In this study, the effect of dual-enzyme hydrolysis on the allergenicity of the major proteins in skimmed milk was evaluated based on the degree of hydrolysis, molecular mass distribution, immunoglobulin G (IgG) binding capacity and allergic patients’ serum IgE binding capacity of hydrolysates. The results showed that after one-step hydrolysis for 80 min at an enzyme/substrate ratio of 3 000 U/g and an Alcalase to Flavourzyme ratio of 3:1, the IgG binding capacity of casein, α-lactalbumin and β-lactoglobulinin in skimmed milk decreased significantly by 74.93%, 97.24% and 93.46%, respectively (P < 0.05).The advanced structure of proteins was damaged, leading to an increase in the α-helical and β-turn contents, and a decrease in the β-sheet content. In addition, the free sulfhydryl content increased significantly to 14.29 μmol/g (P < 0.05), the surface hydrophobicity also increased, and the structure became loose. The particles in skimmed milk were aggregated, causing the average particle size to increase significantly to 346.90 nm (P < 0.05) and reducing the stability of skimmed milk. The binding capacity to serum IgE from patients with milk allergy of the enzymatic hydrolysate decreased significantly by 23.53% (P < 0.05), and the overall allergenicity decreased significantly by 69.60% (P < 0.05). Therefore, dual-enzyme hydrolysis is an effective method to reduce the allergenicity of skimmed milk.

The effects of Alcalase (AT), Protamex (PT) or Flavorzyme (FT) treatment on the antigenicity and molecular mass distribution of proteins, flavor and color of skim milk were investigated by using various techniques such as indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). The results showed that the effect of AT treatment on reducing the antigenicity of the major milk allergens was significantly better than that of PT and FT treatment (P < 0.05). The antigenicity of α-lactalbumin (α-LA), β-lactoglobulin (β-LG) and casein (CN) was inhibited by 64.01%, 76.00% and 69.10% by AT treatment for 20 min at an enzyme/substrate ratio of 500 U/g, respectively. The amount of low molecular mass peptides in skim milk increased significantly after treatment with each of the three enzymes. The bitterness, astringency, aftertaste of bitterness (aftertaste-B) and aftertaste of astringency (aftertaste-A) increased with increasing enzymatic treatment time and with increasing enzyme/substrate ratio. The taste of FT-treated milk was better than that of AT- or PT-treated milk. The brightness value of milk decreased significantly, while the redness value increased significantly after enzymatic treatment (P < 0.05). The transmittance increased as well. The color of skim milk treated by AT was more similar to that of whole milk.