UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The utilization of heat coagulated beer wort protein (trub) Yeow, Tony 1978

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1978_A6_7 Y46.pdf [ 4.03MB ]
Metadata
JSON: 831-1.0094494.json
JSON-LD: 831-1.0094494-ld.json
RDF/XML (Pretty): 831-1.0094494-rdf.xml
RDF/JSON: 831-1.0094494-rdf.json
Turtle: 831-1.0094494-turtle.txt
N-Triples: 831-1.0094494-rdf-ntriples.txt
Original Record: 831-1.0094494-source.json
Full Text
831-1.0094494-fulltext.txt
Citation
831-1.0094494.ris

Full Text

THE UTILIZATION OP HEAT COAGULATED BEER WORT PROTEIN (TRUB). B.Sc. ( A g r ) , U n i v e r s i t y o f B.C., 1975 t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l m e n t the r e q u i r e m e n t s f o r the degree o f THE FACULTY OF GRADUATE STUDIES . DEPARTMENT OF FOOD SCIENCE We ac 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 the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA June, 1978 TONY YEOW MASTER OF SCIENCE i n Tony Yeow, 197 8 In presenting t h i s thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it f r e e l y available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head-of my Department or by his representatives. It is understood that copying or publication of this thesis for f i n a n c i a l gain shall not be allowed without my written permission. V Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i A b s t r a c t Trub o b t a i n e d from the br e w i n g p r o c e s s was e x t r a c t e d u s i n g i s o p r o p a n o l as s o l v e n t t o y i e l d a b i t t e r hop r e s i n f r a c t i o n and an i n s o l u b l e p r o t e i n complex. The p r o t e i n was s u b j e c t e d t o an amino a c i d a n a l y s i s ; l y s i n e was t h e l i m i t -i n g amino a c i d i n t r u b p r o t e i n , f o l l o w e d by i s o l e u c i n e . I t s e s s e n t i a l amino a c i d i n d e x was a p p r o x i m a t e l y e q u i v a l e n t t o t h a t of b a r l e y p r o t e i n . F u n c t i o n a l l y , i t was found t h a t t r u b p r o t e i n had poor s o l u b i l i t y , p a r t i c u l a r l y a t a c i d pH v a l u e s , but t h a t i t s w a t e r - h o l d i n g c a p a c i t y was good. Q u a n t i t a t i v e e v a l u a t i o n of the t r u b r e s i n was c a r r i e d out by l e a d conductometry, the l e a d conductance v a l u e of 68% b e i n g used as a guide t o p o t e n t i a l b r e w i n g v a l u e . The e x t r a c t e d r e s i n was a l s o examined q u a l i t a t i v e l y f o r i t s b i t t e r i n g p o t e n t i a l by TLC. A l t h o u g h many r e s i n components were s e p a r a t e d by means of TLC, d i f f i c u l t i e s w i t h i d e n t i f -i c a t i o n y i e l d e d i n c o n c l u s i v e r e s u l t s i n t h i s a r e a . F i n a l l y , the b i t t e r i n g c a p a c i t y o f the t r u b r e s i n was o r g a n o l e p t i c -a l l y compared t o f r e s h hops i n p r e p a r e d b e e r s . Trub r e s i n was ca p a b l e o f b i t t e r i n g beer but i m p a r t e d a h a r s h e r c h a r -a c t e r t o beer t h a n f r e s h hops. Moreover, the r e s i n tended t o i n h i b i t t he f o r m a t i o n o f a s t a b l e y e a s t head d u r i n g the a l e f e r m e n t a t i o n . F i n a l l y , t he a c t u a l r e s i n e x t r a c t i o n procedure was e m p i r i c a l l y examined t o determine the combined e f f e c t of a g i t a t i o n and s o l v e n t - r a t i o on the y i e l d of r e s i n and p r o t e i n . P r o t e i n y i e l d was unaffected by these 2 f a c t o r s , but r e s i n y i e l d was found to be most e f f i c i e n t when employing maximum a g i t a t i o n and using the minimum amount of so l v e n t . Using more solvent d i d not s i g n i f i c a n t l y increase r e s i n y i e l d and tended to depress the e f f e c t of a g i t a t i o n . i i i LIST OF CONTENTS Page I n t r o d u c t i o n . 1 L i t e r a t u r e r e v i e w 3 M a t e r i a l s and methods 8 Continuous e x t r a c t i o n method 8 B a t c h e x t r a c t i o n method 8 Recovery of p r o t e i n f l o u r , r e s i n and the w a t e r - d i s p e r s i b l e f r a c t i o n 9 M o i s t u r e d e t e r m i n a t i o n 10 Measurement of the weight of r e s i n e x t r a c t e d 10 Lead conductance v a l u e of the t r u b r e s i n 12 T h i n l a y e r chromatography o f the hop and t r u b r e s i n s 12 P r o t e i n d e t e r m i n a t i o n 14 Amino a c i d a n a l y s e s o f the t r u b p r o t e i n f l o u r 14 N i t r o g e n s o l u b i l i t y p r o f i l e o f the t r u b p r o t e i n f l o u r 15 E m u l s i f y i n g c a p a c i t y o f the t r u b p r o t e i n f l o u r 15 W a t e r - h o l d i n g c a p a c i t y o f the t r u b p r o t e i n f l o u r . . . . 16 C o l o r d e t e r m i n a t i o n of the t r u b p r o t e i n f l o u r 17 The p r e p a r a t i o n o f beer f o r t a s t e e v a l u a t i o n s 17 T a s t e e v a l u a t i o n s i n o r d e r t o determine the d i f f e r e n c e i n b i t t e r n e s s v a l u e s 18 i v LIST OP CONTENTS.continued' Page R e s u l t s and d i s c u s s i o n 20 P a r t A: The e x t r a c t i o n 20 P a r t B: E v a l u a t i o n of the p r o t e i n f r a c t i o n 39 P a r t C: E v a l u a t i o n o f the t r u b r e s i n f r a c t i o n 56 Appendix 70 R e f e r e n c e s 77 V LIST OP TABLES Table- Page 1. The dry weight of r e s i n - e x t r a c t e d trub 22 2 . The percent y i e l d of crude r e s i n e x t r a c t e d from wet and • dry trub by the continuous r e f l u x method 24 3. Percent y i e l d of crude r e s i n b a t c h - e x t r a c t e d from wet pressed trub with i s o p r o p a n o l 26 4. The y i e l d of d r i e d trub p r o t e i n f l o u r a f t e r 5 e x t r a c t i o n s at 3 d i f f e r e n t s o l v e n t r a t i o s of volume of i s o p r o p a n o l : weight of wet trub 28 5. A summary of the two-way f a c t o r i a l a n a l y s i s of v a r i a n c e with r e p l i c a t i o n f o r ( i ) the t o t a l y i e l d of r e s i n a f t e r 5 c o n s e c u t i v e e x t r a c t i o n s , ( i i ) y i e l d of r e s i n a f t e r one e x t r a c t i o n 29 6. The number of t h e o r e t i c a l e x t r a c t i o n s r e q u i r e d to o b t a i n 1% and 0.1% of the i n i t i a l r e s i n l e v e l (w/w) at a constant y i e l d of 115 g (23%) of d r i e d p r o t e i n f l o u r , e x t r a c t e d from 500 g wet pressed trub 3 3 7. The d e b i t t e r i n g equations d e r i v e d from the l i n e a r r e g r e s s i o n of log-j_g r e s i n weights (Y) vs. the number of e x t r a c t i o n s (X) 35 8. A r e c i p e f o r the p r e p a r a t i o n of muffins which c o n t a i n trub p r o t e i n f l o u r 36 9 . The c o l o r e v a l u a t i o n of d r i e d trub p r o t e i n f l o u r 37 v i LIST OP TABLES c o n t i n u e d T a b l e Page 10. G e n e r a l y i e l d d a t a showing v a r i a b i l i t y o f the v a r i o u s . a n a l y t i c a l e s t i m a t e s . 4 l 11. The p r o t e i n c o n t e n t (6.25 x N) of v a r i o u s t r u b f r a c t i o n s , soy p r o t e i n i s o l a t e and soy f l o u r 42 12. The amino a c i d c o m p o s i t i o n of t r u b p r o t e i n f l o u r (g / l6g'N) M 13- The n i t r o g e n s o l u b i l i t y p r o f i l e o f t r u b p r o t e i n f l o u r , f r e e z e - d r i e d t r u b , and t r u b p r o t e i n f l o u r i n 1 M NaCI s o l u t i o n (g s o l u b l e p r o t e i n / l O O g p r o t e i n ) 52 14. A comparison of the e m u l s i f y i n g c a p a c i t y of t r u b p r o t e i n f l o u r , soy p r o t e i n f l o u r , soy p r o t e i n i s o l a t e and the w a t e r - d i s p e r s i b l e f r a c t i o n (ml o i l per gram sample ) 54 15. A comparison of the water h o l d i n g c a p a c i t y of t r u b p r o t e i n f l o u r , soy f l o u r and soy i s o l a t e (g w a t e r / g sample) 55 16. Summary of c h e m i c a l s c o r e c a l c u l a t i o n f o r t r u b p r o t e i n f l o u r by o l d and new method ( I & I I ) ^6 17- Summary of the E s s e n t i a l Amino A c i d Index c a l c u l a t i o n f o r t r u b p r o t e i n f l o u r (method I I I ) ^7 18. E s s e n t i a l amino a c i d s of v a r i o u s p r o t e i n s e x p r e s s e d as a p e r c e n t a g e of the c o r r e s p o n d i n g amino a c i d o f s t a n d -a r d egg p r o t e i n (method I ) 9^ v i i LIST OF TABLES c o n t i n u e d T a b l e Page 19. A comparison of the amino a c i d c o m p o s i t i o n o f t r u b p r o t e i n , b a r l e y , wheat, soybean and rapeseed (33,34).. 50 20. T.L.C. comparison o f f r e s h hop components s t o r e d a t 10°C f o r d i f f e r e n t time p e r i o d s 57 21. T.L.C. comparison of t r u b r e s i n components s t o r e d a t 10°C f o r zer o and 6 months..... 58 22. To show t h e number o f ml o f l e a d a c e t a t e r e q u i r e d . t o p r e c i p i t a t e the 4 main t r u b r e s i n f r a c t i o n s & t o show the l e a d conductance v a l u e c a l c u l a t i o n 6l 23. Summary o f p a i r e d comparison r e s u l t s t o determine i f d i f f e r e n c e s i n b i t t e r n e s s i s d e t e c t a b l e between: 1. F r e s h hop samples and 100% t r u b r e s i n samples, 2. F r e s h hop samples and 50% t r u b r e s i n samples, 3. F r e s h hop samples and p o o l e d t r u b r e s i n samples... 65 24. Summary o f Ch i - s q u a r e a n a l y s i s w i t h Y a t e s ' c o r r e c t i o n (47) a p p l i e d t o determine l e v e l o f s i g n i f i c a n c e between 1, 2 and 3 (see t a b l e 23) . . . 6 6 25. Summary o f the t e s t ( C h i - s q u a r e a n a l y s i s w i t h Y a t e s ' c o n t i n u i t y c o r r e c t i o n ) t o determine i f the e x t e n t o f d i f f e r e n c e s ( c h o i c e o f which i s more b i t t e r ) was due to chance o r was s i g n i f i c a n t 67 26. Comparative b i t t e r n e s s l e v e l s i n l o c a l (B.C.) b e e r s . . . 69 v i i i LIST OP FIGURES F i g u r e Page 1. B a t c h e x t r a c t i o n scheme f o r t r u b 11 2. S t a n d a r d curve f o r e s t i m a t i o n of s o l u b l e r e s i n c o n c e n t r a t i o n i n i s o p r o p a n o l 13 3. E f f e c t of s o l v e n t r a t i o on r e s i n y i e l d i n 5 minute e x t r a c t i o n s '30 4. The e f f e c t o f a g i t a t i o n time on r e s i n y i e l d % 1:1 s o l v e n t r a t i o 31 5. Approximate y i e l d s i n weight % of the v a r i o u s f r a c t i o n s e x t r a c t e d from wet p r e s s e d t r u b 40 6. N i t r o g e n s o l u b i l i t y p r o f i l e o f t r u b p r o t e i n f l o u r b e f o r e e x t r a c t i o n , a f t e r e x t r a c t i o n and i n 1 M NaCI.... 53 7. Lead conductance p l o t f o r t r u b r e s i n e x t r a c t . . 62 ix ACKNOWLEDGEMENTS I extend my deepest g r a t i t u d e t o Dr. P. Townsley f o r h i s guidance i n t h e d i r e c t i o n o f t h e t h e s i s and f o r h i s c o n s t a n t encouragement throughout the s t u d y . F u r t h e r m o r e , I thank Molson Brewery B. C. L t d . f o r t h e i r c o o p e r a t i o n and f u n d i n g which made t h i s s t u d y p o s s i b l e . 1 INTRODUCTION I n the f i s c a l y e a r e n d i n g 31st March 1975, 459 m i l l i o n g a l l o n s o f beer were produced i n Canada (28). I t has been e s t i m a t e d t h a t as much as 500 mg o f t r u b ( d r y w e i g h t ) may be p r e c i p i t a t e d per l i t e r of wort b o i l e d (3). Thus, i t may be c r u d e l y e s t i m a t e d t h a t some 1150 tons o f t r u b i n dry weight were produced i n Canada f o r the y e a r 1974-75• T h i s unwanted m a t e r i a l i s d i s p o s e d i n v a r i o u s ways. Where hop j a c k s a re used, the t r u b i s trapped, i n the hop f i l t e r bed and d i s p o s e d t o g e t h e r w i t h the spent hops. Where o t h e r methods f o r hop s e p a r a t i o n a re used, the t r u b i s c o l l e c t e d s e p a r a t e l y from the spent hops, e i t h e r by c e n t r i f u g a t i o n or by the use o f a " w h i r l p o o l " s e p a r a t o r . I n the l a t t e r c a s e , . t h e t r u b i s d i s -posed e i t h e r by i n c o r p o r a t i o n , w i t h , o t h e r brewery waste m a t e r i a l s such as spent g r a i n s and y e a s t or d i v e r t e d . i n t o the brewery e f f l u e n t stream. Thus i t may be seen t h a t at the p r e s e n t t i m e , t r u b i s o f n e g a t i v e economic v a l u e . Where i t i s i n c o r p o r a t e d i n t o spent g r a i n s f o r use as f e e d , the b i t t e r f l a v o r o f the t r u b g e n e r a l l y l o w e r s the f e e d q u a l i t y of the g r a i n . T h e . d i v e r s i o n of t r u b i n t o the e f f l u e n t stream i s an unwarranted s o l u t i o n as t h e r e i s an i n c r e a s i n g t r e n d toward more s t r i n g e n t p o l l u t i o n c o n t r o l s a f f e c t i n g f o o d p l a n t s by o b l i g a t i n g them t o i n s t a l l c o s t l y waste d i s p o s a l systems t o handle m a t e r i a l o f ,no.economic v a l u e . T h e r e f o r e i t i s l o g i c a l t o r e a s s e s s the p o t e n t i a l v a l u e o f 2 waste i n g e n e r a l whether f o r p a r t i a l o r complete waste r e u t i l i z -a t i o n , o r i n o r d e r t o lower the l o n g term c o s t f o r waste t r e a t -ment . A c u r s o r y e x a m i n a t i o n o f t r u b r e v e a l s t h a t i t c e r t a i n l y m e r i t s a t t e n t i o n . I t has been r e p o r t e d (1) t h a t t r u b c o n s i s t s o f 50$ - 60% p r o t e i n , 20 - 30% p o l y p h e n o l s , 16 - 20% hop r e s i n s and 2-3% ash. From a f o o d s o u r c e s t a n d p o i n t , the h i g h p r o t e i n c o n t e n t may be a bonus. A l s o s i n c e hops are the s i n g l e most e x p e n s i v e i t e m on the brewer's l i s t of-raw m a t e r i a l s , the 16--20% r e s i n c o n t e n t d e s e r v e s some a t t e n t i o n i n terms o f i t s p o t e n t i a l b i t t e r i n g v a l u e . Compared w i t h o t h e r n a t u r a l and n o v e l p r o t e i n s o u r c e s such as c o t t o n s e e d , rape and l e a f p r o t e i n , t r u b i s r e l a t i v e l y f r e e of t o x i c components such as t h i o g l u c o s i d e s i n rape or g o s s y p o l i n c o t t o n s e e d . S i n c e t r u b p r o t e i n i s d e r i v e d from b a r l e y t h a t has been a s t a p l e meal f o r c e n t u r i e s , the p o s s i b i l i t y o f t o x i c e f f e c t i s remote. F u r t h e r m o r e , because of the p r o l o n g e d b o i l i n g t r e a t m e n t , t r u b i s l i k e l y t o be f r e e from p a t h o g e n i c m i c r o -organisms . T h i s study was l a u n c h e d w i t h the o b j e c t i v e o f waste r e u t i l i z a t i o n and the r e s u l t s o b t a i n e d suggested t h a t e v e n t u a l l y a l l waste m a t e r i a l s w i l l be i n c o r p o r a t e d i n the p r o d u c t i o n scheme. 