Leuconostoc lactis XP1 for producing pullulanase and application of Leuconostoc lactis XP1

Abstract

The invention provides a new strain XP1 of Leuconostoc lactis, and the preservation number of the new strain XP1 of the Leuconostoc lactis is CGMCC (China General Microbiological Culture Collection Center) No.31789. The invention also provides a preparation method of the new strain XP1 of the Leuconostoc lactis. The novel leuconostoc lactis strain XP1 provided by the invention has relatively good pullulanase production activity and acid production characteristic; in a hemolysis experiment, no hemolysis phenomenon is generated, hemolysis is avoided, and safety and non-toxicity are achieved; the compound has drug resistance to rifampicin and has an inhibition effect on pathogenic bacteria such as staphylococcus aureus and escherichia coli; the strain XP1 has good oxidation resistance, the DPPH free radical scavenging rate is 85.20%, the superoxide anion free radical scavenging rate is 63.50%, the ABTS < + > free radical scavenging rate is 55.12%, and the strain XP1 has good application prospects in food development, health care product preparation and medicine preparation.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and specifically to a strain of Leuconostoc lactis ( Leuconostoc lactis ) The technical field of new strain XP1 and its application. Background Art

[0002] Starch debranching enzymes (SDBEs) are starch hydrolases that can hydrolyze α-1,6-glycosidic bonds. Starch debranching enzymes can be divided into four categories, namely amylo-α-1, 6-glucosidase, saccharification enzyme, pullulanase or restriction dextrinase, and isoamylase. Pullulanase (EC3.2.1.41), as a member of the α-amylase family GH13, is a debranching enzyme that can specifically decompose α-1,6 glycosidic bonds in pullulan, β-dextrin and amylopectin, decomposing pullulan into maltotriose, and decomposing β-dextrin and amylopectin into short α-1,4 glucans and maltooligosaccharides. It is widely used in the production of high glucose syrup, ultra-high maltose syrup, beer and modified starch foods.

[0003] Pullulanase-producing strains are more common in mesophilic bacteria, thermophilic bacteria, and thermophilic bacteria. Bacillus is the main bacteria that produces pullulanase, such as Bacillus acidophilus ( Bacillus acidophilus )、Bacillus stearothermophilus( Bacillus stearothermophilus ) and a few fungi. However, lactic acid bacteria that produce pullulanase are relatively rare.

[0004] Currently, there are few pullulanase-producing strains, and it is urgent to discover new pullulanase-producing strains, which is of great significance for promoting the widespread application of pullulanase. Summary of the invention

[0005] The present invention aims to provide a strain of Leuconostoc lactis ( Leuconostoc lactis ) New strain XP1 and its application. The strain in this application was screened from a sample of Magzi wood produced by a family workshop in Kaladala Town, Tekes County, Yili, and its taxonomic status was determined by bacterial growth status and characteristics and 16S rDNA sequence analysis, confirming that it has typical characteristics of new strains. The strain growth characteristics, hemolysis, acid production, pullulanase production, antioxidant activity, drug resistance, and pathogen inhibition were tested. Lactobacillus lactis ( Leuconostoc lactis ) XP1 has good acid and pullulanase production characteristics, is non-hemolytic, safe and non-toxic, is resistant to rifampicin, and has inhibitory effects on pathogenic bacteria Staphylococcus aureus and Escherichia coli; and has good antioxidant properties, and is effective against DPPH free radicals, superoxide anion free radicals, and ABTS + Free radical scavenger has good scavenging ability.

[0006] Specifically, the present invention provides the following technical solutions: The present invention provides a strain of Leuconostoc lactis ( Leuconostoc lactis ) The new strain XP1 was deposited on August 30, 2024 at the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was named as Lactobacillus lactis ( Leuconostoc lactis ) XP1, the deposit number is CGMCC No.31789.

[0007] The present invention is based on the lactic acid Leuconostoc lactis ( Leuconostoc lactis ) XP1, hereinafter referred to as “Lactobacillus lactis XP1” or “strain XP1”.

[0008] The lactis XP1 provided by the present invention is a Gram-positive bacterium with an oval or round shape and a diameter of about 1 μm; its colonies are off-white, round, smooth and moist, with a raised center, neat edges and low transparency. The 16S rDNA gene sequence of lactis XP1 is shown in SEQID NO.1. Lactis XP1 can produce pullulanase and has good acid production characteristics. In the hemolysis experiment, no hemolysis occurs and it is non-hemolytic; it is resistant to rifampicin and has an inhibitory effect on pathogenic bacteria Staphylococcus aureus and Escherichia coli; and the strain XP1 has good antioxidant properties, with DPPH free radicals, superoxide anion free radicals, and ABTS free radicals. + Free scavenging ability. Leuconostoc lactis XP1 can grow in MRS agar medium, GYP agar medium, and MRS broth medium.

