33(5):1067.

1977, Appl. Environ. Microbiol.

Hackler and L. R. Mattick
T. C. Cronk, K. H. Steinkraus, L. R.

Fermentation
Indonesian Tap Ketan
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A P P L I E D A ND E NV I R O NM E NT A L M I C R O B I O L O G Y , M ay 1 9 7 7 ,
p .
1 0 6 7 - 1 0 7 3 V o l . 3 3 , No . 5
C o p yr i g h t 1 9 7 7 A m e r i c an S o c i e t y f o r M i c r o b i o l o g y P r i n t e d i n U . S . A .
I n d o n e s i an T ap e K e t an F e r m e n t at i o n '
T . C . C R O NK 2 K . H . S T E I NK R A U S , * L . R . H A C K L E R , A ND L . R .
M A I T I C K
Ne w Y o r k S t at e A g r i c u l t u r al E x p e r i m e n t S t at i o n , C o r n e l l U n i v e r s i t y, G e n e v a, Ne w Y o r k 1 4 4 5 6
R e c e i v e d f o r p u b l i c at i o n 22 No v e m b e r 1 9 7 6
I n d o n e s i an t ap 6 k e t an i s a f e r m e n t at i o n i n wh i c h a m o l d , A m yl o m yc e s r o u x i i
C al m e t t e ( C h l am yd o m u c o r o r yz ae We n t an d P r i n s e n G e e r l i g s ) , i n c o m b i n at i o n
wi t h o n e o r m o r e ye as t s s u c h as E n d o m yc o p s i s b u r t o n i i c o n v e r t s s t e am e d r i c e t o
a s we e t - s o u r , s l i g h t l y al c o h o l i c p as t e . A s t u d y was m ad e t o d e t e r m i n e t h e
b i o c h e m i c al c h an g e s t h at o c c u r i n t h e s u b s t r at e d u r i n g f e r m e n t at i o n . I t was
f o u n d t h at t h e p r o d u c t was r e ad y f o r c o n s u m p t i o n af t e r f e r m e n t at i o n at 3 0 C f o r
3 6 t o 4 8 h . A . r o u x i i u s e d ab o u t 3 0 % o f t h e t o t al r i c e s o l i d s , r e s u l t i n g i n a c r u d e
p r o t e i n o f 1 2% i n 9 6 h , wh e r e as t h e c o m b i n at i o n o f t h e m o l d wi t h E . b u r t o n i i
r e d u c e d t o t al s o l i d s b y 5 0 % i n 1 9 2 h , c au s i n g c r u d e p r o t e i n t o i n c r e as e t o 1 6 . 5 % .
S o l u b l e s o l i d s i n c r e as e d f r o m 5 t o ab o u t 6 7 % i n 3 6 h an d d e c r e as e d t o 1 2% at 1 9 2 h
wi t h A . r o u x i i al o n e , wh e r e as s o l u b l e s o l i d s f e l l t o ab o u t 8 % at 1 9 2 h i n t h e
f e r m e n t at i o n wi t h b o t h t h e m o l d an d t h e ye as t . T h e m o l d , b y i t s e l f , r e d u c e d t h e
s t ar c h c o n t e n t o f t h e r i c e f r o m 7 8 t o 1 0 % i n 4 8 h an d t o l e s s t h an 2% i n 1 4 4 h . T h e
m o l d p l u s ye as t r e d u c e d t h e s t ar c h c o n t e n t t o ab o u t 1 8 % i n 4 8 h ; h o we v e r t h e
" s t ar c h " c o n t e n t d i d n o t f al l b e l o w 6 % e v e n at 1 9 2 h , p r e s u m ab l y b e c au s e t h e
ye as t was p r o d u c i n g g l yc o g e n , wh i c h was d e t e r m i n e d al o n g wi t h t h e r e s i d u al
s t ar c h . Wi t h b o t h t h e m o l d an d t h e m o l d p l u s ye as t f e r m e n t at i o n s , r e d u c i n g
s u g ar s i n c r e as e d f r o m l e s s t h an 1 % t o ap p r o x i m at e l y 5 % i n 24 h an d r e ac h e d
m ax i m u m c o n c e n t r at i o n , 1 6 t o 1 7 % , b e t we e n 3 6 an d 4 8 h . A . r o u x i i b y i t s e l f
p r o d u c e d a m ax i m u m o f ab o u t 5 . 6 % ( v o l / v o l ) e t h an o l at 9 6 h . T h e h i g h e s t
c o n c e n t r at i o n o f e t h an o l ( 8 % , v o l / v o l ) was p r o d u c e d b y t h e m o l d p l u s E . b u r t o n i i
at 1 4 4 h . T h e m o l d b y i t s e l f r e d u c e d t h e s t ar t i n g p H f r o m 6 . 3 t o ab o u t 4 . 0 i n 4 8 h .
T h e c o m b i n at i o n o f t h e m o l d an d ye as t r e d u c e d t h e p H t o 4 . 1 i n 1 4 4 h . T h e m o l d
i n c r e as e d t o t al ac i d i t y t o ap p r o x i m at e l y 6 . 2 m e q o f H + p e r 1 0 0 m l , an d t h e
c o m b i n at i o n o f t h e m o l d an d ye as t i n c r e as e d t h e t o t al ac i d i t y t o 7 . 8 m e q o f H +
p e r 1 0 0 m l i n 1 9 2 h . A t 4 8 h t h e r e was p r ac t i c al l y n o d i f f e r e n c e i n t h e v o l at i l e
ac i d i t y ( 0 . 20 ) f o r t h e c o m b i n e d f e r m e n t at i o n c o m p ar e d wi t h 0 . 26 m e q o f H + p e r
1 0 0 m l f o r t h e m o l d f e r m e n t at i o n . T h e m o l d an d at l e as t o n e s p e c i e s o f ye as t we r e
r e qu i r e d t o d e v e l o p t h e r i c h ar o m a an d f l av o r o f t yp i c al I n d o n e s i an t ap 6 .
I n d o n e s i an t ap 6 k e t an i s a f e r m e n t e d , p ar -
t i al l y l i qu e f i e d , s we e t - s o u r , m i l d l y al c o h o l i c
r i c e p as t e . I t i s p r e p ar e d f r o m g l u t i n o u s r i c e
wh i c h h as b e e n s t e am e d , s p r e ad i n t h i n l aye r s
o n wo v e n b am b o o t r ays , an d i n o c u l at e d wi t h
r ag i , d r y f l at t e n e d c i r c u l ar c ak e s , ab o u t 3 c m i n
d i am e t e r , p r e p ar e d f r o m r i c e f l o u r an d d i s t i n c -
t i v e s p i c e s an d c o n t ai n i n g t h e r e qu i r e d o r g a-
n i s m s . T h e s u b s t r at e i s t h e n c o v e r e d , t yp i c al l y
wi t h a b am b o o l e af , an d al l o we d t o f e r m e n t f o r
ap p r o x i m at e l y 24 t o 4 8 h at am b i e n t t e m p e r a-
t u r e ( 25 t o 3 0 C ) ( 3 , 8 , 1 1 , 1 4 ) .
T ap e k e t an i s p r o d u c e d t h r o u g h o u t I n d o n e s i a
o n a h o m e o r c o t t ag e i n d u s t r y s c al e . I t i s c o n -
s u m e d as a d e s s e r t o r s n ac k i t e m wi t h o u t f u r -
'
J o u r n al P ap e r n o . 3 0 23 , Ne w Y o r k S t at e A g r i c u l t u r al
E x p e r i m e n t S t at i o n .
2
P r e s e n t ad d r e s s : T h e P i l l s b u r y C o . , M i n n e ap o l i s , M N
5 5 4 0 3 .
t h e r p r o c e s s i n g af t e r t h e f e r m e n t at i o n . A s an
al t e r n at i v e s u b s t r at e , p e e l e d , d i c e d , an d
s t e am e d c as s av a t u b e r s ar e u s e d ; t h i s p r o d u c t i s
t ap 6 k e t e l l a. B r e m an d ar ak , t wo o t h e r p r o d -
u c t s r e l at e d t o t h e t ap 6 f e r m e n t at i o n , ar e p r e -
p ar e d s i m i l ar l y b u t al l o we d t o f e r m e n t l o n g e r ,
r e s u l t i n g i n g r e at e r l i qu e f ac t i o n o f t h e r i c e .
