1995 Proteolysis during tempe fermentation.pdf

8/18/2019 1995 Proteolysis during tempe fermentation.pdf

1/9

Food M icrobiology

1995 12 39-47

Proteolysis du r ing tem pe ferm en tat ion

U B a u m a n n a n d B B i s p in g t

T h e p r o t e o l y t i c c a p a c i t y o f 3 6 s tr a i n s o f t he g e n u s

Rhizopus

i s o la t e d f r o m I n d o n e s ia n t e m p e

o r t e m p e i n o c u l a w a s e x a m i n e d . N o s i g n i f i c a n t c h a n g e s i n t he t o t a l a m o u n t o r p a t t e r n o f

a m i n o a c i d s c o u l d b e f o u n d , b u t t h e re w a s a d i s t i n c t in c r e a s e in t h e a m o u n t o f f re e a m i n o

a c id s . S t r a i n s w i t h a h ig h p r o t e o l y t i c a c t i v i t y w e r e f o u n d , w h i c h w e r e a b l e t o r el e as e n e a r l y

f iv e t i m e s m o r e a m i n o a c i d s a f t e r s t a n d a r d f e r m e n t a t i o n t h a n o t h e rs . C h a n g e s i n f e r m e n t a -

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

F e r m e n t a t i o n s w i t h m i x e d p o p u l a t i o n s o f b a c te r ia a n d Rhizopus y i e l d e d a l o w e r l e v e l o f f r e e

a m i n o a c i ds , b u t an i n c re a s e d t o t a l a m o u n t o f a m i n o a c i ds . Ex a m i n a t i o n o f p r o t e a s e s y s -

t e m s o f t h r e e

Rhizopus

s p e c i es s h o w e d t h a t t h e p r o t e a s e s o f t h e c e ll w a l l f r a c t io n w e r e

m o s t r e s p o n s i b l e f o r p r o t e o l y t i c c a p a c i t y o f t h e d i f f e r e n t s t r ai n s . O n a v e r a ge t h e i r a c t i v it y

a m o u n t e d t o 7 1 o f t h e t o t a l p r o t e o l y t i c ca p a c it y .

I n t r o d u c t i o n

T e m p e k e d e l a i i s a n I n d o n e s i a n f o o d s t u f f

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

i n g b a c k m a n y c e n t u r i e s i n J a v a . T o d a y i t i s

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

I n d o n e s i a n i s la n d s , i n J a p a n , i n t h e U S A , a n d

i n W e s t e r n E u r o p e . I t i s m a d e b y a t w o - s t e p

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

m a n y d i f f e r e n t b a c t e r i a a n d y e a s t s . T h i s f e r -

m e n t a t i o n i m p r o v e s s o m e f e a t u r e s o f s o y b e a n s

i n c l u d i n g f a t t y a c i d c o m p o s i t i o n ( H e r i n g e t a l .

1 9 91 ) , t h e l e v e l a n d p a t t e r n o f o l i g o sa c c h a -

r i d e s ( B a r z e t a l . 1 9 9 0 ) , a n d t h e a m o u n t o f

s e v e r a l v i t a m i n s , e s p e c i a l ly v i t a m i n B 12 a n d

v i t a m i n D ( K e u t h a n d B i s p i n g 1 9 9 3 , D e n t e r

a n d B i s p i n g 1 9 9 3 ) . T h e p r o d u c t i s a s o l i d c a k e ,

w h i c h i s c o n s u m e d i n t h e f o r m o f f r i e d s li c e s ,

a s a k i n d o f I n d o n e s i a n s a t a y , a s p e p p e r e d

*Dedica ted to Pro fesso r Dr H. -J . Rehm, on the

occasion of his 67 th bir thda y.

tCorrespond ing au thor .

p a s t e ( s a m b a l ) , o r a s v e g e t a r i a n t e m p e b u r g e r .

I t m a y b e a d d e d t o d r i n k s a n d t o s o u p s , o r

o f f e r e d a s c r a c k e r s ( S h u r t l e f f a n d A o y a g i 1 9 79 ,

S t e i n k r a u s 1 9 83 ). T o d a y 7 6 5 0 0 0 t o n n e s a r e

p r o d u c e d b y a r o u n d 4 0 0 0 0 h o m e m a n u f a c t u r -

e r s w i t h 1 3 0 0 0 0 e m p l o y e e s i n I n d o n e s i a a l o n e

( W i n a r n o a n d R e d d y 1 98 6 , K a r t a 1 9 8 7) .

