UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1974_A6_7 S23.pdf [ 16.88MB ]
Metadata
JSON: 831-1.0092968.json
JSON-LD: 831-1.0092968-ld.json
RDF/XML (Pretty): 831-1.0092968-rdf.xml
RDF/JSON: 831-1.0092968-rdf.json
Turtle: 831-1.0092968-turtle.txt
N-Triples: 831-1.0092968-rdf-ntriples.txt
Original Record: 831-1.0092968-source.json
Full Text
831-1.0092968-fulltext.txt
Citation
831-1.0092968.ris

Full Text

BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES OCCURRING  IN CHICKEN PECTORALIS MUSCLE  INOCULATED  WITH  PSEUDOMONAS  TRAGI  by GILBERT SAGE B.Sc.  (Agr.), U n i v e r s i t y o f 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 t h i s t h e s i s as conforming r e q u i r e d standard  t o the  THE UNIVERSITY OF BRITISH COLUMBIA August, 1974.  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at  the U n i v e r s i t y  make  that  it  purposes  for  freely  permission may  representatives. thesis  in p a r t i a l  financial  is  Columbia,  British  by  for  gain  Columbia  shall  the  that  not  requirements I  agree  r e f e r e n c e and copying  t h e Head o f  understood  Depa r t m e n t  Date  of  for extensive  permission.  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  available  be g r a n t e d  It  fulfilment  of  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  ii ABSTRACT Chicken p e c t o r a l i s muscle was i n o c u l a t e d with Pseudomonas f r a g i and incubated a t room temperature. nonprotein n i t r o g e n ,  A l t e r a t i o n s i n the  water-soluble p r o t e i n n i t r o g e n ,  soluble protein nitrogen  fractions.-were  and s a l t -  s t u d i e d and attempts  were made to r e l a t e these changes t o s t r u c t u r a l changes observed by scanning and t r a n s m i s s i o n  e l e c t r o n 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 p r o t e i n was found i n the i n o c u l a t e d muscle during a b i l i t y study.  Results  the p r o t e i n  fragi.  extract-  of the g e l f i l t r a t i o n study i n d i c a t e d  that p r o t e o l y s i s of the sarcoplasmic p r o t e i n s the n o n p r o t e i n n i t r o g e n  solubility  f r a c t i o n increased  occurred and that  due t o growth of P.  A l t e r a t i o n s were observed i n the d i s c g e l e l e c t r o -  phoretic patterns fractions.  of the sarcoplasmic and m y o f i b r i l l a r p r o t e i n  Arginine,  threonine, s e r i n e , p r o l i n e and t y r o s i n e  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 e l e c t r o n micrographs i n d i c a t e d that  proteolysis  of the endomysium occurred a f t e r 2 days o f i n c u b a t i o n  a t 25°C.  P r o t e o l y s i s of the endomysium became more e x t e n s i v e as i n c u b a t i o n time i n c r e a s e d .  A f t e r 4 days of i n c u b a t i o n  the m y o f i b r i l s  showed evidence of d i s r u p t i o n as a r e s u l t o f 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 t o a depth of 2 t o  4 micrometers a f t e r 9 days of i n c u b a t i o n .  B a c t e r i a were ob-  served growing between the muscle f i b e r s .  P r o t e o l y s i s was not  as extensive i n samples incubated a t 5 ° C E l e c t r o n micrographs prepared from t h i n s e c t i o n s  o f myo-  i i i fibrils the  inoculated  myofibrils  due  w i t h P. to  of s t r u c t u r a l d e t a i l ,  fra^i  showed iaarlced d i s r u p t i o n  b a c t e r i a l growth. surrounded the  tissue  myofibrils.  C e l l u l a r p r o t r u s i o n s were o b s e r v e d  bacteria.  zone,  bacterial cell.  of d i s r u p t e d  of the  exists  A clear  between t h e  clear  zone and on  the  of  devoid A region intact surface  TABLE OF CONTENTS Page INTRODUCTION LITERATURE REVIEW  3  Meat Spoilage  3  A s p e t i c Techniques  4  Changes i n Muscle P r o t e i n s postmortem Storage  during 5  Biochemical Changes O c c u r r i n g i n S p o i l e d Muscle U l t r a s t r u c t u r e of S t r i a t e d  10 S k e l e t a l Muscle  21  METHODS P a r t I - Biochemical Studies  28  Sample P r e p a r a t i o n  28  Bacterial  29  Counts  Protein Extraction  29  N i t r o g e n Determination  31  Gel F i l t r a t i o n  31  Disc Gel Electrophoresis  32  Free Amino A c i d A n a l y s i s  33  P a r t I I - E l e c t r o n Microscopy Bacterial  Counts  35  P r e p a r a t i o n of Samples f o r Scanning E l e c t r o n Microscopy  36  P r e p a r a t i o n ofi Samples f o r E l e c t r o n Microscopy  36  Transmission  RESULTS AND DISCUSSION P a r t I - Biochemical  Studies  39  V  I n f l u e n c e o f growth o f Pseudomonas f r a g i organisms on the pH and water h o l d i n g c a p a c i t y o f chicken muscle  39  I n f l u e n c e o f growth o f Pseudomonas f r a g i organisms on the e x t r a c t a b i l i t y o f p r o t e i n s from chicken muscle  42  G-el f i l t r a t i o n o f water-soluble p r o t e i n s i n e x t r a c t s from c o n t r o l and i n o c u l a t e d muscle  52  D i s c g e l e l e c t r o p h o r e s i s o f water-soluble, s a l t - s o l u b l e and u r e a - s o l u b l e p r o t e i n s i n e x t r a c t s from c o n t r o l and i n o c u l a t e d muscles  61  A n a l y s i s of f r e e amino a c i d s i n c o n t r o l and i n o c u l a t e d muscles  86  Part I I - E l e c t r o n Microscopy  92  Scanning e l e c t r o n microscopy o f Pseudomonas fragi  92  Transmission e l e c t r o n microscopy o f Pseudomonas fragi  121  GENERAL DISCUSSION Conclusions BIBLIOGRAPHY  132  133  vi LIST OP TABLES Table I  Page I n f l u e n c e o f i n c u b a t i o n time a t 25°C on the b a c t e r i a l numbers and pH of c o n t r o l muscle and muscle i n o c u l a t e d with P. f r a g i  40  Water-soluble p r o t e i n , s a l t - s o l u b l e p r o t e i n and nonprotein n i t r o g e n 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 chicken p e c t o r a l i s muscle  47  A n a l y s i s of v a r i a n c e 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 c t o r a l i s muscle  48  S o l u b i l i t y of the v a r i o u s ' p r o t e i n f r a c t i o n s from u n i n o c u l a t e d muscle and muscle i n o c u l a t e d with P. f r a g i  49  V  R a t i o of areas under the curves a t Vo and Ye f o r glutamic a c i d  58  VI  Average f r e e amino a c i d c o n c e n t r a t i o n o f i n o c u l a t e d and u n i n o c u l a t e d chicken p e c t o r a l i s muscle incubated at 25 C  87  Average amino a c i d c o n c e n t r a t i o n o f chicken 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 c o r r e c t e d f o r a u t o l y t i c changes "~  89  B a c t e r i a l p o p u l a t i o n i n chicken p e c t o r a l i s major i n o c u l a t e d with P. f r a g i incubated a t 25 0  98  II  III IV  VII  VIII  vii LIST OF FIGURES Figure  Page  1  Structure  2  Flow Sheet of P r o t e i n E x t r a c t i o n Procedure  30  3  Water-Soluble p r o t e i n 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  43  4  S a l t - s o l u b l e p r o t e i n 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  44  5  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  45  6  E l u t i o n p a t t e r n s of w a t e r - s o l u b l e p r o t e i n e x t r a c t s from u n i n o c u l a t e d c h i c k e n p e e t o r a l i s muscle  5$  7  E l u t i o n ' p a t t e r n s of w a t e r - s o l u b l e p r o t e i n e x t r a c t s from chicken, p e e 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 o  56  Densitomater 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 u n i n o c u l a t e d c h i c k e n p e e t o r a l i s muscle  63  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 c h i c k e n 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  65-  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 c h i c k e n p e e t o r a l i s muscle  69  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 c h i c k e n 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  71  Densitometer t r a c i n g s of the u r e a - s o l u b l e 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 c h i c k e n p e e t o r a l i s muscle  74  Densitometer t r a c i n g s of the u r e a - s o l u b l e 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 chicken 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  76  14  Electrophoretic  p a t t e r n s of sarcoplasmic  proteins  79  15  Electrophoretic  p a t t e r n s of m y o f i b r i l l a r p r o t e i n s  81  16  Electrophoretic  patterns of urea-soluble proteins  83  17  Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t a g a r , u n i n o c u l a t e d and incubated at 25 C f o r 24 h r (x 22,000)  93  8  9  10  11  12  13  of a Sarcomere  23  viii LIST OP FIGURES Figure 18  19  20  Page Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t a g a r , i n o c u l a t e d w i t h P* f r a g i incubated 24 h r at 25 C (x 11,400)  94  Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t g e l a t i n i n o c u l a t e d with P. f r a g i i n cubated f o r 24 h r at 25 0 (x l,ro"0.')  96  Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t g e l a t i n i n o c u l a t e d with P. f r a g i and incubated f o r 24 h r at 25 C (x l C O O O )  97  21  L o n g i t u d i n a l view of i n t a c t endomysium i n c r y o f r a c t u r e d p e e t o r a 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 i n o c u l a t e d chicken p e e t o r a l i s muscle at zero time (x 22,200)  101  23  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 at 25 0 (x 9,500) ~ 102  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 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 at 25 C (x 9,500)  104  l o n g i t u d i n a l view of endomysium i n 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 f o r 2 days at 25 C (x 9,500l  105  L o n g i t u d i n a l view of muscle f i b e r from c r y o f r a c t ured 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 f o r 4 days at 25 C (x 9,400)  106  L o n g i t u d i n a l view of muscle f i b e r 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 w i t h P . f r a g i and incubated f o r 8 days at room temperature (x 12,100)  108  25  26  27  28  L o n g i t u d i n a l view of muscle f i b e r 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 at 25 0 f o r 11 days (x 10,300) 110  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 0 (x 21,300) 111  30  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 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 0 (x 10,500)  112  LIST OP FIGURES  L o n g i t u d i n a l view of muscle f i b e r s from c r y o f r a c t u r e d p e c t o r a l i s muscle inocubated w i t h P . f r a g i , incubated at 25 C f o r 10.5 days then r e c r y o f r a c t u r e d (x 1,950) L o n g i t u d i n a l view of muscle f i b e r from 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 with. £ • f r a g i . incubated f o r 10.5 days at 25 0 then r e c r y o f r a c t u r e d (x 19,500) L o n g i t u d i n a l view of 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 at 5 0 f o r 6.5 days (x 4,400) L o n g i t u d i n a l view of 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 f o r 21 days at 5 C (x 11,900) L o n g i t u d i n a l view of u n i n o c u l a t e d c r y o f r a c t u r e d p e c t o r a l i s muscle, incubated at 5 C f o r 21 days  (x 10,800)  L o n g i t u d i n a l view of u n i n o c u l a t e d p e c t o r a l i s muscle incubated at 5 C f o r 10.5 days (x 13,200) L o n g i t u d i n a l view of 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 at 5 0 f o r 10.5 days  (x 68,900)  L o n g i t u d i n a l view of 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 at 5 G f o r 16.5 days  (x 497000)"  Cross s e c t i o n of 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 at 5 C f o r 10.5 days  (x 49,0007"^  ACTOWLEDGEICEOTS I wish t o express my g r a t i t u d e the members o f my committee  t o Dr. W.D. Powrie and  f o r t h e i r s u p e r v i s i o n and guidance  throughout the course o f t h i s work. I a l s o wish to thank Dr. D. S c h a l l e r f o r h i s g u i d a n c e ' i n the scanning e l e c t r o n microscope and to Mrs. Jean Simons f o r the t y p i n g o f t h i s t h e s i s .  INTRODUCTION Spoilage of food myosystems can be d i v i d e d i n t o two namely, b a c t e r i a l decomposition  types;  of amino a c i d s with the form-  a t i o n of p u t r i d odors, and mold growth on the s u r f a c e s of muscle and adipose t i s s u e to form l a r g e masses of mycelium. Only b a c t e r i a l s p o i l a g e w i l l be considered i n t h i s t h e s i s .  In  the past, much r e s e a r c h has centered on the i s o l a t i o n and i d e n t i f i c a t i o n of the b a c t e r i a a s s o c i a t e d w i t h meat s p o i l a g e .  For  many years, r e s e a r c h e r s have s t u d i e d the temperature and growth c h a r a c t e r i s t i c s of s p o i l a g e organisms along w i t h the i n c i d e n c e and s i g n i f i c a n c e of these organisms d u r i n g meat s p o i l a g e , but l i t t l e a t t e n t i o n has been focussed on the chemical and  struct-  u r a l changes d u r i n g s p o i l a g e . Various attempts have been made to u t i l i z e chemical  and  p h y s i c a l techniques to o b t a i n an index of m i c r o b i a l s p o i l a g e . Increase i n pH,  p r o d u c t i o n of ammonia and d e t e r m i n a t i o n of  ex-  t r a c t r e l e a s e volume of muscle have at one time or other been advocated  as a s u i t a b l e index of s p o i l a g e .  G e n e r a l l y , these  parameters c o r r e l a t e roughly with b a c t e r i a l numbers only when h i g h b a c t e r i a l l o a d s have developed  and the meat has  spoiled.  W i t h i n the l a s t decade, numerous papers have appeared on the nature of the b i o c h e m i c a l changes which occur d u r i n g the spoilage process.  The p o s s i b l e r o l e of b a c t e r i a l p r o t e o l y s i s  has r e c e i v e d much a t t e n t i o n .  Some workers (Jay and Kontou,  1967;  believe that m i c r o b i a l pro-  and Lerke et a l . , 1967)  t e o l y s i s d u r i n g meat s p o i l a g e i s i n s i g n i f i c a n t while (Tarrant et a l . , 1971;  others  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 p r o t e o l y s i s d u r i n g  2  meat s p o i l a g e . In a l l of the papers reviewed d u r i n g t h i s study, minced muscle had been used as the substrate Mincing i n c r e a s e s  f o r the inoculum.  the s u r f a c e area f o r r a p i d a e r o b i c 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  structural  changes i n c h i c k e n p e e t o r a l i s muscle as i t undergoes s p o i l a g e due to growth of Pseudomonas f r a g i . o c c u r r i n g as a r e s u l t  The a l t e r a t i o n of p r o t e i n s  of p r o t e o l y s i s by P . f r a g i was a l s o  s t u d i e d 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 t r a n s m i s s i o n e l e c t r o n microscopy.  3 LITERATURE REVIEW Meat Spoilage Microorganisms r e s p o n s i b l e f o r the s p o i l a g e o f meat a r e e i t h e r present p r i o r t o s l a u g h t e r or i n t r o d u c e d as contaminants during p r o c e s s i n g , handling, packaging and storage.  Although  the f l o r a of f r e s h meat may c o n s i s t of a l a r g e number of d i f f e r e n t b a c t e r i a genera, only a few o f these genera predominate i n s p o i l e d meat.  Temperature i s the most important  i n f l u e n c i n g the f l o r a which develop on meat d u r i n g  parameter storage.  When meat undergoes s p o i l a g e a t temperatures between 25 and 40°C t h e predominant genus i s e r o b i c grow w i t h i n the meat. e r a t u r e s i s normally  C l o s t r i d i u m , which being anSpoilage o f meat a t c h i l l e d  temp-  the r e s u l t o f a e r o b i c p s y c h r o p h i l i c s p e c i e s ,  which grow predominantly on the s u r f a c e s .  Ayres (1960a) r e -  ported t h a t , a f t e r c u t t i n g up chicken, 75 t o 80 percent  of the  c o l o n i e s on the s u r f a c e s o f the chicken p a r t s were comprised o f chromogenic b a c t e r i a , spore-forming yeasts.  microorganisms, molds and  When the chicken was allowed  t o s p o i l under r e f r i g -  erated c o n d i t i o n s , the predominant organisms were " p r i m a r i l y s i n g l e , p a i r e d or short chained m o t i l e , Gram-negative, nonsporeforming rods some of which produce f l u o r e s c e n t pigments but most do n o t . " I t has been w e l l documented t h a t s p o i l a g e o f meat under c h i l l temperatures r e s u l t s from growth of b a c t e r i a of the Pseudomonas - Achromobacter types'(Ingram and Dainty, 1971;  Barnes and Impey, 1968; Jay, 1967; Ayres, 1960a and b;  and Ayres e t a l . , 1950).  S p o i l a g e of meat at c h i l l e d temperatures i s s u p e r f i c i a l u n l e s s the meat has been processed i n such a manner ( e . g . mincing) as t o d i s t r i b u t e the b a c t e r i a  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) d e s c r i b e d as " d i r t y dishrag"'.  Ayres (1960b) r e p o r t e d the presence of an o f f -  odor when the b a c t e r i a l p o p u l a t i o n of bovine muscle had 7 reached 10  2 organisms/cm . 8  muscle s u r f a c e s reach 10 s l i m y appearance.  V/hen the b a c t e r i a l numbers o f the 2 organisms/cm , the meat takes on a  Ayres et a l . (1950) and Jay (1970)  reported  t h a t the s l i m y appearance was due to the coalescence o f the bacterial  colonies.  Jay (1970) a l s o s t a t e d that  sliminess  was due i n p a r t to the " l o o s e n i n g of meat s t r u c t u r a l as a r e s u l t  of b a c t e r i a l  growth.  Ayres et a l . (1950)  t h a t a pungent ammoniacal odor developed a f t e r slime  proteins" reported format-  ion. A s e p t i c Techniques Changes r e s u l t i n g from b a c t e r i a l decomposition a r e s u p e r imposed on those r e s u l t i n g from a u t o l y s i s ; i s necessary to s t o r e u n i n o c u l a t e d c o n t r o l s lated- samples.  In the p a s t ,  meat due to b a c t e r i a l  along with i n o c u -  r e s e a r c h on b i o c h e m i c a l changes i n  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 o b t a i n i n g undenatured s t e r i l e control.  f o r t h i s reason i t  tissue  f o r the  V a r i o u s techniques have been employed by r e s e a r c h e r s  to o b t a i n germ-free  tissue.  Hasegawa et a l . (1970a and b) used  an a l c o h o l i c wash to o b t a i n 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 o b t a i n s t e r i l e samples of p o r c i n e muscle; however, t h e 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 at 1 0 ° C .  / organisms/g a f t e r  20 days i n c u b a t i o n  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 o b t a i n s t e r i l e (1969) used a s e p t i c  samples of beef muscle.  Lea et a l .  technique to o b t a i n samples of c h i c k e n  breast muscle as w e l l as t r e a t i n g the f l e s h with a 10 ppm c h l o r o t e t r a c y c l i n e s o l u t i o n presumably to prevent growth.  bacterial  U n f o r t u n a t e l y , t h i s a n t i b i o t i c d i d not prevent  b a c t e r i a l growth, but h e l d the b a c t e r i a l l o a d of the c o n t r o l below 10  organisms/g d u r i n g s t o r a g e .  Jay and Eontou (1967)  i r r a d i a t e d beef muscle a t 1 Mrad to o b t a i n a s t e r i l e  control.  These authors found t h i s dosage was s u f f i c i e n t t o destroy a l l p s y c h r o p h i l i c b a c t e r i a without i n h i b i t i n g the c a p a c i t y of the meat to undergo t y p i c a l s p o i l a g e when i n o c u l a t e d with a mixed f l o r a . tectable  The i r r a d i a t i o n treatment d i d not cause any d e -  a l t e r a t i o n i n samples of u n i n o c u l a t e d muscle.  and C l a r k (1970) u t i l i z e d a s e p t i c  Adamcic  technique and a 0.5 Mrad d o s -  age to o b t a i n s t e r i l e samples of chicken s k i n . Changes i n Muscle P r o t e i n s d u r i n g Postmortem  Storage  Muscle p r o t e i n s are g e n e r a l l y c a t e g o r i z e d i n t o three classes;  sarcoplasmic, m y o f i b r i l l a r and s t r o m a l .  The s a r c o -  plasmic p r o t e i n s c o n s i s t of the g l y c o l y t i c enzymes and are e x t r a c t e d by means o f water or d i l u t e s a l t s .  B r i s k e y and  Pukazawa (1971) r e p o r t e d that the sarcoplasmic p r o t e i n s 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 -  t e i n s are i n v o l v e d i n c o n t r a c t i o n and s o l u t i o n s of h i g h i o n i c s t r e n g t h .  are extracted with s a l t  Helander (1957) s t a t e d  that the stroma f r a c t i o n c o n s i s t e d of c e l l w a l l s , blood nerves, endomysium and lagen and  elastin.  vessels  perimysium; these t i s s u e s c o n s i s t of c o l  The  stroma p r o t e i n s can be e x t r a c t e d  from  minced muscle with sodium hydroxide s o l u t i o n s . Khan (1962) estimated the amount ( n i t r o g e n b a s i s ) of  the  v a r i o u s p r o t e i n f r a c t i o n s f o r the breast muscle of 4 month o l d b r o i l e r s to be's> stroma N nonprotein  3.9 N  - 1.2  mg N/g  muscle  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  - 1.2  i g N/g  muscle  TS 9.4  Postmortem changes o c c u r r i n g i n muscle p r o t e i n s have been s t u d i e d e x t e n s i v e l y i n attempts to understand the mechanism of postmortem tenderness.  Increases i n f r e e amino a c i d s and  p r o t e i n n i t r o g e n d u r i n g postmortem storage bovine muscle K'Parrish et a l . , 1969; Gardner and  Stewart, 1966;  have been shown i n  Davey and G i l b e r t ,  Sharp, 1963;  et a l . , 1970a;  Bowers, 1969), r a b b i t muscle (Suzuki et a l . , 1967)  van den Berg, 1964a and The  1966;  Lawrie et a l . , 1961;  and l o c k e r , I960), porcine muscle (Borton  muscle (Lea et a l . , 1969;  non-  M i l l e r et a l . , 1965;  and  and  poultry  and Khan and  b).  i n c r e a s e i n f r e e amino a c i d s and nonprotein  nitrogen  has been a t t r i b u t e d to the enzymic a c t i o n of n a t u r a l l y occ u r r i n g cathepsins  on the p r o t e i n s ; Mycek (1970) made a com-  prehensive review on the i s o l a t i o n and p r o p e r t i e s of epsins.  Cathepsins A, B and  cath-  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 spectively.  The sarcoplasmic p r o t e i n s a r e thought  s u b s t r a t e f o r the c a t h e p t i c enzymes.  res-  to be  the  Bodwell and Pearson (1964)  d i d not d e t e c t any h y d r o l y s i s 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 e x t r a c t of bovine muscle, c a t h e p s i n s . These authors found that the sarcoplasmic p r o t e i n s were t e n s i v e l y hydrolyzed by the cathepsins and suggested  ex-  t h a t the  sarcoplasmic p r o t e i n s i n s i t u were the s u b s t r a t e s f o r these p r o t e o l y t i c enzymes.  