Phytobacterium plantarum with bacteriostatic effect and application thereof

Abstract

The invention discloses a plant lactobacillus with a bacteriostatic effect and application of the plant lactobacillus. The lactobacillus plantarum is named as lactobacillus plantarum C07, the preservation number is CCTCC NO: M20242086, the preservation unit is China Center for Type Culture Collection, the preservation time is September 26, 2024, and the preservation address is Wuhan University, Wuhan University, No. 299, eight road, Wuchang District, Wuhan City, Hubei Province. The strain can produce lactic acid and indole-3-lactic acid at high yield, has a very strong antibacterial effect on multiple putrefying and pathogenic microorganisms such as staphylococcus aureus, escherichia coli, listeria monocytogenes, pseudomonas aeruginosa, salmonella and mould, and is a broad-spectrum antibacterial microorganism.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus plantarum with antibacterial effect and application thereof. Background Art

[0002] Staphylococcus aureus is a widely distributed Gram-positive food poisoning pathogen and a common contaminant in the dairy industry worldwide. The proportion of multidrug-resistant Staphylococcus aureus (resistant to multiple antibiotics) isolated from dairy products is as high as over 60%, especially in milk and cheese, which are prone to become reservoirs of multidrug-resistant Staphylococcus aureus strains. Currently, the most commonly used method for the prevention and treatment of Staphylococcus aureus is the use of antibiotics. However, it has been widely recognized that the use of antibiotics can lead to the resistance of Staphylococcus aureus to antibiotics such as vancomycin, sulfamethoxazole, and ceftazidime. At the same time, excessive use of antibiotics can induce and accelerate the development of bacterial resistance to antibiotics, promote the spread of resistant bacteria to humans, and form super bacteria. In addition, antibiotic residues may affect the fermentation process of dairy products, resulting in a decline in product quality, thereby causing economic losses to the dairy industry.

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

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

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows: a strain of Lactobacillus plantarum with antibacterial effect ( Lactiplantibacillus plantarum ), the plant lactobacillus was named as plant lactobacillus C07 Lactiplantibacillus plantarum C07, the preservation number is CCTCC NO: M20242086, the preservation unit is China Type Culture Collection, the preservation time is September 26, 2024, and the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] Lactobacillus plantarum Lactiplantibacillus plantarum The 16S rDNA sequence of C07 is shown in SEQ ID NO. 1. Lactiplantibacillus plantarum The colony morphology of C07 observed with the naked eye was: round, translucent, convex, milky white colonies with neat edges and moist surfaces; under an optical microscope, the strain cells were rod-shaped, non-spore-forming, Gram-positive, and negative for catalase.

[0007] The present invention also provides the use of the plant lactobacillus in inhibiting spoilage and pathogenic microorganisms, wherein the use of the plant lactobacillus in inhibiting spoilage and pathogenic microorganisms is used for non-therapeutic purposes; the spoilage and pathogenic microorganisms are one or more of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella and mold.

[0008] Preferably, the mold is Geotrichum candidum; and the diameter of the inhibition zone of the supernatant of Lactobacillus plantarum against Geotrichum candidum is greater than 35 mm.

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

[0010] The present invention also provides the application of the plant lactobacillus in producing lactic acid or indole-3-lactic acid. The strain provided by the present invention can produce high amounts of lactic acid and indole-3-lactic acid, with the lactic acid content reaching more than 100 mmol / L and the indole-3-lactic acid content reaching more than 100 μmol / L.

[0011] The present invention also provides the use of the plant lactobacillus in fermented food. Preferably, the fermented food is dairy products, sausages or bacterial powder.

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

[0013] During the 24-hour storage and preservation of fermented milk cakes, the number of viable bacteria was first increased to 1×10 6 CFU / mL of multidrug-resistant Staphylococcus aureus DC.RB-015 was injected into milk. Lactiplantibacillus plantarum C07 was used as the fermentation agent to prepare the fermented milk cake (experimental group), and the fermented milk cake was not inoculated with Lactobacillus plantarum. Lactiplantibacillus plantarumC07 was the control group. The samples were then stored at 4°C and 25°C. The results showed that during the 24-hour storage period, the number of multidrug-resistant Staphylococcus aureus DC.RB-015 in the fermented milk cakes in the control group was higher than that in the experimental group. Lactiplantibacillus plantarum C07 effectively reduced the growth of Staphylococcus aureus in contaminated fermented milk cakes during storage, which is of great significance for developing a starter culture with the property 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) The present invention isolates and screens a lactic acid bacterium C07 that produces lactic acid and indole-3-lactic acid from a fermented milk sample in Dali, Yunnan. The lactic acid bacterium C07 is identified and its antibacterial substances are quantitatively analyzed by morphological identification, 16SrDNA gene sequencing, and targeted metabolomics. Lactiplantibacillus plantarum , and named it Lactobacillus plantarum C07 Lactiplantibacillus plantarum C07.

