CN116178495A - Novel low molecular weight antibacterial peptide derived from lactobacillus plantarum, and preparation method and application thereof - Google Patents
Novel low molecular weight antibacterial peptide derived from lactobacillus plantarum, and preparation method and application thereof Download PDFInfo
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Images
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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
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- 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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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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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
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Abstract
The invention discloses a novel low molecular weight antibacterial peptide derived from lactobacillus plantarum, and a preparation method and application thereof. The invention provides polypeptide PNMGL2 as shown in SEQ ID NO: 1. The invention also protects the application of the polypeptide PNMGL2 as a microbial inhibitor. The invention also provides a method for preparing the microbial inhibitor, which comprises the following steps: lactobacillus plantarum NMGL2 was cultivated. The polypeptide PNMGL2 provided by the invention can be used as a food preservative and a microbial inhibitor and applied to the fields of food and medicine. The method for preparing the microbial inhibitor provided by the invention has the advantages of simple operation of the technological process, low cost and low equipment requirement, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a novel low molecular weight antibacterial peptide derived from lactobacillus plantarum, and a preparation method and application thereof.
Background
According to world health organization study statistics, about 42 ten thousand people die annually due to food-borne diseases. Pathogenic microorganisms in food products, such as Salmonella (Salmonella), enterobacter sakazakii (Enterobacter sakazakii), staphylococcus aureus (Staphylococcus aureus), enteropathogenic Escherichia coli (Escherichia coli), listeria monocytogenes (Listeria monocytogenes), shigella flexneri (Shigella flexneri), norovirus (Norovirus) and the like, pose a threat to food safety. Long-term intake of preservatives added to inhibit pathogenic microorganisms also presents a health hazard. Therefore, finding a nontoxic, pollution-free and efficient food bacteriostasis method has become an urgent task for food preservation.
The antibacterial peptide (antimicrobial peptides, AMPs) is a bioactive substance of low molecular peptides widely existing in various organisms, is protein in chemical nature, has the characteristics of high-efficiency bacteriostasis, no toxicity, no harm, no drug resistance, difficult residue, strong stability and the like, and is considered as the most potential substitute of antibiotics and food preservative.
Various antimicrobial peptides have been found in animals, plants and microorganisms. The animal-derived and plant-derived antibacterial peptides are limited in source and difficult to realize large-scale production, and the microbial-derived antibacterial peptides have the advantages of short fermentation period, low cost, easiness in culture, easiness in industrial production and the like, and become an important direction for the research of antibacterial peptides. At present, most of the research is that a bacteriocin-Nisin from lactic acid bacteria is commercially used, but most of Nisin from lactic acid bacteria has the problems of narrow antibacterial spectrum, poor stability and the like, so that a new antibacterial peptide needs to be searched.
Disclosure of Invention
The invention aims to provide a novel low molecular weight antibacterial peptide derived from lactobacillus plantarum, and a preparation method and application thereof.
The invention provides a polypeptide named polypeptide PNMGL2, as shown in SEQ ID NO: 1.
SEQ ID NO：1：LNFLKK。
The invention also protects the application of the polypeptide PNMGL2 as a microbial inhibitor.
The invention also provides application of the polypeptide PNMGL2 in inhibiting microorganisms.
The invention also protects the application of the polypeptide PNMGL2 in preparing a microbial inhibitor.
The invention also protects application of lactobacillus plantarum NMGL2 in preparation of polypeptide PNMGL2.
The invention also provides a method for preparing the polypeptide PNMGL2, which comprises the following steps: lactobacillus plantarum NMGL2 was cultivated. Lactobacillus plantarum NMGL2 produces polypeptide PNMGL2, so that the system for completing culture contains polypeptide PNMGL2.
The invention also provides a method for preparing the microbial inhibitor, which comprises the following steps: lactobacillus plantarum NMGL2 was cultivated. The system for completing the culture contains a microbial inhibitor.
The method for culturing any one of the above specifically comprises the following steps: lactobacillus plantarum NMGL2 was inoculated into liquid MRS medium (specifically, the bacterial concentration in the system at the time of completion of inoculation was 10 7 cfu/mL), stationary culture.
Specifically, the culture temperature may be 35 to 39℃and more specifically 37 ℃.
Specifically, the incubation time may be 26 to 30 hours, more specifically 28 hours.
