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Some biochemical and ultrastructural changes in intact and sarcoplasmic reduced, bovine Longissimus dorsi… Yada, Rickey Yoshio 1980

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Some Biochemical Intact  and Ultrastructural  and Sarcoplasmic  Longissimus Inoculated  dorsi with  Changes in  Reduced, Muscle  Bovine  Strips  Pseudomonas  fragi  by  RICKEY YOSHIO B.Sc.  Ag.,  University  of  YADA  British  Columbia,  A T H E S I S SUBMITTED IN PARTIAL FULFILLMENT T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E  STUDIES  D E P A R T M E N T OF FOOD S C I E N C E  We a c c e p t to  this  the  thesis  required  THE U N I V E R S I T Y  Rickey  \  conforming  OF B R I T I S H  August ©  as  standard  Moshio  COLUMBIA  1980 Yada,  1980  1977  OF  In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of this  thesis  for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for f i n a n c i a l gain shall not be allowed without my written permission.  Department n f  F  o  o  d  Science  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  August  2 8 , 1980  / i i  ABSTRACT  Intact mild  washing procedure  sarcoplasmic with  at  fluid.  4°C  for  soluble  order  Intact  12 d a y s .  Aseptic in  and u r e a - i n s o l u b l e  on t h e  m i c r o s c o p y were  Analysis water-soluble  Increases  in  sample.  Alterations  the  were  Total  fvagi.  numbers  majority  in  the  pH o f  Significantly  the  the  in  total  both  of  P.  changes fvagi,.  (non-protein  indicated these  that  nitrogen,  the  washing  components. and  inoculated  salt-soluble muscle  pattern  of  and u r e a - i n s o l u b l e a result  of  muscle occurred  (P < 0 . 0 1 )  higher  intact  transmission  growth of  electrophoretic  intact  urea-  ultrastructural  d e c r e a s e d as  the  similar  as  monitored  intact  urea-soluble  storage  salt-soluble,  the^ w a t e r - s o l u b l e  SDS-gel  carbohydrate  An i n c r e a s e increased.  of  under  S c a n n i n g and monitor  the  inoculated  during  as w e l l  components  observed in  salt-soluble,  P.  samples.  removed the  in  stored  were  a consequence of  water-soluble, evident.  numbers,  a  a sarcoplasmic  spoilage  fractions,  and carbohydrates)  were  of  to  of  samples were  effect  were  subjected  concentration  water-soluble,  protein  the extractability  fractions  the  subsequent  water-soluble  proteins  effectively  the  employed to  as  of  procedure  reduce  controls  pH a n d b a c t e r i a l  muscle surface  protein  and  the  washed i n o c u l a t e d m u s c l e  electron  to  m u s c l e was  and washed m u s c l e  growth  Alterations  carbohydrate,  dovsi  to.evaluate  on b a c t e r i a l  conditions.  and  in  Pseudomonas fvagi  reduction  Longissimus  bovine  growth  the  proteins  growth as  of  bacterial  / i i i  rates muscle  were  observed on the  water-soluble  and  l i t t l e  seen.  the  salt-soluble  salt-soluble  Minor  changes  inoculated  degradation  of  apparent  only  appeared  to  surface  muscle  fibers.  intact  adhesion.  of  not  than  the  washed  content  A slight SDS-gel  was o b s e r v e d i n total  Little  to  the  change  growth  indicated  of  in P.  that  localized colonization. cell  to  cell  B a c t e r i a were  the  mediation  evaginations  of  aseptic muscle  was m i n i m a l  controls.  in  both  but  intact  glycocalyx  intact  were  also  cell  between  inoculated  in  on t h e  and  the  fragi.  samples  bacterial  surface  bacteria. Autolysis  pH o f  surface  observed growing  were p r e s e n t  of  Glycocalyx  attachment,  of  the  carbohydrate  electrophoretograms  apparent. due  nitrogen,  decrease in  electron micrographs  confirmed  Cellular  non-protein  and washed i n o c u l a t e d muscle  only  adhesion.  Transmission tissue  the  electron micrographs  areas  mediate  muscle  the  were  in  protein  sample occurred  both in  in  proteins  Scanning  muscle  change  inoculated muscle tissue.  was  washed  muscle tissue  tissue. Relatively  washed  intact  washed  of  the  to  /iv  TABLE:OF- CONTENTS Page ABSTRACT  • - • •  TABLE OF CONTENTS. ...........  .  i i iv  LIST OF TABLES:  vi  LIST OF FIGURES  vii  ACKNOWLEDGEMENTS  xi  INTRODUCTION  1  LITERATURE REVIEW  4  A. Meat Spoilage B. Aseptic Techniques C. A u t o l y i i c Changes in Postmortem Muscle D. Biochemical Changes During Bacterial Spoilage Muscle E. Bacterial Adhesion F. Inoculation of Intact Muscle Samples  4 5 7 of 8 17 21  METHODS AND MATERIALS A. B. C. D. E. F.  G. H. I. J.  Sarcoplasmic Extraction Preparation of the Inoculum Preparation of the Muscle Samples pH Determination Bacterial Counts Electron Microscopy  23 27 29 31 31 32  1. 2.  32 33  Scanning EM preparation Transmission EM preparation  Extraction of Water- and Salt-Soluble Nitrogen Determination Total Carbohydrate Analysis SDS-Gel Electrophoresis  RESULTS AND DISCUSSION A. B. C.  23  Protein  Fractions.  •  Effect of Gamma Radiation on Colour and Odour Influence of Sarcoplasmic Reduction on Bacterial Growth • • The Effect of Growth of P. fragi on pE, Surface Appearance and Odour  34 36 37 38 43 43 43 48  /v  Page D. E.  Influence of Growth of P. fragi on Total Carbohydrate Content The Effect of P. fragi on the Extractability of NonProtein Nitrogen (NPN) Water-Soluble and SaltSoluble Proteins SDS-Polyacrylamide Gel Electrophoresis  53  3  F.  1. 2. S. 4. G.  Mater-soluble Salt-soluble Urea-soluble Urea-insoluble  Electron 1. 2.  proteins proteins proteins proteins  Microscopy  Scanning electron microscopy Transmission electron microscopy  GENERAL DISCUSSION  59 70 71 75 80 83 86 87 101 I l l  CONCLUSIONS  116  REFERENCES CITED  118  /vi  LIST:OF-TABLES Table 1  ..  Page Quantity  of  markers  protein  used in  preparation  fraction  and m o l e c u l a r  sample p r e p a r a t i o n  applied  to  gel  during  weight  and amount  of  SDS-gel  electrophoresis 2  Molecular weight  3  Influence  of  5  with  at  SDS-gel  4°C  used in  the  of  the  intact  slope  of  the  washed i n o c u l a t e d  The  effect  The  effect  muscle The of  of of  inoculated  P. fragi  of  electrophoresis..  on m u s c l e  slope  content  7  for  reduced  P. fragi intact  and  on the  on t h e  45 comparison of  the  regression with  the  regression  pH o f  (washed)  the  49  intact  and  bovine muscle samples.  total  of  intact  sarcoplasmic reduced  (washed) 54  P. fragi  on t h e  non-protein  and s a r c o p l a s m i c reduced  nitrogen  (washed)  muscle  samples 8 ;  The  60  effect  content muscle 9  The  50  carbohydrate  samples  effect  42  samples  Pseudomonas fragi  Regression statistics  sarcoplasmic 6  markers  incubation  inoculated 4  40  of of  P. fragi intact  on t h e  water-soluble  protein  and s a r c o p l a s m i c reduced  (washed)  samples  effect  content (washed)  of of  63  P. fragi intact  and  on t h e  salt-soluble  sarcoplasmic  muscle samples  protein  r.  reduced 67  /vii  • LIST OF FIGURES Figure  Page  1  Hobart  delicatessen  2  Apparatus intact  3  used for  slicer the  ,  extraction  of  24  sarcoplasm  from  muscle  Bacterial  vents  25 used  to  sterilize  both  air  and  nitrogen  26  4  "Whirl-pak"  5  Plexiglass  incubation  chamber  6  Flow  of  extraction  7  Bacterial  sheet  8  protein  at  pH o f  10  Total  11  of  log  Non-protein  Pseudomonas  35 muscle  fragi,  stored  inoculated at  at  content  ATP t o  data  for  with  4°C  inoculated, stored  population  47  intact  and  washed  4°C of  51 control  samples  ribose  and  stored  inoculated, at  and hypoxanthine  4°C  55  in  muscle nitrogen  inoculated, 4°C  and washed  bacterial  and washed muscle  Conversion of  at  and  samples  postmortem 12  with  procedure  46  carbohydrate  intact  30  intact  and washed muscles  control  muscle  28  4°C  Pseudomonas fragi 9  of  inoculated  Linearization intact  sample  population  samples, stored  muscle  intact  58 content  of  control  and washed muscle  and samples  stored 61  / v i i i  Figure 13  Page Water-soluble inoculated, stored  14  at  at  at  muscle  from  from  from  from  from  water-soluble and  6 and of  intact  samples  and samples  day of  day of  0,  day of  day of  6 and  6 and  6 and  control 6 and  day  and  6 and  73 proteins intact  inoculated  12  76 proteins  and washed  inoculated  12  and  77 proteins intact  inoculated  12  urea-soluble  0,  inoculated  12  urea-soluble  washed c o n t r o l  samples at  proteins  and washed  salt-soluble  0,  muscle 72  control  0,  extracted  12  salt-soluble  0,  proteins  inoculated  water-soluble  intact  Electrophoretograms  muscle  of  washed c o n t r o l  samples at  extracted  control  and washed m u s c l e  intact  Electrophoretograms  muscle  of  washed c o n t r o l  samples at  extracted  20  0,  Electrophoretograms  muscle 19  day  samples at  extracted  content  control  Electrophoretograms  muscle  and  68  samples at  extracted  18  and washed m u s c l e  intact  Electrophoretograms extracted  control  4°C  intact  samples  of  64  Electrophoretograms from  17  intact  Salt-soluble protein  stored  16  content  4°C  inoculated,  15  protein  81 proteins  and washed 12  inoculated 82  /ix  Figure 21  Page Electrophoretograms extracted muscle  22  samples  muscle  (b)  (b)  (b)  (b)  which may  (b)  of  by  slime  day  0, 0,  day  of  inoculated  12  84 proteins  and washed  6 and  inoculated  12  85  uninoculated  intact  (a)  and  12,  88 inoculated  intact  (a)  and  samples day  89  0 inoculated  intact  (a)  and  samples day  90  3 inoculated  samples  showing  intact  (a)  crevices  and (arrow)  bacteria of  muscle of  muscle of  muscle  (S)  6 and  intact  samples  of  trap  SEM m i c r o g r a p h s  and  proteins  urea-insoluble  day  muscle  SEM m i c r o g r a p h s washed  30  (b)  at  muscle  SEM m i c r o g r a p h s washed  29  (b)  of  muscle  SEM m i c r o g r a p h s washed  28  0,  day  muscle  SEM m i c r o g r a p h s washed  27  at  of  SEM m i c r o g r a p h s washed  26  samples  SEM m i c r o g r a p h s washed  25  control  washed c o n t r o l  SEM m i c r o g r a p h s washed  24  from  urea-insoluble  intact  Electrophoretograms extracted  23.  from  of  day  6  92 inoculated  intact  (a)  and  samples day  94  9 inoculated  intact  (a)  and  samples day  12  95  inoculated  intact  (a)  and  samples  showing  the  97 adherence  between muscle  fibrils  of  bacteria  (A). ,  100  /x  Figure. 31  Page TEM m i c r o g r a p h of  the  intact  of  the  adherent  muscle  surface  bacterial after  12  population days  incubation 32  TEM m i c r o g r a p h (P) (X  33  and  for  dorsi days  at  4°C  TEM m i c r o g r a p h  of  intact  for  muscle for  with  primary  polysaccharides  Longissimus  bovine  P. fragi  and  dorsi  incubated  at  of  106 uninoculated  (cross-section)  Longissimus  bovine incubated  for  12  (X 3 3 , 2 0 0 ) of  intact  inoculated;.with  107 bovine  P. fragi  Longissimus and  dorsi  incubated  at  12 d a y s  TEM m i c r o g r a p h  4°C  acidic  showing  12 d a y s  muscle  muscle  36  (S)  bacteria  104  inoculated  TEM m i c r o g r a p h  4°C  adherent  secondary  TEM m i c r o g r a p h  4°C  35  of  51,000)  muscle  34  103  of  109 intact  inoculated 12 d a y s  bovine  w i t h P.  fragi  Longissimus  dorsi  and i n c u b a t e d  at HO  /xi  ACKNOWLEDGEMENTS  I for  wish  his time,  throughout  to  e x p r e s s ray s i n c e r e  patience,  the  o f my g r a d u a t e  and D r .  J . F.  Thanks  technical  in  preparing The of  Dr.  their  to Mr.  I  Dr.  also wish  S. Nakai,  valuable  to  Dr.  his  Skura  criticism thank W. D .  suggestions  S. J . Yee f o r  B. J .  the Powrie,  and a s s i s t a n c e .  invaluable  advice.  patience  Council  study.  committee,  extended  Finally, time,  this  Richards for  are also  to  a s s i s t a n c e and c o n s t r u c t i v e  course of  members  gratitude  and encouragement  the  to  during  S y l v i a Duffek this  study  for  her  and f o r  endless  her  work  manuscript.  support  Canada  acknowledged.  a s p e c i a l thanks  of  through  the Natural  Sciences  post-graduate  and E n g i n e e r i n g Research  scholarships  is  also  /I  INTRODUCTION  The p r i n c i p a l approximately nitrogenous 1975).  component  75% w a t e r ,  substances,  and  1979).  is  the  1.0%  primarily readily  upon  their  extractable  proteins,  strength  extraction.  insoluble  and a r e The  components, myofibrillar involved  of  or  low  however, The  which  proteins  are  contraction.  tissues,  including which  element  (Bodwell  1975;  the  Lawrie,  animal  isolation  are  largest  the  3 major  groups,  walls,  functions  of  based  are The  high  ionic  comparatively  dissociating  agents.  and M c C l a i n ,  major 1971;  The proteins  fraction  blood vessels, the  of  cycle.  and r e g u l a t o r y ,  protein  as  of  (Lawrie,  buffers.  are  al.,  component  s o l i d matter  glycolytic  stromal  et  is  a  nerves,  supportive Forrest  et  al.,  1979).  Microbial myosystems  body  (Forrest  a complex mixture  structural  cell  endomysium and p e r i m y s i u m , of  strong  contains  non-protein  solutions  represent  The  1.5%  strength  proteins  with  the  the  salt  enzymes o f  represent  Muscle  sarcoplasmic proteins  require  stromal  the  into  ionic  sarcoplasmic proteins  in muscle  composite  The  solubilized only  many o f  of  characterized  water  myofibrillar for  constitutes  solubility. in  muscle.  constituents  constituent  are  is  3% l i p i d s ,  inorganic  protein  principal  Muscle proteins  meat  19% p r o t e i n ,  Excluding water,  muscle  of  contamination  problems  that  and the  subsequent  have been w e l l  and c h a r a c t e r i z a t i o n  of  the  spoilage of  documented.  spoilage  flora  food  Although  and the  effect  of  /2  environmental interest  has  spoilage.  shifted  have  and Kontou,  believe  that  spoilage,  while  1967;  Tarrant  significant  nature.  however, 1974; the of  attachment an  minced muscle technique  the  biochemical  to  the  importance  1969;  plays  ( H a s e g a w a et 1971;  g a i n e d much  at., Sage,  attention,  changes of  during  bacterial  Some r e s e a r c h e r s  at.,  (Jay,  1967;  J a y and S h e l e f ,  1976)  an i n s i g n i f i c a n t  role  1970a and b;  Borton  1974;  et  Dainty  during  et  at.,  1975)  at.,  have  degradation.  bacteria about  Recent  to  surfaces  the  is  investigations  of microorganisms  a widespread  mechanisms of  and Kampelmacher,  1975; to  bacterial  (Notermans  M c C o w a n et  myosystems  polysaccharide,  resemble  research prior as the  increases  result  muscle  of  at.,  has  the  adhesion,  and  Kampelmacher,  1979)  at.,  have  phenomenon  into  shown t h e  glycocalyx,  which  presence  appears  to  attachment. Most  that  study  proteolysis  et  of  extra-cellular  mediate  the  protein  limited.  Notermans  organisms  pertinent  Information  is  these  O c k e r m a n et  others  Adhesion in  on  been c o n t r a d i c t o r y .  bacterial  1970a and b; shown  to  Observations  proteolysis Jay  conditions  substrate the  which  occur  this  for  surface  from b a c t e r i a l  those  to  thesis  the  area,  involved  inoculum. thereby  the  Although  maximizing  spoilage,  the  on  (non-mechanically  intact  changes  use  of  this  any  changes  o b s e r v e d may  not  disrupted)  tissue. The o b j e c t  biochemical  changes  of that  the  present  resulted  r e s e a r c h was  from the  to  growth of  observe  some  Pseudomonas  fragi  /3  ATCC  4973 o n i n t a c t  muscle  strips.  microscopy in  attempts  were to  In  and  sarcoplasmic  addition,  employed explain  to  the  reduced  scanning study  Longissimus  and t r a n s m i s s i o n  surface  biochemical  bovine  ultrastructure  changes.  dovsi  electron of  the  muscle  /4  LITERATURE REVIEW A.  Meat  Spoilage The  of  f r e s h meat  Weidemann,  major the  number low  of  different  et  meat  frequency 1969;  Jay,  accepted  that  spoilage,  1978).  spoilage  of  a few  appears  1978;  meat  the  found  Microbacterium  meat  the  (Jay,  most  temperature  1960a;  Jay,  1967;  Ingram  1976;  G i l l  and Newton,  to  Mackey  contains  and  a  predominate.:  psychrotrophic  are  The  large during  bacteria  as major  only  1967;  Vanderzant  1977) .  However,  aerobic,  of  and D a i n t y , 1977;  G i l l  and beef 1971;  Movaxella,  with  regular  and N i c k e l s o n , it  is  gram-negative  predominant spoilage  isolated  of  and c e r t a i n  thermosphactum  have been  a  components  Pseudomonas, Achvomobacter,  of  and Newton,  bacteria  Shelef,  these  Strains  constitutes  Ayres,  of  psychrotrophic,  low  the  Enterobacteriaceae  G i l l  and Newton,  and  Ingram  Many s p e c i e s have been d e s c r i b e d but  Pseudomonas in  namely  (Brown  1970;  at.,  and Newton,  only  spoiling  the  (Gill  spoilage  literature  for  genera,  proportion  1972;  G i l l  bacterial  family  from  1976;  aerobic  R e y et  responsible  Lactobacillus,  the  1967;  flora  floras.  Acinetobacter_, of  Shelef,  of  the  initial  1952).  small  spoilage  genera  and  Jay,  in  the  meat  al.,  relatively  1960a;  Although  temperature  (Kirsch  documented  dressed animals  1979).  of  Jay  characterization  of microorganisms  hides  Derrick,  Ayres,  1971;  source  and  has been w e l l  1958;  and D a i n t y ,  be  isolation  now  generally  genus  important  group  (Wolin  al.,  Jay,  and Newton,  et  1972; 1978).  Jay  of 1957; and  /5  The overall r e s u l t of low temperature b a c t e r i a l spoilage of fresh meat i s the production of " o f f " odours and flavours, accompanied by a slimy appearance.  Incipient spoilage i s measurable  when t o t a l numbers/gram reach approximately 10 Shelef, 1976).  organisms/g  (Jay and  Daub et al. (1979), working with chicken skin, reported  that the f i r s t i n d i c a t i o n of an off-odour and sliminess occurred when the count approached 10  8  organisms/g.  Ayres (1960b) found that 10  7  2 organisms/cm and 10  were required f o r off-odour development of fresh beef,  f o r slime development.  In a similar study, Dainty et al. (1975)  found that spoilage odours were not detected u n t i l microbial numbers 8 reached 4 x 10 /cm u n t i l 2 x 10  2 , while the appearance of slime was not detected  /cm2.  The u t i l i z a t i o n of the soluble non-protein nitrogen components (especially the free amino acids) by the spoilage organisms results i n the production of ammonia, hydrogen s u l f i d e , indole, skatole, and amines, a l l of which contribute to off-odour (Ingram and Dainty, 1971; Jay, 1972).  