[0015] (2) The lactic acid and indole-3-lactic acid produced by the plant lactobacillus provided by the present invention can inhibit bacteria. Lactic acid can adjust the pH value in fermented foods, inhibit the growth of miscellaneous bacteria, and improve product quality.

[0016] (3) Lactobacillus 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 mold. It can inhibit the growth of spoilage and pathogenic microorganisms during food storage, indicating that the strain can be used as a preservative microbial agent to extend the shelf life of food and help maintain the quality and quality of food.

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

[0018] The microbial deposit information is as follows: Classification name: Lactobacillus plantarum C07 Lactiplantibacillus plantarum C07; Deposit number: CCTCC NO: M20242086; Depository: China Center for Type Culture Collection; Deposit date: September 26, 2024; Storage address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figures showing the colony morphology (A), bacterial morphology (B), and 16SrDNA electrophoresis identification (C) of Lactobacillus plantarum C07; Figure 2 The effects of pH (A) and protease (B) on the antibacterial activity of the supernatant of Lactobacillus plantarum C07; Figure 3 is the broad-spectrum antimicrobial ability of the supernatant of Lactobacillus plantarum C07, and AG are the inhibition zones of multidrug-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; Figure 4 The minimum inhibitory concentration (MIC) of the supernatant of Lactobacillus plantarum C07 against Staphylococcus aureus was determined by inhibition growth curve; A is the MIC of the supernatant of Lactobacillus plantarum C07 against two strains of Staphylococcus aureus, B is the inhibition growth curve of the supernatant of Lactobacillus plantarum C07 against multidrug-resistant Staphylococcus aureus DC.RB-015, and C is the inhibition growth curve of the supernatant of Lactobacillus plantarum C07 against Staphylococcus aureus ATCC25923; Figure 5 The figure shows the inhibition test of different MICs of Lactobacillus plantarum C07 supernatant on multidrug-resistant Staphylococcus aureus DC.RB-015 biofilm; A shows crystal violet staining of different MICs of Lactobacillus plantarum C07 supernatant on Staphylococcus aureus DC.RB-015 biofilm, B shows the inhibition rate of different MICs of Lactobacillus plantarum C07 supernatant on Staphylococcus aureus DC.RB-015 biofilm, C shows the laser confocal image of Staphylococcus aureus DC.RB-015 biofilm with the addition of PBS, and D shows the laser confocal image of Staphylococcus aureus DC.RB-015 biofilm with the addition of 1 MIC of Lactobacillus plantarum C07 supernatant; Figure 6 A is a standard chromatogram and regression equation diagram of the antibacterial substance of Lactobacillus plantarum C07; A is a mass spectrum of indole-3-lactic acid and L-lactic acid extracted by UHPLC-MRM-MS / MS, B is a calibration curve diagram of U indole-3-lactic acid, and C is a calibration curve diagram of L-lactic acid; Figure 7 It is the application of Lactobacillus plantarum C07 in fermented milk cake; A is the microbial control of fermented milk cake stored at 4°C for 24 hours, and B is the microbial control of fermented milk cake stored at 25°C for 24 hours. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is 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 solution.

[0021] Example 1 The present invention uses multidrug-resistant Staphylococcus aureus DC.RB-015 as an indicator bacteria, and selects multiple lactic acid bacteria with antibacterial ability from 105 strains by the microtiter plate method. Then, using multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 as indicator bacteria, the antibacterial ability of the lactic acid bacteria supernatant is screened by the punching method to select a lactic acid bacteria with the strongest antibacterial ability. Further, a broad-spectrum antibacterial test was performed using four common bacteria in food contamination (Staphylococcus aureus, Escherichia coli, Salmonella and Listeria monocytogenes) as indicator bacteria. Only the fermentation supernatant of strain C07 has good antibacterial ability, and is then identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum) , named Lactobacillus plantarum C07 Lactiplantibacillus plantarum C07, and the antibacterial properties of its fermentation supernatant and the antibacterial ability of bacterial cells were determined.

