CN102851248B - Lactobacillus jensii for the prevention and treatment of bacterial vaginosis - Google Patents

Abstract

The invention discloses a strain of Lactobacillus jansii and application thereof. The Lactobacillus jensenii provided by the present invention is specifically Lactobacillus jensenii IMJ-1, and its preservation number in the General Microbiology Center of the China Committee for the Collection of Microorganisms is CGMCC No. 6558. Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 of the present invention has a strong ability to produce H ₂ O ₂ , which can effectively inhibit the growth of foreign pathogenic bacteria and other conditional pathogenic bacteria; at the same time, it has strong Adhesion to vaginal epithelial cells ensures the colonization of the strain in the vaginal epithelium. The Lactobacillus jensenii IMJ-1CGMCC No. 6558 provided by the present invention has a certain application prospect in the prevention and treatment of bacterial vaginosis.

Description

Lactobacillus jensii for the prevention and treatment of bacterial vaginosis

technical field

The invention belongs to the field of biotechnology, relates to Lactobacillus jansnii and application thereof, in particular to a strain of Lactobacillus jansii isolated from a human body and used for preventing and treating bacterial vaginosis.

Background technique

Microorganisms inhabit almost every part of the human body, such as the skin, oral cavity, intestinal tract, reproductive tract, etc. There are a large number of bacteria, including many different types of bacteria, which can be roughly divided into "beneficial bacteria" and "conditional treatment". There are two types of germs, and the number and proportion of these bacteria in a healthy human body maintain a stable balance. A variety of bacteria can exist in the vagina of women, among which Lactobacillus is the most common and important beneficial bacteria to maintain a healthy vaginal micro-ecological environment, and Lactobacillus accounts for more than 90% of the total vaginal bacteria in healthy women. They mainly inhibit the growth of pathogenic bacteria and other opportunistic pathogenic bacteria through acid production and hydrogen peroxide. If some factors cause the number of lactobacilli in the vagina to decrease, or even completely disappear, it can lead to abnormal proliferation of many conditional bacteria in the vagina, and bacterial vaginosis is mainly characterized by obvious vaginal microecological environment disturbance.

Bacterial vaginosis is the most common but unappreciated frequently-occurring disease in women of childbearing age. The prevalence rate is about 20% in healthy women and about 15% in pregnant women. Bacterial vaginosis is a "small disease, big trouble", the quality of life of patients is significantly reduced, and the mental burden is heavy; and the risk of contracting AIDS, gonorrhea, non-gonococcal urethritis and other sexually transmitted diseases is significantly increased; pregnant women with the disease may have a series of pregnancies Complications, such as premature birth and chorioamnionitis. At present, the treatment of bacterial vaginosis mainly relies on antibiotics such as metronidazole or lincomycin, but the treatment effect is not good, the cure rate is less than 50%, and the recurrence rate is high. Half of the patients cannot be cured even with a large amount of antibiotics. The clinical symptoms persist, the patients are suffering, and the doctors have no good solution. How to control recurrence and completely cure bacterial vaginosis is a thorny problem that obstetricians and gynecologists urgently need to solve.

In order to improve the therapeutic effect of bacterial vaginosis, new treatment options are urgently needed clinically. Due to the ineffectiveness of antibiotics in the treatment of bacterial vaginosis and the side effects of liver and kidney damage caused by antibiotics, efforts have been made to explore microecological therapies based on probiotic Lactobacillus preparations at home and abroad. Microecological therapy is a new treatment concept aimed at changing the main treatment method of killing microorganisms to increasing probiotics and restoring the normal vaginal microecological environment. That is, after antibiotics are used to remove pathogenic bacteria, live Lactobacillus preparations isolated from the vagina of healthy women are directly supplemented to allow them to adhere and colonize, and promote their natural ability to resist the growth of pathogenic bacteria.

There are many kinds of Lactobacillus in the vagina of healthy women, with individual differences, and the anti-pathogenic bacteria ability of Lactobacillus jansnii varies significantly. When choosing Lactobacillus probiotics, it is necessary to comprehensively consider the type of Lactobacillus, its ability to produce acid and H ₂ O ₂ , and its ability to adhere to vaginal epithelial cells. The basis of the role of Lactobacillus is also a key factor in the efficacy of Lactobacillus.

