Reduction in the antigenicity of beta-lactoglobulin in whole milk powder via supercritical CO2 treatment

Abstract

Cows' milk allergy (CMA) is a common phenomenon experienced in early childhood (<5 years of age) with an average occurrence rate of roughly 2.5%. The most prevalent allergen in cows' milk is believed to be β-lactoglobulin (β-LG). The objective of this study was to evaluate the use of hydrophobic supercritical CO₂ (ScCO₂) to modify the chemical structure β-LG thus impairing its recognition by antibodies. Whole milk powder was selected because of its closest compositional resemblance to bovine fluid milk and its applications in reconstitution and in the beverage (infant, toddler, and adult), confectionary, bakery, and meat industries. For this study, whole milk powder was treated with food-grade CO₂ at temperatures of 50, 63, and 75 °C under operating pressures of 100, 150, 200, 250, and 300 bar. Proteins in whole milk powder were examined using SDS-PAGE, Western blot, and ELISA. Orbitrap Fusion LC/MS-MS and periodic staining was performed to confirm post-translational modifications in β-LG. Functional properties of whole milk powder before and after treatment were assessed by its solubility index, oil holding capacity, emulsion capacity and stability, zeta-potential, particle size, and color analysis. SDS-PAGE of treated samples yielded fuzzy bands (variable mobility of molecules due to different MW results in ill-defined bands) indicative of an increase in molecular weight, presumably due to chemical change in the protein, and demonstrated a maximum of 71.13 ± 0.29% decrease in the band intensity of β-LG under treatment conditions of 75 °C/300 bar for 30 min (P < 0.05). These changes were small with samples treated with heat only. Lighter, diffused bands were observed using Western blot analysis. ELISA tests proved that ScCO₂ treatment specifically and significantly affected the antigenicity of β-LG with a reduction of 42.9 ± 2.83% and 54.75 ± 2.43% at 63 °C/200 bar and 75 °C/300 bar, respectively. Orbitrap fusion detected the presence of fatty acids and sugar moieties bound to β-LG and the latter was confirmed by periodic staining. Functional properties of ScCO₂-treated milk powder yielded a decrease in solubility index and an increase in emulsion capacity of whole milk powder was observed under ScCO₂ treatment at 75 °C/300 bar (P < 0.05), with small and insignificant changes at other treatments producing a decrease in antigenicity. Color changes were small for most samples, except at 63 °C/200 bar, where a significant increase in yellowness was observed. Zeta-potential and particle size measurements indicated that most changes were temperature driven. This study demonstrates 2 approaches to mitigate β-LG antigenicity via fatty acid binding and lactosylation using hydrophobic ScCO₂.

Keywords: antigenicity; beta-lactoglobulin; caprylation; lactosylation.