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The enzymatic in vitro evaluation of protein sources for monogastric animals using the pH-stat method Mann, Jasminder Jason 1988

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THE ENZYMATIC i n v i t r o EVALUATION OF PROTEIN SOURCES FOR MONOGASTRIC ANIMALS USING THE pH-STAT METHOD By JASMINDER JASON MANN B.Sc.<AGR.>, The U n i v e r s i t y o f B r i t i s h C o lumbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Animal 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 Jan u a r y 1988 (c) J a s m i n d e r J a s o n Mann, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Animal Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date January 2 1 , 1 9 8 8 DE-6(3/81) ABSTRACT Three e x p e r i m e n t s were conducted t o s t u d y t h e s e n s i t i v i t y o f t h e p H - s t a t (in_ v i t r o ) method i n t h e p r e d i c t i o n o f t r u e d i g e s t i b i l i t y (TD), as measured by amount o f base added, o f p l a n t p r o t e i n s , e i t h e r a l o n e o r i n t h e presence o f s p e c i f i c a d d i t i v e s ( n i t r o g e n - f r e e m i x t u r e , v i t a m i n m i x t u r e and/or m i n e r a l m i x t u r e ) as p a r t o f a complete d i e t o f p l a n t p r o t e i n s t h a t had been s u b j e c t e d t o v a r i o u s l e v e l s and forms o f h e a t i n g . The i n . v i t r o TD v a l u e s were th e n compared w i t h TD v a l u e s o b t a i n e d in. v i v o ( W i s t a r r a t s ) . In e x p e r i m e n t 1, t h e e f f e c t o f t e m p e r a t u r e ( d r y - h e a t i n g a t 80, 100, 120, 150, 180 and 240-= C or a u t o c l a v i n g a t 121™ C) and t i m e (30, 60, 120 and 240 minutes) o f heat a p p l i c a t i o n on i n v i t r o base consumption (BC) was measured i n 3 g r a i n s (wheat, b a r l e y and sorghum) and whole d e f a t t e d soybeans. The l a r g e s t i n c r e a s e i n BC measured by t h e p H - s t a t method was t h a t o f soybeans i n response t o 30 minutes o f a u t o c l a v i n g . Dry h e a t i n g had v a r i o u s e f f e c t s on t h e BC by soybeans, depending upon t e m p e r a t u r e and time o f a p p l i c a t i o n , but none o f t h e t r e a t m e n t s was as b e n e f i c i a l as a u t o c l a v i n g . M i l d , d r y - h e a t i n g o f g r a i n s a t 80-120 , : 1 C improved BC s l i g h t l y . The improvement was most marked f o r wheat. Both d r y - h e a t i n g o f g r a i n a t t e m p e r a t u r e s above 120,=' C and a u t o c l a v i n g reduced t h e BC s i g n i f i c a n t l y f o r a l l d u r a t i o n s . In e x p e r i m e n t 2, t h e e f f e c t o f i n c l u s i o n o f n o n - p r o t e i n d i e t a r y components ( m i n e r a l s , v i t a m i n s and a n i t r o g e n - f r e e m i x t u r e , s i n g l y and i n c o m b i n a t i o n ) on i n . v i t r o BC measured by t h e p H - s t a t method o f wheat and f a t - e x t r a c t e d soybeans (both p r o t e i n s i n t h e raw and a u t o c l a v e d forms) was m o n i t o r e d . For t h e wheat t r e a t m e n t s , t h e i n c l u s i o n o f a m i n e r a l m i x t u r e s i g n i f i c a n t l y (p<0.001> i n c r e a s e d d i g e s t i b i l i t y . T h i s e f f e c t was g r e a t e s t w i t h a u t o c l a v e d wheat. I t was c o n c l u d e d t h a t , i n g e n e r a l , t h e p r e s e n c e o f m i n e r a l s i n c r e a s e d t h e r a t e o f h y d r o l y s i s . With raw soybeans, t h e d i s t i n c t i o n between t r e a t m e n t s was l e s s w e l l - d e f i n e d . The t r e a t m e n t s c o n t a i n i n g v i t a m i n o r n i t r o g e n - f r e e and m i n e r a l c o m b i n a t i o n m i x t u r e s were d i g e s t e d t o a s i g n i f i c a n t l y g r e a t e r e x t e n t t h a n t h e raw soybeans a l o n e . With a u t o c l a v e d soybeans, a d d i t i v e s had no e f f e c t . T h i s l a c k o f response t o a d d i t i v e s may have been due t o t h e r a t h e r l a r g e amount o f base r e q u i r e d by t h e a u t o c l a v e d soybean p r o t e i n a l o n e . In e x p e r i m e n t 3, a s e r i e s o f r a t - f e e d i n g t r i a l s were conducted i n c o n j u n c t i o n w i t h i n . v i t r o d i g e s t i o n s . D i e t s were f e d t o groups o f W i s t a r r a t s t o d e t e r m i n e TD, B i o l o g i c a l V a l u e <BV), and Net P r o t e i n U t i l i z a t i o n (NPU) i n v i v o . A l t h o u g h BV was measured i t was not r e l e v a n t f o r t h i s work. C o n c u r r e n t l y , t h e same d i e t s were t e s t e d f o r in. v i t r o TD by t h e p H - s t a t method. S p e c i f i c r e g r e s s i o n e q u a t i o n s were d e v e l o p e d f o r each p r o t e i n - t y p e t e s t e d , a f t e r i t was d e t e r m i n e d t h a t a much lower c o r r e l a t i o n c o e f f i c i e n t was o b t a i n e d when one g e n e r a l e q u a t i o n was u t i l i z e d . i i i The n ewly-developed e q u a t i o n s f o l l o w e d t h e f o r m a t y = a + bx, where y = TD (as a p a r t o f o n e ) , a = t h e y - i n t e r c e p t , b = s l o p e o f t h e f u n c t i o n and x = ml 0.10N NaOH added d u r i n g t h e 10-minute d i g e s t i o n . R e g r e s s i o n e q u a t i o n s , c o r r e l a t i o n c o e f f i c i e n t s ( r ) and s t a n d a r d e r r o r s f o r each r e g r e s s i o n (s) between i n . v i t r o and i n . v i v o t r u e d i g e s t i b i l i t y o f p r o t e i n s were as f o l l o w s ; Soybean, soybean ( a u t o c l a v e d ) , soybean/wheat c o m b i n a t i o n s (n = 6) r = 0.93 TD = 0.7868 + 0.2175x s = 0.018 Sorghum (raw, a u t o c l a v e d , 901=> C, 120~ C, 180~ C d r y - h e a t e d , steamed) (n = 6) r = 0.92 TD = 0.4575 + 1.8841x a = 0.058 A l f a l f a p e l l e t s / h a y i n c o m b i n a t i o n w i t h e i t h e r wheat or b a r l e y (n = 13) r = 0.91 TD = 0.3446 + 1.0356x s = 0.046 iv A l f a l f a hay and b a r l e y c o m b i n a t i o n s (n = 5) r = 0.96 TD = 0.2360 + 1.3194x s = 0.048 G r a i n s (19 b a r l e y s , 10 t r i t i c a l e s , 6 sorghums, and 2 wheats) (n = 37) r = 0.74 TD = 0.7419 + 0.4759x s = 0.044 In g e n e r a l , i t can be s t a t e d t h a t t h e p H - s t a t method i s a u s e f u l method f o r s c r e e n i n g p r o t e i n s f o r t h e e f f e c t o f v a r i o u s t r e a t m e n t s on d i g e s t i b i l i t y . Damage due t o a b n o r m a l l y s e v e r e p r o c e s s i n g c o n d i t i o n s ( i . e . h e a t i n g ) i s r e a d i l y d e t e c t e d by t h e p H - s t a t t e c h n i q u e as i n d i c a t e d by a d e c r e a s e i n t h e amount o f base consumed d u r i n g e n z y m a t i c h y d r o l y s i s . v TABLE OF CONTENTS S e c t i o n Page CHAPTER 1 1.0 INTRODUCTION 1 1.1 O b j e c t i v e s and q u e s t i o n s 3 CHAPTER 2 2.0 THEORETICAL BACKGROUND OF THE pH-STAT METHOD 6 2.1 P r i n c i p l e s o f t h e p H - s t a t method 6 2.2 Enzyme s p e c i f i c i t y S 2.3 Enzym a t i c c a t a l y s i s 10 2.4 T i t r a t i o n t h e o r y o f amino a c i d s 13 2.5 B u f f e r i n g c a p a c i t y o f f e e d s t u f f s 18 CHAPTER 3 3.0 EXPERIMENT 1. The e f f e c t o f t e m p e r a t u r e and t i m e o f heat a p p l i c a t i o n on in_ v i t r o ( p H - s t a t ) p r o t e i n d i g e s t i b i l i t y o f g r a i n s and raw < f a t - e x t r a c t e d ) soybeans 25 3.1 I n t r o d u c t i o n 25 3.2 M a t e r i a l s and methods 23 3.2.1 P r o t e i n s o u r c e s and t h e i r p r e p a r a t i o n 28 3.2.2 Enzymes 29 3.2.3 I n v i t r o ( p H - s t a t ) p r o c e d u r e 29 3.2.4 pH e l e c t r o d e maintenance 33 3.3 R e s u l t s 34 3.3.1 E f f e c t s o f d r y h e a t i n g 36 3.3.2 A u t o c l a v i n g vs d r y h e a t i n g e f f e c t s 44 3.3.3 T e s t t o d e t e r m i n e l i m i t i n g f a c t o r i n r e a c t i o n v e s s e l 48 3.3.4 T e s t t o e s t a b l i s h degree o f h y d r o l y s i s t h a t o c c u r s w i t h c a s e i n d u r i n g t h e 10-minute p H - s t a t d i g e s t i o n 48 3.4 D i s c u s s i o n 51 3.4.1 Heat t r e a t m e n t e f f e c t s on p r o t e i n d i g e s t i b i l i t y . . . 51 CHAPTER 4 4.0 EXPERIMENT 2. The e f f e c t o f i n c l u s i o n o f n o n - p r o t e i n d i e t a r y components on iri_ v i t r o <pH-stat) p r o t e i n d i g e s t i b i l i t y o f wheat and raw ( f a t - e x t r a c t e d ) soybeans 58 vi TABLE OF CONTENTS (co n t ' d ) S e c t i o n Page 4.1 I n t r o d u c t i o n 58 4.2 M a t e r i a l s and Methods 59 4.2.1 E x p e r i m e n t a l D e s ign 59 4.2.2 Sample P r e p a r a t i o n and D i g e s t i o n 59 4.2.3 Enzymes 60 4.2.4 I n v i t r o p r o c e d u r e . 60 4.2.5 pH e l e c t r o d e maintenance 60 4.3 R e s u l t s 60 4.4 D i s c u s s i o n 63 CHAPTER 5 5.0 EXPERIMENT 3. A comparison o f p r o t e i n d i g e s t i b i l i t y o f v a r i o u s f e e d s as measured by th e p H - s t a t method and by male W i s t a r r a t s 70 5.1 I n t r o d u c t i o n 70 5.2 M a t e r i a l s and Methods 71 5.2.1 Enzymes. - 71 5.2.2 P r e p a r a t i o n o f p r o t e i n s f o r p H - s t a t t r i a l s 71 5.2.3 In. v i t r o p r o c e d u r e 72 5.2.4 pH e l e c t r o d e maintenance 72 5.2.5 C a l c u l a t i o n o f i n v i t r o t r u e d i g e s t i b i l i t y . . . . . . . . 72 5.2.6 Rat t r i a l s 73 5.2.7 P r e p a r a t i o n o f complete d i e t s f o r r a t t r i a l s 74 5.2.8 P r e p a r a t i o n o f n i t r o g e n - f r e e m i x t u r e 82 5.2.9 A n a l y t i c a l methods 32 5.3 R e s u l t s 83 5.4 D i s c u s s i o n 83 CHAPTER 6 6.0 C o n c l u s i o n s 98 7.0 B i b l i o g r a p h y 100 8.0 Ap p e n d i c e s . 107 v i i L i s t o f T a b l e s T a b l e Page 1. Amino a c i d c o m p o s i t i o n o f c a s e i n 31 2. Sodium h y d r o x i d e consumption by soybeans, wheat, b a r l e y and sorghum, each a u t o c l a v e d f o r v a r i o u s t i m e p e r i o d s 35 3. A b s o l u t e r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n (TD) o f d r y - h e a t e d soybeans 37 4. A b s o l u t e r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n (TD) o f d r y - h e a t e d wheat 38 5. A b s o l u t e r e s u l t s o f i j j . v i t r o p r o t e i n d i g e s t i o n (TD) o f d r y - h e a t e d b a r l e y 39 6. A b s o l u t e r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n (TD) o f d r y - h e a t e d sorghum 40 7. C o m p o s i t i o n o f n i t r o g e n - f r e e m i x t u r e 61 8. C o m p o s i t i o n o f v i t a m i n m i x t u r e 61 9. C o m p o s i t i o n o f m i n e r a l m i x t u r e 62 10. A b s o l u t e and r e l a t i v e r e s u l t s o f i n . v i t r o d i g e s t i o n s conducted on 8 d i e t a r y t r e a t m e n t s d e s i g n e d t o t e s t t h e e f f e c t s o f a d d i t i v e s f o r each o f 4 p r o t e i n s o u r c e s (raw and a u t o c l a v e d wheat and soybeans) 64 11. C o m p o s i t i o n o f sorghum d i e t s 75 12. C o m p o s i t i o n o f b a r l e y d i e t s 76 13. C o m p o s i t i o n o f t r i t i c a l e d i e t s 78 14. C o m p o s i t i o n o f soybean/wheat d i e t s 79 15. C o m p o s i t i o n o f a l f a l f a p e l l e t / h a y / w h e a t d i e t s 80 16. C o m p o s i t i o n o f a l f a l f a h a y / b a r l e y d i e t s 81 17. R e g r e s s i o n e q u a t i o n s d e v e l o p e d , and c o r r e l a t i o n c o e f f i c i e n t s o b t a i n e d between i n . v i t r o and i n . v i v o p r o t e i n d i g e s t i b i l i t i e s (TD) o f v a r i o u s d i e t s 84 18. A comparison o f in. v i v o and i n , v i t r o p r o t e i n d i g e s t i b i l i t i e s (TD) o f v a r i o u s d i e t s 85 vi i i L i s t , o f F i g u r e s F i g u r e Page 1. A p l o t o f v e l o c i t i e s f o r an enzyme r e a c t i o n v e r s u s s u b s t r a t e c o n c e n t r a t i o n s 12 2. S t e p w i s e i o n i z a t i o n f o r a c i d i c and b a s i c amino a c i d s a l o n g w i t h t h e c o r r e s p o n d i n g l o c a t i o n s o f t h e i r i o n i z a t i o n s p e c i e s on a t i t r a t i o n c u r v e 15 3a-d. The e f f e c t o f dry h e a t i n g ( a t d i f f e r e n t t e m p e r a t u r e s ) o f f a t - e x t r a c t e d soybeans, b a r l e y , wheat and sorghum on NaOH consumption d u r i n g 10 minutes o f i n v i t r o d i g e s t i o n 41 4a-d. The e f f e c t s o f a u t o c l a v i n g and dry h e a t i n g o f f a t - e x t r a c t e d soybeans, b a r l e y , wheat and sorghum on NaOH consumption and c o r r e s p o n d i n g TD v a l u e s 45 5. NaOH consumption a f t e r t h e a d d i t i o n o f 1.5 ml enzyme s o l u t i o n t o sodium c a s e i n a t e s u s p e n s i o n s and t h e re s p o n s e t o f u r t h e r a d d i t i o n s t o t h e s u s p e n s i o n a f t e r 10 minutes o f 0.5 ml enzyme s o l u t i o n and o f 5 s u c c e s i v e 2 ml a l i q u o t s o f 1.0 mg N/ml s u s p e n s i o n a t 1 minute i n t e r v a l s 49 6. A comparison o f NaOH consumed by sodium c a s e i n a t e and " a r t i f i c i a l c a s e i n " d u r i n g a 10-minute i n c u b a t i o n t o an e n d p o i n t o f pH 8.00 w i t h t h e p H - s t a t method 50 7. The a b s o l u t e e f f e c t on NaOH a d d i t i o n as a f f e c t e d by t h e i n c l u s i o n o f n i t r o g e n - f r e e mix, m i n e r a l mix and v i t a m i n s i n comparison w i t h t h e p r o t e i n s o u r c e o n l y 65 8. The p e r c e n t a g e change i n NaOH a d d i t i o n as a f f e c t e d by t h e i n c l u s i o n n i t r o g e n - f r e e mix, m i n e r a l mix and v i t a m i n s i n comparison w i t h t h e p r o t e i n s o u r c e o n l y 66 9. R e g r e s s i o n a l r e l a t i o n s h i p between i n v i v o %TD and i n v i t r o base consumption f o r sorghum-based d i e t s . . . 91 10. R e g r e s s i o n a l r e l a t i o n s h i p between i n . v i v o «TD and i n v i t r o base consumption f o r a l f a l f a hay and b a r l e y - b a s e d d i e t s . . . 92 11. R e g r e s s i o n a l r e l a t i o n s h i p between i n . v i v o J«TD and i n v i t r o base consumption f o r soybean-based d i e t s . . . 93 12. R e g r e s s i o n a l r e l a t i o n s h i p between i n v i v o ssTD and i n v i t r o base consumption f o r a l f a l f a - b a s e d d i e t s . . . 94 ix L i s t , o f F i g u r e s ( c o n t ' d ) F i g u r e Page 13- R e g r e s s i o n a l r e l a t i o n s h i p between in. v i v o HTD and i n v i t r o base consumption f o r g r a i n - b a s e d d i e t s 95 14. R e g r e s s i o n a l r e l a t i o n s h i p between i n . v i v o HTD and i n v i t r o base consumption f o r sorghum- and t r i t i c a l e - b a s e d d i e t s 96 15. Overhead view o f t a b l e s a w - t o p , showing s e t - u p p r o c e d u r e t o ensure s a f e t y and s t a b i l i t y w h i l e c u t t i n g t u b i n g t o 15 cm (6") l e n g t h s . . 120 16. Placement o f t h e d r i l l - h o l e on t h e 15 cm (6") cage tube 120 17. Measurement and placement o f d r i l l - h o l e s i n t h e 5 cm (2") e x t e n s i o n p i e c e 125 18. S i m p l e s i d e - v i e w o f m e t a b o l i c cage a f t e r g l u e i n g t h e f o u r p i e c e s (cage compartment, e x t e n s i o n , f e e d e r and bottom) t o g e t h e r 125 19. End-on view o f a cage l i d and i t s two bands o f t u b i n g which h o l d i n p l a c e t h e w i r e mesh s c r e e n . . . . . 128 20. T r a c e p a t t e r n d e s i g n e d f o r making a c e t a t e f u n n e l s , which a r e used t o c h a n n e l t h e u r i n e and f e c e s t o t h e i r s e p a r a t e c o n t a i n e r s 130 21. Completed u n i t s i l l u s t r a t e t h e a b s o l u t e e s s e n t i a l p a r t s o f a m e t a b o l i c cage t h a t f r e q u e n t l y r e q u i r e maintenance 132 x L i s t , o f Appendices Appendix Page 1. O p e r a t i n g g u i d e f o r r o u t i n e p H - s t a t d i g e s t i b i l i t y d e t e r m i n a t i o n s 10S 2. R o u t i n e f o r c o n d u c t i n g r a t t r i a l s I l l 3. Mean XTD v a l u e s o b t a i n e d w i t h r a t s 114 4. C o n s t r u c t i n g m e t a b o l i c cages t o conduct r a t t r i a l s 118 5. M a t e r i a l r e q u i r e d f o r c o n s t r u c t i n g t e n m e t a b o l i c cages f o r r a t s 133 6. Dimensions o f components c o m p r i s i n g t h e r a t m e t a b o l i c cage 134 xi ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e s t g r a t i t u d e t o my l o n g -t i m e A d v i s o r , Dr. R i c h a r d M. Beames, f o r i n i t i a t i n g t h i s s t u d y and f o r h i s c o n t i n u o u s s u p p o r t and gu i d a n c e t h r o u g h o u t my s e v e r a l y e a r s a t UBC. I would a l s o l i k e t o e x p r e s s my a p p r e c i a t i o n t o Dr. Magdalena K i e r c z y n s k a - G o r s k a ( v i s i t i n g Dekaban s c h o l a r from t h e Department o f Animal N u t r i t i o n , Warsaw A g r i c u l t u r a l U n i v e r s i t y ) f o r her h e l p i n many a s p e c t s o f t h i s p r o j e c t . I would l i k e t o acknowledge t h e h e l p p r o v i d e d by Mr. G i l l e a G a l z y , a key pe r s o n i n t h i s s t u d y , as h i s t e c h n i c a l e x p e r t i s e was i n s t r u m e n t a l i n e s t a b l i s h i n g t h e p H - s t a t method, i n a d d i t i o n t o g e n e r a l h e l p i n l a b o r a t o r y p r o c e d u r e s . A d v i c e from Mr. Ted C a t h c a r t was a l s o a p p r e c i a t e d . I a l s o w i s h t o thank t h e members o f t h e committee, Dr. S. N a k a i , Dr. C.R. K r i s h n a m u r t i , Dr. J . Vanderstoep and Dr. R. B l a i r f o r t h e i r s u g g e s t i o n s . I would l i k e t o d e d i c a t e t h i s t h e s i s t o my p a r e n t s who have alw a y s been s u p p o r t i v e i n my s c h o l a s t i c endeavors. F i n a l l y , I would l i k e t o acknowledge t h e N a t u r a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l o f Canada f o r f i n a n c i a l s u p p o r t . CHAPTER 1 1.0 GENERAL INTRODUCTION Measuring p r o t e i n q u a l i t y o f f e e d s t u f f s i n . v i v o i s s l o w , i n c o n v e n i e n t and e x p e n s i v e , and t h e v a l u e o f r e s u l t s o b t a i n e d u s i n g t h e r a t has been c a l l e d i n t o q u e s t i o n ( B o d w e l l and Mara b l e , 1981; M a r i a n i and S p a d o n i , 1979). Thus, t h e r e has been f o r decades a s e a r c h f o r an in. v i t r o a s s a y t h a t a c c u r a t e l y p r e d i c t s p r o t e i n d i g e s t i b i l i t y ( M e l n i c k e_t a l . , 1946; Mauron e t a l . . 1955; S h e f f n e r e t a l . . 1956; Akeson and Stahmann, 1964; Ford and S a l t e r , 1966; Maga et. a l . , 1973; F l o r i d i and F i d a n z a , 1975; Hsu e t a l _ . , 1977; Lynch e_t a l . . , 1979; S t i n s o n and Snyder, 1980; Friedman e t a l . , 1981; G a u t h i e r e t aj^. , 1982; Hahn e t a l . . 1982; Pedersen and Eggum, 1983). In. v i t r o a s s a y s u t i l i z e v a r i o u s c o m b i n a t i o n s o f p r o t e o l y t i c enzymes, e i t h e r i n t h e crud e (eg. p a n c r e a t i n ) , o r p u r i f i e d (eg. t r y p s i n , c h y m o t r y p s i n , p e p t i d a s e , p e p s i n ) forms. I t has been argued by v i r t u a l l y e v e r y a u t h o r o f r e l e v a n t p a p e r s , t h a t t h e i r c o m b i n a t i o n o f enzymes combined w i t h t h e i r p r e s c r i b e d s e t o f c o n d i t i o n s y i e l d s t h e b e s t i j i v i t r o e s t i m a t i o n o f t h e t r u e d i g e s t i b i l i t y (TD) o f the p r o t e i n . However, t h e r e i s some f a u l t w i t h a l m o s t e v e r y proposed method i n t h a t t h e t e c h n i q u e i s e i t h e r t o o t i m e -consuming o r i s p o o r l y c o r r e l a t e d w i t h in. v i v o r e s u l t s . R e c e n t l y , S a v o i e and G a u t h i e r (1986) have used a complex d i a l y s i s c e l l system i n c o n j u n c t i o n w i t h p e p s i n and p a n c r e a t i n enzymes i n o r d e r t o e s t i m a t e p r o t e i n d i g e s t i b i l i t y , c l a i m i n g 1 t h a t t h e i r 24-hour p r o c e d u r e v e r y c l o s e l y s i m u l a t e s i n . v i v o d i g e s t i o n . Whether t h e method e x a c t l y r e s e m b l e s t h e i n . v i v o s i t u a t i o n s h o u l d not be t h e main c o n c e r n , but r a t h e r , t h e c a p a c i t y o f a method t o be a b l e t o a c c u r a t e l y and p r e c i s e l y p r e d i c t d i g e s t i b i l i t y o f a f e e d i s o f prime i m p o r t a n c e ; t h i s i s t h e whole i n t e n t o f t h e development o f an i n . v i t r o method which s h o u l d a l s o be r a p i d and r e l a t i v e l y e c o n o m i c a l . The p H - s t a t method proposed by Pedersen and Eggum (1983) t e n d s t o meet most o f t h e d e s i r a b l e q u a l i t i e s o f an i n . v i t r o method i . e . i t i s r a p i d (10 minute d i g e s t i o n ) , p r e c i s e , a c c u r a t e and can be c o n s i d e r e d q u i t e e c o n o m i c a l because t h e key p i e c e o f equipment which i s used ( a u t o m a t i c t i t r a t o r ) i s e x t r e m e l y v e r s a t i l e and u s e f u l f o r numerous o t h e r t i t r i m e t r i c a p p l i c a t i o n s i n t h e a n a l y t i c a l l a b o r a t o r y . The problem o f b u f f e r i n g by n e w l y - r e l e a s e d amino groups i s e l i m i n a t e d w i t h t h e p H - s t a t method ( S t i n s o n and Snyder, 1980) and t h e d e t r i m e n t a l e f f e c t s o f c h a n g i n g pH on t h e p r o t e o l y t i c enzymes i s m i n i m i z e d whereas i n t h e method o f Hsu et. a l _ . ( 1977), t h e d e c l i n i n g pH caused l e s s t h a n i d e a l c o n d i t i o n s f o r t h e enzymes t o a c t . S u b s t a n t i a l end-product a c c u m u l a t i o n i n a c l o s e d v e s s e l system may i n h i b i t p r o t e o l y s i s ( G a u t h i e r §_t a l . . , 1982). However, i f t h e d i g e s t i o n p e r i o d i s b r i e f as i n t h e p H - s t a t method, i n h i b i t i o n s h o u l d be o f min i m a l c o n c e r n . T h i s method u t i l i z e s r e g r e s s i o n e q u a t i o n s d e v e l o p e d from a comparison o f i n . v i v o and i n v i t r o TD v a l u e s i n o r d e r t o p r e d i c t t h e p r o t e i n q u a l i t y o f a f e e d . Because o f t h e many apparent advantages o f t h e method, the p H - s t a t t e c h n i q u e was chosen f o r t h i s s t u d y . 2 1.1 O b j e c t i v e s With heat t r e a t m e n t b e i n g one o f the most commonly used methods o f p r o c e s s i n g i n t o d a y ' s f e e d i n d u s t r y , i t would be advantageous f o r an i n . v i t r o method t o be a b l e t o d e t e c t changes i n d i g e s t i b i l i t y r e s u l t i n g from heat o r any o t h e r p r o c e s s i n g t r e a t m e n t s . Chapter t h r e e r e p o r t s e x p e r i m e n t s d e s i g n e d t o t e s t t h e s e n s i t i v i t y o f t h e p H - s t a t i n t h e d e t e c t i o n o f d i f f e r e n c e s i n d i g e s t i b i l i t y r e s u l t i n g from v a r i o u s heat t r e a t m e n t s a p p l i e d t o s e v e r a l p r o t e i n s f o r v a r i o u s t i m e p e r i o d s . In any t e s t depending on a measurement o f a c i d p r o d u c t i o n o r pH change, b u f f e r i n g c a p a c i t y o f t h e d i g e s t i o n m i x t u r e c o n t a i n i n g t h e t e s t m a t e r i a l must be c o n s i d e r e d . I t i s r e l a t i v e l y s i m p l e t o p r e d i c t t h e b u f f e r i n g p o t e n t i a l o f pure amino a c i d s . However, i t i s a p p arent t h a t t h e d e t e r m i n a t i o n o f t h e b u f f e r i n g c a p a c i t y o f a complete d i e t i s not s i m p l e . I t has been s u g g e s t e d by v a r i o u s workers t h a t t h e p r e s ence o f m i n e r a l s i n a f e e d s t u f f may i n c r e a s e i t s b u f f e r i n g p o t e n t i a l . C h a pter f o u r d e s c r i b e s an e x p e r i m e n t i n which t h e e f f e c t o f r o u t i n e l y used a d d i t i v e s t o r a t p r o t e i n e v a l u a t i o n t e s t d i e t s ( m i n e r a l s , v i t a m i n s and n i t r o g e n - f r e e p o r t i o n s ) on p H - s t a t r e s p o n s e s was measured. Any s i g n i f i c a n t d e v i a t i o n r e s u l t i n g from t h e i n c l u s i o n o f t h e s e a d d i t i v e s would i n d i c a t e a b u f f e r i n g a c t i o n . The o v e r a l l o b j e c t i v e s o f t h i s s t u d y on t h e p H - s t a t method were t h r e e f o l d . The f i r s t o b j e c t i v e was t o i n v e s t i g a t e t h e s e n s i t i v i t y o f t h e t e c h n i q u e t o changes i n p r o t e i n 3 d i g e s t i b i l i t y due t o heat t r e a t m e n t ( a u t o c l a v i n g o r d r y h e a t i n g f o r d i f f e r e n t t i m e s and t e m p e r a t u r e s ) o f f o u r p r o t e i n s o u r c e s ( E x p e r i m e n t 1 ) . The second o b j e c t i v e was t o examine t h e e f f e c t o f each o f t h e n o n - n i t r o g e n components i n a s t a n d a r d r a t p r o t e i n e v a l u a t i o n d i e t ( n i t r o g e n - f r e e m i x t u r e , m i n e r a l m i x t u r e and v i t a m i n m i x t u r e ) s i n g l y and i n c o m b i n a t i o n on i n  v i t r o TD e s t i m a t e s by t h e p H - s t a t method (Experiment 2 ) . The t h i r d o b j e c t i v e was t o compare in . v i v o and i n v i t r o e s t i m a t e s o f TD o f v a r i o u s f e e d s and s u b s e q u e n t l y d e v e l o p r e g r e s s i o n e q u a t i o n s f o r each f e e d - t y p e t e s t e d ( Experiment 3 ) . With t h e o b j e c t i v e s d e f i n e d , t h e r e s t i l l e x i s t e d s e v e r a l u n d e r l y i n g q u e s t i o n s which were kept i n mind d u r i n g t h e c o u r s e o f t h e s t u d y . These i n c l u d e d ; 1. Does t h e e f f i c i e n c y o f enzyme a c t i v i t y d e c r e a s e w i t h t i m e ? 2. How s u b s t a n t i a l i s a u t o l y s i s i n t h e enzyme s o l u t i o n d u r i n g i t s s t o r a g e on i c e o v e r a day's usage? 