Lactobacillus craw PN5 and application thereof

Abstract

The invention discloses lactobacillus craw PN5 and application thereof, and belongs to the field of microorganisms. The Lactobacillus craw PN5 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 65605. The strain is screened from intestinal tracts of spotted-brown chickens, is good in source safety, has a good inhibition effect on pathogenic bacteria including salmonella pullorum, escherichia coli and the like, has good application potential in the aspect of preventing and treating the pathogenic bacteria, is wide in prospect, can be used for preparing products for preventing and treating the pathogenic bacteria, and has wide application prospects. The strain is a probiotic strain worthy of development.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to crop mucus lactobacillus PN5 and application thereof. Background Art

[0002] As a common foodborne pathogen, Salmonella poses a great risk to human and animal health. Recently, food contamination incidents have become common, especially Salmonella infection, which has caused significant economic losses to the aquaculture industry and public health safety. Similarly, Escherichia coli is another key factor in causing foodborne diseases. At present, antibiotics are still the standard treatment for combating bacterial infections. However, due to long-term reliance on antibiotics, the problem of drug resistance has become more prominent. Therefore, exploring new biological control strategies is an urgent problem to be solved.

[0003] Limosilactobacillus ingluviei is a lactic acid bacterium that is widely found in the intestines of animals and the environment. Studies have found that Lactobacillus ingluviei has certain probiotic functions, such as regulating the balance of intestinal flora and enhancing the body's immunity. In addition, Lactobacillus ingluviei has a certain inhibitory effect on certain pathogens. However, there are still certain limitations in the screening and application of Lactobacillus ingluviei in the field of biological control. Summary of the invention

[0004] The purpose of the present invention is to provide a crop mucus Lactobacillus PN5 and its application to solve the problems existing in the above-mentioned prior art. The crop mucus Lactobacillus PN5 provided by the present invention has a good inhibitory effect on pathogenic bacteria including Salmonella pullorum and Escherichia coli, has good application potential in the prevention and treatment of pathogenic bacteria, has broad prospects, can be used to prepare products for preventing and treating pathogenic bacteria, and is a probiotic strain worthy of development.

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

[0006] The invention provides a crop mucus lactobacillus (Limosilactobacillus ingluviei) PN5. The crop mucus lactobacillus PN5 is stored in Guangdong Province Microbiological Collection Center with a storage number of GDMCC NO: 65605.

[0007] The present invention also provides the use of the crop mucus Lactobacillus PN5 and / or its fermentation product in the preparation of antibacterial products.

[0008] Optionally, the antibacterial product is a product that inhibits foodborne bacteria.

[0009] Optionally, the foodborne bacteria include Salmonella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0010] The present invention also provides an antibacterial product, the active ingredients of which include the crop mucus Lactobacillus PN5 and / or its fermentation product.

[0011] Optionally, the antimicrobial product is used to inhibit foodborne bacteria.

[0012] Optionally, the foodborne bacteria include Salmonella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0013] Optionally, the antibacterial product includes an antibacterial agent, and the antibacterial agent includes a liquid antibacterial agent, a powder antibacterial agent and a granular antibacterial agent.

[0014] The present invention also provides a method for inhibiting foodborne bacteria from infecting food, comprising the steps of using the crop mucus Lactobacillus PN5 and / or its fermentation product, or the antibacterial product in food production and processing.

[0015] The present invention discloses the following technical effects:

[0016] The invention screens out a crop mucus Lactobacillus PN5 from the intestine of ephedra chicken. The strain has good source safety, has good inhibitory effect on pathogenic bacteria including Salmonella pullorum and Escherichia coli, has good application potential in the prevention and treatment of pathogenic bacteria, has broad prospects, can be used for preparing products for preventing and treating pathogenic bacteria, and is a probiotic strain worthy of development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 The colony morphology of strain PN5 (A) and the Gram staining of strain PN5 (B);

[0019] Figure 2 is the phylogenetic tree of strain PN5 based on 16S rDNA sequence;

[0020] Figure 3 is the growth curve of strain PN5;

[0021] Figure 4This is the result of optimizing the culture temperature of strain PN5;

[0022] Figure 5 This is the pH optimization result diagram for the culture of strain PN5;

[0023] Figure 6 This is the effect diagram of strain PN5 inhibiting Salmonella pullorum. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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 those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] In a first aspect, the present invention provides a crop mucus lactobacillus PN5, which is classified and named as Limosilactobacillus ingluviei.

