CN116004465A - Lactobacillus gasseri and application thereof - Google Patents

Abstract

The present invention relates to isolated lactobacillus gasseri-44 (l.gaseri-44) strains, compositions comprising the same and uses thereof.

Description

Lactobacillus gasseri and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to an isolated lactobacillus gasseri strain, a composition comprising the same and application thereof.

Background

The female vaginal system has three lines of defense: anatomical structures, vaginal mucosa and micro-ecological flora, one of the most critical lines of defense is the micro-ecological flora. More than 200 microorganisms are present in the vagina of healthy women, of which 95% are lactic acid bacteria, several Lactobacillus bacteria including Lactobacillus crispatus (Lactobacillus crispatus), lactobacillus gasseri (Lactobacillus gasseri), lactobacillus jensenii (Lactobacillus jensenii) and Lactobacillus inerti (Lactobacillus iners) are dominant. The normal flora in the vagina, the host and the environment keep the coordination and dynamic balance, namely the microecological balance of the vagina. Once the lactic acid bacteria is reduced, the vaginal microecological balance is broken, and the number of pathogenic bacteria is increased or exogenous pathogens invade, vaginal flora is abnormal and vaginal pH is abnormal, possibly resulting in inflammation. In bacterial vaginitis, the balance of intravaginal microecology shifts towards anaerobic bacterial colonisation, and in particular gardnerella vaginalis (Gardnerella vaginalis) and atopoella vaginalis (Atopobium vaginae) are more common (Srinivasan and Fredricks, 2008). If the micro-ecology in the vagina is in an unbalanced state for a long time, vaginitis can repeatedly occur. Numerous documents suggest that vaginitis can lead to a variety of complications and sequelae. For example, vaginitis can lead to pelvic and cervicitis, and presents a more serious threat to the health of pregnant women, such as increased risk of abortion 3 months before pregnancy, premature rupture of membranes, chorioamniosis, and premature delivery. In addition, changes in the vaginal microenvironment have also been associated with urinary tract infections.

Currently, the first-line drug regimen recommended by clinical guidelines of various countries for the treatment of vaginitis is antibiotic drug therapy represented by metronidazole or clindamycin. However, antibiotic drug therapy may cause damage to the beneficial flora and may be more likely to be resistant to the vaginal flora when used for a long period of time.

Probiotics play an important role in the treatment of female colpitis by inhibiting the growth of pathogenic microorganisms by producing lactic acid and various antibacterial compounds and stimulating the immune system by competing for adhesion, thereby achieving the effect of treating colpitis (Kurt Selle and Todd R.Klaenhammer,2013;Craig R.Cohen,2020;Charlotte van der Veer,2019). Exogenous supplementing of probiotics, inhibition of the reproductive growth of pathogenic bacteria and the like by probiotics becomes a new therapy for preventing or treating vaginitis. In addition, by supplementation with probiotics, it is also possible to prevent or treat pathogen-related diseases or disorders, immunoregulatory-related diseases or disorders (Wanil Kim et al, 2020;Peter van Baarlen et al, 2009;Sandra Voltan et al, 2008; F.Blachet al, 2021;Hirotaka Kawanabe-Matsuda et al, 2022;Kyosuke Kobayashi et al, 2019; nuno R Nen et al, 2019;Paola Roggero et al, 2020;Luisa Cervantes-Barragan, 2017), osteoporosis-related diseases or disorders (Claes Olsson et al, 2014; xin Xu et al, 2017; A.G.Nilsson et al, 2018;Abdul Malik Tyagi,2018) or iron deficiency anemia (Susan C.Vondelheid 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), climacteric syndrome or diseases or disorders of the neural system by means of probiotics.

Disclosure of Invention

The present invention provides a novel strain of lactobacillus gasseri having improved properties in antagonizing pathogenic bacteria, tolerating digestive fluids, intestinal colonisation etc.

In one aspect, the invention provides a Lactobacillus gasseri (Lactobacillus gasseri) strain comprising a 16S rRNA sequence with the nucleotide sequence SEQ ID NO. 1. In some embodiments, the strain provided herein is isolated from genital secretions.

In some embodiments, the strains provided herein have one or more of the following properties: (i) Survival rates of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% after 3 hours incubation in simulated gastric fluid; (ii) Survival after 4 hours incubation in simulated intestinal fluid is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or 96%; (iii) It has a higher adhesion capacity to Caco-2 cells, VK2/E6E7 cells, or both, than L.gaseri LBV 150N; (iv) Compared to l.gasser LBV150N, it has a higher bacteriostatic ability against escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atopoella vaginalis, gardnerella vaginalis, malassezia furfur, or any combination thereof; (v) Has remarkable inhibition capability to gardnerella vaginalis, candida albicans or any combination, and can restore the quantity of lactobacillus to above the normal level, simultaneously relieve symptoms such as vulvar edema, multiple secretions and the like, and restore vaginal mucosa injury; (vi) It has a stronger inhibitory effect on antigen-induced histamine release, secretion of Th2 type cytokines (IL-4 and/or IL-5) and/or secretion of IgE than L.gasser LBV 150N; (vii) Reducing dermatitis score, scratching time and skin thickness, significantly reducing atopic dermatitis; (viii) Has therapeutic and prophylactic effects on allergic asthma induced by Ovalbumin (OVA), and exerts its therapeutic and prophylactic effects on allergic asthma by inhibiting secretion of IL-5 and IL-13, which are Th2 type cytokines mediating allergic reactions; and (ix) by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (e.g., eosinophils) in House Dust Mite (HDM) -induced allergic asthma Granulocytes, IL-5 ⁺ CD4 ⁺ T cells and/or IL-13 ⁺ CD4 ⁺ T cells) to exert therapeutic and prophylactic effects on allergic asthma.

In another aspect, the invention provides a lactobacillus gasseri (Lactobacillus gasseri) strain with a collection number of CGMCC No.19529.

In another aspect, the invention provides a method of culturing a strain provided by the invention, comprising culturing the strain in a medium. In some embodiments, the medium is MRS medium. In some embodiments, the culturing is performed under anaerobic conditions. In some embodiments, the culturing is performed at 37 ℃.

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

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

In another aspect, the invention provides a composition comprising an effective amount of a first component, wherein the first component comprises a strain provided by the invention, a derivative thereof, or a medium comprising the same. In some embodiments, the compositions provided herein are food compositions, health food compositions, pharmaceutical compositions, special medical use food compositions, cosmetic compositions, medical device compositions, or feed compositions.

In some embodiments, the compositions provided herein are in the form of a pill, tablet, lozenge, lyophilized powder, granule, 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.

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 compositions provided herein further comprise a second component. In some embodiments, the second component comprises a probiotic, a metagen, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an osteoporosis therapeutic agent, a mental area associated therapeutic agent, a developmental associated therapeutic agent, or a combination thereof. In some embodiments, the weight ratio of the first component to the second component is from 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 invention provides the use of a strain provided by the invention, a derivative thereof, a medium comprising the same or a composition comprising the same in the manufacture of a medicament for antagonizing a pathogen.

In another aspect, the invention provides the use of a strain, derivative thereof, a culture medium comprising the same or a composition comprising the same as provided herein for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with a pathogen.

In some embodiments, the pathogen is selected from the group consisting of: bacteria, fungi, viruses, spirochetes, mycoplasma, rickettsia, chlamydia, and parasites.

In some embodiments, the pathogen is selected from the group consisting of: mycobacterium (Mycobacterium), salmonella (Salmonella), escherichia coli (E.coli), chlamydia (Chlamydia), staphylococcus (Staphylococcus), bacillus (Bacillus), pseudomonas (Pseudomonas), candida (Candida), acetobacter (Atopobium), gardnerella (Gardnerella) and Malaromyces (Pictyrosporum).

In some embodiments, the bacteria include escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atopoella vaginalis, gardnerella vaginalis resistant bacteria, or a combination thereof.

In some embodiments, the fungus comprises candida albicans, malassezia furfur, or a combination thereof.

In some embodiments, the parasite is a trichomonas.

In some embodiments, the pathogen-associated disease or disorder is selected from the group consisting of: female genital tract infections and genital tract flora disorders.

In some embodiments, the pathogen-associated disease or disorder is a malassezia infection-associated skin disease.

In another aspect, the invention provides the use of a strain, derivative thereof, a culture medium comprising the same or a composition comprising the same as provided herein for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with immunomodulation.

In some embodiments, the disease or disorder associated with immune modulation is cancer, allergic disease, or autoimmune disease.

In some embodiments, the cancer is selected from the group consisting of: prostate cancer, stomach-esophagus cancer, lung cancer, liver cancer, pancreas cancer, breast cancer, bronchus 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, stomach 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 scleroderma, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, guillain-Barre 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, wechs granulomatosis, sjogren's syndrome, addison's disease, crohn's disease, white plug disease, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, sphagitis, tonsillitis, bronchitis, pneumonia, gastric ulcers, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, scapulohumeral periarthritis, tendinitis, tenositis, hepatitis, cystitis, nephritis, sepsis, vasculitis, and bursitis.

In another aspect, the invention provides the use of a strain, derivative thereof, a culture medium comprising the same or a composition comprising the same as provided by the invention for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with osteoporosis.

In some embodiments, the osteoporosis related disease or disorder is selected from the group consisting of: juvenile osteoporosis, menopausal osteoporosis, postmenopausal osteoporosis, posttraumatic osteoporosis, and osteoporosis due to aging, corticosteroid therapy, and inactivity.

In another aspect, the invention provides the use of a strain, derivative thereof, medium comprising the same or composition comprising the same as provided herein in the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with iron deficiency anemia, climacteric syndrome or a disease of the central nervous system.

Drawings

FIG. 1 shows that L.gaseri-44 and commercial strain L.gaseri LBV150N significantly inhibited IL-4 secretion compared to the negative control group.

FIG. 2 shows that L.gaseri-44 and commercial strain L.gaseri LBV150N significantly inhibited IL-5 secretion compared to the negative control group.

FIG. 3 shows that L.gaseri-44 and commercial strain L.gaseri LBV150N significantly inhibited IgE secretion compared to the negative control group.

Figure 4 shows that the dermatitis score was significantly reduced in the mice of the l.gaseri-44 dosing group compared to the model control group.

Figure 5 shows that the scratch time was significantly reduced in the mice of the l.gaseri-44 dosing group compared to the model control group.

Figures 6A and 6B show that both ear thickness (figure 6A) and back skin thickness (figure 6B) were significantly reduced in the l.gaseri-44 dosed and dexamethasone mice as compared to the model control group.

Figures 7A and 7B show that both total IL-5 (figure 7A) and total IL-13 (figure 7B) levels were significantly reduced in the mice of the l.gaseri-44 dosing group compared to the model control group.

Figure 8 shows that l.gaseri-44 administration (hdm+44) effectively inhibited airway hyperresponsiveness leading to asthma compared to the asthma model control group (HDM).

FIGS. 9A-9C show that L.gaseri-44 administration (HDM+44) effectively inhibited eosinophils (Eos), IL-5, compared to the asthma model control group (HDM) ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ T cells.

Detailed Description

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

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, dictionary of microbiology and molecular biology (Dictionary of Microbiology and Molecular Biology), 20 th edition (John Willi parent (John Wiley and Sons), new York, 1994), and Hale and Marham, biological dictionary of Halbach (the Harper Collins Dictionary of Biology), permanent Harper Press, new York, 1991, and the like, provide the usual meaning of many terms used herein as general dictionary.

Definition of the definition

As used herein, the articles "a," "an," "the," and "said" are used to refer to one or to multiple (i.e., to at least one) of the grammatical object of the article.

Unless specified otherwise or apparent from context, the term "about" as used herein should be understood to be within normal tolerances in the art, for example, within 2 standard deviations of the mean. "about" may be understood to be 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 gasseri strain

The invention provides an isolated lactobacillus gasseri comprising a 16S rRNA sequence (SEQ ID NO: 1) having the nucleotide sequence SEQ ID NO: 1:

TTAGACGGCTGACTCCTATAAAGGTTATCCCACCGGCTTTGGGTGTTACAGACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAACGGCTTTCAGAGATCCGCTTGCCTTCGCAGGTTCGCTTCTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGATGGCAACTAATGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTCAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGTTTGCACTGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTTCAACAGTTTCTGATGCAATTCTCCGGTTGAGCCGAAGGCTTTCACATCAGACTTATTGAACCGCCTGCACTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTAAGTAATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCTTTCTTCACTACCAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCAGGTCCATCCAAGAGTGATAGCAGAACCATCTTTTAAACTCTAGACATGCGTCTAGTGTTGTTATCCGGTATTAGCATCTGTTTCCAGGTGTTATCCCAGTCTCTTGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTCGCTTGTATCTAGTTTCATTTGGTGCAAGCACCAAATTCATCTAGGCAAGCTCGCTCGACTGCATGTATAG。

the invention provides an isolated lactobacillus which is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) for a year 03 and a month 30 of 2020: beijing, chaoyang area, north Chenxi way No.1, no. 3, post code: 100101, accession number: CGMCC No.19529. The isolated Lactobacillus was designated Lactobacillus gasseri-44 (L.gaseri-44), and the taxonomic designation was Lactobacillus gasseri (Lactobacillus gasseri).

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, the mutant may be a microorganism produced by genetic modification of the 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 that is produced in response to a particular cell culture condition.