T h e i r r e s u l t s are i n agreement w i t h  those obtained by Martins and Whitaker (1968) which i n d i c a t e d that c a t h e p s i n D was  i n c a p a b l e of producing any d e t e c t a b l e  h y d r o l y s i s of actomyosin.  Oaldwell and Grosjean  (1971)  s t u d i e d the a c t i v i t y of cathepsins A, B, C and D on e x t r a c t s from chicken s k e l e t a l muscle.  T h e i r r e s u l t s are i n agreement  with those of l o d i e e et a l . (l966)which i n d i c a t e d t h a t c a t h epsins A, B, and C acted as a peptidase to f u r t h e r hydrolyze the breakdown products produced by c a t h e p s i n D, the major protease i n chicken muscle. Cathepsins are not as s u s c e p t i b l e as b a c t e r i a to d e s t r u c t i o n by i r r a d i a t i o n treatments.  Doty and Wachter (1955) r e p o r t e d  that an i r r a d i a t i o n dosage of 1.6 ately  Mrad r e s u l t e d i n approxim-  50fo r e d u c t i o n i n c a t h e p t i c a c t i v i t y .  have been r e p o r t e d by Schweigert Meegungwan (1962).  Similar results  (1959) and Rhodes and  Mohasseb (1962) concluded  t h a t a 1 Mrad  dose r e s u l t e d i n a t h r e e f o l d d e l a y i n the breakdown of p r o t e i n s to peptides and a t e n f o l d delay i n the breakdown of the peptides of amino a c i d s . 5 Mrad dose reduced  Lawrie  et a l . . (1961) found t h a t a c  the p r o t e o l y t i c a c t i v i t y i n bovine  p o r c i n e muscle by approximately  20 p e r c e n t .  and  These authors  8 concluded t h a t although p r o t e o l y t i c a c t i v i t y was some extent, the i r r a d i a t i o n treatment  reduced  to  d i d not l e a d t o any  sig-  n i f i c a n t d i f f e r e n c e s i n p r o t e o l y t i c breakdown over l o n g p e r i o d s of  storage. Numerous i n v e s t i g a t o r s have s t u d i e d the changes o c c u r r i n g  in  the s o l u b i l i t y of the v a r i o u s p r o t e i n f r a c t i o n s d u r i n g p o s t -  mortem aging of s k e l e t a l muscle.  Khan and van den Berg  (1964a)  s t u d i e d the changes i n p r o t e i n e x t r a c t a b i l i t y from 4- and  8-  month o l d b r o i l e r s s t o r e d a t 0, 2 and 5°C d u r i n g 7 weeks s t o r age.  The authors observed  t h a t storage temperature had  little  e f f e c t on t o t a l n i t r o g e n e x t r a c t a b i l i t y of the b r e a s t muscle. The amount of nonprotein n i t r o g e n and myosin f r a c t i o n s i n creased while the sarcoplasmic f r a c t i o n decreased storage p e r i o d . of  actomyosin.  There was  little  change i n the  over the  extractability  The decrease i n the sarcoplasmic f r a c t i o n  and  accompanying i n c r e a s e i n nonprotein n i t r o g e n suggested  that  p r o t e o l y s i s occurred d u r i n g the storage.  i n the  The decrease  e x t r a c t a b i l i t y of sarcoplasmic n i t r o g e n (100 - 500 mg N/lOO g of  muscle) could not be t o t a l l y accounted  f o r by the i n c r e a s e  i n n o n p r o t e i n n i t r o g e n (50 - 170 mg N/lOO g of muscle) or the l o s s of n i t r o g e n i n d r i p (30  - 90 mg N/lOO g of muscle).  The  authors p o s t u l a t e d t h a t s i n c e the i n c r e a s e i n the myosin f r a c t ion  was  not accompanied by a corresponding decrease  of the a c t o -  myosin f r a c t i o n , there may-be an i n t e r a c t i o n between myosin and the sarcoplasmic p r o t e i n s . van den Berg  (1964a) are s i m i l i a r to those r e p o r t e d by o t h e r  authors; Borton et. a l . et  The r e s u l t s r e p o r t e d by Khan and  (1970a) with p o r c i n e muscle, Ockerman  a l . (1969), Davey and G i l b e r t  (1968) and Locker  (i960) w i t h  9 bovine muscle and Mcintosh (1967) with b o v i n e , p o r c i n e and chicken muscles.  A l l of these authors r e p o r t e d decreases i n  the sarcoplasmic f r a c t i o n , i n c r e a s e s i n n o n p r o t e i n n i t r o g e n and s l i g h t i n c r e a s e s ,  decreases or notchange i n the m y o f i b -  r i l l a r f r a c t i o n d u r i n g low temperature While c o n s i d e r a b l e l i t e r a t u r e i n protein extractability  storage of muscle.  e x i s t s concerning changes  d u r i n g low temperature  storage,  l i t t l e e x i s t s r e g a r d i n g changes o c c u r r i n g d u r i n g h i g h temperature storage (25 to 37°C).  Sharp (1963) s t u d i e d a u t o l y s i s o f  bovine and r a b b i t muscle s t o r e d at 37°C. conducted at low temperatures, extractability  As i n the  t h i s researcher  studies  found t h a t the  of the sarcoplasmic p r o t e i n s decreased and n o n -  protein nitrogen increased.  A f t e r 10 days of storage at 37°C,  the n o n p r o t e i n n i t r o g e n i n c r e a s e d from 10 to 18$ of the  total  e x t r a c t a b l e n i t r o g e n i n bovine muscle and from 13 to 27$ f o r r a b b i t muscle.  The sarcoplasmic p r o t e i n s i n r a b b i t muscle d e -  creased to between 2.5 and 4$ of the t o t a l n i t r o g e n a f t e r days storage at 3 7 ° C bovine samples. ature storage,  The r a t e of decrease was slower i n  U n l i k e r e s u l t s obtained during low temperthe s o l u b i l i t y of the m y o f i b r i l l a r p r o t e i n s d e -  creased by approximately 26$ a f t e r 10 days s t o r a g e . concluded that t h i s ' d e c r e a s e  was the r e s u l t  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 .  The author  of heat d e n a t u r -  R e s u l t s obtained by  Sharp (1963) are i n agreement w i t h those of Lawrie et (1961).  19  al.  These authors a l s o 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 p r o t e i n s was g r e a t l y reduced w i t h h i g h storage of the meat.  temperature  N e i t h e r Sharp (1963) nor l a w r i e et  al.  (1961) found any evidence to suggest that the breakdown of c o l -  10 l a g e n or e l a s t i n occurred d u r i n g h i g h temperature a s e p t i c  stor-  age. Zender et a l . (1958) s t u d i e d a s e p t i c a u t o l y s i s of r a b b i 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 udate i n c r e a s e d throughout the storage p e r i o d .  ex-  The exudate  contained 7% by weight of p r o t e i n and only a s m a l l amount of m a t e r i a l 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  acid.  The p r o -  t e i n s present i n the exudate were s o l u b l e i n s o l v e n t s o f low i o n i c s t r e n g t h or d i s t i l l e d water.  The authors concluded that  the sarcoplasmic p r o t e i n s were present i n the  exudate.  Biochemical Changes O c c u r r i n g i n S p o i l e d Muscle The s i g n i f i c a n c e of b a c t e r i a l p r o t e o l y s i s d u r i n g meat s p o i l a g e has-been i n v e s t i g a t e d by a number of authors w i t h i n the l a s t 10 y e a r s . There i s disagreement as to the importance of b a c t e r i a l p r o t e o l y s i s i n meat s p o i l a g e .  Ingram and Dainty  (1971) r e p o r t e d that there was g e n e r a l agreement i n the  liter-  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  p o p u l a t i o n l e v e l s (10  organisms or g r e a t e r / 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 s p o i l e d on an o r g a n o l e p t i c b a s i s .  'tFay  (1970) s t a t e d that "the p r e c i s e r o l e s played by s p o i l a g e m i c r o organisms that r e s u l t i n the s p o i l a g e of meats are not w e l l understood at t h i s t i m e . " Lerke et, a l .  (1967) i n v e s t i g a t e d the r o l e of p r o t e i n i n  the s p o i l a g e of f i s h .  A nonpigmented Pseudomonas s p . ,  isolated  11 from s p o i l i n g f i s h ,  was used as the i n o c u l u m .  Filtration-  s t e r i l i z e d press j u i c e was used as a s u b s t r a t e . was- d i v i d e d i n t o 3 l o t s ;  a nonprotein f r a c t i o n ,  and the u n f r a c t i o n a t e d press  juice.  This material protein fraction  The authors measured i n -  creases i n v o l a t i l e r e d u c i n g substances,  total volatile n i t -  rogen and trimethylamine n i t r o g e n as c r i t e r i a f o r The b a c t e r i a grew w e l l i n a l l 3 f r a c t i o n s , fractionated increases  spoilage.  but only the u n -  j u i c e and the n o n p r o t e i n n i t r o g e n f r a c t i o n showed  i n the chemical c r i t e r i a  of s p o i l a g e .  The p r o t e i n  f r a c t i o n d i d not g i v e r i s e to s p o i l a g e products when i n o c u l a t e d with s p o i l a g e Jay  organisms.  (1966) s t a t e d that s t u d i e s on p r o t e i n degradation  by b a c t e r i a  d u r i n g s p o i l a g e of bovine muscle i n d i c a t e d that  the f l o r a of r e f r i g e r a t e d meats were i n c a p a b l e of such p r o teolytic  activity.  Jay and Kontou (1967) s t u d i e d the  effect  of b a c t e r i a l , growth on the f r e e amino a c i d s and n u c l e o t i d e s bovine semimembranosus muscle stored at 7°C.  of  When the ground  muscle was allowed to s p o i l w i t h i t s n a t u r a l f l o r a , no decrease was observed i n p r o t e i n l e v e l and there was no change i n amino acid composition.  Ground meat i n o c u l a t e d w i t h a  fluorescent  Pseudomonas s p . had a decrease i n amino a c i d s a f t e r 15 days incubation.  Uninoculated aseptic  i n c o n c e n t r a t i o n of amino a c i d s ,  samples showed due to a u t o l y s i s ,  the authors d i d not d e t e c t any p r o t e i n breakdown. i n o c u l a t e d w i t h a mixed f l o r a c o n s i s t i n g of a  increases although Samples  fluorescent  Pseudomonas s p . and an Achromobacter s p . showed decreases i n amino a c i d composition, although not as extensive sample i n o c u l a t e d w i t h the pure c u l t u r e .  as  the  The f a t e of n u c l e -  o t i d e s i n ground muscle d u r i n g b a c t e r i a l s p o i l a g e was s i m i l a r to that observed f o r amino a c i d s . r e s u l t e d i n a 15$ decrease; decrease;  Spoilage by n a t u r a l f l o r a  f l u o r e s c e n t Pseudomonas s p . 45$  mixed c u l t u r e 43$ and Pseudomonas f r a g i 43$ d e c r e a s e .  The authors concluded t h a t f r e e amino a c i d s and n u c l e o t i d e s are u t i l i z e d by the s p o i l a g e organisms before the are  proteins  attacked. Gardner and Stewart  (1966) i n v e s t i g a t e d the change i n  f r e e amino a c i d s o c c u r r i n g d u r i n g b a c t e r i a l s p o i l a g e of bovine muscle at 15°C with i t s n a t u r a l f l o r a , which c o n s i s t e d p r e dominantly of organisms b e l o n g i n g to the PseudomonasAchromobacter group.  These r e s e a r c h e r s noted that the concen-  t r a t i o n of most f r e e amino a c i d s -increased d u r i n g storage of the muscle.  Greatest i n c r e a s e s 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 i n c r e a s e i n tryptophan was due to a u t o l y s i s , while the i n c r e a s e 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 r e p o r t e d that  the m a j o r i t y of b a c t e r i a i s o l a t e d from s p o i l e d meat possessed glutaminase.  Increases i n ammonia were observed when the Q  b a c t e r i a l p o p u l a t i o n 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.  U n f o r t u n a t e l y , the  authors d i d not run a s e p t i c c o n t r o l s and, t h e r e f o r e ,  they c o u l d  not determine i f the i n c r e a s e i n f r e e amino a c i d s was a r e s u l t of b a c t e r i a l p r o t e o l y s i s or of a u t o l y s i s . Adamcic and C l a r k (1970) s t u d i e d induced chemical changes o c c u r r i n g i n chicken s k i n i n o c u l a t e d with a pigmented Pseudom—  anas, non-pigmented Pseudomonas and Achromobacter s t r a i n s and i n -  cubated a t 5 C.  A l l 3 s p e c i e s of b a c t e r i a brought about a  decrease in. t o t a l e x t r a c t a b l e n i t r o g e n ,  phenol-reagent-positive  and n i n h y d r i n - p o s i t i v e m a t e r i a l s and nonprotein n i t r o g e n d u r i n g l o g a r i t h m i c growth.  A f t e r t h i s phase the v a l u e s f o r the non-  pigment ed Pseudomonas and Achromobacter species remained cons t a n t or slowly i n c r e a s e d t o the l e v e l s i n the c o n t r o l , while the v a l u e s f o r the pigmented Pseudomonas s p e c i e s i n c r e a s e d rapidly.  The authors  the low molecular  concluded  t h a t these organisms u t i l i z e d  weight nitrogenous  compounds d u r i n g l o g -  a r i t h m i c growth then r e p l a c e d them through p r o t e o l y s i s d u r i n g the s t a t i o n a r y growth phase.  Adamcic e t a l . (1970) s t u d i e d  the e f f e c t o f the same 3 species o f p s y c h r o t o l e r a n t b a c t e r i a on the amino a c i d content  of chicken s k i n .  Results indicated  that the Achromobacter sp. and non-pigmented Pseudomonas sp. reduced the l e v e l of a l l f r e e amino a c i d s to below d e t e c t a b l e l e v e l s d u r i n g l o g growth phase.  Leucine,  phenylalanine,  ala-  ni ne , a s p a r t i c a c i d and glutamic a c i d were p r e f e r e n t i a l l y attacked.  These 2 s p e c i e s of b a c t e r i a d i d not r e l e a s e any  f r e e amino a c i d s through p r o t e o l y s i s .  On the other hand, d u r i n g  growth of the pigmented Pseudomonas sp. on chicken s k i n , the t o t a l gontent of f r e e amino a c i d s i n c r e a s e d by 30$.  approximately  The c o n c e n t r a t i o n o f f r e e p r o l i n e and h y d r o x y p r o l i n e i n -  creased 2 and 2.5 f o l d r e s p e c t i v e l y , i n d i c a t i n g p r o t e o l y s i s of c o l l a g e n by c o l l a g e n a s e . and  The nonpigmented Pseudomonas sp.  the Achromobacter sp. grew as r a p i d l y as the pigmented  Pseudomonas sp. on chicken s k i n and produced the c h a r a c t e r i s t i c p u t r i d o f f - o d o r s of s p o i l e d t i s s u e , although these  organisms  were not as p r o t e o l y t i c as the pigmented Pseudomonas sp. authors  concluded  The  t h a t s u p e r i o r p r o t e o l y t i c a c t i v i t y had l i t t l e  or no s i g n i f i c a n c e i n low temperature  s p o i l a g e of p o u l t r y musc-  le. Lea et a l . (1969) i n v e s t i g a t e d changes i n f r e e amino a c i d s of c h i c k e n breast muscle r e s u l t i n g from growth of p s y c h r o p h i l i c bacteria at 1 ° C .  In the u n i n o c u l a t e d c o n t r o l s ,  the n o n p r o t e i n  n i t r o g e n i n c r e a s e d by approximately 10$ while the t o t a l amino a c i d s i n c r e a s e d by 40 to 70$.  These r e s u l t s  free  indicate  that c o n s i d e r a b l e p r o t e o l y s i s had occurred a f t e r 8 days of i n cubation.  When samples were i n o c u l a t e d w i t h Pseudomonas  p u t r e f a c i e n s and a pigmented Pseudomonas s p . and the p o p u l a t i o n 8 xncreased to 10  / organisms/g, the p r o t e o l y t i c changes r e s u l t i n g  from t h e i r growth were s m a l l . changes,  After correcting f o r autolytic  the n o n p r o t e i n n i t r o g e n decreased by an average of  1.0$ while the f r e e amino a c i d s 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 .  Aspartic a c i d , threonine,  serine,  p r o l i n e and g l y c i n e were s e l e c t i v e l y u t i l i z e d by the pigmented Pseudomonas s p . w h i l e Pseudomonas p u t r e f a c i e n s  selectively  u t i l i z e d aspartic  When the p i g -  a c i d , threonine and s e r i n e .  mented Pseudomonas s p . a t t a i n e d a p o p u l a t i o n l e v e l of 10^, large increases sulted.  i n the c o n c e n t r a t i o n of a l l amino a c i d s r e -  Such a l a r g e i n c r e a s e  suggested " a p p r e c i a b l e  bacterial  Q .  p r o t e o l y s i s w i t h growth to 10 / g though none were found a t 10 " .  The authors d i d not d e t e c t any s i g n i f i c a n t i n c r e a s e i n  the q u a n t i t y of p e p t i d e s 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) i n v e s t i g a t e d the e f f e c t s p o i l a g e on the p r o t e i n e x t r a c t a b i l i t y  of b a c t e r i a l  from bovine muscle.  Samples were i n o c u l a t e d w i t h a Pseudomonas s.P. and an  Achromobacter sp. and incubated a t 3 C  Since the sarcoplasmic  f r a c t i o n decreased i n both the i n o c u l a t e d and u n i n o c u l a t e d samples, the authors concluded that a u t o l y s i s r a t h e r than b a c t e r i a l p r o t e o l y s i s was r e s p o n s i b l e f o r the m a j o r i t y 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 p r o t e i n s , but the s o l u b i l i t y of the stroma f r a c t i o n i n c r e a s e d i n the c o n t r o l and decreased 17.5  s i g n i f i c a n t l y i n the i n o c u l a t e d samples a f t e r  days i n c u b a t i o n .  The n o n p r o t e i n n i t r o g e n i n the i n o c u -  lated, samples i n c r e a s e d throughout  the storage p e r i o d but d i d  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 i n c u b a t i o n (25 d a y s ) . Rampton et a l . (1970) used sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , g e l f i l t r a t i o n and d i s c g e l e l e c t r o p h o r e s i s t o study the e f f e c t of Achromobacter l i q u e f a c i e n s , l u t e u s . Pediococcus  Micrococcus  c e r e v i s i a e , Pseudomonas f l u o r e s c e n s .  Streptococcus f a e c a l i s and n a t u r a l f l o r a from commercial hamburger on decomposition and r a b b i t muscle.  of m y o f i b r i l l a r p r o t e i n s i n p o r c i n e  None of the c u l t u r e s used i n the study  any measurable e f f e c t upon the m y o f i b r i l l a r p r o t e i n s .  had  A number  of "non-protein u l t r a v i o l e t l i g h t - a b s o r b i n g components" were present i n the d e n s i t y g r a d i e n t c e n t r i f u g a t i o n f r a c t i o n s of the Weber-Edsall  extracts.  The mixed f l o r a a l t e r e d the peak  h e i g h t s of these components, but the authors d i d not c h a r a c t e r i z e them.  The authors concluded that the b a c t e r i a f i r s t u t -  i l i z e d n o n p r o t e i n nitrogenous compounds, then u t i l i z e d  the  simplest proteinaceous components of the muscle, e s p e c i a l l y the sarcoplasmic p r o t e i n s .  Hasegawa et a l . (1970a) s t u d i e d the e f f e c t o f Pediococcus c e r e v i s i a e . Leuconostoc  mesenteroides.  Micrococcus  l u t e u s and  Pseudomonas f r a g i upon the sarcoplasmic and u r e a - s o l u b l e p r o t e i n f r a c t i o n s of p r o c i n e and r a b b i t muscle. and L. mesenteroides represented the l a c t i c organisms.  P. c e r e v i s i a e acid-producing  M. l u t e u s represented the s a l t t o l e r a n t m i c r o c o c c i  and P. f r a g i was chosen t o represent the p s y c h r o p h i l i c p r o t e o l y t i c organisms.  The pH of the c o n t r o l changed v e r y  little  over the storage p e r i o d , while the pH o f samples i n o c u l a t e d with the l a c t i c a c i d - p r o d u c i n g organisms decreased and, i n those samples i n o c u l a t e d with P. f r a g i o r M. l u t e u s . i n c r e a s e d . f r a g i caused  P.  extensive p r o t e o l y s i s of the sarcoplasmic p r o t e i n s  i n both r a b b i t and p o r c i n e t i s s u e s .  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 r a b b i t muscle but i t s a c t i o n was l e s s extensive on p o r c i n e muscle. Growth of P. c e r e v i s i a e r e s u l t e d i n p r o t e o l y s i s of the s a r c o plasmic p r o t e i n s from r a b b i t , but had no e f f e c t on p o r c i n e tissue.  M. l u t e u s showed minor p r o t e o l y s i s of the sarcoplasmic  f r a c t i o n e x t r a c t e d from r a b b i t muscle and no a c t i o n on p o r c i n e muscle.  Both P. f r a g i and P. c e r i v i s i a e e x h i b i t e d e x t e n s i v e  p r o t e o l y s i s of the u r e a - s o l u b l e f r a c t i o n of porcine muscle and to a l e s s e r extent on r a b b i t muscle.  L. mesenteroides and  M. l u t e u s e x h i b i t e d no d e t e c t a b l e p r o t e o l y t i c a c t i v i t y on the u r e a - s o l u b l e f r a c t i o n e x t r a c t e d from e i t h e r porcine or r a b b i t tissue.  The authors concluded  that the p r o t e o l y t i c  of s p o i l a g e b a c t e r i a was q u i t e s p e c i f i c .  activity  They b e l i e v e d t h a t  the b a c t e r i a produce " h i g h l y s p e c i f i c enzymes which p r e f e r e n t i a l l y a c t upon c e r t a i n p r o t e i n s or enzymes indigenous t o the muscle".  In another  study, Hasegawa  sj^sh.  (l970:b) s t u d i e d  the a c t i o n of C l o s t r i d i u m p e r f r i n g e n s , 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 zopf i  f a e c a l i s and Kurthea  on the u r e a - s o l u b l e and sarcoplasmic p r o t e i n s  from r a b b i t and procine m u s c l e . '  extracted  Spoilage due to growth of C.  p e r f r i n g e n s r e s u l t e d i n c o n s i d e r a b l e p r o t e o l y s i s of both f r a c t i o n s as judged by t h e a l t e r a t i o n s patterns.  i n their gel  electrophoresis  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 t h e r the sarcoplasmic or u r e a - s o l u b l e Borton et a l . coccus c e r e v i s i a e .  fractions.  (1970a) i n v e s t i g a t e d the e f f e c t Leuconostoc mesenteroides.  of P e d i o -  Micrococcus  l u t e u s and Pseudomonas f r a g i on the s o l u b i l i t y of v a r i o u s protein fractions  of p o r c i n e muscle.  f o r 20 days at 2 and 1 0 ° C .  Samples were incubated  I n the c o n t r o l , w a t e r - s o l u b l e  nit-  rogen and i n s o l u b l e p r o t e i n n i t r o g e n decreased while n o n p r o t e i n n i t r o g e n i n c r e a s e d over the storage p e r i o d .  Salt-  s o l u b l e p r o t e i n n i t r o g e n i n c r e a s e d d u r i n g the f i r s t 8 days of storage then remained constant  or decreased  slightly.  There  was no a l t e r a t i o n i n p r o t e i n s o l u b i l i t y p a t t e r n s i n samples i n o c u l a t e d with P . c e r e v i s i a e with L . mesenteroides  or M. l u t e u s •  Samples i n o c u l a t e d  and incubated at 2°C e x h i b i t e d s o l u b i l i t y  p a t t e r n s 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  s o l u b l e p r o t e i n 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 d i f f e r e n c e i n n o n p r o t e i n nitrogen.  There was no d i f f e r e n c e i n the e x t r a c t a b i l i t y  the s a l t - s o l u b l e p r o t e i n u n t i l a f t e r 20 days storage,  of  at which  time a c o n s i d e r a b l e decrease was noted i n the i n o c u l a t e d samples.  The decrease was accompanied by an i n c r e a s e  i n the  insoluble protein nitrogen.  The authors r e p o r t e d t h a t l o s s  i n s o l u b i l i t y of the s a l t - s o l u b l e p r o t e i n was crease i n pH r e s u l t i n g from growth of L.  due to the  de-  mesenteroides.  