【作者】 王敏；

【导师】 申烨华； 李聪；

【作者基本信息】 西北大学 ， 分析化学， 2020， 硕士

【摘要】 抗氧化肽是由2-20个氨基酸组成的活性多肽序列,对清除机体自由基、维持氧化还原动态平衡、防止组织损伤、衰老以及预防心脏病、肿瘤等疾病的产生具有至关重要的作用。油用牡丹籽粕（Po SM）作为榨油后的副产物含有丰富的粗蛋白和膳食纤维,可以作为植物蛋白和抗氧化肽的天然来源。本论文以Po SM为原料,对其进行一般成分和营养成分测定;对油用牡丹籽粕蛋白（Po SMP）和多肽进行制备、功能性质进行研究,成功鉴定出五种新型抗氧化多肽序列;最后,运用量子化学计算对鉴定出的新型多肽进行构效关系研究。研究结果为油用牡丹副产物的利用及新型抗氧化肽的开发提供了依据。具体内容如下:1. 油用牡丹籽粕一般成分和营养成分研究。对Po SM的一般成分、氨基酸、微量元素以及维生素等进行测定。研究结果表明,Po SM中蛋白质的含量为20.2%,天冬氨酸（Asp）和谷氨酸（Glu）的含量分别为1.83 g/100g和4.85 g/100g,其中必需氨基酸的含量为33.7%。此外,微量元素磷P、钾K的含量分别为5.11×10³mg/kg、4.28×10³mg/kg。Po SM富含蛋白质、必需氨基酸及微量元素等。Po SM可作为蛋白和抗氧化肽的优质来源。2. 蛋白质和酶解产物的制备及功能性质研究。采用碱溶酸沉法提取Po SMP,考察料液比（g/m L）、提取时间（min）、p H和提取温度（℃）对蛋白提取率的影响,并采用四因素三水平响应面法进行优化。结果表明在p H=10,52℃,76.92 min,1:14.3 g/m L时Po SMP的提取率最高为55.17%。以水解度和体外抗氧化性为指标,在五种水解酶中,碱性蛋白酶具有最高的水解度和较好的ABTS、OH自由基清除活性。理化性质研究表明,Po SMP相对分子质量主要分布在23k Da和34 k Da附近且23 k Da处的蛋白表现出较强的耐消化性;当p H分别为4.5和3.7时Po SMP和AHs（碱性蛋白酶酶解产物）的溶解度最低,在中性条件下两者均表现出良好的溶解度;Po SMP、AHs的变性温度分别为67℃和83℃。Po SMP具有耐高温、强酸和弱碱性,AHs则具耐高温、强酸和强碱的特性,且两者均没有明显的细胞毒性。3. 抗氧化肽的纯化与鉴定研究。依次选取超滤（10、5、1 k Da）、强阴离子交换色谱(Hi Trip^{T M} Q HP)、反相高效液相色谱（RP-HPLC）对AHs进行纯化富集,通过Nano LC-MS/MS技术鉴定出五种新型的抗氧化多肽,多肽序列分别为FSAP（Phe-Ser-Ala-Pro）,PVETVR（Pro-Val-Glu-Thr-Val-Arg）,QEPLLR（Gln-Glu-Pro-Leu-Leu-Arg）,EAAY（Glu-Ala-Ala-Tyr）和VLRPPLS（Val-Leu-Arg-Pro-Pro-Leu-Ser）。体外抗氧化性实验表明,EAAY多肽序列的抗氧化活性最高,浓度为0.5 mg/m L时对ABTS和OH自由基清除活性分别为98.5±1.1%和61.9±1.3%。4. 量子化学计算对抗氧化多肽的构效关系研究。实验以量子化学计算为基础,对从PEP-FOLD网站上获取五种抗氧化肽的空间分布,采用半经验法PM6和密度泛函理论（DFT）B3LYP/6-31G*基组进行构象优化后,对五种抗氧化肽的前线分子轨道能级和活性位点进行研究。计算结果表明,HOMO（最高占据分子轨道）与LUMO（最低分子未占据轨道）的能隙差值与体外抗氧化自由基活性具有一定程度的吻合,该值可以作为理论评估抗氧化活性的指标之一。此外,自然键轨道（NBO）结果表明与自由基清除相关的氢原子转移的位点主要分布在末端氨基、精氨酸的胍基以及酪氨酸的酚羟基结构上。更多还原