B o t h b r e m an d ar ak ar e p r e p ar e d f r o m t h e
l i qu i d p o r t i o n ; t h e f o r m e r i s s u n - d r i e d an d c o n -
s u m e d as a s o l i d " c an d y, " an d t h e l at t e r i s
c o n s u m e d as an al c o h o l i c b e v e r ag e ( 8 , 1 4 ) .
We n t an d G e e r l i g s ( 1 4 ) we r e t h e f i r s t t o i s o -
l at e an d i d e n t i f y t h e m i c r o o r g an i s m s t h o u g h t
t o b e e s s e n t i al f o r t h e f e r m e n t at i o n . T h e y n o t e d
t h at t h e t ap e f e r m e n t at i o n i s d e p e n d e n t o n t h e
p r e s e n c e o f at l e as t o n e am yl o l yt i c f i l am e n t o u s
f u n g u s an d o n e o r m o r e
al c o h o l - p r o d u c i n g
ye as t s . Nu m e r o u s f u n g i an d ye as t s h av e b e e n
i s o l at e d wh i l e s t u d yi n g t h e m i c r o f l o r a o f
t ap e ,
1 0 6 7

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1 0 6 8 C R O NK E T A L .
r ag i - t ap 6 , an d r e l at e d f e r m e n t at i o n s o f S o u t h -
e as t A s i a ( 2- 4 , 1 1 , 1 3 ) . T h e p r e s e n c e o f t h e
f u n g u s A m yl o m yc e s r o u x i i C al m e t t e ( 5 ) ( f o r -
m e r l y C h l am yd o m u c o r o r yz ae We n t an d P r i n -
s e n G e e r l i g s ) ap p e ar s t o b e e s s e n t i al f o r t h e
p r o d u c t i o n o f t ap 6 k e t an ; l e s s i s k n o wn ab o u t
t h e c o n t r i b u t i o n b y t h e ye as t s . B o t h E n d o m y-
c o p s i s b u r t o n i i ( = E . c h o d at i ) as r e p o r t e d b y
D j i e n ( 3 ) an d C an d i d a l ac t o s a as r e p o r t e d b y
D wi d j o s e p u t r o an d Wo l f ( 4 ) , wh e n c o m b i n e d
wi t h A . r o u x i i , ar e c ap ab l e o f t r an s f o r m i n g
s t e am e d r i c e i n t o g o o d - qu al i t y t ap 6 k e t an .
T h i s i n v e s t i g at i o n was c o n d u c t e d t o d e t e r -
m i n e t h e b as i c b i o c h e m i c al c h an g e s t h at o c c u r
d u r i n g a t yp i c al t ap e k e t an f e r m e n t at i o n u n d e r
p u r e c u l t u r e c o n d i t i o n s . A n at t e m p t was m ad e
t o u n d e r s t an d t h e c h an g e s t h at o c c u r i n t h e
s u b s t r at e as a r e s u l t o f t h e p r e s e n c e o f t h e m o l d
A . r o u x i i an d t h e m o l d i n c o m b i n at i o n wi t h
e i g h t ye as t s t h at h ad b e e n i s o l at e d f r o m r ag i -
t ap e , wi t h p r i n c i p al e m p h as i s o n E . b u r t o n i i ( 3 ,
4 ) .
M A T E R I A L S A ND M E T H O D S
R i c e . Wh i t e p o l i s h e d r i c e ( C ar o l i n a B r an d ) was
u s e d i n al l s t u d i e s e x c e p t f o r t h o s e o n t h i am i n e
p r o d u c t i o n , wh e r e u n e n r i c h e d r i c e was u s e d as s u b -
s t r at e .
C u l t u r e s . T h e m i c r o o r g an i s m s u s e d i n t h i s s t u d y
ar e l i s t e d i n T ab l e 1 . S t o c k c u l t u r e s o f t h e
ye as t s
we r e m ai n t ai n e d o n D i f c o m al t e x t r ac t s l an t s . A .
r o u x i i was m ai n t ai n e d i n S ab o u r au d d e x t r o s e b r o t h .
A l l c u l t u r e s we r e g r o wn at 3 0 C an d s t o r e d at
5 C
b e t we e n t r an s f e r s .
P r e p ar at i o n o f i n o c u l a. T h e ye as t i n o c u l a we r e
p r e p ar e d b y i n t r o d u c i n g 5 m l o f s t e r i l e wat e r o n t o
s l an t s t h at h ad b e e n s t r e ak e d 24 h e ar l i e r an d
g e n t l y
v o r t e x i n g t o f o r m a c e l l s u s p e n s i o n . T wo m i l l i l i t e r s
o f t h e s u s p e n s i o n was d i s t r i b u t e d o v e r t h e r i c e s u b -
s t r at e s u r f ac e .
A f t e r 5 d ays o f i n c u b at i o n , wh i c h al l o we d f o r ad e -
qu at e d e v e l o p m e n t o f t h e ae r i al m yc e l i u m o f A .
T A B L E 1 . C u l t u r e s u s e d i n t h e s t u d i e s
O r g an i s m s
C u l t u r e c o l l e c t i o n
S o u r c e
n o .
A m yl o m yc e s r o u x i i NR R L A - 1 7 , 1 9 9 K . S . D j i e n a
( f o r m e r l y C . o r yz ae )
( 5 )
E n d o m yc o p s i s b u r t o - NR R L - Y - 7 1 4 3 K . S . D j i e n
n i i
E . f i b u l i g e r a NR R L - Y - 7 1 4 5 NR R L b
C an d i d a l ac t o s a NR R L - Y - 7 1 7 0 NR R L
C . m e l i n i i NR R L - Y - 7 1 7 1 NR R L
C . p ar ap s i l o s i s NR R L - Y - 7 1 7 2 NR R L
H an s e n u l a an o m al a NR R L - Y - 7 1 7 4 NR R L
H . m al an g a NR R L - Y - 7 1 7 5 NR R L
H . s u b p e l l i c u l o s a NR R L - Y - 7 1 7 6 NR R L
a
A g r i c u l t u r al U n i v e r s i t y, Wag e n i n g e n , T h e Ne t h e r -
l an d s .
b
No r t h e r n R e g i o n al R e s e ar c h L ab o r at o r y, P e o r i a, I l l .
A P P L . E NV I R O N. M I C R O B I O L .
r o u x i i , m yc e l i al m at s we r e as e p t i c al l y t r an s t e r r e d t o
a c o v e r e d , s t e r i l e , f r i t t e d - g l as s c r u c i b l e . T h e m at s
we r e was h e d an d t r an s f e r r e d t o an O m n i m i x e r c yl -
i n d e r an d c o m m i n u t e d f o r 5 m i n at a m o d e r at e s p e e d
wi t h t h e c yl i n d e r i m m e r s e d i n an i c e b at h . T wo
m i l l i l i t e r s o f t h e i n o c u l u m was ap p l i e d u n i f o r m l y
o v e r t h e r i c e s u b s t r at e .
P r e p ar at i o n o f s u b s t r at e . Nal g e n e s p e c i m e n
d i s h e s ( 1 0 5 b y 4 3 m m ) , f i t t e d wi t h c o v e r s , we r e u s e d
as t h e s u b s t r at e c o n t ai n e r s . F o r t y g r am s o f r i c e an d
1 0 0 m l o f wat e r we r e ad d e d t o e ac h d i s h . T h e r i c e
was s t e am e d f o r 1 5 m i n an d s u b s e qu e n t l y au t o -
c l av e d f o r 1 5 m i n at 1 21 C .
S am p l e p r e p ar at i o n . A s am p l e c o n s i s t e d o f t h e
e n t i r e c o n t e n t s o f a d i s h . F o r we t an al ys e s , d i s h e s
we r e r e m o v e d f r o m t h e i n c u b at o r , c o v e r e d , an d f r o -
z e n at - 20 C u n t i l t h e an al ys e s c o u l d b e p e r f o r m e d .
