Intestine-derived fusobacterium varium having Anti-candida activity, and use thereof
By developing an anti-Candida preparation using intestinal-derived Fusobacterium DA690, the problems of poor biosafety and weak antifungal activity of existing antifungal preparations have been solved, achieving effective Candida killing and colonization prevention.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2026-04-02
AI Technical Summary
Existing antifungal agents have poor biosafety and weak antifungal activity, and cannot effectively prevent and control Candida infections at the source.
A gut-derived Fusobacterium varium DA690 was developed. The bacteria and metabolites were obtained through fermentation culture and used to prepare anti-Candida preparations for application in the intestinal environment to prevent Candida colonization.
It provides biosafety-friendly anti-Candida activity, effectively killing Candida and preventing its colonization, thus curbing infection at its source.
Smart Images
Figure PCTCN2024122509-FTAPPB-I100001 
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Abstract
Description
Intestinal fusobacterium with anti-candida activity and application thereof TECHNICAL FIELD
[0001] The present application relates to a fusobacterium and an application thereof, in particular to an intestinal fusobacterium with anti-candida activity and an application thereof. BACKGROUND
[0002] According to the list of key pathogenic fungi published by the World Health Organization, Candida albicans, Candida parapsilosis, Candida glabrata and other fungi colonized in the human intestine belong to the pathogenic fungi that pose the greatest threat to public health. Currently, the only drugs available in the clinic for treating the above-mentioned pathogenic fungal infections are polyene, triazole and echinocandin drugs. With the long-term use of single drugs, the drug resistance of the above-mentioned pathogenic fungi is becoming more and more serious, further leading to the difficulty in treating such infections.
[0003] The existing research and development of antifungal preparations are mainly based on three types of antifungal drugs, i.e., polyene, triazole and echinocandin, to develop synthetic preparations by additionally adding components to enhance the efficacy of existing antifungal drugs; to develop antifungal preparations by means of compounds with antifungal activity; and to develop antifungal preparations by using bacterial strains with antifungal activity from environments, foods, skins and other habitats. However, the existing research and development strategies of antifungal preparations have the disadvantages of poor biological safety, weak antifungal activity and inability to prevent fungal infections from the source.
[0004] Therefore, it is urgent to develop new antifungal preparations.
[0005] SUMMARY
[0006] The present inventors found in the research that an intestinal source anaerobic bacterium isolated from a stool sample of a healthy volunteer can inhibit and kill Candida albicans, Candida parapsilosis, Candida glabrata and other common intestinal pathogenic fungi. According to 16S rDNA sequence alignment, the 16S rDNA sequence (SEQ ID No. 1) of the strain has a sequence similarity of 99.71% with that of Fusobacterium varium strain NCTC1056, and is determined as Fusobacterium varium of the same species but different strains. In the present application, it is named Fusobacterium varium DA690. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: No. 1, Yikhina, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation date is August 5, 2024, the preservation number is CGMCC No. 1.18079, and the classification and naming is Fusobacterium varium.
[0007] The intestinal source of the anaerobic bacteria Fusobacterium varium DA690 of the present application, i.e. Fusobacterium varium with the preservation number CGMCC No. 1.18079, can effectively kill Candida, has strong antifungal activity, can be used for developing new anti-Candida preparations, has good biological safety, can be applied to the intestinal environment, can prevent infection by stopping the colonization of Candida from the source.
[0008] Therefore, in one aspect, the present application provides a Fusobacterium varium, which has the preservation number CGMCC No. 1.18079.
[0009] In another aspect, the present application also provides a bacterial preparation containing the Fusobacterium varium of the present application (i.e. Fusobacterium varium DA690, i.e. Fusobacterium varium with the preservation number CGMCC No. 1.18079).
[0010] According to specific embodiments of the present application, the bacterial preparation of the present application can be in the form of live bacteria or inactivated bacteria, and can be a liquid preparation or a solid preparation.
[0011] In another aspect, the present application also provides a postbiotic obtained by fermenting and culturing the Fusobacterium varium DA690 of the present application in a culture medium to obtain a postbiotic containing bacterial cells and metabolites, or a postbiotic containing fermentation products.
[0012] According to specific embodiments of the present application, when the Fusobacterium varium DA690 of the present application is fermented and cultured in a culture medium, the culture medium can be one or more of BHIs culture medium, YCFA culture medium, and GAM culture medium.
[0013] According to specific embodiments of the present application, when the Fusobacterium varium DA690 of the present application is fermented and cultured in a culture medium, the culture condition is anaerobic culture.
[0014] According to specific embodiments of the present application, the Fusobacterium varium DA690 of the present application grows in BHIs culture medium, and the optimal growth temperature is 37℃.
