Enhancing fruity aroma of Cabernet Sauvignon wine by mixed culture fermentation with Schizosaccharomyces japonicus, Saccharomyces cerevisiae, and Starmerella bacillaris

The mixed culture fermentation of Saccharomyces cerevisiae and non-Saccharomyces yeast has been reported to bring a higher diversity of volatile compounds and presumably a greater aromatic complexity to wine. This study mainly focuses on the performance of specific indigenous yeasts isolated from local grapes in Guizhou, including killer active S. cerevisiae FBKL2.9126 with high tolerance and good fermentation trait, strong flocculation strain Schizosaccharomyces japonicus FBKL2.9SZJ-29 with good fermentation trait, and high pectinase formation strain Starmerella bacillaris FBKL2.91505. The entire growth cycle of S. japonicus was firstly determined by analyzing the proportions of different cell morphologies of S. japonicus at various stages. Four stages including growth, division, sporulation, and spore release, were defined in the growth cycle of S. japonicus. Three primer pairs were further designed for specific amplification of S. japonicus containing 179For375Rev, 179For388Rev, and 388For518Rev, and were used for the establishment of corresponding quantitative PCR (qPCR) standard curves. Wine fermentations of Cabernet Sauvignon were finally performed with three inoculation modes, including pure fermentation of S. cerevisiae, mixed culture fermentation of S. cerevisiae and S. japonicus, and mixed culture fermentation of S. cerevisiae and S. japonicus with inoculation of S. bacillaris 12 h in advance. The quantity of S. cerevisiae, S. bacillaris, and S. japonicus during wine fermentation was investigated based on qPCR, plating, and cell counting methods. The two non-Saccharomyces yeasts kept the concentration of 10⁶-10⁷ cells/mL during fermentations. The co-inoculation of S. japonicus and S. cerevisiae increased the content of flavor compounds such as isoamyl acetate with OAV>1. The mixed culture fermentation with S. bacillaris inoculated 12 h ahead brought fruitier aromas to Cabernet Sauvignon wine than the other two modes by sensory analysis, and significantly increased the content of fruity substances such as ethyl hexanoate (OAV>1) and hexyl acetate. However, correlation analysis highlighted the role of quantity change of S. cerevisiae in the content change of some related volatile substances. This investigation explored the flavor impact of S. japonicus and S. bacillaris on Cabernet Sauvignon wine by mixed culture fermentation, and provided a reference for quantifying S. japonicus during fermentation.