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Yeast cultivation on natural starches 1974

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YEAST CULTIVATION ON NATURAL STARCHES BY NELIA BENDANA HELBIG . S . Chem. U n i v e r s i t y o f t he P h i l i p p i n e s , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Depar tment o f Food S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A P R I L , 1974 . In p r e sen t ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree tha 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 reference and s tudy. I f u r t he r agree tha t pe rmiss ion f o r ex tens ive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department o f Fo-vd ScCtnt-o The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date 9 Apn'/ ABSTRACT T h i s r e s e a r c h p r o j e c t i s c o n c e r n e d w i t h the use o f an a m y l o l y t i c y e a s t , Endomycops is s p . , f o r s i m u l t a n e o u s p r o d u c t i o n o f y e a s t p r o t e i n and c rude amylase p r e p a r a t i o n s from n a t u r a l s t a r c h m a t e r i a l s . The Endomycops i s y e a s t s were c u l t i v a t e d a l o n e and i n c o m b i n a t i o n w i t h o t h e r y e a s t s w h i c h are unab l e to a t t a c k s t a r c h d i r e c t l y . The p ropaga - t i o n s were c a r r i e d out i n the p r e s e n c e o f u r e a and p h o s p h a t e , under a e r o b i c c o n d i t i o n s , w i t h v i g o r o u s a g i t a t i o n , a t pH 5.0 and 2 8 ° C . At d a i l y i n t e r v a l s , the c u l t u r e s were a n a l y z e d f o r p r o t e i n y i e l d , c e l l d e n s i t y , and amylase a c t i v i t y . The c e l l c rop h a r v e s t e d a f t e r p r o p a g a t i o n o f Endomycops i s y e a s t s on 6.0% p o t a t o media c o n t a i n e d 19% p r o - t e i n and the c u l t u r e f i l t r a t e o b t a i n e d a f t e r b iomass s e p a r a - t i o n had an a c t i v i t y o f 1.5 u n i t s . V a r i a t i o n s i n a c t i v i t y and p r o t e i n c o n t e n t were o b s e r v e d , depend ing on the s t a r c h s u b s t r a t e u s e d , the c o n c e n t r a t i o n o f u r e a added , and a p p a r e n t l y , the amount o f oxygen s u p p l i e d . M i x e d p r e p a r a t i o n s u s i n g C a n d i d a u t i l i s as a n c i l l a r y y e a s t , gave h i g h e r p r o t e i n y i e l d s and amylase a c t i v i t i e s compared to s i n g l e p r o p a g a t i o n s o f Endomycops i s s p . and mixed p r o p a g a t i o n s w i t h Saccharomyces c e r e v i s i a e . P u r p l e yam and c a s s a v a t u b e r s were examined f o r p r o t e i n e n r i c h m e n t and amylase p r o d u c t i o n . I t was o b s e r v e d i i . t h a t the p r o t e i n c o n t e n t o f the c e l l c rops o b t a i n e d from these s u b s t r a t e s c o u l d be i n c r e a s e d about t e n - f o l d b u t t h a t the amylase a c t i v i t i e s o f the c u l t u r e f i l t r a t e s were v e r y low . ACKNOWLEDGEMENTS Thz authoA. zxpftzau gAatvtudz to Vi. P.M. TowniZty, ixndoA uiho6<i t>ULpzh.\)-U>i.0Yi thu> ph.oje.cX wcu> undzmtalizn, ^oh. &wppoHX, kiA advtaz, hoZp^uJi &U£J~cJj>rrtf> and unficuZtng zntkuAtaAm. Appinciiatlon -ci aJU>o o.xtzndtd to Vn.. D.B. BtoLgg, Vh.. W.V. Vowniii, and VKo^Qjaboh, E.L. Wcutdon {oh. theJJi halp In the. pfiupcvtcution tka> tkteU>. i v . TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i i i TABLE OF CONTENTS i v L I S T OF FIGURES v L I S T OF TABLES v i i INTRODUCTION 1 LITERATURE REVIEW 4 MATERIALS AND METHODS 9 I . M i c r o o r g a n i s m s 9 I I . I n o c u l a 10 A. Shaken f l a s k p r o p a g a t i o n s 10 B. S i x l i t e r p r o p a g a t i o n s 10 I I I . M e d i a 11 A. Shaken f l a s k p r o p a g a t i o n s 11 B. S i x l i t e r p r o p a g a t i o n s 11 IV. P r o p a g a t i o n s 11 A. Shaken f l a s k p r o p a g a t i o n s 11 B. S i x l i t e r p r o p a g a t i o n s 12 V. A n a l y s e s 12 A. C e l l c o u n t 12 B. E n z y m a t i c a c t i v i t y 13 C. P r o t e i n 13 D. Y i e l d 13 E . S t a r c h 14 RESULTS 15 Optimum L e v e l s o f N i t r o g e n and P h o s p h o r u s 15 C o n c e n t r a t i o n o f P o t a t o S l u r r y 17 S i x - l i t e r P r o p a g a t i o n s 17 M i x e d P r o p a g a t i o n s 18 C o m p a r i s o n o f V a r i o u s S t a r c h y S u b s t r a t e s 30 DISCUSSION 39 CONCLUSIONS 4 8 BIBLIOGRAPHY 50 V . L I S T OF FIGURES F i g u r e Page 1 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on media c o n t a i n i n g 0.5% s t a r c h and 0.4% ( N H 4 ) 2 H P 0 4 - 16 2 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on media c o n t a i n i n g 0.5% s t a r c h med ia w i t h v a r y i n g l e v e l s o f u r e a . 19 3 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on media c o n t a i n i n g 2.0% p o t a t o s o l i d s , 1.0% u r e a , and o.5% K I ^ P O ^ (shaken f l a s k s ) . 20 4 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on media c o n t a i n i n g 4.0% p o t a t o s o l i d s , 1.0% u r e a , and 0.5% K I ^ P O ^ (shaken f l a s k s ) . 21 5 P r o g r e s s o f c u l t i v a t i o n Of Endomycops i s s p . on med ia c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a , and 0.5% K H 2 P 0 4 (Shaken f l a s k s ) . 22 6 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on media c o n t a i n i n g 10.0% p o t a t o s o l i d s , 1.0% u r e a and 0.5% K H 2 ? 0 4 ( shaken f l a s k s ) . 23 7 P r o g r e s s o f c u l t i v a t i o n o f Endomycops i s s p . on med ia c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a , and 0;5% K H 2 P 0 4 ( V i r . T i s f e r m e n t e r ) . 24 8 F r o g r e s s o f m i x e d c u l t i v a t i o n o f 2 Endomycops i s s p . : 1 C a n d i d a u t i l i s on media c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a , and 0.5% K H 2 P 0 4 (shaken f l a s k s ) . 26 9 P r o g r e s s o f m i x e d c u l t i v a t i o n o f 1 Endomycops i s s p . : 1 C a n d i d a u t i l i s on media c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a and 0.5% K H 2 P 0 4 (shaken f l a s k s ) . 27 10 P r o g r e s s o f m i x e d c u l t i v a t i o n o f 1 Endomycops i s s p . : 2 C a n d i d a u t i l i s on media c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a and 0.5% K H 2 P 0 4 ( shaken f l a s k s ) . 28 11 P r o g r e s s o f c u l t i v a t i o n o f 2 Endomycops i s s p . : 1 C a n d i d a u t i l i s i n media c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a , 0.5% K H 2 P 0 4 ( V i r T i s f e r m e n t e r ) . 2 9 . Progress of mixed c u l t i v a t i o n o f 2 Endomycopsis sp.: 1 Saccharomyces c e r e v i s i a e on media con- t a i n i n g 6.0% potato s o l i d s , 1.0%.urea, and 0.5% KH 2P0 4 (shaken f l a s k s ) . Progress of mixed c u l t i v a t i o n of 1 Endomycopsis sp.: 1 Saccharomyces c e r e v i s i a e on media con- t a i n i n g 6.0% potato s o l i d s , 1.0% urea, and 0.5% KH 2P0 4 (shaken f l a s k s ) . Progress of mixed c u l t i v a t i o n df 1 Endomycopsis sp,: 2 Saccharomyces c e r e v i s i a e on media con- t a i n i n g 6.0% potato s o l i d s , 1.0% urea, 0.5% KH2PC>4 (shaken f l a s k s ) . Progress of s e q u e n t i a l c u l t i v a t i o n of Endomycopsis sp.and Saccharomyces c e r e v i s i a e on media c o n t a i n i n g 6.0% potato s o l i d s , 1.0% urea, 0.5% KH2Pe>4 (shaken f l a s k s ) . Progress of s e q u e n t i a l c u l t i v a t i o n of Endomycopsis sp. and Saccharomyces c e r e v i s i a e on media c o n t a i n i n g 6.0% potato s o l i d s , 1.0% urea, 0.5% KH 2P0 4 . ( V i r T i s f e r m e n t e r ) . Progress o f mixed c u l t i v a t i o n of 2 Endomycopsis sp.: 1 Candida u t i l i s on media c o n t a i n i n g 6.0% pu r p l e yam, 1.0% urea, 0.5% KH 2P0 4 ( V i r T i s fermen t e r ) . Progress o f mixed c u l t i v a t i o n of 2 Endomycopsis sp.: 1 Candida u t i l i s on media c o n t a i n i n g 6.0% cassava, 1.0% urea, 0.5% KH2PC>4 ( V i r T i s f e rmenter). Diagram of the p r i n c i p l e u n d e r l y i n g the u t i l i z a t i o n o f s t a r c h y s u b s t r a t e s by yeast c u l t i v a t i o n L I S T OF TABLES T a b l e I C e l l d e n s i t y and c o n c e n t r a t i o n o f r e s i d u a l s t a r c h a f t e r f i v e d a y s c u l t i v a t i o n o f E n d o m y c o p s i s s p . on m e d i a c o n t a i n i n g 0.5% . s t a r c h , 1.0% u r e a , and v a r y i n g amounts o f K H 2 P 0 4 - I I A m y l o l y t i c a c t i v i t y , and p r o t e i n c o n t e n t a f t e r f i v e d a y s p r o p a g a t i o n , o f E n d o m y c o p s i s s p . on m e d i a c o n t a i n i n g 1.0% u r e a , 0.5% K H 2 P 0 4 and v a r y i n g amounts o f p o t a t o s o l i d s . I l l A m y l o l y t i c a c t i v i t y , and p r o t e i n c o n t e n t a f t e r f i v e d a y s p r o p a g a t i o n o f C a n d i d a u t i l i s w i t h E n d o m y c o p s i s sp.. on media c o n - t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a and 0.5% K H 2 P 0 4 . 