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Biochemical and ultrastructural changes occurring in chicken pectoralis muscle inoculated with pseudomonas… Sage, Gilbert 1974

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BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES OCCURRING IN CHICKEN PECTORALIS MUSCLE INOCULATED WITH PSEUDOMONAS TRAGI by GILBERT SAGE B.Sc. (Agr.), University of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1974. In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thou t my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada Date ABSTRACT i i Chicken pectoralis muscle was inoculated with Pseudomonas  f r a g i and incubated at room temperature. Alterations i n the nonprotein nitrogen, water-soluble protein nitrogen, and s a l t -soluble protein nitrogen fractions.-were studied and attempts were made to relate these changes to structural changes ob-served by scanning and transmission electron microscopy. A s i g n i f i c a n t decrease i n m y o f i b r i l l a r protein s o l u b i l i t y was found i n the inoculated muscle during the protein extract-a b i l i t y study. Results of the gel f i l t r a t i o n study indicated that proteolysis of the sarcoplasmic proteins occurred and that the nonprotein nitrogen f r a c t i o n increased due to growth of P. f r a g i . Alterations were observed i n the disc gel e l e c t r o -phoretic patterns of the sarcoplasmic and m y o f i b r i l l a r protein f r a c t i o n s . Arginine, threonine, serine, proline and tyrosine were s e l e c t i v e l y u t i l i z e d by P. f r a g i . Scanning electron micrographs indicated that proteolysis of the endomysium occurred a f t e r 2 days of incubation at 25°C. Proteolysis of the endomysium became more extensive as incub-ation time increased. After 4 days of incubation the myofibrils showed evidence of disruption as a r e s u l t of b a c t e r i a l growth. Disruption of the muscle f i b e r was l i m i t e d to a depth of 2 to 4 micrometers a f t e r 9 days of incubation. Bacteria were ob-served growing between the muscle f i b e r s . Proteolysis was not as extensive i n samples incubated at 5 ° C Electron micrographs prepared from t h i n sections of myo-i i i f i b r i l s i n o c u l a t e d with P. f r a ^ i showed iaarlced d i s r u p t i o n of the m y o f i b r i l s due to b a c t e r i a l growth. A c l e a r zone, devoid of s t r u c t u r a l d e t a i l , surrounded the b a c t e r i a l c e l l . A r e g i o n of d i s r u p t e d t i s s u e e x i s t s between the c l e a r zone and i n t a c t m y o f i b r i l s . C e l l u l a r p r o t r u s i o n s were observed on the s u r f a c e of the b a c t e r i a . TABLE OF CONTENTS INTRODUCTION Page LITERATURE REVIEW 3 Meat Spoilage 3 Aspetic Techniques 4 Changes i n Muscle Proteins during postmortem Storage 5 Biochemical Changes Occurring i n Spoiled Muscle 10 Ultras t ruc ture of S t r i a t e d S k e l e t a l Muscle 21 METHODS Part I - Biochemical Studies 28 Sample Preparation 28 B a c t e r i a l Counts 29 Prote in Ext rac t ion 29 Nitrogen Determination 31 Gel F i l t r a t i o n 31 Disc Gel Electrophoresis 32 Free Amino A c i d Analys is 33 Part I I - E lec t ron Microscopy B a c t e r i a l Counts 35 Preparation of Samples f o r Scanning Elec t ron Microscopy 36 Preparation ofi Samples for Transmission Elec t ron Microscopy 36 RESULTS AND DISCUSSION Part I - Biochemical Studies 39 V Influence of growth of Pseudomonas f r a g i organisms on the pH and water holding capacity of chicken muscle 39 Influence of growth of Pseudomonas f r a g i organisms on the e x t r a c t a b i l i t y of proteins from chicken muscle 42 G-el f i l t r a t i o n of water-soluble proteins i n extracts from control and inoculated muscle 52 Disc gel electrophoresis of water-soluble, salt-soluble and urea-soluble proteins i n extracts from control and inoculated muscles 61 Analysis of free amino acids i n control and inoculated muscles 86 Part II - Electron Microscopy 92 Scanning electron microscopy of Pseudomonas f r a g i 92 Transmission electron microscopy of Pseudomonas  f r a g i 121 GENERAL DISCUSSION Conclusions 132 BIBLIOGRAPHY 133 v i LIST OP TABLES Table Page I Influence of incubation time at 25°C on the b a c t e r i a l numbers and pH of control muscle and muscle inoculated with P. f r a g i 40 II Water-soluble protein, salt-soluble protein and nonprotein nitrogen extracted from control and inoculated chicken pectoralis muscle 47 III Analysis of variance of protein e x t r a c t a b i l i t y from chicken pectoralis muscle 48 IV S o l u b i l i t y of the various'protein fractions from uninoculated muscle and muscle inoculated with P. f r a g i 49 V Ratio of areas under the curves at Vo and Ye f o r glutamic acid 58 VI Average free amino acid concentration of ino-culated and uninoculated chicken pectoralis muscle incubated at 25 C 87 VII Average amino acid concentration of chicken pectoralis muscle inoculated with P. f r a g i and corrected f o r autolytic changes "~ 89 VIII B a c t e r i a l population i n chicken pectoralis major inoculated with P. f r a g i incubated at 25 0 98 v i i LIST OF FIGURES Figure Page 1 Structure of a Sarcomere 23 2 Flow Sheet of Prote in Extrac t ion Procedure 30 3 Water-Soluble prote in extracted from c o n t r o l and inoculated samples 43 4 S a l t - s o l u b l e prote in extracted from contro l and inoculated samples 44 5 Nonprotein nitrogen compounds extracted from c o n t r o l and inoculated samples 45 6 E l u t i o n patterns of water-soluble prote in extracts from uninoculated chicken p e e t o r a l i s muscle 5$ 7 E l u t i o n ' patterns of water-soluble prote in extracts from chicken, p e e t o r a l i s muscle inoculated with P. f r a g i o 56 8 Densitomater t racings of the sarcoplasmic prote in f r a c t i o n extracted from uninoculated chicken peetora l i s muscle 63 9 Densitometer t racings of the sarcoplasmic p r o t e i n f r a c t i o n extracted from chicken peetor-a l i s muscle inoculated with P. f r a g i 65-10 Densitometer t racings of the m y o f i b r i l l a r p r o t e i n f r a c t i o n extracted from uninoculated chicken p e e t o r a l i s muscle 69 11 Densitometer t racings of the m y o f i b r i l l a r p r o t e i n f r a c t i o n extracted from chicken p e e t o r a l i s muscle inoculated with P. f r a g i 71 12 Densitometer t racings of the urea-soluble p r o t e i n f r a c t i o n extracted from uninoculated chicken peetora l i s muscle 74 13 Densitometer t racings of the urea-soluble prote in f r a c t i o n extracted from chicken peetora l i s muscle inoculated with P . f r a g i 76 14 E lec t rophoret ic patterns of sarcoplasmic proteins 79 15 E lec trophoret ic patterns of m y o f i b r i l l a r proteins 81 16 E lec t rophoret ic patterns of urea-soluble proteins 83 17 Scanning electron micrograph of the surface of nutr ient agar, uninoculated and incubated at 25 C f o r 24 hr (x 22,000) 93 v i i i 94 96 97 LIST OP FIGURES Figure Page 18 Scanning electron micrograph of the surface of nutr ient agar, inoculated with P* f r a g i incubated 24 hr at 25 C (x 11,400) 19 Scanning electron micrograph of the surface of nutr ient g e l a t i n inoculated with P. f r a g i i n -cubated f o r 24 hr at 25 0 (x l,ro"0.') 20 Scanning electron micrograph of the surface of nutr ient g e l a t i n inoculated with P. f r a g i and incubated f o r 24 hr at 25 C (x l C O O O ) 21 Longi tudinal view of i n t a c t endomysium i n c r y o -f rac tured peetora l i s muscle, zero time (x 11,400) 100 22 l o n g i t u d i n a l view of exposed m y o f i b r i l s i n u n -inoculated chicken p e e t o r a l i s muscle at zero time (x 22,200) 101 23 Longi tudinal view of endomysium from cryofractured p e e t o r a l i s muscle inoculated with P. f r a g i and incubated 24 hr at 25 0 (x 9,500) ~ 102 24 Longi tudinal view of m y o f i b r i l s i n cryofractured peetora l i s muscle inoculated with P. f r a g i and incubated 24 hr at 25 C (x 9,500) 25 l o n g i t u d i n a l view of endomysium i n cryofractured peetora l i s muscle inoculated with P. f r a g i and incubated f o r 2 days at 25 C (x 9,500l 26 Longi tudinal view of muscle f i b e r from cryofract -ured peetora l i s muscle inoculated with P. f r a g i and incubated f o r 4 days at 25 C (x 9,400) 27 Longi tudinal view of muscle f i b e r from c r y o -fractured peetora l i s muscle inoculated with P . f r a g i and incubated for 8 days at room temper-ature (x 12,100) 108 28 Longi tudinal view of muscle f i b e r from cryo-fractured peetora l i s muscle inoculated with P. f r a g i and incubated at 25 0 f o r 11 days (x 10,300) 110 29 Longi tudinal view of endomysium from cryofractured peetora l i s muscle, uninoculated and incubated f o r 11 days at 25 0 (x 21,300) 111 30 Longi tudinal view of exposed m y o f i b r i l s from c r y -ofractured peetoral is muscle, uninoculated and incubated for 11 days at 25 0 (x 10,500) 112 104 105 106 LIST OP FIGURES Longi tudinal view of muscle f i b e r s from c r y o -fractured p e c t o r a l i s muscle inocubated with P. f r a g i , incubated at 25 C f o r 10.5 days then recryofractured (x 1,950) Longi tudinal view of muscle f i b e r from c r y o -fractured p e c t o r a l i s muscle inoculated with. £• f r a g i . incubated f o r 10.5 days at 25 0 then recryofractured (x 19,500) Longi tudinal view of cryofractured p e c t o r a l i s muscle inoculated with P. f r a g i and incubated at 5 0 f o r 6.5 days (x 4,400) Longi tudinal view of cryofractured p e c t o r a l i s muscle inoculated with P. f r a g i and incubated f o r 21 days at 5 C (x 11,900) Longi tudinal view of uninoculated cryofractured p e c t o r a l i s muscle, incubated at 5 C f o r 21 days (x 10,800) Longi tudinal view of uninoculated p e c t o r a l i s muscle incubated at 5 C f o r 10.5 days (x 13,200) Longi tudinal view of p e c t o r a l i s muscle inoculated with P . f r a g i and incubated at 5 0 f o r 10.5 days (x 68,900) Longi tudinal view of p e c t o r a l i s muscle inoculated with P. f r a g i and incubated at 5 G f o r 16.5 days (x 497000)" Cross sect ion of p e c t o r a l i s muscle inoculated with P. f r a g i and incubated at 5 C f o r 10.5 days (x 49,0007"^  ACTOWLEDGEICEOTS I wish to express my gratitude to Dr. W.D. Powrie and the members of my committee f o r t h e i r supervision and guidance throughout the course of t h i s work. I also wish to thank Dr. D. Schaller f o r his guidance'in the scanning electron microscope and to Mrs. Jean Simons f o r the typing of t h i s thesis. INTRODUCTION Spoilage of food myosystems can be divided into two types; namely, b a c t e r i a l decomposition of amino acids with the form-ation of putrid odors, and mold growth on the surfaces of muscle and adipose tissue to form large masses of mycelium. Only b a c t e r i a l spoilage w i l l be considered i n t h i s t h e s i s . In the past, much research has centered on the i s o l a t i o n and ident i f i c a t i o n of the bacteria associated with meat spoilage. For many years, researchers have studied the temperature and growth cha r a c t e r i s t i c s of spoilage organisms along with the incidence and significance of these organisms during meat spoilage, but l i t t l e attention has been focussed on the chemical and s t r u c t -u r a l changes during spoilage. Various attempts have been made to u t i l i z e chemical and physical techniques to obtain an index of microbial spoilage. Increase i n pH, production of ammonia and determination of ex-tract release volume of muscle have at one time or other been advocated as a suitable index of spoilage. Generally, these parameters correlate roughly with b a c t e r i a l numbers only when high b a c t e r i a l loads have developed and the meat has spoiled. Within the l a s t decade, numerous papers have appeared on the nature of the biochemical changes which occur during the spoilage process. The possible role of b a c t e r i a l proteolysis has received much attention. Some workers (Jay and Kontou, 1967; and Lerke et a l . , 1967) believe that microbial pro-t e o l y s i s during meat spoilage i s i n s i g n i f i c a n t while others (Tarrant et a l . , 1971; Borton et a l . , 1970a and b; and Hasegawa et a l . , 1970a and b) have shown s i g n i f i c a n t proteolysis during 2 meat s p o i l a g e . In a l l of the papers reviewed during t h i s study, minced muscle had been used as the substrate f o r the inoculum. Mincing increases the surface area f o r r a p i d aerobic growth, thus maximizing any changes r e s u l t i n g from b a c t e r i a l growth. The object of t h i s work i s to determine the s t r u c t u r a l changes i n chicken p e e t o r a l i s muscle as i t undergoes spoilage due to growth of Pseudomonas f r a g i . The a l t e r a t i o n of proteins occurring as a r e s u l t of pro teolys is by P. f r a g i was also studied and attempts were made to r e l a t e these changes to s t r u c t u r a l changes observed by scanning and transmission electron microscopy. LITERATURE REVIEW 3 Meat Spoilage Microorganisms responsible for the spoilage of meat are either present p r i o r to slaughter or introduced as contaminants during processing, handling, packaging and storage. Although the f l o r a of fresh meat may consist of a large number of d i f -ferent bacteria genera, only a few of these genera predominate i n spoiled meat. Temperature i s the most important parameter influencing the f l o r a which develop on meat during storage. When meat undergoes spoilage at temperatures between 25 and 40°C the predominant genus i s Clostridium, which being an-erobic grow within the meat. Spoilage of meat at c h i l l e d temp-eratures i s normally the r e s u l t of aerobic psychrophilic species, which grow predominantly on the surfaces. Ayres (1960a) r e -ported that, a f t e r cutting up chicken, 75 to 80 percent of the colonies on the surfaces of the chicken parts were comprised of chromogenic bacteria, spore-forming microorganisms, molds and yeasts. When the chicken was allowed to s p o i l under r e f r i g -erated conditions, the predominant organisms were "primarily single, paired or short chained motile, Gram-negative, nonspore-forming rods some of which produce fluorescent pigments but most do not." I t has been well documented that spoilage of meat under c h i l l temperatures results from growth of bacteria of the Pseudomonas - Achromobacter types'(Ingram and Dainty, 1971; Barnes and Impey, 1968; Jay, 1967; Ayres, 1960a and b; and Ayres et a l . , 1950). Spoilage of meat at c h i l l e d temperatures i s s u p e r f i c i a l unless the meat has been processed i n such a manner ( e . g . mincing) as to d i s t r i b u t e the bacter ia throughout the t i s s u e mass. The f i r s t subjective i n d i c a t i o n of b a c t e r i a l spoilage i s an o f f - o d o r which Ayres et a l . (1950) described as " d i r t y d i s h r a g " ' . Ayres (1960b) reported the presence of an o f f -odor when the b a c t e r i a l population of bovine muscle had 7 2 reached 10 organisms/cm . V/hen the b a c t e r i a l numbers of the 8 2 muscle surfaces reach 10 organisms/cm , the meat takes on a slimy appearance. Ayres et a l . (1950) and Jay (1970) reported that the slimy appearance was due to the coalescence of the b a c t e r i a l co lonies . Jay (1970) also stated that sl iminess was due i n part to the "loosening of meat s t r u c t u r a l prote ins" as a r e s u l t of b a c t e r i a l growth. Ayres et a l . (1950) reported that a pungent ammoniacal odor developed a f t e r slime format-i o n . Aseptic Techniques Changes r e s u l t i n g from b a c t e r i a l decomposition are super-imposed on those r e s u l t i n g from a u t o l y s i s ; f o r t h i s reason i t i s necessary to store uninoculated controls along with i n o c u -lated- samples. In the past , research on biochemical changes i n meat due to b a c t e r i a l growth has been l i m i t e d due to the d i f f i c u l t y i n obtaining undenatured s t e r i l e t i ssue f o r the c o n t r o l . Various techniques have been employed by researchers to obtain germ-free t i s s u e . Hasegawa et a l . (1970a and b) used an a l c o h o l i c wash to obtain s t e r i l e muscle from p i g and r a b b i t . 