CN117535207A

CN117535207A - Lactobacillus gasseri and application thereof

Info

Publication number: CN117535207A
Application number: CN202410009107.6A
Authority: CN
Inventors: 王琼; 曾婉秋; 高翔
Original assignee: Sichuan Anaerobic Biotechnology Co ltd
Current assignee: Sichuan Anaerobic Biotechnology Co ltd
Priority date: 2024-01-04
Filing date: 2024-01-04
Publication date: 2024-02-09

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus gasseri and application thereof. The lactobacillus gasseri is lactobacillus gass-10 with a preservation number of CCTCC NO: M20231494, the strain is not hemolytic, has NO toxic factor and drug resistance gene, has good safety, can produce lactic acid and inhibit genital tract pathogenic bacteria, namely gardnerella vaginalis, has an important function on adjusting genital tract pH, and has the potential of preventing or/and treating female genital tract diseases.

Description

Lactobacillus gasseri and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus gasseri and application thereof, in particular to application in female genital tract health.

Background

The female genital tract is an open cavity tract, is lodged with a large number of different kinds of microorganisms, is one of main distribution areas of human microorganisms, and is closely related to female reproductive health. About 300 or more microorganisms are symbiotic in the female genital tract, including bacteria, viruses, fungi, etc., with bacteria being the predominant microorganism. They are constrained and balanced with each other to form dynamic balance, and various vaginitis is related to the imbalance of the vaginal microecological environment.

The microbial composition of the genital tract varies from site to site, and in healthy women of childbearing age most bacteria are present in the lower genital tract (vagina and cervix) and the bacteria in the upper genital tract have not been well characterized to date. The vaginal microbiota has low microbial diversity, and is mainly composed of lactobacillus. The vaginal flora can be generally divided into five community (community state types, CST) types. CST-I is mainly composed of Lactobacillus crispatus, CST-II is mainly composed of Lactobacillus gasseri, CST-III is mainly composed of Lactobacillus jensenii, CST-V is mainly composed of Lactobacillus jensenii, and CST-IV has no dominant Lactobacillus group and can be further subdivided into CST IV-A and CST IV-B subtypes, wherein CST IV-B is closely related to bacterial vaginosis (Bacterial Vaginosis, hereinafter referred to as "BV") and mainly composed of anaerobic bacteria such as Bacillus mirabilis, goldebrand bacteria and Gardnerella.

In disease states, the composition of microorganisms and biomass load vary greatly, and multiple pathogens overgrow. The vaginal microecological environment enters a fragile state, is not easy to resist the propagation and invasion of pathogenic bacteria, and causes various vaginal inflammations. Bacterial vaginosis is a common gynecological disease, the infection rate is 15% -52%, and the bacterial vaginosis is a vaginal infectious disease of clinical syndrome caused by dysbacteriosis in vagina due to excessive reproduction of gardnerella vaginalis and other anaerobic bacteria as main substances to replace lactobacillus. BV is reported to be a risk factor for histological choriitis, amniotic fluid infection, post-cesarean endometritis, and other pregnancy defects and pregnancy complications.

Aiming at BV, the clinical treatment method adopts metronidazole or clindamycin, wherein the metronidazole is a precursor drug, and in an anaerobic environment, the nitro of the metronidazole is reduced to amino by the enzymatic reduction in bacterial cells, so that antibiotics are converted into active forms, and then the helical structure of the antibiotics is destroyed by covalent bonding with pathogen DNA, single strand and double strand breaks are caused, so that the pathogen DNA is degraded and dead; clindamycin can bind to the 50S ribosomal subunit on the bacterial ribosome, preventing extension of the peptide chain, thereby inhibiting protein synthesis by bacterial cells, resulting in bacterial death. The treatment with antibiotics is quick in effect, but has great defects, and has the following two aspects: (1) The vaginal microecology is not restored to a healthy equilibrium state which can resist pathogenic bacteria invasion after treatment, and the inhibited or killed pathogenic microorganisms or external pathogenic microorganisms can be further propagated and even pathogenic to cause recurrence or new vaginal inflammation; (2) The microorganisms produce drug resistance, and antibiotics cannot balance the vaginal microecological environment, resulting in refractory BV. Thus, although the antibiotic treatment takes effect quickly, the recurrence rate is high, and the recurrence rate is up to 30% within 3 months.

