Crucial role of early addition of Wickerhamiella versatilis in enhancing aroma formation during soy sauce fermentation.

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In modern Japanese soy sauce production, sealed outdoor fermentation tanks are used to ferment moromi with halotolerant starter cultures: the lactic acid bacterium Tetragenococcus halophilus and yeasts Wickerhamiella versatilis and Zygosaccharomyces rouxii. T.halophilus and W.versatilis are added in the early fermentation stage, while Z.rouxii is introduced about 1 month later to initiate alcoholic fermentation. Both W.versatilis and Z.rouxii contribute to the production of volatile organic compounds (VOC), with 4-ethylguaiacol (4-EG), uniquely produced by W.versatilis, being essential for the characteristic aroma of soy sauce. Prior metagenomic and metabolomic analyses indicated that 4-EG production occurs prior to Z.rouxii fermentation, though the increase in W.versatilis proportion follows it. Additional omics analysis of a production batch confirmed similar microbial and VOC dynamics, with no clear relationship between W.versatilis increase and 4-EG levels. To investigate this, a laboratory-scale experiment was conducted using filter-sterilized moromi supernatant as a medium, with staggered inoculations of the two yeasts. Viable cell density, 4-EG, and ethanol were measured as indicators of fermentation activity. Results showed that when W.versatilis was inoculated before Z.rouxii, 4-EG production commenced earlier, while W.versatilis cell density and ethanol production increased only after Z.rouxii fermentation began. Under these conditions, 4-EG and ethanol production were highest, suggesting that the presence rather than the increase of W.versatilis is crucial for 4-EG production. Consequently, the early addition of W.versatilis was considered an effective strategy to enhance 4-EG and VOC production in moromi.