O n e o f t e m p e ' s m o s t im p o r t a n t q u a l i t i e s is

t h e h ig h p r o t e i n l e v el u p to 4 0 o f t h e d r y

m a s s . D u e t o t h i s h i g h p r o t e i n c o n t e n t , t h e

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

t h e F A O / W H O a m i n o a c i d r e f e r e n c e p a t t e r n

w i t h t h e e x c e p t i o n o f m e t h i o n i n e a n d c y s t e i n e

( M u r a t a e t a l. 1 9 67 , W i n a r n o a n d R e d d y

1 9 8 6 ) . T h i s f a c t e x p l a i n s t h e g r e a t i n t e r e s t i n

t h i s f o o d i n m a n y d e v e l o p i n g c o u n t ri e s , w h i c h

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

t h e y o u n g p o p u l a t i o n .

O n e c h a r a c t e r is t ic o f t e m p e f e r m e n t a t i o n i s

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

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

a m i n o a c i d s ( W a g e n k n e c h t e t al . 1 96 1 , S t i ll -

i n g s a n d H a c k l e r 1 9 6 5 , M u r a t a e t a l . 1 9 6 7 ,

Re ce i ve d :

1 7 M a y 1 9 94

t n s t i tu t f r M i k r o -

b i o l o g ie ,

We s t f l i s ch e Wi l -

h e l m s - U n i v e r s i t t

M S n s t e r ,

Co r r e n ss t r a sse 3 ,

D - 4 8 1 4 9 M S n s t e r ,

G e r m a n y

0 7 4 0 - 0 0 2 0 / 9 5 / 0 1 0 0 3 9 + 0 9 0 8 . 0 0 /0 © 1 9 9 5 A c a d e m i c P r e s s L i m i t e d


2/9

4 0 U B a u m a n n a n d B B is p in g

Heri ng et al. 1991). An increase of the protein

efficiency ratio PER) of tempe was .attr ibute d

to a better availability of amino acids. Mu-

rat a et al. 1971) found in a rat feeding test

that the PER value of tempe was not

significantly different from that of unfer-

mented soybeans, hox~ever Zamora and

Veum 1979) reporte d an increase in the av-

erage daily weight gain for weaning rats fed

soybeans fermented with Rhizopus oligosporus

compared with the daily weight gain for rats

fed soybeans given the same heat treatment

but not fermented. The fermented soybeans

also had a greater apparent biological value

and net protein utilization.

Vitamin levels may also increase during

tempe fermentation. Keuth and Bisping 1993)

found t hat vita min B~2 format ion by Citrobac-

ter freundii during tempe fermentation was

strongly dependent on the metabolic activity

of Rhizopus.

The aim of this work was to find some Rhi-

zopus strains with high proteolytic activity

isolated from Indonesian tempe or tempe

starters, and to assess methods which could

improve the content of free amino acids in

tempe by changing the fermenta tion process.

Some aspects of the Rhizopus protease system

were characterized. Further, the influence of

co-fermenting bacteria on the amount of free

amino acids in tempe was i nvestigated.

a t e r ia l s a n d e t h o d s

Micro o rgan isms and cu l tu re cond i t ions

All Rhizopus strains used were isolated from In-

donesian or Dutch tempe samples, Indonesian

commercial tempe starters, or Indonesian Hibis-

cus leaves used as tempe inoculum. They were

identified as Rhizopus oligosporus synonym R.

microsporus var. oligosporus , R. stolonifer and R.

oryzae synonym R. arrhizus by Hering et al.

1991), Keuth and Bisping 1993), and by the cur-

rent authors, according to the taxonomies of Zycha

et al. 1969) and Schipper 1984). In this work, 27

strains of R. oligosporus, six of R. stolonifer and

three ofR. oryzae were tested Table 1). All Rhizo-

pus strains were cultivated on malt peptone agar

or liquid medium 40 g 1-1 malt, 3 g 1 ~ soypeptone,

17 g 1 l agar) and Czapek-Dox agar or liquid

medium. The pH of all media was adjusted to 5.5.