3. I s t h e 1.5 ml a l i q u o t o f enzyme s o l u t i o n t h a t i s added t o t h e d i g e s t i o n v e s s e l i n e x c e s s o r i s t h e enzyme a l i m i t i n g f a c t o r i n t h e r a t e o f h y d r o l y s i s ? 4. I s t h e r e any a p p a r e n t d i f f e r e n c e i n amount o f base added d u r i n g t h e 10-minute i n . v i t r o d i g e s t i o n by u t i l i z i n g a n i t r o g e n atmosphere i n s t e a d o f a t m o s p h e r i c a i r ? 5. Does t h e l e n g t h o f s o a k i n g t i m e o f a p r o t e i n sample i m m e d i a t e l y p r i o r t o d i g e s t i o n have any i n f l u e n c e on t h e i n  v i t r o d i g e s t i o n v a l u e ? 4 6. I a t h e r e any advantage t o u a i n g a m i c r o e l e c t r o d e i n s t e a d o f a m a c r o e l e c t r o d e ? 7. I s t h e p H - s t a t method r e p e a t a b l e over t i m e ? i . e . does t h e same f e e d sample y i e l d a s i m i l a r r e a d i n g i f d u p l i c a t e s a r e d i g e s t e d on d i f f e r e n t days? 8. Do d i f f e r e n t t y p e s o f p r o t e i n s w a r r a n t t h e use o f s p e c i f i c r e g r e s s i o n e q u a t i o n s . 9. I s b u f f e r i n g by t h e r e l e a s e d amino groups a major problem when w o r k i n g i n t h e pH range o f ""8? I s t h e pH o f 8 a s a t i s f a c t o r y compromise i n t h a t each amino a c i d has a unique t i t r a t i o n c u r v e , w i t h t h e m a j o r i t y h a v i n g m i n i m a l b u f f e r i n g a b i l i t y i n t h i s pH v i c i n i t y ? 10. Does the c o n t r o l band s e t t i n g i n f l u e n c e amount o f base added d u r i n g t h e p H - s t a t d i g e s t i o n ? The f o l l o w i n g c h a p t e r p r o v i d e s i n f o r m a t i o n on t h e p r i n c i p l e s o f p H - s t a t , enzyme a c t i o n and t i t r a t i o n o f amino a c i d s , a knowledge o f which i s n e c e s s a r y t o a d e q u a t e l y i n v e s t i g a t e t h e above p o i n t s . 5 CHAPTER 2 2.0 THEORETICAL BACKGROUND 2.1 P r i n c i p l e s o f t h e pH-atat. method The p H - s t a t method i s based on t h e p r i n c i p l e o f a d d i n g a s e r i e s o f d i s c r e t e q u a n t i t i e s o f base (NaOH) t o a system i n which a c i d i s formed ( J a c o b s e n , 1957). These a d d i t i o n s a r e c o n t r o l l e d by t h e e l e c t r i c a l and m e c h a n i c a l systems o f t h e m e m o t i t r a t o r ( M e t t l e r DL40RC) t o m a i n t a i n pH a t o r v e r y near a p r e - d e t e r m i n e d e n d p o i n t f o r as l o n g as t h e i n . v i t r o d i g e s t i o n method r e q u i r e s . The method o f p r o d u c i n g a c i d may i n c l u d e one where p r o t e o l y t i c enzymes a r e added t o a p r o t e i n sample ( s u s p e n s i o n ) . In t h i s c a s e , t h e a c t i o n o f t h e enzymes h y d r o l y z e s p e p t i d e bonds, l i b e r a t i n g hydrogen i o n s i n t o s o l u t i o n and i n t u r n c a u s i n g a d e c r e a s e i n t h e pH o f t h e s o l u t i o n . I n r e s p o n s e t o t h i s d e c l i n e i n pH, base i s a u t o m a t i c a l l y added from t h e m e m o t i t r a t o r ' s b u r e t t e t i p . A p H - s t a t c o n s i s t s o f a r e f e r e n c e (KC1) e l e c t r o d e c e l l immersed i n t h e r e a c t i o n v e s s e l ( s y s t e m ) . T h i s c e l l i s connected w i t h a pH-meter which i n t u r n i s con n e c t e d w i t h a programmable c o n t r o l l i n g d e v i c e ( m e m o t i t r a t o r ) . T h i s m e m o t i t r a t o r o p e r a t e s a m o t o r - d r i v e n b u r e t t e d r i p p i n g i n t o t h e r e a c t i o n v e s s e l and s u p p l y i n g t h e system w i t h base ( r e a g e n t ) , and i t i s so a d j u s t e d t h a t when t h e p o t e n t i a l o f t h e c e l l d i f f e r s from t h a t c o r r e s p o n d i n g t o t h e d e s i r e d pH v a l u e i t w i l l s t a r t and m a i n t a i n t h e a d d i t i o n o f r e a g e n t u n t i l t h e p o t e n t i a l 6 i s r e s t o r e d t o i t s o r i g i n a l v a l u e . D e v i a t i o n s from t h e d e s i r e d e l e c t r o d e p o t e n t i a l ( o r pH) s h o u l d be v e r y s m a l l i f t h e a p p a r a t u s i s f u n c t i o n i n g p r o p e r l y . C o n s e q u e n t l y , t h e r e a c t i o n may be r e g a r d e d as b e i n g performed a t c o n s t a n t pH. The uptake of r e a g e n t as a f u n c t i o n o f t i m e can be r e a d d i r e c t l y from t h e m e m o t i t r a t o r * a d i s p l a y o r be r e c o r d e d on a m e c h a n i c a l r e c o r d e r . The f u n c t i o n o f t h e m e m o t i t r a t o r ( c o n t r o l l i n g d e v i c e ) i s t o s t a r t t h e m o t o r - d r i v e n b u r e t t e when t h e o u t p u t p o t e n t i a l o f t h e pH-meter d e v i a t e s from t h e f i x e d v a l u e , and s t o p i t when t h e p o t e n t i a l i s r e s t o r e d t o t h e o r i g i n a l v a l u e . An a u t o m a t i c t i t r a t o r has an a d j u s t a b l e p r o p o r t i o n a l band ( c o n t r o l band). T h i s f e a t u r e o f f e r s t h e advantage t h a t , w i t h i n an a d j u s t a b l e pH-range near t h e s e t e n d p o i n t t h e b u r e t t e motor w i l l r u n d i s c o n t i n u o u s l y w i t h s m a l l e r and s m a l l e r c u r r e n t i m p u l s e s t h e c l o s e r t h e pH comes t o 8.00. The motor w i l l s t o p c o m p l e t e l y when t h e s e t pH i s re a c h e d and m a i n t a i n e d f o r a p r e s e t l e n g t h o f t i m e ( i . e . 3 s e c o n d s ) . T h i s p r e v e n t s o v e r s h o o t i n g o f t h e e n d p o i n t . E q u a l l y i m p o r t a n t i s t h e adjustment o f s t i r r i n g speed t o p r o v i d e r a p i d m i x i n g and t o p r e v e n t o v e r s h o o t i n g . T h e o r e t i c a l l y , t h e s t i r r i n g e f f i c i e n c y s h o u l d ensure t h a t any s m a l l q u a n t i t y o f base r e a g e n t added t o t h e r e a c t i o n m i x t u r e i s e v e n l y d i s t r i b u t e d i n a much s h o r t e r t i m e t h a n t h a t o f t h e re s p o n s e o f t h e i n s t r u m e n t ( i . e . l e s s than one s e c o n d ) . The m e c h a n i c a l s t i r r e r which has a r o t a t i n g r o d w i t h b l a d e s , i s one o f t h e most e f f e c t i v e t y p e s o f s t i r r e r s . The n e c e s s i t y o f 7 a v o i d i n g a p l a a h i n g and foaming a e t a a l i m i t t o t h e speeds o f s t i r r i n g . S l i g h t d e f e c t s i n t h e mounting o f t h e r o d may cause u n s t e a d i n e s s o f movement which a t h i g h speeds, i n t h e l i m i t e d space a v a i l a b l e f o r t h e s t i r r e r , may become a danger t o t h e g l a s s e l e c t r o d e o r b u r e t t e t i p , b oth o f which a r e p r e s e n t i n th e r e a c t i o n v e s s e l . 2.2 Enzyme s p e c i f i c i t y I n t h e f o l l o w i n g s e r i e s o f e x p e r i m e n t s ( c h a p t e r s 3-5, i n c l u s i v e ) a multienzyme c o m b i n a t i o n v i z . p o r c i n e t r y p s i n , b o v i n e a - c h y m o t r y p s i n and p e p t i d a s e i n t h e p u r i f i e d forms a r e used. The f i r s t two a r e o f p a n c r e a t i c o r i g i n w h i l e t h e l a s t i s i s o l a t e d from t h e p o r c i n e i n t e s t i n e . T r y p s i n c a t a l y z e s t h e h y d r o l y s i s o f t h e p e p t i d e bond between t h e c a r b o x y l group o f l y s i n e , o r t h e c a r b o x y l group o f a r g i n i n e and t h e amino group o f t h e a d j a c e n t amino a c i d . C l e a v a g e i s s l o w e r when t h e b a s i c r e s i d u e i s a d j a c e n t i n sequence t o an a c i d i c r e s i d u e o r c y s t i n e , and does not o c c u r a t a l l when t h e r e s i d u e i s f o l l o w e d by p r o l i n e ( H i r s e t a l . , 1956). T r y p s i n i s o p t i m a l l y a c t i v e i n t h e pH range 8-9 ( M a c G i l l i v r a y , 1986) and i s s t a b l e i n a c i d s o l u t i o n s above pH 2 but i n s o l u t i o n s more a l k a l i n e t h a n pH 6, undergoes a u t o l y s i s ( K u n i t z and N o r t h r o p , 1934). G o r i n i (1951) found t r y p s i n t o be s t a b l e i n a n e u t r a l pH s o l u t i o n t h a t c o n t a i n e d 0.01 M Ca-*- and was h e l d a t n e a r - f r e e z i n g t e m p e r a t u r e s . C h y m o t r y p s i n c a t a l y z e s t h e h y d r o l y s i s o f t h e p e p t i d e bonds between t h e c a r b o x y l group o f t y r o s i n e , p h e n y l a l a n i n e o r 8 t r y p t o p h a n , and t h e amino group o f t h e a d j a c e n t amino a c i d (Bergmann and F r u t o n , 1937). Slower h y d r o l y s i s has been found t o o c c u r a t t h e c a r b o x y l group o f a s p a r a g i n e , g l u t a m i n e , h i s t i d i n e , l e u c i n e , l y s i n e , m e t h i o n i n e , s e r i n e and t h r e o n i n e , but c l e a v a g e a t t h e s e s i t e s i s u s u a l l y s i g n i f i c a n t o n l y a t a h i g h c o n c e n t r a t i o n o f enzyme o r when p r o l o n g e d d i g e s t i o n i s employed. The r a t e s o f c h y m o t r y p t i c a t t a c k a r e i n f l u e n c e d by t h e n a t u r e o f t h e amino a c i d r e s i d u e a d j a c e n t t o t h e s u s c e p t i b l e bond. Cl e a v a g e a t t h e c a r b o x y l group o f a s p a r a g i n e i s f a v o r e d when t h i s r e s i d u e i s a d j a c e n t i n sequence t o l y s i n e , h i s t i d i n e o r a r g i n i n e (Smyth, 1967). C h y m o t r y p s i n i s o p t i m a l l y a c t i v e a t t h e pH range 8-9 and i s s t a b l e i n s o l u t i o n s h a v i n g pH v a l u e s between 2 and 9. In c o n c e n t r a t e d s o l u t i o n s a t pH 7.8, a c t i v i t y i s not l o s t but t h e predominant a l p h a - f o r m o f t h e enzyme i s c o n v e r t e d by a u t o l y s i s i n t o two new forms; b e t a - and gamma-chymotrypsin. The i n t e s t i n a l p e p t i d a s e i n c l u d e s both t h e amino- and c a r b o x y l - p e p t i d a s e s and has c a t a l y t i c f u n c t i o n i n t h e h y d r o l y s i s o f p e p t i d e bonds from t h e t e r m i n a l amino- and t e r m i n a l c a r b o x y l - e n d s o f a p r o t e i n , r e s p e c t i v e l y . I n t e s t i n a l p e p t i d a s e i s u n s t a b l e below pH 6 (Anson, 1937) and has i t s maximal a c t i v i t y a t pH 7.5. U n l i k e t r y p s i n , and t o a l e s s e r e x t e n t , c h y m o t r y p s i n , p e p t i d a s e i s not prone t o a u t o l y s i s ( D a v i e and N e u r a t h , 1952). In o r d e r t o m i n i m i z e t h e e f f e c t s o f e n z y m a t i c a u t o l y s i s on t h e p H - s t a t v a l u e s , t h e enzyme s o l u t i o n i s made up i m m e d i a t e l y p r i o r t o a s e t o f d e t e r m i n a t i o n s , and, d u r i n g t h e p r o c e d u r e , t h e s o l u t i o n i s ke p t on i c e . 9 2. 3 E n z y m a t i c c a t a l y s i s The b a s i c mechanism by which s u b s t r a t e d e g r a d a t i o n i s c a t a l y z e d by enzyme(s) has been w e l l - d e f i n e d as t h e c o m b i n a t i o n o f t h e enzyme (E> w i t h s u b s t r a t e (S) t o form t h e enzyme-s u b s t r a t e complex ( E S ) . ES t h e n decomposes t o y i e l d p r o d u c t (P) and f r e e ( E ) , which i s a g a i n ready f o r a n o t h e r round o f c a t a l y s i s (Schwimmer, 1981). k + 1 k + 2 CE3 ES] CES] •> EE] CP] , k-1 where [S3 = i n i t i a l s u b s t r a t e c o n c e n t r a t i o n , CED = c o n c e n t r a t i o n o f f r e e enzyme, [ES3 = c o n c e n t r a t i o n o f e n z y m e - s u b s t r a t e complex, k+1 = r a t e c o n s t a n t f o r t h e f o r m a t i o n o f EES], k-1 = r a t e c o n s t a n t f o r t h e r e v e r s a l o f t h e f o r m a t i o n o f CES], k+2 = r a t e c o n s t a n t f o r t h e c o n v e r s i o n o f EES] i n t o EE] and CP]. When a l l t h e enzyme m o l e c u l e s a r e t i e d up as EES], f u r t h e r a d d i t i o n o f s u b s t r a t e w i l l not a c c e l e r a t e t h e r e a c t i o n and t h e v e l o c i t y w i l l be a t a maximum (Vmax) . The enzyme p r o t e i n i s a l a r g e s t r u c t u r e t h a t has a r e l a t i v e l y s m a l l a c t i v e s i t e where c a t a l y s i s o f an o r g a n i c r e a c t i o n o c c u r s . The c o n f o r m a t i o n a l and e l e c t r o c h e m i c a l 10 p r o p e r t i e s o f t h e enzyme a c t i v e s i t e a r e t hought t o c o n t r i b u t e s t r e s s on any s u b s t r a t e m o l e c u l e p r e s e n t i n t h e CES] complex. The s u b s t r a t e t h e n undergoes e f f e c t s o f bond s t r a i n a t c a t a l y t i c a l l y s t r a t e g i c p o i n t s t h a t cause CES] t o d i s s o c i a t e t o EE] and CP] ( Z a p s a l i s and Beck, 1985). S t u d i e s o f most c l o s e d e n z y m e - c a t a l y z e d r e a c t i o n s ( i n c l u d i n g p H - s t a t d i g e s t i o n s ) show a h y p e r b o l i c r e a c t i o n v e l o c i t y (V) when t h i s i s p l o t t e d a g a i n s t CS], w i t h Vmax a s y m p t o t i c a l l y approached by t h e h y p e r b o l a ( F i g u r e 1 ) . G e n e r a l l y , t h e v e l o c i t y o f an enzyme r e a c t i o n i n c r e a s e s i n p r o p o r t i o n t o CS1 o b e y i n g a f i r s t - o r d e r r e a c t i o n (a r e a c t i o n i n which t h e r a t e i s d i r e c t l y p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n o f a s i n g l e s u b s t r a t e ) . However, when CS] i s v e r y l a r g e , t h e o b s e r v e d v e l o c i t y r e a c h e s a maximum. F l a t t e n i n g o f t h e r e a c t i o n c u r v e d i s p l a y s a z e r o - o r d e r r e a c t i o n (a r e a c t i o n where r a t e i s independent o f s u b s t r a t e c o n c e n t r a t i o n ) . O b v i o u s l y , f i r s t - o r d e r r e a c t i o n s a r e c o - m i n g l e d d u r i n g t h e t r a n s i t i o n from a f i r s t - t o z e r o - o r d e r r e a c t i o n . Under z e r o - o r d e r r e a c t i o n c o n d i t i o n s , t h e c a t a l y t i c a c t i v i t y ( p o s s i b l y due t o end-product a c c u m u l a t i o n o r a l a c k o f enzymes) and enzyme c o n c e n t r a t i o n d e t e r m i n e maximum v e l o c i t y ( Z a p s a l i s and Beck, 1985). In t h e p H - s t a t system, t h e enzymes added t o i n i t i a t e t h e 10-minute i n . v i t r o p r o c e d u r e a r e added i n e x c e s s so t h a t an enzyme i n s u f f i c i e n c y w i l l not l i m i t t h e e x t e n t or r a t e o f d i g e s t i o n . 11 2 O M SUBSTRATE CONCENTRATION F i g u r e 1. A p l o t o f v e l o c i t i e s f o r an enzyme r e a c t i o n v e r s u s s u b s t r a t e c o n c e n t r a t i o n s . 2.4 T i t r a t i o n t h e o r y o f amino a c i d s I t i s o f g r e a t i m p o r t a n c e t o u n d e r s t a n d t h e b a s i c p r i n c i p l e o f t i t r a t i o n as i t r e l a t e s t o p r o t e i n s o r , more s p e c i f i c a l l y , t o t h e s m a l l e r u n i t s c o m p r i s i n g them, t h e amino a c i d s . I t i s t h e combined e f f e c t o f t h e t i t r a t i o n c u r v e s o f each o f t h e i n d i v i d u a l amino a c i d s which d e t e r m i n e t h e amount o f base r e q u i r e d t o r e a c h and m a i n t a i n an e n d p o i n t d u r i n g a pH-s t a t d e t e r m i n a t i o n . F u r t h e r , i t i s e s s e n t i a l t o a v o i d a t i t r a t i o n e n d p o i n t ( i . e . pH 9) where s e v e r a l amino a c i d s may have s t r o n g b u f f e r i n g c a p a c i t i e s . Thus, a knowledge o f t i t r a t i o n r e s p o n s e s o f each o f t h e amino a c i d s i s d e s i r a b l e i n t h e u n d e r s t a n d i n g o f t h e r e s p o n s e s o f p r o t e i n s t o e n z y m a t i c h y d r o l y s i s . The twenty amino a c i d s can be grouped i n t o f o u r f a m i l i e s based on one o f t h e p r o p e r t i e s o f t h e i r R-groups, i n p a r t i c u l a r , t h e i r p o l a r i t y ( t h e i r tendency t o i n t e r a c t w i t h water a t a p h y s i o l o g i c a l pH, near 7.0). The R-groups o f t h e amino a c i d s v a r y w i d e l y w i t h r e s p e c t t o t h e i r p o l a r i t y , from t o t a l l y n o n p o l a r t o h i g h l y p o l a r . Amino a c i d s w i t h n o n p o l a r o r h y d r o p h o b i c R-groups a r e a l a n i n e , i s o l e u c i n e , l e u c i n e , m e t h i o n i n e , p h e n y l a l a n i n e , p r o l i n e , t r y p t o p h a n , and v a l i n e ; t h o s e w i t h p o l a r but uncharged R-groupa a r e a s p a r a g i n e , c y s t e i n e , g l u t a m i n e , g l y c i n e , s e r i n e , t h r e o n i n e and t y r o s i n e ; t h o s e w i t h n e g a t i v e l y charged R-groups a r e a s p a r t i c a c i d and g l u t a m i c a c i d ; t h o s e w i t h p o s i t i v e l y c harged R-groups a r e l y s i n e , a r g i n i n e and h i s t i d i n e . 13 Most o f t h e n o n p o l a r R-group amino a c i d s , a r e h y d r o p h o b i c and t h u s , a r e o f t e n found b u r i e d i n t h e i n t e r i o r o f t h e ( g l o b u l a r ) p r o t e i n s i n an aqueous s u s p e n s i o n . The seven amino a c i d s t h a t have uncharged p o l a r R-groups a r e more s o l u b l e i n water because t h e y c o n t a i n f u n c t i o n a l groups t h a t form hydrogen bonds w i t h w a t e r . The net n e g a t i v e charge o f a s p a r t i c a c i d and g l u t a m i c a c i d a t pH 7.0 seems t o be t h e r e s u l t o f t h e p r e s e n c e o f t h e second c a r b o x y l group. The amino a c i d s i n which t h e R-groups have a net p o s i t i v e charge a t pH 7.0 a r e l y s i n e , which has a second amino group a t t h e e p s i l o n - p o s i t i o n on i t s a l i p h a t i c c h a i n ; a r g i n i n e , which has a p o s i t i v e l y c harged g u a n i d i n o group; and h i s t i d i n e , which has t h e weakly i o n i z e d i m i d a z o l e group. Amino a c i d s i n aqueous s o l u t i o n a r e i o n i z e d and can a c t as a c i d s o r bases. When a c r y s t a l l i n e amino a c i d ( a l a n i n e , f o r example) i s d i s s o l v e d i n w a ter, i t o c c u r s as t h e d i p o l a r i o n , which can a c t e i t h e r as an a c i d ( p r o t o n donor) o r as a base ( p r o t o n a c c e p t o r ) . Every amino a c i d has a c h a r a c t e r i s t i c t i t r a t i o n c u r v e . The t i t r a t i o n p l o t s shown i n F i g u r e 2 ( l y s i n e and a s p a r t i c a c i d ) each have t h r e e d i s t i n c t s t e p s , w i t h e v e r y s t e p c o r r e s p o n d i n g t o t h e removal o f one p r o t o n . Each o f t h e t h r e e s t a g e s resembles i n shape t h e t i t r a t i o n c u r v e o f a m o n o p r o t i c a c i d and can be a n a l y z e d i n t h e same manner. At t h e b e g i n n i n g o f t h e t i t r a t i o n o f l y s i n e , f o r example, t h e predominant s p e c i e s i s t h e f u l l y p r o t o n a t e d form. At t h e m i d p o i n t i n t h e f i r s t s t a g e o f t h e t i t r a t i o n , i n which t h e c a r b o x y l group o f 14 Ol Ionization steps for the titration of aspartic acid (acidic amino acid): H O O C C H j — C H — C O O H HOOCCH2—CH—COO" ^ 1.88 I 3.86 NH; NEC* = +1 Species [A] •Net electrical charge on amino acid N H j NEC = O (isoelectric) Species [B] "OOCCH2—CH—COO" *=* "OOCCHa—CH—COO" I 9.60 I NH\ N H i NEC = -1 Species [C] NEC = -2 Species [DJ Ionization steps for titration of lysine (basic amino acid): .H—COOH I H 3NCH2(CH 2 ) j—CH—C *=± H 3NCH 2(CH2)3—CH—COO" <=* NH; NH; NEC=+2 NEC=+1 Species [E] Species [F] H 3NCH2(CH2)3—CH-COO" N H ; NEC = O (isoelectric) Species [G] 10.53 H 2NCH 2(CH2)3—CH—COO" NH 2 NEC = -1 Species [H] Equivalents of base [OH ] added ( Z a p s a l i s and Beck, 1985) F i g u r e 2. Step w i s e i o n i z a t i o n f o r a c i d i c and b a s i c amino a c i d s a l o n g w i t h the c o r r e s p o n d i n g l o c a t i o n s o f t h e i r i o n i z a t i o n s p e c i e s on a t i t r a t i o n c u r v e . l y s i n e l o s e s i t s p r o t o n , t h e same molar c o n c e n t r a t i o n s o f pro t o n - d o n o r and p r o t o n - a c c e p t o r s p e c i e s a r e p r e s e n t . At t h i s same p o i n t , t h e pH i s e q u a l t o t h e pK o f t h e p r o t o n a t e d group b e i n g t i t r a t e d . S i n c e t h e m i d p o i n t pH i s 2.18, t h e c a r b o x y l group o f l y s i n e has a pK o f 2.18. As t h e t i t r a t i o n i s f o l l o w e d f u r t h e r , a n o t h e r i m p o r t a n t p o i n t (F) i s r e a c h e d . Here t h e r e i s a p o i n t o f i n f l e c t i o n , where t h e complete removal o f t h e f i r s t p r o t o n has o c c u r r e d and t h e removal o f t h e second one w i l l b e g i n . The second s t a g e o f t h e t i t r a t i o n c o r r e s p o n d s t o t h e removal o f a p r o t o n from t h e alpha-amino group o f l y s i n e . At t h e m i d p o i n t i n the second s t a g e o f t h e t i t r a t i o n c u r v e o f l y s i n e , e q u i m o l a r c o n c e n t r a t i o n s o f t h e d e p r o t o n a t e d and p r o t o n a t e d forma o f t h e alpha-amino group a r e p r e s e n t . The pH a t t h i s p o i n t i s 8.95, e q u a l t o t h e pK v a l u e f o r t h e a l p h a -amino group. As t h e t i t r a t i o n i s f o l l o w e d f u r t h e r s t i l l , a n o t h e r i m p o r t a n t p o i n t (G> i s r e a c h e d . Here t h e r e i s a p o i n t o f i n f l e c t i o n , where t h e complete removal o f t h e second p r o t o n has o c c u r r e d and t h e removal o f t h e l a s t one w i l l b e g i n . The t h i r d and f i n a l s t a g e o f t h e t i t r a t i o n c o r r e s p o n d s t o t h e removal o f a p r o t o n from t h e e p s i l o n - a m i n o group o f l y s i n e . S i m i l a r t o t h e f i r s t two s t a g e s o f t h e t i t r a t i o n c u r v e , t h e m i d p o i n t o f the t h i r d s t a g e c o r r e s p o n d s t o t h e p o i n t where e q u i m o l a r c o n c e n t r a t i o n s o f t h e d e p r o t o n a t e d and p r o t o n a t e d forms o f t h e e p s i l o n - a m i n o group a r e p r e s e n t . The pH a t t h i s p o i n t i s 10.53, e q u a l t o t h e pK v a l u e f o r t h e 16 e p a i l o n - a m i n o group. From t h e t i t r a t i o n c u r v e o f l y s i n e , i n f o r m a t i o n r e g a r d i n g i t s b u f f e r i n g c a p a c i t y can be o b t a i n e d . The t h r e e r e g i o n s o f t h e c u r v e t h a t a r e r e l a t i v e l y f l a t have a s s o c i a t e d w i t h them a good b u f f e r i n g c a p a c i t y . L y s i n e , l i k e most o t h e r amino a c i d s , i s not a good b u f f e r i n t h e p H - s t a t e n d p o i n t r e g i o n o f pH S.00. The o n l y e x c e p t i o n s a r e c y s t e i n e and c y s t i n e which have p o t e n t i a l - b u f f e r i n g pK v a l u e s o f 8.IS ( s u l f h y d r y l - g r o u p ) and 7.85 (alpha-amino g r o u p ) , r e s p e c t i v e l y . I n g e n e r a l , an amino a c i d w i t h an i o n i z a b l e R-group ( t r i p r o t i c ) has t h r e e pK v a l u e s . The t h i r d s t a g e f o r t h e t i t r a t i o n o f t h e i o n i z a b l e R-group merges t o some e x t e n t w i t h t h e o t h e r s . The i s o e l e c t r i c p o i n t ( t h a t pH where an amino a c i d assumes a net e l e c t r i c a l c harge o f z e r o and s o l u b i l i t y i n water i s a t a minimum) o f an amino a c i d i n t h i s c l a s s r e f l e c t s t h e t y p e o f i o n i z i n g R-group p r e s e n t . For i n s t a n c e , g l u t a m i c a c i d , w i t h two c a r b o x y l groups and one amino group, has an i s o e l e c t r i c p o i n t o f 3.22 (average o f t h e two c a r b o x y l g r o u p s ) . S i m i l a r l y , t h e i s o e l e c t r i c p o i n t o f l y s i n e w i t h two amino groups i s 9.74 (average o f t h e two amino g r o u p s ) . A l t h o u g h t h e d i p r o t i c amino a c i d s ( a l l amino a c i d s h a v i n g a s i n g l e amino group, a s i n g l e c a r b o x y l group, and an R-group t h a t does not i o n i z e , h a v i n g t i t r a t i o n c u r v e s w i t h two i o n i z a b l e groups) d i f f e r i n t h e i r a c i d - b a s e p r o p e r t i e s from t o l y s i n e o r a s p a r t i c a c i d ( f o r which t i t r a t i o n c u r v e s a r e shown), some s i m p l i f y i n g g e n e r a l i z a t i o n s about t h e i r b e h a v i o u r s can be made. T h i s group o f d i p r o t i c amino a c i d s i s c h a r a c t e r i z e d by h a v i n g v e r y s i m i l a r , a l t h o u g h not i d e n t i c a l , 17 pK v a l u e s f o r t h e a l p h a - c a r b o x y 1 groups ( i n t h e range o f 2.0 t o 3.0) and f o r t h e alpha-amino groups ( i n t h e range 9.0 t o 10.0). Owing t o t h e p r e s e n c e o f t h e c a r b o x y l groups o f t h e a c i d i c amino a c i d s ( a s p a r t i c and g l u t a m i c a c i d s ) , p l u s t h e c a r b o x y l group a t t h e t e r m i n a l end o f t h e c h a i n , most p r o t e i n s o l u t i o n s a r e good b u f f e r s below about pH 5 (Ledward, 1979). S i m i l a r l y , due t o t h e e p s i l o n - a m i n o group o f l y s i n e , t h e g u a n i d i n o group o f a r g i n i n e and t h e p h e n o l i c h y d r o x y l group o f t y r o s i n e , most p r o t e i n s a r e a l s o good b u f f e r s a t pH v a l u e s above 9.0. Only c y s t i n e and c y s t e i n e p o s s e s s any a b i l i t y t o b u f f e r a t o r near pH 8.0. Thus, pure p r o t e i n samples w i l l o n l y cause a n e g l i g i b l e amount o f b u f f e r i n g a t t h e pH o f 8 used i n t h e p H - s t a t e s t i m a t i o n s o f p r o t e i n d i g e s t i b i l i t y . 2.5 B u f f e r i n g c a p a c i t y o f f e e d s t u f f s S e v e r a l r e c e n t papers d e a l i n g w i t h i n . v i t r o d i g e s t i o n (0'Hare e_t a l . . , 1984; Pedersen and Eggum, 1983; Hsu et. a l . . 