Rosendal A, Barkholt V
Department of Biochemistry and Nutrition, Technical University of Denmark, Lyngby.
J Dairy Sci. 2000 Oct;83(10):2200-10. doi: 10.3168/jds.S0022-0302(00)75103-4.
Hypoallergenic milk formulas are used as an alternative diet for infants who have allergies to cow's milk when breast-feeding is not possible. These products are based on proteins, which have been heat-treated and hydrolyzed to a different degree in order to cleave antibody-binding structures. Even extensively hydrolyzed products have occasionally been observed to elicit allergic reactions in sensitized infants, however. Therefore, the parameters of relevance to allergenic potential require more investigation. The objective of the present study was to investigate 12 different hydrolyzed milk formulas for their contents of potentially allergenic protein material, i.e. material that may induce allergenicity or elicit allergic responses in already sensitized individuals. Analytical methods applied were gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE, immunoblotting, dot-immunobinding, and ELISA. Care was taken to assure that all protein fractions were investigated, including supernatants and precipitates following centrifugation of the milk formulas. By gel filtration, protein material with apparent molecular masses of 7 to >30 kDa was detected. Analysis by SDS-PAGE of formula precipitates showed that proteins with a molecular mass above 20 kDa were present even in some of the extensively hydrolyzed formulas. Residual antigenic beta-lactoglobulin was found by ELISA in all products. By immunoblotting and dot-immunobinding with antibodies against total whey, caseins, or Kunitz soybean trypsin inhibitor, we observed antigenic material mainly in partially hydrolyzed products. We concluded that SDS-PAGE of formula supernatants and precipitates gave the most differentiated profile of hydrolyzed formulas and that this method is well suited for screening potential allergenicity.
对于无法进行母乳喂养且对牛奶过敏的婴儿，低敏奶粉可作为替代饮食。这些产品以蛋白质为基础，经过不同程度的热处理和水解，以裂解抗体结合结构。然而，即使是深度水解产品，偶尔也会在致敏婴儿中引发过敏反应。因此，与致敏潜力相关的参数需要更多研究。本研究的目的是调查12种不同的水解奶粉中潜在致敏蛋白物质的含量，即可能在已致敏个体中诱导致敏性或引发过敏反应的物质。所应用的分析方法包括凝胶过滤、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、非变性聚丙烯酰胺凝胶电泳（native PAGE）、免疫印迹、斑点免疫结合和酶联免疫吸附测定（ELISA）。我们注意确保对所有蛋白质组分进行研究，包括奶粉离心后的上清液和沉淀物。通过凝胶过滤，检测到表观分子量为7至>30 kDa的蛋白质物质。对奶粉沉淀物进行SDS-PAGE分析表明，即使在一些深度水解奶粉中也存在分子量高于20 kDa的蛋白质。通过ELISA在所有产品中均检测到残留的抗原性β-乳球蛋白。通过用抗总乳清、酪蛋白或库尼茨大豆胰蛋白酶抑制剂的抗体进行免疫印迹和斑点免疫结合，我们主要在部分水解产品中观察到抗原性物质。我们得出结论，对奶粉上清液和沉淀物进行SDS-PAGE能给出水解奶粉最具区分性的图谱，且该方法非常适合筛选潜在致敏性。
