牛乳β-乳球蛋白过敏原表位及其潜在应用

过敏原表位是过敏原直接参与免疫反应的物质基础, 也是导致过敏反应的“元凶”。牛乳β-乳球蛋白是lipocalin家族的代表和常用模式蛋白, 它的表位信息比较完整, 拥有7个人血清IgE和6个IgG的线性表位、3个IgE结合的构象性表位以及7个T细胞表位。另外, 它的动物源血清IgE、IgG和T细胞表位也在不断被发现。牛乳β-乳球蛋白的表位信息为基于表位的应用提供了重要的支撑, 它们的应用主要涉及到预测食物过敏原的致敏性, 食物过敏的交叉反应, 食物过敏的诊断, 食物过敏的免疫治疗以及食物过敏原的检测等5个方面。     

牛乳α-乳白蛋白IgE线性表位的关键氨基酸识别

α-乳白蛋白是引起牛乳过敏的主要过敏原之一。识别α-乳白蛋白作用表位及影响致敏性的关键氨基酸,对于揭示α-乳白蛋白致敏机理及低致敏乳制品的开发具有重要的意义。本研究采用固相合成技术合成α-乳白蛋白系列多肽,以牛乳过敏患者血清为探针,通过酶联免疫吸附分析法识别α-乳白蛋白的作用表位和关键氨基酸。结果表明:免疫球蛋白(immunoglobulins,Ig)E作用表位的氨基酸序列定位为aa1-15、aa6-20、aa46-60、aa71-85和aa101-115。α-乳白蛋白IgE作用表位关键氨基酸为第8位的缬氨酸、第9位的苯丙氨酸、第10位的精氨酸、第103位的酪氨酸、第105位的亮氨酸和第107位组氨酸。本研究可以为过敏原c DNA克隆以激活T细胞、降低IgE结合能力提供重要思路。     

牛乳α-乳白蛋白免疫球蛋白G线性表位的关键氨基酸识别

α-乳白蛋白是牛乳中的主要过敏原,开展α-乳白蛋白表位定位及氨基酸特性研究可深入了解过敏原的致 敏机理,有助于更好地认识免疫球蛋白G（immunoglobulin G,IgG）在乳过敏反应中的作用,为制备低过敏乳制 品提供理论指导。本研究采用丙氨酸免疫表位扫描技术识别α-乳白蛋白的关键氨基酸,即用牛乳过敏患者血清中 IgG识别α-乳白蛋白系列合成的多肽,筛选作用表位,然后用丙氨酸依次取代作用表位的氨基酸合成新的多肽,以 牛乳过敏患者血清池为抗体识别关键氨基酸。结果表明：α-乳白蛋白IgG作用表位的氨基酸序列定位为aa6～20、 aa21～35、aa36～50和aa86～100;关键氨基酸为第9位的苯丙氨酸、第15位的亮氨酸、第24位的脯氨酸、第26位的 色氨酸和第32位的组氨酸。     

牛乳β-乳球蛋白过敏原线性表位串联体的分子设计

本实验以牛乳β-乳球蛋白中与人血清IgE结合的7个B细胞表位(B1、B2、B3、B4、B5、B6和B7)和1个T细胞表位(T)为研究对象,旨在使设计出的牛乳β-乳球蛋白多表位串联体分子中各表位能够保留独立的抗原性。按照表位串联体设计原则,B细胞表位之间的连接序列为甘氨酸(G),T细胞与B细胞表位之间的连接序列为两个赖氨酸(KK)。通过生物信息学的研究,借助DNAStar软件和SOMPA网络服务器,对这8个表位连接顺序进行优化组合后,设计出的串联体分子组合结构为:T-K-K-B1-G-B6-G-B5-G-B3-G-B7-G-B2-G-B4,其氨基酸序列是:KIPAVFKIDALNENKVLVLDTKKLIVTQTMKGEVDDEALEKFDKALKALPGKTKIPAVFKIDAGKPTPEGDLEILLQKGKALPMHIRLSFNGLLDAQSAPLRVYVEELKPGAQKKIIAEKTKI。经过预测,该串联体中各表位均呈现出了抗原性,且没有新的表位产生。研究结果表明,分子设计正确,研究方法值得同类工作借鉴。     