The method of any one of the above further comprising the steps of: after the completion of the culture, the supernatant was collected by centrifugation, filtered and the filtrate was collected.
The parameters of the centrifugation can be specifically as follows: centrifuge at 4℃for 15min with 8000 g.
The filtration specifically adopts a filter membrane with the pore diameter of 0.22 μm.
The method further comprises the steps of: taking the filtrate, adjusting the pH value to 5, then adding a catalase solution, and incubating.
The method further comprises the steps of: taking the filtrate, adjusting the pH value to 5, then adding an equal volume of catalase solution, and incubating for 30min at 37 ℃.
The method further comprises the steps of: extraction is carried out by adopting ethyl acetate, and an extraction phase is collected.
During the extraction, the volume ratio of the liquid sample to the ethyl acetate is 1:1.5.
The method further comprises the steps of: after completion of the extraction, the extract phase was collected, the organic solvent was removed, and then redissolved with PBS buffer (pH 6.5, 20 mol/L) to obtain an extract redissolution.
The method further comprises the steps of: anion exchange chromatography.
Chromatography column: the inner diameter is 16mm and the length is 30mm.
Filling material: DEAE-Sepharose Fast Flow.
And (3) purification: first, the column was equilibrated with Tris-HCl buffer (pH 7.20,0.05 mol/L); then, the extract complex solution (the loading volume is specifically 3 ml) is loaded; then, elution was performed with Tris-HCl buffer (pH 7.2,0.05 mol/L) containing sodium chloride at a flow rate of 1mL/min for a total time of 160min, and the concentration of sodium chloride was linearly increased from 0 to 0.2M from the initial time of elution to the final time of elution.
And collecting the solution after passing through the column corresponding to the second eluting peak.
The post-column solution was collected with a retention volume of 160-200 mL.
The method further comprises the steps of: the solution after passing through the column was freeze-dried, and then dissolved in Tris-HCl buffer (pH 7.2,0.05 mol/L) to obtain a lyophilized product complex solution.
The lactobacillus plantarum NMGL2 has a preservation registration number of CGMCC No.18495.
The microbial inhibitor prepared by any one of the methods belongs to the protection scope of the invention.
Specifically, the microorganism is a pathogenic microorganism.
Specifically, the microorganism is a bacterium.
Specifically, the bacteria are enterobacteriaceae bacteria or staphylococci bacteria or listeriaceae bacteria.
Specifically, the bacteria are escherichia or staphylococcus or listeria or shigella bacteria.
Specifically, the bacterium is enterobacter sakazakii, escherichia coli, staphylococcus aureus, listeria monocytogenes or shigella flexneri.
The polypeptide PNMGL2 provided by the invention can inhibit common food-borne pathogenic bacteria in food, has broad-spectrum tolerance to temperature and pH, has broad-spectrum tolerance to various proteases, and can be used as a food preservative and a microbial inhibitor to be applied to the fields of food and medicine.
The method for preparing the microbial inhibitor has the advantages of simple operation of the technological process, low cost and low equipment requirement, is suitable for industrial production, and has good application prospect.
Drawings
FIG. 1 shows the growth curve and the bacteriostatic activity curve of Lactobacillus plantarum NMGL2.
FIG. 2 is an elution profile of an anion exchange chromatography purification of an antimicrobial peptide.
FIG. 3 is a photograph showing the bacteriostatic activity of the lyophilized product complex solution obtained from different elution peaks.
FIG. 4 is a Tricine-SDS-PAGE electrophoresis of an antibacterial peptide solution.
FIG. 5 is a photograph showing the bacteriostatic activity of polypeptide PNMGL2 against food-borne pathogenic bacteria.
FIG. 6 is a chemical structural formula of polypeptide PNMGL2.
FIG. 7 shows the three-dimensional structure of polypeptide PNMGL2 produced by the PEP-FOLD 3De-novo method.
FIG. 8 shows the results of acid-base stability of the antibacterial peptide.
FIG. 9 shows the results of the thermostability of the antimicrobial peptides.
FIG. 10 shows the results of enzyme sensitivity of the antimicrobial peptides.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. In the examples, the Bradford method was used to detect protein concentration. Model of oxford cup used in the examples: the outer diameter is 7.8mm, the inner diameter is 6.0mm, and the height is 10.0mm. In an embodiment, the diameter of the zone of inhibition is measured using a fully automatic ultra high definition zone of inhibition analyzer Scan 4000.
