Probiotic fermentation product capable of preventing and treating vaginitis as well as preparation and application of probiotic fermentation product

Abstract

The invention belongs to the technical field of biology. More specifically, the invention relates to a fermentation metabolite of clostridium butyricum and a preparation method thereof, and application of the fermentation metabolite in preventing and treating vaginitis. The preparation method of the probiotic fermentation product comprises the following steps: S1, inoculating clostridium butyricum into a basic culture medium containing callicarpa nudiflora polysaccharide and 2 '-fucosyllactose, and carrying out anaerobic fermentation; and S2, collecting the fermentation supernatant, filtering, and concentrating. An in-vitro bacteriostatic test result shows that the probiotic fermentation product provided by the invention has high bacteriostatic activity on candida albicans, staphylococcus aureus and escherichia coli.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to a fermentation metabolite of Clostridium butyricum, a preparation method thereof, and a use thereof in preventing and treating vaginitis. Background Art

[0002] The vaginal microecological environment is a unique and dynamic system, and its destruction will cause a series of problems such as vaginal inflammation. The vaginal microecological environment is affected by many factors, including anatomical structure, microbial flora, local immunity, and endocrine regulation. At present, the treatment trend of vaginal infection has gradually shifted from simply killing various pathogenic microorganisms to treat vaginal inflammation to restoring the vaginal defense function and rebuilding the vaginal microecology. Among them, the establishment of the dominant flora in the vagina is very important for rebuilding the vaginal microecology, and it is also the most widely used method of restoring the vaginal microecological environment.

[0003] Clostridium butyricum, also known as Clostridium butyricum and Clostridium butyricum, is a strictly anaerobic Gram-positive Clostridium that produces acid and gas during anaerobic culture. It mainly exists in the intestines of humans and animals, as well as their feces, sludge, natural yogurt and cheese. It has multiple physiological functions such as regulating the balance of intestinal flora, enhancing the body's immunity, and producing prebiotics. Clostridium butyricum has the effect of promoting the growth of beneficial intestinal bacteria and inhibiting pathogenic bacteria. It can effectively increase the level of immunoglobulins in animal serum and plays an important role in normalizing the host's intestinal flora and improving intestinal health. At present, research on Clostridium butyricum mainly focuses on its effects on animal growth performance and the prevention and treatment of intestinal diseases. For example, Chen Junchao [1] et al. studied the probiotic properties of the metabolites of Clostridium butyricum and found that the fermentation supernatant of Clostridium butyricum had strong tolerance to the simulated digestive tract environment and bile salts. After being treated in the simulated digestive tract environment for 2.5 hours, its survival rate was still as high as more than 90%, and it could tolerate bile salts with a concentration of less than 0.4%; its hydrophobicity and 12h self-aggregation rate were both above 30%; its fermentation supernatant had a significant antibacterial effect on Shigella flexneri (P<0.05), had a certain antibacterial effect on Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes, and had no antibacterial effect on Staphylococcus aureus. The extracellular metabolites of Clostridium butyricum mainly include 12 organic acids, 7 amino acids, 5 nucleosides and 7 other substances. Some active metabolites have certain probiotic effects and potential for application in food production. However, there is no study on the use of its metabolites in the treatment of vaginal inflammation and the effects on the reconstruction of the human vaginal microecological environment.

[0004] 【1】Chen Junchao, Fu Junjie, Liu Jun, et al. Probiotic properties of Clostridium butyricum and its active metabolites[J]. Food Research and Development, 2023, 44(23): 145-152. Summary of the invention

[0005] The present invention provides an extracellular metabolite of Clostridium butyricum that can prevent and treat vaginitis. The metabolite exhibits more excellent antibacterial activity and can promote the restoration of balance of the vaginal microecological environment, which has more advantages than antibiotic medication.

