Primary Metabolites and Microbial Diversity in Commercial Kombucha Products

Kombucha brewers selling non-alcoholic beverages in the United States must ensure that the ethanol content of their products remains below 0.5% (<i>v</i>/<i>v</i>) throughout all stages of production and shelf life. Producers struggle to comply with this regulation in the absence of expensive dealcoholizing equipment if they wish to sell the unpasteurized or minimally pasteurized products that consumers typically expect. To identify which bacterial and/or fungal species contribute to the high ethanol content of commercial kombucha, we analyzed 47 commercial kombucha samples purchased at supermarkets near Cornell University in Ithaca, NY, USA. We analyzed samples for ethanol content via HPLC, microbial load determination, and next-generation amplicon sequencing of the bacterial and fungal populations of those samples. Two brands were found to contain significantly more than 0.5% ethanol (<i>v</i>/<i>v</i>) in the tested samples (<i>t</i>-test, <i>p</i> < 0.05, greater), and three brands were found to contain significantly different amounts of sugar in the tested samples compared to what was reported on the nutrition label (one higher and two lower, <i>t</i>-test, <i>p</i> < 0.05, two-sided). The microbial communities of the samples most significantly varied due to brand (PERMANOVA, <i>p</i> < 0.05). The main bacterial genera observed in the samples were <i>Komagataeibacter</i>, <i>Acetobacter</i>, <i>Gluconobacter</i>, <i>Oenococcus</i>, <i>Lactobacillus</i>, and <i>Bifidobacterium</i>. The main fungal genera observed in the samples were <i>Saccharomyces</i>, <i>Dekkera</i>, <i>Cyberlindnera</i>, <i>Lachancea</i>, <i>Schizosaccharomyces</i>, and <i>Pichia</i>. We did not identify any bacterial or fungal species associated with differences in ethanol content between samples within brands, suggesting significant strain variation in the bacteria and fungi involved in commercial kombucha fermentation. However, we did find that the relative abundance of <i>Lactobacillales</i> and the lactic acid content of the samples were significantly correlated (Kendall correlation test, <i>p</i> < 0.05). These results build upon recent research elucidating the role of lactic acid bacteria in the commercial fermentation of kombucha.