Inorganic salt culture medium for cultivating pseudomonas and its application
By optimizing the carbon and nitrogen source ratio using inorganic salt culture medium, the problem of high cost of nutrient-rich culture medium was solved, resulting in a significant increase in the yield of phenazine compounds and a reduction in cost, which is applicable to the synthesis of phenazine compounds from Pseudomonas.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI JIAOTONG UNIV
- Filing Date
- 2023-08-10
- Publication Date
- 2026-06-23
AI Technical Summary
Existing nutrient-rich culture media are costly for synthesizing phenazine compounds, and a more economical culture medium is needed to replace them in order to reduce the cost of biopharmaceuticals.
An inorganic salt culture medium, comprising a specific ratio of potassium hydrogen phosphate, potassium phosphate, magnesium sulfate, and ferric chloride, was used to optimize the carbon and nitrogen source ratio for culturing Pseudomonas bacteria to synthesize phenazine compounds.
It significantly reduced the cost of raw materials for the fermentation synthesis of phenazine compounds, increased the yield of phenazine compounds from various Pseudomonas aeruginosa subspecies by more than four times, and provided a clear research background.
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Figure CN116987643B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of culture media, specifically relating to an inorganic salt culture medium for culturing Pseudomonas and its applications. Background Technology
[0002] Phenidine compounds are a large class of antibacterial and antitumor active substances in nature. They have broad-spectrum antibacterial and antimicrobial activities and are mainly produced by Pseudomonas, Streptomyces and some microorganisms. Different substituents occupy different positions on heterocycles, resulting in different colors and determining the differences in physicochemical properties and biological activities among different compounds.
[0003] A recent research report indicates that culture media account for 30% of the cost of biopharmaceuticals, significantly impacting overall costs. Existing culture media used for synthesizing phenazine compounds are all nutrient-rich, containing expensive raw materials such as yeast extract and tryptone. Inorganic salt culture media, on the other hand, are less expensive, containing only the essential inorganic salt ions and carbon source necessary for microbial growth; hence, they are also known as basal media. Applying inorganic salt culture media to the industrial production of phenazines could significantly reduce raw material costs, demonstrating substantial industrial application value. Summary of the Invention
[0004] The purpose of this invention is to address the problems existing in the prior art by providing an inorganic salt culture medium for culturing Pseudomonas and its application.
[0005] The objective of this invention can be achieved through the following methods:
[0006] This invention provides an inorganic salt culture medium for culturing Pseudomonas, comprising the following components:
[0007] 8-10 g / L (NH4)2HPO4, 5-7g / L K2HPO4, 3-4 g / L KH2PO4, 0.1-0.5g / L MgSO4, 4-6mg / L FeCl3·6H2O.
[0008] As one embodiment of the present invention, the pseudomonads include *Pseudomonas aeruginosa* (orange needle pseudomonad). Pseudomonas chlororaphis subsp. aurantiaca LX24, causing *Pseudomonas aeruginosa* ( Pseudomonas chlororaphis subsp. aureofaciens GP72 and Pseudomonas aeruginosa ( Pseudomonas chlororaphis subsp. chlororaphis One of HT66.
[0009] The present invention also provides an application of the inorganic salt culture medium in the synthesis of various phenazine compounds by Pseudomonas fermentation.
[0010] In one embodiment of the present invention, when the Pseudomonas fermentation synthesizes various phenazine compounds, the carbon source is glycerol, and the content in the culture medium is 18-22 g / L, preferably 20 g / L.
[0011] As one embodiment of the present invention, the biosynthesis of phenazine compounds includes, but is not limited to, PCA, 2-OH-PHZ, and PCN; specifically, Pseudomonas aeruginosa GP72 synthesizes PCA, Pseudomonas aeruginosa LX24 synthesizes 2-OH-PHZ, and Pseudomonas aeruginosa HT66 synthesizes PCN.
[0012] Compared with the prior art, the present invention has the following beneficial effects:
[0013] (1) This invention optimizes the ratio of carbon and nitrogen sources and simplifies the types of trace elements based on the conventional components of inorganic salt culture medium, thereby significantly increasing the yield of phenazine compounds from various Pseudomonas aeruginosa subspecies. The optimized GDM culture medium yielded four times more than the unoptimized medium and 33.08% more than the traditional Pseudomonas aeruginosa KB medium.
