【摘要】：β-乳球蛋白(β-lactoglobulin)是存在于牛乳機牛乳制品中的主要過敏原之一,母乳中并不存在,絕大多數(shù)的嬰幼兒對其是存在過敏現(xiàn)象的。β-乳球蛋白是一種以二聚體形式存在的球形蛋白,這種空間結(jié)構(gòu)賦予了它很好的穩(wěn)定性以及潛在的致敏特性,同時β-乳球蛋白的二聚體形式對于酸水解以及酶水解具有足夠強的抗拒性。在人體內(nèi)經(jīng)過消化道的消化后仍然保持完整結(jié)構(gòu)進(jìn)而被小腸粘膜吸收,刺激人體的免疫細(xì)胞,最后引起過敏反應(yīng)。β-乳球蛋白的線性表位、構(gòu)象表位和消化水解后的肽段三個方面共同決定了其致敏性,在本課題組前期的研究基礎(chǔ)上我們發(fā)現(xiàn)動態(tài)高壓微射流(Dynamic high pressure microfluidization,DHPM)處理β-乳球蛋白能使其空間構(gòu)象發(fā)生不同程度的去折疊或聚集的變化,促使其過敏表位暴露或被包埋從而導(dǎo)致其理化特性和過敏性發(fā)生變化,但過敏表位改變后β-乳球蛋白對于酶的水解作用的抗拒性以及酶解產(chǎn)物又有何變化仍有待研究。因此,本文擬模擬體內(nèi)消化環(huán)境,研究改性后的β-乳球蛋白消化酶解反應(yīng)后結(jié)構(gòu)與過敏性變化的關(guān)系。為改性后的β-乳球蛋白酶解對其結(jié)構(gòu)和過敏變化提供理論參考。本論文的研究結(jié)果有以下三個方面:(1)動態(tài)高壓微射流處理改性后的β-乳球蛋白體外模擬消化過程中致敏性等功能性質(zhì)的研究。β-乳球蛋白經(jīng)過三種不同的壓力處理(0.1、80、160 MPa)后的體外模擬消化后的樣品,根據(jù)課題組的前期研究我們知道β-乳球蛋白的抗原反應(yīng)性是隨著處理壓力呈現(xiàn)先增加后減少的趨勢的,但是本實驗中消化后其抗原反應(yīng)性是逐漸降低的,水解度和SDS-PAGE都有著不同程度的影響。160MPa條件下的消化酶解效果較好,水解度為21.3%左右。在電泳圖中我們也發(fā)現(xiàn)其小分子段的條帶也較為明顯。(2)動態(tài)高壓微射流處理改性后的β-乳球蛋白體外模擬消化過程前后的微觀結(jié)構(gòu)的研究。結(jié)果表明,隨著實驗過程中的體外消化的進(jìn)行,β-乳球蛋白呈現(xiàn)出較為明顯的變化,在環(huán)境掃描電鏡和原子力顯微鏡中我們能明顯發(fā)現(xiàn)蛋白質(zhì)的聚集結(jié)構(gòu)明顯的有體積減少的趨勢,結(jié)合動靜態(tài)光散射實驗我們發(fā)現(xiàn)蛋白質(zhì)的分子隨著消化的進(jìn)行得到不同分子量段的聚集狀態(tài)。這些都可能和體外消化作用改變了蛋白質(zhì)的結(jié)構(gòu),影響了β-乳球蛋白之間的相互作用有關(guān)。(3)動態(tài)高壓微射流處理改性后的β-乳球蛋白體外模擬消化過程前后的分子結(jié)構(gòu)的研究。通過自由巰基含量、表面疏水性、CD、熒光和質(zhì)譜進(jìn)行分析,實驗結(jié)果表明,自有巰基和表面疏水性都是隨著體外消化呈現(xiàn)逐漸增加的趨勢,熒光譜圖我們發(fā)現(xiàn)熒光強度也有所增加出現(xiàn)了紅移現(xiàn)象,這可能與酶解作用酶切出更多的內(nèi)部的色氨酸有關(guān),同時CD光譜結(jié)果也表明蛋白質(zhì)的結(jié)構(gòu)發(fā)生了明顯的變化,尤其是β-乳球蛋白結(jié)構(gòu)的β-折疊減少以及α-螺旋含量的增加,質(zhì)譜實驗也表明,在不同的壓力條件下的消化能得到不同的肽段,在壓力為160 MPa條件下的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-螺旋結(jié)構(gòu)中,這些都可能影響β-乳球蛋白的CD結(jié)構(gòu)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R151

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