- Email: [email protected]
Optimization and characterization of chitosan-alginate microcapsules containing egg yolk immunoglobulin (IgY)
S258
Abstracts / Journal of Biotechnology 136S (2008) S252–S260
IV5-P-018
IV5-P-019
Pathogenicity of Shewanella marisflavi to Apostichopus japonicus and biological characteristics
Optimization and characterization of chitosan-alginate microcapsules containing egg yolk immunoglobulin (IgY)
Hua Li 1,∗ , Guo Qiao 1 , Qiang Li 1 , Wei Zhou 1 , Soo-Il Park 2
Xiaoyu Li 1,∗ , Liji Jin 1 , Yuhong Zhen 1,2 , Yanan Lu 1 , Linhui Wang 1 , Yongping Xu 1
1
Key Laboratory of Mariculture&Biotechnology, Agriculture Ministry, PRC, Dalian Fisheries University, Dalian 116023, China 2 Department of Aquatic Life Medicine, Pukyong National University, Busan 608737, Republic of Korea E-mail address: [email protected] (H. Li).
1 Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China 2 Department of Pharmacy, Dalian Medical University, Dalian 116044, China
Pathogenicity of Shewanella marisflavi to Apostichopus japonicus was determined by a few challenge tests, and the virulence of S. marisflavi to different hosts (A. japonicus, mouse and Xiphophorus helleri) was evaluated and compared by mean lethal dose 50% (LD50 ). Many researches on S. marisflavi have been done, including morphology, physiological and biochemical characteristics, hemolytic, whole-cell protein and 16S rDNA gene sequence analysis. S. marisflavi could be considered as one of main pathogenes to A. japonicus, of which clinical symptom displayed mouth tumidity, shaking head, skin ulceration and dead finally. A. japonicus could be infected by intraparitoneal and intramuscular injection. LD50 values for A. japonicus, mouse and X. helleri were 3.89 ×106 , 6.8 ×104 and 4.85 ×104 CFU g−1 body weight, respectively. According to the criteria of Santos et al. (1988) and Shideh Khashe and Michael Janda (1998), S. marisflavi was highly virulent strain to X. helleri and mouse. A. japonicus was less sensitive to bacteria than others. Colony of S. marisflavi was light red, 1.2–2 mm size, rod with entire viscosity on marine agar medium (2216E) after incubating at 25 ◦ C for at least 48 h and green on thiosulfatecitrate-bile-salts-sucrose (TCBS) medium. S. marisflavi was with polar flagella and secreting layer. S. marisflavi was Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. The strain exhibited clear hemolysis on sheep blood agar (-hemolysis) and produced H2 S. The survival growth conditions for S. marisflavi were at 4–35 ◦ C, pH 6.0–9.2, 0–8% NaCl, respectively. The whole-cell proteins of strain isolated were identical to that of the type strain of S. marisflavi KCCM 41822. The phylogenetic tree based on 16S rDNA gene sequence revealed that the pathogen had closest phylogenetic affiliations to the S. marisflavi KCCM 41822 [Accession number: AY485224], the similarity and bootstrap value between them was 99.86% and 100%, respectively.
E-mail address: [email protected] (X. Li).
References
References
Santos, Y., Toranzo, A.E., Barja, J.L., Nieto, T.P., Villa, T.G., 1988. Virulence properties and enterotoxin production of Aeromonas strains isolated from fish. Infect. Immun. 56, 3285–3293. Shideh Khashe, Michael Janda, J., 1998. Biochemical and pathogenic properties of Shewanella alga and Shewanella putrefaciens. J. Clin. Microbiol. 36 (3), 783–787.
Carlander, D., Kollberg, H., Wejaker, P.E., Larsson, A., 2000. Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol. Res. 21, 1–6. Li, X.Y., Jin, L.J., McAllister, T.A., Stanford, K., Xu, J.Y., Lu, Y.N., Zhen, Y.H., Sun, Y.X., Xu, Y.P., 2007. Chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (IgY). J. Agric. Food Chem. 55, 2911–2917. Shimizu, M., Fitzsimmons, R.C., Nakai, S., 1988. Anti-E. coli immunoglobulin Y isolated from egg yolk of immunized chickens as a potential food ingredient. J. Food Sci. 53, 1360–1366.
doi:10.1016/j.jbiotec.2008.07.551
Oral administration of specific egg yolk immunoglobulin Y (IgY) presents a promising approach for the passive immunization of animal and human suffering from intestinal diseases (Carlander et al., 2000). However, the activity of IgY may be reduced or destroyed by gastric conditions, particularly low pH (Shimizu et al., 1988). Therefore, it is necessary to find an effective method to preserve the therapeutic value of IgY antibodies during gastric passage. Chitosan-alginate microcapsules have been developed to protect IgY from gastric inactivation (Li et al., 2007). In the present study, in order to obtain the chitosan-alginate microcapsules characterized as having a high encapsulation efficiency (EE%), protecting IgY from gastric inactivation, and providing rapid release in the small intestine, various processing factors were studied. IgY-loaded chitosan-alginate microcapsules were prepared by one-step procedure with air-driven droplet generator. The processing factors investigated included chitosan concentration, CaCl2 concentration, alginate concentration and IgY loading rate. For the IgY-loaded microcapsules, optimum results were obtained under the following conditions: chitosan concentration 0.2% (w/v), alginate concentration 2% (w/v), CaCl2 concentration 0.5% (w/v), and IgY loading rate 25% (w/w). Under the optimum conditions, microcapsules were achieved with high EE% (>75%) and high loading capacity (>20%, w/w). The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and retained greater than 70% activity after 2 h exposure to SGF. 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 4 h. Further in vivo studies are needed to confirm these observations under physiological conditions.