1. INTRODUCTION Today , more than h a l f o f the w o r l d p o p u l a t i o n i s u n d e r n o u r i s h e d , o f t e n i n r e g i o n s where raw m a t e r i a l s are a v a i l a b l e f o r m i c r o b i a l u t i l i z a t i o n . The i m p o r t a n c e o f m i c r o o r g a n i s m s as p o t e n t i a l n u t r i t i o n a l s o u r c e s stems from the f i n d i n g t h a t m i c r o b i a l c e l l m a t t e r i s e s p e c i a l l y r i c h i n most B - v i t a m i n s and i n p r o t e i n s c o n t a i n i n g e s s e n t i a l amino a c i d s . T h e r e f o r e , m i c r o b i a l c e l l s c o n s t i t u t e p o t e n - t i a l e n r i c h m e n t f o r d e f i c i e n t d i e t s . I t i s a g r e e d , c u r r e n t l y , t h a t y e a s t s have the most f a v o r a b l e c h a r a c t e r i s t i c s f o r use as a major food s o u r c e . Not o n l y a re y e a s t s one o f the r i c h e s t s o u r c e s o f B - v i t a m i n s b u t a l s o , they c o n t a i n l a r g e p r o p o r t i o n s o f h i g h q u a l i t y p r o t e i n , c a r b o h y d r a t e , and l i p i d . Y e a s t ' s a b i l i t y to u t i l i z e cheap raw m a t e r i a l s and m a i n t a i n a r a p i d r a t e o f g r o w t h , p a l a t a b i l i t y and l a c k o f p a t h o g e n i c i t y are a d d i t i o n a l a d v a n t a g e s . A l l o f the e s s e n t i a l amino a c i d s e x c e p t m e t h i o n i n e are p r e s e n t i n adequate q u a n t i t i e s i n y e a s t p r o t e i n ( B h a t t a c h a r j e e , 1 9 7 0 ) . Vast q u a n t i t i e s o f cheap , f e r m e n t a b l e s u b s t r a t e s , w h i c h can r e a d i l y be c o n v e r t e d i n t o y e a s t p r o t e i n s and v i t a m i n s are a v a i l a b l e i n c o u n t r i e s w i t h an acu te food s h o r t a g e . The P h i l i p p i n e s i s one o f s e v e r a l c o u n t r i e s where s eve re p r o t e i n d e f i c i e n c y e x i s t s . In these c o u n t r i e s , low p r o d u c t i o n i s u n d e s i r a b l e , the number o f s k i l l e d a g r i c u l t u r a l w o r k e r s i s i n a d e q u a t e , a r a b l e l a n d i s s c a r c e and c a p i t a l 2. i n v e s t m e n t f o r i n t e n s i v e mar ine and a g r i c u l t u r a l p r o d u c t i o n i s u n a v a i l a b l e . Hence , y e a s t p r o t e i n c o n v e r s i o n from abundan t , cheap c a r b o h y d r a t e m a t e r i a l s r e q u i r e s i n v e s t i g a t i o n . S t a r c h y t u b e r s and r o o t c rops are the second most i m p o r t a n t s o u r c e s o f c a r b o h y d r a t e s i n the P h i l i p p i n e s . On the a v e r a g e , 120 grams o f t u b e r s and r o o t c rops are a v a i l a b l e per p e r s o n d a i l y , b u t only- 4 . 2 grams, are a c t u a l l y consumed. Of the t o t a l r o o t c rops a v a i l a b l e , sweet p o t a t o e s , c a s s a v a , yam and t a r o are most abundant ( M o l i n y a w e , 1 9 6 8 ) . In view o f the r e l a t i v e abundance o f s t a r c h y raw m a t e r i a l s i n the P h i l i p p i n e s and the s e v e r e d e f i c i e n c y o f p r o t e i n s and B - v i t a m i n s i n the P i l i p i n o d i e t , C P a s c u a l , 1964) j the B i o l o g i c a l Research . C e n t e r o f the P h i l i p p i n e N a t i o n a l I n s t i t u t e o f S c i e n c e and T e c h n o l o g y has d i r e c t e d much o f i t s e f f o r t s toward the use o f l o c a l m a t e r i a l s as s u b s t r a t e s f o r h i g h p r o t e i n y e a s t p r o d u c t i o n . Large s c a l e p r o p a g a t i o n s o f R h o d o t o r u l a p i l i m a n a e (Wickerham) have been c a r r i e d out on coconu t w a t e r g i v i n g a p r o d u c t c o n t a i n i n g about 50% p r o t e i n and a p p r e c i a b l e amounts o f B - V i t a m i n s . S t u d i e s conduc t ed by the f o r d and N u t r i t i o n Resea r ch C e n t e r o f the i n s t i t u t e have shown the p r o d u c t to be h i g h l y a c c e p t a b l e when used as. a p r o t e i n supplement i n the p r e p a r a t i o n o f c o o k i e s . How.ever, so f a r , none of the r o o t c r o p s have been u t i l i z e d (Baens^ ..Arc'ega, 19 6 8 ) . P a t e n t e d and w o r k a b l e p r o c e s s e s are known f o r the u t i l i z a t i o n o f s t a r c h y s u b s t r a t e s f o r y e a s t p r o d u c t i o n b u t 3. these are ba sed on the p r i n c i p l e o f a c i d h y d r o l y s i s p r i o r to y e a s t p r o p a g a t i o n s i n c e y e a s t s are g e n e r a l l y c o n s i d e r e d unab l e to a s s i m i l a t e s t a r c h ( J a r l , 1969 ; H a t t p r i , 1 9 6 1 ) . By u s i n g amy1 a s e - s y n t h e s i z i n g y e a s t s , w h i c h w i l l c o n v e r t s t a r c h w i t h o u t c o s t l y p r e l i m i n a r y h y d r o l y s i s , a p r o c e s s might be d e v i s e d f o r an e f f i c i e n t c o n v e r s i o n o f i n e x p e n s i v e raw m a t e r i a l s , s u r p l u s p r o d u c t i o n , and was te o r g a n i c m a t t e r i n t o e d i b l e p r o d u c t s . I t i s w e l l known t h a t some o f the was tes from pea p r o c e s s i n g , c o r n s t a r c h m a n u f a c t u r i n g , p o t a t o p r o c e s s i n g , e t c . c o n s i s t o f h i g h c o n c e n t r a t i o n s o f s t a r c h and t h e r e f o r e c o n s t i t u t e p o t e n t i a l s o u r c e s o f ca rbon and energy f o r growth and enzyme s y n t h e s i s o f amylase p r o d u c i n g y e a s t s . Waste d i s p o s a l might be made e c o n o m i c a l l y more a t t r a c t i v e by com-- b i n i n g i t w i t h a p r o c e s s f o r p r o d u c i n g a m y l p l y t i c enzymes. The s t a r c h d e g r a d a t i o n p r o d u c t s may f u r t h e r s e r v e as s u b s t r a t e s f o r the p r o d u c t i o n o f y e a s t b iomass t h a t may be used as food or f o d d e r . A l t e r n a t i v e l y , they may s e r v e as s u b s t r a t e s f o r the g rowth o f s e l e c t e d m i c r o o r g a n i s m s f o r the manufac tu re o f amino a c i d s , v i t a m i n s , f l a v o r e n h a n c e r s , and o t h e r compounds o f n u t r i t i o n a l or m e d i c a l v a l u e . T h i s s t u d y i s m a i n l y c o n c e r n e d w i t h the a p p l i c a t i o n of an amylase p r o d u c i n g y e a s t s p e c i e , Endomycops i s , f o r s i m u l t a n e o u s p r o d u c t i o n o f y e a s t b iomass and crude a m y l o l y t i c enzyme p r e p a r a t i o n s . The e x p e r i m e n t a l p l a n i n c l u d e s a s tudy o f the u t i l i z a t i o n o f p o t a t o was te and o f p r o t e i n e n r i c h m e n t o f r o o t c rops w i d e l y c u l t i v a t e d i n the P h i l i p p i n e s . 4 . LITERATURE RE VIEW Y e a s t s are g e n e r a l l y c o n s i d e r e d unab l e to u t i l i z e s t a r c h and few r e p o r t s are a v a i l a b l e r e g a r d i n g y e a s t fermen- t a t i o n o f s t a r c h y s u b s t r a t e s . A c c o r d i n g t o E b e r t o v a ( 1 9 6 6 ) , o n l y some r e p r e s e n t a t i v e s o f the genera Endomycops is and Endomyces and r a r e l y C a n d i d a ( E b e r t o v a , 1968) have a m y l o l y t i c p r o p e r t i e s . P i o n e e r i n g work on e x t r a c e l l u l a r 1 y p roduced Endomycops i s amylases was pe r fo rmed by Wicke rham, et a l . ( 1 9 4 4 ) A h i g h r a t i o o f OL- to B - a m y l a s e was . found i n c rude p r e p a r a - t i o n s o f the enzyme s y s t e m . The c o m p o s i t i o n and p r o p e r t i e s o f the Endomycops is a m y l o l y t i c complex were i n v e s t i g a t e d more t h o r o u g h l y by M a r r o q u i n and F i t c h ( 1 9 4 6 ) . M a r r o q u i n and S o l o r a n z o ( 1 9 4 7 ) , and M a r r o q u i n and Q a v a r r o n (1947, 1948 ) . M a r r o q u i n and F i t c h (1946) showed, as Wickerham d i d , t h a t the enzyme complex p roduced by Endomycops is f i b u l i g e r was p r i n c i p a l l y d e x t r i n i z i n g . W h i l e i n v e s t i g a t i n g the a m y l o l y t i c and f e r m e n t a t i v e a c t i v i t i e s o f mixed c u l t u r e s o f Endomycops i s f i b u l i g e r and Saccharomyces carb aj a l i under v a r y i n g c o n d i t i o n s o f p H , t e m p e r a t u r e , n a t u r e and c o n c e n t r a - t i o n o f s u b s t r a t e , and p r o p o r t i o n s o f i n o c u l a , M a r r o q u i n and S o l o r a n z o (1946) showed t h a t c e l l g rowth o c c u r r e d most r a p i d l y at 37°C and pH 5 . 