山茶籽多肽制备及其抗老修护功效

Preparation of camellia seed polypeptide and its anti-aging and repairing effect

摘要:

以超临界CO2萃取脱油后山茶籽为原料，通过碱提酸沉工艺对山茶籽蛋白进行提取，然后使用蛋白酶进行酶解制备山茶籽多肽液，采用单因素实验优化山茶籽蛋白提取和酶解工艺，通过液相色谱-质谱联用仪对山茶籽多肽液中多肽及氨基酸组成进行分析，并采用体外测试探究了山茶籽多肽液在化妆品中抗老（抗氧化、抗糖化、抗皱）和修护（划痕）的能力。结果表明，优化的山茶籽蛋白提取工艺条件为：料液比（g∶mL）1∶30、碱提pH＝9.50、提取温度60 ℃、提取时间2 h、酸沉pH＝3.50，在该条件下，山茶籽蛋白提取率为5.05%；优化的山茶籽蛋白酶解的优化条件为：以木瓜蛋白酶为酶解酶，加酶量为3000 U/g、酶解时间2 h、底物质量分数2%，此条件下，山茶籽蛋白酶解物水解率为31.56%。山茶籽多肽液中总肽含量为0.47908%，主要为多肽长度4~8肽的短肽。其总数均相对分子质量（简称分子量）为850.56 Da，相对分子量低于2000 Da的多肽占比为99.56%。山茶籽多肽液含有除异亮氨酸（Ile）外的其余7种成年人必需氨基酸，占总氨基酸含量的40.55%。质量分数8%的山茶籽多肽液对1,1-二苯基-2-三硝基苯肼（DPPH）自由基清除率达到71.76%±0.34%；质量分数1%的山茶籽多肽液对晚期糖化终产物（AGEs）的抑制率为32.63%±0.78%；质量分数0.25%的山茶籽多肽液对HSF细胞中MMP-1的相对表达率为56.96%±0.82%，Ⅲ型胶原蛋白相对表达率为120.45%±0.24%，Ⅳ型胶原蛋白相对表达率为113.24%±0.17%，Ⅴ型胶原蛋白相对表达率为138.33%±2.72%；质量分数0.05%的山茶籽多肽液内，斑马鱼col1a1a基因相对表达率为122.64%±2.07%；质量分数0.4%的山茶籽多肽液内人永生化角质形成细胞（HaCaT）细胞迁移率为40.95%±0.36%。

Abstract:

The Camellia seed protein was extracted by alkali extraction and acid precipitation process with supercritical CO2 extraction and deoiled Camellia seed as raw material, and then the Camellia seed polypeptide solution was prepared by enzymatic hydrolysis with protease. The extraction and enzymatic hydrolysis process of Camellia seed protein was optimized by single factor experiment. The composition of polypeptide and amino acid in camellia seed polypeptide solution was analyzed by liquid chromatography-mass spectrometry. The ability of Camellia seed polypeptide solution to resist aging (anti-oxidation, anti-glycation, anti-wrinkle) and repair (scratch) in cosmetics was investigated by in vitro test. The results showed that the optimum extraction conditions of Camellia seed protein were as follows : solid-liquid ratio (g : mL) 1:30, alkali extraction pH=9.50, extraction temperature 60 °C, extraction time 2 h, acid precipitation pH=3.50. Under these conditions, the extraction rate of Camellia seed protein was 5.05%. The optimal conditions for the enzymatic hydrolysis of Camellia seed protein were as follows : papain was used as the enzyme, the amount of enzyme added was 3000 U/g, the enzymatic hydrolysis time was 2h, and the substrate mass fraction was 2%. Under these conditions, the hydrolysis rate of Camellia seed protein hydrolysate was 31.56%.The total peptide content in the Camellia seed polypeptide solution was 0.47908%, which was mainly a short peptide with a peptide length of 4-8 peptides. The total average molecular weight (molecular weight) was 850.56 Da, and the proportion of polypeptides with relative molecular weight less than 2000 Da was 99.56%. The polypeptide solution of Camellia oleifera seeds contained 7 essential amino acids for adults except isoleucine (Ile), accounting for 40.55% of the total amino acid content. The scavenging rate of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was 71.76%±0.34% when the concentration of Camellia seed polypeptide was 8%.The inhibition rate of 1% Camellia seed polypeptide solution on advanced glycation end products (AGEs) was 32.63%±0.78%. The relative expression rate of MMP-1 in HSF cells was 56.96%±0.82%, the relative expression rate of type III collagen was 120.45%±0.24%, the relative expression rate of type IV collagen was 113.24%±0.17%, and the relative expression rate of type V collagen was 138.33%±2.72%. The relative expression rate of zebrafish col1 a1 a gene was 122.64%±2.07% in 0.05% Camellia seed polypeptide solution. The migration rate of human immortalized keratinocyte (HaCaT) cells in 0.4% Camellia seed peptide solution was 40.95%±0.36%.