【作者】 苏伟；

【导师】 邱树毅；

【作者基本信息】 贵州大学 ， 应用生物技术， 2016， 博士

【摘要】 生物活性肽可作为高效、安全的生物制品,具有重要的研究价值和应用前景。鸭肫属优质蛋白资源,高值化开发利用是产业发展的方向。本论文以鸭肫蛋白为原料,通过可控酶解技术制备抗氧化肽;利用超滤、凝胶过滤色谱、反相高效液相色谱对产物进行逐级分离纯化,采用质谱技术对目标肽段进行一级氨基酸序列的鉴定;建立分子结构模型并验证抗氧化肽的定量构效关系;通过作用于细胞,分析细胞生长抑制、凋亡、生长周期以及细胞内抗氧化酶活性及酶蛋白表达,从分子机制进一步阐明鸭肫肽的抗氧化机理。本文旨为鸭肫蛋白的深度开发和应用,提升其资源高值化利用提供理论依据和技术支撑。本论文的主要研究内容和结果如下:(1)分析了木瓜蛋白酶酶解液中氨基酸含量,其总氨基酸含量高达33594.73 mg/100 g,主要抗氧化性氨基酸含量达9.54%。在单因素实验基础上,以水解度和DPPH清除率为响应值,确定鸭肫蛋白酶解的最佳工艺条件为:酶解时间4.0 h、加酶量3000 U/g、酶解温度55℃、料液比1:3。在此条件下其水解度为28.30%,DPPH清除率为75.22%。其水解度与DPPH自由基清除能力之间不存在明显的相关性。(2)探讨了鸭肫不同分子量段ZT1(<3 kDa)、ZT2(3-10 kDa)和ZT3(>10kDa)产物抗氧化活性。其自由基DPPH清除率和ORAC活性值与其分子量呈显著相关性,分子量小于3kD产物DPPH清除率77.59%,ORAC活性值885.25μmol TE/g。小于3kD鸭肫抗氧化肽清除超氧阴离子、羟基自由基、过氧化氢以及DNA损伤的保护作用具明显的量效关系,其IC50值分别为14.58、0.0163、19.80和1.93mg/mL,鸭肫抗氧化肽对过氧化氢自由基抑制作用最强,对羟自由基活性的抑制作用最弱。(3)分离及鉴定鸭肫抗氧化活性肽。采用中空纤维素膜超滤法对酶解液进行初步分离,将ORAC值活性最高的组分(小于3kDa)使用凝胶色谱和半制备RP-HPLC进一步分离,将纯化的ORAC值活性较高的组分进行氨基酸序列分析鉴定,确定了两个新型的鸭肫抗氧化活性肽,分别为Ser-Ser-Tyr-Glu-Gly-Ile-Glu-Leu-Ile-Ile-Lys和Asn-Lys-Phe-Ile-Leu-Lys。(4)采用ACD/Labs软件对部分已知抗氧化肽化学结构信息进行描述,利用多元线性回归(MLR)、偏最小二乘回归(PLS)和人工神经网络(BPNN)分析方法建模,对抗氧化肽进行活性预测。MLR的线性拟合值为0.9915,PLS的线性拟合值为0.8313,BPNN线性拟合值0.99239,表明构建的神经网络模型具有较高的准确性和重现性。对MLR所建的模型进行F检验,可判定此方程显著。利用MLR、PLS和BPNN所建的模型分别对分离鉴定两个鸭肫抗氧肽DKFILK和SSYEGIELIIK验证,与ORAC活性值和氨基酸结构分析结果一致,进一步证实了组分DKFILK抗氧化活性更高。鸭肫抗氧化肽DKFILK的活性与其氨基酸组成及结构具有一定的关联。(5)鸭肫抗活性肽对人体前列腺增生细胞(BPH1)的抗氧化能力及其机理。不同浓度的鸭肫活性肽在作用48 h后,能明显抑制BPH细胞的增殖,促进其凋亡并且阻滞其细胞周期;当鸭肫活性肽的质量浓度为200310-3 g/L时,细胞中超氧化物岐化酶、过氧化氢酶以及谷胱甘肽过氧化物酶的活力分别提高了26.16%、20.24%和69.41%;采用qRT-PCR检测得出,细胞中CAT和GSH-Px mRNA的表达水平升高,SOD mRNA表达不明显,与鸭肫活性肽浓度呈正相关。鸭肫活性肽通过上调细胞内CAT和GSH-Px mRNA表达水平来削弱细胞自身的氧化损伤作用,发挥其抗氧化作用。更多还原