1977; H i l l e t a l . , 1982; S t i n s o n and Snyder, 1980) have acknowledged t h e p r e s e n c e o f b u f f e r s i n f e e d s t u f f s . Most papers f a i l t o q u a n t i f y t h e b u f f e r i n g c a p a c i t y o f each p r o t e i n sample. Perhaps t h e t a s k o f measuring a b s o l u t e amounts o f b u f f e r p r e s e n t i n t h e p r o t e i n s u s p e n s i o n i s t o o arduous. Be t h a t as i t may, t h e i s s u e has t o be a d d r e s s e d i n o r d e r t o b e g i n t o u n d e r s t a n d t h e b u f f e r i n g s o u r c e s i n t h e samples b e i n g d i g e s t e d i n . v i t r o . I n e v a l u a t i n g t h e pH-drop method f o r measuring p r o t e i n h y d r o l y s i s , Mozersky and P a n n e t t i e r i (1983) c o n c l u d e d t h a t 18 a l t h o u g h t h e t e c h n i q u e can be used t o f o l l o w t h e c o u r s e o f h y d r o l y s i s o f a s i n g l e s u b s t r a t e , pH d e c l i n e a l o n e cannot s e r v e as a measure o f e x t e n t o f p r o t e i n h y d r o l y s i s f o r t h e comparison of d i f f e r e n t s u b s t a n c e s . A l a c k o f c o r r e l a t i o n between d i g e s t i b i l i t y , as c a l c u l a t e d from t h e t e r m i n a l pH, and e x t e n t of h y d r o l y s i s can be a t t r i b u t e d t o t h e f a c t t h a t t h e number o f p e p t i d e bonds r u p t u r e d i s not t h e o n l y d e t e r m i n a n t o f t h e drop i n pH. The l a t t e r i s dependent on t h e number and pK v a l u e s (Chance and N i s h i m u r a , 1967) o f amino groups r e l e a s e d ; i n i t i a l l y b u r i e d b u f f e r i n g s i d e c h a i n s exposed upon h y d r o l y s i s ; b u f f e r i n g s i d e c h a i n s p r e s e n t i n exposed form i n t h e u n h y d r o l y z e d p r o t e i n . An a l t e r n a t i v e t o t h e measurement o f pH-drop (Hsu e t  a l . . 1977) i s t h e measurement o f hydrogen i o n p r o d u c t i o n a t a c o n s t a n t pH by u s i n g a p H - s t a t method (Pedersen and Eggum, 1983). With t h e i m p l e m e n t a t i o n o f t h i s method, t h e e f f e c t o f c h a n g i n g pH on p r o t e o l y t i c enzyme a c t i v i t y was m i n i m i z e d , and, a c c o r d i n g t o S t i n s o n and Snyder (1980), t h e b u f f e r i n g by newly-r e l e a s e d amino groups was e l i m i n a t e d . However, i f t h e p H - s t a t i s f i x e d t o an e n d p o i n t pH 9.0 ( S t i n s o n and Snyder, 1980; H i l l e t a l _ . , 1982) t h e b u f f e r i n g c a p a b i l i t y i s markedly i n c r e a s e d because pK v a l u e s o f t h e alpha-amino groups a r e i n t h i s range. In a p e r s o n a l communication Snyder (1986) suggested t h a t b u f f e r i n g e f f e c t s by p h e n o l i c , s u l f h y d r y l , and e p s i l o n - a m i n o groups may be p r e v a l e n t a t t h i s pH. However, he o v e r l o o k e d t h e f a c t t h a t by h a v i n g a p H - s t a t e n d p o i n t o f pH 9, t h e r e s u l t s o b t a i n e d would 19 be i n f l u e n c e d t r e m e n d o u s l y by b u f f e r i n g amino groups. Pedersen and Eggum (1983), r e a l i z i n g t h e e f f e c t o f b u f f e r i n g alpha-amino groups a t pH 9, used an e n d p o i n t o f pH 8 i n t h e i r e x p e r i m e n t s w i t h t h i s method. A c c o r d i n g t o Pedersen and Eggum (1981), a h i g h b u f f e r c a p a c i t y can be caused by a h i g h m i n e r a l c o n t e n t o f t h e f e e d s t u f f a l o n e ( e x c l u d i n g any m i n e r a l p r e m i x ) . C o i n c i d e n t a l l y , Hsu e t a l _ . (1977) a l s o o b s e r v e d t h a t a h i g h ash c o n t e n t o f a f e e d was d i r e c t l y r e l a t e d t o a h i g h b u f f e r c a p a c i t y . Pedersen and Eggum (1981) used t h e t h r e e - and f o u r -enzyme methods o f Hsu e_t a l . (1977) and S a t t e r l e e e t a l . ( 1 9 79), r e s p e c t i v e l y , t o p r e d i c t p r o t e i n d i g e s t i b i l i t y and b u f f e r i n g c a p a c i t y o f b a r l e y s . I n t h e s e e x p e r i m e n t s , Pedersen and Eggum (1981) noted t h a t b u f f e r c a p a c i t y o f t h e p r o t e i n samples a f f e c t e d t h e pH-drop and t h u s , t h e e s t i m a t e d i n v i t r o d i g e s t i b i l i t y . For b a r l e y s , where i n v i v o d i g e s t i b i l i t i e s were w i t h i n a narrow range, a s i g n i f i c a n t c o r r e l a t i o n between i n  v i t r o d i g e s t i b i l i t y and b u f f e r c a p a c i t y was f o u n d , a l t h o u g h t h e r e was no such c o r r e l a t i o n between i n . v i v o d i g e s t i b i l i t y and b u f f e r c a p a c i t y . However, f o r most p r o t e i n s o u r c e s t h e b u f f e r c a p a c i t y was a c c e p t a b l e ( u s u a l l y below 20 mL 0.0996 N HC1 and d i d not b i a s t h e i n v i t r o d e t e r m i n a t i o n s ) . I n c l u d i n g b u f f e r c a p a c i t y i n t h e m u l t i p l e r e g r e s s i o n e q u a t i o n improved c o r r e l a t i o n s and in . v i t r o e s t i m a t i o n s o n l y s l i g h t l y . Only f o r samples w i t h h i g h b u f f e r c a p a c i t y ( g r e a t e r than 20 mL), was t h e r e a s i g n i f i c a n t improvement i n in . v i t r o e s t i m a t e s by adjustment f o r t h e e f f e c t o f b u f f e r c a p a c i t y . Pedersen and 20 Eggum (1981) s t r o n g l y s t r e s s e d t h a t a s i g n i f i c a n t improvement was o b t a i n e d o n l y w i t h b a r l e y s h a v i n g h i g h b u f f e r c a p a c i t i e s . In 1983, Pedersen and Eggum, w h i l e u s i n g t h e p H - s t a t method t o p r e d i c t p r o t e i n d i g e s t i b i l i t y , o b s e r v e d t h a t p r e t r e a t m e n t w i t h p e p s i n i n c r e a s e s t h e b u f f e r c a p a c i t y o f t h e p r o t e i n s u s p e n s i o n s c o n s i d e r a b l y . T h i s h i g h e r b u f f e r i n g e f f e c t may be p a r t i a l l y e x p l a i n e d by t h e f a c t t h a t because p e p s i n h y d r o l y z e s more o f t h e p e p t i d e bonds, more i o n i z e d groups a r e r e l e a s e d which a r e t h e n added t o t h e p o o l o f b u f f e r i n g c o n s t i t u e n t s . In t h i s same s t u d y , a d d i t i o n o f c a l c i u m i o n s , a t or above t h o s e l e v e l s p r e s e n t i n c a l c i u m - r i c h f e e d s , were found t o s i g n i f i c a n t l y a f f e c t t h e e s t i m a t e d i n . v i t r o d i g e s t i b i l i t i e s . I n a d d i t i o n , t h e s e c a l c i u m i o n s changed t h e s l o p e o f t h e h y d r o l y s i s c u r v e s o f uptake o f t i t r a n t v e r s u s t i m e . D i s t i n c t d i f f e r e n c e s i n m i n e r a l c o n t e n t between d i f f e r e n t groups o f p r o t e i n s e x i s t , and t h i s might c o n t r i b u t e t o t h e d i f f e r e n c e s i n s u s c e p t i b i l i t y t o h y d r o l y s i s o b s e r v e d between d i f f e r e n t p r o t e i n c a t e g o r i e s . However, Pedersen and Eggum (1983) s t a t e d t h a t t h e e f f e c t s o f m i n e r a l s seem r a t h e r complex and t h a t t h e d i f f e r e n c e s i n t h e e f f e c t on h y d r o l y s i s r a t e which were produced by even wide v a r i a t i o n s i n t h e c a l c i u m c o n c e n t r a t i o n , were s m a l l . In t h e o n l y paper w r i t t e n on t h e t o p i c o f b u f f e r i n g c a p a c i t y e f f e c t s on v a r i o u s a s s a y s o f p r o t e i n d i g e s t i b i l i t y , O'Hare e_t a l . (1984) compared t h e e f f e c t o f i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n s on p e p t i d e bond c l e a v a g e w h i l e u s i n g e i t h e r t h e pH-drop or t h e p H - s t a t method. R e s u l t s w i t h t h e 21 l a t t e r method showed t h a t an i n c r e a s e d s u b s t r a t e l e v e l l e d t o an i n c r e a s e d p e p t i d e bond c l e a v a g e r a t e w h i l e w i t h t h e pH-drop method an i n c r e a s e d s u b s t r a t e l e v e l r e s u l t e d i n an ap p a r e n t d e c r e a s e i n r a t e o f c l e a v a g e . The p r o b a b l e e x p l a n a t i o n i s t h a t t h e d e t e c t i o n o f bond c l e a v a g e i s masked i n t h e pH-drop method due t o i n c r e a s e d b u f f e r i n g c a p a c i t y a t h i g h e r p r o t e i n c o n c e n t r a t i o n s (O'Hare e_t a l _ . , 1984) . These workers made d i r e c t comparison between t h e two in . v i t r o a s s a y s (pH-drop and p H - s t a t ) , u s i n g d i g e s t i b i l i t y c a l c u l a t e d a c c o r d i n g t o Hau e t §_1. , (1977) f o r t h e former and "ml NaOH added" f o r t h e l a t t e r . They reasoned t h a t i f both a s s a y s were a c c u r a t e l y measuring p e p t i d e bond c l e a v a g e , a s i m p l e c o r r e l a t i o n would be e x p e c t e d . T h i s , however was not found.. To i n v e s t i g a t e i f t h i s l a c k o f c o r r e l a t i o n was due t o b u f f e r i n g c a p a c i t y , t h e v a l u e s o b t a i n e d w i t h each a s s a y (mol H+ i n t h e pH drop and OH- i n t h e p H - s t a t ) , were e x p r e s s e d as a r a t i o , and t h e v a l u e o b t a i n e d p l o t t e d a g a i n s t b u f f e r i n g c a p a c i t y f o r each p r o t e i n . T h i s showed t h a t systems w i t h a h i g h b u f f e r i n g c a p a c i t y ( d r i e d m i l k ) were u n d e r e s t i m a t e d by t h e pH-drop a s s a y , whereas t h o s e w i t h a low b u f f e r i n g c a p a c i t y ( p a r t i c u l a r l y i n s o l u b l e p r o t e i n s such as wheat g l u t e n ) were c o n s i s t e n t l y o v e r e s t i m a t e d . F u r t h e r m o r e , d e f a t t i n g t h e d r i e d m i l k u s i n g c h l o r o f o r m / m e t h a n o l lowered t h e b u f f e r i n g c a p a c i t y more and t h u s , r a i s e d t h e pH-drop d i g e s t i b i l i t y f i g u r e s , whereas t h e v a l u e o b t a i n e d u s i n g t h e pH-s t a t method remained unchanged. I n 1966, P l a y n e and McDonald d e t e r m i n e d t h e b u f f e r i n g c o n s t i t u e n t s o f s i l a g e s and f o r a g e s . The b u f f e r i n g c a p a c i t y 2 2 v a l u e s o f s e v e r a l herbage s p e c i e s and o f s i l a g e made from each s p e c i e s and t h e c o n t r i b u t i o n s o f p l a n t components t o t h i s b u f f e r i n g , between pH 4 and pH 6, were d e t e r m i n e d , and found t o v a r y w i t h t h e herbage s p e c i e s . The b u f f e r i n g c a p a c i t y o f w i l t e d r e d c l o v e r was 18% lower t h a n t h a t o f f r e s h r e d c l o v e r . Smith <1962) c o n c l u d e d t h a t i n o r g a n i c i o n s a r e l a r g e l y r e s p o n s i b l e f o r b u f f e r i n g e f f e c t s i n a l f a l f a hay. However, he i g n o r e d t h e p o s s i b l e r o l e o f o r g a n i c a c i d s , and used l a c t i c a c i d as t h e t i t r a n t . P l a y n e and McDonald (1966) showed t h a t t h e o r g a n i c a c i d s were r e s p o n s i b l e f o r most o f t h e b u f f e r i n g e f f e c t i n f o r a g e s and s i l a g e s . In r y e g r a s s , t h e main b u f f e r s were found t o be malate and c i t r a t e , but i n r e d c l o v e r , g l y c e r a t e and malate were t h e main b u f f e r s . D u r i n g t h e e n s i l a g e p r o c e s s , m a l a t e , c i t r a t e , and g l y c e r a t e were broken down so t h a t a c e t a t e and l a c t a t e were the cause o f b u f f e r i n g a t t h i s t i m e . L a c t a t e forms a b u f f e r system i n t h e p r e s e n c e o f i n o r g a n i c c a t i o n s ( P l a y n e and McDonald, 1966). The a n i o n f r a c t i o n a c c o u n t e d f o r 80% o f t h e t o t a l b u f f e r i n g c a p a c i t y o f t h e p l a n t m a t e r i a l s and f o r 88% i n s i l a g e s . I n t h i s same s t u d y , b u f f e r i n g caused by p l a n t p r o t e i n s was e s t i m a t e d t o be o n l y 10-20% o f t h e t o t a l b u f f e r i n g c a p a c i t y . I t has been s u g g e s t e d ( N i l s s o n , 1956) t h a t l i b e r a t e d p r o t e i ns a r e t h e major b u f f e r i n g s u b s t a n c e s i n t h e a c i d pH r e g i o n d e s p i t e c o n t r a r y e v i d e n c e ( H u r d - K a r r e r , 1928). F u r t h e r , i t has been shown by McDonald and Henderson (1962) and P l a y n e (1963) t h a t t h e p r o t e i n c o n t e n t o f a s p e c i e s does not d i r e c t l y 23 c o n t r o l t h e b u f f e r i n g c a p a c i t y . With r e s p e c t t o f e e d g r a i n s , Hahn e_t a l . . (1982) have s u g g e s t e d t h a t p h e n o l i c a c i d s , pentosans and o t h e r compounds may a c t as b u f f e r s . 24 CHAPTER 3 EXPERIMENT 1 3.0 The E f f e c t , o f Temperature and Time of A p p l i c a t i o n o f Heat  on I n v i t r o P r o t e i n D i q e a t i b i l i t y o f G r a i n s and Raw ( F a t - e x t r a c t e d ) Soybeans by t h e pH-Stat Method. 3.1 I n t r o d u c t i o n The f o o d and f e e d i n d u s t r i e s r e q u i r e a s i m p l e , r a p i d and i n e x p e n s i v e method f o r t h e a c c u r a t e e s t i m a t i o n o f t h e d i g e s t i b i l i t y o f p r o t e i n o r p a r t i c u l a r amino a c i d s . C l a s s i c a l d i g e s t i b i l i t y t r i a l s , a l t h o u g h t h e most a c c u r a t e , a r e c o s t l y and time-consuming. As a consequence, t h e r e has been c o n s i d e r a b l e e f f o r t expended on t h e development o f i n v i t r o methods. To f u l f i l l a l l t h e r e q u i r e m e n t s o f r o u t i n e a n a l y s i s , i n v i t r o d i g e s t i o n r e s u l t s must be h i g h l y c o r r e l a t e d w i t h i n  v i v o t e s t s , and t h e method s h o u l d be s e n s i t i v e t o a v a r i e t y o f f a c t o r s a f f e c t i n g d i g e s t i v e p r o c e s s e s i n t h e l i v i n g o r g a n i s m . Some r e c e n t methods have used p e p s i n and i n t e s t i n a l f l u i d o f t h e p i g (Furuya a t a l . . , 1979), p e p s i n , p a n c r e a t i n and pronase (Buchmann, 1979), p e p s i n and t r y p s i n o r c h y m o t r y p s i n ( L i e n e r & Thompson, 19S0), p e p s i n d i g e s t i o n i n a d i a f i l t r a t i o n r e a c t o r (Tze-Ming & S h i p e , 1984), and p e p s i n f o l l o w e d by p r o t e o l y s i s w i t h p a n c r e a t i c enzymes i n a c l o s e d system w i t h c o n s t a n t d i a l y s i s o f d i g e s t i o n p r o d u c t s ( G a u t h i e r e_t a l . . , 1982, 1986). Most o f t h e above p r o c e d u r e s which attempt t o , more o r l e s s , s i m u l a t e n a t u r a l c o n d i t i o n s i n t h e d i g e s t i v e t r a c t a r e 25 c o m p l i c a t e d and somewhat time-consuming, a l t h o u g h t h e l a s t mentioned has overcome many o f t h e m a n i p u l a t i o n problems o f former methods. A r a p i d pH-drop method was de v e l o p e d by Hsu e_t a l . (1977) and m o d i f i e d by S a t t e r l e e e t a l . (1982). With t h e s e methods p r o t e i n d i g e s t i b i l i t y i s measured by t h e d e c l i n e o f pH i n t h e p r o t e i n s u s p e n s i o n a f t e r 10 minutes o f d i g e s t i o n w i t h a 3-enzyme s o l u t i o n ( t r y p s i n , c h y m o t r y p s i n and p e p t i d a s e , o r a f t e r an a d d i t i o n a l 10 minutes r e a c t i o n w i t h a f o u r t h enzyme, b a c t e r i a l p r o t e a s e . Pedersen and Eggum (1981), t e s t i n g b oth pH-drop methods, found s i g n i f i c a n t c o r r e l a t i o n s between in. v i t r o r e s u l t s and i n v i v o r e s u l t s w i t h r a t s f o r p l a n t p r o t e i n and p l a n t - a n i m a l p r o t e i n c o m b i n a t i o n s but not f o r a n i m a l p r o t e i n s a l o n e . They o b s e r v e d t h a t t h e methods were s e n s i t i v e t o t h e pr e s e n c e o f t a n n i n s and p r o t e a s e i n h i b i t o r s i n t h e samples t e s t e d . However, t h e r e s u l t s were a f f e c t e d by b u f f e r i n g c a p a c i t y o f t h e p r o t e i n s . A l t h o u g h t h e pH-drop method i s r a p i d and s i m p l e , t h e pH d e c l i n e r e s u l t i n g from t h e r e l e a s e o f amino a c i d s as t h e p r o t e i n h y d r o l y z e s , and t h e a c c u m u l a t i o n o f t h e h y d r o l y s i s p r o d u c t s c o u l d l i m i t t h e p r e c i s i o n o f t h e e s t i m a t i o n (Mozersky & P a n n e t t e r i , 1983). A m o d i f i c a t i o n o f t h e pH-drop p r o c e d u r e , t h e p H - s t a t method, where t h e pH i s ke p t c o n s t a n t and p r o t e i n d i g e s t i o n i s measured from t h e a l k a l i r e q u i r e d t o m a i n t a i n a c o n s t a n t pH 8.00 has been proposed t o a v o i d some d i s a d v a n t a g e s o f t h e o r i g i n a l Hsu e_t (1977) method ( M i l h a l y i , c i t e d by Roth e n b u h l e r & K i n s e l l a , 1985). 26 The p H - a t a t method has been shown t o be s e n s i t i v e t o t h e p r e s e n c e o f t r y p s i n i n h i b i t o r s and t o changes o f p r o t e i n d i g e s t i b i l i t y depending on changes i n t e r t i a r y and q u a r t e r n a r y s t r u c t u r e ( S t i n s o n & Snyder, 1980; O'Hare e_t §_1. 1984; R o t h e n b u h l e r & K i n s e l l a , 1985). These a u t h o r s have found t h e p H - s t a t method p r e c i s e and r e l i a b l e i n d e t e c t i n g d i f f e r e n c e s i n p r o t e i n d i g e s t i b i l i t y . They have a l s o c o n f i r m e d t h a t a m o d i f i c a t i o n o f t h e o r i g i n a l pH-drop method t o t h e p H - s t a t method overcomes t h e problem o f t h e b u f f e r i n g e f f e c t o f p r o t e i n s . Pedersen and Eggum (1983) have found h i g h c o r r e l a t i o n s between p H - s t a t ( i n . v i t r o ) p r o t e i n d i g e s t i b i l i t y and i n v i v o r e s u l t s w i t h r a t s ( r = 0.96). The b e s t p r e d i c t i o n o f p r o t e i n d i g e s t i b i l i t y was o b t a i n e d w i t h a three-enzyme system ( a c c o r d i n g t o Hsu et. a l _ . 1977) and an i n c u b a t i o n p e r i o d o f 10 m i n u t e s . P e p s i n p r e t r e a t m e n t had no p o s i t i v e e f f e c t on t h e r e s u l t s , a l t h o u g h t h e a u t h o r s d i d s u g g e s t t h a t t h i s may be b e n e f i c i a l w i t h p r o t e i n s o u r c e s c o n t a i n i n g p r o t e a s e i n h i b i t o r s . C o n s i d e r i n g t h e advantages o f t h e p H - s t a t method - i t s s i m p l i c i t y , r a p i d i t y and h i g h c o r r e l a t i o n w i t h i n . v i v o r e s u l t s , i t would seem t o be an a p p r o p r i a t e method f o r t h e r o u t i n e measurement o f p r o t e i n d i g e s t i b i l i t y f o r m o n o g a s t r i c a n i m a l s . The o b j e c t i v e o f t h i s e x p e r i m e n t was t o i n v e s t i g a t e t h e s e n s i t i v i t y o f t h e p H - s t a t method t o changes i n p r o t e i n d i g e s t i b i l i t y due t o heat t r e a t m e n t ( a u t o c l a v i n g or d r y h e a t i n g a t d i f f e r e n t t i m e s and t e m p e r a t u r e s ) o f f o u r p r o t e i n s o u r c e s . 27 3.2 M a t e r i a l s and methods 3.2.1 P r o t e i n s o u r c e s and t h e i r p r e p a r a t i o n Four p r o t e i n s o u r c e s , whole soybeans, wheat, b a r l e y and sorghum, were used t o e s t i m a t e t h e e f f e c t o f heat t r e a t m e n t on n i t r o g e n d i g e s t i b i l i t y . G r a i n s were ground i n a C h r i s t i e and N o r r i s hammermill t h r o u g h a 1 mm s c r e e n . Soybeans ( f u l l - f a t ) were f i r s t ground t h r o u g h a 3.6 mm s c r e e n and t h e n t h r o u g h a 1 mm s c r e e n , and t h e n f a t - e x t r a c t e d w i t h p e t r o l e u m e t h e r f o r 12 h r . Ground g r a i n s and f a t - e x t r a c t e d soybeans were exposed t o d r y h e a t i n g i n a Gallenkamp f o r c e d - d r a f t oven a t 80, 100, 120, 150, 180 and 240 , = > C w i t h an a p p l i c a t i o n t i m e f o r each t e m p e r a t u r e o f 30, 60, 120, and 240 m i n u t e s . The m o i s t u r e c o n t e n t changes o f t h e samples due t o heat t r e a t m e n t were r e c o r d e d ; t h i s a l l o w e d t h e c a l c u l a t i o n o f t h e a c t u a l n i t r o g e n c o n t e n t (g/100 g AD) which was e s t i m a t e d o n l y i n unheated f e e d s . N c o n t e n t s (g/100 g DM) were: raw f a t - e x t r a c t e d soybeans 7.62, wheat 2.47, b a r l e y 1.85, sorghum 1.41. The same f e e d s were a u t o c l a v e d a t 121° C (1.5 kg cm~ s) f o r 30, 60, 120, o r 240 minutes (each a p p r o x i m a t e l y 100 g, 1.5 cm l a y e r ) , t h e n d r i e d o v e r n i g h t a t 45° C. A f t e r p r o c e s s i n g , a l l g r a i n s and t h e f a t - e x t r a c t e d soybeans were ground t h r o u g h an 80-mesh (0.18 mm) s c r e e n i n a T e c a t o r - C y c l o t e c m i l l . N i t r o g e n was d e t e r m i n e d on a l l samples by t h e macro-K j e l d a h l method ( A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s , 1975). 28 3.2.2 Enzymes The enzymes used f o r i n . v i t r o d e t e r m i n a t i o n s were p o r c i n e p a n c r e a t i c t r y p s i n (Type IX, 14350 BAEE u n i t s per mg p r o t e i n ) , b o v i n e p a n c r e a t i c c h y m o t r y p s i n (Type I I , 47 u n i t s per mg powder) and p o r c i n e i n t e s t i n a l p e p t i d a s e (Grade I , 22 u n i t s per g powder) purchased from Sigma Ch e m i c a l Company. The three-enzyme a o l u t i o n was p r e p a r e d f r e s h d a i l y . The enzyme a c t i v i t y was d e t e r m i n e d by u s i n g sodium c a s e i n a t e as a r e f e r e n c e p r o t e i n . One mL o f enzyme s o l u t i o n c o n t a i n e d 1.5S mg o f t r y p s i n , 4.54 mg c h y m o t r y p s i n and 2.36 mg i n t e s t i n a l p e p t i d a s e . The pH o f t h e enzyme s o l u t i o n was a d j u s t e d t o 8.00 w i t h 0.10 N NaOH (a C 0 e - f r e e environment f o r t h e base was m a i n t a i n e d by use o f an A s c a r i t e t r a p f i t t e d t o t h e d i s p e n s e r ) a t 37'=' C, i n l e s s t h a n 2 m i n u t e s . The s o l u t i o n was t h e n k e p t on i c e t o m i n i m i z e a u t o l y s i s . 3.2.3 In v i t r o p r o c e d u r e The p H - s t a t method o f Pedersen and Eggum (1983) was used. D e t a i l s o f t h e p r o c e d u r e a r e g i v e n i n Appendix 1. D i g e s t i o n was conducted i n an a u t o m a t i c t i t r a t o r ( H e t t l e r M e m o t i t r a t o r DL 40 RC) which was equipped w i t h a m i c r o - e l e c t r o d e ( I n g o l d , C a t . No. 405-M5-NS) and a r e c o r d e r ( M e t t l e r GA 1 4 ) . The samples o f t h e p r o t e i n s o u r c e s were weighed i n t o t h e g l a s s r e a c t i o n v e s s e l s t o p r o v i d e 1 mg o f n i t r o g e n i n 1 mL of s u s p e n s i o n . D e i o n i z e d water (15 mL) was then added. Samples were kept i n a r e f r i g e r a t o r f o r 3-8 hours b e f o r e i n  v i t r o d i g e s t i o n . 29 The v e s s e l s w i t h p r o t e i n s u s p e n s i o n were p l a c e d i n a 37,:> C w a t e r - b a t h 15-20 minutes p r i o r t o t h e e s t i m a t i o n . The pH was a d j u s t e d t o 8.00 a t 37° C w i t h 0.10 N NaOH ( t i t r a t i n g p a r a m e t e r s : method e n d p o i n t a b s o l u t e , s t i r t i m e p r i o r t o pH adju s t m e n t 300 s e c , c o n t r o l band 0.15 pH u n i t s , d e l a y 3 s e c ) . A 1.5 mL a l i q u o t o f enzyme s o l u t i o n was the n added t o t h e p r o t e i n s u s p e n s i o n v e s s e l and pH 8.00 was m a i n t a i n e d by a u t o m a t i c t i t r a t i o n w i t h 0.10 N NaOH f o r 10 minutes w i t h c o n s t a n t s t i r r i n g (method e n d p o i n t a b s o l u t e , c o n t r o l band 0.10 pH u n i t s ) . A f t e r p r e c i s e l y 10 minutes o f d i g e s t i o n , t h e amount o f a l k a l i consumed i n m a i n t a i n i n g pH 8.00 was r e c o r d e d . The a c t i v i t y o f t h e enzyme was checked each day by u s i n g sodium c a s e i n a t e as a r e f e r e n c e p r o t e i n . A t e s t t o d e t e r m i n e cause o f t h e d i m i n u t i o n i n t h e r a t e o f a l k a l i consumption w i t h t i m e was c a r r i e d out by t h e a d d i t i o n o f e i t h e r s u b s t r a t e o r enzyme a f t e r a 10-minute d i g e s t i o n o f sodium c a s e i n a t e . I n one t e s t , f i v e a d d i t i o n s o f s u b s t r a t e , each o f 2 mL (1 mg N/mL), were made a t 1 minute i n t e r v a l s . I n t h e o t h e r t e s t , t h e r e was a s i n g l e a d d i t i o n o f 0.5 mL enzyme s o l u t i o n . The e f f e c t o f d o u b l i n g s u b s t r a t e c o n c e n t r a t i o n i n t h e o r i g i n a l s u s p e n s i o n was a l s o measured. A t e s t was un d e r t a k e n t o de t e r m i n e t h e amount o f base r e q u i r e d t o c o m p l e t e l y t i t r a t e t o pH 8.00 an amino a c i d m i x t u r e o f a c o m p o s i t i o n s i m i l a r t o t h a t o f c a s e i n . By u s i n g t h e amino a c i d a n a l y s i s o f t h i s c a s e i n which was p r o v i d e d by t h e man u f a c t u r e r (Humko S h e f f i e l d L t d . , T a b l e 1 ) , a 30 TABLE 1. Amino a c i d c o m p o s i t i o n o f C a s e i n * % amino a c i d % N i t r o g e n  i n c a s e i n i n amino a c i d a l a n i n e 2.8 15.72 a r g i n i n e 3.6 32.16 a s p a r t i c a c i d 6.5 10.52 c y s t i n e 0.3 13.08 g l y c i n e 1.7 18.66 g l u t a m i c a c i d 20.8 9.52 h i s t i d i n e 2.5 27.08 i s o l e u c i n e 4.8 10.68 l e u c i n e 8.8 10.68 l y s i n e 7.4 19.17 m e t h i o n i n e 2.5 9.39 p h e n y l a l a n i n e 4.8 8.48 p r o l i n e 12.1 12.17 s e r i n e 5.6 13.33 t r y p t o p h a n 1.1 13.72 t h r e o n i n e 3.9 11.76 t y r o s i n e 5.1 7.73 v a l i n e 5.6 11.96 T o t a l * N i t r o g e n = 12.7 ( A i r - D r y b a s i s ) * a n a l y s e s a c c o r d i n g t o Humko S h e f f i e l d T e c h n i c a l B u l l e t i n . " s y n t h e t i c " c a s e i n was f o r m u l a t e d w i t h pure "L" forms o f each amino a c i d ( e x c e p t DL m e t h i o n i n e ) . An amount o f t h i s m i x t u r e c o n t a i n i n g 15 mg N was t i t r a t e d t o pH 8.00 w i t h o u t enzyme a d d i t i o n . A comparison was made between t h e amount o f base r e q u i r e d t o t i t r a t e sodium c a s e i n a t e a f t e r e n z y m a t i c h y d r o l y s i s and t h a t r e q u i r e d by t h e amino a c i d m i x t u r e b o t h f o r a 10-minute d i g e s t i o n p e r i o d and when each r e a c t i o n was a l l o w e d t o c o n t i n u e t o c o m p l e t i o n . The i n i t i a l pH v a l u e s o f t h e s e two N s o u r c e s were a l s o r e c o r d e d . For t h e a u t o c l a v e d g r a i n s t h e r e s u l t s were a n a l y z e d as a two-way ( g r a i n - t y p e x time) a n a l y s i s o f v a r i a n c e . For d r y -heated g r a i n s , a one-way a n a l y s i s o f v a r i a n c e was used (25 t r e a t m e n t s i n c l u d i n g t h e c o n t r o l , i . e . no h e a t i n g ) f o r each g r a i n s e p a r a t e l y . Means were compared by t h e Stud e n t Newman K e u l s ' m u l t i p l e range t e s t ( B j e r r i n g e_t a_JL_. , 1975). 