A Lactiplantibacillus plantarum with bacteriostatic effect and its application

Abstract

The present invention discloses a Lactiplantibacillus plantarum with antibacterial effect and its application. The Lactiplantibacillus plantarum is named Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07, with the preservation number of CCTCC NO: M20242086, the preservation unit is the China Center for Type Culture Collection, the preservation time is September 26, 2024, and the preservation address is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The strain can produce high yields of lactic acid and indole-3-lactic acid, and has strong antibacterial effects against various spoilage and pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella and molds, and is a broad-spectrum antibacterial microorganism.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to a Lactiplantibacillus plantarum with antibacterial effects and its applications. Background Art

[0002] Staphylococcus aureus is a widely distributed Gram-positive foodborne pathogen and a common contaminant in the dairy industry worldwide. The proportion of multi-drug resistant Staphylococcus aureus (resistant to multiple antibiotics) isolated from dairy products is as high as over 60%, and it is particularly likely to become a reservoir for multi-drug resistant Staphylococcus aureus strains in milk and cheese. Currently, the most commonly used method for preventing and controlling Staphylococcus aureus is the use of antibiotics. However, the use of antibiotics can lead to the development of resistance of Staphylococcus aureus to antibiotics such as vancomycin, sulfamethoxazole, and ceftazidime, which has been widely recognized. At the same time, the overuse of antibiotics can induce and accelerate the development of bacterial resistance to antibiotics, promote the transmission of drug-resistant bacteria to humans to form superbugs. In addition, the residues of antibiotics may affect the fermentation process of dairy products, resulting in a decline in product quality, thereby causing economic losses to the dairy industry.

[0003] Lactic acid bacteria, as a generally recognized as safe (GRAS) strain, have attracted much attention due to their wide application as biological additives in foods. Lactic acid bacteria produce active metabolites through metabolism, such as organic acids, EPS, bacteriocins, SCFAs, vitamins, and some bioactive enzymes. These active metabolites have been proven to have properties such as anti-inflammatory, antibacterial, antioxidant, or immunomodulatory, among which the antibacterial property is one of the most significant properties of lactic acid bacteria. Currently, there is no literature reporting that Lactiplantibacillus plantarum can inhibit multi-drug resistant Staphylococcus aureus. Summary of the Invention

[0004] The present invention isolates a Lactiplantibacillus plantarum (also known as: Lactobacillus plantarum) from fermented whey water in Dali, Yunnan. This strain has high yields of lactic acid and indole-3-lactic acid, and can also significantly inhibit the growth of various spoilage and pathogenic microorganisms, especially can significantly inhibit the growth of multi-drug resistant Staphylococcus aureus and molds, and can extend the shelf life of foods.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows: A Lactiplantibacillus plantarum with antibacterial effects ( Lactiplantibacillus plantarum ) named Lactobacillus plantarum C07 Lactiplantibacillus plantarum C07, with the deposit number of CCTCC NO: M20242086, deposited at the China Center for Type Culture Collection, the deposit time is September 26, 2024, and the deposit address is within Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The Lactiplantibacillus plantarum Lactiplantibacillus plantarum The 16S rDNA sequence of C07 is shown in SEQ ID NO.1. The Lactiplantibacillus plantarum Lactiplantibacillus plantarum The colony morphology of C07 observed by naked eyes is: round, semi-transparent, convex, with neat edges and moist milky white colonies on the surface; under an optical microscope, the strain cells are rod-shaped, do not produce spores, are Gram-positive, and show negative to catalase.

[0007] The present invention also provides the application of the Lactiplantibacillus plantarum in inhibiting spoilage and pathogenic microorganisms, and the application of inhibiting spoilage and pathogenic microorganisms is for non-therapeutic use; the spoilage and pathogenic microorganisms are one or more of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella and molds.

[0008] Preferably, the mold is Geotrichum candidum; the diameter of the inhibition zone of the supernatant of the Lactiplantibacillus plantarum against Geotrichum candidum reaches more than 35 mm.

[0009] As a further description of the above scheme: the Staphylococcus aureus is standard (non-drug-resistant) Staphylococcus aureus and / or multi-drug-resistant Staphylococcus aureus. Preferably, the Staphylococcus aureus is multi-drug-resistant Staphylococcus aureus, and the Lactiplantibacillus plantarum provided by the present invention has a significantly stronger inhibitory effect on multi-drug-resistant Staphylococcus aureus than other strains.