Porcine muscle i n o c u l a t e d with P. f r a g i showed the g r e a t est changes i n p r o t e i n s o l u b i l i t i e s . water-soluble p r o t e i n decreased then i n c r e a s e d and was trol.  The  f o r the f i r s t 4 days at  10°C,  s i g n i f i c a n t l y h i g h e r than i n the con-  s a l t - s o l u b l e p r o t e i n decreased  a t i o n a t 10°0. was  The s o l u b i l i t y o f the  a f t e r 4 days i n c u b -  The i n c r e a s e i n the w a t e r - s o l u b l e  fraction  i n v e r s e l y c o r r e l a t e d (P < .01) with the p r o t e o l y s i s of the  s a l t - s o l u b l e f r a c t i o n ( r = -.37). i o n tended  to decrease  throughout  The i n s o l u b l e p r o t e i n f r a c t the storage p e r i o d while the  nonprotein n i t r o g e n f r a c t i o n g r e a t l y i n c r e a s e d .  The i n c r e a s e  i n nonprotein n i t r o g e n i n d i c a t e d to the authors t h a t extensive p r o t e o l y s i s occurred d u r i n g s p o i l a g e of p o r c i n e muscle by  P.  fragi. Borton et a l . (1970b) i n o c u l a t e d 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 a l t e r e d the e l e c t r o p h o r e t i c p a t t e r n of the s a l t - s o l u b l e proteins.  The r e s u l t s obtained i n t h i s study confirmed  obtained i n t h e i r previous study t h a t of the 4 organisms  those em-  ployed, only P. f r a g i caused a decrease i n the number o f e l e c t r o p h o r e t i c bands of m y o f i b r i l l a r p r o t e i n s . Tarrant et a l . (1971) s t u d i e d the a c t i o n of Pseudomonas f r a g i on the p r o t e i n s of p o r c i n e s k e l e t a l muscle incubated at 10°0.  The r e s u l t s obtained i n t h e i r study were i n agree-  ment with those of Borton et a l . (1970a).  The meat  judged s p o i l e d a f t e r 5 days of i n c u b a t i o n at 10°C,  was but  no  s i g n i f i c a n t changes were observed i n p r o t e i n s o l u b i l i t y 20 days of i n c u b a t i o n .  until  A f t e r 20 days of i n c u b a t i o n , the myofib-  r i l l a r p r o t e i n e x t r a c t decreased by 2/3  of i t s o r i g i n a l  value.  D i s c e l e c t r o p h o r e s i s of the m y o f i b r i l l a r p r o t e i n e x t r a c t showed that the e l e c t r o p h o r e t i c p a t t e r n was a f t e r 20 days s p o i l a g e .  completely  Nonprotein n i t r o g e n i n c r e a s e d approx-  i m a t e l y 3 times because of the i n c r e a s e s i n peptides ammonia.  The  altered  pH i n c r e a s e d from 5.4  the accumulation of ammonia.  to 7.9  There was  no  and  a t t r i b u t a b l e to significant  ference i n the s o l u b i l i t y of the sarcoplasmic  proteins.  authors concluded that p r o t e o l y s i s of the sarcoplasmic t e i n s was  not detected because of decrease i n the  p r o t e i n f r a c t i o n was  o f f s e t by the r e l e a s e of  difThe pro-  sarcoplasmic  water-soluble  f r a c t i o n s 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 protein  fraction.  Numerous authors (Jay and Kontou, 1967; 1969;  l e a .et a l . , 1969;  Qckerman et a l . ,  Adamcic and C l a r k , 1970;  1970a; Hasegawa et al.-, 1970a and  Borton et a l . ,  Tarrant ejt a l . , 1971)  r e p o r t e d t h a t the pH of muscle i n c r e a s e s d u r i n g s p o i l a g e  have un-  l e s s s p o i l a g e i s caused by an a c i d - p r o d u c i n g  organism  (Hasegawa et a l . , 1970a).  (1971) r e p o r t e d  that numerous proposals  Ingram and Dainty  have been made to use  the i n c r e a s e i n  pH as an index of s p o i l a g e , but pH does not c o r r e l a t e w e l l with the b a c t e r i a l l o a d . Jay and Kontou (1967),. Ockerman et §1.(1969) and Adamcic C l a r k (1970) reported decreases i n the e x t r a c t r e l e a s e volume  and  during b a c t e r i a l spoilage.  Jay  (1964)" f i r s t d e s c r i b e d  phenomena termed e x t r a c t r e l e a s e volume (ERV).  the  Homogenates  of s p o i l e d meat were more v i s c o u s than homogenates prepared from meat o f good m i c r o b i a l q u a l i t y . homogenate, the lower the ERV. ERV  The more v i s c o u s a  The author r e p o r t e d  that  was g r e a t e s t between pH 5.0 and 5.8 and e s s e n t i a l l y  above pH 11 and  below pH 4.9.  zero  Jay (1964) t r e a t e d samples o f  bovine muscle with 2 commercial proteases and showed that  the  ERV  d e c l i n e d as i n meat which underwent b a c t e r i a l  spoilage.  Jay  (1966) presented 2 t h e o r i e s t o e x p l a i n the ERV phenomena.  One  theory i s that p r o t e o l y s i s r e s u l t s i n an unmasking o f  water b i n d i n g s i t e s o f the meat p r o t e i n s , thus i n c r e a s i n g water h o l d i n g c a p a c i t y  (WHC).  The second theory  t e n s i v e p r o t e o l y s i s o f the p r o t e i n s does not  i s t h a t ex-  occur but  s t r u c t u r e o f the n a t i v e p r o t e i n i s "loosened".  the  the  The l a t t e r  theory has been expressed by Hamm (i960) i n h i s review on the water h o l d i n g c a p a c i t y o f meat. ported  Ingram and  Dainty  (1971) r e -  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) r e p o r t e d  that the WHC of meat was d i s -  t r i b u t e d i n muscle f i b e r s as f o l l o w s : p r o t e i n s , 5$ due t o water-soluble s o l u b l e nonproteins.  65$ due t o s t r u c t u r a l  p r o t e i n s and 30$ to water-  He a l s o s t a t e d that not more than 5$  of the water i n muscle i s t i g h t l y bound t o the p r o t e i n s and the amount o f bound water i s "hardly i n f l u e n c e d by changes i n s t r u c t u r e and changes of the p r o t e i n s . "  The WHC of meat  i s determined by the amount o f f r e e water which can be imm o b i l i z e d w i t h i n the s p a t i a l s t r u c t u r e o f the p r o t e i n s .  Hamm  (i960) r e f e r s t o changes i n the MHO  i n terms of a " l o o s e n i n g "  or " t i g h t e n i n g " of p r o t e i n s t r u c t u r e . that the WHO  The author r e p o r t e d  of meat was minimal around pH 5 and had  maxima; one at pH 10 and the other at pH 4 . muscle approaches i m a t e l y pH 5)  two  As the pH of the  the i s o e l e c t r i c point of actomyosin  (approx-  the net charge on the p r o t e i n i s decreased.  Thus  the e l e c t r o s t a t i c r e p u l s i o n between adjacent p e p t i d e chains decreases and they can a s s o c i a t e more c l o s e l y with the r e s u l t that l e s s water can be immobilized between them.  As the pH  i s decreased or i n c r e a s e d from the i s o e l e c t r i c p o i n t , the e l e c t r o s t a t i c r e p u l s i o n between the peptide chains i s i n c r e a s e d thus l o o s e n i n g the p r o t e i n s t r u c t u r e r e s u l t i n g i n more imm o b i l i z e d water between the peptide c h a i n s . U l t r a s t r u c t u r e of S t r i a t e d S k e l e t a l Muscle E x c e l l e n t reviews on the u l t r a s t r u c t u r e of s t r i a t e d muscle i n c l u d e those of B r i s k e y and Fukazawa (1971), G a u t h i e r (1970), Peachey (1970), B e n d a l l (1969), S l a u t t e r b a c k (1966) and Lawrie  (1966).  Muscle i s surrounded by a sheath of connective t i s s u e termed the epimysium, which binds the muscle bundles t o g e t h e r to form muscle.  Prom the epimysium f i n e r sheets of connective  t i s s u e , termed the perimysium, form muscle bundles.  surround the muscle f i b r e s t o  The endomysium, which c o n s i s t s of y e t  f i n e r connective t i s s u e , a r i s e s from the perimysium rounds each i n d i v i d u a l muscle f i b e r . posed of three l a y e r s .  Immediately  The endomysium i s comadjacent t o the f i b e r i s  a double l a y e r e d membrane approximately 100 A t h i c k the plasmalemma or sarcolemma.  and s u r -  termed  The middle l a y e r , approximately  500 A t h i c k , i s composed of mucopolysaccharides. l a y e r c o n s i s t s o f a network of collagenous  fibers.  The outer Bendall  (1969) and Lawrie (1966) r e f e r t o t h i s 3 l a y e r e d sheath as the endomysial l a y e r , while S c h a l l e r and Powrie (1971) and Kono et  a l . (1964) c a l l i t the sarcolemma; a term which B e n d a l l  (1969) and Lawrie (1966) reserve f o r the i n n e r plasmalemma. The muscle f i b e r i s a l o n g m u l t i n u c l e a t e d c e l l approxi m a t e l y 20 t o 80 pm  i n diameter and of v a r i a b l e l e n g t h .  The  f i b e r c o n s i s t s of anywhere from 1000 t o 2000 m y o f i b r i l s , each 1 to 2 jjm i n diameter.  The f u n c t i o n a l u n i t o f the m y o f i b r i l  i s the sarcomere, which i s the d i s t a n c e between 2 adjacent Z lines.  In the r e l a x e d s t a t e , the sarcomere i s between 2.5  and~j>yen In l e n g t h . The s t r i a t e d appearance o f s k 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 f i l a m e n t s w i t h i n the sarcomere.  The s t r u c t u r e o f the sarcomere i s represented  diagramatically i n Figure I .  The t h i n a c t i n f i l a m e n t s ,  which a r i s e from the Z l i n e and run between the myosin f i l a m e n t s and some d i s t a n c e i n t o the A Band, account f o r l i g h t I band. The denser A band r e s u l t s from an o v e r l a p p i n g of the a c t i n and t h i c k e r myosin f i l a m e n t s .  The l i g h t e r H zone, present i n  the middle of the A band, c o n s i s t s s o l e l y of myosin f i l a m e n t s . Present  i n the H zone i s a c e n t r a l dark r e g i o n termed the M  band, where the myosin f i l a m e n t s a r e j o i n e d t a i l t o t a i l (Bendall, 1969). The m y o f i b r i l s a r e surrounded by a complex membrane The terminology of B e n d a l l (1969) and Lawrie (1966) w i l l be followed i n t h i s t h e s i s .  23  1  A Band  1 Band  1 Band  H Zone 1  .  1  M Band  Z Line  Z Line Myosin Filament  P i g . 1.  Structure  Actin Filament  o f a Sarcomere  24 bound system of tubules and v e s i c l e s termed the reticulum.  The  sarcoplasmic  sarcoplasmic r e t i c u l u m c o n s i s t s of two p a r t s ;  l o n g i t u d i n a l tubules o r i e n t e d p a r a l l e l to the f i b r i l s transverse tubules. r e t i c u l u m to be two  Peachey (1970) c o n s i d e r s the distinct  and  sarcoplasmic  systems, but r e p o r t s t h a t other  authors c o n s i d e r i t to be one.  Peachey (1970) r e f e r s t o the  l o n g i t u d i n a l t u b u l e s as the sarcoplasmic r e t i c u l u m and  treats  the t r a n s v e r s e tubules ( o r T system) as a separate system.  The  t r a n s v e r s e 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 t r a n s v e r s e l y across  the muscle f i b e r .  The  t r a n s v e r s e tubule i s continuous  with  the saroolemma. Near the H zone, the l o n g i t u d i n a l tubules are f l a t t e n e d forming a p e r f o r a t e d sheet termed the f e n e s t r a t e d c o l l a r .  On  each s i d e of t h i s c o l l a r , the l o n g i t u d i n a l tubules l e a d i n t o a t e r m i n a l cisternum, o f t e n through i n t e r m e d i a t e f l a t t e n e d cisterna.  The t r a n s v e r s e tubule l i e s between two  adjacent  t e r m i n a l c i s t e r n a ; together these three elements form a s t r u c t ure known as a t r i a d .  T r i a d s are l o c a t e d a t the A-I band  j u n c t i o n i n h i g h e r v e r t e b r a t e s and a t the Z l i n e i n the  lower  vertebrates. Mendell muscle.  (1971) s t u d i e d the T system i n chicken p e e t o r a l i s  The t r i a d s were observed  verse tubules were observed back on themselves"  at the Z l i n e and the t r a n s -  to make " l a t e r a l turns or  double  r a t h e r than f o l l o w the u n i n t e r r u p t e d t r a n s -  verse p a t t e r n u s u a l l y observed Fukazawa et al.(1969)  i n s k e l e t a l muscle.  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  z i g z a g c o n f i g u r a t i o n , but such a c o n f i g u r a t i o n was not i n muscle aged 24 h r at  5°C  The authors  evident  reported that  the  m y o f i b r i l s tended to break at the Z - I j u n c t i o n when aged 24 h r .  Davey and G i l b e r t ( 1 9 6 7 ) r e p o r t e d complete  for  dis-  i n t e g r a t i o n of the Z band and apparent l e n g t h e n i n g of the A band i n bovine muscle aged at 1 5 ° G f o r 3 days. S c h a l l e r and Powrie ( 1 9 7 1 ) used the scanning microscope to- study s t r i a t e d t r o u t and t u r k e y .  electron  s k e l e t a l muscle of beef,  F i b r i l s from t r o u t and beef were surrounded  by e l e v a t e d t r a n s v e r s e elements which the authors to c o n s i s t system.  of both the sarcoplasmic  which the authors  considered  r e t i c u l u m and the T  I n p r e - r i g o r bovine muscle,  verse elevation,  rainbow  the most prominent t r a n s -  considered to be the  trans-  verse t u b u l e , had a smaller continuous r i d g e on e i t h e r s i d e of it.  The authors  considered these r i d g e s to be the  terminal  c i s t e r n a which, together with the prominent t r a n s v e r s e r i d g e , form a t r i a d . elevations  In p r e - r i g o r turkey muscle,  these  secondary  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, sarcoplasmic  presumably due to the extensiveness of  reticulum.  The endomysium appeared to  of a network of w e l l d e f i n e d s t r a n d s .  The surface  the  consist of the  endomysium showed pronounced t r a n s v e r s e r i d g e s which the authors b e l i e v e d to be the convex impression on the dneomysium of the u n d e r l y i n g t r a n s v e r s e elements  on the m y o f i b r i l s .  During the postmortem aging of turkey muscle, elements  took on a rough appearance,  the  transverse  i n d i c a t i n g that d i s r u p t i o n  of the t r a n s v e r s e elements was o c c u r r i n g .  A f t e r aging f o r 6  days at 3 G, the t r a n s v e r s e  elements appeared t o 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 s s u e d i s i n t e g r a t i o n had taken p l a c e . Only one paper was found r e g a r d i n g 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  Dutson et a l . (1971) s t u d i e d the u l t r a s t r u c t u r a l  spoilage.  changes  o c c u r r i n g i n porcine muscle i n o c u l a t e d w i t h Pseudomonas f r a g i . The samples were judged to be s p o i l e d a f t e r 8 days of i n c u b ation at 1 0 ° 0 .  A f t e r an 8 day i n c u b a t i o n p e r i o d , the myo-  f i b r i l s appeared d i s r u p t e d i n the A band r e g i o n and the H zone was almost devoid of m a t e r i a l .  Myosin f i l a m e n t s were not  evident and the dense m a t e r i a l c h a r a c t e r i s t i c had been l o s t .  of the Z l i n e  The authors concluded that p r o t e o l y s i s o f 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 m a t e r i a l from the Z l i n e ,  as a r e s u l t of  s p o i l a g e by P . f r a g i . Dutson et a l . (1971) observed p r o t r u s i o n s ,  containing a  dense g r a n u l a r m a t e r i a l ,  on the surface  s p o i l e d porcine muscle,  Globules c o n t a i n i n g a dense m a t e r i a l  was observed adjacent  to the b a c t e r i a .  of P . f r a g i growing on  No p r o t r u s i o n s were  observed on the b a c t e r i a when the organisms were grown on a n o n p r o t e i n media.  The authors p o s t u l a t e d that  proteolytic  enzymes may be s e c r e t e d i n t o the p r o t r u s i o n s , which then break away from the b a c t e r i a l c e l l forming the g l o b u l e s .  These  g l o b u l e s then r e l e a s e the enzymes i n t o the t i s s u e surrounding the b a c t e r i a l  cell.  27 Wiebe and Chapman (1968a and b) r e p o r t e d that some s t r a i n s of Pseudomonas produced e v a g i n a t i o n s or b l e b s on the cell wall. feature,  I n some s t r a i n s ,  while i n o t h e r s ,  b l e b formation was a s t a b l e  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 c o n d i t i o n s . r e p o r t e d that c e r t a i n s t r a i n s  The authors  of marine Pseudomonas produced  the c e l l u l a r evaginations when grown at h i g h ( 2 2 ° C ) i n a h i g h peptane media ( 1 . 0 $ ) .  temperature  The authors were u n -  c e r t a i n as to the f u n c t i o n played by the observed e v a g i n a t i o n s . Smirnova et a l . (1971) level,  s t u d i e d , at the u l t r a s t r u e t u r a l  t o x i n and enzyme s e c r e t i o n i n C l o s t r i d i u m p e r f r i n g e n s .  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 m a t e r i a l protruded through channels which connected the p e r i p l a s m i c space of the c e l l with the e x t e r n a l media.  The amorphous m a t e r i a l formed "microcapsulae"  on the c e l l s u r f a c e .  The m a t e r i a l i n t h i s capsule was separated  from both the cytoplasm and the e x t e r n a l media.  The m a t e r i a l  was s e c r e t e d i n t o 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 m i c r o c a p s u l e s . a l k a l i n e phosphatase. sule s contracted  The authors s t a i n e d B . s u b t i l i s f o r  The amorphous m a t e r i a l i n the m i c r o c a p -  i n t e n s e l y with the c e l l ,  w h i l e i n s t r a i n s which  d i d not produce the enzyme the c e l l was c o n t r a s t e d  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 b a s i s f o r the s e c r e t i o n of macromolecular compounds produced by the b a c t e r i a l  cell.  METHODS Part I - Biochemical S t u d i e s Sample P r e p a r a t i o n Commercial  f r y e r s were obtained immediately a f t e r  slau-  ghter from the packing p l a n t and t r a n s p o r t e d to the l a b o r a t o r y i n crushed i c e . The b r e a s t 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 a t 3°C f o r 24 h r .  P e c t o r a l i s major and minor were e x c i s e d and  ground i n an e l e c t r i c meat g r i n d e r .  The minced muscle  was  then d i v i d e d i n t o 40 g l o t s and wrapped i n Saran f i l m .  The  wrapped samples were p l a c e d 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.) c o n t a i n i n g Co 60.  A f t e r i r r a d i a t i o n , . s a m p l e s were t r a n s -  f e r r e d a s e p t i c a l l y i n t o s t e r i l e 300-ml Erlenmeyer f l a s k s  fitted  with c o t t o n p l u g s . A pure c u l t u r e of Pseudomonas f r a g i (ATCC 4973) was d i l u t e d 200 f o l d w i t h s t e r i l e phosphate b u f f e r .  10 m i l l i l i t e r s  of the d i l u t e d c u l t u r e were t r a n s f e r r e d i n t o 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 f l a s k were mixed to ensure even d i s t r i b u t i o n of the inoculum. f o r 0, 2 . 5 ,  5.5  F l a s k s were incubated at approximately 25°C and 9.5  days.  C o n t r o l samples were t r e a t e d  i n the i d e n t i c a l manner except 10 ml of s t e r i l e b u f f e r was added t o the Erlenmeyer f l a s k s .  phosphate  The a d d i t i o n of  the b u f f e r was necessary to prevent dehydration of the c o n t r o l 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 e n t i r e contents of the  mortar were t r a n s f e r r e d to an 11-ml d i l u t i o n b l a n k , then s t a n d ard  p l a t e counts were determined.  P l a t e counts were determined  on both i n o c u l a t e d and u n i n o c u l a t e d samples.  P l a t e count agar  was used as the p l a t i n g medium and a l l p l a t e s were incubated a t approximately 25°C f o r 48 h r . Protein Extraction The procedures o u t l i n e d by Hasegawa et a l . (1970a) were adopted f o r the e x t r a c t i o n and s e p a r a t i o n of the w a t e r - s o l u b l e , s a l t - s o l u b l e and u r e a - s o l u b l e p r o t e i n f r a c t i o n s and are o u t l i n e d i n F i g u r e 2. A l l e x t r a c t i o n at  procedures were c a r r i e d out  5 ° C F o r t y m i l l i l i t e r s o f 0.3 M sucrose - 0.01 M K C l - 0.01  M T r i s b u f f e r (pH 7.6) S o r v a l l Omni-Mixer. for  were added to 1 0 g of minced t i s s u e i n a  The sample was homogenized a t top speed  10 sec and at h a l f speed f o r 50 s e c .  The homogenate was  c e n t r i f u g e d a t 20,000 x G f o r 15 min i n a r e f r i g e r a t e d The  supernatant,  centrifuge.  which c o n s i s t e d of the sarcoplasmic p r o t e i n  and n o n p r o t e i n n i t r o g e n f r a c t i o n , was decanted and s t o r e d at 5°C.  The p r e c i p i t a t e was r e t u r n e d to the homogenizer and 60 ml  of the sucrose - K C l - T r i s b u f f e r added.  The p r e c i p i t a t e was  homogenized f o r 3 0 sec a t one-quarter speed.  The mixture was  c e n t r i f u g e d at 20,000 x G f o r 15 min and the supernatant carded.  dis-  The p r e c i p i t a t e was then homogenized w i t h 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 ^ ) for  30 sec at one-quarter speed.  The mixture was s t o r e d f o r 2 4  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 c e n t r i f u g e d 15 min a t 20000 x G •Supernatant  - Sarcoplasmic P r o t e i n and n o n protein nitrogen  Precipitate homogenized with 6 v o l of sucrose - KC1 - T r i s centrifuged "Supernatant  -discard  Precipitate homogenized with 6 v o l of Weber-Edsall s o l u t i o n s t o r e d f o r 24 h r 18 v o l of Weber-Edsall s o l u t i o n added and c e n t r i f u g e d 29000 x G f o r 30 min •Supernatant  at  - myofibrillar protein  Precipitate homogenized w i t h 6 v o l of Weber-Edsall s o l u t i o n and c e n t r i f u g e d at 29000 x G f o r 30 min Supernatant  -  discard  Precipitate homogenized with 4 v o l 8 M urea and c e n t r i f u g e d at 29000 x G f o r 30 min •Supernatant Precipitate Pig.  2.  urea s o l u b l e protein  discard  Plow Sheet of P r o t e i n E x t r a c t i o n  Procedure  hours a t 5 ° C was  A f t e r storage, 180 ml of Weber-Edsall  solution  added and the s o l u t i o n mixed with a magnetic s t i r r e r f o r  15 min.  The mixture was  then c e n t r i f u g e d f o r 30 min a t  29,000 x G.  The supernatant c o n s i s t i n g of the s a l t - s o l u b l e  f r a c t i o n was  decanted o f f and s t o r e d at 5 ° C  The  precipitate  was homogenized with 60 ml of Weber-Edsall s o l u t i o n f o r 30 sec at one-quarter speed.  The s o l u t i o n was  and the supernatant d i s c a r d e d .  c e n t r i f u g e d as b e f o r e  The p r e c i p i t a t e was  homogenized  with 40 ml of 8M- urea a t f u l l speed f o r 10 sec and h a l f f o r 50 sec.  speed  The supernatant, c o n t a i n i n g the u r e a - s o l u b l e p r o -  t e i n f r a c t i o n , was  r e t a i n e d and the p r e c i p i t a t e d i s c a r d e d .  N i t r o g e n Determination N i t r o g e n content of the water-soluble e x t r a c t  consisting  of the sarcoplasmic p r o t e i n and nonprotein n i t r o g e n f r a c t i o n , s a l t - s o l u b l e e x t r a c t c o n s i s t i n g of the m y o f i b r i l l a r p r o t e i n s and n o n p r o t e i n n i t r o g e n f r a c t i o n was Kjeldahl analysis.  