Acid and ethanol stress enhance exopolysaccharide production and change membrane vesicle cargo in Oenococcus oeni SD-2a

During malolactic fermentation (MLF), Oenococcus oeni ( O. oeni ) encounters severe stresses. Exopolysaccharides (EPS) and membrane vesicles (MVs) contribute to bacterial stress adaptation, but whether these extracellular components are coordinately remodeled in O. oeni under stress conditions remains unclear. This study investigated how acid (pH4.0) and ethanol (50 mL/L) stress affect EPS production and function, MVs release, and MVs protein cargo in O. oeni SD-2a. EPS composition, thermal properties, microstructure, and lyoprotective effects were characterized, and MVs were examined by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and data-independent acquisition proteomics. Both stresses inhibited growth, but they increased EPS production and altered its composition. Acid stress increased rhamnose molar proportion from 3.6% to 10.5% and decreased glucose from 86.6% to 70.8%. EPS produced at pH 4.0 showed the strongest lyoprotection, maintaining membrane integrity in 80.5% of cells after freeze-drying, compared with 73.3% and 71.2% for control (pH4.8) and ethanol derived EPS, respectively. Acid and ethanol stress increased MVs size from 123.0 nm to 162.7 and 154.5 nm, and also increased MVs concentration. Proteomic profiling identified 1051 proteins shared among all conditions, with acid and ethanol stresses generating 614 and 609 significantly differential abundance proteins (DAPs) compared with control condition. DAPs were enriched in amino sugar and nucleotide sugar metabolism, and six proteins involved in glucose or fructose transport increased under both stresses. These findings reveal O. oeni responds to wine-related stress by coordinately remodeling EPS and MVs cargo composition, providing extracellular targets for improving the stress tolerance of MLF starters.