Phytobacterium plantarum JNL0074, application thereof and microbial agent

Abstract

The invention discloses a plant lactobacillus JNL0074 strain, application thereof and a microbial agent, and belongs to the technical field of microorganisms. The lactobacillus plantarum JNL0074 is preserved in the China General Microbiological Culture Collection Center on June 24, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO: 31047, and the lactobacillus plantarum is classified and named as lactobacillus plantarum. The strain can realize high yield of indole-3-lactic acid in an MRS culture medium. An in-vitro experiment result shows that supernate of the strain remarkably inhibits inflammatory polarization of macrophages stimulated by lipopolysaccharide and secretion of inflammatory factors. Animal experiment results show that the strain can significantly inhibit sodium dextran sulfate induced colonic tissue inflammatory factor secretion and intestinal barrier injury, alleviate ulcerative colitis, and show a huge application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plant lactobacillus JNL0074, an application thereof and a microbial agent. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease with an unknown etiology, mainly including two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Currently, the first-line treatment drugs in clinical practice mainly include aminosalicylicylates, corticosteroids, biological drugs, and immunosuppressants. Not all IBD patients can benefit from these treatments, and these drugs also have a considerable risk of side effects.

[0003] The changes in the intestinal microecology of IBD patients are mainly manifested as dysbiosis, including changes in flora diversity, species and abundance. Probiotics can effectively regulate the imbalance of intestinal flora, improve the microecological environment, enhance the barrier function of the intestinal mucosa, regulate local and systemic immune responses, and provide new options for the treatment of diseases such as IBD. Bifidobacterium, Lactobacillus, VSL#3 probiotics, etc. have been widely used in clinical treatment.

[0004] Short-chain fatty acids (SCFAs), especially butyrate, produced by intestinal bacteria can induce the development of regulatory T cells (Tregs) and promote goblet cells to produce mucus, thereby strengthening the mucosal barrier.

[0005] Indole-3-lactic acid (ILA) is an indole compound produced by intestinal microbial metabolism of tryptophan. Studies have shown that ILA can reduce the polarization of T cells to pro-inflammatory Th17 cells, while also inhibiting the inflammatory polarization of macrophages and the release of pro-inflammatory cytokines, thereby alleviating intestinal inflammatory responses.

[0006] At present, the chemical synthesis of ILA may produce toxic by-products, and the preparation conditions are harsh, the process is complicated, and the cost is high. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a strain of Lactobacillus plantarum JNL0074, an application thereof and a microbial agent.

[0008] In one aspect of the present invention, a strain of Lactobacillus plantarum JNL0074 is provided. The Lactobacillus plantarum JNL0074 was deposited in the China General Microbiological Culture Collection Center on June 24, 2024, with a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO: 31047, and a classification name of Lactobacillus plantarum.

[0009] The present invention also provides the application of the Lactobacillus plantarum JNL0074 in producing indole-3-lactic acid.

[0010] The present invention also provides the use of the Lactobacillus plantarum JNL0074 in reducing the proportion of pro-inflammatory M1 macrophages.

[0011] The present invention also provides the use of the Lactobacillus plantarum JNL0074 in increasing the proportion of anti-inflammatory M2 macrophages.

[0012] The present invention also provides the use of the Lactobacillus plantarum JNL0074 in inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-α.

[0013] The present invention also provides application of the Lactobacillus plantarum JNL0074 in preparing medicine for treating intestinal inflammation.

[0014] The present invention also provides the use of the Lactobacillus plantarum JNL0074 in preparing medicine for treating colitis.

[0015] The present invention also provides a microbial preparation, which contains Lactobacillus plantarum JNL0074.

[0016] Furthermore, the microbial preparation also contains an excipient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention provides a plant lactobacillus JNL0074, which can produce high indole-3-lactic acid in MRS medium, 9-10 times higher than the commercial strain Lactobacillus rhamnosus LGG; the technical solution overcomes many disadvantages of chemical synthesis of ILA, uses microbial metabolism of tryptophan to produce indole-3-lactic acid, has low cost, mild reaction conditions, and is suitable for industrial production needs. In addition, the probiotics producing ILA can be colonized in the human intestine, can continuously secrete ILA, and regulate the host intestinal immune microenvironment.

