CN113512509B - Lactobacillus crispatus and uses thereof - Google Patents

Abstract

The application relates to Lactobacillus crispatus LC-17218 with the preservation number of CGMCC NO. 21222. The lactobacillus crispatus LC-17218 has strong acid production and H production₂O₂Ability, and vaginal epithelial cell adhesion ability. The application also relates to compositions comprising this strain and to the use thereof for the treatment and/or prophylaxis of urogenital diseases.

Description

Lactobacillus crispatus and uses thereof

Technical Field

The application relates to the field of biological medicines, in particular to Lactobacillus crispatus.

Background

The internal environment of human female vagina refers to the inhabitant microbiota in the vaginal cavity of femaleThe formed micro-ecological environment. Normally, women inhabit a large number of microorganisms within their vaginal cavity, which play an important role locally to maintain a healthy state of the woman's vagina. The female vaginal microflora takes lactobacillus as main dominant bacteria, and simultaneously has a small amount of other anaerobic bacteria, and the lactobacillus can produce acid and hydrogen peroxide (H) through the acid₂O₂) The weak acid environment of the vagina is maintained, so that the growth of pathogenic bacteria is inhibited, and the normal internal environment of the vagina is maintained. When the abundance of lactobacillus in the vagina is reduced or the function of lactobacillus is reduced, other anaerobic bacteria in the vagina are excessively proliferated, so that the microecology of the vagina is unbalanced, internal environment disorder is caused, and the infection of pathogens can be promoted to cause local inflammation, pathological changes and even cancers; meanwhile, local pathogen infection of the vagina can increase the risks of pelvic inflammation, premature delivery, procedures and the like.

However, although topical antibiotics can kill some pathogens, the drug resistance associated with the use of antibiotics is becoming more serious, and the antibiotics can kill pathogens and remove lactobacilli in the vagina, so that the environment in the vagina is unbalanced, and after the antibiotics are stopped, local bacterial colonies fall in the remodeling process and still recover the disordered ecological characteristics before treatment, so that the disease of patients is repeated, and further, intractable symptoms appear. In recent years, there has been a study on the use of Lactobacillus for the purpose of improving and remodeling the environment in the vagina by administering to a patient, however, there has been a case where the improvement effect is not added due to the difference in function between different strains.

Disclosure of Invention

The application aims to provide a method for rapidly increasing the value and simultaneously producing acid and H with strong acid production₂O₂The lactobacillus capable of keeping the environment in the vagina in a weakly acidic environment and further resisting the invasion of other pathogenic bacteria to maintain and improve the homeostasis in the vagina, and the preparation method and the application thereof are clear.

In one aspect, the present application provides an isolated Lactobacillus crispatus strain, wherein the biochemical identification result is as follows:

in certain embodiments, the lactobacillus crispatus has one or more of the following properties:

(v) producing at least about 10mg/L hydrogen peroxide under effective culture conditions;

(vi) producing at least about 5mg/L lactic acid under effective culture conditions;

(vii) under the effective culture condition, the concentration of the bacterial liquid in 18h reaches more than about 8 ten thousand times of the initial concentration;

(viii) capable of inhibiting the growth and/or activity of a pathogenic bacterium selected from the group consisting of: streptococcus agalactiae (Streptococcus agalactiae), Prevotella (Prevotella corrpri), Prevotella (Prevotella timenensis), Prevotella bivia (Prevotella bivia), Micrococcus luteus (Micrococcus luteus), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus epidermidis (Staphylococcus epidermidis), and any combination thereof.

In certain embodiments, the nucleotide sequence of the 16s rRNA of Lactobacillus crispatus has at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1.

In certain embodiments, the 16S rRNA of the Lactobacillus crispatus comprises the nucleotide sequence set forth in SEQ ID NO 1.

In certain embodiments, the Lactobacillus crispatus produces at least about 20mg/L hydrogen peroxide under effective culture conditions.

In certain embodiments, the Lactobacillus crispatus produces at least about 6.5mg/L lactic acid under effective culture conditions.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition of streptococcus agalactiae of greater than about 10mm in diameter in an in vitro culture bilayer inhibition assay.

In certain embodiments, the lactobacillus crispatus is lactobacillus crispatus LC-17218 with the accession number CGMCC No. 21222.

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus for the manufacture of a medicament for the treatment and/or prophylaxis of urogenital diseases.

In certain embodiments, wherein the urogenital disease comprises a vaginal disease.

In certain embodiments, the vaginal disease comprises a vaginal infection.

In certain embodiments, the vaginal infection comprises bacterial vaginosis, aerobic vaginosis, viral vaginosis, yeast vaginitis, trichomonas vaginitis, HPV infection, gonorrhea, chlamydia infection, urinary tract infection, or pelvic inflammatory disease.

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus for the preparation of a medicament for the suppression of pathogenic bacteria.

In certain embodiments, the pathogenic bacteria comprise: one or more of streptococcus agalactiae, prevotella diplodiella, microbacterium, enterococcus faecalis, staphylococcus epidermidis, gardnerella vaginalis, candida albicans, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, salmonella paratyphi b and shigella dysenteriae.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the manufacture of a medicament for the treatment and/or prevention of disorders of the vaginal flora.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the preparation of a medicament having a vaginal epithelial cell adhesion function.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the preparation of an antiseptic agent for improving the environment in the vagina.

In certain embodiments, wherein the improving the environment in the vagina comprises maintaining the ph of the vagina or increasing the concentration of vaginal lactate.

In another aspect, the present application provides a composition comprising the aforementioned lactobacillus crispatus and/or a metabolite thereof, and optionally one or more pharmaceutically acceptable carriers.

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus, its metabolites, and/or the aforementioned compositions for the preparation of a sanitary article.

In another aspect, the present application provides a hygiene article comprising the aforementioned lactobacillus crispatus, a metabolite thereof, and/or a composition of the aforementioned.

In certain embodiments, wherein the hygienic article is a medical device.

In certain embodiments, wherein the medical device comprises a colposcope, a gynecological self-examination scope, a guided dilator, a vaginal speculum, a gynecological irrigator, or a female external antibacterial device.

In certain embodiments, wherein the sanitary article comprises a lotion, a panty liner, a sanitary napkin, a tampon, a tissue, a tampon, a gel, an ointment, a cream, a spray, or a body wash.

In another aspect, the present application provides a food product comprising the aforementioned lactobacillus crispatus, a metabolite thereof, and/or a composition of the aforementioned.

In another aspect, the present application provides a dietary supplement comprising the aforementioned lactobacillus crispatus, a metabolite thereof, and/or a composition of the aforementioned.

In another aspect, the present application provides an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO. 1.

