Characteristics of Bacillus amyloliquefaciens Isolated from Soy Sauce and Influence on Putrefaction of Processed Soy Sauce with Less Salt

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1 Original Paper Characteristics of Bacillus amyloliquefaciens Isolated from Soy Sauce and Influence on Putrefaction of Processed Soy Sauce with Less Salt Yoshio MAKINO and Hiroko FUJISAWA Fermentation and Food Research Institute, Kagawa Prefectural Industrial Technology Center, , Koh, Nohma, Uchinomi, Shozu, Kagawa , Japan Characteristics of the bacteria KP-991 and KP-992 strains isolated from soy sauce of which the bacterial spore was stable against heating for 10min at 100 C was investigated. The isolated bacteria were identified to be Bacillus amyloliquefaciens (Fukumoto) Priest et al. from the results of observation of morphology, physiological tests and analysis of 16S rdna. The KP-991 and KP-992 strains were more stable against heating than the type strains of B. amyloliquefaciens, B. subtilis Cohn and B. megaterium de Bary. The KP-991 and KP-992 strains survived in a trial product of dilution-free seasoning because of the high thermostability and remarkably grew in two kinds of dilution-free seasoning on the market. It is suggested that the dilution-free seasoning made from the soy sauce contaminated by the KP-991 or KP- 992 strain putrefies. Key words: Bacillus amyloliquefaciens; seasoning; food hygienics; soy sauce; thermostability 1. Introduction 2. Materials and Methods Processed soy sauce have been much in demand recently affected by the diverse public taste, whereas amount of soy sauce consumed has been decreased annually [1]. Especially, the demand for dilution-free seasoning, Japanese noodle soup used without dilution, has been increased because it is convenient and includes high concentration of various kinds of the seasoning extracted from natural materials. However, the dilution-free seasoning easily putrefies by microorganisms contaminated from soy sauce as a main stuff because concentrations of the contents to depress the growth of microorganisms, for example NaCI, ethanol and others in the dilution-free seasoning, are much lower than those in the condensed seasoning [2,3]. Furthermore, soy sauce manufacturers are obliged to produce seasoning without preservatives because consumers dislike food additives. Therefore, the seasoning without dilution needs to be managed under the severer hygienic condition than the condensed ones. In this study, thermostable bacteria were isolated from soy sauce and investigated. The thermostability, survival and growth of the bacteria in dilution-free seasoning were examined. 2.1 Isolation and identification of thermostable bacteria A 10 ml raw dark-color soy sauce offered by a factory (Uchinomi, Kagawa, Japan) was brought in a test tube (16.5 mmo) and heated for 10 min at 100 Ž. A 0.1 ml heated soy sauce was smeared on a nutrient agar plate composed of peptone, 30.6 g; beef extract, 3.3 g; yeast extract, 1.1 g; NaCI, 39.4 g; glucose, 25.9 g; ethanol, 10.1 ml; agar, 20 g; distilled water to 1000 ml (ph 5.05) so that the contents can be adjusted to average of dilution-free seasoning on the market [4], and incubated for 2 d at 30 C. Two kinds of the grown colonies were taken and incubated under the same condition mentioned above again. The grown bacteria were named KP-991 and KP-992 strains, and stored on slant media of the nutrient agar at 5 Ž. Bacillus amyloliquefaciens (Fukumoto) Priest et al. IFO , B. subtilis Cohn IFO-13719, B. megaterium de Bary ATCC as type strains and the isolated KP-991, KY- 992 strains were tested in the following experiments. Physiological activity tests and analysis of 16S rdna from the isolated bacteria were consigned to NCIMB Japan Co. Ltd. (Shimizu, Shizuoka, Japan). Extraction of DNA from the isolated strains, polymerase chain reaction (PCR) of DNA and purification of the PCR product were operated following the protocol proposed by Applied (Received September ; accepted October ) õ Fax: , makino@itc.pref.kagawa.jp Biosystems Japan Ltd. (Tokyo, Japan) with the MicroSegTM 16S rdna Gene Kit. 16S rdna was analyzed

