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羊皱胃蛋白活性成分的分离纯化、结构鉴定及活性研究
刘 冰
学位类型博士
导师阿布力米提·伊力
2018-06-02
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词羊皱胃 蛋白质部位 提取工艺 分离纯化 抗氧化活性肽
摘要

羊皱胃是羊的第四个胃,也叫羊第四胃,含有丰富的酶类和蛋白质。新疆作为畜牧业大省,羊皱胃来源丰富。国内已有企业以羔羊皱胃有效部位为原料生产制剂,其包含多种酶类和活性多肽可用于治疗多种胃部疾病,具有很好的应用价值。但由于现有提取方法老旧,所得有效部位具有产率低,活性弱,药用物质基础不清楚等缺点。同时,羊皱胃中含有丰富的蛋白类物质,而国内外几乎无相关报道。鉴于此,本文对羔羊皱胃的活性酶有效部位进行了制备工艺和动物实验研究,优化了有效部位的产率和提取酶活性;同时对羊皱胃总蛋白质部位进行了制备、酶解,并对其酶解产物中的抗氧化肽进行了分离纯化、结构鉴定研究。本文旨在为羊皱胃的资源充分利用和科学应用提供技术支撑,为以羔羊皱胃作为原料开发药物和保健品的企业提供物质基础参考,为其建立原料药标准提供科学依据,并为现代多肽类保健食品或药物的开发奠定技术和理论基础。主要研究结果如下:1、羔羊皱胃活性酶有效部位制备工艺、分离纯化及其抗溃疡作用研究以羔羊皱胃作为原料,利用超声波辅助提取法提取其活性酶有效部位提取物(the protease-rich extract from sheep abomasum,SAE)。以酶的活性为主要响应值,通过响应面法分析优化得到最佳提取条件为超声时间18 min、超声功率260 W、液料比22 mL/g,得蛋白酶解活力为 4951.3 ± 71.4 U/g,提取率14.33 ± 2.1%。电泳结果表明SAE中含有以凝乳酶和胃蛋白酶为主的酶类和其他蛋白质,其中凝乳酶活性为26916 Ru/g,胃蛋白酶活性为3685.2 U/g,水溶性蛋白含量为71.5%。液相-二级质谱联用(LC-MS/MS)分析结果显示SAE中含有大量活性肽,包括血清白蛋白,生长因子,核糖体蛋白等,分子量范围为651.39至4248.08 Da,这些物质对胃部疾病的治疗和预防具有一定的作用。以蛋白酶解活力为指标,进一步对SAE进行分离纯化。经过葡聚糖凝胶Sephadex G-25柱层析和超滤(ultrafiltration,UF)两次纯化步骤后,SAE的蛋白酶解活性提高了3.49倍,回收率为36.5%。纯化后SAE凝乳酶和胃蛋白酶活性均显著增加,分别为35291 Ru/g,4023.6 U/g。且纯化后的SAE具有良好的蛋白功能特性。本研究旨在为SAE的大规模纯化和工厂生产提供一定的理论指导。建立了束缚水浸大鼠应激胃溃疡模型,研究了SAE对应激性胃溃疡的治疗作用。结果表明,灌胃给药中剂量(0.1 g/kg/day)和高剂量(0.2 g/kg/day)SAE组可不同程度降低大鼠脑组织单胺类神经递质浓度的升高,维持体液平衡,并显著降低应激性胃溃疡大鼠的溃疡指数,溃疡抑制率分别54.5%,66.9%,与模型组比较,具有显著差异性(P<0.01)。说明SAE可通过神经体液的调节方式,抑制和保护由束缚水浸造成的大鼠应激性胃溃疡和胃黏膜损伤。2、羊皱胃蛋白的制备工艺、成分分析及活性研究以成年羊皱胃为原料,采用超声波辅助提取法提取羊皱胃总蛋白溶液(SAP)。通过响应面法分析优化得到最佳蛋白提取工艺条件为超声时间28 min、超声功率450 W、液料比25 mL/g、提取液pH为10,得到最高水溶性蛋白浓度为320.5 mg/g。利用三种不同沉淀方法制备羊皱胃蛋白浓缩物(sheep abomasum protein concentrates,SAPC),对比测定了其蛋白功能特性、抗菌和抗氧化活性后。结果发现,60%硫酸铵沉淀所得SAPC具有很好的蛋白功能特性和良好的抗氧化活性,其DPPH、HO、ABTS的自由基清除活性IC50值分别为13.02 ± 0.83,15.21 ± 2.02,14.56 ± 1.79 mg/mL,蛋白含量为92.36 g/100g粗羊皱胃蛋白。利用SDS-PAGE和LC-MS分析SAPC的蛋白和多肽分子量范围。结果表明SAPC除了含有多种大分子蛋白(10-100 kDa)外,还包含多种肽类,分子量范围为873.6-7487.8 Da,为羊皱胃蛋白的后续研究提供一定的数据基础。3、羊皱胃蛋白的酶解工艺及其抗氧化活性研究制备抗氧化肽的工艺研究以抗氧化活性为导向,利用不同蛋白酶对SAPC进行酶解,选出最佳酶源为木瓜蛋白酶;通过响应面法分析优化得到最佳酶解工艺条件为:水解温度46 oC、水解时间3.8 h、酶/底物比例为1.5%。该条件下制备的羊皱胃酶解产物(sheep abomasum protein hydrolyses,SAPH)水解度(DH)为16.86%,提取率为40.65%,SAPH的DPPH、HO、ABTS的自由基清除活性IC50值分别为6.92 ± 0.62、8.74 ± 0.23和7.85 ± 1.06 mg/mL。利用LC-MS对SAPH的组成成分进行分析。结果表明,SAPH富含多肽类物质,所含主要肽类的分子量范围为318.5-4043.1 Da,说明木瓜蛋白酶有很好的酶解效果,可以将大分子蛋白酶解为小分子多肽类物质。4、羊皱胃抗氧化肽的分离纯化与结构研究SAPH经一系列现代分离技术,共分离纯化得到8个生物活性肽组分(P1-8)。其中P3和P7具有较强抗氧化活性,其DPPH自由基清除能力IC50值分别为0.63和0.58 mg/mL。采用MALDI-TOF/TOF MS/MS法测得P3和P7的相对分子质量分别为674.37 Da和703.41 Da。对应的一级序列分别为Leu-Glu-Asp-Gly-Leu-Lys(LEDGLK);Ile-Asp-Asp-Val-Leu-Lys(IDDVLK),经检索,P3和P7是两种新型抗氧化肽。

其他摘要