[0006] The object of the present invention is to provide a method for preparing a probiotic fermentation product, comprising the following steps:

[0007] S1, inoculating Clostridium butyricum in a basic medium containing Callicarpa nudiflora polysaccharide and 2'-fucosyllactose for anaerobic fermentation;

[0008] S2. Collect the fermentation supernatant, filter and concentrate to obtain the product.

[0009] In some embodiments of the present invention, the basic culture medium is RCM culture medium. The RCM culture medium described herein specifically comprises: beef extract powder 10.00 g / L, peptone 10.00 g / L, yeast extract powder 3.00 g / L, soluble starch 1.00 g / L, glucose 5.00 g / L, sodium acetate 3.00 g / L, sodium chloride 5.00 g / L, L-cysteine ​​hydrochloride 0.50 g / L, agar 0.15 g / L, pH 6.8±0.1.

[0010] In some embodiments of the present invention, the concentration of Callicarpa nudiflora polysaccharide in the basal culture medium is 1 to 10 mg / L.

[0011] In some embodiments of the present invention, the concentration of 2'-fucosyllactose is 5 to 30 mg / L. 2'-fucosyllactose is the oligosaccharide with the highest content in human milk, and has antiviral and immunomodulatory functions. The present invention has proved through some experiments that the antibacterial activity of the metabolites obtained by fermentation with the addition of 2'-fucosyllactose is significantly improved compared with that without the addition, and the difference is statistically significant (P < 0.05).

[0012] In some embodiments of the present invention, the Callicarpa nudiflora polysaccharide is prepared by the following steps:

[0013] Dry and crush the leaves of Callicarpa nudiflora, add 80-95% ethanol solution 10-15 times of its weight, extract 1-3 times, each time for 3-5 hours, filter and collect the residue;

[0014] Add 10 to 15 times the weight of water at 60 to 80°C to the residue, extract 1 to 3 times, each time for 1 to 3 hours, filter, combine the filtrate, and concentrate to obtain a concentrated solution;

[0015] An equal amount of 80-95% ethanol solution is added to the concentrated solution, and the solution is incubated at 4°C overnight, and the precipitate is collected to obtain a crude polysaccharide; the crude polysaccharide is dissolved in water and dialyzed for 24-48 hours, the dialyzate is passed through a Sephadex G-100 dextran gel column, eluted with water, and the sugar-containing components are collected to obtain Callicarpa nudiflora polysaccharide.

[0016] The present invention has proved through some experiments that the Clostridium butyricum metabolite obtained by fermentation with Callicarpa nudiflora polysaccharide and 2'-fucosyllactose has higher antibacterial activity than the product of adding only one of them, and the average inhibition zone diameters for Candida albicans, Staphylococcus aureus and Escherichia coli are 23.5 mm, 21.0 mm and 22.75 mm, respectively, and the antibacterial activity is significantly higher than that of the fermentation products obtained under the same fermentation conditions by Lactobacillus acidophilus K43, Lactobacillus helveticus, Lactobacillus salivarius and Lactobacillus crispatus.

[0017] In some embodiments of the present invention, the conditions of the anaerobic fermentation are: a temperature of 32 to 37° C. and a fermentation time of 12 to 48 hours.

[0018] In some embodiments of the present invention, the inoculation amount of Clostridium butyricum is 1-5%.

[0019] Another object of the present invention is to provide a probiotic fermentation product prepared by the preparation method.

[0020] Another object of the present invention is to provide the use of the probiotic fermentation product in preparing products for preventing and treating vaginitis.

[0021] In some embodiments of the present invention, the product is prepared in the form of a gel, a lotion, a suppository, an ointment, a spray or a powder.

