Lactobacillus acidophilus and application of lactobacillus acidophilus in preparation of medicine for improving repeated discomfort of female vagina

Abstract

The invention discloses a bacterial strain, the bacterial strain is Lactobacillus acidophilus LA-013, the preservation number of the bacterial strain is CGMCC (China General Microbiological Culture Collection Center) NO.30391, and the whole genome sequence of the bacterial strain comprises a sequence as shown in SEQ No.1-4. The invention also discloses a preparation method of the bacterial strain. The strain disclosed by the invention has the positive effect of regulating the micro-ecology of the female vagina on a human body and can be used for preparing medicines for improving the repeated discomfort of the female vagina.

Description

Technical Field

[0001] The present application relates to the field of medical microbiology technology, and in particular, to a Lactobacillus acidophilus and its application in the preparation of a medicine for improving recurrent vaginal discomfort in women. Background Art

[0002] Lactobacillus acidophilus (LA) is a common symbiotic bacteria in the human body. It exists in the human oral cavity, intestines, reproductive tract and other parts, and plays an important role. Previous studies have shown that Lactobacillus acidophilus can improve and regulate the balance of human intestinal flora, relieve lactose intolerance, constipation, enhance the body's immunity and inhibit vaginal inflammation. It is a probiotic that is currently widely studied and has been included in my country's "List of Bacteria that can be used in Food" and "List of Bacteria that can be used in Infant Food", and has been widely developed into various food products for application.

[0003] However, as the research deepened, it was found that there are large functional differences between different strains of Lactobacillus acidophilus, and they can not only play a positive role in the human intestine. Summary of the invention

[0004] In view of the shortcomings of the existing methods, the present application proposes a Lactobacillus acidophilus and its application in the preparation of a drug for improving recurrent vaginal discomfort in women, so as to solve the technical problem of the limitation of the application of Lactobacillus acidophilus in the related technology.

[0005] In a first aspect, the present application provides a strain, wherein the strain is Lactobacillus acidophilus, LA-013, the preservation number of the strain is CGMCC NO.30391, and the full genome sequence of the strain includes the sequences shown in SEQ Nos.1 to 4.

[0006] Furthermore, its genome sequence includes a biosynthetic gene cluster and does not contain drug resistance genes.

[0007] Furthermore, the biosynthetic gene cluster includes a peptide cleavage / export ABC transporter functional gene and a bacteriocin functional gene.

[0008] In the second aspect, the aforementioned strain is used in the preparation of a drug for improving recurrent vaginal discomfort in women.

[0009] Optionally, the strain has an inhibitory effect on conditional pathogens and / or pathogenic bacteria of the vagina under anaerobic and aerobic conditions, respectively.

[0010] Further, the conditional pathogens include at least one of Atopobium vaginae, Prevotella bivia, Lactobacillus iners, Candida albicans, Staphylococcus epidermidis, Escherichhiacoli, Enterococcus faecalis and Micrococcus luteus; and the pathogenic bacteria include Gardnerella vaginalis.

[0011] Furthermore, the drug can improve the symptoms of abnormal vaginal discharge volume, abnormal vaginal discharge color, abnormal vaginal discharge nature, vulvar itching, vulvar redness and swelling, and / or vulvar odor.

[0012] Optionally, the medicine includes a bacterial agent of the strain and a pharmaceutically acceptable carrier; the bacterial agent includes at least one of a metabolite, a culture solution, a suspension, a fermentation solution or an extract of the strain.

[0013] Furthermore, the drug includes a drug for gastrointestinal administration and / or a drug for parenteral administration.

[0014] The Lactobacillus acidophilus (LA-013) strain of the present application was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short) (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​April 22, 2024, with the deposit number CGMCCNO.30391.

[0015] The beneficial technical effects brought about by the technical solution provided by the embodiment of the present application include: (1) The positive effect of the strain of the present application on the human body is reflected in regulating the female vaginal microecology, and can be used to prepare drugs to improve recurrent vaginal discomfort in women.

[0016] (2) The strain of the present application can inhibit the conditional pathogens and pathogens of the vagina under anaerobic and demanding conditions, respectively, and grows rapidly with low nutritional requirements, which is conducive to the formation of lactobacilli as the dominant bacteria in the vagina after the drug is used.