3 LITERATURE REVIEW There are no d i r e c t r e f e r e n c e s i n the l i t e r a t u r e on the r e u t i l i z a t i o n o f t r u b per s e , not even f o r use as f e e d . The b rewing t e x t by Hough, B r i g g s and Stevens (1) r e f e r s t o t r u b as an i n s o l u b l e coagulum, o c c u r r i n g i n p a r t i c l e s about 30 - 80 m i c r o n s i n d i a m e t e r , w i t h a s p e c i f i c g r a v i t y o f 1.2 -1.25. I t c o n s i s t s of 50 - 60% p r o t e i n , 16 - 20% hop r e s i n s , 20 - 30% p o l y p h e n o l s and 2-3% ash. A l t h o u g h heat i s the p r i n c i p a l p r e c i p i t a t i n g f a c t o r i n t r u b f l o c c u l a t i o n , the p resence of t a n n i n s and p o l y p h e n o l i c compounds d e r i v e d from the added hops a i d t r u b f o r m a t i o n by c o m p l e x i n g w i t h the a v a i l a b l e p r o t e i n . P r o t e i n - p o l y p h e n o l i n t e r a c t i o n s have been s t u d i e d m a i n l y w i t h r e g a r d t o haze f o r m a t i o n but b rewing t e x t s g e n e r a l l y agree t h a t s i m i l a r p r o t e i n - p o l y p h e n o l i c i n t e r a c t i o n s o c c u r d u r i n g wort b o i l i n g . The c h e m i c a l f o r c e r e s p o n s i b l e f o r the i n t e r a c t i o n s i s presumably hydrogen bonds, the s t r o n g e s t b e i n g t h a t formed between N - s u b s t i t u t e d amides and p h e n o l s . Hop . r e s i n s are c l a s s i f i e d as p o l y p h e n o l s and hence as w e l l promote t r u b p r e c i p i t a t i o n by a s i m i l a r mechanism. R e s i n s may a l s o c o p o l y m e r i z e w i t h t a n n i n s . The e s t i m a t e d , p r o d u c t i o n of trub. v a r i e s . B r i g g s et a l (1) r e p o r t e d 75 - 100 lb.wet weight per 1630 h e c t o l i t e r s of b e e r ; Luers (2) e s t i m a t e d 0.25 kg t r u b dry weight, per 100 kg of malt and a c c o r d i n g t o Royston (3) i t was 500 mg dry weight t r u b per l i t e r o f wort b o i l e d . 4 The p r o t e i n p o r t i o n o f t r u b has been o f l i t t l e i n t e r e s t so f a r . No d i r e c t i n f o r m a t i o n on r e u t i l i z a t i o n of t h i s p r o -t e i n i s a v a i l a b l e . The n i t r o g e n o u s c o n s t i t u e n t s o f wort account f o r 3 - 5% o f t h e mash e x t r a c t and i n c l u d e s m a l l m o l e c u l a r weight compounds such as p e p t i d e s and n u c l e i c a c i d s i n a d d i t i o n t o p r o -t e i n s . Guenther & S t u t l e r (4) i n 1965 showed the e f f e c t of b o i l i n g on the m o l e c u l a r weight d i s t r i b u t i o n o f the wort p r o t e i n s (appendix 1). I t i s shown t h a t most o f the h i g h m o l e c u l a r weight p r o t e i n s were p r e c i p i t a t e d as t r u b a f t e r b o i l i n g f o r 95 m i n u t e s . Compared w i t h o t h e r c e r e a l g r a i n s , b a r l e y has a h i g h a l b u m i n or water s o l u b l e p r o t e i n f r a c t i o n (appendix 2). D u r i n g g e r m i n a t i o n , t h e s o l u b l e n i t r o g e n , r e p r e s e n t e d m a i n l y by a l b u m i n , g l o b u l i n and the n o n - p r o t e i n n i t r o g e n f r a c t i o n , show.: an o v e r a l l i n c r e a s e w i t h a concomitant decrease i n h o r d e i n and g l u t e l i n (appendix 3). D u r i n g mashing, t h e s o l u b l e n i t r o g e n i s e x t r a c t e d and becomes p a r t o f t h e wor t . D u r i n g the wort b o i l i n g , most o f the h i g h e r m o l e c u l a r weight n i t r o g e n compounds are p r e c i p i t a t e d (appendix 1) and t o g e t h e r w i t h the spent .hops c o n s t i t u t e , the hot break o r t r u b . As a r e s u l t , t r u b n i t r o g e n c o n s i s t s m a i n l y o f the ( i ) s o l u b l e , ( i i ) heat c o a g u l a b l e and ( i i i ) h i g h m o l e c u l a r weight compounds. Presumably t h e n , more a l b u m i n and g l o b u l i n p r o t e i n s would be p r e c i p i t a t e d t h a n h o r d e i n o r g l u t e l i n . I t i s g e n e r a l l y r e c o g n i z e d t h a t the albumins and g l o b u l i n s f u n c t i o n as enzymes and o t h e r b i o l o g i c a l l y a c t i v e compounds . Munck (5) has mentioned t h a t b a r l e y g r a i n e x t r a c t s p o s s e s s the a c t i v i t i e s o f phosphatase, beta-amylase and p r o t e a s e . Because of t h e i r r o l e as f u n c t i o n a l enzymes, the c e r e a l albumins and g l o b u l i n s g e n e r a l l y demonstrate a more b a l a n c e d amino a c i d p r o f i l e t h a n the i n s o l u b l e p r o t e i n s . Por c e r e a l p r o t e i n s i n g e n e r a l , S h u k l a (6) has n o t e d t h a t the s o l u b l e f r a c t i o n i s r i c h e r i n t r y p t o p h a n and i o r i i z a b l e r e s i d u e s such as a r g i n i n e , l y s i n e and g l u t a m i c a c i d . They a l s o have more s u l f u r c o n t a i n i n g amino a c i d s . A comparison of t h e .amino a c i d p r o f i l e between b a r l e y embryo, where the s o l u b l e p r o t e i n s p r e -dominate, and b a r l e y endosperm, where the i n s o l u b l e m a t r i x p r o t e i n l o c a t e , show t h a t i n g e n e r a l , s o l u b l e b a r l e y p r o t e i n i s r i c h e r t h a n i n s o l u b l e b a r l e y p r o t e i n i n the e s s e n t i a l amino a c i d s (appendix 4 ) . A l l t h e s e f i n d i n g s suggest t h a t p o s s i b l e use may e x i s t f o r t r u b p r o t e i n , e s p e c i a l l y from the n u t r i t i o n a l a s p e c t . W i t h r e g a r d t o the spent b i t t e r s u b s t a n c e s p r e s e n t i n t r u b , some i n v e s t i g a t i o n s have been conducted. S p e t s i g (7) examined the b i t t e r s u b s t a n c e s of spent hops, t r u b and the yeast~,cover u s i n g r e v e r s e d phase p a r t i t i o n chromatography t o s e p a r a t e them. Laws (8) a l s o examined the o x i d a t i o n p r o d u c t s of the a l p h a and b e t a - a c i d s and t h e i r s i g n i f i c a n c e i n b r e w i n g . I t appears t h a t ( i ) a l t h o u g h some o f the t r a n s f o r m a t i o n s which t a k e p l a c e d u r i n g the wort b o i l i n g are t o n o n - b i t t e r end p r o d u c t s , a s i g n i f i c a n t p o r t i o n o f the b i t t e r s u b s t a n c e s remain i n the form of o x i d i s e d p r o d u c t s , ( i i ) t h a t t h e s e o x i d a t i o n p r o d u c t s may have some brewing v a l u e . S p e c i f i c a l l y , . i t i s known t h a t : ( 1 ) the b e t a - a c i d s themselves c o n t r i b u t e l i t t l e b i t t e r n e s s t o beer but t h e i r o x i d i s e d p r o d u c t s (hulupone, i t s analogues and d e r i v a t i v e s } , which may form on b o i l i n g , are b i t t e r , b e i n g a p p r o x i m a t e l y 50% as b i t t e r as t r a n s - i s o - h u m u l o n e (27). (2) h u m u l i n i c a c i d , formed from the h y d r o l y s i s o f a l p h a - a c i d (humulone) i s n o n - b i t t e r , and t h e r e f o r e u n d e s i r a b l e . (3) the o x i d i s e d i s o - a l p h a - a c i d s , formed d u r i n g wort b o i l i n g , have 10 - 20% the b i t t e r i n g e f f e c t o f t r a n s - i s o - h u m u l o n e . (4) the f u r t h e r o x i d a t i o n p r o d u c t s of hulupone c o n t r i b u t e n e g l i g i b l e b i t t e r n e s s t o beer (27). (5) the i s o - a l p h a - a c i d s are e x t r e m e l y b i t t e r and t h e r e f o r e d e s i r a b l e ; on the o t h e r hand, the o x i d a t i o n p r o d u c t s o f humulone are g e n e r a l l y n o n - b i t t e r . I n summary, i t may be seen t h a t l i t t l e work has been done on t r u b u t i l i z a t i o n per se. R e l a t e d work i n d i c a t e s t h a t the r e s i n o u s components may be bound by hydrogen bonding t o the p r o t e i n f r a c t i o n or be c o p o l y m e r i z e d w i t h o t h e r p o l y p h e n o l i c m a t e r i a l s . I f e x t r a c t a b l e , t h i s r e s i n m a t e r i a l w i l l most l i k e l y be o x i d i s e d . S i n c e some o x i d a t i o n p r o d u c t s have been shown t o be b i t t e r , b i t t e r i n g p o t e n t i a l may s t i l l be p r e s e n t i n t h e r e s i n e x t r a c t e d from t r u b . The p r o t e i n f r a c t i o n i s most l i k e l y 7 d e r i v e d from th e s o l u b l e f r a c t i o n of the b a r l e y g r a i n . Thermal d e n a t u r a t i o n i s c e r t a i n t o have t a k e n p l a c e d u r i n g the p r o l o n g e d b o i l i n g t r e a t m e n t . Because i t i s most l i k e l y d e r i v e d from the s o l u b l e p r o t e i n f r a c t i o n , t r u b p r o t e i n may have some n u t r i t i o n a l p r o m i s e . 8 MATERIALS AND METHODS Trub was s u p p l i e d c o u r t e s y o f Molson Brewery L t d . o f 1550 B u r r a r d S t . , Vancouver. The c h e m i c a l s used f o r e x t r a c t i o n were o f a n a l y t i c a l grade ( F i s h e r S c i e n t i f i c Co. L t d . ) . Continuous E x t r a c t i o n Method: c A G o l d f i s l h f a t e x t r a c t o r (Labconco 35001) based on a r e f l u x p r i n c i p l e was used t o c o n t i n u o u s l y e x t r a c t r e s i n from s o l i d t r u b samples. Both wet and dry samples were e x t r a c t e d at the lo w e s t temperature s e t t i n g f o r 5 days u s i n g t h r e e d i f f e r -ent s o l v e n t s , pure i s o p r o p y . l a l c o h o l , an a z e o t r o p i c m i x t u r e o f i s o p r o p y l a l c o h o l and wa t e r , and hexane. . F o r the e x t r a c t i o n o f wet p r e s s e d t r u b c o n t a i n i n g 26.5 - 28.3% dry m a t t e r , a 5 g sample s i z e was used. A 1.25 g sample was used f o r the e x t r a c t i o n o f f r e e z e - d r i e d samples. The d r i e d s o l v e n t chambers were t a r e d p r i o r t o the e x t r a c t i o n . A f t e r e x t r a c t i o n , t he r e m a i n i n g s o l v e n t was e v a p o r a t e d o f f at 50°C and the c o o l e d chambers reweighed t o o b t a i n t o t a l weight o f e x t r a c t p l u s chamber. Net weight o f e x t r a c t was o b t a i n e d by d i f f e r e n c e . B a t c h E x t r a c t i o n Method: P r i o r t o b a t c h e x t r a c t i o n , the excess water was removed from the wet t r u b by s q u e e z i n g t h r o u g h cheese c l o t h . A s t e r i l -i z e d mop s t r a i n e r was employed t o press, out as much excess water as p o s s i b l e . A p p r o x i m a t e l y 50 - 56% water was removed i n t h i s manner thus h a l v i n g the o r i g i n a l weight of t r u b . T h i s wet p r e s s e d t r u b , c o n t a i n i n g 26.5 - 28.3% dry m a t t e r , was the raw m a t e r i a l used i n every b a t c h e x t r a c t i o n . 500 g o f wet p r e s s e d t r u b was weighed i n t o a 4000 ml N a l -gene beaker and a f i x e d volume of i s o p r o p y l a l c o h o l added. The s o l i d - l i q u i d m i x t u r e was a g i t a t e d by means of a top d r i v e p r o p e l l e r t u r n i n g at 700 rpm. The beaker was c o v e r e d d u r i n g a g i t a t i o n to p r e v e n t e v a p o r a t i o n and s p i l l a g e . The s h a f t was m a i n t a i n e d at V from the bottom of the. beaker t o ensure as u n i f o r m an a g i t a t i n g m otion as p o s s i b l e . A f t e r a g i t a t i n g f o r a s p e c i f i e d t i m e , the s l u r r y was v a c u u m . f i l t e r e d t h r o u g h a #4 Whatman f i l t e r paper on a 18 cm d i a m e t e r Buchner f u n n e l . Vacuum was m a i n t a i n e d u n t i l no more s o l v e n t f i l t e r e d t h r o u g h . The e x t r a c t e d t r u b would t h e n be r e - e x t r a c t e d or a i r d r i e d a t room temperature depending on the number of e x t r a c t i o n s r e q u i r e d i n the experiment'. By means of t h i s b a s i c p r o c e d u r e , 60 d i f f e r e n t e x t r a c t i o n s were performed u s i n g 5 s e q u e n t i a l e x t r a c t i o n s , 4 d i f f e r e n t e x t r a c t i o n t i m e s f o r Jg, 1, 2 and 5 m i n u t e s , and t h r e e s o l v e n t volumes of 500, 1000 and 1500 mis f o r 500 g o f wet p r e s s e d t r u b . Each e x t r a c t i o n was r e p e a t e d u s i n g a d i f f e r e n t 500 g t r u b sample g i v i n g a t o t a l o f 120 e x t r a c t i o n s . From each e x t r a c t i o n , , a 10 - 15 ml sample, of r e s i n - c o n t a i n i n g s o l v e n t was saved f o r l a t e r . s p e c t r o p h o t o m e t r y measurement. Recovery of p r o t e i n f l o u r , r e s i n and .the w a t e r - d l s p e r s i b l e  f r a c t i o n : A f t e r e x t r a c t i o n , the d e b i t t e r e d . t r u b was d r i e d i n a stream of a i r f o r 24 hours w i t h o u t h e a t . On d r y i n g , i t became a l i g h t w h i t i s h power. T h i s was c a l l e d Trub P r o t e i n F l o u r . The r e s i n was r e c o v e r e d by vacuum. e v a p o r a t i o n o f the s o l -vent i n a B u c h l e r f l a s h e v a p o r a t o r at 40°C. . Upon complete 10 e v a p o r a t i o n o f the a l c o h o l - w a t e r m i x t u r e , the r e s i d u e , termed the Crude R e s i n E x t r a c t , was r e d i s s o l v e d w i t h a s m a l l q u a n t i t y o f pure i s o p r o p a n o l . I n v a r i a b l y a s m a l l p a r t o f the Crude R e s i n E x t r a c t was a l c o h o l - i n s o l u b l e . However, t h i s l i g h t c o l o r e d a l c o h o l - i n s o l u b l e f r a c t i o n was e a s i l y d i s p e r s i b l e i n water and hence i t was termed the W a t e r - D i s p e r s i b l e F r a c t i o n . T h i s Water-D i s p e r s i b l e F r a c t i o n was u l t i m a t e l y r e c o v e r e d i n a c r y s t a l - l i k e form by f r e e z e d r y i n g . Meanwhile the a l c o h o l - s o l u b l e p o r t i o n was f i l t e r e d and vacuum e v a p o r a t e d a g a i n t o remove the i s o p r o p a n o l . As t h e l a s t t r a c e s o f s o l v e n t were removed, the r e s i n o u s dark brown r e s i d u e became more and more v i s c o u s . Vacuum was m a i n t a i n e d u n t i l no a l c o h o l odour c o u l d be d e t e c t e d . T h i s f i n a l r e s i d u e was termed the Trub R e s i n . A s c h e m a t i c o u t l i n e o f the e n t i r e r e c o v e r y o p e r a t i o n i s shown i n f i g u r e 1. M o i s t u r e d e t e r m i n a t i o n : ' F o r t r u b and p r o t e i n f l o u r samples, m o i s t u r e was d e t e r m i n e d by d r y i n g samples i n a 110°C oven t o c o n s t a n t w e i g h t , the time r e q u i r e d b e i n g u s u a l l y 48 h o u r s . 