determined  Nonprotein n i t r o g e n was  by micro-  obtained by p r e -  c i p i t a t i o n of the water-soluble p r o t e i n s with an equal volume of 20$  (W/V)  N i t r o g e n was  t r i c h l o r o a c e t i c a c i d (Tarrant et a l . , 1971). expressed as mg N/g  fresh tissue.  Gel F i l t r a t i o n G e l f i l t r a t i o n was i n a column 2.5  c a r r i e d out u s i n g Sephadex G-50  cm i n diameter and 32 cm i n l e n g t h .  of the Sephadex column was 1966).  Packing  done a c c o r d i n g t o i n s t r u c t i o n (Anon.  The eluant c o n s i s t e d of d e i o n i z e d water c o n t a i n i n g 2$  sodium a z i d e as a b a c t e r i o s t a t i c agent. min.  fine  Flow r a t e was 1.3  F r a c t i o n s were c o l l e c t e d by means of a drop  counter  ml/  yielding fractions  of 3 . 2 - 0 . 1 m l .  determined with blue d e x t r a n .  The v o i d volume (Vo) was  The e f f l u e n t volume (Ve)  glutamic a c i d was determined by n i n h y d r i n r e a c t i o n Stein  for  of Moore and  (1954).  The w a t e r - s o l u b l e p r o t e i n 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 filtrate  to g i v e 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  u t i o n were l a y e r e d on t o p o f the Sephadex bed.  the sol-  The e l u t i o n  p a t t e r n was analysed u s i n g the n i n h y d r i n r e a c t i o n of Moore and Stein  (1954).  Disc Gel Electrophoresis Polyacylamide d i s c g e l e l e c t r o p h o r e s i s the sarcoplasmic, ions.  was performed on  m y o f i b r i l l a r and u r e a - s o l u b l e p r o t e i n f r a c t -  The procedure o u t l i n e d by Davis (1964) was f o l l o w e d to  produce a 7$ running g e l w i t h a 2 . 5 $ spacer g e l .  For  p h o r e s i s of the m y o f i b r i l l a r and u r e a - s o l u b l e p r o t e i n  electrofractions,  s u f f i c i e n t urea was added to the g e l s to produce running and spacer g e l s c o n t a i n i n g 4 M u r e a .  B u f f e r used f o r  was 0.025 M T r i s - 0 . 1 9 M g l y c i n e pH 8 . 3 . tube was a p p l i e d and e l e c t r o p h o r e s i s  A current  run at room  electrophoresis of 3 ma per temperature.  One m i l l i l i t e r of 0 . 0 0 1 $ bromophenol blue was added to the b u f f e r r e s e r v o i r as a t r a c k i n g dye.  top  E l e c t r o p h o r e s i s was com-  p l e t e d when the t r a c k i n g dye was approximately 0 . 5 cm from the end of the g e l . A f t e r completion of the e l e c t r o p h o r e t i c removed from the e l e c t r o p h o r e s i s dermic s y r i n g e .  r u n , the g e l s were  tubes with the a i d of a hypo-  The g e l s were placed i n t e s t tubes and s t a i n e d  f o r 15 min with a s o l u t i o n of 1.0$ Amido Black 10B i n 10$ acetic acid.  Gels were destained i n 7 $ a c e t i c a c i d u n t i l the  background was c l e a r  (approximately  3 days).  oscan was used to t r a c e the e l e c t r o p h o r e t i c ference  A Joyce Chrompatterns.  An i n t e r -  f i l t e r with a maximum transmittance at 580 nm was u s e d .  R e s u l t s obtained d u r i n g p r e l i m i n a r y i n v e s t i g a t i o n s i n d i c a t e d that the best r e s o l u t i o n of the v a r i o u s could be obtained as f o l l o w s .  The sarcoplasmic  fractions f r a c t i o n was  d i l u t e d 1:4 by volume w i t h 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 u r e a ;  0.1 ml of the  d i l u t e d p r o t e i n s o l u t i o n was then l a y e r e d on top of the spacer gel.  The p r o t e i n c o n c e n t r a 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 gel.  of the u r e a - s o l u b l e  fraction  a p p l i c a t i o n to the polyacrylamide  The u r e a - s o l u b l e 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 e x c l u s i o n l i m i t of 30,000 molecular weight.  After  concentration,  0.1 ml of the concentrate was appled to the g e l . Free Amino A c i d A n a l y s i s Sample p r e p a r a t i o n f o r f r e e amino a c i d a n a l y s i s was c a r r i e d out a c c o r d i n g to the method o u t l i n e d by T a l l a n e_t a l . (1954).  Ten grams of minced t i s s u e were blended with 100 ml  of 1$ p i c r i c a c i d i n a Waring Blendor at top speed f o r 2 m i n . The m a t e r i a l was c e n t r i f u g e d at 5000 x G f o r 5 min.  The s u p e r -  natant was f i l t e r e d through Whatman No. 1 f i l t e r paper t o r e move f a t *  The f i l t r a t e  was then passed through a Dowex 2-X10  ( c h l o r i d e 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 h i g h i n a 2.5 by 20 cm chromatography column.  P r i o r to use, the r e s i n was of IN HOI  regenerated w i t h 6-5 ml p o r t i o n s  then washed with d i s t i l l e d water u n t i l the e f f l u e n t  was n e u t r a l .  The f i l t r a t e was a p p l i e d to the column and  the  f i r s t 25 ml were passed through the column and d i s c a r d e d .  The  next 50 ml of e f f l u e n t were c o l l e c t e d and d r i e d at 55°C i n a r o t a r y evaporator under vacuum.  The r e s i d u e was  taken up i n  10 ml of pH 2.2 amino a c i d d i l u t i o n b u f f e r and s t o r e d a t 5°G until  analysis. Amino a c i d a n a l y s i s was  performed  on a Phoenix Model  M 6800 amino a c i d a n a l y s e r u s i n g the Moore-Stein two  column  system. Part II  - E l e c t r o n Microscopy  Commercial chicken f r y e r s , obtained from a r e t a i l were used throughout  the e l e c t r o n microscopy  lowing procedure was adopted p e c t o r a l i s muscle.  outer 0.5  fol-  removed and the s u r f a c e of  f l o o d e d with a b s o l u t e e t h a n o l .  cm of the muscle was  approximately 0.5  The  to o b t a i n s t e r i l e samples of the  The s k i n was  the b r e a s t muscle was  study.  outlet,  The  removed and s t r i p s of muscle  cm i n width and 3 to 5 cm i n l e n g t h were  removed from the i n t e r n a l p o r t i o n of the p e c t o r a l i s muscle. These s t r i p s were p l a c e d i n a s t e r i l e p e t r i p l a t e and cut i n t o cubes of approximately 0.5  cm.  These cubes were then c r y o -  f r a c t u r e d by the f o l l o w i n g method. An aluminum weighing d i s h c o n t a i n i n g isopentane floated i n l i q u i d nitrogen.  When the isopentane had  was gelled,  the d i s h was  removed and a cube of muscle immersed i n the  isopentane.  A f t e r the t i s s u e had f r o z e n , i t was h i t with a  p l e x i g l a s s r o d , p r e v i o u s l y cooled i n l i q u i d n i t r o g e n , cryofracture  the m a t e r i a l .  to  The r e s u l t i n g muscle fragments  were no g r e a t e r than 2 mm i n diameter. were t r a n s f e r r e d onto s t e r i l e  The f r o z e n fragments  stainless steel tissue  grids within a s t e r i l e p e t r i plate.  culture  The bottom of such p e t r i  p l a t e was covered with 3 MM chromatography paper. The c r y o f r a c t u r e d samples were i n o c u l a t e d , by means of an i n o c u l a t i n g l o o p , from a 3 day o l d c u l t u r e of Pseudomonas f r a g i (ATGO 4973) grown i n n u t r i e n t b r o t h .  The s t e r i l e  t r o l s were i n o c u l a t e d w i t h s t e r i l e phosphate b u f f e r .  con-  In order  to prevent d e s i c c a t i o n of the samples d u r i n g i n c u b a t i o n , 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 . p l a t e s were incubated e i t h e r at room temperature or i n a c o l d room (3 to 5 ° C ) .  The  (23 t o 25°c)  The p l a t e s 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, c r y o f r a c t u r e d samples were t r a n s f e r r e d i n t o tubes of n u t r i e n t b r o t h and incubated at approximately 25°C f o r 48 h r , then observed f o r growth. Absence o f t u r b i d i t y a f t e r a 48-hr i n c u b a t i o n p e r i o d 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 ured samples i n o c u l a t e d with P . f r a g i .  cryofract-  A c r y o f r a c t u r e d sample  was f i r s t weighed, then ground w i t h sand i n a mortar w i t h a pestle.  The contents  of the mortar were t r a n s f e r r e d to a SS-mlk  d i l u t i o n blank then standard p l a t e counts were performed.  Plate  count agar was used as the p l a t i n g medium, and a l l p l a t e s were incubated at approximately 25°C f o r 48 h r . P r e p a r a t i o n of Samples f o r Scanning E l e c t r o n Microscopy A f t e r i n c u b a t i o n , the samples were t r a n s f e r r e d to  5-ml  beakers and t r e a t e d at about 25°C, as o u t l i n e d below: 1)  f i x e d i n 2.5$ g l u t a r a l d e h y d e i n 0.10 M phosphate  buffer,  pH f o r 1 hr 2)  washed 3 times i n 0.24 M sucrose; 5 min f o r each wash  3)  p o s t f i x e d with 1.0$  osmium t e t r o x i d e i n 0.12  M sucrose  f o r about 18 h r 4)  washed 3 times i n 0.25 M sucrose; 5 min f o r each wash  5)  dehydrated c o n s e c u t i v e l y i n 50$, 75$, 95$ and a b s o l u t e ethanol; 5 min i n each s o l u t i o n  6)  3 a d d i t i o n a l changes of a b s o l u t e e t h a n o l ; 5 min f o r each  7)  samples  then d r i e d i n a stream of n i t r o g e n  The d r i e d samples were mounted on aluminum stubs with 'Dag  1  d i s p e r s i o n and allowed to dry f o r approximately 0.5 n r .  The specimens orator.  were then coated with gold i n a Mikros  evap-  Specimens were examined w i t h a Cambridge Stereoscan  scanning e l e c t r o n microscope. P r e p a r a t i o n of Samples f o r Transmission E l e c t r o n Microscopy The double f i x a t i o n procedure o u t l i n e d below was  used  f o r p r e p a r a t i o n of T.E.M. Samples from the c r y o f r a c t u r e d pieces.  A l l procedures were c a r r i e d out a t about  25°C,  u n l e s s s t a t e d otherwise; l)  f i x e d i n 2.5$ g l u t a r a l d e h y d e i n 0.10 M phosphate  buffer,  pH 7.0 f o r 1 h r 2)  2 washes i n 0.10 M phosphate b u f f e r pH 7.0, 5 min each  3)  p o s t f i x e d i n 1.0$ osmium t e t r o x i d e  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 b u f f e r pH 7.0; 5 min f o r each wash  5)  dehydrated c o n s e c u t i v e l y i n 30$, 50$, 70$, 85$, 95$ and a b s o l u t e e t h a n o l ; 5 min f o r each s o l u t i o n  6)  3 changes,  7)  propylene oxide i n f i l t r a t i o n 30 min f o r each step  8)  30 min each of a b s o l u t e e t h a n o l  i)  1 par t propylene oxide to 3 parts  ii)  1 par t propylene oxide to 1 p art  iii)  3 p a r t s propylene oxide to 1 p a r t  ethanol ethanol ethanol  2 changes of 100$ propylene o x i d e , 30 min f o r each change  9)  Bpon 812 i n f i l t r a t i o n ; 30 min f o r each step i)  1 part Epon to 3 p a r t s propylene oxide  ii)  1 par t Epon to 1 p a r t propylene oxide  iii)  3 parts Epon to 1 p art propylene oxide  10)  2 changes,  30 min each,  of 100$ Epon  11)  specimen t r a n s f e r r e d 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)  p l a c e d i n 40°C oven, approximately 12 h r  13)  placed i n 60°C oven approximately 24 hr The specimen blocks were s e c t i o n e d on a S o r v a l MT-2  ultramicrotome.  Sections were picked up on carbon-coated  collodion film grids. saturated  S e c t i o n s were s t a i n e d f o r 15 min i n a  s o l u t i o n of u r a n y l a c e t a t e i n 70$ methanol and 15 min  i n Reynolds l e a d c i t r a t e (Reynolds, 1963).  G r i d s were viewed  with an AEI C o r i n t h 275 t r a n s m i s s i o n e l e c t r o n  microscope.  39 RESULTS AND DISCUSSION Part I - Biochemical S t u d i e s Influence of growth of Pseudomonas f r a g i organisms on the pH and water h o l d i n g c a p a c i t y of chicken muscle A l l control  (uninoculated)  and i n o c u l a t e d 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  inacti-  vate n a t u r a l l y o c c u r r i n g microorganisms  Irradiation  (Table I ) .  at t h i s dosage caused the formation of a s l i g h t pink pigmenta t i o n of the chicken muscle.  This r e s u l t  i s i n agreement with  the f i n d i n g s of Whiting (1970), Hanson et a l . 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 c u l t u r e added to each sample ($0 g) ml of s t e r i l e t r o l samples  (1963) and  phosphate (40 g ) .  of P . f r a g i were  of muscle as an inoculum while 10  s o l u t i o n was added to each of the  V i s i b l e l i q u i d was present i n the  c o n t a i n i n g the u n i n o c u l a t e d c o n t r o l s ;  flasks  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 c o n t a i n i n g the samples a f t e r 2.5 days of i n c u b a t i o n .  con-  inoculated  The absence of f r e e  l i q u i d i n the f l a s k s c o n t a i n i n g the i n o c u l a t e d samples was due to the i n c r e a s e d w a t e r - h o l d i n g c a p a c i t y presumably by the i n c r e a s e i n pH.  caused  Hamm (i960) reported that maximum  w a t e r - h o l d i n g c a p a c i t y 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 i n o c u l a t e d samples at v a r i o u s i n c u b a t i o n times are shown i n Table The c o n t r o l samples h e l d i n a s t e r i l e  I.  environment had no b a c t -  e r i a l growth over an i n c u b a t i o n p e r i o d of 9.5 days.  A rapid  T A B L E  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 . Treatment of muscle a  Incubation time i n days  Control  0  0  0 0  Inoculated  2.5 5.5 9.5 0  3.02 x 1 0  2.5  1.05 x 1 0  5.5  3.98 x 10  9  9.5  3.47 x 1 0  8  24 nr postmortem muscle at zero time  Number of bacteria per g of muscle  pH 5.9 5.3 5.2  0  5.3 6  5.9 1 G  6.95 7.9 8.5  i n c r e a s e i n b a c t e r i a l numbers i n the i n o c u l a t e d samples waso observed d u r i n g the f i r s t a decrease i n b a c t e r i a l The  2.5  days of i n c u b a t i o n , f o l l o w e d  by  population.  change i n pH of the c o n t r o l and i n o c u l a t e d muscle  samples i s shown i n Table I . 24-hr postmortem muscle was t a i n e d by s e v e r a l authors  The i n i t i a l pH of the 5.9.  S i m i l a r r e s u l t s were  (Lea et a l . , 1969;  I960; and deFremery and P o o l , I960). muscle decreased to 5.3  irradiated  a f t e r 2.5  The  ob-  Dodge and  pH of the  Peters,  uninoculated  days of i n c u b a t i o n and  re-  mained at t h i s l e v e l f o r the 9.5-day i n c u b a t i o n p e r i o d .  The  pH of the i n o c u l a t e d samples i n c r e a s e d a p p r e c i a b l y over the 9.5-day i n c u b a t i o n p e r i o d t o 8 . 5 .  An i n c r e a s e i n pH  s p o i l a g e of muscle by P. f r a g i was  a l s o found by Tarrant et a l .  during  (1971), Borton et a l . (1970a) and Hasegawa et a l . (1970a). Tarrant et a l . (1971) r e p o r t e d that the i n c r e a s e i n pH was to  the production of amines and The uninoculated  ammonia.  c o n t r o l s remained a l i g h t pink c o l o r f o r  the d u r a t i o n of the i n c u b a t i o n p e r i o d .  A f t e r 2.5  days of i n -  cubation, the i n o c u l a t e d samples turned to a salmon pink, a f t e r 9.5  due  but  days the c o l o r became a y e l l o w i s h pink.  The i n o c u l a t e d samples a t t a i n e d a p u t r i d odor a f t e r days of i n c u b a t i o n .  The p u t r i d odors a s s o c i a t e d with  2.5  spoiling  meat are caused p r i m a r i l y by hydrogen s u l f i d e from s u l f u r - c o n t a i n i n g amino a c i d s , ammonia from amino a c i d s and r e l a t e d compounds and i n d o l e from tryptophan No p u t r i d or o f f - o d o r was samples.  (Ingram and Dainty,  1971).  noted with the incubated c o n t r o l  The s u r f a c e s ance a f t e r  of the i n o c u l a t e d samples had a slimy appear-  2.5 days of i n c u b a t i o n .  Jay (1970) s t a t e d  slimy appearance of s p o i l e d meat i s due to coalescence  that  the  of the  surface b a c t e r i a l c o l o n i e s and the " s o f t e n i n g or l o o s e n i n g " of meat s t r u c t u r a l p r o t e i n s . detectable  Ayres et a l .  (1950) r e p o r t e d that a  slime was observed when the l o g of the  bacterial  numbers was between 8.0 and 9.0 per g on c u t - u p p o u l t r y . contents  The  of an i n c u b a t i o n f l a s k were mixed w i t h a s p a t u l a b e -  f o r e samples were removed f o r a n a l y s i s . had a l o o s e s t r u c t u r e  i n the respect  The u n i n o c u l a t e d t i s s u e  that the extruded  strands  of muscle formed during the mincing step i n sample p r e p a r a t i o n d i d not adhere to each o t h e r .  A f t e r 2.5 days of i n c u b a t i o n the  i n o c u l a t e d t i s s u e formed a cohesive mass upon m i x i n g .  Jay  (1970)  reported that the slime l a y e r was p r i m a r i l y r e s p o n s i b l e f o r the tacky consistency of s p o i l e d meat. The w a t e r - h o l d i n g c a p a c i t y of muscle homogenate was e v a l u ated by c e n t r i f u g i n g samples at 20,000 x G and measuring the supernatant volume. 39 - 1 m l ; whereas,  With c o n t r o l samples,  the volume was  the volume of the supernatant from i n o c u l -  ated samples was 30 - 1 m l .  The i n c r e a s e d w a t e r - h o l d i n g c a p -  a c i t y i n the i n o c u l a t e d meat i s i n agreement with the of Adamcic and C l a r k (1970), Ockerman et a l . et. a l .  results  (1969) and Borton  (1968).  I n f l u e n c e 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 p r o t e i n s from chicken muscle R e s u l t s of w a t e r - s o l u b l e p r o t e i n , s a l t - s o l u b l e p r o t e i n and n o n p r o t e i n n i t r o g e n f r a c t i o n s e x t r a c t e d from c o n t r o l and i n oculated muscle samples are-.' shown i n F i g u r e s 3, 4 and 5  10  8  c  <D  O) O  k . c  +J  6  ;C <5 **  o »_ 4 a a) a> a u 3  </>  O  E 2  CO 1 1  Control  3  Inoculated  or E 3  2  4  8  10  Incubation Time , Days  P i g . 3.  W a t e r - s o l u b l e p r o t e i n 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  14  12  10  8  Control L  r>  Inoculated  _l_  6  2  Incubation  E i g . 4.  8  10  T i m e , Days  S a l t - s o l u b l e p r o t e i n 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  Control  D  •  Inoculated  •  •  -I  2  1  I  i  i_  4  6  8  10  Incubation Time , Days  N o n p r o t e i n n i t r o g e n compounds e x t r a c t e d f r o m c o n t r o l a n d i n o c u l a t e d samples  respectively. New  Summaries of a n a l y s i s of v a r i a n c e and Duncan's  M u l t i p l e Range Test are presented i n Tables I I I and  IV  respectively. The amounts of w a t e r - s o l u b l e , s a l t - s o l u b l e and  nonprotein  n i t r o g e n e x t r a c t e d from the c o n t r o l chicken muscle a t zero time (Table I I ) were lower than those r e p o r t e d by Khan  (1962).  However, Khan (1962) i n d i c a t e d that the n i t r o g e n content of the v a r i o u s p r o t e i n f r a c t i o n s can vary from 10 to 2 5 $ due t o d i f f e r e n c e s i n breed, n u t r i t i o n and pre-and p o s t - s l a u g h t e r conditions. A significant  (P <  .05) decrease was  noted i n the e x t r a c t -  a b i l i t y of the water-soluble p r o t e i n ( F i g . 3) from the c o n t r o l samples d u r i n g the 9.5 IV, there was  day storage p e r i o d .  no s i g n i f i c a n t  (P >  .05)  As shown i n Table  change 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 p r o t e i n ( F i g . 4) from the c o n t r o l samples d u r i n g the f i r s t 5.5 days of s t o r a g e . (P < The  A  significant  .05) decrease d i d occur a f t e r 9 . 5 days of i n c u b a t i o n . s o l u b i l i t y of the nonprotein n i t r o g e n f r a c t i o n ( F i g . 5)  e x t r a c t e d from the c o n t r o l d i d not change (P >  .05)  sig-  n i f i c a n t l y d u r i n g the storage p e r i o d . Studies on changes i n p r o t e i n e x t r a c t a b i l i t y d u r i n g p o s t mortem aging at h i g h temperatures authors ( P a r r i s h et a l . , 1969; 1963;  Lawrie et a l . , 1961;  (25 to 37°C) by numerous  Suzuki et a l . , 1967;  Sharp,  Locker, I960; Zender et a l . ,  1958)  i n d i c a t e d that there were s i g n i f i c a n t decreases i n the e x t r a c t a b i l i t y of w a t e r - s o l u b l e p r o t e i n and s a l t - s o l u b l e p r o t e i n f r a c t i o n s while the changes i n non-protein n i t r o g e n were some-  TABLE I I Water-soluble p r o t e i n , s a l t - s o l u b l e p r o t e i n and nonprotein n i t r o g e n 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 chicken p e e t o r a l i s muscle.  Treatment Control  Control  Inoculated  Control  Inoculated  Control  Inoculated  Incubation time i n days 0  2.5  2  -5  5.5  5.5  9.5  9.5  Water-soluble protein . nitrogen  Salt-soluble protein . nitrogen  Nonprotein , nitrogen  8.5  12.2  4.1  8.1  13.8  4.0  5.7  11.9  3.9  4.9  7.3  3.5  7.1  9.6  3.6  7.1  9.8  3.3  4.5  13.0  3.4  4.4  10.9  3.4  8.9  8.7  3.4  10.3  7.9  4.8  3.1  7.2  2.5  2.9  6.9  2.6  5.8  4.7  2.9  6.7  5.0  3.5  a  mg N/g  of muscle  b  R e s u l t s are the averages of d u p l i c a t e  ^determinations  48  TABLE I I I Analysis of variance p e e t o r a l i s muscle  o f p r o t e i n e x t r a c t a b i l i t y from  M e a n  sq.  M e a n  chicken  sp.  Source  df  water soluble protein nitrogen  Treatments Zero C o n t r o l Vs. o t h e r Time  6  10.241  17.638 *  0.547  1  9.38  28.262 *  0.678  2  5.97  27.094  0.789  21.924 *  0.429  Inoculated v s . Uninoculated 1 Time x Inoculation Flasks/ Treatment Total:.  * 34.575  saltsoluble protein nitrogen  nonprotein nitrogen  m e a n  sq.  2  2.776 *  0.727  0.3  7  0.269  0.549  0.197  13  S i g n i f i c a n t a t P = 0.05  49  TABLE  M  S o l u b i l i t y o f t h e v a r i o u s p r o t e i n f r a c t i o n s from muscle and muscle i n o c u l a t e d w i t h P. f r a g i . Water-soluble protein nitrogen  Treatment C o n t r o l G time C o n t r o l 2.5 days I n o c u l a t e d 2.5 days C o n t r o l 5.5 days Inoculated  5.5 days  C o n t r o l 9.5 days I n o c u l a t e d 9.5 days  a ed ab d 9.588 e 2.993 f 6.252 be 8.287 5.282 7.102 4.47  Salt-soluble protein nitrogen 13.008 a 11.88 a 9.66 b 11.95  a  be c 7.068 d 4.866  8.262  uninoculated  Nonprotein nitrogen 4.026 a 3.694 a 3.44 ab 3.374 a b 4.088 a 2.556 b 3.23 ab  Treatment means i n t h e same column s h a r i n g t h e same s u p e r s c r i p t s are n o t s i g n i f i c a n t l y d i f f e r e n t a t t h e 5fo l e v e l o r p r o b a b i l i t y .  50 what v a r i a b l e .  R e s u l t s obtained by Suzuki  et a l . (1967),  Sharp (1963), Lawrie et a l . (1961) and Locker (i960) i n d i c a t e d that s i g n i f i c a n t i n c r e a s e s i n nonprotein n i t r o g e n  occurred.  P a r r i s h et a l . (1969) noted a decrease i n nonprotein n i t r o g e n i n bovine muscle s t o r e d f o r 24 h r at 37°C and g 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 may of nonprotein n i t r o g e n .  