| Egg yolk immunoglobulin, also called IgY, is a kind of antibodies transported from IgG in poultry serum into the egg yolk. It is stable, easy-separated, safe and efficient, and it also has strong specificity against its antigen. As a result, IgY has been widely used in food area such as functional food additives, immune detection materials and biological preservatives.The IgY against Shewanella putrefaciens can tagetedly inhibit the growth of Shewanella putrefaciens, which is a common kind of spoilage bacteria in aquatic products. The antibacterial efficiency of this special IgY in liquid and solid medium has been proved, and it can also extend the the shelf life of chilled Paralichthys olivaceus samples. At present, the mechanism of bacterial inhibitory effect of IgY is not clear, which leads to several problems and restrictions in its application area, such as high concentration, limited effective time and certain blindness when searching its synergistic antibacterial factors.In this research, the purified IgY against Shewanella putrefaciens was chosen as object, and bacterial inhibitory mechanism and its action sites were investigated according to the morphological and physiological changes of the bacteria. On the basis of the antibacterial mechanism and in order to improve the efficiency, the antibacterial effect of IgY treated by enzymatic hydrolysis was also studied. Main results are listed as followed:1. An effective gel filtration chromatography method is established and IgY of92.33%purity can be generated with the recovery rate of89.6%. The titer of the purified IgY is more than1:1000, which is increased double compared to the IgY before purification.2. The morphological and physiological changes of Shewanella putrefaciens treated by the special IgY were analyzed. The results demonstrate that the bacterial cells become distorted, broken, and the cell walls of some bacteria become obviously thinner.The hydrophobicity decreases sharply and both the cell walls and membranes of the bacteria are broken. A kind of characteristics components with large size is discovered in the supernatant solution after the dissociation effect of glycine. This characteristic components proves to be a kind of polysaccharide,5.83×105Da with the main monosaccharides of D-(+)-glucosamine, D-(+)-Galactosamine, glucose, D galactose and guluronic acid, including some lauric acid, tetradecanoic acid and hexadecanoic acid. It is presumed to be lipopolysaccharide on the bacterial surface. The changes indicates the possible inhibitory mechanism may be as follows:the Shewanella putrefaciens-specific IgY targetedly binds to the lipopolysaccharide expressed on the Shewanella putrefacien surface, changes the bacterial hydrophobicity and blocks the protective role of lipopolysaccharide, resulting in death of the bacteria followed by destruction of the cell wall and cell membrane.3. On the basis of inhibitory mechanism, the method of improving bacteriostatic effects of IgY against Shewanella putrefaciens by reducing the molecular weight while reserving the antigen-binding sites in the IgY was evaluated. The peptic digestion procedure was monitored by high performance liquid chromatograph (HPLC), and the best condition of generating IgY antigen-binding fragment (IgY-Fab) is mixing IgY and pepsin (m/m=20:1) in acetic acid buffer (pH=4.2) for6hours. The43kD IgY-Fab of90%purity can be obtained using Sephacryl S-100gel filtration chromatography and it retaines antigen-binding and antibacterial activities. It also shows a better preservation effect when used in chilled turbot samples, compared with the whole IgY in equimolar concentrationThis study fills the research gaps of inhibitory mechanism of specific IgY towards Shewanella putrefaciens and proposes a new method to improve the IgY antibacterial effect. It lays a theoretical foundation for the application of IgY against Shewanella putrefaciens and other similar IgY when applied in food preservation field. |
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