'Measurement of the weight of r e s i n e x t r a c t e d : The amount of r e s i n d i s s o l v e d i n i s o p r o p y l a l c o h o l was e s t i m a t e d by measuring r e s i n absorbance at 275 nm a g a i n s t pure i s o p r o p y l a l c o h o l as r e f e r e n c e . P r i o r t o t a k i n g a b s o r -bance r e a d i n g s , samples were f i l t e r e d t h r o u g h Whatman #1 paper and q u a n t i t a t i v e l y d i l u t e d t o measureable range w i t h pure i s o p r o p y l a l c o h o l . Readings were t a k e n as soon as p o s s i b l e 11 FIGURE 1: B a t c h E x t r a c t i o n Scheme For Trub Wet t r u b e x p r e s s excess wort r e p e a t 5 t i m e s y Wet p r e s s e d t r u b Wort ( d i s c a r d e d ) e x t r a c t w i t h i s o p r o p a n o l @ 700 rpm Wet.trub p r o t e i n f l o u r S o l v e n t c o n t a i n i n g crude r e s i n a i r d r y 24 hours w i t h o u t heat vacuum e v a p o r a t e @ l e s s t h a n 40°C D r i e d t r u b p r o t e i n f l o u r Crude r e s i n e x t r a c t d i s s o l v e i n i s o p r o p a n o l A l c o h o l - s o l u b l e r e s i n  i n pure i s o p r o p a n o l A l c o h o l - i n s o l u b l e p o r t i o n vacuum e v a p o r a t e l e s s t h a n 40°C d i s p e r s e i n H^O and f r e e z e - d r y Trub r e s i n e x t r a c t W a t e r - d i s p e r s i b l e f r a c t i o n 12 a f t e r the e x t r a c t i o n , u s u a l l y w i t h i n 2 hours o r l e s s . I t was found t h a t a d i r e c t c o r r e l a t i o n e x i s t s between the weight o f s o l u b l e r e s i n and i t s . a b s o r b a n c e a t 275 nm i n i s o p r o p y l a l c o h o l ( f i g u r e 2). By l i n e a r r e g r e s s i o n , i t was c a l c u l a t e d t h a t : A = 4525 B - 82 where A = absorbance B = c o n c e n t r a t i o n o f s o l u b l e r e s i n i n g/ml. The v a l u e A was o b t a i n e d by d i s s o l v i n g a known weight o f r e s i n i n a f i x e d volume of i s o p r o p y l a l c o h o l , m easuring the absorbance and m u l t i p l y i n g t h i s v a l u e by the d i l u t i o n f a c t o r r e q u i r e d t o a c h i e v e range. Lead conductance v a l u e o f the t r u b r e s i n : The p r o c e d u r e recommended by the J . I . B . A n a l y s i s Committee was f o l l o w e d (9). Both the BIRF v a r i a n t and the Wollmer v a r i a n t -were t r i e d as methods of e x t r a c t i n g the p r e c i p i t a b l e r e s i n . I t was found t h a t the Wollmer v a r i a n t .using e t h e r was more s a t i s f a c t o r y because the t o l u e n e employed i n . t h e BIRF v a r i a n t was a poor s o l v e n t f o r t r u b r e s i n . T h i n l a y e r chromatography, o f the hop•and t r u b r e s i n s : The procedure o u t l i n e d by F r a n i a u & Mussche 197^ (11) was f o l l o w e d . . A uv spectrum, between 200 and 400 nm was measured f o r each s e p a r a t e d . t r u b r e s i n component, u s i n g a l k a l i n e methanol as the e x t r a c t i n g s o l v e n t . . However, c o n c l u s i v e i d e n t i f i c a t i o n o f the t r u b b i t t e r components was not c a r r i e d out s i n c e pure s t a n d a r d s were c o m m e r c i a l l y u n a v a i l a b l e . Comparisons were . made between f r e s h l y e x t r a c t e d r e s i n s and 6-month-old r e s i n e x t r a c t s s t o r e d at 4°C. Comparisons were a l s o made between 13 FIGURE 2: S t a n d a r d curve f o r e s t i m a t i o n o f s o l u b l e r e s i n c o n c e n t r a t i o n i n i s o p r o p a n o l . 14 hop r e s i n components and t r u b r e s i n components. The hops used were o f Brewer's Gol d v a r i e t y and were purchased from the l o c a l Wine A r t s t o r e . P r o t e i n d e t e r m i n a t i o n : The amounts o f p r o t e i n i n f r e e z e - d r i e d t r u b , t r u b p r o t e i n f l o u r and the f r e e z e - d r i e d w a t e r - d i s p e r s i b l e ' f r a c t i o n were e s t i m a t e d by a p p l y i n g a f a c t o r o f 6.25 t o the K j e l d a h l t o t a l n i t r o g e n v a l u e . The r a p i d m i c r o k j e l d a h l p r o c e d u r e o f Concon & S o l t e s s 1973 (12) was used f o r d i g e s t i o n . The d i g e s t was t h e n d i l u t e d t o 25 ml w i t h d i s t i l l e d , w a ter and the t o t a l n i t r o g e n d e termined i n the T e c h n i c o n N i t r o g e n Auto A n a l y s e r . F o r t r u b and the p r o t e i n f l o u r , 20 - 30 'mg o f sample c o n t a i n i n g 2 - 100 ppm n i t r o g e n was r e q u i r e d . A l a r g e r amount, 100 mg o f t h e w a t e r - d i s p e r s i b l e f r a c t i o n was d i g e s t e d because o f i t s lower n i t r o g e n c o n t e n t . Amino a c i d a n a l y s e s o f the t r u b p r o t e i n f l o u r : C y s t e i n e was f i r s t p r o t e c t e d by p y r i d y l e t h y l a t i o n a c c o r d -i n g t o the method o f Cavins eb a l (13). The p r o t e i n sample was th e n h y d r o l y s e d w i t h p a r a - t o l u e n e s u l f o n i c a c i d f o r 24 hours a c c o r d i n g t o the method o f L i u & Chang ( 1 4 ) . A f t e r h y d r o l y s i s , the amino a c i d s were assayed u s i n g a Phoenix M6800 amino a c i d a n a l y s e r , equipped w i t h the Durrum s i n g l e column system. The a r e a o f each amino a c i d peak was c a l c u l a t e d by the HW ( h e i g h t -w e i g h t ) method where the HW v a l u e = peak h e i g h t x dot count o f each peak. The amount of each amino a c i d c a l c u l a t e d from the HW v a l u e was e x p r e s s e d as grams o f amino a c i d per 100 g o f sample. 15 N i t r o g e n s o l u b i l i t y p r o f i l e o f the t r u b p r o t e i n f l o u r : The s t a n d a r d method proposed by Balmaceda et_ a l (15) was a p p l i e d . Por t r u b p r o t e i n f l o u r , the n i t r o g e n s o l u b i l i t y was de t e r m i n e d as f o l l o w s : 200 mg o f f l o u r weighed i n t o a c e n t r i f u g e tube was d i s p e r s e d i n 10 ml d i s t i l l e d w a ter t o make a p p r o x i m a t e l y a 1% p r o t e i n s o l u t i o n . W ith 0.1 N HCI or 0.IN NaOH, the pH o f the s o l u t i o n was a d j u s t e d t o the d e s i r e d v a l u e s (pH 2, 3, 4.5, 7, 8.5, 10, 11 and 12). A f t e r weight e q u a l i z a t i o n w i t h d i s t i l l e d w a t e r , the tubes were c e n t r i f u g e d a t 28000 g f o r 30 m i n u t e s . The s u p e r n a t a n t was decanted or f i l t e r e d i n t o K j e l d a h l f l a s k s f o r t o t a l n i t r o g e n d e t e r m i n a t i o n . The r e s u l t was e x p r e s s e d as: P e r c e n t s o l u b i l i t y = grams o f N i n s u p e r n a t a n t x 100 grams o f N i n 200 mg f l o u r S o l u b i l i t y p r o f i l e s were a l s o e v a l u a t e d f o r f r e e z e - d r i e d u n e x t r a c t e d t r u b and f o r t r u b p r o t e i n f l o u r i n 1 M Na C l . The average o f d u p l i c a t e a s s a y s was r e p o r t e d i n t h i s t h e s i s . E m u l s i f y i n g c a p a c i t y o f the t r u b p r o t e i n f l o u r : The s t a n d a r d p r o c e d u r e i n the G e n e r a l Foods Methodology (15) was m o d i f i e d as f o l l o w s : ( i ) t h e pH o f t h e d i s p e r s i o n was not a d j u s t e d t o 7 ( i i ) p u r e . ' c o r n ; o i l (Mazola) was used r a t h e r t h a n F i s h e r o i l ( i i i ) t he r e s u l t was e x p r e s s e d as mis o f o i l e m u l s i f i e d per' gram o f crude f l o u r r a t h e r t h a n per gram o f p r o t e i n . Thus t h e e m u l s i f y i n g c a p a c i t y o f 1 gram o f t r u b p r o t e i n f l o u r was com-par e d w i t h 1 gram o f soy p r o t e i n f l o u r and 1 gram of soy i s o l a t e 16 on an "as i s " weight b a s i s r a t h e r t h a n a p r o t e i n b a s i s . The proc e d u r e was as f o l l o w s : One gram o f f l o u r was d i s p e r s e d i n 100 ml o f water. The o i l f l o w r a t e was m a i n t a i n e d throughout the exper i m e n t . 70 ml o f the p r o t e i n d i s p e r s i o n was then added t o the mason j a r equipped w i t h : ( i ) an ohm-meter f o r measuring the r e s i s t a n c e o f t h e d i s p e r s i o n ( i i ) a t o p - d r i v e b l e n d i n g u n i t s e t f o r 10,000 rpm ( i i i ) o i l i n t a k e w i t h an o n / o f f clamp A f t e r 30 seconds o f m i x i n g , o i l t i t r a t i o n began and was ti m e d w i t h a stopwatch. End p o i n t was i n d i c a t e d a t i n f i n i t e r e s i s t a n c e , d e n o t i n g a change i n the c o n t i n u o u s phase. T h i s measurement was -repeated at l e a s t 4 t i m e s . E m u l s i f y i n g c a p a c i t y o f the un-> - . t r e a t e d or u n e x t r a c t e d t r u b and o f the w a t e r - d i s p e r s i b l e f r a c t -i o n was a l s o d e t e r m i n e d i n a s i m i l a r manner. W a t e r - h o l d i n g c a p a c i t y o f the t r u b p r o t e i n f l o u r : A g a i n the s t a n d a r d method proposed by Balmaceda e_t a l (15) was adopted. One gram o f p r o t e i n f l o u r was weighed i n t o a p r e v i o u s l y t a r e d p o l y c a r b o n a t e c e n t r i f u g e tube and 30 ml o f d i s t i l l e d w a ter was added and shaken t o d i s p e r s e the f l o u r . The tube was then heated f o r 30 minutes a t 60°C and s u b s e q u e n t l y c o o l e d f o r the same p e r i o d i n tap water.. F o l l o w i n g weight e q u a l -i z a t i o n w i t h d i s t i l l e d w a t e r , the sample was c e n t r i f u g e d a t 18000 g f o r 10 minutes at 25°C. A f t e r d e c a n t i n g the s u p e r -n a t a n t , the tube was reweighed. F o r l o o s e p a c k i n g samples, a f i l t e r paper was used t o r e c o v e r l o o s e p a r t i c l e s w h i c h were .!7 r e t u r n e d t o the tube. To compensate f o r the amount o f s o l u b l e p r o t e i n l o s t i n the s u p e r n a t a n t , the s u p e r n a t a n t was q u a n t i -t a t i v e l y c o l l e c t e d and i t s dry w e i g h t . s u b t r a c t e d from the o r i g i n a l 1 gram of dry p r o t e i n . Thus: Wt of - Wt o f dry s o l u b l e p r o t e i n p r o t e i n Water h o l d i n g c a p a c i t y = Wt o f wet - Wt -per gram of sample sample & of tube tube C o l o r d e t e r m i n a t i o n of the t r u b p r o t e i n f l o u r : The Unicam Spect r o p h o t o m e t e r 800B equipped w i t h the s p e c u l a r r e f l e c t a n c e a c c e s s o r y was used t o p l o t the r e f l e c t a n c e spectrum o f f i n e l y ground samples from 400 - 700 nm. Magnesium o x i d e was employed as the s t a n d a r d f o r 100% r e f l e c t a n c e . Readings at every 10 nm were used t o c a l c u l a t e the CIE t r i s t i m u l u s v a l u e s X, Y & Z by the w e i g h t e d . o r d i n a t e method based on i l l u m i n a n t C (16). Measurement of the b i t t e r n e s s of b e e r : The method f a v o r e d by the I n s t i t u t e o f Brewing A n a l y s i s Committee was f o l l o w e d (9)- A m o d i f i c a t i o n o f the o r i g i n a l method of Rigby & Bethune (17) f o r e s t i m a t i n g i s o - c C - a c i d s i n beer, t h i s p r o c e d u r e e x t r a c t s a c i d i f i e d beer u s i n g i s o - o c t a n e and meas-ure s i t s absorbance a t 275 nm.. The r e s u l t i s e x p r e s s e d as Euro-pean B i t t e r n e s s U n i t s (E.B.U.) which i s 50 x absorbance. The p r e p a r a t i o n of beer f o r t a s t e e v a l u a t i o n s - : Wort of s p e c i f i c g r a v i t y 1.040 was p r e p a r e d by d i s s o l v i n g 55 g of dark John B u l l malt e x t r a c t and 55 g of i n v e r t sugar, per l i t e r of water. The wort was then d i v i d e d . i n t o 3 b a t c h e s . 3 g / l i t e r of N o r t h e r n Brewer hops.were added t o , t h e f i r s t , 0.57 g / l i t e r of t r u b r e s i n e x t r a c t t o . t h e second, and none t o the 18 t h i r d . A l l t h r e e b a t c h e s were b o i l e d f o r e x a c t l y 1 hour t o i n c o r -p o r a t e the b i t t e r n e s s . Losses due t o e v a p o r a t i o n (approx. 50%) were compensated f o r by a d d i n g water a t the end o f the b o i l i n g . Spent hops were d i s c a r d e d . A f t e r the wort had c o o l e d t o 30°C, a l e y e a s t was- p i t c h e d i n and a l l o w e d t o ferment t o c o m p l e t i o n (3 d a y s ) . The f e r m e n t i n g brew was v i g o r o u s l y s t i r r e d t w i c e d a i l y . A f t e r f e r m e n t a t i o n , the beer was f i l t e r e d and the b i t t e r n e s s a s s a y e d . The b i t t e r n e s s l e v e l i n the f i r s t 2 b a t c h e s were a d j u s t e d t o 20 E.B.U. (European B i t t e r n e s s U n i t s ) by b l e n d i n g i n the un-hopped be e r . Then e q u a l volumes o f the a d j u s t e d b a t c h e s were bl e n d e d t o g e t h e r t o form the mixed b a t c h . At the end, t h e r e were 3 b a t c h e s o f beer % 20 E.B.U. each, v i z . , . t h e beer brewed w i t h f r e s h hops, beer brewed w i t h t r u b r e s i n o n l y and beer c o n s i s t i n g o f a 1:1 b l e n d o f the two. Tas t e e v a l u a t i o n s i n o r d e r t o determine the d i f f e r e n c e i n b i t t e r -ness v a l u e s : The t e s t p r o c e d u r e adopted was a p a i r e d comparison (19)-Two samples were p r e s e n t e d s i m u l t a n e o u s l y and the p a n e l i s t was asked t o d e c i d e whether they were the same or d i f f e r e n t i n b i t t e r -n e s s . I f the p a n e l i s t d e c i d e d t h a t the samples were d i f f e r e n t , t h e n the more b i t t e r sample was t o be i d e n t i f i e d on the form s u p p l i e d . The chosen p a n e l c o n s i s t e d ' o f 18 p e r s o n s , 2 women and 16 men. Sample c o n t a i n e r s were l a b e l l e d w i t h random 3 d i g i t numbers. The beer samples c o n t a i n e d no c a r b o n a t i o n . C r a c k e r s and water were p r o v i d e d as p a l a t e r e f r e s h m e n t . 19 S i x p a i r s were p r e s e n t e d t o each p a n e l i s t f o r judgement. Each p a i r always c o n t a i n e d a sample o f beer made from f r e s h hops as the r e f e r e n c e sample. The o t h e r sample i n the p a i r would always be beer made from e i t h e r 100% t r u b r e s i n or 50% t r u b r e s i n a d j u s t e d t o the same l e v e l o f a n a l y t i c a l b i t t e r n e s s as t h e r e f e r e n c e sample. The d a t a o b t a i n e d was a n a l y s e d u s i n g C h i - s q u a r e a n a l y s i s w i t h t h e Y a t e s ' c o n t i n u i t y c o r r e c t i o n a p p l i e d f o r degrees o f freedom = 1 (47). 20 RESULTS AND DISCUSSION The d i s c u s s i o n of t h i s p r o j e c t may be d i v i d e d i n t o t h r e e p a r t s v i z . , A. The e x t r a c t i o n p r o c e d u r e B. E v a l u a t i o n o f the p r o t e i n f r a c t i o n . C. The e x a m i n a t i o n and e v a l u a t i o n o f the r e s i n f r a c t i o n . P a r t A: The e x t r a c t i o n : Prom the s t a r t i t was reasoned t h a t i f . any b r e w i n g c o n c e r n d e c i d e d t o e x t r a c t r e s i n from t r u b , the p r o c e s s would, e n t a i l a b a t c h r a t h e r t h a n a c o n t i n u o u s e x t r a c t i o n because o f the p r o h i b i -t i v e c a p i t a l c o s t i n v o l v e d i n s e t t i n g up the c o n t i n u o u s mode o f e x t r a c t i o n . A l s o , because of the l a r g e energy demand r e q u i r e d i n any t ype of d r y i n g o p e r a t i o n , i t was d e c i d e d t h a t a l l e x t r a c t i o n work be conducted on wet r a t h e r t h a n d r i e d t r u b . Once i t was s e t t l e d t h a t the t ype of e x t r a c t i o n be conducted on wet t r u b u s i n g b a t c h e x t r a c t i o n , the q u e s t i o n on. the c h o i c e o f s o l v e n t a r o s e . Three r e s i n s o l v e n t s were c l o s e l y examined, v i z . , hexane, methylene c h l o r i d e and i s o p r o p y l a l c o h o l , on the b a s i s o f t h e i r a b i l i t y t o d e b i t t e r t r u b . The d e b i t t e r i n g e f f e c t o f each s o l v e n t was t e s t e d s i m p l y be t a s t i n g the d r i e d p r o t e i n r e s i d u e a f t e r s e q u e n t i a l e x t r a c t i o n i n v o l v i n g a g i t a t i o n i n a Waring b l e n d e r f o l l o w e d by vacuum f i l t r a t i o n . A 4:1 (v/w) b a t c h e x t r a c t i o n performed s e q u e n t i a l l y 5 t i m e s f a i l e d t o d e b i t t e r the wet p r e s s e d t r u b when e i t h e r methylene c h l o r i d e or hexane was used. On;.' the o t h e r hand, i s o p r o p a n o l d e b i t t e r e d . the t r u b under t h e s e c o n d i t i o n s . 