Biological material preservation information

The strain Lactobacillus crispatus LC-17218 is screened from vaginal secretion of women of healthy childbearing age in China, and is preserved in China general microbiological culture Collection center (CGMCC for short) in 11-23 months in 2020, and the preservation unit address is as follows: no. 3 of Xilu No. 1 of Beijing, Chaoyang, China academy of sciences, the collection registration number is CGMCC NO.21222, and the classification name of the strain is Lactobacillus crispatus.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIG. 1 shows a photograph of a colony of Lactobacillus crispatus LC17218 strain as described herein;

FIG. 2 shows a morphological diagram of Lactobacillus crispatus LC-17218 according to the present application under an electron microscope;

FIG. 3A shows the API32 biochemical results of Lactobacillus crispatus LC17218 described herein;

FIG. 3B shows the API20 biochemical results of Lactobacillus crispatus LC-17218 described herein;

FIG. 4 shows the results of the Streptococcus agalactiae inhibition assay by Lactobacillus crispatus LC-17218 as described herein;

FIG. 5 shows the results of the inhibition assay of Prevotella corri by Lactobacillus crispatus LC-17218 as described herein;

FIG. 6 shows the results of a test for the inhibition of Prevotella timonensis by Lactobacillus crispatus LC-17218 as described herein;

FIG. 7 shows the results of a test for the inhibition of Prevotella bivia by Lactobacillus crispatus LC-17218 as described herein;

FIG. 8 shows the results of the Lactobacillus crispatus LC-17218 inhibition assay on Micrococcus luteus as described herein;

FIG. 9 shows the results of an inhibition assay of Lactobacillus crispatus LC-17218 against Enterococcus faecalis as described herein;

FIG. 10 shows the results of the inhibition assay of Staphylococcus epidermidis by Lactobacillus crispatus LC-17218 as described herein.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "Lactobacillus crispatus" or "Lactobacillus crispatus" generally refers to the species of the genus Lactobacillus. This species is usually distinguished from other lactobacilli based on the polynucleotide sequence of the ribosomal 16S rRNA gene.

In the present application, the term "sequence identity" generally means that two polynucleotides or amino acid sequences are identical (i.e., in nucleotides, or residues) over a comparison window. The term "% sequence identity" is calculated as follows: the percentage of sequence identity is determined by comparing the two optimally aligned sequences over a comparison window, determining the number of positions in the two sequences at which the same nucleic acid base (e.g., A, T, C, G, U or I) or amino acid residue is present, to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the comparison window size), and multiplying the result by 100. The term "substantial identity" or "substantially identical" generally denotes a property of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid sequence comprises a sequence having at least about 85% sequence identity, e.g., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% sequence identity, more typically at least about 96%, 97%, 98% or 99% sequence identity, when compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, specifically over a window of at least 18-48 nucleotide (6-16 amino acid) positions, often over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence that may comprise deletions or additions over the comparison window, these sequences may include deletions or additions that total 20% or less of the reference sequence.

In the present application, the term "effective culture conditions" generally refers to the environment in which the Lactobacillus crispatus cells are placed or exposed to promote growth of the cells. Thus, the term generally refers to media, temperature, atmospheric conditions, substrates, agitation conditions, etc., that can affect cell growth, allowing for propagation times of about 0.5 to 48 hours.

In this application, the term "vaginal" generally refers to the vaginal region or segment or surrounding area, including the labia, vulva, cervix, uterus, fallopian tubes, ovaries, urethra, bladder, anus, and rectum, including mucosal tissue thereof.

In the present application, the terms "disease" or "disorder" are used interchangeably and generally refer to any change in the state of the body or certain organs that impedes or disturbs the performance of a function and/or causes symptoms such as discomfort, dysfunction, pain or even death in a person who is diseased or in contact with it.

In the present application, the term "pathogenic" (e.g., "pathogenic bacteria") generally refers to substances, microorganisms, or conditions that can cause disease. In certain contexts, pathogens also include microorganisms (e.g., bacteria) that are associated with a disease or disorder, but for which a causal relationship (e.g., a direct causal relationship) has not yet been established or is yet to be established. In some embodiments, microorganisms that are not pathogens and may be commensals may cause or be associated with disease or dysbiosis, depending on various factors (e.g., immune status of the locus, abundance of microbial taxa, etc.). Such microorganisms are called "pathogenic organisms".

In the present application, the term "pathogen" generally refers to all pathogens which may be deposited in the urogenital tract, and in particular gardnerella vaginalis, prevotella bivalens, Neisseria gonorrhoeae, mycoplasma (mycoplasmas), campylobacter mobilis (mobillucus), Candida albicans and Candida glabrata (Candida glabrata), and pathogens which may cause intestinal infections, such as for example Enterobacteriaceae (Enterobacteriaceae), including Escherichia coli (Escherichia coli), Salmonella (Salmonella), Staphylococcus (Staphylococcus), Clostridium difficile (Clostridium difficile) and Shigella (Shigella).

In the present application, the terms "vaginal flora" or "vaginal microbiota" are used interchangeably and generally refer to the microorganisms that colonize the vagina.

In the present application, the term "disorder of vaginal flora" or "vaginal dysbiosis" generally refers to a disorder of the normal dynamic balance of the vaginal flora, such as a reduction in gram-positive bacteria and/or an increase in gram-negative bacteria compared to a healthy vaginal environment (e.g., a condition in which the vaginal mucosa lacks protective lactobacilli and is colonized by a number of different non-lactobacilli, which condition may be symptomatic or asymptomatic). Specific disorders or conditions include, but are not limited to, fungal (e.g., yeast infection), bacterial (e.g., bacterial vaginosis), viral or parasitic (e.g., trichomonas) infections. The infections include those characterized/diagnosed as vaginitis, vaginal candidiasis, and vaginosis. Some examples of microorganisms that cause such infections include Candida species, particularly Candida albicans (Candida albicans) and Candida tropicalis (c. tropicalis), and t.glabrata, gardnerella (vaginal), various mixed anaerobic and digestive streptococcus (Peptostreptococcus) bacteria. These vaginal infections, if left untreated, can result in pathological secretions in the vagina, significant discomfort to female patients, and/or serious medical consequences.

In the present application, the term "inhibition" generally refers to a process that inhibits or interferes with the growth and reproduction of bacteria and their activity.

In the present application, the term "isolated" when applied to a nucleic acid or protein generally means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in its natural state. For example, it may be in a homogeneous state, and may be in a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

The term "isolated" when applied to bacteria generally refers to bacteria that have been (1) separated from at least some of the components with which they are associated at the time of initial production (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by hand, e.g., using hand culture conditions, such as (but not limited to) culture on a plate and/or in a fermentor. Isolated bacteria include those that are cultured, even if such culture is not a single culture. An isolated bacterium may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which it is initially associated. In embodiments, the isolated bacteria are greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. In certain embodiments, the bacterial population provided herein comprises isolated bacteria. In certain embodiments, the compositions provided herein comprise isolated bacteria. In certain embodiments, the administered bacteria are isolated bacteria.

In the present application, the term "pharmaceutically acceptable carrier" generally refers to a substance that facilitates administration of an active agent to and absorption by a subject and that can be included in the compositions of the present application without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline solution, lactated ringer's solution, common sucrose, common glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, dyes (color), and the like. Such preparations may be sterilized and those skilled in the art will recognize that other pharmaceutical carriers may be used herein.

In the present application, the term "CFU (Colony-Forming Units)" generally refers to the total number of colonies of microorganisms such as bacteria, fungi, yeasts, etc. in a product, and is generally used as a viable count.

