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Comparison of proteinase assay methods and identification of pseudomonads in milk Kwan, Kent Kee Ho 1983

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COMPARISON OF PROTEINASE ASSAY METHODS AND IDENTIFICATION OF PSEUDOMONADS IN MILK BY KENT KEE HO KWAN B. S c . , The U n i v e r s i t y of C a l g a r y , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF FOOD SCIENCE) We a c c e p t e d 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 UNIVERSITY OF BRITISH COLUMBIA AUGUST 1983 © Kent Kee Ho Kwan, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Food S c i e n c e The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 0 c t ]# 1983 DE-6 (3/81) i i ABSTRACT T h i s t h e s i s i s d i v i d e d i n t o two c h a p t e r s summarized s e p a r a t e l y b elow. (1) F o u r methods ( a b s o r b a n c e a t 280 nm; t h e Lowry method; t h e f l u o r e s c a m i n e method and t h e t r i n i t r o b e n z e n e s u l f o n i c a c i d method ) f o r d e t e r m i n i n g h y d r o l y s i s o f m i l k p r o t e i n s were compared. E a c h method was a p p l i e d t o t h e t r i c h l o r a c e t i c a c i d s o l u b l e f r a c t i o n o f m i l k p r o t e i n , w h i c h had been d i g e s t e d w i t h t r y p s i n f o r v a r i o u s p e r i o d s of t i m e . D e t e c t a b i l i t y was measured a s t h e r a t i o between s t a n d a r d e r r o r of e s t i m a t e and s l o p e c a l c u l a t e d f r o m t h e l i n e a r r e g r e s s i o n a n a l y s i s o f Deming f o r c a s e s when b o t h v a r i a b l e s were s u b j e c t t o e r r o r . A l t h o u g h i t was n o n d i m e n s i o n a l , t h e d e t e c t a b i l i t y t h u s c a l c u l a t e d was s i m p l e and r e l i a b l e f o r c o m p a r i n g a s s a y methods w h i c h were b a s e d on d i f f e r e n t a n a l y t i c a l p r i n c i p l e s . D e t e c t a b i l i t y a s w e l l a s the. d e t e c t i o n l i m i t m e a sured a c c o r d i n g t o S c h w e r d t f e g e r showed t h a t , o f t h e methods compared, t h e f l u o r e s c a m i n e method was most r e l i a b l e and s e n s i t i v e . (2) P r o t e o l y t i c pseudomonads i s o l a t e d f r o m raw m i l k were c l a s s i f i e d by n u m e r i c a l taxonomy. U n w e i g h t e d p a i r - g r o u p a v e r a g e - l i n k a g e c l u s t e r a n a l y s i s was u s e d t o c l u s t e r 49 b a c t e r i a l s t r a i n s , o f w h i c h 26 were Pseudomonas s p e c i e s a s d e s c r i b e d i n t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y , b a s e d on 52 c h a r a c t e r s . The m i l k i s o l a t e s r e s i d e d i n two c l u s t e r s : one c o n t a i n i n g P\_ f l u o r e s c e n s and t h e o t h e r P_^  f r a g i . i i i The i s o l a t e s i d e n t i f i e d w i t h f l u o r e s c e n s c o u l d h y d r o l y z e m i l k p r o t e i n s , b u t t e r f a t ; c o u l d p r o d u c e p h o s p h o l i p a s e . The P_j_ f r a g i -l i k e i s o l a t e s c o u l d h y d r o l y z e m i l k p r o t e i n s , b u t t e r f a t ; but c o u l d n o t p r o d u c e p h o s p h o l i p a s e and f l u o r e s c e n t p i g m e n t . S t u d i e s , on h y d r o l y t i c c h a r a c t e r s of m i l k i s o l a t e s , showed t h a t t h e n a t u r e o f t h e s u b s t r a t e and c o n d i t i o n s under w h i c h t h e t e s t was b e i n g c o n d u c t e d were c r i t i c a l . No r e l a t i o n s h i p was f o u n d between p r o t e o l y t i c p s y c h r o t r o p h s p o p u l a t i o n s and p r o t e o l y s i s i n m i l k s t o r e d a t 4 C. E l e c t r o p h e r o g r a m s showed t h a t / 3 - c a s e i n was more s u s c e p t i b l e t o d e g r a d a t i o n by m i l k pseudomonads. i v TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i v L I S T OF TABLES v i i i L I S T OF FIGURES i x ACKNOWLEDGEMENTS x i C h a p t e r 1. C o m p a r i s o n o f F o u r Methods f o r D e t e r m i n i n g P r o t e o l y t i c A c t i v i t y i n M i l k 1 INTRODUCTION 2 LITERATURE REVIEW 5 A. A p p l i c a t i o n o f t h e f l u o r e s c a m i n e method f o r a s s a y i n g p r o t e o l y s i s i n m i l k 5 B. C o m p a r i s o n o f methods f o r d e t e r m i n a t i o n of p r o t e i n h y d r o l y s i s 5 MATERIALS AND METHODS 8 A. C o m p a r i s o n o f t h e p r o t e i n a s e a s s a y methods 8 1 . A b s o r b a n c e a t 280 nm 8 2. Lowry method 9 3. T r i n i t r o b e n z e n e s u l f o n i c a c i d method .... 9 4. F l u o r e s c a m i n e method 9 B. S t a t i s t i c a l c o m p a r i s o n o f t h e p r o t e i n a s e a s s a y methods 10 C. D e t e r m i n a t i o n of t h e d e t e c t i o n l i m i t o f t h e TNBS and f l u o r e s c a m i n e methods 10 RESULTS AND DISCUSSION 12 V CONCLUSIONS 24 REFERENCES 25 C h a p t e r 2. I d e n t i f i c a t i o n and St u d y of P r o t e o l y t i c Pseudomonads I s o l a t e d from Raw M i l k 29 INTRODUCTION 30 LITERATURE REVIEW 32 A. Pseudomonas s p e c i e s i n raw m i l k 32 B. A p p r o a c h e s to. i d e n t i f i c a t i o n 32 1. I d e n t i f i c a t i o n a c c o r d i n g t o t h e B e r g e y ' s Manual of D e t e r m i n a t i v e B a c t e r i o l o g y (1974) 33 2. I d e n t i f i c a t i o n a c c o r d i n g t o t h e M a n u a l f o r t h e I d e n t i f i c a t i o n o f M e d i c a l B a c t e r i a (Cowan and S t e e l , 1965) 34 3. I d e n t i f i c a t i o n by t h e API 20E s y s t e m ( A n a l y t a b P r o d u c t s , P l a i n v i e w , NY) 35 4. I d e n t i f i c a t i o n by c l u s t e r a n a l y s i s 36 5. I d e n t i f i c a t i o n w i t h no r e f e r e n c e s p r o v i d e d 38 6. R a p i d c h a r a c t e r i z a t i o n o f B a c t e r i a by u s i n g a m u l t i p o i n t i n o c u l a t o r 39 MATERIALS AND METHODS 42 A. I s o l a t i o n o f p r o t e o l y t i c s t r a i n s f r o m raw m i l k 42 B. P r i m a r y c h a r a c t e r i z a t i o n t e s t s 42 v i 1 . Gram s t a i n 42 2. O x i d a s e r e a c t i o n 42. 3. C a t a l a s e t e s t 43 4. Growth a t pH 4.5 ,. 43 5. O x i d a t i o n o r f e r m e n t a t i o n o f g l u c o s e ... 43 6. M o t i l i t y 43 7. Growth on Pseudomonas i s o l a t i o n a g a r ... 44 C. S e c o n d a r y c h a r a c t e r i z a t i o n t e s t s 44 1. Growth a t 4 C and 41 C , 44 2. P r o d u c t i o n o f f l u o r e s c e n t p i g m e n t s 44 3. N i t r a t e r e d u c t i o n 44 4. A r g i n i n e d i h y d r o l a s e 45 5. I n d o l e p r o d u c t i o n 45 6. M e t h y l Red 45 7. Voges P r o s k a u e r 45 8. Tween 80 h y d r o l y s i s 46 9. B u t t e r f a t h y d r o l y s i s 46 10. P h o s p h o l i p a s e p r o d u c t i o n 46 11. G e l a t i n a s e p r o d u c t i o n 46 12. P r o t e i n a s e p r o d u c t i o n 46 13. A c c u m u l a t i o n o f p o l y - 0 - h y d r o x y b u t y r a t e . 46 14. U t i l i z a t i o n o f t h e d i f f e r e n t c a r b o n s o u r c e s 47 15. U t i l i z a t i o n o f t h e d i f f e r e n t n i t r o g e n o u s compounds 47 D. R a p i d c h a r a c t e r i z a t i o n u s i n g a m u l t i p o i n t i n o c u l a t o r 47 E. N u m e r i c a l taxonomy 50 v i i F. H y d r o l y t i c c h a r a c t e r i s t i c s 51 1. P r o t e i n a s e p r o d u c t i o n a t 4 C, 22 C and 32 C 51 2. G e l a t i n a s e p r o d u c t i o n a t 4 C and 22 C 51 G. P r o t e o l y t i c pseudomonads i n c u b a t e d i n s t e r i l e m i l k 52 H. E l e c t r o p h o r e s i s . 53 RESULTS AND DISCUSSION 55 A. I d e n t i f i c a t i o n of p r o t e o l y t i c pseudomonads i n raw m i l k 55 B. Growth o f p r o t e o l y t i c pseudomonads i n s t e r i l e m i l k . 67 CONCLUSIONS 75 REFERENCES 7 6 v i i i L I S T OF TABLES PAGE C h a p t e r 1 . T a b l e 1 . D e t e c t a b i l i t y o f t h e f l u o r e s c a m i n e method ( F l u o ) , t h e TNBS method, t h e Lowry method and t h e a b s o r b a n c e a t 280 nm (A 2 8 o) a s e s t i m a t e d by Deming's method 1 9 C h a p t e r 2. T a b l e 2. H y d r o l y t i c c h a r a c t e r i s t i c s o f t h e m i l k pseudomonads 64 i x L I S T OF FIGURES PAGE C h a p t e r 1. F i g . 1. C o m p a r i s o n o f t h e f l u o r e s c a m i n e method w i t h t h e TNBS method as a measure of p r o t e i n h y d r o l y s i s by Deming's method 13 F i g . 2. C o m p a r i s o n o f t h e f l u o r e s c a m i n e method w i t h t h e Lowry method as a measure of p r o t e i n h y d r o l y s i s by Deming's method 14 F i g . 3. C o m p a r i s o n o f t h e f l u o r e s c a m i n e method w i t h t h e A 2 B O method as a measure o f p r o t e i n h y d r o l y s i s by Deming's method 15 F i g . 4. C o m p a r i s o n o f t h e TNBS method w i t h t h e Lowry method a s a measure o f p r o t e i n h y d r o l y s i s by Deming's method 16 F i g . 5. C o m p a r i s o n o f t h e TNBS method w i t h t h e A 2 B o method as a measure o f p r o t e i n h y d r o l y s i s by Deming's method 17 F i g . 6. C o m p a r i s o n o f t h e Lowry method w i t h t h e A 2 s o method as a measure o f p r o t e i n h y d r o l y s i s by Deming's method 18 C h a p t e r 2. F i g . 7. The m u l t i p o i n t i n o c u l a t o r 49 F i g . 8. Dendrogram showing r e l a t i o n s h i p o f p r o t e o l y t i c pseudomonads from raw m i l k 56 F i g . 9. Growth and p r o t e i n a s e a c t i v i t y i n 10% RSM i n o c u l a t e d w i t h (a) I s o l a t e 0-0; (b) I s o l a t e 2-1; ( c ) I s o l a t e 2-2 and (d) I s o l a t e 8-1 68 F i g . 10. Growth and p r o t e i n a s e a c t i v i t y i n 10% RSM i n o c u l a t e d w i t h (a) I s o l a t e 4-1; X (b) I s o l a t e 6-3; ( c ) I s o l a t e 8-2 and (d) I s o l a t e 10-0 69 F i g . 11. E l e c t r o p h o r e t i c p r o f i l e s o f m i l k p r o t e i n s c a u s e d by i s o l a t e s 0-0 and 2-1 a f t e r 0, 6, 16 and 19 d a y s of s t o r a g e i n 10% RSM a t 4 C 71 F i g . 12. E l e c t r o p h o r e t i c p r o f i l e s o f m i l k p r o t e i n s c a u s e d by i s o l a t e s 8-1 and 8-2 a f t e r 0, 6, 16 and 19 d a y s o f s t o r a g e i n 10% RSM a t 4 C 72 F i g . 13. E l e c t r o p h o r e t i c p r o f i l e s o f m i l k p r o t e i n s c a u s e d by i s o l a t e s 4-1, 6-3, 10-0 and 2-2 a f t e r 0, 16 and 19 d a y s of s t o r a g e i n 10% RSM a t 4 C 73 x i ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o e x p r e s s h i s d e e p e s t g r a t i t u d e t o h i s R e s e a r c h S u p e r v i s o r , D r . B . J . S k u r a , f o r h i s a s s i s t a n c e and en c o u r a g e m e n t ; D r . S. N a k a i f o r h i s h e l p f u l a d v i c e . A p p r e c i a t i o n i s a l s o due t o members o f h i s s u p e r v i s o r y c o m m i t t e e : D r . W.D. P o w r i e and D r . P.M. T o w n s l e y f o r t h e i r c o n s t r u c t i v e c r i t i c i s m i n t h e p r e p a r a t i o n of t h i s t h e s i s . S p e c i a l t h a n k s a r e due t o A n n e - M a r i e Pham f o r h e r u n d e r s t a n d i n g , e n c o u r a g e m e n t and p r e p a r a t i o n o f t h i s t h e s i s . 1 CHAPTER 1 C o m p a r i s o n of f o u r methods f o r d e t e r m i n i n g p r o t e o l y t i c a c t i v i t y i n m i l k 2 INTRODUCTION As a c o n s e q u e n c e of c h a n g i n g s o c i o - e c o n o m i c c o n d i t i o n s and p r o d u c t i o n methods, t h e r e i s a t r e n d t o w a r d a l o n g e r p e r i o d o f t i m e e l a p s i n g between m i l k i n g and p r o c e s s i n g o f m i l k . T h e s e e x t e n d e d p e r i o d s o f t i m e p e r m i t t h e g r o w t h o f p s y c h r o t r o p h i c b a c t e r i a i n raw m i l k ( C o u s i n , 1 9 82). P r o t e i n a s e s p r o d u c e d by many s t r a i n s o f p s y c h r o t r o p h i c Pseudomonas s p e c i e s a r e e x t r e m e l y h e a t r e s i s t a n t (Adams e t a l . , 1976) and have been shown t o d e c r e a s e t h e s h e l f l i f e of d a i r y p r o d u c t s ( C o u s i n , 1982). The H u l l method ( H u l l , 1947) has been u s e d t o measure p r o t e o l y t i c a c t i v i t y i n m i l k i n most s t u d i e s (Adams e t a l . , 1975; G e b r e - E g z i a b h e r e t a l . , 1980; R i c h t e r e t a l . , 1979). T h i s c o l o r i m e t r i c method measures t h e a c i d - s o l u b l e t y r o s i n e and t r y p t o p h a n r e l e a s e d d u r i n g p r o t e o l y s i s . Cogan (1977) i n d i c a t e d t h a t t h e measurement o f p r o t e o l y s i s by means o f t h e i n c r e a s e i.n t r i c h l o r a c e t i c a c i d (TCA) s o l u b l e t y r o s i n e and t r y p t o p h a n has some d i s a d v a n t a g e s s i n c e l i t t l e T C A - s o l u b l e t y r o s i n e and t r y p t o p h a n may be r e l e a s e d d u r i n g p r o t e o l y s i s . The need f o r a r a p i d , s e n s i t i v e and r e l i a b l e method f o r d e t e r m i n g p r o t e o l y s i s has been s t r e s s e d (Law, 1979; R i c h t e r e t a l . , 1979). J u f f s (1973) e v a l u a t e d t h e Lowry m o d i f i c a t i o n o f t h e F o l i n p r o c e d u r e (Lowry e t a l . f 1951), e x p r e s s i n g p r o t e o l y s i s i n t e r m s of t y r o s i n e c o n c e n t r a t i o n . A l i c h a n i d i s and Andrews (1977) s t u d i e d t h e p r o t e o l y t i c a c t i v i t y o f t h e e x t r a c e l l u l a r p r o t e i n a s e p r o d u c e d by a Pseudomonas s t r a i n u s i n g a m o d i f i c a t i o n of t h e Lowry method. R i c h a r d s o n and Te W h a i t i (1978) u s e d a b s o r b a n c e 3 a t 280 nm ( A 2 B 0 ) t o a s s a y p r o t e i n a s e a c t i v i t y . T r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS), a n o n s p e c i f i c amino a c i d r e a g e n t , was u s e d t o measure p r o t e o l y s i s i n m i l k ( M c K e l l a r , 1981). S n o e r e n e t a l . (1979) and S n o e r e n and B o t h (1981) d e t e r m i n e d t h e n i t r o g e n c o n t e n t i n d i f f e r e n t f r a c t i o n s as ammonia by a r a p i d c o l o r i m e t r i c p r o c e d u r e (Koops e t a l . , 1975). G r i f f i t h s e t a l . (1981) u s e d a p r o t e i n a s e a s s a y b a s e d on t h e a b i l i t y of a v a r i e t y of p r o t e i n a s e s t o r e l e a s e a b l u e dye c o v a l e n t l y a t t a c h e d t o d e n a t u r e d c o l l a g e n . S t a r c h g e l and 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 have been u s e d t o e s t i m a t e d e g r a d a t i o n o f c a s e i n components i n raw m i l k ( J u f f s , 1975; Law e t a l . , 1979). C h i s m e t a l . (1979) d e v e l o p e d a s e n s i t i v e a s s a y f o r p r o t e i n a s e s b a s e d on t h e r e a c t i o n o f amino g r o u p s o f TCA-s o l u b l e p e p t i d e s and amino a c i d s w i t h t h e f l u o r e s c a m i n e . A m i c r o m e t h o d , u s i n g f l u o r e s c a m i n e , f o r t h e a s s a y of p r o t e o l y s i s was d e v e l o p e d by C a s t e l l e t a l . ( 1 9 7 9 ) . F l u o r e s c a m i n e r e a c t s w i t h f r e e amino g r o u p s o f amino a c i d s and p e p t i d e s l i b e r a t e d d u r i n g p r o t e o l y s i s . A l t h o u g h t h e r e have been many p a p e r s p u b l i s h e d f o r a s s a y i n g p r o t e o l y s i s , t h e r e a r e o n l y few p a p e r s i n w h i c h o b j e c t i v e and s t a t i s t i c a l l y r e l i a b l e c o m p a r i s o n of d i f f e r e n t methods a r e d e s c r i b e d . The p a p e r s t o compare d i f f e r e n t methods r e p o r t e d by Schwabe (1973) and by Kas a n d Rauch (1982) a r e a l l w i t h o u t r e l i a b l e s u p p o r t i n g d a t a . S c h w e r d t f e g e r (1977) compared t h e TNBS, F o l i n , t u r b i d i t y , r a d i a l d i f f u s i o n on g e l and t h e a n i l i n o n a p h t h a l e n e s u l f o n a t e f l u o r e s c e n c e methods u s i n g a d e t e c t i o n l i m i t d e t e r m i n e d a s t h e minimum amount o f t h e 4 p r o t e i n a s e s d e t e c t a b l e . However, t h i s method f o r c o m p a r i s o n i s s u b j e c t of t h e a n a l y t i c a l c o n d i t i o n s e s p e c i a l l y i n c u b a t i o n c o n d i t i o n s . T h i s method i s a l s o t e d i o u s s i n c e t h e amount of p r o t e i n a s e s has t o be c h a n g e d u n t i l F - v a l u e becomes s i g n i f i c a n t ( p < 0 . 0 l % ; n = 4 ) . The o b j e c t o f t h i s s t u d y was t o e v a l u a t e f o u r methods f o r m e a s u r i n g p r o t e i n h y d r o l y s i s i n m i l k i n t e rms o f t h e i r r e l i a b i l i t y and d e t e c t a b i l i t y . The methods compared were: a b s o r b a n c e a t 280 nm ( J u f f s , 1973); t h e Lowry method (Lowry e t a l . , 1951); a m o d i f i c a t i o n of t h e m i c r o f l u o r o m e t r i c method o f C a s t e l l e t a l . ( 1 9 7 9 ) ; and t h e TNBS method ( M c K e l l a r , 1981). T h e s e f o u r methods have not been compared b e f o r e i n a s t a t i s t i c a l l y r e l i a b l e way. D e t e c t a b i l i t y c a l c u l a t e d from t h e l i n e a r r e g r e s s i o n a n a l y s i s o f Deming (Wakkers e t a l . , 1975) and t h e d e t e c t i o n l i m i t d e f i n e d by S c h w e r d t f e g e r (1977) were u s e d f o r r e l i a b l e . c o m p a r i s o n . 