Probiotic composition containing lactobacillus gasseri LN32 and lactobacillus crispatus LN70 and application of probiotic composition in reduction of vaginal infection risk

Abstract

The invention provides a probiotic composition containing lactobacillus gasseri LN32 and lactobacillus crispatus LN70 and application of the probiotic composition to reduction of vaginal infection risk, and belongs to the technical field of microorganism application. The probiotic feed additive contains four strains of probiotics, including plant lactobacillus LN66, lactobacillus rhamnosus LN56, lactobacillus gasseri LN32 and lactobacillus crispatus LN70. The probiotic composition realizes vaginal flora reconstruction through oral administration, inhibits reproduction of Gardneria vaginalis and Candida albicans, increases lactobacillus colonization, and eliminates abnormal leucorrhea, vaginal scorching heat and itching, frequent urination, urgent urination, dysuria and the like. The probiotic composition is combined with a conventional carrier, and is applied to preparation of food, dietary supplement or medicine for preventing and / or treating microecological imbalance of vagina and urethra, bacterial vaginitis, colpitis mycotica and urethral infection. Therefore, vaginal harmful bacteria are inhibited, vaginal flora is reconstructed, and the vaginal infection risk and the urethral infection risk are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial application, in particular to a probiotic composition containing Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 and application in reducing the risk of vaginal infection. BACKGROUND

[0002] The incidence of female genital tract infection, including bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), is high. The global incidence of BV among women of childbearing age is between 23% and 29%, and about 75% of women will experience at least one episode of VVC in their lifetime, 40%-45% of women will experience at least two episodes, and the incidence of BV combined with VVC mixed infection is more than 40%. The main symptoms are abnormal leucorrhea, vaginal burning and itching, frequent and painful urination, and so on. If not treated in time, it may lead to adverse pregnancy reactions, increased risk of sexual transmission, and risk of salpingitis, etc. The existing treatment methods mostly use antibiotics or antifungal drugs, including oral and topical administration, but antibiotic treatment often fails, has a high recurrence rate, and has many side effects.

[0003] Research has found that vaginal microorganisms are crucial to maintaining vaginal health, and Lactobacillus is the main component of healthy vaginal microorganisms, accounting for more than 70% of the total amount of vaginal bacteria. The species of Lactobacillus in a healthy vagina are relatively few, mainly including Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners, etc. Lactobacillus can produce lactic acid, acidify the vaginal environment, and inhibit the proliferation of a variety of pathogens. It can also inhibit the transformation of Candida albicans from yeast to hyphae, reduce its pathogenicity, prevent C. albicans from adhering to and invading vaginal epithelial cells, and reduce the risk of infection.

[0004] Although the pathogenesis of vaginitis is not clear, studies have shown that the occurrence of vaginitis is closely related to vaginal microbial imbalance and the decrease of Lactobacillus in the vagina. In recent years, more and more studies have shown that the human body is a whole, and the vagina and the distal organs (including the intestinal tract) realize bidirectional communication through immune, neural, endocrine, and metabolic pathways. Although the intestinal-vaginal axis has the characteristics of distal interaction, the rectum is close to the vagina, and the migration of microorganisms between the rectum and the vagina is a unique feature of the proximal axis. Microorganisms can directly migrate from the anus to the vagina and urethra. In addition, intestinal microorganisms and their metabolites may affect vaginal microorganisms through immune mechanisms. It can be seen that there is interaction between intestinal and vaginal microorganisms, and most vaginal microbial imbalances are related to intestinal microbial imbalances; women with BV also have chronic intestinal diseases.

[0005] At present, there are local use products of directly administering lactobacillus in the vagina at home and abroad, including suppositories and gel preparations, but local use on the one hand increases the risk of infection due to direct contact; on the other hand, even if local use is effective for a short time, it cannot solve the problem of intestinal microbial imbalance, and the recurrence rate is high.

[0006] In addition, plantaricin LN66 and rhamnolactobacillus LN56 are published probiotics, which can refer to invention patents ZL 201911218939.4 and ZL 202011412221.1. The two strains of probiotics have significant inhibitory properties of harmful bacteria, and play an important role in the reconstruction of flora, the reduction of harmful bacteria of halitosis and the risk of helicobacter pylori, but the application in inhibiting harmful bacteria in the vagina and reducing the risk of vaginal infection has not been developed.