Sheep abomasum is the fourth stomach of sheep and rich in active proteases and proteins. Central Asia and Xinjiang are rich in sheep resources. Large amounts of sheep abomasum as a by-product of the sheep meat industry are generated yearly. Active proteases in the young sheep abomasum have long been used to treat chronic gastritis in traditional Chinese medicine because they are rich in chymosin and pepsin which stimulates the mucosal defenses. Freeze-melting and stirring are the most conventional methods for extracting active proteases from materials, which is normally time-consuming and disadvantageous to bioactivity or pharmaceutical values. Meanwhile, no study has thus far researched active proteins from sheep abomasum. Therefore, in this paper, the preparation process and animal experimental study of the proteases-rich extract from young sheep abomasum were performed. At the same time, the high protein part of the sheep abomasum was prepared and hydrolyzed, and the antioxidant peptides were isolated, purified and identified from sheep abomasum protein hydrolysates. The study aimed to provide technical support for the full utilization and scientific application of sheep abomasum, and to provide the technical and theoretical foundation for the development of modern peptide health foods or drugs. The main findings are as follows:1. Extraction and purification the protease-rich extract from young sheep abomasum and its gastroprotective effect against stress-induced gastric ulcers in ratsYoung sheep abomasum was used as material. Ultrasound-assisted extraction method (UAE) was first developed to obtain the protease-rich extract from sheep abomasum (SAE). UAE parameters were firstly optimized using response surface methodology. Ultrasonic time of 18 min, ultrasonic power of 260 W, and a liquid/solid ratio of 22 mL/g resulted in the highest proteolytic activity and yield of the SAE (4951.3 U/g, 14.3% respectively). The result of electrophoresis showed that SAE mainly contained chymosin, pepsin and some proteins. The activity of chymosin and pepsin were 26916 Ru/g and 3685.2 U/g, respectively, and the content of water soluble protein was 71.5%. LC-MS/MS analysis showed that the SAE contains many low molecular weight (MW) peptides, mainly including latent transforming growth factor, pre-pro serum albumin, ribosomal protein, and albumin, which might stimulate gastric motility and protect the gastric mucosa. After two purification steps of Sephadex G-25 column and Ultrafiltration (UF), the proteolytic activity of SAE was increased 3.49-fold with a recovery yield of 36.5 %. And the purified SAE obtained in this way showed good functional properties. Then we investigated SAE’s gastroprotective effect against gastric ulcers induced by water immersion-restraint stress in rats. After 7 days of intragastric administration of the SAE (0.2 g/kg/day), the ulcer index (2.71 ± 0.97; p < 0.01) decreased significantly compared with that in the model group, and the ulcer inhibition ratio was 66.5%. In addition, the response to stress was significantly attenuated by balancing the levels of norepinephrine, 5-hydroxytryptamine, and dopamine in brain regions.2. Preparation process, composition analysis and activities of protein concentrates from sheep abomasumSheep abomasum protein concentrates (SAPC) was extracted by UAE. Response surface methodology and