J. Home Econ. Jpn. Vol. 48 No. 12 1083-1087 (1997)

Note

Isolation of a Collagenase-Producing Bacterium from Traditional Fermented Food and Its Enzyme Production

Kim Anh To, Tadayoshi TANAKA*and Hiroko NAGANO**

Center for Biotechnology, Hanoi University of Technology, 1- Dai coviet, Hanoi, Vietnam * Kyoritsu Women's Junior College , Chiyoda-ku, Tokyo 101, Japan ** Faculty of Education, Gifu University, Gifu 501-11, Japan

The production in Southeast Asian countries of naturally fermentedfoods which are protein rich relies on the proteolytic action of their natural bacterial flora. A bacterium, FS-2, which was isolated from Laotian fish sauce, was identified as Bacillus subtilis. B. subtilis FS-2 is a collagenase-producing bacterium that was aerobically inducible by insoluble collagen and polypeptone. The maximum enzyme activity was detected at the middle of the stationary phase in a medium containing 0.75% polypeptone and 1% NaCl. (Received December 13, 1996)

Keyword: traditional fermented food, collagenase, halotolerant bacterium, Bacillus subtilis.

increased. This has suggested that the collagenase INTRODUCTION was potentially inducible extracellular enzyme whose Collagenase, as defined, can hydrolyze both native production could be regulated. and denatured collagen. It is widely used in biological In Southeast Asian countries, the production of studies (Seifter and Harper 1971; Harper 1980; naturally fermented foods, which have a very Peterkofsky 1982) and in food treatment (Watanabe et important role in nutrition as well as in traditional al. 1994; Suzuki et al. 1990; Miller et al. 1989; Bernal cuisine (fish sauce, fermented meat, shrimp sauce, and Stanley 1986). Collagenase is known by a variety fermented tofu, fermented soybean, etc.), relies on the of microorganisms such as Clostridium histolyticum proteolytic action of their natural bacterial flora. In (Yoshida and Noda 1965), Bacillus alvei DC-1 the course of our study laboratory, we found several (Kawahara et al. 1993), Bacillus licheniformis N22 bacteria from traditional foods which were able to (Adornnithee et al. 1994), Bacillus cereus (Makinen produce collagenases. During the screening of and Makinen 1987) and Vibrio alginolyticus (Robbertse bacteria possessing high collagenase activity, we et al. 1978). Most of these bacteria have been isolated obtained Bacillus subtilis FS-2 from one of the from the soil, and others from cured hides. As has traditional fermented foods, a fish sauce. The present been proven in the studies of Keil-Dlouha et al. study was aimed to determine the optimum conditions (1976) and Reid et al. (1980), a collagenase from for enzyme production in order to subsequently Achromobacter iophagus (later identified as a V. purify the enzyme. alginolyticus) was considered as an inducible enzyme MATERIALS AND METHODS by collagen, due to its high molecular weight fragments and by peptone. On the other hand, the Food samples work of Welton and Woods (1973) has confirmed the The food samples tested in this present study Achromobacter iophagus collagenase as a halotolerant consisted of 26 traditional fermented foods: 13 enzyme. The maximum activity and least-lag period samples were fermented meat (from nem chua, have been observed at an NaCl concentration of Vietnam and from wettha chin, Myanmar); 6 were fish 2.34%. At NaCl concentrations above 2.34%, the sauces (from nuoc mam, Vietnam and from padek, collagenase activity decreased and the lag period Laos); 3 were fermented tofu (from chao, Vietnam); 2

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were shrimp sauces (from mam torn, Vietnam); and 2 Collagenase assay

were fermented soybean (from tuong, Vietnam). All A reaction mixture consisting of 0.8 ml of a 50 mm

bacteria were maintained on nutrient agar slants Tris-HC1 buffer containing 4 mm CaCl2 and 10 mg of

before testing. insoluble collagen had previously been incubated for

Media 10 min at 30 t, after which 0.2 ml of an enzyme was

The medium (Hisano et al. 1989) for screening added. The enzyme reaction was carried out at 30 t

collagenase bacteria was (in gll) KH2PO4 (2), K2HPO4 for 30 min while shaking to suspend the collagen, and

