Development of a novel selective medium for the isolation and enumeration of acetic acid bacteria from various foods

Acetic acid bacteria (AAB) are gram-negative, obligate aerobic bacteria that produce acetic acid from alcohols or sugars. AAB are ubiquitous and found in many food products, including vinegar and wine. Owing to the lack of a suitable selective culture medium and the fastidious nature of AAB in food samples, ecological studies have been fundamentally limited for decades. Here, we developed a novel selective medium (AAB-selective agar; ABS) for AAB from food samples and evaluated its performance in comparison with glucose-yeast extract-calcium carbonate agar (GYC). In inclusivity tests, 16 species of AAB showed good growth on both ABS and GYC. Remarkably, the growth of 21 non-AAB strains was inhibited on ABS but not on GYC, suggesting the high exclusivity of the novel selective medium (p < 0.05). In artificial AAB-positive food samples, including wine, vinegar, yogurt, fruit, and kimchi, ABS provided better visual differentiation of AAB colonies, showing green colonies with a yellow halo surrounded by no competing colonies. Moreover, in ABS, a significantly greater number of AAB colonies were recovered from yogurts, fruits, and kimchi (Acetobacter aceti ATCC 15973 in yogurt, 4.24 +/- 0.17 vs. 3.80 +/- 0.14; Gluconobacter oxydans ATCC 15163 in fruit, 4.87 +/- 0.07 vs. 4.53 +/- 0.15; Gluconacetobacter hansenii ATCC 35959 in kimchi, 4.25 +/- 0.14 vs. 3.85 +/- 0.12; p < 0.05). Finally, a total of five novel AAB strains were isolated from 10 vinegar and 10 kimchi samples using ABS, while unsuccessful using GYC, demonstrating the superior performance of ABS in isolating new AAB strains or detecting AAB contamination.