5 Borton et a l . (1970a) employed t h i s technique i n an attempt to obtain s t e r i l e samples of porcine muscle; however, the samples were not s t e r i l e , although the b a c t e r i a l counts were 4 / seldom i n excess of 10 organisms/g a f t e r 20 days incubation at 1 0 ° C . Ockerman j|t a l . (1969) developed a s u r g i c a l i s o l a t o r technique to obtain s t e r i l e samples of beef muscle. Lea et a l . (1969) used aseptic technique to obtain samples of chicken breast muscle as well as t r e a t i n g the f l e s h with a 10 ppm chlorote t racyc l ine s o l u t i o n presumably to prevent b a c t e r i a l growth. Unfortunately, t h i s a n t i b i o t i c did not prevent b a c t e r i a l growth, but held the b a c t e r i a l load of the contro l below 10 organisms/g during storage. Jay and Eontou (1967) i r r a d i a t e d beef muscle at 1 Mrad to obtain a s t e r i l e c o n t r o l . These authors found t h i s dosage was s u f f i c i e n t to destroy a l l psychrophi l ic bac ter ia without i n h i b i t i n g the capacity of the meat to undergo t y p i c a l spoilage when inoculated with a mixed f l o r a . The i r r a d i a t i o n treatment d i d not cause any de-tectable a l t e r a t i o n i n samples of uninoculated muscle. Adamcic and Clark (1970) u t i l i z e d aseptic technique and a 0.5 Mrad dos-age to obtain s t e r i l e samples of chicken s k i n . Changes i n Muscle Proteins during Postmortem Storage Muscle proteins are general ly categorized into three c lasses ; sarcoplasmic, m y o f i b r i l l a r and stromal . The sarco-plasmic proteins consist of the g l y c o l y t i c enzymes and are extracted by means of water or d i l u t e s a l t s . Briskey and Pukazawa (1971) reported that the sarcoplasmic proteins occupy " a l l the spaces of the muscle c e l l not taken up by formed elements and the c o n t r a c t i l e system." The m y o f i b r i l l a r p r o -teins are involved i n contraction and are extracted with s a l t solutions of high ionic strength. Helander (1957) stated that the stroma f r a c t i o n consisted of c e l l walls, blood vessels nerves, endomysium and perimysium; these tissues consist of c o l lagen and e l a s t i n . The stroma proteins can be extracted from minced muscle with sodium hydroxide solutions. Khan (1962) estimated the amount (nitrogen basis) of the various protein fractions for the breast muscle of 4 month old b r o i l e r s to be's> stroma N 3.9 - 1.2 mg N/g muscle nonprotein N 6,0 - 0.3 iog N/g muscle m y o f i b r i l l a r E 17.0 - 0.6 mg N/g muscle sarcoplasmic TS 9.4 - 1.2 i g N/g muscle Postmortem changes occurring i n muscle proteins have been studied extensively i n attempts to understand the mechanism of postmortem tenderness. Increases i n free amino acids and non-protein nitrogen during postmortem storage have been shown i n bovine muscle K'Parrish et a l . , 1969; Davey and G i l b e r t , 1966; Gardner and Stewart, 1966; Sharp, 1963; Lawrie et a l . , 1961; and locker, I960), porcine muscle (Borton et a l . , 1970a; and Bowers, 1969), rabbit muscle (Suzuki et a l . , 1967) and poultry muscle (Lea et a l . , 1969; M i l l e r et a l . , 1965; and Khan and van den Berg, 1964a and b). The increase i n free amino acids and nonprotein nitrogen has been attributed to the enzymic action of naturally oc-curring cathepsins on the proteins; Mycek (1970) made a com-prehensive review on the i s o l a t i o n and properties of cath-epsins. Cathepsins A, B and C have a pH optimum ranging from 7 pH 5 to 6, while D and E have an optimum of 3.8 and 2.5 r e s -spectively. The sarcoplasmic proteins are thought to be the substrate f o r the catheptic enzymes. Bodwell and Pearson (1964) did not detect any hydrolysis of a c t i n , myosin or actomyosin by a p a r t i a l l y p u r i f i e d extract of bovine muscle, cathepsins. These authors found that the sarcoplasmic proteins were ex-tensively hydrolyzed by the cathepsins and suggested that the sarcoplasmic proteins i n s i t u were the substrates f o r these proteolytic enzymes. Their results are i n agreement with those obtained by Martins and Whitaker (1968) which indicated that cathepsin D was incapable of producing any detectable hydrolysis of actomyosin. Oaldwell and Grosjean (1971) studied the a c t i v i t y of cathepsins A, B, C and D on extracts from chicken sk e l e t a l muscle. Their r e s u l t s are i n agreement with those of lodiee et a l . (l966)which indicated that cath-epsins A, B, and C acted as a peptidase to further hydrolyze the breakdown products produced by cathepsin D, the major protease i n chicken muscle. Cathepsins are not as susceptible as bacteria to destruct-ion by i r r a d i a t i o n treatments. Doty and Wachter (1955) reported that an i r r a d i a t i o n dosage of 1.6 Mrad resulted i n approxim-a t e l y 50fo reduction i n catheptic a c t i v i t y . Similar r e s u l t s have been reported by Schweigert (1959) and Rhodes and Meegungwan (1962). Mohasseb (1962) concluded that a 1 Mrad dose resulted i n a threefold delay i n the breakdown of pro-teins to peptides and a tenfold delay i n the breakdown of the peptides of amino acids. Lawrie et a l . c. (1961) found that a 5 Mrad dose reduced the proteolytic a c t i v i t y i n bovine and porcine muscle by approximately 20 percent. These authors 8 concluded that although proteolytic a c t i v i t y was reduced to some extent, the i r r a d i a t i o n treatment did not lead to any s i g -n i f i c a n t differences i n proteolytic breakdown over long periods of storage. Numerous investigators have studied the changes occurring i n the s o l u b i l i t y of the various protein fractions during post-mortem aging of sk e l e t a l muscle. Khan and van den Berg (1964a) studied the changes i n protein e x t r a c t a b i l i t y from 4- and 8-month old b r o i l e r s stored at 0, 2 and 5°C during 7 weeks st o r -age. The authors observed that storage temperature had l i t t l e effect on t o t a l nitrogen e x t r a c t a b i l i t y of the breast muscle. The amount of nonprotein nitrogen and myosin fractions i n -creased while the sarcoplasmic f r a c t i o n decreased over the storage period. There was l i t t l e change i n the e x t r a c t a b i l i t y of actomyosin. The decrease i n the sarcoplasmic f r a c t i o n and accompanying increase i n nonprotein nitrogen suggested that proteolysis occurred during the storage. The decrease i n the e x t r a c t a b i l i t y of sarcoplasmic nitrogen (100 - 500 mg N/lOO g of muscle) could not be t o t a l l y accounted f o r by the increase i n nonprotein nitrogen (50 - 170 mg N/lOO g of muscle) or the loss of nitrogen i n drip (30 - 90 mg N/lOO g of muscle). The authors postulated that since the increase i n the myosin f r a c t -ion was not accompanied by a corresponding decrease of the acto-myosin f r a c t i o n , there may-be an interaction between myosin and the sarcoplasmic proteins. The r e s u l t s reported by Khan and van den Berg (1964a) are s i m i l i a r to those reported by other authors; Borton et. a l . (1970a) with porcine muscle, Ockerman et a l . (1969), Davey and Gilbert (1968) and Locker (i960) with 9 bovine muscle and Mcintosh (1967) with bovine, porcine and chicken muscles. A l l of these authors reported decreases i n the sarcoplasmic f r a c t i o n , increases i n nonprotein ni t rogen and s l i g h t increases , decreases or notchange i n the myofib-r i l l a r f r a c t i o n during low temperature storage of muscle. While considerable l i t e r a t u r e exis ts concerning changes i n prote in e x t r a c t a b i l i t y during low temperature storage, l i t t l e exis ts regarding changes occurring during high temper-ature storage (25 to 37°C). Sharp (1963) studied a u t o l y s i s of bovine and rabbi t muscle stored at 37°C. As i n the studies conducted at low temperatures, t h i s researcher found that the e x t r a c t a b i l i t y of the sarcoplasmic proteins decreased and non-prote in nitrogen increased. A f t e r 10 days of storage at 37°C, the nonprotein nitrogen increased from 10 to 18$ of the t o t a l extractable nitrogen i n bovine muscle and from 13 to 27$ f o r rabbit muscle. The sarcoplasmic proteins i n rabbi t muscle de-creased to between 2.5 and 4$ of the t o t a l ni trogen a f t e r 19 days storage at 3 7°C The rate of decrease was slower i n bovine samples. Unlike r e s u l t s obtained during low temper-ature storage, the s o l u b i l i t y of the m y o f i b r i l l a r proteins de-creased by approximately 26$ a f t e r 10 days storage. The author concluded that this 'decrease was the r e s u l t of heat denatur-a t i o n of the m y o f i b r i l l a r p r o t e i n s . Results obtained by Sharp (1963) are i n agreement with those of Lawrie et a l . (1961) . These authors also noted that the s o l u b i l i t y of the s t r u c t u r a l proteins was great ly reduced with high temperature storage of the meat. Neither Sharp (1963) nor lawrie et a l . (1961) found any evidence to suggest that the breakdown of c o l -10 lagen or e l a s t i n occurred during high temperature aseptic s t o r -age. Zender et a l . (1958) studied aseptic a u t o l y s i s of rabbi t and lamb muscle during storage at 25 and 3 7 ° C Formation of an exudate from the muscles, at both temperatures, was observed during the f i r s t day of storage and the volume of the ex-udate increased throughout the storage p e r i o d . The exudate contained 7% by weight of prote in and only a small amount of mater ial not p r e c i p i t a b l e by 10$ t r i c h l o r a c e t i c a c i d . The p r o -teins present i n the exudate were soluble i n solvents of low i o n i c strength or d i s t i l l e d water. The authors concluded that the sarcoplasmic proteins were present i n the exudate. Biochemical Changes Occurring i n Spoiled Muscle The s i g n i f i c a n c e of b a c t e r i a l proteolys is during meat spoilage has-been invest igated by a number of authors w i t h i n the l a s t 10 years . There i s disagreement as to the importance of b a c t e r i a l pro teolys is i n meat spoi lage . Ingram and Dainty (1971) reported that there was general agreement i n the l i t e r -ature that b a c t e r i a l p r o t e o l y s i s of meat only occurs at high population l e v e l s (10 organisms or greater/g) and that there i s l i t t l e evidence of s i g n i f i c a n t p r o t e o l y s i s before the product would be regarded as spoi led on an organoleptic b a s i s . 'tFay (1970) stated that "the precise r o l e s played by spoilage micro-organisms that resul t i n the spoilage of meats are not w e l l understood at t h i s t i m e . " Lerke et, a l . (1967) invest igated the r o l e of p r o t e i n i n the spoilage of f i s h . A nonpigmented Pseudomonas s p . , i s o l a t e d 11 from s p o i l i n g f i s h , was used as the inoculum. F i l t r a t i o n -s t e r i l i z e d press ju ice was used as a substrate . This mater ia l was- d iv ided into 3 l o t s ; a nonprotein f r a c t i o n , p r o t e i n f r a c t i o n and the unfractionated press j u i c e . The authors measured i n -creases i n v o l a t i l e reducing substances, t o t a l v o l a t i l e n i t -rogen and trimethylamine ni trogen as c r i t e r i a f o r spoi lage . The bacter ia grew wel l i n a l l 3 f r a c t i o n s , but only the u n -frac t ionated juice and the nonprotein ni trogen f r a c t i o n showed increases i n the chemical c r i t e r i a of spoi lage . The p r o t e i n f r a c t i o n d i d not give r i s e to spoilage products when inoculated with spoilage organisms. Jay (1966) stated that studies on prote in degradation by bacter ia during spoilage of bovine muscle indicated that the f l o r a of r e f r i g e r a t e d meats were incapable of such p r o -t e o l y t i c a c t i v i t y . Jay and Kontou (1967) studied the e f f e c t of b a c t e r i a l , growth on the free amino acids and nucleotides of bovine semimembranosus muscle stored at 7°C. When the ground muscle was allowed to s p o i l with i t s natura l f l o r a , no decrease was observed i n prote in l e v e l and there was no change i n amino ac id composition. Ground meat inoculated with a f luorescent Pseudomonas sp. had a decrease i n amino acids a f t e r 15 days incubat ion . Uninoculated aseptic samples showed increases i n concentration of amino acids , due to a u t o l y s i s , although the authors d i d not detect any prote in breakdown. Samples inoculated with a mixed f l o r a c o n s i s t i n g of a f luorescent Pseudomonas sp. and an Achromobacter sp. showed decreases i n amino a c i d composition, although not as extensive as the sample inoculated with the pure c u l t u r e . The fate of n u c l e -otides i n ground muscle during b a c t e r i a l spoilage was s i m i l a r to that observed f o r amino a c i d s . Spoilage by natural f l o r a resul ted i n a 15$ decrease; f luorescent Pseudomonas sp . 45$ decrease; mixed culture 43$ and Pseudomonas f r a g i 43$ decrease. The authors concluded that f ree amino acids and nucleotides are u t i l i z e d by the spoilage organisms before the proteins are attacked. Gardner and Stewart (1966) invest igated the change i n free amino acids occurring during b a c t e r i a l spoilage of bovine muscle at 15°C with i t s natura l f l o r a , which consisted p r e -dominantly of organisms belonging to the Pseudomonas-Achromobacter group. These researchers noted that the concen-t r a t i o n of most free amino acids -increased during storage of the muscle. Greatest increases were i n glutamic a c i d and t r y -ptophan while a decrease was observed i n glutamine. The authors concluded that the increase i n tryptophan was due to a u t o l y s i s , while the increase i n glutamic a c i d and decrease i n glutamine were due to b a c t e r i a l deamidation. The authors reported that the majori ty of bacter ia i s o l a t e d from spoi led meat possessed glutaminase. Increases i n ammonia were observed when the Q . b a c t e r i a l population reached 10 organisms/g and were a t t r i b u t e d to deamidation by the b a c t e r i a l enzymes. Unfortunately , the authors d i d not run aseptic controls and, therefore , they could not determine i f the increase i n f ree amino acids was a r e s u l t of b a c t e r i a l pro teolys is or of a u t o l y s i s . Adamcic and Clark (1970) studied induced chemical changes occurring i n chicken s k i n inoculated with a pigmented Pseudom— anas, non-pigmented Pseudomonas and Achromobacter s t ra ins and i n -cubated at 5 C. A l l 3 species of bacteria brought about a decrease in. t o t a l extractable nitrogen, phenol-reagent-positive and ninhydrin-positive materials and nonprotein nitrogen during logarithmic growth. After t h i s phase the values for the non-pigment ed Pseudomonas and Achromobacter species remained con-stant or slowly increased to the l e v e l s i n the control, while the values f o r the pigmented Pseudomonas species increased rapi d l y . The authors concluded that these organisms u t i l i z e d the low molecular weight nitrogenous compounds during l o g -arithmic growth then replaced them through proteolysis during the stationary growth phase. Adamcic et a l . (1970) studied the effect of the same 3 species of psychrotolerant bacteria on the amino acid content of chicken skin. Results indicated that the Achromobacter sp. and non-pigmented Pseudomonas sp. reduced the l e v e l of a l l free amino acids to below detectable l e v e l s during l o g growth phase. Leucine, phenylalanine, a l a -nine, aspartic acid and glutamic acid were p r e f e r e n t i a l l y attacked. These 2 species of bacteria did not release any free amino acids through proteolysis. On the other hand, during growth of the pigmented Pseudomonas sp. on chicken skin, the t o t a l gontent of free amino acids increased by approximately 30$. The concentration of free proline and hydroxyproline i n -creased 2 and 2.5 f o l d respectively, i n d i c a t i n g proteolysis of collagen by collagenase. The nonpigmented Pseudomonas sp. and the Achromobacter sp. grew as rapidly as the pigmented Pseudomonas sp. on chicken skin and produced the ch a r a c t e r i s t i c putrid off-odors of spoiled tissue, although these organisms were not as proteolytic as the pigmented Pseudomonas sp. The authors concluded that superior proteolytic a c t i v i t y had l i t t l e or no s i g n i f i c a n c e i n low temperature spoilage of poultry musc-l e . Lea et a l . (1969) invest igated changes i n free amino acids of chicken breast muscle r e s u l t i n g from growth of p s y c h r o p h i l i c bac ter ia at 1 ° C . In the uninoculated contro ls , the nonprotein nitrogen increased by approximately 10$ while the t o t a l f r e e amino acids increased by 40 to 70$. These r e s u l t s i n d i c a t e that considerable p r o t e o l y s i s had occurred a f t e r 8 days of i n -cubation. When samples were inoculated with Pseudomonas putrefaciens and a pigmented Pseudomonas sp . and the population 8 / xncreased to 10 organisms/g, the p r o t e o l y t i c changes r e s u l t i n g from t h e i r growth were s m a l l . A f t e r correc t ing f o r a u t o l y t i c changes, the nonprotein ni trogen decreased by an average of 1.0$ while the free amino acids decreased s l i g h t l y , i n d i c a t i n g u t i l i z a t i o n by the b a c t e r i a . Aspar t ic a c i d , threonine, ser ine , p r o l i n e and glycine were s e l e c t i v e l y u t i l i z e d by the pigmented Pseudomonas sp. while Pseudomonas putrefaciens s e l e c t i v e l y u t i l i z e d aspar t ic a c i d , threonine and s e r i n e . When the p i g -mented Pseudomonas sp . at tained a population l e v e l of 10^, large increases i n the concentration of a l l amino acids r e -s u l t e d . Such a large increase suggested "appreciable b a c t e r i a l Q . p r o t e o l y s i s with growth to 10 / g though none were found at 10 " . The authors d i d not detect any s i g n i f i c a n t increase i n the quantity of peptides produced as a r e s u l t of a u t o l y s i s or b a c t e r i a l growth. Ockerman et a l . (1969) invest igated the ef fec t of b a c t e r i a l spoilage on the prote in e x t r a c t a b i l i t y from bovine muscle. Samples were inoculated with a Pseudomonas s.P. and an Achromobacter sp. and incubated at 3 C Since the sarcoplasmic f r a c t i o n decreased i n both the inoculated and uninoculated samples, the authors concluded that autolysis rather than b a c t e r i a l proteolysis was responsible f o r the majority of the observed decrease i n s o l u b i l i t y . Mo s i g n i f i c a n t change was observed i n the s o l u b i l i t y of the m y o f i b r i l l a r proteins, but the s o l u b i l i t y of the stroma f r a c t i o n increased i n the control and decreased s i g n i f i c a n t l y i n the inoculated samples a f t e r 17.5 days incubation. The nonprotein nitrogen i n the inocu-lated, samples increased throughout the storage period but did not become s i g n i f i c a n t u n t i l the l a t e r stages of incubation (25 days). Rampton et a l . (1970) used sucrose density gradient cent-ri f u g a t i o n , gel f i l t r a t i o n and disc gel electrophoresis to study the effect of Achromobacter liquefaciens, Micrococcus  luteus. Pediococcus cerevisiae, Pseudomonas fluorescens. Streptococcus f a e c a l i s and natural f l o r a from commercial ham-burger on decomposition of m y o f i b r i l l a r proteins i n porcine and rabbit muscle. None of the cultures used i n the study had any measurable effect upon the m y o f i b r i l l a r proteins. A number of "non-protein u l t r a v i o l e t light-absorbing components" were present i n the density gradient centrifugation fractions of the Weber-Edsall extracts. The mixed f l o r a altered the peak heights of these components, but the authors did not character-i z e them. The authors concluded that the bacteria f i r s t ut-i l i z e d nonprotein nitrogenous compounds, then u t i l i z e d the simplest proteinaceous components of the muscle, especially the sarcoplasmic proteins. Hasegawa et a l . (1970a) studied the eff e c t of Pediococcus  cerevisiae. Leuconostoc mesenteroides. Micrococcus luteus and Pseudomonas f r a g i upon the sarcoplasmic and urea-soluble pro-t e i n fractions of procine and rabbit muscle. P. cerevisiae and L. mesenteroides represented the l a c t i c acid-producing organisms. M. luteus represented the s a l t tolerant micrococci and P. f r a g i was chosen to represent the psychrophilic pro-t e o l y t i c organisms. The pH of the control changed very l i t t l e over the storage period, while the pH of samples inoculated with the l a c t i c acid-producing organisms decreased and, i n those samples inoculated with P. f r a g i or M. luteus. increased. P. f r a g i caused extensive proteolysis of the sarcoplasmic proteins i n both rabbit and porcine tissues. 