The term "CFU/dose" means daily or every doseThe amount of bacteria present in the composition/food or dietary supplement/medicament provided by the subject. For example, in certain embodiments, the food product or dietary supplement comprises lactobacillus crispatus at 10⁶To 10¹²The amount of CFU/dose present (e.g., 10)⁸To 10¹²CFU/dose). In such an embodiment, if lactobacillus crispatus is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject each day or each time may contain about 10⁶To 10¹²CFU lactobacillus crispatus. Of course, alternatively, the amount of such bacteria may be divided into multiple administrations, as long as the total amount of lactobacillus crispatus that the subject receives over any particular time (e.g., every 24 hour period) is from about 10⁶To about 10¹²Bacteria of the CFU, i.e. Lactobacillus crispatus or Lactobacillus crispatus in a food product or dietary supplement as described above, to 10⁶To 10¹²The amount of CFU/dose present (e.g., 10)⁸To 10¹²CFU/dose).

In the present application, the term "treatment and/or prevention" includes not only the treatment and/or prevention of a disease, but also generally includes the prevention of the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease and/or any symptoms associated therewith, and/or preventing a further increase in the severity of the disease and/or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease and any pharmacological effects that are generally beneficial to the treated patient. The composition of the present application does not need to achieve a complete cure or eradicate any symptom or manifestation of the disease to form a viable therapeutic agent. As recognized in the relevant art, drugs used as therapeutic agents may reduce the severity of a given disease state, but need not eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically constitutes a viable prophylactic agent and need not be completely effective in preventing the onset of the condition. It is sufficient to simply reduce the impact of the disease in the subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or to reduce the likelihood of the disease occurring or worsening.

In the present application, the terms "subject" and "patient" are used interchangeably and generally refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, among others. Animals include all vertebrates, e.g., mammals and non-mammals, such as dogs, cats, sheep, cows, pigs, rabbits, chickens, etc.

In the present application, the term "about" generally refers to a variation in the range of about 0.5% -10% above or below the specified value, such as a variation in the range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

Detailed Description

Lactobacillus crispatus

In one aspect, the present application provides an isolated Lactobacillus crispatus strain, wherein the biochemical identification result is as follows:

in certain embodiments, the lactobacillus crispatus may have one or more of the following properties:

(i) producing at least about 10mg/L hydrogen peroxide under effective culture conditions;

(ii) producing at least about 5mg/L lactic acid under effective culture conditions;

(iii) under the effective culture condition, the concentration of the bacterial liquid in 18h reaches more than about 8 ten thousand times of the initial concentration;

(iv) capable of inhibiting the growth and/or activity of a pathogenic bacterium selected from the group consisting of: streptococcus agalactiae (Streptococcus agalactiae), Prevotella (Prevotella corrpri), Prevotella (Prevotella timenensis), Prevotella bivia (Prevotella bivia), Micrococcus luteus (Micrococcus luteus), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus epidermidis (Staphylococcus epidermidis), and any combination thereof.

In certain embodiments, the Lactobacillus crispatus can produce at least about 10mg/L, or at least about 11mg/L, or at least about 12mg/L, or at least about 13mg/L, or at least about 14mg/L, or at least about 15mg/L, or at least about 16mg/L, or at least about 17mg/L, or at least about 18mg/L, or at least about 19mg/L, or at least about 20mg/L, or at least about 21mg/L, or at least about 22mg/L, or at least about 23mg/L, or at least about 24mg/L, or at least about 25mg/L of hydrogen peroxide under effective culture conditions. For example, the Lactobacillus crispatus can produce at least about 20mg/L hydrogen peroxide under effective culture conditions.

In certain embodiments, the Lactobacillus crispatus can produce at least about 5mg/L, or at least about 5.1mg/L, or at least about 5.2mg/L, or at least about 5.3mg/L, or at least about 5.4mg/L, or at least about 5.5mg/L, or at least about 5.6mg/L, or at least about 5.7mg/L, or at least about 5.8mg/L, or at least about 5.9mg/L, or at least about 6.0mg/L under effective culture conditions, or at least about 6.1mg/L, or at least about 6.2mg/L, or at least about 6.3mg/L, or at least about 6.4mg/L, or at least about 6.5mg/L, or at least about 6.6mg/L, or at least about 6.7mg/L, or at least about 6.8mg/L, or at least about 6.9mg/L, or at least about 7.0mg/L of lactic acid.

In certain embodiments, the lactobacillus crispatus can have a bacterial fluid concentration that is about 8 ten thousand fold, or 9 ten thousand fold, or 10 ten thousand fold, or 11 ten thousand fold, or 12 ten thousand fold, or 13 ten thousand fold, or 14 ten thousand fold, or 15 ten thousand fold, or 16 ten thousand fold, or 17 ten thousand fold, or 18 ten thousand fold, or 19 ten thousand fold, or 20 ten thousand fold or more of the initial concentration within 18 hours of effective culture conditions.

In certain embodiments, the nucleotide sequence of the 16s rRNA of Lactobacillus crispatus can have at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 1. For example, about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% sequence identity.

In certain embodiments, the 16S rRNA of Lactobacillus crispatus can comprise a nucleotide sequence as set forth in SEQ ID NO 1. For example, the 16S rRNA of Lactobacillus crispatus can be the nucleotide sequence shown in SEQ ID NO 1.

As another example, the 16S rRNA of Lactobacillus crispatus may be the nucleotide sequence shown in SEQ ID NO: 1; and the Lactobacillus crispatus can produce at least about 10mg/L of hydrogen peroxide under effective culture conditions; and the Lactobacillus crispatus can produce at least about 5mg/L of lactic acid under effective culture conditions; the concentration of the lactobacillus crispatus can reach more than about 8 ten thousand times of the initial concentration within 18 hours under the effective culture condition; and the lactobacillus crispatus is capable of inhibiting the growth and/or activity of the following pathogenic bacteria: streptococcus agalactiae (Streptococcus agalactiae), Prevotella (Prevotella corrpri), Prevotella (Prevotella timenensis), Prevotella bivia (Prevotella bivia), Micrococcus luteus (Micrococcus luteus), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus epidermidis (Staphylococcus epidermidis), and any combination thereof.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition of Streptococcus agalactiae (Streptococcus agalactiae) diameter of above about 10mm in an in vitro culture bilayer inhibition assay. For example, the lactobacillus crispatus culture bilayer inhibition assay may have a diameter for the streptococcus agalactiae zone of up to about 10.5mm, or about 11mm, about 11.5mm, or about 12mm, about 12.5mm, or about 13mm, about 13.5mm, or about 14mm, about 14.5mm, or about 15mm, about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm or more.

In certain embodiments, the lactobacillus crispatus has a Prevotella (Prevotella corrpri) zone diameter of up to about 15mm or more in an in vitro culture bilayer inhibition assay. For example, the Lactobacillus crispatus culture bilayer inhibition assay may have a Prevotella timonensis zone diameter of about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm, or about 20.5mm, or about 21mm, or about 21.5mm, or about 22mm, or about 22.5mm, or about 23mm, or about 23.5mm, or about 24mm, or about 24.5mm, or about 25mm or more.

