Preparation and application of micro-ecological regulator for vaginal mucosa bacteriostasis

Abstract

The invention relates to a microecological preparation for vaginal bacteriostasis, which is characterized in that a lactococcus lactis subsp. Lactis KF-22 strain which can produce hydrogen peroxide at high yield and has a pH value of 4.16 at a fermentation end point is matched with five probiotics, and auxiliary materials such as starch, sodium carboxymethyl cellulose and microcrystalline cellulose are used for preparing probiotic tablets or capsules. Bacteriostatic tests prove that the traditional Chinese medicine composition has an inhibiting effect on escherichia coli, staphylococcus aureus and candida albicans, and animal experiments prove that the traditional Chinese medicine composition has no irritation or slight irritation on vaginal mucosa. The formula preparation is used for treating recurrent BV and RVVC, compared with antibiotics and hormone groups, the treatment effects of the formula preparation are similar, but after follow-up visit of recurrent BV and RVVC C1 for months and 2 months, the recurrence rate of a test group is far lower than that of an antibiotic control group. The preparation has a very good vaginosis treatment effect, can be used as a related hygienic product or medicine for treating BV and VVC vaginosis, reduces the recurrence rate of the BV and VVC vaginosis at the same time, and has very important social significance.

Description

Technical Field

[0001] The present invention relates to a Lactococcus lactis KF-22 isolated from kefir yogurt. After being compounded with a variety of probiotics, it can inhibit common pathogenic bacteria in the vagina, and at the same time has no irritation to the vaginal mucosa. It can be used to treat BV and VVC vaginitis, belonging to the field of microecological technology. Background Art

[0002] Women will experience special physiological stages such as menstrual period, pregnancy, puerperium and menopause during their entire life cycle. Coupled with the different physiological structures of men and women, women face more reproductive health problems than men. According to the survey report on women's reproductive health and mental health carried out by the Institute of Occupational Health and Poison Control of the Chinese Center for Disease Control and Prevention and related units, the total prevalence rate of women's reproductive system infections is as high as 50.5%. The general survey results of 73,861 cases of gynecological diseases in Wuxi Maternal and Child Health Hospital show that the prevalence rate of gynecological diseases is as high as 62.97%. Data provided by the China Report Hall, the most authoritative market research report and research report service provider at home and abroad, show that the incidence rate of global gynecological diseases has reached more than 65%, and among women of childbearing age, the incidence rate of gynecological diseases has exceeded 70%; the annual incidence figure of reproductive and urinary tract infections in the world has reached 333 million person-times. The high incidence of gynecological diseases has become an indisputable fact.

[0003] Currently, the treatment of gynecological diseases mainly relies on western medicine supplemented by traditional Chinese medicine. Although it can have good effects in a short time, long-term use will destroy the original microecological environment of the vaginal flora balance, leading to repeated attacks of the disease and being unfavorable for prognosis. The currently recognized dominant vaginal flora is Lactobacillus, which plays a very important protective role in women's vaginal health. Lactobacillus can utilize the glycogen released by vaginal epithelial cells to metabolize and produce a large amount of substances such as lactic acid, hydrogen peroxide, and bacteriocin, creating a slightly acidic environment with a pH of 3.8-4.5 in the female reproductive tract. At the same time, in combination with other normal flora, a thick probiotic biofilm ecological defense barrier (Probiotic Biofilm, PBF) is formed on the surface of the vaginal mucosal tissue. They inhibit the survival of other pathogenic bacteria by competing for nutrient sources, colonization sites, releasing antibacterial substances, etc., and maintain the natural health state of the female reproductive tract.

[0004] When the number of beneficial bacteria in the vagina decreases and the beneficial bacteria film is damaged, pathogenic bacteria will invade and multiply in large numbers, and vaginal diseases will occur. According to the different pathogenic bacteria involved, it can be divided into bacterial vaginitis (BV), vulvovaginal candidiasis (VVC), trichomonal vaginitis (TV), etc. The pathogenic bacteria in the vagina can also ascend to infect parts such as the uterus, fallopian tubes, and ovaries, causing lesions in these parts. By guarding the gateway of the female reproductive tract - the vagina, through in-depth research on the vaginal flora, developing relevant microecological preparations, and intervening and regulating the microecology of the female reproductive tract, it has become a new medical direction for preventing and treating female reproductive system diseases.