suggested that aggre-  have caused some b i n d i n g  Zender et a l . (1958) d i d not observe a  s i g n i f i c a n t i n c r e a s e i n nonprotein n i t r o g e n u n t i l a f t e r  31  days of storage at 25°C. The l a c k of any s i g n i f i c a n t i n c r e a s e i n the e x t r a c t a b i l i t y of nonprotein n i t r o g e n from c o n t r o l samples i n d i c a t e s that a u t o l y s i s of the sarcoplasmic  p r o t e i n s , which are the s u b s t r a t e  f o r c a t h e p t i c enzymes (Martins and Whitaker, 1968$ Bodwell and Pearson, 1964;  Sharp, 1963)  s i g n i f i c a n t a u t o l y s i s may treatment.  was  not e x t e n s i v e .  have been due  t o the  The absence of irradiation  Mohasseb (1962), Schweigert (1959) and Doty and  Wachter (1955) r e p o r t e d that i r r a d i a t i o n dosages of 1 to  1.6  Mrads reduced the p r o t e o l y t i c a c t i v i t y of muscle cathepins approximately  by  50$.  A white p r e c i p i t a t e was  first  observed i n the bottom of  f l a s k s c o n t a i n i n g u n i n o c u l a t e d muscle a f t e r three days i n cubation.  Zender ejb a l . (1958) r e p o r t e d that a l i q u i d  c o n t a i n i n g 7$ by weight water-soluble muscle d u r i n g storage.  Therefore,  c i p i t a t e observed i n t h i s study was t e i n s which were present  exudate,  p r o t e i n s , d r i p s from the  i t i s l i k e l y that the  pre-  i n s o l u b l e sarcoplasmic  i n the muscle exudate.  pro-  T h i s would  account f o r the observed decrease i n the e x t r a c t a b i l i t y of the water-soluble  p r o t e i n without  concomitant i n c r e a s e i n  non-  protein nitrogen. The  extractability  of the water-soluble  p r o t e i n from sam-  p l e s i n o c u l a t e d with P. f r a g i was not s i g n i f i c a n t l y (P > .05) different 0 time.  a f t e r 2.5 and 9.5 days i n c u b a t i o n from the samples a t There was a s i g n i f i c a n t  (P < .05) i n c r e a s e i n the  l e v e l o f e x t r a c t a b l e water-soluble i n c u b a t i o n ( P i g . 3).  p r o t e i n a f t e r 5.5 days o f  The e x t r a c t a b i l i t y  of the water-soluble  p r o t e i n from the i n o c u l a t e d muscle was s i g n i f i c a n t l y (P < .05) higher than from the c o n t r o l a t the v a r i o u s i n c u b a t i o n times (Table I V ) .  As shown i n F i g u r e 4, 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 p r o t e i n from the i n o c u l a t e d samples decreased throughout the i n c u b a t i o n p e r i o d .  The e x t r a c t a b i l i t y o f the  s a l t - s o l u b l e p r o t e i n s was s i g n i f i c a n t l y (P <  .05) lower i n the  i n o c u l a t e d samples compared t o the r e s p e c t i v e c o n t r o l samples (Table I V ) .  No s i g n i f i c a n t  extractability  (P > .05) change was noted i n the  of the nonprotein  n i t r o g e n f r a c t i o n s between the  i n o c u l a t e d and c o n t r o l samples a t the v a r i o u s i n c u b a t i o n 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  times.  o f the s a l t -  s o l u b l e p r o t e i n s from the i n o c u l a t e d samples, a f t e r 9.5 days i n c u b a t i o n , suggested that p r o t e o l y s i s o f the m y o f i b r i l l a r p r o t e i n s 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 f i n d i n g s of both Tarrant et a l . (1971) and Borton et a l . (1970a) u s i n g porcine muscle and the t r a n s m i s s i o n e l e c t r o n micrographs of Buts.on et a l . (1971) which showed degradation  of the f i b r i l s t r u c t u r e as a r e s u l t of  s p o i l a g e of porcine muscle by P. f r a g i . Both T a r r a n t et a l . (1971) and Borton et a l . (1970a) r e -  ported s i g n i f i c a n t i n c r e a s e s i n nonprotein! n i t r o g e n during s p o i l a g e of porcine muscle by P. f r a g i . reported that the i n c r e a s e ammonia.  ejb a l .  (1971)  c o n s i s t e d p r i m a r i l y of peptides and  No s i g n i f i c a n t i n c r e a s e  observed i n the present s t u d y . increase  Tarrant  i n nonprotein n i t r o g e n was  The l a c k of any s i g n i f i c a n t  i n nonprotein n i t r o g e n l e v e l s i n the i n o c u l a t e d  samples was unexpected.  Numerous authors  (Tarrant  et  al..  1971; Adamcic and C l a r k ,  1970; Borton et a l . , 1970a; Ockerman  et a l . , 1969; l e r k e et a l . , 1967) have r e p o r t e d i n c r e a s e s i n nonprotein n i t r o g e n l e v e l s d u r i n g low temperature (between 2 and 10°C) s p o i l a g e of meat by v a r i o u s Pseudomonas  species.  Muscle incubated at h i g h temperatures as used i n t h i s  study  should have g r e a t e r chemical changes than those observed d u r i n g low temperature s p o i l a g e .  Proteolysis  and 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 s of l a r g e p r o t e i n fragments polypeptides,  of the  sarcoplasmic  should r e s u l t  which are  i n formation  f u r t h e r broken down i n t o  peptides and e v e n t u a l l y 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 i n c r e a s e i n nonprotein n i t r o g e n was due mainly to the l a r g e v a r i a t i o n between cates w i t h i n treatments,  e x p e c i a l l y i n the i n o c u l a t e d  replisamples,  which was g r e a t e r 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 w a t e r - s o l u b l e c o n t r o l and i n o c u l a t e d muscle  -proteins i n e x t r a c t s from  The e l u t i o n p a t t e r n s of the w a t e r - s o l u b l e p r o t e i n e x t r a c t s from u n i n o c u l a t e d c o n t r o l and i n o c u l a t e d samples are  shown i n  F i g u r e s 6 and 7 r e s p e c t i v e l y .  The area under the curves were  measured u s i n g a p l a n i m e t e r .  R a t i o s were determined f o r these  areas r e l a t i v e  to the area of the curves  o c c u r r i n g at the v o i d  53  F i g . 6. E l u t i o n p a t t e r n s o f water-soluble p r o t e i n e x t r a c t s from u n i n o c u l a t e d cnicken 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.  2.5 days i n c u b a t i o n  C.  5.5 days i n c u b a t i o n  D.  9 « 5 days i n c u b a t i o n  70  = V o i d Volume  VE  = E f f l u e n t volume f o r glutamic  acid  55  P i g . 7.  E l u t i o n p a t t e r n s of water-soluble p r o t e i n e x t r a c t s from chicken 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 A.  2.5  days i n c u b a t i o n  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  70  = Void  VE  = E f f l u e n t volume f o r glutamic  volume acid  ABSORBANCE  AT o  P  l• •• t •  5 70 O  Oi 1  nm.  o  b  00 1  r~  00  volume (Vo) and. e f f l u e n t volume (Ve) f o r glutamic a c i d i n the c o n t r o l at 0 time.  The r a t i o of the area under the curves i s  presented i n Table V. The e l u t i o n p a t t e r n of the c o n t r o l a t 0 time ( F i g . 6A) was  c h a r a c t e r i z e d by 2 peaks.  The f i r s t peak, o c c u r r i n g at  Vo c o n s i s t s o f the sarcoplasmic p r o t e i n s while the second peak, o c c u r r i n g very c l o s e to Vt, c o n s i s t s of f r e e amino a c i d s and small p e p t i d e s .  The c o l o r i n t e n s i t y of the n i n h y d r i n r e -  a c t i o n i s g r e a t e r f o r f r e e amino a c i d s and peptides than f o r proteins.  Therefore, the area under the 2 curves does not  g i v e the r a t i o of c o n c e n t r a t i o n s . The area under the sarcoplasmic peak (Vo) i n the uninocu l a t e d samples decreased with i n c r e a s e d i n c u b a t i o n time.  This  o b s e r v a t i o n concurs with the r e s u l t s obtained d u r i n g the p r o tein extractability studies. of a u t o l y s i s .  This decrease cannot be the :result  A u t o l y s i s would r e s u l t i n the p r o d u c t i o n of l a r g e  p r o t e i n fragments,  due t o the a c t i o n of c a t h e p s i n D.  These  fragments would be hydrolyzed f u r t h e r to peptides by a c t i o n of cathepsins A, B and 0 ( C a l d w e l l and Grosjean, 1971; et a l . , 1969;  I o d i c e et a l . ,  1966).  Parrish  P r o d u c t i o n of l a r g e p r o t e i n  fragments and p o l y p e p t i d e s would r e s u l t 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 p r o t e i n s e x t r a c t e d from the c o n t r o l samples ( P i g . 6, A, B, C and D) d i d not change shape over the i n c u b a t i o n £egi.od, although the area under the curve (Vo) This decrease was  not due to a u t o l y s i s but r a t h e r r e p r e s e n t s a  decrease i n s o l u b i l i t y of the sarcoplasmic p r o t e i n s . et a l .  decreased.  Zender  (1958) r e p o r t e d l a r g e amounts of sarcoplasmic p r o t e i n s  .TABLE V i l a t i o of areas under the curves a t Yo and Ye f o r glutamic a c i d  Treatment Control  Inoculated  Incubation time i n days  Yo Sarcoplasmic Peak  Ve Nonprotein N i t r o g e n peak  0  1  1  2.5  0.42  0.79  5.5  0.14  0.71  9.5  0.18  0.60  «5  0.62  1.46  5.5  0.55  1.27  9.5  0.41  2.02  2  present  i n the exudate produced d u r i n g storage of muscle  h i g h temperatures (25°G).  I n the present  study, a  at  precipitate  was observed i n the f l a s k s c o n t a i n i n g u n i n o c u l a t e d muscle. This p r e c i p i t a t e  may be i n s o l u b l e sarcoplasmic  was o r i g i n a l l y present  i n the  p r o t e i n which  exudate.  As shown i n Table V, the r a t i o  of the area under the  curve o c c u r r i n g at Ve f o r glutamic a c i d , e p r e s e n t i n g n o n p r o t e i n n i t r o g e n i n the c o n t r o l samples,  decreased  s l o w l y with i n -  creased s t o r a g e . In the i n o c u l a t e d samples ( F i g . 7) the curve at V o , r e p r e s e n t i n g the sarcoplasmic  proteins,  underwent two changes  as opposed to the s i n g l e change o c c u r r i n g i n the c o n t r o l . i n the c o n t r o l , the c o n t r o l ,  the peak height decreased with time b u t ,  the shape of the curve changed.  As  unlike  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 c o n t r o l samples. agree w i t h r e s u l t s  T h i s decrease does not  obtained i n the p r o t e i n  extractability  study, which i n d i c a t e d that the l e v e l of w a t e r - s o l u b l e  protein  was s i g n i f i c a n t l y (P < .05) h i g h e r i n the i n o c u l a t e d  samples  a f t e r 5.5 days i n c u b a t i o n than a?ter 2.5 and 9.5 d a y s .  The  reason f o r t h i s discrepancy must l i e i n the method f o r  deter-  mining n o n p r o t e i n n i t r o g e n as the w a t e r - s o l u b l e p r o t e i n was calculated  from the d i f f e r e n c e between t o t a l  water-soluble  n i t r o g e n and MPN. Bell  (1963) d i d a comparative  study on methods f o r d e t e r -  mination of NPN on blood serum, m i l k ,  bran and f l o u r .  Bell  (1963) found t h a t "no  c o n s i s t e n t d i f f e r e n c e s or  were observed r e l a t i n g method and solutions". used.  Por  similarities  r e s u l t s on one  The-value obtained f o r NPN  i n blood serum by-  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 a c i d y i e l d e d a value of 2.7$, of 52$.  B e l l (1963) concluded t h a t KPN  method of p r e p a r a t i o n  and  the  depended on the method  example, determination of NPN  d i a l y s i s i n d i c a t e d a value of 6.3$;  or a l l of  a difference  must be d e f i n e d by  the  that d i a l y s i s or g e l f i l t r a t i o n  achieves s e p a r a t i o n most c l o s e l y r e l a t e d to molecular s i z e alone.  Prom the work of B e l l (1963) i t i s clear, that the  NPN  v a l u e s obtained by t r i c h l o r o a c e t i c a c i d p r e c i p i t a t i o n during the p r o t e i n s o l u b i l i t y study and  the i n d i c a t i o n of NPN  by g e l f i l t r a t i o n cannot be v a l i d l y compared.  obtained  As the water-  s o l u b l e p r o t e i n was  c a l c u l a t e d d u r i n g the p r o t e i n e x t r a c t a b i l i t y  study from the NPN,  t h i s value may  indicated'by The  gel  a l s o be d i f f e r e n t from that  filtration.  sarcoplasmic p r o t e i n peak i n the c o n t r o l was  m e t r i c a l ; whereas, the curve from the i n o c u l a t e d became skewed to the r i g h t a s . i n c u b a t i o n ( P i g . 7B and  0).  A f t e r 9.5  time  days i n c u b a t i o n  sym-  samples  increased  ( P i g . 7C)  nin-  h y d r i n - p o s i t i v e m a t e r i a l extended between the 2 curves o r i g i n a l l y present at 0 time ( P i g . 6A). s i m i l a r r e a c t i o n i n the ( F i g . 60).  There i s no i n d i c a t i o n of a  c o n t r o l s a f t e r 9.5  days  incubation  Presumably t h i s m a t e r i a l c o n s i s t s of a mixture  of p r o t e i n fragments having a wide range of molecular weights f a l l i n g below the e x c l u s i o n l i m i t of Sephadex G-50 t h a t s o f amino a c i d s . from two  T h i s m a t e r i a l may  and  above  have o r i g i n a t e d  sources; breakdown of the sarcoplasmic p r o t e i n s  and  releaser  of w a t e r - s o l u b l e fragments from the m y o f i b r i l l a r p r o -  t e i n s both as a r e s u l t of p r o t e o l y s i s of P . f r a g i . The curve near the t o t a l volume ( V t . ) ,  r e p r e s e n t i n g low  molecular weight n i n h y d r i n p o s i t i v e compounds i n the i n o c u l a t e d samples increased i n area and changed i n shape w i t h t i m e . seen i n F i g u r e 7 , the peak height remains r e l a t i v e l y  As  constant.  The i n c r e a s e i n area i s due to the change i n shape of the  curve  because of the presence of m a t e r i a l w i t h a lower e l u t i o n volume, therefore,  h i g h e r 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 d u r i n g 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 p r o t e i n s by P . f r a g i • S t a t i s t i c a l a n a l y s i s was not performed on the areas of the curves.  As i n d i c a t e d i n Table "V, the area under the curve near  the Vt p o s i t i o n of the c o n t r o l samples decreased while the area under the curve of the i n o c u l a t e d samples i n c r e a s e d .  After 9 . 5  days of i n c u b a t i o n , the area of the curve o c c u r r i n g near the Vt p o s i t i o n of the i n o c u l a t e d samples was 3 times as great as  the  corresponding area i n the e l u t i o n p a t t e r n of the c o n t r o l .  It  appears that a s i g n i f i c a n t p r o t e i n degradation does occur  as  a r e s u l t of growth of P . f r a g i . D i s c g e l e l e c t r o p h o r e s i s of w a t e r - s o l u b l e , s a l t - s o l u b l e and u r e a - s o l u b l e p r o t e i n s i n e x t r a c t s from c o n t r o l and i n o c u l a t e d muscles 1.  Water-soluble F r a c t i o n E l e c t r o p h o r e c t i c g e l p a t t e r n s and densitometer  tracings  of the sarcoplasmic p r o t e i n e x t r a c t from the u n i n o c u l a t e d c o n t r o l are shown i n F i g u r e s 14 and 8 r e s p e c t i v e l y .  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  incubation  0.  9.5  days  incubation  63  A  .  B  TV  8  ./V  64  Fig.  9.  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 chicken 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 A.  2.5  days i n c u b a t i o n  B.  5.5 days i n c u b a t i o n  C.  9.5  days i n c u b a t i o n  66 F i g u r e 8A, 9 d i s t i n c t bands are apparent mortem) .  The e l e c t r o p h o r e t i c  the i n c u b a t i o n p e r i o d .  at 0 time (24 h r  post-  m o b i l i t y of band 2 i n c r e a s e d  over  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 t e r 5.5-days  incubation this  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 band 1 ( F i g . 8B).  As shown i n F i g u r e 8G, the i n t e n s i t y  band 2 continued to i n c r e a s e  as  of  u n t i l 9.5 days of i n c u b a t i o n .  Bands 3 to 9 tended to decrease -in i n t e n s i t y over the i n c u b a t i o n p e r i o d r e f l e c t i n g a decrease i n The e l e c t r o p h o r e t i c of the sarcoplasmic  extractability.  g e l patterns and densitometer  p r o t e i n s extracted from the i n o c u l a t e d  t i s s u e are shown i n F i g u r e s 14 and 9 r e s p e c t i v e l y . changes occurred i n the e l e c t r o p h o r e t i c sample.  tracings  pattern  A number of  of the  A f t e r 2 . 5 days of i n c u b a t i o n , a new band,  inoculated  designated  A, was observed ( F i g . 9A).  T h i s band may be a p r o t e i n  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 F i g u r e 9B, band 2 disappeared from the p a t t e r n 5.5-days i n c u b a t i o n .  The disappearance  w i t h i n c r e a s e d i n t e n s i t y of band 3 '  after  of band 2 c o i n c i d e d  T h i s suggested  that p r o -  t e o l y s i s r e s u l t e d i n i n c r e a s e d m o b i l i t y of band 2 with the  re-  s u l t that band 2 merged with band 3 forming a s i n g l e peak. The i n t e n s i t i e s  of the other bands i n F i g u r e 9B were lower  than i n F i g u r e 9A.  The e l e c t r o p h o r e t i c  m o b i l i t y of band 9 i n  the u n i n o c u l a t e d c o n t r o l increased a f t e r ' 5 . 5 ( F i g s . 8B and 14E); samples ( F i g s . rophoretic  no such i n c r e a s e  9B and 14F).  days i n c u b a t i o n  occurred i n the  inoculated  Band 4 was m i s s i n g from the  elect-  p a t t e r n o f muscle incubated f o r 9.5 days ( F i g .  There was no change i n number of bands present  i n the  9C).  electrophoretic  g e l p a t t e r n of the sarcoplasmic p r o t e i n s ex-  t r a c t e d from the c o n t r o l samples at v a r i o u s i n c u b a t i o n times while samples i n o c u l a t e d 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)  t h a t growth of P . f r a g i caused extensive  reported  a l t e r a t i o n s i n the  s t a r c h g e l e l e c t r o p h o r e t i c p a t t e r n of the sarcoplasmic  proteins  e x t r a c t e d from r a b b i t and porcine muscle.  reported  The authors  the l o s s of 70 to 80 percent of the bands i n the p a t t e r n due t o p r o t e o l y s i s caused by  fragi.  Such extensive degradation of  the sarcoplasmic p r o t e i n s was not observed i n the present 2.  study.  Salt-soluble Proteins The e l e c t r o p h o r e t i c  g e l p a t t e r n s of the s a l t - s o l u b l e p r o -  t e i n e x t r a c t s are presented i n F i g u r e 15.  Densitometer  tracings  of the g e l s from e x t r a c t s from c o n t r o l and i n o c u l a t e d muscle samples are presented i n F i g u r e s 10 and 11 r e s p e c t i v e l y . F i g u r e 10A shows the densitometer t r a c i n g of the g e l s f o r the m y o f i b r i l l a r p r o t e i n s e x t r a c t e d from the u n i n o c u l a t e d c o n t r o l at 0 time.  Fourteen d i s t i n c t bands were observed.  major bands i n the g e l s are bands 1 and, 8.  The  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) r e p o r t e d by F i s h e r (1963).  Band  14 was absent from the p a t t e r n of c o n t r o l samples incubated f o r 2.5 days or l o n g e r .  Apart from the l o s s of band 14, no  f u r t h e r changes were observed i n the e l e c t r o p h o r e t i c  gel pat-  t e r n o f s a l t - s o l u b l e p r o t e i n e x t r a c t e d from c o n t r o l samples a f t e r 2.5 days i n c u b a t i o n ( 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  B.  2.5  incubation days  time  incubation  70  P i g . 11.  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 c h i c k e n p e c 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 A.  2.5 days i n c u b a t i o n  B.  9.5 days i n c u b a t i o n  B  appearance  of band 14 was a r e s u l t  of i n s o l u b i l i z a t i o n .  As shown i n F i g u r e 11 A , the r e l a t i v e m o b i l i t y of bands 11 and 13 i n c r e a s e d i n the e l e c t r o p h o r e t i c  p a t t e r n of  salt-sol-  uble p r o t e i n s extracted from i n o c u l a t e d muscle a f t e r 2 . 5 - d a y s incubation. 8)  The i n t e n s i t y and s i z e of the major bands ( l and  a f t e r 9.5-days i n c u b a t i o n ( F i g . 11 B) was c o n s i d e r a b l y lower  than i n the c o n t r o l g e l f o r the same i n c u b a t i o n time ( F i g . 10 B) The r e s u l t s  i n d i c a t e that 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 occurred as a r e s u l t Borton et a l .  of growth of P.  fragi.  (1970b) and Tarrant et a l .  the complete breakdown i n the e l e c t r o p h o r e t i c  (1971)  reported  p a t t e r n of the  s a l t - s o l u b l e 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 porcine muscle, i n o c u l a t e d with P. f r a g i » evidence of such extensive present 3.  a f t e r 20 days storage at 1 0 ° C .  No  p r o t e o l y s i s was observed i n the  study.  Urea-soluble Proteins The u r e a - s o l u b l e f r a c t i o n contains those p r o t e i n s which  were i n s o l u b l e i n water or s a l t clude the stroma p r o t e i n s .  s o l u t i o n s , but does not i n -  The e l e c t r o p h o r e t i c . . g e l  patterns  from the u r e a - s o l u b l e p r o t e i n e x t r a c t s of the c o n t r o l and i n o c u l a t e d t i s s u e are presented i n F i g u r e 16. tracings  The densitometer  of the g e l s are shown i n F i g u r e s 12 and 13  respectively  As shown i n F i g u r e 12A, 12 bands were present i n the c o n t r o l g e l s at 0 time.  There was no change i n the g e l  a f t e r 2.5 days of i n c u b a t i o n . ( F i g . 12B),  pattern  A f t e r 5 . 5 days of i n c u b a t i o n  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 t r a c i n g s of the u r e a - s o l u b l e 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 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  C.  9.5 days i n c u b a t i o n  10  75  F i g . 13*  Densitometer t r a c i n g s of the u r e a - s o l u b l e 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 chicken p e c 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 A.  2.5 days i n c u b a t i o n  B.  9.5 days i n c u b a t i o n  78  F i g . 14.  E l e c t r o p h o r e t i c p a t t e r n s o f sarcoplasmic  proteins  A.  Zero i n c u b a t i o n , c o n t r o l  B.  Zero i n c u b a t i o n , i n o c u l a t e d P.  C.  2.5  days of i n c u b a t i o n , u n i n o c u l a t e d  D.  2.5  days of i n c u b a t i o n , i n o c u l a t e d with P.  E.  5.5  days o f i n c u b a t i o n , u n i n o c u l a t e d  P.  5.5  days of i n c u b a t i o n , i n o c u l a t e d with P.  G-.  9.5  days of i n c u b a t i o n , u n i n o c u l a t e d  H.  9.5  days of i n c u b a t i o n , i n o c u l a t e d with P. f r a g i  fragi control fragi  control fragi  control  80  P i g . 15.  Eleetrophoretic patterns of m y o f i b r i l l a r proteins A.  Zero i n c u b a t i o n , u n i n o c u l a t e d  control  B.  Zero i n c u b a t i o n , i n o c u l a t e d with P.  C.  2.5  days of i n c u b a t i o n , u n i n o c u l a t e d  D.  2.5  days of i n c u b a t i o n , i n o c u l a t e d P.  