Use of a Minimal Microbial Consortium to Determine the Origin of Kombucha Flavor.

Microbiological, chemical, and sensory analyses were coupled to understand the origins of kombucha organoleptic compounds and their implication in the flavor of the kombucha beverage. By isolating microorganisms from an original kombucha and comparing it to monocultures and cocultures of two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and an acetic acid bacterium (Acetobacter indonesiensis), interaction effects were investigated during the two phases of production. 32 volatile compounds identified and quantified by Headspace-Solid Phase-MicroExtraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) were classified according to their origin from tea or microorganisms. Many esters were associated to H. valbyensis, while alcohols were associated to both yeasts, acetic acid to A. indonesiensis, and saturated fatty acids to all microorganisms. Concentration of metabolites were dependent on microbial activity, yeast composition, and phase of production. Sensory analysis showed that tea type influenced the olfactive perception, although microbial composition remained the strongest factor. Association of B. bruxellensis and A. indonesiensis induced characteristic apple juice aroma.

Oxygen management during kombucha production: Roles of the matrix, microbial activity, and process parameters.

Oxygen plays a key role in kombucha production, since the production of main organic acids, acetic and gluconic acids, is performed through acetic acid bacteria's oxidative metabolism. Oxygen consumption during traditional kombucha production was investigated by comparing kombucha to mono and cocultures in sugared tea of microorganisms isolated from kombucha. Two yeasts, Brettanomyces bruxellensis and Hanseniaspora valbyensis and one acetic acid bacterium Acetobacter indonesiensis were used. Results showed that tea compounds alone were mainly responsible for oxygen depletion during the first 24 h following inoculation. During the first 7 days phase of production in open vessel, the liquid surface was therefore the only access to oxygen for microorganisms, as anaerobic conditions were sustained below this area. During the 5 days second phase of production after bottling, comparison of cultures with different microbial compositions showed that oxygen was efficiently depleted in the head space of the bottles in 3-6 h if the acetic acid bacterium was present. Lower access to oxygen after bottling stimulated ethanol production in B. bruxellensis and H. valbyensis cocultures with or without A. indonesiensis. This study provides insights into the management of oxygen and the roles of the tea and the biofilm during kombucha production.

Microbial Interactions in Kombucha through the Lens of Metabolomics.

Kombucha is a fermented beverage obtained through the activity of a complex microbial community of yeasts and bacteria. Exo-metabolomes of kombucha microorganisms were analyzed using FT-ICR-MS to investigate their interactions. A simplified set of microorganisms including two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and one acetic acid bacterium (Acetobacter indonesiensis) was used to investigate yeast-yeast and yeast-acetic acid bacterium interactions. A yeast-yeast interaction was characterized by the release and consumption of fatty acids and peptides, possibly in relationship to commensalism. A yeast-acetic acid bacterium interaction was different depending on yeast species. With B. bruxellensis, fatty acids and peptides were mainly produced along with consumption of sucrose, fatty acids and polysaccharides. In opposition, the presence of H. valbyensis induced mainly the decrease of polyphenols, peptides, fatty acids, phenolic acids and putative isopropyl malate and phenylpyruvate and few formulae have been produced. With all three microorganisms, the formulae involved with the yeast-yeast interactions were consumed or not produced in the presence of A. indonesiensis. The impact of the yeasts' presence on A. indonesiensis was consistent regardless of the yeast species with a commensal consumption of compounds associated to the acetic acid bacterium by yeasts. In detail, hydroxystearate from yeasts and dehydroquinate from A. indonesiensis were potentially consumed in all cases of yeast(s)-acetic acid bacterium pairing, highlighting mutualistic behavior.

Evolution in Composition of Kombucha Consortia over Three Consecutive Years in Production Context.

Kombucha is a traditional drink obtained from sugared tea that is transformed by a community of yeasts and bacteria. Its production has become industrialized, and the study of the microbial community's evolution is needed to improve control over the process. This study followed the microbial composition of black and green kombucha tea over three consecutive years in a production facility using a culture-dependent method. Microorganisms were isolated and cultivated using selective agar media. The DNA of isolates was extracted, amplified using 26S and 16S PCR, and sequenced. Identities were obtained after a comparison to the NCBI database. Dekkera/Brettanomyces bruxellensis, Hanseniaspora valbyensis and Saccharomyces cerevisiae were the major yeast species, and the major bacterial genera were Acetobacter and Liquorilactobacillus. Results highlight the persistence of yeast species such as B. bruxellensis detected in 2019. Some yeasts species appeared to be sensitive towards stressful events, such as a hot period in 2019. However, they were resilient and isolated again in 2021, as was the case for H. valbyensis. Dominance of B. bruxellensis was clear in green and black tea kombucha, but proportions in yeasts varied depending on tea type and phase (liquid or biofilm). Composition in acetic acid and lactic acid bacteria showed a higher variability than yeasts with many changes in species over time.

Microbial Dynamics between Yeasts and Acetic Acid Bacteria in Kombucha: Impacts on the Chemical Composition of the Beverage.

Kombucha is a traditional low-alcoholic beverage made from sugared tea and transformed by a complex microbial consortium including yeasts and acetic acid bacteria (AAB). To study the microbial interactions and their impact on the chemical composition of the beverage, an experimental design with nine couples associating one yeast strain and one AAB strain isolated from original black tea kombucha was set up. Three yeast strains belonging to the genera Brettanomyces, Hanseniaspora, and Saccharomyces and three strains of Acetobacter and Komagataeibacter species were chosen. Monocultures in sugared tea were analyzed to determine their individual microbial behaviors. Then, cultivation of the original kombucha consortium and cocultures in sugared tea were compared to determine the interactive microbial effects during successive phases in open and closed incubation conditions. The results highlight the main impact of yeast metabolism on the product's chemical composition and the secondary impact of bacterial species on the composition in organic acids. The uncovered microbial interactions can be explained by different strategies for the utilization of sucrose. Yeasts and AAB unable to perform efficient sucrose hydrolysis rely on yeasts with high invertase activity to access released monosaccharides. Moreover, the presence of AAB rerouted the metabolism of Saccharomyces cerevisiae towards higher invertase and fermentative activities.