[0017] (3) The genome sequence of the strain of the present application includes a biosynthetic gene cluster, and the core marker genes include a peptide cleavage / export ABC transporter functional gene (ctg_196) and a bacteriocin functional gene (biosynthetic (rule-based-clusters) RiPP-like: Bacteriocin Iic function ctg_198 gene), indicating that the strain of the present application achieves antibacterial effect through biosynthetic regulation and has broad application prospects in the fields of biological products, medicine, food industry, agriculture, etc.

[0018] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which: Figure 1 A graph showing the correlation between the strains of the present application and the risk of various female reproductive tract infections; Figure 2 This is a whole genome sequencing analysis diagram of the strain LA-013 of the present application; Figure 3 This is a growth curve diagram of strain LA-013 of the present application; Figure 4 This is a diagram of the acid production of strain LA-013 of the present application; Figure 5 This is the colony growth diagram of strain LA-013 of the present application; Figure 6 This is a Gram staining diagram of the strain LA-013 of the present application; Figure 7 The biochemical profile and summary results of strain LA-013 of the present application; Figure 8 This is a bacterial mass spectrum of the strain LA-013 of the present application based on time-of-flight mass spectrometry; Fig. 9 The drug resistance verification and summary results of strain LA-013 of this application; Fig.10 The 24-hour inhibition zone comparison and summary results of the strain LA-013 and antibiotics against Atopobium vaginae of the present application; Fig.11Comparison of the 24-hour colony pattern of the strain LA-013 of the present application against Candida albicans under anaerobic conditions; Fig.12 Comparison of the 24-hour colony pattern of the strain LA-013 in this application against Candida albicans under aerobic conditions; Fig.13 The 24-hour inhibition zone of the strain LA-013 of the present application against Escherichia coli; Fig.14 The 24-hour inhibition zone of the strain LA-013 of the present application against Staphylococcus epidermidis; Fig.15 The 24-hour inhibition zone of the strain LA-013 of the present application against Enterococcus faecalis; Fig.16 The 24-hour inhibition zone of the strain LA-013 of the present application against Micrococcus luteus; Fig.17 The 24-hour inhibition zone of the strain LA-013 of the present application against Gardnerella vaginalis; Fig.18 The 24-hour inhibition zone of the strain LA-013 of the present application against Prevotella bivia; Fig.19 The 24-hour inhibition zone of the strain LA-013 of the present application against Lactobacillus iners; Fig. 20 Statistics on improvement of vaginal discomfort in women with recurrent vaginal discomfort after using the strain LA-013 bacterial powder of the present application; Fig.21 Statistics on the improvement of various vaginal discomfort symptoms in women with recurrent vaginal discomfort after applying the strain LA-013 powder of the present application. DETAILED DESCRIPTION

[0020] The embodiments of the present application are described below in conjunction with the drawings in the present application. It should be understood that the implementation methods described below in conjunction with the drawings are exemplary descriptions for explaining the technical solutions of the embodiments of the present application and do not constitute a limitation on the technical solutions of the embodiments of the present application.

[0021] Those skilled in the art will appreciate that, unless specifically stated, the "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present application refers to the presence of the features, integers, steps and / or operations, but does not exclude the implementation of other features, information, data, steps, operations, elements, components and / or combinations thereof supported by the technical field. The term "and / or" used herein refers to at least one of the items defined by the term, for example, "A and / or B" can be implemented as "A", or as "B", or as "A and B".

[0022] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

[0023] First, several terms involved in this application are introduced and explained: Bacterial mass spectra based on time-of-flight mass spectrometry (TOF-MS): mainly obtained by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This spectrum has the characteristics of specificity, rapidity, high throughput and accuracy, and has broad application prospects in the fields of clinical microbial identification, environmental monitoring and food quality control.

[0024] Biosynthetic gene cluster (BGC): It is a very important type of gene collection in the microbial genome, which plays a key role in the biosynthesis of microbial secondary metabolites. A BGC usually contains several to hundreds of functional genes, which are clustered on the chromosome. The genes included in the BGC usually produce one or several small molecule compounds together. Most of these compounds are secondary metabolites (SM), most of which are biologically active, usually low molecular weight compounds, produced at specific stages of growth and development. The genes in the BGC usually form several different transcription units and may contain more than one drug resistance gene, and there is a certain degree of mutual regulation between the expression of drug resistance genes and structural genes. The regulation of gene expression in most BGCs occurs at the transcriptional level. In addition to being regulated by specific pathway-specific regulatory factors, it is also regulated by global regulatory factors that simultaneously affect the synthesis of other intracellular products and even cell differentiation.

