Culture method of shiga toxin-producing escherichia coli
Through specific culture media and multiple culture methods, combined with the use of specific immunomagnetic beads, the problem of difficult to efficiently cultivate Shiga toxin E. coli in the prior art is solved, the quality of the strain and the maximum bacterial concentration are improved, and public health safety is enhanced.
Patent Information
- Application Number
- CN202510334315.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-06-06
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to efficiently cultivate Shiga toxin E. coli, which leads to a threat to public health and safety, and is difficult to isolate and culture.
Specific culture medium and multiple culture methods were used, including limiting the nitrogen source in one culture, reducing pH to form acid stress conditions in the secondary culture, and restoring mild conditions in three cultures, combined with the use of specific immunomagnetic beads to improve the quality of the strain and maximum bacterial concentration.
The quality and maximum bacterial concentration of Shiga toxin-producing E. coli are improved, the difficulty of isolation and culture is reduced, and public health safety is enhanced.
Smart Images
Figure BDA0005321282010000101
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial culture, in particular to a method for culturing Shiga toxin-producing Escherichia coli. Background Art
[0002] Shiga toxin-producing Escherichia coli (STEC) is an important class of zoonotic pathogens, with Escherichia coli O157:H7 as a typical representative. These strains cause serious diseases, including hemorrhagic colitis and hemolytic uremic syndrome, by secreting Shiga toxins, posing a major threat to public health. STEC has a wide host range and is mainly transmitted through contaminated food (such as uncooked meat, raw milk, vegetables, etc.) or water sources. Therefore, it is crucial to establish an efficient and reliable STEC culture method in food safety, clinical diagnosis and epidemiological research.
[0003] In summary, in order to solve the above problems, it is of great significance to provide a method for culturing Shiga toxin-producing Escherichia coli. Summary of the invention
[0004] The object of the present invention is to provide a method for culturing Shiga toxin-producing Escherichia coli to solve the problems raised in the prior art.
[0005] To achieve the above object, the present invention provides the following technical solutions:
[0006] A method for culturing Shiga toxin-producing Escherichia coli comprises the following steps:
[0007] S1: After thawing and activating the frozen bacteria, inoculate them into the culture medium, culture once; culture twice; culture three times to obtain bacterial liquid;
[0008] S2: Pipette the bacterial solution into a sterile centrifuge tube, add specific immunomagnetic beads, and continue to slowly flip and adsorb for 30 to 40 minutes. Insert the magnetic plate, continue to flip for 10 to 20 minutes, let it stand for 5 to 10 minutes, wash twice, remove the cleaning solution, and finally add sterile water. Repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain the Shiga toxin-producing Escherichia coli solution.
[0009] Preferably, the frozen bacterial strain includes one of O157:H7 and O128:H2.
[0010] Preferably, during the one culture, the temperature is 10-15°C, the time is 8-12 hours, and the pH is 6-6.5.
[0011] Preferably, during the secondary culture, the temperature is 36-37° C., the time is 12-16 h, and the pH is 4-4.5.
[0012] Preferably, during the three cultivations, the temperature is 36-37°C, the time is 6-8 hours, and the pH is 6.5-7.
[0013] Preferably, the culture medium includes the following substances, calculated in 1000 parts by mass: 10-12 parts of soy peptone, 7-9 parts of sustained-release agent, 3-5 parts of sodium dihydrogen phosphate, 1-2 parts of glucose, 2-3 parts of mannitol, 4-6 parts of sodium chloride, 0.2-1 parts of methyl red, 3-4 parts of glutathione, 2-6 parts of bile salt No. 3, 0.08-0.1 parts of magnesium chloride, 0.05-0.08 parts of calcium sulfate, 0.3-0.7 parts of sodium dihydrogen phosphate, 1-3 parts of sodium hydroxide, 0.03-0.05 parts of sodium carbonate, 30-40 parts of olive oil, 0.003-0.004 parts of cefixime, and the rest is sterile water.
