【作者】 李铮；

【导师】 罗永康； 吴建平；

【作者基本信息】 中国农业大学 ， 食品科学， 2014， 博士

【摘要】 鱼类是食物的主要过敏原之一,主要过敏原蛋白为小清蛋白,是一个分子量10-12KDa的酸性钙结合蛋白。目前国外有很多关于鳕鱼、鲑鱼、沙丁鱼等海水鱼过敏原的报道,国内也有一些关于淡水鱼的报道,但是没有关于草鱼的报道。草鱼是我国主要消费的淡水鱼之一。本研究分离纯化了草鱼主要过敏原小清蛋白,制备了用于ELISA检测的兔抗小清蛋白多克隆抗体,研究了热加工对小清蛋白抗原性、变应原性及消化稳定性的影响,建立了美拉德反应调控小清蛋白抗原性的回归模型,纯化、鉴定并分析了草鱼小清蛋白的三个亚型,研究了鱼糜产品的潜在致敏性,填补了国内关于草鱼过敏原研究的空白。1.草鱼小清蛋白分离纯化及ELISA检测方法的建立。从草鱼鱼肉中分离纯化了小清蛋白,制备了兔抗草鱼小清蛋白多克隆抗体。测定了该抗体用于ELISA实验的条件和标准曲线。分析了兔抗草鱼小清蛋白多克隆抗体和鲤鱼、鲫鱼、鲢鱼及武昌鱼的交叉反应性,结果表明这5种淡水鱼中小清蛋白有高度同源性。收集了鱼过敏患者血清,根据血清收集时间制备了两批血清池用于后续小清蛋白IgE结合能力检测。2.热加工对草鱼小清蛋白抗原性、变应原性及消化稳定性的影响。结果表明,80和100℃热处理在90min内使白肉中小清蛋白的抗原性及变应原性升高,65℃热处理使白肉的变应原性降低。在相同热处理时间,随着温度的升高红肉的抗原性和变应原性降低。蒸、煮、高压及烤四种热加工方式对草鱼鱼肉中小清蛋白有一定影响。高压(121℃,0.15MPa)处理30min对鱼肉中小清蛋白影响最大,小清蛋白更易被消化,抗原性及变应原性显著降低。反之,100℃烘烤使小清蛋白抗原性升高,且模拟胃肠消化后小清蛋白的抗原性及变应原性均高于对照组。3.美拉德反应条件对小清蛋白抗原性和变应原性的影响。建立了草鱼小清蛋白与麦芽糖美拉德反应中抗原性与反应条件的关系的预测模型,研究了美拉德反应条件对小清蛋白抗原性的影响。在研究范围内,反应温度对小清蛋白-麦芽糖的抗原性影响最大。以小清蛋白的抗原性为响应值优化出了获得低抗原性的小清蛋白-麦芽糖的美拉德反应条件,在此条件下得到的小清蛋白-麦芽糖的抗原性的预测值为0.276μg/mL没有发生美拉德反应的小清蛋白的抗原性为26.54μg/mL说明美拉德反应显著降低了小清蛋白的抗原性。在此反应条件制备小清蛋白-麦芽糖,其IgE结合能力显著降低。4.小清蛋白亚型分离及性质分析。采用高效液相色谱从凝胶层析得到的小清蛋白中分离出三种小清蛋白亚型,命名为PVⅠ、PVⅡ和PVⅢ,测定了分子量和氨基酸序列。采用ELISA法分析小清蛋白亚型的IgG和IgE结合能力,IgE结合能力最强的为PVⅡ,且PVⅡ在草鱼小清蛋白中含量最高,这说明PVⅡ是主要过敏原。经过胃蛋白酶和胰蛋白酶消化后,只有PVⅠ仍然具有较高的IgE结合能力。PVⅠ之所以保留部分IgE结合能力,是因为PVⅠ有一部分没有被胃蛋白酶消化。经过热处理,PVⅡ和PVⅢ分子量分布、lgG及IgE结合能力基本保持稳定。加热时间为1h,热处理温度高于70℃时,或者热处理温度99℃,加热时间大于lOmin时,PVI被分解,这导致了其IgG及IgE结合能力下降。5.鱼糜制品潜在致敏性分析。结果表明,-20、-40、-60℃冻藏14周草鱼白肉中小清蛋白含量和抗原性没有明显变化。鱼糜漂洗过程中,第1次的漂洗水中含有大量的小清蛋白,2-5次漂洗的漂洗水中小清蛋白含量逐渐减少,第6次漂洗的漂洗水中小清蛋白基本不可见。采用兔抗小清蛋白抗体测定,十种鱼糜产品除了2-1和7-3,其余的样品中小清蛋白含量均小于0.125μg/mL,2-1和7.3两个样品中小清蛋白含量在0.125-0.25μg/mL之间：采用人血清池测定,10种鱼糜产品的ELISA检测值小于0.1785,小清蛋白含量范围为0.001-0.1μg/mL。更多还原

