Clostridium butyricum CB24011205 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to clostridium butyricum CB24011205 capable of inhibiting growth and colonization of vibrio cholerae and application of the clostridium butyricum CB24011205. According to the present invention, the clostridium butyricum CB24011205 strain is separated from the intestinal tract content of the marine white shrimp, and the preservation number of the clostridium butyricum CB24011205 strain is CGMCC No. 33030; the strain has the capability of efficiently inhibiting vibrio cholerae, can be compounded with daidzein to prepare a microecological preparation, not only has remarkable advantages in colonization and growth of vibrio cholerae, but also can provide certain protection for other pathogenic bacteria (such as escherichia coli and salmonella), can be used for enhancing intestinal immunity and repairing functions, and can be used for preparing the microecological preparation. Good application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Clostridium butyricum ( Clostridium butyricum ) CB24011205 with the ability to inhibit the growth and colonization of Vibrio cholerae and its applications. Background Art

[0002] Vibrio cholerae ( Vibrio cholerae ) is the main pathogen causing cholera, which is usually transmitted through contaminated water sources, leading to acute diarrhea and dehydration. To prevent and treat cholera, the existing prevention and treatment methods are as follows: 1) Oral rehydration salts (ORS): This is the most basic treatment method in cholera treatment. By replenishing electrolytes and fluids, it can relieve dehydration symptoms, especially significantly improving the survival rate of patients in the early stage of cholera.

[0003] 2) Antibiotic treatment: Commonly used antibiotics include tetracycline, ciprofloxacin, erythromycin, etc., which are mainly used to reduce the number of Vibrio cholerae in the body and its infectivity. However, with the widespread use of antibiotics, Vibrio cholerae has gradually developed resistance to some antibiotics, resulting in the uncertainty of treatment effects.

[0004] 3) Recombinant B subunit vaccine against Vibrio cholerae: This vaccine mainly induces an immune response against Vibrio cholerae in the body by inoculating the B subunit protein of Vibrio cholerae for cholera prevention. This vaccine mainly protects the host from Vibrio cholerae by activating specific immune responses (such as antibody generation), but its immune effect may be short-term, and regular booster vaccinations are required to maintain a high level of protection. In addition, oral subunit vaccines also have problems such as the risk of causing local adverse reactions and a narrow scope of action.

[0005] 4) Bacteriophage lysis scheme: Using bacteriophages to specifically lyse Vibrio cholerae as a new treatment method, studies have shown that bacteriophages can effectively kill Vibrio cholerae and reduce its transmission. However, due to the strong specificity of bacteriophages, limited scope of action, and possible influence by the variation of infected strains in clinical applications, this method still faces certain challenges in practical applications.

[0006] Although the existing cholera treatment methods and prevention means are effective to a certain extent, they still have the following obvious deficiencies: 1) Antibiotic resistance issues and the emergence of drug-resistant strains: Currently, antibiotics are one of the main means of treating cholera. However, with the increasing frequency of use, Vibrio cholerae has gradually developed stronger resistance to antibiotics. Common antibiotics such as tetracycline and ciprofloxacin, although effective against Vibrio cholerae in the early stage, over time, the emergence of drug-resistant strains has weakened the effectiveness of these drugs. This not only increases the difficulty of treatment but also may lead to treatment failure and the continuation of the transmission chain. Especially in low-resource areas, the spread of drug-resistant strains is more serious.

[0007] 2) Short-term immune protection of Vibrio cholerae vaccine: The recombinant B subunit vaccine of Vibrio cholerae prevents the infection of Vibrio cholerae by activating the body's immune system and can provide certain protection in the short term. However, this protection is usually short-lived and requires regular booster vaccinations to maintain effective immunity. The immune response of the vaccine is affected by individual differences, and the immune response of some people may be weak, unable to obtain sufficient protection. In addition, the vaccine can only target Vibrio cholerae and cannot provide defense against other pathogens.

[0008] 3) Specificity and limitations of phage therapy: Phage therapy uses phages to specifically kill Vibrio cholerae, with high specificity and selectivity. Although it has been successful in some cases, the effectiveness of phage therapy is limited by the mutation of Vibrio cholerae. If Vibrio cholerae develops resistance to phages, the treatment effect may be greatly reduced. In addition, the screening and application of phages require precise operation, and the treatment plan is relatively complex. Each patient may need to customize the phage selection and dose adjustment according to individual differences, which poses certain challenges to clinical application.

