Method for improving fermentation level of polymyxin B by using active peptide BHL
By using the active peptide BHL in the seeds and fermentation medium of polymyxin B, the problem of unstable polymyxin B fermentation level was solved, the potency of polymyxin B was improved and the fermentation cycle was optimized, reaching the international advanced level and pharmacopoeia standards.
Patent Information
- Application Number
- CN202510720833.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-09-26
AI Technical Summary
The fermentation level of polymyxin B in the existing technology is unstable, which affects its production efficiency and output, and it is difficult to reach the international advanced level and the current Chinese Pharmacopoeia standards.
The active peptide BHL (amino acid sequence: Val-Ala-Arg-Val-Ala-Arg-Val-Ala-Arg) was used in seeds and fermentation medium, and the fermentation level of polymyxin B was improved by shaking culture and pH adjustment.
Effectively improve the fermentation unit of polymyxin B, ensure a short fermentation cycle, and make the potency of polymyxin B reach the international advanced level and the current Chinese Pharmacopoeia standard, thereby generating good social and economic benefits.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial pharmaceutical manufacturing, in particular to a method for improving the fermentation level of polymyxin B by utilizing active peptide BHL. Background Art
[0002] The emergence of drug-resistant bacterial strains poses a serious threat to human safety and health. Among these highly resistant bacteria are Enterococci, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, the latter four of which are all Gram-negative. In the era of multidrug resistance, polymyxins are highly recommended by authoritative medical experts and treatment guidelines both domestically and internationally, becoming the last line of defense against multidrug-resistant Gram-negative infections that are ineffective against antibiotics such as aminoglycosides, quinolones, and carbapenems.
[0003] Polymyxin is a general term for a group of basic polypeptide antibiotics, mainly including five types: A, B, C, D and E, among which polymyxin B is the most active. Their basic structure is a cyclic decapeptide sequence, and only polymyxin B and E are used clinically. The antibacterial mechanism of polymyxin is divided into two stages: first, in a liquid environment, the positively charged polymyxin electrostatically binds to the negatively charged lipid A on the outer membrane, causing the outer membrane to swell; then, it penetrates the outer membrane through a "self-promoted uptake" mechanism, destroying the physical integrity of the cell membrane phospholipid bilayer, leading to osmotic imbalance, causing the leakage of important components such as nucleotides, amino acids, and phosphates in the cell, inhibiting bacterial growth or causing bacterial death.
[0004] Polymyxin B is produced by polymyxin B, a Gram-positive bacterium that produces a variety of bioactive substances, including peptides, proteins, and polysaccharides. Peptides, among them, have antagonistic effects on bacteria and fungi. Strain instability and fermentation performance are key issues in the production of polymyxin B. In recent years, research and development of polymyxin B production have focused primarily on increasing yield and optimizing the production process. Studies have shown that traditional mutagenesis methods can be used to select higher-yielding mutant strains, as well as optimizing fermentation conditions, to effectively increase polymyxin B production.
[0005] Bioactive peptides (BAPs) are small polypeptides produced by protein hydrolysis, with a molecular weight typically below 5,000 Daltons and a structure intermediate between amino acids and proteins. They have multiple functions, including regulating the immune system, antibacterial, lowering blood pressure, antioxidant, and anti-inflammatory, and they also play an important role in promoting the growth of Bacillus. Lipopeptide compounds, such as surfactin, iturin, and fengycin, are an important class of antimicrobial active substances produced by Bacillus. Bioactive peptides promote the growth and biological activity of Bacillus through various mechanisms, including antibacterial, antitumor, and antiviral functions. Their safety, broad spectrum, high efficiency, non-toxicity, and ease of decomposition in the body give them broad application prospects in agriculture, medicine, and environmental protection. Summary of the Invention
[0006] The object of the present invention is to provide a method for improving the fermentation level of polymyxin B by utilizing the active peptide BHL.
[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions: The active peptide BHL has an amino acid sequence of Val-Ala-Arg-Val-Ala-Arg-Val-Ala-Arg.
[0008] The application of the above-mentioned active peptide BHL in improving the fermentation level of polymyxin B.
[0009] A method for improving the fermentation level of polymyxin B using the above-mentioned active peptide BHL comprises the following steps: (1) Activating the polymyxin B-producing bacteria and inoculating them into a seed culture medium containing the active peptide BHL, and then placing them in a constant temperature shake flask incubator at 27-31°C and 220 rpm for 20-24 hours to obtain a seed solution; (2) transferring the seed liquid to a fermentation medium containing the active peptide BHL, and then placing it in a constant temperature shake flask incubator at 27-31°C and 220 rpm for shaking fermentation for 20-65 hours to obtain a fermentation liquid; (3) The pH of the fermentation broth was adjusted to 1.8-2.5 with oxalic acid, and then the polymyxin B titer was tested.