[0030] The crop mucus lactobacillus PN5 of the present invention originates from the chicken intestine. According to the colony morphology test, the crop mucus lactobacillus PN5 colony is milky white, has neat and convex edges, a smooth surface and a uniform texture. The bacterial body is spherical, the Gram staining result is positive, and the bacterial body has no flagella, no movement and no spores.

[0031] The antibacterial experiment found that the crop mucus Lactobacillus PN5 provided by the present invention has a good inhibitory effect on pathogenic bacteria including Salmonella pullorum and Escherichia coli, has good application potential in the prevention and treatment of pathogenic bacteria, and is a probiotic strain worthy of development.

[0032] The method for screening crop mucus Lactobacillus PN5 of the present invention comprises the following steps:

[0033] The strain sampled from the chicken intestine was separated and purified to obtain a single colony, which was then identified to obtain crop mucus Lactobacillus PN5.

[0034] The above-mentioned screening method of crop mucus Lactobacillus PN5 is simple and convenient, and can quickly obtain multiple strains including crop mucus Lactobacillus PN5.

[0035] In some preferred embodiments, the separation and purification methods include but are not limited to the spread plate method and the plate streak method, or bacterial separation and purification methods well known to those skilled in the art.

[0036] In some preferred embodiments, the identification method includes physiological and biochemical tests and 16S rDNA gene sequence analysis. Physiological and biochemical tests are used to perform preliminary identification of the strain. 16S rDNA gene sequence analysis is used to obtain the species of the strain.

[0037] In a second aspect, the present invention provides a bacterial agent, including crop mucus Lactobacillus PN5, and may also include other strains or auxiliary materials, etc. The bacterial agent has all the beneficial effects of the crop mucus Lactobacillus PN5 of the present invention.

[0038] In some preferred embodiments, the dosage form of the bacterial agent includes liquid bacterial agent, powder and granular bacterial agent.

[0039] In a third aspect, the present invention provides a fermentation product obtained by fermentation of crop mucus Lactobacillus PN5 or a bacterial agent.

[0040] Antibacterial experiments have shown that the fermentation products of crop mucus Lactobacillus PN5 have a good inhibitory effect on pathogenic bacteria including Salmonella and Escherichia coli, and can be used to prepare antibacterial products.

[0041] In a fourth aspect, the present invention provides a use of crop mucus Lactobacillus PN5, bacterial agents or fermentation products in the preparation of products for preventing and controlling pathogenic bacteria.

[0042] In a fifth aspect, the present invention provides a product for preventing and treating pathogenic bacteria, including crop mucus Lactobacillus PN5, a bacterial agent or a fermentation product.

[0043] In a sixth aspect, the present invention provides a method for inhibiting foodborne bacteria from infecting food, comprising the steps of using the crop mucus Lactobacillus PN5 and / or its fermentation products, or the antibacterial products in food production and processing.

[0044] The above-mentioned food production includes the production of livestock and poultry related by-products.

[0045] The present invention is further illustrated by specific examples and comparative examples. The strains and culture media used in the examples are shown in Table 1.

[0046] Table 1 Test strains and culture medium

[0047]

[0048] The culture medium and components involved in the embodiments of the present invention are as follows:

[0049] MRS liquid culture medium: glucose 20.0 g, tryptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, deionized water 1 L, pH 6.5 (add 1.5% agar as solid culture medium).

[0050] LB medium: 10.0 g tryptone, 10.0 g sodium chloride, 5.0 g yeast extract powder, 1 L deionized water, pH 7.3 (add 1.5% agar as a solid medium).

