Abstract
The wine ecosystem constitutes a highly selective ecological niche characterized by low pH, high ethanol levels, sulfur dioxide, polyphenols, and nutrient limitation. During malolactic fermentation, this environment becomes dominated by specialized lactic acid bacteria (LAB), particularly Lactiplantibacillus plantarum and Oenococcus oeni, whose persistence under such stressors suggests the presence of adaptive traits relevant to probiotic development. In this study, twenty-three LAB isolates obtained from the spontaneous wine ecosystem were systematically evaluated through a multi-stage screening strategy. Primary single-factor assays revealed pronounced inter- and intraspecies variability in tolerance to acid, lysozyme, and bile salts. As a result, all O. oeni isolates and eight L. plantarum strains were excluded from further consideration. The four selected L. plantarum isolates (M-1, SY-2, XJA2, and XJ14) were subsequently subjected to simulated gastrointestinal challenges. Strains M-1 and XJ14 maintained high viability across both gastric and intestinal phases. In contrast, SY-2 and XJA2 exhibited pronounced gastric sensitivity but demonstrated strong survival in the intestinal phase. Functional characterization further distinguished the isolates: M-1 and XJ14 displayed balanced probiotic profiles, whereas XJA2 exhibited exceptional auto-aggregation and efficient metabolic capacity, suggesting specific colonization potential despite its gastric vulnerability. Comprehensive safety assessments confirmed the absence of hemolytic activity, biogenic amine production, and acquired antibiotic resistance in the tested isolates. Collectively, these findings identify M-1 and XJ14 as promising candidates for direct probiotic application, and XJA2 as a promising functional strain for encapsulation-based delivery. This study highlights the wine ecosystem as a valuable reservoir for novel probiotic development.