[0015] According to specific embodiments of the present application, the Fusobacterium varium DA690 of the present application grows in BHIs culture medium, and after anaerobic static culture for 12h, the bacterial cell density reaches 1.03±0.02, and the pH is 6.0.
[0016] According to a specific embodiment of the present application, the formula of the BHI medium described in the present application is as follows: tryptone 10.0 g, beef heart infusion powder 17.5 g, sodium chloride 5.0 g, glucose 2.0 g, sodium phosphate dibasic 2.5 g, distilled water 1000 mL, agar 20.0 g, sterilization at 121℃ for 15 min; and then, vitamin K3 and hemin solution are added to the above-mentioned sterilized medium to make the final concentration thereof 1 mg / L and 5 mg / L, respectively.
[0017] In the present application, the postbiotic refers to the total metabolic products obtained by fermentation culture of the bacterial strain, which can contain bacterial bodies or further metabolic active substances obtained by removing the bacterial bodies.
[0018] According to a specific embodiment of the present application, in the present application, the fermentation product obtained by fermentation culture of the Fusobacterium varium DA690 in the medium is the postbiotic containing bacterial bodies and metabolic products; or, the fermentation product is further removed of the bacterial bodies by centrifugation and / or filtration, for example, the fermentation supernatant is collected by further centrifugation (for example, centrifugation at 6000-10000 rpm for 10 min), and then filtered by a filter membrane (for example, a 0.22 μm filter membrane) to obtain the postbiotic containing metabolic products and free of bacterial bodies (which can be stored at -80℃ for later use); optionally, the postbiotic can be further dried to prepare a powder.
[0019] On the other hand, the present application also provides a method for culturing the Fusobacterium varium DA690, which comprises:
[0020] The Fusobacterium varium DA690 is inoculated into a culture medium for fermentation culture.
[0021] According to a specific embodiment of the present application, in the present application, preferably, the culture medium is one or more of the BHI medium, the YCFA medium and the GAM medium.
[0022] According to a specific embodiment of the present application, in the present application, more preferably, the culture conditions are as follows: the Fusobacterium varium is transferred into fresh BHI medium at a volume ratio of 1%-5%, and then cultured anaerobically at 37℃ for 24 h.
[0023] On the other hand, the present application also provides the use of the Fusobacterium varium DA690, the bacterial preparation or the postbiotic in resisting pathogenic fungi.
[0024] According to a specific embodiment of the present application, in the present application, the resisting pathogenic fungi refers to inhibiting the growth of pathogenic fungi and / or killing pathogenic fungi.
[0025] According to a specific embodiment of the present application, in the present application, the pathogenic fungi include one or more of Candida albicans, Candida parapsilosis, Candida glabrata and other enteric pathogenic fungi.
[0026] According to a specific embodiment of the present application, in the present application, the Fusobacterium varium, the bacterial preparation or the postbiotic are used for preparing an anti-pathogenic fungal product.
[0027] According to a specific embodiment of the present application, preferably, the anti-pathogenic fungal product is a medicine (such as a microecological medicine), a health food, a feed or an in-vitro disinfectant.
[0028] In another aspect, the present application also provides an anti-pathogenic fungal preparation containing the Fusobacterium varium DA690, the bacterial preparation and / or the postbiotic according to the present application.
[0029] According to a specific embodiment of the present application, in the present application, the anti-pathogenic fungal preparation can further comprise one or more additives: excipients, preservatives, diluents, fillers, absorption promoters, sweeteners or combinations thereof.
[0030] According to a specific embodiment of the present application, in the present application, the anti-pathogenic fungal preparation can be a powder, a tablet, a granule, a suppository, a microcapsule, an ampoule or a liquid spray.
[0031] In summary, the present application provides a Fusobacterium varium with anti-Candida activity of intestinal origin and its application in developing an anti-Candida preparation, which can solve the problems of poor biological safety and weak anti-fungal activity of the existing anti-fungal preparations, and can also curb the infection from the source by preventing the colonization of Candida. BRIEF DESCRIPTION OF DRAWINGS
[0032] FIG. 1 is a bacterial morphology diagram of the Fusobacterium varium CGMCC No. 1.18079 of the present application.
[0033] Figure 2 is a graph showing the results of the detection of the activity of the fermentation broth of Fusobacterium varium CGMCC No. 1.18079 of the present application in BHI medium in inhibiting the growth of pathogenic fungi. Note: BHI-BHIs, adding the supernatant of the BHI medium without bacteria as the negative control; BHI-PBS, adding sterile PBS buffer as the negative control; BHI-B.b., adding the fermentation supernatant of the probiotic strain Bifidobacterium bifidum as the negative control; BHI-F.v., adding the fermentation supernatant of Fusobacterium varium CGMCC No. 1.18079 (F.v.) as the experimental group.