[0010] The present invention also provides the application of the Lactiplantibacillus plantarum in the production of lactic acid or indole-3-lactic acid. The strain provided by the present invention can produce high yields of lactic acid and indole-3-lactic acid, the lactic acid content can reach more than 100 mmol / L, and the indole-3-lactic acid content can reach more than 100 μmol / L.

[0011] The present invention also provides the application of the Lactiplantibacillus plantarum in fermented foods. Preferably, the fermented foods are dairy products, sausages or bacterial powders.

[0012] As a further preference, the dairy product is fermented milk or fermented milk cake, and the obtained fermented milk or fermented milk cake has an antibacterial effect; the viable count of the fermented milk is not less than 1×10 6 CFU / mL.

[0013] During the 24-hour storage and preservation of the fermented milk cake, first inject 1×10 6 CFU / mL of multi-drug-resistant Staphylococcus aureus DC.RB-015 into the milk. Using Lactobacillus bulgaricus, Streptococcus thermophilus and Lactiplantibacillus plantarum Lactiplantibacillus plantarum C07 as a starter to prepare a fermented milk cake (experimental group), without inoculating Lactiplantibacillus plantarum Lactiplantibacillus plantarumC07 was used as the control group. They were then stored at 4°C and 25°C respectively. The results showed that during the 24-hour storage period, there were more multidrug-resistant Staphylococcus aureus DC.RB-015 in the fermented milk cakes of the control group than in the experimental group. Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07 effectively reduced the growth of Staphylococcus aureus in the contaminated fermented milk cakes during storage, which is of great significance for developing a starter with the characteristic of producing antibacterial substances to replace the addition of chemical preservatives.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) A lactic acid bacterium C07 that produces lactic acid and indole-3-lactic acid was isolated and screened from the fermented milk whey samples in Dali, Yunnan. The lactic acid bacterium C07 was identified and the antibacterial substances were quantitatively analyzed through morphological identification, 16S rDNA gene sequencing, and targeted metabolomics. It belongs to Lactiplantibacillus plantarum , and is Lactiplantibacillus plantarum, which was named Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07.

[0015] (2) The Lactiplantibacillus plantarum provided by the present invention produces lactic acid and indole-3-lactic acid, which can inhibit bacteria. Lactic acid can regulate the pH value in fermented foods, inhibit the growth of miscellaneous bacteria, and improve the product quality.

[0016] (3) The Lactiplantibacillus plantarum C07 provided by the present invention Lactiplantibacillus plantarum C07 can inhibit the growth of spoilage and pathogenic microorganisms such as multidrug-resistant Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Salmonella, and molds. It can inhibit the growth of spoilage and pathogenic microorganisms during the storage of food, indicating that the strain can be used as an anti-corrosion microbial agent to extend the shelf life of food and is beneficial to maintaining the quality and quality of food.

[0017] (4) The Lactiplantibacillus plantarum C07 provided by the present invention Lactiplantibacillus plantarum C07 can effectively reduce the growth and biofilm formation of multidrug-resistant Staphylococcus aureus DC.RB-015 in milk cakes during the storage of fermented foods, and extend the storage period of fermented foods.

[0018] The microbial preservation information is as follows:

[0019] Taxonomic name: Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07;

[0020] Preservation number: CCTCC NO: M20242086;

[0021] Preservation unit: China Center for Type Culture Collection;

[0022] Preservation time: September 26, 2024;

[0023] Depository Address: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Description of the Drawings

[0024] Figure 1 It is a figure of the colony morphology (A), cell morphology (B), and 16S rDNA electrophoresis identification (C) of Lactiplantibacillus plantarum C07;

[0025] Figure 2 It is the influence of pH (A) and protease (B) on the antibacterial activity of the supernatant of Lactiplantibacillus plantarum C07;

[0026] Figure 3 It is the broad-spectrum antibacterial ability of the supernatant of Lactiplantibacillus plantarum C07. A - G are the inhibition zones of multi-drug resistant Staphylococcus aureus DC.RB-015, Staphylococcus aureus ATCC25923, Salmonella WX29, Escherichia coli CICC10389, Listeria monocytogenes CMCC21633, Pseudomonas aeruginosa ATCC27853, and Geotrichum candidum BNCC33594 respectively;