As used herein, "progeny" refers to any microorganism produced by propagation or multiplication of a parent strain or mutant, variant thereof, which itself may be identified as the same or substantially the same strain as the parent strain. It will be appreciated that, in view of the asexual propagation process, the progeny strain is almost identical in gene to the parent strain. Thus, in one embodiment, the progeny strain is identical in gene to the parent strain and can be considered a "clone" of the parent strain. In another embodiment, the progeny strain is substantially identical in gene to the parent strain.

The mutant, variant or progeny has at least 90%, 95%, 98%, 99%, 99.5% or 99.9% sequence identity in the bacterial full-length genome as compared to its parent strain. In addition, the mutant, variant or progeny will retain the same phenotype as the parent strain, e.g., the mutant, variant or progeny may exhibit the same or equivalent level of ability to antagonize pathogenic bacteria, tolerate digestive fluids, colonise by the gut, etc., as the parent strain.

"percent (%) sequence identity" in relation to a nucleotide sequence (or amino acid sequence) refers to the percentage of nucleotide (or amino acid) residues in a candidate sequence that are identical to the nucleotide (or amino acid) residues in a reference sequence after aligning the candidate sequence to 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 a residue. Alignment for the purpose of determining the percentage of nucleotide (or amino acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (see, e.g., the website of the national center for biotechnology information (U.S. national Center for Biotechnology Information, NCBI), see also Altschul s.f. et al, journal of molecular biology (j. Mol. Biol.), 215:403-410 (1990), stephen f. et al, nucleic Acids research (Nucleic Acids res.), 25:3389-3402 (1997)), clustalW2 (see, e.g., the website of the european institute of biological information (European Bioinformatics Institute), see also Higgins d.g. et al, enzymology method (Methods in Enzymology), 266:383-402 (1996), larkin m.a. et al, bioinformatics (Oxford, england) 23 (2947)), and software implementation (Megalign (DNASTAR)) or (2007). One of ordinary skill in the art may use default parameters provided by the tool, or may customize parameters suitable for alignment, such as by selecting a suitable algorithm.

[ PREPARATION METHOD ]

The lactobacillus gasseri provided by the invention is isolated.

As used herein, "isolated" refers to a substance that has been altered from a natural state by manual means. If an "isolated" composition or substance is present in nature, 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 animal is not "isolated," but is "isolated" if the strain is sufficiently isolated from coexisting materials in its natural state to thereby exist in a substantially pure state.

The lactobacillus strain of the present invention may be isolated from secretions of the genital tract, in particular female genital tract. As used herein, the female "genital tract" includes the vagina, uterus, cervix, ovary, and the like. In a preferred embodiment, the lactobacillus strain of the invention is isolated from vaginal and/or cervical secretions.

The lactobacillus strains of the present invention may be isolated and purified in a manner conventional in the art, and the culture after the isolation and purification step is substantially free of contaminants other than the lactobacillus strains of the present invention, including microbial contaminants and unwanted chemical contaminants.

In certain embodiments, the secretion at 1/3 of the vaginal side wall of healthy subjects is collected using a cotton swab, placed in a sterile tube, the bacterial suspension after washing the swab with PBS is used as a mother liquor, further diluted to different concentrations with PBS, and coated on freshly prepared Rogosa SL solid medium, respectively, and anaerobically cultured. Single colonies of different forms were then individually picked with an inoculating loop and streaked onto freshly prepared MRS solid medium for anaerobic culture to obtain purified single colonies.

[ PREPARATION METHOD ]

The lactobacillus strains of the present invention may be identified by methods conventional in the art, including but not limited to classical morphological feature tests, physiological biochemical property tests and molecular biological tests. The morphology, staining, culture characteristics, colony characteristics and the like of bacteria are preliminary basis for identifying bacteria, and biochemical reactions of bacteria can be used for distinguishing and identifying the types of bacteria.

In certain embodiments, the lactobacillus strains of the present invention are identified by the following method: observing the colony morphology in the culture medium after culturing, wherein the colony is circular; taking a pure culture smear of the strain for gram staining, and showing gram positive characteristics; microscopic examination shows that the strain is in a short rod shape and can be connected into a long chain.

Based on the above-described staining and morphological characteristics, the strain was preliminarily determined to belong to Lactobacillus by using classical taxonomies, for example, by referring to the relevant description in "berjie systems bacteriology handbook (Bergey's Manual of Systematic Bacteriology)" (Williams and Wilkins company (Williams & Wilkins co.), 1984).

Further, the strain may be identified by a conventional 16S rRNA gene sequence detection method. In certain embodiments, the 16S rRNA gene sequence detection method employed is as follows:

(1) Performing PCR amplification;

(2) Performing gel electrophoresis on the PCR product to determine a 16S rRNA gene segment;

(3) Taking a PCR sample for 16S rRNA sequencing;

(4) BLAST sequence similarity comparison analysis is carried out on the sequences obtained by sequencing and data in NCBI database, and the strain species (species) can be judged when the highest homology score is more than 97%.

Based on the 16S rRNA gene sequence detection result, it was determined that the strain of the present invention was Lactobacillus gasseri.

In certain embodiments, a lactobacillus strain as claimed herein 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 set forth in SEQ ID No. 1, while maintaining the morphological and functional characteristics of lactobacillus gasseri-44 (l.gaseri-44).

[ Property ]

The lactobacillus strain provided by the invention not only can effectively antagonize various pathogenic bacteria (including but not limited to escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atopoella vaginalis, gardnerella vaginalis, candida albicans and malassezia furfur), but also has good gastric acid and bile salt resistant digestive juice resistant capability, is beneficial to survival in gastrointestinal tract environment, and is particularly suitable for being prepared into orally administered products. The lactobacillus strain of the present invention also has improved colonisation properties (e.g. colonisation in the intestine and vagina), is capable of exerting its probiotic effect for a long time, and is a probiotic strain with development potential. The lactobacillus strain of the present invention also has a remarkable therapeutic and/or prophylactic effect on allergic reactions.

The lactobacillus strain of the present invention has one or more of the following beneficial properties:

acid resistance: after 3 hours of incubation in simulated gastric fluid, the lactobacillus strain of the present invention has a viability of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. Any suitable acid tolerance test method may be used to determine the acid tolerance of the lactobacillus strains of the invention. In one embodiment, the acid tolerance of the lactobacillus strains of the present invention is determined in vitro simulated gastric fluid. In one embodiment, the acid tolerance test comprises the re-suspension of the bacterial cells to be tested with simulated gastric fluid (e.g. pH 3.0) and normal physiological saline, respectively, after incubation at 37 ℃ for 3 hours, gradient dilution and plating counting. The acid tolerance can be calculated using the following formula: acid tolerance (%) = (Log 10 CFU/mL in simulated gastric fluid)/(Log 10 CFU/mL in physiological saline) ×100. In one embodiment, the simulated gastric fluid is formulated by: pepsin (Sigma, P7000-25G, 630U/mg) 96.84mg powder was dissolved in 32.3mL of physiological saline at pH 3.0 to give a final concentration of 3g/L; it was filtered through a 0.22 μm sterile filter and ready to use.

Bile tolerance: after 4 hours of incubation in simulated intestinal fluid, the lactobacillus strain of the invention has a viability of at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or 96%. Any suitable bile tolerance test method may be used to determine bile tolerance of the lactobacillus strains of the invention. In one embodiment, bile tolerance of the lactobacillus strains of the invention is determined in vitro simulated intestinal fluid. In one embodiment, the bile tolerance test comprises the re-suspension of the bacteria to be tested with simulated intestinal fluid (e.g. pH 8.0) and normal physiological saline, respectively, after incubation for 4 hours at 37 ℃, gradient dilution and plating counting. Bile tolerance can be calculated using the following formula: bile tolerance (%) = (Log 10 CFU/mL in simulated intestinal fluid)/(Log 10 CFU/mL in physiological saline) ×100. In one embodiment, the simulated intestinal fluid is formulated by: 0.5mL of 100 Xtrypsin and bile salt mother liquor are respectively added into 49mL of physiological saline with pH of 8.0, and the final concentration is 1g/L of trypsin and 0.3% of bile salt (namely 3 mg/mL); it was filtered through a 0.22 μm sterile filter and ready to use.

Colonisation properties: the lactobacillus strain of the present invention has a higher colonisation ability to Caco-2 cells, VK2/E6E7 cells or both than the commercial lactobacillus gasseri strain l.gaseri LBV 150N. Any suitable adhesion capability test method may be used to determine the colonisation performance of the lactobacillus strains of the present invention. In one embodiment, a suitable concentration of bacterial fluid (e.g., about 2.5X10 ⁸ CFU/mL, about 5.0X10 ⁷ CFU/mL or about 5.0X10 ⁶ CFU/mL) was added to the cells to be adhered, and left to adhere for 2 hours at 37 ℃ with the target multiplicity of infection (Multiplicity of Infection, MOI, bacteria: cells) was 10:1. After the incubation and adhesion are finished, the cells are washed and lysed, and the cells and bacteria which are fully shed are obtained and counted by plating. The adhesion rate can be calculated using the following formula: adhesion% = adhered bacterial load CFU/inoculated bacterial load CFU x 100%. Experiments can be performed with appropriate cell lines selected based on the target site of colonization. In one embodiment, the colonisation performance test uses the human intestinal derived cell line Caco-2 cells. In one embodiment, the colonisation performance test uses vaginal epithelial cells VK2/E6E7 cells.

Antibacterial performance: compared to the commercial lactobacillus gasseri strain l.gaseri LBV150N, the lactobacillus strain of the present invention has a higher bacteriostatic ability against escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atoposis vaginalis, gardnerella vaginalis, malassezia furfur, or any combination thereof. Any suitable test method may be used to determine the properties of the lactobacillus strains of the invention to antagonize pathogenic bacteria, such as diffusion methods for qualitative assays and dilution methods for quantitative assays. Specific methods suitable for use include, but are not limited to, double-layered plate methods, tortilla methods (e.g., inverted tortilla methods and banded tortilla methods), sheet diffusion methods, and the like.

In one embodiment, the bilayer plate method comprises the following specific steps: pathogenic bacteria (such as Staphylococcus aureus) are taken in a solid medium cooled to about 45 ℃, and poured onto the probiotic MRS agar to be tested which is cultured for 20-24 hours. After solidification, the plate is placed under atmospheric conditions at 37 ℃ for 1 day until a zone of inhibition appears. Parallel test plates were set up for each probiotic.

In another embodiment, the bilayer plate method comprises the following specific steps: the probiotic bacteria liquid is spotted (spot) on MRS solid culture medium plate, and anaerobic culture is carried out for 24-48 hours at 37 ℃. Seed solution of pathogenic bacteria (such as Malassezia furfur) is inoculated into mYPG liquid culture medium prepared under aerobic condition, and cultured at 37deg.C for 24-48 hr. Preparing a mYPG culture medium, sterilizing for 20 minutes at 115 ℃, cooling to about 40 ℃, mixing 2.5mL of the culture medium with 500 mu L of a standby pathogenic bacteria culture solution, respectively pouring the mixed solution onto MRS solid culture medium of the sample-application probiotics, and standing for solidification of the culture medium. The coagulated medium plates were incubated at 37℃for 24-48 hours with oxygen. The antibacterial activity of the probiotics is determined by measuring the diameter of the inhibition zone. Parallel test plates were set up for each probiotic.

In one embodiment, the inverted cake method comprises the following specific steps: pathogenic bacteria (such as Escherichia coli, pseudomonas aeruginosa or Salmonella typhi) are coated on NA or Columbia blood agar medium and 5% sheep blood plate, after the bacteria are fully absorbed, probiotic bacteria cake is extracted and inverted on pathogenic bacteria plate, and the pathogenic bacteria is cultured for 1 day under 37 ℃ atmosphere until bacteria inhibition zone appears. Parallel test plates were set up for each probiotic.

In one embodiment, the specific steps of the stripy cake method are as follows: the probiotic inoculum was dipped with cotton swab, then spread into a 2cm wide strip on MRS solid medium plate, and anaerobic cultured for 24 hours at 37 ℃. The plate was removed and melted YM solid medium was poured onto the surface of the plate to solidify the thin layer. Uniformly coating pathogenic bacteria liquid on the surface of YM solid culture medium by using cotton stick, after drying, firstly placing the culture medium at 4 ℃ for 4 hours, then at 37 ℃ for 24 hours, and observing the inhibitory effect of probiotics on pathogenic bacteria. Parallel test plates were set up for each probiotic.

The pathogenic bacteria antagonistic properties of the lactobacillus strains 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 bacterial fluid, and sampling for pathogen and probiotic (e.g., lactobacillus) colony counts, respectively, before and after administration; the invention can also be used for observing the vulva before and after administration, recording the conditions of vulva red swelling, vaginal secretion, and the like, and performing vaginal lavage liquid smear (PAS staining), thereby confirming the functions of the lactobacillus of the invention for regulating vaginal flora, inhibiting pathogenic bacteria growth and colonisation. In one embodiment, a bacterial vaginitis model is constructed using gardnerella vaginalis. In one embodiment, candida albicans is used to construct a model of candidal vaginitis.