Box-Behnken design were applied to determine the optimal parameters for UAE. The maximum water-soluble protein concentration was 320.5 mg/g under the optimal conditions with ultrasonic treatment time of 28 min, ultrasonic power of 450 W, liquid/solid ratio of 25 mL/g, and pH of 10. Then, three methods for isolating proteins were constructed and compared depending on the values of protein functional properties, antibacterial and antioxidant activities. The results showed that the method with 60% ammonium sulfate precipitation was found to be the most appropriate one. Its protein content was 92.36 g/100 g crude sheep abomasum proteins, and the IC50 values of the free radical scavenging activities of DPPH, HO and ABTS were 13.02 ± 0.83, 15.21 ± 2.02, and 14.56 ± 1.79 mg/mL, respectively. SDS-PAGE and LC-MS analysis showed that SAPC contains a variety of peptides with a molecular weight range of 873.6-7487.8 Da in addition to a variety of macromolecular proteins (10-100 kDa). 3. Study on hydrolytic conditions and antioxidant activity of protein concentrates from sheep abomasum Sheep abomasum proteins were first hydrolyzed using alcalase, neutrase, or papain. Results from 3 enzyme tests revealed that the hydrolysis process of papain catalyst was most e?ective as SAPH generated by papain exhibited the highest degree of hydrolysis (DH), yield, and antioxidant activity. The e?ect of the conditions for hydrolysis with papain was further optimized using the response surface methodology. The highest DH and yield of SAPH (16.86% and 40.65%, respectively) were obtained under the following conditions: hydrolysis temperature, 46 °C; hydrolysis time, 3.8 h; and enzyme/substrate ratio, 1.5%. The IC50 values of the free radical scavenging activities of DPPH, HO and ABTS were 6.92 ± 0.62,8.74 ± 0.23,7.85 ± 1.06 mg/mL, respectively. SDS-PAGE and LC-MS analysis showed that SAPH is rich in peptides and the molecular weight of the main peptides ranged from 318.5 to 4043.1 Da.4. Isolation, purification and identification of two antioxidant peptides from sheep abomasum protein hydrolysatesEight subfractions were purified from SAPH by ultrafiltration, ion exchange chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography, namely, P1 to P8. Compared with other fractions, P3 and P7 exhibited higher DPPH radical scavenging activities and the IC50 values were 0.63 and 0.58 mg/mL, respectively. Peptide sequences were determined as Leu-Glu-Asp-Gly-Leu-Lys (LEDGLK) and Ile-Asp-Asp-Val-Leu-Lys (IDDVLK) with molecular weights of 674.37 and 703.41 Da, respectively. The results of the present study indicated that peptides purified from SAPH were natural antioxidants and could be used as food additives and pharmaceutical products.

页数123
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/5470
专题资源化学研究室
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刘 冰. 羊皱胃蛋白活性成分的分离纯化、结构鉴定及活性研究[D]. 北京. 中国科学院大学,2018.
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