CN114853849B - Maotai-flavor liquor Daqu antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs - Google Patents

Maotai-flavor liquor Daqu antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs Download PDF

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CN114853849B
CN114853849B CN202210684235.1A CN202210684235A CN114853849B CN 114853849 B CN114853849 B CN 114853849B CN 202210684235 A CN202210684235 A CN 202210684235A CN 114853849 B CN114853849 B CN 114853849B
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polypeptide
staphylococcus aureus
bacteria
gln
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CN114853849A (en
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陈良强
杨帆
胡阳
王莉
靳艳
于洋
于文皓
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Kweichow Moutai Co Ltd
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    • 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
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    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract

The invention discloses a Maotai-flavor liquor Daqu antibacterial polypeptide and application thereof in preparing anti-inflammatory drugs, and belongs to the technical field of biology. The amino acid sequence of the antibacterial polypeptide provided by the invention is Ile-Ile-Gln-Pro-Gln-Gln-Pro-Ala-Gln-Leu. The antibacterial polypeptide has good inhibitory activity on gram-negative bacteria and gram-positive bacteria, can be used as a natural and safe antibacterial agent for preparing feed additives, can be used for preparing foods, cosmetics or sterilizing agents, and has good application prospect in preparing medicines for preventing and/or reducing intestinal tract infection diseases caused by escherichia coli and staphylococcus aureus.

Description

Maotai-flavor liquor Daqu antibacterial polypeptide and application thereof in preparation of anti-inflammatory drugs
Technical Field
The invention relates to a Maotai-flavor liquor Daqu antibacterial polypeptide and application thereof in preparing anti-inflammatory drugs, belonging to the technical field of biology.
Background
Antibiotics are special secondary metabolic organic substances produced by microorganisms, and can inhibit the growth and metabolism of bacteria by inhibiting the formation of nucleic acids of bacteria, inhibiting the synthesis of bacterial proteins, changing the permeability of cell membranes of bacteria, interfering the synthesis of cell walls of bacteria and the like when used at low concentration. Antibiotics are widely used because of their remarkable antibacterial effect, however, overuse also results in the generation of bacterial resistance, making the treatment of bacterial infectious diseases increasingly difficult.
The antibacterial peptide (antimicrobial peptides, AMPs) is used as a novel antibiotic, has very wide sources, and has the characteristics of 10-50 amino acids, strong cationic property, amphipathy, broad-spectrum antibacterial property and the like. Compared with the traditional antibiotics, the antibacterial peptide has the following greatest advantages: the antibacterial peptide is mainly combined to a bacterial cell membrane through electrostatic attraction, and is combined with phospholipid to be inserted into a cytoplasmic membrane through hydrophobic action, so that the structure of the bacterial cell membrane is destroyed. The external force action can not cause the microorganism to generate drug resistance basically. However, the current use of antimicrobial peptides still faces two major challenges: firstly, the antibacterial activity of the natural antibacterial peptide is relatively low, and secondly, the stability and cytotoxicity of the antibacterial peptide are difficult to regulate and control. Thus, the method is applicable to a variety of applications. The development of safe, stable and low-cytotoxicity antibacterial peptide has important significance.
The Daqu is prepared by fermenting grains serving as substrates at high temperature by microorganisms, wherein the antibacterial peptide derived from the grain protein is food-borne, natural and safe, and has good potential as an antibacterial drug. Therefore, the antibacterial peptide derived from the Daqu substrate wheat protein is screened from Daqu, and can be used for preparing and researching medicines and/or health-care products for preventing and/or reducing intestinal tract infection diseases caused by escherichia coli and staphylococcus aureus.
Disclosure of Invention
The invention aims to provide an application of an antibacterial polypeptide derived from Daqu in preparing medicines for preventing and/or treating intestinal diseases caused by bacteria.
In one embodiment, the bacteria include gram positive bacteria and gram negative bacteria.
In one embodiment, the gram positive bacterium is escherichia coli.
In one embodiment, the gram negative bacterium is staphylococcus aureus.
