A Novel High-Throughput Anaerobic Culture Method to Evaluate Food Health Effects via Gut Microbiota Modulation

Abstract

As health demands grow, nutritional evaluation now extends beyond nutrient supply, and health-effect–based methods are gradually gaining traction. However, assessing food impacts on gut microbiota remains complex and costly. This study developed a cost-effective in vitro method for evaluating food effects on individual gut microbiota, integrating 96-well microplates, multilayer liquid sealing, and high-throughput multiomics (16S rRNA sequencing + untargeted metabolomics) to enable large-scale anaerobic culture and dynamic growth monitoring under aerobic conditions. Using this method, the fermented products of Pichia fermentans were shown to significantly boost Bifidobacterium abundance (4.6-fold to 5.6-fold vs. control) by Day 4, though microbial diversity decreased. Testing raw (RA) versus cooked (CO) pecan kernels, RA significantly promoted health-associated genera: Blautia, which showed progressive enrichment (rising 17-fold from Day 2 to Day 4 in RA) and reached 193.8 times the control level (5.1%, p = 0.0127), with potential anti-inflammatory implications; Fusicatenibacter, surging to 1274.3 times the control (3.3%, p = 0.001) and 5.6 times higher than CO (p = 0.0332), suggesting potential cognitive benefits; and Bacteroides, whose reduction was significantly attenuated in RA compared to control (p = 0.0002), potentially supporting bile acid metabolism relevant to ulcerative colitis management. In contrast, CO favored opportunistic pathogens: Raoultella was exclusively elevated in CO relative to both control and RA (p < 0.05); Klebsiella abundance was notably higher in CO than in RA (p < 0.05), though increased in both groups versus control. Untargeted metabolomics indicated RA upregulated C5-branched dibasic acid metabolism and secondary bile acid biosynthesis, accumulating metabolites (e.g., CLA, 7-KDA) associated with these pathways. This method aids in optimizing food processing and guiding precision nutrition strategies.