Strain-level variation in diacetyl production by Lactiplantibacillus plantarum reveals genetic drivers of flavor in fermented dairy.

Strain-level diversity in aroma formation remains undercharacterized in Lactiplantibacillus plantarum, a key adjunct culture in dairy fermentation. We screened 351 isolates from traditional Chinese fermented foods for diacetyl production and identified wide phenotypic variation, with strain WJ108 producing 26.85 ± 0.72 mg/kg, which is more than double that of known high producers. Selected strains representing high, medium, and low diacetyl yields were analyzed using GC-MS, revealing ketone enrichment in high producers and aldehyde and alcohol accumulation in low producers. Electronic nose profiling and principal component analysis confirmed distinct volatile signatures across groups. Quantitative PCR (qPCR) analysis showed that α-acetolactate synthase (alsS) and NADH oxidase (nox) were upregulated in high producers, whereas lactate dehydrogenase, alcohol dehydrogenase, and pyruvate formate lyase were dominant in low producers. Correlation analysis confirmed that W1C, W3C, and W5C electronic nose sensors were highly responsive to ketones. Integrated GC-MS and qPCR data further revealed that nox and alsS genes positively regulate diacetyl and acetoin biosynthesis in L. plantarum. These results establish a genetic and metabolic basis for aroma diversity in L. plantarum and support the precision selection of strains with enhanced flavor-forming potential for dairy applications.