Probiotic composition for treating vaginitis and preparation method thereof

Abstract

The invention provides a probiotic composition for treating vaginitis and a preparation method thereof, and belongs to the technical field of probiotics. The preparation method comprises the following steps: performing water extraction on cortex phellodendri, honeysuckle and cacumen biotae to obtain an extract, inoculating probiotics into medicine residues for fermentation, performing separation to obtain antibacterial peptide, bacterial sludge and a fermentation product, preparing an antibacterial peptide-polysaccharide compound from the antibacterial peptide, embedding the bacterial sludge through an acid-soluble shell layer to obtain an acid-soluble bacterium-loaded microcapsule, mixing the acid-soluble bacterium-loaded microcapsule with the antibacterial peptide-polysaccharide compound, the extract and prebiotics, adding the mixture into hydrogel, and performing freeze-drying to obtain a finished product. And preparing the probiotic composition for treating vaginitis. The prepared probiotic composition for treating vaginitis is good in treatment effect, good in storage stability, good in temperature resistance and moisture resistance, safe to use, free of side effects, good in biocompatibility, free of drug resistance, beneficial to treatment and prevention of relapse of vaginitis and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to a probiotic composition for treating vaginitis and a preparation method thereof. Background Art

[0002] Vaginal infection is the most common gynecological infectious disease in women of childbearing age, accounting for about 50% of gynecological outpatients, mainly including bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), Trichomonal vaginitis (TV) and aerobic vaginitis (AV). BV is caused by the reduction or lack of dominant lactobacilli in the vagina, the excessive proliferation of anaerobic bacteria (such as Gardnerella vaginalis, Mobiluncus, Prevotella and Bacteroides) and Mycoplasma hominis and other pathogenic bacteria. It can be transmitted through sex, with an infection rate of 15%-30%, accounting for 40%-50% of vaginal infections, and is the most common vaginitis. Gardnerella vaginalis is a recognized core pathogen of BV and can be detected in the vagina of 95%-100% of patients. Typical clinical symptoms of BV patients include thin vaginal discharge with a foul or fishy odor, local itching and burning sensation in the vulva, vaginal fluid pH higher than 4.5, and the presence of a large number of clue cells. Pathogenic bacteria associated with bacterial vaginosis include Gardnerella, Atopobium, Prevotella, Streptococcus, and Bacillus mobilis, which are related to the development of BV, whether they act alone or interact with each other.

[0003] Clinically, the treatment of vaginitis mainly relies on a combination of vaginal lavage and antibacterial drugs, mostly using imidazoles, clindamycin hydrochloride and compound preparations. However, the above drugs can cause many adverse reactions, leading to the gradual increase of drug resistance of pathogens, and have disadvantages such as high recurrence rate, imbalance of internal environment microecology and incomplete treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic composition for treating vaginitis and a preparation method thereof, which has good therapeutic effect, good storage stability, good temperature resistance and moisture resistance, is safe to use, has no side effects, has good biocompatibility, will not produce drug resistance, is beneficial to the treatment and prevention of recurrence of vaginitis, and has broad application prospects.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The invention provides a method for preparing a probiotic composition for treating vaginitis. The method comprises the following steps: extracting phellodendron, honeysuckle and platycladus orientalis leaves with water to obtain an extract, inoculating probiotics into the residues for fermentation, separating and obtaining antimicrobial peptides, bacterial mud and fermentation products, preparing antimicrobial peptide-polysaccharide complexes from the antimicrobial peptides, embedding the bacterial mud through an acid-soluble shell layer to obtain acid-soluble bacteria-loaded microcapsules, mixing the antimicrobial peptide-polysaccharide complexes, the extracts and prebiotics into a hydrogel to prepare the probiotic composition for treating vaginitis.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. Extraction of Chinese medicine: Wash Phellodendron chinense, honeysuckle and Platycladus orientalis leaves, add them to water, heat and boil to extract, filter to obtain Chinese medicine residue, dry the filtrate to obtain an extract;