(7), MgSO4•7H20 (0.1), citrate •2H20 (0.05), yeast was stopped by adding 1 ml of 0.1 M acetic acid. The

extract (1) and collagen (3) at pH 7.5. The medium same mixture excluding insoluble collagen was used

for enzyme production (in g/l) was KH2PO4 (2), as a control. The released amount of free a -amino

K2HPO4 (7), MgSO4 •E 7H20 (0.1), citrate •E 2H20 (0.05), groups from the collagen was measured by the

yeast extract (1), CaC12•2H20 (0.1) and gelatin (3) at ninhydrin method (Rosen 1957). Collagenase activity

pH 7.5. is expressed as ,u mol of leucine equivalent per min

Screening method per ml of the culture filtrate. The sample was suspended in a 0.9% NaC1 solution Bacterium growth and enzyme production

and incubated for 2-3 days in a screening medium The bacterium was first grown in a subculture at

(Hisano et al. 1989). The diluted culture was spread 35 •Žthe screening medium already described, in on a plate of the collagen agar medium and then which 0.3 % casein was used to replace the insoluble

incubated for 48 h. Colonies forming halos were collagen due to its high cost. From this subculture,

picked up, each being grown in the collagen medium cultures for enzyme production were inoculated and for 24 h and then spread on plates of the same aerobically incubated at 35•Ž. The influence of

medium in order to produce single colonies. Their substrates on the collagenase production was exam-

collagenase-producing ability was examined, and the ined by replacing 0.3% collagen with 0.3% casein or bacterium with the highest activity was selected. gelatin. Identification of the bacterium Polypeptone was used to replace collagen in the

The bacterium was identified according to the API expectation of its ability to induce enzyme production.

50 CH method (Logan and Berkeley 1984), the In all experiments, the turbidity of the culture fluid

nomenclature being determined by ATB Plus soft- which expressed the growth was measured at 660 nm, ware containing Bergey Manual of Systematic Bac- and collagenase activity was periodically monitored. teriology (Claus and Berkeley 1986).

Table 1 . Positive samples for the collagenase bacterial isolation

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collagenase producer. RESULTS AND DISCUSSION Bacterial growth and enzyme production Isolation of the collagenase bacterium B. subtilis FS-2 was grown aerobically on a From 11 of the 26 samples tested, colonies forming screening medium to which was added different halos after 24-48 h of incubation on the collagen substrates of casein, gelatin or insoluble collagen. plates were observed (Table 1). Only 8 strains There was a minor difference in the bacterial growth isolated from these colonies showed enzyme activity among these cultures (data not shown). In the in the enzyme production medium. Strain FS-2 that subculture, the exponential phase started after 2 h of had been isolated from Laotian fish sauce showed the incubation, and the stationary started at 7 h after highest activity and was selected. It was a Gram incubation. The lag phase was generally almost the positive bacterium, had a rod shape, formed acid from same and lasted for 2 h. Thus, for bacterial growth, D-glucose, fructose and glycerol, and hydrolyzed any of the tested substrates could be used; Insoluble starch and esculin. FS-2 was identified as Bacillus collagen would not be chosen due to its high cost. In subtilis by the API 50 CH identification method, its the subsequent experiments, a 6-h subculture con- microbiological properties being summarized in Table taining 0.3% casein was used as an inoculant for the 2 . To our knowledge, this is the first report on B. enzyme production culture. subtilis that had been isolated from food as a The cultures containing different substrates showed a considerable difference in the accumulation of enzyme activity (Fig. 1). Either very little or no Table 2 . Microbiological properties of Bacillus subtilis collagenase activity was detected in the cultures with FS-2 casein or gelatin as a substrate. On the other hand, the culture with insoluble collagen showed very high activity, indicating that collagenase was a potentially inducible enzyme. Polypeptone was used to search for a suitable inducer to replace collagen. The result showed that, by increasing the polypeptone concentration from

Fig . 1 . Accumulation of collagenase on different substrates Each culture was inoculated from a 6 h subculture containing 0.3% casein. The experiment was done in a 300 ml flask containing 50 ml of the enzyme production medium with different substrates added: 0.3% casein (-), 0.3% gelatin (0) or 0.3% insoluble collagen (0).

