| The study was granted by National 863 Program under Grant research and development on the stability and processing applicability of active protein of pine nut(Pinus koraiensis)(2013AA102206-5).Three short peptides with sequence of Met-Met-Cys-Thr-Asp,(MMCTD),Lys-Cys-His-Lys-Pro(KCHKP)and Gln-Trp-Phe-His(QWFH)obtained from previous studies,would be adopted in this project.The parameters of improving the antioxidant activity of short peptides by PEF were optimized,and the mechanism were futher investigated by means of circular dichroism spectrum,reversed-phase high performance liquid chromatography,Fourier transform infrared spectroscopy,ultraviolet absorption spectrum,1H-NMR and other kinds of analytic techniques.In addition,the verficvation experiments were conducted by using intrinsic fluorescence spectra and Infrared microscopy.This study has provided the technical and theoretical basis for the PEF treatment of antioxidant peptides with different sequences.The results of this study were as follows:(1)The effects of PEF on the antioxidant activity of short peptides were studied.MMCTD,KCHKP and QWFH were taken as experimental objects and the DPPH,ABTS radical scavenging capacity and Oxygen radical absorption capacity were used as evaluation indexe.The effects of electric field strength and electric field frequency on the antioxidant activity of short peptides were studied.The antioxidant activity of MMCTD,KCHKP and QWFH were all improved significantly to some extent after PEF treatment,and reached the maximum values at 2400 Hz,5 k V/cm,1800 Hz,10 k V/cm and 2400 Hz,15 k V/cm,respectively.There,the original samples and samples after PEF treatment under the opimal optimal parameter were selected for further analysis(2)The effects of PEF on secondary structure of the antioxidant peptides were studied.The MMCTD,KCHKP and QWFH without PEF treatment and optimal processing conditions were taken as as experimental objects,and the content of secondary structure,thermodynamic parameter and Zeta potential of three short peptides in aqueous solution were selected as evaluation indexes,and then the effect of PEF on the secondary structure of short peptides was investigated.Results showed that the content of mian secondary structure of MMCTD,KCHKP and QWFH decreased in the aqueous solution,and the random coil structure increased significantly,which indicated that PEF destroyed the secondary structure of short peptides in aqueous solution.As the H bond that maintained the secondary structure was destroyed after PEF treatment,the denaturation temperature of the small peptide decreased,the internal energy of the system decreased and the disorder increased,these results indicated that short peptides can react with free radicals more effectively after PEF.The decrease of the zeta potential of the short peptide induced by PEF also reflected the decrease of the stability.(3)The effects of PEF on primary structure of the antioxidant peptides were studied.MMCTD,KCHKP and QWFH without PEF treatment and optimal processing conditions were taken as as experimental objects,and the effects of PEF on primary structure of short peptides were studied from the peptide chain,peptide bond,functional group and H proton of the three small peptides by using HPLC?UV?FT-IR?and 1H-NMR.RP-HPLC results showed that small peptide chains remained stable but had not cleaved into smaller peptides after PEF treatment.And the UV and FT-IR results showed that PEF treatment not only enhanced the vibration intensity of the peptide bond of the small peptide backbone,but also affected the micro environment of the side chain group.1H-NMR results showed that the short peptide did not produce any new type of H proton after PEF treatment,and the polypeptide chain had a stable chemical structural,which were consistent with the previous results.(4)The functional mechanism of improvement of antioxidant activity of short peptides induced by PEF was verified.The QWFH with highest improvement of antioxidant activity was taken as as experimental objects,and the verification of functional mechanism was conducted through FT-IR imaging and intrinsic fluorescence spectra.The FT-IR imaging result showed that PEF changed the structure of QWFH chain and the spatial distribution of side chain groups,which made QWFH react with free radicals in the system more fully.The result of intrinsic fluorescence spectra also verified that the microenvironment of Try changed to hydrophilicity,and play a better role in scavenging free radical. |
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