[0009] 4) Side effects and adaptability issues of vaccines and phage therapy: The Vibrio cholerae vaccine may cause some side effects after vaccination, such as local reactions (redness, pain) or systemic discomfort (such as mild fever, fatigue). Although the side effects are mild, some people may have an allergic reaction to the vaccine or a weak immune response, resulting in poor vaccine effectiveness. In addition, the immune protection of the vaccine only targets Vibrio cholerae and cannot provide protection against other pathogens. Although phage therapy has high specificity, host immune responses may occur during the treatment process, and some patients may have an immune response to phages, thus reducing the treatment effect. In addition, the use of phages needs to be adjusted according to specific phage selection and individualized treatment, so the treatment process is relatively complex.

[0010] In view of this, the present invention is hereby proposed. Summary of the Invention

[0011] To solve the above technical problems, the present invention provides a Clostridium butyricum ( Clostridium butyricum ) CB24011205 with the ability to inhibit the growth and colonization of Vibrio cholerae and its applications.

[0012] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a Clostridium butyricum ( Clostridium butyricum ) CB24011205, and its preservation number is CGMCC No. 33030.

[0013] In the second aspect, the present invention provides a bacterial agent containing the Clostridium butyricum CB24011205.

[0014] In the third aspect, the present invention provides a fermentation product prepared by fermenting the Clostridium butyricum CB24011205 or the bacterial agent.

[0015] In the fourth aspect, the present invention provides a microecological preparation, and its main active ingredients include component A and component B. Among them, component A is the Clostridium butyricum CB24011205 and / or the fermentation product; component B is daidzein.

[0016] The microecological preparation developed and provided by the present invention is a synbiotic microecological preparation combining Clostridium butyricum and daidzein. This preparation improves the intestinal microecology, mainly targets the colonization and growth of Vibrio cholerae, and has a certain broad-spectrum antibacterial effect.

[0017] Its specific technical principle and advantages are as follows: Prophylaxis and rapid response: This preparation can effectively prevent the colonization of Vibrio cholerae in the intestine by taking it in advance, and can quickly inhibit its growth within 24 hours. This preparation regulates the intestinal flora, enhances the intestinal immune barrier function, and prevents the invasion of pathogenic bacteria such as Vibrio cholerae, thus achieving a dual effect of prevention and treatment.

[0018] Promote the synergistic inhibitory effect on Vibrio cholerae: Clostridium butyricum can promote the growth of beneficial flora in the intestine (such as Bifidobacterium, Lactobacillus, etc.) through its metabolite butyric acid, and then inhibit the colonization of Vibrio cholerae. Compared with the traditional antibiotic treatment method, the microecological preparation can avoid the invasion of Vibrio cholerae by restoring and maintaining the intestinal microbial balance without destroying the beneficial flora in the intestine.

[0019] Enhance intestinal immunity and repair function: The butyric acid produced by Clostridium butyricum can promote the regeneration and repair of intestinal epithelial cells and enhance the intestinal barrier function. In addition, butyric acid can also regulate the function of the intestinal immune system and enhance the body's immune defense ability, thereby helping to resist the invasion of pathogenic bacteria such as Vibrio cholerae.

[0020] Antibacterial effects of daidzein metabolites: Daidzein is difficult to be directly absorbed in the intestine, but it can be metabolized into O - deoxymethylangolensin (O - DMA) and equol by intestinal probiotics, and these metabolites have significant antibacterial effects. Research shows that O - DMA has a significant inhibitory effect on the growth of Vibrio cholerae, while equol shows stronger activity than daidzein in terms of antioxidant and antibacterial properties.

[0021] Broad - spectrum anti - pathogen effect: Although this preparation is mainly targeted at Vibrio cholerae, its components also show certain inhibitory effects on some other common pathogens (such as Escherichia coli, Salmonella, Vibrio parahaemolyticus, etc.). Therefore, the preparation not only has a significant effect against Vibrio cholerae, but also provides certain protection against a variety of other pathogens.

[0022] The synbiotic microecological preparation of Clostridium butyricum combined with daidzein proposed by the present invention has significant advantages in the colonization and growth of Vibrio cholerae, and at the same time can also provide certain protection against other pathogens (such as Escherichia coli, Salmonella, etc.). By regulating the intestinal flora, enhancing immune defense and repairing the intestinal barrier, the preparation demonstrates broad - spectrum, efficient, rapid and preventive anti - pathogen effects, and is a new treatment method for the prevention and treatment of Vibrio cholerae.

[0023] Fifth aspect, the present invention provides the application of the Clostridium butyricum CB24011205, the bacterial agent, the fermentation product or the microecological preparation in bacteriostasis in non - disease treatment methods.

[0024] Preferably, in the above application, the bacteriostasis includes: inhibiting the growth and colonization of Vibrio cholerae.

[0025] Preferably, in the above application, the bacteriostasis also includes: inhibiting the activity of at least one of Escherichia coli, Salmonella, and Vibrio parahaemolyticus.