[0009] The present invention provides a lactobacillus numbered XP1 ( Leuconostoc lactis ) Through the molecular level identification of the strain system well known in the art, combined with multi-phase classification system identification analysis such as morphological identification, the present invention relates to the lactoferrin numbered XP1 ( Leuconostoc lactis ) There are many differences between XP1 and the closest standard type strain of the genus, confirming that the strain numbered XP1 belongs to Leuconostoc lactis A new species of the genus, with characteristics typical of new species.

[0010] The invention provides pullulanase prepared by fermenting and culturing the Leuconostoc lactis XP1.

[0011] As an embodiment of the present invention, the method for preparing pullulanase by fermentation culture comprises the following steps: inoculating strain XP1 into 5 mL of MRS broth medium, and culturing at 37°C in a shaker at 180 r / min for 36 h.

[0012] The MRS broth medium is prepared by heating and dissolving: 10.0 g of peptone, 8.0 g of beef extract powder, 4.0 g of yeast extract powder, 20 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, and 1.0 g of Tween 80 in 1000 mL of distilled water to obtain a pH value of 5.7±0.2.

[0013] The present invention also provides a bacteriostatic agent, which comprises a combination of one or more of the Leuconostoc lactis XP1 and a fermentation culture thereof.

[0014] The active ingredient of the antibacterial agent mentioned above may be composed of a combination of one or more of Leuconostoc lactis XP1 and its fermentation culture, and the antibacterial agent may further include pharmaceutically acceptable excipients.

[0015] Based on the above functions, the present invention provides the use of Leuconostoc lactis XP1, its fermentation culture or the antibacterial agent in inhibiting the activity of Staphylococcus aureus and Escherichia coli.

[0016] The present invention also provides the use of Leuconostoc lactis XP1 in preparing a probiotic preparation with antibiotic resistance. The probiotic preparation comprises Leuconostoc lactis XP1; and the antibiotic comprises rifampicin.

[0017] The invention also provides application of Leuconostoc lactis XP1 in the development of health food.

[0018] The invention provides application of Leuconostoc lactis XP1 and fermentation products thereof in the development of health food.

[0019] The Leuconostoc lactis XP1 provided by the present invention has at least the following beneficial effects: (1) The present invention isolated a strain of Leuconostoc lactis XP1 from the Xinjiang cereal beverage Magazi wood. Its colony is off-white, round, smooth and moist, with a raised center, neat edges and low transparency. It is a Gram-positive bacterium. The 16S rDNA of strain XP1 is similar to Leuconostoc lactis The homology of the strains was 100%. Based on the morphological characteristics and identification results, strain XP1 was confirmed to be Leuconostoc lactis. Screening with the identification medium confirmed that it had pullulanase activity.

[0020] (2) In MRS broth medium, the pullulanase activity of strain XP1 was determined to be 0.0027±0.0009 U / mL. Subsequently, the growth characteristics, safety, and functional characteristics of XP1 were evaluated. The results showed that the acid production characteristics of XP1 tended to stabilize after 36 h, and the final pH value was 3.83; the strain grew best at a pH of 6.5; the enzyme activity of the strain reached a maximum of 0.0027 (U / mL) when the enzyme reaction temperature was 55°C; (3) The strain XP1 provided in this application did not produce hemolysis in the hemolysis experiment, is non-hemolytic, safe and non-toxic, and can be widely used in food development.

[0021] (3) The strain XP1 provided in the present application showed resistance to rifampicin in the antibiotic resistance experiment.

[0022] (4) The strain XP1 provided in this application has a good inhibitory effect on pathogens such as Staphylococcus aureus and Escherichia coli.

[0023] (5) The strain XP1 provided in this application has good antioxidant activity, with a DPPH free radical scavenging rate of 85.20%, a superoxide anion free radical scavenging rate of 63.50%, and ABTS + The free radical scavenging rate is 55.12%, which has good application prospects in food development, health care product preparation, and drug preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is the transparent zone of the strain in the identification medium; Figure A shows a panoramic view of strain XP1; Figure B shows a local view of strain XP1.

