CN117337836A - Method for inhibiting listeria monocytogenes by using mulberry ketone G and application - Google Patents
Method for inhibiting listeria monocytogenes by using mulberry ketone G and application Download PDFInfo
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- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Plant Pathology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Communicable Diseases (AREA)
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- Organic Chemistry (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for inhibiting listeria monocytogenes and application thereof, which comprises the steps of putting a certain amount of mulberry ketone G and water into a spray can for stirring or vibrating to prepare a solution or suspension with the concentration of not less than 4 mug/mL, so that the listeria monocytogenes can be inhibited, the mulberry ketone G can be used for removing the listeria monocytogenes in a refrigerator, and an anti-listeria monocytogenes drug or a disinfection preparation can be prepared. The invention discovers that the mulberry ketone G has stronger sterilization performance on the listeria monocytogenes for the first time, determines the MIC and the MBC of the mulberry ketone G on the listeria monocytogenes, researches the effect of the mulberry ketone G on the growth curve of the listeria monocytogenes, researches the influence of the mulberry ketone G on the cell morphology of the listeria monocytogenes, and invents a method for inhibiting the listeria monocytogenes and application thereof.
Description
Technical Field
The invention belongs to the technical field of daily sterilization and disinfection, and particularly relates to a method for inhibiting listeria monocytogenes by using mulberry ketone G and application thereof.
Background
Listeria monocytogenesListeria monocytogenesLM) is abbreviated as listeria monocytogenes, which is facultative anaerobic gram-positive bacillus-free listeria, and is one of four foodborne pathogenic bacteria commonly existing in human and livestock. It can proliferate under a wide range of adverse environmental conditions including low temperature, low acid and high salt, and can cause serious human diseases such as sepsis, meningitis, and serious complications during pregnancy (abortion and stillbirth), with mortality rates of up to 20% -30%. Although listeria monocytogenes resistance is at a low level worldwide, listeria monocytogenes resistant strains are now being reported in more and more countries with long-term use and abuse of antibiotics in humans and animals.
The refrigerator refrigerating chamber is a food preservation container which is frequently used by people, is also a place where Listeria monocytogenes is easy to breed, and because of the harm of the Listeria monocytogenes, the Listeria monocytogenes needs to be removed regularly, and the use of common antibiotics is easy to generate drug resistance, has unsatisfactory effect and can pollute food stored in the refrigerator.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for inhibiting listeria monocytogenes by using the mulberry ketone G and application thereof on the basis of discovering that the mulberry ketone G has stronger sterilization performance on listeria monocytogenes for the first time through a large number of researches.
The method for inhibiting listeria monocytogenes by using the mulberry ketone G adopts the following steps:
the mulberry ketone G is prepared into a solution or suspension with the concentration not lower than 4 mug/mL, and then the solution or suspension is contacted with the listeria monocytogenes to inhibit the listeria monocytogenes,
if the Listeria monocytogenes in the refrigerator refrigerating chamber is eliminated, a certain amount of mulberry ketone G and water can be placed into a watering can for stirring or shaking to prepare a solution or suspension with the concentration of not less than 4 mug/mL, and then the watering can is aimed at the refrigerator refrigerating chamber for spraying.
Morone G can also be made into various forms of emulsion, oral preparation, etc. for treating diseases related to Streptococcus agalactiae infection, or disinfectant for killing Listeria monocytogenes.
The invention is based on the finding that the mulberry ketone G has stronger sterilization performance on the listeria monocytogenes, and determines the MIC and MBC of the mulberry ketone G on the listeria monocytogenes, researches the effect of the mulberry ketone G on the growth curve of the listeria monocytogenes, researches the influence of the mulberry ketone G on the cell morphology of the listeria monocytogenes, and finally makes the result that the result is simpler, but consumes a great deal of energy and creative labor.
The mulberry ketone G (Kuwanon G) is a flavonoid compound extracted from cortex Mori of Morus, which is root bark of Morus alba L of Morus genus of Moraceae family, and is also called white mulberry bark or root bark of Morus alba. Mulberry is a functional plant for both medicine and food, and is used as a medicine in the east and west. In China, the Chinese medicinal herbs can be traced to Shen nong Ben Cao Jing (Shen nong's herbal), and all the later generation herbal meridians are recorded. Although the mulberry ketone G has strong antibacterial activity on staphylococcus aureus, streptococcus faecalis, bacillus subtilis and certain mould, the mulberry ketone G also has the functions of anti-inflammatory, blood pressure reduction, enzyme activity inhibition, blood sugar reduction and the like. However, the antibacterial effect and the antibacterial mechanism of Guan Sangtong G on listeria monocytogenes are not reported in the domestic and foreign documents, and the killing and inhibiting effects of the mulberone G on listeria monocytogenes cannot be predicted in the prior art, so that the antibacterial effect and the antibacterial mechanism of the mulberone G on listeria monocytogenes are researched.
