BioSMART ISSN: 1411-321X

Volume 5, Nomor 1 April 2003

Halaman: 5-7

Development of Pure Culture Starter for Kecap, an Indonesian Soy Sauce

JOKO SULISTYO1, SAYUKI NIKKUNI 2

1
Research Center for Biology - LIPI, Bogor 16002.
2
Food Science and Technology Division, JIRCAS, Tsukuba, Ibaraki 305-8686, Japan

Received: 4 December 2003. Accepted: 15 January 2003

ABSTRACT

In order to prevent aflatoxin contamination during the production of kecap, an Indonesian soy sauce, a white-spored mutant K-1A strain
induced from an aflatoxin-negative koji mold was applied to koji making process of kecap. While kecap koji making process took 9
days by conventional method without inoculum, the use of K-1A strain as a starter shortened the process to 3 days at room temperature.
The traditional koji sample contained 2.3 x 107 cfu/g of Aspergillus and 5.0 x 106 cfu/g of molds belonging to Mucorales. The koji
prepared with this starter contained 2.3 x 107 cfu/g of Aspergillus sp. K-1A as the dominant species and some contaminants from the
environment such as other species of Aspergillus (2.0 x 106 cfu/g) and Mucorales (1.4 x 105 cfu/g). The mash prepared with kecap koji
inoculated with this starter contained higher formol nitrogen (FN) and water-soluble nitrogen (WN) than those prepared with koji
inoculated without any starter. These results demonstrated that the use of this starter did not only contribute to the prevention of
aflatoxin contamination but also improved the conventional kecap fermentation process.

Key words : Aspergillus, ragi kecap, fermentation, soybean, non-aflatoxin.

INTRODUCTION In this study, we prepared ragi kecap using a white-

spored mutant, K-1A and applied it for kecap production at
Kecap is a fermented soybean food known as Indonesi- Zebra Kecap factory in Bogor, Indonesia. We will analyze
an soy sauce. Whole soybeans, specially the varieties of and evaluate the quality of kecap moromi during
black soybeans, are used as raw material for processing fermentation.
kecap. Kecap is prepared by spreading cooked soybeans on
bamboo trays and leaving them for a period of time until
molded soybeans (koji) are formed. Kecap manufacturers MATERIALS AND METHODS
do not usually use any inoculum (tane-koji, ragi kecap) for
koji preparation. During processing, molds grow on the Preparation of ragi kecap
cooked soybeans as the result of contamination from the Soaked rice (4 kg as raw material) was autoclaved at
environment such as the air and the previously used trays. 121ºC for 30 min, allowed to cool down until 40ºC, inocu-
Judoamidjojo (1986) reported that molds isolated from lated with the ragi kecap that had been prepared at JIRCAS
kecap koji were mostly from the genus of Aspergillus. It is in Tsukuba, 2001, left it overnight at room temperature,
known that agricultural food commodities such as peanut spread on a aluminum tray, and covered with a bamboo
and maize are widely contaminated with aflatoxins, natural tray. After 4-day incubation at room temperature, it was
carcinogens, produced by fungi belong to Aspergillus dried at 42ºC for 3 days.
section Flavi, in Indonesia (Dharmaputra, 1991). It was
also reported that 15 out of 32 samples of Indonesian kecap Kecap production
contained aflatoxin B1 at concentrations of more than 5 Kecap were prepared by the conventional and
µg/kg (Sadjono et al., 1992). Since the possibility of traditional methods without inoculum and newly developed
aflatoxin contamination cannot be ruled out in the methods using the ragi kecap prepared at JIRCAS in 2001
traditional way of koji processing, thus, it is necessary to at Zebra Kecap Factory in Bogor (Figure 1).
use a pure culture starter, ragi kecap from the standpoint of
preventing aflatoxin contamination. Microbial analysis and isolation of fungi
In our previous study (Nikkuni et al., 2002), we Sample (10g) was mixed with 90 ml of autoclaved
induced white-spored mutants from koji molds by UV- 0.05% Tween 80. For counting filamentous fungi, serial
irradiation and used them in kecap fermentation. We dilutions of the samples were prepared in the same solution
evaluated the possibility of distinguishing the mutant from and the diluted suspensions (0.1ml) were spread on the pla-
aflatoxin-producing molds and revealed that white-spored tes of potato dextrose agar (PDA, Difco, USA) containing
mutant can be used as a starter for preparation of kecap koji 100 ppm chloramphenicol. The plates were incubated at
from the standpoint of preventing aflatoxin contamination. room temperature for 1 to 3 days at room temperature.