[0007] Therefore, in order to solve the problems of increased infection risk and high recurrence rate, the oral probiotic composition of intestinal-vaginal axis strain synergistic complementation is studied to reduce the use of antibiotics, balance the intestinal microorganisms, reduce the risk of vaginal infection and the recurrence rate, and improve the overall health of women, which has important social value and economic value. SUMMARY

[0008] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a probiotic composition containing lactobacillus gasseri LN32 and lactobacillus crispatus LN70 and its application in reducing the risk of vaginal infection, which realizes the reconstruction of vaginal flora, inhibits the reproduction of gardnerella vaginalis and candida albicans, increases the colonization of lactobacillus, and eliminates abnormal leukorrhea, vaginal burning and itching, frequent and urgent urination, and pain. The probiotic composition is combined with a conventional carrier and applied to the preparation of food, dietary supplements or drugs for preventing and / or treating vaginal and urethral microecological imbalance, bacterial vaginitis, fungal vaginitis and urethral infection, so that the present application realizes the inhibition of harmful bacteria in the vagina, the reconstruction of vaginal flora to reduce the risk of vaginal infection and the risk of urethral infection.

[0009] To achieve the above-mentioned purpose, the present application provides the following scheme: A probiotic composition contains four strains of probiotics, including plantaricin LN66 and rhamnolactobacillus LN56, and the probiotic composition further comprises lactobacillus gasseri LN32 and lactobacillus crispatus LN70.

[0010] Preferably, in the probiotic composition, the viable bacterial count of the plantaricin LN66, rhamnolactobacillus LN56, lactobacillus gasseri LN32 and lactobacillus crispatus LN70 is greater than 1.0x10 9 CFU / g(ml).

[0011] The application also provides the use of the probiotic composition and a conventional carrier in combination in the preparation of a drug for preventing and treating vaginal infections.

[0012] The application also provides the use of the probiotic composition and a conventional carrier in combination in the preparation of a drug for preventing and treating urinary tract infections.

[0013] The application also provides the use of the probiotic composition in the reconstruction of vaginal flora.

[0014] The application also provides a strain combination of the probiotic composition, and the strain combination comprises Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70. The strain of the Lactobacillus gasseri is LN32, the preservation time is November 18, 2019, the preservation place is the China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.18958. The strain of the Lactobacillus crispatus is LN70, the preservation time is April 22, 2020, the preservation place is the China General Microbiological Culture Collection Center, and the biological preservation number is CGMCC No.19702.

[0015] The application also provides the use of the strain combination of the probiotic composition in the preparation of a product for producing hydrogen peroxide, that is, the use of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 in the production of hydrogen peroxide.

[0016] The application also provides the use of the strain combination of the probiotic composition in the preparation of a product for adhering to intestinal epithelial cells and vaginal epithelial cells, that is, the use of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 in the adhesion to intestinal epithelial cells and vaginal epithelial cells.

[0017] The application also provides the use of the strain combination of the probiotic composition in the preparation of a product for inhibiting the proliferation of Gardnerella, that is, the use of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 in the inhibition of the proliferation of Gardnerella.

[0018] The application also provides the use of the strain combination of the probiotic composition in the preparation of a product for inhibiting the proliferation of Candida albicans, that is, the use of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 in the inhibition of the proliferation of Candida albicans.

[0019] According to the specific embodiments of the application, the following technical effects are disclosed. The present application provides a probiotic composition and application in reducing the risk of vaginal infection, the probiotic composition contains Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70, in addition to the existing health functions of Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56, it also includes inhibiting harmful bacteria, improving oral health and reducing the risk of Helicobacter pylori infection.

[0020] The Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 provided by the present application have the functions of producing hydrogen peroxide, having superior adhesion to intestinal epithelial cells and vaginal epithelial cells, and inhibiting the reproduction of Gardnerella vaginalis and Candida albicans.

[0021] The probiotic composition provided by the present application reduces the risk of vaginal infection by inhibiting Gardnerella, Candida albicans and reconstructing vaginal flora; it is also related to the combination of the probiotic composition with a conventional carrier, which can be applied to prepare food, dietary supplements or drugs for preventing and / or treating vaginal and urethral microecological disorders, bacterial vaginitis, fungal vaginitis and urethral infections, so as to realize the inhibition of harmful bacteria in the vagina, the reconstruction of vaginal flora and the reduction of the risk of vaginal infection by the combination of Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70, and also reduce the risk of urethral infection.

[0022] Deposit Description Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri , the strain number is LN32, the preservation time is November 18, 2019, the preservation place is China General Microbiological Culture Collection Center, the specific address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the biological preservation number is CGMCC No. 18958.

