CN1888051B - Plant lactobacillus strain and its application - Google Patents

Abstract

The invention discloses a plant lactobacillus and application thereof. Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 disclosed in the present invention is isolated from the vaginal secretions of pregnant women in China, and is effective against common pathogenic bacteria in women's vaginas such as Staphylococcus aureus, Candida albicans, Escherichia coli and vaginal Gardner The bacterium has strong antibacterial effect, and the ability to produce acid and H ₂ 0 ₂ is higher than that of other lactobacilli. After 96 hours of culture, the pH of the culture solution can reach 3.96; the surface of the strain has thick non-pilus adhesin , has good adhesion to vaginal epithelial cells, and placed in a preservation solution at a suitable room temperature, the number of viable bacteria increases by 3×10 ⁴ times. Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 can be used to prepare vaginal microecological preparations for preventing and/or treating women's vaginal infectious diseases.

Description

A strain of Lactobacillus plantarum and its application

technical field

The invention relates to a strain of Lactobacillus and its application, in particular to a strain of Lactobacillus plantarum and its application.

Background technique

1. Lactobacillus and women's vaginosis

Lactobacillus (Lactobacillus) is the main and most important normal flora in the vagina of healthy women. Lactobacillus in the vagina is not only non-pathogenic, but also can self-clean the micro-ecological environment of the vagina and restrict the colonization and growth of other pathogenic bacteria. Therefore, clinically, a large number of lactobacilli (several per field of view under a high-power microscope) can be seen in vaginal smears, and no or only a small amount of Gram-negative bacilli can be seen as the standard of normal and no vaginal infection, while lactobacilli in the vagina are absent Or reduction is an important indicator of vaginal infection (Robert P.Nugent, Marijane A.Krohn, and Sharon L.Hillier Reliability of diagnosing bacterial vaginosis improved by a standardized method of Gram stain interpretation Journal of Clincal Microbiology 1991,297-2): 301).

Women's bacterial vaginosis (Bacterial Vaginosis, referred to as BV) mainly refers to vaginitis caused by bacteria, molds, trichomonas and other microorganisms. Clinically, antibiotics are generally used for treatment, but antibiotic treatment has a high recurrence rate, especially for some elderly patients. For patients with vaginitis and multiple vaginitis, antibiotic treatment is often ineffective, because the use of antibiotics to kill pathogenic microorganisms also destroys the normal flora in the body, especially inhibits the growth of Lactobacillus in the vagina, thus losing The restrictive effect on pathogenic microorganisms in the micro-ecological environment allows other pathogenic microorganisms to colonize and grow. In addition, women are prone to vaginal infections due to immune regulation disorders during pregnancy, and the particularity of pregnant women prevents them from being treated with antibiotics.

2. The application of Lactobacillus vaginal preparations

Lactobacillus has been widely used abroad as a microecological agent to treat vaginosis in women (Nigel W. Mclean and Isobel J. Rosenstein Characterization and selection of a Lactobacillus species to re-colonise the vagina of women with recurrent bacterial vaginosis L Med Microbiol 2000, 49: 543-552; Elmer GM. Probiotics: "living drugs" Am J Health Syst Pharm 2001, 58(12): 1101-9), which is an over-the-counter preparation that patients can purchase by themselves. The Lactobacillus in the preparation is generally isolated from the vagina of normal women, and the common ones are Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum. Due to race and individual differences, similar foreign preparations may not be suitable for Asians including Chinese, and similar preparations of Lactobacillus have been developed in China.

The application of probiotics to treat diseases is definitely another bright spot after the glorious era of antibiotics, and it is another pharmaceutical direction that antibiotics cannot replace, with huge economic and social benefits.

