Departments ofMicrobiology' and Physiology,2 FacultyofScience, Mahidol University, and Department of. Science, Ministry ofScience, Technology and Energy,3 ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1980, p. 430-435 0099-2240/80/02-0430/06$02.00/0
Vol. 39, No. 2
Isolation and Analysis of Molds from Soy Sauce Koji in Thailand A. BHUMIRATANA,' * T. W. FLEGEL,' T. GLINSUKON,2 AND W. SOMPORAN3 Departments of Microbiology' and Physiology,2 Faculty of Science, Mahidol University, and Department of Science, Ministry of Science, Technology and Energy,3 Bangkok, Thailand
Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.
Soy sauce fermentation in Thailand is usually carried out by small producers following traditional methods handed down through the generations. The process is of the Chinese rather than the Japanese type (3) and, briefly, it consists of the following steps. Whole soybeans are soaked for various periods (usually overnight) before being cooked (3 to 6 h) in boiling water or with steam. They are then drained and cooled before being mixed with a quantity of wheat or rice flour (Fig. 1). Amounts vary and, in some cases, additions are made until the mixture "looks right." However, the total weight added varies roughly from 15 to 50% of the dry weight of the soybeans used. In some establishments, the flour is roasted before being added. The bean and flour mixture is next placed in woven bamboo trays which are incubated in racks (Fig. 2) for 4 to 7 days (koji stage). Fungi inoculated from the air and from the bamboo trays (these are not sterilized between batches) overgrow the mixture, which is eventually dumped into large earthenware jars (Fig. 3) with sea salt brine (final concentration of salt is approximately 18% [wt/vol] and results from mixing approximately 20 kg of koji with 60 liters of 22% salt brine). Brine incubation (mash, or moromi, stage) usually lasts for 30 to 90 days in open yards in the sun, but some hasty processers use incubations of only 2 weeks. The original brine is drained off and constitutes the first grade of soy sauce. More brine may then be added, and further incubation for 1 or 2 months may follow to yield lowergrade sauces. In other cases, the solid bean sediment is separately bottled and sold as another type of condiment (called tau jeow). The total process is summarized in Fig. 4 and is characterized by no control over the inoculum or the culture conditions. As a result, the quality of the product is variable, and some batches are decid-
edly inferior. This contrasts markedly with Japanese manufacturers who control the inoculum, the culture conditions, and the ageing conditions. The microbiology of soy sauce fermentation has been studied in some detail (12), and one of the most important features of the koji step is the growth of Aspergillus strains with high protease activity. In this regard, interviews with several Thai factory owners revealed that the green kojis common during cool weather were preferable to the black or grey kojis common during hot weather. Our examinations of grey kojis showed that they contained a predominance of zygomycetous fructifications (e.g., Rhizopus spp., Mucor spp., Absidia spp., and a Syncephalastrum sp.), whereas the green ones showed a predominance of Aspergillus conidiophores. The work in this study was therefore directed predominantly towards Aspergillus strains, although a representative of Rhizopus was included for comparative purposes. With the aim of eventually improving the reliability and constancy of local soy sauce manufacture by the introduction of controlled koji inoculum, we have isolated several molds from local factories and analyzed them for suitability as koji fungi. Our criteria for selection were high protease activity, no toxin production, and a good taste and aroma of the final product. This report describes these analyses, which culminated in the recommendation of one of the molds for factory use. MATERIALS AND METHODS Mold strains. Samples of soy sauce koji of various ages were obtained from a number of factories in the Bangkok area. They were collected in plastic bags and taken to the laboratory within 1 h, at which time they were either processed immediately or stored at 4°C 430
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FIG. 1. Factory workers mixing wheat flour with cooked soybeans on a wooden mixing table. The amount of flour added was not weighed but rather added until the mixture looked right.
