Integrated GC-HRAM-MS and UHPLC-QTOF-MS metabolomics reveal mineral-induced metabolic adaptation of Lactiplantibacillus pentosus 9D3 during milk fermentation

Milk fermentation by plant-associated probiotic strains is constrained by poor adaptation to dairy matrices. This study evaluated genome-guided micronutrient supplementation to improve the performance and metabolomic profile of Lactiplantibacillus pentosus 9D3 in milk. Individual supplementation with Mn2+ or Mg2+ significantly enhanced bacterial growth and acidification, whereas Fe2+, Zn2+, and B-group vitamins showed limited effects. Optimal supplementation with 50 mg/L Mn2+ and 100 mg/L Mg2+ increased viable counts from 7.54 to 8.89 log CFU/mL. A cell population increase of ∼1.9 log CFU/mL was achieved despite reducing the inoculum level from 10% to 6%. Integrated metabolomic profiling using GC-HRAM-MS and UHPLC-QTOF-MS identified 299 metabolites across supplemented fermented milk, non-supplemented fermented milk, and unfermented milk, with group separation. Pathway analysis revealed significant enrichment of seven metabolic pathways, including purine, pyrimidine, galactose, propanoate, butanoate, amino sugar and nucleotide sugar, and α-linolenic acid metabolism. These findings support cost-efficient precision fermentation of functional dairy products.