Lactobacillus as an intravaginal microecological preparation is suitable for the following groups of people:

(1) Elderly patients with vaginitis

Senile vaginitis is atrophic vaginitis, which is one of the common diseases of elderly women. The incidence rate of vaginitis in postmenopausal women is 30-50% in domestic reports and 30% in foreign countries. Experts believe that the main cause of senile vaginitis is vaginal microscopic The lack of Lactobacillus in the environment is caused by the colonization of other pathogenic microorganisms. Antibiotics are not effective in treating senile vaginitis, and regular implantation of Lactobacillus preparations can inhibit the growth of other pathogenic bacteria, which is to improve the symptoms of vaginitis in elderly patients and keep them in a healthy state. status, thereby improving the quality of life of elderly women (Reid G, Bruce AW. Urogenital infections in women: can probiotics help? PostgradMed J. 2003 Aug; 79 (934): 428-32).

(2) Pregnant women

Compared with normal women, pregnant women are prone to genitourinary infection due to decreased immunity due to the increase in T-suppressed lymphocytes caused by changes in hormone levels. Antibiotics are generally not used to treat infections in pregnant women, because most antibiotics have an impact on the fetus. For example, the drug "Metronidyl" used to treat trichomonas and anaerobic vaginitis has been shown to have carcinogenic and teratogenic effects in animal experiments. Macrolide drugs used in the treatment of non-specific vaginitis affect fetal bone development. Lactobacillus is a normal flora, non-pathogenic, and has a self-cleaning effect on the vaginal microenvironment. Therefore, implanting lactobacillus preparations into the vagina of pregnant women suffering from vaginitis can safely pass through the pregnancy and perinatal period. Pregnant women infected with BV are likely to cause endometritis and premature delivery. The latest survey in the UK shows that at 20 weeks of pregnancy, pregnant women who have Lactobacillus producing H ₂ O ₂ in their vagina have a much lower risk of developing BV than those without H ₂ Pregnant women with Lactobacillus _O2 had significantly lower endometritis prevalence and premature delivery than the latter (Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, Corfield A, Millar M. Identification and H(2)O(2)production of vaginal lactobacilli from pregnant women at high risk of preterm birth and relation with outcome. J Clin Microbiol. 2004 Feb; 42(2):713-7). Therefore, someone used H ₂ O ₂ producing Lactobacillus bacteria liquid to wash the vagina of 22 pregnant women with BV, aged 20-34, who were 3 months pregnant, once a day, and 7 days was a course of treatment. Start the second course of treatment 1 week later. Results After 3 days of treatment, the symptoms of all pregnant women were improved, and 90% of the pregnant women's vaginal conditions returned to normal after 2 courses of treatment (Tasdemir M, Tasdemir I, Tasdemir S, Tavukcuoglu S. Alternative treatment for bacterial vaginosis in pregnant patients; restoration of vaginal acidity and flora.Arch AIDS Res. 1996;10(4):239-41).

(3) Patients with chronic diseases

For patients with chronic diseases, such as diabetes patients, lupus erythematosus, rheumatism, organ transplantation and other long-term use of immunosuppressants and broad-spectrum antibiotics, normal flora imbalance often occurs; some physically disabled, long-term sitting and lying women are prone to vaginal infections. Oxygen bacteria; For such women, implanting lactobacillus vaginal preparations will relieve their pain and play an effective "cleaning" role.

3. The source and conditions for the strains used as Lactobacillus vaginal preparations

1. Used as a source of Lactobacillus vaginal preparation strains

The strains used as Lactobacillus vaginal microecological preparations are generally derived from the normal vaginal flora of healthy women, and Lactobacillus isolated from the normal vaginal flora of pregnant women with excellent vaginal cleanliness will have a better effect. In 1999, it was reported in the UK that Lactobacillus isolated from pregnant women was used to develop vaginal microecological preparations.

Pregnant women during pregnancy, especially in the early pregnancy, due to changes in hormone levels, the body's immune system is disordered, often due to the hyperactivity of T suppressor cells, resulting in low immunity, and thus prone to genitourinary infection. However, in the state of low immunity, some pregnant women can still maintain good vaginal cleanliness, indicating that the normal vaginal flora, namely Lactobacillus, has a strong ability to inhibit the growth of pathogenic bacteria.

