Bifidobacterium longum subsp. longum, and use thereof in inhibiting bacteria, relieving colitis, and mitigating inflammatory bone loss

Abstract

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

Description

A strain of Bifidobacterium longum and its application in inhibiting bacteria, alleviating colitis, and improving inflammatory bone loss. Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium longum and its application in inhibiting bacteria, relieving colitis, and improving inflammatory bone loss. Background Technology

[0002] Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory bowel disease, primarily including ulcerative colitis (UC) and Crohn's disease (CD). Common symptoms include abdominal pain and diarrhea. The prevalence and incidence of IBD are highest in Western countries, reaching 0.5% and 0.3‰ respectively. IBD is closely related to the level of social modernization, and its incidence is increasing in newly industrialized countries. Currently, the prevalence of UC and CD is growing rapidly. The intestinal inflammation of IBD affects the patient's systemic metabolism. Most patients will experience bone loss and osteoporosis, with incidence rates reaching 67% and 57.6% respectively; correspondingly, the risk of fracture increases to 1.3-1.4 times that of normal individuals.

[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, both immune cells and osteoblasts in the body 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] Current research has linked enteritis to osteoporosis, thus probiotics hold significant potential for mitigating bone loss caused by this condition. Although research on probiotics for bone health is still in its early stages, as a safe and natural intervention, they offer a novel perspective on healthy bone metabolism. Developing probiotic formulations specifically for bone health could not only provide a new treatment approach but also help mitigate the risks associated with traditional medications (such as the potential side effects of alfacalcidol, including hypercalcemia, constipation, and vomiting, and its contraindication for pregnant women), offering more comprehensive health benefits and representing a promising health treatment option. Summary of the Invention

[0005] Purpose of the invention: To address the limitations of existing technologies in improving colitis and its inflammatory bone loss, this invention provides a strain of Bifidobacterium longum subsp. longum that can effectively inhibit pathogens, alleviate colitis, and improve inflammatory bone loss, as well as its application, enabling patients to escape the side effects and limitations of traditional drug treatments.

[0006] Technical Solution: To solve the above-mentioned technical problems, this invention provides a strain of *Bifidobacterium longum* subsp. *longum* isolated from the feces of healthy children. Whole-genome sequencing analysis and BLAST nucleic acid sequence comparison using the NCBI gene database confirmed that this strain is *Bifidobacterium longum* subsp. *longum*. The *Bifidobacterium longum* subsp. *longum* dipro-017 described in this invention was deposited on August 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31575, located at No. 1, Beichen West Road, Chaoyang District, Beijing, China.

[0007] The *Bifidobacterium longum* subsp. *longum* dipro-017 is Gram-positive, with rod-shaped or V-shaped cells of varying length and diameter, and no spores. When streaked on MRS agar, distinct colonies appear after anaerobic incubation at 37°C for 24–48 hours. These colonies are round, white, opaque, raised, with a smooth surface, and a diameter of 0.2–3 mm. Using MRS liquid medium, distinct bacterial precipitation occurs after anaerobic incubation at 37°C for 12–16 hours.

[0008] The strain is able to produce indole-3-lactic acid by metabolizing tryptophan, and the content of indole-3-lactic acid produced is 1000-10000 ng / ml.

[0009] The present invention also includes products containing the aforementioned Bifidobacterium longum subsp. longum dipro-017, including fermented products, food, health products, pharmaceuticals, feed, protective agents, functional additives, auxiliary additives, drug carriers, or pharmaceutical excipients.

[0010] The protective agents, functional additives, auxiliary additives, drug carriers or pharmaceutical excipients include, but are not limited to, albumin, gelatin, soluble starch, dextrin, meat juice, pectin, gum arabic, hydroxymethyl cellulose, algae, vitamin D, vitamin E, protein hydrolysate, sodium thiosulfate, dextran, and natural mixtures such as skim milk and serum.

[0011] The fermented products, foods, health products, pharmaceuticals, and feeds include supernatant, precipitate, or suspension of Bifidobacterium longum subsp. longum dipro-017.

[0012] The dosage forms of the products include powders, capsules, tablets, pills, film-coated tablets, aerosols, granules, liquids, liposomes, transdermal preparations, suppositories, or lyophilized powder injections.

[0013] The present invention also includes the use of the product of Bifidobacterium longum subsp. longum dipro-017 in the preparation of products for the prevention, relief, regulation or treatment of ulcerative colitis and inflammatory bone loss.

[0014] This invention also includes the application of the aforementioned Bifidobacterium longum subsp. longum dipro-017 in the product as a product having at least one of the following functions:

[0015] 1) Products with antibacterial effects;

[0016] 2) Products that improve ulcerative colitis;

[0017] 3) Products that can improve the level of lipopolysaccharide (LPS) in the blood or increase the level of the anti-inflammatory cytokine IL-10;

[0018] 4) Products that improve the levels of blood bone metabolism markers such as anti-tartrate phosphatase (TRAP) or type I collagen N-terminal propeptide (PINP).