0 . A m y l o l y t i c and f e r m e n t a t i v e a c t i v i t i e s were h i g h e s t i n 10% p r e m a l t e d wheat mash w i t h a 2% i n o c u l u m c o n s i s t i n g o f 5 parts Endomycops i s f i b u l i g e r to 5 . 3 p a r t s Saccharomyces c a r b a j a l i . The optimum t e m p e r a t u r e s f o r amylase a c t i v i t y was 65°C f o r a - , and 40°C f o r B- a m y l a s e . The optimum pH range was 5.5 - 6 .5 f o r a - , and 4 .5 - 6 .0 f o r ^ - a m y l a s e . S u b s e q u e n t l y , FUkumoto e t a l . (1960) and H a t t o r i and T a k e u c h i (1961) examined the p r o d u c t i o n o f Endomycops is and Endomyces a m y l o l y t i c enzyme sys tems on v a r i o u s s t a r c h media and the p r o p e r t i e s o f p u r i f i e d amylases o b t a i n e d . Ammonium s u l f a t e and r i v a n o l t r e a t e d p r e p a r a t i o n s were s t u d i e d w i t h r e s p e c t to pH and s t a b i l i t y to hea t and e t h y l e n e - d i a m i n e t e t r a a c e t a t e (EDTA) . The optimum ranges f o r a - and 3 - a m y l a s e were 5.5 - 6 .0 and 4 .5 - 5 .5 r e s p e c t i v e l y , i n c l o s e agreement w i t h the v a l u e s r e p o r t e d e a r l i e r by M a r r o q u i n and G a v a r r o n ( 1 9 4 6 ) . a - A m y l a s e was s t a b l e i n the pH range 5.0 - 7.5 and 3 - a m y l a s e i n the range 5.0 - 9 . 0 . I n s t a b i l i t y to hea t and EDTA was n o t e d . Sys t emmat i c s t u d i e s by E b e r t o v a (1966) on the p r o d u c t i o n o f amylase u s i n g d i f f e r e n t ca rbon s o u r c e s r e v e a l e d t h a t an enzyme e x h i b i t i n g m a l t a s e and t r a n s g l u c o s i d a s e a c t i v i t i e s was r e l e a s e d by Endomycops i s c a p s u l a r i s a t the b e g i n n i n g o f the l o g a r i t h m i c growth phase whereas s t a r c h h y d r o l y z i n g enzymes were r e l e a s e d at the end . G l u c o a m y l a s e was p r o d u c e d a t pH 4 . 0 and a - a m y l a s e a t pH 6 . 0 . Sadova et a l . (1969) found p e n t o s e s , p h o s p h o d e x t r i n s and s t a r c h to be the b e s t c a rbon s o u r c e s f o r the s y n t h e s i s o f a m y l o l y t i c enzymes by Endomycops is s p . 2 0 - 9 . Recent r e p o r t s on the p r o d u c t i o n , s e p a r a t i o n , and p r o p e r t i e s o f e x t r a c e l l u l a r Endomycops is amylases i n c l u d e those o f H o s t i n o v a and Z e l i n k a (1969 ) , Gracheva et a l . (19 69 ) and K o l t s o v a and Sadova ( 1 9 7 0 ) . H o s t i n o v a and Z e l i n k a (1969) o b s e r v e d maximum amylase s y n t h e s i s by Endomycops is f i b u l i g e r d u r i n g the l o g a r i t h m i c phase o f c u l t i v a t i o n . K o l t s o v a and Sadova (1970) p r e c i p i t a t e d 64% o f the t o t a l g l u c o a m y l a s e and 53.5% o f the t o t a l a -amylase at 60% s a t u r a t i o n w i t h ammonium s u l f a t e , whereas a l l o f the g l u c o a m y l a s e , 73% o f the a - a m y l a s e , and 28% o f the m a l t a s e were p r e c i p i t a t e d at 90% s a t u r a t i o n w i t h ammonium s u l f a t e . K u e h n e r ' s work (1953) i s b e l i e v e d to be the f i r s t a t t empt at u s i n g a m y l o l y t i c y e a s t s d e l i b e r a t e l y f o r the p r o d u c t i o n o f a n u t r i t i o n a l m a t e r i a l . Using a mixed c u l t u r e c o n s i s t i n g o f Endomycops is f i b u l i g e r or Endomycops i s c h o d a t i and C a n d i d a u t i 1 i s grown on cooked whea t , c o r n , r i c e , o r p o t a t o e s , a 60% y i e l d was o b t a i n e d c o n t a i n i n g 20 - 30% p r o t e i n , i ndependen t o f the r a t i o o f o rgan i sms u s e d . A 22" X 4" p r o p a g a t o r equ ipped w i t h b a f f l e s , a r i n g s p a r g e r and p r o p e l l e r b l a d e s was o p e r a t e d under the f o l l o w i n g con- d i t i o n s : 650 RPiM a g i t a t i o n r a t e , 2 volumes o f a i r pe r volume o f mash pe r m i n u t e , 28°C and pH 5 - 6. As a r e s u l t o f K u e h n e r ' s s t u d i e s , Wickerham and Kuehner (1956) p r o p o s e d a p r o c e s s f o r y e a s t p r o t e i n p r o d u c - t i o n u s i n g Endomycop s i s f i b u l i g er and Endomycop s i s c h o d a t i b u t the p r o c e s s has r emained u n d e v e l o p e d to d a t e . Y e a s t a m y l o l y t i c p r o p e r t i e s have a l s o been a p p l i e d i n a Swedish p r o c e s s by T v e i t (1967) and J a r l (1969 ) . D e s i g n e d to reduce the b i o l o g i c a l oxygen demand o f s t a r c h y e f f l u e n t s , Endomycops is f i b u l i g e r and C a n d i d a u t i l i s were grown s y m b i o t i c a l l y on s t a r c h media at pH 5.0 and 2 8 ° C . The o n l y a d d i t i o n s made were phospha te and u r e a . Most o f the s t u d i e s done by T v e i t and J a r l were c a r r i e d out i n 100 - 1000 l i t e r f e r m e n t e r s equ ipped w i t h a s i n g l e o r i f i c e s p a r g e r , i m p e l l e r , and b a f f l e s . The h a r v e s t e d p r o d u c t s were s c r e e n e d and c o n c e n t r a t e d . S u b s e q u e n t l y , the s e p a r a t e d b iomass was d r i e d f o r use as a n i m a l feed s u p p l e m e n t . At about the same t i m e , i n C z e c h o s l o v a k i a , P o l i v k a and Z e l i n k a (1969) p roduced y e a s t p r o t e i n on a much s m a l l e r s c a l e . U s i n g c o r n s t a r c h , c o r n s t eep l i q u o r and ammonium s u l f a t e , P o l i v k a and Z e l i n k a (1969) o b t a i n e d Endomycops i s f i b u l i g e r b iomass c o n t a i n i n g more than 40% p r o t e i n s . Sub- s e q u e n t l y , they e x p l o r e d the p o s s i b i l i t i e s o f u s i n g was te p r o d u c t s o f s t a r c h p l a n t s such as p o t a t o s t a r c h e f f l u e n t s . There appears to be no p r o c e s s d e s i g n e d to p roduce y e a s t p r o t e i n from s t a r c h y m a t e r i a l s w h i l e p r o v i d i n g the a m y l o l y t i c enzyme complex as a b y - p r o d u c t . However , a - amylase has been p r e p a r e d from Endomycops is f i b u l i g e r ( D a v i e s , 1 9 6 3 ) . A m y l o l y t i c enzyme p r o d u c t s have been o b t a i n e d by 8. c u l t i v a t i o n o f Endomycops is f i b u l i g e r , Endomycops i s l i n d n e r i , Endomycops is j a v a e n s i s and Endomycops is h o r d e i i n n u t r i e n t media c o n t a i n i n g 0.1 - 15.0% by w e i g h t o f a t l e a s t one h i g h e r f a t t y a c i d , h i g h e r f a t t y a c i d e s t e r o r n a t u r a l f a t o r o i l CMatsutaru. Chem. Co . L t d . , 1 9 6 5 ) . R u t l o f f (1968) has d e s c r i b e d a p r o c e s s o f p r e p a r i n g g l u c o a m y l a s e from Endomycops is y e a s t s . 9 MATERIALS AND METHODS I . M i c r o o r g a n i s m s The a m y l o l y t i c yeas.t used i n t h i s s t u d y was i s o l a t e d by Browne , (1973) from L a o - c h a o , a C h i n e s e fe rmented r i c e p r o d u c t . Lao-chao was made by m i x i n g g l u t i n o u s r i c e and a l o c a l l y av .a i l ah . le c o m m e r c i a l s t a r t e r c a l l e d chiu--yueh or p e h - y u e h . Wang and H e s s e l t i n e (1970) showed t h a t Lao-chao c u l t u r e c o n t a i n e d Mucorangeous f u n g i and a y e a s t s p e c i e Endomycops i s . The Endomycopsis y e a s t s were m a i n t a i n e d on s l a n t s c o n s i s t i n g o f 0.2% s o l u b l e s t a r c h ' , 0.5% p e p t o n e , 0.3% y e a s t e x t r a c t and 1.5% a g a r . S t e r i l i z a t i o n was e f f e c t e d by a u t o - c l a v i n g capped tubes f o r 15 minu te s at 15 l b s p r e s s u r e . A f t e r i n o c u l a t i o n , s l a n t s were i n c u b a t e d at 37°C f o r 48 hours and s t o r e d at 4 ° C . The n o n - a m y l o l y t i c y e a s t s used were C a n d i d a u t i l i s and Saccharomyces c e r e v i s i a e . Pure c u l t u r e s were o b t a i n e d from the U n i v e r s i t y o f B r i t i s h C o l u m b i a , Depar tment o f M i c r o b i o l o g y . The C a n d i d a u t i l i s c u l t u r e o r i g i n a t e d from t h e A m e r i c a n Type C u l t u r e C o l l e c t i o n (ATCC 9256) and the Saccharomyces c e r e v i s i a e from the U n i v e r s i t y o f C a l i f o r n i a ( B e r k e l e y ) . The o rgan i sms were m a i n t a i n e d on s l a n t s c o n s i s - t i n g o f 0.3% y e a s t e x t r a c t , 0.5% peptone and 1.5% a g a r . I n o c u l a t e d s l a n t s were i n c u b a t e d at 30°C f o r 48 hours and s t o r e d at 4 ° C . 