21 The c h o i c e o f i s o p r o p a n o l was based m a i n l y on i t s d e b i t -t e r i n g e f f e c t on the p r o t e i n r e s i d u e . However,, the low t o x i c i t y o f t h i s s o l v e n t as w e l l as i t s p r o f o u n d d e h y d r a t i n g e f f e c t on the wet t r u b (see t a b l e 1) f u r t h e r s u p p o r t e d the c h o i c e o f i s o p r o p a n o l . The s u p e r i o r performance of i s o p r o p a n o l over the n o n - p o l a r s o l v e n t s hexane and methylene c h l o r i d e was p a r t l y e x p l a i n e d by t h e presence of a l a r g e amount of water (71.7 - 73-5%) i n t h e wet p r e s s e d t r u b . The e x c e s s i v e water p r e s e n t may a c t as a p h y s i c a l b a r r i e r t o methylene c h l o r i d e and hexane e x t r a c t i o n s because of t h e i r s t r o n g n o n - p o l a r i t y . On the o t h e r hand, i s o p r o p a n o l i s c o m p l e t e l y m i s c i b l e w i t h water and forms.a s t a b l e b i n a r y a z e o t r o p e w i t h water (see appendix 5 f o r the d i s t i l l a t i o n c o n s t a n t s ) . The a l t e r e d n a t u r e o f the r e s i n s p r e s e n t i n t r u b may a l s o e x p l a i n the e f f e c t i v e n e s s o f the a l c o h o l s o l v e n t . D u r i n g the wort b o i l i n g , v a r i o u s hop components undergo c h e m i c a l t r a n s f o r -mations t o more p o l a r d e r i v a t i v e s . P o r i n s t a n c e , t h e i s o - c C - a c i d s a r e more s o l u b l e t h a n the o c - a c i d s . S i m i l a r l y many s t u d i e s (7, 22, 23, 24) have shown t h a t o x i d i s e d hop components are more w a t e r - s o l u b l e t h a n t h e i r u n o x i d i s e d d e r i v a t i v e s . A s h u r s t (25) has shown t h a t t h e mechanism r e s p o n s i b l e f o r the i n c r e a s e d s o l u b -i l i t y of humulone d u r i n g o x i d a t i o n i s the p r o d u c t i o n o f an < X - g l y c o l from an o l e f i n i c bond. He extended t h i s mechanism t o the o x i d a t i o n o f o t h e r hop r e s i n s as w e l l . S p e t s i g (7) has r e p o r t e d t h a t t r u b r e s i n c o n t a i n s many such p o l a r o x i d i s e d comp-onents . F u r t h e r m o r e , i t has been s t a t e d t h a t the bonds between the r e s i n and p r o t e i n f r a c t i o n o f t r u b are most l i k e l y hydrogen bonds (1). 22 T a b l e 1: The dry weight of r e s i n - e x t r a c t e d t r u b p r o t e i n . * Trub p r o t e i n f l o u r % Dry Weight U n e x t r a c t e d 26. .85 E x t r a c t e d once 85. • 70 E x t r a c t e d t w i c e 92. .50 E x t r a c t e d f o u r times 92, .60 E x t r a c t e d f i v e t imes 93. .20 * E x t r a c t e d w i t h i s o p r o p a n o l % 4:1 v/w f o r 2 minutes 23 Loomis & B a t t a i l e (20) a l l u d e d t o the hydrogen-bond-breaking c a p a b i l i t y o f i s o p r o p a n o l . Hence i t i s almost c e r t a i n t h a t the e x t r a c t i n g power o f i s o p r o p a n o l , a p o l a r s o l v e n t , be g r e a t e r t h a n t h a t o f hexane and methylene c h l o r i d e i n the t r u b system. On the same grounds, the e x t r a c t i n g power o f the more p o l a r a z e o t r o p i c m i x t u r e of 88% i s o p r o p a n o l and 12% water may be even g r e a t e r . Indeed, our r e s u l t s from the c o n t i n u o u s e x t r a c t i o n appears t o c o n f i r m t h i s premise ( t a b l e 2). The a z e o t r o p e e x t r a c t s approx-i m a t e l y 24% more crude r e s i n t h a n i s o p r o p a n o l (11.11% v s . 8.93%). V e r z e l e (27) i n h i s work (appendix 6) p o i n t e d out t h a t w i t h in^."-; , c r e a s i n g s o l v e n t p o l a r i t y , more and more u n s p e c i f i e d m a t e r i a l i s e x t r a c t e d from d r i e d hops. I f t h i s t r e n d o c c u r s i n the t r u b s y s -tem, t h e n i t i s l i k e l y t h a t the use o f the a z e o t r o p e w i l l e x t r a c t more p o l y p h e n o l i c and t a n n i n m a t e r i a l from the t r u b t h a n would the use o f i s o p r o p a n o l as e x t r a c t a n t . Such ex t r a n e o u s m a t e r i a l may not be d e s i r a b l e i n terms o f r e u t i l i s i n g t he r e s i n i n beer but may be d e s i r a b l e from the s t a n d p o i n t o f d e b i t t e r i n g the p r o t e i n r e s i d u e . F o r t h i s s t u d y , pure i s o p r o p a n o l was chosen a s . e x t r a c t a n t because i t l e n t I t s e l f f o r the r e s i n q u a n t i f i c a t i o n p r o c e d u r e w h i c h i n v o l v e d s p e c t r o p h o t o m e t r y . However, i n p r a c t i c e where s o l v e n t r e c y c l i n g i s a s t a n d a r d p r o c e d u r e , the a z e o t r o p i c e x t r a c t -t i o n would be p r e f e r a b l e . B e f o r e b e g i n n i n g the q u a n t i t a t i v e assessment o f e x t r a c t i o n c h a r a c t e r i s t i c s , a b r i e f e x p l a n a t i o n on the w a t e r - d i s p e r s i b l e 24 T able 2: The p e r c e n t y e i l d o f crude r e s i n e x t r a c t e d from wet and dry t r u b by the c o n t i n u o u s r e f l u x method. R e f l u x s o l v e n t Hexane I s o p r o p a n o l I s o p r o p a n o l - w a t e r azeotrope. Wet p r e s s e d t r u b % Y i e l d 0.64 8.93 11.11 (Vet b a s i s ) n 6 6 6 S t d d e v i a t i o n 0.10 1.67 2.28 S t d e r r o r 0.04 0.68 0.93. Coef f , of .'.'variation .14 .90% 18.70% 20.54% % Y i e l d 2.39 33.08 41.19 ( d r y B a s i s * ) F r e e z e - d r i e d t r u b % Y i e l d 1.12 23.98 39.04 (dry b a s i s ) n 2 2 2 St d d e v i a t i o n 0 2.57 0.85 S t d e r r o r 0 1.82 0.60 C o e f f o f v a r i a t i o n 0% 10.73% 2.17% * Based on 27% dry ma t t e r i n wet p r e s s e d t r u b . 25 f r a c t i o n and i t s e f f e c t on the e x t r a c t i o n d a t a may be h e l p f u l . As o u t l i n e d i n f i g u r e 1, the w a t e r - d i s p e r s i b l e f r a c t i o n would p r e c i p i t a t e out as an a l c o h o l - i n s o l u b l e r e s i d u e when the crude r e s i n e x t r a c t was r e d i s s o l v e d i n . p u r e i s o p r o p a n o l . Most of t h i s h y d r o p h i l i c f r a c t i o n would be found i n the f i r s t e x t r a c t because most of the water p r e s e n t i n the t r u b was removed i n the f i r s t e x t r a c t i o n . When an a l i q u o t o f t h i s f i l t e r e d f i r s t e x t r a c t was d i s s o l v e d i n pure i s o p r o p a n o l f o r r e s i n e s t i m a t i o n on the s p e c t r o -photometer, a s l i g h t t u r b i d i t y i n v a r i a b l y d e v e l o p e d , i n d i c a t i n g the presence of the a l c o h o l - i n s o l u b l e , w a t e r - d i s p e r s i b l e f r a c t i o n . Subsequent comparisons of r e s i n y i e l d s e s t i m a t e d g r a v i m e t r i c a l l y ( t a b l e 2) and s p e c t r o p h o t o m e t r i c a l l y ( t a b l e 3) showed t h a t the l a t t e r tended t o be g e n e r a l l y h i g h e r . The d i s c r e p a n c y was a t t r i b u t e d f i r s t l y t o the e f f e c t o f t u r b i d i t y on the s p e c t r o p h o t o -m e t r y r e a d i n g s and s e c o n d l y t o the Incomplete e x t r a c t i o n o f the r e s i n e x t r a c t e d f o r g r a v i m e t r i c e s t i m a t i o n . T h i s p r o b a b l y o c c u r r e d because no a g i t a t i o n was used i n the c o n t i n u o u s r e f l u x r e s i n e x t r a c t i o n . C o n s e q u e n t l y , because of the unexpected p r e s e n c e of the w a t e r - d i s p e r s i b l e f r a c t i o n , the s p e c t r o p h o t o m e t r i c method, o r i g i n a l l y d e v i s e d t o measure o n l y a l c o h o l - s o l u b l e , r e s i n , was extended t o e s t i m a t e the t o t a l crude r e s i n c o n t e n t , which i n c l u d e s b o t h the w a t e r - d i s p e r s i b l e f r a c t i o n and the a l c o h o l - s o l u b l e r e s i n . Prom an economic a s p e c t , the i m p o r t a n t c o n s i d e r a t i o n s i n the e x t r a c t i o n s t e p are.: ( i ) t o maximise the y i e l d o f t r u b p r o t e i n f l o u r ( i i ) t o maximise the y i e l d of t r u b r e s i n ( i i i ) t o m i n i m i s e s o l v e n t u t i l i z a t i o n ( i v ) t o m a x i m a l l y d e b i t t e r the t r u b p r o t e i n f l o u r . 26 T a b l e 3: P e r c e n t y i e l d o f crude r e s i n b a t c h - e x t r a c t e d from wet p r e s s e d t r u b w i t h i s o p r o p a n o l . No. o f e x t r a c t s 1 2 3 4 5 S o l v e n t A g i t a t i o n R a t i o * * time (min) (v/w) 1:1 h 4. 87* 6.49 7.25 7.72 8. 00 1:1 1 6. 39 7.72 8.41 8.81 9.05 1:1 2 7. 31 8.81 9.51 9-96 10.22 1:1 5 8. 63 10.26 11.01 11.40 11.64 2:1 h 6. 42 8.59 9.67 10.18 10.45 2:1 l 6. 30 . 8.47 9-54 9-96 10.17 2:1 2 7. 39 9.22 10.09 10.42 10. 56 2:1 5 . 9- 18 11.13 11.93 12.24 12.40 3:1 h 6. 67 9.36 10.59 11.19 11.46 3:1 l 8. 29 11. 02 12.05 12.48 12.69 3:1 2 7. 66 10. 52 11.53 11.68 11.82 3:1 5 8. 63 11.05 11.79 12.02 12.13 * % Y i e l d based on a b s o r p t i o n a t 275 nm e x p r e s s e d on b a s i s o f wet t r u b . Each v a l u e i s t h e average % y e i l d o f 2 independent e x t r a c t i o n s . * * S o l v e n t r a t i o = volume of i s o p r o p a n o l / w t o f wet t r u b 27 With respect t o the f i r s t c o n s i d e r a t i o n ( i ) : s i n c e n a t i v e b a r l e y p r o t e i n i s known t o have a l a r g e c o n t e n t o f h o r d e i n , t h e a l c o h o l - s o l u b l e p r o t e i n , i t was o r i g i n a l l y s p e c u l a t e d t h a t some s i g n i f i c a n t p r o t e i n l o s s e s would o c c u r by s o l u b i l i z a t i o n i n t h e i s o p r o p a n o l - w a t e r m i x t u r e . However upon a n a l y s i s , l i t t l e p r o t e i n was found e i t h e r i n the r e s i n ( < 5% p r o t e i n ) o r i n the w a t e r -d i s p e r s i b l e f r a c t i o n (2.36% p r o t e i n ) . Moreover, the y i e l d s o f p r o t e i n f l o u r a f t e r 5 s e q u e n t i a l e x t r a c t i o n s were found t o be a p p r o x i m a t e l y the same r e g a r d l e s s o f the amount o f s o l v e n t used or the a g i t a t i o n time i n v o l v e d ( t a b l e 4). C o n s e q u e n t l y , the y e i l d o f t r u b p r o t e i n f l o u r was c o n s i d e r e d as b e i n g a p p r o x i m a t e l y c o n s t a n t (23%) and not s u b j e c t t o m a x i m i z a t i o n . The b a t c h e x t r a c t i o n experiment was performed t o o p t i m i s e the o t h e r t h r e e c o n s i d e r a t i o n s ( i i , i i i & i v ) . The experiment e m p i r i c a l l y examines the e f f e c t o f two f a c t o r s v i z . , the i s o p -r o p a n o l - t r u b r a t i o (v/w) and the a g i t i a t i o n time on t h e y i e l d o f r e s i n . An a n a l y s i s o f v a r i a n c e ( t a b l e 5) on the r e s i n y i e l d d a t a ( t a b l e 3) showed t h a t b o t h s o l v e n t r a t i o and a g i t a t i o n time were s i g n i f i c a n t , t o the 5% l e v e l i n a f f e c t i n g r e s i n y i e l d . The g e n e r a l t r e n d i s f o r t h e - y i e l d o f r e s i n t o i n c r e a s e w i t h more s o l v e n t and l o n g e r a g i t a t i o n ( f i g u r e s 3 & 4). The r e s i n y i e l d d a t a a l s o r e v e a l s the n e g a t i v e . l o g a r i t h m i c c h a r a c t e r o f the e x t r a c t i o n . F i g u r e 3 d e p i c t s t h i s t r e n d i n comparing t h e r e s i n y i e l d o f the t h r e e s o l v e n t r a t i o s at 5 minutes e x t r a c t i o n t i m e . Most o f the r e s i n i s e x t r a c t e d i n the f i r s t 2 e x t r a c t i o n s . A q u i c k c a l c u l a t i o n from t a b l e 3 shows t h a t i n a l l • 12 c a s e s , 8 l ' - 91% o f the r e s i n has been e x t r a c t e d a f t e r 2 e x t r a c t -28 Table 4: The y i e l d o f d r i e d t r u b p r o t e i n f l o u r a f t e r 5 e x t r a c t i o n s at 3 d i f f e r e n t s o l v e n t r a t i o s o f volume o f i s o p r o p a n o l : weight o f wet t r u b . S o l v e n t r a t i o (v/w) 1:1 2:1 3: 1 E x t r a c t i o n time (min) Y i e l d o f d r i e d t r u b (%)* h 21.22 20.55 23. 03 1 24.53 21.35 .21. 35 2 25-72 24.12 . 22. 12 5 23.60 21.86 22. 26 * Y i e l d e x p r e s s e d as % o f wet p r e s s e d t r u b . 29 T a b l e 5: A summary o f the two-way f a c t o r i a l a n a l y s i s o f v a r i a n c e w i t h r e p l i c a t i o n f o r : ( i ) the t o t a l y i e l d o f r e s i n a f t e r 5 c o n s e c u t i v e e x t r a c t i o n s ; ( i i ) y i e l d o f r e s i n a f t e r one e x t r a c t i o n DP SS MS F P 5 e x t r a c t i o n s T o t a l . 23 49.89 R e p l i c a t e 11 43.65 S o l v e n t 2 21.12 10.56 20.31** P 0.0005 Time 3 13.64 4.55 8.74** P 0.0025 S o l v e n t x time 6 8.90 1.48 2.85 P 0.10 E r r o r 12 6.24 0.52 1 e x t r a c t i o n T o t a l 23 40. 03 R e p l i c a t e 11 34.17 S o l v e n t 2 4.11 2.06 4.22*'' P 0.05 Time 3 24.78 8.26 16.94** P 0.0005 S o l v e n t x time 6 5.29 0.88 1.81 P 0.25 E r r o r 12 5-85 0.49 DP = Degrees o f freedom; SS =  Sum o f squares ; MS = Mean Square F = F v a l u e ; P = P r o b a b i l i t y * S i g n i f i c a n t a t the 5% l e v e l . 30 L o g , n R e s i n weight (Y) Number of s e q u e n t i a l e x t r a c t s (X) FIGURE 3: E f f e c t of s o l v e n t r a t i o on r e s i n y i e l d i n 5 minute e x t r a c t i o n s . 31 32 i o n s . Thus, from the s t a n d p o i n t o f s o l v e n t economy, 2 e x t r a c t -Ions may be c o n s i d e r e d t o be the a r b i t r a r y p o i n t at which r e s i n r e c o v e r y t e r m i n a t e s . A f t e r 2 e x t r a c t i o n s , the co n c e r n s h i f t s away from r e s i n r e c o v e r y toward the d e b i t t e r i n g o f the p r o t e i n r e s i d u e . At the p o i n t o f two e x t r a c t i o n s , the e f f e c t o f d i f f e r e n t s o l v e n t r a t i o s on r e s i n y e i l d i s not marked, p a r t i c u l a r l y i f 5 . minute a g i t a t i o n t i m e s are employed ( t a b l e 3). Thus f o r the sake of s o l v e n t economy, i t i s l o g i c a l t o adopt the l o w e s t o f t h e s o l -vent r a t i o s . The use o f the lo w e s t s o l v e n t r a t i o a l s o improves the e f f i c i e n c y o f a g i t a t i o n , due.to the i n t e r a c t i o n e f f e c t between s o l v e n t r a t i o and a g i t a t i o n time ( t a b l e 5). I n p r a c t i c e , the wet t r u b ^ i s o p r o p a n o l m i x t u r e was d i f f i c u l t t o handle a t the 1:1 s o l -vent r a t i o . There was no f r e e s o l v e n t p r e s e n t and u n i f o r m agita-e t i o n and s o l v e n t e x p u l s i o n was d i f f i c u l t . . These d i f f i c u l t i e s may j u s t i f y t he use o f a h i g h e r s o l v e n t r a t i o i n a c t u a l p r a c t i c e . The f i n a l c o n s i d e r a t i o n ( i v ) i s the d e b i t t e r i n g o f the p r o t e i n f l o u r . The r e s i d u a l r e s i n l e v e l at which the p r o t e i n f l o u r may be c o n s i d e r e d " d e b i t t e r e d " i s unknown.. Tannins and p o l y p h e n o l i c compounds may so c o n t r i b u t e t o the b i t t e r n e s s o f ..