CN110882284A - New bacteriostatic application of fresh water fungus clostridium aquaticus - Google Patents
New bacteriostatic application of fresh water fungus clostridium aquaticus Download PDFInfo
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- 239000013505 freshwater Substances 0.000 title claims abstract description 32
- 241000233866 Fungi Species 0.000 title claims abstract description 30
- 230000003385 bacteriostatic effect Effects 0.000 title abstract description 6
- 241000193403 Clostridium Species 0.000 title 1
- 241000588724 Escherichia coli Species 0.000 claims abstract description 13
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 12
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 12
- 241000607768 Shigella Species 0.000 claims abstract description 11
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 11
- 241000607142 Salmonella Species 0.000 claims abstract description 9
- 241000186781 Listeria Species 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 20
- 244000052616 bacterial pathogen Species 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 238000009630 liquid culture Methods 0.000 claims description 10
- 229930000044 secondary metabolite Natural products 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 241000186779 Listeria monocytogenes Species 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 3
- 239000002024 ethyl acetate extract Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 241000355049 Clohesyomyces aquaticus Species 0.000 claims 1
- 238000001035 drying Methods 0.000 claims 1
- 239000002207 metabolite Substances 0.000 abstract description 9
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000012258 culturing Methods 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 6
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- 239000000725 suspension Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 2
- 235000017491 Bambusa tulda Nutrition 0.000 description 2
- 241001330002 Bambuseae Species 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000011425 bamboo Substances 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
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- 241000607149 Salmonella sp. Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
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- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
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- 230000002538 fungal effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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- 210000002429 large intestine Anatomy 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a freshwater fungusClohesyomyces aquaticusThe separated metabolite of the fungus has good bacteriostatic effect on staphylococcus aureus, listeria, bacillus subtilis, escherichia coli, shigella and salmonella.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a new application of a freshwater fungus, specifically to a freshwater fungusClohesyomyces aquaticus) The secondary metabolite of (2) is used for inhibiting food-borne pathogenic bacteria.
Background
According to related reports, the freshwater fungus is considered to have great potential as a natural resource treasury which is hardly developed, and particularly, the freshwater fungus is increasingly concerned due to the complex metabolic pathway and the various metabolic products. According to incomplete statistics, the number of active metabolites isolated from freshwater fungi has reached 8,600, which can be assigned to various structural types.
Statistically, more than 3000 freshwater fungi are identified and isolated, but only a small part of the fungi have been reported to have the records of bacteriostatic activity research. The obtained substance has the advantages of short period, low cost, and good biological activity of resisting bacteria and virus. The fungi are widely distributed in China, the number of species is large, and the method has important significance for comprehensive utilization and deep development of freshwater fungi resources in China.
Disclosure of Invention
The invention aims to provide a freshwater fungusClohesyomyces aquaticusThe new application of the metabolite, namely the application of the secondary metabolite in inhibiting the food-borne pathogenic bacteria.
The purpose of the invention is realized by the following technical scheme:
(1) culturing fresh water fungusClohesyomyces aquaticusCulturing on PDA culture medium, and placing in a thermostat for inversion and amplification culture;
(2) when the freshwater fungi which is enlarged and cultured in the step (1) grows out colonies with a larger area, beating the freshwater fungi into fungus cakes by using a puncher;
(3) preparing a PDB liquid culture medium, inoculating the bacterial cake obtained in the step (2) into the PDB liquid culture medium, and fermenting the liquid culture medium inoculated with the bacterial cake;
(4) and after the fermentation is finished, adding ethyl acetate with the same volume as the liquid culture medium into the fermentation liquor, performing ultrasonic extraction, standing, filtering out thalli, collecting an ethyl acetate extraction layer, and filtering and concentrating to obtain a secondary metabolite of the freshwater fungus.
The application of the invention is that the secondary metabolite is used as an active substance to be prepared into a medicament or other product forms which are convenient to utilize, and one or more pharmaceutically acceptable auxiliary materials can be added, wherein the auxiliary materials comprise conventional filling agents, diluents, adhesives, excipients, absorption promoters, filling agents, surfactants, stabilizers and the like in the pharmaceutical field, and flavoring agents, pigments, sweetening agents and the like can be added as necessary.
The application of the invention can be prepared into various forms such as pills, powder, tablets, granules, oral liquid, injection and the like besides capsules.