5 LITERATURE REVIEW A. A p p l i c a t i o n o f t h e f l u o r e s c a m i n e method f o r a s s a y i n g  p r o t e o l y s i s i n m i l k F l u o r e s c a m i n e ( F l u r a m ) i s a u s e f u l r e a g e n t f o r t h e d e t e c t i o n o f amino a c i d s , p e p t i d e s , p r o t e i n s and o t h e r p r i m a r y a m i n e s . I t s r e a c t i o n w i t h p r i m a r y amines i s s p o n t a n e o u s a t room t e m p e r a t u r e . The r e a c t i o n p r o d u c t s a r e f l u o r e s c e n t , w h i l e f l u o r e s c a m i n e and i t s d e g r a d a t i o n p r o d u c t s a r e n o n - f l u o r e s c e n t ( U d e n f r i e n d e t a l . , 1972). The s i m p l i c i t y and s e n s i t i v i t y o f t h e f l u o r e s c a m i n e method make i t a t t r a c t i v e f o r p r o t e i n a s e a c t i v i t y d e t e r m i n a t i o n . Schwabe (1973) d e s c r i b e d a f l u o r e s c e n t a s s a y f o r d e t e r m i n i n g c a t h e p s i n a c t i v i t y u s i n g f l u o r e s c a m i n e . C h i s m e t a l . (1979) m o d i f i e d t h e method o f Schwabe and a p p l i e d i t t o a s s a y p r o t e i n a s e s i n m i l k . T h i s method i n v o l v e d t h e r e a c t i o n s o f T C A - s o l u b l e p e p t i d e s and amino a c i d s w i t h f l u o r e s c a m i n e . P e a r c e (1979) d e v e l o p e d a method u s i n g f l u o r e s c a m i n e t o s t u d y t h e r e n n e t a c t i o n on m i l k . The change i n f l u o r e s c e n c e i n t e n s i t y b r o u g h t a b o u t by r e n n e t a c t i o n was p r o p o r t i o n a l t o t h e amount o f c a s e i n o - m a c r o p e p t i d e r e l e a s e d . Beeby (1980) a l s o u s e d f l u o r e s c a m i n e t o f o l l o w t h e a c t i o n o f c h y m o s i n on / c - c a s e i n . B. C o m p a r i s o n o f methods f o r d e t e r m i n a t i o n o f p r o t e i n  h y d r o l y s i s 6 When two o r more methods f o r d e t e r m i n a t i o n o f p r o t e i n h y d r o l y s i s e x i s t , t h e r e a r i s e s a t once t h e p r o b l e m o f c o m p a r i n g t h e i r r e l a t i v e m e r i t s . A c c o r d i n g t o Schwabe's (1973) f i n d i n g , t h e f l u o r e s c a m i n e method was one h u n d r e d t i m e s more s e n s i t i v e t h a n t h e Lowry method. D a t a from w h i c h t h e c o n c l u s i o n was drawn were not p u b l i s h e d . N a k a i e t a l . (1974) d e s c r i b e d a s t u d y on t h e a p p l i c a t i o n of f l u o r e s c a m i n e i n t h e a n a l y s e s o f p e p t i d e s i n column c h r o m a t o g r a p h i c , f r a c t i o n s . The f l u o r e s c e n t a s s a y was f o u n d t o be a p p r o x i m a t e l y f i v e t i m e s a s s e n s i t i v e a s t h e n i n h y d r i n method. However t h e r e was no d a t a t o s u p p o r t t h i s c o n c l u s i o n . S p e n c e r and S p e n c e r (1974) d i d a c o m p a r i s o n of t h e n i n h y d r i n a s s a y method and t h e 1 - a n i l i n o - 8 - n a p h t h a l e n e s u l f o n a t e (ANS) a s s a y method. P r o t e o l y t i c a c t i v i t y o f an e x t r a c t f r o m pumpkin s e e d s u s i n g pumpkin s e e d g l o b u l i n a s a s u b s t r a t e was d e t e r m i n e d by t h e two methods. F l u o r i m e t r i c a c t i v i t y was g i v e n a s change i n r e l a t i v e f l u o r e s c e n c e u n i t . A c t i v i t y by t h e n i n h y d r i n method was g i v e n a s change i n a b s o r b a n c e a t 570 nm. The A N S - f l u o r e s c e n c e method was f o u n d t o be a p p r o x i m a t e l y t wenty t i m e s more s e n t i v e i n t e rms o f t h e change i n f l u o r e s c e n c e r e l a t i v e t o t h e change i n a b s o r p t i o n p e r m i l l i g r a m e x t r a c t p r o t e i n . S c h w e r d t f e g e r (1977) compared d i f f e r e n t methods f o r t h e a s s a y p r o t e i n a s e a c t i v i t i e s by d e t e r m i n i n g t h e minimum amount of t h e p r o t e i n a s e s d e t e c t a b l e by e a c h method. The TNBS method was shown t o be t h e most s e n s i t i v e o f a l l t h e methods s t u d i e d . 7 Kas and Rauch (1982) d e t a i l e d a r a d i o m e t r i c method f o r t h e d e t e r m i n a t i o n of p r o t e o l y t i c a c t i v i t i e s i n f o o d . R e s u l t s o b t a i n e d were compared w i t h t h a t d e t e r m i n e d by t h e Lowry method. E n z y m a t i c a c t i v i t i e s were e x p r e s s e d i n a b s o r b a n c e and c o u n t s p e r m i n u t e s . T h r o u g h d i r e c t c o m p a r i s o n o f e n z y m a t i c a c t i v i t i e s m e a s ured by t h e two methods, t h e r a d i o m e t r i c method was r e p o r t e d t o be one h u n d r e d t i m e s more s e n s i t i v e . 8 MATERIALS AND METHODS F l u o r e s c a m i n e ( F l u r a m ) was o b t a i n e d from C h e m i c a l Dynamics C o r p . ( S o u t h P l a i n f i e l d , N J ) . T r i n i t r o b e n z e n e s u l f o n i c a c i d was o b t a i n e d f r o m Eastman Kodak ( R o c h e s t e r , NY) and F o l i n - C i o c a l t e a u p h e n o l r e a g e n t from F i s h e r S c i e n t i f i c ( F a i r Lawn, N J ) . T r y p s i n (Type X I , d i p h e n y l c a r b o n y l c h l o r i d e t r e a t e d , f r o m b o v i n e p a n c r e a s ) was o b t a i n e d f r o m Sigma C h e m i c a l Company ( S t . L o u i s , MO). A l l c h e m i c a l s and r e a g e n t s were o f r e a g e n t g r a d e . A. C o m p a r i s o n o f t h e p r o t e i n a s e a s s a y methods To a s s a y t h e e f f e c t o f t r y p s i n on m i l k p r o t e i n s , 2.5 ml o f 0.02 M p h o s p h a t e b u f f e r (pH 6.6) c o n t a i n i n g t r y p s i n (24.1 y g / m l ) were a d d e d t o 150 ml o f 10% (w/v) r e c o n s t i t u t e d s k i m m i l k (RSM) i n 0.2 M p h o s p h a t e b u f f e r (pH 6.6). The r e a c t i o n was r u n a t 40 C. A t v a r i o u s t i m e i n t e r v a l s (1-7 m i n ) , 5.0 ml o f t h e r e a c t i o n m i x t u r e were w i t h d r a w n and t h e r e a c t i o n s t o p p e d by t h e a d d i t i o n o f an e q u a l volume o f 24% (w/v) TCA. The m i x t u r e was c e n t r i f u g e d (10,000 x g, 5 min) and t h e s u p e r n a t a n t f i l t e r e d (Whatman No. 4 ) . The f i l t r a t e was a s s a y e d w i t h t h e f o l l o w i n g methods. F o r some s a m p l e s i t was n e c e s s a r y t o d i l u t e w i t h 12% (w/v) TCA t o y i e l d o n - s c a l e r e a d i n g s . 1) A b s o r b a n c e a t 280 nm A b s o r b a n c e o f t h e f i l t r a t e s , w h i c h were d i l u t e d when n e c e s s a r y ( u s u a l l y d i l u t e d f i v e t i m e s ) , was d e t e r m i n e d w i t h a 9 Unicam SP-800B S p e c t r o p h o t o m e t e r (Pye Unicam L t d . , C a m b r i d g e , E n g l a n d ) . 2) Lowry method F i l t r a t e (1.0 ml) was m i x e d w i t h 5.0 ml o f t h e a l k a l i n e c o p p e r t a r t r a t e r e a g e n t a s d e s c r i b e d by Lowry e t a l . ( 1 9 5 1 ) . A f t e r 10 min, 0.5 ml o f F o l i n - C i o c a l t e a u r e a g e n t , 1.0 N i n a c i d i t y , was a d d e d . A f t e r i n c u b a t i o n a t room t e m p e r a t u r e f o r 30 min, a b s o r b a n c e was measured a t 700 nm w i t h a Unicam SP-800B S p e c t r o p h o t o m e t e r . 3) T r i n i t r o b e n z e n e s u l f o n i c a c i d method F i l t r a t e (0.2 ml) was m i x e d w i t h 2.0 ml o f 0.2 M sodium b o r a t e b u f f e r (pH 9.2) and 1.0 ml o f 4.0 mM TNBS. A f t e r i n c u b a t i o n a t room t e m p e r a t u r e f o r 30 min, 1.0 ml o f 2.0 M m o n o b a s i c sodium p h o s p h a t e c o n t a i n i n g 18 mM sodium s u l f i t e was a d d e d . A b s o r b a n c e , a t 420 nm, was measured w i t h a Unicam SP-800B S p e c t r o p h o t o m e t e r . 4) F l u o r e s c a m i n e method To 0.1 ml o f f i l t r a t e , 0.3 ml o f 3 M p o t a s s i u m p h o s p h a t e d i b a s i c was a d d e d f o l l o w e d by 0.15 ml o f 0.03% (w/v) f l u o r e s c a m i n e i n a c e t o n e . The m i x t u r e was i m m e d i a t e l y m i x e d . To a d j u s t t h e volume t o c u v e t t e s i z e , 3.0 ml o f d i s t i l l e d w a t e r were a d d e d . F l u o r e s c e n c e i n t e n s i t y ( e x c i t a t i o n w a v e l e n g t h a t 395 nm; e m m i s s i o n w a v e l e n g t h a t 480 nm) was m e a s u r e d w i t h an Aminco Bowman 4-8202 s p e c t r o f l u o r o m e t e r ( A m e r i c a n I n s t r u m e n t 10 Co., I n c . , S i l v e r S p r i n g , MD). B. S t a t i s t i c a l c o m p a r i s o n o f t h e p r o t e i n a s e a s s a y methods D a t a , i n terms o f a b s o r b a n c e ( f r o m A 2 8 o i Lowry and TNBS methods) and f l u o r e s c e n c e i n t e n s i t y ( f r o m t h e f l u o r e s c a m i n e m e t h o d ) , were p o o l e d . Dealing's method (Wakkers e t a l . , 1975), u s i n g a program w r i t t e n f o r a Monroe 1880-88 programmable c a l c u l a t o r , was e m p l o y e d . D a t a o b t a i n e d a t v a r i o u s t i m e i n t e r v a l s w i t h one p r o t e o l y s i s a s s a y method ( e g . f l u o r o m e t r i c ) were compared w i t h t h e d a t a f o r t h e same t i m e i n t e r v a l s f r o m a n o t h e r p r o t e o l y s i s a s s a y method ( e g . TNBS). T h i s a n a l y s i s p r o d u c e d s i x r e g r e s s i o n l i n e s ( e a c h r e g r e s s i o n compared two m e t h o d s ) . W i t h t h i s p r o g r a m , t h e s t a n d a r d e r r o r o f e s t i m a t e and t h e s l o p e o f t h e r e g r e s s i o n l i n e were c a l c u l a t e d . The d e t e c t a b i l i t y of e a c h p r o t e o l y s i s a s s a y method was e x p r e s s e d a s t h e s t a n d a r d e r r o r o f e s t i m a t e of a p a r t i c u l a r a s s a y method d i v i d e d by i t s s l o p e r a t i o . F o r e a c h p a i r o f methods f o r m e a s u r i n g d i f f e r e n t h y d r o l y s i s p r o d u c t s ( i . e . amino g r o u p s and a r o m a t i c g r o u p s ) , t h e d a t a t r a n s f o r m a t i o n o f F u j i i and N a k a i (1980) was a p p l i e d f o r l i n e a r i z a t i o n u s i n g a t r a n s f o r m a t i o n e q u a t i o n o f yB = ax. Then, Deming's method was a p p l i e d f o r c o m p a r i s o n o f t h e o f t h e methods u s i n g yB v s . x a s v a r i a b l e s . C. D e t e r m i n a t i o n o f t h e d e t e c t i o n l i m i t o f t h e TNBS and 11 f l u o r e s c a m i n e methods The method a c c o r d i n g t o S c h w e r d t f e g e r (1977) was f o l l o w e d . To 10 ml o f 0.2 M b o r a t e b u f f e r (pH 7.4) c o n t a i n i n g 0.05% C a C l 2 ,and 1.0 ml o f 10% RSM, 10 ml o f t r y p s i n a t v a r i o u s c o n c e n t r a t i o n s were a d d e d . The m i x t u r e was i n c u b a t e d a t 35 C. A f t e r 24 h, t h e r e a c t i o n was t e r m i n a t e d by t h e a d d i t i o n o f TCA ( 3 . 5 % f i n a l c o n c e n t r a t i o n ) . The m i x t u r e was c e n t r i f u g e d (10,000 x g; 5 min) and t h e s u p e r n a t e n t f i l t e r e d (Whatman #4). The f i l t r a t e was a s s a y e d w i t h t h e f l u o r e s c a m i n e method a n d t h e TNBS method as d e s c r i b e d by S c h w e r d t f e g e r ( 1 9 7 4 ) . The d e t e c t i o n l i m i t was d e f i n e d as t h e t r y p s i n c o n c e n t r a t i o n r e q u i r e d t o y i e l d s i g n i f i c a n t d i f f e r e n c e (P < 0.01%; n = 4) between s a m p l e s b e f o r e and a f t e r i n c u b a t i o n ( S c h w e r d t f e g e r , 1977). 12 RESULTS AND DISCUSSION The r e s u l t s o f c o m p a r i s o n o f t h e methods f o r d e t e r m i n i n g p r o t e i n a s e a c t i v i t y a r e shown i n F i g u r e s 1-6. R e l a t i o n s h i p s between t h e f l u o r e s c a m i n e method v s . t h e TNBS method ( F i g . 1 ) , and t h e Lowry method v s . t h e A 2 B o method ( F i g . 6) a p p e a r t o be l i n e a r . T h i s i s r e a s o n a b l e s i n c e t h e f i r s t p a i r o f methods a r e b o t h d e t e r m i n i n g amino g r o u p s w h i l e t h e s e c o n d p a i r of methods a r e b o t h f o r a r o m a t i c g r o u p s . On t h e c o n t r a r y , t h e r e l a t i o n s h i p between t h e f l u o r e s c a m i n e method and t h e Lowry method ( F i g . 2) does n o t a p p e a r t o be l i n e a r . S i m i l a r n o n l i n e a r r e l a t i o n s h i p s were o b s e r v e d between t h e f l u o r e s c a m i n e method and t h e A 2 8 0 method ( F i g . 3 ) , t h e TNBS method and t h e Lowry method ( F i g . 4 ) , and t h e TNBS method and t h e A 2 8 0 method ( F i g . 5 ) . T h i s c u r v i l i n e a r i t y may be a c o n s e q u e n c e o f d i f f e r e n t h y d r o l y s i s p r o d u c t s b e i n g d e t e c t e d by e a c h method. F o r t h e s e p a i r s o f methods t h e p l o t s were l i n e a r i z e d f i r s t , t h e n t h e methods were compared by Deming's method. L i n e a r i z a t i o n c o n s i d e r a b l y i m p r o v e d t h e d e t e c t a b i l i t y o f t h e Lowry method but n o t f o r t h e o t h e r methods ( T a b l e 1) p r o b a b l y b e c a u s e o f c o n s i d e r a b l e d e v i a t i o n of t h e Lowry method f r o m l i n e a r i t y . T a b l e 1 shows t h a t t h e f l u o r e s c a m i n e method was more r e l i a b l e t h a n t h e o t h e r t h r e e methods ( s m a l l e r s t a n d a r d d e v i a t i o n o f t h e random e r r o r s i n t h e m e t h o d ) . The TNBS method was more r e l i a b l e t h a n t h e Lowry method o r t h e A 2 8 0 method a s a measure o f t h e d e g r e e o f h y d r o l y s i s o f m i l k p r o t e i n s . The 12a F i g . 1. Comparison of t h e f l u o r e s c a m i n e method w i t h the TNBS method as a measure of p r o t e i n h y d r o l y s i s by Deming's method. Absorbance and f l u o r e s c e n c e i n t e n s i t y a r e e x p r e s s e d as t h e apparent absorbance and f l u o r e s c e n c e i n t e n s i t y of t h e u n d i l u t e d s o l u t i o n . 13a F i g . 2. Comparison of the fluorescamine method with the Lowry method as a measure of p r o t e i n h y d r o l y s i s by Deming's method. Absorbance and f l u o r e s c e n c e i n t e n s i t y are expressed as the apparent absorbance and f l u o r e s c e n c e i n t e n s i t y of the u n d i l u t e d s o l u t i o n . The broken l i n e r e p r e s e n t s the transformed data from the Lowry method vs. the fluorescamine method. 14a F i g . 3. Comparison of the fluorescamine method with the A 2 s o method as a measure of p r o t e i n h y d r o l y s i s by Deming's method. Absorbance and f l u o r e s c e n c e i n t e n s i t y are expressed as the apparent absorbance and f l u o r e s c e n c e i n t e n s i t y of the u n d i l u t e d s o l u t i o n . The broken l i n e r e p r e s e n t s the transformed data from the A 2 8 o method vs. the fluorescamine method. r 2 = 0.970 600 1200 1800 2400 3000 3600 FLUORESCAMINE ( F.I. ) 15a F i g . 4. Comparison of the TNBS method with the Lowry method as a measure of p r o t e i n h y d r o l y s i s by Deming's method. Absorbance i s expressed as the apparent absorbance of the u n d i l u t e d s o l u t i o n . The broken l i n e r e p r e s e n t s the transformed data from the Lowry method v s . the TNBS method. 91 16a F i g . 5. C o m p a r i s o n o f t h e TNBS method w i t h t h e A 2 8 0 method a s measure o f p r o t e i n h y d r o l y s i s by Deming's method. A b s o r b a n c e i e x p r e s s e d a s t h e a p p a r e n t a b s o r b a n c e o f t h e u n d i l u t e d s o l u t i o n The b r o k e n l i n e r e p r e s e n t s t h e t r a n s f o r m e d d a t a f r o m t h e A 2 8 method v s . t h e TNBS method. 0.3 r2= 0.958 17a F i g . 6. Comparison of the Lowry method with the A 2 B o method as a measure of p r o t e i n h y d r o l y s i s by Deming's method. Absorbance i s expressed as the apparent absorbance of the u n d i l u t e d s o l u t i o n . 19 i Table 1. Detectability of the fluorescamine method (Fluo), the TNBS method, the Lowry method and the absorbance at 280 nm ( A 2 8 0 ) a s estimated by Deming's procedure. Fluo TNBS Lowry ' A280 Direct comparison Fluo and TNBS 98 (1.0) 155 (1.58) Fluo and Lowry 46 (1.0) 192 (4.17) Fluo and A^gg 72 (1.0) 189 (2.63) TNBS and Lowry 0.45 (1.58) 1.22 (4.28) TNBS and A280 0.64 (1.58) 1.01 (2.49) Lowry and A 2 8 O 0.14 (4.17) 0.08 (2.38) (average) (1.0) (1.6) (4.2) (2.5) After linearization Fluo and Lowry 47 (1.0) 132 (2.81) Fluo and A 2 8 O 64 (1.0) 158 (2.47) TNBS and Lowry 0.37 (1.73) 0.60 (2.81) TNBS and A2go 0.45 (1.28) 0.87 (2.47) (average) (1.0) (1.5) (2.8) (2.5) The figures in parentheses are calculated for direct comparison of detectability by assigning 1.0 to the fluorescamine method. 20 absorbance at 280 nm was more r e l i a b l e than the Lowry method. Schwerdtfeger (1977) claimed that the TNBS method (Schwerdtfeger, 1974) c o u l d d e t e c t t r y p s i n (Merck) a c t i v i t y at a l e v e l as low as 3 ng/ml. However, using the same a n a l y t i c a l c o n d i t i o n s but with a t r y p s i n from a d i f f e r e n t source, t r y p s i n a c t i v i t y was d e t e c t e d a l e v e l of 260'ng/ml (P = 0.005%; n = 4). T h i s d i f f e r e n c e i n s e n s i t i v i t y may be caused by the d i f f e r e n c e i n the p u r i t y of the t r y p s i n s used. In comparison, the fluorescamine method proposed c o u l d d e t e c t t r y p s i n a c t i v i t y at a c o n c e n t r a t i o n of 35 ng/ml (P = 0.003%; n = 4) a c c o r d i n g t o the c o n d i t i o n s s p e c i f i e d by Schwerdtfeger (1974); C a l c u l a t i o n of the degree of d i l u t i o n of TCA f i l t r a t e p r i o r to measurement of absorbance and f l u o r e s c e n c e i n t e n s i t y y i e l d e d v a l u e s of 2.00, 1.82, 0.48, 0.28 ml of TCA f i l t r a t e per 10 ml f i n a l d i l u t e d volume f o r the A 2 9 ( ) , Lowry, TNBS and fluorescamine methods, r e s p e c t i v e l y . T h i s means the fluorescamine method needs the s m a l l e s t amount of sample f o r the best p r e c i s i o n . As i n d i c a t e d a l r e a d y , the two methods f o r comparing d i f f e r e n t p r o t e o l y s i s assay procedures y i e l d e d some d i f f e r e n t r e s u l t s . When the d e t e c t i o n l i m i t of Schwerdtfeger (1977) was used, the fluorescamine method was 7.4 times (260/35) more s e n s i t i v e than the TNBS method. On the other hand, the comparison of d e t e c t a b i l i t y used i n t h i s study showed that the fluorescamine method was 1.5 times more s e n s i t i v e than the TNBS method. T h i s d i f f e r e n c e i n the s e n s i t i v i t y may be due to the d i f f e r e n c e i n the i n c u b a t i o n c o n d i t i o n s (24 h at 35 C vs. 7 min at 40 C) and d i f f e r e n t parameters used ( d e t e c t i o n l i m i t f o r the 21 method o f S c h w e r d t f e g e r v s . d e t e c t a b i l i t y e m p l o y e d i n t h i s s t u d y ). The f o u r methods c h o s e n were t h o s e t h a t c o u l d be u s e d i n most l a b o r a t o r i e s and w h i c h have been u s e d by v a r i o u s r e s e a r c h g r o u p s t o d e t e c t p r o t e o l y s i s i n m i l k and m i l k p r o d u c t s . The m i c r o f l u o r o m e t r i c method ( C a s t e l l e t a l . , 1979) was c h o s e n o v e r o t h e r p u b l i s h e d f l u o r o m e t r i c methods b e c a u s e l e s s f l u o r e s c a m i n e was u s e d i n t h i s method. S t e i n e t a l . (1974) s t u d i e d t h e i n f l u e n c e o f v a r i o u s p a r a m e t e r s on t h e r a t e of t h e r e a c t i o n o f f l u o r e s c a m i n e w i t h p r i m a r y amines and on t h e r a t e o f h y d r o l y s i s o f t h e r e a g e n t . I t was shown t h a t b o t h were d e p e n d e n t on t h e r