• t r u b p r o t e i n f l o u r t h a t even at v e r y l o w . r e s i n l e v e l s b i t t e r -ness may be d e t e c t a b l e . On the o t h e r hand, the p r e s e n c e o f o t h e r f o o d components i n a p a r t i c u l a r f o r m u l a t i o n employing " d e b i t t e r e d " t r u b may mask the b i t t e r n e s s o f the t r u b p r o t e i n f l o u r so much t h a t i t may not be n e c e s s a r y t o e x t r a c t t h e f l o u r r e s i d u e t o e x t r e m e l y low r e s i n l e v e l s . T a b le 6 shows the number o f t h e o r e t i c a l e x t r a c t i o n s n e c e s s a r y to. r e a c h 1% and 0.1% r e s i n l e v e l (w/w) i n the d r i e d p r o t e i n f l o u r . 33 Table 6: The number o f t h e o r e t i c a l e x t r a c t i o n s * r e q u i r e d t o o b t a i n 1% and 0.1% o f the i n i t i a l r e s i n l e v e l (w/w) at a c o n s t a n t y i e l d o f 115 g (23%) o f d r i e d p r o t e i n f l o u r , e x t r a c t e d from 500 g wet p r e s s e d t r u b . S o l v e n t r a t i o Time 1% R e s i n l e v e l 0.1% R e s i n l e v e l (v/w) (min) Number of e x t r a c t i o n s r e q u i r e d 1:1 h 5.04 (6)** 8.35 (9) 1:1 l 4.65 (5) 7.60 (8) 1:1 2 4.83 (5) 7.74 (8) 1:1 5 4. 70 (5) 7.37 (8) 2:1 h 5.07 (6) 8.03 (9) 2 :1 1 4.80 (5) 7.52 (8) 2 :1 2 4.39 (5) 6.77 (7) 2:1 5 4.43 (5) 6.76 (7) 3:1 h 5.18 (6) 8.09 (9) 3:1 1 4.74 (5) . 7.23 (8) . 3:1 2 4.40 (5) 6.66 (7) 3 :1 5:;: 4.14 (5) 6.20 (7) * C a l c u l a t e d from the d e b i t t e r i n g e q u a t i o n s i n t a b l e 7. ** Numbers i n p a r e n t h e s i s i n d i c a t e s the number o f complete e x t r a c t i o n s n e c e s s a r y t o d e b i t t e r the p r o t e i n f l o u r t o l e s s t h a n 1% and 0.1% r e s i n l e v e l s r e s p e c t i v e l y . 34 The d e b i t t e r i n g e q u a t i o n s ( t a b l e 7) used t o c a l c u l a t e the t h e o r e t i c a l number o f e x t r a c t i o n s were d e r i v e d from l i n e a r r e g -r e s s i o n o f the log-^Q r e s i n weight v s . number o f e x t r a c t i o n s . Good c o r r e l a t i o n i n t h e s e e q u a t i o n s j u s t i f i e s t he o v e r a l l assump-t i o n t h a t d e b i t t e r i n g or r e s i n e x t r a c t i o n f o l l o w s a n e g a t i v e l o g a r i t h m i c p a t h . Thus the p r o t e i n f l o u r d e b i t t e r i n g p r o c e s s i s a l s o hampered by the i n c r e a s i n g l y s l o w e r r e s i n removal r a t e , which n e c e s s i t a t e s the use o f much s o l v e n t a t t h i s d e b i t t e r i n g s t age o f the p r o c e s s . Even at 1:1 s o l v e n t r a t i o , as much as 4000 mis of s o l v e n t are r e q u i r e d t o d e b i t t e r 500 grams o f wet t r u b t o l e s s t h a n one p a r t p e r thousand r e s i n . The problem may be a l l e v i a t e d by (a) the use o f c o u n t e r c u r r e n t e x t r a c t i o n , where f r e s h s o l v e n t i s used t o e x t r a c t , say, the l a s t 2 e x t r a c t s b e f o r e b e i n g used on h e a v i e r e x t r a c t i o n s ^ (b) u s i n g an a l t e r n a t i v e met-hod o f d e b i t t e r i n g the p r o t e i n f l o u r such as an a l k a l i n e wash where the f l o u r i s d e b i t t e r e d by d e s t r u c t i o n o f the r e s i n t o n o n - b i t t e r components. T h i s l a t t e r method was t e n t a t i v e l y t r i e d a t 8:1 (v/w) a l k a l i n e w a t e r : f l o u r r a t i o at pH 9.2 and pH 10. Subsequent i n f o r m a l t a s t e e v a l u a t i o n s on m u f f i n s (see t a b l e 8 f o r f o r m u l a -t i o n ) - i n d i c a t e d t h a t the a l k a l i n e - e x t r a c t e d - f l o u r m u f f i n s had b a r e l y d e t e c t a b l e b i t t e r n e s s l e v e l s compared t o the 5 t i m e s - i s o p -r o p a n o l - e x t r a c t e d m u f f i n . The predominant b i t t e r f l a v o r was no t e d as an a f t e r t a s t e r a t h e r t h a n an immediate s e n s a t i o n . A f t e r d r y i n g , the a l k a l i n e - e x t r a c t e d f l o u r appeared d a r k e r (see t a b l e 9 f o r c o m p a r a t i v e luminance d a t a ) , c o a r s e r and h e a v i e r t h a n the 35 Table 7: The d e b i t t e r i n g e q u a t i o n s d e r i v e d from the l i n e a r r e g r e s s i o n o f log-j_Q r e s i n weight's (Y) v s . the ..•.number of e x t r a c t i o n s ( X ) . S o l v e n t A g i t a t i o n D e b i t t e r i n g C o r r e l a t i o n S td e r r o r -r a t i o time (min.) e q u a t i o n * c o e f f i c i e n t o f e s t i m a t e 1 :1 \ Y = = 1 . 5 8 4 - 0 . 3 0 2 X - 0 . 9 8 3 0 . 079 1 :1 1 Y = = 1 . 6 3 3 - 0 . 3 3 8 X -O . 9 6 6 0 . 1 2 8 1 :1 2 Y = = 1 . 7 1 9 - 0 . 3 4 3 X - 0 . 9 6 3 0 . 1 3 6 1 :1 5 Y = = 1 . 8 2 1 - 0 . 3 7 5 X - 0 . 9 6 9 0 . 1 3 6 2 : 1 \ Y = = 1 . 7 7 8 - 0 . 3 3 9 X - 0 . 9 9 5 0.048 2 : 1 1 Y = = 1 . 8 2 2 - 0 . 3 6 7 X - 0 . 9 9 8 0 . 0 3 6 2 : 1 2 Y = = 1.907 - 0.421X - 0 . 9 9 5 . 0.060 2 :1 5 Y = = 1.959 - 0.429X - 0 . 9 8 7 0.097 3:1 \ Y = = 1.841 - 0.344X -0.999 0.019 3:1 1 Y = = 1 . 9 6 9 - 0.402X - 0 . 9 9 7 0.042 3:1 2 Y = = 2 . 0 1 2 - 0.443X - 0 . 9 9 8 0 . 0 3 6 3:1 5 Y = = 2 . 0 6 9 - 0.485X - 0 . 9 9 7 0 . 0 5 2 * D e b i t t e r i n g e q u a t i o n : Y = l o g , n weight o f r e s i n e x t r a c t e d X = number of e x t r a c t i o n s T a b l e 8: 36 A r e c i p e f o r the p r e p a r a t i o n o f m u f f i n s which c o n t a i n t r u b p r o t e i n f l o u r . \ cup s h o r t e n i n g Jg cup brown sugar \ cup molasses 2 eggs, unbeaten 1 cup o f m i l k 1 cup o f a l l - p u r p o s e f l o u r \\ t s p . b a k i n g powder \ t s p . b a k i n g soda \ t s p . s a l t 3/4 cup Quaker b r a n 3/4 cup TRUB PROTEIN FLOUR Jg cup r a i s i n s o r dates Bake a t 400°F f o r 15 - 20 minutes. 37 Table 9: The c o l o r e v a l u a t i o n o f d r i e d t r u b p r o t e i n f l o u r S o l v e n t Time No. o f C h r o m a t i c i t y c o o r d i n a t e s Luminance r a t i o • e x t r a c t s " " . (rv/w) (min)' X y z Y (%) 1:1 '2 5 0.353 0.351 0.296 49 1:1 1 5 0.351 0. 348 0.301 57 1:1 2 5 0.334 0.327 0.338 44 1:1 5 5 0.351 .0.349 0.301 56 2:1 5 0.348 0. 346 0.306 • 57 2:1 1 5 0.349 0.348 0.303 58 2:1 2 5 0.350 0. 347 0.303 58 2:1 5 5 0.350 0.347 0.303 57 3:1 h 5 0. 347 0.345 0.308 59 3:1 1 5 0.347 0.345 0.308 60 3:1 2 5 0. 346 0.345 0.310 60 3:1 5 5 0.348 0.346 0.306 59 8:1 @ pH 9-2 1 0.351 • 0.345 0.305 25 8:1 @ pH 10 1 0.352 0.342 0.307 21 4:1 No a g i t a t i o n 2 O.361 0.355 0.284 30 4:1 No a g i t a t i o n 3 0.365 0.357 0.278 29 4:1 No a g i t a t i o n 4 0.362 0.355 0.283 32 4:1 No a g i t a t i o n 5 0.362 0.355 0.283 31 4:1 No a g i t a t i o n 7 0.354 0.349 0.297 44 38 i s o p r o p a n o l - e x t r a c t e d samples. The a l k a l i n e d e b i t t e r i n g p r o c e d u r e would l i k e l y e n t a i l e x t r a s t e p s i n the f l o u r p r e p a r a t i o n v i z . , a subsequent water-washing s t e p f o l l o w e d by d r y i n g and g r i n d i n g . A l s o , some p r o t e i n l o s s from a l k a l i s o l u b i l i z a t i o n i s e x p e c t e d . The c o n c l u d i n g summary of t r u b e x t r a c t i o n i s as f o l l o w s : 1. I s o p r o p a n o l d e b i t t e r e d b e t t e r t h a n hexane or methylene c h l o r i d e . 2. The i s o p r o p a n o l - w a t e r a z e o t r o p e e x t r a c t e d more crude r e s i n t h a n i s o p r o p a n o l .(table 2). 3. The p r o t e i n f l o u r y i e l d was not g r e a t l y a f f e c t e d by changes i n the s o l v e n t r a t i o from.1:1 t o 3:1 and i n the a g i t a t i o n time from 0.5 t o 5 minutes ( t a b l e 4). 4. Both s o l v e n t r a t i o and a g i t a t i o n t ime a f f e c t e d the r e s i n y i e l d as shown by a n a l y s i s of v a r i a n c e ( t a b l e 5). 5. As much as 8 - 9 e x t r a c t i o n s were r e q u i r e d t o d e b i t t e r the p r o t e i n f l o u r t o l e s s t h a n 1 p a r t p e r thousand ( t a b l e 6). 6. The e x t r a c t i o n s were i n a c h a r a c t e r i s t i c n e g a t i v e l o g a r i t h m i c r e l a t i o n ( t a b l e 7). 7. I n c r e a s i n g , s o l v e n t r a t i o s d i d not s u b s t a n t i a l l y i n c r e a s e r e s i n y e i l d when the a g i t a t i o n was l o n g e r t h a n 2 minutes so t h a t the 1:1 s o l v e n t r a t i o was t h e most e f f i c i e n t method of e x t r a c t i o n i n terms o f s o l v e n t economy. 8. S o l v e n t c o n s e r v a t i o n and p r o t e i n f l o u r d e b i t t e r i n g were the c o n f l i c t i n g f a c t o r s . 9. A l k a l i n e e x t r a c t i o n was m o d e r a t e l y e f f e c t i v e f o r d e b i t t e r i n g but may e n t a i l a d d i t i o n a l f i n i s h i n g t r e a t m e n t s such as washing and d r y i n g as w e l l as p r o t e i n l o s s e s . 39 An o v e r a l l scheme showing the y i e l d s of the v a r i o u s f r a c t i o n s i s p r e s e n t e d i n f i g u r e 5- V a r i a b i l i t y of the i n d i v i d u a l y e i l d e s t i m a t e s i n c l u d i n g d r y m a t t e r e s t i m a t e s i s shown i n t a b l e 10. P a r t B: E v a l u a t i o n of the p r o t e i n f r a c t i o n : P r i o r t o e x t r a c t i o n , t r u b was a dark g r e e n i s h - b r o w n s o l i d w i t h a t e x t u r e s i m i l a r t o c o t t a g e cheese. I t had a s l i g h t l y sweet w o r t - l i k e odour and an e x t r e m e l y b i t t e r t a s t e . A f t e r e x t r a c -t i o n , the s o l i d was t r a n s f o r m e d t o a f l u f f y b l a n d - t a s t i n g , w h i t i s h powder, w i t h no odour. U n l i k e the o r i g i n a l t r u b , which d r i e d i n b r i t t l e clumps, the p r o t e i n f l o u r , when d r i e d under the same . c o n d i t i o n s was e a s i l y c r u s h e d , w i t h a f r i a b i l i t y r e s e m b l i n g t h a t of caked sand. A f t e r i t s e x t r a c t i o n , the t r u b p r o t e i n f l o u r was e v a l u a t e d f o r i t s p r o t e i n l e v e l ( t a b l e 11), c o l o r . ( t a b l e 9), amino a c i d p r o f i l e ( t a b l e 12), n i t r o g e n s o l u b i l i t y ( t a b l e 13), e m u l s i f y i n g c a p a c i t y ( t a b l e 14) and w a t e r - h o l d i n g c a p a c i t y ( t a b l e 15). The p r o t e i n l e v e l o f the e x t r a c t e d t r u b p r o t e i n f l o u r appeared r e l a t i v e l y c o n s t a n t , r e g a r d l e s s of the amount o f e x t r a c -t i n g s o l v e n t used ( t a b l e 11), w i t h an o v e r a l l average o f 49-65% p r o t e i n . C o n v e r s i o n t o a dry w e i g h t - b a s i s (89.65% dry w e i g h t ) , g i v e s a v a l u e of 55-38% p r o t e i n t h a t i n d i c a t e s a s l i g h t i n c r e a s e from t h a t o f the o r i g i n a l u n e x t r a c t e d t r u b (52.99%)- T h i s i n -c r e a s e i s e x p e c t e d s i n c e e x t r a c t i o n o f the r e s i n component may a l t e r the p r o t e i n / n o n - p r o t e i n r a t i o . I t appears t h a t i s o p r o p a n o l - s o l u b l e . m a t e r i a l i s . r e s p o n s i b l e f o r the dark g r e e n i s h - b r o w n c o l o r of trub.. A f t e r e x t r a c t i o n , 40 FIGURE 5: Approximate y i e l d s * i n weight % of the v a r i o u s f r a c t i o n s e x t r a c t e d from wet p r e s s e d t r u b . Wet p r e s s e d t r u b (100%) Water (65%)** Trub p r o t e i n f l o u r (23%) Crude r e s i n e x t r a c t (12%) A l c o h o l - s o l u b l e r e s i n (9%) W a t e r - d i s p e r s i b l e f r a c t i o n (3%) * To the n e a r e s t 1% E s t i m a t e d by d i f f e r e n c e 4 1 T a b l e 1 0 : G e n e r a l y i e l d d a t a showing v a r i a b i l i t y o f the v a r i o u s a n a l y t i c a l e s t i m a t e s . Wet p r e s s e d t r u b D r i e d p r o t e i n f l o u r Mean dry weight (%) 2 6 . 8 5 89.65 n 9 9 S t d d e v i a t i o n 2 . 8 l 1 . 4 8 S t d e r r o r 0.09 0.49 C o e f f o f v a r i a t i o n 10 .46% 1 . 6 5 %  P r o t e i n f l o u r Mean y i e l d (%) 2 3 - 3 5 n 30 S t d d e v i a t i o n 1.8l S t d e r r o r 0 . 3 3 Coeff. of v a r i a t i o n 7 - 7 5 % Crude r e s i n (% 5 min.) W a t e r - d i s p e r s i b l e f r a c t i o n 12.05 6 0.74 0. 30 6.12% 2.89 7 0.68 0. 29 26.49% NB. A l l d a t a e x p r e s s e d on b a s i s o f wet p r e s s e d t r u b . 42 Table 11: The p r o t e i n content (6.25 x N) of various trub f r a c t i o n s , soy p r o t e i n i s o l a t e and soy f l o u r . Dried trub Trub p r o t e i n * * F l o u r @ 1:1, 5 min Trub p r o t e i n f l o u r @ 2:1, 5 min Trub p r o t e i n f l o u r @ 3:1, 5 min Wate r - d i s p e r s i b l e f r a c t i o n Soy p r o t e i n i s o l a t e Soy p r o t e i n f l o u r P r o t e i n n S.D. S.E. C.V.* (.%). (%) 52.99 3 1-36 0.79 2.6 49-95 3 0.26 0.15 0.5 51.37 3 2.53 1.46 4.9 47.63 3 1.22 0.71 2.6 '2„36 3 0.03 0.02 1.3 90.21 3 2.26 1.30 2.5 60.87 3 0.35 0.20 0.6 * C o e f f i c i e n t of v a r i a t i o n . ** A l l trub p r o t e i n f l o u r samples were ext r a c t e d 5 times wi t h isopropanol. 43 the e x t r a c t i n g s o l v e n t demonstrated the s i m i l a r g r e e n i s h hue. W i t h i n c r e a s i n g r e s i n c o n c e n t r a t i o n s , as d u r i n g vacuum e v a p o r a t i o n , the s o l v e n t l i q u i d p r o g r e s s i v e l y became dark g r e e n , l i g h t brown, c l o u d y l i g h t brown, dark brown, and f i n a l l y t u r n e d t o a v e r y d a r k , almost b l a c k c o l o r . Thus, i t i s not s u r p r i s i n g t h a t d e c o l o r i z a t i o n o f the t r u b o c c u r r e d s i m u l t a n e o u s l y w i t h r e s i n e x t r a c t i o n and depended on the same e x t r a c t i n g c o n d i t i o n s , v i z . , a g i t a t i o n and s o l v e n t r a t i o . The l a s t column of t a b l e 9 i n d i c a t e s t h a t t h e w h i t e n i n g e f f e c t ( Y % ) was g e n e r a l l y enhanced by i n t e n s i f y i n g e i t h e r c o n d i t i o n . A l l the b a t c h e x t r a c t i o n s a c h i e v e d a l u m i n -ance > 44 which was e q u i v a l e n t t o a m i l d g r e y i s h c o l o r , q u i t e s u i t a b l e f o r i n c o r p o r a t i o n i n t o f o o d systems. F u r t h e r d e c o l o r -i z a t i o n above luminance 44 may not be n e c e s s a r y . The o n l y c o l o r problem which may a r i s e i s from the a l k a l i d e b i t t e r i n g s t e p , which darkens the f l o u r (luminance 21 - 25), presumably due t o the f o r m a t i o n o f d a r k e r c o l o r e d d e g r a d a t i o n p r o d u c t s . The amino a c i d a n a l y s i s ( t a b l e 12) showed up h i g h v a r i a -b i l i t y i n the a n a l y s i s f o r many of the amino acids,' e s p e c i a l l y P r o l i n e , Tryptophan and A r g i n i n e . T h i s s u g gests t h a t perhaps because t r u b was 'prepared by t h e r m a l p r e c i p i t a t i o n , the amino a c i d c o m p o s i t i o n o f t r u b p r o t e i n f l o u r may be more v a r i a b l e t h a n t h a t o f n a t u r a l l y o c c u r r i n g p r o t e i n s . The e s s e n t i a l amino a c i d s o f t r u b p r o t e i n f l o u r were e v a l u a t e d i n t h r e e d i f f e r e n t ways as shown i n t a b l e s 16 & 17. The c l a s s i c c h e m i c a l s c o r e method ( I ) which e x p r e s s e s each of the e s s e n t i a l amino a c i d s o f the sample p r o t e i n as a p e r c e n t a g e 44 Table 12: The amino a c i d c o m p o s i t i o n o f t r u b p r o t e i n f l o u r Mean St d dev. n S t d e r r o r C .v. c A s p a r t i c a c i d & a s p a r a g i n e 7.75 1.94 9 0.65 25 Threonine 3-55 0.69 9 0.23 19 S e r i n e 4.45 0.51 9 0.17 11 G l u t a m i c a c i d & g l u t a m i n e 17.73 2.25 9 0.75 13 P r o l i n e 6.46 6.54 9 2.18 101 G l y c i n e 4.49 0.33 9 0.11 7 A l a n i n e 7.68 1.37 9 0.46 18 V a l i n e 5.31 1. 