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To our knowledge, this is the first study to be reported on a food collagenase bacterium.

This work was supported in part by Grant-in-Aid (No. 07680014) for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We express our thanks to Mr. Syamphone Phommalyvong, Ministry of Agriculture and Forestry, Laos for his kindness in collecting the samples.

REFERENCES Adornnithee, S., Akiyama, K., Sasaki, T., and Takata, R. (1994) Isolation and Characterization of a Collagenolytic Enzyme frm Bacillus lichenoformis N22. J. Ferment. Fig.2.Influence of polypeptone concentration on Bioeng., 78, 283-287 collagenase production Bernal, V.M., and Stanley, D.W. (1986) Change in the The bacterium was grown aerobically from a 6 h casein Melting Characteristics of Bovine Tendon Collagen subculture at 35•Ž in a 300 ml flask containing 50 ml of the Induced by a Bacterial Collagenase, J. Food Sci., 51, 834- enzyme production medium excluding collagen, each with 835 polypeptone added at different concentrations: •Ÿ, 0% •›, Claus, D., and Berkeley, R.C.W. (1986) Bergeys Manual of 0.25%; •¢, 0.5%; •¡, 0.75%; •¢, 1%; •œ, 2.5%;+, 5%. Systematic Bacteriology, Vol. 2 (ed. by Sneath, P.H.A., Mair, N.S., and Sharpe, M.E.), Williams & Wilkins, Baltimore, 1105-1139 0.25 to 0.75%, the enzyme activity increased Harper, E. (1980) Collagenases, Ann. Rev. Biochem., 49, proportionally. Maximum activity was observed with 1063-1078 0.75% polypeptone. Any further increase in poly- Hisano, T., Abe, S., Wakashiro, M., Kimura, A., and Murata peptone concentration caused a decrease in enzyme K. (1989) Isolation and Properties of Collagenase activity (Fig. 2). It seemed that, polypeptone was a with Caseinolytic Activity from Pseudomonas sp., J. collagenase inducer at 0.75% or lower concentration, Ferment. Bioeng., 68, 399-403 and it was also an enzyme repressor at a concentra- Kawahara, H., Kusumoto, M., and Otaba, H. (1993) Isolation tion above 0.75%. and Characterization of a New Type of Collagenase- To produce collagenase with polypeptone, in the Producing Bacterium, Bacillus alvei DC-1, Biosci. Biotech. case of NaCl being present, the maximum activity was Biochem., 57, 1372-1373 obtained earlier than when NaCl was not present (18 Keil-Dlouha, V., Mishari, R., and Keil, B.(1976) The and 24 h, respectively). An increase in NaCl Induction of Collagenase and Neutral Proteinase by Their High Molecular Weight Substrates in Achromobact- concentration from 0-1% caused a slight increase in er iophagus, J. Mol. Biol., 107, 293-305 collagenase activity, but at an NaC1 concentration Logan, N.A., and Berkeley, R.C.W. (1984) Identification above 1 %, the collagenase decreased (data not of Bacillus Strains Using the API System, J. Gen. shown). The subsequent experiments for collagenase Microbiol., 130, 1871-1882 production utilized a culture containing 0.75% Makinen, K.K., and Makinen, P.L. (1987) Purification and polypeptone and 1% NaCl. In this culture, collagenase Properties of an Extracellular Collagenolytic Protease was accumulated from the end of the exponential Produced by Human Oral Bacterium Bacillus cereus phase and had maximum activity in the middle of the (Strain Soc. 67), J. Biol. Chem., 262, 12488-12495 stationary phase of bacterial growth. Miller, A.J., Strange, E.D., and Whiting, R.C. (1989) B. subtilis FS-2 was isolated from the microbial flora Improved Tenderness of Restructured Beef Steaks by a of traditional fermented foods as one of only a few Microbial Collagenase Derived from Vibrio B-30, J. Food bacteria capable of producing collagenase. Under the Sci., 54, 855-857 Peterkofsky, B. (1982) Bacterial Collagenase, Methods present conditions, B. subtilis FS-2 produced high collagenase activity with maximum activity within a Enzymol., 82, 453-471 Reid, G.C., Woods, D.R., and Robbs, F.T. (1980) Peptone short time. The present bacterium could probably be Induction and Rifampin Insensitive Collagenase Produc- used as a natural collagenase source that could be tion by Vibrio alginolyticus, J. Bacteriol., 142, 447-454 applied to risk-free food making.