[0010] Preferably, in the above method, the polymyxin B-producing bacteria are activated and then inoculated into a seed culture medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 29° C. and 220 rpm for shaking culture for 22 hours to obtain a seed solution.
[0011] Preferably, in the above method, every 400 ml of the seed culture medium containing the active peptide BHL contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract powder, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the rest is distilled water.
[0012] Preferably, in the above method, the seed liquid is transferred to a fermentation medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 29° C. and 220 rpm for shaking fermentation for 24 hours to obtain a fermentation liquid.
[0013] Preferably, in the above method, each 1000 mL of the fermentation medium of the active peptide BHL contains 10 g of active peptide BHL, 20 g of soluble starch, 10 g of defatted soybean powder, 5 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, and the rest is distilled water.
[0014] Preferably, in the above method, the pH of the fermentation broth is adjusted to 2.1 with oxalic acid, and then the polymyxin B titer is detected.
[0015] The significant advantages of the present invention are: The present invention provides a method for increasing the fermentation level of polymyxin B using the active peptide BHL. This method not only effectively increases the fermentation unit of polymyxin B and ensures a short fermentation cycle, but also enables the polymyxin B potency to reach international advanced levels and current Chinese Pharmacopoeia standards, thereby generating positive social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 : Observe the mycelial morphology under a microscope. DETAILED DESCRIPTION
[0017] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0018] The polymyxin B-producing bacteria in the present invention are polymyxin B-producing Paenibacillus polymyxa, which are conventional existing strains available on the market or strains obtained by conventional screening, such as those available from the China General Microorganism Collection Center, and do not involve the development of new strains. Specifically, the polymyxin B-producing bacteria in the following embodiments are provided by Fujian Fukang Pharmaceutical Co., Ltd., and are activated at 29° C. for 2 days using LBA solid medium (each 1000 mL of LBA solid medium contains 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, and the rest is distilled water, pH 7.0).
[0019] The amino acid sequence of the active peptide BHL in the following examples is Val-Ala-Arg-Val-Ala-Arg-Val-Ala-Arg, which was obtained by Fmoc solid phase synthesis.
[0020] Example 1: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300 mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the remainder is distilled water, with a natural pH). The culture was then shaken in a constant temperature shake flask incubator at 29° C. and 220 rpm for 16 hours to obtain a seed solution. The seed solution was sampled for pH testing, and after centrifugation and stratification, the bacterial concentration was determined (bacterial concentration % = (bacterial sediment volume / seed solution volume) × 100%). The mycelial morphology in the seed solution was observed under a microscope.
[0021] The results showed that the pH of the seed solution was 6.13, the bacterial concentration was 4.5%; the mycelial morphology was Figure 1 A: The hyphae are thick, Gram stain is dark, and the amount of hyphae increases.
[0022] Example 2: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300 mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the remainder is distilled water, with a natural pH). The culture was then shaken in a constant temperature shake flask incubator at 29° C. and 220 rpm for 22 hours to obtain a seed solution. The seed solution was sampled for pH testing, and after centrifugation and stratification, the bacterial concentration was determined (bacterial concentration % = (bacterial sediment volume / seed solution volume) × 100%). The mycelial morphology in the seed solution was observed under a microscope.
[0023] The results showed that the pH of the seed solution was 6.09, the bacterial concentration was 6.0%; the mycelial morphology was Figure 1 B: The hyphae are thick, the Gram stain is dark, and the amount of hyphae is large.
[0024] Example 3: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300 mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the remainder is distilled water, with a natural pH). The culture was then shaken and cultured in a constant temperature shake flask incubator at 31° C. and 220 rpm for 20 hours to obtain a seed solution. The seed solution was sampled for pH testing, and after centrifugation and stratification, the bacterial concentration was determined (bacterial concentration % = (bacterial sediment volume / seed solution volume) × 100%). The mycelial morphology in the seed solution was observed under a microscope.
[0025] The results showed that the pH of the seed solution was 6.32, the bacterial concentration was 6.0%; the mycelial morphology was Figure 1 C: There are a few empty cells in the hyphae, the Gram stain is slightly lighter, and the amount of hyphae is large.
[0026] Example 4: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300 mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the remainder is distilled water, with a natural pH). The culture was then shaken and cultured in a constant temperature shake flask incubator at 31° C. and 220 rpm for 24 hours to obtain a seed solution. The seed solution was sampled for pH testing, and after centrifugation and stratification, the bacterial concentration was determined (bacterial concentration % = (bacterial sediment volume / seed solution volume) × 100%). The mycelial morphology in the seed solution was observed under a microscope.
[0027] The results showed that the pH of the seed solution was 6.35, the bacterial concentration was 6.0%; the mycelial morphology was Figure 1 D: The hyphae have empty cells, the Gram stain is light, and the amount of hyphae is large.
