Lactobacillus pocortisone NM3 and application thereof

Abstract

The invention discloses a lactobacillus bornicaule NM3 and application thereof, and belongs to the field of microorganisms. The lactobacillus pocortisone NM3 is preserved in the Guangdong Microbiological Culture Collection Center, and the preservation number is GDMCC NO: 65604. 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 Baltic Lactobacillus NM3 and application thereof. Background Art

[0002] The intestinal microbiota is closely linked to the animal's intake and conversion of various nutrients, and they play a key role in enhancing the host's disease resistance. When the intestinal microecology is balanced, the indigenous flora will not only not harm the host, but will be able to synthesize essential nutrients such as vitamins and assist the host in digesting food. In addition, these flora can also play a role in biological antagonism and immune defense against foreign pathogens and potential pathogens in the intestine. Therefore, maintaining the balance of the intestinal microecology is crucial to the health of the host.

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

[0004] The purpose of the present invention is to provide a Baltic Lactobacillus NM3 and its application to solve the problems existing in the above-mentioned prior art. The Baltic Lactobacillus NM3 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 Baltic Lactobacillus (Limosilactobacillus balticus) NM3. The Baltic Lactobacillus NM3 is stored in Guangdong Microbiological Collection Center with a storage number of GDMCC NO: 65604.

[0007] The present invention also provides the use of the Baltic Lactobacillus NM3 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 Baltic Lactobacillus NM3 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 Baltic Lactobacillus NM3 and / or its fermentation product, or the antibacterial product in food production and processing.

[0015] The present invention also provides the use of the Baltic Lactobacillus NM3 in preparing a probiotic product.

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

[0017] The present invention screens out a Baltic Lactobacillus NM3 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 to prepare products for preventing and treating pathogenic bacteria, 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 NM3;

[0020] Figure 2 is the Gram staining image of strain NM3;

[0021] Figure 3 is the phylogenetic tree of strain NM3 based on 16S rDNA sequence;

[0022] Figure 4 is the growth curve of strain NM3;

[0023] Figure 5 This is the acid resistance result diagram of strain NM3;

[0024] Figure 6 This is the bile salt resistance result diagram of strain NM3;

[0025] Figure 7 This is a diagram of the self-aggregation effect of strain NM3. 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 those skilled in the art. 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] In a first aspect, the present invention provides a Baltic Lactobacillus NM3, the strain is classified and named: Baltic Lactobacillus, Latin name: Limosilactobacillus balticus.

[0032] The Baltic Lactobacillus NM3 of the present invention is derived from chicken intestines. According to the colony morphology test, the colony of the Baltic Lactobacillus NM3 is opaque white, with protrusions in a ring shape and a smooth surface; the cell morphology is rod-shaped, and the result of Gram staining is positive. It has no flagella, no movement and no spores.

[0033] The research found that the Baltic Lactobacillus NM3 provided by the present invention has a good inhibitory effect on pathogenic bacteria including Salmonella and Escherichia coli, has good application potential in the prevention and treatment of pathogenic bacteria, and is a probiotic strain worthy of development.

[0034] The method for screening Baltic Lactobacillus NM3 of the present invention comprises the following steps:

[0035] The strain sampled from the chicken intestine was separated and purified to obtain a single colony, which was then identified to obtain Baltic Lactobacillus NM3.

[0036] The above-mentioned screening method of Baltic Lactobacillus NM3 is simple and convenient, and can quickly obtain multiple strains including Baltic Lactobacillus NM3.

[0037] 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.

[0038] 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.

[0039] In a second aspect, the present invention provides a bacterial agent, including Baltic Lactobacillus NM3, and may also include other strains or auxiliary materials, etc. The bacterial agent has all the beneficial effects of the Baltic Lactobacillus NM3 of the present invention.

[0040] In some preferred embodiments, the dosage form of the bacterial agent includes liquid bacterial agent, powder and granular bacterial agent.

[0041] In a third aspect, the present invention provides a fermentation product obtained by fermentation of Baltic Lactobacillus NM3 or a bacterial agent.

[0042] Antibacterial experiments have shown that the fermentation products of Baltic Lactobacillus NM3 have good inhibitory effects on pathogenic bacteria including Salmonella and Escherichia coli, and can be used to prepare antibacterial products.

[0043] In a fourth aspect, the present invention provides a use of Baltic Lactobacillus NM3, a bacterial agent or a fermentation product in the preparation of a product for preventing and treating pathogenic bacteria.

[0044] In a fifth aspect, the present invention provides a product for preventing and treating pathogenic bacteria, comprising Baltic Lactobacillus NM3, a bacterial agent or a fermentation product.

[0045] In a sixth aspect, the present invention provides a method for inhibiting foodborne bacteria from infecting food, comprising using the Baltic Lactobacillus NM3 and / or its fermentation product, or the antibacterial product in food production and processing.

[0046] The above-mentioned food production includes the production of livestock and poultry related by-products.

[0047] 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.

