Tracking one year of homemade kombucha backslopping: microbial succession, chemical transformations and bioactivity in green and black tea fermentations

Kombucha is a fermented tea beverage commonly produced at household level through backslopping and valued for its potential functional properties. However, information on the long-term effects of repeated backslopping on microbial ecology, biochemical composition, and product safety remains limited.

This study evaluated the impact of twelve consecutive backslopping cycles, performed under controlled homemade conditions, on green and black kombucha. Physicochemical parameters, bioactive compounds, antioxidant activity, and microbial community dynamics were assessed through chemical analyses, antioxidant assays, and metabarcoding. Partial least squares (PLS) modelling was applied to identify the variables most affected by repeated fermentation cycles.

Throughout all fermentation cycles, both green and black kombucha maintained acetic acid and ethanol concentrations within recommended safety limits, confirming the safety of prolonged homemade backslopping. Repeated fermentations promoted microbial activity, as indicated by enhanced sugar consumption and lower gluconic acid production, without causing critical pH variations or excessive ethanol accumulation. Although phenolic profiles changed over time, antioxidant activity remained stable, suggesting a compensatory contribution of fermentation-derived microbial metabolites. Metabarcoding coupled with PLS analysis revealed progressive shifts within acetic acid bacteria populations, characterized by a decline in Acetobacter ASVs and the concomitant enrichment of Komagataeibacter ASVs across fermentation cycles.

Long-term backslopping significantly influences kombucha microbial succession and biochemical characteristics while preserving product safety and antioxidant potential. These findings provide new insights into the stability of homemade kombucha ecosystems and support the optimization of backslopping practices to maintain beverage functionality and quality.