© 2003 Jurusan Biologi FMIPA UNS Surakarta

6 BioSMART Vol. 5, No. 1, April 2003, hal. 5-7

Traditional process New process RESULTS AND

DISCUSSION
black soybeans (40 kg) black soybeans (40 kg)
Preparation of ragi kecap
soaked soaked
To study the capacity of
boiled, for 2-3 hr boiled for 2-3 hr ragi kecap on production of
kecap comparing to the
spread on a bamboo tray spread on a bamboo tray
traditional way of kecap
inoculated with ragi (120 g)
incubated, at room temperature for 6 to 10 days incubated, at room temperature for 3 days production at Zebra Kecap
factory, it was required the
molded soybeans (kecap koji) molded soybeans (kecap koji)
ragi kecap containing less
sun-dried sun-dried than 10% moisture, more
sun-dried koji (ca 25 kg) than 108 spores of Aspergil-
sun-dried koji (ca 26 kg)
30 kg NaCl lus and less than 104 bacteria
70 L hot water 30 kg NaCl + 70 L hot water 58°C for the pilot scale experiment
Kecap moromi Kecap moromi purposes on kecap
fermented at room temperature for ca 1,5 months production at the factory.
The ragi kecap was prepared
extracted with hot water from rice using the white-
palm sugar spored mutant K-1A strain.
spices
cooked Figure 2 shows the mois-
ture contents in ragi kecap
filtered
during drying at 42 ºC after
bottled 4 day-fermentation at room
Kecap Manis temperature. Moisture of
ragi decreased with drying
Figure 1. Kecap production process at Zebra Kecap Factory in Bogor, Indonesia. time and reached the level of
less than 10% after 3 days.
To count bacteria, 1 ml of diluted suspension was mix-
ed with molten plate-count agar consisting of 0.5% peptone 25
(Difco, USA), 0.25% yeast extract (Difco, USA), 0.1%
glucose and 1.5% agar. One to 6 colonies of Aspergillus 20
Moist ure (%)

were isolated from a PDA plate and further purified.

15
Analytical methods
Moisture. Moisture was determined by the aluminum 10
foil cup method. A piece of aluminum foil (15 – 20 cm 5
square) was formed into a cup shape (ca 5 cm in diameter,
ca 5 cm deep) using a bottom of a 100 ml beaker. Five 0
grams of sample were weighed into an aluminum foil cup 0 1 2 3 4
and heated at 105ºC overnight in an oven and cooled in a
desiccator for 30 min. Da ys
Nitrogen. After homogenizing kecap mash (moromi)
preparations, total nitrogen (TN) was determined by Kjel- Figure 2. Moisture contents of ragi kecap during drying at 42ºC.
dahl method. To determine water-soluble nitrogen (WN),
10g of the homogenized moromi was diluted to 250 ml Table 1 shows the yields, moisture contents, mold
with distilled water. The resulting diluted suspension was counts and total viable counts in prepared ragi. Four kg
centrifuged at 18,000 rpm for 10 min and the supernatant rice was converted into 3.3 to 3.7 kg ragi. Ragi contained
was filtered. Nitrogen present in 20 ml of the filtrate was 7.7% to 10.4% moisture, more than 108 spores of
determined by Kjeldahl method. Formol nitrogen (FN) was Aspergillus and less than 103 bacteria.
determined according to Official Method of Miso Analysis
(1986). After neutralizing the supernatant (50 ml) with 0.1 N Table 1. Preparation of ragi kecap using rice as substrate.
NaOH to pH 8.5, formaldehyde (20 ml, pH 8.5) was added
and the mixture was titrated with 0.1 N NaOH to pH 8.5. Ragi Yield* Moisture Aspergillus sp. TVC**
Sodium chloride. Sodium chloride was determined No. (kg) (%) (cfu/g) (cfu/g)
according to the method described in Methods of Soy Sauce 1. 3.7 7.7 2.3 x 108 < 3000
2. 3.4 10.4 3.5 x 108 < 3000
Studies (1985). The supernatant (5 ml) as prepared above
3. 3.3 9.0 3.9 x 108 < 3000
was taken in a porcelain evaporating dish and titrated with 4. 3.3 8.9 5.8 x 108 < 3000
N/20 silver nitrate in presence of 1 ml 2% potassium * Four kg of rice was used as substrate. ** Total viable bacterial
chromate as an indicator. counts.
 SULISTYO and NIKKUNI – Indonesian Soy Sauce of Kecap 7

