Combined preparation for promoting helicobacter pylori tricarboxylic acid cycle to synthesize ATP (adenosine triphosphate) as well as preparation method and application of combined preparation

Abstract

The invention discloses a combined preparation for promoting helicobacter pylori tricarboxylic acid cycle synthesis of ATP (adenosine triphosphate) and a preparation method and application thereof. The preparation is based on a brain heart infusion culture medium and 10wt% of calf serum, 1mM of L-cysteine, 2mM of L-glutamine and 5mu g / mL of heme are added as key nutritional supplement components, and the pH value of the culture medium is adjusted to 7.35. By applying the combined preparation, the in-vitro culture efficiency of the helicobacter pylori is remarkably improved, the helicobacter pylori can be rapidly increased within a short time, the cost is low, and the combined preparation is suitable for isolated culture and rapid passage of the helicobacter pylori in scientific research and clinical samples.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to a combination preparation that promotes the synthesis of ATP by the tricarboxylic acid cycle in Helicobacter pylori, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative bacillus that colonizes the human gastric mucosa. Its infection is closely associated with the development of various digestive tract diseases, including chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer, posing a significant burden on global public health. Therefore, accurate and efficient detection, isolation, and culture of Helicobacter pylori are crucial for clinical diagnosis, epidemiological surveys, antibiotic susceptibility testing, and research into its pathogenic mechanisms.

[0003] Currently, in vitro isolation and culture of Helicobacter pylori remains one of the "gold standards" for diagnosis and provides essential live bacterial material for subsequent research. Traditional culture methods mainly rely on nutrient-rich solid culture media, such as Columbia blood agar and brain heart infusion agar, usually supplemented with animal blood (such as sheep blood or horse blood) or serum, and provided with a microaerophilic environment (typically 85% N2, 10% CO2, and 5% O2). However, Helicobacter pylori has extremely demanding growth conditions; its slow growth and low positive isolation and culture rate remain major technical challenges for clinical and research work. Specifically, Helicobacter pylori grows slowly on traditional culture media, generally requiring 3 to 5 days, or even longer, to form visible colonies. This significantly prolongs the diagnostic cycle and fails to meet the needs of rapid clinical diagnosis. More importantly, this slow growth rate may cause live bacteria to die or enter an unculturable state during culture due to maladaptation to the in vitro environment, resulting in many real infections going undetected by culture methods, leading to false negative results and often unsatisfactory positive detection rates. Furthermore, the success rate of primary isolation of Helicobacter pylori from clinical gastric mucosal biopsy specimens is even lower, because the bacteria may be in a sub-damaged state after endoscopic sampling, transportation and pretreatment, making them more sensitive to the culture environment.

[0004] Although existing technologies have attempted to improve Helicobacter pylori growth by adding different nutrients to basal culture media, these improvements often suffer from problems such as complex composition, high cost, cumbersome preparation, or unstable effects. For example, some modified culture media may focus on providing abundant carbon and nitrogen sources, but neglect the specific metabolic requirements of Helicobacter pylori in a microaerophilic environment for amino acids, reducing agents, and iron sources, which are crucial for its rapid initiation of proliferation and overcoming growth lag. Therefore, developing a combination formulation with clearly defined components, simple preparation, reasonable cost, and the ability to significantly promote the rapid growth and high positive isolation rate of Helicobacter pylori is urgently needed and has significant application value for improving the level of clinical detection and the efficiency of basic research related to Helicobacter pylori. Summary of the Invention

[0005] Technical problem solved: In order to improve the positive rate of Helicobacter pylori isolation and culture, this invention provides a combination preparation that promotes the synthesis of ATP by the tricarboxylic acid cycle of Helicobacter pylori, its preparation method and application, which can promote the rapid proliferation of Helicobacter pylori in any isolation and culture product. It is simple to operate, low in cost, and suitable for widespread use in scientific research and clinical fields.

[0006] Technical solution: A combination preparation for promoting ATP synthesis by the tricarboxylic acid cycle in Helicobacter pylori, the main components of which include: brain heart infusion culture medium and fetal bovine serum, and the pH value of the combination preparation is 7.35.

[0007] The above-mentioned combination preparation also contains 1 mM L-cysteine, 2 mM L-glutamine, and 5 μg / mL heme.

[0008] The amount of the brain and heart extract culture medium used was 13.33g per 400mL of final culture medium, and the amount of the calf serum used was 40mL.

