A novel low molecular weight antimicrobial peptide derived from Lactobacillus plantarum and its preparation method and application

Abstract

The present invention discloses a novel low molecular weight antimicrobial peptide derived from Lactobacillus plantarum, and a preparation method and application thereof. The present invention provides a polypeptide PNMGL2, as shown in SEQ ID NO: 1. The present invention also protects the use of the polypeptide PNMGL2 as a microbial inhibitor. The present invention also protects a method for preparing a microbial inhibitor, comprising the following steps: culturing Lactobacillus plantarum NMGL2. The polypeptide PNMGL2 provided by the present invention can be used as a food preservative and microbial inhibitor and applied to the fields of food and medicine. The method for preparing a microbial inhibitor provided by the present invention has a simple process operation, low cost, low equipment requirements, and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a novel low-molecular-weight antimicrobial peptide derived from Lactobacillus plantarum and a preparation method and application thereof. Background Art

[0002] According to statistics from the World Health Organization, about 420,000 people die from foodborne diseases each year. Pathogenic microorganisms in food, such as Salmonella, Enterobacter sakazakii, Staphylococcus aureus, enteropathogenic Escherichia coli, Listeria monocytogenes, Shigella flexneri, Norovirus, etc., pose a threat to food safety. Long-term intake of preservatives added to inhibit pathogenic microorganisms also poses certain hazards to health. Therefore, finding non-toxic, pollution-free, and efficient food antibacterial methods has become a top priority for food preservation.

[0003] Antimicrobial peptides (AMPs) are a type of low-molecular-weight peptide bioactive substances that are widely present in various organisms. Their chemical nature is protein. They have the characteristics of high antibacterial efficiency, non-toxicity, no drug resistance, low residue and strong stability. Therefore, they are considered to be the most promising alternatives to antibiotics and food preservatives.

[0004] At present, many antimicrobial peptides have been found in animals, plants and microorganisms. Due to the limited sources of animal and plant antimicrobial peptides, it is difficult to achieve large-scale production. Microbial antimicrobial peptides have the advantages of short fermentation cycle, low cost, easy cultivation, and easy industrial production, and have become an important direction of antimicrobial peptide research. At present, the most studied is the bacteriocin derived from lactic acid bacteria, Nisin, which has been used commercially, but most of the Nisin derived from lactic acid bacteria have problems such as narrow antimicrobial spectrum and poor stability. Therefore, there is an urgent need to find new antimicrobial peptides. Summary of the invention

[0005] The purpose of the present invention is to provide a novel low-molecular-weight antimicrobial peptide derived from Lactobacillus plantarum and a preparation method and application thereof.

[0006] The present invention provides a polypeptide, named as polypeptide PNMGL2, as shown in SEQ ID NO: 1.

[0007] SEQ ID NO: 1: LNFLKK.

[0008] The present invention also protects the use of polypeptide PNMGL2 as a microbial inhibitor.

[0009] The present invention also protects the use of polypeptide PNMGL2 in inhibiting microorganisms.

[0010] The present invention also protects the use of the polypeptide PNMGL2 in the preparation of microbial inhibitors.

[0011] The present invention also protects the use of Lactobacillus plantarum NMGL2 in preparing polypeptide PNMGL2.

[0012] The present invention also protects a method for preparing polypeptide PNMGL2, comprising the following steps: culturing Lactobacillus plantarum NMGL2. Lactobacillus plantarum NMGL2 produces polypeptide PNMGL2, so the system after the culturing contains polypeptide PNMGL2.

[0013] The present invention also protects a method for preparing a microbial inhibitor, comprising the following steps: culturing plant lactobacillus NMGL2. The system after culturing contains the microbial inhibitor.

[0014] The method for culturing any of the above specifically comprises the following steps: inoculating Lactobacillus plantarum NMGL2 into a liquid MRS medium (specifically, the bacterial concentration in the system at the time of inoculation is 10 7 cfu / mL) and cultured statically.

[0015] Specifically, the culture temperature may be 35-39°C, more specifically 37°C.

[0016] Specifically, the culture time may be 26-30 hours, more specifically 28 hours.

[0017] Any of the above methods further comprises the following steps: after the cultivation is completed, collecting the supernatant by centrifugation, and filtering and collecting the filtrate.