1. mesenteroides caused extensive a l t e r a t i o n of the sarcoplasmic f r a c t i o n of rabbit muscle but i t s action was les s extensive on porcine muscle. Growth of P. cerevisiae resulted i n proteolysis of the sarco-plasmic proteins from rabbit, but had no effect on porcine tissue. M. luteus showed minor proteolysis of the sarcoplasmic f r a c t i o n extracted from rabbit muscle and no action on porcine muscle. Both P. f r a g i and P. c e r i v i s i a e exhibited extensive proteolysis of the urea-soluble f r a c t i o n of porcine muscle and to a lesser extent on rabbit muscle. L. mesenteroides and M. luteus exhibited no detectable proteo l y t i c a c t i v i t y on the urea-soluble f r a c t i o n extracted from either porcine or rabbit tissue. The authors concluded that the proteolytic a c t i v i t y of spoilage bacteria was quite s p e c i f i c . They believed that the bacteria produce "highly s p e c i f i c enzymes which preferent-i a l l y act upon certain proteins or enzymes indigenous to the muscle". In another study, Hasegawa sj^sh. (l970:b) studied the ac t ion of Clost r idium perfr ingens , Salmonella e n t e r i t i d i s . Achromobacter l i q u e f a c i e n s . Streptococcus f a e c a l i s and Kurthea  zopf i on the urea-soluble and sarcoplasmic proteins extracted from r a b b i t and procine muscle . ' Spoilage due to growth of C. perfringens resul ted i n considerable p r o t e o l y s i s of both f r a c t -ions as judged by the a l t e r a t i o n s i n t h e i r g e l electrophoresis pat terns . None of the other organisms used i n the study showed any detectable i n d i c a t i o n s of p r o t e o l y t i c a c t i v i t y on e i ther the sarcoplasmic or urea-soluble f r a c t i o n s . Borton et a l . (1970a) invest igated the ef fec t of Pedio-coccus c e r e v i s i a e . Leuconostoc mesenteroides. Micrococcus  luteus and Pseudomonas f r a g i on the s o l u b i l i t y of various prote in f r a c t i o n s of porcine muscle. Samples were incubated f o r 20 days at 2 and 1 0 ° C . In the c o n t r o l , water-soluble n i t -rogen and insoluble prote in nitrogen decreased while non-prote in nitrogen increased over the storage p e r i o d . S a l t -soluble prote in ni trogen increased during the f i r s t 8 days of storage then remained constant or decreased s l i g h t l y . There was no a l t e r a t i o n i n prote in s o l u b i l i t y patterns i n samples inoculated with P. cerevis iae or M. luteus• Samples inoculated with L . mesenteroides and incubated at 2°C exhibited s o l u b i l i t y patterns s i m i l a r to the c o n t r o l s . The s o l u b i l i t y of the water soluble prote in was s i g n i f i c a n t l y lower i n samples incubated at 1 0 ° C , but there was no s i g n i f i c a n t di f ference i n nonprotein n i t r o g e n . There was no difference i n the e x t r a c t a b i l i t y of the s a l t - s o l u b l e prote in u n t i l a f te r 20 days storage, at which time a considerable decrease was noted i n the inoculated samples. The decrease was accompanied by an increase i n the insoluble protein nitrogen. The authors reported that l o s s i n s o l u b i l i t y of the salt-soluble protein was due to the de-crease i n pH r e s u l t i n g from growth of L. mesenteroides. Porcine muscle inoculated with P. f r a g i showed the great-est changes i n protein s o l u b i l i t i e s . The s o l u b i l i t y of the water-soluble protein decreased for the f i r s t 4 days at 10°C, then increased and was s i g n i f i c a n t l y higher than i n the con-t r o l . The salt-soluble protein decreased a f t e r 4 days incub-ation at 10°0. The increase i n the water-soluble f r a c t i o n was inversely correlated (P < .01) with the proteolysis of the salt-soluble f r a c t i o n (r = -.37). The insoluble protein f r a c t -ion tended to decrease throughout the storage period while the nonprotein nitrogen f r a c t i o n greatly increased. The increase i n nonprotein nitrogen indicated to the authors that extensive proteolysis occurred during spoilage of porcine muscle by P. f r a g i . Borton et a l . (1970b) inoculated porcine muscle with the same organisms used i n the previous study mentioned above (Borton et a l . , 1970a) to determine i f growth of these organ-isms altered the electrophoretic pattern of the salt-soluble proteins. The results obtained i n t h i s study confirmed those obtained i n t h e i r previous study that of the 4 organisms em-ployed, only P. f r a g i caused a decrease i n the number of electrophoretic bands of m y o f i b r i l l a r proteins. Tarrant et a l . (1971) studied the action of Pseudomonas  f r a g i on the proteins of porcine s k e l e t a l muscle incubated at 10°0. The results obtained i n t h e i r study were i n agree-ment with those of Borton et a l . (1970a). The meat was judged spoiled a f t e r 5 days of incubation at 10°C, but no s i g n i f i c a n t changes were observed i n protein s o l u b i l i t y u n t i l 20 days of incubation. After 20 days of incubation, the myofib-r i l l a r protein extract decreased by 2/3 of i t s o r i g i n a l value. Disc electrophoresis of the m y o f i b r i l l a r protein extract showed that the electrophoretic pattern was completely altered a f t e r 20 days spoilage. Nonprotein nitrogen increased approx-imately 3 times because of the increases i n peptides and ammonia. The pH increased from 5.4 to 7.9 attributable to the accumulation of ammonia. There was no s i g n i f i c a n t d i f -ference i n the s o l u b i l i t y of the sarcoplasmic proteins. The authors concluded that proteolysis of the sarcoplasmic pro-teins was not detected because of decrease i n the sarcoplasmic protein f r a c t i o n was offset by the release of water-soluble fractions r e s u l t i n g from proteolysis of the salt-soluble protein f r a c t i o n . Numerous authors (Jay and Kontou, 1967; Qckerman et a l . , 1969; l e a .et a l . , 1969; Adamcic and Clark, 1970; Borton et a l . , 1970a; Hasegawa et al.-, 1970a and Tarrant ejt a l . , 1971) have reported that the pH of muscle increases during spoilage un-le s s spoilage i s caused by an acid-producing organism (Hasegawa et a l . , 1970a). Ingram and Dainty (1971) reported that numerous proposals have been made to use the increase i n pH as an index of spoilage, but pH does not correlate well with the b a c t e r i a l load. Jay and Kontou (1967),. Ockerman et §1.(1969) and Adamcic and Clark (1970) reported decreases i n the extract release volume during b a c t e r i a l spoilage. Jay (1964)" f i r s t described the phenomena termed extract release volume (ERV). Homogenates of spoiled meat were more viscous than homogenates prepared from meat of good microbial quality. The more viscous a homogenate, the lower the ERV. The author reported that ERV was greatest between pH 5.0 and 5.8 and es s e n t i a l l y zero above pH 11 and below pH 4.9. Jay (1964) treated samples of bovine muscle with 2 commercial proteases and showed that the ERV declined as i n meat which underwent b a c t e r i a l spoilage. Jay (1966) presented 2 theories to explain the ERV phenomena. One theory i s that proteolysis results i n an unmasking of water binding s i t e s of the meat proteins, thus increasing the water holding capacity (WHC). The second theory i s that ex-tensive proteolysis of the proteins does not occur but the structure of the native protein i s "loosened". The l a t t e r theory has been expressed by Hamm (i960) i n h i s review on the water holding capacity of meat. Ingram and Dainty (1971) r e -ported that the true r e l a t i o n s h i p between ERV and WHC has not been determined. Hamm (i960) reported that the WHC of meat was d i s -tributed i n muscle f i b e r s as follows: 65$ due to s t r u c t u r a l proteins, 5$ due to water-soluble proteins and 30$ to water-soluble nonproteins. He also stated that not more than 5$ of the water i n muscle i s t i g h t l y bound to the proteins and the amount of bound water i s "hardly influenced by changes i n structure and changes of the proteins." The WHC of meat i s determined by the amount of free water which can be im-mobilized within the s p a t i a l structure of the proteins. Hamm (i960) refers to changes i n the MHO i n terms of a "loosening" or "tightening" of protein structure. The author reported that the WHO of meat was minimal around pH 5 and had two maxima; one at pH 10 and the other at pH 4 . As the pH of the muscle approaches the i s o e l e c t r i c point of actomyosin (approx-imately pH 5) the net charge on the protein i s decreased. Thus the e l e c t r o s t a t i c repulsion between adjacent peptide chains decreases and they can associate more closely with the r e s u l t that less water can be immobilized between them. As the pH i s decreased or increased from the i s o e l e c t r i c point, the e l e c t r o s t a t i c repulsion between the peptide chains i s increased thus loosening the protein structure r e s u l t i n g i n more im-mobilized water between the peptide chains. Ultrastructure of Striated Skeletal Muscle Excellent reviews on the ultrastructure of s t r i a t e d muscle include those of Briskey and Fukazawa (1971), Gauthier (1970), Peachey (1970), Bendall (1969), Slautterback (1966) and Lawrie (1966). Muscle i s surrounded by a sheath of connective tissue termed the epimysium, which binds the muscle bundles together to form muscle. Prom the epimysium f i n e r sheets of connective tissue, termed the perimysium, surround the muscle f i b r e s to form muscle bundles. The endomysium, which consists of yet f i n e r connective tissue, arises from the perimysium and sur-rounds each i n d i v i d u a l muscle f i b e r . The endomysium i s com-posed of three layers. Immediately adjacent to the f i b e r i s a double layered membrane approximately 100 A thick termed the plasmalemma or sarcolemma. The middle layer, approximately 500 A thick, i s composed of mucopolysaccharides. The outer layer consists of a network of collagenous f i b e r s . Bendall (1969) and Lawrie (1966) re f e r to thi s 3 layered sheath as the endomysial layer, while Schaller and Powrie (1971) and Kono et a l . (1964) c a l l i t the sarcolemma; a term which Bendall (1969) and Lawrie (1966) reserve f o r the inner plasmalemma. The muscle f i b e r i s a long multinucleated c e l l approx-imately 20 to 80 pm i n diameter and of variable length. The f i b e r consists of anywhere from 1000 to 2000 myofibrils, each 1 to 2 jjm i n diameter. The functional unit of the myo f i b r i l i s the sarcomere, which i s the distance between 2 adjacent Z l i n e s . In the relaxed state, the sarcomere i s between 2.5 and ~j> yen In length. The s t r i a t e d appearance of sk e l e t a l muscle r e s u l t s from the arrangement of a c t i n and myosin filaments within the sarcomere. The structure of the sarcomere i s represented diagramatically i n Figure I. The thi n a c t i n filaments, which a r i s e from the Z l i n e and run between the myosin filaments and some distance into the A Band, account f o r l i g h t I band. The denser A band results from an overlapping of the a c t i n and thicker myosin filaments. The l i g h t e r H zone, present i n the middle of the A band, consists solely of myosin filaments. Present i n the H zone i s a central dark region termed the M band, where the myosin filaments are joined t a i l to t a i l (Bendall, 1969) . The myofibrils are surrounded by a complex membrane The terminology of Bendall (1969) and Lawrie (1966) w i l l be followed i n thi s thesis. 23 1 Band A Band 1 Band 1 H Zone 1 . 1 Z Line M Band Myosin Filament Z Line Actin Filament P i g . 1. S t r u c t u r e of a Sarcomere 24 bound system of tubules and v e s i c l e s termed the sarcoplasmic reticulum. The sarcoplasmic reticulum consists of two parts; longitudinal tubules oriented p a r a l l e l to the f i b r i l s and transverse tubules. Peachey (1970) considers the sarcoplasmic reticulum to be two d i s t i n c t systems, but reports that other authors consider i t to be one. Peachey (1970) refers to the longitudinal tubules as the sarcoplasmic reticulum and treats the transverse tubules (or T system) as a separate system. The transverse tubules surround the circumferance of the m y o f i b r i l and are continuous from f i b r i l to f i b r i l transversely across the muscle f i b e r . The transverse tubule i s continuous with the saroolemma. Near the H zone, the longitudinal tubules are flattened forming a perforated sheet termed the fenestrated c o l l a r . On each side of t h i s c o l l a r , the longitudinal tubules lead into a terminal cisternum, often through intermediate flattened cisterna. The transverse tubule l i e s between two adjacent terminal cisterna; together these three elements form a s t r u c t -ure known as a t r i a d . Triads are located at the A-I band junction i n higher vertebrates and at the Z l i n e i n the lower vertebrates. Mendell (1971) studied the T system i n chicken peetoralis muscle. The triads were observed at the Z l i n e and the trans-verse tubules were observed to make " l a t e r a l turns or double back on themselves" rather than follow the uninterrupted trans-verse pattern usually observed i n s k e l e t a l muscle. Fukazawa et al.(1969) reported that the Z l i n e i n chicken p e c t o r a l i s muscle, f i x e d one hour post-mortem, had a zigzag conf igurat ion , but such a configurat ion was not evident i n muscle aged 24 hr at 5 ° C The authors reported that the m y o f i b r i l s tended to break at the Z-I junct ion when aged f o r 24 h r . Davey and G i l b e r t (1967) reported complete d i s -i n t e g r a t i o n of the Z band and apparent lengthening of the A band i n bovine muscle aged at 15°G for 3 days. Schal ler and Powrie (1971) used the scanning elec t ron microscope to- study s t r i a t e d s k e l e t a l muscle of beef, rainbow trout and turkey. F i b r i l s from trout and beef were surrounded by elevated transverse elements which the authors considered to consist of both the sarcoplasmic ret iculum and the T system. In p r e - r i g o r bovine muscle, the most prominent t r a n s -verse e levat ion , which the authors considered to be the t r a n s -verse tubule, had a smaller continuous r idge on e i ther side of i t . The authors considered these ridges to be the terminal c i s terna which, together with the prominent transverse r i d g e , form a t r i a d . In p r e - r i g o r turkey muscle, these secondary elevations could not be d i f f e r e n t i a t e d from the gross t r a n s -verse elements, presumably due to the extensiveness of the sarcoplasmic re t iculum. The endomysium appeared to consist of a network of w e l l defined strands. The surface of the endomysium showed pronounced transverse ridges which the authors believed to be the convex impression on the dneomysium of the underlying transverse elements on the m y o f i b r i l s . During the postmortem aging of turkey muscle, the transverse elements took on a rough appearance, i n d i c a t i n g that d i s r u p t i o n of the transverse elements was occurr ing . A f t e r aging f o r 6 days at 3 G, the transverse elements appeared to have c o l l a p s e d . The endomysium of 6 day postmortem turkey appeared perforated, i n d i c a t i n g t i ssue d i s i n t e g r a t i o n had taken place . Only one paper was found regarding changes i n the u l t r a -structure of s k e l e t a l muscle r e s u l t i n g from b a c t e r i a l spoi lage . Dutson et a l . (1971) studied the u l t r a s t r u c t u r a l changes occurring i n porcine muscle inoculated with Pseudomonas f r a g i . The samples were judged to be spoi led a f te r 8 days of incub-a t ion at 1 0 ° 0 . A f t e r an 8 day incubation per iod , the myo-f i b r i l s appeared disrupted i n the A band region and the H zone was almost devoid of m a t e r i a l . Myosin filaments were not evident and the dense material c h a r a c t e r i s t i c of the Z l i n e had been l o s t . The authors concluded that pro teolys is of the m y o f i b r i l l a r proteins occurred, with s p e c i f i c d i s r u p t i o n of myosin and the dense mater ial from the Z l i n e , as a r e s u l t of spoilage by P. f r a g i . Dutson et a l . (1971) observed protrusions , containing a dense granular mater ia l , on the surface of P. f r a g i growing on spoi led porcine muscle, Globules containing a dense mater ia l was observed adjacent to the b a c t e r i a . No protrusions were observed on the bacter ia when the organisms were grown on a nonprotein media. The authors postulated that p r o t e o l y t i c enzymes may be secreted into the protrusions , which then break away from the b a c t e r i a l c e l l forming the globules . These globules then release the enzymes in to the t i ssue surrounding the b a c t e r i a l c e l l . 27 Wiebe and Chapman (1968a and b) reported that some s t ra ins of Pseudomonas produced evaginations or blebs on the c e l l w a l l . In some s t r a i n s , bleb formation was a s table feature , while i n others, bleb formation occurred only under s p e c i f i c n u t r i t i o n a l and p h y s i o l o g i c a l condi t ions . The authors reported that c e r t a i n s t ra ins of marine Pseudomonas produced the c e l l u l a r evaginations when grown at high temperature ( 2 2 ° C ) i n a high peptane media (1.0$). The authors were u n -c e r t a i n as to the funct ion played by the observed evaginations. Smirnova et a l . (1971) s tudied, at the u l t r a s t r u e t u r a l l e v e l , toxin and enzyme secret ion i n Clost r idium perfr ingens . B a c i l l u s s u b t i l i s and B a c i l l u s l i c h e n i f o r m i s . The authors observed that an amorphous mater ia l protruded through channels which connected the periplasmic space of the c e l l with the external media. The amorphous mater ial formed "microcapsulae" on the c e l l surface . The material i n t h i s capsule was separated from both the cytoplasm and the external media. The mater ial was secreted into the media a f t e r p a r t i a l l y s i s of the c e l l w a l l above the microcapsules. The authors stained B. s u b t i l i s f o r a l k a l i n e phosphatase. The amorphous mater ial i n the microcap-sules contracted intensely with the c e l l , while i n s t ra ins which did not produce the enzyme the c e l l was contrasted uniformly . The authors concluded that formation of the microcapsules on the c e l l surface provides the s t r u c t u r a l basis f o r the secret ion of macromolecular compounds produced by the b a c t e r i a l c e l l . METHODS Part I - Biochemical Studies  Sample Preparation Commercial fryers were obtained immediately a f t e r slau-ghter from the packing plant and transported to the laboratory i n crushed i c e . The breast muscle was removed, s t i l l attached to the breastbone, and placed i n a p l a s t i c bag f o r storage at 3°C for 24 hr. Pectoralis major and minor were excised and ground i n an e l e c t r i c meat grinder. The minced muscle was then divided into 40 g l o t s and wrapped i n Saran f i l m . The wrapped samples were placed i n crushed i c e , then i r r a d i a t e d at 1 Mrad dosage i n a G-ammacell 220 (Atomic Energy of Canada Ltd.) containing Co 60. After irradiation,.samples were trans-ferred a s e p t i c a l l y into s t e r i l e 300-ml Erlenmeyer flasks f i t t e d with cotton plugs. A pure culture of Pseudomonas f r a g i (ATCC 4973) was diluted 200 f o l d with s t e r i l e phosphate buffer. 