Detection of Potentially Allergenic Material in 12 Hydrolyzed Milk Formulas

Abstract

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.

Analysis of trace amounts of bovine β‐lactoglobulin in infant formulas by capillary electrophoresis with on‐capillary derivatization and laser‐induced fluorescence detection

“…Hypoallergenic formulas, based on hydrolyzed cow's milk proteins, have been demonstrated to be the best option for children with cow's milk protein allergy [5]. However, despite the use of hydrolyzed formulas for child nutrition, cases of allergic reactions have been reported for extensively hydrolyzed formulas (eHF) and more commonly for partially hydrolyzed formulas (pHF) [6,7]. This indicates that minute amounts of residual intact proteins and/or peptides with a molecular weight high enough to be allergenic are still present in these formulas.…”

Milk Allergen Detection

“…Several methods especially target allergenic cow's milk proteins in food products and, in particular, in hypoallergenic infant formulas [17]. Among the many methods used, Malmheden Yman et al [18] investigated the presence of milk in various food products analyzed by rocket immunoelectrophoresis (RIE) with a sensitivity of 30 mg/kg.…”

“…In their study, Mariager et al [21] compared polyclonal and monoclonal antibodies and found the polyclonal antibodies they used to be more suitable. In a study comparing several analytical methods (gel filtration, SDS-PAGE, native PAGE, immunoblotting, dot immunoblotting, and ELISA), antibodies were raised against b-lactoglobulin denatured at different temperatures [17]. ELISA was also used for the analysis of residual allergenic milk proteins, mainly casein, used as fining reagent in bottled wine [22].…”

Processing Foods Free from Dairy Proteins

“…Heating of milk may also favor the binding of β -Lg with CN micelles and coaggregation with α -la, which can extensively modify surface availability of epitopes [83] . Walter et al [84] found that pasteurization caused aggregation of β -Lg and α -la inhibiting uptake by intestinal epithelial cells in vitro and in vivo .…”