The Preparation Of Low Antigenic Whey Protein By Enzyme And Flavor Improvement

Whey protein is recognized as one of the high quality protein supplements, and is commonly used in infant formula powder. However, whey protein may cause allergy, which seriously affects the growth and development of infants and young children, and even threaten their lives. To solve this problem, this project carried out the following research:preparation of low antigenic whey protein hydrolysate by proteases, separated the fractions of hydrolysate and explored the mechanisms how enzymatic hydrolysis could reduce antigenicity by LC-MS/MS method, and then improved the flavor of the hydrolysate by microbial fermentation.(1)The results of enzymatic hydrolysis of whey protein showed that among six kinds of proteases from animal, plant and microorganism, Alcalase, PTN and Papain were more suitable for the hydrolysis of whey protein.(2)After comparing with different methods of enzymatic hydrolysis, it was found that Alcalase together with PTN one-step hydrolysis generated lower antigenic product which had more small peptides. Obtained optimized parameters for hydrolysis were [S] of 15% (w/v), [E]/[S] of 0.3% Alcalase+0.3% PTN (on the basis of substrate weight), temperature 55?, pH 8.0, and the hydrolysis time of 3 h. The degree of hydrolysis of this prepared product was 12.2%, and the fraction which molecular weight above 10,000 Da is 8.7%, molecular weight less than 1,000 Da occupied 65.0%, BLG antigenicity decreased 93.2%, ALA antigenicity decreased 95.1%.Compared the properties with imported hydrolyzed whey protein product, this prepared whey protein hydrolysate was medium-hydrolyzed whey protein product with low antigenicity.(3) Various peptides of the whey protein hydrolysate were separated and characterized by ultrafiltration, Sephadex G-15 chromatography and LC-MS/MS detection. Enzymatic hydrolysis destroyed the three main epitopes of BLG (V41-K60, Y102?R124 and L149?I162), and the three main epitopes of ALA (V42? E49, W60?F80 and C91?L96).The root cause that hydrolysate with low antigenicity was that enzymatic hydrolysis destroyed the epitopes of allergen proteins, and there was no direct linear relationship between molecular weight and antigenicity.(4)To improve the flavor of the hydrolysates, Lactobacillus fermentation was carried out, the optimized fermentation condition obtained by Response Surface Optimization was 8.9 hours fermentation, inoculation of 6.02%(on the basis of substrate weight), substrate concentration 9.15%(w/v), pH 7.80. The bitterness of initial concentration was defined as 10 by sensory evaluation, after fermentation the bitterness decrease to 1.875. HS-SPME-GC-MS was used to detect flavor compounds. 29 kinds of volatile substances were detected, including aldehydes, ketones, alcohols, acids, and esters. The type and content of volatile flavor substances before and after fermentation had changed. The content of 2-nonanone and 2-decanone reduced from 2.89% and 2.97% to 1.76% and 1.22%, respectively. In addition,16.22% of hexanol and 28.0% of (all-E)-2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene were generated.This study established solid foundation on further development of hydrolyzed whey protein product.