大豆致敏蛋白致敏机制研究

采用酶联免疫印迹实验对脱糖基前后大豆致敏蛋白的致敏性进行识别,发现脱糖基后大豆致敏蛋白的致敏性依然存在,说明糖基不是引起致敏的主要原因;对模拟胃肠道消化后大豆致敏蛋白的致敏性进行识别.发现消化后大豆致敏蛋白已被水解,水解后的蛋白不与患者血清中的IgE抗体结合,说明正常消化情况下的大豆蛋白不易引起人体过敏.     

大豆制品中致敏蛋白的识别

本实验采用酶联免疫印迹和竞争性抑制实验识别大豆制品中的致敏蛋白。研究结果显示,大豆中的致敏蛋白主要存在于非发酵性豆制品中,且脂肪氧化酶和32.5kD的致敏蛋白在大豆加工过程中很容易失去致敏性,18.9kD的致敏蛋白在组织化大豆蛋白中容易失去致敏性,而β-伴球蛋白的α-亚基虽然存在一定的非特异性吸附,但依然是致敏性较强的蛋白。     

中国花生致敏蛋白的识别

我国对花生过敏方面的研究很少。本实验利用中国常用花生品种识别鉴定了中国主要的致敏蛋白,比较了国内外花生品种致敏蛋白相对含量的差异,期望找到中国花生过敏发病率较低的原因,为临床食物过敏患者的治疗和低过敏花生品种的培育提供理论依据。研究结果表明:Ara h1和三条Ara h3多肽是中国主要的致敏蛋白,并发现了Ara h1的亚基,分子量为58kD的多肽。Ara h1和Ara h3的相对含量各品种之间差异显著,并且低于国外花生品种。因此中国花生主要致敏蛋白相对含量低可能是导致中国花生过敏发病率较低的主要原因。     

致敏大豆蛋白P34及其清除方法的研究进展

大豆作为"八大"过敏食品之一,其过敏患者数量约占食物过敏总人数的25%。如何降低大豆产品的致敏性,保证消费者的安全,已经成为食品安全领域一个热议的话题。大豆中含有多种致敏因子,如Gly-m Bd 30K蛋白、Gly-m Bd 28K蛋白和Gly-m Bd 28K蛋白等。其中Gly-m Bd 30K蛋白作为致敏性最强的过敏原,能被65%的大豆过敏患者血清识别,在几种过敏蛋白中占着较突出的地位。本文综述了Gly-m Bd 30K蛋白的抗原表位、去除方法的研究现状。其中,酶法处理、超高压法、挤压膨化法等降低大豆致敏性的研究已取得了一定的成果,这为进一步提高大豆及其制品的品质质量,保证其在食品工业和畜牧业方面的安全利用提供了科学参考。     

处理方法对大豆致敏蛋白的影响

Gly m Bd 30K、Gly m Bd28K和β-伴大豆球蛋白的α亚基(MW68K)是大豆中的主要致敏蛋白，本文对经过不同处理的大豆进行SDS-PAGE电泳，结果显示，结合盐析和pH值处理可以有效去除Gly m Bd 30K和Glym Bd28K，但要同时去除三种致敏蛋白还需进一步研究。     

湿法糖基化改性大豆分离蛋白在低致敏千页豆腐中的应用

为寻求高效的糖基化改性条件和良好的应用途径，本文以葡萄糖、木糖两种单糖为糖基供体，探究反应时间、反应温度、蛋白与糖比例、蛋白浓度对大豆分离蛋白湿法糖基化反应的影响，分析大豆分离蛋白-葡萄糖复合物(SPI-葡萄糖)、大豆分离蛋白-木糖复合物(SPI-木糖)致敏性的变化，并将SPI-葡萄糖、SPI-木糖应用于低致敏千页豆腐生产。结果表明，木糖相较于葡萄糖更易与大豆分离蛋白发生糖基化反应，但也更容易有类黑素产生。在蛋白浓度为25 mg/mL时，糖基化反应程度最高，并且糖基化反应程度与反应温度、反应时间、糖添加量呈正相关。SDS-PAGE和傅里叶变换红外光谱分析结果表明糖基化改性使大豆分离蛋白和糖分子发生了共价结合，SPI的自由氨基减少。糖基化反应程度越高，SPI致敏性越低，SPI-木糖接枝物致敏性降低程度最高可达50%，以其为原料经谷氨酰胺转氨酶(TG酶)交联所制作的千页豆腐有良好的弹性和咀嚼性。     

