Facultative anaerobic microorganism culture method based on microwell plate and application of facultative anaerobic microorganism culture method

Abstract

The invention belongs to the technical field of microorganism culture, and discloses a facultative anaerobic microorganism culture method based on a microwell plate and application of the facultative anaerobic microorganism culture method. The method comprises the following steps: 1) uniformly mixing gellan gum and a culture medium, and melting to obtain a suspension culture medium; diluting the facultative anaerobic microorganisms subjected to amplification culture and a suspension culture medium to obtain indicator bacterium suspension; and (2) adding a phosphoric acid solution containing glucose into the outermost circle of holes of the microwell plate, adding the indicator bacterium suspension into the inner holes, adding a glucose oxidase solution into the outermost circle of holes, sealing, standing and culturing. According to the invention, the problem of reading error caused by fringe effect and thallus sedimentation in traditional microplate culture is solved, the reliability of data is remarkably improved, and the cost and the operation complexity are reduced. The method disclosed by the invention is used for detecting the activity of the antibacterial substance of the facultative anaerobic bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial cultivation, and in particular relates to a microplate-based facultative anaerobic microbial cultivation method and application. Background Art

[0002] As a model organism, facultative anaerobic microorganisms are easy to operate and have metabolic diversity, making them an indispensable tool for antibacterial activity detection. Through a variety of detection methods, the antibacterial effect of compounds can be evaluated efficiently and accurately, providing an important basis for the development and clinical application of antibacterial drugs. Microplates are used as culture containers, and the changes in the absorbance value of each well before and after the cultivation of facultative anaerobic microorganisms are determined by turbidimetry to achieve the purpose of high-throughput detection.

[0003] Due to the structure of the microplate and the characteristics of turbidimetric detection, the following problems exist in the process of using microplates to culture facultative anaerobic microorganisms: (1) Bacterial sedimentation: Microorganisms tend to settle to the bottom of the wells during static culture, resulting in low absorbance (OD value) measurements or unstable readings, especially during long-term culture or high-density culture. (2) Edge effect: Due to differences in temperature, humidity and evaporation rates, the growth rate of microorganisms in the outer wells of traditional microplates is inconsistent with that in the central wells, resulting in significant deviations in detection data (such as OD value and fluorescence intensity). (3) Insufficient control of facultative anaerobic conditions: Conventional microplates make it difficult to accurately control oxygen concentration, which affects the metabolic state of facultative anaerobic microorganisms.

[0004] The research on the facultative anaerobic microorganism cultivation method mainly focuses on the modification of the cultivation device, such as: Chinese utility model with authorization announcement number CN218755761U, title: A facultative anaerobic microorganism cultivation device; Chinese utility model with authorization announcement number CN218058988U, title: Cultivation device for facultative anaerobic antimony oxidizing bacteria and strictly anaerobic antimony oxidizing bacteria. No relevant literature on the facultative anaerobic microorganism cultivation method is found.

[0005] The research on detecting antibacterial active substances based on microplates mainly focuses on: ① increasing the number of single detections, i.e., high-throughput detection: designing high-throughput turbidimetric detection methods, such as large-scale screening technologies based on 96-well plates or 384-well plates, which can simultaneously detect the antibacterial activities of a large number of samples and accelerate the screening speed of antibacterial active substances. For example, Chinese Invention Publication No. CN117737185A discloses a high-throughput screening method for antibacterial activity, including taking a culture plate, setting a negative control group, a positive control group, and a drug experimental group for the target strain in the culture plate to obtain a sample-carrying plate; culturing the strain on the sample-carrying plate to make the target strain reach a preset concentration; and detecting the antibacterial activity of the cultured sample-carrying plate, which can achieve high-throughput detection. ② Combining with other methods to shorten the detection time, improve the detection sensitivity and accuracy. For example, Chinese Patent Invention No. CN201110127094.5 discloses a method for determining the minimum inhibitory concentration of a drug. This invention selects two high-quality fluorescent dyes and uses fluorescence method to determine the minimum inhibitory concentration of the drug against Enterococcus, successfully shortening the detection time to 4h, and solving the problems of long culture time in the traditional broth dilution method and the artificial factor differences in visual judgment results for easily sedimented bacteria.

[0006] Regarding the sedimentation of bacteria, it mostly relies on shaking culture, but this may interfere with the facultative anaerobic environment and increase the complexity of the equipment. Moreover, due to the small volume of a single well in the microplate, relatively intense shaking cannot be carried out, so shaking culture is not applicable. To make the measurement results more accurate, some experimenters will use a pipette to blow and mix the liquid to be measured before detection, but this method is cumbersome to operate and prone to generating bubbles. Due to the scattering and refraction of bubbles, the overall optical uniformity of the liquid to be measured is changed, making the measurement of absorbance values unstable and inaccurate. In addition, the position and number of bubbles in the liquid are often uncertain, and the state of bubbles may be different each time of measurement. This will lead to differences in absorbance values obtained each time of measurement, reducing the reproducibility and accuracy of the measurement. Regarding the edge effect, currently, it is often solved by discarding the peripheral wells or data correction, but this method only solves the "edge effect" caused by heat transfer and cannot solve the influence brought by the oxygen concentration difference.

[0007] In summary, the existing research does not consider the problems of uneven distribution of bacteria in the statically cultured microplate and the detection errors and poor repeatability caused by the "edge effect", nor does it consider the growth conditions of facultative anaerobic microorganisms under different oxygen concentrations.

