本文關(guān)鍵詞：多酚氧化酶交聯(lián)牛乳α-乳白蛋白的結(jié)構(gòu)變化及其消化性與過(guò)敏原性的評(píng)估　出處：《南昌大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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

【摘要】：牛乳因其營(yíng)養(yǎng)及美味而深受人們的喜愛(ài),但牛乳又是FAO公認(rèn)的八大類主要過(guò)敏食品之一,有高達(dá)8%的嬰幼兒和2%的成年人對(duì)牛乳過(guò)敏。近年來(lái)研究發(fā)現(xiàn),酶法交聯(lián)牛乳可降低牛乳的過(guò)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(guò)敏的主要過(guò)敏原之一,已有研究表明,有75%的牛乳過(guò)敏患者血清中含有α-乳白蛋白的特異性抗體,同時(shí),α-乳白蛋白過(guò)敏原的構(gòu)象性表位是其致敏的最主要原因之一。顯而易見(jiàn),以牛乳α-乳白蛋白為對(duì)象研究交聯(lián)蛋白結(jié)構(gòu)與過(guò)敏原性關(guān)系,具有重要的意義。 本論文工作以牛乳α-乳白蛋白為對(duì)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(duì)其結(jié)構(gòu)、消化性以及過(guò)敏原性的影響。研究?jī)?nèi)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(jié)構(gòu)變化以及牛乳α-乳白蛋白交聯(lián)產(chǎn)物的消化性及過(guò)敏原性評(píng)估。研究的主要方法、結(jié)果及結(jié)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(dá)90%,純化得率為21.18%。同時(shí),從雙孢蘑菇中獲得了多酚氧化酶酶液,酶活達(dá)6,000U/mL。 2.探索了α-乳白蛋白構(gòu)象、酶促交聯(lián)反應(yīng)條件以及底物添加量對(duì)a-乳白蛋白交聯(lián)產(chǎn)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(yīng)體系：無(wú)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(yīng)8小時(shí)。 3.通過(guò)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(xué)活性的牛乳α-乳白蛋白交聯(lián)二聚體。采用遠(yuǎn)紫外圓二色譜、ANS探針熒光光譜以及紫外吸收光譜技術(shù)綜合表征了α-乳白蛋白交聯(lián)二聚體的結(jié)構(gòu),結(jié)果表明,相對(duì)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(jié)構(gòu)。 4.通過(guò)體外模擬胃液、腸液消化,較系統(tǒng)地評(píng)價(jià)了牛乳α-乳白蛋白交聯(lián)產(chǎn)物的消化性,研究發(fā)現(xiàn)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(chǎn)物則相對(duì)耐消化,特別是交聯(lián)的高聚物具有很強(qiáng)的耐酶解能力,值得高度關(guān)注。 5.利用ELISA方法初步評(píng)價(jià)了牛乳α-乳白蛋白交聯(lián)產(chǎn)物的潛在過(guò)敏原性,研究結(jié)果表明,與α-乳白蛋白相比,α-乳白蛋白交聯(lián)產(chǎn)物與IgG和IgE的結(jié)合能力均顯著降低,這說(shuō)明多酚氧化酶交聯(lián)α-乳白蛋白具有降低它的過(guò)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：TS252.2;R155.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 沈星燦,梁宏,何錫文,王新省;圓二色光譜分析蛋白質(zhì)構(gòu)象的方法及研究進(jìn)展[J];分析化學(xué);2004年03期

2 張國(guó)文;王安萍;蔣婷;闕青民;;熒光光譜法研究橙皮苷與牛血清白蛋白相互作用特征[J];分析試驗(yàn)室;2008年01期

3 趙聰敏;李為明;崔守信;;乳糖吸收不良和不耐癥[J];國(guó)外醫(yī)學(xué)(兒科學(xué)分冊(cè));1992年03期

4 邵潔;夏振煒;李云珠;俞善昌;;IgE介導(dǎo)的食物過(guò)敏診斷程序及臨床評(píng)價(jià)[J];臨床兒科雜志;2007年01期

5 劉心偉;呂加平;范貴生;;微生物轉(zhuǎn)谷氨酰胺酶在食品工業(yè)中的研究進(jìn)展[J];內(nèi)蒙古農(nóng)業(yè)大學(xué)學(xué)報(bào)(自然科學(xué)版);2005年04期

6 布冠好;鄭U，

本文編號(hào)：1428951