producción fitasa por aspergillus ficuum mediante ssf en torta canola

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0960-8524/95/ 9.50

P R O D U C T I O N O F P H Y T A S E D U R I N G S O L ID S T A T E

F E R M E N T A T I O N U S I N G SP E RG I L L U S F I C U U M N R R L 3 3 5

I N C A N O L A M E A L

A. Ebune, S. AI-Ash eh Z. Duvnjak*

Department of Chemical Engineering, University o f O ttawa, Ottawa, Ontario, Canada, KI N 6N5

(Received 8 N ovem ber 1994; revised version received 8 March 1995; accepted 10 March 1995)

bs t ra c t

Ef fect s o f mois ture content o f media , inoculum age

and hom ogen i z a ti on on p r oduc t i on o f phy t a s e and

reduct ion o f phyt ic acid con tent in canola me al byAs perg i l l u s f i cuum NRRL 3135 dur ing s ta t ic so l id-

state fermentat ion (SSF) have been considered.

Op t i m um mo i s tu r e con t en t o f med i a f o r t hese p r o -

cesses was 64 . Rate o f phytase produc t ion increased

wi th an increase in inoculum age between 2 and 5

days. B oth the level and the ra te o f phytase product ion

were lower in in i tia l s tages o f SSF when inoculu m was

homogenized for longer per iods o f t ime, whi le a f ter

36 h o f the process an increase in phytase product ionwas noticed with an increase in homogenizat ion t ime.

M athe ma tical correlat ions o f phy tase act ivi ty an d

phyt ic acid content reduct ion wi th mois ture content

and w i t h ti me o f homogen i z a ti on o f i nocu l um are p r o -

posed in this work.

Key words: P hy t as e , s o l i d - s t a t e f e rm en t a t i on , phy t a t e ,

c a n o l a m e a l , Aspergi llus f i cu um .

I N T R O D U C T I O N

C ano l a m ea l , wh i ch i s a by -p roduc t o f cano l a -o i lp roces s i ng , i s a good s ou rce o f p ro t e i n fo r an i m a l s ;

i t c o n t a i n s 3 7 - 4 0 p r o t e i n w i t h a m o r e f a v o r ab l e

p a t t e r n o f e s s e n t i a l a m i n o a c i d s t h a n s o y b e a n m e a l

(Clandin in , 1986) .

C a n o l a m e a l c o n t a i n s a b o u t 4 - 6 p h y t ic a c id .

P hy t i c ac i d r educ es b i oava i l ab il i ty o f d i e t a ry m i ne ra l

e l e m e n t s ( F o r d et al., 1978) and i nh i b i t s enzym es

s uch as a - am y l as e (S harm a et al., 1978), t rypsin, tyr-

o s i nas e and peps i n (Gra f , 1986 ) . There fo re , i t i sn e c e s s a r y t o r e d u c e p h y t i c a c i d c o n t e n t i n c a n o l a

m ea l , i n o rde r t o enhance i t s va l ue .

P h y t a s e is a p h o s p h o m o n o e s t e r a s e c a p a b l e o fhyd ro l yz i ng phy t i c ac i d t o y i e l d i no rgan i c o r t hophos -

*Au thor to w hom corre sponde nce should be addressed.

pha t e and a s e r i e s o f l ower e s t e r s o f m yo- i nos i t o l

and , u l t im a t e l y , f r ee m yo- i nos i t o l ( I rv i ng & C os -

grove, 1972) . This enzyme i s widely d i s t r ibuted in

p l an t s , an i m a l t i s s ues and m any s pec i es o f fung i and

bac t e r i a (C os g rove , 1966 ) . Th i s enzym e re l eas es

i n o r g a n ic p h o s p h a t e s f r o m p h y t ic a c i d i n s e e d s t h a t

have i nhe ren t phy t as e , e . g . beans (C hang et al.,

1977) , co t t on s eeds , s oybean (Han et al., 1987) and

wheat (Peers , 1953) .

However , t he re a re s eeds , s uch as cano l a , t ha t

con t a i n co ns i de rab l e qu an t i t i e s o f phy t ic ac id , bu t do

n o t h a v e p h y t a s e ; h e n c e , o t h e r m e a n s o f r e d u c i n g

phy t i c ac i d con t en t a r e neces s a ry . R ack i s (1974 )repo r t ed t ha t au t oc l av i ng o f s oy is o l a t e fo r 4 h a t

115° C des t royed m os t o f t he phy t i c ac i d i n t h i s

m a t e r i a l . I n t he co t y l edon , phy t i c ac i d was r educed

t o v a l u e s o f 3 2 - 6 8 a n d 1 8 - 6 8 d u r i n g r o a s t in g

and au t oc l av i ng , r e s pec t i ve l y (Hus s a i n et al., 1989).

