A strain of Bifidobacterium longum subspecies longum and its application in inhibiting bacteria or improving diseases related to inflammation of the intestinal skin axis

Abstract

The present invention discloses a strain of Bifidobacterium longum subspecies longum and its application in inhibiting bacteria or improving diseases related to intestinal inflammation, belonging to the field of microbial technology. The strain provided by the present invention is a strain of Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum )dipro‑X. The strain Bifidobacterium longum subspecies longum provided by the present invention has good antibacterial activity, can effectively inhibit the expression of inflammatory signal molecules and inflammatory factors in RAW246.7 cells under inflammatory stress, and increase the expression of anti-inflammatory factors, can effectively improve the symptoms of enteritis and skin inflammation in animal models, reduce the level of inflammatory factors, and improve the disorder of intestinal flora structure. At the same time, it increases the expression of genes related to skin cell proliferation in dermatitis models, promotes skin tissue repair, and has a good effect in improving diseases related to inflammation of the intestinal skin axis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bifidobacterium longum subspecies longum and an application thereof in inhibiting bacteria or improving diseases related to inflammation of the intestinal skin axis. Background Art

[0002] The skin is the largest organ in the human body and is home to a large number of commensal microorganisms (bacteria, fungi, and viruses). The skin usually serves as a first protective barrier to prevent the entry of foreign pathogens. Similar to the skin, the human gut also has countless symbiotic microorganisms. Many intestinal bacteria (such as Lactobacillus, Escherichia coli, Bifidobacterium, Streptococcus thermophilus) help maintain human health, while others (such as Clostridium difficile, Campylobacter, Enterococcus faecalis, Helicobacter pylori) are more common in disease states. The connection between the gut-skin axis operates through the activity of immune components that exist between the intestine and the skin. Modulating the interaction between the host and the microbiota is a fundamental function of the immune system. Areas that are colonized by commensal flora, such as the skin and gastrointestinal tract, contain a large number of immune cells. By regulating the immune system, intestinal microorganisms play an important role in enhancing barrier immunity and protecting their ecological niche.

[0003] Disruption of gut integrity and imbalance within the microbiome can have a significant impact on overall skin homeostasis. The gut microbiota interacts with the skin primarily by interacting with the immune system to manage systemic and local inflammation. Antibiotics, prebiotics, probiotics, lifestyle, long-term diet, and disease can all affect the gut microbiome. Skin inflammation can also be caused by small changes in a single bacterial species in the gut microbiome. These changes, in turn, can lead to diseases such as inflammatory bowel disease (IBD), atopic dermatitis (AD), eczema, acne, alopecia areata, hidradenitis, psoriasis, etc.

[0004] Inflammatory bowel disease (IBD) and atopic dermatitis (AD) are the most common chronic inflammatory diseases today. IBD usually presents with persistent intestinal inflammation, abdominal pain, diarrhea, and weight loss. AD is characterized by dry skin, itching, and rashes, which seriously affect the patient's quality of life. Although these two diseases affect different parts of the body, they still have some commonalities. For example, in terms of pathogenesis, both involve primary and secondary infection by pathogenic microorganisms, abnormal responses of the immune system to its own tissues, and persistent and excessive production of inflammatory cytokines. In IBD patients, nitrification stress and oxidative stress are enhanced in the intestine, resulting in a decrease in the abundance of obligate anaerobic bacteria Bacteroidetes and Firmicutes, an increase in the abundance of facultative anaerobic bacteria Proteobacteria, and a decrease in microbial diversity, leading to intestinal mucosal damage and intestinal inflammation. In AD patients, the diversity and composition of the intestinal microbiota are changed, with an increased abundance of pro-inflammatory bacteria, a decreased abundance of anti-inflammatory bacteria, and an increased abundance of Gram-negative intestinal bacteria (such as Bacteroides), leading to an increased translocation of intestinal lipopolysaccharide (LPS) from the intestine to the systemic circulation, which may contribute to AD pathology by stimulating systemic inflammation.

[0005] Nowadays, treatment for IBD and AD usually requires comprehensive management, including drug therapy, lifestyle adjustments and even surgical treatment. For IBD, treatment may include anti-inflammatory drugs, immunosuppressants and biologics. The treatment of AD is mainly to avoid triggers and anti-inflammatory treatment. The main drugs are local corticosteroids, calcineurin inhibitors, systemic immunosuppressants and antibiotics. Although these drugs can relieve the symptoms of IBD and AD, they cannot form a definite treatment plan due to different efficacy and drug side effects. The recurrence rate is high, especially for infants and young children, who are highly sensitive to drugs, have weak drug metabolism and excretion ability, are prone to side effects and adverse reactions during treatment, and the choice of drugs is limited, which directly affects the treatment effect. In addition, since IBD and AD are both chronic diseases, they pose a major challenge to the long-term health and quality of life of patients. Therefore, it is urgent to seek and study new, safer and more effective treatments. Given that changes in the intestinal microbiota play an important role in the pathogenesis of IBD and AD, and both have intestinal flora imbalance, regulating the intestinal microbiota can be used as a new strategy for the treatment of IBD and AD.