P r i o r t o an al ys i s , t h e s am p l e was b r i e f l y t h awe d at
r o o m t e m p e r at u r e an d s u b s e qu e n t l y h o m o g e n i z e d
u s i n g an O m n i m i x e r . Wh e r e d r i e d s am p l e s we r e
r e qu i r e d f o r an al ys e s , t h e s am p l e was r e m o v e d f r o m
t h e i n c u b at o r , f r o z e n at - 4 0 C , an d f r e e z e - d r i e d . A
Wi l e y m i l l e qu i p p e d wi t h a 20 - m e s h s c r e e n was u s e d
t o r e d u c e t h e f r e e z e - d r i e d s am p l e t o a u n i f o r m p o w-
d e r .
A n al yt i c al m e t h o d s . C h an g e s i n p H we r e f o l -
l o we d u s i n g a B e c k m an Z e r o m at i c p H m e t e r . T o t al
ac i d i t y was d e t e r m i n e d b y t i t r at i o n t o p H 8 . 2. V o l a-
t i l e ac i d s we r e d e t e r m i n e d b y s t e am d i s t i l l at i o n o f 5 -
m l s am p l e s u s i n g a m i c r o - s t e am d i s t i l l at i o n J u f f -
m an n - C o m e s ( M i l an , I t al y) ap p ar at u s . T h e d i s t i l -
l at e was t i t r at e d t o p H 8 . 2 wi t h 0 . 0 2 N NaO H .
T o t al s o l i d s o f t h e f e r m e n t e d p r o d u c t we r e d e t e r -
m i n e d b y d r yi n g we i g h e d s am p l e s o f t h e h o m o g e -
n at e at 5 0 C i n a v ac u u m o v e n f o r 24 h . Ni t r o g e n was
d e t e r m i n e d o n we i g h e d s am p l e s b y t h e m i c r o - K j e l -
d ah l m e t h o d o f an al ys i s ( 1 ) , wi t h t o t al c r u d e p r o t e i n
e s t i m at e d b y t h e c o n v e r s i o n f ac t o r 6 . 25 . S o l u b l e
s o l i d s an d s o l u b l e n i t r o g e n we r e d e t e r m i n e d l i k e -
wi s e o n t h e s u p e r n at an t af t e r c e n t r i f u g at i o n o f t h e
h o m o g e n at e at 1 4 , 0 0 0 x g f o r 1 5 m i n i n a S o r v al l
c e n t r i f u g e , m o d e l S S - 3 . S o l u b l e n i t r o g e n i s r e p o r t e d
as a p e r c e n t ag e o f t o t al n i t r o g e n .
S o l u b l e r e d u c i n g s u g ar s we r e d e t e r m i n e d as D -
g l u c o s e b y a c o l o r i m e t r i c c o p p e r m e t h o d ( 1 2) . A p -
p r o x i m at e l y 20 m l o f t h e ab o v e s u p e r n at an t was
p as s e d t h r o u g h a D o we x 5 O W- X 8 c o l u m n t o r e m o v e
e n z ym at i c ac t i v i t y. T h e l as t 5 m l o f t h e e l u at e was
r e t ai n e d , f r o m wh i c h a s am p l e was d i l u t e d t o
ac h i e v e a r e d u c i n g s u g ar c o n c e n t r at i o n o f 0 . 0 5 t o 3 . 0
m g / m l .
S t ar c h was i s o l at e d qu an t i t at i v e l y f r o m t h e
f r e e z e - d r i e d p r o d u c t b y t h e p r o c e d u r e o f H as s i d an d
Ne u f e l d ( 7 ) as o u t l i n e d b y J o s l yn ( 9 ) . F r o m ap p r o p r i -
at e l y d i l u t e d s am p l e s o f t h e s t ar c h s o l u t i o n s , s t ar c h
was d e t e r m i n e d c o l o r i m e t r i c al l y as D - g l u c o s e b y r e -
ac t i o n wi t h an t h r o n e i n 9 5 % H 2S O 4 ( 1 0 ) .
E t h an o l was s e p ar at e d an d d e t e r m i n e d qu an t i t a-
t i v e l y b y g as c h r o m at o g r ap h y. T h e g as c h r o m at o -
g r ap h i c i n s t r u m e n t u s e d was a B e c k m an m o d e l G C -
5 e qu i p p e d wi t h a f l am e i o n i z at i o n d e t e c t o r . A c o i l e d
s t ai n l e s s - s t e e l c o l u m n , 3 f e e t b y 1 / 8 i n c h ( o u t e r d i -
am e t e r ) ( c a. 9 1 . 4 b y 0 . 3 2 c m ) wi t h a wal l t h i c k n e s s o f
0 . 0 2 i n c h ( 0 . 0 5 c m ) , was f i t t e d i n t o t h e i n j e c t i o n p o r t
t o al l o w o n - c o l u m n i n j e c t i o n . T h e c o l u m n p ac k i n g

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B I O C H E M I C A L C H A NG E S D U R I NG T A P I t F E R M E NT A T I O N 1 0 6 9
was P o r ap ak Q - S , 1 0 0 / 1 20 m e s h . T h e c o l u m n o v e n
was m ai n t ai n e d at 1 1 5 C , wi t h t h e d e t e c t o r o v e n an d
t h e i n j e c t o r p o r t m ai n t ai n e d at 23 5 an d 20 0 C , r e -
s p e c t i v e l y. T h e f l o w r at e o f t h e c ar r i e r g as ( n i t r o -
g e n ) was m ai n t ai n e d at 25 m l / m i n . T h e i n s t r u m e n t
s e n s i t i v i t y was s e t at 2 x 1 0 - 2 A , f u l l s c al e .
A H e wl e t t - P ac k ar d i n t e g r at o r , m o d e l 3 3 7 0 A , was
c o n n e c t e d i n s e r i e s t o t h e g as c h r o m at o g r ap h i c i n -
s t r u m e n t t o d e t e r m i n e t h e p e ak ar e as . A v i s u al
r e c o r d o f t h e c h r o m at o g r am was ac h i e v e d b y u s e o f a
B e c k m an 1 0 - i n c h r e c o r d e r ( 1 m V , 0 . 5 i n c h [ 1 . 27 c m ] /
m i n ) .
S t an d ar d aqu e o u s s o l u t i o n s c o n t ai n i n g 0 . 25 , 0 . 5 0 ,
0 . 7 5 , 1 . 0 0 , 1 . 25 , an d 1 . 5 0 % ( v o l / v o l ) e t h an o l we r e
p r e p ar e d . A 2. 0 % ( v o l / v o l ) ac e t o n e s o l u t i o n was u s e d
as t h e i n t e r n al s t an d ar d . C h r o m at o g r ap h i c p e ak
ar e as we r e d e t e r m i n e d b y e l e c t r o n i c i n t e g r at i o n ,
an d t h e p e ak ar e a r at i o , e t h an o l - i n t e r n al s t an d ar d ,
was c al c u l at e d . R e t e n t i o n t i m e s ( T r ) we r e 1 . 0 1 an d
1 . 7 8 f o r e t h an o l an d ac e t o n e , r e s p e c t i v e l y.
A 1 . 0 - m l am o u n t o f h o m o g e n i z e d , f e r m e n t e d p r o d -
u c t was t r an s f e r r e d t o a g as - t i g h t c e n t r i f u g e t u b e
an d d i l u t e d wi t h an e qu al v o l u m e ( 1 . 0 m l ) o f t h e
i n t e r n al s t an d ar d s o l u t i o n . T h e t u b e was s t o p p e r e d ,
an d t h e c o n t e n t s we r e m i x e d we l l an d c e n t r i f u g e d
f o r 5 m i n at 2, 5 0 0 r p m . F r o m t h e s u p e r n at an t , a 2. 5 -
, u l s am p l e was i n j e c t e d i n t o t h e c o l u m n wi t h a n o .