The treatment of the vaginal microecological imbalance comprises three steps of sterilization, mucous membrane repair and restoration of vaginal microecological balance. Sterilization is the first step in the treatment of vaginal inflammation, and inhibits or kills pathogenic microorganisms including hyperproliferative aerobic and anaerobic bacteria, blastospores or hyphae, trichomonas, and the like. After the pathogenic microorganisms are inhibited or killed, the immune repair of the vaginal mucosa and the recovery of dominant lactobacillus are the final targets for treating the colpitis. During this period, if the repair of the vaginal mucosa, the recovery process of lactobacillus is affected, the physical and chemical environment in the vagina is not restored to normal, the suppressed pathogenic microorganisms or the external pathogenic microorganisms can also reproduce again or even cause diseases to cause recurrence or new vaginal inflammation. And probiotics can rapidly occupy the receptors of vaginal epithelium in the vagina to generate a protective effect on the vagina, thereby promoting the vagina to restore to the normal microenvironment and reducing the recurrence of colpitis. Thus, the use of probiotic micro-ecological agents for the treatment of BV is a more preferred approach.

At present, only 2 vaginal microecological medicines are marketed in China, wherein one active ingredient is streptococcus enteritidisStreptococcus faecalis) The method comprises the steps of carrying out a first treatment on the surface of the The other active component is Lactobacillus delbrueckiiLactobacillus delbrueckii). In addition, some drug lines are in clinical development, e.g. EuropeanLiving fungus capsules containing Lactobacillus crispatus Lc262-1 of Saicoraceae recently completed phase 3 clinical trials, KAL-001 living fungus capsules containing 4 dominant lactobacilli from Sichuan anaerobic organisms were under phase 2 study. In addition to pharmaceuticals, some oral probiotics have emerged in the art, such as UREX and astart developed in the coltson, which have been widely used in various oral products for female genital tract health, the principle of which is reported to be the transmission of probiotics to the vagina via the oral-intestinal-anal route.

Chinese patent document CN102851248A discloses a lactobacillus jensenii for the control of bacterial vaginosis. Chinese patent document CN107794236A discloses a Lactobacillus crispatus and an application thereof. Chinese patent document CN108004187a discloses a lactobacillus gasseri and its use for preparing a vaginal antibacterial drug.

Strains are specific according to the introduction of probiotics by WGO (worldwide gastrointestinal organization) clinical guidelines. Even though the same strain is taxonomically different, its function may have a large difference. Thus, it is necessary to continually make updated iterations of probiotics to provide the desirer with better choices for strains that have been reported to be better performing for the species of efficacy.

Disclosure of Invention

One of the purposes of the invention is to provide a lactobacillus gasseri @ cellLactobacillus gasseri) The strain is selected from lactobacillus gasseri Lgass-10 with a preservation number of CCTCC NO: M20231494.

In some embodiments, the strain has a 16S rDNA sequence as set forth in SEQ ID NO. 1.

The second purpose of the invention is to provide the lactobacillus gasseriLactobacillus gasseri) The method for culturing the strain comprises the steps of inoculating the lactobacillus gasseri strain into a culture medium, and carrying out proliferation culture to obtain the proliferated lactobacillus gasseri strain.

In some embodiments, the medium is MRS medium.

The third object of the present invention is to provide a food, health product or pharmaceutical composition, wherein the active ingredient of the composition comprises the lactobacillus gasseri strain or the lactobacillus gasseri strain obtained by the culture method.

In some embodiments, the active ingredient of the composition further comprises one or more selected from the group consisting of lactobacillus crispatus, lactobacillus jensenii, lactobacillus johnsonii and lactobacillus delbrueckii.

In some embodiments, the lactobacillus gasseri strain is the sole active ingredient.

In some embodiments, the composition comprises 10 in a single formulation ⁶ ~10 ¹⁵ Lactobacillus gasseri strain of CFU.

The fourth object of the invention is to provide the application of the lactobacillus gasseri strain in preparing female genital tract health products.

In some embodiments, the female genital tract health product acts as a bacteriostatic or bacteriocidal agent.

In some embodiments, the female genital tract health product is used to inhibit or kill gardnerella vaginalis.