Materials and reagents: listeria ATCC19115, chinese collection of strains; brain heart infusion broth (Brain Heart Infusion, BHI), inc. Of microbiology, inc; mulberone G (Kuwanon G) is greater than 99% pure. Bacterial protein extraction kit: bacterial Protein Extraction Kit, shanghai worker; the remaining reagents were all analytically pure.
Main instruments and equipment: high-pressure steam sterilizing pot: ZEALWAY, a well-known scientific and technological development Co., ltd; biochemical incubator: SPX type, hangzhou Shuomen professional instrument factory; oscillating shaking table: SY-2230, shanghai Jingqi company; UV spectrophotometers, bioSpectrometer-D30, guangzhou Leider Biotechnology Co., ltd; german Karl Cai Sichang emission scanning electron microscope, model Sigma HD.
Bacterial liquid and liquid medicine preparation: preparing a bacterial suspension: inoculating the glycerol cryopreserved listeria monocytogenes into sterilized BHI broth, culturing at 37deg.C for 16 h to logarithmic phase, and calibrating bacterial concentration to 1×10 with BHI broth 8 CFU/mL, and stored in a refrigerator at 4 ℃ for standby. Morone G was prepared as a mother liquor at a concentration of 1024. Mu.g/mL using dimethyl sulfoxide as a solvent.
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of sannone G on listeria monocytogenes: the assay was performed in 96-well plates using a microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) method. Mulberone G with the concentration of 1024 mug/mL is added into a first column of a sterile 96-well plate, the mass concentration of the medicine is sequentially 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 mug/mL by adding BHI culture medium through a double dilution gradient method, and finally 100 mug of bacterial suspension is added. 200. Mu.L of bacterial suspension is additionally added as a positive control group; 200. Mu.L of medium was added as a negative control. 37. The culture medium is subjected to shaking culture at the temperature of 24 ℃ h, and the minimum concentration corresponding to a clarified culture medium is the MIC of the test liquid. Based on the minimum inhibitory mass concentration test, 100 μl of the culture medium was applied to BHI plate medium from well plate without obvious bacterial growth, and the result was observed after culturing in an incubator at 37 ℃ for 24 h. The minimum drug concentration for sterile drop growth on plate medium was taken as the minimum bactericidal mass concentration (MBC).
The MIC of the mulberone G for Listeria monocytogenes was found to be 4. Mu.g/mL and the MBC was found to be 16. Mu.g/mL. The result shows that the mulberry ketone G has stronger sterilization performance on listeria monocytogenes.
Effect of mulberone G on listeria monocytogenes growth curve: experimental methods As with the MIC and MBC assays, drug concentrations were studied at 1/2 MIC, 2MIC and no-dose groups to study the effect of Morone G on the growth curve of Listeria monocytogenes. The above cultures were incubated at 37℃in a 180r/min shaker, sampled at 2, 4, 6, 8, 10, 12, 16, 24, 36, h, and absorbance at OD600 nm was measured with an ultraviolet spectrophotometer. And (3) drawing a time dose response curve of the mulberone G to the listeria monocytogenes by taking time as an abscissa and a light absorption luminosity value as an ordinate.
The experimental results of the effect of mulberone G on the growth curve of Listeria monocytogenes are shown in FIG. 1, and the OD600 absorbance method is one of the methods for studying the growth of microorganisms in liquid media. The growth curve of the listeria monocytogenes is obviously changed after the listeria monocytogenes is subjected to the action of the mulberry ketone G, and the influence on the growth of the listeria monocytogenes is larger as the concentration of the drug is increased. The mulberry ketone G can inhibit the growth of bacteria at low concentration, delay the arrival of the bacterial log phase and is always smaller than the bacterial concentration of a blank control group; when the concentration of the mulberry ketone G is MIC, the mulberry ketone G is found to have obvious inhibition effect on the growth activity of listeria monocytogenes; and when the drug concentration in the bacterial liquid is 2MIC, the OD value of the bacterial liquid does not have a trend to increase, which indicates that the mulberry ketone G can completely inhibit the growth of Listeria monocytogenes.
