Lactobacillus plantarum LAS3 and application thereof

Abstract

The present invention relates to a plant lactobacillus ( Lactiplantibacillus plantarum ) LAS3 and its application belong to the field of microbial technology. The plant lactobacillus LAS3 disclosed in the present invention has been deposited in the Guangdong Provincial Microbial Culture Collection Center, and its deposit number is GDMCC NO.66440. The strain was isolated, purified and screened from Guizhou sour fish, and experiments showed that the strain has excellent pH tolerance and sodium chloride tolerance, and its fermentation broth has good antibacterial effects on a variety of common pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the bacteriocin produced by the strain has excellent antibacterial and antiseptic effects, as well as excellent thermal stability and pH stability. It can be used as an antibacterial and antiseptic raw material and applied to food, health products or daily chemical products. It has broad application prospects and important translational research value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a lactobacillus plantarum LAS3 and its application. BACKGROUND

[0002] Lactic acid bacteria as a generally recognized safe (GRAS) strain, which produces active metabolites through metabolism, these active metabolites have been proved to have anti-inflammatory, antibacterial, antioxidant or immunomodulatory properties, among which the antibacterial property is one of the most outstanding properties of lactic acid bacteria. Lactic acid bacteria bacteriocin as one of the active metabolites of lactic acid bacteria is a kind of polypeptide or precursor polypeptide with antibacterial activity, which has strong antagonistic properties to the growth of other microorganisms, and its antibacterial range is not limited to homologous bacteria.

[0003] In addition, the characteristics of bacteriocin protein make it can be degraded in the human body, so it is non-toxic, non-residual, and has high efficiency, acid resistance, high temperature resistance, no drug resistance, most of the genes are located on the plasmid, small molecular weight, contains modified amino acids, complex structure, etc. 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 health and regulation of intestinal flora.

[0004] In addition, lactobacillus plantarum is a kind of gram-positive anaerobic or facultative anaerobic, facultative heterofermentative lactic acid bacteria, and the bacteriocin secreted by it has broad-spectrum antibacterial activity and safe degradation, which is a research hotspot of new antifungal products. Therefore, in order to meet the growing market demand for bacteriocin in the fields of food, health care products and daily chemical products, it is urgent to isolate and obtain lactobacillus plantarum producing high antibacterial activity bacteriocin. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a lactobacillus plantarum LAS3 with strong stress resistance and high antibacterial activity bacteriocin production, which is isolated and purified from Guizhou sour fish and has been preserved in Guangdong Microbial Culture Collection Center with the preservation number GDMCC NO. 66440.

[0006] In a first aspect, the present application provides a lactobacillus plantarum (Lactobacillus plantarum) LAS3, which has been preserved in Guangdong Microbial Culture Collection Center on May 29, 2025, with the preservation number GDMCC NO. 66440 and the preservation address being No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou. Lactiplantibacillus plantarum In a first aspect, the present application provides a lactobacillus plantarum (Lactobacillus plantarum) LAS3, which has been preserved in Guangdong Microbial Culture Collection Center on May 29, 2025, with the preservation number GDMCC NO. 66440 and the preservation address being No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou.

[0007] In a second aspect, the present application provides the use of the lactobacillus plantarum LAS3 of the first aspect in the preparation of bacteriocin.

[0008] In a third aspect, the present application provides a preparation method of bacteriocin produced by fermentation of Lactobacillus plantarum LAS3 according to the first aspect, comprising the following steps:

[0009] A1, the frozen Lactobacillus plantarum LAS3 is inoculated into a liquid MRS medium for activation, and after activation, is inoculated into a MRS liquid medium for fermentation culture; after fermentation is completed, centrifugal treatment is performed to remove bacterial bodies, and the fermentation liquor is reserved;

[0010] A2, the fermentation liquor and ethyl acetate are mixed, and after extraction treatment, standing separation is performed; the upper organic phase is collected, and the lower aqueous phase is added with ethyl acetate again, and after extraction treatment, standing separation is performed; the upper organic phase is collected;

[0011] A3, the organic phases extracted twice are mixed, and placed in a rotary evaporator; evaporation and concentration are performed under the condition of 40 DEG C and vacuum; the concentrate is collected with ultrapure water, to obtain a mixture I; after freeze-drying of the mixture I, the bacteriocin is obtained.

