CN118047841B - Fermented mulberry leaf antibacterial peptide Squ and application thereof - Google Patents
Fermented mulberry leaf antibacterial peptide Squ and application thereof Download PDFInfo
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- CN118047841B CN118047841B CN202410421981.0A CN202410421981A CN118047841B CN 118047841 B CN118047841 B CN 118047841B CN 202410421981 A CN202410421981 A CN 202410421981A CN 118047841 B CN118047841 B CN 118047841B
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 53
- 240000000249 Morus alba Species 0.000 title claims abstract description 14
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 10
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- 235000019249 food preservative Nutrition 0.000 claims abstract description 10
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 7
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 5
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- 239000002253 acid Substances 0.000 description 1
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- 239000003674 animal food additive Substances 0.000 description 1
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- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Food Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a fermented mulberry leaf antibacterial peptide Squ, the amino acid sequence of which is RMGAGMAK, and the molecular weight of the antibacterial peptide is 820 daltons. The antibacterial peptide Squ can effectively inhibit the growth of escherichia coli or pseudomonas aeruginosa, is not easy to generate drug resistance, and can be used as a good substitute of antibiotics for the control of escherichia coli or pseudomonas aeruginosa; can also be used for preparing antibacterial drugs or food preservatives for preventing diseases caused by escherichia coli or pseudomonas aeruginosa.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a fermented mulberry leaf antibacterial peptide Squ and application thereof.
Background
Coli is a pathogen with extremely wide distribution, is one of the main factors for inducing food-borne diseases, and constitutes a great threat to human health. Pseudomonas aeruginosa is a common gram-negative bacterium which can cause food poisoning such as vomiting, diarrhea and the like, and is also an important pathogen for nosocomial infection. The use of chemical synthetic preservatives and antibiotics is a main means commonly used in industry for solving food-borne pathogenic bacteria, however, the use of chemical synthetic preservatives can cause the problems of carcinogenesis, teratogenesis, poisoning and the like, so that the development and the utilization of natural preservatives are urgent.
Therefore, the antibacterial peptide which can effectively inhibit the infection of escherichia coli or pseudomonas aeruginosa and can replace chemical preservatives and antibacterial drugs has important research significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a fermented mulberry leaf antibacterial peptide Squ and application thereof, and solves the problems in the prior art.
One of the technical schemes adopted for solving the technical problems is as follows: fermentation Sang Shekang mycopeptide Squ8 is provided. The amino acid sequence of the polypeptide is RMGAGMAK as shown in SEQ ID NO: 1.
The molecular weight of the antibacterial peptide Squ is 820 daltons, the charged electric charge is +2, and the total hydrophobicity ratio is 50%.
Antibacterial peptide Squ is responsible for bacterial destruction by the following principle: in one aspect, the antibacterial peptide Squ has a positive charge that can interact with the bacterial cell membrane, disrupting the bacterial cell membrane resulting in bacterial death. On the other hand, the cell membrane is destroyed while the permeability of the bacterial cell membrane is changed, so that the intracellular substances are extravasated to cause bacterial death.
The second technical scheme adopted by the invention for solving the technical problems is as follows: the application of the fermented mulberry leaf antibacterial peptide Squ in preparing antibacterial drugs or food preservatives for inhibiting and/or killing escherichia coli or pseudomonas aeruginosa is provided.
The third technical scheme adopted by the invention for solving the technical problems is as follows: an antibacterial drug is provided, the active ingredients of which comprise fermentation Sang Shekang mycopeptide Squ8, the amino acid sequence of the antibacterial peptide Squ is SEQ ID NO:1.
Preferably, the antibacterial agent is used for inhibiting and/or killing escherichia coli or pseudomonas aeruginosa.
The fourth technical scheme adopted for solving the technical problems is as follows: provided is a food preservative, the active ingredients of which comprise fermentation Sang Shekang mycopeptide Squ, the amino acid sequence of the antimicrobial peptide Squ is SEQ ID NO:1.
Preferably, the food preservative is used to inhibit and/or kill escherichia coli or pseudomonas aeruginosa.