2. The strains used as Lactobacillus vaginal microecological preparations should generally have the following conditions:

(1) Adhesive space-occupying

The adhesion of lactobacilli to vaginal epithelial cells is very important for use as probiotics. In vitro tests have shown that the growth of lactobacilli on the surface of silica gel can interfere with the adsorption of urethral pathogenic bacteria on the surface of silica gel (Velraeds MM Interference in initial adhesion of uropathogenic bacteria and yeasts to silicone rubber by a Lactobacillus acidophilus J Med Microbiol 1998, 47(12): 1081-5).

During the growth process of Lactobacillus, a non-pilus adhesin is continuously secreted to the outside of the cell. Morphological observation shows that there is a thick, dense, uneven, jagged material around the bacteria, also known as fibritlae ), whose chemical composition is lipoteichoic acid (LTA), which plays a role in adhesion to host cells. The mechanism of adhesion between LTA and host cells is considered to be that, on the one hand, it binds to the host cell receptor as a ligand, and on the other hand, it interacts with the host cell through the charge carried by the free part of LTA (Otero MC, Ocana VS, Elena Nader -Macias M. Bacterial surface characteristics applied to selection of probiotic microorganisms. Methods Mol Biol. 2004; 268: 435-40). Thus, the more hair-like protrusions a Gram-positive bacterium has, the stronger its adhesion to the host cell.

Lactobacilli use non-pilus adhesin, that is, hair-like protrusions, which can be well attached to the vaginal wall, and the formed biological barrier prevents other pathogenic bacteria from forming clones in the vagina, which plays a role in mass effect. The larger the size of Lactobacillus, the better the mass effect. Lactobacillus plantarum is the largest species in the genus Lactobacillus, so it has better space-occupying properties. Regarding the use of Lactobacillus plantarum in probiotics and its good adhesion and space-occupying properties to vaginal epithelial cells, there have been reports in foreign literature and patents (Bonetti A, Morelli L, Campominosi E, Ganora E, Sforza F.Adherence of Lactobacillus plantarum P 17630 in soft-gel capsule formulation versus susderlein's bacillus in tablet formulation to vaginal epithelial cells. Minerva Ginecol. 2003 Jun; 55(3): 279-84, 284-7).

(2) acid production

Due to the acidophilicity of Lactobacillus, it is suitable for growth in an acidic environment, and the metabolites produced by its fermentation of sugars are almost all acetic acid, which reduces the pH of the vaginal environment, which is not conducive to the growth of other pathogenic bacteria. Due to the reduced degree of glycogenization of vaginal epidermal cells and the loss of a large number of lactobacilli in elderly women, the pH of the vaginal microenvironment increases, making elderly women prone to vaginitis. Therefore, many studies believe that the acidic micro-ecological environment formed by the existence of lactobacillus is the most important factor to prevent vaginosis (E.R.Boskey, K.M.Telsch Acid production gyvaginal flora in vitro is consistent with the rate and extent of vaginal acidification Infection and Immunity 1999, 67(10):5170-5).

(3) Production of hydrogen peroxide (H ₂ O ₂ )

