entire muscle, termed the epimysium, and it pro-

tre^es imto the body of the muscle at various locations to

separate the muscle into bundles or fasciculi. The layer (or

sheath) of connective tissue surrounding the fasciculi is

termed the perimysium. Another connective tissue sheath an­

astomoses and surrounds the individual muscle fibers (cells)

called the enâoaysiss, and it liés just outside the suscle

cell membrane or sarcolemma. Hence the functions of the en­

tire connective tissue system are to maintain the organiza­

tion of the muscle, provide greater efficiency of movement

during muscle contraction and support the vast network of

branching blood vessels and nerves throughout the auscle sys-

6

tern.

The muscle fiber is a complete cell which is nombranch-

ing, cylindrical and naltinucleated with tapering conical

ends and having variable diaensioBS. The length of the mus­

cle fiber averages 2 to 3cm and has a diameter that ranges

from 10 to lOOum. Bithin the muscle cell there are loag,

nonbranching threads of proteins, 1 to 3am in diaaster lying

parallel to one another along the long axis of the cell.

These threads of proteins vhich travel the entire length of

the cell and have no membranes surrounding them are termed

myofibrils. They contain the contractile apparatus of the

muscle cell. Hyofibrils exist as structural entities of tha

cell because they are insoluble at the ionic strength of the

cell. Myofibrils are bathed in a solution termed the sarco-

plasm containing many soluble proteins. The muscle cell also

contains mitochondria (sarcosomesj , sarcotabuiai: system, uu-

serous nuclei, Golgi apparatus and lipid droplets.

Myofibrils have segusntial light and dark bands «ith the

direction of banding perpendicular to the long azis of the

myofibril. Because myofibrils lis in register so that their

light and dark bands are aligned at right angles to the long

axis of the fiber, a banded appearance is conferred upon the

entire muscle cell. These banding patterns take their names

fron their appearance under polarized light; the light bands?

or I-baads are weakly birefringeat (isotropic) and the dark

7

bands, or A-bands, are strongly birefringent (anisotropic).

Bisecting the center of the I-band is a dark line, the Z-

line, and in the center of the A-band is a light zone, the H-

zone. Bisecting the H-zone is a thin line of protein, called

the M-line. The region from one Z-line to the next is de­

fined as a sarcomere and this represents the contractile unit

of the myofibril.

Myofibrils are comprised of fine, long filaments of pro­

teins seen only in the electron microscope, lying side by

side and extending longitudinally parallel with the loag axis

of the myofibril. Myofilaments do not continue through the

sarcomere but are divided into two kinds of filaments, termed

thick and thin filaments. Huxley (1953) described the stria­

ted muscle myofibril as a double array of interdigitating

thick and thin filaments. The thin filaments are attached at

nno onti f f kia iina anrt <aw+"o*i(^ i nf-/\ hatwaam a a —

jacent thick filaments whereas the thick filaments comprise

the A-band and are not attached to the Z-line, The I-band

contains only the thin filaments and the Z-line while the A-

band contains the thick filaments, H-zone, M-line and part of

the thin filaments. Thick filaments contain the protein myo­

sin and are approximately 110 A in diameter and 1.5um long,

while the thin filaments contain several proteins, actin,

tropomyosin and troponin and are approximately 65 A in

diameter and I.Oum long. A sarcomere is approximately 2 to

8

3ua long. During contraction thin filaments slide into the

A-band so that a contracted myofibril will have a narrower

I-band than in a relaxed myofibril while the à-band remains

at a constant length.

k review of the sarcotabalar system of the muscle cell

has been made by Franzini-Armstrong (1973). The sarcotubular

system consists of two parts: 1) the T-systsm or T-tubules

and 2) the sarcoplasmic reticulum (SE) or L-system. The T-

tubules are a series of membrane-limited tubules that pass

through the muscle cell generally perpendicular to its long

axis. These tubules are invaginations of the sarcolemma so

the lumen of the tubule is extracellular. T-tubules occur at

regular intervals along the muscle cell at every a-I band

junction or at every Z-line. The function of the T-tubules

is to spread nerve impulses very guickly from the sarcolemma

to ayoiibrilso The 35 consists of an sztsssivs network of

aeabrane-liaitsd tubules which extend laterally from the

center of each sarcomere to meet the T-tubule at either the

â-I band junction or at the Z-line. &s the SR membranes ap­

proach the T-tubule, they form a large sac, termed the later­

al cisternae adjacent to the T-tubule. The T-tubule has lat­

eral cisternae on either side of it which appears as three

closely adhering tubules in cross-section termed the triad.

The SB aeabranes have an enormous ability to accumulate Ca^fr

against a concentration gradient and whenever a nerve impulse

9

occurs, the membrane of the lateral cisternae is depolarized,

releasing Ca2+ into the cell near the myofibrils. When mem­

branes are repolarized, the free intracellular Caz* is ceac-

cumulated in the lateral cisternae. The release and reaccu­

mulation of Ca2+ serves as the switch to initiate or stop

contraction.

Huscle proteins have been classified into three groups,

sarcoplasmic, myofibrillar and stroma proteins, based on

their solubility in salt solutions of various ionic strengths

(Briskey, 1967; Goll et al., 1969b; Szent-Syorgi, 1960).

The sarcoplasmic protein fraction is soluble in a neu­

tral salt solution having an ionic strength below 0.2. This

class of muscle proteins makes up 34% of the total muscle

proteins (Hamm, 1960). Through differential sentrifugation,

inhomogeneoas fractions of the sarcoplasm can be isolated,

xucse fractions induis the szrcoplaszic sapernatant. the n.a-

clear fraction, the mitochondrial fraction and the microsomal

fraction» The sarcoplasmic supernatant contains the sarco­

plasmic proteins which are comprised of over 100 different

proteinso Out of a total of 55ag of sarcoplasmic proteins

approximately UOmg are glycolytic enzymes. Sag creatine kin­

ase, 0.2 to 2mg myoglobin, 3 to 6mg extracellular proteins

and 8 to 12mg of other proteins (Scopes, 1970). Obviously

glycolytic and associated enzymes are not in equal abundance

in the sarcoplasm. Glyceraidehyde phosphate dehydrogenase

10

fGâPDH) alone accounts for over 20% of the sarcoplasmic pro­

teins in rabbit muscle (Czok and Bucher, 1960). G&PDH,

phosphorylase b, aldolase, triose phosphate isomerase, eno-

lase, pyruvate kinase and lactate dehydrogenase make up over

half of the sarcoplasmic proteins eith probably over 100

other proteins in minute quantities making up the remainder

of the sarcoplasmic proteins. The nuclear fraction contains

ribonucleic acids, deoxyribose nucleic acids and lipopro­

teins. The mitochondrial fraction consists of 30% lipopro­

tein and the various enzymes and constituents of the

tricarboxylic acid cycle and the electron transport system,

including flavin nucleotides, various hemes and cytochromes.

The microsomal fraction consisting of lysosomes containing

acid hydrolases, proteolytic enzymes, cathepsins, acid phos­

phatase, and ribonuclease (Laakkonen, 1973) .

•The myofibrillar proteins, shich ars soluble in salt

soletions having an ionic strength of 0.4 to 1.5? constitute

about 50-55% of the total muscle proteins and are therefore

the largest class of muscle proteins (Briskey, 1967). In

this protein fraction there are at least nine myofibrillar

proteins, namely, myosin, actin, tropomyosin, tcoponin,

alpha-actinin, beta-actinin, component c and two H-line pro­

teins (Goll et al., 1974). These proteins are insoluble at

the ionic strength within the cell so that they exist as

structural entities making up the myofibril.

11

Hyosin represents 50-55% of tbe myofibrillar proteins

and is found in the thick filament of the myofibril. Myosin

has a molecular weight of approximately 468,000 daltons

(Losey et al., 1969). Byosin has two classes of subunits: 1)

those naturally existing in the molecule, and 2) those pro­

duced by brief proteolytic digestion. The natural subunits

of myosin consist of two similar molecular weight (KB) chains

of 200,000 to 210,000 daltons each and four small subunits

(light chains} of various weights (Lowey et al., 1969). The

different kinds of small subinits are: 1) tha 5,5'-dithiobis

- (2-nitrobenzoic acid) (DTNB) subunits, HW = 19,000 daltons;

2) the alkali-1 subunits, MB = 21,000 daltons; and 3) the

alkali-2 subunits, BH = 17,000 daltons. Each myosin molecule

has two of the DTNB subunits and two of the alkali-1 or

alkali-2 subunits in addition to the two large subunits.

On the other hand, a 1 to 5 aiadte digestion sith tryp­

sin, chysotrypsis or subtilisa at enzyme to myosin ratios of

1:300 {by weight) sill cleave the myosin molecule at its

center to produce teo fragments called light merosyosin (LMH)

and heavy msroayosia {HES) . The LHH is the tail part of the

molecule and the HKM contains part of the tail and the bul­

bous head of the molecule, fill of the ATPase activity, ;

actin-binding ability and the light chains of the myosin

aoleculs are found in the HSa fragment. The tails of adja­

cent myosin molecules aggregate to fora the shaft of the

12

thick filament and the heads of the myosin molecules are beat

oatvard to form the cross-bridges.

actin represents 15-20% of the total myofibrillar pro­

teins and is found in the thin filaments, àctin exists in. !

two forms in vitro: globular (G) actin and fibrous (F) actin.

Only F-actin is found injyivo and it consists of a double-

stranded fiber of s-actia subunits. G-actin bas a molecular

weight of 42,a00-»4,000 daltons and contains one molecule of

Ca2+ and one molecule of ATP per G-actin molecule and it is

polymerized to F-actin by the addition of 0.1f! KCl or ImH

Hg2+ (Laki, 1971). Actin and myosin together are both neces­

sary and sufficient for a contractile response if Mgz* and

ATP are present. The contractile response continues until

the ATP is depleted. This contractile response can not be

controlled (i.e. started and stopped at will).

•• /mm —» C O ̂ X K. mk ^ mm •• A i ^ « M» u.u xjx, vac la 7 x x xxa &.

protein and is found in thin filaments. The IH molecule

contains two subunits of approximately 33,000 and 36-000

daltons {Cohen et al., 197 3). TH lies along the length of

the thin filament in grooves of the actin helix®

Troponin (TH) constitutes 5-8% of the total myofibrillar

protein and is found in thin filaments. The troponin mole­

cule is a slightly asymmetrical spherical molecule having a

molecular weight of 80,000 daltons. The troponin molecule

consists of three polypeptide chains each having a specific

13

function (Hartshocne and Dreizen, 1973): 1)TN-T, haviag a

Bclecalar weight of 37,000-39,000 daltons, binds tightly to

TH but not actin, 2) TN-C, having a molecular weight of

18,500 daltons, binds to TN-T but not actia aad has four

high-affinity binding sites for Ca2+ and 3) TN-I, having a

molecular weight of 24,000 daltons, binds to the TN-T/TN-C

complex and also weakly binds to TB. TN-I strongly inhibits

the Mg2+-modified ATPase of actomyosin but this inhibition

can be derepressed by TN-C in the presence of Ca2+. Cohen et

al, (1973), Drabikowski et al. (1973) and Greaser et al.

(1973) have found that during the preparation and purifica­

tion of troponin, a neutral protease in muscle partially

degrades troponin yielding a 30,000-dalton molecular weight

protein. Moreover Dayton et al. (1974a, b) have found a

calcium-activated neutral protease isolated from muscle sar-

copiasm that degrades troponin after brief trèa-cméat. to yield

a 3Q,QQ0-daltGn solecalar weight protein» The troponin com­

plex is distributed periodically along the thin filament at

385 A intervals and it is bound to a specific site on TK

(Ohtsuki et al., 1957), The control of the Rg2+-modified

ATPase of actomyosin by troponin is therefore located in the

contraction regions of the myofibril by tropomyosin. The TH-

TN complex therefore regulates muscle contraction (Ebashi ̂

al., 1957, 1968).

14

Alp ha-actinia makes up approximately 155 af the total

myofibrillar protein and has a molecular weight of 200,000

daltons with two identical subunits of 100,000 daltons.

Schollaeyer et al. (1973) have shown that alpha-actin

constitutes the dense, aaorphous material of the Z-line and

Singh (1974), in a review of alpha-actinia, suggests that one

role of alpha-actini% in muscle is to enhance the structure

and stability of the Z-line. Dayton et al. (1974a, b) have

shown that a neutral, calcium-activated-factor (CkF) isolated

from the sarcoplaso of muscle removes alpha-actinin from myo­

fibrils. Purified alpha-actinin, however, is not proteolyti-

cally degraded by C&F.

The stromal proteins, which are insoluble in neutral

aqueous solvents, coaprise the connective tissue located in

the epimysiua, perimysium and endomysium. Goll (1962, 1965a,

bj has described the fibrous coanective tissue proteins to be

principally collages, elastin aad reticulia. Of these, col­

lagen is the principal protein found in connective tissue: it

makes up 1-2% of muscle (Goll, 1965a). Ths following brief

discussion on collagen structure is based oa reviews by Piez

(1966) and Veis (1970) . The basic structural unit of a col­

lagen fiber is the needle-like tropocollagen (TC) molecule

which consists of a group of three polypeptide chains. The

rigidity of the TC solecule is due to a triple helix struc­

ture which makes "ossible the formation of nasiaal amounts of

15

hydrogen bonds. The iaino acids, proline and hydrosyproline

make up about one-fourth of the linkages in the primary

structure of the collagen moïecule. With greater quantities

of proline and hydroxyproline, resistance of the molecule to

heat or chemical denatucation is increased, but the collagen

fiber is easily converted to a soluble form, gelatin, by

heating in aqueous solution. The temperatures required for

this conversion varies with 1) the nature of the solution,

over 60OC being required with water but lower temperatures

sufficing in the presence of acids or bases aad 2) advancing

animal age. Heat breaks hydrogen bonds in the TC molecule

which results in the separation of the peptide chains. One

TC molecule having no covalent bonds will give up three

alpha-chains. Some TC molecules have covalent intramolecu­

lar cross linkages binding together two alpha-chains, called

the beta-component, or binding ail three aipha-chaias toget­

her called, the gamaa-cosposent. â foraatios of interssolecu-

lar covalent bonds betveen adjacent alpha-sabanit chains is a

characteristic of the maturation of collagen. Sature colla­

gen fibrils Bay be represented as assemblies of tropocollagen

molecules joined together by a variety of intsrmolecalar co­

valent cross-linkages to form vast asymmetrical polymer

networkso No significant increase in the amount of collagen

occurs as aaiaal age increases {Goll, 1965a; Hill, 1966),

Bailey and Lister (1968) have shown that a proportion of the

16

covalent iatermolecalar cross-links in native collagen are in

a thermal labile form which gradually become stabilized with

increase in animal age. Stabilization of such a bond would

account both for the decrease in salt solubility and for tha

increase in shrinkage temperature with animal age.

Native aadenatared collagen fibers are relatively resis­

tant to enzysic digestion by enzymes except for collagenase.

Gelatin or denatured collagen, however, is susceptible to the

action of many proteolytic enzymes including papain, elas-

tase, ficin and trypsin. Furthermore elastin is the second

most abundant protein in connective tissue, however, there is

less than one-third as much elastin as collagen (Bilson et

al., 1954) in the muscles where elastin is most abundant.

Elastin fibers are smaller than collagen fibers and branch

freely to form networks (Partridge, 1966). Elastin is resis­

tant to aqueous extractions and not greatly aSfectei by

dilute weak acids or dilute bases at IQQOC for several hours.

The insolubility of elastin sssss to be due to the presence

of the desBosine cross-links among adjacent peptide chains.

Elastin is not hydrolyzed by trypsin or collagenase but it is

affected by ficin and elastase (Partridge, 1966) .

Reticulin is found in almost all connective tissue but

in only small asounts» It is morphologically similar to col­

lages and say possibly be a precursor of collagen; however^

the fibers are smaller than collagen, branch freely, and cos-

17

tain considerable amounts of lipid and carbohydrates not pos­

sessed by collagen. Beticulin is also resistant to aqueous

extraction at lOQoc, pH 7 for 2-3 hsr.

Because auscle contraction has an important bearing and

relationship to postmortem muscle shortening and meat tender­

ness, it seems appropriate to present a brief description of

the events and components associated eith auscle contractioa

(Bendall, 1960, 1966; Huxley, 1960, 1964, 1972; Taylor, 1972).

A nerve impulse transmitted to the muscle cell causes the

external membrane to depolarize. This «ave of depolarization

travels along the T-tubules to the interior of the cell to

the lateral cisternae of tfee SR shere a release of the Ca^+p

bound to the SR, occurs and causes the free intracellular

Ca2+ concentration to increase from about 1 X 10-? to 1 K

Free Caz+ which is bound to troponin and held at the

actin sits bj trcpcsjssin cassss a rslsass of the sspprsssiza

of Sg2+-activated âTPase of actia-ayosino The released sup­

pression permits the ATPase site on one of the myosin heads

to be activated by ggz+ to hydrolyse àTP» hs &TP is

hydrolyzed, the actin-conbining site on myosin and the

myosin-combining site on actin interact to generate a rela­

tive sliding movement of one set of filaments past the other.

Contraction of myofibrils is the result of repetitive cyclic

changes at the sites of interaction between actin and myosin.

During contraction, the sarcoaere length decreases but the

18

length of the thick and thin filaments remain constant. When

repolarization of the sarcolemma occurs and free intracellu­

lar Ca2+ is bound back to the SB leaving a level of 1 X 10-'H

or less free Ca2+ in the cell, the Mg2*-activated ATPase of

the actin-ayosin is suppressed by troponin. If the con­

centration is 8-10mM the act in-myosin complex is dissociated

and relaxation occurs.

Energy is used by auscle in sustaining the life process­

es of the cell and during musclé contraction. Hence the

reactions and events of muscle metabolism are very important

and these events have been reveieed by Kastenschmidt (1970),

Lardy (1966) and Lavrie (1966b) , Glucose, a product of di­

gestion, is carried to the muscle cell by the blood stream.

Glucose is stored in the muscle cell as glycogen uhen the

demand for energy is low. During high muscle activity, how­

ever, glycogen is catabolized back to glucose and âïP either

by aerobic or anaerobic mechanisms. Aerobic glycolysis

yields a 1% fold greater amount of ATP than doss anaerobic

glycolysis, hence aerobic glycolysis is the preferential

pathway of glucose breakdovwn. However, some muscles do not

have sufficient oxygen stores (due to a lower amount of

myoglobin in tha cell) to meet the needs of aerobic glycoly­

sis during vigorous activity. Such muscle cells, when stimu­

lated for heavy activity, rapidly incur an orygen debt and

must resort to anaerobic glycolysis to supply the remainder

19

of their needs. These muscle cells usually store large

amounts of glycogen and produce large amounts of lactic acid

during activity. also aerobic glycolysis occurs exclusively

in the muscle sarcosoaes (mitochondria) so the extent of

aerobic glycolysis is also limited soaevhat by the number of

mitochondria in the cell.

Postmortem Muscle

After the exsanguination of an animal, the muscle cell

attempts to continue its life processes and therefore contia-

ues to use ATP as a source of energy, since the glucose

supply to the muscle cell ceases immediately with

exsanguination, the muscle cell begins to degrade glycogen to

meet its energy needs. Aerobic glycolysis continues until

all oxygen stored in the myoglobin is exhausted after which

anaerobic glycolysis begins (Lardj; 1966). since there is no

longer any blood for transport of lactic acid away from the

cell, it accumulates and pH begins to decrease. The amount

of lactic acid produced (and therefore, the pH of the mus­

cle) depends on the amount of glycogen in the cell at death,

on the amount of glycogen used by aerobic glycolysis asd oa

the rats at ehich the glycolytic enzymes catalyze glycolysis

(Kastenschmidt, 1970)o In starved or exhausted animals, gly­

cogen levels at death may be low and very little acid is pro­

duced. Glycogen levels can also be depleted by antemortem

injection of adrenaline aad insulin (Bouton et al., 1971; de

20

Premery and Pool, 1963; Khan and Nakamara, 1970; Klose et

al.« 1970; Penny et al., 1963)= In estreae situations of

glycogen depletion, muscle pH may not even fall below 7.0,

this is called alkaline rigor. Normally, pH decreases from

7.0 to 7.2 at death to 5.3 to 5,6 24 hours aftec death

(Lavrie, 1966a). &s lactic acid accumulates and pH falls,

activity of some of the glycolytic enzymes decreases, and

glycolysis may stop before all glycogen is completely catabo-

lized (Lardy, 19 66). Bancs the amount of glycogen in muscle

immediately after death is not necessarily directly propor­

tional to the final postmortem pH of that muscle. The final

postmortem pH of muscle is called the ultimate pH and is usu­

ally about pH 5.3 to 5.6 which is near the isoelectric point

of actomyosin (Lawrie, 1966a). Ultimate pH of postmortem

muscle is dependent apon the initial pH of at-death musele,

the glycogen content of meat at the someat of death, the in­

tracellular oxygen available for aerobic glycolysis at the

moment of death and the buffering capacity of the muscle pro­

teins (Kastenschmidt, 1970).

When glycolysis ceases, either because of depletion of

muscle glycogen or becaase of inactivation of some glycolytic

enzymes, no more ATP is produced. The muscle cell has a res­

ervoir of high energy phosphate in creatine phosphate {CP}

which is used to very quickly replenish any ATP used in mus­

cle cells (Lardy, 1966), Creatine kinase rephosphorylates

21

ADP to ATP faster than ADP can be produced, even under tre-

aendoas energy utilization (Kasteaschmidt, 1970) . The ATP

level o£ living muscle cells never drops below the normal

physiological levels until CP concentration drops or until

the acid pH begins to denature creatine kinase. As creatine

phosphate is depleted or creatine kinase becomes inactivated

by lOH pH, ADP caanot be cephospfaorylated at the same rate as

it is produced by dephosphorylation of ATP. consequently,

ATP concentration begins to fall and may be completely de­

pleted or if the &TP-utllizing mechanisms are inactivated,

the postmortem decline in ATP concentration may stop when

muscle ATP concentration is still 10-20% of what it »as in

living muscle (Bendall, 1951).

The rate of pH fall has been shown to be greatly influ­

enced by postmortem storage temperature, but the effect of

temperature can vary among different muscles from the same

animal and among corresponding aascles of different species

(Bate-Smith and Bsndall, 1949; Bendall, 1951; 1960; Bssch ^

al., 1967; Cassens and Hewbold, 1966; 1966a, b; Cook and

Langsuorth, 1966; de Fresery and Pool, 1960; Lasrie, 1966a;

Harsh, 1954; Harsh and Thompson, 1958). As the storage tem­

perature declined from 37oc to Ooc, the rate of pH fall de­

clined in rabbit psoas major muscle (Bendall, 1960) and in

bovine psoas saior and ssEitendinosas sascle (Basck et al,,

1967) . However, ia bovine sternomandibularis muscle the rats

22

of pH fall is greater at loc than at 50C âariag the first fev

hours post-mortem (Cassens and Nevbold, 1967a; Nevbald and

Scopes, 1967).

As long as sufficient quantities of ATP are present in

postmortem muscle, the muscle will contract ( when stimu­

lated) and relax in a manner similar to physiological muscle

contraction and relaxation {Bendall, 1960). A muscle strip

excised from a muscle immediately after death will contract

or shorten because of the stimulatory effects of excision.

The muscle strip can also be easily extended by an applied

force (simulating relaxation). When ATP is greatly dimin­

ished, the muscle becomes fixed in whatever degree of con­

traction it happens to be at the time of ATP depletion

(Newbold and Harris, 1972) . Huxley and Brown (1967) have

shown that during muscle contraction only about 20% of the

cross-bridges on the thick filaments were actually attached

to the thin filaments at any one time. Shea ATP was de­

pleted, however, 100% of the cross-bridges were attached to

the thin filaments causing the muscle to be inextensible

(Huxley, 1968) since the thick and thin filaments are locked

into position and can no longer slide past one another. Sar­

comere length in myofibrils has been shown to be a measure of

the contraction state (degree of shortening) of the muscle

(Herring al», 1967Ô). The extent of postmortem shortening

is influenced by storage temperature, although not all mus-

23

des shov the same temperature dependence. Bate-Smith and

Bendall (1949) first showed this temperature affect when they

found that rabbit mascle stored at 37oc shortened 2,5 times

sore than muscle stored at 170C. Harsh (1954) showed the

amount of work done in terms of shortening against a load in

bovine muscle strips increased progressively as storage tem­

perature was increased from to 37®C. Locker and Hagyard

(1963) showed rabbit psoas major muscle, escised pre-rigor,

shortened 30% at 37oc but only 9% at 2®C. They also found

that postmortem bovine muscle shortened maximally at 2*0,

termed cold shortened muscle, minimally in the range of

14-19GC, and intermediately in the range of 15-370C. rhe ex­

tensive and dramatic shortening in bovine muscle at 2^Z,

termed cold- shortening {Locker and Hagyari, 1963) differed

from normal shortening in two respects: 1) it occurred while

aTP concentration was 1-2mg or more (which is highec thaa iu

normal rigor sortis) and 2) it %as reversible for a short

time after death. Ovins (Cook and Langsïorth, 1966) and por­

cine (Galloway and Goll, 1957; Hendricks et al., 1971) mus­

cles have also been shoen to exhibit some cold-shortening

characteristics.

Furthermore, Stroaer and Goll (1957a, b) have shown that

myofibrils from bovine semitendinosus and psoas manor muscles

escised at-death, yers relased, having aide I-bands and R-

zones in the sarcomere. After these muscles were allowed to

24

shorten at 2 ° C , the sarcomeres were greatly contracted having

no H zone and a very narrow I band. Henderson et al, (1970)

showed that sarcomeres were 1.6 to 1.7um in length at 24 hr

post-mortem in rabbit and porcine muscle stored at 2oc and

160C and in bovine muscle stored at 160C and 250C. Sarco­

meres were 1.3am in length in bovine muscle stored at 2 ° C f o e

24 hr. Sarcomeres were 1.3 to 1.6am in length in rabbit,

porcine and bovine muscles stored at 370C for 24 hr. Even

greater shortening can occur in muscles frozen pre-rigor and

then thawed and stored at 2oc called thaw shortening. Thaw-

shortening in excess of 70S of at-death length has been re­

ported in rabbit psoas major muscle (Lawrie, 1968), bovine

sternomandibularis muscle {Harsh and Leet, 1966b; Scopes and

Hewbold, 1968) and ovine lonqissiaus muscle {Harsh and

Thompson, 1958). The amounts of thaw-shortening and cold-

shortening decrease as the period between death and exposure

to cold or freezing conditions is estended {Locker and

Hagjard, 1963; Sarsh and Leet, 1S56fc; Harsh and Thompson,

1958)o

at some point after death, rigor mortis isvelops in the

muscles of the carcass. The term "rigor mortis" literally

means "stiffness of death" and refers to stiffness or rigidi­

ty of muscles that occurs at some varying time after death.

Host of the early sorfe on rigor sortis originated during the

1930*s from the Low Temperature Research Station at

25

Cambridge, England where E. C. Bate-Smith, J. R. Beadall, R.

A. Lavrie, B. B. Marsh, and ce-workers found that muscle

became inextensible during the onset of rigor mortis (Bate-

Smith, 1939). a kymograph was used to record the time-course

of postmortem muscle ezteasibility changes daring the devel­

opment of rigor mortis. This method consisted of periodic

loading and unloading of a 50gm weight on a muscle strip ex­

cised imaediately post-mortem and recording the resultant

amount of extension which occurred in the strip after

loading. It was shown that as rigor mortis developed, the

degree of muscle extension decreased to a point where the

muscle strip was no longer extensible. At this point the

mascle strip was considered to be in full rigor. The muscle

extensibility measurement was described in three different

phases—delay phase, rapid phase and postrigor phase. The

delay phase was the period of time during whish extensibility

does not change and S7P, being contissally resjsthesised by

glycolysis, sas maintained at antesortss levels. The rapid

phase was the period during which extensibility rapidly

decreases, creatine phosphate becomes depleted and &T? con­

centration falls to the point that all possible cross-links

were formed between the thick and thin filaments. The post-

rigor phase was a continuous phase in which little or no

change is extensibility occurred. This sas aa irreversible

phase since the eaergy stores needed for muscle extensibility

26

were depleted and could not be replenished. The method of

measuring muscle extensibility was later improved by Bate-

Smith and Bendall (1949), »ho developed a procedure foe auto­

matically loading and unloading the muscle strip by electri­

cal means. This instrument was further improved by Briskey

et al. (1962), who added sealed chambers in which humidity, temperature and gaseous atmosphere could be controlled or

changed. Because of the availability and widsspread use of

these instruments, rigor mortis has been almost universally

defined in terms of extensibility; a muscle which has become

inextensible is said to be in rigor mortis.

Muscle inextensibility, caused by the formation of

cross-links between the thick and thin filaments, occurs at

the same time as rigor mortis but does not cause muscle ri­

gidity. Muscle rigidity is caused by attempted shortening

(tension development) by two muscles on opposing sides of the

same bone (Goll, 1368). Since neither of these two auscles

were able to shorten substantially, this attssptsd shortening

amounts to an isometric contraction. The stiffness or rigid­

ity of an isometric contraction is directly responsible for

rigor mortis. Only recently has rigor mortis development

been measured by the amount of isometric tension generated by

postmortem muscles Busch et al. (1967)- Jungk and Harion

(1370) and Jungk et (1967) measured the asount of tension

developed in muscle strips held at a fixed length as it

27

passed into rigor sortis. These investigators showed that

Daziman isometric tension development occurred at approxi­

mately the same time as muscle inextensibility occurred.

However, isometric tension gradually decreased after aaximus

tension development even though the muscle remained inexten­

sible. Gradually increasing isometric tension development

corresponded to the onset of rigor, maximum isometric tension

development corresponds to maximum rigor and gradually

declining isometric tension corresponded to the resolution of

rigor mortis.

During the course of rigor mortis it is relatively well

recognized that muscles in a carcass, become less rigid.

This loss of rigidity is measured by a decline in isometric

tension, however, the postmortem measure of muscle extensi­

bility counterdicts any resolution of rigor mortis. Develop-

ssnt cf suscls inextensibility anâ saxiraaa isometric tension

occur at about the same postmortem time, but each develops by

different causes. The loss of muscle extensibility is di­

rectly related to âTP depletion in that postmortem muscle

loses its extensibility only after the ATP level has declined

to less than 20% of its antemortem level (Davies, 1967).

With such a low level of ATP, the cross-links between the

thick and thin filaments will not split, making the muscle

inextensible. Postmortem isometric tension develops as the

muscle attempts to shorten. This shortening requires ATP

28

(Bendall, 1951), so that shorteaiag and isometric tansion

must precede the loss of extensibility in postmortem muscle.

The onset of rigor mortis occurs as the muscle attempts

to shorten post-mortem. This postmortem contcactioa is

caused by the increase in free, intracellular Ca2+ concentra­

tions, just as an increase in free Ca2+ initiates contraction

in living muscle. The increase ia free Ca2+ in postmortem

muscle is due to the loss of Ca2+ binding ability of the SB

(Greaser et al., 1967) . The SS loses the ability to bind

Ca2+ by; 1) the loss of ATP because the energy of arp is

needed for Caz+ accumulation by the SS, 2) a low pH inhibits

Ca2+ binding by the SS and 3) possibly, proteolysis which

would destroy the ability of the SR to bind Ca2+.

The resolution of rigor mortis was proposed by Goll

(Goll, 1968; Goll et al., 1969b) to result from two altera-

Jkli U« J Ni ̂ CLO. W OJU CL V. JU W UO Ol JL C • I / O. lUO* U. ̂J.

cation of the actin-myosin bond resulting ia some subtle in­

crease in postmortem muscle sarcomere length and 2) a degra­

dation of the Z-line structure sith a weakening and possible

rupture of the bonds between the thin filaments and Z-

filaments.

The modification of the actin-myosin boni may be caused

by traces of residual ATP, A DP or other agents in the muscle

cell resulting in some slippage or partial dissociatioa at

the points where myosin cross-bridges interact eith the actin

29

filament. This slippage would cause slight lengthening of

the sarcomere and result in a lessening of tension in the

muscle (Goll et al., 1971a). Fujimaki et al. (1965a, b) have

shown that in the presence of ImM Mg2+ and 600mH KCl, myosin

B prepared from rabbit muscle after 7 days post-mortem was

dissociated to actin and myosin by only 0.1mM ATP, while

G.6aS &TP was required to cause the same dissociation in myo­

sin B prepared from at-death muscle. Goll and Robson (1967}

and Robson et al. (1967) found that the Bgz*- or Caz*- modi­

fied ATPase activities of myofibrils or myosin B prepared

from bovine psoas major and semitendinosus muscles at 24 hr

post-mortem at 2^Z or 16oc are 20-50% higher than the corre­

sponding activities from at-death muscles. Okitani et al.

(1967) observed that storage of myosin B in 0.6H KCl at pH

6.0 and 250C resulted in a 50-100% increase in Mg2+-modified

Âïfase acfcivityo Hay êt al* (1372) âûu Jones (1572) have

shoss that the Caz*- aad Mg2+-aodified âTPase activities of

chicken actomyosia increases during postmortem storage indi­

cating a change in the actin-ayosin interaction. Strap-dberg

et al. (1973) have reported that alteration of sulfhydrl

groups in at-death actomyosin produced many of the same chan­

ges in nucleoside triphosphatase activity as that caused by

postmortem storage.

Many investigators (Gothard et al=, 1956; Stroraer et al.

