Abstract: The aim of this study was to improve the detection efficiency of spoilage bacteria in Chinese rice wine. By using the traditional culture method based on sensory evaluation and the change in total acid, the target spoilage bacteria ZH-1 and ZH-2 were isolated from rancid Chinese rice wine. Furthermore, the spoilage bacteria were identified by their morphological and biochemical characteristics and 16S rRNA gene sequencing. Meanwhile, one-factor-at-a-time method and Box-Behnken design with response surface methodology were employed to optimize the culture conditions for the target spoilage bacteria. The results showed that the rancid Chinese rice wine was muddy with an off odor and increased total acidity. Both ZH-1 and ZH-2 were identified as Lactobacillus fructivorans. The spoilage bacteria grew slowly on solid medium, and it took as long as 9–10 days to culture them using the method described in GB 4789.35-2016; the optimal enrichment conditions for detecting L. fructivorans were MRS agar medium containing 0.08% L-Cys with pH adjusted to 5.4, addition of 9% (V/V) of anhydrous ethanol to the medium immediately before use, and culture temperature 30 ℃. The time required to detect quantitatively L. fructivorans using the optimized method could be shortened by 3–4 days.

Key words: Chinese rice wine, Lactobacillus fructivorans, isolation, identification, optimization