Improving allergic reaction: compared to the commercial lactobacillus gasseri strain l.gaseri LBV150N, the lactobacillus strain of the invention has a stronger inhibitory effect on antigen-induced histamine release, secretion of Th 2-type cytokines (IL-4 and/or IL-5) and/or secretion of IgE, significantly improving allergic reactions. The effects of the lactobacillus strains of the present invention on allergic reactions may be evaluated using any suitable method, including, but not limited to, inhibition of histamine secretion, inhibition of type 2 helper T cell (Th 2) related cytokines such as IL-4 and IL-5 secretion, and/or inhibition of IgE secretion.

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

In one embodiment, each strain is assayed for its ability to inhibit secretion of Th 2-associated cytokines (e.g., IL-4 and IL-5) by: the amounts of secreted IL-4 and IL-5 were determined separately by kit using the EL4 cell line.

In one embodiment, each strain's ability to inhibit IgE secretion is determined by the following method: igE levels were determined by kit using human B cell U266B 1. IgE inhibition can be calculated according to the following formula: igE inhibition= (IgE content in negative control filtrate-IgE content in treated group filtrate)/IgE content in negative control filtrate.

Reduction of atopic dermatitis: the lactobacillus strain of the present invention significantly alleviates the symptoms of atopic dermatitis, such as dryness, edema, erythema/hemorrhage (erythema/hemorrhage), erosion/abscission (erosion/extraction) and itching of the skin. Any suitable experimental model may be used to determine the effect of the lactobacillus strains of the present invention on atopic dermatitis. In one embodiment, an atopic dermatitis NC/Nga mouse model is used. The impact of the lactobacillus strains of the present invention on atopic dermatitis can be evaluated using any suitable criteria, including but not limited to, dryness, oedema, erythema/bleeding (erythema/hemorrage), erosion/exfoliation (error/extraction) and itching of the skin. The foregoing indicators may be obtained using any suitable means, including but not limited to dermatitis scoring, scratching time measurement, ear/back skin thickness measurement, and the like.

Improving allergic asthma: the lactobacillus strain of the present invention has therapeutic and prophylactic effects on allergic asthma induced by Ovalbumin (OVA), and exerts its therapeutic and prophylactic effects on allergic asthma by inhibiting secretion of IL-5 and IL-13, which are Th2 type cytokines mediating allergic reactions; by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (e.g., eosinophils, IL-5) in House Dust Mite (HDM) -induced allergic asthma ⁺ CD4 ⁺ T cells and/or IL-13 ⁺ CD4 ⁺ T cells) to exert therapeutic and prophylactic effects on allergic asthma. Any suitable experimental model may be used to determine the effect of the lactobacillus strains of the present invention on allergic asthma. In one embodiment, OVA mutagenesis is usedGuided asthma model. In one embodiment, an HDM-induced asthma model is used. Any suitable index may be used to evaluate the effect of the lactobacillus strains of the invention on allergic asthma. In one embodiment, a histopathological examination is performed to observe inflammatory cell infiltration, bronchial tissue (bronchial smooth muscle), epithelial cells, and the like. In one embodiment, IL-5 is determined by counting IL-5 or IL-13 producing cells in lymphocytes having CD45 and CD3 epsilon as markers ⁺ And IL-13 ⁺ And (3) cells. In one embodiment, airway Hyperresponsiveness (AHR) is detected. In one embodiment, the cell expressing the common leukocyte marker CD45 is identified by Siglec-f ⁺ CD11b ⁺ Eosinophils were determined by cell count and by counting CD4 with CD3 epsilon, TCR beta and CD4 as markers ⁺ Counting IL-5 or IL-13 producing cells in T cells to determine IL-5 ⁺ CD4 ⁺ T and IL-13 ⁺ CD4 ⁺ T cell number.

[ derivative ]

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

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

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

As used herein, "lysate" refers to the product of a strain obtained by treatment with an enzyme, sonication, homogenization, or the like.

As used herein, "extract" refers to a product obtained by subjecting a strain to a solvent extraction or the like.

As used herein, "inactivated product" refers to a product obtained by subjecting a strain to heat, pressure, or treatment with a drug or the like.

[ cultivation method ]

The invention also provides a method of culturing the lactobacillus strain of the invention comprising culturing said strain in a medium. The lactobacillus strains of the present invention can be grown in any suitable medium for lactobacillus, without losing their genetic properties, nor their characterization and functional properties during growth. According to the growth requirement of bacteria, the culture medium needs basic nutrient components such as carbon source, nitrogen source, growth factors, inorganic salt, water and the like, and is prepared according to a certain formula and preparation method. In particular, the lactobacillus strains of the present invention may be grown in a medium containing an assimilable organic carbon source, an assimilable nitrogen source, suitable salts and trace metals. Preferred media suitable for the lactobacillus strains of the present invention include MRS media. Exemplary ingredients of the MRS medium include peptone, beef powder, yeast powder, glucose, tween 80, dipotassium hydrogen phosphate, sodium acetate, tri-ammonium citrate, magnesium sulfate, manganese sulfate, agar powder, distilled water, and the like.

The lactobacillus strain of the present invention may be cultured by a conventional culture means under any conventional culture conditions suitable for lactobacillus. Specifically, the lactobacillus strain of the present invention may be cultured by broth fermentation, agar surface culture, or the like. The temperature of the medium may be any temperature suitable for the growth of lactobacillus, preferably at a temperature of about 35-40 ℃, more preferably at a temperature of about 37 ℃. The lactobacillus strain of the present invention may be cultivated under anaerobic or microaerophilic conditions, preferably under anaerobic conditions.

In one embodiment, purified single colonies are obtained on a solid medium and then picked up for inoculation into a liquid medium for further amplification of the lactobacillus strain of the present invention. Cells are grown to a desired density (e.g. 10 ⁹ CFU/ml), the lactobacillus cells are harvested by conventional methods and stored in corresponding cryopreservation or used in experiments. Preferably, the lactobacillus cells of the invention are harvested by centrifugation.

[ Medium ]

The invention also provides a culture medium comprising the strain provided by the invention or a derivative thereof.

As used herein, "medium" refers to any medium used to grow and harvest cells and/or products expressed and/or secreted by the cells. The specific type of medium may be selected according to the type of cells to be cultured, the stage of growth, the object of culture, and the like. The culture medium includes, but is not limited to, a solution, a solid, a semi-solid, or a rigid support. The medium includes a medium for isolation, a medium for purification, a medium for proliferation, a medium for harvesting derivatives, a medium for analysis, and the like. The medium may be selected from liquid or solid media known in the art. Exemplary media suitable for use in the present invention include, but are not limited to: MRS medium, GAM medium, BL medium or SL medium.

Composition and method for producing the same

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

The present invention also provides a composition comprising an effective amount of a first component comprising the strain provided herein, derivatives thereof, a medium comprising the same, or a combination of the foregoing.

[ use of the composition ]

The composition may be a food composition, a health food composition, a pharmaceutical composition, a special medical use food composition, a cosmetic composition, a medical device composition or a feed composition.

The strain, the derivative thereof or the culture medium containing the strain can be singly used in foods, health foods, medicines, foods with special medical purposes, cosmetics, medical devices or feeds and the like, and can also be mixed with suitable components known to the person skilled in the art for use in foods, health foods, medicines, foods with special medical purposes, cosmetics, medical devices or feeds and the like.

[ modes of administration, dosage forms ]

The compositions of the present invention may be administered systemically and/or locally. In some embodiments, the compositions of the 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 presented in the form of a pill, tablet, lozenge, lyophilized powder, granule, 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 mist, effervescent tablet, transdermal therapeutic system, microcapsule, implant, or stick.

In particular, for a particular route of administration, the compositions of the invention are present in a correspondingly suitable form.

In some embodiments, the compositions of the invention are for oral administration, which may be formulated into dosage forms known in the art suitable for delivering the strains of the invention, such as tablets (uncoated or coated tablets, e.g., enteric or controlled release coatings), capsules (e.g., hard or soft gelatin capsules), lyophilized powders, granules, pills, emulsions, suspensions, sprays, aerosols or solutions.

In some embodiments, the compositions of the invention are for topical application and may be formulated into dosage forms known in the art suitable for delivery of the strains of the invention, such as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

In some embodiments, the compositions of the invention are for vaginal administration, which may be formulated into dosage forms known in the art suitable for delivering the strains of the invention, such as pessaries, tampons, emulsions, gels, pastes, foams, or sprays.

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

The compositions of the invention may be administered to a subject in unit dosage form one or more times per day. As used herein, "unit dose" refers to physically discrete units suitable for administration to a subject, and each unit comprises an effective amount of a lactobacillus strain or corresponding amount of derivative of the invention to provide a desired effect, e.g., an edible, therapeutic or health effect.

[ auxiliary component ]

The strain of the invention, its derivatives or a culture medium comprising it may be admixed with suitable auxiliary components, which may be achieved in a manner conventional in the art. Exemplary auxiliary components include:

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

ointment bases such as petroleum gums, waxes, triglycerides, waxes, wool wax alcohols, lanolin, hydrophilic ointments and polyethylene glycols;

suppository bases such as polyethylene glycol, cocoa butter and stearin;

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

surfactants, emulsifiers, dispersants or wetting agents, such as sodium lauryl sulfate, lecithin, phospholipids, fatty alcohols, 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, for example, glucose and sodium chloride;

Adsorbents such as highly dispersed silica;

binders such as polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, starch, carbomer, gelatin and gum arabic;

disintegrants, for example, modified starch, sodium carboxymethyl cellulose, sodium starch glycolate, crosslinked polyvinylpyrrolidone and crosslinked sodium carboxymethyl cellulose;

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

coating materials such as sugar and shellac;

film formers, such as polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethylcellulose, hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, polyacrylates and polymethacrylates;

capsule materials such as gelatin and hydroxypropyl methylcellulose;

synthetic polymers such as polylactic acid, polyglycolide, polyacrylate, polymethacrylate, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, polyethylene oxide, polyethylene glycol and copolymers and block copolymers thereof;

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

Penetration enhancers such as surfactants, dimethyl sulfoxide and analogues thereof, azones, pyrrolidone derivatives, alcohols and fatty acids;

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

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

flavoring agents, sweeteners, flavoring and/or odor masking agents.

It will be appreciated that the composition may contain other conventional components in the corresponding dosage form in addition to the ingredients specifically mentioned above.

[ MEANS FOR SOLVING PROBLEMS ]

In some embodiments, the compositions of the present invention comprise 10 ⁶ CFU/g to 10 ¹² The lactobacillus strain of the invention and its corresponding amount of derivatives in an amount of CFU/g, preferably comprises 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 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 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 The lactobacillus strain of the invention or the derivative thereof in an amount of CFU/g.

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

[ second component ]

In some embodiments, the compositions provided herein further comprise a second component. In some embodiments, the second component comprises a probiotic, a metagen, a prebiotic, an antimicrobial agent, an immunomodulator, an anticancer agent, an osteoporosis therapeutic agent, a mental area associated therapeutic agent, a developmental associated therapeutic agent, or a combination thereof.

As used herein, "probiotic" refers to a microorganism, a preparation comprising it or a culture, lysate, extract, inactivated product, metabolite, etc. that retains its essential properties, that has a beneficial effect on the health or wellbeing of the host. Preferably, the probiotic may be selected from Bifidobacterium (bifidobacteria), lactobacillus (Lactococcus), lactococcus (Lactococcus), enterococcus (Enterococcus), streptococcus (Streptococcus), kluyveromyces (Kluyveromyces), yeast (saccharomycetes), candida (Candida) or mixtures thereof. More preferably, the probiotic is selected from bifidobacteria (Bifidobacterium), lactobacilli (Lactobacillus) or mixtures thereof.

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

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

As used herein, "metagen" refers to an inactive bacterial product and/or metabolite of a probiotic microorganism that is biologically active in a host. The metazoan mainly comprises two main substances of metabolites and thallus components. The metabolite may be selected from organic acids, short chain fatty acids, intracellular polysaccharides, vitamins, proteins, enzymes, lipids or mixtures thereof. The bacterial composition may be selected from lipoteichoic acid, teichoic acid, peptidoglycan, cell surface protein, polysaccharide, cell membrane protein, extracellular polysaccharide, or mixtures thereof.

As used herein, "prebiotic" refers to any compound, nutrient, or additional microorganism used to support or enhance the healthy effect of a probiotic, or to facilitate the growth and/or activity of a probiotic. Typical examples of prebiotics are carbohydrates (e.g. oligosaccharides), but neither are non-carbohydrates excluded. The most common form of prebiotic is nutritionally categorized as soluble fiber, with various forms of dietary fiber exhibiting some level of prebiotic effect.

The prebiotic may be selected from oligosaccharides (e.g. fructose, galactose and mannose), dietary fibres (e.g. soluble fibres and soy fibres), inulin or mixtures thereof. Some examples of prebiotics include fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), isomalto-oligosaccharide (IMO), mannooligosaccharide (MOS), xylo-oligosaccharide (XOS), arabinoxylan (AXOS), inulin, soy xylo-oligosaccharide, lactulose (LA), glycosyl Sucrose (GS), lactosucrose (LS), palatinose-oligosaccharide (PAO), malto-oligosaccharide, gums and/or hydrolysates thereof, pectin and/or hydrolysates thereof.

