| 猪肺炎支原体HN0613株高密度发酵工艺研究及效力评价 |
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| 引用本文： | 猪肺炎支原体HN株高密度发酵工艺研究及效力评价.猪肺炎支原体HN0613株高密度发酵工艺研究及效力评价[J].畜牧与饲料科学,2018,39(11):10-15. |
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| 作者姓名： | 猪肺炎支原体HN株高密度发酵工艺研究及效力评价 |
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| 作者单位： | 国家兽用药品工程技术研究中心,河南洛阳471003 |
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| 基金项目： | 国家重点研发计划（2016YFD0500703）;河南省重大科技专项（171100110200）;河南省省级引智项目. |
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| 摘 要： | 旨在建立猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）HN0613株高密度发酵工艺技术。以培养滴度（CCU）作为考查指标，确定Mhp HN0613株的最佳活力代次以及复苏培养时间；通过四因素三水平正交试验筛选最佳基础培养基配方；以通气量、搅拌转速和接种百分比作为单因素考查指标，优化15L发酵工艺参变量，并确定700L高密度发酵放大工艺；按照确定的高密度发酵工艺技术，制备MhpHN0613株灭活疫苗，评价其对兔和仔猪的免疫效力。结果表明，Mhp HN0613株F11代较其他代次CCU水平高，可达1×10^9CCU／mL，为最佳活力代次；其在复苏72h后，pH值降为7．0左右，CCU水平达到最高，为1×10^9CCU／mL，为最佳复苏培养时间；培养体系中，2％的初始葡萄糖添加量、8％接种百分比、初始培养基pH值7．6、15％猪血清添加量为最适培养条件；通气量100L／h、搅拌转速300r／min和8％接种百分比条件最有利于Mhp HN0613株15L发酵培养；首次建立了平衡kLa联动pH值回调的工艺技术路线，并将优化后发酵罐搅拌叶轮结构改为推进式叶轮，降低了叶轮对菌体的剪切作用力，CCU较优化前提高了10倍，且培养周期较优化前缩短了2-3d；700L高密度发酵放大工艺中，Mhp HN0613株培养滴度不低于5×10^9CCU／mL．最高可达1×10^10CCU／mL；免疫兔血清抗体效价均不低于1：40，最高可达1：160；免疫组实验仔猪肺炎减少率均高于80％，临床观察精神及食欲未见异常。该研究为Mhp疫苗规模化发酵生产提供了一定的理论依据。
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| 关 键 词： | 猪肺炎支原体HN0613株 培养滴度 高密度发酵 免疫效力 |
| 收稿时间： | 2018-09-20 |
High-density Fermentation Process of Mycoplasma hyopneumoniae HN0613 Strain and Its Immune Efficacy Evaluation |
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| LIU Peng,LI Peng-hao,ZHANG Yi,ZHU Qiao-yan,LI Xiang-dong,HUANG Yu-xin,TIAN Ke-gong.High-density Fermentation Process of Mycoplasma hyopneumoniae HN0613 Strain and Its Immune Efficacy Evaluation[J].Animal Husbandry and Feed Science,2018,39(11):10-15. |
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| Authors: | LIU Peng LI Peng-hao ZHANG Yi ZHU Qiao-yan LI Xiang-dong HUANG Yu-xin TIAN Ke-gong |
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| Institution: | National Research Center for Veterinary Medicine,Luoyang 471003,China |
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| Abstract: | The aim of this study was to establish a high-density fermentation process of Mycoplasma hyopneumoniae HN0613 strain （Mhp HN0613）. The passage number with best growth activity and the optimum recovery time of Mhp HN0613 were determined using CCU as assessing index; a four-factor and three-level orthogonal test was conducted to screen the optimum basal culture medium formula; the aeration rate, agitation speed and inoculation percentage were used as single evaluating factor to optimize the parameters of fermentation process with volume of 15 L, respectively; based on the developed 15 L high-density fermentation process, a 700 L scaleup fermentation process was subsequently developed; the immune efficacy of the inactivated vaccine of Mhp HN0613 prepared with the developed high-density fermentation process on rabbit and piglet was evaluated. The results showed that the best growth activity was observed in F11 of Mhp HN0613 with CCU of 1×10^9 CCU/mL; the pH value of the medium of Mhp HN0613 reduced to around 7.0 at 72 h post recovery, and the CCU of Mhp HN0613 reached up to the highest level of 1×10^9 CCU/mL, accordingly, 72 h was considered as the optimum recovery time; the optimized basal culture medium formula was as follows： 2% of initial addition of glucose, 8% of inoculation percentage of Mhp HN0613, 7.6 of pH value of the initial culture medium, and 15% of addition of porcine serum; the optimized the parameters of 15 / fermentation process of Mhp HN0613 was as follows： 100 L/h of aeration rate, 300 r/min of agitation speed, and 8% inoculation percentage; a process technology route of balance k,a linkage pH value back-regulation was initially developed in this study; the agitating impeller in the fermentation tank was altered by impulse impeller, which reduced the shear stress of impeller against the bacteria, and the CCU of Mhp HN0613 was increased ten times and the culture period was shorted by 2-3 days; in the 700 L fermentation scaleup process, the culture titer of Mhp HN0613 was no less than 5x 109 CCU/mL and even reached up to the highest level of 1×10^10 CCU/mL; the serum antibody titer of the immunized rabbits with Mhp HN0613 was no less than 1：40 and the highest level was observed as 1：160; the pneumonia decreasing rate of the immunized piglets were all above 80%, and all of the immunized piglets were clinically healthy. This study provides a theoretical basis for the large-scale Mhp vaccine production. |
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