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UBC Theses and Dissertations

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

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Some Biochemical and Ultrastructural Changes in Intact and Sarcoplasmic Reduced, Bovine Longissimus dorsi Muscle Strips Inoculated with Pseudomonas fragi by R I C K E Y Y O S H I O Y A D A B . S c . A g . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 7 7 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF THE R E Q U I R E M E N T S FOR THE D E G R E E OF M A S T E R OF S C I E N C E i n THE F A C U L T Y OF GRADUATE S T U D I E S 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 t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A A u g u s t 1 9 8 0 © R i c k e y M o s h i o Y a d a , 1 9 8 0 \ In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make i t freely 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 financial gain shall not be allowed without my written permission. Department n f F o o d Science The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 A u g u s t 2 8 , 1 9 8 0 / i i ABSTRACT I n t a c t b o v i n e Longissimus dovsi m u s c l e was s u b j e c t e d t o a m i l d w a s h i n g p r o c e d u r e i n o r d e r t o r e d u c e t h e c o n c e n t r a t i o n o f t h e s a r c o p l a s m i c f l u i d . I n t a c t a n d w a s h e d m u s c l e s a m p l e s w e r e i n o c u l a t e d w i t h Pseudomonas fvagi t o . e v a l u a t e t h e e f f e c t o f a s a r c o p l a s m i c r e d u c t i o n o n b a c t e r i a l g r o w t h a n d s u b s e q u e n t s p o i l a g e d u r i n g s t o r a g e a t 4 ° C f o r 12 d a y s . A s e p t i c c o n t r o l s w e r e s t o r e d u n d e r s i m i l a r c o n d i t i o n s . A l t e r a t i o n s i n t h e w a t e r - s o l u b l e , s a l t - s o l u b l e , u r e a -s o l u b l e a n d 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 , a s w e l l a s t h e t o t a l c a r b o h y d r a t e , p H a n d b a c t e r i a l n u m b e r s , w e r e m o n i t o r e d i n b o t h i n t a c t a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s . S c a n n i n g a n d t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w e r e e m p l o y e d t o m o n i t o r u l t r a s t r u c t u r a l c h a n g e s o n t h e m u s c l e s u r f a c e a s a c o n s e q u e n c e o f t h e g r o w t h o f P. fvagi,. A n a l y s i s o f w a t e r - s o l u b l e c o m p o n e n t s ( n o n - p r o t e i n n i t r o g e n , w a t e r - s o l u b l e p r o t e i n s a n d c a r b o h y d r a t e s ) i n d i c a t e d t h a t t h e w a s h i n g p r o c e d u r e e f f e c t i v e l y r e m o v e d t h e m a j o r i t y o f t h e s e c o m p o n e n t s . I n c r e a s e s i n t h e e x t r a c t a b i l i t y o f t he^ w a t e r - s o l u b l e a n d 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 w e r e o b s e r v e d i n t h e i n t a c t i n o c u l a t e d m u s c l e s a m p l e . A l t e r a t i o n s i n t h e S D S - g e l e l e c t r o p h o r e t i c p a t t e r n o f t h e w a t e r - s o l u b l e , s a l t - s o l u b l e , u r e a - s o l u b l e a n d u r e a - i n s o l u b l e p r o t e i n s w e r e e v i d e n t . T o t a l c a r b o h y d r a t e d e c r e a s e d a s a r e s u l t o f g r o w t h o f P. fvagi. A n i n c r e a s e i n p H o f t h e i n t a c t m u s c l e o c c u r r e d a s b a c t e r i a l n u m b e r s i n c r e a s e d . S i g n i f i c a n t l y (P < 0 . 0 1 ) h i g h e r g r o w t h / i i i r a t e s w e r e o b s e r v e d o n t h e i n t a c t m u s c l e t i s s u e t h a n t h e w a s h e d m u s c l e t i s s u e . R e l a t i v e l y l i t t l e c h a n g e i n t h e n o n - p r o t e i n n i t r o g e n , w a t e r - s o l u b l e a n d s a l t - s o l u b l e p r o t e i n c o n t e n t was o b s e r v e d i n t h e w a s h e d i n o c u l a t e d m u s c l e t i s s u e . A s l i g h t 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 w a s s e e n . M i n o r c h a n g e s i n t h e S D S - g e l e l e c t r o p h o r e t o g r a m s o f t h e s a l t - s o l u b l e p r o t e i n s w e r e a p p a r e n t . L i t t l e c h a n g e i n p H o f t h e w a s h e d i n o c u l a t e d s a m p l e o c c u r r e d d u e t o t h e g r o w t h o f P. fragi. S c a n n i n g e l e c t r o n m i c r o g r a p h s i n d i c a t e d t h a t s u r f a c e d e g r a d a t i o n o f b o t h i n t a c t a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s w e r e a p p a r e n t o n l y i n a r e a s o f l o c a l i z e d c o l o n i z a t i o n . G l y c o c a l y x a p p e a r e d t o m e d i a t e n o t o n l y c e l l t o c e l l a t t a c h m e n t , b u t a l s o c e l l t o m u s c l e s u r f a c e a d h e s i o n . B a c t e r i a w e r e o b s e r v e d g r o w i n g b e t w e e n m u s c l e f i b e r s . T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s o f i n t a c t i n o c u l a t e d m u s c l e t i s s u e c o n f i r m e d t h e m e d i a t i o n o f g l y c o c a l y x i n b a c t e r i a l a d h e s i o n . C e l l u l a r e v a g i n a t i o n s w e r e p r e s e n t o n t h e s u r f a c e o f t h e b a c t e r i a . A u t o l y s i s w a s m i n i m a l i n b o t h i n t a c t a n d w a s h e d a s e p t i c m u s c l e c o n t r o l s . /iv TABLE:OF- CONTENTS Page ABSTRACT • - • • i i TABLE OF CONTENTS. ........... . iv LIST OF TABLES: v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS x i INTRODUCTION 1 LITERATURE REVIEW 4 A. Meat Spoilage 4 B. Aseptic Techniques 5 C. A u t o l y i i c Changes in Postmortem Muscle 7 D. Biochemical Changes During Bacterial Spoilage of Muscle 8 E. Bacterial Adhesion 17 F. Inoculation of Intact Muscle Samples 21 METHODS AND MATERIALS 23 A. Sarcoplasmic Extraction 23 B. Preparation of the Inoculum 27 C. Preparation of the Muscle Samples 29 D. pH Determination 31 E. Bacterial Counts 31 F. Electron Microscopy 32 1. Scanning EM preparation 32 2. Transmission EM preparation 33 G. Extraction of Water- and Salt-Soluble Protein Fractions. 34 H. Nitrogen Determination 36 I. Total Carbohydrate Analysis 37 J. SDS-Gel Electrophoresis 38 RESULTS AND DISCUSSION • 43 A. Effect of Gamma Radiation on Colour and Odour 43 B. Influence of Sarcoplasmic Reduction on Bacterial Growth • • 43 C. The Effect of Growth of P. fragi on pE, Surface Appearance and Odour 48 /v Page D. Influence of Growth of P. fragi on Total Carbohydrate Content 53 E. The Effect of P. fragi on the Extractability of Non-Protein Nitrogen (NPN)3 Water-Soluble and Salt-Soluble Proteins 59 F. SDS-Polyacrylamide Gel Electrophoresis 70 1. Mater-soluble proteins 71 2. Salt-soluble proteins 75 S. Urea-soluble proteins 80 4. Urea-insoluble proteins 83 G. Electron Microscopy 86 1. Scanning electron microscopy 87 2. Transmission electron microscopy 101 GENERAL DISCUSSION I l l CONCLUSIONS 116 REFERENCES CITED 118 / v i LIST:OF-TABLES Table .. Page 1 Q u a n t i t y o f p r o t e i n f r a c t i o n a n d m o l e c u l a r w e i g h t 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 a n d a m o u n t o f p r e p a r a t i o n a p p l i e d t o g e l d u r i n g S D S - g e l e l e c t r o p h o r e s i s 4 0 2 M o l e c u l a r w e i g h t m a r k e r s f o r S D S - g e l e l e c t r o p h o r e s i s . . 4 2 3 I n f l u e n c e o f i n c u b a t i o n a t 4 ° C o n m u s c l e s a m p l e s i n o c u l a t e d w i t h Pseudomonas fragi 4 5 4 R e g r e s s i o n s t a t i s t i c s u s e d i n t h e c o m p a r i s o n o f t h e s l o p e o f t h e i n t a c t i n o c u l a t e d r e g r e s s i o n w i t h t h e s l o p e o f t h e w a s h e d i n o c u l a t e d r e g r e s s i o n 4 9 5 T h e e f f e c t o f P. fragi o n t h e p H o f t h e i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) b o v i n e m u s c l e s a m p l e s . 5 0 6 T h e e f f e c t o f P. fragi o n 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 i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) m u s c l e s a m p l e s 54 7 T h e e f f e c t o f P. fragi o n t h e n o n - p r o t e i n n i t r o g e n o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) m u s c l e s a m p l e s 60 8 T h e e f f e c t o f P. fragi o n 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 o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) m u s c l e s a m p l e s 63 9 T h e e f f e c t o f P. fragi o n t h e s a l t - s o l u b l e p r o t e i n r. c o n t e n t o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) m u s c l e s a m p l e s 67 / v i i • LIST OF FIGURES Figure Page 1 H o b a r t d e l i c a t e s s e n s l i c e r , 24 2 A p p a r a t u s u s e d f o r t h e e x t r a c t i o n o f s a r c o p l a s m f r o m i n t a c t m u s c l e 25 3 B a c t e r i a l v e n t s u s e d t o s t e r i l i z e b o t h a i r a n d n i t r o g e n 26 4 " W h i r l - p a k " m u s c l e s a m p l e 28 5 P l e x i g l a s s i n c u b a t i o n c h a m b e r 3 0 6 F l o w s h e e t o f p r o t e i n e x t r a c t i o n p r o c e d u r e 3 5 7 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 a n d w a s h e d m u s c l e s a m p l e s , 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 4 6 8 L i n e a r i z a t i o n o f l o g b a c t e r i a l p o p u l a t i o n d a t a f o r i n t a c t a n d w a s h e d m u s c l e s 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 4 7 9 p H o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C 51 10 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 c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C 5 5 11 C o n v e r s i o n o f A T P t o r i b o s e a n d h y p o x a n t h i n e i n p o s t m o r t e m m u s c l e 58 12 N o n - p r o t e i n n i t r o g e n c o n t e n t o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C 61 / v i i i Figure Page 13 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 o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C 64 14 S a l t - s o l u b l e p r o t e i n c o n t e n t o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C 68 1 5 E l e c t r o p h o r e t o g r a m s o f w a t e r - s o l u b l e p r o t e i n s e x t r a c t e d f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 72 16 E l e c t r o p h o r e t o g r a m s o f w a t e r - s o l u b l e p r o t e i n s e x t r a c t e d f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 73 17 E l e c t r o p h o r e t o g r a m s o f 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 f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 76 18 E l e c t r o p h o r e t o g r a m s o f 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 f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 77 19 E l e c t r o p h o r e t o g r a m s o f u r e a - s o l u b l e p r o t e i n s e x t r a c t e d f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 81 2 0 E l e c t r o p h o r e t o g r a m s o f u r e a - s o l u b l e p r o t e i n s e x t r a c t e d f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 82 / i x Figure Page 21 E l e c t r o p h o r e t o g r a m s 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 f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 84 22 E l e c t r o p h o r e t o g r a m s 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 f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 12 85 2 3 . S E M m i c r o g r a p h s o f d a y 0 , u n i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s 88 24 SEM m i c r o g r a p h s o f d a y 1 2 , i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d (b ) m u s c l e s a m p l e s 8 9 2 5 S E M m i c r o g r a p h s o f d a y 0 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s 90 2 6 S E M m i c r o g r a p h s o f d a y 3 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d (b ) m u s c l e s a m p l e s s h o w i n g c r e v i c e s ( a r r o w ) w h i c h m a y t r a p b a c t e r i a 92 27 S E M 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 i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s 94 28 S E M m i c r o g r a p h s o f d a y 9 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s 95 29 S E M m i c r o g r a p h s o f d a y 12 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d (b ) m u s c l e s a m p l e s 97 3 0 S E M m i c r o g r a p h s s h o w i n g t h e a d h e r e n c e o f b a c t e r i a b y s l i m e ( S ) b e t w e e n m u s c l e f i b r i l s ( A ) . , 1 0 0 / x Figure. Page 3 1 T E M m i c r o g r a p h o f t h e a d h e r e n t b a c t e r i a l p o p u l a t i o n o f t h e i n t a c t m u s c l e s u r f a c e a f t e r 12 d a y s i n c u b a t i o n 1 0 3 3 2 T E M 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 ) a n d 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 ) 104 3 3 T E M 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 a n d i n c u b a t e d a t 4 ° C f o r 1 2 d a y s 1 0 6 3 4 T E M m i c r o g r a p h o f u n i n o c u l a t e d b o v i n e Longissimus dorsi m u s c l e ( c r o s s - s e c t i o n ) i n c u b a t e d f o r 12 d a y s a t 4 ° C ( X 3 3 , 2 0 0 ) 1 0 7 3 5 T E M 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 a n d i n c u b a t e d a t 4 ° C f o r 1 2 d a y s 1 0 9 3 6 T E M 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 a n d i n c u b a t e d a t 4 ° C f o r 12 d a y s H O / x i ACKNOWLEDGEMENTS I w i s h t o e x p r e s s ray s i n c e r e g r a t i t u d e t o D r . B . J . S k u r a f o r h i s t i m e , p a t i e n c e , a s s i s t a n c e a n d c o n s t r u c t i v e c r i t i c i s m t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . I a l s o w i s h t o t h a n k t h e m e m b e r s o f my g r a d u a t e c o m m i t t e e , D r . S . N a k a i , D r . W. D . P o w r i e , a n d D r . J . F . R i c h a r d s f o r t h e i r v a l u a b l e s u g g e s t i o n s a n d a s s i s t a n c e . T h a n k s a r e a l s o e x t e n d e d t o M r . S . J . Y e e f o r h i s i n v a l u a b l e t e c h n i c a l a d v i c e . F i n a l l y , a s p e c i a l t h a n k s t o S y l v i a D u f f e k f o r h e r e n d l e s s t i m e , p a t i e n c e a n d e n c o u r a g e m e n t d u r i n g t h i s s t u d y a n d f o r h e r w o r k i n p r e p a r i n g t h e m a n u s c r i p t . T h e s u p p o r t o f t h e N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l o f C a n a d a t h r o u g h p o s t - g r a d u a t e s c h o l a r s h i p s i s a l s o a c k n o w l e d g e d . / I INTRODUCTION T h e p r i n c i p a l c o m p o n e n t o f m e a t i s m u s c l e . M u s c l e c o n t a i n s a p p r o x i m a t e l y 75% w a t e r , 19% p r o t e i n , 3% l i p i d s , 1 .5% n o n - p r o t e i n n i t r o g e n o u s s u b s t a n c e s , 1.0% i n o r g a n i c c o n s t i t u e n t s ( F o r r e s t et al., 1 9 7 5 ) . E x c l u d i n g w a t e r , p r o t e i n c o n s t i t u t e s t h e l a r g e s t c o m p o n e n t o f m u s c l e a n d i s t h e p r i n c i p a l c o n s t i t u e n t o f t h e s o l i d m a t t e r ( L a w r i e , 1 9 7 9 ) . M u s c l e p r o t e i n s a r e c h a r a c t e r i z e d i n t o 3 m a j o r g r o u p s , b a s e d p r i m a r i l y u p o n t h e i r s o l u b i l i t y . T h e s a r c o p l a s m i c p r o t e i n s a r e r e a d i l y e x t r a c t a b l e i n w a t e r o r l o w i o n i c s t r e n g t h b u f f e r s . T h e m y o f i b r i l l a r p r o t e i n s , h o w e v e r , r e q u i r e s a l t s o l u t i o n s o f h i g h i o n i c s t r e n g t h f o r e x t r a c t i o n . T h e s t r o m a l p r o t e i n s a r e c o m p a r a t i v e l y i n s o l u b l e a n d a r e s o l u b i l i z e d o n l y w i t h s t r o n g d i s s o c i a t i n g a g e n t s . T h e s a r c o p l a s m i c p r o t e i n s r e p r e s e n t a c o m p l e x m i x t u r e o f c o m p o n e n t s , m a n y o f w h i c h a r e e n z y m e s o f t h e g l y c o l y t i c c y c l e . T h e m y o f i b r i l l a r p r o t e i n s r e p r e s e n t t h e s t r u c t u r a l a n d r e g u l a t o r y , p r o t e i n s i n v o l v e d i n m u s c l e c o n t r a c t i o n . T h e s t r o m a l p r o t e i n f r a c t i o n i s a c o m p o s i t e o f t i s s u e s , i n c l u d i n g c e l l w a l l s , b l o o d v e s s e l s , n e r v e s , e n d o m y s i u m a n d p e r i m y s i u m , w h i c h f u n c t i o n s a s t h e m a j o r s u p p o r t i v e e l e m e n t o f t h e a n i m a l b o d y ( B o d w e l l a n d M c C l a i n , 1 9 7 1 ; F o r r e s t et al., 1 9 7 5 ; L a w r i e , 1 9 7 9 ) . M i c r o b i a l c o n t a m i n a t i o n a n d t h e s u b s e q u e n t s p o i l a g e o f f o o d m y o s y s t e m s a r e p r o b l e m s t h a t h a v e b e e n w e l l d o c u m e n t e d . A l t h o u g h i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f t h e s p o i l a g e f l o r a a n d t h e e f f e c t o f /2 e n v i r o n m e n t a l c o n d i t i o n s o n t h e s e o r g a n i s m s h a s g a i n e d m u c h a t t e n t i o n , i n t e r e s t h a s s h i f t e d t o t h e s t u d y o f t h e b i o c h e m i c a l c h a n g e s d u r i n g s p o i l a g e . O b s e r v a t i o n s p e r t i n e n t t o t h e i m p o r t a n c e o f b a c t e r i a l p r o t e o l y s i s h a v e b e e n c o n t r a d i c t o r y . Some r e s e a r c h e r s ( J a y , 1 9 6 7 ; J a y a n d K o n t o u , 1 9 6 7 ; O c k e r m a n et at., 1 9 6 9 ; J a y a n d S h e l e f , 1 9 7 6 ) b e l i e v e t h a t b a c t e r i a l p r o t e o l y s i s p l a y s a n i n s i g n i f i c a n t r o l e d u r i n g s p o i l a g e , w h i l e o t h e r s ( H a s e g a w a et at., 1 9 7 0 a a n d b ; B o r t o n et at., 1 9 7 0 a a n d b ; T a r r a n t et at., 1 9 7 1 ; S a g e , 1 9 7 4 ; D a i n t y et at., 1 9 7 5 ) h a v e s h o w n s i g n i f i c a n t p r o t e i n d e g r a d a t i o n . A d h e s i o n o f b a c t e r i a t o s u r f a c e s i s a w i d e s p r e a d p h e n o m e n o n i n n a t u r e . I n f o r m a t i o n a b o u t t h e m e c h a n i s m s o f b a c t e r i a l a d h e s i o n , h o w e v e r , i s l i m i t e d . R e c e n t i n v e s t i g a t i o n s ( N o t e r m a n s a n d K a m p e l m a c h e r , 1 9 7 4 ; N o t e r m a n s a n d K a m p e l m a c h e r , 1 9 7 5 ; M c C o w a n et at., 1 9 7 9 ) i n t o t h e a t t a c h m e n t o f m i c r o o r g a n i s m s t o m y o s y s t e m s h a v e s h o w n t h e p r e s e n c e o f a n e x t r a - c e l l u l a r p o l y s a c c h a r i d e , t h e g l y c o c a l y x , w h i c h a p p e a r s t o m e d i a t e a t t a c h m e n t . M o s t r e s e a r c h p r i o r t o t h i s t h e s i s i n v o l v e d t h e u s e o f m i n c e d m u s c l e a s t h e s u b s t r a t e f o r t h e i n o c u l u m . A l t h o u g h t h i s t e c h n i q u e i n c r e a s e s t h e s u r f a c e a r e a , t h e r e b y m a x i m i z i n g a n y c h a n g e s t h a t r e s u l t f r o m b a c t e r i a l s p o i l a g e , t h e c h a n g e s o b s e r v e d m a y n o t r e s e m b l e t h o s e w h i c h o c c u r o n i n t a c t ( n o n - m e c h a n i c a l l y d i s r u p t e d ) m u s c l e t i s s u e . T h e o b j e c t o f t h e p r e s e n t r e s e a r c h w a s t o o b s e r v e some b i o c h e m i c a l c h a n g e s t h a t r e s u l t e d f r o m t h e g r o w t h o f Pseudomonas fragi /3 A T C C 4973 o n i n t a c t a n d s a r c o p l a s m i c r e d u c e d b o v i n e Longissimus dovsi m u s c l e s t r i p s . I n a d d i t i o n , s c a n n i n g a n d t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w e r e e m p l o y e d t o s t u d y s u r f a c e u l t r a s t r u c t u r e o f t h e m u s c l e i n a t t e m p t s t o e x p l a i n t h e b i o c h e m i c a l c h a n g e s . / 4 LITERATURE REVIEW A. Meat Spoilage T h e i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f a e r o b i c s p o i l a g e o f f r e s h m e a t h a s b e e n w e l l d o c u m e n t e d i n t h e l i t e r a t u r e ( B r o w n a n d W e i d e m a n n , 1 9 5 8 ; A y r e s , 1 9 6 0 a ; J a y , 1 9 6 7 ; R e y et at., 1 9 7 0 ; I n g r a m a n d D a i n t y , 1 9 7 1 ; J a y a n d S h e l e f , 1 9 7 6 ; G i l l a n d N e w t o n , 1 9 7 8 ) . T h e m a j o r s o u r c e o f m i c r o o r g a n i s m s r e s p o n s i b l e f o r s p o i l a g e a p p e a r s t o b e t h e h i d e s o f d r e s s e d a n i m a l s ( G i l l a n d N e w t o n , 1 9 7 8 ; M a c k e y a n d D e r r i c k , 1 9 7 9 ) . A l t h o u g h t h e i n i t i a l f l o r a o f m e a t c o n t a i n s a l a r g e n u m b e r o f d i f f e r e n t b a c t e r i a l g e n e r a , o n l y a f e w p r e d o m i n a t e . : d u r i n g l o w t e m p e r a t u r e m e a t s p o i