e a c t i o n c o n d i t i o n s , i n c l u d i n g pH, s o l v e n t i n w h i c h the r e a g e n t was p r e p a r e d , t e m p e r a t u r e , r e a g e n t c o n c e n t r a t i o n , and b u f f e r s a l t . Hence, p h o s p h a t e b u f f e r was c h o s e n t o r a i s e t h e pH o f t h e TCA f i l t r a t e t o 8.5 b e c a u s e i t was shown t o be b e t t e r t h a n b o r a t e b u f f e r f o r t h i s p u r p o s e . C o m p a r i s o n o f methods f o r e s t i m a t i o n o f p r o t e i n h y d r o l y s i s i s d i f f i c u l t . F i r s t , t h e compounds b e i n g a n a l y s e d a r e d i f f e r e n t ; a r o m a t i c g r o u p s ( A 2 a o , Lowry) v s . amino g r o u p s ( f l u o r e s c a m i n e , TNBS). S e c o n d , t h e i n s t r u m e n t s u s e d a r e d i f f e r e n t ; s p e c t r o f l u o r o m e t e r ( f l u o r e s c a m i n e ) v s . s p e c t r o p h o t o m e t e r ( A 2 8 0 , Lowry, TNBS). When t h e w o r k i n g p r i n c i p l e o f t h e i n s t r u m e n t s i s d i f f e r e n t , d i r e c t c o m p a r i s o n o f t h e i n c r e a s e i n f l u o r e s c e n c e i n t e n s i t y and t h e i n c r e a s e i n a b s o r b a n c e i s not a p p r o p r i a t e ( S p e n c e r and S p e n c e r , 1974). S i n c e t h e r e i s no e r r o r - f r e e o r r e f e r e e method, d i r e c t a p p l i c a t i o n of r e g r e s s i o n a n a l y s i s t o compare t h e r e s u l t o f one 22 method v s . t h e r e s u l t of a n o t h e r method i s not t o t a l l y j u s t i f i e d . o Deming's method m i n i m i z e s t h e w e i g h t e d sum o f s q u a r e s : S = Z [ ( x . - X i ) 2 + ( y i - Y i ) 2 X ] (1) i n s t e a d o f S = I [ ( x . - X i ) 2 + ( y i - Y ± ) 2 ] (2) i n t h e c l a s s i c a l l i n e a r r e g r e s s i o n p r o c e d u r e , where X^ and Y^ be t h e e s t i m a t e f o r X^ and Y^; X i s a ' w e i g h t ' : X = V ( e ) / V(M (3) where V ( e ) and V ( 6 ) a r e t h e v a r i a n c e s c o r r e s p o n d i n g t o x and y. T h e r e f o r e , Deming's method i s a w e i g h t e d l i n e a r r e g r e s s i o n a n a l y s i s . The " d e t e c t a b i l i t y " d e s c r i b e d i n t h i s s t u d y was s i m i l a r t o t h e s e n s i t i v i t y r a t i o d i s c u s s e d by Mandel ( 1 9 6 4 ) . I t was e x p r e s s e d as t h e r a t i o of o£ / a 5 t o | dM / dN |, where a e and a 6 were s t a n d a r d d e v i a t i o n s o f methods M and N; | dM / dN | was t h e s l o p e of t h e l i n e y i e l d e d by t h e p l o t o f M v s . N. As t h e s e n t i v i t y r a t i o i n c r e a s e d , t h e t e c h n i c a l m e r i t o f method M d e c r e a s e d w i t h r e s p e c t t o method N. The c o m p a r i s o n of t h e two methods i s t h e r e f o r e t h e c o m p a r i s o n o f t h e i r p r e c i s i o n s . However, o t h e r measures of p r e c i s i o n s , s u c h as s t a n d a r d d e v i a t i o n and c o e f f i c i e n t o f v a r i a t i o n , c a n n o t be u s e d d i r e c t l y f o r c o m p a r i n g a n a l y t i c a l methods. T h i s i s b e c a u s e t h e s e n t i v i t y r a t i o i s t h e o n l y one t h a t e n j o y s t h e p r o p e r t y o f i n v a r i a n c e w i t h r e s p e c t t o t r a n s f o r m a t i o n o f s c a l e . I n t h e c o m p a r i s o n method s u g g e s t e d i n t h i s s t u d y , b o t h t h e s t a n d a r d e r r o r o f e s t i m a t e and t h e s l o p e r a t i o o f a p a r t i c u l a r 23 a s s a y method a r e e x p r e s s e d by e i t h e r a b s o r b a n c e or f l u o r e s c e n c e i n t e n s i t y u n i t s . The d e t e c t a b i l i t y ( r a t i o o f s t a n d a r d e r r o r of e s t i m a t e t o s l o p e r a t i o ) p a r a m e t e r c a n c e l s o u t d i f f e r e n c e i n i n s t r u m e n t as w e l l a s d i f f e r e n c e i n compound a n a l y s e d . 24 CONCLUSIONS The method o f S c h w e r d t f e g e r (1977) has an a d v a n t a g e , by w h i c h t h e d e t e c t i o n l i m i t c a n be e x p r e s s e d as t h e minimum amount of enzymes t h a t c a n be d e t e c t e d . A c c o r d i n g l y , a l o n g i n c u b a t i o n t i m e i s r e q u i r e d f o r m a x i m i z i n g t h e s e n s i t i v i t y o f a s s a y s . However, a drawback o f t h i s method i s l i m i t e d d a t a p o i n t s f o r s t a t i s t i c a l c o m p u t a t i o n . To i n c r e a s e t h e number o f d a t a p o i n t s , many more d i f f e r e n t c o n c e n t r a t i o n s o f enzymes s h o u l d be u s e d f o r a s s a y . The method p r o p o s e d i n t h i s s t u d y i s c o n v e n i e n t f o r c o m p a r i s o n s i n c e more r e l i a b l e d a t a c a n be o b t a i n e d . T h i s i s a c h i e v e d by c h a n g i n g t h e i n c u b a t i o n t i m e o f t h e same r e a c t i o n m i x t u r e u s i n g one enzyme c o n c e n t r a t i o n . N e v e r t h e l e s s , t h e d a t a o b t a i n e d a r e n o n d i m e n s i o n a l and t h u s d i f f i c u l t t o c o n v e r t t o more r e a l i s t i c p a r a m e t e r s , f o r example amount o f amino a c i d s o r enzymes. C o n s i d e r i n g t h e s e s i t u a t i o n s , an a p p r o p r i a t e method s h o u l d be c h o s e n o r , i f n e c e s s a r y b o t h methods s h o u l d be employed t o draw t h e most r e a s o n a b l e c o n c l u s i o n . 25 REFERENCES Adams, D.M., B a r a c h , J . T . , and Speck, M.L. 1975. Heat r e s i s t a n t p r o t e a s e s p r o d u c e d i n m i l k by p s y c h r o t r o p h i c b a c t e r i a of d a i r y o r i g i n . J . D a i r y S c i . 58:828. Adams, D.M., B a r a c h , J . T . , and Speck, M.L. 1976. E f f e c t of p s y c h r o t r o p h i c b a c t e r i a f r o m raw m i l k on m i l k p r o t e i n s and s t a b i l i t y o f m i l k p r o t e i n s t o u l t r a h i g h t e m p e r a t u r e t r e a t m e n t . J . D a i r y S c i . 59:823. A l i c h a n i d i s , E . and Andrews, A.T. 1977. Some p r o p e r t i e s o f t h e e x t r a c e l l u l a r p r o t e a s e s p r o d u c e d by t h e p s y c h r o t r o p h i c b a c t e r i u m Pseudomonas f l u o r e s c e n s s t r a i n AR-11. B i o c h i m . B i o p h y s . A c t a 485:424. Beeby, R. 1980. The use o f f l u o r e s c a m i n e a t pH 6.0 t o f o l l o w t h e a c t i o n o f c h y m o s i n on < - c a s e i n and t o e s t i m a t e t h i s p r o t e i n i n m i l k . N. Z. J . D a i r y S c i . T e c h n o l . 15:99. C a s t e l l , J.V., C e r v e r a , M., and Marco, R. 1979. A c o n v e n i e n t m i c r o m e t h o d f o r t h e a s s a y o f p r i m a r y amines and p r o t e i n s w i t h f l u o r e s c a m i n e . A r e e x a m i n a t i o n o f t h e c o n d i t i o n s o f r e a c t i o n . A n a l . Biochem. 99:379. Chism, G.W., Huang, A.E.,'and M a r s h a l l , J.A. 1979. S e n s i t i v e a s s a y f o r p r o t e a s e s i n s t e r i l e m i l k . J . D a i r y S c i . 62: 1798. Cogan, T.M. 1977. A r e v i e w o f h e a t r e s i s t a n t l i p a s e s and p r o t e i n a s e s and t h e q u a l i t y of d a i r y p r o d u c t s . I r . J . F o o d S c i . T e c h n o l . 1:95. C o u s i n , M.A. 1982. P r e s e n c e and a c t i v i t y o f p s y c h r o t r o p h i c m i c r o o r g a n i s m s i n m i l k and d a i r y p r o d u c t s : A r e v i e w . J . F o o d P r o t . 45:172. F u j i i , S. and N a k a i , S. 1980. O p t i m i z a t i o n o f d a t a t r a n s f o r m a t i o n s f o r l i n e a r i z a t i o n . Can. I n s t . F o o d S c i . T e c h n o l . J . 13:188. G e b r e - E g z i a b h e r , A., Humbert, E.S., and B l a n k e n a g e l , G. 1980. H e a t - s t a b l e p r o t e a s e s from p s y c h r o t r o p h s i n m i l k . J . F o o d P r o t . 43:197. G r i f f i t h s , M.W., P h i l l i p s , J.D., a n d M u i r , D.D. 1981. T h e r m o s t a b i l i t y o f p r o t e a s e s and l i p a s e s f r o m a number of s p e c i e s o f p s y c h r o t r o p h i c b a c t e r i a o f d a i r y o r i g i n . J . A p p l . B a c t e r i o l . 50:289. H u l l , M.E. 1947. C o l o r i m e t r i c d e t e r m i n a t i o n of p a r t i a l h y d r o l y s i s o f t h e p r o t e i n s i n m i l k . J . D a i r y S c i . 30:881. J u f f s , H.S. 1973. P r o t e o l y s i s d e t e c t i o n i n m i l k . I . I n t e r p r e t a t i o n o f t y r o s i n e v a l u e d a t a f o r raw m i l k s u p p l i e s i n r e l a t i o n t o n a t u r a l v a r i a t i o n , b a c t e r i a l c o u n t s and o t h e r f a c t o r s . J . D a i r y Res. 40:371. J u f f s , H.S. 1975. P r o t e o l y s i s d e t e c t i o n i n m i l k . IV. S t a r c h - g e l e l e c t r o p h o r e s i s and f o r m o l t i t r a t i o n . J . D a i r y R es. 42:277. Kas, J . and Rauch, P. 1982. R a d i o i s o t o p i c method f o r t h e d e t e r m i n a t i o n o f low and v e r y low p r o t e o l y t i c a c t i v i t i e s i n f o o d s t u f f s . Z. Lebensm. U n t e r s . F o r s c h . 174:290. Koops, J . , Klomp, H., and E l g e r s m a , R.H.C. 1975. R a p i d d e t e r m i n a t i o n o f n i t r o g e n i n m i l k and d a i r y p r o d u c t s by c o l o r i m e t r i c e s t i m a t i o n o f ammonia f o l l o w i n g an a c c e l e r a t e d d i g e s t i o n p r o c e d u r e . N e t h . M i l k D a i r y J . 29:169. Law, B.A. 1979. Reviews o f t h e p r o g r e s s o f d a i r y s c i e n c e : Enzymes o f p s y c h r o t r o p h i c b a c t e r i a and t h e i r e f f e c t s on m i l k and m i l k p r o d u c t s . J . D a i r y R es. 46:573. Law, B.A., Andrews, A.T., C l i f f e , A . J . , S h a r p e , M.E., and Chapman, H.R. 1979. E f f e c t o f p r o t e o l y t i c raw m i l k p s y c h r o t r o p h s on Cheddar c h e e s e making w i t h s t o r e d m i l k . J . D a i r y Res. 46:497. Lowry, O.H., R o s e b r o u g h , N . J . , F a r r , A.L., and R a n d a l l , R . J . 1951. P r o t e i n measurement w i t h t h e F o l i n p h e n o l r e a g e n t . J . B i o l . Chem. 193:265. M a n d e l , J . 1964. I_n The S t a t i s t i c a l A n a l y s i s o f E x p e r i m e n t a l D a t a . John W i l e y and Sons, I n c . , New Y o r k , pp 366-369. 27 M c K e l l a r , R.C. 1981. Development of o f f - f l a v o r i n u l t r a - h i g h t e m p e r a t u r e and p a s t e u r i z e d m i l k a s a f u n c t i o n of p r o t e o l y s i s . J . D a i r y S c i . 64:2138. N a k a i , N., L a i , C.Y., and H o r e c k e r , B.L. 1974. Use o f f l u o r e s c a m i n e i n t h e c h r o m a t o g r a p h i c a n a l y s i s o f p e p t i d e s f r o m p r o t e i n s . A n a l . Biochem. 58:563. P e a r c e , K.N. 1979. Use o f f l u o r e s c a m i n e t o d e t e r m i n e t h e r a t e of r e l e a s e o f t h e c a s e i n o - m a c r o p e p t i d e i n r e n n e t - t r e a t e d m i l k . N. Z. J . D a i r y S c i . T e c h n o l . 14:233. R i c h a r d s o n , B.C. and Te W h a i t i , I . E . 1978. P a r t i a l c h a r a c t e r i z a t i o n o f h e a t - s t a b l e e x t r a c e l l u l a r p r o t e a s e s of some p s y c h r o t r o p h i c b a c t e r i a f r o m raw m i l k . N. Z. J . D a i r y S c i . T e c h n o l . 14:172. R i c h t e r , R.L., S c h m i d t , R.H., S m i t h , K.L., M u l l , L . E . , and Henry , S.L. 1979. P r o t e o l y t i c a c t i v i t y i n u l t r a - p a s t e u r i z e d a s e p t i c a l l y p a c k a g e d w h i p p i n g c r e a m . J . Food P r o t . 42:43. Schwabe, C. 1973. A f l u o r e s c e n t a s s a y f o r p r o t e o l y t i c enzymes. A n a l . Biochem. 53:484. S c h w e r d t f e g e r , E. 1974. A u t o m a t i s c h - k i n e t i s c h Bestimmung von p f l a n z l i c h e n E n z y m a k t i v i t a t e n und I n h a l t s t o f f e n . 2. Bestimmung von A m i n o s a u r e n . Z. Lebensm. U n t e r s . - F o r s c h . 156:266. S c h w e r d t f e g e r , E. 1977. N a c h w e i s g r e n z e p r o t e o l y t i s c h e r A k t i v i t a t e n . M i k r o c h i m i c a A c t a 11:363. S n o e r e n , T.H.M., van d e r Speck, C.A., De k k e r , R., and B o t h , P. 1979. P r o t e o l y s i s d u r i n g t h e s t o r a g e o f U H T - s t e r i l i z e d whole m i l k . I . E x p e r i m e n t s w i t h m i l k h e a t e d by t h e d i r e c t s y s t e m f o r 4 sec a t 142 C. N e t h . M i l k D a i r y J . 33:31. S n o e r e n , T.H.M., and B o t h , P. 1981. P r o t e o l y s i s d u r i n g t h e s t o r a g e o f U H T - s t e r i l i z e d whole m i l k . I I . E x p e r i m e n t s w i t h m i l k h e a t e d by t h e i n d i r e c t s y s t e m f o r 4 s e c a t 142 C. N e t h . M i l k D a i r y J . 35:113 S p e n c e r , P.W. and S p e n c e r , R.D. 1974. G l o b u l i n - s p e c i f i c p r o t e o l y t i c a c t i v i t y i n g e r m i n a t i n g pumpkin s e e d s a s d e t e c t e d by a f l u o r e s c e n c e a s s a y method. P l a n t P h y s i o l . 54:925. 28 S t e i n , S., B o h l e n , P., and U d e n f r i e n d , S. 1974. S t u d i e s on t h e k i n e t i c s o f h y d r o l y s i s o f f l u o r e s c a m i n e . A r c h . B iochem. B i o p h y s . 163:400. U d e n f r i e n d , S., S t e i n , S., B o h l e n , P., D a i r m a n , W., L e i m g r u b e r , W., and W e i g e l e , M. 1972. F l u o r e s c a m i n e : A r e a g e n t f o r a s s a y of amino a c i d s , p e p t i d e s , p r o t e i n s , and p r i m a r y amines i n t h e p i c o m o l e r a n g e . S c i e n c e 178:871. Wakkers, P.J.M., H e l l e n d o r n , H.B.A., Op de Weegh, G.J., and H e e r s p i n k , W. 1975. A p p l i c a t i o n s o f s t a t i s t i c s i n c l i n i c a l c h e m i s t r y . A c r i t i c a l e v a l u a t i o n o f r e g r e s s i o n l i n e s . C l i n . Chim. A c t a 64:173. CHAPTER 2 I d e n t i f i c a t i o n and S t u d y P r o t e o l y t i c Pseudomonads I s o l a t e d f r o m Raw M i l k 30 INTRODUCTION Pseudomonads a r e t h e p r e d o m i n a n t s p e c i e s o f t h e p s y c h r o t r o p h i c m i c r o f l o r a of r e f r i g e r a t e d raw m i l k (Thomas and Thomas, 1973; Tompkin, 1973; S t a d h o u d e r s , 1975). Many of t h e s e s p e c i e s p r o d u c e e x t r a c e l l u l a r enzymes w h i c h c a n d e g r a d e b o t h m i l k p r o t e i n s and m i l k f a t (Law, 1979; M u i r e t a l . , 1979). Even t h o u g h t h e e f f e c t s o f t h e s e pseudomonads on m i l k and m i l k p r o d u c t s have been e x t e n s i v e l y s t u d i e d , t h e r e i s l i t t l e i n f o r m a t i o n on t h e p h e n o t y p i c c h a r a c t e r i s t i c s of t h e s e s p e c i e s . I d e n t i f i c a t i o n o f t h e s e pseudomonads a c c o r d i n g t o t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y (1974) i s commonly employed (Fox e t a l . , 1976; C o u s i n and M a r t h , 1977; R i c h a r d s o n and Te W h a i t i , 1978; G e b r e - E g z i a b h e r e t a l . , 1 980a). In most o f t h e s e s t u d i e s , t h e b a c t e r i a l i s o l a t e s were i d e n t i f i e d t o t h e genus l e v e l . The , c h a r a c t e r i s t i c s s t u d i e d , f r o m w h i c h i d e n t i f i c a t i o n was made, were o f t e n o m i t t e d i n p u b l i s h e d p a p e r s . I t i s d i f f i c u l t f o r t h e r e a d e r t o a s s e s s t h e v a l i d i t y o f t h e i d e n t i f i c a t i o n . I f t h e number o f c h a r a c t e r s s t u d i e d i s s m a l l , as i n i d e n t i f y i n g t o t h e genus l e v e l , a p e r f e c t match w i t h t h o s e named m i c r o o r g a n i s m s i n an e s t a b l i s h e d c l a s s i f i c a t i o n w i l l be common. I f a l a r g e number o f c h a r a c t e r s a r e s t u d i e d , as i n i d e n t i f y i n g t o t h e s p e c i e s l e v e l , t h e n a p e r f e c t match w i l l s e l d o m be p o s s i b l e . One has t o go w i t h a n e a r match i n w h i c h t h e number of d i v e r g e n c i e s i s s m a l l . The i n c l u s i o n of a c e r t a i n s p e c i e s i n t o a named t a x o n w i l l v e r y much depend on t h e c h o i c e o f c h a r a c t e r s and methods e m p l o y e d . 31 At p r e s e n t , t h e r e i s n o t enough i n f o r m a t i o n , a t t h e s p e c i e s l e v e l , a b o u t pseudomonads i n raw m i l k . The o b j e c t of t h i s r e s e a r c h was t o i d e n t i f y t o t h e s p e c i e s l e v e l , by n u m e r i c a l taxonomy, p r o t e o l y t i c pseudomonads i s o l a t e d f r o m raw m i l k . I t was hoped t h a t t h e s e pseudomonads c o u l d be c l a s s i f i e d i n t o s e p a r a t e g r o u p s . R e p r e s e n t a t i v e i s o l a t e s c o u l d t h e n be s e l e c t e d i n t h e f u t u r e f o r more d e t a i l e d s t u d i e s o f t h e i r b i o c h e m i c a l a c t i v i t i e s r e l e v a n t t o s p o i l a g e . I d e n t i f i c a t i o n by u s i n g d i a g n o s t i c t a b l e s i s of l i m i t e d v a l u e b e c a u s e o f t h e d i f f i c u l t y o f i d e n t i f y i n g s t r a i n s a b e r r a n t i n one or more o f t h e c h a r a c t e r s s t u d i e d . Hence, a n u m e r i c a l taxonomy a p p r o a c h t o i d e n t i f i c a t i o n u s i n g c l u s t e r a n a l y s i s was c h o s e n i n s t e a d . Growth and p r o t e i n a s e a c t i v i t y o f e i g h t p r o t e o l y t i c pseudomonads were a l s o e x a m i n e d . T h i s s e r v e d t o d e t e r m i n e i f t h e r e was any r e l a t i o n s h i p between p r o t e i n a s e a c t i v i t y i n m i l k and p r o t e o l y t i c p s y c h r o t r o p h i c c o u n t s . 