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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
8/18/2019 1995 Proteolysis during tempe fermentation.pdf
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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
8/18/2019 1995 Proteolysis during tempe fermentation.pdf
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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.
8/18/2019 1995 Proteolysis during tempe fermentation.pdf
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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 .
8/18/2019 1995 Proteolysis during tempe fermentation.pdf
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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
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4 4 U B a u m a n n a n d B B is p in g
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 .
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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 .
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4 6 U B a u m a n n a n d B B is p in g
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