Effects of Some Metal Ions on the Production of an Antibacterial Substance byrhizopussp. MKU 24

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1 Biocontrol Science, 2004, Vol. 9, No. 4, Note Effects of Some Metal Ions on the Production of an Antibacterial Substance byrhizopussp. MKU 24 HIROSHI MORITA1*, TSUBASA FUKUDA1, HIDETAKA KAWAKITA2, KAORI MAMITSUKA1, MASAMI SHIOZAWA3, KAZUYA UEZU1 AND IKUO KOMAKI1 'Department of Chemical Processes and Environments,Faculty of Environmental Engineering, The University of Kitakyushu, Hibikino, Wakamatsu-ku, Kitakyushu , 2Advanced Research Institute for Science and Engineering, Waseda University, Hibikino, Wakamatsu-ku, Kitakyushu , and 3Department of Bioengineering, Yatsushiro National College of Technology, Hirayama-shinmachi, Yatsushiro , Japan Received 2 April 2004/Accepted 30 July 2004 Rhizopus sp. MKU 24 selected from 46 Rhizopus strains produced a considerable amount of antibacterial activity in a liquid culture. The concentration and the kind of metal ion have large influence on the production of the antibacterial substance. It is concluded that the SLS medium supplemented with 2ƒÊg/m1 of iron ions, 49 ƒêg/ml of magnesium ions, 2.5 ƒêg/ml of zinc ions, and 100 ƒêg/ml of calcium ions was the best condition for producing the antibacterial substance. Antibacterial activity was not detected against Gram-negative bacteria, yeast, and fungi, but strong activity was detected against Bacillus subtilis and Micrococcus luteus. Key words : Liquid culture/metal ion/rhizopus/antibacterial activity. Rhizopus strains are essential microorganisms in the food and the enzyme industries in South East Asia, Southern China, and Japan. They are mainly used to produce tempeh, a traditional fermented Indonesian food made from soybeans. The Rhizopus mold binds grains of soybean into a compact white cake (Jurus and Sundberg, 1976). Tempeh has been a staple source of protein in Indonesian foods for several hundred years (Hesseltine, 1965; Steinkraus et al., 1960). Usually tempeh is sliced and fried until the surface becomes crisp and golden brown, or it can be used as an ingredient in soups, spreads, salads and sandwiches. The Rhizopus mold, when used for tempeh fermentation, produces natural antibiotics. Wang et al. (1972) reported that Rhizopus oligosporus from tempeh, Rhizopus chinensis from soil, and Rhizopus javanicus from shoyu-koji produced an appreciable amount of antibacterial substances and these strains *Corresponding author. Tel : , Fax : + exhibited very high antibacterial activity when cultured in the soybean medium. Rhizopus chinensis from soil also produced antibacterial substances in the rice medium or the milk medium. R. oligosporus from tempeh produced such substances in the milk medium, but not in the rice. The growth and physiology of mycelia depend on many environmental factors. Among them, metal ions have a great effect on mycelial growth and metabolic activity. Generally, the adequate concentration of a specified metal ion is essential for microbial growth while higher metal ion concentrations inhibit the microbial growth (Abelson and Aldorus, 1950; Ross, 1975). Haska and Ohta (1991) investigated the improvement of glucoamylase production on wheat bran solid medium supplemented with calcium and nickel ions. In this study, we have investigated the effects of various metal ions as a supplementation to increase production of antibiotics by the strain Rhizopus sp. MKU 24, and established a liquid culture system with metal ions for antibiotic production. We also

