A strain of Bifidobacterium longum subsp. longum and its application in antibacterial, alleviating colitis and improving inflammatory bone loss

Abstract

The present invention discloses a strain of Bifidobacterium longum subsp. longum dipro-017. The Bifidobacterium longum subsp. longum ([ Bifidobacterium longum subsp. longum ) dipro-017 used in the present invention has the ability to metabolize tryptophan to produce indole-3-lactic acid, can inhibit pathogenic bacteria such as Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli, can effectively improve the weight loss caused by ulcerative colitis, improve the DAI score, improve the colon length, improve the content of lipopolysaccharide LPS in the blood and increase the level of anti-inflammatory cytokine IL-10, and at the same time improve the content of bone metabolism indexes tartrate-resistant acid phosphatase (TRAP) and N-terminal propeptide of type I collagen (PINP) in the blood.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a strain of Bifidobacterium longum subsp. longum and its application in antibacterial, relieving colitis and improving inflammatory bone loss. Background Art

[0002] Inflammatory bowel diseases (IBD) are idiopathic chronic intestinal inflammatory diseases, mainly including ulcerative colitis (UC) and Crohn's disease (CD), and common symptoms include abdominal pain, diarrhea, etc. The prevalence and incidence of this disease are the highest in Western countries, reaching 0.5% and 0.3‰ respectively. IBD is closely related to the degree of social modernization, and the incidence in each emerging industrialized country is increasing day by day. Currently, the prevalence of UC and CD is increasing rapidly. The intestinal inflammation of IBD will affect the whole body metabolism of patients. Most patients will have concurrent bone mass loss and osteoporosis, with the incidence rates reaching 67% and 57.6% respectively; correspondingly, the fracture risk is increased to 1.3 - 1.4 times that of normal people.

[0003] In patients with autoimmune diseases or other chronic inflammatory diseases, systemic bone loss can occur, and the risk of osteopenia or osteoporosis is significantly increased. Under inflammatory conditions in the body, both immune cells and osteoblasts in the body will release pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6, which can accelerate bone catabolism by promoting osteoclast activity.

[0004] Currently, relevant research has linked enteritis and osteoporosis. Therefore, probiotics have great development value in changing the bone loss caused by enteritis. Although the research on probiotics in bone health is still in its initial stage, as a safe and natural intervention measure, it provides a new perspective for bone metabolism health. Developing probiotic preparations specifically for bone health can not only provide a new treatment method, but also help reduce the risks caused by traditional drugs (such as the side effects of hypercalcemia syndrome, constipation, vomiting, etc. may occur after using the drug alfacalcidol, and it is not suitable for pregnant women), bringing more comprehensive health benefits to patients and becoming a health treatment plan with application prospects. Summary of the Invention

[0005] Object of the Invention: To solve the limitations existing in the prior art for improving colitis and its inflammatory bone loss, the present invention provides a strain of Bifidobacterium longum subsp. longum strain that can effectively inhibit pathogenic bacteria, relieve colitis, and improve inflammatory bone loss, enabling patients to get rid of the side effects and limitations brought by traditional drug treatment.

[0006] Technical solution: To solve the above technical problems, the present invention provides a strain of Bifidobacterium longum subsp. longum isolated from the feces of healthy children. Through whole-genome sequencing analysis and BLAST nucleic acid sequence comparison in the NCBI gene database, the results show that this strain is Bifidobacterium longum subsp. longum. The Bifidobacterium longum subsp. longum dipro-017 of the present invention was deposited on August 14, 2024, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number: CGMCC No. 31575, and the deposit address: No. 1, Beichen West Road, Chaoyang District, Beijing, China.

[0007] Among them, the Bifidobacterium longum subsp. longum dipro-017 is Gram-positive, with rod-shaped or V-shaped cells, uneven length and diameter, and no spores. Streak on MRS agar medium and anaerobic culture at 37°C for 24 - 48 h, and obvious colonies will appear. The colonies are round, white, opaque, convex, with a smooth surface, and the colony diameter is 0.2 - 3 mm. Using MRS liquid medium, obvious cell precipitation will occur after anaerobic culture at 37°C for 12 - 16 h.

[0008] Among them, the strain can utilize tryptophan to metabolize and produce indole-3-lactic acid, and the content of indole-3-lactic acid produced is 1000 - 10000 ng / ml.

[0009] The content of the present invention also includes products containing the Bifidobacterium longum subsp. longum dipro-017, and the products include fermented products, drugs, feeds, protectants, functional auxiliaries, auxiliary additives, drug carriers or pharmaceutical excipients.