[0018] The centrifugation parameters may specifically be: 4° C., 8000 g for 15 minutes.

[0019] The filtration specifically uses a filter membrane with a pore size of 0.22 μm.

[0020] The method further comprises the following steps: taking the filtrate, adjusting the pH value to 5, then adding a catalase solution, and incubating.

[0021] The method further comprises the following steps: taking the filtrate, adjusting the pH value to 5, then adding an equal volume of catalase solution, and incubating at 37° C. for 30 minutes.

[0022] The method further comprises the following steps: extracting with ethyl acetate and collecting the extraction phase.

[0023] During the extraction, the volume ratio of the liquid sample to ethyl acetate is 1:1.5.

[0024] The method further comprises the following steps: after the extraction is completed, the extraction phase is collected, the organic solvent is removed, and then the extraction phase is redissolved with a PBS buffer solution (pH 6.5, 20 mol / L) to obtain an extract redissolved solution.

[0025] The method further comprises the following step: anion exchange chromatography.

[0026] Chromatography column: inner diameter 16mm, length 30mm.

[0027] Filling material: DEAE-Sepharose Fast Flow.

[0028] Purification steps: First, balance the column with Tris-HCl buffer (pH7.20, 0.05mol / L); then, load the extract solution (the specific loading volume is 3ml); then, elute with Tris-HCl buffer (pH7.2, 0.05mol / L) containing sodium chloride, the elution flow rate is 1mL / min, the total elution time is 160min, and the concentration of sodium chloride rises linearly from 0 to 0.2M from the initial time of elution to the end time of elution.

[0029] Collect the post-column solution corresponding to the second elution peak.

[0030] Collect the post-column solution with a retention volume of 160-200 mL.

[0031] The method further comprises the following steps: freeze-drying the solution after passing through the column, and then dissolving it in Tris-HCl buffer (pH 7.2, 0.05 mol / L) to obtain a freeze-dried product reconstituted solution.

[0032] Any of the above-mentioned Lactobacillus plantarum NMGL2 has a deposit registration number of CGMCC No.18495.

[0033] The microbial inhibitors prepared by any of the above methods belong to the protection scope of the present invention.

[0034] Specifically, the microorganism is a pathogenic microorganism.

[0035] Specifically, the microorganism is a bacterium.

[0036] Specifically, the bacteria are Enterobacteriaceae, Staphylococcaceae, or Listeriaceae.

[0037] Specifically, the bacteria are Escherichia bacteria, Staphylococcus bacteria, Listeria bacteria, or Shigella bacteria.

[0038] Specifically, the bacteria is Enterobacter sakazakii, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes or Shigella flexneri.

[0039] The polypeptide PNMGL2 provided by the present invention can inhibit common foodborne pathogens in food, has a broad spectrum of tolerance to temperature and pH, has a broad spectrum of tolerance to various proteases, and can be used as a food preservative and microbial inhibitor in the food and medical fields.

[0040] The method for preparing the microbial inhibitor provided by the present invention has simple process operation, low cost, low requirements on equipment, is suitable for industrial production, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the growth curve and antibacterial activity curve of Lactobacillus plantarum NMGL2.

[0042] Figure 2 This is the elution curve of the antimicrobial peptide purified by anion exchange chromatography.

[0043] Figure 3 The antibacterial activity photos of the lyophilized product reconstitution solutions obtained from different elution peaks.

[0044] Figure 4 This is the Tricine-SDS-PAGE electrophoresis diagram of the antimicrobial peptide solution.

[0045] Figure 5 This is a photo of the antibacterial activity of peptide PNMGL2 against foodborne pathogens.

[0046] Figure 6 is the chemical structural formula of polypeptide PNMGL2.

[0047] Figure 7 The three-dimensional structure of peptide PNMGL2 generated by the PEP-FOLD 3De-novo method.

[0048] Figure 8 It is the result of the acid-base stability of antimicrobial peptides.

[0049] Fig. 9 The results are for the thermal stability of antimicrobial peptides.

[0050] Fig.10 Results for enzyme sensitivity of antimicrobial peptides. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0052] The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following embodiments are set up for three repeated experiments, and the results are averaged. In the embodiment, the Bradford method is used to detect the protein concentration. The model of the Oxford cup used in the embodiment: outer diameter 7.8mm, inner diameter 6.0mm, height 10.0mm. In the embodiment, the diameter of the inhibition zone is measured using a fully automatic ultra-high-definition inhibition zone analyzer Scan4000.

