Preparation And Activity Evaluation Of Composite Egg Yolk Immunoglobulin (IgY) Against Periodontitis Pathogens

Chronic periodontitis is a common chronic periodontal disease,which is different from invasive periodontitis,a rapid acute developmental type.Although periodontitis is not fatal,it affects the quality of life.Periodontitis can cause these symptoms,such as brushing bleeding,loose teeth,tooth displacement,oral odor and so on.The current mainstream treatment is the combination of basic treatment and antibiotics,but the resistance of bacterial strains caused by antibiotic abusing has become a global problem.Once the medication is stopped,pathogen will be replanted in the mouth.In recent years,IgY are widely applied to prevent and treat diseases caused by microorganisms such as bacteria,viruses or fungi.In this paper,IgY against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans was prepared and tested for biological activity.Moreover,IgY also was added to a suitable dosage form for its application,which is expected to be a new method to treat periodontitis.The colony characteristics of Porphyromonas gingivalis and Aggregatibacter actinomycestemcomitans can be observed on blood plate agar medium.Subsequently,the cultured bacteria were identified by gram staining,scanning electron microscopy and transmission electron microscopy.The two strains of the bacterial strain described above were used to immunize the laying hen,whose egg was collected later.IgY in the egg yolk is extracted by the water dilution extraction method and the polyethylene glycol 6000 precipitation method.The IgY was purified by ammonium sulfate salting out or sodium sulfate salting out.The content of protein in the extract was detected by Coomassie blue staining.Furthermore,the relative molecular weight and purity of the yolk antibody were analyzed by SDS-PAGE,and the titer and cross-reaction of IgY were detected by indirect ELISA.Indirect immunofluorescence and Western blotting experiments were performed to analyze the specificity of IgY.The crystal violet staining method was used to detect the inhibitory effect of the specific IgY on the biofilm formation of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.The reason for IgY's function were analyzed by immunoelectron microscopy and bacterial co-aggregation experiments.Finally,the stability of IgY was studied in mouthwash.The experimental results show that the bacterial colony characteristics and bacterial morphology are consistent with the description.The extraction method of water extraction and polyethylene glycol extraction are feasible.The composite IgY extracted by polyethylene glycol extraction method had high titer,and the titer of Porphyromonas gingivalis reached 1:10240 and that of Aggregatibacter actinomycetemcomitans reached 1:5120.The highest titer was achieved in about 48 days and lasted for about 15 days.It had weak cross-reactivity with other bacteria.Indirect immunofluorescence and Western blotting experiments showed that the binding activity of IgY to Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans has strong specificity.The IgY inhibited the formation of biofilms in a dose-dependent manner(p<0.05).Immunoelectron microscopy and co-aggregation experiments showed that IgY can bind to the bacterial cell wall specifically,which affected the bacterial flora.Finally,the IgY has excellent stability and it's comparatively stable in mouthwash.In this experiment,a composite IgY with high titer and specificity was prepared,which can significantly inhibit the formation of biofilm of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.Research results are conductive to finding new treatments in periodontal disease and also provide a reference for the addition of IgY into daily chemicals industry.