[0019] 2. In vitro experiments show that the supernatant of Lactobacillus plantarum JNL0074 provided by the present invention significantly inhibits lipopolysaccharide (LPS)-stimulated macrophage inflammatory polarization and the secretion of inflammatory factors. Animal experimental results show that Lactobacillus plantarum JNL0074 can significantly inhibit the secretion of inflammatory factors and intestinal barrier damage in colon tissue induced by dextran sulfate sodium (DSS), alleviate ulcerative colitis, and show great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 This is the phylogenetic tree diagram of Lactobacillus plantarum JNL0074 in Example 1.

[0022] Figure 2 This is the Gram staining image of Lactobacillus plantarum JNL0074 in Example 1.

[0023] Figure 3 Graph showing the level of indole-3-lactic acid production by Lactobacillus plantarum JNL0074 in Example 4; wherein (a) is the level of ILA in the culture medium, and (b) is the level of ILA per OD 600 The level of ILA produced by bacteria.

[0024] Figure 4 The figure is a diagram showing the effect of the supernatant of Lactobacillus plantarum JNL0074 in Example 5 on inhibiting macrophage inflammatory polarization and secretion of inflammatory factors; wherein, (a) is the proportion of M1 macrophages, (b) is the proportion of M2 macrophages, (c) is the level of TNF-α in the cell supernatant, and (d) is the level of IL-6 in the cell supernatant; * is p<0.05, ** is p<0.01; *** is p<0.001, **** is p<0.0001.

[0025] Figure 5 The results of bacterial hemolytic analysis in Example 7; wherein (a) is α hemolysis, (b) is β hemolysis, and (c) is γ hemolysis.

[0026] Figure 6 The figure shows the effect of Lactobacillus plantarum JNL0074 in alleviating DSS-induced colitis in terms of body weight and disease activity index in Example 8; wherein (a) is body weight and (b) is disease activity index.

[0027] Figure 7This is a graph showing the effect of Lactobacillus plantarum JNL0074 on reducing DSS-induced colitis on colorectal length in Example 8; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

[0028] Figure 8 This is a diagram showing the effect of Lactobacillus plantarum JNL0074 in alleviating DSS-induced colitis in colon tissue H&E staining in Example 8.

[0029] Fig. 9 This is a diagram showing the effect of Lactobacillus plantarum JNL0074 in alleviating DSS-induced colitis in terms of the proportion of colonic immune cells and the level of colonic inflammatory factors in Example 8; * is p<0.05, ** is p<0.01; *** is p<0.001, **** is p<0.0001. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0032] The composition of the MRS culture medium used in the embodiment is as follows:

[0033] Peptone 10.0g / L, beef powder 8.0g / L, yeast powder 4.0g / L, glucose 20.0g / L, dipotassium hydrogen phosphate 2.0g / L, diammonium hydrogen citrate 2.0g / L, sodium acetate 5.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1.0g / L. pH value 5.7±0.2 (25℃).

[0034] The plant lactobacillus JNL0074 provided by the present invention is separated from the fermented pickles collected from farmers in Guizhou Province. The strain can produce high indole-3-lactic acid in MRS medium, which is 9-10 times higher than the commercial strain Lactobacillus rhamnosus LGG. In vitro experiments show that the supernatant of plant lactobacillus JNL0074 significantly inhibits the inflammatory polarization of macrophages and the secretion of inflammatory factors stimulated by lipopolysaccharide (LPS). Animal experiment results show that plant lactobacillus JNL0074 can significantly inhibit the secretion of inflammatory factors and intestinal barrier damage in colon tissue induced by dextran sulfate sodium (DSS), and alleviate ulcerative colitis.

[0035] Example 1: Bacterial separation and purification

[0036] Prepare MRS medium according to the instructions and sterilize at 118℃ for 20min. Take an appropriate amount of fermented yogurt, add physiological saline to dilute it in a gradient, evenly apply it on MRS solid medium, and culture it anaerobically at 37℃ for 48h. Pick a single clone and observe the bacterial morphology by Gram staining. Use primers 27F (5'-AGAGTTTGATCCTGGCT CAG-3') and 1492R (5'-GGCTTACCTTGTTACGACTT-3') to amplify bacterial 16S rRNA. The bacterial 16S rRNA gene sequence is:

[0037] 5’-TTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACT CTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCA-3'

[0038] By comparing the NCBI database, a phylogenetic tree was constructed, such as Figure 1 As shown, the alignment result is Lactiplantibacillus plantarum.