7 0 1 H am i l t o n s yr i n g e . T h e p e r c e n t ag e o f e t h an o l
o b t ai n e d was t h e n m u l t i p l i e d b y t h e c o r r e s p o n d i n g
d i l u t i o n f ac t o r .
T h i am i n e was d e t e r m i n e d b y t h e t h i o c h r o m e p r o -
c e d u r e ( 6 ) . A p h o s p h at as e s o l u t i o n ( M yl as e P , p r e -
p ar e d f r o m A s p e r g i l l u s o r yz ae ) was u s e d t o ai d i n t h e
c o n v e r s i o n o f b o u n d t h i am i n e t o i t s f r e e f o r m , wi t h
ac t i v at e d D e c al s o u s e d i n t h e p u r i f i c at i o n p r o c e -
d u r e . U n e n r i c h e d p o l i s h e d r i c e was u s e d f o r t h i s
p o r t i o n o f t h e s t u d y.
R E S U L T S A ND D I S C U S S I O N
T ap 6 k e t an p r o d u c e d b y f e r m e n t i n g s t e am e d
r i c e wi t h A . r o u x i i ( f o r m e r l y C . o r yz ae [ 5 ] )
i n
c o m b i n at i o n wi t h E . b u r t o n i i h ad a s we e t ,
s l i g h t l y ac i d t as t e wi t h a m i l d l y al c o h o l i c f l av o r .
T h e s e we r e t h e o r g an i s m s t h at D j i e n ( 3 ) i s o l at e d
an d r e p o r t e d as c ap ab l e o f p r o d u c i n g
a t yp i c al ,
g o o d - f l av o r e d t ap 6 . B e t we e n 3 6 an d 4 8 h o f
f e r m e n t at i o n at 3 0 C , t h e p r o d u c t was s o f t an d
j u i c y an d r e ad y f o r c o n s u m p t i o n , t h e i n d i v i d u al
r i c e g r ai n s h av i n g b e c o m e a c o h e s i v e , ye t p o -
r o u s , m as s . B e yo n d 4 8 h t h e r i c e s u b s t r at e was
l ar g e l y l i qu i f i e d , an d t h e p r o d u c t c o u l d e i t h e r
b e c o n s u m e d d i r e c t l y o r u s e d i n t h e p r o d u c t i o n
o f b r e m o r ar ak .
T h e m yc e l i u m o f C . o r yz ae was f i r s t v i s i b l e
af t e r 24 h o f f e r m e n t at i o n at 3 0 C . A f t e r an
ad d i t i o n al 1 2 t o 24 h , t h e r i c e g r ai n s
we r e al -
m o s t t o t al l y c o v e r e d wi t h f i n e , wh i t e m yc e l i u m .
S i n c e A . r o u x i i d o e s n o t f o r m f r e e s p o r e s ,
n o
d i s c o l o r at i o n was o b s e r v e d .
T h e d e v e l o p m e n t o f ye as t was f i r s t n o t i c e d
af t e r 3 6 t o 4 8 h as s m al l , wh i t e , u n e v e n l y
d i s -
t r i b u t e d p at c h e s o n t h e s u r f ac e o f t h e r i c e . O n l y
v e r y s l i g h t , l o c al i z e d l i qu e f ac t i o n o f t h e r i c e
o c c u r r e d wh e n i n o c u l at e d wi t h E . b u r t o n i i
al o n e .
I n r i c e f e r m e n t e d wi t h A . r o u x i i , s t ar c h d e -
c r e as e d o n l y s l i g h t l y ( 7 8 t o 7 3 % ) d u r i n g t h e f i r s t
24 h b u t d e c r e as e d s h ar p l y t o ab o u t 1 2%
t h r o u g h 4 8 h , r e ac h i n g l e s s t h an 2% af t e r 1 4 4 h
( F i g . 1 ) . T h e d e c r e as e i n s t ar c h r e f l e c t s t h e
e x t e n t t o wh i c h l i qu e f ac t i o n o f t h e s u b s t r at e
h ad o c c u r r e d . Wi t h t h e m o l d an d t h e ye as t
c o m b i n e d , t h e d e c r e as e i n s t ar c h t o 4 8 % was
s l i g h t l y m o r e r ap i d d u r i n g t h e f i r s t 3 6 h ( F i g .
2) ; b u t t h e t o t al am o u n t o f s t ar c h r e m ai n e d
h i g h e r ( ab o u t 8 % ) b e yo n d 9 6 h , p r e s u m ab l y as a
r e s u l t o f ye as t - p r o d u c e d g l yc o g e n wh i c h wo u l d
b e e x p e c t e d t o b e r e c o v e r e d as " s t ar c h " i n t h e
an al ys i s ( 7 ) .
Wi t h b o t h t h e m o l d an d t h e m o l d p l u s ye as t
f e r m e n t at i o n s , r e d u c i n g s u g ar s i n c r e as e d f r o m
l e s s t h an 1 % t o ap p r o x i m at e l y 5 % i n 24 h an d
r e ac h e d m ax i m u m c o n c e n t r at i o n , 1 6 t o 1 7 % ,
b e t we e n 3 6 an d 4 8 h . B e yo n d 4 8 h t h e c o n c e n -
t r at i o n o f r e d u c i n g s u g ar s l e v e l e d o f f i n t h e
m o l d f e r m e n t at i o n an d d e c r e as e d i n t h e c o m -
b i n e d f e r m e n t at i o n d u e t o u s e o f t h e g l u c o s e b y
t h e ye as t .
T h e p H o f r i c e f e r m e n t e d wi t h A . r o u x i i d e -
c r e as e d o n l y s l i g h t l y d u r i n g t h e f i r s t 24 h , f o l -
1 0
- 4 8
3
6 X
1
4
2-
2
J
0
, o . , o o
7
s e ~~~~~~~~0 R C E f 3 0 C
8 8 0
~
7 6
0 6 6 0 \- 0 T . 5
a, 4 e 4 0 e / 4
2
0
20 / - R E D U C I . C G s u G A a 3
0 24 4 8 7 2 9 6 1 20 1 4 4 1 6 8 1 9 2
H O U R S
F I G . 1 . C h an g e s i n s t ar c h , r e d u c i n g s u g ar s ,
e t h an o l , p H , an d t o t al an d v o l at i l e ac i d i t y o c c u r r i n g
i n r i c e f e r m e n t e d wi t h A . r o u x i i at 3 0 C .
1 0 , I
8 -
0 6 -
< 4
I
2 - O x
0 0 - - - -
7
7
A m l m c o . . - f E c i d . - - . , , - wo 0 . R I C E - T 3 0 C
1 0
3
6
: .
x +i
4 0 0
1 2
0
F I G . 2. C h an g e s i n s t ar c h , r e d u c i n g s u g ar s ,
e t h an o l , p H , an d t o t al an d v o l at i l e ac i d i t y o c c u r r i n g
i n r i c e f e r m e n t e d wi t h A . r o u x i i i n c o m b i n at i o n wi t h
E . b u r t o n i i at 3 0 C .
V O L . 3 3 , 1 9 7 7

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1 0 7 0 C R O NK E T A L .
l o we d b y a s h ar p d e c r e as e f r o m 6 . 3 t o 4 . 0 d u r i n g
t h e n e x t 24 h . T h e p H t h e n g r ad u al l y i n c r e as e d
t o 4 . 5 wi t h c o n t i n u e d f e r m e n t at i o n . T ap e k e t an
p r o d u c e d b y A . r o u x i i i n c o m b i n at i o n wi t h E .
b u r t o n i i h ad a p H o f 3 . 9 at 4 8 h . B e yo n d 4 8 h
t h e r e was s o m e f l u c t u at i o n , b u t t h e p H r e -
m ai n e d l e s s t h an 4 . 3 t h r o u g h o u t t h e 1 9 2 h . A
p H o f ap p r o x i m at e l y 4 . 0 i s c o n s i d e r e d t yp i c al
f o r t h e f e r m e n t e d p r o d u c t ( 3 ) .
