Preparation Of Microencapsulated Egg Yolk Immunoglobulin (IgY) For Oral Administration And Evaluation Of Its Properties

Chicken egg yolk immunoglobulin (IgY) provides an inexpensive and effective source of antibodies for the passive immunization of animals suffering from intestinal diseases. It is a promising alternative for the treatment and prevention of enteric infections, and has shown to be effective against a number of gastrointestinal pathogens, including Escherichia coli, Salmonella and rotavirus. However, its application is limited by its sensitivity to gastric conditions.Here, IgY-loaded microcapsules were prepared using nature polysaccharides such as alginate and chitosan. The objective of this study was to evaluate chitosan-alginate microcapsules as a method of oral delivery of IgY antibodies in vitro and in vivo.Enterotoxigenic Escherichia coli K88 (ETEC K88) was used as an antigen to immunize laying hens, and then produced specific IgY. Immunogenicity of laying hens was investigated following immunization with different antigen preparations, including crude fimbriae, purified fimbriae and formalin-killed whole cells. The results showed that the antibody titer was much greater when the fimbriae were used as an antigen (480000) compared with that of antibodies with the formalin-killed bacteria (320000). Moreover the purified fimbriae dramatically increased the titer. The optimal concentration of purified fimbriae and formalin-killed bacteria was found to be 2 mg/ml and 10¹⁰ cfu/ml respectively, which resulted in the greatest and most stable antibody titer.The optimal concentrations of salt were investigated for purification of IgY by a two-step procedure using salt precipitation. The results showed that precipitation with 50% saturation ammonium sulfate followed by precipitating with 14% sodium sulfate gave optimum IgY recovery (46%) and purity (77%). Upon ultrafiltration, the purity of final IgY powder was 82%.IgY was fairly heat-stable and most of the antibody activity remained after heating up to 70℃for 15 min. IgY was stable at pH above 3, but was very susceptible to pepsin in low pH, while appreciable activity was retained by IgY after trypsin digestion. Freeze-drying could affect activity of IgY. Moreover, IgY was stable stored at -20℃or 4℃for 12 months.Air-driven droplet generator was used to prepare IgY-loaded chitosan-alginate microcapsules. The effects of physical-chemical factors on the properties of IgY-loaded microcapsules were investigated. The properties investigated included the loading capacity for IgY (expressed as the IgY loading percentage, %, w/w of microcapsules), encapsulation efficiency (EE%), gastric stability and release characteristics of these microcapsules. Optimum physical factors were established for preparation of homogenous, spherical and smooth microcapsules. For the IgY-loaded microcapsules, optimum results were obtained under the following conditions: pH of the encapsulation medium 3.5, chitosan concentration 0.2% (w/v), alginate concentration 2% (w/v), CaCl₂ concentration 0.5% (w/v), IgY loading rate 25% (w/w). Under the optimum conditions, IgY loading% and EE% was above 20% and 75%, respectively. The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and retained 70% activity after 2 h exposure to SGF. Moreover, microencapsulated IgY was significantly resistant to pepsin hydrolysis. Less than 10% IgY was released upon the microcapsules exposure to SGF for 2 h, and more than 80% IgY was released upon the microcapsules exposure to simulated intestinal fluid (SIF, pH 6.8) for 4h.The protective effects of microencapsulated IgY obtained from hens immunized with ETEC K88 fimbrial antigen were evaluated in 40-day-old piglets in which ETEC diarrhea was induced. The results demonstrated that the ETEC K88 induced diarrhea was cured 24 h after treated with microencapsulated IgY, and the body weight gain was the fastest during the period of the experiment while those treated with non-encapsulated IgY were cured within 72 h after infection. Whereas control piglets treated with normal saline had severe diarrhea and dehydration after infection. These results indicated that the stability of IgY under gastric conditions was greatly improved by encapsulation in chitosan-alginate microcapsules, and weaned piglets that received microencapsulated IgY were better protected against ETEC infection.Consequently, it can be said that chitosan-alginate microcapsules can be offered for the temporary protection of IgY against acidic and enzymatic degradation during gastric passage, enabling its use for oral passive immunotherapy.