Study On The Directed Enzymatic Hydrolysis Of Whey Protein And Its Application Characteristics Of Product

In this study,using whey protein as raw material,the hydrolysis conditions of extensively hydrolyzed whey protein and partially hydrolyzed whey protein were studied.Hilmar 8390 and Hilmar 8350 were used as reference controls for extensive hydrolysis and partial hydrolysis,respectively,and the enzymatic hydrolysis process was determined on the basis of hydrolysis degree(DH),molecular weight distribution(MWD),free amino acid content(FAA),electropherogram,and antigenic indicators.The hydrolyzed peptides were detected by LC/MS,and the mechanism of antigenicity decrease was further explored.Under the optimal temperature and pH of each single enzyme,after hydrolysis at a substrate concentration of 5%(w/v)for 3 h,the highest DH was observed for Alcalase(20.11%),followed by Neutrase(19.73%).From the depth of the bands in the SDS-PAGE electrophoresis,it can be found that the product after single-enzyme hydrolysis still contains larger peptide segments.Tricine-SDS-PAGE electrophoresis showed that with the increase of hydrolysis time and enzyme-to-substrate ratio(E/S),the conditions became clearer.In order to reach the state of extensive hydrolysis,for the combination of Alcalase and Neutrase with high degree of hydrolysis,under different E/S,complex enzyme ratio,pH,temperature,time 5 single factors,the response surface experiment was used to select hydrolysis the highest condition.The extensive hydrolysis conditions of E/S 6000 U/g,enzyme mass ratio of 1:1,pH 8.5,and 50℃ co-hydrolysis for 3 h were determined.From the analysis of the degree of hydrolysis of Hilmar 8350,the partial hydrolysis conditions of Neutrase at the E/S6000 U/g,pH 7.0,and 50 ℃ for 0.5 h were determined.Further MWD results illustrated that in the condition of extensive hydrolysis,it can meet the requirement that the proportion of molecular weight <3 k Da is more than 95%;under the condition of partial hydrolysis,the proportion of molecular weight <6 k Da was about 70-80%.It can match with the expected molecular weight index.The FAA content measured under the extensive hydrolysis condition was 18.33%,and the FAA content under the partial hydrolysis condition was 11.51%.From the detection of ELISA,we found the antigenicity reduction rate of extensively hydrolyzed whey protein was 59.41%,and the antigenicity reduction rate of partially hydrolyzed whey protein was 12.06%.From the detection results of LC-MS,the allergen breakage sites were concentrated at positions 41-60 and 102-124.It produced more highly abundant peptides such as VRTPEVDDE and VEELKPTPEGDLE.Through mathematical derivation and experimental analysis,the enzymatic hydrolysis kinetic models and various parameters under the conditions of extensive hydrolysis and partial hydrolysis were obtained.The kinetic constants for extensive hydrolysis and partial hydrolysis were determined to be 31.521 and 11.474,respectively,and the enzyme inactivation constants were 3.442 and 1.831,respectively.By comparing the actual DH with the theoretical DH,the accuracy of the model was verified,and the deviation was kept within10%,indicating that the established kinetic model can be applied to actual production.The molecular weight prediction of the extensively hydrolyzed and partially hydrolyzed hydrolysate can be used to determine the molecular weight distribution under a certain hydrolysis condition.Extensively hydrolyzed and partially hydrolyzed protein powders were prepared by spray drying,and extensively hydrolyzed protein powders without macromolecular peptides are prepared by ultrafiltration process.The volume average diameters of extensively hydrolyzed and partially hydrolyzed whey protein powders were 1.853 μm and 0.512 μm,respectively,indicating that the solution of partially hydrolyzed protein powder was more stable.The wet sinkability and dispersibility of the extensively hydrolyzed whey protein powder are better than those of the partially hydrolyzed whey protein powder,and the color is relatively more yellow.The dissolution effect of them were good.From the sensory evaluation results,it can be seen that the bitterness of the extensively hydrolyzed protein powder is stronger.