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Detoxification of rapeseed protein isolates by an activated carbon treatment Woyewoda, Andrew Dennis 1974

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DETOXIFICATION OF RAPESEED PROTEIN ISOLATES BY AN ACTIVATED CARBON TREATMENT BY ANDREW DENNIS WOYEWODA B.Sc. Hons., Chemistry U n i v e r s i t y o f A l b e r t a , Edmonton A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF. MASTER OF SCIENCE i n the Department of Food Science We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1974. In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f F^ 6-o-t^  Sc t Q.^.CJL^> The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada ABSTRACT Rapeseed p r o t e i n i s o l a t e from pH 10 NaOH e x t r a c t i o n was analyzed by gas chromatography ( i s o t h i o c y a n a t e s ) and UV a b s o r p t i o n ( g o i t r i n ) (Youngs and Wetter, 1967) and found to c o n t a i n g l u c o s i n o l a t e s a t l e v e l s e q u i v a l e n t to 0.75 mg 3- b u t e n y l i s o t h i o c y a n a t e , 0.57 mg 4-pentenyl isothiocyanate,, and 0.51 mg o x a z o l i d i n e t h i o n e ( g o i t r i n ) per g i s o l a t e . A two-stage process was developed to decrease the l e v e l s o f these t o x i n s . I s o l a t e s l u r r y was incubated a t pH 7.2 wit h crude myrosinase e x t r a c t e d from white mustard seed (to c o nvert g l u c o s i n o l a t e s to i s o t h i o c y a n a t e s and g o i t r i n ) , a d j u s t e d to pH 10, and passed through a g r a n u l a r a c t i v a t e d carbon column. Subsequent a n a l y s i s r e v e a l e d o n l y 0.006 mg 4- p e n t e n y l i s o t h i o c y a n a t e per g i s o l a t e . G o i t r i n was not d e t e c t a b l e . I n f r a r e d a n a l y s i s confirmed t h a t the column was a l s o p a r t i a l l y e f f e c t i v e i n n i t r i l e removal. To e l i m i n a t e the need f o r myrosinase p u r i f i c a t i o n , the process was m o d i f i e d t o i n c l u d e ground white mustard seed a d d i t i o n d i r e c t l y to rapeseed meal s l u r r y . A f t e r i n c u b a t i o n , the p r o t e i n was e x t r a c t e d , p u r i f i e d by i s o e l e c t r i c p r e c i p i -t a t i o n , r e - d i s s o l v e d , and t r e a t e d by the a c t i v a t e d carbon column. T h i s m o d i f i c a t i o n was i n c l u d e d i n the "recommended d e t o x i f i c a t i o n procedure". . Subsequent experiments on p r o t e i n e x t r a c t s prepared and carbon t r e a t e d a t pH's from 3 to 12, i n c l u s i v e , r e v e a l e d t h a t a l l treatments i n the range of pH 3 to 10 were at l e a s t 93% e f f e c t i v e i n i s o t h i o c y a n a t e removal. A lower e f f i c i e n c y was observed above pH 10. Storage t e s t s (24 hours) on aglycone c o n t a i n i n g p r o t e i n s o l u t i o n s showed i n c r e a s e d l o s s of i s o t h i o c y a n a t e s w i t h i n c r e a s i n g pH from 5 to 10. T h i s c o u l d be due to t h e i r i n t e r a c t i o n w i t h p r o t e i n (Bjorkman, 1973). The column completely removed c h r o m a t o g r a p h i c a l l y p u r i f i e d g l u c o s i n o l a t e s from aqueous s o l u t i o n . However, the r e s u l t s c o u l d not be d u p l i c a t e d f o r s o l u t i o n s c o n t a i n i n g rapeseed p r o t e i n . G l u c o s i n o l a t e content was determined by t r i m e t h y l s i l a t i o n and gas chromatography (modified method of U n d e r h i l l and K i r k l a n d , 1971). ACKNOWLEDGEMENTS I vdti>h to thank and e.xpn.cA4> my de,e.pe.i>t Qfiati.tu.dz to Vn.. S. Nakat whoAe. pattznt gutdance. and 6upe.n.vtiton made. thti> won.k pobhtbte.. I Mould alto Itke. to thank the. {ollowtng on.gant-zattonb {on. thctn. contn.t buttons to thtt, pn.oje.ct: Canbn.a ¥oodi> Ltd., le.thbn.tdge,, KLbe.n.ta {on n.ape.bze.d meal. Canadtan Gn.atn Commt&Aton, \Janco av cn., {on. hamplci> o fa n.apchccd and muhtand &ccd. Whttco Che.mtc.aZ Company, Wen; Soitk, {on. i>ampla> o{ gn.an.alan. acttvate.dcan.bon. i v . TABLE OF CONTENTS PAGE ABSTRACT i ACKNOWLEDGEMENTS i i i TABLE OF CONTENTS i v LIST OF FIGURES v i i LIST OF TABLES v i i i CHAPTER I 1 LITERATURE REVIEW 1 A. INTRODUCTION 1 1. N a t u r a l Occurrence of G l u c o s i n o l a t e s 1 2. G l u c o s i n o l a t e s i n Rapeseed 2 B. CHEMISTRY OF GLUCOSINOLATES 4 1. C h a r a c t e r i z a t i o n and I s o l a t i o n of G l u c o s i n o l a t e s and t h e i r Aglycones 4 2. Enzymatic H y d r o l y s i s of G l u c o s i n o l a t e s 4 3. P r e p a r a t i v e I s o l a t i o n of G l u c o s i n o l a t e s 7 4 Q u a n t i t a t i v e A n a l y s i s of G l u c o s i n o l a t e s and Aglycones . 8 5. I n t e r a c t i o n o f Aglycones w i t h Rapeseed. P r o t e i n s 10 C. THE MYROSINASE ENZYME SYSTEM 13 D. TOXICITY OF GLUCOSINOLATES AND AGLYCONES 14 E. PRODUCTION OF GLUCOSINOLATE-FREE RAPESEED MEALS, CONCENTRATES, AND ISOLATES 16 1. Removal of v o l a t i l e substances by h e a t i n g without p r i o r breakdown of g l u c o s i n o l a t e s 17 2. D i r e c t chemical d e g r a d a t i o n and m i c r o b i a l d e s t r u c t i o n of g l u c o s i n o l a t e s 17 3. H y d r o l y s i s o f g l u c o s i n o l a t e s and removal of products by d i s t i l l a t i o n 19 4. D e a c t i v a t i o n o f the myrosinase enzyme system 19 5. E x t r a c t i o n of g l u c o s i n o l a t e s and h y d r o l y s i s products 20 F. OBJECTIVE OF THIS STUDY 25 V. TABLE OF CONTENTS CONTINUED PAGE CHAPTER I I 27 DETOXIFICATION OF pH 10 SOLUBLE RAPESEED PROTEIN 27 A. INTRODUCTION 27 B. METHODS 29 1. P r e p a r a t i o n o f Base S o l u b l e P r o t e i n F r a c t i o n s 29 a. P r o t e i n e x t r a c t 29 b. P r o t e i n i s o l a t e 29 2. Myrosinase P r e p a r a t i o n 3,1 , 3. P r o d u c t i o n of Aglycones 31 4. Carbon Column Treatment 33 5. Analyses 34 a. I s o t h i o c y a n a t e s 34 b. G l u c o s i n o l a t e s 35 c. G o i t r i n 40 d. N i t r i l e s 40 6. Carbon Column C a p a c i t y 4 2 C. RESULTS AND DISCUSSION .43 1. E f f i c i e n c y of the Process 43 a. I s o t h i o c y a n a t e and g o i t r i n content 43 b. G l u c o s i n o l a t e content 50 c. N i t r i l e content 58 2. A d s o r p t i o n C a p a c i t y o f the Carbon 61 D. CONCLUSIONS AND GENERAL DISCUSSION 61 CHAPTER I I I 65 MODIFICATION OF THE RAPESEED PROTEIN DETOXIFICATION PROCEDURE 6 5 A. INTRODUCTION 65 B. METHODS ' 65 1. Mustard as Source of Myrosinase 65 2. E f f e c t of pH on Carbon A d s o r p t i o n o f I s o t h i o -cyanates and on I s o t h i o c y a n a t e S t a b i l i t y 66 v i . TABLE OF CONTENTS CONTINUED PAGE B. METHODS CONTINUED 3. Carbon A d s o r p t i o n o f G l u c o s i n o l a t e s 68 a. I s o l a t i o n of g l u c o s i n o l a t e s 68 b. P r e p a r a t i o n o f p r o t e i n i s o l a t e 68 c. A p p l i c a t i o n to carbon column 68 C. RESULTS AND DISCUSSION , 69 1. Mustard 69 2. E f f e c t o f pH 70 a. Isothiocyanate" a d s o r p t i o n 7 0 b. I s o t h i o c y a n a t e s t a b i l i t y 7 2 3. Carbon A d s o r p t i o n o f G l u c o s i n o l a t e s 75 D. RECOMMENDED DETOXIFICATION PROCEDURE 77 E. CONCLUSION 8 0 LITERATURE CITED 8 2 APPENDIX 1 94 v i i . LIST OF FIGURES FIGURES PAGE 1-1 G l u c o s i n o l a t e S t r u c t u r e 1 1-2 Enzymatic Degradation of G l u c o s i n o l a t e s 5 1-3 Formation of G o i t r i n 5 1-4 1-cyano-2-hydroxy-3-butene 6 1- 5 Reactions of Phenyl I s o t h i o c y a n a t e 12 2- 1 P r e p a r a t i o n of Rapeseed P r o t e i n F r a c t i o n s ; 30 2-2 Treatment o f Rapeseed P r o t e i n F r a c t i o n s 32 2-3 U l t r a v i o l e t Spectrum of G o i t r i n 41 2-4 Gas Chromatogram of I s o t h i o c y a n a t e s from Rapeseed Meal 4 4 2-5 Gas Chromatogram of G l u c o s i n o l a t e s from Rapeseed Meal - 51 2-6 Gas Chromatogram of F r a c t i o n "0^+ Q 2" 54 2-7 Gas Chromatogram o f F r a c t i o n "Q-j" 55 2-8 I n f r a - r e d Spectrum of P r o t e i n F r a c t i o n s 59 2- 9 U l t r a - v i o l e t Spectrum of A l l y l I s o t h i o c y a n a t e 60 3- 1 E f f e c t o f pH on Carbon A d s o r p t i o n of I s o t h i o c y a n a t e s 71 3-2 Decrease o f Free I s o t h i o c y a n a t e Content A f t e r 24 hours Storage at 5°C r 73 LIST OF TABLES G l u c o s i n o l a t e s i n Rapeseed Gas Chromatographic O p e r a t i n g C o n d i t i o n s Gas Chromatographic r . f . Values of Is o t h i o c y a n a t e s w i t h r e s p e c t to n- b u t y l i s o t h i o c y a n a t e Aglycone Content of P r o t e i n F r a c t i o n s (mg/g) . Comparison of Aglycone Content Among P r o t e i n F r a c t i o n s Expressed as Percent Change between "a" and "b" Gas Chromatographic r . f . Values of S i l a t e d G l u c o s i n o l a t e s w i t h r e s p e c t to S i l a t e d T r e h a l o s e Gas Chromatographic Molar Response F a c t o r s f o r S i l a t e d G l u c o s i n o l a t e s with r e s p e c t to S i l a t e d T r e halose G l u c o s i n o l a t e Content of P r o t e i n F r a c t i o n s (mg/g.) . E f f e c t of Carbon Treatment on S o l u t i o n s C o n t a i n i n g G l u c o s i n o l a t e s CHAPTER I LITERATURE REVIEW A. INTRODUCTION 1. Natural Occurrence of Glucosinolates The presence of water-soluble glucosides (gluco-sinolates; thioglucosides) capable of undergoing enzymatic hydrolysis to o i l - s o l u b l e isothiocyanates (mustard o i l s ; aglyeones), hydrogen su l f a t e , and D-glucose i n c e r t a i n families of plants has drawn the attention of s c i e n t i s t s since the early 1800's, although medicinal use of these compounds dates back to a much e a r l i e r period of time. Almost a l l members of the family Cruciferae i n -vestigated so far have contained at lea s t one thiogluco-side (Rjaer, 1960; Daxenbichler et a l . , 1964). Other families producing these compounds are Capparidaceae, Moringaceae, and Resedaceae — a l l belonging to the order Rhoeadales. Occasionally thioglucosides have been found in plants outside t h i s order. The general chemical structure of a thioglucoside or glucosinolate established by E t t l i n g e r and Lundeen i n 1956 i s i l l u s t r a t e d i n Figure I - l below: S C 6 H H °5 R C N - 0 - SO • + ' " ' 2 X i s a cation, usually potassium - O X + FIGURE I - l Glucosinolate Structure 2 2. Glucosinolates i n Rapeseed Rapeseed (family Cruciferae) contains the predomi nant glucosinolates p r o g o i t r i n , gluconapin, and glucobrassi canapin with low concentrations of glucoiberin, s i n a l b i n , and gluconasturtiin (Kjaer and Boe Jensen, 1956; Rutkowski 1971). Levels at which these compounds occur i n i n d i v i d u a l plants (0.5% to 6.4% fjnoisture and f a t free basis) reported by Lo and H i l l , 1971)vary with environment and species ; i y (Wetter, 1955; Wetter and Craig, 1959; Youngs and Wetter, 1967; Josefsson and Appelqvist, 1968). Canadian grown rapeseed (Brassica napus and Brassica campestris) contains approximately 40% o i l (Downey, 1965), 24% protein (Anderson and Sabry, 1970), and 1.0% moisture (Eapen et a l . , 1968). The remainder i s made up of carbohydrates and c e l l u l o s e (Wetter, 1965) . Most glucosinolates, or rather t h e i r hydrolytic products are goitrogenic i n nature (Astwood et al.., 1949; B e l z i l e et a l . , 1963;. Rutkowski, 1971; Josefsson and Munck, 1972) and therefore l i m i t the use of rapeseed meal currently being produced as a by-product of the o i l extrac-tion process (Bell and B e l z i l e , 1965). Research aimed at the development of glucosinolate-free strains of rapeseed i s progressing (Downey et a l . , 1967). Structures of "R-components" and respective hydro l y t i c products of rapeseed glucosinolates are shown i n Table I - l . TABLE 1-1 Glucosinolates in Rapeseed Glucosinolate Aglycone Product Name Chemical Structure (R) * Name 3-butenyl CH2 = CHCH2CH2 - Gluconapin Isothiocyanate (Volatile) 4-pentenyl CH 2 = CHCH2CH2CH2 - Glucobrassicanapin Isothiocyanate (Volatile) 2-hydroxy-3-butenyl CH 2 = CHCHCH2 -OH Progoitrin. 