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The identification, characterization, and utilization of the ropy lactic streptococci strains and their… Macura, Dragan Mirkov 1983

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THE IDENTIFICATION, CHARACTERIZATION, AND UTILIZATION OF THE ROPY LACTIC STREPTOCOCCI STRAINS AND THEIR EXTRACELLULAR EXCRETION by DRAGAN MIRKOV MACURA B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (D e p a r t m e n t o f Food S c i e n c e ) We a c c e p t t h i s t h e s i s as 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 December, 1983 Cc) D r a g a n M i r k o v M a c u r a , 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis fo r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of f x »oe/ Sc^'-fe^.ZJL-The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 i i A B S T R A C T T r a d i t i o n a l c u l t u r e d m i l k products have been a source of b e n e f i c i a l l a c t i c a c i d b a c t e r i a whose p o t e n t i a l l s c o n s t a n t l y being e x p l o i t e d i n h e a l t h and n u t r i t i o n by modern s c i e n t i s t s . One such source i s the Scandinavian ropy sour m i l k , whose c o n s t i t u e n t m i c r o f l o r a produces a unique e x t r a c e l l u l a r e x c r e t i o n which prevents s y n e r e s i s and g r a i n i n e s s , and produces a t h i c k e r , smoother, and creamier product than normal sour m i l k . The i n d u s t r i a l p o t e n t i a l of these b a c t e r i a l i e s i n that they could be used i n modern c u l t u r e d d a i r y products i n s t e a d of the b a c t e r i a l and p l a n t gum s t a b i l i z e r s , or e x t r a b u t t e r f a t , which are c u r r e n t l y used to a l t e r the r h e o l o g i c a l p r o p e r t i e s . However, when grown i n pure c u l t u r e , these b a c t e r i a g r a d u a l l y l o s e t h e i r ropy c h a r a c t e r , i n d i c a t i n g c u l t u r e i n s t a b i l i t y . T h i s r e s e a r c h p r o j e c t attempted to provide the b a s i c i n f o r m a t i o n needed to c o n t r o l the ropy c u l t u r e s so that r e l i a b l e r o p i n e s s can be ass u r e d , and thus l a r g e s c a l e i n d u s t r i a l a p p l i c a t i o n s be made p o s s i b l e . I t d e s c r i b e d the growth c h a r a c t e r i s t i c s of f o u r ropy i s o l a t e s , i d e n t i f i e d t h e i r s p e c i e s , i s o l a t e d and c h a r a c t e r i z e d the v i s c o u s m a t e r i a l produced by one of the ropy i s o l a t e s , i n v e s t i g a t e d the a n t i m i c r o b i a l a c t i v i t y a g a i n s t the major milk s p o i l a g e b a c t e r i a , s t u d i e d the c o m p a t i b i l i t y of one ropy s t r a i n with b u t t e r m i l k s t a r t e r b a c t e r i a , and demonstrated the b e n e f i c i a l c o n t r i b u t i o n to the v i s c o s i t y of commercial b u t t e r m i l k by t h i s ropy s t r a i n . I t was found that of four pure c u l t u r e ropy i s o l a t e s , one (LLF) was a ropy v a r i a n t of S. l a c t l s , p r e v i o u s l y named Sj i i i l a c t i s l o n g i . The remaining t h r e e (L416, 701 and 705) were, thus f a r unnamed, v a r i a n t s of S. c r e m o r i s , w i t h a suggested name S.  c r e m o r i s l o n g i . Very good r o p i n e s s was produced i n l i q u i d m i l k , n e u t r a l i z e d cheese whey, M 17 medium of T e r z a g h i and Sandine (1975), and a complex c a s e i n d i g e s t medium, as w e l l as on tomato j u i c e agar and skim milk agar. The c u l t u r e i n s t a b i l i t y was found to be due to the s e r i a l c u l t u r e t r a n s f e r r i n g , and due to the prolonged product i n c u b a t i o n at growth temperature. The a n a l y s e s of the v i s c o u s m a t e r i a l sbowed that i t was composed of 47% t o t a l p r o t e i n , 20% methylpentoses, 9.3% protein-bound hexoses, 2.8% s i a l i c a c i d s , and no hexosamines. The r e s t of the molecle remains to be i d e n t i f i e d . The ropy c u l t u r e L416 was found to be i n h i b i t o r y to ATCC s t r a i n s of E. c o l i , S. a u r e u s , and to P. f l u o r e s c e n s b i o t y p e A, while i t showed no d e l e t e r i o u s e f f e c t s a g a i n s t the standard b a c t e r i a of b u t t e r m i l k s t a r t e r . A d d i t i o n of the ropy c u l t u r e to the commercial b u t t e r m i l k s t a r t e r r e s u l t e d i n s i g n i f i c a n t c o n t r i b u t i o n (° — 0.05) to the e q u i l i b r i u m apparent v i s c o s i t y , and the c o n s i s t e n c y of the product. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 LITERATURE REVIEW 5 A. M i c r o f l o r a of S c a n d i n a v i a n Ropy M i l k 5 B. C u l t u r e S t a b i l i t y 6 C. N u t r i t i o n a l Requirements and Growth Media 8 D. Composition of the Slime 9 E. V i s c o m e t r i c A n a l y s i s 10 MATERIALS AND METHODS 13 A. C u l t u r e C h a r a c t e r i z a t i o n 13 1. General Growth Media 13 a) Skim M i l k ( g rowth c u r v e of S. cremoris l o n g ! L416) lk b) C a s i e n D i g e s t Medium 15 c) Skim M i l k Agar 18 d) Tomato J u i c e Agar 19 2. C u l t u r e I d e n t i f i c a t i o n 20 a) Bromocresol Purple Agar 20 b) B i o c h e m i c a l T e s t 21 3. C u l t u r e S t a b i l i t y 23 4. A n t i b a c t e r i a l A c t i v i t y of S. c r e m o r i s l o n g i L416 2k 5. C o m p a t i b i l i t y of S. c r e m o r i s l o n g i L416 With the Commercial B u t t e r m i l k S t a r t e r 25 B. C h a r a c t e r i s t i c s of the V i s c o u s M a t e r i a l 26 1. Slime P r o d u c t i o n and P u r i f i c a t i o n 26 2. A n a l y s i s of the Slime 28 a) P r o t e i n F r a c t i o n 28 b) Carbohydrate F r a c t i o n 29 C. P r a c t i c a l A p p l i c a t i o n s of S. c r e m o r i s l o n g i L416.. 31 1. O p t i m i z a t i o n of the C o n c e n t r a t i o n of S. c r e m o r i s l o n g i L416 i n B u t t e r m i l k 31 a) Experiment 1 31 b) Experiment 2 32 c) Experiment 3 33 2. V i s c o m e t r i c A n a l y s i s of B u t t e r m i l k 3I+ 3. P r a c t i c a l Uses of V i s c o m e t r i c Screen D e v i c e . . . . 35 RESULTS AND DISCUSSION 37 A. C u l t u r e C h a r a c t e r i s t i c s 37 V 1. Growth Curve of S. c r e m o r i s l o n g i L416 37 2. C u l t u r e I d e n t i f i c a t i o n 2+1+ 3. C u l t u r e S t a b i l i t y 1*7 4. A n t i b a c t e r i a l A c t i v i t y of S. c r e m o r i s l o n g i L416 5. C o m p a t i b i l i t y of S. c r e m o r i s l o n g i L416 With Commercial B u t t e r m i l k S t a r t e r 6k B. C h a r a c t e r i s t i c s of V i s c o u s M a t e r i a l 70 1. Slime P r o d u c t i o n and P u r i f i c a t i o n 70 a) Background 70 b) I s o l a t i o n of the Slime 71 2. Composition of the Slime 73 a) P r o t e i n F r a c t i o n 73 b) Carbohydrate F r a c t i o n 75 C. P r a c t i c a l A p p l i c a t i o n of the S. cremoris l o n g i L416 - P r o d u c t i o n of B u t t e r m i l k 8l 1. O p t i m i z a t i o n of S. c r e m o r i s l o n g i L416 C o n c e n t r a t i o n i n B u t t e r m i l k S t a r t e r 8l 2. V i s c o m e t r i c A n a l y s i s of Improved B u t t e r m i l k . . . . 86 3. New Method f o r Measuring V i s c o s i t y i n B u t t e r m i l k 91 4. Use of V i s c o m e t r i c Screen Device 95 SUMMARY AND CONCLUSIONS 99 A. C u l t u r e C h a r a c t e r i s t i c s 99 B. C h a r a c t e r i s t i c s of the Slime 102 C. P r a c t i c a l A p p l i c a t i o n s of S. c r e m o r i s l o n g i L416.. 103 LITERATURE CITED 105 APPENDIX 1 113 APPENDIX 2 116 APPENDIX 3 119 APPENDIX 4 122 APPENDIX 5 125 APPENDIX 6 128 APPENDIX 7 130 APPENDIX 8 137 APPENDIX 9 139 vi L I S T OF TABLES T a b l e T i t l e Page ! 1 Composition of c a s e i n d i g e s t medium 2.6 2 I d e n t i f i c a t i o n of ropy s t r a i n s from S c a n d i n a v i a n ropy m i l k 5^ 3 S t a b i l i t y of slime p r o d u c t i o n by the ropy s t r a i n s of l a c t i c s t r e p t o c o c c i as percent of ropy CFU on skim milk agar 4 S t a b i l i t y of s l i m e p r o d u c t i o n by S. l a c t i s  l o n g i LLF i n l i q u i d skim m i l k as the percent of ropy CFU on skim milk agar 51 5 A c i d i t y m o n i t o r i n g of S. c r e m o r i s ATCC 19257 a f t e r l a c t i c f e r m e n t a t i o n , and s t o r e d at 10°C f o r 15 days 58 6 A c i d i t y m o n i t o r i n g of S. c r e m o r i s skim m i l k f e r m e n t a t i o n s when c o n t a m i n a t e d w i t h E. c o l i ATCC 25922 a f t e r l a c t i c f e r m e n t a t i o n , and s t o r e d at 10°C f o r 15 d a y s 60 7 A c i d i t y m o n i t o r i n g of S. c r e m o r i s skim milk f e r m e n t a t i o n s when c o n t a m i n a t e d with P. f l u o r e s c e n s b i o t y p e A a f t e r l a c t i c f e r m e n t a t i o n , and s t o r e d at 10°C f o r 15 days... 62 8 Change i n b a c t e r i a l numbers (Log CFU/ml.) i n completed skim m i l k f e r m e n t a t i o n s a f t e r storage at 4°C f o r 15 days 69 9 Composition of the s l i m e produced by S. cremoris l o n g i L416 7 7 7 Ik 10 Amino a c i d composition of the dry slime produced by S. c r e m o r i s l o n g i L416, and dry whey permeate 76 11 B u t t e r m i l k p r e f e r e n c e ranking 83 12 B u t t e r m i l k p r e f e r e n c e t e s t i n g - Nine po i n t hedonic s c a l e mean va l u e s (samples s t o r e d at 4 o f o r 1 day) 85 13 B u t t e r m i l k p r e f e r e n c e t e s t i n g - Nine p o i n t hedonic s c a l e mean values (samples s t o r e d at 4°C f o r 15 days 87 v i i L I S T OF FIGURES F i g u r e T i t l e Page 1 L i g h t m i c r o s c o p y m i c r o g r a p h s of S. cremoris ATCC 19257 (a) and S. c r e m o r i s l o n g i ( b ) 3 2 Growth curve of S. c r e m o r i s l o n g i L416 i n skim m i l k at 18°C 39 3 Apparent v i s c o s i t y at constant shear r a t e 500 per sec. v s . I n c u b a t i o n time at constant temperature of 18°C r e l a t i o n s h i p s i n skim m i l k f o r ropy s t r a i n s , and non-ropy l a c t i c b a c t e r i a hi 4 Growth curve of S. c r e m o r i s l o n g i 701 i n c a s e i n d i g e s t medium at 18"C ^ 3 5 S u r v i v a l c u r v e s of S. a u r e u s 25923 a t 10°C i n p a s t e u r i z e d skim m i l k , skim milk fermented w i t h S. c r e m o r i s ATCC 19257 f o r 18 h o u r s at 22°C, and i n skim m i l k f e r m e n t e d with S. cremoris l o n g i L416 a t 22°C f o r 18 h o u r s 57 6 S u r v i v a l c u r v e s of E. c o l i ATCC 25922 a t 10°C i n p a s t e u r i z e d skim m i l k , i n skim milk fermented by S. c r e m o r i s ATCC 19257 a t 22°C f o r 18 h o u r s , and i n skim m i l k f e r m e n t e d by S. cr e m o r i s l o n g i L416 a t 22°C f o r 18 h o u r s 59 7 S u r v i v a l c u r v e s of P. f l u o r e s c e n s biotype A at 10 C i n p a s t e u r i z e d skim m i l k , skim milk f e r m e n t e d w i t h S. c r e m o r i s ATCC 19257 a t 22°C f o r 18 hours, and i n skim m i l k fermented by S.  cremoris l o n g i L416 a t 22°C f o r 18 h o u r s 6 l 8 R e f r i g e r a t e d s t o r a g e s u r v i v a l curves of S.  cremoris l o n g i L416 and commercial b u t t e r m i l k s t a r t e r b a c t e r i a when grown independently of each other i n skim m i l k 65 9 R e f r i g e r a t e d s t o r a g e s u r v i v a l curves of S.  cremoris l o n g i L416 and a commercial b u t t e r m i l k s t a r t e r when grown together i n skim milk at 10% (v/v) l e v e l of L416 i n the inoculum f o r 18 hours at 22°C 67 10 R e f r i g e r a t e d s t o r a g e s u r v i v a l curves of S.  cremoris l o n g i L416 and a commercial b u t t e r m i l k s t a r t e r when grown toge t h e r i n skim milk at 20% (v/v) l e v e l of L416 i n inoculum 68 11 Apparent v i s c o s i t y - time r e l a t i o n s h i p f o r commercial b u t t e r m i l k (1.5% b u t t e r f a t ) and v i i i buttermilk (1.5% butterfat) containing 10% (by volume of t o t a l inoculum) of 18 hour culture of S. cremoris l o n g i L416, at a constant shear rate of 234 per sec. and a constant temperature of 4°C 89 12 Apparent v i s c o s i t y - time relationship for skim milk fermented with commercial buttermilk sta r t e r and S. cremoris l o n g i L416 at a constant shear rate of 234 per sec. and a constant temperature of 4°C 90 13 Viscometric screen device used for comparative consistency measurements of ropy milk 93 14 A diagramatic representation of viscometric screen device 9^  15 Viscometric screen device consistency curves of commercial buttermilk (1.5% b u t t e r f a t ) , and buttermilk containing 10% (by volume of t o t a l inoculum) of 18 hour c u l t u r e of S. cremoris longi. L416 (1.5% b u t t e r f a t ) 97 16 Viscometric decay curves, as measured by the viscometric screen device at 22°C for 1.5% butterfat buttermilks, commercial buttermilk s t a r t e r , and buttermilk with 15% (by volume) of S. cremoris l o n g i L416 i n inoculum 98 i x ACKNOWLEDGEMENTS I wish to express my sencere g r a t i t u d e to Dr. P. M. Townsley f o r h i s i n v a l u b l e a d v i c e , a s s i s t a n c e , enthusiasm, and p a t i e n c e , throughout the course of t h i s study. I am t h a n k f u l to the members of my graduate committee, Dr. S. Nakai, Dr. W. D. Powrie, and Dr. J . F. Ri c h a r d s f o r t h e i r constant a s s i s t a n c e . My thanks are extended to Mr. S. Yee, Mr. K. A . Campbell, Ms. L. Robinson, and Ms. D. S. Smith f o r t h e i r t e c h n i c a l a s s i s t a n c e . I a l s o wish to thank the Q. C. per s o n n e l of F. V. M. P. A . of Burnaby, B. C , Mr. R. E. Irwin and Ms. N. E. B a i l l i e , f o r the equipment and t h e i r p r o f e s s i o n a l a dvice d u r i n g the i n d u s t r i a l r e s e a r c h , development, and p r o d u c t i o n of b u t t e r m i l k . The f i n a n c i a l support through s c h o l a r s h i p s and b u r s a r i e s by the Canada D a i r y Commission, the U n i v e r s i t y of B r i t i s h Columbia, and the Dr. J . F. Morgan Memorial Fund, are g r e a t l y acknowledged. F i n a l l y , I wish to thank my parents, Mr. and Mrs. Mirko and Mi l k a Macura f o r t e a c h i n g me the value of e d u c a t i o n , and f o r t h e i r c o n s t a n t encouragement and support throughout the years of my s t u d i e s . 1 INTRODUCTION C u l t u r e d milk products have always been an important part of human d i e t ( K o s i k o w s k i , 1980). They are o f t e n c r e d i t e d with v a r i a b l e degrees of t h e r a p e u t i c or p r o p h y l a c t i c value ( W i n k e l s t e i n , 1956; Sandine et a l . , 1972; Speck, 1976; Shahani, 1976; Sandine, 1979; G i l l i l a n d , 1979; Deeth and Tamime, 1981; Amer, 1982). C u l t u r e d milk i s e a s i e r to d i g e s t than f r e s h m i l k (Deeth and Tamime, 1981; Speck, 1981), has more v i t a m i n s (Shahani, 1980; Deeth and Tamime, 1981), and i s a n t a g o n i s t i c to many harmful b a c t e r i a ( R a f l e r , 1956). Many r e s e a r c h e r s recommend t h e i r use i n t r e a t i n g i n t e s t i n a l d i s o r d e r s ( W i n k e l s t e i n , 1956; R a f l e r , 1956; G i l l i l a n d , 1979). The m i c r o f l o r a of c u l t u r e d m i l k s may vary depending on the area of o r i g i n and method of p r e p a r a t i o n . However, the common c h a r a c t e r i s t i c s of a l l b a c t e r i a found i n milk products are that they a l l sour the m i l k , and u s u a l l y produce v a r i o u s amounts of f l a v o u r i n g and a n t i m i c r o b i a l substances. The b a c t e r i a used i n c u l t u r e d milk p r o d u c t i o n u s u a l l y belong to the l a c t i c b a c i l l i or c o c c i , some of which are members of n a t u r a l i n t e s t i n a l m i c r o f l o r a (Draser, 1972; F u l l e r , 1972; W i n k e l s t e i n , 1956). In a d d i t i o n to b a c t e r i a , yeasts are f r e q u e n t l y found i n some of these p r o d u c t s . Yeasts u s u a l l y c o n t r i b u t e to e f f e r v e s c e n c e , f l a v o u r , and the h y d r o l y s i s of l a c t o s e . Well known examples of aforementioned products a r e : yogurt, b u t t e r m i l k , sour cream, biogarde, k e f i r , and to l e s s e r extent leben and koumiss. Less w e l l known i n North America i s the Sc a n d i n a v i a n ropy sour m i l k . T h i s low temperature, m i l d l y a c i d i c f e r m e n t a t i o n , forms a smooth, even curd whose c o n s i s t e n c y resembles a s t i f f , extremely 2 v i s c o u s dough. The f i n a l t h i c k n e s s of the product depends p r i m a r i l y on the c u l t u r e s t r a i n s and the b u t t e r f a t content of the m i l k . T h i s product keeps longer than many fermented m i l k s c u l t i v a t e d under the same c o n d i t i o n s (Sundman, 1953a), p o s s i b l y becouse i t i s a n t a g o n i s t i c to some milk s p o i l a g e b a c t e r i a and molds ( N i l s s o n and N i l s s o n , 1955; 1958). I t i s known as " t a t m j o l k " or "langmjolk" i n Sweden, " p i t k a p i i m a " i n south-western F i n l a n d , and " t a e t t e m e l k " i n Norway (Sundman, 1953a). I t s c h a r a c t e r i s t i c ropy c o n s i s t e n c y i s caused by a s l i m e which i s e x c r e t e d i n t o the m i l k medium du r i n g the e x p o n e n t i a l growth phase of the m i c r o f l o r a . T h i s slime a c t s l i k e a food s t a b i l i z e r p r e v e n t i n g s y n e r e s i s and g r a i n i n e s s and p r o v i d i n g a product with n a t u r a l t h i c k n e s s . The e d i b l e ropy sour m i l k i s always produced by the c a p s u l e - f o r m i n g s t r a i n s of l a c t i c a c i d b a c t e r i a , most o f t e n v a r i a n t s of S. c r e m o r i s ( F i g u r e 1) or S. l a c t l s , and i t i s to be d i f f e r e n t i a t e d from problem ropy m i l k which i s a r e s u l t of slime p r o d u c t i o n by some genera of c o l i - a e r o g e n e s and other b a c t e r i a , mostly gram negative rods (Olson-Sopp, 1912; Thomas and M c Q u i l l i n , 1953; Thomas et a l . , 1960; Cheung and Westhoff, 1983). Even though t h i s product has been produced and consumed by the S c a n d i n a v i a n farming communities f o r c e n t u r i e s , i t has never been produced commercially on a l a r g e s c a l e . W o l f e r s t e t t e r (1969) s t a t e d that the S c a n d i n a v i a n ropy m i l k was easy to s e l l i n Sweden, but that i t was very d i f f i c u l t to produce. He i m p l i e d that c u l t u r e i n s t a b i l i t y was a problem, r e s u l t i n g i n i n c o n s i s t a n t q u a l i t y of the product. This could be the major reason f o r l i m i t e d use of these c u l t u r e s i n the d a i r y i n d u s t r y to date. I f the s t a b i l i t y problem could be solved or, at l e a s t FIGURE 1.Light microscopy micrographs of S. cremoris ATCC 19257 (a), and S. cremoris l o n g i (b), showing chains of normal c e l l s , and chains of ropy c e l l s r e s p e c t i v e l y . S t a i n : Indian ink. Magnification: 1,000 X. It c u r t a i l e d , such that constant l e v e l s of slime could be produced by these b a c t e r i a , d a i r y i n d u s t r y l n S c a n d i n a v i a n c o u n t r i e s could produce the t r a d i t i o n a l ropy m i l k on l a r g e s c a l e e a s i l y . In a d d i t i o n , the d a i r y i n d u s t r y i n North America and elsewhere, where ropy a t t r i b u t e i s not popular, could use the ropy c u l t u r e s i n combination with a l r e a d y e s t a b l i s h e d commercial s t a r t e r s to impart the t h i c k n e s s which ropy c u l t u r e s p r o v i d e , thus reducing or p o s s i b l y r e p l a c i n g the need f o r v e g i t a b l e and m i c r o b i a l gum s t a b i l i z e r s which are c u r r e n t l y used l n many c u l t u r e d d a i r y p r o d u c t s . With t h i s i n mind, the o b j e c t i v e s of t h i s r e s e a r c h p r o j e c t were: a) To i d e n t i f y the s p e c i e s of the pure c u l t u r e ropy i s o l a t e s o b t a i n e d from Swedish D a i r i e s A s s o c i a t i o n , Malmo, Sweden, and the Food Research I n s t i t u t e , Ottawa. b) To d e f i n e the growth media and the growth c h a r a c t e r i s t i c s of the ropy s t r a i n s on hand f o r easy h a n d l i n g of the c u l t u r e s . c) To I d e n t i f y and d e f i n e the cause, or causes, of the c u l t u r e i n s t a b i l i t y and to suggest ways of s t a b i l i z i n g the slime p r o d u c t i o n . d) To i s o l a t e and c h a r a c t e r i z e the s l i m e , i . e . , to answer the q u e s t i o n of whether the slime i s a p o l y s a c c h a r i d e or a g l y c o p r o t e i n . e) To t e s t the a n t i m i c r o b i a l a c t i v i t y of the ropy c u l t u r e a g a i n s t the common mi l k s p o i l a g e b a c t e r i a , and a commercial b u t t e r m i l k s t a r t e r . f ) To suggest and demonstrate the i n d u s t r i a l a p p l i c a t i o n ( s ) f o r the ropy s t r a i n ( s ) i n a d a i r y p r o d u c t ( s ) i n Canada. g) I f s u c c e s s f u l , supply the c u l t u r e ( s ) to the major c u l t u r e d i s t r i b u t i o n houses i n North America f o r d e l i v e r y to the d a i r y i n d u s t r y . 5 LITERATURE REVIEW A. M i c r o f l o r a o f S c a n d i n a v i a n Ropy M i l k The m i c r o f l o r a of the Scandinavian ropy m i l k has been a major s u b j e c t of debate w i t h i n the l i m i t e d l i t e r a t u r e a v a i l a b l e on the s u b j e c t . In 1899, T r o i l i - P e t e r s s o n f i r s t s t u d i e d the microorganisms of Swedish ropy milk "langmjolk". She i s o l a t e d the slime producing bacterium which she named B a c t e r i u m l a c t i c u s l o n g i because i t c l o s e l y resembled B a c t e r i u m l a c t i c u s a c i d i , except f o r i t s slime producing a b i l i t y . Olson-Sopp (1912) s t u d i e d the microorganisms i n F i n n i s h ropy milk "taettemelk", and found that good t a e t t e was produced by a symb i o t i c r e l a t i o n s h i p of c e r t a i n S t r e p t o c o c c u s and S t r e p t o b a c i l l u s w i t h a y e a s t of genus S a c c h a r o m y c e s . When grown i n pure c u l t u r e s e p e r a t e l y , none of these organisms produced good t a e t t e , however, when grown tog e t h e r , a d e s i r a b l e product was o b t a i n e d . Macy (1923) i s o l a t e d a s l i m e - p r o d u c i n g s t r a i n of S t r e p t o c o c c u s which c l o s e l y r e s e m b l e d S. l a c t i s v a r . h o i l a n d i c u s and S. t a e t t e , from F i n n i s h " f i l l i " or " p i i m a " . He d e s c r i b e d i n great d e t a i l the morphology, c u l t u r e c h a r a c t e r i s t i c s , and p h y s i o l o g y of these b a c t e r i a and suggested that they be g i v e n new s p e c i e s , S t r e p t o c o c c u s p i i m a . Subsequent study by N i l s s o n and N i l s s o n (1950) i n v e s t i g a t e d the m i c r o f l o r a of Swedish ropy milk and r e p o r t e d that the s l i m e - p r o d u c i n g bacterium was i d e n t i c a l to S. l a c t i s , e x c e p t f o r i t s slim e - p r o d u c i n g a b i l i t y . These r e s e a r c h e r s named i t S. l a c t i s l o n g i . F u r t h e r s t u d i e s of ropy b a c t e r i a by Sundman (1953a) r e v e a l e d the presence of S.  l a c t i s v a r i a n t i n Swedish ropy m i l k , but she concluded that the ropy v a r i a n t of S. cremoris was the p r e d o m i n a n t b a c t e r i a i n that product. 