Development of Sequential Fermentation Starters and Comparison of Quality Characteristics for Black Barley Vinegar Production

To identify the optimal sequential starter and fermentation conditions, black barley vinegar (BBV) was produced by stepwise inoculation with two acetic acid bacteria (AAB). The optimal fermentation conditions for BBV12-8 (fermentation with <i>Acetobacter ascendens</i> GV-12 followed by <i>A. ascendens</i> GV-8) were pH 3.0 and 20 °C, and for BBV8-22 (fermentation with <i>A. ascendens</i> GV-8 followed by <i>Acetobacter pasteurianus</i> GV-22) were pH 3.0 and 30 °C. During fermentation, the levels of most organic acids decreased in both vinegars, while acetic acid increased significantly. Total free amino acids also decreased, and taste fingerprint analysis revealed increased sourness and umami and decreased saltiness with fermentation. In particular, BBV12-8 exhibits higher ethyl acetate and 3-methylbutyl acetate than BBV8-22. This difference was closely related to fermentation temperature, as highly volatile esters were better preserved at 20 °C than at 30 °C. Therefore, <i>A. ascendens</i> GV-12 followed by <i>A. ascendens</i> GV-8 sequential inoculation at pH 3.0 and 20 °C was selected as the optimal condition for producing black barley vinegar with improved flavor characteristics, suggesting that sequential AAB inoculation can be applied to produce vinegar with enhanced flavor quality.