[0014] Preferably, the method for preparing the sustained-release agent comprises the following steps:
[0015] (1) adding chitosan to 8-10 wt% acetic acid aqueous solution, ultrasonically treating for 30-40 min until the solution is clear, adding fish peptone, stirring for 1-2 h, adjusting the pH to 3-4, dropping 5-6 g / L polyethylene glycol aqueous solution, homogenizing for 10-20 min, filtering, and freeze-drying to obtain fish peptone particles;
[0016] (2) Add fish peptone granules into deionized water and stir evenly, add an activator and lysine, stir at 30-40° C. for 1-2 hours, filter, wash, and freeze-dry to obtain a sustained-release agent.
[0017] Preferably, the fish peptone granules include the following raw materials by weight: 1.3-1.5 parts of chitosan, 100 parts of 8-10 wt% acetic acid aqueous solution, 0.5-0.7 parts of fish peptone, and 3-4 parts of 5-6 g / L polyethylene glycol aqueous solution;
[0018] The sustained-release agent comprises the following raw materials in parts by weight: 3 to 4 parts of fish peptone granules, 70 to 80 parts of deionized water, 1 to 2 parts of an activator, and 1 to 2 parts of lysine.
[0019] Preferably, the volume ratio of the bacterial solution to the specific immunomagnetic beads is 50-100:1.
[0020] It is conceivable that the activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide in a mass ratio of 1:1 to 1.5.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] (1) The present invention uses certain stress conditions to cultivate the strain, which can improve the quality and maximum bacterial concentration of Shiga toxin-producing Escherichia coli, and facilitate subsequent experiments and detection. The present invention first limits the content of nitrogen source in the setting of the culture medium, which can make the microorganisms enter a "starvation" state in the middle of a culture, activating the stress response mechanism. Compared with the target bacteria, other bacteria are relatively weak due to the inhibition of bile salts and cefixime, and the target bacteria are relatively strong due to insufficient nitrogen source; and the relatively low temperature culture conditions can stimulate the cold resistance of Shiga toxin-producing Escherichia coli. In the late stage of a culture, the maximum bacterial concentration will be significantly improved.
[0023] (2) The pH was lowered during the secondary culture process, forming acid stress conditions, which prompted the strain to quickly adjust its virulence proteins to improve its survival ability and enhance its quality; at the same time, as the pH dropped, the fish peptone slow-release agent wrapped in chitosan gradually began to release internal protein peptides, supplementing the nitrogen source for the strain and improving the survival rate. The third culture returned to mild conditions, and the remaining high-quality strains will reproduce rapidly, increasing the maximum bacterial concentration. The existence of stress conditions also makes other bacterial species unadaptable, greatly reducing their quantity and types, and reducing the difficulty of separation and culture. DETAILED DESCRIPTION
[0024] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.
[0025] It should be noted that the purchase manufacturers of all raw materials involved in the present invention are not subject to any special restrictions and exemplarily include: chitosan, with a molecular weight of 100-120 kDa; fish peptone, provided by Shanghai Yuanpeptide Biotechnology Co., Ltd.; polyethylene glycol, with a molecular weight of 1000; lysine, CAS: 56-87-1; soy peptone, provided by Shanghai Shenggong Bioengineering Co., Ltd.; mannitol, CAS No.: 87-78-5; glutathione, CAS No.: 70-18-8; bile salt No. 3, CAS No.: 73163-53-8; olive oil, CAS No.: 8001-25-0; cefixime, provided by Shanghai Hengshan Pharmaceutical Co., Ltd.
[0026] In the following examples, parts are by mass, and the raw materials mentioned above and used but not mentioned below are all commercially available.
[0027] Among them, the bacterial strain used in the following comparative examples is O157:H7.
[0028] Example 1: Step 1: Preparation of sustained-release agent:
[0029] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0030] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0031] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0032] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 15°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours; adjust the pH to 6.5, and culture it at 37°C for 7 hours to obtain the bacterial solution;
[0033] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0034] The culture medium includes the following substances, calculated in 1000 parts by mass: 11 parts of soy peptone, 8 parts of sustained-release agent, 4 parts of disodium hydrogen phosphate, 1.5 parts of glucose, 2.5 parts of mannitol, 5 parts of sodium chloride, 0.4 parts of methyl red, 3.5 parts of glutathione, 4 parts of bile salt No. 3, 0.09 parts of magnesium chloride, 0.06 parts of calcium sulfate, 0.5 parts of sodium dihydrogen phosphate, 2 parts of sodium hydroxide, 0.04 parts of sodium carbonate, 40 parts of olive oil, 0.0035 parts of cefixime, and the rest is sterile water.