[0012] Preferably, the inoculation amount of the activated Lactobacillus plantarum LAS3 in step A1 is 2-4% of the MRS liquid medium.

[0013] Preferably, the centrifugal parameters in step A1 are specifically 4500-5500g, 3-6 DEG C, and the centrifugal time is 15-25min.

[0014] Preferably, the fermentation temperature in step A1 is 34-38 DEG C, and the fermentation time is 18-32h.

[0015] Preferably, the volume ratio of the fermentation liquor to ethyl acetate in step A2 is 1:1, the extraction temperature is 25-30 DEG C, and the extraction time is 10-14h.

[0016] Preferably, the rotating speed of the extraction treatment in step A2 is 130-180r / min.

[0017] Preferably, the specific conditions of freeze-drying in step A3 are as follows: vacuum degree 230Pa, and cold trap temperature-46.8 DEG C.

[0018] In a fourth aspect, the present application provides a product comprising the Lactobacillus plantarum LAS3 according to the first aspect.

[0019] In a fifth aspect, the present application provides application of the bacteriocin extract obtained by the method according to the third aspect in preparation of daily-use articles.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The inventors of the present application obtain the Lactobacillus plantarum LAS3 by separation, purification and screening from Guizhou sour fish.

[0022] 1) The environmental tolerance determination results show that the pH tolerance of Lactobacillus plantarum CMRC 1L is pH 3-9, the OD value (absorbance value) of the bacteria is the highest at pH=5, the OD value of Lactobacillus plantarum LAS3 of the application is the highest at pH=6, and the cell density thereof at pH=9 is still obviously higher than that of Lactobacillus plantarum CMRC 1L; the tolerance of Lactobacillus plantarum LAS3 to sodium chloride is above 100 g / L, while Lactobacillus plantarum CMRC 1L basically stops growing when the mass concentration of sodium chloride increases to 80 g / L; it can be seen from the above that the Lactobacillus plantarum LAS3 isolated in the application has more excellent pH tolerance and sodium chloride tolerance than Lactobacillus plantarum CMRC 1L in the prior art;

[0023] 2) The bacteriostatic test results show that the strain of the application has good bacteriostatic effect on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the diameter of the bacteriostatic circle is greater than 13 mm, and the bacterium has better bacteriostatic effect than Lactobacillus plantarum SXp08 in the prior art;

[0024] 3) The heat stability test results show that the bacteriocin produced by Lactobacillus plantarum LAS3 has an antibacterial circle diameter of more than 20 mm on Escherichia coli after being treated at 36℃, 60℃, 80℃ and 100℃ for 40 min, and the antibacterial circle diameter on Escherichia coli is also more than 18 mm after being treated at 121℃ for 40 min, indicating that the bacteriocin produced by Lactobacillus plantarum LAS3 has excellent heat stability; and the pH stability test results show that the antibacterial circle diameter of the bacteriocin produced by Lactobacillus plantarum LAS3 is more than 19 mm under strong acid and alkaline conditions, indicating that the bacteriocin produced by Lactobacillus plantarum LAS3 isolated from Guizhou sour fish has better pH stability, and has excellent antibacterial activity in the pH=2-9 range; and the heat stability and pH stability of the bacteriocin described in the application are also obviously better than the prior art level;

[0025] 4) The results of the preservative effect show that the bacteriocin produced by the strain of the application has excellent preservative effect on Pseudomonas aeruginosa, Burkholderia cenocepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans, has significant inhibitory effect on Pseudomonas aeruginosa, Burkholderia cenocepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans after 6 hours of action, and the viable count can be reduced to below 100 after 1 day of action, and the viable count of Pseudomonas aeruginosa, Burkholderia cenocepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans is 0 after two days, and can be maintained for more than 28 days, indicating that the bacteriocin extract of Lactobacillus plantarum LAS3 can be applied to the preparation of facial mask liquid and other cosmetics, has excellent bacteriostatic and preservative effect, can be used as a bacteriostatic and preservative raw material, applied to daily chemical products, and has wide application prospect and important transformation research value.