U n f o r t u n a t e l y , t h e s e m e t h o d s a r e n o t s u i t a b l e , d u e

t o am i no ac i d des t ruc t i on . P hy t i c ac i d was a l s o

r e d u c e d f r o m r a p e s e e d f l o u r u s i n g v a r i o u s a m o u n t s

o f e t hy l ened i am i ne- t e t r a ace t i c ac i d (0 .25 -0 .75 M) a t

pH 9 -0 , and C aC I2 (0 .25 -0 .75 M) a t pH 3 -5 , bu t

n i t rogen l o s s es we re obs e rved .

I n o r d e r t o a v o i d t h e m e n t i o n e d d i s a d v a n t a g e s ,m i c r o b ia l e n z y m e s c a n b e u s e d f o r r e d u c t i o n o f p h y -

t i c ac i d con t en t i n t hes e com m od i t i e s .

S evera l m i c roo rgan i s m s we re t e s t ed fo r t he i r ab il -

i ty t o p rod uce phy t as e ; one o f t he m os t e f f i c ien t was

Aspergil lus f icuum (S h i eh & Ware , 1968 ) . Th i s

m i c r o o r g a n i s m w a s u s e d i n a s t u d y o f t h e r e d u c t i o n

o f phy t i c ac i d con t en t i n cano l a m ea l i n s o l i d - s t a t e

fe rm en t a t i on (Na i r & Duvn j ak , 1990 ) .

In t h i s work t he e f f ec t o f i nocu l um age , i t s con -

c e n t r a t i o n a n d h o m o g e n i z a t i o n , a n d t h e m o i s t u r e

c o n t e n t o f c a n o l a m e a l m e d i u m o n p h y t a s e p r o d u c -

t i on u s i ng A . f i c u u m i n an S S F p roces s was s t ud i ed .I n a d d i t io n , m a t h e m a t i c a l m o d e l s w e r e g i v e n t h a t

r e l a te t h e p h y t a s e p r o d u c t i o n a n d r e d u c t i o n o f p h y -

t ic a c i d c o n t e n t w i th t h e m o i s t u r e c o n t e n t l e v e l a n d

t h e t i m e o f i n o c u l u m h o m o g e n i z a t i o n .

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8 A. Eb une S. Al-Asheh Z. Duvnjak

M E T H O D S

M ic r oor gan i sm m e d ia an d c on d i t ion s o f gr ow t h

Aspergillus ficuu m N R R L 3 13 5 w a s u s e d i n t h is w o r k

a s a p r o d u c e r o f p h y t a s e . T h e m i c r o o r g a n i s m w a s

m a i n t a i n e d o n a s o li d m e d i u m c o m p o s e d o f 4 .5 %

ma l t a g a r , 0 . 5 % g lu c o s e , 0 . 5 % y e a s t e x t r a c t a n d d i s -t i l le d w a t e r . T h e m e d i u m w a s s t e r i l i z e d a t 1 1 5° C f o r

1 5 m i n , i n o c u l a t e d a f t e r c o o l i n g a n d i n c u b a t e d a t

3 0 ° C . A f t e r t h e t h i r d o r f o u r th d a y , s p o r e s w e r e

f o r m e d . T h e s l a n t s w e r e s t o r e d a t 4 ° C f o r f u r t h e r

us e .

Aspergillus ficuu m w a s f o u n d t o g r o w w e l l o n a

r o t a r y s h a k e r ( 2 0 0 r p m) a t 3 0 °C in t h e f o r m o f p e l -

l e ts i n a li q u id m e d i u m , c o m p o s e d o f 0 -8 % n u t r i e n t

b r o th , 0 5 % g lu c o s e , 0 . 5 % y e a s t e x t r a c t , a n d d i s t i l l e d

w a t e r . A f t e r g r o w t h , t h e c u l t u r e w a s h o m o g e n i z e d

( b e t w e e n 3 0 - 1 2 0 s ) a n d u s e d a s t h e i n o c u l u m f o r

th e S S F p r o c e s s .