6 Most recent e x t e n s i v e study of the m i c r o f l o r a of the Scandinavian ropy sour milk was done by Forsen (1966). In t h i s i n v e s t i g a t i o n of F i n n i s h " l o n g m i l k " , the s l i m e was p r o d u c e d by v a r i a n t s of S. c r e m o r i s , S.  l a c t i s , S. d i a c t y l a c t i s , and L e u c o n o s t o c s p . . L a t e r work by Forsen et a l . (1973) and S a x e l i n et a l . (1979) showed that S.  cremoris was the predominant ropy s t r a i n i n F i n n i s h ropy milk, however Forsen and Myllymaa (1974) r e p o r t e d i n t e r m e d i a t e S.  l a c t l s / S . c r e m o r i s s t r a i n s , thus a d d i n g to the a l r e a d y e x i s t i n g c o n f u s i o n . B. C u l t u r e S t a b i l i t y Regardless of which of the above mentioned b a c t e r i a were found to be r e s p o n s i b l e f o r the slime p r o d u c t i o n , none were found to produce high l e v e l s of slime i n d e f i n i t e l y . C u l t u r e i n s t a b i l i t y was i m p l i e d by most r e s e a r c h e r s who s t u d i e d these b a c t e r i a , thus the p r o d u c t i o n of the e d i b l e ropy milk was regarded as an a r t , r a t h r than a s c i e n c e . W o l f e r s t e t t e r (1969), i n p r o v i d i n g some t i p s on how to produce good ropy m i l k , r e p o r t e d that a " c r i t i c a l r a t i o " of 60 to 70 % ropy, to 30 to 40 % non-ropy c e l l s had to be maintained i f good q u a l i t y product was to be produced. He emphasized that t h i s r a t i o was very d i f f i c u l t to m a i n t a i n , e s p e c i a l l y at high i n c u b a t i o n temperatures, i e . , above 2 5 ° C . W o l f e r s t e t t e r recommended the growth temperature of 18 to 20°C to b e t t e r s u s t a i n the c u l t u r e s t a b i l i t y . Sundman (1953a) r e p o r t e d that the ropy s t r a i n s r e t a i n e d the a b i l i t y to produce v i s c o s i t y a f t e r a year and a h a l f of r e f r i g e r a t e d s t o r a g e . However, t h i s c o n c l u s i o n was made on the b a s i s of t r a n s f e r r i n g of 10 or l e s s c o l o n i e s per c u l t u r e , a f t e r only 1 p r o p a g a t i o n . I t i s p o s s i b l e t h a t t r a n s f e r r i n g a l a r g e r number of c o l o n i e s and more than 1 p r o p agation would have r e v e a l e d the i n s t a b i l i t y i n the ropy c u l t u r e s . The f a c t o r s r e s p o n s i b l e f o r the l o s s of s l i m e - f o r m i n g a b i l i t y , e s p e c i a l l y by pure c u l t u r e s , were never d e f i n e d , p o s s i b l y because much a t t e n t i o n was g i v e n to the s y m b i o t i c r e l a t i o n s h i p between the the ropy and the non-ropy c u l t u r e s , and between the ropy c u l t u r e s and a mold Oospora l a c t i s (Sundman, 1 9 5 3 a ) . I t was thought that without Oospora l a c t i s and the n o n-ropy v a r i a n t s the a b i l i t y to produce sl i m e was l o s t . Forsen (1966) showed that non-ropy b u t t e r m i l k s t a r t e r p r o v i d e d the v i t a m i n s ( n i c o t i n i c a c i d and b l o t i n ) e s s e n t i a l f o r slime p r o d u c t i o n by the ropy b a c t e r i a . T h i s type of a r e l a t i o n s h i p i s not uncommon among l a c t i c a c i d b a c t e r i a , yogurt being a c l a s s i c example* Furthermore, Forsen et a l . (1973) has demonstrated that the r o p y s t r a i n s of S. c r e m o r i s and S. c r e m o r i s / S . l a c t i s i n t e r m e d i a t e forms l o s t the a b i l i t y to produce the s l i m e a f t e r o n l y 3 t r a n s f e r s to f r e s h media at 3 0 ° C . T h i s demonstrated the importance of the growth temperature, and a l s o showed that the mere change i n temperature was not s u f i c i e n t to o b t a i n the l o s s of the s l i m e - p r o d u c i n g a b i l i t y . P r o p a g a t i o n of the c u l t u r e was necesary to o b t a i n the non-ropy trans f o r m s , s u g g e s t i n g that some f a c t o r r e s p o n s i b l e f o r the s l i m e p r o d u c t i o n may have been l o s t i n t r a n s f e r r i n g . B a c t e r i a are known to l o o s e plasmids more r a p i d l y at e l e v a t e d temperatures (Brock, 1979). Yet another kind of v i s c o s i t y l o s s f a c t o r i n the ropy S. c remoris was r e v e a l e d by S a x e l i n et a l . (1979). They observed sudden l o s s of s l i m e i n some c u l t u r e s of ropy l a c t i c b a c t e r i a , due to 8 the presence of an unusual bacteriophage c a l l e d KSY1. S i m i l a r type of a phage, which has an a b i l i t y to d e p o l l m e r i z e capsules without i n f e c t i n g and l y s i n g the ropy b a c t e r i a was r e p o r t e d by L i n d b e r g (1977). KSY1 phage was found to o n l y a f f e c t the S. cremoris s l i m e , not the slime of S. l a c t i s , i n d i c a t i n g t h a t the m o l e c u l a r composition of the slime may be s p e c i e s p e c i f i c . I t i n f e c t s and l y s e s a non-ropy S. c r e m o r i s s t r a i n 249. Thus, i t appears that the non-ropy host b a c t e r i a has to be present before a l a r g e onset of slime d e p o l i m e r i z a t i o n i s i n c u r r e d . C. N u t r i t i o n a l R e q u i r e m e n t s and Growth Media U n l i k e the s t a b i l i t y of the 6 l i m e p r o d u c t i o n , the n u t r i t i o n a l requirements of the ropy s t r a i n s are r e l a t i v e l y w e l l d e f i n e d . T h i s allows the use of s e v e r a l l i q u i d and s o l i d media f o r the growth of ropy l a c t i c b a c t e r i a . Forsen (1966) r e p o r t e d that the ropy s t r a i n s were more n u t r i t i o n a l l y demanding than t h e i r non-ropy c o u n t e r p a r t s . Between 10 and 13 amino a c i d s , as w e l l as r i b o f l a v i n and n i c o t i n i c a c i d were e s s e n t i a l f o r the s l i m e p r o d u c t i o n . She used a complex c a s e i n d i g e s t medium (Table 1) and by omiting one n u t r i e n t at a time, determined the n u t r i e n t requirements of the ropy s t r a i n s . Sundman (1953a, 1953b, 1953c) found whey to be the best medium f o r the slime p r o d u c t i o n by the ropy S. c r e m o r i s . In p r e s e n t r e s e a r c h , In a d d i t i o n to the above mentioned media and m i l k , very good r o p i n e s s was o b t a i n e d i n M17 medium ( T e r z a g h i and Sandine, 1975). T h i s medium was used by S a x e l i n et a l . (1979) i n t h e i r study of the capsule d e p o l i m e r i z i n g phage. Forsen et a l . (1973) used dextrose b r o t h medium ( D i f c o ) , and dextrose broth agar to grow and i s o l a t e the ropy s t r a i n s from F i n n i s h v i l l i . Of the s o l i d 9 media used f o r i s o l a t i n g and growing the ropy s t r a i n s , tomato j u i c e agar (Sundman, 1953a) was used most o f t e n . Macy (1923) r e p o r t e d no s l i m e p r o d u c t i o n on n u t r i e n t agar. T h e r e f o r e , i t i s e v i d e n t that the ropy s t r a i n s have c o m p l i c a t e d , but r e l a t i v e l y w e l l d e f i n e d n u t r i t i o n a l r e q u i r e m e n t s . D. C o m p o s i t i o n o f t h e Slime While i t i s known with r e l a t i v e c e r t a i n t y which b a c t e r i a produce the s l i m e i n the S c a n d i n a v i a n ropy milk ( T r o i l i - P e t e r s s o n , 1899; Olson-Sopp, 1912; Sundman, 1953a; Forsen, 1966), and which n u t r i e n t s they r e q u i r e f o r the slime p r o d u c t i o n ( F o r s e n , 1966), very l i t t l e i s known about the slime c o m p o s i t i o n . The composition of the s l i m e was s t u d i e d by Sundman (1953b) and by N i l s s o n and N i l s s o n (1958). Both found that i t was unique because i t contained a p r o t e l n a c e o u s component, however the n i t r o g e n contents and the amino a c i d p r o f i l e s r e p o r t e d by these s c i e n t i s t s were d r a s t i c a l l y d i f f e r e n t . Sundman r e p o r t e d a n i t r o g e n content of 8.9 % and an amino a c i d p r o f i l e s i m i l a r to that of c a s e i n , w h i l e N i l s s o n s r e p o r t e d n i t r o g e n content of 2.8 % and amino a c i d p r o f i l e completely d i f f e r e n t from that of c a s e i n , Implying that the whey p r o t e i n s may have been a contaminant of the s l i m e analyzed by Sundman, as the c u l t u r e was grown i n whey. Both found a carbohydrate f r a c t i o n a s s o c i a t e d with the p r o t e i n component, s u g g e s t i n g that i t may be a g l y c o p r o t e i n . The only other r e f e r e n c e to the composition of the slime was made by Forsen and V e i l i - M i e s (1981), where they mentioned the presence of g a l a c t o s e , g l u c o s e , and fucose i n the s l i m e . However, no methods of a n a l y s i s was shown, as i t was u n p u b l i s h e d d a t a . I t i s Important to know the g e n e r a l molecular 10 s t r u c t u r e of the sl i m e to be able to p r e d i c t I t s f u n c t i o n a l p r o p e r t i e s and i t s n u t r i t i o n a l v a l u e . E . V i s c o m e t r i c A n a l y s e s A very good review of the l i t e r a t u r e and developments i n rheology of d a i r y products was p u b l i s h e d by P r e n t i c e (1979). He c a l l s f o r more use of rheology i n d a i r y s c i e n c e as i t pr o v i d e s b e t t e r c o n t r o l of the product q u a l i t y , and b e t t e r communication of the product g u a l i t y among s c i e n t i s t s . K o h l i et a l . (1980) reviewed the i n s t r u m e n t a t i o n used i n measuring the t e x t u r e of d a i r y p r o d u c t s . Among other instruments, he d e s c r i b e d the b a s i c p r i n c i p l e s of o p e r a t i o n f o r Hoeppler r o l l i n g sphere vi s c o m e t e r and the r o t a t i o n a l v i s c o m e t e r s , B r o o k f i e l d and MacMichael. Snoeren et a l . (1982) used Haake R o t o v i s c o RV2 viscometer to measure the e f f e c t s of v a r i o u s p h y s i c a l f a c t o r s on the v i s c o s i t y of skim m i l k c o n c e n t r a t e s . They showed that the v i s c o s i t y of skim m i l k c o n c e n t r a t e s i s governed by the volume f r a c t i o n of the d i s p e r s e d p a r t i c l e s . The volume f r a c t i o n depends upon the h y d r a t i o n of the p r o t e i n , p r o t e i n c o m p o s i t i o n , and the p r o t e i n c o n t e n t . H y d r a t i o n of the m i l k p r o t e i n s can be I n f l u e n c e d by p r e h e a t i n g of the milk s i n c e , as a r e s u l t of heat d e n a t u r a t i o n , the v o l u m i n o s i t y of whey p r o t e i n i n c r e a s e s . T h i s e x p l a i n s the f a c t o r s which i n f l u e n c e the v i s c o s i t y of the "raw m a t e r i a l " f o r c u l t u r e d m i l k p r o d u c t i o n . Even though the i n s t r u m e n t a t i o n and the b a s i c p r i n c i p l e s which govern the v i s c o s i t y of c u l t u r e d m i l k s are adequately d e f i n e d , r e l a t i v e l y l i t t l e has been p u b l i s h e d on v i s c o m e t r i c a n a l y s i s of c u l t u r e d m i l k p r o d u c t s . From the p u b l i s h e d i n f o r m a t i o n i t i s evident that many types of vi s c o m e t e r s have been used, and sometimes inadequate 11 e x p e r i m e n t a l c o n d i t i o n s g i v e n . Nonetheless, v i s c o m e t r i c analyses i s a very good way of measuring p h y s i c a l q u a l i t y of c u l t u r e d d a i r y products. Forsen (1966) used Hoeppler viscometer f o r v i s c o s i t y measurements to r e c o r d s l i m e p r o d u c t i o n by the ropy s t r a i n s a f t e r 4 hours of growth. T h i s provided good means of comparison between the f e r m e n t a t i o n s . S o z z i et a l . (1978) used a g r a v i t a t i o n a l f r e e flow viscometer i n the shape of a b u r e t t e to r e c o r d v i s c o s i t y c o n t r i b u t i o n by ropy S. l a c t i s . The comparative measures of v i s c o s i t y were obtained by measuring the time i t took a sample to flow from a 100 ml. mark to 0 ml. mark through a 2.7 mm. diameter opening. I t was assumed that the time of flow was p r o p o r t i o n a l to the v i s c o s i t y of the sample. S i m i l a r p r i n c i p l e was employed by T s a r i g r a d s k a y a and Grigorchuk (1982) when they used a p i p e t t e with the o u t l e t diameter of 4 mm. and measured the time f o r a 100 ml. sample of ropy c u l t u r e s to be d i s c h a r g e d . They were s u c c e s s f u l i n s e l e c t i n g ropy c u l t u r e s on b a s i s of, among other c h a r a c t e r i s t i c s , comparative v i s c o s i t y . They obtained data on u n d i s t u r b e d curd, broken curd, and an i n d i c a t i o n of t h i x o t r o p y , by l e t t i n g the broken curd stand f o r 15 minutes and r e p e a t i n g the v i s c o m e t r i c procedure. K u r w i j i l a et a l . (1981) used a t o r t i o n viscometer (Rheometer STV) to measure apparent v i s c o s i t y of c u l t u r e d m i l k products of v a r i e d m i l k s o l i d s c o n t e n t . They s t u d i e d the e f f e c t of m i l k s o l i d s content and c u l t u r e s t r a i n on the v i s c o s i t y and product q u a l i t y as judged by t a s t e panel a n a l y s e s . They showed that a d i f f e r e n c e i n milk s o l i d s was e a s i l y d e t e c t a b l e by a v i s c o m e t e r . In a d d i t i o n , these r e s e a r c h e r s could detect the d i f f e r e n c e between high, low, and medium gas-producing c u l t u r e s . Maksimova et a l . (1982) used a r o t a t i o n a l viscometer (Rheotest-2) and i n great d e t a i l d e s c r i b e d v i s c o m e t r i c p r o p e r t i e s of s e v e r a l m e s o p h i l i c 12 and t h e r m o p h i l i c l a c t i c c u l t u r e s In s t e r i l e skim m i l k . On the basis of these p r o p e r t i e s they were able to recommend the use of the c u l t u r e s i n d i f f e r e n t c u l t u r e d d a i r y products, a c c o r d i n g to the v i s c o m e t r i c requirements of the p r o d u c t s . Thus, i t appears t h a t , even though d a i r y s c i e n t i s t s and t e c h n o l o g i s t s do not o f t e n r e l y on v i s c o m e t r i c instruments to judge the q u a l i t y of c u l t u r e d milk products, t h i s proves to be a very r e l i a b l e and very e f f e c t i v e method of q u a l i t y c o n t r o l . 1 3 MATERIALS AND METHODS A. C u l t u r e C h a r a c t e r i z a t i o n V i s c o u s c u l t u r e s LLF, 701, and 705 were s u p p l i e d by the Food Research I n s t i t u t e , Ottawa, while the v i s c o u s s t r a i n L415 was obtained from the C e n t r a l L a b o r a t o r y of the Swedish D a i r i e s A s s o c i a t i o n , Malmo, Sweden. A more s t a b l e ropy s t r a i n was i s o l a t e d from t h i s c u l t u r e and named L 4 1 6 . S t a n d a r d c u l t u r e s S t r e p t o c o c c u s  l a c t l s ATCC 19435 and S t r e p t o c o c c u s c r e m o r i s ATCC 19257 were s u p p l i e d by the American Type C u l t u r e C o l l e c t i o n . The recommended growth temperature f o r the s t r a i n s 701, 705, and L416 was 18°C and that f o r LLF was 2 2 ° C . Upon r e c e i p t a l l c u l t u r e s were propagated i n s t e r i l e skim milk ( a u t o c l a v e d f o r 8-10 min. at 121°C) and s t o r e d i n l i q u i d n i t r o g e n or i n a - 6 5 ° C s t o r a g e u n i t . Every 2 months the c u l t u r e s were v i t a l i z e d , p l a t e d on the tomato j u i c e agar (TJA) or on skim milk agar (SMA); a v i s c o u s colony was p i c k e d and propagated i n s t e r i l e skim milk to develop new c u l t u r e s t o c k s . 1. G e n e r a l Growth Media The o b j e c t of t h i s study was to e l u c i d a t e the growth c h a r a c t e r i s t i c s of the v i s c o u s c u l t u r e s i n v a r i o u s growth media, f o r the purpose of s u c c e s s f u l c u l t u r e h a n d l i n g i n an i n d u s t r i a l s e t t i n g . Two l i q u i d and two s o l i d type media were s t u d i e d and v a r i o u s growth a t t r i b u t e s r e c o r d e d . 11+ a) Skim M i l k ( g r o w t h c u r v e of S. c r e m o r i s l o n g i L 4 1 6 ) M i l k i s the u s u a l growth medium f o r l a c t i c s t r e p t o c o c c i . For the s t u d i e s of g e n e r a l c h a r a c t e r i s t i c s of these microorganisms skim m i l k was used. However, s a t i s f a c t o r y v i s c o s i t y was obtained i n m i l k s of v a r i o u s b u t t e r f a t c o n t e n t . Forsen ( 1 9 6 6 ) s t u d i e d the e f f e c t of inoculum s i z e on the r a t e of sl i m e p r o d u c t i o n . She found that 5 % inoculum gave best r e s u l t s . Thus, i n t h i s study, u n l e s s otherwise i n d i c a t e d , the samples were I n o c u l a t e d with an a c t i v e l y growing c u l t u r e at 5 % by volume. A l l v i s c o u s c u l t u r e s e x h i b i t e d very s i m i l a r growth c h a r a c t e r i s t i c s to the poin t that they could not be d i s t i n g u i s h e d i n skim m i l k . Thus, only the c u l t u r e L 4 1 6 was chosen to repr e s e n t the v i s c o u s c u l t u r e s ' growth i n skim m i l k . P a s t e u r i z e d commercial skim milk was obtained from a l o c a l g r o c e r y s t o r e . E f f o r t was made throughout t h i s study to always buy the milk from the same d a i r y to minimize v a r i a b i l i t y . The milk was f u r t h e r a u t o c l a v e d f o r 8 - 1 0 min. at 1 2 1 ° C , c o o l e d to room t e m p e r a t u r e and i n o c u l a t e d with t e s t c u l t u r e s . I t was then i n c u b a t e d at 1 8 ° C f o r 4 days. The growth was measured by a c i d p r o d u t t i o n (pH), the t o t a l number of the colony forming u n i t s ( C F U ) , and e q u i l i b r a t e d apparent v i s c o s i t y measurements at a constant shear r a t e at v a r i o u s i n c u b a t i o n time i n t e r v a l s ( r e f e r to F i g u r e 2 ) d u r i n g 4 days of i n c u b a t i o n . The v i s c o s i t y measurements were made with a c o a x i a l r o t o v i s c o v i s c o m e t e r , Brabender Rheotron, with t o r s i o n dinamometer s p r i n g A, and a c o a x i a l attachments A l and C 4 were used to monitor the v i s c o s i t y development 36 i n d i c a t i o n of growth i n skim m i l k . Each sample was s u b j e c t e d to 7 d i f f e r e n t shear r a t e s ranging from 1 2 5 7 per s e c . to 1 1 1 per sec. f o r attachment C 4 , and 2 6 7 8 per sec. to 5 9 15 per sec. f o r attachment A l . Each sample was sheared at highest shear r a t e u n t i l the s c a l e r e a d i n g e q u i l i b r a t e d , and then the shear r a t e s were decreased stepwise to cover the above i n d i c a t e d ranges, each time t a k i n g a r e a d i n g a f t e r e q u i l i b r a t i o n . Apparent v i s c o s i t y was c a l c u l a t e d f o r each data po i n t and the rheograms of each sample p l o t t e d (data not shown). I t was found that at the shear r a t e of 500 per sec. the r a t e of change of the apparent v i s c o s i t y was l i n e a r f o r a l l samples. T h e r e f o r e , t h i s shear r a t e was chosen f o r r e l a t i v e v i s c o s i t y comparisons between the samples grown f o r d i f f e r e n t amounts of time. The CFU counts were c a r r i e d out on TJA p l a t e s . An a p p r o p r i a t e d i l u t i o n of each d u p l i c a t e sample was s u r f a c e p l a t e d on 4 TJA p l a t e s . The p l a t e s were i n c u b a t e d at 18°C, and the c o l o n i e s were counted a f t e r 4 days of i n c u b a t i o n . They were averaged to d e r i v e average CFU/ml. A c i d development was monitored by measuring pH of d u p l i c a t e samples with a F i s h e r Accumet model 230 pH meter. b) C a s e i n D i g e s t Medium V i s c o u s c u l t u r e s grew very w e l l i n skim m i l k , however, the t o t a l v i s c o s i t y which r e s u l t e d was c o n t r i b u t e d by two s i g n i f i c a n t f a c t o r s . A c i d p r o d u c t i o n by the b a c t e r i a l e d to the c a s e i n p r e c i p i t a t i o n , and the slime p r o d u c t i o n l e d to the i n c r e a s e d r e s i s t a n c e to f l o w . Together, these two f a c t o r s produced a c o m p l i c a t e d l a t t i c e whose i n d i v i d u a l c o n t r i b u t i o n to t o t a l v i s c o s i t y of the m i l k medium was i m p o s s i b l e to e v a l u a t e . Thus, i n an attempt to study the c o n t r i b u t i o n of the slime alone we chose to grow the v i s c o u s 16 c u l t u r e s on a complex c a s e i n d i g e s t medium. T h i s medium was formulated by Ford et a l . (1958) and mo d i f i e d f o r the s t u d i e s of n u t r i t i o n a l requirements of the v i s c o u s v a r i a n t s by Forsen (1966). Table 1 l i s t s the composition of t h i s medium f o r convenience, s i n c e i t was pu b l i s h e d i n German. Table 1. Composition of c a s e i n d i g e s t medium (Forsen, 1966). N u t r i e n t s / I n g r e d i e n t s g./lOOO ml. B a c t o - c a s i t o n e A Bacto casamino a c i d s 5 Lact o s e 10 3 3 0.25 C i t r i c a c i d 0.5 Sodium a c e t a t e 1 L-C y s t e i n e h y d r o c h l o r i d e 0.5 A s c o r b i c a c i d 0.5 Adenine 5 Guanine 5 ..cont'd 17 Table 1 continued N u t r i e n t s / I n g r e d i e n t s mg./lOOO ml. Xanthine 5 U r a c i l 5 P y r i d o x a l h y d r o c h l o r i d e 2 N i c o t i n i c a c i d 1 Thiamine h y d r o c h l o r i d e 1 R i b o f l a v i n e 1 D-pantothenic a c i d ( C a - s a l t ) 1 ug./lOOO ml. p-Aminobenzoic a c i d 10 B i o t i n 10 F o l i c a c i d 1 Cobalamine 1 M i n e r a l s ' s o l u t i o n * 10 ml • M i n e r a l s ' s o l u t i o n g./lOOO ml. 5 20 0.5 0.5 ... cont'd C a C l 2 M g C l 2 x 6H 20 F e C l 3 x 6H 20 ZnSO, x 7H„0 18 Table 1 cont i n u e d N u t r i e n t / I n g r e d i e n t g./lOOO ml, C o C l 2 x 6H 20 0.25 CuSO^ x 7H 20 0.25 Na 2MoO A 0.25 VS0 4 0.25 These i n g r e d i e n t s were s o l u b i l i z e d i n d i s t i l l e d d e i o n i z e d water, pH ad j u s t e d to 6.8 w i t h IN KOH, dispensed i n t o f e r m e n t a t i o n v e s s e l s (95 ml. i n t o 250 ml Erlenmeyer f l a s k s ) , and a u t o c l a v e d f o r 8-10 min. at 121°C.The f l a s k s were then cooled to room temperature and i n o c u l a t e d w i t h 5% (v/v) of 24 hour c u l t u r e 701. They were Incubated at 18°C f o r 3 days and the growth recorded by m i c r o s c o p i c c o u n t i n g of apparent CFU with the a i d of Levy-Hausser c e l l c o u n t i n g chamber ( C A . Hausser & Son Co., P h i l a . USA). Apparent v i s c o s i t y measurements were done a f t e r e q u i l i b r a t i o n at constant shear r a t e of 500 per s e c , and a c i d development recorded by measuring pH. c) Skim M i l k Agar N u t r i e n t agar, 2.3% (w/v), was rehydrated and au t o c l a v e d at 121°C f o r 15 m i n . L i q u i d skim m i l k was a u t o c l a v e d at 121°C f o r 8-10 min. s e p a r a t e l y . While the agar was s t i l l hot, the skim m i l k (10% v/v) was added and g e n t l y mixed. A f t e r c o o l i n g to 19 45-50°C, the medium was p o u r e d i n t o P e t r i p l a t e s to s o l i d i f y . These p l a t e s were used f o r s u r f a c e s p r e a d i n g , the technique most o f t e n employed throughout t h i s p r o j e c t . One tenth of a m i l l i l i t e r a l i q u o t s of a p p r o p r i a t e d i l u t i o n s were spread p l a t e d and the p l a t e s incubated f o r up to 5 days before c o u n t i n g . d) Tomato J u i c e Agar Tomato j u i c e agar was one of the media used f o r i s o l a t i n g the v i s c o u s b a c t e r i a from o r i g i n a l s t o c k s . I t was formulated by m o d i f y i n g the procedures of Sundman (1953a) and Kulp and White (1932). Tomato j u i c e was e x t r a c t e d from f r e s h r i p e tomatoes. A procedure f o r 1 1. i s o u t l i n e d here, however, any m u l t i p l e of t h i s procedure may be prepared. The f r e s h r i p e tomatoes were washed i n warm water and 450 g. macerated with a household b l e n d e r . They were b o i l e d f o r 5-10 min. and then f i l t e r e d through a f o u r - f o l d cheese c l o t h . In a 2 1. Erlenmeyer f l a s k 333 ml. of f i l t r a t e was mixed with 5 g. of peptone, 3 g. of l a c t o s e , 2 g. o f K H 2 P Q a , 0.5 g. o f MgS0 4, 1 g. of c i t r i c a c i d , and 23 g. of n u t r i e n t agar. The t o t a l volume was then r a i s e d to about 900 ml. with d i s t i l l e d water, and the mixture heated to d i s s o l v e the n u t r i e n t s . The pH was a d j u s t e d to 6.8 and the volume made up to 1 1. with d i s t i l l e d water. I t was t h e r e a f t e r a u t o c l a v e d at 121°C f o r 10 min, and upon c o o l i n g to 4 5 - 5 0 ° C , P e t r i p l a t e s were poured. A f t e r s o l i d i f y i n g the p l a t e s were s l i g h t l y r e d i s h , and they provided a good growth medium f o r s u r f a c e - s p r e a d p l a t i n g of l a c t i c s t r e p t o c o c c i . 