In some embodiments, the female genital tract health product is for modulating the acidic environment of the genital tract.

In some embodiments, the Lgass-10 strain of the invention has the following identifying characteristics:

1. white colonies with round shape, moist surface, translucency and neat edges are formed on the MRS culture medium;

2. gram staining was positive, rod-like, single or paired.

The lactobacillus gasseri Lgass-10 has no virulence factor, no drug resistance gene, no hemolysis and good safety; has strong lactic acid producing capacity, can reduce pH and maintain weak acid healthy environment of genital tract; and can inhibit the growth of pathogenic bacteria Gardnerella vaginalis.

The strain preservation information of the invention is as follows:

strain name: lactobacillus gasseri @Lactobacillus gasseri）Lgass-10；

Preservation date: 2023, 08, 17;

identification of survival date: 2023, 24 th 08;

preservation unit: china center for type culture collection (China Center for Type Culture Collection, CCTCC), address: university of martial arts, hubei province, post code: 430072, telephone: 027-68754052;

preservation number: cctccc No. M20231494.

Drawings

FIG. 1 is a front photograph of a colony form of Lactobacillus gasseri Lgass-10.

FIG. 2 is a photograph of a gram stain of Lactobacillus gass Lgass-10.

Detailed Description

Definition and description

The lactobacillus gasseri strain of a particular accession number claimed in the present invention, the meaning of which includes but is not limited to:

1. lactobacillus gasseri with CCTCC NO: M20231494 deposited in the collection centerLactobacillus gasseri) Lgass-10 strain;

2. a Lactobacillus gasseri strain having the same genome as the Lgass-10 strain;

3. the passaging strain without mutation based on the aforementioned 1 or 2;

4. a passaging strain based on the aforementioned 1, 2 or 3 that accumulates minute mutations in passaging, but has no substantial change in toxicity, immunogenicity and biological activity; and

5. the live or inactivated form of the strain according to any one of the foregoing 1 to 4, and may be a whole cell or a derivative such as a lysate or a fermentation product.

As known in the art, strains inevitably introduce minor mutations by the use of progeny, and when mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect strain toxicity, immunogenicity and biological activity (e.g., residues that may be linked amino acid residues between two domains, or are located within the higher structure of the protein and do not affect toxicity, immunogenicity and biological activity by virtue of not contacting immune cells), it is reasonable to expect that these minor changes do not significantly affect toxicity, immunogenicity and biological activity of the progeny strain, and are derived from the strains contributed by the invention and therefore remain within the substantial technical contribution of the invention. These minor mutations remain insubstantial mutations and should be considered as mutant strains that have no alterations in toxicity, immunogenicity, and biological activity.

There is no substantial change in toxicity, immunogenicity, and biological activity, including, but not limited to, regarding toxicity, immunogenicity, and biological activity as being the same within the limitations and acceptable or unavoidable errors of detection techniques such as detection sensitivity, detection limits, and the like. The toxicity, immunogenicity and biological activity of the Lgass-10 strain offspring were determined by cells, animals, etc., and the expected or unavoidable systematic errors were attributed to insubstantial changes due to differences in cell lines, animal species, age, sex, health conditions, culture conditions, etc.

The composition of the invention contains an active ingredient Lgass-10 strain and other ingredients, such as auxiliary ingredients without physiological effects or other functional ingredients. Functional ingredients include, but are not limited to, other functional strains, or prebiotics, metazoan ingredients, and the like.

As a preferred mode, the composition of the invention contains other active lactobacilli, such as one or more selected from the group consisting of Lactobacillus crispatus, lactobacillus jensenii, lactobacillus johnsonii and Lactobacillus delbrueckii.

The auxiliary materials comprise a drug carrier and an excipient. A pharmaceutical carrier refers to a pharmaceutical carrier that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered probiotic. The pharmaceutically acceptable carrier may enhance or stabilize the composition or may be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers can include solvents, dispersion media, coatings, surfactants, antioxidants, isotonic agents, absorption delaying agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., remington's Pharmaceutical Sciences, 18 th edition MackPrinting Company,1990, pages 1289-1329). Unless the conventional carrier is incompatible with the active ingredient, it is contemplated that it will be used in a therapeutic or pharmaceutical composition. The carrier may be selected to minimize adverse side effects in the subject and/or minimize inactivation of the active ingredient.

