【作者】 张苗；

【导师】 木泰华；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2012， 博士

【摘要】 本研究以甘薯蛋白为原料,经不同蛋白酶酶解制备甘薯蛋白酶解肽,研究了甘薯蛋白酶解肽的抗氧化活性并对其制备工艺进行了优化；对最优工艺条件下得到的甘薯蛋白抗氧化活性肽进行了分离、纯化及结构鉴定；同时,探讨了甘薯蛋白酶解肽抑制结肠癌细胞HT-29增殖及转移活性,并对其作用机理进行了研究,从而为开发抗氧化、抗肿瘤保健产品提供理论依据。研究意义在于提升甘薯生产附加值,为开发具有抗氧化和抗肿瘤功能的肽类食品提供理论支撑。采用6种蛋白酶(alcalase、proleather FG-F、AS1.398、neutrase、papain和pepsin)对甘薯蛋白进行酶解。测定了酶解产物的抗氧化活性及其对DNA氧化损伤的保护作用。Alcalase酶解产物表现出最高的羟自由基清除活性(IC50为1.74mg/mL)和Fe2+螯合力(IC50为1.54mg/mL)(P<0.05)。与其他五种酶解产物相比,alcalase酶解产物中含有最丰富的<3kDa的组分。此外,alcalase酶解产物<3kDa组分显示出了最高的抗氧化活性,并可通过清除·OH自由基和螯合Fe2-保护DNA免受氧化损伤。以碱性蛋白酶alcalase为水解酶,研究了不同预热处理温度和时间对甘薯蛋白alcalase酶解特性的影响。在对甘薯蛋白进行预热处理的基础上,响应面法被用于研究甘薯蛋白抗氧化活性肽的制备。测定了酶与底物浓度比(E/S)、pH值和温度对甘薯蛋白酶解肽·OH自由基清除活性和Fe2+螯合力的影响。采用多元回归分析,对试验数据进行二次多项式模型拟合,并分析了模型的有效性及因子间的交互作用。结果表明,制备甘薯蛋白抗氧化肽的最优工艺参数为：底物浓度为5%,E/S为4%,pH值为8.0,温度为57℃,时间为2h。在此条件下,测得·OH自由基清除活性和Fe2+螯合力分别为40.03±0.63%和74.08±1.53%。将最优工艺条件下得到的甘薯蛋白酶解产物通过超滤分级得到4个组分,低分子量组分F-Ⅳ(<kDa)显示了最强的·OH自由基清除活性和Fe2+螫合力,在浓度为3.0mg/mL时,分别为59.74%和82.27%。然后依次采用大小排阻层析色谱和反相高效液相色谱对F-Ⅳ进行分离纯化,得到两个组分Ⅳ-5c和Ⅳ-5i,在浓度为0.3mg/mL时,其·OH自由基清除活性和Fe2+螯合力分别为33.22%和45.01%。采用LC-MS/MS对Ⅳ-5c和Ⅳ-5i的氨基酸序列进行鉴定,得到5个肽段,其中Thr-Tyr-Gln-Thr-Phe、Ser-Gly-Gln-Tyr-Phe-Leu和Tyr-Met-Val-Ser-Ala-Ile-Trp-Gly来源于sporamin A,而Tyr-Tyr-Ile-Val-Ser和Tyr-Tyr-Asp-Pro-Leu来源于sporamin B。甘薯蛋白酶解肽具有一定的抑制结肠癌细胞HT-29增殖及转移活性。甘薯蛋白酶解肽<3kDa组分可诱导HT-29细胞中细胞周期依赖性激酶抑制因子p21的表达,从而阻滞细胞周期进程：并可诱导HT-29细胞内促凋亡蛋白Bax的上调表达、抗凋亡蛋白Bcl-2的下调表达,来激活线粒体途径,从而诱导细胞凋亡；与此同时,可显著降低HT-29细胞中uPA活性,并通过诱导HT-29细胞内uPA的下调表达,从而抑制细胞迁移。更多还原