1967; Takahashi et al.. 1357) have shoen, ultrastructurally.

a tendency of rigor-shortened sarcomeres to lengthen between

12 and 30 hr post-mortem in the absence of ATP. stromer et

al. (1967) have shown that with bovine muscle, allowed to

cold-shorten, the sarcomeres will lengthen post-rigar by a

sliding of interdigitating filaments past one another caused

by a slippage of the interaction of the myosin cross-bridges

and actin filaments. These studies, however, were conducted

on myofibrils that were supercontracted to sarcomere lengths

of approximately I.Oom, and such shortening does not occur

normally in muscles attached to the carcass during rigor de­

velopment.

Degradation of 2-line structures would cause breaks to

occur in the myofibrils and as a result the tension held by

the muscle would necessarily lessen. Postmortem Z-line deg­

radation has been observed by Davey and Dickson (197 0), Davey

and Gilbert (1967; 1969), Headecsou et_al. (137C), Penny

(1970) and Takahashi et al» (1967) . Haga et ai. (1956) have

Sucwû that a break between the actin filaments and the z-line

oust occur before actin was extracted. Myosin can be solu-

bilized in one hour by extraction with 0.5H KCl solutions at

pH 7.0, but actin requires a considerably longer time for

extraction. Since myosin cross-bridges are bound to actin

during rigor mortis, actin extractability will affect myosin

estraction. If the actin-myosin bonds are not aeakenei or

dissociated, the rate of actosyosin extraction is dependent

31

on the rate of actio extraction or the rate of dissociation

of the thin filaments from the Z-lines (Goll at al,, 1969a).

Complete extraction of myosin without actin is possible when

pyrophosphate or ATP is present to dissociate and prevent

further interaction of myosin and actin (Haga, et al., 1965).

However, if the bonds between the thin filaments and Z-line

are weakened or disrupted actin will be extracted along with

myosin in the pyrophosphate or àTP-containing solutions

(Hihalyi and Rove, 1966).

The amount of actomyosin extracted has been shown to ia-

crease during postmortem storage (Chaudhry et al., 1969;

Davey and Gilbert, 1968a; Valin, 1968). Davey and Gilbert

(1968b) found, by extracting myofibrillar proteins with a

0.6H KCl solution at pH9,2, that extraction was complete in

60 minutes from myofibrils stored 2 days post-mortem, but

only 10 minutes was required to yield the same amount of myo­

fibrillar protein from myofibrils stored 17 days post-mortem.

They also found that by adding magnesium and pyrophosphate

to the extraction solution the total myofibrillar proteins

extracted from myofibrils increased from SUS at 1 day to 75%

at 17 days of postmortem storage. Myosin was almost com­

pletely extracted at 1 day post-mortem while at 17 days post­

mortem myosin, actin plus other myofibrillar proteins were

extracted (Davey and Gilbert, 1968a). These results suggest

that during postmortem storage a weakening of the z-line

32

occurs so that actia becomes soluble in pyrophosphate extrac­

tions.

Penny (1968), using the Ca^^-modified ATPase activity to

determine the amount of myosin, found that pyrophosphate-

containing solutions quantitatively extracted myosin from at-

death myofibrils, but extraction of myofibrils stored for 4

days at 15-180C or for 14 days at 40C caused solubilization

of myosin and tropomyosin. Chaudhry et al. (196 9) found that

increased postmortem storage time at 20C resulted in in­

creased amounts of actin extracted.

A weakening or rupture of the Z-line would also result

in a decrease of the tensile strength of the muscle fiber.

Stanley ̂ al. (1971) found that bovine lonqissimus muscle

strips at 1 day post-mortem required 15% greater force to

break the strips by opposing longitudinal forces than was re­

quired for muscle strips 7 days post-mortem. Bouton and

Harris (1972b) also found a significant reduction in fiber

tensile strength in bovine lonoissiaas muscles from 2 to 1%

days post-mortem. Additionally, during the preparation of

myofibrils by hoisogenization, fewer sarcomeres per myofibril

{myofibril fragmentation) would result if the number of

weakened or ruptured z-lines were greater.

Numerous workers {Da?ey and Gilbert, 1967, 1969;

Fukazaea et al.. 1969; Hay et al., 1973b; Henderson et al.,

1970; Parrish et al.* 1973b; Sayre, 1970; ïakahashi et al..

33

1967) have observed that during postmortem storage greater

fragmentation of myofibrils, breakage of the myofibril at the

Z-line into smaller segments, occurs after hoaogenization of

muscle tissue. Davey and Gilbert (1969) found that as post­

mortem storage time proceeded, muscle fiber pieces lose the

precise register of the myofibrils and finally break down to

single myofibrils. Since fragmentation of the myofibril

reflects the loss of strength or weakness of the z-line, in­

creased myofibril fragmentation during postmortem storage

indicates a weakening of the Z-line during storage.

The postmortem changes in sarcoplasmic proteins primari­

ly involve the proteins which have an integral role in muscle

metabolism. The energy stores, ATP and CP, are utilized re­

sulting in the products adenosine monophosphate (àMP) and

creatine (Scopes, 1970). Several sarcoplasmic proteins such

as creatine kinase are particularly susceptible to precipita­

tion at a low pH (about pS5«5) (Scopes, 196%). Because of

the vast number of different proteins in tàe sarcoplasmic

protein fraction, protein solubility studies have been made

on this fraction to determine gross effects of postmortem

storage. Sarcoplasmic protein solubility is normally

greatest immediately after death and then either remains un­

changed or decreases during postmortem storage (Chaudhry et

al.. 1969; Scopes and Laarie, 1963). If muscle pH falls

below 6.0 while the muscle temperature is 350C or higher.

34

however, sarcoplasmic protein solubility decreases substan­

tially (Sayre and Briskey, 1963; Scopes, 1964) . Chaudhry et

al. (1969) have shown that a large decrease in sarcoplasmic

protein solubility occurred in bovine and rabbit muscle dur­

ing postmortem storage above 30oc. On the other hand, rapid

cooling and storage of at-death muscle to resulted in

sarcoplasmic protein solubility to remain near the at-death

level (Borchert and Briskey, 1965; Goll et_al., 1964). Onder

normal postmortem storage conditions 10-30% of at-death

sarcoplasmic protein solubility is lost. Numerous investiga­

tors (&berle and Berkel, 1966; Borchert et al., 1969; Hay ^

al., 1973a; Lavrie et al., 1963; Haier and Fischer, 1966;

Neelin and Rose, 1964) have shown, however, that gel electro-

phoretic patterns of sarcoplasmic proteins extracted from at-

death muscle were very similar to those extracted from post­

mortem muscle stored at 50C or lower. Even when sarcoplasmic

protein solubility of postmortem muscle was lower than at-

death muscle, fee qualitative differences wers shown

(Borchert et al., 1969).

Heelia and Ecobichon (1966) have suggested that during

the preparation of sarcoplasmic proteins in hypotonic salt

solutions cytoplasmic structures such as the sarcolemma or

the sarcoplasmic reticulum are released into the sarcoplasa.

Furthermore, these proteins were increasingly released during

postmortem storage even when the sarcoplasmic proteins were

35

prepared in hypertonic sucrose solutions, which supposedly

preserve the cytoplasmic structure in at-death muscle. Bason

(1969), Greaser et al. (1967, 1969a, b, c) and Nakamura

(1973) have also found an alteration in the cytoplasmic

structure during postmortem muscle storage. These investiga­

tors have shown that the sarcoplasmic reticular membranes

very quickly lost their calcium sequestering ability after

death, even though their ATPase activity and ultrastructure

did not undergo large postmortem changes. Osner (1965) and

Reed et al. (1966) have also shown that the sarcolemma is a

very labile system that may change rapidly after death.

Postmortem changes in the stroma protein fraction has

been studied by numerous investigators. Winegarden et al.

(1952) reported that strips of collagenous connective tissue

from bovine skeletal muscle required a slightly lower shear-

force to shear theo afc 35 days of postmortem storage at 2^C

than at 10 days of postmortem storage at 2®C. Husaini et al.

(1950) and Hershbsrger et aie (1951) found that collagen and

elastin content, measured by alkali-insoluble nitrogen, of

bovine loaqissimus muscle decreased by 10-30% after 15 days

of postmortem storage at 30C. Prudent (1947) and Kierbicki

et al. (1954), however, reported no change in the alkali-

insoluble protein content of bovine muscle up to 15 days of

postaortes storage. Khan and van den Berg (1964) and Sayre

(1968) found the alkali-insoluble protein content of chicken

36

muscle remained constant during postmortem storage at O^c far

4 days and, in some instances, for several months.

While these studies did not investigate any ultrastruc­

tural changes in the fibrous connective tissue proteins, they

demonstrated that no large change in alkali-insoluble protein

content appears to occur during postmortem storage.

Sharp (1963) reported no change in the estractability of

hydroxyproline (a measure of soluble collagen content) during

postmortem storage of bovine muscle for 172 days at 37oc.

However, Herring et al. (1967a) reported that the percentage

of total muscle hydroxyproline that was solubilized by heat­

ing at 770c for 10 minutes increased significantly after

post-mortem storage at 40c for 10 days. These studies sug­

gested that a subtle change in collagen occurred during post­

mortem storage and this change was not detected during stor­

age at 370c but it was detectable when heated to 50-800C.

HcClain ̂ al. 51965) reported there was no change in total

collagen of bovine longissimus muscle during 7 days of post­

mortem storage at 40c, but that there was a gradual conver­

sion of neutral-soluble collagen to acid-soluble collagen

during this storage period. ScClain (1969) and EcClaia and

Pearson (1969) have also shown that a rapid drop in pH imme­

diately after death in porcine muscle can alter the solubili­

ty of collagen after heating. These results suggested that

postmortem storage affected the structure of the collagen by

37

the alteration of the number and strength of cross-linkages

between collagen molecules. Clayson et al. (1966) have sug­

gested that postmortem changes in cross-linkages between col­

lagen molecules were probably not due to proteolytic enzymes

bat rather caused by traces of oxidized nitrogen in a highly

active form.

These studies indicated that postmortem storage caused

some weakening or rupture of the collagen cross-linkages re­

sulting in increased collagen solubility after heating to

about 500C. De Fremery and Streetar (1969) found no increase

in susceptibility of collagen solubilization by heating dur­

ing postmortem storage at 2°C for 24 hr in poultry muscle nor

was any change found in the amount of alkali-insoluble pro­

tein during postmortem storage at 20C for periods up to 8

days. It is not clear to what extent postmortem storage in­

fluences connective tissue proteins, but it is indicated that

these influences are subtle and probably confined to changes

in the number and strength of collagen cross-linkages.

HcClain et al. (1970) suggested that the postmortem pH

fall, more than prolonged storage, altered the collagen

structure. Furthermore, Kruggel and Field (1971) and

Pfeiffer et al. (1972) suggested that the number of covalent

cross-linkages in collagen could be reduced by pre-rigor mus­

cle stretching. Herring et (1967a) noted, however, that

the amount of postmortem shortening did not significantly

38

affect collagen content nor solubility of bovine Iongissimas

muscle.

Almost sixty years ago Hoagland reported (Hoagland et

al.# 1917) that proteolysis was an important factor contrib­

uting to postmortem changes in muscle proteins. Whitaker

(1964) suggested that the postmortem changes in muscle were

due to proteolytic activity affecting the myofibrillar pro­

teins. Goll (1968) proposed that the alteration of the

actin-myosin interaction and the degradation of the Z-line

during postmortem storage may have been due to very limited

and specific proteolysis. Pairrish and Bailey (1966, 1967)

showed the presence of a proteolytic enzyme, cathepsin D, in

bovine muscle and presented evidence that it was located in

the lysososes. lodice et al. (1966), Parrish and Bailey

(1967) and Suzuki and Fujimaki (1968) found cathepsin D was

the active protease in skeletal muscle alchougâ C&ldwell anS

Grcsjean (1971) found cathepsin A «as more active in chicken

skeletal muscle. Ds Dure {1959, 1963) deaonstrated that ly-

sosomes contained a number of acid hydrolases including ca­

thepsin D and that the function of lysosoces was that of acid

digestion under physiological and pathological conditions (le

Duve and Wattiauz, 1966), such as an increase in catheptic

activity in dystrophic muscle (Tappel et al., 1962; Weinstock

et ale. 1955; Zalkia et al.* 1962). Pellegrino and Franzini

(1953) showed that lysosomes appeared only during muscle

39

atrophy and that no lysosones were present in normal auscle.

Price et al. (1964) and Smith (1964) indicated that lysosomes

of apparent muscle origins were confined to blood vessels and

macrophages. Canonico and Bird (1970), however, demonstrated

the existence of two locations for two distinct groups of ly­

sosomes; one group originated from macrophages and connective

tissue cells and the other group originated in the muscle

cells. Those lysosomes in muscle cells contained cathepsin D

and acid phosphatase» Stauber and Bird (1974) have shown

that lysosomes in skeletal muscles are part of the sarcotubu-

lar system of the cells.

Classically, the extent of proteolysis in postmortem

muscle is determieed by aa increase in free amino acids and

non-protein nitrogen (Parrish, 1971). Increases in free

amino acids and non-protein nitrogen have been observed in

postmortem beef auscle (Davej and Gilbert, 1966; Field and

Chang, 1969; Field at al., 1971; Gardner and Stewart, 1966;

Locker, 1960b; Parrish et al., 1969b) poultry muscle (Khan

and van den Berg, 1964; Miller et al., 1965) and rabbit mus­

cle (Suzuki et al.. 1967). Parrish et ai. (1969b) showed a

four-to-six fold increase in several amino acids but the pro­

tein source of these amino acids could not be determined, al­

though it was suggested that the myofibrillar proteins were

aa unlikely source. Additionally, most of the increase in

free amino acids and non-protein nitrogen occurred from 7 to

40

28 days post-mortem which is the period after which most of

the myofibril structural change had already occurred (Parrish

et al., 1969b) . Bodvell and Pearson (1964) and Sharp (1963)

concluded that the increase in free amino acids largely orig­

inated from the sarcoplasmic protein fraction which has

little influence on meat tenderness. Locker (1960b) using N-

terminal group analysis, found no evidence that myofibrillar

proteins were proteolytically cleaved daring postmortem stor­

age. Friedman et al. (1969) , Fukazawa and ïasui (1967) and

Park and Pennington (1967) have shown no proteolysis of in­

tact myofibrils or thick and thin filaments. From the stud­

ies of several investigators (Clayson et al., 1966;

Etherington, 1971; Soil, 1965b) it was concluded that there

was no significant proteolysis during postmortem storage in

the sarcoplasmic and stroma proteins.

Goll et al. (1971b) and Stromer et al. (1967) treated

supercontracted myofibrils briefly with trypsin in tke

absence of ATP and found that sarcomeres lengthened from

I.OuQ to 1.7UB. Tryptic digestion of at-âaath myofibrils

also caused a 50-8055 increase in the ng2+- and Caz*- modified

ATPase activities, which is similar to ATPase activities of

myofibrils from postmortem muscle. The close similarity be-

tseea the effects of trypsin on myofibrils and the effects of

postmortem storage on myofibrils suggests that the increased

ATPase activity and the lengthening observed in postmortem

41

muscle may be due to a very limited and specific proteolysis

(Soil, 1968). Busch et al. (1972) and Suzuki and Goll (1974)

have shown that many of the characteristic alterations in

Mg2+-modified ATPase activity of actomyosia by postmortem

storage can be duplicated by brief incubation of at-death

actomyosin with a calcium-activated factor (CàF), isolated

from muscle tissue, which sill also selectively degrade Z-

lines- Goll et al. (1969b), in a review, discussed three

sites in the myofibril which are labile to proteolysis, based

on known vulnerability to trypsin. The first site is myosin,

which is rapidly cleaved into light and heavy meromyosin by

trypsin (nihalyi and Szent-Gyorgi, 1953) or chymotrypsin

(Gergely ^ al., 1955). Both Bodwell and Pearson (1964) and

Sartins and Bhitaker (1968) have shown that neither actomyo­

sin nor myosin is affected by partially purified muscle ca-

thepsins. Also, myosin âîPase has not been fDuad to change

appreciably during postsortes storage (Goll and Robson, 1967;

Penny, 1968; Sobson et al., 1957; Su and Sayre, 1971) as

would be expected if myosin had been proteolyticaHy degrad­

ed. However, Suzuki et al. (1969) have shown some hydrolysis

of the myofibrillar proteins by purified cathepsin D from

rabbit muscle. Dayton et_al. (1974a, b) have shown that pur­

ified C&P does not proteolyticaHy degrade myosin even though

the intact myofibril has been degraded. On the other hand,

Suzuki and Goll (197 4) have shown a very slight increase in

42

myosin ATPase after treatment of myofibrils with CAF.

all work considered, it can be stated that the function­

al activity of myosin may be slightly modified by CAF, but no

significant proteolysis of myosin occurs due to CAP or

catheptic action.

The second proteolytically-labile site in the myofibril

is the tropomyosin-troponin comples. This complex has been

shown to be rapidly degraded to small peptides by trypsin

(laki, 1957; Ebashi and Kodama, 1966). However, biochemical

evidence on calcium-sensitivity of the ATPase of postmortem

myofibrils has shown that the tropomyosin-troponin complex

was not proteolytically destroyed, even after 13 days post­

mortem at 160C (Galloway and Goll, 1967; Goll and Robson,

1967), Davey and Gilbert (1966) observed that tropomyosin

remains difficult to extract even after 21 days post-mortem

at 20C indicating no extensive dégradation of trcposyosin^

àrakawa et (1970a, b) showed that yields of alpha-actinin

and the tropomyosin-troponin fraction were not changed by

postmortem storage and that these t^o protein fractions pre­

pared from postmortem muscle stored for 8 days at 25oc were

functionally active and exhibited normal sedimentation pat­

terns. These findings indicated that no extensive degrada­

tion of alpha-actinin, tropomyosin or troponin occurred dur­

ing postmortem storage. However^ ârakawa et al. (1970a)

acknowledged that some weakening in the interaction between

43

the tropoayosin-troponin complex and actin had occurred.

Fobson (1971) and Robson and cohen (1971) found a significant

decrease in the calcium-sensitive response of myofibrils even

after 1 day post-mortem at 2 oc and this decrease continued to

decrease up to 7 days post-mortem in both bovine and porcine

muscles. Additionally, Suzuki and Goll (1974) have shown

that brief treatments with CAF increased the Bg2+-nodfied

ATPase activity of at-death myofibrils indicating that this

sarcoplasmic enzyme diminishes the inhibition of troponin-I

on the Mg2+-modified ATPase activity of actomyosin (Ebashi

and Kodama, 1966). Furthermore, Dayton et al. (1974a, b)

found that CAF degraded troponin and tropomyosin into several

fragments ranging in molecular weight from 10,000 daltons to

30,000 daltons, but did not degrade myosin, actin or alpha-

actinin even after prolonged incubation periods. Alpha-

actinin was shosn to se released from the S-lins vithcut

being proteolyticallj degraded. Hay ^ (1973a) observed

in chicken breast myofibrils stored ap to 168 hr post-mortem

that little change occurred in the migration of the proteins

on SDS-polyacryiamide gels except for the obvious appearance

of a 30,000-dalton molecular weight protein appearing after

48 hr post-mortem. They suggested that this protein may be a

proteolytic breakdown product of myosin or troponin B.

Dayton ̂ al. (1974b) reported that very brief treatment of

purified troponin with CAF yields a 30,000-àalton molecular

4U

weight protein from troponin-T. These findings indicate that

CAP, which is proteoiytically active under normal postmortem

muscle conditions (Dayton et al., 197Ua, b; Suzuki and Goll,

1974) may cause a very limited and specific proteolytic deg­

radation of the troponin-tropomyosin complex which may not,

however, inhibit its functional activity.

The third possible site for postmortem proteolysis of

myofibrils is at or near the Z-line. Goll et al. (1969) and

Stromer et al, (1967) have shown that brief treatments of at-

death myofibrils with trypsin quickly removes the Z-lines and

releases alpha-actinin. Furthermore, Stromer and Goll

(1967a) have shown that at 13 days of postmortem storage at

either 20c or 160C, bovine myofibrils are mors extensively

fragmented into myofibrils of three or four sarcomeres in

length than immediately after death. The fragmentation of

the myofibril occurred due to breaks at the S-lins, In­

creased fragmentation of the myofibril occurred during post­

mortem storage of chicken muscle after 4 days at 2°c

(Takahashi et al., 1967) and after 2 hr at IS^C post-mortem

plus 22 hr at 3oc (Sayre, 1970) . Fukazawa and Yasui (1967)

have reported complete loss of Z-line structure in chicken

muscle after 24 hr at 2°C, however, incubation of at-death

myofibrils, having intact Z-lines, with cathepsins did not

produce any loss of Z-line structure, While Z-line degrada-

tioû of myofibrils was a very dramatic postmortem observa-

45

tion, cathepsins apparently did not influence this degrada­

tion.

Basch et al. (1972) found that a muscle strip held iso-

metrically in a saline solution containing Ca^-s- had a faster

decline in postrigor isometric tension and considerably

greater myofibril fragmentation than a muscle strip held in

only saline or saline plus EDT& solution. Datrey and Gilbert

(1969) prepared myofibrils from bovine sternoaandibuiàris

muscles at 30 hr post-nortea and stored those myofibrils in a

KCl-phosphate, KCl-phosphate-EDTA or clarified sarcoplasmic

extract solution for up to 20 days. This study showed that

1) EDTA-containiag solution inhibited any change in fragmen­

tation for 20 days of storage, 2) myofibrils in the KCl-

phosphate solution were very fragmented after 10 days of

storage and 3) myofibrils in the sarcoplasmic extract were

extremely reagmeated before 10 days of storage^ This stady,

indicated that s<ae factor in the sarcoplasmic extract, which

is inhibited by EDTA? eshances myofibril fragmentation.

Henderson et al. (1970) found Z-line degradation occyrrsd

within 24 hr after death in bovine^ porcine and rabbit muscle

with bovine muscle exhibiting the least degradation. These

muscles were stored at 2°C, 16®C, 25oc or 37oc during the

first 24 hr post-mortem with Z-line degradation being greater

at the higher storage temperatures. This study indicated

that some factor in aascle, activated by higher temperatures.

46

enhanced Z-line degradation causing greater myofibril frag­

mentation. Busch et al. (19 72) sere successful in isolating

a crude calcium-activated-sarcoplasmic factor (CAP) which

selectively degraded 2-liaes. Dayton et al, (1974a, b) have

purified this enzyme and have shown that it completely re­

moved the Z-line of the myofibril in 5 minutes at 25oc.

Suzuki and soil (1974) have shown that crude CiF Has active

at pH 5.5, at low temperatures (50C) and at laH Ca^* levels

{Dayton et al., 1974a); these conditions are typically found

in post-rigor muscle. It is highly possible that z-line deg­

radation observed in postmortem muscle is caused by CAP

action on the myofibril.

Influences on Postmortem Muscle Tenderness

It has been universally recognized for over a century

that the storage of muscle post-mortem above QOC for a period

of several days improves the tenderness of that muscle. The

complexities of the postmortem tenderization of muscle have

been studied by numerous investigators for the past several

decades and it was not until now that these complexities have

been caraveled to a sore snderstaniabls Isvsl.

Both the rate and extent (ultimate pH) oE the postmortem

fall in pH have been associated with the tenderness of meat.

Treatments which increase the rate of pH and ATP fall have

been shown to increase poultry meat toughness (de Fremery and

Pool* 1950; Khan and Nakamura, 1970). Busch et al. (1967),

47

however, did not find a parallel increase in tenderness with

the rate of pH drop in bovine seaitendinosns and psoas major

muscles stored at 20C, 160C or 37oc. Bouton at al. (1957)

found that bovine muscle with an ultimate pH of 6.0 was least

tender whereas tenderness was higher when the ultimate pH was

either higher or lover than 6.0» In contrast, tenderness of

rabbit muscle {Miles aad Lasrie, 1970), ovine muscle (Bouton

et al., 1971) and fish muscle (Kelly et al., 1966) has been

shown to be greater with higher ultimate pH.

While these studies indicate some possible relationships

between the rate and extent of pH fall in postmortem muscle

and meat tenderness, no adequate explanation has been given

for a causal effect of pH on structural changes in the myofi­

bril that would directly influence meat tenderness. The cir­

cumstances surrounding the pH fall probably indicates other

events or conditions in the muscle cell that more directly

influences meat tenderness. Bouton et (1973a) have shown

that the influence of muscle contraction on meat tenderness

was diminished as the ultimate pH of bovine muscle increased.

The rate and extent of pH fail in postmortem muscle may in­

dicate the rate and extent of biochemical reactions occurring

without pH directly affecting postmortem muscle tenderiza-

tion.

Locker (196 0a) attributed seat toughness to two

components: background and actomyosin toughness. Connective

48

tissues constitute the background toughness and the myofi­

brillar proteins constitute the actomyosin toughness. The

relationships of background toughness and actomyosin

toughness to the total meat toughness is not constant since

some muscles have different amounts of connective tissue than

others (Vognarova et al., 1968). Because no objective method

exists for measuring background and actomyosin toughness ac­

curately and independently, the relative contributions of

these tjro components of tenderness to total toughness is dif­

ficult to determine in any given sample of meat. Estimates

of the relative contributions of connective tissue to total

meat toughness can be made by fiber adhesion measurements

(Bouton and Harris, 1972a, c; Pool, 1967) ehich measure the

strength or weakness of the connective tissue between muscle

fibers. W-B shear-force values (Bouton and Harris, 1972b;

Bouton et al., 1973b) or fiber tensile strength measurements

fBouton and Harris, 1972b, c; Stanley et ai., 1972), which

are estimates of actomyosin toughness, measura the strength

or weakness of the myofibril. Purchas (1972) has used such

measurements together with an analysis designed to express

all measurements on the same relative scale and has concluded

that a 1% change in actomyosin toughness produced the same

tenderness change as a 10% change in background toughness.

Paul ̂ al. (1973) also determined that myofibrillar proteins

influenced muscle texture considerably more than collagen.

49

While connective tissue establishes the primary degree of

meat tenderness, it is clear that actooyosin toughness, hence

myofibrillar proteins, have extremely important effects on

postmortem muscle tenderization.

The relationship between the changes in the muscle pro­

teins during postmortem storage and the change in meat ten­

derness has been studied by numerous investigators. Thompson

et al. (1968) found that the solubility of sarcoplasmic pro­

teins of bovine lonqissiaus muscle from a carcass side stored

at 30®C for the first 24 hr post-mortem was slightly higher

than from the carcass side stored at 3oc for 3 days post­

mortem. However, no significant qualitative protein changes

were found to be related to the tenderness differences found

between the treatments. McClain and Hullins (1969) found no

relationship between the amount of extracted sarcoplasmic

proteins of bovine muscle and the muscle tenderness. Dikeman

et al, (1971) found a correlation coefficient of -0.70 be­

tween sarcoplasmic protein solubility and muscle tenderness

using bovine lonqissisus muscles of animals of A and E matur­

ity. However, they also found sarcoplasmic protein solubili­

ty increased with animal age indicating that protein solubil­

ity and tenderness may both be directly related to animal age

and only indirectly related to each other.

From the results of these studies and because the sarco­

plasmic proteins do not constitute a part of the muscle

50

Structure, it is doubtful that the sarcoplasmic protein frac­

tion directly affects meat tenderness. The active role that

this protein fraction plays in the tenderness of meat may be

due to the proteolytic enzymes found in this fraction (Busch

et al.a 1972; Dayton et al., 1974a, b; Randall and HacRae,

1967) .

As mentioned previously, connective tissue is important

in that it establishes the basal level of meat tenderness.

Connective tissue is thus important in the tenderness differ­

ences in muscles from animals of different maturities, Goll

(1965a), Hill (1966) and Shimokomaki et al. (1972) have shown

that the number of intermolécular covalent bonds in collagen

increases as an animal matures and grows older. Giffee

(1962) and Kim et (1967) have shown that the increase ia

these covalent bonds of collagen is reflected in lower muscle

tenderness of older animals. Field et aJ.. (1969) found bo­

vine lonqissimns muscle epimysium to be less tender in older

animals, however, neither hydroxyproline content nor epimysi­

um thickness was significantly related to tenderness measured

by w-B shear. Cross et al» (1973) found a significant corre­

lation between the amount of collagen and elastin and the

evaluation by a sensory panel of the amount of connective

tissue residue reaaiaing at the end of Eastication. However,

no significant correlation was found between the amount of

collagen and elastin and the tenderness the sensory panel at-

51

tribttted to the muscle fiber component. Kruggel ̂ al.

(1970) found more aldehyde-type covalent cross-linkages in

acid soluble collagen in less tender bovine longissiaus mus­

cles than in more tender longissiaus muscles.

Elastin comprises less than one-third of the connective

tissue proteins (Soli et al., 1963) and it is considered to

have only miaisal influence on muscle tenderness (Cross et

al., 1973; Partridge, 1966).

These results indicate that background toughness of meat

is an important part of the total tenderness of meat, howev­

er, the influence that postmortem storage has on the improve­

ment of the connective tissue component of muscle tenderness

is not completely clear.

On a macromolecular level, intramuscular fat (marbling)

has been associated vith meat tenderness. Marbling in beef

longissiaus muscle has been used in D.S.D.&. Beef Quality

Grades to indicate the quality level (of which tenderness is

a principal part) of beef carcasses. Parrish (197%), la a

review, has found that, in only a fee selectively controller

studies, marbling accounted for as high as 36S of the varia­

tion in meat tenderness, but in most studies marbling ac­

counted for less than 10% of the variation in meat tender­

ness» It was concluded that iatranascalar fat has little in­

fluence on meat tenderness especially on the increase in seat

tenderness that occurs during postmortem storage. Parrish et

52

al (1973a) have shown that the degree of internal doneness of

broiled steaks from bovine lonqissiaus was a much more impor­

tant factor of meat tenderness than was marbling. Draudt

(1972) and Hamm (1966) , in reviews, have described the pre­

dominant changes in the two major structural components of

Buscle, the collagenoas and myofibrillar proteins, daring

heating. Heating to an internal temperature of 60oc causes

shrinkage and partial solubilization of collagen and minimum

drying- hardening and coagulation of the myofibrillar pro­

teins resulting in aasimum beef tenderness. Further heating

causes greater collagen solubilization, however, this is

offset by the toughening effect from coagulation of the myo­

fibrillar proteins. At high internal meat temperatures

(80OC-90OC), the meat becomes very tough due to the coagula­

tion of the myofibrillar proteins (Schmidt and Parrish,

1971). Cooking temperature can thus have extremely large in­

fluences on meat tenderness making comparisons between ten­

derness measures difficult when different cooking tempera­

tures are used.

Because the myofibrillar proteins play an integral role

in seat tenderness, (actosyosin toughness), the postmortem

changes in these proteins have been studied in their rela­

tionship to the postmortem tenSerization of meat. Haga et

alo (1965) f Sayre (1968) and Weinberg and Bose (1960) sug­

gested that the increase in postmortem actomyosin extraction.

53

caused by detachaant of thin filaments from z-line, vas re­

lated to the increase in postmortem meat tenderness. Corre­

lation coefficients of -0.69 (Hegarty et al., 1963) and -0.77

(Aberle and Herkel, 1966) have been found between the amount

of myofibrillar protein extracted post-mortem and W-B shear-

force measurements of bovine longissiaus muscles. Di^eman et

al. (1971) found a correlation coefficient of 0,66 between

myofibrillar protein extracted and sensory panel tenderness

of bovine lonqissimns muscle stored U days post-mortem. How­

ever, Goll et al. (1964) found no significant correlation co­

efficients between myofibrillar protein solubility and ten­

derness for muscles removed frost the carcass at-death or for

those left attached to the carcass. Chaudhry et al. (1969)

found no significant differences in the solubility of myofi­

brillar proteins up to 4 days of postmortem storage between

bovine psoas major and sesitendiaosus muscles, both of these

muscles being extremely different in their tenderness proper­

ties (Dumont, 1971; Herring et al., 1965b) . ncClais and

Hullins (1969) found no significant difference between myofi­

brillar proteins extracted from tender and less tender sus-

cles of bovine Ionaissiaus a useles» It appears that the sol­

ubility of myofibrillar proteins is not an accurate and

dependable indicator of meat tenderness, however, the change

in solubility during postmortem storage may indicate a weak­

ening of the Z-line-thin filament attachment which is proba-

54

bly related to postmortem muscle tenderization. Apparently,

however, myofibrillar protein solubility is not a precise

enough measure of that weakening to form a direct relation­

ship with muscle tenderness.

The onset of rigor mortis is caused by the attempted

shortening of the carcass muscles on opposing sides of the

skeleton. The degree to which the muscles actually shorten

could have important effects on the tenderness of those mus­

cles. Locker (1960a) concluded that there was a direct rela­

tionship between postmortem muscle shortening and muscle ten­

derness. Numerous investigators have examined the extent and

significance of postmortem shortening as it influences meat

tenderness (Buck and Black, 1967; Buck et al., 1970; Busch et

al., 1967; Cooper et al., 1968; Davey et al., 1967; Herring

et al.. 1965a, b; 1967b; Howard and Judge, 1968; Harsh and

Leet, 1966a, b; marsh et al., 1968; HcCrae et al., 1971;

Smith et al., 1971; Weidemann et, al., 1967; s sib our ri et al.,

1958) .

