Kombucha meets circular economy: A microbiome and metabolite perspective on second fermentation with plant by-products

Kombucha is a traditional fermented beverage produced through the fermentation of sugared tea by a symbiotic culture of bacteria and yeasts (SCOBY). In recent years, the valorisation of plant-based by-products as fermentation substrates has gained attention as a sustainable approach to improving both the nutritional and economic efficiency of fermented beverages. The present study investigated the production of kombuchas supplemented with pineapple, fennel, and carrot by-products during the secondary fermentation phase, aiming to evaluate their influence on fermentation dynamics, microbial ecology, and the chemical and aromatic profiles of the final products. The experimental design integrated culture-dependent and culture-independent approaches, including amplicon sequencing, to characterize microbial community composition and evolution throughout fermentation. Chemical profiling was carried out using gas chromatography coupled with quadrupole mass spectrometry (GC-qMS) and high-performance liquid chromatography equipped with diode-array and refractive index detectors (HPLC-DAD/RI). The fermentation process was monitored during both the primary and secondary stages, and a shelf-life assessment was conducted over 14 days of refrigerated storage (4 °C) to evaluate product stability. Microbiological results indicated a predominance of Schizosaccharomyces spp., while Komagataeibacter spp. was the only bacterial genus identified. A significant reduction in α-diversity was observed over time, suggesting selective adaptation of the microbial community to the fermentation environment. β-diversity analysis revealed clear differences among samples collected after 8 and 22 days, reflecting the combined influence of time and substrate composition on microbial succession. Chemical analyses demonstrated an increase in acetic acid concentration and a progressive decline in pH throughout fermentation, consistent with the metabolic activity of acetic acid bacteria. Among volatile organic compounds (VOCs), alcohols and organic acids were the most abundant chemical classes detected. Several VOCs were associated with minor yeast genera, including Hannaella, Galactomyces, Aureobasidium, and Millerozyma, whereas Schizosaccharomyces spp. showed a strong correlation with specific aroma-active compounds, highlighting its key role in defining the sensory characteristics of the beverage. Overall, this study provides new evidence on how different vegetable by-products and microbial consortia influence the development of chemical and aromatic compounds in kombucha. The findings highlight the potential of using by-products as a sustainable, value-added strategy for producing fermented beverages, while also supporting the principles of the circular economy and resource-efficient food systems.