Intestinal-derived megasphaera indica having Anti-candida activity and use thereof

Abstract

Disclosed are an intestinal-derived Megasphaera indica having anti-Candida activity and a use thereof. Specifically, provided is a Megasphaera indica, having the accession number CGMCC No.1.18064. The Megasphaera indica can inhibit and kill pathogenic candida, and has good biological safety.

Description

Intestinal Megasphaera indica with anti-candida activity and application thereof TECHNICAL FIELD

[0001] The present application relates to Megasphaera indica and application thereof, in particular to an intestinal Megasphaera indica with anti-candida activity and application thereof. BACKGROUND

[0002] According to the list of priority pathogenic fungi published by the World Health Organization, Candida albicans, Candida parapsilosis, Candida glabrata and the like which colonize in the human intestinal tract belong to pathogenic fungi which pose the greatest threat to public health. At present, the drugs clinically available for treating the infection of the above-mentioned pathogenic fungi only include polyene, triazole and echinocandin drugs. With the long-term use of a single drug, the drug resistance of the above-mentioned pathogenic fungi is increasingly 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 the 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 the like 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 an intestinal anaerobic bacterium which is isolated from a stool sample of a healthy volunteer, can inhibit and kill Candida albicans, Candida parapsilosis, Candida glabrata and the like 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 97.38% with that of Megasphaera indica strain RH115, is determined as Megasphaera indica of the same species but different strains, is named as Megasphaera indica DA18 in the present application, and the strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: No. 1, Yikhinxi Lu, Chaoyang Qu, Beijing, China Academy of Microbiology), the preservation date is August 5, 2024, the preservation number is CGMCC No. 1.18064, and the classification name is Megasphaera indica.

[0007] SEQ ID No. 1:

[0008] The intestinal source of the anaerobic bacteria Megasphaera indica DA18 of the present application, i.e. Megasphaera indica with the preservation number CGMCC No. 1.18064, 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 preventing the colonization of Candida from the source.

[0009] Therefore, in one aspect, the present application provides a Megasphaera indica, which has the preservation number CGMCC No. 1.18064.

[0010] In another aspect, the present application also provides a bacterial preparation containing the Megasphaera indica of the present application (i.e. Megasphaera indica DA18, i.e. Megasphaera indica with the preservation number CGMCC No. 1.18064).

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

[0012] In another aspect, the present application also provides a postbiotic, which is a postbiotic containing bacterial cells and metabolic products obtained by fermentation culture of the Megasphaera indica DA18 of the present application in a culture medium, or a postbiotic containing fermentation products.

[0013] According to a specific embodiment of the present application, when the Megasphaera indica DA18 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.

[0014] According to a specific embodiment of the present application, when the Megasphaera indica DA18 of the present application is fermented and cultured in a culture medium, the culture condition is anaerobic culture.

[0015] According to a specific embodiment of the present application, the Megasphaera indica DA18 of the present application grows in BHIs culture medium, and the optimal growth temperature is 37℃.

[0016] According to a specific embodiment of the present application, the Megasphaera indica DA18 of the present application grows in BHIs culture medium, and after anaerobic static culture for 24h, the bacterial cell density reaches 1.14±0.02, and the pH is 6.0.

[0017] 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-sterilized medium to make the final concentrations thereof 1 mg / L and 5 mg / L, respectively.

[0018] In the present application, the postbiotic refers to the total metabolic products obtained by fermentation culture of the bacterial strain, and can contain bacterial bodies or further removal of the bacterial bodies to obtain metabolically active substances.

[0019] According to a specific embodiment of the present application, in the present application, the fermentation product obtained by fermentation culture of the M. indica DA18 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.

[0020] In another aspect, the present application further provides a method for culturing the M. indica DA18, which comprises:

[0021] The M. indica DA18 is inoculated into a culture medium for fermentation culture.

[0022] 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.

[0023] According to a specific embodiment of the present application, in the present application, more preferably, the culture conditions are as follows: the M. indica is transferred into fresh BHI medium at a volume ratio of 1%-5%, and then cultured anaerobically at 37 ℃ for 24 h.

[0024] In another aspect, the present application further provides the use of the M. indica DA18, the bacterial preparation or the postbiotic in resisting pathogenic fungi.

[0025] 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 the pathogenic fungi.

[0026] 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.

[0027] According to a specific embodiment of the present application, in the present application, the Megasphaera indica, the bacterial preparation or the postbiotic is used for preparing an anti-pathogenic fungal product.

