In-vitro anaerobic culture method of intestinal contents
By using DMEM/F12 liquid culture medium in an anaerobic bag, the problem of complex operation and difficult real-time observation of intestinal microbiota culture in the prior art is solved, and efficient and simple intestinal Microbial culture and fermentation are suitable for industrial applications.
Patent Information
- Application Number
- CN202411831401.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-02-14
AI Technical Summary
In the prior art, the in vitro culture technology of intestinal bacterial flora has the problem of operational complexity and difficult to observe the culture status in real time, which limits its promotion in industrial applications.
Using an in vitro anaerobic culture method of intestinal contents, the intestinal tissue containing intestinal contents or intestinal contents is placed in DMEM/F12 liquid culture medium and anaerobic in an anaerobic bag. Culture achieves efficient culture and fermentation of intestinal microorganisms.
This method significantly improves the culture efficiency of intestinal microorganisms, simplifies the operating process, facilitates industrial application, and provides cultures with rich metabolites and good bacterial diversity.
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Figure CN119432606A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of microbial culture, and in particular relates to an in vitro anaerobic culture method for intestinal contents. Background Art
[0002] With the launch of the Human Microbiome Project, intestinal flora has become a core focus in the field of microbiology research. Given the close and complex relationship between intestinal flora and human health and a variety of diseases, in-depth exploration of its distribution characteristics is crucial to revealing the mysteries of human health. Given the limitations and high costs of in vivo research, it is particularly urgent to explore in vitro culture techniques and fermentation methods for intestinal contents.
[0003] In view of this, the present invention creatively proposes a novel in vitro anaerobic culture method for intestinal contents, aiming to improve the culture efficiency of intestinal microorganisms and / or simplify the operation process to facilitate industrialization. Summary of the invention
[0004] The object of the present invention is to provide an in vitro anaerobic culture method for intestinal contents and a culture prepared by the method. The method has the characteristics of high culture efficiency, simple operation and / or convenient industrialization. The culture prepared by the method has rich metabolites, good flora diversity and / or high total probiotic colony count.
[0005] In a first aspect, the present invention provides an in vitro anaerobic culture method for intestinal contents, the method comprising placing animal intestinal contents or intestinal tissue containing intestinal contents in a container (preferably a centrifuge tube) to which DMEM / F12 liquid culture medium has been added, and placing the container in an anaerobic bag for anaerobic culture.
[0006] In some specific embodiments, the intestinal tissue containing intestinal contents has been freed from the membrane, blood vessels and fat outside the intestine, and both ends of the intestinal tissue are tied, preferably with sterile thread.
[0007] In some specific embodiments, the intestinal contents or intestinal tissue containing intestinal contents are from humans or non-human animals. In some specific embodiments, the non-human animals are selected from rats, mice, pigs, cattle and sheep.
[0008] In some embodiments, the anaerobic culture comprises: opening the anaerobic bag package on a clean bench, placing an oxygen indicator, opening the anaerobic gas production bag, sealing the anaerobic bag, and placing the container (preferably a centrifuge tube) in the anaerobic bag for constant temperature culture.
[0009] In some specific embodiments, the anaerobic culture conditions include: constant temperature culture at 30-45° C. for 24-36 hours, preferably constant temperature culture at 30° C. for 24 hours.
[0010] In some specific embodiments, the anaerobic bag is a round-bottom vertical bag.
[0011] In some specific embodiments, the volume of the anaerobic bag is 2.5-5L, preferably 2.5L.
[0012] In some specific embodiments, the DMEM / F12 liquid culture medium further contains penicillin and streptomycin.
