大肠杆菌O157:H7噬菌体鸡尾酒喷雾干燥微囊粉剂的制备及其应用

Preparation and application of Escherichia coli O157:H7 phage cocktail spray drying microcapsule

摘要:

【目的】提高噬菌体在常温环境下的保存稳定性，解决噬菌体鸡尾酒在体内失活的问题，为噬菌体对肠道疾病的治疗提供参考依据。【方法】本研究采用喷雾干燥技术制备噬菌体鸡尾酒微球粉末，通过单因素试验和正交试验确定最佳制备条件，并对其特性进行研究，比较其与游离噬菌体在常温环境和体内环境的稳定性差异，并通过口服给药的方式对大肠杆菌(Escherichia coli) O157:H7导致的肠道疾病进行治疗。【结果】本研究以海藻糖和亮氨酸组合为保护剂制备了一种具有热稳定性的噬菌体鸡尾酒微球粉末，试验结果显示，海藻糖和亮氨酸质量比为9:1时，设置进料速度为7.5 mL/min、海藻糖浓度为2%、入口温度为130 ℃、噬菌体鸡尾酒悬液与保护剂溶液体积比为1:50，噬菌体滴度损失最小，仅下降(0.623±0.235) log₁₀ PFU/g。其在常温条件下保存6个月，噬菌体鸡尾酒滴度损失(0.862±0.082) log₁₀ PFU/g，较游离噬菌体具有更长的保存稳定性，且其于体内环境的稳定性和治疗效果均优于游离噬菌体鸡尾酒。【结论】采用喷雾干燥法配合合适的保护剂配方可制得具有生物活性和热稳定性的噬菌体鸡尾酒微球粉末，延长其保质期，便于常温条件下的保存运输，使噬菌体制剂从实验室方向转化为工业方向的规模化生产提供参考依据。且噬菌体微球粉末清除肠道内大肠杆菌的能力更强、速度更快，是一种具有体内治疗发展潜力的口服给药剂型。

Abstract:

[Objective] To improve the storage stability of the phage at room temperature, solve the problem of inactivation of phage cocktail in vivo, and provide references for the effective treatment of gastrointestinal diseases by phages. [Methods] This study prepared phage cocktail microsphere powder by spray drying technology. The optimal preparation conditions were determined by single factor experiment and orthogonal experiment. The characteristics were studied, and the stability of the phage was compared with that of free phage at room temperature and in vivo was compared. Gastrointestinal diseases caused by Escherichia coli O157:H7 were treated by oral administration. [Results] In this study, a combination of trehalose and leucine was used to prepare a thermally stable phage cocktail microsphere powder. The results showed that the optimal mass ratio of trehalose and leucine was 9:1, the optimal speed of wriggling pump was 7.5 mL/min, the optimal trehalose concentration was 2%, the optimal inlet temperature was 130 ℃, and the optimal volume ratio of the phage cocktail suspension to the protectant solution was 1:50. Under the optimal conditions above, the titer loss was the lowest, only decreased by (0.623±0.235) log₁₀ PFU/g. After 6 months of storage at room temperature, the titer loss of the phage cocktail microsphere powder was only (0.862±0.082) log₁₀ PFU/g, which had longer storage stability than the free phage, and the stability in vivo and therapeutic effect were much better than the free phage. [Conclusion] Studies have shown that the spray drying method and suitable protective agent formula can be used to prepare phage cocktail dry powder with biological activity and thermal stability, which can extend retention time, facilitating the transportation under normal temperature conditions. This provides references for the transformation of phage preparations from the laboratory to the industrial large-scale production. Moreover, the phage microsphere powder shows an excellent ability to remove E. coli in the intestinal tract with a high speed, indicating its potential as an orally therapeutic agent in vivo.