[0028] Example 5: Control group: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300 mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract powder, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the rest is distilled water, natural pH), and then placed in a constant temperature shake flask incubator at 29°C and 220 rpm for shaking culture for 22 hours to obtain seed liquid. The seed solution was inoculated at a 10% (v / v) inoculum into a 300 mL shake flask containing 30 mL of fermentation medium (per 1000 mL, the fermentation medium contained 20 g of soluble starch, 10 g of defatted soybean flour, 5 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, and the remainder was distilled water, with a natural pH). The flask was then incubated in a constant-temperature shake flask incubator at 29°C and 220 rpm for 24 hours to obtain the fermentation broth. The fermentation broth was sampled after 0, 8, and 24 hours of incubation to measure pH. The bacterial cell concentration (% cell concentration = (cell pellet volume / broth volume) × 100%) was determined after centrifugation and stratification. After 24 hours of incubation, the pH of the fermentation broth was adjusted to 2.1 with oxalic acid, and the polymyxin B titer was determined by HPLC.
[0029] Experimental group: After activating the polymyxin B-producing bacteria, a single colony was picked and inoculated into a 300-mL shake flask containing 30 mL of seed culture medium (each 400 mL of seed culture medium contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract powder, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the rest is distilled water, with a natural pH). The culture was then placed in a constant temperature shake flask incubator at 29°C and 220 rpm for 22 h to obtain the seed solution. The seed solution was transferred at a 10% (v / v) inoculum to a 300 mL shake flask containing 30 mL of fermentation medium (per 1000 mL, the fermentation medium contained 10 g of active peptide BHL, 20 g of soluble starch, 10 g of defatted soybean flour, 5 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, and the remainder was distilled water, with a natural pH). The flask was then placed in a constant temperature shake flask incubator at 29°C and 220 rpm for 24 hours to obtain a fermentation broth. Samples were taken after 8 and 24 hours of fermentation to measure the pH of the fermentation broth. After centrifugation and stratification, the bacterial cell concentration was determined (% bacterial concentration = (volume of bacterial pellet / volume of fermentation broth) × 100%). After 24 hours of fermentation, the pH of the fermentation broth was adjusted to 2.1 with oxalic acid, and the polymyxin B titer was determined by HPLC.
[0030] The results showed that after 8 hours of fermentation, the pH of the fermentation broth in the control group was 6.53, and the bacterial concentration was 15%. After 24 hours of fermentation, the pH of the fermentation broth was 5.78, and the bacterial concentration was 25%. After 8 hours of fermentation, the pH of the fermentation broth in the experimental group was 6.52, and the bacterial concentration was 16%. After 24 hours of fermentation, the pH of the fermentation broth in the experimental group was 5.74, and the bacterial concentration was 24%. After the fermentation, the polymyxin B titer in the fermentation broth in the control group was 478 U / ml, while that in the experimental group was 552 U / ml, a 15.48% increase compared to the control.
[0031] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. Active peptide BHL, characterized by: The amino acid sequence of the active peptide BHL is Val-Ala-Arg-Val-Ala-Arg-Val-Ala-Arg.
2. Use of the active peptide BHL as claimed in claim 1 in improving the fermentation level of polymyxin B.
3. A method for improving the fermentation level of polymyxin B using the active peptide BHL according to claim 1, characterized in that: The following steps are involved: (1) After activating the polymyxin B-producing bacteria, the culture medium was selected and inoculated into a seed culture medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 27-31°C and 220 rpm for 20-24 hours to obtain a seed solution; (2) The seed liquid was transferred to a fermentation medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 27-31°C and 220 rpm for shaking fermentation for 20-65 hours to obtain a fermentation broth; (3) The pH of the fermentation broth was adjusted to 1.8–2.5 with oxalic acid, and then the polymyxin B titer was tested.
4. The method according to claim 3, wherein: After the polymyxin B-producing bacteria are activated, they are picked and inoculated into a seed culture medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 29° C. and 220 rpm for shaking culture for 22 hours to obtain a seed solution.
5. The method according to claim 3, wherein: Each 400 ml of the seed culture medium containing the active peptide BHL contains 4 g of active peptide BHL, 40 g of wheat flour, 0.02 g of amylase, 1.2 g of yeast extract powder, 0.06 g of dipotassium hydrogen phosphate, 1.8 g of ammonium sulfate, 0.3 g of light calcium carbonate, and the remainder is distilled water.
6. The method according to claim 3, wherein: The seed liquid was transferred to a fermentation medium containing the active peptide BHL, and then placed in a constant temperature shake flask incubator at 29° C. and 220 rpm for shaking fermentation for 24 hours to obtain a fermentation liquid.
7. The method according to claim 3, wherein: Each 1000 mL of the fermentation medium of the active peptide BHL contains 10 g of active peptide BHL, 20 g of soluble starch, 10 g of defatted soybean powder, 5 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of sodium chloride, and the remainder is distilled water.
8. The method according to claim 3, wherein: The pH of the fermentation broth was adjusted to 2.1 with oxalic acid, and then the polymyxin B titer was tested.