Lactobacillus plantarum LAS3 and application thereof

Abstract

The invention relates to a lactobacillus plantarum LAS3 strain and an application of the lactobacillus plantarum LAS3 strain, and belongs to the technical field of microorganisms. The lactobacillus plantarum LAS3 disclosed by the invention is preserved in Guangdong Microbial Culture Collection Center, and the preservation number of the lactobacillus plantarum LAS3 is GDMCC NO.66440. The strain is separated, purified and screened from Guizhou sour fish, and experiments show that the strain has excellent pH tolerance and sodium chloride tolerance; fermentation liquor of the strain has a good antibacterial effect on multiple common pathogenic bacteria such as pseudomonas aeruginosa, burkholderia cepacia, pseudomonas putida, staphylococcus aureus, bacillus subtilis and candida albicans, and bacteriocin generated by the strain has excellent antibacterial and preservative effects and also has excellent thermal stability and pH stability. The antibacterial preservative can be used as an antibacterial preservative raw material, is applied to food, health care products or daily chemical products, and has wide application prospects and important conversion research values.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum LAS3 and an application thereof. Background Art

[0002] Lactic acid bacteria, as generally recognized as safe (GRAS) strains, produce active metabolites through metabolism. These metabolites have been shown to possess anti-inflammatory, antibacterial, antioxidant, and immunomodulatory properties. Antibacterial properties are among the most notable properties of lactic acid bacteria. Lactic acid bacteriocins, as active metabolites of lactic acid bacteria, are a class of peptides or precursor peptides with antibacterial activity. They have strong antagonistic properties against the growth of other microorganisms, and their antibacterial activity is not limited to homologous bacteria.

[0003] In addition, the characteristics of bacteriocin protein allow it to be degraded in the human body, so it is non-toxic and residue-free. It is also highly effective, acid-resistant, high-temperature resistant, drug-resistant, with most genes located on plasmids, small molecular weight, containing modified amino acids, and complex structure. These characteristics not only make lactic acid bacteria bacteriocin one of the representatives of natural preservatives, but also make it a good material for molecular genetics, genetic engineering, protein engineering, daily chemical products, skin care, and regulating intestinal flora.

[0004] Furthermore, Lactobacillus plantarum is a Gram-positive anaerobic or facultative anaerobic and heterofermentative lactic acid bacteria. Its secreted bacteriocins exhibit broad-spectrum antibacterial activity and safe degradation, making them a research hotspot for novel antibiotic alternatives. Therefore, to meet the growing market demand for bacteriocins in food, healthcare products, and daily chemical products, there is an urgent need to isolate and obtain bacteriocins that produce high-antibacterial activity. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a strain of Lactobacillus plantarum LAS3 that has strong stress resistance and produces bacteriocin with high antibacterial activity. The strain was isolated and purified from Guizhou sour fish and has been deposited in the Guangdong Provincial Microbial Culture Collection with a deposit number of GDMCC No. 66440.

[0006] In a first aspect, the present invention provides a method for preparing a lactobacillus plantarum ( Lactiplantibacillus plantarum )LAS3, the bacteria was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 29, 2025, with the deposit number GDMCC NO.66440, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In a second aspect, the present invention provides use of the Lactobacillus plantarum LAS3 described in the first aspect in preparing bacteriocins.

[0008] In a third aspect, the present invention provides a method for preparing bacteriocin by fermenting Lactobacillus plantarum LAS3 according to the first aspect, comprising the following steps: A1. Inoculate frozen Lactobacillus plantarum LAS3 into liquid MRS medium for activation, and then inoculate into MRS liquid medium for fermentation. After fermentation, centrifuge to remove the bacteria and retain the fermentation broth. A2. Mix the fermentation broth with ethyl acetate, extract the mixture, allow to stand for separation, collect the upper organic phase, add ethyl acetate to the lower aqueous phase again, extract the mixture, allow to stand for separation, and collect the upper organic phase; A3. The organic phases from the two extractions were mixed, placed in a rotary evaporator, evaporated and concentrated under vacuum conditions at 40° C., and the concentrate was collected with ultrapure water to obtain a mixture I; and the mixture I was freeze-dried to obtain the bacteriocin.

[0009] Preferably, the activated Lactobacillus plantarum LAS3 fermentation inoculation amount in step A1 is 2-4% of the MRS liquid culture medium.

