Intestine-derived fusobacterium variabilis with anti-candida activity and application thereof

Abstract

The invention provides intestinal fusobacterium variabilis with anti-candida activity and application of the intestinal fusobacterium variabilis. Specifically, the invention provides the fusobacterium varium, and the fusobacterium varium has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 1.18079. The invention also provides a preparation method of the fusobacterium varium. The fusobacterium variabilis provided by the invention can inhibit and kill pathogen candida and has good biological safety.

Description

Technical Field

[0001] The present invention relates to a mutable fusobacterium and application thereof, in particular to an enterogenic mutable fusobacterium with anti-Candida activity and application thereof. Background Art

[0002] According to the list of priority pathogenic fungi published by the World Health Organization, Candida albicans, Candida parapsilosis, Candida glabrata, etc., which colonize the human intestine, are pathogenic fungi that pose the greatest threat to public health. Currently, the only clinically available drugs for treating infections caused by the above pathogenic fungi are polyenes, triazoles, and echinocandins. With the long-term use of a single drug, the drug resistance of the above pathogenic fungi has become increasingly serious, further making it difficult to treat such infections.

[0003] The research and development of existing antifungal preparations is mainly based on three types of antifungal drugs: polyenes, triazoles, and echinocandins. Synthetic preparations are developed by adding additional components to enhance the efficacy of existing antifungal drugs; antifungal preparations are developed with the help of compounds with antifungal activity; and antifungal preparations are developed using bacterial strains with antifungal activity from habitats such as the environment, food, and skin. However, the research and development strategies of existing antifungal preparations have shortcomings such as poor biosafety, weak antifungal activity, and the inability to prevent and treat fungal infections from the source.

[0004] Therefore, there is an urgent need to develop new antifungal agents. Summary of the invention

[0005] The inventors of this case discovered a strain of anaerobic bacteria of intestinal origin during their research. The bacteria was isolated from a fecal sample of a healthy volunteer and was able to inhibit and kill common intestinal pathogenic fungi such as Candida albicans, Candida parapsilosis, and Candida glabrata. According to the 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 to be a different strain of the same species of Fusobacterium varium, and is named Fusobacterium varium DA690 in the present invention. The strain has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration (address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a deposit date of August 5, 2024, a deposit number of CGMCC No. 1.18079, and a classification name: Fusobacterium varium.

[0006] SEQ ID No.1:

[0007]

[0008] The intestinal anaerobic bacteria Fusobacterium varium DA690 of the present invention, i.e. Fusobacterium varium with a preservation number of CGMCC No.1.18079, can effectively kill Candida albicans, has strong antifungal activity, can be used to develop new anti-Candida albicans preparations, has good biosafety, can be applied to the intestinal environment, and can curb infection from the source by preventing Candida albicans colonization.

[0009] Thus, in one aspect, the present invention provides a Fusobacterium varium having a deposit number of CGMCC No.1.18079.

[0010] On the other hand, the present invention further provides a bacterial preparation, which contains the mutable Fusobacterium described in the present invention (ie, mutable Fusobacterium DA690, i.e., mutable Fusobacterium with the deposit number CGMCC No. 1.18079).

[0011] According to a specific embodiment of the present invention, the bacterial preparation of the present invention may be in the form of live bacteria or inactivated bacteria, and may be a liquid preparation or a solid preparation.

[0012] On the other hand, the present invention also provides a postbiotic, which is a postbiotic containing bacteria and metabolites obtained by fermenting the mutable Fusobacterium DA690 described in the present invention in a culture medium, or a postbiotic containing a fermentation product.

[0013] According to a specific embodiment of the present invention, when the mutable Fusobacterium DA690 of the present invention is fermented and cultured in a culture medium, the culture medium may be one or more of a BHIs culture medium, a YCFA culture medium, and a GAM culture medium.

[0014] According to a specific embodiment of the present invention, when the variable Fusobacterium DA690 of the present invention is fermented and cultured in a culture medium, the culture condition is anaerobic culture.

[0015] According to a specific embodiment of the present invention, the variable Fusobacterium DA690 of the present invention grows in BHIs medium, and its optimal growth temperature is 37°C.

[0016] According to a specific embodiment of the present invention, the variable Fusobacterium DA690 described in the present invention grows in BHIs medium. After anaerobic static culture for 12 hours, the cell density reaches 1.03±0.02 and the pH is 6.0.

