蛋清蛋白肽的制备与鉴定及其生物活性研究

发布时间：2018-07-04 18:27

本文选题：蛋清蛋白肽 + 结构鉴定　；参考：《吉林大学》2017年博士论文

【摘要】：蛋清蛋白(EWP)作为一种广泛优质的蛋白质来源,具有多种生理功能和营养价值。它经蛋白酶裂解所得到的蛋清蛋白肽,也已被大量研究证实具有抑菌、抗氧化、降血压、降血糖等多种生理活性,而如何有效制备和准确鉴定出高活性的蛋清蛋白肽依旧是目前研究的热点和难点。本文通过体外传统酶解法和体内消化法进行蛋清蛋白肽的制备与鉴定,并研究其抗氧化应激和抗炎活性作用,为预防疾病、保护人类健康提供可能。首先利用体外传统酶解法,辅助以电子束辐照技术(EBI),研究其对EWP酶解效果、结构和性质的影响。结果发现,EWP的水解度随EBI剂量的增加而显著提高(P0.05),且在3.42 k Gy的剂量下,其酶解物的DPPH自由基及羟基自由基清除能力分别提高了2.5%和100%。小鼠经口急性毒性试验、骨髓细胞微核试验和精子畸形试验结果均表明其酶解物无毒、无致畸性,毒理学评价安全。同时,低场核磁共振(LF-NMR)、扫描电子显微镜和中红外光谱(MIR)检测表明,EBI能够破坏EWP表面微观结构,降低EWP的持水性,使其变性起始温度降至25℃,更易变性,为将EBI应用于提高活性肽制备效率提供了可能。其次,将EWP酶解液经超滤、葡聚糖凝胶柱层析分离纯化,并利用氨基酸成分分析(AAA)、MIR以及液相串联质谱(LC-MS/MS)技术进行蛋清蛋白肽的结构鉴定。结果表明,分子量小于1 k Da的EWP酶解液超滤组分(EWPH-III)具有显著的DPPH、ABTS自由基以及氧自由基清除能力,并进一步得到P4、P6分离组分。通过结构鉴定,获得三条分子量分别为627.7 Da,632.9 Da和684.1 Da的蛋清蛋白肽,氨基酸序列分别为DHTKE,FFGFN和MPDAHL,其中MPDAHL的体外自由基清除能力较强。然后,分别建立过氧化氢(H_2O_2)诱导人结肠癌细胞HT-29的氧化应激模型和肿瘤坏死因子(TNF-a)诱导的3T3-L1脂肪细胞炎症模型,考察MPDAHL对肠道细胞氧化应激损伤的保护作用以及对脂肪细胞的抗炎活性作用。结果表明,MPDAHL的细胞抗氧化活性(CAA)随浓度的增加而增强,当浓度为50μM时,能够抑制H_2O_2诱导的HT-29细胞氧化应激损伤,显著抑制IL-8的分泌及脂质氧化MDA的形成(P0.05);同时,MPDAHL对TNF-α诱导的脂肪细胞中促炎因子IL-6和MCP-1的分泌具有显著抑制作用,并促进脂联素的分泌,在50μM浓度下具有一定的体外抗炎活性。综合评价MPDAHL的抗氧化应激和抗炎活性,其最适处理浓度偏高,不利于进一步发挥体内活性作用和后期的开发研究。因此,接下来采用体内消化法,以EWP中卵转铁蛋白(OVT)为原料,在考察其体内抗炎活性作用基础上,预测鉴定出具有体内活性的可吸收蛋清蛋白肽。建立脂多糖(LPS)诱导小鼠慢性炎症模型,利用酶联免疫印迹、基因表达等生物学方法对OVT体内消化后的抗炎活性作用进行研究。结果表明,16周受试期内,相较于仅用300μg/kg BW/day LPS诱导的阳性对照组,150 mg/kg BW/day的OVT高剂量处理组小鼠血液中内毒素含量显著降低,促炎细胞因子TNF-α、MCP-1及IL-6浓度分别降低了59.2%、45%和48%(P0.05);同时,OVT处理组小鼠肠道组织中IL-6的表达受到抑制,紧密连接蛋白ZO-1、Claudin-2、Claudin-4和JAMA的基因表达显著上调,且血液中D-甘露醇透过量显著低于阳性对照组(P0.05),则OVT能抑制LPS引起的肠道炎症,抵御肠道损伤,保护肠道完整性;此外,高剂量OVT显著促进了小鼠脂肪组织中脂联素的分泌,上调了PPAR-γ和IRS-1蛋白的表达,能够改善小鼠胰岛素抵抗。因此,OVT体内消化后,具有良好的抑制慢性炎症作用,对预防二型糖尿病有巨大潜力。最后,对OVT体内消化后的可吸收肽进行预测鉴定。利用MALDI-TOF-MS技术对比分析大鼠OVT灌胃后的门静脉血清样品与对照组血清样品质谱图,发现了16个仅存在于OVT灌胃1 h、2 h或4 h大鼠血清中的离子峰,再经LC-TOF-MS进一步确定了3个m/z分别为462.24 Da,476.26 Da,491.23 Da的离子峰,得到13条OVT源的可吸收蛋清蛋白肽序列。总之,本文利用传统体外酶解,辅助以电子束辐照,分离、纯化、鉴定获得了3条具有一定细胞抗氧化应激和抗炎活性作用的蛋清蛋白肽,再利用体内消化法,通过小鼠慢性炎症模型发现了蛋清蛋白的体内抗炎活性,并直接鉴定出了13条大鼠消化后的的体内可吸收蛋清蛋白肽,为高效制备和准确鉴定生物利用率高的生物活性肽提供了新思路。
[Abstract]:Egg white protein (EWP), as a wide and high-quality source of protein, has a variety of physiological functions and nutritional value. The egg white protein peptide obtained by protease cracking has also been proved to have many physiological activities, such as bacteriostasis, antioxidation, blood pressure reduction and hypoglycemia, and how to effectively prepare and accurately identify the highly active egg white. Protein peptide is still a hot and difficult point in the present study. In this paper, the preparation and identification of egg white protein peptides are carried out by enzymatic hydrolysis and digestion in vivo, and the antioxidative and anti-inflammatory activities are studied in order to prevent diseases and protect human health. First, the enzyme solution method of body external transmission is used to assist the electron beam irradiation technique. The effect of the enzyme