20 2. C u l t u r e I d e n t i f i c a t i o n Current l i t e r a t u r e i s very c o n t r o v e r s i a l about the s p e c i e c l a s s i f i c a t i o n of the v i s c o u s l a c t i c s t r e p t o c o c c i ( r e f e r to the l i t e r a t u r e r e v i e w ) . Thus, the o b j e c t of t h i s experiment was to i d e n t i f y the s p e c i e s of the v i s c o u s c u l t u r e s on hand. Thi s was achieved with v a r i o u s d i f f e r e n t i a l growth media and v a r i o u s d i f f e r e n t i a l growth c o n d i t i o n s , as d e s c r i b e d below. a) B r o m c r e s o l P u r p l e Agar The bromcresol purple agar (BCPA) p l a t e s were prepared a c c o r d i n g to the procedure of Reddy et a l . (1969) with some a l t e r a t i o n s . To 300 ml. of d i s t i l l e d water 5 g. of t r y p t o n e , 5 g. of yeast e x t r a c t , 4 g. of L - a r g i n i n e h y d r o c h l o r i d e , and 1 g. K2HPO4 were added. These c o n t e n t s were s o l u b i l i z e d and pH ad j u s t e d to 6.8. In a separate f l a s k , 23 g. of n u t r i e n t agar and 6 g. of carboxymethyl c e l l u l o s e (CMC) were s o l u b i l i z e d by heating i n 200 ml. of d i s t i l l e d water. T h i s was added to the f i r s t f l a s k and the volume made up to 830 ml with d i s t i l l e d water. The contents were s t e r i l i z e d by a u t o c l a v i n g at 121°C f o r 15 min. In s e p a r a t e i n d i v i d u a l f l a s k s 3 g. of CaC0 3 i n 100 m l . of d i s t i l l e d water, and 0.1 g. of bromcresol purple (BCP) i n 20 ml of d i s t i l l e d water were a l s o a u t o c l a v e d at 121°C f o r 15 min. In a d d i t i o n , 50 ml. of skim milk was a u t o c l a v e d at 121°c f o r 8-10 m i n . s e p a r a t e l y . Upon a u t o c l a v i n g , a l l above i n g r e d i e n t s were poured a s e p t i c a l l y i n t o one f l a s k , s w i r l e d to mix w e l l , and poured i n t o P e t r i p l a t e s . The r e s u l t i n g p l a t e s were dark purple which turned yellow at pll below 5.2. In order to r e g a i n p u r p l e c o l o u r the pH had to be r a i s e d to 6.8. The a p p r o p r i a t e 21 b a c t e r i a l d i l u t i o n s were s u r f a c e - s p r e a d and Incubated at the c o r r e s p o n d i n g growth temperatures f o r 5 days. During f i r s t 4 days of i n c u b a t i o n a l l the c o l o n i e s appeared y e l l o w as a r e s u l t of a c i d p r o d u c t i o n . A f t e r 5 days, the r e l e a s e of ammonium from a r g i n i n e by S. l a c t i s began to show i t s n e u t r a l i z i n g e f f e c t , t u r n i n g the medum around the a r g i n i n e u t i l y z i n g c o l o n i e s back to p u r p l e . S. c r e m o r i s does not u t i l i z e a r g i n i n e , thus, no ammonia was r e l e a s e d , r e s u l t i n g i n no f u r t h e r c o l o u r change. S. c r e m o r i s c o l o n l s stayed y e l l o w , making i t easy to d i f f e r e n t i a t e them from the S. l a c t i s c o l o n i e s a f t e r 5 d a y s . The r e s u l t s were recorded as p o s i t i v e or n e g a t i v e r e a c t i o n f o r a r g i n i n e metabolism ( T a b l e 2 ) . b) B i o c h e m i c a l T e s t s A number of d i f f e r e n t i a l media were prepared to a i d i n i d e n t i f i c a t i o n of the s p e c i e s of the v i s c o u s c u l t u r e s . A v a i l a b l e l i t e r a t u r e i n d i c a t e s that the most commonly found S c a n d i n a v i a n v i s c o u s s t r e p t o c o c c i b e l o n g t o the s p e c i e s l a c t i s and cremoris (Olsen-Sopp, 1912; Macy, 1923; Sundman, 1953a; N i l s s o n and N i l s s o n , 1950; 1958), thus we used the b i o c h e m i c a l t e s t s most commonly used i n d i f f e r e n t i a t i n g between these two s p e c i e s (Buchanan et al.,1974; H a r r i g a n and McCance,1966). Litmus m i l k was prepared i n 14 x 160 mm. t e s t tubes a c c o r d i n g to D l f c o Manual (1953). F i v e ml a l i q u o t s i n t e s t tubes were i n o c u l a t e d with a l o o p f u l of a c t i v e l y growing t e s t c u l t u r e s . They were incubated at t h e i r r e s p e c t i v e growth temperatures. Readings were taken a f t e r 48 hours of i n c u b a t i o n . O b s e r v a t i o n s were taken of c o l o r change ( a c i d p r o d u c t i o n ) , m i l k c o a g u l a t i o n , l i t m u s r e d u c t i o n 22 ( l i t m u s turns c o l o r l e s s ) . The r e s u l t s appear i n Table 2. A r g i n i n e u t i l i z a t i o n t e s t i s the most f r e q u e n t l y employed t e s t i n d i s t i n g u i s h i n g between S. l a c t i s and S. c r e m o r i s . I t can be done on s o l i d support medium (BCPA) or i n a l i q u i d medium ( a r g i n i n e b r o t h ) . The former method rec o r d s a c o l o u r change as a r e s u l t of pH change, while the l a t t e r method d e t e c t s the presence of ammonium i n the growth medium. Ammonium i s detected with N e s s l e r ' s reagent which turns orange to brown i n i t s presence. In t h i s experiment, both t e s t s were used as i t was important to d i s t i n g u i s h between the s p e c i e s . A r g i n i n e broth was prepared by the method of Abd-el-Malek and Gibson (1948) as d e s c r i b e d by H a r r i g a n and McCance (1966). To 100 ml. of d i s t i l l e d w a ter 0.5 g D - g l u c o s e , 0.2 g. K 2HP0^, 0.5 g. t r y p t o n e , 0.25 g. yeast e x t r a c t , and 0.3 g. a r g i n i n e monohydrochloride was added. The contents were s o l u b i l i z e d and pH a d j u s t e d to 7.0. The medium was dispensed i n t o 14 x 160 mm. t e s t tubes, capped, and a u t o c l a v e d at 121°C f o r 15 min. Upon c o o l i n g they were i n o c u l a t e d with a c t i v e l y growing t e s t c u l t u r e s and the a p p r o p r i a t e c o n t r o l s . The tubes were then incubated at t h e i r r e s p e c t i v e growth temperatures f o r 48 hours. The r e s u l t s were recorded with the help of N e s s l e r ' s reagent which was prepared as per H a r r i g a n and McCance (1966). The ammonium d e t e c t i o n was done by mixing 1 ml. of growth medium, a f t e r 48 hours of i n c u b a t i o n w i t h 1 ml. of N e s s l e r ' s reagent. The tubes were recorded as ammonium p o s i t i v e or ammonium ne g a t i v e ( T a b l e 2 ) . Bromcresol p u r p l e agar p r e p a r a t i o n was d e s c r i b e d i n s e c t i o n 2a above. Growth at 40°C was t e s t e d i n the c a s e i n d i g e s t medium of 23 Forsen (1966). S t e r i l e t e s t tubes were I n o c u l a t e d with v i a b l e c u l t u r e s a t IX (v/v) inoculum l e v e l and i n c u b a t e d at 40°c f o r 120 hours. Growth was d e t e c t e d s p e c t r o p h o t o m e t r i c a l l y by r e c o r d i n g absorbance at 520 um i n Cary 210 Spectrophotometer. T h i s wavelength was d e r i v e d from a scan of the v i s i b l e range of growing v i s c o u s c e l l s . The p o s i t i v e growth was a r b i t r a r i l y a s signed to the tubes where absorbance at 520 um Increased by more than 0.27 u n i t s d u r i n g the 120 hours of i n c u b a t i o n . Growth at pH 9.2 was a l s o t e s t e d i n the c a s e i n d i g e s t medium of Forsen (1966). pH was a d j u s t e d to 9.2 w i t h IN NaOH. The growth was recorded s p e c t r o p h o t o m e t r i c a l l y as d e s c r i b e d above. 3. C u l t u r e S t a b i l i t y The purpose of t h i s experiment was to i d e n t i f y , and p o s s i b l y prevent, the f a c t o r s which govern the unsteady p r o d u c t i o n of slime by the ropy b a c t e r i a . The I n v e s t i g a t i o n was completed i n two p a r t s . F i r s t , w i t h a l l ropy c u l t u r e s I n c l u d e d , and t h e n w i t h S. l a c t i s l o n g i LLF a l o n e . The v i s c o u s c o l o n i e s of the c u l t u r e s 705, 701, and L416 were propagated i n skim m i l k at 18°C w h i l e L L F was grown at 22°C. A f t e r 24 hours of growth they were t r a n s f e r e d to f r e s h milk at 5% (v/v) i n o c u l a t i o n l e v e l . T h i s t r a n s f e r was designated t r a n s f e r number 1. Subsequent t r a n s f e r s were done a f t e r 48 hours of i n c u b a t i o n . A f t e r each t r a n s f e r , d i l u t i o n s were made and the c u l t u r e s were p l a t e d on 10Z l i q u i d skim m i l k agar p l a t e s . The p l a t e s were in c u b a t e d at t h e i r r e s p e c t i v e growth temperatures, and counted when the c o l o n i e s could be v i s u a l l y e v a l u a t e d . Each colony was touched with a s t r a i g h t wire 2k and recorded as v i s c o u s or non v i s c o u s . The percent of c o l o n i e s found to be v i s c o u s was c a l c u l a t e d . The data obtained appear i n Table 3. The second part of t h i s experiment was done the same as above, except: a) A l l t r a n s f e r s were done at e x a c t l y 50 hours of I n c u b a t i o n , b) Each p l a t e was counted at e x a c t l y 48, 96, and 114 hours and recorded as 2, 4, 6 day growths r e s p e c t i v e l y . The r e s u l t s appear i n Table 4. 4. A n t i b a c t e r i a l A c t i v i t y o f S. c r e m o r i s l o n g i L416 The a n t i b a c t e r i a l a c t i v i t y o f S. c r e m o r i s l o n g i L416 was determined a c c o r d i n g to the method of Goel et a l . (1971). The contam i n a t i n g t e s t o r g a n i s m s c h o s e n were E s c h e r i c h i a c o l i ATCC 25922, S t a p h y l o c o c c u s a u r e u s ATCC 25923, and Pseudomonas  f l u o r e s c e n s b i o t y p e A. Nine one l i t r e g l a s s c o n t a i n e r s were employed to s t e r i l i z e (10 min. at 121°C a u t o c l a v i n g ) 570 ml. skim milk, three of which were, upon c o o l i n g , i n o c u l a t e d with 30 ml. of 24 hour c u l t u r e of S. cremoris ATCC 19257 , t h r e e were i n o c u l a t e d with 24 hour c u l t u r e of S. cremoris l o n g i L416, and the r e m a i n i n g three ( c o n t r o l s ) were l e f t u n l n o c u l a t e d . The i n o c u l a t e d f l a s k s were incubated at 22°C f o r 18 hours. During t h i s time the c o n t r o l s were held at 4°C to prevent b a c t e r i a l growth. From each f l a s k , 100 ml. was withdrawn f o r pH and a c i d i t y measurements, b r i n g i n g t o t a l volume of the f l a s k s to 500 ml. A l l the f l a s k s were then contaminated w i t h 1.0 ml of a c t i v e l y growing t e s t c u l t u r e s . These f l a s k s were s t o r e d at 10°C f o r 15 days to approximate the domestic storage c o n d i t i o n s . The number of co n t a m i n a t i n g t e s t organisms was d e r i v e d by p l a t i n g a p p r o p r i a t e d i l u t i o n s of inoculums on s e l e c t i v e media f o r those organisms. 25 The average values f o r 5 p l a t e s were: 3.5 x 1 0 8 CFU/ml. f o r S. a u r e u s , 8.4 x 1 0 7 C F U / m l . f o r E . c o l i , 3.1 x 1 0 7 CFU/ml. f o r Pseudomonas. To f a c i l i t a t e u n i f o r m d i s t r i b u t i o n , 1.0 ml. of the contaminating c u l t u r e s was d i l u t e d w i t h 9.0 ml. of s t e r i l e phosphate b u f f e r and a s e p t i c a l l y mixed i n . Immediately a f t e r c ontamination, and every day f o r the next 5 days, as w e l l as on days 10 and 15, 10 ml. samples were withdrawn from each f l a s k and t o t a l number of CFU/ml. of the contaminating b a c t e r i a d e r i v e d on a p p r o p r i a t e s e l e c t i v e media. E. c o l l was p o u r - p l a t e d i n v i o l e t r e d b i l e agar (VRBA). The p l a t e s were o v e r l a y e d with a t h i n l a y e r of VRBA a f t e r i n i t i a l l y poured agar s o l i d i f i e d . They were i n c u b a t e d at 32°C f o r 24 h o u r s . S. aureus was grown on B a i r d - P a r k e r agar (BPA) with t e l l u r i t e enrichment, and the p l a t e s were i n c u b a t e d at 32°C f o r 24 h o u r s . P. f l u o r e s c e n c e was p o u r - p l a t e d i n n u t r i e n t a g a r , and i n c u b a t e d at 10°C f o r 8 days. For a l l the above samples, 5 p l a t e s of a p p r o p r i a t e d i l u t i o n were counted and averaged a c c o r d i n g to the standard methods f o r d a i r y p r o d u c t s . The r e s u l t s appear i n F i g u r e s 5, 6 and 7. For s i m p l i c i t y , the counts f o r days 1,2,3 and 4 were l e f t out s i n c e t h e i r overview i s r e p r e s e n t e d by the r e s u l t s of days 0 and 5. 5. C o m p a t i b i l i t y of S. c r e m o r i s l o n g i L416 With the Commercial  B u t t e r m i l k S t a r t e r The c o m p a t i b i l i t y t e s t s were done to f i n d out i f the v i s c o u s c u l t u r e L416 has any adverse e f f e c t s on the commercial b u t t e r m i l k s t a r t e r d u r i n g c o l d s t o r a g e , and v i c e v e r s a . V a r i e d p r o p o r t i o n s of L416 (10Z, 15% and 20% of the inoculum volume) were grown i n d u p l i c a t e 500 m l . skim m i l k samples, at 22°C u n t i l t o t a l 26 a c i d i t y (T.A.) reached 0.85%. A f t e r breaking the curd ( r e f e r to s e c t i o n C f o r more d e t a i l s on making b u t t e r m i l k ) , the product was s t o r e d at 4-6°C f o r 15 d a y s . E v e r y 5 days two 10 ml. samples were taken from each d u p l i c a t e and s u r f a c e p l a t e d on skim milk agar p l a t e s . The p l a t e s were i n c u b a t e d at 22°C u n t i l the c o l o n i e s could be v i s u a l l y e v a l u a t e d . Each colony was touched with an i n o c u l a t i n g wire to note i f i t was ropy. F i v e p l a t e s were counted f o r each d u p l i c a t e p l a t i n g and the numbers of CFU averaged. The r e s u l t s are presented g r a p h i c a l l y i n F i g u r e s 8-10. B. C h a r a c t e r i s t i c s o f t h e V i s c o u s M a t e r i a l In order to a v o i d the problem of i s o l a t i n g the slime from a p r e c i p i t a t e d c a s e i n matrix i n milk, the v i s c o u s c u l t u r e L416 was grown i n a c i d cheese whey where p r o t e i n p r e c i p i t a t i o n does not o c c u r . 1. Slime P r o d u c t i o n and P u r i f i c a t i o n The f r e s h cottage cheese whey was obtained from a l o c a l d a i r y . I t s pH was a d j u s t e d to 6.8 to 7.0 wit h 2N NaOH. The p r e c i p i t a t e which r e s u l t e d from pH change was separated by c e n t r i f u g i n g at 13,200 x g f o r 10 min. The whey was then d e p r o t e i n i z e d with an u l t r a f i l t r a t i o n u n i t u s i n g a m l l l i p o r e membrane with an e x c l u s i o n m o l e c u l a r weight of 10,000. The r e t e n t a t e was d i s c a r d e d and the f i l t r a t e was f i l t e r s t e r i l i z e d with a s t e r i l e 0.45 um m l l l i p o r e f i l t e r . I t was then i n o c u l a t e d at 5% (v/v) l e v e l with S. cr e m o r i s  l o n g i L416 p r e v i o u s l y grown i n the same medium f o r 24 hours. The inoculum was developed from a v i s c o u s colony grown on m i l k agar. The i n o c u l a t e d bulk c u l t u r e was I n c u b a t e d at 18°C without shaking f o r 24 27 hours, and then t r a n s f e r e d to the c o l d room at 4-6°C f o r a d d i t i o n a l 24 hours. The fermented whey changed from a c l e a r l i g h t green s o l u t i o n to an opalescent green, heavy, e g g w h i t e - l i k e s l i m e . When s w i r l e d i n a f l a s k , or poured i n t o a d i f f e r e n t c o n t a i n e r i t behaved l i k e a heavy, v i s c o u s mucus r e s i s t i n g flow at f i r s t and then " f a l l i n g " out of the f l a s k . The v i s c o u s mucus was c e n t r l f u g e d a t 13,200 x g f o r 30 min. to s e p a r a t e c e l l s . To the Bupernatent f l u i d , equal volumes of 95% e t h a n o l was added s l o w l y , while s t i r r i n g c o n s t a n t l y . The p r e c i p i t a t e d slime was separated by e i t h e r c e n t r l f u g i n g at 13,200 x g f o r 5 min., or by f i l t r a t i o n . I t was then d i s s o l v e d i n d i s t i l l e d water by magnetic s t i r r i n g o v e r n i g h t at 5°C. An e q u a l volume of c o l d 25% TCA was added to the s o l u b i l i z e d sample. The sample was shaken v i g o r o u s l y by hand and l e f t to stand f o r 15 minutes, and then c e n t r l f u g e d at 13,200 x g f o r 30 minutes, and f i l t e r e d with a 0.45 um m i l l i p o r e f i l t e r to s e p a r a t e the TCA i m p u r i t i e s and l e f t o v e r c e l l s . The TCA was separated from the f i l t r a t e by two e x t r a c t i o n s with equal volumes of e t h y l e t h e r . The r e s i d u a l ether was removed by purging with n i t r o g e n gas. The sample was d i a l y z e d a g a i n s t r u n n i n g tap w ater at 5°C f o r 48 hours and f r e e z e d r i e d . T h i s m a t e r i a l was c o n s i d e r e d to be pure s l i m e , and i t was s u b j e c t e d to m o d i f i e d t e s t s f o r serum g l y c o p r o t e i n s ( W i n z l e r , 1955), s i a l i c a c i d s assay (Warren, 1958), t o t a l p r o t e i n content (Lowry et a l . , 1951), and amino a c i d a n a l y s i s (Simpson et a l . , 1975). 2 8 2. A n a l i s i s o f t h e S l i m e a) P r o t e i n F r a c t i o n The purpose of t h i s experiment was to prove, or d i s p r o v e , the presence of p r o t e i n i n the s l i m e . Thus, the F o l i n phenol method and the amino a c i d a n a l y s i s were used. T o t a l p r o t e i n ( F o l i n phenol method) was d e r i v e d by the method o f Lowry et a l . (1951). A s i x poin t standard curve was c o n s t r u c t e d by u s i n g 4 x c r y s t a l l i z e d bovine serum albumin (BSA), and a l i n e a r r e g r e s s i o n equation (1) d e r i v e d . y - 0.022x + 0.0032 r - 0.994 (1) y • absorbance at 750 um x - ug. BSA/0.8 ml. Q u a d r u p l i c a t e samples of pure s l i m e , each c o n t a i n i n g 107 ug. of the s l i m e per 0.8 ml, were prepared and the amount of t o t a l p r o t e i n c a l c u l a t e d from the above e q u a t i o n (Appendix 1 ) . The amino a c i d composition was ob t a i n e d by the methanesulfonic a c i d method (Simpson et a l . , 1975). No a n a l y s i s f o r h a l f - c y s t i n e was done s i n c e only presence or absence of amino a c i d s was of i n t e r e s t . A s i n g l e column system (Duran Chem. Corp., Palo A l t o , C a l i f . ) a t t a c h e d to a phoenix model M6800 amino a c i d a n a l y z e r (Phoenix P r e c i s s i o n Instrument Co., P h i l a d e l p h i a , Penn.) was used f o r the amino a c i d anlysiB. Monroe programmable c a l c u l t o r program based on the summation of amino a c i d s method f o r t o t a l p r o t e i n d e t e r m i n a t i o n i n foods (Ueidelbaugh et a l . , 1975) was used to determine the q u a n t i t a t i v e and the 29 q u a l i t a t i v e amounts of each amino a c i d . The q u a n t i t a t i v e a n a l y s i s was r e s u l t s appear i n Table 10. b) C a r b o h y d r a t e F r a c t i o n The protein-bound hexoses content of the pure slime was determined by the o r c i n o l - s u l f u r i c a c i d method of L u s t i n g and Langer ( W i n z l e r , 1955) usi n g an equal mixture of D-galactose and D-mannose as a st a n d a r d . Dry sl i m e sample was run i n d u p l i c a t e and the percent hexoses d e r i v e d from the f o l l o w i n g l i n e a r r e g r e s s i o n e q u a t i o n : y - 2.234x - 0.055 r - 0.997 (2) y • absorbance at 540 um x - mg. hexose/ml. T h i s equation d e s c r i b e s a 7 p o i n t standard curve c o n s t r u c t e d f o r hexose c o n c e n t r a t i o n s ranging between 0.050 nig./ml. to 0.604 mg./ml. ( r e f e r to Appendix 2 ) . d e r i v e d by adapting the Dische and S h e t t l e s method as d e s c r i b e d by W i n z l e r (1955). The slime sample was run i n d u p l i c a t e and the f i n a l r e s u l t s averaged. Rhamnose was used as a standard as i t g i v e s the same r e s u l t s as f u c o s e . The amount of methylpentose, c a l c u l a t e d as fucose, was d e r i v e d from the f o l l o w i n g e q u a t i o n : used to determine the t o t a l amount of p r o t e i n i n the sample. The The methylpentose ( f u c o s e ) content of the sl i m e was mg. Fucose/lOOml. - 20 x (3) (A, s t . 3 9 6 - A "st. 1*30 ) 30 A • absorbance at 396 um or at 430 um, b l . " water blank plus reagents, s t . • rhamnose standard. The s i a l i c acids content of the 6llme was measured by the modified t h i o b a r b i t u r i c ac id method (Warren, 1959). The slime sample (2 .724 mg.) was hydro l y zed i n 0 . 1 N H^O^ f o r 1 hour at 8 0 ° C . The sample was n e u t r a l i z e d , and the volume made up to 10 ml . with d i s t i l l e d * deionlzed water. From t h i s , three 0.2 m l . samples were treated as per Warren (1959). A 5 point standard curve was constructed from N-acetylneuraminic acid (Sigma, IV) ranging from 0.011 to 0.056 uMoles/.2 m l . (see Appendix 4) , and rgress ion equation 4 der ived : y - 15.43x + 0.031 r - 0.993 (4) y • absorbance at 549 um x - uHoles of N-acetylneuraminic a c i d . Hexosamine composition of the slime was determined by the Elson-Morgan method (Winzler , 1955). A 6 point standard curve was constructed using glucosamine hydrochlor ide ranging from 0.008 to 0.501 mg. free glucosamine/ml. , and fo l lowing l i near regression equation der i ved : y - 3.489x - 0.024 r - 0.995 (5) y - absorbance at 530 um x " mg. hexosamine/ml. 31 F i v e r e p l i c a t e s of the slime sample were run (Appendix 5 ) * C• P r a c t i c a l A p p l i c a t i o n s o f S. c r e m o r i s l o n g i L416 1. O p t i m i z a t i o n of the C o n c e n t r a t i o n of S. cremoris l o n g i L416  i n B u t t e r m i l k The purpose of these experiments was to o p t i m i z e the c o n c e n t r a t i o n of the ropy c u l t u r e i n the commercial b u t t e r m i l k s t a r t e r to produce a smoother, creamier, t h i c k e r and more s t a b l e b u t t e r m i l k , and at the same time a v o i d the ropy appearance and mouthfeel. The commercial b u t t e r m i l k s t a r t e r and the procedure f o r i t s p r e p a r a t i o n were obtained from a l o c a l d a i r y . T h i s procedure was s i m i l a r to that of Kosikowski (1977). The s t a r t e r r e p o r t e d l y was a b l e n d of S . c r e m o r i s , S. l a c t i s v a r . d i a c e t y l a c t i s , and Leuconostoc sp.. The e x p e r i m e n t a l c u l t u r e chosen f o r t h i s experiment was S . cremoris l o n g i L416. C o m m e r c i a l m i l k was obtained from a l o c a l grocery s t o r e . a) Experiment 1 ( B u t t e r m i l k P r o d u c t i o n ) F r e s h , 2% b u t t e r f a t commercial m i l k was blended with f r e s h commercial skim m i l k i n the r a t i o of 3 to 1 by volume, to o b t a i n 1.5% b u t t e r f a t m i l k . The t e m p e r a t u r e of the samples was r a i s e d to 80°C and held f o r 30 minutes f o r p a s t e u r i z a t i o n . I t was then c o o l e d to 22°C and I n o c u l a t e d w i t h a 24 h o u r m i l k c u l t u r e of above mentioned s t a r t e r s at t o t a l inoculum of 3% by volume. The blend of S. cremoris  l o n g i L416 In commercial b u t t e r m i l k s t a r t e r (B.M.S.) v a r i e d a c c o r d i n g to the e x p e r i m e n t a l v a r i a t i o n s l i s t e d below. The samples were 32 incubated f o r 12 to 16 h o u r s at 2 2 ° or u n t i l the t i t r a t a b l e a c i d i t y (T.A.) reached 0.85% as t i t r a t e d a g a i n s t 0.1 N NaOH. The samples were then s a l t e d with 0.1% (w/v) of s a l t (NaCl) and the curd broken with a 5 cm. c r o s s s e c t i o n 3-blade p r o p e l l e r r o t a t i n g at 450 rpm. f o r 2 minutes. They were t h e r e a f t e r s t o r e d at 4°C o v e r n i g h t , or u n t i l t e s t e d . The o p t i m i z a t i o n of the l e v e l of S. c r e m o r i s l o n g i L416 i n the inoculum was done i n three separate experiments. Each time a t a s t e panel a n a l y s i s was done to compare the q u a l i t y of the b u t t e r m i l k with ropy c u l t u r e to that of a c o n t r o l . P r e f e r e n c e t e s t i n g was most o f t e n used, and the samples ranked i n order of p r e f e r e n c e . Thus, i n the f i r s t experiment a 50/50 blend of L416 and B.M.S. were compared to a l a b o r a t o r y prepared B.M.S. sample and to the f r e s h commercial b u t t e r m i l k of the same brand purchased i n a l o c a l g r o c e r y s t o r e . The samples were s u b j e c t e d to a 20 member t a s t e panel comprised of g e n e r a l l a b o r a t o r y - t y p e populace. The p a n e l i s t s were asked to rank the samples i n order of p r e f e r e n c e . The s t a t i s t i c a l a n a l y s i s a p p l i e d to the r e s u l t s was the a n a l y s i s of v a r i a n c e (AN0V) as d e s c r i b e d by F i s c h e r and Yates (1949). When a s i g n i f i c a n t d i f f e r e n c e was found, Tukey's t e s t was a p p l i e d to l o c a t e the d i f f e r e n c e (Larmond, 1977). The r e s u l t s appear i n Table 11. b) Experiment 2 ( P r e l i m i n a r y O p t i m i z a t i o n ) On the b a s i s of the r e s u l t s obtained i n the above experiment, the experiment was repeated with a decreased p r o p o r t i o n of the v i s c o u s c u l t u r e i n the t o t a l inoculum. The decrease was necessary because at 50% L416 l n the t o t a l inoculum r o p i n e s s was e v i d e n t and was r a t e d by the p a n e l i s t s as a n e g a t i v e 33 a t t r i b u t e . Thus, b u t t e r m i l k samples with 25%, 10%, and 5% L416 were prepared. They were e v a l u a t e d v i s u a l l y and o r g a n o l e p t i c a l l y by the author and the Q u a l i t y C o n t r o l (Q. C.) management of the c o o p e r a t i n g d a i r y . A consensus was reached that the a p p r o p r i a t e l e v e l of L416 should l i e between 5% and 15% of the t o t a l Inoculum. No s t a t i s t i c a l a n a l y s i s was done on the r e s u l t s of t h i s experiment as I t was used only as a p r e l i m i n a r y a n a l y s i s l e a d i n g to the more indepth o p t i m i z a t i o n study d e s c r i b e d i n experiment 3. c) Experiment 3 ( F i n a l O p t i m i z a t i o n ) T h i s experiment was done as d e s c r i b e d i n experiment 1 except the l e v e l s of c u l t u r e L416 used were 5%, 10%, and 15% of the t o t a l inoculum. The c o n t r o l (B.M.S.) was prepared i n 2% b . f . m i l k while the blended samples were prepared i n 1% to estimate the p o s s i b i l i t y f o r lowering the b u t t e r f a t content i n b u t t e r m i l k . One l i t e r d u p l i c a t e s of each product were p r e p a r e d and s t o r e d at 4°C. They were then s u b j e c t e d to a 10 member t a s t e panel a n a l y s i s a f t e r 1 and 15 days of r e f r i g e r a t e d s t o r a g e . The panel was picked among the Q.C. and p r o d u c t i o n p e r s o n n e l of the c o o p e r a t i n g d a i r y , who were very f a m i l i a r w i t h the q u a l i t y of b u t t e r m i l k . The p a n e l i s t s were asked to e v a l u a t e the products on the b a s i s of f l a v o u r and a c i d i t y , body and t e x t u r e , and o v e r a l l a c c e p t a b i l i t y . A nine p o i n t hedonic s c a l e with d e s c r i p t o r s ranging from " l i k e extremely" to " d i s l i k e extremely" on each end of the s c a l e was used f o r p r e f e r e n c e t e s t i n g (see Appendix 6 ) . The r e s u l t s were as s i g n e d numerical v a l u e s and su b j e c t e d to the a n a l y s i s of v a r i a n c e . Where a s i g n i f i c a n t d i f f e r e n c e was found, Tukey's t e s t was used to i d e n t i f y that d i f f e r e n c e (Larmond, 1977). 34 The r e s u l t s appear In T a b l e s 12 and 13. 