Lactobacillus plantarum (Lactobacillus plantarum) NMGL2 has a preservation registration number of CGMCC No.18495. Lactobacillus plantarum (Lactobacillus plantarum) NMGL2 is abbreviated as Lactobacillus plantarum NMGL2. Lactobacillus plantarum (Lactobacillus plantarum) NMGL2 is described in patent No. ZL201910916373.6, with grant publication No. CN 110591955B, with grant publication date 2021.06.15, which is a strain that the patent is entitled to protect.
Enterobacter sakazakii for the examples: CICC 21546. Escherichia coli used in the examples: CMCC 44825. Staphylococcus aureus for the examples: CMCC 26071. Listeria monocytogenes for the examples: CMCC 54002. Shigella flexneri for the examples: CICC 21534.CICC: china center for Industrial microorganism culture Collection. CMCC: china medical bacterial preservation management center.
Catalase: soy Biotechnology Co., ltd., product number C8071. Catalase solution: the catalase was dissolved in PBS buffer (pH 6.5, 20 mol/L) and brought to a concentration of 1mg/mL.
Trypsin: beijing Bo Ottoda technologies Co., ltd., product number T6150. Pepsin: beijing Bo Ottoda technologies Co., ltd., product number P6390. Proteinase K was purchased from Soy Biotechnology Inc., cat# P9460. Lysozyme: shanghai Source leaf biology Co., ltd., product No. S10038. Lipase: shanghai Source leaf biology Co., ltd., product No. S10035. Alkaline protease: shanghai Source leaf biology Co., ltd., product No. S10154.
Preparation method of the bacteria-containing medium for example 1, example 2, example 3 and example 6: 100mL of LB medium containing 0.75g/100mL of agar was prepared, sterilized at high temperature, and cooled to 45℃at room temperature, 1mL of Staphylococcus aureus liquid (1 mL of Staphylococcus aureus liquid, the content of Staphylococcus aureus was 1X 10) 8 cfu/mL), and poured into a culture dish after being mixed evenly.
EXAMPLE 1 growth curves and antibacterial Activity curves of Lactobacillus plantarum NMGL2
Inoculating Lactobacillus plantarum NMGL2 frozen in glycerol tube at-80deg.C into liquid MRS culture medium at 1% (the bacterial concentration in the system is 10 at the time of inoculation 7 cfu/mL), stationary culture at 37℃for 48 hours. During the incubation, samples were taken every 2 hours.
Taking each sample, and determining OD 600nm Values and pH.
And respectively taking each sampling sample, detecting antibacterial activity after pretreatment, and determining the optimal culture time of the antibacterial substances.
The pretreatment method comprises the following steps: (1) sampling, centrifuging at 4deg.C and 8000rpm for 15min, collecting supernatant, filtering with a filter membrane with pore size of 0.22 μm, collecting filtrate, and adjusting pH to 5 with HCl aqueous solution or NaOH aqueous solution; (2) taking 1 part by volume of the liquid phase system which completes the step (1), adding 1 part by volume of catalase solution, and incubating for 30min at 37 ℃ to obtain the sample liquid.
Method for detecting antibacterial activity (double-layer agar diffusion method and staphylococcus aureus as indicator bacteria): (1) taking a culture dish with the diameter of 9cm, pouring 8mL of 1.5g/100mL of agar aqueous solution, standing at room temperature until solidification is achieved, and then uniformly placing 3 oxford cups on the surface of a flat plate; (2) pouring 8mL of bacteria-containing culture medium into the culture dish after the step (1) is completed, and pulling out an oxford cup after solidification, wherein a sample adding hole is formed at the position of the oxford cup; (3) after the completion of the step (2), a sample solution (100. Mu.l/well) was added to the well, and the culture was allowed to stand at 4℃for 2 hours, then at 37℃for 12 hours, and then the diameter (mm) of the inhibition zone was measured.
Each sample was set with 3 replicates and the results averaged.