E.  5»5 days of i n c u b a t i o n , u n i n o c u l a t e d  F.  5.5  days o f i n c u b a t i o n , i n o c u l a t e d P.  G-.  9.5  days of i n c u b a t i o n , u n i n o c u l a t e d  H.  *9.5  fragi  days of i n c u b a t i o n , i n o c u l a t e d P.  control fragi control fragi control fragi  81  82  ,Pig. 16.  Electrophoretic patterns of urea-soluble  proteins  A.  Zero i n c u b a t i o n , u n i n o c u l a t e d c o n t r o l  B.  Zero i n c u b a t i o n , i n o c u l a t e d P.. f r a g i  c.  2.5 days of i n c u b a t i o n , u n i n o c u l a t e d c o n t r o l  D.  2.5 days of i n c u b a t i o n , i n o c u l a t e d with P.  E.  5.5 days of i n c u b a t i o n , u n i n o c u l a t e d c o n t r o l  P..  5.5 days of i n c u b a t i o n , i n o c u l a t e d with P.' f r a g i  G.  9.5 days of i n c u b a t i o n , u n i n o c u l a t e d c o n t r o l  H.  9.5 days of i n e u b a t i o n , i n o c u l a t e d w i t h P.  fragi  fragi  83  84 appeared and the m o b i l i t y of band 2 i n c r e a s e d .  A f t e r 9.5 days  of i n c u b a t i o n ( F i g . 12G) 4 . a d d i t i o n a l bands (C, D, E and F) were n o t e d .  The bands present  at 0 time ( F i g . 12A) tended to  become more d i s t i n c t and i n t e n s e as i n c u b a t i o n time  increased.  The major bands present i n the g e l p a t t e r n 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 g e l p a t t e r n of the s a l t - s o l u b l e ' p r o t e i n s e x t r a c t e d from c o n t r o l samples at 0 time ( F i g . 10A). The g e l p a t t e r n from the e x t r a c t of the i n o c u l a t e d sample, a f t e r 2.5 days of i n c u b a t i o n ( F i g . 13A) was f a i n t .  The major  bands present i n the c o n t r o 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 a l s o p r e s e n t .  Uo d i f f e r e n c e  i n the band p a t t e r n of the e x t r a c t s from samples incubated f o r 5.5 days and 9.5 days was observed.  The band p a t t e r n became  more d i s t i n c t and the bands i n c r e a s e d i n i n t e n s i t y , bands 9, 10, 11 and 12, as i n c u b a t i o n time i n c r e a s e d During the e x t r a c t i o n of s a l t - s o l u b l e p r o t e i n s ,  especially ( F i g . 13B). a vis-  cous l a y e r formed between the supernatant,  which contained  the  s a l t - s o l u b l e p r o t e i n s and the p r e c i p i t a t e ,  which contained  the  salt-insoluble proteins.  P r e l i m i n a r y experiments  indicated  that t h i s v i s c o u s m a t e r i a l c o n s i s t e d 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 d u r i n g the i n c u b a t i o n p e r i o d , et a l .  lawrie  (1961) and Sharp ( 1 9 6 3 ) r e p o r t e d 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 p r o t e i n s was g r e a t l y reduced d u r i n g h i g h tempe r a t u r e (25 - 37°G) storage of meat.  These authors concluded  that t h i s decrease was the result of heat d e n a t u r a t i o n of the myofibrillar proteins. first  T h i s i n s o l u b i l i z a t i o n phenomena was  observed i n the c o n t r o l a f t e r 2.5 days of i n c u b a t i o n .  P r o t e i n i n s o l u b i l i z a t i o n was not observed i n the i n o c u l a t e d samples u n t i l 5.5 days of i n c u b a t i o n and then i t was not extensive  as i n the c o n t r o l .  The i n c r e a s e  as  i n i n t e n s i t y of  the bands i n the g e l p a t t e r n of the u r e a - s o l u b l e p r o t e i n  ex-  t r a c t s c o i n c i d e s w i t h the observed i n s o l u b i l i z a t i o n of the myofibrillar proteins.  This f a c t ,  coupled w i t h the s i m i l a r i t y  between the g e l patterns of the s a l t - s o l u b l e and u r e a - s o l u b l e p r o t e i n e x t r a c t s 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 p r o t e i n s comprise a major p o r t i o n of the u r e a - s o l u b l e Hasegawa _et a l .  (1970a) s t u d i e d the- e f f e c t  extract.  of P . f r a g i on  the u r e a - s o l u b l e p r o t e i n s e x t r a c t e d from porcine and r a b b i t muscle.  The authors r e p o r t e d that the i n t e n s i t y and s i z e of  most of the bands g r e a t l y decreased i n d i c a t i n g extensive t e o l y s i s by P. f r a g i .  The authors reported the  changes i n the g e l p a t t e r n and appearance  pro-  following  f o r porcine muscle; l o s s of 9 bands  of 1 new band and f o r r a b b i t muscle, l o s s of 2  bands and appearance  of 4 new bands.  The work of the above  authors would i n d i c a t e that the u r e a - s o l u b l e f r a c t i o n was a w e l l d e f i n e d f r a c t i o n of the muscle p r o t e i n s , electrophoretic  gel pattern.  The r e s u l t s  with a  consistant  of the present  study  i n d i c a t e t h a t the u r e a - s o l u b l e f r a c t i o n i s a heterogenous ture,  mix-  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 i z e of the bands i n F i g u r e 13A are g r e a t l y decreased i n comparison with the u n i n o c u l a t e d c o n t r o l (Fig. 12A).  This could be i n t e r p r e t a t e d  t e n s i v e p r o t e o l y s i s by P . f r a g i .  as evidence f o r  ex-  However, a f t e r 9.5 days of  i n c u b a t i o n ( F i g . 1 3 B ) , the band p a t t e r n became more d i s t i n c t .  I f the e l e c t r o p h o r e t i c p a t t e r n s were r e f l e c t i n g changes i n the u r e a - s o l u b l e p r o t e i n s due to p r o t e o l y s i s by P. f r a g i , then the opposite should be t r u e 3 the bands should decrease i n i n t e n s i t y and  s i z e with i n c r e a s e d i n c u b a t i o n time.  In the present  study,  the u r e a - s o l u b l e 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 a t i o n of the s a l t - s o l u b l e p r o t e i n s and not the e f f e c t of b a c t e r i a l p r o t e o l y s i s upon the p r o t e i n s of the u r e a - s o l u b l e fraction. A n a l y s i s of f r e e amino a c i d s i n c o n t r o l and i n o c u l a t e d muscles R e s u l t s of the f r e e amino a c i d a n a l y s i s of the u n i n o c u l a t e d and i n o c u l a t e d samples are presented obtained  i n Table V I .  The v a l u e s  f o r the c o n t r o l a t 0 time, at which time the muscle  was 24 h r postmortem, are s i m i l a r to the r e s u l t s reported by l e a et a l . (1969) and M i l l e r et a l . (1965) f o r the f r e e amino a c i d a n a l y s i s on the breast muscle of b r o i l e r s . study,  I n the present  s e p a r a t i o n of l y s i n e and h i s t i d i n e was not obtained,  t h e r e f o r e , these 2 amino a c i d s have been reported as a combined lysine-histidine level.  The c o n c e n t r a t i o n of l y s i n e and h i s t -  i d i n e was s i m i l a r to that r e p o r t e d by M i l l e r et a l . (1965) but was 30 times the c o n c e n t r a t i o n r e p o r t e d by Lea et a l . (1969). Lea et a l . (1969) s t a t e d t h a t two d i p e p t i d e s , anserine and carnosine, are present  i n l a r g e c o n c e n t r a t i o n s i n chicken b r e a s t  muscle (52.8 |jmoles/g).  These two d i p e p t i d e s tend t o be e l u t e d  from the i o n exchange column with l y 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 s e p a r a t i o n of these amino a c i d s . to separate  Failure  these two d i p e p t i d e s undoubtedly l e a d to the h i g h  values obtained  f o r 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 f r e e amino a c i d  'TABLE VI Average f r e e amino a c i d c o n c e n t r a t i o n of i n o c u l a t e d and uninoculated chicken p e e t o r a l i s muscle incubated at 25°C  Control  Inoculated Incubation time i n days  Free Amino A c i d Lysine  ''—y  Histidine ' Ammonia Arginine Aspartic acid Threonine Serine Glutamic a c i d Proline Glycine Alanine  0 28.0  -  7.33 0.62 0.44 0.89 0.94 0.98 0.45 0.86  2.5  5. 5  9.5  22.35  2 2 . 65  22.5  --  8.1  0.79 1.45  9 . 01 1. 35 2. 61  2.16  3 . 38  2.02  3 . 28 4 . 89  2.79 0.88  l . 45  1.87  3 . 02  2.38  3 . 84 0 . 18  Half Cystine  0.99 0.11  0.14  Valine  0.61  1.67  Methione  0.63  Isoleucine  0.39 0*26  Leucine  0.48  Tyrosine  0.29 0.21  0.83 0.63  Phenylalanine  cC - Aminobutyric 0.11 Acid  2. 59 1. 11  -  2.5 26  -  5.5  9.5  9.48  4.66  -  8 . 9 8 29.5 113.7 1.46 2.77 1.10 1.93 5.18 0.99 1.74 3.23 0.72' 0.45 4.34 2.51 3.19 1.96 0 . 5 1 0.30 2.76 1.65 8.74 4 . 0 1 1.76 1.52  -  3*55 112.85  -  1.31 0.31 0.24 3.59 0.16 16.38  1.29  0.18  0.09  0.36  2.94  4.17  0.53 3.05  2.06  2.19  1.86  0.87  1. 45 2. 34  1.41  1.95 1.10 1.11  2.41  1.84  3.39  1. 10  1.12  0.63  1. 12  1.11  0.57 0.58  0.55 0.48  1.64 0.17 0.32  0.26  0 . 18  0.16  0.11  3.56  2.59  0.81  \\ mole/g of muscle  1.06  88 c o n c e n t r a t i o n i n the s t e r i l e days of i n c u b a t i o n at 1°G. amino a c i d s except c y s t i n e .  c o n t r o l i n c r e a s e d "by 70$ a f t e r 8 Increases were noted f o r a l l of the As shown i n Table V I , the f r e e amino  a c i d s i n the c o n t r o l i n c r e a s e d on the average by a f a c t o r  of 4  a f t e r 9-5 days of i n c u b a t i o n at 2 5 ° 0 . The b a c t e r i a l l o a d i n the i n o c u l a t e d samples was g r e a t e r 9  /  than 10  organisms/g f o r a l l of the samples.  s u l t i n g from the growth of P. f r a g i are r e s u l t i n g from a u t o l y s i s ; t h e r e f o r e  The changes  superimposed on those  i t i s necessary  to  the i n c r e a s e due to a u t o l y s i s i n order to i s o l a t e the 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 . , 1 9 6 9 ) . results lysis  from the i n o c u l a t e d samples were c o r r e c t e d  (Table V I I ) ,  re-  effect  When the for auto-  the f o l l o w i n g r e s u l t s were f o u n d .  days of i n c u b a t i o n , there were l a r g e i n c r e a s e s ammonia, g l y c i n e and oc - a m i n o b u t y r i c a c i d .  subtract  A f t e r 9.5  i n l e v e l s of  Smaller  occurred i n h a l f c y s t i n e , methionine and v a l i n e .  increases  While a r g i h i n e  i n c r e a s e d i n the c o n t r o l , none was present i n the i n o c u l a t e d samples.  Large decreases were observed f o r t h r e o n i n e ,  p r o l i n e and t y r o s i n e . acid,  serine,  Smaller decreases were noted i n a s p a r t i c  glutamic a c i d , a l a n i n e , i s o l e u c i n e , leu.cine and p h e n y l a l a -  nine. Separation of l y s i n e and h i s t i d i n e was achieved i n the i n o c u l a t e d sample a f t e r  9 . 5 days i n c u b a t i o n .  time i n which such s e p a r a t i o n o c c u r r e d .  T h i s was the only  No s e p a r a t i o n of these  two peaks occurred i n the c o n t r o l samples.  I t would appear  that p r o t e o l y s i s of the two d i p e p t i d e s , anserine and c a r n o s i n e , occurred,  thus e l i m i n a t i n g t h e i r i n t e r f e r e n c e i n the  of the l y s i n e - h i s t i d i n e peak (Lea et a l . ,  1969).  separation  TABLE VII Average f r e e amino a c i d c o n c e n t r a t i o n ' of c h i c k e n p e c 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 c o r r e c t e d f o r a u t o l y t i c changes.  Incubation time i n days Pree Amino A c i d  2.5  Lysine  -  Histidine  -  Ammonia  +21.4  Arginine  -  0.79  Aspartic Acid  -  0.35  Threonine  -  1.17  Serine  -  Glutamic A c i d  5.5  9.5  —  —  -  j i  +104.69  -  +103.87 1.46  1.35 0.68  -  1.46  -  1.64  -  4.87  1.3  -  2.83  -  2.99  -  0.28  -  1.7  Proline  -  0.37  1.15  -  1.8  Glycine  -  -  0.22  5.72  +  13.62  Alanine  -  0.62  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 c o n c e n t r a t i o n p moles/gram  +  . -  +  0.85  2.32  2.72  When the values from the i n o c u l a t e d samples a f t e r 9.5 days of i n c u b a t i o n are compared with the i n i t i a l values from the u n i n o c u l a t e d c o n t r o l ,  arginine,  ( 0 time)  threonine,  serine,  p r o l i n e and t y r o s i n e were the only amino a c i d s which decreased from the i n i t i a l v a l u e s , these amino a c i d s by P. T o t a l concentration  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 fragi. of amino a c i d s a f t e r 9*5 days of i n -  cubation was twice as great i n the i n o c u l a t e d muscle as i n the control.  The i n c r e a s e was due mainly to the i n c r e a s e i n ammonia  and g l y c i n e .  The l a r g e i n c r e a s e i n ammonia was due to  deamination a c t i v i t y  of the b a c t e r i a .  been reported by Tarrant (1966).  et a l .  (1971)  the  S i m i l a r i n c r e a s e s have and Gardner and Stewart  The l a r g e amount of ammonia produced accounts f o r  the  h i g h pH observed i n the s p o i l e d muscle. The complete disappearance  of a r g i n i n e from the  samples was l i k e l y due to the arginase a c t i v i t y which converts a r g i n i n e i n t o o r n i t h i n e and u r e a . reported  of P.  inoculated fragi,  Thornley ( i 9 6 0 )  that t h i s r e a c t i o n was t y p i c a l of pseudomonads.  though the presence of arginase a c t i v i t y disappearance  of a r g i n i n e ,  Al-  would account f o r  the  no t r a c e of o r n i t h i n e was d e t e c t e d .  I t may be that the o r n i t h i n e was u t i l i z e d by P. f r a g i or that it  combined with another amino a c i d peak as the amino a c i d  a n a l y z e r used i n t h i s study was not equipped with a column f o r analysis protein  of p h y s i o l o g i c a l f l u i d s ,  but r a t h e r was set up f o r  hydrolysates.  Gardner and Stewart ( 1 9 6 6 ) allowed ground beef to undergo s p o i l a g e with i t s n a t u r a l f l o r a , which c o n s i s t e d  predominately  of b a c t e r i a belonging These authors  to the Pseudomonas-Achrobacter group.  found that the c o n c e n t r a t i o n of f r e e amino a c i d s  i n c r e a s e d , with the g r e a t e s t i n c r e a s e being i n glutamic a c i d . C y s t i n e and  c>c -aminobutyric  meat, which supports crease i n these two  a c i d were found only i n the s p o i l e d  the f i n d i n g s of t h i s study t h a t the i n amino a c i d s was  due  to b a c t e r i a l p r o t e o l y -  sis. Lea et a l . (1969) showed that 30$  of the f r e e amino a c i d s  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 l o a d was  below 7 x 3.0  organisms/g of muscle.  Changes  i n a s p a r t i c a c i d , threonine, s e r i n e , p r o l i n e and g l y c i n e a c counted f o r 86$  of the observed decrease.  Except f o r g l y c i n e ,  a l l other amino a c i d s r e p o r t e d 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 c o n c e n t r a t i o n i n the i n o c u l a t e d sample, compared to the cont r o l i n the present  study.  Adamcic et a l . (1970) s t u d i e d the growth of a pigmented Pseudomonas species on chicken s k i n .  During the l o g phase of  growth the b a c t e r i a i n c r e a s e d the c o n c e n t r a t i o n of most f r e e amino a c i d s , producing  a net i n c r e a s e of 30$.  i n d i c a t e d that s e r i n e and hydroxyproline imately 50$, proline. was  and  there was  Their r e s u l t s  decreased  by approx-  a s l i g h t decrease i n g l y c i n e and  At the end of the l o g r i t h m i c growth phase, there  an i n c r e a s e i n the c o n c e n t r a t i o n of f r e e hydroxyproline  p r o l i n e i n d i c a t i n g that the organism produced a  and  collagenase  which attacked the c o l l a g e n of the chicken s k i n . According  to V e i s (1970), p r o l i n e and  comprise approximately  hydroxyproline  2 5$ of the amino a c i d r e s i d u e s i n c o l -  l a g e n , while g l y c i n e comprises n e a r l y 3 3 $ *  Glycine i s  the  f i r s t member of the t r i p l e t of r e s i d u e s , which r e s u l t s i n g l y c i n e being at every t h i r d r e s i d u e p o s i t i o n throtighout most of the p o l y p e p t i d e c h a i n .  As shown i n Table V I I , the p r o l i n e c o n -  c e n t r a t i o n decreased w i t h i n c r e a s e d i n c u b a t i o n time and t h e r e was no t r a c e of h y d r o x y p r o l i n e .  It  i s p o s s i b l e that any hydrox-  p r o l i n e or p r o l i n e r e l e a s e d by p r o t e o l y t i c a c t i o n was metaboli z e d by P . f r a g i . t h e r e f o r e , the l a r g e i n c r e a s e  no i n c r e a s e was d e t e c t e d .  However,  i n g l y c i n e , as shown i n Table V I I , may re  f l e e t p r o t e o l y s i s of the endomysium c o l l a g e n network. Part I I - E l e c t r o n Microscopy Scanning e l e c t r o n microscopy of Pseudomonas f r a g i 1.  Growth of P . f r a g i on n u t r i e n t agar and n u t r i e n t  gelatin  The a b i l i t y of b a c t e r i a to l i q u i f y g e l a t i n i s one of the t e s t s used to determine the presence The surface  of p r o t e o l y t i c  activity.  of n u t r i e n t agar and n u t r i e n t 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 c o u l d be o b served on a n o n p r o t e i n and p r o t e i n s u b s t r a t e The surface F i g u r e 17,  respectively.  of u n i n o c u l a t e d n u t r i e n t agar,  i s quite i r r e g u l a r .  shown i n  Growth of P . f r a g i upon n u t r i e n t  agar, a f t e r 24 h r i n c u b a t i o n , i s shown i n F i g u r e 18.  The  b a c t e r i a do not appear to d i s r u p t 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.  the b a c t e r i a are undergoing c e l l d i v i s i o n (A i n F i g .  Many of 18).  Although P . f r a g i has p o l a r f l a g e l l u m , none are v i s i b l e i n any  Fig.  17.  Scanning e l e c t r o n micrograph o f the s u r f a c e of n u t r i e n t agar, u n i n o c u l a t e d and incubated a t 25 C f o r 24 h r (x 22,000).  Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t agar, i n o c u l a t e d with P . f r a g i incubated 24 hr at 25 0 (x 11,400)7  of the scanning e l e c t r o n micrographs due to the low r e s o l u t i o n of the instrument (250 £ maximum) compared to the h i g h i o n obtainable w i t h a - t r a n s m i s s i o n e l e c t r o n Growth of P . f r a g i on the surface  resolut-  microscope.  of n u t r i e n t  a f t e r 24 h r i n c u b a t i o n , i s shown i n F i g u r e 19.  gelatin  As the  extra-  c e l l u l a r proteases produced by the b a c t e r i a 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 b a c t e r i a i s l o s t and they pene-  t r a t e i n t o the g e l a t i n as .shown i n F i g u r e 19. i n F i g u r e 20,  As shown at A  a f i l m of amorphous m a t e r i a l i s present  between  the b a c t e r i a .  Strands o f m a t e r i a l extend between  (B i n F i g . 20)  and between the b a c t e r i a and the surface  n u t r i e n t g e l a t i n (C i n F i g . 20).  bacteria of the  There was no evidence of s i m -  i l a r i n t e r c e l l u l a r m a t e r i a l when the organism was grown on n u t r i e n t agar ( F i g . 1 8 ) . (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 p r e p a r a t i o n . Fig.  20)  Possibly this i n t e r c e l l u l a r material  The filamentous m a t e r i a l  (B and C i n  probably r e s u l t e d 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)  d u r i n g the dehydration steps  of specimen p r e p a r a t i o n . 2.  Growth of P . f r a g i on p e e t o r a l i s major muscle The r e s u l t s  of the p l a t e counts on the i n o c u l a t e d muscle  samples incubated at 25°C are presented i n Table V I I I . was no growth i n the u n i n o c u l a t e d c o n t r o l s .  There  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 l a r g e m i c r o b i a l p o p u l a t i o n would be a t t a i n e d i n a short time p e r i o d .  T h i s was  done to minimize the e f f e c t of an i n c r e a s i n g b a c t e r i a l l o a d from that due to l e n g t h of i n c u b a t i o n .  The b a c t e r i a l l o a d was  Scanning e l e c t r o n micrograph of the surface o f n u t r i e n t g e l a t i n i n o c u l a t e d with P . f r a g i incubated f o r 24 h r at 25 0 (x 1 , 1 0 0 ) .  F i g . 20.  Scanning e l e c t r o n micrograph of the s u r f a c e of n u t r i e n t g e l a t i n i n o c u l a t e d with P. f r a g i and incubated f o r 24 h r at 25 C (x 10, COO).  98  TABLE V I I I B a c t e r i a l p o p u l a t i o n i n chicken p e e t o r a l i s with P. f r a g i incubated at 25 0  Incubation time i n days  ,  inoculated  Number o f b a c t e r i a per g of muscle  0  1.98 x 1 0  1  2.05 x l O  2  2.8  4  1.15 x l O  n  6  5.26 x 1 0  1 0  8  2.36 x 1 0  1 0  11 ~—  major  x 10  4.56 x l O ,  a average of d u p l i c a t e  samples  1  1  7  0  1 0  0  i n the order of 10"""^ organisms per g of muscle f o r the incubation period.  A p u t r i d odor was detected  entire  on a l l of the  i n o c u l a t e d c r y o f r a c t u r e d samples a f t e r i n c u b a t i o n . Kono et a l .  (1964) r e p o r t e d that sarcolemma or  endomysium was composed of three l a y e r s c o n s i s t i n g of an outer network of entangled c o l l a g e n f i b e r s , a middle amorphous l a y e r and an i n n e r plasma membrane.  As r e p o r t e d 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 r e s o l v e d by the scanning e l e c t r o n microscope. (approximately 24 h r postmortem) have a c r i s s - c r o s s  structure  the endomysium appeared to  (Pig. 2 1 ) .  the entangled network s t r u c t u r e by Kono et a l . ()1964).  However, at 0 time  Presumably t h i s was  of c o l l a g e n f i b e r s r e p o r t e d  In F i g u r e 21, the arrows p o i n t to  verse r i d g e s on the endomysium.  trans-  S c h a l l e r and Powrie (1971)  concluded that these r i d g e s were the convex i m p r e s s i o n , on the endomysium, of the u n d e r l y i n g t r a n s v e r s e transverse  elements run from one f i b r i l  elements. to another  Pig.  2 2 ) . Presumably these elevated transverse  sist  of the transverse  form a t r i a d .  Elevated (arrows  in  elements c o n -  tubule and two t e r m i n a l c i s t e r n a e which  A c c o r d i n g to Mendell (1971) the t r i a d s i n chicken  p e e t o r a l i s muscle o v e r l a y the Z d i s c of the m y o f i b r i l s . The surface of a f i b r i l day  from i n o c u l a t e d muscle, a f t e r  of i n c u b a t i o n , i s shown i n F i g u r e 23.  extensive,  graph,  P r o t e o l y s i s i s not  however, the arrows i n d i c a t e areas where the  have begun to penetrate  the endomysium.  bacteria  As shown i n the m i c r o -  the surface of the endomysium has a g r a n u l a r  r a t h e r than the c r i s s - c r o s s  1  appearance  strand network apparent at 0 t i m e .  The g r a n u l a r appearance may be due to p r o t e o l y s i s of the c o l -  Fig.  21  L o n g i t u d i n a l view of i n t a c t endomysium i n c r y o f r a c t u r e d p e e t o r a l i s muscle, zero time (x 1 1 , 4 0 0 ) .  101  Fig.  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 i n o c u l a t e d chicken p e e t o r a l i s muscle at zero time (x 22,200).  