[0009] The preparation method of the above-mentioned combination preparation for promoting the synthesis of ATP by the tricarboxylic acid cycle of Helicobacter pylori includes the following steps: (a) mixing brain heart infusion culture medium with water, sterilizing and cooling to about 50°C; (b) adding fetal bovine serum, L-cysteine, L-glutamine and heme to the culture medium obtained in step (a) and mixing evenly; (c) adjusting the pH of the mixture obtained in step (b) to 7.35.

[0010] Preferably, in step (a), the sterilization is autoclaving.

[0011] Preferably, the order of addition of each component is as follows: first add calf serum, then add L-cysteine, L-glutamine and heme.

[0012] A culture medium for isolating and culturing Helicobacter pylori, comprising the above-described combination preparation.

[0013] The above-mentioned culture media can be liquid or solid.

[0014] The above-mentioned combination preparations or culture media are used in the preparation of products for the isolation, culture or proliferation of Helicobacter pylori.

[0015] Beneficial effects: This invention significantly enhances the growth rate of Helicobacter pylori by simultaneously adding fetal bovine serum, L-cysteine, L-glutamine, and heme to the basal culture medium of brain and heart infusion and precisely adjusting the pH to 7.35. For example... Figure 1 The growth curves show the OD values ​​of the bacterial culture in the culture medium containing the complete formulation during a 24-hour culture period. 600 The growth rate was significantly higher than that of Comparative Example 1, which did not contain any of the combined formulation, and Comparative Example 2, which simultaneously lacked L-cysteine. This confirms that the presence of all components in the combined formulation is necessary for achieving rapid proliferation; the absence of any component prevents the achievement of optimal growth promotion. This synergistic effect is further reflected in colony formation ability and biomass accumulation. Figure 2 As shown, bacterial cultures using the complete combination formulation resulted in a greater number of colonies and earlier appearance after plating at different time points. Meanwhile, as... Figure 3 The bacterial wet weight results showed that after culturing for 24 to 36 hours, the bacterial cell yield of the complete combination formulation was significantly higher than that of the control groups with missing components. L-cysteine ​​provides a reducing environment and may participate in detoxification; L-glutamine serves as an important nitrogen and energy source; heme provides an essential iron source; and calf serum provides various growth factors and lipids. Under suitable pH conditions simulating the gastric microenvironment, these components enhance the substrate supply for the Helicobacter pylori tricarboxylic acid cycle, promote ATP synthesis, and thus accelerate the growth of Helicobacter pylori, effectively solving the common problems of slow growth and poor activity of Helicobacter pylori during in vitro culture. Therefore, this invention provides a combination formulation with clearly defined components and simple preparation, effectively overcoming the drawbacks of traditional methods such as long culture cycles and unstable positive detection rates, and can obtain high abundance and high activity of Helicobacter pylori cultures in a short time. This method is low-cost, highly standardized, and has good reproducibility. It is suitable for the primary isolation of Helicobacter pylori from clinical gastric mucosal biopsy samples, improving the isolation success rate, and is also suitable for the long-term maintenance and large-scale amplification of strains in scientific research, with broad practical value and promising prospects for promotion. Attached Figure Description

[0016] Figure 1 This is a growth curve of the liquid separation culture medium prepared in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention.

[0017] Figure 2 This is a colony growth diagram of solid isolated culture medium from Examples 1, 1, and 2 of the present invention, after being continuously coated onto plates for 24 hours.

[0018] Figure 3 For the analysis of bacterial wet weight results of Example 1, Comparative Example 1, and Comparative Example 2 of this invention (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.). Detailed Implementation

[0019] The present invention will be described in more detail below with reference to specific embodiments, but the implementation of the present invention is not limited to the examples described. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are all conventional methods well known to those skilled in the art, and the materials and reagents used can be obtained through commercial channels.

[0020] Overview of the Plan

[0021] This formulation provides a combined preparation that promotes the synthesis of ATP by the tricarboxylic acid cycle in Helicobacter pylori, comprising the following raw materials in parts by weight: 13.33 g of brain heart infusion culture medium, 1 part of the combined preparation, and 1 part of fetal bovine serum.

[0022] The combination formulation contains the following components at the following concentrations: L-cysteine ​​1mM, L-glutamine 2mM, and heme 5μg / mL, with a culture medium pH of 7.35.

[0023] This solution also provides a method for preparing a combination formulation that promotes ATP synthesis from the tricarboxylic acid cycle in Helicobacter pylori, comprising the following steps:

[0024] (1) Weigh 13.33g of brain and heart extract culture medium and 360 mL of pure water, pour them into a glass bottle, mix well, seal, sterilize in an autoclave for 30 minutes, and cool to about 50℃ for later use.