l a g e , n a m e l y t h e p s y c h r o t r o p h i c b a c t e r i a ( K i r s c h et al., 1 9 5 2 ) . M a n y s p e c i e s h a v e b e e n d e s c r i b e d b u t o n l y a r e l a t i v e l y s m a l l p r o p o r t i o n o f t h e s e a r e f o u n d a s m a j o r c o m p o n e n t s o f m e a t s p o i l a g e f l o r a s . S t r a i n s o f Pseudomonas, Achvomobacter, Movaxella, Acinetobacter_, Lactobacillus, Microbacterium thermosphactum a n d c e r t a i n g e n e r a o f t h e f a m i l y E n t e r o b a c t e r i a c e a e h a v e b e e n i s o l a t e d w i t h r e g u l a r f r e q u e n c y f r o m s p o i l i n g m e a t ( J a y , 1 9 6 7 ; V a n d e r z a n t a n d N i c k e l s o n , 1 9 6 9 ; J a y , 1 9 7 2 ; G i l l a n d N e w t o n , 1 9 7 7 ) . H o w e v e r , i t i s now g e n e r a l l y a c c e p t e d t h a t t h e p s y c h r o t r o p h i c , a e r o b i c , g r a m - n e g a t i v e g e n u s Pseudomonas c o n s t i t u t e s t h e m o s t p r e d o m i n a n t a n d i m p o r t a n t g r o u p o f b a c t e r i a i n t h e l o w t e m p e r a t u r e s p o i l a g e o f b e e f ( W o l i n et al., 1 9 5 7 ; A y r e s , 1 9 6 0 a ; J a y , 1 9 6 7 ; I n g r a m a n d D a i n t y , 1 9 7 1 ; J a y , 1 9 7 2 ; J a y a n d S h e l e f , 1 9 7 6 ; G i l l a n d N e w t o n , 1 9 7 7 ; G i l l a n d N e w t o n , 1 9 7 8 ) . /5 The overall result of low temperature bacterial spoilage of fresh meat is the production of "off" odours and flavours, accompanied by a slimy appearance. Incipient spoilage is measurable when total numbers/gram reach approximately 10 organisms/g (Jay and Shelef, 1976). Daub et al. (1979), working with chicken skin, reported that the f i r s t indication of an off-odour and sliminess occurred when 8 7 the count approached 10 organisms/g. Ayres (1960b) found that 10 2 organisms/cm were required for off-odour development of fresh beef, and 10 for 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 2 reached 4 x 10 /cm , while the appearance of slime was not detected until 2 x 10 /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 in the production of ammonia, hydrogen sulfide, indole, skatole, and amines, a l l of which contribute to off-odour (Ingram and Dainty, 1971; Jay, 1972). The formation of slime is the consequence of bacterial coalescence as well as the loss of muscle integrity due to microbial growth (Ayres et al., 1950; Jay, 1970). B. Aseptic Techniques Changes in myosystems during refrigerated storage may be caused by microorganisms as well as autolytic reactions. In order to obtain an understanding of these changes and their relative importance in meat spoilage, i t is necessary to differentiate 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 sterile tissue. The limitation of achieving this undoubtably stems from the practical 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). Various methods have been employed in obtaining sterile muscle tissue. Zender et al. (1958) used a surgical room technique to aseptically 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 ster i l e . A chlor-tetracycline 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 for autolytic studies. Ockerman et al. (1964) used a gnotobiotic method to study both aseptic muscle and organ tissue. Ockerman et al. (1969) described a surgical isolator technique for removing sterile muscle tissue. Hasegawa et al. (1970a and b), Rampton et al. (1970) and Borton et al. (1970a) used an absolute alcohol rinse in attempts to obtain st e r i l e samples, and discovered that although most control samples were st e r i l e , some were contaminated. Hone et al. (1975), using a core technique to collect aseptic beef tissue, found that of the 23 samples collected, 22% were ste r i l e (< 1 organism/g) but 74% were contaminated with < 5 organisms/g. Ockerman and Cahill (1977), employing the same technique, found that although the control samples were contaminated, bacterial counts / 7 r e m a i n e d r e l a t i v e l y c o n s t a n t o v e r t h e 21 d a y s o f p o s t m o r t e m s t o r a g e . B u c k l e y et at. ( 1 9 7 6 ) u s e d a n a s e p t i c c u t t i n g t e c h n i q u e i n c o n c e r t w i t h a l a m i n a r a i r f l o w u n i t t o o b t a i n a s e p t i c t i s s u e f r o m t h e Longissimus dorsi m u s c l e . J a y a n d K o n t o u ( 1 9 6 7 ) e x p o s e d b e e f t i s s u e t o a 1 M e g a r a d d o s e o f gamma r a d i a t i o n t o o b t a i n s t e r i l e s a m p l e s . T h e a u t h o r s f o u n d t h a t t h i s d o s a g e w a s c a p a b l e o f d e s t r o y i n g a l l p s y c h r o p h i l i c b a c t e r i a w i t h o u t a f f e c t i n g t h e n o r m a l s p o i l a g e o f t h e m e a t w h e n i n o c u l a t e d w i t h a m i x e d f l o r a . S i m i l a r r e s u l t s w e r e r e p o r t e d b y S a g e ( 1 9 7 4 ) w o r k i n g w i t h c h i c k e n m u s c l e . N u m e r o u s i n v e s t i g a t o r s s t u d y i n g t h e e f f e c t o f gamma r a d i a t i o n o n a u t o l y t i c e n z y m e s ( c a t h e p t i c ) h a v e s h o w n t h a t c a t h e p s i n s a r e n o t a s s u s c e p t i b l e a s b a c t e r i a t o i r r a d i a t i o n . D r a k e et at. ( 1 9 5 7 ) , w o r k i n g w i t h p o r c i n e m u s c l e a n d g r o u n d b e e f , f o u n d i n c r e a s e s i n f r e e a m i n o a c i d s a f t e r i r r a d i a t i o n i n d i c a t i n g a g e n e r a l p r o t e o l y s i s . Z e n d e r et at. ( 1 9 5 8 ) , w o r k i n g w i t h r a b b i t a n d l a m b m u s c l e , s h o w e d t h a t i r r a d i a t i o n n o t o n l y p r e v e n t e d b a c t e r i a l c o n t a m i n a t i o n , b u t h a d l i t t l e e f f e c t o n a u t o l y t i c p r o t e o l y s i s . D o t y a n d W a c h t e r ( 1 9 5 5 ) r e p o r t e d t h a t 50% o f t h e c a t h e p t i c a c t i v i t y w a s i n a c t i v a t e d b y a 1 . 5 M e g a r a d d o s e o f gamma r a d i a t i o n . L a w r i e et at. ( 1 9 6 1 ) , i n e x p e r i m e n t s w i t h b o v i n e a n d p o r c i n e m u s c l e , f o u n d t h a t a 5 M e g a r a d d o s e r e d u c e d p r o t e o l y t i c a c t i v i t y b y a p p r o x i m a t e l y 6 0 % . C. Autotytic Changes in Postmortem Musote I n c r e a s e s i n n o n - p r o t e i n n i t r o g e n c o m p o n e n t s i n a s e p t i c b e e f m u s c l e d u r i n g p o s t m o r t e m s t o r a g e h a v e b e e n o b s e r v e d ( Z e n d e r et at., / 8 1 9 5 8 ; L o c k e r , 1 9 6 0 ; T h o m p s o n et al., 1 9 6 1 ; S h a r p , 1 9 6 3 ; D a v e y a n d G i l b e r t , 1 9 6 6 ; G a r d n e r a n d S t e w a r t , 1 9 6 6 ; D a v e y a n d G i l b e r t , 1 9 6 9 ; P a r r i s h et al., 1 9 6 9 ; P e n n y a n d . F e r g u s o n - P r y c e , 1 9 7 9 ) . T h e i n c r e a s e s h a v e b e e n a t t r i b u t e d t o t h e a c t i o n o f e n d o g e n o u s p r o t e o l y t i c e n z y m e s ( c a t h e p s i n s ) k n o w n t o b e p r e s e n t i n r e l a t i v e l y l o w c o n c e n t r a t i o n s i n m u s c l e t i s s u e . P a r r i s h a n d B a i l e y ( 1 9 6 7 ) , w o r k i n g w i t h b e e f , f o u n d t h a t t h e m a j o r p r o t e o l y t i c e n z y m e w a s c a t h e p s i n D . I t h a s a l s o b e e n s u g g e s t e d b y O k i t a n i et al. ( 1 9 7 7 ) t h a t c a t h e p s i n B c o u l d b e t h e e n z y m e r e s p o n s i b l e f o r p o s t m o r t e m d e g r a d a t i o n . I t h a d b e e n b e l i e v e d t h a t m u c h o f t h e " i n c r e a s e i n t h e n o n - p r o t e i n n i t r o g e n r e s u l t e d f r o m t h e d e g r a d a t i o n o f t h e s a r c o p l a s m i c p r o t e i n s a n d p e p t i d e s b y c a t h e p t i c e n z y m e s , w h i l e t h e m y o f i b r i l l a r p r o t e i n s w e r e n o t c l e a v e d d u r i n g p o s t m o r t e m s t o r a g e ( S h a r p , 1 9 6 3 ; B o d w e l l a n d P e a r s o n , 1 9 6 4 ; P a r r i s h et al., 1 9 6 9 ) . D. Biochemical Changes During Bacterial Spoilage of Muscle A l t h o u g h t h e r o l e o f b a c t e r i a i n m e a t s p o i l a g e h a s l o n g b e e n r e c o g n i z e d , t h e m e c h a n i s m o f d e g r a d a t i o n a n d m u s c l e p r o t e i n i n v o l v e m e n t h a s n o t b e e n e l u c i d a t e d ( H a s e g a w a et al., 1 9 7 0 a ) . I t i s g e n e r a l l y a c c e p t e d t h a t n o c l e a r c h a n g e s o c c u r u n t i l b a c t e r i a l p o p u l a t i o n s e x c e e d 1 0 Ig. G a r d n e r a n d S t e w a r t ( 1 9 6 6 ) , s t u d y i n g t h e c h a n g e s i n t h e f r e e a m i n o a c i d s a n d o t h e r n i t r o g e n c o m p o u n d s i n s t o r e d b e e f m u s c l e , f o u n d t h a t g l u t a m i n e d e c r e a s e d d u r i n g s t o r a g e w h i l e m o s t o t h e r a m i n o a c i d s , n o t a b l y t r y p t o p h a n a n d g l u t a m i c a c i d , i n c r e a s e d . T h e i n c r e a s e i n g l u t a m i c a c i d a n d c o r r e s p o n d i n g d e c r e a s e i n g l u t a m i n e was a t t r i b u t e d /9 to the bacterial production of glutaminase, while the increase in tryptophan was attributed to autolytic processes. Increases in ammonia production were not significant u n t i l incipient spoilage 8 9 (10 to 10 organisms/g) had been reached. In a similar study, Jay and Kontou (1967) reported that fresh beef allowed to undergo microbial spoilage at 7°C showed decreases in amino acids and nucleotides when bacterial numbers were high; conversely, when bacterial numbers were low, decreases in both moieties were not detected. The authors concluded that these low molecular weight compounds, rather than the "primary" proteins, were the precursors for the compounds associated with low temperature meat spoilage. Jay (1967) studied the characteristics of low temperature beef spoilage and found that in the presence of many low molecular weight compounds, the attack of the "primary" proteins by the organisms involved (predominantly Pseudomonas spp.) was minimal at most. The author postulated that the breakdown of the proteins was the result of cathepsins which were released due to bacterial action. Lerke et al. (1967) investigated the role of protein during microbial spoilage of fis h muscle. Muscle press juice from English sole was fractionated by gel 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 III), the protein-free fraction spoiled according to organoleptic and chemical c r i t e r i a (volatile reducing substances, total volatile nitrogen and trimethylamine nitrogen). Proteolysis due to bacterial action, as measured by Kjeldahl nitrogen, was evident in both the /io unfractionated muscle juice as well as the protein fraction. However, no significant proteolysis occurred until spoilage was readily evident. Castell and Greenough (1959), studying the relationship between substrate composition and development of odours in fish muscle, commented on the i n a b i l i t y of Pseudomonas fragi- to hydrolyze proteins; yet this organism was able to produce strong off-odours from fish muscle extracts. In another study, Ockerman et al. (1969) investigated the alterations in the various protein fractions of sterile and inoculated beef (inoculated with either a Pseudomonas spp., Aehromobacter or a general inoculum composed of the natural flora of beef) during refrigerated storage (3 ± 2°C). Kjeldahl analysis of the sarcoplasmic and myofibrillar fractions revealed no significant difference between aseptic and inoculated samples. Significant differences were detected in the stromal fraction after 17.5 days incubation, where a definite decrease was exhibited by the inoculated samples with a subsequent increase;in non-protein nitrogen. Rampton et al. (1970) studied the effects of selected bacteria - Aehromobacter liquefaciens, Micrococcus luteus, Pediococcus cerevisiae, Pseudomonas fluorescens, Streptococcus faecalis and a mixed flora (obtained from commercial hamburger) - on rabbit and porcine myofibrillar 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 myofibrillar proteins, It was noted, however, that "although / I I the present study shewed no proteolysis of the m y o f i b r i l l a r fraction, it did not rule out the possibility that other species and strains of bacteria could 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 proteins". T h e p r e s e a r c h e r s p o s t u l a t e d t h a t t h e b a c t e r i a f i r s t u t i l i z e s i m p l e p r o t e i n a c e o u s c o m p o u n d s s u c h a s t h e r s a r c o p l a s m i c p r o t e i n s . B o r t o n et al. ( 1 9 7 0 a ) s t u d i e d t h e e f f e c t s o f f o u r b a c t e r i a l s p e c i e s {Pediococcus cerevisiae, Micrococcus luteus, Leuconostoc mesenteroides a n d Pseudomonas f r a g i ) o n t h e s o l u b i l i t y o f " t h e v a r i o u s p r o t e i n f r a c t i o n s o f p o r c i n e m u s c l e s t o r e d a t 2 a n d 1 0 ° C . T h e s o l u b i l i t i e s o f t h e v a r i o u s p r o t e i n f r a c t i o n s w e r e a f f e c t e d b y t h e i n o c u l a t i o n t r e a t m e n t . S t u d i e s r e v e a l e d a l o s s i n t h e w a t e r - s o l u b l e f r a c t i o n ( s a r c o p l a s m i c p r o t e i n s ) d u r i n g s t o r a g e o f t h e c o n t r o l s a n d t h e M. luteus a n d L. mesenteroides t r e a t e d s a m p l e s . S a m p l e s t r e a t e d w i t h P. fragi e v i d e n c e d a n i n i t i a l l o s s , f o l l o w e d b y a n i n c r e a s e . T h e s o l u b i l i t y o f t h e s a l t - s o l u b l e p r o t e i n s ( m y o f i b r i l l a r f r a c t i o n ) i n c r e a s e d d u r i n g t h e f i r s t 8 d a y s o f s t o r a g e , t h e n d e c r e a s e d o r r e m a i n e d r e l a t i v e l y c o n s t a n t . I n c o m p a r i s o n t o t h e o t h e r i n o c u l a t e d s a m p l e s , i n s o l u b l e p r o t e i n g e n e r a l l y i n c r e a s e d e x c e p t f o r P. fragi i n o c u l a t e d s a m p l e s w h i c h s h o w e d a d e c r e a s e . A l t h o u g h n o n - p r o t e i n n i t r o g e n i n c r e a s e d o n l y s l i g h t l y f o r a l l t r e a t m e n t s , i n c l u d i n g c o n t r o l s , d u r i n g t h e 20 d a y s t o r a g e p e r i o d , t h e P. fragi t r e a t e d s a m p l e s s h o w e d g r e a t i n c r e a s e s . B o r t o n et al. ( 1 9 7 0 b ) , i n a c o m p a n i o n p a p e r , u s e d s t a r c h u r e a - g e l a n d d i s c u r e a - g e l e l e c t r o p h o r e s i s t o s t u d y t h e e f f e c t s o f / 1 2 Pseudomonas fragi, Pediocooous cerevisiae, Leuoonostoc mesenteroides a n d Micrococcus luteus o n p o r c i n e m y o f i b r i l l a r p r o t e i n s s t o r e d a t 2 a n d 1 0 ° C . O f t h e m u s c l e s a m p l e s i n o c u l a t e d , o n l y s a m p l e s i n o c u l a t e d w i t h Pseudomonas fragi s h o w e d a l o s s i n t h e n u m b e r o f p r o t e i n b a n d s , i n d i c a t i n g t h i s o r g a n i s m e x h i b i t e d some p r o t e o l y t i c e f f e c t u p o n t h e m y o f i b r i l l a r p r o t e i n s . H a s e g a w a et al'. ( 1 9 7 0 a ) s t u d i e d t h e e f f e c t s o f Pseudomonas fragi, Micrococcus luteus, Leuoonostoc mesenteroides a n d Pediocooous cerevisiae u p o n t h e s a r c o p l a s m i c a n d u r e a - s o l u b l e p r o t e i n s f r o m p o r c i n e a n d r a b b i t m u s c l e . C o m p a r i s o n , o f s t a r c h g e l p a t t e r n s o f s a r c o p l a s m i c p r o t e i n s f r o m a s e p t i c a n d i n o c u l a t e d m u s c l e s a f t e r s t o r a g e a t 1 0 ° C i n d i c a t e d t h a t d i f f e r e n t m i c r o o r g a n i s m s p r e f e r e n t i a l l y u t i l i z e d s p e c i f i c p r o t e i n s . P. fragi c a u s e d e x t e n s i v e p r o t e o l y s i s i n b o t h r a b b i t a n d p o r c i n e s a r c o p l a s m i c p r o t e i n s . L. mesenteroides c a u s e d e x t e n s i v e a l t e r a t i o n t o t h e s a r c o p l a s m i c p r o t e i n s o f r a b b i t m u s c l e b u t h a d l e s s e f f e c t u p o n p o r c i n e m u s c l e . P. cerevisiae h a d n o e f f e c t o n p o r c i n e m u s c l e b u t e x e r t e d m a j o r p r o t e o l y t i c a c t i v i t y u p o n r a b b i t m u s c l e s a r c o p l a s m . M. luteus c a u s e d o n l y m i n o r b r e a k d o w n o f r a b b i t m u s c l e s a r c o p l a s m i c p r o t e i n s a n d h a d n o a c t i o n u p o n p o r c i n e m u s c l e . B o t h P. fragi a n d P. cerevisiae c a u s e d c o n s i d e r a b l e p r o t e o l y s i s o f t h e u r e a - s o l u b l e p r o t e i n s i n p o r c i n e m u s c l e , h o w e v e r , l i t t l e w a s d e t e c t e d i n r a b b i t m u s c l e . N e i t h e r M. luteus n o r L. mesenteroides e x e r t e d a n y m e a s u r a b l e e f f e c t u p o n t h e u r e a - s o l u b l e p r o t e i n s . T h e a u t h o r s c o n c l u d e d t h a t m e a t s p o i l a g e w a s "a highly complex process involving a number of organisms producing highly specific enzymes, which preferentially act upon only certain proteins or enzymes indigenous to muscle". A 3 I n a s u b s e q u e n t s t u d y , H a s e g a w a et al. ( 1 9 7 0 b ) u s e d s t a r c h - g e l e l e c t r o p h o r e s i s t o i n v e s t i g a t e t h e e f f e c t s o f Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaeiens, Streptococcus faecalis a n d Kurthia zopfii o n t h e s a r c o p l a s m i c a n d u r e a - s o l u b l e p r o t e i n s f r o m r a b b i t a n d p o r c i n e m u s c l e . R e s u l t s i n d i c a t e d t h a t C. perfringens c a u s e d e x t e n s i v e a l t e r a t i o n i n t h e s a r c o p l a s m i c f r a c t i o n o f p o r c i n e m u s c l e , w h e r e a s e s , enteritidis a n d S. faecalis c a u s e d o n l y m i n o r p r o t e o l y s i s . N e i t h e r A. liquefaeiens n o r K. zopfii p r o d u c e d a n y m e a s u r a b l e a m o u n t o f p r o t e o l y s i s i n t h e s a r c o p l a s m i c f r a c t i o n o f p i g m u s c l e . A l t h o u g h m i n o r p r o t e o l y s i s w a s e v i d e n c e d i n t h e e l e c t r o p h o r e t i c p a t t e r n s f o r r a b b i t m u s c l e , n o n e o f t h e t e s t o r g a n i s m s c a u s e d t h e c o m p l e t e d i s a p p e a r a n c e o f a n y p r o t e i n b a n d . E x a m i n a t i o n o f t h e u r e a - s o l u b l e p r o t e i n p a t t e r n s r e v e a l e d t h a t C. perfringens c a u s e d e x t e n s i v e p r o t e o l y s i s i n p o r c i n e m u s c l e , a l t h o u g h l i t t l e was d e t e c t e d i n r a b b i t m u s c l e . N o n e o f t h e o t h e r o r g a n i s m s i n v o l v e d c a u s e d a n y m e a s u r a b l e e f f e c t u p o n t h e u r e a - s o l u b l e p r o t e i n s . T a r r a n t et al. ( 1 9 7 1 ) s t u d i e d t h e a c t i o n o f Pseudomonas fragi o n t h e s a r c o p l a s m i c a n d m y o f i b r i l l a r p r o t e i n s o f p o r c i n e m u s c l e d u r i n g a 20 d a y i n c u b a t i o n p e r i o d . E l e c t r o p h o r e t i c p a t t e r n s i n d i c a t e d t h a t g r o w t h o f P. fragi -was a c c o m p a n i e d b y e x t e n s i v e p r o t e o l y s i s o f m y o f i b r i l l a r p r o t e i n s w i t h a c o r r e s p o n d i n g i n c r e a s e i n n o n - p r o t e i n n i t r o g e n ( p r i m a r i l y p e p t i d e s a n d a m m o n i a ) . M a j o r p r o t e o l y s i s d i d n o t o c c u r b e f o r e s p o i l a g e s i n c e q u a n t i t a t i v e c h a n g e s i n t h e p r o t e i n f r a c t i o n c o u l d n o t b e d e t e c t e d u n t i l a f t e r 8 d a y s o f i n c u b a t i o n . C o m p l e t e b r e a k d o w n o f t h e m y o f i b r i l l a r f r a c t i o n w a s n o t e v i d e n t u n t i l 2 0 d a y s . T h e s e i n v e s t i g a t o r s i n d i c a t e d t h a t , b e c a u s e o f t h e g r o w t h /14 of P. fragi on the surface of the muscle sample, proteolysis might have occurred at an early stage, but was not detected until spoilage had proceeded further and affected the entire mass of muscle. The study revealed that no preferential breakdown of a particular myofibrillar protein occurred. In addition,, no significant change (as measured by micro-Kjeldahl analysis) was observed in the sarcoplasmic protein fraction from the inoculated pork. The authors noted that the loss of sarcoplasmic protein may have been offset by the release of water-soluble fragments from the myofibrillar protein fraction. In an electron microscopy study of porcine muscle, Dutson et al. (1971) showed extreme disruption of myofibrils in the P. fragi. inoculated samples as compared to the uninoculated controls. Tarrant et al. (1973) studied the effect of a proteolytic enzyme preparation from Pseudomonas fragi on porcine muscle. Inoculation of the various protein fractions with the enzyme preparation resulted in rapid hydrolysis of the salt-soluble proteins; the sarcoplasmic proteins resisted hydrolysis. Buckley et al. (1974) studied the proteolytic activity of Pseudomonas perolens on porcine muscle at 10°C for 20 days. Protein solubility studies of the inoculated samples revealed a decrease in the sarcoplasmic protein fraction and a large increase in the myofibrillar protein,fraction. Non-protein nitrogen increased in both aseptic and inoculated samples, however, larger increases were noted in the inoculated samples. The authors concluded that enzyme production coincided with high bacterial numbers and pH increase, resulting in substantial changes in primary protein / 1 5 s o l u b i l i t y . S a g e ( 1 9 7 4 ) c o n d u c t e d a s t u d y i n v e s t i g a t i n g t h e e f f e c t o f Pseudomonas fragi o n m i n c e d c h i c k e n p e c t o r a l i s m u s c l e s t o r e d a t 2 5 ° C . " E l e c t r o p h o r e t i c p a t t e r n s i n d i c a t e d e v i d e n c e o f p r o t e o l y s i s o f t h e s a r c o p l a s m i c , m y o f i b r i l l a r a n d s t r o m a l f r a c t i o n s . T h e p H o f t h e i n o c u l a t e d s a m p l e s i n c r e a s e d a p p r e c i a b l y o v e r t h e 9 . 