[0017] According to a specific embodiment of the present invention, the formula of the BHIs culture medium of the present invention is: 10.0g of tryptone, 17.5g of ox heart powder, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, 1000mL of distilled water, 20.0g of agar to be added to the solid, and sterilized at 121°C for 15min; to the above sterilized culture medium, filter-sterilized vitamin K3 and hemin solution are added to the final concentrations of 1mg / L and 5mg / L, respectively.

[0018] In the present invention, the postbiotics refer to the general term for metabolites obtained by fermentation culture of bacterial strains, and may contain bacterial cells or metabolically active substances obtained by further removing bacterial cells.

[0019] According to a specific embodiment of the present invention, in the present invention, the fermentation product obtained by fermenting and culturing the variable Clostridium DA690 in a culture medium is a postbiotic containing bacteria and metabolites; or, the fermentation product is further centrifuged and / or filtered to remove the product of the bacteria, for example, the fermentation broth supernatant is further collected by centrifugation (for example, centrifugation at 6000-10000 rpm for 10 min), and filtered with a filter membrane (for example, a 0.22 μm filter membrane) to obtain a postbiotic containing metabolites from which the bacteria are removed (which can be stored at -80°C for standby use); optionally, the postbiotic can be further dried to prepare a powder.

[0020] On the other hand, the present invention also provides a method for culturing the variable Fusobacterium DA690, the method comprising:

[0021] The variable Fusobacterium DA690 was inoculated into the culture medium for fermentation culture.

[0022] According to a specific embodiment of the present invention, in the present invention, preferably, the culture medium is one or more of BHIs culture medium, YCFA culture medium and GAM culture medium.

[0023] According to a specific embodiment of the present invention, in the present invention, more preferably, the culture condition is: transferring the variable Fusobacterium to fresh BHIs culture medium at a volume ratio of 1%-5%, and anaerobic culture at 37°C for 24 hours.

[0024] On the other hand, the present invention also provides the use of the mutans DA690, the bacterial preparation or the postbiotic in resisting pathogenic fungi.

[0025] According to a specific embodiment of the present invention, in the present invention, the resistance to pathogenic fungi is to inhibit the growth of pathogenic fungi and / or kill pathogenic fungi.

[0026] According to a specific embodiment of the present invention, in the present invention, the pathogenic fungi include one or more of intestinal pathogenic fungi such as Candida albicans, Candida parapsilosis and Candida glabrata.

[0027] According to a specific embodiment of the present invention, in the present invention, the mutable Fusobacterium, the bacterial preparation or the postbiotic is used to prepare a product against pathogenic fungi.

[0028] According to a specific embodiment of the present invention, preferably, the product against pathogenic fungi is a drug (such as a microecological drug), a health food, a feed or an in vitro disinfectant.

[0029] On the other hand, the present invention also provides an anti-pathogenic fungus preparation, which contains the mutans DA690, the bacterial preparation and / or the postbiotics described in the present invention.

[0030] According to a specific embodiment of the present invention, in the present invention, the anti-pathogenic fungus preparation may further comprise one or more additives: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners or a combination thereof.

[0031] According to a specific embodiment of the present invention, in the present invention, the anti-pathogenic fungus preparation can be a powder, a tablet, a granulation, a suppository, a microcapsule, an ampoule or a liquid spray.

[0032] In summary, the present invention provides an enteric variable Fusobacterium with anti-Candida activity and the use of the same to develop an anti-Candida preparation, which can solve the shortcomings of poor biosafety and weak antifungal activity of existing antifungal preparations, and can curb the infection from the source by preventing the colonization of Candida. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a bacterial morphology diagram of Fusobacterium varium CGMCC No.1.18079 of the present invention.

[0034] Figure 2The results of the activity test of the fermentation liquid of Fusobacterium varium CGMCC No.1.18079 in BHIs medium to inhibit the growth of pathogenic fungi are shown in the figure. Note: BHI-BHIs, the supernatant of BHIs medium without bacteria was added as a negative control; BHI-PBS, the sterile PBS buffer was added as a negative control; BHI-Bb, the fermentation supernatant of the probiotic strain Bifidobacterium bifidum was added as a negative control; BHI-Fv, the fermentation supernatant of Fusobacterium varium CGMCC No.1.18079 (Fv) was added as an experimental group.

[0035] Figure 3 This is a graph showing the results of the activity test of the fermentation liquid of Fusobacterium varium CGMCC No.1.18079 in BHIs medium in killing pathogenic fungi. Note: Initial Candida, the inoculum amount of Candida during co-culture; Candida+Fv, the number of live Candida after co-culture of Candida and the fermentation supernatant of Fusobacterium varium CGMCC No.1.18079 (Fv) for 48 hours.