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention provides a strain of *Bifidobacterium longum* subsp. *longum* capable of inhibiting pathogens and its application in improving colitis and inflammatory bone loss. This invention demonstrates for the first time that strain dipro-017 possesses the ability to inhibit pathogens and metabolize tryptophan to produce indole-3-lactic acid. In a DSS-induced colitis mouse model, dipro-017 significantly improved mouse body weight, colon length, and DAI score, while reducing serum LPS levels and increasing serum levels of the anti-inflammatory cytokine IL-10. This invention also demonstrates for the first time that dipro-017 can improve inflammatory bone loss, specifically by effectively regulating bone metabolism indicators such as anti-tartrate phosphatase (TRAP) and type I collagen N-terminal propeptide (PINP). Therefore, using *Bifidobacterium longum* subsp. *longum* dipro-017 strain to prepare products that improve colitis and inflammatory bone loss has great potential. Attached Figure Description

[0020] Figure 1: Liquid chromatography peak diagram of indole-3-lactic acid produced by tryptophan metabolism in the long subspecies of Bifidobacterium longum, strain dipro-017.

[0021] Figure 2: Statistical results of the inhibition zone diameter of the long subspecies of Bifidobacterium dipro-017 against three pathogens.

[0022] Figure 3: Body weight changes in DSS-induced colitis mouse models in different groups. Figure A shows the percentage change in mouse body weight with DSS administration time, and Figure B shows the percentage change in mouse body weight on the eighth day after DSS administration.

[0023] Figure 4: Colon length of DSS-induced colitis mouse model in different groups. Figure A shows the colon length of different groups in photographs, and Figure B shows the colon length of different treatment groups in statistical analysis.

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

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

[0026] Figure 7: Results of bone metabolism marker detection in DSS-induced colitis mouse models in different groups. Figure A shows the level of anti-tartrate phosphatase (TRAP) in serum, and Figure B shows the level of type I collagen N-terminal propeptide (PINP) in serum. Detailed Implementation

[0027] The present invention will now be described in detail with reference to 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. After isolation, it was identified by MALDI-TOF, 16S rRNA sequencing and whole genome sequencing (ANI value of 98.832%). The species identification results were consistent with the MALDI-TOF results at the species level, and were all Bifidobacterium longum subsp. longum.

[0030] The present invention, Bifidobacterium longum subsp. longum dipro-017, was deposited on August 14, 2024, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31575.

[0031] For functional experiments, glycerol-preserved *Bifidobacterium longum* subsp. *longum* dipro-017 was removed from a -80°C freezer, thawed in a 37°C water bath, and 20 μL was spread onto MRS+ (MRS (Guangdong Huankai Microbial Technology Co., Ltd., catalog number: 027315) + 0.5 g / L cysteine) plates and anaerobically cultured at 37°C for 48 h. Subculturing was then performed using an inoculation loop. After 48 h of culture, single colonies were picked and transferred to MRS broth liquid medium (MRS+) supplemented with 0.5 g / L cysteine, and incubated at 37°C for 48 h for expansion culture, after which subsequent experiments could be conducted.

[0032] Example 2: Determination of indole-3-lactic acid content in dipro-017 culture supernatant

[0033] (1) After resuscitating and expanding the dipro-017 strain according to the method in Example 1, the resulting strain culture was centrifuged at 8000 rpm for 5 min at 4℃, and the supernatant was discarded. The obtained dipro-017 cells were washed once with phosphate-buffered saline (PBS) (Procell, 10 mM, pH 7.2-7.4, catalog number: PB180327), and then resuspended in PBS. The OD600 was adjusted to 2 to prepare a bacterial suspension. 500 μl of the bacterial suspension was added to 4 ml of M9 medium (Shanghai Huzhen Biotechnology Co., Ltd., catalog number: HZ0060-500 ml) supplemented with 1 mM tryptophan and cultured at 37℃ for 48 h. After the culture was completed, the culture was centrifuged at 8000 rpm for 3 min at 4℃, and the supernatant was retained and filtered through a 0.22 μm filter membrane for subsequent detection.

[0034] (2) UV detection conditions: Waters 2498 detector, 280 nm wavelength, column temperature: 25℃, injection volume: 30 μl. A series of gradient concentrations (0, 50, 100, 500, 1000 ng / ml) of indole-3-lactic acid (purchased from Sigma) standard were measured. A standard curve of peak area versus concentration was established, and the regression equation was calculated as y = 92.372x - 1049.6 (R² = 0.9999).

[0035] (3) The content of indole-3-lactic acid in the supernatant obtained in step (1) was determined by Waters HPLC. The chromatographic column was a Kinetex C18 (5μm 150*4.6mm). 30μl of the supernatant obtained in step (1) was taken into the chromatographic column.