L o n g i t u d i n a l v i e w o f endomysium f r o m 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 w i t h P. f r a g i a n d i n c u b a t e d 24 h r a t 25 0  (x 9,5007:  l a g e n f i b e r s , which comprise the outer l a y e r of the endomysium. In F i g u r e 24,  the b a c t e r i a  exposed f i b r i l s verse elements  are present  on 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  The t r a n s -  (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 F i g u r e 24 p o i n t to mitochondria which are present i n the interfibrillar  space.  The surface  of a muscle f i b e r a f t e r 2 days of i n c u b a t i o n  at 25°C i s shown i n F i g u r e 25. below the surface  The b a c t e r i a have  penetrated  of the endomysium, p r e s e n t i n g a p i c t u r e  s i m i l a r to that of growth on n u t r i e n t g e l a t i n ( F i g . 19 and  20).  P r o t e o l y s i s of the endomysium i n d i c a t e s the P. f r a g i produces a collagenase.  Some of the b a c t e r i a have been washed away  d u r i n g the f i x a t i o n stage of specimen p r e p a r a t i o n , l e a v i n g obl o n g shaped holes (A i n F i g . 2 5 ) , which are approximately same s i z e as the b a c t e r i a ,  i n the endomysium.  These h o l e s s u g -  gest that complete s o l u b i l i z a t i o n of the p r o t e i n s occurs the immediate v i c i n i t y of the c e l l s u r f a c e . solubilized,  the b a c t e r i a  s i n k i n t o the t i s s u e .  This  ( l 9 7 l ) which showed a c l e a r  approximately 0.25 pro i n diameter, w a l l and the f i b r i l s  in  As the p r o t e i n i s  a t i o n i s i n accord w i t h the t r a n s m i s s i o n micrographs sented by Dutson et a l .  the  observpre-  zone,  between the b a c t e r i a l  cell  of the muscle.  With i n c r e a s e d i n c u b a t i o n time, domysium becomes more e x t e n s i v e .  p r o t e o l y s i s of the e n -  As shown i n F i g u r e 26,  after  4 days of i n c u b a t i o n l a r g e areas of the endomysium have been digested.  The m a j o r i t y of the b a c t e r i a have been washed away  exposing the u n d e r l y i n g f i b r i l s .  Individual f i b r i l s  cannot  104  Fig.  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 i n c u b a t e d 24 n r a t 25 0 (x 9,500).  105  P i g . 25.  L o n g i t u d i n a l view of endomysium i n 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 f o r 2 days at 25 0 (x 9 , 5 0 0 ^  106  Pig.  26.  L o n g i t u d i n a l view of muscle f i b e r from 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 with P . f r a g i and incubated f o r 4 days a t 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 . examination  The r e a s o n becomes c l e a r upon  of the t r a n s m i s s i o n micrographs ( F i g . 37 and  39). pm  Complete s o l u b i l i z a t i o n o n l y occurs i n a s m a l l zone - 0.08 i n diameter - a d j a c e n t t o the b a c t e r i a .  The  c l e a r zone must  r e p r e s e n t 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 f o r m i n g 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  thus making i t i m p o s s i b l e t o d i s t i n g u i s h the i n d i v i d u a l The Fig.  concave i m p r e s s i o n s v i s i b l e on the exposed f i b r i l s  spaces fibrils  (A i n  26) are i n d i c a t i o n s t h a t 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 d u r i n g s p o i l a g e of muscle by P. The' s l i g h t convex i m p r e s s i o n s  fragi•  on the endomysium remnant i n  the c e n t e r of F i g u r e 26 i n d i c a t e s t h a t t r a n s v e r s e elements are p r e s e n t , a l t h o u g h these elements are not p r e s e n t s u r f a c e thus i n d i c a t i n g t h a t elements of the  on the  fibril  sarcoplasmic  r e t i c u l u m are s u b j e c t t o p r o t e o l y s i s . M i c r o g r a p h s of f i b r i l s from i n o c u l a t e d samples i n c u b a t e d i n excess of 4 days show s i m i l a r s i g n s of d i s i n t e g r a t i o n .  The  s u r f a c e 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 F i g u r e 27.  As i n F i g u r e 26, convex i m p r e s s i o n s  on the endo-  mysium i n d i c a t e t h a t i n t e r a c t t r a n s v e r s e elements are under the endomysium.  present  However, t h e r e i s no i n d i c a t i o n of t r a n s  v e r s e elements i n areas where the endomysium has been a t t a c k e d . Strands  of m a t e r i a l are a s s o c i a t e d w i t h some of the b a c t e r i a •  (A i n F i g . 2 7 ) .  S i m i l a r i n t e r c e l l u l a r s t r a n d s 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 . 2 0 ) . As shown i n F i g u r e 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 m a t t e r s h r i n k s d u r i n g the  de-  108  Fig.  27.  L o n g i t u d i n a l viev. of muscle f i b e r from 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 f o r 8 days at room temperature (x 1 2 , 1 0 0 ) . 1  h y d r a t i o n procedure of sample p r e p a r a t i o n forming shown at A i n F i g u r e 2 7 .  The  the  strands  concave appearance of the exposed  m y o f i b r i l s , i s s i m i l a r to t h a t observed a f t e r 4 days i n c u b a t i o n . The m y o f i b r i l l a r p r o t e i n s are apparently more r e s i s t a n t t o p r o t e o l y t i c a c t i o n of P. f r a g i than was  the collagenous  material  of the endomysium. A f t e r 11 days of i n c u b a t i o n ( F i g . 28) the b a c t e r i a have penetrated  deeper i n t o the m y o f i b r i l s .  measure the extent of p e n e t r a t i o n .  I t i s impossible  to  However, i t appears t h a t  the b a c t e r i a have d i g e s t e d through one f i b r i l and are a t t a c k i n g the u n d e r l y i n g f i b r i l s .  Therefore,  p e n e t r a t i o n i s no  greater  than 3 to 4pfl"i. Micrographs of the u n i n o c u l a t e d  c o n t r o l were taken at the  same time as those of the i n o c u l a t e d samples.  Changes i n the  c o n t r o l s were not excessive and f o r t h i s reason  only photo-  micrographs taken of the c o n t r o l a f t e r 11 days of i n c u b a t i o n w i l l be presented. (Fig.  The  performations  present  i n the endomysium  29) are i n d i c a t i o n s of t i s s u e d e t e r i o r a t i o n . Such i n -  d i c a t i o n s of d e t e r i o r a t i o n was  extremely v a r i a b l e .  Some areas  of the endomysium appeared i n t a c t while other areas, on same sample, showed signs of extreme d i s t u p t i o n .  the  S c h a l l e r and  Powrie (1971) r e p o r t e d t h a t the t r a n s v e r s e elements of turkey m y o f i b r i l s appeared c o l l a p s e d a f t e r 6 days postmortem at  5°C.  at  25°C.  storage  F i g u r e 30 shows i n t a c t m y o f i b r i l s a f t e r 11 days storage The  t r a n s v e r s e elements do not appear c o l l a p s e d but  they do show some signs of d i s i n t e g r a t i o n . The  transverse  elements are not as d i s t i n c t as at 0 time, nor are they tinuous, but they are d i s r u p t e d i n a number of areas  con-  (A i n  Fig.  28.  L o n g i t u d i n a l view of muscle f i b e r from 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 with. P . f r a g i and incubated at 25 0 f o r 11 days.  Tx 10,300).  Ill  Pig.  29.  L o n g i t u d i n a l v i e w o f endomysium f r o m c r y o f r a c t u r e d p e e t o r a l i s m u s c l e , u n i n o c u l a t e d and i n c u b a t e d f o r 11 d a y s a t 25 C (x 2 1 , 3 0 0 ) .  112  Fig.  30.  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 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 1 0 , 5 0 0 ) .  F i g . 30). 3.  •  '  G r y o f r a c t u r e of muscle a f t e r i n c u b a t i o n Inoculated c r y o f r a c t u r e d p i e c e s of chicken p e e t o r a l i s  muscle were c r y o f r a c t u r e d again a f t e r 10.5 days of i n c u b a t i o n at 25°0.  The b a c t e r i a l l o a d was i n excess o f 1 0 ^  per g o f muscle.  organisms  As shown i n F i g u r e 31, the b a c t e r i a grow up  between the muscle f i b e r s when such space i s a v a i l a b l e .  The  top s u r f a c e o f the f i b e r s i n F i g u r e 31 does not show s i g n s o f b a c t e r i a l growth.  Presumably these s u r f a c e s were i n immediate  contact w i t h adjacent f i b e r s .  At h i g h e r m a g n i f i c a t i o n ( F i g .  32) the b a c t e r i a are " s i n k i n g " i n t o the f i b e r ( A ) .  It i s  apparent that a e r o b i c s p o i l a g e organisms can penetrate  into  the i n t e r i o r of the muscle i n cut up p o u l t r y . 4.  Growth of P. f r a g i on chicken p e e t o r a l i s muscle at 5 0  incubated  The b a c t e r i a l p o p u l a t i o n was 7x10*% 2 . 1 x l 0 , and 4.5x10**"° 7  organism per g of muscle a t 0, 6.5 days and 21 days r e s p e c t i v e l y . There was no growth on the u n i n o c u l a t e d  controls.  P u t r i d odor  was detected on the i n o c u l a t e d samples a f t e r 6.5 days of i n c u b ation. A f t e r 6.5 days ( F i g . 3 3 ) there was no i n d i c a t i o n o f extensive proteolysis.  I n the upper r i g h t hand corner of F i g u r e 3 3 ,  the endomysium appears to be s l i g h t l y d i s r u p t e d . A f t e r 21 days of i n c u b a t i o n ( F i g . 3 4 ) the b a c t e r i a have penetrated  the endomysium and p r o t e o l y s i s of u n d e r l y i n g myo-  f i b r i l s has occurred.  D e t e r i o r a t i o n o f the endomysium i n  P i g . 31.  L o n g i t u d i n a l view of muscle f i b e r s from c r y o f r a c t u r e d p e c t o r a l i s muscle inocubated with. P . f r a g i . incubated a t 25 0 f o r 10.5 days then r e c r y o f r a c t u r e d (x 1 , 9 5 0 ) .  115  Fig.  32.  L o n g i t u d i n a l view of muscle f i b e r from c r y o f r a c t u r e d p e c t o r a l i s muscxe i n o c u l a t e d with P . f r a g i . incubated f o r 10.5 days at 25 0 then r e c r y o f r a c t u r e d (x 1 9 , 5 0 0 ) .  116  the. c o n t r o l samples, a f t e r 21 days of i n c u b a t i o n , was not severe as that o c c u r r i n g i n samples s t o r e d at room  as  temperature.  As shown i n F i g u r e 35, the endomysium i s not p e r f o r a t e d and the t r a n s v e r s e  r i d g e s are d i s t i n c t .  These r e s u l t s  disagree  with those of S c h a l l e r and Powrie (1971) who found t h a t endomysium of turkey muscle was e x t e n s i v e l y p e r f o r a t e d 6 days storage at  5°C  the after  In t h e i r work, S c h a l l e r and Powrie  (1971) c r y o f r a c t u r e d the muscle a f t e r postmortem s t o r a g e , while i n t h i s study the t i s s u e was c r y o f r a c t u r e d p r i o r to i n c u b a t i o n . P o s s i b l y the endomysium becomes f r a g i l e d u r i n g postmortem aging and tends to fragment.  I f t h i s i s the case,  cryofracturing  a f t e r postmortem aging would r e s u l t i n g r e a t e r  structural  damage• 5.  Summary V i s u a l evidence obtained on the scanning e l e c t r o n m i c r o -  scope i n d i c a t e s t h a t 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 p r o t e i n s does occur d u r i n g s p o i l a g e of p o u l t r y by P . f r a g i . The endomysial l a y e r 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 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 cannot u n t i l the endomysium has been p e n e t r a t e d .  occur  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 occurred s l o w l y i n d i c a t i n g that  either  they are more r e s i s t a n t  produces  to a t t a c k or that the b a c t e r i a  a l i m i t e d q u a n t i t y of the enzyme r e s p o n s i b l e f o r t h e i r ruption.  The presence  of the oblong shapped h o l e s , which a p -  proximates the s i z e of the b a c t e r i a w i t h the presence  dis-  of i n t a c t  i n the endomysium a l o n g  remnants of the endomysium a d -  jacent to b a c t e r i a l c o l o n i e s ,  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-  e n t l y d i f f u s i o n of the enzyme or enzymes i n t o the muscle o c c u r ed only i n a s m a l l area around the b a c t e r i a l  cell.  As one would expect, the r a t e o f p r o t e o l y s i s was f a r g r e a t e r at the h i g h e r temperature,  which i s i n agreement with  the work of Borton ejb. al. (1970b) and Rey :  The micrographs et  et a l . (1970).  support the r e s u l t s obtained by  a l . (1970a and b) and T a r r a n t et a l . (1971) who  Borton  have shown  p r o t e o l y s i s of m y o f i b r i l l a r p r o t e i n s occurs i n muscle i n o c u l ated with P. f r a g i .  The micrographs  Ockerman et a l . (1969) who  support the r e s u l t s of  found that the stromal p r o t e i n f r a -  c t i o n decreased i n muscle i n o c u l a t e d with a Pseudomonas s p e c i e s . P r o t e o l y s i s of the endomysium would account  f o r p a r t of the  decrease i n i n s o l u b l e p r o t e i n n i t r o g e n observed by et  a l . (l970a) i n porcine muscle i n o c u l a t e d by P.  Borton fragi.  Jay (1966) concluded that s p o i l a g e of Pseudomonas organisms occurs i n the absence of s i g n i f i c a n t p r o t e o l y s i s . the scanning e l e c t r o n microscopy  R e s u l t s of  studies i n d i c a t e that pro-  t e o l y s i s i s l i m i t e d to the s u r f a c e of the muscle f i b e r s does extend beyond a few micrometers.  and  I t seems l i k e l y t h a t the  extent of the 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 p r o teins i s i n s i g n i f i c a n t during spoilage. Off-odors were detected from the i n o c u l a t e d samples incubated a t 25°0 and 5°0 before any m i c r o s c o p i c evidence of extensive p r o t e o l y s i s was  observed.  These r e s u l t s agree with  the c o n c l u s i o n s reached by Lerke et a l . (1967) t h a t p r o t e o l y s i s r e p r e s e n t s an advanced stage of meat s p o i l a g e .  The  cryofract-  121 ured samples were i n o c u l a t e d with a h i g h i n i t i a l s t r a i g h t from n u t r i e n t b r o t h .  inoculum  I t i s l i k e l y t h a t the n u t r i e n t  b r o t h s u p p l i e d the necessary n u t r i e n t s f o r growth which l e a d to  the development of o f f - o d o r s .  F u r t h e r work i s r e q u i r e d to  determine, whether or not p r o t e o l y s i s c o i n c i d e s with the of  onset  s p o i l a g e or r e p r e s e n t s an advanced s t a t e of s p o i l a g e .  This  can best be achieved by i n o c u l a t i n g the muscle with b a c t e r i a suspended i n b u f f e r and u s i n g a low i n i t i a l Transmission  e l e c t r o n mlscroscopy  inoculum.  of Pseudomonas f r a g i  Samples of chicken p e e t o r a l i s muscle were incubated a t for  10.5  i n excess  days.  A f t e r i n c u b a t i o n , the b a c t e r i a l p o p u l a t i o n  of 10"^  organisms per g of muscle.  5°C was  Off-odors were de-  t e c t e d at the end of the i n c u b a t i o n i n d i c a t i n g t h a t the muscle had  spoiled.  No b a c t e r i a l growth or o f f - o d o r s were detected i n  the u n i n o c u l a t e d c o n t r o l s . The Z d i s c on the u n i n o c u l a t e d c o n t r o l s ( P i g . 36) shows s i g n s of d i s r u p t i o n .  The Z d i s c appears d i f f u s e and some of  the dense m a t e r i a l present i n the Z d i s c has been l o s t i n P i g . 36).  (arrows  D i s i n t e g r a t i o n of the Z l i n e d u r i n g postmortem  a g i n g agrees with the r e p o r t s by Davey and Dickson  (1970) on  beef muscle and Fukazawa et a l . (1969) on chicken muscle. There was  complete d i s i n t e g r a t i o n of the m y o f i b r i l s i n  areas adjacent to b a c t e r i a l growth ( F i g . 37).  As r e p o r t e d by  Wiebe and Chapman (1968a and b ) , the c e l l w a l l i s i r r e g u l a r l y undulant.  P r o t r u s i o n s (B i n F i g . 37) were observed  s u r f a c e of the b a c t e r i a .  on the  These p r o t r u s i o n s are s i m i l a r i n  appearance to the " b l e b l i k e e v a g i n a t i o n s " observed  by Dutson  122  L_  Fig.  37.  L o n g i t u d i n a l view of p e c 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 a t 5 C f o r 1 0 . 5 days (x  687900).  et a l . (1971)» on the s u r f a c e o f P. f r a g i growing on s p o i l e d porcine muscle.  Wiebe and Chapman (1968a) reported the  pre-  sence o f c e l l u l a r p r o t r u s i o n s on the c e l l w a l l o f some pseudomads under c e r t a i n 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 c o n d i t i o n s . Smirnova e t a l . (1971) observed c e l l u l a r p r o t r u s i o n s which they termed "microcapsules"  on c e r t a i n s p e c i e s o f b a c t e r i a .  et a l . (1971) and Smirnova e t a l . (1971) concluded  Dutson  that  c e l l u l a r enxymes were secreted, as such h i g h molecular  these weight  substances cannot d i f f u s e through the c e l l membrane. 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 ,  approximately  0.08 pm i n diameter surrounds the b a c t e r i a , i n d i c a t i n g that comp l e t e s o l u b i l i z a t i o n o f the m y o f i b r i l l a r p r o t e i n s has  occurred.  An amorphous r e g i o n (A i n P i g . 37), presumably c o n s i s t i n g o f d i s i n t e g r a t e d sarcomere components, occurs between the c l e a r zone and unattacked  p o r t i o n o f the f i b r i l  (C i n P i g . 37).  average d i s t a n c e between the b a c t e r i a and the i n t a c t i s 0.6 um.  P r o t e o l y s i s i s l i m i t e d t o a s m a l l area  The  fibrils surrounding  the b a c t e r i a . M y o f i b r i l s from muscle i n o c u l a t e d and incubated with P. f r a g i but not adjacent 38.  t o b a c t e r i a l c e l l s are shown i n Pigure  Most o f the dense m a t e r i a l 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 o f the uninoculated  c o n t r o l ( P i g . 36).  The  d i f f e r e n c e i n the M band and the H zone can be accounted f o r by c o n t r a c t i o n . 36)  The mean sarcomere l e n g t h i n the c o n t r o l ( P i g .  was 2.0pm while the sarcomere l e n g t h i n the i n o c u l a t e d  sample ( P i g . 38) i s 1.6pm.  According  t o B e n d a l l (1969), as  the muscle c o n t r a c t s the a e t i n f i l a m e n t s s l i d e up between the  L o n g i t u d i n a l view of 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 at 5 0 f o r 1 6 . 5 days (x  497000).  myosin f i l a m e n t s and i n t o the H zone, thus d e c r e a s i n g i t s  size.  C o n t r a c t i o n a l s o r e s u l t e d i n i n c r e a s e d d e n s i t y of the M band. The r e s u l t s  of t h i s study are not i n agreement  Dutson et a l .  (1971)•  w i t h those of  These authors r e p o r t e d that m y o f i b r i l s  from porcine muscle i n o c u l a t e d M t h P . f r a g i showed a d i s r u p t e d appearance  i n the A band, the H zone was almost devoid of mat-  e r i a l and the dense m a t e r i a l 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 m a t e r i a l from the Z l i n e occurred as a consequence of s p o i l a g e 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 i d not c o n t a i n any b a c t e r i a l  cells.  Re-  s u l t s obtained i n t h i s study i n d i c a t e that p r o t e o l y s i s only occurs i n a l i m i t e d area surrounding the b a c t e r i a . i n f i b r i l s which are not adjacent  The H zone  to b a c t e r i a l c e l l s was  intact.  D i s r u p t i o n of the Z l i n e was probably due to postmortem a g i n g r a t h e r than p r o t e o l y s i s . v M y o f i b r i l s from i n o c u l a t e d t i s s u e are shown i n cross s e c t i o n i n F i g u r e 39.  The t h i c k myosin f i l a m e n t s are s u r r o u n -  ded by t h i n n e r a c t i n f i l a m e n t s . s m a l l zone adjacent  P r o t e o l y s i s occurs only i n a  to the b a c t e r i a .  of s p e c i f i c d i s r u p t i o n of myosin.  There i s no i n d i c a t i o n  127  Fig.  39.  Gross s e c t i o n of 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 at 5 G f o r 10.5 days  (x 497oooT  128 GENERA! DISCUSSION R e s u l t s obtained i n t h i s study i n d i c a t e that of the sarcoplasmic, occurs as a r e s u l t a)  m y o f i b r i l l a r and stroma p r o t e i n  fractions  of s p o i l a g e by Pseudomonas f r a g i .  Evidence of p r o t e o l y s i s of the sarcoplasmic 1)  proteolysis  alterations  proteins:  o c c u r r i n g i n the e l u t i o n p a t t e r n  during  gel f i l t r a t i o n studies. 2)  l o s s of 2 bands i n the d i s c g e l  electrophoresis  patterns. b)  Evidence f o r 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 1)  proteins:  Scanning e l e c t r o n and t r a n s m i s s i o n e l e c t r o n m i c r o 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  2)  The e x t r a c t a b i l i t y  of growth of P .  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  was s i g n i f i c a n t l y (P ^ .05) 3)  fragi.  lower i n i n o c u l a t e d , samples.  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  elect-  r o p h o r e t i c p a t t e r n s was lower i n the i n o c u l a t e d samples and decreased i n samples incubated f o r l o n g e r time periods. c)  Evidence of p r o t e o l y s i s of the stroma p r o t e i n s : 1)  Scanning e l e c t r o n micrographs i n d i c a t e d that  the  endomysium was very s u s c e p t i b l e to degradation by the p r o t e o l y t i c enzymes of P . 2)  fragi.  R e s u l t s of f r e e amino a c i d a n a l y s i s of the i n o c u l a t e d muscle i n d i c a t e d that the i n c r e a s e  i n g l y c i n e was  approximately 15 times g r e a t e r than the i n c r e a s e the other amino a c i d s .  As g l y c i n e composes,  in  approx-  129 i m a t e l y /3 of the amino a c i d r e s i d u e s i n c o l l a g e n 1  ( Y e i s , 1970).  This i n c r e a s e i n the l e v e l of g l y c i n e  may "be a f u r t h e r evidence f o r the p r o t e o l y s i s o f the stroma f r a c t i o n . Results  of the p r o t e i n s o l u b i l i t y s t u d i e s of the i n o c u l a t e d  muscle i n d i c a t e d that the sarcoplasmic not  p r o t e i n s o l u b i l i t y was  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 c o n t r o l , except  a f t e r 5.5  days i n c u b a t i o n when i t was s i g n i f i c a n t l y  T h e r e was no s i g n i f i c a n t i n c r e a s e i n nonprotein the i n o c u l a t e d samples.  Results  nitrogen i n  of g e l f i l t r a t i o n  c o n t r a d i c t e d the p r o t e i n s o l u b i l i t y s t u d i e s .  higher.  studies  Results  obtained  i n the g e l f i l t r a t i o n s t u d i e s i n d i c a t e d that the sarcoplasmic f r a c t i o n e x t r a c t e d from i n o c u l a t e d muscle c o n t i n u a l l y decreased during i n c u b a t i o n , although t h i s decrease was not as extensive as that o c c u r r i n g i n the c o n t r o l and the nonprotein  nitrogen  f r a c t i o n increased  (1963)  and  i n the i n o c u l a t e d samples.  