[0036] Deposit of microorganisms for patent procedures:

[0037] Fusobacterium varium DA690:

[0038] Deposit date: August 5, 2024;

[0039] Depository: China General Microbiology Center (CGMCC);

[0040] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0041] Deposit number: CGMCC No.1.18079;

[0042] Taxonomic nomenclature: Fusobacterium varium. DETAILED DESCRIPTION

[0043] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0044] When embodiment provides numerical range, it should be understood that, unless the present invention indicates otherwise, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected.Unless otherwise defined, all technical and scientific terms used in the present invention are identical with the meanings commonly understood by those skilled in the art.Except the concrete method, equipment, reagent materials used in the embodiment, according to the grasp of prior art by those skilled in the art and the record of the present invention, any method, equipment and material of the prior art similar or equivalent to the method, equipment, reagent materials in the embodiment of the present invention can also be used to realize the present invention.

[0045] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the technical field.

[0046] For easier understanding of the present invention, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art.

[0047] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", "implementations", etc. refer to specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiments / implementations described herein, which are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0048] In each embodiment, the experimental method for killing pathogenic fungi by the strain to be tested is as follows: the pathogenic fungi activated overnight in YPD medium are washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer; the concentration of pathogenic fungal somatic cells is adjusted using a cell counting plate at 1-5×10 5 The treated pathogenic fungal cells were inoculated into a 96-well plate containing fresh BHIs medium at a final concentration of cells / well; the treated bacterial fermentation liquid was added to the 96-well plate at a volume ratio of 1:1, and the plate was placed in a microplate reader and shaken at 37°C and 600rpm for 24 hours; the growth curve of the pathogenic fungi was recorded. The cultured bacterial liquid was diluted and spread on a YPD medium agar plate, and the number of surviving pathogenic fungi was counted after static culture at 37°C for 24 hours. Among them, the formula of the above YPD medium is: 20.0g of tryptone, 10.0g of yeast extract powder, 20.0g of glucose, 1000mL of distilled water, 20.0g of agar was added to the solid, and sterilized at 121°C for 15min.

[0049] Example 1: Screening and identification of Fusobacterium varium strains

[0050] Use a sterile sampler to dig out the middle part of a healthy volunteer's feces that is not in contact with the air, and add an appropriate amount of sterile water to dissolve it. Take out 100μL of the feces aqueous solution, dilute it 10 times in a gradient, and then spread it on the BHIs medium agar plate. Incubate it in an anaerobic box at 37℃ for 48h, pick white round colonies with neat edges, and microscopically examine them as spindle-shaped microscopic bacterial cells. Repeatedly streak to determine pure colonies, and the colony strain is named DA690. Its bacterial morphology is shown in Figure 1 .

[0051] Among them, the formula of the above-mentioned BHIs culture medium is: 10.0g of tryptone, 17.5g of ox heart powder, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, 1000mL of distilled water, 20.0g of agar to be added to the solid, and sterilized at 121°C for 15min; filter-sterilized vitamin K3 and hemin are added to the above-mentioned sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0052] The above strain was inoculated into BHIs liquid culture medium, and the strain was identified as Fusobacterium varium through 16S rRNA gene sequence analysis.

[0053] The above-mentioned Fusobacterium variatum DA690 grows in BHIs medium, and its optimal growth temperature is 37°C.

[0054] The above-mentioned variable Fusobacterium DA690 grew in BHIs medium. After anaerobic static culture for 12 hours, the bacterial density reached 1.03±0.02 and the pH was 6.0.

[0055] The above-mentioned variable Fusobacterium DA690 strain was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration (address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on August 5, 2024, with a deposit date of August 5, 2024, a deposit number of CGMCC No.1.18079, and a classification name: Fusobacterium varium.

[0056] Example 2: Preparation of anti-pathogenic fungal active fermentation broth by Fusobacterium varium in BHIs medium

[0057] The single colony of Fusobacterium varium CGMCC No.1.18079 was inoculated into a fresh BHIs medium and cultured in an anaerobic chamber at 37°C for 24 hours; the activated Fusobacterium varium bacterial solution was transferred into a fresh BHIs medium at a volume ratio of 2%, and cultured in an anaerobic chamber at 37°C for 24 hours; the bacterial culture solution was collected and centrifuged at 10000 rpm for 10 minutes to remove bacterial cells, the supernatant was filtered with a 0.22 μm filter membrane, and the obtained fermentation solution was stored at -80°C for standby use.