Citrobacter freundii and Micrococcus luteus were

cultivated on Standard I agar or liquid medium

Merck, Darmstadt, Germany).

Tabl e 1. Origin of tempe and tempe inoculum samples from which Rhizopus strains were

isolated

Species Place Strain

R. oligosporus Bandar Lampung, Sumatra

Bandung, Java

Bogor, Java

Denpasar, Bali

Jakarta, Java

Medan, Sumatra

Pontianak, Kalimantan

Purwokerto, Java

Samarinda, Kalimantan

Serpong, Java

Surabaya, Java

Tegal, Java

Tulung Agung, Java

Ujung Pandang, Sulawesi

Yogyakarta, Java

Bogor, Java

Enschede, Netherlands

Malang, Java

Bundung, Java

Bogor, Java

Jakarta, Java

R. oryzae

R. stolonifer

Balu

Heba, Hepla

Bogo, CN, IN, Tebo

Bali, Denl, Den2

Jaba, Jap, Liga, Sja, Teja

MS1, MS2, MS5

Pon

Purwo

Sama

Serp

Sur

Tegal

Q1, Tup

ju

CD

Fi

EN

Mala

Hib

IK, J16

CM, GT


3/9

P r o t e o ly s i s d u r i n g t e m p e f e r m e n t a t i o n 4

Fermenta t ion cond i t ions

Tempe fermentations were carried out under stan-

dardized conditions (Hering et al. 1991). Soybeans

were acidified with lactic acid to pH 5.0, cooked for

30 min, hulled and cooked again for 30 min (pH

5.0). After surface drying, beans (300 g wet

weight) were packed into plastic foils (13 × 13 cm),

and autoclaved at 121°C for 20 min. The plastic

bags were then perforated and the beans inocu-

lated with a spore suspension (1.8 ml) of a

Rhizopus st rai n (106 spores ml -l of 0.9 NaC1, cor-

responding to 6 x 103 spores g-1 of beans). In

fermentations with

Citrobacter freundii

or

Micro

coccus luteus

1.8 ml of a suspension (10 T cells m1-1

of 0.9 NaC1, corresponding to 6 × 104 cells g-1 of

beans) was added. Beans were fermented in an incu-

bator (Cytoperm 8088, Heraeus, Hanau, Germany)

at a relative humid ity (RH) of 90 and a tempera-

ture of 32°C for a period of 30 h. For experiments

with reduced RH the incubator was adjusted to 60

RH. In experiments with lowered fermentation

temperatures (24°C) the process was stopped after

40 h, when a sliceable tempe cake was obtained.

na lys is o f am ino ac ids

Tempe samples were frozen at -70°C and lyophilized

at -52°C, 0.16 mbar in a freeze dryer (Lyolab C

3021, LSL Secfroid Sa., Aclens -Lousanne, Swit zer-

land) for 72 h, and pulverized in a household

mixer. For det ermination of free amino acids 1 g

powder was boiled in 30 ml water for 1 h and cen-

trifuged for 10 min at 4000 g (Sorvall RC-5 B

centrifuge, GSA rotor, Du Pont, Wilmington, DE,

USA). The precipitate was diluted with 30 ml of

water and the process repeated. The liquid phases

were combined, concentrated, and calibrated to 10

ml at pH 2.2 with citrate buffer. Total amino acids

were determined by an acid extraction (Allen

1989). Extracts were defatted using acetone/dichloro-

methane (1:1 v/v). Amino acid analysis was feasible

after a pre-column derivatization with the Edman

reagent phenylisothiocyanate (PITC) following the

method of Spatz et al. (1989). With this method all

common amino acids were detectable with the ex-

ception of trypto phane and proline/alanine and

glutamic acid/asparagine, which could only be detected

together. A Merck-Hitachi HPLC (Darmstadt, FRG;

Tokyo, Japan) with a L-620 intelligent pump, a chro-

mato integrator D-2000, a UV-detector L-4000,

and a LiChrospher RP-select B, 5/~m column was

used. A mixture of sodium acetate 70 mM,

tetraethylammonium bromide 3.5 mM, tetrabuty-

lammonium hydrogen sulphat e 3-5 mM, acetonitrile

249.0 ml, and methanol 39.0 ml, distilled water to

1000 ml, pH 6.5 was used. The sample size was 10

/~1 and the flow rate was adjusted to 1 ml min -1.