[0053] Lactobacillus plantarum NMGL2, the deposit registration number is CGMCC No.18495. Lactobacillus plantarum NMGL2 is referred to as Lactobacillus plantarum NMGL2. Lactobacillus plantarum NMGL2 is recorded in the patent with the patent number ZL201910916373.6, the authorization announcement number of the patent is CN 110591955 B, the authorization announcement date of the patent is 2021.06.15, and the strain is the strain protected by the patent.

[0054] Enterobacter sakazakii used in the examples: CICC 21546. Escherichia coli used in the examples: CMCC44825. Staphylococcus aureus used in the examples: CMCC 26071. Listeria monocytogenes used in the examples: CMCC 54002. Shigella flexneri used in the examples: CICC 21534. CICC: China Industrial Microbiological Culture Collection Center. CMCC: China Medical Bacteria Collection Center.

[0055] Catalase: Solebao Biotechnology Co., Ltd., catalog number C8071. Catalase solution: Dissolve catalase in PBS buffer (pH 6.5, 20 mol / L) to a concentration of 1 mg / mL.

[0056] Trypsin: Beijing Bio-Toda Technology Co., Ltd., product number T6150. Pepsin: Beijing Bio-Toda Technology Co., Ltd., product number P6390. Proteinase K was purchased from Solebao Biotechnology Co., Ltd., product number P9460. Lysozyme: Shanghai Yuanye Biotechnology Co., Ltd., product number S10038. Lipase: Shanghai Yuanye Biotechnology Co., Ltd., product number S10035. Alkaline protease: Shanghai Yuanye Biotechnology Co., Ltd., product number S10154.

[0057] Preparation method of bacterial culture medium for Example 1, Example 2, Example 3 and Example 6: Prepare 100 ml of LB culture medium containing 0.75 g / 100 ml agar, sterilize at high temperature, and add 1 ml of Staphylococcus aureus liquid (the content of Staphylococcus aureus in 1 ml of Staphylococcus aureus liquid is 1×10 8 cfu / mL), mix well and pour into the culture dish.

[0058] Example 1. Growth curve and antibacterial activity curve of Lactobacillus plantarum NMGL2

[0059] Take the frozen Lactobacillus plantarum NMGL2 in a -80℃ glycerol tube and inoculate it into the liquid MRS medium at a 1% inoculation amount (the bacterial concentration in the system is 10 at the time of completion of inoculation). 7 cfu / mL) and incubated at 37°C for 48 hours. During the incubation period, samples were taken every 2 hours.

[0060] Take each sample separately and measure OD 600nm value and pH value.

[0061] Each sampling sample was taken separately, and the antibacterial activity was tested after pretreatment to determine the optimal culture time for producing antibacterial substances.

[0062] The pretreatment method is as follows: ① Take a sample, centrifuge it at 4°C and 8000rpm for 15min, collect the supernatant, filter it with a 0.22μm filter membrane and collect the filtrate, and adjust the pH value to 5 with an aqueous HCl solution or an aqueous NaOH solution; ② Take 1 volume of the liquid system completed in step ①, add 1 volume of catalase solution, and incubate at 37°C for 30min to obtain the test sample solution.

[0063] Method for detecting antibacterial activity (using double-layer agar diffusion method and Staphylococcus aureus as indicator bacteria): ① Take a culture dish with a diameter of 9 cm, pour 8 mL of 1.5 g / 100 ml agar aqueous solution, let it stand at room temperature until solidified, and then evenly place 3 Oxford cups on the surface of the plate; ② Pour 8 mL of bacterial culture medium into the culture dish that has completed step ①, pull out the Oxford cup after solidification, and the position of the Oxford cup forms a sample well; ③ After completing step ②, add the test sample solution (100 μl / well) to the sample well, incubate at 4°C for 2 hours, and then incubate at 37°C for 12 hours, and then measure the diameter (mm) of the inhibition zone.

[0064] Three replicates were set for each sample and the results were averaged.