The formation of slime i s the consequence  of b a c t e r i a l coalescence as well as the loss of muscle i n t e g r i t y due to microbial growth (Ayres et al., 1950; Jay, 1970). B.  Aseptic  Techniques Changes i n myosystems during refrigerated storage may  be caused by microorganisms  as well as a u t o l y t i c reactions.  In order  to obtain an understanding of these changes and t h e i r r e l a t i v e importance  i n meat spoilage, i t i s necessary to d i f f e r e n t i a t e between  /6  changes induced by bacteria and those due to muscle autolysis (Buckley et al., 1976).  The success of such a study depends upon a  method to obtain s t e r i l e tissue.  The l i m i t a t i o n of achieving t h i s  undoubtably stems from the p r a c t i c a l d i f f i c u l t y of removing  samples  of internal tissue without introducing contaminants from the abundant microflora of the surface ( G i l l and Newton, 1978; Mackey and Derrick, 1979). tissue.  Various methods have been employed i n obtaining s t e r i l e muscle Zender et al. (1958) used a surgical room technique to  a s e p t i c a l l y remove rabbit and lamb Longissimus  dorsi  muscle.  Sharp  (1963), working with rabbit and beef muscle, used an alcoholic-dye dip method and reported that 50% of the tissue samples taken were sterile.  A c h l o r - t e t r a c y c l i n e procedure was used by Khan and van den  Berg (1964) to obtain s t e r i l e chicken muscle, however, the method could only be used f o r a u t o l y t i c studies.  Ockerman et al. (1964) used  a gnotobiotic method to study both aseptic muscle and organ t i s s u e . Ockerman et al. (1969) described a surgical i s o l a t o r technique f o r removing s t e r i l e muscle tissue.  Hasegawa et al. (1970a and b),  Rampton et al. (1970) and Borton et al. (1970a) used an absolute alcohol rinse i n attempts to obtain s t e r i l e samples, and discovered that although most control samples were s t e r i l e , some were contaminated. Hone et al. (1975), using a core technique to c o l l e c t  aseptic beef  tissue, found that of the 23 samples collected, 22% were s t e r i l e (< 1 organism/g) but 74% were contaminated with < 5 organisms/g. Ockerman and C a h i l l  (1977), employing the same technique, found that  although the control samples were contaminated, b a c t e r i a l counts  /7  remained Buckley with  a  relatively et  to  laminar  air  dorsi  meat  of  used an a s e p t i c  cutting  technique  flow  when  Sage  that  bacteria  inoculated  (1974)  on a u t o l y t i c susceptible with porcine acids  after  (1958), only  autolytic the  muscle  J a y and Kontou  without  prevented  a mixed  with  to  bacterial Doty  activity Lawrie  et  muscle,  found  that  activity  approximately  60%.  Autotytic  beef muscle  during  in  found  a general  (1961),  postmortem  samples.  results  all of  the  were  reported  cathepsins  et  are  (1957),  at.  in  radiation not  as  working  free  amino  Z e n d e r et  showed t h a t  irradiation  (1955)  in  gamma  proteolysis.  but  by  of  had  l i t t l e  reported  a 1.5  effect  that  experiments  with  at.  on  50%  Megarad dose  a 5 Megarad dose reduced  non-protein  tissue  spoilage  increases  lamb m u s c l e ,  Changes in Postmortem  Increases  Drake  inactivated  at.  the  destroying  effect  shown t h a t  and Wachter  was  of  normal  the  contamination,  and p o r c i n e  C.  have  beef,  and  from  muscle.  irradiation.  rabbit  concert  sterile  Similar  studying  indicating  in  obtain  the  flora.  chicken  (catheptic)  proteolysis.  by  affecting  tissue  storage.  exposed beef  capable  d o s a g e was  postmortem  (1967)  this  and ground  with  aseptic  to  irradiation  gamma r a d i a t i o n .  obtain  gamma r a d i a t i o n  as b a c t e r i a  catheptic  to  investigators  enzymes  working  over  unit  with  working  Numerous  of  21 d a y s  muscle.  found  psychrophilic  not  the  a 1 Megarad dose of  The a u t h o r s  by  (1976)  at.  Longissimus  constant  of  bovine  proteolytic  Musote nitrogen  storage  components  in  have been observed  aseptic (Zender  et  at.,  /8  1958;  Locker,  Gilbert,  1960;  1966;  Parrish  et  T h o m p s o n et  Gardner and S t e w a r t , 1969;  al.,  have been a t t r i b u t e d (cathepsins) in  muscle  found been  known  tissue.  that  the  while  postmortem et  of  Biochemical  recognized, not  free  acids, in  the  in  the  relatively (1967),  increases  non-protein  It  D.  It  has  B could  by  cleaved  also  be  the  believed  resulted  and p e p t i d e s not  beef,  had been  nitrogen  were  Bodwell  with  cathepsin  degradation.  enzymes  concentrations  cathepsin  that  proteins  1963;  low  working  sarcoplasmic proteins  (Sharp,  The  and 1969;  endogenous p r o t e o l y t i c  (1977)  postmortem  1 0 Ig.  that  role  of  mechanism o f  no  clear  Bacterial bacteria  from catheptic  during  and P e a r s o n ,  1964;  Spoilage  Muscle  acids  ( H a s e g a w a et  glutamine  and  (1966),  corresponding  studying in  storage acid,  decrease  It  bacterial  compounds  and g l u t a m i c  spoilage  1970a).  until  decreased during  tryptophan  acid  nitrogen  meat  of  has  and muscle p r o t e i n  al.,  changes occur  and o t h e r  in  degradation  Gardner and Stewart  notably  glutamic  the  elucidated  that  amino  found  the  been  accepted exceed  for  Davey  Davey and G i l b e r t ,  enzyme was  al.  Changes During  Although  has  in  proteolytic et  of  Bailey  the m y o f i b r i l l a r  storage  1963;  Parrish  1969).  al.,  D.  action  be present  the"increase  degradation  enzymes,  the  Okitani  enzyme r e s p o n s i b l e much o f  to  to  the major  1966;  Sharp,  P e n n y and. F e r g u s o n - P r y c e , 1 9 7 9 ) .  P a r r i s h and  suggested by  that  1961;  al.,  is  the  most  increased. in  been  involvement  generally  populations  stored  while  long  glutamine  changes beef  in  the  muscle,  other  amino  The  increase  was  attributed  /9  to the b a c t e r i a l production of glutaminase, while the increase i n tryptophan was attributed to a u t o l y t i c processes.  Increases i n  ammonia production were not s i g n i f i c a n t u n t i l incipient spoilage 8 (10  9 to 10  organisms/g) had been reached.  In a s i m i l a r study, Jay  and Kontou (1967) reported that fresh beef allowed to undergo microbial spoilage at 7°C showed decreases i n amino acids and nucleotides when b a c t e r i a l numbers were high; conversely, when b a c t e r i a l numbers were low, decreases i n both moieties were not detected.  The authors  concluded that these low molecular weight compounds, rather than the "primary" proteins, were the precursors f o r the compounds associated with low temperature meat spoilage. Jay (1967) studied the c h a r a c t e r i s t i c s of low temperature beef spoilage and found that i n the presence of many low molecular weight compounds, the attack of the "primary" proteins by the organisms involved (predominantly Pseudomonas most.  spp.) was minimal at  The author postulated that the breakdown of the proteins was  the r e s u l t of cathepsins which were released due to b a c t e r i a l action. Lerke et al. (1967) investigated the r o l e of protein during microbial spoilage of f i s h muscle.  Muscle press juice from English  sole was fractionated by g e l f i l t r a t i o n into a protein and a protein-free fraction.  Upon inoculation with spoilage bacteria {Pseudomonas spp.  Group I I I ) , the protein-free f r a c t i o n spoiled according to organoleptic and chemical  c r i t e r i a ( v o l a t i l e reducing substances, t o t a l v o l a t i l e  nitrogen and trimethylamine  nitrogen).  Proteolysis due to b a c t e r i a l  action, as measured by Kjeldahl nitrogen, was evident i n both the  /io unfractionated muscle j u i c e as well as the protein f r a c t i o n . no s i g n i f i c a n t proteolysis occurred u n t i l spoilage was evident.  However,  readily  C a s t e l l and Greenough (1959), studying the relationship  between substrate composition and development of odours i n f i s h muscle, commented on the i n a b i l i t y of Pseudomonas fragiyet t h i s organism was  to hydrolyze proteins;  able to produce strong off-odours  from f i s h  muscle extracts. In another study, Ockerman et al.  (1969) investigated the  alterations i n the various protein fractions of s t e r i l e inoculated beef (inoculated with either a Pseudomonas  and  spp.,  Aehromobacter  or a general inoculum composed of the natural f l o r a of beef) during refrigerated storage (3 ± 2°C).  Kjeldahl analysis of the sarcoplasmic  and m y o f i b r i l l a r fractions revealed no s i g n i f i c a n t difference between aseptic and inoculated samples. detected  S i g n i f i c a n t differences were  i n the stromal f r a c t i o n after 17.5  d e f i n i t e decrease was  days incubation, where a  exhibited by the inoculated samples with a  subsequent increase;in non-protein nitrogen. Rampton et al. bacteria - Aehromobacter Pediococcus faecalis  cerevisiae,  (1970) studied the effects of selected liquefaciens,  Micrococcus  Pseudomonas fluorescens,  and a mixed f l o r a (obtained  luteus, Streptococcus  from commercial hamburger) - on  rabbit and porcine m y o f i b r i l l a r proteins.  Using sucrose density  gradient centrifugation, gel f i l t r a t i o n and disc gel electrophoresis, the authors found that none of the inocula had any measurable effect upon the m y o f i b r i l l a r proteins,  It was  noted, however, that  "although  /II  the present  study  it did not rule of bacteria The  shewed no proteolysis  out the possibility  could  compounds  B o r t o n et  species  protein  fractions  inoculation fraction  solubility  increased  of  the  of  during great  various  the the  insoluble  inoculated  protein  samples which  20 d a y  first  and  strains  during  8 days In  storage  period,  Leuconostoc  2 and were  10°C.  the  various  The  affected  by  the  storage  of  the  controls  samples.  followed  Samples  by  an  then  other for  Although  treatments,  P. fragi  treated  fraction)  decreased  the  and  increase.  (myofibrillar  increased except  all  of"the  water-soluble  comparison to  for  bacterial  the  showed a d e c r e a s e . slightly  four  solubility  storage,  generally  of  in  proteins of  simple  loss  treated loss,  proteins".  proteins.  luteus,  at  fraction,  utilize  effects  fractions  revealed a  initial  constant.  on t h e stored  protein  salt-soluble first  the  Micrococcus  Studies  increased only  the  bacteria  studied  porcine muscle  e v i d e n c e d an  remained r e l a t i v e l y  nitrogen  (1970a)  L. mesenteroides  and  during  species  such as t h e r s a r c o p l a s m i c  (sarcoplasmic proteins)  P. fragi  samples,  the  cerevisiae,  treatment.  M. luteus  with The  of  that  Pseudomonas f r a g i )  and  solubilities  the  al.  {Pediococcus  mesenteroides  that other  cause 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  presearchers postulated  proteinaceous  of the m y o f i b r i l l a r  or  inoculated  P.  fragi  non-protein including  treated  samples  controls, showed  increases. Borton  urea-gel  et  al.  (1970b),  and d i s c u r e a - g e l  in  a companion paper,  electrophoresis  to  study  the  used  starch  effects  of  /12  Pseudomonas fragi, and  Micrococcus  and  10°C.  with  Pediocooous  luteus  Of  the  this  myofibrillar  muscle  showed a  proteins.  Pediocooous  upon  and r a b b i t  sarcoplasmic  proteins  utilized in  both  10°C  rabbit  the  that  muscle  but  effect  on p o r c i n e muscle but  rabbit  muscle  sarcoplasm.  rabbit  muscle  sarcoplasmic proteins  muscle.  Both  proteolysis l i t t l e  of  P. fragi the  was d e t e c t e d  mesenteroides proteins.  effect  and  in  the  to  gel  microorganisms  effects  muscle.  concluded that  meat  of  rabbit had  activity  upon  of  porcine  considerable  M.  luteus  upon the  nor  "a  producing  act upon only certain  however, L.  urea-soluble  s p o i l a g e was  a number of organisms  no  upon  breakdown  porcine muscle,  Neither effect  after  mesenteroides  minor  action  of  proteolysis  proteolytic  caused in  proteins  preferentially  L.  caused only  of  and  P. cerevisiae  and had no  the  patterns  inoculated muscles  P. cerevisiae  muscle".  bands,  upon  the  sarcoplasmic proteins  luteus  preferentially  effect  starch  2  inoculated  protein  caused extensive  any measurable  involving  enzymes indigenous  and  exerted major  M.  at  mesenteroides  upon p o r c i n e muscle.  rabbit  The a u t h o r s  enzymes, which  Leuoonostoc  urea-soluble proteins  exerted  complex process  to  of  studied  sarcoplasmic proteins.  alteration  stored  samples  number  al'. ( 1 9 7 0 a )  different  extensive  less  only  Comparison, of  caused  had  proteins  some p r o t e o l y t i c  P. fragi  proteins.  the  mesenteroides  s a r c o p l a s m i c and u r e a - s o l u b l e  from a s e p t i c  and p o r c i n e  in  luteus,  muscle.  indicated  specific  loss  exhibited  Micrococcus  cerevisiae  at  inoculated,  H a s e g a w a et  from p o r c i n e  storage  samples  organism  Pseudomonas fragi,  Leuoonostoc  on p o r c i n e m y o f i b r i l l a r  Pseudomonas fragi  indicating  cerevisiae,  proteins  highly  highly or  specific  A3  In starch-gel  a  Salmonella  Streptococcus  proteins  C. perfringens  caused  of  only  and  Kurthia  from  rabbit  minor  muscle,  any measurable  fraction  of  pig muscle.  of  the  complete  urea-soluble  on t h e a  of  None o f upon  the  the  et al.  incubation  growth  of  myofibrillar  nitrogen occur  porcine  other  before  fraction  (1971)  studied  -was  proteins  peptides  spoilage  since  detected  ammonia).  Complete breakdown  of  the  until  investigators  indicated  faecalis K.  nor  zopfii  sarcoplasmic was  evidenced  the  test  in  the  organisms  Examination  l i t t l e  involved  of  was  caused detected  caused  any  Pseudomonas  of  porcine  patterns  extensive  Major  fraction that,  of  during  proteolysis in  non-protein  proteolysis in  8 days  muscle  fragi  indicated  increase  changes  after  myofibrillar  S.  C. perfringens  action  proteins  quantitative  be  These  the  a corresponding and  sarcoplasmic  band.  that  indicated  proteins.  accompanied by  with  of  although  Electrophoretic  could not  20 d a y s .  none  organisms  urea-soluble  period.  P. fragi  (primarily  muscle,  muscle,  s a r c o p l a s m i c and m y o f i b r i l l a r  20 d a y  that  in  the  proteolysis  revealed  muscle.  Tarrant  minor  patterns  the  A. liquefaeiens in  and  Results  and  protein  in  effect  enteritidis  proteolysis  rabbit  sarcoplasmic  in  any p r o t e i n  proteolysis  measurable  of  Clostridium  of  muscle.  alteration  used  liquefaeiens,  on t h e  disappearance of  extensive rabbit  for  effects  and p o r c i n e  Although  patterns  the  zopfii  Neither  amount  (1970b)  al.  Achromobacter  whereases,  proteolysis.  produced  caused the  investigate  caused extensive  porcine  electrophoretic  to  H a s e g a w a et  enteritidis,  faecalis  urea-soluble  fraction  study,  electrophoresis  perfringens,  that  subsequent  the  did  not  protein  incubation.  was n o t  because of  evident the  until  growth  /14  of P. fragi  on the surface of the muscle sample, proteolysis might  have occurred at an early stage, but was not detected u n t i l  spoilage  had proceeded further and affected the entire mass of muscle.  The study  revealed that no p r e f e r e n t i a l breakdown of a p a r t i c u l a r m y o f i b r i l l a r protein occurred. by micro-Kjeldahl  In addition,, no s i g n i f i c a n t change (as measured analysis) was  observed i n the sarcoplasmic  f r a c t i o n from the inoculated pork. of sarcoplasmic water-soluble  protein may  protein  The authors noted that the loss  have been offset by the release of  fragments from the m y o f i b r i l l a r protein f r a c t i o n .  In an electron microscopy study of porcine muscle, Dutson et al.  (1971) showed extreme disruption of myofibrils i n the P.  inoculated samples as compared to the uninoculated Tarrant et al.  fragi.  controls.  (1973) studied the effect of a p r o t e o l y t i c  enzyme preparation from Pseudomonas fragi  on porcine muscle.  Inoculation  of the various protein f r a c t i o n s with the enzyme preparation resulted in rapid hydrolysis of the s a l t - s o l u b l e proteins; the proteins r e s i s t e d hydrolysis.  Buckley et al.  proteolytic a c t i v i t y of Pseudomonas perolens 10°C f o r 20 days.  sarcoplasmic  (1974) studied the on porcine muscle at  Protein s o l u b i l i t y studies of the inoculated  samples revealed a decrease i n the sarcoplasmic  protein f r a c t i o n and  a large increase i n 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 .  Non-protein  nitrogen increased i n both aseptic and inoculated samples, however, larger increases were noted i n the inoculated samples. concluded that enzyme production  The authors  coincided with high b a c t e r i a l numbers  and pH increase, r e s u l t i n g i n substantial changes i n primary protein  /15  solubility. of  Sage  (1974)  Pseudomonas fragi  25°C. the  from  samples  from  5.9  5.9  to  on t h e  slime  et  slime  final  microbial in  flora  more  the  Borton 1971;  al.,  et  that  spoilage  is  caused by  such  as  slime  al.,  the  the  effect  stored  at  proteolysis The pH o f  9.5  aseptic  al.,  the  day  of  the  incubation  controls  (Jay  the  of  of  decreased  ammonia and amines  did  beef  were  et  occur  until  attained"  spoilage  gave  a  one,  it  proteolysis.  genera  Pure  Pseudomonas The  and  authors proportion  proteolytic.  al.,  1967;  O c k e r m a n et  1970a and b;  increases during  (Tarrant  not  inoculum  Ockerman and C a h i l l ,  production  bacterial  gel  samples had a high  an a c i d p r o d u c i n g  microbial  slime  the  and Kontou,  1974;  various  proteolysis.  H a s e g a w a et  muscle  proteolysis  post-spoilage  degree  of  muscle using  "naturally  members  which  effects  contaminated  inoculated  1970a;  pH o f  caused by the  to  that  some o f  the  that  extensive  authors  B u c k l e y et  reported  is  over the  Although  greatest  spp.,  Numerous 1969;  of  fractions.  bovine  naturally  and  indicated  that  Pseudomonas  or  similar  rapid  of  indicated  evident.  caused the  postulated  pH o f  studied  fractions  Results  were  studies  Aeromonas  (1975)  al.  inoculated  and  culture  the  muscle  evidence  and s t r o m a l  while  protein  odour  resulted  indicated  the  5.5.  electrophoresis. the  8.5,  investigating  pectoralis  increased appreciably  to  Dainty species  a study  chicken  patterns  sarcoplasmic, myofibrillar  period  of  on m i n c e d  "Electrophoretic  inoculated  in  conducted  organism.  