CN104293718B - Lactobacillus jensenii and application thereof - Google Patents

Abstract

The invention discloses a strain of Lactobacillus jansii and application thereof. Lactobacillus jensenii (Lactobacillus.jensenii) disclosed by the present invention is named as IMJ‑19, and has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on July 14, 2014, and its preservation number is CGMCC No.9439 . Lactobacillus jensenii (Lactobacillus.jensenii) IMJ-19 disclosed by the present invention has a strong ability to produce H ₂ O ₂ , can effectively inhibit the growth of foreign pathogenic bacteria and other conditional pathogenic bacteria, and has a strong The adhesion to the vaginal epithelial cells ensures the colonization of the strains in the vaginal epithelium. Lactobacillus jensii IMJ‑19 has a certain application prospect in the prevention and treatment of urogenital tract infections.

Description

A strain of Lactobacillus jansii and its application

technical field

The invention relates to the field of microorganisms, in particular to a strain of Lactobacillus jansii and its application.

Background technique

Urogenital tract infection is the most common but not paid attention to frequently-occurring disease of women, and the incidence rate is second only to respiratory tract infection, which can be as high as 40%. Women's physical and mental health will not only affect the happiness and stability of many families, but also affect the population quality and economic development of the whole society. The common pathogens of female urinary tract infection are Gram-negative bacilli, among which Escherichia coli is the most common, followed by Staphylococcus; most of these are normal flora on the human intestinal tract and skin and mucous membranes. Bacteria can migrate from the intestinal tract, skin and mucous membranes to the urinary system and reproduce in large numbers, causing urinary tract infections. Common reproductive tract infection pathogens in women include trichomonas, mold and chlamydia, etc. Most of the reproductive tract infections are caused by vaginal infection; the protracted spread of the disease can cause pelvic inflammatory disease and lead to infertility. Urogenital tract infection is "small disease, big trouble", and it can also increase the risk of sexually transmitted diseases such as AIDS and gonorrhea; the quality of life of patients is obviously reduced, and the mental burden is heavy. Pregnant women may have a series of pregnancy complications, such as premature birth and chorioamnionitis, and other adverse pregnancy outcomes; during delivery, the birth canal can also directly infect newborns with similar pathogens.

At present, the treatment of genitourinary tract infection mainly relies on various antibiotics, but the treatment effect is not good, the cure rate is below 40%, and the recurrence rate is very high, up to more than 40%. The main reason is that the presence of drug-resistant pathogenic bacteria in the patient's genital tract makes antibiotics ineffective; in addition, even though antibiotic treatment can kill pathogenic bacteria, it also kills beneficial bacteria (probiotics), so it is difficult to restore the urogenital tract in a healthy state Dominant flora.

There are a large number of bacteria in the genitourinary tract of healthy women of childbearing age, collectively known as the urogenital tract flora, which includes a variety of different types of bacteria, which can be roughly divided into two categories: "beneficial bacteria" and "pathogenic bacteria". Among them, Lactobacillus It is the most common and important beneficial bacteria (“good bacteria”) for maintaining urogenital health, while Escherichia coli, Staphylococcus, Trichomonas, mold and Chlamydia are pathogenic bacteria (“bad bacteria”). Certain factors lead to the abnormal proliferation of a certain pathogenic bacteria, causing genitourinary tract infection. The change of bacterial species in the vagina is closely related to the health of women and fetuses. Among them, Lactobacillus flexus and Lactobacillus jannasii are the main dominant "just bacteria" to maintain a healthy microecological environment in the vagina, which can account for the total bacterial flora of the healthy urogenital tract. More than 90% of the amount. They mainly maintain a healthy environment of the urogenital flora by inhibiting the growth and invasion of foreign pathogenic bacteria and other conditional pathogenic bacteria ("evil bacteria"), and the mechanism of action of "good bacteria (good bacteria)" overcoming "evil bacteria" It mainly includes the following aspects: (1) produce acidic substances such as lactic acid and acetic acid by decomposing the glycogen stored in the vaginal mucosa epithelium, and maintain the acidic environment of the vagina to inhibit the growth of pathogenic bacteria; (2) produce hydrogen peroxide and lactobacillus (3) Lactobacillus beneficial bacteria will form a preconceived space-occupying protective effect to prevent the invasion of pathogenic microorganisms after they colonize the vaginal mucosa epithelium.