[0023] Lactobacillus crispatus (Lactobacillus crispatus) Lactobacillus crispatus , the strain number is LN70, the preservation time is April 22, 2020, the preservation place is China General Microbiological Culture Collection Center, the specific address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the biological preservation number is CGMCC No. 19702. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below by combining with the specific embodiments provided by the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0025] In the present application, the Lactobacillus gasseri LN32 is directly isolated from the feces of healthy infants, has a rod shape, is gram-positive, does not form spores, produces lactic acid, is contactase-negative, and is facultative anaerobic. The partial 16S rDNA sequence (1313 bp) of the Lactobacillus gasseri LN32 is as follows: The Lactobacillus crispatus LN70 of the present application is directly isolated from the feces of healthy infants, has an elongated rod shape, appears in a slightly curved chain, is positive in Gram staining, does not form spores, produces lactic acid, is negative in contact enzyme, and is facultative anaerobic. The partial 16S rDNA sequence (1416bp) of the Lactobacillus crispatus LN70 is as follows: The technical solutions of the present application are further described below according to specific embodiments.

[0026] Example 1 In this embodiment, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were subjected to strain microscopic examination, MRS plate purification and isolation, strain fermentation, and verification of their hydrogen peroxide production capacity to confirm the characteristics and probiotic functions of the strains. The specific process includes: First, under sterile conditions, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were picked up with inoculation loops, respectively, and smeared on glass slides, and then subjected to staining and microscopic observation of the bacterial morphology.

[0027] Secondly, under sterile conditions, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were picked up with inoculation loops, respectively, and streaked on MRS plates, and then the plates were placed in an incubator for 37℃ culture for 72 h. Single colonies were selected for microscopic examination to achieve strain purification.

[0028] Finally, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were picked up from the slant with inoculation loops, respectively, and two loops were transferred to 2 50ml MRS liquid medium, and then cultured at 37℃ for 24h. Then, 5% inoculation was performed in 50ml MRS liquid medium, and then cultured at 37℃ for 72h to obtain Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 fermentation broth, respectively.

[0029] After the above process, the H2O2 production capacity of the two Lactobacillus strains was determined, which specifically includes: Freshly cultured Lactobacillus (more than 10 8 CFU / ml) was gradient diluted to a certain concentration, and 100μL was inoculated on H2O2 identification plates, i.e. smeared on MRS agar plates containing 0.25 mg / mL TMB (3,3',5,5'-tetramethylbenzidine) and 0.01 mg / mL horseradish peroxidase, and then cultured at 37℃ anaerobically for 48h to 72h. After taking out the MRS agar plate, the MRS agar plate was exposed to air, and the colonies producing H2O2 would turn blue, while the colonies not producing H2O2 would not change color. The H2O2 produced was semi-quantitatively determined according to the color change time, and the results are shown in Table 1.

[0030] Table 1 H2O2 semi-quantitative determination standard

[0031] In Table 1, the strains are scored as weak (i.e. 1 point, time ≥ 20 minutes), medium (i.e. 2 points, time 10-20 minutes), strong (i.e. 3 points, time ≤ 10 minutes) and 0 point indicates no blue color. According to the results shown in Table 2, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 have the same ability to produce H2O2 as Lactobacillus plantarum LN66, which is superior to other Lactobacillus gasseri LN20 and Lactobacillus crispatus LN26, and is also superior to Lactobacillus rhamnosus LN56. The colonies of live Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 start to appear a little blue at 2-3 min, and a large amount of blue appears within 9 min, according to the judgment standard shown in Table 1, the semi-quantitative ability of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 to metabolically produce hydrogen peroxide is 3 points.

[0032] Table 2 Ability to produce H2O2

[0033] It can be found from Table 2 that Lactobacillus gasseri LN32, Lactobacillus crispatus LN70 and Lactobacillus plantarum LN66 have a significant advantage over other strains in terms of the ability to produce H2O2.

[0034] Example 2 In this example, the inhibitory activity of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 on harmful bacteria in the vagina (Gardnerella, Candida albicans) and the adhesion ability to intestinal epithelial cells and vaginal epithelial cells are mainly verified, and the mechanism of action in vaginal flora reconstruction is elucidated.