[0025] (2) In a brain and heart extract culture medium at about 50°C, add 40 mL of fetal bovine serum, 1 mM of L-cysteine, 2 mM of L-glutamine and 5 μg / mL of heme in sequence, adjust the pH to 7.35, shake well, and prepare a brain and heart extract culture medium containing the combined preparation for later use.

[0026] This protocol also provides the application of a combination preparation that promotes ATP synthesis from the tricarboxylic acid cycle in Helicobacter pylori, comprising the following steps:

[0027] (1) Take Helicobacter pylori in the logarithmic growth phase from Columbia solid plates and prepare a bacterial suspension using brain heart infusion medium, and adjust the concentration OD 600 It is 0.3 (1×10 8 CFU / mL), diluted 1000 times, yields 1×10⁻⁶. 5 CFU / mL, for later use.

[0028] (2) Take 9 mL of the culture medium containing the above-mentioned combination preparation and dispense it into sealed centrifuge tubes. Add 1 mL of bacterial suspension, mix well, and incubate in a microaerophilic environment at 37°C for 24 hours. Measure the OD at 0, 6, 12, 18, and 24 hours after incubation. 600 At 6, 12, 18 and 24 hours of incubation, 100 μL of bacterial suspension was spread onto Columbia agar plates, and the growth curves were recorded and the growth of colonies on the plates at different time points was observed.

[0029] Example 1

[0030] A method for preparing a combination formulation that promotes ATP synthesis from the tricarboxylic acid cycle in Helicobacter pylori includes the following steps:

[0031] (1) Weigh 13.33g of brain and heart extract culture medium and 360 mL of pure water, pour them into a glass bottle, mix well, seal, sterilize in an autoclave for 30 minutes, and cool to about 50℃ for later use.

[0032] (2) In a brain and heart extract culture medium at about 50°C, 40 mL of fetal bovine serum, 1 mM of L-cysteine, 2 mM of L-glutamine and 5 μg / mL of heme were added in sequence, the pH was adjusted to 7.35, and the mixture was shaken thoroughly to prepare a brain and heart extract culture medium containing the combined preparation.

[0033] Comparative Example 1

[0034] Compared with Example 1, the difference is that no combination formulation was added, and the steps are as follows:

[0035] (1) Weigh 13.33g of brain and heart extract culture medium and 360 mL of pure water, pour them into a glass bottle, mix well, seal, sterilize in an autoclave for 30 minutes, and cool to about 50℃ for later use.

[0036] (2) Add 40 mL of fetal bovine serum to the brain and heart extract culture medium at about 50°C, adjust the pH to 7.35, shake well, and prepare brain and heart extract culture medium without the combined preparation.

[0037] Comparative Example 2

[0038] Compared to Example 1, the difference is that L-cysteine ​​was not added, and the steps are as follows:

[0039] (1) Weigh 13.33g of brain and heart extract culture medium and 360 mL of pure water, pour them into a glass bottle, mix well, seal, sterilize in an autoclave for 30 minutes, and cool to about 50℃ for later use.

[0040] (2) Add 40 mL of fetal bovine serum, 2 mM of L-glutamine and 5 μg / mL of heme to the brain and heart extract culture medium at about 50°C, adjust the pH to 7.35, shake well, and prepare the brain and heart extract culture medium containing part of the combined preparation.

[0041] Experiment 1: Monitoring the effectiveness of Helicobacter pylori isolation culture medium

[0042] The optimized culture medium, as used in Example 1, is characterized by simulating a nutrient-rich environment in the stomach and containing calf serum and components of a combined preparation.

[0043] The application of a combination preparation that promotes ATP synthesis from the tricarboxylic acid cycle in Helicobacter pylori, used for isolating and amplifying Helicobacter pylori bacterial culture, includes the following steps:

[0044] (1) Take Helicobacter pylori in the logarithmic growth phase from Columbia solid plates and prepare a bacterial suspension using brain heart infusion medium, and adjust the concentration OD 600 It is 0.3 (1×10 8 CFU / mL), diluted 1000 times, yields 1×10⁻⁶. 5 CFU / mL, for later use.

[0045] (2) Prepare the culture medium according to the method in Example 1. Take 9 mL of the culture medium containing the combined preparation, the culture medium without the combined preparation, and the culture medium containing part of the combined preparation and dispense them into sealed centrifuge tubes. Add 1 mL of bacterial suspension, mix well, and incubate in a microaerophilic environment at 37°C for 24 hours. Measure the OD at 0 hours, 6 hours, 12 hours, 18 hours, and 24 hours after incubation. 600 Values ​​are used to record the growth curve.