[0022] The present invention has the following beneficial effects:

[0023] The results of in vitro antibacterial tests and clinical trials show that the probiotic fermentation product provided by the present invention has high antibacterial activity against Candida albicans, Staphylococcus aureus and Escherichia coli, and can promote the restoration of the balance of the vaginal microecological environment. It can be used for candidal vaginitis and has more advantages than antibiotics in clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To compare the antibacterial activities of different probiotic fermentation products against vaginal pathogens. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0026] Preparation of Callicarpa nudiflora polysaccharide:

[0027] The leaves of Callicarpa nudiflora are dried and crushed, and a 95% ethanol solution in an amount of 10 times the weight thereof is added, and the leaves are soaked for 3 times, each time for 3 hours, and the leaves are filtered and the residue is collected; water in an amount of 80°C in an amount of 10 times the weight thereof is added to the residue, and the leaves are soaked for 3 times, each time for 2 hours, and the residue is filtered, the filtrate is combined, and the filtrate is concentrated to obtain a concentrated solution; an equal amount of 95% ethanol solution is added to the concentrated solution, and the solution is incubated at 4°C overnight, and the precipitate is collected to obtain a crude polysaccharide; the crude polysaccharide is dissolved in water and then dialyzed for 48 hours, and the dialyzate is passed through a Sephadex G-100 polysaccharide gel column, and the solution is eluted with water, and the sugar-containing component is collected to obtain Callicarpa nudiflora polysaccharide.

[0028] Test Example 1: Comparison of antibacterial activity of fermentation products of different strains

[0029] This part studies the effect of different bacterial strains on the performance of fermentation products in the same fermentation substrate. The strains used are shown in Table 1 below. The strains in the table are all purchased commercially. The specific detection methods are as follows.

[0030] 1.1 Activation of strains: In a clean bench, the strains were inoculated into the corresponding proliferation medium at an inoculum volume of 2%, wherein Clostridium butyricum was cultured in RCM medium (beef extract powder 10.00 g / L, peptone 10.00 g / L, yeast extract powder 3.00 g / L, soluble starch 1.00 g / L, glucose 5.00 g / L, sodium acetate 3.00 g / L, sodium chloride 5.00 g / L, L-cysteine ​​hydrochloride 0.50 g / L, agar 0.15 g / L, pH 6.8 ± 0.1, water as solvent), and cultured anaerobically at 37°C for 12 h. The remaining strains were cultured in an incubator at 37°C for 12 h using MRS and activated for 2 generations in succession for subsequent experiments.

[0031] 1.2 Fermentation culture of Clostridium butyricum: 15 mg / L 2'-fucosyllactose and 3 mg / L Callicarpa nudiflora polysaccharide were added to RCM medium to prepare a fortified medium, 2% Clostridium butyricum was inoculated into the fortified medium, and anaerobically fermented at 37°C for 24 h. The fermentation liquid was centrifuged at 8000 r / min for 10 min to discard the bacteria, and the supernatant was filtered through a 0.22 μm microporous filter membrane. The filtrate was rotary evaporated to 1 / 3 of the original volume to obtain the fermentation liquid, which was stored for later use. The fermentation culture of other strains refers to the above, except that MRS is used instead of RCM, and the culture is carried out in an incubator at 37°C for 24 hours; the composition of MRS medium: casein peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, sodium acetate 5g / L, diammonium citrate 2g / L, Tween-801g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate heptahydrate 0.05g / L, calcium carbonate 20g / L, 2.0% agar, pH 6.8, and water as solvent.

[0032] Table 1 Microbial sources

[0033] Strain name Commercial sources Clostridium butyricum Guangdong Microbiological Culture Collection Center (GDMCC1.676) Lactobacillus acidophilus K43 Guangdong Yikewei Biotechnology Co., Ltd. Lactobacillus helveticus China General Microbiological Culture Collection Center, CGMCC1.9090 Lactobacillus salivarius Guangdong Microbiological Culture Collection Center (GDMCC1.986) Lactobacillus crispatus Mall Beina Chuanglian Biotechnology Co., Ltd., item number: BNCC319959 Candida albicans Beijing Sanyao Technology Co., Ltd., CMCC(F)98001 Staphylococcus aureus Beijing Sanyao Technology Co., Ltd., CMCC(B)26003 Escherichia coli Beijing Sanyao Technology Co., Ltd., CMCC(B)44102

