Analysis of Absorption Characteristics of Walnut Peptides by Everted Rat Sacs and Identification of Antioxidant Peptides

This study investigated the absorption characteristics of <3 kDa walnut protein peptides simulating gastrointestinal digestion products using the everted rat sacs method. The optimal conditions for walnut peptide absorption in the intestine were selected based on antioxidant capacity (DPPH free radical clearance rate, ABTS⁺ free radical clearance rate, Fe²⁺ chelation rate) and active peptide absorption rate. The absorbed peptide components were screened and identified using simulated molecular docking technology and Nano-HPLC-MS/MS technology to obtain new walnut derived peptides that could be fully absorbed and had antioxidant activity. The results showed that when the absorption concentration of walnut peptide digestion product was 6 mg/mL, and the absorption time was 2 h, its antioxidant activity was the highest and the absorption rate in the small intestine was the highest. Further identification by Nano-HPLC-MS/MS showed that, the 0.5~1 kDa active peptide was the most numerous, with the C-terminal/N-terminal amino acids mainly concentrated in Pro, Thr, and Leu. Thirty-three active peptides were fully absorbed and crossed the intestinal barrier. By combining molecular docking technology with DPP-IV, the three active peptides NLRFPL, NPDDEFRPQ, and KGHLFPN with the lowest binding energies of −8.8, −8.6, and −8.6 kal/mol, respectively were obtained. Among these, KGHLFPN displayed the strongest antioxidant capacity. In summary, simulating gastrointestinal digestion with <3 kDa walnut peptides could release the active peptide KGHLFPN, which had the highest antioxidant activity and could be absorbed by the small intestine. These findings would provide new ideas for studies exploring the intestinal absorption characteristics of exogenous peptides, and the screening and identification of antioxidant peptides.