[0027] Figure 4 It is the determination of the minimum inhibitory concentration (MIC) and growth inhibition curve of the supernatant of Lactiplantibacillus plantarum C07 against Staphylococcus aureus. A is the MIC of the supernatant of Lactiplantibacillus plantarum C07 against two strains of Staphylococcus aureus, B is the growth inhibition curve of the supernatant of Lactiplantibacillus plantarum C07 against multi-drug resistant Staphylococcus aureus DC.RB-015, and C is the growth inhibition curve of the supernatant of Lactiplantibacillus plantarum C07 against Staphylococcus aureus ATCC25923;

[0028] Figure 5 It is the determination of the biofilm inhibition of different MICs of the supernatant of Lactiplantibacillus plantarum C07 against multi-drug resistant Staphylococcus aureus DC.RB-015. A is the crystal violet staining of the biofilm of Staphylococcus aureus DC.RB-015 with different MICs of the supernatant of Lactiplantibacillus plantarum C07, B is the inhibition rate of the biofilm of Staphylococcus aureus DC.RB-015 with different MICs of the supernatant of Lactiplantibacillus plantarum C07, C is the confocal laser scanning microscopy image of the biofilm of Staphylococcus aureus DC.RB-015 with PBS added, and D is the confocal laser scanning microscopy image of the biofilm of Staphylococcus aureus DC.RB-015 with 1 MIC of the supernatant of Lactiplantibacillus plantarum C07 added;

[0029] Figure 6 It is the chromatogram of the standard substance and the regression equation diagram of the antibacterial substance of Lactiplantibacillus plantarum C07. A is the mass spectrometry diagram of extracting indole-3-lactic acid and L-lactic acid by UHPLC-MRM-MS / MS, B is the calibration curve diagram of indole-3-lactic acid, and C is the calibration curve diagram of L-lactic acid;

[0030] Figure 7Application of Lactiplantibacillus plantarum C07 in fermented milk cakes; A is microbial control in fermented milk cakes for 24 h during storage at 4 °C, and B is microbial control in fermented milk cakes for 24 h during storage at 25 °C. Specific implementation manners

[0031] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0032] Example 1

[0033] In the present invention, multi-drug resistant Staphylococcus aureus DC.RB-015 was used as the indicator bacterium, and multiple lactic acid bacteria with antibacterial ability were selected from 105 strains by the microplate method. Then, using multi-drug resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 as the indicator bacteria, a lactic acid bacterium with the strongest antibacterial ability was re-screened by the punching method for the antibacterial ability of the supernatant of the lactic acid bacteria. Further, broad-spectrum antibacterial tests were carried out using 4 common bacteria in food contamination (Staphylococcus aureus, Escherichia coli, Salmonella, and Listeria monocytogenes) as the indicator bacteria. Only the fermentation supernatant of strain C07 had good antibacterial ability, and then it was identified as Lactiplantibacillus plantarum by morphology and 16S rDNA Lactiplantibacillus plantarum) , named Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07, and its antibacterial characteristics of the fermentation supernatant and the antibacterial ability of the bacterial cells were determined.

[0034] 1. Test strains

[0035] Lactic acid bacteria: All were isolated and preserved in the Key Laboratory of the College of Food Science and Technology, Yunnan Agricultural University.

[0036] Indicator bacteria: Staphylococcus aureus ( Staphyloccocus aureus ATCC25923) was purchased from the American Type Culture / Collection Center, and Listeria monocytogenes ( Listeria monocytogene CMCC21633) was provided by the School of Public Health, Dali University. Multi-drug resistant Staphylococcus aureus ( Staphyloccocus aureusDC.RB-015) was screened by Yunnan Agricultural University (reference: Prabakusuma, A.S.; Zhu, J.; Shi, Y.; Ma, Q.; Zhao, Q.; Yang, Z.; Xu, Y. Prevalence and antimicrobial resistance profiling of Staphylococcus aureus isolated from traditional cheese in Yunnan, China. 3Biotech 2021, 12, 1.), and it shows resistance to penicillin, oxacillin, erythromycin, clindamycin, tetracycline, and cefoxitin.