[0028] 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.

[0029] In another aspect, the present application also provides an anti-pathogenic fungal preparation containing the Megasphaera indica DA18, the bacterial preparation and / or the postbiotic according to the present application.

[0030] 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.

[0031] 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.

[0032] In summary, the present application provides a strain of enteric Megasphaera indica with anti-Candida activity and its application in developing an anti-Candida preparation, which can solve the problems of poor biological safety and weak anti-fungal activity of existing anti-fungal preparations, and can also curb the infection from the source by preventing the colonization of Candida. BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 is a bacterial morphology diagram of the Megasphaera indica CGMCC No. 1.18064 of the present application.

[0034] Figure 2 is a graph showing the results of the detection of the activity of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application in BHI medium in inhibiting the growth of pathogenic fungi. Note: BHI-BHIs, BHI medium supernatant without added bacteria as negative control; BHI-PBS, sterile PBS buffer added as negative control; BHI-B.b., fermentation supernatant of probiotic strain Bifidobacterium bifidum as negative control; BHI-M.i., fermentation supernatant of Megasphaera indica CGMCC No. 1.18064 (M.i.) as experimental group.

[0035] Figure 3 is a graph showing the results of the detection of the activity of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application in BHI medium in killing pathogenic fungi. Note: Initial Candida, the amount of Candida added at the beginning of co-culture; Candida+M.i., the viable cell count of Candida after co-culture with the fermentation supernatant of Megasphaera indica CGMCC No. 1.18064 (M.i.) for 48 h.

[0036] Microorganism preservation for patent procedures:

[0037] Megasphaera indica DA18:

[0038] Preservation date: August 5, 2024;

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

[0040] Address of the preservation unit: No. 1, Yihuangyuan, Beichen West Road, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences;

[0041] Preservation number: CGMCC No. 1.18064;

[0042] Classification name: Megasphaera indica. DETAILED DESCRIPTION

[0043] 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 specific specific embodiments described below; it should also be understood that the terms used in the embodiments of the present application are used to describe the specific specific embodiments, but are not intended to limit the scope of protection of the present application.

[0044] 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.

[0045] Unless otherwise indicated, the experimental methods, assays, and preparation methods disclosed herein are conventional techniques in the art.

[0046] For the purposes of the present application, the following terms are defined below.

[0047] 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.

[0048] In each embodiment, the 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 cells 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

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

[0050] 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 medium agar plate. After 48 h of culture at 37℃ in an anaerobic box, a white round and edge neat colony was picked up, and microscopic examination showed a small shuttle-shaped cell. The pure colony was repeatedly streaked to determine the pure colony, and the strain was named DA18. The strain morphology is shown in Figure 1.

[0051] The formula of the above BHIs 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 medium to make the final concentrations 1 mg / L and 5 mg / L, respectively.

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

[0053] The above Megasphaera indica DA18 grew in BHIs medium, and the optimum growth temperature was 37℃.

[0054] The above Megasphaera indica DA18 grew in BHIs medium, and after 24 h of anaerobic static culture, the cell density reached 1.14±0.03, and the pH was 6.0.

[0055] The above Megasphaera indica DA18 strain was preserved in the China General Microbiological Culture Collection Center (CGMCC) (address: No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences) on August 5, 2024, the preservation date was August 5, 2024, the preservation number was CGMCC No. 1.18064, and the classification and naming was Megasphaera indica.

[0056] Example 2: Preparation of anti-pathogenic fungal active fermentation liquor of Megasphaera indica in BHIs medium

[0057] The single colony of Megasphaera indica CGMCC No. 1.18064 was inoculated into fresh BHIs medium and cultured at 37°C for 24h in an anaerobic box; the activated Megasphaera indica was inoculated into fresh BHIs medium at a volume ratio of 2% and cultured at 37°C for 24h in an anaerobic box; the bacterial culture solution was collected and 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 obtained fermentation liquor was stored at -80°C for use.

[0058] The formula of the above BHIs 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 which needs to be added with solid. The medium was sterilized at 121°C for 15 min. Filter-sterilized vitamin K3 and hemin were added to the sterilized medium to make the final concentrations 1 mg / L and 5 mg / L, respectively.