[0025] Figure 2 Shown is the single colony morphology and Gram staining of strain XP1.

[0026] Figure 3 Shown is the phylogenetic tree of strain XP1 based on 16S rDNA sequence analysis.

[0027] Figure 4 Shown is a graph of the glucose standard curve.

[0028] Figure 5 Shown is a graph showing the acid production capacity of strain XP148h.

[0029] Figure 6 Shown is the survival rate of strain XP1 under different pH conditions.

[0030] Figure 7 Shown is the optimal enzyme reaction temperature diagram for strain XP1.

[0031] Figure 8 Shown is a graph showing the antioxidant activity of strain XP1. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0034] The percentages in the following examples are by weight unless otherwise specified.

[0035] The following examples are based on the lactobacillus strain CGMCC No.31789 ( Leuconostoc lactis ) XP1, hereinafter referred to as “Lactobacillus lactis XP1” or “strain XP1”.

[0036] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0037] Example 1: Screening and identification of Leuconostoc lactis XP1 (1) Sample collection The samples of Magzi wood came from a family workshop producing Magzi wood products in Kaladala Town, Tekes County, Yili.

[0038] (2) Strain isolation and purification The plate dilution coating method was used, and MRS, GYP and improved tomato juice agar medium were used as lactic acid bacteria isolation medium. -3 , 10 -4 , 10 -5 100 µL of each of the Magzimu beverages were inoculated on the separation medium, with 3 parallels for each gradient, and inverted cultured at 37 °C for 48 h. Single colonies with obvious differences in colony morphology were picked, streaked twice on the corresponding culture medium, and stored in porcelain bead strain storage tubes at -20 °C. The isolated strains were subjected to Gram staining and microscopic examination to observe the microscopic morphology.

[0039] MRS agar medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, agar 14.0 g, Tween 80 1.0 g, heated and dissolved in 1000 mL distilled water, pH 6.5±0.2.

[0040] GYP agar medium: 10.0 g glucose, 10.0 g yeast extract powder, 5.0 g peptone, 2.0 g beef extract powder, 0.5 g Tween 80, 2.0 g sodium acetate, 0.01 g sodium azide, 0.2 g magnesium sulfate, 0.01 g manganese sulfate, 0.01 g ferrous sulfate, 0.01 g sodium chloride, 5.0 g calcium carbonate, 12.0 g agar, heated and dissolved in 1000 mL distilled water, pH 6.8±0.1.

[0041] Improved tomato juice agar medium: 2.5 g tomato powder, 10.0 g beef extract powder, 5.0 g yeast extract powder, 2.0 g dipotassium hydrogen phosphate, 2.0 g glucose, 5.0 g sodium acetate, 1.0 g Tween 80, 20.0 g lactose, 15.0 g agar, heated and dissolved in 1000 mL distilled water, pH 6.8±0.2.

[0042] (3) Initial screening of enzyme-producing bacteria The isolated strains were cultured in a shaking incubator at 37°C and 170 r / min for 24 h. After vortex mixing, they were streaked on a plate in the identification medium and cultured in a 37°C incubator for 24-48 h. After that, an appropriate amount of iodine test solution was added. After standing for 20 min, the strains containing transparent circles were selected.

[0043] Identification medium: 20.0 g pullulan, 10.0 g peptone, 1.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 15 g agar, heated and dissolved in 1000 mL distilled water, pH 6.0.

[0044] (4) Strain identification The genomic DNA of the isolated strain was extracted using a bacterial DNA extraction kit, and the bacteria were PCR amplified using universal primers 27F / 1492R. The PCR reaction system (50 μL) included 25 μL of 2×Power Taq PCR GreenMix, 2 μL of DNA template, 1 μL of each of the upper and lower primers, and 21 μL of nuclease-free water. PCR amplification was performed according to the instructions, and the PCR products were detected on a 20 g / L agarose gel and then bidirectionally sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were bidirectionally spliced ​​and primers were removed using Dnaman8.0 software, and then submitted to the GenBank database of the National Center of Biotechnology Information (NCBI) website for basic local alignment search tool (BLAST) homology analysis to clarify the taxonomic status of the isolated strain. The phylogenetic tree was then constructed using MEGA7.0 software for the obtained gene sequences.

