Food Microbiology, 1995, 12, 39-47

Proteolysis during tempe fermentation*

U. Baumann and B. Bisping t

The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe
or tempe inocula was examined. No significant changes in the total amount or pattern of
amino acids could be found, but there was a distinct increase in the amount of free amino
acids. Strains with a high proteolytic activity were found, which were able to release nearly
five times more amino acids after standard fermentation than others. Changes in fermentation parameters such as temperature or relative humidity improved these results.
Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free
amino acids, but an increased total amount of amino acids. Examination of protease systems of three Rhizopus species showed that the proteases o f the cell wall fraction were
most responsible for proteolytic capacity of the different strains. On average their activity
amounted to 71% of the total proteolytic capacity.

Introduction

paste (sambal), or as vegetarian tempeburger.

It may be added to drinks and to soups, or
Tempe kedelai is an Indonesian foodstuff offered as crackers (Shurtleff and Aoyagi 1979,
based on soybeans, which has a tradition dat- Steinkraus 1983). Today 765000 tonnes are
ing back many centuries in Java. Today it is produced by around 40 000 home manufacturalso becoming more popular on the other ers with 130 000 employees in Indonesia alone
Indonesian islands, in Japan, in the USA, and (Winarno and Reddy 1986, Karta 1987).
One of tempe's most important qualities is
in Western Europe. It is made by a two-step
fermentation involving a Rhizopus species plus the high protein level up to 40% of the dry
many different bacteria and yeasts. This fer- mass. Due to this high protein content, the
mentation improves some features of soybeans amino acid composition of tempe surpasses
including fatty acid composition (Hering et al. the FAO/WHO amino acid reference pattern
1991), the level and p a t t e r n of oligosaccha- with the exception of methionine and cysteine
rides (Barz et al. 1990), and the amount of (Murata et al. 1967, Winarno and Reddy
several vitamins, especially vitamin B12 and 1986). This fact explains the great interest in
vitamin D (Keuth and Bisping 1993, Denter this food in many developing countries, which
and Bisping 1993). The product is a solid cake, fight against protein deficiency, especially of
which is consumed in the form of fried slices, the young population.
One characteristic of tempe fermentation is
as a kind of Indonesian satay, as peppered
the production of lower molecular weight
*Dedicated to Professor Dr H.-J. Rehm, on the compounds such as free fatty acids and free
amino acids (Wagenknecht et al. 1961, Stilloccasion of his 67th birthday.
tCorresponding author.
ings and Hackler 1965, M u r a t a et al. 1967,
0740-0020/95/010039+09 $08.00/0

Received :
17 May 1994

tnstitut f#r Mikrobiologie,

Westf#lische Wilhelms-Universit#t
MSnster,
Corrensstrasse 3,
D-48149 MSnster,
Germany

1995 Academic Press Limited

40

U. Baumann and B. Bisping

Hering et al. 1991). An increase of the protein

efficiency ratio (PER) of tempe was.attributed
to a better availability of amino acids. Murata et al. (1971) found in a rat feeding test
that the PER value of tempe was not
significantly different from t h a t of unfermented soybeans, hox~ever Zamora and
Veum (1979) reported an increase in the average 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 that vitamin B~2 formation by Citrobacter freundii during tempe fermentation was
strongly dependent on the metabolic activity
of Rhizopus.
The aim of this work was to find some Rhizopus 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 fermentation 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 investigated.

Materials and Methods

Micro-organisms and culture conditions
All Rhizopus strains used were isolated from Indonesian or Dutch tempe samples, Indonesian
commercial tempe starters, or Indonesian Hibiscus 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 current 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 of R. oryzae were tested (Table 1). All Rhizopus 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).

Table 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

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

R. oryzae
R. stolonifer

Uju
CD
Fi
EN
Mala
Hib
IK, J16
CM, GT

Proteolysis during tempe fermentation

Fermentation conditions

Enzyme activity

Tempe fermentations were carried out under standardized 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 121C for 20 min. The plastic
bags were then perforated and the beans inoculated with a spore suspension (1.8 ml) of a
Rhizopus strain (106 spores ml -l of 0.9% NaC1, corresponding to 6 x 103 spores g-1 of beans). In
fermentations with Citrobacter freundii or Micrococcus 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 incubator (Cytoperm 8088, Heraeus, Hanau, Germany)
at a relative humidity (RH) of 90% and a temperature of 32C for a period of 30 h. For experiments
with reduced RH the incubator was adjusted to 60%
RH. In experiments with lowered fermentation
temperatures (24C) the process was stopped after
40 h, when a sliceable tempe cake was obtained.

