Multi-omics analysis of microbial succession and metabolite dynamics in cold-stored walnut kernels

Fresh walnut kernels are highly susceptible to microbial contamination and quality deterioration during cold storage due to their high moisture content and active physiological metabolism, posing challenges to storage safety. This study systematically investigated the microbial community succession, metabolite dynamics, and quality changes of dehusked fresh walnut kernels during 90 days of cold storage at 4 °C. The results showed that the bacterial community was dominated by Proteobacteria (83.35 %-87.13 %) throughout storage, with the initially dominant Paucibacter showing a slight decrease in relative abundance and cold-tolerant Pseudomonas exhibiting a rapid increase. Meanwhile, cold-tolerant saprophytes such as Fusarium and Bipolaris in the fungal community were enriched. Potential pathogenic bacteria (e.g., Staphylococcus aureus, Vibrio parahaemolyticus) were detected, and the contents of FB2 and OTA increased with storage time. A total of 915 metabolites were identified, among which 473 were differential expressed (DEMs). DEMs were mainly enriched in the glycerophospholipid metabolism (ko00561) and glycolysis (ko00010) pathways, indicating that microorganisms maintained growth by decomposing lipids and carbohydrates in the host. The fatty acid composition fluctuated dynamically, especially in linolenic acid (C18:3), which was synergistically regulated by catalytic enzymes (e.g., Lip, LOX) and microorganisms (e.g., Fusarium). Microbe-metabolite correlation analysis confirmed that Pseudomonas, Agathobacter, and other genera had correlations with key metabolites (e.g., azelaic acid, linolenic acid), collectively driving quality changes in fresh walnut kernels. This study is the first to reveal the "microorganism-enzyme-metabolite" cascade regulatory network in fresh walnut kernels during cold storage via multi-omics, providing a theoretical basis for the development of targeted preservation technologies and food safety risk control for fresh walnuts.