CN110881479A - Bacteriostatic application of freshwater fungus Dictyochaeta aquatica - Google Patents
Bacteriostatic application of freshwater fungus Dictyochaeta aquatica Download PDFInfo
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- 241000233866 Fungi Species 0.000 claims abstract description 30
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 18
- 241000588724 Escherichia coli Species 0.000 claims abstract description 16
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 14
- 241000607768 Shigella Species 0.000 claims abstract description 13
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 13
- 241000607142 Salmonella Species 0.000 claims abstract description 10
- 229930000044 secondary metabolite Natural products 0.000 claims abstract description 10
- 241000186781 Listeria Species 0.000 claims abstract description 8
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
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- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a freshwater fungusDictyochaeta aquaticaThe application of the strain is the application of the secondary metabolite of the freshwater fungus in inhibiting food-borne pathogenic bacteria, and the metabolite of the fungus has good bacteriostatic effects on staphylococcus aureus, listeria, bacillus subtilis, escherichia coli, shigella and salmonella, and has market popularization and application values.
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 fungusDictyochaeta aquatica) The secondary metabolite of (2) is used for inhibiting food-borne pathogenic bacteria.
Background
Statistically, more than 3000 species of freshwater fungi are identified and isolated in total. However, only 87 strains identified as genus of freshwater fungi or species isolated from freshwater have been found to report on the record of bacteriostatic activity studies, and 82 strains among them are active in inhibiting fungi or bacteria.
The obtained product can inhibit pathogenic bacteria of food origin, and promote species diversity increase. For example, some fungi are reported to be pathogens —Candida albicans(Candida albicans). Some fungal metabolites have been reported to have antibacterial activity, such as lincomycin; there are also a number of applications in the food industry such as:pigmentmentalmonascus (monascus pigment) lays a foundation for the development and utilization of subsequent fungi.
Disclosure of Invention
The invention aims to provide a new application of freshwater fungi, namely freshwater fungiDictyochaeta aquaticaThe use of the metabolite of (a) in inhibiting food-borne pathogenic bacteria.
The food-borne pathogenic bacteria is staphylococcus aureus (A)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.）。
The purpose of the invention is realized by the following technical scheme:
(1) culturing fresh water fungusDictyochaeta aquaticaCulturing 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 liquid culture medium, and culturing 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, filtering the extracted ethyl acetate, 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 inventionDictyochaeta aquatica) Is obtained by separation from bamboo segments in the small rivers of the division of the Thailand of Bashu, and the contents of the separation and identification thereof are described in the documents "Ming-Jie W, Huang Z, Wei D, et al]Phytotaxa, 2018, 362(2):187- ".
The food-borne pathogenic bacteria are all purchased from Shanghai Lu micro-technology, Inc., Staphylococcus aureus: (S. aureus)Staphylococcus aureus) Strain number ATCC25923, 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 fungiDictyochaeta aquaticaThe 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 foodborne pathogenic bacteria, wherein a is Escherichia coli, b is Shigella, c is Salmonella, d is Staphylococcus aureus, e is Bacillus subtilis, f is Listeria, and g is DMSO blank control.
Detailed Description
The present invention is further illustrated by the following figures and examples, without limiting the scope of the invention thereto, wherein the process is carried out in a conventional manner unless otherwise specified, and wherein reagents are used, such as reagents used or formulated in a conventional manner, unless otherwise specified.
Example 1: fresh water fungusDictyochaeta aquaticaPreparation 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 addedDictyochaeta aquaticaInoculating 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)Dictyochaeta aquaticaBeating the mushroom cakes into fungus cakes with the diameter of 6 mm on a PDA culture medium by using a puncher, and then inoculating the fungus cakes into the liquid culture medium prepared in the step (3) to culture the fungus cakes on a constant temperature shaking bed at the temperature of 27 ℃ and at the speed of 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 1d 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) And Salmonella bacteriaSalmonellasp.) is inoculated on an LB agar culture medium, after 24 hours of culture at 37 ℃, a single colony is picked up and inoculated in a 15 mL centrifuge tube containing 5 mL of LB liquid culture medium, and the culture is carried out overnight at constant temperature of 37 ℃ and 160 rpm;
(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 liquid at the wavelength of 600nm, taking 7 bacterial liquids with different concentrations and absorbance values of 0.2-0.6, adding 100 mu L of each bacterial liquid onto an LB solid culture medium, coating, performing inverted culture in a 37 ℃ incubator for 24h, and counting bacterial colonies on a flat plate with the bacterial 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 10mg of the dried freshwater fungus metabolite in the example 1, adding 1mL of DMSO 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 | 13.02 | 10.42 | 12.77 | 11.83 | 12.38 | 11.3 |
| Mean deviation of | 1.41 | 0.68 | 1.01 | 1.03 | 0.56 | 0.65 |
From the above results, it can be seen that: the present invention relates to a fresh water fungusDictyochaeta aquaticaThe metabolite has good bacteriostatic effect on staphylococcus aureus, listeria, bacillus subtilis, escherichia coli, shigella and salmonella.
Claims (3)
1. Fresh water fungus: (Dictyochaeta aquatica) 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 fermentation, adding ethyl acetate into fermentation liquor for extraction after the fermentation is finished, 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 |
|---|---|---|---|---|
| CN118109317A (en) * | 2024-04-30 | 2024-05-31 | 江西农业大学 | Chloridium gonytrichii DZW4 strain 4 and application thereof in preparation of preparation for inhibiting pathogenic microorganisms |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5091413A (en) * | 1990-02-13 | 1992-02-25 | Merck & Co., Inc. | Antibiotic agent |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5091413A (en) * | 1990-02-13 | 1992-02-25 | Merck & Co., Inc. | Antibiotic agent |
Non-Patent Citations (7)
| Title |
|---|
| WEI MING-JIE，ZHANG HUANG: "《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》 * |
| WEI, MING-JIE，ZHANG, HUANG，DONG, WE，等: "《Introducing Dictyochaeta aquatica sp. nov. and two new species of Chloridium (Chaetosphaeriaceae, Sordariomycetes) from aquatic habitats》", 《PHYTOTAXA》 * |
| WU, HAO，YANG, HONGYAN，YOU, XIANGLING，等: "《Isolation and Characterization of Saponin-Producing Fungal Endophytes from Aralia elata in Northeast China》", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 * |
| 周德庆: "《微生物学教程》", 31 May 2002, 高等教育出版社 * |
| 孙燕: "《微生物学实验指导》", 31 August 2015, 陕西师范大学出版总社有限公司 * |
| 谢明勇，陈绍军: "《食品安全导论》", 30 September 2016, 中国农业大学出版社 * |
| 陕西省农林学校: "《微生物学》", 31 October 1984, 中国林业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118109317A (en) * | 2024-04-30 | 2024-05-31 | 江西农业大学 | Chloridium gonytrichii DZW4 strain 4 and application thereof in preparation of preparation for inhibiting pathogenic microorganisms |
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