Using bovine sternoaandibalaris muscles excised soon

after death, Behnke et al. (1973), Davey et al. (1957), Harsh

(1972), Barsh and Carse {1974, and Harsh and Leet (1966a, b)

have shown that muscle shortening by up to 20% of the excised

length only slightly affects tenderness whereas muscle short­

ening from 20-40% causes a four-to five-fold decrease in ten­

derness. Further muscle shortening from 40-55% causes a

55

progressive increase in seat tenderness until it finally ap­

proximates the same level of tenderness found in muscles

shortened by less than 20%. Muscle shortening has also been

shown to similarly influence tenderness of ovine muscles

(HcCrae et al., 1971), The increase in toughness with short­

ening is considered to be due to the increased density of

myofilaments per fiber area caused by contraction of the sar­

comere. This increased density results in a greater number

of myofilaments which must be sheared across per fiber area

during mastication {flarsh and Leet, 1966b) .

Herring et (1965a, b) and Siskus et al. (1973) re­

ported a highly significant relationship between the amount

of muscle contraction, as evaluated by sarcomere length, and

tenderness in bovine muscles. Herring et al. (1967b) showed

that when a muscle was excised and permitted to severely

shorten during the development of rigor mortis, the sascle

did not have an acceptable sensory panel tsadsraess level

even after ten days of postmortem storage,

Goll ̂ al. (1964) showed that excising a muscle from a

carcass immediately after death stimulated a greater degree

of shorteiniag of the siiscle than in muscles left intact in

the carcass. These excised muscles had increased g-B shear

values (less tender) and after postsortes storage became more

tender, but steaks from these excised suscles zever did

become as tender as steaks from muscles left in the carcass.

56

This was explained on the basis of the effect of cold short­

ening on unrestrained nascles when stored under cold (2-40C)

conditions. Muscles left attached to the carcass, however,

became more tender during postmortem storage with most of

this increase in tenderness occurring the first 24 hr of

postmortem storage. Herring et al. (1974b) have also shown

that bovine semiteadinosus muscle excised at death which

shortened 36% of its excised length still improved in tender­

ness during postmortem storage.

Harsh and Leet (1966b) investigated the effect of muscle

shortening on meat tenderness and found that the excised pra-

rigor muscle stored at 1oc or 5oc had sarcomere lengths 50%

shorter than muscle excised post-rigor. The post-rigor ex­

cised muscle was found to always be more tender than muscle

excised pre-rigor, additionally, Seville et_al. (19 71) re­

ported that post-rigor bovine psoas major muscle had consid­

erably longer sarcomere lengths than post-rigor semitendino-

sus muscle, which is almost always less tender than the psoas

major muscle (Dumont, 1971). These studies indicate that

postmortem muscle shortening is detrimental to post-rigor

muscle tenderness, thus it is clearly desirable to minimize

or prevent postmortem muscle shortening.

Huscle restrained from shortening pre-rigor will not

shorten post-rigor (Harsh and Thompson, 1958) nor will it

become tougher by pre-rigor exposure to cold-shortening

57

conditions (Harsh and Leet, 1966b; Taylor et al., 1972).

Hascles on a suspended carcass are not all restrained to the

same extent because the amount by which a muscle can shorten

depends on the hanging position of the carcass during the de­

velopment of rigor mortis. Herring et al. (1965b) have shown

that the psoas major, latissinus dorsi and rectus femoris

muscles froîâ a bovine carcass side suspended by the ichilles'

tendon were more tender than the corresponding muscles from a

side suspended horizontally; semimembranosus, senitendinosus,

lonqissimus and biceps femoris muscles were less tender and

serrâtus ventralis and triceps brachii muscles were un­

changed. arango et al. (1970), Harris and Hacfarlane (1971)

and Hostetler et al. (1970, 1972, 1973) have investigated the

effects of hanging bovine carcass sides in different ways aad

have found that, compared with the conventional method of

hanging by the àchilles* tendon, hanging a carcass side by

the obturator foramen improves the tenderness of the loagis-

simus. semimembranosus and biceps femoris muscles. Beaton

and Harris {1972c) have shown that the tenderness of ovine

lonqissimus. semimembranosus and biceps femoris muscles can

be improved by hanging the whole carcass from the pelvis

instead of the âchilles* tendon, Davey and Gilbert (1974)

have shown that placing ovine carcasses in a standing posi­

tion pre-rigor prevents toughening due to shortening of most

carcass muscles. The improvement in tenderness results from

58

the reduction in or prevention of shortening in the affected

muscles.

Not only will the method of carcass suspension improve

meat tenderness but postmortem storage temperature also in­

fluences meat tenderness. Postmortem storage of a carcass at

160C or higher immediately after death for at least 12 hr

compared to storage at 2-4®C has been shorn to stimulate a

greater increase in postmortem muscle tenderization (Culp et

al., 1973; Fields et al., 1971; Judge et al., 1971; Hartin et

al., 1971; Parrish et al., 1969a, 1973b; Semlsk and Riley,

1974). The lonqissiaus muscles from intact ovine carcasses

held for various periods at 20oc before being exposed to

freezing conditions have been shown by Harsh et al. (1968) to

be more tender when the storage period exceeds 15 hr than

when it is less than 16 hr. HcCrae et aJ., (1971) have shown

that ovine muscle tenderness is greatly improved if the car­

cass is stored at 18oc froa 10-15 hr before freezing than if

held at a lower temperature for 16 hr. Smith et al. (1971)

concluded that storing a bovine carcass at 1S®C for the first

16-20 hr post-mortem produces as much improvement in lonqis­

siaus muscle tenderness as hanging the carcass at 2^Z by the

obturator foramen to prevent shortening. Bouton et_al.

(1974) have shown that postmortem storage of ovine carcasses

at 7-80C sill prevent the muscle toughness that is caused by

muscle shortening that occars when the carcass was stored at

59

0-1 OC.

Schmidt and Gilbert (1970) have compared the tenderness

of bovine lonqissinas and biceps feaoris muscles excised pre-

rigor and stored at 15®C for 24 or U8 hr Hith the tenderness

of corresponding muscles left attached to the other side of

the carcass and stored at 90C for 24 hr. The biceps femoris

and loaaissiaus muscles stored at IS^C for 24 hr were equiva­

lent in tenderness to the corresponding muscles restrained on

the carcass, stored at 9®C for 24 hr. The excised muscles

stored at 15*0 for 48 hr were significantly more tender than

the excised muscles stored at 15oc for 24 hr or the muscles

left attached to the carcass and stored at 9oc for 48 hr.

Besides reducing toughness by preventing or reducing

shortening by immediate postmortem storage at high tempera­

ture or by unconventional hanging of carcasses to provide

greater aascle restraint. Herring et ai. (1564) suggested

that tenderness may have resulted frcs increased tension

placed on the muscle in the carcass. Gillis and Henrickson

(1967, 1969) studied the influence of tension oa the sarco­

mere length of bovine seaiaeabraaosus and semitendiaosus mus­

cle strips excised at death and weighted with 1000 to SOOOgm

weights at 2°C. The increased tension resulted in increased

sarcomere length, increased muscle tenderness and decreased

fiber diameter. Buck et al. (1970) have sbowa that stretch­

ing muscles in a rabbit carcass by draping the carcass over a

60

hook and tying the feet together resulted in longer sarco­

meres of the lonaissiaus muscle myofibrils. Back and Black

(1967, 1968) indicated that the improvement in tenderness,

from stretching bovine lonqissimus muscle, resulted from a

weakening or thinning of the perimysial connective tissue

causing more denaturation during heating. Kruggel and Field

(1971) and Kruggel et (1970) found that stretching bovine

longissimus muscles reduced the number of aldehyde-type

cross-linkages of collagen. Buege and Stouffsr (1974a, b)

and Quarrier et al. (1972) stretched excised ovine and bovine

longissimus muscles with weights and found that only a 5%

stretch (increase over excised length) produced most of the

tenderness improvement that can be obtained from any applied

degree of muscle tension. These studies also showed that

even the stretched muscles continued to improve in tenderness

during postmortem storage. The rate of postmortem muscle

tenderization was not significantly different between the

stretched and intact muscles.

The following conclusions can be drawn from the studies

on shortening by the aforaentioned investigators: 1) muscle

shortening during rigor development is detrimental to muscle

tenderness, 2) extreme cold-shortening or thaw-shortening

causes an extreme and almost irreversible toughening effect

on muscle, however, these effects do not normally occur under

conventional carcass handling, 3) normal muscle shortening in

61

the carcass can be avoided or reduced by storage of the car­

cass immediately post-mortem at 8-250C for 8-20 hr (depending

on species and muscles) or by hanging the carcass in an

unconventional manner, such as by the obturator foramen, to

apply increased tension on some particular muscles of the

carcass and 4} shortened muscles, except those extremely

shortened by cold-shortening or tha»-shortening, to slightly

stretched muscles still undergo the process of postmortem

muscle tenderization. Hhile shortening or stretching muscle

pre-rigor may influence the post-rigor muscle tenderness, a

post-rigor storage period will normally improve the muscle

tenderness.

As previously discussed, one possible cause of the res­

olution of rigor mortis is the degradation of the Z-line. â

degradation or weakening of the Z-line in postmortem muscle

would also cause a decrease in the muscle tensile strength.

Sakamura (1972) found that muscle fiber tensile strength de­

clined during postmortem storage. Bouton and Harris (1972a)

and Stanley et al. (1972) found significant correlation coef­

ficients of 0.4 to 0.8 between fiber tensile strength and W-B

shear-fores values. These studies indicate that the degrada­

tion of the Z-line was reflected in the reduction of fiber

teasile strength which is correlated with seat tenderness. &

degradation or weakening of the z-lines in postmortem muscle

would also cause shorter myofibril fragments, due to breakage

62

at the Z-line during myofibril preparation and should improve

the tenderness of the muscle. Coaissiong (1974) found that

muscle fibers, treated with dithiothreitol which removed the

Z-lines, fragmented to almost all single sarcomeres during

homogenization indicating that the Z-line is essential for

the structural integrity of the myofibril. Since tke length

of the myofibril fragment should reflect the extent of Z-liae

degradation, myofibril preparations were characterized ac­

cording to numbers of myofibrils having specific lengths,

numbers of sarcomeres per myofibril fragment, by Fukazawa et

al. (1969) and Sayre (1969, 1970). Sayre (1959, 1970) meas­

ured fragmentation by using a scale from & to E, which com­

bined length of the myofibril fragment with state of contrac­

tion. The results of this study showed that fragmeatation

was not a reliable index of tenderness, bat it was noted that

widely distributed breaks in the myofibrils at the z-line may

cause appreciable tenderization that might not be detected by

microscopic examination. Parrish et ai. (1S73u) observed

that smaller myofibril fragments originated from the more

tender meat samples and suggested that myofibril fragmenta­

tion sas a much more important factor in meat tenderness than

was muscle shortening although the degree of fragmentation

was not quantified. Takahashi st al. (1967) and Fukazawa et

al. (1969) used a ratio of the number of myofibril fragments

having 1-4 sarcomeres to the total number of myofibril

63

fragments, although fragmentation as estimated by this pro­

cedure changed with post-mortem time and with different pH

values and temperatures, no relationship to tenderness was

found. Berry et al. (1974) found a significant but low cor­

relation coefficient (0.20) between shear-force values and

the number of sarcomeres per myofibril of bovine loagissimuB

muscle. High correlation coefficients were found between

myofibril fragmentation and B-B shear values in bovine Iqn-

gissiaus muscle (-0.75) (Huiler et al., 1974) and chicken

pectoralis muscle (-0.58) at 6 days of postmortem storage

(Whiting and Richards, 1971) .

Davey and Gilbert (1969) who acknowledged that micro­

scopic measurements of myofibril fragmentation involves some

subjective judgements and was subject to inherent sampling

errors, used turbidity of a myofibril suspension as a measure

of fragsentation. Turbidity results from lig»t scattsriag

froa BYofibrlls and its value sill be related in large part

to the number of fiber pieces in suspension, in increase ia

turbidity value should indicate an increase ia the number of

myofibrils in suspension. If equal protein concentrations

are used? an increase in turbidity should therefore, indicate

an increase in fragmentation, which results ia a larger num­

ber of shorter myofibrils. The advantage of a turbidometric

measure of fragaestatioa is that it allons a larger and more

representative sample than can be used microscopically.

64

Davej attd Gilbert (1969) confirmed that tarbidometric mea­

sures on myofibril suspensions paralleled microscopic obser­

vations of myofibril fragaentation, but the relationships of

these measures to meat tenderness were not investigated.

Holler et al. (1973), using the procedure developed by

Davey and Gilbert (1969), showed that fragmentation, as meas­

ured by percentage emission of a myofibril suspension on a

spectrophotometer, gave a correlation coefficient of -0.78

with W-B shear-force values. This was one of the first re­

ports that Z-line degradation as measured by myofibril frag­

mentation was highly correlated with bovine muscle tender­

ness. Love and Mackay (1962) and Kelly et al. (1966) found a

close relationship between fish muscle tenderness and the

optical density of a whole muscle homogenate without

separating sarcoplasmic or connective tissue from the homoge-

nate. Holler et al. (1973), however, showed that microscopic

measurements of myofibril fragmentation {average number of

sarcomeres per myofibril of "GO myofibrils measured) were not

as highly correlated with tenderness (0.61) as were turbidity

measurements of fragmentation (-0.78). This study did not,

hoeever, investigate the changes in fragmentation with in­

creasing time of postmortem storage to determine whether in­

creasing fragmentation during postaortes storage could be re­

lated to increasing tenderness daring this period.

65

Laakkonen (1973) has stated that the presence of a col­

lagen proteolytic enzyme in skeletal muscle has not been con­

firmed. Clayson et al. (1966) and Etherington (1971) have

shown that any change in collagen during post-mortem storage

can not be attributed to enzymatic action on collagen. How­

ever, numerous investigators (El-Gharbawi and Whitaker, 1963;

Dasson and Bells, 1969; Gottschall and Kies, 1943; Hiyada and

Tappel, 1956; Tappel et al., 1956; Tsen and Tappel, 1959;

Sang et al., 1958) have shown that application of proteolytic

enzymes of plant, animal and microbial origin exogenous to

muscle tissue improves meat tenderness. Wang et al. (19 57)

have microscopically characterized the effect of twslve en­

zymes on muscle components of freeze dried steaks. The

amount of proteolysis on the muscle components varied depend­

ing upon enzyme specificity. Plant and pancreatic enzymes

caused both collagen and myofibrillar degradation ehereas the

microbial enzymes exhibited priaariiy myofibrillar degrada­

tion. Several investigators (Goeser, 1361; Huffman st al.

1967a, b; Robinson and Goeser, 1962) have shown that antemoc-

tem injection of papain in bovine animals improves the ten­

derness of postmortem muscles. Rang and Rice (1970) and Rang

and Earner (1974) have shown that trypsin, bromelin and col-

lagenase were stronger solubilizing agents of the connective

tissue proteins while papain and ficin were more effective in

solubilizing the myofibrillar proteins. While collagenolysis

66

voald obviously increase meat tenderness, there appears to be

no significant postmortem proteolysis by endogenous proteases

of the connective tissue proteins (Clayson et al., 1965;

Etherington, 1971; Goll et al. 1969). While the above stud­

ies have shown that proteolysis of the muscle proteins

improves meat tenderness, these proteases are exogenous to

the suscle tissue, ho»ever, and can not therefore contribute

to natural postmortem meat tenderization.

Recently, Penny et al. (1974) have shown that freeze-

dried steaks from bovine semitendinosus muscle, reconstituted

in a solution containing C&F, were considerably more tender

than steaks reconstituted in solutions without CAP. The Z-

lines of the myofibrils from the steaks reconstituted in CâF-

containing solutions were also extensively degraded indicat­

ing that the tenderness improvement of CAP was due to the

structural weakening of the Z-iine. This study has shown

that C&F, which is an enzyme endogenous to the muscle cell

(Dayton et al., 1974a, b), improves meat tenderness in a sim­

ilar manner as has been shown to occur during postmortem

storage.

67

METHODS &ND MATEBI&LS

Source of Muscle Tissue

Muscle sanpies aad steaks were obtained from bovine ani­

mals Sleighing 400-500kg, 12-18 months of age and on similar

feeding regimes at the Iowa State University Beef Nutrition

Farm, ât-death samples »ere obtained from the anterior por­

tion of the H. longissiaus, B. semitendinosus and H. psoas

laaior within oae hour after exsanguination of the animal at

the Iowa State Oniversity Meat Laboratory, taken to the Food

Research Laboratory and used immediately for myofibril, C&F

or troponin preparation. Subsequent samples of the longissi-

mus. semitendinosus and psoas major muscles were removed from

the companion side of the carcass at 1, 2, 3, 6, 7, 10 or 13

days post-mortem (not all postmortem times were used in each

ezperiment). Samples for each postmortem tiss, sslacted at

random locations eithin each muscle, were used immediately

for myofibril fragmentation determination, CAF activity assay

or Instron fiber tensile strength determination. Steaks,

3.1cm thick, were removed from the muscle, packaged in

freezer paper 24 hr post-mortem and stored at 2®C. The

packaged steaks were randomly selected for each postmortem

storage period. On each particular postmortem day, the se­

lected steaks were frozen and stored at -20®c for 2-4 months

for subsequent sensory panel evaluation or Warner-Bratzler

(w-B) shear-force détermination.

68

Wholesale short loins from thirty-five A-maturity car­

casses grading D.S.D.A. Choice and twelve c-maturity carcas­

ses were randomly selected 24-30 hr after death (1 day post­

mortem) from a commercial packing company (Wilson and

Company, cedar Rapids, Iowa) . The wholesale short loins were

taken to the Iowa State University Heat Laboratory where five

3.1 CD steaks were removed from the anterior end of the loin

and packaged in freezer paper. Two steaks (one for sensory

panel and one for W-B shear) were randomly selected, frozen

and stored at -20°C» Two other steaks were stored at 2-Uoc

for 7 days post-mortem and then frozen and stored at -20oc.

The frozen steaks were stored for 14-40 days for subsequent

sensory panel evaluation and W-B shear-force determination,

a four-gram sample was removed from the fifth steak at 1 and

7 days post-mortem and used immediately for myofibril frag­

mentation determination.

Bovine lonaissimus muscles from the wholesale short loin

of six veal were obtained from animals originating from the

Iowa State University Dairy Farm and slaughtered at the Iowa

State University Meat Laboratory. Muscle samples and steaks

were removed at 1 and 7 days post-mortem and used in the same

manner as previously described for A- and C-maturity bovine

carcasses in the preceding paragraph.

69

Hyofibril Preparation

Myofibrils were isolated from bovine longissiaus, psoas

ma-jor and semitendinosus muscles. Four grams of scissor-

minced sascle vere homogenized in a Baring Blendor for 30

seconds in 10 volumes (V/H) of a 2°c isolating medium con­

taining 100 ma KCl, 20Q5 K phosphate (pS 7.0), 1sS SDTn and

ImH sodium azide. The homogenate was sedimented at 1000 x g

for 15 minutes and the supernatant decanted. The sediment

was resuspe&ded in 5 volumes (V/H) of the same isolating

medium nsing a stir rod, sedimented again at 1300 x g for 15

minutes and the supernatant decanted. The sediment vas re-

suspended in 10 volumes (v/v) of the same isolating medium

and passed through a polyethylene strainer to remove the con­

nective tissue and debris. Ten volumes (v/e) of the isola­

ting medium were used to facilitate passage through the

strainer» The suspension was sedimented at 1000 x g for 15

Binâtes and the supernatant decanted. The sediments were

washed three times more by suspending in 5 volumes (v/w) of

the same is'olating medium and sedimented at 1000 x g for 15

minutes. Finally the sedimented myofibrils were resuspended

in 5 volumes (v/w) of the same isolating medium and the pro­

tein concentration determined by the biuret procedure of

Gornall et al. (19U9).

70

Myofibril Fragmentation Determination

An aliquot of the myofibril suspensions were diluted,

with the same isolating medium in which the myofibrils were

suspended, to 0.5mg/ml protein concentration. Protein con­

centration of the diluted myofibril suspension was determined

by the biuret method of Somali et al. (1949). If the pro­

tein concentration of the diluted myofibril suspension was

not within the range of 0-45 to 0.55mg/ml, a redilution and

protein concentration determination of the myofibril sample

vas made, k 10ml aliquot of the diluted myofibril suspension

vas stirred and absorbance of this suspension was measured at

540am with a Spectronic 20 Bausch S Lomb Colorimeter. Myofi­

bril fragmentation values were recorded as absorbance units

per 0.5mg/ml myofibril protein concentration multiplied by

200. The constant, 200, expanded the absorbance units to

range from 30 to 100. The absorbance units, expanded by the

constant, is called the myofibril fragmentation index.

Light Microscopy

Myofibrils were examined with a Zeiss Photomiccoscope

equipped for phase and polarized light optics. For phase

optics, a 100% neofluar, a 40s neofluar and a 25% phase ob­

jectives were used eith a green interference filter in the

light path. For polarized light optics a lOx objective was

used with polarizing filters in the light path. Results sere

71

recorded on Kodak Panatomic X film.

Troponin Preparation

Troponin was prepared from lOOga of at-dsatk bsvias 1 pri­

ais si m us muscle according to the procedure described by

Aralcava et al. (1970a, b, c) . Myofibrils, swollen by a ser­

ies of water washes, were centrifuged at 15,000 x g for 30

minutes. The supernatant was salted out at 30% to 75%

ammonium sulfate saturation and centrifugea at 15,000 x g for

10 minutes. The precipitate was dissolved in Inn K bicarbon­

ate and dialyzed for 30 hr against ImM K bicarbonate. Enough

3a KCl was added to the dialyzed solution to make the final

solution 1H KCl. This solution was clarified at 105,000 x g

for 1 hr and the pH of the supernatant adjusted to 4.6 and

held at that pH for 1 hr at 20C. The solution was then cen­

trifugea at 15,000 : g for 10 sinates and the supernatant

adjusted to pH 7.5. The pH adjusted supernatant was salted

out at 40% to 60S ammonium sulfate saturation and centrifugea

at 15,000 2 g for 10 minutes. The precipitate was dissolved

ia ImS K bicarbonate, 0.5% 2-MCE and dialyzed for 60 hr

against ImH K bicarbonate. The dialyzed solution was clari­

fied at 105,000 s g for 1 hr to yield a partially purified

troponin preparation. Troponin aas further purified by di-

ethylaminoethyl (DEAE)-cellulose column chromatography as de­

scribed by ran Eerd and Kawasaki (1973). A 1.6 z 20cm column

yas packed sith DSâE-csllulose, equilibrated sit h 20sH Tris-

72

HCl (pH 7.5) and elated with a linear KCl gradient (from 0 to

0.4M) in the same buffer solution. Troponin was eluted at a

single peak at 3.18ÎÎ KCl. DElE-cellulose column chromatogra­

phy was performed at 2®C. Troponin purity was monitored be­

fore and after column chromatography by SDS-polyacrylamide

gel electrophoresis.

Calcium-Activated-Factor (CAP) Preparation and Assays

Crude CAP was prepared from lOOgm of at-death muscle and

at 3 and 6 days postmortem muscle according to the proce­

dure described by Busch et_al. (1972) . Ground muscle was

suspended in 6 volumes (v/w) of 4mH EDTA, pH 7.0-7,6, by usa

of a Waring Blendor for 90 seconds. Additional Tris was

added to the postmortem auscle homogenate to adjust the pH to

7.0-7.6. The suspension was centrifuged at 15,000 z g for 20

niautes and the supernatant filtered through glass wool. The

supernatant was then adjusted to pH 6.2 with acetic acid,

left at O^C for 30 minutes and then centrifuged at 15,000 x g

for 20 minutes. The pH 6.2 supernatant was adjusted to pH

4,9 with acetic acid, left at Qoc for 30 minutes and centri­

fuged at 15,000 X g for 20 minutes. The pH 4.8 sediment was

suspended in 7sl of lOOmH Tris-HCl, pH 8,2, EDTA and the

pK adjusted to 7.0. The suspension was diluted with water to

20H1 final volume and clarified at 105,000 X g for 1 hr. The

clarified solution was dialyzed against ImH K bicarbonate,

503 EDTA, 5ES 2-SC3 for 12-18 hours= After dialysis, the so­

73

lution was clarified at 105,000 x g for 60 minutes. The

snspernatant from this clarification was designated crude

CAP.

assay conditions were as follows: tOOmB KCl, lOOmM

Tris-acetate (pH 7.5), lOnH 2-HCE, 5mH Ca2+, 5ng/ml casein

and 0.25mg/ml CAP (CAP to casein ratio of 1:20) in a 2ml

total assay volume incubated at 25®C for 30 minutes. The re­

action was stopped by precipitating the protein with 2ml of

5S TCAc It was centrifuged at 1000 x g for 20 minutes and

the optical density (OD) of the supernatant was measured at

278nm. CAP activity was calculated as total 3D units per

lOOgm of muscle.

Bovine ayofibrils and purified troponin were treated

with purified porcine CAP (prepared by William Dayton, Dayton

et ale 1974ae b). Myofibrils were treated with porcine CAP

(CAP to myofibril ratio of 1:200 by protein concentration)

under the saae conditions described in the CAP-casein assay

except casein was replaced with lOmg of myofibrillar protein.

The reaction was stopped by addition of 0.22mls of 0.1M

2BTA. Hyofibrils «ere washed with lOOaH NaCl three times at

2°C to remove the Tris» KCl, HCE and caz*.

Myofibrils were then observed with light microscopy and

also run on SDS-polyacrylamide gel electrophoresis. Purified

troponin was also treated with porcine CAP (CAP to troponin

ratio of 1:1000 by protein concentration) for 5, 10, 20 and

74

40 minutes. Incubation conditions were the same as those de­

scribed for CAF-casein assay and terminated in the same way

as described for myofibril treatment. CAF-treated troponin

was dialyzed after incubation against ImS K bicarbonate for 3

hr and then run on SDS-polyacrylamide gel electrophoresis.

Sodium Oodecyl Sulfate (SDS) Polyacrylaaide Gel Electrophoresis

Myofibrils and purified troponin were run on SDS-

polyacrylamide gel electrophoresis according to the method of

Heber and osborn (1969) to determine troponin purity and

alternations of the myofibrillar proteins and troponin by

postmortem storage time and temperature and CAP treatments.

Both 7 1/2% acrylamide and bis-acrylamide in a 75:1

weight ratio and 10% acrylamide and bis-acrylamide in a 37:1

weight ratio were used in gels which were run in 5mm (inner

diameter ) X 120mm tubes. The gels and the upper and lower

reservoirs contained lOOmH Ha phosphate (pH 7.1) and 0.1%

SDS. SDS-protein complexes were prepared by incubating myo­

fibrils and troponin in the SDS-tracking dye solution

consisting of 1.75% SDS, 1.5H 2-aCE, 20mH Sa phosphate (pH

7.0), 0.01% broaophenol blue (tracking dye) and 6.75% glycer­

ol (to increase the density of the solution) at 100®C for 15

minutes. The applied loads to the gels sere 25 to 35ag of

myofibrillar proteins and 5 to 30ug of troponin. Electropho­

resis was performed at 20-25®C in gels 8cm long and run at a

75

constant current of 6 aanp per gel until the tracking dye had

traveled to approximately 1.5cm from the bottom of the tube.

The gels vere stained ia a solution of 0.2% Coomassie

Brilliant Blue in 279B1 of 50% methanol and 21 ml of glacial

acetic acid for 12-16 hr and destained in a quick gel

destainer (Canalco Co.) for 20 minutes containing a solution

of 7.5/0 {?/?} acetic acid and 5% (?/?} methanol. After de-

staining, the gels were stored in destainiag solution and

after the background gel stain had been completely destained

(usually 10-21 days after de staining) the gels were photo­

graphed eith a Nikon single-lens reflex camera on Kodak Pana-

toaic X film. The molecular weights of the protein

components were determined by comparison of their mobilities

with proteins of known molecular weight as described by Heber

and Osborn (1959).

Myofibrils, in preparation for SDS-poiyacryiamide gel

electrophoresis, were washed in IGGmS SaCl three times to

remove the KCl and finally suspended in ICOmû NaCl to a mini­

mum protein concentration of Sag/sl. An aliquot of this sus­

pension was diluted with water to a myofibrillar protein con­

centration of IcSsg/sl and 15-30na HaCl, To 1ml of the di­

luted myofibrils was added 0.5ml of the SDS-tracking dye so­

lution just prior to incubation at IQOoc. Troponin was

dialyzed against ImH K bicarbonate to resove ail salt and

then 5Oui of the SDS-tracking dye solution was added to 100al

76

Of troponin (1.5ug/ul) just prior to incubation at 100*0.

Huscle Fiber Tensile Strength Heasureneat

& TT—B floor sodel Instron universal Testing Machine was

used to determine the tensile strength of strips from sis bo­

vine seai-tendinosus and six psoas major muscles at 1, 3, 7

and 13 days post-mortem stored at Strips were removed

from both raw muscles and muscles cooked in plastic bags sub­

merged in a SQOC water bath for 1 hr and then cooled to 2®C.

The muscle strips were dissected from the muscles by cutting

parallel to the fiber acis. The sample strips were clamped

to a type B load cell and crosshead with type B clamps.

Sample strip dimensions were: length-G.Ocm, length available

for stretching after clamping-3.Ocm, strip weight-1.0 to

2.4gm. All samples were weighed on an analytical balance

prior to testing. Instrssent parameters were: crosshead

speed-lOcm/min., chart speed-IOcm/min., full scale

deflection-0 to lOOOgm. The average breaking strength value

of six strips was used as the measure of fiber tensile

strength for each sample- Results were recorded as grams of

force per gram of muscle strip.

Barner-Bratzler (H-B) Shear-Force and Sensory Panel Determinations

Steaks, 3.1cm thick, from fresh, or frozen and then

thawed for 24 hr at 2°Cf of bovine longissimus, semitendino-

s%s and psoas major suscles of various postmortem times were

77

evaluated for shear-force on a Harner-Bratz1er (»-B) shear

apparatus. Ba* steaks, not previously frozen, «ere cooked la

plastic bags submerged in a 60<*C water bath for 1 he aad the

frozen and thawed steaks were broiled to 65°C in an electric

oven. Steaks were placed 10cm from the heating coil and oven

broiled to half the final temperature (determined by. a ther-

aoaeter placed in the geometric center of the steak) on one

side, turned and broiled to the final temperature on the

other side. Ml steaks were allowed to cool to room tempera­

ture (approximately 25oC) before three 1.27cm diameter cores

were removed and each sheared twice. The average of six

shear values were used as the shear-force for each steak and

recorded as Kg of force per cmz.

à ten-member sensory panel, consisting of 5 men and 5

woaen, having a mean age of 26, were selected for their re­

peatability of detecting broiled beef steak teauerness. Thsj

were suuseguently trained to isproee their sensitivity to

detect tenderness differences.

Steaks were randomly selected, from all steaks frozen,

thawed for 24 hr at 2°C and broiled, in the same manner as

described for the W-B shear-force determinations, to an in­

ternal temperature of 65oc. Panelists sat in separate cubi­

cles isolated from the preparation area and lighted by a red

fluorescent light. Panelists received sis different samples

(1.27cm s 2.5cm z 2.5cm) from the same location im each steak

78

per testing day. Panelists evaluated each meat sample for

tenderness, flavor and juiciness based on a hedonic system

from 8 to 1 with 8 being extremely tender, desirable flavor

or juicy to 1 being extremely tough, undesirable flavor or

dry. The average of the ten panelists* values was used as

the tenderness, flavor and juiciness score for each sample.

Data Analysis

The data from the various experiments were analyzed for

means, standard deviations, standard errors, simple correla­

tions and significant mean differences by the analysis of

variance and students' "t" test according to methods de­

scribed by Snedecor (1967) and Steel and Torrie (1960). The

Statistical Analysis System of the lona State Computation

Center were used for data computations.

79

HESDLTS

Hyofibriis were prepared froa bovine longisslasas sensi-

tendinosus and psoas iaajor ausclss at 1, 3, 5 and 10 days of

postmortem storage at 2°C for microscopic observations. Fig­

ure 1 shovs polarized-light and phase-contrast micrographs of

myofibrils from lonqissimas muscle. Polarized-light micro­

graphs (Figs, la-d) show that the muscle sample separates

into fiber segments or pieces and single myofibrils during

homogenization. The size of the fiber piece become smaller

with postmortem time (Fig, Id). &t higher magnification and

under phase-contrast microscopy, myofibrils at 1 day post­

mortem (Fig. le) are long having many (over 20) sarcomeres

per myofibril. As postmortem time increases myofibrils

(Figs. If-h) become shorter and thus become more fragmented.

This increase in isyofibril fragmentation during postmortem

storage agrees with the observations of Davey and Gilbert

(1967, 1969), Fukazawa et_al. (1969), Hay et_ai. (1973b),

Henderson et al. (1970), Parrish et_al, (1973b), Sayre (1970)

and Takahashi et al, (1967)* Even at higher magnification,

phase-contrast of myofibrils (Figs. 1i-i) shoe that single

syofibrils become shorter with postmortem time. The Z-lines

in myofibrils at 1 day post-mortem (Fig. 1i) are very dis­

tinct and prominent whereas Z-lines become slightly less dis­

tinct as postmortem time increases. Z-lines, however, are

faint, but detectable even at 10 days post-mortem (Fig. 11).