32 3.2.4 pH e l e c t r o d e maintenance The e l e c t r o d e was k e p t c l e a n by s o a k i n g i t weekly i n pH E l e c t r o d e C l e a n e r ( I n g o l d C a t . No. 18508) f o l l o w e d by a t h o r o u g h r i n s i n g w i t h d i s t i l l e d w a t e r . I f p r o t e i n c o n t a m i n a t i o n was s e v e r e , s o a k i n g i n a 5% p e p s i n (W/V) s o l u t i o n o f 0.10 N HC1 f o r one hour removed t h e c o n t a m i n a n t s . Once e v e r y two months t h e 3 M KC1 r e f e r e n c e e l e c t r o l y t e was r e p l a c e d . Very r a r e l y , t h e e l e c t r o d e tended t o respond v e r y s l o w l y t o changes i n pH. T h i s g e n e r a l l y was symptomatic o f a c l o g g e d o r d r y r e f e r e n c e j u n c t i o n and was remedied by immersing t h e e l e c t r o d e i n a beaker o f i t s own e l e c t r o l y t e (3 M KC1). The s o l u t i o n was heated t o a p p r o x i m a t e l y 70™ C and a l l o w e d t o c o o l t o room t e m p e r a t u r e w h i l e t h e e l e c t r o d e soaked i n i t . P r o l o n g e d use o f an e l e c t r o d e under a d v e r s e c o n d i t i o n s a l s o l e a d t o s l u g g i s h r e s p o n s e c h a r a c t e r i s t i c s . To improve e l e c t r o d e performance, R e a c t i v a t i n g S o l u t i o n ( I n g o l d C a t a l o g No. 18895) was used as f o l l o w s ; The t i p o f t h e e l e c t r o d e was p l a c e d i n a s m a l l p l a s t i c beaker and a s u f f i c i e n t amount o f s o l u t i o n was poured i n t o ensure t h a t t h e pH membrane was submerged. The membrane was t h e n a g i t a t e d f o r two minutes i n s o l u t i o n . The e l e c t r o d e was removed and r i n s e d w i t h d i s t i l l e d water and t h e n p l a c e d i n a beaker o f d i s t i l l e d water o v e r n i g h t . The e l e c t r o d e was t h e n r e c a l i b r a t e d . I f t h e response was poor, t h e p r o c e d u r e was r e p e a t e d . When t h e e l e c t r o d e was not i n use, i t s membrane was l e f t s o a k i n g i n a Radiometer b u f f e r s o l u t i o n o f pH 7.00. The 33 c a l i b r a t i o n o f t h e e l e c t r o d e was done b e f o r e each s e t o f d i g e s t i o n s w i t h pH 7.00 b u f f e r . I f t h e pH o f t h e sample was e x t r e m e l y a c i d i c o r b a s i c , t h e n a t w o - p o i n t c a l i b r a t i o n was done ( u s i n g Radiometer b u f f e r s pH 4.01 and 9.18). 3.3 R e s u l t s The 10-minute 0.10N NaOH consumption v a l u e s e s t i m a t e d from i n . v i t r o e n z y m a t i c d i g e s t i o n o f t h e f o u r a u t o c l a v e d p r o t e i n s o u r c e s a r e g i v e n i n T a b l e 2. The a u t o c l a v i n g o f soybeans r e g a r d l e s s o f d u r a t i o n i n c r e a s e d base consumption. The improvement was g r e a t e s t w i t h a u t o c l a v i n g f o r 30-60 minutes but s i g n i f i c a n t l y d e c r e a s e d w i t h t h e 120-minute d u r a t i o n , w i t h a f u r t h e r s i g n i f i c a n t d e c r e a s e when t h e a u t o c l a v i n g was extended t o 240 mi n u t e s . A u t o c l a v i n g reduced base consumed by b a r l e y , wheat and sorghum. The g r e a t e s t d e t r i m e n t a l e f f e c t o c c u r r e d w i t h wheat, w i t h mL base consumed d e c r e a s i n g from 0.440 w i t h no a u t o c l a v i n g t o 0.259 w i t h 30 minutes o f a u t o c l a v i n g . E x t e n d i n g t h e l e n g t h o f a u t o c l a v i n g had o n l y a s l i g h t e f f e c t . A u t o c l a v i n g o f b a r l e y and sorghum a l s o reduced NaOH v a l u e s but t o a l e s s e r e x t e n t . A l t h o u g h NaOH v a l u e s c o n t i n u e d t o d e c r e a s e s i g n i f i c a n t l y w i t h t i m e a f t e r 60 mi n u t e s , t h e s e d e c r e a s e s were s m a l l . 34 TABLE 2. Sodium h y d r o x i d e (mL 0.10N NaOH) consumed by soybean, wheat, b a r l e y and sorghum, each a u t o c l a v e d * f o r v a r i o u s l e n g t h s o f t i m e . P r o t e i n Source Time o f A u t o c l a v i n g (min) 30 60 120 240 P r o t e i n SEM«« Mean SEM Soybean 0.113n Wheat 0.440c B a r l e y 0.321e Sorghum 0.263g 0.479a 0.468b 0.389d 0.259g 0.261g 0.245h 0.252gh 0.242h 0.2281 0.193k 0.197k 0.1671 0.325f 0.003 0.353a 0.254gh 0.292b 0.208J 0.257c 0.151m 0.194d 0.001 Time Mean 0.297a 0.296a 0.292a 0.257b 0.235c 0.001 a-m W i t h i n comparison groups, mean v a l u e s not c o n t a i n i n g a common l e t t e r a r e s i g n i f i c a n t l y (p<0.05) d i f f e r e n t , a c c o r d i n g t o S t u d e n t Newman-K e u l s ' t e s t . * a u t o c l a v i n g c o n d i t i o n s ; 121,;>C, 1.5 kg/cm squared ** SEM = s t a n d a r d e r r o r o f mean 3.3.1 E f f e c t s o f d r y h e a t i n g The changes i n base consumption r e s u l t i n g from t h e d r y -heated soybeans and wheat, b a r l e y and sorghum a r e p r e s e n t e d i n T a b l e s 3, 4, 5, and 6 r e s p e c t i v e l y . The g r a p h i c e f f e c t on a l k a l i consumption and TD ( a c c o r d i n g t o Pedersen and Eggum, 1983; TD = .78 61 • 0.2686x, r e g r e s s i o n e q u a t i o n f o r p l a n t p r o t e i n s ) i s shown i n F i g u r e 3(a-d>. Dry h e a t i n g o f soybeans a t t e m p e r a t u r e s up t o 120° C had no e f f e c t on mL base consumed. At 150'= C, h e a t i n g s i g n i f i c a n t l y improved base consumed, w i t h t h e improvement i n c r e a s i n g t o t h e maximum t i m e o f a p p l i c a t i o n (240 m i n u t e s ) . At ISO*3 C, t h e base consumed was a t a s i m i l a r h i g h l e v e l f o r 30- and 60-minute a p p l i c a t i o n but d i m i n i s h e d w i t h more extended a p p l i c a t i o n s . At 240'= C, o n l y t h e 30-minute a p p l i c a t i o n improved base consumption. The e f f e c t s o f d r y heat t r e a t m e n t s on t h e base consumed by wheat, b a r l e y and sorghum were s i m i l a r . H e a t i n g a t t e m p e r a t u r e s below ISO*3 C had l i t t l e o r no s i g n i f i c a n t e f f e c t . The h i g h e r t e m p e r a t u r e s and l o n g e r t i m e s o f a p p l i c a t i o n d e c r e a s e d base v a l u e s . At t e m p e r a t u r e s o f 150, ISO and 240'= C, i n c r e a s i n g t i m e s o f a p p l i c a t i o n and i n c r e a s i n g t e m p e r a t u r e s d e c r e a s e d base consumed, w i t h , f o r each g r a i n , t h e l o w e s t v a l u e b e i n g o b t a i n e d w i t h 240'= C f o r 240 m i n u t e s . The mL base-added v a l u e s f o r u n t r e a t e d g r a i n and g r a i n heated a t 240'= C f o r 240 minutes were, r e s p e c t i v e l y - wheat 0.440, 0.105; b a r l e y 0.321, 0.118; sorghum 0.263, 0.085. 36 TABLE 3. A b s o l u t e (mL 0.10N NaOH) r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n o f d r y heated soybeans. NaOH consumed by unheated soybeans = 0.113a mL Time o f h e a t i n g (min) Temperature («=• C) 30 60 120 240 SO O.lOSa 0.112a 0.111a 0.105a 100 0.110a 0.114a 0.121a 0.124a 120 0.115a 0.118a 0.124a 0.130a 150 0.161a 0.211b 0.291b 0.347c 180 0.336c 0.312c 0.219b 0.135a 240 0.293b 0.185a 0.114a 0.100a E x p e r i m e n t a l e r r o r mean square = 0.01, n = 50. Mean v a l u e s n o t h a v i n g a common l e t t e r a r e s i g n i f i c a n t l y (P<0.05) d i f f e r e n t , a c c o r d i n g t o St u d e n t Newman-Keula' t e s t . 37 TABLE 4. A b s o l u t e (mL 0.10N NaOH) r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n o f d r y heated wheat. NaOH consumed by unheated wheat = 0.440e mL Time o f h e a t i n g (min) Temperature <° C) 30 60 120 240 80 0.503f 0.497e 0.494e 0.491e 100 0.455e 0.458e 0.463e 0.463e 120 0.463e 0.461e 0.447e 0.438e 150 0.430e 0.394d 0.350d 0.310d 180 0.296c 0.249b 0.205b 0.165a 240 0.228b 0.170a 0.138a 0.105a E x p e r i m e n t a l e r r o r mean square = 0.01, n = 50. Mean v a l u e s n o t h a v i n g a common l e t t e r a r e s i g n i f i c a n t l y <P<0.05) d i f f e r e n t , a c c o r d i n g t o St u d e n t Newman-Keuls' t e s t . 38 TABLE 5. A b s o l u t e (mL 0.10N NaOH) r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n o f d r y heated b a r l e y . NaOH consumed by unheated b a r l e y = 0.321c mL Time o f h e a t i n g (min) Temperature (~ C) 30 60 120 240 80 0.315c 0.318c 0.316c 0.324c 100 0.328c 0.329c 0.340c 0.348c 120 0.319c 0.302c 0.327c 0.328c 150 0.372c 0.351c 0.321c 0.278b 180 0.318c 0.277b 0.233b 0.185b 240 0.276b 0.224b 0.177a 0.118a E x p e r i m e n t a l e r r o r mean squ a r e = 0.01, n = 50. Mean v a l u e s n o t h a v i n g a common l e t t e r a r e s i g n i f i c a n t l y (P<0.05) d i f f e r e n t , a c c o r d i n g t o St u d e n t Newman-Keuls' t e s t . 39 TABLE 6. A b s o l u t e (mL 0.10N NaOH) r e s u l t s o f i n . v i t r o p r o t e i n d i g e s t i o n o f d r y heated sorghum. NaOH consumed by unheated sorghum = 0.263c mL Time o f h e a t i n g (min) Temperature <° C) 30 60 120 240 80 0.263c 0.260c 0.265c 0.273c 100 0.279c 0.272c 0.268c 0.268c 120 0.286c 0.284c 0.284c 0.252c 150 0.236c 0.208c 0.180b 0.137b 180 0.138b 0.114b O.lOlab 0.094ab 240 0.121b 0.095a 0.102b 0.085a E x p e r i m e n t a l e r r o r mean square = 0.01, n = 50. Mean v a l u e s n o t h a v i n g a common l e t t e r a r e s i g n i f i c a n t l y <P<0.05) d i f f e r e n t , a c c o r d i n g t o Stu d e n t Newman-Keuls' t e s t . 40 F i g u r e 3 ( a - d ) . The e f f e c t o f d r y h e a t i n g a t d i f f e r e n t t e m p e r a t u r e s : 80-=C (O), 100~C (•) , 120~C <•) . 150~C (•> , lSO-^C (A) and 240='C <A) o f f a t - e x t r a c t e d soybeans ( a ) , b a r l e y <b), wheat <c> and sorghum (d) on NaOH consumption (blO) d u r i n g 10 minutes o f i n . v i t r o d i g e s t i o n and c o r r e s p o n d i n g TD v a l u e s <TD = 0.7861 • 0.2686-x ). R e s u l t s o b t a i n e d w i t h u n t r e a t e d m a t e r i a l s a r e r e p r e s e n t e d by - - -. 41 H 0.90 00 S a S o 2 O H ft, S P z o o as o z z o 0.80 30 60 120 240 T I M E O F H E A T I N G (min) F i g u r e 3 a , f a t - e x t r a c t e d s o y b e a n s -I 0.90 30 60 120 240 T I M E O F H E A T I N G (min) F i g u r e 3 b , b a r l e y 42 -1 0.94 0.80 F i g u r e 3 c , wheat 30 60 120 240 T I M E O F H E A T I N G (min) 0.80 F i g u r e 3d, sorghum 43 3.3.2 A u t o c l a v i n g va d r y - h e a t i n g e f f e c t a The same t e m p e r a t u r e ( 1 2 0 - 1 2 1 0 C) a p p l i e d f o r t h e same d u r a t i o n s <30-240 m i n u t e s ) , e i t h e r d r y or a u t o c l a v e d ( F i g u r e 4a-d) a f f e c t e d base consumed e s t i m a t i o n s i n d i f f e r e n t ways. In th e c a s e o f soybeans, d r y h e a t i n g a t t h i s t e m p e r a t u r e improved base consumption o n l y s l i g h t l y , whereas a u t o c l a v i n g f o r 30-60 minutes i n c r e a s e d i t s i g n i f i c a n t l y . The h i g h e s t base a d d i t i o n o b t a i n e d w i t h t h e d r y h e a t i n g o f soybeans ( 1 5 0 0 C f o r 240 minutes) o f 0.347 was markedly lower t h a n t h e maximum a u t o c l a v e d v a l u e s o f 0.479 and 0.468 f o r t h e r e s p e c t i v e d u r a t i o n s o f 30 and 60 mi n u t e s , but was s i m i l a r t o t h a t o f soybeans a u t o c l a v e d f o r 240 mi n u t e s , which produced t h e l o w e s t a l k a l i v a l u e f o r a u t o c l a v i n g soybean-Moderate d r y h e a t i n g o f wheat, b a r l e y and sorghum had no s i g n i f i c a n t e f f e c t on base consumption, whereas a u t o c l a v i n g reduced base consumed, w i t h t h i s not b e i n g a f f e c t e d by t i m e o f a p p l i c a t i o n . The a d v e r s e e f f e c t o f a u t o c l a v i n g was g r e a t e s t f o r wheat. With wheat, b a r l e y and sorghum, a u t o c l a v i n g had a d e t r i m e n t a l e f f e c t , which produced base consumption v a l u e s s i m i l a r t o t h o s e o b t a i n e d w i t h d r y h e a t i n g a t 180'= C f o r 60 minutes f o r wheat, ISO™ C f o r 120 minutes f o r b a r l e y and 150° C f o r 120 minutes f o r sorghum. 44 F i g u r e 4(a - d) . The e f f e c t s o f a u t o c l a v i n g a t 121'='C CO) and d r y h e a t i n g a t 120°C <•) o f f a t - e x t r a c t e d soybeans ( a ) , b a r l e y ( b ) , wheat (c) and sorghum (d) on NaOH consumption and c o r r e s p o n d i n g TD v a l u e s <TD = 0.7861 + 0.2686-x ). R e s u l t s o b t a i n e d w i t h u n t r e a t e d m a t e r i a l a r e r e p r e s e n t e d by - - -. 45 60 s E o Z o H D CO Z o u s o to z z o 0.50 -0.45 -0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 K — L 0.92 0.90 0.88 0.86 - 0.84 5 H m W O W D as H - 0.82 0.80 30 60 120 240 T I M E O F H E A T I N G (min) F i g u r e 4 a , f a t - e x t r a c t e d s o y b e a n s I 0.90 - 0.88 > H s H0 .86 p cu O •4 0.84 5 w D -10.82 g 30 60 120 T I M E O F H E A T I N G (min) 240 0.80 F i g u r e 4 b , b a r l e y 4 6 60 E o O H OL, s D CO O (J X o « z 2 o 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 -0.05 -0.00 k—1 30 60 120 240 T I M E O F H E A T I N G (min) 0.40 0.35 0.30 0.25 0.20 0.15 0.10 -0.05 -0.00 L V - 1 30 60 120 240 , T I M E O F H E A T I N G (min) -I 0.92 0.90 0.88 H 0.86 H ffl H CO W O Q 0.84 g H 0.82 0.80 F i g u r e 4 c , w h e a t 0.90 0.88 >-H 5 0.86 P CO UJ 2 0.84 S w 0.82 H 0.80 F i g u r e 4 d , s o r g h u m 4 7 3.3.3 Teat t o de t e r m i n e l i m i t i n g f a c t o r i n r e a c t i o n v e s s e l The r e a c t i o n , w i t h a c i d p r o d u c t i o n e s s e n t i a l l y f o l l o w i n g a f i r s t - o r d e r d i m i n u t i o n c u r v e , i s n o r m a l l y t e r m i n a t e d a f t e r 10 minutes i n t h i s p r o c e d u r e . T e s t s w i t h sodium c a s e i n a t e demonstrated t h a t t h e r e d u c t i o n i n t h e r a t e o f r e a c t i o n o v er t h e 10-minute p e r i o d r e s u l t e d from a d e p l e t i o n o f h y d r o l y z a b l e s u b s t r a t e r a t h e r t h a n an enzyme inadequacy ( F i g u r e 5 ) , as demonstrated by t h e f a c t t h a t a f t e r 10 minutes o f r e a c t i o n , t h e a d d i t i o n o f 0.5 mL enzyme s o l u t i o n had no e f f e c t ; however f i v e s u c c e s s i v e 2.0 mL a d d i t i o n s o f s u b s t r a t e a t 1-minute i n t e r v a l s each r e s u l t e d i n f u r t h e r NaOH consumption. Enzyme e x c e s s was f u r t h e r demonstrated when a d o u b l i n g o f t h e c o n c e n t r a t i o n o f s u b s t r a t e (from 1 mg N/mL s u s p e n s i o n ) i n c r e a s e d t h e 10-minute consumption o f 0.10 N NaOH from 0.839 mL t o 1.542 mL. 3.3.4 T e s t t o e s t a b l i s h degree o f h y d r o l y s i s t h a t o c c u r s w i t h  c a s e i n d u r i n g t h e 10-minute p H - s t a t d i g e s t i o n Over a 10-minute d i g e s t i o n p e r i o d , t h e sodium c a s e i n a t e r e q u i r e d 0.801 mL o f 0.10 N NaOH t o m a i n t a i n a pH o f 8.00 ( F i g u r e 6 ) . In t h e case o f t h e f r e e amino a c i d m i x t u r e ( s i m u l a t i n g t h e amino a c i d c o m p o s i t i o n o f c a s e i n ) , 3.01 mL o f base was r e q u i r e d t o b r i n g t h e pH t o 8.00 from 3.06. There was no d i s t i n c t i o n between t h e p r e - d i g e s t i o n s t a g e ( i . e . t o b r i n g t h e i n i t i a l pH t o 8.00) and t h e d i g e s t i o n s t a g e w i t h t h e amino a c i d m i x t u r e because a l l amino a c i d s were i n a f r e e form a t t h e commencement o f t h e t i t r a t i o n . 48 F i g u r e 5. NaOH consumption a f t e r t h e a d d i t i o n o f 1.5 ml enzyme s o l u t i o n t o sodium c a s e i n a t e s u s p e n s i o n s p r o v i d i n g 1.0 mg N/ml < > and 2.0 mg N/ml (— — — ) . Response t o f u r t h e r a d d i t i o n s t o t h e 1.0 mg N/ml s u s p e n s i o n a f t e r 10 minutes o f 0.5 ml enzyme s o l u t i o n i s denoted by <—• — •—) and o f 5 s u c c e s i v e 2 ml a l i q u o t s o f 1.0 mg N/ml s u s p e n s i o n ( O ) a t 1 minute i n t e r v a l s i s denoted by < ) . 49 F i g u r e 6. A comparison o£ NaOH consumed by sodium c a s e i n a t e ( 1) and " a r t i f i c i a l c a s e i n " (— — —> d u r i n g a 10-minute i n c u b a t i o n t o an e n d p o i n t o f pH 8.00 w i t h t h e p H - s t a t method. A l s o noted was t h e t i m e (minutes) r e q u i r e d f o r each " c a s e i n " t o r e a c h e n d p o i n t 8.00 where no f u r t h e r NaOH a d d i t i o n o c c u r e d (O). 50 3.4 D i s c u s s i o n 3.4.1 Heat t r e a t m e n t e f f e c t s on p r o t e i n d i g e s t i b i l i t y P r o t e i n f e e d s t u f f s s u b j e c t e d t o heat t r e a t m e n t undergo v a r i o u s changes which may be e i t h e r b e n e f i c i a l o r d e t r i m e n t a l as r e g a r d s p r o t e i n d i g e s t i b i l i t y and b i o l o g i c a l v a l u e , depending on t h e form o f t h e f e e d s t u f f and t h e h e a t i n g c o n d i t i o n s , i . e . t e m p e r a t u r e , t i m e o f a p p l i c a t i o n , m o i s t u r e l e v e l . Leguminous s e e d s , e.g. soybeans, f a b a beans and k i d n e y beans a r e known t o be improved i n p r o t e i n d i g e s t i b i l i t y when s u b j e c t e d t o m o i s t heat a t 100-120° C f o r a s h o r t t i m e (30-60 minutes) (Combs e_t a l . 1967; L i e n e r & Thompson, 1980; W i l l i a m s e t a l . 1982; V a n d e r g r i f t , 1985). In t h e s e c o n d i t i o n s a n t i -n u t r i t i o n a l f a c t o r s p r e s e n t i n raw seeds ( e . g . t r y p s i n i n h i b i t o r s ) a r e al m o s t c o m p l e t e l y d e s t r o y e d , w h i l e t h e s l i g h t d e n a t u r a t i o n o f p r o t e i n i n c r e a s e s i t s s u s c e p t i b i l i t y t o e n z y m a t i c h y d r o l y s i s ( P r i e s t l y , 1979; S t i n s o n & Snyder, 1980; Eggum & Beames, 1983). A u t o c l a v i n g o f soybeans i s more e f f e c t i v e t h a n d r y h e a t i n g i n t h e d e s t r u c t i o n o f t r y p s i n i n h i b i t o r s ( W i l l i a m s e t  a l . 1982; V a n d e r g r i f t , 1985). In t h e absence o f r e d u c i n g s u g a r s , soy p r o t e i n can remain u n a f f e c t e d f o r extended d u r a t i o n s o f a u t o c l a v i n g ( K n i p f e l , 1981). However, i n t h i s s t u d y t h e a u t o c l a v i n g o f soybeans f o r more th a n 60 minutes s i g n i f i c a n t l y reduced p r o t e i n d i g e s t i b i l i t y . Dry h e a t i n g o f 51 soybeans a t t e m p e r a t u r e s above IZO™ C improved p r o t e i n d i g e s t i b i l i t y , but i t never reached t h e b e s t r e s u l t o b t a i n e d by a u t o c l a v i n g . H e a t i n g a t 180 and 2 4 0 o C f o r l o n g e r t h a n 30 minutes i n c r e a s e d t h e a d v e r s e e f f e c t u n t i l t h e reduced p r o t e i n d i g e s t i b i l i t y e v e n t u a l l y negated t h e b e n e f i c i a l e f f e c t r e s u l t i n g from t h e d e s t r u c t i o n o f t h e t r y p s i n i n h i b i t o r . S e vere h e a t i n g c o n d i t i o n s cause e x t e n s i v e changes i n p r o t e i n s t r u c t u r e , l e a d i n g t o a d e c l i n e o f i t s d i g e s t i b i l i t y and b i o l o g i c a l v a l u e as a r e s u l t o f r e a c t i o n s between amines, amino a c i d s , p e p t i d e s and s i d e - c h a i n s o f p r o t e i n w i t h o t h e r components i n c l u d i n g r e d u c i n g s u g a r s , f a t s and t h e i r o x i d a t i o n p r o d u c t s , p o l y p h e n o l s , v i t a m i n B e , and a v a r i e t y o f c r o s s -l i n k i n g r e a c t i o n s w i t h i n p r o t e i n c h a i n s ( H u r r e l l & C a r p e n t e r , 1981; Mauron, 1981). The p r e s e n c e o f r e d u c i n g s u g a r s i n a heated p r o d u c t r e s u l t s i n a complex M a i l l a r d non-enzymatic browning r e a c t i o n . The p r o d u c t s o f an advanced M a i l l a r d r e a c t i o n have a l s o been found t o a c t as p r o t e o l y t i c enzymes' i n h i b i t o r s ( H u r r e l l & C a r p e n t e r , 1981; Mauron, 1981; P e r k i n s e t a l . 1982). In t h e absence o f r e d u c i n g s u g a r s t h e r e i s r e l a t i v e l y l i t t l e d e s t r u c t i o n o f l y s i n e and o t h e r amino a c i d s d u r i n g h e a t i n g ( L i -Chan & N a k a i , 1981) but t h e f o r m a t i o n o f c r o s s - l i n k s both w i t h i n and between p e p t i d e c h a i n s d e c r e a s e s p r o t e i n d i g e s t i b i l i t y and b i o l o g i c a l v a l u e ( B j a r n a s o n & C a r p e n t e r , 1970; V a r n i s h & C a r p e n t e r , 1975ab; W a l l e t a l . 1975). 52 Hansen ejt (1975) and Hansen & J o h n s t o n (1976) found a h i g h r a t e o f p r o t e i n a g g r e g a t i o n i n wheat f l o u r s heated a t 108° C t o 1 5 0 0 C f o r 2-10 minutes but l e s s a g g r e g a t i o n w i t h 174-=> c h e a t i n g . T h i s h e a t i n g lowered t h e p e p t i c and t r y p t i c d i g e s t i o n r a t e s even a t 108,=> C. At 174™ C t h e r e was a c l e a v a g e o f p r o t e i n c h a i n s t o p e p t i d e s l e s s a c c e s s i b l e t o c a r b o x y p e p t i d a s e a t t a c k . There was e x t e n s i v e d e s t r u c t i o n o f l y s i n e and c y s t i n e w i t h t h e a d v e r s e e f f e c t b e i n g a g g r a v a t e d as m o i s t u r e i n c r e a s e d from 13 t o 33%. The a u t h o r s suggest t h a t t h e d e c r e a s e d p r o t e i n d i g e s t i o n o f e x t e n s i v e l y heated wheat f l o u r s c o u l d be e x p l a i n e d as a p r o t e i n - s t a r c h i . e . M a i l l a r d -t y p e r e a c t i o n . A c c o r d i n g t o Mauron (1981), t h e r e d u c t i o n i n p r o t e i n v a l u e i n c r e a s e s as t e m p e r a t u r e i n c r e a s e s , but even r e l a t i v e l y low t e m p e r a t u r e s a p p l i e d f o r a l o n g t i m e can produce d e t r i m e n t a l e f f e c t s . M i l d h e a t i n g (below 100'= C) o f c e r e a l g r a i n s has no e v i d e n t e f f e c t on p r o t e i n d i g e s t i b i l i t y , o r may s l i g h t l y i n c r e a s e i t ( G r i s w o l d , 1951) and r e s u l t s i n l i t t l e damage t o amino a c i d s ( W a l l e t a l _ . 1975) . I n t h i s e x p e r i m e n t , d r y h e a t i n g t o a t e m p e r a t u r e o f 120° C had l i t t l e e f f e c t on p r o t e i n d i g e s t i b i l i t y . A c c o r d i n g t o G r i s w o l d (1951), wet h e a t i n g o f wheat c a u s e s l e s s damage t o l y s i n e t h a n d r y h e a t i n g . However, C h e r r y (1982) s t a t e d t h a t wet h e a t i n g o f s t a r c h y p r o d u c t s , r e s u l t e d i n g e l a t i n i z a t i o n , e n a b l i n g amylose and a m y l o p e c t i n t o r e a c t w i t h t h e NH e groups o f g l u t e n , e s p e c i a l l y i n t h e a c i d i c medium. 53 Dahle e t a l . (1975) showed t h e a b i l i t y o f wheat p r o t e i n t o b i n d t o g e l a t i n i z e d wheat s t a r c h and d e x t r i n s ; t h e soy p r o t e i n i s o l a t e s had much l e s s a f f i n i t y f o r t h i s b i n d i n g a c t i o n . L i -Chan & Nakai (1981) s u g g e s t e d t h a t components l i k e s t a r c h would cause some d e s t r u c t i o n o f l y s i n e ( t h u s - p r o t e i n d i g e s t i b i l i t y ) d u r i n g b a k i n g . S i n c e h i g h m o i s t u r e l e v e l s f a v o u r t h e h y d r o l y s i s o f s t a r c h and s u c r o s e , t h i s c o n d i t i o n can a l s o i n c r e a s e t h e e x t e n t o f M a i l l a r d r e a c t i o n s i n s t a r c h - p r o t e i n s ystems. P r o t e i n d i g e s t i b i l i t y o f sorghum can be reduced by t h e p r e s e n c e o f t a n n i n s which can form e n z y m e - r e s i s t a n t p r o t e i n -t a n n i n complexes ( P e r e z & Bourdon, 1984; Hahn & Rooney, 1986). B o i l i n g o f p r o d u c t s r i c h i n t a n n i n s has been shown t o improve p r o t e i n d i g e s t i b i l i t y ( B a r r o g a e_t a l . 1985) . I n t h i s l a t t e r s t u d y w i t h mung beans (V i g n a r a d i a t a ( L ) . W i l c z e k ) , p a r t o f t h e r e s p o n s e t o h e a t i n g o c c u r r e d as a r e s u l t o f i n a c t i v a t i o n o f t r y p s i n i n h i b i t o r s . R e s u l t s o b t a i n e d i n t h i s l a b o r a t o r y w i t h r a t s ( u n p u b l i s h e d ) showed no response i n TD and BV t o d r y h e a t i n g o f sorghum a t 90 and 120,=> C f o r 120 minutes but a marked r e d u c t i o n i n both t h e s e parameters w i t h d r y h e a t i n g a t I S O 0 C f o r 120 minutes or steaming ( a t m o s p h e r i c p r e s s u r e ) o r a u t o c l a v i n g f o r 120 m i n u t e s . These r e s u l t s were i n c o n t r a s t t o t h o s e o f B a r r o g a e_t a l . (1985). The p r e s e n t r e s u l t s w i t h the p H - s t a t p r o c e d u r e show a s i m i l a r r e s p o n s e i n t h a t none o f t h e h e a t i n g t r e a t m e n t s a p p l i e d t o t h e g r a i n s had a b e n e f i c i a l e f f e c t on TD, w h i l e t h e h i g h e r t e m p e r a t u r e s , both d r y and m o i s t , reduced t h i s v a l u e . 