A lactic acid bacteria bacteriocin, its preparation method and application

Abstract

This invention discloses a lactic acid bacteria bacteriocin, its preparation method, and its applications, belonging to the field of microbial technology. The invention extracts a bacteriocin from *Lactobacillus pentosus* with accession number CCTCC NO: M 2022431. This bacteriocin exhibits thermal stability, can withstand low temperatures, and maintains its activity over a wide pH range. Simultaneously, this bacteriocin can inhibit the growth and proliferation of the foodborne pathogen *Listeria monocytogenes*. Because it originates from edible Sichuan pickled vegetables, this bacteriocin is harmless to humans and can provide a new research direction for the prevention or treatment of diseases caused by foodborne pathogens, thus showing promising application prospects.

Description

TECHNICAL FIELD

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

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

[0003] Lactic acid bacteria can secrete metabolites with bacteriostatic activity, such as organic acids, bacteriocins and H2O2, 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] Therefore, the present application is proposed. SUMMARY

[0005] One of the purposes of the present application includes providing a lactic acid bacteria bacteriocin.

[0006] The second purpose of the present application includes providing a preparation method of the above-mentioned lactic acid bacteria bacteriocin.

[0007] The third purpose of the present application includes providing an application of the above-mentioned lactic acid bacteria bacteriocin in the preparation of a bacteriostatic product.

[0008] The fourth purpose of the present application includes providing a bacteriostatic product.

[0009] The present application solves its technical problems by using the following technical solutions:

[0010] The present application provides a lactic acid bacteria bacteriocin, which is extracted from pentose lacto plant bacillus, and the preservation number of the pentose lacto plant bacillus is CCTCC NO: M 2022431.

[0011] Optionally, the molecular weight of the above-mentioned lactic acid bacteria bacteriocin is approximately 5988 Da.

[0012] Optionally, the amino acid sequence of the above-mentioned lactic acid bacteria bacteriocins is shown in SEQ ID NO:1.

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

[0014] Optionally, the above preparation method includes: centrifuging the fermentation broth of Lactobacillus pentosus to obtain the supernatant, adding ammonium sulfate to the supernatant to make the ammonium sulfate saturation in the supernatant 80% to 100%, centrifuging again to obtain the precipitate, and then sequentially resolubilizing, dialyzing and freeze-drying to obtain crude bacteriocin.

[0015] Optionally, the preparation of the Lactobacillus pentosacchari fermentation broth includes: inoculating Lactobacillus pentosacchari into a fermentation medium for culture.

[0016] Optionally, the fermentation medium comprises: 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 includes purifying the crude bacteriocin, which includes: dissolving the crude bacteriocin and then separating it using a Sephadex G-50 gel chromatography column, followed by dialysis and freeze-drying to obtain lactic acid bacteria bacteriocin.

[0018] Furthermore, this invention also proposes an application of the above-mentioned lactic acid bacteria bacteriocin in the preparation of antibacterial products.

[0019] Optionally, the above-mentioned antibacterial products include antibacterial agents that inhibit the growth of Listeria monocytogenes.

[0020] The present invention also proposes an antibacterial product comprising the above-mentioned lactic acid bacteria bacteriocins.

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

[0022] This invention extracts a bacteriocin from *Lactobacillus pentosaceus* with accession number CCTCC NO: M 2022431. This bacteriocin exhibits thermal stability, withstands low temperatures, and maintains its activity over a wide pH range. Furthermore, this bacteriocin inhibits the growth and proliferation of the foodborne pathogen *Listeria monocytogenes*. Because it originates from edible Sichuan pickled vegetables, this bacteriocin is harmless to humans and offers a new research direction for the prevention or treatment of diseases caused by foodborne pathogens, thus demonstrating promising application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The gel filtration chromatography column elution chromatogram (a) and high performance liquid chromatography chromatogram (b) of the Pentosacchari lactobacillus bacteriocin L14 in Example 1 are shown.