10. I I . I n o c u l a A . Shaken f l a s k p r o p a g a t i o n From the s l a n t s , 100 ml o f s t e r i l e media c o n t a i n i n g 0.2% s o l u b l e s t a r c h , 0.3% y e a s t e x t r a c t , and 0.5% peptone i n 250 ml E r l e n m e y e r f l a s k s were i n o c u l a t e d w i t h Endomycops i s s p . u s i n g s t a n d a r d t e c h n i q u e s i n an E n v i r a z o n e module i n o c u l a t i n g h o o d . The c u l t u r e s were i n c u b a t e d f o r 36 hours on a New B r u n s w i c k g y r a t o r y s h a k e r o p e r a t i n g at a speed o f 196 RPM i n an i n c u b a t o r m a i n t a i n e d at 28°C and 85% r e l a t i v e h u m i d i t y . Ten m i l l i l i t e r s o f t hese c u l t u r e s , a s e p t i c a l l y p i p e t t e d , were used to i n o c u l a t e the shaken f l a s k p r o p a g a t o r s . T y p i c a l l y , such c u l t u r e s c o n t a i n e d about 10 c e l l s / m l . For mixed f e r m e n t a t i o n , 100 ml o f s t e r i l e media con - t a i n i n g 0.3% y e a s t e x t r a c t and 0.5% pep tone i n 250 ml E r l e n m e y e r f l a s k s were p r e p a r e d . A s e t o f media was i n o c u l a t e d w i t h Endomycops i s sp.. and a n o t h e r s e t w i t h C a n d i d a u t i 1 i s or Saccharomyces c e r e v i s i a e . The c u l t u r e s were i n c u b a t e d as d e s c r i b e d p r e v i o u s l y . A f t e r 36 h o u r s , s e p a r a t e coun t s were made o f the a m y l o l y t i c and n o n - a m y l o l y t i c y e a s t s . A p p r o p r i a t e amounts o f each were mixed and used to i n o c u l a t e the shaken f l a s k p r o p a g a t o r s . B . S i x l i t e r p r o p a g a t i o n s The e n t i r e c o n t e n t s o f t h r e e f l a s k s c o n t a i n i n g 100 ml each o f the 3 6 - h o u r y e a s t c u l t u r e s were a s e p t i c a l l y t r a n s f e r r e d i n t o a V i r T i s f e r m e n t e r assembly c o n t a i n i n g s i x l i t e r s o f s t e r i l e m e d i a . 1 1 . I I I . M e d i a A . Shaken f l a s k p r o p a g a t i o n s Washed, u n p e e l e d p o t a t o e s were we ighed and homogenized i n a War ing b l e n d e r w i t h some d i s t i l l e d w a t e r . The volume o f the r e s u l t i n g s l u r r y needed to p r e p a r e a medium c o n t a i n i n g the d e s i r e d c o n c e n t r a t i o n o f p o t a t o s o l i d s was a s c e r t a i n e d by d e t e r m i n i n g the s o l i d s c o n t e n t o f the s l u r r y . The s o l i d s c o n t e n t o f the p r e p a r e d p o t a t o s l u r r y was measured u s i n g an Ohaus m o i s t u r e d e t e r m i n a t i o n b a l a n c e . F i v e - g r a m samples were d r i e d under an i n f r a r e d lamp a t 60 w a t t s f o r 25 m i n u t e s . M e d i a so p r e p a r e d were d i s p e n s e d i n 100 ml p o r t i o n s i n t o 250 ml f l a s k s , c o t t o n p l u g g e d , and a u t o c l a v e d f o r 15 minu te s a t 15 l b s p r e s s u r e . A f t e r o v e r n i g h t r e f r i g e r a t i o n at 4 ° C , and b e f o r e i n o c u l a t i o n , pH was a d j u s t e d from 7.5 to 5 .0 u s i n g a u t o c l a v e d IN H C l . B . S i x l i t e r p r o p a g a t i o n s S i x l i t e r s o f media s i m i l a r l y p r e p a r e d were poured i n t o a 12 l i t e r V i r T i s f e r m e n t e r f l a s k w i t h a l i d , a l l p o r t s o f w h i c h were c o t t o n p l u g g e d . S t e r i l i z a t i o n was e f f e c t e d by a u t o c l a v i n g f o r 45 minu tes at 15 l b s p r e s s u r e . A f t e r c o o l i n g o v e r n i g h t i n the f e r m e n t e r , t h e r m o s t a t i c a l l y c o n t r o l l e d a t 1 0 ° C , pH was a d j u s t e d u s i n g a u t o c l a v e d 3N H C l . I V. P r o p a g a t i o n s A . Shaken f l a s k p r o p a g a t i o n s C o t t o n - p l u g g e d 250 ml E r l e n m e y e r f l a s k s c o n t a i n i n g 12 . 100 ml o f c u l t u r e were p l a c e d on a New B r u n s w i c k g y r a t o r y shake r o p e r a t i n g a t a speed o f 196 RPM i n an i n c u b a t o r kep t a t 28°C and 85% r e l a t i v e h u m i d i t y . P e r i o d i c a l l y , samples were a s e p t i c a l l y w i t h d r a w n f o r a n a l y s e s . B. S i x l i t e r p r o p a g a t i o n s A t h r e e - s t a t i o n Vir Tis f e r m e n t e r w i t h 1 2 - l i t e r f e r m e n t e r f l a s k s was used i n t h i s s t u d y . The f e r m e n t e r was equ ipped w i t h s p a r g e r s , a d j u s t a b l e b a f f l e s , m a g n e t i c i m p e l l e r s as w e l l as foam, p H , and t empe ra tu r e c o n t r o l s . M e d i a were a u t o c l a y e d f o r 45 minu te s a t 15 l b s p r e s s u r e i n a 12 l i t e r f e r m e n t e r f l a s k . A f t e r c o o l i n g , i t s l i d was r e p l a c e d w i t h an e t h y l e n e o x i d e g a s - s t e r i 1 i z e d f e r m e n t e r head w i t h d i s s o l v e d o x y g e n , foam, and pH p r o b e s . A 12-hour exposure to e t h y l e n e o x i d e gas ( c r y o x i d e ) was used to ensure s t e r i l i z a t i o n o f the f e r m e n t e r heads and p r o b e s . C u l t i v a t i o n s were c a r r i e d out a t 2 8 ° C . F ive to n i n e p a r t s pe r m i l l i o n o f d i s s o l v e d oxygen was m a i n t a i n e d i n the medium w h i c h was a g i t a t e d at the h i g h e s t r a t e c o m p a t i b l e w i t h foam c o n t r o l . G e n e r a l l y , the magne t i c i m p e l l e r s were o p e r a t e d at 650 - 750 RPM u n l e s s foaming was e x t e n s i v e . V. A n a l y s e s R o u t i n e a n a l y s e s i n c l u d e d : A. C e l l count C e l l s were coun ted w i t h the a i d b f a Haemacytometer w i t h Neubauer r u l i n g , a c c o r d i n g to the method d e v i s e d by the 13 A m e r i c a n S o c i e t y o f Brewing Chemis t s ( 3 4 ) . B . E n z y m a t i c a c t i v i t y Amylase a c t i v i t y was measured f o l l o w i n g the method used by Gracheva e t a l . (1969) on Endomycops is s p . amylase and by Fukumoto (1955) and H a t t o r i (1960) on Endomyces a m y l a s e s . The p r o c e d u r e c o n s i s t e d o f a 30 -minu te h y d r o l y s i s o f a 2.0% s o l u b l e s t a r c h ( L i n t n e r ' s ) s o l u t i o n b u f f e r e d at pH 5 . 6 . Reduc ing suga r s were s p e c t r o p h o t o m e t r y a l l y d e t e r m i n e d b e f o r e and a f t e r h y d r o l y s i s a t 45°C by the N e l s o n - S o m o g y i method (1945) e m p l o y i n g a l o w - a l k a l i n i t y copper r e agen t and a r s e n o m o l y b d a t e . Amylase a c t i v i t y was e x p r e s s e d as grams o f g l u c o s e r e l e a s e d i n 100 ml o f c u l t u r e i n one h o u r . C. P r o t e i n P r o t e i n s were e x t r a c t e d from c e n t r i f u g e d c e l l s and r e s i d u a l p o t a t o s o l i d s by s o a k i n g i n 2N NaOH as d e s c r i b e d by M i t s u d a (19 7 0 ) . A f t e r 100 h o u r s , the s p e c t r o p h o t o m e t r i c method o f Lowry (1951) was used f o r q u a n t i t a t i v e p r o t e i n a n a l y s i s . D. Y i e l d Y i e l d s were measured g r a v i m e t r i c a l l y u s i n g the method recommended by the I n s t i t u t e o f Brewing (20) f o r a n a l y s i s o f y e a s t c o n c e n t r a t i o n . Y i e l d s were e x p r e s s e d as grams o f d ry c e n t r i f u g e d c e l l s o b t a i n e d from 100 ml o f c u l t u r e . 14 E. S t a r c h R e s i d u a l s t a r c h i n the, c u l t u r e f l u i d was measured f o l l o w i n g the p r o c e d u r e ' o u t l i n e d by G i l b e r t and Spragg (1968) a f t e r p e r c h l o r i c a c i d t r e a t m e n t as d e s c r i b e d by McReady et a l . (19 50) . 15 . RESULTS Optimum L e v e l s o f N i t r o g e n and Phosphorus A p r e l i m i n a r y s t u d y was c o n d u c t e d i n o r d e r to * e s t a b l i s h the l e v e l s o f a s s i m i l a b l e n i t r o g e n and phospha te most s u i t a b l e f o r growth and a m y l o l y t i c enzyme s y n t h e s i s by the y e a s t Endomycops is s p . In media c o n t a i n i n g 0.5% s o l u b l e s t a r c h and 0.4% ammonium hydrogen p h o s p h a t e , i t was o b s e r v e d t h a t o v e r a p e r i o d o f f o u r d a y s , the pH o f the c u l t u r e dropped g r a d u a l l y from 5.0 to 3 . 4 . F i g u r e 1 shows the t ime c o u r s e o f the p r o p a g a t i o n . I t i s known t h a t the amylases are i n a c t i v a t e d at pH v a l u e s l e s s than 4 . 