[0008] Therefore, it is necessary to establish a microplate-based facultative anaerobic microorganism culture method. This method is used for detecting the activity of antibacterial substances against facultative anaerobic bacteria, which can not only eliminate the measurement errors caused by uneven distribution of bacteria but also eliminate the uneven growth caused by the "edge effect". This method is used for detecting the activity of antibacterial substances against facultative anaerobic bacteria, with simple operation, accurate results and high-throughput culture. Summary of the Invention

[0009] In order to solve the problems of uneven distribution of bacteria in the static-cultured microplate and the detection errors and poor repeatability caused by the "edge effect", and to establish a method with simple operation, accurate results and high-throughput screening of antibacterial active substances, the present invention provides a microplate-based facultative anaerobic microorganism culture method and its application. In the outermost culture wells (i.e., edge wells) of the microplate, a glucose oxidase solution and a glucose solution are added, and the concentration of oxygen in the plate is regulated by adjusting the concentration of glucose; the suspension medium containing gellan gum is diluted with the bacterial solution to obtain a bacterial suspension; then the bacterial suspension is added to the internal wells and cultured statically. The method of the present invention enables the sensitive indicator bacteria to be suspended and grow stably in the medium, eliminates the influence of the "edge effect" on the growth of facultative anaerobic microorganisms, and at the same time eliminates the detection errors caused by the deposition of bacteria at the bottom of the liquid culture container, achieving the purpose of high-throughput and accurate detection of the growth of facultative anaerobic microorganisms. The method of the present invention is used for detecting the activity of antibacterial substances against facultative anaerobic bacteria.

[0010] The object of the present invention is achieved by the following technical solutions.

[0011] A microplate-based facultative anaerobic microorganism culture method includes the following steps: 1) Mix and melt gellan gum with the medium of facultative anaerobic microorganisms to obtain a suspension medium; dilute the facultative anaerobic microorganisms after enlarged culture with the suspension medium to obtain an indicator bacterial suspension; 2) Add a phosphoric acid solution containing glucose to the outermost culture wells of the microplate, add the indicator bacterial suspension to the internal wells of the microplate, then continue to add a glucose oxidase solution to the outermost culture wells of the microplate, and seal the microplate; 3) Culture statically.

[0012] The gellan gum in step 1) is deacylated gellan gum or low-acyl gellan gum, and the content of the gellan gum in the suspension medium is 0.2 g / L to 0.4 g / L.

[0013] The facultative anaerobic microorganisms are Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida.

[0014] The viable count of the indicator bacterial suspension is 104 ~10 7 CFU / mL; that is, the facultative anaerobic microorganism to be subcultured is diluted with a suspension medium so that the viable count after dilution is 10 4 ~10 7 CFU / mL.

[0015] In the phosphoric acid solution containing glucose described in step 2), the glucose concentration is 50 g / L to 300 g / L. The phosphoric acid solution containing glucose is obtained by dissolving glucose in a phosphate buffer solution with a concentration of 0.02 mol / L to 0.05 mol / L and a pH of 4.0 to 7.0.

[0016] The concentration of the glucose oxidase solution is 0.5 g / L to 50 g / L. The glucose oxidase solution is obtained by dissolving glucose oxidase in a phosphate buffer solution with a concentration of 0.02 mol / L to 0.05 mol / L and a pH of 5.0 to 7.0.

[0017] The volume ratio of the phosphoric acid solution containing glucose to the glucose oxidase solution is 50 to 100:50 to 100, preferably 1:1. The amount of the phosphoric acid solution containing glucose added to the culture well is 50 to 100 μL, and the amount of the indicator bacteria suspension added to the culture well is 50 to 150 μL.

[0018] The conditions for static culture described in step 3) are to culture in an incubator at 28 to 45 °C for 4 to 72 h.

[0019] The melting temperature described in step 1) is 90 to 100 °C. The suspension medium described in step 1) specifically refers to mixing gellan gum with the medium of facultative anaerobic microorganisms, stirring and heating at 90 to 100 °C until melted to be transparent and clear, sterilizing, and cooling. Sterilization conditions: sterilize at 115 to 121 °C for 15 to 30 min.

[0020] The facultative anaerobic microorganism for subculture refers to inoculating the strain of facultative anaerobic microorganism into the sterilized medium, culturing at 28 to 45 °C for 6 to 16 h to obtain an activated bacterial liquid; taking the activated bacterial liquid and inoculating it again into a fresh medium for subculture at 28 to 45 °C.

[0021] The subculture is static culture or shaking culture (rotation speed is 1 to 250 rpm) for 8 to 24 h.

[0022] The method of the present invention is used for detecting the activity of antibacterial substances of facultative anaerobes, including high-throughput screening of antibacterial substances and determination of the minimum inhibitory concentration.

[0023] A method for detecting the activity of antibacterial substances of facultative anaerobes includes the following steps: S1. Mix and melt gellan gum with the medium of facultative anaerobic microorganisms to obtain a suspension medium. S2. Dilute the facultative anaerobic microorganisms after enlarged cultivation with the suspension medium to obtain an indicator bacteria suspension. S3. After preparing the bacteriostatic substance into a stock solution, dilute it with the suspension medium to obtain a bacteriostatic substance suspension. S4. Divide the internal holes of the microplate except the outermost ring holes into a test area, a bacterial liquid growth control area, and a negative control area; add a phosphoric acid solution containing glucose to the outermost ring holes of the microplate, add the suspension medium to the other internal holes, then add the bacteriostatic substance suspension to the holes in the test area, then add the indicator bacteria suspension to the holes in the test area and the holes in the bacterial liquid growth control area, add the suspension medium to the holes in the negative control group area, and finally add glucose oxidase solution to the outermost ring culture holes, and seal the microplate. S5. Incubate statically. S6. Measure the absorbance values of each well at 550 - 600 nm.

[0024] In step S4, the bacteriostatic substance suspension forms different concentrations in the holes of the test area by dilution with the suspension medium.

[0025] Specifically, the bacteriostatic substance suspension in the holes of the test area is serially diluted with the suspension medium so that the bacteriostatic substance forms a concentration gradient in the holes.

[0026] The serial dilution is specifically carried out by the two-fold serial dilution method.

