CN117535208B - Lactobacillus crispatus and application thereof in female genital tract health - Google Patents

Lactobacillus crispatus and application thereof in female genital tract health Download PDF

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CN117535208B
CN117535208B CN202410009279.3A CN202410009279A CN117535208B CN 117535208 B CN117535208 B CN 117535208B CN 202410009279 A CN202410009279 A CN 202410009279A CN 117535208 B CN117535208 B CN 117535208B
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lactobacillus
lactobacillus crispatus
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crispatus
lcris
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CN117535208A (en
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王琼
曾婉秋
高翔
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Sichuan Anaerobic Biotechnology Co ltd
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Sichuan Anaerobic Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Abstract

The invention relates to the field of microorganisms, in particular to lactobacillus crispatus and application thereof in female genital tract health. The Lactobacillus crispatus has no virulence factor, no drug resistance gene, no hemolysis, and good safety. The bacterium has strong lactic acid production capability, can reduce female vagina pH, and inhibit pathogenic bacteria reproduction. In addition, the antibacterial substance hydrogen peroxide generated by the bacterium shows effective antibacterial capacity on gardnerella vaginalis.

Description

Lactobacillus crispatus and application thereof in female genital tract health
Technical Field
The invention relates to the field of microorganisms, in particular to lactobacillus crispatus 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 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. Under the disease state, the microorganism composition and the biomass load change greatly, a plurality of pathogens overgrow, the vaginal microecological environment enters a fragile state, and the infection and the reproduction of the pathogens are not easy to resist, and various vaginal inflammations appear. Bacterial vaginosis is a common gynecological disease, the infection rate is 15% -52%, and the bacterial vaginosis is a vaginal infectious disease which mainly causes clinical syndrome due to dysbacteriosis in vagina because gardnerella vaginalis and other anaerobic bacteria are used as main overpropagation 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 the antibiotics metronidazole or clindamycin, the metronidazole is a precursor, and in an anaerobic environment, the nitro of the metronidazole is reduced to amino by the intracellular enzymatic reduction of bacteria, so that the antibiotics are converted into an active form, and then the helix 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 fungi, 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. Therefore, the probiotic microecological preparation is the most preferable mode for treating BV in the current technical means.
At present, only 2 vaginal microecological medicines are marketed in China, and one of the medicines is streptococcus enteritidisStreptococcus faecalis) The other is Lactobacillus delbrueckiiLactobacillus delbrueckii) Is active ingredient. In addition, some drug lines are in the stage of clinical development, such as the European micro-family of live bacteria capsules containing Lactobacillus crispatus Lc262-1, which has recently been subjected to phase 3 clinical trials, and KAL-001 live bacteria capsules containing 4 dominant lactobacilli from Sichuan anaerobic organisms are in phase 2 research. In addition to pharmaceuticals, some oral probiotics have emerged in the art, such as UREX developed by kehansen ® And ASTART E ® Has been widely used in various oral products for female genital health problems, the principle of which is reported to be the transmission of probiotics to the vagina via the oral-intestinal-anal route.
Lactobacillus crispatus is one of the dominant species of the female genital tract in China, and some published patents and documents report the effect of the lactobacillus crispatus on the female genital tract health, for example, chinese patent CN116515686B discloses a lactobacillus crispatus for treating female genital infection and regulating premenstrual psychological symptoms and application thereof, and Chinese patent CN116286486A discloses a lactobacillus crispatus for relieving various vaginitis based on host immune regulation. However, it is well known in the art that there are differences in the performance of different strains of the same species, and that in selecting probiotics for lactobacillus, a combination of species of lactobacillus and the probiotic capabilities of the different strains is required. Thus, screening for Lactobacillus renaligenes for better probiotics is an unmet need.
Disclosure of Invention
The first aim of the invention is to provide a Lactobacillus crispatus @ withLactobacillus crispatus) The strain is selected from Lactobacillus crispatus-1 with a preservation number of CCTCC NO: M20231544;
in some embodiments, the strain has a 16S rDNA sequence as set forth in SEQ ID NO. 1.