Because different Lactobacillus strains have different adhesion affinities to vaginal epithelial cells, the strains with strong affinity have strong colonization ability, which can inhibit and kill a variety of pathogenic microorganisms. If the Lactobacillus live bacteria preparation cannot colonize the vaginal epithelial cells, no matter how strong the effect of inhibiting and killing various pathogenic microorganisms in vitro experiments is, it will become a blank check.

A perfect and effective probiotic strain must at least meet the following standards: (1) It must be a normal colonization bacteria in the host application site; (2) It can survive and grow in the host application site; (3) It can provide host beneficial bacteria. (4) Relevant products must be able to maintain activity and viability during manufacture and storage.

At present, there is only one kind of vaginal probiotic agent in China, and its main ingredient is live Lactobacillus delbrueckii. Although it is a kind of Lactobacillus isolated from the vagina of healthy women, it is not the main beneficial bacteria in the vaginal flora of Chinese women. The vaginal bacteria of healthy women in my country are dominated by Lactobacillus jansii.

Contents of the invention

The object of the present invention is to provide a strain of Lactobacillus jansii for preventing and treating bacterial vaginosis.

The Lactobacillus jensenii provided by the present invention is specifically Lactobacillus jensenii IMJ-1, which has been preserved in the General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short, address: For: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation registration number is CGMCC No.6558.

The Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 is about 1-2 mm in size, opaque, protruding milky-white colonies, with neat edges, relatively moist colonies, and stable size and shape (Figure 1). The bacterial strain IMJ-1 in the logarithmic growth phase, after smear and Gram staining, observed the morphology of the bacteria under an optical microscope: Gram staining positive, no bacillus, and no flagella. (figure 2). Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 is facultatively anaerobic, with an optimum growth temperature of 37-42°C and an optimum pH of 5.8-6.2; it can ferment glucose, fructose, sucrose, trehalose, maltose, etc. kind of sugar.

The application of Lactobacillus jensenii IMJ-1CGMCC No.6558 in the production of H ₂ O ₂ also falls within the protection scope of the present invention.

In the application, the Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No. 6558 was planted in the culture medium, and cultured by first standing and then shaking culture; the static culture was an anaerobic environment.

In one embodiment of the present invention, the medium is specifically MRS (deMan, Rogosa and Sharpe, MRS) liquid medium; the temperature of the culture is 37°C; Static culture was performed for 21 hours, followed by shaking culture for 3 hours; the rotational speed of the shaking culture was 220 rpm, and the rotation radius was 2.54 cm.

The application of the Lactobacillus jensenii IMJ-1CGMCC No. 6558 in the preparation of products for preventing and/or treating bacterial vaginosis also belongs to the protection scope of the present invention.

The product can be an antibacterial agent or medicine for preventing and/or treating bacterial vaginosis.

The application of the Lactobacillus jensenii IMJ-1CGMCC No.6558 in the preparation of products with vaginal epithelial cell adhesion function also belongs to the protection scope of the present invention.

Another object of the present invention is to provide a bacterial agent.

The bacterial agent provided by the present invention uses the Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 as the active ingredient.

The application of the bacterial agent in the production of H ₂ O ₂ also belongs to the protection scope of the present invention.

The application of the bacterial agent in the preparation of products for preventing and/or treating bacterial vaginosis also belongs to the protection scope of the present invention.

The application of the bacterial agent in the preparation of products with vaginal epithelial cell adhesion function also belongs to the protection scope of the present invention.

Another object of the present invention is to provide a product for preventing and/or treating bacterial vaginosis.

The active ingredient of the product for preventing and/or treating bacterial vaginosis provided by the present invention is the Lactobacillus jensenii IMJ-1CGMCC No.6558.

The product can be an antibacterial agent or medicine for preventing and/or treating bacterial vaginosis.

Another object of the present invention is to provide a product with the function of adhesion of vaginal epithelial cells.