[0026] Sixth aspect, the present invention provides the application of the Clostridium butyricum CB24011205, the bacterial agent, the fermentation product or the microecological preparation in the preparation of bacteriostatic drugs.

[0027] Preferably, the bacteriostatic drug is an oral preparation.

[0028] Seventh aspect, the present invention provides the application of the Clostridium butyricum CB24011205, the bacterial agent, the fermentation product or the microecological preparation in the preparation of drugs for enhancing intestinal immunity and repair function.

[0029] Beneficial effects: The present invention provides a Clostridium butyricum ( Clostridium butyricum)Strain CB24011205, with a preservation number of CGMCC No. 33030. This strain has the ability to efficiently inhibit Vibrio cholerae and can be formulated into a probiotic preparation in combination with daidzein. It not only has significant advantages in the colonization and growth of Vibrio cholerae, but also provides certain protection against other pathogenic bacteria (such as Escherichia coli, Salmonella, etc.). It can be used to enhance intestinal immunity and repair function and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will describe the drawings required for use in the examples or the description of the prior art.

[0031] Figure 1 It shows the results that Clostridium butyricum can significantly inhibit the growth and colonization of Vibrio cholerae in the intestines of mice in Example 2. Among them, Figure A is a schematic diagram of three mixed gavage modes in the experiment; Figure B is a comparison of the inhibitory effects of the three gavage modes on the colonization growth and colonization of Vibrio cholerae.

[0032] Figure 2 It shows the results that the content of daidzein in the intestinal metabolites of mice colonized with Clostridium butyricum significantly decreased in Example 3.

[0033] Figure 3 It shows the results that both daidzein and Clostridium butyricum showed obvious antibacterial effects, and the inhibitory effect on Vibrio cholerae was more obvious after their combination in Example 4.

[0034] Figure 4 It shows the results that daidzein and its downstream metabolite S-equol have a significant inhibitory effect on Vibrio cholerae in Example 4.

[0035] Figure 5 It shows the results that the daidzein-Clostridium butyricum probiotic preparation can significantly inhibit the colonization of Vibrio cholerae in the intestinal model of neonatal mice in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] The present invention isolated a dominant strain CB24011205 with the function of inhibiting the growth and colonization of Vibrio cholerae from the intestinal contents of white shrimp. It was preserved at the China General Microbiological Culture Collection Center (abbreviation: CGMCC, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on December 11, 2024, and was taxonomically named Clostridium butyricum Clostridium butyricum with a preservation number of CGMCC No. 33030.

[0037] The Clostridium butyricum strain CB24011205 provided by the present invention has the following unique advantages: Strong tolerance: It can maintain good activity under high temperature and low pH environments, can pass through the gastrointestinal tract without damage, and ensure its function in the intestine. Its spore form endows it with strong survival ability in harsh environments and can tolerate gastric juice, bile and digestive juices.

[0038] Promote the growth of beneficial bacteria: It can promote the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus, and these beneficial bacteria play an important role in inhibiting pathogenic bacteria and enhancing intestinal immunity.

[0039] Anti-pathogenic bacteria: By competitively inhibiting the colonization of pathogenic bacteria, it prevents the invasion of pathogenic bacteria such as Salmonella and Escherichia coli into the intestine.

[0040] Enhance immune function: It can promote the function of the intestinal immune system through its metabolite butyric acid, enhancing the body's defense ability against pathogens. Butyric acid can also promote the regeneration and repair of intestinal epithelial cells and enhance the function of the intestinal barrier.

[0041] Prebiotic effect: Through the production of butyric acid, it can provide nutrients for other beneficial bacteria in the intestine, further optimizing the intestinal microecological environment.

[0042] The present invention further formulates the Clostridium butyricum strain CB24011205 and daidzein into a probiotic preparation.

[0043] In a more preferred and specific embodiment, the formulation of the probiotic preparation includes a core active ingredient and an excipient ingredient; the core active ingredient is shown in Table 1, and the excipient ingredient is shown in Table 2.

[0044] Table 1 Core active ingredient Table 2 Excipient composition More preferably, the formulation ratio of the probiotic preparation is shown in Table 3 and Table 4.

