Characterization of peptide-enriched walnut meal paste via microbial diversity and metabolite profiling

Walnut meal (WM), a protein-rich byproduct of oil extraction, holds great potential for conversion into value-added food products but remains underutilized. In this study, a two-stage fermentation process involving Aspergillus oryzae inoculation for koji preparation followed by natural brine fermentation was developed to produce a peptide-enriched walnut meal paste (WMP). Physicochemical analysis showed significant increases in total acidity (0.97 g/100 g), amino acid nitrogen (0.59 g/100 g), and reducing sugars (3.83 g/100 g) after fermentation. 16S rRNA sequencing revealed microbial succession from Proteobacteria to Firmicutes, with Bacillus, Lysinibacillus, and Enterococcus emerging as dominant genera. Redundancy analysis confirmed strong correlations between these bacteria and key physicochemical parameters. Untargeted metabolomics identified 410 differential metabolites, including 137 significantly upregulated peptides, which accounted for 48.4 % of all upregulated compounds. Many of these peptides exhibited known bioactivities such as inhibition of angiotensin-converting enzyme and dipeptidyl peptidase IV, antioxidant capacity, and kokumi flavor enhancement. KEGG pathway enrichment highlighted the activation of amino acid metabolism, particularly phenylalanine, tyrosine, and tryptophan pathways. Overall, this study provides a promising strategy for the high-value utilization of WM through microbial fermentation and offers insights into the mechanisms underlying peptide accumulation and functional enhancement in fermented plant-based foods.