In order to achieve the above object, the present invention uses the antimicrobial polypeptide as an active ingredient for inhibiting the growth activity of microorganisms, the antimicrobial polypeptide comprising the following (a) or (b):
(a) The amino acid sequence is shown in SEQ ID NO:1 is shown in the specification;
(b) An antibacterial polypeptide with improved protein and gene levels and activity of inhibiting the growth of microorganisms is prepared on the basis of the antibacterial polypeptide.
The antibacterial polypeptide is an active ingredient of an anti-escherichia coli and staphylococcus aureus drug, and a pharmaceutically acceptable carrier or auxiliary material can be added.
In one embodiment, the adjuvants include solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retarders.
The amino acid sequence of the antibacterial polypeptide with the inhibitory activity on escherichia coli and staphylococcus aureus is Ile-Ile-Gln-Pro-Gln-Gln-Pro-Ala-Gln-Leu. The molecular weight is 1135.33Da, white powder is easy to dissolve in water, and has strong inhibition effect on the growth of escherichia coli and staphylococcus aureus.
The invention also provides a bacterial growth inhibitor, which takes the antibacterial polypeptide as an active substance.
In one embodiment, the bacteria include gram positive bacteria and gram negative bacteria.
In one embodiment, the gram positive bacterium is escherichia coli.
In one embodiment, the gram negative bacterium is staphylococcus aureus.
In one embodiment, the growth inhibitory agent further comprises a chelating agent.
In one embodiment, the chelating agent includes citrate, lactate, pyrophosphate, and EDTA.
The invention also provides application of the antibacterial polypeptide or the growth inhibitor in preparing medicines for resisting gram-positive bacteria and/or gram-negative bacteria.
In one embodiment, the gram positive bacterium is escherichia coli.
In one embodiment, the gram negative bacterium is staphylococcus aureus.
The invention also provides the use of said antimicrobial polypeptide or said growth inhibitory agent for reducing or eliminating bacteria on the surface of an article.
In one embodiment, the articles include food, cutlery, utensils and medical devices that are ingested by the animal as food or drinking water.
In one embodiment, the bacteria include gram positive bacteria and gram negative bacteria.
In one embodiment, the gram positive bacterium is escherichia coli.
In one embodiment, the gram negative bacterium is staphylococcus aureus.
The invention also provides a method for reducing or eliminating bacteria, wherein the antibacterial peptide or the growth inhibitor is applied to the surface of an article for reducing or eliminating bacteria.
In one embodiment, the bacteria include gram positive bacteria and gram negative bacteria.
In one embodiment, the gram positive bacterium is escherichia coli.
In one embodiment, the gram negative bacterium is staphylococcus aureus.
The method is not directed to disease diagnosis or treatment.
The invention also provides application of the antibacterial peptide or the growth inhibitor in preparing foods, cosmetics, feed additives, preservatives or killers.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains and determines the structure of the antibacterial polypeptide from Daqu, and the molecular mass is 1135.33Da. The antibacterial polypeptide has good antibacterial activity, and has good application prospect in preparing medicines and/or health products for preventing and/or reducing intestinal tract infection diseases caused by escherichia coli and staphylococcus aureus.
Drawings
FIG. 1 is a diagram showing the results of inhibition by polypeptide IIQPQQPAQL, and FIG. 1 (A) is a diagram showing the results of an experiment for inhibiting Escherichia coli by polypeptide IIQPQQPAQL; FIG. 1 (B) is a graph showing the experimental results of inhibition of Staphylococcus aureus by polypeptide IIQPQQPAQL.
Detailed Description
The following examples relate to the following media:
LB medium: 10g/L tryptone; 5g/L of yeast extract; sodium chloride 10g/L.
TSB medium: 17g/L of casein pancreatin digest; 3g/L of soybean meal papain digest; 5g/L of sodium chloride; 2.5g/L of dipotassium hydrogen phosphate; glucose 2.5g/L.
