Modulation of cecal microbiota and fecal metabolism in mice by walnut protein

Walnut meal is a by-product of walnut oil pressing, in which the protein content is more than 40%, which is an excellent food raw material, but at present, it is basically used as animal feed or discarded, which results in a great waste of resources, and its modulating effect on the intestinal microbiota is not clear. In this study, we used supercritically extracted walnut meal as a raw material, prepared walnut meal isolate protein (WP) by alkaline extraction and acid precipitation, and systematically analyzed its structure by Fourier infrared spectroscopy (FTIR), Raman spectroscopy (Raman), and scanning electron microscopy (SEM); meanwhile, we explored the effects of WP on the cecal bacterial flora and fecal metabolites of mice by microbiological and metabolomic techniques. The results showed that the protein content of WP prepared using alkaline extraction and acid precipitation was as high as 83.7%, in which arginine and glutamic acid were abundant, and it has the potential to be used as a raw material for weight-loss meal replacement food; FTIR and Raman analyses showed that the absorption peaks of WP's characteristic functional groups were obvious, and that the content of the α-helix and β-fold in the secondary structure was greater than 30%, which indicated that it was structurally stable; differential scanning calorimetry (DSC) and SEM analyses showed that WP is a typical spherical particle, its denaturation temperature is 73.6 °C, and it has good thermal stability. Supplementation of WP significantly altered the composition of the intestinal flora in mice, with an increase in beneficial bacteria and a decrease in harmful bacteria; the strongest modulation of the intestinal flora was achieved by altering the composition of the intestinal flora and by increasing the number of Akkermansia (p < 0.01), which consequently affects the function of the microbiota. Based on LC-MS metabolomic results, we identified a total of 87 WP-regulated metabolites, mainly enriched in the bile secretion pathway, which had the highest relevance, followed by benzoxazine biosynthesis. In summary, walnut protein is an important plant protein and has a positive impact on intestinal health, which may provide new ideas for the development of functional foods.