Lactobacillus arrhizatae and application thereof

Abstract

The invention discloses lactobacillus arrhizatae and application thereof, and relates to the technical field of microorganisms. The lactobacillus arrhizatae is preserved in the Guangdong Microbial Culture Collection Center on December 10, 2024, the preservation number is GDMCC 65603, and the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. According to the invention, a strain of Lactobacillus argenteus is screened from healthy free-ranging adult local partridge chickens from Guangxi Zhuang Autonomous Region Tibetan City, the strain is named as LB19, and verification proves that the Lactobacillus argenteus has a good inhibition effect on food-borne pathogenic bacteria including salmonella and escherichia coli, and can be used for preparing food-borne pathogenic bacteria. Good application potential is realized in the aspect of preventing and treating food-borne pathogenic bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Lactobacillus argenteus and application thereof. Background Art

[0002] The commercial broiler industry occupies a pivotal position in the livestock industry. However, for a long time, broiler production has mainly relied on vaccines and environmental control measures, combined with a veterinary epidemic prevention system of chemical drugs and antibiotics. This practice has led to the problem of chemical drug, hormone and antibiotic residues, which not only make animals resistant to drugs, but also may cause risks such as carcinogenicity, teratogenicity and mutagenicity, and pose a threat to human health through the food chain.

[0003] In recent years, probiotics have received extensive attention as a new feed additive. They can effectively inhibit the attachment and reproduction of pathogens and maintain the microecological balance of the gastrointestinal tract of animals. The metabolites of probiotics are beneficial to the host and can be used as a source of nutrition or promote the digestion and absorption of food by animals. The present invention intends to develop a new probiotic to achieve effective inhibition of foodborne pathogens. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus argenteus and its application to solve the problems existing in the above-mentioned prior art. The Lactobacillus argenteus has a good inhibitory effect on a variety of food-borne pathogens and has good application potential in the prevention and treatment of food-borne pathogens.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a strain of Lactobacillus argentoratensis, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 10, 2024, with a deposit number of GDMCC 65603, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides the use of the above-mentioned Lactobacillus argenteus or its metabolites in the preparation of biological inhibitors of food-borne pathogenic bacteria.

[0008] The present invention also provides the use of the above-mentioned Lactobacillus argenteus or its metabolites in the preparation of an environmental disinfectant for inhibiting food-borne pathogens.

[0009] The present invention also provides the use of the above-mentioned Lactobacillus argenteus or its metabolites in the preparation of medicines for preventing and treating diseases caused by foodborne pathogenic bacteria.

[0010] Furthermore, the foodborne pathogenic bacteria is at least one of Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii and Klebsiella pneumoniae.

[0011] The present invention also provides a biological inhibitor for food-borne pathogenic bacteria, wherein the active ingredient comprises the above-mentioned Lactobacillus argenteus or its metabolites.

[0012] The present invention also provides an environmental disinfectant for inhibiting food-borne pathogenic bacteria, wherein the active ingredient comprises the above-mentioned Lactobacillus argenteus or its metabolites.

[0013] The present invention also provides a medicine for preventing and treating diseases caused by food-borne pathogenic bacteria, wherein the active ingredient comprises the above-mentioned Lactobacillus argenteus or its metabolites.

[0014] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the foodborne pathogenic bacteria is at least one of Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii and Klebsiella pneumoniae.

[0016] The present invention discloses the following technical effects:

[0017] The present invention screened a strain of Lactobacillus argentoratensis LB19 from healthy free-range adult native hemp chickens from Laibin City, Guangxi Zhuang Autonomous Region. It has been verified that the strain has a good inhibitory effect on foodborne pathogens including Salmonella and Escherichia coli, and has good application potential in the prevention and treatment of foodborne pathogens. Lactobacillus argentoratensis LB19 can be used to prepare products for preventing and treating foodborne pathogens, and is a probiotic strain worthy of development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 is the colony morphology of strain LB19;

[0020] Figure 2 is the Gram staining image of strain LB19;

[0021] Figure 3 is the phylogenetic tree diagram of strain LB19;

