Soy protein remnants digested by gastro-duodenal proteases can alter microbial interactions and intestinal cholesterol absorption

Background: Although the food quality of soy protein is known to be as good as that of animal proteins, some soybean proteins are not susceptible to digestion and remain undigested in the intestine. We hypothesized that digestion-resistant soy proteins might interact with the intestinal membrane, microbes, and metabolites, and change the intestinal physiology or the profile of the gut microbiome. Objective: To identify the Protease-Resistant Soy Proteins (PRSPs) and their interaction with intestinal membrane proteins by MS, and to assess the functions of PRSPs in the small intestine. Methods: Soy proteins were sequentially digested with pepsin and pancreatin, and the PRSPs were identified by SDS-PAGE and MS. Intestinal cell membrane proteins interacting with PRSPs were isolated by affinity purification and photo-affinity crosslinking, and identified using MS/MS. Inhibition of cholesterol absorption to lipoprotein-depleted intestinal cells, CaCo-2, and hepatic cells, HepG2, was measured in the presence and absence of PRSPs. FITC-conjugated Gram-positive, Lactobacillus plantarum, and Gram-negative bacteria, Escherichia coli, were incubated with Ca-Co-2 cells in the presence of PRSPs to investigate the regulation of bacterial cell binding to intestinal epithelial cells by PRSPs. Results: MS/MS of PRSPs identified glycinin, β-conglycinin, trypsin inhibitors, lipoxygenase, and sucrose-binding protein. MS analysis also identified the intestinal membrane proteins bound to PR-SPs. The functions of the identified interacting proteins included ion transportation, carbohy-drate-binding, cytoskeleton formation, hydrolysis, cell-cell junction formation, and cholesterol/s-teroid-binding. In particular, apolipoprotein E, aminopeptidase N, and Niemann-Pick C1-like protein 1 are known to be involved in cholesterol absorption in the small intestine. The inhibition of cholesterol absorption by CaCo-2 and HepG2 cells by PRSPs confirmed the MS results. Binding of L. plantarum and E. coli to CaCo-2 cells was efficiently inhibited by PRSPs. Conclusion: PRSPs can interact with intestinal membrane proteins, and regulate cholesterol absorption by intestinal epithelial cell and interactions of the gut microbiome. Soy protein in the intestine acts as a nutrient, and triggers changes in intestinal functions by interacting with intestinal cells, mi-croorganisms, and nutrients. These findings will provide valuable new functional information about the effects of soy proteins on human health.