| Antibiotics are widely used in the prevention and treatment of animal disease.The abuse of antibiotics has led to many problems such as drug residues in animal product and the increase of bacterial resistance.As a new type of environmental protection and pollution-free biological preparation,Chicken yolk antibody(Immunoglobulin,IgY)shows great advantages in oral prevention and treatment of diseases of young livestock.However,IgY is easy to be inactivated in bad gastric acid environment or due to improper storage,which limits its application.Microcapsule technology is one of the new and effective methods to solve this problem in recent years.Previous studies have shown that embedding IgY in sodium alginate/chitosan can solve the problem of inactivation in gastric acid to a certain degree.However,this two-layer entrapment still has the problem of poor gastric acid tolerance,which may be mainly due to the fact that the residual carboxyl group is easy to destroy the chelate bond connection in the presence of water.Therefore,this study with sodium alginate and chitosan as wall materials,IgY as the core material,the use of sodium alginate/chitosan/sodium alginate triple package is process,preparation of sodium alginate/chitosan IgY/sodium alginate microcapsule,and its in vitro gastrointestinal tolerability,storage stability and other key performance,evaluates its application effect in the chicken for sodium alginate/chitosan/alginate sodium IgY microcapsule providing scientific theory basis for the feasibility of substituting antibiotics.The research is divided into two parts:Test 1:screening of the optimal preparation process of sodium alginate/chitosan/sodium alginate IgY microcapsule and study on artificial gastrointestinal tolerance and bacteriostatic properties in vitroAccording to the concentration of IgY 200 mg/mL,high concentration of sodium alginate mass fraction 6%,mass fraction 5%CaCl2,mass fraction 0.8%chitosan,chitosan pH 5,low concentration of sodium alginate 0.1%mass fraction conditions for preparation of sodium alginate/chitosan/alginate sodium IgY microcapsule embedding rate is highest,which can reach 95.52%.At the same time,its drug-loading rate can reach 23.36%,and its shape is more regular before and after lyophilization,and its mechanical strength is higher.The artificial gastric juice protection rate of the prepared sodium alginate/chitosan/sodium alginate IgY microcapsule can reach more than 70%.The release rate of IgY in artificial intestinal fluid was up to 90%at 1h.At the same time,the content of IgY was over 80%when it was stored at room temperature and cold storage for 8 weeks.The results of in vitro bacteriostatic test showed that sodium alginate/chitosan/sodium alginate IgY microcapsule had significant inhibitory effect on salmonella and escherichia coli at 9h and 8h respectively,basically meeting the requirements of oral administration.Test 2:effects of sodium alginate/chitosan/sodium alginate IgY microcapsule on growth performance,intestinal tissue development and immune performance of broilersAccording to the results of in vitro tests,sodium alginate/chitosan/sodium alginate IgY microcapsules with excellent embedding rate,drug loading rate,mechanical strength and other properties,as well as good in vitro gastrointestinal tolerance and strong antibacterial properties were added to broilers’ diets at different levels.The effects of sodium alginate/chitosan/sodium alginate IgY microcapsule on growth performance,intestinal tissue development and immune performance of broilers were compared with those of uncoated IgY and antibiotic groups.100 healthy 1-day-old broilers of similar weight were randomly divided into 5 groups.With the control group(CON group)based diet,feeding trial T2,T3 group respectively on the basis of basic diet added 0.6 g/kg of sodium alginate/chitosan/alginate sodium IgY microcapsule(IgY the net amount of about 50 mg/kg)and 0.7 g/kg of sodium alginate/chitosan/alginate sodium IgY microcapsule(IgY the net amount of about 60 mg/kg),test T1 group on the basis of basic diet added 50 mg/kg bag was not IgY,test the T4 group on the basis of basic diet added 0.5 g/kg aureomycin.The growth performance,small intestinal tissue development,immune organ indexes,intestinal tract,fecal flora,serum biochemical indexes,serum immune indexes,serum antioxidant indexes,and blood physiological indexes of broilers were measured at 7,14,21,and 28 days of age.The test results show that:(1)growth performance:compared with the control group,dietary addition of 0.6g/kg sodium alginate/chitosan/sodium alginate IgY microcapsules,0.7 g/kgsodium alginate/chitosan/sodium alginate IgY microcapsules,50 mg/kg uncoated IgY and 0.5 g/kg chlortetracycline could increase the daily gain and reduce the feed-to-gain ratio of broilers.However,the effect of adding different doses of sodium alginate/chitosan/sodium alginate IgY microcapsules was better than that of adding uncoated IgY.At the same time,the effect of 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was significantly better than that of chlortetracycline group(P<0.05).