Preparation of kecap production at Zebra Kecap Factory value comprises ammonia and amino nitrogen, FN and TN
Kecap koji were prepared by the conventional methods of each kecap moromi were determined. Total nitrogen
and the newly developed methods using the ragi kecap. (TN) contents, water-soluble nitrogen (WN), formol
Moisture contents and microbial counts in koji preparations nitrogen (FN), sodium chloride and pH during fermentation
are shown in Table 2. Both samples contained approxi- are shown in Table 3.
mately 108 cfu/g of bacteria. The traditional koji sample After 2 months submerged fermentation, the ratio of FN
contained 2.3 x 107 cfu/g of Aspergillus sp. as dominant to TN (FN/TN) and the WN to TN (WN/TN) of kecap
mold and 5.0 x 106 cfu/g of Mucorales. After sun-drying, moromi prepared without ragi kecap according to Zebra
these values decrease to one-tenth. The koji prepared using Kecap procedure were 13.1% and 41.9%, respectively.
the ragi kecap contained 2.3 x 107 cfu/g of Aspergillus sp. However, the values of FN/TN and the WN/TN of kecap
K-1A as the dominant mold, even though it contained moromi inoculated with ragi kecap K-1A were 26.1% and
contaminants: 2.0 x 106 cfu/g of the other Aspergillus spp. 56.5%, respectively. Aspergillus strain K-1A apparently
and 5.0 x 106 cfu/g of Mucorales from the environment and contributed well to hydrolysis of the soybean proteins
the moisture contents decreased to 7 to 8%. during the fermentation rather than that of kecap from
Zebra factory.
Table 2. Moisture and microbial counts in kecap koji. The ragi kecap that was prepared using K-1A was the
Moist- Mold count (cfu/g) white-spore mutant of strain K-1 isolated from the Japanese
Cooked and TVC tane-koji for soy sauce production. However, the WN
ure Aspergillus Rhizopus/
Fermented Soybean (cfu/g) content of the moromi samples prepared using both of the
(%) K-1A others Mucor
Cooked soybean 68.5 - - - - original strain (K-1) and the mutant one (K-1A) was not so
Traditional kecap koji much difference. Since the mutants develop white conidia
Fermented (9 d) koji 36.5 - 2.3x107 5.0x106 4.1x109 during koji fermentation and exhibited enough activity for
Sundried koji 7.3 - 2.8x106 1.5x105 2.6x109
K-1A kecap koji
digesting soybean proteins, this white-spored koji of K-1A
Fermented (3 d) koji 54.9 2.3x107 2.0x106 1.4x105 8.6x108 strain was expectedly useful as a ragi kecap for manufac-
Sundried koji 8.1 8.6x106 1.0x106 1.3x105 1.8x109 turing kecap koji from the standpoint of preventing afla-
toxin contamination, since it could be distinguished from
Kecap mash (moromi) fermentation those prepared with the original koji strain and the aflatoxin
Use of the ragi kecap for a large scale preparation of producer by their appearances (Nikkuni et al, 2002).
kecap koji is becoming important procedure by
overgrowing soaked, cooked, cooled soybeans coated with
ground roasted wheat with K-1A strain belonging to the ACKNOWLEDGEMENT
Aspergillus oryzae species. The kecap koji contains
proteases, amylases, and lipases that hydrolyse their We would like to express our sincere thanks to Dr. Arie