As used herein, "antimicrobial agent" refers to a substance or combination of substances that is capable of killing or inhibiting the growth and/or activity of a microorganism. Exemplary antimicrobial agents that may be used in the present invention include, but are not limited to:

macrolides or ketolactones, 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., cefalopram, cefapigenin, ceftioxime, cefazolin, cefamandole, cefuroxime, cefampicillin, cefprozil, cefaclor, chlorocephalexin, cefoxitin, cefmetazole, ceftioxime, ceftriaxone, cefoperazone, ceftazidime, cefpodoxime, cefozoxime, ceftitinib, cefpirome and cefepime), and carbapenems (e.g., carbapenem, imipenem, meram and PZ-601);

monocyclic beta-lactams, such as aztreonam;

Quinolones such as nalidixic acid, octreoic acid, norfloxacin, pefloxacin, enoxacin, ofloxacin, levofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, gratifloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin, gefefloxacin, gemifloxacin, and pazufloxacin;

antibacterial sulfonamides such as p-aminobenzoic acid, sulfadiazine, sulfaisoxazole, sulfamethoxazole, and phthaloyl sulfathiazole;

aminoglycosides, for example streptomycin, neomycin, kanamycin, paromomycin, gentamicin, tobramycin, amikacin, netilmicin, spectinomycin, sisomicin, bicaline and isopalmitin;

tetracyclines, such as tetracycline, aureomycin, demeclocycline, minocycline, oxytetracycline, metacycline, doxycycline, and tigecycline;

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

lincomycin species, such as lincomycin and clindamycin;

glycopeptides such as vancomycin and teicoplanin;

streptomycins, such as quinupristine and duloxetine;

Oxazolidinones, such as linezolid and tedizolid;

polymyxins, colistins and colistins;

trimethoprim and bacitracin;

outflow pump inhibitors, and the like.

As used herein, "immunomodulator" refers to a substance, agent, signaling pathway, or component thereof that modulates an immune response, such as immunosuppressants and immunostimulants, and the like. By "modulating" an immune response is meant any change in the cell type or cellular 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 may be manifested by an increase or decrease in the number of various types of cells, an increase or decrease in cellular activity, or by any other change 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 can interfere with the ability of cancer cells to evade immune system attacks and help the immune system respond more strongly to tumors. The immune checkpoint molecule may mediate co-stimulatory signals to enhance the immune response, or may mediate co-inhibitory signals to inhibit the immune response. Examples of checkpoint modulators include, but are not limited to, modulators of PD-1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGF beta, 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 CD 83. In certain embodiments, the immune checkpoint modulator comprises a PD-1/PD-L1 axis inhibitor.

Adoptive cell transfer attempts to enhance the natural ability of T cells to fight cancer. In this mode of treatment, T cells are taken from the patient and expanded and activated in vitro. In certain embodiments, the T cells are modified in vitro to CAR-T cells. The most active anticancer T cells or CAR-T cells were cultured in bulk in vitro for 2 to 8 weeks. During this time, 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 certain embodiments, the adoptive cell transfer is CAR-T therapy.

Cytokines are used to enhance presentation of tumor antigens to the immune system. Two major types of cytokines used in the treatment of cancer are interferon and interleukin. Examples of cytokines include, but are not limited to, interferons (e.g., interferon- α, interferon- β, and interferon- γ), colony stimulating factors (e.g., macrophage CSF, granulocyte macrophage CSF, and granulocyte CSF), insulin growth factors (IGF-1), vascular Endothelial Growth Factors (VEGF), transforming Growth Factors (TGF), fibroblast Growth Factors (FGF), interleukins (e.g., 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 factors (e.g., 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, resulting in tumor cell lysis, followed by 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 combat cancer by enhancing the immune system's response to cancer cells. The therapeutic vaccine may comprise a non-pathogenic microorganism, a genetically modified virus targeting tumor cells, or one or more immunogenic components.

As used herein, an "anticancer agent" refers to an agent (e.g., a compound, drug, antagonist, inhibitor, modulator) that has anti-tumor properties or inhibits the growth or proliferation of cells. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is a biologic. In some embodiments, the anticancer agent is an immunotherapeutic formulation. In some embodiments, the anticancer agent is an agent approved by a food and drug administration for the treatment of cancer.

Examples of anticancer agents useful in the present invention include, but are not limited to: anastrozole, bicalutamide, bleomycin sulfate, busulfan, capecitabine, N4-pentoxy-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside cytarabine liposome injection, dacarbazine (actinomycin D, cosmegan), daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5-fluorouracil, flutamide, fludarabine tizalcitabine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, L-asparaginase, calcium folinate, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, maillard, paclitaxel, nab-paclitaxel, jetstretin, polifeprosan 20 and carmustine implants, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine and vinorelbine.

Particularly interesting anticancer agents useful in the present invention include, but are not limited to: antitumor antibiotics, tyrosine kinase inhibitors, alkylating agents, antimicrotubule or antimitotic agents and oncolytic viruses.

Exemplary antitumor antibiotics include, but are not limited to: doxorubicin, bleomycin, daunorubicin (daunorubicin hydrochloride, daunorubicin and erythromycins 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, bai Shuti, 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-sarcosinum, or melphalan), altretamine (also known as Hexamethylmelamine (HMM)), carmustine, bendamustine hydrochloride, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine, procarbazine, mechlorethamine hydrochloride, streptozotocin, and thiotepa.

Exemplary antimicrotubule or antimitotic 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, an "osteoporosis therapeutic agent" refers to an agent capable of preventing, alleviating, delaying or eliminating symptoms associated with osteoporosis, reducing the risk of suffering from a disease associated with osteoporosis, curing a disease associated with osteoporosis, or a combination thereof. Exemplary osteoporosis therapeutic agents useful in the present invention include, but are not limited to: bisphosphates (e.g., alendronate, risedronate, ibandronate, and zoledronic acid), raloxifene, desuzumab, and teriparatide.

As used herein, a "mental domain associated therapeutic agent" refers to an agent capable of preventing, alleviating, delaying or eliminating a symptom associated with a mental domain, reducing the risk of suffering from a disease associated with a mental domain, curing a disease associated with a mental domain, or a combination thereof.

As used herein, a "development-related therapeutic agent" refers to an agent that is capable of preventing, alleviating, delaying or eliminating a development-related symptom, reducing the risk of developing a development-related disease, curing a development-related disease, or a combination thereof.

In some embodiments, the weight ratio of the first component to the second component is from 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 administered prior to the second component. In some embodiments, the first component is administered after the second component is administered. In some embodiments, the first component is administered 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 and second components provided herein are formulated into a single therapeutic composition and the first and second components are administered simultaneously. In some embodiments, the first and second components provided herein are separated from one another, e.g., each formulated into a single therapeutic composition, and the first and second components are administered simultaneously. In some embodiments, the first and second components provided herein are separated from one another, e.g., each formulated into a single therapeutic composition, and the first and second components are administered at different times, e.g., the first component is administered before the second component is administered, or the first component is administered after the second component is administered, or the first and second components are administered in an alternating fashion. The first and second components may be administered in a single dose or in multiple doses.

Therapeutic method

The invention also provides a method of treatment comprising administering to a subject in need thereof an effective amount of a strain provided herein, a derivative thereof, a medium comprising the same, or a composition comprising the same, for use in preventing and/or treating a disease or disorder in a subject.

As used herein, "effective amount" refers to an amount that provides the desired effect while not producing serious side effects at the medical discretion. The amount of the microorganism to be administered to a subject by the composition of the present invention may be appropriately adjusted in consideration of the administration route, subject individual difference, and the like.

In certain embodiments, the dosage administered may vary during use. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered may be varied during use, depending on the subject's response.

It will be appreciated that the effective amount of the lactobacillus strains or derivatives thereof of the present invention will depend on various factors known in the art, such as the weight, age, prior medical history, current medication, health status and the likelihood of cross-reactivity, allergies, sensitivity and adverse side effects of the subject, as well as the route of administration and the extent of disease development. Those skilled in the art will be able to consider factors such as those described above to reduce or increase the dosage. The above dosage ranges do not limit the scope of the invention in any way.

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, "preventing and/or treating" a disease or disorder includes preventing or alleviating a condition, slowing the onset or rate of progression of a condition, reducing the risk of developing a condition, preventing or delaying the progression of symptoms associated with a condition, reducing or ending symptoms associated with a condition, producing complete or partial regression of a condition, curing a condition, or some combination thereof.

[ diseases associated with pathogen infection ]

The invention also provides a method of treatment comprising administering to a subject in need thereof a strain provided herein, a derivative thereof, a medium comprising the same, or a composition comprising the same, for antagonizing a pathogen in the subject, or for preventing and/or treating a disease or disorder associated with the pathogen.

Examples of such pathogens include, but are not limited to, bacteria, fungi, viruses, spirochetes, mycoplasma, rickettsia, chlamydia, and parasites.

In some embodiments, the pathogen is selected from the group consisting of: mycobacterium (Mycobacterium), salmonella (Salmonella), escherichia coli (E.coli), chlamydia (Chlamydia), staphylococcus (Staphylococcus), bacillus (Bacillus), pseudomonas (Pseudomonas), candida (Candida), acetobacter (Atopobium), gardnerella (Gardnerella) and Malaromyces (Pictyrosporum).

In some embodiments, the bacteria include escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atopoella vaginalis, gardnerella vaginalis resistant bacteria, or a combination thereof.

In some embodiments, the fungus comprises candida albicans, malassezia furfur, or a combination thereof.

In some embodiments, the parasite is a trichomonas.

In some embodiments, the pathogen-associated disease or disorder includes female genital tract infection and genital tract flora disorder.

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 infection, infections endangering the fetus in pregnant women, premature birth and urinary tract infections.

In some embodiments, the pathogen-associated disease or disorder comprises malassezia infection-associated disease.

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

[ diseases associated with immunomodulation ]

The invention also provides a method of treatment comprising administering to a subject in need thereof a strain provided herein, a derivative thereof, a medium comprising the same, or a composition comprising the same, for the prevention and/or treatment of a disease or disorder associated with immune modulation.

In some embodiments, the disease or disorder associated with immune modulation 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 cancers include, but are not limited to, prostate cancer, stomach-esophagus cancer, lung cancer, liver cancer, pancreatic cancer, breast cancer, bronchus 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, gastrointestinal cancer, skin cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcomas, teratomas, gliomas, adenocarcinomas, leukemias, lymphomas, and myelomas.

As used herein, "allergic disease" refers to any symptom, tissue damage, or loss of tissue function caused by 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, an "autoimmune disease" refers to a disease in which the immune system of a mammal produces a humoral or cellular immune response against the mammal's own tissues, or against antigens that are not harmful to the mammal itself, thereby producing tissue damage in the mammal. Symptoms and severity vary from patient to patient, as well as the clinical characteristics of each patient vary greatly over time.

Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, rheumatic fever, lupus, systemic scleroderma, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, guillain-Barre 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, welch granulomatosis, sjogren's syndrome, addison's disease, crohn's disease, white plug disease, edema, conjunctivitis, periodontitis, rhinitis, otitis, chronic sinusitis, pharyngolaryngitis, gastritis, bronchitis, pneumonia, gastric ulcers, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, scapulohumeral periarthritis, tendinitis, tenosynovitis, hepatitis, cystitis, nephritis, sepsis, bursitis, and sepsis.

[ diseases associated with osteoporosis ]

The present invention also provides a method of treatment comprising administering to a subject in need thereof a strain provided herein, a derivative thereof, a medium comprising the same, or a composition comprising the same, for the prevention and/or treatment of a disease or disorder associated with osteoporosis.

As used herein, "osteoporosis" refers to bone diseases characterized by deterioration of bone strength due to reduction in bone mass and/or deterioration of bone mass, resulting in increased risk of fracture, including primary and secondary.

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

[ other uses ]

The invention also provides a method of treatment comprising administering to a subject in need thereof a strain provided herein, a derivative thereof, a medium comprising the same, or a composition comprising the same, for the prevention and/or treatment of a disease or disorder associated with iron deficiency anemia, climacteric syndrome, a neural system disease.

Pharmaceutical use

The invention also provides the use of a strain of the invention, a derivative thereof, a medium comprising the same or a composition comprising the same in the manufacture of a medicament for antagonizing a pathogen.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same in the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with a pathogen.

The invention also provides the use of a strain of the invention, a derivative thereof, a medium comprising the same or a composition comprising the same in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with immunomodulation.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same in the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with osteoporosis.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with iron deficiency anemia, climacteric syndrome or a disease of the central nervous system.

Therapeutic use

The invention also provides the use of a strain of the invention, a derivative thereof, a medium comprising the same or a composition comprising the same in antagonizing an pathogen.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same for the prevention and/or treatment of a disease or disorder associated with a pathogen.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same for the prevention and/or treatment of a disease or disorder associated with immunomodulation.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same in the prevention and/or treatment of a disease or condition associated with osteoporosis.

The invention also provides the use of a strain of the invention, a derivative thereof, a culture medium comprising the same or a composition comprising the same for the prevention and/or treatment of a disease or disorder associated with iron deficiency anemia, climacteric syndrome or a disease of the central nervous system.

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 described below (including in whole or in part) are encompassed within the scope of the invention. These particular compositions, materials, and methods are not intended to limit the invention but are merely illustrative of specific embodiments that are within the scope of the invention. Equivalent compositions, materials, and methods can be developed by those skilled in the art without the need for the inventive faculty, without departing from the scope of the invention. It should be understood that many variations to the procedure described herein may be made while still being within the scope of the present invention. The inventors intend such variations to be included within the scope of the invention.