[0022] 1. Test strains Lactic acid bacteria: All were isolated and preserved by the Key Laboratory of the College of Food Science and Technology of Yunnan Agricultural University.

[0023] Indicator bacteria: Staphylococcus aureus ( Staphyloccocus aureus ATCC25923) was purchased from the American Type Cell / Strain Collection Center, Listeria monocytogenes ( Listeria monocytogene CMCC21633) was provided by the School of Public Health of Dali University, multidrug-resistant Staphylococcus aureus ( Staphyloccocus aureus DC.RB-015) was screened by Yunnan Agricultural University (reference Prabakusuma, AS; 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.), showing resistance to penicillin, oxacillin, erythromycin, clindamycin, tetracycline and cefoxitin.

[0024] salmonella( Salmonella WX29) was screened by Yunnan Agricultural University and identified as Salmonella by 16s rDNA gene sequence analysis.

[0025] Escherichia coli ( Escherichia coli CICC10389) and Geotrichum candidum ( Geotrichum candidum BNCC335994) were purchased from China Industrial Microbiology Culture Collection Center.

[0026] 2. Experimental culture medium and reagent formulation MRS medium (1L): 20.0g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 1mL Tween, 0.2g magnesium sulfate, 0.04g manganese sulfate, add distilled water to 1L, pH 7.0; add 15.0g agar as a solid culture medium.

[0027] LB medium (1L): 5.0g yeast powder, 10.0g tryptone, 10.0g sodium chloride, add distilled water to 1L, pH 7.0; add 20.0g agar as solid culture medium.

[0028] TSB medium (1L): 17.0 g tryptone, 3.0 g phytone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, pH 7.0; add 20.0 g agar as a solid medium.

[0029] PDA medium (1 L): 200 g peeled potatoes, 20 g glucose and 15 g agar.

[0030] 3. Test methods (1) Determination of antibacterial ability Microplate method: 100 μL of lactic acid bacteria sterile fermentation broth and 100 μL of 10% lactic acid were added to each well of a 96-well plate. 8 CFU / mL of multidrug-resistant Staphylococcus aureus DC.RB-015 suspension was used as indicator bacteria. After culturing at 37°C for 12 hours, the absorbance was measured at 600 nm on a microplate reader. The antibacterial activity of lactic acid bacteria was expressed by its antibacterial rate against the indicator bacteria multidrug-resistant Staphylococcus aureus DC.RB-015.

[0031]

[0032] Punching method: Pour 30mL LB solid culture medium into each plate, blow with sterile air for 2h in a sterile operating table, quickly and evenly spread 180μL indicator bacteria on the plate, use a sterile puncher to press a hole with a diameter of 7mm on the plate, take it out with tweezers, add 200μL of the test solution, put it in a 37℃ incubator and culture it for 10h, then take it out for observation and measure the antibacterial ability, and judge the antibacterial ability of the test solution according to the size of the inhibition zone.

[0033] Inhibition zone diameter (mm) = directly measured zone diameter; Spreading method: When determining the antibacterial properties of fermentation supernatant and bacteria, the present invention uses Staphylococcus aureus, a typical food spoilage bacterium, as an indicator bacterium. After the indicator bacteria are activated, they are inoculated into LB liquid culture medium at a 1% inoculation rate and cultured on a shaking table at 37°C for 12 hours. The cultured indicator bacteria solution is used as the stock solution for gradient dilution, and the live bacteria are counted. The antibacterial experiment is performed using the punching method. The optimal concentration of the indicator bacteria for the antibacterial experiment is determined based on the diameter of the antibacterial circle and the neatness of the edge.

[0034] Double-layer plate streak method: Draw two 3 cm parallel lines in the center of the MRS solid culture medium with the activated lactic acid bacteria strain and culture at 37°C for 48 h. Inoculate the mold spore suspension with a 1% (V / V) inoculation amount into the PDA culture medium cooled to about 50°C, take 5 mL of the PDA culture 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 up three parallel experiments in each group, and evaluate the antibacterial effect according to the diameter of the antibacterial zone around the lactic acid bacteria in each group.