[0048] Table 1 Test strains and culture medium

[0049]

[0050] The culture medium and components involved in the embodiments of the present invention are as follows:

[0051] 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).

[0052] 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).

[0053] Example 1 Isolation of Baltic Lactobacillus NM3 strain

[0054] 1. Sample collection and processing

[0055] Healthy ephedra chickens were slaughtered, and the chicken intestines were removed. The duodenum, jejunum, ileum, and cecum were cut open using sterilized scissors and forceps, and placed in 4 sterile plates. The intestines were cut open with scissors, and the mucosa was scraped with a sterilized spoon. 0.1 g of the scraped material was taken and placed in a pre-labeled sterilized 1.5 mL EP tube, and gradient dilution was performed to 10 -3 ~10 -6 , and then spread on MRS-CaCO3 solid culture medium.

[0056] 2. Isolation and purification of strains

[0057] Select dilutions of different dilutions (dilutions are 10 -2 , 10 -3 and 10 -4 ), take 200 μL of each dilution and spread it evenly on the MRS-CaCO3 plate, then use a vacuum compression bag and deoxidizer to perform anaerobic culture at 37°C for 1 to 2 days. After the culture is completed, observe whether there is a clear calcium-dissolving circle on the plate, pick the colonies with larger calcium-dissolving circles, and then streak them on the MRS-CaCO3 plate and culture them at 37°C for 1 to 2 days. Repeat 2 to 3 times to obtain pure culture (such as Figure 1 The pure culture strain screened was named NM3.

[0058] Example 2 Identification of strains

[0059] 1. Physiological and biochemical tests of strain NM3

[0060] The purified strain NM3 was subjected to Gram staining ( Figure 2 The results of the experiment were compared with the Bergey's Manual of Systematic Bacteriology, 8th Edition, to make a preliminary determination of the strain. The results showed that the screened strain NM3 was purple in Gram stain and positive ( Figure 2 The cell shape is rod-shaped, catalase and oxidase are negative, no spore formation, and the results of physical and chemical tests are shown in Table 2.

[0061] Table 2 Physiological and biochemical identification results of strain NM3

[0062]

[0063] Note: "+" in the table means positive; "-" means negative

[0064] 2. Molecular level identification

[0065] The genome of the strain was extracted using the Tiangen Universal DNA Purification and Recovery Kit (centrifugal column type) DP302. The genomic DNA was used as a template to amplify the 16S rDNA gene fragment of the target strain. The forward primer was 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1); the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The PCR reaction system is shown in Table 3, and the PCR amplification program is shown in Table 4. After the reaction, 4 μL of the PCR reaction product was taken out and electrophoresed on an agarose gel for 30 minutes. Primer synthesis and PCR amplification product sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd. The sequence obtained by sequencing was searched for homology using BLAST on the NCBI website, and the 16S rDNA sequence of the standard strain with higher homology was downloaded from Genebank. Multiple sequence alignment was performed in the MEGA11 software, and the phylogenetic tree was constructed using the neighbor-joining method (NJ). Figure 3 As shown. Figure 3 It can be seen that strain NM3 and Limosilactobacillus balticus BG-AF3-A (MT823145) are clustered together, so the strain is named Limosilactobacillus balticus NM3. The strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 10, 2024, with the deposit number GDMCC NO: 65604, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0066] Table 3 PCR reaction system (50 μL)

[0067]

[0068] Table 4 PCR amplification program

[0069]

[0070] Example 3 Determination of the growth curve of Baltic Lactobacillus NM3

[0071] (1) Preparation of fermentation broth

[0072] The Baltic Lactobacillus NM3 stored at -80°C was inoculated into the MRS liquid medium, cultured at 37°C for 24 hours, and transferred twice continuously to complete the activation of the strain. Then, the activated Baltic Lactobacillus NM3 was inoculated into 200 mL of the MRS liquid medium at a volume of 2%, shaken evenly, and cultured at 37°C for 24 hours to obtain the fermentation liquid.

[0073] (2) Determination of growth curve

[0074] Take the Baltic Lactobacillus NM3 after activation for one generation, inoculate it into 200mL LMRs liquid medium at a volume of 2%, shake it evenly, place it in a static culture at 37°C, take samples every 6 hours, and measure its OD 600nm , draw the growth curve of Baltic Lactobacillus NM3, such as Figure 4 As shown. Figure 4 It can be seen that Baltic Lactobacillus NM3 enters the stable period from about 12 hours.

[0075] Example 4 Determination of acid and bile resistance of Baltic Lactobacillus NM3

[0076] 1. Acid resistance test

[0077] The activated lactic acid bacteria seed liquid was added to the MRS liquid culture medium with pH values ​​of 2.0 and 3.0 at an inoculation rate of 1% (v / v), and an MRS liquid culture medium without acid was set as a control group. After 37°C static culture for 3 hours, 200 μL of bacterial liquid was taken from the acid-treated group and the control group, and then appropriately diluted and evenly spread on the MRS solid culture medium plate. Each dilution was paralleled 3 times. After 24 hours of static culture at 37°C, the plates with colony counts between 30 and 300 were selected for live bacteria count and survival rate calculation. The survival rate calculation formula is:

[0078] X / %=A / A0×100,

[0079] Where: X is the survival rate of the acid-treated group; A is the number of viable bacteria per mL after 3 hours of treatment, lg CFU / mL; A0 is the number of viable bacteria in the control group, lg CFU / mL.