[0034] 1.3 Antibacterial test: The antibacterial test was carried out by double-layer agar diffusion method. 10 mL of 1.8% solid water agar medium was poured into a sterile plate. After cooling and solidification, an appropriate amount of sterile Oxford cups were evenly placed. 5 mL of vaginal pathogen suspension (1×10 7 CFU / mL) of LB medium, pour it on the bottom water agar medium, wait for it to solidify, remove the Oxford cup to form holes; add 100μL of fermentation concentrate with different treatments to each group of holes, diffuse at 4℃ for 30min, and culture at 37℃ for 8h. Observe whether there is an antibacterial zone around the culture well and measure its diameter with a vernier caliper, record the effective diameter (i.e., excluding the size of the Oxford cup itself, where the specifications of the Oxford cup are 6.0mm inner diameter and 7.8mm outer diameter), the results are as follows Figure 1 shown.

[0035] from Figure 1 It can be seen that the fermentation products of Clostridium butyricum have the highest antibacterial activity against vaginal pathogens Candida albicans, Staphylococcus aureus and Escherichia coli, with average inhibition zone diameters of 23.5 mm, 21.0 mm and 22.75 mm, respectively. The antibacterial activity is significantly stronger than that of Lactobacillus fermentation products.

[0036] Experimental Example 2: Comparison of antibacterial activity of Clostridium butyricum fermentation products prepared under different fermentation conditions

[0037] This part studies the effect of adding or not adding 2'-fucosyllactose and / or Callicarpa nudiflora polysaccharide in RCM culture on the activity of Clostridium butyricum fermentation products. The antibacterial activity of the fermentation substrates was compared with reference to Experiment 1.3. The results are shown in Table 2 below.

[0038] Experimental group A1: the fermentation medium was RCM medium;

[0039] Experiment A2 group: fermentation medium was RCM + 15 mg / L 2'-FL;

[0040] Experiment A3 group: fermentation medium was RCM + 3 mg / L Callicarpa nudiflora polysaccharide;

[0041] Experiment A4 group: fermentation medium was RCM + 15 mg / L 2'-FL + 3 mg / L Callicarpa nudiflora polysaccharide;

[0042] Experiment A5 group: the fermentation medium was RCM+15 mg / L 2'-FL+3 mg / L Callicarpa nudiflora extract.

[0043] The preparation of Callicarpa nudiflora extract refers to the extraction method of Example 1 in CN 111671827 A.

[0044] Table 2 Comparison of the diameters of inhibition zones of different fermentation products (mm, )

[0045]

[0046]

[0047] Note: Compared with group A1, * P<0.05, ** P<0.01, *** P<0.001; compared with group A4, △△ P<0.01, △△△ P<0.001.

[0048] As can be seen from the above table, adding 2'-FL or Callicarpa nudiflora polysaccharide to fermentation can improve the antibacterial activity of the product to a certain extent, but the antibacterial activity of the fermentation product with the addition of 2'-FL or Callicarpa nudiflora polysaccharide alone is lower than that with the simultaneous addition of 2'-FL and Callicarpa nudiflora polysaccharide, and the difference is statistically significant (P < 0.01).

[0049] Example 1, Preparation of Clostridium butyricum fermentation broth:

[0050] Strain activation: Clostridium butyricum was inoculated into RCM medium (beef extract powder 10.00 g / L, peptone 10.00 g / L, yeast extract powder 3.00 g / L, soluble starch 1.00 g / L, glucose 5.00 g / L, sodium acetate 3.00 g / L, sodium chloride 5.00 g / L, L-cysteine ​​hydrochloride 0.50 g / L, agar 0.15 g / L, pH 6.8 ± 0.1, water as solvent) at an inoculum size of 2%, anaerobically cultured at 37°C for 12 h, and activated for 2 generations;

[0051] Fermentation culture of Clostridium butyricum: 15 mg / L 2'-fucosyllactose and 3 mg / L Callicarpa nudiflora polysaccharide were added to RCM medium to prepare a fortified medium, 2% activated Clostridium butyricum was inoculated into the fortified medium, and anaerobically fermented at 37°C for 24 hours. The fermentation liquid was centrifuged at 8000 r / min for 10 minutes to discard the bacteria, and the supernatant was filtered through a 0.22 μm microporous filter membrane. The filtrate was rotary evaporated to 1 / 3 of the original volume to obtain a fermentation liquid.

