| To provide market with high quality green food and boost international competitiveness of our animal production, scientists in animal feed additives field are working hard to develop new type of antibiotic growth promoter. There are many other choices substitute for antibiotics additives used in animal feed, such as plant extracts, micro-ecologic agents,enzymes, antimicrobial peptides and etc. Antimicrobial peptides can(1) have broad activities;(2) effect rapidly and stable;(3) develop no drug-resistance;(4) have inhibition or killing effects on enveloped virus, fungus and cancer cells;(5) develop no drug residual.Given these unique advantages, National Development and Reform Commission had issued documents to courage the research and production of antimicrobial peptides product for two consective years. Cecropin was originally identified and isolated from hyalophora cecropia by Sweden scientist Boman in 1980. It is the first animal-origin peptide family been discovered with antimicrobial activity,the name Cecropin was after hyalophora cecropia.The thesis mainly studies the screen of the bacillus subtilis which can efficiently secretive express Cecropin AD, optimization of its pilot scale production process,separation and purification of fermentation broth and animal trials. Four parts including:(1)The comparation of Cecropin AD expression level between different bacillus subtilis strains.The expression vector was transferred into 3 host bacterial strains to heterogeneously express the recombinant Cecropin AD, the expression level was compared with the help of SDS-PAGE and HPLC. The small peptides had been expressed by different host strains were all 3.8 k Da, and the highest express efficacy was found in host WB800 which was up to 826.72 μg/ml.(2) fermented WB800 on a pilot scale and the production process was optimized. WB800 selected as the tested strain and experiments were designed by means of Box-Behnken Design(BBD), which based on Design-Expert response surface analysis, and then evaluated the influence of p H value, ventilation quantity and rotate speed on the final yield of Cecropin AD. The optimum technological conditions of Cecropin AD productionwere then determined to realize the industrial production of Cecropin AD. From our experiment, we found that the expression level of Cecropin AD should be attained a theoretic maximum(979.98 μg/ml) when the p H value of the fermentation broth reached7.60, rotate speed reached 230.00 rpm and ventilation quantity were 5.00 L/h, and after three verification the actual content was as high as 961.52 μg/ml, which means Cecropin AD could be commercially produced on this condition.(3) With WB800 as the host bacterial and the optimized fermentation process, we gained the crude fermentation broth.The purification technology was designed according to the physicochemical properties of Cecropin AD; every step of purification was explored and optimized; the purification steps were then connected; and the purified Cecropin AD was determined and tested. Results showed: with the help of RPC, the purity of fermentation broth after ion-exchange column chromatography is 69.5%, after hydrophobic chromatography is 87.4%, and the final purity of the desired product is 100%. This peptide had bactericidal effect on both gram negative and gram positive bacterial.(4) This Cecropin was diluted to certain concentration in pig feed, and the effects of Cecropin AD on growth and immune performance of weanling piglets were determined. A total of 360 weanling pigs( 8.46± 0.12 kg) were selected to estimate effects of cecropin on growth performance and immune response of pigs. Adopting single factor completely random design, animals were divided into four groups, each group included 6 repeat and each repeat 15 pigs, including Control group: basal diet(cornsoybean meal with no antibiotics additives); Antibiotics group: basal diet+ 25 mg/kg virginiamycin + 100 mg/kg terramycin + 60 mg/kg colistin sulfate(all contents are drug effective component contents, the same below); Cecropin AD group: basal diet+ 400 mg/kg Cecropin AD; Cooperative group: basal diet+ 250 mg/kg Cecropin AD+ 50 mg/kg terramycin + 20 mg/kg colistin sulfate. Compared with control and antibiotics groups, the Cecropin AD group and antibiotics + Cecropin AD group have significantly high feed efficency(F/G)(P < 0.05), and the later two groups were also exhibited improved daily gain and feed intake. On the diarrhea rate and swine fever antibody levels, the tested groups were all significantly better than that of control group(P < 0.05), antibody blocking rate of Cecropin AD group is significantly better than the other three groups(P < 0.05). Part of theserum biochemical indicators : BUN, GLU, TP and Ig G&Ig A of Cecropin AD group has also significantly improved(P < 0.05).(5) The application of Cecropin AD in large-scale piggery(Beijing, Hebei, Henan Province, et al) was very successful: It can significantly improve feed conversion and survival ratio and reduce diarrhea rate in weanling pig, which means an increase in economic benefit up to 13-16 yuan per pig. Consequently, in weanling pigs feed, Cecropin AD can partly replace the G+ antibiotics feed additives such as kitasamycin, virginiamycin and etc. and reduce colistin sulfate use.In summary, the bacillus subtilis WB800 which can efficiently secretive express Cecropin AD was obtained after screening of 3 different bacillus subtilis strains. The technological conditions of Cecropin AD production were optimized to realize the industrial production of Cecropin AD. The purification and separation technology was designed according to the physicochemical properties of Cecropin AD, the purity of the final product was up to 100%. The application of Cecropin AD in weanling pig showed it can be the best substitute for antibiotics used in animal feed. |
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