[0045] Table 3 Preferred formulation ratio of probiotic preparation (core active ingredient) Table 4 Preferred formulation ratio of probiotic preparation (excipient) In a more preferred and specific embodiment, the preparation method of the probiotic preparation includes: (1) Preparation of core active ingredient: Under anaerobic conditions, cultivate Clostridium butyricum CB24011205 using BHI liquid medium (BD, product number 211059) containing 0.1% cysteine until the cell concentration reaches 1×10 11CFU / mL. The bacterial cell precipitate was collected by centrifugation (8000×g, 10 min, 4°C) or membrane filtration. The bacterial cells were resuspended with sterile normal saline or phosphate buffer (PBS, pH 7.2) and then freeze-dried. According to the standard of 1×10¹ 0 ∼1×10¹¹ CFU / g, the freeze-dried Clostridium butyricum powder was quantitatively weighed. Genistein powder was weighed according to the standard of 50∼200 mg / g. The particle size of genistein was reduced to ≤10 μm by ultrafine comminution technology to improve its solubility and absorption rate. Low-speed mixing (50~100 rpm) was used to fully mix the Clostridium butyricum powder and genistein.

[0046] (2) Mixing with excipients: Skim milk powder (5%~10%), trehalose (2%~5%), maltodextrin (20%~40%, dried at 70°C to reduce hygroscopicity and improve fluidity), fructooligosaccharide (5%~10%), dipotassium hydrogen phosphate (1%~2%), ascorbic acid (0.1%~0.5%), microcrystalline cellulose (3%~5%), and magnesium stearate (1%~2%) were weighed according to the ratio. A double-screw mixer was used to mix the bacterial cell powder, genistein and excipients at low speed (50~100 rpm), and the mixing time was controlled within 30 min.

[0047] (3) Granulation or tabletting: If granule agent was prepared, dry granulation (roller compaction granulation method) was used to ensure fluidity. If tablet was prepared, tabletting was carried out after adding magnesium stearate, and the pressure was controlled at 5~10 MPa to avoid rupture of bacterial cells.

[0048] (4) Drying and packaging: Vacuum drying at 50°C was used to reduce the moisture content to ≤5%. Nitrogen filling packaging was used to avoid oxidation and improve the stability of the preparation.

[0049] (5) Quality control: Detection of bacterial cell activity: Plate counting method was used to ensure that the concentration of Clostridium butyricum in the finished product met 1×10¹ 0 ∼1×10¹¹ CFU / g.

[0050] Determination of component content: HPLC method was used to detect the content of genistein to ensure that it was within the range of 50∼200 mg / g.

[0051] Moisture content: Karl Fischer titration method was used to detect to ensure that the moisture was ≤5%.

[0052] Fluidity and stability: Angle of repose measurement method was used to evaluate the fluidity of the powder to ensure excellent fluidity.

[0053] (6) Application method: Aquaculture: Add it to the feed according to the standard of 5×10¹ 0 CFU / g, and add 1 - 5 g per ton of feed.

[0054] Human oral administration: Make it into capsules or tablets according to the standard of 1×10¹ 0 CFU / g, and take 1 - 2 tablets (each tablet is 500 mg) daily.

[0055] Among them, daidzein has the following unique advantages: Strong biological activity: After being metabolized and transformed by intestinal probiotics, it can produce metabolites with stronger biological activity, such as O - DMA and equol. O - DMA and equol can significantly inhibit the growth of pathogenic bacteria such as Vibrio cholerae, and equol has strong antioxidant activity and also has the potential for anti - tumor.

[0056] Broad - spectrum antibacterial effect: Daidzein and its metabolites have significant inhibitory effects on a variety of pathogenic bacteria, including Vibrio cholerae, Escherichia coli, Salmonella, etc.

[0057] Antioxidant and anti - cancer effects: Its metabolite equol has great antioxidant potential, and its antioxidant activity is 100 times that of daidzein, and it has a long in - vivo half - life, which helps to delay the aging process and prevent oxidative damage. In addition, equol is also considered to have potential anti - cancer activity and can reduce the risk of cancer by regulating the endocrine system.

[0058] Promote intestinal health: Its metabolites help to maintain and repair the intestinal barrier by improving the intestinal microecological balance.

[0059] This invention combines the Clostridium butyricum strain CB24011205 with daidzein, and the two have the advantages of synergistic effect: Enhance the anti - pathogenic bacteria effect: Clostridium butyricum competitively inhibits the colonization of pathogenic bacteria, and the metabolites of daidzein (such as O - DMA and equol) further strengthen the antibacterial effect, which can effectively prevent the growth and reproduction of pathogenic bacteria such as Vibrio cholerae and Escherichia coli.

[0060] Dual regulation of the immune system: Clostridium butyricum can enhance intestinal immune function and promote the development of immune organs, while daidzein and its metabolites enhance the overall immunity of the body through antioxidant and anti - inflammatory effects. The interaction between the two helps to comprehensively improve the immune defense ability of the body.

