Abstract: Despite many non-Saccharomyces yeasts being considered spoilage microorganisms, they can increase aroma and flavor diversity in alcoholic beverages. The purpose of this study was to investigate nontraditional inoculation strategies using aroma-producing yeast strains for Kyoho wine fermentation, followed by an instrumental analysis and sensory evaluation. The winemaking process was carried out using Saccharomyces cerevisiae Gr112, Hanseniaspora uvarum Pi235, and Pichia kluyveri Pe114. Multiple inoculation strategies were explored. In instrumental analysis results, mixed culture could promote the formation of esters (5.9-folds) and glycerol (1.3-folds) and reduce the content of ethanol (–0.5% [v/v]) in wine. The sensory analysis results suggested that the three yeast strains sequential inoculation treatment was associated with the aroma attributes “floral,” “red fruity,” and “tropical fruity.” Co-cultivation contributed to an increase in complexity and aromatic intensity, with the three-strain inoculation treatment presenting a more distinctive appearance. Practical Application: The inoculation of S. cerevisiae improved the accumulation of volatile acids and esters by inhibiting the growth of non-Saccharomyces yeast strains. Inoculation of H. uvarum and P. kluyveri would effectively solve the defect of excessive content of higher alcohols in wines produced by S. cerevisiae. The suitable inoculation strategy between non-Saccharomyces yeasts could improve the overall quality of Kyoho wine whose starter might be widely used in fermentation industry.
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