【作者】 李莉；

【导师】 郑捷； 高建忠；

【作者基本信息】 天津科技大学 ， 食品工程（专业学位）， 2020， 硕士

【摘要】 本论文以泥鳅粘液冻干粉为原料,通过酶解技术制备泥鳅粘液抗氧化肽,利用体外化学模型评价泥鳅粘液多肽的抗氧化活性。采用三种分离手段对泥鳅粘液多肽进行分离纯化,并采用反相高效液相色谱(RP-HPLC)及基质辅助激光解析电离飞行时间质谱仪(MALDI-TOF MS)对目标肽段进行一级结构氨基酸序列的鉴定,利用红外光谱分析多肽的二级结构,研究了泥鳅粘液多肽的抗氧化活性及热稳定性。泥鳅粘液冻干粉的蛋白含量为71.48%,氨基酸含量丰富,组成合理,富含必需氨基酸、呈味氨基酸、抗氧化活性氨基酸以及支链氨基酸等多种具有营养价值的氨基酸,可用于蛋白酶解生产生物活性肽。采用胰蛋白酶、木瓜蛋白酶、碱性蛋白酶、中性蛋白酶和风味蛋白酶水解泥鳅粘液蛋白,以水解度以及抗氧化能力为指标,确定了碱性蛋白酶为泥鳅粘液蛋白制备抗氧化肽的最适蛋白酶。在此基础上,分析了温度、pH、底物浓度、酶解时间和加酶量五个因素对碱性蛋白酶酶解泥鳅粘液冻干粉的影响,并利用响应面法对酶解条件进行优化,结果表明,水解度和抗氧化能力达到最优的酶解条件为温度50℃,pH为8.5,底物浓度为8.77 mg/mL,酶解时间为5.5 h,加酶量8625 U/g蛋白,此时水解度35.14%,DPPH自由基清除率值为58.32%,与模型预测值无显著性差异(P>0.05)。采用超滤膜、凝胶过滤色谱、半制备型RP-HPLC等系列技术对泥鳅粘液多肽进行了分离纯化,所得肽段的抗氧化能力比分离前提高了3倍以上,说明经过分离纯化可显著地富集泥鳅粘液抗氧化肽。最后用MALDI-TOF MS对泥鳅粘液多肽的氨基酸序列进行了鉴定,并且对其进行氨基酸组成分析,得到肽段的氨基酸序列为SLIGRTLVVHEK,分子量 1351.59 Da。采用三种体外化学模型综合评价泥鳅粘液多肽的抗氧化活性,结果表明泥鳅粘液多肽具有较强的体外抗氧化活性,肽浓度在1 mg/mL时,DPPH自由基清除能力为44.65±0.23%、羟基自由基清除能力为34.12±0.09%,以及还原力为0.124±0.004,均低于相同质量浓度的抗坏血酸的抗氧化能力。利用FTIR技术对泥鳅粘液多肽进行了二级结构表征,红外光谱结果显示泥鳅粘液多肽的α-螺旋结构相对含量为8.78%,β-折叠结构相对含量为22.32%;β-转角结构相对含量为54.98%;无规则卷曲结构相对含量为13.93%。使用扫描电镜对泥鳅粘液多肽进行微观结构观察,使用TGA对其热稳定性分析。结果显示泥鳅粘液多肽呈现无规则的片状结构和多孔型结构。TGA结果显示泥鳅粘液多肽的失重率和降解温度分别为75.45%和293.46℃,说明泥鳅粘液多肽有较好的热稳定性。更多还原

【Abstract】 In this paper,loach mucus freeze-dried powder was used to prepare a novel antioxidant peptide from enzymatic hydrolysis of loach mucus.Three separation methods were used to isolate and purify the peptide from loach mucus.The RP-HPLC and MALDITOF MS were used to identify the amino acid sequence of the peptide.And FTIR was used to analyze the secondary structure of the peptide.Finally,it was studied that the antioxidant activity and thermal stability of the peptide from loach mucus.The protein content of loach mucus freeze-dried powder is 71.48%.The content of amino acids in loach mucus is rich and the composition is reasonable.It is abundant in essential amino acids,flavor amino acids,antioxidant amino acids and branched chain amino acids.It can be used for protease hydrolysis to produce bioactive peptides.It was used that trypsin,papain,alcalase,neutral protease and flavor protease to hydrolyze loach mucus.It was taken the degree of hydrolysis and antioxidant capacity as indicators.In addition,alcalase was determined to be the most suitable protease for preparing the antioxidant peptide from loach mucus.On this basis,it was analyzed that the effects of temperature,pH,substrate concentration,enzymatic time and enzyme addition on alcalase hydrolysis of loach mucus freeze-dried powder.The results showed that the optimal enzymolysis conditions were temperature 50 ℃,pH 8.5,substrate concentration 8.77 mg/mL,enzymatic time 5.5 h,enzyme addition 8625 U/g protein,the degree of hydrolysis was 35.14%,the DPPH radical scavenging activity was 58.32%,there was no significant difference with the model prediction value(P>0.05).The peptide from loach mucus was isolated and purified by ultrafiltration,gel filtration chromatography and prep-HPLC.The antioxidant capacity of the peptide increased by more than 3 times than that before separation,indicating that isolation and purification can significantly enrich the antioxidant peptide from loach mucus.Finally,it was identified by MALDI-TOF MS that the amino acid sequence of the peptide from loach mucus.And it was analyzed that the amino acid composition of the peptide was analyzed.The amino acid sequence of the purified peptide,an antioxidant 12-mer peptide with molecular weight of 1352.10 Da,was identified as SLIGRTLVVHEK.Three chemical models in vitro were used to evaluate the antioxidant activity of the mucopolysaccharide peptide.The results showed that the peptide had strong antioxidant activity,and the DPPH radical scavenging ability was 44.65 ± 0.23%,the hydroxyl free radical scavenging ability was 34.12 ±0.09%,and the reducing power was 0.124±0.004,which were lower than that of ascorbic acid at a concentration of 1 mg/mL.The secondary structure of the peptide was characterized by FTIR.The results showed that the relative content of α-helix structure,β-fold structure,β-corner structure and irregular curl structure was 8.78%,22.32%,54.98%and 13.93%,separately.The microstructure of the peptide from loach mucus was observed by SEM and its thermal stability was analyzed by TGA.The SEM results showed that the peptide from loach mucus presented irregular lamellar and porous structure.The TGA results showed that the weight loss rate and degradation temperature of myxopeptide were 75.45%and 293.46℃,respectively.It indicates that the peptide from loach mucus had good thermal stability.更多还原