[0009] S2. Preparation of culture medium: The Chinese medicine residue obtained in step S1, desalted whey powder, glucose, yeast extract powder were added to water and sterilized to obtain a culture medium;

[0010] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, activated and cultured to obtain bacterial seed liquid;

[0011] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the medium obtained in step S2, and the culture was fermented, filtered, and the bacterial sludge was collected. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10KDa, and freeze-dried to obtain antimicrobial peptides. The remaining liquid was added with ethanol for precipitation, filtered, washed, and dried to obtain a complex polysaccharide. The filtrate was recovered with ethanol, and freeze-dried to obtain a fermentation product.

[0012] S5. Preparation of antimicrobial peptide-polysaccharide complex: dissolving the antimicrobial peptide obtained in step S4 in water, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activating the reaction, adding the complex polysaccharide obtained in step S4, heating and stirring the reaction, dialyzing, and drying to obtain an antimicrobial peptide-polysaccharide complex;

[0013] S6. Preparation of acid-soluble bacteria-carrying microcapsules: carboxymethyl chitosan and gelatin are added to water, heated to dissolve, tannic acid, lecithin and the bacterial sludge obtained in step S4 are added, dispersed evenly, added dropwise to fish oil, emulsified, the pH value of the solution is adjusted, the reaction is stirred, and freeze-dried to obtain acid-soluble bacteria-carrying microcapsules;

[0014] S7. Preparation of a probiotic composition for treating vaginitis: add sodium alginate, inulin and arabinose into water, add citric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir to react, add silk fibroin peptide, heat and stir to react, then add the extract prepared in step S1, the fermentation product prepared in step S4, the antimicrobial peptide-polysaccharide complex prepared in step S5, and the acid-soluble bacteria-loaded microcapsule prepared in step S6, stir and mix evenly to prepare a probiotic composition for treating vaginitis.

[0015] As a further improvement of the present invention, the mass ratio of Phellodendron chinense, honeysuckle and Platycladus orientalis leaves in step S1 is 5-7:1-2:4-6, and the time of heating and boiling extraction is 3-5h; the mass ratio of Chinese medicine residue, desalted whey powder, glucose, yeast extract powder and water in step S2 is 15-20:5-7:10-12:3-5:200-300.

[0016] As a further improvement of the present invention, the activation culture conditions in step S3 are anaerobic conditions, 36-38°C, 100-200r / min, culture for 18-24h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

[0017] As a further improvement of the present invention, the inoculation amounts of the seed solutions of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 in step S4 are 1-2 v / v%, 2-4 v / v%, 2-3 v / v%, and 2-3 v / v%, respectively, and the fermentation and culture conditions are anaerobic conditions, 36-38° C., 100-200 r / min, and the fermentation and culture is 50-56 h.

[0018] As a further improvement of the present invention, the mass ratio of the antimicrobial peptide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and the complex polysaccharide in step S5 is 10-12:4-7:5-8:12-15, the temperature of the activation reaction is 0-4°C, the time is 30-40min, and the temperature of the heating and stirring reaction is 50-60°C, and the time is 4-6h.

[0019] As a further improvement of the present invention, the mass ratio of carboxymethyl chitosan, gelatin, tannic acid, lecithin, bacterial mud, water and fish oil in step S6 is 12-15:7-10:4-6:0.5-1:20-25:200-250:500, the pH value of the adjusted solution is 8.4-8.6, and the stirring reaction time is 30-60 min.

[0020] As a further improvement of the present invention, the mass ratio of sodium alginate, inulin, arabinose, citric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, silk fibroin peptide, extract, fermentation product, antimicrobial peptide-polysaccharide complex, and acid-soluble bacteria-loaded microcapsule in step S7 is 15-20:3-5:2-3:3-4:4-6:4-6:6-9:4-6:5-7:2-3:7-10, and the temperature of the heating and stirring reaction is 70-80°C, and the time is 4-5h.