10 m i l l i l i t e r s of the di l u t e d culture were transferred into an Erlenmeyer f l a s k to give an i n i t i a l inoculum of approximately 10^ organisms/g. Contents of the flask were mixed to ensure even d i s t r i b u t i o n of the inoculum. Flasks were incubated at approximately 25°C for 0, 2 .5 , 5.5 and 9.5 days. Control samples were treated i n the i d e n t i c a l manner except 10 ml of s t e r i l e phosphate buffer was added to the Erlenmeyer f l a s k s . The addition of the buffer was necessary to prevent dehydration of the control samples. 29 B a c t e r i a l Counts B a c t e r i a l numbers were determined by g r i n d i n g a 1 g sample with sand i n a mortar and p e s t l e . The entire contents of the mortar were t ransferred to an 11-ml d i l u t i o n blank, then stand-ard plate counts were determined. Plate counts were determined on both inoculated and uninoculated samples. Plate count agar was used as the p l a t i n g medium and a l l plates were incubated at approximately 25°C f o r 48 h r . Prote in Ext rac t ion The procedures out l ined by Hasegawa et a l . (1970a) were adopted f o r the extract ion and separation of the water-soluble , s a l t - s o l u b l e and urea-soluble prote in f r a c t i o n s and are out-l i n e d i n Figure 2. A l l ex t rac t ion procedures were c a r r i e d out at 5 ° C Forty m i l l i l i t e r s of 0.3 M sucrose - 0.01 M K C l - 0.01 M T r i s buffer (pH 7.6) were added to 10 g of minced t issue i n a S o r v a l l Omni-Mixer. The sample was homogenized at top speed f o r 10 sec and at h a l f speed f o r 50 sec. The homogenate was centrifuged at 20,000 x G f o r 15 min i n a r e f r i g e r a t e d c e n t r i f u g e . The supernatant, which consisted of the sarcoplasmic prote in and nonprotein nitrogen f r a c t i o n , was decanted and stored at 5 ° C . The p r e c i p i t a t e was returned to the homogenizer and 60 ml of the sucrose - K C l - T r i s buffer added. The p r e c i p i t a t e was homogenized f o r 30 sec at one-quarter speed. The mixture was centrifuged at 20 ,000 x G f o r 15 min and the supernatant d i s -carded. The p r e c i p i t a t e was then homogenized with 60 ml of Weber-Edsall s o l u t i o n (0.6 M K C l , 0.04 M KHCO^, 0.1 M K ^ O ^ ) f o r 30 sec at one-quarter speed. The mixture was stored f o r 24 Muscle Sample homogenized with 4 v o l of 0.3 M sucrose, 0.01 M K G l , 0.01 M T r i s centrifuged 15 min at 20000 x G •Supernatant - Sarcoplasmic Protein and non-prote in ni t rogen P r e c i p i t a t e homogenized with 6 v o l of sucrose - KC1 - T r i s centrifuged "Supernatant - d i s c a r d P r e c i p i t a t e homogenized with 6 v o l of Weber-Edsall s o l u t i o n stored f o r 24 hr 18 v o l of Weber-Edsall s o l u t i o n added and centrifuged at 29000 x G f o r 30 min •Supernatant - m y o f i b r i l l a r prote in P r e c i p i t a t e homogenized with 6 v o l of Weber-Edsall s o l u t i o n and centrifuged at 29000 x G f o r 30 min Supernatant - d iscard P r e c i p i t a t e homogenized with 4 v o l 8 M urea and centrifuged at 29000 x G f o r 30 min •Supernatant urea soluble prote in P r e c i p i t a t e - discard P i g . 2. Plow Sheet of Protein Ext rac t ion Procedure hours at 5 ° C After storage, 180 ml of Weber-Edsall solution was added and the solution mixed with a magnetic s t i r r e r f o r 15 min. The mixture was then centrifuged f o r 30 min at 29,000 x G. The supernatant consisting of the salt-soluble f r a c t i o n was decanted off and stored at 5 ° C The pr e c i p i t a t e was homogenized with 60 ml of Weber-Edsall solution for 30 sec at one-quarter speed. The solution was centrifuged as before and the supernatant discarded. The precipitate was homogenized with 40 ml of 8M- urea at f u l l speed f o r 10 sec and half speed for 50 sec. The supernatant, containing the urea-soluble pro-t e i n f r a c t i o n , was retained and the precipitate discarded. Nitrogen Determination Nitrogen content of the water-soluble extract consisting of the sarcoplasmic protein and nonprotein nitrogen f r a c t i o n , salt-soluble extract consisting of the m y o f i b r i l l a r proteins and nonprotein nitrogen f r a c t i o n was determined by micro-Kjeldahl analysis. Nonprotein nitrogen was obtained by pre-c i p i t a t i o n of the water-soluble proteins with an equal volume of 20$ (W/V) t r i c h l o r o a c e t i c acid (Tarrant et a l . , 1971). Nitrogen was expressed as mg N/g fresh tissue. Gel F i l t r a t i o n Gel f i l t r a t i o n was carried out using Sephadex G-50 f i n e i n a column 2.5 cm i n diameter and 32 cm i n length. Packing of the Sephadex column was done according to i n s t r u c t i o n (Anon. 1966). The eluant consisted of deionized water containing 2$ sodium azide as a ba c t e r i o s t a t i c agent. Flow rate was 1.3 ml/ min. Fractions were collected by means of a drop counter y i e l d i n g f r a c t i o n s of 3 .2 - 0 . 1 m l . The v o i d volume (Vo) was determined with blue dextran. The e f f l u e n t volume (Ve) f o r glutamic ac id was determined by ninhydrin react ion of Moore and Ste in (1954). The water-soluble prote in f r a c t i o n was f i l t e r e d through Whatman Wo. 1 f i l t e r paper and s u f f i c i e n t sucrose added to the f i l t r a t e to give a 1$ s o l u t i o n . F i v e m i l l i l i t e r s of t h i s s o l -u t i o n were layered on top of the Sephadex bed. The e l u t i o n pattern was analysed using the ninhydrin r e a c t i o n of Moore and Ste in (1954). Disc Gel Electrophoresis Polyacylamide disc g e l electrophoresis was performed on the sarcoplasmic, m y o f i b r i l l a r and urea-soluble prote in f r a c t -i o n s . The procedure outl ined by Davis (1964) was followed to produce a 7$ running g e l with a 2.5$ spacer g e l . For e l e c t r o -phoresis of the m y o f i b r i l l a r and urea-soluble prote in f r a c t i o n s , s u f f i c i e n t urea was added to the gels to produce running and spacer gels containing 4 M urea . Buffer used f o r electrophoresis was 0.025 M T r i s - 0 . 1 9 M glycine pH 8 . 3 . A current of 3 ma per tube was applied and electrophoresis run at room temperature. One m i l l i l i t e r of 0.001$ bromophenol blue was added to the top b u f f e r r e s e r v o i r as a t racking dye. Electrophoresis was com-pleted when the t racking dye was approximately 0 . 5 cm from the end of the g e l . A f t e r completion of the electrophoret ic run, the gels were removed from the electrophoresis tubes with the a i d of a hypo-dermic syr inge . The gels were placed i n test tubes and stained f o r 15 min with a s o l u t i o n of 1.0$ Amido Black 10B i n 10$ acet ic a c i d . Gels were destained i n 7 $ acet ic a c i d u n t i l the background was c lear (approximately 3 days) . A Joyce Chrom-oscan was used to trace the electrophoret ic pat terns . An i n t e r -ference f i l t e r with a maximum transmittance at 580 nm was used. Results obtained during preliminary inves t igat ions i n -dicated that the best r e s o l u t i o n of the various f r a c t i o n s could be obtained as f o l l o w s . The sarcoplasmic f r a c t i o n was d i l u t e d 1:4 by volume with 40$ sucrose and the m y o f i b r i l l a r f r a c t i o n d i l u t e d 1:1 by volume with 8 M urea ; 0.1 ml of the d i l u t e d prote in s o l u t i o n was then layered on top of the spacer g e l . The prote in concentration of the urea-soluble f r a c t i o n was i n s u f f i c i e n t f o r d i r e c t a p p l i c a t i o n to the polyacrylamide g e l . The urea-soluble f r a c t i o n was concentrated 4 f o l d using an Amicon U l t r a f i l t r a t i o n c e l l having a membrane with an exclusion l i m i t of 30,000 molecular weight. A f t e r concentration, 0.1 ml of the concentrate was appled to the g e l . Free Amino Acid Analysis Sample preparation f o r f ree amino a c i d analysis was c a r -r i e d out according to the method out l ined by T a l l a n e_t a l . (1954). Ten grams of minced t issue were blended with 100 ml of 1$ p i c r i c ac id i n a Waring Blendor at top speed f o r 2 min. The mater ial was centrifuged at 5000 x G for 5 min. The super-natant was f i l t e r e d through Whatman No. 1 f i l t e r paper to r e -move f a t * The f i l t r a t e was then passed through a Dowex 2-X10 (chloride form) r e s i n bed to remove the p i c r i c a c i d . The r e s i n was packed 3-cm high i n a 2.5 by 20 cm chromatography column. Prior to use, the r e s i n was regenerated with 6-5 ml portions of IN HOI then washed with d i s t i l l e d water u n t i l the effluent was neutral. The f i l t r a t e was applied to the column and the f i r s t 25 ml were passed through the column and discarded. The next 50 ml of effluent were collected and dried at 55°C i n a rotary evaporator under vacuum. The residue was taken up i n 10 ml of pH 2.2 amino acid d i l u t i o n buffer and stored at 5°G u n t i l analysis. Amino acid analysis was performed on a Phoenix Model M 6800 amino acid analyser using the Moore-Stein two column system. Part II - Electron Microscopy Commercial chicken f r y e r s , obtained from a r e t a i l outlet, were used throughout the electron microscopy study. The f o l -lowing procedure was adopted to obtain s t e r i l e samples of the pectoralis muscle. The skin was removed and the surface of the breast muscle was flooded with absolute ethanol. The outer 0.5 cm of the muscle was removed and s t r i p s of muscle approximately 0.5 cm i n width and 3 to 5 cm i n length were removed from the i n t e r n a l portion of the pectoralis muscle. These s t r i p s were placed i n a s t e r i l e p e t r i plate and cut into cubes of approximately 0.5 cm. These cubes were then cryo-fractured by the following method. An aluminum weighing dish containing isopentane was floated i n l i q u i d nitrogen. When the isopentane had gelled, the dish was removed and a cube of muscle immersed i n the isopentane. After the tissue had frozen, i t was h i t with a p l e x i g l a s s rod, previously cooled i n l i q u i d ni t rogen, to cryofracture the m a t e r i a l . The r e s u l t i n g muscle fragments were no greater than 2 mm i n diameter. The frozen fragments were t ransferred onto s t e r i l e s t a i n l e s s s t e e l t i ssue cul ture gr ids within a s t e r i l e p e t r i p l a t e . The bottom of such p e t r i plate was covered with 3 MM chromatography paper. The cryofractured samples were inoculated , by means of an i n o c u l a t i n g loop, from a 3 day old culture of Pseudomonas  f r a g i (ATGO 4973) grown i n nutr ient b r o t h . The s t e r i l e con-t r o l s were inoculated with s t e r i l e phosphate b u f f e r . In order to prevent des icca t ion of the samples during incubat ion, 10 ml of s t e r i l e 0.24M sucrose was added to the p e t r i p l a t e s . The plates were incubated e i ther at room temperature (23 to 25°c) or i n a cold room (3 to 5 °C) . The plates were examined every 3 days to ensure that s u f f i c i e n t sucrose s o l u t i o n was present to prevent any dehydration of the samples. B a c t e r i a l Counts To assess the a s e p t i c technique, cryofractured samples were t ransferred in to tubes of nutr ient broth and incubated at approximately 25°C f o r 48 h r , then observed f o r growth. Absence of t u r b i d i t y a f t e r a 48-hr incubation period was i n -d i c a t i v e of a s t e r i l e technique. B a c t e r i a l numbers were determined only f o r the c r y o f r a c t -ured samples inoculated with P. f r a g i . A cryofractured sample was f i r s t weighed, then ground with sand i n a mortar with a p e s t l e . The contents of the mortar were t ransferred to a SS-mlk d i l u t i o n blank then standard plate counts were performed. Plate count agar was used as the pla t i n g medium, and a l l plates were incubated at approximately 25°C f o r 48 hr. Preparation of Samples f o r Scanning Electron Microscopy After incubation, the samples were transferred to 5-ml beakers and treated at about 25°C, as outlined below: 1) fixed i n 2.5$ glutaraldehyde i n 0.10 M phosphate buffer, pH for 1 hr 2) washed 3 times i n 0.24 M sucrose; 5 min f o r each wash 3) postfixed with 1.0$ osmium tetroxide i n 0.12 M sucrose for about 18 hr 4) washed 3 times i n 0.25 M sucrose; 5 min for each wash 5) dehydrated consecutively i n 50$, 75$, 95$ and absolute ethanol; 5 min i n each solution 6) 3 additional changes of absolute ethanol; 5 min f o r each 7) samples then dried i n a stream of nitrogen The dried samples were mounted on aluminum stubs with 'Dag1 dispersion and allowed to dry f o r approximately 0.5 nr. The specimens were then coated with gold i n a Mikros evap-orator. Specimens were examined with a Cambridge Stereoscan scanning electron microscope. Preparation of Samples for Transmission Electron Microscopy The double f i x a t i o n procedure outlined below was used for preparation of T.E.M. Samples from the cryofractured pieces. A l l procedures were carried out at about 25°C, unless stated otherwise; l ) fixed i n 2.5$ glutaraldehyde i n 0.10 M phosphate buffer, pH 7.0 f o r 1 hr 2) 2 washes i n 0.10 M phosphate buffer pH 7.0, 5 min each 3) postf ixed i n 1.0$ osmium tetroxide i n 0.10 M phosphate b u f f e r , pH 7.0 f o r 1 hr 4) 4 washes i n 0.10 M phosphate buffer pH 7.0; 5 min f o r each wash 5) dehydrated consecutively i n 30$, 50$, 70$, 85$, 95$ and absolute ethanol ; 5 min f o r each s o l u t i o n 6) 3 changes, 30 min each of absolute ethanol 7) propylene oxide i n f i l t r a t i o n 30 min f o r each step i ) 1 part propylene oxide to 3 parts ethanol i i ) 1 part propylene oxide to 1 part ethanol i i i ) 3 parts propylene oxide to 1 part ethanol 8) 2 changes of 100$ propylene oxide, 30 min f o r each change 9) Bpon 812 i n f i l t r a t i o n ; 30 min for each step i ) 1 part Epon to 3 parts propylene oxide i i ) 1 part Epon to 1 part propylene oxide i i i ) 3 parts Epon to 1 part propylene oxide 10) 2 changes, 30 min each, of 100$ Epon 11) specimen t ransferred to beam c e l l , then c e l l f i l l e d 2/3 f u l l with Epon 812 12) placed i n 40°C oven, approximately 12 hr 13) placed i n 60°C oven approximately 24 hr The specimen blocks were sectioned on a Sorval MT-2 ultramicrotome. Sections were picked up on carbon-coated c o l l o d i o n f i l m g r i d s . Sections were stained f o r 15 min i n a saturated s o l u t i o n of uranyl acetate i n 70$ methanol and 15 min i n Reynolds lead c i t r a t e (Reynolds, 1963). Grids were viewed with an AEI Corinth 275 transmission e lec t ron microscope. RESULTS AND DISCUSSION 39 Part I - Biochemical Studies Influence of growth of Pseudomonas f r a g i organisms on the pH  and water holding capacity of chicken muscle A l l control (uninoculated) and inoculated samples were i r r a d i a t e d at 1 Mrad l e v e l , which was s u f f i c i e n t to i n a c t i -vate n a t u r a l l y occurring microorganisms (Table I ) . I r r a d i a t i o n at t h i s dosage caused the formation of a s l i g h t pink pigment-a t ion of the chicken muscle. This r e s u l t i s i n agreement with the f indings of Whiting (1970), Hanson et a l . (1963) and Coleby et a l . (i960). Ten m i l l i l i t e r s of a d i l u t e d culture of P . f r a g i were added to each sample ($0 g) of muscle as an inoculum while 10 ml of s t e r i l e phosphate s o l u t i o n was added to each of the con-t r o l samples (40 g ) . V i s i b l e l i q u i d was present i n the f l a s k s containing the uninoculated c o n t r o l s ; whereas,, no v i s i b l e l i q u i d was observed i n the f l a s k s containing the inoculated samples a f t e r 2.5 days of incubat ion . The absence of free l i q u i d i n the f l a s k s containing the inoculated samples was due to the increased water-holding capacity presumably caused by the increase i n pH. Hamm (i960) reported that maximum water-holding capacity of meat occurred around pH 10 and was minimal around pH 5. The t o t a l numbers of P. f r a g i organisms i n inoculated samples at various incubation times are shown i n Table I . The contro l samples held i n a s t e r i l e environment had no bact -e r i a l growth over an incubation period of 9.5 days. A rapid T A B L E I Influence of incubation time at 25 C on the bacterial numbers and pH of control muscle .and muscle inoculated with P. fragi. Treatment of muscle a Incubation time in days Number of bacteria per g of muscle pH Control 0 0 5.9 2.5 0 5.3 5.5 0 5.2 9.5 0 5.3 Inoculated 0 3.02 x 10 6 5.9 2.5 1.05 x 10 1 G 6.95 5.5 3.98 x 10 9 7.9 9.5 3.47 x 10 8 8.5 24 nr postmortem muscle at zero time increase i n b a c t e r i a l numbers i n the inoculated samples waso observed during the f i r s t 2.5 days of incubation, followed by a decrease i n b a c t e r i a l population. The change i n pH of the control and inoculated muscle samples i s shown i n Table I. The i n i t i a l pH of the i r r a d i a t e d 24-hr postmortem muscle was 5.9. Similar results were ob-tained by several authors (Lea et a l . , 1969; Dodge and Peters, I960; and deFremery and Pool, I960). The pH of the uninoculated muscle decreased to 5 .3 a f t e r 2.5 days of incubation and re-mained at t h i s l e v e l f o r the 9.5-day incubation period. The pH of the inoculated samples increased appreciably over the 9.5-day incubation period to 8 . 