A second object of the present invention is to provide a method for culturing the aforementioned Lactobacillus crispatus strain, which comprises inoculating the Lactobacillus crispatus strain to a culture medium, and performing proliferation culture to obtain a proliferated Lactobacillus crispatus 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, the active ingredient of which comprises the Lactobacillus crispatus strain described above or the Lactobacillus crispatus strain obtained by the above culture method.
In some embodiments, the active ingredient of the composition further comprises one or more selected from the group consisting of lactobacillus jensenii, lactobacillus johnsonii, lactobacillus delbrueckii, and lactobacillus grignard.
In some embodiments, the lactobacillus crispatus strain is the sole active ingredient.
In some embodiments, the composition comprises 10 in a single formulation 6 ~10 15 Lactobacillus crispatus of CFULactobacillus crispatus) Strains.
A fourth object of the present invention is to provide the use of the aforementioned strain of lactobacillus crispatus for the preparation of a product for improving the health of the female genital tract.
In some embodiments, the product that improves female genital tract health is used as a bacteriostatic or bacteriocidal agent.
In some embodiments, the product for improving female genital tract health is used to inhibit or kill gardnerella vaginalis.
In some embodiments, the Lactobacillus crispatus-1 according to the invention has the following morphological characteristics:
(1) The bacterial colony in MRS solid culture medium is in a gray round shape, is full in the middle and dispersed around, and is irregular;
(2) The compound has gram positive and bent rod shape, and can be connected into a long chain.
The lactobacillus crispatus Lcris-1 provided by the invention has no virulence factor, no drug resistance gene and no hemolysis, and has good safety. The lactic acid producing capability is strong, the female vagina pH can be reduced, and the bacteriostatic substance hydrogen peroxide can be produced, so that the pathogenic bacteria reproduction can be effectively inhibited.
The strain preservation information provided by the invention is as follows:
strain name: lactobacillus crispatus @Lactobacillus crispatus）Lcris-1
Preservation date: 2023, 08 and 30 days
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. M20231544.
Drawings
FIG. 1 is a front view of colony morphology of Lactobacillus crispatus-1 of example 1.
FIG. 2 is a gram stain of Lactobacillus crispatus Lcris-1 of example 1.
Detailed Description
Definition and description
Strains for which a particular accession number for a microorganism is claimed herein include, but are not limited to:
1. lactobacillus crispatus with CCTCC NO: M20231544 as the deposited microorganism in the collection centerLactobacillus crispatus) Lcris-1 strain;
2. a Lactobacillus crispatus strain having the same genome as the Lcris-1 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, it is reasonable to expect that minor mutations are unavoidable when the mutation occurs in a non-coding sequence region or synonymous mutation of the coding region or mutation that does not affect the toxicity, immunogenicity and biological activity of the strain (e.g., a linked amino acid residue that may be located between two domains or a residue that is located within the higher structure of the protein and does not affect toxicity, immunogenicity and biological activity by not contacting immune cells), and that the objective of the invention can still be achieved without such minor changes significantly affecting toxicity, immunogenicity and biological activity of the progeny strain, and that it is derived from the strain contributed by the invention and therefore still falls 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 Lcris-1 strain offspring are determined by cells, animals and the like, and the expected or unavoidable systematic errors belong to no substantial change due to differences in cell lines, animal varieties, ages, sexes, health conditions, culture conditions and the like.
The composition contains an active ingredient Lcris-1 strain and other ingredients, such as auxiliary ingredients with no 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 jensenii, lactobacillus johnsonii, lactobacillus delbrueckii and Lactobacillus grignard.
The auxiliary materials comprise additives, drug carriers and excipients. 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 6 ~10 15 CFU、10 7 ~10 13 CFU or 10 7 ~10 12 Lactobacillus crispatus of CFULactobacillus crispatus）。
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 purchased as follows:
MRS broth preparation: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g, dissolving into 1L distilled water; heatingBoiling, cooling to room temperature, adding 0.55. 0.55 g cysteine hydrochloride, stirring for dissolving, and regulating pH value 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.
Preparation of 1% calcium carbonate MRS solid medium: weighing MRS finished product culture medium (Thermo Scientific ™, CM 0359B) powder 52.0 g, agar powder 15.0 g, and calcium carbonate 10.0 g, dissolving into 1L distilled water, heating to boil, adding 0.55 g cysteine hydrochloride after boiling, adjusting pH to 6.5, sterilizing at 118 deg.C under moist heat for 20 min, storing in shade, and keeping away from light for use.