54 In t h e case o f b a r l e y , TD was not a f f e c t e d by h e a t i n g a t t e m p e r a t u r e s from 80-120° C f o r up t o 120 minut e s . Beyond 150° C f o r 30 mi n u t e s , TD was r e d u c e d , w i t h t h e r e d u c t i o n b e i n g i n c r e a s e d w i t h t i m e o f a p p l i c a t i o n . The n e g a t i v e e f f e c t s o f h i g h e r t e m p e r a t u r e s were not as l a r g e as t h o s e f o r wheat and sorghum, a l t h o u g h a u t o c l a v i n g caused a d e c r e a s e which was s i m i l a r t o t h a t found w i t h t h e s e l a t t e r two g r a i n s . B a r l e y s c o n t a i n a p e p s i n - s t a b l e t r y p s i n - i n h i b i t o r , but t h i s i s p r e s e n t a t t o o low a c o n c e n t r a t i o n t o a f f e c t p r o t e i n d i g e s t i b i l i t y (Pedersen & B o i s e n , 1982). Eggum (1978), q u o t i n g h i s own e a r l i e r work, r e p o r t e d a d e c r e a s e i n TD o f b a r l e y d r i e d a t 100° C f o r 30-40 m i n u t e s . Our r e s u l t s do not s u p p o r t t h i s f i n d i n g . In g e n e r a l , a l l r e s u l t s o f t h i s s t u d y c o n f i r m e d t h e e x p e c t e d changes i n p r o t e i n d i g e s t i b i l i t y due t o d i f f e r e n t heat t r e a t m e n t s - b e n e f i c i a l s h o r t t i m e a u t o c l a v i n g o f raw soybeans, a l m o s t no e f f e c t o f m i l d d r y h e a t i n g o f soybeans and g r a i n s , and a d e t r i m e n t a l e f f e c t o f l o n g t i m e exposure t o h i g h e r t e m p e r e a t u r e s , wet and d r y , o f both soybeans and g r a i n s . T h i s c o n f i r m s t h a t t h e p H - s t a t method i s s e n s i t i v e f o r d e t e c t i n g changes i n p r o t e i n d i g e s t i b i l i t y i n d u c e d by heat t r e a t m e n t . G a u t h i e r e t a l . (1982; 1986) p o i n t e d o ut t h a t t h e r a t e o f p r o t e i n h y d r o l y s i s i n c l o s e d , t r a d i t i o n a l i n . v i t r o systems i s i n h i b i t e d by t h e a c c u m u l a t i o n o f l o w - m o l e c u l a r weight d i g e s t i o n p r o d u c t s i n t h e r e a c t i o n m i x t u r e . In our e x p e r i m e n t s , even w i t h c a s e i n , which i s t o t a l l y d i g e s t e d i n 55 v i v o (Beantea & Eggum, 1981), o n l y 0.80 mL 0.10N NaOH was r e q u i r e d t o t i t r a t e 15 mg c a s e i n N o v e r a 10-minute p e r i o d , w i t h l i t t l e h y d r o l y s i s o c c u r r i n g a f t e r t h i s t i m e . I t was e s t i m a t e d t h a t complete h y d r o l y s i s would r e q u i r e a p p r o x i m a t e l y 3.01 mL 0.10 N NaOH. The f a c t t h a t t h e r e was no re s p o n s e t o a d d i t i o n o f enzyme a f t e r 10 minutes but t h a t f u r t h e r a d d i t i o n o f c a s e i n i n i t i a t e d an approach towards a new e q u i l i b r i u m w i t h each s u c c e s s i v e i n c r e m e n t s u g g e s t e d a p r o d u c t i n h i b i t i o n (caused by end-product a c c u m u l a t i o n i n t h e c l o s e d r e a c t i o n v e s s e l ) r a t h e r t h a n a reduced enzyme a c t i v i t y . The a p p r o x i m a t e l y p r o p o r t i o n a l r e s p o n s e i n NaOH r e q u i r e m e n t t o a t w o - f o l d i n c r e a s e i n c o n c e n t r a t i o n o f s u b s t r a t e s u p p o r t e d t h e f a c t t h a t a l a r g e s u r p l u s o f enzyme was p r e s e n t . I n h i b i t i o n i s not n e c e s s a r i l y a problem i n a p r o t e i n d i g e s t i b i l i t y t e s t p r o v i d i n g t h e r e i s , i n g e n e r a l , a s a t i s f a c t o r y c o n s t a n t r e l a t i o n s h i p between t h e i n i t i a l r a t e o f h y d r o l y s i s i n a c l o s e d system and an i n . v i v o r e s u l t . Such a r e l a t i o n s h i p was shown by Pedersen and Eggum (1983) f o r t h e p H - s t a t method. F u r t h e r m o r e , i t i s p o s s i b l e and d e s i r a b l e t o e s t i m a t e p r o t e i n q u a l i t y by u s i n g a r e l a t i v e l y s h o r t and t h u s i n c o m p l e t e e n z y m a t i c h y d r o l y s i s (Menden & Cremer, 1966). W i t h i n p r o t e i n s , some p e p t i d e bonds a r e l e s s s u s c e p t i b l e t o enzymes th a n o t h e r s , so t h a t some amino a c i d s a r e r e l e a s e d r a p i d l y w h i l e o t h e r s a r e s l o w l y d i g e s t e d ( R o b b i n s , 1978). Thus, t o e v a l u a t e p r o t e i n q u a l i t y w i t h an in . v i t r o d i g e s t i o n , t h e p r o t e o l y s i s must be stopped when d i f f e r e n c e s i n h y d r o l y s i s r a t e o f amino a c i d s a r e s t i l l e v i d e n t , as demonstrated w i t h p r o t e i n s such as c a s e i n and soybean (Vachon e_t a l . . , 1983). The r e l a t i v e l y i n c o m p l e t e h y d r o l y s i s t h a t r e s u l t e d w i t h t h e sodium c a s e i n a t e i n comparison t o t h e amino a c i d m i x t u r e may have been due p a r t l y t o t h e i n a c c e s s i b i l i t y o£ t h e p e p t i d e bonds i n t h e c a s e i n a t e . Green and Neurath (1954) r e c o g n i z e d t h a t t h e r a t e o f p r o t e o l y s i s depends on t h e c o n f o r m a t i o n o f t h e p r o t e i n s u b s t r a t e . I t i s w e l l known t h a t i n a p e p t i d e c h a i n , p r o t e o l y s i s depends not o n l y on t h e p r i m a r y s t r u c t u r e around t h e bonds s u s c e p t i b l e t o e n z y m a t i c a t t a c k but a l s o on t h e s econdary and t e r t i a r y s t r u c t u r e o f t h e p e p t i d e c h a i n ( R u p l e y , 1967). In f a c t , t h e f o l d i n g o f t h e p e p t i d e c h a i n may reduce t h e s u s c e p t i b i l i t y t o p r o t e o l y s i s o f p e p t i d e bonds which a r e e a s i l y s p l i t when t h e c h a i n i s u n f o l d e d (Ronca e_t a l . . 1975). With c a s e i n a t e , i t may be t h a t t h e exposed p e p t i d e bonds a r e r a p i d l y h y d r o l y z e d (which c o i n c i d e s w i t h t h e l a r g e amount o f base t h a t i s added d u r i n g t h e f i r s t two minutes f o l l o w i n g enzyme a d d i t i o n ) , w h i l e c l e a v a g e o f t h e i n t e r n a l bonds may be h i n d e r e d because o f t h e i r r e l a t i v e i n a c c e s s i b i l i t y t o t h e m i x t u r e o f p r o t e o l y t i c enzymes used. Indeed, t h e p H - s t a t method was d e v e l o p e d t o overcome t h e c o n f o u n d i n g e f f e c t o f t h e r e d u c t i o n o f pH on enzyme a c t i v i t y ( Haldane, 1965) and, t h u s , on t h e r e l e a s e o f amino a c i d s i n t h e method o f Hsu e t a l . (1977). 57 CHAPTER 4 EXPERIMENT 2 4.0 The E f f e c t , o f I n c l u s i o n o f N o n - p r o t e i n D i e t a r y  Components on In v i t r o ( p H - s t a t ) P r o t e i n D i g e s t i b i l i t y  o f Wheat and Raw. F a t - e x t r a c t e d Soybeans. 4.1 I n t r o d u c t i o n The i n . v i t r o e n z y m a t i c p H - s t a t p r o c e d u r e f o r p r e d i c t i n g p r o t e i n d i g e s t i b i l i t y , based on a 10-minute i n c u b a t i o n o f a p r o t e i n s u s p e n s i o n w i t h a three-enzyme s o l u t i o n , as used by Pedersen and Eggum (1983), i s a p o t e n t i a l r o u t i n e method f o r c h a r a c t e r i z i n g a n i m a l f e e d s . P r e l i m i n a r y i n v e s t i g a t i o n s showed t h a t t h e e x t e n t t o which a t e s t p r o t e i n i s d i g e s t e d ( t h e amount o f base r e q u i r e d t o m a i n t a i n pH c o n s t a n t ) i s m o d i f i e d by t h e p r e s e n c e o f n o n - p r o t e i n components. The a d d i t i o n o f m i n e r a l s , v i t a m i n s and a n i t r o g e n - f r e e m i x t u r e as p r e s e n t i n r a t , p r o t e i n - e v a l u a t i o n d i e t s , changes t h e amount o f NaOH r e q u i r e d t o m a i n t a i n pH 8.00 when t h e p r o t e i n s o u r c e i s s u b j e c t e d t o enzyme h y d r o l y s i s . T h i s s t u d y was d e s i g n e d t o examine t h e e f f e c t o f each o f t h e n o n - n i t r o g e n m i x t u r e s ( n i t r o g e n - f r e e , m i n e r a l , v i t a m i n ) s i n g l y and i n c o m b i n a t i o n on i n . v i t r o True D i g e s t i b i l i t y (TD) e s t i m a t e s by t h e p H - s t a t method. 58 4.2 M a t e r i a l s and methods 4.2.1 E x p e r i m e n t a l D e s ign For each o f f o u r p r o t e i n s o u r c e s (wheat, soybean, a u t o c l a v e d wheat, a u t o c l a v e d s o y b e a n ) , a 2 U F a c t o r i a l e x p e r i m e n t (k = 3; n i t r o g e n - f r e e m i x t u r e , v i t a m i n m i x t u r e and m i n e r a l m i x t u r e ) was d e s i g n e d t o g i v e e i g h t d i g e s t i o n m i x t u r e s . N i t r o g e n c o n t e n t was t h e same f o r a l l d i g e s t i o n m i x t u r e s . Each sample was d i g e s t e d i n d u p l i c a t e w i t h i n each p r o t e i n s o u r c e . To t e s t f o r d i f f e r e n c e s between t h e e i g h t t r e a t m e n t s , s i n g l e degree o f freedom a n a l y s i s was used. 4.2.2 Sample P r e p a r a t i o n and D i g e s t i o n Four p r o t e i n s o u r c e s , wheat and soybeans, b o t h raw and a u t o c l a v e d , were used. The wheat was ground t h r o u g h a 1 mm s c r e e n , t h e n t h r o u g h a 0.18 mm (SO mesh) s c r e e n . The soybeans ( f u l l - f a t ) were ground t h r o u g h a 2.7 mm s c r e e n , t h e n a 1.0 mm s c r e e n , and the n f a t - e x t r a c t e d w i t h p e t r o l e u m e t h e r and f i n a l l y passed t h r o u g h a 0.18 mm (SO mesh) s c r e e n . Coarse g r i n d i n g ( £ 1 mm) was done i n a C h r i s t i e and N o r r i s hammermill, w h i l e f i n e g r i n d i n g (0.18 mm) was done w i t h a Udy T e c a t o r - C y c l o t e c L a b o r a t o r y M i l l . A u t o c l a v i n g was done a f t e r t h e f i n e g r i n d i n g a t 121™ C, 1.5 kg/cm a f o r 2 h o u r s . For each p r o t e i n s o u r c e (raw wheat, a u t o c l a v e d wheat, raw f a t - e x t r a c t e d soybeans, and a u t o c l a v e d f a t - e x t r a c t e d s o y b e a n ) , e i g h t c o m b i n a t i o n s were p r e p a r e d by t h e a d d i t i o n o r not o f a n i t r o g e n - f r e e m i x t u r e , a v i t a m i n m i x t u r e and a m i n e r a l 59 m i x t u r e . P r o p o r t i o n s i n t h e complete m i x t u r e s were: wheat 76, n i t r o g e n - f r e e mix 46, m i n e r a l s 5, v i t a m i n s 1; soybeans 32, n i t r o g e n - f r e e mix 88, m i n e r a l s 5, v i t a m i n s 1. These r a t i o s were m a i n t a i n e d i n t h e i n c o m p l e t e m i x t u r e s . N i t r o g e n c o n t e n t o f a l l f o u r p r o t e i n s o u r c e s and o f a l l m i x t u r e s , a f t e r p r e p a r a t i o n , was de t e r m i n e d by t h e m a c r o - K j e l d a h l ( A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s , 1975) method. The c o m p o s i t i o n s o f t h e n i t r o g e n - f r e e and t h e m i n e r a l m i x t u r e s were t h e same as t h o s e used by Eggum (1973) and a r e g i v e n i n T a b l e s 7 and 9, r e s p e c t i v e l y . The v i t a m i n m i x t u r e was t h e AIN 76 f o r m u l a ( T a b l e 8 ) . 4.2.3 Enzymes The enzymes used i n t h e p H - s t a t method were mentioned i n Experiment 1. 4.2.4 In v i t r o p r o c e d u r e The p H - s t a t method as d e s c r i b e d by Pedersen and Eggum (1983) was used. D e t a i l s were g i v e n i n Experiment 1. 4.2.5 pH e l e c t r o d e maintenance D e t a i l s o f e l e c t r o d e maintenance were g i v e n i n Experiment 1. 4.3 R e s u l t s The a b s o l u t e q u a n t i t i e s o f 0.10 N NaOH added over t h e 10-minute d i g e s t i o n p e r i o d and t h e c a l c u l a t e d TD v a l u e s a r e T a b l e 7. C o m p o s i t i o n o f n i t r o g e n - f r e e m i x t u r e <g/kg N - f r e e mix on a d r y matter b a s i s ) . 89.2 g cane sugar 52.0 g c e l l u l o s e powder 52.0 g soybean o i l 806.7 g p o t a t o s t a r c h ( a u t o c l a v e d ) T a b l e 8. C o m p o s i t i o n o f v i t a m i n m i x t u r e (g/kg v i t a m i n mix on a d r y matter b a s i s ) . 0.600 9 Thiamin HC1 0.600 g R i b o f l a v i n 0.700 g P y r i d o x i n e HC1 3.0 g N i c o t i n i c a c i d 1.6 g D-Calcium P a n t o t h e n a t e 0.200 g F o l i c a c i d 0.020 g D - B i o t i n 0.001 g Cyanocobalamin 0.8 g R e t i n y l p a l m i t a t e pre-mix 20.0 g d l - a - T o c o p h e r y l a c e t a t e p r e 0.0025 g C h o l e c a l c i f e r o l 0.005 g Menaquinone 972.9 g S u c r o s e , f i n e l y powdered 61 T a b l e 9. C o m p o s i t i o n o f m i n e r a l m i x t u r e (g/kg m i n e r a l mix on a d r y matter b a s i s ) . 68.6 g c a l c i u m c a r b o n a t e 308.3 g c a l c i u m c i t r a t e 112.8 g c a l c i u m hydrogen phosphate 218.8 g p o t a s s i u m hydrogen phosphate, secondary 124.7 g p o t a s s i u m c h l o r i d e 77.1 g sodium c h l o r i d e 38.3 g magnesium s u l p h a t e 35.2 g magnesium c a r b o n a t e 15.3 g ammonium f e r r i c i t r a t e 0.201 g manganese s u l p h a t e 0.078 g c u p r i c s u l p h a t e 0.041 g p o t a s s i u m i o d i d e 0.507 g sodium f l u o r i d e 0.090 g aluminum ammonium s u l p h a t e 62 l i s t e d i n T a b l e 10. G r a p h i c a l r e p r e s e n t a t i o n s o f t h e a b s o l u t e e f f e c t and p e r c e n t a g e change i n NaOH a d d i t i o n as a f f e c t e d by the i n c l u s i o n o f n i t r o g e n - f r e e mix, m i n e r a l mix and v i t a m i n s i n comparison w i t h t h e p r o t e i n s o u r c e o n l y a r e seen i n F i g u r e s 7 and 8, r e s p e c t i v e l y . For t h e wheat t r e a t m e n t s , t h o s e t r e a t m e n t s c o n t a i n i n g a m i n e r a l m i x t u r e r e q u i r e d s i g n i f i c a n t l y more (p<0.001) NaOH and had a h i g h e r TD th a n t h o s e t r e a t m e n t s not c o n t a i n i n g t h e m i n e r a l m i x t u r e . T h i s e f f e c t was even g r e a t e r w i t h a u t o c l a v e d wheat. With raw soybeans, t h e d i s t i n c t i o n between t r e a t m e n t s was l e s s w e l l - d e f i n e d . The t r e a t m e n t s c o n t a i n i n g v i t a m i n and n i t r o g e n - f r e e / m i n e r a l m i x t u r e s used s i g n i f i c a n t l y more (p<0.05) NaOH d u r i n g d i g e s t i o n than t h e raw soybeans a l o n e . With a u t o c l a v e d soybeans, a d d i t i v e s had no e f f e c t . 4.4 D i s c u s s i o n From t h e r e s u l t s o b t a i n e d w i t h t h e two soybean p r o t e i n s o u r c e s , i t i s e v i d e n t t h a t t h e i n v i t r o d i g e s t i b i l i t y o f t h e p r o t e i n i t s e l f had a major e f f e c t on t h e res p o n s e o f a d d i t i o n a l m i x t u r e s . S i n c e raw soybeans c o n t a i n soy p r o t e i n s i n t h e i r n a t i v e form which a r e r e l a t i v e l y r e s i s t a n t t o p r o t e o l y s i s (Fukushima, 1969), t h e a b i l i t y o f t h e enzymes t o p e n e t r a t e and h y d r o l y z e t h e compact and r i g i d p r o t e i n s t r u c t u r e i s m i n i m i z e d . C o n s e q u e n t l y , t h e e f f e c t o f any a d d i t i o n ( e . g . m i n e r a l s ) i s more e a s i l y seen w i t h p o o r l y d i g e s t i b l e p r o t e i n s such as raw soybeans. I n c o n t r a s t , s i n c e t h e i n t e r n a l p r o t e i n s i n t h e 63 TABLE 10. fibsolute(a) and relative(b) results obtained with the 8 dietary treatments for each of the 4 protein sources. Added diet component n i l N-free N-free N-free Mineral Vitamin Mineral fill SEM(d) * vitamin * mineral * Vitamin Protein Source wheat 0.453 0.445 0.444 0.492 0.492 0.470 0.500 0.4% 0.005 100.0 98.3 98.2 108.7 108.6 103.9 110.5 109.6 wheat 0.282 0.299 0.288 0.349 0.346 0.294 0.353 0.360 0.003 (autoclaved)c 100.0 106.2 102.0 123.6 122.9 104.3 125.0 127.6 soybeans 0.092 0.094 0.094 0.100 0.098 0.105 0.101 0.101 0.002 100.0 101.6 100.0 107.8 106.2 113.5 108.7 109.5 soybeans 0.522 0.539 0.556 0.540 0.538 0.541 0.522 0.541 0.008 (autoclaved) 100.0 103.4 106.7 103.6 103.1 103.6 100.1 103.7 a - ml NaOH consumed/15 ag N/10 min.; mean of 2 values for each treatment b - relative percentages of NaOH consumption within protein sources (assuming value for no addition for each protein source = 100%) c - autoclaving conditions; 121 C, 1.5 kg per square cm, 2 hours d - standard error of the mean 64 o.6 r AUTOCLAVED SOYBEANS F i g u r e 7. The a b s o l u t e e f f e c t on NaOH a d d i t i o n as a f f e c t e d by the i n c l u s i o n o f n i t r o g e n - f r e e mix <NF), m i n e r a l mix (M) and v i t a m i n s (V) i n comparison w i t h the p r o t e i n s o u r c e o n l y . 130 AUTOCLAVED WHEAT CO CO O N F M V O N F M V O N F M V O N F M V F i g u r e 8. The percentage change i n NaOH a d d i t i o n as a f f e c t e d by t h e i n c l u s i o n n i t r o g e n - f r e e mix (NF>, m i n e r a l mix <M> and v i t a m i n s (V) i n comparison w i t h t h e p r o t e i n s o u r c e o n l y . n a t i v e s t r u c t u r e have been made more a c c e s s i b l e t o enzymes by h e a t i n g , i t i s more d i f f i c u l t t o see t h e e f f e c t s o f a d d i t i o n s ( e . g . m i n e r a l s ) w i t h a h i g h l y d i g e s t i b l e p r o t e i n s o u r c e such as a u t o c l a v e d soybeans. These e x p e r i m e n t s show t h a t , i n g e n e r a l , t h e p r e s ence o f t h e m i n e r a l m i x t u r e i n c r e a s e s t h e r a t e o f p r o t e i n h y d r o l y s i s . M e t a l i o n s a f f e c t p r o t e o l y t i c enzymes i n v a r i o u s ways. C a l c i u m i o n s have been shown t o s t a b i l i z e t r y p s i n and c h y m o t r y p s i n , p r e v e n t a u t o l y s i s and even i n c r e a s e t h e a c t i v i t y o f enzymes (Green and N e u r a t h , 1954). Pedersen and Eggum (1983) found t h a t t h e p H - s t a t i n . v i t r o e s t i m a t i o n was s i g n i f i c a n t l y i n f l u e n c e d by t h e c a l c i u m c o n c e n t r a t i o n o f t h e s u s p e n s i o n . They r e p o r t e d t h a t t h e a d d i t i o n o f 5 and 8 mg o f c a l c i u m i n c r e a s e d t h e p r e d i c t e d TD v a l u e s by 3 t o 4 f . Our r e s u l t s a r e i n g e n e r a l agreement w i t h t h e e f f e c t o f c a l c i u m as i t i s t h e most predominant m i n e r a l c o m p r i s i n g t h e m i n e r a l m i x t u r e . Copper may e l e v a t e o r lower t h e a c t i v i t y o f t r y p s i n ( K i r c h g e s s n e r e t a l . . , 1980). The former o c c u r s when t h e t r a c e element copper i s added i n v e r y s m a l l amounts. The l a t t e r o c c u r s a t h i g h e r c o n c e n t r a t i o n s . The a d d i t i o n o f manganese t o a s u s p e n s i o n p r e v i o u s l y d e f i c i e n t i n t h i s element o r z i n c , has been known t o a c t i v a t e t h e enzyme a m i n o p e p t i d a s e (Smith e t a l . . 1983). B u f f e r i n g c a p a c i t y may be i n c r e a s e d as t h e ash c o n t e n t i s i n c r e a s e d (Hsu e t a l ^ . , 1977). However, t h e problem o f b u f f e r i n g i s not a major c o n c e r n w i t h t h e p H - s t a t method. C o n s e q u e n t l y , pure p r o t e i n samples cause o n l y a n e g l i g i b l e 67 amount o f b u f f e r i n g i n t h e pH-atat e s t i m a t i o n s o f p r o t e i n d i g e s t i b i l i t y because t h e p H - s t a t a u t o m a t i c a l l y adds base t o t h e r e a c t i o n m i x t u r e as t h e pH s t a r t s t o d e c r e a s e , t h u s k e e p i n g the pH c o n s t a n t . By k e e p i n g pH c o n s t a n t , t h e e f f e c t s o f c h a n g i n g pH on t h e p r o t e o l y t i c enzyme a c t i v i t y a r e removed and, a c c o r d i n g t o S t i n s o n and Snyder (1980), b u f f e r i n g by newly r e l e a s e d amino groups i s e l i m i n a t e d . With t h e p H - s t a t method (O'Hare e t a l . , 1984) an i n c r e a s e i n s u b s t r a t e c o n c e n t r a t i o n l e a d s t o a p r o p o r t i o n a l i n c r e a s e i n p e p t i d e bond c l e a v a g e w h i l e t h e pH-drop method d e m o n s t r a t e s an a p parent d e c r e a s e i n c l e a v a g e per mg p r o t e i n . The e x p l a n a t i o n g i v e n by t h e s e a u t h o r s i s t h a t t h e d e t e c t i o n o f bond c l e a v a g e i s masked i n t h e pH-drop method due t o i n c r e a s e d b u f f e r i n g c a p a c i t y a t h i g h e r p r o t e i n c o n c e n t r a t i o n s . With r e s p e c t t o t h e e f f e c t s o f t h e n i t r o g e n - f r e e o r t h e v i t a m i n m i x t u r e s on t h e amount o f base r e q u i r e d t o m a i n t a i n a pH o f 8, no s i g n i f i c a n t i n c r e a s e was a p p arent w i t h t h e d i f f e r e n t p r o t e i n s o u r c e s ( e x c e p t i o n o f raw soybeans which showed a s i g n i f i c a n t i n c r e a s e w i t h t h e a d d i t i o n o f v i t a m i n s ) . No e x p l a n a t i o n i s o f f e r e d f o r t h i s l a t t e r r e s p o n s e . A c c o r d i n g t o a r e c e n t s t u d y by Hagan and V i l l o t a ( 1 9 8 7 ) , t h e p r e s e n c e o f c e r t a i n n o n - p r o t e i n s u b s t a n c e s can e i t h e r enhance o r i n h i b i t t h e r a t e o f h y d r o l y s i s o f soy p r o t e i n s , depending on t h e e n z y m e / s u b s t r a t e r a t i o . These r e s e a r c h e r s found t h a t t h e p r e s ence o f c a r b o h y d r a t e s enhanced the soy p r o t e i n p e p t i c h y d r o l y s i s when the e n z y m e / s u b s t r a t e was 0.5%. However, a t e n z y m e / s u b s t r a t e o f 3.5%, h y d r o l y s i s was 68 i n h i b i t e d by c a r b o h y d r a t e s . The p H - s t a t t e c h n i q u e uses o n l y p r o t e o l y t i c enzymes. T h i s g i v e s l e s s complete h y d r o l y s i s than would be a c h i e v e d w i t h the i n c l u s i o n o f p a n c r e a t i n i n t h e enzyme m i x t u r e ( e . g . G a u t h i e r e t a l _ . , 1986) where bonds i n v o l v i n g g l y c o s i d e s and phosphates a r e c l e a v e d t o g i v e more complete p r o t e i n d i g e s t i o n . However, p a n c r e a t i n may produce a c i d s due t o t h e e f f e c t o f l i p a s e s p r e s e n t and t h u s a f f e c t t h e i n t e r p r e t a t i o n o f t h e pH r e s u l t s . Another major c o n c e r n w i t h t h e p H - s t a t method i s t h e magnitude o f t h e " a " v a l u e i n r e l a t i o n t o "b" i n t h e e q u a t i o n TD = a + bx. P r e l i m i n a r y r e s u l t s i n t h i s l a b o r a t o r y s u g g e s t t h a t d i f f e r e n t e q u a t i o n s s h o u l d be used f o r v a r i o u s c a t e g o r i e s o f p r o t e i n s . 69 CHAPTER 5 EXPERIMENT 3 5.0 A comparison o f p r o t e i n d i g e s t i b i 1 i t l e a o f v a r i o u s f e e d s  as measured by t h e p H - s t a t method and by male W i s t a r r a t s 5.1 I n t r o d u c t i o n The i n . v i t r o e n z y m a t i c p H - s t a t method f o r p r e d i c t i n g p r o t e i n d i g e s t i b i l i t y , based on a 10-minute i n c u b a t i o n o f a p r o t e i n s u s p e n s i o n w i t h a three-enzyme s o l u t i o n , as used by Pedersen and Eggum (1983), i s a p o t e n t i a l r o u t i n e and r a p i d t e c h n i q u e f o r c h a r a c t e r i z i n g a n i m a l f e e d s . I n o r d e r t o v e r i f y r e s u l t s o f i n . v i t r o s t u d i e s , s e v e r a l r e s e a r c h e r s have compared t h e i r r e s u l t s w i t h f i g u r e s o b t a i n e d i n t e s t s w i t h a n i m a l s t u d i e s (Pedersen and Eggum, 1983; Wolzak e t a l _ . , 1981; B o d w e l l e t a l . . 1980; M a r s h a l l e t a l . , 1979). In so d o i n g , t h e y have d e v e l o p e d r e g r e s s i o n e q u a t i o n s a l l o w i n g t h e p r e d i c t i o n o f p r o t e i n d i g e s t i b i l i t y . Once d e v e l o p e d , t h e e q u a t i o n s a l l e v i a t e t h e need f o r t h e l a b o r i o u s but p r e v i o u s l y e s s e n t i a l methods which r e q u i r e t h e f e e d i n g o f a n i m a l s . However, t h e need f o r s p e c i f i c e q u a t i o n s f o r d i f f e r e n t groups o f p r o t e i n s has been s t r e s s e d i n d i f f e r e n t i n . v i t r o s t u d i e s which used multi-enzyme methods (Wolzak e t a l . . , 1981; B o d w e l l et. a l . , 1980; M a r s h a l l e t a l . . 1979). A c c o r d i n g t o Pedersen and Eggum (1983), t h e pH-s t a t p r o c e d u r e does not r e q u i r e t h e use o f s p e c i f i c p r o t e i n c a t e g o r y r e g r e s s i o n e q u a t i o n s . However, i n t h e i r r e s u l t s t h e r e s t i l l appeared t o be d i f f e r e n c e s i n t h e s u s c e p t i b i l i t y t o 70 p r o t e o l y s i s between d i f f e r e n t p r o t e i n c a t e g o r i e s . T h i s s t u d y was d e s i g n e d t o d e v e l o p r e g r e s s i o n e q u a t i o n s f o r s e v e r a l p l a n t - p r o t e i n c a t e g o r i e s v i z . sorghum-based d i e t s , sorghum- and t r i t i c a l e - b a s e d d i e t s , g r a i n - b a s e d d i e t s , a l f a l f a h a y / b a r l e y - b a s e d c o m b i n a t i o n d i e t s , soybean-based d i e t s , and a l f a l f a - b a s e d d i e t s were used. For c o m p a r a t i v e p u r p o s e s , t h e a b s o l u t e v a l u e s (ml NaOH) o b t a i n e d w i t h t h e p H - s t a t were used not o n l y t o d e v e l o p new e q u a t i o n s , but were used i n t h e s i n g l e (common) r e g r e s s i o n s u g g e s t e d f o r a l l p l a n t p r o t e i n s by Pedersen and Eggum (19S3) and t h e one g e n e r a l e q u a t i o n s u g g e s t e d f o r a l l p r o t e i n t y p e s by Pedersen and Eggum (1983). 5.2 M a t e r i a l s and methods 5.2.1 Enzymes The enzymes used f o r i n v i t r o d e t e r m i n a t i o n s were d i s c u s s e d i n Experiment 1 (Chapter 3 ) . 5.2.2 P r e p a r a t i o n o f p r o t e i n s o u r c e s ( d i e t s ) f o r p H - s t a t  t r i a l s The r e f e r e n c e p r o t e i n f o r t h e p H - s t a t d i g e s t i o n s (sodium c a s e i n a t e ) was purchased from Sigma Chemical Company. Sample p r e p a r a t i o n f o r t h e e n z y m a t i c i n v i t r o d i g e s t i o n p r o c e d u r e r e q u i r e d t h e f i n e r g r i n d i n g (80 mesh or 0.18 mm) o f a subsample t a k e n o f t h e c o a r s e r r a t d i e t s (18 mesh or 1 mm). G r i n d i n g f o r t h e i n . v i t r o p r o c e d u r e s was done i n a Udy Cy c l o n e Sample M i l l which was f i t t e d w i t h an 80 mesh s c r e e n . 