[0035] Example 2: Step 1: Preparation of sustained-release agent:
[0036] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0037] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0038] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0039] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 15°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours; adjust the pH to 6.5, and culture it at 37°C for 7 hours to obtain the bacterial solution;
[0040] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0041] The culture medium includes the following substances, calculated in 1000 parts by mass: 10 parts of soy peptone, 7 parts of sustained-release agent, 3 parts of disodium hydrogen phosphate, 1 part of glucose, 2 parts of mannitol, 4 parts of sodium chloride, 0.2 parts of methyl red, 3 parts of glutathione, 2 parts of bile salt No. 3, 0.08 parts of magnesium chloride, 0.05 parts of calcium sulfate, 0.3 parts of sodium dihydrogen phosphate, 1 part of sodium hydroxide, 0.03 parts of sodium carbonate, 30 parts of olive oil, 0.003 parts of cefixime, and the rest is sterile water.
[0042] Example 3: Step 1: Preparation of sustained-release agent:
[0043] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0044] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0045] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0046] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 15°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours; adjust the pH to 6.5, and culture it at 37°C for 7 hours to obtain the bacterial solution;
[0047] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0048] The culture medium includes the following substances, calculated in 1000 parts by mass: 12 parts of soy peptone, 9 parts of sustained-release agent, 5 parts of disodium hydrogen phosphate, 2 parts of glucose, 3 parts of mannitol, 6 parts of sodium chloride, 1 part of methyl red, 4 parts of glutathione, 6 parts of bile salt No. 3, 0.1 parts of magnesium chloride, 0.08 parts of calcium sulfate, 0.7 parts of sodium dihydrogen phosphate, 3 parts of sodium hydroxide, 0.05 parts of sodium carbonate, 40 parts of olive oil, 0.004 parts of cefixime, and the rest is sterile water.
[0049] Comparative Example 1: Based on Example 1, conventional culture conditions were used, and the other processes remained unchanged, as follows: Step 1: Preparation of sustained-release agent:
[0050] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0051] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0052] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0053] S1: After thawing and activating the frozen strain O157, it was inoculated into the culture medium with a pH of 6.5 and cultured at 37°C for 31 hours to obtain a bacterial solution;
[0054] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0055] The culture medium includes the following substances, calculated in 1000 parts by mass: 11 parts of soy peptone, 8 parts of sustained-release agent, 4 parts of disodium hydrogen phosphate, 1.5 parts of glucose, 2.5 parts of mannitol, 5 parts of sodium chloride, 0.4 parts of methyl red, 3.5 parts of glutathione, 4 parts of bile salt No. 3, 0.09 parts of magnesium chloride, 0.06 parts of calcium sulfate, 0.5 parts of sodium dihydrogen phosphate, 2 parts of sodium hydroxide, 0.04 parts of sodium carbonate, 40 parts of olive oil, 0.0035 parts of cefixime, and the rest is sterile water.
[0056] Comparative Example 2: Based on Example 1, a conventional temperature was used for one culture, and the rest of the process remained unchanged, as follows: Step 1: Preparation of sustained-release agent:
[0057] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0058] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0059] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0060] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 37°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours; adjust the pH to 6.5, and culture it at 37°C for 7 hours to obtain the bacterial solution;
[0061] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0062] The culture medium includes the following substances, calculated in 1000 parts by mass: 11 parts of soy peptone, 8 parts of sustained-release agent, 4 parts of disodium hydrogen phosphate, 1.5 parts of glucose, 2.5 parts of mannitol, 5 parts of sodium chloride, 0.4 parts of methyl red, 3.5 parts of glutathione, 4 parts of bile salt No. 3, 0.09 parts of magnesium chloride, 0.06 parts of calcium sulfate, 0.5 parts of sodium dihydrogen phosphate, 2 parts of sodium hydroxide, 0.04 parts of sodium carbonate, 40 parts of olive oil, 0.0035 parts of cefixime, and the rest is sterile water.