2 148 Yoshio MAKINO and Hiroko FUJISAWA using ABI PRISMTM 377 DNA sequencer (Applied Biosystems Japan Ltd.) and the homology of DNA from the isolated bacteria with DNA from the bacteria registered in a data base of MicroSegTM was searched. A platinum loop of the bacterial cells taken from the slant medium was suspended in a small amount of nutrient broth [6]. The cell suspension was added to a 50 ml nutrient broth included in a 250 ml shaking flask. A precultured cell suspension was prepared by shaking (velocity 120 rpm, margin 3 cm) the flask for 17 h at 30 Ž. The dilution-free seasoning produced by the companies A (S-A) and B (S-B) were used for the cultivation of the thermostable bacteria. Total nitrogen, NaCI, direct reducing sugar, ethanol and ph in the seasoning were analyzed by Kjeldahl method, Mohr's method, 3, 5-dinitrosalicylic acid test, gas chromatography and glass electrode test, respectively. Water activity at 25 Ž was determined with a EZ-100 water activity meter (measurable range , 2.2 Test for thermostability of the isolated bacteria Bacterial spore suspensions were produced by the reported method [5] using the prepared bacteria. Four test tubes (16.5 mmo) including a 10 ml suspension per tube were heated for 0, 15, 30 and 45 min at 90 Ž, respectively. Survival spores were counted on the standard plate (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan). This test was operated for five kinds of the bacterial strains. minimum value 0.02) (Freund Industrial Co., Ltd., Tokyo, Japan). A 9.9 ml seasoning S-A or S-B was introduced in an L- form tube. A 0.1 ml precultured cell suspension was added to the tube, and cultured at 50 rpm with a TN-2612 Bio-photo recorder (Advantec MFS, Inc., Tokyo, Japan) at 37 C with the periodical recording of optical density at 660 nm (O.D. 660 nm). Dry cell mass in the seasoning (g. F1) was calculated from the value of O.D. 660 nm using a calibration line. This investigation was operated for 2.3 Investigation of spore counts in the process of seasoning production A dilution-free seasoning was produced following the five kinds of the bacterial strains. 3. Results and Discussion method reported by Noguchi [4]. A condensed seasoning was made by dissolving a 22.5 g sugar and a 1.0 g glutamic acid monosodium salt in the mixture of a 110 ml dark-color soy sauce and a 20 ml sweet sake, and sterilized by filtration (pore size 0.45 um). A 0.1 ml spore suspension previously produced was added to a test tube including a 2.5 ml sterilized condensed seasoning. Survival spores were counted after heating for 1 min at 80 C. Soup was produced by heating a 680 ml tap water including 5 g flakes of dried bonito, 10 g flakes of dried frigate mackerel and 5 g flakes of dried scombroid for 30 min at 85 C. It was sterilized by filtration after cooled at room temperature. Dilution-free seasoning was produced by adding a 7.5 ml soup to a 2.6 ml condensed seasoning including a 0.1 ml spore suspension in a test tube. Survival spores were counted in the dilution-free seasoning after heating for 15 min at 85 Ž and for 30 min at 90 Ž, respectively. This investigation was operated for five kinds of the bacterial strains. Characteristics of the KP-991 and KP-992 strains isolated from raw soy sauce were presented in Table 1. The strains were assumed to be Bacillus group bacteria from the results of Gram's stain, catalase reaction, growth under aerobic condition, morphology and endospore production [7]. Color of colonies, and formation of acid from arbutin, glycogen and salicin were different between both strains. They were assumed to be B. subtilis or B. amyloliquefaciens because they grew at 50 C and on the media including 10% NaCI, also were absolutely aerobic bacteria and reduced nitrate to nitrite [7]. The strains were identified to be B. amyloliquefaciens because J3-galactosidase reaction was negative and did not utilize citrate [7]. The KP-991 and KP- 992 strains share the highest homology (KP %, KP %) with B. amyloliquefaciens among the species registered in the data base of MicroSegTM, according to the sequence data for 16S rdna. Isolation of genus Bacillus from soy sauce has already been reported. Haga & Endo [8] and Sekine & Shigeta [9] reported that soy 2.4 Test for growth of thermostable bacteria in sauce is chiefly contaminated by B. subtilis. No reports dilution-free seasoning have been found concerning contamination of soy sauce by B. amyloliquefaciens. The species had been included in B. subtilis before [10]. Therefore B. amyloliquefaciens isolated from soy sauce might be reported as B. subtilis in the past. Thermostablity of the tested bacterial spores was shown in Fig. 1. The spores of B. megaterium ATCC strain were killed within 15 min. Four digits of the spores of B. subtilis IFO strain were killed after heating for 15 min at 90 Ž, and almost spores were killed after heating for 45 min at 90 Ž. However, the KP-991,

