Driving kombucha fermentation through microbial consortia remains based on scarce scientific elements, especially regarding the consumption of nitrogenous nutrients. This study aimed at analyzing quantitatively proteins and free amino nitrogen (FAN) concentrations, and qualitatively the soluble proteins profiles during a 12-day production process. The effect of microbial interactions was evaluated by comparing the growth of previously isolated major microorganisms in monoculture and cocultures to original kombucha. Protein and FAN relative concentrations kinetics depended on microbial compositions. These parameters were associated to less variations and a positive effect on total microbial population in original kombucha, underlining a complex yet better management of the nutrients. Analysis of extracellular proteins by electrophoresis showed the presence of a microbial 15 kDa fraction common to all cultures. In contrast, a 21 kDa protein fraction was associated with the presence of Acetobacter indonesiensis. Exploratory proteomics analysis revealed the presence of characteristic extracellular moonlighting proteins, originating from this acetic bacteria. Production of extracellular vesicles was successfully confirmed with the isolation and observation of vesicles in A. indonesiensis culture, as well as in kombucha. The results characterize the central role of microbial interactions on FAN consumption and open new perspectives on the function of the bacterial vesicles observed.

The aim of this study was to assess the impact of production parameters on the reproducibility of kombucha fermentation over several production cycles based on backslopping. Six conditions with varying oxygen accessibility (specific interface surface) and initial acidity (through the inoculation rate) of the cultures were carried out and compared to an original kombucha consortium and a synthetic consortium assembled from yeasts and bacteria isolated from the original culture. Output parameters monitored were microbial populations, biofilm weight, key physico-chemical parameters and metabolites. Results highlighted the existence of phases in microbial dynamics as backslopping cycles progressed. The transitions between phases occurred faster for the synthetic consortium compared to the original kombucha. This led to microbial dynamics and fermentative kinetics that were reproducible over several cycles but that could also deviate and shift abruptly to different behaviors. These changes were mainly induced by an increase in the Saccharomyces cerevisiae population, associated with an intensification of sucrose hydrolysis, sugar consumption and an increase in ethanol content, without any significant acceleration in the rate of acidification. The study suggests that the reproducibility of kombucha fermentations relies on high biodiversity to slow down the modulations of microbial dynamics induced by the sustained rhythm of backslopping cycles.

Achieving both the maintenance of the highest live microbial population possible, while limiting the production of ethanol and overpressure in kombucha bottles, constitutes a challenge that requires gaining insights into the shelf life of kombucha. This field study aimed at understanding the impact of biotic and abiotic factors (microbial activity and storage temperature respectively) on bottled unflavored and flavored kombuchas during their shelf life. Changes in the microbial and chemical compositions were monitored over 1 to 8 months of storage at 4 °C or 20 °C. Non-targeted analysis of volatile compounds was carried out to identify biomarkers across chemical families and put into perspective with a descriptive sensory analysis. Results show the efficiency of refrigeration and pasteurization to inhibit alcoholic fermentation. However, flavoring strongly accelerated yeast fermentative activity despite refrigeration leading to excessive ethanol concentrations and in-bottle pressures. This may be caused by the introduction of assimilable nitrogen through flavoring ingredients. Fatty acids were determined as biomarkers for aging during cold storage with or without flavoring. Despite changes in volatile composition, olfactory sensory profiles were mildly impacted. Ammonium supplementation stimulated microbial activities before bottling (aerobic phase) but not after, hinting at an effect of amino acids during the shelf life. This study advises kombucha producers to pay greater attention to the assimilable nitrogen content of flavoring ingredients to improve the control over the quality of their final products during the shelf life.

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.

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.

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 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.

Kombucha is a traditional fermented beverage obtained from the transformation of sugared black tea by a community of yeasts and bacteria. Kombucha production recently became industrialized, but its quality standards remain poorly defined. Metabolomic analyses were applied using FT-ICR-MS to characterize the impacts of production phases and the type of tea on the non-volatile chemical composition of kombucha. Independently from tea type, the first phase of acidification in open vessel was characterized by the release of gluconate and gallate from acetic acid bacteria metabolism and probably from polymeric polyphenols, respectively. The second phase of carbonation in closed vessel induced a consumption or transformation of oleic acid that could be consecutive of oxygen limitation. The first phase had the most impact on molecular diversity, but tea type mainly influenced the global composition in polyphenol profile. Black tea polyphenols were more impacted by microbial activity compared to green tea polyphenols.

Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in the de novo formation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected to be the microbiologically active site for cellulose production and spatial optimization of yeast–AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles.

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.

Kombucha is a beverage made from sugared tea transformed by yeasts and acetic acid bacteria. Being originally homemade, it has become an industrially produced soft drink whose quality standards are poorly defined and whose production process is still not fully controlled. Based on current knowledge in beverages, links between kombucha's chemical composition and sensorial compounds are drawn. Macromolecules create turbidity, whereas uncharacterized tea pigments derivatives participate in the color. Residual sugars bring sweetness and organic acids produced by acetic acid bacteria form its characteristic sour taste. Acetic acid is also part of its aroma profile, although little data are available on the smell of kombucha. Carbon dioxide, potentially polyphenols, and residual ethanol are involved in the mouthfeel. In this review, after defining the key compounds that shape the characteristic sensory properties of kombucha, the impact of different production parameters is discussed. Water composition is determinant in the extraction of tea compounds along with the tea type and infusion duration and temperature. The type and amount of sweeteners play a role in the sweetness and influences the production kinetics. Similarly, the amount of inoculum and its microbial composition have an effect on the production, but the role of the vessels' geometry and temperature are also essential parameters that can be used to adjust the acidification phase's duration. Despite the amount of research carried out, further investigations of kombucha's sensory characteristics are needed. Such research could lead to a better definition of kombucha's quality and to an improved control over its production process.