[0026] Biological material preservation

[0027] A Lactobacillus plantarum LAS3, which is classified and named as Lactiplantibacillus plantarum , has been preserved in the Guangdong Microbial Culture Collection Center on May 29, 2025, with a preservation number of GDMCC NO.66440 and a preservation address of No. 59 Building, 5th Floor, Guangzhou City, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a colony morphology diagram of Lactobacillus plantarum LAS3;

[0029] Figure 2 It is a gram staining diagram of Lactobacillus plantarum LAS3;

[0030] Figure 3 It is a phylogenetic tree of Lactobacillus plantarum LAS3;

[0031] Figure 4 It is the influence of different pH values on the growth of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L, and the absorbance value (OD600nm) of the bacterial liquid under different pH values;

[0032] Figure 5 It is the influence of different sodium chloride mass concentrations on the growth of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L, and the absorbance value (OD600nm) of the bacterial liquid under different sodium chloride mass concentrations;

[0033] Figure 6 It is an Oxford cup bacteriostatic effect diagram of Lactobacillus plantarum LAS3 on Pseudomonas aeruginosa;

[0034] Figure 7 It is an Oxford cup bacteriostatic effect diagram of Lactobacillus plantarum LAS3 on Burkholderia cenocepacia;

[0035] Figure 8 Oxford cup inhibition effect diagram of Pseudomonas putida by Lactobacillus plantarum LAS3. DETAILED DESCRIPTION

[0036] For the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0037] Other materials, reagents, etc. used in the examples can be obtained from commercial channels if not otherwise specified.

[0038] Culture medium involved in the present application:

[0039] MRS liquid medium: 10 g / L proteose peptone, 5 g / L beef extract powder, 4 g / L yeast extract powder, 2 g / L potassium phosphate dibasic, 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, sterilized at 121℃ for 15 min, pH 6.2±0.2;

[0040] MRS agar medium: 10 g / L proteose peptone, 5 g / L beef extract powder, 4 g / L yeast extract powder, 2 g / L potassium phosphate dibasic, 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℃ for 15 min, pH 6.2±0.2.

[0041] Example 1: Strain isolation

[0042] 1) Take 10 g Guizhou sour fish and add 90 mL MRS liquid medium, and enrich culture at 36℃ for 24 h to obtain an enrichment liquid;

[0043] 2) Take 100 µL of the enrichment liquid and spread on 2% CaCO3 MRS agar medium, and culture at 36℃ for 24 h;

[0044] 3) Pick the colonies with obvious calcium-dissolving rings, and repeatedly isolate and purify them on MRS agar medium by plate streaking method until a single strain is isolated, and it is named as LAS3. The purified strain is inoculated in 30% glycerol solution and stored in a-80℃ refrigerator.

[0045] Example 2: Strain identification

[0046] 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".

[0047] 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 2 As 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.

[0048] 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."

[0049] 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.

[0050] Table 1 Physiological and biochemical experimental results

[0051] Identification item Identification result Contact enzyme - Nitrate reduction test - Gelatin liquefaction test - H2S production - Indole production - Esculin + Cellobiose + Maltose + Fructose + Rhamnose - Sucrose + Raffinose + Xylose + Galactose + Melibiose + Mannose + Pinaculin + Arabinose + Mannitol + Salicin + Sorbitol -

[0052] Note: + indicates a positive result, - indicates a negative result.

[0053] Molecular biological identification: 16S rDNA identification was performed. Genomic DNA of 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.

[0054] 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, the strain LAS3 was identified as Lactobacillus plantarum. Lactiplantibacillus plantarum (NBRC 15891).