of  The  "alkalizing  al.,  1971;  Tarrant  1977)  spoilage,  al., et  have  unless  increased  substances"  Jay,: 1972).  pH  /16  During surface  (Gill  spoilage  utilize  carbohydrate 4,  the  Initially, apparent  1978).  15°C,  1971).  together  change i n  samples  there  to  was,  10 in  cases,  by v a r y i n g  bacterial  rate  of  change at  15°C  (1976)  initially  attacked  at  the  the all  total  in  bacteria of  changes beef  meat  in  total  stored  aerobic  samples, with  subsequent  count. no  increases  in  bacterial  at  at  at  effect.  In  the  all  the  three  effect  instead  These secondary substrates  surface.  The  become d e t e c t a b l e  noted  that  when t h e  since  cell  do n o t  that  an  overall  reached a  level  limitation  Pseudomonas glucose,  several  amino  on  spp.  continued acids  become d e p l e t e d  organoleptic  density  with  substrate  on e x h a u s t i o n o f  utilizing  greater  level.  of  and found  a  concentra-  carbohydrate  population this  significant  glucose  increased, with Total  same  glucose  increase without  temperatures,  bacterial  glucose but  author  of  4 and 9°C.  surfaces  same r a t e ,  decrease in  numbers  examined the  meat  overall  Subsequent decreases i n  lactate.  to  in in  decreases occurred past  growth  grow  than  fluctuated  bacterial  to  count.  increase until  Gill  the  degrees  e x p e r i e n c e d as b a c t e r i a l  rapid  studied  an i n i t i a l  were  10 / g ;  The  (1965)  l i t t l e  tion  of  authors.  meat  the  components  counts;  in  to  by  soluble  fell  variation  tendency  on the  weight  changes  produced all  followed  concentrations  only  10  10  concentration  of  g l u c o s e and g l y c o g e n  with  bacterial  4 from  a number  Gardner  value),  occurs  Substrate u t i l i z a t i o n  glycogen concentration  numbers  bacteria  low molecular  (anthrone  9 and  of  has been examined by  (Ingram and D a i n t y ,  at  growth  and Newton,  flora  initially  spoilage,  at  and the  spoilage is reported 8 2 e x c e e d s 10 organisms/cm ,  /17  it  is  start  probable to  that  degrade  weight  substrate  Shelef  (1977),  spoilage slime and  of  amino  studying  beef,  the  were  to  Kampelmacher  spoilage  Gill  bacteria low  molecular  and Newton  g l u c o s e was  spoilage  (1977).  bacterial  characteristic's  the  Incipient  such  as  depleted  (off-odours  and  6.0.  bacteria  (1974)  to  studied  Results  implicated  clearly of  attachment  by  to  flagellated  the  lowering  the  was  d e c r e a s e d due  The  optimal was  that  pH o f to  to  and  the  various  to  (E. coli  to  the  importance the  skirt  the  be  of of  around  21°C.  of The  of the  dealt  with  in  The K  the  low  N 1  +  2  9  7  fact.  important  degree  )  c  o  m  P  a  The parameters.  the  attachment  the  microorganisms.  different  and  chickens.  flagella  {E. coli  were  medium,  attachment  broiler  this  the  flagellated  broilers.  underlined  reduced m o t i l i t y for  of the  bacteria  K^)  attachment  of  skin  pH and t e m p e r a t u r e  the  temperature found  Notermans  non-flagellated  bacteria  also noted  food myosystems. attachment  very  on  has  the  bacteria,  out  l i t t l e  strains  microorganisms  of  r e s e a r c h h a s b e e n carried  marine  by  bacterial  bacteria  no  regarding  g l u c o s e on the  above a pH o f  extensive  non-flagellated  By  of  by  when t h e  Adhesion  adhesion of  authors  reported  effect  increase.  mechanismsuDf a t t a c h m e n t  attachment  were  results  observed u n t i l  became a p p a r e n t  Although  the  the  produced only  Similar  observed that  pH s t a r t e d  Bacterial  are  acids.  utilization  and o f f - o d o u r s  tackiness)  E.  off-odours  rate  flagellated  increasing rate  of  r  e  d  /18  attachment in  from  activity  of  In reported chicken  a  that skin  0 C to  21 C w a s  microorganisms subsequent  actually  skin,  and c o u l d be  noted  that  since  it  the  appeared "to  of  play  chicken skin;  attachment  to  the  bacteria  of  M c M e e k i n et microscopy  investigation  "microtopography" to  contamination  capillary to  size  clean.  surface  the  to  concluded  that  since  the  role"  in  surrounding  rinsing.  film  the  It  was o f  was  of  and i n  the  the  great  attachment  time-dependent  (1975)  associated with  film  adequate water  (1979) of  the  surface  conducted a scanning microorganisms was  importance  bacteria  proportion  skin  an  important  would  developed in  the  c h i c k e n were  extremely of  (1974).  on t h e  observation always  greater  Butler  et  the The  viable  than  in  relation  on  liquid  the  film  authors  as w e l l counts  those  of  difficult  material"  surface,  that  The  s u r f a c e s were  w h i c h may c o r r e s p o n d t o  material  skin.  parameter  a "layer  and .Kampelmacher of  be  electron  on c h i c k e n  and c h a n n e l s on t h e  contaminated,  layer  increase  present.  growth  chicken  flora  a water  the  being  crevices  and once  may e x p l a i n  with macerated or  the  by Notermans  topography,  swabs  of  Microbial  of  referred  al.  in  in  a key  combined  and Kampelmacher  bacterial  removed by  to  number  Notermans  the  flora  the  flagella.  present  easily  bacterial  and  study,  a proportion was  e x p l a i n e d by  as  the  obtained  obtained  with  rinses. In  attachment  another  study,  of microorganisms  to  al.  aseptic  (1979)  investigated  samples of  pork  skin  the and  thin  /19  surface direct the  slices  organisms  concentration organisms.  that  of  of  the  effects  of  attachment the  safety  attachment  organisms,  and  medium  point  contrast  time,  to  Pbhja,  accurately  the  attached  of  The removal  is  (Butler  and t y p e s dependent et  al.,  {Escherichia  spp.  Notermans  found  the  that  and  the  the  numbers  important  the  and t y p e s from a  evaluation  some o f  the  and subsequent  upon the  1979).  the  chicken skin  only  bacteria  and  explained  these  ability  from  forces  Therefore,  food  evaluation  microbial  swabbing, as  (Niskanen  determine  a muscle  a better  of  procedures  population  to  by which  of  aspect.  tissue maceration procedures  of  and  inoculated  such as n o n - d e s t r u c t i v e 16% o f  than  was  from a q u a l i t y  about  attached  This  determine  of  of  was g r e a t e r  of  (1979)  and pH o f  not  of  {Lactobacillus  studies  between  a  the  species  herbicola)  al.  effectiveness  only  number  have been devised f o r  destructive  number  any p r o c e d u r e ,  the  greater  insignificant.  exist  Some t e c h n i q u e s ,  more  1977).  also  techniques  estimates  the et  were  accurately  but  populations,  compared to the  to  on a m u s c l e : s u r f a c e i s view,  the  indicated  and c o n c e n t r a t i o n  the  species  temperature  that  ability  questionable. given  Erwinia  Butler  on attachment  study.  numerous  microbial  higher  gram-positive  the  of  attachment  gram-negative  and  1975),  Results  medium:  the  of motile,  In  immersion  used in  Although  are  test  spp.).  microorganisms  by  the  non-motile,  The  and  in  (1974  carcasses.  between b a c t e r i a l  marked d i f f e r e n c e s  samples  have  lamb  Pseudomonas putrefaciens  Kampelmacher  is  and  Attachment  Staphylococcus  by  beef  relationship  test  coli,  of  surface,  the  microorganisms  understanding  /20  of  the mechanisms  of  a procedure  muscle  for  surfaces,  thereby  behind  but  phase".  also  about  Marshall  al.  et  a surface In  this  (Butler  the  (1971)  2.5%  time-dependent  of  polymers  between  metabolism.  During  attached  to  removable  epithelial  the  the  surface phase,  exhibiting  surfaces  within  the  and  the  reticulo-rumen  electron cells  with  the  "formation  of occasional  carbohydrate adherent  material  by  the  with formation  bacterial firmly  and were  ruthenium  (glycocalyx)  of  of  SEM showed  b a c t e r i a l cells".  bacteria.  surface  were  microscopy  epithelial  presence of  the  of  bacteria  Waals  exhibit  adhesion  the  the  der  involved  bacteria  the  to  a result  of  (in  of  no  longer  NaCl.  scanning  similar  irreversible  London-van able  are  entailed  attachment  Brownian motion  studied  (TEM)  2.5%  no  numbers,  attachment  attachment  phase  as  the  (1978)  morphologically  and the  and  selection on  bacterial  removed by washing  al.  cell  the  bacteria  bacterial  s t i l l  irreversible  latter  surface,  that  were  M c C o w a n et  colonization  of  this  in  1979).  al.,  such as  with  surface  microscopy  forces  readily  bacteria  of  reduce  a weak  by washing  transmission of  stated  bacteria  and were  The  to  et  involved  Brownian motion NaCl.  and types  only  p h a s e " and a "time-dependent  phase  the  not  mechanisms o f  attractive  state  aid,  techniques  reversible  through  may  numbers  shelf-life  The r e v e r s i b l e  forces.  in  Information  an " i n s t a n t a n e o u s  to  estimating  increasing  limited.  attachment  cattle  (SEM).  on b o t h  The g l y c o c a l y x  of  to  using Examination  intermittent  microcolonies  Transmission red)  bacteria  revealed  of  electron the  presence  the  epithelial  the  bacteria  cells appeared  /21  to mediate the attachment of bacteria to the epithelium, to food p a r t i c l e s and to each other. Costerton et al.  (1974b) indicated that acid polysaccharides  were involved i n the adhesion process.  The authors noted that  these polysaccharides could be i n the form of pure polysaccharides, glycoproteins or other mixed polymers. Notermans et al. to  (1979) investigated the attachment of bacteria  s t e r i l e teats of cows at various storage temperatures and reported  that a f t e r i n i t i a l attachment, the strength of attachment increased. The increase appeared to be faster at higher storage temperatures, and was due to the formation of e x t r a c e l l u l a r substances. companion  paper, Firstenberg-Eden  et al.  In a  (1979) used SEM to observe  that polymers, i n the form of thin f i b e r s , were produced during the storage of the inoculated teats. and formed slime.  These f i b e r s eventually thickened  Fletcher and Floodgate  (1973), using a ruthenium  red-alcian blue s t a i n combination, demonstrated an e x t r a c e l l u l a r , a c i d i c polysaccharide which was bacterium  to surfaces.  involved i n the adhesion of marine  According to the workers, the i n i t i a l  stage  of i r r e v e r s i b l e adhesion involves contact between the surface and the primary polysaccharide surrounding the bacteria, p r i o r to the formation of secondary fibrous, a c i d i c polysaccharides.  F.  Inoculation  of Intact  Muscle  Samples  Most research dealing with muscle spoilage has employed ground muscle as the substrate for the inoculum.  Sage (1974) stated that  122  mincing  increases the  surface  from b a c t e r i a l  growth;  of  occur  those  which In  structural dipped  order  to  integrity  into  an  and  carcasses  as  a means  Butler  at.  (1979)  beef  et  and  lamb  attachment  however,  on  intact  of  the  muscle,  (1974)  without  a technique buffer  used the  inoculation.  into  maximizing  any  c h a n g e s may n o t  tissue  embedded s t e r i l e  carcasses)  media.  these  physiological  Kampelmacher for  thus  changes  be  indicative  myosystems.  inoculate  inoculated  Notermans  area,  "Gulf"  disrupting whereby has  dip  been  method  wax  (pork  prior  to  tissue  skin  is  employed.  of  Using a similar tissue  the  whole  broiler  technique, and  dipping  in  surfaces  of  /23  METHODS AND MATERIALS  Bovine Longissimus  dorsi  muscle was excised from a 24 h  postmortem steer, at a l o c a l abattoir, and transported on i c e to the  laboratory.  P r i o r to sectioning, the muscle was sprayed with a  70% ethanol solution i n order to reduce surface b a c t e r i a l contamination (Hasegawa et al., 1970a).  Thin s l i c e s  (3 mm) were  removed from the muscle using a Hobart delicatessen s l i c e r (Don M i l l s , Ont.) (Figure 1) which had been previously sprayed with 70% ethanol, as a b a c t e r i c i d a l agent. avoided.  Obvious adipose tissue was  Upon completion o f every f i f t h s l i c e , the s l i c e r was sprayed  with 70% ethanol.  The above procedure was adopted i n attempts to  minimize the effects of microbial growth p r i o r to gamma radiation.  A.  Sarcoplasmic  Extraction  Extraction o f sarcoplasmic f l u i d was effected by placing the muscle s l i c e s i n a 10.2 cm radius x 30.5 cm high s t e r i l e plexiglass tank (8 l i t e r capacity) f i l l e d with s t e r i l e 0.0100M Na HP0 ~0.119 M 2  NaH P0 2  4  4  pH 5.8 (ionic strength = 0.15) buffer solution (Tu, 1973).  In order to obtain adequate extraction of the water-soluble components, the buffer was circulated by means of a magnetic s t i r r e r . In addition, f i l t e r s t e r i l i z e d nitrogen gas was sparged through the tank at a rate of 100 ml/min (Figure 2).  Sterilization  of nitrogen  was accomplished by passing the gas .through a series of four-37 mm b a c t e r i a l vents (Gelman Instrument Company, Ann Arbor, MI) (Figure 3).  FIGURE  1:  Hobart  delicatessen  slicer.  /25  FIGURE  2:  Apparatus sarcoplasra  used from  for  the  intact  extraction muscle.  of  the  FIGURE 3:  B a c t e r i a l vent used t o s t e r i l i z e a i r and n i t r o g e n .  both  Ill The b u f f e r s o l u t i o n was changed e v e r y hour f o r t h e f i r s t 4 h and t h e n e v e r y 4 h f o r t h e n e x t 20 h.  These samples were termed "washed".  The e x t r a c t i o n was c a r r i e d out a t 4°C. Upon c o m p l e t i o n o f t h e e x t r a c t i o n p r o c e d u r e , a p o l y p r o p y l e n e chromatography c l i p ( F i s h e r S c i e n t i f i c , P i t t s b u r g h , PA) was t o each washed muscle sample a t one e x t r e m i t y . was p l a c e d i n a s t e r i l e 18 oz "Whirl-pale" Guelph, Ont.) ( F i g u r e 4 ) .  Each muscle sample  bag (Arnold-Nasco L t d . ,  S i m i l a r p r o c e d u r e s were c a r r i e d out f o r  muscle samples n o t exposed t o t h e e x t r a c t i o n t r e a t m e n t . were termed " i n t a c t " .  These  samples  The " W h i r l - p a k " samples ( i n t a c t and washed)  were t h e n packed i n i c e and i r r a d i a t e d w i t h a 1 Gammacell  attached  Megarad dose i n t h e  220 (Atomic Energy o f Canada L t d . ) c o n t a i n i n g  ^°Co  (Sage, 1974).  B.  Preparation  of the  Inoculum  A f r e e z e d r i e d pure c u l t u r e o f Pseudomonas fragi  (ATCC 4973)  was p r e p a r e d as p e r American Type C u l t u r e C o l l e c t i o n C a t a l o g u e o f S t r a i n s I ( 1 2 t h E d i t i o n , 1976).  The c u l t u r e was m a i n t a i n e d on  t r y p t i c a s e soy agar (TSA) s l a n t s (BBL, C o c k e y s v i l l e , MD)  a t 4°C.  P r i o r t o i n o c u l a t i o n o f t h e muscle samples, a l o o p f u l o f P.  fragi  was t r a n s f e r r e d from t h e TSA s l a n t and grown i n 200 ml o f t r y p t i c a s e soy b r o t h  (TSB) (BBL, C o c k e y s v i l l e , MD)  i n a s h a k i n g water b a t h . (1000 x g ; 10°C).  a t 25°C f o r 24 h  The c u l t u r e was t h e n c e n t r i f u g e d  f o r 20 min  The p e l l e t was resuspended i n 50 ml o f s t e r i l e  FIGURE  4:  "Whirl-pak"  muscle  sample.  /29  attachment media consisting of 0.15 M NaCl, 0.0062 M Na HP0 , 0.0021 M 2  NaH P0 2  4  and 0.001 M EDTA, adjusted to pH 5.8  4  (Notermans and  Kampelmacher, 1974; Butler et al., 1979) and mixed vigorously to obtain a uniform suspension.  The suspension was then centrifuged  at 10°C f o r 20 min, at 1000 xg.  The resuspension-centrifugation  procedure was repeated twice to ensure complete removal of the TSB. Upon completion, the washed p e l l e t was resuspended i n 4 I of s t e r i l e attachment media.  Bacterial enumeration of the inoculated attachment 7 media on TSA indicated a concentration of 1.88 x 10 organisms/ml.  C.  Preparation  of Muscle  Samples  Gamma s t e r i l i z e d samples (intact and washed) were immersed for 10 min into the 4 1 of attachment media containing P. (Butler et al., 1979).  fragi  After drainage for 10 sec, the muscle  samples were placed into s t e r i l e stomacher bags inverted and hung i n a 38.1 cm x 33.0 cm x 50.8 cm s t e r i l e plexiglass chamber (Figure 5). To ensure aerobic conditions, a p o s i t i v e pressure was applied by passing f i l t e r s t e r i l i z e d medical grade compressed a i r (100 ml/min) through the chamber.  Control samples were treated i n a similar  manner with s t e r i l e attachment- media.  Thus, control uninoculated  samples were available at each storage period so that one could d i f f e r e n t i a t e changes induced by microbial growth with those of autolysis (Rampton et al., 1970).  FIGURE  5:  Plexiglass  incubation  chamber.  /31  D.  pH  Determination The pH o f t h e m u s c l e samples was determined a f t e r each  i n c u b a t i o n p e r i o d by homogenizing a 5 g r e p r e s e n t a t i v e sample w i t h 5 ml o f d i s t i l l e d - d e i o n i z e d water i n an Omni m i x e r ( I v a n S o r v a l l I n c . , Norwalk, CN) a t h a l f speed f o r 15 sec and t o p speed f o r 45 sec ( B u c k l e y et al.,  1974).  The pH o f t h e r e s u l t a n t s l u r r y was t h e n  determined w i t h a F i s h e r Accumet pH/ion meter (Model 230).  E.  Bacterial  Counts  B a c t e r i a l numbers on b o t h i n o c u l a t e d and c o n t r o l samples ( i n t a c t and washed) were enumerated a f t e r each i n c u b a t i o n p e r i o d by 2 p l a c i n g a 4 g ( a p p r o x i m a t e l y 30 cm  t o t a l surface area) r e p r e s e n t a t i v e  sample i n a s t e r i l e stomacher bag.  T h i r t y - s i x ml o f s t e r i l e  0.1%  (w/v) peptone ( D i f c o , D e t r o i t , MI) was t h e n added, and t h e sample stomached f o r 2 min i n a C o l w o r t h stomacher l a b - b l e n d e r 400 (A. J . Seward, London, E n g l a n d ) .  Appropriate s e r i a l decimal d i l u t i o n s of  the macerated sample were p r e p a r e d w i t h s t e r i l e 0.1% peptone b r o t h , and were s u r f a c e s p r e a d , i n d u p l i c a t e , on TSA.  A l l p l a t e s were  i n c u b a t e d a t a p p r o x i m a t e l y 25°C f o r 48 h t o o b t a i n t h e t o t a l count.  viable  B a c t e r i a l enumeration was a l s o c a r r i e d out on t h e attachment  media i n o r d e r t o e v a l u a t e t h e e f f e c t i v e n e s s o f t h e attachment p r o c e d u r e .  /32  F.  Electron 1.  