Enzymatic Hydrolysis Of Milk αs1- Casein For Allergenicity Reduction

Casein, an important ingredient for bovine milk, contain 80% of milk total protein. Casein mainly contain four protein: αs1-、αs2-、β-、κ- casein. αs1-casein is a major allergen to the infants and may induce respiratory, cutaneous and gastrointestinal reactions. However, there was no effective method to degrade αs1-casein. Casein, is rich in nutrients, contain essential amino acids for body. Therefore, eliminating the αs1-casein is critical to produce milk products with low allergenity. In this paper, preferential hydrolysis αs1-casein by proteases was studied. Main results were described as following.(1) In this study, the Baclicus licheniformis CN1 was isolated from farm soil. Research found that the supernatant which fermented by B. licheniformis CN1 can degrade milk allergen αs1-casein.(2) The screening of wild bacteria B. licheniformis CN1 had a bottle of level of ferment optimization. The optimal fermentation medium consisted of(g·L-1): starch, 30; beef extract, 6; Na H2PO4, 0.5; Na2HPO4, 0.5; the initial p H 9.0. The condition of fermentation: fermentation time, 24 hours; fermentation temperature, 37 °C; inoculation volume, 6%.(3) The method for protease purification is simple with ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of the purified enzyme was 30.5 k Da. The optimal p H and temperature was 10.0 and 50~60 °C(4) To determine the ability to degrade milk allergy protein, found that the optimal reaction was as follows, the purified enzyme(100 U·m L-1) and 2%(ratio of mass to volume) skim milk at a ratio of 8 %(ratio of volume to volume) for 20 min with the p H 9.0 and temperature at 50~60 °C. By comparing with commercial enzymes(protease M and peptidase R) which were used to degrade milk casein, the enzyme has enormous advantage to degrade αs1-casein, indicating the enzyme can play a prior characteristic on producing hyposensitization milk products. Western blotting showed αs1-casein was degraded in the beginning, but the allergen fragment was appeared with the time extended. After 20 min hydrolysis, the αs1-casein and its degraded fragments were completely disappeared.(5) Protease was immobilized by sodium alginate then explored its specificity to degrade milk αs1-casein. The optimal condition of immobilization was as follows: sodium alginate 4.0%(ratio of mass to volume), Ca Cl2 3%(ratio of mass to volume), the volume of sodium alginate and protease was 1:3 and 0.5 h for immobilization time. Under the condition, the efficiency of immobilization reached 67.5%, the optimal p H and temperature was 10.0 and 60 °C, respectively. The activity of immobilized enzyme was retained 40% after six cycles. Addition of immobilized protease to 2.0%(ratio of mass to volume) skim milk, the milk allergen αs1-casein was specifically degraded with p H 10.0 and temperature 60 °C after 25 minutes later.