[0010] Preferably, the centrifugation parameters in step A1 are 4500-5500 g, 3-6° C., and the centrifugation time is 15-25 min.

[0011] Preferably, the fermentation temperature in step A1 is 34-38° C., and the fermentation time is 18-32 hours.

[0012] Preferably, the volume ratio of the fermentation liquid to ethyl acetate in step A2 is 1:1, the extraction temperature is 25-30° C., and the extraction time is 10-14 h.

[0013] Preferably, the rotation speed of the extraction treatment in step A2 is 130-180 r / min.

[0014] Preferably, the specific conditions for freeze drying in step A3 are a vacuum degree of 230 Pa and a cold trap temperature of -46.8°C.

[0015] In a fourth aspect, the present invention provides a product comprising the Lactobacillus plantarum LAS3 described in the first aspect.

[0016] Preferably, the product is food, health care product or daily chemical product.

[0017] In a fifth aspect, the present invention provides use of a bacteriocin extract prepared by the Lactobacillus plantarum LAS3 described in the first aspect or the method described in the third aspect in the preparation of foods, health products or daily chemical products.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The inventors of the present application isolated, purified and screened Lactobacillus plantarum LAS3 from Guizhou sour fish.

[0019] 1) The results of the environmental tolerance test showed that the pH tolerance of Lactobacillus plantarum CMRC 1L was pH 3-9. The OD value (absorbance value) of the bacterium was the highest at pH = 5, while the OD value of the Lactobacillus plantarum LAS3 of the present invention was the highest at pH = 6, and its cell density at pH = 9 was still significantly higher than that of Lactobacillus plantarum CMRC 1L; Lactobacillus plantarum LAS3 had a tolerance to sodium chloride of more than 100 g / L, while Lactobacillus plantarum CMRC 1L basically stopped growing when the mass concentration of sodium chloride increased to 80 g / L; In summary, the Lactobacillus plantarum LAS3 isolated by the present invention has better pH tolerance and sodium chloride tolerance than the Lactobacillus plantarum CMRC 1L in the prior art; 2) The results of the antibacterial test showed that the strain of the present invention had good antibacterial effects on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis, and Candida albicans, with the diameters of the inhibition zones all greater than 13 mm. This strain had a superior antibacterial effect compared to the prior art strain Lactobacillus plantarum SXp08. 3) The results of the thermal stability test showed that the bacteriocin produced by Lactobacillus plantarum LAS3 had an inhibition zone diameter of greater than 20 mm against Escherichia coli after being treated at 36°C, 60°C, 80°C and 100°C for 40 minutes, and after being treated at 121°C for 40 minutes, the inhibition zone diameter against Escherichia coli could reach more than 18 mm, indicating that the bacteriocin produced by Lactobacillus plantarum LAS3 has excellent thermal stability; and the results of its pH stability test showed that the inhibition zone diameter of Lactobacillus plantarum LAS3 bacteriocin under strong acid and alkaline conditions was greater than 19 mm, indicating that the bacteriocin produced by Lactobacillus plantarum LAS3 isolated from Guizhou sour fish in this application has better pH stability, and has excellent antibacterial activity in the pH range of 2-9; and the thermal stability and pH stability of the bacteriocin described in this application are also significantly better than the existing technology level; 4) Antiseptic effect The results show that the bacteriocin produced by the strain of the present invention has an excellent antiseptic effect on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans. After 6 hours of action, it has a significant inhibitory effect on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans. The number of viable bacteria can drop to below 100 after one day of action. After two days, the number of viable bacteria in the bacterial solution of Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans is all 0, and can be maintained for more than 28 days. This shows that the bacteriocin extract of Lactobacillus plantarum LAS3 can be used to prepare cosmetics such as facial mask liquid. It has excellent antibacterial and antiseptic effects and can be used as an antibacterial and antiseptic raw material for food, health products or daily chemical products. It has broad application prospects and important translational research value.

