基于蛋白组学的烘焙对花生基质中Ara h 2结构和致敏性影响研究

Peanut (Arachis hypogaea) is listed among the eight major food allergens by FAO. Peanut allergy is common, frequently severe, and typically permanent，which threatens food safety. Ara h 2 is major allergen in peanut, and reducing the allergenicity of Ara h 2 by processing is of especial interest. In present proposal, the change on structure and allergenicity of allergen Ara h 2 in peanut matrix affected by baking processing was to be studied. Although the structure and allergenicity of allergen during processing had been broadly explored, but no way except hydrolysis was found to decrease allergenicity. Because structural changes of purified proteins in processing could not reflect the situation in real processing. For allergen in matrix, the regulation between structure and allergenicity remains unclear. To find the processing that decrease allergenicity of allergen, change of allergen in matrix during processing must be studied, especially the change on structure and allergenicity..Here, we would study changes of Ara h 2 in peanut matrix on structure and allergenicity during total peanut seeds baking. The allergen and its derivatives would be separated after recognized, then their structure would be characterized and their allergenicity would be evaluated. At first, the protein samples from baked peanut would be separated on a gel by two- dimensional electrophoresis, and recognized by sera from patients suffering peanut allergy, and anti-Ara h 2 IgG. After the recognition, these parts derived from Ara h 2, namely Ara h 2 and its derivatives would be separated by immunoaffinity chromatography by aid of simulated digestion. As the polyclone antigen was used, and the antigenicity of allergen would not decrease after thermal processing, most Ara h 2 fragments, derivatives with antigen epitopes could be separated from peanut homogenate for following research.. The structure of purified allergen and its derivatives would be characterized by electrophoresis, mass spectrometry and spectroscopy. Such as mass spectrometry was employed to analysis prime structure, circular dichroism was employed to analysis secondary structure and fluorescence absorption spectrum was employed to analysis tertiary structure. The total protein, separated Ara h 2 and its derivatives would be detected by ELISA with Ig E from patient with peanut allergy and simulation in some animal experiments to evaluate the allergenicity..This research would discover the changes on structure and allergenicity of Ara h 2 in matrix induced by baking, promoting our understanding on processing modification on Ara h 2, including the structure-activity relationship change of Ara h 2 during processing. After the molecular change of allergen Ara h 2 during baking was described, the research could give clues for allergen desensitization in peanut. Additionally, the strategy used in the study, separating sample after recognition, could be reference for other studied related to constituent change in matrix.

花生及其制品是重要的食物过敏原，由于其使用广泛且过敏症状严重，危害食品安全。Ara h 2是花生中主要过敏原蛋白，它的加工改性有助降低花生过敏风险。研究拟利用蛋白组学的技术和方法，揭示烘焙加工对基质中Ara h 2结构和致敏性的影响。首先对烘焙后的花生蛋白组分进行全面识别，利用双向电泳将其分离在凝胶上，再分别采用花生过敏患者的血清和兔抗Ara h 2抗体进行识别，确定加工产物中致敏性组分及其中Ara h 2的衍生物；其次利用免疫亲和层析结合模拟消化将基质中的Ara h 2及其衍生物分离出来；最后结合电泳、质谱和光谱学等技术对分离获得的各组分进行结构表征，结合Ig E体外识别和动物激发实验进行致敏性评估。研究将阐明基质中Ara h 2结构和致敏性的变化，探索烘焙对其构效关系的影响，为花生脱敏研究指示方向。研究引入蛋白组学的方法，对食物组分先识别再分离的思路，可供食品组分变化相关研究借鉴。

花生及其制品是重要的食物过敏原，由于其使用广泛且过敏症状严重，危害食品安全。Ara h 2是花生中主要过敏原蛋白，它的加工改性有助降低花生过敏风险。研究对烘焙加工后基质中的花生过敏原蛋白，尤其是Ara h 2的结构和潜在致敏性进行了分析。研究发现热加工后花生总蛋白含量无显著变化，热加工前后花生蛋白致敏性的变化与蛋白含量无关，但不同方法对加工后的过敏原蛋白提取效率不一，获得的溶液中，蛋白组成也有重大差异，这使得热加工后食物过敏原致敏性评估结果难于相互对比验证。虽然含有高度变性剂的离液剂能够将更多的蛋白提取出来，但获得的蛋白都已经丧失了高级结构。利用质谱技术检测花生加工前后过敏原蛋白Ara h 2的变化，iTRAQ技术比较烘焙样品与未加工花生样品间共有的肽段的数量比值，MALDI-TOF-MS鉴定加工前后花生蛋白酶解片段的差异。这些酶切片段的差异就是加工引起的，在SWISS-MODEL构建的Ara h 2结构模型上，分析这些差异发现，烘焙加工引起最重要的Ara h 2结构变化就是破坏了其二硫键。失去二硫键的Ara h 2消化稳定性下降，更加容易被酶解，更多过敏原表位被破坏，致敏性下降。热加工花生脱脂后，其模拟消化产物与Ig E的结合能力普遍下降。细胞学评估显示，烘焙后过敏原蛋白潜在致敏性没有显著增加，这与动物实验的结果是一致的。研究中，我们发现用嗜碱性粒细胞评价脱脂粉的模拟消化产物的致敏性，是一个较优的加工蛋白致敏性体外评估模型。另外，研究也发现生物膜干涉法也可以用于检测过敏原蛋白与血清IgE结合能力，能节约血清并取得较好效果。这些研究结果将为后续食物过敏原的脱敏加工研究和相关技术应用提供借鉴。