[0027] The holes in the test area are divided into area one and area two. The bacteriostatic substance suspension in the holes of area one is serially diluted with the suspension medium so that the bacteriostatic substance forms a concentration gradient; the holes in area two are parallel test holes, i.e., parallel holes; each hole in area one corresponds to a concentration, and there is at least one parallel hole (such as 1 - 4 parallel holes) in area two for each concentration.

[0028] Specifically, in the test area, the bacteriostatic substance suspension in the holes of one row is serially diluted with the suspension medium so that the bacteriostatic substance forms a concentration gradient in the holes; there are one row, two rows, three rows, or four rows of holes as parallel holes, and the concentration of the bacteriostatic substance in the parallel holes is the same as that in the previous concentration gradient. For example, the bacteriostatic substance suspension in the holes of the first row is serially diluted with the suspension medium so that the bacteriostatic substance forms a concentration gradient in the holes, and the concentration of the bacteriostatic substance in 1 - 4 rows of the holes in the second row, the third row, the fourth row, and the fifth row is the same as that in the first row.

[0029] In step S6, according to the absorbance values, plot a curve with the concentration of the bacteriostatic substance as the abscissa and the OD600 value as the ordinate, and the inflection point is the minimum inhibitory concentration.

[0030] The microplate described in step S4 is a 96-well plate, 48-well plate, 24-well plate, 384-well plate, or 1536-well plate.

[0031] The concentration of the antibacterial substance suspension described in step S3 is 200 - 1000 μg / mL.

[0032] The conditions in steps S1 and S2 are the same as those defined in the previous facultative anaerobic microorganism culture method.

[0033] The phosphoric acid solution containing glucose and the glucose oxidase solution in step S4 are the same as those defined in the previous facultative anaerobic microorganism culture method.

[0034] The conditions for static culture in step S5 are to place it in an incubator at 28 - 45°C for 4 - 72 h.

[0035] In the present invention, gellan gum is added to the liquid medium. After heating and hydration, gellan gum forms a double helix structure and further aggregates and crosslinks through interactions such as hydrogen bonds and van der Waals forces to form a three-dimensional network structure. Suspended macromolecular particles (such as macronutrients and microorganisms) are captured by this three-dimensional network structure and restricted in the pores of the network, unable to freely settle and aggregate, thus achieving a good suspension effect. This network structure can withstand a certain external force, maintain the stability of the system, prevent particles from sinking due to gravity, and thus achieve the suspension culture of microbial cells, eliminating the detection error caused by uneven distribution of cells. In addition, by adjusting the concentration of gellan gum, the liquid has a certain fluidity while ensuring the suspension of particles, and small molecules such as water can freely flow in the gaps of the three-dimensional network. In the outermost ring of the microplate in the present invention, a phosphoric acid solution containing glucose oxidase and glucose is added. By using the reaction of glucose oxidase catalyzing the combination of glucose and oxygen to generate gluconic acid, the concentration of oxygen is regulated by adjusting the concentration of glucose, thereby regulating the growth of facultative anaerobes; at the same time, the outermost ring of holes is filled with the solution, which can ensure the uniformity of the temperature inside the holes, and finally achieve the purpose of eliminating the "edge effect" of the microplate and regulating the oxygen concentration inside the microplate.

[0036] Compared with the prior art, the present invention has the following advantages and effects: The present invention utilizes the characteristic of gellan gum to form a three-dimensional network at low concentrations to prepare a suspension medium. Compared with biological polysaccharides such as agar and carrageenan, gellan gum has the advantages of anti-microbial utilization, high gel transparency, good thermal stability, and a wide applicable pH range. The suspension medium prepared by the present invention, different from ordinary agar gels, has a certain fluidity at room temperature, facilitating experimental operations. The three-dimensional network structure formed by gellan gum can suspend macromolecules such as microbial cells and proteins, preventing them from settling; for small molecules such as water, their movement is not restricted. This enables microorganisms to grow uniformly in the medium, avoiding sedimentation and stratification phenomena, and also maintaining the uniform light transmission of the sample. In addition, for the growth of facultative anaerobic microorganisms affected by oxygen concentration and the "edge effect", the present invention sets glucose oxidase and glucose solution in the outermost circle of the microplate. Through a catalytic reaction, the oxygen in the peripheral wells is consumed and the evaporation rate is balanced, reducing the edge effect, thereby maintaining the consistency of the growth conditions of facultative anaerobic microorganisms.

[0037] The present invention requires no special instruments and reagents, uses common and inexpensive reagents, realizes the suspension culture of facultative anaerobic bacteria in a liquid medium, simultaneously eliminates the "edge effect" and the influence of oxygen concentration on the growth of facultative anaerobic indicator bacteria, solves the problems of complex operation, small single detection volume, and large result error in the traditional microplate culture method, is applicable to high-throughput detection scenarios, significantly improves data reliability, and reduces costs and operation complexity. Description of the Drawings

[0038] Figure 1 Schematic diagram of sample addition in the microplate for Example 1; Figure 2 Positions of each well in the microplate for Examples 1-2 and Comparative Examples 1-3; Figure 3 Well distribution of each group for Example 3 and Comparative Example 4; Figure 4 Schematic diagram of two-fold serial dilution for Example 3. Detailed Description of the Invention

[0039] The following further specifically details the present invention in conjunction with specific embodiments, but the implementation manners of the present invention are not limited thereto.