32 LITERATURE REVIEW A. Pseudomonas s p e c i e s i n raw m i l k The i s o l a t i o n o f Pseudomonas s p e c i e s i n raw m i l k has been w e l l documented ( W i t t e r , 1961; Thomas and Thomas, 1973; S t a d h o u d e r s , 1975; C o u s i n , 1982). In t h e i r s t u d y on t h e t h e r m o s t a b i l i t y o f p r o t e a s e s f r o m p s y c h r o t r o p h i c b a c t e r i a o f d a i r y o r i g i n , G r i f f i t h s e t a l . (1981) r e p o r t e d t h a t 53% of t h e p s y c h r o t r o p h i c p o p u l a t i o n were Pseudomonas s p e c i e s . S p e c i e s of t h e genus S e r r a t i a c o n t r i b u t e d o n l y 16% o f t h e m i c r o f l o r a . M u i r e t a l . (1979) c o n c l u d e d t h a t Pseudomonas was t h e p r e d o m i n a n t genus i n raw m i l k c o l l e c t e d o v e r a p e r i o d o f 14 months. Pseudomonads a c c o u n t e d f o r 65% - 78% o f t h e p s y c h r o t r o p h i c p o p u l a t i o n . They f o u n d t h a t t h e r e was no s e a s o n a l p a t t e r n i n f r e q u e n c y o f i s o l a t i o n o f Pseudomonas g r o u p s . S p e c i e s o f t h e E ' n t e r o b a c t e r i a c e a e f a m i l y were f o u n d more f r e q u e n t l y i n t h e summer months o f May, J une and J u l y . J u f f s (1973) s t u d i e d t h e m i c r o f l o r a o f m i l k p r o d u c e d i n S o u t h E a s t e r n Q u e e n s l a n d , A u s t r a l i a . Of t h e 330 i s o l a t e s s t u d i e d , 167 were i d e n t i f i e d as Pseudomonas s p e c i e s . Samagh and Cunningham (1972) r e p o r t e d t h a t 28% of t h e p s y c h r o t r o p h i c m i c r o f l o r a b e l o n g e d t o t h e genus Pseudomonas. A u d r e y and F r a z i e r (1959) o b t a i n e d 17% Pseudomonas s p e c i e s out o f 220 p s y c h r o t r o p h i c m i c r o o r g a n i s m s i s o l a t e d f r o m raw m i l k c o l l e c t e d f r o m 12 f a r m s . B. A p p r o a c h e s t o i d e n t i f i c a t i o n Pseudomonas f l u o r e s c e n s i s c o n s i d e r e d by numerous 33 r e s e a r c h e r s t o be t h e p r e d o m i n a n t s p e c i e s c o n t a m i n a t i n g raw m i l k ( C o u s i n , 1982). I d e n t i f i c a t i o n o f t h e b a c t e r i a l i s o l a t e s i s most commonly p e r f o r m e d a c c o r d i n g t o t h e B e r g e y ' s Manual of D e t e r m i n a t i v e B a c t e r i o l o g y (1974) o r t h e d i a g n o s t i c t a b l e s o f Cowan and S t e e l (1965) (Fox e t a l . , 1976; C o u s i n and M a r t h , 1977). 1) I d e n t i f i c a t i o n a c c o r d i n g t o t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) . A c c o r d i n g t o B e r g e y ' s M a n u a l , members o f t h e genus Pseudomonas a r e m o t i l e , s t r i c t l y a e r o b i c , G r a m - n e g a t i v e r o d s w h i c h a r e c a t a l a s e and o x i d a s e p o s i t i v e . Most s p e c i e s r e q u i r e no g r o w t h f a c t o r s . D i f f u s i b l e f l u o r e s c e n t p i g m e n t s a r e p r o d u c e d by some s p e c i e s . D e t a i l e d c h a r a c t e r i s t i c s o f 29 s p e c i e s , i n c u b a t e d a t 30 C, a r e a l s o g i v e n . F o r f l u o r e s c e n s , 14 p r i n c i p a l d i s t i n c t i v e c h a r a c t e r i s t i c s a r e t a b u l a t e d and o t h e r p h e n o t y p i c c h a r a c t e r i s t i c s a r e a l s o d e s c r i b e d . K e y s a r e g i v e n f o r t h e p r i m a r y s e p a r a t i o n o f s p e c i e s i n t o g r o u p s t h a t c a n be h a n d l e d c o n v e n i e n t l y . The key t o i d e n t i f y P. f l u o r e s c e n s i s g i v e n a s : g r o w t h f a c t o r s a r e n o t r e q u i r e d , p o l y - / 3 - h y d r o x y b u t y r a t e i s n o t a c c u m u l a t e d , f l u o r e s c e n t p i g m e n t s a r e p r o d u c e d and t h e a r g i n i n e d i h y d r o l a s e s y s t e m i s p r e s e n t . I t a l s o s t a t e s t h a t even i m p o r t a n t d i f f e r e n t i a l t r a i t s may be a b s e n t i n some s t r a i n s when i s o l a t e d , o r may be l o s t a f t e r l a b o r a t o r y c u l t i v a t i o n . To a v o i d t h e r i s k o f m i s i d e n t i f i c a t i o n o f a s t r a i n a b e r r a n t i n one o r more o f t h e d i f f e r e n t i a l t r a i t s , t h e d i a g n o s t i c keys s h o u l d n o t be s o l e l y r e l i e d upon. 34 C h a r a c t e r s g i v e n i n t h e t a b l e and d e s c r i p t i o n f o r t h a t s u s p e c t e d s p e c i e s s h o u l d a l s o be s t u d i e d . T a y f o u r e t a l . (1982) i d e n t i f i e d a p s y c h r o t r o p h i c s t r a i n as P. f l u o r e s c e n s b i o t y p e A a c c o r d i n g t o t h e B e r g e y ' s Manual of D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) . I n f o r m a t i o n on how t h e i d e n t i f i c a t i o n was made was n o t g i v e n . R i c h a r d s o n (1981) p u r i f i e d and c h a r a c t e r i z e d a h e a t - s t a b l e p r o t e a s e f r o m P. f l u o r e s c e n s i s o l a t e d f r o m r e f r i g e r a t e d raw m i l k . No d e t a i l e d d e s c r i p t i o n o f t h e c h a r a c t e r s s t u d i e d were p r o v i d e d . Law e t a l . (1979a) i d e n t i f i e d f i v e p r o t e o l y t i c G r a m - n e g a t i v e i s o l a t e s f r o m raw m i l k a s P^ f l u o r e s c e n s a c c o r d i n g t o t h e schemes o u t l i n e d i n B e r g e y ' s M a n u a l . T h e s e s t u d i e s have one common m i s t a k e : t h e r e i s i n s u f f i c i e n t i n f o r m a t i o n on how t h e i d e n t i f i c a t i o n was done. I t woul d be more r e a s s u r i n g i f t h e c h a r a c t e r s s t u d i e d and t h e i n c u b a t i o n t e m p e r a t u r e employed were i n c l u d e d . 2) I d e n t i f i c a t i o n a c c o r d i n g t o t h e Manual f o r t h e I d e n t i f i c a t i o n o f M e d i c a l B a c t e r i a (Cowan and S t e e l , 1965) In t h i s manual, Pseudomonas a r e d e f i n e d a s G r a m - n e g a t i v e , m o t i l e , a e r o b i c r o d s w h i c h a r e c a t a l a s e and o x i d a s e p o s i t i v e . D i f f u s i b l e y e l l o w o r g r e e n p i g m e n t s may be p r o d u c e d by members of t h i s g e n u s . A d i a g n o s t i c t a b l e o f c h a r a c t e r i s t i c s f o r o n l y t h r e e Pseudomonas s p e c i e s , i n c u b a t e d a t 37 C, was a l s o i n c l u d e d . The t h r e e s p e c i e s d e s c r i b e d a r e P^ a e r u g i n o s a , P. f l u o r e s c e n s and P_;_ p s e u d o m a l l e i . Of t h e t h r e e s p e c i e s , o n l y P_^  f l u o r e s c e n s c a n grow a t 5 C but n o t a t 42 C. O b v i o u s l y , s p e c i e s o t h e r t h a n 35 t h e t h r e e s p e c i e s l i s t e d c a n not be i d e n t i f i e d a c c o r d i n g t o t h i s m a n u a l . T h i s manual c a n o n l y be c o n s u l t e d i f t h e p u r p o s e i s t o i d e n t i f y t h e m i l k i s o l a t e s t o t h e genus l e v e l . M u i r e t a l . (1979) i s o l a t e d p s y c h r o t r o p h i c b a c t e r i a from s u p p l i e s of raw m i l k . The G r a m - n e g a t i v e b a c t e r i a l i s o l a t e s were g r o u p e d by t h e f i r s t - s t a g e d i a g n o s t i c p r o c e d u r e o f Cowan and S t e e l ( 1 9 6 5 ) . They f o u n d t h a t s p e c i e s o f t h e genus Pseudomonas were t h e p r e d o m i n a n t p s y c h r o t r o p h i c b a c t e r i a i n raw m i l k . C o u s i n and M a r t h (1977) examined c h a n g e s i n m i l k p r o t e i n s c a u s e d by p s y c h r o t r o p h i c b a c t e r i a . F i v e o f t h e n i n e b a c t e r i a l i s o l a t e s e x a m i n e d were Pseudomonas s p e c i e s . The pseudomonads were i d e n t i f i e d a c c o r d i n g t o t h e d i a g n o s t i c t a b l e s o f Cowan and S t e e l (1965) and B e r g e y ' s Manual ( 1 9 7 4 ) . M o d i f i c a t i o n of m i l k p r o t e i n s by Pseudomonas s p e c i e s were a l s o i n v e s t i g a t e d by D e B e u k e l a r e t a l . ( 1 9 7 7 ) . I s o l a t e s were c l a s s i f i e d i n t o g e n e r a by t h e d i a g n o s t i c t a b l e s of Cowan and S t e e l (1965) and B r e e d e t a l . ( 1 9 5 7 ) . 3) I d e n t i f i c a t i o n by t h e API 20E s y s t e m ( A n a l y t a b P r o d u c t s ,  P l a i n v i e w , NY) The use o f t h e API 20E s y s t e m t o i d e n t i f y b a c t e r i a l i s o l a t e s f r o m raw m i l k has been r e p o r t e d ( M c K e l l a r , 1981). He s t u d i e d t h e r e l a t i o n s h i p between p r o t e o l y s i s and o f f - f l a v o r d e v e l o p m e n t i n UHT m i l k c a u s e d by t h r e e b a c t e r i a l i s o l a t e s i d e n t i f i e d t o be s t r a i n s of Pseudomonas. He u s e d an i n c u b a t i o n t e m p e r a t u r e of 22 C i n s t e a d o f 35 C as recommended. A d d i t i o n a l i n f o r m a t i o n on t h e a b i l i t y o f t h e i s o l a t e s was a l s o i n c l u d e d . 36 G r i f f i t h s e t a l . (1981) c h a r a c t e r i z e d t h e G r a m - n e g a t i v e r o d -s h a p e d b a c t e r i a , i s o l a t e d f r o m raw m i l k , u s i n g API 20E s t r i p s i n c u b a t e d a t 30 C. The m a j o r i t y o f t h e s t r a i n s r e p o r t e d were P. p u t i d a , P. f l u o r e s c e n s and P^ f r a g i . The API 20E s y s t e m i s a r e a d y - t o - u s e , m i c r o t u b e s y s t e m d e s i g n e d f o r t h e p e r f o r m a n c e o f 23 b i o c h e m i c a l t e s t s . T h i s s y s t e m i s d e s i g n e d m a i n l y f o r t h e i d e n t i f i c a t i o n o f E n t e r o b a c t e r i a c e a e . The d a t a base i s b a s e d on r e s u l t s a f t e r 18 24 h o u r s o f i n c u b a t i o n a t 35 - 37 C. The a p p l i c a t i o n o f t h i s s y s t e m t o i d e n t i f y b a c t e r i a l c u l t u r e s not of c l i n i c a l o r i g i n has y e t t o be e s t a b l i s h e d . The a c c u r a c y of t h i s s y s t e m t o i d e n t i f y n o n f e r m e n t a t i v e b a c i l l i i s b e g i n n i n g t o g a i n a t t e n t i o n ( O t t o and B lachman, 1979). R e s u l t s o b t a i n e d f r o m u s i n g t h i s s y s t e m , e s p e c i a l l y w i t h an i n c u b a t i o n t e m p e r a t u r e o t h e r t h a n 35 - 37 C, s h o u l d be e v a l u a t e d w i t h c a r e . 4) I d e n t i f i c a t i o n by c l u s t e r a n a l y s i s The weakness o f c u r r e n t c l a s s i f i c a t i o n s i s t h a t g e n e r a and s p e c i e s a r e s t i l l d e s c r i b e d on t h e b a s i s o f one o r a few s p e c i f i c c h a r a c t e r s . P r o d u c t i o n o f f l u o r e s c e n t p i g m e n t , a c c u m u l a t i o n o f p o l y - / 3 - h y d r o x y b u t y r a t e and t h e p r e s e n c e o f t h e a r g i n i n e d i h y d r o l a s e s y s t e m a r e a few r e q u i r e d c h a r a c t e r s f o r a s t r a i n t o p o s s e s s t o be i d e n t i f i e d as Pseudomonas. Such c l a s s i f i c a t i o n s c a r r y t h e r i s k o f m i s i d e n t i f i c a t i o n , f o r a s t r a i n may be a b e r r a n t i n one o f t h e s p e c i f i c c h a r a c t e r s u s e d t o make a c l a s s i f i c a t i o n . In o r d e r t o accomodate s t r a i n v a r i a t i o n , i d e n t i f i c a t i o n 37 u s i n g c l u s t e r a n a l y s i s i s u s e d . The b a c t e r i a l i s o l a t e s a r e i d e n t i f i e d on t h e b a s i s o f a l a r g e s e t o f c h a r a c t e r s , w i t h e a c h c h a r a c t e r b e i n g g i v e n e q u a l w e i g h t . The s i m i l a r i t y o f c h a r a c t e r i s t i c s between p a i r s o f o r g a n i s m s i s e x p r e s s e d n u m e r i c a l l y , u s u a l l y i n terms o f a s i m p l e m a t c h i n g c o e f f i c i e n t . T h o s e b a c t e r i a l i s o l a t e s w i t h h i g h s i m i l a r i t y v a l u e s a r e a r r a n g e d t o g e t h e r t o f o r m c l u s t e r s o r g r o u p s . T h e r e a r e t h r e e t y p e s o f c l u s t e r a n a l y s i s : t h e s i n g l e -l i n k a g e , c o m p l e t e - l i n k a g e and a v e r a g e - l i n k a g e . W i t h t h e a v e r a g e - l i n k a g e c l u s t e r i n g methods, t h e u n w e i g h t e d a r i t h m e t i c mean a n a l y s i s i s t h e most common. The t h r e e methods d i f f e r i n t h e c r i t e r i o n f o r a d m i s s i o n of a b a c t e r i a l i s o l a t e t o a c l u s t e r o r f o r f u s i o n o f c l u s t e r s . In c o m p l e t e - l i n k a g e a n a l y s i s , an i s o l a t e i s n o t a d m i t t e d t o a c l u s t e r u n t i l i t s s i m p l e m a t c h i n g c o e f f i c i e n t s w i t h a l l members o f t h e c l u s t e r a r e e q u a l t o o r g r e a t e r t h a n t h e l e v e l s e t f o r c l u s t e r f o r m a t i o n , and two c l u s t e r s c a n f u s e o n l y when t h e s i m p l e m a t c h i n g c o e f f i c i e n t s between a l l i s o l a t e s i n t h e two c l u s t e r s a r e e q u a l t o o r g r e a t e r t h a n t h e s e t c l u s t e r i n g l e v e l . In s i n g l e - l i n k a g e a n a l y s i s , l i n k a g e between an i s o l a t e and any member o f a c l u s t e r a t a g i v e n s i m i l a r i t y l e v e l p r o v i d e s a d m i s s i o n t o t h a t c l u s t e r , and two c l u s t e r s c a n f u s e w i t h l i n k a g e between any p a i r o f i s o l a t e s (one f r o m e a c h c l u s t e r ) . I n a v e r a g e - l i n k a g e a n a l y s i s , an i s o l a t e i s n o t a d m i t t e d t o a c l u s t e r u n t i l t h e mean o f i t s s i m p l e m a t c h i n g c o e f f i c i e n t s w i t h a l l members of t h e c l u s t e r a r e e q u a l t o o r g r e a t e r t h a n t h e l e v e l s e t f o r c l u s t e r f o r m a t i o n , and two c l u s t e r s c a n f u s e o n l y when t h e mean o f a l l t h e 38 s i m i l a r i t y c o e f f i c i e n t s between a l l i s o l a t e s i n t h e two c l u s t e r s a r e e q u a l t o o r g r e a t e r t h a n t h e s e t c l u s t e r i n g l e v e l . J u f f s ( 1 9 7 3 ) , u s i n g c o m p l e t e - l i n k a g e c l u s t e r a n a l y s i s , i d e n t i f i e d Pseudomonas s p e c i e s i s o l a t e d f r o m raw m i l k . R e l e v a n t c h a r a c t e r s of 13 Pseudomonas s p e c i e s as p r o p o s e d by S t a n i e r e t a l . (1966) were a l s o i n c o r p o r a t e d i n t h e a n a l y s i s . Twenty n i n e b i o c h e m i c a l c h a r a c t e r s of 180 b a c t e r i a l i s o l a t e s were examined. A l l c h a r a c t e r i z a t i o n s were p e r f o r m e d a t 28 C. The dendrogram o b t a i n e d by c l u s t e r a n a l y s i s showed t h a t P\_ f l u o r e s c e n s was t h e p r e d o m i n a n t s p e c i e s . The c l u s t e r s , i n w h i c h t h e m a j o r i t y o f t h e m i l k i s o l a t e s r e s i d e d i n , were q u i t e d i s t i n c t f r o m t h e r e f e r e n c e s p e c i e s p r o p o s e d by S t a n i e r e t a l . ( 1 9 6 6 ) . Samagh and Cunningham (1972) u s e d s i n g l e - l i n k a g e c l u s t e r a n a l y s i s t o i d e n t i f y 182 pseudomonads f r o m m i l k . S i x t e e n known r e p r e s e n t a t i v e s of d i f f e r e n t Pseudomonas s p e c i e s , a l o n g w i t h t h e 182 m i l k i s o l a t e s were examined f o r 97 c h a r a c t e r s a t 20 C. T h e i r r e s u l t s i n d i c a t e d t h a t f l u o r e s c e n s , P. a e r u g i n o s a - l i k e o r g a n i s m s and P_^  p u t i d a were t h e m a j o r s p e c i e s . They a l s o s u s p e c t e d t h a t 41 n o n f l u o r e s c e n t c u l t u r e s , w h i c h t h e y d i d n o t i n c l u d e i n t h e a n a l y s i s , were r e l a t e d t o P\_ f r a g i o r P. p u t r e f a c i e n s . 5) I d e n t i f i c a t i o n w i t h no r e f e r e n c e s p r o v i d e d R i c h a r d s o n and Newstead (1979) r e p o r t e d t h e e f f e c t s o f h e a t - s t a b l e p r o t e a s e s , from two P^ f l u o r e s c e n s s t r a i n s , on t h e s t o r a g e l i f e o f u l t r a - h i g h t e m p e r a t u r e t r e a t e d m i l k . The a p p r o a c h u s e d t o i d e n t i f y t h e s e two s t r a i n s was n o t p r o v i d e d . 39 J u f f s (1974) i s o l a t e d t h r e e s t r a i n s of P_^  f l u o r e s c e n s and t h r e e s t r a i n s o f a e r u g i n o s a from raw m i l k . The i d e n t i f i c a t i o n scheme employed was not p u b l i s h e d . I n t e r e s t i n g l y , Adams e t a l . (1975) d e f i n e d i s o l a t e s b e l o n g i n g t o t h e genus Pseudomonas as m o t i l e , a e r o b i c , G r a m - n e g a t i v e r o d s t h a t grew a t 32 C but n o t a t 37 C. The r e f e r e n c e t o s u p p o r t t h i s c l a s s i f i c a t i o n was not r e p o r t e d . 6) R a p i d c h a r a c t e r i z a t i o n o f b a c t e r i a by u s i n g a m u l t i p o i n t  i n o c u l a t o r B e e c h e t a l . (1955) d e s i g n e d a m u l t i p o i n t i n o c u l a t o r f o r p l a t i n g b a c t e r i a r a p i d l y . The d e v i c e c o n s i s t e d of 24 v e r t i c a l s t a i n l e s s s t e e l r o d s a t t a c h e d t o t h e i n n e r b a s a l s u r f a c e of an i n v e r t e d s t a i n l e s s s t e e l c u p . I n o c u l u m (1 ml) was h e l d i n 9 x 50 mm t u b e s p o s i t i o n e d by an i n o c u l u m t u b e h o l d e r . The h o l d e r was a c i r c u l a r p l a t e d r i l l e d w i t h 24 h o l e s , i n t h e same p a t t e r n as t h e i n o c u l a t i n g r o d s , e a c h c a p a b l e o f h o l d i n g a 9 x 50 mm t u b e . The i n o c u l a t o r was d i p p e d i n t o t h e i n o c u l u m t u b e s , and t h e n w i t h d r a w n and t r a n s f e r r e d t o a n u t r i e n t a g a r p l a t e t o a c h i e v e a m u l t i p l e i n o c u l a t i o n . In t h i s way, a p l a t e would be s i m u l t a n e o u s l y i n o c u l a t e d w i t h 24 m i c r o o r g a n i s m s i n 30 s e c o n d s as compared w i t h 8 m i n u t e s r e q u i r e d f o r i n o c u l a t i n g them s i n g l y w i t h a w i r e l o o p . A s i m i l a r m u l t i p l e i n o c u l a t i o n d e v i c e was r e p o r t e d by S m i t h ( 1 9 6 1 ) . The i n o c u l a t o r was made of a c i r c u l a r s t e e l p l a t e , 8.5 cm i n d i a m e t e r , f i t t e d on t h e upper s i d e w i t h a c e n t r a l l y p l a c e d h a n d l e o f s t a i n l e s s s t e e l r o d . I n t o e a c h o f s y m m e t r i c a l l y a r r a n g e d h o l e s b o r e d i n t h e p l a t e was f i t t e d a 1.5 40 cm s t a i n l e s s s t e e l b o l t . T h e s e b o l t s c o n s t i t u t e d t h e i n o c u l a t i o n s t u b s . The c o n t a i n e r f o r i n o c u l a c o n s i s t e d of 8 cm d i a m e t e r d i s c s o f p l e x i g l a s s s h e e t d r i l l e d t o g i v e 16 r o u n d e d w e l l s , c o n c e n t r i c a l l y a l i g n e d w i t h t h e m u l t i p l e i n o c u l a t o r s t u b s . About 0.3 ml o f b a c t e r i a l s u s p e n s i o n t o be t e s t e d were p i p e t t e d i n t o e a c h o f t h e w e l l s . I n o c u l a t i o n was done by i m m e r s i n g t h e i n o c u l a t o r i n t o t h e w e l l s f o l l o w e d by t r a n s f e r f r o m t h e w e l l s t o a p p r o p r i a t e a g a r p l a t e s . Kaneko e t a l . (1977) d e v e l o p e d a m u l t i p l e s y r i n g e i n o c u l a t o r f o r a g a r p l a t e s . The a p p a r a t u s was n o t s i m p l e a s i t c o n s i s t e d o f 5 components: an i n o c u l a t o r h o l d e r ; t h e f r o n t p l a t e ; t h e s y r i n g e h o l d e r s ; t h e s y r i n g e p r e s s u r e p l a t e ; and t h e s y r i n g e i n o c u l a t o r s . I n o c u l a t i o n was q u i t e d i f f e r e n t f r o m t h e two methods d i s c u s s e d a s t h e p l a t e h a d t o be i n t h e i n v e r t e d p o s i t i o n d u r i n g i n o c u l a t i o n . Fung and Hartman (1972) d e v e l o p e d a s i m p l e m u l t i p l e i n o c u l a t i o n d e v i c e c a p a b l e of making 96 i n o c u l a t i o n s a t a t i m e . I t was made by i n s e r t i n g 27 mm p i n s s e p a r a t e l y i n t o 96 wax-f i l l e d w e l l s (8 x 12 c o n f i g u r a t i o n ) of a t i s s u e c u l t u r e p l a t e . A c u l t u r e p l a t e was p r e p a r e d by i n t r o d u c i n g 0.20 ml o f a d i f f e r e n t b r o t h c u l t u r e i n t o e a c h o f t h e w e l l s o f a s t e r i l e t i s s u e c u l t u r e p l a t e . S t e r i l i z a t i o n o f t h e i n o c u l a t i n g d e v i c e was a c h i e v e d by d i p p i n g t h e p i n s i n t o 90% a l c o h o l f o r 20-30 s f o l l o w e d by f l a m i n g . The s t e r i l e i n o c u l a t i o n d e v i c e was c h a r g e d by l o w e r i n g i t i n t o t h e c u l t u r e p l a t e . The c h a r g e d i n o c u l a t i o n d e v i c e was p l a c e d on a d r i e d s u r f a c e o f a p p r o p r i a t e a g a r . The m i c r o t i t e r t e c h n i q u e o f Fung and Hartman (1972) was 41 u s e d by C a s a s e t a l . (1977) t o enumerate m e s o p h i l e s , p s y c h r o t r o p h s and c o l i f o r m s i n raw and p a s t e u r i z e d m i l k . They r e p o r t e d t h a t t h e M i c r o t i t e r Count method was r e l i a b l e when compared w i t h t h e S t a n d a r d P l a t e Count method. The m i c r o t i t e r t e c h n i q u e was a l s o u s e d t o i d e n t i f y , t o t h e genus l e v e l , b a c t e r i a l c u l t u r e s i s o l a t e d f r o m raw m i l k ( O t t e e t a l . , 1979). 