[0046] (3) The standard strain of Helicobacter pylori was cultured in the optimized culture medium of Example 1. It grew rapidly within 24 hours, and the growth rate was significantly faster than that of Comparative Example 1 and Comparative Example 2. The growth curve with OD values ​​is shown in the figure below. Figure 1 As shown.

[0047] Experiment 2

[0048] The culture medium used in Example 1 was optimized as follows: it simulates the nutrient-rich environment of the stomach and contains calf serum and components of the combined preparation. The procedures are the same as in Example 1.

[0049] (1) Take Helicobacter pylori in the logarithmic growth phase from Columbia solid plates and prepare a bacterial suspension using brain heart infusion medium, and adjust the concentration OD 600 It is 0.3 (1×10 8 CFU / mL), diluted 1000 times, yields 1×10⁻⁶. 5 CFU / mL, for later use.

[0050] (2) Prepare the culture medium according to the method of Example 1. Take 9 mL of the culture medium containing the combined preparation, the culture medium without the combined preparation and the culture medium containing part of the combined preparation and dispense them into sealed centrifuge tubes. Add 1 mL of bacterial suspension, mix well and incubate in a microaerophilic environment at 37°C for 24 hours. At 6 hours, 12 hours, 18 hours and 24 hours of incubation, take 100 μL of bacterial suspension and spread it on Columbia agar plates to observe the growth of colonies on the plates at different time points.

[0051] (3) The standard strain of Helicobacter pylori was cultured in the optimized culture medium of Example 1, and it grew rapidly within 24 hours, with a growth rate significantly faster than that of Comparative Example 1 and Comparative Example 2. The growth of the culture plates is shown in the figure. Figure 2 As shown.

[0052] Experiment 3

[0053] The culture medium used in Example 1 was optimized as follows: it simulates the nutrient-rich environment of the stomach and contains calf serum and components of the combined preparation. The procedures are the same as in Example 1.

[0054] (1) Take Helicobacter pylori in the logarithmic growth phase from Columbia solid plates and prepare a bacterial suspension using brain heart infusion medium, and adjust the concentration OD 600 It is 0.3 (1×10 8 CFU / mL), diluted 1000 times, yields 1×10⁻⁶. 5 CFU / mL, for later use.

[0055] (2) Prepare the culture medium according to the method of Example 1. Take 9 mL of the culture medium containing the combined preparation and 9 mL of the culture medium without the combined preparation or containing part of the combined preparation and dispense them into sealed centrifuge tubes. Add 1 mL of bacterial suspension, mix well and incubate in a microaerophilic environment at 37°C for 24-36 hours. Measure the wet weight of bacteria at different time points of 24 hours, 28 hours and 36 hours after incubation.

[0056] (3) The standard strain of Helicobacter pylori was cultured in the optimized culture medium of Example 1. The growth rate of Helicobacter pylori was significantly faster than that of Comparative Example 1 and Comparative Example 2 at 24, 28, and 36 hours. The wet weight results of bacteria at each time point are shown below. Figure 3 As shown.

[0057] The preparation and application of a combination formulation for promoting ATP synthesis by the tricarboxylic acid cycle in Helicobacter pylori, as described in this invention, are further explained in detail through the following experimental procedures.

[0058] 1. Materials

[0059] 1.1 Sample

[0060] (1) Columbia culture medium, purchased from Beijing Bairddi Biotechnology Co., Ltd.;

[0061] (2) Fetal bovine serum, purchased from Zhengzhou Pingrui Biotechnology Co., Ltd.;

[0062] (3) L-cysteine, purchased from Shanghai Maclean Biotechnology Co., Ltd.;

[0063] (4) L-glutamine, purchased from Shanghai Maclean Biotechnology Co., Ltd.;

[0064] (5) Heme, purchased from Shanghai McLean Biotechnology Co., Ltd.

[0065] 1.2 Strains

[0066] Helicobacter pylori strains: standard strain 26695 or strain G27.

[0067] 1.3 Main culture media and reagents: Columbia agar medium, brain heart infusion medium, fetal bovine serum.

[0068] 1.4 Main instruments: Tri-gas incubator, shaker incubator, centrifuge, microplate reader, electronic balance.

[0069] 1.5 Consumables: EP tubes, tip heads, centrifuge tubes.

[0070] 2. Methods and Results

[0071] 2.1 Effect of brain and heart extract culture medium containing combined formulation on the growth of H. pylori

[0072] (1) Preparation of combination preparation: Prepare 1 mM L-cysteine, 2 mM L-glutamine and 5 μg / mL heme.

