Ultrasound-triggered metabolic responses of Lactobacillus acidophilus at different growth phases enhance the functional quality of Citrus aurantium L. juice.

Accumulating evidence has demonstrated that ultrasound-assisted lactic acid bacteria (LAB) fermentation can improve the functional quality of fruit juices. However, the growth phase-dependent metabolic responses of LAB to low-frequency ultrasonication remain poorly understood. In this study, low-frequency ultrasonication (83.6 and 119.17 W/L) was applied during the lag, logarithmic, and stationary phases of Lactobacillus acidophilus LA137 fermentation in Citrus aurantium L. juice. Microbial growth, physicochemical properties, β-glucosidase activity, total phenolic and flavonoid contents, and antioxidant capacity were evaluated in this study. Ultrasonication applied during the logarithmic and stationary phases exerted greater effects on microbial metabolism and functional quality than treatment during the lag phase. Among all treatments, ultrasonication at 119.17 W/L during the logarithmic phase (LU4) and 83.6 W/L during the stationary phase (LU5) showed superior enhancement of β-glucosidase activity, phenolic/flavonoid accumulation, and antioxidant capacity. Integrated untargeted metabolomics revealed distinct metabolic responses between LU4 and LU5. LU4 preferentially promoted phenylpropanoid and flavonoid metabolism, resulting in the accumulation of antioxidant-related metabolites, including gallic acid, taxifolin, and 7-galloylcatechin. In contrast, LU5 mainly enhanced amino acid and organic acid metabolism, leading to increased levels of lactic acid, γ-aminobutyric acid, alanine, and serine. Correlation analysis further linked these metabolites to improved antioxidant properties. These findings demonstrate that low-frequency ultrasonication redirects microbial metabolism toward distinct functional routes depending on the growth phase of L. acidophilus, providing new mechanistic insights into the precise regulation of ultrasound-assisted LAB fermentation.