[0020] Biomaterial Deposit A strain of Lactobacillus plantarum LAS3, classified and named Lactiplantibacillus plantarum , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 29, 2025, with the deposit number GDMCC NO.66440, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the colony morphology of Lactobacillus plantarum LAS3; Figure 2 is the Gram staining image of Lactobacillus plantarum LAS3; Figure 3 is the phylogenetic tree of Lactobacillus plantarum LAS3; Figure 4 Effects of different pH values on the growth of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L, absorbance values (OD600nm) of bacterial solution at different pH values; Figure 5 Effects of different sodium chloride concentrations on the growth of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L, absorbance values (OD600nm) of bacterial solution at different sodium chloride concentrations; Figure 6 This is the Oxford cup antibacterial effect diagram of Lactobacillus plantarum LAS3 against Pseudomonas aeruginosa; Figure 7 This is the Oxford cup antibacterial effect diagram of Lactobacillus plantarum LAS3 against Burkholderia cepacia; Figure 8 This is the Oxford cup antibacterial effect diagram of Lactobacillus plantarum LAS3 on Pseudomonas putida. DETAILED DESCRIPTION

[0022] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.

[0024] The culture medium involved in the present invention is: MRS liquid medium: 10 g / L peptone, 5 g / L beef extract powder, 4 g / L yeast extract powder, 2 g / L potassium dihydrogen phosphate, 2 g / L ammonium citrate tribasic, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, sterilized at 121°C for 15 min, pH 6.2 ± 0.2; MRS agar medium: 10 g / L peptone, 5 g / L beef extract powder, 4 g / L yeast extract powder, 2 g / L potassium dihydrogen phosphate, 2 g / L triammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL / L Tween 80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 15 g / L agar powder, sterilized at 121°C for 15 min, pH 6.2±0.2.

[0025] Example 1: Strain Isolation 1) Take 10g of Guizhou acid fish and add 90mL of MRS liquid culture medium. Incubate at 36℃ for 24h to obtain enrichment solution. 2) Take 100 µL of the enrichment solution, spread it on 2% CaCO3 MRS agar medium, and incubate at 36°C for 24 h; 3) Select colonies with a prominent calcium-solubility zone and repeatedly isolate and purify them on MRS agar using the streak plate method until a single strain is isolated, which is designated LAS3. Inoculate the purified strain into 30% glycerol solution and store at -80°C.

[0026] Example 2 Strain Identification The isolated and preserved LAS3 strain was activated and Gram-stained, and the colony morphology and Gram-staining results were observed. At the same time, the observation and analysis were carried out with reference to the "Berger's Manual of Identification of Bacteriology", and the physiological and biochemical characteristics of the strain were tested with reference to the "Classification, Identification and Experimental Methods of Lactic Acid Bacteria".

[0027] Morphological characteristics: such as Figure 1 As shown in the figure, after the strain was cultured at 36°C for 24 hours, the colony morphology was flat white colonies with a light yellow bulge in the center, the colony diameter was 2-3 mm, the whole was round, moist, opaque, with complete edges, smooth surface, soft texture, and white on the back of the colony; Figure 2As shown in the figure, after Gram staining and microscopic observation, the strain was Gram-positive bacteria, short rod-shaped, non-spore-forming, without flagella, and non-motile.

[0028] Physiological and biochemical tests: Refer to the "Classification and Identification of Lactic Acid Bacteria and Experimental Methods." Physiological and biochemical identification items include: catalase, nitrate reduction test, gelatin liquefaction test, H2S production, indole, esculin, cellobiose, maltose, fructose, rhamnose, sucrose, raffinose, xylose, galactose, melibiose, mannose, melezitose, arabinose, mannitol, salicin, and sorbitol. The selected strains were cultured in MRS liquid medium at 36°C for 24 hours. The bacteria were inoculated with a loop into the corresponding bacterial microreaction tube and incubated at 36°C for 24 hours. The color change of the reaction tube was recorded, and identification was performed according to the "Handbook of Common Bacterial Systematic Identification." The results of physiological and biochemical tests of the strain are shown in Table 1. The results of catalase, hydrogen sulfide, nitrate reduction, indole, and gelatin liquefaction tests were negative, indicating that strain LAS3 had no ability to metabolize and produce gelatinase, hydrogen sulfide, indole, or catalase during the fermentation process, nor had the ability to reduce nitrate; the identification results of esculin, cellobiose, maltose, fructose, sucrose, raffinose, xylose, galactose, melibiose, mannose, melezitose, arabinose, mannitol, and salicin were positive, indicating that strain LAS3 had the ability to ferment esculin, cellobiose, maltose, fructose, sucrose, raffinose, xylose, galactose, melibiose, mannose, melezitose, arabinose, mannitol, and salicin; the identification results of rhamnose and sorbitol were negative, indicating that strain LAS3 had no ability to ferment and utilize rhamnose and sorbitol; based on the morphological characteristics and physiological and biochemical identification tests, strain LAS3 can be preliminarily determined to be a Lactobacillus genus.