Examples

EXAMPLE 1 isolation, purification, and enrichment culture of Lactobacillus gasseri Strain

1. Isolation, purification and enrichment culture of Lactobacillus strains

Several women of healthy childbearing age without vaginal infection or any intestinal disease were enrolled to provide samples, and participants all passed the health examination of the physical examination center and provided information about their age (21-30), menstrual cycle, and other healthy activities, etc. by questionnaires. Starting 2 weeks before sample collection, all participants avoided all types of probiotic-containing formulations, sample collection used the port. A-Cd system of BD company in the united states, collected secretions at 1/3 of the vaginal side wall of the subject with two sterile cotton swabs, placed in sterile tubes, quickly transported to laboratory biosafety cabinets with ice bags, the bacterial suspension after flushing the swabs with a small amount of sterile PBS as a mother liquor, then diluted to different concentrations with sterile PBS, coated on freshly prepared Rogosa SL solid medium, labeled with information, placed in culture boxes, and placed in anaerobic bags, placed in 37 ℃ incubator, and incubated for 48-72 hours.

Single colonies with different forms (surface, edge, color, size and the like) are respectively picked from a cultured Rogosa SL flat plate by an inoculating loop, inoculated onto a freshly prepared MRS solid culture medium according to an octave lineation method, placed in a culture box, placed in an anaerobic gas producing bag, placed in a 37 ℃ incubator for culturing for 24-72 hours to obtain purified single colonies, and inoculated to the MRS liquid culture medium by the inoculating loop, placed in the 37 ℃ incubator for anaerobic culturing for 24 hours to obtain the Lactobacillus gasseri strain.

A probiotic health care product for vaginal use is purchased from a certain naughty store. Opening the packaging box in the biosafety cabinet, taking a capsule, opening the capsule shell, pouring the bacterial powder in the capsule shell into 10mL of sterile PBS solution, and uniformly mixing by vortex oscillation, which is recorded as 10 ^-1 Dilution is then continued with 10-fold gradient dilution to 10 ^-4 From 10 ^-3 And 10 ^-4 Dilution of100 mu L of each was spread on MRS solid medium plates, and cultured anaerobically at 37℃for 24-48 hours. When single colony grows on the MRS solid culture medium, 4 different colony forms are found on the MRS solid culture medium of the Yuehan product, the single colony with different forms is respectively picked by an inoculating loop and inoculated to the MRS liquid culture medium, and the MRS liquid culture medium is placed in a 37 ℃ incubator for anaerobic culture for 16-24 hours; after the cultivation, the culture medium is centrifuged separately, part of the supernatant is removed, the strain is resuspended, and then an equal volume of 20% glycerol is added, mixed by vortex shaking, sub-packaged into a freezing tube, preserved at-80 ℃, and simultaneously the strain sample is sent to carry out 16S rRNA sequencing, and the strain is identified, thus obtaining the commercial strain Lactobacillus gasseri LbV N (L.gaseri LBV 150N).

2. Identification and preservation of Lactobacillus

(1) Culture characteristics, staining microscopy and morphological characteristics

For lactobacillus gasseri of the present invention: the bacterial colony of the lactobacillus obtained after the culture is round, the bacterial colony is smeared with a pure culture of the lactobacillus for gram staining, and the bacterial colony is in a gram positive and short rod shape and can be connected into a long chain, and the lactobacillus is primarily judged.

(2) Identification of 16S rRNA Gene sequence

And (3) adopting a kit for directly carrying out PCR amplification, adopting 27F (5'AGA GTT TGA TCM TGG CTC AG 3') and 1492R (5'TAC GGY TAC CTT GTT ACG ACT T3') as primers to carry out PCR amplification, taking a PCR product to carry out gel electrophoresis, determining a 16S rRNA gene fragment, and if the gel electrophoresis result shows that the PCR is successful, sending a PCR sample to a gene sequencing company to carry out 16S rRNA sequencing. BLAST sequence similarity comparison analysis is carried out on the sequence obtained by sequencing and data in NCBI database, and the strain obtained by separation is determined to be lactobacillus gasseri, namely L.gaseri-44 according to the highest homology score being more than 97%.

EXAMPLE 2 study of gastric acid and bile salt resistance of Lactobacillus gasseri L.gaseri-44

The probiotics can only play the probiotic effect after entering the intestinal tract, so that the capability of the probiotics strain for resisting digestive juice such as gastric acid, bile salt and the like is very necessary to be examined. To verify that L.gaseri-44 meets the conditions, the invention determines its acid and bile salt resistance by an in vitro simulated gastrointestinal fluid method.

L.gaseri-44 and L.gaseri LBV150N (control, commercial strain) were inoculated from glycerol tubes to MRS liquid medium with an inoculating loop, cultured at 37℃for 18-24 hours in an anaerobic working station, centrifuged at 8000rpm for 5 minutes, and then the cells were collected. Each strain was individually adjusted to an appropriate concentration (about 5X 10 by using a turbidity meter ⁸ CFU/mL). The culture was inoculated into fresh MRS liquid medium at an inoculum size of 5%, and anaerobically cultured at 37℃for 6 hours to logarithmic phase. After 6 hours, the mixture was taken out, centrifuged at 8000rpm for 5 minutes, the medium was removed, and physiological saline was added to resuspend the mixture to a predetermined concentration. The suspension was conditioned with a turbidity meter to give a bacterial concentration of about 1X 10 ⁹ CFU/mL. The resuspension was diluted 10-fold in a gradient and 100. Mu.L of the plate was counted.

1. Gastric acid resistance test

Preparing sterilized normal saline: 0.9% w/v, hydrochloric acid to adjust the pH to 3.0.

Preparing simulated gastric juice: pepsin (Sigma, P7000-25G, 630U/mg) 96.84mg of the powder was dissolved in 32.3mL of physiological saline at pH 3.0 to give a final concentration of 3g/L. It was filtered through a 0.22 μm sterile filter and ready to use.

Generally, the pH of human stomach acid is 3.0-3.5. 2mL of the concentrated bacterial suspension was centrifuged again to remove the supernatant, in duplicate. One cell was resuspended in an equal volume of simulated gastric fluid at pH 3.0 and the other cell was resuspended in an equal volume of normal saline, and after incubation at 37℃for 3 hours, each probiotic was subjected to gradient dilution and plating counting. The acid resistance calculation formula is as follows:

acid tolerance (%) = (Log 10 CFU/mL in simulated gastric fluid)/(Log 10 CFU/mL in physiological saline) ×100.

2. Cholate resistance test

Preparing sterilized normal saline: 0.9% w/v, naOH was used to adjust the pH to 8.0.

A mother liquor of 100 Xtrypsin (biological engineering (Shanghai) Co., ltd., A003702-0100, 204U/mg protein) was prepared: 100mg was dissolved in 1mL of physiological saline at pH 8.0. Preparing a mother solution of 100 Xbile salts (Oxoid, LP 0055): 192.54mg was dissolved in 0.640 mL of physiological saline at pH 8.0.

Preparing simulated intestinal juice: 0.5mL of 100 Xtrypsin and bile salt stock solution were added at 49mL of pH 8.0 physiological saline, respectively, at a final concentration of 1g/L trypsin and 0.3% bile salt (i.e., 3 mg/mL), respectively. It was filtered through a 0.22 μm sterile filter and ready to use.

2mL of the concentrated bacterial suspension was centrifuged to remove the supernatant, and the supernatant was removed in duplicate. One cell was resuspended in an equal volume of simulated intestinal fluid at pH 8.0 and the other cell was resuspended in an equal volume of normal saline, and after incubation at 37℃for 4 hours, each probiotic was subjected to gradient dilution and plating counting. The cholate tolerance calculation formula is as follows:

cholate tolerance (%) = (Log 10CFU/mL in simulated intestinal fluid)/(Log 10CFU/mL in physiological saline) ×100.

3. Experimental results

The present invention uses the commercial strain L.gasser LBV150N as a control. The experimental results are shown in tables 1 and 2, and the survival rate of the commercial strains L.gaseri LBV150N and L.gaseri-44 after 3 hours incubation in simulated gastric fluid is about 100%, which shows that they all have good gastric acid tolerance; whereas the survival rate of L.gaseri LBV150N was 92.73% and that of L.gaseri-44 was 96.48% after 4 hours incubation of the 2 strains in simulated intestinal fluid, respectively, indicating that L.gaseri-44 has better bile salt tolerance. In conclusion, L.gaseri-44 has good gastric acid and bile salt resistance.

TABLE 1 gastric acid resistance test results of Lactobacillus gasseri strain

TABLE 2 results of bile salt tolerance experiments with Lactobacillus gasseri strains

EXAMPLE 3 cloning Property study of Lactobacillus gasseri L.gaseri-44

The probiotic bacteria can exert the probiotic effect for a long time if the probiotic bacteria can colonise the intestinal tract and the vagina, so the invention adopts a cell line Caco-2 derived from the human intestinal tract and a vaginal epithelial cell VK2/E6E7 to evaluate the intestinal tract colonisation performance of L.gaseri-44.

The Caco-2 cells were cultured in complete medium EMEM (GNM 11700, jino Biochemical technologies Co., hangzhou) and 10% fetal bovine serum was added. The Caco-2 cell preservation tube was unscrewed slightly and melted rapidly (within 2 minutes) in a 37℃water bath. The outer wall of the storage tube was sterilized with 75% ethanol. The cell cryopreservation solution was transferred to a centrifuge tube containing 9mL of medium with a pipette, centrifuged at 1000rpm for 3-5 minutes, the supernatant removed, and gently resuspended with the appropriate amount of medium added. Put into CO ₂ Culturing in an incubator. When the growth confluence rate reaches 80% -90%, the cells are passaged. After 2-3 passages, old medium was aspirated, washed 2 times with DPBS (without calcium and magnesium ions), 2mL pancreatin was added to the flask and the flask was gently shaken to allow the digest to flow across all cell surfaces. After digestion for 2-5 minutes at 37 ℃, the flask was placed under a microscope for observation, after cytoplasmic retraction and increased cell gap, the side wall of the flask was tapped, and after the cells were separated from the bottom wall of the flask, the digestion was stopped by immediately adding 3 volumes of complete medium. The bottom wall of the bottle is repeatedly and gently blown by a liquid-transferring gun, so that the cells are separated from the bottle wall to form single cell suspension. 1000rpm, and centrifuging for 3-5 minutes. The supernatant was discarded, resuspended in fresh medium, and the actual viable cell count was determined by taking a portion of the cell suspension from the automatic cytometer. Based on the results of viable cell count, the cells were diluted to a cell density of 5X 10 with medium ⁵ 100. Mu.L of cell suspension was added to each well of a cell/mL, 96-well plate, and each well contained 5X 10 ⁴ cell/well。37℃，5％ CO ₂ Culturing in incubator for 20-24 hr.

The VK2/E6E7 cell culture medium was 500mL of serum-free keratinocyte basal medium K-SFM (Invitrogen, 10744-019) and 1mL of keratinocyte growth supplement was added. Before use, the mixture is preheated at 37 ℃.

L.gaseri-44 and L.gaseri LBV150N were inoculated from glycerol tubes to MRS liquid medium with an inoculating loop and incubated in an anaerobic workstation at 37℃for 12-18 hours. After centrifugation at 8000rpm for 5 minutes, the cells were collected. The probiotics were individually adjusted to the appropriate concentration (about 5×10 by turbidity meter ⁸ CFU/mL). According to 5%The inoculum size was inoculated into fresh MRS liquid medium, and anaerobic culture was performed at 37℃for 6 hours to logarithmic growth phase. Centrifugation was performed at 8000rpm for 5 minutes, the medium was removed, and physiological saline was added to resuspend to a certain concentration. The suspension was conditioned with a turbidity meter to give a probiotic concentration of about 1.0X10 ⁹ CFU/mL. Adding 40% glycerol with equal volume into the re-suspension, mixing, transferring 1 mL/branch to cell freezing tube, and placing on dry ice. Transferring to-80deg.C refrigerator after solidification, and long-term storing. And (3) taking 1 tube of frozen bacteria, melting the frozen bacteria in a water bath at 37 ℃ before the experiment starts, performing 10-time gradient dilution, counting 10 mu L of coated plates, and repeating for 3 times.

On the day of the experiment, the frozen tube is taken out and immediately placed in a water bath kettle at 37 ℃ for melting. After transfer to a 2mL centrifuge tube, centrifugation at 8000rpm for 5 minutes, the supernatant was removed and resuspended in medium. The bacterial suspension was diluted to about 2.5X10 with the corresponding medium ⁸ CFU/mL, re-diluted 5-fold, re-diluted 10-fold, giving about 5.0X10 respectively ⁷ CFU/mL and about 5.0X10 ⁶ CFU/mL bacterial suspension for later use.

On the day of the experiment, the cells were removed from the incubator, the medium was discarded, and the corresponding medium was added for washing 1 time. mu.L of each prepared concentration of the bacterial liquid was added to the cells. Placing the plate at 37deg.C, CO ₂ The incubator was left to stand for adhesion for 2 hours with a target multiplicity of infection (Multiplicity of Infection, MOI, bacteria: cells) of 10:1. After the incubation adhesion was completed, the cells were lysed by adding 200. Mu.L of DPBS containing 0.05% Triton X-100 and sucked by a pipette and washed 3 times (washing volume 200. Mu.L, lowest washing speed) with a plate washer (BioTek, 405 TS) to allow the cells and bacteria to fall off sufficiently. 10-fold gradient dilutions were made in physiological saline and 100 μl of the plates were counted and the plates were MRS agar, 3 replicates. The following formula was used to calculate the adhesion rate

Adhesion% = adhered bacterial count CFU/inoculated bacterial count CFU x 100%

TABLE 3 adhesion ability of Lactobacillus gasseri L.gaseri-44 to Caco-2 cells and VK2/E6E7 cells

The results of the experiment are shown in Table 3, and the adhesion of L.gaseri-44 was higher than that of the commercial strain L.gaseri LBV150N for both human colon cancer cells Caco-2 and vaginal epithelial cells VK2/E6E7, indicating that L.gaseri-44 is a probiotic strain with development potential.