[0006] Bifidobacterium is the dominant genus in the human intestinal flora and the most abundant microorganism in the infant intestine. Among them, Bifidobacterium longum, as the dominant bifidobacterium in the infant intestine, has a significant effect on the maturation and stability of infant intestinal microorganisms. It can participate in metabolic processes, regulate the activity and function of immune cells, reduce inflammatory responses, and play an important role in the maintenance and repair of intestinal barrier function and systemic immune balance (see Wu Shiyin, Cai Meiqin. Research progress on Bifidobacterium longum and intestinal health of infants and young children [J]. Journal of Clinical Pediatrics, 2022, 40(09): 715-720.). However, most of the current reports on Bifidobacterium longum are about improving intestinal inflammation, constipation and obesity. For example, in the Chinese invention patent with publication number CN114164134B, a long subspecies of Bifidobacterium longum KLDS K5 is disclosed, which can relieve weight loss during ulcerative colitis, improve colon mucosal damage and intestinal flora, but in the mouse experiment design, the probiotics are fed to mice with skim milk, so the improvement of colitis cannot be attributed entirely to the strain KLDS K5; for example, in the invention patent with publication number CN116790453B, a long subspecies of Bifidobacterium longum BL11 is mentioned, which can produce antimicrobial peptides and can significantly improve intestinal inflammation and impaired intestinal barrier function caused by bacterial infection, but the research results and conclusions are mainly based on Escherichia coli BNCC336902, which may mean that it may not have the same effect on other types of pathogenic strains, and further research is still needed to verify. In addition, according to existing data, there are no reports of long subspecies of Bifidobacterium longum strains that improve diseases related to intestinal skin axis inflammation. Therefore, the present invention aims to provide a strain of Bifidobacterium longum subspecies longum with antibacterial and anti-inflammatory capabilities, and its use in preventing and / or treating diseases related to intestinal inflammation. Summary of the invention

[0007] Purpose of the invention: In order to solve the limitations of the prior art in improving diseases related to intestinal inflammation, the present invention provides a strain of Bifidobacterium longum subspecies longum that can effectively inhibit pathogens, inhibit inflammatory factors, enhance anti-inflammatory factors, and improve diseases related to intestinal inflammation, and its application, so that patients can get rid of the side effects and limitations of traditional drug treatments.

[0008] Technical solution: To solve the above technical problems, the present invention provides a strain of Bifidobacterium longum subspecies long isolated and screened from the feces of healthy children. The strain was analyzed by whole genome sequencing and BLAST nucleic acid sequence comparison was performed through the NCBI gene database. The results showed that the strain was Bifidobacterium longum subspecies long ( Bifidobacterium longum subsp.longum ). The Bifidobacterium longum subspecies longum of the present invention ( Bifidobacterium longum subsp.longum)dipro-X was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No. 29384.

[0009] Wherein, the long subspecies of Bifidobacterium longum (Bifidobacterium longum subsp.longum) dipro-X is Gram-positive, the bacteria are rod-shaped or V-shaped, the length and diameter are uneven, and there are no spores. On MRS agar medium, after streaking, obvious colonies appear after anaerobic culture at 35~39°C for 24~48h. The colonies are round, white opaque protrusions, smooth surface, and the colony diameter is 0.2~3mm. In MRS liquid culture medium, obvious bacterial precipitation appears when anaerobic culture is carried out at 35~39°C for 12~16h. After 48h of culture, the pH of the culture solution is about 3.5.

[0010] The present invention also includes the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X products, including but not limited to fermentation products or medicines.

[0011] The fermented product comprises the fermentation supernatant, fermentation precipitate or bacterial suspension of Bifidobacterium longum subsp. longum dipro-X.

[0012] 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.

[0013] The product includes one or more of protective agents, functional additives, auxiliary additives, drug carriers, and pharmaceutical excipients.

[0014] The present invention also includes the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) Dipro-X, and the use of the above products in the preparation of drugs for inhibiting bacteria and inflammation or improving diseases related to intestinal inflammation.

[0015] Wherein, the diseases associated with inflammation of the gut-dermal axis include one or more of inflammatory bowel disease (IBD), atopic dermatitis (AD), eczema, acne, alopecia areata, hidradenitis or psoriasis.

[0016] The present invention also includes the application of the Bifidobacterium longum subsp. longum dipro-X in the product having at least one of the following functions:

[0017] 1) Products with antibacterial effects;

[0018] 2) Products that reduce inflammatory signaling molecules and pro-inflammatory cytokines;

[0019] 3) Products with the ability to increase anti-inflammatory cytokines;

[0020] 4) Products that improve enteritis;

[0021] 5) Products that can reduce the visual and pathological damage to the skin caused by atopic dermatitis;

[0022] 6) Products that can reduce the levels of serum inflammatory factors in atopic dermatitis;

[0023] 7) Products that reduce the expression level of inflammatory cytokine genes in atopic dermatitis lesions;

[0024] 8) Products that promote the expression of genes related to cell proliferation in atopic dermatitis lesions;

[0025] 9) Products with skin tissue repair function;

[0026] 10) Products that can improve intestinal flora disorders.

[0027] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum )dipro-X has good antibacterial ability against pathogens (Escherichia coli, Staphylococcus aureus, Salmonella, Enterococcus faecalis, Klebsiella pneumoniae).

[0028] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation described above is capable of utilizing tryptophan to produce indole-3-lactic acid (ILA).

[0029] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can inhibit the production of nitric oxide (NO) and tumor necrosis factor α (TNF-α) by RAW264.7 cells induced by LPS, and can induce RAW264.7 and THP-1 cells to produce high levels of interleukin 10 (IL-10).

[0030] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the formulations were able to restore body weight loss in mice with colitis induced by dextran sodium sulfate (DSS).

[0031] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can improve the shortening of colon length in mice with DSS-induced colitis.

[0032] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can reduce the DSS-induced mouse serum inflammatory factor TNF-α.

[0033] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can reduce the serum LPS level of mice induced by DSS.

[0034] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can change the cecal microbial composition of DSS mice.

[0035] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation described above can reduce the ocular lesions of the skin in mice with atopic dermatitis induced by dinitrochlorobenzene (DNCB).

