Integrated Assessment of the Functional Activity of the Gut Environment and Metabolic Profiles in Rats Fed an Insect-Based (Tenebrio molitor) Diet

Edible insects are increasingly proposed as sustainable protein sources in animal nutrition, yet their effects on gut microbial function, intestinal safety, and systemic metabolism require comprehensive evaluation. This study investigated the impact of a diet containing 35% Tenebrio molitor meal on the functional activity of gut environment and host metabolic profiles in rats, in comparison with diets containing chicken hydrolysate or a standard laboratory diet. Female Wistar rats were fed the experimental diets for 60 days, after which fecal short-chain fatty acids (SCFAs), microbial enzyme activity, fecal water genotoxicity, and liver and spleen metabolomes were assessed. Rats fed the T. molitor diet exhibited significantly lower fecal concentrations of major SCFAs, including acetate, butyrate, valerate, and branched-chain fatty acids (BCFAs), indicating altered colonic fermentation. Both β-glucosidase and β-glucuronidase activities differed significantly between groups and were highest in the T. molitor group. β-Glucuronidase activity also decreased over time, whereas no significant group vs. time interaction was observed. Fecal water genotoxicity decreased markedly in the T. molitor group over time. Untargeted UHPLC–HRMS metabolomics revealed pronounced diet-dependent differences in metabolite profiles. In the liver, the insect-based diet was associated with the enrichment of glycerophospholipid metabolism, primary bile acid biosynthesis, and pyrimidine metabolism pathways, as well as lower abundance of metabolites assigned to riboflavin- and phenylalanine-related pathways. In the spleen, diet-associated differences involved metabolites assigned to lipid-related pathways, including glycerophospholipid, linoleic acid, and steroid-related metabolism, as well as nucleotide-associated pathways. These findings support further evaluation of complete insect-based formulations and highlight specific metabolic pathways that should be considered when evaluating the safety and physiological implications of insect-based feeds.