Investigating the molecular mechanisms and microbial drivers underlying dry-cured ham quality: A combined metabolomics and microbiological analysis

• Linoleic acid and Nucleotide metabolism account for the differences in metabolites. • Branched-chain compounds are the primary contributors to the flavor characteristics. • Staphylococcus exhibits a positive correlation with the branched-chain compounds. • Staphylococcus strains effectively increased the yield of aroma compounds. This study aims to reveal the molecular basis of the unique characteristics of dry-cured ham and to deeply explore the microbial driving factors influencing the formation of ham quality. The volatile flavor compounds and non-volatile metabolites present in three kinds of ham were analyzed using GC-MS and LC-MS/MS nontargeted metabolomics technology. A total of 53 volatile flavor compounds were identified in dry-cured hams, including 7 aldehydes, 10 ketones, 5 alcohols, 4 acids, 6 esters, and 21 other compounds. Among these compounds, the branched-chain derivatives such as 2-Methyl-butanal, 3-Methyl-butanal and 3-Methyl-butanol exhibited the highest odor activity values. A total of 2389 metabolites were detected, with Lipids and lipid-like molecules, Organic acids and derivatives as well as Organoheterocyclic aompounds constituting over 70%. Pathway enrichment analysis revealed that differential metabolites primarily originated from processes such as Linoleic acid metabolism and Nucleotide metabolism. The potential pathways for metabolite production were systematically reconstructed. Amino acid metabolism, nucleotide metabolism, and lipid metabolism emerged as the primary pathways contributing to the flavor profile and metabolite generation in dry-cured ham. To investigate the role of microorganisms in the formation of aroma compounds in dry-cured ham, high-throughput sequencing combined with Staphylococcus fermentation experiments was conducted. The results revealed that Staphylococcus was the most predominant genus across various ham varieties and exhibited significant potential in promoting the formation of 2-Methyl-butanol, 3-Methyl-butanol, Heptanal, and non-volatile metabolites. Furthermore, variations were observed among different strains. The synergistic interactions among these strains are believed to contribute to the flavor quality of dry-cured ham.