The antimicrobial peptides of the invention can be synthesized using methods known to those skilled in the art, such as solid phase synthesis, and purified using methods known to those skilled in the art, such as high performance liquid chromatography.
The implementation of the invention has the following beneficial effects:
According to the invention, fermented mulberry leaves are taken as a research object, and a polypeptide Squ with a brand new amino acid sequence is found out by carrying out bioinformatics prediction on the peptide fragment based on a shotgun mass spectrum result of LCMS. Studying the antibacterial activity of the polypeptide Squ on escherichia coli and pseudomonas aeruginosa; and using Escherichia coli and Pseudomonas aeruginosa as examples, and observing Squ damage degree of the strain by using a transmission electron microscope. Experimental results show that the peptide has strong inhibition effect on escherichia coli and pseudomonas aeruginosa. The antibacterial mechanism is that the antibacterial agent is firstly adsorbed on the surface of bacteria, then the cell membrane of the bacteria is destroyed, and the generation of the membrane is inhibited to enable substances in the cells to be extravasated, so that the effect of inactivating the bacteria is achieved. The antibacterial peptide Squ has certain potential in the application of food preservatives and feed additives.
Drawings
FIG. 1 is a schematic structural diagram of an antibacterial peptide Squ.
FIG. 2 is a graph showing a Minimum Inhibitory Concentration (MIC) measurement control of the antibacterial peptide Squ against Escherichia coli.
Wherein,
A: the concentration of the antibacterial peptide is 0 mug/mL;
b: the concentration of the antibacterial peptide is 62.5 mug/mL;
c: the concentration of the antibacterial peptide is 125 mug/mL;
D: the concentration of the antibacterial peptide is 250 mug/mL;
E: the concentration of the antibacterial peptide is 500 mug/mL;
f: the concentration of the antibacterial peptide is 1000 mug/mL.
FIG. 3 is a graph of a Minimum Inhibitory Concentration (MIC) assay of antibacterial peptide Squ against P.aeruginosa.
Wherein,
A: the concentration of the antibacterial peptide is 0 mug/mL;
b: the concentration of the antibacterial peptide is 7.81 mug/mL;
C: the concentration of the antibacterial peptide is 15.63 mug/mL;
d: the concentration of the antibacterial peptide is 31.25 mug/mL;
E: the concentration of the antibacterial peptide is 62.5 mug/mL;
f: the concentration of the antibacterial peptide is 125 mug/mL.
FIG. 4 is a graph showing the time-dependent killing of the antibacterial peptide Squ against E.coli.
Wherein, (. A) blank; (■) MIC; (ζ) 2MIC;
FIG. 5 is a graph showing the time-kill profile of antibacterial peptide Squ against P.aeruginosa.
Wherein, (. A) blank; (■) MIC; (ζ) 2MIC;
FIG. 6 is a transmission electron microscope image of E.coli.
Wherein, (a) a blank group; coli treated with the antibacterial peptide Squ;
FIG. 7 is a transmission electron micrograph of Pseudomonas aeruginosa.
Wherein, (a) a blank group; and (B) Pseudomonas aeruginosa treated with antibacterial peptide Squ.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, and it is to be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 liquid chromatography/Mass Spectrometry Combined technique (LCMS) for fermenting Mulberry leaf
The preparation method of the fermented mulberry leaves comprises the following steps: pulverizing folium Mori, sieving with 40 mesh sieve, inoculating 2% bacterial solution (lactobacillus, candida utilis, and Bacillus subtilis) into the pulverized folium Mori powder, diluting, adding the pulverized folium Mori powder (water content of 50%) and stirring; sealing the fermentation bag by using a heat sealing machine, and fermenting at normal temperature for 5-7 d deg.C until the fermented material has sweet and sour fermentation smell.
Accurately weighing the fermented mulberry leaf 1 g fermented in the previous step, extracting protein by adopting an SDT (sodium dodecyl sulfate) cracking method, and quantifying the protein by adopting a BCA (broadcast and multicast) method. And desalting and freeze-drying, and then performing on-machine testing. And (3) detecting peptide fragments in the fermented mulberry leaves by adopting a shotgun-based protein identification technology.