30 9 0.43 24 M e t h i o n i n e 2.07 0.47 9 0.16 23 I s o l e u c i n e 3.45 0. 40 9 0.13 12 L e u c i n e 7-94 1.47 9 0.49 19 T y r o s i n e 4.19 0.90 9 0.30 21 P h e n y l a l a n i n e 3.65 0.58 9 0.19 16 L y s i n e 3-39 0. 80 8 0.28 24 H i s t i d i n e 1.96 0.33 9 0 .11 17 Tryptophan 1.41 1.26 9 0.42 89 A r g i n i n e 8.04 2.84 8 1.00 35 C y s t i n e & C y s t e i n e 2 .41 0.69 9 0.23 29 * C o e f f i c i e n t o f v a r i a t i o n t o the n e a r e s t p e r c e n t . 45 of the corresponding amino a c i d i n whole egg p r o t e i n as r e f e r e n c e , has been c r i t i c i z e d because i t does not take i n t o account the t o t a l e s s e n t i a l amino a c i d f i g u r e . Thus, e v a l u a t i o n by method (II ) , proposed by the FAO/WHO i n 1965 (37), and which accounts f o r the t o t a l e s s e n t i a l amino a c i d s was a l s o i n c l u d e d . T h i s method (II) giv e s a higher chemical score than method (I) (66.61% vs. 48.60%). However, n e i t h e r method c o r r e l a t e s w e l l with the Net P r o t e i n U t i l i z a t i o n (N.P.U.) value obtained from animal feed t r i a l s or with the pe p s i n d i g e s t i b i l i t y v a l u e . Hence method ( I I I ) which c o r r e l a t e s b e t t e r with the N.P.U. value was a l s o in-e cluded. T h i s method, developed by Oser (38), i n v o l v e s t a k i n g the l o g of the f r a c t i o n s obtained i n method ( I ) , summing them and d i v i d i n g by the number of e s s e n t i a l amino a c i d s , and t a k i n g the a n t i l o g to o b t a i n a new chemical score value subsequently renamed the E s s e n t i a l Amino A c i d Index (EAAI). The EAAI was c a l c u l a t e d ' u s i n g the 8 amino a c i d s c o n s i d e r e d e s s e n t i a l f o r a d u l t s ( l i e , Leu, Lys, Met, Phe, Thr, Trp & V a l ) . For i n f a n t s , Arg and His are i n c l u d e d i n the above l i s t of amino a c i d s ( t a b l e 17). T h i s l a t t e r e v a l u a t i o n f o r i n f a n t s gave a high e r index than f o r a d u l t s (0.74 vs. 0.68) when a p p l i e d to trub p r o t e i n . Based on the amino a c i d values i n t a b l e 12 and the c a l c u l a t i o n s i n t a b l e 16, i t appears that the f i r s t l i m i t i n g amino a c i d i n trub p r o t e i n f l o u r i s l y s i n e , f o l l o w e d by i s o l e u c i n e then methionine. The l i m i t i n g e f f e c t of methionine i s m i t i g a t e d by the r e l a t i v e l y h i g h c y s t e i n e content. T y r o s i n e and a r g i n i n e a l s o occur at comparatively high l e v e l s . A comparison of the l i m i t i n g amino a c i d s i n trub p r o t e i n f l o u r , wheat, b a r l e y , soy-46 Table 16: Summary o f c h e m i c a l s c o r e c a l c u l a t i o n f o r t r u b p r o t e i n f l o u r by o l d and new method ( I & I I ) * * * S t d egg. p r o t e i n (SEP) Trub p r o t e i n f l o u r (TPP) Method I TPF/SEP x 100 Method I I TPP/34.28 X 51.21/SEP (g / l 6g N) (g / l 6g N) (%) Thr 5-120 3-55 69-34 95.03 V a l 6 .848 5.31 77-54 106.27 Met 3.360 2. 07 6 l .6 l 84.44 H e 6.288 3.45 54.87** 75.20** Leu 8.816 7.94 . 90.06 123.43 Tyr 4.160 4.19 100.72 138.04 Phe 5-728 3.65 63.72 87.33 Lys 6.976 3.39 48.60* 66.61* Trp 1 .488 1 .41 94.76 129.87 Cys 2. 432 2 .41 99.10 135.82 T o t a l e s s e n t i a l a . a c i d s 51-216 34.28 * 1st l i m i t i n g amino a c i d and c h e m i c a l s c o r e . * * 2nd l i m i t i n g amino a c i d . *** Re f e r e n c e #36 and 37 r e s p e c t i v e l y . Table 17: 47 Summary o f the E s s e n t i a l Amino A c i d Index c a l c u l a t i o n f o r t r u b p r o t e i n f l o u r (method I I I ) . * * TPP/SEP* Log 1 0TPP/SEP Thr V a l Met H e Leu Phe Lys Trp T o t a l ( A d u l t ) H i s Arg T o t a l ( C h i l d ) 0.6934 0.7754 0 .6161 0.5487 0.9006 0.6372 0.4860 0.9476 0. 8059 1. 3189 -0.1590 -0.1105 -0.2103 -0.2607 -0.0455 -0.1957 -0.3134 -0.0234 -1.3185 -0.0937 0.1202 -1.2920 Mean 0Adult) A n t i l o g EAAI ( A d u l t ) -0.1648 "0.6842 0.68 Mean ( C h i l d ) A n t i l o g E A A I ; ( C h i l d ) •0.1292 0.7427 0.74 * Grams of e s s e n t i a l amino a c i d i n t r u b p r o t e i n f l o u r d i v i d e d by grams o f e s s e n t i a l amino a c i d i n s t a n d a r d egg p r o t e i n . ** Refe r e n c e #38. 48 bean and ra p e s e e d i s g i v e n i n t a b l e 18. Trub p r o t e i n s c o r e s b e t t e r t h a n wheat and i s n e a r l y e q u i v a l e n t t o b a r l e y p r o t e i n a c c o r d i n g t o method ( I ) . T h i s r e s u l t i s s u p p o r t e d by a c o m p a r i -son o f t h e i r EAAI v a l u e s ( l o w e r h a l f o f t a b l e 18) w h i c h p l a c e s t r u b p r o t e i n f l o u r almost on the same l e v e l as b a r l e y p r o t e i n . A comparison o f t r u b p r o t e i n f l o u r w i t h , o t h e r common p r o -t e i n s o u r c e s ( t a b l e 19) i n d i c a t e s t h a t : ( i ) o v e r a l l , t r u b p r o t e i n b e a r s a l l the c h a r a c t e r i s t i c s o f a t y p i c a l c e r e a l p r o t e i n w i t h h i g h G l u , and low Lys and Met. ( i i ) t r u b p r o t e i n bears a g e n e r a l resemblance t o b a r l e y p r o t e i n i n most o f the amino a c i d s , except t h a t t r u b p r o t e i n i s n o t a b l y r i c h e r i n A r g , A l a and T y r , and more d e f i c i e n t i n Pro and Phe t h a n b a r l e y p r o t e i n . I t i s i n t e r e s t i n g t o note t h a t the embryo p r o t e i n o f b a r l e y i s a l s o r i c h e r t h a n the whole k e r n e l b a r l e y p r o t e i n i n A r g , A l a and Tyr and more d e f i c i e n t i n Pro and Phe (appendix 4)\. Thus one i s l e d t o c o n c l u d e t h a t t r u b p r o t e i n appears t o be a composite o f b a r l e y p r o t e i n and i t s embryo p r o t e i n , a f a c t which p o i n t s t o i t s o r i g i n as a t h e r m a l p r e c i p i t a n t . I t i s g e n e r a l l y agreed (29, 30, 31, 32) t h a t any examin-a t i o n of p r o t e i n f u n c t i o n a l i t y s h o u l d b e g i n w i t h the n i t r o g e n s o l u b i l i t y p r o f i l e , as s o l u b i l i t y i s p r o b a b l y the s i n g l e most u s e f u l i n d e x i n j u d g i n g o t h e r f u n c t i o n a l p o t e n t i a l i t i e s and i n a s s e s s i n g the s e v e r i t y of p r i o r t r e a t m e n t g i v e n t o the p r o t e i n . I n t h i s c a s e , the e f f e c t o f u s i n g i s o p r o p a n o l , a known d e n a t u r a n t i n the p r e p a r a t i o n o f f i s h p r o t e i n c o n c e n t r a t e (29), was t e s t e d 49 Table 18: E s s e n t i a l amino a c i d s o f v a r i o u s p r o t e i n s e x p r e s s e d as a pe r c e n t a g e o f the c o r r e s p o n d i n g amino a c i d o f s t a n d a r d egg p r o t e i n (method I ) . Trub p r o t e i n Wheat B a r l e y Soybean Rapeseed Thr 69-34 54.10 68.75 69.14 84.96 V a l 77.54 65.13 79.44 77.83 79.15 Met 61. 61 50.00 47.62 38.39s 62.80 H e 54.87 60.75 60.75. 75.38 69.02 Leu 90.06 76.23 78.72 79-85 76.79 Tyr 100.72 64.18 . 60.82 73.08 62.26 Phe 63.72 83.62 88.51 80.48 68.09 Lys 48.60 33.26 49.74 87.87 66.47* Trp 94.76 i-02.82 95.43 119.62 111.56 Cys 99-10 106.50 101.97 84.70 112.66 H i s 80.59 90.87 87-58 111.84 109.79 Arg 131.89 65.29 71.69 110.73 101.05 * L i m i t i n g amino a c i d E s s e n t i a l amino a c i d i n d e x : Trub p r o t e i n 0.68 Wheat 0.63 B a r l e y 0.69 Soybean 0.75 Rapeseed 0.J6 50 T a b l e 19: A comparison o f the amino a c i d c o m p o s i t i o n o f t r u b p r o t e i n , b a r l e y , wheat, soybean and rapeseed (33,34).. '..Trub P r o t e i n S e l k i r k wheat B a r l e y 6-row Soybean Rapesei Asp 7-75* 4.67 6.11 11.33 7.34 Thr 3.55 2.77 3-52 3-54 4.35 Ser 4.45 5.01 4.24 4.64 4.42 Gl u 17.73 33.12 24.48 18.08 18.08 Pro 6.46 11.14 10.94 5.14 6.13 Gly 4.49 3.76 4.19 4. 21 5. 02 A l a 7.68 3. 26 4.06 4.18 4.34 V a l 5-31 4.46 5.44 5-33 5.42 Met 2.07 1.68 1.60 1. 29 2.11 H e 3.45 3.82 3.82 4.74 4.34 Leu 7-94 6.72 . 6.94 7. 04 6.77 Tyr 4.19 2.67 2.53 3.04 2.59 Phe 3.65 4.79 5.07 4.61 3.90 Lys 3.39 2. 32 3.47 6.13 5.86 H i s 1. 96 2.21 2.13 2. 72 2.67 Trp 1.41 1.53 1. 42 1. 78 1.66 Arg 8.04 3.98 4.37 6.75 6.16 Cys 2. 41 2.59 2.48 2.06 2.74 * A l l v a l u e s i n grams o f amino a c i d / l 6 g N from the s o l u b i l i t y p r o f i l e s b e f o r e and a f t e r e x t r a c t i o n . T a b l e 13 and f i g u r e 6 prove t h a t the e x t r a c t i o n w i t h i s o p r o p a n o l dec-r e a s e d the s o l u b i l i t y s l i g h t l y , but t h a t the o v e r a l l s o l u b i l i t y o f the o r i g i n a l t r u b was a l r e a d y so low t h a t any d e c rease i n s o l u b i l i t y brought about by i s o p r o p a n o l was i n s i g n i f i c a n t . I n -c r e a s i n g the i o n i c s t r e n g t h w i t h IM NaCI r e s u l t e d i n s l i g h t l y i n c r e a s e d s o l u b i l i t y a t a c i d pH but d e p r e s s e d i t a t a l k a l i n e pH. A g a i n the e f f e c t was almost n e g l i g i b l e . Removal of t h e r e s i n appears t o enhance s o l u b i l i t y a t pH 12, p r o b a b l y because o f the n e u t r a l i z i n g e f f e c t o f the b i t t e r r e s i n a c i d s i n the u n e x t r a c t e d t r u b . The e x t r e m e l y low o v e r a l l s o l u b i l i t y of a l l the samples s u g g e s t s t h e i r t h e r m a l h i s t o r y and appears t o be symptomatic o f e x t e n s i v e and i r r e v e r s i b l e d e n a t u r e a t i o n . The e f f e c t o f heat on p r o t e i n s o l u b i l i t y has been t h o r o u g h l y examined i n the l i t e r -a t u r e (32, 39, 40, 41, 42, 43). The above f i n d i n g s e x p l a i n the r e s u l t s o f the e m u l s i f i c -a t i o n experiment ( t a b l e 14), which i n d i c a t e d t h a t , on a weight t o weight b a s i s , t r u b p r o t e i n f l o u r was a b l e t o e m u l s i f y 2/3 as much o i l as soy p r o t e i n f l o u r under i d e n t i c a l c o n d i t i o n s . T h i s r a t h e r f a i r performance was, however, marred by the g r e a t i n s t a b i l i t y o f the e m u l s i o n formed, which u s u a l l y l a s t e d l e s s t h a n 15 seconds b e f o r e s e p a r a t i n g . The w a t e r - h o l d i n g c a p a c i t y o f t r u b p r o t e i n f l o u r appears t o be good, b e i n g 2.3 t i m e s g r e a t e r t h a n soy f l o u r and 0.7 times t h a t o f the i s o l a t e on a weight b a s i s ( t a b l e 15). I t i s thought t h a t p r o t e i n s sorb water by b i n d i n g m o l e c u l e s t o s p e c i f i c h y d r o -p h i l i c s i t e s (43). Moreover, d e n a t u r a t i o n appears t o s l i g h t l y 52 Table 13: The n i t r o g e n s o l u b i l i t y p r o f i l e of trub p r o t e i n f l o u r , f r e e z e -d r i e d t r u b , and trub p r o t e i n f l o u r i n 1 M NaCl s o l u t i o n (g s o l u b l e protein/lOOg p r o t e i n ) . Trub p r o t e i n Freeze-dried Trub p r o t e i n f l o u r flour(49.65% trub (52.99% i n 1 M NaCl(49.65% p r o t e i n ) p r o t e i n ) p r o t e i n ) ;pll:'c.f the % protein-water mixture ^ p r o t e i n (6.25 x N) i n s o l u b l e protein/lOOg . s o l u t i o n ; p r o t e i n ) pH 2 2.52* 2.87 2 .21 pH 3 1.93 2.47 2 .14 pH 4.5 1.93 2.76 2. 36 PH 7 2.56 2.83 2.69 pH 8.5 2.28 2.99 2 .21 PH 10 2.87 3.65 2.53 PH 11 • 4.09 4.53 3.13 PH 12 16.64 7.H 11.46 * Average of d u p l i c a t e runs. N i t r o g e n s o l u b i l i t y (% w/w) B-o--_ Trub p r o t e i n f l o u r -O F r e e z e - d r i e d t r u b -V Trub p r o t e i n f l o u r i n IM NaCI 3 4.5 7 10 11 12 P H FIGURE 6: N i t r o g e n s o l u b i l i t y p r o f i l e o f t r u b p r o t e i n f l o u r b e f o r e e x t r a c t i o n , a f t e r e x t r a c t i o n and i n 1 M NaCI. 54 Table 14: A comparison of the e m u l s i f y i n g c a p a c i t y o f t r u b p r o t e i n f l o u r , soy p r o t e i n f l o u r , soy p r o t e i n i s o l a t e and the w a t e r - d i s p e r s i b l e f r a c t i o n (ml o i l per gram sample) Trub p r o t e i n Soy p r o t e i n Soy p r o t e i n Water-- - - f l o u r ' f l o u r i s o l a t e d i s p e r s i b l e ( d e f a t t e d ) f r a c t i o n (49.65% p r o t ) (60.87% p r o t ) (90.21% p r o t ) (2.36% p r o t ) Mean (ml o f cor n o i l e m u l s i f i e d ) 106.0 S t d dev. 1.6 n - 7 S t d e r r o r 0.6 C o e f f i c i e n t o f v a r i a t i o n (%) 1.6 152.9 3.2 4 1.6 2.1 247.8 21.2 9 7.1 8.6 97.3 2.6 4 1.3 2.6 55 Table 15: A comparison o f the t r u b p r o t e i n f l o u r , (g w a ter/g sample) water h o l d i n g c a p a c i t y o f soy f l o u r and soy i s o l a t e Trub p r o t e i n - Soy p r o t e i n Soy p r o t e i n f l o u r f l o u r i s o l a t e ( d e f a t t e d ) Mean tg H„0/g sample) 2.55 8.25 S t d dev. n S t d e r r o r C o e f f i c i e n t o f v a r i a t i o n {%) 0.18 3 . 0.10 3.03 0. 04 3 0. 02 1. 42 1. 09 2 0.77 13. 20 56 enhance w a t e r - h o l d i n g c a p a c i t y (44, 45). T h i s i s thought t o be due t o an i n c r e a s e i n the number of w a t e r - b i n d i n g or h y d r a t i o n s i t e s , a r i s i n g from the u n f o l d i n g of the p o l y p e p t i d e c h a i n d u r i n g d e n a t u r a t i o n . An o v e r a l l o b s e r v a t i o n o f t r u b p r o t e i n f l o u r l e a d s t o the f o l l o w i n g c o n c l u s i o n : 1 . Trub p r o t e i n has a n u t r i t i o n a l p o t e n t i a l almost e q u i v a l e n t to t h a t o f b a r l e y , j u d g i n g from i t s e s s e n t i a l amino a c i d i n d e x . N u t r i t i o n a l q u a l i t y may be c o n c l u s i v e l y e s t a b l i s h e d i n a c t u a l f e e d t r i a l s . 