Study On Preparation Of Hypoallergenic Hydrolysate By Controllable Enzymatic Hydrolysis Of Milk Protein

Milk protein has high nutritional value and is considered to be the best substitute for breast milk.However,cow’s milk protein allergy is a common food allergy in children,and enzymatic hydrolysis is widely used to reduce the allergenicity of cow’s milk protein.Compared with the free enzyme,the magnetic immobilized enzyme has high catalytic properties and stability,and can realize the recycling of the enzyme and reduce the production cost.In this paper,through the screening of protease,the partial and extensive hydrolysis process of milk protein was determined,and the sensitization of the hydrolyzed product was explored;the kinetic model of partial and extensive controllable enzymatic hydrolysis was established,and the relationship between hydrolysis time and degree of hydrolysis was clarified;Magnetic nanoparticles immobilized composite enzymes to study the effect of immobilized enzymes on hydrolyzing milk protein and reducing sensitization.Neutrase and alcalase were identified as enzymes for enzymolysis experiments by single enzyme screening.Partial and extensive hydrolysis processes are determined by molecular weight distribution and determination of free amino acids.Partial hydrolysis process:neutral protease,E/S 6000 U/g,hydrolysis p H 7,temperature 50°C,hydrolysis time 0.5 h.The degree of hydrolysis of the hydrolyzate is 9.68%;the molecular weight<6000 Da peptides account for 95.02%,the<1200 Da peptides account for 51.66%,and the free amino acids account for 9.42%;the antigenicity reduction rate of casein is 14.93%,and the reduction rate ofβ-lactoglobulin antigenicity was 11.06%.Extensive hydrolysis process:the ratio of compound enzymes（neutrase and alcalase）was 1:1,E/S 6000 U/g,hydrolysis temperature 50℃,hydrolysis p H 8.5,and hydrolysis time 3 h.The degree of hydrolysis of the hydrolyzate was 24.68%;the molecular weight<3000 Da peptides accounted for99.03%,the<1200 Da peptides accounted for 91.59%,and the free amino acids accounted for 15.92%;The antigenicity reduction rate of casein was 33.76%,and the antigenicity reduction rate ofβ-lactoglobulin was 22.38%.Partially and extensively controllable enzymatic hydrolysis kinetic models were obtained by mathematical derivation.Through the model,we could preliminarily judge the degree of hydrolysis.The theoretical value and the actual value have a good fit,which is of practical significance.For the partial hydrolysis kinetic model,kinetic constants k₂=14.531min^-1and k_d=1.9669 min^-1,minimum critical initial protease concentration E₀=c₀S₀,maximum critical substrate initial concentration S₀=E₀/c₀,c₀=2.484×10^-3.For the extensive hydrolysis kinetic model,the kinetic constants k₂=23.752 min^-1and k_d=3.5445 min^-1,c₀=1.495×10^-3.At the same time,the relationship between the degree of hydrolysis and molecular weight distribution was established.When the degree of hydrolysis is not higher than 15%,the proportion of peptides with molecular weight<1000 Da ranges from 29 to60%,and when the degree of hydrolysis is greater than 15%,the distribution of peptides with molecular weight<1000 Da,the range was between 60 and 85%.In view of the instability of free protease and its difficulty in recycling,neutrase and alcalase were co-immobilized on the modified magnetic Fe₃O₄nanoparticles.By FT-IR and SEM analysis,it was found that the immobilized enzyme was successfully immobilized on the magnetic Fe₃O₄nanoparticles.After 5 times of reuse,the reuse rate of the immobilized enzyme still maintained 68.23%of the initial activity.After the milk protein was hydrolyzed by 6000 U/g immobilized enzyme for 3 h at 50℃and p H 8.5,the degree of hydrolysis of the hydrolyzate was 22.76%,and the antigenicity reduction rates ofβ-lactoglobulin and casein were 21.34%,respectively.and 30.89%.Peptides with molecular weight<1000 Da accounted for 82.75%,and free amino acid content accounted for 13.65%.This result indicated that the hydrolysis activity of the immobilized enzyme is similar to that of the free enzyme,and it could be used to prepare the deeply hydrolyzed milk protein.