Detection was at 254 nm at a tempera tur e of 56°C.

Enzyme ac t iv i ty

For enzymatic tests submerged fermentations

were performed in 450 ml Fernbach flasks con-

taining 100 ml malt peptone medium at 32°C for

24-70 h. The cultures were shaken in an incubator

shaker (model G25, New Brunswick Scientific,

Edison, NJ, USA) at 200 r min -1. For intra cell ular

and cell wall bound enzymes, pretreatment was

necessary. Exoproteases were concentrated out of

the medium by precipitation with 70 ammon ium

sulphate. After that the medium was centrifuged

at 26 000 g (Sorvall RC-5B centrifuge, SS-34 rotor,

Du Pont, Wilmington, DE, USA) for 20 min. The

precipitate was solubilized in 2 ml sodium acetate

buffer and desalted using a PD-10 column (Phar-

macia, Piscataway, NJ, USA). For intracellular

enzymes the mycelium was washed at pH 5.5,

then homogenized in a precooled mortar by step-

wise addition of 40 ml sodium acet ate buffer. The

homogenate was centrifuged as described above.

The separated raw extract contained the intracel-

lular enzymes. For cell wall bound enzymes the

precipitate was washed three times and cen-

trifuged, then homogenized with 25 ml buffer.

This suspension was stirred with 0-5, 1.0, 2.0, 3.0

and 4-0 M LiC1 solution and at different pH values

at 4°C for 12 h. Then the suspensions were cen-

trifuged again and the precipitate and solutions

tested. Fractional precipitations were used to fur-

ther characterize the proteases. The precipitates

were desalted using PD-10 columns and used for

the enzymatic tests. Enzy matic activity was tested

by the methods of Bergmeyer (1970) and Kurono

et al. (1971). Casein solution, 0-6 (Hamma rst en

casein, Merck, Darmstadt, Germany) was used as

substrate. Various experimental conditions were

used, i.e. tartarate, citrate, or Tris-HC1 buffer at

pH values from 2.0 to 9-0 and 35°C. EDTA (5

mmol 1-1), pepst atin A from

Streptomyces

spp.

(10 -v mol l-l), and pheny lmeth ylsu lphon yl fluoride

(PMFS) (1 mmol 1-1) were tested as p rotease in-

hibitors. All tests were performed at pH values of

3.0 and 7-0. Protein content was tested with the

method of Bradford (1976).

De te rm ina t ion o f mo lec u la r we igh t

Molecular weights were determined by discontinu-

ous sodium dodecyl sulphate (SDS) gel electro-

phoresis (Lugtenberg et al. 1975) and silver stain-

ing (Merril et al. 1981). Glycoproteins were detected

by the periodic acid sta ining (PAS) method of Kap-

itan y and Zebrowski (1973). A mixture of mar ke r

proteins (Dalton Mark VII-L, Sigma, St Louis,

MO, USA) was included, together with Rhizopus

protease (P-5027, Sigma, St Louis, MO, USA).

Electrophoretic bands were only detected in the 20

and 70 ammoni um sulphate precipitations.


4/9

42 U Baum ann and B B isp ing

R e s u l t s

P r o t e o l y ti c a c t i v i ty o f d i f f e r e n t h zopus

s t ra ins

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

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

t e m p e in c o m p a r is o n w i t h u n f e r m e n t e d b e a n s .

A s l ig h t d e c r e a s e o f 6 - 7 w a s o b s e r v e d f o r

t o t a l a m i n o a c i d s . T h e o p p o s i t e e f f e c t w a s

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

R h i z o p u s s t r a i n s . F r e e a m i n o a c i d c o n c e n t r a -

t i o n s i n c r e a s e d u p t o f i v e -f o l d a f t e r 3 0 h o f

s t a n d a r d f e r m e n t a t i o n w i t h R. o l igosporus

M S 1 . T h i s r e l e a s e c o n t i n u e d a n d t h e a m o u n t

o f f r e e a m i n o a c i d s i n c r e a s e d u p t o 6 . 5 - fo l d

a f t e r 4 5 h a n d u p t o 8 . 3 -f o l d a f t e r 7 0 h .