The active ingredient of the product with vaginal epithelial cell adhesion function provided by the present invention is the Lactobacillus jensenii IMJ-1CGMCC No.6558.

According to the recognized standards of probiotic strains, the present invention isolates the dominant vaginal flora—Lactobacillus jansnii—from healthy women in China, and screens out a strain with high H ₂ O ₂ production capacity and strong affinity with vaginal epithelium. A probiotic strain with strong cell adhesion and colonization ability—Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558. The experiment proved that the Lactobacillus jensenii IMJ-1CGMCC No.6558 was inoculated in the MRS liquid medium at a density of 10 ⁶ cfu/ml, and after static culture at 37°C for 21 hours in an anaerobic environment, the transformation For shaking culture for 3 hours, the rotational speed was 220 rpm, and the final concentration of H ₂ O ₂ in the culture supernatant was as high as 362.3 μM. Efficient production of hydrogen peroxide can effectively inhibit the growth of foreign pathogenic bacteria and other conditional pathogenic bacteria; strong adhesion to vaginal epithelial cells ensures the colonization of bacterial strains in vaginal epithelial cells. The Lactobacillus jensenii IMJ-1CGMCC No.6558 provided by the present invention has a certain application prospect in the prevention and treatment of bacterial vaginosis.

Preservation instructions

Bacteria name: Lactobacillus jansii

Latin name: (Lactobacillus jensenii)

Strain number: IMJ-1

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: September 11, 2012

Registration number of the collection center: CGMCC No.6558

Description of drawings

Figure 1 is a colony of Lactobacillus jensenii IMJ-1CGMCC No.6558 isolated from a healthy person.

Figure 2 shows the morphology of Lactobacillus jensenii IMJ-1CGMCC No.6558 observed under an optical microscope.

Figure 3 shows the adhesion of Lactobacillus jensenii IMJ-1CGMCC No.6558 to vaginal epithelial cells.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, isolation and identification of Lactobacillus jansnii strain

1. Isolation of Lactobacillus jansii strains

MRS medium: peptone 10.0g; beef extract 10.0g; yeast extract 5.0g; diammonium hydrogen citrate 2.0g; glucose 20.0g; Tween 80, 1.0mL; sodium acetate ( _CH3COONa · _3H2O ) 5.0g Dipotassium hydrogen phosphate (K ₂ HPO4·3H ₂ O) 2.0g; Magnesium sulfate (MgSO ₄ ·7H ₂ O) 0.58g; Manganese sulfate (MnSO ₄ ·H ₂ O) 0.25g; Distilled water 1000mL; adjust to pH 6.2 ~6.6.

More than 200 samples of vaginal secretions from healthy women without symptoms and signs of vaginitis were collected. The samples were numbered sequentially, inoculated in MRS liquid medium, and incubated anaerobically at 37°C for 24 hours. For each sample, 5 single colonies were randomly selected and cultured in 2 mL MRS liquid medium for 24 hours, and the colonies were numbered sequentially. Bacteria are amplified for bacterial conservation and DNA preparation. Hundreds of isolated bacteria were tested for bacterial species identification, hydrogen peroxide production and adhesion to vaginal epithelial cells, and one of them was named Lactobacillus jannis with strong adhesion and hydrogen peroxide production for IMJ-1.

2. Identification of Lactobacillus jansii strains

It was identified that the strain IMJ-1 isolated in step 1 was Lactobacillus jensenii. The Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1 was preserved on September 11, 2012 in the General Microbiology Center of the China Committee for the Collection of Microorganisms (CGMCC for short), the address is: Courtyard 3, No. 1 Beichen West Road, Chaoyang District, Beijing No.), the deposit number is CGMCC No.6558. Specifically, the strain IMJ-1 was identified from the following aspects:

1. Morphological identification

On the MRS Lactobacillus medium plate, the strain IMJ-1 isolated from the above step 1 appeared as opaque and protruding milky-white colonies with a size of about 1-2 mm, and the edges of the colonies were neat and moist, and the size and shape were stable (Figure 1) .

The bacterial strain IMJ-1 in the logarithmic growth phase, after smear and Gram staining, observed the morphology of the bacteria under an optical microscope: Gram staining positive, no bacillus, and no flagella. (figure 2).