5 . An increase i n pH during spoilage of muscle by P. f r a g i was also found by Tarrant et a l . (1971), Borton et a l . (1970a) and Hasegawa et a l . (1970a). Tarrant et a l . (1971) reported that the increase i n pH was due to the production of amines and ammonia. The uninoculated controls remained a l i g h t pink color f o r the duration of the incubation period. After 2.5 days of i n -cubation, the inoculated samples turned to a salmon pink, but aft e r 9.5 days the color became a yellowish pink. The inoculated samples attained a putrid odor aft e r 2.5 days of incubation. The putrid odors associated with s p o i l i n g meat are caused primarily by hydrogen s u l f i d e from sulfur-con-taining amino acids, ammonia from amino acids and related com-pounds and indole from tryptophan (Ingram and Dainty, 1971). No putrid or off-odor was noted with the incubated control samples. The surfaces of the inoculated samples had a slimy appear-ance a f t e r 2.5 days of incubat ion . Jay (1970) stated that the slimy appearance of spoi led meat i s due to coalescence of the surface b a c t e r i a l colonies and the "softening or loosening" of meat s t r u c t u r a l prote ins . Ayres et a l . (1950) reported that a detectable slime was observed when the l o g of the b a c t e r i a l numbers was between 8.0 and 9.0 per g on cut-up p o u l t r y . The contents of an incubation f l a s k were mixed with a spatula be-fore samples were removed f o r a n a l y s i s . The uninoculated t issue had a loose structure i n the respect that the extruded strands of muscle formed during the mincing step i n sample preparation did not adhere to each other. A f t e r 2.5 days of incubation the inoculated t issue formed a cohesive mass upon mixing. Jay (1970) reported that the slime l a y e r was p r i m a r i l y responsible f o r the tacky consistency of spoi led meat. The water-holding capacity of muscle homogenate was e v a l u -ated by centr i fuging samples at 20,000 x G and measuring the supernatant volume. With control samples, the volume was 39 - 1 ml ; whereas, the volume of the supernatant from i n o c u l -ated samples was 30 - 1 ml . The increased water-holding cap-a c i t y i n the inoculated meat i s i n agreement with the r e s u l t s of Adamcic and Clark (1970), Ockerman et a l . (1969) and Borton et. a l . (1968). Influence of growth of Pseudomonas f r a g i organisms on the  e x t r a c t a b i l i t y of proteins from chicken muscle Results of water-soluble pro te in , s a l t - s o l u b l e prote in and nonprotein nitrogen f r a c t i o n s extracted from control and i n -oculated muscle samples are-.' shown i n Figures 3, 4 and 5 10 c 8 <D O) O k. +J c 6 ;C <5 ** o »_ a 4 a) a> a u 3 </> 3 O E 2 CO 1 1 or E Control Inoculated 3 2 4 Incubation Time , Days 8 10 P i g . 3. Water-soluble prote in extracted from contro l and inoculated samples 14 12 10 8 Control L Inoculated r> 2 _ l _ 6 8 10 Incubation T ime, Days E i g . 4. S a l t - s o l u b l e prote in extracted from contro l and inoculated samples Control D • Inoculated • • -I 1 I i i _ 2 4 6 8 10 Incubation Time , Days Nonprotein n i t r o g e n compounds e x t r a c t e d from c o n t r o l and i n o c u l a t e d samples respectively. Summaries of analysis of variance and Duncan's New Multiple Range Test are presented i n Tables III and IV respectively. The amounts of water-soluble, salt-soluble and nonprotein nitrogen extracted from the control chicken muscle at zero time (Table II) were lower than those reported by Khan (1962). However, Khan (1962) indicated that the nitrogen content of the various protein fractions can vary from 10 to 2 5 $ due to differences i n breed, n u t r i t i o n and pre-and post-slaughter conditions. A s i g n i f i c a n t (P < .05) decrease was noted i n the extract-a b i l i t y of the water-soluble protein (Fig. 3) from the control samples during the 9.5 day storage period. As shown i n Table IV, there was no s i g n i f i c a n t (P > .05) change i n the extract-a b i l i t y of the salt-soluble protein (Fig. 4) from the control samples during the f i r s t 5.5 days of storage. A s i g n i f i c a n t (P < .05) decrease did occur aft e r 9 . 5 days of incubation. The s o l u b i l i t y of the nonprotein nitrogen f r a c t i o n ( Fig. 5) extracted from the control did not change (P > .05) s i g -n i f i c a n t l y during the storage period. Studies on changes i n protein e x t r a c t a b i l i t y during post-mortem aging at high temperatures (25 to 37°C) by numerous authors (Parrish et a l . , 1969; Suzuki et a l . , 1967; Sharp, 1963; Lawrie et a l . , 1961; Locker, I960; Zender et a l . , 1958) indicated that there were s i g n i f i c a n t decreases i n the extract-a b i l i t y of water-soluble protein and salt-soluble protein fractions while the changes i n non-protein nitrogen were some-TABLE I I Water-soluble protein, salt-soluble protein and nonprotein nitrogen extracted from control and inoculated chicken peetoralis muscle. Treatment Incubation time i n days Water-soluble protein . nitrogen Salt-soluble protein . nitrogen Non-protein , nitrogen Control 0 8.5 12.2 4.1 8.1 1 3 . 8 4.0 Control 2.5 5.7 11.9 3.9 4.9 7.3 3.5 Inoculated 2 - 5 7.1 9.6 3.6 7.1 9 . 8 3.3 Control 5.5 4.5 13.0 3.4 4.4 10.9 3.4 Inoculated 5.5 8.9 8.7 3.4 10.3 7.9 4 . 8 Control 9.5 3.1 7.2 2.5 2.9 6.9 2.6 Inoculated 9.5 5 . 8 4.7 2.9 6.7 5.0 3.5 a mg N/g of muscle b Results are the averages of duplicate ^determinations 48 TABLE I I I A n a l y s i s of variance of p r o t e i n e x t r a c t a b i l i t y from chicken p e e t o r a l i s muscle Source df M e a n sq. water soluble p r o t e i n n i t r o g e n M e a n sp. s a l t -s o l u b l e p r o t e i n n i t r o g e n m e a n sq. nonprotein n i t r o g e n Treatments 6 10.241 17.638 * 0.547 Zero C o n t r o l Vs. other 1 9.38 28.262 * 0.678 Time 2 5.97 27.094 0.789 Inoculated vs. Uninoculated 1 * 34.575 21.924 * 0.429 Time x 2.776 * I n o c u l a t i o n 2 0.727 0.3 F l a s k s / Treatment 7 0.269 0.549 0.197 Total:. 13 S i g n i f i c a n t at P = 0.05 49 TABLE M S o l u b i l i t y of the various p r o t e i n f r a c t i o n s from uninoculated muscle and muscle i n o c u l a t e d with P. f r a g i . Treatment Water-soluble p r o t e i n n i t r o g e n  S a l t - s o l u b l e p r o t e i n n i t r o g e n  Nonprotein n i t r o g e n C o n t r o l G time 8.287 Con t r o l 2.5 days 5.282 Inoculated 2.5 days 7.102 C o n t r o l 5.5 days 4.47 Inoculated 5.5 days 9.588 Co n t r o l 9.5 days 2.993 Inoculated 9.5 days 6.252 a ed ab d e f be 13.008 11.88 9.66 11.95 8.262 7.068 4.866 a a b a be c d 4.026 3.694 3.44 3.374 4.088 2.556 3.23 a a ab a b a b ab Treatment means i n the same column sharing the same s u p e r s c r i p t s are not s i g n i f i c a n t l y d i f f e r e n t at the 5fo l e v e l or p r o b a b i l i t y . 50 what variable. Results obtained by Suzuki et a l . (1967), Sharp (1963), Lawrie et a l . (1961) and Locker (i960) indicated that s i g n i f i c a n t increases i n nonprotein nitrogen occurred. Parrish et a l . (1969) noted a decrease i n nonprotein nitrogen i n bovine muscle stored for 24 hr at 37°C and suggested that aggre-gation of the m y o f i b r i l l a r proteins may have caused some binding of nonprotein nitrogen. Zender et a l . (1958) did not observe a s i g n i f i c a n t increase i n nonprotein nitrogen u n t i l a f t e r 31 days of storage at 25°C. The lack of any s i g n i f i c a n t increase i n the e x t r a c t a b i l i t y of nonprotein nitrogen from control samples indicates that autolysis of the sarcoplasmic proteins, which are the substrate for catheptic enzymes (Martins and Whitaker, 1968$ Bodwell and Pearson, 1964; Sharp, 1963) was not extensive. The absence of s i g n i f i c a n t autolysis may have been due to the i r r a d i a t i o n treatment. Mohasseb (1962), Schweigert (1959) and Doty and Wachter (1955) reported that i r r a d i a t i o n dosages of 1 to 1.6 Mrads reduced the proteolytic a c t i v i t y of muscle cathepins by approximately 50$. A white precipitate was f i r s t observed i n the bottom of fl a s k s containing uninoculated muscle a f t e r three days i n -cubation. Zender ejb a l . (1958) reported that a l i q u i d exudate, containing 7$ by weight water-soluble proteins, drips from the muscle during storage. Therefore, i t i s l i k e l y that the pre-c i p i t a t e observed i n t h i s study was insoluble sarcoplasmic pro-teins which were present i n the muscle exudate. This would account for the observed decrease i n the e x t r a c t a b i l i t y of the water-soluble protein without concomitant increase i n non-protein nitrogen. The e x t r a c t a b i l i t y of the water-soluble protein from sam-ples inoculated with P. f r a g i was not s i g n i f i c a n t l y (P > .05) diffe r e n t a f t e r 2.5 and 9.5 days incubation from the samples at 0 time. There was a s i g n i f i c a n t (P < .05) increase i n the l e v e l of extractable water-soluble protein a f t e r 5.5 days of incubation (Pig. 3). The e x t r a c t a b i l i t y of the water-soluble protein from the inoculated muscle was s i g n i f i c a n t l y (P < .05) higher than from the control at the various incubation times (Table IV). As shown i n Figure 4, the e x t r a c t a b i l i t y of the salt-soluble protein from the inoculated samples decreased throughout the incubation period. The e x t r a c t a b i l i t y of the salt-soluble proteins was s i g n i f i c a n t l y (P < .05) lower i n the inoculated samples compared to the respective control samples (Table IV). No s i g n i f i c a n t (P > .05) change was noted i n the e x t r a c t a b i l i t y of the nonprotein nitrogen fractions between the inoculated and control samples at the various incubation times. The s i g n i f i c a n t decrease i n the e x t r a c t a b i l i t y of the s a l t -soluble proteins from the inoculated samples, a f t e r 9.5 days incubation, suggested that proteolysis of the m y o f i b r i l l a r proteins occurred as a r e s u l t of the growth of P. f r a g i . This i s i n agreement with the findings of both Tarrant et a l . (1971) and Borton et a l . (1970a) using porcine muscle and the trans-mission electron micrographs of Buts.on et a l . (1971) which showed degradation of the f i b r i l structure as a result of spoilage of porcine muscle by P. f r a g i . Both Tarrant et a l . (1971) and Borton et a l . (1970a) re-ported s i g n i f i c a n t increases i n nonprotein! ni trogen during spoilage of porcine muscle by P. f r a g i . Tarrant ejb a l . (1971) reported that the increase consisted p r i m a r i l y of peptides and ammonia. No s i g n i f i c a n t increase i n nonprotein ni trogen was observed i n the present s tudy. The lack of any s i g n i f i c a n t increase i n nonprotein nitrogen l e v e l s i n the inoculated samples was unexpected. Numerous authors (Tarrant et a l . . 1971; Adamcic and Clark , 1970; Borton et a l . , 1970a; Ockerman et a l . , 1969; lerke et a l . , 1967) have reported increases i n nonprotein nitrogen l e v e l s during low temperature (between 2 and 10°C) spoilage of meat by various Pseudomonas species . Muscle incubated at high temperatures as used i n t h i s study should have greater chemical changes than those observed during low temperature spoi lage . Proteolysis of the sarcoplasmic and m y o f i b r i l l a r prote in f r a c t i o n s should r e s u l t i n formation of large p r o t e i n fragments which are further broken down i n t o polypeptides, peptides and eventually f r e e amino a c i d s . The i n a b i l i t y to detect such a s i g n i f i c a n t increase i n nonprotein nitrogen was due mainly to the large v a r i a t i o n between r e p l i -cates wi thin treatments, expecia l ly i n the inoculated samples, which was greater than the v a r i a t i o n between treatment means. G-el f i l t r a t i o n of water-soluble -proteins i n extracts from  control and inoculated muscle The e l u t i o n patterns of the water-soluble prote in extracts from uninoculated control and inoculated samples are shown i n Figures 6 and 7 r e s p e c t i v e l y . The area under the curves were measured using a planimeter. Ratios were determined f o r these areas r e l a t i v e to the area of the curves occurring at the void 53 F i g . 6. E l u t i o n patterns of water-soluble protein extracts from uninoculated cnicken peetoralis muscle A. zero incubation time B. 2.5 days incubation C. 5.5 days incubation D. 9«5 days incubation 70 = Void Volume VE = Effluent volume f o r glutamic a c i d 55 Pig. 7. Elution patterns of water-soluble protein extracts from chicken peetoralis muscle inoculated with P. f r a g i A. 2.5 days incubation B. 5.5 days incubation 0. 9.5 days incubation 70 = Void volume VE = Effluent volume f o r glutamic acid ABSORBANCE AT 5 70 nm. o P O o l • • • t • Oi 1 00 1 b r~ 00 volume (Vo) and. effluent volume (Ve) for glutamic acid i n the control at 0 time. The r a t i o of the area under the curves i s presented i n Table V. The elution pattern of the control at 0 time (Fig. 6A) was characterized by 2 peaks. The f i r s t peak, occurring at Vo consists of the sarcoplasmic proteins while the second peak, occurring very close to Vt, consists of free amino acids and small peptides. The color i n t e n s i t y of the ninhydrin re-action i s greater for free amino acids and peptides than for proteins. Therefore, the area under the 2 curves does not give the r a t i o of concentrations. The area under the sarcoplasmic peak (Vo) i n the uninoc-ulated samples decreased with increased incubation time. This observation concurs with the results obtained during the pro-t e i n e x t r a c t a b i l i t y studies. This decrease cannot be the :result of autolysis. Autolysis would r e s u l t i n the production of large protein fragments, due to the action of cathepsin D. These fragments would be hydrolyzed further to peptides by action of cathepsins A, B and 0 (Caldwell and Grosjean, 1971; P a r r i s h et a l . , 1969; Iodice et a l . , 1966). Production of large protein fragments and polypeptides would result i n a t a i l i n g e f f e c t of the sarcoplasmic peak towards the Vt position, The curve of the sarcoplasmic proteins extracted from the control samples (Pig. 6, A, B, C and D) did not change shape over the incub-ation £egi.od, although the area under the curve (Vo) decreased. This decrease was not due to autolysis but rather represents a decrease i n s o l u b i l i t y of the sarcoplasmic proteins. Zender et a l . (1958) reported large amounts of sarcoplasmic proteins .TABLE V i l a t i o of areas under the curves at Yo and Ye f o r glutamic acid Treatment Incubation time i n days Yo Sarcoplasmic Peak Ve Nonprotein Nitrogen peak Control 0 1 1 2.5 0.42 0.79 5.5 0.14 0.71 9.5 0.18 0.60 Inoculated 2«5 0.62 1.46 5.5 0.55 1.27 9.5 0.41 2.02 present i n the exudate produced during storage of muscle at high temperatures (25°G). In the present study, a p r e c i p i t a t e was observed i n the f l a s k s containing uninoculated muscle. This p r e c i p i t a t e may be insoluble sarcoplasmic prote in which was o r i g i n a l l y present i n the exudate. As shown i n Table V, the r a t i o of the area under the curve occurring at Ve f o r glutamic a c i d , epresenting nonprotein nitrogen i n the control samples, decreased slowly with i n -creased storage. In the inoculated samples ( F i g . 7) the curve at Vo, r e -presenting the sarcoplasmic prote ins , underwent two changes as opposed to the s ingle change occurring i n the c o n t r o l . As i n the c o n t r o l , the peak height decreased with time but, unl ike the c o n t r o l , the shape of the curve changed. As shown i n Table V, the area under the curve at Vo decreased with time. The decrease was not as great with the inoculated samples as that which occurred i n the contro l samples. This decrease does not agree with r e s u l t s obtained i n the prote in e x t r a c t a b i l i t y study, which indicated that the l e v e l of water-soluble prote in was s i g n i f i c a n t l y (P < .05) higher i n the inoculated samples a f te r 5.5 days incubation than a?ter 2.5 and 9.5 days. The reason for t h i s discrepancy must l i e i n the method for deter -mining nonprotein nitrogen as the water-soluble prote in was calculated from the d i f ference between t o t a l water-soluble nitrogen and MPN. B e l l (1963) d id a comparative study on methods f o r d e t e r -mination of NPN on blood serum, milk , bran and f l o u r . B e l l (1963) found that "no consistent differences or s i m i l a r i t i e s were observed r e l a t i n g method and results on one or a l l of the solutions". The-value obtained f o r NPN depended on the method used. Por example, determination of NPN i n blood serum by-d i a l y s i s indicated a value of 6.3$; whereas, p r e c i p i t a t i o n with t r i c h l o r o a c e t i c acid yielded a value of 2.7$, a difference of 52$. B e l l (1963) concluded that KPN must be defined by the method of preparation and that d i a l y s i s or gel f i l t r a t i o n achieves separation most closely r e l a t e d to molecular size alone. Prom the work of B e l l (1963) i t i s clear, that the NPN values obtained by t r i c h l o r o a c e t i c acid p r e c i p i t a t i o n during the protein s o l u b i l i t y study and the indication of NPN obtained by gel f i l t r a t i o n cannot be v a l i d l y compared. As the water-soluble protein was calculated during the protein e x t r a c t a b i l i t y study from the NPN, t h i s value may also be d i f f e r e n t from that indicated'by gel f i l t r a t i o n . The sarcoplasmic protein peak i n the control was sym-metrical; whereas, the curve from the inoculated samples became skewed to the right as.incubation time increased (Pig. 7B and 0). After 9.5 days incubation (Pig. 7C) nin-hydrin-positive material extended between the 2 curves o r i g i n -a l l y present at 0 time (Pig. 6A). There i s no i n d i c a t i o n of a s i m i l a r reaction i n the controls a f t e r 9.5 days incubation (Fig. 60). Presumably t h i s material consists of a mixture of protein fragments having a wide range of molecular weights f a l l i n g below the exclusion l i m i t of Sephadex G-50 and above thatsof amino acids. This material may have originated from two sources; breakdown of the sarcoplasmic proteins and releaser of water-soluble fragments from the m y o f i b r i l l a r p r o -te ins both as a r e s u l t of proteolys is of P. f r a g i . The curve near the t o t a l volume ( V t . ) , representing low molecular weight ninhydrin p o s i t i v e compounds i n the inoculated samples increased i n area and changed i n shape with time. As seen i n Figure 7 , the peak height remains r e l a t i v e l y constant. The increase i n area i s due to the change i n shape of the curve because of the presence of material with a lower e l u t i o n volume, therefore , higher molecular weight. . No attempt -wascmade to i d e n t i f y the compounds present i n t h i s peak but i t probably consists of small peptides which were produced during p r o t e o l y -s i s of the sarcoplasmic and m y o f i b r i l l a r proteins by P. f r a g i • S t a t i s t i c a l analysis was not performed on the areas of the curves. As indicated i n Table "V, the area under the curve near the Vt p o s i t i o n of the control samples decreased while the area under the curve of the inoculated samples increased. A f t e r 9 . 