T h e t o t al ac i d i t y o f t h e r i c e f e r m e n t e d b y A .
r o u x i i i n c r e as e d f r o m 0 . 5 2 t o 4 . 9 4 m e q o f H + p e r
1 0 0 m l an d t o 5 . 3 2 m e q o f H + p e r 1 0 0 m l f o r t h e
c o m b i n e d f e r m e n t at i o n wi t h i n 4 8 h . A t 1 9 2 h ,
t o t al ac i d i t y f o r t h e m o l d f e r m e n t at i o n was
ab o u t 6 . 25 m e q o f H + p e r 1 0 0 m l , an d i t was
n e ar l y 8 m e q o f H + p e r 1 0 0 m l f o r t h e c o m b i n e d
f e r m e n t at i o n ( F i g . 1 an d 2) .
A t 4 8 h , t h e r e was e s s e n t i al l y n o d i f f e r e n c e i n
t h e v o l at i l e ac i d i t y f o r t h e m o l d f e r m e n t at i o n ,
0 . 26 m e q o f H + p e r 1 0 0 m l , c o m p ar e d wi t h 0 . 20
f o r t h e c o m b i n e d f e r m e n t at i o n . A t 9 6 h , t h e
v o l at i l e ac i d i t y was n e ar l y 1 m e q o f H + p e r 1 0 0
m l i n b o t h f e r m e n t at i o n s . A s f e r m e n t at i o n c o n -
t i n u e d , t h e r e was a s l i g h t l y h i g h e r ( 1 . 4 0 m e q o f
H + p e r 1 0 0 m l ) v o l at i l e ac i d i t y i n t h e c o m b i n e d
f e r m e n t at i o n at 1 4 4 t o 1 9 2 h .
A f t e r an i n i t i al 24 - h l ag , r i c e f e r m e n t e d b y
t h e m o l d al o n e c o n t ai n e d 2. 3 % ( v o l / v o l ) e t h an o l
at 4 8 h . A t 9 6 h a m ax i m u m c o n c e n t r at i o n o f
5 . 6 % ( v o l / v o l ) e t h an o l was ac h i e v e d . T h e r e af -
t e r , t h e c o n c e n t r at i o n d e c r e as e d s l i g h t l y, d u e
p r e s u m ab l y t o f u r t h e r m e t ab o l i s m o r l o s s b y
e v ap o r at i o n .
T h e c o n c e n t r at i o n o f e t h an o l i n r i c e f e r -
m e n t e d wi t h A . r o u x i i i n c o m b i n at i o n wi t h E .
b u r t o n i i was 2. 7 % ( v o l / v o l ) at 4 8 h , i n c r e as e d t o
6 . 4 % at 7 2 h , an d r e ac h e d a m ax i m u m c o n c e n -
t r at i o n o f 8 . 0 % ( v o l / v o l ) at 1 4 4 h .
E n d o m yc o p s i s f i b u l i g e r a, C an d i d a l ac t o s a,
C . m e l i n i i , an d C . p ar ap s i l o s i s , e ac h i n c o m b i -
n at i o n wi t h A . r o u x i i , h ad i n c r e as e d t h e
T A B L E 2. E t h an o l p r o d u c t i o n an d ac c u m u l at i o n b y
A . r o u x i i al o n e an d i n c o m b i n at i o n wi t h v ar i o u s
ye as t s o n r i c e at 3 0 C
E t h an o l ( % , v o l / v o l ) a
O r g an i s m s
24 h b 4 8 h 9 6 h 1 4 4 h
A . r o u x i i <0 . 1 2. 3 5 . 6 5 . 6
A . r o u x i i + E . b u r t o n i i <0 . 1 2. 7 6 . 8 8 . 0
A . r o u x i i + E . f i b u l i g e r a <0 . 1 3 . 7 7 . 0 7 . 7
A . r o u x i i + C . l ac t o s a <0 . 1 3 . 3 7 . 4 7 . 3
A . r o u x i i + C . m e l i n i i <0 . 1 3 . 4 7 . 3 7 . 5
A . r o u x i i + C . p ar ap s i l o s i s <0 . 1 3 . 6 7 . 2 7 . 6
A . r o u x i i + H . an o m al a <0 . 1 2. 6 3 . 2 3 . 7
A . r o u x i i + H . m al an g a <0 . 1 2. 8 5 . 6 6 . 0
A . r o u x i i + H . s u b p e l l i c u l o s a <0 . 1 2. 6 4 . 2 4 . 2
a
E ac h e n t r y i s a m e an o f t r i p l i c at e an al ys e s .
b
L e n g t h o f f e r m e n t at i o n .
e t h an o l c o n c e n t r at i o n a m i n i m u m o f 1 . 0 % ( v o l /
v o l ) ab o v e t h at p r o d u c e d b y A . r o u x i i al o n e at
4 8 h ( T ab l e 2) . A t 9 6 h t h e r e we r e s m al l e r
d i f f e r e n c e s b e t we e n t h e e t h an o l c o n c e n t r at i o n s
o f t h e p r o d u c t s f e r m e n t e d b y t h e s e s am e ye as t s
an d E . b u r t o n i i . A t 1 4 4 h , h o we v e r , t h e h i g h e s t
c o n c e n t r at i o n o f e t h an o l was p r o d u c e d b y E .
b u r t o n i i p l u s t h e m o l d .
E t h an o l c o n c e n t r at i o n s s i m i l ar t o t h o s e p r o -
d u c e d b y A . r o u x i i i n c o m b i n at i o n wi t h E . b u r -
t o n i i we r e al s o p r o d u c e d i n c o m b i n at i o n wi t h
H an s e n u l a an o m al a, H . m al an g a, an d H . s u b -
p e l l i c u l o s a at 4 8 h . H o we v e r , b y 9 6 h t h e
e t h an o l c o n c e n t r at i o n s o f t h e r i c e f e r m e n t e d b y
t h e s e c o m b i n at i o n s we r e m ar k e d l y l o we r t h an
t h e c o n c e n t r at i o n s p r e s e n t i n t h e r i c e f e r -
m e n t e d b y t h e o t h e r ye as t s p l u s t h e m o l d .
E t h an o l c o n c e n t r at i o n s r e s u l t i n g f r o m t h e s e
s p e c i e s o f H an s e n u l a we r e n e ar l y as l o w o r
l o we r t h an t h at p r e s e n t i n t h e r i c e f e r m e n t e d
b y t h e m o l d al o n e .
T h e r i c e f e r m e n t e d i n p ar t b y H . an o m al a
an d H . s u b p e l l i c u l o s a d e v e l o p e d a s t r o n g o d o r
o f e t h yl ac e t at e , wh i c h m i g h t d e c r e as e p al at a-
b i l i t y t o s o m e c o n s u m e r s . T h e e s t e r i f i c at i o n o f
ac e t i c ac i d wi t h e t h an o l t o f o r m e t h yl ac e t at e
ac c o u n t s p ar t i al l y f o r t h e d e c r e as e d c o n c e n t r a-
t i o n s o f e t h an o l o b s e r v e d . A l s o , t h e s e s p e c i e s o f
H an s e n u l a m ay p r o d u c e l e s s e t h an o l t h an E n -
d o m yc o p s i s an d C an d i d a s p e c i e s . No e t h yl ac e
t at e ar o m a was p r o d u c e d b y H . m al an g a.
T e m p e r at u r e al s o i n f l u e n c e d t h e c o n c e n t r a-
t i o n o f e t h an o l p r e s e n t i n t ap e k e t an ( T ab l e 3 ) .
I n p r o d u c t f e r m e n t e d wi t h A . r o u x i i al o n e an d
i n c o m b i n at i o n wi t h E . b u r t o n i i , t h e c o n c e n t r a-
t i o n o f e t h an o l at 3 6 h i n c r e as e d as i n c u b at i o n
t e m p e r at u r e was r ai s e d f r o m 25 t o 3 5 C . T h e r e
was al s o an i n c r e as e i n t h e c o n c e n t r at i o n o f
e t h an o l f o r b o t h f e r m e n t at i o n s at 4 8 an d 9 6 h
wi t h a t e m p e r at u r e i n c r e as e f r o m 25 t o 3 0 C .
