WO2024146604A1

WO2024146604A1 - Lactobacillus crispatus and use thereof

Info

Publication number: WO2024146604A1
Application number: PCT/CN2024/070628
Authority: WO
Inventors: 毛毅斌; 孙宁云; 王莎莎; 于鸿晶; 徐晓芬; 王苒君
Original assignee: 上海上药信谊药厂有限公司
Priority date: 2023-01-06
Filing date: 2024-01-04
Publication date: 2024-07-11
Also published as: CN116103199A

Abstract

An isolated Lactobacillus crispatus strain, a composition containing same and the use thereof.

Description

A strain of Lactobacillus crispatus and its application

Field of the Invention

The invention relates to the field of microorganisms, and in particular to an isolated Lactobacillus crispatus strain, a composition containing the strain and application thereof.

Background technique

The female vaginal system has three lines of defense: anatomical structure, vaginal mucosa and microecological flora, of which the most critical line of defense is the microecological flora. There are more than 200 kinds of microorganisms in the vagina of healthy women, of which 95% are lactic acid bacteria, including Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus iners. Several Lactobacillus bacteria, including Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus iners, dominate. The normal flora in the vagina maintains a coordinated and dynamic balance with the host and the environment, that is, the vaginal microecological balance. Once the number of lactic acid bacteria decreases, the vaginal microecological balance is broken, the number of pathogenic bacteria increases or exogenous pathogens invade, and the vaginal flora and vaginal pH are abnormal, it is possible to cause inflammation. In bacterial vaginitis, the balance of the vaginal microecology shifts toward anaerobic colonization, especially Gardnerella vaginalis and Atopobium vaginaae (Srinivasan and Fredricks, 2008). If the vaginal microecology is in an unbalanced state for a long time, vaginitis will recur. A large amount of literature shows that vaginitis can cause a variety of complications and sequelae. For example, vaginitis can cause pelvic inflammatory disease and cervicitis, and it also poses a more serious threat to the health of pregnant women, such as increasing the risk of miscarriage in the first 3 months of pregnancy, premature rupture of membranes, chorioamnionitis, and premature birth. In addition, changes in the vaginal microecological environment are also associated with urinary tract infections.

At present, the first-line medication recommended by clinical guidelines in various countries for the treatment of vaginitis is antibiotic therapy represented by metronidazole or clindamycin. However, antibiotic therapy may cause damage to beneficial flora, and long-term use is more likely to cause vaginal flora resistance.

Probiotics play an important role in the treatment of vaginitis in women. They inhibit the growth of pathogenic microorganisms by producing lactic acid and a variety of antibacterial compounds, and stimulate the immune system through competitive adhesion, thereby achieving the effect of treating vaginitis (Kurt Selle and Todd R. Klaenhammer, 2013; Craig R. Cohen, 2020; Charlotte van der Veer, 2019). Exogenous supplementation of probiotics and the use of probiotics to inhibit the reproduction and growth of pathogenic bacteria have become a new therapy for the prevention or treatment of vaginitis. In addition, through the supplementation of probiotics, it can also be used to prevent or treat pathogen-related diseases or conditions, immune regulation-related diseases or conditions through the brain-gut axis or other mechanisms (Wanil Kim et al., 2020; Peter van Baarlen et al., 2009; Sandra Voltan et al., 2008; F. Blanchet et al., 2021; Hirotaka Kawanabe-Matsuda et al., 2022; Kyosuke Kobayashi et al., 2019; Nuno R Nené et al., 2019; Paola Roggero et al., 2020; Luisa Cervantes-Barragan, 2017), diseases or conditions related to osteoporosis (Claes Ohlsson et al., 2014; Xin Xu et al., 2017; AGNilsson et al., 2018; Abdul Malik Tyagi, 2018) or iron deficiency anemia (Susan C. Vonderheid et al., 2019; Stine Bering, 2006; Zatollah Asemi and Ahmad Esmaillzadeh, 2013; Michael Hoppe et al., 2017; Ulrika Axling et al., 2021; Nathalie Scheers et al., 2016; Michael Hoppe et al., 2015), diseases or conditions related to menopausal syndrome or central nervous system diseases.

Summary of the invention

The present invention provides a novel Lactobacillus crispatus strain having improved properties in terms of antagonism against pathogenic bacteria, tolerance to digestive juices, intestinal colonization and the like.

In one aspect, the present invention provides a Lactobacillus crispatus strain comprising a 16S rRNA sequence having a nucleotide sequence of SEQ ID NO: 1. In some embodiments, the strain provided by the present invention is isolated from reproductive tract secretions.

In some embodiments, the strains provided by the present invention have one or more of the following properties: (i) a survival rate of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% after incubation in simulated gastric fluid for 3 hours; (ii) a survival rate of at least 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55% or 56% after incubation in simulated intestinal fluid for 4 hours; (iii) compared to L. crispatus LBV88, it has a higher adhesion ability to Caco-2 cells, VK2/E6E7 cells or both; (iv) compared to L. crispatus LBV88 has higher antibacterial ability against Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus, vaginal Gardnerella resistant, Atopobium, Malassezia furfur or any combination; (v) has significant inhibitory ability against vaginal Gardnerella, Candida albicans or any combination, and restores the number of Lactobacillus to above normal levels, while relieving symptoms such as vulvar edema and excessive secretions, and restoring vaginal mucosal damage; (vi) compared with L. crispatus LBV88, which has a stronger inhibitory effect on antigen-induced histamine release, secretion of Th2 cytokines (IL-4 and/or IL-5) and/or secretion of IgE; (vii) reduces dermatitis scores, scratching time and skin thickness, and significantly alleviates atopic dermatitis; (viii) has a therapeutic and preventive effect on allergic asthma induced by ovalbumin (OVA), and exerts its therapeutic and preventive effect on allergic asthma by inhibiting the secretion of IL-5 and IL-13, which are Th2 cytokines that mediate allergic reactions; and (ix) exerts a therapeutic and preventive effect on allergic asthma in allergic asthma induced by house dust mite (HDM) by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (such as eosinophils, IL-5 ⁺ CD4 ⁺ T cells and/or IL-13 ⁺ CD4 ⁺ T cells).

On the other hand, the present invention provides a Lactobacillus crispatus strain, whose preservation number is CGMCC No.19531.

On the other hand, the present invention provides a method for culturing the strain provided by the present invention, comprising culturing the strain in a culture medium. In some embodiments, the culture medium is an MRS culture medium. In some embodiments, the culture is carried out under anaerobic conditions. In some embodiments, the culture is carried out at 37°C.

On the other hand, the present invention provides a derivative of the strain provided by the present invention. In some embodiments, the derivative is a culture of the strain provided by the present invention, a lysate of the strain provided by the present invention, an extract of the strain provided by the present invention, an inactivated product of the strain provided by the present invention, or a combination thereof.

In another aspect, the present invention provides a culture medium comprising the strain or a derivative thereof provided by the present invention.

On the other hand, the present invention provides a composition comprising an effective amount of a first component, wherein the first component comprises a strain provided by the present invention, a derivative thereof, or a culture medium comprising the same. In some embodiments, the composition provided by the present invention is a food composition, a health food composition, a pharmaceutical composition, a food composition for special medical purposes, a cosmetic composition, a medical device composition, or a feed composition.

In some embodiments, the compositions provided by the present invention are in the form of pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oily solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapeutic systems, microcapsules, implants or sticks.

In some embodiments, the compositions provided herein are formulated for ocular, otic, intranasal, sublingual, oral, transdermal, topical, nasal, rectal, or parenteral administration.

In some embodiments, the composition provided by the present invention further includes a second component. In some embodiments, the second component includes probiotics, postbiotics, prebiotics, antimicrobial agents, immunomodulators, anticancer agents, osteoporosis therapeutic agents, mental field related therapeutic agents, development related therapeutic agents or combinations thereof. In some embodiments, the weight ratio of the first component to the second component is 1:99 to 99:1. In some embodiments, the first component is administered before, after or simultaneously with the second component.

In another aspect, the present invention provides use of the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same in the preparation of a medicament for antagonizing pathogens.

In another aspect, the present invention provides use of the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same in the preparation of a medicament for preventing and/or treating a disease or condition associated with a pathogen.

In some embodiments, the pathogen is selected from the group consisting of bacteria, fungi, viruses, spirochetes, mycoplasmas, rickettsiae, chlamydiae, and parasites.

In some embodiments, the pathogen is selected from the group consisting of Mycobacterium, Salmonella, E. coli, Chlamydia, Staphylococcus, Bacillus, Psudomonas, Candida, Atopobium, Gardnerella, and Pityrosporum.

In some embodiments, the bacteria include Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Atopobium vaginalis, Gardnerella vaginalis, or a combination thereof.

In some embodiments, the fungus comprises Candida albicans, Malassezia furfur, or a combination thereof.

In some embodiments, the parasite is Trichomonas.

In some embodiments, the pathogen-associated disease or condition is selected from the group consisting of female reproductive tract infection and reproductive tract flora disorder.

In some embodiments, the disease or condition associated with a pathogen is a skin disease associated with Malassezia infection.

On the other hand, the present invention provides use of the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same in the preparation of a medicament for preventing and/or treating diseases or disorders related to immunoregulation.

In some embodiments, the disease or disorder associated with immunomodulation is cancer, an allergic disease, or an autoimmune disease.

In some embodiments, the cancer is selected from the group consisting of prostate cancer, gastro-esophageal cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia, lymphoma and myeloma.

In some embodiments, the allergic disease is selected from the group consisting of allergic rhinitis, allergic asthma, atopic dermatitis, allergic keratoconjunctivitis, urticaria, food allergy, drug allergy, dust mite allergy, and pollen allergy.

In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, rheumatic fever, lupus, systemic sclerosis, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, Guillain-Barré syndrome myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto's thyroiditis, temporal arteritis, juvenile diabetes, alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolytic anemia, Wegener's granulomatosis, Sjögren's syndrome, Addison's disease, Crohn's disease, Behcet's disease, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, pharyngitis, tonsillitis, bronchitis, pneumonia, gastric ulcer, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, periarthritis of the shoulder, tendinitis, tenosynovitis myositis, hepatitis, cystitis, nephritis, sepsis, vasculitis and bursitis.

In another aspect, the present invention provides use of the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same in the preparation of a medicament for preventing and/or treating a disease or condition associated with osteoporosis.

In some embodiments, the disease or disorder associated with osteoporosis is selected from the group consisting of juvenile osteoporosis, menopausal osteoporosis, postmenopausal osteoporosis, post-traumatic osteoporosis, and osteoporosis due to aging, corticosteroid treatment, and inactivity.

On the other hand, the present invention provides use of the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same in the preparation of a medicament for preventing and/or treating diseases or conditions associated with iron deficiency anemia, menopausal syndrome or central nervous system diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that L. crispatus-120 and the commercial strain L. crispatus LBV88 significantly inhibited the secretion of IL-4 compared with the negative control group.

Figure 2 shows that L. crispatus-120 and the commercial strain L. crispatus LBV88 significantly inhibited the secretion of IL-5 compared with the negative control group.

Figure 3 shows that L. crispatus-120 and the commercial strain L. crispatus LBV88 significantly inhibited the secretion of IgE compared with the negative control group.

FIG4 shows that the dermatitis score of mice in the L. crispatus-120 administration group was significantly reduced compared with the model control group.

FIG5 shows that the scratching time of mice in the L. crispatus-120 administration group was significantly reduced compared with the model control group.

Figures 6A and 6B show that compared with the model control group, the ear thickness (Figure 6A) and dorsal skin thickness (Figure 6B) of the mice in the L. crispatus-120 administration group and the dexamethasone group were significantly reduced.

Figures 7A and 7B show the total IL-5 in the mice in the L. crispatus-120 administration group compared with the model control group (Figure 7A) The levels of IL-13 and total IL-13 (Figure 7B) were significantly decreased.

FIG8 shows that L. crispatus-120 administration (HDM+120) effectively inhibits airway hyperresponsiveness causing asthma compared with the asthma model control group (HDM).

9A-9C show that L. crispatus-120 administration (HDM+120) effectively inhibited eosinophils (Eos), IL-5 ⁺ CD4 ⁺ T cells, and IL-13 ⁺ CD4 ⁺ T cells compared with the asthma model control group (HDM).

Detailed ways

The following description of this article is intended only to illustrate the various embodiments of this article. Therefore, the specific transformation discussed should not be interpreted as limiting the scope of this article. It will be apparent to those skilled in the art that various equivalents, changes and modifications can be implemented without departing from the scope of this article, and it should be understood that such equivalent embodiments will be included in this article. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 20th Edition (John Wiley and Sons, New York, 1994), and Hale and Marham, the Harper Collins Dictionary of Biology (Harper Perennial, New York, 1991), etc., provide general meanings of many of the terms used herein as general dictionaries.

definition

As used herein, the articles "a," "an," and "the" are used to refer to one or more than one (ie, to at least one) of the grammatical object of the article.

Unless expressly specified or obvious from the context, as used herein, the term "about" should be understood as within the normal tolerance range in the art, such as within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value.

Lactobacillus crispatus strain

The present invention provides an isolated Lactobacillus crispatus strain, which comprises a 16S rRNA having a nucleotide sequence of SEQ ID NO: 1. Sequence (SEQ ID NO: 1):

The present invention provides an isolated lactobacillus, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on March 30, 2020, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, zip code: 100101, and a deposit number of CGMCC No. 19531. The isolated lactobacillus is named Lactobacillus crispatus-120 (L. crispatus-120), and its classification name is Lactobacillus crispatus (Lactobacillus crispatus).

The lactobacillus strains of the present invention also include mutants, variants and/or progeny of the above lactobacillus strains.

As used herein, "mutant" refers to any microorganism produced by modification of a parent strain. For example, a mutant may be a microorganism produced by genetically modifying a deposited strain.

As used herein, "variant" refers to a naturally occurring microorganism derived from a parent strain. For example, a variant may be a microorganism produced by adapting to specific cell culture conditions.

