Combined protease hydrolysis of whey protein: structure, digestibility, and allergenicity in infant formula

To develop novel infant formula bases that combine low allergenicity and excellent processing properties, this study systematically evaluated the application potential of whey protein hydrolysates prepared by combined protease strategies. The basic composition, amino acid profile, peptide fingerprint of hydrolysate, along with its structural characteristics, digestibility, and allergenicity in infant formula systems were determined. Based on the advantages of high protein recovery, low bitterness, and low antigenicity, the Trypsin-Papain-Flavourzyme (TPF) combination and the Papain-Proteaxh (PP) combination were selected for the preparation of whey protein hydrolysates. Compared to PP, TPF released more long peptides with high net charge and amphiphilic characteristics while effectively reducing antigenicity, enabling the whey protein hydrolysate to maintain high surface hydrophobicity. In infant formula applications, TPF also exhibited stronger surface activity, with a 15% increase in protein adsorption rate and a 5% reduction in average particle size. Furthermore, INFOGEST digestion and KU812 cell experiments indicated that the residual antigenicity of TPF could be effectively eliminated during digestion, with the IgG binding capacity and cellular degranulation levels of the prepared infant formula showing no significant difference compared to the control group after digestion. In summary, this study demonstrated that combined protease hydrolysis strategy can effectively maintain surface activity and digestibility while reducing the allergenicity of whey protein hydrolysates, providing new insights and scientific evidence for the development of novel infant formula bases that combine safety and processing performance.