doi:10.1016/j.jbiotec.2008.07.552
Abstracts / Journal of Biotechnology 136S (2008) S252–S260
IV5-P-018
IV5-P-019
Pathogenicity of Shewanella marisflavi to Apostichopus japonicus and biological characteristics
Optimization and characterization of chitosan-alginate microcapsules containing egg yolk immunoglobulin (IgY)
Hua Li 1,∗ , Guo Qiao 1 , Qiang Li 1 , Wei Zhou 1 , Soo-Il Park 2
Xiaoyu Li 1,∗ , Liji Jin 1 , Yuhong Zhen 1,2 , Yanan Lu 1 , Linhui Wang 1 , Yongping Xu 1
1
Key Laboratory of Mariculture&Biotechnology, Agriculture Ministry, PRC, Dalian Fisheries University, Dalian 116023, China 2 Department of Aquatic Life Medicine, Pukyong National University, Busan 608737, Republic of Korea E-mail address: [email protected] (H. Li).
1 Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China 2 Department of Pharmacy, Dalian Medical University, Dalian 116044, China
Pathogenicity of Shewanella marisflavi to Apostichopus japonicus was determined by a few challenge tests, and the virulence of S. marisflavi to different hosts (A. japonicus, mouse and Xiphophorus helleri) was evaluated and compared by mean lethal dose 50% (LD50 ). Many researches on S. marisflavi have been done, including morphology, physiological and biochemical characteristics, hemolytic, whole-cell protein and 16S rDNA gene sequence analysis. S. marisflavi could be considered as one of main pathogenes to A. japonicus, of which clinical symptom displayed mouth tumidity, shaking head, skin ulceration and dead finally. A. japonicus could be infected by intraparitoneal and intramuscular injection. LD50 values for A. japonicus, mouse and X. helleri were 3.89 ×106 , 6.8 ×104 and 4.85 ×104 CFU g−1 body weight, respectively. According to the criteria of Santos et al. (1988) and Shideh Khashe and Michael Janda (1998), S. marisflavi was highly virulent strain to X. helleri and mouse. A. japonicus was less sensitive to bacteria than others. Colony of S. marisflavi was light red, 1.2–2 mm size, rod with entire viscosity on marine agar medium (2216E) after incubating at 25 ◦ C for at least 48 h and green on thiosulfatecitrate-bile-salts-sucrose (TCBS) medium. S. marisflavi was with polar flagella and secreting layer. S. marisflavi was Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. The strain exhibited clear hemolysis on sheep blood agar (-hemolysis) and produced H2 S. The survival growth conditions for S. marisflavi were at 4–35 ◦ C, pH 6.0–9.2, 0–8% NaCl, respectively. The whole-cell proteins of strain isolated were identical to that of the type strain of S. marisflavi KCCM 41822. The phylogenetic tree based on 16S rDNA gene sequence revealed that the pathogen had closest phylogenetic affiliations to the S. marisflavi KCCM 41822 [Accession number: AY485224], the similarity and bootstrap value between them was 99.86% and 100%, respectively.
E-mail address: [email protected] (X. Li).
References
References
Santos, Y., Toranzo, A.E., Barja, J.L., Nieto, T.P., Villa, T.G., 1988. Virulence properties and enterotoxin production of Aeromonas strains isolated from fish. Infect. Immun. 56, 3285–3293. Shideh Khashe, Michael Janda, J., 1998. Biochemical and pathogenic properties of Shewanella alga and Shewanella putrefaciens. J. Clin. Microbiol. 36 (3), 783–787.
Carlander, D., Kollberg, H., Wejaker, P.E., Larsson, A., 2000. Peroral immunotherapy with yolk antibodies for the prevention and treatment of enteric infections. Immunol. Res. 21, 1–6. Li, X.Y., Jin, L.J., McAllister, T.A., Stanford, K., Xu, J.Y., Lu, Y.N., Zhen, Y.H., Sun, Y.X., Xu, Y.P., 2007. Chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (IgY). J. Agric. Food Chem. 55, 2911–2917. Shimizu, M., Fitzsimmons, R.C., Nakai, S., 1988. Anti-E. coli immunoglobulin Y isolated from egg yolk of immunized chickens as a potential food ingredient. J. Food Sci. 53, 1360–1366.
doi:10.1016/j.jbiotec.2008.07.551
Oral administration of specific egg yolk immunoglobulin Y (IgY) presents a promising approach for the passive immunization of animal and human suffering from intestinal diseases (Carlander et al., 2000). However, the activity of IgY may be reduced or destroyed by gastric conditions, particularly low pH (Shimizu et al., 1988). Therefore, it is necessary to find an effective method to preserve the therapeutic value of IgY antibodies during gastric passage. Chitosan-alginate microcapsules have been developed to protect IgY from gastric inactivation (Li et al., 2007). In the present study, in order to obtain the chitosan-alginate microcapsules characterized as having a high encapsulation efficiency (EE%), protecting IgY from gastric inactivation, and providing rapid release in the small intestine, various processing factors were studied. IgY-loaded chitosan-alginate microcapsules were prepared by one-step procedure with air-driven droplet generator. The processing factors investigated included chitosan concentration, CaCl2 concentration, alginate concentration and IgY loading rate. For the IgY-loaded microcapsules, optimum results were obtained under the following conditions: chitosan concentration 0.2% (w/v), alginate concentration 2% (w/v), CaCl2 concentration 0.5% (w/v), and IgY loading rate 25% (w/w). Under the optimum conditions, microcapsules were achieved with high EE% (>75%) and high loading capacity (>20%, w/w). The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and retained greater than 70% activity after 2 h exposure to SGF. 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 4 h. Further in vivo studies are needed to confirm these observations under physiological conditions.
doi:10.1016/j.jbiotec.2008.07.552