[0036] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can reduce the pathological damage of skin tissue in mice with atopic dermatitis induced by DNCB.

[0037] Among them, Bifidobacterium longum subsp. Bifidobacterium longum subsp.longum ) dipro-X or the preparation can reduce the TNF-α level in the serum of DNCB-induced atopic dermatitis mice.

[0038] Among them, Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can reduce the mRNA expression levels of inflammatory cytokines TNF-α and interleukin 1β (IL-1β) in the skin lesions of DNCB-induced atopic dermatitis mice.

[0039] Among them, Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can promote the expression level of mRNA of cell proliferation-related genes, KRT15, ​​ITGA6 and PCNA in the skin lesions of DNCB-induced atopic dermatitis mice.

[0040] Wherein, the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X or the preparation can change the colonic microbial composition of DNCB-induced atopic dermatitis mice.

[0041] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention provides a strain of Bifidobacterium longum subspecies longum that can inhibit pathogens and inhibit inflammatory responses, as well as an application that effectively improves diseases related to intestinal skin axis inflammation. The present invention proves for the first time the safety of the dipro-X strain, and has the ability to inhibit pathogens and metabolize tryptophan to produce ILA, which can significantly reduce the inflammatory signal molecules and inflammatory factors produced by RAW264.7 cells under LPS induction, and can also activate RAW264.7 and THP-1 cells to produce anti-inflammatory cytokines. The dipro-X of the present invention and its products can effectively inhibit the expression of inflammatory signal molecules and inflammatory factors in RAW264.7 cells under LPS induction, and can stimulate RAW264.7 and THP-1 cells to produce anti-inflammatory cytokines. In the DSS colitis mouse model, dipro-X can significantly improve the weight and colon length of mice, reduce the level of inflammatory factors, enhance the intestinal barrier function, and optimize the intestinal flora structure. The present invention also proves for the first time that dipro-X can improve the symptoms of DNCB-induced atopic dermatitis in mice, and effectively regulate the expression of inflammatory factors, promote skin cell proliferation, and maintain the integrity of the skin barrier, thereby significantly improving the symptoms of atopic dermatitis. Therefore, the use of the long subspecies dipro-X strain of Bifidobacterium longum to prepare products that improve diseases related to inflammation of the intestinal skin axis has a good prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The level of ILA produced by Bifidobacterium longum dipro-X.

[0043] Figure 2 Bifidobacterium longum dipro-X inhibits LPS-induced inflammatory signaling molecule NO in RAW264.7 cells.

[0044] Figure 3 Bifidobacterium longum dipro-X inhibits LPS-induced inflammatory factor TNF-α in RAW264.7 cells.

[0045] Figure 4 Bifidobacterium longum dipro-X can induce RAW264.7 cells to produce IL-10.

[0046] Figure 5 Bifidobacterium longum dipro-X can induce THP-1 cells to produce IL-10.

[0047] Figure 6 Bifidobacterium longum dipro-X can restore DSS-induced weight loss in mice.

[0048] Figure 7 Bifidobacterium longum dipro-X was able to restore colon length in DSS mice.

[0049] Figure 8 Bifidobacterium longum dipro-X can reduce the level of serum inflammatory factor TNF-α in DSS mice.

[0050] Figure 9 Bifidobacterium longum dipro-X can reduce the serum lipopolysaccharide (LPS) level in DSS mice.

[0051] Figure 10 Bifidobacterium longum dipro-X can change the cecal microbial composition of DSS mice.

[0052] Figure 11 Bifidobacterium longum dipro-X can reduce DNCB-induced skin lesions in mice. Ctrl: negative control, PBS gavage; DNCB group: positive control, DNCB solution treatment, PBS gavage; DNCB+dipro-X group: DNCB solution treatment, Bifidobacterium longum dipro-X (1×10 10 CFU) by intragastric administration. A: Appearance of mice; B: Dermatitis score of mice; C: Thickness of mouse ears.

[0053] Figure 12 Bifidobacterium longum dipro-X can alleviate DNCB-induced pathological damage in mice. Ctrl: negative control, PBS gavage; DNCB group: positive control, DNCB solution treatment, PBS gavage; DNCB+dipro-X group: DNCB solution treatment, Bifidobacterium longum dipro-X (1×10 10 CFU) by oral administration. A: Mouse back skin tissue section, 40×; B: Epidermal thickness.

[0054] Figure 13 Bifidobacterium longum dipro-X can reduce the level of TNF-α in the serum of mice with DNCB-induced dermatitis.

[0055] Figure 14 Bifidobacterium longum dipro-X can reduce the mRNA expression levels of inflammatory cytokines TNF-α and IL-1β in the skin lesions of DNCB-induced dermatitis mice.

[0056] Figure 15 Bifidobacterium longum dipro-X can promote the mRNA expression levels of cell proliferation-related genes, KRT15, ​​ITGA6 and PCNA in the skin lesions of DNCB-induced dermatitis mice.