An excipient refers to a substance that is added to a pharmaceutical composition to give the drug a certain shape or a certain concentration. Such as sterile water, physiological saline, polyalkylene glycols (such as polyethylene glycol), vegetable oils or hydrogenated naphthalenes, calcium bicarbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, and the like.

The composition of the present invention may be prepared in any form convenient for use, such as powder, tablet, granule, gel, capsule or liquid, which are common in clinical or food.

The compositions of the invention are administered to a subject in an amount (e.g., therapeutically effective amount) and frequency effective to exert efficacy, preferably in a single dose of 10 ⁶ ~10 ¹⁵ CFU、10 ⁷ ~10 ¹³ CFU or 10 ⁷ ~10 ¹² Lactobacillus gasseri of CFULactobacillus gasseri）。

The specific temperature parameters in the present invention, unless specified otherwise, are understood to be constant temperature treatments and allow for variations within a certain temperature interval. Such as within a range of + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by a person skilled in the art without making creative efforts based on the embodiments in the present invention shall fall within the protection scope of the present invention.

The materials used in the following examples were formulated or commercially available as follows:

MRS broth preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g, dissolving into 1L distilled water; heating to boil, cooling to room temperature, adding 0.55 g cysteine hydrochloride, stirring to dissolve, and adjusting pH to 6.5; mounting and fixing deviceMeasuring and separating device and introducing N ₂ Heating to boil, boiling for 20 min, cooling, packaging into 10 mL anaerobic tube, sterilizing at 118 deg.C under moist heat for 20 min, storing in shade and in dark place.

MRS solid culture medium preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g and agar powder 15.0 g, dissolving into distilled water 1L, boiling, adding cysteine hydrochloride 0.55 g after boiling, adjusting pH to 6.5, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark for use.

Preparation of LBS solid medium: the LBS finished product culture medium (Qingdao high-tech industrial park Haibo biotechnology Co., ltd., HB 0385) powder 84.0 g is weighed, then 1. 1 mL Tween 80 and 1.3 mL glacial acetic acid are absorbed, heated and stirred to be dissolved in 1000 mL distilled water, and the culture medium is used in the same day without high-pressure sterilization. The next day, the autoclave was used at 118℃for 15 minutes.

Hydrogen peroxide semi-quantitative medium preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g, agar powder 15.0 g, dissolving in 1L distilled water, adjusting pH to 6.5, sterilizing at 118 deg.C under high temperature and humidity for 20 min, placing into a 50deg.C water bath after sterilization, maintaining for 30 min, adding 3,3', 5' -tetramethyl benzidine (TMB) (final concentration is 0.25 mg/mL) and horseradish peroxidase (HRP) (final concentration is 0.01 mg/mL), and mixing; after cooling and solidifying, the names and the preparation dates of the culture mediums are marked, and the culture mediums are placed in a refrigerator at 4 ℃ for standby.

Preparation of anaerobic PBS: weighing potassium dihydrogen phosphate 0.27 and g, disodium hydrogen phosphate 1.42 and g, sodium chloride 8 and g, and potassium chloride 0.2 and g, dissolving in distilled water 1 and L, heating and boiling, cooling to room temperature, adding cysteine hydrochloride 0.55 and g, stirring and dissolving, adjusting pH to 6.5, loading into quantitative liquid separator, and introducing N ₂ Heating to boil, boiling for 30 min, cooling, packaging into 10 mL anaerobic tube, sterilizing at 121deg.C under moist heat for 30 min, and storing in shade and in dark place.

Preparing an anaerobic BHI liquid culture medium: weighing 37.0 g of BHI product culture medium (Thermo Scientific ™, CM 1135B) powder, dissolving in 1L distilled water, heating to boil, cooling to room temperature, and adding 0.55 gCysteine hydrochloride, stirring to dissolve, adjusting pH to 6.5, adding quantitative liquid separator, and introducing N ₂ Heating to boil, boiling for 20 min, cooling, and packaging with N ₂ And CO ₂ Packaging into 10 mL anaerobic tubes at a ratio of 1:1, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark for use.