71 M a c r o - K j e l d a h l (AOAC, 1975) a n a l y s e s were done on samples a f t e r f i n e - g r i n d i n g t o d e t e r m i n e n i t r o g e n p e r c e n t a g e . The weight o f d i e t c o n t a i n i n g 150 mg N was used f o r each p H - s t a t d i g e s t i o n . 5.2.3 In v i t r o p r o c e d u r e The p H - s t a t method as d e s c r i b e d by Pedersen and Eggum (1983) was used w i t h some m o d i f i c a t i o n s as d e s c r i b e d i n Experiment 1. 5.2.4 pH e l e c t r o d e maintenance Maintenance o f e l e c t r o d e s i s d e t a i l e d i n Experiment 1. 5.2.5 C a l c u l a t i o n o f i n v i t r o t r u e d i g e s t i b i l i t y I n v i t r o TD was c a l c u l a t e d f i r s t l y , a c c o r d i n g t o t h e r e g r e s s i o n e q u a t i o n g i v e n by Pedersen and Eggum (1983) f o r p l a n t p r o t e i n s e x c e p t f o r t h e x v a l u e which was d i v i d e d by 1.5, i n t h e p r e s e n t e x p e r i m e n t s , s i n c e 15 ml o f s u s p e n s i o n was used i n s t e a d o f 10 ml. I t was assumed t h a t t h e p r o t e i n s i n both s t u d i e s were d i g e s t e d t o t h e same e x t e n t s i n c e t h e s u b s t r a t e : enzyme r a t i o s were t h e same. The e q u a t i o n used was: TD = 0.7861 * 0.2686x (where x i s ml o f O.IO N NaOH consumed by t h e 15 ml s u s p e n s i o n d u r i n g 10 minutes o f d i g e s t i o n ) . S i m i l a r l y , TD was a l s o c a l c u l a t e d u s i n g t h e r e g r e s s i o n e q u a t i o n d e v e l o p e d f o r a l l p r o t e i n s (Pedersen and Eggum, 1983). A g a i n , t h e x v a l u e has been d i v i d e d by 1.5 t o y i e l d : TD = 0.7614 + 0.3185X A f i n a l method f o r c a l c u l a t i n g i n . v i t r o TD i n v o l v e d t h e 72 development and use o f s p e c i f i c r e g r e s s i o n e q u a t i o n s f o r each p r o t e i n t y p e which was s t u d i e d ( T a b l e 17) i n t h e s e e x p e r i m e n t s . 5.2.6 Rat t r i a l s R e s u l t s were o b t a i n e d from r a t p r o t e i n e v a l u a t i o n t r i a l s i n which t r u e d i g e s t i b i l i t y , b i o l o g i c a l v a l u e and net p r o t e i n u t i l i z a t i o n were measured. Complete d i e t samples were r e t a i n e d and used i n t h e p H - s t a t p o r t i o n o f t h i s e x p e r i m e n t . The e x p e r i m e n t a l p r o c e d u r e has been d e s c r i b e d by Eggum (1973). The r o u t i n e s t e p s a r e summarized i n Appendix 2. Groups o f f i v e W i s t a r male r a t s w e i g h i n g a p p r o x i m a t e l y 68 g were used i n each e x p e r i m e n t w i t h p r e l i m i n a r y p e r i o d s o f 4 days and b a l a n c e p e r i o d s o f 5 days. Each r a t was a s s i g n e d t o a s e p a r a t e m e t a b o l i c cage and f e d 10 g DM once d a i l y ( c o n t a i n i n g 150 mg N). The a n i m a l s were g i v e n water ad l i b i t u m . The r e l a t i v e h u m i d i t y and a i r t e m p e r a t u r e were kept c o n s t a n t t h r o u g h o u t t h e 9 day t r i a l (73X, and 24,=* C , r e s p e c t i v e l y ) . The h u m i d i t y and t e m p e r a t u r e were m o n i t o r e d w i t h a Hygrothermograph (Model 594) r e c o r d i n g i n s t r u m e n t . The room was equipped w i t h an a u t o m a t i c 12-hour l i g h t - d a r k c y c l e (0600 t o 1800 hours) . A l t h o u g h TD, BV, and NPU were measured, o n l y TD was used t o compare w i t h i n v i t r o d i g e s t i o n r e s u l t s . D u r i n g t h e 5-day t e s t p e r i o d , a l l f e c e s and u r i n e were c o l l e c t e d . U r i n e was c o l l e c t e d t h r o u g h a g l a s s w o o l - f i 1 t e r e d f u n n e l . Both t h e u r i n e and f e c a l f l a s k s c o n t a i n e d a 5% s u l f u r i c a c i d s o l u t i o n t o i n h i b i t m i c r o b i a l growth. The 73 u r i n e f l a s k c o n t a i n e d 50 ml a c i d w h i l e t h e f e c e s f l a s k c o n t a i n e d 100 ml a c i d . Any f e e d r e s i d u e s were weighed and r e c o r d e d so t h a t an a d j u s t m e n t i n t h e c o r r e c t i o n f a c t o r ( f o r f e c e s ) c o u l d be made. At t h e end o f t h e e x p e r i m e n t , t h e r a t s were e u t h a n i z e d i n a c a r b o n d i o x i d e - s a t u r a t e d atmosphere. 5.2.7 P r e p a r a t i o n o f complete d i e t s f o r r a t t r i a l s Each complete d i e t was c o m p r i s e d o f v a r i o u s c o m b i n a t i o n s o f p r o t e i n , a b a s a l m i n e r a l m i x t u r e , a b a s a l v i t a m i n m i x t u r e and v a r y i n g amounts o f n i t r o g e n - f r e e m i x t u r e (depending on t h e n i t r o g e n c o n t e n t o f t h e p r o t e i n s o u r c e ( s ) i n a p a r t i c u l a r d i e t (see T a b l e s 7, 8 and 9 f o r c o m p o s i t i o n t a b l e s o f v i t a m i n , m i n e r a l , and n i t r o g e n - f r e e m i x t u r e s ) . C o m p o s i t i o n f o r m u l a e o f d i e t s a r e g i v e n i n T a b l e s 11 t o 16, i n c l u s i v e . A f t e r t h e N c o n t e n t o f each o f t h e p r o t e i n s o u r c e s c o m p r i s i n g a p a r t i c u l a r t r e a t m e n t ( d i e t ) was known, t h e i n d i v i d u a l i n g r e d i e n t s a l o n g w i t h t h e n i t r o g e n - f r e e components were mixed i n an Erweka c u b e - s h e l l b l e n d e r f o r 7 m i n u t e s . M i x i n g f o r a l o n g e r t i m e r e s u l t e d i n s t a t i c a t t r a c t i o n s , t h u s , c a u s i n g a poor mix. Each d i e t was f o r m u l a t e d t o p r o v i d e 150 mg N and 10 g DM/rat/day. F i v e hundred grams DM o f each d i e t a r y m i x t u r e was p r e p a r e d . A f t e r t h e d i e t had been mixed t h o r o u g h l y , a N d e t e r m i n a t i o n was c o n d u c t e d . For each r a t , t h e d i e t was weighed i n t o 2 s e p a r a t e Rubbermaid Serve 0' S e a l c o n t a i n e r s (250 ml s i z e ) . The 4-day ( f o r t h e p r e - c o l l e c t i o n p e r i o d ) and 74 Table 11. Composition of sorghun diets (g/500 g diet on a dry natter basis) Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 g N/lOOg diet DM 1.39 1.37 1.37 1.41 1.38 1.38 Sorghum 459.86 — — — — — Sorghum<ft) — 476.00 — — — — Sorghum(120) — — 453.69 — — ' — Sorghum(S) — — — 476.00 — — Sorghum(90) — — — — 463.01 — Sorghum(iaO) — — — — — 466.21 N-free mixture 16.14 — 22.31 — 12.99 9.79 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 4.00 ft - autoclaving conditions; 121°C, 1.5 kg/cm squared, 2 hours. 120 - dry heated for 2 hr. in a forced-draft oven S - steamed for a 2 hour period 90 - dry heated for 2 hr. in a forced-draft oven 180 - dry heated for 2 hr. in a forced-draft oven 75 Table 12. Composition of barley diets (g/500 g diet on a dry ut t e r basis) Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet B Diet 9 Diet 10 g N/lOOg diet DM 1.50 1.52 1.47 1.52 1.51 1.48 1.54 1.55 1.47 1.54 Barley #1 309.09 - - - - - - - -Barley #2 - 274.30 _ _ _ _ _ _ _ _ Barley #3 - - 271.69 - - - - - - -Barley #4 - - - 303.49 - - - - - -Barley #5 - - - - 310.07 _ _ _ _ _ Barley «6 _ _ _ _ _ 271.38 - - - -Barley #7 _ _ _ _ _ _ 274.57 — - — Barley #8 _ _ _ _ _ _ _ 246.31 -Barley #9 _ _ _ _ _ _ _ _ 275,50 _ Barley #10 _ _ _ _ _ _ _ _ _ 329.69 N-free mixture 169.62 205.07 206.57 175.50 169.62 200.22 201.43 229.80 200.41 146.29 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Table 12 (cont'd) -J Components Diet 11 Diet 12 Diet 13 Diet 14 Diet 15 Diet 16 Diet 17 Diet 16 Diet 19 g N/lOOg diet DM 1.54 1.48 1.55 1.52 1.55 1.54 1.53 1.52 1.53 Barley #11 390.63 - - - - - - - -Barley #12 - 373.13 _ _ _ _ _ _ _ Barley #13 - - 460.04 - - _ _ - -Barley #14 - - - 298.83 _ _ _ _ _ Barley #15 - 270.78 -Barley #16 _ _ _ _ _ 382.53 - - -Barley #17 _ _ _ _ _ _ 419.09 - -Barley #18 _ _ _ _ _ _ _ 326.04 — Barley #19 _ _ _ _ _ _ _ _ 259.50 N-free mixture 85.37 102.87 15.96 177.17 205.22 93.42 56.91 149.% 216.50 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Table 13. Composition of triticale diets (g/500 g diet on a dry matter basis). Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 Diet 9 Diet 10 g N/lOOg diet DM 1.50 1.52 1.54 1.54 1.51 1.59 1.52 1.52 1.52 1.50 Triticale #1 289.57 _ _ _ _ _ _ _ _ _ Triticale #2 - 269.78 - - - - - - - -Triticale #3 - - 315.13 _ _ _ _ _ _ _ Triticale #4 - -- - 266.90 - - - - - -Triticale #5 - - — - 269.78 _ _ _ _ _ Triticale #6 - - — - - 286.26 — _ _ _ Triticale #7 _ _ _ _ _ _ 276.75 _ _ _ Triticale #8 — — — — — — — 264.08 — — Triticale #9 — _ _ _ _ _ _ _ 252.53 Triticale #10 — — — — — — — — — 264.08 N-free mixture 186.43 206.22 160.87 209.10 206.22 189.74 199.25 211.92 223.47 211.92 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Table 14. Composition of soybean/wheat diets (g/500 g diet on a dry natter basis) Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 g N/lOOg diet DM 1.49 1.52 1.53 1.56 1.50 1.51 1.49 1.52 Soybeans(A) 123.51 — — — 61.77 — 62.31 — Soybeans — 138.74 — — — 62.89 — — Wheat(fl) — — 293.54 — 144.14 — — — Wheat — — — 298.80 — 146.73 145.39 — (Soybeans and Wheat)A —~ 207.75 (62.33, 145.39) N-free mixture 354.63 352.45 182.46 177.19 205.91 266.39 268.30 268.25 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin aixture 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 A - autoclaving conditions; 121°C, 1.5 kg/cm squared, 2 hours. 79 Table 15. Composition of alfalfa pellet/hay/wheat diets (g/500 g diet on a dry matter basis) Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Diet 7 Diet 8 g N/lOOg diet DM 1.5b 1.54 1.58 1.50 1.53 1.57 1.56 1.55 Alfalfa Pellets 179.34 — — — — — — — Alfalfa Pellets(A) - 180.15 178.71 — — — — — Alfalfa Hay — — — — 164.06 — — — Alfalfa Hay(A) — — — — — 163.64 162.46 — Wheat 119.52 120.09 — — 109.33 109.09 — — Wheat(A) — — 119.31 — — — 108.45 — (Alfalfa Pellets and Wheat)A 298.80 (179.28, 119.52) — (Alfalfa Hay and Wheat)A —"~ 274.72 (164.83, 109.89) N-free mixture 177.14 175.70 178.08 177.13 202.61 203.20 205.18 201.21 Mineral mixture 20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 A - autoclaving conditions; 121°C, 1.5 kg/cm squared, 2 hours. 80 Table 16. Composition of alfalfa hay/barley diets (g/500 g diet on a dry matter basis). Components Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 g N/lOOg diet DM 1.52 1.49 1.51 1.48 1.50 Alfalfa Hay 169.17 — 166.67 — — Alfalfa Hay(A) — 167.29 — 165.44 — Barley 112.78 111.52 — — — Barley(A) — — 111.11 110.29 — (Alfalfa Hay and Barley)A 280.90 (168.54, 112.36) N-free mixture 194.05 197.19 198.22 200.26 195.10 Mineral mixture 20.00 20.00 20.00 20.00 20.00 Vitamin mixture 4.00 4.00 4.00 4.00 4.00 A - autoclaving conditions; 121°C, 1.5 kg/cm squared, 2 hours. 81 5-day ( b a l a n c e p e r i o d ) f e e d s were weighed i n t o s e p a r a t e c o n t a i n e r s . 5.2.8 P r e p a r a t i o n o f n i t r o g e n - f r e e m i x t u r e The p o t a t o s t a r c h which was t h e major i n g r e d i e n t i n t h e n i t r o g e n - f r e e m i x t u r e was t h o r o u g h l y mixed i n a Hobart mixer wi o n e - t h i r d p a r t water and then s p r e a d e v e n l y t o a depth o f 2 cm and a u t o c l a v e d i n a g l a s s Pyrex pan ( c o n d i t i o n s : 2 h r . , 121'=> C , 1.5 kg/cm 6 2) . On r e m o v a l , t h e t h i c k l i q u i d was a l l o w e d t o s e t s l i g h t l y , and was t h e n c u t i n t o cubes w i t h a k n i f e . These were a l l o w e d t o dry o v e r n i g h t i n a Gallenkamp f o r c e d - d r a f t d r y i n g oven a t 50,=>C. The d r i e d s t a r c h was t h e n ground i n t h e C h r i s t y and N o r r i s hammermill ( C h e l m s f o r d , England) t h r o u g h a 1 mm s c r e e n and was mixed i n t h e Hobart mixer w i t h t h e o t h e r components c o m p r i s i n g t h e n i t r o g e n - f r e e m i x t u r e . The amount o f n i t r o g e n - f r e e m i x t u r e added t o each d i e t was v a r i e d , depending on t h e amount o f t h e p r o t e i n component(s) t o ensure t h a t each o f t h e d i e t s was i s o n i t r o g e n o u s . P r i o r t o b e i n g mixed w i t h t h e o t h e r i n g r e d i e n t s , t h e p r o t e i n s o u r c e s were hammermilled i n t h e C h r i s t y and N o r r i s m i l l t h r o u g h a 1 mm s c r e e n . 5.2.9 A n a l y t i c a l methods A l l d r y matter and n i t r o g e n d e t e r m i n a t i o n s were done a c c o r d i n g t o s t a n d a r d methods (AOAC, 1975). A volume o f u r i n e (18 ml) was a n a l y z e d f o r n i t r o g e n c o n t e n t . The f e c e s s u s p e n s i o n was t r a n s f e r r e d i n t o pre-weighed 250 ml square 82 p o l y e t h y l e n e b o t t l e s and ground w i t h a Brinkmann P o l y t r o n Homogenizer (Model no. PT 10-35) w i t h Probe G e n e r a t o r (Model PT-20, s a w - t o o t h e d ) . A sample ( a p p r o x i m a t e l y 15 g) o f t h e r e s u l t a n t s l u r r y was weighed d i r e c t l y i n t o a t a r e d K j e l d a h l f l a s k f o r n i t r o g e n a n a l y s i s . TD v a l u e s were c a l c u l a t e d u s i n g a c o r r e c t i o n f a c t o r o f 102mg n i t r o g e n (Eggum, 1973) f o r t h e 5-day m e t a b o l i c f e c a l n i t r o g e n p r o d u c t i o n . 5.3 R e s u l t s The n e w l y - d e v e l o p e d r e g r e s s i o n e q u a t i o n s f o r t h e v a r i o u s p r o t e i n s t e s t e d a r e shown i n T a b l e 17. The 10-minute a b s o l u t e NaOH consumption, c o r r e s p o n d i n g TD v a l u e s u s i n g n ewly-developed e q u a t i o n s , t h e s i n g l e p l a n t -p r o t e i n e q u a t i o n d e v e l o p e d by Pedersen and Eggum (1983) and t h e u n i v e r s a l e q u a t i o n d e v e l o p e d by Pedersen and Eggum (1983) f o r a l l t y p e s o f p r o t e i n s and t h e mean in . v i v o ( r a t ) v a l u e s a r e g i v e n f o r each p r o t e i n t e s t e d i n T a b l e 18. The mean i n . v i v o TD v a l u e s , s t a n d a r d e r r o r s o f t h e means and % c r u d e p r o t e i n o f t h e p r o t e i n s o u r c e s a r e g i v e n i n Appendix 3. The p l o t s o f t h e r e g r e s s i o n r e l a t i o n s h i p s between i n  v i v o tTD and i n . v i t r o base consumption f o r sorghum-, a l f a l f a h a y / b a r l e y - , soybean-, a l f a l f a - , g r a i n - , and s o r g h u m / t r i t i c a l e -based d i e t s a r e shown i n F i g u r e s 9, 10, 11, 12, 13 and 14, r e s p e c t i v e l y . 5.4 D i s c u s s i o n As seen by t h e r e s u l t s o f t h e c o r r e l a t i o n c o e f f i c i e n t s i n T a b l e 17, i t i s a p parent t h a t a s e p a r a t e e q u a t i o n f o r each 83 T a b l e 17. R e g r e s s i o n e q u a t i o n s d e v e l o p e d and c o r r e l a t i o n c o e f f i c i e n t s (r> o b t a i n e d between in, v i t r o and i n  v i v o p r o t e i n d i g e s t i b i l i t i e s (TD> o f v a r i o u s d i e t s . S t a n d a r d e r r o r o f r e g r e s s i o n <s> and number o f d i e t s (n) per e q u a t i o n a r e a l s o s t a t e d . Soybean, soybean ( a u t o c l a v e d ) , soybean/wheat c o m b i n a t i o n s (n = 6) r = 0.93 TD = 0.7868 + 0.2175x s = 0.018 Sorghum (raw, a u t o c l a v e d , 90° C, 120™ C, 180° C d r y - h e a t e d , steamed) (n = 6) r = 0.92 TD = 0.4575 * 1.8841x s = 0.058 A l f a l f a p e l l e t s / h a y i n c o m b i n a t i o n w i t h e i t h e r wheat o r b a r l e y (n = 13) r = 0.91 TD = 0.3446 + 1.0356x a = 0.046 A l f a l f a hay and b a r l e y c o m b i n a t i o n s (n = 5) r = 0.96 TD = 0.2360 + 1.3194x s = 0.048 G r a i n s (19 b a r l e y s , 10 t r i t i c a l e s , 6 sorghums, and 2 wheats) (n = 37) r = 0.74 TD = 0.7419 + 0.4759x s = 0.044 8 4 T a b l e 18. A comparison o f i n . v i v o and i n v i t r o p r o t e i n d i g e s t i b i l i t y (TD) of v a r i o u s complete d i e t s , w i t h i n v i t r o TD v a l u e s c a l c u l a t e d i n 3 d i f f e r e n t ways. X i i V 1 v u P r o c e d u r e r a t XTD ml base JiTDl *TD2 JSTD3 added B a r l e y s No 1 87.31 0.388 92.65 89.03 88.50 No 2 92.86 0.345 90.61 87.88 87.13 No 3 93.55 0.345 90.61 87.86 87.11 No 4 89.25 0.378 92.18 88.76 88.17 No 5 90.28 0.383 92.42 88.90 88.34 No 6 90.80 0.373 91.94 88.64 88.03 No 7 93.66 0.399 93.18 89.31 88.83 No 8 96.46 0.472 96.65 91.28 91.17 No 9 97.99 0.496 97.79 91.93 91.94 No 10 93.84 0.358 91.23 88.21 87.53 No 11 93.47 0.385 92.51 88.95 88.40 No 12 91.18 0.333 90.04 87.55 86.75 No 13 95.98 0.568 101.22 93.87 94.23 No 14 91.61 0.349 90.80 87.98 87.26 No 15 94.05 0.388 92.65 89.03 88.50 No 16 91.49 0.371 91.85 88.58 87.96 No 17 89.84 0.394 92.94 89.19 88.69 No 18 91.51 0.347 90.70 87.93 87.19 No 19 91.44 0.388 92.65 89.03 88.50 TD1; TD = 0.7419 •»• 0.4759x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r g r a i n s . XTD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 + 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 + 0.3185x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . 85 T a b l e I S . c o n t ' d i n v i v o -P r o c e d u r e r a t %TD ml base added fcTDl %TD2 J4TD3 T r i t i c a l e s No 1 97.57 0.454 95.80 90.80 90.60 No 2 96.11 0.466 96.37 91.13 90.98 No 3 98.14 0.462 96.18 91.02 90.85 No 4 95.54 0.467 96.41 91.15 91.01 No 5 97.76 0.462 96.18 91.02 90.85 No 6 98.51 0.459 96.03 90.94 90.76 No 7 96.70 0.461 96.19 90.99 90.82 No S 95.15 0.473 96.70 91.31 91.21 No 9 96.74 0.436 94.94 90.32 90.03 No 10 97.26 0.408 93.61 89.57 89.13 Wheats No 1 95.94 0.504 98.18 92.13 92.18 No 2 92.95 0.513 98.60 92.39 92.48 TD1; TD = 0.7419 • 0.4759x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r g r a i n s . %TD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 + 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 + 0.31S5x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . 86 T a b l e I S . c o n t ' d - i n v i v o -P r o c e d u r e r a t XTD ml base added XTD1 *TD2 XTD3 Sorghums Raw 96.74 0.231 89.17 84.80 83.48 • A u t o c l a v e d 71.52 0.129 70.11 82.08 80.26 Steamed 79.15 0.220 87.11 84.51 83.13 90~C DH 94.94 0.267 96.11 85.79 84.65 120~C DH 96.35 0.264 95.45 85.70 84.54 180=>C DH 70.62 0.136 71.34 82.26 80.47 TD1; TD = 0.4575 «• l.S841x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r sorghum. «TD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 + 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 • 0.3185x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . » A u t o c l a v i n g c o n d i t i o n s ; 121 0C, 1.5 kg/cm a, 2 hours Steam t r e a t m e n t ; 2 hours DH ( d r y - h e a t i n g ) i n a f o r c e - d r a f t oven f o r 2 hours 87 T a b l e 18. c o n t ' d - i n v i v o - i n v i t r o P r o c e d u r e r a t XTD ml base %TD1 %TD2 XTD3 added Sovbean-baaed d i e t s Raw 79.09 0.090 80.64 81.04 79.02 A u t o c l a v e d 85.48 0.398 87.33 89.29 88.80 Wheat ( a u t o c l a v e d ) + Soybeans ( a u t o c l a v e d ) , 70:30 90.16 0.513 89.84 92.39 92.48 Wheat + Soybeans, 70:30 84.81 0.171 82.41 83.21 81.60 Wheat • Soybeans ( a u t o c l a v e d ) , 70:30 89.84 0.521 90.02 92.61 92.74 Wheat • Soybeans ( a u t o c l a v e d t o g e t h e r ) , 70:30 89.50 0.459 88.66 90.93 90.75 TD1; TD = 0.7868 * 0.2175x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r soybean-based d i e t s . %TD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 * 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 +• 0.3185x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . » A u t o c l a v i n g c o n d i t i o n s ; 121,:=C, 1.5 kg/cm e, 2 hours 88 T a b l e 18. c o n t ' d - i n v i v o - i n v i t r o P r o c e d u r e r a t XTD ml base XTD1 XTD2 XTD3 added A l f a l f a - b a s e d d i e t s Wheat • A l f a l f a P e l l e t s , 60:40 77.20 0.382 74.02 88.67 88.30 Wheat • A l f a l f a P e l l e t s ( a u t o c l a v e d ) , 60:40 61.50 0.303 65.84 86.75 85.79 Wheat ( a u t o c l a v e d ) +• A l f a l f a P e l l e t s ( a u t o c l a v e d ) , 60:40 61.79 0.232 58.49 84.84 83.52 Wheat + A l f a l f a P e l l e t s ( a u t o c l a v e d t o g e t h e r ) 60.81 0.229 58.18 84.76 83.43 Wheat • A l f a l f a Hay, 60:40 82.90 0.489 85.10 91.74 91.71 Wheat + A l f a l f a Hay ( a u t o c l a v e d ) , 60:40 64.51 0.306 66.15 86.84 85.90 Wheat ( a u t o c l a v e d ) + A l f a l f a Hay ( a u t o c l a v e d ) , 60:40 64.47 0.247 60.04 85.25 84.02 Wheat + A l f a l f a Hay ( a u t o c l a v e d t o g e t h e r ) 64.61 0.235 58.80 84.91 83.61 B a r l e y + A l f a l f a Hay, 60:40 79.76 0.393 74.15 89.17 88.66 TD1; TD = 0.3446 • 1.0356x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r a l f a l f a - g r a i n c o m b i n a t i o n d i e t s . XTD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 + 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 + 0.3185x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . * A u t o c l a v i n g c o n d i t i o n s ; 121°C, 1.5 kg/cm 1 2, 2 hours 89 T a b l e I S . c o n t ' d - i n v i v o -P r o c e d u r e r a t scTD ml base added STDl XTD2 %TD3 B a r l e y + A l f a l f a Hay ( a u t o c l a v e d ) , 60:40 52.40 0.265 61.90 85.73 84.58 B a r l e y ( a u t o c l a v e d ) + A l f a l f a Hay, 60:40 79.11 0.436 79.61 90.32 90.03 B a r l e y ( a u t o c l a v e d ) + A l f a l f a Hay ( a u t o c l a v e d ) , 60:40 53.72 0.213 56.51 84.33 82.92 B a r l e y * A l f a l f a Hay ( a u t o c l a v e d t o g e t h e r ) , 60:40 53.31 0.211 56.31 84.28 82.86 TD1; TD = 0.3446 + 1.0356x; e q u a t i o n d e v e l o p e d i n p r e s e n t s t u d i e s s p e c i f i c a l l y f o r a l f a l f a - g r a i n c o m b i n a t i o n d i e t s . %TD o b t a i n e d by m u l t i p l y i n g TD by 100. TD2; TD = 0.7861 • 0.2686x; e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r p l a n t p r o t e i n s . TD3; TD = 0.7614 + 0.3185x; g e n e r a l e q u a t i o n d e v e l o p e d by Pedersen & Eggum (1983) f o r a l l p r o t e i n s . * A u t o c l a v i n g c o n d i t i o n s ; 121 , : 3C, 1.5 kg/cm a, 2 hours 90 100 -i n i 1 1 i 0.10 0.15 0.20 0.25 0.30 0.10N NaOH CONSUMED (ml/10 min/15 mg N) F i g u r e 9. R e l a t i o n s h i p between in. v i v o %TD and in . v i t r o base consumption f o r sorghum-based d i e t s : raw ( a ) , steamed 2 hours (b) , a u t o c l a v e d a t 121 aC, 1.5 kg/cm 1 2 f o r 2 hours <c) , d r y - h e a t e d a t 90-=>C f o r 2 hours (d) , d r y - h e a t e d a t 120~C f o r 2 hours <e) and d r y - h e a t e d a t lSO'^C f o r 2 hours ( f ) . R e g r e s s i o n l i n e d e v e l o p e d : TD = 0.4575 + 1.8841.x ; r = 0.92. 91 90 n "1 I I I i i i i 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.10N NaOH CONSUMED (ml/10 min/15 mg N) F i g u r e 10- R e l a t i o n s h i p between in_ v i v o ssTD and in . v i t r o base consumption f o r a l f a l f a hay + b a r l e y - b a s e d d i e t s where r e g r e s s i o n l i n e d e v e l o p e d : TD = 0.2360 + 1.3194-x; r = 0.96 92 F i g u r e 11. R e l a t i o n s h i p between i n . v i v o %TD and i n v i t r o base consumption f o r soybean-based d i e t s where r e g r e s s i o n l i n e d e v e l o p e d : TD = 0.7868 + 0.2175-x ; r = 0.93. 93 90 - I 50 -) 1 1 1 1 1 1 1 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.10N NaOH CONSUMED (ml/10 min/15 mg N) F i g u r e 12 . R e l a t i o n s h i p between in. v i v o %TD and in. v i t r o base consumption f o r a l f a l f a - b a s e d d i e t s where r e g r e s s i o n l i n e d e v e l o p e d : TD = 0.3446 + 1.0356-x; r = 0.93 94 100 o > C >-•• r— CO Ld o Q Ld Z) £ _ I— Legend O Whea t Diets n=2 ® S o r g h u m Diets n=6 • Tr i t ica le Diets n=10 Bar ley Diets n=19 TD R e g r e s s i o n L ine 0.2 0.3 0.4 0.5 0.10N NaOH CONSUMED (ml/10 min/15 mg N) 0.6 F i g u r e 13 . R e l a t i o n s h i p between i n , v i v o 'iTD and i n v i t r o base consumption f o r g r a i n - b a s e d d i e t s where r e g r e s s i o n l i n e d e v e l o p e d : TD = 0.7419 + 0.4759•x; r = 0.74 95 F i g u r e 14 . R e l a t i o n s h i p between i n v i v o %TD and i n . v i t r o base consumption f o r sorghum- and t r i t i c a l e - b a s e d d i e t s where r e g r e s s i o n l i n e d e v e l o p e d : TD = 0.7128 +• 0.5844«x ; r = 0.80 96 p r o t e i n - t y p e i s w a r r a n t e d . With t h e e x c e p t i o n o f t h e b a r l e y s , i t i s a l s o a p p a r e n t t h a t t h e c l o s e s t s i m i l a r i t i e s between TD i n v i t r o v a l u e s and i n . v i v o v a l u e s a r e o b t a i n e d w i t h t h e newly-developed r e g r e s s i o n e q u a t i o n s as compared t o e i t h e r o f th e e q u a t i o n s d e v e l o p e d by Pedersen and Eggum (1933). T h i s s u g g e s t s t h a t perhaps t h e a p p l i c a t i o n o f e q u a t i o n s d e v e l o p e d i n c e r t a i n r e g i o n s s h o u l d be conducted o n l y i n t h o s e a r e a s (from which t h e samples were d e r i v e d ) . In o b s e r v i n g t h e v a l u e a s s i g n e d t o t h e y - i n t e r c e p t p o r t i o n o f t h e r e g r e s s i o n e q u a t i o n , i t can be seen t h a t by h a v i n g a r e l a t i v e l y l a r g e f i x e d v a l u e , t h e range i n which t h e e q u a t i o n i s a p p l i c a b l e d i m i n i s h e s c o n s i d e r a b l y . I n T a b l e 18, a comparison o f TD1 and TD2 o r TD3 f o r sorghum-baaed d i e t s e x e m p l i f i e s t h e i n a b i l i t y o f t h o s e e q u a t i o n s h a v i n g l a r g e y-i n t e r c e p t v a l u e s (Pedersen and Eggum, 1983) t o a c c u r a t e l y p r e d i c t TD v a l u e s as o b t a i n e d i n v i v o . There i s a major compromise i n t h e p r e d i c t i o n p o t e n t i a l o f a p r o t e i n when an e q u a t i o n i s d e r i v e d by u s i n g samples i n a narrow TD range. A l s o , t h e a p p l i c a t i o n o f a s i n g l e e q u a t i o n t o o b t a i n a c c e p t a b l e p r e d i c t i o n s o f TD i n d i f f e r e n t p r o t e i n s o u r c e s i s d i f f i c u l t t o j u s t i f y . 