仔猪副伤寒活疫苗中耐热保护剂应用相关参数的研究

Study of the application parameters for thermo-stabilizer in the manufacture of swine paratyphoid live vaccine

摘要:

【目的】研究并确定耐热保护剂在仔猪副伤寒活疫苗中应用的各参数，为制备细菌耐热保护剂活疫苗提供参考。【方法】将耐热保护剂与制苗菌液等体积混合，分别采用3种常用冻干曲线(1、2、3)进行冻干，抽样检测确定耐热保护剂配套冻干曲线。在确定合适冻干曲线的基础上，进一步研究耐热保护剂与不同菌龄菌液(发酵培养12、15、18和21 h)、不同浓度菌液(菌液终浓度分别为3×1010、5×1010和7×1010 CFU/mL)、不同感作时间(分别作用0、24和48 h)、耐热保护剂的不同保存时间(2?8 °C分别保存0、10、20、30和40 d)，以及不同分装量(2、3和4 mL)等多种不同参数对疫苗质量的影响。【结果】配套冻干曲线研究表明，曲线2冻干的产品性状、活菌存活率与耐老化指标效果最好；菌龄研究表明，37 °C发酵培养18 h (位于对数生长末期或稳定前期)，其冻干菌存活率达78%?81%，优于其它时间；配苗试验表明，菌液适宜终浓度为3×1010?5×1010 CFU/mL；最适感作时间为2?8 °C感作24 h，冻干菌存活率可达85.3%?90.5%；耐热保护剂保存期试验表明，2?8 °C保存40 d与0 d (配制当日)的保护剂冻干效果基本一致；配苗分装量试验表明，7 mL西林瓶中分装3 mL或4 mL，其冻干菌存活率与2 mL基本一致，但耐老化试验中活菌存活率比2 mL略高。【结论】收获发酵培养18 h的菌液、配苗终浓度采用3×1010?5×1010 CFU/mL，使用2?8 °C保存40 d内的耐热保护剂，让保护剂与制苗用菌液2?8 °C感作24 h，采用3?4 mL进行定量分装，按曲线2冻干为制备仔猪副伤寒耐热保护剂活疫苗的适合参数。

Abstract:

[Objective] To study the application parameters of thermo-stabilizer in the manufacture of swine paratyphoid vaccine, and provide reference for the preparation of thermo-stable bacterial live vaccine. [Methods] The thermo-stabilizer was mixed with same amount of bacteria culture, Salmonella enterica serovar Choleraesuis strain C500. The mixture was lyophilized using freeze-drying curves 1, 2 and 3, and the matched freeze-drying curved was then determined by sampling the survival ratio post lyophilization. Based on the optimal freeze-drying curve, the effect of various factors on the vaccine quality post lyophilization was further investigated. The studied factors included the culture age (C500 culture fermented for 12, 15, 18 and 21 h), the culture concentration (3×1010, 5×1010 and 7×1010 CFU/mL), interaction time for thermo-stabilizer and C500 culture (0, 24 and 48 h), storage time of fresh prepared thermo-stabilizer (0, 10, 20, 30 and 40 d) and the sub-package volume (2, 3 and 4 mL). [Results] The lyophilized product by freezing-drying curve 1 had the properties of surface crystallization and bottom shrinkage. The lyophilized product by freezing-drying curve 3 had a small amount of crystallization and the lower concave on the surface. The lyophilized product by freeze-drying curve 2 had the best properties, highest survival ratio and best aging resistance. The C500 strain cultured at 37 °C for 18 h was optimal for lyophilization with a highest survival ratio of 78%?81%. The optimal culture concentration was 3×1010?5×1010 CFU/mL. The optimal incubation time for the C500 culture and thermo-stabilizer was 24 h at 2?8 °C, with the survival ratio of 85.3%?90.5%. After stored at 2?8 oC for 40 days, the thermo-stabilizer has the same effect with fresh prepared one. The survival ratio after lyophilization was almost the same for the sub-package of 3 mL or 4 mL compared with 2 mL, but the survival ratio was 3?7 percentage points higher than sub-packing with 2 mL after stored at 37 °C for 7 days. [Conclusion] The optimal parameters for the thermo-stabilizer in manufacture of swine paratyphoid live vaccine were the following: freeze-drying curve 2, culture time of 18 h, culture concentration of 3×1010?5×1010 CFU/mL, thermo-stabilizer used within 40 days if storage at 2?8 °C from fresh preparation, incubation time of 24 h for the C500 culture and thermo-stabilizer, sub-package volume of 3?4 mL.