Hydrogen peroxide semi-quantitative medium preparation: weighing MRS finished product culture medium (OXOID CM 1175) powder 52.0 g, agar powder 15.0 g, dissolving in distilled water 1L, adjusting pH to 6.5, sterilizing at 118 deg.C under high temperature and humidity for 20 min, placing into a water bath at 50deg.C after sterilization, maintaining for 30 min, adding 3,3', 5' -Tetramethylbenzidine (TMB) (final concentration is 0.25 mg/mL) and horseradish peroxidase (HRP) (final concentration is 0.01 mg/mL), and mixing; cooling and solidifying, and placing 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: BHI finished medium (Thermo Scientific ™, CM 1135B) powder 37.0 g was weighed,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, 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 6.0 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 MRS solid culture medium added with calcium carbonate, placing in anaerobic incubator, culturing at 37deg.C for 48 h, selecting single colony with calcium fusion ring, culturing in MRS broth culture medium for 24 h, transferring part of the cultured bacterial liquid to continue culturing, extracting bacterial DNA from the other part of the bacterial liquid, sequencing by Beijing Optimago Corp, BLAST comparing PCR result, and finally determining 3 Lactobacillus crispatus%Lactobacillus crispatus) Respectively named as Lcris-1, lcris-2 and Lcris-5.
And (3) streaking bacterial liquid after PCR verification of the Lactobacillus crispatus Lcris-1 strain to an MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 24-48 hours, wherein the bacterial colony is in an off-white round shape, full in the middle and dispersed around, irregular, and a front photograph is shown in figure 1.
Collecting colony of 1-ring lactobacillus crispatus Lcris-1 on glass slide, and dripping appropriate amount of aseptic 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 and photographing, and the bacterial staining mirror is detected as gram-positive campylobacter, and can be connected into long chains, as shown in figure 2.
EXAMPLE 2 Whole genome and novel analysis of strains
Lcris-1 was inoculated into 5 mL anaerobic MRS broth at an inoculum size of 2%, cultured to late logarithmic growth, strain whole genome DNA was extracted, and second and third generation whole genome sequencing was performed simultaneously. 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). 698 published searches were found by searching in GenbankLactobacillus crispatusWhole genome, 2 strains were found to be closest to the Lactobacillus crispatus-1 whole genome and below 99.9% by fastANI (v 1.33) comparison, GCA_022753235.1 (ANI= 99.4954%) and GCA_002861765.1 (ANI= 99.4573%) respectively. Thus, lactobacillus crispatus Lcris-1 can be considered as a novel strain, and its 16SrDNA sequence is shown in SEQ ID NO. 1.
Example 3 Low pH growth tolerance experiment
Activating the preserved Lactobacillus crispatus Lcris-1 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 crispatus Lcris-1 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 crispatus Lcris-1 has the acid-resistant and acid-producing characteristics.
Example 4 oxygen tolerance
To demonstrate the unique properties of Lactobacillus crispatus Lcris-1, the present invention also provides a control of Lactobacillus delbrueckii DS isolated from commercial "Lactobacillus vaginalis live bacteria Capsule" ("Dijunsheng") in a portion of the experiments.
Respectively transferring Lcris-1 and DS into 96-well plates respectively packed with aerobic MRS broth culture medium in an aerobic environment according to 10% of inoculum size, arranging three strains in parallel, adding deoxidized bag into a sealed container for culturing 48 h, and performing OD 600 And (5) monitoring. With DS as a control, 2 was counted, 3 was counted as better than DS tolerance, 1 was counted as slightly weaker than DS tolerance, and 0 was counted as difficulty in growth in an oxygen-containing medium (with addition of an oxygen-scavenging bag).
The results show that: lcris-1 has an oxygen tolerance equivalent to DS and is designated as 2. The lactobacillus crispatus Lcris-1 belongs to facultative anaerobes, the culture operation of the Lcris-1 is not required to be carried out in an anaerobic environment in the whole course, and the preparation of a culture medium is also not required to be strictly deoxidized.
EXAMPLE 5 Hydrogen peroxide production capability
After lactobacillus is activated, 2 mu L of bacteria liquid is inoculated in MRS solid culture medium containing 0.25 mg/mL of 3,3', 5' -tetramethyl benzidine solution and 0.01 mg/mL of horseradish peroxidase by a liquid transfer device, the plates are placed in the same anaerobic sealed tank, an anaerobic gas producing bag is added, the culture is carried out at 37 ℃, at 48 h observation time points, the corresponding plates are taken out, the plates are exposed to air, and after 30 min, the color reaction is observed and recorded by photographing: 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 are shown in Table 1: lactobacillus crispatus Lcris-1 has a certain capacity to produce hydrogen peroxide.
