Study On The Effects Of High Hydrostatic Pressure On Bovine Whey Protein Enzymolysis And Hydrolysates

Ultra-high pressure food processing technology is considered to be the most potential and promising food processing technology,and gets more and more attention from many people.Whey protein is one of the main protein component,which is of high nutrition,and can be used as food additives are widely used in food industry.In order to further the development and utilization of whey protein in the food industry,means of decomposing allergenic components of whey protein by enzymatic hydrolyzation,increasing the digestion of whey protein,enhancing biological activity, have become a current research hot spots in this field both at home and abroad.However,there is no research reports about the effect of high pressure on whey protein of Alcalase enzymatic hydrolyzation and antioxidant activity of the hydrolysates.95%whey protein isolate(WPI) powder was studied in this paper,the effect of high pressure on the structure of different pH WPI solution of 5%(w/v) was studied,the change of hydrolysis degree of WPI at different pH(6.0～11.0) and pressure(100～500MPa) was discussed,the hydrolysate was studied by SDS-PAGE,Tricine-SDS-PAGE and gel chromatography,and pyrogallol auto-oxidation system,DPPH method and fenton reaction were used to determine the free radical scavenging activity of hydrolysates.The results as follows:(1) At same pressure,the degree of hydrolysis of WPI increased with the rising pH of solution, and reached the toppest at pH11.0;at the same pH of solution,the degree of hydrolysis of WPI increased with the rising pressure,and the increasing extent decreased with pH rising and there was no significant difference in high pressure treatments except at pH 6.0 and 7.0.Compared to normal atmosphere,when pH at 6.0,the effect of high pressure on WPI hydrolysis was the most obvious, the degree of hydrolysis is 2.91%at normal atmosphere,the degree of hydrolysis reached to 8.41%, the degree of hydrolysis can rise twice.However the effect of high pressure on the degree of hydrolysis gets downtrend with pH rising,when pH at 11.0 the degree of hydrolysis increased 21.07%compared to normal atmosphere pressure. (2) Ultra high pressure accelerated the change of WPI structure,and there was difference for pH and presure.The denature extent increased with the pressure rising and reached the highest at 500MPa,which is significant at pH6.0 and 7.0.(3) There were some difference on the effects of pressure on the solvent and pressure,but the total current was similar.At pH9.0,the enzymic activity enhanced under 200 and 300MPa,and decreased under 400 and 500MPa,when the enzyme was solved in water;the enzymic activity decreased in general while no significant changes under 200 and 300MPa when enzyme was solved in Tris-HCl buffer(pH9.0).(4) The distinct band of composing protein of WPI was not found in SDS-PAGE pattern of WPI hydrolysates,which meaned most main composing proteinβ-Lg andα-La were hydrolyzed. The area of 3～10kDa in Tricine-SDS-PAGE pattern was stained,and the colour of stained area became light with the degree of hydrolysis increasing,which meaned the hydrolyzation induced the generation of more low molecular weight peptides,maximum low molecular weight peptides was generated at pH6.0,and minimum at pH11.0.A special band of 8.3kDa was checked out of the hydrolysates of during the treatment of 100～300MPa at pH9.0.(5) Ultra high pressure accelerated the hydrolyzation of different pH WPI,and decreased the elution peak area of high molecular substance,and enhance the proportion of low molecular weight substance.The hydrolysates were different with different pH under high pressure treatment by the gel chromatography analysis of hydrolysates.The main molecular weight of hydrolysate of pH6.0 were 7187,3550;the main molecular weight of hydrolysate of pH7.0 were 4707,2019,886;the main molecular weight of hydrolysate of pH8.0 were 4707,2325,1148;the main molecular weight of hydrolysate of pH9.0 were 7187,2677,1322,567,138;the main molecular weight of hydrolysate of pH11.0 were 6241,2019.(6) At same pH and different pressure,the content and varieties of hydrolysates may be different. At pH9.0,there were 5 hydrolysates when pressure under 300MPa and the main molecular weight of hydrolysate were 7187,2677,1322,567,138;there were 4 hydrolysates when pressure was 300MPa and the main molecular weight of hydrolysate were 7187,1322,567,138;there were 4 hydrolysates when pressure was 500MPa and the main molecular weight of hydrolysate were 6241, 997,371,104。(7) The hydrolysates of WPI had stronger free radicals scavenging ability in vitro,and the free radicals scavenging ability changed with the difference of pH and pressure.The WPI hydrolysate prepared by 100MPa treatment at pH6.0 has the highest rate of DPPH·which reached 53.07%;the WPI hydrolysate prepared by 200MPa treatment at pH9.0 has the highest rate of O₂^·- which reached 38.02%;the WPI hydrolysate prepared by 100MPa treatment at pH11.0 has the highest rate of·OH which reached 38.02%.The research indicated that ultra high pressure can enhance the hydrolysis of WPI,affect the enzymic activity of Alcalase,accelerate the change of WPI strcutural protein,affect the consistent of hydrolysates and antioxidant ability,and give some theory reference for the application of high pressure in food industry.