Microscopy  Scanning  EM  preparation  Muscle pieces  tissue  and f i x e d  glutaraldehyde buffer  buffer  2.5%  cut  (v/v)  into  electron  (61 m l  The  (pH 7 . 0 )  0.05  tissue prior  M Na^HEO^  was to  then  3  1 h  times  by  (Sjostrand, with  0.05  dehydration  80%  (v/v)  with  ethanol were  ethanol  90%;  acetate 10 m i n  duration  with  absolute  amyl  acetate.  (Parr with  Samples  were  (Technics  5 min  each;  of  by  25,  Samples were  paste then  50 a n d change,  Co., Moline,  IL)  (Structure  Probe  Alexandria,  of  of  phosphate tetroxide  buffer  (pH  was  10 m i n  one  (v/v) 1 h  50,70  on  in  a  of  Samples amyl of  with  diluted 100%  Parr-bomb  aluminum  Chester, in  All  acetate  duration,  and  each,  change,  amyl  dried  West  followed  water.  series  7.0)  washed  100% e t h a n o l .  NJ):  and o b s e r v e d  M  ethanol:  and mounted Inc.,  4  osmium  This  with  75%  phosphate  subsequently  of  gold-palladium  VA)  0.05  7.0).  critical-point  coated with  2  (w/v)  then  M  M KH PC> )  a graded  each,  and one  in  2 cm  grade  0.05  distilled-deionized  Co., Fairlawn,  ethanol  Inc.,  (pH  each,  infiltration  with  0.05  2 changes,  20 m i n  -  M phosphate  Scientific  Instrument silver  to  0.05  buffer  were made w i t h  subjected  1%  an ascending s e r i e s  and 3 c h a n g e s ,  (Fisher  -  in  Samples were  M phosphate  for  dilutions  then  1967).  through  + 39 ml  post-fixing  2 cm x  microscopic  washed t w i c e  (CAN-EM C h e m i c a l s , G u e l p h , O n t . ) for  approximately  (CAN-EM C h e m i c a l s , G u e l p h , O n t . )  pH 7 . 0  overnight.  in  was  stubs  PA).  a sputter  on a H i t a c h i  coater S-500  /33  scanning electron microscope at an accelerating voltage of 20 KV.  Images were recorded on I l f o r d Pan F 135 fine grain black  and white f i l m ( I l f o r d Ltd., Essex, England). 2.  Transmission  EM  preparation  Samples f o r transmission electron microscopy were prepared according to the method o f McCowan et at. modifications.  (1978)' with s l i g h t  Tissues were fixed i n 2.5% glutaraldehyde -  0.05 M phosphate buffer pH 7.0 (see above buffer) overnight and subsequently placed i n 0.015% (w/v) ruthenium red (Aldrich Chemical Co., Milwaukee, WI) i n 0.05M phosphate buffer f o r 2 h. The samples were placed i n 0.05 M phosphate buffer with 0.05% (w/v) ruthenium red (ruthenium red-phosphate buffer) f o r 2 h at room temperature and then washed i n 0.05% ruthenium red-phosphate buffer 5 times f o r 1 h each time, with one overnight wash included, before being post-fixed f o r 2 h i n 2% osmium tetroxide i n ruthenium red-phosphate buffer.  After f i v e further 1 h washes  i n ruthenium red-phosphate buffer, samples were subjected to an ethanol dehydration series of 20 min each, i n steps of 15, 30, 50, 70, 90 and 100% ethanol.  The ethanol was diluted by  ruthenium red buffer up to the 70% solution.  The 90% ethanol  solution was prepared with d i s t i l l e d - d e i o n i z e d water.  Samples  were washed i n two changes of 100% propylene oxide (Polysciences, Inc., Warrington, PA) f o r 15 min each, then i n f i l t r a t e d with a 1:1 mixture of propylene oxide and Epon 812 (Polysciences, Inc.,  /34  Warrington, PA) overnight.  Samples were f i n a l l y embedded i n  100% Epon 812 (Luft, 1961) and sectioned on a "Porter-Blum" MT-2 ultramicrotome (Ivan Sorvall Inc., Norwalk, CN).  Sections were  mounted on copper grids and stained with uranyl acetate and lead c i t r a t e (Reynolds, 1963; Sjostrand, 1967). were obtained with a Zeiss EM-10  Electron micrographs  (Carl Zeiss, Oberkochen, West  Germany) transmission electron microscope operated at an accelerating voltage of 60-80 kV and recorded on Kodak 70 f i n e grain f i l m (Eastman Kodak Co., Rochester, NY).  mm,  Upon  completion of the above procedures (bacterial counts, pH determination and EM sample preparation), the muscle samples were frozen i n l i q u i d nitrogen and subsequently l y o p h i l i z e d u n t i l ready f o r analysis.  G.  Extraction  of  Water-  and  Salt-Soluble  Protein  Fractions  Procedures s i m i l a r to those outlined by Hasegawa et  al.  (1970a) were adopted f o r the extraction and water- and salt-soluble protein: fractions (Figure 6).  The following procedure was carried  out f o r both intact and washed muscle to evaluate not only the effectiveness of the washing procedure, but also the effect of the reduction of sarcoplasm on growth and p r o t e o l y t i c a c t i v i t y of P.  fragi.  Five ml of 0.3 M sucrose - 0.01 M KC1 - 0.01 M T r i s - c i t r a t e buffer (pH 7.6) was added to 0.5 g of a l y o p h i l i z e d muscle sample and homogenized i n a Polytron mixer (Brinkman Instruments, Rexdale, Ont.)  /35  Lyophilized Muscle homogenized  with'10  Sample  v o l (10 v o l / w ) o f 0,3M s u c r o s e , 0.01M KC1, 0.01M c e n t r i f u g e d a t 2 0 , 0 0 Q x g f o r 15 m i n Supernatant:  water  Tris  soluble  proteins  and n o n - p r o t e i n  nitrogen  Precipitate h o m o g e n i z e d w i t h 6 v o l (6 v o l / w ) of sucrose-KCl-Tris c e n t r i f u g e d a t 2 0 , 0 0 0 j c g f o r 15 m i n Supernatant:  discard  Precipitate homogenized 8 vol  w i t h 6 y o l (6 v o l / w ) of Weber-Edsall solution, stored for 24h, (8 v o l / w ) o f W e b e r - E d s a l l s o l u t i o n added, homogenized and c e n t r i f u g e d a t 2 9 , 0 0 0 x g f o r 30 m i n Supernatant:  salt-soluble  proteins  Precipitate homogenized  with  6 vol  (6 v o l / w )  centrifuged  at  of  Weber-Edsall  29,000xg  for  30  solution  and  min  Supernatant:  discard  Precipitate homogenized  with  6 vol  centrifuged  at  (6 v o l / w )  29,000xg  for  of 30  8M u r e a min  Supernatant; Precipitate:  PIGURE  6:  Flow  sheet  extractions  of  protein  were  urea-soluble  urea-insoluble  extraction  carried  and  o u t .at  proteins  procedure. 4°C,  All  proteins  /36  at 1/4 speed f o r 15 sec and at.1/2 speed f o r 15 sec. was centrifuged (20,000 xg, 15 min, 4°C).  The homogenate  The supernatant  containing the water-soluble protein f r a c t i o n .and the non-protein nitrogen, was decanted and stored at 4°C.  The p r e c i p i t a t e , along  with 3 ml of the sucrose-KCl-Tris buffer, was returned to the Polytron mixer and homogenized f o r 30 sec at 1/2 speed.  The mixture  was centrifuged at 20,000 xg f o r 15 min and the supernatant discarded.  The p r e c i p i t a t e was then homogenized with 3 ml of Weber-  Edsall solution (0.6 M KC1, 0.04 M KHC0 , 0.1 M K C0 ) f o r 30 sec at 3  1/2 speed, and stored f o r 24 h at 4°C.  2  3  After storage, 4  ml of  Weber-Edsall solution were added and the solution mixed with the Polytron mixer at.1/4 speed f o r 15 sec. for 30 min at 29,000 xg.  The mixture was centrifuged  The supernatant consisting of the salt-soluble  protein f r a c t i o n was decanted and stored at 4°C u n t i l ready for analysis, while the p r e c i p i t a t e was homogenized with 3 ml of WeberEdsall solution f o r 15 sec at 1/4 speed.  The mixture was  then  centrifuged f o r 30 min at 29,000 xg and the supernatant discarded. The p r e c i p i t a t e was homogenized with 3 ml of 8 M urea at 1/4  speed  for 15 sec and 1/2 speed f o r 15 sec, and the mixture was centrifuged for 30 min at 29,000 xg.  The supernatant, containing the urea-soluble  protein f r a c t i o n as well as the urea-insoluble p r e c i p i t a t e were retained.  H.  Nitrogen  Determination  Protein content of the water- and salt-soluble protein fractions was determined by the rapid micro-Kjeldahl method of Concon  /37  and  Soltess  (1973)  using  Instruments  Corp.,  Tarrytown,  factor;.of  6.0  mg p r o t e i n was  et  nitrogen  obtained  with  was u s e d  by  1971).  content  of  of  soluble  of  the  Technicon Auto  by  the micro-Kjeldahl  (AOAC,  1975).  per  gram d r y  I.  Total  Carbohydrate  and  In  (w/v)  5 mg o f  2 ml  of  official  nitrogen  final  was  (Tarrant  the  nitrogen  the  sensitivity  was  action  fraction  determined  AOAC  e x p r e s s e d a s mg  47.021  nitrogen  the  of  to  content  the  using various sulfate  (SDS),  successful.  finely ground,  and p l a c e d i n  ground,  various  a c i d method error  tissue,  0.1%  muscle  (Dubois  introduced  attempts  reducing  agents  (v/v)  samples et  by  al., blending  w e r e made (8M u r e a ,  finally  freeze-dried,  washed muscle  in  water  intact  a test bath  muscle  tube. for  to 1.0%  3-mercaptoethanol),  A n a l y s e s were  lyophilized,  a 100°C  the  inherent  homogenized muscle  1 0 mg o f  of  phenol-sulfuric  reduce  tissue  finely  •  d i s t i l l e d - d e i o n i z e d water  stoppered  was b e l o w  nitrogen  protein acid  revealed that  therefore,  Analysis  the  none proved  by m i x i n g or  by  -sodium d o d e c y l  however,  -  conversion  e x p r e s s e d as  water-soluble  nitrogen  nitrogen  carbohydrate  order  pipetting  solubilize  method  was  trichloroacetic  experiments  Analyzer,  protein  Soluble non-protein  the  (w/v)  (Technicon  weight.  determined  1956).  20%  of  to  Nitrogen  weight.  non-protein  Non-protein  Total was  0°C  Analyzer  A nitrogen  1960).  gram d r y  Preliminary  the  NY).  precipitation  an e q u a l volume  al.,  Technicon Auto  (Locker,  per  the  the  samples  The  1 min.  performed  tubes  tissue with were  Preliminary  /38  experiments had shown that t h i s l a t t e r step was necessary i n order for the s u l f u r i c acid to complete the hydrolysis of the muscle tissue. After the samples were cooled to room temperature, (w/v) phenol was added. were then added rapidly.  0.05 ml of 80%  Five ml of concentrated s u l f u r i c acid Vortexing of the mixture was required  immediately after the addition of the s u l f u r i c acid i n order to complete the hydrolysis of the muscle samples.  The tubes were allowed to stand  10 min, then shaken, and subsequently incubated i n a 25°C water bath for 15 min p r i o r to determining the absorbance at 485 nm with a Unicam SP 800B spectrophotometer.  Quadruplicate analyses were  carried out on duplicate muscle samples.  Total carbohydrate content  was estimated from a standard curve f o r glucose. In order to test f o r the p o s s i b i l i t y of a M a i l l a r d reaction occurring between the muscle proteins and endogenous carbohydrates during the heating procedure, a known amount of glucose was added to both washed and intact muscles and compared to untreated samples (no glucose added) f o r t o t a l carbohydrate content. Preliminary results indicated a 96% recovery carbohydrate.  of the added  It was therefore concluded that the heat  treatment  used did not substantially affect the effectiveness of the carbohydrate assay procedure  J.  SDS-Gel  (phenol-sulfuric acid).  Electrophoresis  SDS-gel electrophoresis was performed on a l l (control and inoculated) water-, s a l t - , urea-soluble and urea-insoluble proteins  /39  in  a Pharmacia disc  Fine  gel  electrophoresis  Chemicals, Uppsala,  and Osborn (BioRad  (1969)  Sweden).  a n d W e b e r et  Laboratories,  contained  M sodium phosphate  as w e l l  38.6  g Na HP,0 -7H 0,  water  pure  sodium dodecyl  sulfate  2  Various  4  molecular  weight  containing 0.01  0.1  Kocal,  above  40°C  the  of  one d r o p  prevent  then  The g e l s  10 p i  glycerol  were of  The run  was  were  gel  0.5  0.1  in  of  the  d y e was m a r k e d w i t h  of  ml  were  test  bromophenol  with  0.1  first  a needle  (Deutsch,  were  the  blue  30 m i n  individually (Matheson, pH 7 . 2  1977).  the tip  20-25°C and  blue  gel.  at  5 mA p e r (tracking  The  in  India  Coleman and gels  buffer. a  constant gel dye)  The p o s i t i o n  dipped  a  Various  M sodium phosphate at  kept  presence of  1973).  (Kocal,  bromophenol end o f  tubes  8. M u r e a ,  5 min  applied  England).  known  of  SDS i n  et:al.,  (Hay 1)  Poole,  M sodium phosphate b u f f e r  the  the  electrophoretically  preparations  electrophoresis  for  NaH^PO^H^O,  stoppered 0.9  x  reservoirs,  (w/v)  g SDS f o r  added and mixed  to  cm f r o m  ions  (w/v)  c o m p l e t e d when t h e  approximately  boiled  (Table  overlayed  subjected  2 mA p e r  in  g  and p r o t e i n s  Weber  5 mm ( i d )  lower  (7.8  of  acrylamide  v  in  and 0.1%  and 0 . 0 1 5 - 0 . 0 3  0.001%  OH)  carefully  current  tracking  of  and  M phosphate b u f f e r ,  potassium  w  (BDH C h e m i c a l s L t d . ,  crystallization  of  ( / )  run  pH 7 . 2  extract,  were  were  GE-4, Pharmacia  procedure  7.5%  Salt-soluble protein  Norwood, of  buffer  (Model  the  upper  2 liters)  sample p r e p a r a t i o n  gels;  gels  as the  (SDS)  1),  0.1  1977).  to  of  and B e l l ,  were  ml  to  protein  (Table  concentration  volumes to  of  3-mercaptoethanol  ml  1976;  high  2  quantities  (1972),  R i c h m o n d , CA)  The g e l s ,  0.1  Following  al.  7 5 mm t u b e s .  system  of  ink.  thereafter. was the The  gels  /40  TABLE  Protein  1:  1 Quantity of protein fraction and m o l e c u l a r weight m a r k e r s u s e d i n s a m p l e p r e p a r a t i o n and amount o f preparation applied to gel during SDS-gel electrophoresis.  Quantity used for p r e p a r a t i o n (mg)  fraction  Amount o f p r e p a r a t i o n applied to gel (pi)  Water-soluble  100  10  Salt-soluble  100  50  Urea-soluble  100  50  10  20  10  10  Urea-insoluble Molecular  weight  markers  Protein fractions procedure  were  those  obtained  during  the  protein  extraction  /41  were  fixed  Blue  R (INC  9.2%  (v/v)  destained acetic  and  stained  in  a  solution  glacial by  acetic  diffusion  acid until  the  in  acid  ( W e b e r et  5% ( v / v )  b a c k g r o u n d was  densitometer.  Relative mobilities  densitometric  weights  trace  as the  distance migrated were  the m o b i l i t y  estimated of  the  from  ratio by  the  molecular  L o g Mwt  Mwt  = molecular  a  =  b  = slope of  the  mobility  the  of  for  7.5%  (v/v)  Gels  were MI)  dye.  protein  methanol  -  and  glacial scanned i n  a  scanning the  Apparent  (Table  Brilliant  45 m i n  distance migrated  markers  intercept  Rm = r e l a t i v e  1972)  regression equation  weight  (v/v)  c a l c u l a t e d from  tracking  = a + bRm  line  -  clear.  were  weight  regression  al.,  Coomassie  45.4%  C o . , Ann A r b o r ,  of  the  (w/v)  in  methanol  (German I n s t r u m e n t  the  0.25%  P h a r m a c e u t i c a l s , P l a i n v i e w , NY)  Gelman G e l s c a n  over  of  (1)  by  the  protein  molecular  derived  from  2).  (1)  TABLE  2:  Molecular  weight  markers for  SDS-gel  electrophoresis.  Company  Protein  Bovine  serum  albumin  Bovine  serum  albumin  bovine prepared as p e r Payne (1973) egg  Ovalbumin  Sigma  bovine  white  Molecular  weight''"  67,000  134,000  ICN  Nutritional  43,500  Biochemicals a-chymotrypsinogen  bovine egg  Lysozyme  From S o b e r  A  (1970)  pancreas white  Sigma  23,400  Worthington  14,000  /43  RESULTS  A.  Effect  of  AND  Gamma 'Radiation  DISCUSSION  on  Colour'  and  Odour  S t e r i l e products have been obtained using gamma radiation but off-odours and colours have been undesirable by-products of this process (Batzer and.Doty, 1955). I r r a d i a t i o n of both intact and washed muscle (inoculation or aseptic control) samples, p r i o r to treatment resulted i n a c h a r a c t e r i s t i c "wet dog" odour.  The odour results from the formation  of hydrogen s u l f i d e , mercaptans, carbonyls and aldehydes due to the degradation of free amino acids (Batzer and Doty, 1955; Merritt et al.,  1959). Changes i n colour were also noted a f t e r i r r a d i a t i o n .  The  bright red colour of the intact muscle samples was transformed to a tan brown colour while the pink colour (almost pork-like i n appearance) of the washed muscle samples took on a greyish-brown appearance. Coleby et al.  (1961) noted that the bright red colour of oxymyoglobin  tended to be oxidized to brown metmyoglobin upon i r r a d i a t i o n .  The  colour difference found between the intact and washed samples, before and a f t e r i r r a d i a t i o n , could be explained by the loss i n myoglobin during the washing procedure.  B.  Influence  of  Sarcoplasmic  Reduction  on  Bacterial  Growth  Inoculation of aseptic intact and washed muscle samples was 7  carried out i n attachment media containing approximately 10  P.  fragi  /44  cells/ml.  Enumeration of the samples at day 0 indicated attachment 5  in the range of 10  6 to 10  2 CFU/cm .  Similar results were reported  by Notermans and Kampelmacher (1974) and Butler et al.  (1979).  At each sampling period, representative tissue was removed from both the s t e r i l e and inoculated samples and checked for b a c t e r i a l growth (Table 3).  No growth was  samples during the 12 days of incubation.  observed in.the control Jay and Kontou (1967)  working with beef, and Sage (1974) working with chicken, found that a 1 Megarad dose of gamma r a d i a t i o n was  s u f f i c i e n t to destroy the  naturally occurring microflora. Bacterial growth on the intact samples was i n i t i a l 6 day period; this was  rapid f o r the  followed by a more modest increase  during the next 6 days of storage.  The increase i n b a c t e r i a l numbers  in the washed samples, although moderate, occurred at a steady rate over the 12 day incubation period (Figure 7). In order to s t a t i s t i c a l l y evaluate the effect of a  sarcoplasmic  protein reduction on b a c t e r i a l growth, data from both intact and washed inoculated muscle samples were subjected to a data transformation for l i n e a r i z a t i o n using the modified super-simplex optimization method of F u j i i and Nakai (1980) (Figure 8).  The u t i l i z a t i o n of such  a method allows f o r c a l c u l a t i o n of a regression equation for each set of data, thereby enabling one to use regression techniques  for  comparing two straight lines (Mullen and Stanley, 1979). Linearization of the log b a c t e r i a l cound data yielded the following regression  equations:  TABLE  3:  Influence  of  Pseudomonas  incubation  at  4 C on m u s c l e samples  inoculated  with  fragi.  Average  bacterial  numbers  Incubation  a  (CFU/cm^ period  fresh  muscle  tissue)  (days)  0  3  6  9  12  control  0  0  0  0  0  Washed c o n t r o l  0  0  0  0  0  Sample  Intact  inoculated  4.19 X 1 0  5  5.,20 X i o  Washed i n o c u l a t e d  1.32 X 1 0  6  6.,29 X  Intact  Average  is  based on 4  determinations  V  7  3.,76 X 1 0  9  1.22 X i o  5,,73 X i o  8  1.13 X i o  1 0  9  1.82 X 10 1.43 X i o  1  /46  Incubation period  FIGURE 7:  (days)  B a c t e r i a l p o p u l a t i o n o f i n t a c t and washed muscle samples, i n o c u l a t e d w i t h Pseudomonas fragi, s t o r e d a t 4°C.  