[0061] Intestinal barrier repair and health promotion: The metabolite butyric acid of Clostridium butyricum can promote the regeneration and repair of intestinal epithelial cells, improve intestinal barrier function, and at the same time, the metabolites of daidzein further enhance intestinal health by regulating the intestinal microecology and reduce the colonization opportunity of pathogenic bacteria.

[0062] Broad-spectrum applications: This probiotic preparation can not only effectively combat pathogenic bacteria such as Vibrio cholerae, but also be widely used in the prevention and treatment of various diseases such as diarrhea, enteritis, and immune regulation, showing broad clinical application prospects.

[0063] The probiotic preparation provided by the present invention has good application prospects and market potential: Diarrhea and gut health: The inhibitory effect of this preparation on Vibrio cholerae and other gut pathogenic bacteria makes it a potential treatment option for diseases such as diarrhea and gut infections.

[0064] Antioxidation and anti-tumor: The antioxidant and anti-tumor effects of equol provide broader health protection functions for this preparation.

[0065] Livestock, aquaculture, and pet health: This probiotic preparation can promote the digestion and absorption of feed by animals, enhance immune function, prevent and treat intestinal diseases such as animal diarrhea, and promote animal growth and increase survival rate.

[0066] Therefore, the present invention provides an innovative probiotic treatment option with significant advantages and broad application prospects in inhibiting the growth of Vibrio cholerae and improving gut health.

[0067] In addition, there are obvious deficiencies in existing cholera treatment methods and prevention means.

[0068] Regarding the problems of drug resistance and the emergence of drug-resistant strains in existing means, the probiotic preparation provided by the present invention can enhance the intestinal immune barrier by regulating the balance of the intestinal microbial community and has a broad protective effect against various pathogenic bacteria. Different from antibiotics, probiotic preparations do not directly kill pathogenic bacteria, but inhibit the growth of harmful pathogens by maintaining a healthy intestinal microecology, avoiding the emergence of drug-resistant strains and the transmission of drug resistance. This broad-spectrum protective effect of probiotic preparations is not easily affected by the variation of a single pathogen, so it has more long-term prevention and treatment value.

[0069] Regarding the problems of the short protection period and small protection range of existing Vibrio cholerae vaccines, the probiotic preparation provided by the present invention can enhance the overall function of the body's immune system by long-term regulation of the intestinal microecology and provide continuous support for the intestinal immune barrier. Different from the short-term immune effect of Vibrio cholerae vaccines, probiotic preparations can not only help defend against Vibrio cholerae by restoring the balance of the intestinal flora, but also enhance the body's defense ability against other pathogenic bacteria. Its immune support effect is more persistent and extensive, takes effect faster, reduces the need for regular vaccine supplementation, and is applicable to different individuals, especially those with a weak immune system.

[0070] In view of the highly specific and limited problems of existing phage therapies, the probiotic preparation provided by the present invention can inhibit the colonization of Vibrio cholerae by regulating the overall environment of the gut microbiota. Its mechanism of action is more extensive, being effective not only against Vibrio cholerae but also capable of regulating other bacterial communities in the gut and inhibiting the growth of other harmful pathogens. The probiotic preparation does not require individualized customization like phage therapy and is not affected by the variation of a single pathogen, making it more convenient to use without the need to precisely select phages or adjust treatment regimens.

[0071] In view of the side effects and adaptability problems of existing vaccines and phage therapies, the probiotic preparation provided by the present invention has fewer side effects and is generally well tolerated. It replenishes beneficial gut flora through oral administration, is convenient to use, and does not require injection or special treatment. Different from vaccines and phage therapies, the probiotic preparation can not only enhance the intestinal immune barrier but also improve gut health, providing continuous support to the overall immune system and having better adaptability. The components of the probiotic preparation are natural, less likely to cause allergic reactions, and are suitable for most people, especially those with weakened immune systems.

[0072] In summary, the probiotic preparation provided by the present invention plays a crucial role in preventing pathogen colonization and inhibiting its growth by regulating the balance of the gut microbiota. The microbial community in the gut not only helps maintain the immune function of the host but also defends against pathogen invasion through multiple mechanisms. Probiotics can prevent pathogen attachment and colonization by occupying receptor sites on the intestinal mucosa, reducing pathogen invasion of intestinal epithelial cells. At the same time, probiotics can also directly inhibit the growth of pathogens by secreting antibacterial substances such as organic acids and antimicrobial peptides. In addition, the metabolites of probiotics (such as short-chain fatty acids, amino acids, etc.) can improve the intestinal environment, enhance the function of the intestinal barrier, and further reduce the colonization opportunity of pathogens. These mechanisms make the probiotic preparation of great value in preventing intestinal infections, regulating intestinal immune responses, and improving overall gut health.

