Study On The Preparation And Purification Of Antioxidant Peptides From Whey Protein And Its In Vitro Antioxidant Activities

Whey protein was used as raw material to prepare antioxidant peptides by enzymatic method.The whey protein peptides were fractionated by series of ultrafiltration separation technology,and their antioxdiant activity were evaluated by 5 kinds of in vitro tests.The oxidative stability of the peptides with the strongest activity were studied.On the basis,the peptides were further isolated and purified by gel chromatography,ion chromatography and RP-HPLC.Consequently,the sequences were identified by mass sepctrometry.1.Papain,alkaline protease,neutral protease,bromelain and acid protease were used in hydrolysis whey protein.In addition,through the hydroxyl free radical scavenging rate and the yield of pepetides,the neutral protease was selected as the best enzyme.With regards to single factor experiment and response surface experimental design,the optimal hydrolysis conditions were found as follows: pH at 5.50,temperature at 65 ° C,time at 1.6 h,substrate concentration at 5% and enzyme dosage at 5 000 U/g,resulting in an hydroxyl free radical scavenging rate of 74.54 ? 0.95%.2.Subsequently,the hydrolysates were isolated in four different molecular weight molecular peptides as followed: P-I(>10kDa),P-II(10~5 kDa),P-III(5~3 kDa)and P-IV(<3kDa)by ultrafiltration membrance with molecular weight cut-off 10 kDa,5 kDa and 3kDa.On the basis of 5 kinds of in vitro tests,the hydroxyl free radical,ABTS free radical and superoxide anion free radical scavenging activity were P-IV>P-III>P-II>P-I.The DPPH free radical scavenging capacity was P-I>P-II>P-IV>P-III.There was no significant difference in reducing power between the four peptides(P>0.05).It turned out that P-IV boasted the strongest in vitro antioxidant activity.3.The stability study of the P-IV showed that the P-IV harbor a good resistance to heat and acid resistance,however the alkali resistance was poor.The NaCl inhibited the stability of the P-IV significantly(p<0.05).The stability declined when the irradiation time exceeded 6h(p<0.05).The sugar had no significant effect on the stability of the P-IV at 25℃(p>0.05).4.The P-IV was separated by Sephadex G-25 gel chromatography.The optimum conditions were as follows: ultrapure water as eluent with 2.0mL injection volume at the speed of 0.6 mL/min.Two peaks F1 and F2 were observed.And the hydroxyl free radicals andABTS free radicals scavenging capacity of F1 were stronger than F2.Consequently the F1 was separated by DEAE-52 ion chromatography.The optimum conditions were as follows:1.0mol/L NaCl as the eluent with 2.0 mL injection volume at the speed of 1.5 mL/min.Three peaks F11,F12 and F13 were observed.The hydroxyl free radicals and ABTS radicals scavenging capacity of F13 were stronger than F11 and F12.RP-HPLC was used to separate F13.There was only one peak F131 observed.ESI-MS/MS were used for identification amino acid sequence,and four antioxidant peptides sequence,RTPEVDDEALEK,LVRTPEVDDE,ELKPTPEGDL and VIESPPEINT were identified.