2 112 H. MORITA ET AL. compared the antibiotic production on the present medium with that on a wheat bran solid medium which is widely used for Rhizopus fermentation. Forty six Rhizopus strains in total (33 Rhizopus sp. and 13 Rhizopus oryzae strains) were used in this study (Table 4). Rhizopus sp. strains from tempeh and ragi were obtained by Elegado and Fujio (1993). The MKU series was provided by Kasetsart University, Department of Microbiology, Thailand, and UQM 186F strain was provided by University of Queensland, Australia. The TISTR series was obtained from the Thailand Institute of Scientific Technological Research, Thailand, and the IFO series was obtained from the Institute for Fermentation Osaka, Japan. Bacillus subtllis IFO 3335, Micrococcus luteus IFO 3333, Escherichia coli IFO 3972, Staphylococcus aureus IFO 12732, Pseudomonas fluorescens IFO 3507, Saccharomyces cerevisiae IFO 2363, and Aspergillus oryzae IFO were obtained from the Institute for Fermentation Osaka, Japan. From the preliminary screening of the best producer of antibacterial substances on the potato dextrose agar slant at 30 Ž for 5 days, Rhizopus sp. MKU 24 was selected (data not shown), and we used it in the following experiments. The metal compounds (Nacalai Tesque Co.) tested were silver sulfate (Ag+ ), copper sulfate (Cu2+), nickel chloride hexahydrate (Ni2+), cadmium chloride (Cd2+), iron ( I I ) sulfate heptahydrate (Fe2+), manganese sulfate pentahydrate (Mn2+ ), calcium chloride (Ca2+), lead acetate trihydrate (Pb2+), cobalt chloride hexahydrate (Co2+), magnesium sulfate heptahydrate (Mg2+ ), zinc sulfate heptahydrate (Zn2+), aluminum chloride hexahydrate (A13+), and sodium molybdate dihydrate (Mo6+). SLS medium consisted of 2 g of liquefied cassava starch, 0.4 g of ammonium acetate, 0.1 g of dipotassium hydrogenphosphate, and 0.33 g of citric acid, which were dissolved in 100 ml of deionized water. The ph was adjusted to 6.0 with 1 N sodium hydroxide. The medium was sterilized at 121 Ž for 20 min in a shaken flask. The Rhizopus mycelia and spores were precultured on a slant of potato dextrose agar at 30 Ž for 7 days. Two milliliters of the spore and the mycelia suspension (about 109 cfu per ml) were inoculated into the medium. It was incubated at 30 Ž for 3 days on a reciprocal shaker with agitation at 300 strokes per min. The culture broth was filtrated through filter paper (No.7, Toyo Roshi Ltd.) to remove the fungal mycelia. The filtrate was used for the antibacterial assay. The dry mycelium weight (DMW) was determined by drying the filtrate at 105 Ž for 24 h. DMW was defined as the mycelial weight per 100 ml culture medium. The solid culture was prepared according to the method of Elegado and Fujio (1993). The medium consisted of 20 g of wheat bran, 2 g of cassava starch, and 20 ml of tap water in a 500 ml Erlenmeyer flask stopped with a cotton plug. After the medium was autoclaved, 2 ml of the spore suspension was inoculated into it, and cultivation was carried out at 30 for 2-7 days. Ž After cultivation, 200 ml of deionized water was added to the culture flask. The mixture was stirred for 12 h at 4 Ž, and then centrifuged at 12,000 ~ g for 20 min. The supernatant was used for the antibacterial assay. The assay of the antibacterial activity was done according to the modified paper disk method (Kobayashi et al., 1992) with B. subtllis IFO 3335 as the test organism. A paper disk was placed on the nutrient agar medium with which the bacterium was mixed. Fifty,ƒÊl of test sample were spread on the paper disk (Toyo Roshi Ltd., 8 mm dia.), and the me- TABLE 1. Effects of Fe2+0, Mg', and Zn2+ on the production of antibiotics by Rhizopus sp. MKU 24 in the medium.

