Enteric Megasphaera indica with anti-Candida activity and its application

Abstract

This invention provides an enteric-derived Megasphaera indica with anti-Candida activity and its applications. Specifically, this invention provides a Megasphaera indica with accession number CGMCC No. 1.18064. The Megasphaera indica of this invention can inhibit and kill pathogenic Candida, exhibiting good biosafety.

Description

Technical Field

[0001] This invention relates to a type of giant coccidia in India and its applications, specifically, to an enterogenic giant coccidia in India with anti-Candida activity and its applications. Background Technology

[0002] According to the World Health Organization's list of key pathogenic fungi, Candida albicans, Candida parapsilosis, and Candida glabrata, which colonize the human gut, are among the pathogenic fungi that pose the greatest threat to public health. Currently, the only clinically available drugs for treating infections caused by these pathogens are polyenes, triazoles, and echinocandins. However, with the long-term use of single-drug therapy, drug resistance in these pathogens is becoming increasingly severe, further complicating the treatment of these infections.

[0003] Current antifungal drug development primarily relies on three classes of antifungal drugs: polyenes, triazoles, and echinocandins. This involves developing synthetic formulations by adding additional components to enhance the efficacy of existing antifungal drugs; developing antifungal drugs using compounds with antifungal activity; and developing antifungal drugs using bacterial strains with antifungal activity from the environment, food, skin, and other habitats. However, current antifungal drug development strategies suffer from drawbacks such as poor biosafety, weak antifungal activity, and the inability to prevent fungal infections at their source.

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

[0005] The inventors in this case discovered an anaerobic bacterium from the gut, isolated from a fecal sample of a healthy volunteer, which can inhibit and kill common intestinal pathogenic fungi such as Candida albicans, Candida parapsilosis, and Candida glabrata. Based on 16S rDNA sequence alignment, the 16S rDNA sequence (SEQ ID No. 1) of this strain has a sequence similarity of 97.38% with that of Megasphaera indica strain RH115, thus identifying it as a different strain of the same species of Megasphaera indica. In this invention, it is named Megasphaera indica DA18. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), deposited on August 5, 2024, with accession number CGMCC No. 1.18064, and classified as Megasphaera indica.

[0006] SEQ ID No. 1:

[0007]

[0008] The intestinal-derived anaerobic bacterium Megasphaera indica DA18 of this invention, namely Megasphaera indica with accession number CGMCC No.1.18064, can effectively kill Candida albicans, has strong antifungal activity, can be used to develop novel anti-Candida albicans preparations, and has good biosafety, can be applied to the intestinal environment, and can inhibit infection at the source by preventing Candida albicans colonization.

[0009] Thus, on the one hand, the present invention provides a Megasphaera indica, which has the accession number CGMCC No.1.18064.

[0010] On the other hand, the present invention also provides a bacterial preparation containing the *Macrococcus indicus* described in the present invention (i.e., *Macrococcus indicus* DA18, i.e., *Macrococcus indicus* with accession number CGMCC No. 1.18064).

[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 in the form of liquid preparation or solid preparation.

[0012] On the other hand, the present invention also provides a metabiotic, which is a metabiotic containing bacterial cells and metabolites obtained by fermenting and culturing the Indian giant cocci DA18 described in the present invention in a culture medium, or a metabiotic containing fermentation products.

[0013] According to a specific embodiment of the present invention, when the Indian giant cocci DA18 is fermented in a culture medium, the culture medium can be one or more of BHIs medium, YCFA medium, and GAM medium.

[0014] According to a specific embodiment of the present invention, the Indian giant coccidia DA18 is cultured under anaerobic conditions during fermentation in a culture medium.

[0015] According to a specific embodiment of the present invention, the Indian giant coccidia DA18 is grown in BHIs medium, and its optimal growth temperature is 37°C.

[0016] According to a specific embodiment of the present invention, the Indian giant cocci DA18 of the present invention was grown in BHIs medium, and after anaerobic static culture for 24 h, the cell density reached 1.14±0.02, and the pH was 6.0.

[0017] According to a specific embodiment of the present invention, the formula of the BHIs culture medium of the present invention is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride solution to the above sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0018] In this invention, the term "post-biotic" refers to the collective term for metabolites obtained from the fermentation culture of the bacterial strain, which may contain bacterial cells or metabolically active substances obtained by further removing bacterial cells.

[0019] According to a specific embodiment of the present invention, the fermentation product obtained by fermenting *Gastrococcus indicus* DA18 in a culture medium is a metabiotic containing bacterial cells and metabolites; or, the fermentation product is further centrifuged and / or filtered to remove bacterial cells, for example, the fermentation broth supernatant is collected by further centrifugation (e.g., centrifugation at 6000-10000 rpm for 10 min) and filtered with a filter membrane (e.g., a 0.22 μm filter membrane) to obtain a metabiotic containing metabolites with removed bacterial cells (which can be stored at -80°C for later use); optionally, the metabiotic can be further dried to prepare a powder.

