鸭大肠杆菌O78血清型强毒株分离鉴定及其高密度发酵新工艺

Isolation and identification of duck virulent Escherichia coli O78 serotype strain and its new high-density fermentation technology

依据大肠杆菌(Escherichia coli,E.coli)持家基因PhoA PCR检测,从临床病料中分离鉴定出11株鸭致病性E.coli。经O抗原血清型鉴定以及毒力分析,筛选出1株O78血清型鸭E.coli强毒株EC-KP120427。以此菌株为基础,使用单因素法以及正交法,获得O78血清型鸭E.coli强毒株新型高密度发酵培养基,配方中初糖采用甘油替代葡萄糖,无机盐仅添加磷酸氢二钾,同时补充铁、锰、铝3种微量元素,在普通摇床上37℃培养菌液D600 nm可达到9.0以上。使用赛多利斯5 L发酵罐,对EC-KP120427菌株进行发酵培养试验。使用优化的新型高密度发酵培养基,按1∶10比例接种对数生长期种子菌,菌液D600 nm值为1.0、控制温度37℃、pH 7.0、调节空气通气量1.5~5.0 L/min、转速100~400 r/min、发酵至对数生长期以15 mL·h-1·L-1流速慢速流加50%葡萄糖溶液,培养菌液D600 nm值可高达30.0以上,成功实现了O78血清型鸭E.coli强毒株高密度发酵。本研究为致病性E.coli高密度发酵技术标准的建立奠定基础,同时为水禽E.coli细菌疫苗的改进提供了技术支持。

According to the detection of Escherichia coli(E.coli)housekeeping gene PhoA by PCR,11 strains of duck pathogenic E.coli were isolated and identified from clinical specimens.A strain EC-KP120427 of duck virulent E.coli O78 serotype was screened out by O antigen serotype identification and virulence analysis.Based on this strain,a new high-density fermentation medium for duck virulent E.coli O78 serotype strain was screened out by single factor method and crosscut method.The initial sugar,glucose was replaced by glycerol.K2HPO4 was the only inorganic salt.Supplementation of 3 microelements(iron,manganese,aluminum).The D600 nm of the bacterial solution at 37℃in the constant temperature shaking table could reach more than 9.0.Fermentation experiments of EC-KP120427 strain were carried out in Sartorius 5 L fermentation tank.Using the optimized new high-density fermentation medium,the D600 nm value of bacterial solution could be as high as 30.0 by controlling the inoculation rate 1∶10 of the logarithmic phase seed bacteria,temperature 37℃,pH7.0,air flow 1.5-5.0 L/min,rotary speed 100-400 r/min and slowflow rate 15 mL·h-1·L-1 of 50%glucose solution.The high density fermentation of virulent E.coliO78 serotype strain was successfully realized.This study laid a foundation for the establishment of high-density fermentation technology for standard of pathogenic E.coli,and provided technical support for the waterfowl Ec.oli bacterial vaccine.