[0021] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0022] S1. Chinese medicine extraction: 5-7 parts by weight of Phellodendron, 1-2 parts by weight of honeysuckle, 4-6 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 3-5h, filtered to obtain Chinese medicine residues, and the filtrate was dried to obtain an extract;

[0023] S2. Preparation of medium: 15-20 parts by weight of the Chinese medicine residue obtained in step S1, 5-7 parts by weight of desalted whey powder, 10-12 parts by weight of glucose, 3-5 parts by weight of yeast extract powder are added to 200-300 parts by weight of water and sterilized to obtain a medium;

[0024] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, and activated and cultured at 36-38°C for 18-24h under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0025] S4. Fermentation: The Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 obtained in step S3 were inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1-2v / v%, 2-4v / v%, 2-3v / v%, and 2-3v / v%, respectively, under anaerobic conditions, 36-38°C, 100-200r / min, and fermented for 50-56h, filtered, and the sludge was collected. The filtrate was ultrafiltrated to collect a solution with a molecular weight of 2-10KDa, and freeze-dried to obtain an antimicrobial peptide, and ethanol was added to the remaining liquid to a system ethanol content of 80-85wt%, precipitated for 1-2h, filtered, washed, and dried to obtain a complex polysaccharide, and the filtrate was recovered with ethanol, freeze-dried, and a fermentation product was obtained;

[0026] S5. Preparation of antimicrobial peptide-polysaccharide complex: dissolve 10-12 parts by weight of the antimicrobial peptide prepared in step S4 in 200 parts by weight of water, add 4-7 parts by weight of N-hydroxysuccinimide and 5-8 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activate the reaction, add 12-15 parts by weight of the complex polysaccharide prepared in step S4, heat to 50-60° C., stir to react for 4-6 hours, dialyze, and dry to obtain an antimicrobial peptide-polysaccharide complex;

[0027] S6. Preparation of acid-soluble bacteria-loaded microcapsules: 12-15 parts by weight of carboxymethyl chitosan, 7-10 parts by weight of gelatin are added to 200-250 parts by weight of water, heated to 50-60 ° C, stirred to dissolve, 4-6 parts by weight of tannic acid, 0.5-1 parts by weight of lecithin and 20-25 parts by weight of the bacterial mud obtained in step S4 are added, uniformly dispersed, added dropwise to 500 parts by weight of fish oil, emulsified, the pH value of the solution is adjusted to 8.4-8.6, stirred for 30-60 min, freeze-dried, and acid-soluble bacteria-loaded microcapsules are obtained;

[0028] S7. Preparation of a probiotic composition for treating vaginitis: add 15-20 parts by weight of sodium alginate, 3-5 parts by weight of inulin, and 2-3 parts by weight of arabinose to 200 parts by weight of water, add 3-4 parts by weight of citric acid, 4-6 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-6 parts by weight of N-hydroxysuccinimide, stir and react at 0-4°C for 20-40 minutes, add 6-9 parts by weight of silk fibroin peptide, heat to 70-80°C, stir and react for 4-5 hours, then add 4-6 parts by weight of the extract prepared in step S1, 5-7 parts by weight of the fermentation product prepared in step S4, 2-3 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 7-10 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix evenly, and freeze-dry to prepare a probiotic composition for treating vaginitis.

[0029] The present invention further protects a probiotic composition for treating vaginitis prepared by the above preparation method.

[0030] The present invention has the following beneficial effects:

[0031] Lactobacillus is the dominant bacteria in the vagina. It can maintain the balance of vaginal microecology through multiple mechanisms such as competitive adhesion, lowering vaginal pH, producing inhibitors such as hydrogen peroxide, bacteriocins and surfactants, and stimulating the local immune system, thereby facilitating the growth of Lactobacillus and inhibiting the growth of pathogens.

[0032] The probiotic composition added in the present invention directly acts on the vagina to exert a protective mechanism.