Mulberone G effect on listeria monocytogenes protein synthesis: the bacterial suspensions were treated with various concentrations of mulberone G to reach MIC, 2MIC and blank, shake incubated at 37 ℃ for 16 h to log post growth, centrifuged at 8000 r/min for 10 min, washed three times with sterile PBS (0.1 mol/mL, ph=7.4) and protein extracted as described in bacterial protein extraction kit (Bacterial Protein Extraction Kit, shanghai). Subsequently, 15. Mu.L of the sample was subjected to SDS-PAGE, and after the electrophoresis was completed, the gel was stained with Coomassie Brilliant blue R250 overnight, then decolorized by 4-8 h, and finally photographed by a gel imager.
FIG. 2 shows the effect of mulberone G on the synthesis of listeria monocytogenes cellular protein, analyzed by SDS-PAGE electrophoresis with molecular weights ranging from 10 kDa to 75 kDa. The results show that the strips of the blank group are clear and bright, the influence degree of the mulberone G with different concentrations on the cell protein synthesis of the listeria monocytogenes is different, and the protein strips are less and less clear along with the higher the concentration of the mulberone G. When the concentration of the mulberry ketone G is MIC, the number of protein bands is obviously reduced (shown by a solid line frame), only three bands are clearly visible, and the rest bands are basically disappeared; when the concentration of moridone was 2MIC, the bands were significantly lighter (indicated by the dashed box). The mulberone G can obviously influence the synthesis of listeria monocytogenes protein, and the more obvious the influence on the protein is along with the increase of the drug concentration.
Effect of mulberone G on listeria monocytogenes cell morphology: the bacterial suspension in logarithmic phase was adjusted to 1X 10 with BHI medium 8 CFU/mL, mulberone G was added to give final concentrations of MIC and MBC, and no mulberone G was added as a blank. Shaking table (37deg.C, 180 r/min) to logarithmic phase, centrifuging at 8000 r/min for 10 min, washing with sterile PBS for 3 times, collecting thallus, and fixing with 2.5% glutaraldehyde solution overnight. And then sequentially dehydrating with 30.0%, 50.0%, 70.0%, 90.0% and 100.0% ethanol (for 2 times), drying, and finally spraying gold to prepare the sample.
FIG. 3 shows the effect of mulberry ketone G on the cell morphology of Listeria monocytogenes. The Listeria monocytogenes in the blank (FIG. 3A) was short bar-shaped, smooth and full cell surface, well-defined cell-cell morphology, and normal morphology. The cell membrane structure of listeria monocytogenes added with mulberone G (fig. 3B) was damaged and found to be distorted, and the cell morphology was significantly changed, so that some cells were broken. It was shown that mulberone G would disrupt the cell membrane of Listeria monocytogenes, thereby causing extracellular material to flow out and eventually die.
Compared with the prior art, the invention has the characteristics and beneficial effects that:
the invention discovers that the mulberry ketone G has stronger sterilization performance on the listeria monocytogenes for the first time, determines the MIC and MBC of the mulberry ketone G on the listeria monocytogenes, researches the effect of the mulberry ketone G on the growth curve of the listeria monocytogenes, researches the influence of the mulberry ketone G on the cell morphology of the listeria monocytogenes, and invents a method for inhibiting the listeria monocytogenes by using the mulberry ketone G and application thereof.
The present study initially explored the antibacterial activity of mulberone G against listeria monocytogenes. The minimum inhibitory concentration and the minimum bactericidal concentration of the mulberry ketone G on the listeria monocytogenes are respectively 4 mug/mL and 16 mug/mL, which are measured by a trace broth dilution method, and the mulberry ketone G has stronger antibacterial performance on the listeria monocytogenes in vitro. In recent years, research on natural-source traditional Chinese medicine extracts shows that the natural-source traditional Chinese medicine extracts are obviously different from the traditional antibiotic antibacterial mechanism, and have the advantages of difficult generation of drug resistance, few side effects, more action targets and the like, and become a research hotspot at home and abroad.
