A strain of Lactobacillus pentosus JNL0030, its application and a microbial preparation

Abstract

The present invention discloses a strain of Lactobacillus pentosus JNL0030, its application and a microbial preparation, which belong to the field of microbial technology. The Lactobacillus pentosus JNL0030 was deposited in the China General Microbiological Culture Collection Management Center on June 24, 2024, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC NO:31045, and a classification name of Lactobacillus pentosus. The strain can produce high indole-3-lactic acid in MRS culture medium. In vitro experimental results showed that the supernatant of the strain significantly inhibited the inflammatory polarization of macrophages and the secretion of inflammatory factors stimulated by lipopolysaccharide. Animal experimental results showed that the strain can significantly inhibit the secretion of inflammatory factors and intestinal barrier damage in colon tissue induced by sodium dextran sulfate, alleviate ulcerative colitis, and show great application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus pentosus JNL0030, an application thereof and a microbial preparation. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract caused by multiple factors such as environment, genetics, immunity and microorganisms. It includes two subtypes: ulcerative colitis (UC) and Crohn's disease (CD). At present, traditional treatments for IBD include 5-aminosalicylic acid, glucocorticoids, immunosuppressants, etc. These drugs only relieve clinical symptoms but cannot prevent the disease from worsening. They have poor clinical efficacy, often cause recurrence of the disease, and ultimately lead to problems such as an increase in the rate of surgical operations.

[0003] The typical feature of IBD is intestinal microecological imbalance. Probiotics can inhibit pathogenic bacteria and repair damaged intestinal microecology, and many studies have reported that probiotics have anti-inflammatory effects in vivo and in vitro. Moreover, probiotics colonize in the intestines and can exert a continuous therapeutic effect with low side effects. Therefore, the use of probiotics to treat IBD has great advantages.

[0004] 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, inhibit the inflammatory polarization of macrophages, inhibit the release of pro-inflammatory cytokines, and reduce intestinal inflammatory response. In addition, ILA can be used by intestinal commensal bacteria to produce indole-3-propionic acid, thereby enhancing CD8 + The efficacy of T cell-mediated aPD-1 tumor immunotherapy. Therefore, indole-3-lactic acid can play an anti-inflammatory and anti-tumor role in the body and has great value in drug development.

[0005] At present, chemical synthesis of ILA may produce toxic byproducts, and the preparation conditions are harsh, the process is complicated, and the cost is high. Microbial metabolism of tryptophan to produce indole-3-lactic acid has low cost and mild reaction conditions, which is suitable for industrial production needs. In addition, ILA-producing probiotics can colonize the human intestine, can continuously secrete ILA, and regulate the host intestinal immune microenvironment. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a strain of Lactobacillus pentosaceus JNL0030, an application thereof and a microbial preparation.

[0007] In one aspect of the present invention, a strain of Lactobacillus pentosus JNL0030 is provided. The Lactobacillus pentosus JNL0030 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: 31045, and a classification name of Lactobacillus pentosus.

[0008] The present invention also provides the application of the Lactobacillus pentosus JNL0030 in producing indole-3-lactic acid.

[0009] The present invention also provides the use of the Lactobacillus pentosus JNL0030 in reducing the proportion of pro-inflammatory M1 macrophages.

[0010] The present invention also provides the use of the Lactobacillus pentosus JNL0030 in increasing the proportion of anti-inflammatory M2 macrophages.

[0011] The present invention also provides the use of the Lactobacillus pentosus JNL0030 in inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-α.

[0012] The present invention also provides application of the Lactobacillus pentosus JNL0030 in preparing medicine for treating intestinal inflammation.

[0013] The present invention also provides the use of the Lactobacillus pentosus JNL0030 in preparing medicines for treating colitis.

[0014] The present invention also provides a microbial preparation, which contains Lactobacillus pentosaceus JNL0030.

[0015] Furthermore, the microbial preparation also contains an excipient.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present application provides a strain of Lactobacillus pentosus JNL0030 and its application, which can produce high indole-3-lactic acid in MRS medium, about 22 times higher than the commercial strain Lacticaseibacillus rhamnosus LGG. It overcomes many disadvantages of chemical synthesis of ILA, uses microbial metabolism of tryptophan to produce indole-3-lactic acid, has low cost and mild reaction conditions, and is suitable for industrial production needs. In addition, ILA-producing probiotics can colonize the human intestine, can continuously secrete ILA, and regulate the host intestinal immune microenvironment.