The results are shown in FIG. 1. Lactobacillus plantarum NMGL2 shows a common bacterial growth pattern. During the cultivation, the pH of the cultivation system was lowered from 6.2 to 3.91 and kept stable. Lactobacillus plantarum NMGL2 is capable of detecting antibacterial activity (9.83 mm diameter of the zone of inhibition at 8 hours of cultivation) from 8 hours of cultivation (at this time in logarithmic growth phase), then continues to increase with the increase of cultivation time and reaches the maximum at 28 hours of cultivation (15.80 mm diameter of the zone of inhibition at 28 hours of cultivation), and then decreases with the increase of cultivation time. Therefore, the optimal cultivation time for producing the antibacterial substance is 28 hours.
Example 2 optimization of the method for crude extraction of antibacterial peptides from Lactobacillus plantarum NMGL2 culture broth
1. Culture of Lactobacillus plantarum NMGL2
1. The activated lactobacillus plantarum NMGL2 was inoculated to the liquid MRS medium at an inoculum size of 1% (the bacterial concentration in the system at the time of completion of inoculation was 10) 7 cfu/mL), stationary culture at 37℃for 28 hours, then centrifuging at 4℃for 15 minutes at 8000g, collecting the supernatant, filtering with a filter membrane having a pore size of 0.22 μm, and collecting the filtrate.
2. And (3) taking the filtrate obtained in the step (1), and adjusting the pH value to 5 by using an aqueous solution of NaOH.
3. And (3) taking the liquid phase system which completes the step (2), adding an equal volume of catalase solution, and incubating for 30min at 37 ℃.
2. Crude extraction of antimicrobial peptides by ammonium sulfate precipitation with different saturation levels
1. 8 parts of the liquid phase system (30 mL each) after the completion of the first step was taken and ammonium sulfate precipitation was carried out. The following ammonium sulfate saturation levels were used for the ammonium sulfate precipitation: 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Parameters of ammonium sulfate precipitation: standing at 4 ℃ for 12 hours.
2. After completion of step 1, the supernatant and the pellet were collected by centrifugation at 12000g for 15min at 4℃and the pellet was reconstituted with 1mL of PBS buffer (pH 6.5, 20 mol/L) to obtain a pellet reconstituted solution.
3. The protein concentration of the precipitated complex solution obtained in step 2 was measured, and the results are shown in Table 1.
4. The supernatant obtained in step 2 and the precipitated complex solution obtained in step 2 were used as sample solutions, respectively, and the antibacterial activity was measured (the method was the same as in example 1).
Three replicates were performed and the results averaged.
The results are shown in Table 1. The results show that ammonium sulfate cannot effectively precipitate bacteriostatic substances.
TABLE 1
3. Extraction of crude antibacterial peptide by using different organic solvents
5 parts of the liquid phase system (30 mL each) after completion of the first step was taken.
1 part of the liquid phase system is directly concentrated to 1mL by rotary evaporation at 45 ℃ without any treatment, and is used as a reference sample. The bacteriostatic activity was measured using the reference sample as a sample solution (the method was the same as that in example 1). Detecting the protein concentration of the reference sample. The diameter of the inhibition zone of the reference sample is 30.40+/-0.40 c mm, protein concentration is 108.54 + -1.06 A mg/mL。
Extracting the rest 4 parts of liquid phase system. The organic solvents respectively adopted are: ethyl acetate, n-butanol, n-hexane or chloroform. The extraction conditions are as follows: adding an equal volume of organic solvent, and oscillating and extracting for 2 hours at room temperature and 120 rpm. After completion of the extraction, an aqueous phase (raffinate phase) and an organic phase (extract phase) were collected, respectively. The organic solvent was removed by rotary evaporation at 45℃from the extract phase, and then reconstituted with 1mL of PBS buffer (pH 6.5, 20 mol/L) to give an extract reconstituted solution. The antibacterial activity was measured by using the raffinate phase and the extract complex solution as sample solutions, respectively (the method is the same as that in example 1). The protein concentration of the extract complex solution was measured.
Three replicates were performed and the results averaged.
The results are shown in Table 2. The antibacterial activity of the extract complex solution obtained by adopting n-butanol or ethyl acetate extraction is close to that of a reference sample. In view of the high boiling point of n-butanol during rotary evaporation, which is time consuming, ethyl acetate is ultimately selected as the organic solvent for extraction.
TABLE 2
4. Extraction of crude antibacterial peptide by using ethyl acetate with different volumes
6 parts of the liquid phase system (30 mL each) after the completion of the first step were taken.