bouraud dextrose agar in 250-ml Erlenmeyer flasks at 30°C for 7 days. Thirty milliliters of 0.05 M phosphate buffer (pH 7), containing 2 to 3 drops of Tween 20, was added, and the flasks were shaken vigorously to suspend the spores. Numbers were determined by direct count in a Bright-line hemacytometer (American Optical Corp.) or by plate counts, and appropriate dilutions were made with phosphate buffer before inoculation. The wheat bran cultures were incubated at room temperature (30 to 35°C). Another substrate used consisted of soybean-flour mixtures (described below) of 300 g each, which were sterilized by autoclaving before inoculation and incubation on bamboo trays in the open at ambient temperatures (30 to 35°C). These cultures were used for comparison with the relative enzyme activities obtained using wheat bran substrate. At daily intervals up to 4 days, two flasks for each tested mold strain were individually extracted by stirring for 15 min with 50 ml of 0.04 M phosphate buffer (pH 6.5) for wheat bran cultures and with 200 ml of 0.04 M phosphate buffer for soybean cultures. The extract was then filtered through Whatman no. 2 filter paper, and the filtrate (crude proteolytic enzyme extract) was stored in sealed vials at -20°C until assayed. The proteolytic activity of these crude extracts was determined at 40°C by the casein digestion method of Spies (11). One unit of protease activity was defined as 1 ug of tyrosine equivalent liberated per min. Protease production by the six fungi isolated in f
FIG. 2. Koji stage of soy sauce fermentation consisting of uncleaned, unsterilized bamboo trays to which the soybean flour mixture illustrated in Fig. 1 was transferred. The koji was not purposely inoculated with microorganisms but must have become randomly inoculated by spores on the trays, in the air, and in the uncooked flour. These trays were incubated in the open with no temperature or humidity control. and processed the following day. One gram of each sample was thoroughly mixed with 4.0 ml of sterilized 0.05 M phosphate buffer (pH 7.0), after which the supematant suspension was serially diluted in the same buffer. Then, 0.1 ml of each dilution was plated on Sabouraud dextrose agar (Difco Laboratories), and the plates were incubated at 30°C for 2 days. Predominant mold colonies were transferred by streaking spores on new plates, from which stock cultures were in turn obtained. Six of these were selected for detailed analysis, and they were designated 2a, 4a, 5a, 7a, 8a (Aspergillus spp.), and lc (Mucor sp.). One Japanese commercial strain of Aspergillus oryzae (designated 9a) was used for comparative purposes. Evaluation of protease production. One substrate used to assess protease production by stock cultures consisted of 5 g of wheat bran with 5 ml of 0.5% NaCl solution mixed and autoclaved in 500-ml Erlenmeyer flasks. After cooling to room temperature, the flasks were inoculated with 5.0 ml of spore suspensions (107 spores per ml), which had been prepared as follows. Fungus cultures were grown on 50 ml of Sa-
FIG. 3. Factory worker transferring 5- to 7-day-old koji to an earthenware jar before the addition of salt brine and incubation in the sun.
APPL. ENVIRON. MICROBIOL.
BHUMIRATANA ET AL. Flour (wheat and/or rice)
soaked in water overnight
cooked (by boiling or by steam)
drained and cooled
mixed under nonsterile conditions
Incubated 4-7 days on uncleaned,previously used bamboo trays in the open; no temperature or moisture control
Transferred to earthermare jars
incubated in the
for 60 days (moronm stage)
beans and liquid separated by draining
beans nore brine added and further incubation in the su n for lower grade soy sauc:e or
liquid incubated in th! sun for a further period of variable duration
filtered and bottled
grade ;(y sauce bottled as a condement called tao Jemow (low grade)
FIG. 4. Schematic outline of the process of soy in a traditionally operated factory in Thailand.
