Lactobacillus bacteriocin as well as preparation method and application thereof

Abstract

The invention discloses lactobacillus bacteriocin as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. According to the invention, a bacteriocin is extracted from lactobacillus pentosus with the preservation number of CCTCC NO: M 2022431, and the bacteriocin has thermal stability, can tolerate low temperature, and can keep the activity in a very wide pH range. Meanwhile, the bacteriocin can inhibit the growth and proliferation of the food-borne pathogenic bacteria Listeria monocytogenes, and the bacteriocin is derived from edible Sichuan pickles, so that the bacteriocin is harmless to human bodies, can provide a new research direction for preventing or treating diseases caused by the food-borne pathogenic bacteria, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a lactic acid bacteria bacteriocin and a preparation method and application thereof. Background Art

[0002] Listeria monocytogenes is a Gram-positive zoonotic pathogen and one of the most threatening foodborne pathogens to human health. Listeria monocytogenes can cause listeriosis and gastroenteritis, leading to meningitis, miscarriage, sepsis and other diseases. The infectivity and harmfulness of Listeria monocytogenes make its treatment measures particularly important. The traditional method for treating the above-mentioned foodborne pathogens is antibiotic therapy, but due to the abuse of antibiotics, multidrug-resistant pathogens have shown extensive resistance to antibiotics such as penicillin and ciprofloxacin, which makes the development of new treatment methods urgent. With the continuous development of food industry technology, people prefer green, mild, natural and environmentally friendly antibacterial methods. The emergence of natural antimicrobial agents, such as lactoperoxidase, lactoferrin, lysozyme from animals, essential oils and herbal extracts from plants, and bacteriocins from microorganisms, provides people with new ideas.

[0003] Lactic acid bacteria can secrete metabolites with antibacterial activity, such as organic acids, bacteriocins and H 2 O 2 etc., thereby inhibiting the growth of pathogenic bacteria. Nisin and pediocin are the most studied bacteriocins that can be used as commercial natural preservatives. Bacteriocins are bacteriocins synthesized in the ribosomes of strains such as Lactococcus lactis and Enterococcus faecalis.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the objects of the present invention includes providing a lactic acid bacteria bacteriocin.

[0006] The second object of the present invention includes providing a method for preparing the above-mentioned lactic acid bacteria bacteriocin.

[0007] The third object of the present invention includes providing the use of the above-mentioned lactic acid bacteria bacteriocin in the preparation of antibacterial products.

[0008] A fourth object of the present invention includes providing an antibacterial product.

[0009] The present invention solves the technical problem by adopting the following technical solutions:

[0010] The invention provides a lactic acid bacteria bacteriocin. The lactic acid bacteria bacteriocin is extracted from Lactobacillus pentosus. The preservation number of the Lactobacillus pentosus is CCTCC NO: M 2022431.

[0011] Optionally, the molecular weight of the lactic acid bacteria bacteriocin is about 5988Da.

[0012] Optionally, the amino acid sequence of the lactic acid bacteria bacteriocin is as shown in SEQ ID NO:1.

[0013] The present invention also provides a method for preparing lactic acid bacteria bacteriocin, which is obtained through fermentation products of Lactobacillus pentosus or through chemical synthesis.

[0014] Optionally, the preparation method comprises: centrifuging the fermentation liquid of Lactobacillus pentosus to obtain the supernatant, adding ammonium sulfate to the supernatant so that the saturation of ammonium sulfate in the supernatant is 80% to 100%, centrifuging again to obtain the precipitate, and then redissolving, dialyzing and freeze-drying in sequence to obtain a crude bacteriocin.

[0015] Optionally, the preparation of the Lactobacillus pentosus fermentation broth comprises: inoculating Lactobacillus pentosus into a fermentation medium for culturing.

[0016] Optionally, the fermentation medium contains: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1 ml / L anhydrous sorbitan monooleate, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate trihydrate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.05 g / L manganese sulfate tetrahydrate.

[0017] Optionally, the preparation method further comprises purifying the crude bacteriocin, and the purification comprises: dissolving the crude bacteriocin and then separating it with a Sephadex G-50 gel chromatography column, and then dialyzing and freeze-drying again to obtain the lactic acid bacteria bacteriocin.

[0018] In addition, the present invention also proposes an application of the lactic acid bacteria bacteriocin in the preparation of an antibacterial product.