I n a c o m p a r i s o n o f 3 6 s t r a i n s e x a m i n e d , a l l

s t r a i n s o f

R. s to loni fer

s h o w e d l o w a c t i v i t y

a f t e r 3 0 h o f s t a n d a r d f e r m e n t a t i o n . T h e y

r e l e a s e d f r o m 5 . 0 - 1 0 . 2 m g a m i n o a c i d s g - 1

d r y w e i g h t ( d w ) , w h e r e a s

R. oryzae

s t r a i n s

r e a c h e d a m o u n t s u p t o 1 5 .1 m g g-~ d w , a n d

R. oligosporus u p t o 1 9 . 6 mg g - 1 d w . T h e

R. oryzae

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

1 2 m o s t p r o t e o l y t i c s t r a i n s . R e l a t i n g t o R .

oryzae and R. o l igosporus i t c a n b e s a i d t h a t

t h e p r o t e o l y t ic c a p a c i t y d e p e n d s o n t h e s t r a i n

a n d n o t o n t h e s p e c i e s u s e d . F i g . 1 s h o w s 1 6

o u t o f 3 6 s t r a i n s a s a n e x a m p l e . A n a l y s i s o f

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

d i f f e re n c e s a m o n g t h e s t r a i n s . O n a v e r a g e ,

h i g h e r p r o t e o l y t ic a c t i v i t y r e s u l t s i n a h i g h e r

a m o u n t o f a l l a m i n o a c i d s ( F i g. 2 ) .

V a r ia t io n o f fe r m e n t a t i o n p a r a m e t e r s

T h e l o w e r f e r m e n t a t i o n t e m p e r a t u r e ( 24 ° C)

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

t e m p e c a k e w a s o b t a i n e d o n l y a f t e r 4 0 h .

N e v e r t h e l e s s , t h e a m o u n t o f f r e e a m i n o a c i d s

w a s i m p r o v e d b y u p t o 1 3 0 ( s t r a i n H i b) . T h e

h i g h e s t a m o u n t w a s a g a i n o b t a i n e d w i t h R .

ol igosporus M S 1 ( 2 4 m g g - 11 d w ) ( T a b l e 2 ). A

s i m i l a r s i t u a t io n w a s o b s e r v e d w h e n f e r m e n -

t a t i o n w a s c a r ri e d o u t a t 6 0 R H . F r e e

a m i n o a c i d c o n c e n t r a t i o n s i n c r e a s e d t o 1 1 5

a f t e r 3 0 h a n d t o 1 5 7 a f t e r 4 5 h c o m p a r e d to

f e r m e n t a t i o n a t 9 0 R H f o r t h e s a m e t i m e .

n f lu e n c e o f m i x e d c u l t u r e s o n f re e

a m i n o a c i d c o n c e n tr a t io n

W h e n t h e f e r m e n t a t i o n w a s c a r r ie d o u t w i t h

m i x e d c u l t u r e s o f R . ol igosporus a n d b a c t e r i a ,

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

w i t h u n f e r m e n t e d b e a n s . I n c o n t r a s t t o t h is ,

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

l e v e l o f 4 3 a n d 3 5 , r e s p e c t i v e l y , w h e n Cit

robacter f reundi i

o r

Micrococcus lu teus

w e r e

2 0

16

~ 12

E

8

C M J 1 6 I K

H i b G T

R s to lon i fer

xx

E N F i S u r M S 2 M S 5 T e g a l

M a l a S a m a T e bo T e j a M S 1

R oryzae R o l igosporus

igure

1 . E x e m p l a r y r a n k o f p r o t e o ly t i c a c t i v it y o f 1 6 o u t o f 3 6 R h i z o p u s s t r a i n s . T h e t o t a l

a m o u n t o f a m i n o a c i d s re l e a s e d g -1 d r y w e i g h t ( d w ) a f te r s t a n d a r d f e r m e n t a t i o n i s s h o w n .