[0045] The initial screening results of enzyme-producing strains were used to identify the enzyme-producing strains using identification medium. Figure 1 As shown in the figure, a transparent circle can be seen around the single colony of strain XP1, which preliminarily indicates that strain XP1 has the activity of producing pullulanase. Figure 2 As shown in the figure, the single colony morphology and Gram staining of strain XP1. It can be seen from the figure that the single colony morphology of strain XP1 is grayish white, round, smooth and moist, with a convex center, neat edges and low transparency. After Gram staining, XP1 is a Gram-positive bacterium with an oval or round shape and a diameter of about 1μm.

[0046] (6) Strain identification After BLAST comparison and identification, a phylogenetic tree was constructed based on the 16S rDNA sequencing results of the tested strains. The results of gene sequence homology analysis of the screened strains are shown in Table 1. The sequence similarity between the isolated strains and the standard strains reached 100%. The 16S rDNA results showed that the Figure 3 As shown, strain XP1 isolated from Maggi wood is Leuconostoc lactis ( L. lactis ).

[0047] Table 1: 16S rDNA sequence homology analysis of strains

[0048] Based on the above morphological characteristics and the results of 16S rDNA gene sequence homology comparison analysis, it can be seen that strain XP1 belongs to Lactobacillus lactis ( Leuconostoc lactis), and with the existing Leuconostoc lactis ( Leuconostoc lactis ) strains are different and are a new strain of Leuconostoc lactis ( Leuconostoc lactis ) strain. It can be seen that the lactobacillus strain numbered XP1 provided by the present invention ( Leuconostoc lactis ) Through the molecular level identification of the strain system well known in the art, combined with the multi-phase classification system identification analysis such as morphological identification, the present invention relates to the lactoferrin numbered XP1 ( Leuconostoc lactis ) has many differences from the closest standard type strain of the genus, confirming that the strain numbered XP1 belongs to Lactobacillus lactis ( Leuconostoc lactis ) genus, with typical characteristics of new species. Leuconostoc lactis )XP1 was deposited on August 30, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, China), and was named as Lactobacillus lactis ( Leuconostoc lactis ) The XP1 deposit number is CGMCC No.31789.

[0049] Example 2: Pullulanase production characteristics of Leuconostoc lactis XP1 1. Pullulanase activity assay (1) Drawing of glucose standard curve

[0050] Take 3 mL of the test solution with different glucose content and measure its absorbance value at 540 nm in a UV spectrophotometer. Use the test solution with a glucose content of 0 mg / mL to adjust to zero. Draw a glucose standard curve with the glucose content as the horizontal axis and the absorbance value as the vertical axis.

[0051] (2) Pullulanase activity assay The strains producing pullulanase obtained in the initial screening were inoculated into 5 mL of MRS broth medium and cultured in a 37°C shaker at 180 r / min for 36 h. After the culture was completed, centrifuged at 6000 r / min for 8 min at 4°C, 100 μL of the supernatant was taken into a 10 mL stoppered colorimetric tube, 100 μL of 0.5% pullulan solution was added, mixed evenly, placed in a 55°C water bath for 60 min, the colorimetric tube was taken out, 600 μL of DNS test solution was quickly added and mixed evenly, and immediately taken out after 5 min of boiling water bath, cooled to room temperature in cold water, made up to 10 mL with distilled water, mixed evenly, and its absorbance was measured at 540 nm. The amount of reducing sugar generated was calculated according to the glucose standard curve. The blank control was the reducing sugar content of the supernatant inactivated at 100°C for 10 min.

[0052] MRS broth medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 801.0 g, heat and dissolve in 1000 mL distilled water, pH 5.7±0.2.

[0053] (3) Enzyme activity calculation method Enzyme activity definition: Under the enzyme activity assay conditions, the amount of enzyme required to hydrolyze 0.5% pullulan to produce 1 μmol glucose per minute is 1 enzyme activity unit (U / mL). The calculation formula is as follows:

[0054] Wherein, U is the pullulanase activity unit (U / mL); ODA is the absorbance of the experimental group measured at 540 nm; ODB is the absorbance of the control group measured at 540 nm; 1000 is the conversion factor; n is the dilution factor; k is the slope of the standard curve; V is the volume of the reaction system (mL); 180 is the molecular weight of glucose (g / mol); and 60 is the reaction time (min).

[0055] 2. Pullulanase activity test results See the attached glucose standard curve. Figure 4 shown.

[0056] As shown in Table 2, in MRS broth medium, the pullulanase activity of XP1 was 0.0027±0.0009 U / mL.

