Preparation And Purification Of Antioxidant Peptides From Walnut

Walnut is a kind of food which its nutritional and economic value is very high,containing rich oil and protein, and walnut has been commonly used for oil materials in the industrial production, at present, but the utilization of oil cake containing a lot of protein has certain limitations.In order to fully develop and utilize the resources of walnut protein, increase additional value of walnut products, this paper selects walnut cake as raw material to prepare antioxidant activity peptides, and peptides mixture was separated and purified to obtain small peptides molecules of higher antioxidant activity, and its structure composition was studied. The main findings are as follows.In this paper, we studied the influence factors of walnut protein preparing walnut polypeptides with enzymatic hydrolysis, the experiment selected the DPPH clearance rate of walnut protein Enzymatic hydrolysate as the index, and the neutral protease was screened out from four kinds of commercial protease. The method of single factor and orthogonal test was used to study the process of walnut protein enzymolysis.Through the experiment obtained optimal enzymolysis process:enzymolysis time was3 h, the amount of enzyme was 4%, the temperature was 35℃and pH value was 7.0,substrate concentration was 2.5%, under these condition, the clearance rate of prepared antioxidant peptides on DPPH can reach 95.56%.After the walnut protein extraction liquid of enzymolysis was centrifugated,ultrafiltrated, freezed drying, The molecular weight of walnut polypeptides was mainly concentrated in 3KD. The amino acid content and composition of walnut protein and polypeptides were analyzed, the results showed that walnut protein and polypeptides in the crude product of amino acids were basically consistent, which both contain 7 kinds of essential amino acids, except for tryptophan, but arginine,glutamic acid and aspartic acid content were relatively high.Using column chromatography system, the walnut polypeptides crude product was preliminarily separated by anion column, cation column and G25 gel column inturn. The polypeptides components with strong DPPH scavenging ability were obtained by experiments, and the optimum separation conditions of column chromatography were determined. The optimal separation conditions of sample solution through an anion exchange column: eluent was 0.6mol/L NaCl, pH value was7, the concentration of the sample was 10 mg/mL, the flow rate was 10mL/min, The highest antioxidant activity was obtained after gradient elution. Under the condition of the same concentration,the DPPH clearance rate of A was improved by 2.26%compared with the prior separation.Then A component was purified by cation exchange column, and its optimal separation conditions:elution liquid was0.4mol/LNa Cl, pH value was 7, sample concentration was about 5mg/mL, the flow rate was 10mL/min, obtained H component and the antioxidant activity of H component was highest after gradient elution. Under the condition of the same concentration, the DPPH clearance rate of H was improved by 2.61% compared with the prior separation. Then H component was purified by the G25 gel column and its optimal separation conditions: sample volume was 1mL, sample concentration was about 1mg/mL, the flow rate was 0.5mL/min. obtained I component and the antioxidant activity of I component was highest after gradient elution. Under the condition of the same concentration, the DPPH clearance rate of I was improved by2.26%, 4.87% and 7.15% respectively compared with the prior separation,A component and initial sample solution. I components were detected by LC-MS/MS,the molecular weight range of I was between 713-1620 Da, there were 6 kinds of possible peptides sequences.It was possibility of a maximum 15 peptides compound,and Phe-Val-Asn-Ser-Thr-Val-Val-Ala-Ser-Val-Thr-Ile-Ile-Asp-Arg was sequence segment of peptides.In addition, this paper also explored the determination method of the content of small molecule polypeptides in the low concentration range, the biuret method and Coomassie brilliant blue method and UV points spectrophotometric method were compared and analyzed, The results show that the Kaumas Bradford method on determination of small molecular weight polypeptides content is not feasible. And biuret method only determined the small molecular weight peptides at concentrationsabove the 0.1mg/mL.Therefore it is not suitable for the determination of low concentrations peptides. And ultraviolet spectrophotometry for the determination of small molecular polypeptides content in low concentration range is better.