[0025] Peptide cleavage / export ABC transporter functional gene: It plays a vital role in the body and is mainly responsible for the cleavage and transmembrane transport of peptides. ABC transporters are a class of membrane proteins with ATP hydrolysis activity that can use the energy generated by ATP hydrolysis to drive the transmembrane transport of substances. During the peptide cleavage process, specific domains of ABC transporters (such as peptidase domains) will recognize and bind to the target peptide, and then use the energy generated by ATP hydrolysis to cleave the peptide bond, thereby completing the peptide cleavage process. After peptide cleavage, smaller peptides or amino acid residues will be produced, and these cleaved products may be further used by cells or excreted from the body. ABC transporters can transport peptides or amino acid residues from inside cells to outside cells, or from outside cells to inside cells as needed. This transport process is crucial for maintaining the balance of substances inside and outside cells and cell functions. The functions and characteristics of ABC transporters provide new ideas for drug development. By regulating the activity or expression level of ABC transporters, the concentration and distribution of drugs in cells can be changed, thereby achieving the treatment or prevention of diseases. ABC transporters also have broad application prospects in bioengineering technology. By modifying the structure and function of ABC transporters through genetic engineering, the transmembrane transport and enrichment of specific substances can be achieved, providing new technical means for biomanufacturing and bioseparation.

[0026] Bacteriocin functional gene (biosynthetic (rule-based-clusters) RiPP-like: BacteriocinIic): Bacteriocin Iic is a bacteriocin, which belongs to the class of post-translationally modified ribosomal polypeptides (RiPPs) biosynthetic products. RiPPs are biologically active polypeptides synthesized by ribosomes and post-translationally modified. They are widely present in bacteria and exhibit diverse biological functions. Bacteriocin Iic has significant antibacterial activity and can inhibit or kill bacteria closely related to it, thereby maintaining the balance and stability of the microbial community. The mechanism of action of Bacteriocin Iic may involve binding to specific receptors on the target cell membrane, leading to cell membrane destruction or metabolic disorders inside the cell. In addition, it may also exert antibacterial effects by interfering with the normal physiological processes of bacteria (such as DNA replication, protein synthesis, etc.). Due to the characteristics of Bacteriocin Iic, such as high efficiency, non-toxicity, acid resistance, high temperature resistance, no residue, and no drug resistance, it is considered to be a potential natural preservative, antibacterial agent or drug precursor. It has broad application prospects in food industry, agriculture, medicine and other fields.

[0027] Conditional pathogens: refer to bacteria that can break through the host defense mechanism and cause infection under certain specific circumstances, such as when the body's immune function decreases. They are usually present in the human body's internal and external environment, and are not pathogenic under normal circumstances, but can be transformed into pathogens under certain conditions. When conditional pathogens are transformed into pathogens, a series of clinical manifestations may be caused. The conditional pathogens involved in this application include: Atopobium vaginae, Prevotella bivia, Lactobacillus iners, Candida albicans, Staphylococcus epidermidis, Escherichia coli, Enterococcus faecalis and Micrococcus luteus.

[0028] Inhibition zone diameter: refers to the diameter of the transparent circle (i.e., inhibition zone) that can inhibit bacterial growth after the antibacterial agent or drug diffuses on the agar medium. The size of this diameter can reflect the strength of the inhibitory effect of the antibacterial agent or drug on microorganisms. According to the judgment of the antibacterial effect in the 2002 "Technical Specifications for Disinfection", an inhibition zone diameter greater than 20mm indicates a strong antibacterial effect, an inhibition zone between 10 and 20mm indicates a medium antibacterial effect, and an inhibition zone less than 10mm indicates a weak antibacterial effect; and when the inhibition zone diameter is less than 7mm, it is considered to have no antibacterial effect.

[0029] Bacteriostatic rate: calculated by the antibacterial formula. The antibacterial formula is a mathematical expression used to calculate and evaluate the inhibitory effect of antibacterial agents or drugs on microorganisms. The following are some common antibacterial formulas and their explanations: 1) Antibacterial formula based on colony diameter Formula: Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100% Control colony diameter (Bck or CK): refers to the diameter of the colony growth in the control group without the addition of antibacterial agents or drugs.