Figure 1. polarized-light and phase micrographs of myofi­brils prepared from bovine lonqrissiaus muscle at different times of postmortem storage at 2°C

a. Polarized-light micrograph of myofibrils at 1 day post-mortem. Both single myofibrils and fiber pieces are present. X200

b. Polarized-light micrograph of myofibrils at 3 days post-mortem. Fiber pieces and myofibrils are more fragmented than those in Fig. la. X200

c. Polarized-light micrograph of myofibrils at 5 days post-mortem. Greater myofibril fragmen­tation is shovn than in Fig. la and lb. X200

d. Polarized-light micrograph of myofibrils at 10 days post-mortem. Myofibrils are highly frag­mented. X200

e. Phase micrograph o£ myofibrils at 1 day post­mortem. X800

f. Phase micrograph of myofibrils at 3 days post­mortem. Myofibrils are fragmented more than myofibrils in Fig. le. Z800

g. Phase micrograph of myofibrils at 6 days post-mortesa Myofibrils are more fragmented than those in Fig. 1e and If. 1800

h. Phase micrograph of highly fragmented myofi­brils at 10 days post-mortem. %500

i. Phase micrograph of myofibril at 1 day post­mortem. Sarcomeres are related and Z-lines are prominent. X2000

j. Phase micrograph of myofibrils at 3 days post-mortemo Some fragmentation is observed and z-lines are prominent* X2000

Phase micrograph of myofibrils at 6 days post­mortem. Syofibrils are more fragmented and Z-lines are less prominent. X2000

1. Phase micrograph of myofibrils at 10 days post-mortem. Syofibrils are highly fragment­ed aad %-lines are very faint» 22000

Si

DAT 3

0^ te &AÏ s

DAY 3 SAY Î I5>f -<:%

DAY 10 ifAT O

DAY 6 k DAY 10 1

82

Henderson et al. (1970), on the other hand, have shown that

Z-lines become amorphous and lose their prominent appearance I

daring postmortem storage. Myofibrils prepared from,bovine

semitendinosas muscles at 1, 3, 6 and 10 days of postmortem

storage at 20C are shown in Figure 2. The polarized light

micrographs of myofibrils (Figs. 2a-d) show that fiber pieces

vere similar to those found for loaqissisas muscle (Figs. 1a-

d)• also, size of the fiber pieces greatly decreased with

postmortem time. At higher magnification, the phase-contrast

micrographs (Figs. 2e-h) show a similar increase in myofibril

fragmentation with postmortem time as did myofibrils from

lonqissiaas muscle (Figs, le-h). kt 2000% magnification, the

phase-contrast micrographs of myofibrils (Figs. 2i-l) show

that myofibrils become shorter with postmortem time, Z-lines

appear to become slightly less prominent and more degraded.

Myofibrils were also prepared from bovine psoas maior muscles

at 1, 3, 5 and 10 days of postmortem storage at 2-C, Figure

3 shows polarized-light micrographs of these myofibrils.

Hyofibrils at 1 day postmortem have extremely large fiber

pieces (larger than shown for the longissimus (Fig. laj and

seaiteadinosus (Fig 2a) muscles) and a few single myofibrils.

Bhile the size of these fiber pieces decrease as postmortem

time increased, the fiber pieces at 10 days post-mortem (Fig.

3d) are larger than those shown at 1 day post-sortem for the

lonqissiaus (Fig. la) and semitendinosas (Fig. 2a) muscles.

Figure 2. Polarized-light and phase micrographs of myofi­brils prepared from semitendinosas muscle at dif­ferent times of postmortem storage at 2°C

a. Polarized-light micrograph of myofibrils at 1 day post-mortem. X200

b. Polarized-light micrograph of myofibrils at 3 days post-mortem. X200

c. Polarized-light micrograph of myofibrils at 6 days post-mortem, Hyofibrils are more frag­mented than those shOHn in Fig. 2a aad 2bo X200

d. Polarized-light micrograph of myofibrils at 10 days post-mortem. Myofibrils are highly frag­mented. 1200

e. Phase micrograph of myofibrils at 1 day post­mortem. 1500

f. Phase micrograph of myofibrils a». 3 days post­mortem. Syofibrils are more fragmented than in Fig. 2e. 1500

g. Phase micrograph of myofibrils at 5 days post­mortem. Hyofibrils are very fragmented. X500

h. Phase sicrograph of myofibrils at 10 days post-mortem. Myofibrils are very highly fragmented. X500

i. Phase micrograph of myofibrils at 1 day post­mortem. 12 00 G

j. Phase micrograph of myofibril at 3 days post­mortem. Myofibrils are more fragmented than those in Fig. 2i. X2000

k. Phase micrograph of myofibrils 6 days post­mortem. z-lines are prominent and myofibrils are very fragmented than in Fig, 2i and 2j. 12000

1, Phase micrograph of myofibrils at 10 days post-mortem. Z-iines are faint aad syofi­brils are very highly fragmented. X2000

84

- X

DAY 3 DAY 1

DAY 10 DAY 6

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DAY 1

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DAY 6

DAY 3

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DAY 1 DAY 3

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85

Phase-contrast micrographs of myofibrils from the psoas manor

muscles are shown in Figure 4 and these micrographs further

exemplify their nature not to fragment, kt low magnifica­

tion, the micrograph of myofibrils (Figs. Ua-d) show a slight

increase in myofibril fragmentation from 1 to 10 days post­

mortem. Figures »e-h, at 2000X magnification, show no change

in myofibril length or appearance of the Z-line as postmortem

time increases. The increase in myofibril fragmentation dur­

ing postmortem storage at 2°C, shown in the myofibrils of

lonqissimns (Fig. 1) and semitendinosus (Fig. 2) muscles, was

considerably greater than was shown for myofibrils of £soas

major muscles (Fig. 3 and 4) .

Several investigators (Fukazawa et al., 1969; Sayre,

1970; Takahashi et al., 1967) have attempted to measure the

myofibril fragmentation observed in the light microscope and

relate it to meat tenderness. Bhile these investigators

acknowledged that myofibril fragmentation increased as meat

tenderness increased during postmortem storage, they were

unsuccessful in developing a method to quantitate myofibril

fragmentation. Davey and Gilbert (1969) found that the mi­

croscopic method of measuring myofibril fragmentation was

limited by the sample size that could be measured and inher­

ent sampling errors eristed due to the limited sample size.

These investigators found that a turbidometric aeasure of

myofibril fragmentation was a more accurate and objective

Figure 3. Polarized-light micrographs of myofibrils prepared from bovine psoas major muscle at different times of postmortem storage ât 2®C

a. Syofibrils prepared at 1 day post-mortem. arrow points to a muscle fiber piece whicii did not separate into single myofibrils. X200

b. Myofibrils prepared at 3 days post-mortem. Large fiber pieces are more fragmented than at 1 day post-mortem (Fig. 3a). X200

c. Myofibrils prepared at 6 days post-mortem. Note that large fiber pieces are more frag­mented than those at 1 day post-mortem (Fig. 3a and 3b). X200

d. Myofibrils prepared at 10 lays post-mortem. These sho« somewhat more fragmentation than myofibrils in Fig. 3c. X200

Figure 4. Phase micrographs of myofibrils prepared from bo­vine psoas major muscles at different times of postmortem storage at 2*C

a. Myofibrils at 1 day post-mortem. X800

b. Myofibrils at 3 days post-mortem. Myofibrils are someehat more fragmented than shown in Fig. 4a. 2800

c. Myofibrils at 6 days post-mortem. X500

d. Myofibrils at 10 days post-mortem. ByofibrlLs are slightly fragmented. X500

e. Myofibrils at 1 day post-mortem. Sarcomeres are relaxed and Z-lines are prominent. X2000

f. Myofibrils at 3 days post-mortem. Sarcomeres are relaxed and Z-lines prominent. X2000

g. Myofibrils at 6 days post-mortem, Z-line integrity appears similar to Fig. 4e and 4f. X2000

h. Myofibrils at 10 days post-mortem. Z-lines remain prominent. X2000

89

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measure of fragmentation. Holler et al. (1973) adapted the

turbidometric method of measuring fragmentation (Davey and

Gilbert (1969) and found it to be highly correlated to meat

tenderness. The following is a study made by this author to

utilize a similar method of turbidometric measure of myofi­

bril fragmentation. Myofibrils were prepared from bovine

loaqissiaus muscles at 3^ and 7 days of postmortem storage

at 20C by homogenizing a Wgm, scissor-minced muscle sample in

a Baring Blendor for 5, 10, 15, 20, 30, ^5, 60, 75, and 90

seconds. Absorbance of the myofibril suspensions (0.5mg myo­

fibrillar protein per ml) measured at each homogenizing time

for three postmortem storage periods are shown in Figure 5.

As homogenizing time increased the absorbance of myofibril

suspensions increased. Also absorbance measures of myofibril

fragmentation were higher with increased postmortem storage

time. The 30-second hoaogenizing time was chosen because it

gave the greatest difference in absorbance saeasures between

the three postmortem storage periods and this homogenizing

time was close to the maximum absorbance measure of the 7

days postmortem storage period. All subsequent myofibrils

were prepared according to the procedure as described in the

METHODS AND MATERIALS section of this dissertation using a

30-secoad hoaogenizing time® Because the turbidometric

method measures not only the relative sizes of myofibrils bat

also the relative sizes of fiber pieces, the absorbance val-

Figure 5. Effect of homogenizing (Waring Blendor) and post­mortem storage times on the fragmentation index (absorbance at 540nm z 200) of myofibrils from bo-viae loaqisslaas auscle. Day 1, 3, and 7 refers to 1, 3, and 7 dajs of postmortem storage of the carcass at 2-C

MYOFIBRIL FRAGMENTATION INDEX (ABSORBANCE AT 540nm X 200) ro <u> 0»

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93

aes are transformed (as described in the METHODS AND HATEBI-

ALS section) to myofibril fragmentation index values.

An attempt was also made to develop a procedure for

measuring myofibril fragmentation of cooked meat. Syofibrils

prepared from cooked meat are, suprisingly, altered only

slightly as seen with phase-contrast microscopy (Schmidt and

Parrisa, 1971). However, heating causes the dscaturatioa of

many sarcoplasmic proteins even at SO^C (Hamm, 1966) which

appear to encapsulate and clump to the myofibrils. These de­

natured proteins are impossible to remove from myofibrils

using routine myofibril isolation techniques. Because these

denatured proteins are present in myofibril suspensions, the

suspensions are so cloudy that a turbidometrlc measure does

not accurately reflect the relative sizes of the myofibrils

and fiber pieces. Hence, the method used to isolate myofi­

brils and aeasasre myofibril fragmentation in fresh auscls

tissae is isadeqsate tor cooked meat.

Hyofifcril fragmentation index was determined ou bovine

semitendinosus and psoas laaior muscles at 1, 3, 7 and 13 days

of postmortem storage at 2°C and these results are illustrat­

ed in Figure 6. The means and standard errors of the myofi­

bril fragmentation index values of semiteadingsus and psoas

major muscles at various postmortem storage times ace con­

tained is Table 1. In the semitendiaosus muscle, mvofibril

fragmentation index mean values were significantly different

Figure 6- Effect of postmortem storage time on the fragmen­tation index of myofibrils from bovine samitendi-nosus and psoas major muscles at different times of postmortem storage at 2°C

95

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70

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SEMITENDINOSUS

KSUAS

40

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POSTMORTEM STORAGE (DAYS)

n 13

96

Table 1. Effect of postmortem storage [2°C) on myofibril fragmentation index (PI), Warner-Bratz 1er (W-B) shear-force and fiber tensile strength (TS) (raw and cooked) of seaiteadlnosas (ST) and psoas aaior (PB) muscles»

FI2 ST

PH

4&,8a3ag

Days of postmortem storage

3 7 13

68^±2^ 80.0t22.5

52^11-0 . 5U.4±1.1 56.4±1.3

H-B3 ST 3.ii3i0iiS 2.72^0^22. __2t55&0.1±_ 2.53zOi1S

PH Ia90&0^09 Ii81i0.07_ ._jLi83iOs.11_ __Tz.65tO.10

TS* (raw) ST 266*23 217*18 190±19 179±21

PH 249±15 223^5 198±15 186±16

TS* (cooked) ST Ml±28 383i20 3ii6+21 3tt2±23

?n 250=28 391629 3S5&24

iHeans ± standard errors of five carcasses. Means not underscored by the same dotted line are significantly differ­ent (P<0.05).

zâbsorbance per Q.Ssg myofibril protein 1200.

3Kg of shear-force per cm.

4Gm of breaking force per gm of muscle strip.

97

(P<0-05) between all postmortem times except between 7 and 13

days post-mortem. In the psoas major muscle, myofibril frag­

mentation index mean values were not significantly different

(P<0.05)• In the semitenflinosas muscle, myofibril fragmenta­

tion index values increased from 1 to 13 days post-mortem

with the greatest increase occurring between 1 and 3 days

post-mortem (Fig. 6). In the psoas major muscle, myofibril

fragmentation index values increased only slightly from 1 to

13 days post-mortem when compared to the increase found in

semitendinosus muscle. The increase in the myofibril frag­

mentation index of the semitendinosus muscle appears to

coincide with the observed increases in fragmentation of myo­

fibrils during postmortem storage shown in Figure 2. The

slight increase in the myofibril fragmentation index of nsgis

major muscle also appears to parallel the slight increase in

myofibril fragmentation daring postEortea storage observed in

Figures 3 and %. Additionally, differences is nyofibril

fragmentation index during postmortem storage between semi-

tendiaosus and psoas major muscles (Fig. S and Table 1)

appear to agree with the observed fragmentation differences

in myofibrils of these two muscles during postmortem storage

shown in Figures 2, 3 and U.

In another experiment? myofibril fragmentation index was

detersined on bovine loagissisus, seaitendiaosus and psoas

major muscle stored at 1, 3 and 6 days post-sortem. The

98

means and standard errors of myofibril fragmentation index

values for all three muscles are shown in Table 2. Similar

to that found in the previous experiment, the myofibril frag­

mentation index of longissimus and semitendinosus muscles in-

creased from 1 to 6 days post-mortem with the greatest in­

crease occurring between 1 and 3 days post-mortem and the

means of t&e fragmentation index for each postmortem storage

period were found to be significantly different (P<0.05) from

each other, on the other hand, in psoas major muscle, the

myofibril fragmentation index increased only slightly from 1

to 6 days post-mortem with only the means between 1 and 6

days post-mortem being significantly different (P<0.05).

Fragmentation index results of the three muscles agree with

the increases in myofibril fragmentation determined by micro­

scopic observation during postmortem storage {Figs. 1-4). In

summary, myofibrils froa loaglssiaus and semitendinosus mus­

cles showed almost a doubling in fragmentation index values

whereas myofibrils from psoas aaior muscles showed only a

slight increase during postmortem storage at 2°C.

In another experiment, myofibrils were prepared from bo­

vine lonqissimss« semitendinosus and nsoas major muscles

stored at 2 or 3 days postmortem at 2°C and 25°C and the

results of this experiment are illustrated in Figure 7. In

all three muscles, myofibril fragmentation indez increased

from 1 to 3 days postmortem at both 20C and 250C treatments.

99

Table 2. Effect of postmortem storage (20C) on myofibril fragmentation index (PI) and Warner-Bratzler (H-B) shear-force of lonqissians (L), semitendinosus (SÎ) and psoas major (P5) muscles:

Fic

Days of postmortem storage

1 3 6

L

ST 48.810.8 68^2±1^ ZliiilrO

PM 5U. 7±1. 0

9-B3 L 2^6040^10 2.23tOUZ 2.13&0.12

ST 3.2240^11 2.7220.09 2^64&g^11

PH 2^640^12 liSiLiOill Ii86i0^17

iHeans ± standard errors of five carcasses. Heans not underscored by the same dotted line are significantly differ­ent {P<0.05).

zabsorbance per O.Smg myofibril protein X 200.

3kg of shear-force per cm.

Figure 7, Effect of postmortem storage time and temperature treatments on the fragmentation index of myofi­brils prepared from bovine lonqissimus, semitendi-nosus and psoas maior muscles at different times of postmortem storage at 2®C and 25°C

MYOFIBRIL FRAGMENTATION INDEX (ABSORIiANCE AT 540ntn X 200)

r o cn o ~r

m o "r

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102

àùSitlonally, muscles stored at 250C had a higher myofibril

fragmentation index than muscles stored at 20C. However,

myofibril fragmentation index eas not as high in the psoas

major muscle as in the lonqissiaus and semitendinosus mus­

cles. Increase in myofibril fragmentation index, due to tem­

perature treatment, was also much less in psoas major muscle

than it vas in loaqissisas and sesiteadinosas muscles» High

postmortem storage temperatures (160C) have been shown to

produce accelerated tenderness increases in bovine Icngissi-

mus and semitendinosus muscle (Parrish et al., 1973b). The

25*0 storage temperature in the present study resulted in a

much more rapid increase in myofibril fragmentation index

value in longissimus and semitendinosus muscle with myofibril

fragmentation index value being approximately the same at 3

days postmortem (25°C) as was found in these muscles stored

for 6-13 days post-aortem {2°C) (Tables 1 and 2) . The factor

that causes myofibril fragmentation index to be greater eith

increased storage temperature apparently acts similarly in

lonqissiBus and semitendinos us muscles but is either not

present in similar quantities or is not as active in psoas

major muscle.

Barner-Bratzler (W-B) shear-force values were determined

on bovine semitendinosus and psoas major at 1, 3, 7 and 13

days of postaortes storage at 2®C and cooked to 65oc. The

effect of postmortem storage on E-B shear-force values for

103

the seaitendinosns and psoas major muscles are shown la Fig­

ure 8 and the S-B shear-force means and standard errors are

shown in Table 1. In the seaitendinosus muscle, the mean H-B

shear-force at 1 day post-mortem was found to be significant­

ly different CP<0.05) from the means of 3, 7 and 13 days

post-mortem with the latter three postmortem days not

significantly different {P<0.05) from each other. In the

psoas major muscle, no significant difference (P<0.05) in

mean H-B shear-force between days post-mortem was found. The

decline in H-B shear-force of semitendinosus muscle corres­

ponded to the increase in the myofibril fragmentation index

of semitendinosus muscle {Fig. 6). This suggests that the

increase in myofibril fragmentation may be associated to the

decrease in the W-B shear-force value of the muscle. In con­

trast to the gsqas major muscle (Fig. 8) , the W-B shear-force

only slightly decreased frcs 1 to 13 days pDst-sorten»- The

slight change in H-B shear-force during postmortem storage in

the psoas salor auscle also appears to be associated to the

slight change found in myofibril fragmentation during post­

mortem storage (Fig* 6} »

In another experiment, H-B shear-force was determined on

bovine lonaissimus. semitendinosus and psoas maior muscle at

1, 3 and 6 days of postmortem storage at 2°C, Results from

this experiment are shown in Table 2. In the longissimus,

semitendinosus and psoas major muscles, S-B shear-force de-

Figure 8. Effect of postmortem storage time on Warner-Bra tz 1er (B-B) shear-force values of bovine semi tendinosas and psoas major muscles at different times of postmortem storage at 2°C

WARNIIR-BMTZLER SHEAR-FORCE VALUES (Kg/cm?)

106

creased from 1 to 6 days post-mortem. The mean 0-B shear-

force, for semitendinosas muscle, was significantly (P<0.05)

higher at 1 day than at 3 and 6 days post-mortem; the means

of the later two days were found to not be significantly

(P<0«05) different. Also, no significant (P<0.05) differ­

ences between means of H-B shear-force at 1, 3 and 6 days

post-mortem were found for loacfissiaas and psoas aajor mus­

cles. Parrish et al. (1973b), however, found W-B shear-force

to be significantly (P<0.05) higher in loncrlssisus muscle at

1 day than at 6 days post-mortem.

»-B shear-force was also determined on longissimus,

semitendinosns and psoas major muscles stored for 1, 2 and 3

days post-mortem at 2°c and 25oc (Fig. 9). la all three mus­

cles, W-B shear-force values were lower at 1, 2 and 3 days

post-mortem at 250C storage temperature than at 2oc. B-B

shear-force decreased from 1 to 3 days post-mortem in all

three muscles at both 20C and 25°C storage but the least

change in W-B shear-force, due to either storage time or tem­

perature, was found for psoas major muscle. In both the loa-

aissiaqs and semitendinosus muscles, the decrease in H-B

shear-force from 1 to 3 days post-mortem was greater at 2°C

than at 25oc storage, however, the W-B shear-force was con­

siderably lower at 1 day post-mortem at 250c than at 2® c

storage. Since the W-B shear-force at 1 day post-mortem,

250C, for lonqissimus and semitendinosns muscles appeared to

Figure 9. Effect of postmortem storage time and temperature on Barner-Bratzler (W-B) shear-force values of ba-vine ignaissimus, sem^endi&osus and mso&s_m&ior muscles at different times of postmortem storage at 20C and 25*0

108

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SEMITENDINOSUS, 2*C

SEMITENDINOSUS, 25®C

LONGISSIMUS, 2®C

LONGISSIMUS, 25'C

PSOAS, 2"C

PSOAS, 25®C

1 2

POSTMORTEM STORAGE (DAYS)

109

be approaching a similar value as at 3 days postmortem, 20C,

it appears that a larger decrease in W-B shear-force had al­

ready occurred by 1 day post-mortem at 25oc storage. Busch

et al. (1967) found W-B shear-force of semitendinous muscla

to decrease during postmortem storage and found that at 7

days post-mortem at 2°C the S-B shear-force was approximately

the same as at 2 days post-mortem at 37oc witii most of the

decrease in H-B shear-force in the muscle stored at 37°C oc­

curred by 6 hours post-mortem. They also found a large de­

crease in B-B shear-force in the psoas ma-jor muscle during

postmortem storage, however, the muscle had been excised from

the carcass at death.

In a comparative evaluation of 8-B shear-force (Fig. 9)

and myofibril fragmentation index (Fig. 7) of longissimus,

seaitendinosus and psoas major muscles at 1, 2 and 3 days of

postmortem storage at 2°c and 25°Ct three observations can be

made. 1) In the psoas saior muscle the change in syofibril

fragmentation indes aad W-B shear-force are very slight dur­

ing postmortem storage compared to the longissians and seai­

tendinosus muscle and only slightly affected by storage tem­

perature. 2) la the lonqrissxmus and semitendinosus muscles,

the change in myofibril fragmentation index and W-B shear-

force from 1 to 3 days post-mortem is greater at 2°: than at

250C. 3) The differences in myofibril fragmentation index

and W-B shear-force at 1 day post-mortem between 25°C and 2oc

no

storage is similar between longissimus and seaiteadinosus

muscles. These observations indicate: 1) that some factor

in muscle is influencing both myofibril fragmentation index

and W-B shear-force, 2) that this factor is in similar quan­

tities or has similar activity levels in the longissimus and

semitendinosus muscles but it is lower in quantity or activi­

ty in the psoas major muscle, and 3) that this factor is more

active at higher storage temperatures.

Muscle fiber tensile strength was used to measure the

strength or weakness of the myofibril structure. Muscle

fiber tensile strength was measured by Instron breaking

strength of muscle strips from raw and cooked semitendinosus

and psoas major muscles at 1, 3, 7 and 13 days of postmortem

storage at 2®C. The fiber breaking strength decreased in

both raw and cooked muscle from 1 to 13 days post-mortem with

the greatest decrease occurring from 1 to 3 days post-mortem

for both the semitendinosus and psoas major muscles {Fig.

10). Cooked muscle strips from both muscles required almost

twice the force to break the muscle strip compared with the

force necessary to break raw muscle strips. Also to be noted

is that cooked seaiteadinosus muscle strip required less

breaking force than did the cooked psoas ma-jor muscle strip.

It appears that the decrease in muscle fiber breaking

strength during postmortem storage (Fig 10) occurs in a man­

ner similar to the decrease in H-B shear-force during post-

Figure 10. Effect of postmortem storage time on the fiber breaking strength of raw and water-bath cooked (60®C) bovine semitendinosus and psoas,major mus­cles at different tiaes of postmortem storage at 20C

112

PSOAS, COOKED

SEMITENDINOSUS, COOKED

PSOAS. RAW

SEMITENDINOSUS, RAW

: « a s 5 L. 1 3 5 7 9 11 13

POSTMORTEM STORAGE (DAYS)

113

mortem storage (Fig. 8) for the seaitendinosus muscle. The

means and standard errors of fiber tensile strength of raw

and cooked semitendinosus and psoas major muscles at 1, 3, 7

and 13 days post-mortem are shown in Table 1. The means of

fiber tensile strength for both raw and cooked seaitendinosus

muscle and raw psoas major muscle at 1 day post-mortem were

found to be significantly (P<0.05) higher than the means at 7

and 13 days post-mortem, but they were not significantly dif­

ferent from the means at 3 days post-mortem. No significant

(P<0.05} differences were found among the means at 3, 7 and

13 days post-mortem in raw and cooked seaitendinosus muscle

and raw psoas major muscle. In the cooked psoas aaior mus­

cle, no significant (P<0.05) differences were found among the

means of fiber tensile strength at 1, 3, 7 and 13 days post­

mortem. These results (Fig. 10 and Table 1) indicate that

the finer tensile strength of the semiteiadinosus and psoas

aaior ssscles decrease during postsortss storage and that the

decrease in fiber tensile strength is greater in sssitendino-

SUS muscle, than in psoas aaior muscle= These results also .

suggest that cooking in a eater bath at 60oc causes a decided

increase in force necessary to break fibers. It is puzzling

that the psoas major muscle strips required such a high

breaking force because it is a much more tender muscle than

the seaitendisosus sescle. Since breaking strength measures

primarily strength or weakness of myofibrils, these results

114

substantiate that psoas major muscle myofibril does not frag­

ment as much as semitendinosus muscle myofibrils.

The changes in myofibril fragmentation aad W-B shear-

force during postmortem storage as previously noted, indicate

changes occur in myofibrillar proteins during postmortem

storage. Myofibrils, prepared from bovine lonqissimus muscle

at death and at 1, 3, 6 and 10 days of postmortem storage at

2°C, were electrophoresed on sodioara dodecyl sulfate (SDS) 7

1/2% and 10 % polyacrylamide gels. Figures lla-e show the

SDS-7 1/2% polyacrylamide gels at 0, 1, 3, 6 and 10 days

post-mortem. From 0 to 10 days post-mortem, mo major chan­

ges (appearance or disappearance of protein bands) occurred

in the bands above actin (myosin and alpha-actinin); however,

obvious changes in protein bands occurred below actin. A

gradual decrease and disappearance of the troponin-T proteia

band and a gradual appearance of the protein band la the

30,000-daltoa molecular weight region occarred from 0 to 10

days post-mortem, while these changes in myofibrillar pro­

teins of lonqissimus muscle myofibrils appeared very limited

and selective, it agreed with that reported for chicken

breast muscle (Hay et al., 1973a). Goll et^al. (1969) have

discussed two possible modifications of the myofibril after

rigor development; one being a modification of the actin-

myosin interaction and the other a degradation of the z-lina.

No dégradative changes in myosin, actin or alpha-actinia

Figure 11. SDS-7 1/2% and 10% polyacrylamide gels of myofi­brils prepared from bovine loaqiss|.aus muscle at death and at different times of postmortem stor­age at 2®C

a-e. 7 1/2% gels of myofibrils fro» 0(a), 1(b), 3(c), 6(d), and 10(e) days post-mortem. Note the gradual decrease of the tropoain-T band and the gradual increase of the 30,000-dalton band from 0 to 10 days post­mortem. No other major changes of other bands are noted

f-j. 10% gels of myofibrils from 0(f), 1(g), 3(h), 6(i), and 10 fj) days post-mortem. Bote the decrease of the troponin-T band and the increase of the 30,000-dalton band from 0 to 10 days post-mortem

30,000 DALTONS TRdPONIN-T

ACT IN

i M

# ' i Hi'

30,000 DALTONS l

II mmi :'BiP

lO

UE ̂ i «/>

lis o 50

:a\ H m 3t

117

have been shown to account for these myofibril modifications.

SDS-7 1/2% gels separate the heavy molecular weight proteins

above actin but the lighter proteins below actin have better

resolution in SDS-10% polyacrylamide gels. The SDS-10% poly-

acrylanide gels (Figs, llf-j) more clearly show the gradual

decrease and disappearance of the troponin-T protein band and

the gradual appearance of the 30,OSO-dalton protein band from

0 to 10 days post-mortem. Figure 12 shows the SDS-7 1/2% and

10% polyacrylanide gels of myofibrils from bovine semitendi-

nosQs muscle at death and at 1, 3, 6 and 10 days of post­

mortem storage at 20C. The SDS-7 1/2% (Figs. 12a-e) and 108

(Figs. 12f-j) polyacrylaaide gels show nearly identical chan­

ges in the myofibrillar proteins of the semitendlnosns muscle

myofibrils as found in myofibrils from the longissimns muscle

(Fig. 11). The troponin-T band gradually becomes less

intense and disappears at the same time as a 30,OOO-dalton

protein appears from 0 to 10 days post-mortem while no other

major changes occur in the other myofibrillar proteins.

Figure 13 shows the SDS-7 1/2% and 10% pslyacrylamide

gels of myofibrils from bovine psoas.major muscle at death

and at 1, 3^ 5 and 10 days of postmortem storage at 20C.

Both SDS-7 1/2% (Figs. 13a-e) and 10% (Figs. 13f-j) polyac-

rylamide gels show only ertreaeiy slight changes in the myo­

fibrillar proteins from 0 to 10 days post-mortem. The

troponin-T protein band, which appears very faintly in these

Figure 12. SDS-7 1/2% and 10% polyacrylamide gels of myofi­brils prepared from bovine semitendlnosas muscle at death and at different times of postmortem storage at 2®C

a-e. 7 1/2S gels of myofibrils at 0 (a) ̂ 1(b), 3(c), 6(d), and 10(e) days post-mortem. Rote the gradual decrease of the troponin-T band and the gradual increase of the 30,000-dalton band during postmortem stor­age. Ho other major changes of other bands are noted

f-j. 10% gels of myofibrils at 0(f), 1(g), 3(h), 6(i), and 10 (j) days post-mortem. Bote the decrease of the troponin-T band and the in­crease of the 30,000-dalton band during postmortem storage

119

7 1/2%

DAYS POSTMORTEM 0 1 3 A._ JUL

DAYS POSTMORTEM

o • • m

a b c d e f g h i

Figure 13. SDS-7 1/2% and 10% polyacrylaiaide gels of myofi­brils prepared from bovine psoas major muscles at death and at different times of postmortem stor­age at 2*0

a-e. 7 1/2% gels of myofibrils at 0(a), 1(b), 3(c), 6(d), and 10(e) days post-mortem. No major changes occurred in the bands during postmortem storage

f-g. 10% gels of myofibrils at 0(f), 1(g), 3(h), 6{i) , and 10(j) days post-mortem. Bote only a slight increase occurred in the 30,000-dalton band (j) during postmortem storage

121

7 m%

a. a

p^srmmm

DAYS POSTMORTEM 13 6

DAtS

h

122

gels, does not completely disappear in either the SDS-7 1/2%

or 10% polyacrylaaide gels from 0 to 10 days of postmortem

storage. Likewise only a very faint 30,000-dalton protein

band appears at 10 days post-mortem (Figs, lie and j). These

slight changes in the myofibrillar protein in the myofibrils

of the psoas major muscle during postmortem storage are in

sharp contrast to the obvious changes in the troponin-T pro­

tein band and the 30,000-dalton protein band in myofibrils

from lonqissifflus and semitendinosus muscles during postmortem

storage- Postmortem muscle storage of lonqissiaus and semi-

tendinosus muscles produce striking changes and similarities

in myofibril fragmentation, as observed in the light micro­

scope (Figs. 1 and 2), myofibril fragmentation index (Fig. 7

and Table 2), W-B shear-force (Fig. 9 and Table 2) and pro­

tein changes on SDS-polyacrylamide gels (Figs. 11 and 12).

In contrast, postmortem stored psoas major muscle does not

change substantially; consequently it differs consistsatly

from the longlssiaus and semxtendinosus ausclas with the sass

measures. While no direct relationship can be established

between the degradative protein changes occurring in muscle

during postmortem storage, shown in SDS-polyacrylamide gels,

and the changes in the myofibril fragmentation index or the

5-S shear-force, it is probable that a factor, or group of

factors, causes all of these measured postmortem muscle chan­

ges to occur sisultaneoGSly.

123

To determine if the 25oc postmortem storage temperature

treatment affected the postmortem myofibrillar protein chan­

ges in a manner similar to myofibril fragmentation index

(Fig. 7 and Table 2) and W-B shear-force (Fig. 9 and Table

2 ) f f m y o f i b r i l s f r o m b o v i n e l o n q i s s i m u s m u s c l e s t o r e d f o r 2

and 3 days post-mortem at 20C and 250C were electrophoresed

on SDS-10% polyacrylaaide gels. Ko changes ia the proteins

bands during postmortem storage are shown in the gels (Figs.