[0040] Example 1: Static suspension culture of Escherichia coli in a 96-well plate Step 1: According to the cultivation requirements of Escherichia coli, prepare 500 mL of LB liquid medium. The composition of the medium is as follows: tryptone: 5.0 g, yeast extract: 2.5 g, sodium chloride: 2.5 g. After dissolving in water, adjust the pH to 7.0 ± 0.2 and make up the volume to 500 mL; Weigh 0.20 g of deacylated gellan gum (the addition amount of gellan gum is 0.4 g / L), add it to the above-mentioned culture medium, disperse it evenly, heat and melt it to transparency and clarity at 90 - 100 °C, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121 °C for 20 min. After cooling, a suspension culture medium is obtained; Step 2: Take 50 μL of Escherichia coli stored in a glycerol tube, inoculate it into a 10 mL sterilized ordinary LB medium test tube, place it in a shaker at 37 °C and 180 rpm, and culture it for 12 - 16 h to obtain an activated bacterial solution; Take 500 μL of the activated strain and inoculate it into 100 mL of fresh ordinary LB medium again, and expand the culture for 12 h under the shaker conditions of 37 °C and 180 rpm to obtain an Escherichia coli bacterial suspension; Dilute the activated Escherichia coli bacterial suspension and the suspension culture medium prepared in Step 1 so that the number of viable bacteria after dilution is 1×10 4 CFU / mL, which is the test bacterial suspension; Step 3: Take a 96-well plate, add 100 μL of 300 g / L glucose phosphate solution (the solvent is PBS solution with 0.05 mol / L and pH 5.0) to the outermost ring of wells in the culture plate, and add the diluted test bacterial suspension to other positions (inner wells, i.e., test wells) in the 96-well plate; Then add 100 μL of 50 g / L glucose oxidase phosphate solution (the solvent is PBS solution with 0.05 mol / L and pH 5.0) to the outermost ring of wells in the culture plate; Finally, cover the lid and seal it with a sealing tape on the outer ring of the microplate; Step 4: Place the above-mentioned sample-loaded microplate in an incubator at 37 °C, and use a microplate reader to measure the absorbance values of each well at 600 nm at 0 h, 4 h, 24 h, 48 h, and 72 h respectively.

[0041] In this example, the schematic diagram of adding samples to the microplate is as Figure 1 shown. The solution containing glucose oxidase is added to the outermost ring of wells in the culture plate finally, and the test wells at other positions are added with the test bacterial suspension, Figure 1 and the black circle part in it is the well added with the solution containing glucose oxidase, and the white circle is the test well added with the test bacterial suspension.

[0042] The positions of each well in the microplate of this example are as Figure 2 shown. The microplate includes peripheral wells (i.e., the outermost ring of wells), inner ring wells 1, inner ring wells 2, and a central well.

[0043] Calculate the OD600 increase values of each well at 4 h, 24 h, 48 h, and 72 h. The results are shown in Tables 1 to 4. Compared with Comparative Example 1 (adding phosphoric acid aqueous solution to the outermost circle of a 96-well plate and using ordinary LB medium) and Comparative Example 2 (using ordinary LB medium and inoculating Escherichia coli in all 96 wells), the absorbance values of the Escherichia coli samples in Example 1 after culturing for 4 h, 24 h, 48 h, and 72 h are more stable than those of the other two, and both the variance and coefficient of variation are smaller than those of the comparative examples, indicating that in Example 1, the detection data stability of the Escherichia coli absorbance value is better and the error is small. Comparing the samples at different positions of the 96-well plate, the data deviation between the central well and the inner circle wells in Example 1 is small (the positions of the wells in the microplate in this example are as Figure 2 shown), while the data deviation in Comparative Example 1 and Comparative Example 2 is large (coefficient of variation CV > 5%), indicating that the OD600 data is affected by the position of the well plate where it is located, and Example 1 of the present invention can eliminate this influence.

[0044] Table 1 Absorbance values (△OD600) of Escherichia coli samples after culturing for 4 h △OD600 = OD600(4 h) - OD600(0 h)

[0045] Table 2 Absorbance values (△OD600) of Escherichia coli samples after culturing for 24 h △OD600 = OD600(24 h) - OD600(0 h)

[0046] Table 3 Absorbance values (△OD600) of Escherichia coli samples after culturing for 48 h △OD600 = OD600(48 h) - OD600(0 h)

[0047] Table 4 Absorbance values (OD600) of Escherichia coli samples after culturing for 72 h △OD600 = OD600(72 h) - OD600(0 h)

[0048] Example 2: Static suspension culture of Staphylococcus aureus in a 96-well plate Step 1: According to the culture requirements of Staphylococcus aureus, prepare 1000 mL of nutrient broth medium (NB). The composition of the medium is as follows: peptone: 10 g (w / v), beef extract powder: 3.0 g (w / v), sodium chloride: 5.0 g (w / v). After dissolving in water, adjust the pH to 7.4 ± 0.2 and make up the volume to 1000 mL; Weigh 0.10 g of deacylated gellan gum (the addition amount of gellan gum is 0.2 g / L), add it to the above-mentioned culture medium, disperse it evenly, heat and melt it to transparency and clarity at 90 - 100 °C, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121 °C for 20 min. After cooling, a suspension culture medium is obtained; Step 2: Take 50 μL of Staphylococcus aureus stored in a glycerol tube, inoculate it into a 10 mL sterilized ordinary NB culture medium test tube, place it in a shaker at 37 °C and 150 rpm, and culture it for 12 - 16 h to obtain an activated bacterial solution; Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary NB culture medium, and continue to culture it for 8 hours under the conditions of a shaker at 37 °C and 150 rpm to obtain a Staphylococcus aureus bacterial suspension; Dilute the activated Staphylococcus aureus bacterial suspension and the suspension culture medium so that the number of viable bacteria after dilution is 1×10 5 CFU / mL, which is the test bacterial suspension; Step 3: Take a 96-well plate, leave out the outermost circle of the culture plate, and add 100 μL of 100 g / L glucose phosphate solution (the solvent is a phosphate solution with 0.04 mol / L and pH 5.5) to the outer peripheral wells; Add the diluted test bacterial suspension (the positions of each well are as Figure 2 shown) to other positions (inner circle well 1, inner circle well 2, central well) of the 96-well plate. Subsequently, add 100 μL of 1.0 g / L glucose oxidase phosphate solution (the solvent is a phosphate solution with 0.04 mol / L and pH 5.5) to the outermost circle wells, that is, the outer peripheral wells, cover the lid and seal it with a sealing tape on the outer circle of the microplate; In this example, the positions of each well in the microplate are as Figure 2 shown; Step 4: Place the above sample plate in an incubator at 37 °C and culture it. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance values of each well at 600 nm at 0 h and 48 h respectively.