2. The good w a t e r - h o l d i n g c a p a c i t y of the t r u b p r o t e i n f l o u r s h o u l d be c o n f i r m e d w i t h o t h e r r e l a t e d f u n c t i o n a l t e s t s such as s w e l l i n g b e f o r e t e s t i n g i n a c t u a l p r o d u c t s . K i n s e l l a (31) suggests i n c o r p o r a t i o n i n t o meat systems as an e x c e l l e n t way t o t e s t t h i s p a r t i c u l a r p r o p e r t y . S i n c e the water b i n d i n g c a p a c i t y i s a l s o c l o s e l y r e l a t e d t o v i s c o s i t y (30, 46), i t s performance i n v i s c o u s foods such as soups, purees and even doughs may be e n l i g h t e n i n g . P a r t C: E v a l u a t i o n of the t r u b r e s i n f r a c t i o n : Trub r e s i n was s e p a r a t e d . b y TLC a c c o r d i n g t o the method of F r a n i a u & Mussche ( 1 1 ) . The r e s i n components. of t r u b were s e p a r -a t e d on a s i l i c a g e l G / c e l l u l o s e p l a t e a t pH 5. Under uv l i g h t each component e x h i b i t e d a d i s t i n c t i v e f l u o r e s c e n t c o l o r . These however, d i d not c o r r e l a t e w e l l w i t h Rf v a l u e , and an attempt t o match t r u b r e s i n components w i t h those from f r e s h and aged hops was u n s u c c e s s f u l ( t a b l e 20 and 21). Thus whenever p o s s i b l e , uv scans T a b l e 20: .T.L.C. comparison o f f r e s h hop components s t o r e d at 10°C f o r d i f f e r e n t time p e r i o d s . # uv c o l o r Rf v a l u e X max 1 day 20 days 6 mths (nm) 1. B l a c k > 0.37 230,373b,254s* 2. L i g h t b l u e 0.37 3. Broad r e d 0.44 4. B l a c k (.trace) > 0.44 5. F l u o r e s c e n t y e l l o w 0.46 283b 6. Dark p u r p l e 0 • 51 0.49 7 -8. L i g h t y e l l o w B l a c k 0.50 > 0.50 9. Orange-red > • • 0.55 233,274b 10. B l u e - w h i t e 0.58 11. P i n k 0.58 231,320b 12. B l a c k 0.61 234,322b 13. B l u e - w h i t e 0.61 14. P u r p l e - p i n k > 0.61 15. Y e l l o w 0 .64 16. B l u e 0 .69 0.68 O.67 17. P i n k 0 • 72 0.70 18. B l u e 0.76 19- G r e e n - y e l l o w 0 .80 0.92 O.87 20. V i o l e t 0 .85 *b = b r o a d , s = s h o u l d e r 58 T a b l e 21: T.L.C. comparison o f t r u b r e s i n components s t o r e d at 10°C f o r ze r o a n d . s i x months. # uv c o l o r Rf v a l u e A max. 0 months 6 months, (nm) 1. L i g h t b l u e 2. B l a c k 3. Broad grey 4. B l a c k 5. Orange-red 6. P u r p l e 0. 47 7. White 8. Broad p i n k 9. L i g h t p u r p l e 0. 57 10. P u r p l e - p i n k 0. 62 11. Dark green 12. L i g h t b l u e - p u r p l e 0. 64 13. I 2 o n l y * s > 0. 64 14. I 2 o n l y 0. 68 15- P i n k 0. 72 16. L i g h t b l u e 0. 82 17. G r e e n - y e l l o w 0. 91 18. I 2 o n l Y 0. 98 B l a n k scan o f the s i l i c a g e l 0.23 0.31. 232 s* 0.37 • 255 0.43 0.44 282 b, s 0.48 281 0.51 266 b 0.62 253 O.65 354 0.81 273 207, 220 * s = s h o u l d e r , b = broad peak ** Could be d e t e c t e d o n l y a f t e r s a t u r a t i o n w i t h I ? vapor. 59 were made from the s e p a r a t e d components t o determine t h e i r a b s o r p -t i o n maxima f o r p o s s i b l e i d e n t i f i c a t o n . Maxima at wavelengths l e s s t h a n 220 nm were i g n o r e d because o f g e l a b s o r p t i o n a t 220 and 207 nm. Some d i f f i c u l t y was encountered i n o b t a i n i n g c o n c l u s i v e scans because o f ( i ) the i n s t a b i l i t y o f the s e p a r a t e d components t o a i r and l i g h t , and ( i i ) i n t e r f e r e n c e from t a i l i n g , p a r t i c u l a r l y i n t h e f r e s h hop s e p a r a t i o n . However, the maximum wavelengths o b t a i n e d , when compared t o p u b l i s h e d v a l u e s (appendix 6 and 7), t e n t a t i v e l y i n d i c a t e d the presence o f : (.(.i) the i s o - a l p h a - a c i d s (#11, t a b l e 21) ( i i ) the b e t a - a c i d s (#12, t a b l e 21) ( i i i ) h u m u l i n i c a c i d (#8, t a b l e 21) No a l p h a - a c i d was d e t e c t e d i n t r u b r e s i n , p o s s i b l y because o f o x i d a t i o n l o s s e s d u r i n g the e x t r a c t i o n . Hulupone, the b i t t e r f i r s t o x i d a t i o n p r o d u c t o f the b e t a - a c i d s ( l u p u l o n e s ) may be p r e s e n t at #3, t a b l e 21; the second maximum a t 325 nm was masked p r o b a b l y because o f the s m a l l q u a n t i t y o f hulupone p r e s e n t . An o v e r a l l uv scan o f t h e f u l l m i x t u r e o f t r u b r e s i n comp-onents i n d i c a t e s a maximum r a n g i n g from 226 - 243 nm, w i t h a s h o u l d e r at 275 nm. An o v e r a l l v i s i b l e scan shows a s m a l l peak at 658 nm, w i t h a r a p i d i n c r e a s e as one approaches the uv range. A q u a n t i t a t i v e e s t i m a t i o n o f the s e p a r a t e d t r u b r e s i n components was not attempted s i n c e the pure b i t t e r components were not a v a i l a b l e t o use as r e f e r e n c e s . Any e v a l u a t i o n of the b i t t e r i n g c a p a c i t y o f t h e t r u b r e s i n components would be premature a t t h i s p o i n t and must aw a i t the 60 r e s u l t s of the b i t t e r i n g t r i a l s . However, as mentioned i n the l i t e r a t u r e r e v i e w s e c t i o n , the c o n t r i b u t i o n t o b i t t e r n e s s o f the r e b o i l e d t r u b would be d e r i v e d m a i n l y from the o x i d i s e d b e t a - a c i d s , the o x i d i s e d i s o - a l p h a - a c i d s , and any r e m a i n i n g i s o - a l p h a - a c i d s . Lead conductometry (9) was c a r r i e d out t o o b t a i n a q u a n t i t -a t i v e v a l u e f o r the. b i t t e r i n g p o t e n t i a l o f the t r u b r e s i n . I t i s known t h a t f r e s h hop a n a l y s i s by l e a d conductometry y i e l d s a l e a d conductance v a l u e (LCV) c l o s e l y a p p r o x i m a t i n g i t s a l p h a - a c i d c o n t -e n t , and t h e r e f o r e p r o v i d e s a good e s t i m a t e o f i t s b i t t e r i n g p o t e n t i a l d u r i n g the b o i l i n g . I n the case o f o l d e x t r a c t s o r o l d hops, the LCV i s l e s s r e l a i b l e as a guide t o b i t t e r i n g p o t e n t i a l , s i n c e o t h e r f r a c t i o n s i n t e r f e r e w i t h the a n a l y s i s by a l s o p r e c i p i -t a t i n g the l e a d . N e v e r t h e l e s s , the LCV i s s t i l l used as a guide t o b r e w i n g v a l u e i n t h e s e c a s e s , s i n c e the f r a c t i o n s r e s p o n s i b l e f o r b i t t e r n e s s i n o l d hops a l s o p r e c i p i t a t e l e a d . The LCV o f o l d hops has been s t a t i s t i c a l l y c o r r e l a t e d w i t h i t s b i t t e r i n g power i n b i t t e r n e s s t a s t e t r i a l s (10). The LCV o b t a i n e d f o r t r u b r e s i n e x t r a c t was 68.4% ( t a b l e 22). T h i s v a l u e of 68.4% r e p r e s e n t s the LCV o f the h i g h e s t p o s s i b l e c o n c e n t r a t i o n o f the t r u b r e s i n , a f t e r as much i s o p r o p a n o l and water as p o s s i b l e were removed by vacuum e v a p o r a t i o n a t 40°C. S i n c e no a l p h a - a c i d was d e t e c t e d i n the TLC e x p e r i m e n t , i t appears t h a t the LCV o f 68.4% i s due m a i n l y t o the p r e c i p i t a t i n g a c t i o n of o t h e r b i t t e r f r a c t i o n s , presumably the o x i d i s e d r e s i n f r a c t i o n s . I n c o n t r a s t , a s o l v e n t e x t r a c t o f f r e s h hops, c o n c e n t r a t e d t o an LCV o f 68.4% c o n t a i n s approx. 68.4% a l p h a - a c i d and l i t t l e o r no o x i d i s e d m a t e r i a l . The c o n d u c t i v i t y p l o t ( f i g . 7 ) i n d i c a t e s t h a t the t r u b 61 Table 22: To show the number o f ml * o f l e a d a c e t a t e r e q u i r e d t o p r e c i p i t a t e the 4 main t r u b r e s i n f r a c t i o n s and t o show the l e a d conductance v a l u e c a l c u l a t i o n . Sample # Run # ml of l e a d a c e t a t e p r e c i p i t a t e d (3-778% w/v) 1 1 •3. 493 (!)** 9.608 (2) 1 2 .3. 735 (1) 7.871 (2) 22.918 (4) 1 3 4. 010 (1) 8.164 (2) 14.707 (3) 2 4 4. 784 (1) 9-467 (2) 14.550 (3) 2 5 4. 384 (1) 12.277 (3) 2 6 3. 523 (1) 8.795 (2) 20.617 (4) 3 7 3. 585 (1) 13.880 (3) 27.803 (4) 3 8 3. 931 (1) 10.893 (2) 21.951 (4) 3 9 4. 056. (1) 7.861 (2) 19.651 (4). Mean o f f r a c t i o n (1) = 3. 945 m i s , w i t h n = 9 Mean of f r a c t i o n (2) = 8. 951 m i s , w i t h n = 7 Mean o f f r a c t i o n (3) = 13 .854 mis , w i t h n = 4 Mean o f f r a c t i o n (4) = 22 .588 mis , w i t h n = 5 C a l c u l a t i o n f o r l e a d conductance value. (LCV): Formula: P e r c e n t LCV = ml o f 4% (w/v) PbAc .x. 76.98*** g o f e x t r a c t = 3-9 x (3.778/4.000) x 76.98 4.1298 = 68.41% * The number o f ml o f l e a d a c e t a t e a r e the a b s c i s s a e of the i n t e r s e c t i o n p o i n t s o f the 4 l i n e s o b t a i n e d by l i n e a r r e g r e s s i o n of the c o n d u c t i v i t y p l o t . ** Numbers i n b r a c k e t s s i g n i f y the r e s i n f r a c t i o n eg. (1) = f i r s t r e s i n f r a c t i o n . * * * 76.98 = c o n s t a n t f o r . L C V f o r m u l a when u s i n g Wollmer v a r i a n t . C o n d u c t i v i t y (mho x 10 ) 15 h FIGURE 7 : Lead conductance p l o t f o r t r u b r e s i n e x t r a c t . 63 r e s i n components tended t o p r e c i p i t a t e i n 4 main f r a c t i o n s , v i z . , at 3-9, 9-0, 13-9 and 22.6 mis o f l e a d a c e t a t e . As i n s t a n d a r d l e a d conductance e s t i m a t i o n s , o n l y the 3.9 ml v a l u e was used t o d e r i v e the LCV o f 68.4%, the assumption b e i n g t h a t the c o n t r i b u t i o n s t o b i t t e r n e s s o f the o t h e r f r a c t i o n s a r e n e g l i g i b l e . I t was f e l t t h a t some k i n d o f p r e l i m i n a r y b r e w i n g t e s t was n e c e s s a r y i n o r d e r t o determine ( i ) i f the b i t t e r s u b s t a n c e s I n t r u b r e s i n a re c a p a b l e o f i m p a r t i n g b i t t e r n e s s t o sweet w o r t , ( i i ) whether the i m p a r t e d b i t t e r n e s s I s s i g n i f i c a n t l y d i f f e r e n t t h a n t h a t d e r i v e d from f r e s h hops. Dark malt e x t r a c t was q u a n t i t a t i v e l y hopped i n a model system w i t h f r e s h hops and t r u b r e s i n e x t r a c t (see method s e c t i o n ) and fermented w i t h a l e y e a s t . Dark malt e x t r a c t was used t o m i n i m i z e the c o l o r d i f f e r e n c e between p a i r e d samples i n t h e sub-sequent t a s t e t r i a l , t h e r e b y e l i m i n a t i n g c o l o r b i a s . A l e y e a s t was chosen p a r t l y because an a e r o b i c f e r m e n t a t i o n was s i m p l e r t o c o n t r o l and p a r t l y because o f the p r a c t i c e by l a g e r brewers of not a l l o w i n g t h e i r spent hops t o s t a n d f o r any time i n the wort because i t i s b e l i e v e d t o a f f e c t f l a v o r . Moreover, a l e c h a r a c t e r appears more c o m p a t i b l e w i t h s t r o n g e r hop a d d i t i o n s t h a n l a g e r . D u r i n g f e r m e n t a t i o n , the t r u b r e s i n b a t c h f a i l e d t o form a s t a b l e y e a s t head, making r e g u l a r skimming i m p o s s i b l e . N e v e r t h e l e s s , a f t e r 3 days, f e r m e n t a t i o n completed as the wort was no l o n g e r sweet. 64 The o v e r a l l r e s u l t s o f the t a s t e t r i a l s i n d i c a t e t h a t t h e r e i s a d e t e c t a b l e d i f f e r e n c e between the a l e s brewed w i t h t r u b r e s i n and those brewed w i t h f r e s h hop. S p e c i f i c a l l y however, the d i f f e r e n c e appears t o be s l i g h t , s i n c e w i t h 54 o p i n i o n s , no d i f f e r e n c e c o u l d be d e t e c t e d ( t a b l e 23, 2 4 ) . Moreover, the j u d g e -ments as t o which of the " d i f f e r e n t " samples were more b i t t e r were not a t a l l s i g n i f i c a n t ( t a b l e 25) i n d i c a t i n g t h a t the b i t t e r n e s s l e v e l s i n the f r e s h hop and t r u b r e s i n samples w e r e r v e r y c l o s e t o g e t h e r . F u r t h e r m o r e , the t a s t e t r i a l s have shown t h a t f i r s t l y , t r u b r e s i n can be r e i n c o r p o r a t e d i n t o wort by r e b o i l i i n g , and s e c o n d l y , t h a t t r u b r e s i n does d e f i n i t e l y c o n t r i b u t e t o wort b i t t e r n e s s . An e s t i m a t e o f the amount o f t r u b r e s i n u t i l i z e d i n t h e s e t r i a l s " may be i n t e r e s t i n g a t t h i s p o i n t . A random s u r v e y of l o c a l b eer has r e v e a l e d t h a t most l a g e r s l i e i n the range o f 12 EBU ( t a b l e 2 6 ) . Toby C h a r r i n g t o n , which i s q u i t e b i t t e r t o the Canadian p a l a t e , r e g i s t e r s a t 23 EBU. The t r u b r e s i n a l e was brewed at 20 EBU. I t r e q u i r e s r o u g h l y 1 g o f t r u b r e s i n per l i t e r o f wort b o i l e d t o a c h i e v e 20 EBU. I f the y e i l d o f r e s i n i s c o n s e r v a t i v e l y p l a c e d a t 10% o f wet p r e s s e d t r u b , t h e n 1 kg o f t r u b y i e l d s 100 g o f r e s i n which i s c a p a b l e of b i t t e r i n g 100 l i t e r s o f a l e . P o s s i b l e problems from r e u t i l i z i n g t r u b r e s i n may a r i s e from 3 a r e a s : 1. Yeast m a l f u n c t i o n : top f e r m e n t i n g y e a s t s may cease t o be top f e r m e n t o r s , t h e r e b y c r e a t i n g skimming p r o b l e m s , and 65 Table 23: Summary of p a i r e d comparison r e s u l t s t o determine i f d i f f e r e n c e i n b i t t e r n e s s i s d e t e c t a b l e between: 1. F r e s h hop samples and 100% t r u b r e s i n samples 2. F r e s h hop samples, and 50% t r u b r e s i n samples 3. F r e s h hop samples and p o o l e d * t r u b r e s i n samples D i f f e r e n c e judgement # o f judgements F r e s h hop v s . 100% t r u b r e s i n A d i f f e r e n c e e x i s t s 33 No d i f f e r e n c e e x i s t s . . . . . . . , 2 1 T o t a l 5_4" F r e s h hop v s . 50% t r u b r e s i n A d i f f e r e n c e e x i s t s 34 No d i f f e r e n c e e x i s t s 20_ T o t a l 5 i F r e s h hop v s . p o o l e d t r u b r e s i n A d i f f e r e n c e e x i s t s 67 No d i f f e r e n c e e x i s t s 4l_ T o t a l 108 E x t e n t o f d i f f e r e n c e s (more b i t t e r ) F r e s h hop Trub r e s i n 1. 15 15 30 18 12 37 s Data was s u b j e c t e d t o h e t e r o g e n i e t y t e s t (47), found t o be homogeneous and t h e r e f o r e p o o l e d . 66 T a b l e 24: Summary o f Ch i - s q u a r e a n a l y s i s w i t h Y a t e s ' c o r r e c t i o n (47) a p p l i e d t o determine l e v e l o f s i g n i f i c a n c e between 1, 2 and 3-1. F r e s h hops v s . 100% t r u b r e s i n (.54 o p i n i o n s ) . C h i - s q u a r e v a l u e = 2..2407 P r o b a b l y i s between 10% and 25% T h e r e f o r e not s i g n i f i c a n t at the 5% l e v e l . T h i s i m p l i e s t h e r e i s no d i f f e r e n c e i n b i t t e r n e s s between f r e s h hops and 100% t r u b r e s i n . 2. F r e s h hops v s . 50% t r u b r e s i n (54 o p i n i o n s ) . C h i - s q u a r e v a l u e = 3.1296 P r o b a b i l i t y i s between 5% and 10% T h e r e f o r e not s i g n i f i c a n t a t the 5% l e v e l . T h i s i m p l i e s t h e r e i s no d i f f e r e n c e i n b i t t e r n e s s between f r e s h hops and 50% t r u b r e s i n . 3. F r e s h hops v s . 