As women of childbearing age grow older, the number of Lactobacillus flexi or Lactobacillus jennis in the vagina will gradually decrease. After menopause, ovarian estrogen secretion gradually stops, vaginal epithelial cells lack glycogen reserves, vaginal pH rises, Lactobacillus flexures or Lactobacillus jansii will further decrease, and pathogenic bacteria will increase. Therefore, Lactobacillus flexus or Lactobacillus jannis also plays an important role in the genitourinary health of elderly women.

The theory of microecology holds that adjusting the therapy of ecological preparations to strengthen and protect the composition and proportion of the dominant bacterial flora in the vagina can drive out the intrusion of foreign "evil" pathogenic bacteria and improve women's inherent self-protection of "strengthening and eliminating pathogenic factors" effect. Lactobacillus is the dominant flora of healthy women's vaginal flora, accounting for more than 90% of the total vaginal bacteria. Patients with urogenital tract infection have significantly reduced vaginal Lactobacillus, and some are even completely undetectable, thus losing the ability to resist foreign pathogenic bacteria infection . By supplementing the missing Lactobacillus, it is beneficial to rebuild the dominant flora of the female genitourinary tract and restore its inherent protective effect of "strengthening the body and eliminating pathogenic factors".

Urogenital tract infection is a common disease, frequently-occurring disease, and easy to relapse in women. Although antibiotics have a good short-term effect, they have a recurrence rate of 40%. Studies have shown that the dominant strains with protective effects—the preparations of Lactobacillus flexus or Lactobacillus jansii will be easier to settle and proliferate in the vagina, and produce antibacterial substances to exert a lasting effect. Therefore, the development of specific vaginal lactobacillus preparations adapted to the characteristics of women in my country has a broad market prospect, changing the previous guiding ideology of purely bactericidal medication in the treatment of genitourinary tract infections, and avoiding a series of adverse reactions and complications caused by a large number of antibiotics used as treatment. It is beneficial to restore the dominant flora of female genitourinary tract and rebuild its microecological balance; thereby effectively reducing the recurrence rate of urogenital tract infection and truly curing female genitourinary tract infection.

At present, there is only one kind of probiotic agent for female genitourinary tract infection in China, and its main component 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.

Contents of the invention

The object of the present invention is to provide a strain of Lactobacillus jensenii and its application.

The Lactobacillus jannis provided by the present invention, named IMJ-19, was preserved on July 14, 2014 in the General Microbiology Center of China Microbiological Culture Collection Management Committee (abbreviated as CGMCC, address: Beichen West Road, Chaoyang District, Beijing) No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101), the preservation number is CGMCC No.9439.

Another object of the present invention is to provide a bacterial agent whose active ingredient is the aforementioned Lactobacillus jensenii IMJ-19CGMCC No.9439.

The preparation method of the bacterial agent described above is also the protection scope of the present invention.

The preparation method of the above-mentioned bacterial agent comprises the following steps: using the above-mentioned Lactobacillus jensenii IMJ-19CGMCC No.9439 as an active ingredient to obtain the bacterial agent.

The application of the above-mentioned Lactobacillus jansnii or bacterial agent or the preparation method of bacterial agent in the preparation of products for maintaining the acidic environment of the urogenital tract also belongs to the protection scope of the present invention.

The application of the above-mentioned Lactobacillus jansnii or bacterial agent or bacterial agent preparation method in the preparation of products that inhibit the growth and/or reproduction of pathogenic bacteria and/or opportunistic pathogenic bacteria in the urogenital tract also belongs to the protection scope of the present invention.