[0049] In this example, the positions of each well in the microplate are as Figure 2 shown. The microplate includes outer peripheral wells, inner circle well 1, inner circle well 2, and central well.

[0050] The results are shown in Table 5. Compared with Comparative Example 3 (using ordinary NB culture medium, and inoculating Staphylococcus aureus in all 96 wells), the absorbance value of the sample after culturing Staphylococcus aureus for 48 h in Example 2 is slightly higher than that in Comparative Example 3, and the variance and coefficient of variation are much smaller than those in the comparative example, indicating that in Example 2, the growth of Staphylococcus aureus is better, and the stability of the detected data is better with small errors. Comparing the absorbance values at different positions in the plate, the uniformity of the absorbance values of each well in Example 2 is good; for the absorbance values of the wells at different positions in Comparative Example 3, there is a large difference, and the coefficient of variation is 16.73% > 5%, indicating a high degree of data dispersion.

[0051] Absorbance values of samples after 48 h of Staphylococcus aureus culture △OD600 = OD600 (48 h) - OD600 (0 h)

[0052] Example 3: Determination of the minimum inhibitory concentration of nisin against Enterococcus faecalis Step 1: Prepare 500 mL of LB liquid medium according to the culture requirements of Enterococcus faecalis. According to the mass - volume ratio, the composition of the medium is as follows: Tryptone: 5.0 g (w / v), Yeast extract: 2.5 g (w / v), Sodium chloride: 2.5 g (w / v). After dissolving in water, adjust the pH to 7.0 ± 0.2 and make up the volume to 500 mL; Weigh 0.15 g of deacylated gellan gum (the addition amount of gellan gum is 0.3 g / L), add it to the above - mentioned medium, heat it to transparency and clarity at 90 - 100 °C, dispense it into test tubes and Erlenmeyer flasks, sterilize it at 115 °C for 30 min, and cool it to obtain a suspension medium; Step 2: Weigh 0.2000 g of Nisin (nisin) powder and dissolve it in 100 mL of sterile triple - distilled water to prepare a Nisin mother liquor with a concentration of 2000 μg / mL. Filter and sterilize it with a sterile 0.22 μm fiber filter membrane in a laminar flow hood and then dispense it, and store it at - 20 °C; When conducting the experiment, take it out and thaw it, and dilute it to 400 μg / mL with the suspension medium in Step 1; Step 3: Take 50 μL of Enterococcus faecalis preserved in a glycerol tube, inoculate it into a 10 mL sterilized ordinary LB medium test tube, place it in a shaker at 37 °C and 180 rpm for 6 - 8 h to obtain an activated bacterial solution; Take 500 μL of the activated strain and inoculate it into 100 mL of fresh ordinary LB medium again, and expand the culture under the conditions of a shaker at 37 °C and 180 rpm to obtain an Enterococcus faecalis bacterial suspension; Dilute the activated Enterococcus faecalis bacterial suspension with the suspension medium in Step 1 so that the number of viable bacteria after dilution is 1×10 7 CFU / mL, which is the test indicator bacterial suspension; Step 4: Take a 96 - well plate, add 100 μL of 50 g / L glucose phosphate solution (the solvent is PBS solution with 0.02 mol / L and pH 7.0) to the outermost - circle wells, and add 100 μL of suspension medium to other wells (test wells, negative control wells, bacterial solution growth control wells); In this example, the well distribution of each group is as Figure 3 shown, where the numbers B2 - B11, C2 - C11, D2 - D11, E2 - E11 are experimental groups, F2 - F11 are set as bacterial solution growth control groups, and G2 - G11 are set as negative control groups; Step 5: Take 100 μL of Nisin at 400 μg / mL in Step 2 and add it to well B2. Use the two-fold serial dilution method (that is, each dilution reduces the concentration of the previous solution to 1 / 2 of the original, thus forming a series of concentration gradients with equal ratios), and perform gradient dilution on wells B3 - B11 with the suspension medium (see Figure 4 ). The Nisin concentrations in wells B2 - B11 are shown in the Nisin concentrations in Table 6; perform 4 parallel operations for each sample, that is, the operations for C2 - C11, D2 - D11, and E2 - E11 are the same as those for B2 - B11; the schematic diagram of the two-fold serial dilution is as Figure 4 shown; Step 6: In some of the inner wells of the 96-well plate, such as wells numbered F2 - F11, serve as the wells for the bacterial liquid growth control group, and wells numbered G2 - G11 serve as the wells for the negative control group; add 100 μL of Enterococcus faecalis bacterial suspension to the bacterial liquid growth control group and the experimental group; add 100 μL of sterile suspension medium to the negative control group; finally, add 100 μL of 0.5 g / L glucose oxidase phosphate solution (the solvent is 0.02 mol / L phosphate buffer solution with pH 7.0) to the outer wells, and stick on the sealing tape; Step 7: Incubate the above sample plate in an incubator at 37°C; according to the OD value measurement method, use an enzyme-linked immunosorbent assay (ELISA) reader to read and record the OD600 value of each well before and after 24 h of incubation in the 96-well plate, and then calculate the difference in the OD600 value before and after the drug action. The drug action concentration in the wells with an OD value change less than 0.05 is the minimum inhibitory concentration (MIC).