5 days of incubation, the area of the curve occurring near the Vt p o s i t i o n of the inoculated samples was 3 times as great as the corresponding area i n the e l u t i o n pattern of the c o n t r o l . I t appears that a s i g n i f i c a n t protein degradation does occur as a r e s u l t of growth of P. f r a g i . Disc ge l electrophoresis of water-soluble, s a l t - s o l u b l e and  urea-soluble proteins i n extracts from control and inoculated  muscles 1. Water-soluble Frac t ion Electrophorect ic gel patterns and densitometer tracings of the sarcoplasmic prote in extract from the uninoculated con-t r o l are shown i n Figures 14 and 8 respectively. As shown i n 62 F i g . 8. Densitometer t r a c i n g s of the sarcoplasmic p r o t e i n f r a c t i o n e x t r a c t e d from -uninoculated chicken p e e t o r a l i s muscle. A. zero i n c u b a t i o n time B. 5.5 days i n c u b a t i o n 0. 9.5 days i n c u b a t i o n 63 A . B TV 8 . / V 64 F i g . 9. Densitometer t racings of the sarcoplasmic prote in f r a c t i o n extracted from chicken peetora l i s muscle inoculated with P . f r a g i A . 2.5 days incubation B. 5.5 days incubation C. 9.5 days incubation 66 Figure 8A, 9 d i s t i n c t bands are apparent at 0 time (24 hr post-mortem) . The electrophoret ic m o b i l i t y of band 2 increased over the incubation p e r i o d . I n i t i a l l y band 2 appeared as a shoulder on band 1 ( F i g . 8A) but a f te r 5.5-days incubation t h i s shoulder formed a d i s t i n c t band of approximately the same i n t e n s i t y as band 1 ( F i g . 8B). As shown i n Figure 8G, the i n t e n s i t y of band 2 continued to increase u n t i l 9 .5 days of incubat ion . Bands 3 to 9 tended to decrease -in i n t e n s i t y over the i n c u b -at ion period r e f l e c t i n g a decrease i n e x t r a c t a b i l i t y . The electrophoret ic ge l patterns and densitometer t rac ings of the sarcoplasmic proteins extracted from the inoculated t issue are shown i n Figures 14 and 9 r e s p e c t i v e l y . A number of changes occurred i n the electrophoret ic pattern of the inoculated sample. A f t e r 2 .5 days of incubation, a new band, designated A, was observed ( F i g . 9A). This band may be a protein fragment r e s u l t i n g from p r o t e o l y s i s of the s a l t - s o l u b l e p r o t e i n s . As shown i n Figure 9B, band 2 disappeared from the pattern a f t e r 5.5-days incubat ion . The disappearance of band 2 coincided with increased i n t e n s i t y of band 3 ' This suggested that p r o -t e o l y s i s resul ted i n increased m o b i l i t y of band 2 with the r e -sul t that band 2 merged with band 3 forming a s ingle peak. The i n t e n s i t i e s of the other bands i n Figure 9B were lower than i n Figure 9A. The electrophoret ic m o b i l i t y of band 9 i n the uninoculated control increased a f t e r ' 5 . 5 days incubation ( F i g s . 8B and 14E); no such increase occurred i n the inoculated samples (F igs . 9B and 14F). Band 4 was missing from the e l e c t -rophoretic pattern of muscle incubated f o r 9.5 days ( F i g . 9C). There was no change i n number of bands present i n the electrophoret ic ge l pattern of the sarcoplasmic proteins ex-tracted from the control samples at various incubation times while samples inoculated and incubated with P. f r a g i l o s t 2 bands and gained 1 new band. Hasegawa et a l . (1970a) reported that growth of P. f r a g i caused extensive a l te ra t ions i n the starch ge l e lectrophoret ic pattern of the sarcoplasmic proteins extracted from rabbit and porcine muscle. The authors reported the loss of 70 to 80 percent of the bands i n the pattern due to proteolys is caused by f r a g i . Such extensive degradation of the sarcoplasmic proteins was not observed i n the present study. 2. S a l t - s o l u b l e Proteins The electrophoret ic ge l patterns of the s a l t - s o l u b l e p r o -t e i n extracts are presented i n Figure 15. Densitometer t racings of the gels from extracts from control and inoculated muscle samples are presented i n Figures 10 and 11 r e s p e c t i v e l y . Figure 10A shows the densitometer t r a c i n g of the gels f o r the m y o f i b r i l l a r proteins extracted from the uninoculated con-t r o l at 0 time. Fourteen d i s t i n c t bands were observed. The major bands i n the gels are bands 1 and, 8. Although no attempt was made to i d e n t i f y the bands, the r e l a t i v e m o b i l i t i e s of these 2 bands corresponds to the r e l a t i v e m o b i l i t y of actomyosin (band l ) and myosin (band 8) reported by Fisher (1963). Band 14 was absent from the pattern of control samples incubated for 2.5 days or longer . Apart from the l o s s of band 14, no further changes were observed i n the electrophoret ic ge l pat -tern of s a l t - s o l u b l e protein extracted from contro l samples a f t e r 2.5 days incubation ( F i g . 10B). Presumably the d i s -68 F i g . 10. Densitometer t r a c i n g s of the m y o f i b r i l l a r p r o t e i n f r a c t i o n e x t r a c t e d from u n i n o c u l a t e d chicken p e c t o r a l i s muscle. A. zero i n c u b a t i o n time B. 2.5 days i n c u b a t i o n 70 P i g . 11. Densitometer tracings of the m y o f i b r i l l a r prote in f r a c t i o n extracted from chicken p e c t o r a l i s muscle inoculated with P. f r a g i A. 2.5 days incubation B. 9.5 days incubation B appearance of band 14 was a resul t of i n s o l u b i l i z a t i o n . As shown i n Figure 11 A, the r e l a t i v e m o b i l i t y of bands 11 and 13 increased i n the electrophoret ic pattern of s a l t - s o l -uble proteins extracted from inoculated muscle a f t e r 2 .5-days incubat ion. The i n t e n s i t y and s ize of the major bands ( l and 8) a f t e r 9 .5-days incubation ( F i g . 11 B) was considerably lower than i n the control g e l f o r the same incubation time ( F i g . 10 B) The r e s u l t s indicate that proteolysis of the s a l t - s o l u b l e p r o -teins occurred as a r e s u l t of growth of P. f r a g i . Borton et a l . (1970b) and Tarrant et a l . (1971) reported the complete breakdown i n the electrophoretic pattern of the s a l t - s o l u b l e prote in f r a c t i o n extracted from porcine muscle, inoculated with P. f r a g i » a f t e r 20 days storage at 1 0 ° C . No evidence of such extensive proteolys is was observed i n the present study. 3 . Urea-soluble Proteins The urea-soluble f r a c t i o n contains those proteins which were insoluble i n water or s a l t so lut ions , but does not i n -clude the stroma prote ins . The electrophoret ic . .gel patterns from the urea-soluble prote in extracts of the control and inoculated t issue are presented i n Figure 16. The densitometer t racings of the gels are shown i n Figures 12 and 13 respec t ive ly As shown i n Figure 12A, 12 bands were present i n the con-t r o l gels at 0 time. There was no change i n the gel pattern a f t e r 2 .5 days of incubat ion . A f t e r 5.5 days of incubation ( F i g . 12B), 2 a d d i t i o n a l bands (A and B) of high m o b i l i t y 73 F i g . 12. Densitometer tracings of the urea-soluble prote in f r a c t i o n extracted from uninoculated chicken peetora l i s muscle A. zero incubation time B. 5.5 days incubation C. 9.5 days incubation 10 75 F i g . 13* Densitometer t racings of the urea-soluble prote in f r a c t i o n extracted from chicken p e c t o r a l i s muscle inoculated with P. f r a g i A . 2.5 days incubation B. 9.5 days incubation 78 F i g . 14. Electrophoretic patterns of sarcoplasmic proteins A. Zero incubation, control B. Zero incubation, inoculated P. f r a g i C. 2.5 days of incubation, uninoculated control D. 2.5 days of incubation, inoculated with P. f r a g i E. 5.5 days of incubation, uninoculated control P. 5.5 days of incubation, inoculated with P. f r a g i G-. 9.5 days of incubation, uninoculated control H. 9.5 days of incubation, inoculated with P. f r a g i 80 Pig. 15. Eleetrophoretic patterns of m y o f i b r i l l a r proteins A. Zero incubation, uninoculated control B. Zero incubation, inoculated with P. f r a g i C. 2.5 days of incubation, uninoculated control D. 2.5 days of incubation, inoculated P. f r a g i E. 5»5 days of incubation, uninoculated control F. 5.5 days of incubation, inoculated P. f r a g i G-. 9.5 days of incubation, uninoculated control H. *9.5 days of incubation, inoculated P. f r a g i 81 82 ,Pig. 16. Electrophoretic patterns of urea-soluble proteins A. Zero incubation, uninoculated control B. Zero incubation, inoculated P.. f r a g i c . 2.5 days of incubation, uninoculated control D. 2.5 days of incubation, inoculated with P. f r a g i E. 5.5 days of incubation, uninoculated control P.. 5.5 days of incubation, inoculated with P.' f r a g i G. 9.5 days of incubation, uninoculated control H. 9.5 days of ineubation, inoculated with P. f r a g i 83 84 appeared and the m o b i l i t y of band 2 increased. A f t e r 9.5 days of incubation ( F i g . 12G) 4.additional bands (C, D, E and F) were noted. The bands present at 0 time ( F i g . 12A) tended to become more d i s t i n c t and intense as incubation time increased. The major bands present i n the gel pattern at 0 time ( F i g . 12A) are 9, 10, 11 and 12. These bands correspond i n p o s i t i o n to bands 8, 10, 11 and 12 i n the ge l pattern of the s a l t - s o l u b l e ' proteins extracted from control samples at 0 time ( F i g . 10A). The g e l pattern from the extract of the inoculated sample, a f t e r 2.5 days of incubation ( F i g . 13A) was f a i n t . The major bands present i n the contro l (9, 10, 11 and 12) are evident and 2 a d d i t i o n a l bands (A and F) are also present. Uo difference i n the band pattern of the extracts from samples incubated f o r 5.5 days and 9.5 days was observed. The band pattern became more d i s t i n c t and the bands increased i n i n t e n s i t y , e s p e c i a l l y bands 9, 10, 11 and 12, as incubation time increased ( F i g . 13B). During the extract ion of s a l t - s o l u b l e proteins , a v i s -cous layer formed between the supernatant, which contained the s a l t - s o l u b l e proteins and the p r e c i p i t a t e , which contained the s a l t - i n s o l u b l e p r o t e i n s . Preliminary experiments indicated that t h i s viscous material consisted of s a l t - s o l u b l e proteins which were i n s o l u b i l i z e d during the incubation p e r i o d , lawrie et a l . (1961) and Sharp (1963) reported that the s o l u b i l i t y of the s a l t - s o l u b l e proteins was great ly reduced during high temp-erature (25 - 37°G) storage of meat. These authors concluded that t h i s decrease was the result of heat denaturation of the m y o f i b r i l l a r prote ins . This i n s o l u b i l i z a t i o n phenomena was f i r s t observed i n the control a f te r 2.5 days of incubat ion . Prote in i n s o l u b i l i z a t i o n was not observed i n the inoculated samples u n t i l 5.5 days of incubation and then i t was not as extensive as i n the c o n t r o l . The increase i n i n t e n s i t y of the bands i n the g e l pattern of the urea-soluble prote in ex-t rac ts coincides with the observed i n s o l u b i l i z a t i o n of the m y o f i b r i l l a r prote ins . This f a c t , coupled with the s i m i l a r i t y between the gel patterns of the s a l t - s o l u b l e and urea-soluble prote in extracts suggests that the i n s o l u b i l i z e d m y o f i b r i l l a r proteins comprise a major port ion of the urea-soluble extract . Hasegawa _et a l . (1970a) s tudied the- ef fec t of P. f r a g i on the urea-soluble proteins extracted from porcine and r a b b i t muscle. The authors reported that the i n t e n s i t y and s ize of most of the bands great ly decreased i n d i c a t i n g extensive pro-t e o l y s i s by P. f r a g i . The authors reported the f o l l o w i n g changes i n the gel pattern f o r porcine muscle; loss of 9 bands and appearance of 1 new band and f o r rabbit muscle, loss of 2 bands and appearance of 4 new bands. The work of the above authors would indicate that the urea-soluble f r a c t i o n was a wel l defined f r a c t i o n of the muscle proteins , with a consistant electrophoret ic gel pat tern . The resul t s of the present study indica te that the urea-soluble f r a c t i o n i s a heterogenous mix-ture, the composition of which depends on the treatment that the muscle has received. The i n t e n s i t y and s ize of the bands i n Figure 13A are great ly decreased i n comparison with the uninoculated contro l ( F i g . 12A) . This could be interpretated as evidence f o r ex-tensive proteolysis by P. f r a g i . However, a f t e r 9.5 days of incubation ( F i g . 13B) , the band pattern became more d i s t i n c t . I f the electrophoretic patterns were r e f l e c t i n g changes i n the urea-soluble proteins due to proteolysis by P. f r a g i , then the opposite should be true 3 the bands should decrease i n int e n s i t y and size with increased incubation time. In the present study, the urea-soluble f r a c t i o n r e f l e c t s the extent of i n s o l u b i l i z -ation of the salt-soluble proteins and not the effect of b a c t e r i a l proteolysis upon the proteins of the urea-soluble f r a c t i o n . Analysis of free amino acids i n control and inoculated muscles Results of the free amino acid analysis of the uninoculated and inoculated samples are presented i n Table VI. The values obtained f o r the control at 0 time, at which time the muscle was 24 hr postmortem, are sim i l a r to the results reported by l e a et a l . (1969) and M i l l e r et a l . (1965) f o r the free amino acid analysis on the breast muscle of b r o i l e r s . In the present study, separation of l y s i n e and h i s t i d i n e was not obtained, therefore, these 2 amino acids have been reported as a combined l y s i n e - h i s t i d i n e l e v e l . The concentration of l y s i n e and h i s t -idine was similar to that reported by M i l l e r et a l . (1965) but was 30 times the concentration reported by Lea et a l . (1969). Lea et a l . (1969) stated that two dipeptides, anserine and carnosine, are present i n large concentrations i n chicken breast muscle (52.8 |jmoles/g). These two dipeptides tend to be eluted from the ion exchange column with ly s i n e and h i s t i d i n e , thus i n t e r f e r r i n g with the separation of these amino acids. F a i l u r e to separate these two dipeptides undoubtedly lead to the high values obtained for l y s i n e - h i s t i d i n e i n the present study. Lea et a l . (1969) found that the t o t a l free amino acid 'TABLE VI Average f ree amino a c i d concentration of inoculated and uninoculated chicken peetoral is muscle incubated at 25°C Control Inoculated  Incubation time i n days Free Amino Acid 0 2.5 5. 5 9.5 2.5 5.5 9.5 Lysine ''—y H i s t i d i n e ' 28.0 22.35 22. 65 22.5 26 9.48 4.66 - - - - - - 3*55 Ammonia 7.33 8.1 9 . 01 8.98 29.5 113.7 112.85 Arginine 0.62 0.79 1. 35 1.46 - - -Aspart ic ac id 0.44 1.45 2. 61 2.77 1.10 1.93 1.31 Threonine 0.89 2.16 3 . 38 5.18 0.99 1.74 0.31 Serine 0.94 2.02 3 . 28 3.23 0.72' 0.45 0.24 Glutamic ac id 0.98 2.79 4. 89 4.34 2.51 3.19 3.59 Prol ine 0.45 0.88 l . 45 1.96 0.51 0.30 0.16 Glycine 0.86 1.87 3 . 02 2.76 1.65 8.74 16.38 Alanine 0.99 2.38 3 . 84 4.01 1.76 1.52 1.29 Half Cystine 0.11 0.14 0 . 18 0 .18 0 .09 0.36 0.53 Valine 0 .61 1.67 2. 59 2.94 1.95 4.17 3.05 Methione 0.39 0.63 1. 11 1.06 1.10 2.06 2.19 Isoleucine 0*26 0 .81 1. 45 1.41 1.11 1.86 0.87 Leucine 0.48 0.83 2. 34 2.41 1.84 3.39 1.64 Tyrosine 0.29 0.63 1. 10 1.12 0.57 0.55 0.17 Phenylalanine 0.21 0.63 1. 12 1.11 0.58 0.48 0.32 cC - Aminobutyric Acid 0.11 0.26 0 . 