In certain embodiments, the lactobacillus crispatus has a Prevotella (Prevotella timonensis) zone diameter of greater than about 15mm in an in vitro culture bilayer inhibition assay. For example, the Lactobacillus crispatus culture bilayer inhibition assay may have a Prevotella timonensis zone diameter of about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm, or about 20.5mm, or about 21mm, or about 21.5mm, or about 22mm, or about 22.5mm, or about 23mm, or about 23.5mm, or about 24mm, or about 24.5mm, or about 25mm or more.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition of Prevotella (Prevotella bivia) to greater than about 15mm in diameter in an in vitro culture bilayer inhibition assay. For example, the lactobacillus crispatus culture bilayer inhibition assay may have a Prevotella bivia zone diameter of about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm, or about 20.5mm, or about 21mm, or about 21.5mm, or about 22mm, or about 22.5mm, or about 23mm, or about 23.5mm, or about 24mm, or about 24.5mm, or about 25mm, or about 25.5mm, or about 26mm, or about 26.5mm, or about 27mm, or about 27.5mm, or about 28mm, or about 28.5mm, or about 29mm, or about 29.5mm, or about 30mm or more.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition of micro-bacteria (Micrococcus luteus) of up to about 15mm in diameter in an in vitro culture bilayer inhibition assay. For example, the lactobacillus crispatus culture bilayer inhibition assay may have a microcccus luteus zone diameter of about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm, or about 20.5mm, or about 21mm, or about 21.5mm, or about 22mm, or about 22.5mm, or about 23mm, or about 23.5mm, or about 24mm, or about 24.5mm, or about 25mm or more.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition of Enterococcus faecalis (Enterococcus faecalis) of greater than about 10mm in a two-layer inhibition assay in vitro culture. For example, the lactobacillus crispatus culture bilayer inhibition assay may have a zone of inhibition of Enterococcus faecalis from about 10.5mm, or about 11mm, about 11.5mm, or about 12mm, about 12.5mm, or about 13mm, about 13.5mm, or about 14mm, about 14.5mm, or about 15mm, about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm or more in diameter.

In certain embodiments, the lactobacillus crispatus has a zone of inhibition against Staphylococcus epidermidis (Staphylococcus epidermidis) of up to about 10mm in an in vitro culture bilayer inhibition assay. For example, the lactobacillus crispatus culture bilayer inhibition assay may have a staphylocccus epidermidis zone diameter of about 10.5mm, or about 11mm, about 11.5mm, or about 12mm, about 12.5mm, or about 13mm, about 13.5mm, or about 14mm, about 14.5mm, or about 15mm, about 15.5mm, or about 16mm, about 16.5mm, or about 17mm, about 17.5mm, or about 18mm, about 19.5mm, or about 20mm or more.

In certain embodiments, the lactobacillus crispatus is lactobacillus crispatus LC-17218 with the accession number CGMCC No. 21222.

Use and composition

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus for the manufacture of a medicament for the treatment and/or prophylaxis of urogenital diseases.

In certain embodiments, wherein the urogenital disease comprises a vaginal disease.

In certain embodiments, the vaginal disease comprises a vaginal infection. For example, bacterial vaginosis, aerobic vaginosis, viral vaginosis, yeast vaginitis, trichomonas vaginitis, HPV infection, gonorrhea, chlamydial infection, urinary tract infection or pelvic inflammatory disease.

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus for the preparation of a medicament that can be used to repress a pathogenic bacterium.

In certain embodiments, the pathogenic bacteria may include: one or more of streptococcus agalactiae, prevotella diplodiella, microbacterium, enterococcus faecalis, staphylococcus epidermidis, gardnerella vaginalis, candida albicans, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, salmonella paratyphi b and shigella dysenteriae.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the manufacture of a medicament that can be used for the treatment and/or prevention of disorders of the vaginal flora.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the preparation of a medicament, which may have a vaginal epithelial cell adhesion function. For example, the lactobacillus crispatus strain may have a cohesion value percentage of at least about 50% of Vaginal Epithelial Cells (VECs). In the present application, "percent VEC cohesion value" is generally defined as the percentage of VECs to which at least one lactobacillus cell adheres to the total number of VECs in the identified group. Methods for determining acceptable VEC cohesion values are well known in the art and can be found in U.S. patent No. 6,468,526 and U.S. patent No. 6,093,394. See also Kwok, et al, J.Urol.2006,176: 2050-.

In another aspect, the present application provides a use of the aforementioned lactobacillus crispatus for the preparation of an antiseptic agent that can be used to improve the environment within the vagina.

In certain embodiments, wherein said improving the environment in the vagina may comprise maintaining the ph of the vagina or increasing the concentration of vaginal lactate.

In another aspect, the present application provides a composition which may comprise the aforementioned lactobacillus crispatus and/or a metabolite thereof, and optionally one or more pharmaceutically acceptable carriers.

In certain embodiments, the lactobacillus crispatus is typically present at about 10⁵To about 10¹²CFU/dose (e.g., about 10)⁵CFU/dose, about 10⁶CFU/dose, about 10⁷CFU/dose, about 10⁸CFU/dose, about 10⁹CFU/dose, about 10¹⁰CFU/dose, about 10¹¹CFU/dose, or about 10¹²CFU/dose) or as cell-free supernatant of the corresponding culture in liquid, semi-solid or lyophilized form.

In certain embodiments, the carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, or lubricant. The filler can be one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent can be one or more of ethanol or glycerol; the disintegrant can be one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive can be one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant may be one or more of magnesium stearate, sodium fumarate stearate, talc or silica.

In certain embodiments, the composition may be in the form of a soft capsule, suppository, gel, topical solution, lotion, or liniment.

In certain embodiments, the composition can be configured for oral, vaginal, urethral, and/or rectal administration. For example, the compositions may be instilled in the form of a lyophilizate, cream, paste, gel liquid or suppository, capsule or tablet, or a stylus for enteral, urethral, or vaginal application. Exemplary vaginal administrations include topical, sublabial, intradermal, intramuscular, intracavitary, subcutaneous or insufflation, or may be by direct injection or by providing a spray.

Exemplary dosage forms for vaginal delivery may include suppositories (e.g., pessaries), creams, ointments, gels (lubricants), solutions, suspensions, emulsions, pessaries, tampons, pads, irrigants, sponges, cups, intrauterine devices (IUDs), intravesical infusions, strips, sprays, foams, tablets, capsules, pills, patches, pellets, caps, membranes, fibers, applicators, adhesives, covers (e.g., condoms), or amniotic extraluminal infusions. In some embodiments, a dosage form suitable for vaginal delivery is capable of maintaining a particular shape or consistency following administration. In some embodiments, a dosage form suitable for vaginal delivery may dissolve or change form after administration.

In another aspect, the present application provides the use of the aforementioned lactobacillus crispatus, its metabolites, and/or the aforementioned compositions for the preparation of a sanitary article.

In another aspect, the present application provides a hygiene article which may contain the aforementioned lactobacillus crispatus, a metabolite thereof, and/or the aforementioned composition.

In certain embodiments, wherein the hygienic article may be a medical device. Such as colposcopy, gynecological self-examination scope, guide dilator, vaginal speculum, gynecological irrigator or external female antibacterial device.

In certain embodiments, wherein the hygienic device may comprise a lotion, pad, tissue, tampon, gel, ointment, cream, spray, or body wash.