F i f t y g r a m s o f c a n o l a m e a l a n d 5 0 m l o f d i s t i l l e d

w a t e r ( u n l e s s o t h e r w i s e s t a t e d ) w e r e p u t i n 5 0 0 m l

E r l e n m e y e r f l as k s a n d a u to c l a v e d a t 1 2 1 °C f o r 45

min . T h e f l a s k s w e r e t h e n a l l o w e d t o c o o l , i n o c u -

l a t e d w i t h h o m o g e n i z e d i n o c u l u m a n d i n c u b a t e d

s t a t i c a l l y a t 3 0 ° C . A l l t h e t e s t s w e r e c a r r i e d o u t i n

d u p l i c a t e , a n d t h e r e s u l t s s h o w n a r e a v e r a g e v a lu e s .

A n alys is o f sam p le s

Enzyme preparation and assa y procedure. E n z y m e

a ct iv it y an d a m o u n t o f e n z y m e p r o d u c e d w e r e d e t e r -

m i n e d i n t h e c r u d e e n z y m e e x t ra c t e d f ro m c a n o l a

m e a l c u l t u r e d u r i n g t h e f e r m e n t a t i o n p r o c e s s .F iv e mi l l i l i t e r s o f a 2 % a q u e o u s s o lu t i o n o f

C a C 1 2 . 2 H 2 0 w e r e u s e d f o r e x t r a c t i o n o f p h y t a s e

f r o m a 1 g s a m p l e b y c o n t i n u o u s s h a k i n g o n a r o t a r y

s h a k e r ( 2 0 0 r p m ) f o r 1 h a t 2 5 ° C . T h e l i q u id w a s

s q u e e z e d o u t t h r o u g h d o u b l e - l a y e r c h e e s e - c l o t h a n d

c e n t r i f u g e d ( 5 0 0 0 g ; 1 5 min ; 4 ° C ). T h e c l e a r s u p e r -

n a t a n t w a s d e s i g n a t e d t h e c r u d e e n z y m e .

P h y t a s e a c ti v it y w a s a s s a y e d b y m e a s u r i n g t h e

a m o u n t o f p h o s p h o r u s r e l e a s e d b y u s in g s o d i u m

p h y t a t e a s t h e s u b s t r a t e . A r e a c t i o n m i x t u r e c o n -

s i s t e d o f 0 2 M a c e t a t e b u f f e r ( p H 4 . 7) , 1 ml o f 1 .5

m M s o d i u m p h y t a t e a n d 0 1 m l o f c r u d e e n z y m e .T h e r e a c t i o n s w e r e c a r r i e d o u t a t 6 0° C f o r 1 0 m i n ,

a n d t h e n s t o p p e d b y a d d i n g 5 m l o f 1 0 % t r i c h lo r o

a c e t i c a c id . S p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f t h e

p h o s p h o r u s r e l e a s e d w a s c o n d u c t e d u s i n g T a u s s k y -

S c h o o r r e a g e n t , a s d e s c r i b e d b y H a r l a n d a n d

H a r l a n d ( 1 9 8 0 ) .

O n e u n i t o f p h y t a s e a c ti v it y w a s d e f i n e d a s t h e

a m o u n t o f p h y t a s e r e q u i r e d t o r e l e a s e 1 m g o f p h o s -

p h o r u s f r o m 1 m l o f 1 .5 m M s o d i u m p h y t a t e p e r

h o u r a t a g i v e n t e m p e r a t u r e a n d p H .

Phytic acid. A p p r o x i m a t e l y 3 g o f s a m p l e s w e r e

t a k e n i n 1 5 0 m l E r l e n m e y e r f la s ks , a n d p h y t i c ac i dw a s e x t r a c t e d u s in g 5 0 m l o f 2 .4 % H C I u n d e r c o n -

t i n u o u s s h a k i n g ( 2 00 r p m ) f o r 1 h . A f t e r c e n t r i fu g i n g

( 6 0 0 0 g , 1 5 m i n ) , t h e s u p e r n a t a n t w a s c o l l e c t e d a n d

p h y t i c a c i d m e a s u r e d b y t h e H a u g a n d L a n t z s c h

m e t h o d ( 19 8 3 ).