[0037] Salmonella( Salmonella WX29) was screened by Yunnan Agricultural University and identified as Salmonella through 16s rDNA gene sequence analysis.

[0038] Escherichia coli( Escherichia coli CICC10389) and Geotrichum candidum( Geotrichum candidum BNCC335994) were all purchased from China Center for Industrial Culture Collection.

[0039] 2. Formulas of culture media and reagents for experiments

[0040] MRS medium (1L): Glucose 20.0 g, peptone 10.0 g, beef extract 8.0 g, yeast extract 4.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, Tween 1 mL, magnesium sulfate 0.2 g, manganese sulfate 0.04 g. Add distilled water to 1L, pH 7.0; add 15.0 g of agar to make it a solid medium.

[0041] LB medium (1L): Yeast powder 5.0 g, tryptone 10.0 g, sodium chloride 10.0 g. Add distilled water to 1L, pH 7.0; add 20.0 g of agar to make it a solid medium.

[0042] TSB medium (1L): Tryptone 17.0 g, plant peptone 3.0 g, sodium chloride 5.0 g, dipotassium hydrogen phosphate 2.5, glucose 2.5 g, pH 7.0; add 20.0 g of agar to make it a solid medium.

[0043] PDA medium (1L): Peeled potatoes 200 g, glucose 20 g, and agar 15 g.

[0044] 3. Experimental methods

[0045] (1)Determination of antibacterial ability

[0046] Using the microplate method: Add 100 μL of the cell-free fermentation broth of lactic acid bacteria and 100 μL of a suspension of multi-drug resistant Staphylococcus aureus DC.RB-015 with a concentration of 10 8 CFU / mL as the indicator bacteria into each well of a 96-well plate. After culturing at 37 °C for 12 h, measure the absorbance at 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The antibacterial activity of lactic acid bacteria is expressed as the inhibition rate against the indicator bacteria, multi-drug resistant Staphylococcus aureus DC.RB-015.

[0047]

[0048] Well punching method: Pour 30 mL of LB solid medium into each petri dish, blow it with sterile air for 2 h in a sterile operation bench, quickly and evenly spread 180 μL of the indicator bacteria on the petri dish, use a sterile well punch to press out holes with a diameter of 7 mm on the petri dish, take them out with forceps, add 200 μL of the sample solution to be tested, put it in an incubator at 37 °C for 10 h, then take it out to observe and measure the antibacterial ability, and judge the strength of the antibacterial ability of the sample solution to be tested according to the size of the inhibition zone.

[0049] Diameter of inhibition zone (mm) = directly measured diameter of the zone;

[0050] Coating method: When measuring the antibacterial properties of the fermentation supernatant and bacterial cells in this invention, a typical food spoilage bacterium, Staphylococcus aureus, is selected as the indicator bacteria. After activating the indicator bacteria, inoculate them into LB liquid medium at an inoculation amount of 1%, and culture them on a shaker at 37 °C for 12 h. Gradient dilute the cultured indicator bacteria solution as the stock solution and perform viable cell counting. Use the well punching method to conduct the antibacterial experiment. Determine the optimal concentration of the indicator bacteria for the antibacterial experiment according to the diameter of the inhibition zone and the neatness of the edge.

[0051] Double-layer plate streaking method: Draw two 3-cm parallel lines in the center of the MRS solid medium with the activated lactic acid bacteria strain, and culture it at 37 °C for 48 h. Inoculate the mold spore suspension into the PDA medium cooled to about 50 °C at an inoculation amount of 1% (V / V), take 5 mL of the PDA medium containing the mold spore suspension and pour it on the upper layer of the lactic acid bacteria plate inoculated with 2-cm parallel lines. After it solidifies, culture it at 30 °C for 48 h. Set three parallel experiments for each group, and evaluate the antibacterial effect according to the size of the inhibition zone diameter around the lactic acid bacteria in each group.

[0052] Table 1 Determination of the concentration of the indicator bacteria solution

[0053]

[0054] When the dilution factor reaches 1000, take 180 μL of the indicator bacteria for coating. After diffusion culture, the thickness of the bacterial moss in the inhibition zone is moderate and the size of the inhibition zone is moderate. At this time, the number of indicator bacteria coated on the plate is about 106 CFU / mL. Therefore, in this invention, the concentration of the indicator bacteria for the antibacterial experiment is fixed as follows: the concentration of bacteria such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella, and molds is 10 6 CFU / mL.