2. Physiological and biochemical characteristics analysis

Strain IMJ-1 is facultatively anaerobic, with an optimum growth temperature of 37-42°C and an optimum pH of 5.8-6.2. According to the API bacterial identification standard, use API 50CHL medium and API 50CH reagent strip (bioMe'rieux, Inc., Marcy l'Etoile, France) to conduct sugar fermentation reaction interpretation of the detection strain IMJ-1 to detect the physiological and biochemical characteristics of the strain IMJ-1 . API50CHL is a simple medium consisting of API 50CH test strips of 49 fermentable carbohydrates. Inoculate each vial of the test strip with a suspension made of the bacteria to be tested. When cultured, the pH drops due to acid production from fermented carbohydrates, discoloring the indicator.

The results showed that strain IMJ-1 could ferment glucose, fructose, sucrose, trehalose, maltose and other sugars.

3. 16s rDNA sequence homology analysis

Cultivate the strain IMJ-1 isolated in the above step 1 by conventional methods, extract the total DNA of the strain as a template for gene amplification, and use universal primers 27f: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492r: 5′-TACGGTTACCTTGTTACGACTT-3′ to amplify the bacteria Conserved region of 16S rRNA gene. The 25 μL amplification system includes: 1×PCR reaction buffer, 200 μmol/L dNTPs, 0.2 μmol/L upstream and downstream primers, 1U Taq DNA polymerase, 1 μL template DNA. Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 30 s, a total of 25 cycles; extension at 72°C for 10 min. PCR products were detected by 1% gel electrophoresis, and positive results were sequenced bidirectionally with 27f: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492r: 5'-TACGGTTACCTTGTTACGACTT-3'. Sequence splicing and similarity analysis were completed using DNAStar software, and sequence alignment was completed online through the National Center for Biotechnology Information NCBI database (http://www.ncbi.nlm.nih.gov).

The 16s rDNA sequence of strain IMJ-1 is detailed in sequence 1 in the sequence listing.

Example 2. Functional Analysis of Lactobacillus jensenii IMJ-1CGMCC No.6558

1. Production of H ₂ O ₂ by Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 Detection H ₂ O ₂ standard curve preparation: first use piperazine-N,N'-di-ethane with a concentration of 100mmol/L Sulfonic acid (Piperazine-N, N_-Bis 2-ethanesulfonic acid, PIPES) diluted 30% (v/v) H ₂ O ₂ (equivalent to 9.128mol/L) stock solution to 1mol/L, and then 100mmol/L PIPES dilutes 1mol/L H ₂ O ₂ into 0μmol/L, 20μmol/L, 40μmol/L, 60μmol/L, 80μmol/L, 100μmol/L working solution; then take 100μL of the above H ₂ O ₂ working solution and Mix 100 μL of TMB (tetramethylbenzidine) with a concentration of 20 mmol/L and 2 μL of horseradish peroxidase (1 mg/mL), incubate at 16°C for 10 min, and measure the OD ₆₃₀ value. Finally, the H ₂ O ₂ concentration standard curve was obtained: y=0.0033x-0.0496, R ² =0.992.

Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 liquid culture H ₂ O ₂ concentration detection: pick Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 colony, inoculate 1mL of bacterial solution cultured for 48h in 10mL In the MRS liquid medium, the concentration of the bacterial strain in the culture medium was 10 ⁶ cfu/ml, and after static culture at 37°C for 21 hours in an anaerobic environment, it was switched to shaking culture at 37°C for 3 hours, the rotation speed was 220r/min, and the rotation radius was 2.54cm. After the cultivation, collect the fermentation broth, centrifuge at 12000r/min for 2min, take 100μL of the bacterial supernatant to measure the OD ₆₃₀ value, and calculate the H ₂ O ₂ concentration in the bacterial supernatant according to the standard curve. The experiment was repeated three times, and the results were averaged.

The results are shown in Table 1. Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 can produce a large amount of H ₂ O ₂ in the fermentation medium, and its concentration is as high as 362.3 μM.

