Portner Daniella C, Leuschner Renata G K, Murray Brent S
Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK.
Cryobiology. 2007 Jun;54(3):265-70. doi: 10.1016/j.cryobiol.2007.03.002. Epub 2007 Mar 24.
Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.
空肠弯曲菌的冻干培养物在食品和微生物行业中用作参考材料和菌种保藏。然而，空肠弯曲菌在冻干及随后的储存过程中极易受损，因此拥有更持久的培养物会很有用。为了优化存活率，对空肠弯曲菌在冻干及随后储存过程中的存活情况进行了研究。使用不同菌龄（24 - 120小时）的培养物、各种冻干保护剂（10%植物蛋白胨、蛋白胨、胨化乳、海藻糖、大豆蛋白胨和山梨醇）、各种储存条件（空气、氮气和真空）以及复水条件（培养基、温度和时间）对空肠弯曲菌进行冻干。一日龄培养物冻干后的存活率显著高于菌龄较大的培养物。在肌醇肉汤中添加海藻糖作为冻干保护剂，在4℃储存2个月后存活率几乎提高了2个对数单位（log(10)）。与在4℃储存后在最大恢复稀释液（MRD）中复水相比，在真空气氛中储存并在37℃的肌醇肉汤中复水可使回收率提高1 - 2个对数单位（log(10)）的存活率。当一日龄培养物在含有10%（w/v）海藻糖的肌醇肉汤中冻干，在4℃真空储存，并在相同培养温度（37℃）的肌醇肉汤中复水30分钟时，储存期间的存活率最佳。结果表明，通过优化培养菌龄、冻干保护剂以及储存和复水条件，可显著提高空肠弯曲菌冻干细胞在储存期间的存活率。存活率损失的对数速率（K）与绝对温度呈很好的反比关系，即符合阿累尼乌斯速率定律。将结果外推到更典型的储存温度（4℃）预测出非常低的K值为1.5×10⁻³。这些结果将有助于开发改进的参考材料和保藏在菌种库中的样品。