[0052] Example 2 is different from Example 1 in that 10 mg / L 2'-fucosyllactose and 5 mg / L Callicarpa nudiflora polysaccharide are added to the fortified culture medium, and the other parameters are the same.

[0053] Example 3 is different from Example 1 in that 5 mg / L 2'-fucosyllactose and 10 mg / L Callicarpa nudiflora polysaccharide are added to the fortified culture medium, and the other parameters are the same.

[0054] Examples 4 to 6: A gynecological wash (mass fraction)

[0055] Components Example 4 Example 5 Example 6 Clostridium butyricum fermentation broth Example 1, 10% Example 2, 10% Example 3, 10% Cocamidopropyl Betaine 3.0％ 3.0％ 3.0％ Hyaluronic acid 0.5％ 0.5％ 0.5％ Tetrasodium EDTA 0.1％ 0.1％ 0.1％ Deionized water margin margin margin

[0056] Preparation process: weigh a certain amount of deionized water, add tetrasodium ethylenediaminetetraacetic acid and hyaluronic acid, stir to dissolve, add cocamidopropyl betaine, stir to fully dissolve, then add Clostridium butyricum fermentation liquid, stir evenly to obtain the product.

[0057] Comparative Example 1 is different from Example 4 in that, during the preparation of Clostridium butyricum fermentation broth, the fermentation culture step adopts RCM medium, and the other parameters are the same.

[0058] Comparative Example 2 is different from Example 4 in that, during the preparation of Clostridium butyricum fermentation broth, only 15 mg / L of 2'-fucosyllactose is added to the fortified culture medium, and the other parameters are the same.

[0059] Comparative Example 3 is different from Example 4 in that, during the preparation of the Clostridium butyricum fermentation broth, only 3 mg / L of Callicarpa nudiflora polysaccharide was added to the fortified culture medium, and the other parameters were the same.

[0060] Test 3: Evaluation of vaginal mucosal irritation

[0061] According to the "Technical Specifications for Disinfection", the sample stock solutions of Example 4 and Comparative Examples 1 to 3 were used for the test: New Zealand rabbits were randomly divided into 5 groups, 3 in each group, and were named blank control group (normal saline), Example 4 and Comparative Examples 1 to 3. There was no secretion, congestion or other damage to the vaginal opening of each group of animals. The blunt hose (catheter) was connected to a 5mL syringe. The experimental rabbit was fixed on its back to expose the perineum and vaginal opening. The catheter was moistened with the test solution and gently inserted into the vagina 4-5cm, and 2mL of the test solution was slowly injected with a syringe, and the catheter was gently withdrawn. The control group was treated with normal saline in the same way. After 24h, the animals were killed by gas embolism, the complete vagina was taken out and cut longitudinally, and the naked eye was observed for macroscopic lesions such as bleeding and edema. The two ends and the middle three parts of the vagina were selected for routine paraffin embedding and sectioning. After HE staining, histopathological observation was performed. According to the results of pathological examination, the stimulation reaction of the vaginal mucosa was scored, the average integral of the stimulation reaction of the vaginal mucosa was calculated, the stimulation index was obtained, and the stimulation intensity was graded. The test results are shown in Table 3 below.

