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1995 Proteolysis During Tempe Fermentation PDF
1995 Proteolysis During Tempe Fermentation PDF
1995 Proteolysis During Tempe Fermentation PDF
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
Received :
17 May 1994
40
Table 1. Origin of tempe and tempe inoculum samples from which Rhizopus strains were
isolated
Species
Place
Strain
R. oligosporus
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
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.
41
42
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
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).
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
Protease inhibitor
EDTA
PEP
No.
Cell wall bound
Extracellular
Intracellular
0.21
0,'21
0.11
0.21
0.22
0.11
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
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.
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
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Edited by J. I. Pitt.
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