The Effect Of Different Structural Flax Cyclic Peptides On The Oxidative Stability Of Flaxseed Oil And Mechanism

Flaxseed oil is rich in approximately 50%of alpha-linolenic acid,which is an important source of dietary essential fatty acids.Flaxseed oil contains n-3polyunsaturated fatty acids,which have health benefits for the prevention of various chronic diseases,such as hypertension,hyperlipidemia and obesity,as well as anti-inflammatory and anti-cancer effects.However,processed flaxseed oil is more susceptible to oxidation and rancidity than unprocessed,mainly because the refining process removes the natural antioxidants from flaxseed oil,and the edible oil may also be subject to accelerated oxidation by metal ions during processing.Currently we know that flaxseed contains several natural antioxidant components,including vitamin E,lignans,phenolic acids and flavonoids,as well as flax cyclic peptides.Flax cyclic peptide is a class of hydrophobic cyclic peptides,which was a unique natural component of flaxseed and flaxseed oil.More than 30 cyclic peptides have been discovered,consisting of 8-9 amino acids,with immune,anti-cancer,anti-inflammatory and antioxidant biological activities.The free radicals generated from oil oxidation may react with-OH,-SR and-NH in cyclic peptide amino acids to block the chain reaction of oil oxidation and thus acting as an antioxidant.In addition,whether the unique cavity structure of flax cyclopeptide can encapsulate metal ions,free radicals and aldehydes and ketones products and other polar compounds,thus acting as antioxidant capacity?How do the different structures of cyclic peptides in flaxseed oil act as antioxidants and their mechanisms?Are there any differences in the antioxidant capacity of different structures of flax cyclic peptides?In order to provide scientific experimental data for the reasonable and moderate processing and storage of flaxseed oil and for the high-value utilization of flaxseed industry,this study was conducted to isolate different structures of flaxseed cyclic peptides,and to explore the oxidative stability of flaxseed oil,the relationship between the polar components and cyclic peptides at different oxidation stages by adding different structures of cyclic peptides and metal ions to refined flaxseed oil（excluding cyclic peptides,vitamin E,polyphenols and other natural antioxidant components）.Besides,the antioxidant capacity of flaxseed cyclic peptides in flaxseed oil and the binding/encapsulation of oxidation products such as aldehydes and ketones and metal ions were investigated through a combination of theory and experiments with molecular docking and fluorescence burst.The main findings are as follows:1、The effects of different concentrations of cyclic peptide[1-9-NαC]-Linusorb B3（CLA）on the oxidative stability of refined flaxseed oil（RFO）were analyzed by short-term accelerated oxidation experiments,and it was found that CLA（0.025 m M～0.25 m M）showed a dose-dependent effect on the improvement of its oxidative stability.In refined flaxseed oil,after adding different ratios of cyclic peptides with metal ions Fe²⁺and Zn²⁺,it was found that when the ratio of cyclic peptides to metal ions was increased from 1:1 to 1:2,the OSI values decreased from 0.55±0.07 h to 0.48±0.11h（P>0.05）in the Fe²⁺group and from 0.63±0.04 h to 0.65±0.07 h in the Zn²⁺group（P>0.05）,but there was no significant difference between their respective OSI values.This indicated that CLA still had a strong ability to maintain the stability of flaxseed oil when the ratio of CLA to metal ions was 1:2.When the ratio of CLA to metal ions increased to 1:3,the OSI values of flaxseed oil decreased significantly from 0.55±0.07 h to 0.23±0.04 h（P<0.05）in the Fe²⁺group and from 0.63±0.04 h to 0.25±0.00h（P<0.05）in the Zn²⁺group.This may indicate that the presence of excess metal ions exceeded the metal binding capacity of CLA and rapidly caused oxidation of flaxseed oil.Therefore,on balance,1:2 was chosen as the appropriate ratio of cyclic peptides to metal ions in the subsequent study of the antioxidant mechanism.2.By adding purified CLA to refined flaxseed oil for 24 days with accelerated oxidation experiment,we found that CLA could effectively improve the oxidative stability of refined flaxseed oil compared with the control group（RFO）.The acid value（AV）of RFO increased 24.14 times after accelerated oxidation,while RFO with CLA addition（CLA-RFO）increased only 7.21 times.Similarly,the peroxide value（POV）and anisidine value（P-AV）of CLA-RFO were significantly reduced（P<0.05）.In addition,CLA