Method for rapidly detecting polymyxin B fermentation liquor

Abstract

The invention discloses a method for rapidly detecting polymyxin B fermentation liquor, which belongs to the field of microorganisms and comprises the following steps: taking paenibacillus polymyxa, and detecting the fermentation liquor through activation, plate single colony separation, 48-pore plate culture and 96-pore plate biological turbidimetry. According to the method disclosed by the invention, a 48-pore plate is used for replacing a shake flask to culture a single colony obtained by flat plate separation, and meanwhile, a biological turbidimetric method is used for replacing liquid phase detection, so that the consumption of manpower and material resources is reduced, the single screening sample amount is increased, and the screening and detection efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a method for rapidly detecting polymyxin B fermentation broth. Background Art

[0002] Microorganisms in nature do not have the characteristics of "high yield, stability, strong survival ability, and easy reproduction". For fermented products, the strain is the key to determining the product value of the fermentation enterprise. The enterprise must have a good strain resource library to have the hope of improving the fermentation process and equipment to improve the product value.

[0003] For strain selection, not only does it require mutagenesis and specific genetic modification of the strain, but screening technology is also important. However, there are relatively few studies on the methods for obtaining high-yield strains of polymyxin Bacillus. At the same time, most screening still uses traditional shake flask screening methods, including single colony isolation and culture of culture dishes, shake flask fermentation, and fermentation broth content detection relying on high-performance liquid chromatography. It has the problems of small screening volume, low efficiency, large equipment occupancy rate, time-consuming and labor-intensive, and high cost and long time for liquid phase detection, and small number of test samples. Therefore, it is of great significance to study a method for rapid, efficient and accurate rapid detection of polymyxin B fermentation broth. Summary of the invention

[0004] In order to solve the limitations of the prior art, the present invention provides a method for rapidly detecting polymyxin B fermentation broth. The purpose is to improve the detection efficiency of polymyxin B fermentation broth.

[0005] To solve the above problems, the present invention provides the following technical solutions:

[0006] The present invention provides a well plate culture condition for Paenibacillus polymyxa: a 48-well plate is used instead of a shake flask, and the volume of a single-well culture medium is reduced from 50 mL of a shake flask to 1-1.2 mL. A sterile toothpick is used to gently pick half of the mature single colonies to the 48-well plate, and the culture is shaken at 32° C. and 180 rpm for 56 hours.

[0007] The present invention provides a method for rapidly detecting polymyxin B fermentation broth, comprising the following steps:

[0008] (1) Weigh the polymyxin B standard and dissolve it in ultrapure water to prepare an antibiotic stock solution with a concentration of 1000 U / mL. Then use ultrapure water to dilute the prepared polymyxin B antibiotic stock solution to solutions with concentrations of 0, 60, 70, 80, 90, 100, 110, and 120 U / mL, respectively.

[0009] (2) Take the E. coli cryopreservation tube and add LB medium to adjust the absorbance of the bacterial solution to A. 600nm= 0.200-0.300. The mixed indicator bacteria Escherichia coli liquid was dispensed into two 96-well plates at a volume of 270 μL / well, for establishing a standard curve and fermentation liquid detection, respectively.

[0010] (3) Establish a standard curve. Take a well plate with E. coli solution, add 30 μL of sterile standard solution of different concentrations and mix evenly, set a row of 8 wells in the well plate as parallel. At the same time, blank LB medium is used as the blank control, and bacterial suspension without antibiotics is selected as the positive control. 30 μL of sterile ultrapure water is added to make up the volume. After culturing at 32°C for 4 hours, use an enzyme reader to read the A 600nm The absorbance is measured at the wavelength, and the absorbance value of each concentration of the standard solution is detected. The absorbance value is the horizontal coordinate x, and the solution concentration is the vertical coordinate y. The standard curve is drawn, and the linear relationship between x and y is added so that the correlation coefficient R 2 It can be used when ≥0.99.

[0011] (4) Fermentation broth detection. The supernatant of the fermentation broth after centrifugation was aspirated and diluted 100 times with sterile ultrapure water. 30 μL of the dilution was then aspirated and added to another well plate with E. coli solution. After mixing evenly, the well plate was incubated at 32°C for 4 h and the A 600nm The absorbance was measured at wavelength.