Preparing an anaerobic BHI semi-solid culture medium: weighing BHI finished medium (Thermo Scientific ™, CM 1135B) powder 37.0 g, dissolving into 1L distilled water; heating to boil, cooling to room temperature, adding 6 g agar powder, 0.55 g cysteine hydrochloride, stirring to dissolve, adjusting pH to 6.5, adding quantitative liquid separator, and introducing N ₂ Heating to boil, boiling for 20 min under slight boiling condition, slightly cooling, and packaging with N ₂ And CO ₂ Packaging into 10 mL anaerobic tubes at a ratio of 1:1, sterilizing at 118 deg.C under moist heat for 20 min, and storing in shade and in dark place.

Example 1 isolation and identification of strains

Collecting vaginal secretion samples of 20-40 years old Chinese healthy women by using a vaginal cotton swab; taking 2 mL aseptic and anaerobic PBS buffer solution in an anaerobic tube filled with the cotton swab, fully vibrating and uniformly mixing, and continuously carrying out ten-fold gradient dilution on the buffer solution; coating 100 μL of 10000-fold diluted liquid on LBS solid culture medium, culturing in anaerobic incubator at 37deg.C for 48 h, selecting several single colony of lactobacillus, culturing in MRS broth culture medium for 24 h, transferring part of the cultured bacterial liquid for continuous culture, extracting bacterial DNA from the other part of the bacterial liquid, transferring to Beijing Qingzhu biological science and technology Co., ltd for sequencing, and comparing PCR result with BLAST to obtain 4 strains of Lactobacillus gasseriLactobacillus gasseri) Designated as Lgass-10, G11, G13 and G29, respectively.

And (3) marking bacterial liquid after PCR verification of the Lgass-10 strain to an MRS solid culture medium, and carrying out anaerobic culture for 24-48 hours at 37 ℃, wherein the bacterial colony is a white bacterial colony with round shape, moist surface, translucency and neat edge, and a front photograph is shown in figure 1.

1 loop Lgass-10 strain is taken and dropped on a glass slide, and a proper amount of sterile ddH is added dropwise ₂ O is coated as a thin bacterial liquid layer, and the glass slide is placed on an alcohol lamp and heated until water evaporates, and the glass slide passes through the flame for 2-3 times rapidly to fix the bacterial cells. The staining was then performed according to the gram staining kit instructions (Guangdong CycloKai microorganism technologies Co., ltd., 029010). Microscopic examination and photographing, and the bacterial staining mirror is used for detecting gram staining positive, rod-shaped, single or paired arrangement, as shown in figure 2.

EXAMPLE 2 Whole genome and novel analysis of strains

Lactobacillus gasseri Lgass-10 was inoculated into MRS broth of 5 mL, cultured to the late logarithmic growth phase, and strain whole genome DNA was extracted while performing second and third generation whole genome sequencing. After assembly and annotation, protein sequences were entered into virulence gene libraries Virulence Factor Databases (VFDB) and The Comprehensive Antibiotic Resistance Database (CARD) for virulence factor and drug resistance gene analysis, respectively. The results show that the bacterium does not have virulence factors and drug resistance genes.

The novel analysis of the strain was performed using the average nucleotide similarity (Average Nucleotide Identity, ANI). By searching in Genbank, 119 published results were foundLactobacillus gasseri Whole genome, 2 strains were found to be most similar to L.griseus Lgass-10 whole genome and below 99.9% by fastanI (v 1.33) comparison, GCA_000175055.1 (99.7457%) and GCA_022752855.1 (99.6031%), respectively. Thus, lactobacillus gasseri Lgass-10 was considered to be a novel strain, and its 16S rDNA sequence was shown in SEQ ID NO. 1.

Example 3 Low pH growth tolerance experiment

Activating the preserved Lgass-10 with MRS broth with pH value of 6.5, and culturing overnight at 37 ℃; transferring the activated bacteria liquid into MRS broth culture medium with pH value of 4-5 according to 10% inoculum size, and measuring OD every 2-3 h ₆₀₀ Values.

The results show that: the lactobacillus gasseri Lgass-10 can grow in a low pH environment (pH value is 4-5), and the pH value of the culture solution after the culture is finished is 2.5, so that the lactobacillus gasseri Lgass-10 has the acid-resistant and acid-producing characteristics.