R E S U LT S A N D D I S C U S S I O N

T h e w a t e r c o n t e n t o f t h e s u b s t r a t e p l a y s an i m p o r -

t a n t r o l e i n b o t h c e ll g r o w t h a n d e n z y m e p r o d u c t i o ni n s o l i d - s t a t e f e r m e n t a t i o n . F o r e x a m p l e , H a n et al.( 1 98 7 ) f o u n d t h a t t h e o p t i m u m l e v e l o f m o i s t u r e f o r

p h y t a s e p r o d u c t i o n a n d c e l l g r o w t h i n s o y b e a n m e a l

u s in g A. f icuum i s 2 5 - 3 5 % a n d a b o u t 5 0 % , r e s p e c -

t i v e ly . T h e l e v e l o f p h y t a s e p r o d u c e d w a s d r a s t i c a l l y

r e d u c e d w h e n t h e w a t e r c o n t e n t e x c e e d e d 4 0 % . N a i r

a n d D u v n j a k ( 1 9 9 0 ) r e p o r t e d r e l a t iv e l y f a st r e d u c -

t i o n o f p h y t i c a c i d c o n t e n t i n c a n o l a m e a l u s i n g A .

f icuum i n S S F w h e n t h e m o i s t u r e c o n t e n t w a s 6 4 % .

T h e r e s u l t s f r o m th i s s t u d y [ F ig . l ( a ) ] s h o w th a t t h e

a m o u n t o f e n z y m e p r o d u c e d i n c r e a s e s w i t h a n

i n c r e a s e i n m o i s t u r e c o n t e n t u p t o 6 4 % . T h e s e

r e s u lt s c a n b e c o r r e l a t e d m a t h e m a t i c a l l y u s i n g t h e

lo g i s ti c l a w ty p e o f e q u a t i o n :

v

d t O~mY 1 - ( 1 )

. , . 4

4

3

1

75

5

2 5

0

4

A

I I ~ I

v

o

o.M

a

b

48 72 96 12

r i m e h )

B

Fig. 1. Effect of mo isture conten t on : (a) productionof phytase , (b) reduction of phytic acid content dur ingSSF. (Moisture , %: o - - 31; A - - 39; + - - 45; x - - 50;

o 64.)

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Production of phytase during solid state fermentation 9

w h e r e v i s t h e p h y t a se a c t i v i t y , V m i s the

m a x i m u m p h y t a s e a c t iv i ty w h i c h c a n b e a c h i e v e d

theoretically, 0~m i s a n e mp i r i c a l c o n s t a n t a n d t is t h e

t i m e o f f e r m e n t a t i o n . I n t e g r a t i o n o f e q n ( 1 ) g iv e s

V m

v = (2)

v m )1 + - 1 e . . . .A) o

w h e r e Vo i s t h e i n i ti a l p h y t a se a c t i v it y a f t e r i n o c u l a -

t ion .

T h e v a l u e s o f t h e p a r a m e t e r s vm a n d a m f o r d i f f er -

e n t m o i s t u r e l e v e ls w e r e c a l c u l a t e d u s in g e q n ( 2 )

a n d a p p l y i n g t h e l e a s t s q u a r e s t e c h n i q u e ( M a r -

q u a r d t , 1 96 3) f o r t h e s e t o f e x p e r i m e n t a l d a t a f o r

e a c h m o i s t u r e c o n t e n t ( T a b l e 1 ) . F i g u r e 2 s h o w s

t h a t t h i s e q u a t i o n g i v e s o n l y a r o u g h e s t i m a t e o f t h e

e x p e r i m e n t a l d a t a , s i n c e e q n ( 2 ) d o e s n o t a c c o u n t

f o r t h e p h a s e w h e r e e n z y m e a ct iv i ty d e c r e a s e s .T h e r a t e s o f p h y t i c a c i d r e d u c t i o n i n c a n o l a m e a l

[ Fi g. l ( b ) ] w e r e i n a g r e e m e n t w i th t h e p r o d u c t i o n o f

p h y t a s e [F ig . l ( a ) ] ; h i g h e r m o i s t u r e c o n t e n t s w e r e

b e n e f i c i a l t o b o t h p r o c e s s e s . T h e r a t e o f p h y t i c a c i d

h y d r o ly s i s a t 6 4 m o i s tu r e [ Fig. l ( b ) ] w a s p r o p o r -

t i o n a t e l y m u c h h i g h e r t h a n a t 5 0 m o i s t u r e , w h e n

c o mp a r in g t h e i r e n z y me l e v e l s . I t i s p o s s ib l e t o

a s s u m e t h a t d i ff u s io n o f t h e p r o d u c e d e n z y m e w a s

e n h a n c e d b y i n c r e a s i n g t h e w a t e r c o n t e n t i n s o l i d -

s t a t e c u l t u r e s a n d t h a t , i n a d d i t i o n t o t h e h i g h e r

e n z y m e l e v e l , c o n t r i b u t e d t o t h e m u c h h i g h e r r a t e o f

p h y t i c a c i d r e d u c t i o n i n t h e s y s t e m c o n t a i n i n g 6 4m o i s t u r e .