[0025] Figure 2 The inhibitory effect of Pentosacchari lactobacillus bacteriocin L14 on Listeria monocytogenes in Example 2;

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

[0027] Figure 4 Temperature stability (a), pH stability (b), UV stability (c), and enzyme stability (d) of *Lactobacillus pentosus* L14 in Example 4. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0029] The *Lactiplantibacillus pentosus* L14 used in the following examples is a strain isolated by the inventors from Sichuan pickled vegetables. It was identified as *Lactiplantibacillus pentosus* through physiological, biochemical, and 16S rDNA sequence analysis. This strain has been disclosed in patent CN202311069646.0 – A strain of *Lactiplantibacillus pentosus* that inhibits *Yersinia lipolyticis* and its application; its accession information is as follows:

[0030] It is deposited at the China Center for Type Culture Collection; address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province; deposit date: April 27, 2022; accession number: CCTCC NO: M 2022431; taxonomic name: Lactiplantibacillus pentosus L14.

[0031] This invention yielded a bacteriocin from the fermentation broth of *Lactiplantibacillus pentosus* L14, which was named bacteriocin L14. The amino acid sequence of this bacteriocin is shown in SEQ ID NO:1; it is a class IIa bacteriocin with a molecular weight of approximately 12810 Da.

[0032] The above-mentioned bacteriocin was obtained by the following preparation method according to the present invention, and the steps are as follows:

[0033] S1. Inoculate *Lactobacillus pentosus* into a fermentation medium and culture it to obtain the fermentation broth of *Lactobacillus pentosus*.

[0034] Among them, Lactiplantibacillus pentosus L14 is the above-mentioned Lactiplantibacillus pentosus L14, and its 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.

[0035] The fermentation conditions for the above-mentioned Lactobacillus pentosus were: static incubation at 37℃ for 24 hours.

[0036] S2. Centrifuge the fermentation broth of Lactobacillus pentosaccharide and collect the supernatant. Add an ammonium sulfate solution with a saturation of 80% to 100% to the supernatant, centrifuge again, collect the precipitate, and then perform redissolution, dialysis, and freeze-drying in sequence to obtain crude bacteriocin.

[0037] S3. Dissolve the crude bacteriocin and then separate it using a Sephadex G-50 gel chromatography column. After dialysis and freeze-drying, lactic acid bacteria bacteriocin can be obtained.

[0038] Experiments have shown that the bacteriocin L14 obtained by the above preparation method is thermally stable and can withstand low temperatures, maintaining its activity over a wide pH range; it can inhibit the growth and reproduction of Listeria monocytogenes, therefore this 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 will be further described in detail below with reference to embodiments.

[0040] The Listeria monocytogenes used in this embodiment of the invention is Listeria monocytogenes ATCC19115, which was purchased in the laboratory.

[0041] Example 1

[0042] This embodiment describes the preparation method of Lactobacillus pentosolicus L14 bacteriocin, as follows:

[0043] (1) Activate and expand the culture of Lactiplantibacillus pentosus L14;

[0044] (2) The culture medium prepared in step (1) was inoculated into the fermentation medium at an inoculation rate of 3%, and the culture was incubated at 37°C for 24 hours to obtain a fermentation broth containing bacteriocin from Lactobacillus pentosus.

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

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

[0047] (4) Add (NH4)2SO4 to the supernatant obtained in step (3) to make it saturated to 85%, stir at room temperature for 10 min, let stand at 4℃ for 12 h, centrifuge, and collect the precipitate.

[0048] (5) Dissolve the precipitate obtained in step (4) with pH 6.0 PBS buffer, dialyze with ultrapure water, and freeze dry under vacuum to obtain crude bacteriocin;

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

[0050] (7) Separate the crude bacteriocin solution obtained in step (6) using a Sephadex G-50 gel chromatography column and collect the bacteriocin components.

[0051] (8) The bacteriocin solution obtained in step (7) is dialyzed and then freeze-dried under vacuum 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℃, the flow rate was 1 mL / min, and the gradient used in the experiment was that the mobile phase A increased from 24% to 60% within 20 min. The retention time of Lactobacillus pentosus L14 bacteriocin was 3.2 min.

[0053] Figure 1 The elution chromatogram (a) and high performance liquid chromatogram (b) of Sephadex G-50 gel filtration column for the bacteriocin L14 of Lactobacillus pentosus provided in this invention.