【Abstract】 Fish is one of the major food allergen, and parvalbumin is the main allergen in fish. Parvalbumin is an acidic calcium-binding protein with a molecular weight of10-12KDa. Parvalbumin was reported inseveral kinds of sea fish species, such as cod, salmon and pilchard, and it was also reported in some freshwater fish in China, but there was no research about grass carp, one of the most frequently consumed freshwater fish in china. This study purified the main allergen in grass carp, and prepared the rabbit anti-parvalbumin antibody. This study also investigated the effect of processing methods on the antigenicity, allergenicity and digestibility of parvalbumin. The effect of Maillard reaction on the antigenicity of parvalbumin was studied. Parvalbumin isotypes were purified and characterized. And the pertential allergenicity of surimi products was evaluated in this study.1. Purification of parvalbumin from grass carp and establishment of ELISA methods. Heat treatment and Gel filtration were used to purify parvalbumin from grass carp muscle. The fractions with high IgG-binding were collected. Anti-grass carp parvalbumin antibody was produced in rabbit and then used in ELISA test. The allergen coated concentration was2μg/mL and the sera was diluted160000times in ELISA. The cross reactivity between the sera and carp, crucian, silver carp and bream were investigated, and the results revealed high cross reactivity between them. Different sera from fish allergic patients were collected and used as sera pool to test the IgE-binding capactity of parvalbumin.2. Effects of thermal processing on the antigenicity, allergenicity and digestibility of parvalbumin. Heat treatment caused higher antigenicity and allergenicity of white muscle when heated at80and100℃within90min. However, heat treatment at65℃was able to decrease the allergenicity of white muscle as the heating time increased. For the dark muscle, the antigenicity and allergenicity decreased with the increasing temperature. The effects of steam, boil, high peressure and bake on parvalbumin were different. High pressure (121℃,0.15MPa) was the most effective method to control the antigenicity of parvalbumin, and the parvalbumin in the grass carp muscle which treated by high pressure was more sensitive to digestion and the antigenicity and allergenicity of parvalbumin were decreased significantly during digestion. The water used in boil and the water obtained from steam and high pressure treated grass carp muscle contained parvalbumin, which can cause fish allergy.3.Effect of maillard reaction condition on the antigenicity and allergenicity of parvalbumin. A model for predicting antigenicity of PV and maltose Maillard reaction products was established. On the field of conditions studied, temperature had the most important effect on the antigenicity of PV. The predicted antigenicity of PV was reduced from26.54μg mL-1to0.276μg mL-1under the optimal Maillard reaction condition. Maillard reaction lead significantly suppressed IgE binding to PV and it was an effective method to control the major allergen of grass carp. After Maillard reaction, the essential amino acid content of PV decreased14.50%and the non-essential amino acid content of PV decreased23.21%.4. Purification andcharacterization of parvalbuminisotypes from grass carp. Three PV isotypes were purified and denoted as PⅥ, PⅦ and PⅧ. The molecular weights of PⅥ, PⅦ and PⅧ were determined to be11.968,11.430and11.512kDa, respectively. PⅥ showed74%sequence similarity with PV isotype4a from Daniorerio while PⅦ and PⅧ showed46%sequence similarities with PⅤ isotypes from Hypophthalmichthy smolitrix, respectively. PVII showed the highest IgG-binding and IgE-binding as well as the highest content among the three isotypes. Therefore, PⅦ was demonstrated to be the main allergen. However, PⅦ was liable to gastrointestinal enzymes compared with PⅥ, which was resistant to pepsin digestion. The IgE-binding capacity of pepsin digested PⅥ was significantly higher than other digested PV isotypes, and the undigested PVI in the peptic digest was responsible for its IgE-binding capacity. After thermal treatment, the structure, antigenicity and allergenicity of PⅦ and PⅧ were stable. But PVI was degraded when the heat time was1h, and the heat temperature was higher than70℃or when the heat temperature was99℃, and the heat time was longer than10min. And the degradation of PVI may be was the reason for the dereased IgG and IgE binding capacities.5. Analysis of the potential allergic of surimi products. The content and antigenicity of parvalbumin in grass carp white muscle were stable when stored at-20,-40,-60℃in14weeks. The content of parvalbumin decreased significantly during the surimi processing procedure. Rabbit anti-parvalbumin antibody and human sera pool were used to test parvalbumin in different concentration, and the results were used as a standard to measure the parvalbumin content in ten kinds of surimi products. The parvalbumin content of the surimi products were lower than0.125μg/mL except for2-1and7-3when tested by rabbit anti-parvalbumin antibody. The parvalbumin content of the surimi products were in the range of0.001-0.1μg/mL when tested by the human sera pool.更多还原