Thailand was compared with that of a culture of Aspergillus oryzae sold commercially as a koji mold in Japan. Toxicity assays. Glutinous rice (250 g) mixed with 250 ml of water was autoclaved in two 2,800-ml Fernbach flasks for 15 min at 121°C. The flasks were then inoculated with an appropriate fungus and incubated at room temperature (30 to 35°C) for 10 to 14 days. Then both the whole cultures were extracted twice with 500 ml of chloroform in a Waring blender. The two extracts were pooled (1 liter) and filtered several times through Whatman no. 1 filter paper before being cleared and dehydrated by filtration through anhydrous sodium sulfate. On a vacuum rotary evaporator, the volume was reduced to an oily residue which was taken up in petroleum ether. Insoluble matter was removed by filtration and dried in vacuo. This method was modified from that of Glinsukon et al. (2) for preparing crude toxin, and the final residue was labeled "crude toxin." It was tested for aflatoxin content and toxicity to rats as outlined below. (ii) Duplicate 100-ml samples of soy sauce were thrice extracted for 30 min with 250 ml of chloroform on a wrist action shaker. The pooled extracts (750 ml each sample) were then filtered through anhydrous sodium sulfate to remove water. The filtrate was then reduced to a small volume in a rotary vacuum evaporator, transferred to a small vial, and evaporated to
dryness under N2 gas. This crude extract was then assayed for aflatoxins as described below. (iii) Toxicity to rats was tested for in weanling female rats (40 to 50 g, Fischer-derived strain; Animal Production Centre, Faculty of Science, Mahidol University) with five animals each in all test and control groups. Crude toxin was dissolved in dimethyl sulfoxide and administered intraperitoneally at a dose level equivalent to 40 g of mold rice (-30 mg of crude toxin per rat). Animals were sacrificed 7 days after dosing, and autopsies were performed for gross examination of visceral organs. (iv) The aflatoxin assay was modified from the methods of Eppley (1). Dried extracts [(i) and (ii) above] were dissolved in 0.1 ml of chloroform, and 100- to 200-Ml samples were applied to thin-layer chromatographic plates along with standard aflatoxins B,, B2, G, and G2. The plates were developed in two solvent systems and monitored for aflatoxin under ultraviolet light. Sensitivity was down to 25 ng. Preparation of semicontrolled koji. Commercial dried soybeans (44 kg) were soaked overnight in an excess of tap water. After draining, the beans were steam cooked at 15 lb/in2 for 15 min, cooled to room temperature, and mixed thoroughly with 6 kg of rice flour previously roasted in a hot-air oven. The koji was then inoculated with spores (106 per g [dry weight] of koji). The inoculated koji was then spread on round bamboo trays (-60-cm diameter) like those used in the factories, except for being washed before use. The maximum depth of the koji did not exceed 4 cm. The trays were incubated in the open at room temperature for 7 days before being transported to a factory for incubation in salt brine, parallel to regular factory koji. The object of this experiment was not to work under sterile conditions, but rather to follow a procedure similar to that in the factory except for the care taken to inoculate the koji with spores from a specific mold. Miscellaneous analytical methods. Protein was routinely determined by the method of Lowry et al. (7) with tyrosine as the standard. Soluble nitrogen was determined by the method of the Association of Official Analytical Chemists (5), and reducing sugar was determined by the method of Somoggi (10). Gustatory and olfactory judgments were made by the soy sauce factory owners.
RESULTS The proteolytic activities of wheat bran culture extracts for six Thai isolates of koji fungi are shown in Table 1, along with the activity of a commercial strain of A. oryzae (9a) from Japan. Strain 4a gave the highest sustained activity over the 4-day test period. Similar tests on soybean substrate with fewer isolates also ranked 4a highest in activity (Table 2). As a consequence, 4a was tested for toxicity as a prelude to its use as inoculum in factory-produced koji. Glutinous rice cultures of strain 4a yielded an average of 0.67 g of residue from crude toxin extracts. However, no aflatoxins were detectable in the residue, and toxicity tests with rats were negative (there were no deaths or significant
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changes in body weight, and visceral organs were normal). For identification, 4a was grown on Czapek solution agar and malt extract agar (8) with the following results. Growth on Czapek agar was 4.5 cm in 5 days with colonies that were yellow green, not turning brown with age (2 weeks). Conidiophores arose mostly from the agar and were evenly produced but not densely packed; many conidial heads were columnar. In older parts of the colonies there was occasional overgrowth by aerial mycelium bearing smaller conidiophores. On malt extract agar, growth was 6.5 cm in 5 days with green colonies. Virtually all conidial heads were tightly columnar, arising directly from the agar. Conidiophores were not tightly packed but showed slight zonation. Conidiophores on Czapek agar were not over 300 ,im long, and on malt extract agar they were not over 250 ,Am long. The following macroscopic features were the same on both media. Conidiophores, 6.5 to 10.4,um wide with vesicles 13 to 28 ,um in diameter, were at first clavate but later more or less globose. Sterigmata were mostly on the upper two-thirds to one-half of the vesicles, uniseriate and 7 to 12 ,um long. Spores were globose, 3.5 to 7.5 ,um in diameter, and minutely sculptured (Fig. 5). The isolate was identified as Aspergillus flavus var. columnaris Raper and Fennel (8), and this identification was kindly confirmed by K. B. Raper. Results of tests made on koji and soy sauce from a regular factory run and from one using strain 4a as inoculum are shown in Fig. 6 and Table 3. Figure 6 shows the protease activity of two regular factory runs and a third one using strain 4a as inoculum. The activity with 4a was approximately 4 times higher from days 2 to 5. Analysis of the soy sauce product of these kojis TABLE 1. Proteolytic activity in wheat bran cultures of six isolates compared with A. oryzae (9a) from Japan Strain
Tyrosine equivalent released (jug/g [dry wt] per min) following days of incubation: of substrate
lc 0 103 187 68 170 2a 0 696 811 1,582 224 4a 0 3,313 11,360 6,978 2,488 5a 0 500 432 3,077 1,109 0 6,830 7a 811 947 6,410 0 5,950 8a 892 446 3,624 0 1,353 974 122 9a 439 0 Control' 0 0 0 0 'The control consisted of equally treated but uninoculated substrate.