2. V i s c o m e t r i c A n a l y s i s o f B u t t e r m i l k The purpose of t h i s experiment was not to p r o v i d e a complete v i s c o m e t r i c a n a l y s i s of b u t t e r m i l k . The v i s c o s i t y c o n t r i b u t i o n to skim m i l k and the c a s e i n d i g e s t medium by the ropy c u l t u r e s was shown ( F i g u r e s 2 to 4 ) . Rather, the purpose of t h i s experiment was to compare the r e s i s t a n c e to shear s t r e s s of the b u t t e r m i l k samples with and without the ropy c u l t u r e . T h i s i s important because e x c e s s i v e s h e a r i n g of b u t t e r m i l k r e s u l t s i n t h i n n e r body and lower q u a l i t y l e a d i n g to e x c e s s i v e s y n e r e s i s . A l s o , a simple d e v i c e f o r comparative c o n s i s t e n c y measurements was i n v e n t e d to a v o i d the use of the Brabender v i s c o m e t e r . T h i s may enable the i n d u s t r i a l p l a n t o p e r a t o r s to monitor the v i s c o s i t y development without i n v e s t i n g i n expensive equipment. The data f o r the apparent v i s c o s i t y decay curves were ob t a i n e d by the method of Tung et a l . (1971) w i t h Brabender r o t a t i o n a l v i s c o m e t e r . The samples analyzed were those of commercial b u t t e r m i l k (1.5% b u t t e r f a t ) c o n t a i n i n g 10% of c u l t u r e L416 i n BMS inoculum ( F i g u r e 11), and skim m i l k f e r m e n t a t i o n of S. cremoris l o n g i L416 ( F i g u r e 12) with a p p r o p r i a t e c o n t r o l s . S t a t i s t i c a l s i g n i f i c a n c e was t e s t e d by ANOV (Appendix 7 ) . For the comparative c o n s i s t e n c y measurements of the "high body" commercial b u t t e r m i l k a " v i s c o m e t r i c screen d e v i c e " (V.S.D.) ( F i g u r e s 13 and 14) was d e v i s e d . T h i s d e v i c e c o n s i s t s o f : a) two c l e a r , graduated v o l u m e t r i c c o n t a i n e r s ( c y l i n d e r s ) , both used a l t e r n a t e l y f o r pouring and f o r r e c e i v i n g the sample a f t e r 35 i t has been passed through the standard s i e v e (#3, F i g u r e 13) b) a standard mesh s i e v e which a l l o w s the f l u i d s , whose v i s c o s i t i e s are to be compared, to pass through at a reasonable r a t e so that the t i m i n g of a g i v e n volume of the f l u i d can be done a c c u r a t e l y (#1, F i g u r e 13) c) a "sample v e s s e l " or a f u n n e l which s i t s f l a t on the screen d e f i n i n g the area of the screen through which the sample i s to f l o w (#2, F i g u r e 13) d) a stopwatch to r e c o r d the time i t takes a predetermined volume of the sample to go through the d e f i n e d area of the s c r e e n . e) a constant temperature water bath f o r r i n s i n g VSD p a r t s and e q u i l i z i n g t h e i r temperature (#4, F i g u r e 13) f ) an " a i r w i r e " used to prevent t r a p p i n g of the a i r i n the r e c e i v i n g v e s s e l when the f u n n e l i s f u l l (#5, F i g u r e 13). 3) P r a c t i c a l Uses o f V i s c o m e t r i c Screen Device The VSD can be used to monitor v i s c o s i t y developments i n any m a t e r i a l where v i s c o s i t y can be measured by c o n v e n t i o n a l procedures. In t h i s experiment I t was used i n m o n i t o r i n g b u t t e r m i l k c o n s i s t e n c y d u r i n g c o l d s t o r a g e , and to r e g i s t e r the c o n s i s t e n c y d i f f e r e n c e between the normal commercial b u t t e r m i l k and the commercial b u t t e r m i l k c o n t a i n i n g ropy b a c t e r i a ( F i g u r e 15). In a d d i t i o n , the VSD can be used to study the r e s i s t a n c e to shear by the sample i n q u e s t i o n . T h i s was demonstrated i n t h i s r e s e a r c h by repeated p a s s i n g of the samples through the VSD and r e c o r d i n g the time i t took the same volume of the sample under the same experimental c o n d i t i o n s to go through the s c r e e n . A l i n e a r r e l a t i o n s h i p of the l o g co n v e r s i o n s of the data was obtained ( F i g u r e 16) which was comparable to the data o b t a i n e d by Brabender Rheotron ( F i g u r e s 11 and 1 2 ) . 37 RESULTS AND DISCUSSION A. C u l t u r e C h a r a c t e r i s t i c s In d e f i n i n g the g e n e r a l c h a r a c t e r i s t i c s of the ropy l a c t i c s t r e p t o c o c c i t h i s r e s e a r c h d e a l t with the growth c h a r a c t e r i s t i c s of a s e l e c t e d ropy s t r a i n i n v a r i o u s l i q u i d and s o l i d growth media. I t furthermore d e a l t with s p e c i e s I d e n t i f i c a t i o n and d i f f e r e n t i a t i o n of group N s t r e p t o c o c c i , the c u l t u r e s t a b i l i t y of slime p r o d u c t i o n , a n t i b a c t e r i a l a c t i v i t y of the chosen s t r a i n i n skim m i l k medium, and i t s c o m p a t i b i l i t y with the mixed m i c r o f l o r a of the commercial b u t t e r m i l k s t a r t e r . The purpose of t h i s r e s e a r c h was not to provide great d e t a i l on any of the above c h a r a c t e r i s t i c s , but to p r o v i d e enough i n f o r m a t i o n about these c u l t u r e s to f a c i l i l t a t e t h e i r use i n the d a i r y i n d u s t r y . 1. Growth Curve o f S. c r e m o r i s l o n g i LA16 A m i c r o b i a l growth curve l s d e f i n e d as the graph i n which the i n c r e a s e i n c e l l numbers, or i n biomass, i s p l o t t e d a g a i n s t time ( S i n g l t o n and S a i n s b u r y , 1978). However, i f the a c t u a l number of c e l l s or the amount of biomass are not of i n t e r e s t , very good In f o r m a t i o n about the growth of b a c t e r i a can be o b t a i n e d by measuring the disappearance of a g i v e n n u t r i e n t i n the growth medium, or by measuring the p r o d u c t i o n of a m e t a b o l i c byproduct. In d a i r y i n d u s t r y i t i s u s u a l to measure the amount of a c i d produced by measuring pH, or by measuring t i t r a t a b l e a c i d i t y as an i n d i c a t i o n of m i c r o b i a l growth. However, a c i d p r o d u c t i o n does not always correspond to the v i s c o s i t y development due to the slime p r o d u c t i o n i n ropy b a c t e r i a . 38 I t can be assumed, on the other hand, that the slime p r o d u c t i o n i s always a r e s u l t of m i c r o b i a l growth. T h e r e f o r e , i n t h i s r e s e a r c h , v i s c o s i t y measurements were p l o t t e d as a f u n c t i o n of i n c u b a t i o n time, and c o r r e l a t e d with the more t r a d i t i o n a l methods of measuring growth; c o u n t i n g of colony forming u n i t s , and pH measurements, i n an attempt to show that v i s c o s i t y measurements can be used to p l o t the growth curve of ropy b a c t e r i a along the s i d e of the t r a d i t i o n a l methods. I t was hoped that t h i s i n f o r m a t i o n would be u s e f u l i n p r a c t i c a l a p p l i c a t i o n s of these c u l t u r e s . These forms of r e c o r d i n g growth of microorganisms are a p p l i c a b l e f o r l i q u i d media o n l y . On s o l i d media the s t u d i e s were mostly of m o r p h o l o g i c a l nature f r e q u e n t l y r e q u i r i n g counting c o l o n i e s , or determining the f r a c t i o n of ropy c o l o n i e s where mixed c u l t u r e s were i n v o l v e d . The ropy c o l o n i e s were m o r p h o l o g i c a l l y i n d i s t i n g u i s h a b l e from the non-ropy ones, thus, they were d i s t i n g u i s h e d by touching them with an i n o c u l a t i n g l o o p . A s t r i n g of v i s c o u s m a t e r i a l r e v e a l e d the presence of s l i m e . General growth media s u i t a b l e f o r the measurement of v i s c o s i t y was o b t a i n e d through the use of skim milk, and the c a s e i n d i g e s t media ( F o r s e n , 1966). Other l i q u i d media i n which good v i s c o s i t y development was recorded were the M17 medium ( T e r r a g h i and Sandine, 1975), and n e u t r a l i z e d cheese whey. No attempt was made to r e c o r d the d e t a i l s of the growth c h a r a c t e r i s t i c s i n the cheeBe whey and the M17 medium, other than that they support the s l i m e p r o d u c t i o n . The growth c h a r a c t e r i s t i c s of the ropy s t r a i n L416 i n skim m i l k at 18°C are shown i n F i g u r e 2. I t was not reasonably p o s s i b l e to study the growth r a t e s of a l l the a v a i l a b l e ropy c u l t u r e s , thus 39 a FIGURE 2. Growth curve o f cremoris l o n g i hhl6 i n skim milk at 18 C. • pH (average of 2 r e a d i n g s ) , # apparent v i s c o s i t y at constant shear r a t e of 500 per sec. (average of four r e a d i n g s ) , • l o g of CFU/ml. (average of the counts on 8 TJA p l a t e s ) . 39 o n l y s t r a i n L416 was chosen as i t showed a growth behaviour t y p i c a l of a l l ropy s t r a i n s . F i g u r e 2 shows that i n skim milk most a c i d p r o d u c t i o n and c e l l growth took p l a c e d u r i n g the f i r s t 20 hours of i n c u b a t i o n . The v i s c o s i t y development i n c r e a s e d up to 20 hours, and then g r a d u a l l y d e c r e a s e d . The r a t e of v i s c o s i t y decrease v a r i e d with the s t r a i n of the ropy b a c t e r i a ( F i g u r e 3 ) . Even though F i g u r e 2 shows a minimal decrease i n v i s c o s i t y a f t e r 48 hours (72 to 96 hour r e g i o n on the c u r v e ) , the v i s u a l o b s e r v a t i o n s showed that a f t e r 6 to 8 days of i n c u b a t i o n at room temperature a l l ropy c u l t u r e s l o s t t h e i r v i s c o s i t y . The ropy nature of the m i l k medium was a l s o l o s t . I t was p o s s i b l e to s e l e c t s t r a i n s with v a r i a b l e r a t e s of slime p r o d u c t i o n and v i s c o s i t y l o s s . From the v i s c o m e t r i c a n a l y s i s of the ropy sour m i l k i t i s apparent that there are two major f a c t o r s which c o n t r i b u t e to the t o t a l apparent v i s c o s i t y of a ropy m i l k product* F i r s t , and most important, the s l i m e produced by the b a c t e r i a l m i c r o f l o r a , and second, and very important c o n t r i b u t i o n , the a c i d c o agulated milk p r o t e i n s . These e f f e c t s are shown f o r a l l c u l t u r e s i n F i g u r e 3. T h i s F i g u r e shows that a non-ropy c o n t r o l c o n t r i b u t e d about 12 c p . to the e q u i l i b r a t e d apparent v i s c o s i t y at the shear r a t e of SOO per sec. at 18°C a f t e r 48 hours of i n c u b a t i o n , w h i l e a l l ropy s t r a i n s show much higher apparent v i s c o s i t y when measured under the same experimental c o n d i t i o n s . The d i f f e r e n c e i n e q u i l i b r a t e d apparent v i s c o s i t y between a l l other curves and the non-ropy c o n t r o l curve i n F i g u r e 3 e x e m p l i f i e s the c o n t r i b u t i o n to the v i s c o s i t y of the m i l k medium by the s l i m e of the ropy b a c t e r i a l s t r a i n s . I n attempting to c h a r a c t e r i z e the growth behaviour of the FIGURE 3. Apparent v i s c o s i t y at constant shear r a t e of 500 per sec. vs. i n c u b a t i o n time at constant temperature of 18°C r e l a t i o n s h p s i n skim milk f o r ropy s t r a i n s , and non-ropy l a c t i c b a c t e r i a . # non-ropy S. cremoris, T S_;_ l a c t i s l o n g i LLF, • S_^_ cremoris l o n g i 701, ^> S_;_ cremoris l o n g i 705 > H S_;_ cremoris l o n g i Lkl6. Each p o i n t represents an average of 3 readings. 1+1 ropy s t r a i n i n the medium devoid of the c o a g u l a b l e m i l k p r o t e i n s , the ropy c u l t u r e 701 was grown i n the .casein d i g e s t medium ( T a b l e 1 ) . S t r a i n 701 was chosen as i t produced m a r g i n a l l y b e t t e r v i s c o s i t y i n t h i s medium, than other s t r a i n s . The growth curve i s shown i n F i g u r e 4. I t i s e v i d e n t from t h i s graph that even though t h i s medium supports the slime p r o d u c t i o n , i t i s not as good a n u t r i e n t source as skim m i l k . I t does, however, prov i d e an o p p o r t u n i t y to conduct absorbance s t u d i e s on the ropy s t r a i n s , which can not be done i n m i l k . C l e a r whey permeate and the M17 medium can be used f o r the same purpose. V i s c o m e t r i c a n a l y s i s of the ropy milk has not been very e x t e n s i v e thus f a r , as i n d i c a t e d by the s c a r c i t y of l i t e r a t u r e on t h i s t o p i c . Thus, i t i s d i f f i c u l t to make a comparative e v a l u a t i o n of the c u r r e n t i n v e s t i g a t i o n s . As d i s c u s s e d i n the l i t e r a t u r e review, Forsen (1966) measured the v i s c o s i t y of the ropy c u l t u r e s a f t e r 4 hours of growth, however, no e x p e r i m e n t a l c o n d i t i o n s were p r o v i d e d . S o z i et a l . (1978) measured the c o n s i s t e n c y of a ropy c u l t u r e by u s i n g a g r a v i t a t i o n a l f r e e flow v l s c o s i m e t e r . T h i s instrument could not measure apparent v i s c o s i t y , thus p r o v i d i n g comparative measurements v a l i d o n ly f o r an instrument such as that d e s c r i b e d i n t h e i r study. Somewhat s i m i l a r instrument was used by Tsaregradskaya and G r l g o r c h u k (1982) to measure the v i s c o s i t i e s of ropy c u l t u r e s d u r i n g c u l t u r e s e l e c t i o n f o r sourcream p r o d u c t i o n . They used a 100 ml. p i p e t t e with an o u t l e t diameter of 4 mm, and measured the time of sample d i s c h a r g e , p r o v i d i n g e m p i r i c a l measurements of v i s c o s i t y . The disadvantage of t h i s method i s that t h i c k m i l k obscures the manlscus, as i t has a high a f f i n i t y f o r the w a l l s of the viscometer, making a l a r g e e r r o r i n measurements. The most complete, and the most 1+3 a FIGURE 1+ . Growth curve of cremoris l o n g i 701 i n case i n d i g e s t medium at l8°C. 4} pH (average of 3 r e a d i n g s ) , # apparent v i s c o s i t y at constant shear r a t e of 1500 per sec. (average o f 3 r e a d i n g s ) , B l o g apparent CFU/ml. (average of CFU i n 16 squares on Levy-Hausser chamber). L O G A P P A R E N T C.F.u/ m l . A P P A R E N T V I S C O S I T Y a t 1 5 0 0 s e c ' . 1 , c p . j — P H _L_ a VI standard, study of the v i s c o m e t r i c behaviour of the ropy l a c t i c s t r e p t o c o c c i i n s t e r i l e skim m i l k was c a r r i e d out by Maksimova et a l . (1982). These r e s e a r c h e r s used a r o t a r y viscometer (Rheotest-2) to d e s c r i b e r h e o l o g i c a l c h a r a c t e r i s t i c s of the ropy c u l t u r e s a f t e r they formed curd l n m i l k . However, no v i s c o m e t r i c data was p r o v i d e d on the c u l t u r e s d u r i n g the growth p e r i o d , or when the maximum s l i m e producton took p l a c e . T h i s data i s important to the d a i r y p l a n t o p e r a t o r d u r i n g the I n d u s t r i a l use of the ropy c u l t u r e s . T h i s r e s e a r c h , thus shows, not only that the growth of ropy b a c t e r i a can be f o l l o w e d by v i s c o s i t y measurements i n s t e a d of a c i d p r o d u c t i o n and colony c o u n t i n g ( F i g u r e 2 ) , but t h a t v i s c o s i t y of ropy m i l k i s a combined e f f e c t of m i l k c o a g u l a t i o n and slime p r o d u c t i o n ( F i g u r e 3 ) . Furthermore, d u r i n g t h i s v i s c o m e t r i c study i t was n o t i c e d that v i s c o s i t y was l o s t by ropy s t r a i n s under c e r t a i n growth and s t o r a g e c o n d i t i o n s . F u r t h e r v i s c o m e t r i c a n a l y s i s i s d i s c u s s e d l n s e c t i o n C2. 2. C u l t u r e I d e n t i f i c a t i o n P r e v i o u s r e s e a r c h e r s ( T r o i l i - P e t e r s s o n , 1899; N i l s s o n , 1950; Sundman, 1953a; Fo r s e n , 1966) have found that the ropy v a r i a n t s of S. l a c t i s and S. c r e m o r i s were most o f t e n the major c o n s t i t u e n t s of the m i c r o f l o r a i n S c a n d i n a v i a n ropy m i l k . These b a c t e r i a t o g e t h e r with S. d i a c e t y l a c t i s c o n s t i t u t e what i s c a l l e d group N s t r e p t o c o c c i . T h e r e f o r e , the ropy c u l t u r e s were s u b j e c t e d to the t e s t s which were designed to d i s t i n g u i s h between the members of group N s t r e p t o c o c c i ( T a b l e 2 ) . Of the t e s t s used the key d i f f e r e n t i a t i n g f a c t o r s were the p r o d u c t i o n of ammonia and ot h e r a l k a l i n e substances, such as amines, from a r g i n i n e , the growth at pU 9.2, and the growth i n 4Z N a C l . S. l a c t i s produces ammonia from a r g i n i n e , grows at pH 9.2 and i n the presence of 4Z NaCl, while S.  cremoris does not ( H a r r i g a n and McCance, 1966; Buchanan et a l . , 1974). The gas p r o d u c t i o n from c i t r a t e In s e m i - s o l i d agar medium was used to d i f f e r e n t i a t e between S. l a c t i s and S. d i a c e t y l a c t l s . S. d i a c e t y l a c t l s produces gas from c i t r a t e , w h i l e S. l a c t i s does not. The same d i f f e r e n t i a t i o n c o u ld be made on the b a s i s of a c e t o i n p r o d u c t i o n (Voges-Proskauer r e a c t i o n ) , where S. l a c t i s does not produce a c e t o i n from g l u c o s e , w h i l e S. d i a c e t y l a c t l s does ( H a r r i g a n and McCance, 1 9 6 6 ) . A c e t o i n , C 4 H g 0 2 i s a p r e c u r s o r to the m a j o r b u t t e r f l a v o u r compound d l a c e t y l , C ^ H ^ C ^ ( W i n d h o l z , 1976). S. d i a c e t y l a c t l s p r o d u c e s l a r g e amounts of t h i s compound, thus c o n t r i b u t i n g to the f l a v o u r of the m e s o p h i l i c c u l t u r e d d a i r y p r o d u c t s . Ammonia p r o d u c t i o n from a r g i n i n e was t e s t e d i n two media; a r g i n i n e b r o t h , and B.C.P.A.. In a r g i n i n e b r o t h , the d e t e c t i o n of a l k a l i n e r e a c t i o n was done by a c o l o r change r e a c t i o n of N e s s l e r ' s reagent. B.C.P.A. d e t e c t s pH change. I t i s p u r p l e or dark blue at n e u t r a l pH, however, when I t comes i n contact w i t h a c i u (pH < 5.2), i t turns y e l l o w . Thus, a l l c o l o n i e s on B.C.P.A. appear white at f i r s t , they a l l form y e l l o w zones w i t h i n f i r s t two days of growth, and then a r g i n i n e u t i l i z i n g b a c t e r i a (S.  l a c t i s ) e l i m i n a t e the y e l l o w z o n e s , i n d i c a t i n g an a l k a l i n e r e a c t i o n . T a b l e 2 shows that of the s t r a i n s t e s t e d LLF appears to be a v a r i a n t of S. l a c t i s , and the s t r a i n s L416, 701, and 705 TABLE 2. identification of ropy strains isolated from Scandinavian ropy milk, (all results are based on triplicate readings) Ropy Strains Controls Biochemical Tests LLP L416 701 705 ATCC 19435* ATCC 19257° Litmus ACRC ACR ACR ACR ACR ACR Bilk Arginine - e «• broth BCPA* +f _g _ - • Citrate agar pH 9.2 •* -J 4* NaCl -1 40*C +1 • Identification of S.lact S.crem fl.crera S.crem Ropy Strains a S. lactiB standard culture (American Type Culture Collection). D S. cremoriB standard culture (American Type Culture Collection). c Acid, curd, reduction. d Represents ammonia production from arginine. * Represents no ammonia production from arginine. * Represents alkaline reaction from arginine. 9 Represents no alkaline reaction from arginine. n Represents no gas production from citrate. 1 Represents growth, as recoreded by absorbance >. -27 ults at 520 nm. 3 Represents no growth, as recorded by absorbance at 520 nm. * Bromocresol purple agar. 1 Deviates from Bergey's Manual of Determinative Bacteriology, 8th ed. U7 appear to the ropy v a r i a n t s of S. c r e m o r i s . S i n c e the ropy S.  l a c t i s has a l r e a d y been named S. l a c t i s l o n g i ( T r o i l i - P e t e r s s o n , 1899; N i l s s o n and N i l s s o n , 1950) i t seems a p p r o p r i a t e to name the ropy S. c r e m o r i s v a r i a n t s S t r e p t o c o c c u s c r e m o r i s l o n g i . 3. C u l t u r e S t a b i l i t y The S c a n d i n a v i a n ropy e d i b l e m ilk has probably been produced f o r c e n t u r i e s i n the c o o l c e l l a r s of North European homes. It must have been passed dovn from g e n e r a t i o n to g e n e r a t i o n p r e s e r v i n g i t s v i s c o u s c h a r a c t e r , as the s c i e n c e , or the a r t , of l o c a t i n g the o r i g i n of the c u l t u r e has slowly become remote, or p o s s i b l y vanished a l t o g e t h e r . Current l i t e r a t u r e r e p o r t s a number of supposed methods f o r i n i t i a t i n g r o p i n e s s i n m i l k . These range from u s i n g the black f o r e s t s n a i l s i n milk, to rubbing the i n s i d e s of the m i l k p a i l s with the l e a v e s of butterwort, known as T a e t t e graes i n Norway (Thomas and M c Q u i l l i n , 1953), to the a b i l i t y of S. cremoris to p r o d u c e c a p s u l a r m a t e r i a l when grown at 10-18°C ( N i l s s o n and N i l s s o n , 1 9 5 0 ) , or i n d u c t i o n of slime p r o d u c t i o n by chemical agents ( F o r s e n and V e i l i - M i e s , 1981). N i l s s o n and N i l s s o n (1950) s t a t e d that they were never able to f i n d a person who could "perform the m i r a c l e " of producing the ropy m i l k v i a any other method but i n o c u l a t i o n of milk with p r e e x i s t i n g ropy m i l k . Even though i t i s s t i l l not known with c e r t a i n t y how t h i s product o r i g i n a t e d , i t i s known that microorganisms are r e s p o n s i b l e f o r the observed r o p i n e s s . I t i s a l s o known that the l e v e l of r o p i n e s s i n the product v a r i e s a c c o r d i n g to the r a t i o of the ropy to non-ropy ho b a c t e r i a i n the product, which i s , at l e a s t i n p a r t , i n f l u e n c e d by the temperature of i n c u b a t i o n ( W o l f e r s t e t t e r , 1969). Most authors who d i d r e s e a r c h on S c a n d i n a v i a n ropy m i l k thus f a r , h i n t e d that r e g a r d l e s s of which bacterium or b a c t e r i a were r e s p o n s i b l e f o r the slime p r o d u c t i o n i t d i d not r e t a i n i t s a b i l i t y to produce slime i n d e f i n i t e l y . T h i s may have been the major f a c t o r d e t e r m i n i n g the use of the ropy c u l t u r e s i n d a i r y i n d u s t r y . Our o r i g i n a l i n t e r e s t i n t h i s r e s e a r c h was prompted by a person from a l o c a l Swedish community i n B r i t i s h Columbia, Canada, who brought a sample of ropy m i l k to the l a b o r a t o r y complaining t h a t he could not m a i n t a i n constant v i s c o s i t y p r o d u c t i o n by h i s mixed c u l t u r e . As a r e s u l t he could not m a i n t a i n constant q u a l i t y of the p r o d u c t . We t r i e d , and were u n s u c c e s s f u l i n m a i n t a i n i n g constant v i s c o s i t y i n that product. We then, obtained pure ropy c u l t u r e i s o l a t e s , and found t h a t , to no one's s u r p r i s e , they too l o s t t h e i r a b i l i t y to produce slime even f a s t e r than the mixed c u l t u r e s . The r a t e of l o s s seemed to be p r o p o r t i o n a l to the number of t r a n s f e r s , the l e n g t h of i n c u b a t i o n at growth temperature, and to the temperature of storage f o l l o w i n g the i n c u b a t i o n p e r i o d . An experiment was designed with the purpose to r e c o r d the p r o p o r t i o n of the ropy c e l l s as r e l a t e d to the t o t a l number of CFU i n the fermented ropy m i l k , as a f u n c t i o n of the number of t r a n s f e r s , and the l e n g t h of i n c u b a t i o n at growth temperature. Table 3 shows the percent ropy CFU as r e l a t e d to the number of t r a n s f e r s of the c u l t u r e s to the f r e s h m ilk medium. Ten t r a n s f e r s were made every 4 8 hours and a p p r o p r i a t e d i l u t i o n s s u r f a c e p l a t e d on skim m i l k agar p l a t e s , to determine the percent ropy CFU f o r that t r a n s f e r . The k9 TABLE 3- Stability of al i a * production by the ropy atralna of lactic atreptocoecl •a the percent of ropy C.V.V. on aklm milk agar. Ropy Strains Transfer Number Tlscb 0>r) L416« 701« 70 5« Total f Xopy? tRopy Total Ropy tRopy Total Ropy %Ropy Total Ropy sRopy 1 96 146 147 194 242 265 124 26 _c 552 124 26 103 100 100 19 341 77 341 77 100 100 26 74 26 74 100 100 29 66 279 29 66 279 100 100 100 2 102 146 195 491 394 80 515 515 100 675 675 100 398 398 100 3 102 150 136 64 47 715 715 100 656 656 100 43 43 100 4 105 120 150 362 0 0 455 4 55 100 304 304 100 180 129 72 5 74 103 168 175 288 554 225 41 600 600 100 S91 579 98 - - -503 0 0 - - - - -291 151 257 133 88 88 6 75 97 129 133 291 0 0 531 315 59 427 341 80 375 347 93 7 50 83 170 215 996 87 9 25 12 48 83 62 75 1401 1255 90 8 100 118 120 134 0 0 184 79 43 427 247 58 1207 729 60 9 118 120 1B7 0 0 880 738 84 88 76 86 216 85 39 10 73 118 616 14 2 228 164 72 31 23 74 1833 936 51 * Serial transfer Into skim ailk every 48 hrs. ° Surface spread plate Incubation time. c Mot counted. d Petri plates incubated at 22*C. • Petri plates incubated at 1B*C. ' Total number of colonies oountad. 