[0073] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0074] At the endpoints and any values disclosed in this specification, the exact ranges or values are not limited, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0075] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "specific implementation manners", or "some specific implementation manners", etc. means that the specific features, structures, materials, or characteristics described in connection with this embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0076] In the embodiments provided in this specification, for those without specific technologies or conditions indicated, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0077] In the following embodiments, unless otherwise specified, the Clostridium butyricum mentioned is the Clostridium butyricum strain CB24011205.

[0078] Example 1 This example describes the obtaining process of the Clostridium butyricum strain CB24011205: Under aseptic conditions, the white shrimp was dissected and the intact intestine (including the contents) was taken out, and gently rinsed 3 times with sterile PBS (pH 7.4) to remove surface impurities. The whole intestine was placed in 1 - 5 mL of sterile PBS (ensuring complete immersion of the tissue), and then transferred to a sterile glass grinder and ground thoroughly until the tissue was completely homogenized. The grinding solution was transferred to a sterile centrifuge tube and labeled as the stock solution (10 0 ), and then serially diluted 10-fold to 10 -2 . 100 μL each of the stock solution to 10 -2 dilution was spread on BHI plates (3 replicates for each gradient), and cultured under anaerobic conditions at 37 °C for 24 - 48 hours. Regularly shaped single colonies were picked and identified by MALDI-TOF MS. The identified single colonies were purified by streaking 3 times and then preserved in BHI preservation solution and stored at -80 °C.

[0079] Example 2 This example is used to prove the antibacterial effect of Clostridium butyricum strain CB24011205 against Vibrio cholerae in the mouse intestine.

[0080] FVB / N mice were individually housed in an SPF-class barrier environment and fed a normal diet for one week. Before inoculating the bacteria, streptomycin (5 mg / mL) and aspartame (5 mg / mL) were added to the drinking water of the mice and treated for 12 h; 50 μL of filtered and sterilized 10% NaHCO 3 (pH 8.0) was used to gavage the mice; 15 min later, 100 μL of Vibrio cholerae C6706 bacterial solution at a concentration of 10 9 CFU / ml was used to gavage the mice; then, at three time points, namely 24 h before gavage with C6707, 24 h / 48 h after gavage, Clostridium butyricum strain CB24011205 was inoculated. At the 72nd h, the mice were sacrificed, and the small intestinal contents were taken for isolation and culture on Sm plates, and the colony count of Vibrio cholerae was performed to analyze the effect of these intestinal bacteria on the colonization of Vibrio cholerae. The results showed that in either case, Clostridium butyricum strain CB24011205 significantly inhibited the colonization of Vibrio cholerae ( Figure 1 )

[0081] Example 3 This example is used to prove the relationship between daidzein and Clostridium butyricum strain CB24011205.

[0082] Ten-week-old male FVB / N mice were individually housed in an SPF-class barrier environment and fed a normal diet for one week. Before inoculating the bacteria, aspartame (5 mg / mL) was added to the drinking water of the mice and treated for 12 h; the mice were fasted overnight and water was withheld 2 h in advance the next day. 50 μL of filtered and sterilized 10% NaHCO 3 (pH 8.0) was used to gavage the mice; 15 min later, 100 μL of Vibrio cholerae C6706 at a concentration of 10 9 CFU / ml and 108 CFU / ml of Clostridium butyricum strain CB24011205 bacterial solution were used to gavage the mice; the control was 100 μL of Vibrio cholerae C6706 at a concentration of 10 9 CFU / ml; at the 20th h, the mice were sacrificed, and the small intestinal contents were taken for high-resolution non-targeted metabolomics analysis. An ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometer or a tandem Orbitrap mass spectrometer was used to detect the metabolites in the samples. By matching the retention time, molecular mass (molecular mass error within <10 ppm), secondary fragmentation spectrum, collision energy, etc. of the metabolites in the local database, the structure of the metabolites in the biological samples was identified, and the identification results were strictly manually double-checked and confirmed ( Figure 2 )

[0083] Metabolomic analysis showed that the content of daidzein in the intestines of mice colonized with Clostridium butyricum was significantly lower than that of the control group, suggesting the utilization of daidzein by Clostridium butyricum strain CB24011205.

[0084] Example 4 This example provides an experiment for inhibiting Vibrio cholerae in vitro.