5 d a y i n c u b a t i o n p e r i o d f r o m 5 . 9 t o 8 . 5 , w h i l e t h e p H o f t h e a s e p t i c c o n t r o l s d e c r e a s e d f r o m 5 . 9 t o 5 . 5 . D a i n t y et al. ( 1 9 7 5 ) s t u d i e d t h e e f f e c t s o f v a r i o u s b a c t e r i a l s p e c i e s o n t h e p r o t e i n f r a c t i o n s o f b o v i n e m u s c l e u s i n g g e l e l e c t r o p h o r e s i s . R e s u l t s i n d i c a t e d t h a t p r o t e o l y s i s d i d n o t o c c u r i n t h e s l i m e i n o c u l a t e d o r n a t u r a l l y c o n t a m i n a t e d b e e f u n t i l s p o i l a g e o d o u r a n d s l i m e w e r e e v i d e n t . A l t h o u g h t h e s l i m e i n o c u l u m g a v e a f i n a l m i c r o b i a l f l o r a s i m i l a r t o t h e " n a t u r a l l y a t t a i n e d " o n e , i t r e s u l t e d i n m o r e r a p i d a n d e x t e n s i v e p o s t - s p o i l a g e p r o t e o l y s i s . P u r e c u l t u r e s t u d i e s i n d i c a t e d t h a t m e m b e r s o f t h e g e n e r a Pseudomonas a n d Aeromonas c a u s e d t h e g r e a t e s t d e g r e e o f p r o t e o l y s i s . T h e a u t h o r s p o s t u l a t e d t h a t t h e s l i m e i n o c u l a t e d s a m p l e s h a d a h i g h p r o p o r t i o n o f Pseudomonas s p p . , some o f w h i c h w e r e p r o t e o l y t i c . N u m e r o u s a u t h o r s ( J a y a n d K o n t o u , 1 9 6 7 ; O c k e r m a n et al., 1 9 6 9 ; B o r t o n et al., 1 9 7 0 a ; H a s e g a w a et al., 1 9 7 0 a a n d b ; T a r r a n t et al., 1 9 7 1 ; B u c k l e y et al., 1 9 7 4 ; O c k e r m a n a n d C a h i l l , 1 9 7 7 ) h a v e r e p o r t e d t h a t t h e p H o f m u s c l e i n c r e a s e s d u r i n g s p o i l a g e , u n l e s s s p o i l a g e i s c a u s e d b y a n a c i d p r o d u c i n g o r g a n i s m . T h e i n c r e a s e d p H i s c a u s e d b y t h e m i c r o b i a l p r o d u c t i o n o f " a l k a l i z i n g s u b s t a n c e s " s u c h a s a m m o n i a a n d a m i n e s ( T a r r a n t et al., 1 9 7 1 ; J a y , : 1 9 7 2 ) . / 1 6 D u r i n g s p o i l a g e , g r o w t h o f b a c t e r i a o c c u r s o n l y o n t h e m e a t s u r f a c e ( G i l l a n d N e w t o n , 1 9 7 8 ) . S u b s t r a t e u t i l i z a t i o n b y t h e s p o i l a g e f l o r a h a s b e e n e x a m i n e d b y a n u m b e r o f a u t h o r s . T h e b a c t e r i a i n i t i a l l y u t i l i z e t h e l o w m o l e c u l a r w e i g h t s o l u b l e c o m p o n e n t s o f m e a t ( I n g r a m a n d D a i n t y , 1 9 7 1 ) . G a r d n e r ( 1 9 6 5 ) s t u d i e d t h e c h a n g e s i n t o t a l c a r b o h y d r a t e ( a n t h r o n e v a l u e ) , g l u c o s e a n d g l y c o g e n i n b e e f s t o r e d a t 4 , 9 a n d 1 5 ° C , t o g e t h e r w i t h c h a n g e s i n t h e t o t a l a e r o b i c c o u n t . I n i t i a l l y , g l y c o g e n c o n c e n t r a t i o n f e l l i n a l l s a m p l e s , w i t h n o a p p a r e n t c h a n g e i n b a c t e r i a l c o u n t s ; s u b s e q u e n t i n c r e a s e s i n b a c t e r i a l 4 10 n u m b e r s f r o m 10 t o 10 p r o d u c e d l i t t l e o v e r a l l e f f e c t . I n t h e s a m e s a m p l e s t h e r e w a s , i n a l l c a s e s , a n i n i t i a l d e c r e a s e i n g l u c o s e c o n c e n t r a t i o n f o l l o w e d b y v a r y i n g d e g r e e s o f i n c r e a s e w i t h o u t s i g n i f i c a n t v a r i a t i o n i n b a c t e r i a l c o u n t . S u b s e q u e n t d e c r e a s e s i n g l u c o s e c o n c e n t r a -t i o n w e r e e x p e r i e n c e d a s b a c t e r i a l n u m b e r s i n c r e a s e d , w i t h a g r e a t e r r a t e o f c h a n g e a t 1 5 ° C t h a n a t 4 a n d 9 ° C . T o t a l c a r b o h y d r a t e c o n c e n t r a t i o n s f l u c t u a t e d a t a l l t h r e e t e m p e r a t u r e s , w i t h a n o v e r a l l t e n d e n c y t o i n c r e a s e u n t i l t h e b a c t e r i a l p o p u l a t i o n r e a c h e d a l e v e l o f 10 / g ; r a p i d d e c r e a s e s o c c u r r e d p a s t t h i s l e v e l . G i l l ( 1 9 7 6 ) e x a m i n e d t h e e f f e c t o f s u b s t r a t e l i m i t a t i o n o n b a c t e r i a l g r o w t h a t m e a t s u r f a c e s a n d f o u n d t h a t Pseudomonas s p p . i n i t i a l l y a t t a c k e d g l u c o s e b u t o n e x h a u s t i o n o f g l u c o s e , c o n t i n u e d t o g r o w a t t h e s a m e r a t e , u t i l i z i n g i n s t e a d s e v e r a l a m i n o a c i d s a n d l a c t a t e . T h e s e s e c o n d a r y s u b s t r a t e s d o n o t b e c o m e d e p l e t e d a t t h e s u r f a c e . T h e a u t h o r n o t e d t h a t s i n c e o r g a n o l e p t i c s p o i l a g e i s r e p o r t e d 8 2 t o b e c o m e d e t e c t a b l e w h e n t h e c e l l d e n s i t y e x c e e d s 10 o r g a n i s m s / c m , / 1 7 i t i s p r o b a b l e t h a t o f f - o d o u r s a r e p r o d u c e d o n l y w h e n t h e b a c t e r i a s t a r t t o d e g r a d e a m i n o a c i d s . S i m i l a r r e s u l t s r e g a r d i n g l o w m o l e c u l a r w e i g h t s u b s t r a t e u t i l i z a t i o n w e r e r e p o r t e d b y G i l l a n d N e w t o n ( 1 9 7 7 ) . S h e l e f ( 1 9 7 7 ) , s t u d y i n g t h e e f f e c t o f g l u c o s e o n t h e b a c t e r i a l s p o i l a g e o f b e e f , o b s e r v e d t h a t n o s p o i l a g e c h a r a c t e r i s t i c ' s s u c h a s s l i m e a n d o f f - o d o u r s w e r e o b s e r v e d u n t i l t h e g l u c o s e w a s d e p l e t e d a n d t h e p H s t a r t e d t o i n c r e a s e . I n c i p i e n t s p o i l a g e ( o f f - o d o u r s a n d t a c k i n e s s ) b e c a m e a p p a r e n t a b o v e a p H o f 6 . 0 . E. Bacterial Adhesion A l t h o u g h e x t e n s i v e r e s e a r c h h a s b e e n carried o u t o n t h e mechanismsuDf a t t a c h m e n t b y marine b a c t e r i a , v e r y l i t t l e h a s d e a l t w i t h t h e a d h e s i o n o f b a c t e r i a t o f o o d m y o s y s t e m s . N o t e r m a n s a n d K a m p e l m a c h e r ( 1 9 7 4 ) s t u d i e d t h e a t t a c h m e n t o f v a r i o u s f l a g e l l a t e d a n d n o n - f l a g e l l a t e d b a c t e r i a l s t r a i n s t o t h e s k i n o f b r o i l e r c h i c k e n s . R e s u l t s c l e a r l y i m p l i c a t e d t h e i m p o r t a n c e o f t h e f l a g e l l a i n t h e a t t a c h m e n t o f m i c r o o r g a n i s m s t o t h e skirt o f b r o i l e r s . T h e l o w d e g r e e o f a t t a c h m e n t b y t h e n o n - f l a g e l l a t e d b a c t e r i a {E. coli K 1 2 N 9 7 + ) c o m P a r e d t o f l a g e l l a t e d b a c t e r i a (E. coli K ^ ) u n d e r l i n e d t h i s f a c t . T h e a u t h o r s a l s o n o t e d t h a t p H a n d t e m p e r a t u r e w e r e i m p o r t a n t p a r a m e t e r s . B y l o w e r i n g t h e p H o f t h e a t t a c h m e n t m e d i u m , t h e a t t a c h m e n t r a t e w a s d e c r e a s e d d u e t o t h e r e d u c e d m o t i l i t y o f t h e m i c r o o r g a n i s m s . T h e o p t i m a l t e m p e r a t u r e f o r a t t a c h m e n t o f t h e d i f f e r e n t f l a g e l l a t e d b a c t e r i a w a s f o u n d t o b e a r o u n d 2 1 ° C . T h e i n c r e a s i n g r a t e o f / 1 8 a t t a c h m e n t f r o m 0 C t o 21 C w a s e x p l a i n e d b y t h e c o m b i n e d i n c r e a s e i n a c t i v i t y o f m i c r o o r g a n i s m s a n d f l a g e l l a . I n a s u b s e q u e n t s t u d y , N o t e r m a n s a n d K a m p e l m a c h e r ( 1 9 7 5 ) r e p o r t e d t h a t a p r o p o r t i o n o f t h e b a c t e r i a l f l o r a a s s o c i a t e d w i t h t h e c h i c k e n s k i n w a s a c t u a l l y p r e s e n t i n a w a t e r f i l m s u r r o u n d i n g t h e s k i n , a n d c o u l d b e e a s i l y r e m o v e d b y a d e q u a t e r i n s i n g . I t w a s n o t e d t h a t t h e b a c t e r i a l f l o r a i n t h e w a t e r f i l m w a s o f g r e a t i m p o r t a n c e s i n c e i t a p p e a r e d " t o p l a y a k e y r o l e " i n t h e a t t a c h m e n t o f b a c t e r i a t o c h i c k e n s k i n ; a t t a c h m e n t b e i n g t i m e - d e p e n d e n t a n d i n p r o p o r t i o n t o t h e n u m b e r o f b a c t e r i a p r e s e n t . M c M e e k i n et al. ( 1 9 7 9 ) c o n d u c t e d a s c a n n i n g e l e c t r o n m i c r o s c o p y i n v e s t i g a t i o n o f t h e m i c r o o r g a n i s m s o n c h i c k e n s k i n . T h e " m i c r o t o p o g r a p h y " o f t h e s u r f a c e w a s a n i m p o r t a n t p a r a m e t e r i n r e l a t i o n t o c o n t a m i n a t i o n s i n c e c r e v i c e s a n d c h a n n e l s o n t h e s u r f a c e s w e r e o f c a p i l l a r y s i z e a n d o n c e c o n t a m i n a t e d , w o u l d b e e x t r e m e l y d i f f i c u l t t o c l e a n . M i c r o b i a l g r o w t h d e v e l o p e d i n a " l a y e r o f m a t e r i a l " o n t h e s u r f a c e o f t h e c h i c k e n s k i n w h i c h may c o r r e s p o n d t o t h e l i q u i d f i l m r e f e r r e d t o b y N o t e r m a n s a n d . K a m p e l m a c h e r ( 1 9 7 4 ) . T h e a u t h o r s c o n c l u d e d t h a t t h e l a y e r o f m a t e r i a l o n t h e s u r f a c e , a s w e l l a s t h e t o p o g r a p h y , m a y e x p l a i n t h e o b s e r v a t i o n t h a t v i a b l e c o u n t s o b t a i n e d w i t h m a c e r a t e d c h i c k e n w e r e a l w a y s g r e a t e r t h a n t h o s e o b t a i n e d w i t h s w a b s o r r i n s e s . I n a n o t h e r s t u d y , B u t l e r et al. ( 1 9 7 9 ) i n v e s t i g a t e d t h e a t t a c h m e n t o f m i c r o o r g a n i s m s t o a s e p t i c s a m p l e s o f p o r k s k i n a n d t h i n / 1 9 s u r f a c e s l i c e s o f b e e f a n d l a m b c a r c a s s e s . R e s u l t s i n d i c a t e d a d i r e c t r e l a t i o n s h i p b e t w e e n b a c t e r i a l a t t a c h m e n t a n d c o n c e n t r a t i o n o f t h e t e s t o r g a n i s m s i n t h e a t t a c h m e n t m e d i u m : t h e g r e a t e r t h e c o n c e n t r a t i o n o f t e s t o r g a n i s m s , t h e h i g h e r t h e n u m b e r o f a t t a c h e d o r g a n i s m s . A t t a c h m e n t o f m o t i l e , g r a m - n e g a t i v e s p e c i e s {Escherichia coli, Pseudomonas putrefaciens a n d Erwinia herbicola) w a s g r e a t e r t h a n t h a t o f t h e n o n - m o t i l e , g r a m - p o s i t i v e s p e c i e s {Lactobacillus s p p . a n d Staphylococcus s p p . ) . I n c o n t r a s t t o t h e s t u d i e s o f N o t e r m a n s a n d K a m p e l m a c h e r ( 1 9 7 4 a n d 1 9 7 5 ) , B u t l e r et al. ( 1 9 7 9 ) f o u n d t h a t t h e e f f e c t s o f i m m e r s i o n t i m e , t e m p e r a t u r e a n d p H o f t h e i n o c u l a t e d a t t a c h m e n t m e d i u m o n a t t a c h m e n t w e r e i n s i g n i f i c a n t . T h i s w a s e x p l a i n e d b y t h e m a r k e d d i f f e r e n c e s t h a t e x i s t b e t w e e n c h i c k e n s k i n a n d t h e s a m p l e s u s e d i n t h e s t u d y . T h e a b i l i t y t o a c c u r a t e l y d e t e r m i n e t h e n u m b e r s a n d t y p e s o f m i c r o o r g a n i s m s o n a m u s c l e : s u r f a c e i s n o t o n l y i m p o r t a n t f r o m a f o o d s a f e t y p o i n t o f v i e w , b u t a l s o f r o m a q u a l i t y e v a l u a t i o n a s p e c t . A l t h o u g h n u m e r o u s t e c h n i q u e s h a v e b e e n d e v i s e d f o r t h e e v a l u a t i o n o f m i c r o b i a l p o p u l a t i o n s , t h e e f f e c t i v e n e s s o f s o m e o f t h e s e p r o c e d u r e s i s q u e s t i o n a b l e . Some t e c h n i q u e s , s u c h a s n o n - d e s t r u c t i v e s w a b b i n g , h a v e g i v e n e s t i m a t e s o f o n l y a b o u t 16% o f t h e m i c r o b i a l p o p u l a t i o n a s c o m p a r e d t o t h e m o r e d e s t r u c t i v e t i s s u e m a c e r a t i o n p r o c e d u r e s ( N i s k a n e n a n d P b h j a , 1 9 7 7 ) . T h e r e m o v a l a n d s u b s e q u e n t a b i l i t y t o d e t e r m i n e a c c u r a t e l y t h e n u m b e r a n d t y p e s o f b a c t e r i a f r o m a m u s c l e s u r f a c e , b y a n y p r o c e d u r e , i s d e p e n d e n t u p o n t h e f o r c e s b y w h i c h t h e m i c r o o r g a n i s m s a r e a t t a c h e d ( B u t l e r et al., 1 9 7 9 ) . T h e r e f o r e , a b e t t e r u n d e r s t a n d i n g / 2 0 o f t h e m e c h a n i s m s b e h i n d a t t a c h m e n t m a y a i d , n o t o n l y i n t h e s e l e c t i o n o f a p r o c e d u r e f o r e s t i m a t i n g n u m b e r s a n d t y p e s o f b a c t e r i a o n m u s c l e s u r f a c e s , b u t a l s o i n t e c h n i q u e s t o r e d u c e b a c t e r i a l n u m b e r s , t h e r e b y i n c r e a s i n g s h e l f - l i f e ( B u t l e r et al., 1 9 7 9 ) . I n f o r m a t i o n a b o u t t h e m e c h a n i s m s o f b a c t e r i a l a t t a c h m e n t a r e l i m i t e d . M a r s h a l l et al. ( 1 9 7 1 ) s t a t e d t h a t a t t a c h m e n t e n t a i l e d a n " i n s t a n t a n e o u s r e v e r s i b l e p h a s e " a n d a " t i m e - d e p e n d e n t i r r e v e r s i b l e p h a s e " . T h e r e v e r s i b l e p h a s e i n v o l v e d a w e a k a t t a c h m e n t o f t h e b a c t e r i a t o a s u r f a c e t h r o u g h a t t r a c t i v e f o r c e s s u c h a s L o n d o n - v a n d e r W a a l s f o r c e s . I n t h i s s t a t e t h e b a c t e r i a w e r e s t i l l a b l e t o e x h i b i t B r o w n i a n m o t i o n a n d w e r e r e a d i l y r e m o v e d b y w a s h i n g t h e s u r f a c e w i t h 2 . 5 % N a C l . T h e t i m e - d e p e n d e n t i r r e v e r s i b l e p h a s e i n v o l v e d t h e f o r m a t i o n o f p o l y m e r s b e t w e e n b a c t e r i a a n d s u r f a c e a s a r e s u l t o f 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 t h i s l a t t e r p h a s e , t h e b a c t e r i a w e r e f i r m l y a t t a c h e d t o t h e s u r f a c e , e x h i b i t i n g n o B r o w n i a n m o t i o n a n d w e r e n o l o n g e r r e m o v a b l e b y w a s h i n g w i t h 2 . 5 % N a C l . M c C o w a n et al. ( 1 9 7 8 ) s t u d i e d t h e a d h e s i o n o f b a c t e r i a t o e p i t h e l i a l c e l l s u r f a c e s w i t h i n t h e r e t i c u l o - r u m e n o f c a t t l e u s i n g t r a n s m i s s i o n (TEM) a n d s c a n n i n g e l e c t r o n m i c r o s c o p y ( S E M ) . E x a m i n a t i o n o f t h e s u r f a c e o f t h e e p i t h e l i a l c e l l s b y S E M s h o w e d i n t e r m i t t e n t c o l o n i z a t i o n w i t h t h e "formation of occasional microcolonies of morphologically similar b a c t e r i a l cells". T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y ( i n t h e p r e s e n c e o f r u t h e n i u m r e d ) r e v e a l e d t h e p r e s e n c e o f c a r b o h y d r a t e m a t e r i a l ( g l y c o c a l y x ) o n b o t h t h e e p i t h e l i a l c e l l s a n d t h e a d h e r e n t b a c t e r i a . T h e g l y c o c a l y x o f t h e b a c t e r i a a p p e a r e d /21 to mediate the attachment of bacteria to the epithelium, to food particles and to each other. Costerton et al. (1974b) indicated that acid polysaccharides were involved in the adhesion process. The authors noted that these polysaccharides could be in the form of pure polysaccharides, glycoproteins or other mixed polymers. Notermans et al. (1979) investigated the attachment of bacteria to sterile teats of cows at various storage temperatures and reported that after 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 extracellular substances. In a companion paper, Firstenberg-Eden et al. (1979) used SEM to observe that polymers, in the form of thin fibers, were produced during the storage of the inoculated teats. These fibers eventually thickened and formed slime. Fletcher and Floodgate (1973), using a ruthenium red-alcian blue stain combination, demonstrated an extracellular, acidic polysaccharide which was involved in the adhesion of marine bacterium to surfaces. According to the workers, the i n i t i a l stage of irreversible adhesion involves contact between the surface and the primary polysaccharide surrounding the bacteria, prior to the formation of secondary fibrous, acidic 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 m i n c i n g i n c r e a s e s t h e s u r f a c e a r e a , t h u s m a x i m i z i n g a n y c h a n g e s f r o m b a c t e r i a l g r o w t h ; h o w e v e r , t h e s e c h a n g e s m a y n o t b e i n d i c a t i v e o f t h o s e w h i c h o c c u r o n i n t a c t m y o s y s t e m s . I n o r d e r t o i n o c u l a t e t i s s u e w i t h o u t d i s r u p t i n g t h e s t r u c t u r a l i n t e g r i t y o f t h e m u s c l e , a t e c h n i q u e w h e r e b y t i s s u e i s d i p p e d i n t o a n i n o c u l a t e d p h y s i o l o g i c a l b u f f e r h a s b e e n e m p l o y e d . N o t e r m a n s a n d K a m p e l m a c h e r ( 1 9 7 4 ) u s e d t h e d i p m e t h o d o f w h o l e b r o i l e r c a r c a s s e s a s a m e a n s f o r i n o c u l a t i o n . U s i n g a s i m i l a r t e c h n i q u e , B u t l e r et at. ( 1 9 7 9 ) e m b e d d e d s t e r i l e t i s s u e ( p o r k s k i n a n d s u r f a c e s o f b e e f a n d l a m b c a r c a s s e s ) i n t o " G u l f " w a x p r i o r t o d i p p i n g i n a t t a c h m e n t m e d i a . /23 METHODS AND MATERIALS Bovine Longissimus dorsi muscle was excised from a 24 h postmortem steer, at a local abattoir, and transported on ice to the laboratory. Prior to sectioning, the muscle was sprayed with a 70% ethanol solution in order to reduce surface bacterial contamination (Hasegawa et al., 1970a). Thin slices (3 mm) were removed from the muscle using a Hobart delicatessen slicer (Don Mill s , Ont.) (Figure 1) which had been previously sprayed with 70% ethanol, as a bactericidal agent. Obvious adipose tissue was avoided. Upon completion of every f i f t h s lice, the slicer was sprayed with 70% ethanol. The above procedure was adopted in attempts to minimize the effects of microbial growth prior to gamma radiation. A. Sarcoplasmic Extraction Extraction of sarcoplasmic f l u i d was effected by placing the muscle slices in a 10.2 cm radius x 30.5 cm high sterile plexiglass tank (8 l i t e r capacity) f i l l e d with sterile 0.0100M Na2HP04~0.119 M NaH2P04 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 st i r r e r . In addition, f i l t e r s t erilized 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 bacterial vents (Gelman Instrument Company, Ann Arbor, MI) (Figure 3). F I G U R E 1 : H o b a r t d e l i c a t e s s e n s l i c e r . / 2 5 F I G U R E 2 : A p p a r a t u s u s e d f o r t h e e x t r a c t i o n o f t h e s a r c o p l a s r a f r o m i n t a c t m u s c l e . FIGURE 3: B a c t e r i a l vent used to s t e r i l i z e both a i r and nitrogen. Ill The b u f f e r s o l u t i o n was changed every hour f o r the f i r s t 4 h and then every 4 h f o r the next 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 at 4°C. Upon completion o f the e x t r a c t i o n procedure, a polypropylene 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 attached to each washed