Detection of potentially allergenic material in 12 hydrolyzed milk formulas

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.