[0035] Firstly, the inhibitory activity of the two Lactobacillus strains provided by the present application on Gardnerella (ATCC14018) is verified, and the specific process is as follows: 10 μL of freshly cultured Lactobacillus suspension (more than 10 8 CFU / ml) is inoculated on the surface of MRS agar, and incubated anaerobically at 37°C for 48 hours; 100 μL of freshly cultured Candida albicans suspension is mixed uniformly in 5 mL of soft BHI agar (0.7% agar) in a 50°C water bath, and poured onto the above-mentioned solid plate inoculated with Lactobacillus, and after solidification, incubated anaerobically at 37°C for 12-48 hours. The growth of Gardnerella is evaluated to assess the inhibitory activity on vaginal Gardnerella, and the results are shown in Table 3.

[0036] Table 3 Inhibitory activity on vaginal Gardnerella

[0037] The inhibitory activity is evaluated as follows: +++, high inhibitory activity against Gardnerella vaginalis, i.e. complete inhibition of the growth of Gardnerella vaginalis; ++, medium inhibitory activity against Gardnerella vaginalis; +, low inhibitory activity against Gardnerella vaginalis; and 0, no inhibitory activity, i.e. normal growth of Gardnerella vaginalis. According to the results in Table 3, the inhibitory activity against Gardnerella vaginalis of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 is superior to that of Lactobacillus gasseri LN20 and Lactobacillus crispatus LN26, and is also superior to that of Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56. Therefore, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 have a significant advantage over other strains in inhibiting the activity of Gardnerella vaginalis.

[0038] Secondly, the inhibitory activity of the two Lactobacillus strains provided by the present application against Candida albicans (ATCC90028) is verified. The specific process is as follows: 10 μL of a fresh culture of the Lactobacillus strain (more than 10 8 CFU / ml) is inoculated on the surface of MRS agar, and incubated anaerobically at 37°C for 48 hours; 100 μL of a fresh culture of Candida albicans is mixed uniformly in 5 mL of soft YM agar (0.7% agar) in a water bath at 50°C, and poured on the Lactobacillus MRS agar plate which has been incubated for 48 hours, and then incubated aerobically at 37°C for 12-48 hours. The inhibitory activity against Candida albicans is evaluated by the growth of the yeast, and the results are shown in Table 4.

[0039] Table 4 Inhibitory activity against Candida albicans

[0040] The inhibitory activity is evaluated as follows: +++, high inhibitory activity against Gardnerella vaginalis, i.e. complete inhibition of the growth of Gardnerella vaginalis; ++, medium inhibitory activity against Gardnerella vaginalis; +, low inhibitory activity against Gardnerella vaginalis; and 0, no inhibitory activity, i.e. normal growth of Gardnerella vaginalis. According to the results in Table 3, the inhibitory activity against Gardnerella vaginalis of Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 is superior to that of Lactobacillus gasseri LN20 and Lactobacillus crispatus LN26, and is also superior to that of Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56. Therefore, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 have a significant advantage over other strains in inhibiting the activity of Gardnerella vaginalis.

[0041] Thirdly, the adhesion ability of the two Lactobacillus strains provided by the present application to intestinal epithelial cells is verified. The adhesion performance of different Lactobacillus strains is determined according to the number of Lactobacillus strains adhered to the monolayer of intestinal epithelial cells. The specific process is as follows: after 4 Lactobacillus strains are cultured in MRS liquid medium for 48 hours (more than 10 8CFU / ml), centrifuged, washed three times with PBS, and resuspended in complete DMEM medium for later use. Meanwhile, Caco-2 cells were adjusted to 600,000 cells / mL and seeded into 6-well plates. After 48 hours of culture to form a monolayer, they were washed twice with sterile PBS, trypsinized in the first well, and the cells were counted using a hemocytometer. Separately, 1 mL each of the previously prepared lactobacilli and a blank PBS control were added to five well plates. After incubation for 2 hours, unadhered lactobacilli were washed and lysed with 1 mL of 0.05% Triton X-100 to prepare a bacterial suspension. The suspension was diluted, and 100 μL of the suspension was evenly spread onto MRS plates. After anaerobic incubation for 48 hours, the number of lactobacilli on each plate was counted, and the number of adherent lactobacilli was calculated. The results are shown in Table 5.

[0042] Table 5 Adhesion ability to intestinal epithelial cells

[0043] The adhesion rate to intestinal epithelial cells was calculated by the following formula: Adhesion rate (%) = number of adherent lactobacilli / total number of cells × 100; According to the results in Table 5, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 exhibited superior adhesion to intestinal epithelial cells compared to Lactobacillus gasseri LN20 and Lactobacillus crispatus LN26. Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56 also exhibited excellent adhesion to intestinal epithelial cells. Therefore, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 exhibited significant advantages over their counterparts in terms of intestinal epithelial cell adhesion.

