降低牛乳中β-乳球蛋白致敏性方法的研究进展

摘要：牛乳过敏（CMA）是婴幼儿中一种常见的食物过敏,严重危害婴幼儿的身体健康。β-乳球蛋白（β-LG）是牛乳中重要营养物质,但却是引起牛乳蛋白过敏的主要过敏原。目前,降低牛乳中 β-LG 致敏性已经成为国内外研究热点之一。对CMA机制进行了介绍,综述了降低β-LG致敏性的常用方法,包括物理改性如热处理、高压处理、超声波处理,化学改性如糖基化、β-LG与脂肪酸结合、β-LG与多酚结合,酶法改性以及联合改性如物理改性联合酶水解改性、动态高压微射流技术联合脂肪酸处理、酶水解联合糖基化处理,旨在为生产低致敏性食品提供一定的理论依据。     

降低牛乳蛋白致敏性的改性方法研究进展

近年来,牛乳过敏的发病率增加,引起了国内外的广泛关注,已经成为研究热点之一。牛乳中的蛋白质是引起过敏的主要原因,其中酪蛋白、β-乳球蛋白、以及α-乳白蛋白是主要过敏原。研究概述了牛乳蛋白过敏的免疫病理机制以及引起牛乳蛋白过敏的主要过敏原,主要介绍了目前国内外研究中采用的改性方法的技术进展,包括热处理、糖基化、高压处理、酶法水解等,同时列举了诸多实例,并对其进行分析总结。通过改性方法可以一定程度上降低致敏性,但是关于过敏机理、适用性以及改性过程中是否会出现新的过敏原表位的问题仍然需要进一步的研究。     

山羊乳与牛乳配方乳粉的致敏性比较

山羊乳是一类营养丰富且易于消化的乳品,因其营养成分与牛乳相近而受到广泛关注。本研究通过蛋白质潜在致敏性树状评估策略（生物信息学、消化稳定性、血清学研究、动物模型）对山羊乳配方乳粉的致敏性进行评价,并研究山羊乳配方乳粉与牛乳过敏患者血清中免疫球蛋白（immunoglobulin,Ig）E之间存在的交叉反应性。结果表明,山羊乳配方乳粉的167 种蛋白质中,β-乳球蛋白和α-乳清蛋白含量显著低于牛乳配方乳粉（P＜0.05）,山羊乳配方乳粉在模拟胃液中更易消化,且与过敏患者血清的结合能力弱于牛乳配方乳粉,动物模型结果表明山羊乳配方乳粉致敏小鼠体温下降幅度、临床过敏症状评分、血清中特异性IgE和IgG1抗体水平、血浆中组胺水平、血管渗透性及组织炎症程度等均显著低于牛乳配方乳粉组致敏小鼠（P＜0.05）,交叉反应性结果表明山羊乳配方乳粉能与牛乳过敏患者血清结合,但结合能力明显低于牛乳配方乳粉。结论：与牛乳配方乳粉相比,山羊乳配方乳粉的致敏性较弱,可作为牛乳的替代品,降低牛乳过敏性疾病的发病风险。     

加工对牛乳过敏原的影响

简单介绍了牛乳中主要过敏原成分β-乳球蛋白、酪蛋白、α-乳白蛋白的生化性质及其过敏原表位.详细陈述了加热、加压、酶解、糖基化、发酵等加工方法对牛乳过敏原的影响,对开发无过敏或低过敏的乳制品具有一定指导作用.     

加热对牛乳蛋白过敏原的影响

对牛乳中主要过敏原β-乳球蛋白、α-乳白蛋白和酪蛋白进行了介绍,分析了在加热过程中牛乳过敏原的物理变化和化学改性,详细论述了加热对牛乳过敏原抗原性的影响及作用机理.     

牛乳中主要过敏原致敏机理及其脱敏技术的研究进展

牛乳蛋白过敏是婴幼儿最普遍的一类食物过敏,主要是由于牛乳中αs1-酪蛋白、α-乳白蛋白和β-乳球蛋白引起的免疫应答,可引发呕吐、腹泻甚至特应性皮炎等症状,严重影响了婴幼儿的生长发育。本文主要从牛乳蛋白过敏机制、脱敏方法和应用及其国内外抗过敏配方奶粉的研究进展进行了概述,并展望了牛乳脱敏技术的重点研究方向。     