[0005] At the same time, compared with the treatment of killing both beneficial and harmful bacteria such as antibiotics, microecological preparations regulate the human microecological balance by supplementing beneficial bacteria to the human body, which is beneficial for the body to improve its own immunity and has a lower recurrence rate. Currently, there are only two approved over-the-counter microecological preparations for women. One is "Yanhua" produced by Xi'an Zhenghao Biopharmaceutical Co., Ltd., whose core ingredient is live Enterococcus faecalis, and its mechanism of action is to produce lactic acid to lower the vaginal pH. The other is "Ding Junsheng" produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd., whose core ingredient is Lactobacillus, but its treatment is only for bacterial vaginitis and has no therapeutic effect on vulvovaginal candidiasis caused by Candida albicans, etc. Currently, it is urgent to develop new vaginal microbial strains to make the microecological preparations more selective, broaden the treatment scope of microecological preparations, and play a greater role in female reproductive system diseases. Summary of the Invention

[0006] The present invention provides a new strain Lactococcus lactis KF-22 derived from kefir yogurt, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 5, 2017. During its growth and proliferation process, it will metabolize and produce a large amount of lactic acid, and the pH at the end of fermentation is 4.16, within the normal pH range of women. At the same time, it can also produce a large amount of hydrogen peroxide, exopolysaccharides, etc., which can inhibit a variety of pathogenic bacteria, and it is a beneficial strain that can be used for female vaginal diseases.

[0007] The present invention provides a method for preparing freeze-dried bacterial powder of the above strain. The strain prepared by this method has a viable count as high as 10 10 ~10 11 cfu / g.

[0008] The preparation steps of the bacterial powder of the present invention are as follows: activate the strain to prepare the seed culture medium, then inoculate it into the MRS culture medium, with an inoculation amount of 1%, a culture temperature of 37°C, a pH of 6.0 - 6.4, and a culture time of 18 - 22 h to obtain the fermentation broth. After three centrifugations, washings, and addition of freeze-drying protectants, it is freeze-dried by a vacuum freeze dryer to obtain the Lactococcus lactis bacterial powder.

[0009] The freeze-dried KF-22 bacterial powder is then mixed with five probiotics, Lactobacillus acidophilus, Weissella confusa, Lactobacillus casei, Lactobacillus helveticus, and Lactobacillus plantarum, in a ratio of 5:3:3:3:3:3. These six probiotics together account for 1% - 10% of the preparation, 25 - 30% starch, 8 - 10% carboxymethyl cellulose, 55 - 60% microcrystalline cellulose, and 0.3 - 0.6% magnesium stearate / silica (lubricant).

[0010] The microecological preparation made by the present invention has an effective viable count of 10 7 cfu / g, and this dose can meet the functional requirements.

[0011] The microecological preparation made by the present invention has an inhibitory effect on Candida albicans, Escherichia coli, and Staphylococcus aureus.

[0012] This product can be stored at room temperature for 2 years without losing its probiotic properties.

[0013] The microecological preparation made by the present invention has passed the vaginal mucosa irritation test and has no irritating effect on the vaginal mucosa.

[0014] The clinical trial results of applying this preparation to treat recurrent BV and RVVC show that compared with the control group using only antibiotics and hormones and other preparations, the treatment effects are comparable, and the recurrence rate of vaginal diseases is lower. Description of the Drawings

[0015] Figure 1 It is a detailed process flow chart for the preparation of vaginal topical probiotic tablets.

[0016] Figure 2 It is a detailed process flow chart for the preparation of vaginal topical probiotic powder. Detailed Description of the Invention

[0017] Preparation of bacterial powder.

[0018] This strain was isolated and streaked from kefir-source yogurt to obtain a single strain. The strain was activated to prepare a seed medium, which was then inoculated into MRS medium at an inoculation amount of 1%, cultured at 37°C, pH 6.0 - 6.4, and cultured for 18 - 22 h to obtain a fermentation broth. After three centrifugations, washings, and addition of a freeze-drying protectant, the Lactococcus lactis powder was obtained by freeze-drying using a vacuum freeze-dryer. The viable count was detected to be 2.25×10 10 cfu / g.

[0019] Identification of the probiotic properties of the powder.