[0019] Optionally, the antibacterial product comprises an antibacterial agent that inhibits the growth of Listeria monocytogenes.

[0020] The present invention also provides an antibacterial product, which comprises the lactic acid bacteria bacteriocin.

[0021] The present invention has the following beneficial effects:

[0022] The present invention extracts a bacteriocin from Lactobacillus pentosus with a preservation number of CCTCC NO: M 2022431. The bacteriocin has thermal stability, can tolerate low temperatures, and maintains its activity in a wide pH range. At the same time, the bacteriocin can inhibit the growth and proliferation of foodborne pathogenic bacteria Listeria monocytogenes. Since the bacteriocin is derived from edible Sichuan pickles, it is harmless to the human body and can also provide a new research direction for preventing or treating diseases caused by foodborne pathogenic bacteria, so it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The gel filtration column elution chart (a) and the high performance liquid chromatography chart (b) of the Lactobacillus pentosus bacteriocin L14 of Example 1;

[0025] Figure 2 The inhibitory effect of the Lactobacillus pentosus bacteriocin L14 on Listeria monocytogenes in Example 2;

[0026] Figure 3 The mass spectrometry identification result of Lactobacillus pentosus bacteriocin L14 in Example 3;

[0027] Figure 4 The temperature stability (a), pH stability (b), UV stability (c) and enzyme stability (d) of Lactobacillus pentosus L14 of Example 4 are shown. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0029] Lactiplantibacillus pentosus L14 used in the following examples is a strain isolated by the inventor from Sichuan pickles, and was identified as Lactiplantibacillus pentosus by physiological and biochemical and 16S rDNA sequence analysis. This strain has been disclosed in patent CN202311069646.0-A strain of Lactiplantibacillus pentosus inhibiting Yarrowia lipolytica and its application, and its deposit information is:

[0030] Deposited in China Center for Type Culture Collection; storage address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; storage date: April 27, 2022; storage number: CCTCC NO: M 2022431; taxonomic name: Lactiplantibacillus pentosus L14.

[0031] The present invention obtains a bacteriocin from the fermentation broth of Lactiplantibacillus pentosus L14, and names it bacteriocin L14. After testing, the amino acid sequence of the bacteriocin is shown in SEQ ID NO: 1, which is a class IIa bacteriocin with a molecular weight of about 12810 Da.

[0032] The above bacteriocin is obtained by the following preparation method of the present invention, and the steps are as follows:

[0033] S1. Inoculate Lactobacillus pentosus into a fermentation medium for culturing to obtain a fermentation liquid of Lactobacillus pentosus.

[0034] Wherein, the pentosus lactobacillus is the above-mentioned Lactiplantibacillus pentosus L14, and its fermentation medium comprises: 10 g / L peptone, 8 g / L beef extract powder, 4 g / L yeast extract, 20 g / L glucose, 1 ml / L anhydrous sorbitan monooleate, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate trihydrate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.05 g / L manganese sulfate tetrahydrate.

[0035] The fermentation culture conditions of the above-mentioned Lactobacillus pentosus are: static culture at 37° C. for 24 hours.

[0036] S2. The fermentation liquid of Lactobacillus pentosus is centrifuged to obtain the supernatant, and an ammonium sulfate solution with a saturation of 80% to 100% is added to the supernatant. The precipitate is obtained after centrifugation again, and then redissolved, dialyzed and freeze-dried in sequence to obtain a crude bacteriocin.

[0037] S3. After dissolving the crude bacteriocin, separate it using a Sephadex G-50 gel chromatography column, and then dialyze and freeze-dry to obtain the lactic acid bacteria bacteriocin.

[0038] Experiments have shown that the bacteriocin L14 obtained by the above preparation method is thermally stable, can withstand low temperatures, and maintain its activity within a wide pH range; it can inhibit the growth and reproduction of Listeria monocytogenes, so the bacteriocin can be used to prepare antibacterial products, especially products that inhibit the growth of Listeria monocytogenes, such as antibacterial agents.

[0039] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0040] The Listeria monocytogenes used in the embodiments of the present invention is Listeria monocytogenes ATCC19115, which was purchased from the laboratory.