42 MATERIALS AND METHODS A. I s o l a t i o n o f p r o t e o l y t i c s t r a i n s from raw m i l k P r o t e o l y t i c p s y c h r o t r o p h s i n raw m i l k were i s o l a t e d on s k i m m i l k a g a r (4 C, 7 d) ( L e e , 1976). I s o l a t e d c o l o n i e s e x h i b i t i n g z o n e s o f c l e a r i n g were p u r i f i e d on s k i m m i l k a g a r i n c u b a t e d a t 22 C. They were t h e n s t o r e d a t 4 C on n u t r i e n t a g a r ( D i f c o , MI) s l a n t s a s s t o c k c u l t u r e s w i t h s u b c u l t u r e a t 8 - 10 week i n t e r v a l s . Type s t r a i n s o f f o u r Pseudomonas s p e c i e s , P. f l u o r e s c e n s ATCC 948; Pj_ f l u o r e s c e n s b i o t y p e A ATCC 17397; P. p u t i d a ATCC 12633 and P_;_ f r a g i ATCC 4976) were i n c l u d e d f o r r e f e r e n c e p u r p o s e s . E a c h i s o l a t e was grown i n n u t r i e n t b r o t h ( D i f c o , MI) f o r 24 h a t 22 C b e f o r e c h a r a c t e r i z a t i o n . A l l t e s t s were i n c u b a t e d a t 22 C e x c e p t when s p e c i f i e d . B. P r i m a r y c h a r a c t e r i z a t i o n t e s t s 1) Gram s t a i n The H u c k e r ' s m o d i f i c a t i o n was u s e d (Cowan and S t e e l , 1974). 2) O x i d a s e r e a c t i o n The method o f K o v a c s (1956) was u s e d . The t e s t o r g a n i s m was p i c k e d w i t h a p l a t i n u m w i r e and smeared a c r o s s t h e s u r f a c e o f a p i e c e o f f i l t e r p a p e r i m p r e g n a t e d w i t h 2 t o 3 d r o p s o f 1% 43 t e t r a m e t h y l - p - p h e n y l d i a m i n e d i h y d r o c h l o r i d e p a p e r . A p o s i t i v e r e a c t i o n was shown by t h e d e v e l o p m e n t of a d a r k p u r p l e c o l o r w i t h i n 10 s. 3) C a t a l a s e t e s t ~ I s o l a t e s were s c r a p e d from t h e s k i m m i l k a g a r and s u s p e n d e d i n a d r o p o f 3% H 2 0 2 on a s l i d e . E v o l u t i o n o f b u b b l e s w i t h i n one m i n u t e was r e c o r d e d a s p o s i t i v e . 4) Growth a t pH 4.5 B r a i n h e a r t i n f u s i o n b r o t h ( D i f c o , MI) was i n o c u l a t e d w i t h one l o o p f u l of c u l t u r e grown f o r 24 h i n n u t r i e n t b r o t h . C u l t u r e s were examined f o r growth a f t e r 7 d i n c u b a t i o n . 5) O x i d a t i o n o r f e r m e n t a t i o n o f g l u c o s e The Hugh and L e i f s o n (1953) method was e m p l o y e d . M i n e r a l o i l , a b o u t 1 cm i n d e p t h , was u s e d t o s e a l t h e t u b e . A s t r i c t a e r o b e c a u s e d t h e d e v e l o p m e n t o f a y e l l o w c o l o r i n t h e open t u b e and no change i n c o l o r i n t h e s e a l e d t u b e . 6) M o t i l i t y M o t i l i t y was e xamined by b o t h t h e h a n g i n g d r o p t e c h n i q u e and m o t i l i t y medium (Cowan and S t e e l , 1974). M o t i l e o r g a n i s m m i g r a t e d t h r o u g h t h e medium w h i c h became t u r b i d . Growth o f n o n - m o t i l e o r g a n i s m was c o n f i n e d t o t h e s t a b 1 i n e . 44 7) Growth on Pseudomonas i s o l a t i o n a g a r The a b i l i t y of e a c h m i l k i s o l a t e t o grow on Pseudomonas i s o l a t i o n a g a r ( D i f c o , MI) was e x a m i n e d . Pseudomonas s p e c i e s were d e f i n e d as G r a m - n e g a t i v e , m o t i l e r o d s . They were s t r i c t a e r o b e s and c o u l d n o t grow i n b r a i n h e a r t i n f u s i o n b r o t h a t pH 4.5. They were o x i d a s e and c a t a l a s e p o s i t i v e ( S t a n i e r e t a l . , 1966; Hugh, 1973). C. S e c o n d a r y c h a r a c t e r i z a t i o n t e s t s 1) Growth a t 4 C and 41 C A b i l i t y o f i s o l a t e s t o grow on s k i m m i l k a g a r a t 4 C (7 10 d) a n d 41 C (2 d) was e v a l u a t e d . 2) P r o d u c t i o n of f l u o r e s c e n t p i g m e n t s Medium B o f K i n g e t a l . (1954) was u s e d . F l u o r e s c e n c e was c o n f i r m e d under u l t r a v i o l e t l i g h t (350 nm). 3) N i t r a t e r e d u c t i o n F o l l o w i n g i n c u b a t i o n o f e a c h i s o l a t e i n n i t r a t e b r o t h ( D i f c o , MI) f o r 5 d , n i t r i t e was d e t e c t e d w i t h s u l p h a n i l i c a c i d and a - n a p h t h y l a m i n e . The p r e s e n c e o f a r e d c o l o r i n d i c a t e d t h a t n i t r a t e had been r e d u c e d t o n i t r i t e . I f nb c o l o r d e v e l o p m e n t was r e c o r d e d , z i n c d u s t was ad d e d t o d e t e r m i n e t h e p r e s e n c e o f n i t r a t e n o t r e d u c e d by t h e t e s t o r g a n i s m . A r e d c o l o r showed t h a t n i t r a t e was n o t r e d u c e d , f o r t h e z i n c d u s t r e d u c e d t h e 45 n i t r a t e t o n i t r i t e . The a b s e n c e o f a r e d c o l o r , a f t e r t h e a d d i t i o n o f z i n c d u s t , showed t h a t n i t r a t e had been r e d u c e d but not t o n i t r i t e . 4) A r g i n i n e d i h y d r o l a s e The method o f N i v e n e t a l . (1942) was u s e d . The t e s t o r g a n i s m was i n c u b a t e d i n a r g i n i n e b r o t h f o r 24 h. A r g i n i n e h y d r o l y s i s was i n d i c a t e d by t h e d e v e l o p m e n t o f a brown c o l o r a f t e r t h e a d d i t i o n o f t h e N e s s l e r ' s r e a g e n t . 5) I n d o l e p r o d u c t i o n I n d o l e p r o d u c t i o n f r o m 1% t r y p t o n e b r o t h ( D i f c o , MI) a f t e r 2 d was r e c o r d e d a s p o s i t i v e by t h e p r e s e n c e o f a r e d c o l o r a f t e r a d d i t i o n o f K o v a c s ' r e a g e n t . 6) M e t h y l r e d The method o f C l a r k e and Lubs (1915) was u s e d . M e t h y l r e d s o l u t i o n was a d d e d t o an i n o c u l a t e d m e t h y l r e d - V o g e s - P r o s k a u e r (MR-VP) b r o t h (BBL M i c r o b i o l o g y S y s t e m s , MD) a f t e r i n c u b a t i o n f o r 4 d . A p o s i t i v e r e a c t i o n was i n d i c a t e d by t h e d e v e l o p m e n t of a r e d c o l o r . 7) Voges P r o s k a u e r The method o f B a r r i t t (1936) was f o l l o w e d . To a 48 h c u l t u r e grown i n MR-VP medium, 5% a - n a p h t h o l s o l u t i o n f o l l o w e d by 40% p o t a s s i u m h y d r o x i d e s o l u t i o n were a d d e d . A p o s i t i v e r e a c t i o n was i n d i c a t e d by a s t r o n g r e d c o l o r . 46 8) Tween 80 h y d r o l y s i s The method as d e s c r i b e d i n Cowan and S t e e l (1974) was a d o p t e d . F o r up t o 7 d a y s , p l a t e s were examined f o r t h e p r e s e n c e o f an opaque h a l o a r o u n d i s o l a t e d c o l o n i e s . 9) B u t t e r f a t h y d r o l y s i s The medium d e s c r i b e d by J o n e s and R i c h a r d s (1952) was f o l l o w e d . L i p o l y s i s was r e c o r d e d as p o s i t i v e when a d i s t i n c t b l u e zone was s e e n a r o u n d t h e c o l o n y w i t h i n 7 d . 10) P h o s p h o l i p a s e p r o d u c t i o n L e c i t h i n medium ( C h r i s o p e e t a l . , 1976) was u s e d . P l a t e s were examined, o v e r a p e r i o d o f 7 d a y s , f o r t h e p r e s e n c e o f c o l o n i e s s u r r o u n d e d by an opaque z o n e . 11) G e l a t i n a s e p r o d u c t i o n The method a c c o r d i n g t o Kohn (1953) u s i n g c h a r c o a l g e l a t i n d i s c ( O x o i d , O t tawa, Canada) was f o l l o w e d . H y d r o l y s i s was shown by t h e a p p e a r a n c e o f f r e e c h a r c o a l p a r t i c l e s i n t h e p e p t o n e s o l u t i o n ( 0 . 1 % w/v). 12) P r o t e i n a s e p r o d u c t i o n Skim m i l k a g a r was e m p l o y e d and a c l e a r zone a r o u n d c o l o n i e s , a f t e r f l o o d i n g w i t h 10% a c e t i c a c i d , was t a k e n as an i n d i c a t i o n o f p r o t e i n a s e p r o d u c t i o n . 13) A c c u m u l a t i o n o f poly-/3 - h y d r o x y b u t y r a t e 47 The a b i l i t y t o a c c u m u l a t e p o l y - / 3 - h y d r o x y b u t y r a t e u s i n g t h e medium recommended by S t a n i e r e t a l . (1966) was e v a l u a t e d . The p o l y m e r was d e t e c t e d by m i c r o s c o p i c e x a m i n a t i o n o f b a c t e r i a a f t e r s t a i n i n g w i t h Sudan B l a c k B (Sigma, S t . L o u i s , MO). 14) U t i l i z a t i o n of t h e d i f f e r e n t c a r b o n s o u r c e s The a b i l i t y t o grow on 35 compounds as t h e s o l e s o u r c e of c a r b o n was s t u d i e d . The b a s a l medium was t h a t o f Dowson ( 1 9 4 9 ) . A l l compounds were f i l t e r s t e r i l i z e d b e f o r e a d d i t i o n t o t h e a u t o c l a v e d medium. The s u b s t r a t e c o n c e n t r a t i o n was 0.1% (w/v) e x c e p t f o r s u g a r s (0.2%) a n d p h e n o l ( 0 . 0 2 5 % ) . The s u g a r s t e s t e d were D - r i b o s e , D - x y l o s e , L - a r a b i n o s e , L-rhamnose, D - g l u c o s e , D-mannose, D - g a l a c t o s e , D - f r u c t o s e , s u c r o s e , t r e h a l o s e , m a l t o s e , l a c t o s e , g l u c o n a t e and s a l i c i n . O t h e r compounds t e s t e d i n c l u d e d a c e t a t e , p r o p i o n a t e , b u t y r a t e , s u c c i n a t e , m a l a t e , l a c t a t e , c i t r a t e , p y r u v a t e , m a n n i t o l , i n o s i t o l , a d o n i t o l , g l y c e r o l , p r o p y l e n e g l y c o l , e t h a n o l , m e t h a n o l , g l y c i n e , L - a l a n i n e , L - s e r i n e , L - v a l i n e , L - a r g i n i n e . 15) U t i l i z a t i o n of t h e d i f f e r e n t n i t r o g e n o u s compounds The a b i l i t y t o use a c e t a m i d e , n i c o t i n a t e , and p a n t h o t h e n a t e as s o l e n i t r o g e n s o u r c e was t e s t e d on t h e b a s a l medium o f Dowson ( 1 9 4 9 ) . The s u b s t r a t e c o n c e n t r a i o n was 0.1% (w/ v ) . The t h r e e compounds were f i l t e r s t e r i l i z e d . D. R a p i d c h a r a c t e r i z a t i o n u s i n g a m u l t i p o i n t i n o c u l a t o r 48 A m u l t i p o i n t i n o c u l a t o r , d e v e l o p e d a f t e r t h a t of Fung and Hartman ( 1 9 7 2 ) , was c o n s t r u c t e d by f i x i n g 96 s t a i n l e s s s t e e l p i n s (25 mm l o n g ) i n t o a p i e c e of 8.5 cm x 12.5 cm x 0.5 cm t h i c k b a l s a wood ( F i g . 7 ) . The p i n s were p o s i t i o n e d t o c o r r e s p o n d w i t h t h e c e n t e r s of t h e 96 w e l l s o f a t i s s u e c u l t u r e p l a t e ( F a l c o n , O x n a r d , C A ) . A p i e c e o f 9 cm x 13 cm x 1 cm t h i c k h i g h d e n s i t y p o l y e t h y l e n e b o a r d ( C a d i l l a c P l a s t i c , V a n c o u v e r , B.C., Canada) was p l a c e d on t o p of t h e b a l s a wood. A knob w i t h a 5 cm screw was u s e d t o l o c k t h e b o a r d and t h e b a l s a wood i n p l a c e . The whole s y s t e m was wrapped w i t h aluminum f o i l b e f o r e t h e p i n s were i n s e r t e d . The i n o c u l a t i o n d e v i c e was s t e r i l i z e d by a u t o c l a v i n g (121 C, 15 m i n ) . C u l t u r e s grown o v e r n i g h t i n n u t r i e n t b r o t h (22 C) were i n t r o d u c e d i n t o t h e w e l l s o f a 96 w e l l t i s s u e c u l t u r e p l a t e . M e d i a t o be i n o c u l a t e d were d i s p e n s e d i n t o t h e w e l l s o f a n o t h e r p l a t e . An e x p e r i m e n t a l d e s i g n whereby d i f f e r e n t b r o t h c u l t u r e s were i n t r o d u c e d i n t o d i f f e r e n t rows o f w e l l s (12 w e l l s / row) o f t h e 8 x 12 c o n f i g u r a t i o n t i s s u e c u l t u r e p l a t e was employed. D i f f e r e n t media were t h e n d i s p e n s e d i n t o d i f f e r e n t columns o f w e l l s (8 w e l l s / c o l u m n ) . The s t e r i l e m u l t i p o i n t i n o c u l a t o r was c h a r g e d by d i p p i n g t h e p i n s i n t o t h e w e l l s o f t h e p l a t e c o n t a i n i n g t h e b r o t h c u l t u r e s . The c h a r g e d i n o c u l a t o r was t h e n p l a c e d on t o p o f a t i s s u e c u l t u r e p l a t e c o n t a i n i n g t h e a p p r o p r i a t e media so t h a t a l l t h e p i n s were immersed i n t h e m e d i a . The i n o c u l a t o r was t h e n s t e r i l i z e d , by d i p p i n g t h e p i n s i n t o 90% a l c o h o l f o l l o w e d by f l a m i n g . 48a i F i g . 7 . The m u l t i p o i n t i n o c u l a t o r . 50 T h i s r a p i d c h a r a c t e r i z a t i o n method was u s e d t o s t u d y t h e a b i l i t y of t h e b a c t e r i a i s o l a t e d f r o m m i l k t o use 35 compounds as t h e s o l e s o u r c e of c a r b o n and 3 compounds as t h e s o l e s o u r c e o f n i t r o g e n . F o r t h e r e s t o f t h e c h a r a c t e r i z a t i o n s , t h e c o n v e n t i o n a l methods were f o l l o w e d . E. N u m e r i c a l taxonomy B a c t e r i a l i s o l a t e s i d e n t i f i e d a s Pseudomonas t o g e t h e r w i t h t h e 4 r e f e r e n c e s t r a i n s : P^ f l u o r e s c e n s ATCC. 948; P_j_ f l u o r e s c e n s b i o t y p e A ATCC 17397; E\ p u t i d a ATCC 12633 and E\ f r a g i ATCC 4976 were s u b j e c t e d t o n u m e r i c a l t a x o n o m i c a n a l y s i s . I n a d d i t i o n , r e s u l t s o f t h e r e l e v a n t c h a r a c t e r s s t u d i e d f o r t h e 26 Pseudomonas s p e c i e s a s d e s c r i b e d i n t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y (1974) were a l s o i n c l u d e d . The 26 s p e c i e s i n c l u d e d were: P^ a e r u g i n o s a ; P^ p u t i d a ; P. f l u o r e s c e n s ; P^ c h l o r o r a p h i s ; P_^  c i c h o r i i ; s t u t z e r i ; P. m e n d o c i n a ; P^ a l c a l i g e n e s ; P_^  p s e u d o a l c a l i g e n e s ; P. p s e u d o m a l l e i ; P_;_ m a l l e i ; P_j_ c a r y o p h y l l i ; P_;_ c e p a c i a ; P. m a r g i n a t a ; P_^  l e m o i g n e i ; P_j_ t e s t o s t e r o n i ; P^ a c i d o v o r a n s ; P. d e l a f i e l d i i ; P^ s o l a n a c e a r u m ; P_j_ f a c i l i s ; P_^  s a c c h a r o p h i l a ; P_^  r u h l a n d i i ; P_^  f l a v a and P^ p a l l e r o n i . F o r P^ f l u o r e s c e n s , t h r e e b i o t y p e s were a l s o s t u d i e d . The s p e c i e s E\ m a l t o p h i l i a , P_^  v e s i c u l a r i s and P. d i m i n u t a were not s u b j e c t e d t o t h i s a n a l y s i s b e c a u s e t h e s e s p e c i e s r e q u i r e one o r more g r o w t h f a c t o r s w h i l e no g r o w t h f a c t o r was r e q u i r e d by any o f t h e m i l k i s o l a t e s . 51 The c h a r a c t e r d a t a were c o d e d i n a t w o - s t a t e c o d i n g f o r m a t ; a p o s i t i v e (+) r e a c t i o n was s c o r e d as 1, and a n e g a t i v e (-) r e a c t i o n as 0. A d a t a m a t r i x c o n s i s t i n g of 49 b a c t e r i a l s t r a i n s s c o r e d f o r 52 c h a r a c t e r s was o b t a i n e d . The s i m p l e m a t c h i n g c o e f f i c i e n t s ( S g M ) between any p a i r of b a c t e r i a l s t r a i n s were computed and t h e s t r a i n s were c l u s t e r e d by u n w e i g h t e d p a i r - g r o u p a v e r a g e - l i n k a g e a n a l y s i s ( S n e a t h and S o k a l , 1973). The a n a l y s i s and t h e p r i n t i n g o f t h e d e n d r o g r a m were p e r f o r m e d on t h e M i c h i g a n T e r m i n a l S ystem (MTS) Amdahl 470 V/8 computer a t t h e 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 w i t h t h e NT-SYS p r o g r a m (1980) o b t a i n e d f r o m t h e S t a t e U n i v e r s i t y o f New York a t S t o n y B r o o k . F. H y d r o l y t i c c h a r a c t e r i s t i c s 1) P r o t e i n a s e p r o d u c t i o n a t 4 C, 22 C and 32 C A b i l i t y of i s o l a t e s t o p r o d u c e zones o f c l e a r i n g on s k i m m i l k a g a r a t 4 C (7 - 10 d ) , 22 C (2 d) and 32 C (2 d) were examined by s u r f a c e p l a t i n g on s k i m m i l k a g a r f o l l o w e d by i n c u b a t i o n a t t h e a p p r o p r i a t e t e m p e r a t u r e . 2) G e l a t i n a s e p r o d u c t i o n a t 4 C and 22 C A b i l i t y o f t h e i s o l a t e s t o h y d r o l y z e g e l a t i n c h a r c o a l d i s c a t 4 C (30 d) and 22 C ( 1 4 - 2 0 d) were e v a l u a t e d . D a t a f r o m b u t t e r f a t , Tween 80 and l e c i t h i n h y d r o l y s i s s t u d i e s (22 C) were a l s o i n c l u d e d i n t h i s e v a l u a t i o n . 