[0058] Among them, the formula of the above-mentioned BHIs culture medium is: 10.0g of tryptone, 17.5g of ox heart powder, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, 1000mL of distilled water, 20.0g of agar to be added to the solid, and sterilized at 121°C for 15min; filter-sterilized vitamin K3 and hemin are added to the above-mentioned sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0059] Example 3: Application of active fermentation broth of Fusobacterium varium to inhibit the growth of pathogenic fungi

[0060] The pathogenic fungi Candida albicans, Candida parapsilosis and Candida glabrata isolated from the human intestine were streaked and revived on YPD medium agar plates, and cultured at 37°C for 48 hours; single colonies of the above pathogenic fungi were picked up in fresh YPD liquid culture medium, and cultured at 37°C and 200 rpm for 18 hours; the activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer, and the speed of centrifugation was 3000 rpm for 1 minute; the pathogenic fungal suspension resuspended in sterile PBS buffer was diluted tenfold twice in a row, 10 μL of the bacterial suspension with a dilution factor of 100 was aspirated into the technical room of the cell counting plate, and the cell concentration was read under an ordinary optical microscope; the concentration was 1-5×10 5 The diluted pathogenic fungus suspension was inoculated into a 96-well plate containing fresh BHIs medium at a final concentration of 1:1 cells / well; the bacterial active fermentation broth treated in Example 2 was added to the 96-well plate at a volume ratio of 1:1, and the plate was placed in a microplate reader and shaken at 37°C and 600 rpm for 24 hours to record the growth curve of the pathogenic fungus.

[0061] Among them, according to 1-5×10 5After inoculating the diluted pathogenic fungus suspension into a 96-well plate containing fresh BHIs medium at a final concentration of 1:1 cells / well, fresh BHIs medium, sterile PBS buffer and fermentation supernatant of the probiotic strain Bifidobacterium bifidum were added to the 96-well plate at a volume ratio of 1:1, respectively, as a negative control group.

[0062] The formula of the YPD medium is as follows: 20.0 g of tryptone, 10.0 g of yeast extract powder, 20.0 g of glucose, 1000 mL of distilled water, 20.0 g of agar to solids, and sterilized at 121°C for 15 min.

[0063] Figure 2 The fermentation broth of Fusobacterium varium CGMCC No.1.18079 in BHIs medium shows the activity of inhibiting the growth of pathogenic fungi. Note: BHI-BHIs, adding the supernatant of BHIs medium without bacteria as a negative control; BHI-PBS, adding sterile PBS buffer as a negative control; BHI-Bb, adding the fermentation supernatant of the probiotic strain Bifidobacterium bifidum as a negative control; BHI-Fv, adding the fermentation supernatant of Fusobacterium varium CGMCC No.1.18079 (Fv) as an experimental group. It can be seen that the fermentation broth of Fusobacterium varium CGMCC No.1.18079 of the present invention has the activity of inhibiting the growth of pathogenic fungi.

[0064] Example 4: Application of Fusobacterium varium active fermentation broth in killing pathogenic fungi

[0065] The pathogenic fungi Candida albicans, Candida parapsilosis and Candida glabrata isolated from the human intestine were streaked and revived on YPD medium agar plates, and cultured at 37°C for 48 hours; single colonies of the above pathogenic fungi were picked up in fresh YPD liquid culture medium, and cultured at 37°C and 200 rpm for 18 hours; the activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer, and the speed of centrifugation was 3000 rpm for 1 minute; the pathogenic fungal suspension resuspended in sterile PBS buffer was diluted tenfold twice in a row, 10 μL of the bacterial suspension with a dilution factor of 100 was aspirated into the technical room of the cell counting plate, and the cell concentration was read under an ordinary optical microscope; the concentration was 1-5×10 5The diluted pathogenic fungus suspension was inoculated into a 96-well plate containing fresh BHIs medium at a final concentration of 1:1 cells / well; the bacterial active fermentation broth treated in Example 2 was added to the 96-well plate at a volume ratio of 1:1, and the plate was placed in a microplate reader and shaken at 37°C and 600 rpm for 24 hours; the cultured bacterial solution was diluted and spread on a YPD medium agar plate, and the plate was statically cultured at 37°C for 24 hours, and then counted to count the number of surviving pathogenic fungi.

[0066] Among them, according to 1-5×10 5 After inoculating the diluted pathogenic fungus suspension into a 96-well plate containing fresh BHIs medium at a final concentration of 1:1 cells / well, fresh BHIs medium and sterile PBS buffer were added to the 96-well plate at a volume ratio of 1:1, respectively, as a negative control group.