As used herein, "progeny" refers to any microorganism produced by breeding or proliferation of a parent strain or its mutant, variant, and the progeny strain itself can be identified as a strain identical or substantially identical to the parent strain. It is understood that, in view of the asexual reproduction process, the progeny strain is almost identical to the parent strain in genes. Therefore, in one embodiment, the progeny strain is identical to the parent strain in genes, and can be considered as a "clone" of the parent strain. In another embodiment, the progeny strain is substantially identical to the parent strain in genes.

In the full-length genome of the bacteria, the mutant, variant or progeny has at least 90%, 95%, 98%, 99%, 99.5% or 99.9% sequence identity compared to its parent strain. In addition, the mutant, variant or progeny will retain the same phenotype as the parent strain, for example, the mutant, variant or progeny can show the same or equivalent level of antagonism to pathogenic bacteria, tolerance to digestive juices, intestinal colonization and other aspects as the parent strain.

"Percentage (%) of sequence identity" in relation to a nucleotide sequence (or amino acid sequence) refers to the percentage of nucleotide (or amino acid) residues in the candidate sequence that are identical to the nucleotide (or amino acid) residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to maximize the number of identical nucleotides (or amino acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignments for the purpose of determining the percentage of identity of nucleotide (or amino acid) sequences may be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402). (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007))), and ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art may use the default parameters provided by the tools, or may customize parameters suitable for alignment, such as by selecting an appropriate algorithm.

【Separation】

The Lactobacillus crispatus provided by the present invention is isolated.

As used herein, "isolated" means that the substance has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, then the composition or substance has been altered from its original environment or removed from its original environment, or both. For example, a strain naturally occurring in a living organism is not "isolated", but if the strain is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state, then the strain is "isolated".

The lactobacillus strains of the present invention can be isolated from secretions of the reproductive tract, especially the female reproductive tract. As used herein, the female "reproductive tract" includes the vagina, uterus, cervix and ovary, etc. In a preferred embodiment, the lactobacillus strains of the present invention are isolated from secretions of the vagina and/or cervix.

The lactobacillus strain of the present invention can be obtained by separation and purification in a conventional manner in the art. The culture after the separation and purification steps substantially contains no contaminants other than the lactobacillus strain of the present invention, including microbial contaminants and unwanted chemical contaminants.

In certain embodiments, a cotton swab is used to collect secretions from 1/3 of the vaginal side wall of a healthy subject and placed in a sterile tube. The bacterial suspension after washing the swab with PBS is used as the mother solution, which is further diluted to different concentrations with PBS and spread on freshly prepared Rogosa SL solid medium for anaerobically cultured. Subsequently, single colonies of different morphologies are picked up with an inoculation loop and inoculated on freshly prepared MRS solid medium by streaking, and anaerobically cultured to obtain purified single colonies.

【Characterization】

The lactobacillus strain of the present invention can be identified by conventional methods in the art, including but not limited to classical morphological characteristics detection, physiological and biochemical characteristics detection and molecular biological detection. The morphology, staining, culture characteristics and colony characteristics of bacteria are the preliminary basis for bacterial identification, and the biochemical reaction of bacteria can be used to distinguish and identify the types of bacteria.

In certain embodiments, the lactobacillus strain of the present invention is identified by the following method: observing the colony morphology in the culture medium after culture, and the colonies are round; taking a smear of the pure culture of the strain for Gram staining, showing Gram-positive characteristics; microscopic examination shows that the strain is short rod-shaped and can be connected into long chains.

Based on the above staining and morphological characteristics, by using classical taxonomy, for example, by referring to the relevant description in Bergey’s Manual of Systematic Bacteriology (Williams & Wilkins Co., 1984), it was preliminarily determined that the strain belonged to the genus Lactobacillus.

Furthermore, the strain can be identified by conventional 16S rRNA gene sequence detection methods. In certain embodiments, the 16S rRNA gene sequence detection method used is as follows:

(1) PCR amplification;

(2) Perform gel electrophoresis on the PCR product to determine the 16S rRNA gene fragment;

(3) Take PCR samples for 16S rRNA sequencing;

(4) The sequence obtained by sequencing was compared with the data in the NCBI database for BLAST sequence similarity analysis. When the highest homology score was greater than 97%, the species of the strain could be determined.

Based on the 16S rRNA gene sequence detection results, it can be determined that the strain of the present invention is Lactobacillus crispatus.

In certain embodiments, the Lactobacillus strain claimed for protection comprises a 16S rRNA sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the nucleotide sequence shown in SEQ ID NO:1, while maintaining the morphological and functional characteristics of Lactobacillus crispatus-120 (L. crispatus-120).

【nature】

The lactobacillus strain of the present invention can not only effectively antagonize multiple pathogenic bacteria (including but not limited to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Atopobium vaginalis, vaginal Gardnerella resistant bacteria, Candida albicans and Malassezia furfur), but also has good tolerance to digestive juices such as gastric acid and bile salts, which helps its survival in the gastrointestinal environment, and is particularly suitable for being prepared into products for oral administration. The lactobacillus strain of the present invention also has improved colonization performance (for example, colonization in the intestinal tract and vagina), can play its beneficial effect for a long time, and is a probiotic strain with development potential. The lactobacillus strain of the present invention also has significant treatment and/or preventive effects on allergic reactions.

The lactobacillus strains of the present invention have one or more of the following beneficial properties:

Acid tolerance: After incubation in simulated gastric fluid for 3 hours, the survival rate of the lactobacillus strain of the present invention is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. Any suitable acid tolerance test method can be used to determine the acid tolerance of the lactobacillus strain of the present invention. In one embodiment, the acid tolerance of the lactobacillus strain of the present invention is determined in simulated gastric fluid in vitro. In one embodiment, the acid tolerance test includes resuspending the thalli to be tested with simulated gastric fluid (e.g., pH 3.0) and normal saline, incubating at 37°C for 3 hours, gradient dilution and plate counting. Acid tolerance can be calculated using the following formula: Acid tolerance (%) = (Log10 CFU/mL in simulated gastric fluid)/(Log10 CFU/mL in normal saline) × 100. In one embodiment, the preparation method of simulated gastric fluid is: dissolve 96.84 mg of pepsin (Sigma, P7000-25G, 632 U/mg) powder in 32.3 mL of pH 3.0 saline to a final concentration of 3 g/L; filter with a 0.22 μm sterile filter membrane and use it immediately after preparation.

Bile tolerance: After incubation in simulated intestinal fluid for 4 hours, the survival rate of the Lactobacillus strain of the present invention is at least 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55% or 56%. Any suitable bile tolerance test method can be used to determine the bile tolerance of the Lactobacillus strain of the present invention. In one embodiment, the bile tolerance of the Lactobacillus strain of the present invention is determined in simulated intestinal fluid in vitro. In one embodiment, the bile tolerance test includes resuspending the bacteria to be tested in simulated intestinal fluid (e.g., pH 8.0) and conventional saline, incubating at 37°C for 4 hours, gradient dilution and plate counting. Bile tolerance can be calculated using the following formula: Bile tolerance (%) = (Log10 CFU/mL in simulated intestinal fluid)/(Log10 CFU/mL in saline) × 100. In one embodiment, the simulated intestinal fluid is prepared by adding 0.5 mL of 100× trypsin and bile salt stock solutions to 49 mL of pH 8.0 saline, with final concentrations of 1 g/L trypsin and 0.3% bile salt (i.e., 3 mg/mL), respectively; filtering with a 0.22 μm sterile filter membrane and preparing for immediate use.

Colonization performance: Compared with the commercial strain L. crispatus LBV88, the lactobacillus strain of the present invention has a higher colonization ability on Caco-2 cells, VK2/E6E7 cells or both. Any suitable adhesion ability test method can be used to determine the colonization performance of the lactobacillus strain of the present invention. In one embodiment, a bacterial solution of a suitable concentration (e.g., about 2.5×10 ⁸ CFU/mL, about 5.0×10 ⁷ CFU/mL or about 5.0×10 ⁶ CFU/mL) is added to the cells to be adhered, and the cells are allowed to adhere at 37°C for 2 hours, and the target multiplicity of infection (MOI, bacteria: cells) is 10:1. After the incubation and adhesion are completed, the cells are washed and lysed, and the fully detached cells and bacteria are obtained, and the plates are plated and counted. The adhesion rate can be calculated using the following formula: Adhesion rate % = adhered bacterial amount CFU/inoculated bacterial amount CFU×100%. A suitable cell line can be selected for experiment according to the target site of colonization. In one embodiment, the colonization performance test uses human intestinal cell line Caco-2 cells. In one embodiment, the colonization performance test uses vaginal epithelial cells VK2/E6E7 cells.

Antibacterial performance: Compared with the commercial strain L. crispatus LBV88, the lactobacillus strain of the present invention has a higher antibacterial ability against Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus, vaginal Gardnerella, Atopobium, Malassezia furfur or any combination. Any suitable test method can be used to determine the performance of the lactobacillus strain of the present invention against pathogenic bacteria, such as the diffusion method for qualitative determination and the dilution method for quantitative determination. Specific methods suitable for use include, but are not limited to, the double-layer plate method, the cake method (such as the inverted cake method and the strip cake method), the paper diffusion method, etc.

In one embodiment, the specific steps of the double-layer plate method are as follows: take pathogenic bacteria (such as Staphylococcus aureus) in a solid culture medium cooled to about 45°C, and pour it on the MRS agar of the probiotics to be tested that has been cultured for 20-24 hours. After solidification, the plates were placed in an atmospheric environment at 37°C for 1 day until an inhibition zone appeared. Parallel test plates were set for each probiotic.

In another embodiment, the specific steps of the double-layer plate method are as follows: the probiotic bacterial liquid is spotted on the MRS solid culture medium plate and cultured anaerobically at 37°C for 24-48 hours. The pathogenic bacteria (e.g., Malassezia furfur) seed liquid is inoculated into the mYPG liquid culture medium prepared under aerobic conditions and cultured at 37°C for 24-48 hours. Prepare the mYPG culture medium, sterilize at 115°C for 20 minutes, wait for the temperature to cool to about 40°C, take 2.5mL of the culture medium and mix it with 500μL of the pathogenic bacteria culture solution to be used, pour the mixed solution into the MRS solid culture medium with the probiotics spotted, and let it stand until the culture medium solidifies. The solidified culture medium plate is cultured aerobically at 37°C for 24-48 hours. The antibacterial activity of the probiotics is determined by measuring the diameter of the inhibition zone. Parallel test plates are set for each probiotic.

In one embodiment, the specific steps of the inverted cake method are as follows: take pathogenic bacteria (such as Escherichia coli, Pseudomonas aeruginosa or Salmonella typhi) and spread them on NA or Columbia blood agar medium + 5% sheep blood plate. After they are fully absorbed, take out the probiotic cake and invert it on the pathogenic bacteria plate, and culture it under 37°C atmospheric conditions for 1 day until an inhibition zone appears. Set up parallel test plates for each probiotic.

In one embodiment, the specific steps of the strip cake method are as follows: dip the probiotic inoculum with a cotton swab, and then spread it into a 2 cm wide strip along the diameter on the MRS solid culture medium plate, and culture the plate anaerobically at 37°C for 24 hours. Take out the plate, pour the melted YM solid culture medium on the surface of the plate, and wait for a thin layer to solidify. Use a cotton swab to evenly spread the pathogenic bacteria liquid on the surface of the YM solid culture medium, wait for it to dry, first culture it at 4°C for 4 hours, and then culture it at 37°C for 24 hours, and observe the inhibitory effect of probiotics on pathogenic bacteria. Set up parallel test plates for each probiotic.

The performance of the antagonistic pathogenic bacteria of the lactobacillus strain of the present invention can also be determined by any suitable in vivo experiment. In one embodiment, an animal disease model is constructed by, for example, perfusing a pathogenic bacteria liquid, and sampling is performed before and after administration to count pathogenic bacteria and probiotics (for example, lactobacillus) colonies; vulva observation can also be performed before and after administration to record situations such as vulva redness and swelling, vaginal discharge, and vaginal lavage smear (PAS staining), thereby confirming that lactobacillus of the present invention regulates vaginal flora, inhibits the growth of pathogenic bacteria and the effect of colonization. In one embodiment, a bacterial vaginitis model is constructed using vaginal Gardnerella. In one embodiment, a candidal vaginitis model is constructed using Candida albicans.

Improvement of allergic reactions: Compared with the commercial strain L. crispatus LBV88, the lactobacillus strain of the present invention has a stronger inhibitory effect on antigen-induced histamine release, Th2 cytokine (IL-4 and/or IL-5) secretion and/or IgE secretion, and significantly improves allergic reactions. Any suitable method can be used to evaluate the effect of the lactobacillus strain of the present invention on allergic reactions. The invention relates to the effects of the cytokine inhibitory factor on the sexual response of rats, including but not limited to the inhibition of histamine secretion, the inhibition of the secretion of type 2 helper T cells (Th2) related cytokines (such as IL-4 and IL-5) and/or the inhibition of IgE secretion.

In one embodiment, the ability of each strain to inhibit histamine secretion is determined by the following method: after culturing the RBL-2H3 cell line, degranulation is induced, and then the o-phthalaldehyde post-column conversion method is used to determine the histamine content in the filtrate by high performance liquid chromatography. The histamine release inhibition rate can be calculated according to the following formula: histamine inhibition rate = (histamine content in the negative control filtrate - histamine content in the treatment group filtrate) / histamine content in the negative control filtrate.

In one embodiment, the ability of each strain to inhibit the secretion of Th2-related cytokines (such as IL-4 and IL-5) is determined by the following method: using the EL4 cell line, the amount of secreted IL-4 and IL-5 is determined by a kit.

In one embodiment, the ability of each strain to inhibit IgE secretion is determined by the following method: human B cells U266B1 are used to measure the level of IgE by a kit. The IgE inhibition rate can be calculated according to the following formula: IgE inhibition rate = (IgE content in negative control filtrate - IgE content in treatment group filtrate) / IgE content in negative control filtrate.

