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

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.