The application of the above-mentioned Lactobacillus jansnii or bacterial agent or the preparation method of bacterial agent in the preparation of a product capable of adhering and colonizing epithelial cells of the urogenital tract also falls within the protection scope of the present invention.

The application of the above-mentioned Lactobacillus jansnii or bacterial agent or the preparation method of bacterial agent in the preparation of hydrogen peroxide-producing genitourinary tract beneficial bacteria also belongs to the protection scope of the present invention.

The application of the preparation method of the above-mentioned Lactobacillus jansnii or bacterial agent or bacterial agent in the preparation of products for the prevention and/or treatment of genitourinary tract infections or in the preparation of products for the prevention and/or treatment of diseases caused by genitourinary tract infections also belongs to protection scope of the present invention.

Lactobacillus jensenii (Lactobacillus.jensenii) IMJ-19 provided by the invention has a strong ability to produce H ₂ O ₂ , can effectively inhibit the growth of foreign pathogenic bacteria and other conditional pathogenic bacteria, and has a strong Adhesion to vaginal epithelial cells ensures the colonization of the strain in the vaginal epithelium. Lactobacillus jennis IMJ-19 has a certain application prospect in the prevention and treatment of genitourinary tract infection.

Description of drawings

Figure 1 is a colony of Lactobacillus jansii IMJ-19.

Figure 2 shows the adhesion of Lactobacillus jansii IMJ-19 to vaginal epithelial cells.

detailed description

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.

The vaginal secretion samples of healthy women were provided by Hansi Kang Health Management Institution.

Preparation of MRS liquid medium: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 801.0 mL of Tween, sodium acetate (CH ₃ COONa 3H ₂ O) 5.0g, dipotassium hydrogen phosphate (K ₂ HPO ₄ 3H ₂ O) 2.0g, magnesium sulfate (MgSO ₄ 7H ₂ O) 0.58g, manganese sulfate (MnSO ₄ .H ₂ O) 0.25g were dissolved in distilled water, and used Distilled water to 1000mL, adjusted to pH 6.2-6.6.

Embodiment 1, isolation and identification of Lactobacillus jansnii strain

1. Isolation of Lactobacillus jansii strains

The vaginal secretion samples collected from more than 200 cases of healthy women without symptoms and signs of vaginitis were numbered sequentially, inoculated into 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. 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-19.

2. Identification of Lactobacillus jansii strains

(1) Morphological identification

Inoculate the bacterial strain IMJ-19 isolated in the above step 1 on the MRS Lactobacillus medium plate and cultivate it. The results showed that the colonies were opaque and protruding milky-white colonies with a diameter of about 1-2 mm, and the edges of the colonies were neat and moist, and the size and shape were stable (as shown in Figure 1). The Lactobacillus jansnii in logarithmic growth phase was identified as: Gram-positive, non-spore-forming bacilli, without flagella.

(2) Physiological and biochemical characteristics analysis

Strain IMJ-19 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 strips (purchased from bioMe'rieux, Inc., Marcy l'Etoile, France) to carry out sugar fermentation reaction interpretation of the detection strain IMJ-19 to detect strain IMJ-19 Physiological and biochemical characteristics. API50CHL is a simple medium consisting of API 50CH test strips of 49 fermentable carbohydrates. Each small tube of the test strip was inoculated with a suspension made of the test strain IMJ-19. When cultured, the pH drops due to acid production from fermented carbohydrates, discoloring the indicator.

The results showed that the strain IMJ-19 could ferment glucose, fructose, sucrose, trehalose, maltose and other sugars.

(3) 16s rDNA sequence homology analysis

The total DNA of the strain IMJ-19 was extracted as a template, and the general primers 27f and 1492r were used for PCR amplification to obtain a fragment containing the conserved region of the rRNA gene of the strain IMJ-1916S. The primer sequences are as follows:

27f (upstream primer): 5'-AGAGTTTGATCCTGGCTCAG-3';

1492r (downstream primer): 5'-TACGGTTACCTTGTTACGACTT-3'.