5-vinyl-2-Oxazolidinethione (goitrin) (non-Volatile) or 1-cyano- 2-hydroxy- 3-butene (non-Volatile) 3-methylsulphinylpropyi CH3SCH2CH2CH2 -0 Glucoiberin Isothiocyanate (non-Volatile) 2-phenylethyl p-hydroxybenzyl CCH_-CH0CH-D D 2 2 H0C6H4CH2 -Gluconasturtiin Sinalbin Isothiocyanate (Volatile) Isothiocyanate (Volatile) * See F i g u r e 1-1 B. CHEMISTRY OF GLUCOSINOLATES 1. C h a r a c t e r i z a t i o n and I s o l a t i o n o f G l u c o s i n o l a t e s  and t h e i r AgXycones E a r l y attempts at s e p a r a t i o n o f the steam d i s t i l l -a b l e i s o t h i o c y a n a t e s produced on enzymatic de g r a d a t i o n of g l u c o s i n o l a t e s i n c l u d e d f r a c t i o n a l d i s t i l l a t i o n (Schmalfuss, 1938; Schwarze, 1949; E t t l i n g e r and Hodgkins, 1955; Kjaer and Boe Jensen, 1956) and d i s t r i b u t i o n between p a r t i a l l y , m i s c i b l e s o l v e n t s (Schmid and K a r r e r , 1948a,b). A combination of i s o t h i o c y a n a t e (Kjaer e t a l . , 1953), t h i o u r e a (ammonia t r e a t e d i s o t h i o c y a n a t e ) (Kjaer and R u b i n s t e i n , 1953), and g l u c o s i n o l a t e (Gmelin, 1954; S c h u l t z and Gmelin, 1952 ; S c h u l t z and Wagner, 1956; Wagner, 1956) paper chromatographic techniques r e s u l t e d i n the s e p a r a t i o n and c h a r a c t e r i z a t i o n of s e v e r a l o f the parent g l u c o s i d e s and t h e i r aglycones. Over f i f t y g l u c o s i n o l a t e s have now been i s o l a t e d i n some form and s e v e r a l more i d e n t i f i e d (VanEtten, e t a l . , 1969). The f i r s t r e p o r t e d chemical s y n t h e s i s o f a n a t u r a l l y o c c u r r i n g g l u c o s i n o l a t e was t h a t of E t t l i n g e r and Lundeen (1957). T h e i r product, g l u c o t r o p a e o l i n , i s the major g l u c o s i n o l a t e o f Tropaeolum majus L. 2. Enzymatic H y d r o l y s i s o f G l u c o s i n o l a t e s The g e n e r a l h y d r o l y s i s r e a c t i o n of g l u c o s i n o l a t e s by the enzyme myrosinase ( t h i o g l u c o s i d e ( g l u c o s i n o l a t e ) g l u c o h y d r o l a s e ; EC 3.2.3.1) i n v o l v e s f i r s t an enzymatic cleavage of the glucose and sulfate moieties followed by a Lossen rearrangement (Ettlinger and Lundeen, 1957) to produce the isothio-cyanate as illustrated i n Figure 1-2 below: R C - S N 0 -glucose S 0 3 K myrosinase enzyme R - S - C e-N Thiocyanate N C - S - H " f t -S^N - 0 S 0 3 K + glucose Lossen rearrangement R - N = C = S + KHSO^ Isothiocyanate FIGURE 1-2 Enzymatic Degradation of Glucosinolates * A l t e r n a t e pathway i n s p e c i a l cases If a B-hydroxyl group i s present i n "R" of the enzyma-t i c a l l y produced isothiocyanate, a cyclization inrnediately occurs, producing the oxazolidinethione. This reaction i s illustrated i n Figure 1-3 for 2-hydroxy-3-butenyl glucosinolate (progoitrin) isolated from rutabaga seeds by Greer i n 1956: — N H 2C : NH H 2C a-L = a-i - c 2 A H OH c = s CH - C C = S H 0 2- hydroxy-3-butenyl Isothiocyanate 5-vinyl-2-oxazolidinethione (goitrin) FIGURE 1-3 Formation of Goitrin The a g l y c o n e , g o i t r i n , was i s o l a t e d from y e l l o w t u r n i p by Astwood e t a l . , i n 1949 and the s t r u c t u r e p r o v e d by E t t l i n g e r i n 1950 by c h e m i c a l s y n t h e s i s . Other h y d r o l y s i s mechanisms have been i n v e s t i g a t e d ( K j a e r , 1960) . Some p l a n t s c o n t a i n enzymes c a p a b l e o f p r o -d u c i n g t h i o c y a n a t e s i n s t e a d o f i s o t h i o c y a n a t e s and g o i t r i n s (Gmelin and V i r t a n e n , 1 9 5 9 a , b ) . D a x e n b i c h l e r e t a l . (1966) and V a n f i t t e n , . e t a l . (1966,;, 1969) s t u d i e d f o r m a t i o n o f the . n i t r i l e l - c y a n o - 2 - h y d r o x y - 3 - b u t e n e ( F i g u r e 1-4) as an a l t e r n a t e p r o d u c t to g o i t r i n i n the e n z y m a t i c d e g r a d a t i o n o f p r o g o i t r i n and c o n c l u d e d t h a t the r e s p e c t i v e amounts o f each produced depended on t e m p e r a t u r e , p H , and source o f m y r o s i n a s e . CH 2 = CH - CH - CH 2 - C = N • I OH FIGURE 1-4 1- cr/ano-2-hydroxy-3-butene I n v e s t i g a t i o n s on crambe seed meal (Crambe a b y s s i n i c a ) by V a n E t t e n , e t a l . (1966) showed t h a t p r e f e r e n -t i a l e n z y m a t i c f o r m a t i o n o f g o i t r i n o c c u r r e d w i t h i n c r e a s e d t e m p e r a t u r e and p H , d i l u t i o n o f the meal w i t h w a t e r , d r y h e a t i n g the m e a l , and s t o r a g e o f the seed under ambient c o n d i t i o n s . D a x e n b i c h l e r e t a l . (1966) r e p o r t e d t h a t a t d e c r e a s i n g p H ' s from pH 7 to 3, n i t r i l e f o r m a t i o n was increased during hydrolysis of p r o g o i t r i n by myrosihase derived from white mustard seed (Sinapis alba). 3. Preparative I s o l a t i o n of Glucosinolates Attempts at separation of glucosinolates on a preparative scale have generally met with l i m i t e d success. I n i t i a l separations were performed by paper (Schultz.and Gmelin, 1952; Schultz and Wagner, 1956; Kjaer and Boe Jensen, 1956) and t h i n layer (Wagner et a l . , 1965; Matsuo,• 19 70) chromatographic.procedures. Chromatographic column techniques have resulted i n only p a r t i a l separation and then only from plants extremely abundant i n one p a r t i c u l a r glucosinolate. Greer i s o l a t e d p r o g o i t r i n from rutabaga seed (alumina; 195 6) and Brassica seed (anion exchange on Amberlite IR-4B i n the Cl form; 1962). Bachelard and Trikojus (1963) used.cellu-lose and alumina columns to i d e n t i f y glucosides present i n pasture weeds. Bjorkman (1972) employed DEAE Sephadex A-25. anion exchange chromatography for preliminary separation of rapeseed glucosinolates and a f f i n i t y chromatography (arginine coupled Sephadex G-10) for further p u r i f i c a t i o n . In 1970, Aleksiejczyk et a l . were moderately successful i n i s o l a t i n g these compounds by high voltage paper electrophoresis. However, complete separation of gluco-sinolates from rapeseed has never been s a t i s f a c t o r i l y achieved on a preparative scale. 4. Quantitative Analysis of Glucosinolates and  Aglycones Quantitation of glucosinolates has usually been dependent on analysis of released products of enzymatic hydrolysis (Youngs and Wetter, 1967). Total thioglucoside content has been determined by sul f u r t i t r a t i o n (VanEtten, et a l . , 1965) or glucose analysis (Schultz and Gmelin, 1954) aft e r hydrolysis. Glucose determination by the anthrone procedure following s u l f u r i c acid cleavage of thioglucosides (Gmelin, 1954; Schultz and Gmelin, 1954) has met with limited success due to the presence of other carbohydrates i n plant material (Kjaer, 1960). V o l a t i l e isothiocyantes have been determined by arge.ntimetric methods (Wetter, 1955) . After production of thioureas by steam d i s t i l l a t i o n into aqueous ammonia, s i l v e r n i t r a t e was added to f a c i l i t a t e decomposition of the product to a mono-substituted car bod iimide and insoluble s i l v e r s u l f i d e . The unreacted s i l v e r was then determined by the Volhard method using potassium thiocyanate. Thioureas (Kjaer et a l . , 1953; Moll, 1963; Appelqvist and Josefsson, 1965, 1967) and g o i t r i n (Astwood et a l . , 1949; Wetter, 1957) have been quantitated by t h e i r absorbance i n the u l t r a - v i o l e t region. Szewczuk et a l . (1969a) used the g o i t r i n c a t a l y s i s of iodine reduction by azide ion as a base for a chemical method of g o i t r i n determination where excess iodine was t i t r a t e d with sodium arsenite. Recently, gas chromatography of isothiocyanates and other v o l a t i l e hydrolysis products has been accomplished (Jart, 1961; Kirk et a l . , 1964; Youngs and Wetter, 1967). Daxenbichler et a l . (1970) applied t h i s method to the determination of g o i t r i n s and n i t r i l e s . . . . In 1971, Underhill and Kirkland devised a procedure for glucosinolate analysis without myrosinase hydrolysis by employing t r i m e t h y l s i l a t i o n and gas chroma-tography of the corresponding der i v a t i v e s . However, t h i s method was li m i t e d by the u n a v a i l a b i l i t y of pure standards. 5. Interaction of Aglycones with Rapeseed . Proteins In 1973 Bjorkman reported the occurrence of an isothiocyanate-protein reaction ; at •. pH values higher than 35 6. By incubation of S l a b e l l e d gluconapin and gluco-a l y s s i n with rapeseed meal at 40°C for 1 hour and subse-quent fra c t i o n a t i o n by Sephadex G-50 and G-200 chromatography i t was determined that 36% of the amount of added i s o t h i o -cyanate was incorporated by the protein at pH 9. Incorpora-t i o n was pH dependent, increased l i n e a r l y with amount of isothiocyanate added, and did not increase with a longer reaction time. From the Sephadex elut i o n patterns, i t was 10. found t h a t the r a d i o a c t i v e i s o t h i o c y a n a t e s r e a c t e d mainly w i t h low molecular weight b a s i c p r o t e i n s ( i s o e l e c t r i c p o i n t s approximately 11) r i c h i n l y s i n e . Only a very s m a l l amount of g o i t r i n was a s s o c i a t e d w i t h the p r o t e i n and t h i s i n t e r a c t i o n was independent of pH. A s s o c i a t i o n o f i n t a c t g l u c o s i n o l a t e s with rapeseed p r o t e i n s was not d e t e c t e d . An i s o t h i o c y a n a t e i s used i n p r o t e i n sequencing procedures (Edman, 1970). In t h i s method, phenyl i s o t h i o -cyanate couples w i t h the f r e e amino group of the N t e r m i n a l amino a c i d of a p e p t i d e a t b a s i c pH to produce the phenyl thiocarbamyl- amino a c i d . T h i s d e r i v a t i v e i s subsequently c l e a v e d from the r e s t of the p e p t i d e by an aqueous a c i d treatment a t e l e v a t e d temperature thereby producing the t h i a z o l i n o n e , which i n t u r n can be converted t o the more s t a b l e 3-phenyl-2-thiohydantoin-amino a c i d . T h i s r e a c t i o n and s i m i l a r r e a c t i o n s i n v o l v i n g i s o t h i o c y a n a t e s are i l l u s t r a t e d i n F i g u r e 1-5. The i s o t h i o c y a n a t e - p r o t e i n r e a c t i o n r e p o r t e d by Bjorkman (1973) must be the form a t i o n o f the th i o c a r b a m y l -amirio a c i d and the e f f e c t o f ammonia on the r e a c t i o n can be e x p l a i n e d by the form a t i o n of the s u b s t i t u t e d t h i o u r e a (see F i g u r e 1-5). 11 1. With ammonia ( N o l l e r , 1965) H S i I I C,H C - N = C =. S + NH -> C rH N - C - NH„ 6 5 3 6 5 2 phenyl t h i o u r e a 2. With 1° and 2° amines . ( N o l l e r , page 349, 1965) H S H C,H._ - N = C = S + H_NR -»• C^HCN - C - N - R bo Z bo s u b s t i t u t e d t h i o u r e a 3. With p e p t i d e s (Edman, 1965) H H O H H S H H O H C,H -N=C=S + N-C-C-N.^ '-»• C rH cN-C-N-C-C-N^ v 6 5 , , ^X 6 5 X H R phenylthiocarbamyl d e r i v a t i v e H + ? -> C rH N - C = NH I I 1 1 H + NH - X S-CVC „ \ pe p t i d e R 0 2 - a n i l i n o - 5 - t h i a z o l i n o n e derivative (ammonium form) + H 20 H S H H C CH C - N - C - N - C - COOH + H~ bo , R phenylthiocarbamyl a c i d 12. p h e n y l t h i o h y d a n t o i n amino a c i d FIGURE 1-5 Reactions o f Phenyl I s o t h i o c y a n a t e 13. Konigsberg (Edman, 1970) reported a possible reaction of isothiocyanate with sulfhydryl groups of proteins, p a r t i c u l a r l y cysteine. However, t h i s was not observed by Edman (1970). C. THE MYROSINASE ENZYME SYSTEM Myrosinase (thioglucoside (glucosinolate) glucohydrolase EC 3.2.3.1) responsible for the hydrolysis of glucosinolates to aglycones has been found i n plants (Kjaer, 1960; VanEtten,. et a l . , 1969; Vaughan et a l . , 1968; MacGibbon and A l l i s o n (1970), bacteria (Oginsky et a l . , 1965) and mammals (Goodman et a l . , 1959). Bjorkman and Janson (1972) characterized the myrosinase system of white mustard seed and demonstrated the existence of a number of isoenzymes. Lonnerdal and Janson (19.73) i s o l a t e d the main component of thi s enzyme from Brassica napus L. and reported that the myrosinase content of rapeseed was only one f i f t e e n t h that of white mustard seed (Sinapis alba). Myrosinase i s active over the pH range of 3 to 11 (VanEtten, et a l . , 1966), although maximum release of i s o -thiocyanates and oxazolidinethiohes occurs at pH 6-9 and pH 7-9, respectively (Appelqvist and Josefsson, 1967). This observed decrease i n g o i t r i n production below pH 7 could be due to n i t r i l e formation (Daxenbichler et a l . , 1966). 14 . Maximum h y d r o l y s i s r a t e was observed a t 70°C. A d d i t i o n of a s c o r b i c a c i d to the system i n c r e a s e d myrosinase a c t i v i t y by a f a c t o r of 2.9 and 3.8 f o r i s o t h i o c y a n a t e and g o i t r i n r e l e a s e , r e s p e c t i v e l y ( A p p e l q v i s t and J o s e f s s o n , 1967). Ascorbate a c t i v a t i o n of myrosinase was o r i g i n a l l y r e p o r t e d by Nagashima and Uchiyama (1959a) who claimed t h a t the cleavage r a t e of s i n i g r i n was i n c r e a s e d by 260 p e r c e n t on a d d i t i o n of 0.001 M L - a s c o r b a t e . A c c o r d i n g to Henderson and McEwen (1972), maximum a c c e l e r a t i o n was achieved between pH 5.5 and 6.5 ( c i t r i c acid-sodium phosphate b u f f e r ) . The enzyme was a l s o found to be i n h i b i t e d by s u l p h y d r y l b l o c k i n g agents (Nagashima and Uchiyama, 1959b). Mode of enzyme p r e p a r a t i o n a f f e c t s ascorbate a c t i v a t i o n ( E t t l i n g e r et a l . , 1961). A d d i t i o n of 0.002 M L-ascorbate to a standard p r e p a r a t i o n of myrosinase and to whole mustard seed enhanced the r a t e of h y d r o l y s i s of s i n i g r i n by a f a c t o r of f o u r (Nagashima and Uchiyama, 195 9a; Schwimmer, 1960) and e i g h t y r e s p e c t i v e l y , w h i l e a m o d i f i e d method of enzyme p r e p a r a t i o n i n c r e a s e d the r a t e by 4 00 ( E t t l i n g e r e t a l . , 1961). D. TOXICITY OF GLUCOSINOLATES AND AGLYCONES When g l u c o s i d e c o n t a i n i n g seeds are crushed, the myrosinase system i s a c t i v a t e d , decomposing b i o l o g i c a l l y i n a c t i v e t h i o g l u c o s i d e s to g o i t r o g e n i c v i n y l o x a z o l i d i n e -t h i o n e s , n i t r i l e s , i s o t h i o c y a n a t e s , and t h i o c y a n a t e s (Rutkowski, 1971). V i n y l o x a z o l i d i n e t h i o n e , the most a c t i v e compound of the group, a c t s by b l o c k i n g the i r r e v e r s i b l e mechanism i n v o l v e d w i t h the o r g a n i c b i n d i n g of i o d i n e i n the t h y r o i d , thereby sup p r e s s i n g thyroxine s y n t h e s i s (Rutkowski, 1971). The r e s u l t i n g imbalance s t i m u l a t e s s e c r e t i o n o f e x c e s s i v e amounts of thyrotropine by the hypophysis which i n t u r n causes i n c r e a s e d t h y r o i d growth ( C l a n d i n i n and Bayly, 1960). T h i s e f f e c t cannot be a l l e v i a t e d by i o d i n e supplementation (Rutkowski, 1971), although a f t e r prolonged f e e d i n g of v i n y l o x a z o l i d i n e t h i o n e c o n t a i n i n g meal to c h i c k e n s , p h y s i o l o g i c a l e q u i l i b r i u m was a p p a r e n t l y achieved w i t h i n c r e a s e d t h y r o i d s i z e ( C l a n d i n i n , 1965). N i t r i l e s have been shown to be about 8 times as t o x i c as g o i t r i n , a c t i n g p r i m a r i l y