respective substrates in the subsequent submerged Budiman, Director, Research Center for Biology, LIPI, for
fermentation in approximately 20% weight per volume salt opportunity and encouragement given to carryout the colla-
brine. During the submerged fermentation, some halophilic boration research on development of ragi kecap between
microorganisms such as Pediococcus cerevisiae, Research Center for Biology and Japan International
Lactobacillus delbruekii, and salt-tolerant Saccharomyces Research Center for Agricultural Sciences (JIRCAS). We
rouxii naturally developed, since in a fact that kecap wish to thank Yati Sudaryati Soeka, Elidar Naiola, and
fermentation depends on proteolytic enzymes derived from Devira Verina of Microbiology Division Research, Center
those halophilic microbial strains to hydrolyse the proteins for Biology, LIPI, for their great help, support, kind
in the substrate to the constituent amino acids and peptides. hospitality and collaborative assistance in carrying out
Kecap moromi were prepared with the kecap koji and experiments.
allowed to ferment in order to have more favorable kecap
product enriched with amino acid constituents. Free amino REFERENCES
acids are known to contribute the taste of kecap and
glutamic acid is especially important for flavor. As FN Dharmaputra, O.S., H.S.S. Tjtrosomo, and Sulaswati. 1991. Aspergillus
flavus and aflatoxin in peanuts collected from three markets in Bogor,
West Java, Indonesia. In Naewbanij, J.O. (ed.). Proceedings of the
Table 3. Formol nitrogen (FN), water-soluble nitrogen (WN), Twelfth ASEAN Seminar on Grain Postharvest Technology. Bangkok:
total nitrogen (TN), NaCl contents, and pH values of kecap The ASEAN Grain Post-Harvest Programme.
moromi. Editorial Committee for Society for Shoyu Shiken-Ho and Japan Soy
Sauce Research Institute. 1985. Shoyu Shiken-Ho (Methods of Soy
Fermentation NaCl FN WN TN FN/TN WN/TN FN/WN Sauce Studies). Tokyo: Nihon Shoyu Kenkyujo.
pH
Period (days) (%) (%) (%) (%) (%) (%) (%) Institute of Miso Technologist. 1986. Official Method of Miso Analysis.
Control*(42) 5.53 20.8 0.17 0.68 1.65 10.3 41.21 25.00 Tokyo: Institute of Miso Technologist.
Control (60) 5.54 21.0 0.26 0.83 1.98 13.1 41.92 31.33 Judoamidjojo, M. 1986. The studies on kecap – indigenous seasoning of
K-1A** (2) 5.73 21.0 0.15 0.40 1.12 13.4 35.7 37.5 Indonesia Memoirs of the Tokyo University of Agriculture 28: 100-159.
K-1A (14) 5.64 20.9 0.24 0.65 1.35 17.8 48.2 36.9 Nikkuni, S., J.S. Utomo, S.S. Antarlina, E. Ginting, and T. Goto. 2002.
K-1A (42) 5.51 20.3 0.41 0.77 1.57 22.3 49.0 45.5 Application of white-spored mutants induced from koji molds for the
K-1A (60) 5.45 20.6 0.42 0.91 1.61 26.1 56.5 46.2 production of Indonesian soy sauce (kecap). Mycotoxins 52 (1): 13-22
Sadjono, R. Kapti, and S. Sudarmadji. 1992. Growth and aflatoxin
* Traditionally processed moromi. ** Newly processed moromi
production by Aspergillus flavus in mixtured culture with
with ragi K-1A. Aspergillus oryzae. ASEAN Food Journal 7: 30-33.
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