[0057] Table 2: Results of strains in MRS medium

[0058] Example 3: Determination of strain characteristics 1. Determination method (1) Acid-producing characteristics of the strain After activating the strain stored at -20℃ for 2-3 generations, add it to the sterilized MRS broth medium at a ratio of 1%. Perform three parallel tests in each group, measure the pH value of the bacterial solution every 2 hours, record the data, mark the horizontal axis as time, and the vertical axis as pH value, and use Origin 2021 software to draw the acid production curve.

[0059] (2) Optimal pH value of the strain The optimal growth pH of the strain was determined with a pH gradient of 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5. After activating the strain stored at -20°C for 2-3 generations, it was added to the sterilized MRS broth medium at a ratio of 1% and the OD of the bacterial solution at 0 h and 16 h was measured.600 nm value, calculate the absorbance ratio (ΔOD 600 nm value).

[0060] (3) Optimal enzyme reaction temperature of the strain The pullulanase activity was determined at different enzyme reaction temperatures according to the pullulanase activity determination method, with a gradient of 25°C, 35°C, 45°C, and 55°C for 60 min, and the enzyme activity was calculated using the pullulanase activity calculation formula.

[0061] (4) Hemolytic activity The blood agar plate method was used to detect the hemolytic activity of the strain. 3 μL of the bacterial suspension was spotted on the blood agar plate, with Staphylococcus aureus as the control, and cultured at 37 °C for 1 day to observe whether a hemolytic zone appeared around the colony.

[0062] (5) Antibiotic resistance This experiment uses the drug sensitivity paper (diameter 6.0 mm) agar diffusion method. The results are determined according to the "CLSI Antimicrobial Susceptibility Test Standards".

[0063] (6) Antibacterial activity The Oxford cup method was used and Escherichia coli and Staphylococcus aureus were selected as the test pathogens.

[0064] (7) Antioxidant properties ①Superoxide anion clearance rate The superoxide anion radical scavenging ability was determined by the pyrogallol auto-oxidation method. A certain amount of bacterial suspension was centrifuged at 10000 r / min for 5 min, and the supernatant was taken for testing. 4.5 mL of 0.05 mol / L Tris-HCl buffer (pH= 8.2) was placed in a colorimetric tube, preheated in a 25 ℃ constant temperature water bath for 20 min, 2 mL of sample solution was added, and then 0.1 mL of 25 mmol / L pyrogallol solution was added. After mixing, the mixture was reacted in a 25 ℃ constant temperature water bath for 5 min, and then 1 mL of 8 mol / L concentrated hydrochloric acid was added to terminate the reaction, and the absorbance of the solution at 320 nm was measured. The following treatments were used: A0 (control): 2 mL pure water + 4.5 mL Tris-HCl buffer + 0.1 mL pyrogallol solution; A1 (test sample): 2 mL sample + 4.5 mL Tris-HCl buffer + 0.1 mL pyrogallol solution; A2 (blank control): 2 mL sample + 4.5 mL Tris-HCl buffer + 0.1 mL HCl solution. The calculation formula is as follows:

[0065] ②ABTS free radical scavenging rate Prepare 7.4 mmol / L ABTS stock solution and 2.45 mmol / L potassium persulfate solution, and mix them in a 1:1 (v / v) ratio. Let stand at room temperature in the dark for 12-16 hours to prepare ABTS stock solution. Dilute the ABTS stock solution with PBS to an absorbance of 0.7±0.02 at 734 nm wavelength, and use it as ABTS free radical working solution. Use the following treatments: A0 (control): 2 mL anhydrous ethanol + 2 mL sample; A1 (sample to be tested): 2 mL ABTS free radical working solution + 2 mL sample; A2 (blank control): 2 mL anhydrous ethanol + 2 mL ABTS free radical working solution; measure the absorbance of each group at 734 nm wavelength. The calculation formula is as follows:.

[0066]

[0067] ③ DPPH free radical scavenging rate The test was carried out on a 96-well plate. A certain mass of DPPH free radicals was weighed, and 0.1 mmol / L DPPH ethanol solution was prepared with ethanol as solvent. A certain amount of bacterial suspension was centrifuged at 12000 r / min for 10 minutes, and the supernatant was taken for testing. The following treatments were used: A0 (control): 2mL anhydrous ethanol + 2mL DPPH ethanol solution; A1 (sample to be tested): 2mL sample + 2mL DPPH ethanol solution; A2 (blank control): 2mL sample + 2mL anhydrous ethanol. After standing at 37°C in the dark for 30 minutes, the absorbance of each treatment was measured at 517 nm. Three replicates were set for each sample, and the average value was taken. The results are expressed as DPPH free radical scavenging rate%. The calculation formula is as follows:

[0068] 2. Measurement results (1) Acid-producing characteristics of the strain See attached Figure 5 It can be seen that the pH value of strain XP1 showed a downward trend and stabilized after 36 hours. At 48 hours, the final pH value was 3.83.