[0044] Finally, the adhesion ability of the two lactobacilli provided by the present invention to vaginal epithelial cells was verified, and the adhesion performance of different lactobacilli was determined according to the number of lactobacilli adhered to the vaginal epithelial cell monolayer. The specific process was as follows: 4 lactobacilli were cultured in MRS liquid medium for 48 hours (more than 10 8 CFU / ml) for later use. Meanwhile, human vaginal epithelial Vk2 / E6E7 cells were seeded at a density of 5 million per well in a 6-well culture plate and cultured at 37°C, 5% CO₂ for 48 hours to form a monolayer. The cells were then washed twice with sterile PBS, trypsinized in the first well, and counted using a hemocytometer. Separately, 1 mL each of the previously prepared lactobacilli and a blank PBS control were added to five well plates. After incubation for 2 hours, unadhered lactobacilli were washed and lysed with 1 mL of 0.05% Triton X-100 to prepare a bacterial suspension. The suspension was diluted, and 100 μL of the suspension was evenly spread on MRS plates. After anaerobic incubation for 48 hours, the number of lactobacilli on each plate was counted, and the number of adherent lactobacilli was calculated. The results are shown in Table 6.

[0045] Table 6 Adhesion ability to vaginal epithelial cells

[0046] wherein the adhesion rate to vaginal epithelial cells is calculated by the following formula: Adhesion rate (%) = number of adhered Lactobacillus / total number of cells x 100; According to the results obtained in Table 6, the adhesion ability to vaginal epithelial cells of L. gasseri LN32 and L. crispatus LN70 is superior to that of other L. gasseri LN20 and L. crispatus LN26, and also superior to that of L. plantarum LN66 and L. rhamnosus LN56. Thus, L. gasseri LN32 and L. crispatus LN70 have a significant advantage over other strains in terms of vaginal epithelial cell adhesion ability.

[0047] Example 3 In this example, the effectiveness of the probiotic composition containing L. gasseri LN32, L. crispatus LN70, L. plantarum LN66, and L. rhamnosus LN56 in reducing the risk of vaginal infection was verified through animal models and clinical experiments.

[0048] First, an animal experiment was conducted to reduce the risk of Gardnerella infection by orally administering a probiotic composition of L. plantarum LN66, L. rhamnosus LN56, L. gasseri LN32, and L. crispatus LN70, and the risk of bacterial vaginosis was determined based on the inhibition of the number of Gardnerella in the vagina of the animal and the increase in the number of Lactobacillus, according to the following method: A total of 50 female BALB / c mice were randomly divided into 5 groups of 10 each: a blank control group, a model group, a combination of 2 public strains group: L. plantarum LN66 + L. rhamnosus LN56 (i.e., the LN66 + LN56 group), a combination of 2 new strains group: L. gasseri LN32 + L. crispatus LN70 (i.e., the LN32 + LN70 group), and a combination of 4 probiotics group: L. plantarum LN66, L. rhamnosus LN56, L. gasseri LN32, and L. crispatus LN70 (i.e., the LN66 + LN56 + LN32 + LN70 group).

[0049] The animal experimental model was established according to the model of bacterial vaginosis described by Sabbatini et al. (Sabbatini S, Monari C, Ballet N, et al. Saccharomyces cerevisiae–based probiotic as novel antimicrobial agent for therapy of bacterial vaginosis, Virulence, 2018, 9(1): 954-966). Three days before infection (i.e. day -3) and on the day of infection (i.e. day 0), 100 µL of estradiol valerate was subcutaneously injected into the mice except for the control group, i.e. 0.5 mg of estradiol valerate was dissolved in 100 µL of medium-chain glycerides. On day 0, the mice were vaginally inoculated with 20 µL of a Gardnerella vaginalis suspension at a concentration of 10 8 CFU / mL, for 5 consecutive days, until the end of the vaginal Gardnerella vaginalis inoculation on day 4, to establish the mouse model of bacterial vaginosis. After infection (i.e. day 6), the control group was administered 200 µL of PBS buffer once a day by gavage, and the other three groups were administered 200 µL of a probiotic suspension at a concentration of 10 10 CFU / mL once a day by gavage until the end of the experiment, for a total of 14 days.

