CN117535206B

CN117535206B - Lactobacillus salivarius and application thereof

Info

Publication number: CN117535206B
Application number: CN202410004784.9A
Authority: CN
Inventors: 曾婉秋; 王琼; 高翔
Original assignee: Sichuan Anaerobic Biotechnology Co ltd
Current assignee: Sichuan Anaerobic Biotechnology Co ltd
Priority date: 2024-01-03
Filing date: 2024-01-03
Publication date: 2024-03-29
Anticipated expiration: 2044-01-03
Also published as: CN117535206A

Abstract

The invention relates to the field of microorganisms, in particular to lactobacillus salivarius and application thereof. The Lactobacillus casei is characterized by having a preservation number of CCTCC NO: M20231492Limosilactobacillus urinaemulieris) The Lvagi-36 strain has no virulence factor, no drug resistance gene, no hemolysis and good safety. And has the capability of producing lactic acid and hydrogen peroxide, can resist low pH, has good antibacterial capability for genital tract pathogenic bacteria such as Gardner vagina bacteria, and can be applied to preparing female genital tract health products.

Description

Lactobacillus salivarius and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to lactobacillus salivarius and application thereof, and particularly relates to lactobacillus salivarius Lvagi-36 and application thereof 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 differentiation of dominant lactobacilli in healthy women of different territories and ethnicities can generally divide the vaginal flora 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 and 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 useIn the initiation phase, such as the living bacteria capsule containing Lactobacillus crispatus Lc262-1 of the European micro-family, the KAL-001 living bacteria capsule containing 4 dominant lactobacilli of Sichuan anaerobic organisms is in phase 2 study just recently completed in phase 3 clinical trial. 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.

Because of the abundant diversity of lactobacillus in the vagina of human body, different lactobacillus show different probiotics ability, and the synergy plays a role in the vaginal microenvironment. And the individual difference exists, and the dominant strains in different female vaginas are slightly different, so that the types of the lactobacillus and the probiotics of different strains need to be comprehensively considered when the corresponding lactobacillus for the vagina is selected. Lactobacillus salivarius is a lactobacillus isolated from the genital tract of healthy females and has not been reported in terms of female genital tract health to date.

Disclosure of Invention

The invention firstly provides a lactobacillus mucilaginosus which is a kind of lactobacillus mucilaginosusLimosilactobacillus urinaemulieris) The strain is selected from Lvagi-36 with a preservation number of CCTCC NO: M20231492.

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

The invention further provides a culture method of the lactobacillus casei strain, which comprises inoculating lactobacillus casei to a culture medium, and performing proliferation culture to obtain the proliferated lactobacillus casei strain.

In some embodiments, the medium is MRS medium.

The invention further provides a food, a health-care product or a pharmaceutical composition, and the active ingredient of the food, health-care product or pharmaceutical composition contains the lactobacillus casei strain of urinary tract mucus 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, lactobacillus delbrueckii, and lactobacillus grignard.

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

In addition, the invention also provides application of the lactobacillus urous or the composition 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 lactobacillus salivarius of the present invention has the following identifying characteristics:

1. microscopic examination of the strain to gram-positive Brevibacterium;

2. the colony forms in MRS culture medium are light white semitransparent circular colonies, the middle is convex, and the surface is smooth and moist.

The lactobacillus casei has no virulence factor, no drug resistance gene and no hemolysis, and has good safety. The lactic acid producing capability is strong, the pH value can be reduced, the acid resisting capability is strong, and the reproduction of pathogenic bacteria in genital tract can be inhibited.

The strain preservation information of the invention is as follows:

strain name: lactobacillus mucilaginosus in urinary tractLimosilactobacillus urinaemulieris）Lvagi-36；

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. M20231492.

Drawings

FIG. 1 is a front view of the colony morphology of Lactobacillus salivarius Lvagi-36.

FIG. 2 is a photograph of a gram stain of Lactobacillus salivarius Lvagi-36.

Description of the embodiments

Definition and description

Lactobacillus salivarius strains of a particular accession number as claimed in the present invention include, but are not limited to:

1. lactobacillus casei of urinary tract with CCTCC NO: M20231492 as the deposited microorganismLimosilactobacillus urinaemulieris) Lvagi-36 strain;

2. a strain of lactobacillus casei having the same genome as the lvagei-36 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;

5. based on the live or inactivated form of the strain according to any of the foregoing 1-4, it may be whole cells or derivatives such as lysates or fermentation products.

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 L vagi-36 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 varieties, age, sex, health conditions, culture conditions, etc.