on the l i v e r and kidneys (VanEtten, e t a l . , 1969). The mechanism of t h e i r a c t i o n i s hot c l e a r s i n c e r e p r o d u c i -b i l i t y of r e s u l t s has not always been p o s s i b l e (Rutkowski, 1971). I s o t h i o c y a n a t e a c t i v i t y which can be e l i m i n a t e d by d i e t a r y i o d i n e supplementation (Rutkowski, 1971) c o n s i s t s of b l o c k i n g i o d i n e uptake by the t h y r o i d gland and l i b e r a t i n g the i o d i n e a l r e a d y accumulated t h e r e (Fertman and C u r t i s , 1951; Gmelin and V i r t a n e n , 1960). Thiocyanates e x h i b i t only m i l d g o i t r o g e n i c p r o p e r t i e s (Rutkowski, 1971). Monogastric mammals are the most s u s c e p t i b l e to the d e t r i m e n t a l e f f e c t s of aglycones. Negative e f f e c t s observed i n swine have, been i n maturation, r e p r o d u c t i o n , and l a c t a t i o n (Bowland, 1965; B e l l , 1965; Rutkowski, 1971). P o u l t r y e x h i b i t a d e p r e s s i o n p e r i o d i n development i n which t h y r o i d s i z e i n c r e a s e s r a p i d l y . A f t e r t h i s i n i t i a l adjustment, near normal growth i s g e n e r a l l y observed ( C l a n d i n i n e t a l . , 1959; C l a n d i n i n and Bayly, 1960; C l a n d i n i n , 1965). While ruminants seemed almost u n a f f e c t e d by go i t r o g e n s (Apslund and McElroy, 1961; Whiting, 1965), v i n y l o x a z o l i d i n e t h i o n e was thought to be t r a n s m i t t e d t o the milk from these animals (Clements and Wishart, 1956, ; V i r t a n e n e t a l . , 1958; C l a n d i n i n and Bayly, 1960). However, i n g e s t i o n of m i l k from B r a s s i c a f ed cows r e v e a l e d no d i s -turbance i n the accumulation of r a d i o a c t i v e i o d i n e uptake i n the human t h y r o i d gland (V i r t a n e n e t a l . , 1959; V i r t a n e n , 1961). In s i m i l a r experiments Hoppe e t a l . , (1971) r e p o r t e d the presence of t h i o c y a n i d e s which a f f e c t e d cheese r i p e n i n g . Such compounds were c o n s i d e r e d u n d e s i r a b l e c o n s t i t u e n t s i n mi l k (Rutkowski, 1971). E. PRODUCTION OF GLUCOSINOLATE-FREE RAPESEED MEALS, CONCENTRATES, AND ISOLATES. Meal and f l o u r d e r i v e d from rapeseed i s an e x c e l l e n t source of p r o t e i n , approximately 4 0 and 55%, r e s p e c t i v e l y (Lo and H i l l , 1971; Tape e t a l . , 1970). However the presence of " g o i t r o g e n i c compounds" ( g l u c o s i n o l a t e s and aglycones) i n rapeseed l i m i t s i t s p o t e n t i a l a t the prese n t time ( B e l l , 1955, 1957; B e l l and B e l z i l e , 1965). D e t o x i f i c a t i o n procedures r e p o r t e d i n the l i t e r a -t u r e can be d i v i d e d i n t o f i v e major c a t e g o r i e s (Rutkowski, 17. 1970) and w i l l be presented i n t h a t manner here. 1. Removal of v o l a t i l e substances by h e a t i n g without "• p r i o r breakdown of g l u c o s i n o l a t e s . The most d i r e c t s o l u t i o n to the problem i s steaming o r t o a s t i n g . B e l l and B e l z i l e (1965) r e p o r t e d t h a t d u r i n g steaming, the amount of p r e s s u r e had a marked e f f e c t on the r a t e of d i s -appearance of i s o t h i o c y a n a t e and g o i t r i n producing compounds. In one case, a f t e r 2 hours of treatment, 90% and 75% o f these r e s p e c t i v e compounds had been e l i m i n a t e d although a corresponding decrease i n p r o t e i n q u a l i t y was a l s o observed. Jakubowski e t a l . , (1970) r e p o r t e d t h a t superheated steam t r e a t -ment f o r over 3 hours completely e l i m i n a t e d g o i t r o g e n i c compounds. In Poland, i n d u s t r y has adopted a t o a s t i n g process to reduce g l u c o s i n o l a t e content by 75%. However, a 50% decrease i n t o x i n content by t h i s method i s accompanied by a corresponding decrease of about o n e - t h i r d i n s o l u b l e p r o t e i n (Rutkowski, 1970) . 2. D i r e c t chemical d e g r a d a t i o n and m i c r o b i a l d e s t r u c t i o n o f g l u c o s i n o l a t e s . A d i r e c t ammoniation process r e s u l t i n g i n the t r a n s f o r m a t i o n of g l u c o s i n o l a t e s to t h i o u r e a 18. derivatives has been described by Chanet (1970) (see Figure 1-5). The process has not been widely accepted (Rutkowski, 1970) and is currently under investigation (Bell et a l . , 1970) . Bel l et a l . , (1970) patented a process for the degradation of glucosinolates to their corres-ponding steam volat i le n i t r i l e s by treatment with copper and iron sa l ts . However, the n i t r i l e of go i t r in , l-cyano-2-hydroxy-3-butene, was not volat i le and the process was therefore limited to meals of low progoitrin content. The reaction of iron salts with glucosinolates has been invest i -gated by Austin et a l . , 1968. Dilute sul fur ic acid degradation of gluco-sinolates was proposed by Szewczuk et al . (1969b, 1970) . However, the process resulted in considerable degradation of lys ine. In a microbial approach described by Starton (1970) i t was found that after a fermentation period of 85 hours at 37°C with the culture Geotrichum candidum, complete degradation of gluco-sinolates in rapeseed meal and elimination of their products had occurred along with a 9 0% so lub i l i za -tion of the available protein. Final pH of the process was pH 4.0. 19. Faruga et a l . (1973) found that silage made from steamed potatoes and rapeseed meal was free of goitrogenic properties. Hydrolysis of glucosinolates and removal of products by d i s t i l l a t i o n . Preliminary degradation of glucosinolates to aglycones followed by steam d i s t i l l a t i o n was pro-:, posed by Schwarze (1949), Andre (1955) , and Goering (1961). Unfortunately, g o i t r i n remained i n the meal (Bell and B e l z i l e , 1965). A si m i l a r process was outlined by,Mustakas et a l . (1962, 1963) for the d e t o x i f i c a t i o n of mustard seed (Brassica juncea) which i s known to contain only isothiocyanate producing glucosides. A similar process to be e f f e c t i v e for rapeseed would require too much heat and would therefore be economically unfeasible (Rutkowski, 1970). Deactivation of the myrosinase enzyme system. If myrosinase were inactivated before the decomposition of glucosinolates to aglycones, the meal would not be considered toxic since gluco-sinolates are not goitrogenic themselves. However, the presence of enzymes i n the g a s t r o i n t e s t i n a l t r a c t (Greer, 1962), capable of degrading gluco-sinolates to t h e i r aglycones and the a b i l i t y of 20. E. c o l i and A. aerogenes to produce these same enzymes ( B e l l , 1955), l i m i t s the usefulness of t h i s approach. Currently, myrosinase i n a c t i v a t i o n i s con-sidered b e n e f i c i a l during production of rapeseed o i l and meal to minimize s u l f u r contamination of the o i l from glucosinolate hydrolysis at the crushing stage ( B e l l , et a l . , 1963). Although dry heating was i n e f f i c i e n t i n myrosinase i n a c t i v a t i o n (Bell and B e l z i l e , 1965), complete success was obtained with a wet-heat process consisting of a 2 or 3 minute treatment at 100°C (Eapen et a l . , 1968). This method has been applied by other researchers as a preliminary step i n t h e i r d e t o x i f i c a t i o n procedures, (e.g., Sosulski, et a l . , 1972). Extraction of glucosinolates and hydrolysis products. Although simple extraction of rapeseed meal by aqueous and ethanolic solutions increased feeding value (Allen and Dow, 1952; B e l l , 1957), hot water extraction resulted i n a 20% loss of s o l i d s (Bell and B e l z i l e , 1965). Tookey et a l . (1965) produced a b i o l o g i c a l l y improved meal from Crambe abyssinica by allowing myrosinase hydrolysis-to proceed and subsequently e x t r a c t i n g w i t h 88 to 98% acetone (aqueous) . Eapen e t a l . (1968, 1969) p u b l i s h e d a f e a s i -b i l i t y s tudy f o r the d e t o x i f i c a t i o n and d e h u l l i n g o f rapeseeds i n v o l v i n g p r e l i m i n a r y i n a c t i v a t i o n o f m y r o s i n a s e , wet g r i n d i n g , and t h r e e 30 minute hot o r c o l d water e x t r a c t i o n s . The d e t o x i f i e d r e s i d u e was d r i e d , f l a k e d , d e f a t t e d , and a i r c l a s s i f i e d i n t o two p r o d u c t s w i t h 33 and 60% p r o t e i n r e s p e c -t i v e l y . When the p r o c e s s was a p p l i e d to m e a l , a 27 to 39% l o s s o f p r o t e i n o c c u r r e d to the aqueous phase compared to 14 to 26% w i t h ground s e e d . B a l l e s t e r e t a l . (1970) found t h a t d o u b l e water e x t r a c t i o n (14 hours + 1 hour) a t room temperature r e d u c e d o x a x o l i . d i n e t h i o n e and i s o t h i o c y a n a t e p r o d u c i n g compunds by 84 and 77%, r e s p e c t i v e l y , and i n c r e a s e d b i o l o g i c a l q u a l i t y o f the m e a l . The method o f Eapen e t a l . (1969) was m o d i f i e d by Tape e t a l . (1970) by the i n c o r p o r a t i o n o f two t h i r t y minute e x t r a c t i o n s a t ambient temperature and a f i l t e r wash ( v i b r a t o r y screen), t o produce r a p e s e e d f l o u r and meal c o n t a i n i n g 50% and 30% p r o t e i n r e s p e c t i v e l y , with o n l y t r a c e s o f g l u c o -s i n o l a t e s . E k l u n d e t a l . (1971) o b t a i n e d 3 l i p i d - r i c h rapeseed p r o t e i n c o n c e n t r a t e s ( a p p r o x i m a t e l y 48% p r o t e i n , 31% f a t ) by a 28% e t h a n o l , M N a C l extraction and centrifugation. Although 67% of the oxazolidinethiones were liberated during the i n i t i a l extraction, the f i n a l product contained only 5% of the originally- present goitrogens. The major disadvantages of the process were the high l o s s of material to the solvent (.30% and 5Q% of the o r i g i n a l f a t and protein) and the high, l i p i d content of the f i n a l product. In 1972, B a l l e s t e r et a l . shortened t h e i r o r i g i n a l 15 hour batch extraction procedure (Ballester et a l . , 197 0) to a 2 hour continuous process capable of removing 100% and 97% of the isothiocyanate and oxazolidinethione producing glucosides. However, feeding t r i a l s of the r e s u l t i n g meal indicated the presence of other p o t e n t i a l l y harmful factors i n rapeseed meal. High concentra-tions of tannins have been reported (Durkee, 1971; Yapar and Clandinin, 1972) . Owen and Chichester (1971) produced a protein i s o l a t e containing 84% protein and only 0 to 3% of the o r i g i n a l l y present oxazolidinethione producing glucosinolates from rapeseed meal by 6% NaCl extraction, removal of supernatant, four water washes, addition of calcium carbonate (to increase curd strength and accelerate p r e c i p i t a t i o n ) , and adjustment to pH 5. The supernatant was siphoned of f and preci p i t a t e d protein centrifuged out. When the method was duplicated by G i r a u l t (1973), an 18% y i e l d was obtained. A similar procedure (Lo and H i l l , 1970) employing 10% NaCl extraction of Brassica napus meal, f i l t r a t i o n , and a 3-day d i a l y s i s , yielded a product containing 61 to 64% protein (approximately 75% of the o r i g i n a l meal nitrogen) and 30% of the o r i g i n a l glucosinolate content. A unique approach to the problem was taken by Sosulski et a l . (1972) who used d i f f u s i o n extraction of whole rapeseeds at pH's from 1.1 to 12.3. Five or six 1 hour extractions with 0.01 N NaOH at 60°C proved most e f f i c i e n t . However, unless the seeds were boiled before treatment, a high s u l f u r content resulted i n the o i l . Bhatty and Sosulski (1972) modified the method by using 0.01 N NaOH i n 50% ethanol for 4 two hour extractions at 70°C. Only low lev e l s of thioglucosides were detected i n the pro-duct. The major disadvantage of the process was reduced protein s o l u b i l i t y of the meal because of p a r t i a l denaturation. Kozlowska et a l . (1972) duplicated f i v e previously reported glucosinolate extraction pro-24. cedures: aqueous extraction of meal (Ballester,et a l . 