超高压处理对大豆蛋白致敏原P34免疫活性的影响研究

利用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western-Blot和ELISA法研究了超高压处理不同压力及保压时间对大豆蛋白致敏原P34免疫活性的影响。结果表明:超高压处理能够降低大豆分离蛋白中大豆蛋白致敏原P34的致敏性。蛋白浓度为5%的大豆分离蛋白溶液的最佳处理条件为压强600MPa、处理时间20min,该条件下大豆蛋白致敏原P34的致敏性降低了21.69%。     

BALB/c小鼠动物模型评价食物过敏性的可行性研究

目的:动物模型对于模拟人体食物过敏的可能性以及用来评价食品致敏性的可行性一直存在争议。本研究中使用BALB/c小鼠研究了其作为食物过敏动物模型的可行性,并对几种食物中蛋白的致敏性进行评价。方法:选择大豆球蛋白、卵清白蛋白、马铃薯碱性磷酸酶作为受试物,对各蛋白进行BALB/c小鼠模型研究,模拟胃液消化试验和生物信息学分析。取3~4周龄雌性BALB/c小鼠,经口给予蛋白诱发致敏。大刺激后,测定动物血浆中组胺水平,血清中特异性Ig E、Ig G1水平,m MCP-1水平和肠道渗透性,并对主要脏器进行病理组织学观察。结果:给予BALB/c小鼠一次经口致敏各蛋白,各组间特异性抗体Ig E、Ig G1水平、m MCP-1水平、组胺水平以及白蛋白水平均未表现出明显差异;而给BALB/c小鼠多次接触过敏原时,特异性抗体Ig E、Ig G1水平、组胺以及白蛋白水平都有显著性升高,产生明显的Th2型免疫反应,且BALB/c小鼠能区分这些蛋白的致敏性,即大豆球蛋白卵清白蛋白马铃薯碱性磷酸酶。模拟胃液消化试验也验证了动物试验对受试蛋白致敏性的分析结果。通过生物信息学对抗原指数分析,同样发现大豆球蛋白致敏性显著高于卵清白蛋白和马铃薯碱性磷酸酶。结论:BALB/c小鼠用于研究食品过敏原中的大豆球蛋白、卵清白蛋白、马铃薯碱性磷酸酶的致敏性是可行的,能够区分这些食物蛋白的致敏性。     

致敏蛋白α亚基缺失型大豆氨基酸组成及营养评价

结合模糊识别方法评价的数学模型法和联合国粮农组织/世界卫生组织（Food and Agriculture Organization/World Health Organization,FAO/WHO）、鸡蛋蛋白两种模式下的化学分析法评价两个致敏蛋白α亚基缺失型大豆蛋白质氨基酸营养价值,解析α亚基缺失特性对大豆氨基酸组分及营养品质的影响。用氨基酸分析仪测定氨基酸的组分和含量,α亚基缺失特性用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecylsulfate polyacrylamide gelelectrophoresis,SDS-PAGE）确认,蛋白和脂肪含量用Perten 8620近红外谷物分析仪测定。结果表明：1）致敏蛋白α亚基缺失型大豆的氨基酸总量、必需氨基酸总量、蛋白质、油分含量不因α亚基的缺失而降低;2）致敏蛋白α亚基缺失型大豆的11S/7S比值在4.65以上,高于目前普遍报道的2.0～3.0;3）致敏蛋白α亚基缺失型大豆必需氨基酸含量接近或高于FAO/WHO标准;两种模式下,α亚基缺失型大豆的5种化学评分及贴进度值都很高,接近标准蛋白。致敏蛋白α亚基缺失型大豆蛋白质氨基酸组分平衡,11S/7S比值更优,营养品质更高。     

低致敏大豆蛋白制备关键技术研究

正大豆含有丰富的蛋白质和平衡的氨基酸,是优质的植物性蛋白源,但其中含有的抗原蛋白会导致人和动物的过敏反应。大豆抗原蛋白又称为致过敏因子,其中大豆球蛋白和β-伴大豆球蛋白是免疫原性最强的两种蛋白质。自20世纪30年代Duke首次发现大豆蛋白可引起婴儿腹泻、虚脱和肠道炎症反应以来,科学家们对大豆蛋白的研究未曾间断,现已逐渐深入到大豆抗原蛋白的致过敏机理研究。如郑环宇等(2015)研究超高压对风味蛋白酶处理大豆分离蛋白中致敏原P34免疫活性的影响,并对脱敏后的SPI功能特性进行了研究。     