[0057] Figure 16 Bifidobacterium longum dipro-X can change the colonic microbial composition of dermatitis mice. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0059] The comparative strains involved in the following embodiments: Bifidobacterium breve M-16V (M-16V) is a well-known strain, which was isolated from the probiotic product "M16V" of Difuluo (Shanghai) Biological Co., Ltd.; Lactobacillus rhamnosus GG (LGG) strain publication number ATCC 53103 is a well-known strain, which was isolated from the probiotic product "BG" of Difuluo (Shanghai) Biological Co., Ltd.; Pediococcus pentosaceus CECT 8330 (8330) is a well-known strain, which was isolated from the probiotic product "8330 Ambino" of Difuluo (Shanghai) Biological Co., Ltd.; Lactobacillus reuteri DSM 17938 (17938) is from Bio Company, Sweden; MRS culture medium was purchased from Guangdong Huankai Microbiology; RAW264.7 cell line was purchased from Shanghai Cell Bank of the Chinese Academy of Sciences, and THP-1 cell line was purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; DMEM culture medium, RPMI-1640 culture medium, fetal bovine serum, penicillin and streptomycin and 0.25 % pancreatic enzyme was purchased from Gibco; LPS and phorbol ester (PMA) were purchased from Beijing Solebao Technology Co., Ltd.; NO detection kit was purchased from Beyotime Biotechnology; ELISA detection kit (TNF-α, IL-10) was purchased from Wuhan Fine Biotechnology Co., Ltd.; 7-week-old male C57BL / 6J mice and 7-week-old female BALB / c mice, both SPF grade, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.; DSS was purchased from MP Biochemicals; DNCB was purchased from Sigma-Aldrich; PrimeScript™ RT reagent Kitwith gDNA Eraser was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.; ChamQ Universal SYBR qPCRMaster Mix kit was purchased from Nanjing Novozyme Biotechnology; primers were from Beijing Qingke Biotechnology Co., Ltd.

[0060] The Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) The isolation and identification methods of dipro-X and control strains are as follows:

[0061] 1. Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) Isolation and identification of dipro-X

[0062] Fecal samples were obtained from healthy children aged 3 years. The fecal samples were resuspended and diluted with sterile phosphate buffered saline (PBS, 10 mM, pH 7.2-7.4, the same below) or normal saline. -4 , 10 -5 , 10 -6The dilution gradient was spread on MRS+ agar medium (MRS agar medium supplemented with 0.05% cysteine), and cultured anaerobically at 37°C for 48 hours. The colonies that met the morphology of Bifidobacterium were picked and streaked and purified for three generations. After that, they were preliminarily identified by MALDI mass spectrometry. Then, a single colony was picked and inoculated in MRS+ liquid medium (MRS liquid medium supplemented with 0.05% cysteine), and cultured anaerobically at 37°C for 48 hours. Part of the bacterial liquid was sent for whole genome sequencing. The results showed that the strain was Bifidobacterium longum, and it was named Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) dipro-X and evaluate the genomic safety of the strain, and the remaining bacterial liquid was retained.

[0063] 2. Purification and identification of comparative strains

[0064] The inoculation loop was dipped into the bacterial suspension of Bifidobacterium breve M-16V, Lactobacillus rhamnosus GG (LGG), Pediococcus pentosaceus CECT 8330 and Lactobacillus reuteri DSM17938 products. Bifidobacteria were cultured on MRS+ agar medium, while Lactobacillus and Pediococcus were streaked on MRS agar medium. Single colonies were streaked and purified for three generations and then preliminarily identified by MALDI mass spectrometry. Single colonies were picked and inoculated into MRS+ / MRS liquid culture medium and cultured anaerobically at 37°C for 48 hours. Part of the bacterial solution was sent for whole genome sequencing to identify the species, and the rest of the bacterial solution was retained.

[0065] Example 1 Safety Analysis of Bifidobacterium longum

[0066] The genomic safety of the dipro-X strain was evaluated based on whole genome sequencing, and the results showed that the dipro-X genome contained no virulence genes.

[0067] Example 2 Hemolytic activity of Bifidobacterium longum

[0068] Use an inoculation loop to dip the bacterial liquid of the dipro-X strain after 3 generations of activation and streak it on the Columbia blood plate. Incubate it anaerobically at 37°C for 48 hours. If there is no hemolytic ring around the colony, dipro-X has no hemolytic activity.

[0069] Example 3 Antibacterial ability of Bifidobacterium longum dipro-X

[0070] Escherichia coli, Staphylococcus aureus, and Salmonella were activated in LB liquid medium, Enterococcus faecalis in TSA liquid medium, and Klebsiella pneumoniae in NB medium for three generations, and the OD625nm value was adjusted to 0.16-0.20 with sterile saline (the concentration of bacteria in the bacterial suspension was about 3×10 8CFU / mL) for later use; dipro-X in the exponential growth phase was inoculated into MRS+ liquid culture medium at a 2% inoculation rate and cultured anaerobically for 48h for later use. Agar plates containing pathogens (Escherichia coli, Staphylococcus aureus, Salmonella, Enterococcus faecalis and Klebsiella pneumoniae) were prepared by the pouring method. Specifically, the corresponding agar culture medium was first prepared, sterilized and cooled to 40°C, and an appropriate amount of pathogenic bacteria solution was added and mixed. After each plate was poured, 30mL of the plate was poured. After the agar solidified, a hole was punched with an 8 mm puncher, and finally 200μL of the cultured dipro-X was added to the hole, and cultured at 37°C for 16h, and the diameter of the inhibition zone was measured.

[0071] Comparative Example 1

[0072] The method is basically the same as Example 3, except that the antibacterial activity of Bifidobacterium breve M-16V against 5 pathogens was tested.

[0073] The results are shown in Table 1, which show that the diameters of the inhibition zones of dipro-X against Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae are 14.46, 14.32, 14.61 and 16.38 mm, respectively, which are 10.72%, 10.13%, 14.56% and 5.81% higher than those in Comparative Example 1, respectively. The diameter of the inhibition zone against Enterococcus faecalis is 12.57 mm, which is similar to Comparative Example 1. Dipro-X has excellent antibacterial properties.