Example 1 preparation and identification of polypeptide IIQPQQPAQL
A method of combining LC-MS/MS with a bottom-up proteomics technique is adopted. The method is characterized in that Maotai-flavor high-temperature Daqu is taken as a raw material, and peptide fragments with inhibition effect on escherichia coli and staphylococcus aureus are screened by combining structure-activity relationship characteristics through gradient extraction of water and ethanol, centrifugation, ultrafiltration and LC-MS/MS analysis.
The specific method comprises the following steps:
(1) Sample preparation
1g of the Daqu sample was mixed with 20mL of ultrapure water, centrifuged at 200rpm for 15min at 37℃and 4℃and 15000 Xg, the supernatant (designated as supernatant A) was removed, 20mL of 50% ethanol-water solution was added to the precipitate (designated as precipitate A), the supernatant (designated as supernatant B) was removed by shaking at 200rpm for 2h at 37℃and 15000 Xg at 4℃and 15min, the precipitate (designated as precipitate B) was added to 20mL of ethanol, and the supernatant (designated as supernatant C) was removed by centrifugation at 200rpm for 2h and 15000 Xg at 37 ℃. The supernatant A, B, C was ultrafiltered with an ultrafiltration membrane having a molecular weight cut-off of 10000Da, and the obtained filtrate A, B, C was desalted with a C18-SPE cartridge (Waters Oasis HLB SPE), respectively. After desalting, the samples were lyophilized and stored at-80 ℃ to obtain the corresponding lyophilized and stored samples A, B, C.
(2) LC-MS/MS analysis
The freeze-dried and preserved sample A, B, C is respectively dissolved in 0.1% (v/v) FA-H2O solution, and LTQ-orbitrapVelos (double partial pressure linear trap and electrostatic field orbitrap combined high-resolution mass spectrum) is used for mass spectrum analysis of the sample. The loading amounts were 2. Mu.g, respectively, and mobile phase A of the liquid chromatograph was an aqueous solution containing 0.1% (v/v) formic acid, and mobile phase B was acetonitrile (v/v) containing 0.1% formic acid. The gradient elution procedure was as follows:
0-80min,7% -27% B (v/v); 80-95min,27% -40% B;95-97min, 40-90% B;97-107min,90% B;107-109min,90% -0% B;109-126min,0% B, flow rate 60. Mu.L/min.
The mass spectrum ion source is ESI, the ion transmission temperature is 250 ℃, the spraying voltage is 2.2kV, the normalized collision energy is 35%, the primary spectrum resolution is 60000, the scanning range is 400-2000m/z, and the data acquisition mode is a data dependent mode (DDA). Selecting 20 strongest parent ions in the primary spectrum for secondary fragmentation, wherein dynamic exclusion is set as repeated counting, 2; repeating time, 30s; the exclusion time was 60 seconds.
(3) Data retrieval
RAW file use MaxQuant TM The (v.1.5.3.30) software was retrieved in the wheat (Wheat, 379 proteins) database (http:// www.uniprot.org /). The search parameters are as follows: no enzyme cleavage, maximum number of missed cleavage and fixed modification were set, and the variable modification was set to methionine oxidation (+ 15.9949 Da). The mass tolerance deviation of the parent ion was 20ppm and the fragment ion was 0.5Da. Controlling PSM false positive rate (FDR)<1% of the polypeptides were analyzed as valid data. 475 polypeptides from 39 proteins were identified in the experiment. Since antibacterial peptides generally have the following characteristics, such as generally containing hydrophobic amino acids, basic amino acids; has strong cationic property, amphipathy and the like. Therefore, the structure-activity relationship of the combined antibacterial peptide is used for screening partial potential antibacterial peptide from the identification result, and the result is thatAs shown in table 1:
table 1 results of partial identification of potential antimicrobial peptides
(4) Analysis of polypeptide IIQPQQPAQL Properties
Polypeptide IIQPQQPAQL is derived from wheat seed as main storage protein, gamma gliadin (P21292, f 253-262) with peptide isoelectric point of 5.52 and molecular mass of 1135.33Da. Biological information of the polypeptide is obtained by an online tool Expasy, and the polypeptide is shown to be neutral, the instability coefficient is 105.30, the aliphatic amino acid index and the hydrophilicity are 127.00 and-0.260 respectively, so that the polypeptide is proved to have stronger hydrophobicity and amphipathy.