[0041] Example 1

[0042] This embodiment is a method for preparing bacteriocin of Lactobacillus pentosus L14, which is as follows:

[0043] (1) activating and expanding the culture of Lactiplantibacillus pentosus L14;

[0044] (2) inoculating the culture medium with the expansion solution prepared in step (1) at an inoculum amount of 3%, and culturing at 37° C. for 24 hours to obtain a fermentation solution containing bacteriocin of Lactobacillus pentosus;

[0045] The fermentation medium is MRS liquid medium, which consists of the following components: peptone 10g / L, beef extract 8g / L, yeast extract 4g / L, glucose 20g / L, anhydrous sorbitan monooleate 1ml / L, dipotassium hydrogen phosphate 2g / L, sodium acetate trihydrate 5g / L, triammonium citrate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate tetrahydrate 0.05g / L. Sterilize at 121℃ for 15min;

[0046] (3) centrifuging the liquid fermentation broth obtained in step (2) and collecting the supernatant;

[0047] (4) Add (NH 4 ) 2 SO 4 The saturation was 85%, stirred at room temperature for 10 min, allowed to stand at 4 °C for 12 h, centrifuged, and the precipitate was collected;

[0048] (5) dissolving the precipitate obtained in step (4) with pH 6.0 PBS buffer, dialyzing with ultrapure water, and vacuum freeze-drying to obtain crude bacteriocin;

[0049] (6) dissolving the crude bacteriocin obtained in step (5) in water to obtain a crude bacteriocin solution;

[0050] (7) separating the crude bacteriocin solution obtained in step (6) through a Sephadex G-50 gel chromatography column to collect bacteriocin components;

[0051] (8) dialyzing and vacuum freeze-drying the bacteriocin solution obtained in step (7) to obtain purified bacteriocin;

[0052] (9) The purified bacteriocin obtained in step (8) was dissolved in UP water and analyzed by high performance liquid chromatography. The mobile phase A was acetonitrile, the mobile phase B was 5 mM HCl, the column temperature was 39° C., the flow rate was 1 mL / min, and the gradient used in the experiment was increased from 24% to 60% of the mobile phase A within 20 min. The retention time of the bacteriocin of Lactobacillus pentosus L14 was 3.2 min.

[0053] Figure 1 The present invention provides a Sephadex G-50 gel filtration column elution diagram (a) and a high performance liquid chromatography diagram (b) of the Lactobacillus pentosus bacteriocin L14.

[0054] Example 2

[0055] This example verifies the effect of Lactobacillus pentosus L14 bacteriocin on the growth of Listeria monocytogenes

[0056] (1) The L14 obtained in Example 1 was dissolved in a pH 6.0 PBS buffer solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm to obtain a sterile L14 solution.

[0057] (2) Preparation of Listeria monocytogenes culture medium

[0058] Listeria monocytogenes was inoculated into TSA-YE liquid culture medium, and statically cultured at 37°C for 24 hours to prepare a seed solution; the prepared seed solution was inoculated into TSA-YE liquid culture medium at an inoculation amount of 3% (v / v), and statically cultured at 37°C for 24 hours to prepare a culture solution.

[0059] (3) Antibacterial experiment of Lactobacillus pentosus L14 bacteriocin

[0060] 1) L14 was co-cultured with Listeria monocytogenes to investigate the effect of bacteriocin L14 of Lactobacillus pentosus L14 on the growth of Listeria monocytogenes.

[0061] 2) Detection method: Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid culture medium at a 3% inoculum volume, shake evenly, and pipette 2 mL of the bacterial solution into a 5 mL sterile centrifuge tube.

[0062] 6) Add 2mL of 1mg / mL Lactobacillus pentosus L14 bacteriocin L14 solution respectively, seal the centrifuge tube, culture at 37℃ for 16h, and measure the optical density value at 600nm. Compare with the optical density value of Listeria monocytogenes at 0h. Add 2mL of sterile pH6.0 PBS buffer to 2mL of Listeria monocytogenes culture as the control group. The results are shown in Figure 2 shown.

[0063] Figure 2 The results show that the bacteriocin L14 of Lactobacillus pentosus L14 provided by the present invention has an inhibitory effect on the growth of Listeria monocytogenes.

