Phytobacterium pentosus JNL0030, application thereof and microbial preparation

Abstract

The invention discloses a strain of lactobacillus pentosus JNL0030, application thereof and a microbial preparation, and belongs to the technical field of microorganisms. The lactobacillus pentosus JNL0030 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 (China General Microbiological Culture Collection Center) NO: 31045, and the lactobacillus pentosus is classified and named as lactobacillus pentosus. 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 particularly relates to a strain of Lactobacillus pentosaceus JNL0030, an application thereof and a microbial preparation. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disease caused by a combination of environmental, genetic, immune, and microbial factors. It includes two subtypes: ulcerative colitis (UC) and Crohn's disease (CD). Currently, traditional treatments for IBD include 5-aminosalicylic acid, glucocorticoids, and immunosuppressants. These medications only alleviate clinical symptoms but are unable to prevent disease progression. Their clinical efficacy is poor, often leading to recurrences and ultimately increasing surgical procedures.

[0003] IBD is typically characterized by an imbalanced intestinal microbiome. Probiotics can inhibit pathogenic bacteria and repair a damaged intestinal microbiome. Numerous studies have reported probiotics' anti-inflammatory effects both in vivo and in vitro. Furthermore, probiotics colonize the intestine, exerting their therapeutic effects continuously and with minimal side effects. Therefore, the use of probiotics in the treatment of IBD offers significant advantages.

[0004] Indole-3-lactic acid (ILA) is an indole compound produced by intestinal microbial metabolism of tryptophan. Studies have shown that indole-3-lactic acid 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 reducing intestinal inflammatory responses. In addition, indole-3-lactic acid can also 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] Currently, chemical synthesis of ILA can produce toxic byproducts, and its preparation requires demanding conditions, complex processes, and high costs. Microbial metabolism of tryptophan to produce indole-3-lactic acid offers low cost and mild reaction conditions, making it suitable for industrial production. Furthermore, ILA-producing probiotics can colonize the human intestine, continuously secrete ILA, and modulate the host's intestinal immune microenvironment. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides a strain of Lactobacillus pentosaceus JNL0030, its application 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 is classified as Lactiplantibacillus pentosus.

[0008] The present invention also provides the use of the Lactobacillus pentosaceus 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 the use of the Lactobacillus pentosus JNL0030 in preparing medicines 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 further comprises an excipient.

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

[0017] 1. This application provides a strain of Lactobacillus pentosaceus JNL0030 and its applications. This strain can produce high levels of indole-3-lactic acid in MRS culture medium, with a yield approximately 22 times higher than that of the commercial strain Lacticaseibacillus rhamnosus LGG. This method overcomes many of the drawbacks of chemically synthesizing ILA by using microbial metabolism of tryptophan to produce indole-3-lactic acid, which is low-cost, has mild reaction conditions, and is suitable for industrial production needs. Furthermore, ILA-producing probiotics can colonize the human intestine, continuously secrete ILA, and modulate the host intestinal immune microenvironment.

[0018] 2. In vitro experiments showed that the supernatant of Lactobacillus pentosus JNL0030 provided by the present invention significantly inhibited lipopolysaccharide (LPS)-stimulated macrophage inflammatory polarization and the secretion of inflammatory factors. Animal experiments also showed that Lactobacillus pentosus JNL0030 could significantly inhibit dextran sulfate sodium (DSS)-induced colonic tissue inflammatory factor secretion and intestinal barrier damage, alleviating ulcerative colitis, demonstrating great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, 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 The graph of the indole-3-lactic acid production level of Lactobacillus pentosus JNL0030 in Example 4; wherein (a) is the ILA level in the culture medium, and (b) is the level of each OD 600 The level of ILA produced by the 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, and **** is p < 0.0001.

[0024] Figure 5 The results of bacterial hemolysis analysis in Example 7 are shown; (a) is α hemolysis, (b) is β hemolysis, and (c) is γ hemolysis.

[0025] Figure 6 These are graphs showing the effects of Lactobacillus pentosus JNL0030 on alleviating DSS-induced colitis in terms of body weight and disease activity index in Example 8; wherein (a) represents body weight and (b) represents disease activity index.

[0026] Figure 7This is a graph showing the effect of Lactobacillus pentosus JNL0030 on alleviating DSS-induced colitis on colorectal length in Example 8; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** 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] Figure 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; * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as 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 the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate 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 10.0 g / L, beef powder 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L. pH 5.7 ± 0.2 (25°C).

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

[0034] Example 1, Bacterial separation and purification

[0035] Prepare MRS medium according to the instructions and sterilize at 118°C for 20 minutes. Take an appropriate amount of fermented yogurt, add physiological saline to dilute it in a gradient, evenly spread it on MRS solid medium, and culture it anaerobically at 37°C for 48 hours. Pick a single colony 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 being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC NO: 31045, and the classification name being 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 Gram-positive.

[0040] Example 2, bacterial culture and counting

[0041] Prepare MRS medium according to the instructions and sterilize at 118°C for 20 minutes. After cooling to room temperature, inoculate Lactobacillus pentosus JNL0030 at a 1% inoculum volume and culture anaerobically at 37°C for 24 hours. After culture, the bacterial solution was diluted with physiological saline gradients, and 100 μL was evenly spread on MRS solid medium and cultured anaerobically at 37°C for 48 hours. The bacterial growth was calculated based on the number of colonies grown. OD value of the bacterial solution after 24 hours of culture of Lactobacillus pentosus 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 value of bacterial solution after 48 hours 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.