muscle sample at one extremity. Each muscle sample was placed i n a s t e r i l e 18 oz "Whirl-pale" bag (Arnold-Nasco L t d . , Guelph, Ont.) (Figure 4 ) . S i m i l a r procedures were c a r r i e d out f o r muscle samples not exposed to the e x t r a c t i o n treatment. These samples were termed " i n t a c t " . The "Whirl-pak" samples ( i n t a c t and washed) were then packed i n i c e and i r r a d i a t e d w i t h a 1 Megarad dose i n the Gammacell 220 (Atomic Energy o f Canada Ltd.) c o n t a i n i n g ^°Co (Sage, 1974). B. Preparation of the Inoculum A fr e e z e d r i e d pure c u l t u r e o f Pseudomonas fragi (ATCC 4973) was prepared as per American Type Culture C o l l e c t i o n Catalogue of S t r a i n s I (12th E d i t i o n , 1976). The c u l t u r e was maintained 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) at 4°C. P r i o r to i n o c u l a t i o n o f the 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 the TSA s l a n t and grown i n 200 ml of t r y p t i c a s e soy broth (TSB) (BBL, C o c k e y s v i l l e , MD) at 25°C f o r 24 h i n a shaking water bath. The c u l t u r e was then c e n t r i f u g e d f o r 20 min (1000 xg; 10°C). The p e l l e t was resuspended i n 50 ml of s t e r i l e F I G U R E 4 : " W h i r l - p a k " m u s c l e s a m p l e . /29 attachment media consisting of 0.15 M NaCl, 0.0062 M Na2HP04, 0.0021 M NaH2P04 and 0.001 M EDTA, adjusted to pH 5.8 (Notermans and Kampelmacher, 1974; Butler et al., 1979) and mixed vigorously to obtain a uniform suspension. The suspension was then centrifuged at 10°C for 20 min, at 1000 xg. The resuspension-centrifugation procedure was repeated twice to ensure complete removal of the TSB. Upon completion, the washed pellet was resuspended in 4 I of sterile 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 sterilized samples (intact and washed) were immersed for 10 min into the 4 1 of attachment media containing P. fragi (Butler et al., 1979). After drainage for 10 sec, the muscle samples were placed into s t e r i l e stomacher bags inverted and hung in 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 positive pressure was applied by passing f i l t e r s t erilized medical grade compressed air (100 ml/min) through the chamber. Control samples were treated in a similar manner with st e r i l e attachment- media. Thus, control uninoculated samples were available at each storage period so that one could differentiate changes induced by microbial growth with those of autolysis (Rampton et al., 1970). F I G U R E 5: P l e x i g l a s s i n c u b a t i o n c h a m b e r . /31 D. pH Determination The pH of the muscle samples was determined a f t e r each in 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 with 5 ml of d i s t i l l e d - d e i o n i z e d water i n an Omni mixer (Ivan S o r v a l l Inc., Norwalk, CN) at h a l f speed f o r 15 sec and top speed f o r 45 sec (Buckley et al., 1974). The pH of the r e s u l t a n t s l u r r y was then 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 both 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 (approximately 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 of 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 then added, and the sample stomached f o r 2 min i n a Colworth stomacher lab-blender 400 (A. J . Seward, London, England). Appropriate s e r i a l decimal d i l u t i o n s of the macerated sample were prepared with s t e r i l e 0.1% peptone br o t h , and were surface spread, i n d u p l i c a t e , on TSA. A l l p l a t e s were incubated at approximately 25°C f o r 48 h to o b t a i n the t o t a l v i a b l e count. B a c t e r i a l enumeration was a l s o c a r r i e d out on the attachment media i n order to evaluate the e f f e c t i v e n e s s of the attachment procedure. / 3 2 F. Electron Microscopy 1. Scanning EM preparation M u s c l e t i s s u e was c u t i n t o a p p r o x i m a t e l y 2 cm x 2 cm p i e c e s a n d f i x e d i n 2 . 5 % ( v / v ) e l e c t r o n m i c r o s c o p i c g r a d e g l u t a r a l d e h y d e ( C A N - E M C h e m i c a l s , G u e l p h , O n t . ) - 0 . 0 5 M p h o s p h a t e b u f f e r p H 7 . 0 ( 6 1 m l 0 . 0 5 M N a ^ H E O ^ + 3 9 m l 0 . 0 5 M K H 2 P C > 4 ) o v e r n i g h t . T h e t i s s u e w a s t h e n w a s h e d t w i c e i n 0 . 0 5 M p h o s p h a t e b u f f e r ( p H 7 . 0 ) p r i o r t o p o s t - f i x i n g i n 1% ( w / v ) o s m i u m t e t r o x i d e ( C A N - E M C h e m i c a l s , G u e l p h , O n t . ) - 0 . 0 5 M p h o s p h a t e b u f f e r ( p H 7 . 0 ) f o r 1 h ( S j o s t r a n d , 1 9 6 7 ) . S a m p l e s w e r e t h e n s u b s e q u e n t l y w a s h e d 3 t i m e s w i t h 0 . 0 5 M p h o s p h a t e b u f f e r ( pH 7 . 0 ) . T h i s w a s f o l l o w e d b y d e h y d r a t i o n t h r o u g h a n a s c e n d i n g s e r i e s o f e t h a n o l : 5 0 , 7 0 a n d 80% ( v / v ) e t h a n o l f o r 5 m i n e a c h ; 2 c h a n g e s , o f 10 m i n e a c h , w i t h 9 0 % ; a n d 3 c h a n g e s , o f 20 m i n e a c h , w i t h 100% e t h a n o l . A l l e t h a n o l d i l u t i o n s w e r e m a d e w i t h d i s t i l l e d - d e i o n i z e d w a t e r . S a m p l e s w e r e t h e n s u b j e c t e d t o i n f i l t r a t i o n b y a g r a d e d s e r i e s o f a m y l a c e t a t e ( F i s h e r S c i e n t i f i c C o . , F a i r l a w n , N J ) : o n e c h a n g e , o f 10 m i n d u r a t i o n e a c h , w i t h 2 5 , 50 a n d 75% ( v / v ) a m y l a c e t a t e d i l u t e d w i t h a b s o l u t e e t h a n o l a n d o n e c h a n g e , o f 1 h d u r a t i o n , w i t h 100% a m y l a c e t a t e . S a m p l e s w e r e c r i t i c a l - p o i n t d r i e d i n a P a r r - b o m b ( P a r r I n s t r u m e n t C o . , M o l i n e , I L ) a n d m o u n t e d o n a l u m i n u m s t u b s w i t h s i l v e r p a s t e ( S t r u c t u r e P r o b e I n c . , W e s t C h e s t e r , P A ) . S a m p l e s w e r e t h e n c o a t e d w i t h g o l d - p a l l a d i u m i n a s p u t t e r c o a t e r ( T e c h n i c s I n c . , A l e x a n d r i a , V A ) a n d o b s e r v e d o n a H i t a c h i S - 5 0 0 /33 scanning electron microscope at an accelerating voltage of 20 KV. Images were recorded on Ilford Pan F 135 fine grain black and white film (Ilford Ltd., Essex, England). 2. Transmission EM preparation Samples for transmission electron microscopy were prepared according to the method of McCowan et at. (1978)' with slight modifications. Tissues were fixed in 2.5% glutaraldehyde -0.05 M phosphate buffer pH 7.0 (see above buffer) overnight and subsequently placed in 0.015% (w/v) ruthenium red (Aldrich Chemical Co., Milwaukee, WI) in 0.05M phosphate buffer for 2 h. The samples were placed in 0.05 M phosphate buffer with 0.05% (w/v) ruthenium red (ruthenium red-phosphate buffer) for 2 h at room temperature and then washed in 0.05% ruthenium red-phosphate buffer 5 times for 1 h each time, with one overnight wash included, before being post-fixed for 2 h in 2% osmium tetroxide in ruthenium red-phosphate buffer. After five further 1 h washes in ruthenium red-phosphate buffer, samples were subjected to an ethanol dehydration series of 20 min each, in 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 distilled-deionized water. Samples were washed in two changes of 100% propylene oxide (Polysciences, Inc., Warrington, PA) for 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 in 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 citrate (Reynolds, 1963; Sjostrand, 1967). Electron micrographs were obtained with a Zeiss EM-10 (Carl Zeiss, Oberkochen, West Germany) transmission electron microscope operated at an accelerating voltage of 60-80 kV and recorded on Kodak 70 mm, fine grain film (Eastman Kodak Co., Rochester, NY). Upon completion of the above procedures (bacterial counts, pH determination and EM sample preparation), the muscle samples were frozen in liquid nitrogen and subsequently lyophilized until ready for analysis. G. Extraction of Water- and Salt-Soluble Protein Fractions Procedures similar to those outlined by Hasegawa et al. (1970a) were adopted for the extraction and water- and salt-soluble protein: fractions (Figure 6). The following procedure was carried out for 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 proteolytic activity of P. fragi. Five ml of 0.3 M sucrose - 0.01 M KC1 - 0.01 M Tris-citrate buffer (pH 7.6) was added to 0.5 g of a lyophilized muscle sample and homogenized in a Polytron mixer (Brinkman Instruments, Rexdale, Ont.) / 3 5 L y o p h i l i z e d M u s c l e S a m p l e h o m o g e n i z e d w i t h ' 1 0 v o l ( 1 0 v o l / w ) o f 0 , 3 M s u c r o s e , 0 . 0 1 M K C 1 , 0 . 0 1 M T r i s 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 S u p e r n a t a n t : w a t e r s o l u b l e p r o t e i n s a n d n o n - p r o t e i n n i t r o g e n P r e c i p i t a t e h o m o g e n i z e d w i t h 6 v o l ( 6 v o l / w ) o f s u c r o s e - K C l - T r i s 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 S u p e r n a t a n t : d i s c a r d P r e c i p i t a t e h o m o g e n i z e d w i t h 6 y o l (6 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 , s t o r e d f o r 2 4 h , 8 v o l (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 a d d e d , h o m o g e n i z e d a n d 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 S u p e r n a t a n t : s a l t - s o l u b l e p r o t e i n s P r e c i p i t a t e h o m o g e n i z e d w i t h 6 v o l ( 6 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 a n d c e n t r i f u g e d a t 2 9 , 0 0 0 x g f o r 3 0 m i n S u p e r n a t a n t : d i s c a r d P r e c i p i t a t e h o m o g e n i z e d w i t h 6 v o l (6 v o l / w ) o f 8M u r e a a n d c e n t r i f u g e d a t 2 9 , 0 0 0 x g f o r 3 0 m i n S u p e r n a t a n t ; u r e a - s o l u b l e p r o t e i n s P r e c i p i t a t e : u r e a - i n s o l u b l e p r o t e i n s P I G U R E 6 : F l o w s h e e t o f p r o t e i n e x t r a c t i o n p r o c e d u r e . A l l e x t r a c t i o n s w e r e c a r r i e d o u t . a t 4 ° C , /36 at 1/4 speed for 15 sec and at.1/2 speed for 15 sec. The homogenate was centrifuged (20,000 xg, 15 min, 4°C). The supernatant containing the water-soluble protein fraction .and the non-protein nitrogen, was decanted and stored at 4°C. The precipitate, along with 3 ml of the sucrose-KCl-Tris buffer, was returned to the Polytron mixer and homogenized for 30 sec at 1/2 speed. The mixture was centrifuged at 20,000 xg for 15 min and the supernatant discarded. The precipitate was then homogenized with 3 ml of Weber-Edsall solution (0.6 M KC1, 0.04 M KHC03, 0.1 M K 2C0 3) for 30 sec at 1/2 speed, and stored for 24 h at 4°C. After storage, 4 ml of Weber-Edsall solution were added and the solution mixed with the Polytron mixer at.1/4 speed for 15 sec. The mixture was centrifuged for 30 min at 29,000 xg. The supernatant consisting of the salt-soluble protein fraction was decanted and stored at 4°C un t i l ready for analysis, while the precipitate was homogenized with 3 ml of Weber-Edsall solution for 15 sec at 1/4 speed. The mixture was then centrifuged for 30 min at 29,000 xg and the supernatant discarded. The precipitate was homogenized with 3 ml of 8 M urea at 1/4 speed for 15 sec and 1/2 speed for 15 sec, and the mixture was centrifuged for 30 min at 29,000 xg. The supernatant, containing the urea-soluble protein fraction as well as the urea-insoluble precipitate 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 a n d S o l t e s s ( 1 9 7 3 ) u s i n g t h e T e c h n i c o n A u t o A n a l y z e r ( T e c h n i c o n I n s t r u m e n t s C o r p . , T a r r y t o w n , N Y ) . A n i t r o g e n t o p r o t e i n c o n v e r s i o n f a c t o r ; . o f 6 . 0 w a s u s e d ( L o c k e r , 1 9 6 0 ) . N i t r o g e n w a s e x p r e s s e d a s mg p r o t e i n n i t r o g e n p e r g r a m d r y w e i g h t . S o l u b l e n o n - p r o t e i n n i t r o g e n w a s o b t a i n e d b y t h e p r e c i p i t a t i o n o f t h e w a t e r - s o l u b l e p r o t e i n f r a c t i o n w i t h a n e q u a l v o l u m e o f 0 ° C 20% ( w / v ) t r i c h l o r o a c e t i c a c i d ( T a r r a n t et al., 1 9 7 1 ) . P r e l i m i n a r y e x p e r i m e n t s r e v e a l e d t h a t t h e n i t r o g e n c o n t e n t o f t h e s o l u b l e n o n - p r o t e i n n i t r o g e n w a s b e l o w t h e s e n s i t i v i t y o f t h e T e c h n i c o n A u t o A n a l y z e r , t h e r e f o r e , n i t r o g e n was d e t e r m i n e d b y t h e m i c r o - K j e l d a h l m e t h o d - o f f i c i a l f i n a l a c t i o n AOAC 4 7 . 0 2 1 ( A O A C , 1 9 7 5 ) . N o n - p r o t e i n n i t r o g e n w a s e x p r e s s e d a s mg n i t r o g e n p e r g r a m d r y w e i g h t . I. Total Carbohydrate Analysis • 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 t h e v a r i o u s m u s c l e s a m p l e s w a s d e t e r m i n e d b y t h e p h e n o l - s u l f u r i c a c i d m e t h o d ( D u b o i s et al., 1 9 5 6 ) . I n o r d e r t o r e d u c e t h e i n h e r e n t e r r o r i n t r o d u c e d b y b l e n d i n g a n d p i p e t t i n g o f h o m o g e n i z e d m u s c l e t i s s u e , a t t e m p t s w e r e m a d e t o s o l u b i l i z e t h e t i s s u e u s i n g v a r i o u s r e d u c i n g a g e n t s ( 8 M u r e a , 1.0% ( w / v ) - s o d i u m d o d e c y l s u l f a t e ( S D S ) , 0 . 1 % ( v / v ) 3 - m e r c a p t o e t h a n o l ) , h o w e v e r , n o n e p r o v e d s u c c e s s f u l . A n a l y s e s w e r e f i n a l l y p e r f o r m e d b y m i x i n g 10 mg o f f i n e l y g r o u n d , f r e e z e - d r i e d , w a s h e d m u s c l e t i s s u e o r 5 mg o f f i n e l y g r o u n d , l y o p h i l i z e d , i n t a c t m u s c l e s a m p l e s w i t h 2 m l o f d i s t i l l e d - d e i o n i z e d w a t e r i n a t e s t t u b e . T h e t u b e s w e r e s t o p p e r e d a n d p l a c e d i n a 1 0 0 ° C w a t e r b a t h f o r 1 m i n . P r e l i m i n a r y /38 experiments had shown that this latter step was necessary in order for the sulfuric acid to complete the hydrolysis of the muscle tissue. After the samples were cooled to room temperature, 0.05 ml of 80% (w/v) phenol was added. Five ml of concentrated sulfuric acid were then added rapidly. Vortexing of the mixture was required immediately after the addition of the sulfuric acid in order to complete the hydrolysis of the muscle samples. The tubes were allowed to stand 10 min, then shaken, and subsequently incubated in a 25°C water bath for 15 min prior 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 for glucose. In order to test for the possibility of a Maillard 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) for total carbohydrate content. Preliminary results indicated a 96% recovery of the added carbohydrate. It was therefore concluded that the heat treatment used did not substantially affect the effectiveness of the carbohydrate assay procedure (phenol-sulfuric acid). J. SDS-Gel Electrophoresis SDS-gel electrophoresis was performed on a l l (control and inoculated) water-, salt-, urea-soluble and urea-insoluble proteins / 3 9 i n a P h a r m a c i a d i s c g e l e l e c t r o p h o r e s i s s y s t e m ( M o d e l G E - 4 , P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , S w e d e n ) . F o l l o w i n g t h e p r o c e d u r e o f W e b e r a n d O s b o r n ( 1 9 6 9 ) a n d W e b e r et al. ( 1 9 7 2 ) , 7 . 5 % ( w / v ) a c r y l a m i d e ( B i o R a d L a b o r a t o r i e s , R i c h m o n d , C A ) g e l s w e r e r u n i n 5 mm ( i d ) x 75 mm t u b e s . T h e g e l s , a s w e l l a s t h e u p p e r a n d l o w e r r e s e r v o i r s , c o n t a i n e d 0 . 1 M s o d i u m p h o s p h a t e b u f f e r p H 7 . 2 ( 7 . 8 g N a H ^ P O ^ H ^ O , 3 8 . 6 g N a 2 H P , 0 4 - 7 H 2 0 , w a t e r t o 2 l i t e r s ) a n d 0 . 1 % ( w / v ) e l e c t r o p h o r e t i c a l l y p u r e s o d i u m d o d e c y l s u l f a t e ( S D S ) (BDH C h e m i c a l s L t d . , P o o l e , E n g l a n d ) . V a r i o u s q u a n t i t i e s o f p r o t e i n e x t r a c t , a n d p r o t e i n s o f k n o w n m o l e c u l a r w e i g h t ( T a b l e 1 ) , w e r e b o i l e d i n s t o p p e r e d t e s t t u b e s c o n t a i n i n g 0 . 1 m l o f 0 . 1 M p h o s p h a t e b u f f e r , 0 . 9 m l o f 8. M u r e a , 0 . 0 1 m l 3 - m e r c a p t o e t h a n o l a n d 0 . 0 1 5 - 0 . 0 3 g SDS f o r 5 m i n ( D e u t s c h , 1 9 7 6 ; K o c a l , 1 9 7 7 ) . S a l t - s o l u b l e p r o t e i n p r e p a r a t i o n s w e r e k e p t a b o v e 4 0 ° C t o p r e v e n t c r y s t a l l i z a t i o n o f t h e SDS i n t h e p r e s e n c e o f a h i g h c o n c e n t r a t i o n o f p o t a s s i u m i o n s ( H a y et:al., 1 9 7 3 ) . V a r i o u s v o l u m e s o f s a m p l e p r e p a r a t i o n ( T a b l e 1) w e r e a p p l i e d i n d i v i d u a l l y t o t h e g e l s ; 10 p i o f 0 . 0 0 1 % ( w / v ) b r o m o p h e n o l b l u e ( M a t h e s o n , C o l e m a n a n d B e l l , N o r w o o d , OH) i n 0 . 1 M s o d i u m p h o s p h a t e b u f f e r p H 7 . 2 a n d o n e d r o p o f g l y c e r o l w e r e a d d e d a n d m i x e d ( K o c a l , 1 9 7 7 ) . T h e g e l s w e r e t h e n c a r e f u l l y o v e r l a y e d w i t h 0 . 1 M s o d i u m p h o s p h a t e b u f f e r . T h e g e l s w e r e s u b j e c t e d t o e l e c t r o p h o r e s i s a t 2 0 - 2 5 ° C a t a c o n s t a n t c u r r e n t o f 2 mA p e r g e l f o r t h e f i r s t 3 0 m i n a n d 5 mA p e r g e l t h e r e a f t e r . T h e r u n w a s c o m p l e t e d w h e n t h e b r o m o p h e n o l b l u e ( t r a c k i n g d y e ) was a p p r o x i m a t e l y 0 . 5 cm f r o m t h e e n d o f t h e g e l . T h e p o s i t i o n o f t h e t r a c k i n g d y e w a s m a r k e d w i t h a n e e d l e t i p d i p p e d i n I n d i a i n k . T h e g e l s /40 1 T A B L E 1 : Q u a n t i t y o f p r o t e i n f r a c t i o n a n d m o l e c u l a r w e i g h t 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 a n d a m o u n t o f p r e p a r a t i o n a p p l i e d t o g e l d u r i n g S D S - g e l e l e c t r o p h o r e s i s . Q u a n t i t y u s e d f o r A m o u n t o f p r e p a r a t i o n P r o t e i n f r a c t i o n p r e p a r a t i o n (mg) a p p l i e d t o g e l ( p i ) W a t e r - s o l u b l e 1 0 0 10 S a l t - s o l u b l e 1 0 0 50 U r e a - s o l u b l e 1 0 0 5 0 U r e a - i n s o l u b l e 10 20 M o l e c u l a r w e i g h t m a r k e r s 10 10 P r o t e i n f r a c t i o n s w e r e t h o s e o b t a i n e d d u r i n g t h e p r o t e i n e x t r a c t i o n p r o c e d u r e / 4 1 w e r e f i x e d a n d s t a i n e d i n a s o l u t i o n o f 0 . 2 5 % ( w / v ) C o o m a s s i e B r i l l i a n t B l u e R ( I N C P h a r m a c e u t i c a l s , P l a i n v i e w , N Y ) i n 4 5 . 4 % ( v / v ) m e t h a n o l -9 . 2 % ( v / v ) g l a c i a l a c e t i c a c i d ( W e b e r et al., 1 9 7 2 ) f o r 4 5 m i n a n d d e s t a i n e d b y d i f f u s i o n i n 5% ( v / v ) m e t h a n o l - 7 . 