[0013] In some specific embodiments, the method further comprises preparing the DMEM / F12 liquid culture medium: weighing DMEM / F12 culture medium powder and sodium bicarbonate powder at a ratio of 10-15 g / L (preferably 11 g / L) and 2-5 g / L (preferably 2.4 g / L) respectively to prepare DMEM / F12 liquid culture medium, and adjusting the pH of the culture medium to 7-8 (preferably 7.2). In some specific embodiments, the method further comprises preparing a mixed solution of penicillin and streptomycin, and adding the mixed solution to the DMEM / F12 liquid culture medium. Preferably, the volume ratio of the mixed solution to the DMEM / F12 liquid culture medium is 1:
[0014] (50-100), preferably 1:50, or 1:100. In some specific embodiments, the method further comprises, under aseptic operation conditions, using a filter membrane with a pore size of 0.2-0.4 μm to sterilize the DMEM / F12 liquid culture medium and / or the mixed solution of penicillin and streptomycin by positive pressure filtration, and packaging after filtration, and storing at 2-8°C.
[0015] In some specific embodiments, the preparation of the mixed solution of penicillin and streptomycin includes: uniformly mixing the penicillin solution and the streptomycin solution at a volume ratio of 1:(1-3), preferably 1:1, or 1:1.5.
[0016] In some specific embodiments, the concentration of the penicillin solution is 100-150 U / mL, preferably 100 U / mL or 150 U / mL.
[0017] In some specific embodiments, the concentration of the streptomycin solution is 0.1-0.5 mg / mL, preferably 0.1 mg / mL or 0.5 mg / mL.
[0018] In a second aspect, the present invention also provides a culture prepared by the aforementioned in vitro anaerobic culture method of intestinal contents.
[0019] Beneficial Effects
[0020] 1. The anaerobic culture method proposed in the present invention solves the technical bottlenecks faced by current intestinal flora culture technology, such as operational complexity and difficulty in real-time observation of culture status, and effectively realizes the in vitro culture and fermentation of intestinal flora.
[0021] 2. The method described in the present invention uses anaerobic bags as the culture environment. The anaerobic bags can efficiently absorb oxygen in the bags and simultaneously generate about 20% to 21% of carbon dioxide, accurately simulating the anaerobic ecology required by microorganisms and providing an ideal habitat for the in vitro growth of intestinal microorganisms.
[0022] 3. The method of the present invention is easy to operate and significantly reduces the difficulty of experimental operation. The method can not only efficiently culture intestinal contents or other beneficial strains and fermentation products, but also provides the possibility of intuitively observing the culture status in real time, so that researchers can more accurately monitor the growth dynamics of microorganisms.
[0023] 4. In terms of the selection of culture medium, the method of the present invention adopts DMEM / F12 culture medium, which has a wider range of applications and higher availability than other types. Its nutrients are balanced and rich, including key nutrients such as glucose, amino acids, vitamins and L-glutamine, which provide ideal nutritional support for the growth and reproduction of strains. More importantly, the osmotic pressure of DMEM / F12 culture medium is highly close to the internal environment of animals, which helps to significantly improve the survival rate and growth efficiency of strains.
[0024] 5. The culture prepared according to the method of the present invention has the advantages of rich metabolites, good bacterial diversity and / or high total probiotic colony count. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 : Place the intestinal contents of humans or mice into an anaerobic bag;
[0026] Figure 2 : The supernatant obtained by centrifugation after anaerobic culture of human or mouse intestinal contents. DETAILED DESCRIPTION
[0027] In order to better understand the present invention, specific embodiments will be given to further illustrate the present invention, but it should be understood that the embodiments described are exemplary embodiments, and the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.
[0028] Example 1
[0029] A method for achieving in vitro culture and in vitro intestinal content fermentation by anaerobic culture, comprising the following steps:
[0030] (1) Prepare DMEM / F12 liquid culture medium: weigh DMEM / F12 culture medium powder at a ratio of 11 g / L and sodium bicarbonate powder at a ratio of 2.4 g / L to prepare DMEM / F12 liquid culture medium. Use hydrochloric acid solution to adjust the pH to 7.2. Use a 0.2 μm pore size filter membrane to sterilize under aseptic operation conditions. The filtered culture medium liquid is dispensed into culture medium bottles and stored at 2°C in a sealed and dark place.