[0014] (2) In view of the high price of Pseudomonas aeruginosa culture medium, this invention uses inorganic salt culture medium for fermentation culture, which can significantly reduce the raw material cost of future industrial fermentation synthesis. It provides methodological guidance for future improvements in the industrial synthesis of PCA and has great industrial application value. At the same time, the inorganic salt culture medium has a clear and simple composition, which also provides a clear research background for future studies on the metabolic regulation of phenazine compounds. Attached Figure Description
[0015] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:
[0016] Figure 1 The growth curves of GP72 in different KB and GDM media;
[0017] Figure 2 Graphs showing the product synthesis of GP72 in KB and GDM media;
[0018] Figure 3 Bar chart showing the product synthesis of GP72 in ME and GDM media;
[0019] Figure 4 This is a bar chart showing the fermentation of GP72 in ammonium concentration gradient media, where a represents the PCA yield at different ammonium concentrations, and b represents the OD of the fermentation broth at different ammonium concentrations. 600 value;
[0020] Figure 5The bar chart shows the PCA synthesis of GP72 under different iron ion valence states. Detailed Implementation
[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.
[0022] Example 1
[0023] This embodiment provides an inorganic salt culture medium for culturing Pseudomonas aeruginosa. The formula of this inorganic salt culture medium is as follows: 9.9 g / L (NH4)2HPO4, 5.8 g / L K2HPO4, 3.7 g / L KH2PO4, 0.12 g / L MgSO4, and 5.4 mg / L FeCl3·6H2O. The preparation of the culture medium and the steps for culturing the strain are as follows:
[0024] (1) Preparation of inorganic salt culture medium GDM
[0025] Add the corresponding masses of (NH4)2HPO4, KH2PO4, KH2PO4, MgSO4FeCl3·6H2O and 20g glycerol (glycerol is used as the carbon source in this experiment) to a graduated cylinder containing 950 mL of distilled water. Mix thoroughly until completely dissolved, then bring the volume to 1 L with distilled water. After dispensing, autoclave at 121℃ for 20 minutes.
[0026] (2) Fermentation process of the strains used in the experiment
[0027] The strain GP72 (He Yanjing, Liu Haiming, Hu Hongbo, et al. Isolation and identification of a Pseudomonas antagonistic to Phytophthora capsici [J]. Acta Microbiologica Sinica, 2006(04):516-521.) stored at -80℃ was streaked twice on LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl and 20% agar) for activation. A single colony from the plate was picked and cultured overnight in 5 mL LB liquid medium (containing 100 mg / L Amp). Fermentation was carried out in 250 mL concave fermentation flasks containing 60 mL of sterilized inorganic salt medium at a 1% inoculum rate. All fermentation experiments were performed in triplicate.
[0028] (3) Detection of bacterial growth
[0029] The growth rate of the strains in this experiment was measured using OD. 600The characterization process was performed using the following steps: Samples were taken four times, at 24h, 48h, 72h, and 96h. 1 mL of the bacterial culture that had grown to the target detection point was taken in a clean bench, centrifuged at 12,000 rpm for 1 min, and the waste liquid was discarded. The culture was then resuspended in 1 mL of distilled water. To ensure the detection value was between 0.2 and 0.8, the culture was diluted to a suitable concentration with distilled water, and the bacterial biomass was measured using a spectrophotometer at a wavelength of λ=600 nm.
[0030] (4) Detection of the yield of phenazine compounds
[0031] The product detection method in this experiment was high-performance liquid chromatography (HPLC). The specific operation steps are as follows: 400 μL of bacterial culture at the target time point was taken in a clean bench, and 20 μL of 6 mol / L hydrochloric acid was added for acidification. The mixture was allowed to stand for 20 s. 3.6 mL of ethyl acetate was added, and the mixture was thoroughly shaken and mixed for 5 min for extraction. After centrifugation at 6,000 rpm for 5 min, 400 μL of the supernatant was collected and dried overnight in a fume hood. The supernatant was dissolved in 1 mL of HPLC-grade acetonitrile and filtered through a 0.22 μm filter into an HPLC sample vial, which was then stored at 4℃.
[0032] Taking PCA, a secondary metabolite synthesized by strain GP72, as an example, the specific chromatographic operating parameters are as follows. The chromatographic column used in this experiment was a reverse-flow C18 column (Agilent Technologies, 5 μm, 4.6*250 mm). The mobile phase A was 0.1% formic acid in water (1 mL formic acid solution dissolved in 999 mL of ultra-high pressure water), and the mobile phase B was HPLC-grade acetonitrile solution. The detection method was as follows: elution rate 1.0 mL / min; elution conditions set to 0-2 min, A:B = 80%:20%; 2-15 min, A:B = 50%:50%; 15-20 min, A:B = 20%:80%. The UV monitoring wavelength was 254 nm, and the PCA peak time was approximately 10.2 minutes. A standard curve for the determination of phenazine compounds was prepared using PCA standards from Shanghai Nongle Company; the sample concentration was directly proportional to the peak area detected by HPLC.