[0022] Figure 4 is a growth curve graph of strain LB19 based on 16S rRNA sequence;

[0023] Figure 5 This is the Oxford Cup antibacterial effect diagram of strain LB19 inhibiting Salmonella pullorum;

[0024] Figure 6 This is the Oxford cup antibacterial effect diagram of strain LB19 inhibiting Escherichia coli;

[0025] Figure 7 This is a graph showing the results of the self-aggregation assay of strain LB19. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Screening, identification and preservation of lactic acid bacteria

[0032] 1. Isolation and purification of lactic acid bacteria

[0033] First, healthy free-range adult native hemp chickens from Laibin City, Guangxi Zhuang Autonomous Region were bled until death, and then the cecum, jejunum, duodenum, and ileum were taken out with sterilized tweezers and placed in separate culture dishes. A sterilized spoon was used to scrape 0.1 g of the contents on the mucosa of the cut chicken intestines, and a 1.5 mL EP tube with a cap containing 900 μL of sterile saline was prepared, and the scraped contents were added to the tubes. The mixture was fully shaken and evenly mixed. The dilution gradient at this time was 10 -1 In the clean bench: use a 200μL pipette to take 100μL of the chicken intestinal secretion mixture that has been fully shaken by the oscillator and add it to a 1.5mL centrifuge EP tube with a cap containing 900μL sterile saline, so that the secretion diluted with saline is fully mixed with the mixture. -3 , 10 -4 , 10 -5 and 10 -6 Take out 200 μL of each of the four different concentrations of dilution and spread it evenly on the MRS-CaCO3 medium. Then, repeat the test three times for each concentration and culture it in an anaerobic environment using the candle jar method for 1-2 days. In order to obtain a single colony of pure culture, use an inoculation loop to scrape the colony containing a relatively clear white calcium-dissolving circle, streak it on the CaCO3-MRS medium for separation and purification, repeat 2 to 3 times, and culture it at 37°C for 1-2 days. The strains that are Gram-positive and catalase-negative are preliminarily judged as lactic acid bacteria, and the strains preliminarily judged as lactic acid bacteria are preserved using the glycerol tube preservation method.

[0034] The colony morphology of a lactic acid bacteria strain (named LB19) obtained in the initial screening is shown in Figure 1 The colony has neat edges, a smooth and moist surface, is translucent, has small, round, bag-like protrusions, and a uniform texture; Gram staining results are shown in Figure 2 The bacterial body is short and thick rod-shaped, Gram staining is positive, without flagella, no movement and no spores.

[0035] 2. Molecular Biological Identification of Lactic Acid Bacteria

[0036] The strain LB19 was subjected to molecular biological identification, as detailed below:

[0037] According to the instructions of the Tiangen kit, bacterial genomic DNA was extracted, and then the 16S rRNA gene fragment of the target strain genome was amplified using universal primers 27F and 1492R as forward and reverse primers.

[0038] The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2.

[0039] Table 1 PCR reaction system (50 μL)

[0040]

[0041] Table 2 PCR amplification program

[0042]

[0043] The results of 16S rRNA gene sequencing of strain LB19 are as follows:

[0044]

[0045] The phylogenetic tree of strain LB19 was constructed, and the results are shown in Figure 3 The results showed that the strain LB19 was classified as: Lactobacillus argentoratensis, and its Latin name was: Lactiplantibacillus argentoratensis.

[0046] The growth curve of strain LB19 based on 16S rRNA sequence is shown in Figure 4 .

[0047] 3. Strain preservation

[0048] Lactiplantibacillus argentoratensis LB19 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 10, 2024, with the deposit number GDMCC 65603, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0049] Example 2 Antibacterial activity test of Lactobacillus argenteus LB19

[0050] The antibacterial ability of Lactobacillus argentulatus LB19 was determined by the Oxford cup double-layer plate method. The bacterial liquids of 8 indicator bacteria (Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii, and Klebsiella pneumoniae) were spread on LB culture medium respectively. Four Oxford cups were placed in each plate. 200 μL of Lactobacillus argentulatus LB19 bacterial liquid supernatant was added to 3 Oxford cups, and 200 μL of MRS liquid culture medium was added to 1 as a control. The plate was placed upright at 37°C incubator and cultured for 24 hours. After the end, whether there was an inhibition zone around the colony was observed and the diameter of the inhibition zone was measured. The results are shown in Table 3. The antibacterial effect diagram of some Oxford cups is shown in Figure 5-Figure 6 The results showed that Lactobacillus argenteus LB19 had different degrees of inhibitory effects on 8 indicator bacteria, among which the inhibitory effects on Salmonella pullorum and Micrococcus luteus were stronger.

