Study On Extraction And Phosphorylation Of Walnut Protein Isolate

This study aims to study the effects of walnut polyphenols on the extraction rate,physicochemical properties,and functional properties of walnut protein isolate（WPI）.To eliminate the influence of walnut polyphenols,pellicles-free walnut flour was prepared from the walnut kernel.Meanwhile,walnut flour with pellicles was prepared from the whole walnut kernel to serve as a control.Walnut protein fractions（albumins,globulins,prolamins,and glutelins）were prepared according to the Osborne method.The interactions between walnut polyphenols and the walnut protein fractions were studied by fluorescence quenching.The results showed that walnut polyphenols could bind to walnut albumin,globulin,and glutelin with the apparent binding constants of 5.61×10³ M^-1,5.73×10³·M^-1,and 8.41×10³ M^-1,respectively.However,UV turbidity and particle size distribution experiments showed that the binding of polyphenols led to severe precipitation of walnut albumin and globulin but not glutelin,which could explain the low solubility of the walnut proteins in salt solution in the presence of walnut polyphenols.Then,the differences in composition,structure,and physicochemical properties of WPI prepared by alkaline-extraction and acid precipitation and salt-extraction method were investigated.Polyacrylamide gel electrophoresis（SDS-PAGE）and size exclusion chromatography（SEC-HPLC）showed that the presence of walnut polyphenols resulted in high molecular weight aggregation of WPI prepared by alkaline-extraction/acid precipitation from walnut flour with pellicles.Fourier transform infrared spectroscopy（FTIR）and circular dichroism spectroscopy（CD）showed that the WPI prepared from walnut flour with pellicles had moreα-helical structure and lessβ-sheet structure than that from pellicles-free walnut flour.The structure and protein composition of WPI prepared by salt-extraction and alkaline-extraction/acid precipitation method from pellicles-free walnut flour were similar.Finally,the solubility and emulsification properties of walnut protein isolates were studied.The results showed that the solubility of WPI prepared by salt-extraction was the lowest（10%）,while the emulsification activity（EAI）and emulsification stability（ESI）were the best.Due to the existence of walnut polyphenols,the solubility and emulsifying properties of WPI prepared by alkaline-extraction/acid precipitation from walnut flour with pellicles were higher than that from pellicles-free walnut flour.However,the solubility of walnut protein isolate obtained by either salt-extraction or alkaline-extraction/acid precipitation is relatively poor,so the development of other protein modification methods is necessary to improve the solubility of the walnut protein.Next,we tried to improve the solubility and functional properties of walnut protein isolates by phosphorylation modification.Phosphorylated walnut protein isolate（P-WPI）was prepared from pellicles-free walnut flour with sodium trimetaphosphate（STMP）at different p H values.The results of X-ray photoelectron spectroscopy（XPS）,FTIR spectrum,and phosphorus content determination confirmed the phosphorylation of extracted WPI.The results of FTIR and CD spectra showed that the phosphorylated walnut isolates had moreα-helical structure and lessβ-sheet structure than the WPI prepared by alkaline-extraction and acid precipitation.Phosphorylation decreased the surface hydrophobicity（H₀）and increased the surface charge of the extracted protein,and enhanced the solubility of the WPI.The emulsifying properties of phosphorylated walnut protein isolate were also significantly increased compared with that prepared by alkaline-extraction and acid precipitation.The phosphorylated WPI stabilized emulsion had smaller particle sizes and larger surface charges.These results confirmed that the phosphorylation can effectively balance the surface hydrophobicity and surface charge of the protein,thus improving the solubility and emulsion properties of the protein.In addition,the in vitro digestibility of phosphorylated walnut protein isolates was also studied,and the results showed that the digestibility of phosphorylated walnut protein isolates was significantly decreased in simulated gastric and intestinal juices.These results proved that phosphorylation was a very efficient method,which could significantly improve the solubility and functional properties of walnut protein isolate,and provided a theoretical basis for the development of walnut products.Walnut protein may be used as food materials to add to neutral or alkaline soft drinks.Besides,the emulsion prepared by walnut protein and other food accessories can also form dairy products rich in walnut protein,which greatly promotes the application of walnut protein in the food industry.