M ax i m u m e t h an o l p r o d u c t i o n ( 8 . 5 % , v o l / v o l )
was ac h i e v e d b y t h e m o l d p l u s ye as t i n c u b at e d
at 25 C f o r 1 4 4 h .
T A B L E 3 . I n f l u e n c e o f t e m p e r at u r e o n e t h an o l
p r o d u c t i o n an d ac c u m u l at i o n b y A . r o u x i i al o n e an d
i n c o m b i n at i o n wi t h E . b u r t o n i i o n r i c e
T e m p E t h an o l ( % , v o l / v o l ) a
O r g an i s m s
O C
P
( aC ) 3 6 h b 4 8 h 9 6 h 1 4 4 h
A . r o u x i i 25 0 . 2 2. 0 5 . 3 5 . 6
3 0 1 . 4 2. 7 5 . 3 5 . 2
3 5 1 . 9 3 . 2 3 . 5 3 . 5
A . r o u x i i + E . 25 0 . 2 2. 4 6 . 0 8 . 5
b u r t o n i i 3 0 1 . 3 3 . 0 6 . 9 7 . 2
3 5 1 . 5 2. 4 5 . 1 4 . 6
a
E ac h e n t r y i s a m e an o f t r i p l i c at e an al ys e s .
b L e n g t h o f f e r m e n t at i o n .
A P P L . E NV I R O N. M I C R O B I O L .

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B I O C H E M I C A L C H A NG E S D U R I NG T A P E F E R M E NT A T I O N 1 0 7 1
A n i n c u b at i o n t e m p e r at u r e o f 3 0 C ap p e ar s
o p t i m al f o r m ax i m u m p r o d u c t i o n an d ac c u m u -
l at i o n o f e t h an o l i n t ap e f e r m e n t e d f o r 4 8 h b y
A . r o u x i i an d E . b u r t o n i i . H o we v e r , t o ac h i e v e
t h e m ax i m u m c o n c e n t r at i o n o f e t h an o l f o r t h e
p r o d u c t i o n o f ar ak , 1 4 4 h at 25 C wo u l d b e m o s t
e f f e c t i v e .
I n r i c e f e r m e n t e d b y A . r o u x i i , t o t al s o l i d s
c h an g e d l i t t l e d u r i n g t h e f i r s t 3 6 h ; b y 4 8 h
t h e r e was a 1 0 % r e d u c t i o n i n s o l i d s an d b y 9 6 h
t h e r e was n e ar l y a 3 0 % r e d u c t i o n ( F i g . 3 ) . B e -
yo n d 9 6 h n o f u r t h e r d e c r e as e i n t o t al s o l i d s was
o b s e r v e d . A s i m i l ar r e d u c t i o n i n t o t al s o l i d s
was o b s e r v e d f o r t h e c o m b i n e d f e r m e n t at i o n i n
t h e i n i t i al 4 8 h ( F i g . 4 ) ; h o we v e r , b e yo n d 4 8 h ,
t h e l o s s o f s o l i d s was g r e at e r f o r t h e c o m b i n e d
f e r m e n t at i o n t h an f o r t h e f e r m e n t at i o n b y t h e
m o l d , ap p r o x i m at e l y 4 0 % o f t h e s o l i d s h av i n g
b e e n h yd r o l yz e d b y t h e c o m b i n e d ac t i o n o f t h e
m o l d an d ye as t , b e t we e n 9 6 an d 1 4 4 h ( F i g . 4 ) .
S o l u b l e s o l i d s i n c r e as e d s h ar p l y, f r o m ap -
p r o x i m at e l y 5 t o 6 7 % d u r i n g t h e f i r s t 3 6 h o f t h e
f e r m e n t at i o n wi t h A . r o u x i i al o n e an d i n c o m -
b i n at i o n wi t h E . b u r t o n i i . T h e d e c r e as e i n s o l u -
b l e s o l i d s b e yo n d 4 8 h r e f l e c t s t h e u s e o f s u g ar s
f o r t h e p r o d u c t i o n o f e t h an o l . T h e d e c r e as e was
g r e at e r wi t h t h e ye as t p r e s e n t , wi t h s o l u b l e
s o l i d s f i n al l y r e ac h i n g a l e v e l o f ab o u t 4 0 % ( 1 9 2
h ) .
O n a d r y- we i g h t b as i s , t h e t o t al c r u d e p r o t e i n
o f t h e A . r o u x i i - f e r m e n t e d r i c e i n c r e as e d ap -
p r o x i m at e l y 1 % b e t we e n 3 6 an d 4 8 h ( F i g . 3 ) .
C r u d e p r o t e i n c o n t i n u e d t o i n c r e as e b y n e ar l y
3 . 5 % d u r i n g t h e f o l l o wi n g 4 8 h . T h i s i n c r e as e
r e f l e c t s t h e c o r r e s p o n d i n g d e c r e as e i n t o t al
s o l i d s . A s a r e s u l t o f a g r e at e r d e c r e as e i n t o t al
s o l i d s i n t h e r i c e f e r m e n t e d b y t h e m o l d an d
ye as t o c c u r r i n g af t e r 4 8 h ( F i g . 4 ) , t h e i n c r e as e
i n t o t al c r u d e p r o t e i n was al s o g r e at e r t h an
t h at f o r t h e r i c e f e r m e n t e d b y t h e m o l d al o n e .
T o t al c r u d e p r o t e i n e x c e e d e d 1 6 % at 1 9 2 h f o r
t h e c o m b i n e d f e r m e n t at i o n v e r s u s 1 2% f o r t h e
r i c e f e r m e n t e d wi t h o u t t h e ye as t .
1 0 0
A . yl o . yc e o . . , O N R I C E A T 3 0 C
20
80 ~~~~~~~~~~~1 6 0 )
T O T A L S O L I D S
r
V I S - = =
ws
S O 1
6 0~
o 1 0 1 2 0
4 0 1 1
O ) 20 O T E I N
0 o 20 4
c o
0
0
0 24 4 8 7 2 9 6 1 20 1 4 4 1 6 8 1 9 2
H O U R S
F I G . 3 . C h an g e s i n t o t al an d s o l u b l e s o l i d s an d
t o t al an d s o l u b l e c r u d e p r o t e i n o c c u r r i n g i n r i c e f e r -
m e n t e d wi t h A . r o u x i i at 3 0 C .
c o
0
20
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0
' 1 6 m '
0
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' 8
0
41 2m
I L
H O U R S
F I G . 4 . C h an g e s i n t o t al an d s o l u b l e s o l i d s an d
t o t al an d s o l u b l e p r o t e i n o c c u r r i n g i n r i c e f e r m e n t e d
wi t h A . r o u x i i i n c o m b i n at i o n wi t h E . b u r t o n i i at
3 0 0 C .
Wi t h A . r o u x i i - f e r m e n t e d r i c e , t h e s o l u b l e
c r u d e p r o t e i n d e c r e as e d d u r i n g t h e f i r s t 24 h
an d t h e r e af t e r c o n t i n u al l y i n c r e as e d . T h e r ap i d
as s i m i l at i o n o f n i t r o g e n b y t h e d e v e l o p i n g m o l d
d u r i n g t h e e ar l y s t ag e s o f t h e f e r m e n t at i o n
m ay ac c o u n t f o r t h e i n i t i al l o s s i n t h e s o l u b l e
p r o t e i n . A t 1 9 2 h , s o l u b l e c r u d e p r o t e i n f o r t h e
m o l d was ab o u t 9 % v e r s u s 1 2% i n t h e m o l d p l u s
ye as t f e r m e n t at i o n .
C h an g e s i n t o t al an d s o l u b l e s o l i d s , t o t al an d
s o l u b l e c r u d e p r o t e i n , r e d u c i n g s u g ar s , p H , an d
t o t al an d v o l at i l e ac i d i t y we r e i n g e n e r al s i m i -
l ar f o r r i c e f e r m e n t e d wi t h E . f i b u l i g e r a, C .
l ac t o s a, C . m e l i n i i , an d C . p ar ap s i l o s i s ( T ab l e
4 ) . T o t al s o l i d s d e c r e as e d at a s l o we r r at e i n r i c e
f e r m e n t e d wi t h H . an o m al a, H . m al an g a, an d
H . s u b p e l l i c u l o s a, an d c o n s e qu e n t l y t h e p e r -
c e n t ag e s o f t o t al an d s o l u b l e p r o t e i n s at e ac h
i n t e r v al we r e al s o l o we r . I n c o n t r as t , t h e p e r -
c e n t ag e o f s o l u b l e s o l i d s at e ac h i n t e r v al was
h i g h e r .