The freshwater fungus of the inventionClohesyomyces aquaticus) Is obtained by separating bamboo segments from the small river of Sichuan city, Thailand, and the separation and identification of the bamboo segments are disclosed in the literature Zhang H, Dong W, Hyde K D, et al]Fungal diversity, 2017, 85(6):1-36 ".
The food-borne pathogenic bacteria are all purchased from Shanghai Lu micro-technology, Inc., Staphylococcus aureus: (S. aureus)Staphylococcus aureus) Strain number ATCC29213, Listeria (Listeria monocytogenes) Strain number ATCC19115, Bacillus subtilis ((Bacillus subtilis))Bacillus subtilis) Strain number ATCC6633, Escherichia coli: (Escherichia coli) Strain number ATCC25922, Shigella (A)Shigella Castellani) Strain number CMCC51105, Salmonella (Salmonella)Salmonellasp.) Strain number CMCC 50093.
The invention has the advantages and technical effects that:
the invention provides a new method for preventing and inhibiting food-borne pathogenic bacteria, and the invention provides a new method for preventing and inhibiting freshwater fungiClohesyomyces aquaticusThe secondary metabolite has good bacteriostatic effect on staphylococcus aureus, listeria, bacillus subtilis, escherichia coli, shigella and salmonella, and the secondary metabolite is simple to prepare, short in period, low in cost and wide in market application prospect.
Drawings
FIG. 1 shows the results of experiments on the inhibition of the metabolites of freshwater fungi against six food-borne pathogenic bacteria, wherein a is Escherichia coli, b is Staphylococcus aureus, c is Bacillus subtilis, d is Listeria, e is Salmonella, f is Shigella, and g is DMSO blank.
Detailed Description
The present invention is further illustrated by the following figures and examples, without limiting the scope of the invention thereto, in which the process is carried out in a conventional manner unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared in a conventional manner unless otherwise specified.
Example 1: fresh water fungusClohesyomyces aquaticusPreparation of secondary metabolites
(1) Preparation of solid medium: weighing PDA culture medium, adding distilled water for dissolving, sterilizing at 121 deg.C for 20 min in a high pressure steam sterilizing pot, and uniformly pouring the sterilized culture medium into a culture dish;
(2) fresh water fungus amplification culture: after the PDA culture medium in the culture dish in the step (1) is completely solidified, the freshwater fungi are addedClohesyomyces aquaticusInoculating the mycelium onto culture medium, sealing with sealing film, placing upside down in constant temperature incubator, and culturing at 27 deg.C;
(3) preparing a liquid culture medium: weighing PDB culture medium, placing in a 250 mL conical flask, adding distilled water for dissolving, and sterilizing in a high-pressure steam sterilization pot at 121 ℃ for 20 min to obtain PDB liquid culture medium;
(4) liquid fermentation of freshwater fungi: culturing the freshwater fungi cultured in the step (2)Clohesyomyces aquaticusBeating a fungus cake with the diameter of 6 mm on a PDA culture medium by using a puncher, and inoculating the fungus cake into the liquid culture medium prepared in the step (3), and culturing on a constant temperature shaking table at the condition of 27 ℃ and 160 r/min;
(5) preparation of a freshwater fungus metabolite: adding ethyl acetate with the same volume as the liquid culture medium into the fermentation liquor obtained by the liquid fermentation in the step (4), ultrasonically extracting for 30 min, standing for 1 day after extraction, filtering the thalli by using a filter screen, separating liquid by using a separating funnel, and taking an upper ethyl acetate layer to obtain an extract liquid;
(6) concentrating the fresh water fungus fermentation extract: and (4) filtering the ethyl acetate extract liquor obtained in the step (5) by using filter paper, and evaporating on a rotary evaporator at the temperature of 38 ℃ at the speed of 80 r/min to obtain a metabolite of the freshwater fungus.