Liquid phase conditions: timsTOF PRO systems (Bruker); the analytical Column was a C18 reverse phase chromatography Column (Thermo SCIENTIFIC EASY Column, 75 μm. Times.10 cm); mobile phase a was 0.1% formic acid, B was 84% acetonitrile and 0.1% formic acid, flow rate was 300 nL/min;
Mass spectrometry conditions: peptide fragment analysis was performed using timsTOF Pro mass spectrometers (Bruker), which perform full scan acquisitions (m/z 100-1700). The library searching software is MaxQuant, and the mass spectrum results of the protein and peptide fragments are identified by using a Morus alba database in Uniprot.
Example 2 screening of fermentation Sang Shekang mycopeptides
The 14 amino acid sequences obtained through LCMS result, the antibacterial peptide sequences possibly existing in the fermented mulberry leaf protein sequences are predicted by utilizing antibacterial peptide prediction on-line servers APD3 and CAMP, charges and hydrophobicity possibly having the antibacterial sequences are analyzed, and finally amino acid sequence RMGAGMAK (shown as SEQ ID NO: 1) is screened out for chemical synthesis (synthesized by Beijing-middle family matte biotechnology Co., ltd.) and antibacterial activity verification is carried out. The antibacterial peptide sequence RMGAGMAK predicted by screening consists of 8 amino acids, and has total charge of +2, hydrophobicity of 50 percent, molecular mass of 820 Da and is named Squ. The structural schematic of the antibacterial peptide Squ is shown in fig. 1.
Example 3 Minimum Inhibitory Concentration (MIC) determination
Coli and Pseudomonas aeruginosa were cultured at 37℃in a 12 h to logarithmic phase and diluted to 10 6-7 CFU/mL in 0.01M pH 7.2 phosphate buffer. The peptides were dissolved in phosphate buffer and mixed with bacteria in equal volumes at 37 ℃ 2 h. The Minimum Inhibitory Concentration (MIC) refers to the minimum concentration of antimicrobial peptide at which no bacterial growth is visible from the microtiter plate after overnight incubation at 37 ℃. As shown in FIGS. 2 and 3, the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide Squ against Escherichia coli was 125. Mu.g/mL, and the Minimum Inhibitory Concentration (MIC) of Pseudomonas aeruginosa was 15.63. Mu.g/mL.
Example 4 time kill Curve TIMEKILL determination
Coli and Pseudomonas aeruginosa were cultured at 37℃for 12 hours to the logarithmic phase, and diluted to 10 3-4 CFU/mL in 0.01 mM phosphate buffer, pH 7.2. MIC and 2 XMIC concentration peptides were mixed with bacteria at 37℃in equal volumes and incubated separately, plated plates were sampled every 30 minutes, and the total number of colonies was recorded after incubation at 37℃overnight. The result shows that the antibacterial peptide Squ has obvious effect on the escherichia coli and the pseudomonas aeruginosa in 1 hour; and then continue to decrease in trend. Under the action of the antibacterial peptide, the bacterial count is reduced more rapidly. The antibacterial peptide Squ has obvious inhibition effect on escherichia coli and pseudomonas aeruginosa along with the increase of the action time (shown in figure 4 and figure 5).
EXAMPLE 5 Transmission Electron microscopy analysis
Bacteria at 10 6-7 CFU/mL were treated with 2×mic of antimicrobial peptide Squ for 2h at 37 ℃, then centrifuged at 2700 g for 10 min and washed twice with phosphate buffer (pH 7.2). After fixation with 1% osmium acid, dehydration was performed with 95% ethanol and then acetone treatment was performed on 20 min. The samples were baked 24 h at 70 ℃, sheets of 70-90 nm were prepared on copper grids and then stained with lead citrate and uranium acetate. Ultrastructural observation and capture were performed using an H-7650 transmission electron microscope.
As shown in a in fig. 6, a in fig. 7, the intracellular tissue and structural integrity of bacterial cells was good for untreated bacteria. However, as shown in B in FIG. 6 and B in FIG. 7, after the antibacterial peptide Squ was treated, it was seen that the structure of the bacterial cell membrane began to be blurred with cavitation of the cells, the shape of the cells became irregular, the cell membrane was completely collapsed, and the cytoplasmic contents were discharged. The result of a transmission electron microscope shows that the antibacterial peptide Squ has a destructive effect on cell membranes and internal structures of escherichia coli and pseudomonas aeruginosa.