The composition contains the active ingredient lactobacillus salivarius 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 prebiotic ingredients, post-prebiotic 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, lactobacillus delbrueckii and Lactobacillus grignard.

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 present invention are administered to a subject in an amount (therapeutically effective amount) and frequency effective to exert efficacy, preferably in a single dose of 10 ² ~10 ¹⁵ CFU、10 ⁴ ~10 ¹³ CFU or 10 ⁵ ~10 ¹² Urinary tract mucus lactobacillus of CFULimosilactobacillus urinaemulieris）。

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 configured 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; installing quantitative liquid separator 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.

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 BHI product culture medium (Thermo Scientific ™, CM 1135B) powder 37.0 g, dissolving in distilled water 1L, heating to boil, cooling to room temperature, adding cysteine hydrochloride 0.55 g, stirring to dissolve, adjusting pH to 6.5, loading into quantitative dispenser, 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 mu L of liquid diluted 10000 times on an MRS solid culture medium, placing the solid culture medium in an anaerobic incubator for culture at 37 ℃, after culturing at 48 and h, selecting a plurality of lactobacillus single colonies to respectively culture in an MRS broth culture medium for 24 and h, transferring one part of the cultured bacterial liquid for continuous culture, and extracting bacterial DNA from the other part of the bacterial liquid; amplifying and sequencing bacterial 16S rDNA gene, and comparing the sequencing result with BLAST to determine 3 strains of urinary tract mucus lactobacillusLimosilactobacillus urinaemulieris) Designated as lvagei-35, lvagei-36 and lvagei-42, respectively, and were each preserved with glycerol.

And (3) streaking Lvagi-36 bacterial liquid to an MRS solid culture medium, and carrying out anaerobic static culture at 37 ℃ for 24-48 h, wherein the colony form is a light white semitransparent circular colony, the middle part is convex, the surface is smooth and moist, and the front photograph is shown in figure 1. Taking 1-ring colony on glass slide, and dripping proper amount of sterile ddH 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, observation and photographing, and the bacterial staining mirror is used for detecting gram-positive Brevibacterium, and the gram-positive Brevibacterium is shown in FIG. 2.

EXAMPLE 2 Whole genome and novel analysis of strains

Lactobacillus salivarius Lvagi-36 was inoculated into 5 mL MRS broth medium, cultured to logarithmic growth phase, and then strain whole genome DNA was extracted and subjected to whole genome sequencing by using Illumina high throughput sequencing platform Novageq 6000. After assembly and annotation by conventional methods, protein sequences were aligned to VFDB (Virulence Factor Databases) and CARD (The Comprehensive Antibiotic Resistance Database) databases for development of virulence factors and drug resistance gene analysis. 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, 5 published results were foundLimosilactobacillus urinaemulierisWhole genome, 2 strains were found to be closest to the Lvagi-36 whole genome and below 99.9% by fastanI (v 1.33) comparison, GCA_014838745.1 (98.8562%) and GCA_932750685.1 (97.5773%), respectively. Thus, lvagi-36 is considered to be a novel strain, and the 16S rDNA sequence thereof is shown in SEQ ID NO. 1.

Lactobacillus urinary tract mucus lvegi-36 was preserved in China center for type culture Collection (China Center for Type Culture Collection, CCTCC), with a preservation number of: cctccc No. M20231492.

Example 3 Low pH growth tolerance experiment

Activating: the preserved lactobacillus salivarius was cultured overnight at 37 ℃ with MRS broth activation at pH 6.5.

And (3) switching: transferring the activated bacteria liquid into MRS broth culture medium with pH value of 4-5, and measuring OD every 2-3 h ₆₀₀ Values.

Comparison of OD ₆₀₀ Value: screening for rapid proliferation or OD ₆₀₀ Higher value strains from different samples.

The results show that Lactobacillus salivarius Lvagi-36, lvagi-35 and Lvagi-42 are all capable of growing in low pH environments.

Example 4 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.

TABLE 1 lactic acid producing ability of strains and 48 h pH value of culture

Microorganism	Average lactic acid content (mg/L)	Average pH (48 h)
			Lvagi-36	13957.5347	3
Lvagi-35	11797.2532	3.5
			Lvagi-42	10413.3975	3.5

The results show that Lactobacillus salivarius Lvagi-36 produces lactic acid to lower pH. Under the same experimental conditions, the lactic acid producing capacity of Lvagi-36 is stronger than that of the homologous strains Lvagi-35 and Lvagi-42.