[0055] Primary screening of lactic acid bacteria with antibacterial ability

[0056] 105 strains of lactic acid bacteria preserved were inoculated into MRS liquid medium at an inoculation amount of 2%, cultured at 37°C for 24 h, and activated for three generations. Centrifuged at 8000 r / min at 4°C for 15 min, filtered through a 0.22 μL filter membrane, and the supernatant was taken for standby. Using multidrug-resistant Staphylococcus aureus DC.RB-015 as the indicator bacteria, the antibacterial ability was detected by the microplate method. In this invention, 105 strains of lactic acid bacteria preserved in the laboratory were used for primary screening, and the results showed that a total of 7 strains of lactic acid bacteria had an antibacterial effect on the indicator bacteria greater than 50%.

[0057] Rescreening of lactic acid bacteria with antibacterial ability

[0058] The lactic acid bacteria with stronger antibacterial activity screened in the primary screening were inoculated into MRS liquid medium at an inoculation amount of 2%, cultured at 37°C for 24 h, and activated for three generations. Centrifuged at 8000 r / min at 4°C for 15 min, filtered through a 0.22 μL filter membrane, and the supernatant was taken. The antibacterial diameters of the supernatant against multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 were measured by the punching method respectively.

[0059] The antibacterial circle method is a way to measure the bactericidal effect, mainly used to determine the antibacterial effect, and is a qualitative or semi-quantitative method. By measuring the antibacterial ability of lactic acid bacteria against multidrug-resistant Staphylococcus aureus DC.RB-015 and the standard quality control strain Staphylococcus aureus ATCC25923 through the antibacterial circle measurement, it can be seen from Table 2 that the antibacterial ability of strain C07 is the strongest, and strain C07 has the best inhibitory ability against multidrug-resistant Staphylococcus aureus.

[0060] Table 2 Rescreening of lactic acid bacteria with antibacterial ability

[0061]

[0062] Note: Antibacterial circle diameter (mm)

[0063] (4) Classification and identification of lactic acid bacteria with antibacterial ability

[0064] ① Strain morphological identification

[0065] Strain 07 isolated from fermented whey water in Dali, Yunnan was observed for colony morphology and Gram stained. 3% hydrogen peroxide solution was added to the colony, and if bubbles were produced within 30 s, it indicated that the catalase test result was positive.

[0066] Strain C07 grew well on the MRS solid medium plate. (As shown in Figure 1 A), the microscopic morphology of the strain was spherical (1-2 μm in diameter), milky white in color, viscous in texture, with typical growth characteristics of lactic acid bacteria; without flagella, Gram-positive staining under the microscope, and no bubbles were produced by catalase test (as shown in Figure 1 B).

[0067] ② 16S rDNA identification

[0068] PCR amplification of the 16S rDNA gene was carried out using genomic DNA as a template. The PCR amplification used 27F (the sequence is shown in SEQ ID No. 1, 5’-AGTTTGATCMTGGCTCAG-3’) and 1492R (the sequence is shown in SEQ ID No. 2; 5’-GGTTACCTTGTTACGACTT-3’) as primers. The PCR reaction conditions were initial denaturation at 98 °C for 2 min, followed by 35 amplification cycles, including denaturation at 98 °C for 10 s, annealing at 56 °C for 10 s, and extension at 72 °C for 10 s. The final extension step was carried out at 72 °C for 5 min. The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 minutes.

[0069] PCR amplification using the strain genomic DNA as a template yielded a PCR fragment of approximately 1500 bp (the 16S rDNA nucleotide sequence is shown in SEQ ID No. 3). The result of the amplified fragment on 2% agarose gel electrophoresis is shown in Figure 1 C. The PCR product was sent to Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing result (No. NR_042057.1: 82-1487) was input into the online alignment at www.NCBI.nlm.nih.gov. The homology of strain C07 with Lactiplantibacillus plantarum was 98.56%. The strain was identified as Lactiplantibacillus plantarum and named Lactiplantibacillus plantarum C07 Lactiplantibacillus plantarum C07, and it was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M20242086.

