Abstract:[Background] Kombucha is a traditional tea beverage fermented by a symbiotic consortium of bacteria and yeasts. A number of metabolites in kombucha are beneficial to human health such as promoting digestion, anti-inflammatory, antibacterial and anti-diabetes. The beneficial metabolites are mainly produced by complex microbial interactions dominated by acetic acid bacteria and yeasts. Therefore, kombucha is an excellent model for studying metabolic process in a symbiotic microbial consortium. [Objective] This study aims to study the effects of interactions among different microorganism on metabolites in kombucha by culturing microbial strains isolated from kombucha singly or in combination under different culture conditions. [Methods] Dominant microbial strains were isolated from kombucha consortium by agar plate dilution method. The isolated microbes were identified by morphological observation and molecular biology methods. Different culture combinations were designed to investigate the effects of single-culture and mixed-culture on the pH value, acidity, reducing sugar and total polyphenol contents in fermentation broth. [Results] The isolated microbial strains grew well in tea sugar medium singly or in combination, but with different growth rate. The microbial growth rate was closely related to the fermentation ability of the tested strain to produce reducing sugar. pH value of fermentation broth was significantly affected by Komagataeibacter sp. strain C6, which showed a strong ability to produce organic acids. The fermentation process by microbes in the broth significantly increased the contents of total polyphenols in the fermentation broth. Compared to the single-culture, the mixed-culture of three strains contributed to the stability of the microbial community, and significantly promoted the production of reducing sugar, organic acid and decreasing of pH value, and slight increase of polyphenols in kombucha. [Conclusion] Kombucha is a symbiotic consortium-fermented system with complex interactions between bacteria and yeasts. The multi-strains co-culturing contributes to formation of the stable microbial communities, and also promotes the metabolism of organic acids, reducing sugars and polyphenols in kombucha. The symbiotic fermentation with different microbes is the biochemical basis for the popular kombucha drinks with flavor taste and beneficial physiological activity.