0 ( T s u c h i y a e t a l . , 1950; M a r r o q u i n , 1947) and t h e r e i s e v i d e n c e i n the l i t e r a - t u r e i n d i c a t i n g b e t t e r pH c o n t r o l and h i g h e r p r o t e i n accumu- l a t i o n by u s i n g u r e a i n s t e a d o f ammonium s a l t s i n p r o p a g a t i o n s o f t h i s type (Keuhne r , 1 9 5 3 ) . T h e r e f o r e , i t was d e c i d e d t h a t u r e a w o u l d be used w i t h a phospha te s o u r c e , i e i . K H ^ P O ^ i n subsequen t e x p e r i m e n t s . . U n l i k e u r e a , ammonium s a l t s gave r e s i d u a l a c i d as the n i t r o g e n - c o n t a i n i n g p o r t i o n s o f the m o l e c u l e s were u t i l i z e d . V a r i o u s amounts o f KH^PO^ were t r i e d and i t was i n f e r r e d from the r e s u l t s o f t he se e x p e r i m e n t s , shown i n T a b l e I , t h a t the phospha te l e v e l d i d not e x e r t a s i g n i f i c a n t e f f e c t on e i t h e r g rowth r a t e o r s t a r c h h y d r o l y . s i s . S e v e r a l p r o p a g a t i o n s i n w h i c h the amount o f u r e a used was v a r i e d showed a r e m a r k a b l e dependence o f r a t e o f y e a s t STARCH CONCENTRATION (mg/ml) 17 . TABLE I . C e l l d e n s i t y and c o n c e n t r a t i o n o f r e s i d u a l s t a r c h a f t e r f i v e days c u l t i v a t i o n o f Endomycops is sp . on media c o n t a i n i n g 0.5% s t a r c h , 1.0% u r e a , and v a r y i n g amounts o f K l U P O , . K H 2 P 0 4 c o n c e n t r a t i o n Y e a s t p o p u l a t i o n S t a r c h c o n c e n t r a t i o n (%) (X 108 c e l l s / m l ) (mg/ml) 0.1 1.20 0 . 19 0 .2 1.12 0 .17 0 . 5 1 . 20 0 . 20 1.0 1.15 0 .18 grow th and e x t e n t g f s t a r c h d i s a p p e a r a n c e on u r e a c o n c e n t r a - t i o n . The r e s u l t s , which , a re p r e s e n t e d i n f i g u r e 2 , i n d i c a t e t h a t 1.0% i s opt imum. L e v e l s o f 2 . 0 , 3 .0 and 4.0% u r e a were i n h i b i t o r y . C o n c e n t r a t i o n o f P o t a t o S l u r r y Growth r a t e , a m y l o l y t i c a c t i v i t y , and p r o t e i n con- t e n t were s u b s e q u e n t l y e v a l u a t e d w h i l e v a r y i n g the amount o f p o t a t o s o l i d s s u p p l i e d i n the media from 2.0% to 10.0%. In acco rdance w i t h the r e s u l t s o f the p r e l i m i n a r y e x p e r i m e n t s , 1.0% u r e a was added w i t h 0.5% K l ^ P O ^ . P igures 3-6 show the t ime cou r se o f the p r o p a g a t i o n s i n 2 . 0 , 4 . 0 , 6 . 0 , and 10.0% p o t a t o s o l i d s . A summary o f the d a t a i s p r e s e n t e d i n T a b l e I I . S i x - l i t e r P r o p a g a t i o n s The 100 ml shaken f l a s k e x p e r i m e n t s were then 1 8 . s c a l e d up to s i x l i t e r s u s i n g media c o m p o s i t i o n o b s e r v e d to be o p t i m a l f o r p r o t e i n a c c u m u l a t i o n and enzyme a c t i v i t y . The t ime cou r se o f p r o p a g a t i o n i n s i x l i t e r s o f medium c o n - t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a , and 0.5% KH^PO^ i s shown i n F igure 1. Wi th oxygen t e n s i o n m a i n t a i n e d at l e v e l s much h i g h e r than i n the shaken f l a s k s ; a s i g n i f i c a n t r i s e i n p r o t e i n v a l u e s was o b s e r v e d . M i x e d P r o p a g a t i o n s Two n o n - p r o t e o l y t i c , n o n - a m y l o l y t i c y e a s t s , C a n d i d a u t i 1 i s and Saccharomyces c e r e v i s i a e ( A h e a r n , 1968 ; S y l v e n , 1958) were grown s y m b i o t i c a l l y w i t h Endomycops i s s p . C o m p a r i - sons were made of y e a s t g r o w t h s , amylase a c t i v i t y i n the c u l t u r e f l u i d s , and p r o t e i n c o n c e n t r a t i o n i n the c e l l c rops i n Endomycops i s - Cand i da and Endomycops is - Saccharomyces c u l t u r e s . The r e s u l t s o f e x p e r i m e n t s w i t h v a r y i n g C a n d i d a : Endomycops is r a t i o s ( 1 : 2 , 1 :1 , 2 :1) gave no i n d i c a t i o n t h a t a m y l o l y t i c a c t i v i t y , g rowth r a t e , and p r o t e i n a c c u m u l a t i o n c o u l d be c o r r e l a t e d w i t h the p r o p o r t i o n o f the two y e a s t s i n the i n o c u l u m . F igures 8-10 and Tab l e I I I summarize these ob s e r v a t i o n s . As e x p e c t e d , the l a g phase of y e a s t growth was s h o r t e n e d c o n s i d e r a b l y , the amylase a c t i v i t y i n c r e a s e d s i g n i f i c a n t l y , and the p r o t e i n l e v e l r o se s h a r p l y when the p r o p a g a t i o n was c a r r i e d out under s u f f i c i e n t a e r a t i o n and v i g o r o u s a g i t a t i o n STARCH CONCENTRATION (mg/ml). 20. O Population n A c t i v i t y A P ro te in i l 1 , , , :—i 1 1 0 20 60 100 140 180 TIME (hours) FIGURE 3. Progress of c u l t i v a t i o n of Endomycopsis sp. on media containing 2.0% potato s o l i d s , 1.0% urea, and 0.5% KH„PO. (shaken f l a s k s ) . 21 0 Population • Activity (soluble starch inoculum) A Protein 0 20 60 100 140 180 TIME (hours) FIGURE 4. Progress of cultivation of Endomycopsis sp. on media containing 4.0% potato solids, 1.0% urea, and 0.5% KH-PO. (shaken flasks). 22. O 20 60 100 140 180 TIME (hours) FIGURE 5. Progress of cultivation of Endomycopsis sp. on media containing 6.0% potato solids, 1.0% urea, and 0.5% KI^PG^ (shaken flasks). 23. 0 Population o A c t i v i t y A Protein 0 20 60 100 140 180 TIME (hours) FIGURE 6. Progress of cul t ivat ion of Endcflycopsis sp. on media containing 10.0% potato sol ids , 1.0% urea and 0.5% KH PO. (shaken f lasks) . 24. 0 Population D A c t i v i t y A Protein 0 20 60 100 . 140 180 TIME (hours) FIGURE 7. Progress of c u l t i v a t i o n of Endomycopsis sp. on media containing 6.0% potato s o l i d s , 1.0% urea, and 0.5% KH„PO. (Vir Tis fermenter). 25 . TABLE I I . A m y l o l y t i c a c t i v i t y , and p r o t e i n c o n t e n t a f t e r < f i v e days p r o p a g a t i o n , . o f Endomycops is s p . on media c o n t a i n i n g 1.0% u r e a , 0.5% K H 2 P 0 4 and v a r y i n g amounts o f p o t a t o s o l i d s . P o t a t o S o l i d s (%) Amylase A c t i v i t y (gm g l u c o s e / 1 0 0 m l / h r ) P r o t e i n i n S o l i d s (%) 2 . 0 0 . 1 7 5 2 .0 4 .0 . 0 . 2 5 1 2 .6 6 . 0 0.29 2 2 . 0 10 .0 0 .273 2 .0 i n a 1 2 - l i t e r f e r m e n t e r . The t ime c o u r s e o f p r o p a g a t i o n o f Endomycops i s s p . w i t h C a n d i d a u t i 1 i s ( 2 : 1 ) i n 6.0% p o t a t o s o l i d s , 1.0% u r e a , and 0.5% KH^PO^ i s p r e s e n t e d i n F i g u r e 1 1 . Using v a r i o u s r a t i o s o f Saccharomyces c e r e v i s i a e to Endomycops i s s p . ( 1 : 2 , 1 : 1 , 2 : 1 ) , as i n o c u l a , g rowth r a t e s and p r o t e i n l e v e l s o b t a i n e d d i d not d i f f e r s i g n i f i c a n t l y from those o b s e r v e d i n c u l t u r e s o f C a n d i d a u t i l i s w i t h Endomycops i s s p . However , amylase a c t i v i t i e s were c o n s i d e r - a b l y l o w e r . F igu re s 12-14 show the r e s u l t s o f t he se e x p e r i - ments . These o b s e r v a t i o n s are d i f f i c u l t to i n t e r p r e t i n view o f r e p o r t s i n the l i t e r a t u r e i n d i c a t i n g the absence o f a p p r e c i a b l e p r o t e o l y t i c a c t i v i t y i n Saccharomyces c e r e v i s i a e c u l t u r e s , ( A h e a r n , 1968; S y l v e n , 1 9 5 8 ) . A f u r t h e r a t t empt was made to a s se s s the p o s s i b i l i t y o f o b t a i n i n g a p r o t e i n - r i c h p r o d u c t w i t h an e n z y m a t i c a l l y - a c t i v e l i q u o r from s e q u e n t i a l p r o p a g a t i o n s o f Endomycops i s s p . 26. 0 Population 0 20 60 100 140 180 TIME (hours) FIGURE 8. Progress of mixed cul t ivat ion of 2 Endomycopsis s p . : 1 Candida u t i l i s on media containing 6.0% potato sol ids , 1.0% urea, and 0.5% KH2pC»4 (shaken f l a s k s ) . 27. O Population 0 20 60 100 140 . 180 TIME (hours) FIGURE 9. Progress of mixed cul t ivat ion of 1 Endomycopsis s p . : 1 Candida u t i l i s on media containing 6.0% potato sol ids , 1.0% urea and 0.5% KH 2P0 4 (shaken f lasks ) . 28. O Population n Activity A Protein 0 20 60 100 140 180 TIME (hours) FIGURE 10. Progress of mixed cultivation of 1 Endomycopsis sp.: 2 Candida u t i l i s on media containing 6.0% potato solids, 1.0% urea and 0.5% KĤ PÔ (shaken flasks). O 20 60 100 140 180 TIME (hours) FIGURE 11, Progress of cultivation of 2 Endomycopsis sp.: 1 Candida u t i l i s i n media containing 6.0% potato solids, 1.0% urea, 0.5% KH2PC>4 (Vir Tis ferjinenters). 