Effect Of Ultra-high Pressure Combined With Enzymatic Hydrolysis On The Physicochemical And Sensitization Of Whey Protein Isolate

Whey is a by-product of cheese production,whey contains rich whey protein with high nutritional value,known as the king of protein,plays a good role in promoting the growth and development of infants and young children,and is widely used in processed food.However,some people will have allergic reactions due to eating whey protein,mainly because ?-lactoglobulin(?-LG)and ?-lactoglobulin(?-LA)contained in whey protein have potential allergenicity.At present,the treatment of milk allergy is relatively rare.If allergy is diagnosed,it most can only be prevented by avoiding food completely.However,always avoiding the intake of milk and its products may lead to malnutrition and even affect the growth of infants and young children.Therefore,it is necessary to study appropriate whey protein modification technology to degrade these sensitized whey proteins.In many protein modification technologies,the application of enzymatic modification is more mature,enzyme-catalyzed hydrolysis of macromolecular proteins into small molecular peptides and amino acids,destroying the linear or spatial conformation of allergen proteins,thus significantly reducing its allergenicity.Moreover,it also has the advantages of mild action conditions,economy,the production of functional new peptides and so on.At present,most of the hypoallergenic formula milk powder in China are imported from abroad,and there are few hypoallergenic dairy products on the market at home.In this paper,taking whey protein isolate as the research object,the effects of different levels of ultra-high pressure(UHP)on the structure and allergenicity of whey protein isolate were studied.The protease with the highest hydrolysis degree of whey protein isolate was selected from five kinds of biological enzymes commonly used in the laboratory.On the basis of single factor,the hydrolysis conditions were further optimized by Box-Behken response surface experiment,combined with the decrease of allergenicity,the optimal hydrolysis conditions of protease were obtained.Through the design of ultra-high pressure compound enzyme hydrolysis process,the reduction of allergenicity of whey protein isolate after treatment and the situation of residual peptides were explored.The main conclusions are as follows:(1)After different ultra-high pressure treatment of WPI,it is found that ultra-high pressure can significantly change the surface morphology of WPI.Ultra-high pressure treatment can change the content of ?-helix,?-fold and irregular crimp of WPI,and affect the secondary structure of the protein.It can significantly increase the content of sulfhydryl group and surface hydrophobic group,increase the fluorescence intensity and change the position of maximum absorption wavelength;ultra-high pressure has no significant effect on the degradation of allergenic protein,even because of pressure treatment,expose the allergenic sites embedded in the protein,resulting in the increase of allergenicity.(2)Among the set ultra-high pressure treatment conditions,the treatment group that had the most significant effect on the structure of WPI was 400 Mpa for 20 min.(3)Through the single factor experiment,it was found that the hydrolysis effect of alkaline protease was the best when WPI,was hydrolyzed by five kinds of protease.The hydrolysis process of alkaline protease was optimized by response surface experiment.The optimum hydrolysis process was as follows: enzyme addition 6.4%,initial p H11,enzymatic hydrolysis time 4h,temperature 60°C.Under these conditions,the degree of hydrolysis of WPI reached 20.11%.(4)Alkaline protease hydrolysis had a significant effect on the antigenicity of allergenic proteins in WPI.The residual antigenicity of ?-LG and ?-LA in WPI decreased by 40.22% and 29.71%,respectively.(5)Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Tricine-SDS-PAGE were used to analyze the molecular weight of the hydrolyzed product.It was found that the protein above 10 k Da in WPI could be hydrolyzed completely by alkaline protease hydrolysis alone,but there were residues near 5k Da.However,after ultra-high pressure pretreatment and alkaline protease hydrolysis,it was found that the peptides above 1.7k Da were basically degraded.Therefore,ultra-high pressure compound enzyme hydrolysis can effectively reduce the molecular weight of WPI,thus reducing the allergenicity.(6)The effect of ultra-high pressure on ?-LA was more significant,and that of enzymatic hydrolysis on ?-LG was more significant.After ultra-high pressure pretreatment and alkaline protease hydrolysis,the residual antigenicity of ?-La and?-LG in WPI decreased by 84.98% and 79.89%,respectively(7)Fourteen allergenic peptides were identified by NANO LC/Q exactive HF orbitrap UHR-MS,and the ratios were 17-24?18-26?93-99?93-101?61-71,91-98?93-99?93-99?93-101 in ?-LA and 17-24?18-26?138-147?112-118?52-58?138-147 in ?-LG.Accounted for 5.6% of the total number of peptides detected.This suggests that the hydrolysate still has the risk of sensitization.