【Abstract】 Bioactive peptides are biological,active,and safe biological products that have important research value and application prospects.Duck gizzard is a high-quality protein resource.High value of the development and utilization are the direction of industrial development.In this study,antioxidant peptides from duck gizzard protein were prepared by controlled enzymatic hydrolysis technology.The product was purified by ultrafiltration,gel filtration chromatography,and reversed-phase high performance liquid chromatography.The first-order amino acid sequence of the target peptide was identified by mass spectrometry.Quantitative structure-activity relationships of the antioxidant peptides based on molecular structure were established.The anti-oxidation mechanism of the antioxidant peptides was further elucidated from the molecular mechanisms of cellular activity,analysis of cell growth inhibition,apoptosis detection,growth cycle,intracellular antioxidant enzyme activity,and enzyme protein expression.In this study,the development and application of duck gizzard proteins were discussed,providing a theoretical basis and technical support for the development and utilization of duck gizzard protein resources.The main contents and results of this paper were as follows:(1)The amino acid content in the papain hydrolysate was 33594.73 mg/100 g,and the content of the main antioxidant amino acid was 9.54%.Based on the single factor experiment,the degree of hydrolysis and DPPH clearance were taken as the response values,and the optimal hydrolysis conditions were as follows: hydrolysis time 4 h,enzyme dosage 3000 U/g,hydrolysis temperature 55°C,and a material to liquid ratio of 1:3.Under these conditions,the degree of hydrolysis was 28.30%,and the DPPH removal rate was 75.22%.There was no significant correlation between the degree of hydrolysis and the DPPH free radical scavenging ability.(2)The antioxidant activities of ZT1(<3 kDa),ZT2(3-10 kDa),and ZT3(> 10 kDa)with different molecular weights were investigated.The DPPH scavenging rate and ORAC activity of the free radicals were significantly correlated with their molecular weight.The DPPH scavenging rate of the products with molecular weights less than 3 k Da was 77.59% and the ORAC activity was 885.25 μmol TE/g.The antioxidant peptides with higher antioxidant activities were less than 3 kDa.The protective effect of the antioxidant peptides on scavenging the superoxide anions,hydroxyl radicals,hydrogen peroxide,and DNA damage showed an obvious dose-effect relationship.The IC50 values were 14.58,0.0163,19.80,and 1.93 mg/ml,respectively.Therefore,the duck gizzard antioxidant peptide had the strongest inhibitory effect on hydrogen peroxide radicals and the weakest inhibition on hydroxyl radical activity.(3)Separation and identification of antioxidant peptides from duck gizzard protein.The enzymolysis solution was first separated by hollow cellulose membrane ultrafiltration.The fraction with the highest ORAC activity(less than 3 kDa)was further separated by gel chromatography and semi-preparative RP-HPLC.Purified peptides with the highest ORAC activity were identified as Gly-Ile-Glu-Leu-Ile-Ile-Lys and Asn-Lys,respectively.These novel antioxidant peptides have never been identified before.(4)The chemical structure information of some known antioxidant peptides was described using the ACD/Labs software.Activity prediction of antioxidant peptides was performed by using multivariate linear regression(MLR),partial least squares regression(PLS),and artificial neural network(BPNN)analyses.The linear fitting value of MLR was 0.9915,the linear fitting value of PLS was 0.8313,and the linear fitting value of BPNN was 0.99239,indicating that the constructed neural network model had high accuracy and reproducibility.The F-test was performed on the model constructed by MLR,and showed that the equation was significant.The models of MLR,PLS,and BPNN were used to separate and identify two duck gizzard antioxidant peptides,DKFILK and SSYEGIELIIK.The ORAC activity was in agreement with the results of the amino acid structure analysis,further confirming the higher antioxidant activity of component DKFILK.The mechanism of the antioxidant peptide,DKFILK from the duck gizzard had a certain correlation with its amino acid composition and structure.(5)Anti-oxidative capacity and mechanism of duck gizzard antioxidant peptide in human benign prostatic hyperplasia cells.Different concentrations of duck gizzard peptide after treatment for 48 h could significantly inhibit B-cell proliferation by promoting apoptosis and preventing cell cycle progression.When the mass concentration of the duck gizzard active peptide was 200 × 10-3 g/L,the superoxide dismutase,catalase,and glutathione peroxidase activities increased by 26.16%,20.24%,and 69.41%,respectively.The expression of CAT and GSH-Px mRNA was increased and SOD mRNA expression was not significantly correlated with the concentration of the active peptide of duck gizzard as observed by qRT-PCR.Duck gizzard active peptides played an important role in antioxidant activity by upregulating the expression of CAT and GSH-Px mRNA in cells,and attenuating the oxidative damage in cells.更多还原