The Research Of Stability For Paenibacillus Polymyxa And Fermentation Process Of Polymyxin E

Polymyxin E is a multi-component alkaline polypeptide antibiotic produced by Paenibacillus polymyxa,Mainly used in the treatment of multi-drug resistant gram-negative infection.A key problem in the process of producing polymyxin E was faced:the strains were not stable,easy to death in the process of preservation,easy to autolysis in producing,and production level was too low.In this dissertation,the preservation method and rejuvenation method of Paenibacillus polymyxa,polymyxin E damage mechanism and protective measures of producing bacteria,and Polymyxin E fermentation processes were studied.The preservation method and rejuvenation method of Paenibacillus polymyxa were studied.The result showed that using 20%skimmed milk powder as protective agent,the survival rate of the strains in-80oC preservation was 17.3 times higher than in the freeze-drying preservation.In the freeze-drying preservation,adding thehalose into 20%skimmed milk,the survival rate increased by 5.0 times.The strains preserved in-80oC for six months,separated and purified for three times,the highest antibiotic production capacity returned to 93.0%,and using resistance plate to separate and purify for one time,the highest antibiotic production capacity returmed to 81.0%.Polymyxin E damage mechanism and protective measures of producing bacteria were studied.The result showed that high concentrations of polymyxin E have damage to G+ bacteria.Then preliminary speculation increasing the cell membrane permeability was one of the mechanisms of polymyxin E to inhibit G+ bacteria.In addition,divalent cations Ca²⁺ ion and Mg²⁺ ion can protect the damage.The protective effects of Ca²⁺ was better than Mg²⁺.Polymyxin E fermentation processes were studied.Firstly,the basic formula was optimized,new formula using 20%glucose and 80%soluble starch as a composite carbon source,the synthesis of polymyxin E in Shake flask level increased by 77.2%.Based on the original and new formula fed-batch fermentation,Polymyxin E synthesis were 7.6 times and 5.4 times higher than batch fermentation,respectively.