Fermentation of second cheese whey by Propionibacterium freudenreichii: Metabolomic profiling and pathway insights for valorization.

To investigate the key metabolic pathways activated by Propionibacterium freudenreichii during the fermentation of second cheese whey (SCW), five strains previously selected for physiological, technological, and functional traits were used to ferment three SCW samples from dairies differing in technological level and whey cheese coagulation technique. Despite variations in SCW composition, all strains exhibited robust growth, reaching up to 9.65 Log₁₀ CFU/mL. Untargeted metabolomic analysis grouped both fermented and unfermented SCW samples into three main clusters, primarily driven by differences among the unfermented SCWs. This highlights the key role of SCW composition in shaping the metabolome, although the contribution of the different strains in the metabolomic profile of fermented SCW was also evident. The analysis further revealed the consistent accumu- lation of 45 metabolites across all fermented samples, including derivatives of vitamins B9 and B12, essential amino acids, as well as prebiotic and antioxidant compounds. Eleven metabolites were identified as potential biomarkers of SCW fermentation by P. freudenreichii, such as dopamine 4-sulfate and N-(1-deoxy-1-fructosyl) phenylalanine. Pathway analysis showed consistent enrichment of the “one-carbon pool by folate” pathway, along with glycine, serine, and threonine metabolism and vitamin B6 pathways. These findings demonstrate the metabolic versatility of P. freudenreichii in SCW and its suitability for SCW valorization through the production of nutritionally relevant compounds. Further experiments are currently in progress to assess the in vivo probiotic potential of the resulting microbial biomass and to confirm the suitability of fermented SCW as a sustainable and cost-effective food and feed additive