[0012] (5) Calculation of biological activity data. Substitute the absorbance data of the fermentation broth obtained by the test into the standard curve equation. The calculated solution concentration multiplied by the dilution factor equals the biological activity data of the fermentation broth. Select strains with high biological activity for preservation.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. The present invention adopts a 48-well plate for strain initial screening fermentation culture, expands strain screening throughput, reduces the amount of culture medium used, and the screening throughput of a single person in a single experiment reaches 288 single colonies. It effectively solves the problems of high work intensity, small number of samples picked, low screening possibility, high equipment occupancy rate, small amount of liquid phase detection samples, and long time consumption, and improves detection efficiency.

[0015] 2. The present invention utilizes biological turbidimetry, and can efficiently and quickly determine the ability of the screened strain to produce the target product by detecting the turbidity of the plate according to the degree of inhibition of the strain fermentation broth on Escherichia coli, thereby increasing the possibility of screening out excellent strains.

[0016] 3. The screening process of the present invention is simple to operate, accurate and rapid, and provides a reference for the screening of other strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a verification diagram of the detection method of Example 1.

[0018] Figure 2 Flow chart of the method for rapid detection of polymyxin B fermentation broth.

[0019] Figure 3 This is the standard curve of polymyxin B sulfate content-absorbance value. DETAILED DESCRIPTION

[0020] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It will be understood that the specific embodiments described herein are only used to explain the relevant inventions, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the invention are shown in the accompanying drawings. It should be noted that, in the absence of 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 accompanying drawings and in conjunction with the embodiments.

[0021] Isolation and culture of single colonies: Take the strain to be screened and use an inoculation needle to pick up an appropriate amount of bacterial liquid on the slant solid culture medium on the clean bench, streak evenly and culture at 32°C. After the culture matures, add an appropriate amount of sterile water for gradient dilution and culture on the solid plate.

[0022] Well plate screening: Use 48-well plates instead of shake flasks, and reduce the volume of medium per well from 50 mL per shake flask to 1-1.2 mL. Use a sterile toothpick to gently pick half of the mature single colonies to the 48-well plate, and shake and culture at 32°C, 180 rpm for 56 hours.

[0023] Well plate detection: Weigh the polymyxin B standard, dissolve it in ultrapure water to prepare an antibiotic stock solution with a concentration of 1000U / mL, and then use ultrapure water to dilute the prepared polymyxin B antibiotic stock solution to solutions with concentrations of 0, 60, 70, 80, 90, 100, 110, and 120U / mL.

[0024] Take the E. coli cryopreservation tube and add LB medium. Adjust the absorbance of the bacterial solution to A. 600nm = 0.200-0.300. The mixed indicator bacteria Escherichia coli liquid was dispensed into two 96-well plates at a volume of 270 μL / well, for establishing a standard curve and fermentation liquid detection, respectively.

[0025] Establish a standard curve. Take a well plate with E. coli solution, add 30 μL of sterile standard solution of different concentrations and mix evenly, set 8 wells in a row of the well plate as parallel. At the same time, blank LB medium is used as blank control, and bacterial suspension without antibiotics is selected as positive control, and 30 μL of sterile ultrapure water is added to make up the volume. After culturing at 32℃ for 4 hours, use an enzyme reader to read the A 600nmThe absorbance is measured at the wavelength, and the absorbance value of each concentration of the standard solution is detected. The absorbance value is the horizontal coordinate x, and the solution concentration is the vertical coordinate y. The standard curve is drawn, and the linear relationship between x and y is added so that the correlation coefficient R 2 It can be used when ≥0.99.

[0026] Table 1 Standard curve establishment and sample addition method

[0027]

[0028] Fermentation broth detection. Pipette the supernatant of the fermentation broth after centrifugation, add sterile ultrapure water to dilute 100 times, and then pipette 30 μL of the dilution into another well plate with E. coli solution. Mix well, incubate at 32°C for 4 hours, and use an enzyme reader to read the A 600nm The absorbance was measured at wavelength.

[0029] Calculation of biological activity data. Substitute the detected absorbance data of the fermentation broth into the standard curve equation, and the calculated solution concentration multiplied by the dilution factor equals the biological activity data of the fermentation broth, thus realizing rapid detection of the fermentation broth.

[0030] Example 1: Validation of a fermentation broth for rapid detection of polymyxin B

[0031] Eight Paenibacillus polymyxa (strain number CICC 21779) fermentation broths with different product concentrations after liquid phase detection were selected, and the feasibility of the method was verified by well plate detection.