[0031] (2) Preparation of dual antibody solution: Add PBS buffer to penicillin and streptomycin respectively, stir evenly to fully dissolve penicillin and streptomycin. Mix the dissolved 100U / mL penicillin solution and 0.1mg / mL streptomycin solution in a ratio of 1:1, filter under aseptic conditions using a 0.2μm pore size filter membrane for positive pressure sterilization, and dispense the filtered dual antibody solution into centrifuge tubes and store at -80°C.
[0032] (3) Prepare cell tissue culture medium: add double antibody solution in a ratio of 1:50 into DMEM / F12 liquid culture medium.
[0033] (4) Place in cell tissue culture medium: Take the cecum tissue from the mouse digestive tract, remove the membrane, blood vessels and fat outside the intestine, and tie knots at both ends of the intestine with sterile fine thread. Place the obtained cecum tissue in a centrifuge tube containing cell tissue culture medium.
[0034] (5) Anaerobic culture: In a clean and sterile clean bench, open the anaerobic bag, put in the oxygen indicator, open the anaerobic gas production bag, and seal the anaerobic bag. Tie the centrifuge tube with a rubber band and quickly place the centrifuge tube in a 2.5L round-bottom vertical anaerobic bag. Incubate at a constant temperature of 30°C in a water bath for 24 hours.
[0035] Example 2
[0036] A method for achieving in vitro culture and in vitro intestinal content fermentation by anaerobic culture, comprising the following steps:
[0037] (1) Prepare DMEM / F12 liquid culture medium: weigh DMEM / F12 culture medium powder at a ratio of 11 g / L and sodium bicarbonate powder at a ratio of 2.4 g / L to prepare DMEM / F12 liquid culture medium. Use hydrochloric acid solution to adjust the pH to 7.2. Use a 0.2 μm pore size filter membrane to sterilize under aseptic operation conditions. The filtered culture medium liquid is dispensed into culture medium bottles and stored at 2°C in a sealed and dark place.
[0038] (2) Preparation of dual antibody solution: Add PBS buffer to penicillin and streptomycin respectively, stir evenly to fully dissolve penicillin and streptomycin. Mix the dissolved 150U / mL penicillin solution and 0.5mg / mL streptomycin solution in a ratio of 1:1.5, filter under aseptic conditions using a 0.2μm pore size filter membrane for positive pressure sterilization, and dispense the filtered dual antibody solution into centrifuge tubes and store at -20°C.
[0039] (3) Prepare cell tissue culture medium: add double antibody solution in a ratio of 1:50 into DMEM / F12 liquid culture medium.
[0040] (4) Place in cell tissue culture medium: Take the cecum tissue from the mouse digestive tract, remove the membrane, blood vessels and fat outside the intestine, and tie knots at both ends of the intestine with sterile fine thread. Place the obtained cecum tissue in a centrifuge tube containing cell tissue culture medium.
[0041] (5) Anaerobic culture: In a clean and sterile clean bench, open the anaerobic bag, put in the oxygen indicator, open the anaerobic gas production bag, and seal the anaerobic bag. Tie the centrifuge tube with a rubber band and quickly place the centrifuge tube in a 2.5L round-bottom vertical anaerobic bag. Incubate at a constant temperature of 30°C in a water bath for 24 hours.
[0042] Example 3
[0043] A method for achieving in vitro culture and in vitro intestinal content fermentation by anaerobic culture, comprising the following steps:
[0044] (1) Prepare DMEM / F12 liquid culture medium: weigh DMEM / F12 culture medium powder at a ratio of 11 g / L and sodium bicarbonate powder at a ratio of 2.4 g / L to prepare DMEM / F12 liquid culture medium. Use hydrochloric acid solution to adjust the pH to 7.2. Use a 0.2 μm pore size filter membrane to sterilize under aseptic operation conditions. The filtered culture medium liquid is dispensed into culture medium bottles and stored at 2°C in a sealed and dark place.