[0033] Among them, Lactobacillus rhamnosus LRYZU021 lowers the pH value of the vagina by metabolizing glycogen into lactic acid. Under normal circumstances, the pH value in the vagina is less than or equal to 4.5, and the low pH of the vagina can protect it from pathogens.

[0034] Lactobacillus reuteri FPHC2951 produces antimicrobial factors including hydrogen peroxide and bacteriocins, which are toxic to catalase-negative bacteria (such as most anaerobic microorganisms). Bacteriocins form pores in the cytoplasmic membrane, interfere with cellular enzyme reactions (such as cell wall synthesis) and nuclease activity, and can inhibit the growth of pathogens such as Staphylococcus aureus, Klebsiella and Escherichia coli.

[0035] Lactobacillus crispatus LCP051 can produce surfactants, including peptidoglycan, phosphatidic acid, bacterial polysaccharides and biosurfactants, which directly adhere to the pattern recognition receptors of epithelial cells and mucosal layers to mediate the host's physiological responses, and inhibit the adhesion and colonization of pathogens through anti-biofilm, antioxidant, antiviral, pathogen inhibition and immunomodulatory effects.

[0036] Lactobacillus gasseri LG019 and its metabolites stimulate the immune function of vaginal epithelial cells, inhibit the growth and reproduction of pathogenic bacteria by producing mucin, immunoglobulins (secretory IgA and IgG), secretory leukocyte protease inhibitors, neutrophil gelatinase-associated lipid deposition protein, interleukin-1 receptor antagonists, β-defensins and other antimicrobial proteins, and inhibit proinflammatory mediators such as IL-6, IL-8 and tumor necrosis factor α. Various lactobacilli have the ability to adhere to and compete for vaginal epithelial adhesion sites, participate in inhibiting the colonization of pathogens, and hinder the colonization of pathogens on the mucosa by occupying or masking (through spatial hindrance) their potential binding sites on the mucosa, thereby playing a role in treating vaginitis and improving the symptoms of vaginitis.

[0037] Different lactobacilli have different mucosal colonization or antagonistic microbial abilities. The four lactobacilli of the present invention act synergistically, can adhere to human epithelial cells, produce hydrogen peroxide, have antibacterial activity against vaginal Gardnerella and vulvovaginal Candida, and coaggregate with pathogens.

[0038] Phellodendron chinense, honeysuckle and Platycladus orientalis leaves contain good antibacterial flavonoids, triterpenes and other ingredients, which greatly improve the therapeutic effect of vaginitis caused by bacteria. Further fermentation of the Chinese medicine residues promotes the proliferation of probiotics and obtains fermentation products containing more antibacterial active substances.

[0039] Antimicrobial peptides with a molecular weight of 2-10 kDa were separated by ultrafiltration, and complex polysaccharides were separated from the remaining active products. The filtrate was freeze-dried to obtain the fermentation product, which also had good antimicrobial activity. The antimicrobial peptides were activated and coupled with the complex polysaccharides to obtain an antimicrobial peptide-polysaccharide complex, which significantly improved the antibacterial and antiviral properties. It can catalyze the virus to produce oxidative stress response, leading to the death of the virus, and also has a good killing effect on Trichomonas vaginalis.

[0040] The present invention reacts carboxymethyl chitosan, gelatin and tannic acid, tannic acid has a unique polyphenolic hydroxyl structure, and the gelatin molecular chain has rich amino acid residues, and multi-point hydrogen bonds can be formed between the two molecules; at the same time, the phenolic hydroxyl on the tannic acid is easily oxidized to quinone compounds, which can form Schiff base bonds with the amino groups of the gelatin segments. Since the Schiff base bonds formed are dynamic covalent bonds, they have pH responsiveness, so that the microcapsules can be stored intact in a neutral environment, and the survival rate of probiotics is improved. The microcapsules are added to the gel structure, and the structure of the gel can protect the microcapsules from passing through the stomach environment, so that the microcapsules can be broken in the acidic environment of the vagina, and the purpose of controlling the release of probiotics is achieved. Adding prebiotics to the gel raw material can promote the formation of gel on the one hand, and on the other hand, prebiotics can also provide raw materials for probiotics, promote the proliferation and growth of probiotics, help to inhibit pathogenic bacteria in the vaginal environment, and better play the effect of treating vaginitis.