[0053] In this example, the well distribution of each group is as Figure 3 , the glucose oxidase phosphate solution is added to the outermost circle of wells in the microplate at the end. The outermost circle of wells is denoted as the glucose oxidase solution wells. The wells in the microplate are also divided into test wells, negative control wells, and bacterial liquid growth control wells.

[0054] Figure 4 It is the schematic diagram of the two-fold serial dilution in Example 3.

[0055] The results are shown in Table 6. The minimum inhibitory concentrations of Nisin in Example 3 and Comparative Example 4 are the same, both being 6.25 μg / mL. The CV values of the absorbance values in the examples are all less than those in the comparative examples, indicating that the data in the examples have good stability and small deviations; when the concentration of Nisin is lower than the MIC, the absorbance values in the examples are greater than those in the comparative examples, indicating that in the examples of the present invention, the growth condition of Staphylococcus aureus is better than that in the comparative examples.

[0056] Table 6 Comparison table of data before and after 24 h of culture in Example 3 and Comparative Example 4

[0057] In the table: ΔOD600 = OD600 (24 h) - OD600 (0 h) Note: CV is the coefficient of variation, that is, CV = standard deviation / mean * 100%, which is used to reflect the stability of the data. It is determined that CV ≥ 5% means the data is unstable.

[0058] Comparative Example 1: Static culture of Escherichia coli in a 96-well plate (Method 1) Step 1: According to the cultivation requirements of Escherichia coli, prepare 500 mL of LB liquid medium. The composition of the medium is as follows: Tryptone: 5.0 g (w / v), Yeast extract: 2.5 g (w / v), Sodium chloride: 2.5 g (w / v). After dissolving in water, adjust the pH to 7.0 ± 0.2 and make up the volume to 500 mL; After dissolving the above medium, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121 °C for 20 min. After cooling, obtain the suspended medium; Step 2: Take 50 μL of Escherichia coli preserved in a glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube. Place it in a shaker at 37 °C and 180 rpm for 12 - 16 h to obtain an activated bacterial solution; Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture for 12 h under the conditions of a shaker at 37 °C and 180 rpm; Dilute the activated Escherichia coli bacterial suspension with the suspended medium prepared in Step 1 so that the number of viable bacteria after dilution is 1×10 4 CFU / mL, which is the bacterial suspension to be tested; Step 3: Take a 96-well plate, leave out the outermost circle (peripheral wells) of the culture plate, and add 200 μL of phosphoric acid solution (0.05 mol / L, pH 5.0); Add the diluted bacterial suspension to be tested to other positions of the 96-well plate (inner circle well 1, inner circle well 2, and central well), cover the lid and seal it with sealing tape on the outer circle of the microplate; Step 4: Place the above sample plate in an incubator at 37 °C, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance values of each well at 600 nm at 0 h, 24 h, 48 h, and 72 h respectively.

[0059] In this comparative example, the positions of each well in the microplate are as Figure 2 shown. The microplate includes peripheral wells, inner circle well 1, inner circle well 2, and central well.

[0060] Comparative Example 2: Static culture of Escherichia coli in a 96-well plate (Method 2) Step 1: Prepare 500 mL of LB liquid medium according to the cultivation requirements of Escherichia coli. The composition of the medium is as follows: Tryptone: 5.0 g (w / v), Yeast extract: 2.5 g (w / v), Sodium chloride: 2.5 g (w / v). After dissolving in water, adjust the pH to 7.0 ± 0.2, and make up the volume to 500 mL; After dissolving the above medium, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121°C for 20 min. After cooling, obtain the suspension medium; Step 2: Take 50 μL of Escherichia coli preserved in a glycerol tube and inoculate it into a 10 mL sterilized ordinary LB medium test tube. Incubate it in a shaker at 37°C and 180 rpm for 12 - 16 h to obtain the activated bacterial liquid. Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary LB medium, and expand the culture under the conditions of a shaker at 37°C and 180 rpm for 12 h. Dilute the activated Escherichia coli suspension with the suspension medium prepared in Step 1 so that the number of viable bacteria after dilution is 1×10 4 CFU / mL, which is the test bacterial suspension; Step 3: Take a 96-well plate and add the diluted test bacterial suspension to all wells (peripheral wells, inner ring wells, and central well). Cover the plate and seal it with sealing tape on the outer circle of the microplate; Step 4: Incubate the above sample plate in an incubator at 37°C, and use a microplate reader to measure the absorbance values at 600 nm of each well at 0 h, 24 h, 48 h, and 72 h respectively.

[0061] In this comparative example, the positions of each well in the microplate are as Figure 2 shown. The microplate includes peripheral wells, inner ring well 1, inner ring well 2, and central well.

[0062] Comparative Example 3: Static culture of Staphylococcus aureus in a 96-well plate Step 1: Prepare 1000 mL of nutrient broth medium (NB) according to the cultivation requirements of Staphylococcus aureus. The composition of the medium is as follows: Peptone: 10 g (w / v), Beef extract powder: 3.0 g (w / v), Sodium chloride: 5.0 g (w / v). After dissolving in water, adjust the pH to 7.4 ± 0.2, and make up the volume to 1000 mL; After dissolving the above medium, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121°C for 20 min. After cooling, obtain the ordinary liquid medium; Step 2: Take 50 μL of Staphylococcus aureus stored in a glycerol tube and inoculate it into a 10 mL sterilized ordinary NB medium test tube. Incubate it in a shaker at 37°C and 150 rpm for 12 - 16 h to obtain an activated bacterial solution. Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh ordinary NB medium, and continue to culture it for 8 hours under the conditions of a shaker at 37°C and 150 rpm. Dilute the activated Staphylococcus aureus bacterial suspension with a suspension medium so that the number of viable bacteria after dilution is 1×10 5 CFU / mL, which is the test bacterial suspension; Step 3: Take a 96-well plate and add the diluted test bacterial suspension to all wells (peripheral wells, inner ring wells, and central well). Cover the plate and seal it with sealing tape on the outer circle of the microplate; Step 4: Incubate the above sample plate in an incubator at 37°C, and use a microplate reader to measure the absorbance values of each well at 600 nm at 0 h and 48 h respectively.