Relieve atopic dermatitis: The lactobacillus strains of the present invention significantly reduce the symptoms of atopic dermatitis, such as dryness, edema, erythema/hemorrhage, erosion/excoriation, and itching of the skin. Any suitable experimental model can be used to determine the effect of the lactobacillus strains of the present invention on atopic dermatitis. In one embodiment, the atopic dermatitis NC/Nga mouse model is used. Any suitable index can be used to evaluate the effect of the lactobacillus strains of the present invention on atopic dermatitis, including but not limited to dryness, edema, erythema/hemorrhage, erosion/excoriation, and itching of the skin. Any suitable method can be used to obtain the aforementioned indexes, including but not limited to dermatitis scores, scratching time measurements, ear/back skin thickness measurements, etc.

Improve allergic asthma: The lactobacillus strain of the present invention has a therapeutic and preventive effect on allergic asthma induced by ovalbumin (OVA), and exerts its therapeutic and preventive effect on allergic asthma by inhibiting the secretion of IL-5 and IL-13, which are Th2 cytokines that mediate allergic reactions; in allergic asthma induced by house dust mites (HDM), it exerts a therapeutic and preventive effect on allergic asthma by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (e.g., eosinophils, IL-5 ⁺ CD4 ⁺ T cells and/or IL-13 ⁺ CD4 ⁺ T cells). Any suitable experimental model can be used to determine the effect of the lactobacillus strain of the present invention on allergic asthma. In one embodiment, an OVA-induced asthma model is used. In one embodiment, an HDM-induced asthma model is used. Any suitable index can be used to evaluate the effect of the lactobacillus strain of the present invention on allergic asthma. In one embodiment, a histopathological examination is performed to observe inflammatory cell infiltration, bronchial tissue (bronchial smooth muscle), epithelial cells, etc. In one embodiment, IL-5 ⁺ and IL-13 ⁺ cells are determined by counting cells producing IL-5 or IL-13 in lymphocytes with CD45 and CD3ε as markers. In one embodiment, airway hyperresponsiveness (AHR) is detected. In one embodiment, In the embodiment, eosinophils are determined by counting Siglec-f ⁺ CD11b ⁺ cells among cells expressing the common leukocyte marker CD45, and the number of IL-5 ⁺ CD4 + T and IL-13 + CD4 ⁺ ^T cells is determined by counting IL-5 or IL-13 producing cells among CD4 ⁺ T cells having CD3ε, TCRβ ^and CD4 as markers.

【derivative】

The present invention also provides derivatives of the strain provided by the present invention. As used herein, "derivatives" refer to products derived from the strain, including cultures, lysates, extracts, inactivated products, and the like.

In some embodiments, the derivative is a culture of the strain provided by the present invention, a lysate of the strain provided by the present invention, an extract of the strain provided by the present invention, an inactivated product of the strain provided by the present invention, or a combination thereof.

As used herein, "culture" refers to a product obtained by culturing a strain in a culture medium, and the product may include the strain itself.

As used herein, "lysate" refers to the product obtained by treating a strain with enzymes, ultrasound, homogenization, etc.

As used herein, "extract" refers to the product obtained by subjecting the strain to treatment such as solvent extraction.

As used herein, "inactivated product" refers to a product obtained by treating a strain with heat, pressure or drugs.

【Cultivation method】

The present invention also provides a method for culturing the lactobacillus strain of the present invention, including culturing the strain in a culture medium. The lactobacillus strain of the present invention can grow in any culture medium suitable for lactobacillus, and the strain does not lose its genetic characteristics during growth, nor does it lose its characterization and functional characteristics. According to the required bacterial growth, the culture medium needs to have basic nutrients such as carbon source, nitrogen source, growth factor, inorganic salt and water, and is prepared according to a certain formula and method. Specifically, the lactobacillus strain of the present invention can grow in a culture medium containing assimilable organic carbon source, assimilable nitrogen source, appropriate salt and trace metal. The preferred culture medium suitable for the lactobacillus strain of the present invention includes MRS culture medium. The exemplary components of MRS culture medium include peptone, beef powder, yeast powder, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate, agar powder, distilled water, etc.

The lactobacillus strain of the present invention can be cultured by conventional culture methods under any conventional culture conditions suitable for lactobacillus. Specifically, the lactobacillus strain of the present invention can be cultured by methods such as broth fermentation, agar surface culture, etc. The temperature of the culture medium can be any temperature suitable for the growth of lactobacillus, preferably cultured at a temperature of about 35-40° C., more preferably cultured at a temperature of about 37° C. The lactobacillus strain of the present invention can be cultured under anaerobic or microaerobic conditions, preferably cultured under anaerobic conditions.

In one embodiment, a purified single colony is first obtained on a solid medium, and then a single colony is picked and inoculated into a liquid medium to further amplify the lactobacillus strain of the present invention. After the cells grow to a desired density (e.g., 10 ⁹ CFU/ml), the lactobacillus cells are harvested using conventional methods and correspondingly frozen for storage or used in experiments. Preferably, centrifugation is used to harvest the lactobacillus cells of the present invention.

【Culture medium】

The present invention also provides a culture medium, which comprises the strain or its derivative provided by the present invention.

As used herein, "culture medium" refers to any culture medium for growing and collecting cells and/or products expressed and/or secreted by the cells. The specific culture medium type can be selected according to the type of cells to be cultured, the growth stage, the culture target, etc. The culture medium includes, but is not limited to, a solution, a solid, a semisolid or a rigid support. The culture medium includes a culture medium for separation, a culture medium for purification, a culture medium for proliferation, a culture medium for harvesting derivatives, a culture medium for analysis, etc. The culture medium can be selected from a liquid culture medium or a solid culture medium known in the art. Exemplary culture media suitable for the present invention include, but are not limited to, MRS culture medium, GAM culture medium, BL culture medium or SL culture medium.

combination

The lactobacillus strain of the present invention, its derivative or culture medium containing the same can be administered alone or as part of a product. The product can contain auxiliary components well known to those skilled in the art.

The present invention also provides a composition, which comprises an effective amount of a first component, wherein the first component comprises the strain provided by the present invention, its derivative, a culture medium containing the same, or a combination of the aforementioned components.

[Use of the composition]

The composition can be a food composition, a health food composition, a pharmaceutical composition, a food composition for special medical purposes, a cosmetic composition, a medical device composition or a feed composition.

The strains, derivatives thereof or culture media containing the same provided by the present invention can be used alone for food, health food, medicine, food for special medical purposes, cosmetics, medical devices or feed, etc., or can be mixed with suitable components well known to those skilled in the art for use in food, health food, medicine, food for special medical purposes, cosmetics, medical devices or feed, etc.

【Administration method, dosage form】

The compositions of the present invention can act by systemic and/or local administration. In some embodiments, the compositions of the present invention are formulated for ocular, otic, intranasal, sublingual, oral, transdermal, topical, nasal, rectal or parenteral administration.

The compositions of the present invention may be present in suitable forms known in the art. In some embodiments, the compositions of the present invention may be present in the form of pills, tablets, lozenges, lyophilized powders, granules, capsules, aqueous solutions, alcoholic solutions, oily solutions, syrups, emulsions, suspensions, suppositories, solutions for injection or infusion, ointments, gels, tinctures, creams, patches, lotions, sprays, aerosols, powder sprays, effervescent tablets, transdermal therapeutic systems, microcapsules, implants or sticks.

In particular, for a particular administration route, the composition of the present invention is in a corresponding suitable form.

In some embodiments, the composition of the present invention is for oral administration and can be formulated into dosage forms known in the art suitable for delivering the strain of the present invention, such as tablets (uncoated or coated tablets, such as enteric coatings or controlled release coatings), capsules (such as hard gelatin capsules or soft gelatin capsules), lyophilized powders, granules, pills, emulsions, suspensions, sprays, aerosols or solutions.

In some embodiments, the composition of the present invention is for topical administration and can be formulated into dosage forms known in the art suitable for delivering the strain of the present invention, such as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

In some embodiments, the composition of the present invention is for vaginal administration and can be formulated into dosage forms known in the art suitable for delivering the strain of the present invention, such as vaginal rings, tampons, creams, gels, pastes, foams or sprays.

In some embodiments, the compositions of the present invention are for rectal administration and can be formulated into dosage forms known in the art suitable for delivering the strains of the present invention, such as suppositories or enemas.

The composition of the present invention can be administered to a subject in the form of a unit dose once or more per day. As used herein, "unit dose" refers to a physically discrete unit suitable for administration to a subject, and each unit contains an effective amount of the lactobacillus strain of the present invention or a corresponding amount of derivative to provide an expected effect, such as edible, therapeutic or health-care effects.

【Auxiliary components】

The strain of the present invention, its derivative or culture medium containing it can be mixed with suitable auxiliary components, which can be achieved by conventional means in the art. Exemplary auxiliary components include:

fillers, such as cellulose, microcrystalline cellulose, lactose, mannitol, and starch;

ointment bases such as petroleum jelly, paraffin, triglycerides, waxes, wool wax, wool alcohol, lanolin, hydrophilic ointments and polyethylene glycols;

suppository bases such as polyethylene glycol, cocoa butter, and hard fat;

solvents, such as water, ethanol, isopropanol, glycerol, propylene glycol, liquid polyethylene glycols, and paraffin;

Surfactants, emulsifiers, dispersants or wetting agents, such as sodium lauryl sulfate, lecithin, phospholipids, fatty alcohols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters and poloxamers;

buffers, acids and bases, such as phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, tromethamine, and triethanolamine;

isotonic agents, such as dextrose and sodium chloride;

Adsorbents, such as highly dispersed silica;

Binders such as polyvinylpyrrolidone, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethylcellulose, starch, carbomer, gelatin and gum arabic;

disintegrants such as modified starch, sodium carboxymethylcellulose, sodium starch glycolate, cross-linked polyvinyl pyrrolidone, and cross-linked sodium carboxymethylcellulose;

lubricants, such as magnesium stearate, stearic acid, talc, and highly dispersed silica;

coating materials, such as sugar and shellac;

film formers, for example polyvinyl pyrrolidone, polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylates and polymethacrylates;

capsule materials, such as gelatin and hydroxypropyl methylcellulose;

Synthetic polymers, such as polylactic acid, polyglycolide, polyacrylates, polymethacrylates, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene oxide, polyethylene glycol, and copolymers and block copolymers thereof;

plasticizers, such as polyethylene glycol, propylene glycol, glycerol, triacetin, triacetyl citrate, and dibutyl phthalate;

Penetration enhancers, such as surfactants, dimethyl sulfoxide and its analogs, azone compounds, pyrrolidine derivatives, alcohol compounds and fatty acid compounds;

Stabilizers, for example antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, preservatives such as parabens, sorbic acid, thimerosal, benzalkonium chloride, chlorhexidine acetate and sodium benzoate;

colorants, such as inorganic pigments, such as iron oxide and titanium dioxide;

Flavoring, sweetening, flavor and/or odor masking agents.

It should be understood that in addition to the ingredients specifically mentioned above, the composition may also contain other conventional components in corresponding dosage forms.

【content】

In some embodiments, the composition of the present invention comprises an amount of 10 ⁶ CFU/g to 10 ¹² CFU/g of the lactobacillus strain of the present invention and a corresponding amount of its derivatives, preferably 10 ⁶ CFU/g to 10 ¹¹ CFU/g, 10 ⁶ CFU/g to 10 ¹⁰ CFU/g, 10 ⁶ CFU/g to 10 ⁹ CFU/g, 10 6 CFU/g to 10 8 CFU/g, 10 ⁶ CFU/g to 10 ⁷ CFU/g, 10 ⁷ CFU/g to 10 ¹² CFU/g, 10 ⁷ CFU/g to 10 ¹¹ CFU/g, 10 ⁷ CFU/g to 10 ¹⁰ CFU/g, 10 ⁷ CFU/g to 10 ⁷ CFU/g, 10 7 CFU/g to 10 ⁹ CFU/g, 10 ⁷ CFU/g to 10 ⁸ CFU/g, 10 8 CFU/g to 10 ¹² CFU/g, ¹⁰ ⁸ CFU/g to 10 ¹³ CFU/g. The lactobacillus strain of the present invention in an amount of 10 CFU/g to 10 ¹¹ CFU/g, 10 ⁸ CFU/g to 10 ¹⁰ CFU/g, 10 ⁸ CFU/g to 10 ⁹ CFU/g, 10 ⁹ CFU/g to 10 ¹² CFU/g, 10 ⁹ CFU/g to 10 ¹¹ CFU/g, 10 ⁹ CFU/g to 10 ¹⁰ CFU/g, 10 ¹⁰ CFU/g to 10 ¹² CFU/g, 10 ¹⁰ CFU/g to 10 ¹¹ CFU/g or 10 ¹¹ CFU/g to 10 ¹² CFU/g, or a corresponding amount of a derivative thereof.

As used herein, "CFU" stands for "colony forming unit."

【Second component】

In some embodiments, the composition provided by the present invention further comprises a second component. In some embodiments, the second component comprises a probiotic, a postbiotic, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an osteoporosis therapeutic agent, a psychiatric field-related therapeutic agent, a development-related therapeutic agent, or a combination thereof.

As used in the present invention, "probiotics" refers to microorganisms that have a beneficial effect on the health or well-being of the host, preparations containing them, or cultures, lysates, extracts, inactivated products, metabolites, etc. that retain their basic properties. Preferably, probiotics can be selected from Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Preferably, the probiotics are selected from Bifidobacterium, Lactobacillus or a mixture thereof.

In some embodiments, the strain belonging to the genus Lactobacillus can be any lactobacillus with a probiotic effect, including but not limited to: Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus johnsonii and Lactobacillus plantarum.

In some embodiments, the strain belonging to the genus Bifidobacterium can be any bifidobacterium with a probiotic effect, including but not limited to: Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis and Bifidobacterium adolescentis.