[0069] (2) Optimal pH value of the strain See attached Figure 6 As shown, the OD600nm ratio of the strain was the highest at pH 6.5, and there was no significant difference at pH 5.5 ( p <0.05). That is, the optimum pH value is 6.5.

[0070] (3) Optimal enzyme reaction temperature of the strain See attached Figure 7As shown, when the strain was cultured at 37°C and the enzyme reaction temperature was 55°C, the highest enzyme activity was 0.0027 (U / mL), that is, the optimal enzyme reaction temperature was 55°C.

[0071] (4) Hemolytic activity According to the color of the hemolysis zone, hemolysis is divided into α hemolysis and β hemolysis. The former is a grass green hemolysis zone; the latter is a transparent hemolysis zone. If there is no hemolysis zone, it is γ hemolysis, which is not pathogenic to the human body. The hemolysis of the strain is shown in the table. As shown in Table 3, the positive control Staphylococcus aureus has a transparent hemolysis zone, which is β hemolysis. The screening strain has no hemolysis, indicating that it is not hemolytic.

[0072] Table 3: Hemolytic activity of strains

[0073] (5) Antibiotic resistance The test results are shown in Table 4. Among them, XP1 was only resistant to rifampicin (5 μg / tablet), but the result was 18.5 (I), which indicated that XP1 had limited sensitivity to rifampicin.

[0074] Table 4: Results of strain sensitivity test to 10 antibiotics (mm)

[0075] Note: - indicates the drug resistance result is 0 (R).

[0076] (6) Antibacterial activity The antibacterial effect of the strain on pathogenic bacteria is shown in Table 5, indicating that the strain has an inhibitory effect on Staphylococcus aureus and Escherichia coli.

[0077] Table 5: Antibacterial diameter of strains (mm)

[0078] (7) Antioxidant properties See attached Figure 8 It can be seen that XP1 has a certain antioxidant capacity. Among them, the DPPH free radical scavenging rate is the highest at 85.20%, followed by the superoxide anion free radical scavenging rate of 63.50%, and the last is ABTS + The free radical scavenging rate was 55.12%.

[0079] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A pullulanase-producing strain of Leuconostoc lactis ( Leuconostoc lactis ) XP1, characterized in that, The lactic acid Leuconostoc lactis ( Leuconostoc lactis )XP1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31789 and the deposit date August 30, 2024. The 16S rDNA gene sequence is shown in SEQ ID No.

1.

2. A lactobacillus according to claim 1 ( Leuconostoc lactis ) A method for preparing pullulanase by fermentation culture of XP1.

3. The lactobacillus lactis according to claim 2 ( Leuconostoc lactis ) A method for preparing pullulanase by fermentation culture of XP1, characterized in that: The method for preparing pullulanase obtained by fermentation culture comprises the following steps: Leuconostoc lactis ) XP1 was inoculated into 5 mL of MRS broth and cultured at 37°C in a shaker at 180 rpm for 36 h.

4. The MRS broth medium as claimed in claim 3 is prepared by heating and dissolving: 10.0 g of peptone, 8.0 g of beef extract powder, 4.0 g of yeast extract powder, 20 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, and 1.0 g of Tween 80 in 1000 mL of distilled water to obtain a pH of 5.7±0.

2.

5. An antibacterial agent, characterized in that It contains the lactobacillus described in claim 1 ( Leuconostoc lactis ) XP1, or a combination of one or more of their fermentation cultures.

6. An antibacterial agent according to claim 5, characterized in that The antibacterial agent also includes pharmaceutically acceptable excipients.

7. Use of the antibacterial agent according to any one of claims 5 to 6 in inhibiting the activity of Staphylococcus aureus and Escherichia coli.

8. The lactobacillus lactis according to claim 1 ( Leuconostoc lactis ) Application of XP1 in the preparation of probiotic preparations with antibiotic resistance.

9. The lactobacillus lactis according to claim 1 ( Leuconostoc lactis ) Application of XP1 in the development of health foods.

10. The lactobacillus lactis according to claim 1 ( Leuconostoc lactis ) Application of XP1 and its fermentation products in the development of health foods.

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