[0035] Table 1 Determination of indicator bacteria solution concentration

[0036] When the dilution factor reaches 1000, 180 μL of indicator bacteria is applied. After diffusion culture, the thickness of the inhibition zone is moderate and the size of the inhibition zone is moderate. At this time, the number of indicator bacteria applied on the plate is about 10 6 Therefore, the concentration of indicator bacteria in the antibacterial experiment of the present invention is fixed as follows: the concentration of Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Salmonella and mold is 10 6 CFU / mL.

[0037] Initial screening of lactic acid bacteria with antibacterial ability The 105 preserved lactic acid bacteria were inoculated with MRS liquid culture medium at a 2% inoculation rate, cultured at 37°C for 24h, and activated for three generations. 8000r / min, 4°C centrifuged for 15min, filtered with a 0.22μL filter membrane, and the supernatant was taken for use. Multidrug-resistant Staphylococcus aureus DC.RB-015 was used as the indicator bacteria, and the antibacterial ability was detected by the microtiter plate method. The present invention used 105 lactic acid bacteria preserved in the laboratory for preliminary screening, and the results showed that a total of 7 lactic acid bacteria had an antibacterial result of greater than 50% on the indicator bacteria.

[0038] Rescreening of lactic acid bacteria with antibacterial ability The lactic acid bacteria with strong antibacterial activity in the initial screening were inoculated into MRS liquid culture medium at a 2% inoculation rate and cultured at 37℃ for 24h to activate the third generation. The culture was centrifuged at 8000r / min and 4℃ for 15min, filtered with a 0.22μL filter membrane, and the supernatant was taken. The antibacterial diameter of the supernatant against multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 was determined by the punching method.

[0039] The inhibition zone method is a way to measure the bactericidal effect. It is mainly used to determine the antibacterial effect. It is a qualitative or semi-quantitative method. The inhibition zone determination was used to determine the antibacterial ability of lactic acid bacteria against multidrug-resistant Staphylococcus aureus DC.RB-015 and the standard quality control strain Staphylococcus aureus ATCC25923. It can be seen from Table 2 that strain C07 has the strongest antibacterial ability, and strain C07 has the best inhibitory ability against multidrug-resistant Staphylococcus aureus.

[0040] Table 2 Rescreening of lactic acid bacteria with antibacterial ability

[0041] Note: Diameter of inhibition zone (mm) (4) Classification and identification of lactic acid bacteria with antibacterial ability ①Strain morphological identification Strain 07 was isolated from fermented whey water in Dali, Yunnan, and its colony morphology was observed and Gram staining was performed. 3% hydrogen peroxide solution was added to the colony. If bubbles were generated within 30 seconds, the catalase test result was positive.

[0042] Strain C07 grows well on MRS solid medium plates. Figure 1 As shown in A), the strain has a spherical shape (1-2 μm in diameter), is milky white in color, has a viscous texture, and has typical growth characteristics of lactic acid bacteria; it has no flagella, is Gram-positive under a microscope, and catalase shows no bubble production. (e.g. Figure 1 B).

[0043] ②16S rDNA identification PCR amplification of 16SrDNA gene was performed using genomic DNA as template. PCR amplification used 27F (sequence as shown in SEQ ID No.1, 5'-AGTTTGATCMTGGCTCAG-3') and 1492R (sequence as 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 performed 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.

[0044] The genomic DNA of the strain was used as a template to amplify a PCR fragment of about 1500 bp (the nucleotide sequence of 16SrDNA is shown in SEQ ID No. 3). The results of electrophoresis of the amplified fragment on 2% agarose gel were as follows: Figure 1 C. The PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results (No.NR_042057.1:82-1487) were entered into www.NCBI.nlm.nih.gov for online comparison. Lactiplantibacillus plantarum The homology was 98.56%, and the strain was identified as Lactobacillus plantarum and named Lactobacillus plantarum C07. Lactiplantibacillus plantarum C07 and deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20242086.