【Abstract】 In this study, sweet potato proteins were isolated,and then hydrolyzed by different proteases to get sweetpotato protein hydrolyzates, of which the antioxidant activities were investegted and the enzymatic conditions were optimized; Under the optimum conditions,the antioxidative peptides from sweetpotato protein were separation, purification and identification;Meanwhile, the inhibition effect on the proliferation and metastasis of colon cancer cells HT-29for sweetpotato protein hydrolyzates were studied, as well as its mechanism, so to provide a theoretical basis for the development of antioxidant and antitumor health products.The aim of this research is to increase the additional value of sweet potato production,and provide theoretic support for the development of functional peptides foods with antioxidant and antitumor activities.Sweetpotato protein hydrolysates were prepared by six enzymes (alcalase, proleather FG-F, AS1.398,neutrase, papain and pepsin). The antioxidant activities and protective effect against oxidative DNA damage of sweetpotato protein hydrolysates were investigated. Alcalase hydrolysates exhibited the highest hydroxyl radical scavenging activity (1C501.74mg/mL) and Fe2+-chelating ability(IC501.54mg/mL)(P<0.05). Compared to other five hydrolysates, the hydrolysates obtained by alcalase had the most abundant<3kDa fractions. In addition,<3kDa fractions of alcalase hydrolysates showed the highest antioxidant activities and protective effects against DNA damage through both scavenging hydroxyl radicals and chelating Fe2+ion.The effect of pre-heating temperature and time on enzymolysis characteristics of sweetpotato protein by alcalase was investigated.On the basis of pre-heating treatment of sweetpotato protein, the response surface methodology was employed to study the preparation of antioxidative peptides from sweetpotato protein.The effects of enzyme to substrate concentration ratio, pH value and temperature on hydroxyl radicals scavenging activity and Fe2+-chelating ability were investigated. Experimental data were fitted to two quadratic polynomial models using multiple regression analysis.The validity of the model and the interactions of impact factors were analyzed.The results showed that the optimum conditions for preparing antioxidative peptides from sweetpotato protein were as follows:substrate concentration5%,enzyme to substrate concentration ratio4%, pH8.0, temperature57℃and time2h. Under the optimum conditions, the hydroxyl radicals scavenging activity and Fe2+-chelating ability were40.03±0.63%and74.08±1.53%.Sweetpotato protein hydrolysates obtained under the optimum conditions were separated into four fractions by ultrafiltration, and low-molecular-weight fraction F-IV (<3kDa) exhibited the strongest hydroxyl radical scavenging activity and Fe2+-chelating ability, which was59.74%and82.27%at concentration of3.0mg/mL, respectively. Fraction F-IV was then purified with size exclusion chromatography followed by reverse-phase high-performance liquid chromatography, by which two fractions IV-5c and IV-5i were obtained showing the highest hydroxyl radical scavenging activity and Fe2+-chelating ability,33.22%and45.01%at0.3mg/mL, separately. The amino acid sequence determination by LC-MS/MS identified five peptides in antioxidative fractions IV-5c and IV-5i,of which Thr-Tyr-Gln-Thr-Phe, Ser-Gly-Gln-Tyr-Phe-Leu and Tyr-Met-Val-Ser-Ala-Ile-Trp-Gly matched the sequence of sporamin A, while Tyr-Tyr-Ile-Val-Ser and Tyr-Tyr-Asp-Pro-Leu matched the sequence of sporamin B.Enzymatic peptides from sweetpotato protein exhibited certain inhibition effect on the proliferation and metastasis of colon cancer cells HT-29.<3kDa fractions of sweetpotato protein peptides could induced the expression of cyclin-dependent kinase inhibitory factor p21in HT-29cells, thereby blocking cell cycle progression, and promoted apoptosis in HT-29cells through activation of mitochondrial pathways as evidenced by increased expression of Bax and decreased expression of Bcl-2.At the same time,<3kDa fractions could significantly reduce the activity and decreased expression of uPA in HT-29cells, thereby inhibiting cell migration.更多还原