As used herein, "postbiotics" refers to non-viable bacterial products and/or metabolites of probiotic microorganisms that are biologically active in the host. Postbiotics mainly include two major categories of substances: metabolites and bacterial components. The metabolites can be selected from organic acids, short-chain fatty acids, intracellular polysaccharides, vitamins, proteins, enzymes, lipids or mixtures thereof. The bacterial components can be selected from lipoteichoic acid, teichoic acid, peptidoglycan, cell surface proteins, polysaccharides, cell membrane proteins, extracellular polysaccharides or mixtures thereof.

As used herein, "prebiotic" refers to any compound, nutrient, or other microorganism that is used to support or enhance the health effects of probiotics or is beneficial to the growth and/or activity of probiotics. Typical examples of prebiotics are carbohydrates (e.g., oligosaccharides), but non-carbohydrates are not excluded. The most common form of prebiotics is nutritionally classified as soluble fiber, and various forms of dietary fiber show a certain level of prebiotic effect.

Prebiotics can be selected from oligosaccharides (e.g., fructose, galactose, and mannose), dietary fibers (e.g., soluble fibers and soy fibers), inulin, or mixtures thereof. Some examples of prebiotics include fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), isomaltooligosaccharides (IMO), manno-oligosaccharides (MOS), xylo-oligosaccharides (XOS), arabinoxy-oligosaccharides (AXOS), inulin, soy oligoglucans, lactulose (LA), glycosylsucrose (GS), lactosucrose (LS), palatinose oligosaccharides (PAO), maltooligosaccharides, gums and/or hydrolyzates thereof, pectins and/or hydrolyzates thereof.

As used herein, "antimicrobial agent" refers to a substance or combination of substances that can kill microorganisms or inhibit the growth and/or activity of microorganisms. Exemplary antimicrobial agents that can be used in the present invention include, but are not limited to:

macrolides or ketolides, such as erythromycin, azithromycin, clarithromycin, and telithromycin;

beta-lactams, such as penicillins (e.g., penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, and temocillin), cephalosporins (e.g., cephalothin, cefpiroxine, cephradine, cefotaxime, cefazolin, cephalosporin), mendol, cefuroxime, cephalexin, cefprozil, cefaclor, loracarb, cefoxitin, cefmetazole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, cefbutin, ceftriaxone, cefpirome, and cefepime) and carbapenems (such as carbapenem, imipenem, meropenem, and PZ-601);

Monocyclic β-lactams, such as aztreonam;

quinolones (such as nalidixic acid, octramox, norfloxacin, pefloxacin, enoxacin, ofloxacin, levofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grefloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin, gemifloxacin, gemifloxacin, and pazufloxacin);

antibacterial sulfonamides and antibacterial sulfonamides, such as p-aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, and phthalylsulfathiazole;

aminoglycosides, such as streptomycin, neomycin, kanamycin, paromomycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, bikaline, and isepamicin;

Tetracyclines, such as tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, metacycline, doxycycline, and tigecycline;

rifamycins, such as rifampin, rifapentine, rifabutin, benzoxazine rifamycins, and rifaximin;

Lincomycins, such as lincomycin and clindamycin;

glycopeptides, such as vancomycin and teicoplanin;

Streptomycins, such as quinupristin and dalopristin;

oxazolidinones, such as linezolid and tedizolid;

polymyxins, colistin, and colistin;

trimethoprim and bacitracin;

Outflow pump inhibitors, etc.

As used herein, "immunomodulator" refers to a substance, agent, signal transduction pathway or component thereof that modulates an immune response, such as an immunosuppressant and an immunostimulator, etc. "Modulating" an immune response refers to any change in the cell type or cell activity of the immune system, including an increase or decrease relative to a reference level. The modulation includes stimulation or inhibition of the immune system, which can be manifested by an increase or decrease in the number of various types of cells, an increase or decrease in cell activity, or by any other changes occurring within the immune system.

Exemplary immunomodulators include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

Checkpoint modulators interfere with the ability of cancer cells to evade immune system attack and help the immune system to respond more strongly to tumors. Immune checkpoint molecules can mediate co-stimulatory signals to enhance immune responses or can mediate co-inhibitory signals to suppress immune responses. Examples of checkpoint regulators include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16, and CD83 regulators. In certain embodiments, immune checkpoint regulators include PD-1/PD-L1 axis inhibitors.

Adoptive cell transfer attempts to enhance the natural ability of T cells to fight cancer. In this treatment, T cells are taken from the patient and expanded and activated in vitro. In some embodiments, T cells are modified in vitro into CAR-T cells. The most active T cells or CAR-T cells against cancer are cultured in large quantities in vitro for 2 to 8 weeks. During this period, the patient will receive treatments such as chemotherapy and radiation therapy to reduce the body's immunity. After these treatments, the T cells or CAR-T cells cultured in vitro will be returned to the patient. In some embodiments, adoptive cell transfer is CAR-T therapy.

Cytokines are used to enhance the presentation of tumor antigens to the immune system. Two main types of cytokines used to treat cancer are interferons and interleukins. Examples of cytokines include, but are not limited to, interferons (such as interferon-α, interferon-β and interferon-γ), colony stimulating factors (such as macrophage CSF, granulocyte macrophage CSF and granulocyte CSF), insulin growth factor (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FGF), interleukins (such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12), tumor necrosis factor (such as TNF-α and TNF-β) or any combination thereof.

Oncolytic viruses are genetically modified viruses that can kill cancer cells. Oncolytic viruses can specifically infect tumor cells, causing tumor cell lysis, followed by the release of large amounts of tumor antigens, triggering the immune system to target and eliminate cancer cells with such tumor antigens. Examples of oncolytic viruses include, but are not limited to, talimogene laherparepvec (T-Vec, Imlygic).

Therapeutic vaccines fight cancer by boosting the immune system's response to cancer cells. Therapeutic vaccines may contain non-pathogenic microorganisms, genetically modified viruses that target tumor cells, or one or more immunogenic components.

As used herein, "anticancer agent" refers to an agent (e.g., compound, drug, antagonist, inhibitor, modulator) that has anti-tumor properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anticancer agent is a chemotherapeutic agent. In some embodiments, the anticancer agent is a biologic. In some embodiments, the anticancer agent is an immunotherapy agent. In some embodiments, the anticancer agent is an agent approved by the Food and Drug Administration for the treatment of cancer.

Examples of anticancer agents that can be used in the present invention include, but are not limited to, anastrozole, bicalutamide, bleomycin sulfate, busulfan, capecitabine, N4-pentyloxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin (actinomycin D, Cosmegan), daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludalafil phosphate 5-fluorouracil, flutamide, tezacitabine, gemcitabine (difluorodeoxycytidine), hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, leucovorin, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, mylotarg, paclitaxel, nab-paclitaxel, pentostatin, polyphenprosan 20 with carmustine implant, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine.

Anticancer agents of particular interest for use in the present invention include, but are not limited to, antitumor antibiotics, tyrosine kinase inhibitors, alkylating agents, anti-microtubule or anti-mitotic agents, and oncolytic viruses.

Exemplary antitumor antibiotics include, but are not limited to, doxorubicin, bleomycin, daunorubicin (daunorubicin hydrochloride, daunomycin and daunorubicin hydrochloride, daunorubicin liposomes (daunorubicin citrate liposomes), mitoxantrone, epirubicin, idarubicin, mitomycin C, geldanamycin and herbimycin.

Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib hydrochloride, sunitinib malate, bosutinib, dasatinib, pazopanib, sorafenib, vandetanib, imatinib, and imatinib mesylate.

Exemplary alkylating agents include, but are not limited to, oxaliplatin, temozolomide, dactinomycin (also known as actinomycin-D), Melphalan (also known as L-PAM, L-sarcolysin, or phenylalanine mustard), altretamine (also known as hexamethylmelamine (HMM)), carmustine, bendamustine, bendamustine hydrochloride, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine, procarbazine, mechlorethamine, mechlorethamine hydrochloride, streptozotocin, and thiotepa.

Exemplary anti-microtubule or anti-mitotic agents include, but are not limited to, vinca alkaloids (such as vinorelbine tartrate, vincristine, and vindesine), taxanes (such as paclitaxel and docetaxel), and estramustine.

As used herein, "osteoporosis therapeutic agent" refers to an agent that can prevent, alleviate, delay or eliminate symptoms associated with osteoporosis, reduce the risk of suffering from osteoporosis-related diseases, cure osteoporosis-related diseases or a combination thereof. Exemplary osteoporosis therapeutic agents that can be used in the present invention include, but are not limited to, bisphosphonates (e.g., alendronate, risedronate, ibandronate and zoledronic acid), raloxifene, denosumab and teripartate.

As used herein, "mental disease-related therapeutic agent" refers to an agent that can prevent, alleviate, delay or eliminate symptoms related to the mental field, reduce the risk of developing a mental disease-related disease, cure a mental disease-related disease, or a combination thereof.

As used herein, a "developmental therapeutic agent" refers to an agent that can prevent, alleviate, delay or eliminate developmental-related symptoms, reduce the risk of developing a developmental-related disease, cure a developmental-related disease, or a combination thereof.

In some embodiments, the weight ratio of the first component to the second component is 1:99 to 99: 1. In certain embodiments, the weight ratio of the first component to the second component is 1:99, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5 or 99:1.

In some embodiments, the first component is applied before the second component is applied. In some embodiments, the first component is applied after the second component is applied. In some embodiments, the first component is applied simultaneously with the second component. In some embodiments, the first component and the second component are applied in an alternating manner.

In some embodiments, the first component and the second component provided by the present invention are formulated into a single therapeutic composition, and the first component and the second component are administered simultaneously. In some embodiments, the first component and the second component provided by the present invention are separated from each other, for example, each is formulated into a single therapeutic composition, and the first component and the second component are administered simultaneously. In some embodiments, the first component and the second component provided by the present invention are separated from each other, for example, each is formulated into a single therapeutic composition, and the first component and the second component are administered at different times, for example, the first component is administered before the administration of the second component, or the first component is administered after the administration of the second component, or The first component and the second component are administered in an alternating manner. The first component and the second component may be administered in a single dose or in multiple doses.

treatment method

The present invention also provides a treatment method, which comprises administering an effective amount of the strain provided by the present invention, its derivative, a culture medium containing the same, or a composition containing the same to a subject in need thereof, thereby preventing and/or treating a disease or condition in the subject.

As used herein, "effective amount" refers to an amount that can provide the desired effect without causing serious side effects under medical judgment. Considering the administration route, individual differences of subjects, etc., the amount of microorganisms administered to the subject through the composition of the present invention can be appropriately adjusted.

In certain embodiments, the dosage can be changed during use. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage can be changed during use, depending on the reaction of the subject.

It is to be understood that the effective amount of lactobacillus strains of the present invention or derivatives thereof depends on various factors known in the art, such as experimenter's weight, age, past medical history, current drug therapy, health status and the possibility of cross-reaction, allergy, sensitivity and adverse side effects, and route of administration and degree of disease development. Those skilled in the art can consider that for example above-mentioned factors reduce or increase dosage. The above dosage range does not constitute any form of limitation to the scope of the present invention.

As used herein, "subject" refers to any animal, preferably a mammal, such as a human, monkey, mouse, rat, rabbit, more preferably a human.

As used herein, "prevention and/or treatment" of a disease or condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

【Diseases related to pathogen infection】

The present invention also provides a treatment method, which comprises administering the strain provided by the present invention, its derivative, culture medium containing the same, or composition containing the same to a subject in need, thereby antagonizing pathogens in the subject, or preventing and/or treating diseases or conditions associated with the pathogens.

Examples of such pathogens include, but are not limited to, bacteria, fungi, viruses, spirochetes, mycoplasmas, rickettsiae, chlamydiae, and parasites.

In some embodiments, the parasite is Trichomonas.

In some embodiments, the pathogen-associated diseases or conditions include female reproductive tract infections and reproductive tract flora disorders.

The pathogen-associated diseases or conditions include diseases or conditions associated with vaginal inflammation, including but not limited to bacterial vaginitis, fungal vaginitis (e.g., candidal vaginitis), viral vaginitis, yeast vaginitis, Trichomonas vaginitis, infections in the vagina, sexually transmitted diseases such as HIV and chlamydia infections, infections that endanger the fetus in pregnant women, premature births, and urinary tract infections.

In some embodiments, the disease or condition associated with a pathogen comprises a disease associated with Malassezia infection.

The Malassezia infection-related diseases include, but are not limited to, dandruff, seborrheic dermatitis, atopic dermatitis and psoriasis.

【Diseases related to immune regulation】

The present invention also provides a treatment method, which comprises administering the strain provided by the present invention, its derivative, culture medium containing the same or composition containing the same to a subject in need thereof, thereby preventing and/or treating a disease or condition related to immune regulation.

In some embodiments, the disease or disorder associated with immune regulation is cancer or an autoimmune disease.

As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis.

Examples of cancer include, but are not limited to, prostate cancer, gastroesophageal cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, bronchial cancer, duct cancer, bone cancer, liver and bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia, lymphoma and myeloma.

As used herein, "allergic disease" refers to any symptoms, tissue damage, or loss of tissue function caused by an allergy.

Allergic diseases include, but are not limited to, allergic rhinitis, allergic asthma, atopic dermatitis, allergic keratoconjunctivitis, urticaria, food allergy, drug allergy, dust mite allergy, and pollen allergy.

As used herein, "autoimmune disease" refers to a disease in which the immune system of a mammal produces a humoral immune response or a cellular immune response against the mammal's own tissues, or against antigens that are harmless to the mammal itself, thereby producing tissue damage in the mammal. Symptoms and severity vary from patient to patient, and the clinical features of each patient vary greatly over time.

Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, rheumatic fever, lupus, systemic sclerosis, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, Guillain-Barré syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto's thyroiditis, temporal arteritis, juvenile diabetes, alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolytic anemia, Wechsler's musculoskeletal disease, and schizophrenia. Granulomatosis, Sjögren's syndrome, Addison's disease, Crohn's disease, Behcet's disease, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, pharyngitis, tonsillitis, bronchitis, pneumonia, gastric ulcer, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, periarthritis of the shoulder, tendinitis, tenosynovitis myositis, hepatitis, cystitis, nephritis, sepsis, vasculitis, and bursitis.