1970); enzyme i n a c t i v a t i o n and aqueous extraction of crushed seeds (Tape et a l . , 1970); d i f f u s i o n extraction (0.01 N NaOH) of in t a c t seeds and d i f f u s i o n extraction (0.01 NaOH) of in t a c t seeds with preliminary enzyme i n a c t i v a t i o n (Sosulski et a l . , 1972); and d i f f u s i o n extraction (50% v/v ethanolic NaOH adjusted to pH 12) of i n t a c t seeds, (Bhatty and Sosulski, 1972). The aqueous extraction (Ballester et a l . , 1970) was the most e f f e c t i v e i n glucosinolate removal. However, as i n the method of Tape et a l . (1970) almost one-third of the meal proteins were l o s t to the solvent. The ethanolic process (Bhatty and Sosulski, 1972), although preventing myrosinase action and most completely detoxifying rapeseed samples tested, required long extraction times and resulted i n denaturation of meal proteins. G i r a u l t (1973) outlined optimum conditions for extraction and p r e c i p i t a t i o n of proteins from rapeseed meals. Most e f f e c t i v e extraction was obtained with 10% NaCl or 0.1 N NaOH and maximum p r e c i p i t a t i o n at pH 3 for NaCl and pH 6.5 for NaOH extraction. Sodium hydroxide extracted 20% more of the t o t a l nitrogen than sodium chloride, and did not seem to have any detrimental e f f e c t on extracted proteins. The actual y i e l d obtained with the 0.1 N NaOH method was 30%. Decrease of t h i o -glucoside content was not investigated. F. OBJECTIVE OF THIS STUDY Kodagoda et a l . (1973a) produced acid, neutral, and base soluble protein i s o l a t e s (average 91% protein content) from rapeseed f l o u r prepared by Tape et a l . (1970). However, the process was not considered i n d u s t r i a l l y a t t r a c t i v e since traces of glucosinolates were s t i l l detectable i n the f i n a l product (personal communication) and dependence on rapeseed flou r as a raw material was economically unfavourable. The object of t h i s study was to develop a method of preparation of glucosinolate-free rapeseed protein (either concentrate or isolate) similar to that of Kodagoda et a l . (1973a) d i r e c t l y from commercial rapeseed meal. The process was to be adaptable to other rapeseed protein extraction pro-cedures such as those of G i r a u l t (1973) to y i e l d a completely detoxified protein product acceptable for human consumption. The proposed method of d e t o x i f i c a t i o n entailed preliminary glucosinolate hydrolysis and subsequent removal of aglycone products by activated carbon treatment of the protein solution. Processes involving activated carbon adsorption currently i n use by the food industry are for the elimination of contaminating pigments (from beer, wine, sugar, o i l s and f a t s , water) and odoriferous compounds(from brandy,wine, beer, water) characterized by a high degree of resonance, usually due to pi-unsaturation and the presence of e l e c t r o -negative groupings sometimes containing nitrogen and sul f u r atoms (Mantell, 1928, 1968; Smisek and Cerny, 1970). Since aglycones seemed to be i n t h i s category, prospects of success seemed favorable. CHAPTER I I DETOXIFICATION OF pH 10 SOLUBLE RAPESEED PROTEIN A. INTRODUCTION P r e l i m i n a r y experiments confirmed t h a t the mustard odor bf e n z y m a t i c a l l y t r e a t e d rapeseed meal decreased a f t e r carbon treatment i n d i c a t i n g t h a t t h e r e was indeed p a r t i a l or t o t a l e l i m i n a t i o n o f aglycones by carbon a d s o r p t i o n . T h e r e f o r e , an experiment was designed to determine three c r i t i c a l f a c t o r s : f i r s t , the e f f i c i e n c y of myrosinase con-v e r s i o n of g l u c o s i n o l a t e s to i s o t h i o c y a n a t e s and 5 - v i n y l -2 - o x a z o l i d i n e t h i o n e ( g o i t r i n ) i n rapeseed meal; second, the a c t u a l decrease i n c o n c e n t r a t i o n o f l i b e r a t e d aglycones by carbon treatment; and t h i r d , the a d s o r p t i o n c a p a c i t y of the carbon a v a i l a b l e . Success of the proposed process would r e s u l t i n the p r o d u c t i o n of a g l y c o n e - f r e e rapeseed p r o t e i n s u i t a b l e f o r human consumption. I n d u s t r i a l adoption o f such a p r o c e s s , however, would depend on the t o x i c i t y o f p r o t e i n - i n c o r p o r a t e d i s o t h i o -cyanates r e p o r t e d by Bjorkman (1973) d u r i n g the course o f t h i s study (see s e c t i o n IB5). Although these products would l i k e l y remain a t t a c h e d t o the i n t e g r a l s t r u c t u r e o f the p r o t e i n a t the p r e p a r a t i o n stage, they would be r e l e a s e d d u r i n g d i g e s t i o n . The most t o x i c aglycone, g o i t r i n , would be u n a f f e c t e d by the i n t e r a c t i o n ( s e c t i o n .IB5) . 28. The pH 10 s o l u b l e r a p e s e e d . p r o t e i n , s i m i l a r , to t h a t prepared by Kodagoda e t a l . (1973a), was chosen f o r the f i r s t d e t o x i f i c a t i o n t r i a l s i n c e a h i g h p r o p o r t i o n of rapeseed p r o t e i n should be s o l u b l e a t t h a t pH, and success w i t h t h i s f r a c t i o n would t h e r e f o r e a l l o w e x t e n s i o n of the process to most rapeseed p r o t e i n s . pH 7.2 was chosen f o r myrosinase treatment to maximize i s o t h i o c y a n a t e and g o i t r i n p r o d u c t i o n ( A p p e l q v i s t and J o s e f s s o n , 1967), e l i m i n a t e the formation of n i t r i l e s which would occur a t lowerpH (Daxenbichler e t a l . , 1966), and minimize i s o t h i o c y a n a t e p r o t e i n i n t e r a c t i o n (Bjorkman, 1973). Because the p o s s i b i l i t y o f n i t r i l e produc-t i o n c o u l d not be overlooked, an experiment was designed to study n i t r i l e a d s o r p t i o n by a c t i v a t e d carbon. For t h i s study, myrosinase treatment was performed a t pH 3.3. For use i n a l l experiments d e s c r i b e d i n Chapter I I , a semi-pure form of the enzyme myrosinase was prepared from white mustard seed. Carbon column c a p a c i t y was not determined under a c t u a l experimental c o n d i t i o n s used in. the process; i . e . , i n the presence of p r o t e i n . Instead, an aqueous s o l u t i o n of pure i s o t h i o c y a n a t e was used. The e n t i r e experiment w i l l be f u l l y d e s c r i b e d i n the next s e c t i o n . B. METHODS 1. Preparation of Base Soluble Protein Fractions a. Protein extract As outlined i n the flow chart of Figure 2-1, commercial rapeseed meal (Brassica campestris var. Span) was obtained from Canbra Foods Ltd., Lethbridge, Alberta; defatted by petroleum ether extraction; ground i n a Wiley M i l l with a 20-mesh screen; and sieved through a 60-mesh screen to y i e l d a product termed "meal f l o u r " . Preliminary investigation of the meal indicated complete lack of myrosinase a c t i v i t y . NaOH (0.1 N, 600 ml) was added to 100 gm of meal f l o u r ; the mixture was blended i n the Sorvall Multimixer at maximum setting for 3 minutes; pH was adjusted to 10 with N NaOH by au t o t i t r a t i o n (Radiometer); and the mixture was centrifuged. The s o l i d s fraction-was c o l l e c t e d and the extraction repeated twice at pH 10 with 600 ml of NaOH solution. The pooled supernatant (1700 ml collected) was f i l t e r e d and l a b e l l e d protein extract "R". The insoluble h u l l - r i c h s o l i d s residue was l y o p h i l i z e d pending further study. b. Protein i s o l a t e Protein extract "R" (1.1 l i t r e s ) was adjusted to pH 5.3 to i s o e l e c t r i c a l l y p r e c i p i t a t e the base soluble - f r a c t i o n (Kodagoda et a l . , 1973a) and centrifuged (Figure 2-1). The supernatant was decanted; sedimented protein i s o l a t e was col l e c t e d and suspended i n 20 0 ml of water; and the mixture FIGURE1 2-1 Preparation of Rapeseed Protein Fractions 30. DEFATTED RAPESEED MEAL 3 times WILEY MILL (20 mesh) 4 SIEVE (60 mesh) 4- meal flour pH 10 EXTRACTION (Sorvall mulldmixer) CENTRIFUGE — 4- supernatant hull fraction (discard) insoluble residue PROTEIN EXTRACT "R" PROTEIN EXTRACT "R" ADJUST pH 5.3 + Supernatant ^ — CENTRIFUGE 4- protein isolate ADD WATER (discard) HOMOGENIZE (Sorvall multimixer) 4 PROTEIN ISOLATE "U" was blended f o r 1 minute i n the multimixer a t maximum s e t t i n g to produce a f i n e l y d i v i d e d p r o t e i n suspension or s l u r r y which was l a t e r a d j u s t e d to a t o t a l volume of 600 mis. . C e n t r i f u g e d p r o t e i n i s o l a t e was not washed a f t e r the d e c a n t a t i o n step to minimize p r o t e i n l o s s . The s l u r r y was l a b e l l e d p r o t e i n i s o l a t e "U". 2. Myrosinase P r e p a r a t i o n A crude form of the enzyme, myrosinase, was prepared a c c o r d i n g to the method used by A p p e l q v i s t and J o s e f s s o n (1967). White mustard seed, o b t a i n e d from the Canadian G r a i n Commission, Vancouver, was ground on the Wiley M i l l (20-mesh); blended w i t h three volumes of c o l d water (4°C) h e l d a t t h a t temperature f o r 1 hour; and then c e n t r i f u g e d . The super-natant was f i l t e r e d i n t o an equal volume of i c e c o l d 90% e t h a n o l to p r e c i p i t a t e the enzyme; c e n t r i f u g e d ; c o l l e c t e d on a buchner f u n n e l ; .washed w i t h c o l d 70% e t h a n o l ; and d i s s o l v e d i n a volume of water equal to t h a t of the weight of the o r i g i n a l mustard. T h i s aqueous s o l u t i o n was f u r t h e r p u r i f i e d by c e n t r i f u g a t i o n and f i l t r a t i o n . The f i l t r a t e was f r e e z e - d r i e d to y i e l d the crude myrosinase powder which was subsequently s t o r e d f r o z e n i n a d e s i c c a t o r . 3. P r o d u c t i o n of Aglyicones At t h i s stage, p r o t e i n e x t r a c t "R" and p r o t e i n i s o l a t e "U" were t r e a t e d i d e n t i c a l l y as shown i n F i g u r e 2-2. The p r o t e i n s o l u t i o n was a d j u s t e d to pH 7.2, 50 to 100 mg of crude myrosinase powder was added, and the s o l u t i o n was 32. FIGURE 2-2 TREATMENT OF RAPESEED PROTEIN FRACTIONS PROTEIN ISOLATE "U" PROTEIN EXTRACT "R" ADJUST AND MAINTAIN pH 7.2 4 • 4 ADD MYROSINASE 4- 4 STIR OVERNIGHT (approximately 35 - 4 0°C) 4 +. ADJUST pH 10 "X" 4-'S' 4-PASS THROUGH WHITCO 2 09 CARBON i ^ II II ip II 4- 4 ADJUST pH 6.5 4 4 FREEZE-DRY PROTEIN ISOLATE . . Z II PROTEIN EXTRACT M I J I I s t i r r e d and maintained a t pH 7.2 by auto t i t r a t i o n (0.5- N NaOH) f o r a period o f approximately 12 hours a t 35-40°C. During t h i s p e r i o d , g l u c o s i n o l a t e s were converted to i s o t h i o -cyanates, g o i t r i n , b i s u l f a t e , and glucose.by the a c t i o n o f myrosinase (see S e c t i o n IB2). P r o t e i n s o l u t i o n s were l a b e l l e d "S" (from "R") and "X" (from "U"). A sample analogous to "R" was t r e a t e d o v e r n i g h t w i t h myrosinase a t pH 3.3 to serve as a " n i t r i l e - r i c h " sample f o r the n i t r i l e a d s o r p t i o n experiment. At t h i s pH, i n s t e a d of complete c o n v e r s i o n o f 2-hydroxy-3-butenyl i s o t h i o c y a n a t e t o 5 - v i n y l - 2 - o x a z o l i d i n e t h i o n e ( g o i t r i n ) , a s i g n i f i c a n t amount of the n i t r i l e , l-cyano-2-hydroxy-3- butene, i s formed (Daxenbichler e t a l . , 1966). 4. Carbon.Column Treatment Grade 209, Whitco g r a n u l a r a c t i v a t e d carbon, 18 x 40 mesh, was obta i n e d , c o u r t e s y of the Whitco Chemical Company, New York, N.Y. The carbon was s l u r r i e d i n water to remove f i n e s and a d j u s t e d to the pH of maximum s o l u b i l i t y o f the p r o t e i n s o l u t i o n being t r e a t e d . In t h i s experiment, pH 10. was used. The carbon was packed w i t h water i n a 2 x 20 cm g l a s s column. As shown i n the flow c h a r t o f F i g u r e 2-2, the p r o t e i n s o l u t i o n s , "S" and "X", were s o l u b i l i z e d a t pH 10 and passed through the column a t a flow r a t e of approximately 15 to 20 ml per minute. Constant flow r a t e was not s t r i c t l y m aintained 34. E f f l u e n t s were l a b e l l e d "T" (from "S") and "Z" (from "X"). The " n i t r i l e - r i c h " sample was a l s o passed through the column at pH 10. 5. Analyses a. I s o t h i o c y a n a t e s I s o t h i o c y a n a t e content o f p r o t e i n samples was d e t e r -mined by gas chromatographic a n a l y s i s (Youngs and Wetter, 1967) w i t h n - b u t y l i s o t h i o c y a n a t e as i n t e r n a l standard. Peak areas were determined by the h e i g h t - w i d t h method. Response f a c t o r s f o r e t h y l {C^Y and a l l y l (C^) i s o -t h i o c y a n a t e s r e l a t i v e to n - b u t y l (C^) i s o t h i o c y a n a t e agreed w i t h the t h e o r e t i c a l v a l u e s of 2 and 1.33 r e s p e c t i v e l y . Since d e t e c t o r response can be assumed to be independent of u n s a t u r a t i o n i n the case o f flame i o n i z a t i o n d e t e c t o r s (from the above o b s e r v a t i o n and as r e p o r t e d by Youngs and Wetter, 1967), response f a c t o r s of 1.00 and 0.80 were used f o r 3-butenyl (C.) and 4-pentenyl (C..) i s o t h i o c y a n a t e s , r e s p e c -t i v e l y r e l a t i v e to n - b u t y l i s o t h i o c y a n a t e . Procedure: For samples a l r e a d y t r e a t e d w i t h myro-s i n a s e ("S, T, X, Z"), 2 o r 3 ml a l i q u o t s of p r o t e i n s o l u t i o n were withdrawn i n t o g l a s s stoppered t e s t tubes immediately a f t e r p r e p a r a t i o n and shaken wi t h 3 to 5 ml of methylene c h l o r i d e c o n t a i n i n g i n t e r n a l standard n - b u t y l i s o t h i o c y a n a t e . The methylene c h l o r i d e phase was withdrawn f o r gas chroma-t o g r a p h i c a n a l y s i s . Samples c o n t a i n i n g g l u c o s i n o l a t e s (e.g. "R,U") were t r e a t e d as above except t h a t a f t e r methylene c h l o r i d e a d d i t i o n 1 ml of 0.1N sodium c i t r a t e - p h o s p h a t e b u f f e r pH 7.0 was added f o l l o w e d by about 5 mg of myrosinase. The r e a c t i o n v e s s e l s were shaken p e r i o d i c a l l y d u r i n g a span of a t l e a s t 3 hours bef o r e a l i q u o t s of the methylene c h l o r i d e phase c o u l d be withdrawn f o r subsequent a n a l y s i s . Operating c o n d i t i o n s f o r the gas chromatograph are g i v e n i n Table 2-1. b. G l u c o s i n o l a t e s G l u c o s i n o l a t e content of p r o t e i n f r a c t i o n s was determined by t r i m e t h y l s i l a t i o n and gas chromatography ( U n d e r h i l l and K i r k l a n d , 1971) w i t h t r e h a l o s e as i n t e r n a l standard. Only the predominantly o c c u r r i n g g l u c o s i n o l a t e s : gluconapin, g l u c o b r a s s i c a n a p i n , and p r o g o i t r i n were q u a n t i t a t e d Peak areas were determined by h e i g h t - w i d t h measurement. G l u c o s i n o l a t e peaks on the gas chromatograms were i d e n t i f i e d by f i r s t p r e p a r i n g a stock s o l u t i o n of g l u c o s i n o l a t e from rapeseed meal by 80% methanol e x t r a c t i o n , and then par-t i a l l y s e p a r a t i n g them by column chromatography u s i n g f i r s t , DEAE Sephadex A-25 f o l l o w e d by a f f i n i t y chromatography on a r g i n i n e coupled Sephadex G-10 a c c o r d i n g .to the method o f Bjorkman (1972). R e s o l u t i o n o