[0064] Example 3

[0065] This example is the identification of bacteriocin of Lactobacillus pentosus

[0066] (1) Electrophoresis of Bacteriocin L14 of Lactobacillus pentosus L14

[0067] 1) L14 obtained in Example 1 was dissolved in UP water, and a separation gel, an interlayer gel and a concentrated gel were prepared using a Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320).

[0068] 2) Place the electrophoresis tank in a 4°C water bath, add anode buffer (T1225) to the outer tank and cathode buffer (T1215) to the inner tank, perform pre-electrophoresis at 30V for 10 min, use an ultra-low molecular weight protein marker (Solarbio, PR1300), add L14 aqueous solution to the spotting wells and perform electrophoresis at 30V for 1h, then stop electrophoresis at 100V until bromophenol blue reaches the bottom of the gel, and perform Coomassie Brilliant Blue staining.

[0069] (2) Enzymatic hydrolysis and mass spectrometry identification steps

[0070] 1) Cut the protein bands stained with Coomassie Brilliant Blue in (1) and store them at 4°C. 2 O for 3 times, add 150 μL of decolorizing solution for decolorization, wash with water for 4 times, and then wash the strips with 300 μL of 25 mM ammonium bicarbonate, 50% acetonitrile and 100% acetonitrile for 2 times in sequence, and finally dehydrate the gel until the gel blocks turn white.

[0071] 2) Add 50 μL of 10 mM DTT solution and reduce in a 56°C water bath for 30 min. When the temperature drops to room temperature, add an equal volume of 50 mM IAA solution for alkylation in the dark for 15 min. Then wash the strips twice with 300 μL of 25 mM ammonium bicarbonate, 50% acetonitrile and 100% acetonitrile. Finally, dehydrate the gel until the gel blocks turn white.

[0072] 3) Add 15-20 μL of 0.01 μg / μL proteomics-grade trypsin, and after it is fully absorbed on ice and becomes transparent, add 50 mM NH 4 HCO 3 Cover with 30-40 μL of solution (containing 10% ACN); digest overnight in a 37°C water bath.

[0073] 4) After enzymatic hydrolysis, the supernatant was transferred to another new EP tube, and 100 μL of extraction solution (67% acetonitrile, containing 2% formic acid) was added to the remaining gel block, incubated at 37°C for 30 min, then ultrasonicated for 15 min, centrifuged, the supernatant was combined, concentrated by centrifugation, and dried for mass spectrometry analysis.

[0074] (3) Mass spectrometry operation and database retrieval

[0075] The cleaved polypeptide sample was centrifugally dried, redissolved in Nano-LC mobile phase A (0.1% formic acid / water), loaded into a bottle, and subjected to online LCMS analysis.

[0076] The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, ), and then 20 μL of volume was used for washing and desalting. The liquid phase was Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA). The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ), the gradient used in the experiment was from 5% to 38% of mobile phase B (80% acetonitrile, 0.1% formic acid) within 30 min.

[0077] The mass spectrometer used a ThermoFisher Q Exactive system (ThermoFisher, USA) combined with a nanospray NanoFlex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 275 °C. The mass spectrometer scan mode was in the information-dependent acquisition mode (DDA, Data Dependent Analysis), with a primary mass spectrometer scan resolution of 70000, a scan range of 350-2000 m / z, and a maximum injection time of 100 ms. In each DDA cycle, up to 20 secondary spectra with charges of 2+ to 5+ were collected, and the maximum injection time of the secondary mass spectrometer ions was 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 seconds.

[0078] The original raw spectrum files collected by mass spectrometry were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) software. The database was the Lactiplantibacillus pentosus protein database downloaded from Uniprot, and the search parameters were set as follows: chymosin digestion, primary mass spectrometry mass tolerance of 10 ppm, and secondary mass spectrometry of 0.05 Da.

[0079] Comparison of LC-MS / MS with the universal protein database (UniProt) revealed that bacteriocin L14 was similar to the protein numbered G0M498·G0M498_LACPE in the database and belonged to the same genus as Lactobacillus pentosus.

[0080] The amino acid sequence is:

[0081] MNNALSFEQQFTDFSTLSDSELESVEGGRNKLAYNMGHYAGKATIF GLAAWALLA (SEQ ID NO: 1), the mass spectrum result is as follows Figure 3 shown.