14a, c and e) of myofibrils from the lonqissimus muscle stor­

ed at 20C, However, gels (Figs. 14b, d and f) of myofibrils

from muscle stored at 250C, show a 30,000-dalton protein band

appearing slightly at 2 and 3 days post-mortem. Troponin-T

is not clearly resolved in these gels (Fig. 14), however, it

appears that troponin-T is present in myofibrils from muscle

stored for 3 days post-mortem at 2oc but not at 250C. The

higher postmortem storage temperature (250C) has no apparent

effect on the other myofibrillar proteins (Fig. 14). The gel

in Figure 14f (3 days post-mortem at 23*0) shows the same

protein changes as in myofibrils from longissimns muscle

stored for 10 days post-mortem at 2oc (Fig. 11]). The higher

storage temperature appeared to only accelerate the changes

in the myofibrillar proteins and not cause additional degra-

dative changeso The same high temperature acceleratioa of

postmortem changes was also found in myofibril fragmentation

index (Fig. 7 and Table 2) and in H-B shear-force (Fig. 9 and

Figure 14. SDS-10% polyacrjlaaide gels of myofibrils pre­pared from bovine lonqissipgs muscle at different times of postmortem storage at 2®C and 25®c

a. Gel of myofibrils at 1 day post-mortem, 2®C

b. Gel of myofibrils at 1 day post-mortem, 25°:. Bote the decreased intensity of the troponin-T band compared with Fig. 14a

c. Gel of myofibrils at 2 days post-mortem, 2°^

d. Gel of myofibrils at 2 days post-mortem, 25*0. Note the slight appearance of the 30,000-dalton band and the decreased intensi­ty of the troponin-T band

e. Gel of myofibrils at 3 days post-mortem, 2^C

f. Gel of myofibrils at 3 days post-mortem, 25®C. Note the absence of the troponin-T band and the presence of the 30,000-dalton band

:25

POSTMORTEM TIME DAY 1

TEMPERATURE Z°C 25^C DAY 2

2°C 25°C DAY 3

2*C 25*C

a-ACTIRIN

AGTÏN TROPONIM-T

30.000 OALTONS

TROPONIN-I *^5SF

Figure 15- SDS-IOS polyacrylaraide gels of myofibrils pre­pared from bovine semitendinosus muscle at dif­ferent times of postmortem storage at 2®C and 25°C

a. Gel of myofibrils at 1 day post-aortem, 2°Co Note the presence of the troponin-T band and the absence of the 30,000-dalton band

b. Gel of myofibrils at 1 day post-mortem, 25°C. Note the decreased intensity of the troponin-T band and the presence of the 30^000-dalton band

c. Gel of myofibrils at 2 days post-mortem, 2®C. Note the slightly visible 30,000-daltott band

d. Gel of myofibrils at 2 days post-mortem, 25®C, Note the absence of the troponin-T band and the greater intensity of the 30,000-dalton band compared with the gel in Fig. 15b

e. Gel of myofibrils at 3 days post-mortem, 2oc. Note the decreased intensity of the troponin-T band and the increased intensity of the 30,000-dalton band compared with the gel in Fig. 15c

f= Gel of myofibrils at 3 days post-mortem, 250c, Note the absence of the troponia-r band and the decreased intensities of the tropoaia-I band coaparsd sith the gel ia Fig. 15d or 15e

127

POSTMORTEM TIME DAY 1 TEMPERATURE 2°C 25°C

DAY 2 2°C 25^C

DAY 3 2°C 25*C

«-ACT1NIN

ACTIN TROPONÎN-T

30.000 DALTOMS

TROPONÏN-I

m 1 TV

-*v-

•Sï"^ asÂ

ft s m ÉSBS

® #

apaw»,

«C-

2 b c d e

128

Table 2) •

Figure 15 shows the SDS-10% polyacrylaaiie gels of myo­

fibrils from bovine seiaitendinosas muscle at 1, 2 and 3 days

of postmortem storage at 20C and 25oc. The gels of myofi­

brils from muscles stored at 2°C (Figs. 15a, c and e) show

that the intensity of the troponin-T band (which is not

clearly resolved) decreased slightly and the 30,000-dalton

band increased slightly from 1 to 3 days post-mortem. Howev­

er, the gels of myofibrils from seaitendinosuis muscle stored

at 250C (Figs. 15b, d and f) show that the troponin-T band

decreased in intensity and then disappeared while the

30,000-dalton band increased in intensity from 1 to 3 days

post-mortem. The 25oc storage temperature treatment caused

similar myofibrillar protein degradation as the 20C storage

temperature treatment from 1 to 3 days post-mortem but the

higher storage temperature caused the effects to occur sooner

in both the longissimus and semitendinosus muscles. Gels of

myofibrils from semitendinosus muscle stored at 3 days post­

mortem at 250C appeared to have the same degree of protein

degradation as shown in the gels of myofibrils from the seai-

teadiaosas muscle stored at 10 days post-mortem at 2°C (Fig,

12j).

Figure 16 shows the SDS-10% polyacrylassida gels of myo­

fibrils from bovine psoas maior muscles at 1, 2 and 3 days of

postmortem storage at 2°C and 250C. No major differences are

Figure 16. SDS-10% polyacrylanide gels of myofibrils pre­pared from bovine psoas mai or muscle at different times of postmortem storage at 2®C and 25®C

a-b. Gels of myofibrils at 1 day post-mortem, 2®C {a> aad 25*0 {b). No differeace be­tween gels appear (a and b)

c-d. Gels of myofibrils at 2 days post-mortem, 2®C (c) and 25*0 (d) . Note that ao major difference in the protein bands occur be­tween gels (c and d)

e-f« Gels of myofibrils at 3 days post-mortem, 2°C (e) and 25®C {f). Note that no major difference in the protein bands occur be­tween gels (e and f)

130

POSTMORTEM TIME DAY I TEMPERATURE 2°C 25°C

DAY 2 Z°C 25°e

t-ACTÎNîN

ACTIw TROPONIN-T

30,000 DALTOK$<

TROPONI#-!

b

DAY 3 2°C 25°C

M M

e f

131

shown between the gels of myofibrils either among days of

post-mortem storage or between storage temperatures at each

postmortem period. The apparent lack of significant changes

in the myofibrillar proteins due to either postmortem storage

time or temperature is similar to the relatively slight

change in myofibril fragmentation index (Pig. 7 and Table 2)

and H-B shear-force (Pig. 9 and Table 2) during postmortem

storage at 2°c or 25oc storage temperature. These results on

SDS-polyacrylamide gels indicate that some factor, or fac­

tors, causing postmortem muscle changes in the lonqissimus

and semitendinosus muscles has a greater activity at higher

storage temperatures and that this factor is apparently

lacking or suppressed in the psoas major muscle.

A calcium-activated-factor (CAP), a protease endogenous

to muscle tissue, has been shown to selectively remove Z-

lines and degrade the myofibrillar proteins troponia,

tropomyosin and coapoasat-C (Dayton et al,, 197%a. b). CAP

was prepared from bovine Isaqissimus, psoas major and semi-

tendinosus muscles at death and at 1, 3 and 5 days of post­

mortem storage at 20C and its activity was measured on the

proteolytic degradation of denatured casein (Fig. 17). cap

activity is highest at-death for all three muscles and its

activity decreases from O to 6 days post-mortsm. CâP activi­

ties of lonqissisas and semitendinossas muscles were nearly

identical at-death and decreased at about the same rate by 5

Figure 17. Effect of postmortem storage on C&F activity of bovine laaaiSSiiaS» 2S2âs_major, and semitendino-sns muscles at different times of postmortem storage at 2®c

CAP ACTIVITY (ABS0R13ANCE UNITS AT 278nm/100g MUSCLE)

o o ——y

134

days post-mortem to less than half of the at-death level with

the greatest decrease occurring between 0 and 1 day post­

mortem. In the psoas major muscle, the CAF activity at-death

is less than half the at-death C&F activity of the longissi-

mus and semitendiaosns muscles. caF activity in the psoas

major muscle decreases to near 0 at 1 day post-mortem and re­

mains at that level from 1 to 6 days post-mcrtes. No direct

relationship can be made between postmortem C&F activity and

the postmortem changes in myofibril fragmentation index and

W-B Shear-force because the proteolytic action of C&P should

increase if it caused the increase in postmortem myofibril

fragmentation index (Figs. 6 and 7 and Tables 1 and 2) and

the decrease in postmortem ff-B shear-force (Figs. 8 and 9 and

Tables 1 and 2) . The causal effect of CAF proteolytic activ­

ity on increasing myofibril fragmentation index and decreas­

ing W~B shear-force can not, however, be discounted even

though no direct linear relationship can be made, because an

active CAP fraction can be prepared from muscle stored 6 days

post-mortem in the longissiaus and semitendinosus muscles and

1 day post-mortem in the psoaS-iajgr muscle (Fig. 17). It is

possible that even a postmortem CAF activity level, loser

than the at-death level, is sufficient to cause the observed

postmortem muscle changes» It is also possible that the CAF

proteolytic activity from 0 to 1 day post-mortem causes most

of the degradation of the myofibrillar proteins at that time

135

but the effects on myofibril fragmentation index and W-B

shear-force are not completely revealed until a later post­

mortem time. It is also interesting to note that differences

in postmortem CAP activity betveen the psoas„major muscle and

that of the lonaissimus and semitendinosus muscles (Fig. 17)

follows the pattern of differences in these muscles for the

postmortem myofibril fragmentation index (Figs. 6 and 7 and

Tables 1 and 2) and W-B shear-force (Figs, 8 and 9 and Tables

1 and 2) measurements. The coincidental occurrence of the

similar changes in CftF activity, myofibril fragmentation in­

dex and B-B shear-force during postmortem storage of the lon-

qissimus and semitendinosus muscles while the changes of

these measures are considerably less in the psoas major mus­

cle indicates on integral relationship among these three

characteristics of postmortem muscle exists.

To determine the effect of CâF on myofibrils, purified

CAF from porcine muscle (prepared by W. Dayton, Dayton et

al., 1974a, b) was incubated with myofibrils from at-death

bovine lonqissimus. semitendinosus and psoas Bailor muscles

(Fig. 18). Myofibrils from the lonqissimus muscle incubated

in the absence of CAF (Fig. 18a) had relaxed sarcomeres with

Z-lines present, however, myofibrils incubated in the pres­

ence of CAF (Fig. 18b) show that Z-lines were completely re­

moved with little other apparent change occurring in the myo­

fibrils. The myofibrils from semitendinosus (Figs. 18c and

Figure 18. Phase micrographs of at-death {control) and C&F-treated at-death myofibrils of bovine longissi-mus. seaitendinosus. and psoas,major muscles

a. Control lonaissiaus myofibrils. Note the presence of Z-lines. X2GG0

b. CâF-treated at-death lonaissiaus myofibrils. Note the absence of Z-lines. X2000

c. Control semitendinosus myofibrils. Note the presence of prominent z-lines. X2000

d. cap-treated at-death seaitendino sus myofi­brils. Hyofibrils are fragmented and Z-lines are absent. 12000

e. Control psoas majoy myofibrils. Note the presence of prominent z-lines. X2000

f« cap-treated at-death psoas major myofibrils. Bote absence of Z-lines. 12000

'37

CONTROL CAF-TREATED * \ m •

"5 m-

CONTROL LOMGISSI^US

CAF-TREATED

t f f % f

mtm: »*

SEKITEMBlooses CONTROL CAF-TREATED

"CZar-

.. '.3S:

& '

'T **##

PSOAS MAJOR

138

d) and psoas major muscle (Figs, 18e and f) also show that

CAF apparently only selectively removes the Z-lines from myo­

fibrils. The ability of CAF to remove the Z-lines from myo­

fibrils (Fig, 18) implicates CAF as the causal factor

influencing a weakening of the z-line in postmortem muscle;

consequently, greater postmortem myofibril fragmentation re­

sulted (Figs. 1 and 2). To test this implication, at-death

myofibrils from the lonqissimus. semitendinosus and psoas

major muscles were incubated in the absence and presence of

CAF and electrophoresed on SDS-7 1/2% and 10% polyacrylamide

gels (Figs. 19-21). The SDS-7 1/2% polyacrylamide gels Figs.

19a and b) demonstrated that CAF-treated at-daath lonqissimas

muscle myofibrils removed alpha-actinin, troponin-T and

troponin-I bands and a 30,000-dalton protein band appears.

The SDS-10% polyacrylamide gels (Figs. 19c and d) showed more

clearly the removal of the troponin-T and troponia-l bands

and the appearance of the 30,000-dalton band in the CAF-

treated myofibrils. These results agree with those reported

by Dayton et al. (1974a, b) in that purified porcine CAP

selectively removes, after brief incubation with CAF, alpha-

actinia, troponin-T and troponin-I while a 30,000-dalt3n band

appears from porcine myofibrils. The removal of alpha-

actinin from CAF-treated myofibrils accounts for the removal

of Z-iines in the myofibril (Fig, 18b), Also, since only the

troponin subunits (troponin-T and I) were proteolytically da-

Figure 19. SDS-7 1/2% and 10S polyacrylanide gels of at-death bovine longissinus myofibrils incubated in the absence and presence of CAF

a. 7 1 /2% gel of control myofibrils. Note the presence of alpha-actinin, troponin-T and troponin-I bands

b. 7 1 /2% gel of CAF-treated myofibrils. Note the absence or decrease intensity of alpha-actinin, troponin-T and troponin-I bands and the presence of bands in the 30,000-dalton region

c. 10% gel of control myofibrils. Note the presence of alpha-actinin, troponin-T and troponin-I bands

d- 10% gel of CAF-treated myofibrils. Note the absence or decreased intensity of alpha-actinin, troponin-T and troponin-I bands and the presence of bands in the 30,000-dalton region

140

7 1/2% 10%

CAF-COHTRGi TREATED

CAP-CunTRCi. TREATED

«-ACTININ-

ACTIN

TROPONIN-T

30,CGC DALTONS'

TROPONIN-I-

TROPONIN-G'

* ̂

•«-ACTIKIN

<*-ACTIN -*-TROPONIN-T

^ -<-30,000 DALTONS

-TROPONIN-!

•TROPONÎN-C

Figure 20. SDS-7 1/2% and 10% polyacrylamide gels of at-deatfe bovine seaitendinosus myofibrils incubated in the absence and presence of CAF

a. 7 1/2% gel of control myofibrils. Note the presence of alpha-actinin, tropoain-T and troponin-I bands

b. 7 1/2% gel of CAF-treated myofibrils. Note the absence or decreased intensity of alpha-actiain, troponin-T and tropoain-I bands and the presence of bands in the 30,000-dalton region

c. 10% gel of control myofibrils. Note the presence of alpha-actinin, troponin-T and troponin-I bands

d. 10% gel of CâF-treated myofibrils. Note the absence or decreased intensity of alpha-actinin, troponin-T a;nd troponin-I bands and the presence of bands in the 30,000-dalton region

142

7 1/2% 10%

CAP-CONTROL TREATED

CAF-CONTROL TREATED

i i c-ACTI#!#"

ACTING m ̂ M * a# v _ IR v r wMt m— r

30,000 rtit-s' wn t . I vrt^r -

TROPONIN-!-

TROPONIN-C" :

1

.-aw;'.

##

•«-ACTISÎN

1«»-ACTIN "«f^TROPONIN-T

30»000 DAiTONS

•TROPON1N-Î

•TROPONIN-C

a b c d

Figure 21. SDS-7 1/2% and 10% polyacrylamide gels of at-death bovine psoas major myofibrils incubated ia the absence and presence of C&F

a. 7 1/2% gel of control myofibrils. Note the presence of alpha-actinia, troponin-T and troponin-I bands

b. 7 1/2% gel of CAF-treated myofibrils. Note the absence or decreased intensity of alpha-actinin, troponin-T and troponin-I bands and the presence of the bands in the SOfOOO-dalton region

c. 10% gel of control myofibrils. Mote the presence of the alpha-actinin, troponin-T and troponin-I bands

d. 10% gel of CAF-treated myofibrils. Note the absence or decreased intensity of the alpha-actinin, troponin-r and troponin-I bands and the presence of bands in the 30,000-dalton region

144

7 1/2%

CAP-CONTROL TREATED

w# I

a-ACTININ-^ ̂ Sm

ACTIN TROPONIN-T

30,000 DALTONS

TROPONIN.I

TROPONIN-C

10%

CAF-CONTROL TREATED

•«-ACTÎNÎN me m

4#

•ACTIN TROPONIN-T

•30,000 DALTONS

TROPONIN-!

TROPONIN-C

a b c d

145

graded, the myofibril (other than the z-line) remained appar­

ently unaffected by CâF treatment, SDS-7 1/2% and 10% poly-

acrylamide gels of myofibrils incubated in the absence and

presence of C&F from semitendinosus (Fig. 20) and psoas major

(Fig. 21) muscles show the same C&F proteolytic effects as

shown in lonqissians muscle (Fig. 19). SDS-10S polyacryla-

mide gels of mvofibrils from lonaisslmus. semitendinosus and

psoas major muscles incubated in the presence of CâF (Figs.

19, 20 and 21) show similar results as those shown in gels of

myofibrils from postmortem muscle (Figs. 11, 12 and 13)

except that alpha-actinin was present in the gels of myofi­

brils from postmortem muscle. Dayton et al. (1974a, b) have

shown that while C&F removes the Z-lines from myofibrils and

alpha-actinin in SDS-polyacrylanide gels of C&F-treated myo­

fibrils, C&F does not proteolytically degrade purified alpha-

actinin. Aipha-actinin is thus apparently released from the

myofibril but is not itself proteolytically degraded by CkF.

The close SXwXlarx.t^es of gels of C&F—treated myofxbjrxls

(Figs. 19, 20 and 21) and gels of myofibrils from muscle

Stored over 3 days post-mortem (Figs. 11, 12 and 13) clearly

implicate C&F as the causal factor producing the changes in

the myofibrillar proteins in postmortem muscle.

The gels of myofibrils from postmortem muscle and from

C&F-treated myofibrils have all shown the common alterations

in myofibrils to be primarily removal of the troponin-T band

146

an? appearance of the 30,000-aalton band. To determine

whether the 30,000-dalton band originated from troponin-T,

purified troponin prepared from at-death bovine longissiaus

muscle was incubated with purified CAP from porcine muscle

for 0, 5, 10, 20 and 40 minutes. SDS-10% polyacrylamide gels

of control and CAF-treated troponin are shown in Figure 22.

After 5 Einutes incubation with CAP, troponin showed some

apparent proteolytic degradation by the appearance of bands

below the troponin-T band, in the 30,000-ialton region, and

the bands below the troponin-C subunits. As incubation time

increased from 5 to 40 minutes (Figs, 22b-e), the troponin-T

band decreased in intensity and almost disappeared at 40

minutes incubation. The troponin-I band decreased slightly

as incubation time with CAF increased, but the troponin-C

band appeared to be unaffected by CAF treatment. The

30,000-dalton band increased in intensity up to lO minutes

incubation and then decreased in intensity as incubation time

increases beyond 10 minutes. The bands below troponin-C

became increasingly greater in intensity during the 40-sinute

CAF treatment. The 30,000-daiton band apparently originated

from the degradation of troponin-T by CAF treatment, however,

the 30,000-dalton protein is not a final product of troponia-

T degradation because it apparently is further degraded by

CAF into smaller molecular weight proteins. Troponin, before

and after CAF treatment, was electrophoresed along with myo-

Figure 22. SDS-10X polyacrylamide gels of purified troponin prepared from the at-death bovine loagissimus muscle and incubated with CAF for 0, 5, 10, 20 and %0 ainutes

a« Troponin with no CAF treatment. Note the troponin subunits T, I and c

b. Troponin after 5 minutes incubation with CAP. Note the proteolytic breakdown products in the 30,000-dalton band region and below the I and C subunits

c. Troponin after 10 minutes incubation with CAF, Note the increased intensity of the proteolytic breakdown products and the de­creased intensity of the troponin-T band

d. Troponin after 20 minutes incubation with CAF. Note the increase in intensity of the bands below the I and c subunits and the de­crease in the troponin-T band and the bands in the 30,OOO-daltoa region

e. Troponin after 40 minutes incubation with CAFo Note the faint bands in troponin-T and in the 30,000-dalton region and the increased intensity in the bands below the I and c subunits. The troponin-I band has also de­creased compared to the gel in Fig. 17b

148

TIME OF CAP TREATMENT (MIN) 0 5 10 20 40

TROPININ-T

30,000 DALTONS

TROPONIN-!

TROPONIN-C

a b c d e

• • • •

149

fibrils from at-fleath bovine longissiiaus muscle and at 10

days of postmortem storage on SDS-10% polyacrylamida gels

(Fig, 23). The gel of at-death (control) myofibrils (Fig.

23a) shoved the presence of troponin-T and the absence of the

30,000-dalton band. When troponin (with no CAF treatment)

was electrophoresed along with the at-death myofibrils, (Fig.

23b) troponin sabunit bands showed increased intensity but

the 30,000-dalton band was not present. Bhen CAF-treated

troponin was electrophoresed with the at-death myofibrils

(Fig, 23c) , the troponin sab unit bands showed greater inten­

sity and the 30,000-dalton band appeared. The gel of myofi­

brils from muscle stored 10 days post-mortem (Fig. 23d)

showed the absence of the troponin-T band and the presence of

the 30,000-dalton band. When troponin (sith no CAF treat­

ment) was electrophoresed with myofibrils from postmortem

muscle (Fig. 23e), the troponin-T band appeared in the same

location as that sheen in the gel of at-death sy©fibrils

(Fig. 23a), the tropoain-I and c bands ssrs of greater inten­

sity and the 30,000 dalton band was of similar intensity as

shown in Figure 23d. When CAF-treated troponin was electro­

phoresed with myofibrils from postmortem muscle (Fig. 23f),

the troponin-T band appeared and the troponin-I, traponin-C

and 30,000-dalton bands increased in intensity. It is clear

that the band in at-death sycflbrils (Fig. 23a) that

disappears from the gel of myofibrils after postmortem muscle

Figure 23. SDS-10% polyacrylamide gels of myofibrils pre­pared from at-death bovine longissimus muscle and at 10 days of postmortem storage in the absence (control) and presence of troponin (?K) and C&P-treated troponin (CAF-TN)

a. Gel of at-death control myofibrils

b. Gel of at-death myofibrils combined with tro­ponin. Note the increased intensities in the troponin subunit bands

c. Gel of at-death myofibrils combined with CâF-treated troponin. Note the presence of the 30,000-dalton band

d. Gel of myofibrils at 10 days post-mortem CcontrolJ. Note the abseaee of troponin-T and the presence of the 30,000-dalton band

e. Gel of myofibrils at 10 days post-mortem com­bined with troponin. Note the troponin-T band is in the same location as shown in the at-death myofibrils (Fig. 23a)

f. Gel of myofibrils at 10 days post-mortem com­bined with C&F-treated troponin. Note the 30,000-dalton band and the bands below troponin-c are of greater intensity than when CAF-treated troponin was not combined with myofibrils (Fig. 25q and 23e)

151

AT-DEÂTH MYOFIBRILS CONTROL TN CAF-TK

10 DAYS POSTMORTEM MYOFIB RIL S

CQNTROi TN CAF-TH

ACTIlt TROPONIM-

30,000 DALTOmS

TRGPOMIK-Î

TROPOfîîK-C

#

a b c d e f

152

storage (Fig. 23d) is troponin-T and the 30,000-dalton band

that appears in the gel of myofibrils from postmortem muscle

(Fig. 23d) originates from the degradation of troponin-T.

Figures 24a and b show the SDS-10X polyacrylamide gels

of myofibrils from at-dsath bovine Ipngissimus muscle and at

10 days of postmortem storage at 2oc. The major changes in

the myofibrillar proteins during postmortem storage is the

selective degradation of troponin-T yielding a 30,000-dalton

molecular «eight protein. Figure 2Uc and d show the SDS-10%

polyacrylamide gels of troponin from at-dsath bovine longis-

simus muscle with and without CàF treatment. C&F treatment

of troponin shows troponin-T degraded partially to a

30,000-dalton molecular weight protein and troponin-I being

also degraded. It is interesting to note that the major

change occurring naturally in postmortem muscle (Figs. 21a

and b) can be manufactured by CâF treatment of purified tro­

ponin (Figso 24c and d) «

Since the myofibrillar proteins constitute the major

portion of the muscle structure, myofibril fragmentation

should play an integral role in meat tenderness and quality.

To test this, loin steaks from lonaissiaus muscles from veal,

^-maturity and C-maturity were examined at 1 and 7 days post­

mortem. Table 3 shows the means and standard errors of myo­

fibril fragmentation index, W-B shear-force and sensory panel

tenderness, flavor and juiciness of loin steaks from lonqis-

Figure 2U. SDS-10X polyacrylamide gels of myofibrils pre­pared from at-death (control) bovine longissimus muscle and at 10 days of postmortem storage and gels of purified troponin (TN) and troponin incubated with CAF (CAF-TN)

a. Gel of at-death myofibrils (control). Note presence of troponin-T bands and absence of 30p000-dalton band

b. Gel of myofibrils 10 days post-mortem. Note absence of troponin-T band and presence of 30,000-daiton band

c. Gel of CAP-treated troponin (CAF-TN) . Note presence of bands in 30,000-dalton region

d. Gel of purified troponin (TN). Note absence of 30,000-dalton band

154

DAY CAP-CONTROL 10 IN TN

ACTIN-TROPONIN-T

30»000 DALTONS

TROPONIN-!

TROPONÎN-C'

a b c d

155

simns muscle from six veal carcasses at 1 and 7 days of post­

mortem storage at 20C. Myofibril fragmentation index was

significantly {P<0.05) higher at 7 days than at 1 day post­

mortem. However, W-B shear-force and sensory panel tender­

ness, flavor and juiciness were not significantly (P<0.0 5)

different between 1 and 7 days post-mortem. Even though ten­

derness (measured by H-B shear-force and sensory panel) was

considerably higher at 7 days than at 1 day post-mortem, the

standard errors were too large to make these differences sig­

nificant.

Table H contains the means and standard errors of myofi­

bril fragmentation index, »-B shear-force and sensory panel

tenderness, flavor and juiciness of bovine longissimus muscle

loin steaks from thirty-five A-maturity carcasses at 1 and 7

days of postmortem storage at 2oc. Myofibril fragmentation

index was significantly (P<0.05) higher at 7 days than at 1

day post-mortem, whereas sensory panel flavor was signifi­

cantly CP<0.05) but only slightly lower at 7 days than at 1

day post-mortem. R-B shear-force and sensory panel tender­

ness and juiciness were not significantly (?<0.05/ different

between 1 and 7 days post-mortem. In comparison with results

of a previous experiment, the mean myofibril fragmentation

index of 64.8 at 1 day post-sortes (Table U) was nearly the

same as the mean myofibril fragmentation indez of 69.8 at 3

days post-mortem (Table 2) measured on longissimus muscle of

156

Table 3. Effect of postmortem storage (20C) on myofibril fragmentation index (PI), Warner-Bratz 1er (W-B) shear-force and sensory panel tenderness (TEND), flavor (FLA) and juiciness (JO) of longissimus muscle from six veal carcasses^

FI2

W-B3

TEND*

FLA*

jn*

Days of postmortem storage

1 7

4^5310.55 3^62^0.49

3i91+0^54 5il7tOi55

3.3610.18 3^22±0 j.17

5i.32i0̂ 27 5.34±0i23

iQeans ± standard errors. Means not underscored by the same dotted line are significantly different {P<0.05).

zAbsorbance per O.Smg myofibril protein S 200,

3Kg of shear-force per cm.

^Hedônic scale of 1 to 8 «fith 3 being eztremely tender, flavorful or juicy.

157

Table U. Effect of postmortem storage (20C) on myofibril fragmentation index (FI) , Warner-Bratz 1er (B-B) shear-force and sensory panel tenderness (TEND), flavor (FLA) and juiciness (JD) of lonaissimus muscles from thirty-five A-maturity carcasses*

Days of postmortem storage

1 7

PI2 64.8t0.7 71.4&0,6

*-B3 2.85^0.08 2j,72±0.07

TEND* 5.29±0. 20 5u52±0.15

Fia* 5.86^0. 08 5.62*0.08

JO* 5^94&0.09 5.78^0.09

iReans ± standard errors. Means not underscored by the same dotted line are significantly different (P<0.05).

2&bsorbance per 0.5mg myofibril protein X 200.

3Kg of shear-force per cm.

nëuoûic acaie St ; 8 with 8 being extrsaely tender* -Sedonic scale -« 4-- q

flavorful or juicy.

158

animals having similar maturities as those reported in Table

4. The bovine carcasses in the two separate experiments

originated from different sources and samples were handled

differently (See METHODS AND HâTEHIULS section) which may ac­

count for part of the differences in the postmortem myofibril

fragmentation index means. The ff-B shear-force mean of

2.72kg/CE2 {Table at 7 days post-mortem was also higher

than that shown for the longisslmns muscle in Table 2 of

2.60kg/cm2 at 1 day post-mortem.

Table 5 shows the means and standard errors of myofibril

fragmentation index, a-B shear-force and sensory panel ten­

derness, flavor and juiciness of the bovine lonqissimus mus­

cle from twelve C-matarity carcasses at 1 and 7 days of post­

mortem storage at 2°C, Myofibril fragmentation index was

significantly {P<0.05) higher at 7 days than at 1 day post­

mortem, but W-B shear-force and sensory panel tenderness,

flavor and juiciness were not significantly {P<0.05) differ­

ent between 1 and 7 days post-mortem. h larger difference in

myofibril fragmentation index means between 1 and 7 days

post-mortem was found in the lonqissimus muscle of the C-

aaturity carcasses {Table 5) than was found in the â-œaturity

(Table 4) or veal (Table 3) carcasses. However, the tender­

ness of the loacfissimus muscle at respective days post-mortem

was lower in the C-maturity carcasses than in the â-maturity

carcasses, but higher than in the veal carcasses.

159

Table S.

FI2

W-B3

TEND*

Plâ*

JO*

Effect of postmortem storage (20C) on myofibril fragmentation index (PI), Warner-Bratz 1er (W-B) shear-force and sensory panel tenderness (TEND), flavor (FL&) and juiciness (JD) of loncrissimus muscles from twelve C-maturity carcassesi

Days of postmortem storage

1 7

Z4i2±g.8

3i36t0.16 2i94&0.13

5^42i0^27

5.57+0,14 5^U2i0^17

5.73±0.20 5.56±0.18

iHeans ± standard errors. Means not underscored by the same dotted line are significantly different (P<0.05).

2&bsorbance per 0.5mg myofibril protein X 200.

3Kg of shear-force per cm.

•Hedonic scale of 1 to 8 with 8 being extremely tender.

160

Simple correlation coefficients (Table 6) were deter­

mined among myofibril fragmentation index, W-B shear-force

and sensory panel tenderness, flavor and juiciness of longis-

simus muscles from veal, &-aaturity and c-maturity bovine

carcasses at 1 and 7 days of postmortem storage at 2^C. The

correlation coefficients between myofibril fragmentation in­

dex and S-B shear-force were significant (PoOoOS, 9,0=01) far

both 1 and 7 days post-mortem for all three maturity groups

with the lowest coefficient being -0.85 for the a-maturity

group at 1 day post-mortem. Like-wise, the correlation coef­

ficient between myofibril fragmentation index and sensory

panel tenderness gas significant (P<0.05, P<0.01) for both 1

and 7 days post-mortem for all three maturity groups with the

lowest coefficient being 0.6 5 for the C-maturity group at 7

days post-mortem. In addition, the correlation coefficient

between %-B shear"fores aad sszscry panel tsnisrnsss for veal

steak sa s significant CP<0,01) at 7 days post-mortem (-0.94)

but not at 1 day post-mortem (-0.76)« Also the correlation

coefficient between sensory panel tenderness and juiciness

for veal was significant (P<0.05) at 7 days post-mortem

(0.82) but not at 1 day post-mortem (0.75). &11 other corre­

lation coefficients for veal carcasses were not significant

(P<0.05}. The correlation coefficients between sensory ten­

derness and sensory flavor and juiciness were moderately high

{0.62-0.69} for the lonqissimus muscle of â-maturity carcas-

161

Table 6. Effect of postmortem storage (2QC) OQ correlation coefficients among myofibril fragmentation index (PI), Warner-Bratzler (R-B) shear-force and sen­sory panel tenderness (lEHD) , flavor (FLâ) and juiciness (JO) of longissiaus muscles from veal, A-maturity and C-maturity carcasses

ÏSàii àzmatnritzz S^maturitzi, Days of postmortem storage

17 17 17

FI VS S-B -0. 95*$ -0. 97** -0. 65** -0.75** -0. 68* -0. 72* FI VS TEHD 0. 88* 0. 95** 0. 67** 0.73** 0. 68* 0. 65* FI VS FLA Oo 52 0. 06 0. 43** 0.54** 0. 57* 0. 06 FI VS JO 0. 58 0. 78 0. 50** 0.57** 0. 36 0. 52 W-B VS TEHD -0. 76 -0. 94** -0. 86** -0.62** -0. 59* -0. 48 S-5 VS FL£ -0, 5U -Oo 01 -0 = 50** -0,49** -0. 47 0. 01 B-B VS JO -0. 59 -0. 79 -0. 47** -0.31 0. 04 -0. 32 TEND VS FLA 0. 55 0. 06 0. 63** 0.69** 0. 75** 0. 55 TEND VS JO 0. 75 0. 82* 0. 64** 0.62** 0. 66* 0. 83** FLA VS JO 0. 22 -0. 27 0. 49** 0.49** 0. 46 0. 66*

^Correlation coefficients of six veal carcasses.

^Correlation coefficients of thirty-five A-maturity car casses.

^Correlation coefficients of twelve C-maturity carcas-

•Significant at the 5% level.

*$Si3nificant at the IS level.

162

ses. Sensory flavor and juiciness were also moderately cor­

related to H-B shear-force and myofibril fragmentation index,

however, these correlations were probably not direct correla­

tions, but resulted from their mutual relationship to sensory

tenderness. The correlation coefficients between sensory

tenderness and flavor, sensory tenderness and juiciness and

sensory juiciness and flavor were higher in the loaqissimus

muscle of the C-maturity carcasses than in either the A-

maturity or veal carcasses.