18 0.16 0.11 3.56 2.59 \\ mole/g of muscle 88 concentration i n the s t e r i l e control increased "by 70$ a f t e r 8 days of incubation at 1°G. Increases were noted f o r a l l of the amino acids except c y s t i n e . As shown i n Table V I , the free amino acids i n the control increased on the average by a fac tor of 4 a f t e r 9-5 days of incubation at 25°0. The b a c t e r i a l load i n the inoculated samples was greater 9 / than 10 organisms/g f o r a l l of the samples. The changes r e -s u l t i n g from the growth of P. f r a g i are superimposed on those r e s u l t i n g from a u t o l y s i s ; therefore i t i s necessary to subtract the increase due to a u t o l y s i s i n order to i s o l a t e the ef fec t due to b a c t e r i a l p r o t e o l y s i s (Lea et a l . , 1969) . When the r e s u l t s from the inoculated samples were corrected for auto-l y s i s (Table V I I ) , the fo l lowing r e s u l t s were found. A f t e r 9.5 days of incubation, there were large increases i n l e v e l s of ammonia, g lycine and oc -aminobutyric a c i d . Smaller increases occurred i n hal f cyst ine , methionine and v a l i n e . While argihine increased i n the c o n t r o l , none was present i n the inoculated samples. Large decreases were observed f o r threonine, ser ine , prol ine and tyros ine . Smaller decreases were noted i n aspart ic a c i d , glutamic a c i d , a lanine , i s o l e u c i n e , leu.cine and phenylala -n i n e . Separation of l y s i n e and h i s t i d i n e was achieved i n the inoculated sample a f t e r 9.5 days incubat ion . This was the only time i n which such separation occurred. No separation of these two peaks occurred i n the control samples. I t would appear that pro teolys is of the two dipept ides , anserine and carnosine, occurred, thus e l iminat ing t h e i r interference i n the separation of the l y s i n e - h i s t i d i n e peak (Lea et a l . , 1969). TABLE VII Average free amino ac id concentration ' of chicken p e c t o r a l i s muscle inoculated with P. f r a g i and corrected f o r a u t o l y t i c changes. Pree Amino A c i d 2.5 Incubation time 5.5 i n days 9.5 Lysine - — — H i s t i d i n e - - j Ammonia +21.4 +104.69 i +103.87 Arginine - 0.79 - 1.35 1.46 Aspart ic A c i d - 0.35 - 0.68 - 1.46 Threonine - 1 .17 - 1.64 - 4.87 Serine - 1.3 - 2.83 - 2.99 Glutamic A c i d - 0 .28 - 1.7 0.85 Prol ine - 0.37 - 1.15 - 1.8 Glycine - 0.22 + 5.72 + 13.62 Alanine - 0.62 . - 2.32 2.72 Half Cystine - 0.05 + 0.12 + 0.35 Valine + 0 .28 + 1.58 + 0.11 Methionine + 0.47 + 0.95 + 1.13 Isoleucine + 0.3 + 0 .41 - 0.53 Leucine + 1.01 + 1.05 - 0.77 Tyrosine - 0 .06 - 0.55 - 0.95 Phenylalanine - 0.05 - 0.64 - 0.79 oc-Aminobutyric Acid - 0.11 + 3.38 + 2.43 a concentration p moles/gram When the values from the inoculated samples a f t e r 9.5 days of incubation are compared with the i n i t i a l values (0 time) from the uninoculated c o n t r o l , a rg inine , threonine, ser ine , prol ine and tyrosine were the only amino acids which decreased from the i n i t i a l values , i n d i c a t i n g s e l e c t i v e u t i l i z a t i o n of these amino acids by P. f r a g i . T o t a l concentration of amino acids a f t e r 9*5 days of i n -cubation was twice as great i n the inoculated muscle as i n the c o n t r o l . The increase was due mainly to the increase i n ammonia and g l y c i n e . The large increase i n ammonia was due to the deamination a c t i v i t y of the b a c t e r i a . S imi lar increases have been reported by Tarrant et a l . (1971) and Gardner and Stewart ( 1 9 6 6 ) . The large amount of ammonia produced accounts f o r the high pH observed i n the spoi led muscle. The complete disappearance of arginine from the inoculated samples was l i k e l y due to the arginase a c t i v i t y of P. f r a g i , which converts arginine into ornithine and urea. Thornley ( i 9 6 0 ) reported that t h i s reac t ion was t y p i c a l of pseudomonads. A l -though the presence of arginase a c t i v i t y would account f o r the disappearance of a rg inine , no trace of ornithine was detected. It may be that the ornithine was u t i l i z e d by P. f r a g i or that i t combined with another amino acid peak as the amino ac id analyzer used i n t h i s study was not equipped with a column f o r analysis of p h y s i o l o g i c a l f l u i d s , but rather was set up f o r prote in hydrolysates . Gardner and Stewart (1966) allowed ground beef to undergo spoilage with i t s natura l f l o r a , which consisted predominately of bacteria belonging to the Pseudomonas-Achrobacter group. These authors found that the concentration of free amino acids increased, with the greatest increase being i n glutamic acid. Cystine and c>c -aminobutyric acid were found only i n the spoiled meat, which supports the findings of t h i s study that the i n -crease i n these two amino acids was due to b a c t e r i a l proteoly-s i s . Lea et a l . (1969) showed that 30$ of the free amino acids were u t i l i z e d by a pigmented Pseudomonas species when the b a c t e r i a l load was below 7 x 3.0 organisms/g of muscle. Changes i n aspartic acid, threonine, serine, proline and glycine ac-counted for 86$ of the observed decrease. Except for glycine, a l l other amino acids reported by Lea et a l . (1969) as being used s e l e c t i v e l y by Pseudomonas organisms were at lower concentration i n the inoculated sample, compared to the con-t r o l i n the present study. Adamcic et a l . (1970) studied the growth of a pigmented Pseudomonas species on chicken skin. During the log phase of growth the bacteria increased the concentration of most free amino acids, producing a net increase of 30$. Their results indicated that serine and hydroxyproline decreased by approx-imately 50$, and there was a s l i g h t decrease i n glycine and proline. At the end of the logrithmic growth phase, there was an increase i n the concentration of free hydroxyproline and proline i n d i c a t i n g that the organism produced a collagenase which attacked the collagen of the chicken skin. According to Veis (1970), proline and hydroxyproline comprise approximately 2 5$ of the amino acid residues i n c o l -lagen, while glycine comprises nearly 3 3 $ * Glycine i s the f i r s t member of the t r i p l e t of residues , which r e s u l t s i n g l y -cine being at every t h i r d residue p o s i t i o n throtighout most of the polypeptide chain . As shown i n Table V I I , the p r o l i n e con-centrat ion decreased with increased incubation time and there was no trace of hydroxyproline. I t i s possible that any hydrox-p r o l i n e or p r o l i n e released by p r o t e o l y t i c ac t ion was metabol-i z e d by P. f r a g i . therefore, no increase was detected. However, the large increase i n g l y c i n e , as shown i n Table V I I , may re f l e e t pro teolys is of the endomysium collagen network. Part II - E lec t ron Microscopy Scanning electron microscopy of Pseudomonas f r a g i 1 . Growth of P. f r a g i on nutr ient agar and nutr ient g e l a t i n The a b i l i t y of bacter ia to l i q u i f y g e l a t i n i s one of the tests used to determine the presence of p r o t e o l y t i c a c t i v i t y . The surface of nutr ient agar and nutr ient g e l a t i n was i n o c u l -ated by P. f r a g i so that the growth of P . f r a g i could be ob-served on a nonprotein and prote in substrate r e s p e c t i v e l y . The surface of uninoculated nutr ient agar, shown i n Figure 17, i s quite i r r e g u l a r . Growth of P . f r a g i upon nutr ient agar, a f t e r 24 hr incubat ion, i s shown i n Figure 18. The bacter ia do not appear to disrupt the surface of the agar nor i s there any i n d i c a t i o n of an e x t r a c e l l u l a r exudate. Many of the bacter ia are undergoing c e l l d i v i s i o n (A i n F i g . 18). Although P. f r a g i has polar f l a g e l l u m , none are v i s i b l e i n any F i g . 17. Scanning electron micrograph of the surface of nutrient agar, uninoculated and incubated at 25 C f o r 24 hr (x 22,000). Scanning e lec t ron micrograph of the surface of nutr ient agar, inoculated with P. f r a g i incubated 24 hr at 25 0 (x 11,400)7 of the scanning electron micrographs due to the low r e s o l u t i o n of the instrument (250 £ maximum) compared to the high r e s o l u t -i o n obtainable with a-transmission electron microscope. Growth of P. f r a g i on the surface of nutr ient g e l a t i n a f t e r 24 hr incubat ion, i s shown i n Figure 19. As the ex t ra -c e l l u l a r proteases produced by the bacter ia l i q u i f y the g e l a t i n , the s t r u c t u r a l support f o r the bacter ia i s l o s t and they pene-trate in to the g e l a t i n as .shown i n Figure 19. As shown at A i n Figure 20, a f i l m of amorphous material i s present between the b a c t e r i a . Strands of mater ia l extend between bacter ia (B i n F i g . 20) and between the bacter ia and the surface of the nutr ient g e l a t i n (C i n F i g . 20). There was no evidence of s im-i l a r i n t e r c e l l u l a r mater ial when the organism was grown on nutr ient agar ( F i g . 18). Poss ibly t h i s i n t e r c e l l u l a r mater ial (A i n F i g . 20) was l i q u i f i e d g e l a t i n which was f i x e d during specimen preparat ion. The filamentous mater ial (B and C i n F i g . 20) probably resul ted from shrinkage of the f i x e d l i q u i -f i e d g e l a t i n f i l m (A i n F i g . 20) during the dehydration steps of specimen preparat ion. 2. Growth of P. f r a g i on peetora l i s major muscle The r e s u l t s of the plate counts on the inoculated muscle samples incubated at 25°C are presented i n Table V I I I . There was no growth i n the uninoculated c o n t r o l s . A high i n i t i a l inoculum was used i n order that a r e l a t i v e l y large microbia l population would be at tained i n a short time p e r i o d . This was done to minimize the e f f e c t of an increas ing b a c t e r i a l load from that due to length of incubat ion. The b a c t e r i a l load was Scanning electron micrograph of the surface of nutr ient g e l a t i n inoculated with P. f r a g i incubated f o r 24 hr at 25 0 (x 1 , 1 0 0 ) . F i g . 20. Scanning elec t ron micrograph of the surface of nutr ient g e l a t i n inoculated with P. f r a g i and incubated f o r 24 hr at 25 C (x 10, COO). 98 TABLE VIII B a c t e r i a l population i n chicken peetora l i s major inoculated with P. f r a g i incubated at 25 0 Incubation time i n days Number of bac ter ia per g of muscle 0 1.98 x 10 7 1 2.05 x l O 1 0 2 2.8 x 1 0 1 0 4 1.15 x l O n 6 5.26 x 1 0 1 0 8 2.36 x 1 0 1 0 11 ~ — , , 4.56 x l O 1 0 a average of duplicate samples i n the order of 10"""^  organisms per g of muscle f o r the ent ire incubation p e r i o d . A p u t r i d odor was detected on a l l of the inoculated cryofractured samples af ter incubat ion. Kono et a l . (1964) reported that sarcolemma or endomysium was composed of three layers c o n s i s t i n g of an outer network of entangled collagen f i b e r s , a middle amorphous l a y e r and an inner plasma membrane. As reported by S c h a l l e r and Powrie (1971) such u l t r a s t r u c t u r a l d e t a i l cannot be resolved by the scanning electron microscope. However, at 0 time (approximately 24 hr postmortem) the endomysium appeared to have a c r i s s - c r o s s structure (P ig . 21) . Presumably t h i s was the entangled network structure of collagen f i b e r s reported by Kono et a l . ()1964). In Figure 21, the arrows point to t rans -verse ridges on the endomysium. Schal ler and Powrie (1971) concluded that these ridges were the convex impression, on the endomysium, of the underlying transverse elements. Elevated transverse elements run from one f i b r i l to another (arrows i n P i g . 22). Presumably these elevated transverse elements con-s i s t of the transverse tubule and two terminal cisternae which form a t r i a d . According to Mendell (1971) the t r i a d s i n chicken peetora l i s muscle overlay the Z disc of the m y o f i b r i l s . The surface of a f i b r i l from inoculated muscle, a f t e r 1 day of incubation, i s shown i n Figure 23. P ro teolys is i s not extensive, however, the arrows indicate areas where the bacter ia have begun to penetrate the endomysium. As shown i n the micro-graph, the surface of the endomysium has a granular appearance rather than the c r i s s - c r o s s strand network apparent at 0 time. The granular appearance may be due to proteolys is of the c o l -F i g . 21 Longi tudinal view of in tac t endomysium i n cryofractured peetoral is muscle, zero time (x 11,400). 101 F i g . 22. Longi tudinal view of exposed m y o f i b r i l s i n uninoculated chicken peetora l i s muscle at zero time (x 22,200). L o n g i t u d i n a l view of endomysium from c r y o f r a c t u r e d p e e t o r a l i s muscle i n o c u l a t e d with P. f r a g i and incubated 24 h r a t 25 0 (x 9,5007: lagen f i b e r s , which comprise the outer layer of the endomysium. In Figure 24, the bac ter ia are present on exposed f i b r i l s . The exposed f i b r i l s show no i n d i c a t i o n of p r o t e o l y s i s . The t rans -verse elements (A) are i n r e g i s t r y across the f i b r i l s and can be seen surrounding the f i b r i l s (B i n F i g . 24). The arrows (C) i n Figure 24 point to mitochondria which are present i n the i n t e r f i b r i l l a r space. The surface of a muscle f i b e r a f t e r 2 days of incubation at 25°C i s shown i n Figure 25. The bacter ia have penetrated below the surface of the endomysium, presenting a pic ture s i m i l a r to that of growth on nutr ient g e l a t i n ( F i g . 19 and 20). Pro teolys is of the endomysium indicates the P. f r a g i produces a collagenase. Some of the bacter ia have been washed away during the f i x a t i o n stage of specimen preparation, l e a v i n g ob-long shaped holes (A i n F i g . 2 5 ) , which are approximately the same s ize as the b a c t e r i a , i n the endomysium. These holes sug-gest that complete s o l u b i l i z a t i o n of the proteins occurs i n the immediate v i c i n i t y of the c e l l surface. As the prote in i s s o l u b i l i z e d , the bacter ia sink into the t i s s u e . This observ-a t i o n i s i n accord with the transmission micrographs p r e -sented by Dutson et a l . ( l97l) which showed a c lear zone, approximately 0.25 pro i n diameter, between the b a c t e r i a l c e l l wal l and the f i b r i l s of the muscle. With increased incubation time, proteolys is of the en-domysium becomes more extensive. As shown i n Figure 26, a f t e r 4 days of incubation large areas of the endomysium have been digested. The majority of the bacter ia have been washed away exposing the underlying f i b r i l s . I n d i v i d u a l f i b r i l s cannot 104 F i g . 24. L o n g i t u d i n a l view of m y o f i b r i l s i n c r y o f r a c t u r e d p e c t o r a l i s muscle i n o c u l a t e d w i t h P. f r a g i and incubated 24 nr at 25 0 (x 9,500). 105 P i g . 25. Longi tudinal view of endomysium i n cryofractured peetora l i s muscle inoculated with P. f r a g i and incubated f o r 2 days at 25 0 (x 9 ,500^ 106 P i g . 26. Longitudinal view of muscle f i b e r from cryofractured pectoralis muscle inoculated with P . f r a g i and incubated for 4 days at 25 C (x 9,400T7~^ ,be d i s t i n g u i s h e d i n t h i s r e g i o n . The reason becomes c l e a r upon examination of the transmission micrographs ( F i g . 37 and 39). Complete s o l u b i l i z a t i o n only occurs i n a small zone - 0.08 pm i n diameter - adjacent to the b a c t e r i a . The c l e a r zone must represent the f i n a l stage of p r o t e o l y s i s . Beyond t h i s c l e a r zone, the f i b r i l s have been broken down forming an amorphous mass. Presumably t h i s m a t e r i a l f i l l s the i n t e r f i b r i l l a r spaces thus making i t impossible to d i s t i n g u i s h the i n d i v i d u a l f i b r i l s The concave impressions v i s i b l e on the exposed f i b r i l s (A i n F i g . 26) are i n d i c a t i o n s that p r o t e o l y s i s of the m y o f i b r i l l a r p r o t e i n s does occur during spoilage of muscle by P. f r a g i • The' s l i g h t convex impressions on the endomysium remnant i n the center of Figure 26 i n d i c a t e s that transverse elements are present, although these elements are not present on the f i b r i l surface thus i n d i c a t i n g that elements of the sarcoplasmic r e t i c u l u m are subject to p r o t e o l y s i s . Micrographs of f i b r i l s from i n o c u l a t e d samples incubated i n excess of 4 days show s i m i l a r signs of d i s i n t e g r a t i o n . The surface of a f i b e r a f t e r 8 days of i n c u b a t i o n i s shorn i n Figure 27. As i n Figure 26, convex impressions on the endo-mysium i n d i c a t e that i n t e r a c t transverse elements are present under the endomysium. However, there i s no i n d i c a t i o n of trans verse elements i n areas where the endomysium has been attacked. Strands of m a t e r i a l are associated with some of the b a c t e r i a • (A i n F i g . 27). S i m i l a r i n t e r c e l l u l a r strands of m a t e r i a l were seen'when b a c t e r i a were grown on n u t r i e n t g e l a t i n ( F i g . 20). As shown i n Figure 39, amorphous m a t e r i a l (A) r e s u l t i n g from p r o t e o l y t i c breakdown of the f i b r i l s surrounds the b a c t e r i a . Presumably some of t h i s amorphous matter shrinks during the de-108 F i g . 27. Longi tudinal viev.1 of muscle f i b e r from c r y o -fractured pec tora l i s muscle inoculated with P. f r a g i and incubated f o r 8 days at room temperature (x 12,100). hydration procedure of sample preparation forming the strands shown at A i n Figure 2 7 . The concave appearance of the exposed myofibrils, i s similar to that observed a f t e r 4 days incubation. The m y o f i b r i l l a r proteins are apparently more resistant to pro-t e o l y t i c action of P. f r a g i than was the collagenous material of the endomysium. After 11 days of incubation (Fig. 28) the bacteria have penetrated deeper into the myofibrils. I t i s impossible to measure the extent of penetration. However, i t appears that the bacteria have digested through one f i b r i l and are attacking the underlying f i b r i l s . Therefore, penetration i s no greater than 3 to 4pfl"i. Micrographs of the uninoculated control were taken at the same time as those of the inoculated samples. Changes i n the controls were not excessive and for t h i s reason only photo-micrographs taken of the control af t e r 11 days of incubation w i l l be presented. The performations present i n the endomysium (Fig. 29) are indications of tissue deterioration. Such i n -dications of deterioration was extremely variable. Some areas of the endomysium appeared int a c t while other areas, on the same sample, showed signs of extreme distuption. Schaller and Powrie (1971) reported that the transverse elements of turkey myofibrils appeared collapsed af t e r 6 days postmortem storage at 5°C. Figure 30 shows intact myofibrils a f t e r 11 days storage at 25°C. The transverse elements do not appear collapsed but they do show some signs of disintegration. The transverse elements are not as d i s t i n c t as at 0 time, nor are they con-tinuous, but they are disrupted i n a number of areas (A i n F i g . 28. Longi tudinal view of muscle f i b e r from c r y o -frac tured p e c t o r a l i s muscle inoculated with. P. f r a g i and incubated at 25 0 f o r 11 days. Tx 10,300). I l l P ig. 29. L o n g i t u d i n a l view of endomysium from c r y o f r a c t u r e d p e e t o r a l i s muscle, u n i n o c u l a t e d and incubated f o r 11 days at 25 C (x 2 1 , 3 0 0 ) . 112 F i g . 30. Longi tudinal view of exposed m y o f i b r i l s from cryofractured peetoral is muscle, uninoculated and incubated f o r 11 days at 25 C (x 10,500). F i g . 30). • ' 3. Gryofracture of muscle a f t e r incubation Inoculated cryofractured pieces of chicken peetoralis muscle were cryofractured again a f t e r 10.5 days of incubation at 25°0. The b a c t e r i a l load was i n excess of 1 0 ^ organisms per g of muscle. As shown i n Figure 31, the bacteria grow up between the muscle f i b e r s when such space i s available. The top surface of the f i b e r s i n Figure 31 does not show signs of b a c t e r i a l growth. Presumably these surfaces were i n immediate contact with adjacent f i b e r s . At higher magnification (Fig. 32) the bacteria are "sinking" into the f i b e r (A). I t i s apparent that aerobic spoilage organisms can penetrate into the i n t e r i o r of the muscle i n cut up poultry. 