Phytobacterium parabarium LN43 and application thereof
By isolating Lactobacillus paraplantatus LN43 from pickled vegetables, the problem of Salmonella pullorum infection in chickens has been solved, providing a solution for inhibiting various pathogens, especially effective against multidrug-resistant strains. It also exhibits acid resistance and bile salt tolerance, making it suitable for the preparation of bioinhibitors and drugs.
Patent Information
- Application Number
- CN202511398425.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2025-11-18
AI Technical Summary
Salmonella pullorum infection leads to high morbidity and mortality rates in chicks, and drug-resistant strains are spreading seriously. Current technology lacks effective antibacterial measures.
Lactobacillus paraplantatus LN43 was isolated from pickled vegetables and its preservation number is GDMCC No. 66615. It has antibacterial effects against a variety of pathogenic bacteria, and its metabolites are used to prepare biological inhibitors, environmental disinfectants and drugs.
Lactobacillus paraplantatus LN43 exhibits good antibacterial effects against a variety of pathogens, especially against multidrug-resistant Salmonella pullorum. It is acid-resistant and bile salt resistant, making it suitable for preparing products that inhibit foodborne pathogens and possessing significant potential as an antibiotic alternative.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial technology, in particular to a strain of Lactiplantibacillus paraplantarum LN43 and its application. BACKGROUND
[0002] Salmonella pullorum is a gram-negative pathogen of Salmonella, which can cause pullorum disease in chicks. Infection can cause systemic infection and white diarrhea symptoms, and the morbidity and mortality of 2-3 week-old chicks are extremely high. Some chicks resistant to Salmonella pullorum will become long-term carriers and eventually develop into subclinical and persistent infections, causing horizontal and vertical transmission. With the popularity of intensive farming mode and the growth of global poultry trade, the spread of Salmonella pullorum has expanded in scope and impact, causing huge economic losses to the farming industry.
[0003] Lactic acid bacteria are an important part of the normal microecology of the human and animal body, mainly as symbiotic bacteria inhabiting the digestive tract. This genus has significant acid tolerance and bile salt tolerance, which enables most strains to successfully colonize the intestinal environment and form dominant populations. After colonization, lactic acid bacteria exert their effects of inhibiting pathogenic bacteria and regulating intestinal flora homeostasis by secreting various beneficial metabolites. Lactiplantibacillus paraplantarum, as a kind of lactic acid bacteria, exhibits unique antibacterial properties. SUMMARY
[0004] The present application aims to provide a strain of Lactiplantibacillus paraplantarum LN43 and its application to solve the problems existing in the prior art. The present application isolates a strain of Lactiplantibacillus paraplantarum LN43 from sour pickles, with a preservation number of GDMCC No. 66615. The strain has good antibacterial effects on Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Micrococcus luteus, and has good antibacterial effect on multi-drug resistant Salmonella pullorum.
[0005] To achieve the above-mentioned purpose, the present application provides the following solutions:
[0006] The present application provides a strain of Lactiplantibacillus paraplantarum LN43 with antibacterial effect, the preservation number of the Lactiplantibacillus paraplantarum is GDMCC No. 66615, the preservation unit is Guangdong Microbial Culture Collection Center, the preservation time is June 30, 2025, and the address of the preservation unit is Building 59, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard.
[0007] The application also provides application of the Lactobacillus paraplantarum LN43 or the metabolite thereof in preparation of a biological inhibitor of food-borne pathogenic bacteria.
[0008] The application also provides application of the Lactobacillus paraplantarum LN43 or the metabolite thereof in preparation of an environmental disinfectant for inhibiting food-borne pathogenic bacteria.
[0009] The application also provides application of the Lactobacillus paraplantarum LN43 or the metabolite thereof in preparation of a medicine for preventing and treating diseases caused by food-borne pathogenic bacteria.
[0010] Preferably, the food-borne pathogenic bacteria are at least one of Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium and Micrococcus luteus.