可视化膜芯片技术检测常见食源性过敏原成分

目的 建立包括鱼、虾、鸡、鸭、花生、核桃、小麦以及大豆过敏原成分的基因膜芯片技术, 实现对这几种食源性过敏原的同步可视化检测。方法 针对常见食源性过敏物质的成分特异性基因或者过敏原蛋白基因设计特异性的带生物素标记的引物和探针。采用反向斑点杂交(reversedot blot, RDB)结合多重聚合酶链式反应(multiplex polymerase chain reaction, MPCR)技术, 最终通过化学显色直观显示检测结果, 实现对多种食源性过敏原的同步可视化检测。结果 所建立的可视化基因膜芯片方法准确性强, 通过单目标物以及多目标物特异性实验, 显示本方法仅对鱼、虾、花生、核桃、大豆等靶标食源性过敏原有特异性反应, 非靶标物检测均为阴性; 通过制备不同质量浓度比的模拟样品考核方法的灵敏度, 经检测, 方法检测灵敏度可达0.1%(质量分数)。结论 所建立的方法可达到可视化、快速、准确地鉴别食品中鱼、虾、花生、核桃、大豆等常见食源性过敏原。     

Purification, Thermal Stability, and Antigenicity of the Immunodominant Soybean Allergen P34 in Soy Cultivars, Ingredients, and Products

ABSTRACT:  Protein P34 ( Gly m Bd 30K) is the immunodominant allergen in soybean ( Glycine max L.). The objectives of this study were (1) to study the effect of thermal treatment on P34 antigenicity and secondary structure after isolation and purification of P34 from soybean by chromatographic techniques; (2) to identify the variability of P34 allergen within 138 accessions from a diverse USDA soybean germplasm collection by ELISA; and (3) to quantify P34 immunoreactivity in various commercial soy ingredients and products. Thermal processing decreased P34 antigenicity. Soybean accessions with the highest P34 content were ancestral (12 mg/g defatted flour) followed by modern (10 mg/g defatted flour) and exotic (8 mg/g defatted flour). The cultivar that emerged as the lowest-expressing P34 accession was PI548657 (2.3 mg/g defatted flour). Among commercial soy ingredients, soy flour yielded the highest P34 antigenicity (32 mg/g extracted protein) followed by soy protein isolate (29 mg/g extracted protein) and soy protein concentrate (24 mg/g extracted protein). Among soy consumer products, soymilk presented the highest P34 antigenicity, ranging from 7 to 23 mg/g extracted protein, followed by tempeh (8 mg/g extracted protein), soy infant formula (3.4 mg/g extracted protein), soy powder (2 mg/g extracted protein), and soy cheese products (0.50 mg/g extracted protein). Korean miso, soy sauce, soy chili mix, soy nuts, soy cream cheese, soy meat patty, texturized soy protein, and soy cereal exhibited undetectable P34 antigenicity (detection limit = 0.45 ng). Selecting soybean varieties with low levels of this allergen, or via processing, could potentially make soybean products less antigenic.     

限制性酶解对豆粉相关性质的影响

为降低豆粉的致敏性,扩大其应用,采用限制性酶解制备豆粉。以木瓜蛋白酶、风味蛋白酶和碱性蛋白酶为限制性酶解用酶,研究不同的酶制剂及酶解时间对豆粉的致敏性、溶解性、表面疏水性及乳化性的影响,并对豆粉进行感官评价。结果表明:随着酶解时间的延长,豆粉的致敏性降低,溶解性、表面疏水性、乳化活性和乳化稳定性增加;其中利用木瓜蛋白酶酶解30 min制备的豆粉致敏原含量最低,为1. 95%,溶解性最高,为88. 55%,乳化活性及乳化稳定性最大,分别为182. 4 m2/g和120. 8 min;利用SDS-PAGE电泳发现,酶解作用使豆粉蛋白质中的7S和11S降解,生成小分子的肽类,从而降低豆粉的致敏性;利用木瓜蛋白酶酶解20 min制备的豆粉口感与传统市售豆粉相似。     