T h e f o l l o w i n g e m p i r i c a l c o r r e l a t i o n c a n f i t t h e

r e s u lt s f o r p h y t i c a c id r e d u c t i o n f o r v a r i o u s m o i s t u r e

c o n t e n t s :

P = P o e x p ( - k t 2 (3)

w h e r e P i s t h e n o r m a l i z e d p h y t i c a c i d c o n c e n t r a t i o n

a t t ime t , P o i s t h e i n i t i a l n o r ma l i z e d p h y t i c a c id

c o n c e n t r a t i o n a n d k i s a n e m p i r i c a l c o n s t a n t . T o u s e

t h i s e q u a t i o n t o c a l c u l a t e p h y t i c a c i d c o n t e n t r e d u c -

t i o n d u r i n g S S F , t h e k c o n s t a n t w a s e v a l u a t e d f o r

e a c h m o i s t u r e c o n t e n t ( T a b l e 1 ) u s i n g t h e e x p e r i -

m e n t a l d a t a f r o m t h i s w o r k , a n d a p p l y i n g t h e l e a s t

s q u a r e s t e c h n i q u e . C o m p a r i s o n b e t w e e n t h e e x p e r i -

T a b l e 1 . M a x i m u m e n z y m e a c t iv i t y V m s p e c i f i c r a t e c o n -

s t a n t o f e n z y m e a c ti v i ty C ~m a n d t h e e m p i r i c a l c o n s t a n t

k f o r v a r i o u s m o i s t u r e c o n t e n t s

M oisture Maximum enzym e Specif ic ra te Empir icalconte nt activity, v_ constant, constant, k

( ) (units g ~') ctm (h -1 ) (h -2 )

31 2.53 0.057 0.0005839 3.22 0.075 0.0013945 3.48 0-077 0-0021950 4-29 0.082 0-0031864 4.71 0.096 0.01133

m e n t a l d a t a a n d t h o s e c a l c u l a t e d f r o m e q n ( 3 ) f o r

v a r i o u s m o i s t u r e c o n t e n t s ( F i g . 3 ) s h o w s t h a t t h e

s u g g e s t e d e m p i r i c a l m o d e l p r e d i c t s t h e p h y t i c a c i d

l e v e l i n c a n o l a m e a l w i t h v a r i o u s m o i s t u r e c o n t e n t s

v e r y w e l l .

T h e a g e o f i n o c u l u m u s e d i n t h i s s o l i d s t a t e c u l -

t u r e a f f e c te d th e a m o u n t o f e n z y m e p r o d u c e d . T h e5 a n d 2 d a y i n o c u l u m p r o d u c e d t h e l a r g e s t a n d t h e

l e a s t a mo u n t o f e n z y me , r e sp e c t i v e ly ( F ig . 4 ) .

Aspergillus ficuu m g r e w i n t h e l i q u i d m e d i u m i n

th e f o r m o f p e l l e t s , a n d i t w a s d i f f i c u l t t o c o n s i s -

t e n t l y t r a n s f e r t h e s a m e a m o u n t o f i n o c u l u m i n t o

t h e s o l i d m e d i u m . T h e r e f o r e , h o m o g e n i z a t i o n o f

i n o c u l u m w a s c o n s i d e r e d . E f f e c t s o f i n o c u l u m

h o m o g e n i z a t i o n t i m e o n p h y t a s e p r o d u c t i o n a n d

p h y t i c a c id r e d u c t io n a r e sh o w n in F ig . 5 ( a ) a n d ( b ) ,

r e sp e c t i v e ly . I n t h e f i r s t 2 4 h , t h e mo s t p h y t a se w a s

p r o d u c e d i n t h e m e d i a i n o c u l a t e d w i t h A. f i cuum

t h a t w e r e h o m o g e n i z e d f o r a s h o r t e r p e r io d o f t im e[ F ig . 5 ( a ) ] . H o w e v e r , a f t e r 4 8 h t h e s i t u a t i o n

r e v e r s e d . T h i s i m p l i e s t h a t t h e i n o c u l a e x p o s e d t o

h o m o g e n i z a t i o n f o r a s h o r t e r p e r i o d o f t i m e w a s le s s

d a m a g e d , w h i c h r e s u l t e d i n a h i g h e r i n i t i a l p h y t a s e

a c t i v i t y c o m p a r e d t o t h e i n o c u l a h o m o g e n i z e d f o r a

l o n g e r p e r i o d o f t i m e . S i n c e t h e i n o c u l a h o m o g e -

n i z e d f o r l o n g e r p e r i o d s h a d m o r e g r o w t h c e n t e r s ,

t h e y b e g a n t o e x h ib i t h ig h e r p h y t a se a c t i v i ty a f t e r a

5

3

0Dcn 2

0

5

~ 3

m 2

0

5

~ 3&1)

m 2

F i g . 2 .

