| Egg yolk immunoglobulin(IgY)is a natural antibody.IgY extracted from eggs immunized by Helicobacter pylori(HP)has strong anti-HP activity and can effectively treat gastric ulcers.However,the application of anti-HP IgY is still limited by two factors:one is the low efficiency of the IgY extraction method,which needs to be further optimized;the other is that IgY is easily inactivated in gastric fluid,so choosing a suitable drug delivery method is necessary.Most of the proteins in egg yolk are lipoproteins but IgY is a water-soluble protein.Therefore,the first step of IgY isolation is removing the lipoproteins from the yolks to obtain water-soluble components.In order to solve the above problems,this paper explored the effect of freeze-thaw-water dilution method on the extraction of IgY,optimization of conditions and extraction mechanism,and studied the anti-HP IgY composite tablet formulation and its release characteristics in stomach.Firstly,this thesis studied the effect of freezing conditions on the extraction of IgY,which was investigated by macrograph,protein concentration,soluble solid content and transmittance.The results showed that the clearer and purer IgY extracts were obtained from frozen-thawed yolks(frozen at-18? for more than 4h and frozen at-40? for more than 2h)with water dilution method.In order to optimize the conditions for extracting IgY by freeze-thaw-water dilution method,this thesis studied the effects of ion concentration,p H,flocculant dosage and dilution ratio on the extraction of IgY from frozen-thawed egg yolk(frozen at-18? for 8h).The results showed that the best isolation conditions were:using deionized water,dilution ratio of 1:5(egg yolk:deionized water,m/m),original p H 6.2,adding 0.6%sodium alginate and0.18%calcium chloride(sodium alginate:calcium chloride fixed at 10:3).Under these optimizing conditions,the yield of crude IgY product was 99?122 mg/egg,the purity was 27.5±0.7%,and the activity retention rate was 91.87±2.91%.The freeze-thaw-water dilution method solved the problems of poor degreasing capacity and low purity of IgY product(15%?18%)in the traditional water dilution method without p H adjustment,and reduced water quality requirement and dilution ratio.The IgY extracts could be processed with ultrafiltration without adding flocculants.Therefore,freeze-thaw treatment of egg yolk combined with water dilution method could reduce the costs of IgY production process and optimize production processes.In order to reveal the mechanism of extracting IgY by freeze-thaw-water dilution method,rheological properties,yolk protein distribution,water mobility,interaction between yolk proteins and laser confocal microscopy graphs of frozen-thawed egg yolk were analyzed.The results showed that the frozen-thawed egg yolks(frozen at-18? and-40? for 8h)formed solid gels,in comparison,the egg yolks frozen at-18? formed harder gels,which might due to larger uneven ice crystals formation during slow freezing,causing proteins(especially lipoproteins)to lose water,aggregate and flocculate.The intermolecular force test results showed that the hydrophobic interaction and electrostatic interaction between the egg yolk proteins were enhanced after freeze-thaw treatment,and a dense gel structure was formed between the protein molecules.Therefore,freeze-induced egg yolk gelation led to low-density lipoproteins and other heteroproteins remaining in a dense gel structure,while IgY was released into the supernatant,which was conducive to obtaining a clear and pure IgY aqueous extract.In order to expand the drug delivery method of anti-HP IgY in stomach,this thesis studied the gastric release characteristics of konjac glucomannan(KGM),calcium alginate and anti-HP IgY compound tablets.The results showed that in simulated gastric fluid,calcium alginate-IgY tablets no longer released IgY after 1h digestion while KGM-IgY tablets could continuously release IgY.The higher the proportion of KGM in KGM-IgY tablets,the slower the release rate of IgY.Studies on the structure and rehydration-corrosion characteristics of KGM-IgY tablets found that the surface of KGM-IgY tablets hydrated to form a gel layer when exposed to water,which could hinder the release of IgY.The release mechanism was mainly diffusion and corrosion.This thesis explored the best process conditions and mechanism for extracting IgY by freeze-thaw-water dilution method,as well as the gastric release characteristics of anti-HP IgY tablets,which provide a research basis for the optimization of industrial IgY production and the application of anti-HP IgY in the stomach. |
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