3 B. amyloliquefaciens from soy sauce 149 Table 1 Characteristics of the bacteria isolated from soy sauce

4 150 Yoshio MAKING and Hiroko FUJISAWA Fig. 1 Thermostability of the bacterial spores KP-991, KP-992, Bacillus amyloliquefacience IFO , Bacillus subtilis IFO-13719, ž Bacillus megaterium ATCC Fig. 3 Growth of the bacterial cells in dilution-free seasoning KP-991, KP-992, Bacillus amyloliquefacience IFO , Bacillus subtilis IFO-13719, ž Bacillus megaterium ATCC Open or closed symbol denotes the seasoning produced by the company A or B, respectively. KP-992 and B. amyloliquefaciens IFO strains were more stable against heating than the other ones because the degree of the spores killed after heating for 45 min at 90 Ž was within a digit. It is suggested that the spores of the strains except B. megaterium ATCC survive in a product of dilution-free seasoning because the proposed pasteurization method of the seasoning is heating for 30 min at 90 Ž [4]. The counts of surviving spores in the process of seasoning production were shown in Fig. 2. The spores of the B. megaterium ATCC and B. subtilis IFO strains were killed by heating for 15 min at 85 Ž and for 30 min at 90 Ž, respectively. However, the degree of the spores of the KP-991, KP-992 and B. amyloliquefaciens IFO Fig. 2 Survival of the bacterial spores in the process of seasoning production KP-991, KP-992, Bacillus amyloliquefacience IFO-15535, Bacillus subtilis IFO-13719, Bacillus strains killed until the final product was within ca. two digits. Thermostability of the isolated strains was higher than the type strain of B. amyloliquefaciens from the results of Figs. 1 and 2. megaterium ATCC The growth of the tested strains in two kinds of the seasoning was shown in Fig. 3. According to the results in Table 2 Contents in dilution-free seasoning used for the cultivation of bacteria Table 2, the isolated KP-991 and KP-992 strains properly grew in S-A because they were isolated on a nutrient agar plate of ph 5.05, of water activity 0.96, with 3.65 g 100 mf 1 NaCI and with 0.72 g 100 mf 1 ethanol. Although the isolated strains grew in both kinds of the seasoning, the lag phase in S-B was longer than that in S-A. This result may * The unit is mg 100ml 1 be obtained because ph and water activity values in S-B