In another aspect, the present application provides a food product which may comprise the aforementioned lactobacillus crispatus, a metabolite thereof, and/or a composition of the aforementioned. The food product may be in the form of a liquid, solid, suspension or powder.

In certain embodiments, the lactobacillus crispatus may be encapsulated or coated.

In certain embodiments, the food product comprises a carrier material selected from the group consisting of oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fiber, sugars, proteins, and glycosylated proteins. For example, the food product is selected from the group consisting of bread, dairy products (e.g., yogurt, flavored fermented milks, lactic acid bacteria beverages, cheese), juices, health bars (health bars), spreads, biscuits, candies, and cereals.

In another aspect, the present application provides a dietary supplement that may comprise the aforementioned lactobacillus crispatus, a metabolite thereof, and/or a composition of the aforementioned. In certain embodiments, the dietary supplement may be formulated for oral administration.

In certain embodiments, the dietary supplement may be in the form of a pill, powder, capsule, tablet, granulated powder, film-covering agent (opercula), orally soluble granule, sachet, dragee, or liquid.

In certain embodiments, the food product or dietary supplement may further include, but is not limited to, one or any combination of the following: a probiotic (e.g. a probiotic bacterium), a dietary fibre, a prebiotic, a protein (e.g. an enzyme), a carbohydrate, a lipid material (e.g. a fat), a vitamin, a mineral, a plant component (e.g. a plant extract), an amino acid, an immunomodulator, a milk substitute, or a metabolite or extract of bifidobacterium breve or progeny thereof. In certain embodiments, the compositions of the present application may also be combined with various food acceptable adjuvants such as sweetening or flavoring agents, coloring substances, stabilizers, glidants, fillers, and the like.

In another aspect, the present application provides an isolated nucleic acid, which may comprise the nucleotide sequence set forth in SEQ ID NO. 1.

In another aspect, the present application further provides the following embodiments:

1. an isolated Lactobacillus crispatus (Lactobacillus crispatus) having the biochemical identification result as follows:

2. the isolated lactobacillus crispatus of embodiment 1, having one or more of the following properties:

(i) producing at least about 10mg/L hydrogen peroxide under effective culture conditions;

(ii) producing at least about 5mg/L lactic acid under effective culture conditions;

(iii) under the effective culture condition, the concentration of the bacterial liquid in 18h reaches more than about 8 ten thousand times of the initial concentration;

(iv) capable of inhibiting the growth and/or activity of a pathogenic bacterium selected from the group consisting of: streptococcus agalactiae (Streptococcus agalactiae), Prevotella (Prevotella corrpri), Prevotella (Prevotella timenensis), Prevotella bivia (Prevotella bivia), Micrococcus luteus (Micrococcus luteus), Enterococcus faecalis (Enterococcus faecalis), Staphylococcus epidermidis (Staphylococcus epidermidis), and any combination thereof.

3. The isolated lactobacillus crispatus of any one of embodiments 1-2, having at least about 99% sequence identity of the nucleotide sequence of the 16s rRNA of lactobacillus crispatus to the nucleotide sequence set forth in SEQ ID No. 1.

4. The isolated lactobacillus crispatus according to any one of embodiments 1-3, wherein the 16S rRNA of the lactobacillus crispatus comprises a nucleotide sequence as set forth in SEQ ID No. 1.

5. The isolated lactobacillus crispatus of any one of embodiments 1-4, which produces at least about 20mg/L hydrogen peroxide under effective culture conditions.

6. The isolated lactobacillus crispatus of any one of embodiments 1-5, which produces at least about 6.5mg/L lactic acid under effective culture conditions.

7. The isolated Lactobacillus crispatus of any one of embodiments 1-6, having a zone of inhibition of Streptococcus agalactiae (Streptococcus agalactiae) of up to about 10mm in diameter, Prevotella corri of up to about 15mm in diameter, Prevotella timonensis of up to about 15mm in diameter, Prevotella bivia of up to about 15mm in diameter, Micrococcus luteus of up to about 15mm in diameter, Enterococcus faecalis of up to about 10mm in diameter, and/or Staphylococcus epidermidis of up to about 10mm in diameter in an in vitro culture double layer inhibition assay.

8. The isolated lactobacillus crispatus according to any one of embodiments 1-7, being lactobacillus crispatus LC-17218 with accession number CGMCC No. 21222.

9. Use of lactobacillus crispatus according to any one of embodiments 1-8 for the preparation of a medicament for the treatment and/or prevention of urogenital diseases.

10. The use according to embodiment 9, wherein the urogenital condition comprises a vaginal condition.

11. The use of embodiment 10, wherein the vaginal disease comprises a vaginal infection.

12. The use of embodiment 11, the vaginal infection comprising: bacterial vaginosis, aerobic vaginosis, viral vaginosis, yeast vaginitis, trichomonas vaginitis, HPV infection, gonorrhea, chlamydial infection, urinary tract infection or pelvic inflammatory disease.

13. Use of lactobacillus crispatus of any of embodiments 1-8 in the preparation of a medicament for suppressing a pathogenic bacteria.

14. The use of embodiment 13, wherein the pathogenic bacteria comprise: one or more of streptococcus agalactiae, prevotella diplodiella, microbacterium, enterococcus faecalis, staphylococcus epidermidis, gardnerella vaginalis, candida albicans, staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, salmonella paratyphi b and shigella dysenteriae.

15. Use of lactobacillus crispatus according to any of embodiments 1-8 for the preparation of a medicament for the treatment and/or prevention of a disorder of vaginal flora.

16. Use of lactobacillus crispatus of any of embodiments 1-8 in the preparation of a medicament having vaginal epithelial cell adhesion function.

17. Use of lactobacillus crispatus according to any of embodiments 1-8 in the preparation of an antiseptic formulation for improving the environment within the vagina.

18. The use of embodiment 17, wherein the improving the environment in the vagina comprises maintaining vaginal ph or increasing vaginal lactate concentration.

19. A composition comprising lactobacillus crispatus and/or a metabolite thereof according to any of embodiments 1-8, and optionally one or more pharmaceutically acceptable carriers.

20. Use of a lactobacillus crispatus of any one of embodiments 1-8, a metabolite thereof, and/or a composition of embodiment 19 in the manufacture of a hygiene product.

21. A hygiene article comprising a lactobacillus crispatus of any of embodiments 1-8, a metabolite thereof, and/or a composition of embodiment 19.

22. The sanitary article according to embodiment 21, wherein the sanitary article is a medical device.

23. The sanitary article of embodiment 21, wherein the sanitary article comprises a lotion, a panty liner, a sanitary napkin, a tampon, a tissue, a tampon, a gel, an ointment, a cream, a spray, or a body wash.

24. A food product comprising lactobacillus crispatus of any one of embodiments 1-8, a metabolite thereof, and/or a composition of embodiment 19.

25. A dietary supplement comprising lactobacillus crispatus, a metabolite thereof, of any of embodiments 1-8, and/or a composition of embodiment 19.