The number of lactobacilli parasitic in the vagina of some women is not small, but inflammation still occurs, because these lactobacilli do not produce H ₂ O ₂ , and most of the lactobacillus parasitic in the vagina of women with good cleanliness produce H ₂ O ₂ . For example, Gallia investigated the vaginal flora of 40 healthy women and 72 women infected with BV in 2003, and found that 92.5% (37/40) of healthy women had Lactobacillus producing _H2O2 in their vaginal flora _. However, only 4.1% (3/72) of women infected with BV had Lactobacillus producing H ₂ O ₂ in their vaginal flora. The author believes that the H ₂ O ₂ produced by Lactobacillus plays a non-specific role in protecting the vaginal microenvironment (Shopova E. Hydrogenperoxide-producing Lactobacillus species in healthy women and in women with bacterial vaginosis Akush Ginekol (Sofiia). 2003; 42 Suppl 1 :12-5). H ₂ O ₂ can inhibit the growth of some aerobic bacteria and molds. It has been reported in the literature that the lack of Lactobacillus producing H ₂ O ₂ is the main factor for the recurrence of vaginitis and urethritis caused by Escherichia coli (Juarez Tomas MS, Ocana VS, WieseB, Nader-Macias ME. Growth and lactic acid production by vaginalLactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli. J Med Microbiol. 2003 Dec;52(Pt 12):1117-24). Some people use H ₂ O ₂ solution to wash the vagina to treat BV infection, and compare it with metronidazole and clindamycin treatment: 58 female patients aged 18-42 years old infected with BV were clinically treated with 30ml 3% H ₂ O ₂ solution Wash the vagina once a day for a total of 7 days. Compared with metronidazole and clindamycin treatment after 3 months, the main symptoms of BV were cleared, including elimination of malodor in 98% of patients, 98% of vaginal pH<4.5, 97.7% negative amine test, 100% of vaginal discharge Clue cells were not detected and a normal flora containing Lactobacillus producing H ₂ O ₂ was established (Cardone A, Zarcone R, Borrelli A, Di Cunzolo A, Russo A, Tartaglia E. Utilization of hydrogen peroxide in the treatment of recurrent bacterial vaginosis . Minerva Ginecol. 2003 Dec;55(6):483-92).

(4) Antibacterial effect

Some lactobacilli can secrete lactobacillus bacteria in vitro during the growth process. These bacteria can inhibit the growth of many vaginal pathogenic microorganisms. The composition and mechanism of these bacteria are still unclear. Studies have shown that lactic acid Bacillus bacteriocin is a substance with antibacterial protein components, and some people think that the so-called lactobacillus bacteriocin is the acidic metabolite of Lactobacillus. However, no matter what kind of ingredients Lactobacillus is, the Lactobacillus strain used as vaginal microecological preparation must have antibacterial effect (Aroucheva AA. Simoes JA, Faro S. Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inbibits Gardnerella vaginalis Infect Dios Obstet gynecol 2001, 9(1): 33-9; Tomas MS, Claudia Otero M, OcanaV, Elena Nader-Macias M. Production of antimicrobial substances by lactic acid bacteria I: determination of hydrogen peroxide. Methods Mol Biol. 2003.268 46).

(5) Growth conditions and stability

Lactobacillus growth has very different nutritional needs. Most Lactobacillus have strict nutritional requirements, require high-cost nutritional additives, and grow slowly, so they are not suitable for preparation production. Lactobacillus strains used as vaginal microecological preparations must be able to adapt to ordinary growth conditions, and can reproduce in large numbers under ordinary growth conditions; in addition, the strain can survive for a long time in a suitable preservation solution, which is also a must for vaginal microecological preparation strains condition.

Contents of the invention

The purpose of the present invention is to provide a new plant lactobacillus.

The plant Lactobacillus provided by the present invention is Lactobacillus plantarum (Lactobacillus plantarum) L323, and this bacterial strain has been preserved in the General Microorganism Center (abbreviated as CGMCC) of China General Microorganism Culture Collection Management Committee (CGMCC) on March 14, 2005, and the preservation number is CGMCC No. 1329.

Lactobacillus plantarum (Lactobacillus plantarum) L323CGMCC No.1329 is isolated from human body with high vaginal cleanliness and good microenvironment. It shows a strong antibacterial effect, and the ability to produce acid and H ₂ O ₂ is higher than that of other lactobacilli. After 96 hours of cultivation, the pH of the culture solution can reach 3.96; the surface of the strain has thick non-pilus adhesin , has good adhesion to vaginal epithelial cells, and placed in a suitable preservation solution at room temperature for half a year, the number of viable bacteria increased by 3×10 ⁴ times. Lactobacillus plantarum (Lactobacillus plantarum) L323CGMCC No.1329 can be used to prepare vaginal microecological preparations for preventing and/or treating women's vaginal infectious diseases.