B

i I n I i I i

I ~ I , I ,

2 4 48 7 96

T ime( I I )

o

i I F I i

2 4 4 8 7 2 9 6

Tim e (h)

Predicted (solid l ines) and experimental (sym-bols) results for phytase activity for different moisturecontents. (Moisture , : A -- 31; B - - 39; C - - 45; D --

50; E -- 64. )

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10 A. Ebu ne S. Al-Asheh Z. Duvnjak

I 0 0

7 5

5 O

0

1 0 0

75

~ 5 0

x~ e~

1

7 5

~o

.~ e s

I I I r I ,

i t . I f

E

.o

2 4 4 8 7 2 9 6 1 2 0

T i m e (1 1 )

, I I , I n

I i I L I I i

2 4 4 8 7 2 9 6 1 2 0

T i m e ( h )

Fig. 3 . Pred icted (sol id l ines) and expe r imen tal (sym-bo l s) r esu lt s fo r phy t i c ac id con t en t r educ t i on fo r d i f f e ren tmoi s tu re con t en t . (Moi s tu re , : A - - 31 ; B - - 39; C - -

45; D -- 50; E - - 64. )

v

0

e~

Fig . 4 .

6 0

0

4 0

30y2 0

1 0

0 . 0 I , I , I ,

0 g 4 4 8 7 2 9 6 1 2 0

T i m e h )

Effec t o f i nocu lum age on p roduct i on o f phy t asedur ing SSF. (Age, days: © - - 2; zx - - 3 ; + - - 4 ; x - - 5 . )

5

~

A

, I , I , I ,

2 4 4 a 7 2 9 a

T i m e h )007

O 5 0

0 2 4 4 8 7 2

T i m e h )

Fig . 5 . Ef fec t o f t he t ime o f hom ogen i za t i on o f i nocu lumon : ( a ) phy t ase p roduct i on , (b ) r educ t i on o f phy t i c ac idcon t en t i n cano l a meal du r ing SSF. (Time, seconds : o - -

10; m - - 30; + - - 60; X -- 120; O - - 240.)

T a b l e 2 . M a x i m u m e n z y m e a c t iv i ty , V m, s p e c i f ic r a t e c o n -

s t a n t o f e n z y m e a c t iv i ty , ~ m , a n d t h e e m p i r i c a l c o n s t a n t ,k , fo r v a r io u s t i m e s o f h o m o g e n i z a t i o n

Tim e o f Ma x imum Specif ic r a t e Em pi r i ca lhom ogen i za t i on en zyme cons t an t , cons t an t , k

(s) activity, v~ ~ m (h - l ) (h -3 × 10 -5 )(un i t s g - f )

10 2.43 0.1439 1.4730 3.15 0-0920 1.7260 3.50 0.0805 2.17

120 3.80 0.0778 2-39240 3-14 0.0787 3.09

r e c o v e r y p e r i o d o f a b o u t 3 6 h . A l t h o u g h t h e e n z y m e

c o n c e n t r a t i o n i n t h e f i r st 2 4 h w a s t h e l o w e s t i n t h e

s y s t em i n o c u l a t e d w i th t h e m o s t h o m o g e n i z e d i n o c u -

l u m , p h y t i c a c i d h y d r o l y s i s w a s s t il l t h e f a s t e s t o f a l l

s y s t e m s [ F i g . 5 ( b ) ] . W e a s s u m e t h a t t h e i n o c u l u m

w a s m o r e e v e n l y d i s t ri b u t e d t h r o u g h o u t t h e m e d i u m

a n d t h e r e f o r e p h y t a s e w a s i n b e t t e r c o n t a c t w i th

p h y t i c a c i d .