具有T细胞口服免疫耐受作用的β-乳球蛋白水解物的制备及鉴定

以牛乳中主要过敏原β-乳球蛋白为研究对象,制备和鉴定具有T细胞表位和口服免疫耐受性的水解物,旨在为牛乳过敏患者口服免疫治疗方法提供理论依据。采用生物信息学方法预测β-乳球蛋白的T细胞表位,通过质谱分析6种蛋白酶水解β-乳球蛋白水解物的氨基酸序列,用T细胞增殖实验鉴定水解物中多肽免疫耐受作用,体内实验测定小鼠血清特异性抗体(IgE、IgG₁、IgG_2a)、Th1细胞因子(IFN-γ、IL-17)、Th2细胞因子(IL-4、IL-5、IL-13)、组胺、几丁质酶-3样蛋白1的水平及小鼠脾脏细胞亚群的变化水平,验证β-乳球蛋白水解物的口服免疫治疗活性。研究结果表明：胰蛋白酶、复合蛋白酶和木瓜蛋白酶水解物具有口服耐受性,其中复合蛋白酶和木瓜蛋白酶水解物具有口服耐受性是创新性发现。中性蛋白酶水解物虽然含有T细胞表位,但是仍然具有致敏性。因此含有T细胞表位的水解物不一定具有口服免疫耐受性,需要体内实验验证。研究结论以期为新型抗过敏乳基料的开发和临床治疗牛乳过敏提供参考数据。     

糖基化修饰牛乳清蛋白过敏原性研究进展

牛乳含有丰富的营养物质,是除母乳外婴儿最理想的食品来源。但是,部分婴儿对牛乳会产生过敏反应。通过美拉德反应对乳清蛋白进行糖基化改性是一种比较新的方法,但已经成为食品中研究的热点。本文对牛乳中主要过敏原的结构特征、致敏性、表位等进行阐述,详细地介绍乳清蛋白中α-乳白蛋白和β-乳球蛋白糖基化改性的国内外研究进展。糖基化法作为降低乳清蛋白致敏性的方法之一,能较好地保持其原有结构和功能特性。     

牛乳中主要蛋白过敏原研究现状

牛乳蛋白过敏原的研究成为近几年国内外的研究热点之一.依据有关牛乳中过敏原的研究报道.对牛乳中的主要过敏原酪蛋白、B-乳球蛋白(p-LG)及á一乳白蛋白(á-LA)的结构特征、表位、改性等的研究现状进行介绍,并对牛乳蛋白过敏原的研究进行展望.     

生物法制备低过敏乳的研究进展

牛乳中的乳蛋白绝大多数潜在过敏原,致使部分过敏体质的人群在摄入牛乳时发生过敏现象.降低牛乳蛋白过敏性的方法主要是通过物理法、化学法和生物法破坏蛋白质的过敏表位.与物理法、化学法比较,生物法不仅能更有效降低牛乳的过敏性,还可以赋予食品更多典型风味.文章主要介绍牛乳中过敏蛋白的组成及过敏表位,重点阐述两种制备低过敏乳的生物法(酶法水解和乳酸菌发酵法)及其研究进展,为制备低过敏乳制品提供参考依据.     

水牛乳中主要过敏原的分离纯化

牛乳和水牛乳存在免疫交叉反应,牛乳过敏患者可能对水牛乳过敏。因此,分离纯化出水牛乳中各种过敏原将为进一步的工作奠定物质基础。实验中以摩拉水牛乳为原料,通过等电点沉淀、凝胶柱层析法和阴离子交换法分离纯化水牛乳的酪蛋白、β-乳球蛋白α-乳白蛋白,得到了SDS-PAGE纯(纯度≥90%)的β-乳球蛋白α-乳白蛋白。离子交换层析分离β-乳球蛋白、α-乳白蛋白的得率分别为50.26%、52.86%;凝胶层析分离β-乳球蛋白、α-乳白蛋白的得率分别为49.39%、84.19%。实验结果表明,等电点沉淀、凝胶柱层析法和阴离子交换法适合于分离水牛乳中主要过敏原成分。     

牛乳β-乳球蛋白变异体检测方法研究进展

β-乳球蛋白(β-LG)是牛乳中重要的天然活性营养物质,也是牛乳中主要的过敏原之一。β-乳球蛋白约为乳清蛋白总量的50%,是牛乳中主要的乳清蛋白,具有较强的热敏感性和致敏性,在优质乳品工业中起关键作用。其不仅作为牛奶热处理强度的评估指标,还作为食品中牛乳过敏原的标志蛋白,有效识别和检测牛乳β-乳球蛋白非常重要。已报道的牛乳β-乳球蛋白有13种变异体,其中A和B是牛乳β-LG的常见变异体,且含量最高。根据不同的检测原理,文章简述近年来牛乳β-乳球蛋白变异体的三大类检测方法,总结电泳法、色谱法和免疫法的原理、优缺点及部分应用实例,重点介绍液相色谱法和毛细管电泳法两种定量方法,并展望牛乳β-乳球蛋白未来的发展方向。     

牛奶过敏原表位研究进展

详细地综述了牛乳酪蛋白、α-乳白蛋白、β-乳球蛋白、牛血清白蛋白的过敏原B细胞表位以及T细胞表位的研究进展,介绍了牛乳过敏原之间构象性表位的相似形以及线性表位序列位置的特异性,同时也简述了牛乳过敏原表位定位的方法及表位定位的应用。     