Because myofibril fragmentation index is related to the

degradation of the myofibrillar proteins during postmortem

storage, myofibrils were selected from two loagissiaus mus­

cles of veal. A-maturity and C-maturity carcasses at 1 and 7

days postmortem at 2 0C which had estreme differences in myo­

fibril fragmentation index, W-B shear-force and sensory ten­

derness scores» These selected myofibrils were electropho-

ressd on SDS-1G% polyacry lass ids gels and are shown in Figures

25-27. The gels of the myofibrils of lonqissimus muscles of

both veal carcasses {Fig. 25) show that at 1 day post-mortem

the troponin-T band is present but the 30,000-dalton band is

absent which coincided with a low myofibril fragmentation in­

dex and muscle tenderness (measured by H-B shear-force and

sensory panel evaluation). The gels of the myofibrils form

the lonqissimas muscle at 7 days post-mortem show the absence

of the troponin-T band and the presence of the 30,000-daltOQ

Figure 25. SDS-10X polyacrylamide gels of myofibrils pre­pared from lonaissimus muscles of two vaal car­casses at 1 and 7 days of postmortem storage at 2®C having different Rarner-Bratzler (W-B) shear-force, sensory tenderness and myofibril fragmen­tation index vaines

a. Gel of myofibrils at 1 day post-mortem having a H-B shear-force of 4.42 and a fragmentation index of 60 (carcass 1) . Bote the faint 30,000-darton band

b. Gel of myofibrils at 7 days post-mortem having a B-B shear-force of 2,85 and a frag­mentation index of 74 (carcass 1) . Note the intense 30,000-dalton band

c. Gsl of ayofibrils at 1 day post-mortem having a W-B shear-force of 3.42 and a fragmentation index of 63 (carcass 2) . Note the faint 30,000-dalton band

d. Gel of myofibrils at 7 days post-mortem having a W-B shear-force of 3.01 and a frag­mentation index of 70 (carcass 2). Note the intense 30,000-dalton band

16+

CARCASS V 2 DAYS POSTMORTEM 1 7 1 7

W-B SHEAR-FORCE 4.42 2.85 3.42 3.01 SENSORY TENDERNESS 4.10 5.30 4.43 5.30

FRAGMENTATION INDEX 60 74 63 70

ACTÎN TROPONIN-T

Figure 26. SDS-10% polyacrylamide gels of myofibrils pre­pared from lonqissimus muscles of two ft-aaturity bovine carcasses at 1 and 7 days of postmortem storage at 2°C having different Waraer-Bratzler (W-B) shear-force, sensory tenderness and myofi­bril fragmentation index values

a. Gel of myofibrils at 1 day post-mortem having a H-B shear-force of 2.90 and a fragmentation index of 58 (carcass 1) . Note the faint 30,000-dalton band

b. Gel of myofibrils at 7 days post-mortem having a ff-B shear-force of 2.74 and a frag­mentation index of 62 (carcass 1). Note the faint 30,000-dalton band

c. Gel of myofibrils at 1 day post-mortem having a W-B shear-force of 2.83 and a fragmentation index of 64 (carcass 2). Note the slightly intense 30,000-dalton band

d. Gel of myofibrils at 7 days post-mortem having a H-B shear-force of 2.51 and a frag­mentation index of 68 (carcass 2), Note the intense 30,000-dalton band

166

CARCASS

DAYS POSTMORTEM

W-B SHEAR-FORCE SENSORY TENDERNESS FRAGMENTATION INDEX

? t 7

ACTIN TROPONIN-T

30,000 DALTONS

2.83 5.70

64

2.51 6.10

68

a b c d

Figure 27. SDS-10% polyacrylanide gels of myofibrils pre­pared from lonqissimus muscles of two C-maturity bovine carcasses at 1 and 7 days of postmortem storage at 2^0 having different Barner-Bratzler (H-B) shear-force, sensory tenderness and myofi­bril fragmentation index values

a. Gel of myofibrils at 1 day post-mortem having a W-B shear-force of 3.79 and a fragmentation index of 46 (carcass 1). Note the absence of the 30,000-dalton band

b. Gel of myofibrils at 7 days post-mortem having a W-B shear-force of 3.35 and a frag­mentation index of 66 (carcass 1). Note the presence of the 30,000-dalton band

c. Gel of myofibrils at 1 day post-mortem having a H-B shear-force of 3,56 and a fragmentation index of 54 (carcass 2). Note the very faint 30,000-dalton band

d. Gel of myofibrils at 7 days post-mortem having a H-B shear-force of 2.96 and a frag­mentation index of 74 (carcass 2). Note the intense 30,0 00-dalton band

168

CARCASS

DAYS POSTKORTEM

W-B SHEAR-FORCE 3,79 3.35 SENSORY TENDERNESS 4.60 5.30

FRAGMENTATION INDEX 46 65

•>4 a

ACT IH TROPONTN-

30,000 DALTONS—M

169

band, which coincided with higher myofibril fragmentation and

muscle tenderness. These results in the veal carcass (Fig.

25) further indicate the relationships between the myofibril

fragmentation index, S-B shear-force, sensory tenderness and

the postmortem changes in the myofibrillar proteins.

The gels of the myofibrils from the two a-maturity car­

casses at 1 and 7 days post-mortem are shown in Figure 26.

Lonaissimus muscles of both carcasses have large differences

in myofibril fragmentation index, W-B shear-force, sensory

tenderness between only carcass 1, 1 day post-mortem and car­

cass 2, 7 days post-mortem. The gels of myofibrils from the

lonqissimus muscle shoe that troponin-T is not degraded in

myofibrils from carcass 1, 1 day post-mortem, but, in carcass

2,7 days post-mortem, troponin-T is degraded. These results

show that troponin-T degradation coincides with greater myo­

fibril fragmentation inde: and muscle tenderness.

The gels of myofibrils, tenderness measures and syofi-

bril fragmentation index of lonqlssiîaus susclss from tsc C-

Eaturity carcasses are shown in Figure 27. As has been pre­

viously observed in the veal and ^-maturity lonqissimus mus­

cles {Figso 25 and 26) , the presence of the troponin-T band

in the gels generally parallels high W-B shear-force and low

sensory tenderness and eyofibril fragmentation index, while

absence of troponin-? and the presence of the 30,000-dalton

protein parallels the opposite of these measures in the Ion-

170

gissimus muscle oE the t*o c-maturity carcasses. From the

data shown in Figures 25-27 of longissimus muscles of veal,

A-maturity and c-maturity carcasses, it appears that inter­

relationships exist between muscle tenderness, myofibril

fragmentation and myofibrillar protein degradation.

171

DI SCnSSION

The objective of this investigation uas to provida un­

derstanding about some of the important cellular and subcel­

lular events occurring in postmortem bovine skeletal muscle

and to show how these events relate and contribute to post­

mortem muscle tenderization. To elucidate these events and

determine something about their relationships two relatively

new techniques were used to characterize some of these

postmortem muscle events. The tecuûiqses, tarbidosetric

measure of myofibril fragmentation and SDS-polyacrylamide gel

electrophoresis, yielded significant results and gave impetus

to the direction of this investigation.

The method of determining myofibril fragmentation by

measuring the absorbance of myofibril suspensions was valu­

able in quantitating the postmortem changes in the fragmenta­

tion of the myofibril and associating those changes with

postmortem muscle tenderization. The turbidometric measure

of myofibril fragmentation is based on the premise that the

amount of light absorbance of a myofibril suspension will in­

dicate the relative size of myofibrils and fibes pieces is

the suspension. For example, in two myofibril suspensions

having the same protein concentration, the suspension having

shorter myofibrils and smaller fiber pieces, will haVe a more

turbid suspension and a higher absorbance value than the

other suspension with longer myofibrils and larger fiber

172

pieces. Several investigators (Berry et al.. 1974; Fukazawa

et al.. 1969; Sayre, 1969, 1970; Takahashi 6t_al., 1967) have

used microscopic methods, for counting sarcomeres par myofi­

bril or determining myofibril lengths, as a measure of myofi­

bril fragmentation. Bhile these investigators found myofi­

bril fragmentation changed during postmortem muscle storage

similar to the change ia muscle tenderness, a high relation­

ship to tenderness was not found. Parrish et al. (1973b)

followed both changes in sarcomere lengths and myofibril

fragmentation during postmortem storage and they reported

that improved tenderness seemed more a function of fragmenta­

tion of myofibrils than a change in sarcomere length of lon-

gissimus muscle stored in the carcass. These results sug­

gested that an objective method of measuring myofibril frag­

mentation would be a potentially useful means of determining

tenderness, âa objective Bsasurs of sycfibril fragsantation

as opposed to siccoscopic methods provides the advantage of a

larger and more representative sample and removes personal

bias and subjectivity. Measurement of a suspension of myofi­

brils and fiber pieces is recorded in optical density units,

but to give it a more descriptive expression the optical den­

sity value was multiplied by 200 and termed myofibril frag­

mentation index. Hence, the myofibril fragmentation index is

not an absolute value of myofibril lengths but a relative

value of the degree of disintegration of muscle fibers by fa3-

173

mogenization.

The technigue of SDS-polyacrylamide gel electrophoresis

was valuable in separating and identifying myofibrillar pro­

teins because of its sensitivity to small molecular subunit

changes. The capability of preparing intact myofibrils from

muscles after postmortem storage or other treatments and then

identifying the proteins from the myofibrils was particularly

useful. Changes in the myofibrils after postmortem muscle

storage (to be discussed later) were found to be principally

the degradation of one myofibrillar protein sabunit. Also,

the degradation of myofibrillar proteins in the presence of

CàF (to be discussed later) was found to be similar to the

natural degradation of myofibrillar proteins in postmortem

auscle. These two results yhich srere easily discernible on

SDS-polyacrylamide gels, would probably have gone undetected

if this technique had not been utilized, a distinct advan­

tage of SDS-polyacrylaeide gel electrophoresis over

biochemical ssasurss is that the myofibrillar proteins can be

visually seen and degradative changes (disappearance or ap­

pearance of proteins bands} can be viewed and identified.

These visual observations greatly assisted in interpretation

of the changes occurring in the myofibril and muscle.

The disintegration of myofibrils into shorter myofibrils

during postmortem muscle storage has been observed by many

investigators with the light microscope but few have attempt­

171»

ed to address the more important practical issues of 1)

quantifying and identifying the changes which occur in myofi­

brils, 2) investigating possible causative factors responsi­

ble for fragmentation and 3) relating these changes to meat

tenderness. Use of a system to objectively measure fragmen­

tation must be carefully controlled and some understanding of

what honogenization does to muscle tissue is imperative if it

is to be effectively related to tenderness. Brief homogeni-

zation of muscle tissue does not separate it into hoaogeneous

units and size, but muscle disintegrates into short,

heterogeneous units moderate and long single myofibrils and

small, moderate and large fiber pieces. Observing myofibrils

under the light microscope at high magnifications (i.e. using

100 X objective) prohibits accurate appraisal of other than

single myofibrils or very small fiber pieces. Bith low

magnifications (i.s. usiag 10 X objective) fiber pieces of

almost all sizes are discernible, however, sarcomeres in myo­

fibrils cannot be clearly distinguished. The degree to which

the postmortem muscle tissue is broken (severity of grinding

and homogenizing) will have a tremendous impact on the size

of myofibrils and fiber pieces. Extremes in grinding and

homogenizing will result in very small fiber pieces and short

single myofibrils, while minimal breaking of the muscle tis­

sue will yield large fiber pieces and mostly long single myo­

fibrils. To determine differences in the fragaentatioa of

175

muscle fibers and myofibrils between different muscles or

treatments on the same muscle, the myofibril preparation pro­

cedure must be closely controlled. Severe methods of

rupturing the muscle tissue may eliminate or mask the tissue

differences while gentle rupturing techniques make the tissue

differences undetectable. In this study, it was found that a

30-second hosogenization tine in a Baring Bleador of a ^gm,

scissor-minced muscle sample was an adequate compromise to

sufficiently rupture the muscle tissue without eliminating

the tissue differences. In all muscles measured for myofi­

bril fragmentation, the control sample was taken from 1 day

post-aortea muscle.

It was noted that myofibrils and fiber pieces from bo­

vine lonqissimus and semitendinosns muscles broke into small­

er sizes during homogenization from 1 to 10 days post-mortem

at 2-C. Syofibrils appear tc brsak at or near the Z-lir.e in­

dicating that postmortem storage results in a weakening of

the myofibril structure at or near the Z-line, However, even

at 10 days of post-mortem storage, the Z-line appears promi­

nent although less distinct than at 1 day post-mortem. These

observations are substantiated by the work of Davey and

Gilbert (1967, 1969), Fukazawa et al. (1969), Hay et al.

(1973b), Parrish et al. (197 3b), Sayre (1970) and Takahashi

et al. (1967). Myofibrils and fiber pieces from the psoas

major muscle appear to break in only slightly smaller sizes

176

during homogenization from 1 to 10 days postmortem. Obvious­

ly, muscle makes a difference in the way its myofibrils frag­

ment during postaortes storage. Although there vas a differ­

ence in amount of fragmentation due to aascle, myofibril

fragmentation index, determined by absorbance of a myofibril

suspension closely agreed with the myofibril changes observed

microscopically in lonaissin us, seaitendinosgs and psoas

major muscles. The major increase in myofibril fragmentation

occurred between 1 and 3 days post-mortem with lesser in­

creases being observed at 6 days post-mortem and only slight

increase after 6 days postmortem in lonqissimus and semiten-

dinosus muscles. In addition to following myofibril fragmen­

tation index to determine weakening of the myofibril struc­

ture, possibly at the Z-line, two mechanical devices were

used, the W-B shear-force apparatus and the Instron universal

Tasting Sachins, 5-5 shear-force is highly correlated with

meat tenderness (correlation coefficients of -0.5 to -0.8

(Parrish et al., 1973a;) and measures primarily the toughness

of meat associated with the myofibrillar proteins (Purchas,

1972). W-B shear-force, which measures myofibril resistance

to shearing force, decreased during postmortem storage in

lonqissimus and semitendinosus but not in psoas major muscle.

The decrease in H-B shear-force coincided with the increase

in myofibril fragaentation indes in loagissiaus and seaiten-

diaosus muscle. The Instron Universal Testing Machine nsa-

177

sures the force required to break a muscle strip (opposing

forces are applied longitudinally to the muscle strip). The

breaking force or strength indicates the strength or weakness

of myofibrils in muscle strips to resist the opposing forces

(Bouton and Harris, 1972b; Stanley et_al., 1971, 1972).

Breaking strength was much greater in strips from cooked mus­

cle {60OC) than in strips from rae muscle in both semitendi-

nosus and psoas major muscle, probably due to the coagulation

and hardening of the muscle proteins (Bouton and Harris,

1972a). In strips from both cooked and raw semitendinosus

and psoas major muscles, breaking strength decreased during

postmortem storage with the greatest decrease occurring in

semitendinosus muscle. The postmortem decrease in breaking

strength in semitendinosus muscle coincided with postmortem

changes in myofibril fragmentation index and B-B shear-force

indicating that significant change cr yeaknsss in the myofi­

bril occurs during postmortem storage.

Lonaissimus. semitendinosus and psoas major muscles were

also stored for 3 days post-mortem at 25oc. High temperature

(250C) storage of the three muscles was implemented in this

study to further determine what occurs to muscle sturcture at

higher temperatures than at conventional postmortem storage

temperatures and to ascertain how higher temperatures effect

these changes- Hyofibril fragmentation index greatly in­

creased from 1 to 3 days post-mortem, with the greatest in­

178

crease occurring from 1 to 2 days post-mortem in longissimus

and semitendinosus muscles stored at 25®C, Myofibril frag­

mentation index, in the longissimus and Semitendinosus mus­

cles at 3 days post-mortem at 25oc was nearly the same as

that at 10 days post-mortem at 2°c, This indicates that

higher storage temperature accelerates postmortem increase in

myofibril fragmentation index. Koreover? at 1 day post­

mortem, in longissimus and semit_endinosus muscle, myofibril

fragmentation index was higher in muscles stored at 25oc than

at 20C indicating that an accelerated increase in myofibril

fragmentation had started even before 1 day post-mortem. In

psoas major muscle, however, higher storage temperature

{250C) only slightly increased myofibril fragmentation com­

pared with 20C storage temperature. Hence, this study is the

first to quantitatively show that high postmortem storage

temperature (25-0) accelerates sycfifcril fragmentation and

that nyofibers and myofibrils from different muscles do not

fragment in a similar manner. High postmortem storage tem­

perature (250C} results in lower W-B shear-force from 1 to 3

days post-mortem more than 2 oc postmortem storage temparatace

and g-B shear-force at 3 days post-mortem, 25oc, was nearly

the same as at 10 days post-mortem, 2®C, in longissimus and

semitendinosus muscles which agreed with that reported for

longissimus and semitendinosus muscles (Parrish et al.,

1973b). Again, psoas major muscle reacted differently to

179

shear-force in that it decreased only slightly more daring

post-mortem storage at 250C than at 20C, These observations

further substantiate the view that myofibril fragneatation is

an iaportant modifier of neat tenderness.

Although sample numbers were too small in these studies

to determine meaningful correlation coefficients between the

measures, it is obvious that weakening of the Z-line during

post-mortem storage measured by myofibril fragmentation index

and fiber tensile strength, is directly related to muscle

tenderness. Similar changes in myofibril fragmentation index

and »-B shear-force during post-mortem storage at both 2oc

and 250C and consistent differences in these measures between

lonqissiBUs and semitendinosas muscles and the psoas major

muscle practically exclude the possibility of an unrelated

coincidence of these results. The accelerated changes in

frigsentation and teadecmess at 25»C storage tempecatuce

seems to imply that a specific and limited proteolysis, more

active at higher temperatures, may be causing the weakening

of the Z-lines and increased myofibril fragmentation.

SDS-polyacrylaoide gel electrophoresis was a most useful

tool to characterize the changes in myofibrils of longissi-

mus, semitendinosus and psoas major muscles during postmortem

storage at 2°C and 25°C, Through the use of this highly sen­

sitive technique to protein changes, it was shown that the

only major alteration occurring in myofibrils of loagissiaas

180

and semitendinosas muscles from 1 to 10 days of post-mortem

storage at 20C »as the disappearance of troponin-T and ap­

pearance of a 30,000-dalton molecular weight protein. This

agrees with that found during postmortem storage of chicken

breast muscle (Hay et al., 1973a). Alpha-actinin, which is

found exclusively in the Z-line (Scholloeyer et al., 1973),

does not appear to decrease in intensity during post-mortem

storage of longissimus and semitendinosus muscles, even

though ayofibrils from these muscles appear to increasingly

break into shorter segments at the z-line during postmortem

storage. However, even though myofibril fragmentation index

increased during postmortem storage, Z-lines did not disap­

pear indicating that the Z-line may be weakened without

alpha-actinin being degraded or released from the myofibril.

In myofibrils from psoas major muscle, very little change

occurs ia sjofitrillar proteins d'lsring postmortem storage at

20C with only a subtle decrease in troponin-T and a faint ap­

pearance of a 30,000-dalton protein at 10 days post-mortem.

At 250c postmortem lonaissimus and semitendinosus muscle

storage, disappearance of troponin-T and appearance of a

30,000-dalton protein are still the only major changes occur­

ring in myofibrils, but these changes occur more quickly

post-mortem at 25oc than at 2°C. So major changes occur in

myofibrils from psoas major muscles during postmortem storage

even at 250C. Disappearance of troponin-T and appearance of

181

a 30,000-dalton protein during postmortem storage is a very

limited and selective alteration of the myofibril. Because

higher storage temperature (250C) appears to only accelerate

these alterations and not produce other alterations, suggests

that a very specific protease is responsible for these alter­

ations in the myofibril. Also, because these alterations in

the myofibril are much greater in seaitendinosus and longis-

Simus muscles than in psoas major muscle, saggests that the

alterations are related to postmortem myofibril fragmentation

index increase (Z-line weakening and breakage to yield

shorten myofibrils) and W-B shear-force decrease. Because

the location of troponin is on tropomyosin in thin filaments

of the myofibril and not in the Z-line (Ebashi et al., 1968)

precludes a conclusion that Z-liae Heakening is caused by

degradation of troponin-T. However, proteolytic degradation

of %-lines may coincide with proteolytic dégradation of

troponin-T. It aay be speculated that bonds holding thin

filaaents to the Z-line say be cleaved proteolytically with­

out degrading proteins at or in the Z-liae. It is also pos­

sible that troponin-T may have some stabilizing effect on the

thin filament which when degraded weakens the myofibril

structure. No evidence, however, is presented in this inves­

tigation to substantiate either of these speculations.

Hay et al. (1973a, b) have shown in chicken breast mus­

cle that myofibrils become more fragmented during postmortem

182

storage, a 30,000-daltoii protein appears and an unspecified

protein, which appears to be troponin-T, disappears on SDS-

polyacrylanide gels daring postmortea storage. In chicken

leg muscle, however, no apparent increase in myofibril

fragmentation, decrease in troponin-T or appearance of a

30,000-dalton protein was found during postmortem storage.

While the precise relationship between myofibril fragmenta­

tion and alterations in myofibrillar proteins during post­

mortem storage is not clear, they occur at the same relative

time postmortem and to the same relatively high degree in

lonaissimos and semitendinosus muscles but consistently to a

lesser degree in psoas major muscle. It appears very likely

that both postmortem myofibril fragmentation and myofibrillar

protein alteration is caused by the same factor, a protease.

A calcium-activated protease (C&F) has been isolated

from skeletal muscle and has been fousi to sslsctiveiy remove

S-liass fro3 ayofibrils (Basch et al., 1972J« These findings

led to studies to determine possible roles CAP may have as a

causal factor in postmortem changes in muscle. First, CAP

was isolated from longissigas^ semitendinosus and psoas major

muscle at 0, 1, 3 and 6 days post-mortem to determine if an

active enzyme fraction existed in postmortem muscle. In Ion-

gisslmus and semitendinosus muscles, similar CAP activity

levels were found with the highest activity being found in

preparations from at-death muscle, bet total activity of CAF

183

diminished during postmortem storage. This is the first

study to show that an active C&F preparation can be made from

postmortem bovine muscle. The postmortem decline in CAP ac­

tivity indicates that it is not released from some cellular

organelle, such as lysosomes, during postmortem storage. C&F

may also gradually be denatured by low postmortem muscle pH

or it may be autolytic and gradually degrade itself. In

psoas major muscle, however, at-death CAP activity level was

about half of the at-death level in longissimas and sesiten-

dinosus muscles, and CAP activity decreased to near zero at 1

day post-mortem. It is unknown why CAP activity is lower in

preparations from psoas major muscle than samitendlnosus and

lonqissimus muscles, however, it may be related to the dif­

ferent physiological functions these muscles perform. Be­

cause an active CAP fraction can be isolated from postmortem

muscle? it seems reasoaable to conclude that GIF is tha

protease involved with postmortem muscle tenderization. Fur­

thermore, Dayton et al, C197Ua; b) and Suzuki and Goll (197%)

have shown that CAP is active in conditions which normally

esist in postmortem muscle (i.e= pH5.5-5.7, 0-4oc, 1 mH Ca2+).

More importantly the postmortem CAP activity results show

that CAP activity in longissimus and semitendinosus muscles

are high while CAP activity is considerably lower in psoas

major muscle* Because differences in CAP activity levels be­

tween longissimus and semitendinosus muscles and EiSas^Bajor

184

muscle are of the same magnitude as differences in postmortem

myofibril fragmentation index, B-B shear-force and myofibril­

lar protein alteration between these muscles, it is clear

that CâF is the likely causal factor affecting these post­

mortem muscle characteristics. To test this further, myofi­

brils from at-death longissimus, semitendinosus and psoas

major auscles Here briefly treated with CAP to see if z-lines

would be removed and if myofibril fragmentation occurred.

Microscopic observation showed that C&F-traated myofibrils

from all three muscles had Z-lines removed and were more

fragmented than controls but had no other apparent

disruption» In addition, SDS-polyacrylamide gels of CAF

treated myofibrils from all three muscles showed the absence

of alpha-actinin and troponin-T, decreased intensity of

troponin-I and the presence of a 30,000-dalton protein.

Dajtoa et al. (19742, b) have also shonn that aipha-acfciain

is released from myofibrils (porcine muscle) by CAP and also

that purified alpha-actinin is not degraded by CAF treatment.

Degradation of troponin-? and appearance of a 30,000-dalton

protein after CAF treatment closely resembles changes in myo­

fibrils from postmortem longissiaus and semitendinosus mus­

cles.

It appeared obvious that the 30,000-daiton protein orig­

inated from troponin-T degradation, however, to substantiate

this, purified troponin from lonaissigus muscle was mildly

185

treated with C&F for 5 to 40 minutes and electrophoresad on

SDS-polyacrylamids gels. Troponin-T was quickly degraded to

a 30,000-dalton protein and other low molecular weight pro­

teins. Troponin-I was slightly degraded at longer incubation

times, but troponin-C appeared to not be degraded. The

30,000-dalton protein, originating from tropoain-T degrada­

tion, was also eventually degraded so that at the end of the

40 minutes incubation period almost no protein appeared

larger than troponio-I. During postmortem muscle storage of

lonqissiaus and semitendinosus muscles, gels of myofibrils

show the 30,000-dalton protein increases in intensity and

alpha-actiain appears unaltered. Thus? effects observed in

these myofibrils from post-mortem muscles can result from

only very brief and diluted CftF activity, otherwise more ex­

tensive degradation in gels of myofibrils would be observed

and Z-liaes is myofibrils --culd all bs cosplstsly remo^edc

Myofibrils from lonqissiaus muscle at-death and at 10

days post-mortem at 2°C were electrophoresed along with puri­

fied troponin and CâF-treated troponin on SDS-polyacrylamids

gels. These gels clearly demonstrated that the protein

thought to be troponin-T was in fact tropoain-T which disap­

peared from 0 to 10 days post-mortem and that the

30,000-dalton protein appearing at 10 days post-mortem did in

fact originate from troponin-T degradation. It is very in­

teresting to note that alterations in myofibrillar proteias

186

on gels of myofibrils from at-death and at 10 days post­

mortem lonaissimus muscle can be almost duplicated by very

brief treatment of troponin with C&F. It is clear that CAF,

while active in postmortem muscle, probably weakens the Z-

line (as evidenced by its ability to remove alpha-actinin

from myofibrils) and at the same time degrades troponin-T.

Proteolysis by CâF in muscle appears to be very limited and

specific and is probably responsible for postmortem changes

of the muscle characteristics: myofibril fragmentation index

increase, W-B shear-force decrease and troponin-T degrada­

tion. The subtle postmortem changes in these characteristics

in psoas major muscle is probably due to a considerably lower

level of C&F activity than that found in longissimus and

seaitendinosus muscles.

Myofibrillar protein change, increase in myofibril frag­

mentation index aad decrease iû w-5 shear-force during post­

mortes suscle storage have been suggested to be caused by C&F

and have also been shown to be integrally related to the

postmortem change in seat tenderness. Because the number of

samples and animals in previous experiments were too few to

make adequate correlations, an experiment was conducted to

determine the relationship between myofibril fragmentation

index and meat tenderness and quality (H-B shear-force and

sensory tenderness, flavor and juiciness) on lonaissimus mus­

cles of thirty-five â-maturity bovine carcasses. In addi­

187

tion, six veal and twelve c-maturity carcasses were used in

the same experiment to determine effect of animal maturity on

postmortem muscle attributes. Means of myofibril fragmenta­

tion index were significantly (P<0.05} higher at 7 days post­

mortem than at 1 day post-mortem for all maturity groups.

Lonaissimus muscles from veal carcasses had the lowest myofi­

bril fragmentation index mean and muscles from à-maturity

carcasses had the highest means at 1 day post-mortem. At 7

days post-mortem, loaqissimus muscles from veal carcasses had

the lowest myofibril fragmentation index means and muscles

from A and c-maturity groups had similarly higher means.

Evidently lonqissimas muscles from immature animals (veal)

are more resistant to rupture and breaking during preparation

of myofibrils than from mature animals. The explanation for

this observation, however, has not been investigated and no

reasons caa thus bs given. Differences ir. myofibril fragmen­

tation index means of longissimus muscles from â-maturity

carcasses between 1 and 7 days post-mortem was about 5-fold

greater in previous experiments than in this experiment.

Also myofibril fragmentation index means at 1 day post-mortem

for A-maturity longissimus muscles in this experiment were

closer to means found at 3 days post-mortem in previous ex­

periments. Muscles in this experiment were handled differ­

ently than in previous experiments (see HETHODS AHD MATERIALS

section) resulting, evidently, in some fragmentation changes

188

that had occurred after 1 day post-mortem in previous experi­

ments, occurred before the 1 day post-mortem sample could be

measured in this experiment. However, significant (P<0.05)

differences in myofibril fragmentation index of muscles be­

tween 1 and 7 days post-mortem could be measured. On the

other hand, differences in W-B shear-force and sensory ten­

derness in lonqissiaus muscles from veal, &-aatarity aad C-

aaturity carcasses between 1 and 7 days post-mortem were not

large enough to be significantly (P<0.05) different. Evi­

dently, some of the postmortem increase in tenderness had oc­

curred before the 1 day post-mortem sample could be evaluat­

ed. Myofibril fragmentation index yas sensitive enough to

detect the differences between 1 and 7 days post-mortem,

while B-S shear-force and sensory tenderness could not detect

the differences.

Simple correlation coeffieieats «ers detsrzizsd azczg

myofibril fragsestatioa iBdex? H-B shear-force and sensory

tenderness, flavor and juiciness of longissimus muscles of

veal, I-Baturity and C-satarity carcasses at 1 and 7 days

post-aortea at 2oc. Significant correlation coefficients be­

tween myofibril fragmentation index and w-B shear-force

ranged from -0.65 to -0.97 and between myofibril fragmenta­

tion index and sensory tenderness ranged from 0.65 to 0.95 in

all three aaturity groups and at 1 and 7 days post-mortem.

The highest correlation coefficients were found in muscles

189

from the veal maturity group which say be misleading because

only 6 carcasses from this maturity group were used in this

experiment. Significant correlation coefficients between

myofibril fragmentation index and ï-B shear-force ranged from

-0.65 to -0.75 and between myofibril fragmentation index and

sensory tenderness ranged from 0.65 to 0.75 in the & and C-

maturity groups at 1 and 7 days post-mortem. The postmortem

increase in tenderness is therefore highly related to the in­

crease in myofibril fragmentation. These results indicate

that structural weakening of the myofibril enhances meat ten­

derness. Because more carcasses were used in the A and c-

maturity groups, these correlations are probably closer to

the true correlations of the population. Additionally, imma­

ture animals {veal) may not reflect the same conditions exis­

ting in the population of mature carcasses. These results

closely agree with Boiler et ai. {1373) who found, using a

similar method of measuring myofibril fragmentation as was

used in this investigation, a correlation coefficient of

-0.78 between myofibril fragmentation and S-S shear-force of

lonqisslBus muscles from A-maturity bovine carcasses at 7

days post-mortem at 2oc. Correlation coefficients between H-

3 shear-force and sensory tenderness ranged from -0.48 to

-0.9% in the three maturity groups at 1 and 7 days and these

correlations eere all sigsifleast except in the veal at 1 day

post-mortem and C-maturity group at 7 days post-mortem.

190

These results clearly show that myofibrils isolated from

postmortem muscle and determined as a fragmentation index is

a good indicator of cooked muscle tenderness. Obviously,

myofibril fragmentation index is one of the most highly cor­

related measures on raw muscle attributes that indicates

cooked meat tenderness and it accounts for approximately U 5 %

of the tenderness of seat broiled to 65oc internally. Evi­

dently, the changes in the myofibril during postmortem muscle

storage is not greatly altered during heating and enhances

the tenderness of cooked meat.

Hyofibrils from longissimus muscles of veal, a-maturity

and C-maturity carcasses at 1 and 7 days post-mortem at 2 ° C

were selected on the basis of extremes in myofibril fragmen­

tation index, W-B shear-force and seasory tenderness of the

muscles and electrophoresed on SDS-polyaerylamide gels. Gels

of myofibrils from all three aatarity groiips at 1 and 7 days

post-sortes shoe a general relationship between high muscle

tenderness {high myofibril fragmentation index and sensory

tenderness and low 5-B shsar-force) and the dsgradatioa of

myofibrillar proteins (absence of troponia-T and appearance

of 30,000-dalton protein). Conversely, less tender muscles

showed less myofibril degradation. Comparisons between ma­

turity groups in muscle tenderness and myofibril degradation

do not show similar changes in magnitude in that veal myofi­

brils show as such myofibril degradation in the gels at 7

191

days post-nortea as shown in gels of i and C-naturity myofi­

brils even though veal muscles are less tender. Immature

muscle (veal), however, may impart influences on muscle ten­

derness different fros mature muscle (& and C-aaturity)«

Clearly, myofibril degradation is integrally related to

muscle tenderness. As previously discussed, CAF activity ia

postmortes muscle appears to be responsible for dégradative

changes in myofibrils which apparently result in increased

muscle tenderness daring postmortem storage. Penny et al.

(1974) has shown that freeze-dried steaks, rehydrated with a

CAF-containing solution was considerably more tender than

controls and that this tenderness increase was due to a

structural weakening of the myofibril at or near the Z-line.