f g l u c o s i n o l a t e s by the Sephadex columns was determined by s i l a t i o n and gas chroma-t o g r a p h i c a n a l y s i s of e f f l u e n t f r a c t i o n s . From the e l u t i o n p a t t e r n s , three samples l a b e l l e d "Q-^r Q 2 a n ^ Q-j"/ were chosen. Each sample co n t a i n e d predominantly one g l u c o s i n o l a t e TABLE 2-1 Gas Chromatographic Operating Conditions Instrument Microtech MT-220 Analysis Isothiocyanates Glucosinolates Carrier gas Flow rate (exit) Column dimensions Nitrogen 45-50 ml/min 1/4 i n , X 6 feet stainless steel Nitrogen 45-50 ixi/min 1/4 in, X 6 feet stainless steel Solid support Liquid phase Temperatures: Inlet Detector Column Chromosorb W A/W DMCS 20% FFAP 175 C 200 C 130 C Chromosorb W 80/100 mesh 5% 07-1 250 C 275 C 220 C and was contaminated by o n l y low l e v e l s of o t h e r s . Myrosinase was added to the samples and r e l e a s e d aglycones were i d e n t i f i e d and q u a n t i t a t e d by gas chromatography ( i s o t h i o c y a n a t e s ; s e c t i o n IIB5a) , and u l t r a - v i o l e t Spectroscopy ( g o i t r i n ; s e c t i o n I I B 5 c ) . In the a n a l y s i s , " 0 ^ " and"Q 2" were pooled s i n c e they c o n t a i n e d g l u c o n a p i n and g l u c o b r a s s i c a n a p i n , both r e l e a s i n g o n l y i s o t h i o c y a n a t e s on enzyme h y d r o l y s i s (Table I-I) . I d e a l l y , , i t had been hoped t h a t a 100% s e p a r a t i o n of g l u c o s i n o l a t e s could be ob t a i n e d i n the three "Q" f r a c t i o n s , thereby a l l o w i n g q u a n t i t a t i o n o f g l u c o s i n o l a t e s i n a chemical manner without the use of the myrosinase enzyme system.. One such method would have been chemical d e g r a d a t i o n o f g l u c o s i n o l a t e s by s u l p h u r i c a c i d f o l l o w e d by glucose d e t e r m i n a t i o n by the anthrone procedure (Gmelin, 1954)." However, s i n c e time d i d not permit f u r t h e r work on i s o l a t i o n , q u a n t i t a t i o n o f g l u c o s i n o l a t e content o f the standard s o l u t i o n s was dependent on e f f i c i e n c y o f the enzyme system (see s e c t i o n IC) . The o b t a i n e d data was used i n the c a l c u l a t i o n o f response f a c t o r s f o r the i n d i v i d u a l g l u c o s i n o l a t e s w i t h r e s p e c t to the i n t e r n a l standard, t r e h a l o s e . These were compared t o response f a c t o r s d e r i v e d under a c t u a l experimental c o n d i t i o n s ; i . e . , from p a r a l l e l analyses of g l u c o s i n o l a t e s and r e l e a s e d aglycones i n p r o t e i n s o l u t i o n s . For example, molar p r o g o i t r i n c ontent o f "R" was e q u i v a l e n t t o molar g o i t r i n c o ntent of "S". S i m i l a r l y , p r o t e i n s o l u t i o n s "U" and "X" were compared. 38. E q u i v a l e n t i s o t h i o c y a n a t e c o n t e n t o f "R" and "U" c o u l d not be d e t e r m i n e d from r e s p e c t i v e a n a l y s e s o f "S" and "X" s i n c e s i g n i f i c a n t e v a p o r a t i o n o f i s o t h i o c y a n a t e s had o c c u r r e d d u r i n g o v e r n i g h t m y r o s i n a s e h y d r o l y s i s ( s e c t i o n I I B 3 ) . There was a l s o t h e p o s s i b i l i t y o f p r o t e i n - i s o t h i o -c y a n a t e i n t e r a c t i o n (Bjorkman, 1973) w h i c h would f u r t h e r d e c r e a s e d e t e c t a b l e i s o t h i o c y a n a t e c o n t e n t . T h e r e f o r e , i n t h i s a n a l y s i s , m y r o s i n a s e was added t o a l i q u o t s o f "R" and "U" as d e s c r i b e d , l a t e r i n t h i s s e c t i o n . A n a l y s e s were performed on f r e e z e - d r i e d p r o t e i n f r a c t i o n s "R" and "U" as w e l l as on i n d i v i d u a l a l i q u o t s o f p r o t e i n s o l u t i o n s t o ensure a c c u r a c y o f d e t e r m i n e d response f a c t o r s . P r o c e d u r e : I n t e r n a l s t a n d a r d , t r e h a l o s e was added t o 2 o r 3 ml a l i q u o t s o f n e u t r a l i z e d p r o t e i n i n ground g l a s s s t o p p e r e d t e s t t u b e s . S i n c e i n t h e f i n a l gas chromatogram, a low b r o a d i n t e r f e r i n g peak was p r e s e n t i n t h e v i c i n i t y o f the t r e h a l o s e peak, I t was n e c e s s a r y t o use a c o n c e n t r a t i o n o f t r e h a l o s e w h i c h would produce a peak o f l a r g e r s i z e t h a n t h a t o f t h e g l u c o s i n o l a t e peaks, even t o the p o i n t o f r e q u i r i n g a change o f a t t e n u a t i o n . By t h i s p r o c e d u r e , e r r o r from the i n t e r f e r e n c e peak was m i n i m i z e d . A f t e r a d d i t i o n o f the i n t e r n a l s t a n d a r d , t h e s o l u t i o n s were mixed t h o r o u g h l y and e v a p o r a t e d t o d r y n e s s under a stream o f n i t r o g e n . Remaining t r a c e s o f m o i s t u r e were removed by vacuum d e s i c c a t i o n . For a n a l y s i s of dry samples, approximately 5 to 10 mg of myrosinase-free sample was a c c u r a t e l y weighed i n t o a ground g l a s s stoppered t e s t tube f o l l o w e d by 2 to 3 ml of d i l u t e d t r e h a l o s e standard. The mixture was then t r e a t e d as the " p r o t e i n s o l u t i o n " a l r e a d y d e s c r i b e d . The d r i e d samples were s i l a t e d by a d d i t i o n of 1 to 2 ml of anhydrous p y r i d i n e , 0.5 ml hexamethyl d i s i l a z a n e , and 0.25 ml c h l o r o t r i m e t h y l s i l a n e . The r e a c t i o n v e s s e l s were immediately stoppered and p l a c e d i n a 97 °C o i l bath o v e r n i g h t . A f t e r t h i s r e a c t i o n p e r i o d , p y r i d i n e and excess s i l a t i o n reagents were removed by vacuum d i s t i l l a t i o n a t room tempera-t u r e ; 1 to 2 ml of i s o - o c t a n e was added to take up s i l a t e d p r oducts; the sample was f i l t e r e d ; and gas chromatographic a n a l y s i s was performed on the f i l t r a t e . I n j e c t i o n s i z e varxed between 2 to 7 m i c r o l i t r e s . Operating c o n d i t i o n s f o r the gas chromatograph are given i n Table 2-1. Q u a n t i t a t i o n of g l u c o s i n o l a t e s was achieved by the f o l l o w i n g formula: m i l l i m o l e s g l u c o s i n o l a t e i n sample = Millimoles trehalose added X response factor X area of gluco-sinolate peak (area of trehalose peak) 40. c. G o i t r i n G o i t r i n content of p r o t e i n samples was determined a f t e r f r e e z e - d r y i n g , by u l t r a - v i o l e t a n a l y s i s a c c o r d i n g to a m o d i f i e d method o f Youngs and Wetter (1967). Log molar a b s o r p t i v i t y c o e f f i c i e n t f o r g o i t r i n was assumed to be 4.25 as r e p o r t e d by Tookey e t a l . (1965). Since samples being analyzed ("S, T, X, Z") had a l r e a d y been t r e a t e d with myrosinase a t pH 7.2 d u r i n g p r e p a r a t i o n , no f u r t h e r treatment was necessary (see s e c t i o n II5Cb). Procedure: Approximately 20. to 40 mg of sample was weighed i n t o a g l a s s stoppered t e s t tube f o l l o w e d by 3 drops of water. When the sample was thoroughly wetted, 1 ml of methylene c h l o r i d e was added t o the t e s t tube and the contents mixed on the Vortex mixer f o r 1 minute. When the methylene c h l o r i d e phase c l e a r e d , 0.5 ml was withdrawn and mixed w i t h 5 ml of a b s o l u t e e t h a n o l , the mixture was allowed t o stand f o r 3 hours, and absorbance was determined i n the range of 200 to 275' nm :>n the Unicam U l t r a v i o l e t Spectrophotometer SP 1800 a g a i n s t a blank of 0.5 ml methylene c h l o r i d e i n 5 ml et h a n o l . Absorbance was c a l c u l a t e d by drawing a b a s e l i n e to the curve (Figure 2-3) and measuring the net absorbance a t the maximum (24 3-24 5 nm). d. N i t r i l e s The change i n n i t r i l e content due to carbon treatment WAVELENGTH (nm) FIGURE 2-3 U l t r a v i o l e t Spectrum of G o i t r i n of the " n i t r i l e - r i c h " sample ( p r e p a r a t i o n d e s c r i b e d i n s e c t i o n -IIB3) was determined by measurement of the n i t r i l e peak h e i g h t a t 2257 cm 1 (4.43 urn) (Daxenbichler et a l . , 1966) o b t a i n e d from the i n f r a - r e d s p e c t r a run b e f o r e and a f t e r • treatment. The instrument used was the P e r k i n Elmer Model 475 G r a t i n g Spectrophomometer equipped w i t h 0.5 mm sodium c h l o r i d e c e l l s . Procedure: Samples b e f o r e and a f t e r treatment were c o l l e c t e d , i s o e l e c t r i c a l l y p r e c i p i t a t e d , and c e n t r i f u g e d . A l i q u o t s of supernatant (50 to 100 ml) were withdrawn and e x t r a c t e d 3 times w i t h 3 volumes of c h l o r o f o r m . The e x t r a c t was d r i e d over sodium s u l f a t e , f i l t e r e d , evaporated to dryness under a stream of n i t r o g e n and vacuum d e s s i c a t e d . The o i l r e s i d u e was d i s s o l v e d i n 1 ml o f c h l o r o f o r m f o r i n f r a - r e d a n a l y s i s . Chloroform was used i n the r e f e r e n c e c e l l . 6. Carbon Column C a p a c i t y Dry a c t i v a t e d carbon (Whitco 209, 3 g.) , was packed i n a 1 x 7 cm glass- column as d e s c r i b e d i n s e c t i o n IIB4.. An aqueous s o l u t i o n of a l l y l i s o t h i o c y a n a t e (approximately 50 mg per l i t r e ) , was passed through the column a t a r a t e averaging 12 ml per minute. Aglycone was d e t e c t e d i n the e f f l u e n t by measurement of absorbance i n the range of 200 to 250nm on the Unicam U l t r a V i o l e t Spectrophotometer. Although a l l y l i s o t h i o c y a n a t e i s not predominant i n rapeseed, i t was chosen as the aglycone f o r t h i s experiment due to i t s a v a i l a b i l i t y 43. and i t s c h e m i c a l s t r u c t u r e . The p r e s e n c e o f an o l e f i n i c d o u b l e bond i n t h i s compound s h o u l d make i t s a d s o r p t i o n c h a r a c t e r i s t i c s somewhat analogous t o t h o s e o f t h e n a t u r a l l y o c c u r r i n g a g l y c o n e s , 3 - b u t e n y l , and 4 - p e n t e n y l i s o t h i o c y a n a t e s . A l t h o u g h t h e c a r b o n c a p a c i t y may be a f f e c t e d by p r o t e i n o r o t h e r s u b s t a n c e s p r e s e n t i n rapeseed, t h e v a l u e o b t a i n e d from t h e d e s c r i b e d p r o c e d u r e s h o u l d s t i l l p r o v i d e ., some i n d i c a t i o n o f t h e t r u e c a p a c i t y o f t h e c a r b o n . C. RESULTS AND DISCUSSION 1. E f f i c i e n c y o f t h e P r o c e s s a. I s o t h i o c y a n a t e and g o i t r i n c o n t e n t A t y p i c a l gas chromatogram o f i s o t h i o c y a n a t e s r e l e a s e d from r a p e s e e d meal i s shown i n F i g u r e 2-4. C o r r e s p o n d i n g r . f . v a l u e s o f t h e s e compounds w i t h r e s p e c t t o n - b u t y l i s o t h i o c y a n a t e s appear i n T a b l e 2-2 below. The s t a n d a r d was e l u t e d a p p r o x i m a t e l y 8 1/2 minutes a f t e r i n j e c -t i o n . TABLE 2-2 Gas Chromatographic r . f . v a l u e s o f I s o t h i o c y a n a t e s with respect to n-butyl Isothiocyanates A g l y c o n e r . f . + S.D.* 3- b u t e n y l i s o t h i o c y a n a t e 1.319 ± 0.003 4- p e n t e n y l i s o t h i o c y a n a t e 1.911 + 0.009 n - b u t y l i s o t h i o c y a n a t e ( s t a n d a r d ) 1.000 * S.D. c a l c u l a t e d from f o r m u l a g i v e n i n T a b l e 2-3. TIME (minutes) FIGURE 2-4. Gas Chromatogram of I s o t h i o c y a n a t e s from Rapeseed Meal Sampling of p r o t e i n s o l u t i o n s was performed as soon as p o s s i b l e a f t e r p r e p a r a t i o n of minimize l o s s of i s o t h i o -cyanates by e v a p o r a t i o n . T r i p l i c a t e or q u a d r u p l i c a t e a n a l yses of two samples were normally s u f f i c i e n t to p r o v i d e a c c e p t a b l e r e s u l t s . Aglycone content i n mg per gram dry weight of p r o t e i n f r a c t i o n s (see F i g u r e 2-1, 2-2 or Appendix 1) a t d i f f e r e n t stages o f p r e p a r a t i o n appears i n Table 2-3. Both median and average v a l u e s w i t h standard d e v i a t i o n s are presented. Average va l u e s were used f o r c a l c u l a t i o n of " T o t a l Aglycone Content" and f o r comparison purposes. Aglycone l e v e l s were found to be near the d e t e c t i o n l i m i t i n some f r a c t i o n s . For example, i n p r o t e i n e x t r a c t "R" n e i t h e r 3-butenyl nor 4-pentenyl i s o t h i o c y a n a t e were d e t e c t a b l b e f o r e myrosinase treatment. A f t e r treatment i n the presence of methylene c h l o r i d e t o e x t r a c t l i b e r a t e d i s o t h i o c y a n a t e s , a v a l u e of 16.84 mg aglycone per gram dry weight was o b t a i n e d T h i s f i g u r e appears i n Table 2-3 o p p o s i t e "R+" myrosinase". The value f o r g o i t r i n , 6.37 mg/ml i n c l u d e d i n t h i s t o t a l was d e r i v e d from the a n a l y s i s of g o i t r i n i n f r a c t i o n "S". Since "S" was prepared from "R" by the a d d i t i o n of myrosinase a t pH 7.2, the molar g o i t r i n content of both was assumed to be i d e n t i c a l . S i m i l a r l y , the v a l u e f o r g o i t r i n of ,rU+ myrosinase" was d e r i v e d from the a n a l y s i s of g o i t r i n i n "X". The d i s c r e p a n c y i n i s o t h i o c y a n a t e content between p r o t e i n e x t r a c t s "R+ myrosinase" and "S" and p r o t e i n i s o l a t e s TABLE 2-3 Aglycone Content of Protein Fractions (mg/g) t 3-butenyl Isothiocyanate 4-pentenyl Isothiocyanate 5-vinyl-2-oxazolidinethione (goitrin) Total Median Average. Median Average Median Average Average R N.D. N.D. N.D. N.D. 0.07 + 0.07 0.01 0.07 S 1.30 1.33 - 0.10 1.49 + 1.52 0.07 6.37 + 6.37 0.04 9.22 T 0.034 0.036 + 0.008 0.023 + 0.025 0.003 0.350 + 0.356 0.007 0.417 X 0.156 0.161 + 0.012 0.116 + 0.118 0.020 0.502 + 0.506 0.015 . 