[0082] Example 4

[0083] This example shows the properties of Lactobacillus pentosus L14 bacteriocin

[0084] (1) Temperature stability of Lactobacillus pentosus L14 bacteriocin

[0085] 1) The L14 obtained in Example 1 was dissolved in a pH 6.0 PBS buffer solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm to obtain a sterile L14 solution.

[0086] 2) Take equal amounts of the L14 solution in 1) in centrifuge tubes and heat them in a -20, 40, 60, 80, 100°C water bath and a 121°C autoclave for 10 min and 30 min, then immediately cool to room temperature in an ice water bath. Store the bacteriocin as a control.

[0087] 3) Preparation of Listeria monocytogenes culture medium

[0088] Listeria monocytogenes was inoculated into TSA-YE liquid culture medium, and statically cultured at 37°C for 24 hours to prepare a seed solution; the prepared seed solution was inoculated into TSA-YE liquid culture medium at an inoculation amount of 3% (v / v), and statically cultured at 37°C for 24 hours to prepare a culture solution.

[0089] 4) Antibacterial experiment of bacteriocin of Lactobacillus pentosus L14

[0090] Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid culture medium at a 3% inoculum, shake evenly, and pipette 2 mL of the bacterial solution into a 5 mL sterile centrifuge tube. Add 2 mL of the Lactobacillus pentosus L14 bacteriocin L14 solution treated in 2) respectively, seal the centrifuge tube, culture at 37° C. for 16 h, and measure the optical density value at a wavelength of 600 nm.

[0091] (2) pH stability of Lactobacillus pentosus L14 bacteriocin

[0092] 1) The L14 obtained in Example 1 was dissolved in a pH 6.0 PBS buffer solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm to obtain a sterile L14 solution.

[0093] 2) Take equal amounts of L14 solution prepared in 1) in centrifuge tubes and adjust the pH to 2, 3, 4, 5, 7, 8, 9, 10, 11, and 12 with 5M HCl and 5M NaOH. After incubating in a 37°C water bath for 1 hour, adjust the pH of each solution to 6. The bacteriocin solution without pH adjustment was used as a control.

[0094] 3) Preparation of Listeria monocytogenes culture medium

[0095] Listeria monocytogenes was inoculated into TSA-YE liquid culture medium, and statically cultured at 37°C for 24 hours to prepare a seed solution; the prepared seed solution was inoculated into TSA-YE liquid culture medium at an inoculation amount of 3% (v / v), and statically cultured at 37°C for 24 hours to prepare a culture solution.

[0096] 4) Antibacterial experiment of bacteriocin of Lactobacillus pentosus L14

[0097] Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid culture medium at a 3% inoculum, shake evenly, and pipette 2 mL of the bacterial solution into a 5 mL sterile centrifuge tube. Add 2 mL of the Lactobacillus pentosus L14 bacteriocin L14 solution treated in 2) respectively, seal the centrifuge tube, culture at 37° C. for 16 h, and measure the optical density value at a wavelength of 600 nm.

[0098] (3) UV stability of Lactobacillus pentosus L14 bacteriocin

[0099] 1) The L14 obtained in Example 1 was dissolved in a pH 6.0 PBS buffer solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm to obtain a sterile L14 solution.

[0100] 2) Take equal amounts of the L14 solution in 1) in centrifuge tubes and expose them to ultraviolet light for 10, 20, 30, 40, 50, and 60 min, respectively. The bacteriocin solution without ultraviolet light was used as a control.

[0101] 3) Preparation of Listeria monocytogenes culture medium

[0102] Listeria monocytogenes was inoculated into TSA-YE liquid culture medium, and statically cultured at 37°C for 24 hours to prepare a seed solution; the prepared seed solution was inoculated into TSA-YE liquid culture medium at an inoculation amount of 3% (v / v), and statically cultured at 37°C for 24 hours to prepare a culture solution.

[0103] 4) Antibacterial experiment of bacteriocin of Lactobacillus pentosus L14

[0104] Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid culture medium at a 3% inoculum, shake evenly, and pipette 2 mL of the bacterial solution into a 5 mL sterile centrifuge tube. Add 2 mL of the Lactobacillus pentosus L14 bacteriocin L14 solution treated in 2) respectively, seal the centrifuge tube, culture at 37° C. for 16 h, and measure the optical density value at a wavelength of 600 nm.

