Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice <i>Koji</i> Cultivation, in Japanese Sake Brewing

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice <i>koji</i> samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 μg/kg dry mass <i>koji</i>, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass <i>koji</i>. Guaiacol forming microorganisms were isolated from four rice <i>koji</i> samples. They were identified as <i>Bacillus subtilis</i>, <i>B. amyloliquefaciens/subtilis</i>, and <i>Staphylococcus gallinarum</i> using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of <i>koji</i> fungi tested produced vanillic acid at the mg/kg dry mass <i>koji</i> level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of <i>koji</i> cultivation in the sake brewing process