[0041] The probiotic composition for treating vaginitis prepared by the present invention has good therapeutic effect, good storage stability, good temperature resistance and moisture resistance, is safe to use, has no side effects, has good biocompatibility, will not produce drug resistance, is beneficial to the treatment and prevention of recurrence of vaginitis, and has broad application prospects. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Lactobacillus rhamnosus LRYZU021 is classified and named as Lactobacillus rhamnosus LRYZU021, the preservation number is CCTCC NO.M2017640, the preservation date is October 30, 2017, and the preservation unit is China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, Postal Code: 430072.

[0044] Lactobacillus reuteri FPHC2951 is classified and named as Lactobacillus reuteri, the preservation number is GDMCC NO.62946, the preservation date is November 3, 2022, and the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code: 510070.

[0045] Lactobacillus crispatus LCP051 is classified and named as Lactobacillus crispatus, the preservation number is CGMCC NO.25132, the preservation date is June 20, 2022, and the preservation unit is the General Microbiology Center of China Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0046] Lactobacillus gasseri LG019 is classified and named Lactobacillus gasseri, with a preservation number of CGMCC NO.30508 and a preservation date of May 7, 2024. The preservation unit is the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101.

[0047] Example 1

[0048] This embodiment provides a method for preparing a probiotic composition for treating vaginitis, which specifically comprises the following steps:

[0049] S1. Chinese medicine extraction: 5 parts by weight of Phellodendron, 1 part by weight of honeysuckle, 4 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 3h, filtered to obtain Chinese medicine residue, and the filtrate was dried to obtain an extract;

[0050] S2. Preparation of medium: 15 parts by weight of the Chinese medicine residue obtained in step S1, 5 parts by weight of desalted whey powder, 10 parts by weight of glucose, 3 parts by weight of yeast extract powder were added to 200 parts by weight of water and sterilized to obtain a medium;

[0051] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, and activated and cultured for 18 hours at 36°C and 100 r / min under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0052] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1v / v%, 2v / v%, 2v / v% and 2v / v%, respectively, under anaerobic conditions, 36°C, 100r / min, fermented and cultured for 50h, filtered, the bacterial sludge was collected, the filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10KDa, freeze-dried, and antimicrobial peptides were obtained, ethanol was added to the remaining liquid to a system ethanol content of 80wt%, precipitated for 1h, filtered, washed, dried, and a complex polysaccharide was obtained, ethanol was recovered from the filtrate, freeze-dried, and a fermentation product was obtained;

[0053] S5. Preparation of antimicrobial peptide-polysaccharide complex: 10 parts by weight of the antimicrobial peptide prepared in step S4 was dissolved in 200 parts by weight of water, 4 parts by weight of N-hydroxysuccinimide and 5 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated. 12 parts by weight of the complex polysaccharide prepared in step S4 was added, and the mixture was heated to 50° C., stirred for reaction for 4 hours, dialyzed, and dried to obtain an antimicrobial peptide-polysaccharide complex.

[0054] S6. Preparation of acid-soluble bacteria-carrying microcapsules: 12 parts by weight of carboxymethyl chitosan and 7 parts by weight of gelatin were added to 200 parts by weight of water, heated to 50°C, stirred to dissolve, 4 parts by weight of tannic acid, 0.5 parts by weight of Tween-40 and 20 parts by weight of the bacterial mud obtained in step S4 were added, stirred and mixed for 20 minutes, added dropwise to 500 parts by weight of fish oil, emulsified at 10000 r / min for 10 minutes, the pH value of the solution was adjusted to 8.4, stirred for 30 minutes, and freeze-dried to obtain acid-soluble bacteria-carrying microcapsules;

