Study On Preparation Of Anti-Oxidant Peptides By Hydrolysis Of Rice Protein

The preparation technology of anti-oxidant peptides,which derived from hydrolysis of rice protein by enzyme,and the peptides's characteristics were investigated in this study.Three commercial proteases,flavor,neutral,and papain,were selected from seven ones according to the elimination rates of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical of hydrolytes.Orthogonal tests were performed to determin the respective optimum hydrolysis condition of each protease.And the elimination rate of DPPH free radical of each optimum hydrolyzed solution were 75.2%,89.5%and 58.8%,respectively.The values of EC₅₀ of the anti-oxidant peptide products were 2.92 mg/mL,2.17 mg/mL,and 3.86 mg/mL,respectively.The neutral protease was preferred because the hydrolate by the neutral protease showed the highest anti-oxidant activity in the three ones.And the optimal enzymatic hydrolysis technologic parameters were as followings:the concentration of substrate 10g/100ml, the quantity of protease 8%,pH 6.0 at 60℃for 30 min.The combinations of two enzymatic hydrolysis were tested based on single enzymatic hydrolysis for rice protein.It was found that the adding order of enzymes affected the anti-oxidant activity of hydrolyte obviously.It is advantageous to de-active the enzyme after the first enzymatic hydrolysis.For enzymatic hydrolysis of rice protein,the optimal combination was:firstly neutral protease hydrolysis for 30 min,then papain hydrolysis for 15min after de-active neutral protease.The EC₅₀ of hydrolysate solution was 1.23mg/mL,decreased 43.3%compared with single enzymatic hydrolysis.The raw material of rice protein was pretreated by ultrasound(power 1500W,at initial temperature 40℃for 20min),then hydrolyzed with neutral protease.The results showed that the elimination rate of DPPH and EC₅₀ were 74.8%and 0.932 mg/mL,respectively.The EC₅₀ dropped 57.1%compared with non-pretreated sample. The peptides were applied for the eliminations of O^2。,DPPH and OH°free radicals and the EC₅₀ were 0.0678 mg/mL,0.932 mg/mL and 0.117 mg/mL,respectively.732 cation exchange resin chromatography,Sephadex-G25 chromatography,and HPLC were employed for the purification of enzymatic hydrolyte.Two anti-oxidant peptides were obtained after the purification.According to the structural analysis by ABI 4700 TOF/TOF,the amino acid sequences of the two peptides were Gly-Val-Pro-Ser-Ala-Val-Ser-Arg-Asn-Met-Thr-Arg-Ser-Arg and Leu-Ala-Ser-Gln-His-Leu-Val-Ala-Ile-Leu-Asp-Pro-Pro-Arg. The EC₅₀ were 4.91μM and 5.39μM, respectively.