超高压-限制性酶解法降低豆乳粉致敏性工艺优化

以传统湿法工艺技术制备豆乳粉为基础,对浆渣分离后的豆乳进行超高压-限制性酶解处理,降低豆乳粉致敏性。在单因素试验基础上,采用响应面分析法对超高压-限制性酶解制备低致敏性豆乳粉工艺进行优化,确定最优超高压-限制性酶解的工艺参数为超高压处理压强320.00?MPa、超高压处理时间15?min、酶解时间60?min、酶添加量0.3?U/g,此条件下致敏性降低率为88.09%,但制备的豆乳粉具有微苦味,需要进一步调配以改善口感。超高压处理与酶解具有协同效应,可显著提高豆乳粉蛋白质体外消化率、显著降低豆乳粉致敏性,对不同加工工艺豆乳粉进行蛋白质体外消化率、蛋白质分散指数、过敏原含量及蛋白电泳分析,进一步证明超高压与酶解的协同效应。     

A DNA method for qualitative identification of plant raw materials in feedstuff

This research developed a simple and not expensive DNA method for the qualitative identification of plant raw materials used as feed mixtures. Specific simple sequence tagged (STS) markers were developed to detect faba bean (Lectin A gene), field pea (Convicilin A gene), grain sorghum (UDP-glucosyltransferase gene) and barley (Hordoindoline-a gene), whereas identification of durum and common wheat (lipid transfer protein gene), soybean (Gly m Bd 30K allergen gene) and maize (invertase gene) was carried out using markers available from the literature. Cross-reactivity of the primer pairs was also checked against oat, rye, kidney bean and lentil. The method was effectively applied to the analysis of flour mixtures and extruded feedstuff. It could be included in traceability and certification of animal feeding systems within high quality animal production chains which are strictly related to the production area by the valorisation of locally grown raw materials.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

烤鳗酱油中大豆、小麦过敏原的ELISA检测

烤鳗酱油的最初生产原料中包括了被许多国家和地区认定为过敏物的大豆和小麦,研究应用ELISA法对烤鳗酱油样品进行了大豆、小麦过敏原的测定.结果表明,对于大豆过敏原:绝大部分烤鳗酱油样品中检测不到,少数样品中其含量很低;对于小麦过敏原:绝大部分烤鳗酱油样品中的含量很低,少数样品检测不到.极微量的过敏原就可引起严重的过敏反应,因此应加强烤鳗酱油中过敏原的管理.     

Extraction and mass spectrometry identification of a major peach allergen Pru p 1

BACKGROUND: Peach allergy can be caused by the allergen Pru p 1. This occurs by cross‐reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)‐binding protein extracted from peach fruit has never been reported. RESULTS: Phosphate‐buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach‐allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two‐dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. CONCLUSION: Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1‐homologous peach allergen Pru p 1. Copyright © 2011 Society of Chemical Industry     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

Soybean Allergens Affecting North American Patients Identified by 2D Gels and Mass Spectrometry

In this study, we compared the immunoglobulin E immunological reactions of 23 soy-allergic, nine soy-sensitive, and four non-allergic human sera to soybean proteins separated by 1D and 2D gel electrophoresis and screened by Western blot. This method led to novel allergen identification in soybean proteins by tandem mass spectrometry analysis of reactive proteins. Soybean is one of the eight most significant foods which provoke allergic reactions among North Americans. Previous studies have identified several putative allergens present in soybean seeds; however, many of these reports did not employ mass spectrometry to conclusively identify the allergenic proteins. Reviews have suggested that soybean seeds may contain between 10 and 20 different proteins responsible for provoking allergic reactions among sensitive patients. We report, in this study of North American patients, a total of 19 potentially allergenic proteins including ten identified by mass spectrometry and five novel allergens. We have also made extensive use of soybean lines lacking various subunits of the major seed storage proteins, glycinin and β-conglycinin. By using these knockout lines in Western blots with patient serum, it was conclusively demonstrated that some patients react predominantly to only a few proteins, while most react to four to six proteins. The findings herein describe the main allergic proteins present in soybean seeds, the relative significance of these allergens among North Americans, and some genetic lines of soybean lacking individual allergens.