[0080] The results of the acid resistance test of strain NM3 are as follows Figure 5 The results showed that the survival rate was 63.37% at pH = 2 and 86.87% at pH = 3, indicating that strain NM3 has a strong acid resistance.

[0081] 2. Bile salt tolerance test

[0082] The activated lactic acid bacteria seed liquid was inoculated into MRS liquid culture medium with pig bile salt concentration of 0.1% and 0.3% at an inoculum volume of 1% (v / v). Three parallel tests were set for each concentration. The control group used MRS liquid culture medium without pig bile salt. The live bacteria were counted after static culture at 37°C for 3 hours. The survival rate calculation formula is:

[0083] Y / %=B / B0×100

[0084] Where: Y is the survival rate of the pig bile salt group; B is the number of viable bacteria per mL after 3 hours of treatment, lg CFU / mL; B0 is the number of viable bacteria in the control group, lg CFU / mL

[0085] The results of the bile salt resistance test of strain NM3 are shown in Figure 6 The results showed that the strain could not survive when the bile salt mass fraction was 0.30%, while the survival rate was 72.16% when the bile salt mass fraction was 0.1%, indicating that the strain NM3 had a certain bile salt tolerance.

[0086] Example 5 Application of Baltic Lactobacillus NM3 in antibacterial

[0087] Baltic Lactobacillus NM3 was fermented according to the method of Example 3(1), and the supernatant was collected by centrifugation for antibacterial experiment. The results are shown in Table 5.

[0088] Table 5 Inhibitory effect of strain NM3 on different indicator bacteria

[0089]

[0090]

[0091] 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).

[0092] Example 6 Drug Sensitivity Test

[0093] The drug sensitivity paper diffusion method (also known as the drug paper agar diffusion method) was used. The seed solution was prepared according to the method of Example 3 (1), and 200 μL of the bacterial solution was evenly spread on the MRS solid culture medium plate. Three similar antibiotic drug sensitivity paper discs were made in parallel for each culture medium. After culturing at 37°C for 24 hours, the diameter of the inhibition zone was measured. The 20 commonly used antibiotic drug sensitivity paper discs used included: penicillins (ampicillin, amoxicillin, penicillin G); cephalosporins (cefotaxime, ceftazidime, cephradine, cefepime); aminoglycosides (gentamicin, streptomycin, kanamycin); vancomycin; erythromycin; tetracycline; ciprofloxacin; chloramphenicol; sulfisoxazole; rifampicin; polymyxin B and lincomycin.

[0094] The determination was made according to the "2010CLSIM100-S20 Antimicrobial Susceptibility Test Implementation Standard" of the American Clinical Laboratory Standards Institute. The results are shown in Table 6: NM3 is sensitive to cephalosporins, penicillins (except penicillin G), erythromycin, chloramphenicol and rifampicin, but is resistant to aminoglycosides (except gentamicin), vancomycin, tetracycline, ciprofloxacin, sulfamethoxazole and lincomycin; the sensitivity to polymyxin B is intermediate. Probiotics may become carriers of antibiotic resistance and lead to the failure of antibiotic treatment. Strain NM3 is sensitive to multiple antibiotics, so it is less likely to transmit resistance genes to pathogens.

[0095] Table 6 Drug sensitivity test results of strain NM3

[0096]

[0097]

[0098] Note: S: sensitive; I: intermediate; R: resistant.

[0099] Example 7 Self-aggregation determination

[0100] Cultured in MRS broth at 37°C for 24 h, the cells were collected by centrifugation at 8000 r / min for 10 min. After washing twice in sterile PBS, the cells were resuspended in PBS to make A 600nm When the OD was between 1.0±0.2, the initial absorbance A0 at 600 nm was recorded. The suspension was incubated at 37°C for 5 h. The final absorbance A1 of the supernatant was measured at 600 nm.

[0101] Self-polymerization formula: Self-polymerization (%) = (A0-A1) / A0×100%

[0102] like Figure 7 As shown, the self-aggregation of Lactobacillus balticus NM3 was 16.97%.

[0103] 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 Baltic Lactobacillus (Limosilactobacillus balticus) NM3, characterized in that: The Baltic Lactobacillus NM3 is stored in Guangdong Provincial Microbiological Collection Center with a storage number of GDMCC NO: 65604.

2. Use of the Baltic Lactobacillus NM3 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 active ingredients include the Baltic Lactobacillus NM3 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 Baltic Lactobacillus NM3 and / or its fermentation product described in claim 1, or the antibacterial product described in any one of claims 5 to 8 in food production and processing.

10. Use of the Baltic Lactobacillus NM3 as claimed in claim 1 in the preparation of a probiotic product.

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