[0065] Results Figure 1 . Lactobacillus plantarum NMGL2 exhibited a common bacterial growth pattern. During the culture process, the pH value of the culture system decreased from 6.2 to 3.91 and remained stable. The antibacterial activity of Lactobacillus plantarum NMGL2 was detectable from 8 hours of culture (when it was in the logarithmic growth phase) (the diameter of the inhibition zone at 8 hours of culture was 9.83 mm), and then the antibacterial activity continued to increase with the increase of culture time and reached the maximum value at 28 hours of culture (the diameter of the inhibition zone at 28 hours of culture was 15.80 mm), and then the antibacterial activity decreased with the increase of culture time. Therefore, the optimal culture time for producing antibacterial substances is 28 hours.

[0066] Example 2: Optimization of the method for crude extraction of antimicrobial peptides from Lactobacillus plantarum NMGL2 culture fluid

[0067] 1. Cultivation of Lactobacillus plantarum NMGL2

[0068] 1. Inoculate the activated Lactobacillus plantarum NMGL2 into the liquid MRS medium at a 1% inoculation rate (the bacterial concentration in the system is 10 at the time of inoculation). 7 cfu / mL), incubated at 37°C for 28 hours, and then centrifuged at 4°C and 8000 g for 15 minutes to collect the supernatant, which was filtered through a 0.22 μm filter membrane and the filtrate was collected.

[0069] 2. Take the filtrate obtained in step 1 and adjust the pH value to 5 with NaOH aqueous solution.

[0070] 3. Take the liquid phase system completed in step 2, add an equal volume of catalase solution, and incubate at 37°C for 30 minutes.

[0071] 2. Crude extraction of antimicrobial peptides using ammonium sulfate precipitation with different saturations

[0072] 1. Take 8 portions of the liquid phase system (30 mL each) obtained in step 1 and perform ammonium sulfate precipitation. The ammonium sulfate precipitation uses the following ammonium sulfate saturation: 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. The parameters of ammonium sulfate precipitation: stand at 4°C for 12 hours.

[0073] 2. After completing step 1, centrifuge at 4°C and 12000g for 15 min, collect the supernatant and precipitate respectively, and redissolve the precipitate with 1 mL PBS buffer (pH 6.5, 20 mol / L) to obtain a precipitate resolution.

[0074] 3. The protein concentration of the precipitate reconstituted solution obtained in step 2 was tested. The results are shown in Table 1.

[0075] 4. The supernatant obtained in step 2 and the precipitate reconstituted solution obtained in step 2 were respectively used as test sample solutions to detect the antibacterial activity (the method is the same as the corresponding method in Example 1).

[0076] The experiment was repeated three times and the results were averaged.

[0077] The results are shown in Table 1. The results showed that ammonium sulfate could not effectively precipitate antibacterial substances.

[0078] Table 1

[0079]

[0080]

[0081] 3. Extraction of Antimicrobial Peptides Using Different Organic Solvents

[0082] Take 5 portions (30 mL each) of the liquid phase system obtained in step 1.

[0083] One portion of the liquid phase system was not treated in any way and was directly concentrated to 1 mL by rotary evaporation at 45°C as a reference sample. The reference sample was used as the test sample liquid to detect the antibacterial activity (the method was the same as the corresponding method in Example 1). The protein concentration of the reference sample was detected. The diameter of the antibacterial zone of the reference sample was 30.40±0.40 c mM, protein concentration was 108.54±1.06 A mg / mL.

[0084] The remaining 4 liquid phase systems were extracted. The organic solvents used were: ethyl acetate, n-butanol, n-hexane or chloroform. The extraction conditions were: add an equal volume of organic solvent, oscillate and extract at room temperature and 120rpm for 2h. After the extraction was completed, the aqueous phase (raffinate phase) and the organic phase (extraction phase) were collected separately. The extract phase was rotary evaporated at 45°C to remove the organic solvent, and then redissolved with 1mL PBS buffer (pH6.5, 20mol / L) to obtain an extract re-solution. The raffinate phase and the extract re-solution were used as test sample solutions to detect the antibacterial activity (the method is the same as the corresponding method in Example 1). The protein concentration of the extract re-solution was detected.

[0085] The experiment was repeated three times and the results were averaged.

[0086] The results are shown in Table 2. The antibacterial activity of the extract resolution obtained by extraction with n-butanol or ethyl acetate was close to that of the reference sample. Considering that n-butanol has a high boiling point and is time-consuming during rotary evaporation, ethyl acetate was finally selected as the organic solvent for extraction.