EXAMPLE 4 Effect of Lactobacillus gasseri L.gaseri-44 on antagonizing pathogenic bacteria

The ability of lactobacillus to antagonize pathogenic bacteria is also one of the mechanisms of the lactobacillus to exert the effect of probiotics, so the invention researches the ability of L.gaseri-44 to antagonize pathogenic bacteria by a double-layer plate method and a bacterial cake method, and the commercial strain L.gaseri LBV150N is used as a control bacterium.

L.gaseri-44 and L.gaseri LBV150N were inoculated from glycerol tubes to MRS liquid medium with an inoculating loop and incubated in an anaerobic workstation at 37℃for 12-18 hours. After centrifugation at 8000rpm for 5 minutes, the cells were collected. The probiotics were individually adjusted to the appropriate concentration (about 5×10 by turbidity meter ⁸ CFU/mL). The culture was inoculated into fresh MRS liquid medium at an inoculum size of 5%, and anaerobically cultured at 37℃for 6 hours to logarithmic phase. After 6 hours, the mixture was taken out, centrifuged at 8000rpm for 5 minutes, the medium was removed, and physiological saline was added to resuspend the mixture to a predetermined concentration. The suspension was conditioned with a turbidity meter to give a bacterial concentration of about 1.0X10 ⁸ CFU/mL. The resuspension was diluted 10-fold in a gradient and 10. Mu.L of the plate was counted. mu.L of the prepared bacterial solution was spotted onto MRS agar medium prepared in advance (1 bacteria/plate, 3 replicates) using a pipette. After the flat plate is dried, the flat plate is placed under anaerobic condition at 37 ℃ for 20-24 hours for standby.

6 pathogenic bacteria (Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atoposis vaginalis and Gardner resistance bacteria) were inoculated with the respective solid medium one day in advance according to Table 4, and cultured overnight at 37 ℃. The test methods are summarized in Table 5.

TABLE 4 culture Medium used for culturing Lactobacillus gasseri strains and respective pathogenic bacteria strains

TABLE 5 bacterial cake method test method and conditions

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On the day of the experiment, the pathogenic bacteria are taken out, and for 6 pathogenic bacteria, 3-6 single colonies are picked into physiological saline by an inoculating loop to prepare bacterial suspension. Adjusted to about 0.2 with a turbidity meter to a bacterial concentration of about 1.0X10 ⁸ CFU/mL。

Double-layer plate method: 100 μl of the prepared 1 strain of pathogenic bacteria (Staphylococcus aureus) was poured onto a probiotic MRS agar to be tested, which was cultured for 20-24 hours, in 5mL of a solid medium (see Table 4) cooled to about 45 ℃. After solidification, the plate is placed under atmospheric conditions at 37 ℃ for 1 day until a zone of inhibition appears. 3 parallel test plates per probiotic.

Inversion bacterial cake method: 100 mu L of prepared pathogenic bacteria (escherichia coli, pseudomonas aeruginosa and salmonella typhi) are respectively coated on NA or Columbia blood agar medium and 5% sheep blood flat plates, and after the bacteria are fully absorbed, all probiotic bacterial cakes are picked up and inverted on the pathogenic bacteria flat plates. The Escherichia coli, pseudomonas aeruginosa and Salmonella typhi plates were incubated at 37℃for 1 day until a zone of inhibition appeared. 3 parallel test plates per probiotic.

The diameter of the inhibition zone is measured by a vernier caliper or a colony counter.

Statistical analysis was performed with GraphPad Prism software. Each set of data was calculated using One-way ANOVA and each probiotic was compared separately using Dunnett's multiple comparisons test. When P-value <0.05, a significant difference is considered.

The inhibitory effect of L.gaseri-44 on 6 pathogenic bacteria is shown in tables 6 to 9. The L.gasser-44 inhibits copper Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, altobermoria vaginalis and Gardnerella vaginalis more than the commercial strain L.gasser LBV150N.

TABLE 6 diameter of zone of inhibition (mm) of Lactobacillus gasseri against Pseudomonas aeruginosa

TABLE 7 diameter of zone of inhibition (mm) of Lactobacillus gasseri against Salmonella typhi and E.coli

Table 8 diameter of zone of inhibition (mm) of Lactobacillus gasseri against Staphylococcus aureus

TABLE 9 inhibition of Lactobacillus gasseri against Gardner resistance bacteria and Altobose

Example 5 effects on mouse Gardner vaginal model

SPF-class 4-6 week ICR female mice were taken in 40 randomly divided groups of 4: healthy control, infected control, metronidazole and L.gaseri-44 groups of 10 animals each. Definition of day of infection as day 0, subcutaneous injection of estradiol at a certain concentration into mice at day 3 and day 0, intraperitoneal injection of ketamine hydrochloride to anesthetize mice at day of infection, and vaginal injection of 20 μl gardnerella vaginalis solution (5×10) ⁷ CFU/mL), 1 time per day, 3 days of continuous inoculation, 4 days of dipping small amounts of mucus from the mouse vagina with sterile swabs, detecting gardnerella, ensuring that gardnerella is continuously colonized in each mouse vagina, and injecting the same volume each time per day into healthy control groupIs a physiological saline solution.

Picking freshly cultured L.gaseri-44 single colony from MRS plate, inoculating into MRS liquid culture medium, standing anaerobic culturing at 37deg.C for 24 hr, centrifuging, re-suspending bacterial mud with PBS, and adjusting concentration to 1×10 with flow cytometry ⁹ CFU/mL. On day 1 after infection, 20 mu L of freshly prepared bacterial liquid is respectively infused into the vagina of a Lactobacillus gasseri-44 group of mice for 3 consecutive days, 1 time per day; mice in the metronidazole group were vaginally injected with 20 μl of metronidazole solution for 3 consecutive days, 1 time per day; the healthy control group and the mice infected with the control group were vaginally administered the same volume of physiological saline daily for 3 consecutive days, 1 time daily.

1. Determination and analysis of vaginal flora in mice

The mice were repeatedly rinsed 5-6 times with 50. Mu.L of physiological saline by a microsampler before administration after molding, 1 day after administration, and 6 days after administration, and 30. Mu.L of the above vaginal lavage fluid was used for counting the colony of Gardner vaginalis and Lactobacillus respectively, and the results are shown in Table 10. The gardnerella vaginalis was counted using Columbia platelets supplemented with gentamicin sulfate (4 mg/L), nalidixic acid (30 mg/L) and amphotericin B (2 mg/L).

And (5) performing preliminary identification according to colony morphology of the selective medium, smear color-staining and detection.

Table 10 results of colony counts of lavage fluids from each group

As can be seen from the above table, the number of gardnerella in the vagina of the L.gaseri-44 mice is reduced by about 30-40 times compared with that of the infection control group on the first day after treatment, and the number of the lactobacillus is about 3 orders of magnitude higher than that of the metronidazole group and the infection control group; the number of gardnerella bacteria colonised in the vagina of the mice of the L.gaseri-44 group on day 6 after treatment was slightly higher than that of the metronidazole group, but 2 orders of magnitude lower than that of the infected control group, while the number of lactobacillus still was about 3 orders of magnitude higher than that of the metronidazole group and the infected control group; it was demonstrated that Lactobacillus gasseri-44 was able to colonize the vagina well and restore the amount of Lactobacillus in the vagina to above normal levels, while stably inhibiting the proliferation of Gardner bacteria in the vagina.

2. Mouse vulva observation before and after treatment

The condition of vulva redness and swelling and vaginal secretion of each group of mice was observed and recorded, and a vaginal lavage liquid smear (PAS staining) was made for each group of typical mice.

Table 11 inflammation conditions such as vulva inflammation and secretion of mice after treatment

Group of	Edema (edema)	Secretion of
			Healthy control group	-	Not obvious
Infection control group	+++	Multiple/thin
			Metronidazole group	+	A small amount of
Lactobacillus gasseri group	+	A small amount of

Table 11 shows the inflammatory reaction caused by the colonisation of the vagina by Gardner vaginalis, the vulva of the infected mice in the control group has a large amount of oedema, more secretion and thin foam, and the PAS dyeing result shows that inflammatory cell infiltration appears on the surface layer of the vaginal mucosa, thus showing that the molding is successful; after the treatment of lactobacillus gasseri-44 bacterial liquid, the symptoms of vulva edema, multiple secretions and the like of the mice are obviously relieved, and the staining result of the vaginal lavage liquid PAS shows that the number of leucocytes in the vaginal secretions of the mice is obviously reduced, and most of leucocytes are vaginal epithelial cells, which indicates that the damage of the vaginal mucosa of the mice has been recovered to a great extent.

The results show that the lactobacillus gasseri-44 has the functions of regulating vaginal flora balance and inhibiting the growth and colonisation of gardnerella vaginalis, and can be used for preventing and treating bacterial vaginitis.

Example 6 Effect on the model of Candida albicans in the mouse vagina

40 female C57BL/6 mice with SPF grade of 6-8 weeks were randomly divided into 4 groups: healthy control, infected control, clotrimazole and L.gaseri-44 groups of 10 animals each. Except for healthy control groups, 50. Mu.L of lincomycin hydrochloride solution with a certain concentration was taken out by a microscale sampler for each group to carry out vaginal irrigation on mice, 1 time per day for 5 consecutive days, and then candida albicans (2.5X10) ⁷ CFU/mL) 20 μl was inoculated into the vagina of the mice, and the inoculation was continued for 6 days, 1 time per day, resulting in a model of vaginal candida albicans infection of the mice. The healthy control group was injected with the same volume of physiological saline every day for 11 consecutive days.

Inoculating freshly cultured Lactobacillus gasseri L.gaseri-44 from MRS plate into MRS liquid culture medium, standing at 37deg.C for anaerobic culture for 24 hr, centrifuging, re-suspending bacterial mud with PBS, and adjusting concentration to 1×10 with flow cytometer ⁹ CFU/mL. The lactobacillus gasseri group mice are perfused with 20 mu L of bacterial liquid for 3 consecutive days, 1 time per day; the clotrimazole group mice were vaginally injected with 20 μl of clotrimazole solution for 3 consecutive days, 1 time per day; the healthy control group and the mice infected with the control group were vaginally administered the same volume of physiological saline daily for 3 consecutive days, 1 time daily.

1. Determination and analysis of vaginal flora in mice

The mice were repeatedly rinsed 5-6 times with 50. Mu.L of physiological saline by a microsampler, before administration after molding, on day 1 after administration, and on day 6 after administration, respectively, and 30. Mu.L of the vaginal lavage fluid was used as colony counts of Candida albicans and Lactobacillus respectively, and the results are shown in Table 12.

And (5) performing preliminary identification according to colony morphology of the selective medium, smear color-staining and detection.

TABLE 12 colony count results for lavage fluids of each group

From the table above, it can be seen that: the number of candida albicans colonisation in the vagina of mice in the lactobacillus gasseri-44 treatment group was reduced by one order of magnitude compared to the infection control group on the first day after treatment, but the number of lactobacillus in the vagina was about 3 orders of magnitude higher than both the infection control group and the clotrimazole group; on day 6 after treatment, the number of candida albicans colonised in the vagina of the lactobacillus gasseri-44 mice is equal to that of the clotrimazole group and is far smaller than that of the infection control group, and the number of lactobacillus in the vagina of the mice is still about 3 orders of magnitude higher than that of the clotrimazole group and the infection control group; it was demonstrated that Lactobacillus gasseri-44 was able to colonise the vagina well and to restore the amount of Lactobacillus in the vagina to above the normal level, while stably inhibiting the proliferation of Candida albicans.

2. Mouse vulva observation before and after treatment

The condition of vulva redness and swelling and vaginal secretion of each group of mice was observed and recorded, and a vaginal lavage liquid smear (PAS staining) was made for each group of typical mice.

Table 13 inflammation conditions such as vulva inflammation and secretion of mice after treatment

Group of	Red and swollen	Secretion of	Vaginal congestion
				Healthy control group	-	Not obvious	-
Infection control group	+++	Multiple/block	+++
				Clotrimazole group	+	A small amount of	+
Lactobacillus gasseri group	+	A small amount of	+

Table 13 shows the inflammatory response caused by candida albicans colonisation in the vagina of mice, and the vulva of mice infected with the control group showed a greater degree of redness and swelling, more secretions, and typical candida symptoms such as lumping, severe vaginal congestion, etc., indicating successful modeling; after the treatment by the lactobacillus gasseri bacterial liquid, the symptoms of vulva inflammation, vaginal congestion, secretion and the like of the mice are obviously relieved.