[0051] Example 1 Isolation of crop mucus Lactobacillus PN5 strain

[0052] Free-range silkie chickens (Pingnan County, Guigang City, Guangxi Zhuang Autonomous Region) were bled to death, and the jejunum, ileum, cecum, and duodenum were removed. A small section of each intestine was placed in a glass dish, which was cut open and the intestinal wall was rinsed 2-3 times with saline. 100 μL of saline was added to the glass dish, and the intestinal mucosa was scraped with a sterile spoon. The contents of the glass dish were sucked and placed in 1.5 mL EP tubes, and 900 μL of sterile saline was diluted to 10 -1 Repeat the previous step and dilute the stock solution into 10 -2 , 10 -3 , 10 -4 Dilution, pipette 10 -3 , 10 -4 , 10-5 200 μL of the intestinal content dilution was evenly spread on the MRS-CaCO3 medium, and 3 replicates were set for each dilution. Deoxygenator was used during culture and vacuum was drawn with a vacuum pump. Anaerobic culture was carried out at 37°C for 48 hours. Single colonies with obvious calcium melting circles were selected with an inoculation loop, and after streaking and purification on the MRS-CaCO3 medium 2 to 3 times, a pure culture of the selected single colonies was obtained. It was preliminarily identified as lactic acid bacteria and named strain PN5, and stored in 60% (v / v) glycerol and refrigerated at -80°C.

[0053] Example 2 Identification of crop mucus Lactobacillus PN5 strain

[0054] (1) Physiological and biochemical tests

[0055] The purified strain PN5 was subjected to Gram staining ( Figure 1 The results of the test were compared with the "Classification and Identification of Lactic Acid Bacteria and Test Methods" to make a preliminary determination of the strain. The test showed that the Gram stain of the screened strain PN5 was purple and positive. Its bacterial morphology was rod-shaped cells, and the catalase and oxidase were negative, without spore formation. The results of its physical and chemical tests are shown in Table 2.

[0056] Table 2 Biochemical identification results of strain PN5

[0057]

[0058] Note: "+" in the table means ≥90% of the strains are positive; "-" means ≥90% of the strains are negative

[0059] (2) 16S rDNA molecular biological identification

[0060] The bacterial genomic DNA was extracted according to the instructions of the Tiangen kit, and then the 16S rDNA gene fragment of the target strain genome was amplified using universal primer 27F (5'-AGAG TTTGATCCTGGCTCAG-3', SEQ ID NO.2) and universal primer 1492R (5'-GGTTACCTTGTTACGACT T-3', SEQ ID NO.3) as forward and reverse primers.

[0061] The PCR reaction system is shown in Table 3, and the PCR amplification program is shown in Table 4. After the reaction, 3 μL of the PCR reaction product was taken for electrophoresis detection. The electrophoresis time was 30 min, and the agarose gel content was 1%.

[0062] Table 3 PCR reaction system (50 μL)

[0063]

[0064]

[0065] Table 4 PCR amplification program

[0066]

[0067] The results of 16S rDNA gene sequence determination are shown in SEQ ID NO.1.

[0068] SEQ ID NO.1:

[0069]

[0070] According to the results of 16S rDNA gene sequencing, the phylogenetic tree of strain PN5 was constructed, as shown in Figure 2 As shown, strain PN5 is clustered with Limosilactobacillus ingluviei DSM 15946 (AZFK01000041). Therefore, strain PN5 is identified as crop mucus Lactobacillus, with the taxonomic name Limosilactobacillus ingluviei. It was deposited in Guangdong Microbiological Culture Collection Center on December 10, 2024, with the deposit address on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC NO: 65605.

[0071] Example 3 Determination of the growth curve of crop mucus Lactobacillus PN5

[0072] (1) Preparation of fermentation broth

[0073] The crop mucus Lactobacillus PN5 stored at -80°C was inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, and transferred twice continuously to complete the activation of the strain.

[0074] (2) Determination of growth curve

[0075] Take the crop mucus Lactobacillus PN5 after activation for the first generation, inoculate it into 200mL MRS liquid medium at a volume of 1%, shake it evenly, and culture it in a 37℃ incubator. Take samples every 6h and measure the OD 600nm , make appropriate gradient dilutions according to the measured absorbance values. Then use the live bacteria counting method to spread 200μL of the dilution on the MRS-CaCO3 plate, and make 3 parallels for each gradient. After anaerobic culture at 37℃ for 1 day, select the plate with CFU between 30-300 for counting. Draw the growth curve of crop mucus Lactobacillus PN5, as shown in Figure 3 shown.

[0076] Example 4 Optimization of strain culture conditions

[0077] (1) Temperature optimization

[0078] Use an inoculation loop to pick a single colony and inoculate it into 10 mL of MRS liquid medium that has been sterilized in advance, and incubate it at 37°C for 24 hours. Use the cultured fresh bacterial suspension as the seed liquid, take 100 μL of it according to the inoculation amount of 1% and add it to a centrifuge tube containing 10 mL of MRS liquid medium, and incubate it in a constant temperature water bath at 25°C, 30°C, 37°C, 42°C, and 45°C for 24 hours. After the culture is completed, use a spectrophotometer to measure the absorbance value.