hydrolysis, structure and properties of EWP was studied. The results showed that the degree of hydrolysis of EWP increased significantly with the increase of the dose of EBI (P0.05). At the dosage of 3.42 K Gy, the DPPH free radical and hydroxyl radical scavenging ability of the enzyme hydrolysate increased by 2.5% and 100%. mice by oral acute toxicity test, and the micronucleus test of bone marrow cells. The test and sperm abnormality test showed that the enzyme hydrolysate was nontoxic, non teratogenicity and toxicological safety. At the same time, low field nuclear magnetic resonance (LF-NMR), scanning electron microscope and mid infrared spectroscopy (MIR) test showed that EBI could destroy the microstructure of EWP surface, reduce the water holding property of EWP, and make its denaturing starting temperature to 25 C, more easily denatured, and E BI was used to improve the efficiency of the preparation of active peptides. Secondly, the EWP enzymolysis solution was purified by ultrafiltration, Sephadex gel column chromatography, and the structure identification of egg white protein peptides was carried out by amino acid composition analysis (AAA), MIR and liquid phase tandem mass spectrometry (LC-MS/MS). The results showed that the ultrafiltration group with a molecular weight less than 1 K Da was found in the ultrafiltration group of EWP enzyme solution. EWPH-III has significant DPPH, ABTS radical and oxygen free radical scavenging ability, and further obtain P4, P6 separation components. Through structural identification, three layers of egg white protein peptides with molecular weights of 627.7 Da, 632.9 Da and 684.1 Da are obtained, and the amino acid sequences are DHTKE, FFGFN and MPDAHL, respectively, in which the free radical scavenging ability of MPDAHL in vitro is more Then, the oxidative stress model of human colon cancer cell HT-29 induced by hydrogen peroxide (H_2O_2) and the inflammatory model of 3T3-L1 adipocyte induced by tumor necrosis factor (TNF-a) were established respectively. The protective effect of MPDAHL on the oxidative stress damage of intestinal cells and the anti-inflammatory activity of MPDAHL on the adipocytes were investigated. The results showed that the anti oxygen cell of MPDAHL cells was anti oxygen. The chemical activity (CAA) increased with the increase of concentration. When the concentration was 50 M, it could inhibit the oxidative stress damage of HT-29 cells induced by H_2O_2, significantly inhibit the secretion of IL-8 and the formation of MDA in lipid oxidation (P0.05). At the same time, MPDAHL has a significant inhibitory effect on the secretion of IL-6 and MCP-1 in the fat cells induced by TNF- alpha, and promotes lipid association. The secretion of the hormone has a certain anti inflammatory activity in vitro under the concentration of 50 M. The antioxidant stress and anti-inflammatory activity of MPDAHL are evaluated synthetically, its optimum treatment