[0018] 2. In vitro experiments show that the supernatant of Lactobacillus pentosus JNL0030 provided by the present invention significantly inhibits inflammatory polarization of macrophages and secretion of inflammatory factors stimulated by lipopolysaccharide (LPS). Animal experimental results show that Lactobacillus pentosus JNL0030 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

[0019] 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.

[0020] Figure 1 This is a phylogenetic tree diagram of the Lactobacillus pentosus JNL0030 strain in Example 1.

[0021] Figure 2 This is a morphological diagram of the Lactobacillus pentosus JNL0030 strain in Example 1.

[0022] Figure 3 Figure 4 shows the level of indole-3-lactic acid produced by Lactobacillus pentosus JNL0030 in Example 4; (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.

[0023] Figure 4 The figure shows the effect of the supernatant of Lactobacillus pentosus JNL0030 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.

[0024] 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.

[0025] Figure 6 The figures are diagrams showing the effects of Lactobacillus pentosus JNL0030 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.

[0026] Figure 7This is a graph showing the effect of Lactobacillus pentosus JNL0030 in 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.

[0027] Figure 8 This is a diagram showing the effect of Lactobacillus pentosus JNL0030 in alleviating DSS-induced colitis in colon tissue H&E staining in Example 8.

[0028] Fig. 9 This is a graph showing the effect of Lactobacillus pentosus JNL0030 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

[0029] 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.

[0030] 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.

[0031] The composition of the MRS culture medium used in the embodiment is as follows:

[0032] 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℃).

[0033] The Lactobacillus pentosus JNL0030 provided by the present invention is isolated from fermented yogurt collected in Yunnan Province. The strain can produce high indole-3-lactic acid in MRS culture medium, which is about 22 times higher than the commercial strain Lactobacillus rhamnosus LGG. In vitro experiments show that the supernatant of Lactobacillus pentosus JNL0030 significantly inhibits the inflammatory polarization of macrophages and the secretion of inflammatory factors stimulated by lipopolysaccharide (LPS). Animal experimental results show that Lactobacillus pentosus JNL0030 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.

[0034] Example 1, Bacterial Isolation and Purification

[0035] 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'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTTACCTTGTTACGACTT-3') to amplify bacterial 16S rRNA. The bacterial 16S rRNA gene sequence is:

[0036]

[0037] By comparing the NCBI database, a phylogenetic tree was constructed, such as Figure 1 As shown, the alignment result is Lactiplantibacillus pentosus.

[0038] The Lactobacillus pentosus JNL0030 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: 31045, and the classification name is Lactiplantibacillus pentosus.

[0039] The morphological characteristics of the Lactobacillus pentosus JNL0030 strain are analyzed as follows: Figure 2 As shown, a single colony was picked for Gram staining and observed under a microscope. Lactobacillus pentosus JNL0030 was short rod-shaped, 1.1-1.4 μm (length) × 0.6-0.7 μm (width), and was Gram-positive.

[0040] Example 2, bacterial culture and counting

[0041] Prepare MRS medium according to the instructions for use and sterilize at 118℃ for 20min. After cooling to room temperature, inoculate Lactobacillus pentosaceus JNL0030 at a 1% inoculation rate and culture anaerobically at 37℃ for 24h. After culture, the cultured bacterial solution was gradient diluted with physiological saline, and 100μL was evenly spread on MRS solid medium and cultured anaerobically at 37℃ for 48h. The bacterial growth was counted according to the number of colonies grown. OD value of bacterial solution after 24h culture of Lactobacillus pentosaceus JNL0030 600 =4.21, the number of viable bacteria is 3.1×10 9 CFU / mL. OD of bacterial solution after 36h of culture 600 =3.77, OD of 48h bacterial solution 600 =3.40.

[0042] Example 3, bacterial supernatant separation

[0043] 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.

[0044] Example 4, Analysis of bacterial indole-3-lactic acid production levels

[0045] Prepare the single standard stock solution of indole-3-lactic acid with 50% methanol and dilute it with 10% methanol. Add 200μL of the bacterial supernatant of Lactobacillus pentosaceus JNL0030 to a final concentration of 10% methanol and shake vigorously to mix, then centrifuge at 12000rpm for 5min. Inject and analyze the extracted samples of the standard and bacterial supernatant. Targeted quantitative mass spectrometry 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 ion 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 pentosaceus JNL0030 was quantitatively analyzed according to the ion intensity of the standard.

[0046] The same number of Lactobacillus pentosus JNL0030 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 36-48h of culture, the ILA content in the culture medium of Lactobacillus pentosus JNL0030 was 5.1-5.3μg / mL, while the highest ILA content in the culture medium of Lactobacillus rhamnosus LGG was about 0.23μg / mL. Figure 3 As shown in (a) and (b).