[0010] Among them, the protectants, functional auxiliaries, auxiliary additives, drug carriers or pharmaceutical excipients include but are not limited to albumin, gelatin, soluble starch, dextrin, meat extract, pectin, gum arabic, carboxymethyl cellulose, algae, vitamin D, vitamin E, protein hydrolysate, sodium thiosulfate, dextran, and natural mixtures such as skim milk, serum, etc.

[0011] Among them, the fermented products, drugs, and feeds include the bacterial supernatant, bacterial precipitate or bacterial suspension of Bifidobacterium longum subsp. longum dipro-017.

[0012] Among them, the dosage forms of the product include powder, capsule, tablet, pill, film coating agent, aerosol, granule, liquid agent, liposome, transdermal agent, suppository or freeze-dried powder injection.

[0013] The present invention also includes the application of the product of Bifidobacterium longum subsp. longum dipro-017 in the preparation of a product for preventing, alleviating, regulating or treating ulcerative colitis and inflammatory bone loss.

[0014] The present invention also includes that the application of Bifidobacterium longum subsp. longum dipro-017 in the product is a product having at least one of the following functions:

[0015] 1) A product having an antibacterial effect;

[0016] 2) A product having an improved ulcerative colitis;

[0017] 3) A product having an improved content of lipopolysaccharide (LPS) in the blood or an increased level of anti-inflammatory cytokine IL-10;

[0018] 4) A product having an improved content of tartrate-resistant acid phosphatase (TRAP) or N-terminal propeptide of type I collagen (PINP), which are bone metabolism indexes in the blood.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention provides a strain of Bifidobacterium longum subsp. longum that can inhibit pathogenic bacteria and its application in improving colitis and inflammatory bone loss. The present invention proves for the first time that the dipro-017 strain has the ability to inhibit pathogenic bacteria and metabolize tryptophan to produce indole-3-lactic acid. In a DSS-induced colitis mouse model, dipro-017 can significantly improve the body weight, colon length and DAI score of mice, while reducing the level of LPS in the serum and enhancing the level of anti-inflammatory cytokine IL-10 in the serum. The present invention also proves for the first time that dipro-017 can improve inflammatory bone loss, specifically manifested as effectively regulating the bone metabolism indexes tartrate-resistant acid phosphatase (TRAP) and N-terminal propeptide of type I collagen (PINP). Therefore, using the Bifidobacterium longum subsp. longum dipro-017 strain to prepare a product for improving colitis and its inflammatory bone loss has good prospects. Description of the Drawings

[0020] Figure 1 : Liquid chromatogram of the production of indole-3-lactic acid by the Bifidobacterium longum subsp. longum strain dipro-017 using tryptophan metabolism.

[0021] Figure 2 : Statistical results of the inhibition zone diameters of Bifidobacterium longum subsp. longum strain dipro-017 against three pathogenic bacteria.

[0022] Figure 3 : Weight changes of DSS-induced colitis mouse models in different groups. Figure A shows the percentage change in mouse weight over time during DSS administration, and Figure B shows the weight percentage results of mice on the eighth day of DSS administration.

[0023] Figure 4 : Colonic lengths of DSS-induced colitis mouse models in different groups. Figure A shows photos of the colonic lengths of different groups, and Figure B shows the statistically analyzed colonic lengths of different treatment groups.

[0024] Figure 5 : DAI scores of DSS-induced colitis mouse models in different groups.

[0025] Figure 6 : Blood inflammation indexes of DSS-induced colitis mouse models in different groups. Figure A shows the level of lipopolysaccharide (LPS) in serum, and Figure B shows the level of IL-10 in serum.

[0026] Figure 7 : Detection results of bone metabolism indexes of DSS-induced colitis mouse models in different groups. Figure A shows the level of tartrate-resistant acid phosphatase (TRAP) in serum, and Figure B shows the level of N-terminal propeptide of type I collagen (PINP) in serum. Specific embodiments

[0027] The present invention will be specifically described below in conjunction with the accompanying drawings and embodiments.

[0028] Example 1 Microbiological identification of strain dipro-017

[0029] Bifidobacterium longum subsp. longum dipro-017 was isolated from the feces of healthy children. Since isolation, MALDI-TOF identification, 16S rRNA sequencing identification, and whole-genome sequencing identification (ANI value of 98.832%) have been carried out respectively. The sequencing identification results of the strain are consistent with the MALTI-TOF results at the species level, both being Bifidobacterium longum subsp. longum.