牛乳过敏原β-乳球蛋白检测方法的研究进展

目的食物过敏已成为全球性的食品安全问题,欧美等发达国家均要求对食品中的过敏原成分进行标识,其中就包括作为八大过敏性食物之一的牛乳及其乳制品。β-乳球蛋白是牛乳中的主要过敏原,约占乳清蛋白的50%,牛乳总蛋白的10%,并且约有82%的牛乳过敏患者对β-乳球蛋白过敏,因此其可以作为检测食品中是否含牛乳蛋白的一个有效的标志物。建立灵敏、可靠的β-乳球蛋白检测方法,对牛乳过敏原标识及保障牛乳过敏人群的安全消费具有重要意义。本文主要综述了牛乳β-乳球蛋白的高效液相色谱法、超高效液相色谱法、液相色谱-质谱联用法、酶联免疫吸附法、免疫层析法、电化学免疫法、蛋白微阵列法、等离子体共振法等色谱学和免疫学检测方法的研究进展,并对未来发展方向作出展望,以期促进牛乳β-乳球蛋白等过敏原检测方法的研究与开发。     

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。     

水牛乳蛋白质的组成

分析了摩拉水牛（M）、尼里-拉菲水牛（N）、一代杂交水牛（F1）、二代杂交水牛（F2）和高代杂交水牛（Fh）5个品代水牛的乳蛋白主要组分的相对百分比含量．同时分析了总氨基酸组成及钙、磷含量。结果表明，水牛乳蛋白的主要组分有：α-乳清蛋白（α-LA）、β-乳球蛋白（β-LG）、免疫球蛋白轻链（IgG—L）和重链（IgG—H）、αs1-酪蛋白（αs1-CN）、αs2-酪蛋白（αs2-CN），β-酪蛋白（β-CN）、κ-酪蛋白（κ—CN）、血清白蛋白（SA）和乳铁蛋白（LF）等；CN在水牛乳蛋白中占优势，与荷斯坦牛乳相比，水牛乳中CN的质量分数稍低，而且各品代水牛乳中的CN有显著性差异（P〈0．05）；乳清蛋白中β-LG含量最高；杂交水牛乳蛋白高于纯种摩拉水牛和尼里一拉菲水牛，差异显著（P〈0．05）：各品代水牛乳的氨基酸比例比较接近；不同品代水牛乳中钙、磷含量没有显著性差异。     

不同热加工工艺对巴氏杀菌乳中乳清蛋白的影响

以牛乳乳清蛋白为研究对象,探究不同热加工工艺（72 ℃/15 s、75 ℃/15 s、80 ℃/15 s、85 ℃/15 s）对巴氏杀菌乳乳清蛋白中3 种活性蛋白（α-乳白蛋白、β-乳球蛋白和乳铁蛋白）的影响,以及测定并分析杀菌温度对各样品菌落总数和嗜冷菌的影响。结果表明：随着热加工强度的提升,牛乳中的菌落总数随之减少,当杀菌温度在80 ℃以上时牛乳中的菌落总数小于10 CFU/mL;当杀菌温度在72 ℃以上时样品中的嗜冷菌数均小于10 CFU/mL;72 ℃/15 s 和75 ℃/15 s对α-乳白蛋白、β-乳球蛋白和乳铁蛋白影响较小,当杀菌温度达到80 ℃以上时,巴氏杀菌乳中的α-乳白蛋白、β-乳球蛋白和乳铁蛋白含量显著下降（P＜0.05）。综上,热加工的时间和温度与乳清蛋白的关系密切,72～75 ℃/15 s 的热加工工艺能更好地保留乳清蛋白中的3 种活性蛋白。     

Concentrations of undeclared allergens in food products can reach levels that are relevant for public health

Food products can become contaminated with food allergens due to cross-contact. Precautionary ‘may contain’ labelling may alert to the possible presence of an allergen, but guidance for such labelling is lacking. As a result, allergy information on the packaging may not be reliable and allergic consumers might be at risk of allergic reactions after consuming unlabelled, but indeed contaminated, products. Recently, a cow's milk protein allergic patient experienced a severe allergic reaction to a dark chocolate product containing undeclared milk proteins. This case induced the authors to investigate to what extent allergen concentrations of unlabelled products reach levels that are of public health relevance. The concentrations of milk proteins in the complaint sample and a collection of products of other batches and brands purchased from different stores were analysed. Together with appropriate threshold and food consumption data, the risks of allergic reactions and the severity of these reactions within the adult milk-allergic population were determined using probabilistic risk assessment techniques. The results show that milk protein concentrations in unlabelled products reach levels that may elicit allergic reactions in up to 68% of the adult allergic consumers. Therefore, concentrations of allergens in unlabelled products could reach levels that are of public health relevance. Application of probabilistic risk assessment can be an aid in revealing the public health consequences of undeclared allergens in food, in risk management decision-making and developing guidance in terms of risk-based concentration limits for precautionary labelling.     