[0050] The vaginal flora of the mice was then detected. Using a micro-pipette with 50 µL of normal saline, the vaginal flora of the mice was repeatedly washed 5-6 times at the following time points, i.e. after modeling (i.e. day 5), 1 day after gavage (i.e. day 20), and 6 days after gavage (i.e. day 25). 30 µL of the above-mentioned vaginal lavage fluid was used for colony counting of Gardnerella vaginalis and Lactobacillus, respectively, and compared with the model group and the blank control group, respectively, to calculate the percentage of Gardnerella vaginalis and Lactobacillus in each group, i.e. the Lactobacillus in the blank control group was 100%, and the Lactobacillus in the model group (%) = the number of Lactobacillus in the model group x 100 / the number of Lactobacillus in the blank control group, and the Lactobacillus in each experimental group was calculated in the same way. Similarly, the Gardnerella vaginalis in the model control group was 100%, and the Gardnerella vaginalis in the blank control group (%) = the number of Gardnerella vaginalis in the blank control group x 100 / the number of Gardnerella vaginalis in the model group, and the Gardnerella vaginalis in each experimental group was calculated in the same way. The results are shown in Table 7.

[0051] According to the results in Table 7, it is shown that on the first day after the end of gavage (i.e. the 20th day), the number of Gardnerella colonization in the vagina of mice in the three probiotic groups is significantly reduced compared with the model group, which is 25.48%, 11.72% and 3.65% respectively in the LN66+LN56 group, the LN32+LN70 group and the LN66+LN56+LN32+LN70 group; while the number of Lactobacillus colonization in the vagina is significantly increased compared with the blank control group, which is 1346.56%, 1428.23% and 1834.15% respectively. On the sixth day after the end of gavage, the number of Gardnerella colonization in the three probiotic groups is less than 1% of the model group, while the number of Lactobacillus colonization is still increasing. It is shown that oral administration of LN66+LN56, LN32+LN70 and LN66+LN56+LN32+LN70 combined strains alone can inhibit Gardnerella infection and increase the number of Lactobacillus. Comparison of the results of the three probiotic groups shows that the combination of LN66+LN56+LN32+LN70 is superior to the other two combinations, i.e. LN66+LN56 and LN32+LN70, in terms of inhibiting Gardnerella infection.

[0052] Table 7 Percentage of Gardnerella and Lactobacillus in the vaginal lavage fluid of each experimental group

[0053] Secondly, an animal experiment of oral administration of the probiotic composition of Lactobacillus gasseri LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 is carried out to reduce the risk of Candida albicans infection, and the number of Candida albicans in the vagina of animals is inhibited and the number of Lactobacillus is increased to determine whether the probiotic can reduce the risk of vaginal Candida albicans infection.

[0054] Specific methods are as follows: 50 SPF C57BL / 6 female mice are randomly divided into 5 groups, 10 in each group: a blank control group, a model group, a combination of 2 public strains group: Lactobacillus gasseri LN66+Lactobacillus rhamnosus LN56 (i.e. the LN66+LN56 group), a combination of 2 new strains group: Lactobacillus gasseri LN32+Lactobacillus crispatus LN70 (i.e. the LN32+LN70 group) and a combination of 4 probiotics group: Lactobacillus gasseri LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 (i.e. the LN66+LN56+LN32+LN70 group). Five days before infection (i.e. the -5th day), 50 μL of lincomycin hydrochloride solution of a certain concentration is used to perform vaginal irrigation of mice by using a micro-sampler once a day for 5 consecutive days, except for the control group. On the 0th day, 2.0×10 7CFU / mL of C. albicans suspension, once a day for 6 consecutive days, until the end of the vaginal inoculation of C. albicans on the 5th day, to cause the C. albicans vaginal infection model of mice. After the infection (i.e. on the 7th day), except for the model group, the blank control group was given 200 μL of PBS buffer once a day by gavage, and the other three groups were given 200 μL of probiotic suspension with a concentration of 10 10 CFU / mL of C. albicans suspension, once a day for 6 consecutive days, until the end of the vaginal inoculation of C. albicans on the 5th day, to cause the C. albicans vaginal infection model of mice. After the infection (i.e. on the 7th day), except for the model group, the blank control group was given 200 μL of PBS buffer once a day by gavage, and the other three groups were given 200 μL of probiotic suspension with a concentration of 10

