In 2015, Bacillus paralicheniformis was separated from B. licheniformis on the basis of phylogenomic and phylogenetic studies, and urease activity was reported as a phenotypic property that differentiates between the two species. Subsequently, we have found that the urease activity of B. paralicheniformis is strain-specific, and does not reliably discriminate between species, as strains having the same urease gene cluster were identified in B. licheniformis and B. sonorensis, the closest relatives of B. paralicheniformis. We developed a multilocus sequence typing scheme using eight housekeeping genes, adk, ccpA, glpF, gmk, ilvD, pur, spo0A, and tpi to clearly identify B. paralicheniformis from closely related Bacillus species and to find a molecular marker for the rapid identification of B. paralicheniformis. The scheme differentiated 33 B. paralicheniformis strains from 90 strains formerly identified as B. licheniformis. Among the eight housekeeping genes, spo0A possesses appropriate polymorphic sites for the design of a B. paralichenofomis-specific PCR primer set. The primer set designed in this study perfectly separated B. paralicheniformis from B. licheniformis and B. sonorensis.
Several types of fermented soybean food are consumed in Korea and representative examples are ganjang (soy sauce) and doenjang (soybean paste). The traditional production of ganjang and doenjang starts from the manufacture of meju. Meju is made by soaking, steaming, crushing, and molding soybeans into blocks, followed by spontaneous fermentation for 2-3 months. The ripened meju is mixed with brine and ripened for a further 2-3 months, then the liquid portion of the mixture is separated, resulting in a traditional type of ganjang. The remaining solid portion is subsequently mashed and fermented for >6 months and becomes quality doenjang [1]. Ripened meju is used as a starter culture as well as a nutrient and flavor source for fermented food production in Korea [2].Understanding of traditional fermented soybean foods has long been a research theme of food scientists in Korea. Several studies including microbial community analysis have provided basic insight for accelerated ripening, quality assurance, and flavor enhancement of fermented soybean foods. Early microbial studies isolated and identified microorganisms exhibiting strong amylase, protease, and lipase activities that can contribute to degradation of soybean macromolecules [3][4][5][6]. More recent culture-independent microbial community analysis techniques have revealed the presence of a wider variety of microorganisms in the food matrices [7][8][9][10][11][12][13][14][15]. However, regardless of the analytical methods used, most microbial studies reported that the bacterial genus Bacillus and the fungal genus Aspergillus were the most populous microorganisms.Recently, molecular biology techniques and sequence databases have contributed to identification, characterization, and typing of bacteria and increased the number of novel bacterial species [16]. Rapid advances in whole-genome sequencing technologies and analysis pipelines have further enhanced the resolution power of bacterial species and influenced the taxonomic status of closely related identities. This advanced bacterial identification methodology has affected the consolidation and assignment of new taxa in the genus Bacillus [17][18][19]. In this context, we isolated and identified Bacillus spp. from 12 meju samples collected from five regions of Korea to determine the predominant Bacillus species in meju based on current bacterial taxonomic standards [20]. One hundred and fifty-one Bacillus isolates were identified, in the following order of predominance: B. velezensis, B. sonorensis, B. subtilis, and B. licheniformis. Strains of four Bacillus spp. were respectively inoculated into sterilized soybeans and the free amino acid profiles of the resulting cultures were analyzed to discern their metabolic traits. After 30 days of culture, B. licheniformis showed the highest production of serine, threonine, and glutamic acid; B. subtilis exhibited the highest production of alanine, asparagine, glycine, leucine, proline, tryptophan, and lysine. B. velezensis increased the γ-aminobutyric acid (GABA) ...
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