FIGURE  8:  Linearization of log bacterial population data f o r i n t a c t and washed muscle samples inoculated w i t h Pseudomonas fragi s t o r e d at 4°C.  /48  Intact  inoculated:  (y  -  5.40)  y  = log  CFU/cm  x  = incubation  2  of  the  -  3.47  r  =  0.97  r  =  0.94  3  J  Washed i n o c u l a t e d :  = 10.66x  J  C,u -  5.90)  = 3.03v  + 0.76  intact  inoculated  muscle  tissue-  washed  inoculated  muscle  tissue  period 2  u  = log  CFU/cm  v  = incubation  Reduction  of  growth rate (slope  of  period  the  = 10.66)  (Table  reduced  and  (Figure  C.  (Zar,  4).  Kontou,  8).  The Effect  resulted  compared  to  indicated  by  the  compounds.  Gill,  1976;  the of  in  the  utilization  slopes  limited  at  low  the in  authors  using  the  P <  0.01  the  (Jay,  1977)  molecular  bacterial  sample  sarcoplasmic  availability  and Newton,  of  muscle  experienced  Several  Gill  a decreased  intact  significance  growth rate  explained  a preferential  muscle  fraction  A comparison  lower  soluble 1976;  as  1974)  The  weight,  suggested during  = 3.03)  s a m p l e s may be  molecular Jay  sarcoplasmic  (slope  Student'sttest level  the  of  low  1967;  have  weight  components  spoilage.  of Growth of P. fragi  on pE  Surface  }  Appearance  and Odour The samples controls  is  and  in  pH o f  presentedriin Table  (intact  throughout 5.56  change  the  5.78  and washed)  experiment, to  5.72  for  the  control  5 and  Figure  remained  although intact  and  inoculated  9.  The pH o f  relatively  slight  muscle the  aseptic  constant  decreases  and washed c o n t r o l s ,  in  pH  (5.67  to  respectively)  TABLE  Sample  4:  Regression inoculated  statistics regression  Slope  used with  in the comparison of the slope of the intact t h a t of the washed i n o c u l a t e d regression.  Residual  DF  Residual  SS  -Ex  Intact  inoculated  10.66  18  2567.68  360  Washed  inoculated  3.03  18  409.97  360  **P  0.01  <  t  value  11.22**  TABLE  5:  T h e e f f e c t o f P . fragi on t h e pH o f (washed) b o v i n e m u s c l e s a m p l e s .  intact  and s a r c o p l a s m i c  reduced  pH Incubation Sample  Intact  0  control  5.67  a  + 0. 0 3  b  period  (days)  3  6  9  12  5 . 6 5 + 0 ,. 0 4  5 .. 6 3 + 0 . . 0 1 .  5 ., 5 7 +  5 . 7 9 + 0 .. 0 1  5 ,. 7 8 + 0 .. 0 1  5 ., 7 2 + 0 ., 0 2  5 ., 7 2 +  0 ., 0 4  5 ,. 5 6 + 0 . 0 1  5.78  + 0 . 01  inoculated  5.66  + 0 . 01  5 . 7 9 + 0 ,. 0 1  6 ,. 8 6 + 0 . . 0 1  7 ., 1 6 + 0 ., 0 6  7 ,. 2 4 + 0 . 0 1  Washed i n o c u l a t e d  5.79  + 0 . 01  5 . 8 0 + 0 .. 0 1  5 .. 7 8 + 0 .. 0 1  5 .. 7 3 + 0 .. 0 2  5 ,. 7 3 + 0 . 0 1  Washed c o n t r o l Intact  Average  'Standard  of  duplicate  deviation  analysis  .  0 . 01  /51  FIGURE 9:  pH o f c o n t r o l and i n o c u l a t e d , i n t a c t and washed muscle samples s t o r e d a t 4°C. Each p o i n t r e p r e s e n t s the mean o f d u p l i c a t e determinations.  /52  were n o t e d .  A s i m i l a r d e c r e a s e i n pH o f the a s e p t i c c o n t r o l was  r e p o r t e d by Sage (1974) w o r k i n g w i t h gamma i r r a d i a t e d , minced chicken muscle. Gardner and S t e w a r t (1966) s t a t e d t h a t a u t o l y t i c o f ammonia i n b e e f muscle was  v e r y low o r n o n - e x i s t e n t .  production  The  intact  i n o c u l a t e d sample d i s p l a y e d a d r a m a t i c r i s e i n pH d u r i n g the days o f i n c u b a t i o n (pH 5.66 Several authors B o r t o n et at., Sage, 1974)  12  t o 7.24). ( J a y and Kontou, 1967;  1970a; Hasegawa et at.,  Ockerman et al.,  1970a; T a r r a n t et at.,  1971;  have r e p o r t e d an i n c r e a s e i n pH i n v a r i o u s myosystems  t o the growth o f Pseudomonas f r a g i . t h a t t h e i n c r e a s e i n pH was and ammonia.  T a r r a n t et al.  1969;  due  (1971) i n d i c a t e d  a t t r i b u t e d t o the p r o d u c t i o n o f amines  The pH o f t h e washed i n o c u l a t e d sample, however,  e x h i b i t e d l i t t l e change d u r i n g i n c u b a t i o n , d e c r e a s i n g s l i g h t l y from an i n i t i a l pH o f 5.79 incubation.  The  a f t e r 12 days o f  l a c k o f i n c r e a s e i n pH e x p e r i e n c e d  i n o c u l a t e d sample may precursors  t o an u l t i m a t e pH o f 5.73  i n t h e washed  be e x p l a i n e d by assuming t h a t most o f the  ( f r e e amino a c i d s , p e p t i d e s and r e l a t e d compounds) f o r  ammonia and amines which would u l t i m a t e l y i n c r e a s e the pH, removed d u r i n g t h e washing p r o c e d u r e .  The p o s s i b i l i t y a l s o e x i s t s  t h a t t h e b u f f e r i n g c a p a c i t y o f the: s a r c o p l a s m i c was  were  extraction buffer  s u f f i c i e n t t o compensate f o r any i n c r e a s e i n pH t h a t r e s u l t e d  from b a c t e r i a l m e t a b o l i s m d u r i n g growth.  /53  By muscle slimy  the  appearance of  slime  as  growth.  formation  8  both  appearance.  intact Jay  the  loss  Ingram  of  muscle  and D a i n t y  o c c u r r e d when b a c t e r i a l  .  No e v i d e n c e o f  washed c o n t r o l  inoculated muscle different more  from  intense  tissue.  indole  and washed  (1970)  inoculated  reported  that  the  bacterial  integrity  (1971)  as  a  reported  numbers  were  result that  odour  and  approximately  low molecular The  difference  weight in  precursors  (intact dog"  both  evident day  "wet  (1967)  stated  throughout  intensity  the  Influence  Results  of  inoculated muscle  the  in  the  retained the  was  intact  their  sulfide,  with and  other  proteins".  and  washed  a reduction The  much  washed muscle  amino acids  in  aseptic  putrid controls  characteristic  "wet  experiment.  on Total  carbohydrate  samples are  the  odour  associated  washing procedure.  total  washed  "ammonia, hydrogen  that  e x p l a i n e d by  of Growth of V. fragi  and  than to the larger  of  intact  study.  The p u t r i d  than  between  duration  either  characteristically  o r i g i n to the free  the  intact  compounds usually  s a m p l e s may be during  was  tissue  compounds rather  odour  present  dog" odour.  and washed muscle t i s s u e )  odour  in  6 that  muscle  foul-odour  inoculated muscle odour  was  intact  meats owe their  was s e e n i n the  initial  J a y and Kontou  formation  samples during  samples by  the  and other  spoiling  odour  the  in  slime  muscle  A putrid  and  of  2  10 / c m  D.  surfaces  s p o i l i n g m u s c l e was due t o  as w e l l  microbial  or  6,  samples had a s l i m y  coalescence of  day  presented  Carbohydrate  Content  analysis  in  in  6 and F i g u r e  Table  the  control 10.  TABLE  6:  The and  e f f e c t o f P . fragi on t h e t o t a l c a r b o h y d r a t e c o n t e n t o f s a r c o p l a s m i c r e d u c e d (washed) b o v i n e m u s c l e s a m p l e s .  Total Sample  Intact  0  control  Washed c o n t r o l  3  +  1. l  3.9  +  10.2  a  c a r b o h y d r a t e c o n t e n t (mg/g d r y w e i g h t Incubation period (days) 6  15. 5 + 0. 9  0. 2  3 , . 8 + 0 ., 2  4 ,, 0 + 0 . 5  3 . , 5 + 0 . ,2  3. 9 + 0. 2  8 , , 0 + 2 . ,1  7. 4 + 0. 6  3 ,, 6 +  3 . 1 + 0. 1  +  2. 2  1 2 , , 7 + 0 ,, 9  1 0 ,. 4 + 1 . 7  Washed  inoculated  4.1  +  0. 6  3 , , 3 + 0 ., 5  3 ,, 3 + 0 . 2  Standard  readings  deviation  12  1 3 , .1 + 0 . , 9  14.4  8  9  tissue)  1 4 ,, 2 + 0 . 9  inoculated  of  muscle  1 3 , , 9 + 2 ., 4  b  Intact  Average  intact  0 . ,2  /55  Washed c o n t r o l  'O  3  A  Washed i n o c u l a t e d •  •  6 Incubation period  FIGURE 10:  a  9 (days)  T o t a l c a r b o h y d r a t e content o f c o n t r o l and i n o c u l a t e d , i n t a c t and washed muscle samples s t o r e d at 4 ° C . Each p o i n t r e p r e s e n t s the mean o f 8 d e t e r m i n a t i o n s .  12  /56  The a b i l i t y (i.e.. the  describe  specific  and t h e i r  include  glycogen,  (1977)  spoilage  of  could not  studied  beef,  the  mean v a l u e .  to  a site  treatment  the  al.  variation  the  total  glycogen  sources by  standard  inoculated  9,  variability  within  the  and b ) ,  the  encountered to  extracted.  the  majority  for 6)  well  Gill  and  bacterial  as w e l l  as  of  within  the  protein  content  The  authors  found  than  a  analysis.  problem  between  treatments.  approximately  was r e m o v e d d u r i n g the  all  attributable  used for  a similar  for  a p p r o a c h e d 27%  muscle,  strip  greater  of  as  some s a m p l e s  c o u l d be  day 0 r e v e a l e d that  carbohydrate :;fraction  would  determined  Table  determine  at  data  1978).  many  glucose-6-phosphate  were  total  same m u s c l e  attempts  that  with  Pseudomonas.  deviation  day  since  meat  during  and  values  of  was  easily  utilization  s a m p l e s was  indicating  in  and Newton,  between  procedure,  reacts  carbohydrate"  (Gill  that  extreme  in  utilization  impossible  1956)  and i n o c u l a t e d m u s c l e homogenates.  Analysis 70% o f  the  within  (1970a  was  al.,  carbohydrate  variability  variability  control  that  site  et  "Total  substrate  of  The  carbohydrate  g l u c o s e - 6 - p h o s p h a t e .and r i b o s e  (intact  variability  H a s e g a w a et  (Dubois  carbon  total  magnitude  of  to  as  of  being utilized)  and r e p o r t e d  treatments  of  the  be u t i l i z e d  the  nature  intermediates  within  location  reagent  glucose,  Although samples,  exact  derivatives.  some g l y c o l y t i c  Newton  the  carbohydrate  phenol-sulfuric  sugars  as  to  total  the  washing  carbohydrate  /57  The i n t a c t a s e p t i c c o n t r o l showed a g e n e r a l i n c r e a s e i n t o t a l c a r b o h y d r a t e d u r i n g t h e experiment, p r o b a b l y due t o t h e p r o d u c t i o n o f r i b o s e from adenosine t r i p h o s p h a t e (ATP) d u r i n g postmortem s t o r a g e ( H u l t i n , 1976)  ( F i g u r e 11).  c a r b o h y d r a t e was  In c o n t r a s t , a d e c r e a s e i n t o t a l  seen i n t h e i n t a c t i n o c u l a t e d samples.  Gill  (1976)  and G i l l and Newton (1977) r e p o r t e d the use o f g l u c o s e as a carbon source by a Pseudomonas i n beef.  The d e c r e a s e i n t o t a l c a r b o h y d r a t e observed i n t h e i n t a c t  i n o c u l a t e d sample may samples.  spp. d u r i n g t h e i n i t i a l s t a g e s o f s p o i l a g e  be r e l a t e d t o t h e i n c r e a s e i n pH o f t h e s e  The amino a c i d c a t a b o l i s m o f b a c t e r i a i s known t o be  a f f e c t e d by t h e presence o f a v a i l a b l e c a r b o h y d r a t e s - t h e more c a r b o h y d r a t e i n t h e medium, t h e l o w e r t h e amount o f ammonia produced (Gardner and S t e w a r t , 1966).  Jacoby  (1964) found t h a t i n t h e p r e s e n c e  of g l u c o s e and o t h e r energy s o u r c e s , amino a c i d c a t a b o l i s m i n Pseudomonas fluoresoens  was  l a r g e l y repressed.  Both washed c o n t r o l and washed i n o c u l a t e d muscle samples remained  r e l a t i v e l y constant during incubation although a  slight  decrease i n t o t a l c a r b o h y d r a t e was n o t e d i n t h e i n o c u l a t e d sample. The s t a t i c c o n d i t i o n o f t h e washed . c o n t r o l samples may  be e x p l a i n e d  by assuming t h a t t h e a u t o l y t i c enzymes as w e l l as t h e p r e c u r s o r s f o r r i b o s e were removed d u r i n g washing t h e r e b y p r e v e n t i n g any i n c r e a s e i n t o t a l carbohydrate.  Any d e c r e a s e i n t o t a l c a r b o h y d r a t e noted  i n t h e washed i n o c u l a t e d sample may by P. fragi procedure.  be a t t r i b u t a b l e t o t h e u t i l i z a t i o n  o f r e s i d u a l c a r b o h y d r a t e not removed d u r i n g t h e  washing  2ATP  -E^  .  2ADP  IMP  AMP  ^ ! Deaminase  *  |  Nucleoside Hydrolase  ^  n o s i n e  FIGURE  11:  R  j  b  o  A  P h  s  e  +  ^ y' Kinase  »  °P  »  e n  h a t a S e  H y p o x a n t  Conversion of  ATP t o  in  meat.  postmortem  q t e  hi  ribose  ATP  +  AMP  Inosine  n e  and  hypoxanthine  /59  E.  The Effect Nitrogen  of P. fragi (NPN)  soluble during and  protein  Figures  12,  The  the  fractions  that  of  contained  in  followed  reported  by  80% o f  (intact  control  inoculated  6 days  after  which  8 and  (NPN) a n a l y s i s  (Table  7,  Figure  this  fraction  that  increase. working  This  fragi.  explain  the  increases  in  The  after  fractions  in  be  from  indicative  postulated  that  initiation  of  the of  of  of  the  12)  during  NPN was  decrease in  NPN f o r  The i n c r e a s e i n rate  the  of  rate  of  the  and  inoculated muscle R a m p t o n et material  The first  NPN w o u l d  production utilization may  NPN c o r r e s p o n d e d  water-soluble  bacteria.  were  and u t i l i z a t i o n  decrease in  nitrogenous  non-protein  porcine muscle.  day 6 the  proteolysis.  by  most  with  production  the  intact  non-protein  proteolysis  in  latter  extractability  protein turn  inequality  decrease.  9  0.6% N P N , w a s h e d  an i n c r e a s e i n  a decrease occurred. however,  was r e m o v e d  Similar results  NPN c o m p o n e n t s may h a v e b e e n e x c e e d e d b y  b y P.  samples  7,  showed an i n i t i a l  sample e x h i b i t e d  proteolysis,  and i n o c u l a t e d  fraction,(sarcoplasm).  (1970a)  al.  salt-  Tables  samples contained  a slight  B o r t o n et  intact  indicate  by  control  and  in  0.1% N P N ) , i n d i c a t i n g  intact  Proteins  watervsoluble  are presented  nitrogen  water-soluble  nitrogen  from  Non-Protein  respectively.  approximately  the  The  14,  non-protein  contained  nitrogen,  extracted  incubation  13 a n d  of  and Salt-Soluble  non-protein  washing procedure  samples  of  of  the... 12 d a y s  revealed  Water-Soluble  3  Results  on the Extractability  to  salt-soluble sample which  al.  may  (1970)  may be r e q u i r e d  for  TABLE  7:  T h e e f f e c t o f P. fragi on t h e s a r c o p l a s m i c r e d u c e d (washed)  Non-protein nitrogen  non-protein nitrogen of bovine muscle samples.  content  (mg n i t r o g e n / g  Incubation  0  Sample  control  5.86  Washed c o n t r o l  1.57  Intact  inoculated  5.51  Washed  inoculated  1.05  Intact  Average  'Standard  of  duplicate  deviation  a  3  period  intact  dry  and  weight  muscle)  (days)  6  12  9  5,,18  +  0.,06  5.,21  +  0.,02  5,.35  0,,04  5,.43  +  0.,07  + 0.,02  1,,23  +  0.,05  1,,20  +  0.,00  1,.43 + 0.,00  1,.16  +  0.,02  +  0. ,04  5,,71  +  0.,11  6. ,41  +  0.,11  5,.79  +  0.,13  5,.64  +  0.,08  +  0. ,00  1,.09  +  0.,02  1,.22  +  0, ,02  1,.40  +  0.,05  1,.30  +  0, .05  +  readings  0. , o o  b  +  /61  °-°0  3  6 Incubation  FIGURE  12:  period  9  12  (days)  N o n - p r o t e i n n i t r o g e n content o f c o n t r o l and i n o c u l a t e d , i n t a c t and washed m u s c l e samples s t o r e d at 4 C. E a c h p o i n t r e p r e s e n t s t h e mean of duplicate determinations.  /62  No d r a s t i c washed c o n t r o l decrease any  NPN.  in  the  However,  during  the  the  in  condition  during  the  the  technique  muscle  easily  removed,  to  myofibrillar  cores,  to  found  however,  Tu  remove that  the  proteins)  any  this  in  experienced:'in  sample would  8,  would  be  to  13)  removed  a repetitive  imbibition-  sarcoplasmic proteins of  the  removed  processes.  Figure was  from  s a r c o p l a s m was  bound r e l a t i v e l y (total indicate  a substantial  be  enzymes,  autolytic  fraction  using  tend  of  inoculated  catheptic  (Table  "apparently  would  a  and u t i l i z a t i o n  1975)  al.,  analysis  (1973),  of  processes.  preventing  50% o f  slight  signify  change  source of et  the  The absence  washed c o n t r o l  major  a  since both  any  Results obtained  washing procedure used r e s u l t e d  strongly  carbohydrate, that  the  decrease in  water-  components. Intact  water-soluble results  that  another,  a majority  some was  matrix".  NPN a n d w a t e r - s o l u b l e  soluble  proteolysis  exists  the  protein 40 t o  sample.  autolytic  thus  washing procedure.  intact  samples, although  sarcoplasm (Forrest  approximately  centrifugation  the  of  lysosomes, the  washing procedure,  that  one  s a m p l e s was d u e t o  The w a t e r - s o l u b l e revealed  also  either  NPN c o u l d p o s s i b l y  between b a c t e r i a l  static  experienced in  washed i n o c u l a t e d  washed i n o c u l a t e d  since the  reside  NPN were  washed i n o c u l a t e d  samples p a r a l l e l l e d  The expected  in  the . p o s s i b i l i t y  inoculated  which  in  equilibrium  and c o n t r o l the  the  was n o t e d  change  dynamic  or  changes  were  control  protein reported  samples showed a g r a d u a l  fraction by  during  incubation  several authors  decrease in (Figure  ( O c k e r m a n et  13).  at.,  the Similar  1969;  TABLE  8:  T h e e f f e c t o f P. fragi on t h e w a t e r - s o l u b l e p r o t e i n c o n t e n t and s a r c o p l a s m i c r e d u c e d (washed) b o v i n e m u s c l e s a m p l e s .  Water-soluble Sample  Intact  0  control  Washed c o n t r o l  134.5  a  + 0 ., 9  b  protein  c o n t e n t (mg p r o t e i n / g d r y Incubation p e r i o d (days)  3  6  of  weight  intact  muscle  9  tissue)  12  1 0 4 . .1 + 2 . , 3  1 0 5 . . 2 . + 0 .. 7  1 0 8 . 6 + 0 ,. 7  9 3 .. 8 + 4 ,. 2  76.3  + 0 .. 4  5 4 . . 3 + 1 .. 8  4 6 . . 0 + 3 .,7  5 3 . 3 + 0 .. 9  1 7 ., 8 + 2 ,. 9  Intact  inoculated  155.8  + 0 ., 7  1 0 4 . .1 + 0 . , 9  1 0 2 .,8 + 2 . 2  1 5 4 . 5 + 1 . .1  1 5 5 . . 9 + 1 .. 2  Washed  inoculated  65.3  + 1. 8  5 5 ..5 + 1. 8  4 8 . ,1 + 1 . 1  4 9 . 1 + ; 1 .. 3  1 9 . , 9 + 0 ., 7  Average  'standard  of  triplicate  deviation  readings  /64  FIGURE  13:  Water-soluble inoculated, stored mean o f  at  protein  intact  4 C.  content  Each point  triplicate  of  control  and washed muscle represents  determinations.  and  samples the  /65  Borton P.  et  al.,  fragi.  1970a;  Bodwell  to  increase  during  storage.  in  Intact water-soluble  The  initial  sarcoplasmic protein  in  involved.  increase  Although  it  in is  extractability salt-soluble previously insoluble  protein  samples  fraction  al.,  during  possible  that  the  insoluble proteins)  the  possibility  in  water  were  not  the  non-soluble .fractions  1970a).  Goll  stromal in  the  was p r o b a b l y  (Mcintosh, et  proteins  al.  1967;  (1970)  may c a u s e t h e  a gradual  protein  also  that  exists  decrease  values  increased.  the  O c k e r m a n et  al.,  that  the  appearance of  new  of  that for  experiment.  