[0020] On the other hand, the present invention also provides a method for culturing the aforementioned *Macrococcus indicus* DA18, the method comprising:

[0021] The aforementioned giant coccidia DA18 was inoculated into a culture medium for fermentation.

[0022] According to a specific embodiment of 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, more preferably, the culture conditions are as follows: *Gastrococcus indicus* is transferred to fresh BHIs medium at a volume ratio of 1%-5% and cultured anaerobically at 37°C for 24 hours.

[0024] On the other hand, the present invention also provides the application of the aforementioned *Gastrococcus in India* DA18, the aforementioned bacterial preparation, or the aforementioned metabiotic in the fight against pathogenic fungi.

[0025] According to a specific embodiment of the present invention, the anti-pathogenic fungus refers to inhibiting the growth of pathogenic fungi and / or killing pathogenic fungi.

[0026] According to a specific embodiment of the present invention, the pathogenic fungi include one or more of the following enteric pathogenic fungi: Candida albicans, Candida parapsilosis, and Candida glabrata.

[0027] According to a specific embodiment of the present invention, the *Macrococcus indicus*, the bacterial preparation, or the metabiotic are products used to prepare antifungal agents.

[0028] According to a specific embodiment of the present invention, preferably, the antifungal product is a drug (e.g., a microecological drug), a health food, feed, or an in vitro disinfectant.

[0029] On the other hand, the present invention also provides an antifungal preparation containing the *Macrococcus indicus* DA18 described in the present invention, the fungal preparation described therein, and / or the metabiotic described therein.

[0030] According to a specific embodiment of the present invention, the antifungal preparation may further include one or more additives: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

[0031] According to a specific embodiment of the present invention, the antifungal preparation can be a powder, tablet, granule, suppository, microcapsule, ampoule, or liquid spray.

[0032] In summary, this invention provides an enterogenic strain of *Macrococcus indicus* with anti-Candida activity and its application in developing into an anti-Candida preparation. This invention addresses the shortcomings of existing antifungal preparations, such as poor biosafety and weak antifungal activity, and can inhibit infection at its source by preventing Candida colonization. Attached Figure Description

[0033] Figure 1 This is a morphological diagram of Megasphaera indica CGMCC No. 1.18064, which is the subject of this invention.

[0034] Figure 2This image shows the results of detecting the inhibitory activity of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 on pathogenic fungal growth in BHIs medium. Note: BHI-BHIs: BHIs medium supernatant without bacterial inoculation served as a negative control; BHI-PBS: sterile PBS buffer served as a negative control; BHI-Bb: fermentation supernatant containing the probiotic strain Bifidobacterium bifidum served as a negative control; BHI-Mi: fermentation supernatant containing Megasphaera indica CGMCC No. 1.18064 (Mi) served as the experimental group.

[0035] Figure 3 This image shows the results of the activity test of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 in BHI medium against pathogenic fungi. Note: Initial Candida, the amount of Candida inoculated during co-culture; Candida+Mi, the number of viable Candida cells after co-culturing the fermentation supernatant of Candida and Megasphaera indica CGMCC No. 1.18064 (Mi) for 48 hours.

[0036] Microbial preservation for patented procedures:

[0037] Megasphaera indica DA18:

[0038] Date of deposit: August 5, 2024;

[0039] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

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

[0041] Accession number: CGMCC No.1.18064;

[0042] Classification and nomenclature: Megasphaera indica. Detailed Implementation

[0043] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and reagents used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described in the embodiments of this invention may be used to implement this invention.

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

[0046] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly 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, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment / implementation, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0048] In each embodiment, the experimental method for the test strain to kill pathogenic fungi was as follows: the pathogenic fungi activated overnight in YPD medium were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer; the concentration of pathogenic fungal cells was adjusted using a cell counting chamber at 1-5 × 10⁻⁶. 5 The final cell / well concentration of the treated pathogenic fungal cells was inoculated into 96-well plates containing fresh BHI medium. The treated bacterial fermentation broth was added to the 96-well plates at a 1:1 volume ratio, and the plates were incubated at 37°C and 600 rpm for 24 hours with shaking. The growth curve of the pathogenic fungus was recorded. The cultured bacterial solution was diluted and spread onto YPD agar plates, incubated statically at 37°C for 24 hours, and the number of surviving pathogenic fungi was counted. The YPD medium formulation was as follows: 20.0 g tryptone, 10.0 g yeast extract, 20.0 g glucose, 1000 mL distilled water, and 20.0 g agar for solids. The medium was sterilized at 121°C for 15 minutes.