52 G. P r o t e o l y t i c pseudomonads i n c u b a t e d i n s t e r i l e m i l k Growth and p r o t e i n a s e a c t i v i t y o f e i g h t p r o t e o l y t i c pseudomonads i s o l a t e d from raw m i l k were s t u d i e d . The e i g h t i s o l a t e s s t u d i e d were s t r a i n s 0-0, 2-1, 8-1, 10-0, 2-2, 4-1, 6-3, and 8-2. P r i o r t o c u l t u r i n g , t h e e i g h t p r o t e o l y t i c i s o l a t e s were grown s e p a r a t e l y (22 C, 48h) i n n u t r i e n t b r o t h . The c u l t u r e s were t h e n t r a n s f e r r e d s e p a r a t e l y t o 150 ml s t e r i l i z e d (15 min, 121 C) 10% (w/v) r e c o n s t i t u t e d s k i m m i l k (RSM) t o g i v e a f i n a l p o p u l a t i o n o f 10 3 - 10" c f u / m l m i l k . Growth and enzyme a c t i v i t y were m o n i t o r e d o v e r a 19 day s t o r a g e p e r i o d a t 4 C w i t h s h a k i n g . Samples were w i t h d r a w n i m m e d i a t e l y a f t e r i n o c u l a t i o n and e v e r y 2 o r 3 d a y s d u r i n g t h e s t o r a g e p e r i o d f o r d e t e r m i n a t i o n o f b a c t e r i a l c o u n t s and p r o t e i n a s e a c t i v i t y . S e r i a l d i l u t i o n s o f m i l k i n 0.1% p e p t o n e b r o t h were d r o p p l a t e d on SMA f o l l o w e d by i n c u b a t i o n a t 4 C f o r 7 d a y s . P r o t e i n a s e a c t i v i t y f o r e a c h i s o l a t e was d e t e r m i n e d by t h e f l u o r e s c a m i n e method ( C a s t e l l e t a l . , 1979). On e a c h s a m p l i n g day, 2 ml o f t h e i n o c u l a t e d m i l k sample were mixed w i t h 2 ml o f 24% (w/v) t r i c h l o r a c e t i c a c i d ( T C A ) . A n o t h e r 10 ml sample were w i t h d r a w n and p l a c e d i n a t e s t t u b e . I t was t h e n i n c u b a t e d a t 40 C f o r 2 h. To p r e v e n t f u r t h e r b a c t e r i a l g r o w t h , 0.01 ml o f 2% (w/v) sodium a z i d e was ad d e d t o t h e m i l k sample. An i n c u b a t i o n t e m p e r a t u r e o f 40 C was employed b e c a u s e i t was f o u n d t o be t h e o p t i m a l t e m p e r a t u r e f o r most p r o t e i n a s e s from p s y c h r o t r o p h i c b a c t e r i a i s o l a t e d f r o m raw m i l k ( G e b r e - E g z i a b h e r e t a l . , 1980a). A f t e r i n c u b a t i o n t h e r e a c t i o n was s t o p p e d by a d d i n g an 53 e q u a l volume of 24% (w/v) TCA. The T C A - 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 was s e p a r a t e d from t h e p r e c i p i t a t e d c a s e i n by c e n t r i f u g a t i o n (10,000 x g, 5 m i n ) . The s u p e r n a t a n t was d e c a n t e d and f i l t e r e d t h r o u g h Whatman No. 54 f i l t e r p a p e r . A 0.1 ml p o r t i o n o f t h e f i l t r a t e was mixed w i t h 0.3 ml 3 M p o t a s s i u m * p h o s p h a t e d i b a s i c f o l l o w e d by t h e a d d i t i o n of 0.15 ml of 0.03% (w/v) f l u o r e s c a m i n e i n a c e t o n e . The m i x t u r e was i m m e d i a t e l y m i x e d . To a d j u s t t h e volume t o c u v e t t e s i z e , 3.0 ml of d i s t i l l e d w a ter were a d d e d . R e l a t i v e f l u o r e s c e n c e ( e x c i t a t i o n w a v e l e n g t h = 395 nm; e m i s s i o n w a v e l e n g t h = 480 nm) was d e t e r m i n e d "with an Aminco Bowman s p e c t r o f l u o r o m e t e r 4-8202 ( A m e r i c a n I n s t r u m e n t Co., S i l v e r S p r i n g , MD) w i t h a xenon lamp. F l u o r e s c e n c e i n t e n s i t i e s were c o n v e r t e d t o m i c r o m o l e s of f r e e amino g r o u p s p e r m i l l i l i t e r o f m i l k by a t y r o s i n e s t a n d a r d c u r v e . P r o t e o l y s i s was d e f i n e d as t h e i n c r e a s e i n t h e c o n c e n t r a t i o n of T C A - s o l u b l e f r e e amino g r o u p s p e r m i l l i l i t e r of m i l k a f t e r i n c u b a t i o n a t 40 C f o r 2 h. H. E l e c t r o p h o r e s i s The pH o f t h e 10% RSM s a m p l e s t a k e n d u r i n g s t o r a g e were a d j u s t e d t o 4.6 w i t h t r i c h l o r a c e t i c a c i d b u f f e r ( N a k a i e t a l . , 1964) t o p r e c i p i t a t e t h e c a s e i n s . The p r e c i p i t a t e was r e d i s s o l v e d t w i c e i n t h e b u f f e r and f r e e z e d r i e d . The c a s e i n s were s a v e d f o r e l e c t r o p h o r e s i s . An A t t o v e r t i c a l g e l e l e c t r o p h o r e s i s u n i t ( A t t o C o r p . , J a p a n ) was u s e d . A 7.5% s e p a r a t i o n g e l was p r e p a r e d by m i x i n g 54 2.33 g o f a c r y l a m i d e (Sigma, S t . L o u i s , MO) and 0.06 g N, N' -m e t h y l e n e - b i s - a c r y l a m i d e ( B i s ) ( B i o - R a d , Richmond, CA) w i t h 8 ml of water and 4 ml o f 0.38 M T r i s - H C l b u f f e r (pH 8.9). P o l y m e r i z a t i o n was i n d u c e d by t h e a d d i t i o n o f 16 ml of 1.07% (w/v) ammonium p e r s u l f a t e ( B i o - R a d , Richmond, CA) and 0.03% (w/v) TEMED (Sigma, S t . L o u i s , MO). U r e a and 2 - m e r c a p t o e t h a n o l were added t o a f i n a l c o n c e n t r a t i o n o f 7 M and 22 mM. A l a y e r of w a t e r was added t o promote p o l y m e r i z a t i o n . The s p a c e r g e l was p r e p a r e d by m i x i n g 0.5 g a c r y l a m i d e and 0.12 g B i s w i t h 5 ml of water and 2 ml o f 0.06 M T r i s - H C l b u f f e r (pH 6 . 7 ) . U r e a was added t o a c o n c e n t r a t i o n o f 7 M. P o l y m e r i z a t i o n was i n d u c e d by a d d i t i o n o f 3 ml of 0.004% (w/v) r i b o f l a v i n and 0.06% (w/v) TEMED. A f l u o r e s c e n t l i g h t was p l a c e d d i r e c t l y b e h i n d t h e e l e c t r o p h o r e s i s c e l l a f t e r a comb, t o fo r m t h e sample s l o t s , was i n s e r t e d . A f t e r 2 h t h e comb was removed and t h e s l o t s washed w i t h e l e c t r o d e b u f f e r (0.38 M g l y c i n e , 0.05 M T r i s pH 8.3) b e f o r e t h e samples were a p p l i e d . C a s e i n s amples ( 0 . 5 % , w/v) were s u s p e n d e d i n pH 6.8 b u f f e r (0.05 M T r i s - g l y c i n e , 7 M u r e a , 70 mM 2 - m e r c a p t o e t h a n o l ) . F o r t y m i c r o l i t e r s o f sample w e r e . i n t r o d u c e d i n t o e a c h sample w e l l . E l e c t r o p h o r e s i s was p e r f o r m e d w i t h a c u r r e n t o f 3 mA/cm o f g e l f o r a b o u t 4 h. The g e l was f i x e d and s t a i n e d w i t h C o o m a s s i e B l u e R-250 (0.05%, w/v; B i o - R a d , Richmond, CA) i n m e t h a n o l : w a t e r : a c e t i c a c i d (50:40:10 v / v ) f o r 1 h. The g e l s were d e s t a i n e d i n m e t h a n o l : w a t e r : a c e t i c a c i d (25:68:7 v/v) f o r a t l e a s t 24 h. The g e l s were d r i e d u n d e r vacuum (55 C, 1 h ) . 55 RESULTS AND DISCUSSION A. I d e n t i f i c a t i o n o f p r o t e o l y t i c pseudomonads i n raw m i l k The d e n d r o g r a m c o n s t r u c t e d f r o m t h e u n w e i g h t e d p a i r - g r o u p a v e r a g e - l i n k a g e c l u s t e r a n a l y s i s i s shown i n F i g . 8. S i n c e t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y (1974) u s e d a m o n o t h e t i c c l a s s i f i c a t i o n o f t h e 29 Pseudomonas s p e c i e s , no a t t e m p t was made t o compare t h a t c l a s s i f i c a t i o n w i t h t h e one shown i n F i g . 8. Emphasis was p u t on t h e c l u s t e r where t h e p r o t e o l y t i c pseudomonads r e s i d e d i n . The f i r s t c l u s t e r c o n s i s t e d of m i l k i s o l a t e s 0-0, 8-1, 10-6, 2-1, 2-2, 2-4, 6-0, B16, 10-1, and t h e r e f e r e n c e s t r a i n s P. f l u o r e s c e n s (ATCC 948) and P^ f l u o r e s c e n s b i o t y p e A (ATCC 17397). A l s o i n t h i s g r o u p were t h e s p e c i e s P^ f l u o r e s c e n s b i o t y p e A, a u r e o f a c i e n s , P. f l u o r e s c e n s b i o t y p e C, P. f l u o r e s c e n s b i o t y p e B and 1E\ c h l o r o r a p h i s as d e s c r i b e d i n t h e B e r g e y ' s Manual of D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) . T h e s e s p e c i e s formed a c l u s t e r a t t h e 82% S g M . The m i l k i s o l a t e s 4-1, 6-3, A14, 10-7, 8-2, 8-4, and 10-0 were i n a n o t h e r c l u s t e r a l o n g w i t h t h e r e f e r e n c e s t r a i n P^ f r a q i (ATCC 4 9 7 6 ) . T h i s c l u s t e r was d i s t i n c t and d i d n o t j o i n t h e o t h e r c l u s t e r s u n t i l t h e 67% s i m i l a r i t y l e v e l ( % S S M ) . A l l i s o l a t e s b e l o n g i n g t o t h e f i r s t c l u s t e r were f l u o r e s c e n t Pseudomonas s p e c i e s . P^ c h l o r o r a p h i s and P. a u r e o f a c i e n s were r e g a r d e d as P_^  f l u o r e s c e n s b i o t y p e s D a n d E r e s p e c t i v e l y by S t a n i e r e t a l . ( 1 9 6 6 ) . B e s i d e s p r o d u c i n g 55a F i g . 8. Dendrogram showing r e l a t i o n s h i p of p r o t e o l y t i c pseudomonads from raw m i l k . S t r a i n s with a s t e r i k s are the s t r a i n s as d e s c r i b e d i n the Bergey's Manual of Determinative B a c t e r i o l o g y (1974). •V. Mruginosa ap. putida I ap. (totsaxl I I •p. MBdecina p. putida •p. alcali9»n«i ap. paaudoalcaliganaa •p. taatoataroni •p. aeldovorana I I . ap. ruhlandi I I . - t — L op. pallaronil : I •P. lMBlgnal •p. aolanaeaarua •p. fluoraacana blotypa A I x ap. auraofaciana I 0-0 1 I I '.I •-1 I I 1 .1- 10-6 1 I 1 I . 2-1 1 I I 1 1.1 2-2 1 II I .LL- 2-4 t I .1-1 1 6-0 i •16 80 100 57 f l u o r e s c e n t p i g m e n t , P. c h l o r o r a p h i s p r o d u c e s a g r e e n i n s o l u b l e p i g m e n t ( c h l o r o r a p h i n ) whereas a u r e o f ac i e n s p r o d u c e s an o r a n g e d i f f u s i b l e p i g m e n t ( p h e n a z i n e - 1 - c a r b o x y l i c a c i d ) . T h e s e two p i g m e n t s were a b s e n t from a l l o f t h e m i l k i s o l a t e s on b o t h medium A and B o f K i n g e t a l . ( 1 9 5 4 ) . A l l t h e m i l k i s o l a t e s , r e s i d i n g i n t h e f i r s t c l u s t e r , s h a r e d t h e a b i l i t y t o use 25 o f t h e 38 compounds t e s t e d a s t h e s o l e s o u r c e s o f c a r b o n and n i t r o g e n . They c o u l d n o t u t i l i z e m a l t o s e , l a c t o s e , b u t y r a t e , p r o p y l e n e g l y c o l , p h e n o l as t h e s o l e s o u r c e o f c a r b o n , and n i c o t i n a t e and p a n t o t h e n a t e as t h e s o l e s o u r c e o f n i t r o g e n . E v e r y member o f t h e f i r s t c l u s t e r , i n c l u d i n g s t r a i n s d e s c r i b e d as i n t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) , c o u l d u s e t h e f o l l o w i n g compounds a s t h e s o l e s o u r c e o f c a r b o n : r i b o s e , g l u c o s e , mannose, t r e h a l o s e , g l u c o n i c a c i d , a c e t a t e , p r o p i o n a t e , s u c c i n a t e , m a l a t e , l a c t a t e , c i t r a t e , p y r u v a t e , m a n n i t o l , g l y c e r o l , a l a n i n e , v a l i n e and a r g i n i n e . They c o u l d n o t use m a l t o s e , l a c t o s e , p h e n o l as t h e s o l e s o u r c e o f c a r b o n , and n i c o t i n a t e as t h e s o l e s o u r c e of n i t r o g e n . A l l m i l k i s o l a t e s c o u l d use g a l a c t o s e as a c a r b o n s o u r c e whereas t h e s p e c i e s d e s c r i b e d as P_j_ f l u o r e s c e n s b i o t y p e A and P. c h l o r o r a p h i s , by S t a n i e r e t a l . ( 1 9 6 6 ) , c o u l d n o t . However P. f l u o r e s c e n s b i o t y p e s B, C, and a u r e o f a c i e n s c o u l d u t i l i z e g a l a c t o s e . The a b i l i t y t o d e n i t r i f y was d e m o n s t r a t e d by P. f l u o r e s c e n s b i o t y p e s B, C, and P_^  c h l o r o r a p h i s , b u t n o t by P. f l u o r e s c e n s b i o t y p e A and P^ a u r e o f a c i e n s ( S t a n i e r e t a l . , 58 1966). T h i s p r o p e r t y was n o t d e m o n s t r a t e d by a l l o f t h e m i l k i s o l a t e s . I t i s not s u r p r i s i n g t h a t t h e m i l k i s o l a t e s had a few n u t r i t i o n a l p r o p e r t i e s d i f f e r e n t f r o m t h o s e d e s c r i b e d by S t a n i e r e t a l . (1966) o r i n t h e B e r g e y ' s Manual of D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) . J u f f s (1973) r e p o r t e d t h a t 103 o f t h e 121 i s o l a t e s f r o m raw m i l k were u n a b l e t o d e n i t r i f y . A l t h o u g h u t i l i z a t i o n o f t r e h a l o s e was c o n s i d e r e d by S t a n i e r e t a l . (1966) t o be an i m p o r t a n t c h a r a c t e r i s t i c o f P_j_ f l u o r e s c e n s , n i n e p e r c e n t o f t h e i s o l a t e s J u f f s (1973) examined were u n a b l e t o u t i l i z e t r e h a l o s e . The i n t e r n a l s u b d i v i s i o n o f P_j_ f l u o r e s c e n s i s f a r from s a t i s f a c t o r y . The s u b d i v i s i o n i n b i o t y p e s i s p r i m a r i l y b a s e d on two s a l i e n t p r o p e r t i e s namely t h e s y n t h e s i s o f l e v a n f r o m s u c r o s e and c a p a c i t y f o r d e n i t r i f i c a t i o n . Some c h a r a c t e r s c o r r e l a t e f a i r l y w e l l w i t h t h e s e two, b u t t h e d e l i m i t a t i o n o f t h e b i o t y p e s i n some c a s e s i s r a t h e r u n c l e a r ( P a l l e r o n i , 1975). The s t r a i n s o f b i o t y p e A a r e c a p a b l e o f l e v a n s y n t h e s i s but n o t o f d e n i t r i f i c a t i o n . The s t r a i n s a s s i g n e d t o b i o t y p e B a r e p h e n o t y p i c a l l y more h e t e r o g e n o u s t h a n t h o s e of b i o t y p e A, and a r e c a p a b l e o f b o t h l e v a n f o r m a t i o n and d e n i t r i f i c a t i o n . The s t r a i n s i n c a p a b l e o f l e v a n p r o d u c t i o n b u t c a p a b l e o f d e n i t r i f i c a t i o n a r e a s s i g n e d t o b i o t y p e C. The b i o t y p e i s h e t e r o g e n o u s and c a n be d i v i d e d i n t o s e v e r a l s u b g r o u p s ( P a l l e r o n i and D o u d o r o f f , 1972). The g r o u p o f m i s c e l l a n e o u s s t r a i n s a s s i g n e d t o b i o t y p e G i s h e t e r o g e n o u s i n n u t r i t i o n a l p r o p e r t i e s . They a r e a l s o i n c a p a b l e o f d e n i t r i f i c a t i o n and l e v a n s y n t h e s i s . S t r a i n s o f o t h e r b i o t y p e s w h i c h have l o s t , by 59 m u t a t i o n , one o r b o t h o f t h e b a s i c p r o p e r t i e s o f c h a r a c t e r i z a t i o n w o u l d be p l a c e d i n t h i s b i o t y p e . The s p e c i e s P. c h l o r o r a p h i s was o r i g i n a l l y d e s c r i b e d as P_j_ f l u o r e s c e n s b i o t y p e D by S t a n i e r e t a l . ( 1 9 6 6 ) . S t r a i n s o f t h i s s p e c i e s a r e c a p a b l e o f l e v a n f o r m a t i o n a n d d e n i t r i f i c a t i o n . The s p e c i e s P. a u r e o f a c i e n s was o r i g i n a l l y a s s i g n e d as P^ f l u o r e s c e n s ' b i o t y p e E ( S t a n i e r e t a l . , 1 9 66). S t r a i n s o f t h i s s p e c i e s a r e c a p a b l e o f l e v a n p r o d u c t i o n b u t i n c a p a b l e o f d e n i t r i f i c a t i o n . The r e a s o n why P^ c h l o r o r a p h i s and P^ a u r e o f a c i e n s were g r a n t e d s p e c i e s s t a t u s was b e c a u s e t h e y p r o d u c e d v e r y c h a r a c t e r i s t i c p h e n a z i n e p i g m e n t s ( S t a n i e r e t a l . , 1966). N u c l e i c a c i d homology s t u d i e s showed t h a t t h e s e two s p e c i e s s h a r e d a h i g h l e v e l o f DNA homology ( P a l l e r o n i and D o u d o r o f f , 1972). L i u ( 1 9 6 0 ) , i n a s e r o l o g i c a l s t u d y on t h e e x t r a c e l l u l a r a n t i g e n s o f Pseudomonas s t r a i n s , f o u n d t h a t P^ a u r e o f a c i e n s and P. c h l o r o r a p h i s showed e x t e n s i v e c r o s s - r e a c t i o n s , w h i c h s u g g e s t e d a c l o s e r e l a t i o n s h i p among t h e s e s p e c i e s . P. f l u o r e s c e n s i s p r o b a b l y t h e most complex s p e c i e s o f t h e genus Pseudomonas. Most e x t e n s i v e s t u d i e s done on P^ f l u o r e s c e n s were o f s o i l , p l a n t o r w a t e r o r i g i n . I n f o r m a t i o n on P_^  f l u o r e s c e n s i n m i l k i s s t i l l l a c k i n g . The c l a s s i f i c a t i o n of t h e Pseudomonas s p e c i e s p r e s e n t e d i n t h i s t h e s i s i s s t i l l i n c o m p l e t e . A s t u d y o f more c h a r a c t e r s a n d on a d d i t i o n a l s t r a i n s w o u l d p e r h a p s r e s u l t i n a more p r e c i s e c l a s s i f i c a i o n o f i s o l a t e s and t h e c r e a t i o n o f new s u b g r o u p s . Above a l l , d a t a f r o m n u c l e i c a c i d homology s t u d i e s w i l l p r o v i d e more v a l u a b l e i n f o r m a t i o n t o t h e c u r r e n t c h a r a c t e r i z a t i o n p r e s e n t e d h e r e . 