[0030] Treated colony diameter (Db or treated colony diameter): refers to the diameter of the colony growth in the treated group after the addition of antibacterial agents or drugs.

[0031] Application: This formula is often used to evaluate the inhibitory effect of antibacterial agents or drugs on microbial growth. The inhibition rate is calculated by comparing the difference in colony diameters between the treatment group and the control group.

[0032] 2) Antibacterial formula based on OD value: Antibacterial rate = [(blank bacterial solution OD value - sample bacterial solution OD value) / blank bacterial solution OD value] × 100% Blank bacterial solution OD value: refers to the optical density value of the bacterial solution without the addition of antibacterial agents or drugs at a specific wavelength.

[0033] OD value of sample bacterial solution: refers to the optical density value of the bacterial solution at the same wavelength after adding antibacterial agents or drugs.

[0034] Application: This formula calculates the antibacterial rate by comparing the OD value changes of the bacterial solution before and after the addition of the antibacterial agent or drug. This method is often used in the quantitative antibacterial effect evaluation in microbiological experiments.

[0035] 3) Antibacterial formula based on the number of colonies: Antibacterial rate = (number of single colonies on the control culture plate - number of single colonies on the treatment culture plate) / number of single colonies on the control culture plate × 100% The number of single colonies on the control culture plate (Bck or CK) refers to the number of single colonies on the culture plate in the control group without the addition of antibacterial agents or drugs.

[0036] The number of single colonies on the treated culture plate (Db) refers to the number of single colonies on the culture plate in the treated group after the addition of antibacterial agents or drugs.

[0037] Application: This formula is often used to evaluate the inhibitory effect of antibacterial agents or drugs on microbial growth. The inhibition rate is calculated by comparing the number of single colonies on the culture plate between the treatment group and the control group.

[0038] 4) Antibacterial formula based on area Formula: Antibacterial rate = (S-SM1) / (S-SM0) × 100% S: Total plate area, refers to the total area of ​​agar culture medium in the culture dish.

[0039] SM1: The mold growth area in the test group refers to the growth area of ​​the mold on the culture medium after the addition of antibacterial agents or drugs.

[0040] SM0: The mold growth area in the control group refers to the growth area of ​​the mold on the culture medium without adding antibacterial agents or drugs.

[0041] Application: This formula calculates the inhibition rate by comparing the difference in the growth area of ​​mold on the culture medium between the treatment group and the control group. This method is often used to evaluate the inhibitory effect of antibacterial agents or drugs on mold growth.

[0042] The research and development ideas of this application include: Previous studies have found that Lactobacillus acidophilus has a significant negative correlation with the risk of various female reproductive tract infections (such as Figure 1As shown in Figure 2, it is believed that Lactobacillus acidophilus has a potential role in protecting reproductive tract health. The present application separates and identifies Lactobacillus acidophilus from healthy women, and investigates the interaction between Lactobacillus acidophilus and vaginal pathogens to confirm the possible mechanism of action of Lactobacillus acidophilus. Further preliminary clinical application is carried out to investigate the prospect of Lactobacillus acidophilus as a drug for improving recurrent vaginal discomfort in women.

[0043] Example 1, Basic Characteristics of Lactobacillus acidophilus (LA-013) 1. Bacterial gene cluster annotation: Through bacterial whole genome sequencing analysis, the whole genome sequence of the strain includes sequences as shown in SEQ No. 1 to 4, SEQ No. 1 to 4 are respectively continuous sequence blocks (Contigs) of the whole genome, and it was identified as Lactobacillus acidophilus, self-numbered as LA-013, and LA-013 contains a biosynthetic gene cluster (Biosynthetic Gene Cluster, BGC) of 13045nt. The details are as follows Figure 2 The core marker genes include ctg_196 with peptide cleavage / export ABC transporter function and ctg_198 with biosynthetic (rule-based-clusters) RiPP-like: Bacteriocin Iic function. This suggests that LA-013 can play a specific role through gene editing or post-transcriptional regulation.

[0044] 2. Cultivation and growth characteristics: (1) Growth curve (such as Figure 3 LA-013 enters the logarithmic growth phase in 6 hours under anaerobic conditions in MRS medium, and its optimal growth time is 6 to 15 hours. The concentration of bacterial solution OD after 15 hours of in vitro culture is 600 The value reaches 1.16.

