醤油の耐塩,好塩性細菌に関する研究
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概要
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Soy sauce is the typical condiment in Japan and the contents is characterized by high salt concentration. Since the first report on the gelatin liquefactive bacteria in soy sauce mash (Nishimura 1897), several groups of workers have been studied on soy sauce bacteria, however, little attention has been paid to halophilism. The present investigation was undertaken to demonstrate the presence of halophile in soy sauce and to elucidate their physiological characters and the mechanism of halophilism. I. Isolation of halophilic bacteria from soy sauce mash. For the isolation of halophilic aerobic (anaerobic) bacteria from soy sauce mash, the bouillon agar containing koji ext. 20 % (10 %), soy sauce 10 %, liver ext. 5 % (10 %), yeast ext. 0.4 % (0.2 %), (sodium thioglycollate 0.1 %) and sodium chloride 10 % was the suitable medium, and Novy's method with alkaline pyrogallol or vacuum culture method (30-60 mm Hg) were the best technique for the isolation of anaerobic bacteria. Using the above medium and method, 44 strains of aerobic halotolerant bacteria, 8 of halotolerant and 4 of halophilic anaerobic bacteria were isolated. II. Aerobic halotolerant bacteria Among the 44 aerobic bacteria, 10 strains of color-producing bacteria and 5 strains of decolorizing bacteria were obtained. Especially, 3 strains of these color-producing bacteria turned the color of medium to deep brown. These strains were considered to belong to genus Micrococcus and the two of them were identified as Micrococcus luteus var. and Micrococcus candidus var. Other isolate was not yet identified. Coloring of the strain No. 46 was closely relative to its growth, and it was considered that the color might be formed by the reaction between metabolic products from sugar and some compound in the medium. III. Anaerobic halotolerant and halophilic bacteria. All anaerobic bacteria isolated from soy sauce mash were lactic acid bacteria, and these halophilic lactic acid bacteria were almost similar in their morphological and physiological characteristics to Pediococcus soyae, but halotolerant one were recognized to be a different species belonging to the genus Tetracoccus, The name Tetracoccus soyae was given to the species in 1961. On the nutritional requirements of these bacteria, koji ext. and liver ext. were effective in promoting the growth of Pc. soyae and Tc. soyae. The effect of koji ext. was very remarkable in the salted media. In the salted media, Pc. soyae required folinic acid which was known as "unique characteristic" of genus Pediococcus. As to the vitamin requirements of Pc. soyae and Tc. soyae, pantothenic acid and nicotinic acid were essential for both when they grew in the unsalted media. In addition, Tc. soyae required tween-80, biotin and riboflavin, and Pc. soyae also required pyridoxine, thiamine and riboflavin. On the other hand, especially biotin was more effective on the growth of Tc. soyae and choline was essential for Pc. soyae in the related media. This paper is the first report on the choline requirement for lactic acid bacteria. In view of the results of non-efficacy of choline relative compounds except betaine and efficacy of acetylcholine for growth of Pc. soyae, it seemed to assumed that the route to nucleic acid metabolism via betaine-methionine transmethylation and the possibility of control of ion transport through cell wall via production of phospholipid might be comprised in the halo-philism of Pc. soyae. On the base requirements, Tc. soyae in the unsalted media and Pc. soyae in the salted media as well as unsalted media required uracil as the pyrimidine base. Uracil could not be substituted by cytosine or thymine, however, in a medium containing uracil as the only factor belonging to base, the growth of Pc. soyae was not good. In addition to uracil, xanthine or guanine as purine base was shown to be required for good growth of Pc. soyae. On the amino acid requirements, Pc. soyae required 8 amino acids (glutamic acid, arginine, tryptophan, serine, leucine, isoleucine, histidine, valine) in the unsalted media and 6 amino acids (glutamic acid, arginine, tryptophan, leucine, histidine, valine) in the salted one. Tc. soyae showed some difference from Pc. soyae, namely, in the unsalted media, among the essential amino acids for Pc. soyae, glutamic acid and valine were stimulatory, and tryptophan, serine, leucine, isoleucine were not required for growth, but proline was required specifically. In general, it was observed that number of amino acid required for halophilic lactic acid bacteria in salted media was reduced in unsalted media. Tc. soyae required peptide for its maximum growth, but Pc. soyae showed the maximum growth in the presence of tryptophan (0.01 to 0.1 % as nitrogen) even in the absence of peptide. Pc. soyae and Tc. soyae are belong to homofermenter, but the fermentation products of them in the unsalted media were somewhat different from ordinal lactic acid bacteria, namely, the yield of lactic acid was lower and acetic acid was higher than the other. However, in the salted media this tendency was not observed. The above facts might be due to the strong decomposition of lactic acid to acetic acid and carbon dioxide compared with the other lactic acid bacteria and this activity showed the maximum in the sodium chloride concentration of one per cent. IV. Mechanism of halophilism in Pc. soyae and Tc. soyae On the halophilism of soy sauce lactic acid bacteria, the intracellular salt concentrations of these halophiles were higher than non-hlaophile but always lower than extracellular salt concentrations. Moreover, the cell wall of these halophiles was not easily broken by the usual physical and chemical methods. From the results of intracellular salt concentration, stable cell wall, halophilic enzyme and choline requirement, it seemed reasonable to conclude that the osmotic barrier and active transport system might be concerned with the mechanism of halophilism in soy sauce lactic acid bacteria.
- 大阪府立大学の論文
- 1964-03-31