【Abstract】 Antioxidant peptides are active peptide sequences composed of 2-20 amino acids.They are momentous for scavenging free radicals,maintaining redox homeostasis,preventing tissue damage,aging,and preventing heart disease,tumors,and other diseases.Paeonia ostii seed meal(Po SM)as a by-product after oil extraction is rich in crude protein and dietary fiber,and can be used as a natural source of vegetable protein and antioxidant peptides.In this paper,Po SM is used as a raw material to determine the general and nutrient components;the preparation and functional properties of oil peony seed meal protein(Po SMP)and peptides are studied,and five novel antioxidant peptide sequences are successfully identified.Finally,quantum chemical calculations were operated to research the structure-activity relationship of the identified novel peptides.The results provided a basis for the utilization of Paeonia ostii by-products and the development of new antioxidant peptides.The details are as follows:1. Study on the general and nutritional components of Paeonia ostii seed meal.The general components,amino acids,trace elements,and vitamins of Po SM were measured.The results show that the content of protein in Po SM is 20.2%,the content of aspartic acid(Asp)and glutamic acid(Glu)are 1.83 g/100g and 4.85 g/100g,respectively,and the content of essential amino acids is 33.7%.In addition,the contents of trace elements phosphorus P and potassium K were 5.11×10~3 mg/kg and4.28×10~3 mg/kg,respectively.Po SM is rich in protein,essential amino acids and trace elements.Po SM can be operated as an excellent source of protein and antioxidant peptides.2. Study on the Preparation and Functional Properties of Proteins and Enzymatic Products.In the experiment,Po SMP was extracted by alkali-soluble acid precipitation method.The effects of material-liquid ratio(g/m L),extraction time(min),p H,and extraction temperature(℃)on protein extraction rate were investigated.The results showed that the maximum extraction rate of Po SMP was 55.17%at p H=10,52℃,76.92 min,and 1:14.3 g/m L.Taking the degree of hydrolysis and in vitro oxidation resistance as indicators,among the five hydrolases,alkaline protease has the highest degree of hydrolysis and good ABTS and OH radical scavenging activity.Physicochemical properties show that Po SMP is mainly distributed near 23 k Da and34 k Da and the protein at 23 k Da sho ws strong digestibility;Po SMP and AHs have the lowest solubility at p H 4.5 and 3.7,respectively,under neutral conditions Both showed good solubility;the denaturation temperatures of Po SMP and AHs were67℃ and 83℃,respectively.Po SMP is resistant to high temperature,strong acid and weak alkali,while AHs is resistant to high temperature,strong acid and strong alkali,and neither of them has obvious cytotoxicity.3. Purification and identification of antioxidant peptides.Ultrafiltration,strong anion exchange chromatography,and re versed-phase high-performance liquid chromatography were sequentially used to purify and enrich AHs.Five ne w antioxidant peptides were identified by Nano LC-MS/MS technology.The peptide sequences were FSAP(Phe-Ser-Ala-Pro),PVETVR(Pro-Val-Glu-Thr-Val-Arg),QEPLLR(Gln-Glu-Pro-Leu-Leu-Arg),EAAY(Glu-Ala-Ala-Tyr)and VLRPPLS(Val-Leu-Arg-Pro-Pro-Leu-Ser).In vitro antioxidant experiments showed that the EAAY peptide sequence had the highest antioxidant activity,with ABTS and OH radical scavenging activities of 98.5±1.1%and 61.9±1.3%at 0.5 mg/m L,respectively.4. Study on Structure-activity Relationship of Antioxidant Peptides by Quantum Chemistry Calculations.The experiment is based on quantum chemical calculations.After the spatial distribution of five antioxidant peptides obtained from PEP-FOLD,the semi-empirical method PM6 and the density functional theory(DFT)B3LYP/6-31G*basis set are used for conformation optimization The orbital energy levels and active sites of the five antioxidant peptides were studied.The calculation results indicate that the difference in energy gap between HOMO(highest occupied molecular orbital)and LUMO(lowest molecular unoccupied orbital)has a certain degree of agreement with in vitro antioxidant free radical activity,and this value can be operated as one of the indicators for theoretical evaluation of antioxidant activity.In addition,the natural bond orbital(NBO)results indicate that the sites of hydrogen atom transfer related to free radical scavenging are mainly distributed on the terminal amino group,the guanidine group of arginine,and the phenolic hydroxyl structure of tyrosine.更多还原