[0055] The 16S rDNA sequence of the strain is shown in SEQ ID NO.1:

[0056]

[0057] Example 3 Determination of environmental tolerance of Lactobacillus plantarum LAS3

[0058] The pH tolerance and sodium chloride tolerance of Lactobacillus plantarum LAS3 and Lactobacillus plantarum CMRC 1L (see CN119552784A) were determined respectively: two strains of Lactobacillus plantarum activated for two generations were respectively inoculated into MRS liquid medium with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10) and sodium chloride mass concentrations (0, 20, 40, 60, 80, 100 g / L) at a volume fraction of 3%; cultured at 36°C, 130 rpm for 20 h, and the OD value (absorbance value) at 600 nm was measured using an enzyme marker.

[0059] The pH tolerance results are shown in Table 1. Figure 4 As shown in Table 1, the OD values of the two strains at different pH values all showed a trend of first increasing and then decreasing, and the growth and reproduction of the strains were inhibited by excessive acid or excessive alkali. Among them, the pH tolerance of Lactobacillus plantarum CMRC 1L was pH 3-9, and the OD value of the strain was the highest at pH=5, while the OD value of Lactobacillus plantarum LAS3 was the highest at pH=6, and the cell density of Lactobacillus plantarum LAS3 in the pH 3-9 range was significantly higher than that of Lactobacillus plantarum CMRC 1L; indicating that the pH tolerance of Lactobacillus plantarum LAS3 is better than that of Lactobacillus plantarum CMRC 1L.

[0060] The sodium chloride tolerance results are shown in Table 2. Figure 5 As shown in Table 2, when the mass concentration of sodium chloride was less than 40 g / L, it had basically no effect on the growth of the two strains; when the mass concentration of sodium chloride was greater than 40 g / L, the OD values of the two strains gradually decreased; among them, Lactobacillus plantarum CMRC 1L basically stopped growing when the mass concentration of sodium chloride increased to 80 g / L; while the sodium chloride tolerance of Lactobacillus plantarum LAS3 was more than 100 g / L, indicating that the sodium chloride tolerance of Lactobacillus plantarum LAS3 was significantly better than that of Lactobacillus plantarum CMRC 1L.

[0061] Example 4 Determination of bacteriostatic properties

[0062] In this embodiment, the Oxford cup method was used to determine the bacteriostatic activity of the fermentation broth of Lactobacillus plantarum LAS3.

[0063] Indicator bacteria: Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis, and Candida albicans.

[0064] Test samples: Lactobacillus plantarum LAS3 fermentation broth dilution group, acid removal treatment group, proteinase K treatment group; Lactobacillus plantarum SXp08 (see CN115612637B) fermentation broth dilution group; the preparation steps of the test samples are as follows (the experimental steps of the Lactobacillus plantarum LAS3 and SXp08 fermentation broth dilution groups are completely the same, and the only difference is the strain):

[0065] 1) Dilution group: Take Lactobacillus plantarum activated for two generations, inoculate in 100 mL of MRS liquid medium at a volume fraction of 3%, and ferment at 36°C for 24 h. After fermentation, centrifuge at 5000g and 4°C for 20 min, discard the bacterial body, and retain the fermentation supernatant. Dilute with an equal volume of MRS liquid medium to obtain the fermentation broth dilution;

[0066] 2) Acid removal treatment group: Take part of the fermentation broth dilution prepared in step 1), add 1 mol / L sodium hydroxide solution to adjust the pH to 6.0, and remove the interference of organic acids;

[0067] 3) Proteinase K treatment group: Take part of the acid-removed fermentation broth dilution prepared in step 2), add proteinase K to make the final concentration of proteinase K in the mixed solution 10 mg / mL, and treat at 37°C water bath for 2 h;

[0068] The specific experimental steps are as follows:

[0069] Under sterile conditions, pour the high-temperature sterilized medium into sterile petri dishes with a diameter of 9 cm, so that each petri dish has 15-20 mL of medium. After cooling, add 0.2 mL of Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans (the viable bacterial count of the indicator bacteria is 10 9 CFU / mL), evenly spread the surface of the medium with a spreader, vertically place the oxford cups on the surface of the corresponding medium with tweezers, gently press to make them contact with the medium without gaps, add 0.1 mL of each test sample to each tube, and mark it. After 24 h of incubation in a 37°C incubator, observe the results. Each group of experiments was repeated three times. During incubation, on the one hand, the test bacteria began to grow, and on the other hand, the test sample spread outward from the oxford cup as the center, forming a circular area where bacteria could not grow, i.e. "bacteriostatic circle". The larger the bacteriostatic circle, the better the bacteriostatic effect. Measure the diameter of the bacteriostatic circle in each group of petri dishes and take the average value;

[0070] The bacteriostasis results of the Lactobacillus plantarum LAS3 group are shown in Table 2: the diameters of the bacteriostasis circle of the Lactobacillus plantarum LAS3 fermentation liquid diluent on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans are 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 liquid diluent has good bacteriostasis effect; and after the interference of organic acids is excluded by sodium hydroxide, the diameters of the bacteriostasis circle of the Lactobacillus plantarum LAS3 fermentation liquid diluent on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans are 12.01 mm, 11.11 mm, 11.04 mm, 14.23 mm, 19.25 mm and 12.79 mm respectively, and the bacteriostasis activity is slightly decreased compared with the diluent group; and after being treated by protease K, the Lactobacillus plantarum LAS3 has no bacteriostasis effect, indicating that the bacteriostasis substance produced by the Lactobacillus plantarum LAS3 is bacteriocin.

[0071] Table 2-Table 3, the diameters of the bacteriostasis circle of the Lactobacillus plantarum LAS3 fermentation liquid diluent on Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis and Candida albicans are all significantly larger than those of the Lactobacillus plantarum SXp08 group, indicating that the Lactobacillus plantarum LAS3 separated from the Guizhou sour fish in the application has better bacteriostasis performance.

[0072] Table 2, the diameters of the bacteriostasis circle of the Lactobacillus plantarum LAS3 group

[0073] Group Pseudomonas aeruginosa Burkholderia cenocepacia Pseudomonas putida Staphylococcus aureus Bacillus subtilis Candida albicans Dilution group 14.92 14.12 13.92 16.81 22.71 15.64 Acid discharge treatment group 12.01 11.11 11.04 14.23 19.25 12.79 Proteinase K treatment group - - - - - -

[0074] Note: “-” indicates no bacteriostasis effect, and the diameter of the bacteriostasis circle (mm).

[0075] Table 3, the diameters of the bacteriostasis circle of the Lactobacillus plantarum SXp08 group

[0076] Group Pseudomonas aeruginosa Burkholderia cenocepacia Pseudomonas putida Staphylococcus aureus Bacillus subtilis Candida albicans Dilution group 9.07 8.16 10.28 11.34 13.49 9.32

[0077] Note: the diameter of the bacteriostasis circle (mm).

[0078] Example 5, preparation of the bacteriocin of the Lactobacillus plantarum LAS3

[0079] A preparation method of a bacteriocin produced by fermentation of Lactobacillus plantarum LAS3, comprising the following steps:

[0080] A1, take the activated culture of Lactobacillus plantarum LAS3 for two generations, inoculate in 1000 mL of MRS liquid medium at 3% of the total volume of the medium, and ferment at 36°C for 24 h; after fermentation, remove the bacterial bodies by centrifugation at 5000 g and 4°C for 20 min, and collect the supernatant;

[0081] A2, mix the fermentation supernatant and ethyl acetate at a volume ratio of 1:1, extract at 28°C and 150 r / min for 12 h, separate by standing, collect the upper organic phase, and add ethyl acetate to the lower aqueous phase at a volume ratio of 1:1, extract at 28°C and 150 r / min for 12 h, separate by standing, and collect the upper organic phase;

[0082] A3, mix the organic phases extracted twice, place in a rotary evaporator, evaporate and concentrate under vacuum at 40°C, collect the concentrate with 15 mL of ultrapure water to obtain mixture I; freeze-dry mixture I under a vacuum of 230 Pa and at a cold trap temperature of -46.8°C to obtain the bacteriocin.

