研究核桃蛋白中最具有抗炎活性的组分及胃肠消化对其性质和抗炎活性的影响。利用LPS诱导的小鼠巨噬细胞RAW 264.7模型，研究6种核桃蛋白酶解物的抗炎活性；利用超滤技术将抗炎活性最强的酶解物进行分级提取，研究不同分子量超滤组分的抗炎活性；建立体外模拟胃肠消化模型，评价体外模拟消化对活性最好组分水解度、多肽含量和抗炎活性的影响。研究发现，6种蛋白酶酶解物均可抑制LPS诱导的RAW 264.7巨噬细胞的NO释放，其中碱性蛋白酶酶解物抑制效果最好，当其剂量为800 μg/mL时NO释放抑制率为18.33%；碱性蛋白酶酶解物中，分子量<1 ku组分对NO释放抑制效果最好，当其剂量为800 μg/mL时NO释放抑制率为25.25%；与未消化组相比，<1 ku胃肠模拟消化组的多肽含量从5.56 g/100 g增加到10.93 g/100 g，水解度从1.82%增加到7.54%，NO释放抑制率从24.75%增加到46.50%。结果表明，核桃碱性蛋白酶解物中，分子量<1 ku组分具有最强的抗炎活性，经胃肠消化后其抗炎活性增加。研究结果可为核桃抗炎肽分离纯化的研究奠定基础，也为核桃副产物高值化利用提供理论依据。

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.

云南省重大科技专项计划项目（202102AE090027-2）；云南省万人计划产业技术领军人才项目（YNWR-CYJS-2020-010）；云南省省市一体化专项（202302AN360002）；云南省农业基础研究联合专项项目（202101BD070001-120）