concentration is high, which is not conducive to the further development of the activity of the body and the development of the later stage. Therefore, the body digestion method is used in the study of the egg transferrin (OVT) in EWP as the raw material. On the basis of its anti-inflammatory activity in vivo, the absorbable egg white protein peptide, which has the activity of the body, was predicted and identified. The model of chronic inflammation induced by lipopolysaccharide (LPS) in mice was established. The anti inflammatory activity of OVT after digestion was studied by biological methods such as enzyme linked immunoblotting and gene expression. The results showed that in the 16 week period, it was compared with that in the experimental period. In the positive control group induced by 300 g/kg BW/day LPS, the content of endotoxin in the blood of 150 mg/kg BW/day was significantly decreased, and the concentrations of proinflammatory cytokines TNF- a, MCP-1 and IL-6 decreased by 59.2%, 45% and 48% (P0.05) respectively. Meanwhile, the expression of IL-6 in the intestinal tissue of the OVT treatment group was inhibited and the close connexin was found. The gene expression of ZO-1, Claudin-2, Claudin-4 and JAMA was significantly up-regulated, and the transmission of D- mannitol in the blood was significantly lower than that of the positive control group (P0.05). OVT could inhibit intestinal inflammation caused by LPS, resist intestinal damage and protect intestinal integrity. In addition, high dose OVT significantly promoted the secretion of adiponectin in the adipose tissue of mice and up regulation of PPAR- gamma. The expression of IRS-1 protein and protein can improve insulin resistance in mice. Therefore, after digestion in OVT, it has a good inhibitory effect on chronic inflammation and has great potential for preventing type two diabetes. Finally, the absorbable peptide of OVT after digestion in vivo is predicted and identified. MALDI-TOF-MS technique is used to compare and analyze the portal vein serum of rat after OVT gavage. The samples and the serum samples of the control group have found 16 ion peaks in the serum of 1 h, 2 h or 4 h rats, which exist only in OVT gavage, and then further determined the ion peaks of 3 m/z, 462.24 Da, 476.26 Da, 491.23 Da, and the absorbable egg white protein peptide sequences of 13 OVT sources. 3 egg white protein peptides were obtained by electron beam irradiation, separation, purification and identification. The anti-inflammatory activity of egg white protein in vivo was detected by the digestive method in vivo, and the absorption of egg white eggs in the body after digestion of 13 rats was identified. White peptide provides a new idea for efficient preparation and accurate identification of bioactive peptides with high bioavailability.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ936.16

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