[0055] S7. Preparation of a probiotic composition for treating vaginitis: add 15 parts by weight of sodium alginate, 3 parts by weight of inulin, and 2-3 parts by weight of arabinose to 200 parts by weight of water, add 3 parts by weight of citric acid, 4 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4 parts by weight of N-hydroxysuccinimide, stir and react at 0°C for 20 minutes, add 6 parts by weight of silk fibroin peptide, heat to 70°C, stir and react for 4 hours, then add 4 parts by weight of the extract prepared in step S1, 5 parts by weight of the fermentation product prepared in step S4, 2 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 7 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix for 20 minutes, and swell for 12 hours to prepare a probiotic composition for treating vaginitis.

[0056] Example 2

[0057] This embodiment provides a method for preparing a probiotic composition for treating vaginitis, which specifically comprises the following steps:

[0058] S1. Chinese medicine extraction: 7 parts by weight of Phellodendron, 2 parts by weight of honeysuckle, 6 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 5h, filtered to obtain Chinese medicine residue, and the filtrate was dried to obtain an extract;

[0059] S2. Preparation of medium: 20 parts by weight of the Chinese medicine residue obtained in step S1, 7 parts by weight of desalted whey powder, 12 parts by weight of glucose, 5 parts by weight of yeast extract powder were added to 300 parts by weight of water and sterilized to obtain a medium;

[0060] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, and activated and cultured for 24 hours at 38°C and 200 r / min under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0061] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the culture medium obtained in step S2, and the inoculation amounts were 2v / v%, 4v / v%, 3v / v% and 3v / v%, respectively, under anaerobic conditions, 38°C, 200r / min, fermented for 56h, filtered, and the sludge was collected. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10KDa, freeze-dried, and antimicrobial peptides were obtained. Ethanol was added to the remaining liquid until the ethanol content of the system was 85wt%, precipitated for 2h, filtered, washed, and dried to obtain a complex polysaccharide, and the filtrate was recovered with ethanol, freeze-dried, and a fermentation product was obtained;

[0062] S5. Preparation of antimicrobial peptide-polysaccharide complex: 12 parts by weight of the antimicrobial peptide prepared in step S4 was dissolved in 200 parts by weight of water, 7 parts by weight of N-hydroxysuccinimide and 8 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated. 15 parts by weight of the complex polysaccharide prepared in step S4 was added, and the mixture was heated to 60° C., stirred for reaction for 6 hours, dialyzed, and dried to obtain an antimicrobial peptide-polysaccharide complex;

[0063] S6. Preparation of acid-soluble bacteria-carrying microcapsules: 15 parts by weight of carboxymethyl chitosan and 10 parts by weight of gelatin were added to 250 parts by weight of water, heated to 60 ° C, stirred to dissolve, 6 parts by weight of tannic acid, 1 part by weight of Tween-40 and 25 parts by weight of the bacterial mud obtained in step S4 were added, stirred and mixed for 20 minutes, added dropwise to 500 parts by weight of fish oil, emulsified at 10000r / min for 10 minutes, the pH value of the solution was adjusted to 8.6, stirred for 60 minutes, and freeze-dried to obtain acid-soluble bacteria-carrying microcapsules;

[0064] S7. Preparation of a probiotic composition for treating vaginitis: add 20 parts by weight of sodium alginate, 5 parts by weight of inulin, and 3 parts by weight of arabinose to 200 parts by weight of water, add 4 parts by weight of citric acid, 6 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-6 parts by weight of N-hydroxysuccinimide, stir and react at 4°C for 40 minutes, add 9 parts by weight of silk fibroin peptide, heat to 80°C, stir and react for 5 hours, then add 6 parts by weight of the extract prepared in step S1, 7 parts by weight of the fermentation product prepared in step S4, 3 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 10 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix for 20 minutes, and swell for 15 hours to prepare a probiotic composition for treating vaginitis.