[0085] Vibrio cholerae C6706 and Clostridium butyricum cultured in the logarithmic phase were adjusted to an OD600 of about 0.75 respectively to prepare standby bacterial suspensions. Four groups were set up for the experiment: 1. Control group: 1 mL of Vibrio cholerae C6706 bacterial suspension was added with 1 mL of BHI liquid medium; 2. 1 mL of Vibrio cholerae C6706 bacterial suspension was added with 1 mL of BHI liquid medium containing 200 mM daidzein; 3. 1 mL of Vibrio cholerae C6706 bacterial suspension was added with 1 mL of Clostridium butyricum strain CB24011205 bacterial suspension; 4. 1 mL of Vibrio cholerae C6706 bacterial suspension was added with 1 mL of Clostridium butyricum strain CB24011205 bacterial suspension containing 200 mM daidzein. The experimental groups and the control group were left standing under anaerobic conditions and cultured at 37 °C for 24 h and then diluted and spotted on plates. The results showed that both daidzein and Clostridium butyricum exhibited obvious antibacterial effects, and the inhibitory effect on Vibrio cholerae was more obvious after the combination of the two ( Figure 3 ).

[0086] Similarly, metabolites of daidzein were added to the Vibrio cholerae bacterial suspension respectively to make the final concentrations 5, 25, 50, 100, 200, 300 μM, left standing under anaerobic conditions, and cultured at 37 °C for 24 h and then diluted and spotted on plates. The experimental results showed that daidzein and its downstream metabolites (such as equol) had a significant inhibitory effect on the growth of Vibrio cholerae. Daidzein is difficult to be directly absorbed in the intestine, but can be metabolized into O - deoxymethylangolensin (O - DMA) and equol under the action of intestinal probiotics, and these metabolites have significant antibacterial effects ( Figure 4 )

[0087] Example 5 This example provides an experimental verification of a neonatal mouse intestinal model.

[0088] Six-day-old C57BL / 6N strain suckling mice, five in each group, were used for the experiment on the inhibition of Vibrio cholerae by the probiotic preparation. Similar to the in vitro experiment, Vibrio cholerae C6706 and Clostridium butyricum cultured to the logarithmic phase were adjusted to OD600 0.75. Vibrio cholerae was mixed with BHI, 200 μM daidzein, the bacterial suspension of Clostridium butyricum strain CB24011205, and the bacterial suspension of Clostridium butyricum strain CB24011205 containing 200 μM daidzein at a volume ratio of 1:1, and anaerobically pre-cultured at 37 °C for 2 h. After 3 h of laboratory adaptation, each suckling mouse was gavaged with 50 μL of the bacterial solution. After 24 h, the intestinal tissues of the mice were ground, serially diluted, and counted using TCBS plates. The results showed that the probiotic preparation of daidzein and Clostridium butyricum could significantly inhibit the colonization of Vibrio cholerae in the intestinal model of suckling mice. In the intestinal model of suckling mice, the antibacterial effect of the combination of daidzein and Clostridium butyricum was verified. The results showed that the combined agent was more effective than the individual application in inhibiting the colonization of Vibrio cholerae ( Figure 5 ).

[0089] In summary, the present invention provides a Clostridium butyricum strain CB24011205 and a synbiotic microecological preparation thereof combined with daidzein. The microecological preparation has the following remarkable technical advantages and benefits: 1) Broad-spectrum antibacterial effect: The microecological preparation in this technical solution can not only effectively inhibit the growth and colonization of Vibrio cholerae, but also has a certain inhibitory effect on other common pathogenic bacteria such as Escherichia coli and Salmonella, demonstrating the characteristics of broad-spectrum antibacterial. 2) Enhance intestinal immune function: Butyric acid produced by Clostridium butyricum can promote the regeneration and repair of intestinal epithelial cells, enhance the intestinal barrier function, and at the same time regulate the function of the intestinal immune system, enhancing the body's immune defense ability. 3) Preventive and rapid response: By taking it in advance, the preparation can effectively prevent the colonization of Vibrio cholerae in the intestine and can quickly inhibit its growth within 24 hours, achieving the dual effects of prevention and treatment. 4) Avoid the problem of drug resistance: Different from traditional antibiotic treatment, the microecological preparation inhibits the growth of harmful pathogens by maintaining the balance of intestinal microorganisms, avoiding the emergence of drug-resistant strains and the transmission of drug resistance. 5) Long-term immune protection: The microecological preparation enhances the overall function of the body's immune system by long-term regulating the intestinal microecology, providing continuous support for the intestinal immune barrier, and has a more lasting protection effect compared with vaccines with short-term immune protection. 6) Reduce side effects and improve adaptability: The microecological preparation has fewer side effects, is usually well tolerated, and does not require injection or special treatment, is easy to use, and is suitable for most people, especially those with weak immunity. 7) Promote intestinal health: The metabolites of daidzein help maintain and repair the intestinal barrier by improving the intestinal microecological balance, further enhancing intestinal health. 8) Antioxidant and anti-tumor potential: Daidzein and its metabolites such as equol have strong antioxidant activity, which helps to delay the aging process and prevent oxidative damage, and at the same time have potential anti-cancer activity. 9) Wide application prospects: The microecological preparation not only has wide application prospects in the field of human health, but can also be applied to the fields of animal husbandry, aquaculture and pet health, promoting the digestion and absorption of feed by animals, enhancing immune function, and preventing and treating intestinal diseases such as diarrhea in animals. 10) Economic and social benefits: This technical solution provides a cost-effective and easy-to-implement means for the prevention and treatment of cholera and other intestinal diseases, helping to reduce medical costs and improve public health levels, and having significant economic and social benefits.