26. An isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO. 1.

Without wishing to be bound by any theory, the following examples are only for illustrating lactobacillus crispatus, preparation method and use, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

Reagents and materials

Reagent

Instrument for measuring the position of a moving object

Name of instrument	Model number	Brands or manufacturers
			Multifunctional enzyme mark instrument	SynergyHTX	BioTek
Vacuum pump	A30	Gene science
			Low-temperature gas bath shaking table	Forma Shake	Forma Shake
Desk type high-speed centrifuge	3K15	Sigma
			Anaerobic/microaerophilic incubator	A35	Whitley Science
Aerobic culture box	Precision	Thermo Scientific

Example 1: screening and strain identification of lactobacillus crispatus

Collecting vaginal secretion of healthy people by adopting a disposable sterile swab, inoculating the vaginal secretion to an MRS culture medium by adopting a direct coating method, and carrying out anaerobic culture at 37 ℃ for 18-36h to obtain a plurality of bacterial colonies; as shown in fig. 1, individual colonies are round, white opaque, large and flat, matt. And respectively extracting DNAs of a plurality of single colonies, amplifying and determining a 16S rDNA sequence of the single colonies, comparing the determination result with the 16S rDNA sequence of lactobacillus, and screening through a series of functional experiments (lactic acid production experiments and hydrogen peroxide production experiments) to obtain the lactobacillus crispatus capable of improving the internal environment of the human vagina, wherein the lactobacillus crispatus is named as LC-17218, the 16S sequencing sequence of the lactobacillus crispatus is shown as SEQ ID NO:1, and the morphogram under an electron microscope is shown as figure 2.

The screening method of lactobacillus crispatus specifically comprises the following steps:

1. proliferation potency assay

a. Scraping lactobacillus lawn from MRS solid culture medium by using a sterile inoculating loop, and transferring the lactobacillus lawn into MRS liquid culture medium to prepare bacterial suspension;

b. pipetting 200. mu.L of the suspension into a 96-well microplate (taking care not to have air bubbles to affect the absorbance measurement);

c. the absorbance of the suspension in a 96-well plate was measured at a wavelength of 570nm using a microplate reader, and the absorbance of the bacterial suspension was adjusted to 1.0 (in this case, the bacterial suspension concentration was 10)⁹CFU/mL); then, adding the bacterial suspension with the determined concentration into MRS liquid culture according to the volume ratio of 1:20 (conventionally, 1mL of bacterial suspension is added into a 50mL centrifuge tube filled with 19mL of liquid culture medium), gently mixing uniformly, and measuring the absorbance (OD:0h) of the bacterial suspension again;

d. screwing a cover of a centrifugal pipe, putting the centrifugal pipe into an anaerobic tank, fixing the anaerobic tank, putting an anaerobic generating bag into an anaerobic tank, covering the anaerobic tank, taking out oxygen in the anaerobic tank by using a vacuum pump, finally putting the anaerobic tank into a Forma Shake shaking table, and culturing for 18 hours at 37 ℃ under the condition of 180 revolutions per minute;

e. the absorbance (OD: 18h) of the 18 h-cultured bacterial culture was measured in a 96-well plate at a wavelength of 570nm by pipetting 200. mu.L.

As a result of comparison of OD570 values before and after the culture, Lactobacillus crispatus LC-17218 was able to proliferate rapidly, as shown in Table 1. Under MRS condition, the concentration of the bacteria liquid reaches 8 ten thousand times of the initial concentration within 18h (about 10)⁹CFU/mL) above.

TABLE 1

2. Lactic acid production test

Detection was performed by using a D-lactate test kit provided by bosch biotechnology: preparing lactic acid premix according to a preparation method of a test kit specification, and performing gradient dilution to obtain a series of standard substance solutions with gradient concentration; preparing enough reagent working solution (paying attention to setting a sample control group); mixing and reacting 20 mu L of sample and 80 mu L of working solution in each hole; measuring the optical density (OD0) (520 and 600nm) at 565nm when the reading time is 0 and the optical density (OD20) after 20min of culture; and fifthly, determining the concentration of the lactic acid in the sample by comparing the OD value with the standard curve.

3. Test for Hydrogen peroxide production

The ability of the strain to produce hydrogen peroxide was judged by comparing its color development with a color chart using a german merck hydrogen peroxide test strip: the bacterial culture which is cultured for 18h by shaking in a shaking table is centrifuged for 10min at the rotating speed of 5500r/min, 100 mu L of culture solution supernatant is absorbed by using a sterile disposable pipette, and is dripped into a sample hole of a German Merck hydrogen peroxide test strip, the color is observed after 30s, and the color is compared with a colorimetric card to judge the hydrogen peroxide production capacity of the strain.

The results of the lactic acid production test and the hydrogen peroxide production test of Lactobacillus crispatus LC-17218 are shown in Table 2, and compared with the control bacterium 1 (the 16S sequencing sequence is shown in SEQ ID NO: 2) and the control bacterium 2 (the 16S sequencing sequence is shown in SEQ ID NO: 3), the lactobacillus crispatus LC-17218 has good acid production and H production₂O₂Capability.

TABLE 2

Sample (I)	D-lactic acid concentration (mg/L)	H2O2(mg/L)	In vitro culture of metabolic pH
				LC-17218	6.59	25	3.1
Reference bacterium 1	3.80	0	4.7
				Control bacterium 2	6.01	1	4.7

Example 2: biochemical identification of lactobacillus crispatus

The results of biochemical identification of strain LC-17218 using API32 and API20 from Meliea, France are shown in FIGS. 3A-3B, and are as follows:

1. the bacterium does not produce urease and decompose urea;

2. the strain does not produce arginine double hydrolase and L-arginine

3. The bacterium does not produce alpha galactosidase and does not decompose 4-o-nitrobenzene-alpha D-galactopyranose;

4. the strain produces beta galactosidase and can decompose 4-o-nitrobenzene-beta-galactopyranose;

5. the strain does not produce beta galactosidase-6-phosphate and does not decompose 4-o-nitrobenzene-beta D-galactopyranoside-6-phosphate-2-Cyclohexylamine (CHA);

6. the strain produces alpha glucosidase and can decompose 4-o-nitrobenzene-alpha;

7. the strain does not produce beta glucosidase and decompose 4-o-nitrobenzene-beta D-glucopyranoside;

8. the bacterium does not produce alpha arabinosidase and decompose 4-o-nitrobenzene-alpha L-arabinofuranoside;

9. the bacterium does not produce beta glucuronidase and does not decompose 4-o-nitrobenzene-beta D-glucuronide;

10. the bacterium does not produce beta-N-acetyl-beta D-glucosaminidase and does not decompose 4-o-nitrobenzene-N-acetyl-beta D-glucosaminide;

11. the strain can produce mannose fermentation (MNE) and decompose D-mannose;

12. the strain can produce mannose fermentation (RAF) and decompose D-raffinose;

13. the bacterium does not produce glutamate decarboxylase and does not decompose glutamate;

14. the strain does not produce alpha fucosidase and does not decompose 4-o-nitrobenzene-alpha L-fucopyranoside;

15. the bacterium does not produce nitrate reduction and decompose potassium nitrate;

16. the bacterium produces indole a little and can decompose L-tryptophan;

17. the strain produces alkaline phosphatase and can decompose 2-naphthol-phosphoric acid;

18. the bacterium produces arginine aromatase and can decompose L-arginine-beta-naphthylamine;

19. the bacterium produces proline aromatic enzyme and can decompose L-proline-beta-naphthylamine;