The influence of the mulberry ketone G on the protein synthesis of the Listeria monocytogenes is researched by a polyacrylamide gel electrophoresis technology, the polyacrylamide gel electrophoresis has the advantages of high resolution, good repeatability, rapidness and the like, and the combination of SDS and protein is high-density, namely, the influence of different net charges carried by various proteins on the electrophoresis mobility is eliminated, so that the method has rapid development and wide application, and is a powerful tool for protein research at present. According to the analysis of SDS-PAGE results, the protein band of the bacteria which are not treated by the mulberone G is obviously different from that of the bacteria which are treated by the mulberone G, when the mass concentration of the mulberone G is MIC, the protein band is obviously reduced and becomes shallow, and the deeper the concentration of the mulberone G is increased, the more the influence on the listeria monocytogenes protein is, so that the mulberone G has an inhibition effect on the listeria monocytogenes protein. This may be because mulberone G affects the normal expression of the protein of the cell or because protease is not normally synthesized, protease is inactive, etc., thereby greatly affecting the growth and reproduction of the cell. Proteins are important components of organisms, are enzymes involved in biochemical reactions, and are one of important markers for understanding the metabolic reproduction of organisms.
The influence of the mulberone G on the morphology of the listeria monocytogenes is observed by a scanning electron microscope, and the structural damage of the cell membrane of the listeria monocytogenes after 24 hours of treatment of the mulberone G MIC is found to be expressed as follows: irregular morphology, distortion, and even collapse and shrinkage. This may be due to the mulberone G affecting the cell membrane and cytoplasm of the cell, thereby inhibiting or preventing the growth of the cell.
In conclusion, the mulberry ketone G has stronger antibacterial activity, and the antibacterial mechanism of the listeria monocytogenes mainly causes outflow of intracellular substances by destroying the cell membrane structure of the thallus, so that the normal growth of bacteria is influenced, and the death of the bacteria is caused. Morone G has the potential to be developed against Listeria monocytogenes more than the MIC of cinnamaldehyde, eugenol, thymol, and tannic acid for Listeria monocytogenes studied by Wang Fan et al. While Guan Sangtong G has other antibacterial mechanisms for Listeria monocytogenes, further detailed research is needed. Therefore, mulberone G can also be a potential antimicrobial agent for preventing diseases associated with infection by Listeria monocytogenes.
Drawings
FIG. 1 is a graph of the effect of mulberone G on the growth curve of Listeria monocytogenes.
FIG. 2 is a graph of the effect of mulberone G on listeria monocytogenes cellular protein synthesis, wherein 1: MIC;2:2MIC; m: a marker;3: control group.
FIG. 3 is a graph of the effect of mulberone G on listeria monocytogenes cell morphology, wherein A: a control group; b: MIC group.
Detailed Description
The invention will now be further described in detail with reference to specific examples.
Example 1
A certain amount of mulberry ketone G and water are placed into a spray can to be stirred or vibrated to prepare a solution or suspension with the concentration of 4.5 mug/mL, and then the solution or suspension is contacted with listeria monocytogenes, so that the effect of inhibiting the listeria monocytogenes can be achieved.
Example 2
And (3) putting a certain amount of mulberry ketone G and water into a spray can for stirring or vibrating to prepare 8.3 mug/mL solution or suspension, and spraying the spray can to a refrigerating chamber of a refrigerator, so that the effect of killing and inhibiting listeria monocytogenes is stronger.
Example 3
A certain amount of mulberry ketone G and auxiliary materials are prepared into a disinfection preparation, so that vessels and sites can be disinfected, the propagation chain of Listeria monocytogenes can be effectively blocked, and the occurrence of related diseases is reduced.
Example 4
The mulberry ketone G is prepared into a tablet with a certain specification, and can be orally taken by people and animals infected with the listeria monocytogenes, so that the related diseases caused by the listeria monocytogenes can be effectively prevented and treated.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Many possible variations and modifications of the disclosed technology can be made by anyone skilled in the art, or equivalent embodiments with equivalent variations can be made, without departing from the scope of the invention. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention shall fall within the scope of the technical solution of the present invention.
Claims (3)
1. A method for inhibiting listeria monocytogenes using mulberone G, comprising: morone G is formulated into a solution or suspension having a concentration of not less than 4. Mu.g/mL, and this solution or suspension is contacted with Listeria monocytogenes.
2. A method of eliminating listeria monocytogenes in a refrigerator compartment, comprising: and (3) putting a certain amount of mulberry ketone G and water into a watering can, stirring or vibrating to prepare a solution or suspension with the concentration of not less than 4 mug/mL, and spraying the watering can to a refrigerating chamber of a refrigerator.
3. The application of the mulberry ketone G in the field of anti-listeria monocytogenes medicines or disinfectant preparations.
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