For enzymatic tests submerged fermentations

were performed in 450 ml Fernbach flasks containing 100 ml malt peptone medium at 32C 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 intracellular
and cell wall bound enzymes, pretreatment was
necessary. Exoproteases were concentrated out of
the medium by precipitation with 70% ammonium
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 (Pharmacia, Piscataway, NJ, USA). For intracellular
enzymes the mycelium was washed at pH 5.5,
then homogenized in a precooled mortar by stepwise addition of 40 ml sodium acetate buffer. The
homogenate was centrifuged as described above.
The separated raw extract contained the intracellular enzymes. For cell wall bound enzymes the
precipitate was washed three times and centrifuged, 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 4C for 12 h. Then the suspensions were centrifuged again and the precipitate and solutions
tested. Fractional precipitations were used to further characterize the proteases. The precipitates
were desalted using PD-10 columns and used for
the enzymatic tests. Enzymatic activity was tested
by the methods of Bergmeyer (1970) and Kurono
et al. (1971). Casein solution, 0-6% (Hammarsten
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 35C. EDTA (5
mmol 1-1), pepstatin A from Streptomyces spp.
(10 -v mol l-l), and phenylmethylsulphonyl fluoride
(PMFS) (1 mmol 1-1) were tested as protease inhibitors. All tests were performed at pH values of
3.0 and 7-0. Protein content was tested with the
method of Bradford (1976).

Analysis of amino acids

Tempe samples were frozen at -70C and lyophilized
at -52C, 0.16 mbar in a freeze dryer (Lyolab C
3021, LSL Secfroid Sa., Aclens-Lousanne, Switzerland) for 72 h, and pulverized in a household
mixer. For determination of free amino acids 1 g
powder was boiled in 30 ml water for 1 h and centrifuged 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/dichloromethane (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 exception of tryptophane 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 chromato 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, tetrabutylammonium hydrogen sulphate 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 temperature of 56C.

Determination of molecular weight

Molecular weights were determined by discontinuous sodium dodecyl sulphate (SDS) gel electrophoresis (Lugtenberg et al. 1975) and silver staining (Merril et al. 1981). Glycoproteins were detected
by the periodic acid staining (PAS) method of Kapitany and Zebrowski (1973). A mixture of marker
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% ammonium sulphate precipitations.

41

42

U. Baumann and B. Bisping

Results

differences among the strains. On average,

higher proteolytic activity results in a higher
amount of all amino acids (Fig. 2).

Proteolytic activity of different Rh izopus

strains
There was no important change in total amino
acids as well as in the~amino acid pattern of
tempe in comparison with unfermented beans.
A slight decrease of 6-7% was observed for
total amino acids. The opposite effect was
observed after release of amino acids by
Rhizopus strains. Free amino acid concentrations increased up to five-fold after 30 h of
standard fermentation with R. oligosporus
MS1. This release continued and the amount
of free amino acids increased up to 6.5-fold
after 45 h and up to 8.3-fold after 70 h.
In a comparison of 36 strains examined, all
strains of R. stolonifer showed low activity
after 30 h of standard fermentation. They
released from 5.0-10.2 mg amino acids g-1
dry weight (dw), whereas R. oryzae strains
reached amounts up to 15.1 mg g-~ dw, and
R. oligosporus up to 19.6 mg g-1 dw. The
R. oryzae strains belonged to the group of the
12 most proteolytic strains. Relating to R.
oryzae and R. oligosporus it can be said that
the proteolytic capacity depends on the strain
and not on the species used. Fig. 1 shows 16
out of 36 strains as an example. Analysis of
the free amino acids pattern showed great

Variation of fermentation parameters

The lower fermentation temperature (24C)
reduced fermentation velocity and a good
tempe cake was obtained only after 40 h.
Nevertheless, the amount of free amino acids
was improved by up to 130% (strain Hib). The
highest amount was again obtained with R.
oligosporus MS1 (24 mg g-11 dw) (Table 2). A
similar situation was observed when fermentation was carried out at 60% RH. Free
amino acid concentrations increased to 115%
after 30 h and to 157% after 45 h compared to
fermentation at 90% RH for the same time.