Description of drawings

Fig. 1 is the Gram stain photo of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329

Figure 2A is the antibacterial effect of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 on Staphylococcus aureus ATCC6035

Figure 2B is the antibacterial effect of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 on Candida albicans ATCC1023

Figure 2C is the antibacterial effect of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 on Escherichia coli ATCC25922

Figure 2D shows the antibacterial effect of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 on Gardnerella vaginalis ATCC14018

Figure 3 is a plate photo of blue-green colonies of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 producing H ₂ O ₂

Fig. 4 is the electron micrograph of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329

Detailed ways

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

Embodiment 1, isolation and identification of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329

1. Isolation of Lactobacillus plantarum L323 CGMCC No.1329

Firstly, the vaginal vaginal fornix secretions of more than 300 healthy Chinese pregnant women undergoing regular pregnancy checkups in the Obstetrics and Gynecology Department of the 301 Hospital were collected, and the following tests were carried out while using MRS medium for bacterial isolation: (1) Direct smear , according to the number of lactobacilli and gram-negative bacteria to evaluate the cleanliness (0-2 points, normal; 4-6 points, mild inflammation; 8-10 points, severe inflammation), among them, the evaluation criteria of vaginal cleanliness Reference literature (Robert P.Nugent, Marijane A.Krohn, and Sharon L.Hillier Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation Journal of Clincal Microbiology1991, 29(2):297-301); (2) vagina Secretion pH determination; (3) Quantitative determination of the number of lactobacilli in vaginal secretions. As a result, a strain of Lactobacillus isolated from vaginal secretions with a vaginal cleanliness rating of 0-2 points, the number of lactobacilli in vaginal secretions greater than 1×10 ⁸ CFU/ml, and a pH value of 3.25 was obtained, and the strain number was L323.

2. Bacteria identification

Bacterial morphology was observed by Gram staining microscope according to the conventional method. Gram staining showed that the L323 strain of Lactobacillus was a Gram-positive thick bacillus with a size of about 1×3-8um (Figure 1).

The biochemical reaction adopts the API 50 bacterial identification system of French Mérieux Biotechnology Company, including API 50 medium, test strips, McFarland standard points, mineral oil, culture box and API Plus software. The identification results of the API 50 bacterial identification system showed that L323 strain Lactobacillus was Lactobacillus plantarum (Lactobacillus plantarum), and its biochemical reaction results are shown in Table 1.

Table 1 Biochemical reactions of Lactobacillus plantarum L323

Aesculin ESC + Xylitol XLT - Glycerin GLY - Gentiobiose GEN + Erythrasol ERY - D-turanose TUR + D-Arabinose DARA - D-lyxose LYX - L-Arabinose LARA + D-tagatose TAG - Ribose RIB + D-rock sugar DFUC - D-xylose DXYL - L-rock sugar LFUC - L-xylose LXYL - D-Arabitol DARL + Adon sugar ADO - L-Arabitol LARL - β-Methyl-D-xyloside MDX - Gluconate GNT + Galactose GAL + 2-keto-gluconate 2KG - Glucose GLU + 5-keto-gluconate 5KG + Fructose FRU + Glycogen GLYG - Mannose MNE + Starch AMD - Sorbose SBE - Raffinose RAF + Rhamnose RHA - Songsan sugar MLZ + Wermaol DUL - Inulin INU - Inositol INO - Trehalose TRE + Mannitol MAN + Sucrose SAC + Sorbitol SOR + Melibiose MEL + α-Methyl-D-mannoside MDM + Lactose LAC +

Aesculin ESC + Xylitol XLT - α-Methyl-D-glucoside MDG - Maltose MAL + N-acetyl-glucosamine NAG + Cellobiose CEL + Laetrile AMY + Willow Alcohol SAL + Arbutin ARB +

Note: "+" means positive reaction, "-" means negative reaction.

Embodiment 2, the characteristic of plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329

Material

Strains Staphylococcus aureus ATCC6035, Candida albicans ATCC1023, Escherichia coli ATCC25922, Gardnerella vaginalis ATCC14018

Reagent

MRS medium and LM broth medium were purchased from Mérieux Biological Company of France;

Tryptone, yeast extract, and agar powder were purchased from Oxide;

Tetramethylbenzidine (TMB), dimethylformamide, and horseradish peroxidase (HRP) were purchased from Sigma.

