Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.