[0055] Then the vaginal flora of mice was detected, using a micro-sampler with 50 μL of normal saline, repeatedly flushing the vagina of mice 5-6 times at the following time periods, i.e. after modeling (i.e. on the 6th day), 1 day after the end of gavage (i.e. on the 21st day), and 6 days after the end of gavage (i.e. on the 26th day), and taking 30 μL of the above vaginal lavage fluid to do the colony counting of C. albicans and lactobacilli, respectively, and comparing with the model group and the blank control group, to calculate the percentage of C. albicans and lactobacilli of each group, i.e. the lactobacilli of the blank control group is 100%, then the lactobacilli (%) of the model group = the number of lactobacilli of the model group x 100 / the number of lactobacilli of the blank control group, and the lactobacilli (%) of each experimental group is calculated in the same way; similarly, the C. albicans of the model control group is 100%, then the C. albicans (%) of the blank control group = the number of C. albicans of the blank control group x 100 / the number of C. albicans of the model group, and the C. albicans (%) of each experimental group is calculated in the same way, and the results are shown in Table 8.

[0056] Table 8 Percentage of C. albicans and lactobacilli in the vaginal lavage fluid of each experimental group

[0057] As can be seen from the results provided in Table 8, 1 day after the end of gavage (i.e. on the 21st day), the number of C. albicans colonization in the vagina of mice of the three probiotic groups was significantly reduced compared with the model group, which was 15.49%, 10.36% and 5.78% for the LN66+LN56 group, the LN32+LN70 group and the LN66+LN56+LN32+LN70 group, respectively; while the number of lactobacilli colonization in the vagina was significantly increased compared with the blank control group, which was 1143.27%, 1317.45% and 1816.34%, respectively. 6 days after the end of gavage, the number of C. albicans colonization of the three probiotic groups was less than 10% of the model group, while the number of lactobacilli colonization was still increasing. It is indicated that oral administration of LN66+LN56, LN32+LN70 and LN66+LN56+LN32+LN70 combined strains alone can inhibit C. albicans infection and increase the number of lactobacilli. Comparing the results of the three probiotic groups shows that, in terms of inhibiting C. albicans infection, the combination of LN66+LN56+LN32+LN70 is superior to the other two combinations, i.e. LN66+LN56 and LN32+LN70.

[0058] Example 4 In this example, the clinical trials of the present application were mainly carried out. First, two kinds of probiotic composition products prepared by the present application were respectively applied, one was the probiotic composition (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were 1.0 x 10 9 CFU / day, and the total viable bacterial content was 4.0 x 10 9 CFU / day) combined with a conventional carrier (referred to as 4.0 x 10 8 CFU / day), and the other was a low-dose probiotic composition (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were 1.0 x 10 8 CFU / day, and the total viable bacterial content was 4.0 x 10 8 CFU / day) combined with a conventional carrier (referred to as 4.0 x 10 9 CFU / day), which were used to treat bacterial vaginosis. The changes of BV-related indicators before and after oral administration of the probiotic composition of the present application were evaluated by bacterial vaginosis (BV) clinical diagnosis Amsel "gold standard" and Gram staining Nugent scoring standard. The specific results are shown in Table 9.

[0059] Table 9 Therapeutic effect of two kinds of probiotic compositions on BV

[0060]

[0061] Among them, the number of lactobacilli was scored according to the average number of lactobacilli observed under each 10 oil lens field, 0+, no lactobacilli; 1+, <1 lactobacilli; 2+, 1-4 lactobacilli; 3+, 5-30 lactobacilli; 4+, >30 lactobacilli.

[0062] According to the results shown in Table 9, according to the clinical diagnosis Amsel "gold standard" and Gram staining Nugent scoring standard, the clinical symptoms and the number of lactobacilli of the patients with bacterial vaginosis were improved after taking the probiotic composition of 4.0 x 10 8 CFU / day of the present application for 4 weeks. At the same time, the improvement effect on bacterial vaginosis of the probiotic composition with a dose lower than that of the present application, i.e. taking the probiotic composition of 4.0 x 10 9 CFU / day of the present application, was also significantly lower than that of the probiotic composition of the present application. The results showed that the viable bacterial content in the probiotic composition of the present application, i.e. Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70, was not less than 1.0 x 10CFU / day, total viable bacteria count not less than 4.0×10 9 CFU / day.