[0045] (2) Preparation of dual antibody solution: Add PBS buffer to penicillin and streptomycin respectively, stir evenly to fully dissolve penicillin and streptomycin. Mix the dissolved 100U / mL penicillin solution and 0.1mg / mL streptomycin solution in a ratio of 1:1, filter under aseptic conditions using a 0.2μm pore size filter membrane for positive pressure sterilization, and dispense the filtered dual antibody solution into centrifuge tubes and store at -80°C.
[0046] (3) Prepare cell tissue culture medium: Add the double antibody solution in a ratio of 1:100 to DMEM / F12 liquid culture medium.
[0047] (4) Place in cell tissue culture medium: Take the cecum tissue from the mouse digestive tract, remove the membrane, blood vessels and fat outside the intestine, and tie knots at both ends of the intestine with sterile fine thread. Place the obtained cecum tissue in a centrifuge tube containing cell tissue culture medium.
[0048] (5) Anaerobic culture: In a clean and sterile clean bench, open the anaerobic bag, put in the oxygen indicator, open the anaerobic gas production bag, and seal the anaerobic bag. Tie the centrifuge tube with a rubber band and quickly place the centrifuge tube in a 2.5L round-bottom vertical anaerobic bag. Incubate at a constant temperature of 30°C in a water bath for 24 hours.
[0049] Comparative Example 1
[0050] An anaerobic culture method for intestinal contents comprises the following steps:
[0051] (1) Preparation of culture medium: Weigh 3 g / L of beef extract, 10 g / L of peptone, and 5 g / L of sodium chloride to prepare the culture medium. Adjust the pH to 7.2 with hydrochloric acid solution.
[0052] (2) Collection of intestinal contents: The cecal tissue in the mouse digestive tract was taken out, and the intestinal contents in the cecal tissue were removed using PBS buffer. The tube was placed in a sterile centrifuge tube, and CO2 gas was introduced. The tube was diluted with sterile saline, vortexed for 5 minutes, and then filtered.
[0053] (3) Anaerobic culture: Place the intestinal filtrate and sterilized culture medium into a high-temperature sterilized culture bottle, bubbling with high-purity CO2 gas for 20 minutes, cover with a rubber stopper and wax seal. Culture in a 30°C constant temperature shaking incubator at 40 rpm for 12 hours.
[0054] Comparative Example 2
[0055] An anaerobic culture method for intestinal contents comprises the following steps:
[0056] (1) Preparation of culture medium: Weigh 3 g / L of beef extract, 10 g / L of peptone, and 5 g / L of sodium chloride to prepare the culture medium. Adjust the pH to 7.2 with hydrochloric acid solution.
[0057] (2) Collection of intestinal contents: The cecal tissue in the digestive tract of mice was taken out, and the intestinal contents in the cecal tissue were removed using PBS buffer and placed in a sterile centrifuge tube. After CO2 gas was introduced, it was diluted with sterile saline, vortexed for 10 minutes, and then filtered.
[0058] (3) Anaerobic culture: Place the intestinal filtrate and sterilized culture medium into a high-temperature sterilized culture bottle, bubbling with high-purity CO2 gas for 20 minutes, cover with a rubber stopper and wax seal. Culture in a 30°C constant temperature shaking incubator at 40 rpm for 12 hours.
[0059] Comparative Example 3
[0060] An anaerobic culture method for intestinal contents comprises the following steps:
[0061] (1) Preparation of culture medium: Weigh 3 g / L of beef extract, 10 g / L of peptone, and 5 g / L of sodium chloride to prepare the culture medium. Adjust the pH to 7.2 with hydrochloric acid solution.
[0062] (2) Collection of intestinal contents: The cecal tissue in the mouse digestive tract was taken out, and the intestinal contents in the cecal tissue were removed using PBS buffer. The tube was placed in a sterile centrifuge tube, and CO2 gas was introduced. The tube was diluted with sterile saline, vortexed for 5 minutes, and then filtered.
