A kind of polymyxin b sulfate production strain, preparation method and application of polymyxin b sulfate
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A technology for producing polymyxin sulfate and producing strains, which is applied to the preparation methods of peptides, polymyxins, and methods based on microorganisms, etc. The effect of the strong ability of prime B
Active Publication Date: 2021-03-23
黄石曼菲特生物科技有限公司
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Patent application number 201710280429.4 "Cultivation Medium for Fermentation of Polymyxin B Sulfate and Method for Fermentation and Production of Polymyxin B Sulfate". Although the invention partially controls the fermentation components, the purity of the product cannot reach 90% %
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Embodiment 1
[0037] Example 1 The acquisition of production strains
[0038]Use sterile water to elute the well-grown polymyxin B strains from the slope, and then use an ultraviolet lamp for 20 to 40 seconds of mutagenesis. DES) concentration is 0.05-0.5 mol / L, in this embodiment, 0.25 mol / L sterile water is used for 5-30 min, and in this embodiment, 20 min is selected. Then it is coated with polymyxin B 0.6 ~ 1.0g / L in the dark, and in the present embodiment, 0.7 g / L slant medium (a) is selected, and a single colony is picked and inoculated into the shake flask seed medium (a) after cultivation. b) Fermentation in medium, screening out strains that are higher than the control unit and less heterogeneous, passed to the third generation, the strains are more stable, and the mutagenized polymyxin B strains are obtained, the strains are Bacteroides pilaris Bacillus ( Paenibacillus peoriae ), single colony morphology is smooth surface, no wrinkle, milky white, oval, rod-shaped under the mic...
Embodiment 2
[0041] The preparation of embodiment 2 polymyxin B sulfate
[0042] 1. Strain fermentation
[0043] (1) Inoculate the mutagenized polymyxin B strain obtained in Example 1 into the shake flask seed medium (b) for cultivation to obtain shake flask seed liquid.
[0044] (2) Inoculate the shake flask seed liquid into a 10L seed tank for cultivation to obtain seed liquid.
[0045] (3) Transplant the seed liquid from the seed tank into a 50L fermenter for cultivation to obtain a fermented liquid.
[0046] Wherein: step (2) the formula of the seed tank culture medium is the same as that of the shake flask seed culture medium (b). Step (3) Fermentation medium formula: soluble starch 5.0~7.0 (6.5 in this example), yeast powder 0.5~0.8 (0.55 in this example), dipotassium hydrogen phosphate 0.01~0.05 (in this example 0.04), ammonium sulfate 0.2~0.4 (0.3 in this embodiment), calcium sulfate 0.2~0.4 (35 in this embodiment), defoamer 0.05, and the balance is water.
[0047] Carbon sourc...
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Abstract
The invention belongs to the technical field of biological fermentation, and in particular relates to a polymyxin B sulfate production strain, a preparation method and application of polymyxin B sulfate. The present invention obtains a polymyxin B sulfate production strain through mutagenesis breeding, and optimizes the carbon source and nitrogen source of the fermentation medium, precisely regulates the fermentation conditions such as the inoculum amount, fermentation temperature, fermentation time, and tank pressure, and controls the The metabolite components finally utilize the bacterial strain obtained by mutagenesis of the present invention to produce polymyxin B sulfate in a tank unit of 2.0 g/L, exceeding the industry average level by 20%. The proportions of several impurity peaks specified by the USP/EP Pharmacopoeia in the fermented liquid in tanks are far lower than the levels recorded in domestic literature. Separation and purification of the fermentation broth to obtain high-purity and high-content polymyxin B sulfate, the biometric titer of the finished product is greater than 8100u/mg, the purity and content are both greater than 90%, the single impurities and components are all qualified, and the total impurities are less than 8 %, product quality is much higher than USP/EP standard.
Description
technical field [0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a polymyxin B sulfate production strain, a preparation method and application of polymyxin B sulfate. Background technique [0002] Polymyxins are produced by Bacillus polymyxa ( Paenibacillus polymyxa ) produced a group of polypeptide antibiotics. Polymyxin B is used medicinally and its sulfate salt is commonly used. In addition to Proteus, polymyxin B sulfate has antibacterial effect on almost all Gram-negative bacteria, and is effective against bacteria in the growth and reproduction phases and in the stationary phase. It is mainly used clinically for infections caused by sensitive bacteria and chlorosis. Urinary system infection caused by Pseudomonas, as well as eye, trachea, meningitis, sepsis, burn infection and skin and mucous membrane infection. [0003] Polymyxin B sulfate is mainly biosynthesized by microbial fermentation, and then extract...
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