[0070] Example 2 Determination of the antibacterial properties of the supernatant of Lactiplantibacillus plantarum C07

[0071] Effects of pH and protease treatment on the antibacterial activity of the supernatant of Lactiplantibacillus plantarum C07

[0072] Effect of pH on the antibacterial activity of the supernatant of Lactiplantibacillus plantarum C07: The pH of the supernatant of Lactiplantibacillus plantarum C07 cultured for 24 h was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 with 1 mol / L NaOH and HCl solutions, and then the antibacterial activity was determined by the spread plate method.

[0073] Effect of protease on the antibacterial activity of the supernatant of Lactiplantibacillus plantarum C07: Pepsin, trypsin, and proteinase K were added to the supernatant of C07 cultured for 24 hours at a final concentration of 1 mg / mL, and the mixture was incubated in a constant temperature water bath at 37 °C for 2 h, and then the enzyme was inactivated at 80 °C for 10 min. Then the samples of each group were adjusted to the original pH of 6.0 - 6.2, and the antibacterial activity against multidrug-resistant Staphylococcus aureus DC.RB-015 was determined by the spread plate method.

[0074] When the pH value of the supernatant of Lactiplantibacillus plantarum C07 was 3, the antibacterial activities against multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 were the highest, and the inhibition diameters were 26.15 ± 0.06 mm and 29.62 ± 0.17 mm, respectively; as the pH gradually increased, the antibacterial activity of the supernatant of Lactiplantibacillus plantarum C07 decreased. When the pH value was 5, the antibacterial activities of the supernatant of Lactiplantibacillus plantarum C07 against the two strains of Staphylococcus aureus decreased to 0% (as Figure 2 A). Proteinase K, pepsin, and trypsin were used to treat the supernatant of Lactiplantibacillus plantarum C07 to determine whether the antibacterial substance was a peptide. The results proved that CFS (supernatant) was not sensitive to all proteases except pepsin (as Figure 2 B). Therefore, it is speculated that the antibacterial substances produced by Lactiplantibacillus plantarum C07 may be the antibacterial effects of organic acids and bacteriocins.

[0075] Determination of the broad-spectrum antibacterial activity of Lactiplantibacillus plantarum C07

[0076] After activating multidrug-resistant Staphylococcus aureus DC.RB-015, Staphylococcus aureus ATCC25923, Listeria monocytogenes CMCC21633, Escherichia coli CICC10389, Salmonella WX29, Pseudomonas aeruginosa ATCC27853, and Geotrichum candidum BNCC33594 as target strains, the inhibition zone diameters of the supernatant of C07 were measured by the spread plate method and the well diffusion method.

[0077] The inhibition zones of Lactiplantibacillus plantarum C07 against different Gram-positive and negative bacteria, such as Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Gram-negative Salmonella, Pseudomonas aeruginosa, and Geotrichum candidum, which are foodborne pathogens Figure 3As shown, the diameters of the inhibition zones of the supernatant of Lactiplantibacillus plantarum C07 against multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 were 19.83±0.91 and 20.58±0.36 mm, respectively. The inhibition diameters against Escherichia coli CICC10389, Listeria monocytogenes CMCC21633, Salmonella WX29, and Pseudomonas aeruginosa ATCC27853 were 14.26±0.23 mm, 18.16±0.25 mm, 19.94±0.20 mm, and 18.16±0.25 mm, respectively. The inhibition zone against Geotrichum candidum BNCC33594 was elliptical, with the major and minor axes being (40 mm, 35 mm). It can be seen that Lactiplantibacillus plantarum C07 had the strongest inhibitory ability against Geotrichum candidum. There were significant differences between the control groups PBS and MRS (P<0.001 for both). These results indicate that Lactiplantibacillus plantarum C07 has broad-spectrum antibacterial activity and can inhibit foodborne pathogens.

[0078] Determination of the minimum inhibitory concentration (MIC) and growth inhibition curve of the C07 supernatant against Staphylococcus aureus

[0079] The activated C07 strain bacterial suspension of the third generation was centrifuged at 8000 r / min at 4°C for 15 min, filtered through a 0.22 μm filter membrane, the supernatant was collected, and freeze-dried for 3 - 4 days to form a freeze-dried powder. The MIC and antibacterial growth curve of the supernatant were determined by the two-fold dilution method and the microplate method, respectively.