(2)Small intestinal tissue development:compared with the control group,dietary addition of 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsules,0.7 g/kg sodium alginate/chitosan/alginate IgY microcapsules,50mg/kg uncoated IgY and 0.5 g/kg chlortetracycline could increase the villus height of duodenum,jejunum and ileum and reduce the crypt depth of duodenum,jejunum and ileum.Increase the ratio of villus height to crypt depth.At the same time,the effect of adding different doses of sodium alginate/chitosan/sodium alginate IgY microcapsules was significantly better than that of adding uncoated IgY(P<0.05).Moreover,the effect of adding 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was similar to that of chlortetracycline group(P>0.05).(3)immune performance:① Indexes of immune organs:adding 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,0.7 g/kg sodium alginate/chitosan/alginate IgY microcapsule,50 mg/kg uncoated IgY and 0.5 g/kg chlortetracycline could significantly increase the bursa weight,bursa index,spleen weight and spleen index of broilers(P<0.05).At the same time,the effect of adding different doses of sodium alginate/chitosan/sodium alginate IgY microcapsules was significantly better than that of adding uncoated IgY(P<0.05).Moreover,the effect of adding 0.7g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was similar to that of chlortetracycline group(P>0.05).② Intestinal and fecal microflora:adding 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,50 mg/kg uncoated IgY and 0.5 g/kg chlortetracycline could significantly reduce the number of Escherichia coli and salmonella in duodenum,jejunum,ileum,cecum and feces of broilers.All of them could significantly increase the number of lactic acid bacteria and bifidobacteria in duodenum,jejunum,ileum,cecum and feces of broilers(P<0.05).Among them,the effect of adding different doses of sodium alginate/chitosan/sodium alginate IgY microcapsules was significantly better than that of adding uncoated IgY(P<0.05).At the same time,the effect of 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was significantly better than that of chlortetracycline group(P<0.05).③ Serum biochemical indexes:dietary addition of 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsules,0.7 g/kg sodium alginate/chitosan/alginate IgY microcapsules,50 mg/kg uncoated IgY,0.5 g/kg chlortetracycline could improve the serum albumin of broilers.At the same time,the effect of adding 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was significantly better than that of adding chlortetracycline group,adding 0.6g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule group and adding uncoated IgY group(P<0.05).The contents of serum total protein,total cholesterol,triglyceride and blood glucose in each group were all within the normal range,and there was no significant difference between them(P>0.05).④ Serum immune index:dietary addition of 0.6g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,0.7 g/kg sodium alginate/chitosan/alginate IgY microcapsule,50 mg/kg uncoated IgY,0.5 g/kg chlortetracycline could increase the content of IgG and IgM in serum of broilers.At the same time,the effect of adding 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was significantly better than that of adding chlortetracycline group,adding 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule group and adding uncoated IgY group(P<0.05).The content of alkaline phosphatase in serum of each group was within the normal range,and there was no significant difference between them(P>0.05).⑤ Serum antioxidant index:dietary addition of 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule,50 mg/kg uncoated IgY and 0.5 g/kg chlortetracycline could improve the total antioxidant capacity of serum,the activity of superoxide dismutase and the activity of glutathione peroxidase in serum,and reduce the content of malondialdehyde in serum of broilers.In addition,the effect of adding 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was significantly better than that of adding chlortetracycline group,adding 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule group and adding uncoated IgY group(P<0.05).⑥ Blood physiological indexes:dietary addition of 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsules,0.7 g/kg sodium alginate/chitosan/alginate IgY microcapsules,50 mg/kg uncoated IgY,0.5 g/kg chlortetracycline could increase blood hemoglobin content,average erythrocyte volume,average hemoglobin concentration and decrease blood erythrocyte distribution density and lymphocyte count.Moreover,the effect of adding 0.7 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule was better than that of adding chlortetracycline group,adding 0.6 g/kg sodium alginate/chitosan/sodium alginate IgY microcapsule group and adding uncoated IgY group.To sum up,sodium alginate/chitosan/sodium alginate triple entrapment method can be used to effectively entrap IgY,and the sodium alginate/chitosan/sodium alginate IgY microcapsules prepared according to the best preparation process have excellent properties,such as excellent entrapment rate,drug loading rate,mechanical strength,good gastrointestinal tolerance and strong bacteriostasis in vitro.When it is used in the feeding of broilers,it can improve the growth performance of broilers,improve the development of small intestinal tissue,enhance immunity,and has the potential to replace antibiotics. |
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