5/9

P r o te o ly s is d u r in g t e m p e f e r m e n t a t i o n 4

V] :Gr.1

:Gr.2

~q : Gr.3

R H E/D S G T PIA Y V M L F K

A m i n o acids

F i g u r e 2 , P a t t e r n o f a m i n o a c i d s r e l ea s e d b y s t r ai n s o f

h i z o p u s

s p. T o s h o w t h a t a h i g h e r

h y d r o ly t i c c a p a c i t y l e a d s t o a n e q u a l r e l e a s e o f a ll a m i n o a c i ds , t h e a v e r a g e p e r f o r m a n c e s a r e

g i v e n a s G r o u p 1 [ 4 - 2 - 7 - 8 m g a m i n o a c i ds r e l ea s e d g - 1 d r y w e i g h t d w )] , G r o u p 2 7 . 9 -1 2 . 1 m g

a m i n o a c i d s r e l e a s e d g - 1 d w ) , a n d G r o u p 3 1 2 . 5- 1 9 .6 m g a m i n o a c i d s r e l ea s e d g - ~ d w ) . T h e

a m i n o a c i d s a r e c o d e d w i t h t h e i n t e r n a t i o n al o n e l e tt e r a b b r ev i a t i on s .

added to the fermentation. These species

were selected from the wide range of bacteria

isolated from tempe because they have been

described previo usly as good vitam in B12 pro-

ducers (Keuth and Bisping 1993).

haracteristics of the protease system

Temperature optima for the protease systems

were found to be 55°C for R o l i g o s p o r u s and

R o r y z a e

and 50°C for

R s t o l o n i f e r

No differ-

ences were found between extracellular, cell

wall bound and intracellular proteins. Maxi-

mum protease activity was observed to have

two peaks at pH 2.5-3 and pH 6-7, for all

species and fractions. An additional maxi-

mum was observed at pH 4.5-5 for intra-

cellular and cell wall bound proteases. The

highest protease activities were found after

fermentation times of 45-70 h.

After enrichment of the proteases of the

three fractions SDS gel electrophoresis indi-

cated presence of protein band s of 68 and

48 kDa for the cell wall bound fraction, 48 kDa

and 36 kDa for the ex tracellul ar fraction, and

36 kDa for the intracellular fraction. PAS

indicated that the 68 kDa protein was a gly-

coprotein. All enriched proteases were total ly

inhibited by pepstatin A of

S t r e p t o m y c e s

spp.

PMFS reduced activities from 63 to 45 .

EDTA produced no effect or even a slight in-

crease of activity (Table 3).

To check the relevance of the different

protease fractions for the total prdteolytic

capacity of the strains, the activities were

T a b l e 2 Comparison of the amou nt of free amino acids (mg g-' dry weight) found in tempe

fermented at two different temperatu res

Fermentation Stain

temperature

R oligosporus R oryzae R stolonife r

MS1 CN En Fi Hib IK

32°C 19.7 8.3 13.6 15-4 5.4 10.0

24°C 24.0 13.0 17-5 17.2 12.5 12.9


6/9


T a b l e 3. Effect of differ ent protease inhibito rs on t he activi ty (U ml -~) of cell wall bound, ex-

tracellular, and intra cellular prote~ses o f R h i z o p u s o l i g o sp o r u s (MS 1)

Enzyme Protease inhibitor

No. EDTA PEP PMFS

Cell wall bound 0.21 0.21 0.00 0.15

Extracellular 0,'21 0.22 0.00 0.13

Intracellular 0.11 0.11 0.00 0.05

relat ed to protein g-~ dw of each fraction. The

cell wall bound proteases (debris) were most

important for the proteolytic capacity of R h i z o -

p u s . In Fig. 3 the tur nover ra te of the three

fractions in relation to the dw is shown for

seven strains. On an average of all strains

76 of the total proteolytic capacity belonged

to this fraction, 14 to the extracel lular sys-

tem, and 10 to the intr acel lula r proteases.

iscuss ion

We were able to find several

R. o l igos por us

and R. o r yz ae strains with high proteolytic

capacities. R. s to lon i f e r strains were less

active. The superiority of

R. o l igos por us

found in this work agrees with reports of

Wang and Hesseltine (1965) and Winarno

and Reddy (1986), who described the impor-

tance of this species for tempe fermenta tion.

We showed that tempe with a good amino

acid release can be produced also with R.

or yz ae

and with regard to taste and slice-

ability even with R. s to lon i f e r .

The efficiency of proteolytic activity could

be improved by reducing fermentation tem-

perature and RH. This was connected with

an increased production of proteases, which

can be observed when molds are forced to

grow under suboptimal temperature and

wat er activit y (aw). This effect was fir st

described by Maxwell (1952) and Yamamoto

(1957) for A s p e r g i l l u s o r y z a e and A. s o jae

and by Wang et al. (1974) for R. o l igos por us .