[0047] Example 5, Anti-inflammatory Level Analysis

[0048] RAW264.7 cells were implanted into 12-well plates (1×106 cells / well) and incubated at 37°C in a CO2 incubator 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 pentosaceus JNL0030 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.

[0049] After 6 hours of LPS stimulation of RAW264.7 cells, the bacterial supernatants of Lactobacillus pentosus JNL0030 and Lactobacillus rhamnosus LGG were added and incubated for 24 hours. Flow cytometry analysis showed that compared with the LPS treatment group, Lactobacillus pentosus JNL0030 had a tendency to reduce the proportion of pro-inflammatory M1 macrophages (p = 0.0971), while significantly increasing the proportion of anti-inflammatory M2 macrophages, such as Figure 4 As shown in (a) and (b) of Figure 2, ELISA analysis results showed that Lactobacillus pentosus JNL0030 significantly inhibited the secretion of pro-inflammatory cytokines IL-6 and TNF-α. Figure 4 As shown in (c) and (d). Moreover, Lactobacillus pentosus JNL0030 has a better inhibitory effect on inflammation than Lactobacillus rhamnosus LGG.

[0050] Example 6, bacterial antibiotic sensitivity analysis

[0051] Lactobacillus pentosus JNL0030 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 pentosus JNL0030 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. Anaerobic culture was performed at 37°C for 24 h. 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.

[0052] Table 1 Antibiotic inhibition zone of Lactobacillus delbrueckii JNL0010

[0053]

[0054] Example 7, Bacterial Hemolytic Analysis

[0055] Prepare Columbia blood agar plates: 23.0g animal tissue hydrolysate, 1.0g starch, 5.0g NaCl, 15g agar, 1000mL distilled water, pH7.3. 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 pentosus JNL0030 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 5 As shown in (a), (b), and (c), A. viridis was used as an α-hemolytic control strain, and S. pyogenes CICC10373 was used as a β-hemolytic control strain. Lactobacillus pentosus JNL0030 did not show a hemolytic ring. Example 8: Probiotics alleviate DSS-induced colitis

[0056] After 1 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. Lactobacillus pentosus JNL0030 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. The daily oral dose for each mouse was 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.

[0057] Disease activity index (DAI) scoring criteria for colitis mice (Table 2):

[0058] Table 2 DAI scoring criteria for colitis model mice

[0059]

[0060] The DAI score is the sum of the scores of weight loss, diarrhea, and blood in stool.

[0061] The colitis mouse model was induced by DSS aqueous solution, and 2×10 9CFU of Lactobacillus pentosus JNL0030 or Lactobacillus rhamnosus LGG. Lactobacillus pentosus JNL0030 significantly slowed down the weight loss of colitis mice and reduced the disease activity index 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 pentosus JNL0030 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 8 As shown. 5-ASA can inhibit colonic inflammatory cell infiltration and protect the integrity of the intestinal barrier, but the protective effect of Lactobacillus rhamnosus LGG on the intestinal tissue structure is weak. Flow cytometry analysis results showed that Lactobacillus pentosus JNL0030 increased the proportion of M2 macrophages (anti-inflammatory) in the intestinal lamina propria, reduced the proportion of M1 macrophages (pro-inflammatory), and significantly upregulated the proportion of Treg immune cell populations. RT-qPCR analysis results showed that Lactobacillus pentosus JNL0030 can significantly inhibit the levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α in colon tissue, indicating that Lactobacillus pentosus JNL0030 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 pentosus JNL0030.

[0062] Example 9, a microbial preparation

[0063] The supernatant containing Lactobacillus pentosaceus JNL0030 prepared in Example 3 can be used to prepare drugs.

[0064] As an embodiment, in the medicine, Lactobacillus pentosus JNL0030 is the only effective active ingredient.

[0065] The supernatant may be freeze-dried to prepare a lyophilized powder.

[0066] 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.

[0067] Alternatively, as another embodiment, Lactobacillus pentosus JNL0030 is combined with other active ingredients for treating colitis.

[0068] 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 pentosus JNL0030, characterized in that: The Lactobacillus pentosus JNL0030 was deposited in the China General Microbiological Culture Collection Center on June 24, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO: 31045, and the classification name is Lactobacillus pentosus Lactiplantibacillus pentosus .

2. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in producing indole-3-lactic acid in vitro.

3. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in the preparation of a medicament for treating colitis.

4. A microbial preparation, characterized in that: The method comprises the Lactobacillus pentosus JNL0030 according to claim 1.

5. The microbial preparation according to claim 4, characterized in that Also included are excipients.

Images