【作者】 陈琪；

【导师】 吉日木图；

【作者基本信息】 内蒙古农业大学 ， 食品科学， 2023， 硕士

【摘要】 机体细胞内积累了过量自由基和活性氧将导致氧化应激,进而损害生物体的健康。尽管内源性的抗氧化防御系统起着重要作用,但外源性抗氧化剂,特别是膳食天然抗氧化剂,对于维持人体细胞中的氧化还原稳态非常重要。通过蛋白水解从食物蛋白质中获取的生物活性肽已被充分证实具有良好的抗氧化能力,对促进人类健康作出了一定的贡献。与化学合成的抗氧化化合物相比,食源性抗氧化肽几乎不存在副作用。越来越多的研究从动物和植物等食物蛋白中提取出多种抗氧化肽。鉴于粮食安全生产成本的挑战,近年来对来源于不可食用或非传统食物的生物活性肽的研究逐渐兴起。为充分利用资源,创造骆驼产业附加值,本研究采用酶解法,从骆驼胎盘中获取生物活性肽,并对其抗氧化活性进行探究,主要研究结果总结如下:1.骆驼胎盘基本营养成分:匀浆后的骆驼胎盘中水分、蛋白质、灰分、脂肪含量分别为88.66±0.02%、8.76±0.12%、0.64±0.11%和1.94±0.15%。2.酶解制备骆驼胎盘抗氧化肽的最优工艺条件:酶解时间3h、酶解温度57.5℃,酶添加量4770U/g,酶解p H6.3,在此条件下的实际水解度为40.21±2.51%。3.骆驼胎盘抗氧化肽的超滤分离及稳定性研究:对骆驼胎盘抗氧化肽酶解液进行超滤分离,得到抗氧化能力最好的组份为:分子量<3KDa;其对DPPH·、ABTS⁺、·OH和O₂^–的清除率分别为72.47%、71.77%、46.50%和44.62%。再对其进行稳定性研究表明:骆驼胎盘抗氧化肽在100℃,2h条件下仍具有较好的活性;在p H3～7的酸性和中性环境下具有良好的抗氧化活性,但在p H9～11的碱性环境中活性显著降低（P<0.05）;对金属离子Cu²⁺、Mg²⁺和K⁺敏感,且抗氧化活性随金属离子浓度的增大显著降低（P<0.05）;在胃-肠消化中对胃蛋白酶耐受,且能提升其抗氧化活性,但在肠消化阶段抗氧化能力显著下降。4.骆驼胎盘抗氧化肽的多肽鉴定及计算机虚拟筛选:利用LC-MS/MS从骆驼胎盘抗氧化粗肽液中共鉴定出894条生物活性肽,通过计算机虚拟筛选出评分接近“1”、水溶性良好、无毒性且不致敏以及ADMET性质综合评估最优的5条具有潜在抗氧化活性的肽段:EFW、SRW、GFDL、FFLE和WRE。分子对接分析结果显示:EFW、SRW、GFDL、FFLE和WRE与Keap1蛋白的结合能分别为-43.23kcal/mol、-46.68kcal/mol、-64.34 kcal/mol、-48.73kcal/mol和-40.12kcal/mol,均可稳定结合。Keap1蛋白与多肽相互作用表明:5条多肽均可对占据Keap1上的Nrf2结合位点产生竞争性抑制作用,保护细胞免受氧化应激的损伤。更多还原

【Abstract】 Excessive accumulation of free radicals and reactive oxygen species in the cells of the organism will lead to oxidative stress,which in turn will damage the health of the organism.Although endogenous antioxidant defense systems play an important role,exogenous antioxidants,especially dietary natural antioxidants,are important for maintaining redox homeostasis in human cells.Bioactive peptides obtained from food proteins through protein hydrolysis have been well proven to have good antioxidant capacity and have contributed to the promotion of human health.Compared to chemically synthesized antioxidant compounds,food-derived antioxidant peptides are virtually free of side effects.More and more research is being done to extract a variety of antioxidant peptides from food proteins such as animals and plants.Given the challenges of food security production costs,research on bioactive peptides derived from inedible or non-traditional foods has emerged in recent years.In order to make full use of the resources and create added value in camel industry,this study used enzymatic digestion to obtain bioactive peptides from camel placenta and investigate their antioxidant activity,and the main findings are summarized as follows:1.Basic nutrients of camel placenta:The moisture,protein,ash,fat and amino acid contents of the homogenized camel placenta were 88.66±0.02%,8.76±0.12%,ash 0.64±0.11%,and 1.94±0.15%,respectively.2.Optimal process conditions for the enzymatic preparation of camel placenta antioxidant peptides:Based on the single-factor experiment,the optimal extraction conditions were obtained by combining response surface optimization analysis:enzymatic digestion time 3h,enzymatic digestion temperature 57.5℃,enzyme addition 4770U/g,enzymatic digestion p H 6.3,and the actual hydrolysis degree under these conditions was 40.21±2.51%.3.Ultrafiltration separation and stability study of antioxidant peptides from camel placenta:The camel placenta antioxidant peptide enzymatic digest was separated by ultrafiltration,and the components with the best antioxidant capacity were obtained:molecular weight<3KDa;Its clearance of DPPH-,ABTS⁺,-OH and O₂^-was 72.47%,71.77%,46.50%and 44.62%,respectively.Further stability studies showed that the camel placenta antioxidant peptide crude still had good activity at 100℃2h;Good antioxidant activity in acidic and neutral environments from p H 3 to 7,but significantly lower activity in alkaline environments from p H 9 to 11（P<0.05）;Sensitive to metal ions Cu²⁺,Mg²⁺and K⁺and antioxidant activity significantly decreased with increasing metal ion concentration（P<0.05）;It is pepsin-tolerant in gastro-intestinal digestion and enhances its antioxidant activity,but its antioxidant capacity decreases significantly in the intestinal digestion phase.4.Peptide identification and computerized virtual screening of antioxidant peptides from camel placenta:A total of 894 bioactive peptides were identified from the antioxidant crude peptide solution of camel placenta by LC-MS/MS,and five peptides with potential antioxidant activity were identified by virtual computer screening:EFW,SRW,GFDL,FFLE and WRE,which had a peptide Rank score close to"1",good water solubility,non-toxic and non-sensitizing,and the best overall assessment of ADMET properties.Molecular docking analysis showed that EFW,SRW,GFDL,FFLE and WRE were stably bound to Keap1 protein with binding energies of-43.23kcal/mol,-46.68kcal/mol,-64.34 kcal/mol,-48.73kcal/mol and-40.12kcal/mol,respectively.Keap1 protein-peptide interactions showed that all five peptides competitively inhibited the Nrf2 binding site occupying Keap1 and protected cells from oxidative stress damage.更多还原