It thus follows that CAF is the probable factor in muscle re­

sponsible for the postmortem degradation of myofibrils and

192

SOMMaBÏ

HeasureœentS to detect postmortem physical and chemical

events have been made on bovine longissimas, semitendisnosas

and psoas major muscles during postmortem storage at 20C and

250c. Hyofibril fragmentation from these muscles was deter­

mined by a method that measured the absorbance of a myofibril

suspension. This method utilized a larger and more repre­

sentative sample size and afforded greater objectivity than

could have resulted in a microscopic method of myofibril

fragmentation determination. Myofibril fragmentation index

(tnrbidometric myofibril fragmentation measure) closely

agreed to microscopically observed changes in myofibril frag­

mentation. Myofibril fragmentation index greatly increased

from 1 to 10 days post-mortem at 2°C in longissimus and semi-

ten dinos us muscles but only slightly increased in psoas malar

muscle. Z-lines in myofibrils remained intact during post­

mortem storage in all three muscles, W-B shear-force greatly

decreased from 1 to 10 days post-mortem at 2^Z in longissimus

and semitendinosus muscles but only slightly decreased in

psoas gaior «useles. postmortem storage at 25oc resulted in

accelerated changes in myofibril fragmentation index and B-B

shear-force in longissimus and seaitendinosus muscles but

these changes eere only slightly Increased in psoas aaior

muscle compared to 20C postmortem storage. Hyofibril Z-lins

weakening during postmortem storage, indicated by myofibril

193

fragmentation index, was greatest in lonqissians and semiten-

dinosus muscles and least in psoas ma^or muscle and appeared

to be associated with the postmortem decrease in R-B shear-

force of these muscles.

Byofibrils from muscles stored at 2oc and 25®C for vari­

ous time periods were electrophoresed on SDS-polyacrylamide

gels to detersin® postaortem changes in myofibrillar pro­

teins. Postmortem storage causes a very limited and specific

change in the myofibrillar proteins. Troponin-T is degraded

to a 30,000-daltoa protein but all other myofibrillar pro­

teins appear unaffected by post-mortem storage in longissiaus

and seaiteadinosus auscles. Only a very slight degradation

of troponin-T occurs at 10 days postmortem in psoas major

muscles. The greatest postmortem changes in myofibril frag­

mentation index, »-B shear-force and troponin-T degradation

occuE iu loaûlsslsus and ssaitesdlsosas while the least chan­

ges occur in psoas major muscle. The higher storage tempera­

ture *2500} causes troponin-T degradation to occur sooner

post-mortem but does sot caase other degradative changes in

the myofibril in lonaissimus and semitendinosus muscles.

Higher storage temperature (250C) has little effect on myofi­

brils of psoas major muscle.

Active CAP (calcium-activated muscle protease) fractions

«ere found in muscles at 0, 1, 3 and 6 days post-mortem. C&F

fractions in lonaissimus and semitendinosus muscle sere siai-

194

larly active but were considerably higher at-ieath and at all

postmortem periods than in psoas major muscle. Rt-death myo­

fibrils treated with CAF had fewer Z-lines and greater frag­

mentation than controls. SDS-polyacrylamide gels of CAF-

treated myofibrils showed the absence of alpha-actinin, deg­

radation of troponin-T to a 30,000-dalton protein and de­

creased intensity of troponin-I. Absence of alpha-actinin in

gels corresponded to Z-line removal in myofibrils. Degrada­

tion of troponin-T by CA? is the same as degradation of tro-

ponin-T by post-mortem storage. Because CAP affects the z-

line, causes the same degradation of troponin-T as occurs ia

postmortem muscle and has considerably higher activity in

longissimus and seaitendinosus muscles than in psoas maior

muscle? it is likely that CAP is directly responsible for the

postmortem increase in myofibril fragmentation index in lon-

gissiisus asd ssaitsndinosas Buscies,

Longissimus muscles from veal, A-maturity and C-maturity

carcasses were investigated for myofibril fragmentation in­

dex, W-B shear-force and sensory tenderness, flavor and jui­

ciness at 1 and 7 days post-mortem at 20C. Significant cor­

relation coefficients between myofibril fragmentation index

and a-B shear-force were found to be for muscles in veal car­

casses at 1 day post-mortem -0.95 and at 7 days post-mortem

-0.97p in A-maturity carcasses at 1 day post-aortem -0.65 and

at 7 days post-mortea -0.75 and ia C-naturity carcasses at 1

195

day post-mortem -0.58 and at 7 days post-mortem -0.72. Sig­

nificant correlation coefficients between myofibril fragmen­

tation index and sensory tenderness were found to be for mus­

cles in veal carcasses at 1 day post-mortem 0.88 and at 7

days post-mortem 0.95, in a-naturity carcasses at 1 day post­

mortem 0.67 and at 7 days post-mortem 0.73 and in C-maturity

carcasses at 1 day post-sort em 0.68 and at 7 days post-mortem

0.65. flyofibril fragmentation index is one of the highest

correlated measures of raw muscle found with cooked meat ten­

derness. Hyofibrils from selected lonqissimus muscles from

veal, a-maturity and c-maturity carcasses were electropho-

resed on SDS-polyacrylaaide gels. Within each maturity

group, high myofibril fragmentation index and muscle tender­

ness accompanied troponin-T degradation while low index and

tenderness values accompanied no troponin-T degradation.

Hyofibrils fros sasclss in issaturs (veal) animais showed

sisilar myofibril degradation as in the mature animal groups,

but myofibril fragmentation index and muscle tenderness was

loyer in the immature (7eal) animals.

The increase in muscle tenderness during postmortem

storage has been shown to be highly correlated with myofibril

fragmentation index. &s discussed previously, proteolytic

cap activity is probably responsible for postmortem increase

in myofibril fragmentation index. Since postmortem increase

in myofibril fragmentation index is highly correlated to

196

postmortem increase in muscle tenderness, it is also likely

that CAP is responsible for postmortem muscle tenderization.

197

COHCLUSIONS

& aeasare of a myofibril suspension by light absorbance

'ayofibril fragmentation index) was found to closely par­

allel the degree of fragmentation of myofibrils observed

in the light microscope.

Myofibril fragmentation index is muscle and storage tem­

perature dependent. Myofibril fragmentation index in­

creased considerably during postmortem storage at 20C and

252c in longissiaas and ssaitendinosus muscles with the

higher storage temperature (25oc) resulting in a greater

increase in myofibril fragmentation index. Only a slight

increase in myofibril fragmentation index during post­

mortem storage at either 2°c or 25°C occurred in psoas

ma1or muscle.

?-B shear-force is muscle and storage temperature depen­

dent, H-B shear-force was highest in semitendiaosus mus­

cle, lowest in psoas aaior muscle and intermediate in

lonqissimus muscle, H-B shear-force decreased consider­

ably during postmortem storage at 2oc and 25oc in longis-

siaiis aad sëâltesdisGsus susclss with the higher storage

temperature {25®C) resulting in a greater decrease in W-B

shear-force. Only a slight decrease in W-B shear-force

during postmortem storage at either 2oc or 250C occurred

in psoas major muscle.

A very Halted and specific proteolytic degradation of

198

myofibrillar proteins occurred during postmortem muscle

storage at 2^C and 25oc. Disappearance of troponin-T and

appearance of a 30,000-daiton molecular weight protein

were the only major changes in myofibrils from lonqissi-

Bus and semitendinosns muscles during postmortem storage

at 20C and 25°C with the higher storage temperature

(250C) resulting in more rapid changes in the myofibrils.

In the psoas major muscle, a 30,000-dalton protein did

not appear and troponin-T appeared to not be degraded,

although troponin-T was not clearly separated, during

postmortem storage at 2oc or 25oc.

C&F activity is muscle dependent and decreases with

postmortem time. Similar CAF activity was found in at-

death lonaissimus and seaitendinosus muscles, but less

than half of that C&F activity was found in at-death

psoas aajor aascis. C&F activity in ion^issimus and

semiteadinosns muscles decreased to less than half of at-

death level at 5 days post-mortem, but C&F activity in

psoas major muscle decreased to near zero level at 1 day

post-mortem and stayed at that level.

Myofibrils after miid treatment with CSF, had Z-lines

removed and greater fragmentation than control myofi­

brils. SDS-polyacrylaaide gels of CAF-treated myofibrils

showed alpha-actinin and troponin-T removed, tropoQin-I

slightly degraded and a 30,000-dalton protein appeared.

199

Purified troponin from bovine ^onaissimus muscle and

treated with CKF showed on SDS-polyacrylamide gels rapid

degradation of troponin-T yielding a 30,000-dalton pro­

tein which was also degraded by CàF after longer incuba­

tion and slight degradation of troponin-I after prolonged

incubation.

Lonaissiaas and sesitendinosus muscles were shown to

have similar myofibril fragmentation index, myofibrillar

protein degradation and CAF activity levels at all post­

mortem times. W-B shear-force values were higher in

semi ten dimos us muscle than in lonqissimus muscle, howev­

er, both showed sisilar changes in R-B shear-force during

postmortem storage. Lower myofibril fragmentation index,

S-B shear-force, estent of myofibrillar protein degrada­

tion and CâF activity level were found in psoas major

muscle t&an £ouqci iu a, on uxssxsus âûu se s x ts u d jLIxS s »âs mus­

cles aad only a slight change in these measures occurred

during pcstaorteis storage in psoas aaior muscle.

Correlation coefficients between myofibril fragmentation

index and S-B shear-force ranged from -0.55 to -0=97 and

between myofibril fragmentation index and sensory tender­

ness ranged from 0.63 to 0.94 for lonqissimus muscles

from veal, &-maturity and C-maturity carcasses at 1 and 7

days post-sortes»

Greater degradation of troponia-T ia myofibrils from Ion-

200

gissiaas muscles which were more tender and had greater

myofibril fragmentation index was found in selected veal,

A-maturity and C-maturity carcasses.

Lonaissimus muscles from mature (&-maturity and C-

maturity) and immature (veal) carcasses showed similar

myofibril degradation and changes in myofibril fragmenta­

tion index during postmortem storage but lower means in

R-B shear-force, sensory tenderness and myofibril frag­

mentation index were found in the immature (veal) carcas­

ses.

201

BIBLIOGR&PBÏ

&berle, E. D. aad R. A. "erkel. 1966. Stability and eiectcophoretic behavior of some proteins of post-mortem aged bovine suscle. J. Food Sci. 31:151-156.

Arakaaa, H., D. E. Goil, and J. Temple. 1970a. Molecular properties of postmortem muscle. 8. Effect of post­mortem storage on alpha-actinin and the tropomyosin-troponin complex. J. Food Sci- 35:703-711.

Arakaea, H., D. E. Goll, and J. Temple, 1970b. Molecular properties of postmortem muscle. 9. Effect of tempera­ture and pH on tropomyosin-troponin and purified alpha-actinin from rabbit muscle. J. Food Sci. 35:712-715.

Arakawa, N., B. M. Robson, and D. E. Goll. 1970c. An im­proved method for the preparation of alpha-actinin from rabbit striated muscle. Biochim. Biophys. Acta 200:2811-295.

Arango, T. C., G. C. Smith, and z. L. Carpenter. 1970. Methods for natural tenderization of beef. Texas Agris. Exp. Sta. Bull. 1098:3-12.

Bailey, A. J. and D. Lister. 1968. Thermally labile cross­links in native collagen. Mature 220:280.

Bate-Smith, E= C= 1939= Changes in elasticity of mammalian saascle undergoing rigor mortis. J. Physiol. 96:i76-i93.

Bate-Smith, E. c. and J. S. Bandall. 1949. Factors deter­mining the time course of rigor mortis. J. Physiol. 110:47-65,

Behnke, J. fi., 0. Fennema, and S. G, Cassens. 1973. Quality changes in pre-rigor beef muscle at 30C. J. Food Sci. 38:539-541.

Bendall, J® S. 1951. The shortening of rabbit muscle during rigor mortis: Its relation to the break down of adeno­sine triphosphate and creatine phosphate and to muscular contractions. J. Physiol. 114:71-88.

Bendall, J. B. 1960. Post-mortem changes in muscle. Pages 227-274 in G. H. Bourne, ed. T&e structure and functisn of muscle. Vol. 2. Academic Press, Inc., He» York, New York.

202

Bendall, J. R. 1966. Hascle as a contractile machine. Pages 7-16 in E. J. Briskey, R. G. Cassens and J. C. Trautman, eds. The physiology and biochemistry of mus­cle as a food. Vol. I. Oaiversity of Wisconsin Press, Hadison, trisconsin.

Berry, B. B., G. c. Smith, and z. L. Carpenter. 1974. Rela­tionships of certain muscle, cartilage and bone traits to tenderness of the beef longissimus. J. Food Sci. 39:819-824.

Bodwell, C. E. and &. a. Pearson. 1964. The activity of partially purified bovine catheptic enzvaes on various natural and synthetic substrates. J. Food Sci. 29:602-607.

Borchert, L. L. and E. J. Briskey. 1965. Protein solubility and associated properties of porcine muscle as influ­enced by partial freezing with liquid nitrogen. J. Food Sci. 30:138-143.

Borchert, L. L., a. D. Powrie, and E. J. Briskey. 1969. A study of the sarcoplasmic proteins of porcine muscle by starch gel electrophoresis. J. Food Sci, 34:148-153.

Bouton, P. E. and P. V. Harris. 1972a. The affects of cook­ing temperature and tiee on some mechanical properties of meat. J. Food Sci. 37:140-144.

Bouton, P. E. and P. V. Harris. 1972b. & comparison of some objective methods to assess meat teaderaess» J, rood Sci. 31:218-221.

Bouton, P. E. and P. V. Harris. 1972c. The effects of some post-slaughter treatments on the mechanical properties of bovine and ovine muscle. J. Food Sci. 37:539-543.

Bouton, P. E., P. V. Harris, and w. S. Shorthose. 1971. Effect of ultimate pH upon the water-holding capacity ââd têâdêrûëSS Of iâûttOîî. J. FûOd Scl. 36; 435-439.

Bouton, p. E., A. Howard, and a. a. Lawrie. 1957. Studies on beef quality. 6. Effects on weight losses and eat­ing quality of further pre-slaaghter treatments. CSIRO Australian Div. Food Preserv. Tech. Paper No. 6.

203

Bouton, P. E., F. D. Carroll, P. v. Harris, and W. R. Shorthose. 1973a. Influence of pH and fiber contrac­tion upon factors affecting the tenderness of bovine suscle. J. Food Sci, 38:%0y-y07.

Bouton, P. 2., ?. ?. Harris, H. R. Shorthose, and R. I. Baxter. 1973b. k comparison of the effects of aging, conditioning and skeletal restraint on the tenderness of mutton. J. Food Sci. 38:932-937.

Bouton, P. E., P. 7. Harris, 9. B. Shorthose, and H. G. Smith. 197 4. Evaluation of methods affecting mutton tenderness. J. Food Technol. 9:31-41.

Briskey, E. J. 1957. Myofibrillar proteins of skeletal mus­cle. Proc. Heat Ind. Res. Conf. 20:1-28.

Bciskey, 2. J. and x. Fukazawa. 1571. Hyofibrillac proteias of skeletal muscle, Adv. Food Res. 19:279-360.

Briskey, E. J., R. N. Sayre, and B. G. Cassens. 1962. De­velopment and application of an apparatus for continuous measurement of muscle extensibility and elasticity be­fore and during rigor mortis. J. Food Sci. 27:560-556.

Buck, E. a. and D, L. Black. 1967. The effect of stretch-tension during rigor on certain physical characteristics of bovine muscle. J. Food Sci. 32:539-543.

Buck- B- 5, and D, L, Black, 1968, Microscopic characteris­tics of cooked muscle subjected to stretch-tension dur­ing rigor. J. Food Sci. 33:464-467,

Buck, E. fi., D. B. Stanley? and E. A, Commissiong. 1970. Fhysxcal and chemical characteristics of free and stretched rabbit muscle. J. Food Sci- 35:100-102.

Buege, D. B. and J. B. Stouffer. 1974a. Effects of pre-rigor tension on tenderness of intact bovine and ovine muscle. J, Food Sci. 39:396-401.

Buege, D. B. and J. B. Stouffer. 1974b. Tenderness varia­tion in ovine longissimus muscle. J. Food Sci. 39: 402-405.

Busch, 8. A., F. C. Parrish and D. E. Goll, 1967. Molecular properties of post-sortea muscle. 4. Effect of temper­ature on adenosine triphosphate degradation, isometric tension parameters, and shear resistance of bovine mus­cle. J. Food Sci. 32:3 90-394.

204

Buschr W. a., B. H. Stromer, D. E. Goll, and &. Suzuki. 197 2- Ca2+ specific removal of Z-lines from rabbit skeletal muscle. J. cell Biol. 52:367-381.

Caldwell, K. â. and 0. K. Grosjean. 1971, lysosomal cathep-sins of chicken skeletal muscle: Distribution and prop­erties. J. &gr. Food Chem. 19:108-115.

Canonico, P. G. and J. P. C. Bird. 1970. Lysosomes in skel­etal muscle tissue. J. Cell Biol. 45:321-333.

Cassensf R= G= and R. P. New bold. 1966. Effects of tempera­ture on post-moirtem metabolism in beef muscle, J. Sci. Food &gric. 17:254-256.

Cassens, R. G. and R. P. Newbold. 1967a. Temperature de­pendence of pH changes in ox muscle post mortem. J. Food Sci. 32:13-14.

Casseas, H. G. and E. P. Newbold. 1967b. Effect of temper­ature on the time course of rigor mortis in ox muscle. J. Food Sci. 32:269-272.

Chaudhry, H- fi., F. C. Parrish» and D. E. Goll. 196 9. Ho-lecular properties of post-mortem muscle. 6. Effect of temperature on protein solubility of rabbit and bovine muscle. Food Sci. 34:183-191.

Claysos; F,- J, R, Beesley- and B. B. Blood. 1966. The chemistry of the natural tenderisation or saturation of meat. J. Sci. Food Agric. 17:220-225.

Cohen, c., D. L. D. Caspar, J. P. Johnson, K. Rauss, S. s. Sargossian and S. A. D. Parry. 1973. Tropcmyosia-troponin assembly. Cold Spring Harbor Symposia on Quan­titative Biol. 37:287-297.

Comissiong, E. a. 1974. The study of the effects of actoay-osin and z-iine interaction on the extensibility of chicken breast muscle postmortem. Ph.D. thesis. Uni­versity of Massachusetts, àmherst, Massachusetts.

Cook, C. F. and E. F. Langsworth. 1966. The effect of pre-slaughter environment temperature and post-mortem treat­ment upon some characteristics of ovine muscle. 1. Shortening and pH. J. Food Sci. 31:497-503.

205

Cooper, C. C.f B. B- Breidenstein, R. G. Cassens, G. Evans, and R. W. Bray. 1968. Influence of marbling and matur­ity on the palatability of beef muscle. 2. Histologi­cal consideration. J. &nia. Sci. 27:15^2-15^6.

Cross, H. R., Z. L. Carpenter, and G. C, Saith. 1973. Ef­fects of intramuscular collagen and elastin on bovine muscle tenderness. J. Food Sci. 38:998-1003.

Culp, G. 5., Z. L. Carpenter, G. c. Smith, and G. 9. Davis. 1973. Postrigor aging effects on beef tenderness. J. Anim. Sci. 37:258. (Abstr.)

Czok, R. and T. Bûcher. 196 0. crystallized enzymes from the myogen of rabbit skeletal muscle. Adv. Protein Chem. 15:315-328.

Davey, C. L- and H. a. Dickson. 1970. Studies in meat ten­derness. 8. nitrastructural changes in meat during aging. J. Food Sci. 35:56-60.

Davey, C. L. and K. 7. Gilbert. 1966. Studies in meat ten­derness. II. Proteolysis and the aging of beef. J. Food Sci. 31:135-140.

Davey, C. L. and K. 1. Gilbert. 1967. structural changes in meat during aging. J. Food Technol. 2:57-59.

Davey, C. L. and K. V. Gilbert. 1968a. Studies in meat ten-dsrnsss, '4. Changes in the extractabiiity of myofi­brillar proteins during meat aging. J. Food Sci. 33:2-7.

Davey, C. L. and K. 7. Gilbert. 1968b. Studies in meat ten­derness. 6. The nature of myofibrillar proteins ex­tracted from meat during aging. J. Food Sci. 33:343-348.

Davey, C. L. and K. 7. Gilbert. 1969. Studies in meat ten­derness. 7. Changes in the fine structure of seat dur­ing aging. Jo Food Sci. 34:69-74.

Davey, C. L. and K. 7. Gilbert. 1974. Carcass posture and tenderness in frozen lamb. J. Sci, Food Agric. 25:923-930.

Davey, C- L., H. Kuttel, and K. 7. Gilbert. 1967. Shorten­ing as a factor in meat aging. J. Food Technol. 2:53-56.

206

Davies, R. E. 1967. Recent theories on the mechanism of nascle contraction and rigor mortis. Proc- Beat Ind. Ses. Conf. 20:39-53.

Dawson, L. E. and G. H- Bells. 1969. Tenderness of freeze-dried chicSea treated sith proteolytic enzymes. Poultry Sci- 48:64-70.

Dayton, W. R., 0. E. Goll, W.J. Reville, H. S. Zeece, M. H. Stroaer, and B. H. Bobson. 1974a. Purification and some properties of a muscle enzyme that degrades myofi­brils. Fed. Proc. 33:1580. (Abstr.)

Dayton, ». S., D. E. Goll, M. H. stromer, H. J. Reville, H. G. Zeece, and R. H. Eobson. 1974b. Some properties of a Ca2+-activated protease that may be involved in myofi­brillar protein turnover. Symposia on Quantitative Biol., Cold Springs Harbor, Sew York.

de Duve, C. 1959. Lysosomes, a new group of cytoplasmic particles. Pages 128-149 in T. Hayaski, ed. Subcellu­lar particles. The Ronald Press, Co., New York, New York.

de Dave, C. 1963. The lysosome concept. Pages 1-27 in M. P. Cameron, ed. Ciba Foundation symposium lysosomes. Little, Brown, and Co., Boston, Massachusetts.

de Duve, C. and R. Battiauz. 1966. Function of lysosomes. Ann- Reva Physiolc 28:435-449,

de Freaery, D. and a. F. Pool. 1960. Biochemistry of chick­en muscle as related to rigor mortis and tenderizatioa. Food Res, 25:73-87.

de Fremery, D. and M. F. Pool. 1963. The influence of post­mortem glycolysis on poultry tenderness, J. Food Sci. 28:173-176.

de Fremery, D. and I. ?. strsster. 1969. Te&âêci2âtià& of chicken muscle: The stability of alkali-insoluble con­nective tissue during post-mortem aging. J, Food Sci. 34:176-180.

Dikeaan, H. E., H. J. Tama, and G. R. Beecher. 1971. Bovine muscle tenderness as related to protein solubility. J. Food Sci. 36:190-193.

207

Drabitcovski, 9.» E. Nowak, B. Barylko and R. Dabrovska. 1973. Troponin—its composition and interaction with tropomyosin and f-actin. Cold Spring Harbor Symposia on Quantitative Bioio 37:245-249.

Draodtp Ho Ne 1972, changes in meat during cooking. Annual Reciprocal Heat Conf. 25:243-259.

Dumont, B. L. 1971. Seat toughness index of beef carcasses. Proc. Heat Ses. Workers Conf. 17:91-94.

Eason, B. &. 1969. purification and properties of skeletal mascle microsomes. Ph.D. Thesis. Iowa State Dniversi-ty, Ames, Iowa.

Ebashi, S. and &. Kodoma. 1966. Native tropomyosin-like action of troponin on trypsin-treated myosin B. J. Bicches. 50:733-734.

Ebashi, S., F. Ebashi, and &. Kodama. 1967. Troponin as the Ca++ receptive protein in the contractile system. J. Biochem. 62:137-138.

Ebashi, S., â. Kodama, and F. Ebashi. 1968. Troponin. I. Preparation and physiological function. J. Biochem. 64: 465-477.

El-Gharbawi, JS. and J. B. Whitaker. 1963. Factors affecting enzymatic solubilization of beef proteins. J. Food Sci. 28:168-172.

Etherington, D. J. 1971. The enzymic breakdown of collagen. Proco «eat Ses. Borkers conf. 17:632-635.

Field, H. and 1» Chang, 1969= Free amino acids in bovine muscle and their relationships to tenderness. J. Food Sci. 34:329-331.

Field, H. à., H. L. Hiley, and ï. Chang. 1969. Epiaysial côEnëctlve tissue as related to beef tenderness. J. Pood Sci. 3 4:514-518.

Field, B. &., H. L. Riley, and Y. Chang. 1971. Free amino acid changes in different aged bovine muscles and their relationship to shear values. J. Food Sci. 36:611-612.

Fields, P. &o, Ho La Best, z. L. carpenter^ and G. Smith. 1971. Elevated storage temperatures and beef palatabil-ity. J. anim. Sci. 33:217, (&bstr.)

208

Franzini-Armstrong, C. 1973. Membranous systems in muscle fibers. Pages 531-619 in G. H. Bourne, ed. The struc­ture and function of muscle. Vol. II. 2nd ed. Aca­demic Press? Inc. Ne* Yorkp New York.

Friedman, I., A. Laufer, and A. H. Davies. 1969. Studies on lysosomes in rat heart cultures. II, Tke effect of exogenous lysosomes. Experientia 25:1092-1093.

Pujimaki, S., N. Arakawa, A. Okitani, and 0. Takagi. 1965a. The changes of "myosin B" during storage of rabbit mus­cle. Part II. The dissociation of "myosin B" into myo­sin A and actin, and its interaction uith ATP. J. Food Sci. 30:937-943.

Fujimaki, H., A, Okitani, and N. Arakawa. 1965b, The chan­ges of "myosin B" during storage of rabbit muscle. Part I. Physicochsmical studies on "syosin B". ?.gric= Biol, Chem. 29:581-583.

Fukazawa, T. and T. lasai. 1967. The change in zigzag con­figuration of the 2-line of myofibrils. Biochim. Biophys. Acta 140:534-537.

Fukazawa, T., E. J. Briskey, K. Takahashi, and T. Yasui. 1969. Treatment and postmortem aging effects on the Z-line from chicken pectoral muscle. J. Food sci. 34:606-610.

Galloway, D. E, and D. E. Goll. 1967. Effect of temperature OÛ molecular properties of post-zortsz porcine suscls. J. Anim. Sci. 26: 130 2-1 308.

Gardner, G. A. and S. J. Stewart. 1966. Changes in the free amino and other nitrogen compounds in stored beef mus­cle. J. Sci. Food agric. 17:491-496.

Gergely, J., H. A. Gouveer, and D. Karibian. 1955. Fragmen­tation of myosin by chymotrypsin, J. Biol. Chem. 212 J165—169 e

Giffee, J. y. 1962. Status of connective tissue research. Annual Reciprocal Seat Conf. Proc. 15:265-269.

Gillis, 8. A. and R. L. Henrickson. 1967. Structural varia­tions of bovine muscle fiber in relation to tenderness. Annual Reciprocal Seat Conf. Proc. 20:17-29.

209

Gillis, ». &. aad R. L. Henricksoa. 1969. The influence of tension on pee-rigor excised bovine muscle. J. Food Sci. 34:375-377.

Goeser, P. A. 1961. Tendered meat through aatemortea vascu­lar injection of proteolytic enzymes. Proc. Heat Ind. Res. Conf. 13:55-73.9

Goll, D. S. 1962. Characterization and determination of muscle connective tissue components, annual Reciprocal Meat Conf. Proc. 15:242-254.

Goll, D. E. 1965a. Collagen fractions from loose connective tissue in muscle. Proc. Heat Ind. Res. Conf. 18:18-19.

Goll, D. E. 1965b. Post-mortem changes in connective tis­sue. Annual Reciprocal Heat Conf. Proc. 18:161-172.

Goll, D. E. 1968. The resolution of rigor mortis. Annual Reciprocol Heat Conf. Proc. 21:16-46.

Goll, D. E. and R. H. Robson. 1967. Molecular properties of postmortem muscle. 1. Myofibrillar nucleoside triphos­phatase activity of bovine muscle. J, Pood Sci. 32:323-329.

Goll, D, E., R. Bray, and w. G. Hoekstra. 1963. Age-associated changes in muscle composition. The isolation and properties of a collagenous residue from bovine mus­cle. J. Food Sci» 28:503-509»

Goll, D. E., D. H. Henderson, and E. A. Kline. 1964. Post-sortea chaages in physical and chemical properties of bovine ssscle. J. Food Sci. 29:590-596.

Goll, D. E., B. F. H. M. Bommaerts, S- K. Reedy, and K. Seraydarioa. 1969a. Studies on alpha-actinin-like pro­teins liberated during trysin digestioa of alpha-actinia and myofibrils. Biochim. Biophys. Acta 175:174-194.

Goll, D. E., R. H. Robson, J. Temple, and H. H. stromer. 1971a. An effect of trypsin on the actin-ayosin inter­action. Biochim. Biophys. Acta. 226:433-452.

210

Goll, 0. E., N. Arakawa, H. H. Stromer, w. A. Busch, and R. M. Bobson. 196 9b. Chemistry of muscle proteins as a food. Pages 755-800 in E. J. Briskey, 8. G. Cassens, and Bo B. Marsh, eds. """The physiology and biochemistry of muscle as a food. Vol. 2. Univ. of Wisconsin, Hadison, Wisconsin.

Goll, D. E., a. H. Stromer, D. G. Olson, 9. B. Dayton, A. Suzuki, and a. a. Bobson. 1974. The role of myofibril­lar proteins in neat tenderness. Proc. Beat Ind. Res. Conf., Chicago, Illinois.

&

Goll, D. E., H. H. Stromer, R. H. Bobson, Jo Temple, B. A. Eason, and V. A. Busch. 1971b. Tryptic digestion of muscle components simulates many of the changes caused by postmortem storage. J. Anim. Sci. 33:963-982.

Gcrnall, à. G-, C. J. Bardatîill and 5= David® 19 49. De­termination of serum proteins by means of the biuret reageant. J. Biol. Chem. 177:7 51-766.

Gothard, B. H., à. H. Hullins, R. F. Boulware and S. L. Hansard. 1966. Histological studies of postmortem changes in sarcomere length as related to bovine muscle tenderness. J. Food Sci. 31:825-8 28.

Gottschall, G. Y. and H. W. Kies. 1943. Digestion of beef by papain. Food Res. 7:373-378.

Gould, R. P. 1973. The microanatomy of muscle. Pages 199-241 ̂ G. H. Sosrae, ed. The strzctcre aai fanction of muscle. Vol. II 2nd Ed. Academic Press, Inc. New York, Hea York.

Greaser? H» L,, R. G. Cassens. and S- G. Hoekstra. 1967. Changes in ozalate-stimulated calcium accumulation in particulate fractions from postmortem muscle. J. Agri. Food Chem. 15:1112-1117.

Greaser, %. L., E. G. Cassens, 2^ J* Briskey? and ?= G= Hoekstra. 1969a. Postmortem changes in subcellular fractions from normal and pale, soft, exudative porcine muscle. 1. Calcium accumulation and adenosine triphosphatase activities. J. Food Sci. 34:120-124.

Greaser, H. L., S. G. Cassens, E. J. Briskey, and B. G. Hoekstra. 1969b. Postmortem changes in subcellular fractions from normal and pale, soft, esudativs porcine muscle. 2. Electron microscopy. J» Food Sci. 39:125-132.

211

Greaser, H. L., B. 6. Cassens, W. G. Hoekstra, and E. J. Briskey. 1969c. The effect of pH-teoperature treat­ments on the calcium-accnmnlating ability of purified sarcoplasîsic reticalam. J« Food Sci. 34:633-63 7.

Greaser, B. L., M. Yamaguchi, C. Brekke, J. Potter and J. Gergely. 1973. Troponin sufaunits and their interac­tions. cold spring Harbor Symposia on Quantitative Biol. 37:235-244.

Haga, T., K. Haruyaaa, and H. Roda. 1965. Formation of actomyosin during the extraction of muscle mince with Weber-Edsall solution. Biochis. Siophys. Acta 94:226-237.

Haga, T., a. Yamamoto, K. Raruyama, and a. Noda. 1966. The effect of myosin and calcium on the solubilization of F-actiu from sascle sines. BiochiE. BiophySo Acta 127:128-139.

Hamm, E. 1960. Biochemistry of meat hydration. Adv. Food Res. 10:355-463.

Hamm, B. 1966. , Heating of muscle systems. Pages 363-385 in E. J. Briskey, E. G. Cassens and J. C. Trautman, eds. The physiology and biochemistry of muscle as a food. Vol. I. Ottiversity of Wisconsin Press, Madison, Biscon-sin.

Harris. P. 7. and J. J. Hacfarlane. 1971. Effects of some post-s laugh ter treatments or. the sschanical properties of meat. Proc. Heat Bes. Workers conf. 17: 102-105.

Hartshorne, D. J- and P. Dreizen. 1973. Studies on the sub-unit composition of troponin. Cold Spring Harbor Symposia on Quantitative Biol. 37:225-234.

Hay, J. D., S. w. Currie, and F. H. Bolfe. 1972. The effect of aging on physicocheaical properties of actomyosin fros chicken breast asd leg suscla. J. Food Sci; 37:346-350.

Hay, J. D., B. H. Currie, and F. H. Bolfe. 1973a. Polyac-rylamide disc gel electrophoresis of fresh and aged chicken muscle proteins in sodium dodecylsulfate. J. Food Sci. 3 8:987-990.

Hay, J. 0., B. S. Carrie, F. E. Sfolfe, and E. J. Sanders. 1973b. Effect of postaortea aging on chicken myofi­brils. J. Food Sci. 38:981-986.

212

Hegarty, G. R., 1. J. Bcatzier, and A. H. Pearson, 1963. The relationship of some intracellular protein charact­eristics to beef muscle tenderness. J. Food Sci. 28:525-530.

Henderson^ D= W»- D. S. Goll. and fl- H. Stromer. 1970. A comparison of shortening and Z-line dégradation in post­mortem bovine, porcine, and rabbit muscle. Amer. J. Anat. 128:117-135.