1 part of the liquid phase system is directly concentrated to 1mL by rotary evaporation at 45 ℃ without any treatment, and is used as a reference sample. The bacteriostatic activity was measured using the reference sample as a sample solution (the method was the same as that in example 1). The diameter of the inhibition zone of the reference sample is 30.40+/-0.40 c mm。
The remaining 5 parts of the liquid phase system were extracted with ethyl acetate. Different volume ratios are set. The volume ratio of the liquid phase system to the ethyl acetate is respectively set to be 1:0.5 or 1:1 or 1:1.5 or 1:2 or 1:2.5. The extraction conditions are as follows: the mixture was extracted at room temperature with shaking at 120rpm for 2h. After completion of the extraction, the organic phase (extract phase) was collected. The organic solvent was removed by rotary evaporation at 45℃from the extract phase, and then reconstituted with 1mL of PBS buffer (pH 6.5, 20 mol/L) to give an extract reconstituted solution. The antibacterial activity was measured using the extract complex solution as a sample solution (the method was the same as that in example 1).
Three replicates were performed and the results averaged.
The results are shown in Table 3. At a volume ratio of 1:1.5, the antibacterial activity of the extract complex solution is highest.
TABLE 3 Table 3
| Volume ratio of liquid phase system to ethyl acetate | Diameter (mm) of inhibition zone of extract complex solution |
| 1：0.5 | 22.17±0.31 c |
| 1：1 | 28.25±0.35 b |
| 1：1.5 | 30.35±0.49 a |
| 1：2 | 30.07±0.40 a |
| 1：2.5 | 29.90±0.10 a |
Example 3 crude extraction of the antibacterial peptide and further purification according to the optimization method of example 2
1. Culture of Lactobacillus plantarum NMGL2
1. The activated lactobacillus plantarum NMGL2 was inoculated to the liquid MRS medium at an inoculum size of 1% (the bacterial concentration in the system at the time of completion of inoculation was 10) 7 cfu/mL), stationary culture at 37℃for 28 hours, then centrifuging at 4℃for 15 minutes at 8000g, collecting the supernatant, filtering with a filter membrane having a pore size of 0.22 μm, and collecting the filtrate.
2. And (3) taking the filtrate obtained in the step (1), and adjusting the pH value to 5 by using an aqueous solution of NaOH.
3. And (3) taking the liquid phase system which completes the step (2), adding an equal volume of catalase solution, and incubating for 30min at 37 ℃.
2. Crude extraction of antibacterial peptide
Taking 30 parts by volume of the liquid phase system which is subjected to the step one, and extracting by adopting ethyl acetate.
The volume ratio of the liquid phase system to the ethyl acetate is 1:1.5.
After completion of the extraction, the organic phase (extract phase) was collected. The organic solvent was removed by rotary evaporation at 45℃from the extract phase, and then reconstituted with 1 part by volume of PBS buffer (pH 6.5, 20 mol/L) to give an extract reconstituted solution.
3. Purification of antimicrobial peptides by anion exchange chromatography
Chromatography column: the inner diameter is 16mm and the length is 30mm.
Filling material: DEAE-Sepharose Fast Flow (Shanghai Source leaf biology Co., ltd., LOT: D30GS172880, CAS# 57407-08-6).
And (3) purification: first, the column was equilibrated with Tris-HCl buffer (pH 7.20,0.05 mol/L); then, the extract compound solution obtained in the second loading step (the loading volume is 3 ml); then, elution was performed with Tris-HCl buffer (pH 7.2,0.05 mol/L) containing sodium chloride at a flow rate of 1mL/min for a total time of 160min, and the concentration of sodium chloride was linearly increased from 0 to 0.2M from the initial time of elution to the final time of elution.
The elution profile is shown in FIG. 2. The abscissa of FIG. 2 corresponds to the retention volume, measured from the equilibrated column, whereas FIG. 2 shows the elution step profile, so that the abscissa of the retention volume is between 100mL and 260mL.
The post-column solutions were collected for 4 elution peaks, respectively. The retention volume corresponding to the first elution peak is 100-160mL, the retention volume corresponding to the second elution peak is 160-200mL, the retention volume corresponding to the third elution peak is 200-220mL, and the retention volume corresponding to the fourth elution peak is 220-250mL.