[0011] The application also provides a biological inhibitor of food-borne pathogenic bacteria, wherein an active ingredient of the biological inhibitor comprises the Lactobacillus paraplantarum LN43 or the metabolite thereof.
[0012] The application also provides an environmental disinfectant for inhibiting food-borne pathogenic bacteria, wherein an active ingredient of the environmental disinfectant comprises the Lactobacillus paraplantarum LN43 or the metabolite thereof.
[0013] The application also provides a medicine for preventing and treating diseases caused by food-borne pathogenic bacteria, wherein an active ingredient of the medicine comprises the Lactobacillus paraplantarum LN43 or the metabolite thereof.
[0014] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.
[0015] Preferably, the food-borne pathogenic bacteria are at least one of Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium and Micrococcus luteus.
[0016] The application discloses the following technical effects:
[0017] The application discloses a Lactobacillus paraplantarum LN43 isolated from pickled vegetables, and the preservation number is GDMCC No.66615. The Lactobacillus paraplantarum LN43 has good bacteriostatic effects on Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii and Klebsiella pneumoniae. Especially, the Lactobacillus paraplantarum LN43 has good bacteriostatic effects on five kinds of multi-drug resistant Salmonella pullorum preserved in a laboratory.
[0018] The acid resistance test result of the Paraplantococcus plantarum LN43 provided by the application shows that the survival rate is 85.66% at pH 2.5 and the survival rate is 94.00% at pH 3, which indicates that the strain LN43 has strong acid resistance. The bile salt resistance test result shows that the survival rate is 60.78% when the mass fraction of bile salt is 0.1%, which indicates that the strain LN43 has certain bile salt tolerance. And the bacterium is sensitive to various antibiotics and has hemolytic safety. Based on its natural bacteriostatic property, safety and environmental friendliness, the Paraplantococcus plantarum LN43 or its metabolites are used for preparing a biological inhibitor of food-borne pathogenic bacteria, an environmental disinfectant for inhibiting food-borne pathogenic bacteria and a medicine for preventing and treating diseases caused by food-borne pathogenic bacteria, which has important significance in antibiotic substitution and the like. BRIEF DESCRIPTION OF DRAWINGS
[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.
[0020] Figure 1 Gram staining chart for colony morphology and Gram staining;
[0021] Figure 2 Phylogenetic tree of Paraplantococcus plantarum LN43;
[0022] Figure 3 Growth curve and acid production curve of Paraplantococcus plantarum LN43;
[0023] Figure 4 Acid and bile salt resistance result chart of Paraplantococcus plantarum LN43;
[0024] Figure 5 Self-aggregation and co-aggregation determination result chart of Paraplantococcus plantarum LN43;
[0025] Figure 6 Hemolytic experiment result chart of Paraplantococcus plantarum LN43. DETAILED DESCRIPTION
[0026] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, a parameter, an individual value or subrange within that range is also specifically disclosed. Each of the smaller ranges is also individually and specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also endpoints of the range, subject to any specifically excluded endpoint.
[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials which are related to the present application. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.
[0029] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.
[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.
[0031] Example 1 Screening and identification of lactic acid bacteria
[0032] 1. Isolation and purification of lactic acid bacteria
[0033] (1) Strain source: sour pickles, collected on March 20, 2025 in Shenyang, Liaoning Province;
[0034] (2) Preparation of sample:
[0035] ① Take 9 mL of sterilized PBS buffer solution into a sterile flask, then add 1 g of sour pickles, shake and wait for use;
[0036] ② Dilute the solution of step ① to prepare samples of different concentration gradients, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6Each of 200 μL was taken and uniformly spread on MRS-CaCO3 medium and cultured in anaerobic environment for 1 to 2 days. In order to obtain pure cultured single colony, the colony containing clear calcium-dissolving ring was scraped with an inoculation loop, and streaked on CaCO3-MRS medium for purification, repeated for 2 to 3 times, and the purified strain was stored in a glycerol tube for freezing preservation.