[0087] Table 2

[0088]

[0089] 4. Extraction of Antimicrobial Peptides Using Different Volumes of Ethyl Acetate

[0090] Take 6 portions of the liquid phase system obtained in step 1 (30 mL each).

[0091] One portion of the liquid phase system was not treated in any way and was directly concentrated to 1 mL by rotary evaporation at 45°C as a reference sample. The reference sample was used as the test sample liquid to detect the antibacterial activity (the method was the same as the corresponding method in Example 1). The diameter of the antibacterial zone of the reference sample was 30.40±0.40 c mm.

[0092] The remaining 5 portions of the liquid phase system were extracted with ethyl acetate. Different volume ratios were set. The volume ratio of the liquid phase system to ethyl acetate was set to 1:0.5 or 1:1 or 1:1.5 or 1:2 or 1:2.5, respectively. The extraction conditions were: room temperature, 120rpm oscillation extraction for 2 hours. After the extraction was completed, the organic phase (extraction phase) was collected. The extraction phase was rotary evaporated at 45°C to remove the organic solvent, and then redissolved with 1mL PBS buffer (pH6.5, 20mol / L) to obtain an extract resolution. The extract resolution was used as the test sample solution to detect the antibacterial activity (the method was the same as the corresponding method in Example 1).

[0093] The experiment was repeated three times and the results were averaged.

[0094] The results are shown in Table 3. At a volume ratio of 1:1.5, the antibacterial activity of the extract reconstituted solution was the highest.

[0095] Table 3

[0096] Volume ratio of liquid phase system to ethyl acetate Diameter of inhibition zone of extract reconstitution solution (mm) 1：0.5 <![CDATA[22.17±0.31 c ]]> 1：1 <![CDATA[28.25±0.35 b ]]> 1：1.5 <![CDATA[30.35±0.49 a ]]> 1：2 <![CDATA[30.07±0.40 a ]]> 1：2.5 <![CDATA[29.90±0.10 a ]]>

[0097] Example 3: Crude extraction of antimicrobial peptides according to the optimized method of Example 2 and further purification

[0098] 1. Cultivation of Lactobacillus plantarum NMGL2

[0099] 1. Inoculate the activated Lactobacillus plantarum NMGL2 into the liquid MRS medium at a 1% inoculation rate (the bacterial concentration in the system is 10 at the time of inoculation). 7 cfu / mL), incubated at 37°C for 28 hours, and then centrifuged at 4°C and 8000 g for 15 minutes to collect the supernatant, which was filtered through a 0.22 μm filter membrane and the filtrate was collected.

[0100] 2. Take the filtrate obtained in step 1 and adjust the pH value to 5 with NaOH aqueous solution.

[0101] 3. Take the liquid phase system completed in step 2, add an equal volume of catalase solution, and incubate at 37°C for 30 minutes.

[0102] 2. Crude Extraction of Antimicrobial Peptides

[0103] Take 30 parts by volume of the liquid phase system prepared in step 1 and extract with ethyl acetate.

[0104] The volume ratio of the liquid phase system to ethyl acetate is 1:1.5.

[0105] After the extraction is completed, the organic phase (extraction phase) is collected, the extraction phase is subjected to rotary evaporation at 45°C to remove the organic solvent, and then redissolved with 1 volume of PBS buffer (pH 6.5, 20 mol / L) to obtain an extract reconstituted solution.

[0106] 3. Purification of antimicrobial peptides by anion exchange chromatography

[0107] Chromatography column: inner diameter 16mm, length 30mm.

[0108] Filling material: DEAE-Sepharose Fast Flow (Shanghai Yuanye Biotechnology Co., Ltd., LOT: D30GS172880, CAS#57407-08-6).

[0109] Purification steps: First, balance the column with Tris-HCl buffer (pH 7.20, 0.05 mol / L); then, load the extract reconstituted solution obtained in step 2 (loading volume is 3 ml); then, elute with Tris-HCl buffer (pH 7.2, 0.05 mol / L) containing sodium chloride, the elution flow rate is 1 mL / min, the total elution time is 160 min, and the concentration of sodium chloride rises linearly from 0 to 0.2 M from the initial time of elution to the end time of elution.

