In this study, we used Chinese chestnut as the main raw material to develop a novel type of whiskey. First, 16 yeasts were isolated and identified for producing aroma using olfactory plate assay. Of these, we screened nine yeast strains based on their fermentation capacity, aroma profile, and sensory evaluation. The results demonstrated the combination of strains HN006 ( Saccharomyces cerevisiae ) and HN010 ( Wickerhamomyces anomalus ) provided satisfactory wine fermentation with an interesting flavor profile, as strain HN010 was highly aromatic and had elevated sensory scores with comparatively low ethanol yield, while strain HN006 had a poor flavor profile but produced the largest amount of ethanol. Subsequently, we co-cultured strains HN006 and HN010 to optimize the fermentation system. The results revealed the following optimum parameters: a mixed inoculum of 6% (v/v) at an HN006/HN010 ratio of 1:2 (v/v), a raw material ratio of 5:3:2 (chestnut: malt: glutinous rice), and yeast extract concentration of 6?g/L. Additionally, this fermentation system was successfully scaled-up to a 1000?L pilot-scale system. The results of this study showed that strains HN006 and HN010 could be used as alternatives for whiskey fermentation, as well as provided a generalized experimental scheme to assess other microorganisms.
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