5 % ( v / v ) g l a c i a l a c e t i c a c i d u n t i l t h e b a c k g r o u n d was c l e a r . G e l s w e r e s c a n n e d i n a G e l m a n G e l s c a n ( G e r m a n I n s t r u m e n t C o . , A n n A r b o r , M I ) s c a n n i n g d e n s i t o m e t e r . R e l a t i v e m o b i l i t i e s w e r e c a l c u l a t e d f r o m t h e d e n s i t o m e t r i c t r a c e a s t h e r a t i o o f t h e d i s t a n c e m i g r a t e d b y t h e p r o t e i n o v e r t h e d i s t a n c e m i g r a t e d b y t h e t r a c k i n g d y e . A p p a r e n t m o l e c u l a r w e i g h t s w e r e e s t i m a t e d f r o m t h e r e g r e s s i o n e q u a t i o n ( 1 ) d e r i v e d f r o m t h e m o b i l i t y o f t h e m o l e c u l a r w e i g h t m a r k e r s ( T a b l e 2 ) . L o g Mwt = a + bRm ( 1 ) Mwt = m o l e c u l a r w e i g h t a = i n t e r c e p t b = s l o p e o f r e g r e s s i o n l i n e Rm = r e l a t i v e m o b i l i t y o f t h e p r o t e i n T A B L E 2 : M o l e c u l a r w e i g h t m a r k e r s f o r S D S - g e l e l e c t r o p h o r e s i s . P r o t e i n C o m p a n y M o l e c u l a r w e i g h t ' ' " B o v i n e s e r u m a l b u m i n b o v i n e S i g m a 6 7 , 0 0 0 B o v i n e s e r u m a l b u m i n b o v i n e p r e p a r e d a s p e r P a y n e ( 1 9 7 3 ) 1 3 4 , 0 0 0 O v a l b u m i n e g g w h i t e I C N N u t r i t i o n a l B i o c h e m i c a l s 4 3 , 5 0 0 a - c h y m o t r y p s i n o g e n A b o v i n e p a n c r e a s S i g m a 2 3 , 4 0 0 L y s o z y m e e g g w h i t e W o r t h i n g t o n 1 4 , 0 0 0 F r o m S o b e r ( 1 9 7 0 ) /43 RESULTS AND DISCUSSION A. Effect of Gamma 'Radiation on Colour' and Odour Sterile products have been obtained using gamma radiation but off-odours and colours have been undesirable by-products of this process (Batzer and.Doty, 1955). Irradiation of both intact and washed muscle (inoculation or aseptic control) samples, prior to treatment resulted in a characteristic "wet dog" odour. The odour results from the formation of hydrogen sulfide, mercaptans, carbonyls and aldehydes due to the degradation of free amino acids (Batzer and Doty, 1955; Merritt et al., 1959). Changes in colour were also noted after irradiation. The bright red colour of the intact muscle samples was transformed to a tan brown colour while the pink colour (almost pork-like in 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 irradiation. The colour difference found between the intact and washed samples, before and after irradiation, could be explained by the loss in 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 in attachment media containing approximately 10 P. fragi /44 cells/ml. Enumeration of the samples at day 0 indicated attachment 5 6 2 in the range of 10 to 10 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 st e r i l e and inoculated samples and checked for bacterial growth (Table 3). No growth was observed in.the control samples during the 12 days of incubation. Jay and Kontou (1967) working with beef, and Sage (1974) working with chicken, found that a 1 Megarad dose of gamma radiation was sufficient to destroy the naturally occurring microflora. Bacterial growth on the intact samples was rapid for the i n i t i a l 6 day period; this was followed by a more modest increase during the next 6 days of storage. The increase in bacterial 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 bacterial growth, data from both intact and washed inoculated muscle samples were subjected to a data transfor-mation for linearization 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 for calculation 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 bacterial cound data yielded the following regression equations: T A B L E 3: I n f l u e n c e o f i n c u b a t i o n a t 4 C o n m u s c l e s a m p l e s i n o c u l a t e d w i t h Pseudomonas fragi. A v e r a g e b a c t e r i a l n u m b e r s a ( C F U / c m ^ f r e s h m u s c l e t i s s u e ) I n c u b a t i o n p e r i o d ( d a y s ) S a m p l e 0 3 6 9 12 I n t a c t c o n t r o l 0 0 0 0 0 W a s h e d c o n t r o l 0 0 0 0 0 I n t a c t i n o c u l a t e d 4.19 X 10 5 5. ,20 X i o 7 3. ,76 X 10 9 1.22 X i o 1 0 1.82 X 10 W a s h e d i n o c u l a t e d 1.32 X 10 6 6. ,29 X V 5, ,73 X i o 8 1.13 X i o 9 1.43 X io 1 A v e r a g e i s b a s e d o n 4 d e t e r m i n a t i o n s /46 Incubation p e r i o d (days) FIGURE 7: B a c t e r i a l p o p u l a t i o n of 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, stored at 4°C. F I G U R E 8: L i n e a r i z a t i o n o f l o g b a c t e r i a l p o p u l a t i o n d a t a f o r i n t a c t a n d w a s h e d m u s c l e s a m p l e s 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 . / 4 8 I n t a c t i n o c u l a t e d : ( y - 5 . 4 0 ) J = 1 0 . 6 6 x - 3 . 4 7 r = 0 . 9 7 J 3 W a s h e d i n o c u l a t e d : C,u - 5 . 9 0 ) = 3 . 0 3 v + 0 . 7 6 r = 0 . 9 4 2 y = l o g C F U / c m o f t h e i n t a c t i n o c u l a t e d m u s c l e t i s s u e -x = i n c u b a t i o n p e r i o d 2 u = l o g C F U / c m o f t h e w a s h e d i n o c u l a t e d m u s c l e t i s s u e v = i n c u b a t i o n p e r i o d R e d u c t i o n o f t h e s a r c o p l a s m i c f r a c t i o n r e s u l t e d i n a d e c r e a s e d b a c t e r i a l g r o w t h r a t e ( s l o p e = 3 . 0 3 ) a s c o m p a r e d t o t h e i n t a c t m u s c l e s a m p l e ( s l o p e = 1 0 . 6 6 ) ( F i g u r e 8 ) . A c o m p a r i s o n o f t h e s l o p e s u s i n g t h e S t u d e n t ' s t t e s t ( Z a r , 1 9 7 4 ) i n d i c a t e d s i g n i f i c a n c e a t t h e P < 0 . 0 1 l e v e l ( T a b l e 4 ) . T h e l o w e r g r o w t h r a t e e x p e r i e n c e d i n t h e s a r c o p l a s m i c r e d u c e d s a m p l e s m a y b e e x p l a i n e d b y t h e l i m i t e d a v a i l a b i l i t y o f l o w m o l e c u l a r w e i g h t , s o l u b l e c o m p o u n d s . S e v e r a l a u t h o r s ( J a y , 1 9 6 7 ; J a y a n d K o n t o u , 1 9 7 6 ; G i l l , 1 9 7 6 ; G i l l a n d N e w t o n , 1 9 7 7 ) h a v e s u g g e s t e d a p r e f e r e n t i a l u t i l i z a t i o n o f l o w m o l e c u l a r w e i g h t c o m p o n e n t s d u r i n g m u s c l e s p o i l a g e . C. The Effect of Growth of P. fragi on pE} Surface Appearance  and Odour T h e c h a n g e i n p H o f t h e c o n t r o l a n d i n o c u l a t e d m u s c l e s a m p l e s i s p r e s e n t e d r i i n T a b l e 5 a n d F i g u r e 9 . T h e p H o f t h e a s e p t i c c o n t r o l s ( i n t a c t a n d w a s h e d ) r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e e x p e r i m e n t , a l t h o u g h s l i g h t d e c r e a s e s i n p H ( 5 . 6 7 t o 5 . 5 6 a n d 5 . 7 8 t o 5 . 7 2 f o r i n t a c t a n d w a s h e d c o n t r o l s , r e s p e c t i v e l y ) T A B L E 4 : R e g r e s s i o n s t a t i s t i c s u s e d i n t h e c o m p a r i s o n o f t h e s l o p e o f t h e i n t a c t i n o c u l a t e d r e g r e s s i o n w i t h t h a t o f t h e w a s h e d i n o c u l a t e d r e g r e s s i o n . S a m p l e S l o p e R e s i d u a l DF R e s i d u a l S S -Ex t v a l u e I n t a c t i n o c u l a t e d 1 0 . 6 6 18 2 5 6 7 . 6 8 3 6 0 W a s h e d i n o c u l a t e d 3 . 0 3 18 4 0 9 . 9 7 3 6 0 1 1 . 2 2 * * * * P < 0 . 0 1 T A B L E 5 : T h e e f f e c t o f P . fragi o n t h e p H o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) b o v i n e m u s c l e s a m p l e s . p H I n c u b a t i o n p e r i o d ( d a y s ) S a m p l e 0 3 6 9 12 I n t a c t c o n t r o l 5 . 6 7 a + 0 . 0 3 b 5 . 65 + 0 , . 0 4 5 . . 6 3 + 0 . . 0 1 . 5 . , 57 + 0 . ,04 5 , . 5 6 + 0 . 01 W a s h e d c o n t r o l 5 . 7 8 + 0 . 01 . 5 . 79 + 0 . . 01 5 , . 7 8 + 0 . .01 5 . , 7 2 + 0 . , 02 5 . , 7 2 + 0 . 01 I n t a c t i n o c u l a t e d 5 . 6 6 + 0 . 01 5 . 79 + 0 , . 01 6 , . 8 6 + 0 . .01 7 . , 1 6 + 0 . , 0 6 7 , . 2 4 + 0 . 01 W a s h e d i n o c u l a t e d 5 . 7 9 + 0 . 01 5 . 80 + 0 . . 01 5 . . 7 8 + 0 . .01 5 . . 7 3 + 0 . . 0 2 5 , . 7 3 + 0 . 01 A v e r a g e o f d u p l i c a t e a n a l y s i s ' S t a n d a r d d e v i a t i o n / 5 1 FIGURE 9: pH of control and inoculated, i n t a c t and washed muscle samples stored at 4°C. Each point represents the mean of duplicate determinations. /52 were noted. A s i m i l a r decrease i n pH of the a s e p t i c c o n t r o l was reported by Sage (1974) working with gamma i r r a d i a t e d , minced chicken muscle. Gardner and Stewart (1966) s t a t e d t h a t a u t o l y t i c production of ammonia i n beef muscle was very low or non-existent. The i n t a c t i n o c u l a t e d sample d i s p l a y e d a dramatic r i s e i n pH during the 12 days of i n c u b a t i o n (pH 5.66 to 7.24). Several authors (Jay and Kontou, 1967; Ockerman et al., 1969; Borton et at., 1970a; Hasegawa et at., 1970a; Tarrant et at., 1971; Sage, 1974) have reported an increase i n pH i n v a r i o u s myosystems due to the growth of Pseudomonas f r a g i . Tarrant et al. (1971) i n d i c a t e d t h a t the i n c r ease i n pH was a t t r i b u t e d t o the production of amines and ammonia. The pH of the 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 during i n c u b a t i o n , decreasing s l i g h t l y from an i n i t i a l pH of 5.79 to an u l t i m a t e pH of 5.73 a f t e r 12 days of i n c u b a t i o n . The l a c k o f i n c r e a s e i n pH experienced i n the washed i n o c u l a t e d sample may be explained by assuming that most of the precursors ( f r e e amino a c i d s , peptides 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 increase the pH, were removed during the washing procedure. The p o s s i b i l i t y a l s o e x i s t s that the b u f f e r i n g c a p a c i t y of the: sarcoplasmic e x t r a c t i o n b u f f e r was s u f f i c i e n t to compensate f o r any increase i n pH that r e s u l t e d from b a c t e r i a l metabolism during growth. /53 B y d a y 6 , t h e s u r f a c e s o f b o t h i n t a c t a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s h a d a s l i m y a p p e a r a n c e . J a y ( 1 9 7 0 ) r e p o r t e d t h a t t h e s l i m y a p p e a r a n c e o f s p o i l i n g m u s c l e w a s d u e t o b a c t e r i a l c o a l e s c e n c e a s w e l l a s t h e l o s s o f m u s c l e i n t e g r i t y a s a r e s u l t o f m i c r o b i a l g r o w t h . I n g r a m a n d D a i n t y ( 1 9 7 1 ) r e p o r t e d t h a t o d o u r a n d s l i m e f o r m a t i o n o c c u r r e d w h e n b a c t e r i a l n u m b e r s w e r e a p p r o x i m a t e l y 8 2 10 / c m . No e v i d e n c e o f s l i m e f o r m a t i o n was s e e n i n e i t h e r i n t a c t o r w a s h e d c o n t r o l m u s c l e s a m p l e s d u r i n g t h e p r e s e n t s t u d y . A p u t r i d o d o u r w a s e v i d e n t i n b o t h i n t a c t a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s b y d a y 6 t h a t w a s c h a r a c t e r i s t i c a l l y d i f f e r e n t f r o m t h e i n i t i a l " w e t d o g " o d o u r . T h e p u t r i d o d o u r was m u c h m o r e i n t e n s e i n t h e i n t a c t m u s c l e t i s s u e t h a n i n t h e w a s h e d m u s c l e t i s s u e . J a y a n d K o n t o u ( 1 9 6 7 ) s t a t e d t h a t "ammonia, hydrogen sulfide, indole and other foul-odour compounds usually associated with spoiling meats owe their o r i g i n to the free amino acids and other low molecular weight compounds rather than to the larger proteins". T h e d i f f e r e n c e i n o d o u r i n t e n s i t y b e t w e e n t h e i n t a c t a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s m a y b e e x p l a i n e d b y a r e d u c t i o n i n p u t r i d o d o u r p r e c u r s o r s d u r i n g t h e w a s h i n g p r o c e d u r e . T h e a s e p t i c c o n t r o l s ( i n t a c t a n d w a s h e d m u s c l e t i s s u e ) r e t a i n e d t h e i r c h a r a c t e r i s t i c " w e t d o g " o d o u r t h r o u g h o u t t h e d u r a t i o n o f t h e e x p e r i m e n t . D. Influence of Growth of V. fragi on Total Carbohydrate Content R e s u l t s o f t h e t o t a l c a r b o h y d r a t e a n a l y s i s i n t h e c o n t r o l a n d i n o c u l a t e d m u s c l e s a m p l e s a r e p r e s e n t e d i n T a b l e 6 a n d F i g u r e 1 0 . T A B L E 6 : T h e e f f e c t o f P . fragi o n 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 i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) b o v i n e m u s c l e s a m p l e s . T o t a l c a r b o h y d r a t e c o n t e n t ( m g / g d r y w e i g h t m u s c l e t i s s u e ) I n c u b a t i o n p e r i o d ( d a y s ) S a m p l e 0 3 6 9 12 I n t a c t c o n t r o l 1 0 . 2 a + 1 . l b 1 3 , , 9 + 2 . ,4 1 4 , ,2 + 0 . 9 1 3 , .1 + 0 . ,9 1 5 . 5 + 0 . 9 W a s h e d c o n t r o l 3 . 9 + 0 . 2 3 , .8 + 0 . ,2 4 , , 0 + 0 . 5 3 . , 5 + 0 . ,2 3 . 9 + 0 . 2 I n t a c t i n o c u l a t e d 1 4 . 4 + 2 . 2 1 2 , ,7 + 0 , ,9 1 0 , .4 + 1 . 7 8 , , 0 + 2 . ,1 7 . 4 + 0 . 6 W a s h e d i n o c u l a t e d 4 . 1 + 0 . 6 3 , ,3 + 0 . ,5 3 , , 3 + 0 . 2 3 , ,6 + 0 . ,2 3 . 1 + 0 . 1 A v e r a g e o f 8 r e a d i n g s S t a n d a r d d e v i a t i o n /55 Washed contro l a A Washed inoculated • • 'O 3 6 9 12 Incubation period (days) FIGURE 10: Total carbohydrate content of control and inoculated, i n t a c t and washed muscle samples stored at 4 ° C . Each point represents the mean of 8 determinations. / 5 6 T h e a b i l i t y t o d e s c r i b e t h e e x a c t n a t u r e o f c a r b o h y d r a t e u t i l i z a t i o n ( i . e . . s p e c i f i c c a r b o h y d r a t e b e i n g u t i l i z e d ) was i m p o s s i b l e s i n c e t h e p h e n o l - s u l f u r i c r e a g e n t ( D u b o i s et al., 1 9 5 6 ) r e a c t s w i t h m a n y s u g a r s a n d t h e i r d e r i v a t i v e s . " T o t a l c a r b o h y d r a t e " i n m e a t w o u l d i n c l u d e g l y c o g e n , g l u c o s e , g l u c o s e - 6 - p h o s p h a t e . a n d r i b o s e a s w e l l a s some g l y c o l y t i c i n t e r m e d i a t e s ( G i l l a n d N e w t o n , 1 9 7 8 ) . G i l l a n d N e w t o n ( 1 9 7 7 ) s t u d i e d t h e s u b s t r a t e u t i l i z a t i o n d u r i n g b a c t e r i a l s p o i l a g e o f b e e f , a n d r e p o r t e d t h a t g l y c o g e n a n d g l u c o s e - 6 - p h o s p h a t e c o u l d n o t b e u t i l i z e d a s c a r b o n s o u r c e s b y Pseudomonas. A l t h o u g h t o t a l c a r b o h y d r a t e v a l u e s w e r e d e t e r m i n e d f o r a l l s a m p l e s , t h e m a g n i t u d e o f t h e s t a n d a r d d e v i a t i o n f o r some s a m p l e s w i t h i n t r e a t m e n t s ( i n t a c t i n o c u l a t e d d a y 9 , T a b l e 6 ) a p p r o a c h e d 27% o f t h e m e a n v a l u e . T h e e x t r e m e v a r i a b i l i t y c o u l d b e a t t r i b u t a b l e t o a s i t e t o s i t e v a r i a b i l i t y w i t h i n t h e t o t a l m u s c l e , a s w e l l a s a l o c a t i o n v a r i a b i l i t y w i t h i n t h e s a m e m u s c l e s t r i p u s e d f o r a n a l y s i s . H a s e g a w a et al. ( 1 9 7 0 a a n d b ) , e n c o u n t e r e d a s i m i l a r p r o b l e m o f w i t h i n t r e a t m e n t v a r i a b i l i t y i n a t t e m p t s t o d e t e r m i n e t h e p r o t e i n c o n t e n t o f c o n t r o l a n d i n o c u l a t e d m u s c l e h o m o g e n a t e s . T h e a u t h o r s f o u n d t h a t t h e v a r i a t i o n b e t w e e n s a m p l e s w a s g r e a t e r t h a n b e t w e e n t r e a t m e n t s . A n a l y s i s o f d a t a a t d a y 0 r e v e a l e d t h a t a p p r o x i m a t e l y 70% o f t h e t o t a l c a r b o h y d r a t e : ; f r a c t i o n was r e m o v e d d u r i n g t h e w a s h i n g p r o c e d u r e , i n d i c a t i n g t h a t t h e m a j o r i t y o f t h e t o t a l c a r b o h y d r a t e w a s e a s i l y e x t r a c t e d . /57 The i n t a c t a s e p t i c c o n t r o l showed a general increase i n t o t a l carbohydrate during the experiment, probably due to the production of r i b o s e from adenosine triphosphate (ATP) during postmortem storage ( H u l t i n , 1976) (Figure 11). In c o n t r a s t , a decrease i n t o t a l carbohydrate was seen i n the i n t a c t i n o c u l a t e d samples. G i l l (1976) and G i l l and Newton (1977) reported the use of glucose as a carbon source by a Pseudomonas spp. during the i n i t i a l stages of s p o i l a g e i n beef. The decrease i n t o t a l carbohydrate observed i n the i n t a c t i n o c u l a t e d sample may be r e l a t e d to the increase i n pH of these samples. The amino a c i d catabolism of b a c t e r i a i s known to be a f f e c t e d by the presence of a v a i l a b l e carbohydrates - the more carbohydrate i n the medium, the lower the amount o f ammonia produced (Gardner and Stewart, 1966). Jacoby (1964) found t h a t i n the presence of glucose and other energy sources, amino a c i d catabolism 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 i n c u b a t i o n although a s l i g h t decrease i n t o t a l carbohydrate was noted i n the 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 of the washed . c o n t r o l samples may be explained by assuming t h a t the a u t o l y t i c enzymes as w e l l as the precursors f o r r i b o s e were removed during washing thereby preventing any increase i n t o t a l carbohydrate. Any decrease i n t o t a l carbohydrate noted i n the washed i n o c u l a t e d sample may be a t t r i b u t a b l e to the u t i l i z a t i o n by P. fragi of r e s i d u a l carbohydrate not removed during the washing procedure. 2ATP -E^ . 2ADP A ^ e n y ' q t e » ATP + AMP Kinase AMP ^ ! * IMP P h ° P h a t a S e » Inosine Deaminase | n o s i n e Nucleoside ^ R j b o s e + H y p o x a n t h i n e Hydrolase F I G U R E 1 1 : C o n v e r s i o n o f A T P t o r i b o s e a n d h y p o x a n t h i n e i n p o s t m o r t e m m e a t . / 5 9 E. The Effect of P. fragi on the Extractability of Non-Protein  Nitrogen (NPN) 3 Water-Soluble and Salt-Soluble Proteins R e s u l t s o f n o n - p r o t e i n n i t r o g e n , w a t e r v s o l u b l e a n d 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 f r o m c o n t r o l a n d i n o c u l a t e d s a m p l e s d u r i n g the. . . 12 d a y s o f i n c u b a t i o n a r e p r e s e n t e d i n T a b l e s 7 , 8 a n d 9 a n d F i g u r e s 1 2 , 13 a n d 1 4 , r e s p e c t i v e l y . T h e n o n - p r o t e i n n i t r o g e n (NPN) a n a l y s i s ( T a b l e 7 , F i g u r e 12 ) r e v e a l e d t h a t a p p r o x i m a t e l y 80% o f t h i s f r a c t i o n w a s r e m o v e d d u r i n g t h e w a s h i n g p r o c e d u r e ( i n t a c t s a m p l e s c o n t a i n e d 0 . 6 % N P N , w a s h e d s a m p l e s c o n t a i n e d 0 . 1 % N P N ) , i n d i c a t i n g t h a t m o s t o f t h e N P N w a s c o n t a i n e d i n t h e w a t e r - s o l u b l e f r a c t i o n , ( s a r c o p l a s m ) . T h e i n t a c t c o n t r o l s h o w e d a n i n i t i a l d e c r e a s e i n n o n - p r o t e i n n i t r o g e n f o l l o w e d b y a s l i g h t i n c r e a s e . S i m i l a r r e s u l t s w e r e r e p o r t e d b y B o r t o n et al. ( 1 9 7 0 a ) w o r k i n g w i t h p o r c i n e m u s c l e . T h e i n t a c t i n o c u l a t e d s a m p l e e x h i b i t e d a n i n c r e a s e i n N P N f o r t h e f i r s t 6 d a y s a f t e r w h i c h a d e c r e a s e o c c u r r e d . T h e i n c r e a s e i n N P N w o u l d i n d i c a t e p r o t e o l y s i s , h o w e v e r , a f t e r d a y 6 t h e r a t e o f p r o d u c t i o n o f N P N c o m p o n e n t s m a y h a v e b e e n e x c e e d e d b y t h e r a t e o f u t i l i z a t i o n b y P. fragi. T h i s i n e q u a l i t y i n p r o d u c t i o n a n d u t i l i z a t i o n m a y e x p l a i n t h e d e c r e a s e . T h e l a t t e r d e c r e a s e i n N P N c o r r e s p o n d e d t o i n c r e a s e s i n e x t r a c t a b i l i t y o f t h e w a t e r - s o l u b l e a n d 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 f r o m t h e i n t a c t i n o c u l a t e d m u s c l e s a m p l e w h i c h m a y i n t u r n b e i n d i c a t i v e o f p r o t e o l y s i s . R a m p t o n et al. ( 1 9 7 0 ) p o s t u l a t e d t h a t n o n - p r o t e i n n i t r o g e n o u s m a t e r i a l m a y b e r e q u i r e d f o r i n i t i a t i o n o f p r o t e o l y s i s b y b a c t e r i a . T A B L E 7: T h e e f f e c t o f P. fragi o n t h e n o n - p r o t e i n n i t r o g e n o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) b o v i n e m u s c l e s a m p l e s . N o n - p r o t e i n n i t r o g e n c o n t e n t (mg n i t r o g e n / g d r y w e i g h t m u s c l e ) I n c u b a t i o n p e r i o d ( d a y s ) S a m p l e 0 3 6 9 12 I n t a c t c o n t r o l 5 . 