[0039] The plant lactobacillus JNL0074 in the present invention was deposited in the China General Microbiological Culture Collection Center on June 24, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 31047, and the classification name is Lactiplantibacillus plantarum.

[0040] Morphological characteristics of the Lactobacillus plantarum JNL0074 strain were analyzed by picking a single colony for Gram staining and observing under a microscope. Figure 2 As shown, Lactobacillus plantarum JNL0074 is short rod-shaped, 1.1-1.9 μm (length) × 0.6-0.9 μm (width), and is Gram-positive.

[0041] Example 2: Bacterial culture and counting

[0042] Prepare MRS medium according to the instructions for use and sterilize at 118℃ for 20min. After cooling to room temperature, inoculate Lactobacillus plantarum JNL0074 at a 1% inoculum and culture anaerobically at 37℃ for 24h. After culture, dilute the cultured bacterial solution with physiological saline gradient, take 100μL and evenly spread it on MRS solid medium, and culture anaerobically at 37℃ for 48h. Count the bacterial growth according to the number of colonies grown. OD value of bacterial solution after 24h culture of Lactobacillus plantarum JNL0074 600 =4.35, the number of viable bacteria is 1.5×10 9 CFU / mL. OD of bacterial solution after 36h of culture 600 =3.71, OD of bacterial solution at 48h 600 =3.75.

[0043] Example 3: Bacterial supernatant separation

[0044] The cultured bacteria were centrifuged at 12000 rpm and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane.

[0045] Example 4: Analysis of indole-3-lactic acid production by probiotics

[0046] Indole-3-lactic acid single standard stock solution was prepared with 50% methanol and diluted with 10% methanol. 200μL of Lactobacillus plantarum JNL0074 bacterial supernatant was added with 10% methanol at a final concentration and vigorous shaking was performed, and centrifuged at 12000rpm for 5min. The standard and the sample after bacterial supernatant extraction were injected and analyzed. TSQ Altis targeted quantitative mass spectrometer analysis. ACQUITY The column temperature of HSS T3 chromatographic column was 40℃, and the mobile phases were A-0.1% formic acid water, B-0.1% formic acid methanol. The gradient elution conditions were: 0-1min, 5% B; 1-2min, 5%-50% B; 2-5min, 50%-95% B; 6-8min, 95%-5% B. Mass spectrometry conditions: electrospray ionization (ESI) source, positive ionization mode. The ion source temperature was 500℃, the ion source voltage was 5500V, the collision gas was 6psi, the air curtain gas was 30psi, and the nebulizer gas and auxiliary gas were both 50psi. Multiple reaction monitoring (MRM) and retention time locking were used for scanning. The concentration of indole-3-lactic acid in the bacterial supernatant of Lactobacillus plantarum JNL0074 was quantitatively analyzed according to the ion intensity of the standard.

[0047] The same number of Lactobacillus plantarum JNL0074 and Lactobacillus rhamnosus LGG were inoculated in MRS liquid culture medium and cultured anaerobically at 37°C. The bacterial OD600 and the ILA content in the fermentation broth were detected at 12h, 24h, 36h and 48h. The results showed that after 48h of culture, the ILA content in the culture medium of Lactobacillus plantarum JNL0074 was 2.22μg / mL, while the ILA content in the culture medium of Lactobacillus rhamnosus LGG was about 0.23μg / mL. Figure 3 As shown in (a) and (b).

[0048] Example 5: Analysis of in vitro anti-inflammatory levels of probiotic supernatant

[0049] RAW264.7 cells were implanted into 12-well plates (1×106 cells / well) and incubated in a CO2 incubator at 37°C for 12 hours to adhere to the wall. Lipopolysaccharide LPS (5μg / mL) was added to stimulate the inflammatory response of RAW264.7 cells. After 6 hours, 50μL of blank MRS medium and Lactobacillus plantarum JNL0074 supernatant were added, respectively, and incubated at 37°C in a CO2 incubator for 24 hours. Mouse TNF-α (Biyuntian, PT512) and IL-6 (Biyuntian, PI326) ELISA kits were used to detect the levels of inflammatory factors TNF-α and IL-6 in the cell supernatant, respectively. Antibodies against mouse F4 / 80, CD206 and CD86 were used to label cells, and cell populations were analyzed by flow cytometry.

