Method for rapidly detecting polymyxin B pore plate fermentation liquor

Abstract

The invention provides a method for rapidly detecting pore plate fermentation liquor of polymyxin B. The method belongs to the field of microorganisms, and comprises the following steps: taking paenibacillus polymyxa to be screened, respectively carrying out activation, solid culture medium colony separation and strain 96-pore plate fermentation culture, pre-treating the pore plate fermentation liquor to precipitate a product, and carrying out solid-liquid separation on the product to obtain the polymyxin B. The invention also provides a method for rapidly detecting the pore plate fermentation liquor of the polymyxin B. A coomassie brilliant blue method is used for replacing liquid phase detection, operation is easy, the labor cost is effectively reduced, the investment of experimental consumables is reduced, the sample amount of single screening is greatly increased, and then the overall efficiency of screening and detection is remarkably improved. By innovating the detection method and process, not only is the detection efficient, but also the time waste of repeated detection and screening caused by inaccurate primary screening detection data is avoided, so that the whole screening process is comprehensively accelerated, the speed of screening the target strain from a large number of strains is accelerated, and the screening efficiency is improved. A large amount of time is saved for subsequent research and development and application, and the whole research and development efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to a method for rapidly detecting polymyxin B in plate fermentation broth. Background Art

[0002] As a highly valuable antibiotic, polymyxin B has a unique antibacterial mechanism that can effectively damage the integrity of the bacterial cell membrane, thereby achieving a bactericidal effect. From the perspective of market supply and demand, due to its irreplaceability in combating drug-resistant bacterial infections, the market demand for polymyxin B continues to climb. In terms of market share, although new antibacterial drugs are constantly being developed and launched, polymyxin B maintains a relatively high market share in the market for drugs treating drug-resistant bacterial infections due to its unique antibacterial activity. Especially in the field of severe infections that are ineffective in treatment with other antibiotics, polymyxin B has become a key treatment option. However, limited by the current relatively limited production technology and the difficulties in strain breeding, the growth rate of its production is difficult to fully meet the clinical demand, resulting in a tight market supply situation.

[0003] For the fermentation industry, the quality of the strain is the core factor determining the product value of fermentation enterprises. Only by building a high-quality strain resource library can enterprises hope to improve the product value by improving the fermentation process and equipment. In the field of strain breeding, not only is it crucial to perform mutagenesis treatment and specific genetic modification on the strain, but the screening technology also plays an important role. However, at present, there is relatively little research on the methods for obtaining high-yield strains of Paenibacillus polymyxa. At the same time, most of the screening work still relies on the traditional shake-flask screening method, including the isolation and culture of single colonies in petri dishes, shake-flask fermentation, and the detection of the content of fermentation broth using a high-performance liquid chromatograph. This traditional screening mode has many drawbacks, such as limited screening volume, low efficiency, high equipment occupancy rate, time-consuming and laborious operation process. In addition, liquid-phase detection not only has high costs, long detection time, but also can handle a relatively limited number of samples. In view of this, developing a rapid, efficient, and accurate detection method for polymyxin B fermentation broth is of great significance for promoting the production of polymyxin B and meeting market demand. Summary of the Invention

[0004] In view of the above defects or deficiencies in the prior art, the present invention provides a method for rapidly detecting polymyxin B in plate fermentation broth to solve the problems existing in the above prior art.

[0005] Its main technical solution is: a method for rapidly detecting polymyxin B in plate fermentation broth, the method comprising the following steps:

[0006] (1) Preparation of standard solution: Weigh the polymyxin B standard, prepare an antibiotic stock solution with a concentration of 2000 μg / mL, and then dilute it to solutions with concentrations of 220, 240, 260, 280, and 300 μg / mL respectively;

[0007] (2) Plotting of standard curve: Dispense Coomassie Brilliant Blue G-250 staining solution into a 96-well analysis plate with a loading volume of 200 μL per well. Set 8 wells in one column of the well plate as parallel samples. Use an 8-channel pipette to sequentially add 40 μL of standard solutions with different concentrations, mix well, and let it stand at room temperature for 10 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at 595nm wavelength. Detect the absorbance values of standard solutions with different concentrations. Use the standard concentration (μg / mL) as the abscissa x and the absorbance value 595nm as the ordinate y to plot the standard curve. Add the linear relationship between x and y so that the correlation coefficient R2 ≥ 0.99 can be used;