[0054] Example 2

[0055] This embodiment verifies the effect of Lactobacillus pentosolicus L14 bacteriocinolone acetonide on inhibiting the growth of Listeria monocytogenes.

[0056] (1) Take the L14 obtained in Example 1 and dissolve it in pH 6.0 PBS buffer to a final concentration of 1 mg / mL, and filter it 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 medium and cultured statically at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into TSA-YE liquid medium at an inoculation rate of 3% (v / v) and cultured statically at 37°C for 24 h to prepare a culture medium.

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

[0060] 1) The effects of L14 bacteriocinolone L14 on the growth of Listeria monocytogenes were investigated by co-culturing L14 with Listeria monocytogenes.

[0061] 2) Detection method: Inoculate Listeria monocytogenes into a sufficient amount of liquid TSA-YE liquid medium at an inoculation rate of 3%, shake well, and aspirate 2 mL of bacterial solution into a 5 mL sterile centrifuge tube.

[0062] 6) Add 2 mL of 1 mg / mL *Lactobacillus pentosaceus* L14 bacteriocin L14 solution to centrifuge tubes, seal the tubes, and incubate at 37°C for 16 h. Measure the optical density at 600 nm. Compare with the optical density of *Listeria monocytogenes* at 0 h. Use *Listeria monocytogenes* culture with 2 mL of sterile pH 6.0 PBS buffer added to 2 mL of *Listeria monocytogenes* culture as a control group. Results are as follows. Figure 2 As shown.

[0063] Figure 2 The results showed that the L14 bacteriocinolite from Lactobacillus pentosus provided in this invention has an inhibitory effect on the growth of Listeria monocytogenes.

[0064] Example 3

[0065] This example demonstrates the identification of bacteriocins from *Lactobacillus pentosaceus*.

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

[0067] 1) Dissolve L14 obtained in Example 1 in UP water and prepare separating gel, sandwich gel and stacking gel using Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320).

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

[0069] (2) Enzymatic digestion and mass spectrometry identification procedures

[0070] 1) Cut the protein bands stained with Coomassie Brilliant Blue in (1) and store them at 4°C. The Coomassie stained bands were rinsed 3 times with ddH2O, destained with 150 μL of destaining solution, washed 4 times with water, and then washed twice with 300 μL of 25 mM ammonium bicarbonate, 50% acetonitrile and 100% acetonitrile in sequence. Finally, the gel was dehydrated until the gel block turned white.

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

[0072] 3) Add 15-20 μL of 0.01 μg / μL proteomics grade trypsin, allow it to fully swell and become transparent on ice, then add 30-40 μL of 50 mM NH4HCO3 (containing 10% ACN) solution to cover it; digest overnight in a water bath at 37°C.

[0073] 4) After enzymatic hydrolysis, transfer the supernatant to another new EP tube, add 100 μL of extraction buffer (67% acetonitrile, containing 2% formic acid) to the remaining gel block, incubate at 37℃ for 30 min, then sonicate for 15 min, centrifuge, combine the supernatants, centrifuge to concentrate and dry, and prepare for mass spectrometry analysis.

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

[0075] After centrifugation and drying, the enzyme-digested peptide samples were redissolved in Nano-LC mobile phase A (0.1% formic acid / water), bottled, and loaded for online LCMS analysis.

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

[0077] Mass spectrometry was performed using a ThermoFisher Q Exactive system (ThermoFisher, USA) combined with a nano-spray NanoFlex ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the ion transmission tube heating temperature was 275 °C. The mass spectrometry scan was performed in information-dependent acquisition mode (DDA). The primary mass spectrometry resolution was 70,000 m / s, the scan range was 350–2000 m / s, and the maximum injection time was 100 ms. A maximum of 20 secondary mass spectra with charges 2+ to 5+ were acquired per DDA cycle, with a maximum secondary mass spectrometry ion injection time of 50 ms. The collision chamber energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, suitable for all precursor ions, and the dynamic exclusion was set to 25 seconds.