[0020] Identification of the ability to produce hydrogen peroxide. Lactococcus lactis is a facultative anaerobe. It can glycolyze sugars, and the final fermentation product is lactic acid. In the presence of oxygen, it can reduce oxygen to hydrogen peroxide. The hydrogen peroxide standard curve uses the H2O2 standard solution as the abscissa and OD510 as the ordinate, and the regression equation is: y = 0.1159x - 0.0085 (R2 = 0.9991). According to the standard curve, the amount of hydrogen peroxide produced by each strain was determined by measuring the OD510 value of the supernatant of each strain and the phenol reagent after color development under the action of horseradish peroxidase. After measurement, the hydrogen peroxide produced by KF-22 was 23.7 μg / mL.

[0021] Identification of the ability to produce lactic acid. Weighed 0.1 g of Lactococcus lactis KF-22 powder and vortexed it in 10 ml of sterile water until evenly mixed. Absorbed 100 μl and incubated it in 5 ml of MRS liquid medium at 37°C for 24 h. Then poured 5 ml of MRS liquid medium into 100 ml of MRS and cultured it at 40°C for 48 hours. Detected the pH of the bacterial liquid. Three groups of detections were performed for each strain, and the average value was taken. Its pH was 4.16, within the normal pH range of women.

[0022] Strain compounding and viable count identification. According to the method of "Food Safety Standards - Microbiological Examination of Foods - Examination of Lactic Acid Bacteria" (GB 4789.35-2016), weigh 25 g of Lactococcus lactis, 15 g each of Lactobacillus acidophilus, Weissella cibaria, Lactobacillus casei, Lactobacillus helveticus, and Lactobacillus plantarum, mix them together with an electric blender and set aside. Weigh 25 g of the sample aseptically, place it in a sterile homogenization cup containing 225 mL of normal saline, homogenize at 8000 r / min to 10000 r / min for 1 min to 2 min to prepare a 1:10 sample homogenate and set aside. According to the estimated viable count of lactobacilli in the sample to be tested, select 2 to 3 consecutive appropriate dilution factors, pipette 1 mL of the sample homogenate into a sterilized petri dish for each dilution factor, and make two petri dishes for each dilution factor. After the diluent is transferred into the petri dish, pour about 15 mL of MRS agar medium cooled to 48 °C into the petri dish and rotate the petri dish to mix evenly. Incubate anaerobically at 36 °C ± 1 °C for 72 h ± 2 h. It is required to complete the process from sample dilution to plate pouring within 15 min. The measured viable count of the sample is 10 10 cfu / g. The viable count detection data prove that the combination of these bacteria has a mutual synergistic effect and can be used as raw materials for the same microecological preparation.

[0023] Preparation of vaginal topical probiotic tablets. The core ingredient of this product is freeze-dried probiotic powder. Weigh 10 g to 50 g of the mixed probiotic powder, mix it with 250 to 300 g of starch (filler), 80 to 100 g of carboxymethyl cellulose as a binder, 570 to 590 g of microcrystalline cellulose, and 3 to 6 g of magnesium stearate. After granulation, press tablets and detect using the sensory properties, hardness, tablet weight, and viable count of the preparation as indicators. Through repeated adjustment and trial, the preparation with the best ratio and meeting the requirements is obtained.

[0024] The detailed process flow chart for the preparation of vaginal topical probiotic tablets is as shown in the appendix of the instruction manual Figure 1 as follows.

[0025] Preparation of vaginal topical probiotic powder. The core ingredient of this product is freeze-dried probiotic powder. Weigh 10 g to 50 g of the mixed probiotic powder, mix it with 250 to 300 g of starch, 80 to 100 g of carboxymethyl cellulose as a binder, and 570 to 590 g of microcrystalline cellulose. After mixing, filling, and polishing, perform aluminum-plastic packaging.

[0026] The detailed process flow chart for the preparation of vaginal topical probiotic powder is as shown in the appendix of the instruction manual Figure 2 as follows.

[0027] According to the standard of "Disinfection Technical Specification" (2002 edition), 2.1.8.2, perform the antibacterial performance test.

[0028] Test strains: Escherichia coli (8099), and its 4th-generation fresh slant culture was used for the test; Staphylococcus aureus (ATCC6538), and its 4th-generation fresh slant culture was used for the test; Candida albicans (ATCC10231), and its 6th-generation fresh slant culture was used for the test. All the above strains were provided by the Guangdong Food Microbiology Safety Engineering Technology Research and Development Center.