[0062] Table 3 Evaluation of vaginal mucosal irritation

[0063] Group Average score Stimulation Index Example 4 1.35 0.35 Comparative Example 1 2.72 1.32 Comparative Example 2 1.67 0.67 Comparative Example 3 2.46 1.16

[0064] Stimulation index = average score of experimental group - average score of control group

[0065] It can be seen from the above table that the irritation intensity grading standard of the gynecological wash of Example 1 and Comparative Example 2 to the vaginal mucosa is non-irritating, and Comparative Example 1 and Comparative Example 3 are extremely mildly irritating. Analysis of the overall test results shows that adding 2'-fucosyllactose during fermentation can reduce the irritation of the fermentation liquid to the vaginal mucosa, while adding Callicarpa nudiflora polysaccharide does not have the above effect.

[0066] Experiment 4: Treatment of fungal vaginitis

[0067] 4.1 General data: 150 patients with candidal vaginitis were selected and divided into a positive control group (clotrimazole vaginal tablets, produced by Zhejiang Suken Pharmaceutical Co., Ltd., specification: 0.5g), Example 1 and comparative examples 1 to 3 according to the random number expression method, with 30 cases in each group. There was no significant difference in the general data of each group. Inclusion criteria: ① meet the diagnostic criteria for candidal vaginitis; ② no sexual life in the past 2 weeks; ③ no history of antibiotic use in the past 4 weeks, and no guided irrigation treatment; Exclusion criteria: ① patients with other gynecological diseases; ② patients with other serious diseases; ③ patients who are allergic to the drugs in this study.

[0068] 4.2 Treatment methods: Medication method for the clotrimazole group: the patient should wash the vulva before going to bed, then take a clotrimazole vaginal tablet and insert it deep into the vagina, once a day, stop taking the medicine after 1 day, and take the medicine again after 4 days, for a total of 2 times; Medication method for the experimental group: first wipe the vagina with a sterilized cotton ball to remove secretions, then take 10mL of this product, dilute it with 100mL of clean water, put it in a vaginal douche, and rinse the vulva and vagina, 2 times / d, and continue the medication for 7 days.

[0069] 4.3 Observation indicators: (1) The clinical symptoms of patients before and after treatment were evaluated using 7 indicators in the self-made TCM symptom score scale (leucorrhea with yellow and white color, fatigue and poor appetite, vulvar itching, vaginal redness, swelling and ulceration, urethral burning pain, yellow and scanty urine, red tongue with yellow greasy or thick fur). The score (10 points / item) was positively correlated with the severity of clinical symptoms. (2) Analysis of vaginal microecological recovery: An appropriate amount of vaginal secretions from patients were collected before and after treatment, and their pH value was tested. The vaginal microecological status was then observed using a transmission electron microscope. Evaluation criteria ① Normal: vaginal secretion pH 3.8-4.5, flora density and diversity are 10-999 bacteria / oil immersion mirror, catalase and sialidase are both negative, Nugent score ≤3 points, AV score ≤2 points; ② Imbalance: Gram-negative bacilli, cocci or miscellaneous bacteria are the dominant flora, flora density and diversity are 1-9 or ≥1000 bacteria / oil immersion mirror, vaginal secretion pH>4.5, Nugent score>3 points, sialidase and catalase are positive, AV score>2 points. (3) Inflammatory factor secretion level: 5 mL of fasting venous blood was drawn from the patients in the morning before and after treatment, and the blood was centrifuged at 3000 r / min for 10 min. The supernatant was collected and stored at low temperature for testing. The C-reactive protein (CRP) level was detected by transmission immunoturbidimetry using a HITACHI 7600 fully automatic biochemical analyzer and its supporting reagents. The procalcitonin (PCT) level was detected by electrochemiluminescence using a Roche ECL1010 fully automatic electrochemiluminescence analyzer and its supporting reagents.