[0063] Comparative Example 3: Based on Example 1, three cultures were not performed, and the other processes remained unchanged, as follows: Step 1: Preparation of sustained-release agent:
[0064] (1) 1.4 parts of chitosan were added to 100 parts of 8 wt % acetic acid aqueous solution, and ultrasonically treated for 30 min until the solution was clear, 0.6 parts of fish peptone were added, and stirred for 1.5 h. The pH was adjusted to 3.5, and 3.5 parts of 5.5 g / L polyethylene glycol aqueous solution were added dropwise, homogenized for 15 min, filtered, and freeze-dried to obtain fish peptone particles;
[0065] (2) adding 3.5 parts of fish peptone granules to 75 parts of deionized water and stirring evenly, adding 1.5 parts of activator and 1.5 parts of lysine, stirring at 35° C. for 1.5 h, filtering, washing, and freeze-drying to obtain a sustained-release agent;
[0066] Step 2: Preparation of Shiga toxin-producing Escherichia coli solution:
[0067] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 15°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours to obtain the bacterial solution;
[0068] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0069] The culture medium includes the following substances, calculated in 1000 parts by mass: 11 parts of soy peptone, 8 parts of sustained-release agent, 4 parts of disodium hydrogen phosphate, 1.5 parts of glucose, 2.5 parts of mannitol, 5 parts of sodium chloride, 0.4 parts of methyl red, 3.5 parts of glutathione, 4 parts of bile salt No. 3, 0.09 parts of magnesium chloride, 0.06 parts of calcium sulfate, 0.5 parts of sodium dihydrogen phosphate, 2 parts of sodium hydroxide, 0.04 parts of sodium carbonate, 40 parts of olive oil, 0.0035 parts of cefixime, and the rest is sterile water.
[0070] Comparative Example 4: Based on Example 1, no sustained-release agent was added, and the rest of the process remained unchanged, as follows: Step 1: Preparation of Shiga toxin-producing Escherichia coli solution:
[0071] S1: After thawing and activating the frozen strain O157, inoculate it into the culture medium, adjust the pH to 6.5, and culture it at 15°C for 10 hours; adjust the pH to 4.5, and culture it at 37°C for 14 hours; adjust the pH to 6.5, and culture it at 37°C for 7 hours to obtain the bacterial solution;
[0072] S2: Pipette 1.5mL of bacterial solution into a sterile centrifuge tube, add 20μL O157 specific immunomagnetic beads, continue to slowly flip and adsorb for 30 minutes, insert into the magnetic plate, continue to flip for 15 minutes, let stand for 5 minutes, wash, remove the cleaning solution, and finally add 1.5mL of sterile water, repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain Shiga toxin-producing Escherichia coli solution.
[0073] The culture medium includes the following substances, calculated in 1000 parts by mass: 15 parts of soy peptone, 4 parts of disodium hydrogen phosphate, 1.5 parts of glucose, 2.5 parts of mannitol, 5 parts of sodium chloride, 0.4 parts of methyl red, 3.5 parts of glutathione, 4 parts of bile salt No. 3, 0.09 parts of magnesium chloride, 0.06 parts of calcium sulfate, 0.5 parts of sodium dihydrogen phosphate, 2 parts of sodium hydroxide, 0.04 parts of sodium carbonate, 40 parts of olive oil, 0.0035 parts of cefixime, and the rest is sterile water.
[0074] Performance test: The samples of the comparative examples of each embodiment were placed in a culture medium for pure culture, diluted, and counted. The Gompertz model in the primary growth model was used to fit the microbial risk model website to obtain the maximum bacterial concentration. The experimental data are shown in Table 1.