β-乳球蛋白IgE抗原决定簇的识别

以β- 乳球蛋白氨基酸序列为模板,错位合成β- 乳球蛋白多肽,以收集到的牛乳过敏患者血清为抗体,鉴定β- 乳球蛋白IgE 抗原决定簇,分析影响致敏性的关键氨基酸,探讨牛乳过敏机理。结果表明：β- 乳球蛋白IgE抗原决定簇有4 条,氨基酸序列分别为17～31、72～86、92～106、152～166,并且苏氨酸(AA20)、蛋氨酸(AA23)、天冬氨酸(AA27)是影响β- 乳球蛋白致敏性的关键氨基酸。说明特异性水解抗原决定簇或定点修饰氨基酸可以实现过敏原脱敏的目的。     

Proteolytic activities of combined fermentation with Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 reduce antigenic response to cow milk proteins

Cow milk protein is one of the leading food allergens. This study aimed to develop an effective method for reducing milk sensitization by evaluating antigenicity of fermented skim milk protein using Lactobacillus helveticus KLDS 1.8701, Lactobacillus plantarum KLDS 1.0386, and a combination of both strains. The proteolytic systems of strains in terms of genotype and phenotype are characterized by complete genome sequence, and evaluation the antigenicity of skim milk proteins was determined by ELISA and liquid chromatography with tandem mass spectrometry. Our results showed that the genomes encoded a variety of peptidase genes. For fermented skim milk, the degree of hydrolysis of the combined strains was higher than that of individual strain. Electrophoresis showed that the band color density of α-casein (α-CN) by fermentation of the combined strains was reduced when compared with control group. The fermentation process of the combined strains inhibited α-CN, β-lactoglobulin, and α-lactalbumin antigenicity by 69.13, 36.10, and 20.92, respectively. Major allergic epitopes of α-CN and β-lactoglobulin were cleaved by abundant proteases of combined strains. In all, this study showed that the fermentation process involving both L. helveticus and L. plantarum strains could reduce cow milk protein allergenicity through the combination of cell-envelope proteinase and peptidase on α-CN.     

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92–100, 125–135, 125–138, and 149–162) in β-LG, 2 peptides in α-LA (AA 80–93 and 63–79), 2 peptides in α_S1-casein (AA 84–90 and 125–132), and 1 peptide (AA 25–32) in α_S2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.     

Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes

The purpose of this study was to determine the best preparation condition of β-lactoglobulin hydrolysate-iron complexes and characterize its structural transformation both before and after binding using the UV-visible absorption spectrum, Fluorescence spectrum, and Fourier transform infrared spectroscopy. Results showed that β-lactoglobulin hydrolysates obtained with alcalase after hydrolysis for 6h possessed the highest iron-binding capacity. The highest yield of complexes was obtained when the mass ratio between β-lactoglobulin hydrolysate and Fe(3+) reached 40:1, with the optimal pH value of 7.0. All of the spectra indicated that some sites such as amido bonds transformed during chelation, and nitrogen atoms could chelate with Fe(3+) to form coordinate bonds by offering electron pairs. Therefore, β-lactoglobulin hydrolysate-iron complexes may be good carriers for iron and possess great potential to be used as iron supplements.     

鸡蛋致敏原溶菌酶的B细胞IgG线性表位定位

目的 探究鸡蛋致敏原溶菌酶的B细胞线性表位。方法 先使用生物信息学工具对其氨基酸序列分析，预测其潜在的B细胞线性表位，同时合成覆盖其完整氨基酸序列的重叠肽，然后利用斑点杂交法（dot-blot）筛选出与抗溶菌酶兔血清IgG特异性结合的多肽片段，从而定位出溶菌酶的B细胞IgG线性表位。结果 通过生物信息学工具综合分析预测出了鸡蛋溶菌酶的4个B细胞线性表位，分别为AA18-21（DNYR）、AA39-51（NTQATNRNTDGST）、AA62-78（WWCNDGRTPGSRNLCNI）、AA109-117（VAWRNRCKG）；利用dot-blot定位出5个鸡蛋溶菌酶的B细胞IgG线性表位，分别为AA1-15（KVFGRCELAAAMKRH）、AA28-30（WVC）、AA70-78（PGSRNLCNI）、AA103-117（NGMNAWVAWRNRCKG）、AA124-126（IRG）。研究结果表明，采用生物信息学工具预测的B细胞线性表位与已知的溶菌酶的B细胞IgE线性表位的重合率达75%，与用dot-blot定位的B细胞IgG线性表位的重合率达40%，一定程度上证实了利用生物信息学预测致敏原表位的可行性，但预测结果的准确性仍需进一步验证。结论 本研究确定的溶菌酶B细胞线性表位可为进一步开展溶菌酶的精准检测和低致敏食品等研发工作提供关键的结构信息。     