20. the strain does not produce leucyl glycine aryl enzyme and does not decompose L-leucyl-L-glycine-beta-naphthylphenol amine;

21. the bacterium produces phenylalanine aromatase and can decompose L-phenylalanine-beta-naphthol amine;

22. the bacterium produces leucine aromatic enzyme and can decompose L-leucine-beta-naphthol amine;

23. the bacterium does not produce pyroglutamic acid aromatase and does not decompose pyroglutamic acid-beta-naphthol amine;

24. the strain produces tyrosine aromatase without decomposing L-tyrosine-beta-naphthylamine;

25. the bacterium produces alanine aromatase, and can decompose L-alanyl-L-alanine-4-nitrobenzene;

26. the bacterium produces glycine aryl enzyme a little and can decompose L-glycine-beta-naphthol amine;

27. the bacterium produces histidine aromatic enzyme slightly, and can decompose L-histidine-beta-naphthylphenol amine;

28. the bacterium produces glutamyl glutamate aromatase a little, and can decompose L-glutamyl-L-glutamic acid-beta-naphthylamine;

29. the strain produces serine aromatic enzyme and can decompose L-serine-beta-naphthol amine.

29. The bacteria can decompose glucose to produce acid;

30. the bacteria can decompose mannitol to produce acid;

31. the bacteria can decompose lactose (source-cow) to produce acid;

32. the strain can decompose sucrose to produce acid;

33. the bacteria can decompose maltose to produce acid;

34. the bacteria can decompose saligenin to produce acid;

35. the bacterium can decompose xylose to produce acid;

36. the bacterium can decompose arabinose to produce acid;

37. the bacterium does not produce hydrolysis (protease) (gelatin), does not decompose gelatin (source-bovine);

38. the bacterium produces beta-glucosidase, which can hydrolyze esculin and ferric citrate;

39. the bacterium can decompose glycerol to produce acid;

40. the bacteria can decompose cellobiose to produce acid;

41. the bacterium can differentially decompose tripanase to produce acid;

42. the bacterium decomposes sorbitol to produce acid;

43. the bacterium decomposes rhamnose to produce acid;

44. the bacterium can decompose trehalose to produce acid.

Example 3: bacteriostatic test of Lactobacillus crispatus

Lactobacillus crispatus LC-17218 is tested for its ability to inhibit Streptococcus agalactiae, Prevotella corrisporus, Prevotella timonensis, Prevotella bivia, Micrococcus luteus, Enterococcus faecalis and Staphylococcus epidermidis.

(1) Inoculating lactobacillus to a sterile paper sheet, and carrying out anaerobic culture for 18 h:

a. scraping lactobacillus lawn from MRS solid culture medium by using sterile inoculating loop, transferring into sterile PBS buffer solution to obtain bacterial suspension, measuring absorbance of the bacterial suspension at wavelength of 570nm by using microplate reader, and adjusting bacterial liquid concentration to 10⁸CFU/mL(OD＝0.5)；

b. Uniformly sticking high-pressure sterile paper sheets on an MRS flat plate by using a sterile sharp-pointed forceps, sucking 10 mu L of bacterial suspension by using a pipettor and a sterile Tip head, dripping the bacterial suspension on the paper sheets, and inversely placing the paper sheets in an anaerobic box for anaerobic culture for 18h after the bacterial suspension is completely absorbed by the MRS flat plate;

(2) and (3) mixing pathogenic bacteria into a BHI culture medium layer for 18 h:

a. scraping pathogenic bacteria lawn from blood plate with sterile inoculating loop, transferring into sterile PBS buffer solution to obtain bacterial suspension, measuring absorbance of the bacterial suspension at wavelength of 570nm with microplate reader, and adjusting bacterial liquid concentration to 10⁸CFU/mL(OD＝0.5)；

b. The concentration of the bacterial liquid is 10⁸Fully mixing the BHI culture medium (which is cooled and insulated to 50-55 ℃ in advance) with the bacterial liquid according to the proportion that the CFU/mL pathogenic bacteria are added into 400 mu L of bacterial liquid per 100mL of BHI agar culture medium;

c. pouring 15 mL/BHI agar culture medium mixed with pathogenic bacteria onto MRS plate cultured for 18h in advance and having lactobacillus grown, inverting after the culture medium is solidified, culturing in a 37 ℃ incubator according to aerobic requirement of the pathogenic bacteria, measuring size of inhibition zone after 18h, and registering data. As a result, as shown in fig. 4 to 10 and table 3, lactobacillus crispatus LC-17218 of the present application was able to significantly inhibit various pathogenic bacteria.

TABLE 3

Example 4: antibiotic sensitivity test of Lactobacillus crispatus

a. Scraping lactobacillus lawn from MRS solid culture medium by using sterile inoculating loop, transferring to MRS liquid culture medium to obtain bacterial suspension, measuring absorbance of the bacterial suspension at wavelength of 570nm by using microplate reader, and adjusting bacterial liquid concentration to 10⁸CFU/mL(OD＝0.5)；

b. Sucking 100 mu L of bacterial suspension by using a pipettor and an aseptic Tip head, dripping the bacterial suspension on an MRS flat plate, and uniformly coating the bacterial liquid by using an aseptic bacteria coating rod until the bacterial liquid is completely absorbed by the flat plate;

c. sticking OXOID drug sensitive tablet (clindamycin, metronidazole, tinidazole and ampicillin) on a plate coated with the strain to be detected, culturing in an anaerobic box at 37 deg.C for 24h, observing the plate, measuring the plate inhibition zone, and registering data.

The results show that the lactobacillus crispatus LC-17218 can be inhibited by clindamycin, metronidazole, tinidazole and ampicillin in vitro.

Example 5: sequencing analysis of bacterial drug-resistant gene by third-generation sequencing technology

a. Preparing single bacteria: carrying out anaerobic culture on lactobacillus crispatus LC-17218 in an MRS culture medium at 37 ℃ for 18-27h to obtain a plurality of colonies; scraping the bacterial colony for one-step analysis;

b. cracking and extracting DNA of bacteria by using a nucleic acid extraction kit, wherein the whole genome DNA of the extracted bacteria is subjected to steps of breaking, ExoVII digestion, damage repair, end repair, connection with a joint, enzyme reaction digestion, fragment sorting and the like to finally obtain a dumbbell-shaped library, and after the dumbbell-shaped library is detected by a Qubit device and an Agilent 2100 bioanalyzer, PacBio sequence is carried out;

c. the sequencing data (fastq files) are assembled into contigs by using SPAdes3.15.2 software and default parameters through steps of misreading correction, iterative k-mer value-based assembly, mismatch correction and the like. The ResFinder4.1 software was used, with reference to its own database of drug-resistant genes, and the drug-resistant genes in contigs were retrieved and annotated with default parameters (90% sequence similarity and 60% minimum length).

The analysis results are shown in Table 4, and Lactobacillus crispatus LC-17218 does not carry related drug resistance genes and has inhibition capacity by various antibacterial agents.