[0063] Secondly, two probiotic composition products prepared by the present invention were respectively applied, one of which was a probiotic composition (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 were 1.0×10 9 CFU / day, total viable bacteria content is 4.0×10 9 CFU / day) combined with a conventional carrier (referred to as 4 billion CFU / day), and the other is a low-dose probiotic composition (Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70, respectively, 1.0×10 8 CFU / day, total viable bacteria content is 4.0×10 8 The above two probiotic compositions were used to treat candidal vaginitis. According to the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Vulvovaginal Candidiasis in China (2024 Edition)", the changes in the indicators related to candidal vaginitis before and after oral administration of the probiotic composition of the present invention were evaluated. The specific results are shown in Table 10.

[0064] Table 10 The therapeutic effects of two probiotic compositions on fungal vaginitis

[0065]

[0066] As shown in Table 10, based on clinical symptoms and vaginal Candida albicans detection, after taking the probiotic composition of the present invention at 4 billion CFU / day for 4 weeks, the clinical symptoms of patients with fungal vaginitis were improved, and Candida albicans turned from positive to negative. At the same time, a probiotic composition with a lower dose than the prescribed dose of the present invention was used as a control, that is, taking the probiotic composition of the present invention at 4.0×10 8 CFU / day probiotic composition also showed a significantly lower improvement effect on fungal vaginitis after taking it for 4 weeks than the probiotic composition of the present invention. The results showed that the content of viable bacteria in the probiotic composition of the present invention, namely Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70, was not less than 1.0×10 9 CFU / day, total viable bacteria count not less than 4.0×10 9 CFU / day.

[0067] Therefore, the probiotic composition containing Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 and the application in reducing the risk of vaginal infection are used to achieve the reconstruction of vaginal flora, inhibit the reproduction of Gardnerella vaginalis and Candida albicans, increase the colonization of Lactobacillus, eliminate abnormal leukorrhea, vaginal burning and itching, frequent urination, urgent urination and painful urination, etc. by the oral route. The probiotic composition is combined with a conventional carrier and applied to the preparation of food, dietary supplements or drugs for preventing and / or treating vaginal and urethral microecological disorders, bacterial vaginitis, mycotic vaginitis and urethral infection, so that the present application realizes the inhibition of harmful bacteria in the vagina, the reconstruction of vaginal flora and the reduction of the risk of vaginal infection and the risk of urethral infection.

[0068] The various embodiments are described in the present specification in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be mutually referred to.

[0069] The principles and implementation manners of the present application are described by using specific examples in the present specification. The above description of the embodiments is only used to help understand the method of the present application and its core idea. Meanwhile, for those skilled in the art, the specific implementation manners and application ranges can be changed according to the idea of the present application. In conclusion, the content of the present specification should not be understood as a limitation of the present application.

Claims

1. A probiotic composition comprising four probiotic strains, including Lactobacillus plantarum LN66 and Lactobacillus rhamnosus LN56, characterized in that: The probiotic composition further comprises Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70.

2. A probiotic composition according to claim 1, characterized in that In the probiotic composition, the viable counts of Lactobacillus plantarum LN66, Lactobacillus rhamnosus LN56, Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70 are all greater than 1.0×10 9 CFU / g(ml).

3. Use of the probiotic composition according to any one of claims 1 to 2 in combination with a conventional carrier in the preparation of a medicament for preventing and treating vaginal infection.

4. Use of the probiotic composition according to any one of claims 1 to 2 in combination with a conventional carrier in the preparation of a drug for preventing and treating urinary tract infection.

5. Use of the probiotic composition according to any one of claims 1 to 2 in rebuilding vaginal flora.

6. A strain combination of a probiotic composition, characterized in that: The bacterial strain combination includes Lactobacillus gasseri LN32 and Lactobacillus crispatus LN70; The strain of Lactobacillus gasseri is LN32, which was deposited on November 18, 2019, at the General Microbiology Center of China Culture Collection Administration, and the biological deposit number is CGMCC No. 18958; The strain of the Lactobacillus crispatus is LN70, the preservation time is April 22, 2020, the preservation location is the General Microbiology Center of the China Culture Collection Administration, and the biological preservation number is CGMCC No.19702.

7. Use of the strain combination of the probiotic composition according to claim 6 in preparing a product for producing hydrogen peroxide.

8. Use of the strain combination of the probiotic composition according to claim 6 in preparing a product for adhering to intestinal epithelial cells and vaginal epithelial cells.

9. Use of the strain combination of the probiotic composition according to claim 6 in preparing a product for inhibiting the proliferation of Gardnerella.

10. Use of the strain combination of the probiotic composition according to claim 6 in preparing a product for inhibiting the proliferation of Candida albicans.

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