[0070] 4.4 Efficacy evaluation criteria: Vaginal secretions were taken for microscopic examination before treatment and one week after drug withdrawal. Markedly effective: After treatment, the patient's accompanying symptoms such as vaginal itching basically disappeared, the total improvement of TCM syndrome score was ≥80%, and the microscopic examination result of vaginal secretions was negative; Effective: Most of the patient's accompanying symptoms were significantly relieved, and 20% ≤ the total improvement of TCM syndrome score was <80%; but the microscopic examination result of vaginal secretions was still positive, and the pseudohyphae was significantly reduced compared with before treatment; Ineffective: There was no significant improvement or aggravation of the accompanying symptoms, microscopic examination results, etc., and the total improvement of TCM syndrome score was <20%. Total effective rate = (markedly effective + effective) / total number of cases × 100%.

[0071] 4.5 Statistical analysis: SPSS24.0 was used to process the data. (χ±s) and (%) represent measurement and counting data. t value and χ2 test were used respectively. P<0.05 was considered statistically significant.

[0072] 5 Results

[0073] 5.1 Comparison of therapeutic efficacy: After treatment, the total efficacy of Example 4 group was significantly higher than that of the clotrimazole group, and the total efficacy of only adding 2'-fucosyllactose or Callicarpa nudiflora polysaccharide was also significantly lower than that of adding both at the same time.

[0074] Table 4 Comparison of clinical efficacy among groups

[0075] Group Significant effect efficient invalid Total Effectiveness Clotrimazole 8(26.67) 13(43.33) 9(30.00) 21(70.00) Example 4 21(70.00) 7(23.33) 2(6.67) 28(93.33) Comparative Example 1 0 4(13.33) 26(86.67) 6(13.33) Comparative Example 2 8(26.67) 8(26.67) 14(46.67) 16(53.33) Comparative Example 3 6(20.00) 7(23.33) 17(56.67) 13(43.33)

[0076] 5.2 Comparison of vaginal microecological recovery: There was no statistical difference in vaginal pH values ​​of each group before treatment (P>0.05). After treatment, the vaginal pH values ​​of each group were lower than those before treatment, among which the reduction in Example 4 group was significantly greater than that in the clotrimazole group, and the difference was statistically significant; and after treatment with Example 4, the number of patients with vaginal microecological environment restored to normal was the largest.

[0077] Table 5 Comparison of vaginal microecological recovery in each group (n=30, χ±s)

[0078]

[0079] Compared with Example 4, * P<0.05, ** P<0.01.

[0080] 5.3 Comparison of inflammatory factor levels: After treatment, the levels of C-reactive protein and procalcitonin in each group were significantly reduced compared with those before treatment, and the reduction in Example 4 group was the largest.

[0081] Table 6 Comparison of inflammatory factor levels in each group (n=30, χ±s)

[0082]

[0083] Compared with the same group before treatment, * P<0.05, ** P<0.01, *** P<0.001.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a probiotic fermentation product, characterized in that: The following steps are involved: S1. inoculating Clostridium butyricum into a basic medium containing Callicarpa nudiflora polysaccharide and 2'-fucosyllactose for anaerobic fermentation; S2. Collect the fermentation supernatant, filter and concentrate to obtain the product.

2. The method for preparing the probiotic fermentation product according to claim 1, characterized in that: The basic culture medium is RCM culture medium.

3. The method for preparing the probiotic fermentation product according to claim 1 or 2, characterized in that: In the basic culture medium, the concentration of Callicarpa nudiflora polysaccharide is 1-10 mg / L.

4. The method for preparing the probiotic fermentation product according to claim 1 or 2, characterized in that: The concentration of the 2'-fucosyllactose is 5-30 mg / L.

5. The method for preparing the probiotic fermentation product according to claim 1, characterized in that: The conditions of the anaerobic fermentation are: temperature of 32-37° C., and fermentation time of 12-48 hours.

6. The preparation method according to claim 1, characterized in that: The inoculation amount of the Clostridium butyricum is 1-5%.

7. Use of the probiotic fermentation product according to any one of claims 1 to 6 in the preparation of a product for treating vaginitis.

8. A gynecological wash, characterized in that: The invention comprises a probiotic fermentation product prepared by the preparation method according to any one of claims 1 to 6.

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