[0110] Elution curve Figure 2 . Figure 2 The horizontal axis corresponds to the retention volume, which is measured from the equilibrium column. Figure 2 Shown is an elution step curve so that the retention volume is 100 mL - 260 mL on the abscissa.

[0111] Collect the post-column solutions of the four elution peaks respectively. The retention volume corresponding to the first elution peak is 100-160 mL, the retention volume corresponding to the second elution peak is 160-200 mL, the retention volume corresponding to the third elution peak is 200-220 mL, and the retention volume corresponding to the fourth elution peak is 220-250 mL.

[0112] Each post-column solution was freeze-dried and then dissolved in Tris-HCl buffer (pH 7.2, 0.05 mol / L) to obtain a lyophilized product reconstitution solution. The volume ratio of the post-column solution to the Tris-HCl buffer used for reconstitution was 50:2.

[0113] Each reconstituted solution of the lyophilized product was used as the test sample solution to detect the antibacterial activity. The test was repeated three times and the results were averaged. Method for detecting antibacterial activity: ① Take a culture dish with a diameter of 9 cm, pour 8 mL of 1.5 g / 100 ml agar aqueous solution, let it stand at room temperature until solidified, and then evenly place 5 Oxford cups on the surface of the plate; ② Pour 8 mL of bacterial culture medium into the culture dish that has completed step ①, pull out the Oxford cup after solidification, and the position of the Oxford cup forms the sample well; ③ After completing step ②, add the test sample solution (50 μl / well) to the sample well, incubate at 4°C for 2 hours, then incubate at 37°C for 12 hours, and then measure the diameter of the inhibition zone (mm). Set up a control well (Control) and replace the test sample solution with an equal volume of Tris-HCl buffer. See exemplary photos. Figure 3 The second elution peak resulted in the highest antibacterial activity of the lyophilized product reconstituted solution.

[0114] The reconstituted solution of the lyophilized product obtained from the second elution peak was named antimicrobial peptide solution.

[0115] 4. Quantitative detection of enzyme activity and calculation of specific activity and purification multiple

[0116] The test samples are: culture medium (ie, the liquid phase system after completing step one), the extract reconstituted solution obtained in step two, and the antimicrobial peptide solution obtained in step three.

[0117] Take the test sample and dilute it twice with Tris-HCl buffer (pH 7.2, 0.05 mol / L) to obtain dilutions. Use each dilution as a test sample solution to detect the antibacterial activity.

[0118] Method for detecting antibacterial activity: ① Pour 8 mL of bacterial culture medium into a culture dish with a diameter of 9 cm and place it at room temperature until solidified; ② After completing step ①, evenly place 8 sterile filter papers with a diameter of 6 mm on the plate, and then drop the test sample solution on the filter paper (10 μl per filter paper); ③ After completing step ②, incubate at 4°C for 2 hours, then incubate at 37°C for 12 hours, and then measure the diameter of the inhibition zone (mm).

[0119] The reciprocal of the highest dilution factor without an inhibition zone is 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 the sample volume in milliliters.

[0120] Take the test sample and detect the protein concentration.

[0121] Specific activity (AU / mg) is the ratio of enzyme activity (AU / ml) to protein concentration (mg / mL).

[0122] The experiment was repeated three times and the results were averaged.

[0123] The results are shown in Table 4.

[0124] Table 4

[0125]

[0126]

[0127] 5. Identification of antimicrobial peptides

[0128] Take the antimicrobial peptide solution obtained in step 3 and perform Tricine-SDS-PAGE electrophoresis. Figure 4 , only one band is shown, and the molecular weight is below 1.7 kDa.

[0129] The gel was cut and the bands were recovered, and after in-gel enzymatic digestion and peptide extraction, MALDI-TOF-MS was performed to obtain the amino acid sequence of the polypeptide, as shown in SEQ ID NO: 1, which was named polypeptide PNMGL2.

[0130] The polypeptide PNMGL2 is composed of 6 amino acid residues (LNFLKK) and has a molecular weight of 761.95Da.

[0131] Example 4: Antibacterial activity of polypeptide PNMGL2 against foodborne pathogens

[0132] Test bacteria: Enterobacter sakazakii, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Shigella flexneri. All the test bacteria are foodborne pathogens.