9 Total number of ropy colonies ln f. 50 r e s u l t s In T a b l e 3 show that percent ropy CFU decreased c o n s i d e r a b l y w i t h i n ten t r a n s f e r s , e s p e c i a l l y f o r the c u l t u r e LLF. T h i s c u l t u r e was incubated at somewhat h i g h e r t e m p e r a t u r e (22°C) on the recommendation of i t s s u p p l i e r (Food Research I n s t i t u t e , Ottawa), thus, i t was not p o s s i b l e to determine whether the i n c r e a s e d i n s t a b i l i t y could be a t t r i b u t e d 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 temperature, or to the d i f f e r e n c e i n the s t r a i n s ' a b i l i t y to maintain constant v i s c o s i t y p r o d u c t i o n . In any case, the purpose of the experiment was to study the e f f e c t of t r a n s f e r r i n g the c u l t u r e s on t h e i r a b i l i t y to m a i n t a i n constant v i s c o s i t y p r o d u c t i o n i n m i l k . I t was found (Table 3) that percent v i s c o u s CFU decreases with the number of c u l t u r e t r a n s f e r s . I t may be argued that t h i s c o n c l u s i o n i s i n v a l i d because the percent ropy CFU was determined on s o l i d medium, while the t r a n s f e r s were done i n l i q u i d medium, c o n d i t i o n s of which d i f f e r c o n s i d e r a b l y . T h i s i s a v a l i d argument, however i t i s the experience of the author that the v i s c o s i t y of the ropy m i l k decreases with time as a r e s u l t of the c u l t u r e t r a n s f e r r i n g . T h i s phenomenon was noted by p r e v i o u s r e s e a r c h e r s ( T r o i l i - P e t e r s s o n , 1899; Olson-Sop, 1912; N i l s s o n and N i l s s o n , 1950; Sundman,1953a; Forsen, 1966; W o l f e r s t e t t e r , 1969), however, nobody r e l a t e d i t to the number of c u l t u r e t r a n s f e r s . Forsen (1973, 1974, 1979) showed that the slime forming a b i l i t y was l o s t immediately when the ropy b a c t e r i a were grown at 30°C. T h i s was not the e x p e r i e n c e of the present author. I t was p o s s i b l e t o p r o d u c e s l i m e at 37 t o 39°C however, n e v e r at 40°C (da t a not shown). I t i s p r e d i c t e d that at these temperatures the s l i m e forming a b i l i t y would be l o s t w i t h i n two or three t r a n s f e r s . The p r a c t i c a l i m p l i c a t i o n s of these f i n d i n g s are that i n 51 TABLE U. Stability of «ll»e production by 8. lactis longi LI? In liquid skin ailk M the percent of the ropy C.r.u. on akin s i l k agar". Days of Incubation at 22*C Transfer Number8 2 4 6 Total 0 Ropyc % Ropy Total Ropy % Ropy Total Ropy % Ropy 0 482 455 94 516 6 1 350 3 1 1 1032 983 86 946 0 0 969 0 0 2 3 542 d 441 81 550 3 1 520 0 0 4 5 576 290 50 621 0 0 660 0 0 6 691 71 10 643 0 0 677 0 0 7 944 90 10 996 0 0 894 0 0 8 633 61 10 590 0 0 812 0 0 9 68B 44 6 691 49 7 832 0 0 10 438 42 10 356 12 3 344 0 0 * Transfers into fresh skim s i l k were made at exactly 50 h. Intervals using a 5t (v/v) Inoculum. D Total number of colonies counted. c Total number of ropy colonies in b. d Not determined. 52 order to m a i n t a i n high and constant v i s c o s i t y p r o d u c t i o n i n ropy c u l t u r e s , which i s very important i n m a i n t a i n i n g the product q u a l i t y and the commercial I n t e g r i t y , one should s u r f a c e p l a t e the c u l t u r e s a f t e r approximately f i v e c o n s e c u t i v e t r a n s f e r s and develop new c u l t u r e s t o c k s from the ropy c o l o n i e s taken o f f the p l a t e s . T h i s method runs a r i s k of the e v e n t u a l l o s s of other g e n e t i c homogeneity among the s t r a i n s , such as the l e v e l of a c i d p r o d u c t i o n , and the p r o d u c t i o n of f l a v o u r i n g compounds, however, i t does assure constant l e v e l of v i s c o s i t y p r o d u c t i o n . The other problems c o u l d be c u r t a i l e d by simple t a s t e t e s t s e l e c t i o n of the s t a r t e r s , before they are used i n l a r g e s c a l e p r o d u c t i o n . The second h a l f of the c u l t u r e s t a b i l i t y study d e a l t w i t h the e f f e c t of the p l a t e i n c u b a t i o n on the p r o p o r t i o n of the ropy CFU on the s u r f a c e of the skim milk agar p l a t e s , as w e l l as the e f f e c t of the number of t r a n s f e r s on the p r o p o r t i o n of ropy CFU i n the milk medium (T a b l e A ) . The r e s u l t s show that the p r o p o r t i o n of the CFU decreased with the time of c u l t u r e i n c u b a t i o n at the i n c u b a t i o n temperature of 22°C. They a l s o c o n f i r m the c o n c l u s i o n , drawn from the r e s u l t s i n T a b l e 3, that the percent ropy c o l o n i e s decreased w i t h the i n c r e a s e d number of t r a n s f e r s . A n o t a b l e drawback of t h i s experiment was that the i n c u b a t i o n of the chosen c u l t u r e (LLF) was done on s o l i d medium, not i n l i q u i d m i l k . In t h i s case, the d i f f e r e n c e i n growth c o n d i t i o n s between the two media may have a more severe a f f e c t than i n the p r e v i o u s experiment. Thus, i t i s recommended t h a t , i n f u t u r e , the c u l t u r e be kept i n l i q u i d m i l k at i n c u b a t i o n temperature, and the p l a t i n g s be done at given time i n t e r v a l s , every two days f o r example, 53 on s o l i d medium. Regardless of t h i s drawback, these r e s u l t s show that there e x i s t s an agent which d i s t r o y s the slime a f t e r i t has been produced by the c e l l s . T h i s p a r a l l e l s the v i s c o s i t y l o s s i n a l i q u i d m i l k medium i f the milk i s Incubated at the growth temperature f o r a few days. The v i s c o s i t y l o s s l s h a l t e d or completely e l i m i n a t e d i f the product i s incubated below 10°C a f t e r the maximum slime p r o d u c t i o n . In c o l d storage the v i s c o u s nature of the product i s p r e s e r v e d i n d e f i n i t e l y . These o b s e r v a t i o n s have very important p r a c t i c a l i m p l i c a t i o n s . I t i s now p o s s i b l e to produce the ropy m i l k with c o n s t a n t l y high v i s c o s i t y c h a r a c t e r , even i f one i s working wi t h u n s t a b l e c u l t u r e s . To m a i n t a i n high v i s c o s i t y i n the product one should p e r i o d i c a l l y develop new ropy c u l t u r e stocks from a ropy c o l o n y . The manufactured products should be r e f r i g e r a t e d immediately a f t e r the d e s i r a b l e l e v e l of the slime p r o d u c t i o n i s a c h i e v e d . The r e a l s o l u t i o n to t h i s problem, most l i k e l y , l i e s i n the g e n e t i c m a n i p u l a t i o n of these c u l t u r e s . I t has been known f o r sometime that such t r a i t s as the l a c t i c a c i d p r o d u c t i o n , p r o t e o l y t i c a c t i v i t y , and the p r o d u c t i o n of f l a v o u r and b a c t e r i o c i d a l compounds, may be coded f o r by the g e n e t i c m a t e r i a l impregnated i n the extrachromosomal, double s t r a n d e d , DNA fragments c a l l e d plasmids, i n l a c t i c a c i d b a c t e r i a (Davies and Gasson, 1981; McKey, 1982; R e f s t r u p and Vogensen, 1980). Plasmids may be l o s t by the b a c t e r i a d u r i n g c e l l d i v i s i o n , r e s u l t i n g i n l o s s of the phenotype which was coded f o r by t h a t p l a s m i d . They can a l s o be t r a n s f e r r e d to g e n e t i c a l l y r e l a t e d b a c t e r i a l c e l l s ( h o s t s ) to induce the phenotype which they code f o r 5^ i n the host c e l l (Brock, 1979). However u n t i l the g e n e t i c m a t e r i a l of i n t e r e s t becomes the I n t e g r a l part of the chromosomal genome, the t r a i t i n q u e s t i o n i s never s t a b i l i z e d i n the c e l l . The i n t e g r a t i o n of the plasmid DNA i n t o the chromosomal DNA can be achieved v i a a v i r u s (temperate b a c t e r i o p h a g e ) , or by an a p p r o p r i a t e n u c l e a s e / l y g a s e enzyme combination (Brock, 1979). E i t h e r way, the g e n e t i c m a t e r i a l becomes a permanent part of the b a c t e r i a l chromosome, s t a b i l i z i n g the t r a i t permanantlty. In the case of the S. l a c t i s l o n g i and S. cremoris  l o n g i , i t i s p o s s i b l e that the s l i m e p r o d u c t i o n i s coded f o r by plasmid DNA. Flasmids have been i s o l a t e d from these s t r a i n s by the author (unpublished d a t a ) , however, no c o n n e c t i o n with the slime p r o d u c t i o n has yet been demonstrated. T h i s work should be continued, and i f i t i s found that the slime p r o d u c t i o n i s indeed coded f o r by the plasmid DNA, t h i s plasmid should be impregnated i n t o the chromosomal DNA, thus s t a b i l i z i n g slime p r o d u c t i o n . A . A n t i b a c t e r i a l A c t i v i t y of S. c r e m o r i s l o n g i LA16 Most b a c t e r i a produce agents that i n h i b i t or k i l l other b a c t e r i a , o f t e n those of the same or r e l a t e d s p e c i e s . These agents are u s u a l l y p r o t e i n s i n nature, and they are c a l l e d b a c t e r i o c i n s to d i s t i n g u i s h them from a n t i b i o t i c s , which have wider spectrum of a c t i v i t y (Brock, 1979). B e t t e r known b a c t e r i o c i n s a r e : n i c i n , produced by S. l a c t i s ; d i p l o c o c c i n , produced by S.  cremoris (Lawrence and Thomas 1979 ) ; and a c i d o p h i l i n (Shahani, 1979) or a c i d o l i n ( M i k o l a j c i k and Hamdan, 1975) produced by L.  a c i d o p h i l u s . Other l a c t i c a c i d b a c t e r i a such as S. d i a c e t y l a c t l s , Leuconostoc c 1 t r o v o r u m . ( B r a n e n et a l . , 1 9 7 5 ) , S. t h e r m o h i l u s 55 ( P u l u s a n i et a l . , 1979), and L. b u l g a r i c u s ( S i ngh et a l . , 1979) produce b a c t e r i o c i n s which are heat s t a b l e low mol e c u l a r weight p e p t i d e s with v a r i e d a n t i b a c t e r i a l a c t i v i t y . In a d d i t i o n to b a c t e r i o c i n s , l a c t i c a c i d , low pH, a c e t i c a c i d , and hydrogen peroxide are o f t e n I m p l i c a t e d i n a n t i m i c r o b i l a c t i v i t y . These are produced i n v a r y i n g q u a n t i t i e s by l a c t i c a c i d b a c t e r i a . S. l a c t i s l o n g i was shown to be a n t a g o n i s t i c to many f u n g i ( N i l s s o n and N i l s s o n , 1955), mostly to those of the genus P e n i c i l l i u m . In a l a t e r r e p o r t ( 1 9 5 8 ) , t h e s e authors demonstrated the a n t i b a c t e r i a l a c t i v i t y of S. l a c t i s l o n g i to E. c o l i , S.  aureus, and C l o s t r e d i u m s p e c i e s . They found t h a t the i n h i b i t o r y substance d i d not d i f f u s e through agar, but the slime i t s e l f was not as i n h i b i t o r y as the b a c t e r i a l s u s p e n s i o n s . The a n t i b a c t e r i a l a c t i v i t y of the ropy c u l t u r e s has been i m p l i c a t e d l n the long s h e l f l i f e of the product ( N i l s s o n and N i l s s o n , 1958), thus, we used a method s i m i l a r to that of Goel et a l . (1971) to study the f a t e of E . c o l l ATCC 25922, S. a u r e u s ATCC 25923, and P. f l u o r e s c e n s b l o p t y p e A, i n an 18-hour f e r m e n t a t i o n of S.  crem o r i s l o n g i . These organisms a r e the u s u a l p o e t p a s t e u r i z a t i o n contaminants of m i l k . They have been used as t e s t organisms i n s t u d i e s of the a n t i b a c t e r i a l a c t i v i t y by l a c t i c a c i d b a c t e r i a by many authors (Amster and J o s t , 1980; Branen et a l . , 1975; Shahani et a l . , 1977; P u l u s a n i et a l . , 1979). A no n - r o p y S. c r e m o r i s ATCC 19257 was used as a c o n t r o l . The purpose of t h i s s t u d y was to f i n d out whether S.  cremoris l o n g i i n h i b i t s the t e s t organisms i n a s i m u l a t e d home r e f r i g e r a t i o n environment ( 1 0 ° C ) and i f so, to compare the i n t e n s i t y of i t s i n h i b i t i o n w i t h t h a t of the non-ropy S. cr e m o r i s s t r a i n . 56 F i g u r e 5 shows t h a t S. a u r e u s was c o m p l e t e l y i n h i b i t e d w i t h i n 5 days of s t o r a g e at 10°C by bo t h S. c r e m o r i s s t r a i n s , w h i l e without S. c r e m o r i s ( c o n t r o l ) t h i s b a c t e r i a grew c o n s t a n t l y d u r i n g the 15 days of c o l d s t o r a g e . The pU and the a c i d l e v e l s were s i m i l a r f o r both s t r a i n s of S. c r e m o r i s (Table 5). F i g u r e 6 shows t h a t E . c o l i was a l s o i n h i b i t e d e q u a l l y by both S. cr e m o r i s s t r a i n s . The r a t e of i n h i b i t i o n was lower than t h a t of S. aureus, t a k i n g i n e x c e s s of 10 days f o r complete i n h i b i t i o n . The a c i d development was m a r g i n a l l y b e t t e r f o r the ropy s t r a i n ( T a ble 6), but i t had no s i g n i f i c a n t e f f e c t on the d i f f e r e n c e i n i n h i b i t i o n . I t i s n o t e w o r t h y t h a t i t took t h i s s t r a i n of E. c o l l 5 days of i n c u b a t i o n to adapt to the adverse environment, as i t s t a r t e d dying d u r i n g the f i r s t 5 days of c o l d s t o r a g e , but i t recovered and grew s u b s t a n t i a l l y t h e r e a f t e r ( r e f e r to c o n t r o l curve, F i g u r e 6). T h i s phenomenon was observed by Goel et a l . (1971), who found that i n b u t t e r m i l k 4 out o f 20 samples produced g r e a t e r than 50% r e d u c t i o n i n E. c o l i numbers w i t h i n 24 hours of storage at 7.2°C. A f t e r 4 days the p o p u l a t i o n of t h i s b a c t e r i a n e a r l y always decreased by 50%. The most s e n s i t i v e to the b a c t e r i a l i n h i b i t i o n by the S. cre m o r i s s t r a i n s was P. f l u o r e s c e n s , the b a c t e r i a most o f t e n a s s o c i a t e d with the s p o i l a g e of f r e s h m i l k d u r i n g c o l d s t o r a g e . Both s t r a i n s of S. cr e m o r i s a c h i e v e d c o m p l e t e i n h i b i t i o n w i t h i n 1 day of sto r a g e at 10°c ( F i g u r e 7) . No p s y c h r o t r o p h s were d e t e c t e d when 1 ml. of the t e s t f e r m e n t a t i o n was p l a t e d on n u t r i e n t agar, during the r e s t of the storage p e r i o d . T h i s was expected, as Branen et a l . (1975) found th a t P. f l u o r e s c e n s was the most s e n s i t i v e of the b a c t e r i a 57a FIGURE 5- S u r v i v a l curve o f S. aureus ATCC 25923 at 10°C i n p a s t e u r i z e d skim milk ( • ), i n skim m i l k fermented by cremoris ATCC 19257 f o r 18 hours at 22°C ( • ), and i n skim milk fermented w i t h cremoris l o n g i Lkl6 f o r 18 hours at 22°C ( • ). Each p o i n t on the graph represents an average count o f 5 BPA p l a t e s incubated at 32°C f o r 2k hours. 8r 0 5 10 D A Y S at 10°C 58 Table 5. A c i d i t y m o n i t o r i n g of the S. c r e m r i s skim milk f e r m e n t a t i o n s when c o n t a m i n a t e d w i t h S. aureus ATCC 25923 a f t e r l a c t i c f e r m e n t a t i o n and s t o r e d at 10°C f o r 15 days. A l l numbers r e p r e s e n t an average of 2 r e a d i n g s . C o n t r o l 8 ATCC 1 9 2 5 7 b L A 1 6 C Days of - — — — — — — — - — Storage pH T . A . d ( % ) pH T.A.(%) pH T.A.(Z) 0 6.60 0.16 A.30 0.87 A.30 0.89 10 6.55 0.2A A.25 0.92 A.30 0.96 15 6.AO 0.37 A.AO 0.91 A.30 0.96 a) Skim milk c o n t r o l . I t was not f e r m e n t e d by S. cremoris b e f o r e i t was c o n t a m i n a t e d w i t h S. a u r e u s ATCC 25923 and s t o r e d a t 1 0°C. b) Standard c u l t u r e of S. c r e m o r i s (American Type C u l t u r e C o l l e c t i o n ) i n o c u l a t e d i n skim m i l k at 5% (v/v) l e v e l of inoculum,to complete f e r m e n t a t i o n at 22°C before contamination and s t o r a g e a t 1 0°C. c) S. cre m o r i s l o n g i LA16 i n o c u l a t e d i n skim m i l k at 5% (v/v) l e v e l of Inoculum to c o m p l e t e f e r m e n t a t i o n at 22°C before c o n t a m i n a t i o n and s t o r a g e at 10°C. d) T i t r a t a b l e a c i d i t y as determined by t i t r a t i o n a g a i n s t 0.1 N NaOH. 59 a FIGURE 6 . S u r v i v a l curves of E^ c o l i ATCC 25922 at 10°C i n pasteurized skim milk ( • ), i n skim milk fermented by cremoris ATCC 19257 at 22°C for 18 hours ( • ), and i n skim milk fermented by cremoris longi LUl6 at 22°C f or 18 hours ( • ). Each point on the graph represents an average count of 5 VRBA plates incubated at 32°C f o r 2h hours. 59 D A Y S at 10°C 6 0 Table 6. A c i d i t y m o n i t o r i n g of S. c r e m o r i s skim m i l k f e r m e n t a t i o n s when c o n t a m i n a t e d w i t h E. c o l i ATCC 25922 a f t e r l a c t i c f e r m e n t a t i o n and s t o r e d at 10°C f o r 15 days. The numbers represent an average of 2 r e a d i n g s . C o n t r o l 3 ATCC 1 9 2 5 7 b L 4 1 6 c Days of - — — — — — Storage pH T . A . d ( % ) pH T.A.(%) pH T.A.(%) 0 6.65 0.17 4.30 0.88 4.30 0.91 10 6.85 0.19 4.30 0.89 4.30 0.94 15 7.20 0.19 4.30 0.93 4.35 0.96 a) Skim m i l k c o n t r o l . I t was not f e r m e n t e d by S. cremoris b e f o r e i t was c o n t a m i n a t e d w i t h S. a u r e u s ATCC 25923 and s t o r e d a t 1 0 ° C . b) Standard c u l t u r e of S. c r e m o r i s (American Type C u l t u r e C o l l e c t i o n ) i n o c u l a t e d i n skim milk at 5% (v/v) l e v e l of inoculum to complete f e r m e n t a t i o n at 22°C before contamination and s t o r a g e a t 1 0 ° C . c) S. cremoris l o n g i L416 i n o c u l a t e d i n skim m i l k at 5% (v/v) l e v e l of inoculum to c o m p l e t e f e r m e n t a t i o n at 22°C before c o n t a m i n a t i o n and s t o r a g e at 10°C. d) T i t r a t a b l e a c i d i t y as determined by t i t r a t i o n a g a i n s t 0.1 N NaOH. 6 l a FIGURE 7 . S u r v i v a l curves of fluorescens biotype A at 10 C i n pas t e u r i z e d skim m i l k ( • ), i n skim milk fermented by cremoris ATCC 19257 at 22°C f o r 18 hours and i n skim m i l k fermented'by cremoris  l o n g i Lkl6 at 22°C f o r 18 hours ( • ). Each p o i n t on the graph represents an average count of 5 n u t r i e n t agar p l a t e s incubated at 10°C f o r 8 days. 61 62 T a b l e 7. A c i d i t y m o n i t o r i n g of S. c r e m o r i s skim m i l k f e r m e n t a t i o n s when c o n t a m i n a t e d w i t h P. f l u o r e s c e n s biotype A a f t e r l a c t i c f e r m e n t a t i o n and s t o r e d at 10°C f o r 15 d a y s . The numbers r e p r e s e n t an average of 2 r e a d i n g s . Days of Storage C o n t r o l 3 ATCC 1 9 2 5 7 b L 4 1 6 c pH T . A . d ( % ) pH T .A.(%) PH T.A.(%) 0 6.60 0.16' A.30 0.87 4.35 0.91 10 6.80 0.20 4.30 0.93 4.30 0.92 15 7 .00 0.27 4.35 0.91 4.35 0.98 a) Skim milk c o n t r o l . I t was not f e r m e n t e d by S. cremoris b e f o r e i t was c o n t a m i n a t e d w i t h S. a u r e u s ATCC 25923 and s t o r e d a t 1 0 ° C . b) Standard c u l t u r e of S. c r e m o r i s (American Type C u l t u r e C o l l e c t i o n ) i n o c u l a t e d i n skim milk at 5% (v/v) l e v e l of inoculum to complete f e r m e n t a t i o n at 2 2 ° b e f o r e contamination and s t o r a g e a t 1 0 ° C . c) S. c r e m o r i s l o n g ! L416 i n o c u l a t e d i n skim milk at 5% (v/v) l e v e l of inoculum to c o m p l e t e f e r m e n t a t i o n at 22°C before c o n t a m i n a t i o n and s t o r a g e at 10°C. d) T i t r a t a b l e a c i d i t y as determined by t i t r a t i o n a g a i n s t 0.1 N NaOH. 63 which he t e s t e d , to the a n t i m i c r o b i a l a c t i v i t i e s of Leuconostoc i c i t r o v o r u m and S. d i a c e t y l a c t i s , t h u s , he chose i t f o r a t e s t organism i n h i s s t u d i e s of the a n t i b a c t e r i a l a c t i v i t i e s i n l a c t i c c u l t u r e s . From these experiments i t a p p e a r s that even though S.  c r e m o r i s l o n g i i s very i n h i b i t o r y to the chosen r e p r e s e n t a t i v e s of the common mi l k s p o i l a g e b a c t e r i a , i t i s not more i n h i b i t o r y than the non-ropy s t r a i n of S. c r e m o r i s (ATCC 19257). S i m i l a r r e s u l t s were obtained when t h i s b a c t e r i a was used i n c u l t u r e d creamed c o t t a g e cheese d r e s s i n g ( d a t a not shown). In t h i s product, no s i g n i f i c a n t i n h i b i t i o n of y e a s t s and molds was observed, which i s c o n t r a d i c t o r y to N i l s s o n and N i l s s o n (1955). Perhaps none of the contaminating molds were of the genus t e s t e d by these a u t h o r s . In p r a c t i c a l terms t h i s study showed that i f the b a c t e r o c i d a l e f f e c t s of S. c r e m o r i s l o n g i L416 are taken i n c o n s i d e r a t i o n , i t i s not more e f f e c t i v e than a non-ropy s t r a i n ATCC 19257. However, Sundman (1953a) r e p o r t e d that the s h e l f l i f e of the ropy m i l k was l o n g e r than th a t of other sour m i l k s . T h i s may perhaps be a t t r i b u t e d to the a b i l i t y of the ropy b a c t e r i a to b e t t e r s u r v i v e a c i d i c c o n d i t i o n s and the c o l d temperature storage (see s e c t i o n 5 ) . In a recent experiment, i t took l e s s than 24 h o u r B to c o a g u l a t e milk at 22°C f o r a ropy b u t t e r m i l k sample which was k e p t a t 4-6°C f o r 53 days. The sample was i n i t i a l l y i n o c u l a t e d w i t h 3Z (v/v) of commercial b u t t e r m i l k s t a r t e r , 15% o f which was S. c r e m o r i s l o n g i . A f t e r 53 days of c o l d storage i t was t r a n s f e r e d to f r e s h . s t e r i l e m i l k at 3% i n o c u l a t i o n l e v e l . Ropy b a c t e r i a predominated i n t h i s f e r m e n t a t i o n , as the product was completely ropy. By comparison, the 61+ normal buttermilk which was treated in the same manner took over 48 hours to coagulate. Even then the curd was very soft, which indicated r e l a t i v e l y low acid production, and possibly low b a c t e r i a l numbers. 5. Compatibility of S. cremoris l o n g i L416 With the Commercial  Buttermilk S t a r t e r As mentioned in section 4, most bacteria produce a n t i b a c t e r i a l substances which are antagonistic to other bacteria. Thus, i t was important to study the compatibility of S. cremoris  longi with the mixed cultures of commercial buttermilk, i f the ropy s t r a i n was to be used i n commercial buttermilk production. From the author's general experience with this culture i t was observed that they grew together without any v i s i b l e deleterious e f f e c t s , however, microbiological proof was needed. Thus, an experiment was designed where the culture L416 was used at a varied percent of the t o t a l buttermilk sta r t e r inoculum. The purpose of the experiment was to study the rel a t i o n s h i p between the CFU of the ropy culture and those of the non-ropy buttermilk starter mixture, during fermentation and during cold storage for the duration of the shelf l i f e of buttermilk. Figure 8 shows the t o t a l CFU of the culture L416 and the buttermilk starter cultures i n skim milk when grown and stored under i d e n t i c a l conditions independently of each other. It shows that BMS CFU constantly decreased upon the onset of cold temperature storage. The ropy culture survived the cold storage well for 10 days and then decreased i n numbers by greater than one log cycle, i n d i c a t i n g that when grown by i t s e l f i t sta r t s dying before the end of the shelf l i f e of buttermilk. The observed changes in b a c t e r i a l numbers during 65a FIGURE 8 . R e f r i g e r a t e d s t o r a g e s u r v i v a l c u r v e s o f §_;_ c r e m o r i s l o n g i Lhl6 ( • ) and c o m m e r c i a l b u t t e r m i l k s t a r t e r ( # ), when grown i n d e p e n d e n t l y o f each o t h e r i n s k i m m i l k . Each p o i n t r e p r e s e n t s an average o f 20 p l a t e s (5 p l a t e s f o r each o f 2 d u p l i c a t e s a m p l e s ) . 7 I •—» • » 0 5 10 15 D A Y S OF S T O R A G E at 4-6°C 66 s t o r a g e at 4°c a r e shown i n T a b l e 8 . F i g u r e s 9 and 10 show that when L416 was grown with the commercial BMS, L416 was able to s u r v i v e c o l d storage b e t t e r than when i t was grown a l o n e , while the numbers of non-ropy CFU decreased a t about the same r a t e as when these b a c t e r i a were grown a l o n e . Both graphs show that the percent ropy CFU i n c r e a s e d c o n s t a n t l y d u r i n g the storage p e r i o d . T h i s may be a t t r i b u t e d to the improved s u r v i v a l of the ropy b a c t e r i a i n the mixed c u l t u r e , not to t h e i r growth at c o l d s t o r a g e . These F i g u r e s a l s o show that a f t e r the f e r m e n t a t i o n p e r i o d , or at the zero time of c o l d s t o r a g e , what s t a r t e d as 10Z inoculum ( F i g u r e 9) proved to be 15% ropy CFU. S i m i l a r l y , 20% Inoculum ( F i g u r e 10) has shown to be g r e a t e r than 60% ropy CFU. There i s no j u s t i f i a b l e e x p l a n a t i o n of t h i s o b s e r v a t i o n except t h a t the ropy b a c t e r i a are the dominant s t r a i n i n t h i s m i x t u r e . They were e i t h e r m u l t i p l y i n g f a s t e r , or the non-ropy c e l l s w e r e a c q u i r i n g the ropy c h a r a c t e r d u r i n g the 18 hour i n c u b a t i o n p e r i o d . T h i s i n d i c a t e s that an imbalance between these c u l t u r e s would soon o c c u r . T h i s phenomenon deserves f u r t h e r i n v e s t i g a t i o n . I t i s not unusual to f i n d imbalance i n the r a t i o of s t r a i n s i n mixed l a c t i c c u l t u r e f e r m e n t a c i o n s . I t i s not always c e r t a i n why the imbalance o c c u r s , but c o m p e t i t i v e I n h i b i t i o n and the p r o d u c t i o n of b a c t e r i o c i n s may be i m p l i c a t e d . There are very few, i f any, s t a b l e l a c t i c mixed c u l t u r e s ( R e l t e r and Moller-Madsen, 1963). The i n s t a b i l i t y occurs a f t e r few t r a n s f e r s , with one s t r a i n immerging as a dominant s t r a i n . FIGURE 9 . R e f r i g e r a t e d storage s u r v i v a l curves of cremoris l o n g i Lkl6 ( • ), and a commercial b u t t e r m i l k s t a r t e r ( • ) when grown together i n skim mi l k at 10% (v/v) l e v e l of L l i l 6 i n the inoculum f o r 18 hours at 22°C. • l o g of t o t a l CFU/ml., • % viscous CFU. Each p o i n t represents an average of 20 p l a t e s (5 p l a t e s f o r each of 2 d u p l i c a t e samples). 