Preparation And Anti-diarrhea Effect Of Immunoglobulin Yolk Enteric Microcapsules

Piglets diarrhea is always one of the enormous challenges that the farming industry confront to.Immunoglobulin yolk(IgY)which is an effective substitute for antibiotics,can prevent piglets diarrhea and improve growth performance.However,some research has also found that IgY added in feeds could not improve growth performance of piglets.The activity of IgY would be rapidly inactivated under the condition at a pH below 3.5 and accelerated by the presence of pepsin at the same time.The study found that,the microencapsulation technology used to coat protein could reduce release rate and improve protein activity in gastric juices.The protein could rapidly release intestinal fluid which could improve application effect.In this study,chitosan-alginate microcapsules and Eudragit microcapsules are used to coating IgY.By optimizing the preparation conditions,we look forward to reduce the release ratio of IgY in gastric juices,increase the release rate in intestinal fluid and improve stability in gastric juice.Meanwhile,the model of mouse diarrhea model by Enterotoxigenic Escherichia coli(ETEC)challenge was established;the effect of the anti-diarrhea in vivo would be verified.The results are as follows:1.The experimental material was the specific anti-ETEC 3FliC spray-dried eggs which was laboratory pre-prepared.The IgY was coated by chitosan-alginate microcapsules.The purified IgY of spray-dried eggs was using by ammonium sulfate precipitation.Then the concentration of CaCl2,alginate,chitosan and protein loading ratio was optimized by single factor experiments and orthogonal experiments.The effect of four factors in the encapsulation efficiency,drug loading,release rate in simulated gastric fluid and simulated intestinal fluid were studied.The optimal conditions for preparing microcapsules was that the concentration of CaCl2,alginate,chitosan and protein loading ratio were 0.5%,2%,0.3%and 8:1,respectively.The IgY loading and encapsulation efficiency of the optimal microcapsules were 61.93%and 8.52%,respectively.No more than 22%IgY was released upon the microcapsules exposure in simulated gastric fluid for 2h.More than 87%and 100%IgY was released upon the microcapsules exposure in simulated intestinal fluid for 4h and 6h,respectively.The results of ELISA found that chitosan-alginate microcapsules improved the stability of IgY in simulated gastric fluid(pH=3.0),and retained 88%activity after 3h exposure to simulated gastric fluid.2.The experimental materials were the specific anti-ETEC 3FliC spray-dried eggs.The spray-dried eggs were granulated by extrusion-spheronization machine.The shell was Eudragit ?.The particles of spray-dried eggs were coated by bottom spray fluidized bed.The experiments found that oil was secreted from particles in the process of coating because of high content of oil in spray-dried eggs.It would lead to particles adhered and affect the coating effects.Oil was adsorpted by adding white carbon black.The effect of supplementation of white carbon black to the degree of oil secret,starch and microcrystalline cellulose to recovery and molding was investigated by single factor experiment.The optimal supplementation of spray-dried eggs,white carbon black and starch were 100 g,12 g,15 g and 8 g,respectively.The particles of different coating weight(8%,12%,16%,20%)were coated by bottom spray fluidized bed.Effect of coating weight on IgY release of Eudragit microcapsules in SGF and SIF,and the IgY stability in simulated gastric fluid were investigated.The results found that the optimal coating weight was 20%.No more than 0%and 25%IgY were released upon the microcapsules exposure in simulated gastric fluid for 1h and 3h,respectively.The IgY was completely released upon the microcapsules exposure in simulated intestinal fluid for 1h.The results of ELISA found that Eudragit microcapsules improved the stability of IgY in simulated gastric fluid(pH=3.0)and retained 83%activity of IgY after 2h exposure in simulated gastric fluid.3.The mouse diarrhea model was established by using K88ac ETEC to gavage ICR mouse.The anti-diarrhea effect of IgY-microcapsules was investigated.The results found that specific spray-dried eggs could significantly reduce the morbidity and the frequency of diarrhea after ETEC challenge(P<0.05;P<0.05).The morbidity of challenge with K88ac group,the groups of feeding medium dose,high dose microcapsuled IgY and Non-microcapsuled IgY were 90%,40%,30%and 40%,respectively.The frequency of diarrhea were 13 ± 18.07,0.71±1.25,1 ± 1.70 and 1.14±1.67,respectively.The adhesion population of K88ac of feeding medium dose and high dose Microcapsuled IgY could significantly reduce in jejunum and ileum(P<0.05;P<0.05).After ETEC challenge,the structure of jejunum and ileum were damaged;intestinal villus was swelled and villus height was shortened(P<0.05;P<0.05).The specific IgY could relieve the injury of intestinal structure and significantly increase villus height/crypt depth ratio of mouse(P<0.05).The specific IgY could increase villus height,but the difference was not significant(P>0.05).In conclusion,Chitosan-alginate microcapsules and Eudragit microcapsules coating IgY could significantly improve the IgY stability in gastric fluid.The feature of two types microcapsules was enteric.IgY microcapsules could effectively relieve diarrhea of mouse by ETEC challenge,inhibit ETEC colonizing in intestinal and relieve the injury of intestinal structure.It might be a new strategy to improve the effect of IgY applied in piglets and prevent piglets diarrhea.