牛奶过敏原制备方法的研究和免疫活性鉴别

目的探索牛奶主要过敏原的制备工艺。方法采用等电点沉淀、凝胶层析和分子筛等技术纯化牛奶中主要过敏原组分;采用SDS-PAGE鉴定蛋白纯度,采用双抗原夹心-ELISA鉴定免疫活性。结果成功获得牛奶中的四种主要过敏原组分:酪蛋白、β乳球蛋白、α乳白蛋白和分子量较高的P1组分,并经过免疫实验证实这四种组分均能与过敏血清产生反应,而其中β乳球蛋白和P1组分反应性较高。结论本研究探索出一种简单、实用的牛奶主要过敏原制备工艺,并证实牛奶中的β乳球蛋白和高分子量蛋白质为主要过敏原。     

Bovine Milk Allergens: A Comprehensive Review

Cow milk allergy is one of the most common food allergies in early childhood and often persists through adult life, forcing an individual to a complete elimination diet. Milk proteins are present in uncounted food products, such as cheese, yogurt, or bakery item, exposing allergic persons to a constant threat. Many efforts have been made to overcome this global problem and to improve the life quality of allergic individuals. First, proper and reliable food labeling is fundamental for consumers, but the verification of its compliance is also needed, which should rely on accurate and sensitive analytical methods to detect milk allergens in processed foods. At the same time, strategies to reduce milk allergenicity, such as immunotherapy or the use of food processing techniques to modify allergen structure, have to be extensively studied. Recent research findings on the applicability of food processing, such as heat treatment, fermentation, or high pressure, have revealed great potential in reducing milk allergenicity. In this review, significant research advances on cow milk allergy are explored, focusing on prevalence, diagnosis, and therapy. Molecular characterization of cow milk allergens and cross‐reactivity with other nonbovine milk species are described, as well as the effects of processing, food matrix, and digestibility on milk allergenicity. Additionally, analytical methods for the detection of milk allergens in food are described, from immunoassays and mass spectrometry methods for protein analysis to real‐time polymerase chain reaction for DNA analysis.     

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.     

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。     

Milk allergens, their characteristics and their detection in food: A review

Cow's milk allergy (CMA) is one of the most common food allergies in childhood. This allergy is normally outgrown in the first year of life, however 15% of allergic children remain allergic. Many studies have been carried out to define and characterise the allergens involved in CMA and described two major allergens: casein (αs1-CN) and β-lactoglobulin. In addition to this, many other milk proteins are antigenic and capable of inducing immune responses. Milk from sheep or goats differs from cow's milk (CM) in terms of composition and allergenic properties. Food processing such as heating affects the stability, structure and intermolecular interactions of CM proteins, thereby changing the allergenic capacity. Chemical and proteolytic treatments of milk to obtain milk hydrolysates have been developed to reduce allergic reactions. Prevention of CMA largely relies on avoidance of all food products containing cow's milk. To achieve this, interest has focused on the development of various technologies for detecting and measuring the presence of milk allergens in food products by immunoassays or proteomic approaches. This review describes the technologies implemented for the analysis of milk allergens (allergenicity, biochemistry) as well as their potential detection in food matrices.     

牛奶过敏原的分离、鉴定与纯化

对牛奶过敏原进行分离、鉴定与纯化。通过SDS-PAGE电泳分离牛奶的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对牛奶过敏原进行初步纯化。结果表明,鲜牛奶粗提液SDS-PAGE显示蛋白条带有12条,奶粉粗提液SDS-PAGE显示出的蛋白条带与鲜牛奶的蛋白条带基本一致。鲜牛奶Western-Blotting显示14ku的阳性条带。离子交换层析可初步纯化出14ku的过敏原蛋白。本研究对牛奶过敏原进行了分离和鉴定,并初步纯化出牛奶的主要过敏原。     

Update on optimized purification and characterization of natural milk allergens

Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.     

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins

BACKGROUND: The main whey proteins α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of α‐LA and β‐LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS‐PAGE electrophoresis. RESULTS: Fermentation by lactic acid bacteria could significantly reduce the antigenicity of α‐LA and β‐LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, α‐LA and β‐LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS‐PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. CONCLUSION: The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity. Copyright © 2010 Society of Chemical Industry     

牛乳中主要过敏原的分离纯化研究进展

牛乳中含有3 种主要的过敏原蛋白：酪蛋白、β- 乳球蛋白和α- 乳白蛋白。本文详细叙述沉淀法、离子交换层析法、凝胶过滤层析法、羟基磷灰石层析法、疏水相互作用层析法、高效液相色谱法以及膜技术等分离纯化技术在牛乳主要过敏原分离纯化中的研究进展。其中,离子交换层析与凝胶层析已被广泛使用,而沉淀法一般作为粗提纯过的步骤。羟基磷灰石层析与疏水相互作用层析法也较为常见,既可单独分离过敏原,又可与其他方法结合来分离过敏原。另外高效液相色谱法与膜技术则是进一步纯化的后续工作,以提高过敏原的纯度。