[0083] Example 6: Preservative efficacy test

[0084] Preparation of bacteriocin extract: weigh 2 g of the bacteriocin prepared in Example 5, add 15 mL of ultrapure water to dissolve, and obtain the bacteriocin extract.

[0085] Add the bacteriocin extract of Lactobacillus plantarum LAS3 prepared as described above to cosmetics as a natural preservative, and perform a preservative challenge test according to the microbial challenge test method of the Cosmetic, Toiletry, and Fragrance Association (CTFA) and the United States Pharmacopoeia;

[0086] First, take twelve portions of a base formula without preservative, and randomly divide them into two groups, six portions in each group; add the bacteriocin extract of Lactobacillus plantarum LAS3 prepared in this example to the test group to make the final content of the bacteriocin extract in the sample 0.05% by mass fraction, and add an equal amount of ultrapure water instead of the bacteriocin extract to the blank control group, and then add Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Staphylococcus aureus, Bacillus subtilis, and Candida albicans in the logarithmic growth phase to make the final bacterial content in each test sample 5×10 6cfu / mL; then mix well, and incubate at 28℃; 6h, 1 day, 2 days, 7 days, 14, 21 days and 28 days after inoculation, the viable bacteria count is determined according to the experimental method recommended by the Cosmetic and Fragrance Association (CTFA) to determine the preservative efficacy of the cosmetic; the judgment standard is: when each sample is inoculated once, the number of surviving bacteria is reduced to not more than 0.1% of the initial concentration on the 7th day, and then gradually reduced, and no bacteria grow on the 28th day; then the preservative is effective and passes the test; otherwise, the preservative is ineffective and fails the test;

[0087] Table 4 Preservative efficacy test results

[0088]

[0089] The preservative challenge results are shown in Table 4. The viable cell counts of P. aeruginosa, B. cepacia, P. putida, S. aureus, B. subtilis and C. albicans all showed a downward trend after 6h of action of the test sample groups; after 1 day of action, the viable cell counts of all test sample groups were less than 100; after 2 days to 28 days of action, the viable cell counts of all test sample groups were 0; indicating that the bacteriocin extract of L. plantarum LAS3 has excellent preservative performance when applied to the preparation of facial mask liquid and other cosmetics, and has broad application prospects and important transformation research value.

[0090] Example 7 Property verification of L. plantarum LAS3 bacteriocin

[0091] Test substance: L. plantarum LAS3 bacteriocin prepared in Example 5 of the present application; bacteriocin prepared according to the preparation method of Example 5 of the present application using L. plantarum W3-2 (see CN117683675B).

[0092] Test substance treatment: 2g of bacteriocin was weighed into 15mL ultrapure water to prepare a bacteriocin extract;

[0093] Indicator bacteria: E. coli;

[0094] 1) Heat stability test:

[0095] The bacteriocin was treated at temperatures of 36℃, 60℃, 80℃, 100℃ and 121℃ for 40min, cooled to room temperature, and the inhibition zone diameter was determined by the inhibition experiment in Example 4.

[0096] 2) pH stability test:

[0097] The test sample was adjusted to pH 2, 2.5, 3, 7, 8, 9 with 1mol / L HCL and 1mol / L NaOH respectively, and then 2h water bath at 37℃, and then the pH was adjusted back to 6. The inhibition zone diameter was determined by the inhibition experiment in Example 4.

[0098] Table 5 Inhibition zone diameter data of thermal stability test

[0099] 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

[0100] Note: Diameter of inhibition zone (mm).

[0101] Table 6 pH stability test inhibition zone diameter data

[0102] 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

[0103] Note: Diameter of inhibition zone (mm).

[0104] 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.

[0105] 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.

[0106] 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. The preparation method according to claim 3, wherein 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.

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

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