PCR system (25 μL): PCR amplification buffer with a final concentration of 200 μmol/L dNTPs, upstream and downstream primers with a final concentration of 0.2 μmol/L, 1 U Taq DNA polymerase, 1 μL template DNA, and ddH ₂ O as the balance .

PCR 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.

The sequence of the 16s rDNA of strain IMJ-19 obtained by PCR amplification is shown in SEQ ID No.1.

Based on the above identification and analysis, the classification of strain IMJ-19 is named as Lactobacillus jensenii (Lactobacillus jensenii). No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), the preservation number is CGMCC No.9439.

Example 2, Functional analysis of Lactobacillus jansnii IMJ-19

1. Detection of H ₂ O ₂ produced by Lactobacillus jannis IMJ-19

(1) Preparation of H ₂ O ₂ standard curve: first use an aqueous solution of piperazine-N,N'-di-ethanesulfonic acid (Piperazine-N,N-Bis 2-ethanesulfonic acid, PIPES) with a concentration of 100mmol/L Dilute the stock solution of 30% by volume _H2O2 solution (equivalent to _9.128mol /L) to 1mol/L, and then dilute the above 1mol/L H2O2 with _100mmol /L _PIPES solution respectively 20μmol/L, 40μmol/L, 60μmol/L, 80μmol/L, 100μmol/L, the above dilutions and the 100mmol/L PIPES aqueous solution are recorded as H2O2 working solution _; then take _100μL of the above _{H2O 2} Mix the working solution with 100 μL of TMB (tetramethylbenzidine) aqueous solution with a concentration of 20 mmol/L and 2 μL of horseradish peroxidase with a concentration of 1 mg/mL (purchased from TaKaRa Company), incubate at 16°C for 10 min, and measure the OD ₆₃₀ values. With the concentration of H ₂ O ₂ (unit: μmol/L) as the abscissa and OD ₆₃₀ as the ordinate, a standard curve was prepared to obtain the standard curve formula y=0.0033x-0.0496, R ² =0.992.

(2) Detection of H ₂ O ₂ concentration in liquid culture of Lactobacillus jansnii IMJ-19: pick the colony of Lactobacillus jansnii IMJ-19, inoculate it in MRS liquid medium, culture it under anaerobic conditions for 48 hours, and take 1mL Inoculate in 10mL MRS liquid medium to make the concentration of the bacteria in the culture medium to 10 ⁵ cfu/ml, in an anaerobic environment, after static culture at 37°C for 21h, switch to shaking culture at 37°C for 3h, with a rotation speed of 220r/min , the radius of rotation is 2.54cm. After the cultivation, the culture solution was collected and centrifuged at 12000r/min for 2min to obtain the supernatant. Take 100 μL supernatant, mix with 100 μL TMB (tetramethylbenzidine) aqueous solution with a concentration of 20 mmol/L, and 2 μL horseradish peroxidase with a concentration of 1 mg/mL (purchased from TaKaRa Company), incubate at 16°C for 10 min, measure Put the OD ₆₃₀ value into the standard curve formula obtained in step (1) to calculate the H ₂ O ₂ concentration in the supernatant. The experiment was repeated three times, and the results were averaged. The results are shown in Table 1.

Table 1 Detection of the ability of Lactobacillus jansii IMJ-19 to produce H ₂ O ₂

repeat 1 repeat 2 repeat 3 average value H ₂ O ₂ yield 310.8 357.8 298.3 322.3

The results showed that: Lactobacillus jansnii IMJ-19 could produce a large amount of H ₂ O ₂ in the fermentation medium, and its concentration reached 322.3μM.

2. Adhesion of Lactobacillus jannis IMJ-19 to vaginal epithelial cells

(1) Vaginal epithelial cells were obtained from more than 20 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 centrifugation Twice, wash off the surface bacteria, and adjust the concentration of vaginal epithelial cells to 10 ⁵ cells/mL with MEM medium.

