Purification And Identification Of Protein From Camellia Oleifera Seed Cake

Camellia oleifera is a kind of typical woody oil plant in the south of China,which is famous for producing edible oil.However,after oil extracted from Camellia oleifera,the comprehensive development and utilization of the by-product resources of Camellia oleifera is low,which has become a bottleneck restricting the development of the Camellia oleifera industry.In order to improve the comprehensive utilization of by-products,broadening the utilization of Camellia oleifera.In this study,we studied the extraction,purification,physical and chemical properties and structure feature of protein from Hainan tea seed cake,the aim is to improve the economic benefits of the region and provide a theoretical basis for the research and development of products related to protein.Objective:To obtain high purity protein from tea seed cake,and carry out physical and chemical properties testing and structural identification,aiming to broaden the research and development of protein functional food,providing some experimental basis for the development and utilization of tea seed cake.Methods:Single factor experiment was used to optimize material-liquid ratio,pH,extraction temperature and extraction time.The optimal extraction process of protein was determined by response surface experiment.The single factor experiment was used to optimize the addition of HZOZ,temperature,time and pH on protein decolorization,the eH,eects of decolorization rate and protein retention rate were determined by orthogonal experiment.The effects of HZOZ on the structure and functional properties of protein were investigated.Sephadex G-100 and DEAE-Sepharose FF were used to separate and purify the protein.The purified protein(P1-2)was qualitatively detected and was characterized by measuring physical and chemical properties.LC-MS-MS and CD were used to identify its structure,analysising of protein component P1-2 amino acid information by Mascot software and comparatively analysising in the NCBI database.Results:(1)Determination of the separation and purification conditions of protein form tea seed cake.The response surface optimization experiment showed that when the pH was 10,the temperature was 50?,time was 60 min,and material-liquid ratio was 1:40,the protein yield of tea seed cake can reach 6.56%±0.53%.The best process for decolorization was determined by orthogonal experiment:the amount of HZOZ added was 4%,the temperature was 40?,the time was 60 min,and pH was 8,under these conditions,the decolorization rate and protein retention were respectively 66.72%±1.26%and 76.31%±0.72%.The decolorization of H2O2 had little effect on the structure of the protein,H2O2 can be used to remove the dark brown color of the protein.After two-step column chromatography,the main component P1-2 with high antioxidant activity was obtained,and its purity was 92.91%by RP-HPLC.(2)The properties and structure of the component PI-2 were determined.The molecular weight of component P1-2 was about 15 kDa,the peak temperature of phase change was 120.06?,the crystallinity was low,and it had a sheet morphology.The six peptide sequences identified by LC-MS-MS respectively were ADQLTDDQISEFK,LTDEEVDEMIR,VFDKDQNGFISAAELR,DTDSEEELKEAFR,SLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARK,EADVDGDGQINYDEFV-K,the amino acid sequence similarity to the protein encoded as gi|194716545|gb|ACF931-34.1| was 72%in the NCBI database.CD analysis showed that there ?-helix was 24.6%,?-fold was 20.0%,?-turn was 25.5%and random coil was 30.1%in P1-2.Conclusion:The protein was extracted from tea seed cake,the main component P1-2 was obtained by decolorization and chromatography,and its properties and structure were identified.