0.785 Z* N.D. N.D. 0.006 + 0.006 0.0002 N.D. N.D. 0.006 R + myrosinase 6.68 6.68 + 0.097 3.75 + 3.79 0.099 6.37tt + 6.37tt 0.04 16.84 U + myrosinase 0.744 0.753 + 0.02 0.578 + 0.573 0.038 0.502tt + 0.506tt 0.015 1.83 Flour ** 0.22 0.21 0.15 0.58 SD =/ (£x) z \ n ) 1/2 V n- 1 / N.D. Not detected ** Concentrations at detection limit Flour prepared by Tape et a l . , 1970. Equivalent Aglycone content. t Median and average + S.D. tt Assumed equal to respective "S" and "X" values. "U+ myrosinase" and "X" can be a t t r i b u t e d to two f a c t o r s : e v a p o r a t i o n of i s o t h i o c y a n a t e s from "S" and "X" d u r i n g t h e i r p r e p a r a t i o n which e n t a i l e d o v e r n i g h t s t i r r i n g a t pH 7.2 i n the presence o f myrosinase; and to p r o t e i n - i s o t h i o c y a n a t e interaction (see s e c t i o n IB5 and I1A) . The f i n a l product of the process, "Z", con t a i n e d o n l y t r a c e amounts o f f r e e aglycones. G o i t r i n and 3-butenyl i s o t h i o c y a n a t e c o n c e n t r a t i o n s were below the d e t e c t i o n l i m i t o f the methods employed i n the an a l y s e s , and on l y 0.006 mg 4-pentenyl i s o t h i o c y a n a t e per gram of p r o t e i n m a t e r i a l was d e t e c t a b l e . For comparison purposes, rapeseed f l o u r prepared by the Food Research I n s t i t u t e (Ottawa) a c c o r d i n g t o the method o f Tape e t a l . (1970) was analyzed by gas chromato-graphy of g l u c o s i n o l a t e s ( s e c t i o n IIB5b) and the r e s u l t s converted t o an e q u i v a l e n t aglycone content. The value from Table 2-3 i s 0.58 mg aglycones per gram o f f l o u r . A complete comparison o f aglycone content among a l l f r a c t i o n s i s g i v e n i n Table 2-4. Values i n the t a b l e below the d o t t e d l i n e were d e r i v e d by d i v i d i n g d e t e c t e d aglycone content o f p r o t e i n f r a c t i o n s i n column "a" ( l e f t column of Table 2-4) by d e t e c t e d aglycone content o f p r o t e i n f r a c t i o n s i n row "b" (top row of Table 2-4) and m u l t i p l y i n g by 100 to c o n v e r t to pe r c e n t . For example, the value of 10.9% appearing o p p o s i t e "U+ myrosinase" and under "R+ myrosinase" was c a l c u l a t e d by d i v i d i n g 1.83 mg/gm by 16.84 mg/gm and 48. TABLE 2-4 Comparison of Aglycone Content Among Protein Fractions Expressed as Percent Change* (between "a" and "b") \ b R + \ myrosinase a \ u + myrosinase X X " R + myrosinase 7*-k 45.2 89.1 95.3 97.5 - V -99.96 54.8 \ 80.2 91.5 95.5 99.94 \ U + myrosinase 10.9 19.8 \ 57.1 77.2 99.67 __ _^ . X 4.7 8.5 42.9 \ 46.9 99.24 \ ^ 2.5 4.5 22.8 53.1 98.56 Z** 0.04 ' 0.06 0.33 0.76 1.44 Values i n this table calculated by: 1. Below dotted line'= (^ ) X 100 2. Above dotted line = (100 - |) X 100 Concentrations at detection limit. multiplying by 100. These numbers (1.83 and 16.84) were obtained from the "Total Aglycone Content" column of Table 2-3 opposite the respective protein f r a c t i o n s . The r a t i o , 10.9% can be interpreted as the percent of o r i g i n a l aglycone remaining after i s o e l e c t r i c p r e c i p i t a t i o n of protein extract "R" to produce protein i s o l a t e "U". In t h i s case, the figure a c t u a l l y represents "equivalent aglycone change" since the aglycones are i n the glucosinolate form during the preparation of "U". The f r a c t i o n of o r i g i n a l isothiocyanates and g o i t r i n remaining aft e r column treatment can be determined by comparing "S" and "T" or "X" and "Z". The values from Table 2-4 are 4.5% and 0.76% respectively and represent the e f f i c i e n c y of the carbon column step. The potential e f f i c i e n c y of the entire process can be determined by comparing "Z" to "R+ myrosinase". The value from the lower half of the table i s 0.04%, which means that in t h i s case, the entire process of i s o e l e c t r i c p r e c i p i t a t i o n followed by myrosinase addition and f i n a l l y carbon treatment was 99.96% e f f e c t i v e i n aglycone removal. This value includes evaporation and possible protein-isothiocyanate reaction products. A similar comparison eliminating t h i s error could be made by comparing "S" to "Z". From the table, the process evaluated i n t h i s manner i s 99.94% e f f e c t i v e . These figures can also be derived from the top of Table 2-4 by reading the value opposite "R+ myrosinase" and under "Z". 50. The top half of Table 2-4 represents percent removal of aglycones between any two f r a c t i o n s and was calculated by the formula 100%-b/a. Comparing "R" and "U" using the top of Table 2-4, 89.1% of the aglycones are l o s t i n the i s o e l e c t r i c p r e c i p i -t a t i o n step. Also from the table, column treatment of "S" and "X" resulted i n 95.5% and 99.2% aglycone removal, respectively. Column treated protein extract "T" was not . i s o e l e c t r i c a l l y p recipitated to produce an i s o l a t e analogous to "U". Although such a step would have reduced aglycone content considerably, i t i s not possible to accurately pre-d i c t the actual reduction which can be expected since aglycones have d i f f e r e n t s o l u b i l i t y properties than those of glucosinolates. Carbon adsorption of protein-isothio-cyanate reaction products mentioned i n section IlA should also be investigated. b. G1ucosinolate content A t y p i c a l gas chromatogram of s i l a t e d glucosino-lates derived from rapeseed meal appears i n Figure 2-5. Corresponding r . f . values for the s i l a t e d compounds with respect to s i l a t e d standard trehalose appear i n Table 2-5. It was found that on the OV-1 column, i n t e r n a l standard hexacosane, used by Underhill and Kirkland (1971), was inadequately resolved from glucosinolates and could there-fore not be used i n the available system. After only limited success with a range of other compounds, trehalose was found to be most acceptable as an i n t e r n a l standard, even though TIME (minutes) FIGURE 2-5 Gas Chromatogram of G l u c o s i n o l a t e s from Rapeseed Meal there was a s l i g h t interference problem as described i n section IIB5b. Only peaks 2, 3, 4, and 7, corresponding to the compounds 3-butenyl glucosinolate (gluconapin), 4-pentenyl glucosinolate (glucobrassicanapin), 2-hydroxy-3-butenyl glucosinolate (progoitrin), and trehalose were determined qua n t i t a t i v e l y . The standard was eluted approxi-mately 30 minutes a f t e r i n j e c t i o n . TABLE 2-5 Gas Chromatographic r . f . Values of S i l a t e d Gluco-sinolates with respect to S i l a t e d Trehalose Glucosinolate r . f . ± S.D. Gluconapin 0.2961 + 0.0015 Glucobrassicanapin 0.3568 + 0.0010 Progo i t r i n 0.4298 ± 0.0009 Trehalose (standard) 1.0000 * S.D. calculated from formula given i n Table 2-3 From Figure 2-5, peaks 1 and 5 were glucosinolates as determined by t h e i r s u s c e p t i b i l i t y to myrosinase hydroly-s i s , but were not present.in high enough concentrations to f a c i l i t a t e i s o l a t i o n by the employed separation techniques. Peak 6 was a naturally occurring carbohydrate of rapeseed meal which, from i t s p o s i t i o n i n the gas chromatogram, was assumed to be a disac.charide. The concentration of t h i s 53. compound was very low i n p r o t e i n i s o l a t e f r a c t i o n s . E x p e r i m e n t a l l y d e r i v e d response f a c t o r s of g l u c o -s i n o l a t e s w i t h r e s p e c t to t r e h a l o s e appear i n Table 2-6. Comparison of g l u c o i n o l a t e peak areas ( r e l a t i v e to the t r e h a l o s e standard peak area) w i t h e n z y m a t i c a l l y r e l e a s e d i s o t h i o c y a n a t e s and g o i t r i n i n a l i q u o t s of p r o t e i n s o l u t i o n s r e s u l t e d i n some v a r i a t i o n of determined response f a c t o r s . Accuracy was l i m i t e d by number of steps r e q u i r e d i n prepara-t i o n of samples f o r a n a l y s i s ( s e c t i o n s IIB5a, b and c ) , nature of the p r o t e i n s o l u t i o n , and dependence on the enzyme system ( s e c t i o n IIB5b). Samples analyzed f o r response f a c t o r d e t e r m i n a t i o n appear to the l e f t of Table 2-6. U n f o r t u n a t e l y , the c o n c e n t r a t i o n of standard t r e h a l o s e used i n "Q1 + Q 2" a n d "Q3" ( s e c t i o n IIB5b) was too low to e l i m i n a t e e r r o r caused by the presence of i n t e r f e r i n g compounds, the peaks of which appeared a t p o s i t i o n s i n the gas chromatogram corresponding to t h a t of t r e h a l o s e . A d d i t i o n a l "Q" samples were not a v a i l a b l e f o r f u r t h e r analyses i n c o r p o r a t i n g h i g h e r c o n c e n t r a t i o n s of t r e h a l o s e to e l i m i n a t e the problem. T h e r e f o r e , i t was decided to use average response f a c t o r s d e r i v e d from analyses on "R" and "U" s i n c e these r e p r e s e n t e d r e s u l t s o b t a i n e d under t y p i c a l experimental c o n d i t i o n s . Gas chromatograms of "Q^ + Q 2" and "Q3" appearing i n F i g u r e s 2-6 and 2-7 demonstrate the degree of s e p a r a t i o n of g l u c o s i n o l a t e s achieved by the Sephadex column s e p a r a t i o n TIME fminutes) FIGURE 2-6. Gas Chromatogram of Fraction "Q1 + Q2". (Attenuation notation (e.g. X 2) signifies further increase i n sensit i v i t y ) . 80 60| LU co Z o LU 20 x l 0 0 FIGURE 2-7. 4. Progoitrin 7. Trehalose 8 2 0 24 28 Gas Chromatogram of Fraction "Q^ ". increase i n sensitivity. 12 16 TIME (minutes) (Attenuation notation (e.g. X 5) signifies further 3 2 U l technique described i n section IIB5b. TABLE 2-6 Gas Chromatographic Molar Response Factors for S i l a t e d Glucosinolates with respect to S i l a t e d Trehalose Source 3-butenyl Glucosinolate (Gluconapin) 4-pentenyl Glucosinolate (Glucobrassica-napin) 2-hydroxy-3-butenyl Glucosinolate (Progoitrin) " R" 1.54 1.32 2.00 "U" 1.48 1.37 2.01 "Qi + Q2" "Q3" 1.6 1.4 1.7 Accepted 1.51 1.34 2.00 Glucosinolate content of protein extract "R", protein i s o l a t e "U" , protein extract "S", and rapeseed f l o u r prepared by the Food Research I n s t i t u t e , Ottawa, by the method of Tape et a l . (1970), appears i n Table 2-7. Values of the gluco-sinolate content of "U" and "R" were derived from the conver-sion of molar values of released aglycones l i s t e d i n Table 2-4. Protein extract "S" contained trace amounts of gluconapin (gas chromatogram) immune to further myrosinase hydrolysis. I t was assumed that t h i s residual l e v e l was due TABLE 2-7 GLUCOSINOLATE CONTENT OF PROTEIN FRACTIONS (mg/g)' Gluconapin (3-butenyl) Glucobrassicanapin (4-pentenyl) Progoitrin (2-hydroxy-3-butenyl) Total R* 24.3 12.7 21.2 58.2 U* 2.7 1.9 1.7 6.3 g** Trace N.D. N.D. Trace Meal Flour 10.1 + 0.10 + 5.6 0.07 9.1 + 0.18 24.8 Extracted Residue 0.2 + 0.01 + 0.2 0.06 0.2 + 0.01 ' 0.6 Flour (Tape et a l . , 1970) 0.8 + 0.06 + 0.7 0.07 0.5 + 0.03 2.0 t . As potassium salts average + S.D. S.D. calculated from formula, Table 2-3. * From aglycone content (Table 273). ** Not degradable by myrosinase N.D. Not detected 58. to the presence of the desulfonated form of the glucosinolate reported by Underhill and Kirkland (1971) which cannot be converted to i t s respective aglycone by myrosinase hydrolysis. Glucosinolates of a l l other fractions were susceptible to myrosinase treatment. The r e s u l t of -this analysis therefore proved the myrosinase treatment of "R", described i n section IIB3, to y i e l d protein extract "S", to be e s s e n t i a l l y complete. The h u l l - r i c h residue remaining a f t e r protein extraction contained only 0.60 mg/g t o t a l glucosinolates ' compared to 58.2 i n protein extract "R". Meal f l o u r , from which "R" was derived, contained 24.8 mg/g: glucosinolates. This value i s approximately half that of "R", indicating that there was a 2-fold concentration of glucosinolates during the preparation of "R". This figure i s dependent on the extent of extraction of glucosinolates from the meal f l o u r (almost 100%; see value for extracted residue, Table 2-7), and the t o t a l y i e l d of extracted material from the meal, (approxi-mately 4 5-50% of the meal f l o u r ) . The greater the y i e l d of protein material i n any process, the lower w i l l be the con-centration e f f e c t on the glucosinolate content of the extract. Detoxified rapeseed f l o u r prepared by the Food Research I n s t i t u t e , Ottawa, (Tape et a l . , 1970) had a gluco-sinolate content approximately 1/3 that of untreated protein i s o l a t e "U". c. N i t r i l e content Infra-red spectra of chloroform extracts of " n i t r i l e - r i c h " protein extracts (preparation described PERCENT T R A N S M I T T A N C E 60 . W A V E L E N G T H (nm) FIGURE 2-9. Ultra-violet Spectrum of A l l y l Isothiocyanate s e c t i o n IIB3) b e f o r e and a f t e r c a r b o n column t r e a t m e n t appear i n F i g u r e 2-8 . The s i z e o f the weak n i t r i l e peak a t 4.43um c h a r a c t e r i s t i c o f the C=N s t r e t c h was used to d e t e r m i n e change o f n i t r i l e c o n t e n t due to c a r b o n column t r e a t m e n t . From the d e c r e a s e d h e i g h t o f t h i s p e a k , i t was assumed t h a t . p a r t i a l n i t r i l e a d s o r p t i o n by the c a r b o n had o c c u r r e d . Peak h e i g h t d e c r e a s e d by a p p r o x i m a t e l y 40%. I n t e n s e peaks a t 6.63ymand 8 . 5 7 y m c h a r a c t e r i s t i c o f g o i t r i n d i s a p p e a r e d o r d e c r e a s e d markedly a f t e r t r e a t m e n t i n d i c a t i n g t h a t g o i t r i n was i n d e e d adsorbed by the c o l u m n . The degree o f p u r i t y o f the e x t r a c t would not j u s t i f y the f o r m u l a t i o n o f f u r t h e r c o n c l u s i o n s from the o b t a i n e d s p e c t r a . 