[0030] The Bifidobacterium longum subsp. longum dipro-017 of the present invention was deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 14, 2024, and the deposit number is: CGMCC No. 31575.

[0031] When conducting functional experiments, the glycerol-preserved bacteria of Bifidobacterium longum subsp. longum dipro-017 were taken out from the -80°C refrigerator, melted in a 37°C water bath, and then 20 μL was taken and spread on an MRS+

[0032] (MRS (Guangdong Huankai Microbial Science and Technology Co., Ltd., product number: 027315) + 0.5 g / L cysteine) plate and anaerobically cultured at 37°C for 48 h; then, a inoculation loop was used to streak for subculture. After culturing for 48 h, a single colony was picked and transferred to an MRS broth liquid medium (MRS+) supplemented with 0.5 g / L cysteine, and incubated in a constant temperature culture at 37°C for 48 h for scale-up culture, and then subsequent experiments could be carried out.

[0033] Example 2 Determination of the content of indole-3-lactic acid in the culture supernatant of dipro-017

[0034] (1) After the dipro-017 strain was resuscitated and expanded according to the method in Example 1, the obtained strain culture solution was centrifuged at 8000 rpm for 5 min at 4°C, and the supernatant was removed. The obtained dipro-017 cells were washed once with phosphate buffer PBS (Procell Company, 10 mM, pH 7.2 - 7.4, product number: PB180327), and then resuspended with phosphate buffer PBS. The OD600 was adjusted to 2 to prepare a cell suspension. 500 μl of the cell suspension was added to 4 ml of M9 (Shanghai Huzhen Biotechnology Co., Ltd., product number: HZ0060-500 ml) medium supplemented with 1 mM tryptophan and cultured at 37°C for 48 h. After culturing, it was centrifuged at 8000 rpm for 3 min at 4°C, and the supernatant was retained, filtered through a 0.22 μm filter membrane, and used for subsequent detection.

[0035] (2) UV detection conditions: The detector model is Waters 2498, the detector wavelength: 280 nm, the column temperature: 25°C, and the injection volume: 30 μl. A series of gradient concentrations (0, 50, 100, 500, 1000 ng / ml) of indole-3-lactic acid (purchased from Sigma Company) standard products were measured. The standard curve of the peak area and concentration of the peak was established, and the regression equation y = 92.372x - 1049.6 (R2 = 0.9999) was calculated.

[0036] (3) Determine the content of indole-3-lactic acid in the supernatant obtained in step (1) by Waters HPLC. The chromatographic column model is Kinetex C18 (5μm 150*4.6mm). Take 30 μl of the supernatant obtained in step (1) and inject it into the chromatographic column;

[0037] The eluent consists of mobile phase A and mobile phase B:

[0038] Mobile phase A: 0.05% trifluoroacetic acid / water, that is, an aqueous solution of trifluoroacetic acid with a volume concentration of 0.05%;

[0039] Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile, that is, an acetonitrile solution of trifluoroacetic acid with a volume concentration of 0.05%;

[0040] The elution program is as follows in the table.

[0041] Table 1 HPLC elution program

[0042]

[0043] At a UV detection wavelength of 280 nm, perform peak area integration using the chromatographic peak corresponding to the retention time of the indole-3-lactic acid standard. As Figure 1 shown, the obtained peak area is 121793 AU*min, that is, y = 121793. Substitute it into the regression equation y = 92.372x - 1049.6 (R2 = 0.9999) for calculation, and the content of indole-3-lactic acid in the dipro-017 fermentation supernatant is 1329.86 ng / ml.

[0044] Example 3 Antibacterial ability experiment of dipro-017

[0045] Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 25922 were continuously activated for 3 generations in LB (Apti, product number: F05932) medium, and then centrifuged and adjusted to an OD600 nm value range of 1.0 - 1.2 with sterile physiological saline (the concentration of bacteria in the bacterial suspension is about 3×10 8CFU / mL) for later use; the dipro-017 bacterial solution in the exponential growth phase was inoculated at 2% in MRS+ liquid culture medium for anaerobic culture for 48h for later use; similarly, the control strain Lactobacillus rhamnosus LGG (Chr. Hansen Ltd., Denmark) in the exponential growth phase was inoculated at 2% in MRS liquid culture medium for anaerobic culture for 24h for later use. Agar plates containing pathogens (Salmonella typhimurium, Staphylococcus aureus, Escherichia coli) were prepared by the pouring method. Specifically, the corresponding LB solid culture medium was first prepared, sterilized and cooled to 40°C, and the pathogenic bacteria solution was added and mixed in a certain proportion to make the number of viable bacteria of the pathogen 10 5 CFU / mL, pour 30mL of the mixture into each plate, and after the agar solidifies, punch holes with an 8mm puncher. Finally, add 200μL of the cultured dipro-017 into the holes, culture at 37℃ for 16h, and measure the diameter of the inhibition zone.