H i g h e r c o n c e n t r at i o n s o f r e d u c i n g s u g ar s
we r e at t ai n e d wi t h t h e H an s e n u l a s p e c i e s t h an
wi t h t h e E . f i b u l i g e r a an d t h e C an d i d a s p e c i e s
s t u d i e d . T h e r e we r e n o ap p r e c i ab l e d i f f e r e n c e s
i n p H . F o r al l ye as t s s t u d i e d , t o t al an d v o l at i l e
ac i d i t y f o l l o we d s i m i l ar p at t e r n s o f i n c r e as e
t h r o u g h 9 6 h . A t 9 6 h , an d al s o at 1 9 2 h , t h e
H an s e n u l a s p e c i e s p r o d u c e d h i g h e r l e v e l s o f
v o l at i l e ac i d i t y t h an t h at o b s e r v e d f o r o t h e r
ye as t s . T o t al ac i d i t y was ap p r e c i ab l y h i g h e r
o n l y i n r i c e f e r m e n t e d f o r 1 9 2 h wi t h H . an o m -
al a an d H . s u b p e l l i c u l o s a.
I n r i c e f e r m e n t e d wi t h E . b u r t o n i i p l u s A .
r o u x i i ( F i g . 4 ) , t h e r e was l e s s d e c r e as e i n t o t al
s o l i d s , wh i c h wo u l d ac c o u n t f o r a l o we r p e r c e n t -
ag e o f c r u d e p r o t e i n c o m p ar e d wi t h f e r m e n t a-
t i o n s i n v o l v i n g c e r t ai n ye as t s i n T ab l e 4 . T h e
h i g h e s t c o n c e n t r at i o n o f b o t h s o l u b l e s o l i d s an d
r e d u c i n g s u g ar s o c c u r r e d i n r i c e f e r m e n t e d
wi t h E . b u r t o n i i p l u s A . r o u x i i . I n t yp i c al t ap e
k e t an f e r m e n t e d f o r 4 8 h , t h e r e we r e n o ap p r e -
V O L . 3 3 , 1 9 7 7

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T A B L E 4 . C h an g e s i n t o t al an d s o l u b l e s o l i d s ( T S ; S S ) , t o t al an d s o l u b l e c r u d e p r o t e i n ( T C P ; S C P ) , r e d u c i n g
s u g ar s ( R S ) , p H , an d t o t al an d v o l at i l e ac i d i t y ( T A ; V A ) o c c u r r i n g i n r i c e f e r m e n t e d wi t h A . r o u x i i i n
c o m b i n at i o n wi t h v ar i o u s ye as t s a
T A
( m e q
V A
( m e q
R i c e f e r m e n t e d wi t h : T i m e ( h ) T S ( % ) S S ( % ) T C P ( % ) S C P ( % ) R S ( % ) p H o f H +/ 1 0 0 o f H +/ 1 0 0
g ) g )
0 9 8 . 7 2. 2 8 . 4 7 . 8 0 . 1 6 . 8 3 . 0 0 0 . 26
A . r o u x i i + E . f i b u l i g e r a
A . r o u x i i + C . l ac t o s a
A . r o u x i i + C . m e l i n i i
3 6 9 2. 8 4 8 . 3 8 . 6 1 . 1 1 0 . 9 4 . 3
4 8 7 9 . 1 4 9 . 5 1 0 . 0 1 . 6 1 1 . 8 4 . 2
9 6 5 0 . 5 3 2. 9 1 6 . 1 5 . 8 7 . 9 4 . 3
1 9 2 4 3 . 9 3 0 . 9 1 8 . 4 1 0 . 6 7 . 0 4 . 4
3 6 9 2. 8 4 7 . 2 8 . 8 1 . 1 1 1 . 2 4 . 3
4 8 7 8 . 1 4 6 . 4 1 0 . 4 1 . 6 1 0 . 8 4 . 3
9 6 5 4 . 5 3 7 . 0 1 4 . 8 5 . 6 9 . 0 4 . 4
1 9 2 4 7 . 4 3 3 . 3 1 7 . 0 1 0 . 4 8 . 0 4 . 5
3 6 9 3 . 3 4 6 . 6 8 . 8 1 . 1 1 0 . 9 4 . 4
4 8 7 7 . 7 4 7 . 8 1 0 . 2 1 . 6 1 1 . 4 4 . 2
9 6 5 5 . 7 3 7 . 5 1 4 . 6 6 . 8 9 . 2 4 . 5
1 9 2 4 3 . 7 3 0 . 7 1 7 . 7 1 1 . 4 7 . 5 4 . 5
A . r o u x i i + C . p ar ap s i l o s i s 3 6 9 3 . 5 4 5 . 6 8 . 6 1 . 1 1 0 . 4 4 . 4
4 8 8 0 . 0 4 9 . 5 1 0 . 2 1 . 6 1 1 . 9 4 . 2
9 6 5 4 . 2 3 6 . 8 1 4 . 9 6 . 0 9 . 0 4 . 3
1 9 2 5 1 . 2 3 7 . 0 1 6 . 5 1 0 . 0 8 . 9 4 . 4
3 . 4 0
4 . 1 8
5 . 23
5 . 8 1
0 . 3 6
0 . 6 2
0 . 8 1
0 . 8 5
3 . 4 2 0 . 3 3
3 . 9 5 0 . 6 8
5 . 3 3 0 . 8 0
5 . 7 4 0 . 9 9
3 . 3 3 0 . 3 1
4 . 0 1 0 . 5 5
5 . 1 1 0 . 7 1
5 . 7 3 0 . 9 1
3 . 20
4 . 4 8
5 . 6 7
6 . 4 4
0 . 3 6
0 . 5 2
0 . 7 1
0 . 9 5
A . r o u x i i + H . an o m al a
A . r o u x i i + H . m al an g a
3 6 9 6 . 3 5 2. 1 8 . 2 0 . 9 1 1 . 2 4 . 4
4 8 8 4 . 6 5 4 . 1 9 . 4 1 . 2 1 3 . 0 4 . 4
9 6 6 7 . 9 5 1 . 5 1 1 . 8 4 . 1 1 2. 9 4 . 4
1 9 2 5 7 . 0 4 6 . 0 1 4 . 0 7 . 7 1 1 . 4 4 . 3
3 6 9 6 . 4 5 1 . 8 8 . 2 1 . 0 1 0 . 2 4 . 2
4 8 8 5 . 8 6 2. 7 9 . 4 1 . 2 1 2. 9 4 . 2
9 6 6 7 . 4 5 1 . 4 1 2. 1 4 . 2 1 2. 7 4 . 4
1 9 2 5 8 . 7 4 8 . 7 1 3 . 8 8 . 0 1 1 . 9 4 . 4
A . r o u x i i + H . s u b p e l l i c u - 3 6 9 6 . 2 6 2. 3 8 . 4
l o s a 4 8 8 7 . 0 6 0 . 5 9 . 1
9 6 6 4 . 0 4 8 . 8 1 2. 6
1 9 2 5 0 . 4 3 9 . 2 1 5 . 6
1 . 0 1 2. 4 4 . 2
1 . 1 1 4 . 4 4 . 3
3 . 8 1 2. 4 4 . 3
8 . 0 1 0 . 1 4 . 3
3 . 3 8 0 . 3 3
3 . 6 4 0 . 6 5
5 . 6 9 1 . 8 8
8 . 1 1 3 . 6 8
3 . 8 5 0 . 28
4 . 20 0 . 4 7
5 . 6 8 1 . 3 3
6 . 8 5 1 . 5 8
3 . 8 3 0 . 3 3
3 . 7 7 0 . 4 9
6 . 24 1 . 5 5
7 . 0 5 2. 7 8
a
E ac h e n t r y i s a m e an o f t r i p l i c at e an al ys e s .