[0036] The eluent consists of mobile phase A and mobile phase B:

[0037] Mobile phase A: 0.05% trifluoroacetic acid / water, i.e., an aqueous solution of trifluoroacetic acid with a volume concentration of 0.05%;

[0038] Mobile phase B: 0.05% trifluoroacetic acid / acetonitrile, i.e., a trifluoroacetic acid / acetonitrile solution with a volume concentration of 0.05%;

[0039] The elution procedure is shown in the table below.

[0040] Table 1 HPLC elution program

[0041] At a UV detection wavelength of 280 nm, the peak area of ​​the chromatographic peak corresponding to the retention time of indole-3-lactic acid standard was integrated, as shown in Figure 1. The obtained peak area was 121793 AU*min, i.e., y = 121793. Substituting into the regression equation y = 92.372x - 1049.6 (R2 = 0.9999), the indole-3-lactic acid content in the fermentation supernatant of dipro-017 was calculated to be 1329.86 ng / ml.

[0042] Example 3: Antibacterial activity test of dipro-017

[0043] Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 25922 were activated for three consecutive generations in LB (Appliedi, catalog number: F05932) medium. After centrifugation, the OD600 nm value was adjusted to 1.0–1.2 with sterile physiological saline (the bacterial concentration in the suspension was approximately 3 × 10⁻⁶). 8 (CFU / mL) was prepared for use; dipro-017 bacterial suspension in the exponential growth phase was inoculated at a 2% inoculation rate into MRS+ liquid medium and anaerobically cultured for 48 h for later use. Similarly, the control strain *Lactobacillus rhamnosus* LGG (Chr. Hansen, Denmark) in the exponential growth phase was inoculated at a 2% inoculation rate into MRS liquid medium and anaerobically cultured for 24 h for later use. Agar plates containing pathogens (*Salmonella cytomegalovirus*, *Staphylococcus aureus*, and *Escherichia coli*) were prepared using the casting method. Specifically, the corresponding LB solid medium was first prepared, sterilized, and cooled to 40°C. Pathogen bacterial suspensions were then added and mixed in a certain proportion to ensure a viable count of 10-1. 5 After mixing CFU / mL, pour 30mL of the mixture into each plate. Once the agar has solidified, punch holes with an 8mm punch. Finally, add 200μL of cultured dipro-017 to each hole and incubate at 37℃ for 16h. Measure the diameter of the inhibition zone.

[0044] As shown in Figure 2, the average inhibition zone of dipro-017 against *Salmonella cytomegalovirus* was 16.65 mm, significantly greater than the 15.05 mm inhibition zone of *Lactobacillus rhamnosus* LGG. The average inhibition zone of dipro-017 against *Staphylococcus aureus* was 14.27 mm, significantly greater than the 12.48 cm inhibition zone of *Lactobacillus rhamnosus* LGG. The average inhibition zone of dipro-017 against *Staphylococcus aureus* was 14.69 mm, significantly greater than the 13.40 mm inhibition zone of *Lactobacillus rhamnosus* LGG. In summary, dipro-017 showed good antibacterial effects against all three pathogens.

[0045] Example 4: dipro-017 can improve body weight in a mouse model of colitis.

[0046] After activating dipro-017, it was cultured at 37℃ for 48 hours, and the concentration was adjusted to 1×10⁻⁶ using phosphate-buffered saline (PBS). 9 CFU / mL. Seven-week-old SPF-grade male C57BL / 6J mice were selected and randomly divided into three groups: normal control group (NC group), DSS model group (PBS group), and DSS+dipro-017 treatment group (dipro-017 group), with six mice in each group. Mice in the NC group and PBS group were administered 200 μL of PBS by gavage daily, while mice in the dipro-017 group were administered 200 μL of dipro-017 bacterial suspension by gavage daily (5 × 10⁻⁶ bacteria per gavage). 8 For 15 consecutive days, mice were administered CFU via gavage. After 7 days of gavage, the NC group had free access to ultrapure water, while the PBS and dipro-017 groups had free access to 3% DSS aqueous solution (by mass percentage, the same below) for 8 consecutive days. Changes in mouse body weight were monitored.

[0047] The results are shown in Figure 3. Figure 3A shows the changes in body weight of mice in each group after 8 consecutive days of 3% DSS modeling. It can be seen that as time goes on, the body weight of mice in the PBS group decreases rapidly, while the body weight loss of mice in the dipro-017 treatment group is significantly alleviated (P<0.001). Similarly, from the body weight data of mice with colitis on the 8th day of the colitis modeling endpoint in Figure 3B, it can be seen that dipro-017 can significantly improve the sharp weight loss of mice with colitis (P<0.001), indicating that dipro-017 has a protective effect against DSS-induced colitis.