[0045] Example 2 Determination of antibacterial properties of Lactobacillus plantarum C07 supernatant Effects of pH and protease treatment on the antibacterial activity of supernatant of Lactobacillus plantarum C07 Effect of pH on the antibacterial activity of the supernatant of Lactobacillus plantarum C07: The pH of the supernatant of Lactobacillus plantarum C07 cultured for 24 h was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 using 1 mol / L NaOH and HCl solutions, and then the antibacterial activity was determined by the coating method.

[0046] Effect of protease on the antibacterial activity of Lactobacillus plantarum C07 supernatant: Pepsin, trypsin and proteinase K were added to the C07 supernatant after culturing for 24 hours at a final concentration of 1 mg / mL, mixed, kept in a constant temperature water bath at 37°C for 2 hours, and then inactivated at 80°C for 10 minutes. The samples in each group were then 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 coating method.

[0047] When the pH value of the supernatant of Lactobacillus plantarum C07 was 3, the antibacterial activity against multidrug-resistant Staphylococcus aureus DC.RB-015 and Staphylococcus aureus ATCC25923 was 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 Lactobacillus plantarum C07 decreased. When the pH value was 5, the antibacterial activity of the supernatant of Lactobacillus plantarum C07 against the two strains of Staphylococcus aureus decreased to 0% (as shown in Table 1). Figure 2 A). The supernatant of Lactobacillus plantarum C07 was treated with proteinase K, pepsin and trypsin to determine whether the antimicrobial active substance is a peptide. The results showed that CFS (supernatant) was insensitive to all proteases except pepsin (e.g. Figure 2 B). Therefore, it is speculated that the antibacterial substances produced by Lactobacillus plantarum C07 may be the antibacterial effects of organic acids and bacteriocins.

[0048] Determination of the broad-spectrum antibacterial activity of Lactobacillus plantarum C07 After the 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 were activated as target strains, the inhibition zone diameter of C07 supernatant was measured by coating method and punching method.

[0049] The inhibition zones of Lactobacillus 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 were shown in Figure 2. Figure 3 As shown. The diameters of the inhibition zones of the supernatant of Lactobacillus 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, and those 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 an ellipse with the major and minor axes being (40 mm, 35 mm). It can be seen that Lactobacillus plantarum C07 had the strongest antibacterial ability against Geotrichum candidum. There was a significant difference between the control groups PBS and MRS (P<0.001 for both). These results suggest that Lactobacillus plantarum C07 has broad-spectrum antimicrobial activity and has inhibitory effects on foodborne pathogens.

[0050] Determination of the minimum inhibitory concentration (MIC) and inhibition growth curve of C07 supernatant against Staphylococcus aureus The activated three-generation C07 strain suspension was centrifuged at 8000r / min and 4°C for 15min, filtered through a 0.22μm filter membrane, the supernatant was collected, and freeze-dried for 3-4 days to form a lyophilized powder. The supernatant MIC and the inhibitory growth curve were determined by the two-fold dilution method and the microplate method, respectively.

[0051] like Figure 4 As shown in A, the higher the concentration of C07 supernatant lyophilized powder, the smaller the OD600nm, indicating a stronger antibacterial effect on Staphylococcus aureus. When the final mass concentration of the supernatant lyophilized powder 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. It was determined that the MIC of C07 supernatant lyophilized powder against multidrug-resistant Staphylococcus aureus DC.RB-015 was 3.5 mg / mL, and the MIC against Staphylococcus aureus ATCC25923 was 3 mg / mL. Figure 4 As shown in B and 4C, compared with the control group (PBS), the supernatant freeze-dried powder of Lactobacillus 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 hours. The Staphylococcus aureus in the control group remained on the typical microbial growth curve. Among the two strains of Staphylococcus aureus treated, 1MIC could inhibit the growth of the two strains of Staphylococcus aureus. 1 / 2MIC reduced the vitality of the two strains of Staphylococcus aureus and delayed the logarithmic phase.

[0052] Minimum inhibitory assay of supernatant of Lactobacillus plantarum C07 against biofilm of multidrug-resistant Staphylococcus aureus DC.RB-015 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.

[0053]

[0054] Figure 5 A through OD 600The 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).