[0074] Table 1

[0075]

[0076] Example 4 Ability of Bifidobacterium longum dipro-X to utilize tryptophan

[0077] The exponentially growing dipro-X was enriched and cultured with MRS+ liquid culture medium, then washed once with PBS (10 mM, pH 7.2-7.4), adjusted to OD600 = 2.5, and then inoculated at a 2% inoculum in M9 culture medium containing 0.12 g / L tryptophan and cultured at 37°C for 48 h. The production of indole derivatives was then detected using a high performance liquid chromatography system (Waters 2498, Waters Corporation, USA).

[0078] Comparative Example 2

[0079] The strain LGG in the exponential growth phase was enriched and cultured in MRS liquid medium, and the subsequent experiments were basically the same as those in Example 4 to test its level of producing ILA using tryptophan.

[0080] The results of Example 4 and Comparative Example 2 are shown in Figure 1The ILA production of dipro-X metabolizing tryptophan was 13357.04 ng / mL, which was significantly higher than the ILA level of comparative example 2 ( P <0.05), and the amount of ILA produced was 5 times that of comparative example 2 (2614.28 ng / mL).

[0081] Example 5 Bifidobacterium longum dipro-X inhibits LPS-induced inflammatory signaling molecule NO levels in RAW264.7 cells

[0082] 1. Activation of Bifidobacterium longum subsp. longum dipro-X: Take out the cryopreserved tube, streak on MRS+ agar medium for anaerobically culture for 48 h, then pick a single colony and culture it anaerobically in MRS+ liquid medium for 48 h, and store it as seed liquid for later use. When using, take the bacterial liquid and inoculate it into MRS+ liquid medium at 2%. After the culture is completed, take the bacterial suspension and centrifuge it at 6000 rpm / min for 5 min, discard the supernatant, resuspend it with PBS, centrifuge and wash it twice for later use.

[0083] 2. RAW264.7 cells were frozen in our laboratory. Recovery: Take out RAW264.7 cells from the liquid nitrogen tank, thaw them quickly in a 37℃ constant temperature water bath, transfer the thawed cells to 4 ~ 6 mL DMEM culture medium, add 10% fetal bovine serum and 1% penicillin-streptomycin, mix well, and culture in a 37℃, 5% CO2 incubator. After the cells are completely attached to the wall, change the medium and continue to culture. When the cell density reaches about 80% ~ 90%, pass the cells. Passage: Discard the old culture medium, wash 3 times with 2 mL PBS solution, gently blow the attached cells with a pipette to suspend them, add 4 ~ 6 mL of fresh culture medium to the new culture bottle, add the cell suspension and mix well, place in a 37℃, 5% CO2 incubator to continue culture, and pass the test after 3 generations in this way. Plating and testing: When the RAW264.7 cells are in good growth condition and reach the logarithmic growth phase, press 1×10 5 / well were inoculated into 96-well plates and cultured until adhered to the wall. The culture medium was discarded, 100 μL of DMEM solution was added to the negative wells, 100 μL of DMEM solution containing 0.5 μg / mL LPS was added to the positive wells, and 100 μL of 1×10 7 The DMEM solution of CFU Bifidobacterium longum dipro-X and 0.5 μg / mL LPS was placed in a 37°C, 5% CO2 incubator for 18 h. The supernatant after the above culture was collected and the NO concentration was detected using a kit. NO inhibition rate = (positive well - sample well) / (positive well - negative well).

[0084] Comparative Example 3

[0085] MRS medium was used for the activation and culture of Pediococcus pentosaceus 8330. The strain activation, passage and cell experimental methods were consistent with those in Example 5, and the level of inhibition of the production of inflammatory signal molecule NO by RAW264.7 cells induced by LPS was tested.

[0086] The results of Example 5 and Comparative Example 3 are as follows Figure 2 As shown. After treatment, dipro-X can inhibit the release of NO in RAW264.7 macrophages induced by LPS, with an inhibition rate of 100%, and the inhibition rate of dipro-X on NO is significantly higher than that of comparison example 3 ( P <0.05), the inhibition rate increased by 10.74%.

[0087] Example 6 Bifidobacterium longum dipro-X inhibits LPS-induced inflammatory factor TNF-α level in RAW264.7 cells

[0088] The results are basically the same as those in Example 5, except that the kit tests the concentration of TNF-α in the supernatant after culture, and the TNF-α inhibition rate = (positive well - sample well) / (positive well - negative well).

[0089] Comparative Example 4

[0090] Except that MRS medium was used for activation and culture of Lactobacillus reuteri 17938, the strain activation, passage and cell test methods were basically the same as those in Example 6 to test its ability to inhibit LPS-induced production of inflammatory factor TNF-α in RAW264.7 cells.

[0091] The results of Example 6 and Comparative Example 4 are as follows Figure 3 After treatment, dipro-X could inhibit LPS-induced TNF-α production in RAW264.7 macrophages, with an inhibition rate of 100%, and the inhibitory effect on TNF-α was significantly better than that of the comparative example 4 strain 17938 ( P <0.001), and the TNF-α inhibition rate was 2.51 times that of Comparative Example 4.

[0092] Example 7 Bifidobacterium longum dipro-X induces RAW264.7 cells to produce anti-inflammatory factors

[0093] The culture and cell culture and plating methods of Bifidobacterium longum dipro-X were basically the same as those in Example 5, except that RAW264.7 cells (1×10 5 / well) in a 96-well plate and cultured until adhered to the wall, the culture medium was discarded, 100 μL of DMEM solution was added to the negative wells (Ctrl), and 100 μL of 1×10 7 CFU dipro-X in DMEM solution, incubate for 18 h. Collect the supernatant after the above culture and detect the IL-10 concentration using a kit.