[0048] Example 5: dipro-017 improves DAI score in colitis mice

[0049] The animal experimental treatment was basically the same as in Example 4, except that, following the experimental method described in the article "Treatment of dextran sulfate 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 assess the severity of colitis in mice. As shown in Figure 4, in this invention, the DAI scores of mice after drinking DSS water changed significantly, with the PBS group scoring over 2 points, showing a significant difference compared to the healthy NC group (P<0.001). Meanwhile, the DAI scores of colitis mice treated with dipro-017 were significantly improved (P<0.05).

[0050] Example 6: dipro-017 improves colon length in mice with colitis

[0051] The animal experimental treatment was basically the same as in Example 4, except that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, they were sacrificed by cervical dislocation. The colon from the anus to the ileocecal junction was dissected, and the colon length was measured with calipers, as shown in Figure 5. Figure 5A shows photographs of the colon tissue length of each treatment group. It can be seen that compared with the normal NC group, the colon of the mice in the PBS group was significantly shortened, indicating that the colon morphology of the mice was damaged. After dipro-017 treatment, the colon length of the mice increased, which can restore this damage to some extent. Further quantitative analysis of colon length results is shown in Figure 5B. Compared with the PBS group, dipro-017 can significantly improve the DSS-induced colon length shortening (P<0.001).

[0052] Example 7: dipro-017 improves blood inflammatory markers in colitis mice

[0053] The animal experimental treatment was basically the same as in Example 4, except that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, blood was collected from the ocular venous plexus, and the serum LPS concentration was detected using an ELISA kit. Lipopolysaccharide (LPS) is an effective stimulant of the immune system; the more LPS that enters the bloodstream through the intestines, the more severe the inflammatory response. As shown in Figure 6A, compared with the NC group, the serum LPS content in the PBS group was significantly increased (P<0.05), indicating that the DSS-induced colitis model leads to an inflammatory response. Compared with the PBS group, dipro-017 can reduce the serum LPS level in colitis mice to some extent, thereby reducing the inflammatory response in the mice.

[0054] The concentration of IL-10 in serum was simultaneously 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 believed to block macrophage metabolism and promote impaired mitophagy, thereby alleviating inflammation. Therefore, high expression of IL-10 is generally considered helpful in suppressing intestinal inflammation. The results, as shown in Figure 6B, indicate that compared with the PBS group, dipro-017 significantly increased the level of IL-10 in mouse serum (P<0.05), demonstrating that Bifidobacterium longum dipro-017 can exert an anti-inflammatory effect, thereby improving the inflammation level in mice with colitis.

[0055] Example 8: dipro-017 improves blood bone metabolism indicators in colitis mice

[0056] The animal experimental treatment was basically the same as in Example 4, except that after mice drank ultrapure water or 3% DSS aqueous solution for 8 days, blood was collected from the ocular venous plexus and the bone metabolism index in the mouse serum was detected using an ELISA kit.

[0057] During bone resorption, osteoclasts secrete tartrate-resistant acid phosphatase (TRAP) as an active enzyme into the bloodstream. Therefore, TRAP is often used as an important indicator of osteoclast count. As shown in Figure 7A, compared with the PBS group, the high expression of TRAP in DSS colitis mice was significantly reduced after treatment with dipro-017 (P<0.05).

[0058] PINP (N-terminal propeptide of type I procollagen) is an N-terminal propeptide of type I collagen, commonly used to reflect the efficacy of osteoporosis treatment, especially for evaluating treatments that promote bone formation. It is an important clinical indicator of bone formation. The ELISA results, as shown in Figure 7B, indicate that compared to the PBS group, dipro-017 treatment significantly increased the low levels of PINP in the serum of colitis-affected mice. Therefore, overall, dipro-017 can improve bone loss caused by inflammation.

Claims

1. A strain of *Bifidobacterium longum* subsp. *longum* dipro-017, characterized in that, The Bifidobacterium longum subsp. longum dipro-017 was deposited on August 14, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31575.

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

3. A product containing *Bifidobacterium longum subsp. longum* dipro-017 as described in claim 1, characterized in that, The products include fermented products, food, health products, pharmaceuticals, or feed.

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

5. The product according to claim 3, characterized in that, The dosage forms of the products include powders, capsules, tablets, pills, film-coated tablets, aerosols, granules, liquids, liposomes, transdermal preparations, suppositories, or lyophilized powder injections.

6. The product according to claim 3, characterized in that, The product also includes protective agents, functional auxiliaries / or auxiliary additives, drug carriers and / or pharmaceutical excipients.

7. The use of the product of claim 1, Bifidobacterium longum subsp. longum dipro-017, and any one of claims 2 to 5, in the preparation of products for the prevention, relief, regulation, or treatment of ulcerative colitis and inflammatory bone loss.

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

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