[0055] Example 3 Determination of the ability of Lactobacillus plantarum C07 to produce L-lactic acid and indole-3-lactic acid Determination of specific antibacterial substances using targeted metabolomics The seed liquid of Lactobacillus plantarum C07 activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at a 1% inoculation volume (V / V). After culturing at 37℃ for 24 h, the supernatant was centrifuged at 8000 rpm and 4℃ for 15 min and filtered with a sterile 0.22 μm filter to obtain the cell-free supernatant. The target compounds were chromatographed on a Waters ACQUITY H-class plus UPLC System ultra-high performance liquid chromatograph using a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The A phase of the liquid chromatography was 0.1% formic acid solution, and the B phase was methanol. The mobile phase flow rate was 300 μL / min, the column oven temperature was 35℃, the sample plate temperature was 10℃, and the injection volume was 1 μL.

[0056] The extracted ion chromatograms (EICs) of L-lactic acid and indole-3-lactic acid are shown in Figure 2. Figure 6 As shown in A, it can be seen that the target compounds in the experimental samples can obtain good chromatographic peaks and are well separated on the chromatogram; the linear coefficients (R 2 ) were all greater than 0.9969, indicating that there was a good linear correlation between the chromatographic peak area and the compound concentration. Quantitative analysis was performed according to the calibration curve calculation formula of the standard. ( Figure 6B) Calculation formula of L-lactic acid in supernatant: y=846.28x+18547.3 ( R 2 =0.998168), (x, unit is μmol / L), ( Figure 6 C) Calculation formula of indole-3-lactic acid in supernatant: y=53951.4x+194.37, (R 2 =0.999178) (x, unit is μmol / L). As shown in Table 3, the content of L-lactic acid in Lactobacillus plantarum C07 is 100818.61±0.4 μmol / L, and the content of indole-3-lactic acid is: 119.25±0.4 μmol / L.

[0057] Table 3

[0058] Example 4 Application of Lactobacillus plantarum in fermented food The experimental group was composed of Lactobacillus bulgaricus: Streptococcus thermophilus: Lactobacillus plantarum C07 in a ratio of 1:1:1, and the starter culture without Lactobacillus plantarum C07 was used as the control group (the same ratio of Lactobacillus bulgaricus and Streptococcus thermophilus). After the experimental group was inoculated into pasteurized milk under a sterile environment, 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, and then the curd temperature was raised to 60°C-70°C for 25-30 min to remove the whey. The experimental and control samples were treated at 4°C and 25°C, respectively. At 0, 6, 12, 18 and 24 hours, 1 mL of samples were collected and cultured on LB agar, incubated at 37°C for 24 hours, and the number of viable bacteria (expressed as log CFU / mL) was recorded. All experiments were repeated three times.

[0059] like Figure 7 As shown, during the 24h storage period, after continuous incubation at 4°C and 25°C, the number of live bacteria in the fermented milk cake samples of the experimental group was generally reduced compared with the number of live bacteria in the control group, with an average reduction of 0.69±0.27 log CFU / mL under 4°C storage and an average reduction of 1.32±1.95 log CFU / mL under 25°C storage. In summary, the inoculation of Lactobacillus plantarum C07 can significantly reduce the count of live Gram-positive foodborne pathogens in fermented milk cakes. This shows that Lactobacillus plantarum C07 can be used as a fermentation agent with a preservative effect in the production of fermented milk, thereby reducing the economic losses of dairy products. The present invention ferments yogurt in the same way and also has similar technical effects.

[0060] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the 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 was named as plant lactobacillus C07 Lactiplantibacillus plantarum C07, the preservation number is CCTCC NO: M20242086, the preservation unit is China Type Culture Collection, the preservation time is September 26, 2024, and the preservation 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 used for non-therapeutic purposes; the spoilage and pathogenic microorganisms are one or more of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella and mold.

3. The use of plant lactobacillus according to claim 2 in inhibiting corruption and pathogenic microorganisms, characterized in that: The mold is Geotrichum candidum.

4. The use of plant lactobacillus according to claim 2 in inhibiting corruption and pathogenic microorganisms, characterized in that: The Staphylococcus aureus is standard Staphylococcus aureus and / or multi-drug resistant Staphylococcus aureus.

5. Use of the Lactobacillus plantarum according to claim 1 in producing lactic acid or indole-3-lactic acid.

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

7. The use of plant lactobacillus in fermented food according to claim 6, characterized in that: The fermented food is dairy products, sausages or bacterial powder.

8. The use of plant lactobacillus in fermented food according to claim 7, characterized in that: The dairy product is fermented milk or fermented milk cake.

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