[0032] Well plate detection: Weigh the polymyxin B standard, dissolve it in ultrapure water to prepare an antibiotic stock solution with a concentration of 1000U / mL, and then use ultrapure water to dilute the prepared polymyxin B antibiotic stock solution to solutions with concentrations of 0, 60, 70, 80, 90, 100, 110, and 120U / mL.

[0033] Take an appropriate amount of E. coli cryopreservation tube and add it to LB medium so that the absorbance value of the medium is A 600nm =0.291. The mixed E. coli culture was dispensed into two 96-well plates at a volume of 270 μL / well, which were used to establish the standard curve and fermentation broth detection respectively.

[0034] Establish a standard curve. Take a well plate with E. coli solution, add 30 μL of sterile standard solution of different concentrations and mix evenly, set 8 wells in a row of the well plate as parallel. At the same time, blank LB medium is used as blank control, and bacterial suspension without antibiotics is selected as positive control, and 30 μL of sterile ultrapure water is added to make up the volume. After culturing at 32℃ for 4 hours, use an enzyme reader to read the A 600nmThe absorbance is measured at the wavelength, and the absorbance value of each concentration of the standard solution is detected. The absorbance value is the horizontal coordinate x, and the solution concentration is the vertical coordinate y. The standard curve is drawn, and the linear relationship between x and y is added so that the correlation coefficient R 2 The standard curve equation is y = -1041.1x + 364.39, R2 = 0.994.

[0035] Table 2 Standard curve equation to establish test values

[0036]

[0037]

[0038] Fermentation broth detection. Centrifuge the fermentation broth to obtain 30 μL of the supernatant, add sterile ultrapure water to dilute 100 times, and then take 30 μL of the dilution and add it to another well plate with E. coli solution. After mixing evenly, incubate at 32°C for 4 hours and use an enzyme reader to read the A 600nm The absorbance was measured at the wavelength. According to the analysis of the test data, the trend of the well plate rapid detection method is consistent with that of the liquid phase detection method, and there is a certain correlation.

[0039] Table 3 Detection method validation

[0040]

[0041] Example 2: A method for rapid detection of polymyxin B fermentation broth

[0042] Isolation and culture of single colonies: Take Bacillus polymyxa (strain number CICC 21779) and use an inoculation needle to pick up an appropriate amount of bacterial liquid on the slant solid culture medium on the clean bench, streak evenly and culture at 32°C. After the culture matures, add 50mL of sterile water for gradient dilution and culture on the solid plate.

[0043] Well plate screening: Prepare the fermentation medium and dispense it into a 48-well plate with a volume of 1-1.2 mL. Number the single colonies in the plate in the order of inoculation, use a sterile toothpick to gently pick half of the mature single colonies into a 48-well plate, and culture at 32°C, 180 rpm for 56 hours.

[0044] Well plate detection: Weigh the polymyxin B standard, dissolve it in ultrapure water to prepare an antibiotic stock solution with a concentration of 1000U / mL, and then use ultrapure water to dilute the prepared polymyxin B antibiotic stock solution to solutions with concentrations of 0, 60, 70, 80, 90, 100, 110, and 120U / mL.

[0045] Take an appropriate amount of E. coli cryopreservation tube and add it to LB medium so that the absorbance value of the medium is A 600nm=0.247. The mixed E. coli solution was dispensed into two 96-well plates at a volume of 270 μL / well, which were used to establish a standard curve and fermentation solution detection respectively.

[0046] Establish a standard curve. Take a well plate with E. coli solution, add 30 μL of sterile standard solution of different concentrations and mix evenly, set 8 wells in a row of the well plate as parallel. At the same time, blank LB medium is used as blank control, and bacterial suspension without antibiotics is selected as positive control, and 30 μL of sterile ultrapure water is added to make up the volume. After culturing at 32℃ for 4 hours, use an enzyme reader to read the A 600nm The absorbance is measured at the wavelength, and the absorbance value of each concentration of the standard solution is detected. The absorbance value is the horizontal coordinate x, and the solution concentration is the vertical coordinate y. The standard curve is drawn, and the linear relationship between x and y is added so that the correlation coefficient R 2 The standard curve equation is y = -543.33x + 191.8, R2 = 0.9908.