[0029] Table 1 Physiological and biochemical experimental results Identification items Identification results Catalase - Nitrate reduction test - Gelatin liquefaction test - <![CDATA[Generation of H2S]]> - Producing indole - Esculin + Cellobiose + maltose + fructose + Rhamnose - sucrose + Raffinose + Xylose + Galactose + melibiose + Mannose + melezitose + arabinose + Mannitol + salicin + sorbitol - Note: + indicates a positive result, - indicates a negative result.

[0030] Molecular biological identification: 16S rDNA identification was performed. Genomic DNA from strain LAS3 was extracted according to the instructions of the bacterial genomic DNA extraction kit and used as a template. 16S rDNA was amplified by PCR using universal 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-CTACGGCTACCTTGTTACGA-3'). The PCR product was examined on an agarose gel and photographed before being sent to the Guangzhou branch of Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The resulting sequence information (SEQ.ID.NO.1) was aligned and analyzed for homology in the NCBI database, and a phylogenetic tree was constructed using MEGA 12 software. Phylogenetic tree Figure 3 As shown, the results showed that strain LAS3 and Lactiplantibacillus plantarum (NBRC 15891) clustered in the same clade, and their homology reached more than 99%. Combined with their morphological characteristics, strain LAS3 was identified as Lactobacillus plantarum. Lactiplantibacillus plantarum (NBRC 15891).

[0031] The 16S rDNA sequence of the strain is shown in SEQ ID NO.1:

[0032] Example 3 Determination of environmental tolerance of Lactobacillus plantarum LAS3 The pH and sodium chloride tolerance of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L (see CN119552784A) were determined: two activated generations of Lactobacillus plantarum strains were inoculated at a volume fraction of 3% in MRS liquid culture medium with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10) and sodium chloride concentrations (0, 20, 40, 60, 80, 100 g / L). The cultures were incubated at 36°C and 130 rpm for 20 h, and the OD (absorbance) at 600 nm was measured using a microplate reader.

[0033] pH tolerance results such as Figure 4 As shown, the OD values of both strains showed a trend of increasing first and then decreasing at different pH levels. Excessive acidity or alkalinity inhibited the growth and reproduction of the strains. Lactobacillus plantarum CMRC 1L had a pH tolerance range of pH 3-9, with its OD value reaching its highest at pH 5, while Lactobacillus plantarum LAS3 had its highest OD value at pH 6. Furthermore, its cell density within the pH range of 3-9 was significantly higher than that of Lactobacillus plantarum CMRC 1L, indicating that Lactobacillus plantarum LAS3 has a better pH tolerance than Lactobacillus plantarum CMRC1L.

[0034] Sodium chloride tolerance results Figure 5 As shown in the data, when the sodium chloride mass concentration is less than 40 g / L, it basically has no effect on the growth of the two strains; when the sodium chloride mass concentration is greater than 40 g / L, the OD values of the two strains gradually decrease; among them, Lactobacillus plantarum CMRC 1L basically stops growing when the sodium chloride mass concentration increases to 80 g / L; and Lactobacillus plantarum LAS3 has a sodium chloride tolerance of more than 100 g / L, indicating that Lactobacillus plantarum LAS3 has significantly better sodium chloride tolerance than Lactobacillus plantarum CMRC1L.