E q u a t i o n ( 2 ) w a s u s e d t o c o r r e l a t e t h e r e s u l t s o f

p h y t a s e a c t i v it y [F i g. 5 ( a )] w i t h v a r i o u s t i m e s o f

h o m o g e n i z a t i o n • T h e v a l u e s o f t h e p a r a m e t e r s ~ m

a n d vm w e r e e v a l u t e d ( T a b l e 2 ) f o r d i f f e r e n t t i m e s

o f h o m o g e n i z a t i o n u s in g th e e x p e r i m e n t a l d a t a a n d

t h e l e a s t s q u a r e s t e c h n i q u e , a s m e n t i o n e d p r e v i -

o u s l y . I t a p p e a r s ( F i g . 6 ) t h a t e q n ( 2 ) f it s t h e

e x p e r i m e n t a l d a t a r e a s o n a b l y w e ll .

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5

3

aj

2

0

5

~ 3

m 2

0

5

~ 3

1 )

~ 2

i l l l

1 0 0

O

I I i I

75

50O

x~ 2s

Co

I i l ~ I i

0

, I h I , I

24 48 72 96

T i m e h )

i I I ,

24 48 7 2 96

Time (h)

Predicted (solid lines) and experimental (sym-bols) resul ts for phytase act iv i ty for various t imes ofinoculum homogenization. (Time, seconds: A - - 10; B - -

30; C - - 60; D - - 120; E - - 240. )

°

0

100 ,

75

0

5O0

~ z6

100 0 24 48

T i m e (h)

7 5

N~ 50o

x ~ 26

o C

24 48 72

Time (h)

P r o d u c t i o n o f p h y t a s e d u r i n g s o l i d s t a te f e r m e n t a t i o n 1 1

F i g 6

72

F i g 7. Pre dicted (solid lines) and experim ental (sym-bols) results for phytic acid content reduction for varioustimes of inoculum homogenization. (Time, seconds: A - -

10; B - - 30; C -- 60; D - - 120; E -- 240.)

T o s h o w t h e r e l a t io n s h i p b e t w e e n t i m e o f h o m o g -

e n i z a t io n a n d r e d u c t i o n o f p h y ti c a c id c o n t e n t , t h e

fo l l o wi n g e m p i r i c a l e q u a t i o n i s s u g g e s t e d :

P = P o e x p ( - k t 3 (4 )

T h e e m p i r i c a l c o n s t a n t , k , f o r d i f f e r e n t t i m e s o f

h o m o g e n i z a t i o n w a s c a l c u l a t e d ( T a b l e 2 ) u s i n g e q n

(4 ) , a n d a p p l y i n g t h e l e a s t s q u a re s t e c h n i q u e fo r t h e

d a t a f r o m F i g . 5 ( b ) . F i g u r e 7 s ho w s g o o d a g r e e m e n t

b e t w e e n t h e e x p e r i m e n t a l r e s u l t s a n d t h o s e c a l c u -l a t e d u s i n g e q n (4 ) fo r d i f f e r e n t t i m e s o f

h o m o g e n i z a t i o n .

C O N C L U S I O N S

O p t i m u m m o i s t u r e c o n t e n t o f t h e s o li d m e d i u m f o r

t h e p r o d u c t i o n o f p h y t a s e a n d h y d r o ly s i s o f p h y ti c

a c i d b y A . f i c u u m N R R L 3 1 35 is 6 4 . A n i n c r e a s e

i n i n o c u l u m a g e b e t w e e n 2 a n d 5 d a y s e n h a n c e d t h e

r a t e o f e n z y m e p r o d u c t i o n . I n t h e e a r l y st a g e s o f

S S F , l o w e r r a te s o f p h y t a s e p r o d u c t i o n w e r e n o t i c e d

i n t h e c u l t u r e s i n o c u l a t e d w i t h i n o c u l u m h o m o g e -

n i z e d fo r a l o n g e r p e r i o d o f t i m e , b u t a t l a t e r s t a g e s

t h e o p p o s i t e w a s o b s e r v e d .

T h e p r o p o s e d m o d e l s f o r p h y ta s e a c t iv i ty a n d

p h y t i c a c i d c o n t e n t r e d u c t i o n f o r v a r i o u s

m o i s t u r e l ev e ls o f s o li d m e d i u m a n d t i m e s o f

h o m o g e n i z a t i o n f i t t h e e x p e r i m e n t a l d a t a r e a s o n a b l y

well .

R E F E R E N C E S

Chang, R., Schwimmer, S. & Burr, H. K. (1977). Phytate:removal from whole dry beans by enzymatic hydrolysis

and diffusion. J. Food Sci ., 42 (5), 1098-101.Clandinin, D. R. (1986). Ca n o la M ea l fo r L ives to ck a n d

Poultry. Can ola Council of Can ada, Winnipeg, M ani-toba, pp. 1-19.