[0073] (2) Culture medium preparation: Weigh 13.33g of brain heart extract culture medium and 360 mL of pure water, pour them into a glass bottle, mix well, seal, sterilize in an autoclave for 30 min, cool to about 50℃ and add 40 mL of fetal bovine serum; then add 1 mL of prepared cysteine, 2 mL of L-glutamine and 5 μg / mL of heme respectively, and adjust the pH to 7.35.

[0074] (3) Preparation of bacterial suspension: Take Helicobacter pylori (G27 or 26695) growing in the logarithmic phase on a solid plate, prepare a bacterial suspension using brain heart infusion medium, and adjust the bacterial suspension concentration to 1×10⁻⁶. 8 CFU / mL (OD600 is 0.3), diluted 1000 times, for later use.

[0075] (4) Inoculation with bacterial suspension: Take 9 mL of the culture medium containing the above-mentioned combination preparation and dispense it into sealed centrifuge tubes. Add 1 mL of bacterial suspension, mix well, and incubate in a microaerophilic environment at 37°C for 24 hours. Measure the OD at 0, 6, 12, 18, and 24 hours of incubation. 600 Record the growth curve; at 6, 12, 18 and 24 hours of culture, take 100 μL of bacterial suspension and spread it on Columbia agar plates to observe the growth of bacterial colonies at different time points; at 24, 28 and 36 hours of culture, measure the wet weight of bacteria.

[0076] (5) Result Interpretation: The test results are meaningful only when bacteria grow significantly in the positive control wells (i.e., with or without the combination preparation) while no growth occurs in the negative control wells (sterile). If bacterial growth occurs in the negative control wells, the test must be repeated. The results show that the growth rate of the group containing the combination preparation is significantly faster than that of the group without the combination preparation and the group containing a partial combination preparation; the bacterial colony count and bacterial wet weight are significantly higher in the group containing the combination preparation than in the group without the combination preparation and the group containing a partial combination preparation.

[0077] (6) Results: The optimal total system containing 400 mL of the combined preparation medium showed that 13.33 g of brain heart extract medium, 40 mL of fetal bovine serum, 1 mM L-cysteine, 2 mM L-glutamine, 5 μg / mL heme, and an optimal pH of 7.35 all promoted the growth of Helicobacter pylori more effectively than the group without the combined preparation and the group containing part of the combined preparation (see [link to relevant documentation]). Figures 1-3 ).

[0078] The liquid culture medium containing the combined preparation for Helicobacter pylori prepared using this scheme 1 can rapidly promote the growth of Helicobacter pylori in large quantities, quickly obtain a large amount of logarithmic phase bacterial suspension, and improve the growth rate and culture positivity rate of Helicobacter pylori.

Claims

1. A combination preparation that promotes the synthesis of ATP from the tricarboxylic acid cycle in Helicobacter pylori, characterized in that, The main components include: brain and heart infusion culture medium and calf serum, and the pH value of the combined preparation is 7.

35.

2. The combination formulation according to claim 1, characterized in that, It also contains 1 mM L-cysteine, 2 mM L-glutamine, and 5 μg / mL heme.

3. The combination formulation according to claim 1, characterized in that, The amount of the brain and heart infusion culture medium used is 13.33g per 400mL of final culture medium, and the amount of the calf serum used is 40mL.

4. The method for preparing the combined formulation for promoting ATP synthesis from the tricarboxylic acid cycle of Helicobacter pylori according to any one of claims 1-3, characterized in that, Includes the following steps: (a) Mix brain and heart infusion culture medium with water, sterilize and cool to about 50°C; (b) Add fetal bovine serum, L-cysteine, L-glutamine and heme to the culture medium obtained in step (a) and mix well; (c) Adjust the pH of the mixture obtained in step (b) to 7.

35.

5. The preparation method according to claim 4, characterized in that, In step (a), the sterilization is autoclaving.

6. The preparation method according to claim 5, characterized in that, In step (b), the order of adding the components is as follows: first add calf serum, then add L-cysteine, L-glutamine and heme.

7. A culture medium for isolating and culturing Helicobacter pylori, characterized in that, It comprises the combination formulation according to any one of claims 1-4.

8. The culture medium according to claim 7, characterized in that, It can be a liquid culture medium or a solid culture medium.

9. Use of the combination formulation according to any one of claims 1-4 or the culture medium according to any one of claims 7-8 in the preparation of products for the isolation, culture or proliferation of Helicobacter pylori.

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