3 ANTIBIOTIC PRODUCTION OF RHIZOPUS SP. MKU dium was incubated at 30 Ž for 12 h. The antibacterial activity was assessed by the number of dilutions that gave a clear zone around the diluted sample on the paper disk of 9 mm in diameter. If the number of dilutions of the sample was 2, the antibacterial activity of the sample per ml was calculated as 40 units (= 2 ~ 1,000/50). Table 1 shows the effects of iron, magnesium, and zinc ion supplementation on the antibacterial activity and the DMW of Rhizopus sp. MKU 24. Rhizopus sp. MKU 24 did not grow or produce antibacterial substances in the SLS medium (no metal ions supplemented). The addition of magnesium ions to the SLS medium was essential both to the growth and to the antibiotic production. Zinc and iron ions enhanced strongly both the antibiotic production and the fungal growth. Addition of all three ions to the basal medium resulted in better antibiotic production than that produced by any combination of two of the three metal ions. The greater the mycelial growth was, the higher the antibacterial activity was obtained. The maximum antibacterial activity was obtained from 2 ƒêg/ml of iron ions, 49 LƒÊg/ml of magnesium ions, and 2.5 ƒêg /ml of zinc ions (data not shown). Foster and Waksman (1939) mentioned that zinc ions were essential to the growth of Rhizopus nigricans. The optimum zinc ion concentration for maximum growth was reported to be 1.2 ƒêg/ml. Although tested microorganisms differ, this result is similar to the results for Rhizopus sp. MKU 24 in our study. Table 2 shows the effects of the supplementation of other metal ions on the production of antibiotics by Rhizopus sp. MKU 24 when the SLS medium was supplemented with 2 ƒê/ml of iron ions, 49 ƒê/ml of magnesium ions, and 2.5 ƒê/ml of zinc ions. Of TABLE 2. Effects of other metals on the antibacterial activity of Rhizopus sp. MKU 24 in the medium. FIG. 1. Effects of concentrations of calcium ions on the production of antibiotics by Rhizopus sp. MKU 24. The basal medium was SLS medium supplemented with Mg2+49,ƒÊg /ml, Zn2+2.5,ƒÊg/ml, and Fe2+2,ƒÊg/ml. The cultivation was carried out at 30 Ž for 3 days., Antibacterial activity; œ DMW. the 10 metal ions tested, calcium ions had the greatest influence on production of the antibacterial substance. Except in the case of calcium ions, production of the antibacterial substance did not improve by addition of 50 ƒêg/ml or more (data not shown). Addition of silver ions or cadmium ions decreased DMW, and brought about the reduction of antibacterial activity. Figure 1 shows the optimum concentration of calcium ions for antibiotic production. The addition of 100 and 125 ƒêg/ml of calcium ions seemed to be the optimum to produce the antibacterial substance, and this resulted in antibacterial activity of 220 U/mI. Mchargue and Calfee (1931) reported that manganese and copper ions in combination with zinc ions also stimulated Rhizopus nigricans growth. In our study we found that copper ions did not stimulate the growth of Rhizopus sp. MKU 24. However manganese ions slightly enhanced the growth (data not shown). The antibacterial spectrum of the culture supernatant of Rhizopus sp. MKU 24 is shown in Table 3. The activities of the filtrate were detected against the Gram-positive bacteria, B. subtilis IFO 3335, M. luteus IFO 3333, and S. aureus IFO Numbers indicate the antibacterial activity (U/m1). The basal medium was SLS medium with Fe2+ 2ƒÊg/ml, Mg2+ 49ƒÊg/ml, and Zn2+2.5 ƒêg/ml. Antibacterial activity did not appear against the Gram-negative bacteria, E. coil IFO 3972 and P. fluorescens IFO 3507, the yeast, S. cerevisiae IFO