[0029] Inoculation of test bacteria: Use a sterile cotton swab to dip into the test bacterial suspension with a concentration of 5×10 5 cfu / ml~5×10 6 cfu / ml (Staphylococcus aureus (ATCC 6538) bacterial suspension, Escherichia coli (8099) bacterial suspension, Candida albicans (ATCC 10231) bacterial suspension), and evenly smear it 3 times on the surface of the nutrient agar medium plate. Each time it is smeared, the plate should be rotated 60°. Finally, the cotton swab is smeared around the edge of the plate for one week. Cover the petri dish and place it at room temperature to dry for 5 min.

[0030] Preparation of bacteriostatic agent tablets: The preparation is made into circular tablets with a diameter of 5 mm and a thickness not exceeding 4 mm, and each group consists of 4 tablets.

[0031] Placement of bacteriostatic samples: Each time, 1 contaminated plate is used for the test. 4 test tablets and 1 negative control tablet are placed on each plate, a total of 5 tablets. Use sterile forceps to pick up the tablets and place them on the surface of the plate. The distance between the centers of each tablet is more than 25 mm, and the distance from the periphery of the plate is more than 15 mm. After placement, gently press the tablets with sterile forceps to make them adhere to the surface of the plate. Cover the plate and place it in an incubator at 37°C for 16 h~18 h to observe the results. Use a vernier caliper to measure the diameter of the inhibition zone (including the patch) and record it. The test is repeated 3 times. When measuring the inhibition zone, a uniform and completely sterile growth inhibition zone should be selected. When measuring its diameter, the outer edge of the inhibition zone should be used as the boundary.

[0032] Evaluation criteria: The bacteriostatic agent continuously dissolves and diffuses through the agar to form different concentration gradients to show its bacteriostatic effect. The test judges whether it has bacteriostatic ability through the size of the inhibition zone. If the diameter of the inhibition zone is greater than 7 mm, it is judged to have a bacteriostatic effect; if the diameter of the inhibition zone is less than or equal to 7 mm, it is judged to have no bacteriostatic effect.

[0033] The bacteriostatic zone data of this preparation against Escherichia coli (8099), Staphylococcus aureus (ATCC 6538), and Candida albicans (ATCC10231) are shown in Table 1, Table 2, and Table 3.

[0034] The average inhibition zone diameters of this preparation against Escherichia coli were 12.28 mm, 11.99 mm, and 12.46 mm, showing antibacterial effects; those against Staphylococcus aureus were 18.13 mm, 18.29 mm, and 18.50 mm, showing antibacterial effects; and those against Candida albicans were 31.72 mm, 31.60 mm, and 31.39 mm, showing antibacterial effects.

[0035] Vaginal mucosa irritation test of the preparation. According to the "Disinfection Technical Specification" (2002 edition), 2.3.5 Vaginal Mucosa Irritation Test, the irritation of this preparation to the vaginal mucosa was tested.

[0036] Operation procedure.

[0037] Connect a blunt-tipped hose about 8 cm long to a 2 mL syringe. Fill the syringe and catheter with the test solution for standby. Prepare one set for each animal. Fix the animal on its back, expose the perineum and vaginal orifice, moisten the catheter with the test solution and gently insert it into the vagina (4 cm - 5 cm), and slowly inject 2 mL of the test solution with the syringe, then withdraw the catheter to complete the dosing.

[0038] At 24 h after dosing, the animals were sacrificed by air embolism method, the intact vagina was dissected and taken out, longitudinally incised, and no manifestations such as congestion and edema were observed macroscopically. Then the vagina was fixed in 10% neutral buffered formalin solution for more than 24 h, and tissues from 3 parts at both ends and the middle of the vagina were selected for section preparation. After HE staining, histopathological examination was carried out.

[0039] Evaluation criteria: The results of histopathological examination were scored for the vaginal mucosa irritation reaction according to Table 4.

[0040] After adding up the irritation reaction scores of 3 parts of 3 animals in the test group and then dividing by the total number of observations (number of animals × 3), the average score of the vaginal mucosa irritation reaction in the test group was obtained. The scoring method for the control group was the same as above.

[0041] After obtaining the irritation index by subtracting the average score of the control group from the average score of the test group, the irritation intensity was graded according to Table 5.