Bell  Synge (1955) i n d i c a t e d that d u r i n g determinations of non-  p r o t e i n n i t r o g e n by a c i d p r e c i p i t a t i o n , small  nonprotein  molecules may be r e t a i n e d on the p r e c i p i t a t e d p r o t e i n as a r e s u l t of a b s o r p t i o n  or i o n exchange.  I n t h i s event, r e s u l t s  of p r o t e i n s o l u b i l i t y would i n d i c a t e lower values n i t r o g e n and higher values  f o r nonprotein  f o r water s o l u b l e p r o t e i n .  p a r i s o n o f the r e s u l t s obtained  during p r o t e i n  Com-  solubility  s t u d i e s with those of the g e l f i l t r a t i o n study i n d i c a t e d that t h i s s i t u a t i o n probably Results  obtained  occurred.  by Hasegawa ejb a l . (1970a) i n d i c a t e d that  p r o t e o l y s i s of the sarcoplasmic extensive  p r o t e i n s by P. f r a g i was more  i n porcine muscle than i n r a b b i t .  The authors con-  eluded  that the b a c t e r i a produced " h i g h l y s p e c i f i c enzymes  which p r e f e r e n t i a l l y a c t upon only c e r t a i n p r o t e i n s or enzymes indigenous to muscle." of the sarcoplasmic  R e s u l t s of the d i s c g e l e l e c t r o p h o r e s i s  p r o t e i n s i n t h i s study showed that 2 of  the p r o t e i n bands disappeared of i n o c u l a t e d t i s s u e and decreased. al.  Therefore,  from the e l e c t r o p h o r e t i c p a t t e r n  the i n t e n s i t y of the remaining bands  the theory put forward by Hasegawa et,  (1970a) r e g a r d i n g p r e f e r e n t i a l a t t a c k could be p o s s i b l e . Dutson et a l . (1971) concluded that s p e c i f i c d i s r u p t i o n  of myosin and m a t e r i a l from the Z l i n e occurred uence of s p o i l a g e by P. f r a g i .  as a conseq-  Disc g e l e l e c t r o p h o r e 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 e x t r a c t e d from i n o c u l a t e d t i s s u e r e ported i n t h i s study d i d not i n d i c a t e such p r e f e r e n t i a l a t t a c k . Transmission  e l e c t r o n miscrographs presented  i n t h i s study i n -  d i c a t e d t o t a l d i s r u p t i o n of the m y o f i b r i l i n areas immediately adjacent  to b a c t e r i a l c e l l s r a t h e r than s p e c i f i c d i s r u p t i o n as  r e p o r t e d by Dutson et a l . (1971). As has been reported i n previous 1971;  Borton et a l . 1970  t e o l y s i s was Jay  s t u d i e s (Tarrant et a l . ,  a and b; Hasegawa e_t a l . 1970a) pro-  not detected u n t i l a f t e r s p o i l a g e was  evident.  (1966) and Jay and Kontou (1967) have s t a t e d that  low  temperature s p o i l a g e occurs without causing s i g n i f i c a n t teolysis.  R e s u l t s obtained  i n the present  pro-  study showed that  s i g n i f i c a n t p r o t e o l y s i s d i d occur d u r i n g s p o i l a g e by P.  fragi,  a species of b a c t e r i a commonly a s s o c i a t e d with meat s p o i l a g e . While q u a n t i t i v e changes i n the p r o t e i n f r a c t i o n s could not detected u n t i l a f t e r s p o i l a g e was  be  evident, the development of  p r o t e o l y t i c a c t i v i t y before the onset of s p o i l a g e may  be s i g n -  ificant. Researchers  s t u d y i n g " b a c t e r i a l p r o t e o l y s i s of meat d u r i n g  s p o i l a g e have, without e x c e p t i o n , used minced t i s s u e i n order  to  produce maximum b a c t e r i a l growth so t h a t changes r e s u l t i n g from such growth could be detected by b i o c h e m i c a l means.  The b i o -  chemical changes evaluated i n t h i s manner may not be the r e p r o d u c t i o n of those caused by s p o i l a g e of n a t u r a l  real  tissue  without mechanical d e f o r m a t i o n . The use of the scanning e l e c t r o n 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 i n t a c t tissue.  R e s u l t s obtained i n t h i s study i n d i c a t e d 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 p r o t e i n s 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 i n t a c t  t i s s u e was i n -  oculated with a h i g h l e v e l inoculum and samples were judged to be s p o i l e d a f t e r 1 day. samples incubated f o r 1 day.  P r o t e o l y s i s was not d e t e c t a b l e T h i s probably r e f l e c t s  in  a lag in  growth r e s u l t i n g from the t r a n s f e r from the l i q u i d medium to the muscle.  Minute p r o t e o l y s i s of the endomysium was  detected  a f t e r 2 days i n c u b a t i o n by SEM, which would be d i f f i c u l t detect by o r d i n a r y b i o c h e m i c a l t e c h n i q u e s .  I n order to  to deter-  mine i f p r o t e o l y s i s does occur p r i o r to i n c i p i e n t s p o i l a g e , w i l l be necessary  to use a low l e v e l inoculum.  In other s t u d i e s 1967;  it  (Ockerman et a l . 1969; Jay and Kontou,  Lerke et a l . , 1967) meat was i n o c u l a t e d w i t h a c u l t u r e of  unknown genera or with a pure c u l t u r e of u n c l a s s i f i e d s p e c i e s . Lack of r e p r o d u c i b i l i t y i n t h i s p r a c t i c e brought about  incon-  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 .  It  is  important to use known pure c u l t u r e s to o b t a i n r e p r o d u c i b l e results,  although i t i s u n l i k e l y t h a t meat undergoes n a t u r a l  low-temperature  s p o i l a g e caused., by a s i n g l e s p e c i e s of  bacteria  The Pseudomonas Achromobacter group i s r e s p o n s i b l e i n most cases of meat s p o i l a g e . Conclusions 1.  P r o t e o l y s i s of the s a r c o p l a s m i c ,  stroma p r o t e i n f r a c t i o n s P.  m y o f i b r i l l a r and  occurs as a r e s u l t of s p o i l a g e by  fragi. 2.  The n o n p r o t e i n n i t r o g e n f r a c t i o n i n c r e a s e d  consider-  a b l y d u r i n g the 9.5-day i n c u b a t i o n p e r i o d of the i n o c u l a t e d samples. 3.  The endomysium was more s u s c e p t i b l e to  proteolysis  than 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 . 4.  T h i s study d i d not i n d i c a t e 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 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 p r o t e i n s  only occurred i n a l i m i t e d area surrounding the 6.  bacteria.  A e r o b i c s p o i l a g e organisms can grow up between the  muscle f i b e r s , thus p e n e t r a t i n g i n t o the i n t e r i o r of the muscle 7. spoilage.  P r o t e o l y s i s probably r e p r e s e n t s an advanced stage o f  BIBLIOGRAPHY Adamcic, M. and C l a r k , D.S. 1970. B a c t e r i a induced b i o c h e m i c a l changes i n c h i c k e n s k i n s t o r e d a t 5 C. J . Food S c i . 35: 103-108. Adamcic, M., C l a r k , D.S. and Y a g u c h i , M. 1970. E f f e c t of p s y c h r o t o l e r a n t b a c t e r i a on the amino a c i d content o f c h i c k e n s k i n . J . Food S c i . 35: 272-275.. Anonymous 1966. Sephadex-gel f i l t r a t i o n i n t h e o r y and p r a c t i c e . Pharmacia F i n e Chemicals Handbook. A y r e s , J.C. 1960a. The r e l a t i o n s h i p of organisms of the gemis Pseudomonas t o the s p o i l a g e of meat, p o u l t r y and eggs. J . A p p l . B a c t e r i o l . :23: 471-486. A y r e s , J.C. 1960b. Temperature r e l a t i o n s h i p s and some o t h e r c h a r a c t e r i s t i c s of the m i c r o b i a l f l o r a d e v e l o p i n g on r e f r i g e r a t e d beef. Food Res. 25: 1-18. A y r e s , J . C , O g i l v y , W.S. and Stewart, G.F., 1950. Post mostem changes i n s t o r e d meats. 1. Microorganisms a s s o c i a t e d w i t h development of s l i m e on e v i s c e r a t e d cutr-up p o u l t r y . Food T e c h n o l . 4: 199-205. Barnes, E.M. and Impey, C.S. 1968. Psychrophilic spoilage b a c t e r i a of p o u l t r y . J . A p p l . B a c t e r i o l . 31: 97-107. B e l l , P.M. 1963. A c r i t i c a l study of methods f o r the d e t e r m i n a t i o n of n o n p r o t e i n n i t r o g e n . A n a l . Biochem .5: 443-451. B e n d a l l , J.R. 1969. "Muscles, M o l e c u l e s and Movement" Heinemann E d u c a t i o n a l Books L t d . , London. B o d w e l l , C.E. and Pearson, A.M. 1964. The a c t i v i t y of p a r t i a l l y p u r i f i e d bovine c a t h e p t i c enzymes on v a r i o u s n a t u r a l and s y n t h e t i c s u b s t r a t e s . J . Food S c i . 29: 603-607. B o r t o n , R.J., B r a t z l e r , L . J . and P r i c e , J.F. 1970a. E f f e c t s of f o u r s p e c i e s of b a c t e r i a on p o r c i n e muscle. 1. Prot e i n s o l u b i l i t y and e m u l s i f y i n g c a p a c i t y . J . Food S c i . 35: 779-782. B o r t o n , R.J., B r a t z l e r , L . J . and P r i c e , J.F. 1970b. E f f e c t s of f o u r s p e c i e s of b a c t e r i a on p o r c i n e muscle. 2. E l e c t r o p h o r e t i c p a t t e r n s of e x t r a c t s of s a l t - s o l u b l e p r o t e i n . J . Food S c i . 35: 783-786. B o r t o n , R.J., Webb, N.B. and B r a t z l e r , L . J . 1968. The e f f e c t of microorganisms on the e m u l s i f y i n g c a p a c i t y and e x t r a c t r e l e a s e volume of f r e s h p o r c i n e t i s s u e s . Food T e c h n o l . 22: 94-96.  Bowers, J.A. 1969. Free amino a c i d s i n p o r c i n e muscle aged one o r e i g h t days. J . A g r i . Food Ohem. 17: 902-903. B r i s k e y , E . J . and Fukazawa, T. 1971. M y o f i b r i l l a r p r o t e i n s of s k e l e t a l muscle. Adv. Food Res. 19: 279-360. C a l d w e l l , K.A. and G r o s j e a n , O.K. 1971. Lysosomal c a t h e p s i n s of c h i c k e n s k e l e t a l muscle d i s t r i b u t i o n and p r o p e r t i e s . J . Agr. Chem. 19: 108-111. Coleby, B., Ingram, M and Shepherd, H.J. I960. Treatment o f meats w i t h i o n i z i n g r a d i a t i o n s . I l l R a d i a t i o n p a s t e u r i z a t i o n o f whole e v i s c e r a t e d c h i c k e n c a r c a s s e s . J . S c i . Food A g r i c . 11: 61-71. Davey, C L . and D i c k s o n , M.R. 1970. S t u d i e s i n meat t e n d e r ness. 8. U l t r a - s t r u c t u r a l changes i n meat d u r i n g a g i n g . J . Pood S c i . 3 5 : 56-60. Davey, C L . and G i l b e r t , K.V. 1966. S t u d i e s i n meat tenderness 2. P r o t e o l y s i s and the a g i n g o f beef. J . Food S c i . 31 135-140.  Davey, C L . and G i l b e r t , K.V. 1967. S t r u c t u r a l changes i n meat d u r i n g a g i n g . J . Food Technol. 2: 57-59. Davey, C l . and G i l b e r t , K.V. 1968. S t u d i e s i n meat t e n d e r n e s s . 4. Changes i n the e x t r a c t a b i l i t y of m y o f i b r i l l a r p r o t e i n s d u r i n g meat a g i n g . J . Food S c i . 3 3 2-11. :  D a v i s , B . J . 1964. D i s k e l e c t r o p h o r e s i s . I I Method and app l i c a t i o n t o human serum p r o t e i n s . Ann. N.Y. Acad. S c i . 121: 404-437. deFremery, D. and P o o l , M.J. i 9 6 0 . B i o c h e m i s t r y o f c h i c k e n muscle as r e l a t e d t o r i g o r m o r t i s and t e n d e r i z a t i o n . Food Res. 25: 78-87. Dodge, J.W. and P e t e r s , F.E. I960. Temperature and pH changes i n p o u l t r y b r e a s t muscles a t s l a u g h t e r . P o u l . S c i . 39: 765-768. Doty, D.M. and Wachter, J.P. 1955. I n f l u e n c e o f gammar a d i a t i o n on p r o t e o l y t i c enzyme a c t i v i t y i n beef muscle. J . A g r i . Ghem. 3 61-63. :  Dutson, T.R., Pearson, A.M., P r i c e , J.F., Spink, G.C and T a r r a n t , P.J.V. 1971. O b s e r v a t i o n s by e l e c t i o n m i c r o scopy on p i g muscle i n o c u l a t e d and i n c u b a t e d w i t h Pseudomonas f r a g i . A p p l . M i c r o b i o l 22: 1152-1158. F i s c h e r , R.L. 1963. Changes i n t h e c h e m i c a l and p h y s i c a l p r o p e r t i e s of p r o t e i n d u r i n g a g i n g o f meat. P r o c . Meat Tenderness Symposium. Campbell So\ip Company, Camden, New Y o r k .  135 Fukazawa, T., B r i s k e y , E . J . , Takahashi, F. and Yastii, T. 1969. Treatment and post-mortem aging e f f e c t s on the Z l i n e of m y o f i b r i l s from chicken p e c t o r a l i s muscle. J . Food S c i . 34: 606-610. Gardner, G.A. and Stewart, D.J. 1966. Changes i n the f r e e amino and other n i t r o g e n compounds i n s t o r e d beef muscle. J . S c i . Food A g r i c . 17: 491-496. Gauthier, G.F. 1970. The u l t r a s t r u c t u r e of three f i b e r types i n mammalian s k e l e t a l muscle. In "The Physiology and Biochemistry of Muscle as a Food, 2", eds. B r i s k e y , E . J . , Cassens, E.G. and Marsh, B.B. Univ. of Wisconsin Press, Madison. Hamm, R. I960. R s . 10: e  Biochemistry of meat h y d r a t i o n . 355-463.  Advances Food  Hanson, H.L., Brushway, M.J., Pool, M.F. and Lineweaver, R. 1963. F a c t o r s causing c o l o r and t e x t u r e d i f f e r e n c e s i n r a d i a t i o n s t e r i l i z e d c h i c k e n . Food Technol. 17: 1188-1194. Hasegawa, T., Pearson, A.M., P r i c e , J.F., Rampton, J.H. and Lechowich, R.Y. 1970a. E f f e c t of m i c r o b i a l growth upon sarcoplasmic and u r e a - s o l u b l e p r o t e i n s from muscle. J . Food S c i . 35: 720-724. Hasegawa, T., Pearson, A.M., P r i c e , J.F. and Lechowish, R.V. 1970b. A c t i o n of B a c t e r i a l growth on the sarcoplasmic and u r e a - s o l u b l e p r o t e i n s from muscle. I . E f f e c t s of C l o s t r i d i u m p e r f r i n g e n s , Salmonella e n t e r i t i d e s . Achromobacter l i a u i f a c i e n s , Streptococcus f a e c a l i s and K u r t h i a z o p f i i . Appl. M i c r o b i o l 20: 117-122. Helander, E. 1957. On q u a n t i t a t i v e muscle p r o t e i n determination; Sarcoplasmic and m y o f i b r i l p r o t e i n content of normal and a t r o p h i c s k e l e t a l muscle. A c t a P h y s i o l . Scand. 41, Suppl. 141. Ingram, M. and Dainty, R.H. 1971. Changes caused by microbes i n s p o i l a g e of meats. J . A p p l . B a c t e r i o i . 34: 21-39. l o d i c e , A.A., Leong, V. and Weinstock, I.M. 1966. Separation of cathepsins A and D of s k e l e t a l muscle. Arch. Biochem. Biophys. 117: 477-486. Jay, J.M. 1964. Release of Aqueous extracts of beef homogenates and f a c t o r s a f f e c t i n g r e l e a s e volume. Food Technol. 18: 1633-1636. Jay, J.M. 1966. I n f l u e n c e of postmortem c o n d i t i o n s on muscle m i c r o b i o l o g y . In "The Physiology and Biochemistry of Muscle as a Food," eds. B r i s k e y , E . J . , Cassens, R.G. and Trautman, J.C. Univ. of Wisconsin Press, Madison. Jay, J.M. 1967. Nature, c h a r a c t e r i s t i c s and p r o t e o l y t i c prope r t i e s of beef s p o i l a g e b a c t e r i a a t low and h i g h tempera t u r e s . A p p l . M i c r o b i o l . 15: 943-944  136 Jay, J.M. 1 9 7 0 . "Modern Food M i c r o b i o l o g y " . Go., New York.  v  a n Nostrand  Reinhold  Jay, J.M. and Kontou, K.S. 1967. Fate o f f r e e amino acicts and n u c l e o t i d e s i n s p o i l i n g beef. A p p l . M i c r o b i o l . 1 5 J 759-764.  Khan, A.W. 1 9 6 2 . E x t r a c t i o n and f r a c t i o n a t i o n o f p r o t e i n s i n f r e s h chicken muscle. J . Food S c i . 2 7 * 4 3 0 - 4 3 4 . Khan, A.W. and vanden Berg, 1. 1 9 6 4 a . Changes i n chicken muscle p r o t e i n s d u r i n g a s e p t i c storage a t above f r e e z i n g temperatures. J . Food S c i . 2 9 4 9 - 5 2 . :  Khan, A.W. and van den Berg, L. 1 9 6 4 b . Some p r o t e i n changes d u r i n g postmortem t e n d e r i z a t i o n i n p o u l t r y meat. J . Food Sci.  29: 597-601.  Kono, T., Kakuraa, F., Jamma, M. and Fukuda, S. 1 9 6 4 . The e l e c t r o n microscope s t r u c t u r e and chemical composition of the i s o l a t e d saroolemma of the r a t s k e l e t a l muscle cell. Biochin. Biophys.. - A c t a 8 8 : 1 5 5 - 1 7 6 . Lawrie, R.A. 1 9 6 6 .  "Meat Science" Pergamon Press, N.Y.  Lawrie, R.A., Sharp, J.G., B e n d a l l , J.R. and Colbey, B. 1 9 6 1 . Treatments of meats with i o n i z i n g r a d i a t i o n s . V I I I , pH, water-^binding ca.pacity and p r o t e o l y s i s of i r r a d i a t e d raw beef and pork during storage and the ATP-ase a c t i v i t y of i r r a d i a t e d r a b b i t muscle. J . S c i . Food A g r i c l 1 2 : 742-751.  Lea, C.H.jStevens, J.H. and Smith, M.J. 1 9 6 9 . Chemical and o r g a n o l e p t i c changes i n p o u l t r y r e s u l t i n g from the growth of p s y c h r o p h y l i c s p o i l a g e amino a c i d s . Br. P o u l . S c i . 10:  203-217.  Lerke, P.,Farbeir, L . and Adams, R. 1 9 6 7 . B a c t e r i o l o g y o f s p o i l a g e of f i s h muscle. I V . Role of p r o t e i n . Appl. M i c r o b i o l . 1 5 : 770-776. Locker, R.H. i 9 6 0 . P r o t e o l y s i s i n the storage o f beef. S c i . Food A g r i c . 1 1 : 5 2 0 - 5 2 6 .  J.  Mcintosh, R.N. 1 9 6 7 . Post-mortem changes i n p r o t e i n e x t r a c t a b i l i t y i n beef, pork and chicken muscle. J . Food S c i . 32:  208-209.  M a r t i n s , C.B. and Whitaker, J.R. 1968. C a t h e p t i c enzymes and meat tenderness. I . P u r i f i c a t i o n of c a t h e p s i n D and i t s a c t i o n on actomyosin. J . Food S c i . 33:59^-64. Mendall, J.R. 1 9 7 1 . Unusual f e a t u r e s of the T system of the p e e t o r a l i s muscle o f the chicken. J . U l t r a s t r u c t u r e Res. 3 7 : 3 8 3 - 3 8 7 .  137 M i l l e r , J.H., Dawson, L.W. and Bauer, D.H. 1965. Free amino a c i d content o f chicken muscle from b r o i l e r s and hens. J . Food S c i . 30: 406-411. Mohasseb, Z.S. 1962. E l e c t r o p h o r e t i c p a t t e r n s of f r e s h i r r a d i a t e d - p a s t e u r i z e d and i r r a d i a t e d - s t e r i l i z e d beef s t o r e d a t 34 F. M. Sc. t h e s i s . Oregon State U n i v e r s i t y C o r v a l l i s , Oregon. Moore, S. and S^ein, W.H. 1954. A modified n i n h y d r i n reagent f o r the photometric determination of amino a c i d s and r e l a t e d compounds. J . B i o l . Ohem. 211: 909-913. Mycek, M.J. 1970. Cathepsins. 315.  Methods i n Enzymol. 19: 385-  Ockerman, H.W., O a h i l , V.R., Weiser, H.H., Davis, G.E. and S i e f k e r , J.R. 1969- Comparison of s t e r i l e and i n o c u l a t e d beef t i s s u e . J . Food S c i . 34: 93-99. P a r r i s h , J r . , F.C., G o l l , D.E., Newcomb I I W.J., de lumen, B.O., Chaudhry, H.M. and K l i n e , E.D. 1969. Molecular propert i e s of post-mortem muscle. 7. Changes', in n o n p r o t e i n n i t r o g e n and f r e e amino a c i d s o f bovine muscle. J . Food S c i . 34: 196-202. Peachey, C D . 1970. Form of the sarcoplasmic r e t i c u l u m and T system of s t r i a t e d muscle. I n "The Physiology and B i o chemistry of Muscle as a Food, 2". eds. B r i s k e y , E . J . , Cassens, R.G. and Marsh, B.B. Univ. of Wisconsin Press, Madison. Rampton, J.H., Pearson, A.M., P r i c e , J.F., Hasegawa, T . and lechowich, R.V. 1970. E f f e c t of M i c r o b i a l growth upon m y o f i b r i l l a r p r o t e i n s . J . Food S c i . 35: 510-513. Rey, C.R., K r a f t , A.A., Walker, H.W. and P a r r i s h , F.C. 1970. M i c r o b i o l changes i n meat during aging a t elevated temperature and l a t e r r e f r i g e r a t e d storage. Food Technol. 24: 67-71. Reynolds, E . S . - 1963. • The use of l e a d c i t r a t e a t h i g h pH as an electron-opaque s t a i n i n e l e c t r o n microscopy. J . C e l l . ' B i o l . 17: 208-212. n  Rhodes, D.1T. and Meegungwan, C. 1962. Treatment of meats with i o n i s i n g r a d i a t i o n . IX I n a c t i v a t i o n of l i v e r a u t o l y t i c enzymes. J . S c i . Food A g r i c . 1 3 : 279-282. S c h a l l e r , D.R. and Powrie, W.D. 1971. Scanning e l e c t r o n microscopy of s k e l e t a l muscle from rainbow t r o u t , turkey and beef. J . Food S c i . 36: 552-559. Schweigert, B.S. 1959. The e'ffects of r a d i a t i o n on p r o t e i n s . I n t e r n a t i o n a l J o u r n a l of A p p l i e d R a d i a t i o n and Isotopes. 6: 76-77.  138 Sharp, J.G. 1963. A s e p t i c a u t o l y g i s i n r a b b i t and bovine muscle d u r i n g s t o r a g e a t 37 . J . S c i . Food A g r i c . 14* 468-479. S l a u t t e r b a c k , D.B. 1966. The u l t r a s t r u c t u r e o f c a r d i a c and s k e l e t a l muscle. I n "The P h y s i o l o g y and B i o c h e m i s t r y o f M u s c l e as a Food," eds. B r i s k e y , E . J . , Cassens, R.G. and Trautman, J.C. U n i v . o f W i s c o n s i n P r e s s , Madison. Smirnova, T.A., Hushnarev, V.M. and T s h a i k o v s k a j a , S.M. 1971. E l e c t r o n M i s c o s c o p i c Study o f t h e s e c r e t i o n o f some b a c t e r i a l enzymes and t o x i n s . J . U l t r a s t r u c t u r e Res. 37: 269-278. S u z u k i , A., 1'Takazato, M. and F u j i m a k i , M. 1967. S t u d i e s on p r o t e o l y s i s i n s t o r e d muscle. P a r t I . Changes i n nonp r o t e i n n i t r o g e n o u s compounds o f r a b b i t muscle d u r i n g s t o r a g e . A g r . B i o l . Chem. 31: 953-957. Synge, R.L.M. 1955. P e p t i d e s (bound amino a c i d s ) and f r e e amino a c i d s . I n "Modern Methods o f P l a n t A n a l y s i s "Vol. I , " eds. Paech, K. and Tracey, M.V. S p r i n g e r P u b l i s h i n g Co., Berlin. T a l l a n , H.H., Moore, S. and S t e i n , W.H. 1954. F r e e amino a c i d s i n t i s s u e o f t h e c a t . J . B i o l . Chem. 211: 927-936"; T a r r a n t , P.J.V., Pearson, A.M., P r i c e , J.F. and Lechowich, R.V. 1971. A c t i o n of Pseudomonas f r a g i on the p r o t e i n s o f p i g muscle. A p p l . M i c r o b i o l . 22: 224-228. T h o r n l e y , M.J. I960. The d i f f e r e n t i a t i o n o f Pseudomonas from o t h e r gram-negatie b a c t e r i a on the b a s i s o f a r g i n i n e metabolism. J . A p p l . B a c t e r i o l . 23: 37-52. V e i s , A. 1970. C o l l a g e n . I n "The P h y s i o l o g y and B i o c h e m i s t r y of Muscle a s a Food, 2". eds. B r i s k e y , E . J . , Cassens, E.G. and Marsh, B.B. U n i v . of W i s c o n s i n P r e s s , Madison. Wiebe, W.J. and Chapman, G.B. 1968a. F i n e s t r u c t u r e o f s e l e c t e d marine Pseudomonas and Achromobacters. J . B a c t e r i o l 95: 1862-1873. Wiebe, W.J. and Chapman, G.B. 1968b. V a r i a t i o n i n f i n e s t r u c t u r e i n a marine Achromobacter and a marine Pseudomonad grown under s e l e c t e d n u r i t i o n a l and temperature regimes. J . B a c t e r i o l . 95: 1874-1886. W h i t i n g , R.C. 1970. T e x t u r a l and c o l o r responses o f c h i c k e n muscle o f s u b s t e r i l i z i n g doses o f gamma i r r a d i a t i o n . M. Sc. T h e s i s . U n i v e r s i t y o f B r i t i s h Columbia. Zender, R., L a t a s t e - D o r o l l e , C , C o l l e t , R.A., R o w i n s k i , P. and Mouton, R.P. 1958. A s e p t i c a u t o l y s i s o f muscle; b i o c h e m i c a l and m i c r o s c o p i c m o d i f i c a t i o n s o c c u r r i n g i n r a b b i t and lamb muscle d u r i n g a s e p t i c and a n e r o b i c s t o r a g e . Food Res. 23: 305-326.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092968/manifest

Comment

Related Items