Abstract
This study aimed to screen high extracellular polysaccharides (EPS) producing lactic acid bacteria and systematically characterize the temporal metabolic dynamics underlying mixed-culture fermentation of Munage grape juice (MGJ). Lactobacillus acidophilus and Lactiplantibacillus plantarum were stepwise selected from ten commercial strains and co-fermented at a 1:1 ratio. Response surface methodology was employed to establish optimal fermentation parameters: an inoculum size of 2.5% (v/v), initial pH of 5.9, temperature of 37 °C, and fermentation duration of 32 h. Under these conditions, EPS production reached 1.26 ± 0.02 g/L. Untargeted metabolomics analysis revealed distinct temporal metabolic reprogramming. During the early stage (0-16 h), pyruvate metabolism pathways were significantly enriched, resulting in the rapid accumulation of organic acids (lactate, citrate), which drove a decline in pH and suppressed undesirable microorganisms. In the mid-stage (16-32 h), phenylpropanoid biosynthesis, glutathione metabolism, and related pathways were cooperatively activated, leading to markedly increased levels of phenolic acids (coumaric acid, isoeugenol), flavonoids, and amino acid derivatives (N-acetylglutamate, His-Trp-Phe), thereby conferring enhanced antioxidant activity and flavor precursors. Metabolic activity plateaued during the late stage (32-40 h) with few differential metabolites observed, confirming 32 h as the optimal fermentation endpoint. This study provides the first comprehensive temporal metabolic atlas of mixed LAB fermentation in MGJ, offering both technical parameters and theoretical support for the development of functional fermented beverages.