[0037] 2. Physiological and biochemical identification
[0038] The strain LN43 was subjected to Gram staining and hydrogen peroxide enzyme test, and the physiological and biochemical indexes were determined. The test results were compared with the eighth edition of the Bergey's Manual of Systematic Bacteriology for preliminary determination of the strain. The Gram staining of the strain LN43 was positive, the cell shape was rod-shaped (as shown in FIG. 1), the contact enzyme and oxidase were negative, and no spore was formed. The physicochemical test results are shown in Table 1. Figure 1
[0039] Table 1. Physiological and biochemical identification results of LN43
[0040] Substrate name LN43 assay result Escin + Cellobiose - Maltose - Mannitol - Salicin - Sorbitol - Sucrose - Raffinose + Lactose + Inulin - Hydrogen peroxide -
[0041] Note: "+" is positive, "-" is negative.
[0042] 3. Molecular biology identification
[0043] According to the operation instruction of the reagent kit, the bacterial genomic DNA was extracted, and identified by Shengong Bioengineering (Shanghai) Co., Ltd. The identification result was Lactiplantibacillus paraplantarum. The nucleotide sequence (SEQ ID NO. 1) of the 16S rDNA of the strain is as follows:
[0044]
[0045] The phylogenetic tree was constructed by Neighbor joining (NJ) method, and the phylogenetic tree result of strain LN43 is shown in FIG. 1. Strain LN43 was clustered with Lactiplantibacillus paraplantarum DSM 10667, and therefore the classification and naming of strain LN43 was Lactiplantibacillus paraplantarum, and the Latin name was Lactiplantibacillus paraplantarum. Figure 2
[0046] Example 2 Bacteriostatic activity test of Lactiplantibacillus paraplantarum LN43
[0047] The bacteriostatic activity of Lactiplantibacillus paraplantarum LN43 was determined by Oxford cup method. 10 mL of NA medium was first laid on the bottom of the plate as the bottom culture medium. Then 1% (v / v) of the indicator bacteria liquid was added to 10 mL of sterilized NA medium cooled to about 50°C, and mixed uniformly, and then poured into the plate as the upper layer culture medium. After the upper layer medium solidified, 5 Oxford cups were placed in each plate, 4 of which were added with 200 μL of Lactiplantibacillus paraplantarum LN43 supernatant, and the other one was added with 200 μL of MRS liquid medium as negative control. After incubation at 37°C for 24 h, the formation of inhibition zone was observed and the diameter was measured, and the results are shown in Table 2.
[0048] Table 2 Bacteriostatic effect of LN43 on different indicator bacteria
[0049] Indicator bacteria Gram staining Strain number Average diameter of inhibition zone / mm Salmonella pullorum Negative ATCC 10398 15.47±0.52 Salmonella typhimurium Negative CICC 10420 14.80±0.16 Salmonella enteritidis Negative CICC 10982 15.27±0.74 Escherichia coli Negative CICC 20547 12.37±0.33 Pseudomonas aeruginosa Negative CICC 23683 13.24±0.48 Staphylococcus aureus Positive CICC 23926 13.82±0.42 Micrococcus luteus Positive CICC 10269 24.40±0.22 Klebsiella pneumoniae Negative ATCC 19606 12.49±0.38 Acinetobacter baumannii Negative ATCC 13884 12.44±0.19
[0050] Note: The data shown in the table includes the diameter of the Oxford cup (7.8 ± 0.2 mm), and all the measured data includes 3 biological replicates, and is presented as mean ± standard deviation (SD).
[0051] Example 3 Antibiotic sensitivity experiment of Lactiplantibacillus paraplantarum LN43
[0052] The antibiotic sensitivity experiment of Lactiplantibacillus paraplantarum LN43 was detected by K-B disc diffusion method recommended by World Health Organization (WHO), and the results are shown in Table 3.
[0053] Table 3 Results of antibiotic sensitivity experiment of LN43
[0054]
[0055]
[0056] Note: S is sensitive; I is intermediate; R is resistant.
