Physicochemical Modification Of Egg Yolk Granules And Its Application In Delivery Of Quercetin

In recent years,due to the pursuit of healthy life,natural bioactive substances such as quercetin,curcumin and apigenin were becoming the focus of research.They possess many physiological activities,such as anti-inflammatory,anti-depressant,anti-cancer and antioxidant,etc.However,these bioactive substances usually have strong hydrophobicity,low water solubility and poor stability,resulting in low bioavailability.Therefore,many researches have focused on developing delivery systems to improve their water solubility and functional properties.Natural protein is a safe component with amphiphilic structure.It has been used to load hydrophobic active substances by forming nanoparticles.Egg yolk granules were obtained from egg yolk by simple dilution centrifugation.It was composed of 70%high density lipoprotein,16%phosvitin and 12%low density lipoprotein,which showed rich nutritional value.It also had the potential to encapsulate hydrophobic active substances.However,The dense structure and low water solubility of yolk granules due to the presence of calcium phosphate bridges,which result in difficulty in exerting their functional properties.In this study,based on the assembly mechanism of yolk granules,calcium chelating agent was used to achieve the dissociation of yolk granules.And induced the self-assembly of granules by Ca²⁺,as an effective carrier of quercetin.Finally,the Octenyl Succinic Anhydride（OSA）was used to modify the granules to increase its stability,and the specific findings were as follows:（1）Yolk granules can be dissociated into 100 nm nanoparticles by sodium tripoly-phosphate,EDTA-2Na and sodium citrate under neutral conditions.Fourier transform infrared spectroscopy showed that the chemical bond of yolk granules was not significantly affected by calcium chelating agent.The concentration of nonprotein-bound Ca²⁺in yolk granules before and after dissociation was determined.It was found that the structure of yolk granules was destroyed by the calcium chelating agent by binding to the calcium phosphate bridge in yolk granules.SDS-PAGE shows that no new protein subunits were formed after the egg yolk granules were dissociated by calcium ion chelating agent.These results indicate that the dissociation of yolk granules by calcium chelating agent is a physical effect.Finally,the endogenous fluorescence spectra verified that more tryptophan was exposed after the yolk granules were dissociated,which made them have the potential to carry hydrophobic active substances.（2）Encapsulation of quercetin was accomplished by precisely regulating the degree of self-assembly of yolk granules through the addition of Ca²⁺.The embedding rate of quercetin increased first and then decreased when the addition of Ca²⁺increased from 0 to 300μg/m L.When the addition of calcium chloride was 200μg/m L,the entrapment efficiency of yolk granule to quercetin was the highest,which was 81.1±0.7%.Fourier transform infrared spectroscopy and X-ray diffraction confirmed that quercetin was successfully encapsulated and contained in yolk granules in an amorphous state.DPPH and ABTS free radical scavenging experiments showed that quercetin carried by yolk granules had strong free radical scavenging activity and its structural properties were not damaged.Meanwhile,the photostability,thermal stability,and storage stability of quercetin encapsulated by yolk granules were significantly improved,and the stability was closely related to the size of the nanoparticles.Similar size effects were also shown in the bioaccessibility and inhibitory activity of HT-29 cells.Nanoparticles with moderate size had higher bioaccessibility,among them,the Gra-Que1 group had the highest bioaccessibility,reaching 46.3±0.01%,and nanoparticles with smaller size were more easily absorbed and utilized by cells,showing excellent inhibitory activity of cancer cell proliferation.（3）Simple granules-quercetin nanoparticles are easy to precipitate when they encounter calcium ions again in food processing.Therefore,OSA modification was used to improve the stability of dissociated yolk particles and quercetin nanoparticles.OSA modified yolk granules with different degrees of substitution were prepared by adjusting the mass ratio of OSA to yolk granules.The particle size of the modified yolk particles did not produce an obvious change,while after nucleophilic substitution reaction with OSA,a large number of carboxyl groups were generated,making its potential drop dramatically.Quercetin was encapsulated in OSA modified yolk granules by reverse-solvent combined with ultrasound.When the content of quercetin was 0.5 mg/m L,the encapsulation efficiency of modified yolk granules for quercetin was 81.0%.The results of X-RD verified that quercetin was successfully loaded into OSA modified yolk granules in an amorphous state.It was found that the strong electrostatic repulsion brought about by the grafting of OSA,together with the steric hindrance provided by the hydrophobic long chain,were able to effectively inhibit the Ca²⁺induced aggregation of yolk granules,elevating their stability.Antioxidant activity experiment and HT-29 cell experiment showed that quercetin carried by OSA modified yolk granules did not damage its structural properties,and promoted the activity of inhibiting HT-29 cell proliferation by improving its water solubility and stability.