4. Growth of P. f r a g i on chicken peetoralis muscle incubated at 5 0 The b a c t e r i a l population was 7x10*% 2.1xl0 7, and 4.5x10**"° organism per g of muscle at 0, 6.5 days and 21 days respectively. There was no growth on the uninoculated controls. Putrid odor was detected on the inoculated samples a f t e r 6.5 days of i n c u b -ation. After 6.5 days (Fig. 33) there was no in d i c a t i o n of exten-sive proteolysis. In the upper right hand corner of Figure 33, the endomysium appears to be s l i g h t l y disrupted. After 21 days of incubation ( F i g . 34) the bacteria have penetrated the endomysium and proteolysis of underlying myo-f i b r i l s has occurred. Deterioration of the endomysium i n P i g . 31. Longi tudinal view of muscle f i b e r s from c r y o -frac tured p e c t o r a l i s muscle inocubated with. P. f r a g i . incubated at 25 0 f o r 10.5 days then recryofractured (x 1,950). 115 F i g . 3 2 . Longi tudinal view of muscle f i b e r from c r y o -fractured p e c t o r a l i s muscxe inoculated with P . f r a g i . incubated f o r 10.5 days at 25 0 then recryofractured (x 19,500). 116 the. contro l samples, a f t e r 21 days of incubat ion, was not as severe as that occurring i n samples stored at room temperature. As shown i n Figure 35, the endomysium i s not perforated and the transverse r idges are d i s t i n c t . These r e s u l t s disagree with those of Schal ler and Powrie (1971) who found that the endomysium of turkey muscle was extensively perforated a f t e r 6 days storage at 5 ° C In t h e i r work, Schal ler and Powrie (1971) cryofractured the muscle a f t e r postmortem storage, while i n t h i s study the t issue was cryofractured p r i o r to incubat ion . Poss ibly the endomysium becomes f r a g i l e during postmortem aging and tends to fragment. I f t h i s i s the case, c ryofrac tur ing a f t e r postmortem aging would r e s u l t i n greater s t r u c t u r a l damage• 5 . Summary V i s u a l evidence obtained on the scanning electron micro-scope indica tes that p r o t e o l y s i s of the stromal and m y o f i b r i l l a r proteins does occur during spoilage of poultry by P. f r a g i . The endomysial layer surrounding the muscle f i b e r was r a p i d l y s o l u b i l i z e d i n d i c a t i n g the P. f r a g i produces a collagenase. N a t u r a l l y pro teolys is of the m y o f i b r i l l a r proteins cannot occur u n t i l the endomysium has been penetrated. P r o t e o l y s i s of the m y o f i b r i l l a r proteins occurred slowly i n d i c a t i n g that e i ther they are more res is tant to attack or that the bacter ia produces a l i m i t e d quantity of the enzyme responsible f o r t h e i r d i s -r u p t i o n . The presence of the oblong shapped holes , which ap-proximates the s ize of the bacter ia i n the endomysium along with the presence of in tac t remnants of the endomysium a d -jacent to b a c t e r i a l colonies , suggests that p r o t e o l y s i s occurs 119 120 only i n the immediate v i c i n i t y of the b a c t e r i a l c e l l . Appar-ently d i f f u s i o n of the enzyme or enzymes into the muscle occur-ed only i n a small area around the b a c t e r i a l c e l l . As one would expect, the rate of proteolysis was f a r greater at the higher temperature, which i s i n agreement with the work of Borton ejb.:al. (1970b) and Rey et a l . (1970). The micrographs support the r e s u l t s obtained by Borton et a l . (1970a and b) and Tarrant et a l . (1971) who have shown proteolysis of m y o f i b r i l l a r proteins occurs i n muscle i n o c u l -ated with P. f r a g i . The micrographs support the results of Ockerman et a l . (1969) who found that the stromal protein f r a -ction decreased i n muscle inoculated with a Pseudomonas species. Proteolysis of the endomysium would account f o r part of the decrease i n insoluble protein nitrogen observed by Borton et a l . (l970a) i n porcine muscle inoculated by P. f r a g i . Jay (1966) concluded that spoilage of Pseudomonas organisms occurs i n the absence of s i g n i f i c a n t proteolysis. Results of the scanning electron microscopy studies indicate that pro-t e o l y s i s i s l i m i t e d to the surface of the muscle f i b e r s and does extend beyond a few micrometers. I t seems l i k e l y that the extent of the proteolysis of the stromal and m y o f i b r i l l a r pro-teins i s i n s i g n i f i c a n t during spoilage. Off-odors were detected from the inoculated samples incubated at 25°0 and 5°0 before any microscopic evidence of extensive proteolysis was observed. These results agree with the conclusions reached by Lerke et a l . (1967) that proteolysis represents an advanced stage of meat spoilage. The cryofract-121 ured samples were inoculated with a high i n i t i a l inoculum straight from nutrient broth. I t i s l i k e l y that the nutrient broth supplied the necessary nutrients f o r growth which lead to the development of off-odors. Further work i s required to determine, whether or not proteolysis coincides with the onset of spoilage or represents an advanced state of spoilage. This can best be achieved by inoculating the muscle with bacteria suspended i n buffer and using a low i n i t i a l inoculum. Transmission electron mlscroscopy of Pseudomonas f r a g i Samples of chicken peetoralis muscle were incubated at 5°C for 10.5 days. After incubation, the b a c t e r i a l population was i n excess of 10"^ organisms per g of muscle. Off-odors were de-tected at the end of the incubation i n d i c a t i n g that the muscle had spoiled. No b a c t e r i a l growth or off-odors were detected i n the uninoculated controls. The Z disc on the uninoculated controls (Pig. 36) shows signs of disruption. The Z disc appears diffuse and some of the dense material present i n the Z disc has been l o s t (arrows i n Pig. 36). Disintegration of the Z l i n e during postmortem aging agrees with the reports by Davey and Dickson (1970) on beef muscle and Fukazawa et a l . (1969) on chicken muscle. There was complete disintegration of the myofibrils i n areas adjacent to b a c t e r i a l growth (Fig. 37). As reported by Wiebe and Chapman (1968a and b), the c e l l wall i s i r r e g u l a r l y undulant. Protrusions (B i n F i g . 37) were observed on the surface of the bacteria. These protrusions are s i m i l a r i n appearance to the "bleblike evaginations" observed by Dutson 122 L_ F i g . 37. Longi tudinal view of p e c t o r a l i s muscle inoculated with P . f r a g i and incubated at 5 C for 10.5 days (x 687900). et a l . (1971)» on the surface of P. f r a g i growing on spoiled porcine muscle. Wiebe and Chapman (1968a) reported the pre-sence of c e l l u l a r protrusions on the c e l l wall of some pseudo-mads under certain n u t r i t i o n a l and physiological conditions. Smirnova et a l . (1971) observed c e l l u l a r protrusions which they termed "microcapsules" on certain species of bacteria. Dutson et a l . (1971) and Smirnova et a l . (1971) concluded that these c e l l u l a r enxymes were secreted, as such high molecular weight substances cannot diffuse through the c e l l membrane. A clear zone, devoid of stru c t u r a l d e t a i l , approximately 0.08 pm i n diameter surrounds the bacteria, i n d i c a t i n g that com-plete s o l u b i l i z a t i o n of the m y o f i b r i l l a r proteins has occurred. An amorphous region (A i n Pig. 37), presumably consisting of disintegrated sarcomere components, occurs between the clear zone and unattacked portion of the f i b r i l (C i n Pig. 37). The average distance between the bacteria and the intact f i b r i l s i s 0.6 um. Proteolysis i s l i m i t e d to a small area surrounding the bacteria. Myofibrils from muscle inoculated and incubated with P. f r a g i but not adjacent to b a c t e r i a l c e l l s are shown i n Pigure 38. Most of the dense material from the Z l i n e has been l o s t . The H zone i s not as d i s t i n c t and the M band appears denser than i n the micrograph of the uninoculated control (Pig. 36). The difference i n the M band and the H zone can be accounted f o r by contraction. The mean sarcomere length i n the control (Pig. 36) was 2.0pm while the sarcomere length i n the inoculated sample (Pig. 38) i s 1.6pm. According to Bendall (1969), as the muscle contracts the aetin filaments s l i d e up between the Longi tudinal view of peetora l i s muscle inoculated with P. f r a g i and incubated at 5 0 f o r 16 .5 days (x 4 9 7 0 0 0 ) . myosin filaments and i n t o the H zone, thus decreasing i t s s i z e . Contraction also resul ted i n increased density of the M band. The r e s u l t s of t h i s study are not i n agreement with those of Dutson et a l . (1971)• These authors reported that m y o f i b r i l s from porcine muscle inoculated M t h P. f r a g i showed a disrupted appearance i n the A band, the H zone was almost devoid of mat-e r i a l and the dense mater ial from the Z l i n e had been l o s t . The authors concluded that s p e c i f i c d i s r u p t i o n of myosin and mater ia l from the Z l i n e occurred as a consequence of spoilage of P. f r a g i . The micrograph of i n t a c t f i b r i l s presented by Dutson et a l . (1971) d id not contain any b a c t e r i a l c e l l s . Re-s u l t s obtained i n t h i s study i n d i c a t e that pro teolys is only occurs i n a l i m i t e d area surrounding the b a c t e r i a . The H zone i n f i b r i l s which are not adjacent to b a c t e r i a l c e l l s was i n t a c t . Disrupt ion of the Z l i n e was probably due to postmortem aging rather than p r o t e o l y s i s . v M y o f i b r i l s from inoculated t i ssue are shown i n cross sect ion i n Figure 39. The t h i c k myosin fi laments are surroun-ded by thinner a c t i n f i laments . P r o t e o l y s i s occurs only i n a small zone adjacent to the b a c t e r i a . There i s no i n d i c a t i o n of s p e c i f i c d i s r u p t i o n of myosin. 127 F i g . 39. Gross sec t ion of peetoral is muscle inoculated with P. f r a g i and incubated at 5 G f o r 10.5 days (x 497oooT 128 GENERA! DISCUSSION Results obtained i n t h i s study indicate that p r o t e o l y s i s of the sarcoplasmic, m y o f i b r i l l a r and stroma protein f r a c t i o n s occurs as a r e s u l t of spoilage by Pseudomonas f r a g i . a) Evidence of pro teolys is of the sarcoplasmic p r o t e i n s : 1) a l t e r a t i o n s occurring i n the e l u t i o n pattern during g e l f i l t r a t i o n s tudies . 2) loss of 2 bands i n the disc ge l electrophoresis patterns . b) Evidence f o r proteolys is of the m y o f i b r i l l a r p r o t e i n s : 1) Scanning electron and transmission electron micro-graphs showed that breakdown of the m y o f i b r i l s occurred as a r e s u l t of growth of P. f r a g i . 2) The e x t r a c t a b i l i t y of the m y o f i b r i l l a r prote in f r a c t i o n was s i g n i f i c a n t l y (P ^ .05) lower i n inoculated, samples. 3) The s t a i n i n g i n t e n s i t i e s of the bands i n the e l e c t -rophoretic patterns was lower i n the inoculated samples and decreased i n samples incubated f o r longer time per iods . c) Evidence of proteolys is of the stroma prote ins : 1) Scanning electron micrographs indicated that the endomysium was very susceptible to degradation by the p r o t e o l y t i c enzymes of P. f r a g i . 2) Results of free amino a c i d analysis of the inoculated muscle indicated that the increase i n glycine was approximately 15 times greater than the increase i n the other amino a c i d s . As glycine composes, approx-129 imately 1/3 of the amino acid residues i n collagen (Yeis, 1970). This increase i n the l e v e l of glycine may "be a further evidence f o r the proteolysis of the stroma f r a c t i o n . Results of the protein s o l u b i l i t y studies of the inoculated muscle indicated that the sarcoplasmic protein s o l u b i l i t y was not s i g n i f i c a n t l y d i f f e r e n t from the zero time control, except afte r 5.5 days incubation when i t was s i g n i f i c a n t l y higher. There was no s i g n i f i c a n t increase i n nonprotein nitrogen i n the inoculated samples. Results of gel f i l t r a t i o n studies contradicted the protein s o l u b i l i t y studies. Results obtained i n the gel f i l t r a t i o n studies indicated that the sarcoplasmic f r a c t i o n extracted from inoculated muscle continually decreased during incubation, although this decrease was not as extensive as that occurring i n the control and the nonprotein nitrogen f r a c t i o n increased i n the inoculated samples. B e l l (1963) and Synge (1955) indicated that during determinations of non-protein nitrogen by acid p r e c i p i t a t i o n , small nonprotein molecules may be retained on the precipitated protein as a resul t of absorption or ion exchange. In t h i s event, results of protein s o l u b i l i t y would indicate lower values for nonprotein nitrogen and higher values for water soluble protein. Com-parison of the results obtained during protein s o l u b i l i t y studies with those of the gel f i l t r a t i o n study indicated that t h i s s i t u a t i o n probably occurred. Results obtained by Hasegawa ejb al. (1970a) indicated that proteolysis of the sarcoplasmic proteins by P. f r a g i was more extensive i n porcine muscle than i n rabbit. The authors con-eluded that the bacteria produced "highly s p e c i f i c enzymes which p r e f e r e n t i a l l y act upon only certain proteins or enzymes indigenous to muscle." Results of the disc gel electrophoresis of the sarcoplasmic proteins i n this study showed that 2 of the protein bands disappeared from the electrophoretic pattern of inoculated tissue and the intensity of the remaining bands decreased. Therefore, the theory put forward by Hasegawa et, a l . (1970a) regarding p r e f e r e n t i a l attack could be possible. Dutson et a l . (1971) concluded that s p e c i f i c disruption of myosin and material from the Z l i n e occurred as a conseq-uence of spoilage by P. f r a g i . Disc gel electrophoresis of the m y o f i b r i l l a r proteins extracted from inoculated tissue r e -ported i n t h i s study did not indicate such p r e f e r e n t i a l attack. Transmission electron miscrographs presented i n t h i s study i n -dicated t o t a l disruption of the myofibril i n areas immediately adjacent to b a c t e r i a l c e l l s rather than s p e c i f i c disruption as reported by Dutson et a l . (1971). As has been reported i n previous studies (Tarrant et a l . , 1971; Borton et a l . 1970 a and b; Hasegawa e_t a l . 1970a) pro-t e o l y s i s was not detected u n t i l a f t e r spoilage was evident. Jay (1966) and Jay and Kontou (1967) have stated that low temperature spoilage occurs without causing s i g n i f i c a n t pro-t e o l y s i s . Results obtained i n the present study showed that s i g n i f i c a n t proteolysis did occur during spoilage by P. f r a g i , a species of bacteria commonly associated with meat spoilage. While quantitive changes i n the protein fractions could not be detected u n t i l after spoilage was evident, the development of proteolytic a c t i v i t y before the onset of spoilage may be sign-i f i c a n t . Researchers studying "bacterial proteolysis of meat during spoilage have, without exception, used minced t i ssue i n order to produce maximum b a c t e r i a l growth so that changes r e s u l t i n g from such growth could be detected by biochemical means. The b i o -chemical changes evaluated i n t h i s manner may not be the r e a l reproduction of those caused by spoilage of natural t i ssue without mechanical deformation. The use of the scanning electron microscope enables one to study the changes r e s u l t i n g from b a c t e r i a l growth i n in tac t t i s s u e . Results obtained i n t h i s study indicated that the endomysium was r e a d i l y attacked by the p r o t e o l y t i c enzymes produced by P. f r a g i , while the m y o f i b r i l l a r proteins appeared more r e s i s t a n t to such p r o t e o l y s i s . The intac t t issue was i n -oculated with a high l e v e l inoculum and samples were judged to be spoi led a f t e r 1 day. Proteolys is was not detectable i n samples incubated f o r 1 day. This probably r e f l e c t s a l a g i n growth r e s u l t i n g from the t ransfer from the l i q u i d medium to the muscle. Minute pro teolys is of the endomysium was detected a f t e r 2 days incubation by SEM, which would be d i f f i c u l t to detect by ordinary biochemical techniques. In order to deter -mine i f proteolys is does occur p r i o r to i n c i p i e n t spoi lage , i t w i l l be necessary to use a low l e v e l inoculum. In other studies (Ockerman et a l . 1969; Jay and Kontou, 1967; Lerke et a l . , 1967) meat was inoculated with a culture of unknown genera or with a pure culture of u n c l a s s i f i e d species . Lack of r e p r o d u c i b i l i t y i n t h i s pract ice brought about i n c o n -s i s t e n c i e s ; ' among r e s u l t s reported i n the l i t e r a t u r e . I t i s important to use known pure cultures to obtain reproducible r e s u l t s , although i t i s u n l i k e l y that meat undergoes n a t u r a l low-temperature spoilage caused., by a s ingle species of bac ter ia The Pseudomonas Achromobacter group i s responsible i n most cases of meat spoi lage . Conclusions 1. P ro teolys is of the sarcoplasmic, m y o f i b r i l l a r and stroma prote in f rac t ions occurs as a r e s u l t of spoilage by P. f r a g i . 2. The nonprotein ni trogen f r a c t i o n increased consider-ably during the 9.5-day incubation period of the inoculated samples. 3. The endomysium was more susceptible to pro teolys is than the m y o f i b r i l l a r protein f r a c t i o n . 4. This study d i d not indica te p r e f e r e n t i a l p r o t e o l y s i s of a p a r t i c u l a r m y o f i b r i l l a r p r o t e i n . 5. P ro teolys is of the stromal and m y o f i b r i l l a r proteins only occurred i n a l i m i t e d area surrounding the b a c t e r i a . 6. Aerobic spoilage organisms can grow up between the muscle f i b e r s , thus penetrating into the i n t e r i o r of the muscle 7. P ro teolys is probably represents an advanced stage of spoi lage . BIBLIOGRAPHY Adamcic, M. and C l a r k , D.S. 1970. B a c t e r i a induced biochemical changes i n chicken s k i n stored at 5 C. J . Food S c i . 35: 103-108. Adamcic, M., Clark, D.S. and Yaguchi, M. 1970. E f f e c t of psychrotolerant b a c t e r i a on the amino a c i d content of chicken s k i n . J . Food S c i . 35: 272-275.. Anonymous 1966. 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