8 6 a + 0. , o o b 5, ,18 + 0. ,06 5. ,21 + 0. ,02 5, .35 + 0, ,04 5, .43 + 0. ,07 W a s h e d c o n t r o l 1.57 + 0. ,02 1, ,23 + 0. ,05 1, ,20 + 0. ,00 1, .43 + 0. ,00 1, .16 + 0. ,02 I n t a c t i n o c u l a t e d 5.51 + 0. ,04 5, ,71 + 0. ,11 6. ,41 + 0. ,11 5, .79 + 0. ,13 5, .64 + 0. ,08 W a s h e d i n o c u l a t e d 1.05 + 0. ,00 1, .09 + 0. ,02 1, .22 + 0, ,02 1, .40 + 0. ,05 1, .30 + 0, .05 A v e r a g e o f d u p l i c a t e r e a d i n g s ' S t a n d a r d d e v i a t i o n / 6 1 °-°0 3 6 9 12 I n c u b a t i o n p e r i o d ( d a y s ) F I G U R E 1 2 : N o n - p r o t e i n n i t r o g e n c o n t e n t o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 C . E a c h p o i n t r e p r e s e n t s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s . / 6 2 No d r a s t i c c h a n g e s i n N P N w e r e e x p e r i e n c e d i n e i t h e r t h e w a s h e d c o n t r o l o r t h e w a s h e d i n o c u l a t e d s a m p l e s , a l t h o u g h a s l i g h t d e c r e a s e was n o t e d i n t h e w a s h e d i n o c u l a t e d s a m p l e . T h e a b s e n c e o f a n y c h a n g e i n t h e w a s h e d i n o c u l a t e d N P N c o u l d p o s s i b l y s i g n i f y a d y n a m i c e q u i l i b r i u m b e t w e e n b a c t e r i a l p r o t e o l y s i s a n d u t i l i z a t i o n o f N P N . H o w e v e r , 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 s i n c e b o t h i n o c u l a t e d a n d c o n t r o l s a m p l e s p a r a l l e l l e d o n e a n o t h e r , a n y c h a n g e e x p e r i e n c e d : ' i n t h e i n o c u l a t e d s a m p l e s w a s d u e t o a u t o l y t i c p r o c e s s e s . T h e s t a t i c c o n d i t i o n o f t h e w a s h e d c o n t r o l s a m p l e w o u l d b e e x p e c t e d s i n c e t h e l y s o s o m e s , t h e m a j o r s o u r c e o f c a t h e p t i c e n z y m e s , w h i c h r e s i d e i n t h e s a r c o p l a s m ( F o r r e s t et al., 1 9 7 5 ) w o u l d b e r e m o v e d d u r i n g t h e w a s h i n g p r o c e d u r e , t h u s p r e v e n t i n g a n y a u t o l y t i c p r o c e s s e s . T h e w a t e r - s o l u b l e p r o t e i n a n a l y s i s ( T a b l e 8 , F i g u r e 1 3 ) r e v e a l e d t h a t a p p r o x i m a t e l y 4 0 t o 50% o f t h i s f r a c t i o n w a s r e m o v e d d u r i n g t h e w a s h i n g p r o c e d u r e . T u ( 1 9 7 3 ) , u s i n g a r e p e t i t i v e i m b i b i t i o n -c e n t r i f u g a t i o n t e c h n i q u e t o r e m o v e t h e s a r c o p l a s m i c p r o t e i n s f r o m i n t a c t m u s c l e c o r e s , f o u n d t h a t a m a j o r i t y o f t h e s a r c o p l a s m w a s e a s i l y r e m o v e d , h o w e v e r , some w a s " a p p a r e n t l y b o u n d r e l a t i v e l y s t r o n g l y t o t h e m y o f i b r i l l a r m a t r i x " . R e s u l t s o b t a i n e d ( t o t a l c a r b o h y d r a t e , N P N a n d w a t e r - s o l u b l e p r o t e i n s ) w o u l d t e n d t o i n d i c a t e t h a t t h e w a s h i n g p r o c e d u r e u s e d r e s u l t e d i n a s u b s t a n t i a l d e c r e a s e i n w a t e r -s o l u b l e c o m p o n e n t s . I n t a c t c o n t r o l s a m p l e s s h o w e d a g r a d u a l d e c r e a s e i n t h e w a t e r - s o l u b l e p r o t e i n f r a c t i o n d u r i n g i n c u b a t i o n ( F i g u r e 1 3 ) . S i m i l a r r e s u l t s w e r e r e p o r t e d b y s e v e r a l a u t h o r s ( O c k e r m a n et at., 1 9 6 9 ; T A B L E 8 : T h e e f f e c t o f P. fragi o n 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 o f i n t a c t a n d s a r c o p l a s m i c r e d u c e d ( w a s h e d ) b o v i n e m u s c l e s a m p l e s . 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 (mg p r o t e i n / g d r y w e i g h t m u s c l e t i s s u e ) I n c u b a t i o n p e r i o d ( d a y s ) S a m p l e 0 3 6 9 12 I n t a c t c o n t r o l 1 3 4 . 5 a + 0 . , 9 b 1 0 4 . .1 + 2 . , 3 1 0 5 . . 2 . + 0 . .7 1 0 8 . 6 + 0 , .7 9 3 . . 8 + 4 , . 2 W a s h e d c o n t r o l 7 6 . 3 + 0 . .4 5 4 . . 3 + 1. .8 4 6 . . 0 + 3 . ,7 5 3 . 3 + 0 . . 9 1 7 . ,8 + 2 , . 9 I n t a c t i n o c u l a t e d 1 5 5 . 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 W a s h e d i n o c u l a t e d 6 5 . 3 + 1 . 8 5 5 . .5 + 1 . 8 4 8 . ,1 + 1 . 1 4 9 . 1 + ; 1. .3 1 9 . ,9 + 0 . ,7 A v e r a g e o f t r i p l i c a t e r e a d i n g s ' s t a n d a r d d e v i a t i o n / 6 4 F I G U R E 1 3 : 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 o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 C . E a c h p o i n t r e p r e s e n t s t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s . /65 B o r t o n et al., 1 9 7 0 a ; S a g e , 1 9 7 4 ) d u r i n g s p o i l a g e o f m u s c l e b y P . fragi. A n y d e c r e a s e i n t h e w a t e r - s o l u b l e p r o t e i n f r a c t i o n m a y h a v e b e e n a t t r i b u t a b l e t o a u t o l y s i s b y c a t h e p t i c e n z y m e s ( S h a r p , 1 9 6 3 ; B o d w e l l a n d P e a r s o n , 1 9 6 4 ) . T a r r a n t et al. ( 1 9 7 1 ) , h o w e v e r , f o u n d a s l i g h t i n c r e a s e i n s a r c o p l a s m i c p r o t e i n i n a s e p t i c p o r c i n e m u s c l e d u r i n g s t o r a g e . I n t a c t i n o c u l a t e d s a m p l e s e x h i b i t e d a n i n i t i a l d e c r e a s e i n t h e w a t e r - s o l u b l e p r o t e i n f r a c t i o n a f t e r w h i c h p r o t e i n v a l u e s i n c r e a s e d . T h e i n i t i a l d e c r e a s e m a y h a v e r e s u l t e d f r o m b a c t e r i a l p r o t e o l y s i s o f t h i s f r a c t i o n ( H a s e g a w a et al., 1 9 7 0 a ) ; i t i s a l s o p o s s i b l e t h a t a u t o l y s i s w a s i n v o l v e d . P r o t e o l y s i s m a y h a v e a l s o a c c o u n t e d f o r t h e i n c r e a s e i n p r o t e i n d u r i n g t h e l a t t e r s t a g e s o f t h e e x p e r i m e n t . A l t h o u g h i t i s p o s s i b l e t h a t b a c t e r i a l p r o t e o l y s i s i n c r e a s e d t h e e x t r a c t a b i l i t y o f w a t e r - s o l u b l e p r o t e i n s t h a t w e r e c o m p l e x e d w i t h s a l t - s o l u b l e p r o t e i n s , 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 p r o t e i n s p r e v i o u s l y i n s o l u b l e i n w a t e r ( s a l t - s o l u b l e , u r e a - s o l u b l e a n d u r e a -i n s o l u b l e p r o t e i n s ) w e r e n o t s o l u b i l i z e d . T h e i n c r e a s e i n e x t r a c t a b l e p r o t e i n was p r o b a b l y d u e t o t h e e n h a n c e d s o l u b i l i t y o f t h e i n i t i a l n o n - s o l u b l e . f r a c t i o n s b y t h e a c t i o n o f l a r g e n u m b e r s o f m i c r o o r g a n i s m s ( M c i n t o s h , 1 9 6 7 ; O c k e r m a n et al., 1 9 6 9 ; B o r t o n et al., 1 9 7 0 a ) . G o l l et al. ( 1 9 7 0 ) 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 s t r o m a l p r o t e i n s m a y c a u s e t h e a p p e a r a n c e o f new s o l u b l e p e p t i d e s i n t h e s a r c o p l a s m i c p r o t e i n f r a c t i o n . B o t h w a s h e d c o n t r o l a n d i n o c u l a t e d s a m p l e s e x h i b i t e d s i m i l a r p a t t e r n s s h o w i n g a g r a d u a l d e c r e a s e i n t h e w a t e r - s o l u b l e p r o t e i n / 6 6 f r a c t i o n f o r t h e f i r s t 9 d a y s , a f t e r w h i c h a d e c r e a s e o c c u r r e d . T h e l a t t e r d e c r e a s e w a s u n e x p l a i n a b l e . S i n c e s i m i l a r e x t r a c t a b i l i t y p a t t e r n s w e r e o b s e r v e d , t h i s w o u l d s u g g e s t t h a t i n o c u l a t i o n o f t h e w a s h e d s a m p l e h a d n o e f f e c t o n p r o t e i n e x t r a c t a b i l i t y , a n d t h a t a n y c h a n g e s t h a t d i d o c c u r m a y h a v e b e e n a u t o l y t i c i n n a t u r e . T h e p r o t e i n c o n t e n t : o f t h e s a l t - s o l u b l e f r a c t i o n f o r t h e v a r i o u s s a m p l e s i s d e p i c t e d i n T a b l e 9 , F i g u r e 1 4 . R e l a t i v e l y l i t t l e c h a n g e o c c u r r e d i n t h e i n t a c t c o n t r o l d u r i n g s t o r a g e i n d i c a t i n g v e r y l i t t l e a u t o l y s i s . T h e a b s e n c e o f a n y d r a s t i c c h a n g e i n t h e c o n t r o l i s i n a g r e e m e n t w i t h t h e r e s u l t s o f T a r r a n t et al. ( 1 9 7 1 ) , a l t h o u g h o t h e r a u t h o r s ( B o r t o n et al., 1 9 7 0 a ; S a g e , 1 9 7 4 ) h a v e r e p o r t e d v a r y i n g d e g r e e s o f d e c r e a s e , p r e s u m a b l y d u e t o a u t o l y s i s . T h e a m o u n t o f e x t r a c t a b l e s a l t - s o l u b l e p r o t e i n i n t h e i n t a c t i n o c u l a t e d s a m p l e d e c r e a s e d s l i g h t l y d u r i n g t h e f i r s t 6 d a y s o f s t o r a g e , a f t e r w h i c h t h e p r o t e i n c o n t e n t i n c r e a s e d b y a f a c t o r o f a p p r o x i m a t e l y 2 . 5 , a n d t h e n r e m a i n e d c o n s t a n t t h r o u g h o u t t h e r e m a i n d e r o f t h e s t o r a g e p e r i o d . H i g h e r e x t r a c t a b i l i t y v a l u e s w e r e a s s o c i a t e d w i t h t h e h i g h e r l e v e l s 9 2 o f b a c t e r i a l c o n t a m i n a t i o n ( 1 0 C E U / c m ) . S e v e r a l a u t h o r s ( I n g r a m a n d D a i n t y , 1 9 7 1 ; D a i n t y et al., 1 9 7 5 ; G i l l a n d N e w t o n , 1 9 7 7 ) h a v e s t a t e d t h a t g e n e r a l l y n o s i g n i f i c a n t c h a n g e s i n p r o t e i n c o n t e n t w e r e 8 2 o b s e r v e d u n t i l b a c t e r i a l n u m b e r s e x c e e d e d 10 / c m . E x t r a c t i o n 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 i n t h e i n t a c t i n o c u l a t e d m u s c l e s a m p l e a f t e r 9 d a y s i n c u b a t i o n r e q u i r e d a n i n c r e a s e i n t h e r e l a t i v e c e n t r i f u g a l f o r c e f r o m 2 9 , 0 0 0 x g t o 4 0 , 0 0 0 x g . S i m i l a r r e s u l t s w e r e r e p o r t e d " b y T a r r a n t et al. ( 1 9 7 1 ) d u r i n g a t t e m p t s t o s e p a r a t e t h e TABLE 9: The e f f e c t of P. fragi on the s a l t - s o l u b l e protein content of in t a c t and sarcoplasmic reduced (washed) bovine muscle samples. S a l t - s o l u b l e protein content (mg protein/g dry weight muscle tissue) Incubation period (days) Sample 0 3 6 9 12 Intact control 87. i 5 a + 3, . l b 95, .2 + 3, .9 105, .4 + 1, .6 101, .2 + 3.1 99, .9 + 2, .6 Washed control 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 inoculated 112. .6 + 1. ,1 101. 8 + 0. ,3 90. ,6 + 2. ,2 112. ,6 + 2.8 83. ,5 + 1. .2 "Average of t r i p l i c a t e readings Standard deviation /68 F I G U R E 1 4 : S a l t - s o l u b l e p r o t e i n c o n t e n t o f c o n t r o l a n d i n o c u l a t e d , i n t a c t a n d w a s h e d m u s c l e s a m p l e s s t o r e d a t 4 ° C . E a c h p o i n t r e p r e s e n t s t h e m e a n o f t r i p l i c a t e d e t e r m i n a t i o n s . / 6 9 r e s i d u a l j n y o f i b r i l l a r p r o t e i n s f r o m t h e w a t e r - s o l u b l e f r a c t i o n i n p o r c i n e m u s c l e i n o c u l a t e d w i t h P. fragi. No i n c r e a s e i n c e n t r i f u g a l f o r c e w a s r e q u i r e d f o r a n y o t h e r t r e a t m e n t ( i n t a c t c o n t r o l , w a s h e d c o n t r o l o r w a s h e d i n o c u l a t e d ) i n t h e p r e s e n t s t u d y . A n i n c r e a s e d p e r m e a b i l i t y o f t h e m y o f i b r i l s t o t h e W e b e r - E d s a l l s o l u t i o n m a y h a v e a c c o u n t e d f o r t h e i n c r e a s e i n s a l t - s o l u b l e p r o t e i n e x t r a c t a b i l i t y ( D a v e y a n d G i l b e r t , 1 9 6 8 ) . T h e i n c r e a s e i n 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 i n t h e i n t a c t i n o c u l a t e d s a m p l e c o r r e s p o n d e d t o t h e i n c r e a s e i n t h e w a t e r -s o l u b l e p r o t e i n f r a c t i o n ( a f t e r 6 d a y s i n c u b a t i o n ) i n t h e c o r r e s p o n d i n g s a m p l e . B o r t o n et at. ( 1 9 7 0 a ) , d u r i n g a s t u d y o f p o r c i n e m u s c l e i n o c u l a t e d w i t h P. fragi, r e p o r t e d a d e c r e a s e i n t h e a m o u n t o f i n s o l u b l e p r o t e i n n i t r o g e n w i t h a s u b s e q u e n t i n c r e a s e d s o l u b i l i t y o f t h e w a t e r - a n d 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 a n d t h e N P N f r a c t i o n . E x a m i n a t i o n o f t h e w a s h e d s a m p l e s ( c o n t r o l a n d i n o c u l a t e d ) r e v e a l e d t h a t t h e a m o u n t o f s a l t - s o l u b l e p r o t e i n was a p p r o x i m a t e l y t h e s a m e . T h e a b s e n c e o f a n e x t r e m e c h a n g e i n t h e i n o c u l a t e d s a m p l e may i n d i c a t e t h a t a n y c h a n g e t h a t d i d o c c u r w a s a g a i n d u e t o a u t o l y t i c p r o c e s s e s . I t i s a l s o p o s s i b l e t h a t t h e p o p u l a t i o n o f P. fragi g r o w i n g o n t h e w a s h e d i n o c u l a t e d m u s c l e t i s s u e was b e l o w t h e c r i t i c a l l e v e l n e c e s s a r y f o r p r o t e o l y s i s t o b e d e t e c t e d . D a i n t y et al. ( 1 9 7 5 ) , w o r k i n g w i t h s l i m e - i n o c u l a t e d b e e f , r e p o r t e d t h a t p r o t e o l y s i s was n o t 9 2 d e t e c t e d u n t i l m i c r o b i a l n u m b e r s w e r e i n e x c e s s o f 3 . 2 x 10 / c m . / 7 0 F. SDS-Polyacrylamide Gel Electrophoresis 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 o f a d e c r e a s e i n t h e c o n c e n t r a t i o n o f m u s c l e w a t e r - s o l u b l e c o m p o n e n t s o n t h e a b i l i t y o f Pseudomonas fragi t o u t i l i z e v a r i o u s p r o t e i n f r a c t i o n s o f b o v i n e Longissimus dorsi, v a r i o u s e l e c t r o p h o r e t i c t e c h n i q u e s ( A C I a g a r o s e f i l m c a s s e t t e , A n a l y t i c a l C h e m i s t s I n c . , P a l o A l t o , C A ; L i t h i u m d o d e c y l s u l f a t e ( L D S ) g e l e l e c t r o p h o r e s i s ( Y a d a et al., 1 9 7 9 ) ; P h a r m a c i a p o l y a c r y l a m i d e g r a d i e n t g e l P A A 4 / 3 0 , P h a r m a c i a F i n e C h e m i c a l s , U p p s a l a , S w e d e n ) w e r e a t t e m p t e d . O f t h e t e c h n i q u e s t e s t e d , o n l y S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( S D S - P A G E ) , e x h i b i t e d a p p r e c i a b l e r e s o l u t i o n o f p r o t e i n b a n d s . S D S - P A G E was c o n d u c t e d o n f r a c t i o n a t e d i n t a c t a n d w a s h e d m u s c l e s a m p l e s ( c o n t r o l a n d i n o c u l a t e d ) o n d a y s 0 , 6 a n d 1 2 . M o b i l i t y o f p r o t e i n s i n S D S - P A G E i s s i m p l y r e l a t e d t o t h e i r m o l e c u l a r w e i g h t ( W e b e r et al., 1 9 7 2 ) . T h e u s e o f d e n a t u r i n g a n d d i s a g g r e g a t i n g a g e n t s ( u r e a , 3 - m e r c a p t o e t h a n o l , a s w e l l a s S D S ) i n t h e p r e p a r a t i o n o f p r o t e i n s a m p l e s f o r S D S - P A G E r e s u l t s i n t h e d e t e r m i n a t i o n o f t h e m o n o m e r m o l e c u l a r w e i g h t o f s u b u n i t p r o t e i n s ( W i l l i a m s a n d G r a t z e r , 1 9 7 1 ) . D u e t o t h e a p p a r e n t +10% a c c u r a c y i n i S D S - P A G E ( W e b e r a n d O s b o r n , 1 9 6 9 ) i t m a y b e p o s s i b l e t h a t s l i g h t p r o t e o l y s i s ( e . g . c l e a v a g e o f a t e r m i n a l a m i n o a c i d f r o m a p o l y p e p t i d e c h a i n ) o f t h e s u b u n i t p r o t e i n s m a y b e u n d e t e c t a b l e e i t h e r d u r i n g a u t o l y s i s o r p r o t e o l y s i s d u e t o P. fragi. T h e u s e o f c o n v e n t i o n a l e l e c t r o p h o r e s i s i n t h e a b s e n c e o f SDS m a y h a v e p r o v e n t o b e a r e l a t i v e l y m o r e s e n s i t i v e t e c h n i q u e i n d e t e c t i n g c h a n g e s w i t h i n p r o t e i n s , s i n c e s e p a r a t i o n o f p r o t e i n s i s b a s e d b o t h o n c h a r g e a n d s i z e o f t h e p o l y p e p t i d e ( T h o r u n a n d M a u r e r , 1 9 7 1 ) . H o w e v e r , w h e t h e r e l e c t r o p h o r e s i s i s c o n d u c t e d w i t h o r w i t h o u t S D S , t h e a p p e a r a n c e o f a s i n g l e b a n d s h o u l d n o t b e i n t e r p r e t e d a s u n e q u i v o c a l e v i d e n c e o f p r o t e i n h o m o -g e n e i t y s i n c e p r o t e i n s w i t h d i f f e r e n t c h a r a c t e r i s t i c s m a y m i g r a t e t o t h e s a m e p o s i t i o n i n t h e g e l ( T h o r u n a n d M a u r e r , 1 9 7 1 ) . I n a d d i t i o n , t h e u s e o f b a n d i n t e n s i t y a s a c o m p a r a t i v e m e a n s o f p r o t e i n q u a n t i t a t i o n m u s t b e t a k e n i n t o c o n s i d e r a t i o n s i n c e t h e c o m p l e x f o r m a t i o n b e t w e e n p r o t e i n a n d d y e i s n o n - s t o i c h i o m e t r i c , a n d t h u s g i v e s o n l y s e m i -q u a n t i t a t i v e r e s u l t s ( B o s s h a r d a n d D a t y n e r , 1 9 7 7 ) . T h e r e f o r e , some c a u t i o n m u s t b e a p p l i e d w h e n i n t e r p r e t i n g e l e c t r o p h o r e t i c r e s u l t s . 1. Water-soluble proteins C o m p a r i s o n o f d a y 0 s a m p l e s i n d i c a t e d t h a t t h e w a s h i n g p r o c e d u r e w a s n o t s e l e c t i v e i n t h e r e m o v a l o f p r o t e i n s s i n c e s i m i l a r b a n d i n g p a t t e r n s w e r e o b s e r v e d i n b o t h i n t a c t ( c o n t r o l a n d i n o c u l a t e d ) ( F i g u r e 1 5 ) a n d w a s h e d ( c o n t r o l a n d i n o c u l a t e d ) ( F i g u r e 1 6 ) m u s c l e s a m p l e s . I t d i d , h o w e v e r , i n d i c a t e t h a t t h e w a s h i n g p r o c e d u r e w a s e f f e c t i v e i n t h e r e m o v a l o f t h e w a t e r -s o l u b l e p r o t e i n f r a c t i o n ( e q u i v a l e n t v o l u m e s o f t h e w a t e r - s o l u b l e p r o t e i n e x t r a c t e d f r o m b o t h i n t a c t a n d w a s h e d m u s c l e s a m p l e s w e r e a p p l i e d t o t h e g e l s ) , a s e v i d e n c e d b y t h e d e c r e a s e d s t a i n i n g i n t e n s i t y . T h e s e r e s u l t s s u p p o r t t h o s e o b t a i n e d i n p r o t e i n n i t r o g e n a n a l y s i s w h i c h i n d i c a t e d a s u b s t a n t i a l d e c r e a s e i n t h e 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 27,000 i 14,000 106,000 81,000 70,000 55,000 47,000 37,000 27,000 116,000 14,000 F I G U R E 1 5 : E l e c t r o p h o r e t o g r a m s o f w a t e r - s o l u b l e p r o t e i n s e x t r a c t e d f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 1 2 . 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 F I G U R E 1 6 : E l e c t r o p h o r e t o g r a m s o f w a t e r - s o l u b l e p r o t e i n s e x t r a c t e d f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y s 0 , 6 a n d 1 2 . /74 amount of water-soluble protein in the washed muscle samples as compared to the intact muscle tissue. Tu (1973) used polyacrylamide disc gel electrophoresis to examine the effect of a repetitive imbibition-centrifugation procedure for the removal of the water-soluble protein fraction 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 in the number or pattern of bands in either the control or inoculated samples for both intact and washed muscle tissue. By day 12, although bacterial numbers were approximately 10 9 2 10 and 10 CFU/cm 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 in either sample. The only evidence of proteolysis was the appearance of a band with an apparent molecular weight of 116,000 in the intact inoculated muscle sample. Dainty et al. (1975), using SDS-PAGE, reported that proteolysis was not detected in the sarcoplasmic proteins extracted from slime inoculated beef, even though bacterial numbers 10 2 had reached 10 CFU/cm . In.rcontrast, Hasegawa et al. (1970a) reported extensive alterations in 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 in the pattern due to proteolysis caused by P. f r a g i . The apparent discrepancy in the extent of proteolysis reported by the above two studies, could possibly be attributed to a myosystem specificity 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 in it s a b i l i t y to detect proteolysis as compared to starch gel-electrophoresis. It is possible that similar situations were encountered in the present study. No changes were apparent in 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 radiation reduced the proteolytic activity of muscle cathepsins by approximately 50%. 