Diseases related to osteoporosis

The present invention also provides a treatment method, which comprises administering the strain provided by the present invention, its derivative, culture medium containing the same, or composition containing the same to a subject in need thereof, thereby preventing and/or treating a disease or condition associated with osteoporosis.

As used herein, "osteoporosis" refers to a bone disease, including primary and secondary, characterized by deterioration of bone strength due to a decrease in bone mass and/or deterioration of bone quality, leading to an increased risk of fracture.

Examples of diseases or conditions associated with osteoporosis include, but are not limited to, juvenile osteoporosis, menopausal osteoporosis, postmenopausal osteoporosis, post-traumatic osteoporosis, and osteoporosis due to aging, corticosteroid therapy, and inactivity. of osteoporosis.

【Other uses】

The present invention also provides a treatment method, which comprises administering the strain provided by the present invention, its derivative, culture medium containing the same, or composition containing the same to a subject in need, thereby preventing and/or treating diseases or conditions associated with iron deficiency anemia, menopausal syndrome, and central nervous system diseases.

Pharmaceutical use

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in the preparation of a medicament for antagonizing pathogens.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in the preparation of a medicament for preventing and/or treating a disease or condition associated with a pathogen.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in the preparation of a medicament for preventing and/or treating a disease or condition associated with immunoregulation.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in the preparation of a medicament for preventing and/or treating a disease or condition associated with osteoporosis.

The present invention also provides the use of the strain of the present invention, its derivative, culture medium containing it or composition containing it in preparing a drug for preventing and/or treating diseases or conditions related to iron deficiency anemia, menopausal syndrome or central nervous system diseases.

Therapeutic Uses

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in antagonizing pathogens.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same, or a composition containing the same in preventing and/or treating a disease or condition associated with a pathogen.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same or a composition containing the same in preventing and/or treating diseases or disorders related to immunoregulation.

The present invention also provides use of the strain of the present invention, its derivative, a culture medium containing the same, or a composition containing the same in preventing and/or treating a disease or condition associated with osteoporosis.

The present invention also provides the use of the strain of the present invention, its derivative, culture medium containing the same or composition containing the same in preventing and/or treating diseases or conditions related to iron deficiency anemia, menopausal syndrome or central nervous system diseases.

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials and methods (including whole or part) described below are included within the scope of the invention. These specific compositions, materials and methods are not intended to limit the present invention, but are only used to illustrate specific embodiments within the scope of the present invention. Without departing from the scope of the present invention, those skilled in the art can develop equivalent compositions, materials and methods without the ability of invention. It should be understood that many changes can be made to the procedures described herein, but still within the limits of the present invention. The inventor intends to include such variations within the scope of the present invention.

Example

Example 1. Isolation, purification and enrichment of Lactobacillus crispatus strains

1. Isolation, purification and enrichment of Lactobacillus strains

Several healthy women of childbearing age without vaginal infection or any intestinal diseases were recruited to provide samples. All participants passed the health checkup at the physical examination center and provided information about their age (21-30), menstrual cycle, and other health behaviors through questionnaires. Starting 2 weeks before sample collection, all participants avoided all types of formulas containing probiotics. Samples were collected using the Port.A-Cd system of BD Company in the United States. Two sterile cotton swabs were used to collect secretions from the 1/3 of the side wall of the subject's vagina. The swabs were placed in sterile tubes and quickly transported to the laboratory biosafety cabinet with ice packs. The bacterial suspension after rinsing the swabs with a small amount of sterile PBS was used as the mother solution, and then diluted with sterile PBS to different concentrations, spread on the freshly prepared Rogosa SL solid culture medium, marked with information, and the culture dish was placed in a culture box, and an anaerobic gas-producing bag was placed in a 37°C incubator for 48-72 hours.

From the cultured Rogosa SL plates, single colonies of different shapes (surface, edge, color, size, etc.) were picked up with an inoculation loop and inoculated onto freshly prepared MRS solid culture medium according to the eight-zone streak method. The culture dish was placed in a culture box and an anaerobic gas-producing bag was placed in a 37°C incubator for incubation for 24-72 hours to obtain purified single colonies. Single colonies were picked up with an inoculation loop and inoculated into MRS liquid culture medium, which was placed in a 37°C incubator for anaerobically cultured for 24 hours to obtain the crispatus Lactobacillus strain.

I bought Luna vaginal probiotics from a Taobao store. I opened the package in a biosafety cabinet and took out a The capsules were opened and the bacterial powder in the capsule shell was poured into 10mL sterile PBS solution, vortexed and mixed, and recorded as ^10-1 dilution, and then continued to be diluted 10 times to ^10-4 , and 100μL was taken from ^10-3 and ^10-4 dilutions and spread on MRS solid culture medium plates, and cultured anaerobically at 37℃ for 24-48 hours. When single colonies grow on MRS solid culture medium, it is found that there are 4 different colony morphologies on the MRS solid culture medium of Luna product. Single colonies of different morphologies are picked with an inoculation loop and inoculated into MRS liquid culture medium, placed in a 37℃ incubator, and cultured anaerobically for 16-24 hours; after the culture is completed, centrifuge them respectively, remove part of the supernatant, resuspend the bacteria, add an equal volume of 20% glycerol, vortexed and mixed, and dispensed into cryopreservation tubes, stored at -80℃, and sent strain samples for 16S rRNA sequencing to identify the strains, and obtain the commercial strain Lactobacillus crispatus LbV88.

2. Identification and preservation of Lactobacillus

(1) Culture characteristics, staining microscopy and morphological characteristics

Regarding the Lactobacillus crispatus of the present invention: the lactobacillus colonies obtained after culture are round, and a smear of the pure culture of the bacteria is taken for Gram staining, showing Gram-positive, short rod-shaped, and can be connected into long chains, and is preliminarily determined to be of the genus Lactobacillus.

(2) 16S rRNA gene sequence identification

A kit for direct PCR amplification was used, and primers 27F (5'AGA GTT TGA TCM TGG CTC AG 3') and 1492R (5'TAC GGY TAC CTT GTT ACG ACT T 3') were used for PCR amplification. The PCR product was subjected to gel electrophoresis to determine the 16S rRNA gene fragment. If the gel electrophoresis results showed that the PCR was successful, the PCR sample was sent to a gene sequencing company for 16S rRNA sequencing. The sequence obtained by sequencing was compared with the data in the NCBI database for BLAST sequence similarity analysis. According to the highest homology score greater than 97%, the isolated strain was determined to be Lactobacillus crispatus, and was named Lactobacillus crispatus-120, i.e. L. crispatus-120.

Example 2. Study on gastric acid and bile salt resistance of Lactobacillus crispatus-120

Oral probiotics can only play their beneficial effects when they enter the intestines, so it is very necessary to examine the ability of probiotic strains to tolerate digestive juices such as gastric acid and bile salts. In order to verify whether L. crispatus-120 meets the conditions, the present invention determines its acid and bile salt resistance properties by in vitro simulated gastrointestinal fluid method.

L. crispatus-120 and L. crispatus LBV88 (control bacteria, commercial strain) were inoculated from glycerol tubes into MRS liquid medium using an inoculation loop, cultured at 37°C for 18-24 hours in an anaerobic workstation, centrifuged at 8000 rpm for 5 minutes, and then the cells were collected. Each strain was adjusted to an appropriate concentration (about 5×10 ⁸ CFU/mL) using a turbidometer. Inoculate 5% of the inoculum into fresh MRS liquid medium and culture anaerobically at 37°C for 6 hours until the logarithmic growth phase. Take out after 6 hours, centrifuge at 8000rpm for 5 minutes, remove the medium, add physiological saline to resuspend to a certain concentration. Use a turbidity meter to adjust the resuspension so that the bacterial concentration is about 1×10 ⁹ CFU/mL. Dilute the resuspension 10 times in a gradient manner, and take 100μL to plate for counting.

1. Gastric acid resistance test

Prepare sterile saline: 0.9% w/v, adjust pH to 3.0 with hydrochloric acid.

Prepare simulated gastric fluid: Dissolve 96.84 mg of pepsin (Sigma, P7000-25G, 632 U/mg) powder in 32.3 mL of pH 3.0 saline to a final concentration of 3 g/L. Filter with a 0.22 μm sterile filter membrane and prepare for immediate use.

Generally speaking, the pH of human gastric acid is 3.0-3.5. Take 2mL of the adjusted bacterial suspension and centrifuge again to remove the supernatant. Repeat in duplicate. Resuspend one portion of the bacteria with an equal volume of simulated gastric fluid at pH 3.0, and resuspend the other portion of the bacteria with an equal volume of normal saline. After incubation at 37°C for 3 hours, dilute each probiotic in a gradient manner and count on a plate. The acid resistance calculation formula is as follows:

Acid tolerance (%) = (Log10 CFU/mL in simulated gastric fluid)/(Log10 CFU/mL in normal saline) × 100.

2. Bile tolerance test

Prepare sterile saline: 0.9% w/v, adjust pH to 8.0 with NaOH.

Prepare 100× trypsin (Shanghai Biotechnology Co., Ltd., A003702-0100, 204U/mg protein) stock solution: 100mg dissolved in 1mL pH 8.0 saline. Prepare 100× bile salt (Oxoid, LP0055) stock solution: 192.54mg dissolved in 0.642mL pH 8.0 saline.

Prepare simulated intestinal fluid: add 0.5 mL of 100× trypsin and bile salt stock solution to 49 mL of pH 8.0 saline, respectively, to a final concentration of 1 g/L trypsin and 0.3% bile salt (i.e. 3 mg/mL). Filter with a 0.22 μm sterile filter membrane and prepare for use.

Take 2 mL of the adjusted bacterial suspension and centrifuge to remove the supernatant. Repeat in duplicate. Resuspend one portion of the bacteria with an equal volume of simulated intestinal fluid at pH 8.0, and resuspend the other portion of the bacteria with an equal volume of normal saline. After incubation at 37°C for 4 hours, dilute each probiotic in a gradient manner and count on a plate. The formula for calculating bile salt tolerance is as follows:

Bile salt tolerance (%)=(Log10 CFU/mL in simulated intestinal fluid)/(Log10 CFU/mL in normal saline)×100.

3. Experimental results

The present invention uses the commercial strain L. crispatus LBV88 as a control. The experimental results are shown in Tables 1 and 2. The survival rates of the commercial strains L. crispatus LBV88 and L. crispatus-120 after incubation in simulated gastric fluid for 3 hours were about 100%, indicating that they both had good gastric acid tolerance; while after the two strains were incubated in simulated intestinal fluid for 4 hours, the survival rate of L. crispatus LBV88 was low, and the survival rate of L. crispatus-120 was 56.34%, indicating that L. crispatus-120 had good bile salt tolerance. In summary, L. crispatus-120 has good gastric acid and bile salt resistance.

Table 1 Results of gastric acid resistance test of Lactobacillus crispatus strains

Table 2 Results of bile salt tolerance test of Lactobacillus crispatus strains

Example 3. Study on the colonization performance of Lactobacillus crispatus-120

Probiotics can only exert their probiotic effects for a long time if they can colonize in the intestines and vagina. Therefore, the present invention uses human intestinal cell line Caco-2 vaginal epithelial cells VK2/E6E7 to evaluate the intestinal colonization performance of L. crispatus-120.

The culture medium for Caco-2 cells uses complete culture medium EMEM (Hangzhou Jinuo Biomedical Technology Co., Ltd., GNM 11700), supplemented with 10% fetal bovine serum. Slightly loosen the lid of the Caco-2 cell preservation tube and quickly (within 2 minutes) thaw in a 37°C water bath. Disinfect the outer wall of the preservation tube with 75% ethanol. Use a pipette to transfer the cell cryopreservation solution to a centrifuge tube containing 9 mL of culture medium, centrifuge at 1000 rpm for 3-5 minutes, remove the supernatant, add an appropriate amount of culture medium and gently resuspend. Place in a _CO2 incubator for culture. When the growth confluence rate reaches 80%-90%, the cells are passaged. After 2-3 subcultures, remove the old culture medium, wash twice with DPBS (without calcium and magnesium ions), add 2mL of trypsin to the bottle and gently shake the culture bottle to make the digestion solution flow over all cell surfaces. After 2-5 minutes of digestion at 37°C, place the culture bottle under a microscope for observation. After the cytoplasm shrinks and the intercellular space increases, tap the side wall of the culture bottle. When the cells detach from the bottom wall of the culture bottle, immediately add 3 times the volume of trypsin to terminate the digestion. Use a pipette to repeatedly and gently blow the bottom wall of the bottle to form a single cell suspension after the cells detach from the bottle wall. Centrifuge at 1000rpm for 3-5 minutes. Discard the supernatant, resuspend in fresh culture medium, and take part of the cell suspension to determine the actual number of live cells on an automatic cell counter. According to the live cell count results, dilute with culture medium to a cell density of 5×10 ⁵ cell/mL, add 100μL of cell suspension to each well of the 96-well plate, and each well contains 5×10 ⁴ cell/well. Incubate at 37°C, 5% CO ₂ for 20-24 hours before use.

VK2/E6E7 cell culture medium was prepared by adding 1 mL of keratinocyte growth supplement solution to 500 mL of serum-free keratinocyte basal culture medium K-SFM (Invitrogen, 10744-019) and preheating at 37°C before use.