[0050] After 6 hours of LPS stimulation of RAW264.7 cells, the supernatants of Lactobacillus plantarum JNL0074 and Lactobacillus rhamnosus LGG were added and incubated for 24 hours. Flow cytometry analysis showed that compared with the LPS treatment group, Lactobacillus plantarum JNL0074 reduced the proportion of pro-inflammatory M1 macrophages and significantly increased the proportion of anti-inflammatory M2 macrophages. Figure 4As shown in (a) and (b). The results of ELISA analysis showed that Lactobacillus plantarum JNL0074 significantly inhibited the secretion of pro-inflammatory cytokines IL-6 and TNF-α. Figure 4 As shown in (c) and (d). Moreover, Lactobacillus plantarum JNL0074 has a better inhibitory effect on inflammation than Lactobacillus rhamnosus LGG.

[0051] Example 6, bacterial antibiotic sensitivity analysis

[0052] Lactobacillus plantarum JNL0074 was inoculated in liquid MRS medium and cultured anaerobically at 37°C for 24 hours to obtain seed liquid. The bacterial liquid was diluted to 0.5 McFarland turbidity with 0.85% saline, 100 μL of bacterial liquid was accurately aspirated, and evenly coated on the surface of MRS agar medium. After the surface bacterial liquid was dried, a drug-sensitive paper was placed on the surface of the culture medium. The sensitivity of Lactobacillus plantarum JNL0074 to six antibiotics, including ampicillin (10 μg / piece), gentamicin (10 μg / piece), kanamycin (30 μg / piece), streptomycin (10 μg / piece), clindamycin (2 μg / piece) and chloramphenicol (30 μg / piece), was detected by KB paper diffusion method. The sensitivity of each bacterial strain to each antibiotic was repeated three times. Cultured anaerobically at 37°C for 24 hours. The diameter of the inhibition zone was measured and recorded, and compared with the diameter of the inhibition zone of the reference strain in the instruction manual. The results are shown in Table 1.

[0053] Table 1 Antibiotic inhibition zone of Lactobacillus plantarum JNL0074

[0054]

[0055] Example 7, Bacterial Hemolytic Analysis

[0056] Prepare Columbia blood agar plates: 23.0g animal tissue hydrolysate, 1.0g starch, 5.0g NaCl, 15g agar, 1000mL distilled water, pH 7.3. Sterilize at 121℃ for 15min. When cooled to 50℃, add 5% defibrinated sheep blood. Cool and set aside. Keep at room temperature for no more than 4h, and store at 4℃ for no more than 7d. Streak Lactobacillus plantarum JNL0074 and control strains on Columbia blood agar plates. Incubate anaerobically at 37℃ for 18-24h, and observe the colony morphology. α-hemolysis is manifested as a narrow, grass-green hemolysis ring; β-hemolysis is manifested as a wide, well-defined, completely transparent hemolysis ring; γ-hemolysis is non-hemolysis, that is, no hemolysis ring appears. Figure 5As shown in (a), (b), and (c), A. viridis was used as an α-hemolytic control strain, Streptococcus pyogenes CICC10373 was used as a β-hemolytic control strain, and Lactobacillus plantarum JNL0074 did not show a hemolytic ring.

[0057] Example 8: Probiotics alleviate DSS-induced colitis

[0058] After one week of adaptive feeding, 6-8 week old male C57Bl / 6N mice were allowed to drink freely 2.5% DSS (molecular weight 36,000-50,000Da) aqueous solution to induce colitis mouse model. Plant Lactobacillus plantarum JNL0074 cultured for 24 hours was centrifuged at 5000rpm and 4℃ for 5 minutes, the supernatant was discarded, and the cells were resuspended in physiological saline. Each mouse was gavaged with 2×10 9 CFU. 5-Amino Salicylic Acid (5-ASA, 100 mg / kg / d) was used as a positive drug control. The experimental period was 7 days, during which the weight, diarrhea, blood in the stool and activity status of the mice were monitored daily. The mice were anesthetized and killed by cervical dislocation, and the colon tissues were collected and analyzed by hematoxylin-eosin (H&E) staining. Flow cytometry was used to analyze the proportion of regulatory T cells (Treg) and macrophages in the colon tissue of mice, and RT-qPCR was used to detect the transcription levels of inflammatory factors IL-β, IL-6 and TNF-α in the colon tissue of mice.