[0094] Comparative Example 5

[0095] MRS medium was used for activation and culture of Lactobacillus rhamnosus LGG. The strain activation, passage and cell test methods were basically the same as those in Example 7, and the level of anti-inflammatory cytokine IL-10 produced by RAW264.7 cells was tested.

[0096] The experimental results of Example 7 and Comparative Example 5 are as follows Figure 4 Compared with the negative wells, dipro-X treatment can significantly induce the production of high levels of IL-10 in RAW264.7 macrophages, with a concentration as high as 1733pg / mL ( P <0.001, and the IL-10 level induced by dipro-X was significantly higher than that of comparative example 5 ( P <0.001), which is 2.33 times that of Comparative Example 5.

[0097] Example 8 Bifidobacterium longum dipro-X induces THP-1 cells to produce anti-inflammatory factors

[0098] 1. The cultivation method of Bifidobacterium longum dipro-X is basically the same as that in Example 5.

[0099] 2. THP-1 cells were cryopreserved in our laboratory. Recovery: Take out the cells from the liquid nitrogen tank, put them in a 37℃ constant temperature water bath to thaw quickly, transfer the thawed cells to 4 ~ 6 mL RPMI-1640 culture medium with 10% fetal bovine serum and 1% penicillin-streptomycin, mix well, and place them in a 37℃, 5% CO2 incubator to culture. The culture density is maintained at 5×10 5 -1×10 6 cells / mL, more than 2×10 6 / mL for subculture. Subculture: Half-medium exchange method, stand the T25 culture flask upright and let the cells settle to the bottom, aspirate 2.5mL of medium, transfer to a centrifuge tube, centrifuge at 1000rpm for 3-5min, suspend the precipitate in the original bottle, and add 2.5mL of fresh medium to the original bottle. After confirming that the cells are in good growth condition, conduct the test. Plating and test: When THP-1 cells are in good growth condition and reach the logarithmic growth phase, press 5×10 5 / well were inoculated into 96-well plates, and 200ng / mL PMA was added to induce the cells for 20h to differentiate. When cell adhesion was observed (conventional THP-1 was in a suspended state, the cell morphology after PMA treatment was flat and elongated or protruded until the cells adhered to the wall, that is, the cells differentiated into M1 type), the culture medium was discarded, and 100μL containing 1×10 7The RPMI-1640 solution of CFU Bifidobacterium longum dipro-X was incubated for 18 h, and the supernatant after the above culture was collected and the IL-10 concentration was detected using a kit.

[0100] Comparative Example 6

[0101] Except that MRS medium was used for the activation and culture of strain LGG, the strain activation, passage and cell test methods were basically the same as those in Example 8 to test the level of anti-inflammatory cytokine IL-10 induced by THP-1 cells.

[0102] The experimental results of Example 8 and Comparative Example 6 are as follows Figure 5 Compared with the control group, dipro-X significantly promoted the production of IL-10 in THP-1 cells, with the concentration reaching 537 pg / mL ( P <0.001). And dipro-X induced significantly higher IL-10 production than comparative example 6 ( P <0.001), which is 74 times the concentration of Comparative Example 6.

[0103] Example 9 Bifidobacterium longum dipro-X restores body weight in mice with enteritis

[0104] After activation of dipro-X, culture at 37°C for 24-48 hours and adjust the concentration to 1×10 9 CFU / mL. 7-week-old SPF male C57BL / 6J mice were selected and randomly divided into a control group (Ctrl), a DSS model group (DSS), and a dipro-X treatment group (DSS+dipro-X), with 6 mice in each group. Each mouse in the Ctrl and DSS groups was intragastrically gavaged with 200 μL PBS per day, and each mouse in the dipro-X group was intragastrically gavaged with 200 μL dipro-X bacterial suspension (5×10 8 CFU) for 23 consecutive days. After 14 days of intragastric administration, the Ctrl group freely drank ultrapure water, and the DSS group and dipro-X group freely drank 3% DSS aqueous solution for 9 consecutive days. The weight changes of mice were monitored at the same time. The results are shown in Figure 6 , Bifidobacterium longum dipro-X can significantly improve the body weight loss of enteritis mice ( P <0.05).

[0105] Example 10 Bifidobacterium longum dipro-X improves colon length in enteritis mice

[0106] The animal experiment treatment was basically the same as in Example 9, except that after 9 days of DSS treatment, the mice were killed by cervical dislocation, the colon from the anus to the ileocecal region was dissected, and the colon length was measured with a vernier caliper. Figure 7As shown, Bifidobacterium longum dipro-X was able to significantly improve DSS-induced colon shortening ( P <0.05).

[0107] Example 11 Bifidobacterium longum dipro-X reduces the level of inflammatory factor TNF-α in the serum of mice with enteritis

[0108] The animal experiment treatment was basically the same as in Example 9, except that after 9 days of DSS treatment, blood was collected from the ocular venous plexus of mice and the serum TNF-α concentration was detected using an ELISA kit. The results are shown in Figure 8 As shown, Bifidobacterium longum dipro-X can reduce the level of inflammatory factor TNF-α in the serum of mice with enteritis, but the difference is not significant ( P >0.05).

[0109] Example 12 Bifidobacterium longum dipro-X reduces serum LPS levels in mice with enteritis

[0110] The animal experiment treatment was basically the same as in Example 9, except that after 9 days of DSS treatment, blood was collected from the ocular venous plexus of mice and the serum LPS concentration was detected using an ELISA kit. The results are shown in Figure 9 As shown, Bifidobacterium longum dipro-X can significantly reduce the serum LPS level of enteritis mice ( P <0.05).