1. Antibacterial test

Inoculate Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 strain in MRS medium, culture at 37°C, 5% CO ₂ for 24 hours, wash the bacterial lawn with sterile saline, turbidity and dilute to 1×10 ⁸ CFU/ml concentration of bacterial liquid, soak a circular sterile filter paper piece with a diameter of 5mm in the bacterial liquid and set aside.

Staphylococcus aureus ATCC6035, Candida albicans ATCC1023 and Escherichia coli ATCC25922 were inoculated on common nutrient agar medium (beef extract 3.0 g/L, peptone 10.0 g/L, NaCl 5.0 g/L, agar 15 g/L, pH 7.0), cultured at 37°C for 24 hours, Gardnerella vaginalis ATCC14018 was inoculated on GC blood agar, cultured at 37°C with 5% CO ₂ for 24 hours, washed with sterile saline, turbidimetric and diluted to 1× 10 ⁸ CFU/ml concentration of bacteria solution, each take 0.1ml coated with common nutrient agar plate, the above-mentioned circular filter paper soaked with Lactobacillus plantarum (Lactobacillus plantarum) L323CGMCC No.1329 were placed on the flat plate coated with the above bacteria Put the plates coated with Staphylococcus aureus ATCC6035, Candida albicans ATCC1023, and E. Incubate at 37°C for 48 hours in a 5% CO ₂ incubator, measure and record the diameter of the inhibition zone and take pictures.

The result of bacteriostasis test is shown in table 2 and Fig. 2A-Fig. 2D, shows that plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.132 is to Staphylococcus aureus, Candida albicans, escherichia coli and vaginal Gardnerella (golden yellow Staphylococcus, Candida albicans, Escherichia coli and Gardnerella vaginalis are common pathogenic bacteria that cause vaginitis) have antibacterial effect. It shows that Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.132 has a good antibacterial effect on common pathogenic bacteria in women's vagina.

Table 2 Antibacterial effect of L323 strain Lactobacillus on common pathogenic bacteria

2. Acidity and component analysis test of acid production

Inoculate Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 in MRS medium, culture at 37°C 5% CO ₂ for 24 hours, wash the bacterial lawn with physiological saline, and make 1×10 ⁸ CFU/ml bacterial suspension , take 0.1ml of bacterial suspension and inoculate it in 5ml of LM broth medium, culture at 37°C with 5% _CO2 for 24 hours, 48 hours, 72 hours, and 96 hours respectively, and use a pH meter to measure the pH value of the cultured bacterial solution at different times .

The variation of the pH value of the culture solution of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 at different culture times is shown in Table 3.

Table 3. Test results of acid production of Lactobacillus plantarum L323 at different times

Take 10ml of the Lactobacillus LM broth medium cultured for 24 hours, centrifuge at 8000rpm for 15 minutes, discard the bacterial sediment, collect the supernatant, and analyze the short-carbon organic acids in the bacterial metabolites: acetic acid, propionic acid, butyric acid, valeric acid Perform gas chromatographic analysis. The results are shown in Table 4, indicating that the short-carbon organic acid produced by Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 is mainly acetic acid, which can reach 1160ug/ml in 24 hours of cultivation.

Table 4. Composition and yield analysis of acid produced by Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329

Organic acid components Yield (ug/ml) Acetic acid 1160 propionic acid ＜50 butyric acid ＜50 Valeric acid ＜20

3. Hydrogen peroxide (H ₂ O ₂ ) test

Weigh 12.5mg of tetramethylbenzidine (TMB) and dissolve it in 1ml of dimethylformamide, mix it with 50ml of MRS medium, sterilize at 121°C for 15 minutes, and plate it to obtain a TMB-MRS medium plate. Store away from light for later use.