Influence of mixed cultures on free

amino acid concentration
When the fermentation was carried out with
mixed cultures ofR. oligosporus and bacteria,
total amino acids increased slightly compared
with unfermented beans. In contrast to this,
the amount of free amino acids decreased to a
level of 43 and 35%, respectively, when Citrobacter freundii or Micrococcus luteus were

20

16

"~ 12
E

Figure 1.

xx

CM J16
IK
Hib
GT

EN
Fi
Mala

Sur
MS2 MS5 Tegal
Sama Tebo Teja MS1

R. stolonifer

R. oryzae

R. oligosporus

Exemplary rank of proteolytic activity of 16 out of 36 Rhizopus strains. The total

amount of amino acids released g-1 dry weight (dw) after standard fermentation is shown.

Proteolysis during tempe fermentation

V] :Gr.1
:Gr.2
~q : Gr.3

E/D S

T PIA Y
V
acids

Amino

Figure 2, Pattern of amino acids released by strains of Rhizopus sp. To show that a higher
hydrolytic capacity leads to an equal release of all amino acids, the average performances are
given as Group 1 [4-2-7-8 m g amino acids released g-1 dry weight (dw)], Group 2 (7.9-12.1 m g
amino acids released g-1 dw), and Group 3 (12.5-19.6 m g amino acids released g-~ dw). The
amino acids are coded with the international one letter abbreviations.
added to the fermentation. These species
were selected from the wide range of bacteria
isolated from tempe because they have been
described previously as good vitamin B12 producers (Keuth and Bisping 1993).

Characteristics of the protease system

Temperature optima for the protease systems
were found to be 55C for R. oligosporus and
R. oryzae and 50C for R. stolonifer. No differences were found between extracellular, cell
wall bound and intracellular proteins. Maximum protease activity was observed to have
two peaks at pH 2.5-3 and pH 6-7, for all
species and fractions. An additional maximum 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 indicated presence of protein bands of 68 and
48 kDa for the cell wall bound fraction, 48 kDa
and 36 kDa for the extracellular fraction, and
36 kDa for the intracellular fraction. PAS
indicated t h a t the 68 kDa protein was a glycoprotein. All enriched proteases were totally
inhibited by pepstatin A of Streptomyces spp.
PMFS reduced activities from 63 to 45%.
EDTA produced no effect or even a slight increase of activity (Table 3).
To check the relevance of the different
protease fractions for the total prdteolytic
capacity of the strains, the activities were

Table 2. Comparison of the amount of free amino acids (mg g-' dry weight) found in tempe
fermented at two different temperatures
Fermentation
temperature
32C
24C

R. oligosporus
MS1
CN

Stain
R. oryzae
En
Fi

R. stolonifer
Hib
IK

19.7
24.0

13.6
17-5

5.4
12.5

8.3
13.0

15-4
17.2

10.0
12.9

43

44

U. Baumann and B. Bisping

T a b l e 3. Effect of different protease inhibitors on the activity (U ml -~) of cell wall bound, ex-

tracellular, and intracellular prote~ses ofRhizopus oligosporus (MS 1)

Enzyme

Protease inhibitor
EDTA
PEP

No.
Cell wall bound
Extracellular
Intracellular

0.21
0,'21
0.11

0.21
0.22
0.11

related to protein g-~ dw of each fraction. The

cell wall bound proteases (debris) were most
important for the proteolytic capacity of Rhizopus. In Fig. 3 the turnover rate 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 extracellular system, and 10% to the intracellular proteases.

Discussion
We were able to find several R. oligosporus
and R. oryzae strains with high proteolytic
capacities. R. stolonifer strains were less
active. The superiority of R. oligosporus
found in this work agrees with reports of

240
200

0.00
0.00
0.00

PMFS
0.15
0.13
0.05

Wang and Hesseltine (1965) and Winarno

and Reddy (1986), who described the importance of this species for tempe fermentation.
We showed that tempe with a good amino
acid release can be produced also with R.
oryzae and with regard to taste and sliceability even with R. stolonifer.
The efficiency of proteolytic activity could
be improved by reducing fermentation temperature 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
water activity (aw). This effect was first
described by Maxwell (1952) and Yamamoto
(1957) for Aspergillus oryzae and A. sojae,
and by Wang et al. (1974) for R. oligosporus.
The optimal temperature for Rhizopus is in

El : Debris
[] : Extracellular
~ : Intracellular

160
120
80
40

Jap

'~

Mala

Teja

MS1

MS2

Tegal

MS5

Rhizopus strains
Figure 3. Turnover rates of the seven most active strains of our collection. On an average 76%
of the total proteolytic capacity of Rhizopus belonged to the cell wall bound fraction (debris).
With the exception of the strain coded Mala, which was a member of R. oryzae, all the other
strains given in this figure belonged to R. oligosporus.