[0034] Figure 3 is a graph showing the results of the detection of the activity of the fermentation broth of Fusobacterium varium CGMCC No. 1.18079 of the present application in BHI medium in killing pathogenic fungi. Note: Initial Candida, the inoculation amount of Candida albicans when co-cultured; Candida+F.v., the viable cell count of Candida albicans after co-culturing with the fermentation supernatant of Fusobacterium varium CGMCC No. 1.18079 (F.v.) for 48 h.
[0035] Microorganism preservation for patent procedures:
[0036] Fusobacterium varium DA690:
[0037] Preservation date: August 5, 2024;
[0038] Preservation unit: China General Microbiological Culture Collection Center (CGMCC);
[0039] Address of the preservation unit: No. 3, Yikhuan 1st Road, Beijing City, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences;
[0040] Preservation number: CGMCC No. 1.18079;
[0041] Classification and naming: Fusobacterium varium. DETAILED DESCRIPTION
[0042] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application.
[0043] When the embodiments give numerical ranges, it is understood that every numerical range given throughout this specification will include every narrower numerical range that falls within the broader numerical range, as well as the upper and lower limits of the broader numerical range. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present application, unless specifically stated otherwise. Unless otherwise indicated, the experimental methods, assays, and preparation methods disclosed herein are conventional techniques in the art.
[0044] Unless otherwise indicated, the experimental methods, assays, and preparation methods disclosed herein are conventional techniques in the art.
[0045] For the purposes of the present application, the following terms are defined below.
[0046] In this specification, the use of "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", "aspects", etc. means that a particular element described in connection with the embodiment / aspects is included in at least one embodiment described herein, and can or can not be present in other embodiments. In addition, it is to be understood that the described elements can be combined in any suitable manner in the various embodiments.
[0047] In each embodiment, the experimental method for testing the pathogenic fungi killing ability of the bacterial strain is as follows: the pathogenic fungi activated overnight in YPD medium is washed once with sterilized PBS buffer and resuspended in an equal volume of sterilized PBS buffer; the concentration of pathogenic fungi cell is adjusted using a cell counting plate, and 1-5x10 5 The pathogenic fungi cells treated above are inoculated into a 96-well plate containing fresh BHIs medium at a final concentration of 1-5x10
[0048] Example 1: Screening and identification of Fusobacterium varium strain
[0049] A portion of the middle of the stool of a healthy volunteer which was not in contact with air was dug up using a sterile sampler and was dissolved with an appropriate amount of sterile water. 100 μL of the stool water solution was taken out, diluted by 10 times gradient, and then coated on a BHIs culture medium agar plate. After being cultured in an anaerobic box at 37℃ for 48h, white round and edge neat colonies were picked up, and microscopic examination showed that the bacterial cells were extremely small and fusiform. The pure colony was determined by repeated streaking, and the strain was named DA690. The bacterial morphology is shown in Figure 1.
[0050] The formula of the above BHIs culture medium is as follows: 10.0 g of tryptone, 17.5 g of beef heart juice powder, 5.0 g of sodium chloride, 2.0 g of glucose, 2.5 g of disodium hydrogen phosphate, 1000 mL of distilled water, and 20.0 g of agar for solidification, sterilized at 121℃ for 15 min; and then vitamin K3 and hemin were added to the above sterilized culture medium to make the final concentrations 1 mg / L and 5 mg / L respectively.
[0051] The above strain was inoculated into BHIs liquid culture medium, and 16S rRNA gene sequence analysis showed that the strain was identified as Fusobacterium varium.
[0052] The above Fusobacterium varium DA690 grows in BHIs culture medium, and the optimum growth temperature is 37℃.
[0053] The above Fusobacterium varium DA690 grows in BHIs culture medium, and after anaerobic static culture for 12h, the bacterial density reaches 1.03±0.02, and the pH is 6.0.
[0054] The above Fusobacterium varium DA690 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Yikhina West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences) on August 5, 2024, the preservation date is August 5, 2024, the preservation number is CGMCC No. 1.18079, and the classification and naming is Fusobacterium varium.