2. A d s o r p t i o n C a p a c i t y o f the Carbon The o b t a i n e d u l t r a - v i o l e t spectrum o f a l l y l i s o t h i o -c y a n a t e appears i n F i g u r e 2 - 9 . Carbon c a p a c i t y was d e t e r m i n e d to be a p p r o x i m a t e l y 380 to 400 mg a l l y l i s o t h i o c y a n a t e per gram o f c a r b o n . U s i n g "R+ m y r o s i n a s e " and U+ m y r o s i n a s e " a g l y c o n e v a l u e s , the carbon s h o u l d be c a p a b l e o f d e t o x i f y i n g a p p r o x i m a t e l y 20 g o f p r o t e i n e x t r a c t o r 200 g o f i s o l a t e per gram o f c a r b o n . , D . CONCLUSIONS AND GENERAL DISCUSSION The f o l l o w i n g c o n c l u s i o n s can be drawn from the r e s u l t s : a . The m y r o s i n a s e c o n v e r s i o n o f g l u c o s i n o l a t e s to i s o t h i o -c y a n a t e s and g o i t r i n was e s s e n t i a l l y complete except for the de-sulfo analogues of glucosinolates which are not considered harmful due to t h e i r immunity to myrosinase hydrolysis. Since only p a r t i a l carbon adsorption of n i t r i l e s occurred, n i t r i l e formation i n the d e t o x i f i c a t i o n process should be.avoided by incubation (VanEtten. et a l . , 1966) and maintenance of neutral or basic pH during hydrolysis (Daxenbichler et a l . , 1966). Free isothiocyanates and g o i t r i n were adsorbed by the carbon column with'greater than 95% and 99% e f f i c i e n c y for protein extract and protein i s o l a t e solutions respectively at pH 10. Presumably, i s o -e l e c t r i c p r e c i p i t a t i o n of the carbon treated protein extract "T" would lower aglycone content to a l e v e l comparable to carbon treated protein i s o l a t e "U" since i s o e l e c t r i c p r e c i p i t a t i o n was found to decrease glucosinolate l e v e l s by 89% i n the above reported experiment. Treatment of i s o l a t e compared to extract treatment would have the advantage of increasing carbon l i f e t i m e due to the lower aglycone content of the i s o l a t e compared to that of the extract.. The l e v e l s of free aglycones i n the finished product "Z" were very low, 0.006 mg 4-pentenyl isothiocyanate per gram dry weight. Since t h i s l e v e l was near the detection l i m i t of the methods used for analysis, there i s a p o s s i b i l i t y that trace amounts of 3-butenyl isothiocyanate and g o i t r i n were s t i l l present. However, these l e v e l s should be considered safe for human consumption since according to Downey et a l . (1967), 3-butenyl isothiocyanate and g o i t r i n occur naturally i n cabbage (Brassica oleracea) at le v e l s of 0 to 3 and 0 to 7 mg/g respectively. These compounds are also found i n other commonly ingested foods, (VanEtter., et a l . , 1969). The by-product of the process, the extracted residue., contained only 0.6 mg/g. glucosinolates and would therefore be an excellent material for incorpora-t i o n into livestock feed providing the material was s u f f i c i e n t l y digestable. The'composition of t h i s material should be further investigated. T o x i c i t y of isothiocyanate-protein i n t e r a c t i o n products (section IB5) should be investigated and t h e i r formation further minimized by decreasing myro-sinase hydrolysis time. This could be achieved by an increase i n temperature (Appelqvist and Josefsson, 1967) and possible addition of ascorbate (section IC). Duration of high pH t r e a t -ments should also be s t r i c t l y c ontrolled. 64. g. In addition to the removal of aglycones, the carbon treatment removed natural rapeseed odor present in the protein extract and i s o l a t e s . P a l a t a b i l i t y was also improved. However, no change i n protein color ( l i g h t brown to brown-green) was observed during carbon treatment. CHAPTER I I I MODIFICATION OF THE RAPESEED PROTEIN DETOXIFICATION PROCEDURE A. INTRODUCTION Although the process d e s c r i b e d i n Chapter I I was s u c c e s s f u l , a number of economic " s h o r t c u t s " were deemed p o s s i b l e . Proposed changes were: 1. D i r e c t i n c o r p o r a t i o n of mustard seed as a source of myrosinase to e l i m i n a t e the need f o r enzyme e x t r a c -t i o n and p u r i f i c a t i o n . 2. A p p l i c a t i o n o f carbon treatment a t v a r i o u s pH's to al l o w e x t e n s i o n o f the carbon d e t o x i f i c a t i o n process to a l l rapeseed p r o t e i n . i s o l a t e s and decrease or e l i m i n a t e i s o t h i o c y a n a t e - p r o t e i n i n t e r a c t i o n ( s e c t i o n I B 5 ) . 3 . P o s s i b l e d i r e c t a p p l i c a t i o n of g l u c o s i n o l a t e s o l u t i o n s to a c t i v a t e d carbon, thereby completely e l i m i n a t i n g the need f o r myrosinase treatment. Experiments were designed t o i n v e s t i g a t e the above p o s s i b i l i t i e s . As w e l l , the pH experiment i n c l u d e d a study of i s o t h i o c y a n a t e s t a b i l i t y a t v a r i o u s pH's. A f i n a l "recommended d e t o x i f i c a t i o n p r o c e s s " was e l u c i d a t e d d e f i n i n g c o n d i t i o n s f o r maximum d e t o x i f i c a t i o n of rapeseed p r o t e i n i s o l a t e s . B. METHODS 1. Mustard as Source of Myrosinase Although i t would be p o s s i b l e to use i n t a c t rapeseed as a source of myrosinase, i t was decided to use mustard seed because of i t s higher enzyme a c t i v i t y (Lonnerdal and Janson, 1973). Procedure: Rapeseed meal f l o u r prepared as i n section IIBla was s l u r r i e d i n ten volumes of water and blended for 3 minutes i n the Sorvall Multimixer at maximum sett i n g . The mixture was adjusted to pH 7.2 and freshly ground undefatted white mustard seed, 1% by weight of meal fl o u r was added. pH 7.2 was maintained overnight (35-4 0°C) by s t i r r i n g and a u t o t i t r a t i o n with N NaOH. The s l u r r i e d meal was then extracted at pH 10, i s o e l e c t r i c a l l y p r e c i p i t a t e d at pH 5.3 to y i e l d a protein i s o l a t e ("MSI") analogous to "U" or "X" of Chapter I I , dissolved at pH 11, and treated by the activated carbon procedure described i n Section IIB4. Glucosinolate content of "MSI" was determined by gas chromatography (section IIB5b) to evaluate extent of myrosinase hydrolysis. Isothiocyanate analyses (section IIB5a) were performed before and after column treatment to determine e f f i c i e n c y of carbon adsorption at pH 11. Treated "MSI" was l a b e l l e d "MS2". The h u l l - r i c h extracted residue was also analyzed for isothiocyanate content. 2. E f f e c t of pH on Carbon Adsorption of Isothiocyanates  and on Isothiocyanate S t a b i l i t y . Procedure: Commercial rapeseed meal was s l u r r i e d with ground white mustard seed and incubated overnight at pH 7.2 and 35-4G°C, i n the manner described i n section I I I B l above. The meal s l u r r y was divided into 5 equal portions which were subsequently extracted at pH 12, 10, 7, 5, or 3 to y i e l d c h a r a c t e r i s t i c protein extracts for each of the pH's. The extracts corresponded i n nature to "R" or "S" prepared i n section I I B l . Each extract was then carbon treated (section IIB4) at i t s respective pH. Sampling before and a f t e r column treatment was performed as follows:. a. 3 ml protein solution + 5 drops pH 5.0, N sodium c i t r a t e buffer + 5 ml methylene chloride*. b. Same as "a" but without c i t r a t e buffer. c. Unbuffered protein solution (no carbon treatment), stored i n cold room i n stoppered Erlenmeyer f l a s k . Sample sets "a" and "b" were analyzed for i s o t h i o -cyanate content (section IIB51) immediately a f t e r preparation and again 24 hours l a t e r . Set "c" was sampled 24 hours after preparation and isothiocyanate content determined i n a manner analogous to the method used for set "b". The experiment was performed only once i n i t s en t i r e t y and must therefore be considered preliminary. G o i t r i n l e v e l s were not investigated. Methylene chloride containing standard n-butyl isothiocyanate 68. 3. Carbon Adsorption of Glucosinolates a. I s o l a t i o n of glucosinolates A stock solution of glucosinolates was prepared according to the method of Greer (1962) from rapeseed meal by employing 75% acetone extraction of rapeseed meal. The extract was concentrated by f l a s h evaporation and subse-quently applied to a column of Amerlite IR-4B i n the chloride form. After organic material was eluted from the column by exhaustive water washing, glucosinolates were eluted with 0.1 N NaCl. The e f f l u e n t was evaporated to dryness on the f l a s h evaporator and taken up i n a small volume of ethanol. This solution served as the gluco-sinolate stock solution. b. Preparation of protein i s o l a t e Base soluble rapeseed protein i s o l a t e equivalent to "U" was prepared as i n section IIBlb. This protein solution was further i s o e l e c t r i c a l l y p r e c i p i t a t e d , centrifuged and re-dissolved i n d i s t i l l e d water. The entire procedure was repeated a t o t a l of f i v e times to remove a l l traces of glucosinolates from the protein which served as the "protein i s o l a t e f r a c t i o n " for the experiment to follow. c. Application to carbon column Diluted glucosinolate stock solutions at pH 3, 7, and 10 were applied to carbon columns of corresponding pH's as described i n section IIB4 and change i n glucosinolate content determined by gas chromatography (section IIB5b). Protein i s o l a t e was added to dil u t e d glucosinolate stock solution and.the mixture was applied to carbon columns at. pH 10 and 7. The pH 7 sample was i n the form of a protein s l u r r y since the protein was only s l i g h t l y soluble at t h i s pH. Glucosinolate content was determined as described above. A basic protein extract s i m i l a r to "R" (section IIBla) and whey from the pH 5.3 i s o e l e c t r i c p r e c i p i t a t i o n of that extract were also applied to the carbon column at pH 10 and 5.3, respectively to determine e f f i c i e n c y of carbon adsorption of glucosinolates from native rapeseed protein solutions. C. RESULTS AND DISCUSSION 1. Mustard Glucosinolate analysis of "MSI" (see section IIIB1 or Appendix) revealed no detectable myrosinase degradable glucosinolate content, in d i c a t i n g that ground mustard seed was an e f f e c t i v e source of myrosinase for the process. An investigation of the minimum l e v e l of mustard or minimum time required for complete glucosinolate degradation was not c a r r i e d out since these factors would depend on the qua l i t y of mustard seed used. Free 3-butenyl and 4-pentenyl isothiocyanates were present i n "MS2" at 20.6% and 12.6% of t h e i r respective l e v e l s i n "MSI" ind i c a t i n g that carbon treatment at pH 11 was less e f f i c i e n t than the pH 10 experiment described i n Chapter I I . Besides i n e f f i c i e n c y of carbon adsorption of 70. i s o t h i o c y a n a t e s a t t h i s h i g h pH, d e t e r i o r a t i o n of p r o t e i n q u a l i t y c o u l d a l s o occur. T h e r e f o r e , pH 11 cannot be recommended f o r the d e t o x i f i c a t i o n p r o c e s s . The i n s o l u b l e r e s i d u e remaining a f t e r e x t r a c t i o n c o n t a i n e d 3-butenyl and 4-pentenyl isothiocya!nates a t l e v e l s o f 0.04 and 0. 085 -mg/g dry weight r e s p e c t i v e l y . T h i s m a t e r i a l would t h e r e f o r e be 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 animal feeds p r o v i d i n g i t was p a l a t a b l e and d i g e s t a b l e . 2. E f f e c t of pH a. I s o t h i o c y a n a t e a d s o r p t i o n As shown i n the graph of F i g u r e 3-1, the column was g r e a t e r than 93% e f f e c t i v e i n removal of i s o t h i o c y a n a t e s from rapeseed p r o t e i n e x t r a c t s at; pH v a l u e s below 10. pH 3 treatment was g r e a t e r than 98% e f f e c t i v e i n t o t a l i s o t h i o -cyanate removal. However, a t pH 12, 27% and 46% of the o r i g i n a l 3-butenyl and 4-pentenyl i s o t h i o c y a n a t e s remained i n the column e f f l u e n t . Although the above r e s u l t s must be c o n s i d e r e d t e n t a -t i v e due to l a c k of r e p l i c a t i o n and the f a c t t h a t a change of flow r a t e or temperature would a f f e c t r e p r o d u c i b i l i t y of the r e s u l t s , the o b s e r v a t i o n t h a t the e f f i c i e n c y of the column i s g r e a t e r than 93% over the range o f pH 3 to pH 10 must be con-s i d e r e d s i g n i f i c a n t . The low i s o t h i o c y a n a t e removal a t pH 12 i s c r e d i b l e s i n c e the v a l u e s o b t a i n e d i n the p r e v i o u s l y d e s c r i b e d experiment f o r the pH 11 treatment o f p r o t e i n i s o l a t e agree c l o s e l y (Figure 3-1). However, pH 10 carbon treatment of p r o t e i n e x t r a c t "S" to y i e l d "T" ( s e c t i o n IIB4) r e s u l t e d i n O 241 Z z < 221 UJ 20 18 CO UJ J— < Z 2 161 u O x 14! i— o 12S 10 < z o O 8 Z 6 LU u a. H v 3-Butenyl • 4 -Pentenyl • 3-Buteny| H 4-Penteny| 3 5 7 9 11 TREATMENT PH FIGURE 3-1. Effect of pH-on Carbon Adsorption of Isothiocyanates • H From MS2 protein . isolate o n l y 2.7% and 1.6% r e s i d u a l 3-butenyl and 4-pentenyl i s o t h i o -cyanates i n "T" compared to the o r i g i n a l l e v e l i n "S". These values are low compared to 4.4% and 6.8% o b t a i n e d i n the p r e s e n t study. Since the curve r i s e s s t e e p l y a f t e r pH 10, t h i s r e g i o n i s probably extremely s e n s i t i v e to o p e r a t i n g parameters such as i o n i c s t r e n g t h of the s o l u t i o n , flow r a t e , temperature and other unknown f a c t o r s and would t h e r e f o r e produce s l i g h t l y v a r i a b l e r e s u l t s . A commercial process o p e r a t i n g a t t h i s pH would have to be c a r e f u l l y c o n t r o l l e d . b. I s o t h i o c y a n a t e s t a b i l i t y No change i n i s o t h i o c y a n a t e content of i n d i v i d u a l samples was observed a f t e r 24 hours i f the samples were s t o r e d over methylene c h l o r i d e as i n s e t s "a" and "b". A l s o , complete agreement was o b t a i n e d f o r i s o t h i o c y a n a t e content of c o r r e s -ponding samples of "a" and "b" i n d i c a t i n g t h a t i n the t e s t e d cases, b u f f e r i n g had no e f f e c t on i s o t h i o c y a n a t e e x t r a c t -a b i l i t y i f e x t r a c t i o n was performed immediately. However, sample s e t 11 c" s t o r e d i n the c o l d room and e x t r a c t e d a f t e r 24 hours, showed a decrease i n i s o t h i o c y a n a t e content from t h a t of "a" and "b". The decrease was a f u n c t i o n of pH as shown i n graph of F i g u r e 3-2. Decrease i n d e t e c t a b l e content of both 3-butenyl and 4-pentenyl i s o t h i o c y a n a t e s was l i n e a r between pH 5 and pH 10, c o n f i r m i n g the f i n d i n g s of Bjorkmann (1973) who r e p o r t e d the i s o t h i o c y a n a t e - p r o t e i n i n t e r a c t i o n to be l i n e a r l y c o r r e l a t e d w i t h pH i n the range of pH 6 to'pH 10. Highest remaining i s o t h i o c y a n a t e c o n t e n t , i . e . , r e g i o n of h i g h e s t i s o t h i o c y a n a t e s t a b i l i t y was pH 5. 