30 TABLE I I I . A m y l o l y t i c a c t i v i t y and p r o t e i n c o n t e n t a f t e r f i v e days p r o p a g a t i o n o f C a n d i d a u t i - l i s w i t h Endomycops i s s p . on media c o n t a i n i n g 6.0% p o t a t o s o l i d s , 1.0% u r e a and 0.5% K H 2 P 0 4 . C a n d i d a : E n d o m y c o p s i s Amylase A c t i v i t y P r o t e i n i n S o l i d s R a t i o (gm g . lucose /100 m l / h r ) (%) 1:2 0 .560 17 .0 1:1 0 .394 17.8 2:1 0 .278 16 .6 and Saccharomyces c e r e v i s i a e . T h i s p o s s i b i l i t y was demon- s t r a t e d by i n t r o d u c i n g the a n c i l l a r y y e a s t a f t e r the Endomycops i s amylases had b u i l t u p . A l t h o u g h no a t t empt was made to a s c e r t a i n the b e s t t ime f o r Saccharomyces c e r e v i s i a e i n o c u l a t i o n , the r e s u l t s shown i n F i g u r e 15 i n d i c a t e d t h a t the i n t r o d u c t i o n o f the a n c i l l a r y y e a s t a t t h e - p o i n t o f maximum Endomycops i s p o p u l a t i o n , was accompanied by f u r t h e r i n c r e a s e i n y i e l d , amylase a c t i v i t y and p r o t e i n c o n t e n t . However , a d i f f e r e n t p a t t e r n was o b s e r v e d when a s i x l i t e r p r o p a g a t i o n was c a r r i e d o u t . The i n t r o d u c t i o n o f Saccharmyces c e r e v i s i a e r educed the amylase a c t i v i t y c o n s i d e r a b l y a l t h o u g h i t i n c r e a s e d the c e l l d e n s i t y and the p r o t e i n c o n t e n t s l i g h t l y . Compar i son o f V a r i o u s S t a r c h y S u b s t r a t e s On the b a s i s o f y e a s t g r o w t h , amylase a c t i v i t y and p r o t e i n y i e l d , an a t t empt was made to compare w i t h p o t a t o , 3 1 . two s t a r c h y s u b s t r a t e s , grown w i d e l y i n the P h i l i p p i n e s , namely p u r p l e yam and c a s s a v a tubers . . The p r o g r e s s o f mixed c u l t i v a t i o n o f tw 6 ' p a r t s E n d o niyc 6 p" s j s to one p a r t C a n d i d a u t i 1 i s i n a medium c o n s i s t i n g o f 6.0% p u r p l e yam, 1.0% u r e a , and 0.5% KH2PO4 i s g i v e n i n F i g u r e 17 . Whereas maximum c e l l d e n s i t y was r eached i n l e s s than two days i n a medium con- t a i n i n g 6.0% p o t a t o s o l i d s , i t took about t h r e e days i n medium c o n t a i n i n g an e q u i v a l e n t c o n c e n t r a t i o n o f the yam. The yam medium a l s o gave lower p r o t e i n y i e l d and amylase a c t i v i t y . I t can be seen from F igure 18 t h a t p r o t e i n accumu- l a t i o n i n mixed c u l t u r e o f Endomycops is s p . and C a n d i d a u t i l i s ( 2 :1 ) on 6.0% c a s s a v a medium was comparab le to t h a t i n 6.0% p u r p l e yam. However , amylase a c t i v i t y i n the c a s s a v a medium was e x t r e m e l y l o w . 32. 0 Population 0 A c t i v i t y _ i , , , 1 , — 0 20 60 100 140 180 TIME (hours) FIGURE 12, Progress of mixed cul t ivat ion of 2 Endomycopsis s p . : 1 Saccharomyces cerevisiae on media con- taining 6.0% potato sol ids , 1.0% urea, and 0.5% KH„PO. (shaken f lasks) . 33. 0 Population n Activity i J 2^ ^ ^ ' TIME (hours) FIGURE 13. Progress of mixed cultivation of 1 Endomycopsis sp.: 1 Saccharomyces cerevisiae on media con- taining 6.0% potato solids, 1.0% urea, and 0.5% KHoP0„ (shaken flasks). 3 4 . O Population D Activity 1 t 0 20 60 100 140 180 TIME (hours) FIGURE 14. Progress of mixed cultivation of 1 Endomycopsis sp.: 2 Saccharomyces cerevisiae on media con- taining 6.0% potato solids, 1.0% urea, 0.5%. KĤ PO,, (shaken flasks). 35. TIME (hours) FIGURE 15. Progress of sequential cultivation of Endcmycopsis sp. and Saccharomyces cerevisiae on media contain- ing 6.0% potato solids, 1.0% urea, 0.5% KH2P04 (shaken fl a s k s ) . 36. 0 Population D A c t i v i t y TIME (hours) FIGURE 16. Progress of sequential c u l t i v a t i o n of Endomycopsis sp.and Saccharomyces c e r e v i s i a e on media contain- ing 6.0% potato s o l i d s , 1.0% urea, 0.5% KH2pC>4 (Vir T i s fermenter). 37. 0 Population • Activity A Protein TIME (hours) FIGURE 17. Progress of mixed cultivation of 2 Endomycopsis sp.: 1 Candida u t i l i s on media containing 6.0% purple yam, 1.0% urea, 0.5% KH2PC>4 (Vir Tis fermenter). 38. H 0,20 f o o CD w o J ] Cn h 0.10 a! 1.2 l . o4 0 20 O P o p u l a t i o n o A c t i v i t y A P r o t e i n 60 100 140 TIME (hours) 180 f 20.0 16.0 12.0 8.0 4.0 FIGURE 18, P r o g r e s s o f rnixed c u l t i v a t i o n o f 2 Endomycopsis ' \sp_.: 1 C a n d i d a u t i l i s o n medi a c o n t a i n i n g 6.0% c a s s a v a , 1.0% u r e a , 0.5% K H 2 P 0 4 ( V i r T i s f e r m e n t e r ) o\o 39 DISCUSSION The p r i n c i p l e u n d e r l y i n g the u t i l i z a t i o n o f s t a r c h y s u b s t r a t e s i s shown d i a g r a m m a t i c a l l y i n F i g u r e 19 . S t a r c h s e r v e s as the ca rbon and energy s o u r c e f o r the g rowth o f two o r g a n i s m s . By means o f the a m y l o l y t i c a c t i v i t y o f the Endomycops is y e a s t , s t a r c h i s degraded i n t o l o w e r s a c c h a r i d e s , p r e d o m i n a n t l y g l u c o s e , w h i c h i n t u r n are a s s i m i l a t e d by an a n c i l l a r y o r g a n i s m . Though unablle to u t i l i z e s t a r c h d i r e c t l y , the a n c i l l a r y o r g a n i s m w i l l u t i l i z e the suga r s for the s y n t h e s i s o f c e l l s u b s t a n c e and the p r o d u c t i o n o f v i t a m i n s , s p e c i f i c amino a c i d s , o r o t h e r compounds o f n u t r i t i o n a l or m e d i c a l v a l u e . As C a n d i d a u t i 1 i s and Saccharomyces c e r e - v i s i a e have been known i n terms o f n u t r i t i o n a l , o r g a n o l e p t i c and p a t h o l o g i c a l p r o p e r t i e s , t hese y e a s t s were employed as a n c i l l a r y o rgan i sms f o r the p r o d u c t i o n o f y e a s t p r o t e i n s and v i t a m i n s . P r e l i m i n a r y e x p e r i m e n t s w i t h the Endomycops is y e a s t i n d i c a t e d t h a t v a r i a t i o n s i n the q u a n t i t y o f a s s i m i - l a b l e phospha te added to the s t a r c h y s u b s t r a t e ( i . e . KH2PO4) d i d not s i g n i f i c a n t l y a f f e c t the p r o p a g a t i o n . T h i s seems r e a s o n a b l e i n view o f the phosphorus c o n t e n t o f p o t a t o w h i c h ranges from 6.83% to 27.14% P 2 0 5 i n p o t a t o ash ( T a l b u r t , 1 9 6 7 ) . The w a t e r s o l u b l e phosphorus compounds i n p o t a t o c o u l d c o n c e i v a b l y e x i s t i n a s s i m i l a b l e form e s p e c i a l l y i n acid m e d i a . P a s t e r n a k , (1951) has shown t h a t the phosphorus i n POTATO, YAM, CASSAVA, ETC. Urea, KH2PO °2 DEXTRINS STARCH <T MALTOSE Endomycopsis sp. GLUCOSE Candida u t i l i s or Saccharomyces cerevisiae YEAST BIOMASS AMYLOLYTIC ENZYMES FIGURE 1 9 . Diagram o f the p r i n c i p l e u n d e r l y i n g the u t i l i z a t i o n o f s t a r c h y s u b s t r a t e s by y e a s t c u l t i v a t i o n . 41 . p o t a t o i s p r e s e n t as o r t h o p h o s p h a t e e s t e r i f i e d w i t h the C^-OH o f a g l u c o s y l r e s i d u e o f the a m y l o p e c t i n m o i e t y o f the p o t a t o s t a r c h . The Endomycops i s p r o p a g a t i o n s e x h i b i t e d s e n s i t i v i t y to v a r i a t i o n s i n the amount o f u r e a i n c o r p o r a t e d i n the s t a r c h y m e d i a . One p e r c e n t u r e a was o p t i m a l ; l e v e l s above 1.0% were i n h i b i t o r y and c o u l d be due i n p a r t to the p o s s i b l e p r o d u c t i o n o f t o x i c s u b s t a n c e s o r c r e a t i o n o f h y p e r o s m o t i c e n v i r o n m e n t s . I t i s e v i d e n t , from the d a t a shown i n T a b l e I I I , t h a t the y i e l d s o f p r o t e i n were v e r y low f o r a l l p o t a t o con - c e n t r a t i o n s u s e d . These r e s u l t s c o u l d be t r a c e a b l e to low oxygen t e n s i o n and h i g h g l u c o s e c o n c e n t r a t i o n i n the p r o p a - g a t i o n m e d i a . C u r r e n t v i ews c o r r e l a t e oxygen and g l u c o s e c o n c e n t r a t i o n s to r e g u l a t i o n o f y e a s t r e s p i r a t o r y s t r u c t u r e s d u r i n g g r o w t h . S e v e r a l r e p o r t s w h i c h have been e x c e l l e n t l y r e v i e w e d by M o s s , e t a l . (1969) i n d i c a t e i n c r e a s e d cy tochrome c o n c e n t r a t i o n s i n m i c r o o r g a n i s m s grown i n l i m i t e d o x y g e n . " G l u c o s e e f f e c t " , i . e . the o b s e r v a t i o n t h a t h i g h g l u c o s e l e v e l s b r i n g about r e p r e s s i o n o f c e r t a i n enzymes, has been d e m o n s t r a t e d w i t h the y e a s t r e s p i r a t o r y enzymes and m i t o - c h o n d r i a . I t has been s u g g e s t e d (Moss , e t a l . 