97 CHAPTER 6 6.0 CONCLUSIONS The s u b j e c t o f i n . v i t r o d i g e s t i o n methods t o p r e d i c t p r o t e i n d i g e s t i b i l i t y i s d i v e r s e and e n t a i l s numerous p o t e n t i a l t e c h n i q u e s which may be used. In t h i s t h e s i s , t h e p H - s t a t method was used because o f i t s many a p p a r e n t advantages. An att e m p t t o d e t e r m i n e whether t h i s method would be s e n s i t i v e t o changes i n p r o t e i n d i g e s t i b i l i t y due t o heat t r e a t m e n t o f v a r i o u s p l a n t p r o t e i n s was made i n Experiment 1. In Experiment 2, t h e s e n s i t i v i t y o f t h e pH-atat method t o t h e p r e s e n c e o r absence o f n o n - n i t r o g e n components, i n a d d i t i o n t o t h e p r o t e i n component, was t e s t e d . F i n a l l y , i n Experiment 3, a s e r i e s o f r e g r e s s i o n e q u a t i o n s were d e v e l o p e d f o r v a r i o u s f e e d s as a r e s u l t o f a comparison o f in. v i t r o and i n v i v o p r o t e i n d i g e s t i b i l i t y f i g u r e s . Experiment 1 demonstrated t h e c a p a b i l i t y o f t h e p H - s t a t t o d e t e c t t h e e f f e c t o f t e m p e r a t u r e and t i m e o f heat a p p l i c a t i o n on in . v i t r o d i g e s t i b i l i t y as measured i n wheat, b a r l e y , sorghum and d e f a t t e d soybeans. A l a r g e i n c r e a s e i n d i g e s t i b i l i t y was d e t e c t e d by t h e p H - s t a t method o f soybeans which had been a u t o c l a v e d f o r 30 minutes. M i l d , d r y - h e a t i n g o f g r a i n s a t t e m p e r a t u r e s l e s s than 120°C improved d i g e s t i b i l i t y s l i g h t l y w h i l e d r y - h e a t i n g above 120 , =C reduced i t s i g n i f i c a n t l y . The r e s p o n s e s o f i n c r e a s e d d i g e s t i b i l i t y o f a u t o c l a v e d soybeans and d e c r e a s e d d i g e s t i b i l i t y o f g r a i n 98 m a t e r i a l which had been s u b j e c t e d t o d r y heat f o r extended p e r i o d s o f t i m e r e - a f f i r m s t h e p o t e n t i a l c a p a c i t y o f t h e pH-s t a t method f o r s c r e e n i n g p u r p o s e s . The r e s u l t s o b t a i n e d w i t h wheat i n Experiment 2 p r o v i d e f u r t h e r e v i d e n c e t h a t t h e p r e s e n c e o f a d d i t i v e s such as m i n e r a l s i n c r e a s e d i g e s t i b i l i t y by i n c r e a s i n g t h e c a t a l y t i c a c t i o n o f c e r t a i n p a n c r e a t i c enzymes. The u n c l e a r r e s p o n s e seen w i t h raw soybeans may have been due t o t h e c o n f o u n d i n g e f f e c t s o f t r y p s i n i n h i b i t o r s p r e s e n t i n t h e p r o t e i n s o u r c e . In Experiment 3, a s e r i e s o f d i e t s were f e d t o r a t s and were a l s o d i g e s t e d by t h e p H - s t a t method. As a r e s u l t , d i f f e r e n t r e g r e s s i o n e q u a t i o n s were d e v e l o p e d . The use o f a s p e c i f i c e q u a t i o n f o r each p r o t e i n - t y p e gave a h i g h e r c o r r e l a t i o n c o e f f i c i e n t than would be o b t a i n e d i f o n l y one g e n e r a l e q u a t i o n was d e v e l o p e d f o r a l l p r o t e i n s s t u d i e d . The p o t e n t i a l f o r t h e p H - s t a t method t o be used i n t h e f e e d m a n u f a c t u r i n g i n d u s t r y , where p r o t e i n s a r e s u b j e c t e d t o h e a t i n g p r o c e s s e s , i s c o n s i d e r a b l e . 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S a v o i e , L. and G a u t h i e r , S.F. (1986). D i a l y s i s c e l l f o r t h e i n v i t r o measurement o f p r o t e i n d i g e s t i b i l i t y . J o u r n a l o f  Food S c i e n c e 51, 494-498. Schwimmer, S. (1981). Source Book o f Food Enzymology. AVI p u b l i s h i n g Co., W e s t p o r t , C t . pages 27-62, 624. S h e f f n e r , A.L., E c k f e l d t , G.A. and S p e c t o r , H. (1956). The p e p s i n - d i g e s t r e s i d u e (PDR) amino a c i d i n d e x o f net p r o t e i n u t i l i z a t i o n . J . N u t r . 60, 105. S m i t h , E., R. H i l l , I . Lehman, R. L e f k o w i t z , P. H a n d l e r . White, A. (1983). In P r i n c i p l e s o f B i o c h e m i s t r y :  Mammalian B i o c h e m i s t r y . 7 t h E d i t i o n . M c G r a w - H i l l I n c . pages 336-339. S m i t h , L.H. (1962). Agron. J . 54, 291. Snyder, H.E. (1986). P e r s o n a l communication. S t i n s o n , C.T. and Snyder, H.E. (1980). E v a l u a t i o n o f heated soy f l o u r s by measurement o f t r y p t i c h y d r o l y s i s u s i n g a p H - s t a t . J o u r n a l o f Food S c i e n c e 45, 936-945. Vachon, C , G a u t h i e r , S.F., J o n e s , J.D. and S a v o i e , L. (1983). In v i t r o e n z y m a t i c r e l e a s e o f amino a c i d s from a l k a l i -t r e a t e d p r o t e i n s c o n t a i n i n g l y s i n o a l a n i n e . N u t r i t i o n  R e p o r t s I n t e r n a t i o n a l 27, 1303-1313 105 V a n d e r g r i f t , W.L. (1985). Use o f soybeans i n p i g d i e t s . P i g News and I n f o r m a t i o n 6, 281-285. V a r n i s h , S.A. and C a r p e n t e r , K . J . (1975a). B r i t i s h J o u r n a l o f  N u t r i t i o n 34. 325-337. V a r n i s h , S.A. and C a r p e n t e r , K .J. (1975b). B r i t i s h J o u r n a l o f  N u t r i t i o n 34. 339-349. W a l l , J.S., James, C. and Donaldson, G.L. (1975). C e r e a l  C h e m i s t r y 54, 779-790. W i l l i a m s , K.C., N e i l l , A.R. and Magee, M.H. (1982). Animal  P r o d u c t i o n i n A u s t r a l i a 15, 635-638. Z a p s a l i s , C. and Beck, R.A. (1985). Food C h e m i s t r y and N u t r i t i o n a l B i o c h e m i s t r y . Ch. 2-4, 31-188. John W i l e y and Sons, New York. 1 0 6 APPENDICES APPENDIX 1 A d e t a i l e d o p e r a t i n g g u i d e f o r r o u t i n e p H - s t a t d i g e s t i b i l i t y <TD> d e t e r m i n a t i o n s . 1. Sample must be ground t h r o u g h an SO-mesh (0.13 mm) s c r e e n . 2. N i t r o g e n c o n t e n t o f sample must be known. 3. Turn Thermomix w a t e r b a t h h e a t e r on and have heat s e t t i n g a t 40'=C (by h a v i n g i t a t t h i s s e t t i n g , t h e v e s s e l / w a t e r j a c k e t t e m p e r a t u r e i s 37,=>C) . 4. Weigh t h a t amount o f f e e d t h a t c o r r e s p o n d s t o 15 mg n i t r o g e n i n t o c u s t o m - b u i l t v e s s e l . 5. Add 15 ml o f d e i o n i z e d water and p l a c e sample v e s s e l i n r e f r i g e r a t o r f o r 1-2 hours t o a l l o w s o a k i n g . 6. In t h e meantime, weigh o ut t h e enzymes i n a p p r o p r i a t e q u a n t i t i e s such t h a t t h e i r f i n a l c o n c e n t r a t i o n i n 1 ml o f enzyme s o l u t i o n i s 1.61 mg t r y p s i n , 3.96 mg c h y m o t r y p s i n and 2.36 mg p e p t i d a s e . A f t e r w e i g h i n g o u t t h e d r y enzymes, d e i o n i z e d water i s added from a b u r e t t e . T h i s m i x t u r e i s a l l o w e d t o s i t f o r about 10 mi n u t e s , a l l o w i n g t h e enzymes t o d i s s o l v e ( i t must be noted t h a t a minimum 14 ml o f enzyme s o l u t i o n be made up as t h i s depth o f l i q u i d i s t h e minimum amount t h a t c o v e r s t h e m i c r o - e l e c t r o d e ' s membrane). The s o l u t i o n i s d i p p e d i n t o t h e w a t e r b a t h f o r a c o u p l e o f minutes a f t e r which i t i s a t t a c h e d t o t h e t i t r a t i o n head o f t h e p H - s t a t t i t r a t o r . The w a t e r j a c k e t i s wrapped around t h e enzyme v e s s e l . The programmed method (#71) t h a t b r i n g s t h e pH o f t h e enzyme s o l u t i o n t o 8.00 i s RUN. Once t h e enzyme s o l u t i o n ' s pH has reached 3.10 ( i t w i l l o v e r s h o o t 3.00 f o r a b r i e f moment w i t h t h e r a p i d a d d i t i o n o f b a s e ) , p r e s s RESET and q u i c k l y d e t a c h t h e v e s s e l and s t o r e i n an i c e - b a t h w h i l e k e e p i n g i t c o v e r e d w i t h p l a s t i c . NB. P r i o r t o u s i n g t h e p H - s t a t t i t r a t o r a f t e r a p e r i o d o f non-use, i t i s i m p e r a t i v e t h a t i t i s c a l i b r a t e d w i t h a b u f f e r o f pH 7.00 ( o r a t w o - p o i n t c a l i b r a t i o n w i t h 2 b u f f e r s i . e . pH 4.01 and 9.13, may be used f o r more a c c u r a t e pH r e a d i n g s o f samples w i t h extreme i n i t i a l pH v a l u e s ) . I n s t r u c t i o n s f o r c a l i b r a t i n g t h e t i t r a t o r a r e g i v e n on pages 58-60 o f t h e ^ O p e r a t i n g I n s t r u c t i o n s . ' 7. Each o f t h e samples t o be d i g e s t e d ( h a v i n g been soaked a t 4,=>C f o r 1-2 hr) a r e a l s o k e p t i n an i c e - b a t h u n t i l about 15 minutes p r i o r t o i t s i n . v i t r o d i g e s t i o n a t which t i m e t h e sample i s soaked i n t h e warm (40'=C) w a t e r b a t h . 108 APPENDIX 1 ( c o n t ' d ) 8. The pre-programmed parameters f o r g r a i n s a r e s t o r e d as method #23 and f o r sodium c a s e i n a t e , method #22. These programs can e a s i l y be changed ( t e m p o r a r i l y o r permanently) by u s i n g a c o m b i n a t i o n o f t h e f o l l o w i n g k e y s : MODIFY, SAVE METHOD, and RUN. S i m i l a r l y , new methods can be c r e a t e d and saved f o r use w i t h d i f f e r e n t p r o t e i n t y p e s ( i . e . a n i m a l p r o t e i n s ) . 9. A f t e r t h e enzyme s o l u t i o n has been brought t o pH 8.00, t h e method f o r d i g e s t i n g sodium c a s e i n a t e (#21) i s e n t e r e d by t y p i n g "71" and t h e n p r e s s i n g RUN. As a s t a n d a r d p r o c e d u r e , sodium c a s e i n a t e i s i n c l u d e d i n each day's s e t o f d i g e s t i o n s t o m o n i t o r any i r r e g u l a r i t i e s i n t h e system. A q u a n t i t y i n t h e amount o f 0.1061 g (AD) c o r r e s p o n d s t o 15 mg N/15 ml s o l u t i o n . 10. The p r o c e d u r e f o r f o l l o w i n g one complete d i g e s t i o n from s t a r t t o f i n i s h i s l i s t e d h e r e ; a) Assuming t h a t t h e sample v e s s e l has been soaked f o r 1 hour a t 4-=C and warmed i n t h e 40°C w a t e r b a t h f o r 15 m i n u t e s , i t i s t h e n a t t a c h e d t o t h e t i t r a t i o n head. b) The RUN b u t t o n i s p r e s s e d t w i c e t o s t a r t t h e p H - s t a t d i g e s t i o n . A f t e r s t i r r i n g f o r 5 minutes ( o r however l o n g t h e parameter has been s t a t e d i n t h e program), 0.10N NaOH i s a u t o m a t i c a l l y added t o b r i n g t h e s u s p e n s i o n pH up t o 8.00. The i n i t i a l pH o f t h e p r o t e i n s u s p e n s i o n and t h e ml o f a l k a l i r e q u i r e d t o b r i n g t h e s o l u t i o n up t o 8.00 a r e r e c o r d e d . c) At p r e c i s e l y t h e moment when t h e b u r e t t e p i s t o n has r e f i l l e d , 1.5 ml o f enzyme s o l u t i o n i s added t o t h e p r o t e i n s u s p e n s i o n i n o r d e r t o a c t i v a t e t h e i n v i t r o d i g e s t i o n . d) In o r d e r t o f o l l o w t h e e n z y m a t i c h y d r o l y s i s f o r 10 m i n u t e s , a s t o p w a t c h i s used. In o r d e r t o r e c o r d t h e ml o f base added d u r i n g t h e 10 minutes ( o r a t any t i m e d u r i n g t h e d i g e s t i o n ) , t h e ROTATE/LIST b u t t o n can be p r e s s e d t o g i v e t h i s i n f o r m a t i o n . e) I f a r e c o r d e r p r i n t o u t f o l l o w i n g t h e d i g e s t i o n (base a d d i t i o n due t o h y d r o l y s i s ) i s d e s i r e d , t h e GA 14 M e t t l e r Recorder i s used. To o p e r a t e i t , i t s power s w i t c h i s t u r n e d on, t h e a p p r o p r i a t e c h a r t speed i s s e l e c t e d , t h e r e c o r d e r pen i s a l l i g n e d t o z e r o and t h e o n / o f f b u t t o n i s p r e s s e d t o s t a r t t h e d r i v e motor. 109 APPENDIX 1 (c o n t ' d ) f ) At 10 minutes post-enzyme a d d i t i o n , t h e volume o f base added i s r e c o r d e d from t h e t i t r a t o r and can a l s o be o b t a i n e d d i r e c t l y from t h e r e c o r d e r p r i n t o u t . g) The v e s s e l i s removed from t h e t i t r a t i o n head and t h e c o n t e n t s a r e d i s p o s e d o f . The same p r o c e d u r e i s f o l l o w e d f o r g r a i n s w i t h t h e e x c e p t i o n t h a t method #23 i s e n t e r e d . The custom-made v e s s e l s a r e v e r y f r a g i l e and e x p e n s i v e and s h o u l d be s t o r e d i n t h e drawer (beneath t h e p H - s t a t a p p a r a t u s ) when not i n use. h) The t i t r a t o r may be t u r n e d o f f when not i n use f o r extended p e r i o d s as t h e memory r e t a i n s t h e programs. i ) I t i s a b s o l u t e l y c r i t i c a l t o en s u r e t h a t t h e e l e c t r o d e membrane does not d r y out when not i n use. Thus, i t s h o u l d be kept immersed a t a l l t i m e s i n a pH 7.00 b u f f e r when not b e i n g used. A l s o , t h e rubb e r p l u g on t h e e l e c t r o d e s h o u l d be c l o s e d t o p r e v e n t l e a k a g e o f t h e 3 M KC1 s o l u t i o n d u r i n g p e r i o d s when not i n use. 110 APPENDIX 2 R o u t i n e f o r c o n d u c t i n g r a t t r i a l a . 1. Order 5 r a t a f o r each t r e a t m e n t t o be t e s t e d per t r i a l ( p l u s 2 e x t r a s ) . These a r e o r d e r e d from t h e Animal Care C e n t e r and may be from t h e i r own c o l o n y o r o r d e r e d i n from C h a r l e s R i v e r , Quebec. Order a t l e a s t one week ahead o f t r i a l d a t e . 2. Weigh t h e a p p r o p r i a t e d i e t f o r each r a t i n t o two Rubbermaid c o n t a i n e r s (one c o n t a i n i n g r e q u i r e m e n t f o r 4 days, t h e second t h e r e q u i r e m e n t f o r 5 days. S t o r e i n r e f r i g e r a t o r u n t i l t h e t r i a l commences. 3. Tape a s h e e t ( s t a t i n g A i r - D r y grams per r a t t o f e e d per day f o r each t e s t d i e t ) above t h e weigh b a l a n c e i n t h e r a t room. 4. C o n d i t i o n s i n t h e r a t - r o o m a r e a d j u s t e d t o ; 72X r e l a t i v e h u m i d i t y (RH) and 23°C. An e l e c t r i c f a n i s used t o ensure t h a t even RH and t e m p e r a t u r e a r e m a i n t a i n e d t h r o u g h o u t t h e room. A hygrothermograph r e c o r d e r i s p l a c e d near t h e cage l e v e l t o p r o v i d e a c o n s t a n t r e c o r d o f t h e c o n d i t i o n s i n the room a t any t i m e . The i n k and paper s h o u l d be checked a t t h e b e g i n n i n g o f t h e a c t u a l c o l l e c t i o n p e r i o d (days 5-9) . 5. P r i o r t o p l a c i n g r a t s i n t o i n d i v i d u a l c a g e s , a p l a s t i c b u c ket i s p l a c e d under each cage t o c a p t u r e t h e f e c a l and u r i n a r y wastes e x c r e t e d d u r i n g t h e p r e - c o l l e c t i o n p e r i o d (days 1-4) o f t h e t r i a l . 6. Upon a r r i v a l o f r a t s , weigh each r a t i n a t a r e d , p l a s t i c beaker w i t h a l i d and t h e n p l a c e i t i n t o i t s i n d i v i d u a l cage and f i n d t h e average o f each s e t o f f i v e r a t s . The mean o f each group o f r a t s s h o u l d be t h e same ( t o t h e n e a r e s t gram) f o r each t r e a t m e n t . 7. B a r l e y (10 grams o f 1 mm g r i n d ) i s f e d t o t h e r a t s d u r i n g th e day t h a t t hey a r e f i r s t put i n t o i n d i v i d u a l cages (day 0 ) . T h i s day i s c o n s i d e r e d an adjustment p e r i o d . 8. Water i s p r o v i d e d ad l i b i t u m v i a n i p p l e - b o t t l e s . M e t a l p l a t e s must be p l a c e d on t h e r u b b e r t o p s t o p r e v e n t r a t s from chewing. The water b o t t l e s s h o u l d be checked t o e n s u r e t h a t e v e r y r a t i s a b l e t o o b t a i n water from h i s b o t t l e - t h e r e i s a tendency f o r a i r b u b b l e s t o lodge and b l o c k t h e f l o w o f water. Water b o t t l e s must be r e f i l l e d on day 5 ( f i r s t day o f c o l l e c t i o n p e r i o d ) . 11 1 APPENDIX 2 ( c o n t ' d ) 9. R a t s s h o u l d be f e d a t t h e same ti m e each day o f t h e p r e -c o l l e c t i o n (days 1-4) and c o l l e c t i o n (days 5-9) p e r i o d s . Morning f e e d i n g i s c o n v e n i e n t . A f t e r f e e d i s weighed i n t o a t a r e d weigh cup, i t i s poured i n t o t h e cage f e e d e r . A f t e r t h i s has been r e p e a t e d f o r a l l r a t s , a p l a s t i c tube w i t h a s o l i d end i s used t o p r e s s t h e f e e d down. Then a b r a s s r i n g i s p l a c e d on t h e f e e d . A f t e r t h i s has been done, t h e r a t i s g i v e n a c c e s s t o t h e f e e d . The Rubbermaid c o n t a i n e r s c o n t a i n i n g t h e 4-day p r e - c o l l e c t i o n f e e d a r e k e p t on t h e s h e l f above t h e r e s p e c t i v e r a t s . The 5-day f e e d f o r t h e c o l l e c t i o n t r i a l i s k e p t i n t h e r e f r i g e r a t o r u n t i l day 5. 10. P r i o r t o commencing t h e a c t u a l c o l l e c t i o n p e r i o d , each r a t i s weighed. T h i s r e q u i r e s two p e o p l e - one weighs and r e c o r d s t h e r a t w e i ght w h i l e t h e second i n s e r t s t h e a c e t a t e f u n n e l + n y l o n mesh under t h e cage, g r e a s e s t h e t o p cage l i d w i t h v a s e l i n e and exchanges i t w i t h t h e lower l i d (which i s c o n t a m i n a t e d w i t h waste p r o d u c t s from t h e f i r s t f o u r days o f t h e o v e r a l l t r i a l ) . 11. Two e r l e n m e y e r f l a s k s c o n t a i n i n g 5% HeS0^. a r e p l a c e d under t h e a c e t a t e f u n n e l and t h e n y l o n mesh n e t t i n g ( e r l e n m e y e r c o n t a i n i n g 50 ml o f a c i d c a p t u r e s u r i n e and has a s m a l l p l a s t i c f u n n e l w i t h g l a s s wool a c t i n g as a f i l t e r w h i l e an e r l e n m e y e r c o n t a i n i n g 100 ml o f a c i d and h a v i n g a PVC b a c k s t o p , c a p t u r e s f e c a l p e l l e t s , r e s p e c t i v e l y ) . Feces may be c o l l e c t e d w i t h o u t a c i d p r e s e r v a t i o n and removed d a i l y and kept a t 4-=C. T h i s method must be used i f d r y m a t t e r and energy d i g e s t i b i l i t y i s t o be measured. 12. Each day o f t h e c o l l e c t i o n p e r i o d i n v o l v e s t h e s p r a y i n g o f t h e a c e t a t e f u n n e l and t h e n y l o n n e t t i n g w i t h a 20% (W/V) s o l u t i o n o f c i t r i c a c i d and t h e washing-down o f t h e g l a s s wool w i t h 5% HaS0*.. T h i s m i n i m i z e s v o l a t i l i z a t i o n l o s s e s o f ammonia. T h i s i s done p r i o r t o f e e d i n g . 13. D u r i n g t h e a f t e r n o o n (4:00 pm) o f each o f t h e c o l l e c t i o n d a ys, t h e b r a s s r i n g on each f e e d e r s h o u l d be a d j u s t e d so t h a t t h e r a t has easy and c o n t i n u o u s a c c e s s t o t h e f e e d . 14. On t h e eve o f t h e f i n a l day o f t h e c o l l e c t i o n p e r i o d , t h e b r a s s r i n g s a r e removed t o a l l o w t h e r a t s t o c o m p l e t e l y c l e a n t h e f e e d e r s . 15. A f t e r t h e c o l l e c t i o n p e r i o d i s c o m p l e t e d , t h e r a t s and f e e d r e s i d u e a r e weighed and t h e r e s u l t s a r e r e c o r d e d . In a l l , t h r e e s e t s o f r a t w e i g h t s s h o u l d have been r e c o r d e d . 1 1 2 APPENDIX 2 (con t ' d ) 16. Weighed r a t s a r e p l a c e d i n t o a p l a s t i c garbage can ( w i t h l i d ) . When 20 o r so r a t s have been ac c u m u l a t e d , they a r e e u t h a n i z e d w i t h C 0 a o v e r d o s e . The c a r c a s s e s a r e d o u b l e -bagged and p l a c e d i n t h e b i o l o g i c a l waste p i c k - u p l o c a t e d b e s i d e t h e beef b a r n . 17. P r o c e d u r e s t o q u a n t i t a t i v e l y t r a n s f e r f e c e s and u r i n e from t h e e r l e n m e y e r f l a s k s t o l e a k p r o o f Nalgene c o n t a i n e r s t a k e s 2-3 hours w i t h 2 p e o p l e . A l l e r lenmeyer f l a s k s c o n t a i n i n g t h e f e c e s a r e brought i n t o t h e r a t - l a b , w h i l e t h e u r i n e e r l e n m e y e r s a r e kep t i n t h e ra t - r o o m so t h a t t h e two pe o p l e have enough bench space. To ens u r e t h a t u r i n e has been t r a n s f e r r e d as q u a n t i t a t i v e l y as p o s s i b l e , a l l f u n n e l s f l o o r i n g and n y l o n mesh n e t t i n g a r e brushed c l e a n w i t h hot water. M a t e r i a l r e q u i r e d t o do t h i s i n c l u d e s a l a r g e r i n g s t a n d w i t h a r i n g clamp i n which s i t s a 10" p l a s t i c f u n n e l . A n y l o n - b r i s t l e d p a i n t b r u s h (2-3") i s used w i t h l i b e r a l amounts o f hot water t o wash a l l s u r f a c e s . The u r i n e f l a s k i s p l a c e d under t h e l a r g e f u n n e l . I t i s c r i t i c a l t h a t t h e f i n a l volume does not exceed t h e 250 ml mark. On c o m p l e t i o n t h e u r i n e i s t r a n s f e r r e d t o a 250ml v o l u m e t r i c f l a s k t h r o u g h a g l a s s wool f i l t e r and t h e s e a r e the n brought up t o volume. At t h i s p o i n t , caps a r e p l a c e d on t h e v o l u m e t r i c f l a s k s and each f l a s k i s i n v e r t e d s e v e r a l t i m e s t o ensure u n i f o r m m i x i n g . The u r i n e s o l u t i o n i s t r a n s f e r r e d i n t o round Nalgene b o t t l e s but i t does not have t o be q u a n t i t a t i v e l y as t h e s o l u t i o n i s a l l r e p r e s e n t a t i v e f o r a n a l y s e s ( t h i s i s c o n t r a r y t o f e c a l t r a n s f e r where a l l must be t r a n s f e r r e d ) . The f e c a l p e l l e t s i n a c i d a r e t r a n s f e r r e d q u a n t i t a t i v e l y i n t o s q u are Nalgene b o t t l e s . D i s t i l l e d water i s used t o remove r e s i d u e from t h e e r l e n m e y e r s . The t a r e w e i ght o f t h e empty square b o t t l e s has been r e c o r d e d b e forehand and w i l l a l l o w t h e t o t a l s l u r r y w e i g h t - o n l y t o be de t e r m i n e d by d i f f e r e n c e a f t e r t h e p e l l e t s a r e blended i n a P o l y t r o n p r o b e - b l e n d e r (where s m a l l amounts o f d i s t i l l e d water a r e used t o r i n s e o f f r e s i d u e from t h e p r o b e ) . I f no a c i d i s used, t h e f e c e s a r e d r i e d a t 70 , =C. Both u r i n e and f e c a l b o t t l e s a r e s t o r e d i n t h e r e f r i g e r a t o r u n t i l r e q u i r e d f o r a n a l y s e s . 18. Cage components a r e l e f t t o soak o v e r n i g h t and a r e washed w i t h a m i l d d e t e r g e n t . The Rubbermaid f e e d c o n t a i n e r s a r e o n l y wiped c l e a n w i t h a damp c l o t h . 