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ISOLATION OF ACETOIN-PRODUCING Bacillus STRAINS FROM JAPANESE TRADITIONAL FOOD—NATTO

In this study, Bacillus strains with an ability to produce acetoin were isolated from a Japanese traditional food, natto, on the basis of the Voges–Proskauer (VP) reaction, and strain SF4-3 was shown to be a predominant strain in acetoin production. Based on a variety of morphological, physiological, and biochemical characteristics as well as the nucleotide sequence analysis of 16S rDNA, the strain SF4-3 was identified as Bacillus subtilis. When it was incubated at 37°C with a speed of 180 rpm for 96 hr in the flasks, the maximum acetoin concentration was up to 33.90 g/L. The fermentation broths were determined by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) analyses; the results showed that the major metabolite was acetoin, and the purity could reach more than 95% without butanedione and 2,3-butanediol, which were usually produced together with acetoin in other strains. A novel aqueous two-phase system (ATPS) composed of hydrophilic solvents and inorganic salts was developed for the extraction of acetoin from fermentation broths. The ethanol and dipotassium hydrogen phosphate system could be used to extract acetoin from fermentation broths. The influences of phase composition on partition of acetoin were investigated. The maximum partition coefficient (9.68) and recovery (94.6%) of acetoin were obtained, when 25% (w/w) dipotassium hydrogen phosphate and 24% (w/w) ethanol were used.