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

[0060] The pathogenic fungi Candida albicans, Candida parapsilosis and Candida glabrata isolated from human intestinal tract were streaked on YPD medium agar plates and incubated at 37°C for 48h. Single colonies of the pathogenic fungi were picked into fresh YPD liquid medium and incubated at 37°C and 200 rpm for 18h. The activated pathogenic fungi were washed once with sterilized PBS buffer and resuspended in an equal volume of sterilized PBS buffer. The resuspended pathogenic fungi were diluted ten times successively, and 10 μL of the suspension with a dilution factor of 100 was taken to the technical chamber of a cell counting plate. The cell concentration was read under a general optical microscope. The pathogenic fungi were inoculated into fresh BHIs medium in a 96-well plate at a final concentration of 1-5×10 5 The diluted pathogenic fungi suspension was inoculated into the 96-well plate containing fresh BHIs medium at a final concentration of 1-5×10

[0061] The pathogenic fungi were inoculated into the 96-well plate containing fresh BHIs medium at 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.

[0062] 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.

[0063] FIG. 2 shows the detection results of the activity of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application in BHIs culture medium in inhibiting the growth of pathogenic fungi. Among them, BHI-BHIs, the supernatant of BHIs culture medium without bacteria added, is the negative control; BHI-PBS, the sterile PBS buffer added, is the negative control; BHI-B.b., the fermentation supernatant of probiotic strain Bifidobacterium bifidum added, is the negative control; BHI-M.i., the fermentation supernatant of Megasphaera indica CGMCC No. 1.18064 (M.i.) added, is the experimental group. It can be seen that the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application has the activity of inhibiting the growth of pathogenic fungi.

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

[0065] 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 pathogenic fungus suspension with a dilution of 100 times was inoculated in a 96-well plate containing fresh BHIs culture medium at a final concentration of 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.

[0066] wherein, the pathogenic fungus was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well, and then 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.

[0067] wherein, the pathogenic fungus was inoculated in a 96-well plate containing fresh BHIs medium at a final concentration of cells / well, and then 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.

[0068] FIG. 3 shows the detection results of the pathogenic fungus-killing activity of the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application in BHIs medium. Wherein: Initial Candida, the inoculation amount of Candida during co-culture; Candida + M.i., the viable cell count of Candida after co-culturing with the fermentation supernatant of Megasphaera indica CGMCC No. 1.18064 (M.i.) for 48 h. It can be seen that the fermentation broth of Megasphaera indica CGMCC No. 1.18064 of the present application has pathogenic fungus-killing activity.

[0069] 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 M. indicum CGMCC No. 1.18064 and other antifungal active substances, such as other M. indicum strains (such as using other M. indicum strains from intestinal source 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 other culture media can be used to prepare anti-candida active fermentation broth based on M. indicum (such as using YCFA medium, GAM medium, chemically defined medium and other culture media that can culture M. indicum 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 be used to inhibit other pathogenic fungi (such as being applied to inhibit or kill other Candida and pathogenic fungi other than C. albicans, C. parapsilosis and C. glabrata). Therefore, all equivalent technical solutions also belong to the protection scope of the present application.

Claims

1. A Megasphaera indica having a deposit number CGMCC No. 1.18064.

2. A bacterial preparation comprising the Megasphaera indica of claim 1.

3. The bacterial preparation of claim 2, which is a liquid or solid preparation in a viable or inactivated form.

4. A postbiotic, which is a postbiotic comprising bacterial cells and metabolites obtained by fermenting the Megasphaera indica 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 fermenting the Megasphaera indica 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 Megasphaera indica of claim 1, the method comprising: inoculating the Megasphaera indica of claim 1 in a culture medium for fermentation; 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 Megasphaera indica of claim 1, the bacterial preparation of claim 2 or 3, or the postbiotic of claim 4 in anti-pathogenic fungi.

7. Use according to claim 6, wherein, The anti-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 Megasphaera indica of claim 1, the bacterial preparation of claim 2 or 3, or the postbiotic of claim 4 is used for preparing an anti-pathogenic fungi product; Preferably, the anti-pathogenic fungi product is a medicine (e.g., a microecological medicine), a health food, a feed, or an in-vitro disinfectant.

10. An anti-pathogenic fungi preparation comprising the Megasphaera indica of claim 1, the bacterial preparation of claim 2 or 3, and / or the postbiotic of claim 4; Preferably, the anti-pathogenic fungi 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 fungi preparation is a powder, a tablet, a granule, a suppository, a microcapsule, an ampoule, or a liquid spray.

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