[0090] The technical solution of this application provides a brand-new, effective and broad-spectrum means for the prevention and treatment of cholera and other intestinal diseases through its unique synbiotic microecological preparation combining Clostridium butyricum and daidzein, and has remarkable technical advantages and social value.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. For example: 1) In addition to the synbiotic microecological preparation of Clostridium butyricum and daidzein, those skilled in the art can consider developing a microecological preparation of a single strain (i.e., a preparation of a single Clostridium butyricum strain CB24011205), and this solution may be more applicable in certain specific situations, such as the inhibition of specific pathogenic bacteria or the maintenance of intestinal health in specific populations. 2) Directly use the metabolites of Clostridium butyricum strain CB24011205 or the metabolites of daidzein (such as butyric acid, O-DMA, and equol, etc.) for compounding as antibacterial and immunomodulatory agents. This method can skip the cultivation and metabolism processes of probiotics and directly utilize the effective active ingredients. 3) Genetically engineered strains, further modify Clostridium butyricum strain CB24011205 through genetic engineering technology to enhance its ability to produce specific metabolites or enhance its inhibitory effect on specific pathogenic bacteria. This solution may provide a more powerful antibacterial effect, but requires more safety evaluations. 4) Personalized customization of microecological preparations, customize personalized microecological preparations according to the intestinal microbiome characteristics and health status of individuals. This solution can provide more precise health interventions, but may involve higher costs and more complex preparation processes. 5) Combined application with other treatment methods, consider combining microecological preparations with other treatment methods (such as antibiotics, vaccines, etc.) to improve the treatment effect and reduce side effects. This solution requires detailed clinical research to determine the optimal combined treatment plan. 6) Delivery systems for microecological preparations, develop new delivery systems for microecological preparations, such as nanoparticles, microcapsules, etc., to improve the stability and bioavailability of the preparations. This solution can improve the storage and transportation conditions of the preparations and enhance their application potential in different environments. 7) Environmental and animal health applications, apply microecological preparations to the fields of environmental purification and animal health, such as aquaculture, animal husbandry, etc., to prevent and control the spread of pathogens, improve animal welfare and production efficiency. The above alternative solutions provide various possibilities for the present technical solution, which can be selected and adjusted according to different application scenarios and requirements. The exploration and implementation of these solutions will further enhance the market competitiveness and social benefits of the present patented technology. The above-mentioned modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Clostridium butyricum ( Clostridium butyricum )CB24011205, its deposit number is CGMCC No. 33030.

2. A bacterial agent containing the Clostridium butyricum CB24011205 according to claim 1.

3. A fermentation product, characterized in that It is obtained by fermenting the Clostridium butyricum CB24011205 described in claim 1 or the bacterial agent described in claim 2.

4. A microecological preparation, characterized in that: The main active ingredients include component A and component B, wherein component A is the Clostridium butyricum CB24011205 described in claim 1 and / or the fermentation product described in claim 3; and component B is daidzein.

5. Use of the Clostridium butyricum CB24011205 according to claim 1, the bacterial agent according to claim 2, the fermentation product according to claim 3 or the probiotic preparation according to claim 4 in the antibacterial treatment of non-disease treatment methods.

6. The use according to claim 5, characterized in that: The bacteriostasis includes: inhibiting the growth and colonization of Vibrio cholerae.

7. The use according to claim 5 or 6, characterized in that: The antibacterial method includes: inhibiting the activity of at least one of Escherichia coli, Salmonella and Vibrio parahaemolyticus.

8. Use of the Clostridium butyricum CB24011205 according to claim 1, the bacterial agent according to claim 2, the fermentation product according to claim 3 or the microecological preparation according to claim 4 in the preparation of antibacterial drugs.

9. The use according to claim 8, characterized in that: The antibacterial drug is an oral preparation.

10. Use of Clostridium butyricum CB24011205 according to claim 1, the bacterial agent according to claim 2, the fermentation product according to claim 3 or the microecological preparation according to claim 4 in the preparation of a drug for enhancing intestinal immunity and repair function.

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