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

[0045] A single-labeled stock solution of indole-3-lactic acid was prepared in 50% methanol and serially diluted with 10% methanol. To 200 μL of the supernatant of Lactobacillus pentosaceus JNL0030, methanol was added to a final concentration of 10%, and the mixture was shaken vigorously. Centrifuged at 12,000 rpm for 5 minutes. The standard and the supernatant extract were injected and analyzed. Targeted quantitative mass spectrometry analysis was performed. ACQUITY The column temperature was 40°C on an HSS T3 column, and the mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in methanol. The gradient elution conditions were: 5% B (0-1 min); 5%-50% B (1-2 min); 50%-95% B (2-5 min); and 95%-5% B (6-8 min). Mass spectrometry conditions included an electrospray ionization (ESI) source in positive ionization mode. The ion source temperature was 500°C, the ion source voltage was 5500 V, the collision gas was 6 psi, the curtain gas was 30 psi, and the nebulizer and auxiliary gases were both 50 psi. Multiple reaction monitoring (MRM) scanning was performed with retention time lock. The concentration of indole-3-lactic acid in the supernatant of Lactobacillus pentosaceus JNL0030 was quantified based on the ion intensity of the standard.

[0046] The same number of Lactobacillus pentosus JNL0030 and Lactobacillus rhamnosus LGG were inoculated into 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 respectively. 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 maximum 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 seeded into 12-well plates (1×106 cells / well) and incubated in a CO2 incubator at 37°C for 12 hours to adhere. Lipopolysaccharide (LPS) (5 μg / mL) was added to stimulate the inflammatory response of RAW264.7 cells. Six hours later, 50 μL of blank MRS medium and supernatant of Lactobacillus pentosaceus JNL0030 were added, respectively, and incubated in a CO2 incubator at 37°C for 24 hours. Mouse TNF-α (Biyuntian, PT512) and IL-6 (Biyuntian, PI326) ELISA kits were used to measure the levels of inflammatory factors in the cell supernatant, respectively. Cells were labeled with antibodies against mouse F4 / 80, CD206, and CD86, 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-treated group, Lactobacillus pentosus JNL0030 had a trend of reducing 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) in 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), Lactobacillus pentosus JNL0030 has a better anti-inflammatory effect than Lactobacillus rhamnosus LGG.

[0050] Example 6, bacterial antibiotic sensitivity analysis

[0051] Lactobacillus pentosus JNL0030 was inoculated into liquid MRS medium and incubated anaerobically at 37°C for 24 hours to obtain a seed solution. The bacterial solution was diluted with 0.85% saline to a McFarland turbidity of 0.5. 100 μL of the solution was accurately pipetted and evenly spread on the surface of the MRS agar medium. After the surface solution dried, a drug sensitivity paper disc was placed on the surface of the medium. The susceptibility of Lactobacillus pentosus JNL0030 to six antibiotics, including ampicillin (10 μg / slice), gentamicin (10 μg / slice), kanamycin (30 μg / slice), streptomycin (10 μg / slice), clindamycin (2 μg / slice), and chloramphenicol (30 μg / slice), was tested using the KB disc diffusion method. The sensitivity of each bacterial strain to each antibiotic was determined in triplicate. Incubate anaerobically at 37°C for 24 hours. Measure and record the diameter of the inhibition zone and compare it with the diameter of the inhibition zone of the reference strain listed in the instructions. 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 manifests as a narrow, grass-green hemolysis ring; β-hemolysis manifests 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 Streptococcus 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 one week of adaptive feeding, 6-8 week-old male C57Bl / 6N mice were given free access to a 2.5% DSS (molecular weight 36,000-50,000 Da) aqueous solution to induce colitis. Lactobacillus pentosus JNL0030, cultured for 24 hours, was centrifuged at 5000 rpm at 4°C for 5 minutes, the supernatant discarded, and the cells resuspended in saline. Each mouse received 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 mice were monitored daily for body weight, diarrhea, hematochezia, and activity. Mice were anesthetized and sacrificed by cervical dislocation. Colon tissue was 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 mouse colon tissue, and RT-qPCR was used to detect the transcription levels of inflammatory cytokines IL-β, IL-6, and TNF-α in mouse colon tissue.

[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 bloody stools.

[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 effect of Lactobacillus rhamnosus LGG and 5-ASA on the body weight and disease activity index of mice was weak. Treatment with Lactobacillus pentosus JNL0030 can 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, as shown in (a) and (b). Figure 7 and Figure 8 As shown. 5-ASA can inhibit the infiltration of inflammatory cells in the colon and protect the integrity of the intestinal barrier, but the protective effect of Lactobacillus rhamnosus LGG on the intestinal tissue structure is weak. The results of flow cytometry analysis 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. The results of RT-qPCR analysis 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 Figure 9 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 medicines.

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

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

[0066] It can also be combined with solvents or other excipients for the treatment of colitis. When combined with solvents, injections 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 foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC NO: 31045, and the classification name is Lactobacillus pentosus.

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

3. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in reducing the proportion of pro-inflammatory M1 macrophages.

4. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in increasing the proportion of anti-inflammatory M2 macrophages.

5. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in inhibiting the secretion of pro-inflammatory cytokines IL-6 and TNF-α.

6. Use of the Lactobacillus pentosus JNL0030 according to claim 1 in preparing medicines for treating intestinal inflammation.

7. Use of the Lactobacillus pentosus JNL0030 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 pentosus JNL0030 according to any one of claims 1 to 7.

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

Images