The optimal temperature for

R h i z o p u s

is in

120

240 El : Debris

[] : Extracellular

200 ~ : Intracellular

160

80

40

~

~

J a p Ma l a

Teja MS1 MS2

Rhizopus strains

Tegal MS

F i g u r e

3 Turno ver rate s of the seven mos t active strain s of our collection. On an ave rage 76

of the total proteolytic capacity of R h i z o p u s belonged to the cell wall bound fraction (debris).

With the exception of the str ain coded Mala, which was a memb er of R. o r yz ae all the other

str ains given in this figure belonged to R. o l igos por us .


7/9

Proteolysis during tem pe fermen tation 5

t h e r a n g e o f 3 0 ° C d e p e n d e n t o n t h e s p e c i es ,

a n d t h e a w s h o u l d b e h i g h e r t h a n 0 . 9 4 G e r -

v a i s e t a l . 1 9 8 8 ) . H i g h e r e x p r e s s i o n o f

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

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

a c id s r e l e a se d . F u r t h e r m o r e , f e r m e n t a t i o n a t

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

t e r ia l c o n t a m i n a t i o n , b e c a u s e b a c t e r i a a r e

m o r e s e n s i t i v e t o r e d u c e d aw t h a n f u n g i.

T o i n c r e a s e t h e f r e e a m i n o a c i d s i n p r a c -

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

e i t h e r u s e f a n s t o c o ol f e r m e n t a t i o n s h e l v e s ,

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

w o u l d re d u c e t h e t e m p e r a t u r e i n s id e t h e

c a k e b y 3 °C p e r c e n t i m e t r e R a t h b u n a n d

S h u l e r 1 9 83 ) . T h e r e d u c t i o n o f f e r m e n t a t i o n

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

H e r i n g e t al . 1 9 9 1 ), w h o o b s e r v e d a b e t t e r

p r o d u c t i o n o f ~ , -l in o le n ic a c i d u n d e r t h e s e

c o n d i t i o n s .

I n c r e a s e d r e l e a s e o f a m i n o a c id s s h o w n

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

t e m p e i n c o m p a r i s o n t o u n f e r m e n t e d s oy -

b e a n s M u r a t a e t a l. 1 9 67 , I s m a i l 1 9 81 ). T h e

f a c t t h a t b a c t e r i a s u c h a s Citrobacter freundii

o r Micrococcus luteus w h i c h f o r m v i t a m i n B ~ 2

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

a c t i v i t y o f Rhizopus s p p . h a s p r e v i o u s l y b e e n

s h o w n K e u t h a n d B i s p i n g 19 9 3) . N o w w e

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

a m i n o a c i d s r e l e a s e d b y

Rhizopus

a s s i s t

g r o w t h a n d v i t a m i n B~2 f o r m a t i o n , a s s h o w n

b y th e l o w e r e d a m o u n t o f f r e e a m i n o a c i d s in

t e m p e a f t e r c o - f er m e n t a t i o n

of Rhizopus

w i t h

C. freundii

o r

M. luteus.

T h e p r o t e a s e s y s t e m w a s i n v e s t i g a t e d t o

g a i n a b e t t e r i n s i g h t i n t o t h e m e c h a n i s m s o f

t h e p r o t e o l y t i c a c t i v i t y o f

Rhizopus.

T h e

b e h a v i o u r o f c e ll w a l l b o u n d , i n t r a c e l l u l a r ,

a n d e x t r a c e l l u l a r p r o t e a s e s w a s s i m i l a r i n

r e la t io n t o p H , t e m p e r a t u r e a n d p r o t e a s e

i n h i b i t o r s . Rhizopus s t r a i n s i n v e s t i g a t e d

s e e m e d t o p o s s e s s o n l y o n e p r o t e a s e t y p e .