[0055] Example 2: Preparation of anti-pathogenic fungal active fermentation broth of Fusobacterium varium in BHIs culture medium
[0056] The single colony of the above-mentioned Fusobacterium varium CGMCC No. 1.18079 was inoculated into fresh BHIs culture medium, and cultured at 37°C for 24h in an anaerobic box; the activated Fusobacterium varium bacterial liquid was transferred into fresh BHIs culture medium at a volume ratio of 2%, and cultured at 37°C for 24h in an anaerobic box; the bacterial culture liquid was collected, centrifuged at 10000rpm for 10min to remove bacterial cells, and the supernatant was filtered through a 0.22μm filter membrane; the obtained fermentation liquid was stored at -80°C for use.
[0057] The formula of the above-mentioned BHIs culture medium is as follows: 10.0g of tryptone, 17.5g of beef heart juice powder, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, 1000mL of distilled water, and 20.0g of agar for solidification, which is sterilized at 121°C for 15min; the sterilized culture medium is added with filtered and sterilized vitamin K3 and hematin chloride, so that the final concentrations thereof are 1mg / L and 5mg / L respectively.
[0058] Example 3: Application of Fusobacterium varium active fermentation liquid in inhibiting growth of pathogenic fungi
[0059] The pathogenic fungi Candida albicans, Candida parapsilosis and Candida glabrata isolated from human intestinal tract were respectively streaked for recovery on YPD culture medium agar plates, and incubated at 37°C for 48h; single colonies of the above-mentioned pathogenic fungi were respectively inoculated into fresh YPD liquid culture medium, and incubated at 37°C, 200rpm for 18h; the activated pathogenic fungi were washed once with sterilized PBS buffer, and resuspended in an equal volume of sterilized PBS buffer, and centrifuged at 3000rpm for 1min; the pathogenic fungi suspension resuspended in sterilized PBS buffer was diluted by 10 times successively for two times, 10μL of the suspension with a dilution of 100 times was taken in the technical chamber of a cell counting plate, and the cell concentration was read under a general optical microscope; the pathogenic fungi suspension with a final concentration of 1-5×10 5 The diluted pathogenic fungi suspension was inoculated into a 96-well plate containing fresh BHIs culture medium at a final concentration of 1-5×10
[0060] The pathogenic fungi suspension with a final concentration of 1-5×10 5The pathogenic fungus suspension diluted above was inoculated in a 96-well plate containing fresh BHIs culture medium, and then fresh BHIs culture medium, sterilized PBS buffer and fermentation supernatant of probiotic strain Bifidobacterium bifidum were added into the 96-well plate at a volume ratio of 1:1, respectively, as negative control groups.
[0061] The YPD culture medium has the following formula: 20.0 g of tryptone, 10.0 g of yeast extract powder, 20.0 g of glucose, 1000 mL of distilled water, and 20.0 g of agar, sterilized at 121°C for 15 min.
[0062] Figure 2 shows the activity of fermentation broth of Fusobacterium varium CGMCC No. 1.18079 in inhibiting the growth of pathogenic fungi in BHIs culture medium. Note: BHI-BHIs, addition of BHI culture medium supernatant without bacteria as negative control; BHI-PBS, addition of sterile PBS buffer as negative control; BHI-B.b., addition of fermentation supernatant of probiotic strain Bifidobacterium bifidum as negative control; BHI-F.v., addition of fermentation supernatant of Fusobacterium varium CGMCC No. 1.18079 (F.v.) as experimental group. It can be seen that the fermentation broth of Fusobacterium varium CGMCC No. 1.18079 of the present application has the activity of inhibiting the growth of pathogenic fungi.
[0063] Example 4: Application of active fermentation broth of Fusobacterium varium in killing pathogenic fungi
[0064] The pathogenic fungi Candida albicans, Candida parapsilosis and Candida glabrata isolated from human intestinal tract were respectively streaked on YPD culture medium agar plates and incubated at 37°C for 48 h. Single colonies of the pathogenic fungi were respectively picked on fresh YPD liquid culture medium and incubated at 37°C and 200 rpm for 18 h. The activated pathogenic fungi were washed once with sterilized PBS buffer and resuspended in an equal volume of sterilized PBS buffer at a speed of 3000 rpm for 1 min. The pathogenic fungus suspension resuspended in sterilized PBS buffer was diluted by 10 times for two times, 10 μL of the suspension with a dilution of 100 times was taken in the technical chamber of a cell counting plate, and the cell concentration was read under a general optical microscope. The cell concentration of the pathogenic fungus suspension was adjusted to 1-5 x 10 5The above diluted pathogenic fungus suspension was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well; fresh BHIs medium and sterilized PBS buffer were added to the above 96-well plate at a volume ratio of 1:1, respectively, as a negative control group.
