Preparation,Structure Characterization And Interaction With Whey Protein Of Camellia Oleifera Cake Polysaccharide

Camellia oleifera belongs to the camellia family and is an important woody oil plant in south China.After the camellia fruit and tea seeds are pressed for oil,the remaining camellia meal is rich in a variety of biologically active substances,such as polyphenols,tea saponin and polysaccharide.In this paper,and the camellia oleiferal cake polysaccharides were extracted and purified.In vitro antioxidant capabilities of different purification process on the scavenging capacity of free radicals(DPPH,ABTS),iron reduction capacity(FRAP)and oxidative radical absorption capacity(ORAC)were also investigated.The purified Camellia oleifera cake polysaccharides were identified by high performance liquid exclusion chromatography-diode array detector-evaporative light scattering detector(HPLC-DADELSD),for preliminarily exploring the relationship between polysaccharide structure and biological activity.Finally,the interaction between Camellia oleifera cake polysaccharide and whey protein is studied to provide a theoretical basis for further processing and utilization in the future.The research results are as follows:In the first part,different ultrasonic power,extraction time,extraction temperature,and solid-liquid ratio have certain influence on the yield of polysaccharide.On the basis of single factor,the response surface optimization experiment was used to determine the optimal extraction process of polysaccharide:ultrasonic power 322 W,extraction time 20 min,the ratio of solid to liquid is 1:25,and the extraction temperature is 60℃.Under these conditions,the yield of polysaccharide is 23.1%.In the second part,the crude polysaccharides were purified by different types of macroporous resins:D101(non-polar),AB-8(weak polar),NKA-9(polar)and XAD-6(ion exchange resin),according to the adsorption effect,the removal effect of pigment>the removal effect of tea saponin>the removal effect of protein,using the comprehensive scoring method,the most suitable macroporous resin for the purification of Camellia oleifera cake polysaccharide was D101.Then the crude polysaccharide of Camellia oleifera cake was deproteinized,and it was found that the TCA and Sevage reagent have their own advantages.So the in vitro antioxidant activity of Camellia oleifera cake polysaccharides obtained by different preparation methods showed that the polysaccharides prepared by TCA method had better reduce ability(FRAP)to ions,and the polysaccharides prepared by Sevage method had better resistance to free radicals(DDPH and ABTS),with high scavenging efficiency and strong oxidative radical absorption(ORAC)ability,among which the scavenging rate of DPPH was 70.9%,the scavenging rate of ABTS was 87.3%,and the ORAC value was 0.29 μg TE/mL.In the third part,high performance liquid chromatography-diode array detectorevaporative light scattering detector(HPLC-DAD-ELSD)were used to identify the structue of polysaccharides.The results showed that:the peak pattern of polysaccharides prepared by Sevage method was sharp and narrow,indicating that the molecular weight distribution was relatively concentrated.However,the peak shape of the polysaccharide chromatogram prepared by TCA method is short and wide,it is speculated that the degree of polymerization of the polysaccharide structure decreases and the branch chain is destroyed,indicating that TCA has a certain damage to the configuration of the polysaccharide in camellia oleifera cake polysaccharides.The polysaccharides were first separated by DEAE-Sephadex A-25 ion exchange column chromatography,and then the polysaccharides were separated according to the molecular weight by Sephacryl S-200 gel column chromatography.Finally,a neutral polysaccharide(COCP-1)was prepared with 92.1%sugar content,84.7%purity,2.0%protein and 0.14%tea saponin.The constitution of COCP-1 is mannose(553.53μg/mg),galactose(54.18 μg/mg),xylose(23.49 μg/mg)and arabinose(56.85 μg/mg)in a molar ratio of 20.6:1.90:1:1.91.The molecular weight of COCP-1 was 278 358 Da.The COCP-1 has typical absorption peaks of sugars,including O-H,C-H vibration,C-O-C vibration and C-O stretching of sugar ring,denaturation vibration of pyran ring,and βglycosidic bond and α-Glycosidic bond characteristic absorption peak.In the fourth part,the interaction and structural stability of Camellia oleifera cake polysaccharide(COCP)and whey protein(WP)under different pH conditions were studied from particle size,Zeta-potential,UV spectroscopy,fluorescence spectroscopy and Fourier transform infrared spectroscopy.The results showed that when the pH was 3.0-4.0,COCP neutralized part of the positive charge of WP,the particle size increased,and the Zetapotential became negative charge,WP and COCP had electrostatic attraction,at this time,whey protein and Camellia oleifera cake polysaccharide formed WP-COCP complex.Therefore,the electrostatic interaction between the WP-COCP complexes can be used to prepare physical hydrogels,and use them to encapsulate flavor substances in the process of forming complexes to prepare microcapsules.COCP changes the α-helix structure and βsheet of whey protein,and transforms the secondary structure of the protein,and promotes the unfolding of protein peptide chains and the exposure to amino acid residues,enhances the hydrophobic interaction between groups,and promotes the rearrangement of hydrophobic and hydrophilic groups.The complex of polysaccharide and protein will form a layer of film on the interface.Because polysaccharide has a thickening effect,it will increase the viscoelasticity of the interface film,and it can be used as an emulsifier.When the pH was 6.0-9.0,the electrostatic repulsion between COCP and WP increased,resulting in the phase separation of protein-rich phase and polysaccharide-rich phase due to thermodynamic incompatibility.Besides electrostatic interactions,there are also hydrogen bonds,intermolecular and intramolecular interactions between WP and COCP.