大菱鲆鳗弧菌灭活疫苗原液发酵条件的优化
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摘要
为实现大菱鲆鳗弧菌灭活疫苗中试生产,通过对大菱鲆鳗弧菌菌株VAM003二级种子培养时间、盐度、培养基、接种量、补料等发酵条件的优化筛选,确定大菱鲆鳗弧菌菌株VAM003灭活疫苗发酵原液的发酵工艺条件。鳗弧菌菌珠VAM003接种于含2. 5%Na Cl的TSB液体发酵培养基,28℃振荡培养12～14 h,制备二级种子液,按发酵罐培养基总量的10%接种二级种子液,28℃补料发酵10～12 h。在该条件下鳗弧菌菌株VAM003发酵活菌数达到1. 20×1010cfu/m L,比优化前提高120%以上。
In order to realize pilot production of inactivated vaccine of turbot Vibrio anguillarum,fermentation technology of ferment initial liquid of inactivated V. anguillarum VAM003 vaccine was determined by optimizing the fermentation conditions of V. anguillarum VAM003 adopting secondary seed culture time,salinity,medium,inoculum volume,and addition feeding etc in this experiment. The inoculum of V. anguillarum VAM003 contained 2. 5% Na Cl with TSB liquid fermentation medium,28 ℃ shaking for 12-14 h to prepare secondary seeds liquid,and inoculated according to 10% of the total fermenter tank volume of the secondary seed liquids,28 ℃,addition feeding fermented for 10-12 hours. Under this optimized conditions,the viable count of the fermented liquid of V. anguillarum VAM003 living cells reached above 1. 20 × 1010 cfu/m L,increased by 120% higher than that before optimization.
In order to realize pilot production of inactivated vaccine of turbot Vibrio anguillarum,fermentation technology of ferment initial liquid of inactivated V. anguillarum VAM003 vaccine was determined by optimizing the fermentation conditions of V. anguillarum VAM003 adopting secondary seed culture time,salinity,medium,inoculum volume,and addition feeding etc in this experiment. The inoculum of V. anguillarum VAM003 contained 2. 5% Na Cl with TSB liquid fermentation medium,28 ℃ shaking for 12-14 h to prepare secondary seeds liquid,and inoculated according to 10% of the total fermenter tank volume of the secondary seed liquids,28 ℃,addition feeding fermented for 10-12 hours. Under this optimized conditions,the viable count of the fermented liquid of V. anguillarum VAM003 living cells reached above 1. 20 × 1010 cfu/m L,increased by 120% higher than that before optimization.
引文
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[13]梁永增,魏冬梅,王磊. 1株鱼类水霉病原真菌拮抗菌的发酵条件优化[J].江苏农业科学,2017,45(1):141-145.
[14]刘润泽,王海飙,常乐,等.一株产聚-β-羟基丁酸酯(PHB)的嗜盐菌Halomonas sp.发酵条件的优化及产物特性鉴定[J].黑龙江大学自然科学学报,2017,1:89-94.
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[17]马红叶,郑仁朝,赵川东,等.重组大肠埃希菌产疏绵状嗜热丝孢菌脂肪酶分批补料发酵工艺[J].生物加工过程,2015,13(2):9-12.
[18]文瑶,周玮,付相敏,等. D-乳酸大肠埃希菌工程菌的驯化及其补料发酵研究[J].湖北农业科学,2016,55(18):4771-4775.
[2] Miltion DL,Norqvist A,Wolf watz H. Cloning of ametalloprotease gene involved in the Virulence menchanism of vibrio anguillarum[J]. J. Bacteriol,1992,174(22):7235-7244.
[3] Rock J L,Nelson D R. Identification and characterization of a Hemolysin gene cluster in Vibrio anguillarum[J]. Infect Immun,2006,74(5):2777-2786.
[4]康玉军,杨姣姣,苟彦龙,等.鳗弧菌灭活疫苗对杂交鲟的免疫效果初探[J].水产养殖,2016,46(17):126-128.
[5]刘瑞,赵明君,杨慧,等.副溶血弧菌(Vibrioparahaemolyticus)tdh2基因和鳗弧菌(V. anguillarum)omp U基因二联DNA疫苗制备及其对大菱鲆免疫保护作用[J].海洋与湖沼,2011,4:580-586.
[6]肖鹏,莫照兰,邹玉霞,等.鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究[J].高技术通讯,2007,9:979-985.
[7]曹宏梅,李健,战文斌.鳗弧菌和溶藻弧菌二联疫苗对大菱鲆的免疫效果[J].中国水产科学,2006,13(3):397-402.
[8]宋晓青,邢婧,战文斌.牙鲆经注射和浸泡免疫鳗弧菌灭活疫苗后7种免疫相关基因表达的变化[J].中国水产科学,2014,21(4):747-758.
[9]罗凯,艾克特,夏理海,等. 3种水产致病弧菌的生长速度对比研究[J].长江大学学报(自然科学版),2017,1,14(2):25-28.
[10]赵鲁宁,李贵阳,李杰,等.海水养殖鱼类鳗弧菌(Vibrio anguillarum)临床分离株的血清型和抗生素耐药谱[J].海洋与湖沼,2015,46(5):1109-1117.
[11]陶家发,赖迎迢,任燕,等.弧菌菌株发酵工艺初步研究[J].工业微生物,2011,41(3):86-89.
[12]商伟伟,侯路红.海洋来源荧光性假单胞菌高产脓菌素培养基及发酵条件优化[J].应用海洋学学报,2016,35(2):166-173.
[13]梁永增,魏冬梅,王磊. 1株鱼类水霉病原真菌拮抗菌的发酵条件优化[J].江苏农业科学,2017,45(1):141-145.
[14]刘润泽,王海飙,常乐,等.一株产聚-β-羟基丁酸酯(PHB)的嗜盐菌Halomonas sp.发酵条件的优化及产物特性鉴定[J].黑龙江大学自然科学学报,2017,1:89-94.
[15] Salehmin MNI,Annuar MSM,Chisti Y. High cell density fedbatch fermentation for the production of a microbial lipase[J].Biochem Eng J,2014,85(15):8-14.
[16] Lee J,Lee SY,Park S,et al. Control of fed-batch fermentations[J]. Biotechnol Adv,1999,17(1):29-48.
[17]马红叶,郑仁朝,赵川东,等.重组大肠埃希菌产疏绵状嗜热丝孢菌脂肪酶分批补料发酵工艺[J].生物加工过程,2015,13(2):9-12.
[18]文瑶,周玮,付相敏,等. D-乳酸大肠埃希菌工程菌的驯化及其补料发酵研究[J].湖北农业科学,2016,55(18):4771-4775.