effectively slowed down the oxidation of oils induced by metal ions.In the Cu²⁺group,the addition of CLA reduced its AV by 18%and POV by 20%.In order to explore the antioxidant mechanism of cyclic peptides,molecular docking experiments and fluorescence bursting experiments showed that CLA could bind to three metal ions and propionaldehyde（one of the main products ofα-linolenic acid oxidation）in a relatively stable manner,with the following order of binding strength:CLA-C₃H₆O>CLA-Cu>CLA-Fe>CLA-Zn.This indicates that the special cyclic structure of CLA,may preferentially bind to propionaldehyde,and secondly may encapsulate metal ions,thus effectively slowing down the oxidation of flaxseed oil.3.In the study of the effect of cyclic peptide[1-9-NαC]-Linusorb B2（CLB）on the oxidation stability of flaxseed oil,CLB can play a certain role in the early stage of flaxseed oil oxidation.On the ninth day of accelerated oxidation,the addition of CLB reduced the AV by 12%and the POV by 5%compared with the control RFO.In contrast,the CLB group made the oxidation of flaxseed oil more drastic in the late stage of accelerated oxidation.After 15 days of accelerated oxidation,the P-AV of RFO increased approximately 12.9 times,whereas the RFO with CLB addition（CLB-RFO）increased 15 times（P<0.05）.Similarly,the addition of CLB in the metal ions group accelerated the oxidative rancidity of flaxseed oil in the late oxidation stage.For example,the addition of CLB(Cu²⁺-CLB group)increased its AV value by about 8%and POV value by about 16%after 15 days of accelerated oxidation compared to the Cu²⁺group（P<0.05）.Since the CLB molecule contains methionine（Met）,it is very easily oxidized in flaxseed oil,which converts it to methionine sulfoxide（Mso）,i.e.oxidized to cyclic peptide[1-9-NαC],[1-Met O]-linusorb B2（CLC）;and can be further oxidized to methionine sulfone’s（Msn）,i.e.to cyclic peptide[1-9-NαC],[1-Met O2]-linusorb B2（CLK）.Therefore,it is speculated that the pro-oxidative effect of CLB addition on flaxseed oil at the late stage of oxidation may be caused by the oxidation of its Met structure to sulfone（Mso）and sulfoxide（Msn）.The oxidized cyclic peptides（CLC and CLK）have a significantly reduced ability to encapsulate/bind metal ions as well as aldehyde and ketone products,etc.,making their antioxidant effect reduced.4.Molecular docking experiments were performed to investigate the binding ability between different metal ions and aldehydes with three cyclic peptides,CLB,CLC and CLK.As the methionine residues of CLB were oxidized to methionine sulfoxide and methionine sulfone forms,their binding ability with metal ions showed a decreasing trend.Among the docking with the three metal ions,the binding ability of CLB with metal ions was the strongest,followed by CLC and the weakest by CLK.In addition,the binding ability of CLK with aldehydes was also the worst.Therefore,it was speculated that in the late stage of oxidation of flaxseed oil,when CLB was oxidized to CLC and CLK,their binding ability to metal ions was weakened,and also the encapsulation ability of CLK to aldehydes was reduced,which affected the oxidation inhibition ability of cyclic peptide K on flaxseed oil.5.The purified cyclic peptides C and K were added to refined flaxseed oil for accelerated oxidation experiments,which verified their oxidation inhibition effect on flaxseed oil.During the oxidation of flaxseed oil,the presence of CLC improved the oxidative stability of refined flaxseed oil.After 15 days of accelerated oxidation,the AV of RFO group was increased by 2.82 times and RFO group with the addition of CLC（CLC-RFO）was increased by 2.6 times.In addition,CLC has some ability to form more stable complexes with metal ions,thus slowing down the oxidation of flaxseed oil.This ability may be related to the presence of a more polar sulfoxide group in CLC.In the Cu²⁺group,the addition of CLC reduced the AV by about 26%and the POV by about 25%（P<0.05）.Interestingly,CLK not only had no antioxidant effect,but also promoted the oxidation of flaxseed oil.At the end of the accelerated oxidation,the AV of CLK-RFO was increased by 4.8 times,while the control group（RFO）was increased by only 2.8 times（P<0.05）.The presence of CLK accelerated the oxidative rancidity of flaxseed oil.Similarly,in the Cu²⁺group,the addition of CLK increased its AV by about 25%and POV by about 33%（P<0.05）,which suggesting that the sulfone group of CLK may not be able to bind to metal ions,which is consistent with the molecular docking results,while the sulfone group of CLK may also have a pro-oxidant effect.