禽多杀性巴氏杆菌不同培养方法制备的种子液对高密度发酵培养菌数的影响
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摘要
本试验用扁瓶固体培养法、摇床液体培养法和10L发酵罐培养法制备禽多杀性巴氏杆菌种子液,采用细菌比浊法和活菌计数法,计算高密度发酵菌液浓度,比较3种方法制备的种子液对禽多杀性巴氏杆菌抗原生产的影响。经过3批次试验,扁瓶固体培养法制备的种子液经高密度发酵培养后测得的活菌数为1.56×1010~1.65×1010 CFU/mL,比浊计数为2.10×1010~2.59×1010 CFU/mL;而摇床液体培养法制备的种子液经高密度发酵培养后测得的活菌数为1.54×1010~1.74×1010 CFU/mL,比浊计数为2.10×1010~2.45×1010 CFU/mL。发酵罐培养制备的禽多杀性巴氏杆菌种子接种200L发酵罐进行高密度发酵培养后测得的活菌数可达1.85×1010~2.05×1010 CFU/mL,比浊计数为2.45×1010~2.80×1010 CFU/mL。结果表明,10L发酵罐培养制备的种子液优于扁瓶固体培养法和摇床液体培养法制备的种子液,而后两者无明显差别;从3批次平均值看,前者比后两者活菌计数结果提高约21.60%,比浊计数结果提高约15.52%。说明采用10L发酵罐培养制备的禽多杀性巴氏杆菌种子进行高密度发酵培养可显著提高菌液浓度,增加单位抗原含量(P<0.01)。
The liquid seeds of avian Pasteurella multocide were cultivated with the solid culture in flat bottles,and the liquid culture on the shaking table and the 10-liter fermentation tank,respectively.Then the avian Pasteurella multocide antigen was prepared with high density fermentation in 200-liter fermentation tank by using the bacterial seeds.The bacterial density was evaluated by the viable cell count method and the turbidimetric method to compare the influences of bacterial seeds on the antigen production.After three batch tests,the viable cell count results showed that the amounts of living bacterial reached 1.56×1010-1.65×1010 CFU/mL,1.54×1010-1.74×1010 CFU/mL and 1.85×1010-2.05×1010 CFU/mL,respectively,with the seeds cultivated by the solid culture in flat bottles,the liquid culture on the shaking table and the 10-liter fermentation tank.The turbidimetry results showed that the amount of bacteria reached2.10×1010-2.59×1010 CFU/mL,2.10×1010-2.45×1010 CFU/mL and 2.45×1010-2.80×1010 CFU/mL,respectively.The conclusion:the seeds prepared with the 10-liter fermentation tank were superior to that with other two methods,and thereafter improved the quality of unit antigen significantly(P<0.01).
The liquid seeds of avian Pasteurella multocide were cultivated with the solid culture in flat bottles,and the liquid culture on the shaking table and the 10-liter fermentation tank,respectively.Then the avian Pasteurella multocide antigen was prepared with high density fermentation in 200-liter fermentation tank by using the bacterial seeds.The bacterial density was evaluated by the viable cell count method and the turbidimetric method to compare the influences of bacterial seeds on the antigen production.After three batch tests,the viable cell count results showed that the amounts of living bacterial reached 1.56×1010-1.65×1010 CFU/mL,1.54×1010-1.74×1010 CFU/mL and 1.85×1010-2.05×1010 CFU/mL,respectively,with the seeds cultivated by the solid culture in flat bottles,the liquid culture on the shaking table and the 10-liter fermentation tank.The turbidimetry results showed that the amount of bacteria reached2.10×1010-2.59×1010 CFU/mL,2.10×1010-2.45×1010 CFU/mL and 2.45×1010-2.80×1010 CFU/mL,respectively.The conclusion:the seeds prepared with the 10-liter fermentation tank were superior to that with other two methods,and thereafter improved the quality of unit antigen significantly(P<0.01).
引文
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[2]Lutz C,Erken M,Noorian P,et al.Environmental reservoirs and mechanisms of persistence of Vibrio cholerae[J].Front Microbiol,2013(4):375.
[3]宁宜宝.兽用疫苗学[M].北京:中国农业出版社,2008:411-418.
[4]Haley B J,Choi S Y,Grim C J,et al.Genomic and phenotypic characterization of Vibrio cholerae non-O1isolates from a US Gulf coast cholera outbreak[J].PLoS One,2014,9(4):e86264.
[5]中华人民共和国农业部.兽用生物制品规程2000年版[S].2000.
[6]Akoachere J F T K,Mbuntcha C K P.Water sources as reservoirs of Vibrio cholerae O1and non-O1strains in Bepanda,Douala(Cameroon):relationship between isolation and physico-chemical factors[J].BMC Infect Dis,2014,14(1):421.
[7]中国兽药典委员会.中华人民共和国兽药典(2010年版3部)[S].2010.
[8]Quinn M J,Resch C T,Sun J,et al.NhaP1is a K(Na)/H antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH[J].Microbiology,2012,158:1094-1105.
[9]吴柏春.微生物学[M].湖北武汉.华中师范大学出版社,2006.
[10]中国兽药监察所.兽用生物制品制造工[M].北京:中国农业出版社,2013.
[11]杨帆,李秀娟,鞠洪涛.禽多杀性巴氏杆菌(G190E40株)菌液发酵参数对细菌菌数影响的探讨[J].家禽科学,2010(9):11-13.
[12]Akoachere J F T K,Masalla T N H N.Multi-drug resistant toxigenic Vibrio cholerae O1is persistent in water sources in New Bell-Douala,Cameroon[J].BMC Infect Dis,2013,13:366.
[13]付强,程立坤,苗立中,等.补料培养在猪链球菌疫苗生产中的应用[J].中国动物医学进展,2013,34(3):133-136.
[14]肖敏,杨峰,王旭荣,等.分光光度法测定金黄色葡萄球菌菌液浓度方法的建立[J].动物医学进展,2014,35(11):40-43.