[0047] Table 4 Standard curve equation to establish test values

[0048]

[0049] Fermentation broth detection. Pipette the supernatant of the fermentation broth after centrifugation, add sterile ultrapure water to dilute 100 times, and then pipette 30 μL of the dilution into another well plate with E. coli solution. Mix well, incubate at 32°C for 4 hours, and use an enzyme reader to read the A 600nm The absorbance was measured at wavelength.

[0050] Table 5 Well plate fermentation broth test values

[0051]

[0052] Calculation of biological activity data. Substitute the detected absorbance data of the fermentation broth into the standard curve equation, and the calculated solution concentration multiplied by the dilution factor equals the biological activity data of the fermentation broth, which can achieve rapid detection of the fermentation broth and improve detection efficiency.

[0053] Table 6 Calculated values ​​of biological activity

[0054]

[0055] As can be seen from the above examples, the present invention provides a method for rapid detection of polymyxin B fermentation broth. The method transforms the traditional shake flask culture of the strain into a high-throughput well plate culture, and uses biological turbidimetry to replace the high-performance liquid phase instrument to achieve rapid and large-scale detection, confirming a complete set of well plate rapid detection methods, greatly improving the screening efficiency of polymyxin Bacillus and the detection efficiency of fermentation broth.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapid detection of polymyxin B fermentation broth, characterized in that: The method comprises the following steps: (1) Single colony culture: After the polymyxa bacillus is cultured to maturity on a slant medium, sterile water is added to scrape off the bacteria to make a bacterial suspension, which is then graded and spread on a solid culture dish for single colony isolation and culture. After the culture is mature, the outer surface of the culture dish is numbered and marked; (2) Initial screening of well plates: Use 48-well plates to ferment mature single colonies to obtain fermentation broth, and use 96-well plates to quickly detect the product concentration in the fermentation broth by bioturbidimetry.

2. A method for rapid detection of polymyxin B fermentation broth according to claim 1, characterized in that: In step (2), the volume of culture medium per well is 1-1.2 mL, and half of the mature single colonies are picked up with a sterile toothpick to a 48-well plate, and cultured at 32° C. and 180 rpm for 56 hours under shaking conditions.

3. A method for rapid detection of polymyxin B fermentation broth according to claim 1, characterized in that: In the step (2), the biological turbidimetry method uses Escherichia coli as the indicator bacteria.

4. A method for rapid detection of polymyxin B fermentation broth according to claim 1, characterized in that: The rapid detection in step (2) consists of the following steps: (1) Weigh the polymyxin B standard, dissolve it in ultrapure water to prepare an antibiotic stock solution with a concentration of 1000 U / mL, and then dilute the prepared polymyxin B antibiotic stock solution with ultrapure water to solutions with concentrations of 0, 60, 70, 80, 90, 100, 110, and 120 U / mL respectively; (2) Take the E. coli cryopreservation tube and add LB medium to adjust the absorbance of the bacterial solution to A. 600nm =0.200-0.300, the mixed indicator bacteria Escherichia coli liquid was dispensed into two 96-well plates at a volume of 270 μL / well, for establishing a standard curve and fermentation liquid detection respectively; (3) To establish a standard curve, take a well plate with E. coli solution, add 30 μL of sterile standard solution of different concentrations and mix them evenly. Set 8 wells in a row of the well plate as parallel. At the same time, blank LB medium is used as the blank control, and bacterial suspension without antibiotics is selected as the positive control. Add 30 μL of sterile ultrapure water to make up the volume. After incubation at 32°C for 4 h, use an enzyme reader to read the A 600nm The absorbance was measured at the wavelength, and the absorbance value of each concentration of the standard solution was detected. The absorbance value was taken as the horizontal coordinate x and the solution concentration was taken as the vertical coordinate y to draw a standard curve. The linear relationship between x and y was added so that the correlation coefficient R 2 ≥0.99 can be used; (4) Fermentation broth detection: After centrifugation, the supernatant of the fermentation broth was aspirated and diluted 100 times with sterile ultrapure water. 30 μL of the dilution was then aspirated and added to another well plate with E. coli solution. After mixing evenly, the well plate was incubated at 32°C for 4 h and the A 600nm The absorbance is measured at the wavelength; (5) Calculation of biological activity data: Substitute the absorbance data of the fermentation broth obtained by the test into the standard curve equation. The calculated solution concentration multiplied by the dilution factor is equal to the biological activity data of the fermentation broth.

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