Prepare a horseradish peroxidase (HRP) solution at a concentration of 0.01 mg/ml, and filter it through a 0.45 μm membrane to sterilize it. Before inoculating the strain, take 0.1 ml and evenly coat the TMB-MRS medium plate, dry it, and place it in the dark at 4°C .

Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 was inoculated on the above-mentioned TMB-MRS medium coated with HRP, and cultured in a 5% _CO2 incubator at 37°C in the dark for 24 hours, and the culture plate was taken out and exposed to light for 3 minutes. , observe the color change of the colonies on the culture plate and record the results. Interpretation of the results: In the presence of H ₂ O ₂ , HRP will oxidize TMB to form a blue-green oxide. Therefore, if the lactic acid bacteria produce H ₂ O ₂ , the colonies grown on the medium containing TMB and coated with HRP will turn blue-green, while the colonies of the non-H ₂ O ₂ producing lactic acid bacteria strains will be off-white. The results are shown in FIG. 3 , the colonies of Lactobacillus plantarum L323 CGMCC No.1329 producing H ₂ O ₂ were blue-green. The production of H ₂ O ₂ by Lactobacillus can make the vaginal microenvironment hypoxic, thereby inhibiting the growth of aerobic pathogenic bacteria. Trials have shown a lack of _{H2O2 -} producing lactobacilli in the vagina and urethra of patients with recurrent vaginitis and urethritis caused by Escherichia coli (Wilks M, Wiggins R, Whiley A, Hennessy E, Warwick S, Porter H, Corfield A, Millar M. Identification and H(2)O(2)production of vaginal lactobacilli from pregnant women at high risk of preterm birth and relation with outcome. J Clin Microbiol. 2004 Feb; 42(2): 713-717).

4. Electron microscope observation of strain ultrastructure

Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 was inoculated with MRS medium at 37°C with 5% CO ₂ and cultured for 24 hours, and the bacterial lawn was swabbed with a sterile cotton swab and inoculated into 5ml LM broth medium at 37°C with 5% CO ₂ Cultivate for 24 hours, centrifuge at 5000rpm/min for 10 minutes, discard the supernatant, and separate the serum immediately after taking blood from the eyeballs of normal mice. Take 1 drop of freshly separated mouse serum and drop it into the bacterial pellet, and place it at room temperature for 10 minutes to fix the bacterial block , add 1ml of glutaraldehyde fixative, and place at 4°C for preparation of electron microscope specimens. Electron microscope observation results are shown in Figure 4, Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCC No.1329 has thick hair-like protrusions on the surface of the bacteria, that is, non-pilus adhesin, which is the basis for bacteria to adhere to epithelial cells.

5. Stability test of strain preservation

Observe the survival of Lactobacillus in different preservation solutions.

Preservation solution preparation:

Preservation solution 1: MRS medium + 5% (mass percentage) skimmed milk

Preservation solution 2: MRS medium + 5% (mass percentage content) skim milk + trehalose 3.78g

Preservation solution 3: MRS medium

Centrifuge the Lactobacillus plantarum L323 CGMCC No.1329 cultivated for 24 hours at 4800rpm/min for 5 minutes, suspend and precipitate the bacteria in different preservation solutions, the bacterial count is 2.2×10 ⁵ CFU/ml, and store them separately Tube 0.5ml/tube, put them in different temperature (37℃, room temperature) environments and store them away from light. Take 1 tube each month, suck out 0.1ml of bacterial solution, dilute it with 1ml LB broth to different concentrations, and apply MRS The medium plates were cultured at 37° C. in 5% CO ₂ for 24 hours, counted, and the preservation effects of different preservation solutions, that is, the survival of bacteria, were observed and recorded. The survival of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCCNo.1329 is shown in Table 5.

Table 5. Survival number (CFU/ml) of Lactobacillus plantarum (Lactobacillus plantarum) L323 CGMCCNo.1329 in different preservation solutions

Claims (2)

1. plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329.

2. plant lactobacillus (Lactobacillus plantarum) L323 CGMCC No.1329 prevents and/or treats application in the woman vagina infectious disease medicament in preparation.

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