5 B. amyloliquefaciens from soy sauce 151 were lower and concentration of ethanol was higher than those in S-A. The lag phase of the growth of the isolated Acknowledgement strains was longer than that of B. subtilis IFO in S-B different from the result in S-A. B, amyloliquefaciens IFO as type strain of the species including the isolated strains did not grow in S-B. These results may be caused by the difference of characteristics between the strains. However, no certain evidence has obtained in this study. Cell mass of B. megaterium ATCC strain on the stationary phase in S-A was lower than that of the other strains, and the strain did not grow in S-B. It was also confirmed that the strain is more difficult to grow in the seasoning than the other strains. From the results mentioned above, it is suggested that seasoning made from the soy sauce contaminated by the KP-991 and KP-992 strains putrefy. The isolated strains have different characteristics, especially thermostability and growth in seasoning from three type strains. Terasawa et al. [11] reported that the growth of genus Bacillus in dilution-free seasoning was prevented by pasteurization over 80 Ž. However, the seasoning made from the soy sauce contaminated by the KP-991 or KP-992 strain should be pasteurized at much higher temperature than 80 Ž to prevent the putrefy judging from the results of survival in the final product (Fig. 2) and the growth in the seasoning (Fig. 3) of the isolated strains. Kusumegi et al. [12] proposed the addition of sodium citrate to pasteurize bacterial spores in seasoning. Killing spores in soy sauce is effective for preventing contamination of seasoning products by bacteria. The methods to pasteurize spores in soy sauce as a main stuff of dilution-free seasoning have been reported. Filtration is effective for removing spores from soy sauce, while the running cost is high [13]. Though the spores were killed by heating at Ž for a few seconds, secondary sediment is easily produced in soy sauce [3]. Yamamoto et al. [14] described that 105 CFU. ml-1 spores included in soy sauce of ph 4.4 and with 3.0% ethanol were killed by heating at 87 Ž for 28 min. Hayakawa et al. [15] reported the method to kill spores by repeating addition and remove of high pressure. Kato et al. [16] mentioned that fermented soybeans without B. subtilis was produced by inoculation of nisin-producing Lactococci. This method may be applied to inhibit growth of Bacillus sp. in soy sauce koji. However, nisin has not been approved as a food additive by the ministry of health, labour and welfare in Japan yet. Procedures to prevent the contamination and growth of bacteria stable at high temperature by the combination of various methods have to be continuously investigated in the future with reference to the published research mentioned above. I wish to express my sincere thanks to Mr. Atsumi Yamada, the vice head of the laboratory in Takesan Co. Ltd., for his contribution of the materials for seasoning production. References 1) T. Ikeda; Extreme decrease of consignment, soy sauce companies come to a crisis. The Beverage & Food Statistics Monthly, 42 (1), (2000). 2) H. Chiba; Contamination of soy sauce koji by bacteria and the counterplan. J.Brew. Soc. Japan, 72, (1977). 3) Y. Hanaoka, N. Saito, T. Yokotsuka; Kikkoman special soy sauce for processing. Seasoning Sci.,19 (4),11-16 (1972). 4) M. Noguchi; Japanese noodle soup production of seasoning for soba. Food Sci., extra volume in spring, (1979). 5) T. Tsuchido, K. Kanda, I. Shibasaki; Enhancement of sporicidal activity of an amphoteric surfactant by heating. J. Ferment. Technol., 53, (1975). 6) D. Claus, R. C. W. Berkeley; gbergey's Manual of Systematic Bacteriology Vol. 2 h, P. H. A. Sneath ed, Williams & Wilkins,1986, p ) G. I. Barrow, R. K. A. Feltham; gcowan and Steel's Manual for the Identification of Medical Bacteria Third ed. h, Cambridge University Press, 1995, p ) H. Haga, K. Endo; On the bacteria and the spore-forming bacteria of shoyu. Seasoning Sci.,18 (2),14-20 (1971). 9) K. Sekine, S. Shigeta; Microbiological studies on Bacillus species occurring in soy sauce-manufacturing processes. J. Japan Soy Sauce Research Institute, 13, (1987). 10) F. G. Priest, M. Goodfellow, L. A. Shute, R. C. W. Berkeley; Bacillus amyloliquefaciens sp. nov., nom. rev. Int. J. Systematic Bacteriology, 37, (1987). 11) M. Terasawa, A. Matsuwaka, K. Yamashita, Y. Mori; Effect of temperature on sterilization of gtsuyu h. J. Japan Soy Sauce Research Institute, 19, (1993). 12) K, Kusumegi, T. Takahashi, M. Miyagi; Effects of addition of sodium citrate on the pasteurizing conditions in g Tuyu h, Japanese noodle soup. Nippon Shokuhin Kagaku Kogaku Kaishi, 43, (1996). 13) H. Haga; Pasteurization and removal of bacteria in soy sauce and storage. J.Brew. Soc. Japan, 66, (1971). 14) Y. Yamamoto, S. Kobayashi, H. Yoshii; Heat sterilization

6 152 Yoshio MAKING and Hiroko FUJISAWA of bacterial spores in soy sauce. J. Japan Soy Sauce Research Institute, 6,19-23 (1980). 15) I. Hayakawa, T. Kanno, M. Tomita, Y. Fujio; Application of high pressure for spore inactivation and protein denaturation. J. Food Sci., 59, (1994). 16) T. Kato, K. Maeda, H. Kasuya, T. Matsuda; Complete growth inhibition of Bacillus subtilis by nisin-producing Lactococci in fermented soybeans. Biosci. Biotechnol. Biochem., 63, (1999).

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