[0111] Example 13 Bifidobacterium longum dipro-X changes the cecal microbial composition of enteritis mice

[0112] The animal experiment treatment was basically the same as in Example 9, except that after 9 days of DSS treatment, the mice were killed by cervical dislocation, the cecal contents were dissected, quickly frozen in liquid nitrogen, and then the cecal microbiome of the mice was analyzed by 16S rDNA amplicon sequencing. Figure 10 Compared with the DSS group, Bifidobacterium longum dipro-X was able to change the composition of cecal microorganisms in mice with enteritis. Bifidobacterium longum dipro-X increased the α diversity index (Chao1 index, number of OTUs, Shannon index, and Simpson index) of the colonic microbial community in mice. PCA analysis showed that there were significant differences in the microbial community structure among the three experimental groups: Ctrl, DNCB, and DNCB + diproX ( P <0.01). According to LEFSe analysis, diproX significantly increased the abundance of Clostridiaceae and Peptostreptococcus. Therefore, dipro-X may promote the recovery of colitis by increasing the diversity and richness of the intestinal microbial community in mice and changing the composition of the intestinal microbial community in mice.

[0113] Example 14 Bifidobacterium longum dipro-X improves skin tissue damage in mice with atopic dermatitis

[0114] Take the Dipro-X freeze-dried bacterial powder and resuspend it in PBS to make the bacterial concentration 5×10 10 CFU / mL. Seven-week-old SPF female BALB / c mice were selected and randomly divided into three groups: control group (Ctrl), allergy model group (DNCB), and dipro-X treatment group (DNCB+dipro-X), with 6 mice in each group. Each mouse in the Ctrl and DNCB groups was intragastrically gavaged with 200 μL PBS per day, and each mouse in the dipro-X treatment group was intragastrically gavaged with 200 μL dipro-X freeze-dried bacterial suspension (1×10 10 CFU) for 24 consecutive days. On day 0, the back of the mice was depilated in an area of ​​about 2 cm × 4 cm. On days 1 and 4, 100 μL of 2% DNCB solution (prepared with a matrix solution of acetone: olive oil = 3:1) was evenly applied to the hairless area of ​​the back of the DNCB and DNCB+dipro-X groups for the first sensitization. At the same time, the Ctrl group was smeared with an equal amount of matrix solution. On days 8, 11, 14, 17, 20, and 23 of the experiment, 100 μL and 20 μL of 1% DNCB solution were applied to the back and right ear of the mice, respectively. μL was used for stimulation, and at the same time, the Ctrl group was smeared with an equal amount of matrix solution; on the 25th day, the skin symptoms of the mice were observed and the degree of skin damage was evaluated. The skin tissue symptom scores were erythema (bleeding), edema (papules), scaling (dryness), and epidermal exfoliation (scratches). Severe erythema, edema, scaling, and epidermal exfoliation were scored as 3 points; moderate erythema, edema, scaling, and epidermal exfoliation were scored as 2 points; mild erythema, edema, scaling, and epidermal exfoliation were scored as 1 point; normal skin was scored as 0 points; the total dermatitis score was evaluated as the sum of the scores of each aspect, with a maximum score of 12 points. In addition, the ear thickness of the right ear of the mouse was measured. Results are shown in Figure 11 The DNCB-induced AD mouse model group showed obvious erosion, erythema, dryness and thick scales on the back skin, and the dermatitis score and ear thickness increased significantly ( P <0.05), after dipro-X treatment, the back skin of mice showed significant improvement, with reduced edema, less scales, and significantly reduced dermatitis scores and ear thickness ( P <0.05). Bifidobacterium longum dipro-X can improve skin tissue damage in DNCB-induced atopic dermatitis in mice.

[0115] Example 15 Bifidobacterium longum dipro-X reduces pathological damage to skin tissues in mice with atopic dermatitis

[0116] The animal experiment treatment was basically the same as in Example 14, except that on the 25th day, the mice were killed by cervical dislocation, the skin tissue was collected and fixed in 4% paraformaldehyde solution, and then the tissue was embedded in paraffin and made into slices (5 μm) for hematoxylin-eosin (HE) staining and skin tissue pathology examination. The thickness of the epidermis was measured and statistically analyzed using ImageJ. The results are shown in Figure 12 The DNCB-induced AD mouse model group showed typical features of dermatitis, including hyperkeratosis, epidermal thickening, and inflammatory cell infiltration, while the epidermal thickness of dipro-X-treated mice was thinner than that of untreated mice, and the difference was significant ( P <0.001). In addition, dipro-X treatment also reduced the infiltration of inflammatory cells. Bifidobacterium longum dipro-X was able to improve the skin tissue pathological damage of DNCB-induced AD mice.

[0117] Example 16 Bifidobacterium longum dipro-X reduces TNF-α levels in serum of mice with atopic dermatitis

[0118] The animal experiment treatment was basically the same as that in Example 14, except that on the 25th day, the mice were killed by cervical dislocation, and the mouse serum was collected and the tumor necrosis factor-α (TNF-α) level in the mouse serum was detected by ELISA. Figure 13 The results showed that the serum TNF-α concentration of AD mice treated with dipro-X was significantly reduced compared with that of DNCB dermatitis mice ( P <0.01). dipro-X improved the systemic inflammation level in DNCB-induced AD mice.

