Long-term Trends in Microbial Metabarcoding, Chemical and Bioactivity Profiles of Green and Black Homemade Kombucha During One Year of Backslopping

Abstract Kombucha is a fermented beverage widely produced at household level through backslopping and appreciated for its functional properties. Limited information is available on how repeated backslopping affects microbial dynamics, biochemical composition, and product safety over time. This study investigated the impact of twelve consecutive backslopping cycles mimicking controlled homemade conditions on green and black kombucha. Physicochemical analyses, bioactive components, antioxidant activity assays, and metabarcoding analyses were carried out to evaluate fermentation performance, safety-related parameters, functional compounds, and microbial community dynamics. Partial least squares (PLS) modelling was applied to identify key variables influenced by fermentation cycles. Both green and black kombucha maintained acetic acid and ethanol concentrations within recommended safety limits throughout all fermentation cycles, supporting the safety of homemade backslopping. Repeated fermentation cycles were associated with increased microbial activity, reflected by enhanced sugar consumption and reduced gluconic acid production, without inducing critical pH shifts nor excess of ethanol production. Functional profiling revealed variability in phenolic composition; however, antioxidant activity remained stable, suggesting a compensatory role of microbial metabolites. PLS analysis of metabarcoding data highlighted shifts within Amplicon Sequence Variants of acetic acid bacteria, with a decreasing trend along the fermentation cycles in Acetobacter in favour of Komagataeibacter . This study provides new insights into long-term backslopping effects and supports process optimization to ensure kombucha functionality and safety.