[0046] like Figure 2 As shown, the average inhibition zone of dipro-017 against Salmonella typhimurium is 16.65 mm, which is significantly better than the inhibition zone of 15.05 mm of Lactobacillus rhamnosus LGG; the average inhibition zone of dipro-017 against Staphylococcus aureus is 14.27 mm, which is significantly better than the inhibition zone of 12.48 cm of Lactobacillus rhamnosus LGG; the average inhibition zone of dipro-017 against Staphylococcus aureus is 14.69 mm, which is significantly better than the inhibition zone of 13.40 mm of Lactobacillus rhamnosus LGG. In summary, dipro-017 showed good antibacterial effects on these three pathogens.

[0047] Example 4: Dipro-017 can improve body weight in colitis mouse model

[0048] After activation of dipro-017, the mixture was incubated at 37°C for 48 h and the concentration was adjusted to 1 × 10 9 CFU / mL. Seven-week-old SPF male C57BL / 6J mice were selected and randomly divided into a normal control group (NC group), a DSS model group (PBS group), and a DSS+dipro-017 treatment group (dipro-017 group), with 6 mice in each group. Each mouse in the NC group and the PBS group was intragastrically gavaged with 200 μL PBS per day, and each mouse in the dipro-017 group was intragastrically gavaged with 200 μL dipro-017 suspension per day (gavage amount of bacteria 5×10 8 CFU) for 15 consecutive days. Seven days after intragastric administration, the NC group drank ultrapure water freely, and the PBS group and dipro-017 group drank 3% DSS aqueous solution (mass percentage, the same below) freely for 8 consecutive days. Meanwhile, the weight changes of mice were monitored.

[0049] The results are as followsFigure 3 As shown Figure 3 A shows the changes in the body weights of mice in each group during 8 consecutive days of 3% DSS-induced modeling. It can be seen that as time extended, the body weights of mice in the PBS group decreased rapidly, while the decrease in the body weights of mice in the dipro-017 treatment group was significantly alleviated (P<0.001). Similarly, from Figure 3 the body weight data of colitis mice at the end point of the 8th day of modeling in B, it can be known that dipro-017 can significantly improve the sharp decrease in the body weights of colitis mice (P<0.001), indicating that dipro-017 has a protective effect on DSS-induced colitis.

[0050] Example 5: dipro-017 improves the DAI score of colitis mice

[0051] The animal experiment treatment was basically the same as that in Example 4. The difference was that, referring to the experimental method in the article Treatment of dextransulfate sodium-induced murine colitis by intracolonic cyclosporine[J].Digestive and Liver Disease, 1993, 38(9):1722-1734., the Disease Activity Index (DAI) of mice was calculated, which can be used to preliminarily evaluate the onset degree of murine colitis. As Figure 4 shown, in the present invention, after mice drank DSS drinking water, the DAI scores changed significantly. The score of the PBS group exceeded 2 points, showing a significant difference compared with healthy mice in the NC group (P<0.001). At the same time, the DAI scores of colitis mice treated with dipro-017 were significantly improved (P<0.05).

[0052] Example 6: dipro-017 improves the colon length of colitis mice

[0053] The animal experiment treatment was basically the same as that in Example 4. The difference was that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, the mice were sacrificed by cervical dislocation, and the colon from the anus to the ileocecal part was dissected and the colon length was measured with a vernier caliper. As Figure 5 shown, where Figure 5 A shows the photos of the colon tissue lengths of each treatment group. It can be seen that compared with the normal NC group, the colon of mice in the PBS group was significantly shortened, indicating that the colon morphology of mice was damaged. After dipro-017 treatment, the colon length of mice increased, and to a certain extent, this damage could be restored. At the same time, the further quantitative analysis results of the colon length are as Figure 5As shown in Figure B, compared with the PBS group, dipro-017 could significantly improve DSS-induced shortening of colon length (P<0.001).