73. STORAGE PH FIGURE 3-2. Decrease of Free Isothiocyanate Content After 24 hours Storage at 5°C, Although much of the isothiocyanate loss can be attributed to evaporation, th.e isothiocyanate-protein i n t e r -action would be expected to proceed more completely at high pH where there i s increased a c c e s s i b i l i t y of sulphydryl, e-amino, and terminal a-amino groups of the rapeseed protein (Bjorkmann, 1973). In t h i s experiment the extent of i n t e r -action or loss of isothiocyanates was determined by comparing samples of "a" and "b" to samples "c" stored i n the cold room fqr 24 hours. However, samples "a" and "b" had been prepared for approximately 1 hour 'before methylene chloride extraction was performed and i t i s not therefore known to what extent the i n t e r a c t i o n had proceeded i n these solutions before sampling was complete. Recognizing t h i s f a c t , and comparing i t to the observation that a f t e r 24 hours storage, only 5% of the o r i g i n a l free isothiocyanate content was detectable i n the pH 10 sample of "c" due to assumed i n t e r a c t i o n , i t may be speculated that an i n d u s t r i a l process operating at pH 10 and requiring a t o t a l time i n t e r v a l of less than 1 hour would be r e l a t i v e l y free from isothiocyanate-protein i n t e r a c t i o n product formation. The presence of ammonium s a l t s i n the meal would compete with the isothiocyanate-protein i n t e r a c t i o n at high pH where free ammonia would be l i b e r a t e d from the s a l t s to form thioureas with the isothiocyanates (section IB5). The entire problem of complex formation can be minimized by decreasing the myrosinase incubation time and minimizing the duration of high pH treatments. Such modifications would be p o s s i b l e i n h i g h speed i n d u s t r i a l s i t u a t i o n s . More work remains to be done i n t h i s area. The f a c t t h a t any pH i n the range of pH 3 to 10 was f e a s i b l e f o r i s o t h i o c y a n a t e removal from p r o t e i n i s o l a t e s by the carbon column permits the e x t e n s i o n o f the process to a l l s o l u b l e rapeseed i s o l a t e s without the pH 10 s o l u b i l i z a -t i o n of a l l f r a c t i o n s p r e v i o u s l y advocated. E f f e c t of pH on carbon a d s o r p t i o n of g o i t r i n must be i n v e s t i g a t e d . As shown i n the experiments i n Chapter I I , no improvement i n p r o t e i n c o l o r was observed a t any pH. Presum-abl y the a d s o r p t i o n process would be more e f f i c i e n t a t higher temperatures. 3. Carbon A d s o r p t i o n o f G l u c o s i n o l a t e s The r e s u l t s of carbon column treatment of g l u c o s i n o -l a t e s o l u t i o n s i s shown i n Table 3-^ -1. Stock g l u c o s i n o l a t e s o l u t i o n was completely adsorbed by the column a t pH 10, and 7 w h i l e pH 3 column treatment o n l y p a r t i a l l y decreased g l u c o -s i n o l a t e content. When p r o t e i n was added to the system, the column had a b s o l u t e l y no e f f e c t on g l u c o s i n o l a t e content i n any of the t e s t e d cases. The o n l y l o g i c a l e x p l a n a t i o n f o r t h i s phenomenon i s t h a t some type of i n t e r a c t i o n e x i s t s between p r o t e i n and g l u c o s i n o l a t e thereby i n t e r f e r i n g w i t h carbon a d s o r p t i o n . However, Bjorkmann (1973) r e p o r t e d no i n t e r a c t i o n between p r o t e i n and g l u c o s i n o l a t e . In the above r e p o r t e d experiment, the g l u c o s i n o l a t e i n t e r a c t i n g p r o t e i n TABLE 3-1 E f f e c t o f Carbon Treatment on S o l u t i o n s C o n t a i n i n g G l u c o s i n o l a t e s SOLUTION DECREASE OF GLUCOSINOLATE CONTENT None P a r t i a l Complet Stock G l u c o s i n o l a t e s o l u t i o n a t : pH 10 pH 7 pH 3 Stock G l u c o s i n o l a t e s o l u t i o n & p r o t e i n i s o l a t e a t : pH 10 pH 7 Rapeseed p r o t e i n e x t r a c t a t : pH 10 Rapeseed p r o t e i n whey a t : pH 5.3 77 . o r substance i s pr e s e n t i n both the pH 5.3 i s o e l e c t r i c a l l y p r e c i p i t a t e d base s o l u b l e p r o t e i n f r a c t i o n and i n the whey from t h i s p r o t e i n , s i n c e carbon treatment f a i l e d to decrease g l u c o s i n o l a t e content i n e i t h e r case (Table 3-1). U n t i l some way i s found to overcome t h i s a s s o c i a t i o n , a c t i v a t e d carbon treatment cannot be a p p l i e d d i r e c t l y to rapeseed p r o t e i n s o l u t i o n s without p r i o r d e g r a d a t i o n of g l u c o s i n o l a t e s to t h e i r r e s p e c t i v e a g l y c o n e s by myrosinase treatment. D. RECOMMENDED DETOXIFICATION PROCEDURE A flow c h a r t f o r the recommended d e t o x i f i c a t i o n process appears i n F i g u r e 3-3. Two products are produced: d e t o x i f i e d , p r o t e i n i s o l a t e ; and, a r e s i d u e f r a c t i o n s u i t a b l e f o r animal feed. The o n l y l o s s o f p r o t e i n d u r i n g i s o l a t e p r e p a r a t i o n occurs from incomplete i s o e l e c t r i c p r e c i p i t a t i o n . T h i s l o s s c o u l d be decreased by subsequent i s o e l e c t r i c treatment of the whey a t another pH or a p p l i c a t i o n o f u l t r a f i l t r a t i o n methods. The recovered p r o t e i n c o u l d be d e t o x i f i e d by the o u t l i n e d procedure as w e l l . A t no time i n the process i s i t recommended t h a t the i s o l a t e be washed, although i n c o r p o r a t i o n o f t h i s step would f u r t h e r decrease aglycone content. A p o t e n t i a l p r o c e s s o r r e q u i r i n g pure p r o t e i n i s o l a t e c o u l d add t h i s step a t the expense o f p r o t e i n y i e l d . An i s o l a t e t r e a t e d i n , t h i s manner would have more w e l l d e f i n e d and r e p r o d u c i b l e f u n c t i o n a l p r o p e r t i e s due to i t s i n c r e a s e d homogeneity (e.g. Kodagoda e t a l . , 1973b). FIGURE 3.-3 78. RECOMMENDED PROCEDURE FOR DETOXIFICATION OF RAPESEED PROTEIN ISOLATES RAPESEED MEAL + WATER HOMOGENIZE (Multimixer) I ADJUST pH 7.2 ADD £ 1 % WHITE MUSTARD SEED (ground) STIRA. AT pH 7.2 AND INCUBATE, (minimum time) • + ->ADJUST TO pH DESIRED FOR PROTEIN EXTRACTION 3 times ( a r b i t r a r y ) 1 CENTRIFUGE + supernatant ISOELECTRIC PRECIPITATION SOLIDS ( c a t t l e f e e d ) CENTRIFUGE WHEY ' ( d i s c a r d o r i s o -\ s o l i d s e l e c t r i c a l l y p r e c i p i t a t e a t REDISSOLVE ISOLATE AT ORIGINAL pH n e W p H ) \ PASS THROUGH CARBON COLUMN NEUTRALIZE DRY P r o d u c t i o n of d e t o x i f i e d rapeseed p r o t e i n concen-t r a t e (not i s o e l e c t r i c a l l y p r e c i p i t a t e d ) i s p o s s i b l e with t h i s p r o c e s s . However, t h i s product would be h i g h i n ash and o r g a n i c i m p u r i t i e s which would a f f e c t f u n c t i o n a l p r o p e r t i e s . A c c o r d i n g to Yapar and C l a n d i n i n (1965), rape-seed meal c o n t a i n s h i g h l e v e l s of t a n n i n s . T h i s f a c t o r would have to be c o n s i d e r e d i n the p r o d u c t i o n of p r o t e i n c o n c e n t r a t e . A c t u a l v a l u e s such as. e x t r a c t i o n volumes, amount of mustard seed added, d u r a t i o n of myrosinase treatment, and method of d r y i n g the p r o t e i n i s o l a t e w i l l have to be determined f o r a p a r t i c u l a r i n d u s t r i a l s i t u a t i o n and cannot be m e a n i n g f u l l y d e f i n e d here. L e v e l of mustard seed and h y d r o l y s i s time r e q u i r e d w i l l depend on enzyme a c t i v i t y of the a v a i l a b l e seed, temperature and pH of h y d r o l y s i s , and presence o f a c t i v a t o r s (ascorbate; s e c t i o n IC) or i n h i b i t o r s (SH b l o c k i n g ) (Nagashima and Uchiyama, 1959b). I f whole mustard, seed i s not used, mode of p r e p a r a t i o n w i l l have to be c o n s i d e r e d i n d e t e r m i n a t i o n of optimum h y d r o l y s i s time ( s e c t i o n I C ) . Removal o f l i p i d from the ground seed may a l s o be a d v i s a b l e . R e s o l u b i l i z a t i o n of p r o t e i n i s o l a t e a f t e r i s o e l e c t r i c p r e c i p i t a t i o n of e x t r a c t should be c a r r i e d out i n as s m a l l a volume of water as p o s s i b l e to f a c i l i t a t e the d r y i n g step at the end. With proper d e s i g n , i t may be p o s s i b l e to t r e a t 80. p r o t e i n i s o l a t e s i n a homogenized p r o t e i n s l u r r y without complete s o l u b i l i z a t i o n . T h i s would e l i m i n a t e i s o t h i o -c y a n a t e - p r o t e i n i n t e r a c t i o n i n base s o l u b l e f r a c t i o n s . E f f e c t s of v a r y i n g flow r a t e , temperature, and s i z e o f column were not determined. Decreasing f l o w . r a t e , r a i s i n g temperature, and i n c r e a s i n g column l e n g t h should i n c r e a s e e f f i c i e n c y of carbon a d s o r p t i o n , although d e t o x i f i - , c a t i o n e f f i c i e n c i e s of 100% were never achieved i n the d e s c r i b e d experiments on p r o t e i n s o l u t i o n s , r e g a r d l e s s of c o n d i t i o n s used. The observed r e s i d u a l aglycone content may be due to a weak p r o t e i n - a g l y c o n e i n t e r a c t i o n . S i n c e such an a s s o c i a -t i o n would l i k e l y be an e q u i l i b r i u m , i n c o r p o r a t i o n o f a h o l d i n g step f o r r e - e q u i l i b r a t i o h o f the system a f t e r carbon treatment and a second treatment c o u l d be c o n s i d e r e d . However, the low l e v e l s o f aglycones p r e s e n t i n the product from the porposed process and the added expense of adding another step would not warrant such a m o d i f i c a t i o n . A p o s s i b l e f u t u r e i n c o r p o r a t i o n i n t o t h i s d e t o x i f i c a -t i o n process would be the use of a system i n which myrosinase was bound t o a s o l i d support. Such an advance would a l l o w the process to be c a r r i e d out i n a continuous automated system. E. CONCLUSION A r e l a t i v e l y s u c c e s s f u l d e t o x i f i c a t i o n process f o r rapeseed p r o t e i n e x t r a c t s and i s o l a t e s s o l u b l e i n the 81. range of pH 3 to pH 7 has been d e v i s e d . E x t e n s i o n of the process to a h i g h e r pH range depends on t o x i c i t y of i s o t h i o -c y a n a t e - p r o t e i n r e a c t i o n products and on the a b i l i t y o f a p o t e n t i a l p r o c e s s o r to p r o v i d e a h i g h speed e x t r a c t i o n system to minimize d u r a t i o n of h i g h pH treatments. Besides the. above mentioned f a c t o r s , i n d u s t r i a l adoption of the proposed process w i l l depend on the f o l l o w i n g f a c t o r s : 1. Economics: - c o s t of raw m a t e r i a l s - c o s t of energy, e t c . 2. Demand f o r product: improvement of c o l o r of the product - w e l l d e f i n e d f u n c t i o n a l p r o p e r t i e s — p o s s i b l y unique to the product - acceptance by food p r o c e s s o r s i n t o t r a d i t i o n a l products - acceptance by the p u b l i c . 82. LITERATURE CITED A l e k s i e j c z y k , Z. and Rutkowski, A. 197 0. I n v e s t i g a t i o n o f h i g h v o l t a g e paper e l e c t r o p h o r e s i s a p p l i c a t i o n f o r p u r i f i c a t i o n and s e p a r a t i o n of rape-seed t h i o g l u c o s i d e s . Zesz. P r o b l . Post. Nauk. Roln. 91: 505. A l l e n , C E . and D.S. Dow. 1952. B i o l o g i c a l assessment of the v a l u e of rapeseed o i l meal as a d i e t a r y component. S c i . 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APPENDIX 1 KEY TO PROTEIN FRACTIONS R pH 10 protein extract S Myrosinase treated protein extract (R) T pH 10 carbon treated protein extract (S) U pH 5.3 i s o e l e c t r i c a l l y p recipitated and p u r i f i e d protein i s o l a t e from pH 10 protein extract (R) X Myrosinase treated protein i s o l a t e (U) Z pH 10 carbon treated protein i s o l a t e (X) MSI Protein i s o l a t e (similar to X) prepared from rapeseed meal slurry incubated with ground mustard seed MS2 . pH 11 carbon treated protein i s o l a t e (MSI). 

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