Example 2: experiment for inhibiting food-borne pathogenic bacteria by secondary metabolites of freshwater fungi
(1) Preparation of bacterial liquid culture medium: weighing LB broth culture medium, adding distilled water for dissolving, sterilizing at 121 deg.C for 20 min in high pressure steam sterilizing pot, and cooling;
(2) activation of pathogenic bacteria: staphylococcus aureus (A), (B), (C)Staphylococcus aureus) Listeria (Listeria monocytogenes)Listeria monocytogenes) Bacillus subtilis preparation (B)Bacillus subtilis) Escherichia coli (E.coli)Escherichia coli) Shigella (A) and (B)Shigella Castellani) Or Salmonella bacteria (A), (B)Salmonella sp.) Inoculating on LB agar culture medium, culturing at 37 deg.C for 24h, picking single colony, inoculating in 15 mL centrifuge tube containing 5 mL LB liquid culture medium, and culturing at 37 deg.C and 160rpm overnight;
(3) standard curve for pathogenic bacterial suspension: placing 4 mL of activated pathogenic bacteria liquid into 10000 r/min, centrifuging for 1min at 4 ℃, adding physiological saline into the precipitate, uniformly mixing, centrifuging for 1min at 4 ℃ and 10000 r/min, removing supernatant, repeatedly suspending with 5 mL of physiological saline twice to obtain bacterial suspension, uniformly mixing by vortex oscillation, performing gradient dilution with the physiological saline, measuring the absorbance of the diluted bacterial solution at the wavelength of 600 nm, taking 7 bacterial solutions with different concentrations and absorbance values of 0.2-0.6, adding 100 mu L of each bacterial solution onto an LB solid culture medium, coating, performing inverted culture in a 37 ℃ incubator for 24h, and counting colonies on a flat plate with the colony number of 30-300; drawing a standard curve according to the colony number and the absorbance value to obtain a calculation formula:
gram-positive bacteria (gold dextran example) colonies: y = 613.68 x-10.921;
gram-negative bacteria (large intestine as an example) colonies: y = 142.4x + 110.1;
(4) preparation of bacterial suspension: adjusting the concentration of pathogenic bacteria to 0.8-2 × 10 with physiological saline according to standard curve of pathogenic bacteria suspension5cfu/mL；
(5) Bacteriostatic test of fermentation extract: weighing 10 mg of the dried freshwater fungus metabolite in the example 1, adding 1 mLDMSO for dissolving, and preparing a sample with the concentration of 10 mg/mL; adding 80 mu L of pathogenic bacterium liquid on a flat plate of LB agar, and uniformly coating; 3 Oxford cups were placed per plate and 10. mu.L of sample was added to each Oxford cup; horizontally placing, culturing at 37 deg.C for 18-24 h, observing, measuring the diameter of the zone with vernier caliper, averaging the three groups of data for each pathogenic bacteria, and obtaining the experimental results shown in FIG. 1 and Table 1;
TABLE 1 metabolite diameter (unit: mm) for inhibition zone of six foodborne pathogenic bacteria
| Bacterial strain | Blank space | Escherichia coli | Staphylococcus aureus | Shigella | Salmonella | Listeria monocytogenes | Bacillus subtilis |
| Mean value of | 8 | 11.11 | 10.40 | 12.72 | 11.33 | 12.00 | 13.73 |
| Mean deviation of | 0.31 | 0.49 | 1.31 | 0.53 | 1.15 | 0.43 |
From the above results, it can be seen that: the present invention relates to a fresh water fungusClohesyomyces aquaticusThe metabolite has good bacteriostatic effect on staphylococcus aureus, listeria, bacillus subtilis, escherichia coli, shigella and salmonella.
Claims (3)
1. Fresh water fungus: (Clohesyomyces aquaticus) The secondary metabolite of (2) is used for inhibiting food-borne pathogenic bacteria.
2. Use according to claim 1, characterized in that: the secondary metabolite of the freshwater fungus is prepared by inoculating the freshwater fungus into a PDB liquid culture medium for liquid fermentation, adding ethyl acetate into fermentation liquor for extraction after the fermentation is finished, filtering out thalli after standing, collecting ethyl acetate extract, filtering, concentrating and drying.
3. Use according to claim 1, characterized in that: the food-borne pathogenic bacteria is Staphylococcus aureus (S.aureus)Staphylococcus aureus) Listeria (Listeria monocytogenes)Listeria monocytogenes) Bacillus subtilis preparation (B)Bacillus subtilis) Escherichia coli (E.coli)Escherichia coli) Shigella (A) and (B)Shigella Castellani) Or Salmonella bacteria (A), (B)Salmonellasp.）。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022174313A1 (en) * | 2021-02-22 | 2022-08-25 | Loam Bio Pty Ltd | Methods for carbon capture and increasing yield of crop plants |
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2019
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Non-Patent Citations (1)
| Title |
|---|
| WEI MING-JIE等: "Screening of antimicrobial secondary metabolites from freshwater fungi", 《中国菌物学会2018年学术年会论文汇编中国菌物学会会议论文集》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022174313A1 (en) * | 2021-02-22 | 2022-08-25 | Loam Bio Pty Ltd | Methods for carbon capture and increasing yield of crop plants |
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