[0063] In this comparative example, the positions of the wells in the microplate are as Figure 2 shown. The microplate includes peripheral wells, inner ring well 1, inner ring well 2, and a central well.

[0064] Comparative Example 4: Determination of the minimum inhibitory concentration of nisin against Enterococcus faecalis Refer to the commonly used laboratory operation method to determine the minimum inhibitory concentration of nisin against Enterococcus faecalis. The steps are as follows: Step 1: According to the culture requirements of Enterococcus faecalis, prepare 500 mL of LB liquid medium. According to the mass-volume ratio, the composition of the medium is as follows: tryptone: 5.0 g (w / v), yeast extract: 2.5 g (w / v), sodium chloride: 2.5 g (w / v). After dissolving in water, adjust the pH to 7.0 ± 0.2, and make up the volume to 500 mL. Heat it to melt and become transparent and clear at 90 - 100°C, dispense it into test tubes and Erlenmeyer flasks, and sterilize it at 121°C for 20 min. After cooling, obtain the LB medium; Step 2: Weigh 0.2000 g of Nisin (nisin) powder and dissolve it in 100 mL of sterile triple-distilled water to prepare a Nisin mother liquor with a concentration of 2000 μg / mL. Filter and sterilize it with a sterile 0.22 μm fiber filter membrane in a laminar flow hood and then dispense it, and store it at -20°C. Take it out and thaw it during the experiment, and dilute it to 400 μg / mL with LB medium; Step 3: Take 50 μL of Enterococcus faecalis stored in a glycerol tube and inoculate it into a 10 mL sterilized common LB medium test tube. Incubate it in a shaker at 37°C and 180 rpm for 6 - 8 h to obtain an activated bacterial solution. Take 500 μL of the activated strain and inoculate it again into 100 mL of fresh common LB medium, and perform enlarged culture under the conditions of a shaker at 37°C and 180 rpm. Dilute the activated Enterococcus faecalis bacterial suspension with a suspension medium so that the number of viable bacteria after dilution is 1×10 7 CFU / mL, which is the test indicator bacterial suspension; Step 4: Take a 96-well plate and add 200 μL of sterile phosphate solution (0.02 mol / L, pH 7.0) to the outermost circle (outer peripheral wells), and add 100 μL of LB medium to other wells (inner circle wells 1, inner circle wells 2, and the central well). Among them, F2 - F11 are set as the bacterial solution growth control groups, G2 - G11 are set as the negative control groups, and the remaining wells are experimental groups. The well distribution of each group in this comparative example is as Figure 3 shown; Step 5: Take 100 μL of 400 μg / mL Nisin in Step 2 and add it to well B2. Use the two-fold serial dilution method (that is, each step of dilution reduces the concentration of the previous step solution to 1 / 2 of the original, thus forming a series of concentration gradients with equal ratios) to perform gradient dilution with a suspension medium. The Nisin concentrations in wells B2 - B11 are shown in the Nisin concentration in Table 6. Make 4 parallels for each sample, that is, the operations of C2 - C11, D2 - D11, and E2 - E11 are the same as those of B2 - B11; Step 6: Add 100 μL of Enterococcus faecalis bacterial suspension to the bacterial solution growth control groups and experimental groups; add 100 μL of sterile LB medium to the negative control group; stick on the sealing tape.

[0065] Step 7: Place the above sample plate in an incubator at 37°C for cultivation. According to the OD value determination method, use an enzyme-labeled instrument to read and record the OD600 value of each well before and after 24 h of cultivation of the 96-well plate, and then calculate the difference in OD600 values before and after the drug action. The drug action concentration in the well with an OD value change less than 0.05 is the minimum inhibitory concentration (MIC).

[0066] The well distribution of each group in this comparative example is as Figure 3 shown. Add glucose oxidase phosphate solution to the outermost circle of wells in the microplate last. The outermost circle of wells is recorded as the glucose oxidase solution wells. The wells in the microplate are also divided into test wells, negative control wells, and bacterial solution growth control wells.

Claims

1. A culture method for facultative anaerobic microorganisms based on a microplate, characterized in that: It includes the following steps: 1) Mix and melt gellan gum with the medium of facultative anaerobic microorganisms to obtain a suspension medium; dilute the facultative anaerobic microorganisms after enlarged culture with the suspension medium to obtain an indicator bacteria suspension; 2) Add a phosphoric acid solution containing glucose to the outermost culture wells of the microplate, add the indicator bacteria suspension to the inner wells of the microplate, then continue to add a glucose oxidase solution to the outermost culture wells of the microplate, and seal the microplate; 3) Incubate statically; In the phosphoric acid solution containing glucose described in step 2), the glucose concentration is 50 g / L to 300 g / L; the concentration of the glucose oxidase solution is 0.5 g / L to 50 g / L; in each outermost culture well, the volume ratio of the phosphoric acid solution containing glucose to the glucose oxidase solution is 50 to 100:50 to 100.

2. The facultative anaerobic microorganism culture method based on a microplate according to claim 1, wherein: The gellan gum described in step 1) is deacylated gellan gum or low-acyl gellan gum, and the content of the gellan gum in the suspension medium is 0.2 g / L to 0.4 g / L; The facultative anaerobic microorganisms are Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida; In each outermost culture well, the volume ratio of the phosphoric acid solution containing glucose to the glucose oxidase solution is 1:

1.

