松茸蛋白抗氧化肽制备及构效关系研究

Research on preparation and structure-activity relationship of Tricholoma matsutake protein antioxidant peptide

目的制备松茸(Tricholoma matsutake)蛋白抗氧化肽,并探究其构效关系。方法利用响应面试验优化松茸蛋白提取工艺,并进行氨基酸分析,质谱鉴定超滤后的松茸蛋白模拟胃肠消化产物,固相合成两条肽评分最高的肽段,通过化学方法和H2O2诱导建立氧化损伤HepaRG细胞模型,评价松茸多肽抗氧化活性,同时结合红外光谱和圆二光谱分析其构效关系。结果松茸蛋白提取较佳工艺参数为提取温度48℃、提取时间120 min、pH 10,此条件下的松茸蛋白提取率高达(72.56±0.45)%;松茸蛋白氨基酸种类齐全,必需氨基酸占总量氨基酸的27.65%;SHSFFLP的1,1-二苯基苦基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐[2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt,ABTS]阳离子自由基清除率显著高于SFPPIWQ(P<0.05),SHSFFLP和SFPPIWQ能显著降低受氧化损伤HepaRG细胞中活性氧和丙二醛含量,提升细胞内超氧化物歧化酶活力。结论本研究采用优化的松茸蛋白提取工艺筛选出两条抗氧化肽HSFFLP和SFPPIWQ,SHSFFLP抗氧化能力强于SFPPIWQ可能与二级结构β折叠的相对含量较高有关。

Objective To prepare matsutake(Tricholoma matsutake)antioxidant peptides and explore thier structure-activity relationship.Methods Matsutake protein extraction was optimized by response surface and amino acids content was analyzed,then 2 peptides were synthesized after mass spectrum identification on matsutake protein simulated gastrointestinal digestion product with ultrafitration.Antioxidant activity of 2 peptides were evaluated by chemical method and oxidative damaged cell model induced by H2O2.And structure-activity relationship was analysed by infrared spectroscopy and circular binary spectroscopy.Results The optimized parameter of matsutake protein extraction technology was extraction temperature 48℃,extraction time 120 min,pH 10.Under these conditions,the extration rate of matsutake protein was(72.56±0.45)%.Matsutake protein had a variety of amino acids and essential amino acid content was 27.65%.1,1-diphenyl-2-picrylhydrazyl(DPPH)free radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt(ABTS)cationic free radical scavenging rates of SHSFFLP were better than SFPPIWQ significantly(P<0.05).Both SHSFFLP and SFPPIWQ could reduce reactive oxygen species and malondialdehyde content and increase superoxide dismutase activity in oxidative damaged HepaRG cells.Conclusion In this study,2 antioxidant peptides,HSFFLP and SFPPIWQ,have been screened by optimized matsutake protein extraction technology.The antioxidant capacity of SHSFFLP was stronger than SFPPIWQ,which may be related to the higher relative content of secondary structureβ-pleated sheet.