Use an inoculation loop to inoculate L.crispatus-120 and L.crispatus LBV88 from the glycerol tube into MRS liquid culture medium, and culture at 37°C in an anaerobic workstation for 12-18 hours. After centrifugation at 8000rpm for 5 minutes, collect the bacteria. Use a turbidity meter to adjust each probiotic to an appropriate concentration (about 5×10 ⁸ CFU/mL). Inoculate into fresh MRS liquid culture medium at 5% inoculation amount, and culture anaerobically at 37°C for 6 hours until the logarithmic growth phase. Centrifuge at 8000rpm for 5 minutes, remove the culture medium, and add physiological saline to resuspend to a certain concentration. Use a turbidity meter to adjust the resuspended solution so that the concentration of probiotics is about 1.0×10 ⁹ CFU/mL. Add an equal volume of 40% glycerol to the resuspended bacterial solution, mix well, transfer 1mL/tube to a cell cryopreservation tube, and place on dry ice. After solidification, transfer to a -80°C refrigerator for long-term storage. Before the experiment begins, take 1 tube of frozen bacteria and place it in a 37°C water bath to thaw, then perform 10-fold gradient dilutions, plate 10 μL for counting, and repeat 3 times.

On the day of the experiment, take out the cryovials and immediately thaw them in a 37°C water bath. Transfer to a 2 mL centrifuge tube, centrifuge at 8000 rpm for 5 minutes, remove the supernatant, and resuspend with culture medium. Dilute the bacterial suspension to about 2.5 × 10 ⁸ CFU/mL with the corresponding culture medium, then dilute 5 times, and then dilute 10 times to obtain bacterial suspensions of about 5.0 × 10 ⁷ CFU/mL and about 5.0 × 10 ⁶ CFU/mL, respectively, for later use.

On the day of the experiment, remove the cells from the incubator, discard the culture medium, and add the corresponding culture medium to wash once. Take 100 μL of each prepared bacterial solution and add it to the cells. Place the plate in a 37°C, _CO2 incubator for 2 hours of adhesion, and the target multiplicity of infection (MOI, bacteria: cells) is 10:1. After the incubation and adhesion are completed, wash three times with a plate washer (BioTek, 405TS) (washing volume 200 μL, minimum washing speed), add 200 μL of DPBS containing 0.05% Triton X-100 to lyse the cells, and use a pipette to blow and aspirate to allow the cells and bacteria to fully fall off. Use physiological saline for 10-fold gradient dilution and take 100 μL to spread on the plate for counting, and the count average is 20. The plate was MRS agar, with 3 replicates. The adhesion rate was calculated using the following formula

Adhesion rate % = adhered bacteria CFU / inoculated bacteria CFU × 100%

Table 3 Adhesion ability of Lactobacillus crispatus-120 to Caco-2 cells and VK2/E6E7 cells

The experimental results are shown in Table 3. For human colon cancer cells Caco-2 and vaginal epithelial cells VK2/E6E7, the adhesion of L. crispatus-120 was higher than that of the commercial strain L. crispatus LBV88, indicating that L. crispatus-120 is a probiotic strain with development potential.

Example 4. Study on the effect of Lactobacillus crispatus-120 on antagonism of pathogenic bacteria

The ability of Lactobacillus to antagonize pathogenic bacteria is also one of the mechanisms for its probiotic effect. Therefore, the ability of Lactobacillus crispatus-120 to antagonize pathogenic bacteria was studied in the present invention by double-layer plate method and bacterial cake method, and the commercial strain L. crispatus LBV88 was used as the control bacteria.

Use an inoculation loop to inoculate L.crispatus-120 and L.crispatus LBV88 from the glycerol tube into MRS liquid culture medium, and culture at 37°C in an anaerobic workstation for 12-18 hours. After centrifugation at 8000rpm for 5 minutes, collect the bacteria. Use a turbidity meter to adjust each probiotic to an appropriate concentration (about 5×10 ⁸ CFU/mL). Inoculate into fresh MRS liquid culture medium at a 5% inoculation rate, and culture anaerobically at 37°C for 6 hours until the logarithmic growth phase. Take out after 6 hours, centrifuge at 8000rpm for 5 minutes, remove the culture medium, and add physiological saline to resuspend to a certain concentration. Use a turbidity meter to adjust the resuspension so that the bacterial concentration is about 1.0×10 ⁸ CFU/mL. Dilute this resuspension 10 times in a gradient, and take 10μL to plate for counting. Use a pipette to take 5μL of the adjusted bacterial solution and spot it on the pre-prepared MRS agar medium (1 bacterium/plate, 3 parallels). After the plate is dried, it is incubated under anaerobic conditions at 37°C for 20-24 hours before use.

Seven pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Candida albicans, Atopobium vaginalis and Gardnerella vaginalis) were inoculated into their respective solid culture media one day in advance according to Table 4 and cultured at 37°C overnight. The test methods are summarized in Table 5.

Table 4 Culture medium used for culturing Lactobacillus crispatus strains and various pathogenic bacteria strains

Table 5 Test method and conditions of mushroom cake method

On the day of the experiment, the pathogenic bacteria were removed. For the 7 pathogenic bacteria, 3-6 single colonies were picked up with an inoculation loop and placed in physiological saline to prepare a bacterial suspension. The turbidity was adjusted to about 0.2 using a turbidity meter, and the bacterial concentration was about 1.0×10 ⁸ CFU/mL; the turbidity of Candida albicans was about 0.2, and the concentration was about 2.0×10 ⁶ CFU/mL.

Double-layer plate method: Take 100 μL of a prepared pathogenic bacteria (Staphylococcus aureus) in 5 mL of solid culture medium (see Table 4) cooled to about 45°C, and pour it on the MRS agar of the probiotics to be tested that has been cultured for 20-24 hours. After solidification, place the plate in 37°C atmospheric conditions for 1 day until an inhibition zone appears. Three parallel test plates are used for each probiotic.

Inverted cake method: Take 100 μL of the prepared pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi), spread them on NA or Columbia blood agar medium + 5% sheep blood plate, and after they are fully absorbed, take out each probiotic cake and invert it on the pathogenic bacteria plate. Incubate the Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi plates at 37°C for 1 day until an inhibition zone appears. Test 3 parallel plates for each probiotic.

Strip cake method: Dip a cotton swab into the probiotic inoculum and spread it on a plate containing 10 mL of MRS solid culture medium to form a 2 cm wide strip along the diameter. Then incubate the plate anaerobically at 37°C for 24 hours. Pour 5mL YM solid medium onto the surface of the plate and wait for a thin layer to solidify. Use a cotton swab to spread the Candida albicans liquid evenly on the surface of the YM solid medium. After it dries, first culture it at 4℃ for 4 hours, then culture it at 37℃ for 24 hours to observe the inhibitory effect of probiotics on Candida albicans. Three parallel test plates are used for each probiotic.

Measure the diameter of the inhibition zone using a vernier caliper or a colony counter.

Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used to calculate the data for each group, and Dunnett’s multiple comparisons test was used to compare each probiotic. When P-value < 0.05, it was considered to be significantly different.

The inhibitory effects of L. crispatus-120 on 7 pathogenic bacteria are shown in Tables 6-10. L. crispatus-120 has stronger inhibitory effects on Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus, vaginal resistant Gardnerella and Atopobium than the commercial strain L. crispatus LBV88. Both L. crispatus-120 and the commercial strain L. crispatus LBV88 have inhibitory effects on the growth of Candida albicans.

Table 6 Diameter of inhibition zone of Lactobacillus crispatus against Pseudomonas aeruginosa (mm)

Table 7 Diameter of inhibition zone of Lactobacillus crispatus against Salmonella typhi and Escherichia coli (mm)

Table 8 Diameter of inhibition zone of Lactobacillus crispatus against Staphylococcus aureus (mm)

Table 9 Inhibitory effect of Lactobacillus crispatus on Candida albicans

Note: “-” indicates no inhibition; “+/-” indicates partial inhibition; “+” indicates significant inhibition.

Table 10 Inhibitory effect of Lactobacillus crispatus on vaginal Gardnerella resistant bacteria and Atopobium

Example 5. Effect on the mouse vaginal Gardnerella model

Forty SPF 4-6-week-old ICR female mice were randomly divided into four groups: healthy control group, infection control group, metronidazole group and Lactobacillus crispatus-120 group, with 10 mice in each group. The infection day was defined as day 0. A certain concentration of estradiol was subcutaneously injected into the mice on day 3 and day 0. A certain amount of ketamine hydrochloride was intraperitoneally injected to anesthetize the mice on the day of infection. Then, 20 μL vaginal Gardnerella solution (5×10 ⁷ CFU/mL) was injected into the vagina of the mice once a day for 3 consecutive days. On the fourth day, a small amount of mucus was taken from the vagina of the mice with a sterile swab to detect Gardnerella to ensure that Gardnerella was continuously colonized in the vagina of each mouse. The healthy control group was injected with the same volume of normal saline every day.

Pick a single colony of freshly cultured Lactobacillus crispatus-120 from the MRS plate and inoculate it into the MRS solution. The cells were incubated in a culture medium at 37°C for 24 hours, centrifuged, and the bacterial sludge was resuspended in PBS and the concentration was adjusted to 1×10 ⁹ CFU/mL using a flow cytometer. On the first day after infection, 20 μL of freshly prepared bacterial solution was injected into the vagina of the mice in the Lactobacillus crispatus-120 group once a day for 3 consecutive days; 20 μL of metronidazole solution was injected into the vagina of the mice in the metronidazole group once a day for 3 consecutive days; the mice in the healthy control group and the infection control group were given the same volume of normal saline vaginally every day for 3 consecutive days, once a day.

1. Determination and Analysis of Mouse Vaginal Microbiota

50 μL of physiological saline was taken with a micropipette, and the vagina of the mice was repeatedly flushed 5-6 times before administration after modeling, on the first day after administration, and on the sixth day after administration. 30 μL of the above vaginal lavage fluid was taken for colony counts of vaginal Gardnerella and Lactobacillus, respectively. The results are shown in Table 11. The Columbia blood plate with gentamicin sulfate (4 mg/L), nalidixic acid (30 mg/L) and amphotericin B (2 mg/L) was used to count vaginal Gardnerella.

Preliminary identification is carried out based on colony morphology on selective culture medium and smear staining and microscopic examination.

Table 11 Colony count results of lavage fluid in each group

As can be seen from the above table, on the first day after treatment, the number of Gardnerella colonization in the vagina of mice in the Lactobacillus crispatus-120 group was significantly reduced compared with that in the infected control group; while the number of Gardnerella in the metronidazole group decreased by about 2 orders of magnitude, but the number of Lactobacillus in the vagina of mice in the Lactobacillus crispatus-120 group was about 3 orders of magnitude higher than that in the metronidazole group and the infected control group; on the 6th day after treatment, the number of Gardnerella colonization in the vagina of mice in the Lactobacillus crispatus-120 group was slightly higher than that in the metronidazole group, but 2 orders of magnitude lower than that in the infected control group, while the number of Lactobacillus in the vagina of mice in the Lactobacillus crispatus-120 group was still about 3 orders of magnitude higher than that in the metronidazole group and the infected control group; this indicates that Lactobacillus crispatus-120 can colonize well in the vagina and increase the number of Lactobacillus in the vagina The amount can be restored to above normal levels, and at the same time the reproduction of vaginal Gardnerella can be stably inhibited.

2. Observation of the mouse vulva before and after treatment

The swelling and redness of the vulva and the amount of vaginal discharge of each group of mice were observed and recorded, and vaginal lavage fluid smears (PAS staining) were taken from typical mice in each group.

Table 12 Inflammation conditions such as swelling and secretion of the vulva of mice after treatment

Table 12 shows the inflammatory response caused by vaginal colonization of Gardnerella vaginalis in the mouse vagina. The mice in the infection control group had a large amount of edema on the vulva, and the secretions were abundant and thin and foamy. The PAS staining results showed that inflammatory cells infiltrated the surface of the vaginal mucosa, indicating that the model was successful. After treatment with the bacterial solution of the Lactobacillus crispatus-120 group, the symptoms of vulvar edema and abundant secretions in the mice were significantly alleviated. The PAS staining results of the vaginal lavage fluid showed that the number of white blood cells in the vaginal secretions of the mice was significantly reduced, and most of them were vaginal epithelial cells, indicating that the damage to the vaginal mucosa of the mice had been restored to a large extent.

The above results show that Lactobacillus crispatus-120 in the present invention has the effects of regulating the balance of vaginal flora and inhibiting the growth and colonization of Gardnerella vaginalis, and can be used to prevent and treat bacterial vaginitis.

Example 6. Effect on the mouse vaginal Candida albicans model

Forty C57BL/6 female mice of SPF grade 6-8 weeks were randomly divided into 4 groups: healthy control group, infection control group, clotrimazole group and Lactobacillus crispatus-120 group, with 10 mice in each group. Except for the healthy control group, 50 μL of lincomycin hydrochloride solution of a certain concentration was taken by micropipette to wash the vagina of mice in other groups once a day for 5 consecutive days, and then 20 μL of Candida albicans (2.5×10 ⁷ CFU/mL) was taken by micropipette to inoculate the vagina of mice for 6 consecutive days, once a day, to create a mouse vaginal Candida albicans infection model. The healthy control group was injected with the same volume of normal saline every day for 11 consecutive days.

A single colony of freshly cultured Lactobacillus crispatus-120 was picked from the MRS plate and inoculated into MRS liquid medium. The culture was kept at 37°C for 24 hours, centrifuged, and the bacterial sludge was resuspended in PBS. The concentration was adjusted to 1×10 ⁹ CFU/mL using flow cytometry. 20 μL of bacterial solution was infused into the mice in the Lactobacillus crispatus group once a day for 3 consecutive days; 20 μL of clotrimazole solution was injected into the vagina of the mice in the clotrimazole group once a day for 3 consecutive days; the mice in the healthy control group and the infection control group were given the same volume of normal saline vaginally every day for 3 consecutive days, once a day.

1. Determination and Analysis of Mouse Vaginal Microbiota

Use a micropipette to take 50 μL of normal saline, and take some mice to repeatedly rinse the mouse vagina 5-6 times before administration after modeling, on the first day after the end of administration, and on the sixth day after the end of administration. Take 30 μL of the above vaginal lavage fluid for colony counts of Candida albicans and Lactobacillus, respectively. The results are shown in Table 13.