The respective column-passing solutions were freeze-dried, and then dissolved in Tris-HCl buffer (pH 7.2,0.05 mol/L) to obtain freeze-dried complex solutions. The volume ratio of the solution after passing through the column to the Tris-HCl buffer solution for redissolution is as follows: 50:2.
And respectively taking each freeze-dried product compound solution as a sample solution for testing the antibacterial activity. Three replicates were performed and the results averaged. The method for detecting the antibacterial activity comprises the following steps: (1) taking a culture dish with the diameter of 9cm, pouring 8mL of 1.5g/100mL agar aqueous solution, standing at room temperature until solidification is achieved, and then uniformly placing 5 oxford cups on the surface of a flat plate; (2) pouring 8mL of bacteria-containing culture medium into the culture dish after the step (1) is completed, and pulling out an oxford cup after solidification, wherein a sample adding hole is formed at the position of the oxford cup; (3) after the completion of the step (2), a sample solution (50. Mu.l/well) was added to the well, and the culture was allowed to stand at 4℃for 2 hours, then at 37℃for 12 hours, and then the diameter (mm) of the inhibition zone was measured. Control wells (controls) were set and the sample stock was replaced with an equal volume of Tris-HCl buffer. An exemplary photograph is shown in fig. 3. The second eluting peak resulted in the highest antimicrobial activity of the lyophilized product complex solution.
The lyophilized product complex solution obtained from the second elution peak was designated as the antibacterial peptide solution.
4. Quantitatively detecting enzyme activity and calculating specific activity and purification times
The test samples are respectively as follows: and (3) culturing the solution (namely completing a liquid phase system of the first step), wherein the extract complex solution obtained in the second step and the antibacterial peptide solution obtained in the third step are obtained.
Taking a test sample, and carrying out double gradient dilution by adopting Tris-HCl buffer solution (pH 7.2 and 0.05 mol/L) to obtain a diluent. And respectively taking each diluent as a sample liquid for testing the antibacterial activity.
The method for detecting the antibacterial activity comprises the following steps: (1) pouring 8mL of the bacteria-containing culture medium into a culture dish with the diameter of 9cm, and standing at room temperature until solidification; (2) after the step (1) is completed, uniformly placing 8 sterile filter papers with the diameter of 6mm on a flat plate, and then dripping sample liquid (10 mu l of each piece of filter paper) on the filter papers; (3) after the completion of the step (2), stationary culture was performed at 4℃for 2 hours, then at 37℃for 12 hours, and then the diameter (mm) of the zone of inhibition was measured.
The reciprocal of the highest dilution without zone of inhibition was defined as 1AU. The enzyme activity calculation formula of the test sample is AU/ml=2 n ×(1000/x), n represents the highest dilution factor; x represents milliliters of loading volume.
Taking a test sample, and detecting the protein concentration.
Specific activity (AU/mg) is the ratio of enzyme activity (AU/mL) to protein concentration (mg/mL).
Three replicates were performed and the results averaged.
The results are shown in Table 4.
TABLE 4 Table 4
5. Identification of antimicrobial peptides
And (3) taking the antibacterial peptide solution obtained in the step (III) and carrying out Tricine-SDS-PAGE electrophoresis. The results are shown in FIG. 4, showing only one band, and molecular weights below 1.7 kDa.
And cutting the gel to recover strips, and carrying out MALDI-TOF-MS after enzymolysis in protein gel and extraction of peptide fragments. The amino acid sequence of the polypeptide is obtained, as shown in SEQ ID NO:1, which is designated as polypeptide PNMGL2.
Polypeptide PNMGL2 consists of 6 amino acid residues (LNFLKK) and has a molecular weight of 761.95Da.
EXAMPLE 4 bacteriostatic Activity of polypeptide PNMGL2 against food-borne pathogenic bacteria
Test bacteria: enterobacter sakazakii, escherichia coli, staphylococcus aureus, listeria monocytogenes and Shigella flexneri. The test bacteria are food-borne pathogenic bacteria.
The preparation method of the bacteria-containing medium used in this example: preparing 100mL LB culture medium containing 0.75g/100mL agar, sterilizing at high temperature, cooling to 45deg.C, and adding 1mL strain solution (the strain content is 10) 8 cfu/mL), and poured into a culture dish after being mixed evenly.