(5) SEQ ID NO:1 information:
(a) Sequence characterization
* Length: 10 amino acids;
* Type (2): amino acids;
* Chain type: a linear single strand;
(b) Molecular type: proteins
Sequence description: SEQ ID NO:1, IIQPQQPAQL
EXAMPLE 2 detection of bacteriostatic Activity of polypeptide IIQPQQPAQL
Three potential antimicrobial peptides IIQPQQPAQL, IFWGIPALLK and AAFSPVSLHSALSLLAAGAGS screened in example 1 were selected and were commissioned to be synthesized by solid phase methods at purities of 95.63%, 99.73% and 95.31%, respectively, by the south-jie peptide biotechnology limited company. The antibacterial activity of three potential antibacterial peptides is examined by taking gram-positive bacteria escherichia coli and gram-negative bacteria staphylococcus aureus as target strains.
(1) Strain and resuscitation
Coli (Escherichia coli K) and staphylococcus aureus (Staphylococcus aureus) were both stored at-80 ℃ by glycerol storage. Before the experiment, one loop of escherichia coli is inoculated into 5mL of liquid LB medium, one loop of staphylococcus aureus is inoculated into 5mL of liquid TSB medium, and the strain is revived by culturing for 12 hours at 37 ℃ and 200 rpm.
After 12h, 100. Mu.L of E.coli bacterial liquid was transferred to 5mL of liquid LB medium, 100. Mu.L of Staphylococcus aureus bacterial liquid was transferred to 5mL of liquid TSB medium, and the mixture was again cultured at 37℃for 12h for resuscitation at 200 rpm. The resuscitating operation was repeated 2-3 times to revive the strain.
(2) Culture medium
The antibacterial experiment used a round petri dish with a diameter of 90 mm. The culture medium is LB solid culture medium and TSB solid culture medium, and the two culture media respectively contain agar with the mass concentration of 1.5%. LB solid medium was mixed to a final concentration of 1X 10 5 CFU/mL of E.coli, TSB solid medium was mixed with a final concentration of 1X 10 5 CFU/mL staphylococcus aureus. The culture medium mixed with the bacterial liquid is respectively poured into six different round culture dishes (the escherichia coli and the staphylococcus aureus are respectively poured into three culture dishes), the filling height of the culture medium in the culture dishes is 4mm, and the culture medium is preserved at 4 ℃ after being sealed, so that six flat plates are obtained.
(3) Antibacterial experiments
3 holes with the diameter of 3mm are respectively manufactured on six flat plates, and the hole spacing is 3cm. Six plates were divided into three groups, one plate containing E.coli and one plate containing Staphylococcus aureus were grouped, and 1.5mg of the same target peptide dissolved in 50. Mu.L of sterile water was added to each of two wells in each group (three target peptides, three groups of experiments), each of which was performed in duplicate. A third well was blank with sterile water and 50. Mu.L was added as well. Standing the plate after sample addition in a refrigerator at 4 ℃ for 4 hours, taking out the plate after the sample liquid in the hole is completely absorbed and diffused, culturing the plate for 12 hours in an inverted mode in a constant-temperature incubator at 37 ℃, and observing and measuring and recording the size of a bacteriostasis zone.
(4) Experimental results
The diameters of the inhibition zones of the three target peptides on escherichia coli and staphylococcus aureus are shown in table 2. The polypeptides IFWGIPALLK and AAFSPVSLHSALSLLAAGAGS have no inhibition effect on escherichia coli and only have weak inhibition effect on staphylococcus aureus; IIQPQQPAQL has inhibiting effect on Escherichia coli and Staphylococcus aureus, and has significantly better inhibiting effect on Staphylococcus aureus than Escherichia coli. The results indicate that polypeptide IIQPQQPAQL has inhibitory activity against both gram-negative and gram-positive bacteria and has higher inhibitory activity against gram-positive bacteria.