Meanwhile, the staining result of the vaginal lavage liquid PAS shows that the mice in the healthy control group have fewer vaginal epithelial cells and fewer white blood cells, while the mice in the infected control group colonized by candida albicans have more white blood cells, which indicates that the vaginal mucosa of the mice is seriously damaged, the number of white blood cells in the vagina of the mice is obviously reduced after the treatment by lactobacillus gasseri L.gaseri-44 bacterial liquid, and the epithelial cells have fewer, which indicates that the damage of the vaginal mucosa of the mice has been recovered.

The results show that the lactobacillus gasseri-44 has the functions of regulating vaginal flora, inhibiting the growth and colonisation of candida albicans in the vagina, and can be used for preventing and treating candidal vaginitis.

EXAMPLE 7 analysis of the action of Lactobacillus gasseri L.gasser-44 against Malassezia furfur

The antifungal effect of L.gaseri-44 was determined by the double-layer plate method, with the commercial strain L.gaseri LBV150N as a control bacterium. L.gaseri-44 and commercial strain L.gaseri LBV150N were inoculated at 3% (V/V) to MRS liquid medium, cultured anaerobically at 37℃for 8-16 hours for use. mu.L of the bacterial liquid was spotted (spot) onto MRS solid medium plates and cultured anaerobically at 37℃for 24-48 hours. 1% Malassezia furfur (ATCC 14521) seed solution was inoculated into a mYPG liquid medium prepared under aerobic conditions, and cultured at 37℃for 24 to 48 hours for use. Preparation of mYPG medium, sterilization at 115℃for 20 minutes, cooling to about 40℃and mixing 2.5mL of the medium with 500. Mu.L of Malachitum furfur (M.fursur) medium to be used, pouring the mixture into MRS solid medium spot-like with L.gaseri-44 and commercial strain L.gaseri LBV150N, respectively, and standing until the medium solidifies. The coagulated medium plates were incubated aerobically at 37℃for 24 to 48 hours. The activity of L.gasser-44 and the commercial strain L.gasser LBV150N against malassezia furfur was determined by measuring the diameter of the zone of inhibition, and the experiments were repeated in triplicate. As shown in Table 14, L.gaseri-44 and commercial strain L.gaseri LBV150N each had an effect of inhibiting growth of malassezia furfur, but L.gaseri-44 had a stronger antagonistic effect on malassezia furfur.

Table 14 diameter of zone of inhibition (mm) of Lactobacillus gasseri against malassezia furfur

EXAMPLE 8 Lactobacillus gasseri L.gaseri-44 inhibition of antigen-induced histamine release Studies

In allergic reactions, a large amount of histamine is released in tissues, which causes inflammatory reactions, and clinically improves symptoms of allergic reactions in patients by blocking secretion of histamine. In order to screen for probiotic strains that inhibit histamine secretion, the present invention evaluated the ability of Lactobacillus gasseri L.gaseri-44 from the human vagina to inhibit histamine secretion using the commercial strain L.gaseriLBV 150N as a control strain. The ability of each strain to inhibit histamine secretion was determined by the following method: after culturing RBL-2H3 cell line, degranulation is induced, then the content of histamine in the filtrate is measured by high performance liquid chromatography by adopting a o-phthalaldehyde post-column conversion method.

L.gaseri-44 and commercial strain L.gaseri LBV150N were cultured in MRS medium, respectively, and after 2-3 times of activation subculture, were used. At 37℃with 5% CO ₂ RBL-2H3 cells (ATCC No. CRL-2256, available from Nanjing, bai Biotechnology Co., ltd.) were cultured in alpha-MEM medium supplemented with 10% FBS Fetal Bovine Serum (FBS), penicillin 100. Mu.g/mL, and streptomycin 100. Mu.g/mL, and after 2-3 times of subculture, 0.5mL of freshly cultured RBL-2H3 cells were inoculated onto 24-well plates at an inoculation concentration of 1X 10 ⁵ Individual wells/well, 37 ℃, 5% co ₂ Culturing for 24 hr, removing supernatant, adding 5% FBS-containing alpha-MEM culture medium and IgE (0.1-0.7 μg/mL), incubating for 1-8 hr, centrifuging at 15000g for 3 min, removing supernatant, adding 1mL HEPES buffer (140mM NaCl,5mM KCl,0.6mM MgCl) ₂ ，1.0mM CaCl ₂ Cells were washed 2-4 times with 5.5mM glucose, 0.1% bovine serum albumin, 5mM HEPES), treated with 100-400. Mu.L of the previously prepared probiotic culture solution or 5-40. Mu.g/mL positive control ketotifen per well for 5-30 minutes, then treated with 100. Mu.L of antigen (DNP-BAS, 100. Mu.g/mL) for 20-40 minutes at 37℃and degranulation was induced by antigen-antibody reaction. Antigen treatment was replaced with 100. Mu.L HEPES buffer to induce degranulation as negativeAnd (3) controlling. After the antigen treatment was completed, the reaction was terminated by placing the 24-well plate in an ice-water bath, adding 0.6mL of ice HEPES buffer, taking the supernatant from each well, adding 20. Mu.L of perchloric acid, centrifuging at 12000rpm for 30 minutes, and filtering the supernatant through a 0.45 μm filter membrane. The content of histamine in the filtrate was determined by high performance liquid chromatography using a post-column conversion of o-phthalaldehyde.

The histamine release inhibition rate after the treatment of lactobacillus gasseri was compared with the negative control group, and was calculated according to the following formula:

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

The results of the experiment are shown in Table 15, and L.gaseri-44 shows a higher histamine release inhibition rate than ketotifen and L.gaseri LBV150N, and thus can effectively alleviate allergic symptoms caused by excessive histamine secretion.

TABLE 15 inhibition of histamine release by Lactobacillus gasseri

Strain	Inhibition of histamine release (%)
		Negative control HEPES buffer	100.0±0
Positive control ketotifen	43.0±0.85
		L.gasseri LBV150N	41.0±3.5
L.gasseri-44	52.0±4.1

EXAMPLE 9 analysis of the Effect of Lactobacillus gasseri L.44 on inhibition of Th 2-type cytokines in T cells

Type 2 helper T cell (Th 2) -associated cytokines (e.g., IL-4 and IL-5) are capable of enhancing IgE production through a Th 2-associated immune response, thereby promoting chronic allergic reactions. The invention further employs the EL4 cell line to evaluate the inhibition of chronic allergic reactions by L.gaseri-44, commercial strain L.gaseri LBV150N as a control strain.

At 37℃with 5% CO ₂ EL4 cells (ATCC TIB 181, purchased from Nanjing Corp. Bai Biotechnology Co., ltd.) were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), penicillin 100. Mu.g/mL, and streptomycin 100. Mu.g/mL, and subcultured for 2-3 times for use; l.gaseri-44 and commercial strain L.gaseri LBV150N were cultured in MRS medium, respectively, passaged 2-3 times, fermentation broth was centrifuged at 12000g for 5 minutes, the supernatant was discarded and the pellet was washed with PBS, and the number of viable bacteria was measured with a flow cytometer for use.

EL4 cells with good viability were cultured at 1X 10 ⁵ Concentration of individual cells/well was seeded on 48-well plates, 37 ℃, 5% co ₂ Culturing for 12-24 hr, adding PMA (final concentration 10 ng/mL), adding 200 μl of previously prepared lactobacillus gasseri bacteria solution, CO-culturing at 37deg.C and 5% CO ₂ After 24 hours incubation in the incubator, supernatants were collected and the amounts of secreted IL-4 and IL-5 were determined using the Mouse IL-4ELISA kit (PI 613, beyotime) and the Mouse IL-5ELISA kit (PI 620, beyotime).

As shown in FIGS. 1 and 2, both L.gaseri-44 and the commercial strain L.gaseri LBV150N significantly inhibited secretion of IL-4 and IL-5 (p < 0.001), and the inhibition by L.gaseri-44 was stronger. Thus, l.gaseri-44 can provide therapeutic and prophylactic effects on allergies by inhibiting secretion of Th 2-type cytokines that mediate allergic reactions.

EXAMPLE 10 analysis of IgE inhibition by Lactobacillus gasseri L.gaseri-44

Immunoglobulin E (IgE) is one of immunoglobulins, a major factor involved in allergic diseases. In general, the total amount of IgE in serum can be measured as one of methods for diagnosing allergic diseases. Thus, the present invention verifies the inhibitory effect of L.gaseri-44 on IgE secretion by using human B cell U266B1, commercial strain L.gaseri LBV150N as a control strain.

At 37℃with 5% CO ₂ U266B1 cells (ATCC No. TIB-196, available from Nanjac, bai Biotechnology Co., ltd.) were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (100. Mu.g/mL) and streptomycin (100. Mu.g/mL) under culture conditions, and subcultured 2-3 times. U266B1 cells were cultured at 5X 10 ⁵ The individual cell/well concentrations were seeded onto 24-well plates and then incubated for 12 to 18 hours for use.

In 24-well plates inoculated with U266B1 cells, each well was treated with 100. Mu.L of LPS (10. Mu.g/mL) and IL-4 (5 ng/mL). Then, 300. Mu.L of the previously prepared Lactobacillus gasseri strain solution or PBS,5% CO, was added to each well in a 24-well plate ₂ Co-incubation at 37 ℃. After 24-48 hours of incubation, the supernatants were collected and IgE levels were determined using the Human IgE ELISA kit (70-EK 175-48, multisciences).

IgE levels after lactobacillus gasseri treatment were compared to negative control groups and IgE inhibition was calculated according to the following formula:

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

As shown in FIG. 3, both L.gaseri-44 and the commercial strain L.gaseri LBV150N showed a significant IgE secretion inhibiting effect, and the inhibition effect of L.gaseri-44 was stronger than that of the negative control group. This experiment shows that L.gaseri-44 can provide therapeutic and prophylactic effects on allergic diseases by inhibiting IgE.

EXAMPLE 11 action of Lactobacillus gasseri L.gaseri-44 to reduce atopic dermatitis

The invention further studies the effect of L.gaseri-44 on reducing atopic dermatitis using animal models. 15 NC/Nga mice were followed by an atopic dermatitis NC/Nga mouse modelThe machine was divided into 3 groups, 5 each, model control group, positive control group and l.gaseri-44 dosing group, each with the hair removed from both ears and back of each mouse. Then, 200 μl of 1% dncb (dinitrochlorobenzene) solution (acetone: olive oil=1:3) was applied to the dehairing part of the mice 1 time a week for 6 times a total to induce atopic dermatitis. Mice in the model control group were perfused daily with 200 μl of PBS from the previous week of dermatitis induction; mice in the dosing group were gavaged daily with L.gaseri-44 1X 10 ⁸ CFU/only; meanwhile, 200. Mu.L of dexamethasone (60. Mu.g/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.gaseri-44 dosing group were measured weekly, and the scratch time and skin thickness of the mice were measured at weeks 3, 7 after the gavage of the probiotic bacteria, respectively.

The skin condition was monitored 1 time every 1 week for 4 weeks from week 3 of the gavage of probiotics. Four indicators of skin dryness, edema, erythema/hemorrhage (erythema/hemorrhage) and erosion/exfoliation (error/extraction) were examined. The status with no lesions was scored for 0, the mild status was scored for 1, the moderate status was scored for 2, the severe status was scored for 3, and the total score was evaluated. The results are shown in figure 4, showing that the dermatitis score of the l.gaseri-44 dosed group was significantly reduced compared to the model control group, indicating that l.gaseri-44 has an effect in treating atopic dermatitis.

To further verify whether the itching symptoms of the model mice could be alleviated after gavage of l.gaseri-44, the scratch time was measured by taking a video of the mouse model for 30 minutes 3 weeks after gavage of the probiotic bacteria. As a result, as shown in FIG. 5, the scratch time of the L.gasser-44 gavage mice was significantly reduced compared to the model control group, indicating that gavage L.gasser-44 significantly reduced the itching symptoms of atopic dermatitis.

After week 4 of gazeri-44, ear thickness and back skin thickness of mice were measured with calipers (calipers), and relief of edema symptoms was observed for each group of mice. The experimental results are shown in fig. 6A and 6B, and the ear thickness (fig. 6A) and back skin thickness (fig. 6B) of both the l.gaseri-44-dosed and dexamethasone-dosed mice are significantly reduced compared to the model control group.

EXAMPLE 12 evaluation of the therapeutic and prophylactic Effect of L.gasser-44 on asthma Using an Ovalbumin (OVA) -induced asthma mouse model

To verify the effect of L.gaseri-44 on allergic asthma, histopathological examination was performed using an OVA-induced asthma mouse model, while measuring the expression levels of IL-5 and IL-13.

Balb/c mice of 5-7 weeks of age were selected and adapted for 1 week and randomly divided into 3 groups of 10 per group, namely normal control group (control group-PBS; not inhaled OVA), OVA asthma model control group (OVA-PBS; inhaled OVA) and probiotic administration group (OVA-44). The normal control group and the asthma-induced control group were perfused with 200 μl of PBS daily and the dosing group was perfused with 200 μl of the. Gasser-44 bacteria solution daily before mice were sacrificed from the start of the experiment to day 31.

Mice were primed by starting the formal experiment after 1 week of acclimation, day 1 on the first day of the formal experiment, and injecting intraperitoneally 200 μl of phosphate buffer (pH 7.4) suspended with 2mg of aluminum hydroxide (ImjectTM Alum Adjuvant, thermosusher) and 20 μg ova (ovalbumin, sigma-aldrich) at day 7 and day 21, respectively. At day 28 to day 30, mice were stimulated by intranasal instillation with 1% OVA inhalation into the lungs for a total of 3 times. Pentobarbital treatment was performed 24 hours after the last stimulation (i.e. day 31) and then bronchial incisions were performed to collect lung tissue samples.