113 APPENDIX 3 Mean v a l u e s <n = 5) f o r XTD as o b t a i n e d w i t h i n . v i v o ( r a t ) t r i a l s f o r each o f t h e p r o t e i n s o u r c e s t e s t e d . S t a n d a r d e r r o r o f t h e mean (SEM), r a t t r i a l number (R ) and cru d e p r o t e i n v a l u e s o f t h e s o u r c e s a r e s t a t e d . P r o t e i n s o u r c e T r i a l # Crude P r o t e i n (X o f DM) TD Mean X SEM b a r l e y #1 R12 15.30 87.31 1.07 b a r l e y #2 R12 17.33 92.86 0.43 b a r l e y #3 R12 17.45 93.55 0.85 b a r l e y #4 R12 15.68 89.25 0.85 b a r l e y #5 R12 15.32 90.28 0.69 b a r l e y #6 R12 17.01 90.80 0.61 b a r l e y #7 R13 17.06 93.66 0.35 b a r l e y #8 R13 19.07 96.46 0.71 b a r l e y #9 R13 17.02 97.99 0.62 b a r l e y #10 R13 14.19 93.84 0.27 b a r l e y #11 R16 12.00 93.47 1.15 b a r l e y #12 R16 12.56 91 .18 0.60 b a r l e y #13 R21 10.19 95.98 0.48 b a r l e y #14 R21 15.69 91.61 1.18 b a r l e y #15 R21 17.31 94.05 0.45 b a r l e y #16 R21 12.25 91.49 1.04 b a r l e y #17 R21 11.19 89.84 0.63 b a r l e y #18 R21 14.38 91 .51 0.50 b a r l e y #19 R21 18.06 91 .43 1.16 114 APPENDIX 3 (co n t ' d ) P r o t e i n s o u r c e T r i a l # Crude P r o t e i n (X o f DM) Mean X TD SEM a u t o c l a v e d soybeans soybeans a u t o c l a v e d wheat wheat R14 R14 R14 R14 38.62 37.94 15.74 15.69 85.48 79.09 92.95 95.94 0.53 0.38 0.43 0.37 a u t o c l . soybeans + a u t o c l . wheat (30:70) R14 90.16 0.97 soybeans + wheat (30:70) R14 84.76 1.26 a u t o c l . soybeans • wheat (30:70) R14 soybeans •- wheat a u t o c l . t o g e t h e r (30:70) R14 a l f a l f a p e l l e t s * wheat (60:40) R15 a u t o c l . a l f a l f a p e l l e t s * wheat (60:40) R15 22.56 15.59/ 15.69 15.59/ 15.69 90.03 89.50 77.20 61.50 0.63 1.93 1.04 0.34 a u t o c l . a l f a l f a p e l l e t s * a u t o c l . wheat (60:40) R15 15.59/ 15.54 61.79 0.52 a l f a l f a p e l l e t s + wheat a u t o c l . t o g e t h e r (60:40) R15 a l f a l f a hay + wheat (60:40) R15 15.66 19.75/ 15.69 60.81 82.90 0.53 0.80 a u t o c l . a l f a l f a hay + wheat (60:40) R15 20.00/ 15.69 64.51 1.25 1 1 5 APPENDIX 3 (c o n t ' d ) P r o t e i n s o u r c e T r i a l # Crude P r o t e i n (X o f DM) TD Mean X SEM a u t o c l . a l f a l f a hay • a u t o c l . wheat (60:40) R15 20.00/ 15.54 64.47 0.77 a l f a l f a hay + wheat a u t o c l . t o g e t h e r (60:40) R15 17.06 64.61 0.67 a l f a l f a hay • b a r l e y (60:40) R16 19.75/ 12.00 79.76 0.33 a u t o c l . a l f a l f a hay +• b a r l e y (60:40) R16 20.00/ 12.00 52.40 0.68 a l f a l f a hay + a u t o c l . b a r l e y (60:40) R16 19.75/ 12.56 79.11 1.20 a u t o c l . a l f a l f a hay + a u t o c l . b a r l e y (60:40) R16 20.00/ 12.56 53.72 0.70 a l f a l f a hay + b a r l e y a u t o c l . t o g e t h e r (60:40) R16 16.69 53.31 1.51 116 APPENDIX 3 ( c o n t ' d ) P r o t e i n s o u r c e T r i a l # Crude P r o t e i n (X o f DM) TD • Mean X SEM sorghum R17 9.19 96.74 1.00 a u t o c l a v e d sorghum R17 8.88 71.52 2.47 steamed (2 h r ) sorghum R17 8.88 79.15 0.39 d r y - h e a t e d (90=>C> sorghum R17 9.13 94.94 0.35 d r y - h e a t e d (120 C >C) sorghum R17 9.31 96.35 0.69 d r y - h e a t e d (laO-^C) sorghum R17 9.06 73.50 0.93 t r i t i c a l e #1 R1S 16.19 97.57 1.01 t r i t i c a l e #2 R18 17.38 96.11 0.37 t r i t i c a l e #3 R18 14.88 98.14 0.58 t r i t i c a l e #4 R18 17.56 95.74 0.35 t r i t i c a l e #5 R1S 17.38 97.76 0.44 t r i t i c a l e #6 R1S 16.38 98.51 0.68 t r i t i c a l e #7 R18 16.94 96.70 0.74 t r i t i c a l e #8 R19 17.75 95.15 0.36 t r i t i c a l e #9 R19 18.56 96.74 0.21 t r i t i c a l e #10 R19 17.75 97.26 0.40 1 1 7 APPENDIX 4 C o n s t r u c t i n g M e t a b o l i c Cages t o Conduct Rat T r i a l s T h i s s e c t i o n i s t o p r o v i d e i n f o r m a t i o n on t h e c o n s t r u c t i o n o f m e t a b o l i c cages f o r r a t s t u d i e s . The b a s i c d e s i g n o f t h e r a t cage i s adapted from Eggum (1973). B u i l d i n g cages i n s t e a d o f p u r c h a s i n g them a l l o w s f o r e a s i e r maintenance. The c a s t a c r y l i c t u b i n g must be o r d e r e d w e l l i n advance from t h e s u p p l i e r ( C a d i l l a c P l a s t i c s ) because o f t h e l o n g d e l i v e r y t i m e ( s e v e r a l months). The amount o f m a t e r i a l t h a t i s r e q u i r e d f o r t h e c o n s t r u c t i o n o f 10 complete cages w i l l be assumed t h r o u g h o u t t h i s paper. A l i s t o f m a t e r i a l s i s g i v e n i n Appendix 5. The body o f t h e cage i s made from 5.25" ( o u t s i d e d i a m e t e r , 0D) c a s t a c r y l i c t u b i n g o f w a l l t h i c k n e s s o f 0.125". The tube i s s u p p l i e d i n 9 f t . l e n g t h s . The c u t t i n g can be done w i t h a t a b l e saw. Because a 9' p i e c e i s awkward and dangerous t o h a n d le on t h e t a b l e saw, t h e l e n g t h s h o u l d be c u t i n h a l f w i t h a s h a r p handsaw. The s m a l l e r h a l f - l e n g t h s can be e a s i l y h a n d l e d . The t a b l e saw s h o u l d be f i t t e d w i t h a new ( s h a r p ) c a r b i d e - t i p p e d , g r i n d i n g - t y p e b l a d e . T h i s s h a r p , g r i n d i n g b l a d e i s c r i t i c a l as t h e c a s t a c r y l i c t u b i n g w i l l s h a t t e r i f a d u l l b l a d e i s used. A l s o , i f a c o a r s e saw ( 4 4 - b i t or r i p p i n g b l a d e , e t c . ) b l a d e i s used, t h e t u b i n g w i l l be grabbed by t h e t e e t h , y i e l d i n g a v e r y rough c u t and c a u s i n g f a t i g u e t o t h e o p e r a t o r . To o b t a i n a p r e c i s i o n c u t f o r a l l 10 118 cage l e n g t h s ( o f 6" e a c h ) , a j i g s h o u l d be f i t t e d on two s i d e s o f t h e saw. A s c r a p p i e c e o f plywood i s screwed i n t o t h e a d j u s t a b l e m e t a l , f o r m i n g a b a c k s t o p so t h a t t h e p i e c e o f t u b i n g w i l l have something t o s u p p o r t i t ( l e n g t h w i s e ) . To t h e r i g h t o f t h e saw-blade ( p e r p e n d i c u l a r ) , a d i s t a n c e o f 6" i s measured and a t t h i s p o i n t , a n o t h e r s c r a p p i e c e o f plywood i s screwed i n t o t h e metal frame o f t h e saw (see F i g u r e 15 f o r e x p l a n a t i o n ) , t h u s , f o r m i n g t h e g u i d e o r f e n c e . The two arrows i n F i g u r e 15 a r e meant t o show t h e d i r e c t i o n s i n which t h e p i e c e o f t u b i n g must be p r e s s e d i n o r d e r t o produce p r e c i s e c u t s . That i s , by p r e s s i n g t h e t u b i n g toward t h e b a c k s t o p and t h e f e n c e , t h e r e i s no w o b b l i n g motion a l l o w e d so t h a t a p e r f e c t r i g h t a n g l e c u t w i l l be made. The o p e r a t o r would be s t a n d i n g on t h e h a n d l e - s i d e o f t h e t a b l e s a w . Once t h e t u b i n g has been c u t i n t o two l e n g t h s ("4.5 f e e t e a c h ) , and t h e b a c k s t o p and f e n c e has been s e c u r e d , t h e t u b i n g i s ready t o be c u t i n t o r a t - c a g e s i z e l e n g t h s . With t h e t u b i n g p u l l e d t a u t toward t h e b a c k s t o p and f e n c e , t h e saw i s s t a r t e d and t h e handle i s c r a n k e d c l o c k w i s e t o r a i s e t h e b l a d e . The o p e r a t o r must h o l d t h e t u b i n g w i t h one hand and c r a n k t h e b l a d e h a n d l e w i t h t h e o t h e r . The b l a d e o n l y needs t o be r a i s e d about 0.5" above t h e t a b l e s u r f a c e because w i t h t h e t u b i n g b e i n g h o l l o w , o n l y t h e t h i n w a l l (0.125") has t o be c u t . But, because t h e c a s t t u b i n g has a l a r g e d i a m e t e r , t h e t u b i n g must be r e v o l v e d 360'= t o a l l o w t h e e n t i r e c i r c u m f e r e n c e t o be c u t . T h i s r e q u i r e s s t e a d y hands and c o n s i d e r a b l e s k i l l as any 119 saw b l a d e i-- 6 - - - L ence j c a s t t u b i n g j f-. i j. I I J I M I M I M I I t " ^ b a c k s t o p h a n d l e t o r a i s e / l o w e r saw-blade F i g u r e 15. Overhead view o f t a b l e s a w - t o p , showing s e t - u p p r o c e d u r e t o ensure s a f e t y and s t a b i l i t y w h i l e c u t t i n g t u b i n g t o 15 cm <6"> l e n g t h s . — 2.75" — 1 . 7 5 " d i a m e t e r h o l e -1. 50 " F i g u r e 16. Placement o f t h e d r i l l - h o l e on t h e 15 cm <6") cage tub e . 120 rough l a t e r a l movement may cause c h i p p i n g i n t h e t u b i n g , or worse, s e v e r e k i c k b a c k i f t h e t u b i n g i s not h e l d / p r e s s e d f i r m l y . The d i r e c t i o n o f r o t a t i o n must be i n t o t h e b l a d e w i t h t h e t o p moving so t h a t t h e d i r e c t i o n o f t h e b l a d e w i l l t e n d t o d r i v e t h e t u b i n g i n t o t h e t a b l e and b a c k s t o p . W h i l e c u t t i n g t h e tube i n t o s h o r t e r l e n g t h s , much a c r y l i c s h a v i n g s a r e produced. Some o f t h i s m a t e r i a l i s needed i n making a bonding agent by m i x i n g i t i n w i t h 1,2 d i c h l o r o e t h a n e s o l v e n t . The s o l v e n t a l o n e i s e x t r e m e l y t h i n so t h a t t h e a d d i t i o n o f some a c r y l i c s h a v i n g s w i l l make i t more v i s c o u s . The s h a v i n g s a r e s o l u b i l i z e d i n t h e s o l v e n t and t h e t h i c k n e s s o f t h e agent depends on how much a c r y l i c i s added. Once t h e 6" l e n g t h s o f t h e 5.25" (0D> t u b i n g i s c u t , s m a l l e r l e n g t h s a r e r e q u i r e d f o r t h e cage ends ( t o p s and bottoms, both s i m i l a r ) . The e x a c t l e n g t h s a r e l i s t e d i n Appendix 6, as a r e a l l o t h e r d i m e n s i o n s f o r t h e v a r i o u s p a r t s o f each cage. The s m a l l e s t bands t h a t a r e needed t o h o l d i n p l a c e t h e 2-mesh w i r e , a r e e x t r e m e l y d i f f i c u l t t o c u t . To ease t h i s d i f f i c u l t y somewhat i t i s recommended t h a t a p i e c e o f t u b i n g from which t h e bands a r e t o be c u t be a minimum o f one f o o t l o n g . O t h e r w i s e , t h e r e i s d i f f i c u l t y i n s t a b i l i z i n g t h e tube w h i l e r o t a t i n g t h r o u g h t h e c u t . A f t e r c u t t i n g t h e l a r g e d i a m e t e r t u b i n g , t h e f e n c e and b a c k s t o p can be a d j u s t e d t o accomodate t h e 2.00" (0D) c a s t a c r y l i c t u b i n g (which i s t o be used f o r t h e e x t e n s i o n t o t h e f e e d e r and f o r t h e f e e d e r i t s e l f ) . The method f o r c u t t i n g i s 1 21 e x a c t l y t h e same as t h a t used w i t h t h e 5.25" m a t e r i a l . The f e n c e i s s e t a t t h a t d i s t a n c e from t h e b l a d e which w i l l y i e l d t h e needed l e n g t h s o f t u b i n g . For t h e round, f l a t p i e c e o f a c r y l i c ( o r p l e x i g l a s ) needed f o r t h e bottom o f each f e e d e r , 2" x 2" s q u a r e s a r e c u t w i t h t h e t a b l e s a w . T h i s 2" x 2" p i e c e i s g l u e d w i t h 1,2-d i c h l o r o e t h a n e and a l l o w e d t o s e t . Then a d i s t a n c e o f 2" i s measured from t h e g r i n d i n g b l a d e t o t h e f e n c e . The b l a d e i s r a i s e d t o " 1 " above t h e t a b l e and then each 2" x 2" square (now g l u e d t o t h e f e e d e r e x t e n s i o n p i e c e which can be used as a h a n d l e w h i l e r o u n d i n g - o f f t h e 2" x 2" > i s r o t a t e d c l o c k w i s e ( w h i l e f l a t on t h e t a b l e ) between t h e f e n c e and t h e g r i t on t h e s i d e o f t h e s p i n n i n g b l a d e . The r e s u l t i s a c i r c u l a r bottom cap f o r t h e f e e d e r . A f t e r t h e t u b i n g has been c u t t o t h e r e q u i r e d l e n g t h s t h e f e e d e r e x t e n s i o n w i t h t h e f e e d e r must be a t t a c h e d t o t h e main cage. H o l e s must be d r i l l e d i n t o t h e 6" p i e c e o f t h e 5.25" d i a m e t e r t u b i n g , and i n t o t h e f e e d e r e x t e n s i o n . A l s o , t h e l a t t e r must be shaped (by s a n d i n g ) t o f i t t h e c o n f o r m a t i o n o f t h e f e e d e r and t h e cage compartment. The h o l e s a r e d r i l l e d on a d r i l l p r e s s f i t t e d w i t h a s p e c i a l b r a c e t h a t p r e v e n t s t h e c y l i n d r i c a l tube from moving w h i l e t h e d r i l l i s used. On t h e 6" l e n g t h o f t h e 5.25" (OD) t u b e , a 1.75" d i a m e t e r h o l e i s d r i l l e d by u s i n g a 1.75" G r e e n l e e holesaw ( t h e holesaw i s mounted on a chuck b i t ) . P r i o r t o u s i n g t h e holesaw, a s m a l l e r h o l e i s d r i l l e d by t h e 1 2 2 chuck assembly t o s e r v e as a g u i d e f o r t h e saw. A f t e r d r i l l i n g t h e g u i d e h o l e , t h e a p p l i c a t i o n o f f o r c e must be v e r y g e n t l e (such t h a t t h e saw t e e t h c u t t i n g a c t i o n i s t h e o n l y downward movement) as any sudden o r s u b s t a n t i a l f o r c e by t h e o p e r a t o r would cause f l e x i o n i n t h e tub e ' s p e r f e c t c i r c u l a r shape and may break t h e t u b e . T h i s s t e p r e q u i r e s p a t i e n c e as t h e holesaw t a k e s a c o n s i d e r a b l e p e r i o d o f ti m e t o c u t t h r o u g h . To d e t e r m i n e where t o s t a r t d r i l l i n g w i t h t h e holesaw, measure 1.50" from one end o f t h e 6" l e n g t h . The p e r i m e t e r o f th e 1.75" d i a m e t e r o f t h e holesaw s h o u l d end 1.50" from one end o f t h e tube and 2.75" from t h e o t h e r end (see F i g u r e 16). S i m i l a r l y , a 1.75" d i a m e t e r h o l e i s made 1.00" from e i t h e r end o f t h e v e r t i c a l ( f e e d e r ) p i e c e . The e x t e n s i o n p i e c e t h a t c o n n e c t s t h e cage compartment w i t h t h e f e e d e r r e q u i r e s t h a t two h o l e s a r e d r i l l e d i n t o i t . On t h e lower s i d e o f t h e e x t e n s i o n p i e c e , a 1.25" d i a m e t e r h o l e i s d r i l l e d 1.25" from e i t h e r o f t h e two ends ( l e n g t h o f e x t e n s i o n p i e c e i s 3.75"). The f u n c t i o n o f t h i s h o l e i s t o p r e v e n t t h e r a t from p u l l i n g back any f e e d from t h e f e e d e r i n t o t h e main compartment. On t h e t o p o f t h e e x t e n s i o n p i e c e , a 0.50" di a m e t e r h o l e i s d r i l l e d ( t h e l o c a t i o n o f t h e d r i l l e d h o l e i s e x a c t l y h a l f way from e i t h e r end o f t h e tube l e n g t h ) . The f u n c t i o n o f t h i s h o l e i s t o a l l o w an a l l i g a t o r c l i p t o be a t t a c h e d t o t h e 4-mesh w i r e c y l i n d e r t h a t w i l l be i n s e r t e d i n the e x t e n s i o n p i e c e . The forward/backward adjustment o f t h e mesh a l l o w s t h e r a t t e c h n i c i a n t o e a s i l y p l a c e f e e d i n w i t h o u t 1 23 t h e p o s s i b i l i t y o f b e i n g b i t t e n and t h e r e b y s p i l l i n g f e e d . F i g u r e 17 shows t h e r e g i o n s where h o l e s a r e d r i l l e d i n t h e e x t e n s i o n p i e c e . A f t e r a l l o f t h e h o l e s have been d r i l l e d , b o t h ends (which a t t h i s p o i n t a r e p e r p e n d i c u l a r t o t h e l e n g t h o f t h e tube) o f t h e e x t e n s i o n p i e c e must be shaped t o f i t t h e c u r v a t u r e o f t h e f e e d e r (on one s i d e ) and t h e cage compartment (on t h e o t h e r ) . Two wooden drums ( o u t s i d e d i a m e t e r s ; 2" and 5.25") have been d e s i g n e d so t h a t they can be a t t a c h e d t o t h e d r i l l p r e s s and t h u s , made t o r e v o l v e a t t h e speed o f t h e d r i l l . The wood drums have been d r i l l e d t h r o u g h t h e i r c e n t e r ( a x i s ) , t h u s a l l o w i n g t h e i r attachment t o t h e d r i l l . To t h e o u t s i d e o f t h e drum, a a t r i p o f 40 (60 or 80) g r i t emery c l o t h (aluminum o x i d e sandpaper) i s i n s e r t e d , t o form a r e v o l v i n g g r i n d e r drum. T h i s d e v i c e i s used t o shape t h e ends o f t h e e x t e n s i o n p i e c e . O b v i o u s l y , t h e l a r g e r drum i s used t o shape t h a t end o f t h e p i e c e which w i l l be a t t a c h e d t o t h e cage compartment, w h i l e t h e s m a l l e r drum i s used t o shape t h a t end o f t h e p i e c e which w i l l j o i n w i t h t h e v e r t i c a l f e e d e r p i e c e . A f t e r t h e d r i l l i n g and s h a p i n g o f t h e a c r y l i c t u b i n g has been c o m p l e t e d , t h e a s s e m b l i n g / j o i n i n g p r o c e s s can b e g i n . The s o l v e n t 1,2 d i c h l o r o e t h a n e i s c a p a b l e o f f u s i n g two s e p a r a t e p i e c e s o f a c r y l i c by l i t e r a l l y s o l u b i l i z i n g t h e m a t e r i a l . Because t h e s o l v e n t a l o n e i s so t h i n , t h e a d d i t i o n o f some s h a v i n g s / b i t s o f a c r y l i c (from t a b l e s a w i n g ) cause t h e s o l v e n t t o t h i c k e n . The t h i c k e r s o l v e n t s e r v e s t o make a b e t t e r a d h e s i o n between t h e two p i e c e s b e i n g g l u e d . The 1 24 -1. 68 -1. 68' 0. 50" 1. 25" -1. 25' - i . 25"-F i g u r e 17. Measurement and placement o f d r i l l - h o l e s i n t h e 5 cm <2") e x t e n s i o n p i e c e . cage compartment 4 d r i l l e d h o l e s : 1 ) 2) 3) 4> 1. 75" 0. 50" 1. 25" 1. 75" 2 -f e e d e r e x t e n s i o n — 3 — bottom F i g u r e 18. S i m p l e s i d e - v i e w o f m e t a b o l i c cage a f t e r g l u i n g t h e f o u r p i e c e s (cage compartment, e x t e n s i o n , f e e d e r and bottom) t o g e t h e r . 125 s u r f a c e a r e a t h a t i s t o connect t h e e x t e n s i o n p i e c e w i t h t h e f e e d e r and t h e cage p a r t i s q u i t e s m a l l . Thus, i t i s advantageous t o use a t h i c k e r , more v i s c o u s s o l v e n t as i t a l l o w s one t o p l a c e i t e x a c t l y where t h e s o l v e n t needs t o be ( w i t h o u t t h e worry o f s o l v e n t r u n n i n g ) . A s m a l l r u b b e r s p a t u l a i s used as t h e s o l v e n t a p p l i c a t o r . To a l l o w t h e f r e s h l y " g l u e d " p i e c e s t o s e t , wide e l a s t i c bands a r e used t o clamp t h e j o i n t s i n t o p l a c e . A minimum o f two hours i s needed t o a l l o w a s t r o n g bond. The round d i s k - b o t t o m o f t h e f e e d e r i s g l u e d i n a s i m i l a r manner but no e l a s t i c bands a r e r e q u i r e d . Thus f a r , t h e cage s h o u l d l o o k somewhat as shown i n F i g u r e I S . The l i d s a r e made by t h e i n s e r t i o n o f a 2-mesh w i r e s c r e e n between two narrow bands (0.25") o f a c r y l i c t u b i n g . Because t h e 2 l i d s have t o f i t around t h e 5.25" o u t s i d e d i a m e t e r o f t h e r a t cage, t h e 1.25" ( l o n g ) band o f 5.25" 0D has t o be c u t l e n g t h w i s e (so t h a t t h e c i r c l e i s opened up and a l l o w s t h e band t o be a d j u s t e d ) . The two s m a l l e r bands a r e a l s o c u t i n t h e same manner so t h a t t h e y a r e a l l o w e d t o be widened p r i o r t o g l u i n g . The manner i n which each l i d i s assembled i s l o g i c a l . The f i r s t 0.25" band i s g l u e d t o t h e i n s i d e o f t h e ( s t r e t c h e d t o f i t t h e 5.25" 0D cage) 1.25" band. The 1.25" p i e c e i s wrapped around t h e main r a t compartment t o a l l o w a p e r f e c t l y snug f i t o f t h e l i d . About 0.5" o f t h e 1.25" band i s l e f t p r o t r u d i n g above t h e 6" l e n g t h o f t h e cage so t h a t t h e 0.25" band can be g l u e d t o t h e i n s i d e o f t h e l i d d i a m e t e r . S i n c e t h e t u b i n g f o r t h e l i d s has been c u t l o n g i t u d i n a l l y ( t o 1 26 a l l o w f o r e x p a n s i o n f o r f i t t i n g t h e OD), i t i s n e c e s s a r y t o e n s u r e t h a t t h e c u t i s p u r p o s e l y m i s a l i g n e d i n each o f t h e t h r e e bands t h a t c o m p r i s e th e l i d . F i g u r e 19 shows t h e l i d components. The 2-mesh w i r e s c r e e n i s c u t i n t o a c i r c u l a r shape (5.25" d i a m e t e r ) w i t h t h e use o f p l i e r s . The s c r e e n i s o n l y sandwiched between t h e two t h i n bands (which a r e g l u e d ) and i s not a c t u a l l y s e c u r e d ( i t s e l f ) t o t h e l i d . The 4-mesh s c r e e n tube t h a t i s r e q u i r e d f o r t h e i n s i d e o f t h e e x t e n s i o n tube i s made by c u t t i n g a 4.5" x 5.5" p i e c e o f mesh and r o l l i n g i t up i n t o a c y l i n d e r (4.5" i n l e n g t h ) t h a t f i t s s n u g l y i n s i d e t h e e x t e n s i o n t u b e . The i d e a l f i t t i n g c y l i n d e r i c a l tube (made from 2-mesh) would be l a r g e enough i n d i a m e t e r so t h a t a 100 g r a t c o u l d e a s i l y pass t h r o u g h y e t s m a l l enough t h a t i t doesn't b i n d t h e 1.75" i n s i d e d i a m e t e r (ID) o f t h e e x t e n s i o n t u b e . A f t e r r o l l i n g t h e mesh, t h e w i r e i s s o l d e r e d a t each o f t h e w i r e s . An endcap o f w i r e s c r e e n a l s o has t o be s o l d e r e d t o t h e end o f t h e newly c r e a t e d c y l i n d e r shape. T h i s c i r c l e - s h a p e d endcap s e r v e s t o keep th e r a t back as f e e d i s put i n t o t h e f e e d e r . F i n a l l y , a 1.50" l o n g n o t c h (and about o n e - t h i r d o f t h e d i a m e t e r o f t h e c y l i n d e r ) i s t o be c u t out o f t h e w i r e c y l i n d e r t o a l l o w t h e r a t a c c e s s t o t h e f e e d (when the w i r e c y l i n d e r i s p u l l e d f o r w a r d ) . To f u r t h e r i n h i b i t t h e r a t from c a r r y i n g f e e d back t o t h e cage compartment w i t h i t , a 1.75" o u t s i d e d i a m e t e r b r a s s 127 '0.60" l i p w i t h 5.25" ID 1. 25" band o f tube TT 25" band o f t u b i n g •2-mesh w i r e s c r e e n 0.25" band o f t u b i n g ro oo F i g u r e 19. End-on view of a cage l i d and i t s two bands o f t u b i n g which h o l d i n p l a c e t h e w i r e mesh s c r e e n . r i n g ( w i t h 2-mesh s c r e e n i n g ) i s p l a c e d on t h e f e e d . With t h i s r i n g i n p l a c e , t h e r a t must c a r e f u l l y l i c k s m a l l amounts o f f e e d t h r o u g h t h e mesh o p e n i n g s . The b r a s s r i n g s a r e made by c u t t i n g 0.50" segments from t h e b r a s s t u b e . To t h e h o l l o w i n s i d e o f t h e b r a s s r i n g a 2-mesh w i r e s c r e e n i s s o l d e r e d . P r i o r t o s o l d e r i n g g a l v a n i z e d s t e e l o r b r a s s , both m a t e r i a l s m a t e r i a l s must be p i c k l e d i n a s t r o n g a c i d ( h y d r o c h l o r i c ) s o l u t i o n t o c l e a n t h e s u r f a c e s , o t h e r w i s e , t h e s o l d e r w i l l not adhere t o t h e d e s i r e d s u r f a c e b u t , r a t h e r , w i l l remain on t h e s o l d e r i n g i r o n t i p . A s t a i n l e s s s t e e l f l u x ( y e l l o w l i q u i d ) i s recommended. S i n c e t h e cage i s t o be used f o r s e p a r a t e c o l l e c t i o n o f u r i n e and f e c e s , a f u n n e l t h a t f i t s s n u g l y i n s i d e t h e p r e c u t h o l e s i n t h e bench (where t h e complete m e t a b o l i c cage w i l l s i t ) i s n e c e s s a r y . The f u n n e l i s made by f i r s t l y , t r a c i n g t h e o u t l i n e p a t t e r n o n t o t h e a c e t a t e s h e e t . S e c o n d l y , t h e m a t e r i a l i s c u t w i t h s c i s s o r s . Then two h o l e s (0.125") a r e d r i l l e d i n each p a t t e r n t o a l l o w f o r r i v e t t i n g . To form a f u n n e l , an e y e l e t i s used t o h o l d t h e a c e t a t e t o g e t h e r . P r i o r t o a c t u a l l y s h a p i n g t h e s e f u n n e l s , t h e o p e r a t o r must become f a m i l i a r w i t h t h e use o f a heatgun. The a c e t a t e i s purchased i n a 22" x 51" s h e e t s , w i t h each s h e e t y i e l d i n g 10 f u n n e l s . The f u n n e l p a t t e r n / o u t l i n e f o r t r a c i n g onto t h e f l a t (0.030" t h i c k ) a c e t a t e s h e e t i s shown i n F i g u r e 20. The e a s i e s t way t o shape a f l a n g e on t h e upper 0.5" o f t h e f u n n e l i s by i n s e r t i n g t h e r i v e t t e d a c e t a t e i n t o a h o l e ( h a v i n g t h e same d i a m e t e r as t h e 129 4 28.5 cm- 8.0 cm 22.5 cm 2.5/ 19.5 cm 2 -1.5 cm 7.0 cm\ F i g u r e 20. T r a c e p a t t e r n d e a i g n e d f o r making a c e t a t e f u n n e l s , which a r e used t o c h a n n e l t h e u r i n e and f e c e s t o t h e i r s e p a r a t e c o n t a i n e r s . 130 h o l e s i n t h e r a t - r o o m benches i n t h e r a t l a b ) . With about 0.5" p r o t r u d i n g above th e s u r f a c e o f t h e bench, th e a c e t a t e i s heated w i t h a heatgun. As t h e a c e t a t e s t a r t s t o droop, th e heat s o u r c e i s removed and t h e f u n n e l i s p r e s s e d r e p e a t e d l y w i t h a scrapwood p i e c e u n t i l i t c o o l s . W h i l e t h e f u n n e l i s s t i l l i n s e r t e d i n t h e h o l e , a s m a l l d r i p - s p o u t i s formed a t t h e bottom o f t h e f u n n e l w i t h a p e n c i l - l i k e o b j e c t . The p r e s e n c e o f a spout a l l o w s t h e u r i n e t o d r i p o f f t h e a c e t a t e f u n n e l and i n t o a s e p a r a t e f l a s k when t h e cage i s i n use. To keep t h e f e c e s s e p a r a t e from th e u r i n e , a n y l o n net meshing i s used. I t i s c u t i n t h e shape o f a t e n n i s r a c k e t -head. A p a t t e r n f o r t h i s can be made by t r a c i n g an e x i s t i n g meshing and t h e n t r y i n g t h e new mesh i n a f u n n e l p r i o r t o mass-p r o d u c i n g t h e " p r o t o t y p e . " T h i s net has a l o n g e r " s p i l l w a y " as compared t o t h e a c e t a t e f u n n e l i t s e l f . T h i s a l l o w s t h e p e l l e t s o f f e c e s t o drop i n t o a s e p a r a t e f l a s k from t h e u r i n e . The n y l o n mesh i s c u t i n t o such a shape so as t o a l l o w t h e a c e t a t e f u n n e l t o be e n t i r e l y c o v e r e d and t h e chances o f f e c e s g e t t i n g under t h e net a r e m i n i m i z e d . S c i s s o r s a r e a l s o adequate f o r c u t t i n g t h i s n y l o n n e t t i n g . Completed cage components a r e shown i n F i g u r e 21. 131 F i g u r e 21. Completed u n i t s i l l u s t r a t e t h e a b s o l u t e e s s e n t i a l p a r t s o f a m e t a b o l i c cage t h a t f r e q u e n t l y r e q u i r e maintenance (not shown i s t h e n y l o n mesh n e t t i n g t h a t f i t s i n s i d e t h e a c e t a t e f u n n e l ) . 132 APPENDIX 5 M a t e r i a l f o r t h e c o n s t r u c t i o n o f t e n metabolism cages, Description of raw material Size purchased Supplier Cost 5.25" (outside diameter) cast acrylic tubing with 0.125" nail thickness Cadillac Plastics 2294 Douglas St., Burnaby, B.C. 299-8937 *13/foot 2" (0D) cast acrylic tubing K i t h 0.125" wall thickness $10/foot 0.125" (3 mm) plexiglass flat piece (feeder bottoms) 0.030" thick acetate sheet galvanized steel (square) Hire meshing; 2 x 2 mesh, 0.072" diameter wire, 0.428" opening. galvanized steel (square) wire meshing; 4 x 4 mesh, 0.030" diameter wire, 0.220" opening 1.75" (0D) brass tubing 1.25° wall thickness 4" x 10" 22" x 51" 6 square feet 3 square feet 1' length C « E Mesh Products, #201 - 204 Cayer St., Coquitlam, B.C. 524-3606 $5 $35/sheet N.A. plumbing shop metal flashing for water bottles 1 square foot building yard $8 1, 2 dichloroethane solvent (toxic) for joining acrylic tubing 8" table saw, d r i l l press 1.75" Greenlee holesaw small bottle Technician (Soil Science) Dept. of Soil Science ftcklands Ltd., Vancouver, B.C. $14 chuck bit for holesaw mount BioResource Engineering workshop 1000 watt heatgun 40- and 60-grit aluminum oxide paper (emery cloth) cylindrical wooden drums (5.25° and 2") for shaping tube joints Physical Plant Stores stored in cupboards in Rat laboratory at South Campus (Swine Unit) 1 3 3 APPENDIX 6 Dimensions o f t h e r a t - m e t a b o l i s m cage components. Component Description OD ID Length Modifications cast acrylic tubing for main cage compartment 5.25° 5.00" 6.00" 1.75" hole drilled with hole edge 1.5" from one end cast acrylic tubing for feeder section 2.00" 1.75° 3.88" 1.75° hole drilled with hole edge 1" from either end Cast acrylic tubing for 2.00° 1.75° extension piece 3.75" 0.5" hole drilled on top, 1.25° hole drilled on bottom. Holes centered in lengthwise direction. Band cut from cast 5.25° 5.00° acrylic tubing (for lid) 1.25" tube cut to allow expansion of band 2 bands cut from 5.25" cast acrylic tubing (for lid) 5.00° 0.25" both bands cut to allow expansion 2-mesh wire screen cut to f i t inside 5.25" diameter (inside lid) 4-mesh wire screen cut to 4.5" x 5.5", soldered into a cylinder with diameter that will f i t inside 1.75° ID tubing and fitted with an endcap flat piece of 0.125" acrylic for feeder cut into 2° x 2" squares, then ground into circles 0.030" thick acetate sheeting for funnels pattern traced, cut out and 2 holes drilled (0.125"), then eyelets rivetted. 1.75" 0D brass tube for feeder rings cut in 0.5" lengths, pickled, fitted and soldered with 2-mesh screen. 1 34 

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