[0045] (2) Acid production capacity (such as Figure 4LA-013 was cultured in MRS medium under anaerobic conditions for 15 hours, and its metabolite pH was 4.5; after 24 hours, its metabolite pH was 4.0.

[0046] (3) Colony morphology (e.g. Figure 5 The colonies cultured in MRS medium for 15 hours were milky white, round, flat around and convex and moist in the center, without any pigmentation.

[0047] (4) Gram staining properties (such as Figure 6 Figure 3 shows the morphology and staining characteristics of the strain after Gram staining in the field of view of the objective lens at 20X and 100X. LA-013 is Gram-positive and has a long rod shape without flagella.

[0048] 3. Biochemical characteristics: (1) Experimental materials: API 20A anaerobic bacteria identification kit (Mérieux Diagnostics).

[0049] (2) Experimental method: Add 55 μL of LA-013 bacterial suspension with a turbidity of 3McFarland into the corresponding reaction wells according to the instructions and interpret the results.

[0050] (3) Results: Figure 7 It can be seen that LA-013 does not produce indole or urease when reacting with tryptophan, but can utilize glucose, mannitol, lactose, sucrose, maltose, isoflavone, xylose, arabinose, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose to produce acid. It does not secrete protease to hydrolyze gelatin, but can secrete β-glucosidase to hydrolyze esculin citrate ferric. The catalase test is negative, and no H2O2 is produced.

[0051] 4. Bacterial mass spectra based on time-of-flight mass spectrometry (e.g. Figure 8 As shown): Using the time-of-flight mass spectrometry microbial identification method for detection, it was found that LA-013 had typical mass spectrum peaks at 6254.92m / z and 4408.67m / z, providing further evidence for the identification of the strain species.

[0052] 5. Drug resistance analysis (such as Fig. 9 As shown in the figure, LA-013 is sensitive to a variety of commonly used clinical antibiotics, and no drug-resistant genes were found in BGC.

[0053] Example 2: Testing the antibacterial effect in vitro 1. Inhibitory effect on Atopobium vaginnae (24 hours). Atopobium vaginae is an anaerobic or microaerophilic Gram-positive bacterium that is part of the female vaginal microbiome and may be associated with bacterial vaginosis in some cases. LA-013 was co-cultured with Atopobium vaginae to produce an inhibition zone around LA-013. The inhibition effects were compared with a variety of other commonly used antibiotics. It was found that LA-013 had a better inhibitory effect on Atopobium vaginae than a variety of antibiotics (such as Fig.10 shown).

[0054] 2. Inhibitory effect on Candida albicans.

[0055] Candida albicans mainly reproduces by budding and requires oxygen. It grows well on ordinary agar, blood agar and SDA agar. Candida albicans is a conditional pathogen and usually does not cause disease. It can cause candidiasis when the body's resistance is reduced or there are other inducements (such as long-term use of antibiotics, immunosuppressants, etc.). It can cause acute and chronic infections of the skin, oral cavity, mucous membranes and internal organs, including vulvar and vaginal infections.

[0056] The inhibition rate of LA-013 against Candida albicans under anaerobic conditions was 67.94% ( Fig.11 shown).

[0057] ① Antibacterial formula: [N C.a -N (LA-C.a ) / N C.a ]×100%; N C.a Indicates the number of single colonies in the culture plate of the Ca group; N (LA-C.a) Represents the number of single colonies in the culture plates of the La+Ca group.

[0058] ② Experimental method: Two groups were set up in the experiment, Ca group and La+Ca group. The Ca group was the positive control group, containing OD 600 =0.5, 1 mL of Candida albicans suspension was added to 1 mL of basal culture medium; the La+Ca group was the LA-013 and Candida albicans inhibition group, including OD 600 = 0.5 LA-013 1mL bacterial suspension and OD 600= 0.5, and the two culture systems were placed in a DWS A35 anaerobic box at 37°C for 24 hours. CCP medium was used for bacterial liquid coating, and the number of single colonies was calculated. The inhibitory effect of LA-013 on Candida albicans was calculated using the antibacterial formula.

[0059] The inhibition rate of LA-013 against Candiida albicans under aerobic conditions was 99.61% ( Fig.12 shown).

[0060] ① Antibacterial formula: [N C.a -N (LA-C.a) / N C.a ]×100%; N C.a Indicates the number of single colonies in the culture plate of the Ca group; N (LA-C.a) Represents the number of single colonies in the culture plates of the La+Ca group.