Artificially synthesizing SEQ ID NO:1, i.e. the polypeptide PNMGL2. The polypeptide PNMGL2 is dissolved in sterile water to make the concentration of the polypeptide PNMGL2 be 200mg/mL, and the sample solution is obtained. And detecting the antibacterial activity of the sample solution.
The method for detecting the antibacterial activity comprises the following steps: (1) taking a culture dish with the diameter of 9cm, pouring 8mL of 1.5g/100mL of agar aqueous solution, standing at room temperature until solidification is achieved, and then uniformly placing 3 oxford cups on the surface of a flat plate; (2) pouring 8mL of bacteria-containing culture medium into the culture dish after the step (1) is completed, and pulling out an oxford cup after solidification, wherein a sample adding hole is formed at the position of the oxford cup; (3) after the completion of the step (2), a sample solution (50. Mu.l/well) was added to the well, and the culture was allowed to stand at 4℃for 2 hours, then at 37℃for 12 hours, and then the diameter (mm) of the inhibition zone was measured. Control wells were set and the sample stock solution was replaced with an equal volume of sterile water. Control wells CFS were set up and an equal volume of culture broth (i.e., the liquid phase system completing step one in example 3) was used instead of the sample broth.
Three replicates were performed and the results averaged.
Exemplary photo results are shown in fig. 5.
The diameter of the antibacterial ring of the sample solution for Enterobacter sakazakii is 18.9mm. The diameter of the antibacterial circle of the sample solution for Escherichia coli is 17.5mm. The diameter of the antibacterial circle of the sample solution for staphylococcus aureus is 18.0mm. The diameter of the inhibition zone of the sample solution for Listeria monocytogenes is 19.1mm. The diameter of the antibacterial circle of the sample solution for shigella flexneri is 19.0mm.
The result shows that the polypeptide PNMGL2 has wide application prospect in the field of food biological preservation.
Example 5 structural analysis of polypeptide PNMGL2
Artificially synthesizing SEQ ID NO:1, i.e. the polypeptide PNMGL2.
Polypeptide PNMGL2 was subjected to physicochemical analysis.
The chemical structural formula is shown in figure 6.
The physicochemical properties analyzed by the PepDrw and ProtParam tools are shown in table 5.
Three-dimensional (3D) structures were generated by the PEP-FOLD 3De-novo method, see fig. 7. Is of a spiral structure.
TABLE 5
Example 6 stability analysis of antibacterial peptides
The test sample is: the antibacterial peptide solution obtained in step three of example 3.
1. Acid-base stability of antimicrobial peptides
The test sample was dispensed into centrifuge tubes (0.5 mL/tube) for a total of 8 centrifuge tubes. The centrifuge tube was then taken and the pH was adjusted to 2, 3, 4, 5, 6, 7, 8 or 9 with aqueous HCl or aqueous NaOH, respectively. Then, the mixture was water-bath at 37℃for 3 hours. The pH is then adjusted back to the original pH of the test sample with aqueous HCl or aqueous NaOH. Then, as a sample solution for a sample, the antibacterial activity was examined (the method was the same as that in example 1).
Three replicates were performed and the results averaged.
The results are shown in FIG. 8.
2. Thermal stability of antimicrobial peptides
The test sample was dispensed into centrifuge tubes (0.5 mL/tube) for a total of 8 centrifuge tubes. Then, the centrifuge tubes were taken, respectively placed under different temperature conditions for 30min, and then cooled at room temperature. Then, as a sample solution for a sample, the antibacterial activity was examined (the method was the same as that in example 1).
The different temperature conditions are respectively as follows: -80 ℃, 20 ℃,37 ℃, 65 ℃, 80 ℃, 100 ℃ or 121 ℃. Wherein-80 ℃ and-20 ℃ are realized by placing in a refrigerator, 121 ℃ is realized by 0.4Mpa steam, and the rest temperature is realized by water bath. The control treatment was placed at 4 ℃.
Three replicates were performed and the results averaged.
The results are shown in FIG. 9.
3. Enzyme sensitivity of antibacterial peptides
The test sample was dispensed into centrifuge tubes (0.5 mL/tube) for a total of 8 centrifuge tubes. Then, the centrifuge tube was taken and enzyme solutions (0.5 mL/tube) were added separately. Then, the mixture was water-bath at 37℃for 3 hours. Then, the enzyme was inactivated by boiling water bath for 5 min. Then, as a sample solution for a sample, the antibacterial activity was examined (the method was the same as that in example 1).