60 A l l t h e m i l k i s o l a t e s r e s i d i n g i n t h e c l u s t e r c o n t a i n i n g t h e r e f e r e n c e f r a g i (ATCC 4976) d i d n o t p r o d u c e any f l u o r e s c e n t p i g m e n t . They were a l l a b l e t o u t i l i z e x y l o s e , m a l a t e , l a c t a t e , c i t r a t e , p y r u v a t e , a l a n i n e , s e r i n e , v a l i n e and a r g i n i n e as a s o l e c a r b o n s o u r c e . They c o u l d n o t u t i l i z e p a n t o t h e n a t e as t h e s o l e n i t r o g e n s o u r c e . The n u t r i t i o n a l s p e c t r u m o f t h i s c l u s t e r was not a s b r o a d as t h e f l u o r e s c e n s c l u s t e r . They c o u l d not u t i l i z e rhamnose, m a l t o s e , l a c t o s e , p r o p i o n a t e , b u t y r a t e , i n o s i t o l , a d o n i t o l , p r o p y l e n e g l y c o l , m e t h a n o l and e t h a n o l . Of a l l t h e s t r a i n s i n t h i s c l u s t e r , o n l y one c o u l d d e n i t r i f y . The P_;_ f r a g i r e l a t e d m i l k i s o l a t e s were a l l a b l e t o breakdown m i l k c a s e i n s a t 4 C and h y d r o l y z e b u t t e r f a t . C o l o n i e s formed on s k i m m i l k a g a r by members of t h i s c l u s t e r were more s l i m y i n a p p e a r a n c e t h a n t h o s e o f P. f l u o r e s c e n s s p e c i e s . P. f r a g i i s n o t a s s i g n e d a s a Pseudomonas s p e c i e s i n t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y ( 1 9 7 4 ) . I t i s l i s t e d u n der addendum I t o t h e g e n u s . In t h e s e v e n t h e d i t i o n o f t h e B e r g e y ' s Manual ( B r e e d e t a l . , 1957) P^ f r a g i was d e s c r i b e d a s p o s s e s s i n g t h e f o l l o w i n g c h a r a c t e r i s t i c s : no p r o d u c t i o n o f s o l u b l e p i g m e n t s , l i q u e f a c t i o n o f g e l a t i n , g r o w t h a t 25 C but n o t a t 37 C, a b s e n c e o f t h e n i t r a t e r e d u c t i o n p r o p e r t y , and a c i d p r o d u c t i o n from g l u c o s e , g a l a c t o s e and a r a b i n o s e . The s o u r c e s o f i s o l a t i o n were d e s c r i b e d as f r o m m i l k and o t h e r d a i r y p r o d u c t s , d a i r y u t e n s i l s and w a t e r . T h i s s t u d y showed t h a t h a l f o f t h e p r o t e o l y t i c m i l k pseudomonads b e l o n g e d t o t h e P_j_ f l u o r e s c e n s g r o u p and t h e o t h e r 61 h a l f t o t h e P_^  f r a g i g r o u p . M u i r e t a l . (1979) r e p o r t e d t h a t on a v e r a g e 43% o f m i l k pseudomonads were o f t h e f l u o r e s c e n t t y p e . C h a r a c t e r i z a t i o n of t h e s e f l u o r e s c e n t s t r a i n s were not done. J u f f s (1973) r e p o r t e d t h a t 72% o f t h e pseudomonads i n raw m i l k were f l u o r e s c e n s . Samagh and Cunningham (1972) r e p o r t e d t h a t o f t h e 182 pseudomonads s t u d i e d , 98 s t r a i n s were i d e n t i f i e d a s P. f l u o r e s c e n s and P^ a e r u g i n o s a - 1 i k e o r g a n i s m s . I s o l a t i o n o f P. f r a g i - l i k e o r g a n i s m s f r o m m i l k was o n l y r e p o r t e d by a few r e s e a r c h e r s . Law e t a l . (1976) r e p o r t e d t h a t t h e h i g h e s t i n c i d e n c e o f l i p o l y t i c a c t i v i t y among t h e p s y c h r o t r o p h i c Gram-n e g a t i v e f l o r a o f c o m m e r c i a l raw m i l k was f o u n d i n s t r a i n s o f P. f l u o r e s c e n s and f r a g i . Samagh and Cunningham (1972) s u s p e c t e d t h a t 41 n o n - f l u o r e s c e n t c u l t u r e s ( o u t o f 182 pseudomonads) were r e l a t e d t o I*\ f r a g i and p u t r e f a c i e n s . They d i d n o t i n c l u d e t h e s e s t r a i n s a s r e f e r e n c e s i n t h e i r s t u d y . I s o l a t i o n of P_^  f r a g i from m i l k was a l s o r e p o r t e d by O v e r c a s t ( 1 9 6 8 ) . The f i n d i n g o f t h e d i s t i n c t c l u s t e r c o n s i s t i n g of t h e m i l k i s o l a t e s 4-1, 6-3, A14, 10-7, 8-4, 8-2, 10-0, and t h e r e f e r e n c e s t r a i n P^ f r a g i (ATCC 4976) i s q u i t e i n t e r e s t i n g . Taxonomic s t u d y o f P_^  f r a g i - l i k e o r g a n i s m s from m i l k has n o t been p u b l i s h e d . P^ f r a g i i s t h e majo r pseudomonad r e s p o n s i b l e f o r meat s p o i l a g e . Shaw and L a t t y (1982) p e r f o r m e d a n u m e r i c a l t a x o n o m i c s t u d y o f Pseudomonas s t r a i n s f r o m s p o i l e d meat. The Pseudomonas s t r a i n s were shown t o r e s i d e i n f o u r c l u s t e r s . Non-f l u o r e s c e n t s t r a i n s were c o n t a i n e d i n two c l o s e l y r e l a t e d c l u s t e r s w h i c h were i d e n t i f i e d w i t h P^ f r a g i (NCIB 8 5 4 2 ) . The 62 o t h e r two c l u s t e r s were d i s t i n c t f r o m f l u o r e s c e n s (NCIB 9 0 4 6 ) , P_j_ p u t i d a (NCIB 9494) and o t h e r r e f e r e n c e s t r a i n s e x a m i n e d . A l l members of t h e f o u r c l u s t e r s p r o d u c e d a c i d o x i d a t i v e l y , grew a t 4 C b u t n o t a t 41 C, d i d n o t a c c u m u l a t e p o l y - / 3 - h y d r o x y b u t y r a t e , d i d n o t d e n i t r i f y and d i d n o t p r o d u c e l e v a n from s u c r o s e . I t i s q u i t e s u r p r i s i n g t h a t t h e r e f e r e n c e P. f l u o r e s c e n s s t r a i n (NCIB 9046, b i o t y p e A) t h a t t h e y e x amined d i d n o t p r o d u c e l e v a n f r o m s u c r o s e . The P_j_ f r a g i - l i k e s t r a i n s were a l l n o n - f l u o r e s c e n t , a b l e t o h y d r o l y z e g e l a t i n (20 C) but d i d n o t show p h o s p h o l i p a s e a c t i v i t y . The two c l u s t e r s , c o n s i s t i n g o f P_j_ f r a g i - l i k e s t r a i n s , were d i s t i n g u i s h e d f r o m one a n o t h e r by t h e i r p a t t e r n o f c a r b o n s o u r c e u t i l i z a t i o n . Most o f t h e members i n t h e c l u s t e r , where t h e r e f e r e n c e s t r a i n P^ f r a g i (NCIB 8542) r e s i d e d i n , c o u l d h y d r o l y z e c a s e i n b u t c o u l d n o t u t i l i z e g a l a c t o s e as s o l e c a r b o n s o u r c e . Members of t h e o t h e r c l u s t e r , r e g a r d e d as a b i o t y p e o f P. f r a g i , were a b l e t o u t i l i z e g a l a c t o s e but few c o u l d h y d r o l y z e c a s e i n . O n l y t h r e e s t r a i n s , f r o m t h e two c l u s t e r s , c o u l d h y d r o l y z e Tween 80 (Shaw and L a t t y , 1982). R e s u l t s p r e s e n t e d i n t h e p r e s e n t s t u d y c o n f i r m t h e h e t e r o g e n o u s n u t r i t i o n a l p r o p e r t i e s o f t h e P^ f r a g i g r o u p . O n l y one s t r a i n i n t h i s s t u d y c o u l d d e n i t r i f y and few c o u l d use g a l a c t o s e as s o l e c a r b o n s o u r c e . L y s e n k o (1961) a t t e m p t e d t o c h a r a c t e r i z e 126 s t r a i n s r e p r e s e n t i n g 46 d i f f e r e n t Pseudomonas s p e c i e s . The s t r a i n s were i s o l a t e d m a i n l y f r o m humans, a n i m a l s , p l a n t s , s o i l and w a t e r . L y s e n k o ' s (1961) r e s u l t s i n d i c a t e d t h a t P\ f r a g i was d i s t i n c t 63 f r o m f l u o r e s c e n s , P. p u t i d a , and P_j_ a e r u g i n o s a . The s p e c i e s P. f r a g i showed t h e f o l l o w i n g c h a r a c t e r i s t i c s : g r o w t h a t 5 C but n o t a t 42 C, a b s e n c e o f f l u o r e s c e n t p i g m e n t s , i n a b i l i t y t o h y d r o l y z e c a s e i n and g e l a t i n a t 28 C, a b i l i t y t o h y d r o l y z e o l i v e o i l , l a c k o f d e n i t r i f i c a t i o n p r o p e r t y , u t i l i z a t i o n o f g a l a c t o s e and c i t r a t e a s c a r b o n s o u r c e s , f a i l u r e t o use g l u c o s e , l a c t o s e , m a l t o s e and l a c t a t e as s o l e c a r b o n s o u r c e . An i d e n t i f i c a t i o n key t o t h e s p e c i e s P_^  f r a g i i s not g i v e n i n t h e e i g t h e d i t i o n o f t h e B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y (1974) nor i n t h e d i a g n o s t i c t a b l e s o f . Cowan and S t e e l ( 1 9 6 5 ) . C h a r a c t e r i z a t i o n of P_;_ f r a g i - l i k e s t r a i n f r o m raw m i l k would be i m p o s s i b l e u n l e s s i d e n t i f i c a t i o n k e y s o t h e r t h a n B e r g e y ' s Manual (1974) o r Cowan and S t e e l (1965) a r e u s e d . R e s u l t s p r e s e n t e d h e r e w a r r a n t f u t u r e r e s e a r c h on t h e t a x o n o m i c s t a t u s o f P_^  f r a g i , e s p e c i a l l y f r o m f o o d s o u r c e s . T a b l e 2 shows some h y d r o l y t i c c h a r a c t e r i s t i c s o f t h e m i l k i s o l a t e s . The m i l k i s o l a t e s i d e n t i f i e d w i t h P^ f l u o r e s c e n s were a l l c a p a b l e o f h y d r o l y z i n g m i l k p r o t e i n s a t 4 C and 22 C. T h i s p r o p e r t y was d e m o n s t r a t e d a t 32 C by a l l t h e m i l k i s o l a t e s e x c e p t s t r a i n 10-1. G e l a t i n a s e a c t i v i t y a t 22 C was shown by a l l b u t one o f t h e i s o l a t e s . However h y d r o l y s i s o f g e l a t i n a t 4 C was o n l y e x h i b i t e d by s t r a i n s 10-6, 2-1, 2-4, 6-0, and B16. A l l t h e i s o l a t e s a l s o showed l i p a s e and p h o s p h o l i p a s e a c t i v i t i e s a t 22 C. The i s o l a t e s i d e n t i f i e d w i t h P^ f r a g i were. somewhat d i f f e r e n t i n c h a r a c t e r . P r o t e o l y s i s was d e t e c t e d w i t h a l l i s o l a t e s a t 4 C b u t n o t a t 32 C. The m a j o r i t y o f t h e s e i s o l a t e s Table 2. Bydrolytic characteristics of the milk pseudomonads C H A R A C T E R I S T I C S S T R A I N S MILK PROTEINS HYDROLYSIS (4*C) MILK PROTEINS HYDROLYSIS (22«C) MILK PROTEINS HYDROLYSIS (32«C) GELATIN HYDROLYSIS (4*C) GELATIN HYDROLYSIS (22»C) BUTTERFAT HYDROLYSIS (22*C) TWEEN 80 HYDROLYSIS (22*C) LECITHIN HYDROLYSIS (22*C) to *d *d M • • 8 M *| •v a a W O O H) _ _ f > S M S t-> M 0 tn o K J K J K J w o w —v c> O C D O D O * 1 ) 1 i i i i i i t» 8 i 8 o i i >> i i i i 5 _ H Z Z f— M O O H < T I I - » U _ » O O « w M w o H W ^ - J & O J O H + +•• + + + • + + + + + 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . + + + + + + + + • + + • + 4 . - . 4 . 4 . . 4 . + + + + + + + • . . . _ _ _ _ _ _ _ _ _ - - + + - + + + - - - - _ + _ - + + . _ _ + + + + + + - + 4- + + - -+ + • + + + +• + + + + 4- 4 . 4 . 4 . 4 . 4 . 4 . 4 . 4 . + • + 4- + + + + - +• + 4- - - 4 . - 4 . - 4 . . + + + +•+• + + + - + + - _ _ _ _ _ _ _ _ 65 showed p r o t e i n a s e a c t i v i t y a t 22 C. G e l a t i n a s e a c t i v i t y was d e t e c t e d w i t h t h r e e of t h e i s o l a t e s a t 4 C but none were g e l a t i n a s e p o s i t i v e a t 22 C. T h e s e f r a g i - l i k e i s o l a t e s showed l i p a s e a c t i v i t y but no p h o s p h o l i p a s e a c t i v i t y . From t a b l e 2, we c a n see t h a t t h e n a t u r e o f t h e s u b s t r a t e and t h e c o n d i t i o n s under w h i c h t h e t e s t i s b e i n g c o n d u c t e d a r e c r i t i c a l . I f p r o t e i n a s e a c t i v i t y was t e s t e d a t 32 C, a l l t h e P. f r a g i - l i k e i s o l a t e s would be n e g a t i v e . P_;_ f r a g i - l i k e s t r a i n s would be r e g a r d e d as n o n - p r o t e o l y t i c , i f g e l a t i n was u s e d i n s t e a d o f m i l k p r o t e i n s as t h e s u b s t r a t e w i t h an i n c u b a t i o n t e m p e r a t u r e o f 22 C. The P^ f r a g i r e l a t e d m i l k i s o l a t e s were a l l a b l e t o h y d r o l z e b u t t e r f a t b u t not Tween 80. Numerous s t u d i e s on t h e l i p a s e a c t i v i t y o f P_j_ f r a g i have been r e p o r t e d ( N a s h i f and N e l s o n , 1953; Law e t a l . , 1976; G r i f f i t h s e t a l . , 1981). However o n l y s t r a i n s A14, 8-4, and 10-0 were a b l e t o h y d r o l y z e Tween 80. T h i s a g r e e s w i t h Shaw and L a t t y (1982) t h a t few o f t h e P^ f r a g i - l i k e s t r a i n s were a b l e t o h y d r o l y z e Tween 80. I t i s a p p a r e n t t h a t b u t t e r f a t h y d r o l y s i s i s a b e t t e r t e s t f o r t h e p r e s e n c e o f l i p a s e t h a n h y d r o l y s i s o f Tween 80. The c h a r a c t e r i s t i c s d i s c u s s e d and t a b u l a t e d i n t h i s s t u d y a r e f o r c o l l e c t i o n o f i n f o r m a t i o n on t h e g e n e r a l p r o p e r t i e s o f t h e s e m i l k i s o l a t e s . More work on a l a r g e r c o l l e c t i o n o f i s o l a t e s w i l l be n e c e s s a r y b e f o r e a key f o r t h e d i f f e r e n t i a t i o n o f t h e c l u s t e r s o f m i l k i s o l a t e s c a n be d e t e r m i n e d . I t s h o u l d be remembered t h a t t h e s t r u c t u r e o f a d e t e r m i n a t i v e key may change when new s p e c i e s o r s p e c i e s f r o m o t h e r o r i g i n s a r e i n c l u d e d . 66 R i c h a r d s o n (1981) c l a s s i f i e d t h e m i l k i s o l a t e as P. f l u o r e s c e n s B52 b e c a u s e i t c o u l d u t i l i z e t r e h a l o s e and 0-a l a n i n e as s o l e c a r b o n s o u r c e , and i t c o u l d grow a t 4 C but not a t 41 C. A l l P_^  f l u o r e s c e n s r e l a t e d i s o l a t e s i n t h i s s t u d y c o u l d use t r e h a l o s e a s t h e s o l e c a r b o n s o u r c e . J u f f s (1973) r e p o r t e d t h a t 91% o f t h e P_;_ f l u o r e s c e n s r e l a t e d s t r a i n s c o u l d u t i l i z e t r e h a l o s e . O t h e r s t u d i e s i n d i c a t e d t h a t 67% (Shaw and L a t t y , 1982) and 52% ( K l i n g e , 1960) o f t h e s t r a i n s c o u l d u t i l i z e t r e h a l o s e as a c a r b o n s o u r c e . S t r a i n s l a c k i n g t h e a b i l i t y t o grow on t r e h a l o s e w i l l n o t be a s s i g n e d t o t h e P_j_ f l u o r e s c e n s g r o u p . A c c o r d i n g t o S t a n i e r e t a l . (1966) t h e s p e c i e s P. a e r u g i n o s a i s e a s i l y d i s t i n g u i s h e d f r o m o t h e r f l u o r e s c e n t pseudomonads. The c h a r a c t e r s most commonly u s e d f o r i t s r e c o g n i t i o n a r e : p y o c y a n i n e p r o d u c t i o n ; g r o w t h a t 41 C b u t n o t a t 4 C; and i t s n a r r o w e r n u t r i t i o n a l s p e c t r u m a s compared t o P. f l u o r e s c e n s . P. a e r u g i n o s a s t r a i n s a r e r e m a r k a b l y u n i f o r m i n p h e n o t y p i c p r o p e r t i e s and i n n u c l e i c a c i d homology ( S t a n i e r e t a l . , 1966). Many phages a r e s p e c i f i c f o r t h e s t r a i n s o f t h i s s p e c i e s . However, Samagh and Cunningham (1972) r e p o r t e d an a e r u g i n o s a - l i k e g r o u p w h i c h was r e l a t e d t o P_j_ f l u o r e s c e n s . They c o n c l u d e d t h a t members o f t h i s g r o u p were p s y c h r o t r o p h i c a e r u g i n o s a - l i k e o r g a n i s m s t h a t l o s t t h e a b i l i t y t o grow a t 41 C and t o p r o d u c e p y o c y a n i n e . F u t u r e r e s e a r c h i s needed t o c o n f i r m t h e p r e s e n c e o f P_j_ a e r u g i n o s a - l i k e s t r a i n s . P. p u t i d a , a s d e s c r i b e d by v a r i o u s a u t h o r s , i s not a b l e t o d e m o n s t r a t e p r o t e i n a s e and l i p a s e a c t i v i t i e s (Rhodes, 1959; 67 L y s e n k o , 1961; S t a n i e r e t a l . , 1966). However s t u d i e s on t h e p r o t e i n a s e a c t i v i t y o f P_j_ p u t i d a s t r a i n s i s o l a t e d f r o m raw m i l k have been p u b l i s h e d (Law e t a l . , 1979 a, b ) . Ej\ p u t i d a r e l a t e d s t r a i n s i s o l a t e d f r o m raw m i l k were r e p o r t e d t o be non-p r o t e o l y t i c ( J u f f s , 1973). Shaw and L a t t y (1982) o b s e r v e d t h e same p r o p e r t y f r o m t h e i r s t r a i n s i s o l a t e d f r o m meat. Whether a f a l s e i d e n t i f i c a t i o n had been made o r a mutant o f t h e s p e c i e s P. p u t i d a was i s o l a t e d f r o m m i l k r e m a i n s t o be s e e n . N e v e r t h e l e s s c a r e must be t a k e n when an i d e n t i f i c a t i o n key i s needed f o r r a p i d c h a r a c t e r i z a t i o n . o B. G rowth o f p r o t e o l y t i c pseudomonads i n s t e r i l e m i l k E i g h t p r o t e o l y t i c i s o l a t e s , f r o m raw m i l k , i d e n t i f i e d t o be Pseudomonas s p e c i e s were grown i n 10% RSM a t 4 C. P r o t e i n a s e a c t i v i t i e s were d e t e c t e d i n 10% RSM i n o c u l a t e d w i t h s t r a i n s 0-0, 2-1, 8-1, and 8-2 when t h e b a c t e r i a l p o p u l a t i o n s r e a c h e d 10 8 -10 9 c f u / m l ( F i g . 9 a, c, d and F i g . 1 0 c ) . No p r o t e i n a s e a c t i v i t y was d e t e c t e d from s t r a i n s 4-1, 6-3, and 10-0 even t h o u g h t h e p r o t e o l y t i c p o p u l a t i o n s r e a c h e d 1 0 s c f u / m l ( F i g . 10 a, b, d ) . A s l i g h t i n c r e a s e i n p r o t e i n a s e a c t i v i t y was d e t e c t e d f r o m s t r a i n 2-2 a f t e r 19 d a t 4 C ( F i g . 9 b ) . I t i s e v i d e n t t h a t b a c t e r i a l c o u n t a l o n e c a n n o t i n d i c a t e when p r o t e o l y s i s w i l l o c c u r . R e p o r t s on p o p u l a t i o n s n e e d e d t o d e t e c t p r o t e o l y s i s v a r i e d , r a n g i n g from 1 0 5 - 10 9 c f u / m l . No r e l a t i o n s h i p between t h e l e v e l of b a c t e r i a l p o p u l a t i o n and p r o t e o l y t i c a c t i v i t y was a l s o o b s e r v e d by E l l i o t e t a l . ( 1 9 7 4 ) , Law ( 1 9 7 9 ) , C o u s i n (1982) 67a F i g . 9. Growth and p r o t e i n a s e a c t i v i t y ( A ) i n 10% RSM i n o c u l a t e d with (a) I s o l a t e 0-0; (b) I s o l a t e 2-1; (c) I s o l a t e 2-2; (d) I s o l a t e 8-1. T o t a l p s y c h r o t r o p h s ( • ) ; p r o t e o l y t i c psychrotrophs ( • ). P r o t e i n a s e a c t i v i t y i s d e f i n e d as the i n c r e a s e i n the c o n c e n t r a t i o n of TCA-soluble f r e e amino groups per m i l l i l i t e r of milk a f t e r i n c u b a t i o n a t 40 C f o r 2 h. PROTEINASE ACTIVITY C JJ. m o l e s / m l mi lk ) I1" / ndD . O O l 68a F i g . 10. Growth and proteinase a c t i v i t y ( A ) in 10% RSM inoculated with (a) Isolate 4-1; (b) Isolate 6-3; (c) Isolate 8-2; (d) Isolate 10-0. Total psychrotrophs ( • ) ; p r o t e o l y t i c psychrotrophs ( • ). Proteinase a c t i v i t y i s defined as the increase in the concentration of TCA-soluble free amino groups per m i l l i l i t e r of milk af t e r incubation at 40 C for 2 h. cn PROTEINASE ACTIVITY ( ji. mo Ies / ml milk) CN O k—. o 0» 00 CN CN - M l O O CN O t o CN CN CN O CN CO — o o-o CN CN < CO co ))I!UJ | U J / ndD O O T 70 and C l i f f e and Law ( 1 9 8 2 ) . P r o t e o l y s i s was n o t d e t e c t e d f r o m s t r a i n s 4-1, 6-3, and 10-0 b e c a u s e t h e y e i t h e r had n o t p r o d u c e d s u f f i c i e n t p r o t e i n a s e s t h a t c o u l d be d e t e c t e d by t h e f l u o r e s c a m i n e method o r t h e y had no t s t a r t e d t o p r o d u c e p r o t e i n a s e s . I t has been shown t h a t t h e k e e p i n g q u a l i t y of m i l k i s d e p e n d e n t on t h e number of b a c t e r i a p r e s e n t i n t h e m i l k , l e n g t h o f t h e l a g phase o f g r o w t h , r a t e o f g r o w t h a t s t o r a g e t e m p e r a t u r e and t y p e of b a c t e r i a p r e s e n t ( S m i t h e t a l . , 1972). A l o n g e r s t o r a g e t i m e a t 4 C i s n e e d e d b e f o r e .a marked i n c r e a s e i n p r o t e i n a s e a c t i v i t y c o u l d be d e t e c t e d f r o m s t r a i n 2-2. E l e c t r o p h o r e g r a m s showed t h a t /3-casein was more s u s c e p t i b l e t o p r o t e o l y s i s c a u s e d by s t r a i n s 0-0, 2-1, 8-1, and 8-2 ( F i g . 11 and 1 2 ) . E x t e n s i v e breakdown o f 0 - and a - c a s e i n s were d e m o n s t r a t e d by s t r a i n s 0-0, 2-1, and 8-1 ( F i g . 11 and 12), w h i c h a l s o showed t h e l a r g e s t i n c r e a s e i n p r o t e i n a s e a c t i v i t y ( F i g . 9 ) . S t r a i n s 2-2, 4-1, 6-3, and 10-0 d i d n o t c a u s e any n o t i c e a b l e c h a n g e s on t h e a - and 0 - c a s e i n s ( F i g . 