[0054] Example 7. dipro-017 improves blood inflammatory indexes in mice with colitis

[0055] The animal experiment treatment was basically the same as that in Example 4. The difference was that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, blood was collected from the orbital venous plexus, and the LPS concentration in serum was detected using an ELISA kit. Lipopolysaccharide LPS is an effective stimulator of the immune system. The more LPS enters the blood through the intestine, the more severe the inflammatory response. The results are as Figure 6 shown in Figure A. Compared with the NC group, the content of serum lipopolysaccharide LPS in the PBS group increased significantly (P<0.05), indicating that the DSS-induced colitis model could lead to an inflammatory response. Compared with the PBS group, dipro-017 could reduce the LPS level in the serum of mice with colitis to a certain extent, thereby reducing the inflammatory response of mice.

[0056] Meanwhile, the concentration of IL-10 in serum was detected using an ELISA kit. As an anti-inflammatory cytokine, IL-10 plays a crucial role in controlling and preventing intestinal inflammation. IL-10 is considered to be able to block the metabolism of macrophages and promote damaged mitophagy, thereby being able to reduce inflammation. Therefore, high expression of IL-10 is generally considered to be helpful in inhibiting intestinal inflammation. The results are as Figure 6 shown in Figure B. Compared with the PBS group, dipro-017 could significantly increase the content of IL-10 in the serum of mice (P<0.05), indicating that Bifidobacterium longum dipro-017 could exert an anti-inflammatory effect, thereby improving the inflammatory level of mice with colitis.

[0057] Example 8. dipro-017 improves blood bone metabolism indexes in mice with colitis

[0058] The animal experiment treatment was basically the same as that in Example 4. The difference was that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, blood was collected from the orbital venous plexus, and the bone metabolism indexes in the serum of mice were detected using an ELISA kit.

[0059] In bone resorption activities, osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) as an active enzyme into the blood. Therefore, TRAP is often used as an important index to reflect the number of osteoclasts. The results are as Figure 7 shown in Figure A. Compared with the PBS group, the highly expressed TRAP in DSS colitis mice decreased significantly after treatment with dipro-017 (P<0.05).

[0060] PINP (N-terminal propeptide of type I procollagen) is the N-terminal propeptide of type I collagen, which is commonly used to reflect the therapeutic effect of osteoporosis treatment, especially applicable to the evaluation of bone formation-promoting treatment, and is an important clinical detection index reflecting bone formation. As shown in the ELISA results Figure 7 shown in B, compared with the PBS group, dipro-017 treatment significantly increased the low level of PINP content in the serum of colitis mice. Therefore, overall, dipro-017 can improve the bone loss caused by inflammation.

Claims

1. A strain of Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) dipro-017, characterized in that The Bifidobacterium longum subsp. Bifidobacterium longum subsp.longum ) dipro-017 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 14, 2024, with the deposit number: CGMCC No. 31575.

2. The Bifidobacterium longum subspecies longum according to claim 1 ( Bifidobacterium longum subsp.longum ) dipro-017, characterized in that The dipro-017 can utilize tryptophan metabolism to produce indole-3-lactic acid, and the content of the produced indole-3-lactic acid is 1000-10000 ng / ml.

3. Containing the Bifidobacterium longum subspecies longum according to claim 1 ( Bifidobacterium longum subsp.longum ) dipro-017 product, characterized in that, The products include fermented products, medicines or feeds.

4. The product according to claim 3, wherein the fermented product comprises Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) fermentation supernatant, fermentation precipitate or bacterial suspension of dipro-017.

5. The product according to claim 3, characterized in that The dosage forms of the product include powder, capsule, tablet, pill, film-coated agent, aerosol, granule, liquid, liposome, transdermal agent, suppository or lyophilized powder injection.

6. The product according to claim 3, characterized in that The product also includes a protective agent, a functional auxiliary agent / or an auxiliary additive, a drug carrier and / or a pharmaceutical excipient.

7. The Bifidobacterium longum subspecies longum of claim 1 ( Bifidobacterium longum subsp.longum ) Dipro-017, use of the product according to any one of claims 2 to 5 in the preparation of a product for preventing, alleviating, regulating or treating ulcerative colitis and inflammatory bone loss.

8. The use according to claim 7, characterized in that: The application of the product is a product that has at least one of the following functions: 1) Products with antibacterial effects; 2) Products that improve ulcerative colitis; 3) Products that improve the content of lipopolysaccharide (LPS) in the blood or increase the level of anti-inflammatory cytokine IL-10; 4) Products that can improve the content of tartrate-resistant phosphatase or type I collagen N-terminal propeptide, an indicator of bone metabolism, in the blood.

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