1969) t h a t a s u b s t a n c e w h i c h stems from g l u c o s e i n h i b i t s b o t h p r o t e i n and l i p o p r o t e i n s y n t h e s i s and t h a t i n h i b i t i o n o f l i p o p r o t e i n s y n t h e s i s l e a d s to d i m i n i s h e d a c t i v i t y o f 1 i p o p r o t e i n - b o u n d 42 . enzymes. I t seems c o n c e i v a b l e t h a t b o t h low oxygen t e n s i o n and h i g h g l u c o s e c o n c e n t r a t i o n s may have been r e s p o n s i b l e f o r the low y i e l d s o f p r o t e i n i n t h i s e x p e r i m e n t . In f a c t , the shaken f l a s k s d e v e l o p e d s t r o n g a l c o h o l i c and m i l d e s t e r - l i k e odors by the second day o f p r o p a g a t i o n . However , the r e s u l t s o f p r o p a g a t i o n c a r r i e d out i n the V i r t i s f e r m e n t e r i n w h i c h h i g h e r oxygen t e n s i o n s c o u l d be m a i n t a i n e d , i n d i c a t e d i n c r e a s e d p r o t e i n p r o d u c t i o n . However , i t s h o u l d be no t ed t h a t the l a t t e r p r o p a g a t i o n s were a l s o s u b j e c t e d to more v i g o r o u s m e c h a n i c a l a g i t a t i o n . The i m p o r t a n c e o f i n t i m a t e c o n t a c t between m i c r o - o rgan i sms and t h e i r s u b s t r a t e s f o r m e t a b o l i c a c t i v i t y i s w e l l e s t a b l i s h e d . Gaden (1952) has shown t h a t m e c h a n i c a l a g i t a t i o n f ragments m y c e l i a l o r g a n i s m s . T h u s , a t h i g h a g i t a t i o n r a t e s , f r a g m e n t a t i o n may o c c u r , i n c r e a s i n g the s u r f a c e exposed to the m e d i a , whereas at low a g i t a t i o n r a t e s , c l u m p i n g may o c c u r , d e c r e a s i n g the c o n t a c t s u r f a c e be tween the o rgan i sms and the s u b s t r a t e s . And at e x t r e m e l y low a g i t a t i o n r a t e s , a u t o l y s i s may o c c u r as c e l l s s e t t l e to the b o t t o m , o f the p r o p a g a t o r . I t i s b e l i e v e d t h a t G a d e n ' s o b s e r v a t i o n s c o u l d be o p e r a t i v e i n t h i s sytem as Endomycops is y e a s t s a re m y c e l i a l o r pseudo- m y c e l i a l , (Keuhne r , 1953; L o d d e r , 1 9 5 2 ) . E n z y m a t i c a s says o f the c e n t r i f u g e d c u l t u r e f l u i d i n d i c a t e d t h a t a m y l o l y t i c a c t i v i t y o f the E n d o m y c o p s i s ' enzyme sys tem was r e l a t e d to i t s growth p a t t e r n . The growth c u r v e s f o r d i f f e r e n t c o n c e n t r a t i o n s o f p o t a t o s u b s t r a t e , 4 3 . shown i n F i g u r e s 3 - 6 , i n d i c a t e two e x p o n e n t i a l phases s e p a r a t e d by a phase o f l i t t l e o r no g r o w t h . As the concen- t r a t i o n o f p o t a t o s o l i d s s u p p l i e d i n the media was i n c r e a s e d from 2 . 0 % to 1 0 . 0 % , the magni tude o f the second e x p o n e n t i a l phase i n c r e a s e d . R a p i d i n c r e a s e s i n amylase a c t i v i t y o c c u r r e d d u r i n g the second e x p o n e n t i a l p h a s e . The d i p s i n the c o r r e s - p o n d i n g amylase a c t i v i t y c u r v e s c o i n c i d e d w i t h the f i r s t e x p o n e n t i a l g rowth phase and are thus s u g g e s t i v e o f an a d a p t i v e p e r i o d . E v i d e n c e f o r t h i s i s the absence o f a d i p i n the a c t i v i t y cu rve when the i n o c u l u m was grown on p o t a t o s l u r r y i n s t e a d o f s o l u b l e s t a r c h as was done p r e v i o u s l y . The a c t i v i t y cu rve showing the absence o f a d a p t a t i o n i s g i v e n i n F i g u r e 4 . I t i s b e l i e v e d t h a t the c o m p l e x i t y o f the c h e m i c a l c o m p o s i t i o n o f b o t h the c u l t u r e f i l t r a t e as w e l l as t h a t o f the enzyme sys tem i t s e l f a d v e r s e l y a f f e c t e d the a c c u r a c y o f the e n z y m a t i c a s s a y s . Hence , the a c t i v i t i e s o b t a i n e d must be r e g a r d e d as o n l y a p p r o x i m a t e . By c a r r y i n g out a n a l y s e s o f h i g h l y p u r i f i e d e x t r a c t s , such e s t i m a t i o n s c o u l d be r e n d e r e d more m e a n i n g f u l . N e v e t h e l e s s , the r e s u l t s o f t h i s e x p e r i m e n t show t h a t a c t i v i t y ro se s t e e p l y as the y e a s t c e l l s d i v i d e d r a p i d l y . D u r i n g mixed c u l t i v a t i o n s o f Endomycops i s s p . and C a n d i d a u t i l i s , g l u c o s e i n the media d i d no t accumula t e as i t was consumed by the C a n d i d a u t i l i s f o r m e t a b o l i c a c t i v i t y as soon as i t was p roduced from s t a r c h breakdown by the 44 Endomycops i s s p . I t i s pe rhaps due to t h i s p r o d u c t e x h a u s t i o n t h a t the s y m b i o t i c c u l t u e s e x h i b i t e d amylase a c t i v i t i e s m a r k e d l y h i g h e r than s i n g l e c u l t u r e s on media o f s i m i l a r c o m p o s i t i o n ( F i g u r e s 8 ^ 1 0 ) . The a c t i v i t y o f the c u l t u r e f i l t r a t e was h i g h e r i n media i n o c u l a t e d w i t h a l a r g e r p r o p o r - t i o n o f Endomycops is s p . , as shown i n T a b l e I I I . In the absence o f " g l u c o s e e f f e c t " , mixed c u l t u r e s a l s o gave h i g h e r p r o t e i n y i e l d s . Growth o f C a n d i d a u t i 1 i s p r e d o m i n a t e d and as C a n d i d a c e l l s are known to have h i g h e r p r o t e i n c o n t e n t than Endomycops is c e l l s , p r o t e i n y i e l d s from mixed c u l t u r e s were n e c e s s a r i l y h i g h e r . In agreement w i t h these r e s u l t s , i t was o b s e r v e d t h a t under c o n d i t i o n s o f s u f f i c i e n t a e r a t i o n and v i g o r o u s a g i t a t i o n , V i r T i s p r o p a g a t i o n s o f mixed c u l t u r e s , c o n s i s t i n g o f two p a r t s Endomycops i s to one p a r t C a n d i d a , gave v e r y h i g h p r o t e i n y i e l d s and h i g h l y a c t i v e l i q u o r s , as shown i n F igu re 1 1 . In c o n t r a s t , i n V i r T i s p r o p a g a t i o n s o f s i n g l e c u l t u r e s o f Endomycops i s s p . , the p r o t e i n l e v e l was about 15% lower and amylase a c t i v i t y was about two u n i t s l o w e r , as shown i n F igure 7. Mixed c u l t u r e s o f Endomycops is s p . and S a c c h a r o - myces c e r e v i s i a e gave somewhat h i g h e r p r o t e i n y i e l d s b u t s u r p r i s i n g l y l o w e r amylase a c t i v i t i e s i n c o m p a r i s o n to mixed c u l t u r e s o f Endomycops i s s p . and C a n d i d a u t i l i s , as i n d i c a t e d i n Figures 1 2 - 1 4 . Among o t h e r f a c t o r s such as r a t e 45 . o f enzyme s y n t h e s i s by the o r g a n i s m and enzyme e x c r e t i o n from the c e l l s , the c o n c e n t r a t i o n o f enzymes i n the c u l t u r e w i l l depend on the enzyme 's s t a b i l i t y i n the medium. I t i s l i k e l y t h a t the drop i n pH o f the media from 5.5 to 2 .5 e x e r t e d an u n f a v o r a b l e i n f l u e n c e on the s t a b i l i t y o f the amylases and c o n s e q u e n t l y on the c o n c e n t r a t i o n o f enzymes i n the m e d i a . T s u c h i y a , e t a l . (1950) i n d i c a t e d the i m p o r t a n c e o f a v o i d i n g growth pH v a l u e s be low 4 . 5 d u r i n g amylase p r o d u c t i o n by A s p e r g i l l u s n i g e r . The p o s s i b i l i t y t h a t Saccharomyces c e r e v i s i a e migh t have c o n s i d e r a b l e p r o t e o l y t i c a c t i v i t y was e l i m i n a t e d i n view o f the r e s u l t s o f s e q u e n t i a l p r o p a g a t i o n s w i t h Endomycops i s s p . In t he se e x p e r i m e n t s , Endomycops i s s p . was c u l t i v a t e d by i t s e l f and amylase a c t i v i t y was a l l o w e d to i n c r e a s e u n t i l the end o f the e x p o n e n t i a l g rowth p h a s e . At t h i s p o i n t Saccharomyces c e r e v i s i a e was i n t r o d u c e d i n t o the c u l t u r e . No d e c r e a s e o f amylase a c t i v i t y was o b s e r v e d . In f a c t , f u r t h e r i n c r e a s e s i n a c t i v i t y , c e l l d e n s i t y and p r o t e i n c o n t e n t were o b t a i n e d , as i n d i c a t e d i n F igu re 15 . The pH o f the c u l t u r e changed from 5.0 to 6 . 4 . However , an e n t i r e l y d i f f e r e n t p a t t e r n , shown i n F i g u r e 16 , was o b s e r v e d when a p a r a l l e l s i x - l i t e r p r o p a g a t i o n was c a r r i e d o u t . An e x t r e m e l y l a r g e v o l u m e . o f s i l i c o n e a n t i f o a m was r e q u i r e d due to p e r s i s t e n t , e x c e s s i v e f o a m i n g . T h i s c o u l d have i n h i b i t e d c e l l g rowth and enzyme s y n t h e s i s . 