[0079] The results are as follows Figure 4 As shown, strain PN5 grew best at 42°C.

[0080] (2) pH optimization

[0081] Use an inoculation loop to pick a single colony and inoculate it into a pre-sterilized 10 mL MRS liquid culture medium, and incubate it at 37°C for 24 hours. Use the cultured fresh bacterial suspension as the seed liquid, take 100 μL of it according to the inoculation amount of 1% and add it to a 10 mL MRS liquid culture medium centrifuge tube adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, and incubate it at 37°C for 24 hours. After the culture is completed, use a spectrophotometer to measure the absorbance value.

[0082] The results are as follows Figure 5 As shown, strain PN5 grew best at pH 7.

[0083] Example 5: Experimental determination of antibiotic sensitivity of crop mucus Lactobacillus PN5

[0084] The drug sensitivity test of 20 antibiotics was tested by referring to the KB disk diffusion method recommended by the World Health Organization (WHO). The results are shown in Table 5.

[0085] Table 5 Drug sensitivity test determination table

[0086]

[0087] Note: All the data shown in the table are measured with 3 biological replicates, and are expressed as mean ± standard; S is sensitive, I is intermediate, and R is resistant.

[0088] According to Table 5, strain PN5 is sensitive to cephalosporins, penicillins, macrolides, tetracyclines, phenicols and rifamycins; strain PN5 has an intermediate sensitivity to gentamicin; strain PN5 is resistant to vancomycin, streptomycin, kanamycin, ciprofloxacin, sulfamethoxazole, polymyxin and lincomycin.

[0089] Example 6 Experimental determination of antibacterial activity of crop mucus Lactobacillus PN5

[0090] The antibacterial ability of crop mucus Lactobacillus PN5 was determined by the Oxford cup double-layer plate method. Eight 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. Four Oxford cups were placed in each plate. 200 μL of crop mucus Lactobacillus PN5 bacterial supernatant was added to three Oxford cups, and 200 μL of MRS liquid culture medium was added to one plate as a control. The plates were placed in a 37°C incubator and cultured for 24 h. 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 6.

[0091] Table 6 Antibacterial effect of strain PN5 on different indicator bacteria

[0092]

[0093] 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).

[0094] According to Table 6, strain PN5 can inhibit a variety of Gram-negative and Gram-positive bacteria. This shows that strain PN5 and its fermentation products can be used to prepare products for the prevention and treatment of foodborne pathogens including Salmonella pullorum and Escherichia coli ( Figure 6 ).

[0095] 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 crop mucus lactobacillus (Limosilactobacillus ingluviei) PN5, characterized in that: The crop mucus Lactobacillus PN5 is stored in Guangdong Microbiological Culture Collection Center with a storage number of GDMCC NO: 65605.

2. Use of the crop mucus Lactobacillus PN5 and / or its fermentation product as claimed in claim 1 in the preparation of antibacterial products.

3. The use according to claim 2, characterized in that: The antimicrobial product is a product that inhibits foodborne bacteria.

4. The use according to claim 3, characterized in that: The foodborne bacteria include Salmonella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Acinetobacter baumannii.

5. An antibacterial product, characterized in that: The effective ingredients include the crop mucus Lactobacillus PN5 and / or its fermentation product as described in claim 1.

6. The antibacterial product according to claim 5, characterized in that: The antimicrobial product is used to inhibit foodborne bacteria.

7. The antibacterial product according to claim 6, characterized in that: The foodborne bacteria include Salmonella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Klebsiella pneumoniae and Acinetobacter baumannii.

8. The antibacterial product according to claim 5, characterized in that: The antibacterial product comprises an antibacterial agent, and the antibacterial agent comprises a liquid antibacterial agent, a powder antibacterial agent and a granular antibacterial agent.

9. A method for inhibiting foodborne bacteria from infecting food, characterized in that: The method comprises the steps of using the crop mucus Lactobacillus PN5 and / or its fermentation product as described in claim 1, or the antibacterial product as described in any one of claims 5 to 8 in food production and processing.

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