[0033] (5) Implementation process and efficacy verification
[0034] The operational results of this study:
[0035] Growth comparison of GP72 strain
[0036] Using KB medium (tryptone 20 g / L, glycerol 18 g / L, MgSO4 0.732 g / L, K2HPO4 0.5142 g / L) as a control, the procedure was the same as for GDM. The growth curve of GP72 was plotted as follows: Figure 1As shown. From a biomass perspective, the strain exhibits a greater advantage in biological growth in KB medium, with an OD of 48 hours. 600 The value is twice that of GDM.
[0037] PCA Production Comparison
[0038] Using KB medium as a control, a PCA yield curve for GP72 was plotted as follows: Figure 2 As shown, strain GP72 can achieve a PCA yield of 1.07 g / L in GDM, which is 33.08% higher than that in the traditional nutrient-rich KB medium, thus increasing the yield of phenazine compounds.
[0039] Example 2
[0040] This embodiment provides an inorganic salt culture medium for culturing Pseudomonas bacteria. The formulation of this inorganic salt culture medium is as follows: 9.9 g / L (NH4)2HPO4, 5.8 g / L K2HPO4, 3.7 g / L KH2PO4, 0.12 g / L MgSO4, 20 μM FeCl3·6H2O. The preparation steps of the culture medium and the culture steps of the bacterial strain are as follows:
[0041] (1) Preparation of inorganic salt culture medium GDM
[0042] Add 9.9g (NH4)2HPO4, 5.8g KH2PO4, 3.7g KH2PO4, 0.12g MgSO4, 20 μM FeCl3·6H2O, and 20g glycerol (glycerol was used as the carbon source in this experiment) to a graduated cylinder containing 950 mL of distilled water. Mix thoroughly until completely dissolved, then bring the volume to 1 L with distilled water. After dispensing, autoclave at 121℃ for 20 minutes.
[0043] The experimental procedure is the same as in Example 1.
[0044] Comparative Example 1
[0045] The culture medium used in this comparative example was the Clinton Fuller 1988 modified E* medium, which consisted of 1.1 g / L (NH4)2HPO4, 5.8 g / L K2HPO4, 3.7 g / L KH2PO4, 12 g / L MgSO4, and 1 mL microelement solution. The experimental procedures were the same as in Example 1. In this inorganic salt medium, the yield of phenazine compounds synthesized by *Pseudomonas* was low. The advantage of this invention is that it can significantly increase the yield of phenazine compounds in *Pseudomonas*. Figure 3As shown, the PCA yield of GP72 in E* medium was only 251.9 mg / L, while the yield in the GDM medium of this invention was 1.07 g / L, which is nearly four times higher.
[0046] Comparative Example 2
[0047] This comparative example provides an inorganic salt culture medium for culturing Pseudomonas aeruginosa. The formulation and experimental methods of this inorganic salt culture medium are basically the same as those in Example 1, except that the concentration of (NH4)2HPO4 is configured as 10 mM, 20 mM, 30 mM, 50 mM, 80 mM, 100 mM, 120 mM, 150 mM, 180 mM, and 200 mM.
[0048] The results are as follows Figure 4 As shown, PCA production increases with increasing ammonium concentration. At a very low ammonium concentration (10 mM), PCA production is only 42 mg / L. As ammonium concentration increases, PCA production gradually increases as well. However, once the ammonium concentration exceeds 150 mM, further increases in ammonium concentration no longer lead to increased PCA production. Therefore, in GDM medium, 8-10 g / L of (NH4)2HPO4 is the optimal concentration for phenazine synthesis.
[0049] Comparative Example 3
[0050] This comparative example provides an inorganic salt culture medium for culturing Pseudomonas aeruginosa. The formulation and experimental methods of this inorganic salt culture medium are basically the same as those in Example 1, except that FeCl3·6H2O is replaced with FeSO4·7H2O.
[0051] The results are as follows Figure 5 As shown, FeCl3·6H2O is more favorable for the synthesis of phenazine compounds.
[0052] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.
Claims
1. An inorganic salt culture medium for culturing Pseudomonas, characterized in that, It consists of the following components: (NH4)2HPO4 9.9 g / L, K2HPO4 5.8g / L, KH2PO4 3.7 g / L, MgSO4 0.12 g / L, FeCl3·6H2O 5.4 mg / L; The Pseudomonas species mentioned is *Pseudomonas auricula-judae* (…). Pseudomonas chlororaphis subsp. aureofaciens GP72.
2. The application of the inorganic salt culture medium as described in claim 1 in the fermentation and synthesis of phenazine compounds by Pseudomonas, characterized in that, The phenazine compound is phenazine-1-carboxylic acid PCA.
3. The application according to claim 2, characterized in that, When the Pseudomonas ferments to synthesize various phenazine compounds, the carbon source is glycerol, and its content in the inorganic salt culture medium is 18-22 g / L.