[0008] (3) Fermentation culture and pretreatment of strains: Pick a single colony of Paenibacillus polymyxa into a 96-well plate for fermentation culture to obtain a fermentation broth, and perform pretreatment after centrifugation of the fermentation broth;

[0009] (4) Select a sterile fermentation medium for the blank control solution, and the treatment method is the same as that of the fermentation broth;

[0010] (5) Sample determination and calculation of fermentation broth concentration: Sequentially pipette 40 μL of the pretreated dissolution solution into a 96-well plate containing 200 μL of Coomassie Brilliant Blue working solution per well, mix well, let it stand at room temperature for 10 minutes, and use an ELISA reader to measure the absorbance value at 595nm wavelength.

[0011] Furthermore, in step (3), the fermentation broth is obtained by inoculating Paenibacillus polymyxa into a solid medium for single colony isolation culture, and after maturity, gently pick half of the mature single colony with a sterile toothpick into a 96-well plate for culture.

[0012] Furthermore, in step (3), the loading volume of the medium in a single well of the 96-well plate is 0.2 mL, and the medium formula is that per 100 mL of the medium contains 3.0 - 5.0 g of soluble starch, 0.3 - 0.6 g of refined soybean cake powder, 0.01 - 0.03 g of dipotassium hydrogen phosphate, and 0.3 - 0.4 g of ammonium sulfate.

[0013] Furthermore, in step (3), the culture conditions of the fermentation broth in the 96-well plate are shaking culture at 30 °C and 180 rpm for 26 h.

[0014] Furthermore, in step (3), the centrifugation conditions of the fermentation broth in the 96-well plate are 3500 rpm for 10 min.

[0015] Further, in the step (3), the fermentation broth pretreatment method is as follows: Take 0.15 mL of the fermentation supernatant, add 0.15 mL of saturated ammonium sulfate solution, perform salting out for 3 hours, centrifuge at 3000 rpm for 5 min, discard the supernatant, and dissolve the precipitate with 200 μL of phosphate buffer solution with a pH of 6.0.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. In the present invention, the primary screening fermentation culture of the strain uses a 96-well plate, breaking the limitation of the number of samples that can be picked under the traditional shaking flask mode of Paenibacillus polymyxa. A single person can complete a screening task that is several times or even dozens of times that of the traditional method in a single experiment, significantly increasing the probability of discovering effective strains and improving the screening efficiency.

[0018] 2. By utilizing the characteristics of the fermentation broth and innovating the detection method and process, the present invention not only makes the detection itself efficient but also avoids the time waste caused by repeated detection and screening due to inaccurate primary screening detection data. Thus, the entire screening process is comprehensively accelerated, the speed of screening target strains from a large number of strains is increased, a large amount of time is saved for subsequent research and application, and the efficiency of the entire research and development is improved.

[0019] 3. The screening process of the present invention is simple in operation, can obtain accurate results quickly, and significantly improves efficiency, providing a reference for the screening of other strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objectives, and advantages of the present application will become more apparent:

[0021] Figure 1 It is a standard curve graph of the content of polymyxin B sulfate - absorbance value. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following further elaborates on the present application with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are merely for explaining the relevant invention and do not limit the invention. Additionally, it should be noted that for the sake of description, only parts related to the invention are shown in the drawings.

[0023] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the drawings and embodiments.

[0024] The present invention provides a method for rapidly detecting the fermentation broth of polymyxin B in a 96-well plate, including the following steps:

[0025] (1) Preparation of standard solution: Weigh the polymyxin B standard, prepare an antibiotic stock solution with a concentration of 2000 μg / mL, and then dilute it to solutions with concentrations of 220, 240, 260, 280, and 300 μg / mL respectively.

[0026] (2) Plotting of standard curve: Dispense 200 μL of Coomassie Brilliant Blue G-250 staining solution into each well of a 96-well analysis plate. Set 8 wells in one column of the well plate as parallel samples. Use an 8-channel pipette to sequentially add 40 μL of standard solutions with different concentrations, mix well, and let it stand at room temperature for 10 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at wavelength A 595nm and detect the absorbance of standard solutions with different concentrations. Take the standard concentration (μg / mL) as the abscissa x and the absorbance A 595nm as the ordinate y to plot the standard curve, add the linear relationship between x and y, and it can be used when the correlation coefficient R2 ≥ 0.99.