'pooled* t r u b r e s i n (.108 o p i n i o n s ) . P o o l e d C h i - s q u a r e v a l u e = 5-7870 P r o b a b i l i t y i s between 1% and 2.5% T h e r e f o r e s i g n i f i c a n t a t the 5% l e v e l . T h i s i m p l i e s t h e r e i s _ a d i f f e r e n c e between f r e s h hops and t r u b r e s i n (50% and 100% b a t c h e s ) w i t h r e s p e c t t o b i t t e r n e s s . * D a t a was s u b j e c t e d t o h e t e r o g e n i e t y t e s t (.47), found t o be homogeneous and t h e r e f o r e p o o l e d . 67 Table 25: Summary o f the t e s t ( C h i - s q u a r e a n a l y s i s w i t h Y a t e s ' c o n t i n u i t y c o r r e c t i o n ) t o determine i f the e x t e n t o f d i f f e r e n c e s (.choice of which i s more b i t t e r ) was due t o chance o r was s i g n i f i c a n t . 1. F r e s h hop v s . 100% t r u b r e s i n (34 o p i n i o n s ) C h i - s q u a r e v a l u e = 0.0606 P r o b a b i l i t y between 90% and 75% T h e r e f o r e not s i g n i f i c a n t at the 5% l e v e l . T h i s i m p l i e s t h a t the b i t t e r n e s s c h o i c e was due t o chance. 2. F r e s h hop v s . 50% t r u b r e s i n (.34 o p i n i o n s ) C h i - s q u a r e v a l u e = 0.2650 P r o b a b i l i t y between 75% and 50% T h e r e f o r e not s i g n i f i c a n t at the 5% l e v e l . T h i s i m p l i e s t h a t t h e b i t t e r n e s s c h o i c e was due t o chance. 3. F r e s h hop v s . p o o l e d * t r u b r e s i n (67 o p i n i o n s ) C h i - s q u a r e v a l u e = 0.5373 P r o b a b i l i t y between 50% and 25% T h e r e f o r e not s i g n i f i c a n t at the 5% l e v e l . T h i s i m p l i e s t h a t the b i t t e r n e s s c h o i c e was due t o chance. * Data was s u b j e c t e d t o h e t e r o g e n i e t y t e s t (47) and found t o be homogeneous b e f o r e p o o l i n g . 68 a s s o c i a t e d f l a v o r problems. A s h u r s t ( 2 6 ) mentions t h a t the . a d d i t i o n o f a s m a l l amount o f hops or i t s water extract'-'may remedy t h i s tendency, which a p p a r e n t l y a l s o o c c u r s w i t h the use o f i i s o m e r i z e d hop e x t r a c t s . 2 . The p o l y p h e n o l i c c o n s t i t u e n t s o f the t r u b r e s i n may a c t as haze p r e c u r s o r s , l e a d i n g t o c h i l l o r permanent haze a t the f i n i s h i n g s t a g e s o f p r o d u c t i o n . On the o t h e r hand, the p o l y p h e n o l i c s may a c t b e n e f i c i a l l y as p r o t e i n p r e c i p i t a n t s d u r i n g the b o i l i n g o f the wor t . 3. The l i t e r a t u r e g e n e r a l l y a l l u d e s tb..:the h a r s h e r f l a v o r n o tes a s s o c i a t e d w i t h aged o r o x i d i s e d r e s i n s ; thus o v e r -s p a r g i n g o f the spent hops or a p r o - a l k a l i n e wort b o i l i s g e n e r a l l y a v o i d e d i n the p r o d u c t i o n o f the f i n e r t a s t i n g b e e r s , a l t h o u g h the h a r s h e r c h a r a c t e r i s d e s i r a b l e i n the manufacture o f the more b i t t e r s t o u t s and a l e s . 69 Table 26: Comparative b i t t e r n e s s l e v e l s i n l o c a l (B.C.) b e e r s a . European b i t t e r n e s s u n i t s (EBU) L a b a t t ' s b l u e ( l a g e r ) 12.3* Cream S t o u t ( L a b a t t ) 15-7 Toby C h a r r i n g t o n ( C a r l i n g 0 'Keefe) 22.7 Guinness ( L a b a t t ) 4l.O Average o f d u p l i c a t e r e a d i n g s N.B. E.B.U. = 50 x absorbance % 275 nm. 70 Appendix 1: The e f f e c t o f b o i l i n g on t h e m o l e c u l a r weight . d i s t r i b u t i o n of wort p r o t e i n s ( 4 ) . M W < 5000 5-10,000 10-50,000 50-100,000 >1,000,000 B o i l e d * 0.0175 0.0125 0.0040 0.0010 0 Not B o i l e d 0.0336 0.0185 , 0.0101 0.0023 0.0028 95 minutes 71 Appendix 2: The p r o t e i n c o m p o s i t i o n o f v a r i o u s g r a i n seed p r o t e i n s (6). % T o t a l P r o t e i n Albumin G l o b u l i n Prolamine, G l u t e l i n T o t a l P r o t e i n (% dry wt) R i c e 5.0 10.0 5.0 80. 0 8 - 10 Oat 1.0 78.0 16. 0 5.0 8 - 14 B a r l e y 13.0 12. 0 52.0 23.0 (normal) ( H o r d e i n ) B a r l e y 18.0 14.0 46.0 22.0 10 - 16 ( H i p r o l y ) Wheat 3 - 5 10. 0 69.0 16.0 Rye 5 - 1 0 5 - 1 0 30 - 50 30 - 50 9 - 14 P e a r l M i l l e t 13. 2 • 9.4 40.0 28.0 12 - 18 Maize 4.0 2.0 55.0 39.0 7 - 13 Sorghum 8.0 8.0 52.0 32.0 9 - 13 72 Appendix 3: The n i t r o g e n c o n t e n t o f the 'crude p r o t e i n ' f r a c t i o n s o f 'Garton improved' b a r l e y and malt (48)-N i t r o g e n , g/lOOg dry weight B a r l e y M a l t & Roots K i l n m alt w i t h o u t r o o t s T o t a l N 1.703 1.684 1.597 S a l t s o l u b l e N 0.563 1.048 0.935 H o r d e i n 0.631 0.261 0.269 G l u t e l i n 0.509 0.375 0.393 G l o b u l i n 0.159 0.210 Albumin 0.186 0.183 0.184 P r o t e o s e 0.093 O.198 N o n - p r o t e i n N 0.130 0.546 0.507 Appendix 4: The amino a c i d c o m p o s i t i o n o f b a r l e y .05). BARLEY (g/lOOg p r o t e i n ) Amino a c i d Whole k e r n e l Endosperm Embryo Lys 3. .90 2. ,80 6. ,20 H i s 2. . 20 2. , 00 3. .30 Arg 4. .40 3. .90 9-,80 Asp 6. .80 4. ,80 10. • 30 Thr 3-.40 2. .80 4. ,10 Ser 3. .70 3. . 20 4. .40 G l u 26. .10 29-.50 15. .80 Pro 11. ,40 14. . 00 4. .90 G l y 4. , 20 2. .90 6. .40 A l a 4. ,40 3. .20 6. .40 Cys 1. .25 1. . 20 0. .70 V a l 5. .30 5. . 00 5. • 70 Met 2. , 60 2. • 90 2. .10 H e 3. .80 3. .70 3. .50 Leu 7. . 10 6. .70 . 6. .50 Tyr 1. • 90 2, . 60 2, .70 P - a l a 5. .40 5. .40 4, .20 74 Appendix 5: The d i s t i l l a t i o n c o n s t a n t s f o r methylene c h l o r i d e , hexane and i s o p r o p a n o l (21). Methylene c h l o r i d e Hexane I s o p r o p a n o l B.P. o f s o l v e n t 1(°C) % C o m p o s i t i o n (v/v) o f water a z e o t r o p e % H 20 % S o l v e n t B.P. o f a z e o t r o p e (°C) # o f l a y e r s % S o l v e n t i n each l a y e r Upper l a y e r Lower l a y e r 40. 0 1 99 38.8 2 2. 99-9 61.0 5.8 9.4.4 61.6 2. 96.2 3.8 82.4 12.2 87.8 80.4 m i s c i b l e 75 Appendix 6: Wavelength o f hop b i t t e r s u b s t a n c e s i n a l k a l i n e methanol (11)• Component 1st maximum 2nd max. B e t a - a c i d s 355 A l p h a - a c i d s 324 360 1st grade d e g r a d a t i o n p r o d u c t s of the i s o - a l p h a - a c i d s 255 271 I s o - a l p h a - a c i d s 255 271 H u m u l i n i c a c i d s 257 266 A l l o - i s o - a l p h a - a c i d s 248 271 Hydrated i s o - a l p h a - a c i d s 255 271 Hulupones 255 325 Humulinone 259 273 76 Appendix J: B i t t e r s u b s tances of hot t r u b i s o l a t e d by r e v e r s e d phase chromatography (7). pH o f e l u a t e Name of compound A b s o r p t i o n max 5.1 cohulupone 255, 325 5.4 hulupone 255, 325 5-6 adhulupone 255, 325 5.7 isocohumulone 255 6.0 isohumulone 255 6.4 isoadhumulone 255 8.9 272, 335 9.1 272 9.2 272 9-5 272 9-5 xanthohumol 380 9.6 272, 375 9.6 325, 370 9.8 320, 375 10.0 c o l u p u l o n e 355 10.0 272 10.2 l u p u l o n e 355 10.3 a d l u p u l o n e 355 77 REFERENCES 1. Hough, J.S., B r i g g s , D.E. and S t e v e n s , R., M a l t i n g & Brewing S c i e n c e , Chapman & H a l l L t d , London 1971. 2. L u e r s , H., Die w i s s e n s c h a f t l i c h e n Grundlagen von M a l z e r e i und B r a u e r e i , V e r l a g Hans Huber, Nurnberg 1950. 3. R o y s t o n , M.G., "Wort b o i l i n g and c o o l i n g " i n Modern Brewing Technology e d i t e d by F i n d l a y , W.P.K. p6l , M a c m i l l a n P r e s s 1971. 4. Guenther, K.R. and S t u t l e r , J.R., P r o c . Ann. M e e t i n g , Am. Soc. Brew. Chem. p30, 1965-5. Munck, L., B a r l e y Seed P r o t e i n s i n Symposium: Seed P r o t e i n s , e d i t e d by I n g l e t t , G.E. pl44, A v i , Conn. 1972. 6. S h u k l a , T.P., C e r e a l P r o t e i n s : C h e m i s t r y and Food A p p l i c a -t i o n s , CRC C r i t . Rev. Food Sc. & N u t r . , V o l 6, p i , June 1975. 7. - S p e t s i g , L.O., The b i t t e r s u b s t a n c e s o f spent hops, t r u b and y e a s t c o v e r : A chroma t o g r a p h i c s t u d y , J . I n s t . Brew. 74:346, 1968. 8. Laws, D.R.J., Hop r e s i n s and beer f l a v o u r V. The s i g n i f i c a n c e o f o x i d i s e d hop r e s i n s i n b r e w i n g , J . I n s t . Brew. 74:178, 1968. 9. Howard, C.A., Chairman o f I n s t . Brew. A n a l . Committee, "Recommended Methods o f A n a l y s i s " , J . I n s t . Brew. 77: l8 l , 1971. 10. Howard, G.A. and M a r t i n , -P.A., B i t t e r i n g power o f s t o r e d hops, J . I n s t . Brew. 70:424, 1964. 11. F r a n i a u , R. & Mussche, R. , Q u a n t i t a t i v e d e t e r m i n a t i o n o f hop b i t t e r s u b s t a n c e s and t h e i r d e r i v a t i v e s i n hop e x t r a c t s by t h i n l a y e r chromatography, J . I n s t . Brew. 80:59, 1974. 12. Concon, J.M. & S o l t e s s , D., Ra p i d m i c r o k j e l d a h l d i g e s t i o n o f c e r e a l g r a i n s and o t h e r b i o l o g i c a l m a t e r i a l s , A n a l . B i o c h . 53:35, 1973. 13. C a v i n s , J .F., K r u l l , L.H., Friedman, M., G i b b s , D.E. & I n g l e t t , G.E., S p e c t r o p h o t o m e t r i c c y s t e i n e a n a l y s i s , J . Agr. Food Chem. 20:1124, 1972. 14. L i u , T.Y. & Chang, Y.H., H y d r o l y s i s o f p r o t e i n s w i t h p - t o l u e n e s u l f o n i c a c i d , J . B i o l . Chem. 246:2842, 1971. 15. Balmaceda, E.A., Kim, M.K., F r a n z e n , R., Mardones, B. & Lugay, J . C , P r o t e i n F u n c t i o n a l i t y Methodology - S t a n d a r d T e s t s , G e n e r a l Foods Corp. Tech. C e n t e r , T a r r y t o w n , N.Y. 1976. 16. F r a n c i s , F . J . and C l y d e s d a l e , F.M., Food C o l o r i m e t r y : Theory and A p p l i c a t i o n s , p 76. A v i , Conn. 1976. 17. R i g b y , F.L. & Bethune, J . L . , J . I n s t . Brew. 61:325, 1955-18. R o b b i n s , G.S. and Pomeranz, Y., C o m p o s i t i o n and u t i l i z a t i o n o f m i l l e d b a r l e y p r o d u c t s I I I . Amino a c i d c o m p o s i t i o n , C e r e a l Chem. 49:240, 1972. 19. Kramer, A. & Twigg, B., Q u a l i t y c o n t r o l f o r the fo o d i n d u s t r y , p 140. A v i , Conn. 1970. 20. Loomis, W.D. and B a t t a i l e , J . , P l a n t p h e n o l i c compounds and i s o l a t i o n o f p l a n t enzymes, Phytochem. 5:423, 1966. 21. Handbook o f P h y s i c s and C h e m i s t r y . , Chemical Rubber Co., Ohio, 47th Ed, 1966-67. 22. N i l s s o n , T.R. and Sandegren, K.E., Swedish P a t e n t 150:997, 1955-23. S p e t s i g , . L.O., The b i t t e r s u b s t a n c e s o f hops. A chroma-t o g r a p h i c s t u d y , J . I n s t . Brew. 72:266, 1966. 24. A s h u r s t , P.R., Laws, D.R.J, and Pinnegar,. M.A., F u r t h e r s t u d i e s on the o x i d a t i o n o f hop r e s i n s , J . I n s t . Brew. 72:561, 1966. 25. A s h u r s t , P.R. & E l v i d g e , J.A., J . Chem. Soc. C , p675, 1966. 26. A s h u r s t , P.R., Hops and t h e i r Use i n Bre w i n g , i n Modern Brewing Technology ed. by W.P.K. F i n d l a y , M a c m i l l a n 1971. 27. V e r z e l e , M., Hop e x t r a c t s - c h e m i s t r y , a n a l y s i s and u t i l i z -a t i o n , p 95, European Brewery C o n v e n t i o n 1971. 28. S t a t i s t i c s Canada, C a t a l o g u e #63202. The c o n t r o l and s a l e of a l c o h o l beverages, i n Canada, f o r f i s c a l y e a r e n d i n g Mar 31, 1975. 29. M a t t i l , K.F., The f u n c t i o n a l r e q u i r e m e n t s o f p r o t e i n s i n f o o d s , J . Am. O i l Chem. Soc. 48:477, 1971. 30. Hermansson, A.M., D e t e r m i n a t i o n o f F u n c t i o n a l P r o p e r t i e s o f P r o t e i n Foods, i n Problems - Human N u t r i t i o n , ed. by P o r t e r , J . & R o l l s , B. Acad.. P r e s s 1973-31. K i n s e l l a , J.E., F u n c t i o n a l p r o p e r t i e s o f p r o t e i n s i n foods -a s u r v e y , C r i t . Rev. Fd Sc. & N u t r . p 219, V . 7 , A p r i l 1976. 32. B e t s c h a r t , A.A., N i t r o g e n s o l u b i l i t y o f a l f a l f a p r o t e i n c o n c e n t r a t e as i n f l u e n c e d by v a r i o u s f a c t o r s , J . Food Sc. 39:1110, 1974. 33. Burrows, V.D., Greene, A.H.M., K o r o l , M.A., Melnychyn, P., Pea r s o n , G.G. & S i b b a l d , I.R., Food P r o t e i n from G r a i n s and O i l s e e d s . , O f f i c e o f the M i n i s t e r r e s p o n s i b l e f o r the Canadian Wheat Board, Ottawa.1972. 34. Tkachuk, R. and I r v i n e , G.N., Amino a c i d c o m p o s i t i o n s o f c e r e a l s and o i l s e e d m eals., C e r e a l Chem. 46:206, 1969. 35- Amino A c i d Content o f Foods and B i o l o g i c a l Data on P r o t e i n s by the Food P o l i c y and Science.--Service, N u t r . D i v . , FAO o f UN, Rome, 1970. 36. M i t c h e l l , H.H. and B l o c k , R.J., Some r e l a t i o n s h i p s between the amino a c i d c o n t e n t o f p r o t e i n s and t h e i r n u t r i t i v e v a l u e s f o r the r a t , J . B i o l . Chem. 163:599, 1946. 37- FAO/WHO, P r o t e i n r e q u i r e m e n t s . , FAO N u t r i t i o n meetings r e p o r t ; s e r i e s no. 37, FAO/OMS, 1965. 38. Oser, B.L., An i n t e g r a t e d e s s e n t i a l amino a c i d i n d e x f o r p r e d i c t i n g , the: b i o l o g i c a l v a l u e o f p r o t e i n s , i n P r o t e i n & Amino A c i d N u t r i t i o n ed. by A l b a n e s e , A.A. Acad. P r e s s , N.Y. 1959. 39- B e t s c h a r t , A.A. & K i n s e l l a , J.E., I n f l u e n c e o f s t o r a g e on c o m p o s i t i o n , amino a c i d c o n t e n t and s o l u b i l i t y o f l e a f p r o t e i n c o n c e n t r a t e , J . Agr. Food. Chem. 22:116, 1974. 40. Lawhon, J . T. and C a t e r , CM., E f f e c t o f p r o c e s s i n g method and pH o f p r e c i p i t a t i o n on the y i e l d s and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n i s o l a t e s from g l a n d l e s s c o t t o n s e e d , J . Food Sc. 36:372, 1971. 41. Tybor, P.T., D i l l , C.W. & Landmann, W.A., F u n c t i o n a l p r o p e r t i e s o f p r o t e i n s i s o l a t e d from b o v i n e b l o o d by a co n t i n u o u s p i l o t p l a n t p r o c e s s , J . Food Sc. 40:155, 1975. 42. Wolf, W., Soybean p r o t e i n : t h e i r f u n c t i o n a l , c h e m i c a l and p h y s i c a l p r o p e r t i e s . J . Agr. Food Chem. 18:969, 1970,, 43. P a u l i n g , L., The a d s o r p t i o n o f water by p r o t e i n s , J . Am. Chem. Soc. 67=557, 1945. 44. Huffman, V.L., Lee, C.K., Bu r n s , E.E., S e l e c t e d f u n c t i o n a l p r o p e r t i e s o f s u n f l o w e r meal, J . Food Sc. 40:70, 1975. 45. K u n t z , I.D. and Kanzmann, W., H y d r a t i o n o f p r o t e i n s and p o l y p e p t i d e s , Adv. P r o t . Chem. 29:239, 1974. 46. B r i s k e y , E . J . , F u n c t i o n a l e v a l u a t i o n o f p r o t e i n i n fo o d systems, i n E v a l u a t i o n o f N o v e l P r o t e i n P r o d u c t s , p 303, ed. by Bender, H.E., K i h l b e r g , R., L o f q v i s t , B. and Munck, L.,.Pergamon P r e s s , London 1970. 47. Zar, J.H., B i o s t a t i s t i c a l A n a l y s i s , P r e n t i c e - H a l l , 1974. 48. B i s h o p , L.R. , J . I n s t . Brew. 74:178, 1929-

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094494/manifest

Comment

Related Items