本文關鍵詞：多酚氧化酶交聯(lián)牛乳α-乳白蛋白的結構變化及其消化性與過敏原性的評估　出處：《南昌大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 牛乳過敏 酶交聯(lián) α-乳白蛋白 多酚氧化酶 模擬消化 過敏原性

【摘要】：牛乳因其營養(yǎng)及美味而深受人們的喜愛,但牛乳又是FAO公認的八大類主要過敏食品之一,有高達8%的嬰幼兒和2%的成年人對牛乳過敏。近年來研究發(fā)現,酶法交聯(lián)牛乳可降低牛乳的過敏原性,為低致敏牛乳的研發(fā)提供了新的思路。牛乳a-乳白蛋白是引發(fā)牛乳過敏的主要過敏原之一,已有研究表明,有75%的牛乳過敏患者血清中含有α-乳白蛋白的特異性抗體,同時,α-乳白蛋白過敏原的構象性表位是其致敏的最主要原因之一。顯而易見,以牛乳α-乳白蛋白為對象研究交聯(lián)蛋白結構與過敏原性關系,具有重要的意義。 本論文工作以牛乳α-乳白蛋白為對象,利用蘑菇多酚氧化酶催化交聯(lián)牛乳α-乳白蛋白,探索α-乳白蛋白交聯(lián)對其結構、消化性以及過敏原性的影響。研究內容包括：牛乳α-乳白蛋白的分離純化及蘑菇多酚氧化酶的提取,多酚氧化酶催化交聯(lián)牛乳α-乳白蛋白的加工工藝研究,牛乳α-乳白蛋白交聯(lián)后的結構變化以及牛乳α-乳白蛋白交聯(lián)產物的消化性及過敏原性評估。研究的主要方法、結果及結論如下。 1.建立了離子交換色譜層析聯(lián)合凝膠色譜層析分離牛乳a-乳白蛋白的方法,純度達90%,純化得率為21.18%。同時,從雙孢蘑菇中獲得了多酚氧化酶酶液,酶活達6,000U/mL。 2.探索了α-乳白蛋白構象、酶促交聯(lián)反應條件以及底物添加量對a-乳白蛋白交聯(lián)產物生成的影響,并建立了多酚氧化酶催化交聯(lián)a-乳白蛋白的最適反應體系：無鈣狀態(tài)下的α-乳白蛋白終濃度1mg/m,多酚氧化酶終濃度1,000U/mL,咖啡酸終濃度1mM,在50℃,pH7.0下,酶促反應8小時。 3.通過Western-Blotting鑒定了牛乳α-乳白蛋白交聯(lián)二聚體,并且利用Native-PAGE切膠回收制備出純度為80%以上的具有生物學活性的牛乳α-乳白蛋白交聯(lián)二聚體。采用遠紫外圓二色譜、ANS探針熒光光譜以及紫外吸收光譜技術綜合表征了α-乳白蛋白交聯(lián)二聚體的結構,結果表明,相對于牛乳α-乳白蛋白,α-乳白蛋白交聯(lián)二聚體具有更高疏水性以及更加松散的蛋白結構。 4.通過體外模擬胃液、腸液消化,較系統(tǒng)地評價了牛乳α-乳白蛋白交聯(lián)產物的消化性,研究發(fā)現牛乳α-乳白蛋白極易被模擬胃液和腸液消化,而牛乳a-乳白蛋白交聯(lián)產物則相對耐消化,特別是交聯(lián)的高聚物具有很強的耐酶解能力,值得高度關注。 5.利用ELISA方法初步評價了牛乳α-乳白蛋白交聯(lián)產物的潛在過敏原性,研究結果表明,與α-乳白蛋白相比,α-乳白蛋白交聯(lián)產物與IgG和IgE的結合能力均顯著降低,這說明多酚氧化酶交聯(lián)α-乳白蛋白具有降低它的過敏原性的作用。
[Abstract]:Milk because of its nutritional and delicious and loved by the people, but the milk is FAO recognized as one of the eight kinds of allergic food, up to 8% of infants and 2% adults of milk allergy. Recent studies showed that enzymatic cross-linking of milk can reduce raw milk allergy, provides new ideas for the development of hypoallergenic milk. Milk a- lactalbumin is a major allergen of milk allergy, studies have shown that 75% of milk allergic patients serum containing alpha lactalbumin specific antibody, at the same time, alpha lactalbumin allergen conformational epitope is one of the major causes of the sensitization the object of study. Obviously, protein structure and allergenicity relationship with a-lactalbumin protein, has important significance.
The work of this paper to a-lactalbumin protein as the object, using catalytic crosslinking mushroom polyphenol oxidase a-lactalbumin protein, explore the alpha lactalbumin crosslinking on the structure, digestibility and allergenicity effect. The research contents include: extraction and purification and separation of mushroom polyphenol oxidase a-lactalbumin protein, processing research the process of polyphenol oxidase catalyzed cross-linked a-lactalbumin protein, structural changes of a-lactalbumin cross-linked protein and a-lactalbumin crosslinked protein digestibility and allergenicity assessment. The main research methods, results and conclusions are as follows.
1., an ion exchange chromatography and gel chromatography for the separation of milk a- lactalbumin was established. The purity was 90% and the yield was 21.18%.. Meanwhile, polyphenol oxidase was obtained from Agaricus bisporus, and the enzyme activity reached 6000U/mL..
2. to explore the alpha lactalbumin conformation, affecting the enzymatic conditions of crosslinking reaction and substrate addition on white a- protein cross-linking products generated, and established the optimum reaction system catalyzed cross-linking of polyphenol oxidase a- lactalbumin: calcium free condition alpha lactalbumin concentration 1mg/m, polyphenol oxidase 1000U/mL final concentration, coffee the final concentration of 1mM acid, pH7.0 at 50 C, the enzymatic reaction for 8 hours.
The 3. were identified by Western-Blotting a-lactalbumin cross-linked protein two precursor, and the use of Native-PAGE prepared at least 80% purity with the biological activity of a-lactalbumin cross-linked protein two polymer gel extraction system. By far UV circular two chromatography, ANS fluorescence spectroscopy and UV absorption spectroscopy comprehensive characterization alpha lactalbumin two cross-linked dimer structure, results show that, compared with bovine alpha lactalbumin, alpha lactalbumin cross-linked two dimer has a higher hydrophobicity and protein structure is more loose.
4. by in vitro simulated gastric and intestinal digestion, was systematically evaluated by digestion of a-lactalbumin protein cross-linking products, the study found that a-lactalbumin protein can easily be simulated gastric and intestinal digestion, and milk white a- protein cross-linking products are relatively resistant to digestion, especially the cross-linked polymer has a strong resistance the ability of enzyme, worthy of attention.
5. using ELISA method, the preliminary evaluation of the a-lactalbumin protein cross-linking products of potential allergenicity, research results show that, compared with the binding ability of alpha lactalbumin, alpha lactalbumin cross-linked product with IgG and IgE were significantly decreased, indicating that the polyphenol oxidase cross-linked a-la has reduced its egg white allergens the role of.

【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：TS252.2;R155.5

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