67 68 a FIGURE 1 0 . R e f r i g e r a t e d storage s u r v i v a l curves of S. cremoris l o n g i Lkl6 ( • ) and a commercial b u t t e r m i l k s t a r t e r ( • ) when grown together i n skim m i l k at 20% (v/v) l e v e l of LUl6 i n the inoculum. l o g of t o t a l CFU/ml., • % viscous CFU. Each p o i n t represents an average of 20 p l a t e s (5 p l a t e s f o r each of 2 d u p l i c a t e samples). 68 Table 8. Change i n b a c t e r i a l numbers (Log CFU a/ml.) i n completed skim m i l k f e r m e n t a t i o n s a f t e r storage at 4°C f o r 15 days ( D a t a o b t a i n e d from F i g u r e s 8,9,10 by s u b t r a c t i n g Log CFU/ml. at 15 days from Log CFU/ml. at 0 d a y s ) . Change i n Log CFU/ml. Sample V i s c o u s Non-viscous T o t a l C o l o n i e s C o l o n i e s C o l o n i e s L 4 1 6 b - 1 . 4 8 e 0 -1.48 BMS C 0 -0.97 -0.97 10% dL416 -0.08 -0.97 -0.66 15% L416 +0.04 f -1.45 -0.40 20% L416 -0.03 -1.25 -0.23 a) Colony forming u n i t s . b) Pure c u l t u r e of S. c r e m o r i s l o n g i L416 grown i n skim milk at 3% (v/v) inoculum l e v e l . c) Commercial b u t t e r m i l k s t a r t e r grown i n skim milk at 3% (v/v) Inoculum l e v e l . d) I n d i c a t e s the p e r c e n t of S. c r e m o r i s l o n g i L416 i n the t o t a l inoculum of 3% ( v / v ) . e) I n d i c a t e s n e g a t i v e change, i . e . l o s s i n CFU/ml.. f ) I n d i c a t e s p o s i t i v e change, i . e . i n c r e a s e i n CFU/ml. 7 0 B. C h a r a c t e r i s t i c s o f V i s c o u s M a t e r i a l 1. Slime P r o d u c t i o n and P u r i f i c a t i o n a) B a c k g r o u n d The ropy l a c t i c a c i d b a c t e r i a from ropy Scandinavian m i l k r e q u i r e a good supply of amino a c i d s to produce slime ( N i l s s o n and N i l s s o n , 1958). Forsen (1966) found t h a t , to produce r o p i n e s s , the f o u r ropy c u l t u r e s s t u d i e d r e q u i r e d between 10 and 13 e s s e n t i a l amino a c i d s . Most c r i t i c a l , and completely r e q u i r e d by a l l ropy c u l t u r e s s t u d i e d by Forsen, were: s e r i n e , t h r e o n i n e , p h e n y l a l a n i n e , g l u t a m i c a c i d , h i s t i d i n e , p r o l i n e . V a l i n e was e s s e n t i a l f o r 3 out of 4 ropy c u l t u r e s s t u d i e d . Macy (1923) recorded very good r o p i n e s s i n media such as peptone, skim m i l k , and whole m i l k , but no r o p i n e s s i n IX aqueous s o l u t i o n of g l u c o s e , l a c t o s e , or s a c c h a r o s e . He d i d , however, rep o r t r o p i n e s s i n 1% aqueous s o l u t i o n of maltose. T h i s i n d i c a t e s t h a t amino a c i d s could be the e s s e n t i a l p r e c u r s o r s , f o r the b i o s y n t h e s i s of the s l i m e . Thus, i t was not s u r p r i s i n g that whey was one of the most s u i t a b l e media f o r the slime p r o d u c t i o n . However, because of i t s p r o t e i n a c e o u s c o m p o s i t i o n i t was very d i f f i c u l t to p u r i f y the 6lime a f t e r f e r m e n t a t i o n . Whey was used as a s u b s t r a t e f o r a p o l y s a c c h a r i d e s l i m e p r o d u c t i o n by C o r y n e b a c t e r i u m #98 (Shams and Tynes, 1983), and f o r the s l i m e p r o d u c t i o n by a ropy S. c r e m o r i s (Sundman, 1953b). There was no attempt to i s o l a t e the s l i m e from Corynebacterium f e r m e n t a t i o n , but Sundman uBed a l c o h o l p r e c i p i t a t i o n , d i a l y s i s , and c e n t r i f u g a t i o n to i s o l a t e the pure slime from a whey f e r m e n t a t i o n . 71 N i l s s o n and N i l s s o n (1958) que s t i o n e d Sundman's method of s l i m e p u r i f i c a t i o n . They grew S. l a c t i s l o n g i i n skim m i l k , c e n t r i f u g e d the c o a g u l a t e d p r o t e i n s , p r e c i p i t a t e d the s l i m e w i t h e t h a n o l , d r i e d i t i n a i r on f i l t e r paper, d i s s o l v e d i n water at pH A.6 to prevent the c a s e i n s from d i s s o l v i n g , f i l t e r e d to separate p r e c i p i t a t e d p r o t e i n s , p r e c i p i t a t e d the slime with a l c o h o l , and a i r d r i e d on f i l t e r paper. When they analyzed the p u r i f i e d s l i m e , both Sundman and N i l s s o n s found a p r o t e i n component. Upon amino a c i d a n a l y s i s , Sundman r e p o r t e d s i m i l a r i t i e s w i t h c a s e i n s , while N i l s s o n s r e p o r t e d no s i m i l a r i t i e s with c a s e i n s d i s p u t i n g Sundman's f i n d i n g s . They showed a unique amino a c i d p r o f i l e of the s l i m e , thus q u e s t i o n i n g Sundman's i s o l a t i o n t e c h n i q u e . b) I s o l a t i o n o f t h e S l i m e In order to av o i d s i m i l a r c r i t i c i s m s t h i s author d e p r o t e i n l z e d the whey before f e r m e n t a t i o n . The d e p r o t e i n i z a t i o n was done by f i r s t n e u t r a l i z i n g the a c i d cheese whey, c e n t r i f u g i n g the p r e c i p i t a t e , and f i l t e r i n g the supernatent l i q u i d through an u l t r a f i l t r a t i o n membrane with an e x c l u s i o n c o e f f i c i e n t of 10,000 m o l e c u l a r weight (MW). T h i s method e l i m i n a t e d the high MW p r o t e i n s from the s u b s t r a t e i n which the ropy b a c t e r i a were subsequently grown. Upon f e r m e n t a t i o n , the v i s c o u s whey was c e n t r i f u g e d to separate c e l l s , and the slime was p r e c i p i t a t e d w i t h 95% e t h a n o l , and then c e n t r i f u g e d or f i l t e r e d . Thus f a r , d i s r e g a r d i n g the < 10,000 MW f r a c t i o n , the p u r i t y of the p r e c i p i t a t e depended upon the f u n c t i o n a l i t y of the 72 u l t r a f i l t r a t i o n membrane. To check the f i l t r a t e f o r high MW p r o t e i n s , 5 ml. p o r t i o n s were mixed w i t h 5 ml. o f 25% c o l d TCA. A s l i g h t haze f o r m a t i o n was observed which was n o t i c e a b l e with a naked eye only when the TCA t r e a t e d sample was compared s i d e by s i d e with a f i l t r a t e sample which was d i l u t e d with equal volumes of d i s t i l l e d water. T h i s t e s t was c o n s i d e r e d adequate f o r p r o v i n g t h a t the f i l t r a t e c o n t a i n e d very l i t t l e or no p r o t e i n s of MW > 10,000. The only other contaminant i n the p r e c i p i t a t e d s l i m e c o u l d be the b a c t e r i a l c e l l s , i f the c e n t r i f u g a t i o n was not adequate. Thus, to separate the c e l l s the p r e c i p i t a t e d slime was d i s s o l v e d i n d i s t i l l e d water and t r e a t e d with equal volumes of c o l d 25% TCA. T h i s step a l s o p r e c i p i t a t e d high MW p r o t e i n s , i f they were not separ a t e d by the u l t r a f i l t r a t i o n membrane. T h i s was f o l l o w e d by c e n t r i f u g a t i o n and by f i l t r a t i o n of the supernatant f l u i d through a 0.45 um m i l l i p o r e membrane to assure the complete c e l l s e p a r a t i o n . The TCA was then e x t r a c t e d twice with equal volumes of e t h y l e t h e r . The remaining e t h e r was evaporated by n i t r o g e n p u r g i n g . The aqueous f r a c t i o n , which was ropy, was d i a l y z e d a g a i n s t running water, i n the c o l d room (about 10°C), f o r 48 hours. The d i a l y s i s bag used r e t a i n e d the m a t e r i a l of MW g r e a t e r than 12,000. Thus, u n l e s s 48 hours of d i a l y z i n g was not lon g enough to d i a l i z e out the low MW whey pe p t i d e s and amino a c i d s , t h i s procedure has assured that the slime was f r e e of the whey and c e l l contaminants. The ropy m a t e r i a l was then f r e e z e d r i e d and subsequently s u b j e c t e d to the a n a l y s i s f o r serum g l y c o p r o t e i n s ( W i n z l e r , 1955), t o t a l p r o t e i n content (Lowry et a l . , 1951), and the amino a c i d a n a l y s i s (Simpson et a l . , 1975). 73 2. Composition of the Slime a) P r o t e i n f r a c t i o n The purpose of this study was not to provide a detailed analysis of the slime composition, but to test the controversy about the protein content of this material. The results show that the slime was composed of 47Z t o t a l protein, 20% methyl pentoses, 9.3% protein-bound hexoBes , and 2.8% s i a l i c acids (Table 9)* No hexosamlnes were found when the method of Morgan and Elson (1933, 1934), as modified by Winrler (1955) was employed (refer to Appendix 5). The same method for the determination of hexosamlnes was used by Sundman (1953b) with negative r e s u l t s . The t o t a l amount of protein In slime found i n this project was s l i g h t l y less than that found by Sundman (1953b) who reported a nitrogen content of 8*9% (55.6% protein) as determined by microkjeldahl method. However, Kjeldahl method determines both protein and non-protein nitrogen, which may, at least in part, account for the di f f e r e n c e . In addition, because of the non--quantltative nature of this experiment, no determination of cystine and tryptophan was done. The amino acid summation method (Heidelbagh et a l . , 1975) was used as the more accurate method of determining the t o t a l protein content of the slime. This method was also used by Neukom (1982, personal communication), who found 55.6 Z protein In the same slime •ample (see Appendix 8) . He found the same amino acid p r o f i l e , showing r e l a t i v e l y high concentrations of glutamic and aspartic •elds, serine, and threonine. Similar general p r o f i l e s were recorded for e x t r a c e l l u l a r proteins of B a c i l l u s b r a v i s (Miyashiro et a l *, Ik T a b l e 9. Composition of the S l i m e P r o d u c e d by S. cremoris l o n g i L416 Component % Dry Weight T o t a l p r o t e i n 46.7 Fucose 19.7 Protein-bound hexoses 9.3 S i a l i c a c i d s 2.8 Hexosamines 0 78.5 1960), egg ovoaucins ( M i l l e r , 1981; Kato and Sato, 1971), and whey proteins (Evans and Gordon, 1980). The s i m i l a r i t y with the egg and whey proteins warrant the Investigation of Its functional properties. Sundman (1953b) reported that the amino acid p r o f i l e of the slime was simi l a r to that of casein hydrolysste. Our results are i n agreement with these findings, as shown l n Table 10. The results i n Table 10 are i n disagreement with those of Nilsson and Nilsson (1958), who subsequent to Sundman's work proved that the slime produced by S.  l a c t i s longi was completely d i f f e r e n t from the amino acid p r o f i l e of unspeciffied milk proteins. The l a t t e r authors used a colorimetrlc method of Moore and Stein for the quantitative determination of the amino acid composition of the slime and the milk proteins, while Sundman (1953b) used a paper chromatography method. Furthermore, Nilsson and Nilsson (1958) found the nitrogen content of the slime to be 2.8Z, much lower than 9.8Z reported by Sundman (1953b). This finding, and the fact that Sundman was working with a ropy s t r a i n of S. cremoris, while N i l s s o n and N i l s s o n were working with a ropy s t r a i n of S. l a c t i s , i n d i c a t e that these two organisms may be producing two d i f f e r e n t types of slime, amino acid p r o f i l e s of which nay be species s p e c i f i c . Thus, the findings of current research were expected to agree with those of Sundman (1953b) since both analyzed the slime produced by a ropy s t r a i n of S. cremoris. Amino acid analysis of the slime produced by S. l a c t i s long! needs to be done before accurate judgement of Nilssons' work can be made. b) Carbohydrate f r a c t i o n Since convenient and quick methodology for the analysis TABLE 1-0. Amino acid composition of the dry slime produced by B. cremoris longi L416 and dry whey permeate (numbers Indicate calculations of one sample). Amino Acids SLIME PS MEATS •g/g" %b •g/g % MP 34.60 7.4 0.96 6.2 Thr SS.30 11.8 0.48 4.1 6er 40.80 8.7 0.40 3.4 Glu 138.30 29.5 3.02 25.7 Pro 23.60 5.1 0.72 6.1 Gly 9.00 1.0 0.22 1.9 Ala 19.96 4.3 0.37 3.2 Val 23.90 5.1 0.52 4.4 Met 6.28 1.4 0.21 1.8 He 32.00 6.9 0.39 3.3 Leu 26.80 5.7 1.42 12.1 Tyr 4.36 0.9 0.33 2.8 Phe 11.40 2.4 0.23 2.0 Lys 27.50 5.9 1.70 14.5 Bis 3.72 0.8 0.38 3.2 Trp 0 0 0 0 Arg 9.76 2.1 0.38 3.2 Cys _c - -* Milligrams of amino acid per gram of freexe dried pure sample. D Amino acids composition of pure sample expressed as percent. c Mot determined. 77 of this slime have not been developed, and since conventional methods for the analysis of the complex molecules such as glycoproteins are too extensive and too Involved for the time span of thi s project, the author chose to adapt the methods for determination of serum glycoproteins as published by Winzler (1955). Adaptations were made for the determination of protein-bound hexoses, hexosamlnes, and fucose, while for determination of s i a l i c acids the method of Warren (1959) was used. The purpose of this research was not to provide absolute determination of these substances In the slime, which are common components of glycoproteins found in blood serum, but to find out i f they, or the substances similar to them, are components of the slime. In her paper chromatography analysis of the carbohydrate f r a c t i o n , Sundman (1953b) reported t o t a l carbohydrate content of 4Z dry weight before d i a l y s i s . After d i a l y s i s , the carbohydrate content decreased to less than IX. It was mostly composed of galactose, with some glucose always present. Nilsson and Nilsson (1958) did not find any sugars upon acid hydrolysis of the slime. They reported that "some sort of carbohydrate not yi e l d i n g reducing sugar on hydrolysis seemed to be bound to the protein". Forsen and Veli-Mies (1981) reported that "the e x t r a c e l l u l a r capsular material of the l a c t i c streptococci contains galactose, glucose, and probably fucose". The protein-bound hexoses content of the pure slime was determined by the o r c i n o l - s u l f u r i c acid method of Lusting and Langer, as described by Winzler (1955). This i s a spectrophotometry method whose accuracy r e l i e s upon the correct choice and the correct r a t i o 78 of the sugars l n the c o n t r o l samples, i . e . i f the r a t i o of g i v e n sugars (eg. g a l a c t o s e and mannose) i n a g l y c o p r o t e i n i s known, and i f i t i s known that there are no other hexoses p r e s e n t , t h i s method can determine the c o n c e n t r a t i o n of these hexoses a c c u r a t e l y . However, i f the above mentioned f a c t o r s are unknown, the method i s s u b j e c t to many e r r o r s ( W i n z l e r , 1955). Thus, the r e s u l t s obtained i n t h i s r e s e a r c h should be i n t e r p r e t e d as approximate s i n c e i t l s not known which hexoses, and i n which p r o p o r t i o n , are present i n the carbohydrate p o r t i o n of the s l i m e m o l e c u l e . However, the t e s t d i d prove p o s i t i v e f o r protein-bound hexoses, t h e r e f o r e , l n f u t u r e a n a l y s i s hexoses should be t e s t e d l n more d e t a i l . The p r e l i m i n a r y carbohydrate a n a l y s e s of the slime by Neukom (1982, p e r s o n a l communication) by gas l i q u i d chromatography (GLC) a f t e r the sample was hydrolyzed by t r i f l u o r o a c e t i c a c i d (IN TFA, 1 h r . , 120°C) and converted to a l d o n o n i t r l l a c e t a t e s showed the presence of g l u c o s e and g a l a c t o s e (see Appendix 9 ) . ' Fucose i s a methylpentose which may be i n v o l v e d i n O - g l y c o s l d i c l i n k a g e s with t h r e o n i n e p r o v i d i n g an important l i n k a g e between a p o l y s a c c h a r i d e c h a i n and a p r o t e i n , or a p e p t i d e (Sharon and L i s , 1981). I t was mentioned to be present i n the slime of the l a c t i c s t r e p t o c o c c i (Forsen and V e i l - M i e s , 1981), however the work was not p u b l i s h e d , thus no method which was used f o r the a n a l y s i s was communicated. The presence of fucose may be of i n t e r e s t to p h a r m a c e u t i c a l I n d u s t r y , as I t may have t h e r a p e u t i c p r o p e r t i e s . I t was r e f e r r e d to as a t h e r a p e u t i c sugar by S h e l l e n b u r g e r and B i r c h (1975), ss I t was found to s i g n i f i c a n t l y reduce the growth of c h e m i c a l l y Induced mammary tumor l n r a t s ( S e l t z e r et a l . , 1969). 79 In t h i s r e s e a r c h , a t e s t f o r methylpentoses was done a c c o r d i n g to the method of Dische and S h e t t l e s (1948) as d e s c r i b e d by W i n z l e r (1955)* Rhamnose was used as a standard s i n c e i t gi v e s the same r e s u l t s as f u c o s e . The t e s t i n v o l v e d a timed (3 min.) s u l f u r i c a c i d h y d r o l y s i s (6 p a r t s c o n c e n t r a t e d H^SO^/l p a r t d e i o n i z e d water) i n b o i l i n g water, a d d i t i o n of 3% c y s t e i n e s o l u t i o n , and d e t e r m i n i n g the absorbance at two wavelengths (396 um and 430 um) to account f o r i n t e r f e r e n c e by other sugars. S i n c e o n l y L - f u c o 6 e has been a s s o c i a t e d w i t h mucoid g l y c o p r o t e i n s ( W i n z l e r , 1955), the methylpentose content was expressed i n terms of percent f u c o s e . However, other pentoses ( a r a b i n o s e and x y l o s e ) may be a s s o c i a t e d with g l y c o p r o t e i n s (Sharon and L i s , 1981). Hexosamines are most o f t e n found components of g l y c o p r o t e i n s . They are Involved l n the f o r m a t i o n of the N - g l y c o s i d i c l i n k a g e between car b o h y d r a t e s and p r o t e i n s . T h i s l i n k a g e occurs between N- a c e t y l g l u c o s a m i n e and asparagine (Sharon and L i s , 1981), making glucosamines the e s s e n t i a l components of N - l i n k e d g l y c o p r o t e i n s . Thus, i t was necessary to determine the hexosamine content of the s l i m e l n p e r s u l t of i t s g e n e r a l c o m p o s i t i o n . The t e s t used i n t h i s r e s e a r c h was that of E l s o n and Morgan (1933) aB d e s c r i b e d by W i n z l e r (1955). The method Involved a c i d h y d r o l y s i s of the slime to r e l e a s e hexosamines, a c e t y l a t l o n of hexosamines with a c e t y l a c e t o n e , f o r m a t i o n of c y c l i c oxazole or p y r r o l e by treatment w i t h a l k a l i , and c o u p l i n g with E r l i c h ' s reagent 80 (p-dlmethylaminobencaldehyde) to form a coloured complex for spectrophotometry determination of Its concentration. The absorbance readings at 530 um are d i r e c t l y proportional to the amount of hexosamlne, i f the hexosamlne concentration i s less than 0.25 mg./ml. (Winzler, 1955). Thus, a 6-point standard curve was formed with the glucosamine hydrochloride standard concentrations between 0.08 and 0.50 mg./ml. The absorbance was recorded at the wavelength of 530 um, and a regression c o e f f i c i e n t of 0.995 obtained (see Appendix 5). The slime sample was treated i n f i v e r e p l i c a t e s and a l l of them showed negative absorbance readings. This was not surpr i s i n g i n view of the fact that the fucose concentration was found to be 20Z of dry slime weight (Table 9, Appendix 3). Also, threonine and serine concentrations were found to be substantial (Table 10). Thus, i t appears that, i f the slime i s a glycoprotein, i t most l i k e l y contains O-linkages between L-fucose and threonine. No N-linkages are l i k e l y to be present. S i a l i c acids i s a c o l l e c t i v e term re f e r r i n g to the derivatives of neuraminic a c i d . They are negatively charged, and are found i n a variety of tissues and body f l u i d s , as well as in bacteria, as parts of conjugative proteins (Warren, 1959). They are usually located on the terminal end of a polysaccharide chain determining the length of the chain. As such, they are involved i n determination of protein configuration, and serve as recognition s i t e s for some functional conjugative proteins (Sharon and L i s , 1981). The presence of s i a l i c acids In the slime was suspected because of i t s glycoprotein-like nature. Thus, the th i o b a r b i t u r i c 81 acid assay for the s i a l i c acids (Warren, 1959) was used for their determination. This method was chosen because i t was developed for the s i a l i c acid determination i n b i o l o g i c a l tissues. It was used ln this laboratory by M i l l e r (1981) for the s i a l i c acid determination i n egg ovomucin, and by G i l l (1976) for the s i a l i c acid determination of rapeseed protein. In this experiment, the s i a l i c acid concentration of the slime was found to be 2.8 Z of the t o t a l dry weight of the slime (see Appendix 4). This concentration i s much higher than the s i a l i c acid concentration of whole egg white (0.03 %) ( M i l l e r , 1981), or whole casein (0.04 Z) (Cayen et a l . , 1962), but i t l s comparable with that of k-casein, 2.2 - 2.6 Z (Cayen et a l . , 1962), and egg ovomucin, 4.6 Z ( M i l l e r , 1981). C. P r a c t i c a l Application of S. cremoris longi L416 - Production  of B u t t e r m i l k a) Optimization of S. cremoris l o n g i L416 Concentration i n  Buttermilk S t a r t e r Cultured b u t t e r l l k was used as a model system for the p r a c t i c a l a p p l i c a t i o n of S. cremoris l o n g i , because i t uses S.  cremoris i n the s t a r t e r , and because of expressed desire by the dairy industry representatives i n Canada to produce thicker buttermilk. The thicker buttermilk was p a r t i c u l a r l y wanted by the da i r i e s which expose their product to excessive shear due to the use of improper equipment, or due to long distance piping before packing. Excessive shearing, causes s t r u c t u r a l breakdown of curd and release of whey, leading to unwanted weeping or syneresls. In addition, i t 8 2 vas hoped that low fat buttermilk (1Z b u t t e r f a t ) , or ekltn milk buttermilk can be produced by using a ropy culture l n the s t a r t e r to replace v i s c o s i t y which l s contributed by butterfat. The use of high concentrations of ropy cultures (50Z of the s t a r t e r or more) resulted l n a very thick and ropy product with very mild d i a c e t y l flavour, thus i t was necessary to find the optimum concentration of S. cremoris l o n g i i n the starter which would contribute the desired thickness, without s i g n i f i c a n t loss of flavour l n the product. This WSB achieved with taste panel analyses. The panel members were chosen among the general research laboratory populace i n the departments of Food and Animal Science at the University of B r i t i s h Columbia. The only c h r i t e r i a for the panelists was that they did not detest buttermilk. It can be assumed, therefore, that t h i s panel was representative of general adult population who consume buttermilk. Table 11 shows that when 4 types of buttermilk were compared on the basis of flavour and a c i d i t y , body and texture, and o v e r a l l a c c e p t a b i l i t y , commercial control (fresh buttermilk purchased from a grocery store) was judged to be s i g n i f i c a n t l y I n f e r i o r ( o £ 0.05) to a l l other samples. Of the laboratory prepared samples the product which contained an equal mixture of the ropy culture and commercial buttermilk s t a r t e r (50/50, Table 11) scored consistently higher than the laboratory-prepared control and the ropy culture alone, however, no s t a t i s t i c a l difference was found. Even though most panel members preferred the "body" and the consistency of the 50/50 sample many of them commented on the unappealing ropy texture of the product. Thus, the Q.C. management 83 Table 11. B u t t e r m i l k P r e f e r e n c e R a n k i n g (Mean V a l u e s 8 of S c o r e s 1 * ) T r a i t s Commercial C o n t r o l Samples Lab C o n t r o l L 4 1 6 c 50/50 d F l a v o u r / A c i d i t y Body/Texture O v e r a l l A c c e p t . - 0.63a - 0.52a - 0.57a + 0.15b + 0.05ab + 0.16b + 0.06b + 0.31b + 0.16b + 0.42b + 0.15b + 0.26b a) Any two means of each g i v e n t r a i t not f o l l o w e d by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l of s i g n i f i c a n c e (number of r e p l i c a t e s , n=20). b) Scores f o r ranked data: o r d i n a l numbers 1,2,3,4 correspond to the scores 1.03, 0.30, -0.30, -1.03 r e s p e c t i v e l y (Larmond, 1977). c) S. cremoris l o n g i L416 u s e d at 3%, by volume, l e v e l of inoculum. ( d) An equal mixture, by volume, of the commercial b u t t e r m i l k s t a r t e r and S. c r e m o r i s l o n g i L416. T o t a l inoculum l e v e l was 3% by volume. 