多酚氧化酶交联牛乳α-乳白蛋白的结构变化及其消化性与过敏原性的评估

发布时间：2018-01-15 15:27

本文关键词：多酚氧化酶交联牛乳α-乳白蛋白的结构变化及其消化性与过敏原性的评估　出处：《南昌大学》2012年硕士论文　论文类型：学位论文

【摘要】：牛乳因其营养及美味而深受人们的喜爱,但牛乳又是FAO公认的八大类主要过敏食品之一,有高达8%的婴幼儿和2%的成年人对牛乳过敏。近年来研究发现,酶法交联牛乳可降低牛乳的过敏原性,为低致敏牛乳的研发提供了新的思路。牛乳a-乳白蛋白是引发牛乳过敏的主要过敏原之一,已有研究表明,有75%的牛乳过敏患者血清中含有α-乳白蛋白的特异性抗体,同时,α-乳白蛋白过敏原的构象性表位是其致敏的最主要原因之一。显而易见,以牛乳α-乳白蛋白为对象研究交联蛋白结构与过敏原性关系,具有重要的意义。本论文工作以牛乳α-乳白蛋白为对象,利用蘑菇多酚氧化酶催化交联牛乳α-乳白蛋白,探索α-乳白蛋白交联对其结构、消化性以及过敏原性的影响。研究内容包括：牛乳α-乳白蛋白的分离纯化及蘑菇多酚氧化酶的提取,多酚氧化酶催化交联牛乳α-乳白蛋白的加工工艺研究,牛乳α-乳白蛋白交联后的结构变化以及牛乳α-乳白蛋白交联产物的消化性及过敏原性评估。研究的主要方法、结果及结论如下。 1.建立了离子交换色谱层析联合凝胶色谱层析分离牛乳a-乳白蛋白的方法,纯度达90%,纯化得率为21.18%。同时,从双孢蘑菇中获得了多酚氧化酶酶液,酶活达6,000U/mL。 2.探索了α-乳白蛋白构象、酶促交联反应条件以及底物添加量对a-乳白蛋白交联产物生成的影响,并建立了多酚氧化酶催化交联a-乳白蛋白的最适反应体系：无钙状态下的α-乳白蛋白终浓度1mg/m,多酚氧化酶终浓度1,000U/mL,咖啡酸终浓度1mM,在50℃,pH7.0下,酶促反应8小时。 3.通过Western-Blotting鉴定了牛乳α-乳白蛋白交联二聚体,并且利用Native-PAGE切胶回收制备出纯度为80%以上的具有生物学活性的牛乳α-乳白蛋白交联二聚体。采用远紫外圆二色谱、ANS探针荧光光谱以及紫外吸收光谱技术综合表征了α-乳白蛋白交联二聚体的结构,结果表明,相对于牛乳α-乳白蛋白,α-乳白蛋白交联二聚体具有更高疏水性以及更加松散的蛋白结构。 4.通过体外模拟胃液、肠液消化,较系统地评价了牛乳α-乳白蛋白交联产物的消化性,研究发现牛乳α-乳白蛋白极易被模拟胃液和肠液消化,而牛乳a-乳白蛋白交联产物则相对耐消化,特别是交联的高聚物具有很强的耐酶解能力,值得高度关注。 5.利用ELISA方法初步评价了牛乳α-乳白蛋白交联产物的潜在过敏原性,研究结果表明,与α-乳白蛋白相比,α-乳白蛋白交联产物与IgG和IgE的结合能力均显著降低,这说明多酚氧化酶交联α-乳白蛋白具有降低它的过敏原性的作用。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TS252.2;R155.5

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