[0080] As Figure 4 shown in A, the higher the concentration of the freeze-dried powder of the C07 supernatant, the smaller the OD600nm, indicating a stronger antibacterial effect against Staphylococcus aureus. When the final mass concentration of the freeze-dried powder of the supernatant was 3.5 mg / mL and 3 mg / mL, the growth of multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 was almost completely inhibited. Thus, the MIC of the freeze-dried powder of the C07 supernatant against multidrug-resistant Staphylococcus aureus DC.RB-015 was determined to be 3.5 mg / mL, and the MIC against Staphylococcus aureus ATCC25923 was 3 mg / mL. As Figure 4 shown in B and 4C, compared with the control group (PBS), the freeze-dried powder of the supernatant of Lactiplantibacillus plantarum C07 in the experimental groups (1 / 2MIC, 1MIC, and 2MIC) inhibited the growth of the two strains of Staphylococcus aureus to varying degrees during 24 h of growth. The Staphylococcus aureus in the control group remained on the typical microbial growth curve. Among the two strains of Staphylococcus aureus after treatment, 1MIC could inhibit the growth of the two strains of Staphylococcus aureus. 1 / 2MIC reduced the viability of the two strains of Staphylococcus aureus and delayed the logarithmic phase.

[0081] Minimum inhibitory assay of supernatant of Lactobacillus plantarum C07 against biofilm of multidrug-resistant Staphylococcus aureus DC.RB-015

[0082] The crystal violet quantitative analysis method was used to analyze about 10 6 CFU / mL suspensions of multidrug-resistant Staphylococcus aureus DC.RB-015 and different concentrations of supernatant of Lactobacillus plantarum C07 at 2×MIC, 1×MIC, 1 / 2×MIC and blank control groups were added to each well of a 96-well polystyrene plate and incubated at 37°C for 24 h. After biofilm formation, sterile PBS was added to each well to remove planktonic cells, and methanol was added for 15 min to fix the biofilm. The fixed biofilm was stained with 1% crystal violet solution for 15 min. Excess stain was removed, the wells were rinsed twice with sterile water, and crystal violet was dissolved in 200 μL of 33% glacial acetic acid to calculate the biofilm inhibition rate.

[0083]

[0084] Figure 5 A through OD 600 The values ​​and crystal violet staining images intuitively demonstrate the inhibitory effect of different concentrations of Lactobacillus plantarum C07 supernatant freeze-dried powder on the growth of Staphylococcus aureus. Figure 5 B shows that the biofilm formation of Staphylococcus aureus was inhibited by >90% at 2×MIC concentration, 66.90% inhibition was observed at 1×MIC concentration, and 26.40% activity was observed at 1 / 2×MIC concentration, which was used for further biofilm-related studies. In the laser confocal images, compared with the untreated control ( Figure 5 C), the biofilm formed on the treated glass slide is poorly visualized ( Figure 5 D). COMSTAT software analysis showed that the observed images showed that the biofilm thickness of the culture treated with 1×MIC Lactobacillus plantarum C07 (14.5±0.3μm) was significantly different from that of the untreated culture (42.94±1.8μm). Among them, the biofilm thickness was reduced by 66.23% (p<0.05).

[0085] Example 3 Determination of the ability of Lactobacillus plantarum C07 to produce L-lactic acid and indole-3-lactic acid

[0086] Determination of specific antibacterial substances using targeted metabolomics

[0087] The Lactiplantibacillus plantarum C07 seed solution activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), cultured at 37 °C for 24 h, centrifuged at 8000 rpm and 4 °C for 15 min, filtered through a sterile 0.22 μm filter to obtain a cell-free supernatant, and a Waters ACQUITY H-class plus UPLC System ultra-high performance liquid chromatograph was used to perform chromatographic separation of the target compound through a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) liquid chromatography column. The mobile phase A was 0.1% formic acid solution, and the mobile phase B was methanol. The flow rate of the mobile phase was 300 μL / min, the column oven temperature was 35 °C, the sample tray temperature was 10 °C, and the injection volume was 1 μL for determination.