[0078] The raw spectral files acquired by mass spectrometry were processed and analyzed using PEAKS Studio 8.5 (BioinformaticsSolutions Inc., Waterloo, Canada) software. The database used was the Lactiplantibacillus pentosus protein database downloaded from Uniprot. The search parameters were set as follows: chymotrypsin digestion, mass tolerance of 10 ppm for primary mass spectrometry, and 0.05 Da for secondary mass spectrometry.

[0079] Comparison of LC-MS / MS with the UniProt protein database revealed that bacteriocin L14 is similar to the protein numbered G0M498·G0M498_LACPE in the database, and both belong to the genus *Lactobacillus pentosus*.

[0080] The amino acid sequence is as follows:

[0081] The mass spectrum results are as follows: MNNALSFEQQFTDFSTLSDSELESVEGGRNKLAYNMGHYAGKATIF GLAAWALLA (SEQ ID NO:1). Figure 3 As shown.

[0082] Example 4

[0083] This example describes the properties of bacteriocin L14 from Lactobacillus pentosaceus.

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

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

[0086] 2) Take equal amounts of L14 solution from 1) into centrifuge tubes and place them in water baths at -20, 40, 60, 80, and 100°C and in autoclaves at 121°C for 10 min and 30 min respectively. Then immediately cool them to room temperature in an ice-water bath. 4. Store the bacteriocin as a control.

[0087] 3) Preparation of Listeria monocytogenes culture medium

[0088] Listeria monocytogenes was inoculated into TSA-YE liquid medium and cultured statically at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into TSA-YE liquid medium at an inoculation rate of 3% (v / v) and cultured statically at 37°C for 24 h to prepare a culture medium.

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

[0090] Listeria monocytogenes was inoculated at a 3% inoculum into a sufficient amount of liquid TSA-YE medium, shaken well, and 2 mL of the bacterial culture was transferred to a 5 mL sterile centrifuge tube. 2 mL of the pentosaccharin L14 bacteriocin solution treated in step 2) was added to each tube. The centrifuge tubes were sealed and incubated at 37°C for 16 h. The optical density was measured at 600 nm.

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

[0092] 1) Dissolve the L14 obtained in Example 1 in pH 6.0 PBS buffer to a final concentration of 1 mg / mL, and filter it 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 from 1) into centrifuge tubes, and adjust the pH to 2, 3, 4, 5, 7, 8, 9, 10, 11 and 12 with 5M HCl and 5M NaOH respectively. After incubating in a water bath at 37℃ for 1 hour, adjust the pH of each solution to 6. The bacteriocin solution without pH adjustment is used as a control.

[0094] 3) Preparation of Listeria monocytogenes culture medium

[0095] Listeria monocytogenes was inoculated into TSA-YE liquid medium and incubated at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into TSA-YE liquid medium at an inoculation rate of 3% (v / v) and incubated at 37°C for 24 h to prepare a culture medium.

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

[0097] Listeria monocytogenes was inoculated at a 3% inoculum into a sufficient amount of liquid TSA-YE medium, shaken well, and 2 mL of the bacterial culture was transferred to a 5 mL sterile centrifuge tube. 2 mL of the pentosaccharin L14 bacteriocin solution treated in step 2) was added to each tube. The centrifuge tubes were sealed and incubated at 37°C for 16 h. The optical density was measured at 600 nm.

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

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

[0100] 2) Take equal amounts of L14 solution from 1) into centrifuge tubes and expose them to ultraviolet light for 10, 20, 30, 40, 50 and 60 min respectively. The bacteriocin solution that is not exposed to ultraviolet light is used as a control.

[0101] 3) Preparation of Listeria monocytogenes culture medium

[0102] Listeria monocytogenes was inoculated into TSA-YE liquid medium and cultured statically at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into TSA-YE liquid medium at an inoculation rate of 3% (v / v) and cultured statically at 37°C for 24 h to prepare a culture medium.

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

[0104] Listeria monocytogenes was inoculated at a 3% inoculum into a sufficient amount of liquid TSA-YE medium, shaken well, and 2 mL of the bacterial culture was transferred to a 5 mL sterile centrifuge tube. 2 mL of the pentosaccharin L14 bacteriocin solution treated in step 2) was added to each tube. The centrifuge tubes were sealed and incubated at 37°C for 16 h. The optical density was measured at 600 nm.