3. The facultative anaerobic microorganism culture method based on a microplate according to claim 1, wherein: The viable count of the indicator bacterial suspension described in step 1) is 10 4 ~10 7 CFU / mL; the facultative anaerobic microorganism to be subcultured is diluted with a suspension medium so that the viable count after dilution is 10 4 ~10 7 CFU / mL; The phosphoric acid solution containing glucose described in step 2) refers to glucose dissolved in a phosphate buffer solution with a concentration of 0.02 mol / L to 0.05 mol / L and a pH of 4.0 to 7.0; The glucose oxidase solution described in step 2) refers to glucose oxidase dissolved in a phosphate buffer solution with a concentration of 0.02 mol / L to 0.05 mol / L and a pH of 5.0 to 7.

0.

4. The facultative anaerobic microorganism culture method based on a microplate according to claim 1, wherein: The addition amount of the phosphoric acid solution containing glucose in the culture well is 50 to 100 μL, and the addition amount of the indicator bacteria suspension in the culture well is 50 to 150 μL; In step 2), the addition amount of the suspension medium in the culture well is 50 to 150 μL; The conditions for the static incubation described in step 3) are to incubate in an incubator at 28 to 45 °C for 4 to 72 h; The melting temperature in step 1) is 90 to 100 °C.

5. The facultative anaerobic microorganism culture method based on a microplate according to claim 1, characterized in that: The suspension medium described in step 1) specifically refers to mixing gellan gum with the medium of facultative anaerobic microorganisms, stirring and heating at 90 to 100 °C until it melts into a transparent and clear state, sterilizing, and cooling; The facultative anaerobic microorganisms after enlarged culture refer to inoculating the strains of facultative anaerobic microorganisms into the sterilized medium, culturing at 28 to 45 °C for 6 to 16 h to obtain an activated bacterial liquid; taking the activated bacterial liquid and inoculating it again into a fresh medium for enlarged culture at 28 to 45 °C; The enlarged culture is static incubation or shaking culture on a shaker for 8 to 24 h.

6. The method according to any one of claims 1 to 5 is used for detecting the activity of antibacterial substances against facultative anaerobes.

7. A method for detecting the activity of an antibacterial substance of a facultative anaerobe, characterized in that: It includes the following steps: S1. Mix and melt gellan gum with the medium of facultative anaerobic microorganisms to obtain a suspension medium; S2. Dilute the facultative anaerobic microorganisms after enlarged culture with the suspension medium to obtain an indicator bacteria suspension; S3. After preparing the antibacterial substance into a stock solution, dilute it with a suspension medium to obtain an antibacterial substance suspension; S4. Divide the inner wells of the microplate except for the outermost wells into a test area, a bacterial growth control area, and a negative control area; add a phosphoric acid solution containing glucose to the outermost wells of the microplate, add a suspension medium to the other inner wells, then add the antibacterial substance suspension to the wells in the test area, subsequently add an indicator bacteria suspension to the wells in the test area and the wells in the bacterial growth control area, add a suspension medium to the wells in the negative control group area, and finally add a glucose oxidase solution to the outermost culture wells, and seal the microplate; S5. Incubate statically; S6. Measure the absorbance values of each well at 550 - 600 nm.

8. The method for detecting the antibacterial substance activity of the facultative anaerobe according to claim 7, characterized in that: The antibacterial substance suspension described in step S4 is diluted with a suspension medium to form different concentrations in the wells of the test area.

9. The method for detecting the antibacterial substance activity of the facultative anaerobe according to claim 8, wherein: In step S4, the different concentrations refer to gradient dilution of the antibacterial substance suspension in the wells of the test area with a suspension medium, so that the antibacterial substance forms a concentration gradient in the wells; The gradient dilution is specifically carried out by the two-fold gradient dilution method.

10. The method for detecting the antibacterial substance activity of the facultative anaerobe according to claim 7, characterized in that: The wells in the test area are divided into area one and area two. The antibacterial substance suspension in the wells of area one is gradient diluted with a suspension medium so that the antibacterial substance forms a concentration gradient; the wells in area two are parallel test wells, i.e., parallel holes; each well in area one corresponds to a concentration, and there is at least one parallel hole in area two for each concentration; the concentration gradient is 0.1 - 400 μg / mL; In step S6, according to the absorbance values, plot a curve with the antibacterial substance concentration as the abscissa and the OD600 value as the ordinate, and the inflection point is the minimum inhibitory concentration; The viable count of the indicator bacteria suspension described in step S2 is 10 4 ~10 7 CFU / mL; In the phosphoric acid solution containing glucose described in step S4, the glucose concentration is 50 g / L - 300 g / L; the concentration of the glucose oxidase solution is 0.5 g / L - 50 g / L; in each outermost well, the volume ratio of the phosphoric acid solution containing glucose to the glucose oxidase solution is 50 - 100:50 - 100; The phosphoric acid solution containing glucose described in step S4 refers to glucose dissolved in a phosphate buffer solution with a concentration of 0.02 mol / L - 0.05 mol / L and a pH of 4.0 - 7.0; The glucose oxidase solution described in step S4 refers to glucose oxidase dissolved in a phosphate buffer solution with a concentration of 0.02 mol / L - 0.05 mol / L and a pH of 5.0 - 7.0; The concentration of the antibacterial substance suspension described in step S3 is 200 - 1000 μg / mL; The amount of the phosphoric acid solution containing glucose added to the culture wells is 50 - 100 μL, and the amount of the indicator bacteria suspension added to the culture wells is 50 - 150 μL; The conditions for the static incubation described in step S5 are to incubate in an incubator at 28 - 45 °C for 4 - 72 h; The melting temperature described in step S1 is 90 - 100 °C; The gellan gum described in step S1 is deacylated gellan gum or low-acyl gellan gum, and the content of the gellan gum in the suspension medium is 0.2 g / L - 0.4 g / L; The facultative anaerobic microorganisms are Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Enterococcus faecalis, Streptococcus thermophilus, Listeria, yeast or Candida.

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