混菌发酵茶籽抗氧化肽的分离纯化及其功能活性研究

Isolation and purification of antioxidant peptide from tea seed by mixed fermentation and its functional properties

为促进茶叶籽资源的深度开发利用,采用不同截留分子质量的超滤膜对混菌发酵茶籽多肽产物进行分级分离,比较茶籽多肽的抗氧化活性与其分子质量的对应关系;利用凝胶过滤色谱技术对茶籽多肽逐级纯化,获得特征性茶籽抗氧化肽,对其二级结构组成及相对含量进行分析,并考察其热稳定性和抗消化稳定性;以H_(2)O_(2)诱导小鼠胚胎成纤维细胞(MEF细胞)建立氧化损伤模型,评价茶籽抗氧化肽的细胞氧化损伤保护功能。结果表明:经超滤分级后,茶籽多肽的抗氧化活性与其分子质量呈负相关,分子质量小于1 kDa的TSP4组分具有最高的自由基清除能力;TSP4分子质量分布范围在90~849 Da之间,凝胶过滤色谱纯化得到的TSP4-b亚组分平均分子质量为446 Da,其二级结构中的β-折叠的相对含量从未发酵茶叶籽的16.59%上升至44.43%,α-螺旋则由未发酵茶叶籽的37.61%降至17.57%;TSP4-b经20~60℃热处理以及模拟胃肠消化后,自由基相对清除率仍可分别保持在90%及80%以上,具备良好的热稳定性及抗消化能力;中(0.5 mg/mL)、高(5.0 mg/mL)剂量的TSP4-b的介入可使H_(2)O_(2)诱导的MEF细胞存活率分别达到73.8%、82.4%。综上,所分离的茶籽抗氧化肽具有较强的抗氧化活性,对H_(2)O_(2)诱导的细胞损伤具有保护作用,在医药、保健食品等领域具有良好的发展潜力。

To promote the in-depth development and utilization of tea seed resources,tea seed peptides(TSP)from mixed fermentation were classified by ultrafiltration membranes with different molecular weight cut off.Then the corresponding relationship between antioxidant activity and molecular weight of tea seed peptides was compared.Gel filtration chromatography techniques was used to sequentially purify the TSP to obtain the characterized antioxidant peptide.The composition and content of secondary structure,thermal stability and anti-digestion stability of the antioxidant peptide were studied.The oxidative damage model was established with H_(2)O_(2) induced mouse embryonic fibroblasts(MEF)to evaluate the cellular oxidative damage protection function of tea seed antioxidant peptide.The results showed that the antioxidant activity of TSP was negatively correlated with their molecular weight and TSP4 with molecular weight less than 1 kDa exhibited the highest radical scavenging capacity.The molecular weight distribution of TSP4 ranged from 90 Da to 849 Da.The average molecular weight of the TSP4-b subcomponent purified by gel filtration chromatography was 446 Da,and theβ-fold in the secondary structure increased from 16.59% of no fermented tea seed to 44.43%,theα-helix decreased from 37.61% of no fermented tea seed to 17.57%.The relative radical scavenging rate of TSP4-b remained above 90% and 80%,respectively after heat treatment at 20-60℃ and simulated gastrointestinal digestion,which showed the excellent thermal stability and anti-digestion ability.After the addition of TSP4-b with medium doses(0.5 mg/mL)and high doses(5.0 mg/mL),the H_(2)O_(2)-induced MEF survival rate reached 73.8% and 82.4%,respectively.In conclusion,the isolated antioxidant peptide from tea seed has strong antioxidant activity and protective effect against H_(2)O_(2)-induced cellular damage,and has good potential for development in the fields of medicine and health food.