[0042] The scoring standard for the mucosa irritation reaction is shown in Table 4. It shows that the vaginal mucosa irritation index of this product for New Zealand rabbits is 0.00, and the reaction intensity of the single vaginal mucosa irritation test for New Zealand rabbits is non-irritating, meeting the requirements of the "Disinfection Technical Specification" (2002 edition).

[0043] Clinical trial verification. Rugao Family Planning Guidance Station used this product to treat recurrent BV and RVVC. From January 2008 to December 2009, 100 patients diagnosed with recurrent BV and RVVC in the outpatient department were selected and randomly divided into a treatment group and a control group. In the treatment group, patients with recurrent BV and RVVC were given Shanyujun freeze-dried active factor capsules with this probiotic combination as the active ingredient (each capsule contains 1 million Shanyujun lactic acid bacteria), 1 capsule was placed deep in the vagina every night for 7 consecutive days; Control group: For BV patients, metronidazole (produced by Heilongjiang Jiren Pharmaceutical Co., Ltd., each capsule contains 0.5 g of metronidazole) was placed deep in the vagina 1 capsule every night for 7 consecutive days. For RVVC patients, Daktarin suppository (produced by Janssen Pharmaceutical Co., Ltd., each suppository contains 0.2 g of miconazole nitrate) was placed deep in the vagina 1 suppository every night for 7 consecutive days. The treatment effects and recurrence rates were observed 7 days, 1 month, and 2 months after stopping the drug. The results are shown in Tables VII and VIII.

[0044] As can be seen from Table VII, the total effective rate of the treatment group for recurrent BV was 98%, and that of the control group was 98%; the total effective rate of the treatment group for RVVC was 94%, and that of the control group was 96%. There was no significant difference in the treatment effects between the two groups.

[0045] As can be seen from Table VIII, the recurrence rates of the treatment group for recurrent BV at 1 month and 2 months were 2% and 8% respectively, and those of the control group at 1 month and 2 months were 30% and 36% respectively. The recurrence rates of the treatment group for RVVC at 1 month and 2 months were 4% and 10% respectively, and those of the control group at 1 month and 2 months were 32% and 34% respectively. There were significant differences between the two groups.

[0046] The microecological preparation prepared by the present invention can be used to treat BV and VVC and reduce the recurrence rates of these two vaginal diseases, which is an invention with great social benefits.

Claims

1. A Lactococcus lactis subsp. lactis KF-22 derived from kefir, which can produce lactic acid, with a pH of 4.16 at the end of fermentation, and can produce 23.7 μg / mL of hydrogen peroxide, can be used as an active ingredient in sanitary products and drugs for treating bacterial and mycotic vaginitis and regulating the vaginal microecological balance.

2. A microecological preparation made from a mixture of Lactococcus lactis subsp. lactis KF-22, Lactobacillus acidophilus, Weissella confusa, Lactobacillus casei, Lactobacillus helveticus and Lactiplantibacillus plantarum in a ratio of 5:3:3:3:3:3, with these six probiotics accounting for 0.1% - 1% in total, 25 - 30% starch, 8 - 10% sodium carboxymethylcellulose, 55 - 60% microcrystalline cellulose, and 0.3 - 0.6% magnesium stearate / silica dioxide, can be used for inhibiting bacteria on the vaginal mucosa.

3. In the present invention, only the optimal probiotic ratio is selected for the probiotic combination, but the microecological preparation combination is not limited to these six probiotics, and can also be one or several combinations within the scope of the "List of Strains That Can Be Used in Foods" issued by the National Health Commission.

4. The active ingredient of the present invention is a probiotic combination preparation, and the others are excipients, including one or more of fillers, binders, disintegrants, flavoring agents and lubricants, including but not limited to these several used in the present invention.

5. The dosage forms of the microecological preparation made from the present probiotic as the active ingredient include granules, powders, tablets, pills, suppositories or oral liquids, etc.

6. The microecological preparation in the present invention has inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida albicans.

7. The microecological preparation in the present invention has no irritation or slight irritation to the vaginal mucosa and can be used as an external bacteriostatic agent for the vaginal mucosa.

8. The microecological preparation in the present invention has been verified through clinical trials and can be used for the treatment of bacterial and mycotic vaginitis.

9. The microecological preparation prepared by the present invention is stored at room temperature and can maintain the viable bacteria count of not less than 10 6 cfu / g and good antibacterial effect within the shelf life of 2 years.

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