Change Of Bitter Peptide Content Of Cold-Pressed Flaxseed Oil During Accelerated Oxidation

The main bitter substance of flaxseed oil,cyclolinopeptide E?CLE?,also known as bitter peptide,is formed by oxidation of its precursor peptide.Based on the establishment of CLE quantitative determination method,the change of CLE content of flaxseed oil during oxidation was fitted,in order to find out the oxidation variation law of CLE,thus providing theoretical guidance for the reasonable processing and storage of flaxseed oil.The main research contents are as follows.Firstly,the pretreatment method for quantitative determination of CLE in flaxseed oil by HPLC was studied.The pretreatment effects of solvent extraction method and solid phase extraction?SPE?method were compared,and the established method was evaluated.The results showed that the pretreatment methods did not affect the peak time and peak shape of CLE,but there were differences in impurity removal effects of the two methods.Compared with the solvent extraction method,the SPE method has better purification and enrichment effect,flaxseed oil sample pretreated by SPE has fewer peaks of triglycerides and other impurities.Furthermore,the pretreatment effect of SPE silica gel cartridges with different filler specifications was compared,the results showed that when the loading amount was fixed,the CLE content decreased with the increase of filler amount,and the separation and enrichment effect of SPE silica gel cartridges with 500 mg filler content was the best.Qualitative analysis by UPLC-Q-TOF/MS confirmed that the material purified by SPE silica gel cartridge was indeed CLE.According to the above,the SPE pretreatment-HPLC quantitative determination method of CLE was established.The recovery of the method was92.62%,the inter-group precision was 1.79%,and the intra-group precision was 0.62%.The LOD was 0.052?g/mL,and LOQ was 0.174?g/mL.Secondly,three representative cold-pressed flaxseed oils of first grade oil,second grade oil and virgin oil were used as raw materials.The initial values of their physiochemical indexes,CLE content,fatty acid composition and antioxidant content were analyzed.The results showed that the quality of the raw oils was good and there was no obvious oxidative rancidity.The highest CLE content in virgin oil was 299.99 mg/kg,followed by second grade oil,which was 295.42 mg/kg,and the lowest in first grade oil,only 1.26 mg/kg.The content of UFA in the three kinds of flaxseed oil reached 87.13%⁹1.08%.They were also rich in trace antioxidants,among which the tocopherol content was 289.95⁴91.00 mg/kg,the polyphenol content was 6.18¹4.33 mg GAE/kg,and the phytosterol content was2724.81³637.48 mg/kg.Thirdly,the Rancimat test was used as accelerated oxidation model.The changes of CLE content during oxidation process of the three cold-pressed flaxseed oils were investigated,and their changes were fitted.The results showed that the CLE content of the first grade oil during accelerated oxidation process at different temperatures has been at an extremely low level,and has never reached the human bitterness threshold,its highest value was only 3.22 mg/kg,while the CLE content of second grade oil and virgin oil were several hundred times that of the first grade oil,moreover,they both increased first and then decreased under five different temperatures with the prolongation of heating time.Change of CLE content of the two oils were divided into two stages of rising and falling for fitting,respectively.The fitting results were in line with zero-order reaction.The variation equation for rising stage of virgin oil was[P_t]=[P₀]+2.57×10¹⁴×[exp?-11377/T?]×t,and for falling stage was[P_t]=[P₀]-3.88×10¹¹×[exp?-8855/T?]×t.The variation equation for rising stage of second grade oil was[P_t]=[P₀]+3.88×10¹³×[exp?-10301/T?]×t,and for falling stage was[P_t]=[P₀]-6.98×10¹⁰×[exp?-8423/T?]×t.The predicted values of the equations were highly consistent with measured values.Finally,the apparent rate constant,activation energy and thermodynamic parameters of the change of CLE content during accelerated oxidation by Rancimat were calculated.The results show that the rate constant k of CLE content increases rapidly with increasing temperature.The rising rate of CLE content in virgin oil was slower than the falling rate,while second grade oil was the opposite.The Ea and?H values of the two oils in rising stages were larger than that in the falling stage,and the Ea and?H value of virgin oil was larger than that of the second grade oil,and both of them were positive values.The?S value was positive during the rising stage but negative during the falling stage.According to the Gibbs function criterion?G=?H-T?S,it can be speculated that the oxidation formation reaction of CLE can only take place spontaneously at high temperature,low temperature can effectively inhibit the formation of CLE.The falling stage of CLE content cannot take place spontaneously,it can be brought into falling stage by reducing the activation energy.Comparing the oxidation process between CLE and flaxseed oil,it was found that the oxidation rate of CLE content in rising stage was slower than that of virgin oil,in falling stage was the opposite.The oxidation rate of CLE content in rising and falling stages were faster than that of second grade oil.