[0057] Example 4 Inhibition effect of Paraplantococcus plantarum LN43 on multi-drug resistant Salmonella pullorum
[0058] The same method as in Example 2 was used to determine the bacteriostatic effect of Paraplantococcus plantarum LN43 on 5 kinds of multi-drug resistant Salmonella pullorum preserved in the laboratory, and the results are shown in Table 4.
[0059] Table 4 Inhibition effect of LN43 on multi-drug resistant Salmonella pullorum
[0060] Indicator bacteria Average diameter of inhibition zone / mm SP13-1 15.99±0.75 SP13-8 16.30±0.33 SP13-9 16.56±0.20 SP13-16 15.55±0.53 SP18-3 12.28±0.45
[0061] Note: The data shown in the table include the Oxford cup diameter (7.8 ± 0.2 mm), and all measured data include 3 biological replicates, presented as mean ± standard deviation (SD).
[0062] Example 5 Growth and acid production curve determination of Paraplantococcus plantarum LN43
[0063] The activated seed liquid of strain LN43 was inoculated into MRS broth medium, inoculated into 200 mL MRS liquid medium at a volume of 1%, uniformly shaken, and placed at 37°C for static culture, and sampling was performed every 2 h until 30 h. The OD600 value of LN43 bacterial liquid and the pH value of sterile supernatant were detected, and the growth curve and pH value change curve were plotted, and the results are shown in Figure 3 Within 4 h of fermentation, it was the lag phase of strain LN43, and then it was the logarithmic growth phase for 10 h, and the stationary phase lasted to 30 h without decline, indicating that strain LN43 had good growth performance. Within the fermentation interval, the pH of the culture solution continuously decreased from the start of fermentation to 22 h, and then stabilized between 3.7-3.9, indicating that the strain had strong acid-producing ability.
[0064] Example 6 Acid and bile salt tolerance determination of Paraplantococcus plantarum LN43
[0065] (1) Acid tolerance determination
[0066] The activated LN43 seed liquid was added to MRS liquid medium with pH values of 2.5 and 3.0 at an inoculation amount of 1% (v / v), and MRS liquid medium without the addition of acid was set as a control group. After 3 h of static culture at 37 ℃, 200 μL of bacterial liquid was taken from the acid treatment group and the control group, and then uniformly coated on MRS solid medium plates after appropriate dilution. Three parallel samples were prepared for each dilution. After 24 h of static culture at 37 ℃, viable cell counting was performed and the survival rate was calculated. The survival rate calculation formula is:
[0067] X% = A / A0 x 100
[0068] In the formula, X is the survival rate; A is the viable cell count per mL after 3 h of treatment, lg CFU / mL; A0 is the viable cell count of the control group, lg CFU / mL.
[0069] The acid resistance test results of strain LN43 are shown in Table 1. Figure 4 The results show that the survival rate is 85.66% at pH 2.5 and 94.00% at pH 3, indicating that strain LN43 has strong acid resistance.
[0070] (2) Cholate salt tolerance determination
[0071] The activated lactic acid bacteria seed liquid was inoculated into MRS liquid medium with a pig cholate salt concentration of 0.1% at an inoculation amount of 1% (v / v), and three parallel tests were set up. The control group used MRS liquid medium without pig cholate salt. After 3 h of static culture at 37 ℃, 200 μL of bacterial liquid was taken from the treatment group and the control group, and then uniformly coated on MRS solid medium plates after appropriate dilution. Viable cell counting was performed. The survival rate calculation formula is:
[0072] Y% = B / B0 x 100
[0073] In the formula, Y is the survival rate; B is the viable cell count per mL after 3 h of treatment, lg CFU / mL; B0 is the viable cell count of the control group, lg CFU / mL.
[0074] The cholate salt tolerance test results of strain LN43 are shown in Table 2. Figure 4 The results show that the survival rate is 60.78% at a cholate salt mass fraction of 0.1%, indicating that strain LN43 has certain cholate salt tolerance.