[0088] The extracted ion chromatograms (EICs) of L-lactic acid and indole-3-lactic acid are as Figure 6 shown in A. It can be seen from this that good chromatographic peaks and good chromatographic separation can be obtained for the target compounds in the experimental samples; the linear coefficients (R 2 ) of all target compounds are greater than 0.9969, indicating a good linear correlation between the chromatographic peak area and the compound concentration. Quantitative analysis was carried out according to the calibration curve calculation formula of the standard product. ( Figure 6 B) The calculation formula for L-lactic acid in the supernatant: y = 846.28x + 18547.3 (R 2 = 0.998168), (x, unit: μmol / L). ( Figure 6 C) The calculation formula for indole-3-lactic acid in the supernatant: y = 53951.4x + 194.37, (R 2 = 0.999178) (x, unit: μmol / L). As shown in Table 3, the content of L-lactic acid in Lactiplantibacillus plantarum C07 is 100818.61 ± 0.4 μmol / L, and the content of indole-3-lactic acid is: 119.25 ± 0.4 μmol / L.

[0089] Table 3

[0090]

[0091] Example 4 Application of Lactiplantibacillus plantarum in fermented foods

[0092] Lactobacillus bulgaricus: Streptococcus thermophilus: Lactiplantibacillus plantarum C07 at a ratio of 1:1:1 was used as the experimental group, and a starter culture without Lactiplantibacillus plantarum C07 (with the same ratio of Lactobacillus bulgaricus and Streptococcus thermophilus) was used as the control group. After inoculating the pasteurized milk in a sterile environment in the experimental group, 100 μL of 10 6 CFU / mL of multidrug-resistant Staphylococcus aureus DC.RB-015 was added to a test tube containing 5 mL of milk, and co-fermented at 37 °C for 5 hours until the pH value dropped to 4.7 - 5.0. Then the curdling temperature was raised to 60 °C - 70 °C and maintained for 25 - 30 min to remove whey. The experimental group and control group samples were treated at 4 °C and 25 °C respectively. At 0, 6, 12, 18, and 24 hours, 1 mL of sample was collected and cultured on LB agar, incubated at 37 °C for 24 hours, and the viable cell count (expressed as log CFU / mL) was recorded. All experiments were repeated three times.

[0093] As Figure 7 shown, during the 24-hour storage period, after continuous incubation at 4 °C and 25 °C, the viable cell count in the fermented milk cake samples of the experimental group was generally lower than that of the control group. Among them, the average decrease was 0.69 ± 0.27 log CFU / mL under 4 °C storage and 1.32 ± 1.95 log CFU / mL under 25 °C storage. In summary, the treatment with inoculation of Lactiplantibacillus plantarum C07 can significantly reduce the count of live Gram-positive foodborne pathogens in fermented milk cakes. It shows that Lactiplantibacillus plantarum C07 can be used as a preservative starter culture in the production of fermented milk, thereby reducing the economic losses of dairy products. The present invention ferments yogurt by the same method and also has similar technical effects.

[0094] It should be understood that the above specific embodiments of the present invention are only used for exemplary illustration or explanation of the principle of the present invention, and do not constitute a limitation to the present invention. Therefore, any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and scope of the present invention shall be included within the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modification examples falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A strain of Lactobacillus plantarum with antibacterial effect ( Lactiplantibacillus plantarum ), characterized in that, The plant lactobacillus is named plant lactobacillus ( Lactiplantibacillus plantarum ) C07, the deposit number is CCTCC NO: M20242086, the depositor is China Center for Type Culture Collection, the deposit time is September 26, 2024, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A use of plant lactobacillus according to claim 1 in inhibiting corruption and pathogenic microorganisms, characterized in that: The application of inhibiting spoilage and pathogenic microorganisms is for non-therapeutic purposes; the spoilage and pathogenic microorganisms are one or more of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella and Geotrichum candidum; the Staphylococcus aureus is standard Staphylococcus aureus and / or multidrug-resistant Staphylococcus aureus.

3. Use of the plant lactobacillus according to claim 1 in preparing fermented food.

4. The use of plant lactobacillus in preparing fermented food according to claim 3, characterized in that: The fermented food is dairy products, sausages or bacterial powder.

5. The use of plant lactobacillus in preparing fermented food according to claim 4, characterized in that: The dairy product is fermented milk or fermented milk cake.

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