Dephenolization And Enzyme Assisted Aqueous Extraction Of Walnut

Walnut kernel is rich in oil and protein,and its oil is an important source of polyunsaturated fatty acids,and its also contains much polyphenols with unique physiological activities.Walnut polyphenols may have an adverse effect on the enzyme assisted aqueous extraction processing(EAEP)because of they may be oxidized or cross-linked with the protein.Basing on the preliminary research of our laboratory,a process for removing phenols from walnut kernel was developded by acidified ethanol ultrasonic treatment.The polyphenols removal rate in the endothelium could reach to 96%.The dephenolic walnut kernels were used to extract oil and protein by EAEP.The walnut protein was whiter,it has better instant dissolution and slower protein oxidation.However,the dephenolization process has low efficiency,both the structure and functional properties of the protein processed by EAEP were not fully revealed,and the oil extraction rate could be improved.Therefore,in the dissertation,the muti-frequency ultrasonic dephenolization processing was used to simplify the dephenolization process and reduce the water consumption of EAEP.Exploring the structure and functional properties of the walnut protein obtained from the EAEP of the dephenolic walnut kernel.Finally,the walnut pellicle polyphenols were enriching by macroporous resin and identified with Ultra-performance liquid chromatography coupled with orbitrap fusion tribrid mass spectrometer(UPLC-TOF-MS/MS)to enhance the industrial application value of this technology.The main content and results are as follows:Firstly,the muti-frequency ultrasonic dephenolization treatment of walnut kernels was studied.Under the frequency of 40 and 28 kHz combination ultrasonic for 60 minutes,for once time,showed that the total phenol content was as high as 19.82 mg/g.The phenols’removal rate was increased to 97.64%,which was increasing the number of extractions effectively.The results of confocal laser scanning microcopy(CLSM)showed that the oil was aggregated from small droplets to flakes,and the oil droplets became larger after the muti-frequency ultrasonic dephenolization treatment,which is beneficial to the research of EAEP.In addition,the UPLC-TOF-MS/MS was used to identify free and bound phenols in walnut pellicles and peeled kernals.The results showed that many types of phenolic substances,mainly including monomeric phenolic acids such as(+)-catechin,gallic acid and ellagic acid,and a large amount of ellagitannin and its derivatives.AB-8 resin was used to purify the crude walnut pellicle polyphenols.The ideal enrichment parameters were as following:sample concentration of 2.00 mg/mL,pH value 3.0,the static adsorption time of 3 h,and 70%was the eluent solvent.Dynamic adsorption study showed that maximum loading amount was 14 times resins bed volume(BV)and the amount of eluent was 4 BV.The purity of walnut pellicle polyphenols was increased from 24.24%to 70.52%.Secodedly,the dephenolic walnut kernels were processed as raw materials of EAEP.Under the condition of low water consumption(solid-liquid ratio 1:3),pH 9.0,reacted at 60℃for 1 hour,the yield of free oil was 43.79%higher than walnuts without dephenolization.Then the emulsion was demulsified with alkaline protease 2709,the demulsification rate could reach to 98.02%.Compared with the original process of our laboratory,the total oil extraction rate of dephenolization walnut kernel was increased from 90.63%to 95.06%,and the water consumption was saved by 40%(solid-liquid ratio 1:3 was reduced from 1:5 to 1:3).Lastly,the water-phase protein processed by EAEP was studied.The results showed that the muti-frequency ultrasonic dephenolization treatment increased the distribution of walnut protein in the water phase and the color was improved.After dephenolization,the purity of the water phase protein increased to 75.54%,the total phenol content was reduced,and α-helix and β-turn content was significantly reduced,while β-sheet was increased;the content of free sulfhydryl groups was reduced,and the content of disulfide bonds was increased;the hydrophobicity of walnut protein increased from 107.57 μmol/g to 122.22 μmol/g;endogenous fluorescence spectroscopy showed that polyphenols would quench the fluorescence of walnut protein;SDS-PAGE results showed that polyphenols can aggregate the protein;the solubility was improved with different pH conditions,the water holding capacity,oil holding capacity,emulsification,emulsification stability,foaming and foaming stability under neutral conditions were better than walnut protein without dephenolization.In general,the muti-frequency ultrasonic dephenolization treatment can effectively increase the polyphenol content in the dephenolization solution.The purity of the pellicle polyphenols purified by AB-8 resins could reach to 70.52%,which was 2.91 times higher than before.The total oil extraction rate reaches 95.06%after process of EAEP,and the water consumption can be saved by 40%.The functional properties such as the solubility of the water-phase protein are significantly improved,it’s good for walnuts’industrial application potential.