In conclusion, the invention provides a brand new antibacterial peptide Squ, wherein the Minimum Inhibitory Concentration (MIC) of the antibacterial peptide Squ on escherichia coli and pseudomonas aeruginosa is 125 mug/mL and 15.63 mug/mL respectively, and the antibacterial peptide has strong inhibition effect on escherichia coli and pseudomonas aeruginosa and good thermal stability. The antibacterial peptide Squ of the invention achieves the effect of inactivating bacteria by adsorbing on the surface of the bacteria, destroying the cell membrane of the bacteria and inhibiting the generation of the membrane to enable the substances in the cells to extravasate.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (6)
1. A fermented mulberry leaf antibacterial peptide Squ has an amino acid sequence shown in SEQ ID NO: 1.
2. Use of the fermented Sang Shekang mycopeptide Squ8 of claim 1 for the preparation of an antibacterial drug or a food preservative, characterized in that: the antibacterial drug or food preservative is used for inhibiting and/or killing escherichia coli or pseudomonas aeruginosa.
3. An antibacterial agent characterized in that: the active ingredients of the polypeptide comprise fermentation Sang Shekang mycopeptide Squ, and the amino acid sequence of the antimicrobial peptide Squ is SEQ ID NO:1.
4. An antimicrobial agent as claimed in claim 3 wherein: the antibacterial drug is used for inhibiting and/or killing escherichia coli or pseudomonas aeruginosa.
5. A food preservative characterized by: the active ingredients of the polypeptide comprise fermentation Sang Shekang mycopeptide Squ, and the amino acid sequence of the antimicrobial peptide Squ is SEQ ID NO:1.
6. The food preservative according to claim 5, characterized in that: the food preservative is used for inhibiting and/or killing escherichia coli or pseudomonas aeruginosa.
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| CN202410421981.0A CN118047841B (en) | 2024-04-09 | 2024-04-09 | Fermented mulberry leaf antibacterial peptide Squ and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519442A (en) * | 2009-03-31 | 2009-09-02 | 安徽省农业科学院蚕桑研究所 | Method for inducing silkworms to produce antibacterial peptides |
| WO2021126960A1 (en) * | 2019-12-16 | 2021-06-24 | Manus Bio, Inc. | Microbial production of mogrol and mogrosides |
| WO2023274024A1 (en) * | 2021-06-28 | 2023-01-05 | 厦门大学 | Antibacterial peptide scyampcin44-63 and application thereof |
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| DE102009007381A1 (en) * | 2009-01-29 | 2010-08-05 | Amp-Therapeutics Gmbh & Co. Kg | Antibiotic peptides |
| KR101734183B1 (en) * | 2016-06-03 | 2017-05-11 | 군산대학교 산학협력단 | Antibacterial peptide derived from Crassostrea gigas and antibacterial pharmaceutical composition containing the same |
| CN111087460B (en) * | 2020-01-14 | 2021-07-30 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Broad-spectrum antibacterial peptide and application thereof |
| CN117106034A (en) * | 2023-09-18 | 2023-11-24 | 山东大学 | Antibacterial peptide and application thereof in preparation of antibacterial drugs |
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| CN101519442A (en) * | 2009-03-31 | 2009-09-02 | 安徽省农业科学院蚕桑研究所 | Method for inducing silkworms to produce antibacterial peptides |
| WO2021126960A1 (en) * | 2019-12-16 | 2021-06-24 | Manus Bio, Inc. | Microbial production of mogrol and mogrosides |
| WO2023274024A1 (en) * | 2021-06-28 | 2023-01-05 | 厦门大学 | Antibacterial peptide scyampcin44-63 and application thereof |
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| 家蚕抗菌肽cecropin A的表达特征及其体内外抑菌研究;吕丁丁等;《基因组学与应用生物学》;20160625;35(第06期);第1368-1376页 * |
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