The fractions of walnut proteins with the highest anti-inflammatory activity and the effect of gastrointestinal digestion on their properties and anti-inflammatory activity were studied.Six walnut protein protease hydrolysates were investigated for their anti-inflammatory activity using the lipopolysaccharide(LPS)-induced RAW 264.7 cell model.The protease hydrolysate demonstrating the strongest anti-inflammatory activity underwent fractionation using ultrafiltration technology,and the anti-inflammatory potential of fractions with different molecular weights was investigated.An in vitro simulated gastrointestinal digestion model was established to evaluate the effects on the degree of hydrolysis,peptide content,and anti-inflammatory activity of the most active fraction.Results indicated that all six protease hydrolysates inhibited nitric oxide(NO)release from LPS-induced RAW 264.7 cells.Notably,the alkaline protease hydrolysate exhibited the highest inhibitory effect,achieving an inhibition rate of 18.33%at a dose of 800 μg/mL.Within the alkaline protease hydrolysate,the fraction with molecular weight＜1 ku demonstrated the most effective inhibition of NO release,with an inhibition rate of 25.25%at the same dose.Comparative analysis revealed that the peptide content of the＜1 ku-simulated gastrointestinal digestion group increased from 5.56 g/100 g to 10.93 g/100 g.Likewise,the degree of hydrolysis increased from 1.82%to 7.54%,with an increase in the NO release inhibition rate from 24.75%to 46.50%.These results underscore the highest anti-inflammatory activity observed in the alkaline protease hydrolysate of walnuts with molecular weight＜1 ku,which was further enhanced post-gastrointestinal digestion.This study provides foundational insights for the isolation and purification of anti-inflammatory peptides from walnuts,and offers a theoretical basis for the valorization of walnut by-products.
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