Courtois , J . & Joseph, G. (1947). Recherches sur la phy-tase 8 . Teneur en inosi tophosphate et act iv i t6phytasique de divers grains. Bull. Soc. Chem. Biol. , 30,195-8.

Cosgrove, D. J. (1966). The chemistry and bioch emis try ofinositol polyphosphates. Re v. Pure Appl. Chem. , 16,209-12.

Ford, J. R., Mustakas, G. C. & Schmutz, R. D. (1978).Phytic acid removal from soybeans by a l ip id proteinconcentrate process . J . Am . O il Chem. Soc. , 55, 371-4.

Graf, E. (1986). Phytic Acid-Chemistry and Applica tion .

The Pillsbury Co., Pilatus Press, Minneapolis, pp. 42-4.Ha n, Y. W., Gallagher, D. J. & Wilfred, A. G. (1987).

Phytase production by Aspergil lus f icuum on semi-solidsubstrate. J. Ind. Microbiol. , 2, 195-200.

Harland, B. F. & Harland, J . (1980). Fermentat ive reduc-

8/13/2019 Producción Fitasa Por Aspergillus ficuum Mediante SSF en Torta Canola

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1 2 A . E b u n e S . A l - A s h e h Z . D u v n j a k

t i o n o f p h y t a t e i n r ye , w h i t e a n d w h o l e w h e a t b r e a d s .Cereal Chem. 5 7 ( 3 ) , 2 2 6 - 9 .

Ha u g , W . L a n tz s c h , H . J . ( 1 9 8 3 ) . S e n s i t iv e m e th o d f o rr a p i d d e t e r m i n a t i o n o f p h y t a te i n c e r e a ls a n d c e r e a lp r o d u c t s . J . Sci . F ood Agric . 3 4 , 1 4 2 3 - 6 .

Hu ssa in , B. , Kh an , S . , I smail , M . Sa t ta r , A. (1989) .E f f e c t o f r o a s t in g a n d a u to c la v in g o n p h y t i c a c id c o n -te n t o f c h ic k p e a . Die Nahrung 3 3 ( 4 ) , 3 4 5 - 8 .

I rv ing , G. C. J . Co sgrov e , D. J . (1972) . Inos i to l pho s-p h a t e p h o s p h a t a s e o f m i c r o b i o lo g i c a l o r ig i n: t h e i n o s i to lp e n t a p h o s p h a t e p r o d u c t s o f Aspergil lus f icuu m p h y ta s e .J. Bacteriol. 1 1 2 , 4 3 4 - 8 .

M a r q u a r d t , D . W . ( 1 9 6 3 ) . A n a l g o r i t h m f o r l e a s t s q u a r e se s t i m a t i o n o f n o n l i n e a r p a r a m e t e r s . J . Soc. Ind. Appl.

Ma th . 1 1 , 4 3 1 - 4 1 .Na i r , V . C . Du v n ja k , Z . ( 1 9 9 0 ) . Re d u c t io n o f p h y t i c

a c i d c o n t e n t i n c a n o l a m e a l b y A . f i c u u m i n s o l id - s t a t ef e r m e n t a t i o n . Appl. Microbiol. Biotechnol. 3 4 ( 2 ) , 1 8 3 - 8 .

P e e r s , F . G . ( 1 9 5 3 ) . P h y ta s e o f wh e a t . Biochem. J . , 53 ,1 0 2 - 1 0 .

Ra c k i s , J . J . ( 1 9 7 4 ) . M in e r a l a v a i l a b i li ty in s o y p r o te inp r o d u c t s . J . Am . O il Chem. Soc. 5 1 , 1 6 1 - 4 .

S h a r m a , C . B . , Go e l , M . I r s h a d , M . ( 1 9 7 8 ) . M y o - in o s i -to l h e x a p h o s p h a te a s a p o te n t i a l in h ib i to r o f o r- amy la seo f d i f f e r e n t o r ig in s . Phytochemistry 1 7 , 2 0 1 - 4 .

S h ie h , T . R . W a r e , J . H . ( 1 9 6 8 ) . S u r v e y o f mic r o -o r g a n i s ms f o r th e p r o d u c t io n o f e x t r a c e l lu la r p h y ta s e .Appl. Microbiol. 1 6 ( 9 ) , 1 3 4 8 - 5 1 .