[0133] Preparation method of the bacterial culture medium used in this example: prepare 100 ml of LB culture medium containing 0.75 g / 100 ml agar, sterilize at high temperature, and add 1 mL of the test bacterial solution (the content of the test bacteria is 10 8 cfu / mL), mix well and pour into the culture dish.

[0134] The polypeptide shown in SEQ ID NO: 1, namely, polypeptide PNMGL2, was artificially synthesized. The polypeptide PNMGL2 was dissolved in sterile water to a concentration of 200 mg / mL, namely, the test sample solution. The antibacterial activity of the test sample solution was detected.

[0135] Method for detecting antibacterial activity: ① Take a culture dish with a diameter of 9 cm, pour 8 mL of 1.5 g / 100 ml agar aqueous solution, let it stand at room temperature until solidified, and then evenly place 3 Oxford cups on the surface of the plate; ② Pour 8 mL of bacterial culture medium into the culture dish that completes step ①, pull out the Oxford cup after solidification, and the position of the Oxford cup forms a sample well; ③ After completing step ②, add the test sample solution (50 μl / well) to the sample well, let it stand at 4°C for 2 hours, then let it stand at 37°C for 12 hours, and then measure the diameter of the inhibition zone (mm). Set up a control well Control, and replace the test sample solution with an equal volume of sterile water. Set up a control well CFS, and replace the test sample solution with an equal volume of culture solution (i.e., the liquid phase system that completes step one in Example 3).

[0136] The experiment was repeated three times and the results were averaged.

[0137] See the example photo results Figure 5 .

[0138] The diameter of the inhibition zone of the test sample solution for Enterobacter sakazakii is 18.9 mm. The diameter of the inhibition zone of the test sample solution for Escherichia coli is 17.5 mm. The diameter of the inhibition zone of the test sample solution for Staphylococcus aureus is 18.0 mm. The diameter of the inhibition zone of the test sample solution for Listeria monocytogenes is 19.1 mm. The diameter of the inhibition zone of the test sample solution for Shigella flexneri is 19.0 mm.

[0139] The results showed that the peptide PNMGL2 has broad application prospects in the field of food biopreservation.

[0140] Example 5: Structural analysis of polypeptide PNMGL2

[0141] The polypeptide shown in SEQ ID NO: 1, namely, polypeptide PNMGL2, was artificially synthesized.

[0142] The peptide PNMGL2 was subjected to physicochemical analysis.

[0143] Chemical structure Figure 6 .

[0144] The physicochemical properties analyzed by PepDrw and ProtParam tools are shown in Table 5.

[0145] The three-dimensional (3D) structure was generated by the PEP-FOLD 3De-novo method, see Figure 7 . It is a spiral structure.

[0146] Table 5

[0147]

[0148] Example 6. Stability analysis of antimicrobial peptides

[0149] The test sample is: the antimicrobial peptide solution obtained in step 3 of Example 3.

[0150] 1. Acid-base stability of antimicrobial peptides

[0151] The test sample was divided into centrifuge tubes (0.5 mL / tube), a total of 8 centrifuge tubes. Then, take the centrifuge tubes and adjust the pH value to 2, 3, 4, 5, 6, 7, 8 or 9 with HCl aqueous solution or NaOH aqueous solution. Then, bathe at 37°C for 3 hours. Then, adjust the pH back to the original pH value of the test sample with HCl aqueous solution or NaOH aqueous solution. Then, use it as a test sample solution to detect antibacterial activity (the method is the same as the corresponding method in Example 1).

[0152] The experiment was repeated three times and the results were averaged.

[0153] Results Figure 8 .

[0154] 2. Thermal stability of antimicrobial peptides

[0155] The test sample was divided into centrifuge tubes (0.5 mL / tube), with a total of 8 centrifuge tubes. Then, the centrifuge tubes were taken and placed under different temperature conditions for 30 minutes, and then cooled at room temperature. Then, the antibacterial activity was detected as a test sample solution (the method is the same as the corresponding method in Example 1).

[0156] The different temperature conditions were: -80℃, -20℃, 37℃, 65℃, 80℃, 100℃ or 121℃. Among them, -80℃ and -20℃ were achieved by placing in a refrigerator, 121℃ was achieved by 0.4Mpa steam, and the remaining temperatures were achieved by water bath. 4℃ was placed as a control treatment.

[0157] The experiment was repeated three times and the results were averaged.

[0158] Results Fig. 9 .