[0061] ② Experimental method: Two groups were set up in the experiment, Ca group and La+Ca group. The Ca group was the positive control group, containing OD 600 =0.5, 1 mL of Candida albicans suspension was added to 1 mL of basal culture medium; the La+Ca group was the LA-013 and Candida albicans inhibition group, including OD 6000 = 0.5 LA-013 bacterial suspension 1mL and OD 600 = 0.5, and the two culture systems were placed in a common carbon dioxide incubator at 37°C for 24 hours. CCP medium was used for bacterial coating, and the number of single colonies was calculated. The inhibitory effect of LA-013 on Candida albicans was calculated using the antibacterial formula.

[0062] Therefore, it is confirmed that LA-013 of the present application can exert an antibacterial effect under both anaerobic and aerobic conditions, and the antibacterial effect under aerobic conditions is more obvious.

[0063] 3. Inhibitory effect on Escherichia coli (ATCC25922), the antibacterial diameter is 25mm (such as Fig.13As shown). Escherichia coli is a facultative anaerobe. According to biological characteristics, Escherichia coli can be divided into six categories, namely enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), enteroadherent Escherichia coli (EAEC) and diffusely adherent Escherichia coli (DAEC). For a long time, Escherichia coli was regarded as a component of normal intestinal flora and was identified as a non-pathogenic bacterium. However, studies in the mid-20th century found that certain serotypes of Escherichia coli are pathogenic to humans and animals, especially to infants and young livestock (poultry), and often cause severe diarrhea, sepsis, urinary tract infections and other diseases. Among them, 0157:H7 type Escherichia coli has strong pathogenicity and resistance to gastric acid, and is highly destructive to cells. Therefore, many countries classify infectious diarrhea caused by 0157:H7 Escherichia coli as a foodborne disease.

[0064] Antibacterial test method: OD 600 100 μL of Escherichia coli suspension with a pH of 5.0 = 0.5 was evenly spread on a BHI culture plate and allowed to stand for 5 minutes. A defatted paper sheet soaked with 20 μL of LA-013 solution was placed in the center of the BHI culture plate and cultured in a normal carbon dioxide incubator at 37°C for 24 hours. The diameter of the sterile area was measured with a ruler centered on the paper sheet. The experiment was repeated 3 times and the average of the three antibacterial diameters was taken to confirm the inhibitory effect of LA-013 on Escherichia coli.

[0065] Therefore, it is confirmed that LA-013 of the present application also has a significant inhibitory effect on at least part of the non-vaginal flora.

[0066] 4. The inhibitory effect on surface Staphylococcus epidermidis is 23.5 mm in diameter (e.g. Fig.14As shown). Staphylococcus epidermidis is aerobic or facultative anaerobic. Staphylococcus epidermidis generally does not produce exotoxins (hemolytic toxins), leukocidin and enterotoxins, so it has weak invasiveness and is a non-pathogenic staphylococcus. However, the pathogenicity of Staphylococcus epidermidis strains is related to their ability to form biofilms. When the body's immune function is reduced and the site of residence changes (such as damaged skin or mucous membranes), the bacteria can cause disease, causing suppurative infections and even sepsis. In particular, newborns, premature infants, infants, the elderly and patients with certain chronic diseases, malnourished people, immunocompromised people, underdeveloped people, or patients with severe trauma, burns, major surgery, long-term bed rest, and coma are more susceptible to infection with Staphylococcus epidermidis. Staphylococcus epidermidis is prone to drug resistance and resistance to commonly used antibiotics that it was sensitive to in the past. This is also an important reason for the increased incidence of Staphylococcus epidermidis infection and sepsis, which brings difficulties to clinical treatment.

[0067] Antibacterial test method: OD 6000＝ 100 μL of 0.5% Staphylococcus epidermidis bacterial suspension was evenly spread on the BHI culture plate and left to stand for 5 minutes. Three defatted paper sheets soaked with 20 μL of LA-013 bacterial solution were evenly attached to the BHI culture plate and cultured in a normal carbon dioxide incubator at 37°C for 24 hours. The diameter of the sterile area was measured with a ruler centered on the paper sheet, and the average of the three antibacterial diameters was taken to confirm the inhibitory effect of LA-013 on Staphylococcus epidermidis.

[0068] Therefore, it is confirmed that LA-013 of the present application has a significant inhibitory effect on at least some drug-resistant pathogenic bacteria.