2. Salt-soluble proteins Electrophoretograms of the salt-soluble protein.fraction extracted from intact and washed muscles are presented in Figures 17 and 18, respectively. Tentative identification of some of the myofibrillar proteins was made (Figures 17 and 18). Examination of the day 0 electrophoretograms indicated no apparent differences in banding pattern between intact (Figure 17) and washed (Figure 18) muscle samples. This would suggest that the washing procedure used did not result in myofibrillar protein / 7 6 Day Apparent I n t a c t T e n t a t i v e I n t a c t Apparent m o l e c u l a r c o n t r o l i d e n t i f i c a t i o n i n o c u l a t e d m o l e c u l a r weight w e i g h t 163,030 126,000 108,003 75,030 55,000 • 43,000 A c t i n 39,003 H Tropomyosin 25,000 21,000 17,000 163,000 126,003 108,000 75,003 25,000 21,000 17,000 163,00C Troponins and L i g h t myosins 55,000 45,003 M A c t i n 39,000 • Tropomyosin • 10,020 • 187,000 loelooc • - K t l n l n I Hf0l 75,000 • 55,000 48,000 H 44,000 39,000 I _ 21.000 17,000 13,000 F I G U R E 1 7 : E l e c t r o p h o r e t o g r a m s o f 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 f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 1 2 . Ill Day Apparent Washed Tentative Washed Apparent molecular control i d e n t i f i c a t i o n inoculated molecular weight weight 163,000 mm 126,000 mm 108,000 mm ,,-actinin 75,000 I 55,000 H 48,000 • Actin 39,000 Tropomyosin 25,000 21,000 17,000 163,000 126,000 108,000 75,000 39,000 25,000 21,000 17,000 163,000 126,000 108,000 75,000 55,000 25,000 21,000 17,000 Troponins and Light myosins 55,000 M 48,000 • Actin Tropomyosin 40,000 48,000 H Actin 39,000 • WM 40,000 3528,000 F I G U R E 1 8 : E l e c t r o p h o r e t o g r a m s o f 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 f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 1 2 . /78 denaturation. Previous work (Tu, 1973) indicated that removal of sarcoplasmic f l u i d from bovine muscle by prolonged c e n t r i f u g a t i o n and imbibition with 0.9% NaCl may r e s u l t i n m y o f i b r i l l a r denaturation. No changes were observed i n the electrophoretic gel patterns of the s a l t - s o l u b l e proteins extracted from e i t h e r the i n t a c t control (Figure 17) and washed control (Figure 18) during the 12 days of incubation. By day 6, the appearance of a new band below a c t i n with an apparent molecular weight of 40,000 was evident i n both i n t a c t (Figure 17) and washed (Figure 18) inoculated samples. No other changes i n banding pattern were evident i n e i t h e r i n t a c t inoculated or washed inoculated electrophoretograms. At day 12, extensive a l t e r a t i o n s i n the banding pattern of the i n t a c t inoculated sample (Figure 17) were observed. Increases i n components with apparent molecular weights of 187,000, 92,000 and 83,000 were evident. The l a t t e r two components may have been breakdown products of a - a c t i n i n (apparent molecular weight of1108,000) since the i n t e n s i t y of t h i s band seemed to have decreased. An extreme breakdown i n the i n t e n s i t y of the band representing a c t i n occurred with a subsequent increase of a band having an apparent molecular weight of 44,000. The l a t t e r band obscured the presence of tropomyosin; tropomyosin had o r i g i n a l l y been i d e n t i f i e d with an apparent molecular weight of 39,000. A change i n banding pattern was also noted i n the lower molecular / 7 9 w e i g h t p r o t e i n s w h e r e c o m p o n e n t s w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 3 1 , 0 0 0 a n d 2 7 , 0 0 0 w e r e e v i d e n t . T h e r e s u l t s i n d i c a t e t h a t p r o t e o l y s i s o f t h e 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 f r o m t h e i n t a c t i n o c u l a t e d m u s c l e s a m p l e o c c u r r e d a s a r e s u l t o f t h e g r o w t h o f P. fragi. B o r t o n et al. ( 1 9 7 0 b ) r e p o r t e d t h a t s a l t - s o l u b l e p r o t e i n e x t r a c t s f r o m p o r c i n e m u s c l e i n o c u l a t e d w i t h Pseudomonas fragi s h o w e d a l o s s i n t h e n u m b e r o f p r o t e i n b a n d s o n e l e c t r o p h o r e s i n d i c a t i n g t h a t t h i s o r g a n i s m e x h i b i t e d some p r o t e o l y t i c e f f e c t u p o n m y o f i b r i l l a r p r o t e i n s . T a r r a n t et al. ( 1 9 7 1 ) , u s i n g d i s c g e l - e l e c t r o p h o r e s i s , o b s e r v e d c o n s i d e r a b l e s a l t - s o l u b l e p r o t e i n d e g r a d a t i o n i n p o r k m u s c l e i n o c u l a t e d w i t h P. fragi d u r i n g a 20 d a y i n c u b a t i o n a t 1 0 ° C . A l t h o u g h c h a n g e s i n b a n d i n g p a t t e r n w e r e a l s o n o t e d f o r t h e w a s h e d i n o c u l a t e d m u s c l e s a m p l e , t h e s e w e r e n o t a s e x t e n s i v e a s t h o s e o b s e r v e d f o r t h e i n t a c t i n o c u l a t e d s a m p l e . New b a n d s w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 3 5 , 0 0 0 a n d 2 8 , 0 0 0 w e r e e v i d e n t . No o t h e r c h a n g e s w e r e a p p a r e n t . T h e l a c k o f e x t e n s i v e c h a n g e s i n t h e w a s h e d i n o c u l a t e d s a m p l e w o u l d s u g g e s t t h a t t h e r e d u c t i o n o f s a r c o p l a s m r e s t r i c t e d b a c t e r i a l n u m b e r s f r o m r e a c h i n g a l e v e l n e c e s s a r y f o r p r o t e o l y s i s . D a i n t y et al. ( 1 9 7 5 ) , u s i n g p o l y a c r y l a m i d e g e l - e l e c t r o p h o r e s i s , r e p o r t e d t h a t 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 l a r p r o t e i n f r a c t i o n e x t r a c t e d f r o m s l i m e i n o c u l a t e d b e e f w a s n o t d e t e c t e d u n t i l b a c t e r i a l n u m b e r s w e r e i n e x c e s s o f 3 . 2 x 1 0 9 / c m 2 . / 8 0 3. ' Urea-soluble proteins T h e u r e a - s o l u b l e f r a c t i o n c o n t a i n s t h o s e p r o t e i n s w h i c h w e r e i n s o l u b l e i n w a t e r o r s a l t s o l u t i o n s b u t d o e s n o t i n c l u d e t h e s t r o m a l p r o t e i n s ( S a g e , 1 9 7 4 ) . T h e 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 t h e u r e a - s o l u b l e p r o t e i n e x t r a c t s f r o m i n t a c t a n d w a s h e d m u s c l e s a m p l e s ( c o n t r o l a n d i n o c u l a t e d ) a r e p r e s e n t e d i n F i g u r e s 19 a n d 2 0 , r e s p e c t i v e l y . B a n d s c h a r a c t e r i z e d w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 4 8 , 0 0 0 , 3 9 , 0 0 0 , 2 5 , 0 0 0 a n d 1 7 , 0 0 0 i n b o t h i n t a c t a n d w a s h e d d a y 0 m u s c l e s a m p l e s ( F i g u r e s 19 a n d 2 0 ) c o r r e s p o n d i n a p p a r e n t m o l e c u l a r w e i g h t t o b a n d s i n t h e g e l p a t t e r n 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 e x t r a c t e d f r o m t h e same s a m p l e s ( F i g u r e s 17 a n d 1 8 ) . T h i s w o u l d s u g g e s t t h a t t h e u r e a - s o l u b l e p r o t e i n f r a c t i o n m a y h a v e b e e n c o n t a m i n a t e d w i t h some 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 . E x a m i n a t i o n o f t h e 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 t h e u r e a - s o l u b l e p r o t e i n f r a c t i o n e x t r a c t e d f r o m t h e i n t a c t c o n t r o l m u s c l e s a m p l e s r e v e a l e d n o c h a n g e i n b a n d i n g p a t t e r n d u r i n g t h e 12 d a y s i n c u b a t i o n . S i m i l a r r e s u l t s w e r e o b s e r v e d f o r t h e S D S - P A G E g e l s o f t h e w a s h e d c o n t r o l s a m p l e s . No a p p a r e n t c h a n g e s w e r e o b s e r v e d i n t h e d a y 6 e l e c t r o p h o r e t o g r a m s o f t h e u r e a - s o l u b l e p r o t e i n s e x t r a c t e d f r o m e i t h e r t h e i n t a c t i n o c u l a t e d ( F i g u r e 1 9 ) o r t h e w a s h e d i n o c u l a t e d ( F i g u r e 2 0 ) s a m p l e . B y d a y . 1 2 , a n a l m o s t c o m p l e t e b r e a k d o w n o f t h e e l e c t r o p h o r e t i c p a t t e r n o f t h e u r e a - s o l u b l e p r o t e i n f r a c t i o n f r o m 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 74,000 64,000 48,000 39,000 187,000 142,000 119,000 104,000 48,000 43,000 25,000 17,000 14,000 F I G U R E 1 9 : E l e c t r o p h o r e t o g r a m s o f u r e a - s o l u b l e p r o t e i n s e x t r a c t e d f r o m i n t a c t c o n t r o l a n d i n t a c t i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 1 2 . / 8 2 Day Apparent Mashed Washed molecular control inoculated 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 74,000 64,000 48,000 39,000 25,000 17,000 14,000 F I G U R E 2 0 : E l e c t r o p h o r e t o g r a m s o f u r e a - s o l u b l e p r o t e i n s e x t r a c t e d f r o m w a s h e d c o n t r o l a n d w a s h e d i n o c u l a t e d m u s c l e s a m p l e s a t d a y 0 , 6 a n d 1 2 . /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 in 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 in the gel pattern of the urea-soluble protein fraction extracted from the day 12 washed inoculated sample were evident. 4. Urea-insoluble proteins The final fraction of the protein extraction procedure was the urea-insoluble protein fraction.< It was assumed that this fraction represented the stromal proteins. 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 at day 0, 6 and 12. FIGURE 22; Electrophoretograms of urea-insoluble proteins extracted from washed control and washed inoculated muscle samples at day 0, 6 and 12. / 8 6 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 f r a c t i o n e x t r a c t e d f r o m t h e i n t a c t c o n t r o l m u s c l e s a m p l e s i n d i c a t e d n o a p p a r e n t c h a n g e i n b a n d i n g p a t t e r n d u r i n g t h e 12 d a y s o f i n c u b a t i o n ( F i g u r e 2 1 ) . S i m i l a r r e s u l t s w e r e o b s e r v e d f o r t h e w a s h e d c o n t r o l g e l s ( F i g u r e 2 2 ) . E x a m i n a t i o n o f t h e 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 t h e 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 e x t r a c t e d f r o m t h e i n t a c t i n o c u l a t e d s a m p l e i n d i c a t e d n o c h a n g e i n p a t t e r n u p t o d a y 6 ( F i g u r e 2 1 ) . H o w e v e r , e x a m i n a t i o n o f t h e d a y 12 e l e c t r o p h o r e t o g r a m r e v e a l e d e x t r e m e a l t e r a t i o n s i n t h e b a n d i n g p a t t e r n ( F i g u r e 2 1 ) . T h e m o s t o b v i o u s c h a n g e s w e r e t h e a l m o s t c o m p l e t e d i s a p p e a r a n c e o f t h e b a n d s w i t h a p p a r e n t m o l e c u l a r w e i g h t s o f 1 6 9 , 0 0 0 a n d 5 0 , 0 0 0 . T h e r e s u l t s o b t a i n e d w o u l d i n d i c a t e t h a t p r o t e o l y s i s 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 o c c u r r e d a s a r e s u l t o f t h e g r o w t h o f P. fragi. Some m e m b e r s o f t h e g e n u s Pseudomonas h a v e b e e n f o u n d t o p r o d u c e c o l l a g e n a s e s ( O c k e r m a n et al., 1 9 6 9 ) . G e l p a t t e r n s 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 f r o m t h e w a s h e d i n o c u l a t e d m u s c l e l i k e t h o s e o f t h e i n t a c t i n o c u l a t e d i n d i c a t e d n o o b v i o u s c h a n g e i n p a t t e r n a s o f d a y 6 , h o w e v e r , u n l i k e t h e i n t a c t i n o c u l a t e d g e l s , n o a p p a r e n t c h a n g e s w e r e n o t e d i n t h e d a y 12 e l e c t r o p h o r e t o g r a m s . G. Electron Microscopy B a c t e r i a l s p o i l a g e o f m e a t a t c h i l l t e m p e r a t u r e s i s g e n e r a l l y r e g a r d e d a s a s u r f a c e p h e n o m e n o n ( G i l l a n d P e n n e y , 1 9 7 7 ) . I n o r d e r t o e x a m i n e t h e e f f e c t s o f Pseudomonas fragi o n t h e s u r f a c e u l t r a s t r u c t u r e / 8 7 o f b o t h w a s h e d a n d i n t a c t m u s c l e s a m p l e s , s c a n n i n g a n d t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y w e r e e m p l o y e d . 1. Scanning electron microscopy M i c r o g r a p h s o f t h e u n i n o c u l a t e d c o n t r o l s ( i n t a c t a n d w a s h e d ) w e r e t a k e n a t t h e s a m e t i m e a s t h o s e o f t h e i n o c u l a t e d s a m p l e s . S i n c e c h a n g e s i n t h e s u r f a c e u l t r a s t r u c t u r e o f t h e c o n t r o l s w e r e n o t e x t e n s i v e d u r i n g t h e 12 d a y s o f i n c u b a t i o n o n l y m i c r o g r a p h s o f d a y 0 ( F i g u r e 2 3 ) a n d d a y 12 ( F i g u r e 2 4 ) c o n t r o l s a r e s h o w n . P e r f o r a t i o n s d i d a p p e a r o n t h e s u r f a c e o f b o t h m u s c l e s a m p l e s ( a r r o w F i g u r e 2 4 ) , w h i c h m a y b e i n d i c a t i v e o f a u t o l y t i c t i s s u e d e t e r i o r a t i o n . S u c h i n d i c a t i o n s o f t i s s u e d e t e r i o r a t i o n w e r e e x t r e m e l y v a r i a b l e f r o m s i t e t o s i t e o n t h e s a m e s a m p l e . S c h a l l e r a n d P o w r i e ( 1 9 7 1 ) , i n a SEM s t u d y o f t h e s k e l e t a l m u s c l e f r o m v a r i o u s s o u r c e s , f o u n d some s u r f a c e u l t r a s t r u c t u r a l d e g r a d a t i o n o n t h e Longissimus dorsi m u s c l e d u r i n g t h e c o m m e r c i a l a g i n g o f b e e f . E x a m i n a t i o n o f t h e i n o c u l a t e d s a m p l e s ( i n t a c t a n d w a s h e d ) m i c r o g r a p h s a t d a y 0 f a i l e d t o r e v e a l t h e p r e s e n c e o f a n y b a c t e r i a ( F i g u r e 2 5 ) a l t h o u g h s t a n d a r d p l a t e c o u n t i n d i c a t e d 5 6 2 b a c t e r i a l n u m b e r s i n t h e r a n g e o f 10 t o 10 C F U / c m ( s e e T a b l e 3 ) . T h e l a c k o f d e t e c t a b l e b a c t e r i a m a y b e d u e t o t h e r e m o v a l o f t h e m i c r o o r g a n i s m s f r o m t h e m u s c l e s u r f a c e d u r i n g f i x a t i o n . M a r s h a l l et al. ( 1 9 7 1 ) " , s t u d y i n g t h e a t t a c h m e n t o f a m a r i n e Pseudomonas s p p . t o g l a s s s l i d e s , o b s e r v e d t h a t d u r i n g t h e p r i m a r y s t a g e s o f F I G U R E 2 3 : SEM micrographs of day 0 , uninoculated intact (a) and washed (b) muscle samples. Collagen and reticular f i b r i l s (arrow) cover the muscle surface. F I G U R E 2 4 : S E M 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 i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s . A r e a s o f t i s s u e d e t e r i o r a t i o n h a v e o c c u r r e d ( a r r o w ) . F I G U R E 2 5 : S E M m i c r o g r a p h s o f d a y 0 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d (b ) m u s c l e s a m p l e s . /91 attachment, bacteria were held weakly to the surface by such forces as London-van der Waals interactions andncould thus be easily removed. McMeekin et al. '(1979), using SEM to examine the 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 fixation in glutaraldehyde, "a scum formed on the surface of the fixative which was likely to have been unfixed lipid material or material washed from the surface by the fixative, and presumably contained many microorganisms". By day 3, limited growth of P. fragi was evident on micrographs of both intact and washed inoculated muscle samples (Figure 26). The observance of bacteria at day 3 and not at day 0 may be due to the increase in bacterial numbers during incubation, such that the probability of bacteria remaining on the surface during fixation would have increased. Although not eyident in the micrographs, i t is 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 "fine slime fibers" that mediate bacterial adhesion were below the limit of resolution of the SEM and were not seen unless they were aggregated. After 6 days of incubation, production of what was tentatively identified as extracellular 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 extracellular acidic polysaccharides by primary attached bacteria (Marshall et al., 1971; Fletcher and Floodgate,1973). Deinema and Zevenhuizen (1972) reported exocellular cellulose f i b r i l s on many gram-negative bacteria. McCowan et al. (1978) reported the presence of "carbohydrate coats" surrounding adherent bacteria, which appeared to mediate the attachment of the bacteria to the epithelium of the reticulo-rumen of cattle, to food particles, and to each other to form microcolonies. Costerton et al. (1978) had earlier termed this carbohydrate material "glycocalyx". By day 6, proteolysis of muscle tissue in areas of bacterial growth in both intact and washed inoculated samples (D in Figure 27) became apparent. The amount of glycocalyx and surface degradation in 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 bacterial population was a complex mass of cells that was apparently held together by slime fibers F I G U R E 2 7 : 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 i n t a c t ( a ) a n d w a s h e d (b ) m u s c l e s a m p l e s . G l y c o c a l y x ( a r r o w ) c a n b e s e e n o n t h e s u r f a c e o f b a c t e r i a . A r e a s o f b a c t e r i a l p r o t e o l y s i s a r e e v i d e n t o n t h e m u s c l e s u r f a c e (D) . / 9 5 b F I G U R E 2 8 : S E M m i c r o g r a p h s o f d a y 9 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s . B a c t e r i a a p p e a r t o b e h e l d t o g e t h e r b y s l i m e f i b e r s ( a r r o w ) . L a r g e a r e a s o f s u r f a c e d e g r a d a t i o n a r e e v i d e n t ( D ) . /96 (arrow in Figure 28). Large areas of surface degradation were evident in both intact and washed inoculated samples (D in Figure 28). After 12 days of incubation, the distribution of the bacteria on the muscle surface was non-uniform and exhibited "intermittent colonization" (McCowan et al., 1978), i.e. areas of high bacterial density (Figure 29). A similar type of growth pattern was reported by McCowan et al. (1979) during a study of colonization of the: bovine tongue. A higher proportion of cells in a given microenvironment could suggest that the physical and chemical conditions of the environment are more favourable for active uptake of nutrients (ZoBell, 1943; Stotzky and Rem, 1966; Fletcher, 1979). The microcolony of the washed inoculated sample was held together by a well defined glycocalyx network while that of the intact inoculated sample was amorphous in nature (Figure 29). This may have resulted from differences in available nutrients (Firstenberg-Eden et al., 1979). Although the washed muscle samples were an adequate growth medium (as indicated by bacterial 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 bacterial growth. When provided with complex and simple nutrient sources, microorganisms wi l l invariably, or always, u t i l i z e the simpler constituents (amino acids, nucleotides, or carbohydrates) preferentially to the more complex ones such as proteins (Jay and Shelef, 1976). Costerton et al. (1978) stated F I G U R E 2 9 : S E M m i c r o g r a p h s o f d a y 12 i n o c u l a t e d i n t a c t ( a ) a n d w a s h e d ( b ) m u s c l e s a m p l e s . /98 that polymers produced by bacteria may not only position the bacteria, but also conserve and concentrate the digestive enzymes and serve as a food reservoir. ZoBell (1943) found slime production, by attached bacteria, to be influenced primarily by the microecosystem. He observed that more slime was produced in ecosystems in which i t was d i f f i c u l t for bacteria to survive. Physical degradation of the muscle tissue in both intact and washed inoculated muscle samples was extensive in areas of colonization. The degradation of proteins, presumably the stromal and myofibrillar fractions, would explain the changes in the SDS-PAGE patterns of the urea-insoluble, urea-soluble and salt-soluble protein fractions of the intact inoculated muscle samples after 12 days incubation. Borton et al. (1970a and b) and Tarrant et al. (1971) reported the proteolysis of myofibrillar proteins in bovine muscle inoculated with P. fragi. Ockerman et al. (1969) observed that the stromal protein fraction was degraded in muscle inoculated with a Pseudomonas spp. Localized degradation of proteins also appeared in the washed inoculated samples with no apparent proteolysis as indicated by SDS-PAGE. It is conceivable that because proteolysis was restricted to localized areas, i t was not detected by SDS-PAGE; bacterial numbers were not sufficient to affect the entire muscle mass. Dainty et al. (1975), studying the protein changes in slime inoculated beef, reported that proteolysis was not detected until microbial numbers were imexcess /99 9 2 of 3.2 x 10 /cm . That the bacterial population of the washed inoculated samples never reached this level may account for the lack of detectable proteolysis. In addition to attractive forces, the microtopography of the muscle surface may play an important role in attachment. Crevices and channels (arrow in Figure 26) between the muscle fibers were in most cases larger than the bacteria, thus allowing for physical entrapment. Once trapped, bacterialcould then produce exopolysaccharides. Firstenberg-Eden et ali (1979), studying the attachment of bacteria to the teats of cows, reported that bacterial adherence may result from organisms becoming locked in small holes or in skin tissue. Similar results were reported by McMeekin et al. (1979). Figure 30a demonstrates the irregularity of the muscle surface. A closer examination of the area between the muscle f i b r i l s (A in Figure 30a) revealed a microcolony of bacteria which had produced extracellular substances (S in Figure 30b). By this process bacterial may encapsulate themselves in surface irregularities, thereby making detachment d i f f i c u l t . Bacterial adherence may explain the practical d i f f i c u l t y in obtaining accurate estimates of bacterial numbers using non-destructive methods (swabbing and rinsing). Several authors (Avens and Miller, 1970; Patterson, 1972; Notermans et al., 1975) observed that viable