[0059] Disease activity index (DAI) scoring criteria for colitis mice (Table 2):

[0060] Table 2 DAI scoring criteria for colitis model mice

[0061]

[0062] The DAI score is the sum of the scores of weight loss, diarrhea, and blood in stool.

[0063] The colitis mouse model was induced by DSS aqueous solution, and 2×10 9 CFU of Lactobacillus plantarum JNL0074 or Lactobacillus rhamnosus LGG. Lactobacillus plantarum JNL0074 has a tendency to slow down the weight loss of colitis mice and reduce the disease activity index (DAI score) of mice, such as Figure 6 As shown in (a) and (b). The improvement effects of Lactobacillus rhamnosus LGG and 5-ASA on the body weight and disease activity index of mice were weak. Treatment with Lactobacillus plantarum JNL0074 could improve the colon shortening caused by DSS, significantly enhance the integrity of the intestinal barrier, and inhibit the infiltration of inflammatory cells in the colon tissue and the destruction of the mucosa, such as Figure 7 and Figure 8As shown. 5-ASA can inhibit colonic inflammatory cell infiltration and protect the integrity of the intestinal barrier, but Lactobacillus rhamnosus LGG has a weaker protective effect on the intestinal tissue structure. Flow cytometry analysis results showed that Lactobacillus plantarum JNL0074 significantly increased the proportion of Treg cell populations in the colon propria, while reducing the proportion of proinflammatory M1 macrophages; RT-qPCR analysis results showed that Lactobacillus plantarum JNL0074 could significantly inhibit the levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in colon tissue, indicating that Lactobacillus plantarum JNL0074 can effectively inhibit intestinal tissue inflammation, such as Fig. 9 As shown in the figure, the inhibitory effect of Lactobacillus rhamnosus LGG on intestinal inflammation is weaker than that of Lactobacillus plantarum JNL0074.

[0064] Example 7, a microbial preparation

[0065] The supernatant containing Lactobacillus plantarum JNL0074 prepared in Example 3 can be used to prepare drugs.

[0066] As an embodiment, in the medicine, Lactobacillus plantarum JNL0074 is the only effective active ingredient.

[0067] The supernatant may be freeze-dried to prepare a lyophilized powder.

[0068] It can also be combined with a solvent or other excipients for the treatment of colitis. When combined with a solvent, an injection can be prepared. When combined with other solid excipients, other conventional dosage forms such as tablets and capsules can be prepared.

[0069] Alternatively, as another embodiment, Lactobacillus plantarum JNL0074 is combined with other active ingredients for treating colitis.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum JNL0074, characterized in that: The plant lactobacillus JNL0074 was deposited in the China General Microbiological Culture Collection Center on June 24, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 31047, and the classification name is Lactiplantibacillus plantarum.

2. Use of the plant lactobacillus JNL0074 according to claim 1 in producing indole-3-lactic acid.

3. Use of Lactobacillus plantarum JNL0074 according to claim 1 in reducing the proportion of pro-inflammatory M1 macrophages.

4. Use of Lactobacillus plantarum JNL0074 according to claim 1 in increasing the proportion of anti-inflammatory M2 macrophages.

5. Use of the Lactobacillus plantarum JNL0074 according to claim 1 in inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-α.

6. Use of Lactobacillus plantarum JNL0074 according to claim 1 in preparing medicines for treating intestinal inflammation.

7. Use of the Lactobacillus plantarum JNL0074 according to claim 1 in the preparation of a medicament for treating colitis.

8. A microbial preparation, characterized in that: The method comprises the Lactobacillus plantarum JNL0074 according to any one of claims 1 to 7.

9. The microbial preparation according to claim 8, characterized in that Also included are excipients.

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