Hendricks, H. B., D. T. Lafferty, E. D. Aberle, H. D. Judge, and J. C. Forrest. 1971. Relation of porcine muscle fiber type and size to postmortem shortening. J. Anis. Sci. 32:57-61.

Herring, H. K., B. G. Cassens, and E. J. Briskey. 1964. Sarcomere length of free and restrained bovine muscles after thaw rigor contracture. Biodynamica 9:257-264*

Herring, H. K., R. G. Cassens, and E. J. Briskey. 1965a. Sarcomere length of free and restrained muscle at low temperature as related to tenderness. J. Sci. Food Agric. 16:379-38%.

Herring, H. K., B. 6. Cassens, and E. J. Briskey. 1965b. Further studies on bovine muscle tenderness as influ­enced by carcass position, sarcomere length and fiber diameter. J. Food Sci. 30:1049-1054.

Herring, H- K., S. G. cassens, and E. J. Briskey. 1967a. Factors affecting collage» solubility in boTine suscls. J. Food Sci. 32:534-538.

Herring, H. K., R. G. cassens, G. G. Suess, ?. H. Brungardt, and E. J. Briskey, 1967h= Tenderness and associated characteristics of stretched and contracted bovine mus­cles. J. Food Sci. 32: 317-323.

Bershberger, T., B. Deans, L. S. Kunkle, P. Gerlaugh, and F. S. Deatherage. 1951. studies cn seat. III. Ths bio­chemistry and guality of meat in relation to certain feeding management practices. Food Technol. 5:523-527.

Hill, F. 1966. The solubility of intramuscular collagen ia meat animals of various ages. J, Food Sci, 31:161-166.

Hoaglandf B.^ C. N, ScBryde and w. C. Powick. 1917. Changes in fresh beef during storage above freezing. U.S. Dept. of Agric. Bull. 433. 18pp.

213

Hostetler, R. L., B. A. Landmann, B. A. link, and H. &. Fitzhugh, Jr. 1970. Influence of carcass position dur­ing rigor mortis on tenderness of beef muscles: compar­ison of tHO treatments. J. Anim. Sci. 31:47-50.

Hostetler, R. L.- B. A. link, w. A. Landman, and H. A. fitzhugh, Jr. 1972. Effect of carcass suspension on sarcomere length and shear force of some major bovine muscles. J. Food Sci. 37:132-135.

Hostetler, R. L., B. A. Link, w. A. Landman, and H. A. Fitzhugh, Jr. 1973. Effect of carcass suspension method on sensory panel scores for some major bovine muscles. J. Food Sci. 38:264-267.

Howard, R. D. and H. D. Judge. 1968. Comparison of sarco­mere length to other predictors of beef tenderness. J, Food Sex. 33:45S''"60.

Huffman, D. L., A. Z. Palmer, J. W. Carpenter, D. D. Hargrove, and a. Roger. 1967a. Effect of breeding, level of feeding and antemortem injection of papain on the tenderness of yeanling calves. J. Anim. Sci. 26:290-293.

Huffman, D. L., A. Z. Palmer, J. H. carpenter, J. F. Hentges, Jr., and R. L. Shirley, 1967b. Effect of antemortem injection of sodium chloride, papain and papain deriva­tives on the tenderness of beef. J. Anim. Sci. 26:285-289.

Husaini, S. A., F. E. Deatherage, and L. E. Kunkle. 1950. Studies OS seats II. Observations on ralatioa of biochemical factors to changes in tenderness. Food Techncl. 4:356-369»

Huxley, H. S. 1953. Electron microscope studies of the or­ganization of the filaments in striated muscle. Siochim. Biophys. Acta 12:387-394.

Huxley, H. E. 1960. Huscle cells. Pages 365-392 in J. Brachet and A. E. Birsky, eds. The cell. Vol. 4. Aca­demic Press, Inc., New York, New York.

Huxley, H. E. 1964. Evidence for the continuity between the central elements of the triads and extracellular space in frog satorius mascle. Nature 202:1067-1071.

214

Huxley, H. E. 1968. Structural difference between resting and rigor muscle; evidence from intensity changes in the low-angle equatorial X-ray diagram. J. Mol. Biol. 37:507-520.

Haxley- H. E. 1972. Molecular basis of contraction. Pages 301-387 in G. H. Bourne, ed. The structure and functisn of muscle Vol. I. 2nd ed. Academic Press, Inc. New York, Hev York.

Huxley, H. E. and w. Brown. 1967. The low-angle X-ray dia­gram of vertebrate striated muscle and its behavior dur­ing contraction and rigor. Jo Hoi. Biol, 30:384-434.

lodice, a. A., 7- Leong, and I. M. weinstock. 1966. Separa­tion of cathepsins A and D of skeletal muscle. Arch. Biocfaem. Biophys. 117:477-486.

Jones, J. B. 1972. Studies on chicken actomyosin. I. Effect of storage on muscle enzymic and physico-chemical properties. J. Sci. Food Agric. 23:1009-1019.

Judge, H. D., H. B. Hendricks? D. T. Lafferty, E. D. Aberle, and J. c. Forrest. 1971. Muscle fiber characteristics, postmortem shortening and tenderness of pock. Proc* Beat Bes. Workers Conf. 17:98-101.

Jungk, R. A. and H. w. Marion. 1970. Postmortem isometric tension changes and shortening in turkey muscle strips held at various temperatures. J. Food Sci. 35:143-145.

Jungk, K. A., H. S. Snyder, D. E. Soil, and K. G. McCoanell. 1967. Isometric tension changes and shortening in mus­cle strips during postmortem aging. J. Food Sci. 32:158-161=

Kang, C- K. and E. E. Rice. 1970. Degradation of various meat fractions by tenderizing enzymes. J, Food Sci. 35:563-565.

Kang, C. K. and W. D. Sarner. 1974. Tenderization of meat with papaya latex proteases. J. Food Sci. 39:812-818.

Kastenschmidt, L. L. 1970. The metabolism of muscle as a food. Pages 735-753 in E. J. Briskey, R. G. Cassens and B. B. Harsh, eds. The physiology and biochemistry of muscle as a food. Vol. II. Oniversity of Wisconsin Press, Madison, Wisconsin.

215

Kelly, K., N- R. Jones, B. H. Love, and J. olley. 1965. Texture and pH In fish muscle related to "cell fragili­ty" measurements. J. Food Technol. 1:9-15.

Khan, A , f. and E. Nakamura. 1970. Effects of pre-and post­mortem glycolysis on poultry tenderness. J. Food Sci. 35:266-267.

Khan, &. W. and L. van den Berg. 1964. Changes in chicken muscle proteins during aseptic storage at above-freezing temperatures. J- Food Sci. 29:49-52.

Kim, C. w., G. ?. Ho, and s. J. Sitchey. 1967. Collagen content and subjective score for tenderness of connec­tive tissue in animals of different ages. J. Food Sci. 32:586-588.

Klose, A. â., 2. J. Luyst, and L. J. Senz- 1970= Effect of contraction on tenderness of poultry muscle cooked in prerigor state. J. Food Sci. 35:577-581.

Kruggel, H. G. and R. &. Field. 1971. soluble intramuscular collagen characteristics from stretched and aged muscle. J. Food Sci. 36:1114-1117.

Kruggel, W. G., R. A. Field, and G. J. ailler. 1970. Physi­cal and chemical epimysial acid-soluble collagen from meats of varying tenderness. J. Food Sci. 35:106-110.

laakkonen* E. 1973. Factors affecting tenderness during hê&tiûg of meat. àuv. rood Sss. 20:257-323.

Laki, K. 1957. The composition of contractile muscle pro­teins. J. Cell and coap. Physiol. 49:249-265.

Laki, K. 1971. Actin. Pagas 97-133 in K. Laki, ed. Contractile proteins and muscle. Harcel Dekker, Inc. Sew York, Hew York.

Lardy, K. k= 1366. Regulation of snsrgy-yislding processes in muscle. Pages 31-38 in E. J. Briskey, R. G. Cassens and J. C. Trautaan, eds. The physiology and biochemis­try of muscle as a food. Vol, I. University of Biscon-sin Press, Hadison, Wisconsin.

Lavrie, R. A, 1966a. Meat Science. Pergamon Press, Oxford. pp« 368=

216

lawrie, R. A. 1966b. Metabolic stresses which affect mus­cle- Pages 137-164 in E. J. Briskey, R. G. Casseas and J. C. Trautman, eds. The physiology and biochamistry of aascle as a foad. Vol. I. Oniversity of Wisconsin Press, Hadison, Wisconsin.

lavrie, R. à. 1968. Thaw-rigor and cold-shortening in rab­bit muscle. J. Food Technol. 3:203-205.

Lawrie, R. &., I. F. Penny, R. K, Scopes, and C. à. Voyle. 1963. Sarcaplasmic proteins in pale, exudative pig mus­cles. Hature 200:673.

Locker, R. H. 1960a. Degree of muscular contraction as a factor in tenderness of beef. Food Res. 25:304-307.

Locker, R. H. 1960b. Proteolysis in the storage of beef. <3. Scx. Food Agrz. 11:520—525.

Locker, B. H. and C. J. Hagyard. 1963. A cold shortening effect in beef muscles. J. Sci. Food Agric. 14:787-793.

Love, R. S. and E. 5. sackay. 1962. Protein denaturation in frozen fish, 7. Development of the cell fragility method for measuring cold-storage changes in the muscle. J- Sci. Food Agric. 13: 200-212.

Lowey, s . , H . S. Slayter, A. G. Seeds and H. Baker. 1969. Substructure of the myosin molecule. I. Subfragments of mvosin bv enzymatic degradation. J. Hoi. Biol. 42:1-10.

Saler, G. s. and S» L. Fischer- 1966= Acrylaeide gel disc electrophoretic patterns and estractability of chicken breast aascla proteins during postsortes aging, Pood Sci. 31:482-487.

Harsh, B. B. 1954. Rigor mortis in beef. J. Sci. Food Agric. 5:70-75.

Sarsh, 6. B. 1972. Postmortem muscle shortening and meat tenderness. Proc. Heat Ind. Res. Conf. 25:109-124.

Harsh, B. B. and B. A. Carse. 1974. Heat tenderness and the sliding-filament hypothesis. J. Food Technol. 9:129-139.

217

Harsh, B. B. and N. G. Leet. 1966a. Resistance to shearing of heat-denatured muscle in relation to shortening. Rature 211;535-636.

Harsh, B. B. and S, G. Leet. 1966b. Studies in meat tender­ness. 3. The effects of cold shortening on tenderness, J. Food Sci. 31:450-459.

Marsh, B. B. and J, F. Thompson. 1958. Rigor mortis and thaw rigor in lamb. J. Sci. Food Agric. 9:417-424,

Harsh, B. B., P. a. Woodhams, and N« G. Leet. 1968. Studies in meat tenderness, 5= The effects on tenderness of carcass cooling and freezing before the completion of rigor mortis. J. Food Sci. 33:12-18.

Hartin, A. H., H. T. Fredeen, and G. H. Weiss. 1971. Ten­derness of beef longissimus dorsi muscle from steers, heifers, and bulls as influenced by source, postmortem aging and carcass characteristics. J. Food sci. 36:619-623.

aartins, C. B. and J- R. 0hltaker. 1968. Catheptic enzymes in meat tenderization. I. Purification of cathepsin D and its action on actooyosin. J. Food Sci, 33:59-64.

Scciain, P. E. 1969. Isolation of intramuscular connective tissue. Nature 221:181.

ScClais, Pi S- and â= Mullins, "969. Relationship of interacellular proteins and muscle pigments to the ten­derness of bovine muscles. J. Anim. Sci. 29:42 3-425.

HcClain, P. E. and A. K. Pearson. 1969. Connective tissues from normal and pale, soft asd ssudative (PSE) porcine muscles. 2. Physical characterization. J. Food Sci. 34:306-308.

Hcclain, P. E., G. J. Creed, E. R. Riley, and I. Rornsteia. 1970. Effect of postaorteia aging on isolation of intramuscular connective tissue. J. Food Sci. 35:258-259.

ScClain, Po E.g lo He Hullins, S. L. Hansard* J. 0. Fos, and E. F. Soulsare. 1965. Acid and salt-soluble collagen in bovine muscle. Proc. Soc. Exptl. Biol. Bed. 119:492-S95.

218

HcCrae, s. E., C. G. seccombe, B. B. Marsh, and R. &. Carse. 1971. Studies in meat tenderness. 9. The tenderness of various lamb muscles in relation to their skeletal restraint and delay before freezing. J, Food sci. 36:566-570.

Hihalyi, E. and &. J. Rove. 1966. Studies on the extraction of actomyosin from rabbit muscle. Biochem. J. 345:267-285.

Rihalyi, E. and A. G. Szent-Gyorgi. 1953. Trypsin digestion of muscle proteins. I. nltracentrifagal analysis of the process. J. Biol. Chea. 201:189-196.

Hiles, c. L. and R. &. Lavrie. 1970. Relation between pH and tenderness in cooked muscle. J. Fool lechnol. 5:325-330.

Miller, J. H., L. £. Dawson, and D. H. Bauer. 1965. Free amino acid content of chicken muscle from broilers and hens. J. Food Sci. 30:406-411.

Hiyada, D. S. and &. L. Tappel. 1956. The hydrolysis of beef proteins by various proteolytic enzymes. Food Res. 21:217-221.

Holler, Â. J., T. vestergaard, and J. Sismer-Pedersen. 1973. Myofibril fragmentation in bovine longissimus dorsi as an index of tenderness. J. Food Sci. 38:824-825.

auller, L., R. H. Best, &. Z. Palmer, and J. s. Carpenter. 1974. Indices of tenderness in cow carcasses. J. &nim. Sci. 39:173. (Âbstr.)

Nakamura, H. 1972. Measurement of tensile strength of mus­cle fibers and its change during postmortem aging of chicken breast muscle. J. &gric. Food Chem. 20: 1167-1172.

Nakamura, R. 1973. Factors associated with postmortem in­crease of extractable Ca++ in chicken breast muscle. J. Food Sci. 38:1113-1114.

Neelin, J. H. and D« J. Ecobichono 1966. Enzymatic activi­ties in pre-rigor and post-rigor sarcoplasmic estracts of chicken pectoral muscle. Canadian J. Biochem. 44:735-741.

219

Neelin, J. H. and D. Rose. 1964. Progressive changes in starch gel electrophoretic patterns of chicken muscle proteins during "aging" post-mortem. J. Pood Sci, 29:5%%-55%.

Eeebold, E. P. and P. 7. Harris» 1972= The effect of pre-rigor changes on meat tenderness, à review. J. Food Sci. 37:337-339.

Newbold, B. P. and P. K. Scopes. 1967. Post-mortem glycoly­sis in ox skeletal muscle. Effect of temperature on the concentrations of glycolytic intermediates and cofac-torso Biochea. J« 105:127-132.

Ohtsuki, I., T. Hasaki, T. Nonomura, and S. Ebashi. 1967. Periodic distribution of troponin along the thin fila­ment. J. Biochem. 61:817-819.

Okitani, A., 0. Takagi, and H. Fujimaki. 1967. The changes of "myosin B" during storage of rabbit muscle. Part IV, Effect of temperature, pH, and ionic strength on denaturation of "myosin B" solution. &gric. Biol. Chem. 31:939-946.

Osner, R. C, 1966. Influence of processing procedures on the post-mortem permeability of chicken muscle sarcolea-mas to protein. J. Food Sci. 31:832-837.

Park, D. C. and R. J. Pennington. 1967. Proteinase activity in muscle particles. Eazym. Biol, Clin. 8:149-160.

Parrish, F. C., Jr. 1971. Extent and role of proteolysis in post-mortes muscle. Saaual Seciprocal Seat Coaf. Proc. 24:97-133.

Parrish, F. C., Jr. 1974. Relationship of marbling to meat tenderness. Heat Ind. Res. Conf., Chicago, Illinois.

Parrish, F. C., Jr. and S, E. Bailey, 1966, Physicochsmical properties and partial purification of porcine sstiscle cathepsin, J. &gric. Food Chem. 14:232-237,

Parrish, F, C,, Jr. and M, E, Bailey. 1967. Physicochemical properties of bovine muscle particulate cathepsin. J. AgriCo Food Cfeeso 15:88-94,

220

Parrish, F. C., Jr., D. G. Olson, B. £. Hiner and R. E. Rust. 1973a. Effect on degree of marbling and internal tem­perature of doneness on beef rib steaks. J. Anim. Sci. 37:430-434.

Parrish, P. C., Jr., R. E. Rust, G. R. Popenhagen, and B. E. Hiaer. 1969a. Effect of postmortem aging time and tem­perature on beef muscle attributes. J. ftnim. Sci. 29:398-403.

Parrish, P. C., Jr., R. B. Young, 6. E. Hiner, and L. 0. Anderson. 1973b. Effect of postmortem conditions on certain chemical, morphological and organoleptic proper­ties of bovine muscle. J. Food Sci. 38:690-695.

Parrish, F. C., Jr., D. E. Goll, g. J. Nevcomb II, B. 0. de Lumen, H. S. Chaudhry, and E. A. Kline. 1969b. Solecii-lar properties of post-mortem muscle. 7. Changes in non-protein nitrogen and free amino acids of bovine mus­cle. J. Food Sci. 34:196-202.

Partridge, S. H. 1966. Elastin. Pages 327-339 in E. J. Briskey, R. G. Cassens, and J. C. Trautman, eds. The physiology and biochemistry of muscle as a food. Vol. 1. University of Bisconsin Press, Madison, Wisconsin.

Paul, P. C., S. E. flcCrae, and L. H. Hofferber. 1973. Heat-induced changes in extractability of beef muscle colla­gen. J. Food Sci. 38:66-68.

Pcllsgrino, aad C= Franzinio 1565- hn electron micro­scope study of denervation atrophy in red and white skeletal fibers. J. Cell Bid. 17:327-349.

Penny, I. F. 1968. Effect of aging on the properties of myofibrils of rabbit muscle. J. Sci. Food Agric. 19:518-524.

Penny, I. F. 1970. Conditioning of bovine muscle. II. Changes ia the composition of extracts of myofibrils after conditioning. J. Sci. Food Agric. 21:303-306.

Penny, I. F., C. A. 7oyie, and E. Dransfield. 1974. The tenderising effect of a muscle proteinase on beef. J. Sci. Food Agric. 25:703-708.

Penny, I. F., c. A. Voyle, and 8. A. Lasrie. 1963. A com­parison of freeze-dried beef muscles of high and low ul­timate pH. J. Sci. Food Agric. 14:535-543.

221

Pfeiffer, N. E., B. A. Field, T. R. Varnell, H. G. Kruggel, sad I. I. Kaiser, 1972- Effects of postmortem aging and stretching on the macromolecular properties of col­lagen» J« Food Scio 37:897-900*

PieZff K= 1966. Collagen. Pages 315-326 ia E. J. Briskey, R. G. cassens and J. C. Traatman, eds. The physiology and biochemistry of muscle as a food. Vol. I. University of Wisconsin Press, Madison, Wisconsin.

Pool, a. F. 1967. Objective measurement of connective tis­sue tenacity in poultry meat- J. Food Sci. 32: 550-553.

Price, H- H-, E. L. Howes and J. H. Blumberg. 1964. Oltra-structaral alterations in skeletal muscle fibers injured by cold. 11. Cells of the sarcolemmal tube: observa­tions on discontinuous regeneration and myofibril forma­tion- Lab. larsst. 13: 1279-1237.

Prudent, I. 1947. The collagen and elastin content of four beef muscles aged varying periods of time. Ph.D. thesis. Iowa State University, âmes, Iowa.

Purchas, B. W. 1972. The relative importance of some deter­minants of beef tenderness. J. Food Sci. 37:341-345.

Quarrier, E., Z. L. Carpenter, and G- C- Smith. 1972. A physical method to increase tenderness in lamb carcas­ses. J. Food Sci- 37:130-131.

Sandall, C. J. aad H. ?. HacSas- 1357. Sydrolytic enzysss in bovine skeletal muscle. II. Proteolytic activity of the eater-soluble proteins separated by starch-gel elec­trophoresis. J. Food Sci. 32:182-184.

Reed, R., T. H. Houston, and P. H. Todd. 1966. Structure and function of the sarcoleasa of skeletal muscle. Hature 211: 534-536.

neviiiS, So Jo, So !• e uOSvpu and M- S - Harrington. 1971» Structural changes during the post rigor storage of some large beef muscles. Proc. Seat Res. Workers Conf. 17:692-697.

Robinson, H. E. and P. a. Goeser. 1962. Enzymatic tenderi-zation of meat. J. Home Econ. 54: 195-200.

222

Eobson, B. H. 1971. Calcium-sensitive response of post­mortem bovine myofibrils. J. Anim. Sci. 33:1144-1145. (Abstr.)

Rob son, B. H. and H. B, Cohen. 1971. Postmortem myofibrils from light and dark muscle. J. Anim. Sci. 33:223-224. (Abstr.)

Eobson, E. a.f D. E. Soil, and M. J. Rain. 1967. Holeculac properties of postmortem muscle. 5. Nucleoside tri­phosphatase activity of bovine myosin B. J. Food Sci. 32:594-549.

Sayre, a. N, 1968. Postmortem changes in extractability of myofibrillar protein from chicken pectoralis. J. Food Sci. 33:609-612.

Sayre, S. S. 1969. Fragmentation of chicken myofibrils. Annual Beciprocal Beat Conf. Proc. 22:230-237.

Sayre, B. K. 1970. Chicken myofibril fragmentation in re­lation to factors influencing tenderness. J. Food Sci. 35:7-10.

Sayre, B. H. and E. J. Briskey. 1963. Protein solubility as influenced by physiological conditions ia muscle. J. Food Sci. 2 8:675-679.

Schmidt, G. B. and K. 7. Gilbert. 1970. The effect of mus­cle escision before the onset of rigor mortis on the pâlâtâbjLjkity of beef. G. ?gcu Tecunol. 5î331°338.

Schsidt; J. Ge and F. C. Parrish. 1971. Holecular proper­ties of postmortem muscle. 10. Effect of internal tes-perature and carcass satarity on structure of bovine longissimus. J, Food Sci, 36:110-119,

Schollmeyer, J. E., D. E. Goll, B. M. Eobson and 5. H. Stromer. 1973. Localization of alpfea-actinin and tropoâycsiû in different muscles. J. Cell Biol. 55:305. (Abstr.)

Scopes, B. K. 1964. The influence of postmortem conditions on the solubilities of muscle protein. Biochea. j. 91:201-207.

223

Scopes, E. K. 1970. Characterization and study of sarco­plasmic proteins. Pages 471-508 in E. J. Briskey, R. 3. Cassens, and B. B. Marsh, eds. The physiology and bio­chemistry of muscle as a food. Vol. 2. Uaiversity of Wisconsin Press, Hadison, Wisconsin.

Scopes, B. K. and B. &. Lavrie. 1963. Postmortem lability of skeletal muscle proteins. Nature 197:1202-1203.

Scopes, R. K. and R. P. Newbold. 1968. Post-mortem glycoly­sis in ox skeletal muscle: Effect of pre-rigor freezing and thawing on the intermediary metabolism. Biochem. J. 109:197-202.

Semlek, M. A. and H. L. Riley. 1974. The effect of post slaughter aging on tenderness. J. Anim. Sci. 38:1328. (nbstr.)

Sharp, J. G. 1963. Aseptic autolysis in rabbit and bovine muscle during storage at 37oc. J. Sci, Food Agric. 14: 468-479.

Shimokomaki, M., Do F. Elsden? and A. J. Bailey. 1972. Meat tenderness: Age related changes in bovine intramuscular collagen. J. Food Sci. 37:892-896.

Singh, I. 1974. N- and C- terminal amino acids of alpha-actinin and studies on the effects of alpha-actinin on F-actin. Ph.D. Thesis. Iowa State Dnivscsity, Ames, Iowa.

Smith, B. 1964. Histological and histochemical changes in the muscles of rabbits given the corticosterioi triam­cinolone. Neurology 14:857-863.

Smith, G. C., T. C. Arango, and Z. L. Carpenter. 1971. Ef­fects of physical and mechanical treatments on the ten­derness of the beef longissimus. J. Food Sci. 36:445-449.

Snedecor, G. W. and 9. G. Cochran. 1967. Statistical meth­ods. Iowa State University Press, Ames, Iowa.

Stanley, D. H., G. P. Pearson, and V. E. Coxworth. 1971. Evaluation of certain physical properties of meat using a universal testing machine. J. Food Sci. 36:255-250.

224

Stanley, D. *., L. B, HcKnight, w. G. S. Hines, w. B. Osborne, and J. M. Deman. 1972. Predicting meat ten­derness from muscle tensile properties. J. Texture Studc 3:51-68.

Stauber. H. T. and J, ï, c. Bird. 1974. S-P zonal fraction­ation studies of rat skeletal muscle lysosooe-ricii fractions. Biochim. Biophys. Acta 338:234-245.

Steel, E. D. G. and J. H. Torrie. 1950. Principles and procedures of statistics. McGraw-Hill Book Company, Inc., Hew York, New York.

Strandberg, K., F. c. Parrish, Jr., D. E. Goll, and S. &. Josephson. 1973. Molecular properties of postmortem muscle: Effect of sulfhydryl reagents and postmortem storage on changes in myosin B. J. Food Sci. 38:69-74.

Stromer, H. H. and D. E. Goll. 1967a. Molecular properties of postmortem muscle. II. Phase microscopy of myofi­brils from bovine muscle. J. Food Sci, 32:329-331.

Stromer, M. H. and D. E. Goll. 1967b. Molecular properties of postmortem muscle. III. Electron microscopy of myo­fibrils. J. Food Sci. 32:386-389.

Stromer, H. H., D. E. Goll, and L. E. Roth. 1967. Morpholo­gy of rigor-shortened bovine muscle and the effect of trypsin on pre-and post-rigor myofibrils, J. Cell Biol. 34:431-445.

Stromer, M. H., D. E. Goll, W. J. Reville, D. G. Olson, W. R. DaytoBf and a. S. Bobson. 1974= Structural changes ia muscle during postmortem storage. Proc. Internat. Congress Food Sci. Technol.. Madrid, Spain.

Suzuki, K, and E. Fujisaki. 1968. Studies on proteolysis in stored msscle. 11. Purification and properties of a proteolytic enzyme, cathepsin D. from rabbit muscle. AgriCe Bid. Chss- 32:973-932.

Suzuki, km and D. E. Goll. 1974. Quantitative assay for caSF (Ca2+-activated sarcoplasmic factor) activity, and effect of CâSF treatment on &TPase activities of rabbit myofibrils. Agric. Biol. Chem. 38:2167-2175.

225

Snzaki, A., H. Nakazato, and H. Fujiaaki. 1967, studies on proteolysis in stored muscle. I. Changes in non­protein nitrogenous compounds of rabbit muscle during storage. &gric. Biol. Chea. 31:95 3-957.

Suzuki, &. Okitani, and M. Fujiaaki. 1969. Studies on proteolysis in stored muscle. IV. Effect of cathepsin D treatment on ftTPase activity of myosin B. Agric. Biol. Chen. 33: 1723-172 6.

Szent-Gyorgi, A. G. 1960. Proteins of the myofibril. Pages 1-49 in G. H. Bourne, ed. The structure and function of muscle. Vol. 2. Academic Press, Inc. New York, New York.

Takahashi, K.* T. Fukazawa, and T. Yasui. 1967. Formation of myofibrillar fragments and reversible contraction of sarcomere in chicken pectoral muscle. J- Food Sci-32:409-413.

Tappel, A- L., D. S. Hiyada, C. Sterling, and V. p. Haier. 1956. Heat tenderization II. Factors affecting the tenderization of beef by papain. Food Res. 21:375-379.

Tappel, A. L., H. Zalkin, K. A. Caldwell, I. D. Desai, and S. Shibko. 1962. Increased lysosomal enzymes in genetic muscular dystrophy. Arch. Biochem. Biophys. 92:340-346.

Taylor, A. A., B. B. Chrystall, and D. N. Rhodes. 1972. Toughness in lamb induced by rapid chilling. J. Food

Taylor? E. H= 1972. Chemistry of muscle contraction. Ann. Rev. Biochem. 41:577-616.

Thompson, G. B., W. D. Davidson, H. W. Hontgomery, and A. F. Anglemier. 1968. Alterations of bovine sarcoplasmic proteins as influenced by high temperature aging. J. Food Sci. 33:68-72.

Tsen, C. c. and A. L. Tappel. 1959. Meat tenderization III. Hydrolysis of actomyosin, actin and collagen by papain. Food Res. 24:362-365.

Valin, c. 1968. Post-mortem changes in myofibrillar protein solubility. J. Food Technol. 3:171-173.

226

van Eerd, J. P. and I. Kawasaki. 1973. Effect of calcium (II) on the interaction between the subunits of troponin and tropomyosin. Biochemistry 12:4972-4980.

Veis, A. 1970. Collagen. Pages 455-470 in E. J. Briskey, B. 6. Cassens and B. B. Harsh, eds. The physiology and biochemistry of mmscle as a food. Vol. II. University of Wisconsin Press, Sadison, Wisconsin.

Tognarova, I., Z. Dvorak, and R. Bohm. 1968. Collagen and elastin in different cuts of veal and beef. J. Food Sci. 33:339-343.

Bang, a., C. E. Veir, N. L. Birkner, and 6. Ginger. 1957. The influence of enzyme tenderizers on the stracture and tenderness of beef. Proc. Heat Ind. Res. Conf. 9:72-74.

Hang, 5.* C« S« Seir? 1= Birkner. and B. Ginger, 1958. Studies on enzymatic tenderization of meat. III. His­tological and panel analysis of enzyme preparations from three distinct sources. Food Res. 23:423-438.

Beber, K. and H. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamida gel electrophoresis. J. Biol. Chem. 244:4406-4412.

Veidemann, J. F., G. Kaess and L. D. Carruthers. 19 67. The histology of pre-rigor and post-rigor ox muscle before and after cooking and its relation to tenderness, J. Food Sci. 32:7-13.

Weinberg, B. and 0. Rose. 1960. Changes in protein extrac-tability during post-rigor tenderization of chicken breast muscle. Pood Technoio 14:376-379.

Weinstock, I. a., &. D. Goldrich, and A. T. Bilhorat. 1955. Enzyme studies in aoscular dystrophy. I. Muscle prote­olytic activity and vitamin E deficiency- Proc. Soc. Esptl. Biol. Bed. 88:257-260.

Welbourn, J. L., R. B. Harrington, and w. J. Stadeloan. 1968. BelatioQShips among shear values, sarcomere lengths and cooling procedures in turkeys. J. Food Sci. 33:450-452.

Whitaker, J. R. 1964. Post mortem proteolytic changes af­fecting myofibrillar proteins. Annual Reciprocal Heat Conf. Proc. 17:153-160.

227

Rhiting, H. C. and J. F. Richards. 1971. Effect of raduri-zation on shear resistance and fragmentation of chicken muscle. J. Food Sci, 36:752-755.

Wierbicki, E., L. E. Kunkle* V. B. Cahill, and P. E. Deatherage. 1954. The relation of tenderness to pro­tein alterations daring post-mortem aging. Food Technol. 8; 506- 511.

Rilson, G. D., Re i. Bray and P. H. Phillips. 1954. The effect of age and grade on the collagen and elastin con­tent of beef and veal. J. &nim. Sci. 13:826-832.

winegarden, S. w., B. Lome, J. Kastelic, 2. A. Kline, &. E. Plagge, and P. S. Shearer. 1952. Physical changes in connective tissues of beef during heating. Food Res. 17: 172-184.

Wiskus, K. J., p. B. Addis, and B. T-I. Ha. 1973. Post­mortem changes in dark turkey muscle. J. Food Sci. 38: 313-315.

iu, C- S. c. and E. H. Sayre. 1971. Myosin stability in in­tact chicken muscle and a protein component released after aging. J. Food Sci. 36: 133-137.

Zalkin, a., à. L. Tappel, K. &. Caldeell, S. Shibko, I. D. Desai, and T. &. Holliday. 1962. Increased lysosomal enzymes in muscular dystrophy of vitamin E deficient rabbits. J. Biol. Chea. 237:2678-2682.

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ACKNOWLEDGERENTS

I express ay appreciation to Dr. F. C. Parrish, Jr.

for serving as my major professor and for assisting and

encouraging me during the preparation of this disserta­

tion. I would also like to thank Dr. D. E. Soli, Dr. R.

a. Robson, Dr. H. H. Stromer and Mr. John C. Olson for

taking an interest in my research program.

k special thank you to B. E. Rust, D. P. co% and

Walker for serving on ay graduate comsittee. Appre­

ciation is also extended to firs. Sherri Josephson, Dr.

Randy Snell, Sr. Doug De Tries, Hiss Pat Chen and Mrs.

Ruth Smith for their invaluable technical assistance

during my research program and also to Mrs. Mary Stimmel

for typing this dissertation. I would also like to ex­

press my appreciation to my fellov graduate stadeats for

their interest and encouragement and especially to

Billiam H. Dayton and Ronald S. alien whose aaay

discussions have immeasurably helped my research plan­

ning, execution and interpretation.

Spscxa^ apprcc^s.«.Xcn xs ».c sy parents Sr.

and Mrs Johan c« Olson and parents-in-law Mr. and Mrs F.

M. Booster for their continual encouragement, moral sup­

port and financial assistance throughout my graduate

career.

229

Finally and most importantly my deepest apprecia­

tion is extended to my wife, Jane, for her patience,

encouragement and infallible support and unfaltering

belief in me during my graduate career.