[0051] Table 3 Antibacterial effect of strain PN5 on different indicator bacteria

[0052]

[0053]

[0054] Note: The data shown in the table include the Oxford cup diameter (7.8±0.2mm). All measured data include 3 biological replicates and are presented as mean±standard deviation (SD).

[0055] Example 3 Antibiotic sensitivity test of Lactobacillus argenteus LB19

[0056] The sensitivity of Lactobacillus argenteus LB19 to 20 antibiotics was detected by referring to the KB disk diffusion method recommended by the World Health Organization (WHO).

[0057] The results of the drug sensitivity test are shown in Table 4, which show that Lactobacillus argentulatus LB19 is resistant to rifamycins rifampicin, cephalosporins (cephradine, ceftriaxone, ceftriaxone, ceftriaxone), macrolides, penicillins, glycopeptides, aminoglycosides, tetracyclines, quinolones, sulfonamides, polymyxins and lincosamides, moderately sensitive to chloramphenicol, and highly sensitive to cephalosporin ceftazidime.

[0058] Table 4 LB19 drug sensitivity test results

[0059]

[0060]

[0061] Note: S means highly sensitive; I means moderately sensitive; R means resistant.

[0062] Example 4 Determination of the self-aggregation of Lactobacillus argenteus LB19

[0063] Lactobacillus argenteus LB19 was cultured in MRS broth at 37°C for 24 hours, and the cells were collected by centrifugation at 8000r / min for 10 minutes. After washing twice in sterile PBS, it was resuspended in PBS so that the OD at A600nm was between 1.0±0.2, and the initial absorbance A0 at 600nm was recorded. The suspension was incubated at 37°C for 5 hours. The final absorbance A1 of the supernatant was measured at 600nm. Self-aggregation formula: Self-aggregation (%) = (A0-A1) / A0×100%.

[0064] like Figure 7 As shown, the self-aggregation of Lactobacillus argenteus LB19 was 19.41%.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactiplantibacillus argentoratensis, characterized in that: The Lactobacillus argenteus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 10, 2024, with the collection number GDMCC 65603, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Lactobacillus argenteus or its metabolites as claimed in claim 1 in the preparation of bioinhibitors of foodborne pathogens.

3. Use of the Lactobacillus argenteus or its metabolites as claimed in claim 1 in the preparation of an environmental disinfectant for inhibiting foodborne pathogens.

4. Use of the Lactobacillus argenteus or its metabolites as claimed in claim 1 in the preparation of drugs for preventing and treating diseases caused by foodborne pathogenic bacteria.

5. The use according to any one of claims 2 to 4, characterized in that: The foodborne pathogenic bacteria is at least one of Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii and Klebsiella pneumoniae.

6. A biological inhibitor for foodborne pathogenic bacteria, characterized in that: The active ingredient comprises the Lactobacillus argenteus or its metabolites as described in claim 1.

7. An environmental disinfectant for inhibiting foodborne pathogens, characterized in that: The active ingredient comprises the Lactobacillus argenteus or its metabolites as described in claim 1.

8. A drug for preventing and treating diseases caused by foodborne pathogenic bacteria, characterized in that: The active ingredient comprises the Lactobacillus argenteus or its metabolites as described in claim 1.

9. The drug according to claim 8, characterized in that The drug also includes pharmaceutically acceptable excipients.

10. The bio-inhibitor according to claim 6, the environmental disinfectant according to claim 7 or the drug according to claim 8, characterized in that: The foodborne pathogenic bacteria is at least one of Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus, Acinetobacter baumannii and Klebsiella pneumoniae.

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