TABLE 1 Hydrogen peroxide production ability of Lactobacillus crispatus Lcris-1
Microorganism Hydrogen peroxide production score-48 h
Lcris-1 1
DS 3
EXAMPLE 6 hemolysis experiment
The deposited lactobacillus was inoculated into 5 mL MRS broth medium with enterococcus faecalis (beta hemolysis, cic 23658, purchased from the chinese industrial microbiological bacterial collection center) as positive control and 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. After anaerobic cultivation at 37℃for 48 h, a completely transparent hemolytic ring with a clear boundary was formed around the colony of the positive control strain, which was beta hemolysis. The culture medium around the colony of Lactobacillus crispatus Lcris-1 was not changed, and was not hemolyzed, i.e., was not hemolyzed.
Example 7 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 an agar culture medium to obtain bacterial cakes; after activation and transfer of gardnerella vaginalis (purchased from Beijing North Injury Biotechnology institute, BNCC 337545), 10 was taken with 10-fold gradient using sterile PBS buffer -1 Mixing the diluted solution 0.5 mL with molten BHI solid culture medium containing 5% horse serum, pouring into 9 cm plate, and completely solidifying to obtain lactobacillus cakeThe method comprises the steps of lightly placing the bacteria on the surface of a BHI solid culture medium, symmetrically placing 4 bacterial cakes on each dish, arranging 2 bacteria in parallel, placing the bacteria into an anaerobic sealed tank, adding an anaerobic gas generating bag, vertically placing the dishes for culturing 48 and h, measuring by a vernier caliper, and calculating the average value of the size of a bacteriostasis zone.
As shown in Table 2, lactobacillus crispatus-1 has an antibacterial effect on Gardnerella vaginalis, and the effect is equivalent to Lactobacillus delbrueckii. Under the same experimental conditions, the inhibition effect of Lcris-1 on gardnerella is stronger than that of the control strains Lcris-2 and Lcris-5 of the same genus.
TABLE 2 capacity of Lactobacillus crispatus-1 to inhibit Gardnerella vaginalis
Microorganism Diameter (mm) -average value of inhibition zone
DS 14.29
Lcris-1 14.11
Lcris-2 10.185
Lcris-5 11.915
Example 8 lactic acid production test
After the lactobacillus is activated, the lactobacillus is transferred into MRS broth culture medium, each strain is set to be 2 times in parallel, and is cultivated at 37 ℃ for 48 h, the pH value of the lactobacillus strain liquid cultivated for 48 h is measured and recorded by using pH 0.5-5.0 test paper, and the strain is selected for liquid chromatography according to 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 average value was calculated, and the results are shown in Table 3, that Lactobacillus crispatus Lcris-1 has a characteristic of high lactic acid production, the lactic acid production is about twice as high as that of the reference strain DS, and higher than that of the control strains Lcris-2 and Lcris-5 of the same genus.
TABLE 3 lactic acid producing ability
Microorganism Average lactic acid content (mg/L)
DS 12964.12
Lcris-1 24487.29
Lcris-2 16098.44
Lcris-5 21997.7078
In conclusion, the lactobacillus crispatus-1 produces lactic acid with high yield and has tolerance to an acidic environment, and can adapt to and maintain the acidic environment of female genital tracts; the bacteria can also produce a certain amount of hydrogen peroxide, and the hydrogen peroxide is a self-cleaning substance with antagonism among bacteria, can inhibit harmful bacteria in genital tracts and promote the growth of beneficial bacteria; the gardnerella vaginalis has a good effect on inhibiting gardnerella vaginalis and has the potential of preventing and/or treating diseases related to gardnerella vaginalis.

Claims (8)

1. Lactobacillus crispatus @ tLactobacillus crispatus) The strain is selected from Lactobacillus crispatus Lcres-1 with a preservation number of CCTCC NO: M20231544.
2. The Lactobacillus crispatus of claim 1Lactobacillus crispatus) The strain culturing method comprises inoculating Lactobacillus crispatus into culture medium, and performing proliferation culture to obtain proliferated Lactobacillus crispatus 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 crispatus of claim 1 as an active ingredientLactobacillus crispatus) A strain or a strain of lactobacillus crispatus obtainable by the cultivation method of claim 2.
5. The composition of claim 4, wherein the active ingredient of the composition further comprises one or more selected from the group consisting of lactobacillus jensenii, lactobacillus johnsonii, lactobacillus delbrueckii, and lactobacillus grignard.
6. The composition of claim 4, wherein the lactobacillus crispatus is the only active ingredient.
7. The composition of claim 4, wherein the composition comprises 10 in a single formulation 6 ~10 15 CFU lactobacillus crispatus.
8. The Lactobacillus crispatus of claim 1Lactobacillus crispatus) Use of a strain in the manufacture of a medicament for inhibiting or killing gardnerella vaginalis.
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