| 李映,吴春光,付静芸,等.副溶血弧菌噬菌体的发酵优化及中试制备工艺探索[J].中国海洋药物,2023,42(4):38-44. |
| 副溶血弧菌噬菌体的发酵优化及中试制备工艺探索 |
| Fermentation Optimization and Pilot-scale Engineering Preparation of Vibrio parahaemolyticus phage |
| 投稿时间：2022-04-08 修订日期：2022-05-12 |
| DOI：10.13400/j.cnki.cjmd.2023.04.008 |
| 中文关键词: 副溶血弧菌噬菌体 发酵优化 中试生产 |
| English Keywords:Vibrio parahaemolyticus phage fermentation optimization pilot production |
| Fund Project: |
|
| 摘要点击次数: 2038 |
| 全文下载次数: 1 |
| 中文摘要: |
| 目的 为了以低成本的方法制备出高效价的噬菌体制剂，对副溶血弧菌噬菌体Vpas_PP24的发酵条件和关键工程化制备工艺进行探索。方法 采用单因素实验法，研究噬菌体Vpas_PP24发酵体系的不同培养基、金属离子、pH、接种量及培养时间对其效价的影响；在中试规模对优化发酵工艺进行了工程化放大；并以膜过滤结合热除菌法进行了下游工程化噬菌体分离及终端除菌工艺探索。结果 确立了副溶血弧菌噬菌体Vpas_PP24的最优培养基及最佳发酵条件。摇瓶水平最适出发培养基为2216E液体培养基；Mg2+和Ca2+可促进Vpas_PP24的增殖，且最适浓度为30 mmol·L-1；最适pH为8；VP8最适接种量为1%；最适培养时间为16 h。在摇瓶水平优化后的效价达到3.0×1010 PFU/mL，较优化前提高了14倍。随之，基于该优化条件，成功将噬菌体发酵放大到50 L中试规模，并建立上游液体深层发酵工程化生产Vpas_PP24的上游工艺，效价达到3.2×1010 PFU/mL，与此同时，建立了下游噬菌体除菌工艺，得到效价为2.5×108 PFU/mL的噬菌体Vpas_PP24批量液体制剂。结论 系统优化了噬菌体Vpas_PP24的发酵条件，并在中试规模进行了工艺放大，成功建立了中试上游噬菌体发酵工艺及下游噬菌体分离工程化工艺及末端去除宿主菌的技术手段，为噬菌体类替抗产品的工程化生产提供了可参考的范例。 |
| English Summary: |
| Objective In order to explore phage Vpas_PP24 as a potent agent against the pathogenic Vibrio parahaemolyticus, optimization of the phage fermentation conditions and engineering process conditions were conducted. Methods The effects of different mediums, metal ions, pH, inoculation, and culture time on the titer of phage Vpas_PP24 were tested by single factor experiment, respectively. And the optimized fermentation conditions were attempted in pilot scale, whereas the downstream phage isolation techniques together with final safety-proof killing of the pathogenic host bacteria were conducted by membrane filtration combined and thermal sterilization, respectively. Results The optimal culture medium and optimal fermentation conditions of phage Vpas_PP24 were established. The optimum starting medium for shake flask level was 2216E liquid medium. It was found that Mg2+ and Ca2+ could promote the proliferation of Vpas_PP24, at their optimum concentration at 30 mmol·L-1. The optimum pH, the optimum inoculum rate of VP8 and the optimum culture time was at 8, 1%, and 16 h, respectively. By applying the optimized conditions, the phage fermentation titer was elevated to 3.0×1010 PFU/mL, which was 14 times higher than that before optimization. Furthermore, the optimized phage fermentation conditions were successfully scaled up to 50 L pilot scale, with upstream pilot submerged fermentation techniques established and phage titer amounting to 3.2×1010 PFU/mL. In addition, based on the membrane filtration combined with thermal sterilization, a downstream process was successfully established for the isolation and formulation of the phage from fermentate, with phage Vpas_PP24 at 2.5×108 PFU/mL at harvest. Conclusion The fermentation conditions of phage Vpas_PP24 were systematically optimized, and the process was scaled up on the pilot scale. The systematic techniques of upstream phage fermentation, downstream phage isolation, as well as terminal host bacteria removal were successfully established on the pilot scale, respectively. The research findings of this work would set a successful model for exploring bacteriophage agents as the alternatives to antibiotics. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
|
| 关闭 |
|
|
|