4 114 H. MORITA ET AL. TABLE 3. Antibacterial spectrum of the cultivated broth from Rhizopus sp. MKU 24. Rhizopus strains in the metal ion-regulated liquid medium (SLS medium supplemented with 2 ƒêg/ml of iron ions, 49 ƒêg/ml of magnesium ions, 2.5 ƒêg/ml of zinc ions, and 100 ƒêg/ml of calcium ions). Twenty six of the 46 Rhizopus strains tested produced antibacterial substances. Among these strains, Rhizopus sp. MKU 24, MKU 38, F 60, F 61, F 64, F 67, Rh 3, G 82, UQM 186F, TISTR 3001, and TISTR 3324 showed high antibacterial activity against B. subtilis IFO 3335 over 100 U/mI. Rhizopus sp. MKU 24 The culture medium was SLS medium with Fe2+2 ƒêg/ml, Mg2+ 49 ƒêg/ml, Zn ƒêg/ml, and Ca ƒêg/ml. The cultivation was carried out at 30 Ž for 3 days. 2363, and fungi, A. oryzae IFO Wang et al. (1972) and Kobayashi et al. (1992) reported that the antibacterial substance from R. oligosporus did not show activity against M. luteus. Solid wheat bran medium is widely used in Rhizopus fermentation. The production of antibiotics by Rhizopus sp. MKU 24 in this medium was investigated. It was found that Rhizopus sp. MKU 24 did not produce any antibacterial substance in spite of giving vigorous mycelial growth (data not shown). Table 4 shows the antibacterial activity of various showed the highest antibacterial activity. Kobayashi et al. (1992) obtained an antibacterial substance from R. oligosporus IFO 8631 in the submerged cultivation broth composing of 5% casamino acid (Difco technical grade). This resulted in an antibacterial activity of 25 U/mI against B. subtilis. Du et al. (2003) reported nitrogen sources greatly affected the production of antibiotics by R. chinesis. The medium containing corn steep liquor resulted in high antibacterial activity (a 16 mm inhibitory zone) against B. subtilis IFO In our study using a metal ion-regulated liquid medium (SLS medium supplemented with 2 ƒêg/ml of iron ions, 49 ƒêg/ml of magnesium ions, 2.5 ƒêg/ml of zinc ions, 100 ƒêg/ ml of calcium ions, and 4 g/i of ammonium acetate) with Rhizopus sp. MKU 24, a 20 mm inhibitory zone TABLE 4. Production of antibiotics by variuos Rhizopus strains. The culture medium was SLS medium with Fe2+ 2 ƒêg/ml, Mg2+ 49 ƒêg/ml, Zn ƒêg/ml, and Ca ƒêg/ml. The cultivation was carried out at 30 Ž for 3 days.

5 ANTIBIOTIC PRODUCTION OF RHIZOPUS SP. MKU was formed. The concentration and the kind of metal ion have a large influence on the production of the antibacterial substance. These results should contribute to improvement in the production of the antibacterial substances in the liquid culture system. ACKNOWLEDGMENTS The authors thank Kaori Morimoto, Tomoko Ohmori, Kanako Sugimoto, and Makiko Miyano for their experimental work. REFERENCES Abelson, P.H. and Aldorus, E. (1950) Ion antagonisms in microorganisms. J. Bacteriol., 60, Du, L., Jia, S., and Lu, F. (2003) Morphological changes of Rhizopus chinesis 12 in submerged culture and its relationship with antibiotic production. Process Biochem., 38, Elegado, F. and Fujio, Y. (1993) Selection of raw-starch digestive glucoamylase producing Rhizopus strain. J. Gen. Appl. Microbiol., 39, Foster, J.W. and Waksman, S.A. (1939) The specific effect of zinc and other heavy metals on growth and fumaric acid production by Rhizopus. J. Bacteriol., 39, Haska, N. and Ohta, Y. (1991) Determination of optimum solid culture condition for the raw sago starch-digesting amylase production by Penicillium brunneum (in Japanese). Starch Sci., 38, Hesseltine C.W. (1965) A millennium of fungi, food, and fermentation. Mycologia, 57, Jurus, A. M and Sundberg, W.J. (1976) Penetration of Rhizopus oligosporus into soybean in tempeh. Appl. Environ. Microbiol., 32, Kobayashi, S., Okazaki, N., and Koseki, T. (1992) Purification and characterization of an antibiotic substance produced from Rhizopus oligosporus IFO Biosci. Biotech. Biochem., 56 (1), Mchargue, J.S. and Calfee, R.K. (1931) Effect of manganese, copper, and zinc on growth and metabolism of Aspergillus flavas and Rhizopus nigricans. Botanical Gazette, 91, Ross, I.S. (1975) Some effects of heavy metals on fungal cells. Trans. Br. Mycol. Soc., 64, Steinkraus, K.H., Hwa, Y.B., Van Buren, J.P., Provvidenti, M.I., and Hand. D.B. (1960) Studies on tempeh - An Indonesian fermented soybean food. Food Res., 25, Wang, H.L., Ellis, J.J., and Hesseltine, C.W. (1972) Antibacterial activity produced by molds commonly used in oriental food fermentations. Mycologia, 64,