不同构象下β-乳球蛋白体外消化前后结构与过敏性的关系

发布时间：2019-05-19 18:17

【摘要】：β-乳球蛋白(β-lactoglobulin)是存在于牛乳机牛乳制品中的主要过敏原之一,母乳中并不存在,绝大多数的婴幼儿对其是存在过敏现象的。β-乳球蛋白是一种以二聚体形式存在的球形蛋白,这种空间结构赋予了它很好的稳定性以及潜在的致敏特性,同时β-乳球蛋白的二聚体形式对于酸水解以及酶水解具有足够强的抗拒性。在人体内经过消化道的消化后仍然保持完整结构进而被小肠粘膜吸收,刺激人体的免疫细胞,最后引起过敏反应。β-乳球蛋白的线性表位、构象表位和消化水解后的肽段三个方面共同决定了其致敏性,在本课题组前期的研究基础上我们发现动态高压微射流(Dynamic high pressure microfluidization,DHPM)处理β-乳球蛋白能使其空间构象发生不同程度的去折叠或聚集的变化,促使其过敏表位暴露或被包埋从而导致其理化特性和过敏性发生变化,但过敏表位改变后β-乳球蛋白对于酶的水解作用的抗拒性以及酶解产物又有何变化仍有待研究。因此,本文拟模拟体内消化环境,研究改性后的β-乳球蛋白消化酶解反应后结构与过敏性变化的关系。为改性后的β-乳球蛋白酶解对其结构和过敏变化提供理论参考。本论文的研究结果有以下三个方面:(1)动态高压微射流处理改性后的β-乳球蛋白体外模拟消化过程中致敏性等功能性质的研究。β-乳球蛋白经过三种不同的压力处理(0.1、80、160 MPa)后的体外模拟消化后的样品,根据课题组的前期研究我们知道β-乳球蛋白的抗原反应性是随着处理压力呈现先增加后减少的趋势的,但是本实验中消化后其抗原反应性是逐渐降低的,水解度和SDS-PAGE都有着不同程度的影响。160MPa条件下的消化酶解效果较好,水解度为21.3%左右。在电泳图中我们也发现其小分子段的条带也较为明显。(2)动态高压微射流处理改性后的β-乳球蛋白体外模拟消化过程前后的微观结构的研究。结果表明,随着实验过程中的体外消化的进行,β-乳球蛋白呈现出较为明显的变化,在环境扫描电镜和原子力显微镜中我们能明显发现蛋白质的聚集结构明显的有体积减少的趋势,结合动静态光散射实验我们发现蛋白质的分子随着消化的进行得到不同分子量段的聚集状态。这些都可能和体外消化作用改变了蛋白质的结构,影响了β-乳球蛋白之间的相互作用有关。(3)动态高压微射流处理改性后的β-乳球蛋白体外模拟消化过程前后的分子结构的研究。通过自由巯基含量、表面疏水性、CD、荧光和质谱进行分析,实验结果表明,自有巯基和表面疏水性都是随着体外消化呈现逐渐增加的趋势,荧光谱图我们发现荧光强度也有所增加出现了红移现象,这可能与酶解作用酶切出更多的内部的色氨酸有关,同时CD光谱结果也表明蛋白质的结构发生了明显的变化,尤其是β-乳球蛋白结构的β-折叠减少以及α-螺旋含量的增加,质谱实验也表明,在不同的压力条件下的消化能得到不同的肽段,在压力为160 MPa条件下的m/z 955.488β-乳球蛋白f(1 8)(LIVTQTMK)、m/z 837.444β-乳球蛋白f(142-148)(ALPMHIR)等,尤其是f(142-148)存在于β-乳球蛋白的α-螺旋结构中,这些都可能影响β-乳球蛋白的CD结构和致敏性。
[Abstract]:Human milk-lactoglobulin is one of the main allergens in milk cow milk products, and is not present in breast milk, and most of the infants are allergic to them. The L-lactoglobulin is a spherical protein that is present in the form of a dimer, which gives it a good stability and a potential sensitization characteristic, while the dimer form of the L-lactoglobulin has a sufficiently strong resistance to acid hydrolysis and enzymatic hydrolysis. After the digestion of the digestive tract in the human body, the whole structure is still kept to be absorbed by the intestinal mucosa of the small intestine, and the immune cells of the human body are stimulated, and finally, the allergic reaction is caused. The sensitivity of dynamic high pressure microfluidics was found on the basis of the previous study of the research group. The DHPM treatment of the L-lactoglobulin can cause different degree of defold or aggregation change of its spatial conformation, and cause the allergic epitope to be exposed or embedded to cause its physical and chemical properties and allergic reaction to change, However, the resistance to the hydrolysis of the enzyme and the change of the product of the enzyme are still to be studied after the change of the allergic epitope. Therefore, this paper is to simulate the in vivo digestion environment, and to study the relationship between the structure and the allergic reaction after the dereaction of the modified antigen-lactoglobulin digestive enzyme. To provide a theoretical reference for the structural and allergic changes of modified yeast-milk-ball proteolysis. The results of this study have the following three aspects: (1) the study of the functional properties of the sensitivity and the like in the in vitro simulation and digestion of the modified L-lactoglobulin in the dynamic high-pressure micro-jet treatment. After three different pressure treatments (0.1,80,160 MPa), the L-lactoglobulin is subjected to three different pressure treatment (0.1,80,160 MPa), and the sample is simulated and digested in vitro, and according to the previous study of the research group, we know that the antigen reactivity of the L-lactoglobulin is decreased after the treatment pressure is increased, However, the degree of hydrolysis and SDS-PAGE of the digestive enzyme in the experiment were decreased gradually, and the hydrolysis degree of the digestive enzyme was about 21.3% at 160 MPa. In the electropherogram, we also found that the bands of their small molecular segments were also significant. (2) The micro-structure of the modified L-lactoglobulin in vitro was studied by dynamic high-pressure micro-jet treatment. The results showed that, with the in-vitro digestion in the course of the experiment, the L-lactoglobulin showed a significant change, and in the environment scanning electron microscope and the atomic force microscope we can clearly find that the aggregation structure of the protein obviously has the tendency of reduced volume, In combination with dynamic and static light scattering experiments, we found that the molecules of the protein get together with the digestion to get the aggregation state of the different molecular weight segments. These and in vitro digestion changes the structure of the protein, affecting the interaction of the antigen-lactoglobulin. (3) The molecular structure of the modified L-lactoglobulin in vitro was studied by dynamic high-pressure micro-jet treatment. Through the analysis of the free base content, surface hydrophobicity, CD, fluorescence and mass spectrum, the experimental results show that the self-active matrix and the surface hydrophobicity are gradually increasing with the in vitro digestion, and the fluorescence spectrum shows that the fluorescence intensity also has an increase in the red shift phenomenon. This may be related to the enzymatic cleavage of more of the internal tryptophan, while the CD spectrum results also indicate a significant change in the structure of the protein, in particular the reduction of the level of the antigen-fold reduction and the increase in the content of the HCO3-helix, and the mass spectrum experiment also shows that, The digestion under different pressure conditions can result in different peptide segments, m/ z 955.488--lactoglobulin f (1 8) (LIVTQTMK), m/ z 837.444--lactoglobulin f (142-148) (ALPMHIR), and the like at a pressure of 160 MPa, in particular f (142-148), These may affect the CD structure and sensitization of the L-lactoglobulin.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

【参考文献】