[关键词]
[摘要]
为探究不同蛋白酶预酶解对玛咖蛋白胃肠消化程度及消化产物免疫调节活性的影响,该研究选用中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、菠萝蛋白酶以及复合蛋白酶分别对玛咖蛋白进行预酶解,再经模拟胃肠消化得到五组消化产物。利用TNBS法、福林酚法等测定消化产物的得率、水解度、可溶性肽含量及游离氨基酸含量,再以小鼠巨噬细胞RAW 264.7为模型探究消化产物的免疫调节活性。结果表明,经五种蛋白酶的预酶解,玛咖蛋白消化产物的水解度、可溶性肽含量和游离氨基酸含量分别提高了35%、5%~16%及37%~92%,且消化产物促进巨噬细胞分泌TNF-α的作用无明显降低。经菠萝蛋白酶预酶解得到的消化产物的免疫调节活性最高,该消化产物含有15 154条分子量小于3 000 u的肽段;在经活性评分及分子对接预测的与TLR2和TLR4受体存在相互作用最强的10个肽段中,NPYPFFGFSI的免疫调节活性最高;HOMO分析结果表明该肽的活性位点位于酪氨酸上。上述结果表明预酶解可在不影响玛咖蛋白免疫调节活性的条件下提高玛咖蛋白的可消化性。
[Key word]
[Abstract]
The effects of pre-hydrolysis using different proteases on the degree of gastrointestinal digestion of maca proteins and the immunomodulatory activity of their digestion products were explored. Dispase, alcalase, papain, bromelaine, and protamex were selected to pre-hydrolyze maca proteins, and the final five digestion products after subsequent in vitro simulated gastrointestinal digestion were obtained. The yield, degree of hydrolysis, soluble peptide content, and free amino acid content of the digested products were determined using the TNBS method and Folin-Ciocâlteu assay. Mouse macrophages RAW 264.7 were used as the model to explore the immunomodulatory activity of the digestion products. Following pre-hydrolysis with the five proteases, the degree of hydrolysis, soluble peptide content, and free amino acids content of the maca protein digestion products were augmented by respectively 35%, 5%~16% and 37%~92%, while no significant inhibitory effect of their promotion on the secretion of TNF-α by macrophages was observed. The digestion products obtained via bromelain pre-hydrolysis showed the highest immunomodulatory activity and comprised 15 154 peptides, with a molecular weight of <3 000 u. Among the top 10 peptides with the strongest interactions with TLR2 and TLR4 predicted based on activity scores and molecular docking, NPYPFFGFSI exhibited the highest immunomodulatory activity. HOMO analysis revealed that the active site of this peptide is located at tyrosine. Therefore, the digestibility of maca proteins can be improved via pre-hydrolysis without compromising their immunomodulatory activity.
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[基金项目]
中国博士后科学项目基金(2019M662931);广州市科学技术局基础与应用基础研究项目(202102020773)