The goal of this study was to determine the impact of industrial processes on the digestion of six milk protein matrices using the harmonized INFOGEST in vitro static digestion protocol. First, this method was optimized to simple protein matrices using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) to compare the intestinal protein hydrolysis obtained with increasing quantities of pancreatin. Similar results were achieved with the originally required pancreatin amount (trypsin activity of 100 U.mL⁻¹) and with a quantity of pancreatin equivalent to a trypsin activity of 27.3 U.mL⁻¹, which was thus used to perform the in vitro digestion of the milk matrices. Molecular weight profiles, peptide heterogeneity from LC-MS/MS data, calcium, free amino acid, and peptide concentrations were determined in the gastric and intestinal phases to compare the milk protein digests. Results showed that the industrial process affected not only the protein distribution of the matrices but also most likely the protein structures. Indeed, differences arose in terms of peptide populations generated when the caseins were reticulated or when their calcium concentrations were reduced.

Objective: Dietary intakes must cover protein and essential amino acid (EAA) requirements. For this purpose, different methods have been developed such as the nitrogen balance method, factorial method, or AA tracer studies. However, these methods are either invasive or imprecise, and the Food and Agriculture Organization of the United Nations (FAO, 2013) recommends new methods and, in particular, metabolomics. The aim of this study is to determine total protein/EAA requirement in the plasma and urine of growing rats. Methods: 36 weanling rats were fed with diets containing 3, 5, 8, 12, 15, and 20% protein for 3 weeks. During experimentation, urine was collected using metabolic cages, and blood from the portal vein and vena was taken at the end of the experiment. Metabolomics analyses were performed using LC-MS, and the data were analyzed with a multivariate analysis model, partial least Squares (PLS) regression, and independent component-discriminant analysis (ICDA). Each discriminant metabolite identified by PLS or ICDA was tested by one-way ANOVA to evaluate the effect of diet. Results: PLS and ICDA allowed us to identify discriminating metabolites between different diet groups. Protein deficiency led to an increase in the AA catabolism enzyme systems inducing the production of breakdown metabolites in the plasma and urine. Conclusion: These results indicate that metabolites are specific for the state of EAA deficiency and sufficiency. Some types of biomarkers such as AA degradation metabolites appear to be specific candidates for protein/EAA requirement.

Long-term adherence to plant-based diets can modify gut bacterial microbiota composition and metabolite profiles, which may be particularly relevant for postmenopausal women who frequently adopt such diets and experience age-related changes in nutrient absorption and metabolism. Fermented foods, commonly consumed in vegetarian diets, enhance dietary diversity and nutritional quality. This study compared gut bacterial microbiota and fecal metabolomes between vegetarians (VGs) and omnivores (OMs) and evaluated the contribution of fermented food intake. Thirty-two healthy postmenopausal Thai women (>55 years; 16 VGs, 16 OMs) were enrolled. Gut bacterial microbiota and fecal metabolites were analyzed using 16S rRNA metagenomic and untargeted ¹H-NMR metabolomics. The five most frequently consumed fermented foods were microbiologically characterized. Fermented food consumption was found to be significantly different between groups. OM participants reported infrequent consumption (60% of weekly meals). VG participants exhibited enrichment of Prevotella, Faecalibacterium, and Blautia, while OM participants showed higher abundances of Bacteroides and Escherichia–Shigella. LEfSe identified Weissella as a bacterial taxon associated with the VG group. Functional prediction and metabolomic analyses indicated enhanced carbohydrate fermentation and increased short-chain fatty acid (SCFA) production in VGs, whereas OM profiles reflected greater protein catabolism. Fermented foods consumed by VGs shared microbial biomarkers with the VG gut bacterial microbiota and were rich in SCFAs and essential amino acids, supporting their potential role as microbial and metabolic contributors within the gut ecosystem and nutritional adequacy in postmenopausal vegetarians.