[0049] Example 1: Screening and identification of Megasphaera indica strains

[0050] Using a sterile sampler, the air-free portion of a healthy volunteer's stool was collected and dissolved in an appropriate amount of sterile water. 100 μL of the stool solution was serially diluted 10-fold and spread onto BHIs agar plates. The plates were incubated at 37°C for 48 hours in an anaerobic environment. White, round colonies with well-defined edges were picked and examined under a microscope. Microscopic examination revealed extremely small, spindle-shaped bacterial cells. Repeated streaking was used to confirm pure colonies, and the resulting bacterial strain was named DA18. See attached image for strain morphology. Figure 1 .

[0051] The formula for the BHIs culture medium is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride to the sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0052] The strain was inoculated into BHI liquid medium, and after 16S rRNA gene sequence analysis, the strain was identified as Megasphaera indica.

[0053] The above-mentioned giant coccidia DA18 was grown in BHI medium, and its optimal growth temperature was 37℃.

[0054] The aforementioned giant coccidia DA18 was grown in BHIs medium. After 24 hours of anaerobic static culture, the cell density reached 1.14 ± 0.03, and the pH was 6.0.

[0055] The aforementioned strain, Megasphaera indica DA18, was deposited on August 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences). The deposit date is August 5, 2024, the accession number is CGMCC No. 1.18064, and the classification name is Megasphaera indica.

[0056] Example 2: Preparation of active fermentation broth against pathogenic fungi from Megasphaera indica in BHI medium

[0057] A single colony of Megasphaera indica CGMCC No. 1.18064 was inoculated into fresh BHI medium and cultured in an anaerobic chamber at 37°C for 24 h. The activated Megasphaera indica culture was then transferred to fresh BHI medium at a volume ratio of 2% and cultured in an anaerobic chamber at 37°C for 24 h. The bacterial culture was collected, centrifuged at 10,000 rpm for 10 min to remove bacterial cells, and the supernatant was filtered through a 0.22 μm filter membrane. The resulting fermentation broth was stored at -80°C for later use.

[0058] The formula for the BHIs culture medium is as follows: 10.0g tryptone, 17.5g beef heart powder, 5.0g sodium chloride, 2.0g glucose, 2.5g disodium hydrogen phosphate, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min. Add filtered and sterilized vitamin K3 and heme chloride to the sterilized culture medium to make their final concentrations 1mg / L and 5mg / L, respectively.

[0059] Example 3: Application of Megasphaera indica active fermentation broth in inhibiting the growth of pathogenic fungi

[0060] Pathogenic fungi *Candida albicans*, *Candida parapsilosis*, and *Candida glabrata* isolated from the human gut were streaked onto YPD agar plates for resuscitation and incubated at 37°C for 48 h. Single colonies of these pathogenic fungi were picked and cultured in fresh YPD liquid medium at 37°C and 200 rpm for 18 h with shaking. The activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer. The suspension was centrifuged at 3000 rpm for 1 min. The resuspended pathogenic fungal suspension was serially diluted 10-fold twice. 10 μL of the 100-fold diluted suspension was transferred to the cell counting chamber, and the cell concentration was read under a regular optical microscope. The concentration was then determined by adding 1-5 × 10⁻⁵ cells per cell. 5 The final cell / well concentration was inoculated into the diluted pathogenic fungal suspension in a 96-well plate containing fresh BHIs medium; 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 an enzyme-linked immunosorbent assay (ELISA) reader and cultured at 37°C and 600 rpm for 24 h with shaking, and the growth curve of the pathogenic fungus was recorded.

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

[0062] The YPD culture medium formula is as follows: 20.0g tryptone, 10.0g yeast extract, 20.0g glucose, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min.

[0063] Figure 2 This paper presents the results of an inhibitory activity test on pathogenic fungal growth by the fermentation broth of *Megasphaera indica* CGMCC No. 1.18064 in BHIs medium. The results show that: BHI-BHIs (BHIs medium supernatant without bacterial inoculation) served as a negative control; BHI-PBS (sterile PBS buffer) served as a negative control; BHI-Bb (fermentation supernatant of the probiotic strain *Bifidobacterium bifidum*) served as a negative control; and BHI-Mi (fermentation supernatant of *Megasphaera indica* CGMCC No. 1.18064 (Mi)) served as the experimental group. It can be seen that the fermentation broth of *Megasphaera indica* CGMCC No. 1.18064 of this invention exhibits inhibitory activity against pathogenic fungal growth.