Infiltration of inflammatory cells consisting of eosinophils, neutrophils and macrophages was observed in antigen-treated bronchi. To verify the effect of l.gasser-44 on asthma, histopathological examination was performed on each group of lung tissue samples. Pathological results showed that in OVA asthma model control group (OVA-PBS), many inflammatory cells including eosinophils infiltrated around bronchioles, and hyperproliferative epithelial cells and thickened bronchial smooth muscle were also found; in the L.gaseri-44 administration group (OVA-44), infiltration of inflammatory cells was significantly reduced, bronchial tissue thickness was also reduced, and epithelial cells were hardly damaged, indicating that L.gaseri-44 had therapeutic and prophylactic effects on OVA-induced allergic asthma.

The number of immune cells from lung tissue samples collected from each group was determined using a flow cytometer (FACSAria III, BD). IL-5 was stained with antibodies directed against several markers (anti-mouse CD45, bioLegend; anti-mouse CD 3. Epsilon., BD; anti-mouse/human IL-5, bioLegend; anti-mouse IL-13, invitrogen) ⁺ CD4 ⁺ T、IL-13 ⁺ CD4 ⁺ T cells. IL-5 determination by counting IL-5 or IL-13 producing cells in lymphocytes having CD45 and CD3 epsilon as markers ⁺ And IL-13 ⁺ And (3) cells.

As shown in fig. 7A and 7B, both IL-5 and IL-13 levels were significantly increased in mice of OVA asthma model control group (OVA-PBS) compared to normal control group (PBS); the significantly reduced levels of both total IL-5 and total IL-13 in mice of the L.gaseri-44 dosing group (OVA-KBL 693) compared to the asthma model control group, demonstrates that the L.gaseri-44 strain of the invention can exert its therapeutic and prophylactic effects on allergic asthma by inhibiting secretion of IL-5 and IL-13, th2 type cytokines that mediate the allergic response.

Example 13 evaluation of the therapeutic and prophylactic effects of L.gasser-44 on asthma Using Dermatophagoides pteronyssinus (HDM) induced asthma model

House dust mites are allergens that are the primary cause of extrinsic asthma. To study the effect of L.gaseri-44 on allergic asthma, the mouse model of HDM-induced asthma was used to evaluate airway hypersensitivity and determine CD45 ⁺ Ratio of eosinophils in cells, CD4 ⁺ IL-5 in T cells ⁺ CD4 ⁺ Ratio of T cells and CD4 ⁺ IL-13 in T cells ⁺ CD4 ⁺ T cell ratio.

Balb/c mice, 5-7 weeks old, were purchased and adapted for 1 week, randomized into 3 groups, normal control group (nasal drop PBS), asthma-induced control group (nasal drop HDM-PBS), and L.gaseri-44 dosing group (HDM-44), 6 mice in each group were used to evaluate Airway Hyperresponsiveness (AHR), and 10 mice in each group were used to evaluate immune cells in the lungs.

After 1 week of adaptation, mice in the normal control group and asthma model control group were daily perfused with PBS (day 1-18) and mice in the L.gaseri-44 dosing group were daily perfused with L.gaseri-44 (day 1-18), and 10. Mu.g HDM (House test mite, greer) in 50. Mu.L phosphate buffer (pH 7.4) was suspended intranasally in day7 for sensitization. 1 week after autopsy, 50. Mu.L of phosphate buffer suspended with 10. Mu.g HDM was inhaled into the lungs by intranasal drops for 5 days (day 14-18). Mice were treated with pentobarbital 24 hours after the last stimulus sensitization (day 19) and then evaluated for Airway Hyperresponsiveness (AHR) and bronchoincisions were performed to collect lung tissue samples.

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

As shown in fig. 8, as methacholine concentration increases, AHR (RL) in the normal control group (CTRL) increases slowly, while AHR in the model control group (HDM) increases rapidly; in the L.gaseri-44 gavage group (HDM+44), AHR was significantly reduced compared to the asthma model control group (HDM), and the reduction in AHR was significant when treated with high concentrations of methacholine, indicating that L.gaseri-44 is effective in inhibiting AHR causing asthma and thus can be effectively used for the treatment and prevention of allergic asthma.

Immune cells in the lung were determined using a flow cytometer (FACSAria III, BD). Eosinophils and IL-5 were treated with murine antibodies (anti-mouse CD45, bioLegend; rat anti-mouse Siglec-F, BD; anti-mouse CD11b, BD; anti-mouse CD3 ε, BD; anti-mouse TCRβ, bioLegend; anti-mouse CD4, bioLegend; anti-mouse IL-5, bioLegend; anti-mouse IL-13, invitrogen) ⁺ CD4 ⁺ T、IL-13 ⁺ CD4 ⁺ T cells were stained. By Siglec-f in cells expressing the common leukocyte marker CD45 ⁺ CD11b ⁺ Eosinophils were determined by cell count and by counting CD4 with CD3 epsilon, TCR beta and CD4 as markers ⁺ Counting IL-5 or IL-13 producing cells in T cells to determine IL-5 ⁺ CD4 ⁺ T、IL-13 ⁺ CD4 ⁺ T cell number.

The experimental results are shown in FIGS. 9A-9C, which show that immune cells, i.e., eosinophils, IL-5, were present in lung tissue of the asthma model control (HDM) mice as compared to the normal control (PBS) ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ The proportion of T cells is obviously improved; in contrast to the asthma model control group, the gavage L.gasser-44 group had a reduced tendency to IL-5 in addition to eosinophils ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ The proportion of T cells is significantly reduced, indicating that L.gaseri-44 can inhibit inflammatory cells, i.e. eosinophils, IL-5 ⁺ CD4 ⁺ T cells and IL-13 ⁺ CD4 ⁺ T cells exert therapeutic and prophylactic effects on allergic asthma.

Claims (36)

1. A lactobacillus gasseri (Lactobacillus gasseri) strain comprising the 16S rRNA sequence with the nucleotide sequence of SEQ ID No. 1.

2. The strain of claim 1 isolated from genital secretions.

3. The strain of claim 1 having one or more of the following properties: (i) Survival rates of at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% after 3 hours incubation in simulated gastric fluid; (ii) Survival after 4 hours incubation in simulated intestinal fluid is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% or 96%; (iii) It has a higher adhesion capacity to Caco-2 cells, VK2/E6E7 cells or both than L.gasteriLBV 150N; (iv) Compared to l.gasser LBV150N, it has a higher bacteriostatic ability against escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atopoella vaginalis, gardnerella vaginalis, malassezia furfur, or any combination thereof; (v) Has remarkable inhibitory effect on gardnerella vaginalis, candida albicans or any combination thereof, and can restore the lactobacillus quantity to above normal level and relieve vulva Symptoms such as edema, excessive secretion and the like, and can recover the damage of the vaginal mucosa; (vi) It has a stronger inhibitory effect on antigen-induced histamine release, secretion of Th2 type cytokines (IL-4 and/or IL-5) and/or secretion of IgE than L.gassseriLBV 150N; (vii) Reducing dermatitis score, scratching time and skin thickness, significantly reducing atopic dermatitis; (viii) Has therapeutic and prophylactic effects on allergic asthma induced by Ovalbumin (OVA), and exerts its therapeutic and prophylactic effects on allergic asthma by inhibiting secretion of IL-5 and IL-13, which are Th2 type cytokines mediating allergic reactions; and (ix) by inhibiting airway hyperresponsiveness and/or inhibiting inflammatory cells (e.g., eosinophils, IL-5) in House Dust Mite (HDM) -induced allergic asthma ⁺ CD4 ⁺ T cells and/or IL-13 ⁺ CD4 ⁺ T cells) to exert therapeutic and prophylactic effects on allergic asthma.

4. A Lactobacillus gasseri (Lactobacillus gasseri) strain with a preservation number of CGMCC No.19529.

5. A method of culturing the strain of any one of claims 1-4, comprising culturing the strain in a medium.

6. The method of claim 5, wherein the medium is MRS medium.

7. The method of claim 5, wherein the culturing is performed under anaerobic conditions.

8. The method of claim 5, wherein the culturing is performed at 37 ℃.

9. A derivative of the strain of any one of claims 1-4.

10. The derivative of claim 9, which is a culture, lysate, extract, inactivation product, or a combination thereof.

11. A culture medium comprising the strain of any one of claims 1-4 or the derivative of claim 9 or 10.

12. A composition comprising an effective amount of a first component, wherein the first component comprises the strain of any one of claims 1-4, the derivative of claim 9 or 10, or the culture medium of claim 11.

13. The composition of claim 12, wherein the composition is a food composition, a health food composition, a pharmaceutical composition, a special medical use food composition, a cosmetic composition, a medical device composition, or a feed composition.

14. The composition of claim 12, wherein the composition is in the form of a pill, tablet, lozenge, lyophilized powder, granule, 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 mist, effervescent tablet, transdermal therapeutic system, microcapsule, implant, or stick.

15. The composition of claim 12, wherein the composition is formulated for ocular, otic, intranasal, sublingual, oral, transdermal, topical, nasal, rectal, or parenteral administration.

16. The composition of claim 12, wherein the composition further comprises a second component.

17. The composition of claim 16, wherein the second component comprises a probiotic, a metagen, a prebiotic, an antimicrobial agent, an immunomodulator, an anti-cancer agent, an osteoporosis therapeutic agent, a mental area associated therapeutic agent, a developmental associated therapeutic agent, or a combination thereof.

18. The composition of claim 16, wherein the weight ratio of the first component to the second component is from 1:99 to 99:1.

19. The composition of claim 16, wherein the first component is administered before, after, or simultaneously with the second component.

20. Use of a strain according to any one of claims 1 to 4, a derivative according to claim 9 or 10, a medium according to claim 11 or a composition according to any one of claims 12 to 19 for the preparation of a medicament for antagonizing pathogens.

21. Use of a strain according to any one of claims 1 to 4, a derivative according to claim 9 or 10, a medium according to claim 11 or a composition according to any one of claims 12 to 19 for the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with a pathogen.

22. The use of claim 20 or 21, wherein the pathogen is selected from the group consisting of: bacteria, fungi, viruses, spirochetes, mycoplasma, rickettsia, chlamydia, and parasites.

23. The use of claim 22, wherein the pathogen is selected from the group consisting of: mycobacterium (Mycobacterium), salmonella (Salmonella), escherichia coli (E.coli), chlamydia (Chlamydia), staphylococcus (Staphylococcus), bacillus (Bacillus), pseudomonas (Pseudomonas), candida (Candida), acetobacter (Atopobium), gardnerella (Gardnerella) and Malaromyces (Pictyrosporum).

24. The use of claim 22, wherein the bacteria comprises escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhi, atoposi's colporus, gardnerella vaginalis resistant bacteria, or a combination thereof.

25. The use according to claim 22, wherein the fungus comprises candida albicans, malassezia furfur, or a combination thereof.

26. The use of claim 22, wherein the parasite is a trichomonas.

27. The use of claim 21, wherein the pathogen-associated disease or disorder is selected from the group consisting of: female genital tract infections and genital tract flora disorders.

28. The use of claim 21, wherein the pathogen-associated disease or disorder is a malassezia infection-associated skin disorder.

29. Use of a strain according to any one of claims 1 to 4, a derivative according to claim 9 or 10, a medium according to claim 11 or a composition according to any one of claims 12 to 19 for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with immunomodulation.

30. The use of claim 29, wherein the disease or disorder associated with immune modulation is cancer, allergic disease, or autoimmune disease.

31. The use of claim 30, wherein the cancer is selected from the group consisting of: prostate cancer, stomach-esophagus cancer, lung cancer, liver cancer, pancreas cancer, breast cancer, bronchus 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia, lymphoma, and myeloma.

32. The use of 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.

33. The use of claim 30, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, rheumatic fever, lupus, systemic scleroderma, atopic dermatitis, psoriasis, psoriatic arthritis, asthma, guillain-Barre 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, wechs granulomatosis, sjogren's syndrome, addison's disease, crohn's disease, white plug disease, edema, conjunctivitis, periodontitis, rhinitis, otitis media, chronic sinusitis, sphagitis, tonsillitis, bronchitis, pneumonia, gastric ulcers, gastritis, colitis, gout, eczema, acne, contact dermatitis, seborrheic dermatitis, ankylosing spondylitis, fibromyalgia, osteoarthritis, scapulohumeral periarthritis, tendinitis, tenositis, hepatitis, cystitis, nephritis, sepsis, vasculitis, and bursitis.

34. Use of a strain according to any one of claims 1-4, a derivative according to claim 9 or 10, a medium according to claim 11 or a composition according to any one of claims 12-19 for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with osteoporosis.

35. The use of claim 34, wherein the osteoporosis related disease or disorder is selected from the group consisting of: juvenile osteoporosis, menopausal osteoporosis, postmenopausal osteoporosis, posttraumatic osteoporosis, and osteoporosis due to aging, corticosteroid therapy, and inactivity.

36. Use of a strain according to any one of claims 1 to 4, a derivative according to claim 9 or 10, a medium according to claim 11 or a composition according to any one of claims 12 to 19 for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder associated with iron deficiency anemia, climacteric syndrome or a disease of the central nervous system.

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