46 . C l e a r l y , the d i s c r e p a n c i e s i n the two p r o p a g a t i o n s r e q u i r e f u r t h e r s t u d y . I t may be o f i n t e r e s t to d e t e r m i n e whe the r the r e d u c t i o n i n growth and amylase a c t i v i t y c o u l d be r e l a t e d to oxygen t e n s i o n , degree o f a g i t a t i o n , e t c . Compared to p o t a t o s u b s t r a t e s , p u r p l e yam ( D i o s c o r e a a l a t a ) and c a s s a v a (Man iho t e s c u l e n t a ) gave low p r o t e i n y i e l d s and amylase a c t i v i t i e s . These d i f f e r e n c e s c o u l d be due to i n t r i n s i c p r o p e r t i e s o f the t u b e r s t h e m s e l v e s . I t i s known t h a t a s u b s t a n t i a l number o f the members o f the genus D i o s c o r e a c o n t a i n v a r y i n g amounts o f a l k a l o i d s , t a n n i n s , and s a p o n i n s , ( C o u r s e y , 1966; M a r t i n , 1 9 7 0 ) . Cas sava c u l t i v a r s c o n t a i n v a r y i n g amounts o f t o x i c c y a n o g e n i c g l u c o - s i d e s m a i n l y l i n a m a r i n . The s u p e r i o r i t y o f p o t a t o as a s u b s t r a t e f o r p r o d u c t i o n o f a m y l o l y t i c enzymes and y e a s t p r o t e i n c o u l d a l s o be a s s o c i a t e d w i t h the p r e s e n c e o f n a t u r a l enzyme s t i m u l a t o r s and growth f a c t o r s and the absence o f n a t u r a l i n h i b i t o r s . I t i s w e l l known t h a t the a c t i v i t y o f some e x t r a c e l l u l a r amylases i s i n h i b i t e d by s p e c i f i c i n h i b i t o r s p r o b a b l y p r o t e i n s or m u c o p r o t e i n s o f u n d e t e r m i n e d c o m p o s i t i o n . The a - amyla se o f B a c i l l u s sub t i l i s , f o r example , i s i n h i b i t e d by w a t e r - s o l u b l e i n h i b i t o r s e x t r a c t e d from wheat endosperm, r y e , and sorghum, w h i c h a l s o i n h i b i t s a l i v a r y and p a n c r e a t i c a m y l a s e s , ( D a v i e s , 1 9 6 3 ) . T race m e t a l s a re e s s e n t i a l f o r g r o w t h , some o f them b e i n g e s s e n t i a l f o r the a c t i v i t y and s t a b i l i t y o f many e x t r a c e l l u l a r enzymes. 47 . Many cases o f f a i l u r e to s y n t h e s i z e a c t i v e enzymes i n growth f a c t o r - d e f i c i e n t media a re known and the e f f e c t o f g rowth f a c t o r s on e x t r a c e l l u l a r enzyme f o r m a t i o n as d i s t i n c t from e f f e c t s o f g rowth as a w h o l e , has been d e m o n s t r a t e d , ( D a v i e s , 1963) . 48 . CONCLUSIONS The r e s u l t s o f t h i s s t udy demons t ra t e the a p p l i c a - b i l i t y o f Endomycops is s p . f o r u t i l i z a t i o n o f p o t a t o was te and f o r p r o t e i n e n r i c h m e n t o f some r o o t c r o p s t h a t are w i d e l y c u l t i v a t e d i n the P h i l i p p i n e s , such as yam and c a s s a v a . In e x p e r i m e n t s pe r fo rmed w i t h p u r p l e yam, w h i c h p e r se c o n t a i n s o n l y 2% p r o t e i n , a y e a s t p r o d u c t was o b t a i n e d c o n t a i n i n g about 22% p r o t e i n , r e p r e s e n t i n g about t e n f o l d p r o t e i n e n r i c h m e n t . The o t h e r p r o d u c t c o n s i s t e d o f a c rude enzyme p r e p a r a t i o n o f the a m y l o l y t i c complex r e c o v e r e d from the c u l t u r e a f t e r h a r v e s t i n g the c e l l c r o p s . The amylase a c t i v i t y o f the enzyme p r e p a r a t i o n was about 1.1 u n i t s . The p r o p a g a t i o n s were c a r r i e d out i n the p r e s e n c e o f 1.0% u r e a and 0.5% K H 2 P 0 4 , a t pH 5 .0 and 2 8 ° C , where s y n t h e s i s and a c t i v i t y o f the a m y l o l y t i c enzyme complex were known to be g r e a t e s t . M i x e d and d u a l p r o p a g a t i o n s o f Endomycops is s p . w i t h C a n d i d a u t i l i s and Saccharomyces c e r e v i s i a e r e s u l t e d i n i n c r e a s e s i n amylase a c t i v i t i e s and p r o t e i n y i e l d s . V a r i a - t i o n s were o b s e r v e d , depend ing on the s t a r c h s u b s t r a t e u s e d , the c o n c e n t r a t i o n o f u r e a added and a p p a r e n t l y the amount o f oxygen s u p p l i e d . As food i n d u s t r i a l was te s h o l d c o n s i d e r a b l e r e s e r v e s o f s t a r c h , they may s e r v e as s u b s t r a t e s f o r the s i m u l t a n e o u s p r o d u c t i o n o f a m y l o l y t i c enzymes and y e a s t p r o t e i n . 49. By s e l e c t i v e methods , a v a r i e t y o f c u l t u r e s can be t r i e d i n c o m b i n a t i o n w i t h the s t a r c h - h y d r o l y z i n g y e a s t s p e c i e , i n f a v o r o f some s p e c i f i c p r o d u c t , such as amino a c i d s , v i t a m i n s f l a v o r e n h a n c e r s , and o t h e r compounds o f n u t r i t i o n a l or m e d i c a l v a l u e . 50 . BIBLIOGRAPHY , A m e r i c a n S o c i e t y o f Brewing C h e m i s t s . Methods o f A n a l y s i s o f the A m e r i c a n S o c i e t y o f B r e w i n g C h e m i s t s . D . E . West , C h a i r m a n , W i s c o n s i n : ASBC, 1958. A h e a r n , D . G . , M e y e r s , S . P . and N i c h o 1 s , R . A . E x t r a c e l l u l a r p r o t e i n a s e s o f y e a s t s and y e a s t - l i k e f u n g i . A p p l . M i c r o b i o l . 1_6, 1370 , 1968. B a e n s - A r c e g a , L . 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B u n k e r , H . " M i c r o b i a l f o o d " . B i o c h e m i s t r y o f I n d u s t r i a l M i c r o o r g a n i s m s . E d i t e d by C . Rainbow and A . R o s e . New Y o r k : Academic P r e s s , 1963 . C o u r s e y , D . G . Yams. London : Longmans Green and C o . , 19 66 . D a v i e s , R. " M i c r o b i a l e x t r a c e l l u l a r enzymes, t h e i r uses and some f a c t o r s a f f e c t i n g t h e i r f o r m a t i o n " . B i o c h e m i s t r y o f I n d u s t r i a l M i c r o o r g a n i s m s . E d i t e d by C. Rainbow and A . R o s e , New Y o r k : Academic P r e s s , 1963. E b e r t o v a , H . S tudy o f the f o r m a t i o n and p r o p e r t i e s o f the a m y l o l y t i c sys tem o f C a n d i d a j a p o n i c a . F o l i a M i c r o b i o l o g i y a 8_, 333 , 1963. 11 . E b e r t o v a , H . A m y l o l y t i c enzymes o f Endomycops is c a p s u l a r i s . F o l i a M i c r o b i o l o g i y a 1_1_, 15 , 1966. 12 . Enebo , L . " S i n g l e C e l l P r o t e i n " . E v a l u a t i o n o f N o v e l P r o t e i n P r o d u c t s . E d i t e d by A . B e n d e r , B . K i h l b e r g , R. L o f q v i s t and L . Munk. New Y o r k : Pergamon P r e s s , 1968. 10 51 . F e n c l , Z . P r o d u c t i o n o f m i c r o b i a l p r o t e i n from ca rbon s o u r c e s . B i o t e c h n o l o g y and B i o e n g i n e e r i n g Symposium No. 1 , 19 69 . Fukumoto, J . T s i y i s a k e , Y and A r a k i , M. S t u d i e s o f amylase o f E n d o m y c o p s i s . Kagaku to Kogyo (Japan) 34 , 423 , 1960 . c f . C A . 5_5, 24917, 1961 . G i l b e r t , G . A . and S p r a g g , S . P . " I o d i m e t r i e d e t e r m i n a t i o n s o f a m y l a s e " . Methods i n C a r b o h y d r a t e C h e m i s t r y . E d i t e d by R. W h i s t l e r and M. W o l f r o m . London : Academic P r e s s , 1968. G r a c h e v a , M . , Sadova , A . , G a i d e n k o , b. and B r o v a r e t , T . 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A c t i v i d a d a m i l o l i - t i c a y f e r m e n t a t i v a en d i v e r o s s u b s t r a t o s ami- laseps . C i e n c i a ( M e x i c o ) 7 , 119 , 1946. M a r r o q u i n , A . and S o l o r a n z o , M . E s t u d i o s sobre l a a m i l a s a de Endomycops i s f i b u l i g e r . T I . Poder a m i l o l i t i c o y f e r m e n t a t i v o en c u l t i v o s mixos . A n a l e s e s c u e l a n a c l . c i e n c . b i o l . ( M e x i c o ) 4_, 311 , 1947 . M a r r o q u i n , A . and G a v a r r o n , F. E s t u d i o s sobre l a a m i l a s a de Endomycops i s f i b u l i g e r . I I I . P r i n c i p a l e s c a r a c - t e r i s t i c a s de l a s p r e p a r a c i o n e s e n z y m a t i c a s c rudas y p u r i f i c u d a s . A n a l e s e s c u e l a n a c l . c i e n c . b i o l . ( M e x i c o ) 4_,325, 1947. M a r r o q u i n , A . and G a v a r r o n , F. E s t u d i o s sob re l a a m i l a s a de Endomycops is f i b u l i g e r . I V . 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