[0035] Example 4 Determination of antibacterial properties In this example, the antibacterial activity of Lactobacillus plantarum LAS3 fermentation broth was determined using the Oxford cup method. Indicator bacteria: Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis, and Candida albicans; The following samples were used: Lactobacillus plantarum LAS3 fermentation broth dilution group, acid removal treatment group, and proteinase K treatment group; Lactobacillus plantarum SXp08 (see CN115612637B) fermentation broth dilution group. Sample preparation steps were as follows (the experimental steps for the Lactobacillus plantarum LAS3 and SXp08 fermentation broth dilution groups were identical, differing only in the strains): 1) Dilution group: Two generations of activated Lactobacillus plantarum were inoculated into 100 mL of MRS liquid medium at a volume fraction of 3%. Fermentation was carried out at 36°C for 24 h. After fermentation, the cells were centrifuged at 5000g and 4°C for 20 min. The supernatant was retained and diluted with an equal volume of MRS liquid medium to obtain fermentation broth dilutions. 2) Acid removal treatment group: Take part of the fermentation broth dilution prepared in step 1) and add 1 mol / L sodium hydroxide solution to adjust the pH to 6.0 to eliminate organic acid interference; 3) Proteinase K treatment group: Take a portion of the fermentation broth dilution after acid removal prepared in step 2) and add proteinase K to a final concentration of 10 mg / mL in the mixed solution. Incubate in a 37°C water bath for 2 h. The specific experimental steps are as follows: Aseptically, pour the culture medium that has been sterilized by high temperature into a sterile culture dish with a diameter of 9 cm, so that each culture dish has 15-20 mL of culture medium. After cooling, add 0.2 mL of Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans (the viable count of indicator bacteria is 10 9 CFU / mL), spread the culture medium surface evenly with a spreading rod, place the Oxford cup vertically on the corresponding culture medium surface with tweezers, and gently press it so that it is in contact with the culture medium without gaps. Add 0.1 mL of various test samples to each small tube, mark them, and incubate them in a 37°C incubator for 24 hours. Observe the results. Repeat each experiment three times. During the incubation period, the test bacteria begin to grow, while the test sample spreads around the Oxford cup as the origin, forming a circular area where colonies cannot grow, namely the "inhibition zone". The larger the inhibition zone, the better the antibacterial effect. Measure the diameter of the inhibition zone in each group of culture dishes and take the average value. The antibacterial results of the Lactobacillus plantarum LAS3 group are shown in Table 2: the diameters of the inhibition zones of the Lactobacillus plantarum LAS3 fermentation broth dilution against Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans were 14.92 mm, 14.12 mm, 13.92 mm, 16.81 mm, 22.71 mm and 15.64 mm, respectively, indicating that the Lactobacillus plantarum LAS3 fermentation broth dilution had a good antibacterial effect; after the interference of organic acids was eliminated by sodium hydroxide, the diameters of the inhibition zones against Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans were 12.01 mm, 11.11 mm, 11.04 mm, 14.23 mm, 19.25 mm and 18.80 mm, respectively. mm and 12.79 mm, and the antibacterial activity was slightly decreased compared with the dilution group; after further treatment with proteinase K, it had no antibacterial effect, indicating that the antibacterial substance produced by Lactobacillus plantarum LAS3 was bacteriocin.

[0036] Table 2-Table 3 inhibition zone diameter data of Lactobacillus plantarum LAS3 group and Lactobacillus plantarum SXp08 group. The inhibition zone diameters of Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans groups of Lactobacillus plantarum LAS3 fermentation broth dilution liquid are significantly larger than those of Lactobacillus plantarum SXp08 group, indicating that Lactobacillus plantarum LAS3 isolated from Guizhou sour fish of the present invention has better antibacterial performance.

[0037] Table 2 Inhibition zone diameter data of Lactobacillus plantarum LAS3 group Group Pseudomonas aeruginosa Burkholderia cepacia Pseudomonas putida Staphylococcus aureus Bacillus subtilis Candida albicans diluent group 14.92 14.12 13.92 16.81 22.71 15.64 Acid removal treatment group 12.01 11.11 11.04 14.23 19.25 12.79 Proteinase K treatment group - - - - - - Note: “-” indicates no antibacterial effect, diameter of inhibition zone (mm).

[0038] Table 3 Inhibition zone diameter data of Lactobacillus plantarum SXp08 group Group Pseudomonas aeruginosa Burkholderia cepacia Pseudomonas putida Staphylococcus aureus Bacillus subtilis Candida albicans diluent group 9.07 8.16 10.28 11.34 13.49 9.32 Note: Diameter of inhibition zone (mm).