Effect of ultraviolet irradiation on molecular properties and immunoglobulin production-regulating activity of β-lactoglobulin

To utilize ultraviolet (UV) irradiation as a means to prepare hypo-allergenic food, we investigated the molecular weight profile, secondary structure content, fluorescence spectrum, and immunoglobulin (Ig) production-regulating activity of β-lactoglobulin after UV-irradiation. UV-irradiation caused a change on the molecular size distribution, disruption of the ordered structure, and decrease of emission intensity of β-lactoglobulin. The alteration on Igs production regulating activity of β-lactoglobulin by UV-irradiation was observed in the mouse spleen lymphocytes system. These results suggest that UV-irradiation is effective for alteration of molecular properties and antigenicities of β-lactoglobulin and such treatment should be useful for the preparation of low allergenic foods.     

Genetic variants of bovine β- and κ-casein result in different immunoglobulin E-binding epitopes after in vitro gastrointestinal digestion

Immunoglobulin E-mediated allergy to cow milk is a common allergy in industrialized countries, mainly affecting young children and infants. β-Casein (CN) and κ-CN belong to the major allergens in cow milk. Within these milk proteins, genetic polymorphisms occur, which are characterized by substitutions or deletions of AA, resulting in different variants for each protein. Until now, these variants have not been considered when discussing the allergenic potential of bovine milk. In this study, the focus was placed on the arising peptide pattern after in vitro gastrointestinal digestion of several β- and κ-CN variants to determine resistant fragments containing IgE-binding epitopes and to identify potential differences between these variants. β-Casein A¹, A², and B, as well as κ-CN A, B, and E, were separated and isolated from milk of cows homozygous for these variants and digested with an in vitro gastrointestinal digestion model. The resulting peptides were identified using mass spectrometry and compared with previously determined epitopes. Seven β-CN and 4 κ-CN peptides, common in all β- or κ-CN variants, remained of sufficient size to harbor IgE-binding epitopes. In addition, some peptides and, consequently, epitopes differ from each other due to the AA substitution occurring in the individual variants. The distinct peptides AA 108 to 129 of β-CN A¹ and A², AA 103 to 123 of β-CN B, as well as AA 59 to 72, AA 59 to 80, and AA 58 to 80 of all 3 β-CN variants correspond to the IgE-binding epitopes AA 107 to 120 and AA 55 to 70, respectively. In κ-CN, the 2 variant-specific peptides AA 136 to 149 (κ-CN A, E) and AA 134 to 150 (κ-CN B) are congruent with the IgE-binding epitope AA 137 to 148. The present study shows that genetic polymorphisms affected the arising peptide pattern of the caseins and thus modifications in the IgE-binding epitopes occurred. As a consequence, the casein variants could show differences in their allergenicity. Studies investigating the allergenic potential of these different peptides are currently in progress.     

Kinetics of Formation and Physicochemical Characterization of Thermally-Induced β-Lactoglobulin Aggregates

Abstract: The kinetics of heat denaturation and aggregation for β-lactoglobulin dispersions (5% w/v) were studied at 3 pHs (6, 6.4, and 6.8) and at a heating temperature of 80 °C. Protein aggregates were characterized for hydrodynamic diameter, microstructure, and molecular weight by means of dynamic light scattering, transmission electron microscopy, and polyacrylamide gel electrophoresis, respectively. Concentration of native β-lactoglobulin decreased with holding time and with a decrease in the pH. Apparent rate constants were calculated for β-lactoglobulin denaturation applying the general kinetic equation solved for a reaction order of 1.5. Values of the apparent reaction rate constant k = 7.5, 6.3 and 5.6 × 10⁻³ s⁻¹ were found for pH 6, 6.4, and 6.8, respectively. Decreasing the pH of the dispersions produced higher aggregate sizes. After a holding time of 900 s, average hydrodynamic diameters for β-lactoglobulin aggregates at pH 6, 6.4, and 6.8 were 96, 49, and 42 nm, respectively. These results were confirmed by transmission electron microscopy images, where a shift in the size and morphology of aggregates was found, from large and spherical at pH 6 to smaller and linear aggregates at pH 6.8. β-Lactoglobulin formed disulfide-linked intermediates (dimers, trimers, tetramers) and so on) which then formed high molecular weight aggregates. From the results obtained by DLS, TEM, and SDS-PAGE a mechanism for β-lactoglobulin aggregation was proposed. This study shows that heat treatment can be used to produce protein aggregates with different sizes and morphologies to be utilized as ingredients in foods.     

Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.