The enzyme solutions were as follows:
trypsin solution: trypsin was dissolved in sterile water (pH 7.8) and brought to a concentration of 5mg/mL.
Pepsin solution: pepsin was dissolved in sterile water (pH 2.0) and brought to a concentration of 5mg/mL.
Proteinase K solution: proteinase K was dissolved in sterile water (pH 7.5) and brought to a concentration of 5mg/mL.
Lysozyme solution: lysozyme was dissolved in sterile water (pH 5.0) and brought to a concentration of 5mg/mL.
Catalase solution: the catalase was dissolved in sterile water (pH 6.0) and brought to a concentration of 5mg/mL.
Lipase solution: the lipase was dissolved in sterile water (pH 7.0) and brought to a concentration of 5mg/mL.
Alkaline protease solution: the alkaline protease was dissolved in sterile water at pH (pH 8.9) and brought to a concentration of 5mg/mL.
In preparing the enzyme solution, the pH value of the sterile water is adjusted by using an aqueous HCl solution or an aqueous NaOH solution, and then the enzyme is dissolved.
An equal volume of sterile water was used in place of the enzyme solution as a control treatment.
Three replicates were performed and the results averaged.
The results are shown in FIG. 10.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. A polypeptide as set forth in SEQ ID NO: 1.
2. Use of the polypeptide of claim 1 as a microbial inhibitor.
3. Use of the polypeptide of claim 1 for inhibiting a microorganism.
4. Use of the polypeptide of claim 1 for the preparation of a microbial inhibitor.
5. Use of lactobacillus plantarum NMGL2 for the preparation of a polypeptide according to claim 1; lactobacillus plantarum NMGL2 has a preservation registration number of CGMCC No.18495.
6. Use of lactobacillus plantarum NMGL2 for the preparation of a microbial inhibitor; lactobacillus plantarum NMGL2 has a preservation registration number of CGMCC No.18495.
7. A method of making the polypeptide of claim 1, comprising the steps of: culturing lactobacillus plantarum NMGL2; lactobacillus plantarum NMGL2 has a preservation registration number of CGMCC No.18495.
8. A method of preparing a microbial inhibitor comprising the steps of: culturing lactobacillus plantarum NMGL2; lactobacillus plantarum NMGL2 has a preservation registration number of CGMCC No.18495.
9. The method of claim 7 or 8, wherein: the method further comprises the steps of: extraction is carried out by adopting ethyl acetate, and an extraction phase is collected.
10. A microbial inhibitor prepared by the method of claim 8 or 9.
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| CN110241052A (en) * | 2019-07-08 | 2019-09-17 | 北京工商大学 | One plant height produces folic acid lactobacillus plantarum GSLP-7 and its application |
| CN110591955A (en) * | 2019-09-26 | 2019-12-20 | 北京工商大学 | Lactobacillus plantarum tolerant to low-temperature acidic double stress and application thereof |
| CN114438058A (en) * | 2022-03-09 | 2022-05-06 | 北京工商大学 | Preparation method of immobilized beta-galactosidase and application of immobilized beta-galactosidase in preparation of galactooligosaccharides |
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| CN110241052A (en) * | 2019-07-08 | 2019-09-17 | 北京工商大学 | One plant height produces folic acid lactobacillus plantarum GSLP-7 and its application |
| CN110591955A (en) * | 2019-09-26 | 2019-12-20 | 北京工商大学 | Lactobacillus plantarum tolerant to low-temperature acidic double stress and application thereof |
| CN114438058A (en) * | 2022-03-09 | 2022-05-06 | 北京工商大学 | Preparation method of immobilized beta-galactosidase and application of immobilized beta-galactosidase in preparation of galactooligosaccharides |
Non-Patent Citations (1)
| Title |
|---|
| QINGXIA REN等: "Purification and characterization of a novel low-molecular-weight antimicrobial peptide produced by Lactiplantibacillus plantarum NMG", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 248, 21 July 2023 (2023-07-21), pages 125932, XP087401239, DOI: 10.1016/j.ijbiomac.2023.125932 * |
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