[0065] Example 3

[0066] This embodiment provides a method for preparing a probiotic composition for treating vaginitis, which specifically comprises the following steps:

[0067] S1. Chinese medicine extraction: 6 parts by weight of Phellodendron, 1.5 parts by weight of honeysuckle, 5 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 4h, filtered to obtain Chinese medicine residue, and the filtrate was dried to obtain an extract;

[0068] S2. Preparation of medium: 17 parts by weight of the Chinese medicine residue obtained in step S1, 6 parts by weight of desalted whey powder, 11 parts by weight of glucose, 4 parts by weight of yeast extract powder were added to 250 parts by weight of water and sterilized to obtain a medium;

[0069] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium respectively, and activated and cultured for 21 hours at 37°C and 150 r / min under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0070] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1.5 v / v%, 3 v / v%, 2.5 v / v% and 2.5 v / v%, respectively, under anaerobic conditions, 37 ° C, 150 r / min, fermented and cultured for 53 h, filtered, the bacterial mud was collected, the filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10 KDa, freeze-dried, and antimicrobial peptides were obtained, ethanol was added to the remaining liquid to a system ethanol content of 82 wt%, precipitated for 1.5 h, filtered, washed, dried, and a complex polysaccharide was obtained, and the filtrate was recovered with ethanol, freeze-dried, and a fermentation product was obtained;

[0071] S5. Preparation of antimicrobial peptide-polysaccharide complex: 11 parts by weight of the antimicrobial peptide prepared in step S4 were dissolved in 200 parts by weight of water, 5 parts by weight of N-hydroxysuccinimide and 6 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated. 13 parts by weight of the complex polysaccharide prepared in step S4 were added, and the mixture was heated to 55° C., stirred for reaction for 5 h, dialyzed, and dried to obtain an antimicrobial peptide-polysaccharide complex.

[0072] S6. Preparation of acid-soluble bacteria-loaded microcapsules: 13.5 parts by weight of carboxymethyl chitosan and 8.5 parts by weight of gelatin were added to 230 parts by weight of water, heated to 55 ° C, stirred to dissolve, 5 parts by weight of tannic acid, 0.7 parts by weight of Tween-80 and 22 parts by weight of the bacterial mud obtained in step S4 were added, stirred and mixed for 20 minutes, added dropwise to 500 parts by weight of fish oil, emulsified at 10000 r / min for 10 minutes, the pH value of the solution was adjusted to 8.5, stirred for 45 minutes, and freeze-dried to obtain acid-soluble bacteria-loaded microcapsules;

[0073] S7. Preparation of a probiotic composition for treating vaginitis: add 17 parts by weight of sodium alginate, 4 parts by weight of inulin, and 2.5 parts by weight of arabinose to 200 parts by weight of water, add 3.5 parts by weight of citric acid, 5 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5 parts by weight of N-hydroxysuccinimide, stir and react at 2°C for 30 minutes, add 7.5 parts by weight of silk fibroin peptide, heat to 75°C, stir and react for 4.5 hours, then add 5 parts by weight of the extract prepared in step S1, 6 parts by weight of the fermentation product prepared in step S4, 2.5 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 8.5 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix for 20 minutes, and swell for 13 hours to prepare a probiotic composition for treating vaginitis.

[0074] Comparative Example 1

[0075] Compared with Example 3, the difference is that Lactobacillus rhamnosus LRYZU021 is not inoculated in step S4.

[0076] The details are as follows:

[0077] S4. Fermentation: The Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 obtained in step S3 were inoculated into the culture medium obtained in step S2, with the inoculation amounts being 3 v / v%, 2.5 v / v%, and 2.5 v / v%, respectively; under anaerobic conditions, at 37°C, 150 r / min, the fermentation was cultured for 53 h, filtered, the bacterial sludge was collected, the filtrate was ultrafiltrated to collect a solution with a molecular weight of 2-10 KDa, and freeze-dried to obtain antimicrobial peptides; the remaining liquid was freeze-dried to obtain a fermentation product.