[0119] Example 17 Bifidobacterium longum dipro-X reduces the expression level of inflammatory factor-related genes in skin lesions of atopic dermatitis mice

[0120] The animal experiment treatment was basically the same as that in Example 14, except that on the 25th day, the mice were killed by cervical dislocation, and the back skin tissue of the mice was collected. The total RNA was extracted by Trizol method, reverse transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser, and quantitative real-time PCR (qPCR) detection was performed using ChamQ Universal SYBR qPCR Master Mix Kit, and GAPDH was used as an internal reference. −ΔΔ The expression levels of TNF-α and IL-1β gene mRNA were analyzed by Ct method. Figure 14 The results showed that compared with the DNCB group, the expression of TNF-α and IL-1β mRNA in the skin lesions of AD mice treated with dipro-X was significantly downregulated (P <0.05). In conclusion, dipro-X can improve AD and downregulate the overexpression of cytokine genes related to skin inflammation.

[0121] Example 18 Bifidobacterium longum dipro-X promotes the expression of cell proliferation-related genes in skin lesions of atopic dermatitis mice

[0122] The animal experiment treatment was basically the same as in Example 14. On the 25th day, the mice were killed by cervical dislocation, and the skin tissue from the back of the mice was collected. The subsequent total RNA extraction, reverse transcription, and qPCR detection methods of the skin were basically the same as in Example 15. The difference was that GAPDH was used as an internal reference during qPCR detection to analyze the expression levels of cell proliferation-related genes KRT15, ​​ITGA6, and PCNA gene mRNA. The results are shown in Figure 15 The results showed that compared with the DNCB group, the expression of KRT15, ​​ITGA6 and PCNA in the lesional tissues of dermatitis mice treated with dipro-X was significantly increased ( P <0.05). dipro-X can promote the proliferation of skin cells, thereby repairing damaged skin and promoting the recovery of AD.

[0123] Example 19 Results of Bifidobacterium longum dipro-X changing the colonic microbiota of mice with atopic dermatitis

[0124] The animal experiment treatment was basically the same as in Example 14, except that on the 25th day, the mice were killed by cervical dislocation, the colon contents were dissected, quickly frozen in liquid nitrogen, and sent for metagenomic sequencing to analyze the changes in the colon microbial community in each group. Figure 16 Compared with the DNCB group, Bifidobacterium longum dipro-X increased the α diversity index (Chao1 index, OTU number, Shannon index and Simpson index) of the mouse colonic microbial community. PCA analysis showed that there were significant differences in the microbial community structure among the three experimental groups: Ctrl, DNCB and DNCB + dipro-X ( P = 0.002). At the phylum level, dipro-X significantly increased the abundance of Bacteroidetes and reduced the abundance of Firmicutes. At the species level, dipro-X treatment increased Bacteroides caecimuris The abundance of Dipro-X has been shown to play a role in inhibiting liver inflammation and improving intestinal health. Therefore, dipro-X may promote the recovery of AD by increasing the diversity and richness of the intestinal microbial community in mice and changing the composition of the intestinal microbial community in mice.

Claims

1. A strain of Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.longum ) dipro-X, characterized in that The Bifidobacterium longum subspecies longum dipro-X was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC NO. 29384.

2. Containing the Bifidobacterium longum subspecies longum according to claim 1 ( Bifidobacterium longum subsp.longum ) dipro-X product, characterized in that, The product includes a fermented product or a medicine, wherein the fermented product includes Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp.longum ) Fermentation precipitate or bacterial suspension of dipro-X.

3. The product according to claim 2, characterized in that The dosage form of the drug includes powder, capsule, tablet, pill, film-coated agent, granule, liquid or liposome.

4. The product according to claim 2, characterized in that The medicine also includes a protective agent.

5. The Bifidobacterium longum subspecies longum of claim 1 ( Bifidobacterium longum subsp.longum ) Use of dipro-X, the product according to any one of claims 2 to 4, in the preparation of a medicament for inhibiting Escherichia coli, Staphylococcus aureus, Salmonella and Klebsiella pneumoniae, and in a medicament for treating inflammatory bowel disease or atopic dermatitis.

6. The use according to claim 5, characterized in that: The product is a product having at least one of the following functions: 1) Products with antibacterial effects; 2) Products that reduce the inflammatory signaling molecule NO and the pro-inflammatory cytokine TNF-α; 3) Products that increase the anti-inflammatory cytokine IL-10; 4) Products that improve colitis; 5) Products that can reduce the visual and pathological damage to the skin caused by atopic dermatitis; 6) Products that can reduce the levels of serum inflammatory factors in atopic dermatitis; 7) Products that reduce the expression level of inflammatory cytokine genes in atopic dermatitis lesions; 8) Products that promote the expression of genes related to cell proliferation in atopic dermatitis lesions; 9) Products that promote the proliferation of skin cells in atopic dermatitis lesions and repair damaged skin; 10) Products that can improve intestinal flora disorders.

7. The use according to claim 6, characterized in that: The product for improving colitis includes at least one of the following functions: Ameliorate the shortened colon length in mice with DSS-induced colitis; or Can reduce the inflammatory factor TNF-α in the serum of mice induced by DSS; or Can reduce the serum LPS level of mice induced by DSS; or Can change the cecal microbial composition of DSS mice.

8. The use according to claim 5, characterized in that: The product specifically includes at least one of the following functions: Can reduce the visual lesions of the skin in mice with DNCB-induced atopic dermatitis; or Can reduce the pathological damage of skin tissue in mice with atopic dermatitis induced by DNCB; or Able to reduce the level of TNF-α in the serum of mice with DNCB-induced atopic dermatitis; or It can reduce the mRNA expression levels of inflammatory cytokines TNF-α and interleukin-1β in the skin lesions of DNCB-induced atopic dermatitis mice; or It can promote the expression level of mRNA of cell proliferation-related genes, KRT15, ​​ITGA6 and PCNA in the skin lesions of DNCB-induced atopic dermatitis mice; or It can change the colonic microbial composition of mice with DNCB-induced atopic dermatitis.

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