increased  the  complexed  with  that  proteins and  urea-  in  enhanced s o l u b i l i t y of  in  proteolysis  increase  action  a  muscle  urea-soluble  The  the  the  were  found  accounted  of  have  1963;  also possible  stages  also  to  by  initial  may  large  1969;  numbers  Borton  proteolysis soluble  of  of  of  et  al., the  peptides  fraction.  Both washed c o n t r o l showing  due  postulated  sarcoplasmic protein  patterns  is  solubilized.  porcine  bacterial  (salt-soluble,  protein  microorganisms  an  proteolysis  proteins  extractable initial  it  latter  bacterial  water-soluble  proteins,  however,  which  1970a);  fraction  (1971), aseptic  from  by  (Sharp,  P r o t e o l y s i s may h a v e  protein  of  resulted  muscle  enzymes  exhibited after  of  protein  catheptic  Tarrant  ( H a s e g a w a et  was  by  al.  1964).  d e c r e a s e may h a v e  fraction  spoilage  water-soluble  autolysis  inoculated  the  autolysis  the  during  et  and P e a r s o n ,  slight  the  1974)  Any decrease i n  been a t t r i b u t a b l e  this  Sage,  and  inoculated  decrease  in  the  samples  exhibited  water-soluble  similar  protein  /66  fraction latter  for  the  first  washed  were  observed,  s a m p l e h a d no  changes  that  did  various change  occurred  l i t t l e  autolysis.  other  authors  degrees  of  decreased protein remained Higher  content: of  the  in  intact  the  (Borton  et  salt-soluble  slightly  of  due  protein the  in  first  the  v a l u e s were  contamination 1971;  stated  generally  observed u n t i l the  after  no  bacterial  salt-soluble  sample  Dainty  protein  9 days  centrifugal  force  reported" by  Tarrant  et  (10  et  Sage, to  6 days  in  have  very  although  storage,  of  sample  after 2.5,  storage the  varying  amount  inoculated  associated with  l i t t l e  reported  The  the  the  control  (1971),  al.  approximately of  the  which and  the  then  period.  higher  CEU/cm ).  Several authors  1975;  and Newton,  numbers fraction  29,000  of  of  for  indicating  change  intact  any  Relatively  autolysis.  the  and t h a t  fraction  storage  et  the  nature.  14.  1974)  remainder  significant  al.  Tarrant  of  levels  2  al.,  incubation  from  during  a factor  throughout  inoculation  in  Figure  any d r a s t i c  1970a;  al.,  increased by  and D a i n t y , that  that  The  extractability  extractability,  9,  control  results  during  extractability  bacterial  similar  salt-soluble  Table  The absence o f  with  constant  the  9 of  a decrease occurred.  suggest  on p r o t e i n  depicted  in  content  Since  would  decrease, presumably  extractable  which  o c c u r may h a v e b e e n a u t o l y t i c  samples i s  agreement  this  effect  The p r o t e i n  in  after  d e c r e a s e was u n e x p l a i n a b l e .  patterns  is  9 days,  Gill  changes i n 8  e x c e e d e d 10 / c m in  the  required xg  (1971)  protein 2  to  intact  1977)  content  inoculated in  Similar  attempts  have  Extraction  an i n c r e a s e  40,000 x g .  during  .  (Ingram  to  the  were  of  muscle relative  results  separate  were the  TABLE 9:  The e f f e c t o f P. fragi on the s a l t - s o l u b l e p r o t e i n content o f i n t a c t and s a r c o p l a s m i c reduced (washed) bovine muscle samples.  S a l t - s o l u b l e p r o t e i n content (mg p r o t e i n / g d r y weight muscle I n c u b a t i o n p e r i o d (days) Sample  0  Intact control  87. i5  + 3,. l  3  6  9  tissue) 12  95,.2 + 3,.9  105,.4 + 1,.6  101,.2 + 3.1  99,.9 + 2,.6  82,.9 + 1,.0  100.,7 + 0,.7  103,.2 + 0.,4  109,.3 + 1.8  99,.4 + 0.,9  Intact inoculated  130.,3 + 1.,8  99.,2 + 3.,9  98.,3 + 0.,2  259.,2 + 1.1  256.,6 + 4.,6  Washed i n o c u l a t e d  112..6 + 1.,1  101. 8 + 0.,3  90.,6 + 2.,2  112.,6 + 2.8  83.,5 + 1..2  Washed c o n t r o l  "Average o f t r i p l i c a t e Standard  deviation  a  readings  b  /68  FIGURE  14:  S a l t - s o l u b l e protein content of control i n t a c t and washed muscle samples s t o r e d p o i n t r e p r e s e n t s t h e mean o f t r i p l i c a t e  and i n o c u l a t e d , at 4°C. Each determinations.  /69  residual jnyofibrillar porcine muscle force  inoculated  was r e q u i r e d  control  or  (Davey  of  for  the  the  intact soluble  protein  sample.  Borton  insoluble water-  et  at.  w i t h P.  the  same.  processes.  level  the  The  may i n d i c a t e  growing  It  on the  is  detected  with  in  the  to  present  the  in  An  washed  increased  Weber-Edsall solution protein  salt-soluble  protein  in  centrifugal  control,  study.  salt-soluble  may  have  extractability  during  a study  reported with  of  amount  the of  a subsequent  salt-soluble  an extreme did  also possible that  (control  in  to  microbial  numbers  was  the  were  in  corresponding muscle  amount  and  of  of  fraction. inoculated)  to  P.  Dainty  of  of  approximately  was b e l o w  that  excess  NPN  a g a i n due  be d e t e c t e d . reported  the  water-  inoculated  population  washed i n o c u l a t e d muscle t i s s u e proteolysis  the  and t h e  o c c u r was the  the  porcine the  protein  change  in  in  increased solubility  fractions  washed samples  change t h a t  of  a decrease in  protein  fraction  increase in  (1970a),  nitrogen  any  the  incubation)  slime-inoculated beef,  until  (intact  6 days  absence of  that  treatment  (after  fragi,  necessary for  working  the  and s a l t - s o l u b l e  that  No i n c r e a s e  fragi.  sample corresponded to  Examination revealed  other  the water-soluble f r a c t i o n  1968).  fraction  protein  P.  myofibrils  increase in  inoculated  inoculated  any  increase in  and G i l b e r t , The  the  for  from  with  washed i n o c u l a t e d )  permeability accounted  proteins  the et  x  autolytic  fragi  al.  proteolysis 3.2  sample  critical (1975), was  9 2 10 / c m .  not  /70  F.  SDS-Polyacrylamide In  order  concentration  of  film  to  dorsi,  cassette,  dodecyl  to  evaluate  utilize  various  Analytical  sulfate  (LDS)  Sweden)  were  SDS-polyacrylamide appreciable  on days  0,  gel  intact  6 and  weight  disaggregating the  determination (Williams  proteolysis chain)  of  autolysis  of  or  of  of  of  Of  protein  et  techniques  Palo  the  on t h e  Alto,  ( Y a d a et  of  bovine  (ACI  agarose  Lithium 1979);  al.,  Pharmacia Fine  (SDS-PAGE),  tested,  Chemicals,  only  exhibited  SDS-PAGE  samples  of  CA;  techniques  bands.  the  ability  fractions  PAA 4 / 3 0 ,  SDS-PAGE  1972).  was  conducted  (control  samples  1971).  for  Due t o  and Osborn,  cleavage of  the  due  simply  The u s e  of  1969)  SDS-PAGE weight the it  a terminal  proteins  proteolysis  is  3-mercaptoethanol,  monomer m o l e c u l a r  subunit  in  in  al.,  (urea,  protein  the  (e.g.  electrophoresis  gel  electrophoresis  (Weber  (Weber  the  Inc.,  electrophoresis  proteins  and G r a t z e r ,  i n i SDS-PAGE  protein  electrophoretic  gradient  a decrease in  components  and washed muscle  agents  preparation  of  and  on  inoculated)  12.  Mobility molecular  effect  various  attempted.  resolution  fractionated  the  Chemists  gel  Pharmacia polyacrylamide Uppsala,  Electrophoresis  muscle water-soluble  Pseudomonas fragi Longissimus  Gel  to  of  absence of  fragi.  to  denaturing  their  and  as w e l l  as  SDS)  results  in  the  subunit  apparent  +10%  accuracy that  amino  a  acid  The use  from either of  SDS m a y h a v e p r o v e n  in  proteins  may be p o s s i b l e  may be u n d e t e c t a b l e P.  related  slight  polypeptide during  conventional to  be  a  relatively  more  sensitive  separation  of  polypeptide is  proteins  (Thorun  conducted with  should not geneity to  technique  the  the must  be  is  or  same p o s i t i o n  be  protein  taken  and dye  is  c a u t i o n must  be  different  the  gel  applied  was n o t  (Figure  muscle  washing procedure soluble  protein  protein  extracted  applied  intensity. nitrogen  to  day  patterns  (Figure  were  and thus  interpreting  selective  inoculated) 16)  protein  homo-  may  migrate  1971).  of  complex  electrophoresis band  characteristics  since the  the  a single  evidence of  and M a u r e r ,  In  protein  gives  only  addition,  quantitation  formation  1977).  since  between semi-  Therefore,  electrophoretic  some  results.  proteins  Comparison of  banding  appearance of  (Thorun  of  whether  a s a c o m p a r a t i v e means  when  proteins,  and s i z e  However,  (Bosshard and D a t y n e r ,  Water-soluble  similar  SDS, the  non-stoichiometric,  results  procedure  on c h a r g e  1971).  consideration  quantitative  1.  in  changes w i t h i n  as u n e q u i v o c a l  with  intensity  into  based both  without  interpreted  band  detecting  and Maurer,  since proteins  use of  in  15)  0 samples i n d i c a t e d in  were  from  the  It  effective  fraction  did, in  both  These r e s u l t s  intact as  support  indicated  since  both  intact  (control  and  volumes  of  of  the  the  and washed m u s c l e  those  the  that  in  the  water-  water-soluble samples  decreased  obtained  a substantial  and  inoculated)  indicate  removal  evidenced by  washing  proteins  however,  the  the  of  (control  (equivalent  gels),  analysis which  removal  observed in  and washed  samples. was  the  that  staining  protein  decrease in  the  Ill  Day  Apparent molecular wei ght  Intact control  Intact inoculated  Apparent molecular weight  106,000 81,000 70,000 55,000 47,000 37,000 27,000  14,000  106,000 81,000 70,000 55,000 47,000 37,000  i  27,000  14,000  106,000  116,000  81,000 70,000 55,000 47,000 37,000 27,000  14,000  FIGURE  15:  Electrophoretograms of water-soluble proteins e x t r a c t e d from i n t a c t c o n t r o l and intact i n o c u l a t e d muscle samples at day 0, 6 and 12.  Day  Apparent molecular weight  Washed control  Washed i noculated  106,000 81,000 70,000 55,000 47,000 37,000 27,000  14,000  106,000 81,000 70,000 55,000 47,000 37,000  . .....  27,000  14,000  106,000 81,000 70,000 55,000 47,000 37,000 27,000  FIGURE  16:  Electrophoretograms of water-soluble proteins e x t r a c t e d f r o m washed c o n t r o l and washed i n o c u l a t e d muscle samples at days 0, 6 and 12.  /74  amount of water-soluble  protein i n the washed muscle samples  as compared to the intact muscle tissue. polyacrylamide  Tu (1973) used  disc gel electrophoresis to examine the effect  of a r e p e t i t i v e imbibition-centrifugation procedure for the removal of the water-soluble  protein f r a c t i o n from intact bovine  muscle and reported a non-specific, uniform reduction of sarcoplasmic  proteins during the procedure.  Comparative analysis of day 6 electrophoretograms (Figures 15 and 16) indicated no apparent difference i n the number or pattern of bands i n either the control or inoculated samples for both intact and washed muscle tissue. By day 12, although b a c t e r i a l numbers were approximately 10  10  and 10  9  CFU/cm  2  for the intact inoculated (Figure  15)  and washed inoculated (Figure 16) muscle samples, respectively, v i r t u a l l y no proteolysis had occurred i n either sample. evidence of proteolysis was  The  only  the appearance of a band with an  apparent molecular weight of 116,000 i n the intact inoculated muscle sample.  Dainty et al.  (1975), using SDS-PAGE, reported  that proteolysis was not detected i n the sarcoplasmic  proteins  extracted from slime inoculated beef, even though b a c t e r i a l numbers 10 had reached 10  2 CFU/cm .  In.rcontrast, Hasegawa et al.  (1970a)  reported extensive a l t e r a t i o n s i n the starch-gel electrophoretic pattern of the sarcoplasmic  proteins extracted from rabbit and  porcine muscle inoculated with P. f r a g i .  The authors reported  the  /75  loss of 70 to 80% of the bands i n the pattern due to proteolysis caused by P. f r a g i .  The apparent discrepancy  i n the extent of  proteolysis reported by the above two studies, could possibly be attributed to a myosystem s p e c i f i c i t y displayed by P.  fragi.  It may, however, also be the result of the type of electrophoresis used to detect the changes within the proteins, in that SDS-PAGE may be much less sensitive i n i t s a b i l i t y to detect proteolysis as compared to starch gel-electrophoresis. It i s possible that s i m i l a r situations were encountered i n the present study. No changes were apparent i n the day 12 controls (Figures 15 and 16).  This would indicate that autolysis was minimal during  the 12 days of incubation.  Doty and Wachter (1955) reported  that a 1.6 Megarad dose of gamma r a d i a t i o n reduced the p r o t e o l y t i c a c t i v i t y of muscle cathepsins by approximately 50%. 2.  Salt-soluble  proteins  Electrophoretograms 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 extracted from intact and washed muscles are presented i n Figures 17 and 18, respectively.  Tentative i d e n t i f i c a t i o n of some  of the m y o f i b r i l l a r proteins was made (Figures 17 and 18). Examination of the day 0 electrophoretograms indicated no apparent differences i n banding pattern between intact (Figure 17) and washed (Figure 18) muscle samples.  This would suggest that the  washing procedure used did not result i n m y o f i b r i l l a r protein  /76  Day  Apparent molecular weight  Intact control  Tentative identification  Intact inoculated  Apparent molecular weight  163,030 126,000 108,003 75,030 55,000 43,000 39,003  • Actin Tropomyosin  H  25,000  T r o p o n i n s and Light myosins  21,000 17,000  163,000 126,003 108,000 75,003 55,000 45,003 39,000  M •  Actin Tropomyosin  •  10,020  25,000 21,000 17,000  • 187,000  163,00C  loelooc  •  75,000  - K t l n l n •  I  Hf l 0  55,000 48,000 39,000  H I  44,000 _  21.000 17,000 13,000  FIGURE  17:  Electrophoretograms of salt-soluble proteins e x t r a c t e d from i n t a c t c o n t r o l and intact i n o c u l a t e d m u s c l e samples at day 0, 6 and 12.  Ill  Day  Apparent molecular weight  Washed control  163,000  Tentative identification  Washed inoculated  Apparent molecular weight  mm  126,000  mm  108,000  mm  75,000  I  ,,-actinin  H  55,000 48,000 39,000 25,000 21,000 17,000  •  Actin Tropomyosin Troponins and Light myosins  163,000 126,000 108,000 75,000 55,000 48,000 39,000  M •  Actin Tropomyosin  40,000  25,000 21,000 17,000  163,000 126,000 108,000 75,000 55,000 48,000  H  39,000  •  25,000 21,000 17,000  FIGURE  18:  Actin WM  35,000 40,000 28,000  Electrophoretograms of salt-soluble proteins e x t r a c t e d from washed c o n t r o l and washed i n o c u l a t e d muscle samples at day 0, 6 and 12.  /78  denaturation.  P r e v i o u s work (Tu, 1973)  of sarcoplasmic f l u i d  indicated that  from bovine muscle by  c e n t r i f u g a t i o n and i m b i b i t i o n w i t h 0.9% m y o f i b r i l l a r denaturation.  No  removal  prolonged  NaCl may  result in  changes were observed  i n 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 o f 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 e i t h e r the i n t a c t c o n t r o l  ( F i g u r e 17) and washed c o n t r o l  ( F i g u r e 18) d u r i n g the 12 days o f i n c u b a t i o n . By day 6, the appearance o f a new an apparent m o l e c u l a r weight  o f 40,000 was  band below a c t i n w i t h e v i d e n t i n both  ( F i g u r e 17) and washed ( F i g u r e 18) i n o c u l a t e d samples. changes i n banding p a t t e r n were e v i d e n t i n e i t h e r i n o c u l a t e d o r washed i n o c u l a t e d  No  intact other  intact  electrophoretograms.  At day 12, e x t e n s i v e a l t e r a t i o n s i n the banding p a t t e r n o f the i n t a c t  i n o c u l a t e d sample ( F i g u r e 17) were observed.  i n components w i t h apparent m o l e c u l a r weights and 83,000 were e v i d e n t .  The  l a t t e r two  been breakdown p r o d u c t s o f a - a c t i n i n  Increases  o f 187,000, 92,000  components may  have  (apparent m o l e c u l a r  weight  of1108,000) s i n c e the i n t e n s i t y o f t h i s band seemed t o have decreased.  An extreme breakdown i n the i n t e n s i t y o f the band  r e p r e s e n t i n g a c t i n o c c u r r e d w i t h a subsequent h a v i n g an apparent m o l e c u l a r weight  o f 44,000.  obscured the presence o f tropomyosin;  i n c r e a s e o f a band The  tropomyosin  been i d e n t i f i e d w i t h an apparent m o l e c u l a r weight change i n banding p a t t e r n was  l a t t e r band  had  originally  o f 39,000.  A  a l s o noted i n the lower m o l e c u l a r  /79  weight  proteins  weights that  of  31,000  proteolysis  intact of  where  P.  and of  protein  the  showed a  indicating  from  loss  that  this  upon m y o f i b r i l l a r  20 d a y  in  pork  incubation  as  with  evident. changes  at  in  level  the of  3.2 x  was n o t  in  10 /cm . 9  2  bands  growth  on  electrophores  some p r o t e o l y t i c  effect  (1971),  disc  al.  using  salt-soluble  banding pattern  were  the  sample,  intact  weights  changes were  of  35,000  protein  during  a  fraction  until  and  reported  extracted  lack  of  suggest  that  numbers  extensive that  from  (1975),  al.  from  extensive  were  numbers  et  the reaching using  proteolysis slime  were  for  New b a n d s  28,000  The  bacterial Dainty  as  sample.  sample would  bacterial  also noted  were not  apparent.  proteolysis.  protein  these  inoculated  gel-electrophoresis,  detected  the  Pseudomonas  with  fragi  washed i n o c u l a t e d  the m y o f i b r i l l a r  et  the  salt-soluble  w i t h P.  sarcoplasm restricted  polyacrylamide  beef  exhibited  inoculated  changes  necessary for  that  of  from  10°C.  molecular  No o t h e r  reduction a  muscle  a result  protein  observed considerable  observed for  apparent  of  indicate  extracted  inoculated  Tarrant  washed i n o c u l a t e d m u s c l e those  as  reported  muscle  number  proteins.  Although the  the  organism  gel-electrophoresis, degradation  (1970b)  al.  molecular  The r e s u l t s  proteins  sample occurred  porcine  in  apparent  evident.  salt-soluble  B o r t o n et  extracts  with  27,000 were  inoculated muscle fragi.  fragi  components  in  of  inoculated excess  of  /80  3. ' Urea-soluble  insoluble stromal of  the  muscle  The u r e a - s o l u b l e  fraction  in  solutions  samples  protein  of  in  salt-soluble (Figures  salt-soluble  samples  12 d a y s  revealed  SDS-PAGE  gels No  of  electrophoretograms  (Figure  the  intact  20) By  extracted  change  washed  apparent  the  in  results control  changes  of  patterns washed in  Figures  in  19  20)  19  and  both  in  the  gel  from  the  same  suggest  been  the  17,000  bands  electrophoretic  Similar  the  l  were  apparent  and  extracted  would  e  presented with  to  g  which  that  pattern  the  contaminated  with  some  fraction.  