[0063] (3) Anaerobic culture: Place the intestinal filtrate and sterilized culture medium into a high-temperature sterilized culture bottle, bubbling with high-purity CO2 gas for 20 minutes, cover with a rubber stopper and wax seal. Culture in a 35°C constant temperature shaking incubator at 60 rpm for 24 hours.
[0064] Test Case
[0065] The choline, TMA, and TMAO contents measured in the examples of the present invention were compared with the choline, TMA, and TMAO contents measured in the control examples. The results are shown in Table 1.
[0066] Table 1 Comparison of choline, TMA, TMAO contents measured by the present invention and those measured by the control example
[0067]
[0068] Note: The control example is the common anaerobic culture method.
[0069] The results showed that the contents of choline, TMA and TMAO in the intestinal contents of the examples of the present invention were generally higher than those of the control examples.
[0070] The total number of probiotics such as Bifidobacterium and Akkermansia measured in the embodiment is 5 times the total number of probiotics such as Bifidobacterium and Akkermansia measured in the control example (the total number of colonies measured in the embodiment is 10.68 lg CFU / mL, and the total number of colonies measured in the control example is 2.38 lg CFU / mL). At the genus level structure, a total of 116 genera were screened out in the intestinal contents of mice described in the embodiment of the present invention.
[0071] The above descriptions are only specific embodiments of the present invention. These examples are not intended to limit the present invention to the precise forms disclosed. Various choices and improvements made by those skilled in the art on the basis of the present invention are within the protection scope of the present invention.
Claims
1. An in vitro anaerobic culture method for intestinal contents, characterized in that: The method comprises placing the intestinal contents of an animal or intestinal tissue containing the intestinal contents in a container having been added with a DMEM / F12 liquid culture medium, and placing the container in an anaerobic bag for anaerobic culture.
2. The method according to claim 1, characterized in that The intestinal contents or intestinal tissue containing intestinal contents are from humans or non-human animals. Preferably, the non-human animals are selected from rats, mice, pigs, cattle and sheep.
3. The method according to claim 1, characterized in that The intestinal tissue has been freed of the membrane, blood vessels and fat outside the intestinal tract, and both ends of the intestinal tissue are tied, preferably with sterile thread.
4. The method according to claim 1, characterized in that The anaerobic culture comprises: opening the anaerobic bag package on a clean bench, putting in an oxygen indicator, opening the anaerobic gas production bag, sealing the anaerobic bag, and placing the container in the anaerobic bag for constant temperature culture.
5. The method according to claim 1, characterized in that The anaerobic culture conditions include: constant temperature culture at 30-45° C. for 24-36 hours, preferably constant temperature culture at 30° C. for 24 hours.
6. The method according to claim 1, characterized in that The anaerobic bag is a vertical round-bottomed bag. Preferably, the volume of the anaerobic bag is 2.5-5L.
7. The method according to claim 1, characterized in that The DMEM / F12 liquid culture medium also contains penicillin and streptomycin.
8. The method according to claim 1 or 7, characterized in that The method further comprises preparing the DMFM / F12 liquid culture medium: DMEM / F12 culture medium powder and sodium bicarbonate powder are weighed at a ratio of 10-15 g / L and 2-5 g / L respectively to prepare DMEM / F12 liquid culture medium, and the pH of the culture medium is adjusted to 7-8; preferably, the method further comprises preparing a mixed solution of penicillin and streptomycin, and adding the mixed solution to the DMEM / F12 liquid culture medium; more preferably, the volume ratio of the mixed solution to the DMEM / F12 liquid culture medium is 1:(50-100); more preferably, the method further comprises performing positive pressure filtration sterilization on the DMEM / F12 liquid culture medium and / or the mixed solution of penicillin and streptomycin using a filter membrane with a pore size of 0.2-0.4 μm under aseptic operating conditions.
9. The method according to claim 8, characterized in that The preparation of the mixed solution of penicillin and streptomycin comprises: uniformly mixing the penicillin solution and the streptomycin solution in a volume ratio of 1:(1-3).
10. A culture prepared according to the method according to any one of claims 1 to 9.