Table 13 Colony count results of lavage fluid in each group

It can be seen from the above table that on the first day after treatment, the number of Candida albicans colonized in the vagina of mice in the Lactobacillus crispatus-120 group was reduced by one order of magnitude compared with the infection control group, but the number of Lactobacillus in the vagina was about 3 orders of magnitude higher than that of the infection control group and the clotrimazole group; on the 6th day after treatment, the number of Candida albicans colonized in the vagina of mice in the Lactobacillus crispatus-120 group was equivalent to that of the clotrimazole group and much smaller than that of the infection control group, while the number of Lactobacillus in the vagina of mice was still about 3 orders of magnitude higher than that of the clotrimazole group and the infection control group; This indicates that Lactobacillus crispatus-120 can colonize well in the vagina and restore the number of Lactobacillus in the vagina to above normal levels, and can stably inhibit the reproduction of Candida albicans.

2. Observation of the mouse vulva before and after treatment

Table 14 Inflammation conditions such as swelling and secretion of the vulva of mice after treatment

Table 14 shows the inflammatory response caused by Candida albicans colonization in the mouse vagina. The mice in the infected control group showed typical Candida symptoms such as a greater degree of redness and swelling on the vulva, a lot of lumpy secretions, and severe vaginal congestion, indicating that the model was successful; after treatment with Lactobacillus crispatus liquid, the symptoms of vulvar redness and swelling, vaginal congestion and secretions in the mice were significantly alleviated.

At the same time, the results of PAS staining of vaginal lavage fluid showed that the vaginal epithelial cells were in the majority and the number of white blood cells was relatively small in the healthy control group, while the vaginal epithelial cells were relatively small and the number of white blood cells was relatively large in the infected control group colonized with Candida albicans, indicating that the vaginal mucosa of the mice was severely damaged. After treatment with Lactobacillus crispatus-120 bacterial fluid, the number of white blood cells in the vagina of the mice was significantly reduced, and epithelial cells were in the majority, indicating that the damage to the vaginal mucosa of the mice had been restored.

The above results show that Lactobacillus crispatus-120 in the present invention has the effects of regulating vaginal flora and inhibiting the growth and colonization of vaginal Candida albicans, and can be used to prevent and treat candidal vaginitis.

Example 7. Analysis of the effect of Lactobacillus crispatus against Malassezia furfur

The antifungal effect of L. crispatus-120 was determined by the double-layer plate method. L. crispatus-120 and commercial strain L. crispatus LBV88 were inoculated into MRS liquid medium at 3% (V/V) and cultured anaerobically at 37°C for 8-16 hours for standby use. 10 μL of bacterial solution was spotted onto MRS solid medium plates and cultured anaerobically at 37°C for 24-48 hours. 1% seed solution of Malassezia furfur (ATCC14521) was inoculated into mYPG prepared under aerobic conditions. In liquid culture medium, culture at 37°C for 24 to 48 hours and set aside. Prepare mYPG culture medium, sterilize at 115°C for 20 minutes, wait until the temperature cools to about 40°C, take 2.5mL of the culture medium and mix it with 500μL of the standby Malassezia furfur (M. furfur) culture solution, pour the mixture onto MRS solid culture medium spotted with L. crispatus-120 and commercial strain L. crispatus LBV88, and let it stand until the culture medium solidifies. The solidified culture medium plate is cultured aerobically at 37°C for 24 to 48 hours. The antifungal activity of L. crispatus-120 and commercial strain L. crispatus LBV88 was determined by measuring the diameter of the inhibition zone, and the experiment was repeated three times in parallel. The results are shown in Table 15, and L. crispatus-120 has a stronger effect on inhibiting the growth of Malassezia furfur than the commercial strain L. crispatus LBV88.

Table 15 Inhibition zone diameter of two strains of Lactobacillus crispatus against Malassezia furfur (mm)

Example 8. Study on the effect of Lactobacillus crispatus on inhibiting antigen-induced histamine release

In allergic reactions, a large amount of histamine is released from tissues, which in turn causes an inflammatory reaction. Clinically, the symptoms of allergic reactions in patients are improved by blocking the secretion of histamine. In order to screen probiotic strains that can inhibit histamine secretion, the present invention evaluated the ability of Lactobacillus crispatus strain L. crispatus-120 derived from the human vagina to inhibit histamine secretion, and used the commercial strain L. crispatus LBV88 as a control strain. The ability of each strain to inhibit histamine secretion was determined by the following method: after culturing the RBL-2H3 cell line, degranulation was induced, and then the o-phthalaldehyde post-column conversion method was used to determine the histamine content in the filtrate by high performance liquid chromatography.

L. crispatus-120 and commercial strain L. crispatus LBV88 were cultured in MRS medium, activated and subcultured 2-3 times before use. RBL- _2H3 cells (ATCC NO.CRL-2256, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in α-MEM medium supplemented with 10% FBS fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO 2 . After 2-3 subcultures, 0.5 mL of freshly cultured RBL-2H3 cells were inoculated into a 24-well plate at a concentration of 1×10 ⁵ cells/well. The cells were cultured at 37°C and 5% CO ₂ for 24 hours, the supernatant was removed, and then α-MEM medium containing 5% FBS and IgE (0.1-0.7 μg/mL) were added. The cells were incubated for 1 to 8 hours, centrifuged at 15000 g for 3 minutes, the supernatant was removed, and 1 mL of HEPES buffer (140 mM NaCl, 5 mM KCl, 0.6 mM MgCl ₂ , 1.0 mM CaCl ₂ , 5.5 mM glucose, 0.1% bovine serum albumin The cells were washed 2-4 times with 100-400 μL of previously prepared bacterial culture fluid or positive control group ketotifen (5-40 μg/mL)/well for 5-30 minutes, and then treated with 100 μL of antigen (DNP-BAS, 100 μg/mL) at 37°C for 20-40 minutes to induce degranulation through antigen-antibody reaction. 100 μL of HEPES buffer was used to induce degranulation instead of antigen treatment as a negative control. After the antigen treatment, the 24-well plate was placed in an ice water bath, 0.6 mL of ice HEPES buffer was added to terminate the reaction, the supernatant was taken from each well, 20 μL of perchloric acid was added, and after centrifugation at 12000 rpm for 30 minutes, the supernatant was filtered through a 0.45 μm filter membrane. The histamine content in the filtrate was determined by high performance liquid chromatography using the o-phthalaldehyde post-column conversion method.

The histamine content after Lactobacillus crispatus treatment was compared with that of the negative control group, and the histamine release inhibition rate was calculated according to the following formula:

Histamine inhibition rate = (histamine content in negative control filtrate - histamine content in treatment group filtrate) / histamine content in negative control filtrate

The experimental results are shown in Table 16. Compared with ketotifen and L. crispatus LBV88, L. crispatus-120 showed a higher histamine release inhibition rate and had excellent degranulation prevention activity, which can effectively alleviate allergic symptoms caused by excessive histamine secretion.

Table 16 Histamine release inhibition rate of Lactobacillus crispatus

Example 9. Analysis of the effect of Lactobacillus crispatus on inhibiting Th2 cytokines in T cells

Type 2 helper T cell (Th2)-related cytokines (e.g., IL-4 and IL-5) can increase the production of IgE through Th2-related immune responses, thereby promoting chronic allergic reactions. The present invention further uses the EL4 cell line to evaluate the inhibitory effect of L. crispatus-120 on chronic allergic reactions, and the commercial strain L. crispatus LBV88 is used as a control strain.

EL4 cells (ATCC TIB 181, purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured at 37°C and 5% _CO2. Co., Ltd.) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin, and was subcultured 2-3 times before use; L. crispatus-120 and commercial strain L. crispatus LBV88 were cultured in MRS medium, subcultured 2-3 times, and the fermentation culture was centrifuged at 12000g for 5 minutes, the supernatant was discarded, and the precipitate was washed with PBS, and the number of live bacteria was determined by flow cytometry before use.

EL4 cells with good vitality were inoculated into 48-well plates at a concentration of 1×10 ⁵ cells/well and cultured at 37°C, 5% CO ₂ for 12 to 24 hours. PMA (final concentration 10 ng/mL) was then added, followed by 200 μL of the previously prepared Lactobacillus crispatus culture for co-culture. After incubation in a 37°C, 5% CO ₂ incubator for 24 hours, the supernatant was collected and the amount of secreted IL-4 and IL-5 was determined using Mouse IL-4 ELISA kit (PI613, Beyotime) and Mouse IL-5 ELISA kit (PI620, Beyotime).

The results are shown in Figures 1 and 2. Compared with the commercial strain L. crispatus LBV88, the Lactobacillus crispatus strain L. crispatus-120 showed a stronger inhibitory effect on the secretion of IL-4 and IL-5, and can provide therapeutic and preventive effects on allergic reactions by inhibiting the secretion of Th2 type cytokines that mediate allergic reactions.

Example 10. Analysis of the inhibitory effect of Lactobacillus crispatus on IgE

Immunoglobulin E (IgE) is a type of immunoglobulin that is involved in the development of allergic diseases. Generally, the total amount of IgE in serum can be measured as one of the methods for diagnosing allergic diseases. Therefore, the present invention verifies the inhibitory effect of L. crispatus-120 on IgE secretion by using human B cells U266B1, and the commercial strain L. crispatus LBV88 is used as a control strain.

U266B1 cells (ATCC NO.TIB-196, purchased from Nanjing Kebai Biotechnology Co., _Ltd. ) were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100 μg/mL) and streptomycin (100 μg/mL) at 37°C and 5% CO2, and subcultured 2-3 times. U266B1 cells were seeded into 24-well plates at a concentration of 5×10 ⁵ cells/well, and then cultured for 12 to 18 hours before use.

In a 24-well plate inoculated with U266B1 cells, 100 μL of LPS (10 μg/mL) and IL-4 (5 ng/mL) were added to each well for treatment. Then, 300 μL of the previously prepared Lactobacillus crispatus strain solution or PBS was added to each well of the 24-well plate and incubated at 5% CO ₂ and 37° C. After incubation for 24-48 hours, the supernatant was collected and the IgE level was determined using a Human IgE ELISA kit (70-EK175-48, MultiSciences).

The IgE levels after Lactobacillus crispatus treatment were compared with those of the negative control group, and the IgE inhibition was calculated according to the following formula:

IgE inhibition rate = (IgE content in negative control filtrate - IgE content in treatment group filtrate) / IgE content in negative control filtrate.

The experimental results are shown in Figure 3. Compared with the negative control group, both L. crispatus-120 and the commercial strain L. crispatus LBV88 showed the effect of inhibiting IgE secretion, and the inhibitory effect of L. crispatus-120 was stronger. This experiment shows that L. crispatus-120 can provide therapeutic and preventive effects on allergic diseases by inhibiting IgE (the main factor involved in allergic reactions).

Example 11. Effect of L. crispatus-120 on alleviating atopic dermatitis

Based on the results of in vitro screening tests, L. crispatus-120 with the best immunomodulatory effect was selected for animal efficacy studies. Using the atopic dermatitis NC/Nga mouse model, 15 NC/Nga mice were randomly divided into 3 groups, model control group, positive control group and L. crispatus-120 administration group, 5 mice in each group, and the hair of each mouse from both ears and back was removed. Then, 200 μL of 1% DNCB (dinitrochlorobenzene) solution (acetone: olive oil = 1:3) was applied to the depilated part of the mouse once a week for a total of 6 times to induce atopic dermatitis. Since the week before the induction of dermatitis, 200 μL of PBS was gavaged to the mice in the model control group every day; L. crispatus-120 1x10 ⁸ CFU/mouse was gavaged to the mice in the administration group every day; at the same time, 200 μL of dexamethasone (dexamethasone, 60 μg/mL) was applied to the mice in the positive control group. During the experiment, the dermatitis scores of mice in the control group and the L. crispatus-120-administered group were measured weekly, and the scratching time and skin thickness of the mice were measured at 3 and 7 weeks after oral administration of probiotics.

Starting from the third week of oral administration of probiotics, skin conditions were monitored by taking photos every week for 4 weeks. Four indicators of skin dryness, edema, erythema/hemorrhage, and erosion/excoriation were examined. The condition without lesions was scored as 0 points, the mild condition was scored as 1 point, the moderate condition was scored as 2 points, the severe condition was scored as 3 points, and the total score was evaluated. As shown in Figure 4, the dermatitis score of the L. crispatus-120 administration group was significantly reduced compared with the model control group, indicating that L. crispatus-120 has the effect of treating atopic dermatitis.

In order to further verify whether oral administration of L. crispatus-120 can alleviate the itching symptoms of model mice, the scratching time was measured by shooting a 30-minute video of the mouse model 3 weeks after oral administration of probiotics. The results are shown in Figure 5. Compared with the model control group, the scratching time of mice in the L. crispatus-120 oral administration group was significantly reduced, indicating that oral administration of L. crispatus-120 greatly alleviated the itching symptoms of atopic dermatitis.

After oral administration of L. crispatus-120 for 4 weeks, the ear thickness and back skin thickness of mice were measured with a caliper, and the relief of edema symptoms in each group of mice was observed. The experimental results are shown in Figures 6A and 6B. Compared with the model control group, the ear thickness (Figure 6A) and back skin thickness (Figure 6B) of mice in the L. crispatus-120 group and the dexamethasone group were significantly reduced.

Example 12: Evaluation of the therapeutic and preventive effects of L. crispatus-120 on asthma using an ovalbumin (OVA)-induced asthma mouse model

In order to verify the effect of L. crispatus-120 on allergic asthma, an OVA-induced asthma mouse model was used, and histopathological examination was performed, and the expression levels of IL-5 and IL-13 were determined.

5-7 week old Balb/c mice were selected and adapted for 1 week, and then randomly divided into 3 groups, namely normal control group (control group-PBS; no OVA inhalation), OVA asthma model control group (OVA-PBS; inhaled OVA) and probiotic administration group (OVA-120), with 10 mice in each group. From the beginning of the experiment to the 31st day before the mice were killed, 200 μL PBS was gavaged into the normal control group and the asthma-induced control group every day, and 200 μL L. crispatus-120 bacterial solution was gavaged into the administration group every day.