TABLE 2. Proteolytic activity of three Aspergillus strains on soybean substrate
Tyrosine equivalent released (,ug/ g [dry wt] of substrate per min) at following no. tion: of days of incuba1
4a 728 8,008 5a 270 520 9a 208 728 Controla 624 1,040 a The control consisted of equally
6,344 1,352 2,264 832 treated
5,606 1,144 2,288 208 but un-
after 45 days' incubation in salt brine is shown in Table 3. Several features are apparent. Levels of the tyrosine equivalents, sugar, and soluble nitrogen were all higher in the soy sauce produced from 4a-inoculated koji; the soluble nitrogen, in particular, was much higher. In the opinion of the factory owners, the organoleptic qualities of the soy sauce resulting from the semicontrolled koji using strain 4a as inoculum made it superior to that resulting from the regular factory koji.
DISCUSSION Because of its superior protease production and flavor-inducing qualities, we recommend strain 4a for use as a koji mold in Thailand. The fact that it is a locally occurring fungus may lend it some ecological advantage over imported koji molds, and this could be particularly important in factories where the control over koji culture is limited. To our knowledge, this is the first report of the use of a variety of A. flavus as a koji mold in the soy sauce industry. This may cause some concern to those who are worried about the danger of aflatoxin production, but we feel there are several reasons to allay any fears and justify its use. First, A. flavus and A. oryzae are closely related, and the distinctions between them are not sharply defined. If one considers a range of characteristics between two extremes, each representing one of these species, somewhere in the shady middle one must choose one species over the other. In the absence of genetic data, one is forced to use limited morphogenetic and physiological criteria, and the authority we chose to follow considers the strain we have selected as a variety of A. flavus. However, a look at the synonyms of this taxon (8) shows that others have called it various things, including A. oryzae. Second, we have tested this strain for aflatoxins on the substrate (i.e., glutinous rice) considered ideal for their production by species of A. flavus, and the tests were negative. So were tests with soybean koji and soy sauce.
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BHUMIRATANA ET AL.
D FIG. 5. Photomicrographs of strain 4a identified as A. flavus var. columnaris. (A) Young conidiophores which arose directly from the agar- (180x), (B) old conidiophores and mature spores (180x), (C) mature spores (1,660X), (D) young conidiophore showing uniseriate sterigmata (1,660X).
Third, soybean has been shown to be a poor substrate for aflatoxin production, even for strains of Aspergillus which are high producers on other substrates (4, 9; C. W. Hesseltine, in P. MatangKasombut, ed., Proc. Fifth Int. Conf. Global Impacts Appl. Microbiol.). On the strength of these arguments, we feel confident in recommending 4a as a koji mold and in subjecting it to further improvement by mutagenesis. Large-scale tests are presently being conducted at a new factory where inoculum and
culture conditions can be controlled. Analysis of these tests will be the subject of further reports. In addition, work is under way to select a high amylase-producing mutant of strain 4a that can be co-inoculated with the high protease strain. Our preliminary tests with laboratory koji inoculated on bamboo trays and then removed to a factory for the moromi, or mash, stage gave superior results to regular factory runs (judgment of the manufacturer). However, an attempt to inoculate koji at the factory site (inoculation
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TABLE 3. Qualities of soy sauce produced from semicontrolled koji inoculated with strain 4a compared with those of soy sauce produced from regular factory koji Soy sauce prepared from:
Semicontrolled koji After filtering Aged
nitroSoluble gen (g/liter)
Sugar (g of liter)
Factory-prepared koji OK ND 32.1 22.9 4.65 64 After filtering OK ND 35.4 28.2 4.9 126 Aged a Sensitivity was down to 25 ng. b The bioassay was done in weanling female rats with a dose level equivalent to 40 g of mold rice per rat. 'ND, Not detectable.