[0064] Example 4: Application of Megasphaera indica active fermentation broth in killing pathogenic fungi

[0065] Pathogenic fungi *Candida albicans*, *Candida parapsilosis*, and *Candida glabrata* isolated from the human gut were streaked onto YPD agar plates for resuscitation and incubated at 37°C for 48 h. Single colonies of these pathogenic fungi were picked and cultured in fresh YPD liquid medium at 37°C and 200 rpm for 18 h with shaking. The activated pathogenic fungi were washed once with sterile PBS buffer and resuspended in an equal volume of sterile PBS buffer. The suspension was centrifuged at 3000 rpm for 1 min. The resuspended pathogenic fungal suspension was serially diluted 10-fold twice. 10 μL of the 100-fold diluted suspension was transferred to the cell counting chamber, and the cell concentration was read under a regular optical microscope. The concentration was then determined by adding 1-5 × 10⁻⁵ cells per cell. 5The final cell / well concentration was inoculated into the diluted pathogenic fungal suspension in a 96-well plate containing fresh BHIs medium. The bacterial active fermentation broth treated in Example 2 was added to the 96-well plate at a 1:1 volume ratio, and the plate was incubated in a microplate reader at 37°C and 600 rpm with shaking for 24 h. The cultured bacterial solution was diluted and spread on YPD agar plates, and after incubation at 37°C for 24 h, the number of surviving pathogenic fungi was counted.

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

[0067] The YPD culture medium formula is as follows: 20.0g tryptone, 10.0g yeast extract, 20.0g glucose, 1000mL distilled water, and 20.0g agar for solids. Sterilize at 121℃ for 15min.

[0068] Figure 3 This paper presents the results of the fungicidal activity test of the fermentation broth of *Megasphaera indica* CGMCC No. 1.18064 in BHI medium. Wherein: Initial Candida, the inoculum amount of *Candida* during co-culture; Candida+Mi, the viable count of *Candida* after co-culturing the fermentation supernatant of *Candida* and *Megasphaera indica* CGMCC No. 1.18064 (Mi) for 48 hours. It can be seen that the fermentation broth of *Megasphaera indica* CGMCC No. 1.18064 of this invention has fungicidal activity.

[0069] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. For example, *C. praecoxibacillus indicus* CGMCC can be used. No. 1.18064 can be used to prepare anti-Candida preparations with other antifungal active substances, such as other strains of *Macrococcus ininus* (e.g., using *Macrococcus ininus* from other intestinal sources to prepare anti-Candida probiotics, or to prepare probiotics, health foods, feeds, or in vitro disinfectants based on its fermentation supernatant or metagener). It can also be used to prepare anti-Candida active fermentation broth based on *Macrococcus ininus* using other culture media (e.g., using YCFA medium, GAM medium, chemically defined medium, or other culture media capable of culturing *Macrococcus inininus* to prepare anti-Candida probiotics, or to prepare probiotics, health foods, feeds, or in vitro disinfectants based on its fermentation supernatant or metagener). It can also be used to inhibit other pathogenic fungi (e.g., to inhibit or kill Candida albicans, *Candida parapsilosis*, and *Candida glabrata*, and other Candida species and pathogenic fungi) to achieve the same purpose. Therefore, all equivalent technical solutions also fall within the scope of protection of this invention.

Claims

1. A species of giant coccidia in India ( Megasphaera indica It has the accession number CGMCC No. 1.18064.

2. A bacterial preparation containing the *Macrococcus indicus* as described in claim 1.

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

4. An epigenetic agent, which is an epigenetic agent containing bacterial cells and metabolites obtained by fermenting and culturing the Indian giant cocci of claim 1 in a culture medium.

5. The epigenetic agent according to claim 4, wherein, The culture medium is BHIs medium.

6. The epigenetic agent according to claim 5, wherein, The fermentation product obtained by fermenting the Indian giant cocci in a culture medium according to claim 1 is a metabiotic containing bacterial cells and metabolites.

7. The epigenetic agent according to claim 6, wherein, The post-genetic agent is further dried to prepare a powder.

8. A method for culturing the *Macrococcus indicus* of claim 1, the method comprising: The Indian giant cocciformis according to claim 1 is inoculated into a culture medium for fermentation culture.

9. The method according to claim 8, wherein, The culture medium is one or more of BHIs medium, YCFA medium, and GAM medium.

10. The method according to claim 9, wherein, The culture conditions are anaerobic.

11. The use of the *Macrococcus indicus* of claim 1, the fungal preparation of claim 2 or 3, or the metabiotic of claim 4 in the preparation of a pharmaceutical product or in vitro disinfectant against pathogenic fungi; in, The pathogenic fungus is Candida albicans ( Candida albicans ), Candida parapsilosis ( Candida parapsilosis ) and Candida glabrata ( Candida glabrata One or more of the following.

12. An antifungal preparation comprising the *Macrococcus indicus* of claim 1, the fungal preparation of claim 2 or 3, and / or the metabiotic of claim 4.

13. The antifungal preparation according to claim 12, wherein the antifungal preparation further comprises one or more additives: excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof.

14. The antifungal preparation according to claim 12, wherein the antifungal preparation is a powder, tablet, suppository, microcapsule, ampoule or liquid spray.

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