Table 1 Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 ability to produce H ₂ O ₂ Detection unit: μM

Repeat 1 Repeat 2 Repeat 3 Average H ₂ O ₂ yield 300.8 378.0 408.1 362.3

2. Adhesion of Lactobacillus jansonii to vaginal epithelial cells

Vaginal epithelial exfoliated cells were obtained from more than 200 healthy women of childbearing age (clinical tests showed no reproductive system infection). Each cell sample was labeled and added to MEM medium (pH 4.0), centrifuged at 120g for 10 minutes, and then repeated centrifuged twice. Wash off the surface bacteria, adjust the concentration of exfoliated vaginal epithelial cells to 10 ⁵ cells/mL with MEM medium; activate the overnight culture of Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 so that the concentration is (10 ⁸ CFU/mL) . The vaginal epithelial exfoliated cells were mixed with the Lactobacillus jensenii IMJ-1CGMCC No.6558 in equal volumes of 500 μL each, and then placed on a shaker at 37°C (rotating speed: 100r/min, radius of rotation: 2.54cm) and incubated for 1h , and then filtered with a 0.8 μm filter membrane to remove free Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558, and the filter membrane was lightly pressed on the cover glass, and the cells were fixed for Gram staining and observed with an oil microscope. Count the number of bacteria attached to three cells, and calculate the average number of bacteria attached to each cell. At the same time, another strain of Lactobacillus jensenii, which was also isolated and identified by the method of Example 1, but had weak adhesion and colonization ability to vaginal epithelial cells, was used as a negative control.

The results showed that Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 had strong adhesion to vaginal epithelial cells isolated from more than 200 healthy women of reproductive age. Figure 3 is the gran-stained oil microscope observation of the adhesion between two strains of Lactobacillus jansnii with different adhesion and colonization ability to vaginal epithelial cells. exfoliated cells; the lower part of the figure shows the adhesion of Lactobacillus jensenii exfoliated cells of the vaginal epithelium, the lower left figure shows strong adhesion (Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558), and the lower right figure shows relatively strong adhesion. Weak adhesion (negative control). From more than 200 cases, 4 cases were randomly selected for statistical analysis, and the average number of Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 adhered to each vaginal epithelial cell reached 243 (see Table 2).

Table 2 Statistics on the number of vaginal epithelial cells adhered to Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558

Based on the results of Example 1 and Example 2, the Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 of the present invention has a strong ability to produce H ₂ O ₂ (effectively inhibiting pathogenic bacteria and other conditional pathogens) bacteria growth), and has a strong adhesion to vaginal epithelial cells (guaranteeing the colonization of the strains in the vaginal epithelium), which makes Lactobacillus jensenii (Lactobacillus jensenii) IMJ-1CGMCC No.6558 an effective bacterial control Candidate probiotic strains for vaginosis.

Claims (8)

1. Lactobacillus Jensenii (Lactobacillus jensenii) IMJ-1, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.6558.

2. the described Lactobacillus Jensenii of claim 1 (Lactobacillus jensenii) IMJ-1CGMCC No.6558 prevents and/or treats application in the product of bacterial vaginosis in preparation.

3. the described Lactobacillus Jensenii of claim 1 (Lactobacillus jensenii) IMJ-1CGMCC No.6558 has application in the product of vaginal epithelial cell adhesion function in preparation.

4. the microbial inoculum take the described Lactobacillus Jensenii of claim 1 (Lactobacillus jensenii) IMJ-1CGMCC No.6558 as activeconstituents.

5. the described microbial inoculum of claim 4 prevents and/or treats application in the product of bacterial vaginosis in preparation.

6. the described microbial inoculum of claim 4 has application in the product of vaginal epithelial cell adhesion function in preparation.

7. prevent and/or treat the product of bacterial vaginosis, it is characterized in that: the activeconstituents of described product is Lactobacillus Jensenii claimed in claim 1 (Lactobacillus jensenii) IMJ-1CGMCC No.6558.

8. have the product of vaginal epithelial cell adhesion function, it is characterized in that: the activeconstituents of described adhesion product is Lactobacillus Jensenii claimed in claim 1 (Lactobacillus jensenii) IMJ-1CGMCC No.6558.

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