TABLE 4

In conclusion, the lactobacillus crispatus has strong in-vitro proliferation capacity, stable genetic characteristics (stable passage, consistent morphological, biochemical, metabolite and drug sensitive characteristics of each generation), easy industrial production and storage, and high-efficiency acid and H production₂O₂The lactobacillus crispatus and the cell-free supernatant thereof can inhibit the proliferation of various pathogenic bacteria in vivo/in vitro, and the bacteria have good vaginal epithelial cell adhesion capacity, can remarkably improve and regulate the vaginal microenvironment and inhibit the vaginal pathogenic bacteria, and simultaneously have good biocompatibility, safety and no toxicity. Therefore, the Lactobacillus crispatus LC-17218 and/or the metabolite thereof has good application prospect in preparing products (such as medicines, medical instruments or sanitary products and the like) for preventing and/or treating urogenital diseases.

Sequence listing

<110> Zhujiang Hospital of southern medical university

<120> Lactobacillus crispatus and uses thereof

<130> 0133-PA-006

<160> 3

<170> SIPOSequenceListing 1.0

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ggataccact tggaaacagg tgctaatacc ggataagaaa gcagatcgca tgatcagctt 180

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aggtaaaggc ttaccaaggc gatgatgcat agccgagttg agagactgat cggccacatt 300

gggactgaga cacgctccaa actcatacgg gaggcagcag tagggaatct tccacaatgg 360

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aagtcacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg 540

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cttaaccgag gaactgcatc ggaaactgtt tttcttgagt gcagaagagg agagtggaac 660

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tggtagtcca tgccgtaaac gatgagtgct aagtgttggg aggtttccgc ctctcagtgc 840

tgcagctaac gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag 900

gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag 960

aaccttacca ggtcttgaca tctagtgcca tttgtagaga tacaaagttc ccttcgggga 1020

cgctaagaca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080

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ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcatcatgc cccttatgac 1200

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gtgtagccca ggtcataagg ggcatgatga cttgacgtca tccccacctt cctccggttt 300

gtcaccggca gtctcattag agtgcccaac ttaatgctgg caactaataa caagggttgc 360

gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacagc catgcaccac 420

ctgtcttagc gtccccgaag ggaactttgt atctctacaa atggcactag atgtcaagac 480

ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 540

cccgtcaatt cctttgagtt tcaaccttgc ggtcgtactc cccaggcgga gtgcttaatg 600

cgttagctgc agcactgaga ggcggaaacc tcccaacact tagcactcat cgtttacggc 660

atggactacc agggtatcta atcctgttcg ctacccatgc tttcgagcct cagcgtcagt 720

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ctacacatgg agttccactc tcctcttctg cactcaagaa aaacagtttc cgatgcagtt 840

cctcggttaa gccgagggct ttcacatcag acttattctt ccgcctgcgc tcgctttacg 900

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agccgtgact ttctggttga ttaccgtcaa ataaaggcca gttactacct ctatccttct 1020

tcaccaacaa cagagcttta cgatccgaaa accttcttca ctcacgcggc gttgctccat 1080

cagacttgcg tccattgtgg aagattccct actgctgcct cccgtaggag tttgggccgt 1140

gtctcagtcc caatgtggcc gatcagtctc tcaactcggc tatgcatcat cgccttggta 1200

agcctttacc ttaccagcta gctaatgcac cgcggggcca tcccatagcg acagcttacg 1260

ccgcctttta aaagctgatc atgcgatctg ctttcttatc cggtattagc acctgtttcc 1320

aagtggtatc ccagactatg gggcaggttc cccacgtgtt actcacccat ccgccgctcg 1380

ctttcctaac gtcattaccg aagtaaatct gttagttccg ctcgctcgac ttgca 1435

<210> 3

<211> 945

<212> DNA

<213> Lactobacillus crispatus

<400> 3

tggctggcgg cgtgctatac atgcaagtcg agcgagcgga actaacagat ttacttcggc 60

aatgacgtta ggaaagcgag cggcggatgg gtgagtaaca cgtggggaac ctgccccata 120

gtctgggata ccacttggaa acaggtgcta ataccggata agaaagcaga tcgcatgatc 180

agcttttaaa aggcggcgta agctgtcgct atgggatggc cccgcggtgc attagctagt 240

tggtaaggta aaggcttacc aaggcgatga tgcatagccg agttgagaga ctgatcggcc 300

acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 360

aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa 420

gctctgttgt tggtgaagaa ggatagaggt agtaactggc ctttatttga cggtaatcaa 480

ccagaaagtc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540

gtccggattt attgggcgta aagcgagcgc aggcggaaga ataagtctga tgtgaaagcc 600

ctcggcttaa ccgaggaact gcatcggaaa ctgtttttct tgagtgcaga agaggagagt 660

ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc 720

ggctctctgg tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga 780

taccctggta gtccatgccg taaacgatga gtgctaagtg ttgggaggtt tccgcctctc 840

agtgctgcag ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact 900

caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtt 945

Claims (13)

1. Isolated lactobacillus crispatus, said lactobacillus crispatus being lactobacillus crispatus: (a)Lactobacillus crispatus) LC-17218 with preservation number CGMCC NO. 21222.

2. Use of lactobacillus crispatus according to claim 1 for the preparation of a medicament for the treatment and/or prevention of vaginal infections.

3. Use of lactobacillus crispatus as claimed in claim 1 for the preparation of a medicament for the suppression of pathogenic bacteria.

4. The use according to claim 3, wherein the pathogenic bacteria comprise: streptococcus agalactiae (Streptococcus agalactiae）、Prevotella corpri、Prevotella timonensisPrevotella bifida: (II)Prevotella bivia) Micro-bacterium (A), (B), (C)Micrococcus luteus) Enterococcus faecalis (C)Enterococcus faecalis) Surface staphylococci bacterium (A)Staphylococcus epidermidis) Gardnerella vaginalis (A) and (B)Gardnerella raginal) Candida albicans (C.albicans) (C.albicans)Candida albicans) Staphylococcus aureus (1)Staphylococcus aureus) Pseudomonas aeruginosa (A), (B)Pseudomonas aeruginosa) Escherichia coli (E.coli)Escherichia coli) Salmonella paratyphi b (a), (bsalmonella paratyphi b) And Shigella dysenteriae: (Shigella Castellani) Any one or more of them.

5. Use of lactobacillus crispatus according to claim 1 for the preparation of a medicament for the treatment and/or prevention of disorders of the vaginal flora.

6. Use of lactobacillus crispatus according to claim 1 for the preparation of a medicament having vaginal epithelial cell adhesion function.

7. Use of lactobacillus crispatus according to claim 1 for the preparation of an antiseptic formulation for improving the environment in the vagina.

8. The use of claim 7, wherein the improvement of the intra-vaginal environment comprises maintaining vaginal ph or increasing vaginal lactate concentration.

9. A composition comprising lactobacillus crispatus according to claim 1 and optionally one or more pharmaceutically acceptable carriers.

10. Use of a lactobacillus crispatus according to claim 1 or a composition according to claim 9 for the preparation of a sanitary article.

11. A hygiene article comprising the lactobacillus crispatus of claim 1 or the composition of claim 9.

12. The sanitary article according to claim 11, wherein the sanitary article is a medical device.

13. The sanitary product of claim 11, wherein the sanitary product comprises a lotion, a panty liner, a sanitary napkin, a tampon, a tissue, a tampon, a gel, an ointment, a cream, a spray, or a body wash.

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