[0105] (4) Enzyme stability of Lactobacillus pentosus L14 bacteriocin

[0106] 1) The L14 obtained in Example 1 was dissolved in a pH 6.0 PBS buffer solution to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm to obtain a sterile L14 solution.

[0107] 2) Take equal amounts of L14 solution prepared in 1) in centrifuge tubes, adjust the pH to 1.9, 8.0, and 10.3 with 5M HCl and 5M NaOH, add pepsin, trypsin, and proteinase K, respectively, to a final concentration of 20 mg / mL, incubate in a 37°C water bath for 1 h, and then adjust the pH of each solution to 6. A bacteriocin solution without the addition of protease was used as a control.

[0108] 3) Preparation of Listeria monocytogenes culture medium

[0109] Listeria monocytogenes was inoculated into TSA-YE liquid culture medium, and statically cultured at 37°C for 24 hours to prepare a seed solution; the prepared seed solution was inoculated into TSA-YE liquid culture medium at an inoculation amount of 3% (v / v), and statically cultured at 37°C for 24 hours to prepare a culture solution.

[0110] 4) Antibacterial experiment of bacteriocin of Lactobacillus pentosus L14

[0111] Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid culture medium at a 3% inoculum, shake evenly, and pipette 2 mL of the bacterial solution into a 5 mL sterile centrifuge tube. Add 2 mL of the Lactobacillus pentosus L14 bacteriocin L14 solution treated in 2) respectively, seal the centrifuge tube, culture at 37° C. for 16 h, and measure the optical density value at a wavelength of 600 nm.

[0112] Figure 4 The results showed that L14 could still maintain about 40% of its activity when treated at -20℃ and 60℃ for 30 minutes, which indicates that the bacteriocin has low-temperature stability and thermal stability; L14 maintains activity in a wide pH range, especially under acidic conditions; L14 maintains high activity within 40 minutes of ultraviolet irradiation; trypsin and proteinase K have a greater effect on the activity of L14.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lactic acid bacteria bacteriocin, characterized in that The lactic acid bacteria bacteriocin is extracted from Lactobacillus pentosus, and the preservation number of the Lactobacillus pentosus is CCTCC NO: M2022431.

2. The lactic acid bacteria bacteriocin according to claim 1, characterized in that The molecular weight of the lactic acid bacteria bacteriocin is about 5988Da.

3. The lactic acid bacteria bacteriocin according to claim 1, characterized in that The amino acid sequence of the lactic acid bacteria bacteriocin is shown in SEQ ID NO:

1.

4. The method for preparing the lactic acid bacteria bacteriocin according to any one of claims 1 to 3, characterized in that: The lactic acid bacteria bacteriocin is extracted from the fermentation product of the lactobacillus pentosaceus or obtained by chemical synthesis.

5. The preparation method according to claim 4, characterized in that: include: The fermentation liquid of the lactobacillus pentosaceus is centrifuged to obtain a supernatant, ammonium sulfate is added to the supernatant to make the saturation of ammonium sulfate in the supernatant 80% to 100%, the precipitate is obtained after centrifugation again, and then redissolved, dialyzed and freeze-dried in sequence to obtain a crude bacteriocin.

6. The preparation method according to claim 5, characterized in that: The preparation of the lactobacillus pentosus fermentation liquid comprises: inoculating the lactobacillus pentosus into a fermentation medium for culturing; The fermentation medium is MRS medium, and its ingredients include: 10 g / L peptone, 8 g / L beef extract powder, 4 g / L yeast extract, 20 g / L glucose, 1 mL / L anhydrous sorbitan monooleate, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate trihydrate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, and 0.05 g / L manganese sulfate tetrahydrate.

7. The preparation method according to claim 4, characterized in that: The preparation method further comprises purifying the crude bacteriocin, wherein the purification comprises: dissolving the crude bacteriocin and then separating it with a Sephadex G-50 gel chromatography column, and then dialyzing and freeze-drying again to obtain the lactic acid bacteria bacteriocin.

8. Use of the lactic acid bacteria bacteriocin according to any one of claims 1 to 3 in the preparation of antibacterial products.

9. The use according to claim 8, characterized in that: The antibacterial product includes an antibacterial agent that inhibits the growth of Listeria monocytogenes.

10. An antibacterial product, characterized in that: The invention comprises the lactic acid bacteria bacteriocin according to any one of claims 1 to 3.

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