[0069] 5. Inhibition of Enterococcus faecalis, the inhibition diameter is 20mm (such as Fig.15 As shown). Enterococcus faecalis is a facultative anaerobic Gram-positive lactic acid bacterium. In most cases, Enterococcus faecalis is harmless to the body, but recent studies have found that some Enterococci have developed virulence genes during long-term evolution, which can cause widespread infection. The most common infection caused by Enterococcus faecalis is urinary tract infection, most of which are related to urethral instrumentation, catheterization, etc. In addition, it can also cause trauma and postoperative surgical infections, sepsis, endocarditis, etc. in the abdomen and pelvis. In a few cases, Enterococcus faecalis can also cause wound infections, cellulitis, meningitis, etc.

[0070] Antibacterial test method: OD 600100 μL of Enterococcus faecalis suspension with a pH of 5.0 = 0.5 was evenly spread on a BHI culture plate and left to stand for 5 min. Three defatted paper sheets soaked with 20 μL of LA-013 bacterial solution were evenly attached to the BHI culture plate and cultured in a normal carbon dioxide incubator at 37°C for 24 hours. The diameter of the sterile area was measured with a ruler centered on the paper sheet, and the average of the three inhibition diameters was taken to confirm the inhibitory effect of LA-013 on Enterococcus faecalis.

[0071] Therefore, it is suggested that LA-013 of the present application can assist in preventing and treating postoperative infection.

[0072] 6. Inhibitory effect on Micrococcus luteus, the inhibition diameter is 26mm (such as Fig.16 As shown in Figure 1.2, Micrococcus luteus is an aerobic or facultative anaerobic bacterium. Micrococcus luteus is generally not pathogenic, but it can be a conditional pathogen, causing local tissue infections such as wounds, and can also cause serious infections such as endocarditis. People with low immunity are more susceptible to infection with this type of bacteria.

[0073] Antibacterial test method: OD 600 = 0.5 Micrococcus luteus suspension 100 μL was evenly spread on the BHI culture plate, and left to stand for 5 minutes. Three degreased paper sheets soaked with 20 μL LA-013 bacterial solution were evenly pasted on the BHI culture plate, and placed in a normal carbon dioxide incubator at 37°C for 24 hours. The diameter of the sterile area was measured with a ruler at the center of the paper sheet, and the average of the three antibacterial diameters was taken to confirm the inhibitory effect of LA-013 on Micrococcus luteus. u The inhibitory effect of teus.

[0074] Therefore, it is confirmed that LA-013 of the present application has a significant inhibitory effect on at least some conditional pathogens.

[0075] 7. Gardnerell vaginalis a vaginalis), the inhibition diameter is 36mm (such as Fig.17 Gardnerella vaginalis is a facultative anaerobic bacterium and the main pathogen causing Gardnerella vaginitis (also known as Haemophilus vaginitis). Therefore, it is confirmed that LA-013 of the present application has a significant inhibitory effect on at least some vaginal pathogens.

[0076] 8. The inhibitory effect on Prevotella bivia is 31 mm in diameter (e.g. Fig.18 As shown in Figure 2). Prevotella bivia is an anaerobic bacterium that can grow and reproduce in an anaerobic or hypoxic environment. Under normal circumstances, Prevotella bivia exists as a probiotic and helps maintain intestinal health. In some cases, Prevotella bivia may become a conditional pathogen, leading to intestinal inflammation, bacterial vaginitis and other diseases. This may be related to factors such as the host's immune status and intestinal microecological imbalance.

[0077] 9. The inhibitory effect on Lactobacillus iners is 23mm in diameter (such as Fig.19 As shown). Lactobacillus iners is a fermentative bacterium, and its main metabolites include lactic acid. In the vagina, Lactobacillus iners may be part of a healthy microbiome and work with other lactobacilli to inhibit the growth of pathogens. In some cases, Lactobacillus iners may have an immunomodulatory effect, helping to maintain the balance of the host immune system. However, Lactobacillus iners may become a conditional pathogen under certain conditions and is associated with reproductive tract infections such as bacterial vaginosis (BV). Studies have shown that an increase in the abundance of Lactobacillus iners may be related to the occurrence and development of vaginal inflammation. Lactobacillus iners may also affect the therapeutic effect of radiotherapy on cancer and drive the drug resistance of cancer cells.