EXAMPLE 5 Hydrogen peroxide production capability

After the strain was activated, 2. Mu.L of the bacterial solution was spotted in MRS solid medium containing 0.25 mg/mL TMB and 0.01 mg/mL HRP with a pipette, the plates were placed in the same anaerobic sealed tank, an anaerobic gas bag was added, the culture was performed at 37℃and the corresponding plates were removed at 48 h, exposed to air, and after 30 min the color development reaction was observed and recorded by photographing: the blue color produced by Lactobacillus delbrueckii was 4 points, the blue color produced by Lactobacillus delbrueckii was 3 points, the blue color produced by Lactobacillus delbrueckii was 2 points, the blue color was very weak, and the color was 1 point (slight color reaction) and the color was not changed, respectively, 0 points.

TABLE 2 Hydrogen peroxide production capability

Microorganism	Hydrogen peroxide production score-48 h
		Lvagi-36	1
Lvagi-35	1
		Lvagi-42	1

The results showed that lactobacillus salivarius produced hydrogen peroxide in both cultures 24 h and 48 h.

Example 6 Gardnerella vaginalis inhibition test

After lactobacillus is activated, 0.1 mL bacterial liquid is evenly mixed with a melted MRS solid culture medium, poured into a 6 cm plate, after complete solidification, the plate is cultivated at 37 ℃ for 48 h, and a puncher with the inner diameter of 6 mm is used for punching holes on the MRS solid culture medium to obtain bacterial cakes; after the gardnerella vaginalis (purchased from Beijing North Injury Biotechnology institute, BNCC 337545) is activated and transferred, the mixture is diluted with 10 times of sterile PBS buffer solution to obtain 10 ^-1 Mixing diluent 0.5 mL with molten BHI solid culture medium containing 5.25 mL horse serum 5%, pouring into 9 cm plate, completely solidifying, placing lactobacillus cakes on the surface of BHI solid culture medium, symmetrically placing 4 cakes on each plate, placing each strain of bacteria in 2 parallel, placing into anaerobic sealed tank, adding anaerobic gas producing bag, culturing in plate forward direction 48 h, and measuring bacteriostasis zone size with vernier caliper to calculate average value.

TABLE 3 Gardnerella vaginalis inhibition ability

Microorganism	Diameter (mm) -average value of inhibition zone
		Lvagi-36	9.12
Lvagi-35	8.59
		Lvagi-42	8.66

The results show that Lactobacillus salivarius Lvagi-36 can effectively inhibit the growth of Gardnerella. Under the same experimental conditions, the antibacterial capacity of the Lvagi-36 is stronger than that of the homologous strains Lvagi-35 and Lvagi-42.

EXAMPLE 7 hemolysis experiment

The deposited lactobacillus salivarius lvagei-36 was inoculated into 5 mL of MRS broth medium at an inoculum size of 10% with enterococcus faecalis (beta hemolysis, CICC23658, purchased from the chinese industrial microorganism strain deposit management center) as a positive control and a blank medium as a negative control. All strains were anaerobically cultured in MRS broth culture medium at 37℃for 12 h to give activated strains. 2.5. Mu.L of each 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 result shows that a completely transparent hemolytic ring with obvious limit is formed around the colony of the positive control strain, and the colony is beta hemolysis; the medium surrounding the colony of lactobacillus salivarius lvegi-36 was unchanged and was gamma-hemolytic, i.e. not hemolytic.

The present invention can be well implemented according to the above-described embodiments. It should be noted that, based on the above structural design, even if some insubstantial modifications or color-rendering are made on the present invention, the essence of the adopted technical solution is still the same as the present invention, so it should be within the protection scope of the present invention.

Claims

1. Lactobacillus salivarius with urinary tract mucusLimosilactobacillus urinaemulieris) The strain is shown as lactobacillus helveticus Lvagi-36 with the preservation number of CCTCC NO: M20231492.

2. The urinary tract lactobacillus mucilaginosus of claim 1Limosilactobacillus urinaemulieris) A method for culturing a strain, which comprises inoculating Lactobacillus curvatus to a culture medium, and performing proliferation culture to obtain a proliferated Lactobacillus curvatus strain.

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

4. A food, health product or pharmaceutical composition comprising the Lactobacillus salivarius as defined in claim 1 as active ingredientLimosilactobacillus urinaemulieris) A strain or a strain containing Lactobacillus salivarius obtained by the culture method as claimed in 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, lactobacillus delbrueckii, and lactobacillus grignard.

6. The food, nutraceutical or pharmaceutical composition of claim 4, wherein the lactobacillus salivarius strain is the sole active ingredient.

7. The urinary tract lactobacillus mucilaginosus of claim 1Limosilactobacillus urinaemulieris) Use of a strain in the preparation of a medicament for inhibiting or killing gardnerella vaginalis.