[0075] Table 1
[0076]
[0077] Conclusion: As shown in Table 1, in Comparative Example 1, conventional culture conditions were used for culture, and the maximum bacterial concentration was significantly lower than that in Example 1. It can be seen that under certain stress conditions, the culture of the strain was promoted; in Comparative Example 2, conventional temperature was used for one culture, and the stress conditions were general, and the maximum bacterial concentration decreased; in Comparative Example 3, three cultures were not performed, and the strain activity was poor, the number was small, and the maximum bacterial concentration decreased; in Comparative Example 4, no slow-release agent was added, and the nitrogen source stress conditions were lacking, and the maximum bacterial concentration was not as good as that in Example 1.
[0078] Conclusion: The present invention adopts a culture medium containing a sustained-release agent and a multiple culture method to successfully provide a method for culturing Shiga toxin-producing Escherichia coli, and the maximum bacterial concentration after culture is high and the quality is good.
[0079] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for culturing Shiga toxin-producing Escherichia coli, characterized in that: The following steps are involved: S1: After thawing and activating the frozen bacteria, inoculate them into the culture medium, culture once; culture twice; culture three times to obtain bacterial liquid; S2: Pipette the bacterial solution into a sterile centrifuge tube, add specific immunomagnetic beads, and continue to slowly flip and adsorb for 30 to 40 minutes. Insert the magnetic plate, continue to flip for 10 to 20 minutes, let it stand for 5 to 10 minutes, wash twice, remove the cleaning solution, and finally add sterile water. Repeatedly blow and pipette to resuspend the immunomagnetic beads to obtain the Shiga toxin-producing Escherichia coli solution.
2. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The frozen bacterial strain includes one of O157:H7 and O128:H2.
3. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: During the primary culture, the temperature is 10-15° C., the time is 8-12 hours, and the pH is 6-6.
5.
4. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: During the secondary culture, the temperature is 36-37° C., the time is 12-16 hours, and the pH is 4-4.
5.
5. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: During the three cultivations, the temperature is 36-37° C., the time is 6-8 hours, and the pH is 6.5-7.
6. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The culture medium comprises the following substances, calculated in 1000 parts by mass: 10-12 parts of soy peptone, 7-9 parts of sustained-release agent, 3-5 parts of disodium hydrogen phosphate, 1-2 parts of glucose, 2-3 parts of mannitol, 4-6 parts of sodium chloride, 0.2-1 parts of methyl red, 3-4 parts of glutathione, 2-6 parts of bile salt No. 3, 0.08-0.1 parts of magnesium chloride, 0.05-0.08 parts of calcium sulfate, 0.3-0.7 parts of sodium dihydrogen phosphate, 1-3 parts of sodium hydroxide, 0.03-0.05 parts of sodium carbonate, 30-40 parts of olive oil, 0.003-0.004 parts of cefixime, and the rest is sterile water.
7. The method for culturing Shiga toxin-producing Escherichia coli according to claim 6, characterized in that: The preparation method of the sustained-release agent comprises the following steps: (1) adding chitosan to 8-10 wt% acetic acid aqueous solution, ultrasonically treating for 30-40 min until the solution is clear, adding fish peptone, stirring for 1-2 h, adjusting the pH to 3-4, dropping 5-6 g / L polyethylene glycol aqueous solution, homogenizing for 10-20 min, filtering, and freeze-drying to obtain fish peptone particles; (2) Add fish peptone granules into deionized water and stir evenly, add an activator and lysine, stir at 30-40° C. for 1-2 hours, filter, wash, and freeze-dry to obtain a sustained-release agent.
8. The method for culturing Shiga toxin-producing Escherichia coli according to claim 7, characterized in that: The fish peptone granules include the following raw materials by weight: 1.3-1.5 parts of chitosan, 100 parts of 8-10 wt% acetic acid aqueous solution, 0.5-0.7 parts of fish peptone, and 3-4 parts of 5-6 g / L polyethylene glycol aqueous solution; The sustained-release agent comprises the following raw materials in parts by weight: 3 to 4 parts of fish peptone granules, 70 to 80 parts of deionized water, 1 to 2 parts of an activator, and 1 to 2 parts of lysine.
9. The method for culturing Shiga toxin-producing Escherichia coli according to claim 1, characterized in that: The volume ratio of the bacterial liquid to the specific immunomagnetic beads is 50-100:1.