counts obtained with macerated samples were always greater than those obtained with swabs or rinses. / 1 0 1 2. ' Transmission electron microscopy T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (TEM) c o m b i n e d w i t h a p p r o p r i a t e s t a i n s , h a s b e e n u s e d t o p r o v i d e i n f o r m a t i o n a b o u t t h e s u r f a c e c h a r a c t e r i s t i c s ( C o s t e r t o n et al., 1 9 7 4 a ; C o s t e r t o n et al., 1 9 7 4 b ; P a t e r s o n et al., 1 9 7 5 ) a n d t h e m e c h a n i s m s o f b a c t e r i a l a d h e s i o n ( J o n e s et al., 1 9 6 9 ; F l e t c h e r a n d F l o o d g a t e , 1 9 7 3 ; F u l l e r a n d B r o o k e r , 1 9 7 4 ; C h e n g et al., 1 9 7 7 ; M c C o w a n et al., 1 9 7 8 ) . I n t h e p r e s e n t s t u d y , s a m p l e s f o r T E M w e r e s t a i n e d w i t h r u t h e n i u m r e d a n d o s m i u m t e t r o x i d e . R u t h e n i u m r e d h a s b e e n w i d e l y u s e d i n e l e c t r o n m i c r o s c o p y t o l o c a t e a c i d i c p o l y s a c c h a r i d e - l i k e m a t e r i a l ( L u f t , 1 9 6 8 ; F l e t c h e r a n d F l o o d g a t e , 1 9 7 3 ) . U s i n g a r u t h e n i u m r e d - o s m i u m t e t r o x i d e s t a i n c o m b i n a t i o n t o i n v e s t i g a t e a b a c t e r i a l s l i m e l a y e r f r o m a s i m u l a t e d s t r e a m , J o n e s et al. ( 1 9 6 9 ) , d e t e c t e d a p o l y s a c c h a r i d e - l i k e m a t i n w h i c h t h e b a c t e r i a w e r e l o c a t e d . M i c r o g r a p h s t a k e n o f s e c t i o n s o b t a i n e d f r o m s a m p l e s s t a i n e d w i t h r u t h e n i u m r e d a l o n e o r w i t h o s m i u m t e t r o x i d e a l o n e d i d n o t r e v e a l a n e l e c t r o n d e n s e a r e a s u r r o u n d i n g b a c t e r i a l c e l l s . I n c o n t r a s t , m i c r o g r a p h s t a k e n o f s a m p l e s i n w h i c h r u t h e n i u m r e d -o s m i u m t e t r o x i d e was u s e d s h o w e d d e n s e d e p o s i t s o f p o l y s a c c h a r i d e a r o u n d b a c t e r i a l c e l l s . O n l y s a m p l e s a t d a y 12 w e r e e x a m i n e d b y T E M s i n c e i t was f e l t t h a t b a c t e r i a l a d h e s i o n w o u l d b e b e s t e x e m p l i f i e d d u r i n g 9 2 a c t i v e s p o i l a g e ( 1 0 C F U / c m ) . A l t h o u g h b a c t e r i a l n u m b e r s w e r e i n 9 2 e x c e s s o f 10 C F U / c m , n o b a c t e r i a c o u l d b e s e e n o n t h e w a s h e d / 1 0 2 i n o c u l a t e d s a m p l e . O w i n g t o t h e l i m i t e d a r e a o f m u s c l e t i s s u e t h a t c a n b e v i e w e d u s i n g T E M , i t i s p o s s i b l e t h a t t h e r e g i o n o f t h e w a s h e d m u s c l e t i s s u e o b s e r v e d was v o i d o f b a c t e r i a . ( i n t e r m i t t e n t c o l o n i z a t i o n ) . T h e r e f o r e , o n l y m i c r o g r a p h s o f t h e d a y 12 i n t a c t m u s c l e s a m p l e s ( c o n t r o l a n d i n o c u l a t e d ) a r e p r e s e n t e d h e r e . T E M m i c r o g r a p h s i n d i c a t e d t h a t t h e a d h e r e n t b a c t e r i a l p o p u l a t i o n w a s h e l d t o g e t h e r b y s l i m e f i b e r s ( F i n F i g u r e 3 1 ) w h i c h m e d i a t e d t h e a t t a c h m e n t o f t h e b a c t e r i a t o t h e m u s c l e s u r f a c e . T h i s c o n f i r m e d t h e r e s u l t s o b t a i n e d f r o m t h e SEM s t u d y . R u t h e n i u m r e d s t a i n i n g d e m o n s t r a t e d t w o t y p e s o f e x t r a -c e l l u l a r p o l y m e r s a s s o c i a t e d w i t h a t t a c h m e n t . One t y p e o f p o l y m e r , d e s i g n a t e d p r i m a r y a c i d i c p o l y s a c c h a r i d e (P i n F i g u r e 3 2 ) , f o r m e d a t h i n . c o a t o n t h e s u r f a c e o f t h e b a c t e r i a l c e l l . T h e p r i m a r y p o l y s a c c h a r i d e i s p r o b a b l y i n i t i a l l y r e s p o n s i b l e f o r b a c t e r i a l a d h e s i o n ( F l e t c h e r a n d F l o o d g a t e , 1 9 7 6 ) . T h e s e c o n d t y p e , s e c o n d a r y p o l y s a c c h a r i d e , ( S i n F i g u r e 3 2 ) , was a n a m o r p h o u s s u b s t a n c e t h a t s t r e t c h e d b e t w e e n a n d a r o u n d a d j a c e n t b a c t e r i a o r b e t w e e n b a c t e r i a a n d t h e m u s c l e s u r f a c e . F l e t c h e r a n d F l o o d g a t e ( 1 9 7 3 ) f o u n d t h a t t h e s e c o n d a r y a c i d i c p o l y s a c c h a r i d e p r e d o m i n a t e d i n p r e p a r a t i o n s o f a t t a c h e d b a c t e r i a a n d w a s u s u a l l y a s s o c i a t e d w i t h g r o u p s o f o r g a n i s m s . T h e s e c o n d a r y p o l y s a c c h a r i d e , h o w e v e r , w a s n o t n e c e s s a r y f o r i r r e v e r s i b l e a d h e s i o n ; t h e i n i t i a l s t a g e o f i r r e v e r s i b l e s o r p t i o n o c c u r s p r i o r v t o t h e p r o d u c t i o n o f s e c o n d a r y p o l y s a c c h a r i d e a n d i n v o l v e s t h e c o n t a c t b e t w e e n t h e m u s c l e s u r f a c e a n d t h e p r i m a r y p o l y s a c c h a r i d e s u r r o u n d i n g t h e b a c t e r i a . /103 FIGURE 31: TEM micrograph of the adherent b a c t e r i a l population of the i n t a c t muscle surface a f t e r 12 days incubation. Slime f i b e r s (F) mediate c e l l to c e l l as well as c e l l to muscle tis s u e adhesion (X 25,000). / 1 0 4 F I G U R E 3 2 : T E M 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 ) a n d 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 ) . / 1 0 5 Z o B e l l ( 1 9 4 3 ) s u g g e s t e d t h a t t h e p o i n t o f a t t a c h m e n t b e t w e e n a b a c t e r i a l c e l l a n d s o l i d s u r f a c e m a y s e r v e a s a c o n c e n t r a t i o n p o i n t f o r b o t h n u t r i e n t s a n d e x o - e n z y m e s t h a t p r o m o t e t h e a s s i m i l a t i o n o f t h e n u t r i e n t m a t e r i a l . B l e b - l i k e e v a g i n a t i o n s o r p r o t r u s i o n s o b s e r v e d o n t h e s u r f a c e o f t h e b a c t e r i a (E i n F i g u r e 3 3 ) m a y b e i n v o l v e d i n p r o t e o l y s i s . D u t s o n et al. ( 1 9 7 1 ) r e p o r t e d s i m i l a r p r o t r u s i o n s o n t h e s u r f a c e o f P. fragi g r o w i n g o n p i g m u s c l e . T h e s u g g e s t i o n was made t h a t t h e s e e v a g i n a t i o n s , w h i c h m a y c o n t a i n p r o t e o l y t i c e n z y m e s , r e l e a s e t h e i r c o n t e n t s i n t o t h e m u s c l e t i s s u e a n d a r e s u b s e q u e n t l y r e s p o n s i b l e f o r m y o f i b r i l l a r d i s r u p t i o n . T h e a u t h o r s o b s e r v e d t h a t P. fragi o r g a n i s m s g r o w n o n n o n - m u s c l e m e d i a d i d n o t c o n t a i n s u r f a c e e v a g i n a t i o n s . W i e b e a n d C h a p m a n ( 1 9 6 8 ) f o u n d t h a t c e r t a i n n u t r i t i o n a l a n d p h y s i o l o g i c a l c o n d i t i o n s i n d u c e d t h e f o r m a t i o n o f s u r f a c e p r o t r u s i o n s o n t h e c e l l w a l l o f some p s e u d o m o n a d s . S m i r n o v a et al. ( 1 9 7 1 ) o b s e r v e d c e l l u l a r p r o t r u s i o n s o n c e r t a i n s p e c i e s o f b a c t e r i a w h i c h t h e y t e r m e d " m i c r o c a p s u l e s " . T h e i n a b i l i t y t o c l e a r l y r e s o l v e t h e r a d i a l p a t t e r n o f a c t i n a n d m y o s i n f i l a m e n t s was e v i d e n t i n a c r o s s - s e c t i o n o f t h e d a y 12 i n t a c t c o n t r o l m u s c l e s a m p l e ; ( F i g u r e 3 4 ) a n d p r e s u m a b l y r e s u l t e d f r o m a u t o l y s i s . I n a n e l e c t r o n m i c r o s c o p i c s t u d y o f p o s t m o r t e m b o v i n e m u s c l e , S t r o m e r a n d G o l l ( 1 9 6 7 ) r e p o r t e d t h a t i n m y o f i b r i l s s t o r e d f o r 13 d a y s a t 2 ° C , "structural d e t e r i o r a t i o n of the sarcomere was so severe that detailed examinations were impossible". 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 s i n c e n o m i c r o g r a p h s / 1 0 6 F I G U R E 3 3 : T E M 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 a n d i n c u b a t e d a t 4 ° C f o r 12 d a y s . B l e b - l i k e ( E ) e v a g i n a t i o n s c a n b e s e e n o n t h e c e l l s u r f a c e ( X 3 1 , 4 0 0 ) . F I G U R E 3 4 : T E M m i c r o g r a p h o f u n i n o c u l a t e d b o v i n e Longissimus dorsi m u s c l e ( c r o s s - s e c t i o n ) i n c u b a t e d f o r 12 d a y s a t 4 ° C (X 3 3 , 2 0 0 ) . /108 were taken of the day 0 intact control muscle sample, the lack of resolvability may have been an artifact induced by the fixation procedures. Muscle tissue adjacent to bacterial growth tended to be extremely disrupted (A in Figures 35 and 36) and presumably consisted of disintegrated sarcomere components. The area contiguous to this region was amorphous and resembled that seen in the control sample :(U in Figure 35 and 36). It is possible that proteolysis during bacterial spoilage is limited to an area surrounding the bacteria. Sage (1974), in a study of P. fragi inoculated chicken pectoralis, reported similar results and stated that "the average distance between the bacteria and the intact f i b r i l was 0.6 um". Dutson et al. (1971) found an extremely disrupted appearance in myofibrils from pig muscle inoculated and incubated with P. fragi (8 days at 10°C) as compared to the uninoculated controls. The extreme disruption of the myofibrils adjacent to bacterial growth evidenced in the day 12 intact inoculated sample, would tend to support the electrophoretic results of the salt-soluble protein extract obtained from this sample. /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). / n o F I G U R E 3 6 : T E M 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 a n d i n c u b a t e d a t 4 ° C f o r 12 d a y s . D i s i n t e g r a t e d 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 ) . / I l l GENERAL DISCUSSION Intact bovine Longissimus dorsi muscle was subjected to a mild washing procedure in order to reduce the concentration of sarcoplasmic f l u i d . Intact and washed muscle samples were inoculated with Pseudomonas fragi to evaluate the effect of sarcoplasmic reduction on bacterial growth and subsequent spoilage. The reduction of soluble low molecular weight components was reflected in a significantly 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 preferentially u t i l i z e low molecular weight, soluble components of meats (Jay, 1972; G i l l and Newton, 1978). G i l l (1976) and G i l l and Newton (1977) reported a preferential u t i l i z a t i o n of muscle substrates by pseudomonads during spoilage such that some amino acids and lactic acid were ut i l i z e d when glucose was exhausted. The catabolism of glucose may inhibit and/or suppress the enzymes involved in the catabolism of amino acids and lactic acid (Paigen and Williams, 1970). If we assume that glucose and amino acids are representative of the total carbohydrate and the non-protein nitrogen (NPN) content, respectively, the decrease in the total carbohydrate content of the intact inoculated muscle by day 6, may have contributed to the decrease in NPN content seen after day 6 due to a / 1 1 2 r e l a x a t i o n i n a m i n o a c i d c a t a b o l i t e r e p r e s s i o n . T h e d e c r e a s e i n N P N m a y a l s o h a v e r e s u l t e d f r o m t h e u t i l i z a t i o n o f n o n - p r o t e i n n i t r o g e n o u s c o m p o n e n t s f o r t h e i n i t i a t i o n o f b a c t e r i a l p r o t e o l y s i s ( R a m p t o n et al., 1 9 7 0 ) . T h e i n c r e a s e i n n o n - p r o t e i n n i t r o g e n p r i o r t o d a y 6 m a y b e r e l a t e d t o t h e d e c r e a s e i n t h e w a t e r - s o l u b l e p r o t e i n e x t r a c t a b i l i t y d u r i n g t h e s a m e t i m e p e r i o d . S D S - g e l e l e c t r o p h o r e s i s i n d i c a t e d t h a t e x t e n s i v e p r o t e o l y s i s o f t h e s a l t - s o l u b l e , u r e a - s o l u b l e a n d 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 e x t r a c t e d f r o m t h e i n t a c t m u s c l e o c c u r r e d a s a r e s u l t o f t h e P. fragi'.. T h e s e c h a n g e s w e r e n o t a p p a r e n t u n t i l a f t e r d a y 6", w h e n c e l l d e n s i t y 10 2 w a s a p p r o a c h i n g 1 0 C F U / c m . I t i s p o s s i b l e t h a t a c r i t i c a l b a c t e r i a l p o p u l a t i o n m u s t b e a c h i e v e d i n o r d e r f o r t h e r e s u l t s o f p r o t e o l y s i s t o b e d e t e c t e d b y e l e c t r o p h o r e s i s . ( D a i n t y et al. , " . 1 9 7 5 ) . T h e s y n t h e s i s o f p r o t e a s e s a n d o t h e r e x o e n z y m e s a r e u s u a l l y r e p r e s s e d u n t i l l a t e e x p o n e n t i a l p h a s e o f g r o w t h ( B o e t h l i n g , 1 9 7 5 ) . S e v e r a l w o r k e r s ( B o r t o n et al., 1 9 7 0 b ; T a r r a n t et al., 1 9 7 1 ; D a i n t y et al., 1 9 7 5 ) u s i n g e l e c t r o p h o r e s i s h a v e b e e n u n a b l e t o d e m o n s t r a t e p r o t e o l y s i s u n t i l s p o i l a g e was w e l l a d v a n c e d . T h e e x t e n s i v e p r o t e o l y s i s i n t h e e l e c t r o p h o r e t o g r a m s i s c o n s i s t e n t w i t h t h e s u r f a c e d e g r a d a t i o n o b s e r v e d i n t h e SEM m i c r o g r a p h s . L i t t l e p r o t e o l y s i s w a s a p p a r e n t i n t h e e l e c t r o p h o r e t o g r a m s o f t h e w a t e r - s o l u b l e p r o t e i n f r a c t i o n f r o m t h e i n t a c t i n o c u l a t e d s a m p l e . S i m i l a r r e s u l t s w e r e r e p o r t e d b y D a i n t y et al. ( 1 9 7 5 ) , who o b s e r v e d t h a t e v e n w h e n b a c t e r i a l n u m b e r s h a d 10 2 r e a c h e d 10 / c m , t h e e l e c t r o p h o r e t i c p a t t e r n s o f t h e s a r c o p l a s m i c p r o t e i n s r e m a i n e d u n c h a n g e d . /113 Increases i n water-soluble and s a l t - s o l u b l e proteins extracted from the i n t a c t inoculated muscle sample were evident during the l a t t e r stages of the experiment (after day 6). Although increases i n e x t r a c t a b i l i t y of the water-soluble protein f r a c t i o n were evident, l i t t l e change i n banding pattern occurred i n the electrophoretograms of t h i s f r a c t i o n . Therefore, i t i s conceivable that increases i n e x t r a c t a b i l i t y of a protein 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 of a f r a c t i o n not s p e c i f i c a l l y extracted f o r . G o l l et al. (1970) postulated that the prote o l y s i s of the stromal' proteins may cause the: appearance of new soluble peptides'in the sarcoplasmic protein f r a c t i o n . Results of the present study may support t h i s hypothesis since extensive breakdown of the electrophoretogram o f the urea-insoluble proteins extracted from the i n t a c t inoculated muscle t i s s u e was observed. However, the p o s s i b i l i t y also exists that hydrolysis products of the s a l t - s o l u b l e protein f r a c t i o n may have been extracted i n the water-soluble protein f r a c t i o n . The i n a b i l i t y to detect possible hydrolysis products from the s a l t - s o l u b l e , urea-soluble and urea-insoluble proteins extracted from the int a c t inoculated muscle i n the SDS-PAGE patterns of the water-soluble proteins may be due to a masking e f f e c t (hydrolysis products migrating to the same p o s i t i o n as proteins indigenous to that f r a c t i o n ) and/or the i n a b i l i t y of the gel to resolve the hydrolysis product (protein i s of such high molecular weight that i t i s unable to enter the g e l , or of such low molecular weight that 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 of 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 of 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 detected i n any other p r o t e i n f r a c t i o n (water-soluble, 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 correspondowell w i t h those obtained 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 the 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 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 over the 12 day p e r i o d . SEM micrographs o f the washed i n o c u l a t e d muscle, however, i n d i c a t e d areas of extensive surface degradation during 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 of P. f r a g i . This i n a b i l i t y to detect s u b s t a n t i a l p r o t e o l y s i s using 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 hypothesis that a c r i t i c a l b a c t e r i a l p o p u l a t i o n i s r e q u i r e d 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 . Tarrant et al. (1973), working with porcine muscle i n o c u l a t e d w i t h P. fragi, reported t h a t growth and spoilage occurred on the muscle s u r f a c e , and only at l a t e r stages d i d the e f f e c t s of spoilage penetrate the e n t i r e meat sample. SDS-gel electrophoretograms 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 extracted 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 the number or p a t t e r n of bands during the experiment suggesting that degradative changes due to a u t o l y s i s were minimal. 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 experienced i n both i n t a c t and washed c o n t r o l samples during the 12 days of i n c u b a t i o n . Evidence of some / 1 1 5 a u t o l y t i c processes was i n d i c a t e d by increases i n t o t a l carbohydrate of the i n t a c t c o n t r o l sample, p o s s i b l y due to the breakdown of ATP to r i b o s e ( H u l t i n , 1976). Sage (1974), working with gamma r a d i a t e d chicken muscle, reported l i t t l e a u t o l y s i s and s t a t e d that "the absence of significant autolysis may have been due to the • r a d i a t i o n treatment". Several authors (Doty and Wachter, 1955; Schweigert, 1959) reported t h a t r a d i a t i o n dosages of 1 to 1.6 Megarad seve r e l y decreased the p r o t e o l y t i c a c t i v i t y o f muscle cathepsins. Jay (1967) and Jay and Kontou (1967) s t a t e d that low temperature sp o i l a g e of myosystems occurs without causing s i g n i f i c a n t p r o t e o l y s i s . R e sults obtained i n the present study i n d i c a t e d that surface p r o t e o l y s i s occurred i n both i n t a c t and washed muscle samples, as evidenced by SEM, due to the growth of P. fragi. However, only i n the i n t a c t i n o c u l a t e d muscle sample was t h i s p r o t e o l y s i s r e a d i l y detected u s i n g SDS-gel e l e c t r o p h o r e s i s . Results of the SEM study i n d i c a t e d that surface ^degradation was r e s t r i c t e d t o areas of l o c a l i z e d c o l o n i z a t i o n i n both 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 6. Glycocalyx seemed to mediate not only c e l l to c e l l attachment, but a l s o c e l l to muscle surface attachment. Transmission e l e c t r o n micrographs confirmed the presence of g l y c o c a l y x i n the mediation of b a c t e r i a l adhesion. /116 CONCLUSIONS The results of the present study indicated that although inoculation of sarcoplasmic reduced bovine Longissimus dorsi muscle with Pseudomonas fragi resulted in extensive surface degradation (SEM micrographs), limited proteolysis was detected using SDS-gel electrophoresis (minor alteration in the salt-soluble protein electrophoretograms). No apparent proteolysis was observed in the electrophoretic patterns of the water-soluble, urea-soluble and urea-insoluble protein fractions extracted from the washed inoculated muscle tissue. Extractability studies indicated l i t t l e change in non-protein nitrogen, water-soluble and salt-soluble 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 slight decrease in total carbohydrate was evident due to the growth of P. f r a g i . The growth rate of the bacteria was significantly lower (P < 0.01) 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 alterations were observed in the SDS-gel electrophoretograms of the salt-soluble, urea-soluble and urea-insoluble proteins, however, only minor changes were observed in the gel patterns of the water-soluble proteins. Large increases in extractability of the water- and salt-soluble proteins occurred after day 6 in the intact inoculated muscle tissue. The pH of the'intact inoculated muscle / 1 1 7 t i s s u e increased as the b a c t e r i a l population increased. A decrease i n t o t a l carbohydrate content was observed due to the growth of P. f r a g i . 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