[0039] Example 5 Preparation of Lactobacillus plantarum LAS3 bacteriocin A method for preparing bacteriocin by fermenting Lactobacillus plantarum LAS3 comprises the following steps: A1, take activation culture two generations of Lactobacillus plantarum LAS3, by 3% of the total volume of MRS liquid medium inoculated into 1000mL of MRS liquid medium fermentation culture, fermentation temperature is 36 ℃, fermentation time is 24h; After fermentation is complete, centrifugation is removed bacterium, centrifugation parameters are specifically 5000g, 4 ℃, centrifugation time is 20min, collect the fermentation supernatant after centrifugation; A2. The fermentation supernatant and ethyl acetate were mixed in a volume ratio of 1:1, extracted at 28°C and 150 rpm for 12 h, allowed to stand for separation, and the upper organic phase was collected. The lower aqueous phase was again added with ethyl acetate in a volume ratio of 1:1, extracted at 28°C and 150 rpm for 12 h, allowed to stand for separation, and the upper organic phase was collected. A3. The organic phases of the two extractions were mixed, placed in a rotary evaporator, evaporated and concentrated under vacuum conditions at 40° C., and the concentrate was collected with 15 mL of ultrapure water to obtain a mixture I; the mixture I was freeze-dried at a vacuum degree of 230 Pa and a cold trap temperature of -46.8° C. to obtain the bacteriocin.

[0040] Example 6 Antiseptic Efficacy Test Preparation of bacteriocin extract: 2 g of the bacteriocin prepared in Example 5 was weighed and added into 15 mL of ultrapure water to reconstitute the bacteriocin extract.

[0041] The bacteriocin extract of Lactobacillus plantarum LAS3 prepared above was added to cosmetics as a natural preservative, and an antiseptic challenge test was conducted with reference to the well-known microbial challenge test methods of the Cosmetic, Toiletries and Fragrance Association (CTFA) and the United States Pharmacopoeia; First, twelve portions of a certain amount of a basic formula preservative-free facial mask liquid were taken and randomly divided into two groups, with six portions of facial mask liquid in each group; the experimental group was added with the bacteriocin extract of Lactobacillus plantarum LAS3 prepared in this example so that the final bacteriocin extract content in the sample was 0.05% by mass fraction; the blank control group was replaced with an equal amount of ultrapure water instead of the bacteriocin extract, and then Pseudomonas aeruginosa bacterial solution, Burkholderia cepacia, Pseudomonas putida bacterial solution, Staphylococcus aureus bacterial solution, Bacillus subtilis bacterial solution and Candida albicans bacterial solution in the logarithmic growth period were added respectively so that the final bacterial content of each test sample was 5×10 6 cfu / mL; then mix thoroughly and incubate at 28°C; determine the viable bacterial count 6 hours, 1 day, 2 days, 7 days, 14 days, 21 days, and 28 days after inoculation according to the experimental method recommended by the Cosmetic and Flavor Association (CTFA) to judge the preservative efficacy of the cosmetics; the judgment standard is: after each sample is inoculated once, the number of surviving bacteria decreases to no more than 0.1% of the initial concentration on the 7th day, then gradually decreases, and no sterile growth is observed on the 28th day; the preservative is effective and passes the test; otherwise, the preservative is ineffective and fails the test; Table 4 Antiseptic efficacy test results

[0042] The antiseptic challenge results are shown in Table 4. After 6 hours of exposure, the viable counts of Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis, and Candida albicans in the sample groups all showed a downward trend; after 1 day of exposure, the viable counts of all sample groups were less than 100; and after 2 to 28 days of exposure, the viable counts of all sample groups were 0. This indicates that the bacteriocin extract of Lactobacillus plantarum LAS3 has excellent antiseptic properties when used in the preparation of cosmetics such as facial mask liquids. This indicates that the bacteria, its fermentation broth, and the bacteriocin prepared can be used as antibacterial and antiseptic raw materials for food, health products, or daily chemical products, and have broad application prospects and important translational research value.

[0043] Example 7 Verification of the properties of Lactobacillus plantarum LAS3 bacteriocin Test substance: Lactobacillus plantarum LAS3 bacteriocin prepared in Example 5 of the present invention; bacteriocin prepared using Lactobacillus plantarum W3-2 (see CN117683675B) according to the preparation method of Example 5 of the present invention.

[0044] Treatment of test substances: Weigh 2 g of bacteriocin and add 15 mL of ultrapure water to reconstitute the bacteriocin to obtain the bacteriocin extract; Indicator bacteria: Escherichia coli; 1) Thermal stability test: The bacteriocin was treated at 36° C., 60° C., 80° C., 100° C., and 121° C. for 40 min, cooled to room temperature, and the diameter of the inhibition zone was determined using the antibacterial experiment in Example 4.