the  no  include  and  (Figures  weight  may h a v e  fraction  incubation.  samples  This  fraction  protein  are  25,000  fraction  protein  proteins  not  intact  characterized  18).  of  from  39,000,  protein  protein  does  inoculated)  molecular  17 a n d  but  those  The e l e c t r o p h o r e t i c  0 muscle  apparent  urea-soluble  either  and  48,000,  day  contains  extracts  Bands  Examination  muscle  1974).  (control  weights  urea-soluble the  (Sage,  and washed  samples  of  salt  respectively.  correspond the  or  urea-soluble  molecular  of  water  proteins  and 20,  intact  proteins  were  patterns  from the  banding were  intact  pattern  observed  of  the  control  during  for  the  day  6  the  samples. observed  urea-soluble  inoculated  gel  (Figure  19)  in  the  proteins or  the  extracted washed  from  inoculated  sample. day. 1 2 ,  electrophoretic  an almost  pattern  of  the  complete  breakdown  urea-soluble  of  protein  the fraction  from  Apparent molecular weight  Intact control  Intact inoculated  Apparent molecular weight  104,000 74,000 64,000 48,000 39,000 25,000 17,000 14,000  104,000 74,000 64,000 48,000 39,000 25,000 17,000 14,000  104,000  187,000 142,000 119,000 104,000  74,000 64,000 48,000 39,000  48,000 43,000  25,000 17,000 14,000  FIGURE  19:  Electrophoretograms of urea-soluble proteins e x t r a c t e d from i n t a c t c o n t r o l and intact i n o c u l a t e d muscle samples at day 0, 6 and 12.  /82  Day  Apparent molecular weight  Mashed control  Washed inoculated  104,000 74,000 64,000 48,000 39,000 25,000 17,000 14,000  104,000 74,000 64,000 48,000 39,000 25,000 17,000 14,000  104,000 74,000 64,000 48,000 39,000 25,000 17,000 14,000  FIGURE  20:  Electrophoretograms of urea-soluble proteins e x t r a c t e d from washed c o n t r o l and washed i n o c u l a t e d m u s c l e samples at day 0, 6 and 12.  /83  the intact inoculated sample was  evident (Figure 19).  Additional  highmoleeular weight proteins (apparent molecular weight of 187,000, 142,000 and 119,000) that were not previously characterized were noted.  This would suggest that very high mole-  cular weight proteins (of such high molecular weight that they were unable to enter the gel) were being degraded to lower molecular weight components.  The near complete absence of low  molecular weight components (less than 40,000 molecular weight) was  also apparent.  Results of the present study would indicate  that the growth of P. fragi-  caused considerable breakdown of the  urea-soluble proteins i n the intact inoculated muscle sample. Hasegawa et al.  (1970a) studied the effect of P. fragi  on the  urea-soluble proteins extracted from porcine and rabbit muscle. The authors reported that the electrophoretic pattern was greatly disrupted, indicating considerable protein breakdown.  No changes  i n the gel pattern of the urea-soluble protein f r a c t i o n extracted from the day 12 washed inoculated sample were evident.  4.  Urea-insoluble  proteins  The f i n a l f r a c t i o n of the protein extraction procedure was the urea-insoluble protein fraction.<  It was  this f r a c t i o n represented the stromal proteins.  assumed that The  electro-  phoretograms of the urea-insoluble protein extracts from intact and washed muscle samples (control and inoculated) are presented in Figures 21 and 22, respectively. Examination of the gel patterns  /84  FIGURE 21:  Electrophoretograms of u r e a - i n s o l u b l e proteins e x t r a c t e d from i n t a c t c o n t r o l and i n t a c t i n o c u l a t e d muscle samples a t day 0, 6 and 12.  FIGURE 22;  Electrophoretograms o f u r e a - i n 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 washed c o n t r o l and washed i n o c u l a t e d muscle samples at day 0, 6 and 12.  /86  of  the  urea-insoluble protein  control during  muscle samples the  observed  12 d a y s  for  the  of  sample  indicated  washed c o n t r o l of  protein  indicated  no  the  the  day  alterations  in  the  banding  were  apparent would  et  indicate  Pseudomonas  al.,  1969).  G.  pattern  pattern  complete of  no  the  Gel patterns  of  growth  of  intact  (Figure  pattern  Similar  results  were  22). gel  patterns  day  6  the of to  produce  bands  in  pattern  gels,  no  apparent  of  as  day  the  the  extracted inoculated  however,  changes were  of  (Ockerman  intact  6,  obtained  proteins  proteins  those of  with  The r e s u l t s  collagenases  urea-insoluble like  obvious  Some m e m b e r s  fragi.  However,  extreme  urea-insoluble P.  21).  The most the  the  inoculated  (Figure  revealed  21).  of  intact  and 50,000.  change  inoculated  banding  from the to  intact  in  disappearance of  have been found  obvious  up  169,000  proteolysis of  (Figure  extracted  washed i n o c u l a t e d muscle  indicated  12  that  21).  electrophoretogram  weights  as a r e s u l t  from the  the  almost  molecular  occurred genus  the  12  (Figure  from the  change  electrophoretic  change i n  of  extracted  apparent  gels  fraction  examination  changes  no  incubation  Examination urea-insoluble  fraction  unlike  noted  in  the  day  temperatures  is  generally  In  order  electrophoretograms.  Electron  Microscopy Bacterial  regarded examine  as the  spoilage of  a s u r f a c e phenomenon effects  of  meat  at  (Gill  Pseudomonas fragi  chill  and Penney, on the  1977).  surface  to  ultrastructure  /87  of  b o t h washed and  electron  1.  intact  microscopy  Scanning  muscle  were  electron  were  samples.  taken  were  not  of  at  the  of  are  Perforations  samples tissue were  (arrow  Schaller  Figure  degradation of  the  12 d a y s  and day  appear  from  found  site in  12  on t h e  to  lack  at  day  (Figure  of  25)  incubation  (Figure  24)  only  controls  of  of  deterioration  tissue on the  a SEM s t u d y  of  both  the  muscle  autolytic  same  sample.  skeletal  muscle  ultrastructural  muscle during  inoculated to  although  numbers of  in  the  detectable from  et al.  studying  (1971)",  glass  the  indicative  some s u r f a c e  the  0 failed  microorganisms  to  of  of  of  reveal standard  samples  the  the  commercial  slides,  range  of  10  b a c t e r i a may be  the muscle the  surface  attachment  observed that  during  (intact  presence of  plate  5  The  inoculated  beef.  micrographs  bacterial  the  and  surface  site  dorsi  of  (intact  ultrastructure  w h i c h may be  (1971),  Examination  bacteria  23)  Longissimus  the  transmission  controls  those  Such i n d i c a t i o n s  sources, on  as  surface  during  did  24),  variable  and P o w r i e  from various  the  (Figure  deterioration.  extremely  aging  0  uninoculated  same t i m e  in  micrographs shown.  the  extensive  day  and  microscopy  S i n c e changes  controls  scanning  employed.  Micrographs washed)  samples,  count  10  due  during of the  any  2 CFU/cm  to  washed)  indicated  6 to  and  the  (see Table  removal  fixation.  a marine primary  of  the  Marshall  Pseudomonas stages  3).  of  spp.  FIGURE  23:  S E M micrographs of day 0 , uninoculated intact (a) and washed (b) muscle samples. Collagen and r e t i c u l a r f i b r i l s (arrow) cover the muscle surface.  FIGURE  24:  SEM m i c r o g r a p h s o f d a y 1 2 , u n i n o c u l a t e d intact (a) and w a s h e d (b) m u s c l e s a m p l e s . Areas of t i s s u e d e t e r i o r a t i o n have o c c u r r e d (arrow).  FIGURE  25:  SEM m i c r o g r a p h s o f (a) a n d w a s h e d (b)  day 0 i n o c u l a t e d muscle samples.  intact  /91  attachment, bacteria were held weakly to the surface by such forces as London-van der Waals interactions andncould thus be easily removed. the  McMeekin et al. '(1979), using SEM to examine  microorganisms on chicken skin, found a 10-fold discrepancy  between counts obtained on nutrient agar and those calculated from the density of organisms on a micrograph.  The authors  stated that during f i x a t i o n i n glutaraldehyde, "a scum formed on the surface lipid  of the fixative material  and presumably  which was likely  or material contained  to have been  washed from the surface many  by the  fixative,  microorganisms".  By day 3, limited growth o f P. fragi  was evident on  micrographs of both intact and washed inoculated muscle (Figure 26).  unfixed  samples  The observance of bacteria at day 3 and not at day 0  may be due to the increase i n b a c t e r i a l numbers during incubation, such that the p r o b a b i l i t y of bacteria remaining on the surface during f i x a t i o n would have increased. the  Although not eyident i n  micrographs, i t i s also possible that some slime fibers were  produced by P. fragi  that mediated the attachment of the organisms  to the muscle surfaces.  McCowan et al. (1978) observed that the  " f i n e slime f i b e r s " that mediate b a c t e r i a l adhesion were below the  l i m i t o f resolution o f the SEM and were not seen unless they  were aggregated. After 6 days of incubation, production of what was t e n t a t i v e l y i d e n t i f i e d as e x t r a c e l l u l a r material could be seen on the  surface of the bacteria present on both intact and washed  FIGURE 26:  SEM micrographs of day 3 inoculated intact (a) and washed (b) muscle samples showing crevices (arrow) which may trap bacteria.  /93  inoculated samples (arrow Figure 27).  Marshall et al. (1971)  stated that the mechanism of attachment involved two  consecutive  steps, namely a primary attachment followed by a time-dependent secondary attachment.  During secondary attachment, the strength  of attachment increases due to the production of e x t r a c e l l u l a r a c i d i c polysaccharides by primary attached bacteria (Marshall et al., 1971;  Fletcher and Floodgate,1973).  Deinema and  Zevenhuizen (1972) reported exocellular c e l l u l o s e f i b r i l s on many gram-negative bacteria. of "carbohydrate  McCowan et al.  (1978) reported the presence  coats" surrounding adherent bacteria, which  appeared to mediate the attachment of the bacteria to the epithelium of the reticulo-rumen of c a t t l e , to food p a r t i c l e s , and to each other to form microcolonies. had e a r l i e r termed t h i s carbohydrate  Costerton et al. (1978)  material "glycocalyx".  By day 6, proteolysis of muscle tissue i n areas of b a c t e r i a l growth i n both intact and washed inoculated samples (D i n Figure 27) became apparent. The amount of glycocalyx and surface degradation i n both washed and intact inoculated muscle samples increased during storage.  After 9 days of incubation, i t could be seen from  the micrographs (intact and washed) that progressively more bacteria were attached to each other and not to the muscle surface (Figure 28).  The adherent b a c t e r i a l population was a complex  mass of c e l l s that was  apparently held together by slime f i b e r s  FIGURE  27:  SEM m i c r o g r a p h s o f d a y 6 i n o c u l a t e d intact (a) a n d w a s h e d (b) m u s c l e s a m p l e s . Glycocalyx (arrow) can be seen on t h e s u r f a c e o f b a c t e r i a . Areas of b a c t e r i a l proteolysis are evident on t h e m u s c l e s u r f a c e (D) .  /95  b  FIGURE  28:  SEM m i c r o g r a p h s  of  and washed  (b)  to  together  be h e l d  Large (D).  areas  of  day  muscle by  surface  9  inoculated  samples. slime  intact  Bacteria  fibers  degradation  (a) appear  (arrow). are  evident  /96  (arrow i n Figure 28).  Large areas of surface degradation were  evident i n both intact and washed inoculated samples (D i n Figure 28). After 12 days of incubation, the d i s t r i b u t i o n of the bacteria on the muscle surface was non-uniform and exhibited "intermittent colonization" (McCowan et al., high b a c t e r i a l density (Figure 29). pattern was  A s i m i l a r type of growth  reported by McCowan et al.  colonization of the bovine tongue. :  1978), i . e . areas of  (1979) during a study of  A higher proportion of c e l l s  in a given microenvironment could suggest that the physical and chemical conditions of the environment are more favourable f o r active uptake of nutrients (ZoBell, 1943; Fletcher, 1979).  Stotzky and Rem,  1966;  The microcolony of the washed inoculated  sample was held together by a well defined glycocalyx network while that of the intact inoculated sample was (Figure 29).  This may  amorphous i n nature  have resulted from differences i n available  nutrients (Firstenberg-Eden  et al., 1979).  Although the washed  muscle samples were an adequate growth medium (as indicated by b a c t e r i a l numbers), the majority of the water-soluble  components  were removed, making the washed samples a more d i f f i c u l t ecosystem on which to support  b a c t e r i a l growth.  When provided with complex  and simple nutrient sources, microorganisms w i l l invariably, or always, u t i l i z e the simpler constituents (amino acids, nucleotides, or carbohydrates) p r e f e r e n t i a l l y to the more complex ones such as proteins (Jay and Shelef, 1976).  Costerton et al.  (1978) stated  FIGURE  29:  SEM m i c r o g r a p h s (a)  and washed  of (b)  day  12 i n o c u l a t e d  muscle  samples.  intact  /98  that polymers produced by bacteria may not only p o s i t i o n the bacteria, but also conserve and concentrate and serve as a food reservoir.  the digestive enzymes  ZoBell (1943) found slime  production,  by attached bacteria, to be influenced primarily by the microecosystem.  He observed that more slime was produced i n  ecosystems i n which i t was d i f f i c u l t f o r bacteria to survive. Physical degradation of the muscle tissue i n both intact and washed inoculated muscle samples was extensive i n areas of colonization.  The degradation of proteins, presumably the  stromal and m y o f i b r i l l a r f r a c t i o n s , would explain the changes i n the SDS-PAGE patterns of the urea-insoluble, urea-soluble and salt-soluble protein fractions of the intact inoculated muscle samples a f t e r 12 days incubation.  Borton et al. (1970a and b)  and Tarrant et al. (1971) reported the proteolysis of m y o f i b r i l l a r proteins i n bovine muscle inoculated with P. fragi.  Ockerman et al.  (1969) observed that the stromal protein f r a c t i o n was degraded i n muscle inoculated with a Pseudomonas spp.  Localized degradation  of proteins also appeared i n the washed inoculated samples with no apparent proteolysis as indicated by SDS-PAGE.  It i s conceivable  that because proteolysis was r e s t r i c t e d to localized areas, i t was not detected by SDS-PAGE; b a c t e r i a l numbers were not s u f f i c i e n t to affect the entire muscle mass.  Dainty et al. (1975), studying  the protein changes i n slime inoculated beef, reported that proteolysis was not detected u n t i l microbial numbers were imexcess  /99  9 2 of 3.2 x 10 /cm .  That the b a c t e r i a l population of the washed  inoculated samples never reached this level may  account for the  lack of detectable p r o t e o l y s i s . In addition to a t t r a c t i v e forces, the microtopography of the muscle surface may  play an important  r o l e i n attachment.  Crevices and channels (arrow i n Figure 26) between the muscle fibers were i n most cases larger than the bacteria, thus allowing for physical entrapment. exopolysaccharides.  Once trapped, bacterialcould then produce  Firstenberg-Eden  et ali  (1979), studying the  attachment of bacteria to the teats of cows, reported that b a c t e r i a l adherence may  result from organisms becoming locked i n small holes  or i n skin tissue. et al.  Similar results were reported by McMeekin  (1979). Figure 30a demonstrates the i r r e g u l a r i t y of the muscle  surface.  A closer examination of the area between the muscle f i b r i l s  (A i n Figure 30a) revealed a microcolony  of bacteria which had  produced e x t r a c e l l u l a r substances (S i n Figure 30b). b a c t e r i a l may  By this process  encapsulate themselves i n surface i r r e g u l a r i t i e s ,  thereby making detachment d i f f i c u l t .  Bacterial adherence may  explain the p r a c t i c a l d i f f i c u l t y i n obtaining accurate  estimates  of b a c t e r i a l numbers using non-destructive methods (swabbing and rinsing). 1972;  Several authors  (Avens and M i l l e r , 1970;  Notermans et al., 1975)  Patterson,  observed that viable counts obtained  with macerated samples were always greater than those obtained with swabs or rinses.  /101  2. ' Transmission  electron  Transmission  microscopy electron  appropriate  stains,  the  characteristics  et  surface  1974b;  al.,  bacterial 1973; 1978).  In  ruthenium used  in  and  the red  present  slime  to  Fletcher tetroxide  layer  a polysaccharide-like  located.  Micrographs  ruthenium  reveal  an  contrast,  taken  red  alone  or  electron  dense  area  micrographs  taken  osmium  tetroxide  was u s e d  around  bacterial  cells.  Only was  felt  of  that  samples bacterial  at  active  spoilage  excess  of  sections  12 w e r e  in  has  al.,  with  been  widely  deposits  investigate al.  bacteria  were  from  samples  alone  did  cells.  ruthenium of  (1969),  stained not In  red-  polysaccharide  e x a m i n e d b y TEM s i n c e be b e s t  a  J o n e s et  bacterial which  Using  to  tetroxide  surrounding samples  the  obtained  osmium  M c C o w a n et  1973).  stream,  which  Floodgate,  stained  red  combination  in  of  exemplified  it during  2  (10  9 10  mat  Costerton  polysaccharide-like  and F l o o d g a t e ,  adhesion would  9  TEM w e r e  acidic  showed dense  day  1977;  Ruthenium  stain  and  with about  mechanisms  al.,  for  locate  with  of  1974a;  al.,  Fletcher  C h e n g et  samples  combined  information  and the  from a simulated  detected  with  et  1969;  al.,  tetroxide.  microscopy  red-osmium  a bacterial  et  (TEM)  provide  1975)  al.,  study,  1968;  to  (Costerton  1974;  and osmium  (Luft,  ruthenium  (Jones  Brooker,  electron  material  been used  P a t e r s o n et  adhesion  Fuller  has  microscopy  CFU/cm ).  Although  bacterial  numbers  were  2 CFU/cm  ,  no  bacteria  could  be  seen on the  washed  in  /102  inoculated that  sample.  Owing  can be v i e w e d u s i n g  the  washed muscle  colonization). muscle  to  tissue  (control  mediated This  was  the  held  only  and  the  cellular  polymers primary  a thin.coat  on  adhesion  is  the of  muscle  secondary attached  organisms. for  and  bacteria  the  polysaccharide  32),  was  contact  the  between  surrounding  the  extra-  One t y p e  of  polymer,  Figure The  formed  primary  for  bacterial  or  between  (1973) in  however, of  secondary  substance  that  bacteria  found  that  preparations  associated with  bacteria.  