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

[0106] 1) Dissolve the L14 obtained in Example 1 in pH 6.0 PBS buffer to a final concentration of 1 mg / mL, and filter it 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 from 1) into centrifuge tubes, adjust the pH to 1.9, 8.0 and 10.3 with 5M HCl and 5M NaOH respectively, add pepsin, trypsin and proteinase K respectively to make their final concentration 20mg / mL, incubate at 37℃ for 1h, and then adjust the pH of each solution to 6. The bacteriocin solution without added proteinase is used as a control.

[0108] 3) Preparation of Listeria monocytogenes culture medium

[0109] Listeria monocytogenes was inoculated into TSA-YE liquid medium and cultured statically at 37°C for 24 h to prepare a seed culture. The prepared seed culture was then inoculated into TSA-YE liquid medium at an inoculation rate of 3% (v / v) and cultured statically at 37°C for 24 h to prepare a culture medium.

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

[0111] Listeria monocytogenes was inoculated at a 3% inoculum into a sufficient amount of liquid TSA-YE medium, shaken well, and 2 mL of the bacterial culture was transferred to a 5 mL sterile centrifuge tube. 2 mL of the pentosaccharin L14 bacteriocin solution treated in step 2) was added to each tube. The centrifuge tubes were sealed and incubated at 37°C for 16 h. The optical density was measured at 600 nm.

[0112] Figure 4 The results showed that L14 retained about 40% of its activity after treatment at -20℃ and 60℃ for 30 min, indicating the bacteriocin's low-temperature and thermal stability. L14 maintained its activity over a wide pH range, especially under acidic conditions. L14 maintained high activity for 40 min under UV irradiation. Trypsin and proteinase K had a significant impact on L14 activity.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a bacteriocin of lactic acid bacteria in the preparation of an antibacterial product, characterized in that, The lactic acid bacteria bacteriocin is extracted from the pentose lacto plant bacillus, and the preservation number of the pentose lacto plant bacillus is CCTCC NO: M 2022431. The amino acid sequence of the lactic acid bacteria bacteriocin is shown as SEQ ID NO:

1. The bacteriostatic product is a bacteriostatic agent for inhibiting the growth of Listeria monocytogenes.

2. Use according to claim 1, characterized in that, The lactic acid bacteria bacteriocin is extracted from the fermentation product of the pentose lacto plant bacillus or obtained by chemical synthesis.

3. Use according to claim 2, characterized in that, The preparation method of the lactic acid bacteria bacteriocin comprises the following steps: centrifuging the fermentation liquid of the pentose lacto plant bacillus to obtain supernatant, adding ammonium sulfate to the supernatant, so that the saturation of ammonium sulfate in the supernatant is 80%-100%, centrifuging again to obtain a precipitate, and then sequentially performing resolubilization, dialysis and freeze-drying to obtain a bacteriocin crude product.

4. Use according to claim 3, characterized in that, The preparation of the fermentation liquid of the pentose lacto plant bacillus comprises the following steps: inoculating the pentose lacto plant bacillus into a fermentation medium for culture. The fermentation medium is an MRS medium, and the components comprise: 10 g / L of proteose peptone, 8 g / L of beef infusion powder, 4 g / L of yeast extract, 20 g / L of glucose, 1 mL / L of anhydrous sorbitol monooleate, 2 g / L of dipotassium hydrogen phosphate, 5 g / L of sodium acetate trihydrate, 2 g / L of triammonium citrate, 0.2 g / L of magnesium sulfate heptahydrate and 0.05 g / L of manganese sulfate tetrahydrate.

5. Use according to claim 4, characterized in that, The preparation method further comprises purifying the bacteriocin crude product, and the purification comprises the following steps: dissolving the bacteriocin crude product and then separating the bacteriocin crude product by using a Sephadex G-50 gel chromatography column, and then performing dialysis and freeze-drying to obtain the lactic acid bacteria bacteriocin.

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