[0027] (3) Strain fermentation culture and pretreatment: Inoculate Paenibacillus polymyxa into a solid medium for single colony isolation and culture. After maturation, gently pick half of the mature single colonies with a sterile toothpick into a 96-well plate, and culture it with shaking at 30 °C and 180 rpm for 26 h. Set the first column of the well plate as the blank control group without picking colonies. After maturation, centrifuge the fermentation broth in the well plate at 3500 rpm for 10 min. Add 0.15 mL of saturated ammonium sulfate solution to 0.15 mL of the fermentation supernatant, perform salting out for 3 hours, then centrifuge at 3000 rpm for 5 min, discard the supernatant, and dissolve the precipitate with 200 μL of pH 6.0 phosphate buffer solution.

[0028] (4) Sample determination and calculation of fermentation broth concentration: Sequentially pipette 40 μL of the pretreated dissolved solution into a 96-well plate containing 200 μL of Coomassie Brilliant Blue working solution in each well, mix well, and let it stand at room temperature for 10 minutes. Use an ELISA reader to measure the absorbance at wavelength A 595nm Calculate the average absorbance of the blank group. The actual absorbance detected is the difference between the measured absorbance and the average absorbance of the blank. Substitute it into the standard curve to obtain the product content of the fermentation broth.

[0029] Example 1:

[0030] A method for rapid detection of polymyxin B fermentation broth in a well plate

[0031] (1) Preparation of standard solution: Weigh 0.20 g of polymyxin B standard, dissolve it in 100 mL of pure water to prepare an antibiotic stock solution with a concentration of 2000 μg / mL, and then dilute it to solutions with concentrations of 220, 240, 260, 280, and 300 μg / mL respectively.

[0032] Table 1 Dilution of standard solution

[0033]

[0034] (2) Preparation of standard curve: Dispense 200 μL of Coomassie Brilliant Blue G-250 staining solution into each well of a 96-well analysis plate, with 8 wells in a column of the plate as parallel samples. The first column is the blank control. Use an 8-channel pipette to add 40 μL of pure water, and then sequentially add 40 μL of standard solution with different concentrations by column. Mix well and let stand at room temperature for 10 minutes. Use a microplate reader to measure the absorbance at 595nm wavelength, and detect the absorbance of each concentration of standard solution, as shown in Table 2. The difference between the measured absorbance and the average absorbance of the blank is the actual measured absorbance. With the standard concentration (μg / mL) as the abscissa x and the actual absorbance 595nm as the ordinate y, draw a standard curve, add the linear relationship between x and y, and obtain the standard curve equation as y = 0.0017x + 0.0067, R2 = 0.9949 (the curve graph is as Figure 1 ).

[0035] Table 2 Establishment of standard curve

[0036]

[0037]

[0038] (3) Fermentation culture and pretreatment of strains: Paenibacillus polymyxa (strain number CICC 21779) is inoculated into a solid culture dish for single colony isolation culture. After maturity, gently pick half of the mature single colonies with a sterile toothpick into a 96-well plate and culture them at 30 °C and 180 rpm for 26 h. The first column of the plate is the blank control group without picking colonies. After maturity, centrifuge the liquid in the plate at 3500 rpm for 10 min. Take 0.15 mL of the supernatant and add 0.15 mL of saturated ammonium sulfate solution. After salting out for 3 hours, centrifuge at 3000 rpm for 5 min, discard the supernatant, and dissolve the precipitate with 200 μL of pH 6.0 phosphate buffer.

[0039] (4) Sample determination and calculation of fermentation broth concentration: Sequentially pipette 40 μL of the dissolved solution after pretreatment into a 96-well plate containing 200 μL of Coomassie Brilliant Blue working solution in each well, mix well, and let stand at room temperature for 10 minutes. Use a microplate reader to measure the absorbance at 595nm wavelength, as shown in Table 3. Calculate the average absorbance of the blank group, and the actual measured absorbance is the difference between the measured absorbance and the average absorbance of the blank, as shown in Table 4. Substitute it into the standard curve to obtain the product content of the fermentation broth, as shown in Table 5.