32),  second type,  predominated  the muscle  surface.  of  cell.  of  which  types  and F l o o d g a t e  production  31)  two  in  stage  bacterial  study.  bacteria  initial  intact  SEM  The  polysaccharide,  12  Figure  an amorphous  adjacent  the  in  responsible  1976).  and was u s u a l l y  priorvto  (P  of  here.  muscle  attachment.  polysaccharide  adhesion;  (F  the  bacterial  Fletcher  secondary  occurs  involves  and around  acidic  The  the  day  adherent  from the  initially  Figure  the  demonstrated  and F l o o d g a t e ,  surface.  irreversible  sorption  probably  (S i n  between  of  region  presented  fibers  polysaccharide  surface  (Fletcher  stretched and the  acidic  the  polysaccharide,  staining  the  tissue  bacteria . (intermittent  of  to  muscle  that  the  bacteria  obtained  of  of  are  that  slime  associated with  designated  polysaccharide  red  was v o i d  micrographs  by  the  results  Ruthenium  possible  indicated  of  area  inoculated)  together  attachment  confirmed  is  observed  TEM m i c r o g r a p h s population  limited  TEM, i t  Therefore,  samples  the  groups  was n o t  of necessary  irreversible  secondary surface  polysaccharide and  the  primary  /103  FIGURE 31:  TEM micrograph o f the adherent b a c t e r i a l p o p u l a t i o n o f the i n t a c t muscle s u r f a c e a f t e r 12 days i n c u b a t i o n . Slime f i b e r s (F) mediate c e l l to c e l l as w e l l as c e l l t o muscle t i s s u e adhesion (X 25,000).  /104  FIGURE  32:  TEM m i c r o g r a p h o f a d h e r e n t b a c t e r i a s h o w i n g p r i m a r y (P) and s e c o n d a r y (S) a c i d i c p o l y s a c c h a r i d e s (X 5 1 , 0 0 0 ) .  /105  ZoBell between  a bacterial  concentration promote  the  point  bacteria  (E  or  P. fragi  cell  and  for  Figure  of  the  33)  similar  evaginations,  w h i c h may  contain  contents  the  for  myofibrillar  organisms  disruption.  grown  evaginations.  and p h y s i o l o g i c a l protrusions al.  of  bacteria  actin day  12  resulted  from  cell  wall  they  termed to  filaments  control  postmortem  bovine muscle,  myofibrils  stored  of the sarcomere impossible".  are  did  surface  in  of  the  the  was made  observed  that  these their  responsible  P.  fragi  surface  that  certain  formation  on  of  release  that  contain  found  Dutson  surface  enzymes,  protrusions  that  proteolysis.  on  not  the  a  Bleb-like  subsequently  (1968)  of  nutritional  surface  Smirnova  certain  species  "microcapsules". resolve  evident  in  the  an  electron  13 d a y s  was so severe  at  exists  and  (1967)  "structural  that detailed also  34)  microscopic  and G o l l 2°C,  radial  pattern  a cross-section  sample;(Figure  Stromer  The p o s s i b i l i t y  as  some p s e u d o m o n a d s .  was  In  the  authors  clearly  muscle  autolysis.  for  of  serve  suggestion  induced  cellular  inability  and myosin intact  and Chapman  observed  which  The  The  conditions  on t h e  (1971)  et  and  on n o n - m u s c l e m e d i a Wiebe  on  proteolytic  tissue  attachment  material.  involved  The  of  exo-enzymes  protrusions  muscle.  muscle  may  and  nutrient  may b e  point  surface  observed  on p i g  into  the  both nutrients  reported  growing  that  solid  protrusions  in  (1971)  al.  suggested  assimilation  evaginations  et  (1943)  the  presumably study  reported  of that  in  deterioration  examinations that  of  of  s i n c e no  were micrographs  /106  FIGURE  33:  TEM m i c r o g r a p h  dorsi  muscle  incubated  at  evaginations (X  31,400).  of  intact  inoculated 4°C  for  c a n be  bovine with  P.  12 d a y s . seen on the  Longissimus fragi and Bleb-like cell  (E)  surface  FIGURE  34:  TEM m i c r o g r a p h  Longissimus incubated  of  uninoculated bovine muscle (cross-section) 12 d a y s a t 4 ° C (X 3 3 , 2 0 0 ) .  dorsi for  /108  were taken of the day 0 intact control muscle sample, the lack of r e s o l v a b i l i t y may  have been an a r t i f a c t induced by the f i x a t i o n  procedures. Muscle tissue adjacent to b a c t e r i a l growth tended to be extremely disrupted  (A i n Figures 35 and 36) and presumably  consisted of disintegrated sarcomere components. to t h i s region was  The area contiguous  amorphous and resembled that seen in the  control sample :(U i n Figure 35 and 36).  It i s possible that  proteolysis during b a c t e r i a l spoilage i s limited to an area surrounding the bacteria.  Sage (1974), i n a study of P.  fragi  inoculated chicken p e c t o r a l i s , reported s i m i l a r r e s u l t s and stated that "the average f i b r i l was 0.6 um".  distance  Dutson et al.  between the bacteria  and the  (1971) found an extremely  disrupted appearance i n myofibrils from pig muscle inoculated and incubated with P. fragi uninoculated  controls.  (8 days at 10°C)  as compared to the  The extreme disruption of the myofibrils  adjacent to b a c t e r i a l growth evidenced i n the day 12 intact inoculated sample, would tend to support the electrophoretic results of the s a l t - s o l u b l e protein extract obtained from this sample.  intact  /109  FIGURE 35:  TEM micrograph of intact bovine Longissimus dovsi muscle inoculated with P. fragi and incubated at 4°C for 12 days. Disintegrated regions (A) of muscle tissue separate bacteria from amorphous tissue (U) (X 40,000).  /no  FIGURE  36:  TEM m i c r o g r a p h o f i n t a c t b o v i n e Longissimus dorsi m u s c l e i n o c u l a t e d w i t h P. fragi and i n c u b a t e d a t 4 ° C f o r 12 d a y s . Disintegrated r e g i o n s (A) o f m u s c l e t i s s u e s e p a r a t e b a c t e r i a f r o m a m o r p h o u s t i s s u e (U) (X 4 0 , 0 0 0 ) .  /Ill  GENERAL DISCUSSION Intact bovine Longissimus  dorsi  muscle was  subjected to a  mild washing procedure i n order to reduce the concentration sarcoplasmic  fluid.  with Pseudomonas fragi  of  Intact and washed muscle samples were inoculated to evaluate the effect of  sarcoplasmic  reduction on b a c t e r i a l growth and subsequent spoilage. The reduction of soluble low molecular weight components was  r e f l e c t e d i n a s i g n i f i c a n t l y lower (P < 0.01)  growth rate of  P. fragi  on the washed muscle sample as compared to the intact muscle  sample.  Enumeration of P. fragi  indicated that the ultimate  population  of the organism never achieved that obtained on the intact muscle sample.  It has been suggested that during low temperature spoilage,  bacteria p r e f e r e n t i a l l y u t i l i z e low molecular weight, soluble components of meats (Jay, 1972; Gill  G i l l and Newton, 1978).  (1976) and G i l l and Newton (1977) reported a p r e f e r e n t i a l  u t i l i z a t i o n of muscle substrates by pseudomonads during  spoilage  such that some amino acids and l a c t i c acid were u t i l i z e d when glucose was  exhausted.  The catabolism  of glucose may  i n h i b i t and/or suppress  the enzymes involved i n the catabolism of amino acids and acid (Paigen and Williams, 1970).  I f we assume that glucose and amino  acids are representative of the t o t a l carbohydrate and the nitrogen (NPN)  lactic  non-protein  content, respectively, the decrease i n the t o t a l  carbohydrate content of the intact inoculated muscle by day 6, may contributed to the decrease i n NPN  have  content seen a f t e r day 6 due to a  /112  relaxation NPN m a y  in  amino  a l s o have  acid  resulted  nitrogenous  components  ( R a m p t o n et  al.,  to  day  1970).  during  SDS-gel of  the  extracted These  the  The i n c r e a s e i n to  the  the  from the  intact  changes were not  same t i m e  apparent  approaching  population be  detected  synthesis late  by  of  et  using  phase of 1970b;  was w e l l  intact  inoculated  et  (1975),  al.  10 reached proteins  10  It  Tarrant  water-soluble  that  as  extensive  a result  after  is  day  prior protein  proteolysis  of  the  6", w h e n c e l l  order  possible that for  fractions  P.  fragi'..  density  is  of  et  al.  of  ,".1975) .  al.,  1971; to  1975).  with  et  Dainty  proteolysis  water-soluble  was  were  repressed  al.,  workers 1975)  in  degradation  fraction reported  e v e n when b a c t e r i a l  ,  the  electrophoretic  remained unchanged.  patterns  of  the  until  the  in  the  from by  numbers  observed  the  Dainty had  2 /cm  to  until  proteolysis  apparent  protein  Similar results  who o b s e r v e d t h a t  surface  proteolysis  Several  demonstrate  the  bacterial  The  The e x t e n s i v e p r o t e o l y s i s  Little  sample.  et  results  (Boethling,  consistent  the  the  a critical  exoenzymes are u s u a l l y  have been unable  SEM m i c r o g r a p h s .  electrophoretograms  nitrogen  and u r e a - i n s o l u b l e p r o t e i n  growth  advanced.  electrophoretograms in the  .  achieved in  electrophoresis  spoilage  indicated  until  p r o t e a s e s and o t h e r  al.,  proteolysis  period.  electrophoresis. (Dainty  exponential  (Borton  CFU/cm  be  non-protein  bacterial  the  in  2  10  must  of  non-protein  muscle occurred  10 was  of  decrease in  urea-soluble  The d e c r e a s e  utilization  initiation  electrophoresis  salt-soluble,  repression.  from the  for  6 may be r e l a t e d  extractability  catabolite  sarcoplasmic  /113  Increases  i n water-soluble  e x t r a c t e d from t h e i n t a c t d u r i n g t h e l a t t e r stages  and s a l t - s o l u b l e p r o t e i n s  i n o c u l a t e d muscle sample were o f t h e experiment  ( a f t e r day 6 ) .  increases i n e x t r a c t a b i l i t y o f the water-soluble were e v i d e n t ,  little  Therefore,  Although  protein fraction  change i n banding p a t t e r n o c c u r r e d  electrophoretograms o f t h i s f r a c t i o n . that increases  evident  i n the  i t i s conceivable  i n e x t r a c t a b i l i t y o f a p r o t e i n f r a c t i o n may r e f l e c t  the p r o t e o l y s i s o f a f r a c t i o n n o t s p e c i f i c a l l y e x t r a c t e d f o r .  Goll  et al. (1970) p o s t u l a t e d t h a t t h e p r o t e o l y s i s o f t h e stromal' p r o t e i n s may cause the: appearance o f new s o l u b l e p e p t i d e s ' i n the sarcoplasmic  protein fraction.  t h i s hypothesis  Results  o f the present  s i n c e e x t e n s i v e breakdown o f t h e  study may  electrophoretogram  o f t h e u r e a - i n 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 t h e i n t a c t muscle t i s s u e was observed.  support  inoculated  However, t h e p o s s i b i l i t y a l s o e x i s t s t h a t  h y d r o l y s i s p r o d u c t s o f t h e 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 may have been e x t r a c t e d  i n the water-soluble  protein fraction.  The i n a b i l i t y  to d e t e c t p o s s i b l e h y d r o l y s i s p r o d u c t s from the s a l t - s o l u b l e , u r e a s o l u b l e and u r e a - i n 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 the i n t a c t i n o c u l a t e d muscle i n t h e SDS-PAGE p a t t e r n s p r o t e i n s may be due t o a masking e f f e c t  o f the water-soluble  ( h y d r o l y s i s products  migrating  t o the same p o s i t i o n as p r o t e i n s indigenous t o t h a t f r a c t i o n ) and/or the i n a b i l i t y o f t h e g e l t o r e s o l v e t h e h y d r o l y s i s product i s o f such h i g h m o l e c u l a r or o f such low m o l e c u l a r  (protein  weight t h a t i t i s unable t o e n t e r t h e g e l , weight t h a t i t passes throughL:the g e l ) .  /114  SDS-gel e l e c t r o p h o r e s i s o f the washed i n o c u l a t e d muscle i n d i c a t e d l i m i t e d p r o t e o l y s i s o f the s a l t - s o l u b l e p r o t e i n s , however, no apparent p r o t e o l y s i s was  d e t e c t e d i n any o t h e r p r o t e i n f r a c t i o n  ( w a t e r - s o l u b l e , u r e a - s o l u b l e and u r e a - i n s o l u b l e p r o t e i n f r a c t i o n s ) . These r e s u l t s c o r r e s p o n d o w e l l  w i t h those o b t a i n e d i n the p r o t e i n  e x t r a c t a b i l i t y s t u d i e s which i n d i c a t e d l i t t l e change i n t h e  non-protein  n i t r o g e n , w a t e r - s o l u b l e 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 e x t r a c t e d from ;the washed i n o c u l a t e d muscle sample o v e r t h e 12 day p e r i o d .  SEM  m i c r o g r a p h s o f t h e washed i n o c u l a t e d m u s c l e , however, i n d i c a t e d areas o f e x t e n s i v e s u r f a c e d e g r a d a t i o n  d u r i n g the l a t e r stages  of  the experiment (days 9 and 12) as a r e s u l t o f the growth o f P. f r a g i . T h i s i n a b i l i t y t o d e t e c t s u b s t a n t i a l p r o t e o l y s i s u s i n g SDS-gel e l e c t r o p h o r e s i s may  f u r t h e r support  the h y p o t h e s i s  that a c r i t i c a l  b a c t e r i a l population i s required to detect p r o t e o l y s i s e l e c t r o p h o r e t i c a l l y . T a r r a n t et al. P. fragi,  (1973), w o r k i n g w i t h p o r c i n e muscle i n o c u l a t e d w i t h  r e p o r t e d t h a t growth and s p o i l a g e o c c u r r e d on the muscle  s u r f a c e , and o n l y a t l a t e r stages d i d the e f f e c t s o f s p o i l a g e p e n e t r a t e t h e e n t i r e meat sample. SDS-gel e l e c t r o p h o r e t o g r a m 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  e x t r a c t e d from the c o n t r o l samples ( i n t a c t and washed) i n d i c a t e d no apparent change i n e i t h e r t h e number o r p a t t e r n o f bands d u r i n g the experiment s u g g e s t i n g t h a t d e g r a d a t i v e were m i n i m a l .  changes due t o a u t o l y s i s  Minor decreases i n non-protein n i t r o g e n , water-soluble  and s a l t - s o l u b l e p r o t e i n were e x p e r i e n c e d  i n b o t h i n t a c t and washed  c o n t r o l samples d u r i n g the 12 days o f i n c u b a t i o n .  E v i d e n c e o f some  /115  a u t o l y t i c processes  was  i n d i c a t e d by i n c r e a s e s i n t o t a l  carbohydrate  o f the i n t a c t c o n t r o l sample, p o s s i b l y due t o the breakdown o f ATP  t o r i b o s e ( H u l t i n , 1976).  Sage (1974), w o r k i n g w i t h gamma  r a d i a t e d c h i c k e n m u s c l e , r e p o r t e d l i t t l e a u t o l y s i s and s t a t e d t h a t "the  absence  of significant  r a d i a t i o n treatment". Schweigert,  1959)  autolysis  may  Several authors  have been due  to the •  (Doty and Wachter,  1955;  r e p o r t e d t h a t r a d i a t i o n dosages o f 1 t o 1.6  Megarad  s e v e r e l y d e c r e a s e d t h e p r o t e o l y t i c a c t i v i t y o f muscle c a t h e p s i n s . J a y (1967) and J a y and Kontou (1967) s t a t e d t h a t temperature s p o i l a g e o f myosystems o c c u r s w i t h o u t c a u s i n g proteolysis.  Results obtained i n the present  low significant  study i n d i c a t e d t h a t  s u r f a c e p r o t e o l y s i s o c c u r r e d i n b o t h i n t a c t and washed muscle samples, as evidenced  by SEM,  due  t o the growth o f P. fragi.  t h e i n t a c t i n o c u l a t e d muscle sample was  However, o n l y i n  this proteolysis readily  d e t e c t e d u s i n g SDS-gel e l e c t r o p h o r e s i s . R e s u l t s o f the SEM was  study i n d i c a t e d t h a t s u r f a c e ^degradation  r e s t r i c t e d t o areas o f l o c a l i z e d c o l o n i z a t i o n i n b o t h i n t a c t  and washed i n o c u l a t e d muscle samples and was  not apparent u n t i l day  G l y c o c a l y x seemed t o m e d i a t e not o n l y c e l l t o c e l l attachment, but a l s o c e l l t o muscle s u r f a c e attachment. micrographs confirmed bacterial  adhesion.  Transmission  electron  t h e p r e s e n c e o f g l y c o c a l y x i n the m e d i a t i o n  of  6.  /116  CONCLUSIONS The results of the present study indicated that although inoculation of sarcoplasmic reduced bovine Longissimus with Pseudomonas fragi (SEM micrographs),  resulted i n extensive surface  limited proteolysis was  dorsi  muscle  degradation  detected using SDS-gel  electrophoresis (minor a l t e r a t i o n i n the s a l t - s o l u b l e protein electrophoretograms).  No apparent proteolysis was observed i n the  electrophoretic patterns of the water-soluble, urea-soluble and urea-insoluble protein fractions extracted from the washed inoculated muscle tissue.  E x t r a c t a b i l i t y studies indicated l i t t l e change i n  non-protein nitrogen, water-soluble and s a l t - s o l u b l e protein content during the 12 day incubation period.  The pH of the washed inoculated  sample remained v i r t u a l l y the same over the duration of the experiment. A s l i g h t decrease i n t o t a l carbohydrate of P. f r a g i . (P < 0.01)  was  evident due to the growth  The growth rate of the bacteria was  s i g n i f i c a n t l y lower  on the washed muscle than on the intact muscle tissue. Scanning electron micrographs indicated surface  degradation  of the intact muscle tissue as a result of the growth of P. f r a g i . Major a l t e r a t i o n s were observed i n the SDS-gel  electrophoretograms  of the salt-soluble, urea-soluble and urea-insoluble proteins, however, only minor changes were observed i n the gel patterns of the water-soluble proteins.  Large increases i n e x t r a c t a b i l i t y of the  water- and s a l t - s o l u b l e proteins occurred after day 6 i n the intact inoculated muscle tissue.  The pH of the'intact inoculated muscle  /117  t i s s u e i n c r e a s e d as t h e b a c t e r i a l p o p u l a t i o n  increased.  A decrease  i n t o t a l c a r b o h y d r a t e c o n t e n t was observed due t o t h e growth o f P. f r a g i . Glycocalyx  appeared t o mediate c e l l t o c e l l as w e l l as  c e l l t o muscle s u r f a c e attachment i n b o t h i n t a c t and washed muscle tissue.  P r o t e o l y s i s o f t h e m y o f i b r i l s was l i m i t e d t o a r e a s o f b a c t e r i a l  growth. The use o f a s i n g l e s p e c i e s o f b a c t e r i a and c o n t r o l l e d c o n d i t i o n s i n t h e s t u d y o f low temperature muscle s p o i l a g e b a c t e r i a l attachment, may occurring spoilage. i t may in  not be t r u l y i n d i c a t i v e o f n a t u r a l l y  The importance o f such a s t u d y , however, i s t h a t  a i d i n t h e i d e n t i f i c a t i o n o f some o f t h e parameters  spoilage.  and  involved  /118  REFERENCES CITED  American Type Culture C o l l e c t i o n Catalogue of Strains I. Twelfth Edition.  1976. Gherna, R. L. and Hatt, H. 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