(2) Lactobacillus jensii IMJ-19 cultured overnight was activated to a concentration of 10 ⁸ CFU/mL.

(3) The vaginal epithelial cells obtained in step (1) at a concentration of 10 ⁵ cells/mL and the concentration of Lactobacillus jansii IMJ-19 obtained in step (2) at a concentration of 10 ⁸ CFU/mL were mixed in equal volumes of 500 μL each and placed in Incubate on a shaker at 37°C (100r/min, 2.54cm radius of rotation) for 1h, then filter with a 0.8μm filter membrane to remove free Lactobacillus jensii IMJ-19, and gently press the filter membrane on the cover slip , fixed the cells for Gram staining and observed with an oil microscope, respectively counted the number of Lactobacillus jansii IMJ-19 adhered to three vaginal epithelial cells, and calculated the average number of Lactobacillus jansii IMJ-19 adhered to each vaginal epithelial cell. 19 bacteria count. At the same time, the vaginal epithelial cells were observed with oil microscope after Gram staining, as a control.

The results are shown in Figure 2, the right in Figure 2 is the vaginal epithelial cells adhered to Lactobacillus jansii IMJ-19; the left is the vaginal epithelial cells isolated from healthy women of childbearing age (control). The results showed that: Lactobacillus jannis IMJ-19 had strong adhesion to vaginal epithelial cells isolated from more than 20 cases of healthy women of childbearing age.

From more than 20 cases, 4 cases were randomly selected for statistical analysis. The results are shown in Table 2. The results showed that the average number of adhered Lactobacillus jensii IMJ-19 per vaginal epithelial cell was 65.

Table 2 Statistics on the number of vaginal epithelial cells adhered to Lactobacillus jensii IMJ _- 19

Based on the above results, Lactobacillus jannis IMJ-19 not only has a strong ability to produce H ₂ O ₂ (which can effectively inhibit the growth of pathogenic bacteria and other opportunistic pathogens), but also has a strong adhesion to vaginal epithelial cells effect (ensuring the colonization of the strain in the vaginal epithelium), which makes Lactobacillus jensii IMJ-19 a candidate probiotic strain for effective prevention and treatment of urogenital tract infections.

Claims (8)

1. A strain of Lactobacillus jensenii (Lactobacillus.jensenii), which is named as IMJ-19, has been preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on July 14, 2014, and its preservation number is CGMCCNo. 9439. 2. A bacterial agent whose active ingredient is Lactobacillus jensenii (Lactobacillus.jensenii) IMJ-19CGMCC No.9439 according to claim 1. 3. The preparation method of the microbial agent according to claim 2, comprising the following steps: using Lactobacillus jensenii (Lactobacillus.jensenii) IMJ-19CGMCC No.9439 according to claim 1 as an active ingredient to obtain the bacterial agent. 4. The application of the preparation method of the Lactobacillus jansnii described in claim 1 or the bacterial agent described in claim 2 or the microbial agent described in claim 3 in the product for preparing the acidic environment that maintains the genitourinary tract. 5. the preparation method of Lactobacillus jansii described in claim 1 or the bacterium agent described in claim 2 or the bacterium agent described in claim 3 inhibits the growth of pathogenic bacteria and/or conditional pathogenic bacteria in the urogenital tract and / or application of multiplied products. 6. The preparation method of Lactobacillus jansnii described in claim 1 or the bacterial agent described in claim 2 or the microbial agent described in claim 3 is in the product that has adhesion colonization ability to the epithelial cell of urogenital tract Applications. 7. the application of the preparation method of Lactobacillus jansnii described in claim 1 or the bacterium agent described in claim 2 or the bacterium agent described in claim 3 in the genitourinary tract beneficial bacterium that produces hydrogen peroxide. 8. The preparation method of Lactobacillus jansii described in claim 1 or the bacterium agent described in claim 2 or the bacterium agent described in claim 3 is in the preparation prevention and/or the product of treatment genitourinary tract infection or in the preparation prevention and/or in products for the treatment of diseases caused by genitourinary tract infections.