[0040] Table 3 Measured absorbance of fermentation broth in 96-well plate

[0041]

[0042]

[0043] Table 4 Actual absorbance values of fermentation broth in 96-well plates

[0044]

[0045] Table 5 Calculated values of product concentration in fermentation broth in 96-well plates

[0046]

[0047]

[0048] As can be seen from the above examples, the present invention provides a method for rapidly detecting polymyxin B fermentation broth. This method transforms the traditional shake flask culture of strains into high-throughput 96-well plate culture, and at the same time uses the Coomassie brilliant blue method to replace high-performance liquid chromatography instruments to achieve rapid and large-scale detection, confirming a complete set of rapid detection methods for 96-well plates, greatly improving the screening efficiency of Paenibacillus polymyxa and the detection efficiency of fermentation broth.

[0049] The above description is only a preferred embodiment of the present application and an explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A method for rapidly detecting polymyxin B in a microtiter plate fermentation broth, characterized in that: The method comprises the following steps: (1) Preparation of standard solution: Weigh the polymyxin B standard and prepare an antibiotic stock solution with a concentration of 2000 μg / mL, and then dilute it to solutions with concentrations of 220, 240, 260, 280, and 300 μg / mL respectively; (2) Preparation of standard curve: Dispense 200 μL of Coomassie Brilliant Blue G-250 staining solution into each well of a 96-well assay plate. Set 8 wells in a column of the well plate as parallel samples. Use an 8-channel pipette to sequentially add 40 μL of standard solutions with different concentrations, mix well, and let stand at room temperature for 10 minutes. Use a microplate reader to measure the absorbance at wavelength A 595nm . Detect the absorbance of standard solutions with different concentrations. Take the standard concentration as the abscissa x and the absorbance A 595nm as the ordinate y to draw a standard curve, and add the linear relationship between x and y so that the correlation coefficient R 2 ≥ 0.99 can be used; (3) Fermentation culture and pretreatment of strains: Pick a single colony of Paenibacillus polymyxa into a 96-well plate for fermentation culture to obtain a fermentation broth, and perform pretreatment after centrifugation of the fermentation broth; (4) Select a sterile fermentation medium for the blank control solution, and the treatment method is the same as that of the fermentation broth; (5) Sample determination and calculation of fermentation broth concentration: Sequentially pipette 40 μL of the pretreated dissolved solution into each well of a 96-well plate containing 200 μL of Coomassie Brilliant Blue working solution, mix evenly, let stand at room temperature for 10 minutes, and use an ELISA reader to measure the absorbance value at 595nm the wavelength.

2. The method for rapidly detecting polymyxin B in a microplate fermentation broth according to claim 1, wherein: In step (3), the fermentation broth is obtained by inoculating Paenibacillus polymyxa into a solid medium for single colony isolation culture, and after maturation, half of the mature single colonies are picked with a sterile toothpick and cultured in a 96-well plate.

3. A method for rapidly detecting polymyxin B in a microtiter plate fermentation broth according to claim 1, characterized in that: In step (3), the loading volume of the medium in each well of the 96-well plate is 0.2 mL, and the medium formula is that each 100 mL of the medium contains 3.0 - 5.0 g of soluble starch, 0.3 - 0.6 g of refined soybean cake powder, 0.01 - 0.03 g of dipotassium hydrogen phosphate, and 0.3 - 0.4 g of ammonium sulfate.

4. A method for rapidly detecting polymyxin B in a microtiter plate fermentation broth according to claim 1, characterized in that: In step (3), the culture conditions of the fermentation broth in the 96-well plate are shaking culture at 30 °C and 180 rpm for 26 h.

5. A method for rapidly detecting polymyxin B in a microtiter plate fermentation broth according to claim 1, characterized in that: In step (3), the centrifugation conditions of the fermentation broth in the 96-well plate are 3500 rpm for 10 min.

6. A method for rapidly detecting polymyxin B in a microtiter plate fermentation broth according to claim 1, characterized in that: In step (3), the pretreatment method of the fermentation broth is to take 0.15 mL of the fermentation supernatant, add 0.15 mL of saturated ammonium sulfate solution, perform salting out for 3 hours, then centrifuge at 3000 rpm for 5 min, discard the supernatant, and dissolve the precipitate with 200 μL of pH 6.0 phosphate buffer.

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