Example 4 oxygen tolerance

Respectively transferring each strain in aerobic environmentPlacing into 96-well plate containing aerobic MRS broth culture medium, arranging three bacteria in parallel, adding deoxidized bag into sealed container, culturing 48 h, and OD ₆₀₀ And (5) monitoring. The value of DS (Lactobacillus delbrueckii DS isolated from commercial "live Lactobacillus vaginalis capsule" ("Dijunsheng") was calculated as 2, 3 better than DS tolerance, 1 slightly weaker than DS tolerance, and 0 difficulty in growth in oxygen-containing medium (added oxygen-scavenging bag).

The results show that: lgass-10 is equivalent to DS oxygen tolerance, and is designated as 2. It is explained that Lactobacillus gasseri Lgass-10 belongs to facultative anaerobes, the culture operation of Lgass-10 is not required to be carried out in an anaerobic environment in the whole course, and the preparation of a culture medium is not required to strictly remove oxygen.

EXAMPLE 5 Hydrogen peroxide production capability

Test strain: lactobacillus delbrueckii DS isolated from commercial "live Lactobacillus vaginalis capsule" ("Dijunsheng"), lactobacillus gass-17 in patent CN110656060B, and another 3 strains of Lactobacillus gasseri G11, G13 and G29 isolated according to the present invention.

After lactobacillus is activated, 2 mu L of bacteria liquid is absorbed by a liquid transfer device to be inoculated in MRS agar containing 0.25 mg/mL of 3,3', 5' -tetramethyl benzidine solution and 0.01 mg/mL of horseradish peroxidase, the dishes are placed in the same anaerobic sealed tank, an anaerobic gas producing bag is added, the culture is carried out at 37 ℃, the corresponding dishes are taken out at 48 h, the dishes are exposed to air, the color reaction is observed after 30 min, and the record is photographed: with Lactobacillus delbrueckii DS as a positive control, the blue color produced by Lactobacillus delbrueckii was measured as 4 points, the blue color produced by Lactobacillus delbrueckii was measured as 3 points, the blue color produced by Lactobacillus delbrueckii was measured as 2 points, the developed blue color was measured as 1 point (slight color development reaction) very weakly, and the color was measured as 0 point.

The results show that: lactobacillus gasseri Lgass-10 has the ability to produce hydrogen peroxide.

TABLE 1 Hydrogen peroxide production capability

Strain	Hydrogen peroxide production score-48 h
		Lgass-10	2
G11	2
		G13	0
G29	1
		Lgass-17	1
DS	3

EXAMPLE 6 hemolysis experiment

The deposited lactobacillus gass Lgass-10 was inoculated into an MRS broth of 5 mL with enterococcus faecalis (beta hemolysis, cic 23658, purchased from the chinese industrial microbiological bacterial collection center) as positive control and a blank medium as negative control. Anaerobic culture of 12 h in MRS broth culture medium at 37deg.C gave an activated strain. 2.5. Mu.L of the activated strain was inoculated onto Columbia blood plates (Shanghai family, majia biotechnology Co., ltd.) and 3 replicates were set per group. Observations were made after anaerobic incubation at 37℃for 48 h.

The results show that: the colony of the positive control strain forms a completely transparent hemolytic ring with obvious limit, which is beta hemolysis. The medium surrounding the colony of Lactobacillus gasseri Lgass-10 was unchanged, and was gamma-hemolyzed, i.e.not hemolyzed.

Example 7 Gardnerella vaginalis inhibition test

After the strain is activated, 0.1 mL bacterial liquid is evenly mixed with a melted MRS solid culture medium, poured into a 6 cm plate, fully solidified and cultured at 37 ℃ for 48 h, the plate is taken out, and a puncher with the inner diameter of 6 mm is used for punching holes on the solid culture medium to obtain bacterial cakes; after the gardner vaginal bacteria (purchased from Beijing Bei Naghuang biological technology institute, BNCC 337545) are activated and transferred, the bacteria are diluted with an anaerobic sterile PBS buffer solution in a gradient of 10 times to obtain 10 ^-1 The dilution liquid 0.5 mL is evenly mixed with molten BHI solid culture medium containing 5% horse serum, poured into a 9 cm plate, after complete solidification, lactobacillus cakes are lightly placed on the surface of the BHI solid culture medium, 4 cakes are symmetrically placed on each plate, 2 strains are arranged in parallel, the plates are placed in an anaerobic sealed tank, an anaerobic gas producing bag is added, the plate is vertically placed for culturing 48 h, and the size of a bacteriostasis zone is measured by a vernier caliper.