[0078] The inhibitory effect of LA-013 on Prevotella bivia and Lactobacillus iners suggests that LA-013 at least partially affects the structural composition of the vaginal flora or the abundance of each flora.

[0079] Example 3, clinical verification The LA-013 of the present application was industrially fermented and the bacterial powder was pressed into tablets. 35 female patients were selected and administered vaginally. One tablet was used daily for eight consecutive days, which significantly improved the recurrent vaginal discomfort of women. Fig. 20 and 21 The significant improvement rate reached 77%, and there was a significant improvement in the symptoms of vaginal discharge volume, color abnormalities and vulvar itching, with improvement rates of 71%, 43% and 40.9% respectively.

[0080] LA-013 is used in the preparation of drugs for improving recurrent vaginal discomfort in women. Usually, a bacterial agent containing LA-013 is used in combination with a pharmaceutically acceptable carrier to prepare a drug that is convenient for storage, transportation and use. Optionally, the bacterial agent of LA-013 includes at least one of a metabolite, a culture solution, a suspension, a fermentation solution or an extract of the strain, and the bacterial agent is used as the main active ingredient of the drug. Drugs can be classified as drugs for gastrointestinal administration or drugs for parenteral administration according to the route of administration.

[0081] Gastrointestinal dosage forms mainly refer to the dosage forms of drug preparations that enter the gastrointestinal tract after oral administration and exert local or systemic effects through absorption. Common dosage forms include powders, tablets, granules, capsules, solutions, emulsions, suspensions, etc.

[0082] Non-gastrointestinal dosage forms mainly refer to all other dosage forms except oral administration routes, which can act locally at the site of administration or exert systemic effects after being absorbed. Including: Injection dosage forms: such as injections, including multiple injection routes such as intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavitary injection. Respiratory tract dosage forms: such as sprays, aerosols, powder sprays, etc. Skin dosage forms: such as external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc. Mucosal dosage forms: such as eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets and patches, etc. Cavity dosage forms: such as suppositories, effervescent tablets, drops and pills, etc., used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.

[0083] In summary, the present application provides a strain, wherein the strain is Lactobacillus acidophilus, LA-013, the deposit number of the strain is CGMCC NO.30391, and the whole genome sequence of the strain includes the sequences shown in SEQ No.1 to 4. The positive effect of the strain of the present application on the human body is reflected in regulating the female vaginal microecology, and can be used to prepare a drug for improving recurrent vaginal discomfort in women.

[0084] The above is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical concept of the scheme of the present application, other similar implementation methods based on the technical ideas of the present application are also within the protection scope of the embodiments of the present application.

Claims

1. A strain, characterized in that The strain is Lactobacillus acidophilus, LA-013, the preservation number of the strain is CGMCC NO.30391, and the full genome sequence of the strain includes sequences shown in SEQ No.1-4.

2. The strain according to claim 1, characterized in that Its genome sequence includes biosynthetic gene clusters and does not contain drug resistance genes.

3. The strain according to claim 2, characterized in that The biosynthetic gene cluster includes a peptide cleavage / export ABC transporter functional gene and a bacteriocin functional gene.

4. Use of the strain according to any one of claims 1 to 3 in the preparation of a drug for improving recurrent vaginal discomfort in women.

5. The use according to claim 4, characterized in that The strain has an inhibitory effect on conditional pathogens and / or pathogenic bacteria of the vagina under anaerobic and aerobic conditions respectively.

6. The use according to claim 5, characterized in that The conditional pathogens include at least one of Atopobium vaginae, Prevotella bivia, Lactobacillus iners, Candida albicans, Staphylococcus epidermiais, Escherichia coli, Enterococcus faecalis and Micrococcus luteus; and the pathogenic bacteria include Gardnerella vaginalis.

7. The use according to claim 4, characterized in that The drug can improve the symptoms of abnormal vaginal discharge volume, abnormal vaginal discharge color, abnormal vaginal discharge nature, vulvar itching, vulvar redness and swelling, and / or vulvar odor.

8. The use according to claim 4, characterized in that The medicine comprises a bacterial agent of the strain and a pharmaceutically acceptable carrier; the bacterial agent comprises at least one of a metabolite, a culture solution, a suspension, a fermentation solution or an extract of the strain.

9. The use according to claim 8, characterized in that The drugs include drugs for parenteral administration and / or drugs for parenteral administration.

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