T A B L E 5 . C h an g e s i n t h e t h i am i n e c o n c e n t r at i o n o f T A B L E 6 . T h i am i n e c o n c e n t r at i o n o f r i c e f e r m e n t e d
r i c e f e r m e n t e d wi t h A . r o u x i i al o n e an d i n f o r 4 8 h wi t h A . r o u x i i i n c o m b i n at i o n wi t h v ar i o u s
c o m b i n at i o n wi t h E . b u r t o n i i at 3 0 C ye as t s at 3 0 C
T h i am i n e ( m g / 1 0 0 g ) a
O r g an i s m s
F e r m e n t at i o n wi t h :
0
h 4 8 h 7 2 h 1 9 2 h
U n f e r m e n t e d r i c e
A . r o u x i i 0 . 0 4 0 . 0 7 0 . 1 0 0 . 1 2 A . r o u x i i + C . l ac t o s a
A . r o u x i i + E . b u r t o n i i 0 . 0 4 0 . 1 1 0 . 1 2 0 . 1 2 A . r o u x i i + C . m e l i n i i
a
E ac h
e n t r y i s
a m e an
o f d u p l i c at e an al ys e s .
A . r o u x i i + C . p ar ap s i l o s i s
b K L e n t r
i s
*
o f
e
A .
r o u x i i
+ H . an o m al a
L e n g t h o f f e r m e n t at i o n . A . r o u x i i + H . m al an g a
A . r o u x i i + H . s u b p e l l i c u l o s a
T h i am i n e ( m g / 1 0 0
g ) a
0 . 0 4
0 . 1 3
0 . 1 3
0 . 1 3
0 . 1 2
0 . 1 1
0 . 1 2
c i ab l e d i f f e r e n c e s b e t we e n t h e r i c e f e r n e n t e d
a E ac h e n t r y i s a m e an o f d u p l i c at e an al ys e s .
c i ab l e d i f f e r e n c e s b e t we e n t h e r i c e f e r m e n t e d
wi t h A . r o u x i i s e p ar at e l y an d t h at f e r m e n t e d
b y t h e m o l d i n c o m b i n at i o n wi t h t h e s e v ar i o u s ab l e b u t l ac k e d t h e d e s i r ab l e f l av o r as s o c i at e d
ye as t s , o r wi t h E . b u r t o n i i . O r g an o l e p t i c al l y, wi t h t ap e k e t an p r e p ar e d b y u s i n g E . b u r t o n i i .
t h e p r o d u c t s p r e p ar e d wi t h t h e C an d i d a s p e - T h e t h i am i n e c o n t e n t o f t h e r i c e i n c r e as e d
c i e s , E . f i b u l i g e r a, an d H . m al an g a we r e p al at - n e ar l y t h r e e t i m e s as a r e s u l t o f f e r m e n t at i o n
U n f e r m e n t e d
A P P L . E NV I R O N. M I C R O B I O L .
1 0 7 2 C R O NK E T A L .

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B I O C H E M I C A L C H A NG E S D U R I NG T A P t F E R M E NT A T I O N 1 0 7 3
b y A . r o u x i i i n c o m b i n at i o n wi t h E . b u r t o n i i
( T ab l e 5 ) . T h e f e r m e n t at i o n o f r i c e b y o t h e r
ye as t s , e ac h i n c o m b i n at i o n wi t h A . r o u x i i , al s o
r e s u l t e d i n s i m i l ar i n c r e as e s i n t h e t h i am i n e
c o n c e n t r at i o n ( T ab l e 6 ) .
L I T E R A T U R E C I T E D
1 . A s s o c i at i o n o f O f f i c i al A n al yt i c al C h e m i s t s . 1 9 7 0 . O f f i -
c i al m e t h o d s o f an al ys i s , 1 1 t h e d . A s s o c i at i o n o f O f i -
c i al A n al yt i c al C h e m i s t s , Was h i n g t o n , D . C .
2. B o e d i j n , K . B . 1 9 5 8 . No t e s o n t h e M u c o r al e s o f I n d o n e -
s i a. S yd o wi a A n n . M yc o l . 1 2: 3 21 - 3 6 2.
3 . D j i e n , K . S . 1 9 7 2. T ap e f e r m e n t at i o n . A p p l . M i c r o b i o l .
23 : 9 7 6 - 9 7 8 .
4 . D wi d j o s e p u t r o , D . , an d F . T . Wo l f . 1 9 7 0 . M i c r o b i o l o g i -
c al s t u d i e s o f I n d o n e s i an f e r m e n t e d f o o d s t u f f s . M yc o -
p at h o l . M yc o l . A p p l . 4 1 : 21 1 - 222.
5 . E l l i s , J . J . , L . J . R h o d e s , an d C . W. H e s s e l t i n e . 1 9 7 6 .
T h e g e n u s A m yl o m yc e s . M yc o l o g i a L X V I I I : 1 3 1 - 1 4 3 .
6 . F r e e d , M . 1 9 6 6 . M e t h o d s o f v i t am i n as s ay. A s s o c i at i o n
o f V i t am i n C h e m i s t s , I n c . , 3 r d e d . I n t e r s c i e n c e P u b -
l i s h e r s , Ne w Y o r k .
7 . H as s i d , W. Z . , an d E . F . Ne u f e l d . 1 9 6 4 . Q u an t i t at i v e
d e t e r m i n at i o n o f s t ar c h i n p l an t t i s s u e s , p . 3 3 - 3 6 . I n
R . L . Wh i s t l e r ( e d . ) , M e t h o d s i n c ar b o h yd r at e c h e m -
i s t r y, v o l . 4 . A c ad e m i c P r e s s I n c . , Ne w Y o r k .
8 . H e s s e l t i n e , C . W. 1 9 6 5 . A m i l l e n n i u m o f f u n g i , f o o d an d
f e r m e n t at i o n . M yc o l o g i a 5 7 : 1 4 9 - 1 9 7 .
9 . M c C r e ad y, R . M . 1 9 7 0 . S t ar c h an d d e x t r i n , p . 5 5 4 - 5 5 6 .
I n M . A . J o s l yn ( e d . ) , M e t h o d s i n f o o d an al ys i s ,
2n d e d . A c ad e m i c P r e s s I n c . , Ne w Y o r k .
1 0 . M o r r i s , D . L . 1 9 4 8 . Q u an t i t at i v e d e t e r m i n at i o n o f c ar -
b o h yd r at e wi t h D r e ywo o d ' s an t h r o n e r e ag e n t . S c i -
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1 1 . S ao n o , S . , I . G an d i ar , T . B as u k i , an d H . K ar s o n o . 1 9 7 4 .
M yc o f l o r a o f " r ag i " an d s o m e o t h e r t r ad i t i o n al f e r -
m e n t e d f o o d s o f I n d o n e s i a. A n n . B o g o r . 5 : 1 8 7 - 20 4 .
1 2. S h al l e n b e r g e r , R . S . , an d R . G . M o o r e s . 1 9 5 7 . Q u an t i -
t at i v e d e t e r m i n at i o n o f r e d u c i n g s u g ar s an d s u c r o s e
s e p ar at e d b y p ap e r c h r o m at o g r ap h y. A n al . C h e m .
29 : 27 - 29 .
1 3 . S o e d ar s o n o , J . 1 9 7 2. S o m e n o t e s o n " r ag i t ap e , " an
i n o c u l u m f o r " t ap e " f e r m e n t at i o n . I l m u P e r t an i an
1 : 23 5 - 24 1 .
1 4 . We n t , F . A . F . C . , an d H . C . P . G e e r l i g s . 1 8 9 5 . B e o -
b ac h t u n g e n u b e r d i e H e f e ar t e n u n d Z u c k e r b i l d e n d e n
P i l z e d e r A r ac k f ab r i k at i o n . V e r h . K . A k ad . We t .
A m s t e r d am S e r . I I 4 : 3 - 3 1 .
V O L . 3 3 , 1 9 7 7

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