1 3 ) . T h i s i s t h e r e a s o n why no p r o t e i n a s e a c t i v i t i e s , u s i n g t h e f l u o r e s c a m i n e method, were d e t e c t e d f r o m t h e s e s t r a i n s . M i l k p r o t e i n d e g r a d a t i o n by p r o t e o l y t i c p s y c h r o t r o p h s has been e x t e n s i v e l y r e v i e w e d (Law, 1979; C o u s i n , 1982). G e b r e -E g z i a b h e r e t a l . ( 1980 b) r e p o r t e d t h a t K- and /3-caseins were more s u s c e p t i b l e t o p r o t e o l y s i s by Pseudomonas s p e c i e s w h i l e t h e a s ~ c a s e i n was l e s s a f f e c t e d . A t t h e end o f 15 d a y s a t 7 C, e l e c t r o p h o r e s i s showed no r e m a i n i n g t r a c e s o f K- and 0 - c a s e i n s w h i l e a c o n s i d e r a b l e l o s s o f a s - c a s e i n . Law e t a l . (1977) 7 0a F i g . 11. E l e c t r o p h o r e t i c p r o f i l e s of milk p r o t e i n s caused by i s o l a t e s 0-0 and 2-1 a f t e r 0, 6, 16 and 19 days of storage i n 10% RSM at 4 C. DAY 0-0 0 6 16 19 ORIGIN p - Casein *s" Casein 71a F i g . 12. E l e c t r o p h o r e t i c p r o f i l e s of milk p r o t e i n s caused by i s o l a t e s 8-1 and 8-2 a f t e r 0, 6, 16 and 19 days of storage i n 10% RSM a t 4 C. 8-1 DAY 0 6 16 ORIGIN p-Casein « s- Casein 72a F i g . 13. E l e c t r o p h o r e t i c p r o f i l e s of milk p r o t e i n s caused by i s o l a t e s 4-1; 6-3; 10-0 and 2-2 a f t e r 0, 16 and 19 days of storage i n 10% RSM a t 4 C. ORIGIN p - Casein *s" Casein GO 74 s t u d i e d g e l a t i o n o f u l t r a - h i g h - t e m p e r a t u r e - s t e r i l i z e d m i l k by p r o t e a s e s from f l u o r e s c e n s , and r e p o r t e d t h a t j3- and K-c a s e i n s were e x t e n s i v e l y d e g r a d e d i n g e l l e d m i l k s a m p l e s and some l o s s of a s ~ c a s e i n was s e e n . C o u s i n and M a r t h (1977) n o t e d t h a t Pseudomonas s p e c i e s h y d r o l y z e d b o t h a - and /3-caseins w h i l e K - c a s e i n was n o t a f f e c t e d . E l e c t r o p h o r e s i s showed b o t h a s - and /3-caseins d i s a p p e a r e d a f t e r 9 d a y s a t 7 C. D e B e u k e l a r e t a l . (1977) o b s e r v e d t h a t a s - and /3-caseins were d e g r a d e d i n skim m i l k i n o c u l a t e d w i t h Pseudomonas s p e c i e s . In t h e i r s t u d y on t h e c h a n g e s of m i l k p r o t e i n by Pseudomonas s p e c i e s , Y a n a g i y a e t a l . (1973) a l s o f o u n d t h a t a s ~ c a s e i n d i s a p p e a r e d a f t e r 14 days a t 5 C. 75 CONCLUSIONS In c o n c l u s i o n , t h e p r o t e o l y t i c pseudomonads i n m i l k c o u l d be c l a s s i f i e d i n t o two c l u s t e r s . The f i r s t c l u s t e r c o n t a i n e d P. f l u o r e s c e n s ( a t 82% S S M ) w h i l e t h e s e c o n d c o n t a i n e d P_j_ f r a g i ( a t 80% S S M ) . The i s o l a t e s i d e n t i f i e d w i t h P_^  f l u o r e s c e n s c o u l d : grow a t 4 C but n o t a t 41 C; h y d r o l y z e m i l k p r o t e i n s , b u t t e r f a t ; c o u l d p r o d u c e p h o s p h o l i p a s e . They p r o d u c e d d i f f u s i b l e p i g m e n t but f a i l e d t o d e n i t r i f y . They c o u l d n ot use l a c t o s e a s s o l e c a r b o n s o u r c e b u t c o u l d use g a l a c t o s e . The c l u s t e r c o n t a i n i n g P_^  f r a g i c o u l d : grow a t 4 C but n o t a t 41 C; h y d r o l y z e m i l k p r o t e i n s and b u t t e r f a t b u t p o s s e s s e d no p h o s p h o l i p a s e a c t i v i t y . No f l u o r e s c e n t p igment was p r o d u c e d and o n l y one s t r a i n c o u l d d e n i t r i f y . No members i n t h i s c l u s t e r c o u l d u t i l i z e l a c t o s e a s s o l e c a r b o n s o u r c e ' b u t few c o u l d use g a l a c t o s e . No r e l a t i o n s h i p was f o u n d between p r o t e o l y t i c p s y c h r o t r o p h s p o p u l a t i o n s and p r o t e o l y s i s i n m i l k s t o r e d a t 4 C. P r o t e i n a s e a c t i v i t i e s were d e t e c t e d , when p r o t e o l y t i c p s y c h r o t r o p h i c c o u n t s r e a c h e d 10 8 c f u / m l , from s t r a i n s 0-0, 2-1, 8-1, and 8-2 but n o t f r o m s t r a i n s 2-2, 4-1, 6-3, and 10-0. 76 REFERENCES Adams, D.M., B a r a c h , J . T . , and Speck, M.L. 1975. Heat r e s i s t a n t p r o t e a s e s p r o d u c e d i n m i l k by p s y c h r o t r o p h i c b a c t e r i a o f d a i r y o r i g i n . J . D a i r y S c i . 58:828. , A u d r e y , J . , J r . and F r a z i e r , W.C. 1959. P s y c h r o p h i l e s i n m i l k h e l d two d a y s i n farm m i l k t a n k s . J . D a i r y S c i . 42:1781. B a r r i t t , M. 1936. The i n t e n s i f i c a t i o n o f t h e Voges - P r o s k a u e r method by t h e a d d i t i o n of a l p h a - n a p h t h o l . J . P a t h . B a c t e r i o l . 42:441. Beech, F.W., C a r r , J.G., and C o d n e r , R.C. 1955. A m u l t i p o i n t i n o c u l a t o r f o r p l a t i n g b a c t e r i a o r y e a s t s . J . Gen. M i c r o b i o l . 13:408. B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y . 1974. 8 t h e d . Buchanan, E.R. and G i b b o n s , N.E. e d s . W i l l i a m s and W i l k i n s Co., B a l t i m o r e , MD. pp 217-243. B r e e d , R.S., M u r r a y , E.G.D., and S m i t h , N.R. 1957. I_n B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y . 7 t h e d . W i l l i a m s and W i l k i n s Co., B a l t i m o r e , MD. pp 90-152. C a s a s , I.A., L e o n , N., and I z q u i e r d o , P. 1977. M i c r o t i t e r t e c h n i q u e f o r e n u m e r a t i o n o f m e s o p h i l e s , p s y c h r o t r o p h s and c o l i f o r m s i n raw and p a s t e u r i z e d m i l k . J . F o o d P r o t . 40:795. C a s t e l l , J.V., C e r v e r a , M., a n d Marco, R. 1979. A c o n v e n i e n t m i c r o m e t h o d f o r t h e a s s a y o f p r i m a r y amines and p r o t e i n s w i t h f l u o r e s c a m i n e . A r e e x a m i n a t i o n o f t h e c o n d i t i o n s o f r e a c t i o n . A n a l . Biochem. 99:379. C h r i s o p e , G.L., Fox, C.W., and M a r s h a l l , R.T. 1976. L e c i t h i n a g a r f o r d e t e c t i o n o f m i c r o b i a l p h o s p h o l i p a s e s . A p p l . E n v i r o n . M i c r o b i o l . 31:784. C l a r k , W.M. and L u b s , H.A. 1915. The d i f f e r e n t i a t i o n o f b a c t e r i a o f t h e c o l o n - a e r o g e n e s f a m i l y by t h e use o f i n d i c a t o r s . J . I n f e c t . D i s . 17:160. 77 C l i f f e , A . J . and Law, B.A. 1982. A new method f o r t h e d e t e c t i o n o f m i c r o b i a l p r o t e o l y t i c enzymes i n m i l k . J . D a i r y Res. 49:209. C o u s i n , M.A. 1982. P r e s e n c e and a c t i v i t y of p s y c h r o t r o p h i c m i c r o o r g a n i s m i n m i l k and d a i r y p r o d u c t s : A r e v i e w . J . Fo o d P r o t . 45:172. C o u s i n , M.A. and M a r t h , E.H. 1977. Changes i n m i l k p r o t e i n s c a u s e d by p s y c h r o t r o p h i c b a c t e r i a . M i l c h w i s s e n s c h a f t 32:337. Cowan, S.T. and S t e e l , K . J . 1965. In Manual f o r t h e I d e n t i f i c a t i o n o f M e d i c a l B a c t e r i a . 1st e d . Cambridge U n i v e r s i t y P r e s s , E n g l a n d , pp 61-82. Cowan, S.T. and S t e e l , K . J . 1974. In Manual f o r t h e I d e n t i f i c a t i o n of M e d i c a l B a c t e r i a . 2nd e d . Cambridge U n i v e r s i t y P r e s s , E n g l a n d , pp 161-180. D e B e u k e l a r , N . J . , C o u s i n , M.A., B r a d l e y , R.L., J r . , and M a r t h , E.H. 1977. M o d i f i c a t i o n o f m i l k p r o t e i n s by p s y c h r o t r o p h i c b a c t e r i a . J . D a i r y S c i . 60:857. Dowson, W.J. 1949. I_n Manual o f B a c t e r i a l P l a n t D i s e a s e s . 1 st e d . B l a c k , A. and B l a c k , C. London, E n g l a n d , p 36. E l l i o t t , J.A., Emmons, D.B., and Y a t e s , A.R. 1974. The i n f l u e n c e of b a c t e r i a l q u a l i t y o f m i l k on t h e p r o p e r t i e s of d a i r y p r o d u c t s . A r e v i e w . Can. I n s t . F o o d S c i . T e c h n o l . J . 7:32. Fox, C.W., C h r i s o p e , G.L., and M a r s h a l l , R.T. 1976. I n c i d e n c e and i d e n t i f i c a t i o n o f p h o s p h o l i p a s e C - p r o d u c i n g b a c t e r i a i n f r e s h and s p o i l e d h o m o g e n i z e d m i l k . J . D a i r y S c i . 59:1857. Fung, D.Y.C. and Hartman, P.A. 1972. R a p i d c h a r a c t e r i z a t i o n o f b a c t e r i a , w i t h e m p h a s i s on S t a p h y l o c o c c u s a u r e u s . Can. J . M i c r o b i o l . 18:1623. G e b r e - E g z i a b h e r , A., Humbert, E.S., and B l a n k e n a g e l , G. 1980a. Heat s t a b l e p r o t e a s e s f r o m p s y c h r o t r o p h s i n m i l k . J . Fo o d P r o t . 43:197. G e b r e - E g z i a b h e r , A., Humbert, E.S., and B l a n k e n a g e l , G. 1980b. 78 H y d r o l y s i s o f m i l k p r o t e i n s by m i c r o b i a l enzymes. J . Food P r o t . 43:709. G r i f f i t h s , M.W., P h i l l i p s , J.D., and M u i r , D.D. 1981. T h e r m o s t a b i l i t y o f p r o t e a s e s and l i p a s e s f r o m a number of s p e c i e s of p s y c h r o t r o p h i c b a c t e r i a o f d a i r y o r i g i n . J . A p p l . B a c t e r i o l . 50:289. Hugh, R. 1973. I_n Manual o f C l i n i c a l M i c r o b i o l o g y . 2nd e d . A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y , B e t h e s d a , MD. pp 175-190. Hugh, R. and L e i f s o n , E . 1953. The t a x o n o m i c s i g n i f i c a n c e o f f e r m e n t i v e v e r s u s o x i d a t i v e m e t a b o l i s m o f c a r b o h y d r a t e s by v a r i o u s G r a m - n e g a t i v e b a c t e r i a . J . B a c t e r i o l . 66:24. J o n e s , A. and R i c h a r d s , T. 1952. N i g h t b l u e and V i c t o r i a b l u e a s i n d i c a t o r s i n l i p o l y s i s m e d i a . P r o c . S o c . A p p l . B a c t e r i o l . 15:82. J u f f s , J . S . 1973. I d e n t i f i c a t i o n o f Pseudomonas s p e c i e s i s o l a t e d f r o m m i l k p r o d u c e d i n s o u t h e a s t e r n Q u e e n s l a n d . J . A p p l . B a c t e r i o l . 36:585. J u f f s , J . S . 1974. I n f l u e n c e of- p r o t e i n a s e s p r o d u c e d by Pseudomonas a e r u g i n o s a and Pseudomoas f l u o r e s c e n s on m a n u f a c t u r e and q u a l i t y o f Cheddar c h e e s e . A u s t . J . D a i r y T e c h n o l . 29:74. Kaneko, T., H o l d e r - F r a n k l i n , M., and F r a n k l i n , M. 1977. M u l t i p l e s y r i n g e i n o c u l a t o r f o r a g a r p l a t e s . A p p l . E n v i r o n . M i c r o b i o l . 33:982. K i n g , E.O., Ward, M.K., and Raney, D.E. 1954. Two s i m p l e media f o r t h e d e m o n s t r a t i o n o f p y o c y a n i n and f l u o r e s c i n . J . L a b . C l i n . Med. 44:301. K l i n g e , K. 1960. D i f f e r e n t i a l t e c h n i q u e s and methods o f i s o l a t i o n o f Pseudomonas . J . A p p l . B a c t e r i o l . 23:442. Kohn, J . 1953. A p r e l i m i n a r y r e p o r t o f a new g e l a t i n l i q u e f a c t i o n method. J . C l i n . M i c r o b i o l . 6:249. K o v a c s , N. 1956. I d e n t i f i c a t i o n o f Pseudomonas p y o c y a n e a by 79 t h e o x i d a s e r e a c t i o n . N a t u r e , London 178:703. Law, B.A. 1979. Reviews o f t h e p r o g r e s s of d a i r y s c i e n c e : Enzymes o f p s y c h r o t r o p h i c b a c t e r i a and t h e i r e f f e c t s on m i l k and m i l k p r o d u c t s . J . D a i r y Res. 46:573. Law, B.A., S h a r p e , M.E., and Chapman, H.R. 1976. The e f f e c t of l i p o l y t i c G r a m - n e g a t i v e p s y c h r o t r o p h s i n s t o r e d m i l k on t h e d e v e l o p m e n t o f r a n c i d i t y ' i n C h e d d a r c h e e s e . J . D a i r y Res. 43:259. Law, B.A., Andrews, A.T., and S h a r p e , M.E. 1977. G e l a t i o n o f u l t r a - h i g h - t e m p e r a t u r e - s t e r i l i z e d m i l k by p r o t e a s e s f r o m a s t r a i n o f Pseudomonas f l u o r e s c e n s i s o l a t e d f r o m raw m i l k . J . D a i r y Res. 44:145. Law, B.A., Andrews, A.T., C l i f f e , A . J . , S h a r p e , M.E., and Chapman, H.R. 1979a. E f f e c t o f p r o t e o l y t i c raw m i l k p s y c h r o t r o p h s on Cheddar c h e e s e - m a k i n g w i t h s t o r e d m i l k . J . D a i r y Res. 46:497. Law, B.A., C o u s i n s , C M . , S h a r p e , M.E., and D a v i e s , F . L . 1979b. In C o l d T o l e r a n t M i c r o b e s i n S p o i l a g e and t h e E n v i r o n m e n t . R u s s e l l , A.D. and F u l l e r , R. e d s . Academic P r e s s , New Y o r k , pp 137-152. L e e , J . S . 1976.1_n Compendium of Methods f o r t h e M i c r o b i o l o g i c a l E x a m i n a t i o n o f F o o d s . Speck, M.L. e d . A.P.H. I n c . , W a s h i n g t o n , DC. pp 180-183. L i u , P.V. 1960. I d e n t i f i c a t i o n o f p a t h o g e n i c pseudomonads by e x t r a c e l l u l a r a n t i g e n s . J . B a c t e r i o l . 81:28. L y s e n k o , 0. 1961. Pseudomonas - An a t t e m p t a t a g e n e r a l c l a s s i f i c a t i o n . J . Gen. M i c r o b i o l . 25:379. M c K e l l a r , R.C. 1981. Development o f o f f - f l a v o r s i n u l t r a - h i g h t e m p e r a t u r e and p a s t e u r i z e d m i l k a s a f u n c t i o n o f p r o t e o l y s i s . J . D a i r y S c i . 64:2138. M u i r , D.D., P h i l l i p s , J.D., and D a l g l e i s h , D.G. 1979. The l i p o l y t i c and p r o t e o l y t i c a c t i v i t y of b a c t e r i a i s o l a t e d f r o m raw m i l k . J . S o c . D a i r y T e c h n o l . 32:19. 80 N a k a i , S., W i l s o n , H.K., and H e r r e i d , E.O. 1964. A s s a y i n g s t e r i l e c o n c e n t r a t e d m i l k f o r n a t i v e p r o t e o l y t i c enzymes. J . D a i r y S c i 47:754. N a s h i f , S.A. and N e l s o n , F . E . 1953. The l i p a s e o f Pseudomonas  f r a g i . I. C h a r a c t e r i z a t i o n of t h e enzyme. J . D a i r y S c i . 36:459. N i v e n , C.F., J r . , S m i l e y , K.L., and Sherman, J.M. 1942. The h y d r o l y s i s of a r g i n i n e by s t r e p t c o c c i . J . B a c t e r i o l . 43:651. N u m e r i c a l Taxonomy S y s t e m o f M u l t i v a r i a t e S t a t i s t i c a l ( NT-SYS ) P r o g r a m s . 1980. R o h l f , F . J . , K i s h p a u g h , J . , and K i r k , D. D e p a r t m e n t of E c o l o g y and E v o l u t i o n . The S t a t e U n i v e r s i t y o f New Y o r k a t S t o n y B r o o k , NY. O t t e , I . , T o l l e , A., and Hahn, G. 1979. M i c r o b i a l a n a l y s i s o f m i l k and m i l k p r o d u c t s . 2. M i n i a t u r i z e d p r i m a r y t e s t s f o r i d e n t i f i c a t i o n o f g e n e r a . M i l c h w i s s e n s c h a f t 34:152. O t t o , L.A. and Blachman, U. 1979. N o n f e r m e n t a t i v e b a c i l l i : E v a l u a t i o n of t h r e e s y s t e m s f o r i d e n t i f i c a t i o n . J . C l i n . M i c r o b i o l . 10:147. O v e r c a s t , W.W. 1968. P s y c h r o t r o p h i c m i c r o o r g a n i s m s and k e e p i n g q u a l i t y of m i l k and i t s p r o d u c t s . J . D a i r y S c i . 51:1336. P a l l e r o n i , N . J . 1975. I_n G e n e t i c s and B i o c h e m i s t r y o f Pseudomonas . C l a r k e , P.H. and Richmond, M.H. e d s . J o h n W i l e y and Sons L t d . , London, E n g l a n d , pp 1-36. P a l l e r o n i , N . J . and D o u d o r o f f , M. 1972. Some p r o p e r t i e s and t a x o n o m i c s u b d i v i s i o n s o f t h e genus Pseudomonas . An. Rev. P h y t o p a t h o l . 10:73. Rhodes, M.E. 1959. The c h a r a c t e r i z a t i o n o f Pseudomonas f l u o r e s c e n s . J . Gen. M i c r o b i o l . 21:221. R i c h a r d s o n , B.C. 1981. The p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f a h e a t - s t a b l e p r o t e a s e from Pseudomonas f l u o r e s c e n s B52. N. Z. J . D a i r y S c i . T e c h n o l . 16:195. 81 R i c h a r d s o n , B.C. and Te W h a i t i , I . E . 1978. P a r t i a l c h a r a c t e r i z a t i o n o f h e a t - s t a b l e e x t r a c e l l u l a r p r o t e a s e s o f some p s y c h r o t r o p h i c b a c t e r i a f r o m raw m i l k . N. Z. J . D a i r y S c i . T e c h n o l . 13:172. R i c h a r d s o n , B.C. and Newstead, D.F. 1979. E f f e c t o f h e a t - s t a b l e p r o t e a s e s on t h e s t o r a g e l i f e o f UHT m i l k . N. Z. J . D a i r y S c i . T e c h n o l . 14:273. Samagh, B.S. and Cunningham, J.D. 1972. N u m e r i c a l taxonomy o f t h e genus Pseudomonas f r o m m i l k and m i l k p r o d u c t s . J . D a i r y S c i . 55:19. Shaw, B.G. and L a t t y , J.B. 1982. A n u m e r i c a l t a x o n o m i c s t u d y of Pseudomonas s t r a i n s from s p o i l e d meat. J . A p p l . B a c t e r i o l . 52:219. S m i t h , D.A. 1961. A m u l t i p l e i n o c u l a t i o n d e v i c e f o r use w i t h f l u i d s . J . A p p l . B a c t e r i o l . 24:131. S m i t h , K.L., M u l l , L . E . , Lane, C.B., and B a g g o t t , A . J . , J r . . 1972. K e e p i n g q u a l i t y of m i l k e x p o s e d t o h i g h t e m p e r a t u r e as e x p e r i e n c e d d u r i n g t r a n s p o r t i n a u t o m o b i l e s . J . M i l k Food T e c h n o l . 35:588. S n e a t h , P.H.A. and S o k a l , R.R. 1973. I_n N u m e r i c a l Taxonomy. W.H. Freeman and Co., San F r a n c i s c o , CA. pp 230-234. S t a d h o u d e r s , J . 1975. M i c r o b e s i n m i l k and d a i r y p r o d u c t s . An e c o l o g i c a l a p p r o a c h . N e t h . M i l k D a i r y J . 29:104. S t a n i e r , R.Y., P a l l e r o n i , N .J., and D o u d o r o f f , M. 1966. The a e r o b i c pseudomonads: a t a x o n o m i c s t u d y . J . Gen. M i c r o b i o l . 43:159. T a y f o u r , A., M i l l i e r e , J.B., and V e i l l e t - P o n c e t , L . 1982. Growth and p r o t e o l y t i c a c t i v i t y o f Pseudomonas f l u o r e s c e n s 28P12 on m i l k r e t e n t a t e s s t o r e d a t low t e m p e r a t u r e . M i l c h w i s s e n s c h a f t 37:720. Tompkin, R.B. 1973. R e f r i g e r a t i o n t e m p e r a t u r e a s an e n v i r o n m e n t a l f a c t o r i n f l u e n c i n g t h e m i c r o b i a l q u a l i t y o f f o o d . A r e v i e w . F o o d T e c h n o l . 27:54. 82 Thomas, S.B. and Thomas, B.F. 1973. P s y c h r o t r o p h i c b a c t e r i a i n r e f r i g e r a t e d b u l k - c o l l e c t e d raw m i l k . D a i r y I n d . 38:61. W i t t e r , L.D. 1961. P s y c h r o p h i l i c b a c t e r i a . A r e v i e w . J . D a i r y S c i . 44:983. Y a n a g i y a , T., M i k a m i , M., and M i u r a , H. 1973. Changes o f m i l k p r o t e i n by p s y c h r o t r o p h i c o r g a n i s m s . J . A g r . Chem. S o c . J a p . 47:259. 

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