81* of the c o o p e r a t i n g d a i r y was c o n s u l t e d and. on the b a s i s of t a s t e t e s t i n g , a d e c i s s i o n was Bade t h a t the p r o p o r t i o n of the ropy c u l t u r e i n the b u t t e r m i l k s t a r t e r should not exceed 25Z (by volume) of the t o t a l b u t t e r m i l k s t a r t e r . Above 25Z, the product was d e s c r i b e d as " f l a t " , and the ropy c h a r a c t e r became n o t i c e a b l e . T h e r e f o r e , a 10 member t a s t e panel was chosen among the d a i r y p l a n t Q . C . p e r s o n n e l , who were very f a m i l i a r with the t a s t i n g procedures and the q u a l i t y of b u t t e r m i l k to f i n d the optimum c o n c e n t r a t i o n of the ropy c u l t u r e In t h i s product. B u t t e r m i l k samples with the ropy c u l t u r e c o n c e n t r a t i o n s of 5Z, 10Z, and 15Z (by volume) of t o t a l inoculum were prepared i n IZ b u t t e r f a t m i l k . The c o n t r o l was prepared with commercial b u t t e r m i l k s t a r t e r (32 by volume), without any ropy c u l t u r e , i n 2Z b u t t e r f a t m i l k . These samples were s u b j e c t e d to the t a s t e panel a n a l y s e s a f t e r 1 day of c o l d storage ( 4 ° C ) , and a f t e r 15 days o f c o l d s t o r a g e . The purpose of the experiment was to f i n d the optimum c o n c e n t r a t i o n of the ropy c u l t u r e i n the s t a r t e r , and to f i n d out whether a lower b u t t e r f a t b u t t e r m i l k can be produced by u s i n g the ropy c u l t u r e . The r e s u l t s i n Table 12 show that no s i g n i f i c a n t d i f f e r e n c e was found among any of the samples prepared a f t e r 1 day of c o l d s t o r a g e . T h i s means t h a t , when b u t t e r m i l k i s f r e s h , a l l 3 l e v e l s of ropy c u l t u r e t e s t e d c o u l d be used to r e p l a c e IZ b u t t e r f a t i n the prod u c t . However, no 1Z b u t t e r f a t c o n t r o l was run, thus, i t was not p o s s i b l e to t e l l i f the t a s t e panel c o u l d d i f f e r e n c l a t e between IZ b u t t e r f a t b u t t e r m i l k and 2Z b u t t e r f a t b u t t e r m i l k . Nonetheless, a f t e r 15 days of c o l d s t o r a g e , I t was found that the "body" and the t e x t u r e of the 10Z and the 15Z ropy c u l t u r e samples 85 Table 12. B u t t e r m i l k P r e f e r e n c e T e s t i n g - Nine P o i n t Hedonlc S c a l e 3 Mean V a l u e s * * ( S a m p l e s were s t o r e d at 4°C f o r 1 day) T r a i r s 57.L416 Samples 10% L416 2% b . f . 15% L416 C o n t r o l F l a v o u r / A c i d i t y Body/Texture O v e r a l l A c c e p t . 6 .6a 6.9a 6.9a 6.1a 6 .6a 6.1a 4.3a 6.2a 5.8a 6.9a 5.0a 5.8a a) Hedonic s c a l e v a l u e s : 1 d i s l i k e extremely, 2 d i s l i k e very much, 3 d i s l i k e moderately, 4 d i s l i k e s l i g h t l y , 5 n e i t h e r l i k e nor d i s l i k e , 6 l i k e s l i g h t l y , 7 l i k e moderately, 8 l i k e very much, 9 l i k e e x t r e m e l y . b) Any two means of each given t r a i t not f o l l o w e d by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t a t 5% l e v e l of s i g n i f i c a n c e (number of r e p l i c a t e s , n=10). c) I n d i c a t e s the p e r c e n t of S. c r e m o r i s l o n g i L416 i n the 3%, by volume, t o t a l inoculum of 1% b . f . b u t t e r m i l k . 8 6 were s i g n i f i c a n t l y b e t t e r ( a 10.05) than the c o n t r o l (Table 13), while f l a v o u r and a c i d i t y were not found to be s t a t i s t i c a l l y d i f f e r e n t . O v e r a l l a c c e p t a b i l i t y was a l s o not found to be s t a t i s t i c a l l y d i f f e r e n t , however, i t was f e l t that improvements could be made i n f l a v o u r by longer i n c u b a t i o n and higher a c i d i t y . Thus, i t was decided that 10% ( v / v ) of S. c r e m o r i s l o n g i L416 should be used i n b u t t e r m i l k s t a r t e r and e s c a l a t e d to I n d u s t r i a l s c a l e . T h i s proved to be a good e s t i m a t i o n , r e s u l t i n g i n a p r o d u c t i o n run of 4,000 l i t r e s , with no adverse e f f e c t s on the consumer acceptance. V i s c o m e t r i c measurements of t h i s product were monitored and compared with iLhoseof non-ropy b u t t e r m i l k f o r the d u r a t i o n of i t s s h e l f l i f e ( F i g u r e 15). I t was found that the new product was s i g n i f i c a n t l y t h i c k e r ( a 1 0.05) throughout the s h e l f l i f e o f 15 days. T h i s area deserves more a t t e n t i o n , as i t appears th a t s u b s t a n t i a l savings could be made by producing a low f a t b u t t e r m i l k with s u p e r i o r q u a l i t i e s by u s i n g S. cr e m o r i s l o n g i i n c o m m e r c i a l b u t t e r m i l k s t a r t e r . 2) V i s c o m e t r i c A n a l y s i s o f Improved B u t t e r m i l k Apparent v i s c o s i t y development by the v i s c o u s c u l t u r e L416 as a f u n c t i o n of f e r m e n t a t i o n time i n skim m i l k , was shown to reach maximum w i t h i n 24 hours of f e r m e n t a t i o n ( F i g u r e 2 ) . F i g u r e 3 shows that the d i f f e r e n c e i n the apparent v i s c o s i t y development between the ropy and the non-ropy l a c t i c s t r a i n s i n skim m i l k was a p p r e c i a b l e . Because of t h i s , the ropy b a c t e r i a are used i n the c u l t u r e d d a i r y products to induce higher v i s c o s i t y and b e t t e r c o n s i s t e n c y (Vedamuthu and Shah, 1983). I t was thus necessary to i n v e s t i g a t e the a b i l i t y of the ropy product to withstand 87 Table' 13. B u t t e r m i l k P r e f e r e n c e T e s t i n g - Nine P o i n t Hedonic S c a l e 8 Mean V a l u e s * 5 ( S a m p l e s were s t o r e d at 4°C f o r 15 days) T r a i t s 5% CL416 Samples 10% L416 15% L416 2% b.f, C o n t r o l F l a v o u r / A c i d i t y 4.8a Body/Texture 5.8a O v e r a l l A c c e p t . 4.8a 5.3a 6.4b 5.8a 4.8a 6.4b 5.0a 6.8a 4.4a 6.1a a) Hedonic s c a l e v a l u e s : 1 d i s l i k e extremely, 2 d i s l i k e very much, 3 d i s l i k e moderately, 4 d i s l i k e s l i g h t l y , 5 n e i t h e r l i k e nor d i s l i k e , 6 l i k e s l i g h t l y , 7 l i k e moderately, 8 l i k e very much, 9 l i k e extremely. b) Any two means of any g i v e n t r a i t not f o l l o w e d by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l of s i g n i f i c a n c e (number of r e p l i c a t e s , n=10). c) I n d i c a t e s the p e r c e n t of S. c r e m o r i s l o n g i L416 i n the 3%, by volume, t o t a l inoculum of 1% b . f . b u t t e r m i l k . 88 shear s t r e s s as compared to the non-ropy product, to e v a l u a t e i t s c o n t r i b u t i o n to the p h y s i c a l p r o p e r t i e s of the p r o d u c t . F i g u r e 11 shows the apparent v i s c o s i t y decay curve of normal commercial b u t t e r m i l k , and the commercial b u t t e r m i l k whose inoculum c o n t a i n e d 10% ( v / v ) S. c r e m o r i s l o n g i L416. I t shows a l o g t r a n s f o r m a t i o n data of the d e c r e a s i n g apparent v i s c o s i t y as a f u n c t i o n of time of s h e a r i n g at a c o n s t a n t shear r a t e of 234 per s e c . Thus, i t was named the v i s c o s i t y decay cutve (Tung et a l . , 1971). The data p o i n t s were obtained from a s t r i p c h a r t r e c o r d e r , which was connected to the r o t a t i o n a l viscometer d u r i n g the v i s c o s i t y measurements, at equal l o g time i n t e r v a l s as suggested by Cramer and M a r c h e l l o (1968). F i g u r e 11 t h e r e f o r e shows the r e s p e c t i v e r a t e s of r e s i s t a n c e s of the b u t t e r m i l k samples to the s t r u c t u r a l breakdown due to constant s h e a r i n g ( s l o p e of the l i n e s ) . A l s o , i t shows the r e s p e c t i v e apparent v i s c o s i t i e s of the samples at any given time d u r i n g the experiment ( l e v e l of the l i n e at any g i v en t i m e ) . T h i s graph r e v e a l s that even though both products show r h e o d i s t r u c t i v e behaviour the l e v e l of the "high body" b u t t e r m i l k curve i s always s i g n i f i c a n t l y h i gher ( a 1 0.05). The same r e l a t i o n s h i p was observed when commercial b u t t e r m i l k f e r m e n t a t i o n was compared to that of pure c u l t u r e L416 f e r m e n t a t i o n i n skim m i l k , except the d i f f e r e n c e was more e x p l i c i t ( F i g u r e 12) i n d i c a t i n g that i t may be p o s s i b l e to r e p l a c e b u t t e r f a t with ropy c u l t u r e . I t i s a l s o e v i d e n t that there i s a d i r e c t r e l a t i o n s h i p between the p r o p o r t i o n of ropy c u l t u r e i n the Inoculum and the "body" c o n t r i b u t i o n to the p r o d u c t . Furthermore, i t appears ( F i g u r e 11) that the i n c o r p o r a t i o n of the ropy b a c t e r i a i n the b u t t e r m i l k s t a r t e r r e s u l t s i n a product which i s b e t t e r able to 89 a FIGURE 11. Apparent v i s c o s i t y ( l | , c e n t i p o i s e ) - time ( t , sec.) r e l a t i o n s h i p f o r commercial b u t t e r m i l k (1.5% b u t t e r f a t ) • , and b u t t e r m i l k (1.5% b u t t e r f a t ) c o n t a i n i n g 10% (by volume of t o t a l inoculum) of 18 hour c u l t u r e of cremoris  l o n g i L*4l6 • , at a constant shear r a t e of 23k per sec. and a constant temperature of k°C. The samples were h e l d at i+°C f o r 15 days before shearing. n represents e q u i l i b r i u m apparent v i s c o s i t y , c e n t i p o i s e . 8 9 -1.1 •1.3 0.5 1.0 1.5 2.0 2.5 L OG t 90a FIGURE 12. Apparent v i s c o s i t y ( r| , centipoise) - time ( t , sec.) r e l a t i o n s h i p f o r skim milk fermented with commercial buttermilk s t a r t e r • , and cremoris longi Lkl6 • , at a constant shear rate of 23^ per sec. and a constant temperature of h°C, a f t e r 1 day of storage at t h i s temperature.n indicates equilibrium apparent v i s c o s i t y , c e n t i p o i s e ) . 9 0 91 w i t h s t a n d r h e o d i s t r u c t i o n due to Bhear. The same e f f e c t c o u l d be expected i n other c u l t u r e d d a i r y products when ropy c u l t u r e s are used. 3) New Method f o r M e a s u r i n g V i s c o s i t y i n B u t t e r m i l k During the i n i t i a l stages of t h i s p r o j e c t , t h i s author used the Brabender r o t a t i o n a l viscometer f o r the v i s c o s i t y m o n i t o r i n g of the ropy and non-ropy c u l t u r e d m i l k s . T h i s instrument i s very v e r s i t i l e , a c c u r a t e , and r e l i a b l e , however i t i s complicated and i t demands c o n s i d e r a b l e amount of e x p e r t i e s i n o p e r a t i o n . In a d d i t i o n , i t l s r e l a t i v e l y e x pensive. Thus, t h i s instrument was not found p r a c t i c a l f o r the use i n i n d u s t r y f o r comparative v i s c o m e t r i c measurements. Assuming that i n the d a i r y i n d u s t r y the prime concern of the q u a l i t y c o n t r o l s t a f f are the comparative v i s c o s i t y v a l u e s , not the a b s o l u t e or the apparent v i s c o s i t y v a l u e s , t h i s author experimented with a l t e r n a t i v e ways of r e c o r d i n g the c o n s i s t e n c y of the ropy b u t t e r m i l k . Of the i n s t r u m e n t a t i o n f o r the v i s c o s i t y measurements i n the d a i r y products which was reviewed by P r e n t i c e (1979) and K o h l i et a l . (1980), and used by S o z z i et a l . (1978), K u r w i j i l a et a l . (1981) , Tsaregradskaya and G r i g o r c h u k (1982), and Maksimova et a l . (1982) , the f r e e g r a v i t a t i o n a l flow v i s c o s i m e t e r d e s c r i b e d by S o z z i et a l . (1978), or the f r e e flow p i p e t t e method of T s a r i g r a d s k a y a and G r i g o r c h u k (1982) appeared most appropreate f o r t h i s purpose. However, the ropy m i l k was so t h i c k and v i s c o u s that i t stuck to the w a l l s of the p i p e t t e making i t very d i f f i c u l t to see the meniscus, thus i n t r o d u c i n g l a r g e e r r o r i n the measurements. The same problem was 92 a n t i c i p a t e d with the viscome t e r d e s c r i b e d by S o z z i et a l . ( 1 9 7 8 ) . T h i s author thus used a standard s i e v e which c o u l d accommodate the range of c o n s i s t e n c i e s which were to be compared and de v i s e d an "instrument" which he c a l l e d "the v i s c o m e t r i c s c r e e n d e v i c e " (VSD). I t was used f o r comparative v i s c o s i t y measurements of t h i c k and normal b u t t e r m i l k s . The d e s c r i p t i o n of t h i s instrument i s pr o v i d e d i n the exp e r i m e n t a l s e c t i o n of t h i s t h e s i s , and the d e t a i l e d p a r t s are shown i n F i g u r e 13. I t s schematic r e p r e s e n t a t i o n i s shown i n F i g u r e 14. Because i t has not been s t a n d a r d i z e d with the standard v i s c o s i t y f l u i d s , i t was only used i n t h i s r e s e a r c h f o r comparative v i s c o s i t y measurements, or c o n s i s t e n c y measurements, not f o r apparent v i s c o s i t y measurements. For the v i s c o m e t r i c s c r e e n d e v i c e to be e f f e c t i v e as an instrument f o r comparative c o n s i s t e n c y measurements, i t has to be used i n compliance with a number of f a c t o r s which govern v i s c o s i t y . These f a c t o r s a r e : a) the temperature of the product and the equipment has to be constant, and p r e f e r a b l y the same, f o r a l l the samples which are to be compared f o r v i s c o s i t y , b) the same screen should be used, and the same e x p e r i m e n t a l c o n d i t i o n s p r o v i d e d , f o r a l l samples which are to be compared, c) the e f f e c t i v e area of the screen should be constant throughout the experiment, d) the volume of the sample to be poured through the sc r e e n should be c o n s t a n t , and l a r g e r than the volume which i s c o l l e c t e d and timed, e) the sample, d u r i n g emptying of v e s s e l #3 ( F i g u r e 13a), should 93 a FIGURE 13-Viscometric Screen Device (VSD) used f o r comparative consistency measurements of ropy milk, a) Assembled VSD. b) Disassembled VSD showing a l l components, l ) Standard mesh sieve. 2) Sample vessel (funnel). I t defines the e f f e c t i v e area of the sieve. 3) Pouring/receiving vessel (graduated c y l i n d e r ) , k) Constant temperature water bath f o r r i n s i n g VSD parts and e q u i l i z i n g t h e i r temperature. 5) " A i r wire" used to prevent trapping of a i r i n r e c e i v i n g vessel when sample ve s s e l i s f u l l . 6) Stop watch. FIGURE lh . A digramatic representation of viscometric screen device (VSD). l ) Sample v e s s e l . 2) Viscometric standard mesh screen, which f i t s on the r e c e i v i n g vessel such that i t provides an a i r vent. 3) Receiving (graduated) vessel which can accurately measure the volume of the passing sample. always h i t the sample v e s s e l (#2 F i g u r e 13), i n the same pl a c e with the same amount |Of f o r c e to minimize the e r r o r i n t r o d u c e d by the i n i t i a l f o r c e of p o u r i n g . It was hoped that t h i s instrument can r e p l a c e g r a v i t a t i o n a l flow v i s c o m e t e r s such as the wide mouth p i p e t e or the instrument d e s c r i b e d and used by S o z z i et a l . (1978). The advantages of the v i s c o m e t r i c screen d e v i c e over e x i s t i n g v i s c o m e t e r s a r e : a) I t should be r e l a t i v e l y i n e x p e n s i v e to make, b) i t i s simple to use and r e q u i r e s no e x p e r t i e s of o p e r a t i o n , c) i t i s b e t t e r than the g r a v i t a t i o n a l f r e e - f l o w v i s c o m e t e r because the operator observes an ascending meniscus r a t h e r than a d i s c e n d i n g one, making i t easy to see r e g a r d l e s s of the sample adhesion to the w a l l s of the v i s c o m e t e r , or I t s opaqueness. I t i s suggested that the screens of d i f f e r e n t mesh should be provided with the VSD to cover a l a r g e range of v i s c o s i t i e s . Another way of p r o v i d i n g l a r g e r range of c a p a b i l i t y of the v i s c o s i t i e s f o r the instrument would be to make the screens of given mesh of d i f f e r e n t e f f e c t i v e a r e a . Thus, the screens should be made a t t a c h a b l e to and removable from the sample v e s s e l . T h i s instrument could be s t a n d a r d i z e d with standard v i s c o s i t y o i l s to prov i d e approximations of apparent v i s c o s i t y of the sample. 4. Use o f V i s c o m e t r i c S c r e e n Device The demonstrations of the use of VSD are shown i n F i g u r e s 15 and 16. F i g u r e 15 shows the changes i n VSD c o n s i s t e n c y d u r i n g the c o l d s t o r a g e (4°C) of commercial b u t t e r m i l k c o n t a i n i n g 10% (by volume) of 96 S. cremoris l o n g i . T h i s d a t a shows t h a t the ropy b u t t e r m i l k regains c o n s i s t e n c y ( t h i c k n e s s ) d u r i n g the s h e l f l i f e of the product b e t t e r than i t s non-ropy c o u n t e r p a r t . I t a l s o shows v i s c o m e t r i c d i f f e r e n c e s between these two products at every time i n t e r v a l throughout the IS days of s t o r a g e . Amount of v a r i a n c e can be shown ( F i g u r e I S ) , as w e l l as s t a t i s t i c a l a n a l y s i s can be a p p l i e d to these r e s u l t s (Appendix 7 ) . I t was found that the ropy b u t t e r m i l k had s i g n i f i c a n t l y h i gher c o n s i s t e n c y ( a — 0 . 0 5 ) throughout the storage p e r i o d . F i g u r e 16 shows t h a t , i n a d d i t i o n to v i s c o m e t r i c comparisons between two or more samples, t h i s instrument can measure the v i s c o s i t y decay, as the l o g t r a n s f o r m a t i o n of the data obtained by repeated p a s s i n g of the samples through the VSD ( p r o v i d i n g i n c r e a s e d s h e a r i n g at the constant shear r a t e ) e x h i b i t s a s t r a i g h t l i n e r e l a t i o n s h i p . A s t a t i s t i c a l comparison of the l i n e s i n F i g u r e 16 (Appendix 7) shows that the i n c o r p o r a t i o n of ropy c u l t u r e i n b u t t r m i l k s i g n i f i c a n t l y improves ( a 1 . 0 . 0 5 ) the product's r e s i s t a n c e to constant shear. These r e s u l t s are comparable to those obtained with r o t a t i o n a l v i s cometer, Brabender Rheotron ( F i g u r e s 11 and 12). 97 a FIGURE15. Viscometric screen device (VSD) consistency curves of commercial buttermilk (1.5% b u t t e r f a t ) O , and buttermilk containing 10$ (by volume, of t o t a l inoculum) of 18 hour culture of S. cremoris l o n g i Lkl6 (l.5% butter f a t ) • . 100 ml. samples were passed through 2.5 cm. area of Uo mesh (1+20 -um) Canadian Standard Sieve, and the time f o r 50 ml. to go through was recorded.at 5 day i n t e r v a l s during a 15 day storage period at U-6°C. A l l readings were taken at k°C. Points on the graph represent mean values. Bars represent standard deviation from the mean. Numbers above points represent sample s i z e (n). C O N S I S T E N C Y ( s e c . ) VO 9 8 a FIGURE 16. Viscometric decay curves, as measured by the viscometric screen device at 22°C, f o r 1.5% b u t t e r f a t buttermilks. ( # ) 3% (by volume) commercial buttermilk s r a r t e r , ( • ) 15% cremoris l o n g i Lkl6 / 85% commercial buttermilk s t a r t e r of the 3% t o t a l inoculum, ( • ) 3% (by volume) §_. cremoris l o n g i I_Al6. Each point represents an average of the number of r e p e t i t i o n s i n d i c a t e d by numbers above points. 98 2T to L O G No. PASSES THRU 20 MESH SIEVE 99 SUMMARY AND CONCLUSIONS A. C u l t u r e C h a r a c t e r i s t i c s The c h a r a c t e r i s t i c s of the ropy c u l t u r e s were expressed i n terms of growth curve, c u l t u r e s t a b i l i t y , a n t i m i c r o b i a l a c t i v i t i e s , and the c o m p a t a b i l i t y with the b a c t e r i a of commercial b u t t e r m i l k s t a r t e r . In most of th e s e e x p e r i m e n t s S. c r e m o r i s l o n g i 1416 was s e l e c t e d , as i t was not convenient to t e s t a l l ropy c u l t u r e s on hand. An attempt was made to pro v i d e only enough d e t a i l to enable i n d u s t r i a l a p p l i c a t i o n s of the ropy b a c t e r i a . Growth curve of S. c r e m o r i s l o n g i L416 was c o n s t r u c t e d ( F i g u r e 2) as e q u i l i b r i u m apparent v i s c o s i t y at constant shear r a t e of 500 per s e c , b a c t e r i a l numbers as CFU/ml., and a c i d p r o d u c t i o n as pH, were recorded as a f u n c t i o n of i n c u b a t i o n time i n skim milk at 18°C over 4 days. As expected, i t was found that most of the a c t i v i t y took p l a c e d u r i n g the f i r s t 20 hours of growth, as the v i s c o s i t y i n c r e a s e d d r a s t i c a l l y between 5 and 24 hours of growth. The i n c r e a s e i n v i s c o s i t y was preceeded by the i n c r e a s e i n b a c t e r i a l numbers and the drop i n pH. T h i s experiment showed that v i s c o s i t y measurements can be used i n c o n s t r u c t i o n of a growth curve, i n s t e a d of more t r a d i t i o n a l CFU counts or pH development. In a d d i t i o n , v i s c o s i t y measurement i s the best i n d i c a t o r of v i s c o s i t y due to slime p r o d u c t i o n , s i n c e a c i d p r o d u c t i o n and i n c r e a s e i n CFU do not always guarantee s l i m e p r o d u c t i o n . The c o n t r i b u t i o n to the v i s c o s i t y by slime i s shown i n F i g u r e 3. T h i s f i g u r e shows that the t o t a l v i s c o s i t y of the ropy milk 1 0 0 was an a d d i t i v e e f f e c t of the v i s c o s i t y due to the m i l k c o a g u l a t i o n and the slime p r o d u c t i o n by the b a c t e r i a . The v i s c o s i t y due to slime was s u b s t a n t i a l , but i t v a r i e d among s t r a i n s ( F i g u r e 3 ) . The t e s t s f o r the i d e n t i f i c a t i o n of the s p e c i e s of the c u l t u r e s on hand showed t h a t L L F was a s t r a i n of S. l a c t i s , while the s t r a i n s L416, 701, and 705 were shown to be the ropy v a r i a n t s of S. c r e m o r i s ( T a b l e 2 ) . S i n c e the ropy v a r i a n t s of S. l a c t i s have been named S. l a c t i s l o n g ! ( N i l s s o n and N i l s s o n , 1950) and s i n c e the ropy v a r i a n t s of S. c r e m o r i s have not been named, i t was suggested t h a t t h e y be named S. c r e m o r i s l o n g i . C u l t u r e s t a b i l i t y was s t u d i e d by determining percent ropy c o l o n i e s as a f u n c t i o n of a number of t r a n s f e r s and the l e n g t h of i n c u b a t i o n at growth temperature ( T a b l e s 3 and 4 ) . I t was found th a t the s t a b i l i t y of slime p r o d u c t i o n decreased with an i n c r e a s e l n each of the above mentioned f a c t o r s . Percent ropy colony decreased to 74% f o r c u l t u r e 701, 72% f o r c u l t u r e L416, 51% f o r c u l t u r e 705, and 2% f o r c u l t u r e LLF a f t e r 10 c o n s e c u t i v e t a n s f e r s i n skim milk ( T a b l e 3 ) . Signs of v i s c o s i t y l o s s due to prolonged i n c u b a t i o n at growth temperature were a l s o observed. Thus, t h i s experiment was repeated w i t h c u l t u r e LLF where the time of i n c u b a t i o n was e x a c t l y equal at c o u n t i n g f o r a l l t r a n s f e r s , and a l l t r a n s f e r s were done at e x a c t l y 50 hours. I t was found that these b a c t e r i a l o s t v i s c o s i t y w i t h i n 4 days of i n c u b a t i o n at 22°C r e g a r d l e s s of how many times they were t r a n s f e r r e d ( T a b l e 4 ) . A l s o , a f t e r 10 s e r i a l t r a n s f e r s t h i s c u l t u r e showed only 10% ropy c o l o n i e s when counted a f t e r 96 hours, and no ropy c o l o n i e s a f t e r 144 hours on skim milk agar. Thus, i t appears that a g e n e t i c f a c t o r r e s p o n s i b l e f o r slime p r o d u c t i o n 101 ( p o s s i b l y a plasmid) was l o s t d u r i n g s e r i a l t r a n s f e r r i n g , while a "chemical agent" may 'have been r e s p o n s i b l e f o r the slime d i s t r u c t i o n d u r i n g prolonged s t o r a g e at i n c u b a t i o n temperature. The l a t t e r f a c t o r was i n h i b i t e d d u r i n g c o l d s t o r a g e , l e . < 10°C (da t a not shown). I t l s recommended t h a t , to m a i n t a i n constant l e v e l of slime p r o d u c t i o n by the stock c u l t u r e s , a " s a f e " number of c u l t u r e t r a n s f e r s should be d e r i v e d f o r a g i v e n c u l t u r e , and a f t e r that number of t r a n s f e r s i s reached d u r i n g the c u l t u r e use, new c u l t u r e stocks should be developed from a ropy c o l o n y . In a d d i t i o n , as soon as a d e s i r a b l e l e v e l of r o p i n e s s i s achieved the product should be r e f r i g e r a t e d to pre s e r v e the v i s c o s i t y . A n t i b a c t e r i a l a c t i v i t y of S. c r e m o r i s l o n g i L416 was s t u d i e d by c h a l l e n g i n g t h i s b a c t e r i a with ATCC s t r a i n s 25923 (S. aureus) ( F i g u r e 5 ) , 25922 ( E . c o l i ) ( F i g u r e 6 ) , and P. f l u o r e s c e n s b i o t y p e A ( F i g u r e 7 ) . A l l the t e s t organisms were d r a s t i c a l l y i n h i b i t e d and completely e l i m i n a t e d w i t h i n ten days of r e f r i g e r a t e d s t o r a g e , while the c o n t r o l s showed growth. However, S. cr e m o r i s  l o n g i L416 was not found to be a b e t t e r i n h i b i t o r of the t e s t organisms than the ATCC s t r a i n of S. cremoris (19257). The c o m p a t i b i l i t y of S. c r e m o r i s l o n g i L416 w i t h the b a c t e r i a of the commercial b u t t e r m i l k s t a r t e r was s t u d i e d by growing known p o r t i o n s of ropy b a c t e r i a with the commercial s t a r t e r , s t o r i n g i t at 4-6°C f o r 15 days, and e v e r y 5 days d e t e r m i n i n g the numbers of t o t a l CFU/ml. and the number of ropy CFU/ml. ( F i g u r e s 8, 9 and 10). I t was found that no d e l e t e r i o u s e f f e c t was e x h i b i t e d by e i t h e r c u l t u r e . Moreover, the ropy b a c t e r i a seemed to s u r v i v e the c o l d s t o r a g e b e t t e r when grown with the b u t t e r m i l k s t a r t e r . The ropy 102 b a c t e r i a e x h i b i t ted s i g n s of dominance, however i t c o u l d not be determined whether the ropy b a c t e r i a were s u r v i v i n g b e t t e r , or the non-ropy b a c t e r i a were a c q u i r i n g the ropy c h a r a c t e r . B. C h a r a c t e r i s t i c s o f t h e Slime The slime was p r o d u c e d by S. c r e m o r i s l o n g i L416 i n d e p r o t e i n i z e d whey. The n e u t r a l i z e d sheese whey was passed through a m i l l i p o r e membrane with an e x c l u s i o n c o e f f i c i e n t of 10,000 MW. The ropy b a c t e r i a were grown i n the < 10,000 MW f r a c t i o n and a f t e r s l i m e p r o d u c t i o n , the b a c t e r i a l c e l l s and contaminating p r o t e i n s were s e p a r a t e d . The pure s l i m e was f r e e z e d r i e d and analysed f o r t o t a l p r o t e i n , protein-bound hexoses, hexosamines, and s i a l i c a c i d s . The e stimate of t o t a l p r o t e i n content of the slime was made by F o l i n phenol method (Lowry et a l . , 1951), while the amino a c i d a n a l y s i s was done with an amino a c i d a n a l y z e r a f t e r sample h y d r o l y s i s w i t h m e t h a n e s u l f o n i c a c i d (Simpson et a l . , 1975). An amino a c i d summation method (Heidelbagh et a l . , 1975) was used to o b t a i n the t o t a l p r o t e i n content of the sample. The amino a c i d p r o f i l e of the s l i m e was found to be s i m i l a r to that of the d r i e d whey permeate i n which the b a c t e r i a were grown (T a b l e 10). The t o t a l p r o t e i n content was found to be 47% of the d r i e d s l i m e . No d e t e r m i n a t i o n of c y s t i n e , t y r o s i n e and moisture were done, s i n c e the purpose of t h i s I n v e s t i g a t i o n was only to prove the presence or absence of p r o t e i n i n the s l i m e . Thus, t h i s a n a l y s i s confirmed that the s l i m e contained a p r o t e i n f r a c t i o n , which compared f a v o r a b l y with the f i n d i n g s of Sundman (1953b). The carbohydrate a n a l y s e s r e v e a l e d 9.3% protein-bound 103 hexoses, 20% methylpentoses ( f u c o s e ) , 2.8% s i a l i c a c i d s , and no hexosamlnes ( T a b l e 9 ) . From the amino a c i d p r o f i l e ( T a b l e 10) and the carbohydrate a n a l y s i s ( T a b l e 9 ) , i t appears that the slime i s a n e g a t i v e l y charged, high molecular weight molecule, most l i k e l y an 0 - l i n k e d g l y c o p r o t e i n , with approximately equal amounts of p r o t e i n and p o l y s a c c h a r i d e . C. P r a c t i c a l A p p l i c a t i o n s o f S. c r e m o r i s l o n g i L416 The p r a c t i c a l a p p l i c a t i o n of S. c r e m o r i s l o n g i L416 was demonstrated i n b u t t e r m i l k . I t was found that the optimum c o n c e n t r a t i o n of the ropy s t a r t e r was 10 to 15% of t o t a l b u t t e r m i l k Inoculum. At 50% (v/v) ropy s t a r t e r i n the inoculum the product was judged to be s i g n i f i c a n t l y b e t t e r on b a s i s of f l a v o u r / a c i d i t y , body/texture and o v e r a l l a c c e p t a b i l i t y ( a 1. 0.05) than the commercial b u t t e r m i l k ( T a b l e 11), however i t was v i s i b l y ropy. At 10% ropy c u l t u r e the v i s c o s i t y c o n t r i b u t i o n was s i g n i f i c a n t ( a 1. 0.05) a f t e r p r o d u c t i o n , and throughout i t s s h e l f l i f e ( F i g u r e 15). In a d d i t i o n to p r o v i d i n g s i g n i f i c a n t l y higher t h i c k n e s s , the ropy c u l t u r e induced s i g n i f i c a n t l y b e t t e r r e s i s t a n c e to shear ( o i 0.05) ( F i g u r e 11). In a d d i t i o n to above d e s c r i b e d experiments, a new instrument f o r measuring comparative v i s c o s i t y i n b u t t e r m i l k was i n v e n t e d . I t was c a l l e d the v i c s c o m e t r i c s creen d e v i c e (VSD) ( F i g u r e s 13 and 14), as i t employed a standard mesh s i e v e to d i s t i n g u i s h between b u t t e r m i l k samples of d i f f e r e n t t h i c k n e s s . T h i s instrument was found to produce r e s u l t s comparable to those obtained by a much more expensive and e l a b o r a t e r o t a t i o n a l v i s c o m e t e r , Brabender Rheotron. VSD was f a s t e r , and e a s i e r and s i m p l e r to use f o r comparative v i s c o s i t y measurements of 104 b u t t e r m i l k . 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T r o i l i - P e t e r s s o n , G., 1899, Schwedische Langmilch. M i l c h - Z i e t u n g , 28:438. Tsaregradskaya, I . V., V. R. Grigorchuk, 1982, S e l e c t i o n of v i s c o u s s t a r t e r s f o r sour cream. 21st I n t e r n . D a i r y Congress B r i e f Communication, V o l . 1, Book 2, Mir P u b l i s h e r s , Moscow, 381. Tung, M. A., B. C. M o r r i s o n , E. L. Watson, 1970, Rheology of f r e s h , aged, and gamma-irradiated egg white. J . Food S c i . 35: 872. Tung, M. A., E. L. Watson, J . F. R i c h a r d s , 1971, Rheology of egg albumen. Trans. A.S.A.E. 14: 17. Vedamuthu, E. R., 1977, E x o t i c fermented d a i r y foods. J . Food P r o t . 40: 801. Vedamuthu, E. R., R. B. Shah, 1983, Method f o r the p r e p a r a t i o n of n a t u r a l l y t h i c k e n e d and s t a b i l i z e d fermented m i l k p r o d u c t s and products produced thereby, U.S. Patent 4,382,097. Warren, L., 1959, The t h i o b a r b l t u r i c a c i d assay of s i a l i c a c i d s , J . B i o l . Chem. 234: 1971. W i n k e l s t e i n , A., L a c t o b a c i l l u s a c i d o p h i l u s t a b l e t s i n the therapy of f u n c t i o n a l i n t e s t i n a l d i s o r d e r s . Am. P r a c t . D i g . T r e a t . 7: 1637. 112 Windholz, M., 1976, The Merck Index - An E n c y c l o p e d i a of Chemicals and Drugs, 9 t h E d i t i o n , Merck and Co. Inc. pp. 8, 389. W i n z l e r , R. J . , 1955, D e t e r m i n a t i o n of serum g l y c o p r o t e i n s , i n Methods of B i o c h e m i c a l A n a l y s i s , V o l . 2, I n t e r s c i e n c e P u b l i s h e r s Inc. p. 279. W o l f e r s t e t t e r , E., 1969, Langmllch, e i n product aus Lappland. DTSC M i l c h w i r s c h . H i l d e r s h e i m , 30: 1473. 113 APPENDIX 1 D e t e r m i n a t i o n of T o t a l P r o t e i n C o n c e n t r a t i o n i n Slime D e t e r m i n a t i o n of T o t a l P r o t e i n C o n c e n t r a t i o n l n the Slime Produced by S. cremoris l o n g i - F o l i n Phenol Method A c c o r d i n g to Lowry et a l . (1951), the F o l i n phenol method can be used f o r determining of as l i t t l e as 0.2 ug./ml. of p r o t e i n . a) P r e p a r i n g s o l u t i o n s i ) Standard: C r y s t a l l i z e d (Ax) bovine serum albumin (BSA), 5.076 mg., was d i s s o l v e d i n 10 ml. of d i s t i l l e d d e l o n i z e d warter, g i v i n g f i n a l p r o t e i n c o n c e n t r a t i o n of 507.6 ug./ml. F u r t h e r d i l u t i o n s were made to o b t a i n the BSA c o n c e n t r a t i o n s shown i n the Table below. i i ) Slime sample: Dry, pure slime (2.142 mg.) was d i s s o l v e d i n 2 ml. of d i s t i l l e d d e l o n i z e d water, g i v i n g the f i n a l 6lime c o n c e n t r a t i o n of 1.071 mg./ml. F u r t h e r d i l u t i o n s were made to o b t a i n the c o n c e n t r a t i o n s shown i n the Table below. Standard curve d i l u t i o n s ug. BSA/0.8 ml. Sample A Sample B Sample C Sample D X S.D. 10.2 0.029 0.027 0.027 0.030 0.028 0.002 20.3 0.054 0.054 0.055 0.051 0.054 0.002 40.6 0.088 0.091 0.087 0.090 0.089 0.002 60.9 0.120 0.121 0.120 0.130 0.123 0.005 81.2 0.186 0.194 0.188 0.194 0.191 0.004 101.5 0.229 0.225 0.235 0.231 0.230 0.004 Slime/0.8 ml. 107.1 0.093 0.106 0.111 0.111 0.105 0.009 b) C a l c u l a t i o n s i ) L i n e a r r e g r e s s i o n a n a l y s e s (Monroe programmable c a l c u l a t o r ) : Y - mX + b Y • absorbance at 750 um. X - ug. BSA/0.8 ml. m • the slope b - the Y i n t e r c e p t S.E.E. - st a n d a r d e r r o r of estimate r • r e g r e s s i o n c o e f f i c i e n t i i ) R e g r e s s i o n a n a l y s e s readout: 115 m X Y b S • E • E • r 52.442 0.1191 0.0022 0.0032 0.0082 0.9935 111) L i n e a r r e g r e s s i o n e q u a t i o n : Y 0.0022X + 0.0032 r 0.994 (1) i v ) C a l c u l a t i n g p r o t e i n content i n pure slime from eq. 1: 0.105 - 0.0022X + 0.0032 0.105 - 0.0032 X 0.0022 X - 46.273 ug. BSA/0.8 ml. T h e r e f o r e , % p r o t e i n i n the slime - 46. 27/107.5 x 100% 43.0%. 116 APPENDIX 2 D e t e r m i n a t i o n of Protein-Bound Hexoses i n Slime 117 D e t e r m i n a t i o n of Protein-Bound Hexoses In the Slime Produced by S.  cremoris l o n g i L416 - L u s t i n g and Langer Method A c c o r d i n g to W i n z l e r (1955) t h i s method, when used f o r blood serum, shows l i n e a r absorbance at 540 um to amounts as high as 0.5 mg of galactose-mannose. a) P r e p a r i n g s o l u t i o n s 1) Standard: Galactose-mannose, 7.604 mg. mannose, and 7.493 mg. g a l a c t o s e was d i s s o l v e d i n 25 ml. of d i s t i l l e d d e i o n i z e d water, g i v i n g f i n a l hexose c o n c e n t r a t i o n of 0.604 mg./ml. i i ) S l i m e s a m p l e : The pure slime samples were d i s s o l v e d i n 1 ml. of 0.1 N NaOH. i i i ) Standard curve d i l u t i o n s : A 5 4 0 mg• Hexose/ml. Sample A Sample B X S.D. 0.050 0.085 0.088 0.087 0.002 0.101 0.165 0.176 0.171 0.008 0.201 0.350 0.370 0.360 0.014 0.302 0.610 0.595 0.603 0.011 0.403 0.860 0.850 0.855 0.007 0.503 1.150 1.000 1.075 0.106 0.604 1.300 1.300 1.300 0 mg. Slime/ml. 1.818 0.326 0.914 0.130 b) C a l c u l a t i o n s i ) L i n e a r r e g r e s s i o n a n a l y s i s (Monroe programmable c a l c u l a t o r ) : Y - mX + b Y " absorbance at 540 um X • mg. hexose/ml. m • the s l o p e b • the Y i n t e r c e p t S.E.E. • Standard e r r o r of es t i m a t e r • C o r r e l a t i o n c o e f f i c i e n t i i ) R e g r e s s i o n a n a l y s i s readout: X Y 0.309 0.636 118 m - 2.234 b - -0.055 S.E.E. - 0.035 r - 0.997 L i n e a r r e g r e s s i o n e q u a t i o n : Y - 2.234 X - 0.055 r - 0.997 11) C a c u l a t l n g Hexose Content i n Pure Slime Sample A: 0.326 - 2.234X - 0.055 0.326 + 0.055 X 2.234 X - 0.171 mg. hexose/ml. 0.171 mg. hexose/ml. % Hexose i n sl i m e - — — x 100% 1.818 mg. s l i m e / m l . • 9.410% hexose In s l i m e . Sample B: 0.130 - 2.234X - 0.055 0.130 + 0.055 X  2.234 • 0.083 mg. hexose/ml 0.083 mg. hexose/ml. % Hexose i n sl i m e x 100% 0.914 mg. sl i m e / m l . - 9.080% hexose i n s l i m e . (2) T h e r e f o r e , the average % hexose i n slime i s 9.25%. 119 APPENDIX 3 D e t e r m i n a t i o n of Methylpentoses i n Slime 120 D e t e r m i n a t i o n of M e t h y l p e n t o s e s l n t h e Pure Slime Produced by S. c r e m o r i s l o n g i L416 A c c o r d i n g to W i n z l e r (1955) fucose content Is c a l c u l a t e d from the d i f f e r e n c e s i n the readings at 396 and 430 um and s u b t r a c t i n g the va l u e s without c y s t e i n e ( e q u a t i o n 3 ) . a) P r e p a r i n g s o l u t i o n s i ) Standard: Rhamnose ('DIFCO' s t a n d a r d i z e d ) was used to prepare 4.047 mg/100 ml. s o l u t i o n i n d i s t i l l e d d e l o n i z e d water. A 10 ml. volume of t h i s s o l u t i o n was f u r t h e r d i l u t e d w i t h 10 ml. of d i s t i l l e d d e l o n i z e d water. i i ) Slime sample: Two samples of d r i e d pure s l i m e , sample A weighing 0.987 mg, and sample B weighing 1.219 mg, were d i s s o l v e d i n 1 ml. of 0.1 N NaOH. A f t e r other reagents were added ( W i n z l e r , 1955), the samples A and B were each d i v i d e d l n two to accommodate the r e a c t i o n with and without c y s t e i n e . Absorbance values of rhamnose standard and slime s o l u t i o n s Sample A 4 3 0 Av . A ^ 3 0 A 3 9 6 A v * A 3 9 6 Standard A +cys t . 0.015 0.015 0.391 0.396 (20.23 ug./ml.) +cys t . 0.015 0.400 - c y s t . 0.000 0.000 0.000 0.000 - c y s t . 0.000 0.000 Standard B +cys t . 0.038 0.036 0.800 0.790 (40.47 ug./ml.) +cys t . 0.034 0.780 - c y s t . 0.000 0.000 0.000 0.000 - c y s t . 0.000 0.000 Slime sample A +cys t . 0.196 0.405 (0.494 mg./ml.) - c y s t . 0.018 0.038 Slime sample B +cyst. 0.226 0.453 (0.610 ug./ml.) - c y s t . 0.019 0.026 Note: Standard A was the o n l y one used i n the c a l c u l a t i o n s because the procedure recommended t h i s c o n c e n t r a t i o n of st a n d a r d s o l u t i o n to be used ( W i n z l e r , 1955). Standard B was run to see I f the absorbance at these wavelengths i s d i r e c t l y p r o p o r t i o n a l to the amount of methylpentose. b) C a l c u l a t i o n s : 1) Slime sample A: (0.405 - 0.196) - (0.038 - 0.018) mg. Fucose/100 ml. - 20.2 x (0.396 - 0.015) - 10.02 mg. Fucose/100 ml. - 0.1002 mg. Fucose/ml. The amount of d r i e d sample used was 0.494 mg./ml., t h e r e f o r e , percent fucose i n sl i m e sample i s : 0.100 % Fucose i n slime sample A - x 100% - 20.2% 0.494 i i ) Slime sample B: (0.453 - 0.226) - (0.026 - 0.019) mg. Fucose/lOOml. - 20.2 x • (0.396 - 0.015) • 11.66 mg. Fucose/100 ml. - 0.117 mg. Fucose/ml. The amount of d r i e d s l i m e used was 0.610 mg./ml., t h e r e f o r e , percent fucose i n sl i m e l s : 0.117 % Fucose i n slime sample B • — x 100% - 19.2% 0.610 T h e r e f o r e , the average percent fucose i n slime sample i s 19.7%. 122 APPENDIX 4 D e t e r m i n a t i o n of S i a l i c A c i d s i n Slime 123 D e t e r m i n a t i o n of S i a l i c A c i d s i n t h e Slime Produced by S. cr e m o r i s  l o n g i L416 - T h i o b a r b i t u r i c A c i d Method A c c o r d i n g to Warren (1959) the t h i o b a r b i t u r i c a c i d assay f o r s i a l i c a c i d s l s more s e n s i t i v e than the most s e n s i t i v e r e s o r c i n o l method. I t i s s p e c i f i c enough to use f o r the s i a l i c a c i d d e t e r m i n a t i o n i n t i s s u e s , and the c o l o r development i s l i n e a r with the c o n c e n t r a t i o n of N - a c e t y l n e u r a m i n i c a c i d ranging from 0.01 to 0.06 uMoles. a) P r e p a r i n g s o l u t i o n s 1) Standard: N - a c e t y l n e u r a m i n i c a c i d (Sigma I V ) , 0.913 mg., was d i s s o l v e d i n 10 ml. of d i s t i l l e d d e i o n i z e d water. T h i s corresponds to 0.276 uMoles of 100% pure N - a c e t y l n e u r a m i n i c a c i d per ml. From t h i s s o l u t i o n , f o l l o w i n g d i l u t i o n s were made: Standard curve d i l u t i o n s uMoles/0.2 ml. A 5 4 9 Sample A Sample B Sample C X S.D. 0.011 0.022 0.034 0.045 0.056 Slime sample 0.206 0.362 0.527 0.799 0.909 0.098 0.205 0.356 0.540 0.721 0.870 0.096 0.236 0.375 0.517 0.686 0.929 0.100 0.216 0.364 0.528 0.735 0.903 0.098 0.017 0.010 0.012 0.058 0.030 0.002 i i ) Slime sample: A d r i e d slime sample, 2 .724 mg., was hydrolyzed i n 10 ml. of 0.1 N H S 0 4 a t 80 C f o r 1 hour b e f o r e 0.2 m l . a l i q u o t s were s u b j e c t e d to the assay f o r s i a l i c a c i d s . T h e r e f o r e , the amount of the sl i m e present i n 0.2 ml. was 0.0485 mg.. b) C a l c u l a t i o n s 1) L i n e a r r e g r a t i o n a n a l y s e s (Monroe programmable c a l c u l a t o r ) : Y - mX + b Y • absorbance at 549 um X • uMoles N - a c e t y l n e u r a m i n i c acid/0.2 ml. m - s l o p e b • Y i n t e r c e p t S.E.E. - Standard e r r o r of estimate r • r e g r e s s i o n c o e f f i c i e n t i i ) L i n e a r r e g r e s s i o n a n a l y s e s readout: X 0.034 12k Y - 0.549 m - 15.435 b - 0.031 S.E.E. - 0.030 r - 0.993 i l l ) L i n e a r r e g r e s s i o n e q u a t i o n : Y - 15.435X + 0.031 r - 0.993 (4) i i ) C a l c u l a t i n g s i a l i c a c i d content i n s l i m e : 0.098 - 0.031 x  15.435 X • 4.340 x 10"^ uMole N - a c e t y l n e u r a m i n i c a c i d . 4.340 x 1 0 " 9 mole x 309 g./mole Percent s i a l i c a c i d i n sl i m e - — — — x 100% 4.854 x 1 0 " 5 g. - 2.76 %. 125 APPENDIX 5 D e t e r m i n a t i o n of Hexosamlnes i n Slime 126 D e t e r m i n a t i o n of Hexosamines In the Slime Produced by S. cremoris  l o n g i L416 - E l s o n - M o r g a n Method A c c o r d i n g to W i n z l e r (1955) absorbance at 530 um i s l i n e a r with hexosamine to amounts as high as 0.25 mg. a) P r e p a r i n g s o l u t i o n s i ) Standard: Glucosamine h y d r o c h l o r i d e , 15.016 mg was d i s s o l v e d i n 25 ml. of d i s t i l l e d d e l o n i z e d water, to g i v e f i n a l c o n c e n t r a t i o n of 0.6006 mg./ml.. T h i s i s e q u i v a l e n t to 0.5005 mg. f r e e glucosamine/ml. Standard curve d i l u t i o n s mg. Free Glucosamine/ml. Sample A Sample B X S.D. 0.501 1.600 1.750 1.675 0.106 0.250 0.920 1.000 0.960 0.057 0.083 0.226 0.243 0.235 0.012 0.050 0.128 0.136 0.132 0.006 0.025 0.051 0.056 0.054 0.004 0.008 0.005 0.002 0.004 0.002 b) C a l c u l a t I o n s : i ) L i n e a r r e g r e s s i o n a n a l y s i s (Monroe programmable c a l c u l a t o r ) : Y - mX + b Y • absorbance at 530 um X • mg. hexosamine/ml. m • the slope b • the Y I n t e r c e p t S.E.E. « standard e r r o r of estimate r • r e g r e s s i o n c o e f f i c i e n t i i ) L i n e a r r e g r e s i o n analyses readout: X - 0.153 Y - 0.510 m " 3.489 b - -0.024 S.E.E. - 0.062 r - 0.995 i i i ) L i n e a r r e g r e s s i o n e q u a t i o n : Y - 3.489X - 0.024 r - 0.995 (5) 127 11) S l i m e s a m p l e ; A d r i e d slime sample, 0.472 mg., was h y d r o l i z e d i n 3N HC1 l n b o i l i n g water bath f o r 4 hours. The sample was n e u t r a l i z e d with 3N NaOH, and d i l u t e d to 10 ml. From t h i s , 1 ml. .samples were f u r t h e r analyzed as per Wi n z l e r (1955). Thus, the c o n c e n t r a t i o n of slime i n these samples was 0.0472 mg./ml. Slime sample data Slime samples A A B C D E - 0.015 - 0.008 - 0.016 - 0.020 - 0.013 i i ) C a l c u l a t i n g hexosamine content i n pure slime S i n c e a l l values f o r the sl i m e sample proved to be ne g a t i v e , i t i s concluded that there are no hexosamines i n the sample. 128 APPENDIX 6 Q u e s t i o n n a i r f o r Hedonic S c a l e 129 QUESTIONNAIRE FOR HEDONIC SCALE NAME DATE PRODUCT Taste these samples and check how much you l i k e or d i s l i k e each one on the basis of f l a v o u r / a c i d i t y . 2 148 l i k e extremely l i k e very much l i k e moderately ~ l i k e s l i g h t l y neither l i k e nor d i s l i k e d i s l i k e s l i g h t l y d i s l i k e moderately d i s l i k e very much d i s l i k e extremely 914 l i k e extremely l i k e very much l i k e moderately l i k e s l i g h t l y niether l i k e nor d i s l i k e d i s l i k e s l i g h t l y d i s l i k e moderately d i s l i k e very much d i s l i k e extremely 579 l i k e extremely l i k e very much l i k e moderately l i k e s l i g h t l y neither l i k e nor d i s l i k e d i s l i k e s l i g h t l y d i s l i k e moderately d i s l i k e very much d i s l i k e extremely Comments: EXAMPLE. The ratings f o r each sample are given numerical values ranging from l i k e extremely (9) to d i s l i k e extremely (1). The r e s u l t s are analyzed by a n a l y s i s of variance. I f only two treatments are evaluated, the mean score received by each can be compared using t - t e s t . Adapted from Larmond, 1977. Same questionnair was used f o r "body/texture" and " o v e r a l l a c c e p t a b i l i t y " with these words repla c i n g " f l a v o u r / a c i d i t y " . APPENDIX 7 S t a t i s t i c a l Analysis of Viscometric Data FIGURE LINE 1 VI ¥2 X2 LINE 2 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A--0.22692E+00 A--0.31093E+OQ B--0.34489E+00 B--0.39764E+00 SIMREG FOR POOLED DATA A--O.20556E+O0 8--0.41176E+00 SEY-0.3612E-01 SEV-0.2595E-01 SEY-0.9447E-01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 6./ 6." O.194E+01 TEST FOR SLOPES F, 1/ 12.« 0.270E+01 TEST FOR LEVELS F. 1/ 13.- 0.988E+02 OVERALL TEST F, 2./ 12.- 0.572E+02 S t a t i s t i c a l a n a l y s i s of the l i n e s i n F i f u r e 11. CORRELATION DEGREES OF COEFFICIENT FREEDOM R- -0.9857 DF- 7. R- -0.9924 DF- 7. R- -0.9227 DF- 15. FIGURE LINE 1 n LINE 2 XI V2 X2 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE A « O.13719E+01 A = 0.26779E*00 B--0.67786E+00 B--0.46979E+00 SEY-0.6485E-01 SEV-0.7339E-01 SIMREG FOR POOLED DATA A" 0.61668E+00 B--0.4G76GE+00 SEY-0.3756E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 14./ 14.« 0.781E+00 TEST FOR SLOPES F. 1/ 28." O.158E+02 TEST FOR LEVELS F. 1/ 29.« 0.556E+03 OVERALL TEST F. 2./ 28. - 0.427E+03 THERE ARE 4 VARIABLES AND 1 PAIRS OF LINES (4F7.3) S t a t i s t i c a l analysis of the lines i n Figure 12. CORRELATION DEGREES OF COEFFICIENT FREEDOM R- -0.9767 DF- 15. R- -0.9684 DF- 15. R- -0.5377 DF- 31. FIGURE LINE 1 VI XI LINE 2 V2 X2 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE 1 A» 0.209B5E*01 2 A* O.10855E+01 8« 0.17600E+00 B- 0.72189E-01 SIMREG FOR POOLED DATA A- 0.15920E*01 B- O.12410E+00 SEY-O.1388E+00 SEY-O. 1431E+00 SEY-O.1102E+01 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 2./ 2.- 0.941E+00 TEST FOR SLOPES F. 1/ 4.» 0.30OE+O2 TEST FOR LEVELS F. 1/ 5.- 0.488E+02 OVERALL TEST F. 2./ 4.-0.181E+03 S t a t i s t i c a l a n a l y s i s o f the l i n e s i n F i g u r e 15. CORRELATION COEFFICIENT DEGREES OF FREEDOM R« 0.9944 DF- 3. R- 0.9663 DF- 3. R- 0.5648 DF« FIGURE LINE 1 VI XI LINE 2 V2 X2 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A- 0.25300E+00 A» 0.92309E+00 B--0.16442E+00 B--0.63169E+00 SEV-O.1173E-01 SEY-O.1786E-01 SIMREG FOR POOLED DATA A- 0.50188E+00 B--0.19901E+00 SEY-O.1823E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 2./ 8. - 0.430E+00 TEST FOR SLOPES F. 1/ 10.- 0.586E+02 TEST FOR LEVELS F. 1/ 11.- 0.215E+03 OVERALL TEST F. 2./ 10.- O.700E*O3 R- -0.9473 R- -0.9939 R- -O.2G70 DF" DF« 3. 9 . DF- 13. S t a t i s t i c a l a n a l y s i s of the l i n e s 1 ( • ) and 2 ( • ) i n F i g u r e 16. FIGURE LINE 1 Y1 X1 LINE 2 Y3 X3 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A- 0.25300E+00 A» O.15521E+01 B«-0.16442E+00 B—0.2B176E+00 SEY-O.1172E-01 SEY-O.3027E-01 SIMREG FOR POOLED DATA A- 0.55000E+00 B- 0.41962E+00 SEY-O.4786E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 2./ 9 . - 0.150E+00 TEST FOR SLOPES F. 1/ 11.- 0.151E+01 TEST FOR LEVELS F. 1/ 12.- 0.367E+04 OVERALL TEST F. 2./ 11.- 0.192E+04 R- -0.9473 R- -0.9747 R- 0.3940 DF- 3. DF- 10. OF- 14. S t a t i s t i c a l a n a l y s i s of the l i n e s 1 ( # ) and 3 ( 4 ^ ) i n F i g u r e 16. FIGURE LINE 1 Y2 LINE 2 X2 Y3 X3 INDIVIDUAL SIMREG LINES STANDARD ERROR OF ESTIMATE CORRELATION COEFFICIENT DEGREES OF FREEDOM A- 0.92309E+O0 A- O.15521E+01 B--0.63169E+00 B—0.28176E+00 SEY-O.1786E-01 SEY-O.3027E-01 SIMREG FOR POOLED DATA A- 0.67843E+00 B- O.19069E+00 SEY-O.4331E+00 COVARIANCE ANALYSIS TEST FOR HOMOGENEITY OF RESIDUAL VARIANCES F. 8./ 9 . - 0.348E+00 TEST FOR SLOPES F. 1/ 17.» 0.792E+02 TEST FOR LEVELS F. 1/ 18.» 0.103E+04 OVERALL TEST F. 2./ 17.- 0.2BOE+04 R- -0.9939 R- -0.9747 R- 0.1767 OF- 9. OF- 10. DF- 20. S t a t i s t i c a l a n a l y s i s o f the l i n e s 2 ( B ) and 3 ( + ) i n F i g u r e 16. O N APPENDIX 8 Amino Acid Analysis by Neukom 138 AMINOSAUERENANALYSE ILW/ETHZ ««**«**«#««*««***«* PROBE SLIME/S. CREMORIS LONGI U.B.C. 26 Aug. 82. ANALYSENNUMMER: 2742 UND 27^2 Dragan Macura AMINOSAUERE % AS GRAMM AS IN IN PROTEIN 100 GRAMM PROBE 1. HYDROXIPROLIN .00 .00 2. ASPARAGINSAUERE+ASPARAGIN 8.52 U.737 3. THREONIN 11.60 6.1*51 k. SERIN 7.83 4.357. 5. GLUTAMINSAUERE+GLUTAMIN 26.02 1^.475 6. PROLIN 10.83 6.022 7. GLYCIN 1.62 - .903 8. ALANIN U.21 2.342 9. CYSTEIN .00 .000 10. VALIN 5.26 2.925 11. METHIONIN 1.34 • 71+5 12. ISOLEUCIN 7-90 U.393 13. LEUCIN 3.90 2.171 lh. TYROSIN .00 .000 15. PHENYLALANIN 1.80 1.004 16. HYDROXILYSIN .00 .000 17. LYSIN 6.36 3.538 18. HISTIDIN 1.02 .567 19. ARGININ 1.79 .996 PROTEINGEHALT * (ALS SUMME DER AS): 55-626 GRAMM PRO 100 GRAMM PROBE BEMERKUNGEN: Protein content, sum of amino acids. DATUM: UNTERSCHRIFT: (Neukom, 1982, personal communication) APPENDIX 9 Carbohydrate Analysis of Neukom 140 GLC Carbohydrate Analysis of the Slime Produced by S. cremoris l o n g i L416 (Neukom, 1982, personal communication). ALDONITRILACETATE 1982 Oct. 06. 10:42:56 CHANNEL 1 RUN 3 FILE 1 METHOD 1 INDEX 2 SAMLE S. cremoris l o n g i L416 XF = 100 NAME CONC RT AREA KF 1 84 5544 RHAMNOSE 7.28 90 16673 1 3 95 1024 4 103 19163 FUCOSE 9.309 107 21316 1 6 120 4041 7 127 3807 ARABINOSE 1.025 135 2347 1 XYLOSE 7.118 146 16301 1 XYLIT 2.488 170 5697 1 11 181 17790 12 191 3578 MANNOSE 3.634 200 8321 1 14 209 2157 15 214 2989 GLUCOSE 28.9 225 66186 1 GALACTOSE 40.25 240 92172 1 18 261 11875 TOTALS 99.99 300981 

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