Proteolysis during tempe fermentation 45

the range of 30C dependent on the species,
and the aw should be higher than 0.94 (Gervais et al. 1988). Higher expression of
proteases combined with a reduced growth
velocity leads to a higher amount of amino
acids released. Furthermore, fermentation at
a lower RH should partly protect against bacterial contamination, because bacteria are
more sensitive to reduced aw than fungi.
To increase the free amino acids in practice, Indonesian tempe producers should
either use fans to cool fermentation shelves,
or reduce the thickness of tempe cakes. This
would reduce the t e m p e r a t u r e inside the
cake by 3C per centimetre (Rathbun and
Shuler 1983). The reduction of fermentation
temperature and aw is also recommended by
Hering et al. (1991), who observed a better
production of ~,-linolenic acid under these
conditions.
Increased release of amino acids shown
here could improve the nutritional value of
tempe in comparison to unfermented soybeans (Murata et al. 1967, Ismail 1981). The
fact that bacteria such as Citrobacter freundii
or Micrococcus luteus which form vitamin B~2
in tempe are dependent on the metabolic
activity of Rhizopus spp. has previously been
shown (Keuth and Bisping 1993). Now we
have found that high concentrations of free
amino acids released by Rhizopus assist
growth and vitamin B~2 formation, as shown
by the lowered amount of free amino acids in
tempe after co-fermentation of Rhizopus with
C. freundii or M. luteus.
The protease system was investigated to
gain a better insight into the mechanisms of
the proteolytic activity of Rhizopus. The
behaviour of cell wall bound, intracellular,
and extracellular proteases was similar in
relation to pH, temperature and protease
inhibitors. Rhizopus strains investigated
seemed to possess only one protease type.
Inhibition of proteases by pepstatin A and
lack of effect of EDTA confirmed that the Rhizopus strains studied possess an aspartyl
protease. Our results agree with those of
Fukumoto et al. (1967), Bott et al. (1982) and
Takahashi (1988) with R. chinensis. The
molecular weight of proteases described by
these authors was reported to be 35 kDa and

a pH optimum of 2-5-3-3 was indicated. The

t e m p e r a t u r e optima ranged from 50-60C for
this species. We also found a pH maximum in
the neutral area, which was described by
Wang and Hesseltine (1965) and Schindler et
al. (1982)for R. chinensis and R. oligosporus,
respectively. This additional pH maximum is
necessary for degradation of soy protein at
pH values of 6-7 that are found in tempe j u s t
a few hours after the start of fermentation.
The electrophoretic bands at 45 and 68 kDa,
respectively, should be further investigated.
We suggest that the large molecules are glycoproteins, which was verified for the 68 kDa
protein by PAS staining. Tsujita and Endo
(1980) described high molecular weight proteases of Aspergillus oryzae with a high
amount of covalently bound sugars, which fix
the protease at the cell wall. The same effect
could be true in our case.
Enzymatic tests and analysis of the
turnovcr rate showed that cell wall bound
proteases are primarily responsible for the
proteolytic capacity of fermenting Rhizopus.
This fact is a great advantage for Rhizopus in
solid substrate fermentations such as the
tempe fermentation. The main benefit of the
proteolytic activity is made by the direct contact between the soybean and the mycelium
while the fungus is growing through the surface of the soybeans. Determination of the
specific activity of cell wall bound proteases
with a simple test system should give an easy
method to find more strains with a high
proteolytic activity, and help to control fermentation qualities of inocula used in
Indonesia.

Acknowledgements
We acknowledge the work of Dr Mien Mahmud and Dr H e r m a n a (Nutrition Research
and Development Centre, Bogor), of Dr
Suyanto Pawiroharsono and Mr Effendi Siregar (BPP Teknologi, Jakarta), and of Mr B.
Kleinsteuber (T~-V-Rheinland, KSln), who
collected a large proportion of the tempe samples. We thank the Federal Ministry of
Research and Technology in Bonn for supporting these investigations.

46

U. Baumann and B. Bisping

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