[0159] 3. Enzyme sensitivity of antimicrobial peptides

[0160] The test sample was divided into centrifuge tubes (0.5 mL / tube), a total of 8 centrifuge tubes. Then, the centrifuge tubes were taken and the enzyme solution (0.5 mL / tube) was added respectively. Then, the sample was placed in a 37°C water bath for 3 hours. Then, the enzyme was inactivated in a boiling water bath for 5 minutes. Then, the antibacterial activity was detected as a test sample solution (the method is the same as the corresponding method in Example 1).

[0161] The enzyme solutions are as follows:

[0162] Trypsin solution: Dissolve trypsin in sterile water (pH 7.8) to a concentration of 5 mg / mL.

[0163] Pepsin solution: Dissolve pepsin in sterile water (pH 2.0) to a concentration of 5 mg / mL.

[0164] Proteinase K solution: Dissolve proteinase K in sterile water (pH 7.5) to a concentration of 5 mg / mL.

[0165] Lysozyme solution: Lysozyme was dissolved in sterile water (pH 5.0) to a concentration of 5 mg / mL.

[0166] Catalase solution: Dissolve catalase in sterile water (pH 6.0) to a concentration of 5 mg / mL.

[0167] Lipase solution: Lipase was dissolved in sterile water (pH 7.0) to a concentration of 5 mg / mL.

[0168] Alkaline protease solution: Alkaline protease was dissolved in sterile water at pH 8.9 to a concentration of 5 mg / mL.

[0169] When preparing the enzyme solution, first adjust the pH of sterile water with HCl aqueous solution or NaOH aqueous solution, and then dissolve the enzyme.

[0170] An equal volume of sterile water was used instead of the enzyme solution as a control treatment.

[0171] The experiment was repeated three times and the results were averaged.

[0172] Results Fig.10 .

[0173] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of polypeptides in the preparation of microbial inhibitors; The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; The microorganism is Enterobacter sakazakii, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes or Shigella flexneri.

2. Application of Lactobacillus plantarum NMGL2 in the preparation of polypeptides; The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; Lactobacillus plantarum NMGL2, its deposit registration number is CGMCC No.18495.

3. A method for preparing the polypeptide of claim 1, comprising the following steps: culturing Lactobacillus plantarum NMGL2; Lactobacillus plantarum NMGL2, whose deposit registration number is CGMCC No.18495; The culturing of Lactobacillus plantarum NMGL2 is carried out by inoculating Lactobacillus plantarum NMGL2 into a liquid MRS culture medium, and the bacterial concentration in the system is 10 7 cfu / mL, static culture at 37°C for 28 h; After the cultivation is completed, the supernatant is collected by centrifugation, and the filtrate is collected by filtration; the centrifugation parameters are: 4°C, 8000g centrifugation for 15 minutes; the filtration uses a filter membrane with a pore size of 0.22 μm; The filtrate was taken, the pH value was adjusted to 5, and then an equal volume of catalase solution was added, and the mixture was incubated at 37° C. for 30 min to obtain a liquid sample; The method further comprises the following steps: extracting with ethyl acetate and collecting the extract phase; during the extraction, the volume ratio of the liquid sample to ethyl acetate is 1:1.5; After the extraction is completed, the extract phase is collected, the organic solvent is removed, and then it is redissolved with pH 6.5, 20 mol / L PBS buffer to obtain an extract resolution; The method further comprises the following steps: anion exchange chromatography; chromatography column: inner diameter 16 mm, length 30 mm; filling material: DEAE-Sepharose Fast Flow; Purification steps: First, equilibrate the column with pH 7.20, 0.05 mol / L Tris-HCl buffer ； Then, 3 ml of the extract re-solution was loaded; eluted with pH 7.2, 0.05 mol / L Tris-HCl buffer containing sodium chloride, the elution flow rate was 1 mL / min, the total elution time was 160 min, and the concentration of sodium chloride increased linearly from 0 to 0.2 M from the initial moment of elution to the end moment of elution; the post-column solution corresponding to the second elution peak was collected; Collect the post-column solution with a retention volume of 60-200 mL; The solution after passing through the column is freeze-dried and then dissolved in pH 7.2, 0.05 mol / L Tris-HCl buffer to obtain a freeze-dried product reconstituted solution, which is the polypeptide solution.

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