Figure 1 shows the inhibitory effect of polypeptide IIQPQQPAQL on escherichia coli and staphylococcus aureus.
Table 2 bacteriostatic effects of polypeptide IIQPQQPAQL
Example 3 Minimum Inhibitory Concentration (MIC) detection of polypeptide IIQPQQPAQL
The minimum inhibitory concentration of the antibacterial peptide IIQPQQPAQL was detected.
(1) Preparation of bacterial liquid and antibacterial peptide solution
The E.coli and Staphylococcus aureus of example 2 were diluted with LB medium and TSB medium, respectively, to prepare 2X 10 5 The corresponding bacterial liquid of CFU/mL and the antibacterial peptide are prepared to be 51.2mg/mL.
(2) Experimental operation
Sterile 96-well plates were used for experiments. Positive control (Positive) 100. Mu.L 2X 10 5 Adding 100 mu L of blank culture medium into CFU/mL bacterial liquid, wherein the final concentration of the bacterial liquid is 1 multiplied by 10 5 CFU/mL; the negative Control (Control) was 200. Mu.L of blank medium. 10 experimental points are added, 160 mu L of blank culture medium is added to 1 st hole, 100 mu L of blank culture medium is added to 2-10 holes, 40 mu L of antibacterial peptide solution is added to 1 st hole, and then 100 mu L to 2 nd hole are sucked, 100 mu L to 3 rd hole are sucked after uniform mixing, and the mixture is diluted to 10 th hole by continuous multiple ratio, and 100 mu L is sucked from 10 th hole and discarded. Then add 2X 10 into each well 5 The concentration of the CFU/mL bacterial liquid is 100 mu L, and the final bacterial liquid concentration of each tube is about 1 multiplied by 10 5 CFU/mL. The concentration of the antibacterial peptide from the 1 st hole to the 10 th hole is 5120, 2560, 1280, 640, 320, 160, 80, 40, 20 and 10 mug/mL respectively. The above-mentioned passThe process was completed within 15min and 2 times per well. The 96-well plate was incubated in an incubator at 37℃for 18h.
(3)OD 600 Measurement
After incubation, taking out the pore plate to blow the liquid in each pore uniformly, and measuring OD by using an enzyme-labeling instrument 600 The lowest concentration in the well where no turbidity occurred was the minimum inhibitory concentration.
(4) Analysis of results
The minimum inhibitory concentration of the escherichia coli is not measured under the experimental concentration of the invention, and the minimum inhibitory concentration of the antibacterial peptide staphylococcus aureus is 2.56mg/mL.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> Guizhou Maotai liquor stock Co., ltd
<120> an antibacterial polypeptide of Maotai-flavor liquor Daqu and its application in preparing antiinflammatory drugs
<130> BAA220174A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213> artificial sequence
<400> 1
Ile Ile Gln Pro Gln Gln Pro Ala Gln Leu
1 5 10

Claims (4)

1. The application of an antibacterial polypeptide in preparing a medicament for resisting staphylococcus aureus and/or escherichia coli, wherein the amino acid sequence of the antibacterial polypeptide is shown in SEQ ID NO: 1.
2. Use of an antimicrobial polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 is shown in the specification; the articles are tableware, utensils for animals to ingest food/drinking water and medical appliances; the bacteria are staphylococcus aureus and/or escherichia coli.
3. A method for reducing or eliminating bacteria, wherein the bacteria are reduced or eliminated by applying an antimicrobial polypeptide to the surface of an article, wherein the method is not for disease diagnosis or treatment purposes, and wherein the antimicrobial polypeptide has an amino acid sequence as set forth in SEQ ID NO:1 is shown in the specification; the articles are tableware, utensils for animals to ingest food/drinking water and medical appliances; the bacteria are staphylococcus aureus and/or escherichia coli.
4. Use of an antimicrobial polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1.
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