+Pure Cultured Koji
E0 tFctory Prepard Koji
DAYS FIG. 6. A comparison of protease activity in laboratory-prepared and factory-prepared koji. The laboratory-prepared koji differed from the factory koji in the care taken to clean the bamboo trays and to purposely inoculate the substrate before incubation. Protease activity is given in kilounits per gram (dry weight) of koji. at the flour-mixing step) did not produce the desired green koji but an undesired black one. This may have resulted because of higher temperatures at the factory (the temperature in the koji was measured as high as 50°C on the second day of incubation, whereas the optimum temperature for growth with this fungus as measured by colony diameter tests on agar was 30°C) or because of contamination arising from the previously used bamboo trays. It may also have resulted from improper moisture content or poor ventilation. In any case, it was recommended to the owner that some form of control be exerted over temperature and moisture during the koji culture. The influence and importance of these variables have been reviewed by Yong and Wood
(12). We have not yet made a detailed study of the nonmold microorganisms also isolated from the koji in local factories. However, in spite of an earlier study recommending co-inoculation of fungus, yeast, and bacteria (6), it appears that good results can be obtained by using only mold
inoculum (12). For this reason, it may be more profitable to concentrate on the yeasts and bacteria in the mash, or moromi, stage. Because the amylase production by strain 4a is relatively low, work is currently under way to select a high-amylase mutant by ultraviolet mutagenesis. Such a mutant will then be tested as a co-inoculant with wild-type 4a. ACKNOWLEDGEMENT We acknowledge the excellent technical assistance of Watanalai Panbangred and Attawut Impolsap. This work was supported by a research grant from the Association of South East Asian Nations-Australian Economic Cooperation Programme Protein Project. LITERATURE CITED 1. Eppley, R. M. 1968. Screening method of zearalenone, aflatoxin and ochratoxin. J. Assoc. Off. Anal. Chem. 51: 74-78. 2. Glinsukon, T., S. S. Yuan, R. Wightman, Y. Kitaura, G. Buchu, R. C. Shaule, G. N. Wojan, and C. M. Christensen. 1974. Isolation and purification of Cytochalasin E. and two tremorgens from Aspergillus clavatus. Plant Food for Man 1:113-119. 3. Hesseltine, C. W. 1965. A millennium of fungi, food and fermentation. Mycologia 57:149-197. 4. Hesseltine, C. W., 0. L. Shotwell, J. J. Ellis, and R. D. Stubblefield. 1966. Aflatoxin formation by Aspergillus flavus. Bacteriol. Rev. 30:795-805. 5. Horowitz, W. (ed.). 1975. Total nitrogen-kjeldahl method, p. 15-16. In Official methods of analysis, 12th ed., Association of Official Analytical Chemists, Washington, D.C. 6. Lockwood, L. B. 1947. The production of Chinese soya sauce. Soybean Dig. 7:10-11. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. Raper, K. B., and D. L. Fennel. 1965. The genus AspergiUlus. The Williams & Wilkins Co., Baltimore. 9. Shotwell, 0. L., E. E. Vandergraft, and C. W. Hesseltine. 1978. Aflatoxin formation on sixteen soybean varieties. J. Am. Oil Chem. Soc. 61:574-577. 10. Somoggi, M. 1945. A new reagent for the determination of sugars. J. Biol. Chem. 160:61-68. 11. Spies, J. R. 1957. Colorimetric procedures for amino acids. Methods Enzymol. 3:467-477. 12. Yong, F. M., and B. J. B. Wood. 1974. Microbiology and biochemistry of soy sauce fermentation. Adv. Appl. Microbiol. 17:157-194.