[0067] The formula of the YPD medium is as follows: 20.0 g of tryptone, 10.0 g of yeast extract powder, 20.0 g of glucose, 1000 mL of distilled water, 20.0 g of agar to solids, and sterilized at 121°C for 15 min.

[0068] Figure 3 The activity of the fermentation broth of Fusobacterium varium CGMCC No.1.18079 in BHIs medium in killing pathogenic fungi is shown. Note: Initial Candida, the inoculum amount of Candida during co-culture; Candida+Fv, the number of live Candida after co-culture of Candida and the fermentation supernatant of Fusobacterium varium CGMCC No.1.18079 (Fv) for 48 hours. It can be seen that the fermentation broth of Fusobacterium varium CGMCC No.1.1807 of the present invention has the activity of killing pathogenic fungi.

[0069] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Those skilled in the art may make various changes and modifications without departing from the essence and scope of the present invention. For example, Fusobacterium mutans CGMCC may be used. No.1.18079 and other antifungal active substances such as other variable Fusobacterium strains are used to prepare anti-Candida preparations (such as using variable Fusobacterium from other intestinal sources to prepare anti-Candida microecological drugs, or preparing microecological drugs, health foods, feeds or in vitro disinfectants based on its fermentation supernatant or postbiotics, etc.), and other culture media can also be used to prepare anti-Candida active fermentation broth based on variable Fusobacterium (such as using YCFA culture medium, GAM culture medium, chemical composition-defined culture medium and other culture media that can culture variable Fusobacterium, culture variable Fusobacterium to prepare anti-Candida microecological drugs, or prepare microecological drugs, health foods, feeds or in vitro disinfectants based on its fermentation supernatant or postbiotics, etc.), and the anti-Candida preparation can also be used to inhibit the application of other pathogenic fungi (such as being applied to inhibit or kill other Candida albicans, Candida parapsilosis and Candida glabrata and pathogenic fungi), to achieve the same purpose. Therefore, all equivalent technical solutions also belong to the protection scope of the present invention.

Claims

1. A Fusobacterium varium having a deposit number of CGMCC No. 1.18079.

2. A bacterial preparation comprising the Fusobacterium mutans according to claim 1.

3. The bacterial preparation according to claim 2, which is a liquid or solid preparation in the form of live bacteria or inactivated bacteria.

4. A postbiotic, which is a postbiotic containing bacterial cells and metabolites obtained by fermenting the mutable Fusobacterium according to claim 1 in a culture medium, or a postbiotic containing a fermentation product; Preferably, the culture medium is BHIs medium; More preferably, the fermentation product obtained by fermenting the variable Fusobacterium according to claim 1 in a culture medium is a postbiotic containing bacteria 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 the postbiotic containing metabolites from which the bacteria are removed; optionally, the postbiotic can be further dried to prepare a powder.

5. A method for culturing the variable Fusobacterium according to claim 1, the method comprising: inoculating the variable Fusobacterium according to claim 1 into a culture medium for fermentation; Preferably, the culture medium is one or more of BHIs culture medium, YCFA culture medium and GAM culture medium; More preferably, the culture condition is anaerobic culture.

6. Use of the mutable Fusobacterium according to claim 1, the bacterial preparation according to claim 2 or 3, or the postbiotic according to claim 4 in resisting pathogenic fungi.

7. The use according to claim 6, wherein: The anti-pathogenic fungi is to inhibit the growth of pathogenic fungi and / or kill pathogenic fungi.

8. The use according to claim 6 or 7, wherein: The pathogenic fungi include one or more of intestinal pathogenic fungi such as Candida albicans, Candida parapsilosis and Candida glabrata.

9. The use according to any one of claims 6 to 8, wherein: The Fusobacterium mutans described in claim 1, the bacterial preparation described in claim 2 or 3, or the postbiotic described in claim 4 is used to prepare a product for resisting pathogenic fungi; Preferably, the product against pathogenic fungi is a drug (such as a microecological drug), a health food, a feed or an in vitro disinfectant.

10. An anti-pathogenic fungus preparation comprising the Fusobacterium mutans according to claim 1, the bacterial preparation according to claim 2 or 3, and / or the postbiotic according to claim 4; Preferably, the anti-pathogenic fungus preparation further comprises one or more additives: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners or combinations thereof; Preferably, the anti-pathogenic fungus preparation is in the form of powder, tablet, granulation, suppository, microcapsule, ampoule or liquid spray.

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