Desensitization Of Bovine Whey Protein Based On Bio-enzyme And Its Mechanism

Whey protein in cow's milk protein is an important source of food for infants and young children,but it can easily cause allergic reactions and seriously threaten physical health.In order to solve this problem,the following research has been carried out in this subject: on the basis of bio-enzymatic hydrolysis of whey protein,the methods of pre-treating whey protein and post-treating whey protein hydrolysate were used to reduce the antigenicity of the whey protein hydrolysate product.And the mechanism of antigenicity reduction was analyzed by liquid chromatography-mass spectrometry(LCMS / MS).The main research process and results are as follows:(1)Screening enzymes that suitable for hydrolyzing whey protein,and using ultrasound-ionic liquid(US-IL)pretreatment to reduce the antigenicity of whey protein.First,two kinds of papain,two kinds of trypsin,neutral protease and alcalase were selected as the screening objects,the degree of hydrolysis and the molecular weight distribution of SDS-PAGE electrophoresis were used as indicators to select a suitable one for whey protein hydrolysis.Papain and alcalase worked best.The US-IL was used to pretreat the whey protein solution,and the mixed solution was hydrolyzed with the selected protease.After hydrolysis,the ionic liquid was recovered by centrifugation.The degree of hydrolysis and molecular weight distribution make a comprehensive evaluation of the effect of the pretreatment method.After US-IL treatment,the degradation rates of ?-LA and ?-LG of the two hydrolysates were significantly increased compared to the direct hydrolysis group.The two antigenic decline rates of the alcalase group were 77.73% and 83.16%,papain group reached 76.48% and 83.24%,respectively.(2)The method of transglutaminase re-crosslinking after enzymolysis was used to reduce the antigenicity of whey protein,and compared with the US-IL pretreatment method.First use the thermolysin,complex protease,trypsin,papain,alcalase,and neutral protease to perform partial hydrolysis of whey protein,and then use transglutaminase to perform the second step on the hydrolysate.Catalyzed crosslinking,thereby achieving the purpose of cleavage,masking epitopes and reducing antigenicity.Combined with the properties and molecular weight distribution of the product,the effect of this post-treatment method is evaluated.The Protease-TG twostep method can increase the antigenic decline rate of ?-LG and ?-LA in whey protein.The trypsin-TG group had the highest decline rates of ?-LG and ?-LA antigens,which were 89.37% and 70.97%,respectively.The papain-TG group had the highest content of small molecular weight peptides.The decline rates of ?-LG and ?-LA antigenicity were 82.26% and 76.36%,respectively.(3)Probe into the mechanism of reducing the antigenicity of whey protein by the combined use of protease and transglutaminaseThe ultrafiltration method was used to fractionate the product of transglutaminase re-crosslinking method to explore the relationship between antigenicity and molecular weight.Then,some of the products with lower antigenicity were purified and separated by AKTA.Collect samples by molecular weight,determine the antigenicity of each part,analyze the peptides and their sequences present in the product by LC-MS / MS,and compare with the reported epitopes of the main allergen proteins in whey protein.The changes of antigenic epitopes after hydrolysis treatment were explored to explain the mechanism of antigenic degradation of enzymatic products.And it is believed that in the part with a molecular weight below 3k Da,there is no linear correlation between the antigenicity and the molecular weight.The key factor that determines the antigenicity is not the ability to hydrolyze into smaller peptides,but whether the antigenic epitope is still exists,and how much it has been destroyed.