本文選題：蛋清蛋白肽 + 結(jié)構(gòu)鑒定��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：蛋清蛋白(EWP)作為一種廣泛優(yōu)質(zhì)的蛋白質(zhì)來源,具有多種生理功能和營養(yǎng)價值。它經(jīng)蛋白酶裂解所得到的蛋清蛋白肽,也已被大量研究證實(shí)具有抑菌、抗氧化、降血壓、降血糖等多種生理活性,而如何有效制備和準(zhǔn)確鑒定出高活性的蛋清蛋白肽依舊是目前研究的熱點(diǎn)和難點(diǎn)。本文通過體外傳統(tǒng)酶解法和體內(nèi)消化法進(jìn)行蛋清蛋白肽的制備與鑒定,并研究其抗氧化應(yīng)激和抗炎活性作用,為預(yù)防疾病、保護(hù)人類健康提供可能。首先利用體外傳統(tǒng)酶解法,輔助以電子束輻照技術(shù)(EBI),研究其對EWP酶解效果、結(jié)構(gòu)和性質(zhì)的影響。結(jié)果發(fā)現(xiàn),EWP的水解度隨EBI劑量的增加而顯著提高(P0.05),且在3.42 k Gy的劑量下,其酶解物的DPPH自由基及羥基自由基清除能力分別提高了2.5%和100%。小鼠經(jīng)口急性毒性試驗(yàn)、骨髓細(xì)胞微核試驗(yàn)和精子畸形試驗(yàn)結(jié)果均表明其酶解物無毒、無致畸性,毒理學(xué)評價安全。同時,低場核磁共振(LF-NMR)、掃描電子顯微鏡和中紅外光譜(MIR)檢測表明,EBI能夠破壞EWP表面微觀結(jié)構(gòu),降低EWP的持水性,使其變性起始溫度降至25℃,更易變性,為將EBI應(yīng)用于提高活性肽制備效率提供了可能。其次,將EWP酶解液經(jīng)超濾、葡聚糖凝膠柱層析分離純化,并利用氨基酸成分分析(AAA)、MIR以及液相串聯(lián)質(zhì)譜(LC-MS/MS)技術(shù)進(jìn)行蛋清蛋白肽的結(jié)構(gòu)鑒定。結(jié)果表明,分子量小于1 k Da的EWP酶解液超濾組分(EWPH-III)具有顯著的DPPH、ABTS自由基以及氧自由基清除能力,并進(jìn)一步得到P4、P6分離組分。通過結(jié)構(gòu)鑒定,獲得三條分子量分別為627.7 Da,632.9 Da和684.1 Da的蛋清蛋白肽,氨基酸序列分別為DHTKE,FFGFN和MPDAHL,其中MPDAHL的體外自由基清除能力較強(qiáng)。然后,分別建立過氧化氫(H_2O_2)誘導(dǎo)人結(jié)腸癌細(xì)胞HT-29的氧化應(yīng)激模型和腫瘤壞死因子(TNF-a)誘導(dǎo)的3T3-L1脂肪細(xì)胞炎癥模型,考察MPDAHL對腸道細(xì)胞氧化應(yīng)激損傷的保護(hù)作用以及對脂肪細(xì)胞的抗炎活性作用。結(jié)果表明,MPDAHL的細(xì)胞抗氧化活性(CAA)隨濃度的增加而增強(qiáng),當(dāng)濃度為50μM時,能夠抑制H_2O_2誘導(dǎo)的HT-29細(xì)胞氧化應(yīng)激損傷,顯著抑制IL-8的分泌及脂質(zhì)氧化MDA的形成(P0.05);同時,MPDAHL對TNF-α誘導(dǎo)的脂肪細(xì)胞中促炎因子IL-6和MCP-1的分泌具有顯著抑制作用,并促進(jìn)脂聯(lián)素的分泌,在50μM濃度下具有一定的體外抗炎活性。綜合評價MPDAHL的抗氧化應(yīng)激和抗炎活性,其最適處理濃度偏高,不利于進(jìn)一步發(fā)揮體內(nèi)活性作用和后期的開發(fā)研究。因此,接下來采用體內(nèi)消化法,以EWP中卵轉(zhuǎn)鐵蛋白(OVT)為原料,在考察其體內(nèi)抗炎活性作用基礎(chǔ)上,預(yù)測鑒定出具有體內(nèi)活性的可吸收蛋清蛋白肽。建立脂多糖(LPS)誘導(dǎo)小鼠慢性炎癥模型,利用酶聯(lián)免疫印跡、基因表達(dá)等生物學(xué)方法對OVT體內(nèi)消化后的抗炎活性作用進(jìn)行研究。結(jié)果表明,16周受試期內(nèi),相較于僅用300μg/kg BW/day LPS誘導(dǎo)的陽性對照組,150 mg/kg BW/day的OVT高劑量處理組小鼠血液中內(nèi)毒素含量顯著降低,促炎細(xì)胞因子TNF-α、MCP-1及IL-6濃度分別降低了59.2%、45%和48%(P0.05);同時,OVT處理組小鼠腸道組織中IL-6的表達(dá)受到抑制,緊密連接蛋白ZO-1、Claudin-2、Claudin-4和JAMA的基因表達(dá)顯著上調(diào),且血液中D-甘露醇透過量顯著低于陽性對照組(P0.05),則OVT能抑制LPS引起的腸道炎癥,抵御腸道損傷,保護(hù)腸道完整性;此外,高劑量OVT顯著促進(jìn)了小鼠脂肪組織中脂聯(lián)素的分泌,上調(diào)了PPAR-γ和IRS-1蛋白的表達(dá),能夠改善小鼠胰島素抵抗。因此,OVT體內(nèi)消化后,具有良好的抑制慢性炎癥作用,對預(yù)防二型糖尿病有巨大潛力。最后,對OVT體內(nèi)消化后的可吸收肽進(jìn)行預(yù)測鑒定。利用MALDI-TOF-MS技術(shù)對比分析大鼠OVT灌胃后的門靜脈血清樣品與對照組血清樣品質(zhì)譜圖,發(fā)現(xiàn)了16個僅存在于OVT灌胃1 h、2 h或4 h大鼠血清中的離子峰,再經(jīng)LC-TOF-MS進(jìn)一步確定了3個m/z分別為462.24 Da,476.26 Da,491.23 Da的離子峰,得到13條OVT源的可吸收蛋清蛋白肽序列�？傊�,本文利用傳統(tǒng)體外酶解,輔助以電子束輻照,分離、純化、鑒定獲得了3條具有一定細(xì)胞抗氧化應(yīng)激和抗炎活性作用的蛋清蛋白肽,再利用體內(nèi)消化法,通過小鼠慢性炎癥模型發(fā)現(xiàn)了蛋清蛋白的體內(nèi)抗炎活性,并直接鑒定出了13條大鼠消化后的的體內(nèi)可吸收蛋清蛋白肽,為高效制備和準(zhǔn)確鑒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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ936.16

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