I n h i b i t io n o f p r o t e a s e s b y p e p s t a t i n A a n d

l a c k o f e f f e c t o f E D T A c o n f i r m e d t h a t t h e Rhi-

zopus s t r a i n s s t u d i e d p o s s e s s a n a s p a r t y l

p r o t e a s e . O u r r e s u l t s a g r e e w i t h t h o s e o f

F u k u m o t o e t a l . 1 9 6 7 ) , B o t t e t a l . 1 9 8 2 ) a n d

T a k a h a s h i 1 9 8 8 ) w i t h R. chinensis. T h e

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

t h e s e a u t h o r s w a s r e p o r t e d t o b e 3 5 k D a a n d

a p H o p t i m u m o f 2 - 5- 3 -3 w a s i n d i ca t e d . T h e

t e m p e r a t u r e o p t i m a r a n g e d f ro m 5 0 - 6 0 ° C fo r

t h i s s p e ci e s . W e a ls o f o u n d a p H m a x i m u m i n

t h e n e u t r a l a r e a , w h i c h w a s d e s c r i b e d b y

W a n g a n d H e s s e l t i n e 1 9 65 ) a n d S c h i n d l e r e t

a l . 1 9 8 2 ) f o r

R. chinensis

a n d

R. oligosporus

r e s p e ct iv e l y . T h i s a d d i t io n a l p H m a x i m u m i s

n e c e s s a r y f o r d e g r a d a t i o n o f s o y p ro t e i n a t

p H v a l u e s o f 6 - 7 t h a t a r e f o u n d in t e m p e ju s t

a fe w h o u r s a f t e r t h e s t a r t o f f e r m e n t a t i o n .

T h e e l e c t r o p h o r e t ic b a n d s a t 4 5 a n d 6 8 k D a ,

r e s p e c t i v e l y , s h o u l d b e f u r t h e r i n v e s t i g a t e d .

W e s u g g e s t t h a t t h e l a r g e m o l e c u l e s a r e g l y -

c o p r o t e i n s , w h i c h w a s v e r i f ie d f o r t h e 6 8 k D a

p r o t e i n b y P A S s t a i n i n g . T s u j i t a a n d E n d o

1 9 8 0 ) d e s c r i b e d h i g h m o l e c u l a r w e i g h t p r o -

t e a s e s o f

Aspergillus oryzae

w i t h a h i g h

a m o u n t o f c o v a l e n tl y b o u n d s u g a r s , w h i c h f ix

t h e p r o t e a s e a t t h e c e l l w a l l . T h e s a m e e f f e c t

c o u l d b e t r u e i n o u r c a s e .

E n z y m a t i c t e s t s a n d a n a l y s i s o f t h e

t u r n o v c r r a t e s h o w e d t h a t c e ll w a l l b o u n d

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

p r o t e o l y t ic c a p a c i t y o f f e r m e n t i n g Rhizopus.

T h i s f a c t i s a g r e a t a d v a n t a g e f o r

Rhizopus

i n

s o li d s u b s t r a t e f e r m e n t a t i o n s s u c h a s t h e

t e m p e f e r m e n t a t i o n . T h e m a i n b e n e f i t o f t h e

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

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

w h i l e th e f u n g u s i s g r o w i n g t h r o u g h t h e s u r -

f a c e o f t h e s o y b e a n s . D e t e r m i n a t i o n o f t h e

s p e c if i c a c t i v i t y o f c e ll w a l l b o u n d p r o t e a s e s

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

m e t h o d t o f in d m o r e s t r a i n s w i t h a h i g h

p r o t e o l y t i c a c t i v i t y , a n d h e l p t o c o n t r o l f e r -

m e n t a t i o n q u a l i t i e s o f i n o c u l a u s e d i n

I n d o n e s i a .

A c k n o w l e d g e m e n t s

W e a c k n o w l e d g e t h e w o r k o f D r M i e n M a h -

m u d a n d D r H e r m a n a N u t r it io n R e s e a rc h

a n d D e v e l o p m e n t C e n t r e , B o g o r) , o f D r

S u y a n t o P a w i r o h a r s o n o a n d M r E f fe n d i S ir e -

g a r B P P T e k n o l o g i, J a k a r t a ) , a n d o f M r B .

K l e i n s t e u b e r T ~ - V - R h e i n l an d , K S ln ) , w h o

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

p le s . W e t h a n k t h e F e d e r a l M i n i s tr y o f

R e s e a r c h a n d T e c h n o l o g y in B o n n f o r s u p -

p o r t i n g t h e s e i n v e s t i g a t i o n s .


8/9


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