[0075] Example 7 Aggregating property and co-aggregating property determination of Lactobacillus paraplantarum LN43
[0076] (1) Self-aggregation rate determination
[0077] LN43 was inoculated in MRS liquid medium and incubated at 37°C for 24 hours. The bacteria were collected by centrifugation at 8000 rpm for 10 min at 4°C, washed twice with PBS, and resuspended to an absorbance of 0.6±0.5 at 600 nm wavelength with PBS. 4 mL of the bacterial suspension was vortexed for 10 s and incubated at 37°C for 4 h. The absorbance of the supernatant OD600was measured. The test was repeated three times.
[0078] Self-aggregation rate (%) = (1-A1 / A0) x 100
[0079] Wherein, A1 is the absorbance after incubation for 4 hours; A0 is the initial absorbance.
[0080] (2) Co-aggregation rate determination
[0081] LN43 was inoculated in MRS liquid medium and incubated at 37°C for 24 hours. The bacteria were collected by centrifugation at 8000 rpm for 10 min at 4°C, washed twice with PBS, and resuspended to an absorbance of 0.6±0.5 at 600 nm wavelength with PBS. At the same time, chicken white diarrhea Salmonella (SP) was cultured in LB at 37°C. The bacteria were collected by centrifugation at 8000 rpm for 15 min at 4°C, washed twice with PBS, and resuspended to an absorbance of 0.6±0.5 at 600 nm wavelength with PBS. 2 mL of each of the lactic acid bacteria suspension and the pathogenic bacteria suspension were mixed for 10 s, and then incubated at 37°C for 4 h. Subsequently, the absorbance of the mixture was measured at 600 nm wavelength (OD mix ). The experiment was repeated three times.
[0082] Co-aggregation rate (%) = 100 x [(OD LAB + OD SP ) - 2(OD mix )] / (OD LAB + OD SP )
[0083] Wherein, OD LAB is the absorbance of the LN43 bacterial suspension, OD SP is the absorbance of the pathogenic bacteria suspension, and OD mix is the absorbance of the mixture.
[0084] The aggregation determination results of strain LN43 showed that the self-aggregation rate was 21.07%, and the co-aggregation rate with chicken white diarrhea Salmonella was 15.06%, indicating that strain LN43 had certain intestinal colonization ability.
[0085] Example 8 Hemolytic experiment of Paraplantibacterium LN43
[0086] The strain LN43 was streaked on the surface of a Columbia blood agar plate to test its hemolytic activity. The results are shown in Table 1. Figure 6 As shown, LN43 hemolytic activity was negative, which indicated that LN43 was hemolytic safe.
[0087] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.
Claims
1. A strain of *Lactiplantibacillus paraplantarum* LN43 with antibacterial activity, characterized in that... The Lactobacillus paraplantatus LN43 has the accession number GDMCC No. 66615, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, was deposited on June 30, 2025, and is located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. The use of Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1 in the preparation of bioinhibitors of foodborne pathogens.
3. The application of Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1 in the preparation of environmental disinfectants that inhibit foodborne pathogens.
4. The use of Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1 in the preparation of medicaments for the prevention and treatment of diseases caused by foodborne pathogens.
5. The application as described in any one of claims 2-4, characterized in that, The foodborne pathogens mentioned are at least one of Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Micrococcus luteus.
6. A bioinhibitor of foodborne pathogens, characterized in that, The active ingredient of the bioinhibitor includes Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1.
7. An environmental disinfectant for inhibiting foodborne pathogens, characterized in that, The active ingredient of the environmental disinfectant includes Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1.
8. A drug for preventing and treating diseases caused by foodborne pathogens, characterized in that, The active ingredient of the drug includes Lactobacillus paraplantatus LN43 or its metabolites as described in claim 1.
9. The medicament as described in claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.
10. The medicament as claimed in claim 8, characterized in that, The foodborne pathogens mentioned are at least one of Staphylococcus aureus, Salmonella pullorum, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Micrococcus luteus.