The formal experiment began after the mice adapted for 1 week. The first day of the formal experiment was recorded as day 1. On day 7 and day 21, the mice were sensitized by intraperitoneal injection of 200 μL phosphate buffer (pH 7.4) suspended with 2 mg aluminum hydroxide (ImjectTM Alum Adjuvant, Thermofisher) and 20 μg OVA (ovalbumin, Sigma-aldrich). From day 28 to day 30, the mice were stimulated by inhalation of 1% OVA into the lungs through intranasal drops for a total of 3 times. Pentobarbital treatment was performed 24 hours after the last stimulation (i.e., day 31), and bronchial incision was subsequently performed to collect lung tissue samples.

Infiltration of inflammatory cells consisting of eosinophils, neutrophils and macrophages was observed in the antigen-treated bronchi. In order to verify the effect of L. crispatus-120 on asthma, histopathological examination was performed on each group of lung tissue samples. Pathological results showed that in the OVA asthma model control group (OVA-PBS), many inflammatory cells including eosinophils infiltrated around the bronchioles, and overproliferated epithelial cells and thickened bronchial smooth muscle were also found; while in the L. crispatus-120 administration group (OVA-120), the infiltration of inflammatory cells was significantly reduced, the thickness of bronchial tissue was also reduced, and the epithelial cells were almost undamaged, indicating that L. crispatus-120 has a therapeutic and preventive effect on allergic asthma induced by OVA.

The immune cells from lung tissue samples collected from each group were measured by flow cytometry (FACSAria III, BD). Cell number. Antibodies against several markers (anti-mouse CD45, BioLegend; anti-mouse CD3ε, BD; anti-mouse/human IL-5, BioLegend; anti-mouse IL-13, Invitrogen) were used to stain IL-5 ⁺ CD4 ⁺ T, IL-13 ⁺ CD4 ⁺ T cells. IL-5 ⁺ and IL-13 ⁺ cells were determined by counting cells producing IL-5 or IL-13 in lymphocytes with CD45 and CD3ε as markers.

The experimental results are shown in Figures 7A and 7B. Compared with the normal control group (PBS), the IL-5 and IL-13 levels in the mice of the OVA asthma model control group (OVA-PBS) were significantly increased; compared with the asthma model control group, the total IL-5 and total IL-13 levels in the mice of the L. crispatus-120 administration group (OVA-120) were significantly decreased, indicating that the L. crispatus-120 strain of the present invention can exert its therapeutic and preventive effects on allergic asthma by inhibiting the secretion of IL-5 and IL-13, which are Th2 cytokines that mediate allergic reactions.

Example 13: Evaluation of the therapeutic and preventive effects of L. crispatus-120 on asthma using the HDM-induced asthma model

House dust mites are allergens that are the main cause of extrinsic asthma. To investigate the effect of L. crispatus-120 on allergic asthma, a HDM-induced asthma mouse model was used to evaluate airway hypersensitivity, and the proportion of eosinophils in CD45 ⁺ cells, the proportion of IL-5 ⁺ CD4 ⁺ T cells in CD4 ⁺ T cells, and the proportion of IL-13 ⁺ CD4 ⁺ T cells in CD4 ⁺ T cells were determined.

5-7 week old Balb/c mice were purchased, adapted for 1 week, and randomly divided into 3 groups, including a normal control group (nasal drops of PBS), an asthma-induced control group (nasal drops of HDM-PBS), and a L. crispatus-120 administration group (HDM-120). Six mice were selected from each group for the evaluation of airway hyperresponsiveness (AHR), and 10 mice were selected from each group for the evaluation of immune cells in the lungs.

After 1 week of acclimatization, the normal control group and the asthma model control group were gavaged with PBS daily (day 1-18) throughout the experiment, while the L. crispatus-120 group was gavaged with L. crispatus-120 daily (day 1-18). On day 7, the mice were sensitized by intranasal instillation of 10 μg HDM (House dust mite, Greer) suspended in 50 μL phosphate buffer (pH 7.4). One week after sensitization, 10 μg HDM suspended in 50 μL phosphate buffer was inhaled into the lungs by intranasal instillation for 5 days (day 14-18). The mice were treated with pentobarbital 24 hours after the last stimulation (day 19), and then airway hyperresponsiveness (AHR) was evaluated and bronchial incision was performed to collect lung tissue samples.

Mice anesthetized with pentobarbital were tested with the animal lung function-airway resistance and lung compliance system (FinePointe Resistance and Compliance, DSI-Buxco) was connected to the mouse, and different concentrations of methacholine PBS solution (0, 5, 10, 20 and 40 mg/mL) were administered to the mice. Then, the volume of air passing through the airway was measured to calculate the AHR value.

The experimental results are shown in Figure 8. As the concentration of methacholine increased, the AHR (RL) in the normal control group (CTRL) increased slowly, while the AHR in the model control group (HDM) increased rapidly. Compared with the asthma model control group (HDM), the AHR in the L. crispatus-120 gavage group (HDM+120) was significantly reduced, and when treated with high concentrations of methacholine, the reduction in AHR was significant, indicating that L. crispatus-120 can effectively inhibit the AHR that causes asthma, and thus can be effectively used to treat and prevent allergic asthma.

Immune cells in the lungs were determined using a flow cytometer (FACSAria III, BD). Mouse antibodies (anti-mouse CD45, BioLegend; rat anti-mouse Siglec-F, BD; anti-mouse CD11b, BD; anti-mouse CD3ε, BD; anti-mouse TCRβ, BioLegend; anti-mouse CD4, BioLegend; anti-mouse IL-5, BioLegend; anti-mouse IL-13, Invitrogen) were used to stain eosinophils and IL-5 ⁺ CD4 ⁺ T, IL-13 ⁺ CD4 ⁺ T cells. Eosinophils were determined by counting Siglec-f ⁺ CD11b ⁺ cells in cells expressing the common leukocyte marker CD45, and IL-5 ⁺ CD4 ⁺ T, IL-13 ⁺ CD4 ^{+ T cell numbers were determined by counting cells producing IL-5 or IL-13 in CD4 +} T cells with CD3ε, TCRβ and ^CD4 as markers.

The experimental results are shown in Figures 9A-9C. Compared with the normal control group (PBS), the proportions of immune cells, namely eosinophils, IL-5 ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ T cells in the lung tissue of mice in the asthma model control group (HDM) were significantly increased, while compared with the asthma model control group, the proportions of eosinophils, IL-5 ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ T cells in the L. crispatus-120 gavage group were significantly decreased, indicating that L. crispatus-120 can exert its therapeutic and preventive effects on allergic asthma by inhibiting inflammatory cells, namely eosinophils, IL-5 ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ T cells.

Claims

A Lactobacillus crispatus strain, which contains a 16S rRNA sequence with a nucleotide sequence of SEQ ID NO:1.
The strain according to claim 1, which is isolated from reproductive tract secretions.
The strain of claim 1, which has one or more of the following properties: (i) a survival rate of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% after incubation in simulated gastric fluid for 3 hours; (ii) a survival rate of at least 10%, 20%, 30%, 40%, 50%, 51%, 52%, 53%, 54%, 55% or 56% after incubation in simulated intestinal fluid for 4 hours; (iii) compared to L. crispatus LBV88, it has a higher adhesion ability to Caco-2 cells, VK2/E6E7 cells or both; (iv) compared to L. crispatus LBV88 has higher antibacterial ability against Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Staphylococcus aureus, vaginal Gardnerella resistant, Atopobium, Malassezia furfur or any combination; (v) has significant inhibitory ability against vaginal Gardnerella, Candida albicans or any combination, and restores the number of Lactobacillus to above normal levels, while relieving symptoms such as vulvar edema and excessive secretions, and restoring vaginal mucosal damage; (vi) compared with L. crispatus LBV88, which has a stronger inhibitory effect on antigen-induced histamine release, secretion of Th2 cytokines (IL-4 and/or IL-5) and/or secretion of IgE; (vii) reduces dermatitis scores, scratching time and skin thickness, and significantly alleviates atopic dermatitis; (viii) has a therapeutic and preventive effect on allergic asthma induced by ovalbumin (OVA), and exerts its therapeutic and preventive effect on allergic asthma by inhibiting the secretion of IL-5 and IL-13, which are Th2 cytokines that mediate allergic reactions; and (ix) exerts a therapeutic and preventive effect on allergic asthma in allergic asthma induced by house dust mite (HDM) by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (such as eosinophils, IL-5 + CD4 + T cells and/or IL-13 + CD4 + T cells).
A Lactobacillus crispatus strain, whose preservation number is CGMCC No.19531.
A method for culturing the strain according to any one of claims 1 to 4, comprising culturing the strain in a culture medium.
The method of claim 5, wherein the culture medium is MRS medium.
The method according to claim 5, wherein the culturing is carried out under anaerobic conditions.
The method of claim 5, wherein the culturing is performed at 37°C.
A derivative of the strain according to any one of claims 1 to 4.
The derivative according to claim 9, wherein the derivative is a culture, a lysate, an extract, an inactivated product or a combination thereof.
A culture medium comprising the strain according to any one of claims 1 to 4 or the strain according to claim 9 or 10 Derivatives of the above.
A composition comprising an effective amount of a first component, wherein the first component comprises the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, or the culture medium according to claim 11.
The composition of claim 12, wherein the composition is a food composition, a health food composition, a pharmaceutical composition, a food composition for special medical purposes, a cosmetic composition, a medical device composition or a feed composition.
The composition of claim 12, wherein the composition is in the form of a pill, tablet, lozenge, lyophilized powder, granules, capsule, aqueous solution, alcoholic solution, oily solution, syrup, emulsion, suspension, suppository, solution for injection or infusion, ointment, gel, tincture, cream, patch, lotion, spray, aerosol, powder spray, effervescent tablet, transdermal therapeutic system, microcapsule, implant or stick.
The composition of claim 12, wherein the composition is formulated for ocular, otic, intranasal, sublingual, oral, transdermal, topical, nasal, rectal or parenteral administration.
The composition of claim 12, wherein the composition further comprises a second component.
The composition of claim 16, wherein the second component comprises a probiotic, a postbiotic, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an osteoporosis therapeutic agent, a psychiatric field-related therapeutic agent, a developmental-related therapeutic agent, or a combination thereof.
The composition of claim 16, wherein the weight ratio of the first component to the second component is 1:99 to 99:1.
The composition of claim 16, wherein the first component is applied before, after or simultaneously with the second component.
Use of the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, the culture medium according to claim 11 or the composition according to any one of claims 12 to 19 in the preparation of a medicament for antagonizing pathogens.
Use of the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, the culture medium according to claim 11 or the composition according to any one of claims 12 to 19 in the preparation of a medicament for preventing and/or treating a disease or condition associated with a pathogen.
The use according to claim 20 or 21, wherein the pathogen is selected from the group consisting of bacteria, fungi, viruses, spirochetes, mycoplasmas, rickettsiae, chlamydiae and parasites.
The use according to claim 22, wherein the pathogen is selected from the group consisting of: Mycobacterium, Salmonella, Escherichia coli, Chlamydia The most common spores in the body are Chlamydia, Staphylococcus, Bacillus, Psudomonas, Candida, Atopobium, Gardnerella and Pityrosporum.
The use according to claim 22, wherein the bacteria include Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi, Atopobium vaginalis, Gardnerella vaginalis or a combination thereof.
The use according to claim 22, wherein the fungus comprises Candida albicans, Malassezia furfur or a combination thereof.
The use according to claim 22, wherein the parasite is Trichomonas.
The use according to claim 21, wherein the disease or condition associated with a pathogen is selected from the group consisting of female reproductive tract infection and reproductive tract flora disorder.
The use according to claim 21, wherein the disease or condition associated with the pathogen is a skin disease associated with Malassezia infection.
Use of the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, the culture medium according to claim 11 or the composition according to any one of claims 12 to 19 in the preparation of a medicament for preventing and/or treating a disease or condition associated with immunoregulation.
The use according to claim 29, wherein the disease or condition associated with immunomodulation is cancer, allergic disease or autoimmune disease.
The use of claim 30, wherein the cancer is selected from the group consisting of: prostate cancer, gastro-esophageal cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia, lymphoma and myeloma.
The use according to claim 30, wherein the allergic disease is selected from the group consisting of allergic rhinitis, allergic asthma, atopic dermatitis, allergic keratoconjunctivitis, urticaria, food allergy, drug allergy, dust mite allergy and pollen allergy.
The use as claimed in claim 30, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, rheumatic fever, lupus, systemic sclerosis, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, Guillain-Barré syndrome, myasthenia gravis, dermatomyositis, polymyositis, multiple sclerosis, autoimmune encephalomyelitis, polyarteritis nodosa, Hashimoto's thyroiditis, temporal arteritis, juvenile diabetes, alopecia areata, pemphigus, aphthous stomatitis, autoimmune hemolytic anemia, Wegener's granulomatosis, Sjögren's syndrome, Addison's disease, Crohn's disease, Behcet's disease, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, Pharyngitis, tonsillitis, bronchitis, pneumonia, gastric ulcer, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, periarthritis of the shoulder, tendinitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, sepsis, vasculitis and bursitis.
Use of the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, the culture medium according to claim 11 or the composition according to any one of claims 12 to 19 in the preparation of a medicament for preventing and/or treating a disease or condition associated with osteoporosis.
The use of claim 34, wherein the disease or condition associated with osteoporosis is selected from the group consisting of juvenile osteoporosis, menopausal osteoporosis, postmenopausal osteoporosis, post-traumatic osteoporosis, and osteoporosis caused by aging, corticosteroid treatment and inactivity.
Use of the strain according to any one of claims 1 to 4, the derivative according to claim 9 or 10, the culture medium according to claim 11 or the composition according to any one of claims 12 to 19 in the preparation of a medicament for preventing and/or treating diseases or conditions associated with iron deficiency anemia, menopausal syndrome or central nervous system diseases.