[0045] 2) pH stability test: The pH of the sample was adjusted to 2, 2.5, 3, 7, 8, and 9 with 1 mol / L HCl and 1 mol / L NaOH, respectively, and the sample was placed in a 37°C water bath for 2 h. The pH was then adjusted back to 6, and the diameter of the inhibition zone was determined using the antibacterial test in Example 4. Table 5 Inhibition zone diameter data of thermal stability test Group / Temperature 36℃ 60℃ 80℃ 100℃ 121℃ LAS3 bacteriocin 22.65 22.32 22.08 20.74 18.39 W3-2 bacteriocin 18.12 18.26 18.03 15.89 13.47 Note: Diameter of inhibition zone (mm).

[0046] Table 6 pH stability test inhibition zone diameter data Group / pH 2 2.5 3 7 8 9 LAS3 bacteriocin 23.82 24.90 22.47 21.95 20.03 19.16 W3-2 bacteriocin 20.97 22.45 20.86 18.78 17.42 15.71 Note: Diameter of inhibition zone (mm).

[0047] The results of the thermal stability test are shown in Table 5. The Lactobacillus plantarum LAS3 bacteriocin prepared in Example 5 of the present application was treated at 36°C, 60°C, 80°C and 100°C for 40 minutes. The diameter of the inhibition zone against Escherichia coli was greater than 20 mm. After treatment at 121°C for 40 minutes, the diameter of the inhibition zone against Escherichia coli could reach more than 18 mm. Compared with the bacteriocin produced by Lactobacillus plantarum W3-2, the inhibition zone diameter of the Lactobacillus plantarum LAS3 bacteriocin group was larger under each temperature treatment condition. It can be seen that the bacteriocin produced by Lactobacillus plantarum LAS3 has excellent thermal stability.

[0048] The results of the pH stability test are shown in Table 6. The Lactobacillus plantarum LAS3 bacteriocin prepared in Example 5 of the present application can maintain strong antibacterial properties under strong acid and alkaline conditions, and compared with the bacteriocin produced by Lactobacillus plantarum W3-2, the bacteriocin produced by Lactobacillus plantarum LAS3 has stronger antibacterial activity under various pH conditions, indicating that the bacteriocin produced by Lactobacillus plantarum LAS3 isolated from Guizhou sour fish in the present application has better pH stability and has excellent antibacterial activity in the pH range of 2-9.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) LAS3, characterized in that Its deposit number is GDMCC NO.66440.

2. Application of plant lactobacillus LAS3 as claimed in claim 1 in preparing bacteriocin.

3. A method for producing bacteriocin by fermenting Lactobacillus plantarum LAS3 according to claim 1, characterized in that: The following steps are involved: A1. Inoculate frozen Lactobacillus plantarum LAS3 into liquid MRS medium for activation, and then inoculate into MRS liquid medium for fermentation and culture. After fermentation, centrifuge to remove the bacteria and retain the fermentation broth. A2. Mix the fermentation broth with ethyl acetate, extract the mixture, allow to stand for separation, collect the upper organic phase, add ethyl acetate to the lower aqueous phase again, extract the mixture, allow to stand for separation, and collect the upper organic phase; A3. The organic phases from the two extractions were mixed, placed in a rotary evaporator, evaporated and concentrated under vacuum conditions at 40° C., and the concentrate was collected with ultrapure water to obtain a mixture I; and the mixture I was freeze-dried to obtain the bacteriocin.

4. The preparation method according to claim 3, wherein The activated Lactobacillus plantarum LAS3 fermentation inoculation amount in step A1 is 2-4% of the MRS liquid culture medium.

5. The preparation method according to claim 3, wherein The centrifugation parameters in step A1 are specifically 4500-5500 g, 3-6° C., and a centrifugation time of 15-25 min.

6. The preparation method according to claim 3, wherein In step A1, the fermentation temperature is 34-38° C. and the fermentation time is 18-32 hours.

7. The preparation method according to claim 3, wherein In step A2, the volume ratio of the fermentation liquid to ethyl acetate is 1:1, the extraction temperature is 25-30° C., and the extraction time is 10-14 hours.

8. A product, characterized in that The method comprises the Lactobacillus plantarum LAS3 as claimed in claim 1.

9. The product according to claim 8, characterized in that The product is food, health care product or daily chemical product.

10. Use of the bacteriocin prepared by the Lactobacillus plantarum LAS3 according to claim 1 or the method according to claim 3 in preparing food, health products or daily chemical products.

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