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Robbertse, P.J., Woods, D.R., Reay, A.H., and Robb, R.T. Proteolytic Enzyme (in Japanese), Nippon Shokuhin (1978) Simple and Sensitive Procedure for Screening Kagaku Kogaku Kaishi (J. Jpn. Soc. Food Sci. Technol.), 37, Collagenolytic Bacteria and the Isolation of a Col- 104-110 lagenase Mutant, J. Gen. Microbiol., 106, 373-376 Watanabe, M., Ikezawa, Z., and Arai S. (1994) Fabrication Rosen, H. (1957). A Modified Ninhydrin Colorimetric and Quality Evaluation of Hypoalergenic Wheat Prod- Analysis for Amino Acids, Arch. Biochem. Biophys., 67, ucts, Biosci. Biotech. Biochem., 58, 2061-2065 10-15 Welton, R.L., and Woods, D.R. (1973) Halotolerant Seifter, S., and Harper, E.(1971) The Enzymes,Vol. 3 (ed. by Collagenolytic Activity of Achromobacter iophagus, J. Gen. Boyer, P.D.), Academic Press, New York, 649-697 Microbiol., 75, 191-196 Suzuki, A., Shimakura, A., Miki, T., Shimizu, M., Koyama, Yoshida, E., and Noda, H. (1965) Isolation and Characteri- G., Saito, M., and Ikeuchi, Y. (1990) Tenderization of zation of Collagenase I and II from Clostridium Culled Chicken Meat with Post-Mortem Injection of a histolyticum, Biochim. Biophys.Acta, 105, 562-574

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トウ キ ム ア イ ン, 田 中 直 義 *, 長 野 宏 子 **

(ハ ノ イ工 科 大 学 バ イ オ テ ク ノ ロ ジ ー セ ン タ ー,* 共 立 女 子 短 期 大 学,** 岐 阜 大 学 教 育 学 部)

平 成8年12月13日 受 理

東 南 ア ジ ア で は,魚,肉,大 豆 等 の 高 た ん ぱ く質 食 品 は 自然 発 酵 に よ る もの が 多 い.味 の 良 い 食 品,ま た は 調 味 料 に な る過 程 に 関 与 して い る た ん ぱ く質 分 解 酵 素 コ ラ ー ゲ ナ ー ゼ 生 産 菌 の 検 索 を行 っ た.コ ラ ー ゲ ナ ー ゼ 生 産 菌 は ラ オ ス の 魚 醤 よ り分 離 し,Bacillus subtilisと 同 定 した.Bacillus subtilisは 耐 塩 性 細 菌 で,コ ラ ー ゲ ン とポ リペ プ トン に よ っ て 誘 導 され る 好 気 性 コ ラ ー ゲ ナ ー ゼ 生 産 菌 で あ っ た.0.75%ポ リペ プ ト ン と,1.0%NaClを 含 有 す る培 地 に よ り, 高 い 酵 素 活 性 を 示 した.

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