Unlocking Microbial Interactions: Multi-Plant-based-Substrate Fermentation with Water Kefir Starters for Functional Beverage Innovation

The specificity of ingredients and fermenting microorganisms is critical in designing fermented plant-based beverages and maximizing their protective properties. We aimed to replicate the kefir microbial ecosystem in a novel multi-plant formulation, investigating synergistic interactions between water kefir-derived starters (Leuconostoc mesenteroides, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, and Pichia bruneiensis) and plant substrates. Starter screening using growth kinetics modelling and acidification identified 16-h fermentation at 30 °C as optimal. Various fermentation methods were employed, including the use of single starters, a ternary lactic acid bacteria (LAB) mixture, and a final combination of all LAB strains with the yeast starter. Subsequently, a comprehensive analysis was conducted, including: microbial growth and acidification; HPLC for microbial metabolites; proteolysis (OPA method, amino acid analyzer); phenolic profiling (LC-MS) and antioxidant activity (DPPH/ABTS); antinutrient quantification (Megazyme kits); texture and syneresis analysis; and VOC profiling (GC-MS). Compared to the beverage without microbial inoculum and incubation (Raw-MPBB), fermentation with Lc. paracasei alone or the ternary LAB culture showed superior microbial growth, strong acidification through the lactic acid production, and enhanced proteolysis as indicated by peptide and free amino acid concentrations. Although phenolic compounds metabolism was starter composition dependent, the same beverages promoted a significant release of phenolics (chlorogenic acid, epicatechin, catechin, and rutin). Other phenolics with enhanced antioxidant potential (such as caffeic, gallic, and protocatechuic acids) were released mainly by the beverage fermented with Leuc. mesenteroides. Only the ternary LAB culture achieved phytic acid degradation, while the beverage fermented with Lc. paracasei not only exhibited the lowest fructan and raffinose contents among all beverages but, together with the beverage fermented with P. bruneiensis, developed more complex volatile organic compound profiles. Beverage stability was improved especially by P. bruneiensis, which effectively minimized syneresis after fermentation. All beverages showed higher firmness than dairy yogurt, regardless of the fermentation effect. Overall, our findings highlight the potential of kefir microbial starters combined with plant-based formulations to create nutritious, stable, and sustainable functional dairy alternatives.