The results show that: lactobacillus gasseri Lgass-10 has antibacterial effect on gardnerella vaginalis, and has better effect than other strains.

TABLE 2 Gardnerella vaginalis inhibition ability

Strain	Diameter (mm) -average value of inhibition zone
		Lgass-10	17.00
G11	14.27
		G13	13.67
G29	12.71
		Lgass-17	16.63
DS	14.29

Example 8 lactic acid production test

After the strain was activated, it was transferred to MRS broth, 2 strains were set in parallel, and cultured at 37℃for 48 h, the pH of the Lactobacillus strain solution cultured for 48 h was measured and recorded with a pH 0.5-5.0 test paper, and the strain was selected for liquid chromatography under the following two conditions. The supernatant was diluted 5-fold, pretreated with concentrated sulfuric acid, and filtered through a 0.22 μm needle filter before loading. The liquid chromatography related parameters are as follows:

instrument model: agilent, analytical liquid chromatography 1200

Chromatographic column model: bere, aminex HPX-87H

Mobile phase: 0.005 M H ₂ SO ₄ At a speed of 0.6 mL/min

Detector and detection wavelength: DAD,207 nm; RID, differential refractive signal

Sample injection amount: 20. mu L.

The results show that: lactobacillus gasseri Lgass-10 has the property of high lactic acid production, which is about twice the lactic acid production capacity of the reference strain DS, and is significantly superior to other control strains of the same genus.

TABLE 3 lactic acid producing ability

Strain	Average lactic acid content (mg/L)
		Lgass-10	20258.61
G11	11936.10
		G13	12192.32
G29	11822.66
		Lgass-17	14219.11
DS	12964.12

In conclusion, the lactobacillus gasseri Lgass-10 strain has acid resistance and strong lactic acid production capacity, and can adapt to and maintain the slightly acidic environment of the genital tract; the strain has better tolerance to oxygen and stronger tolerance in fermentation production or colonisation in genital tract; has good antibacterial activity to gardnerella which is a main pathogenic bacteria of vagina, is superior to other homologous strains and positive control DS strains, and can prevent or/and treat genital tract diseases related to gardnerella.

Claims

1. Lactobacillus gasseri @Lactobacillus gasseri) The strain is selected from lactobacillus gasseri Lgass-10 with a preservation number of CCTCC NO: M20231494.

2. The lactobacillus gasseri according to claim 1Lactobacillus gasseri) The strain culturing method comprises inoculating the strain into a culture medium, and performing proliferation culture to obtain the proliferated lactobacillus gasseri strain.

3. The culture method according to claim 2, wherein the medium is an MRS medium.

4. A food, health product or pharmaceutical composition containing the Lactobacillus gasseri strain of claim 1 as active ingredientLactobacillus gasseri) A strain or a strain of lactobacillus gasseri obtained by the culture method of claim 2.

5. The food, nutraceutical, or pharmaceutical composition of claim 4, wherein the active ingredient of the composition further comprises one or more selected from the group consisting of lactobacillus crispatus, lactobacillus jensenii, lactobacillus johnsonii, and lactobacillus delbrueckii.

6. The food, health product or pharmaceutical composition according to claim 4, characterized in that the lactobacillus gasseri isLactobacillus gasseri) The strain was used as the sole active ingredient.

7. The food, nutraceutical, or pharmaceutical composition of claim 4, wherein the composition comprises 10 in a single formulation ⁶ ~10 ¹⁵ Lactobacillus gasseri strain of CFU.

8. The Lactobacillus gasseri strain according to claim 1Lactobacillus gasseri) Use of a strain or a strain of lactobacillus gasseri obtained by the culture method of claim 2 for the preparation of a healthy product of the female genital tract.

9. The use according to claim 8, wherein the female genital tract health product is for inhibiting or killing gardnerella vaginalis or for modulating the acidic environment of the genital tract.