[0065] wherein the pathogenic fungus was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well, and fresh BHIs medium and sterilized PBS buffer were added to the above 96-well plate at a volume ratio of 1:1, respectively, as a negative control group. 5 The above diluted pathogenic fungus suspension was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well; fresh BHIs medium and sterilized PBS buffer were added to the above 96-well plate at a volume ratio of 1:1, respectively, as a negative control group.
[0066] wherein the pathogenic fungus was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well, and fresh BHIs medium and sterilized PBS buffer were added to the above 96-well plate at a volume ratio of 1:1, respectively, as a negative control group.
[0067] FIG. 3 shows the pathogenic fungus-killing activity of the fermentation broth of Fusobacterium varium CGMCC No. 1.18079 in BHIs medium. Note: Initial Candida, the inoculation amount of Candida albicans in co-culture; Candida + F.v., the viable cell count of Candida albicans after co-culture with the fermentation supernatant of Fusobacterium varium CGMCC No. 1.18079 (F.v.) for 48 h. As can be seen, the fermentation broth of Fusobacterium varium CGMCC No. 1.1807 of the present application has pathogenic fungus-killing activity.
[0068] The above embodiments are only used for illustrating the present application, but not limiting the present application, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application, for example, the anti-candida preparation can be prepared by using the variable fusobacterium CGMCC No. 1.18079 and other antifungal active substances, such as other variable fusobacterium strains (such as using other variable fusobacterium strains from intestinal tract to prepare anti-candida microecological drugs, or to prepare microecological drugs, health food, feed or in-vitro disinfectant based on the fermentation supernatant or postbiotic thereof, etc.), other culture media can also be used to prepare the anti-candida active fermentation broth based on the variable fusobacterium (such as using YCFA medium, GAM medium, chemically defined medium and other culture media that can culture the variable fusobacterium to prepare anti-candida microecological drugs, or to prepare microecological drugs, health food, feed or in-vitro disinfectant based on the fermentation supernatant or postbiotic thereof, etc.), and the anti-candida preparation can also be used to inhibit other pathogenic fungi (such as being applied to inhibit or kill other candida and pathogenic fungi other than Candida albicans, Candida parapsilosis and Candida glabrata). Therefore, all equivalent technical solutions also belong to the protection scope of the present application.
Claims
1. A Fusobacterium varium having a deposit number of CGMCC No. 1.18079.
2. A bacterial preparation comprising the Fusobacterium varium of claim 1.
3. The bacterial preparation of claim 2, which is a liquid or solid preparation in a live or inactivated form.
4. A postbiotic, which is a postbiotic comprising bacterial cells and metabolites obtained by fermentation culture of the Fusobacterium varium of claim 1 in a culture medium, or a postbiotic comprising fermentation products; Preferably, the culture medium is a BHIs culture medium; More preferably, the fermentation product obtained by fermentation culture of the Fusobacterium varium of claim 1 in a culture medium is a postbiotic comprising bacterial cells and metabolites; or, the fermentation product is further centrifuged to collect the supernatant of the fermentation broth, and filtered with a filter membrane to obtain a postbiotic comprising metabolites without bacterial cells; optionally, the postbiotic can be further dried to prepare a powder.
5. A method for culturing the Fusobacterium varium of claim 1, the method comprising: inoculating the Fusobacterium varium of claim 1 into a culture medium for fermentation culture; Preferably, the culture medium is one or more of a BHIs culture medium, a YCFA culture medium, and a GAM culture medium; More preferably, the culture condition is anaerobic culture.
6. Use of the Fusobacterium varium of claim 1, the bacterial preparation of claim 2 or 3, or the postbiotic of claim 4 in resisting pathogenic fungi.
7. Use according to claim 6, wherein, The resisting pathogenic fungi is inhibiting growth of and / or killing pathogenic fungi.
8. Use according to claim 6 or 7, wherein, The pathogenic fungi include one or more of Candida albicans, Candida parapsilosis, and Candida glabrata, and the like intestinal pathogenic fungi.
9. Use according to any one of claims 6 to 8, wherein, The Fusobacterium varium of claim 1, the bacterial preparation of claim 2 or 3, or the postbiotic of claim 4 are used for preparing a product for resisting pathogenic fungi. Preferably, the product for resisting pathogenic fungi is a medicine (e.g., a microecological medicine), a health food, a feed, or an in-vitro disinfectant.
10. An anti-pathogenic fungal preparation comprising the Fusobacterium varium of claim 1, the bacterial preparation of claim 2 or 3, and / or the postbiotic of claim 4. Preferably, the anti-pathogenic fungal preparation further comprises one or more additives: an excipient, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, or a combination thereof. Preferably, the anti-pathogenic fungal preparation is a powder, a tablet, a granule, a suppository, a microcapsule, an ampoule, or a liquid spray.