[0078] Comparative Example 2

[0079] Compared with Example 3, the difference is that Lactobacillus reuteri FPHC2951 is not inoculated in step S4.

[0080] The details are as follows:

[0081] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 prepared in step S3 was inoculated into the culture medium prepared in step S2, with the inoculum amount being 1.5 v / v%, 2.5 v / v% and 2.5 v / v%, respectively. Under anaerobic conditions, the temperature was 37°C and 150 r / min, and the fermentation was carried out for 53 h. The culture was filtered and the bacterial sludge was collected. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10 KDa, and freeze-dried to obtain antimicrobial peptides. Ethanol was added to the remaining liquid until the ethanol content of the system was 82 wt%, and the mixture was precipitated for 1.5 h. The mixture was filtered, washed and dried to obtain a complex polysaccharide. The filtrate was used to recover ethanol, and freeze-dried to obtain a fermentation product.

[0082] Comparative Example 3

[0083] Compared with Example 3, the difference is that Lactobacillus crispatus LCP051 is not inoculated in step S4.

[0084] The details are as follows:

[0085] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951 and Lactobacillus gasseri LG019 prepared in step S3 was inoculated into the culture medium prepared in step S2, with the inoculation amount being 1.5 v / v%, 3 v / v% and 2.5 v / v%, respectively. Under anaerobic conditions, at 37°C and 150 r / min, the fermentation culture was carried out for 53 hours, and the culture was filtered to collect the bacterial sludge. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10 KDa, and freeze-dried to obtain antimicrobial peptides. Ethanol was added to the remaining liquid until the ethanol content of the system was 82 wt%, and the mixture was precipitated for 1.5 hours. The mixture was filtered, washed and dried to obtain a complex polysaccharide. The filtrate was used to recover ethanol, and freeze-dried to obtain a fermentation product.

[0086] Comparative Example 4

[0087] Compared with Example 3, the difference is that Lactobacillus gasseri LG019 is not inoculated in step S4.

[0088] The details are as follows:

[0089] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951 and Lactobacillus crispatus LCP051 obtained in step S3 was inoculated into the culture medium obtained in step S2, with the inoculation amounts being 1.5 v / v%, 3 v / v% and 2.5 v / v%, respectively. Under anaerobic conditions, the temperature was 37°C and 150 r / min, and the fermentation was carried out for 53 h. The culture was filtered and the bacterial sludge was collected. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10 KDa, and the solution was freeze-dried to obtain antimicrobial peptides. Ethanol was added to the remaining liquid until the ethanol content of the system was 82 wt%, and the mixture was precipitated for 1.5 h. The mixture was filtered, washed and dried to obtain a complex polysaccharide. The filtrate was used to recover ethanol, and the solution was freeze-dried to obtain a fermentation product.

[0090] Comparative Example 5

[0091] Compared with Embodiment 3, the difference is that step S5 is not performed.

[0092] The details are as follows:

[0093] S1. Chinese medicine extraction: 6 parts by weight of Phellodendron, 1.5 parts by weight of honeysuckle, 5 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 4h, filtered to obtain Chinese medicine residue, and the filtrate was dried to obtain an extract;

[0094] S2. Preparation of medium: 17 parts by weight of the Chinese medicine residue obtained in step S1, 6 parts by weight of desalted whey powder, 11 parts by weight of glucose, 4 parts by weight of yeast extract powder were added to 250 parts by weight of water and sterilized to obtain a medium;

[0095] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium respectively, and activated and cultured for 21 hours at 37°C and 150 r / min under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0096] S4. Fermentation: The Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 obtained in step S3 were inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1.5 v / v%, 3 v / v%, 2.5 v / v%, and 2.5 v / v%, respectively, under anaerobic conditions, 37 ° C, 150 r / min, and the fermentation culture was carried out for 53 h, filtered, and the bacterial sludge was collected. The filtrate was ultrafiltrated to collect a solution with a molecular weight of 2-10 KDa, and freeze-dried to obtain an antimicrobial peptide, and the remaining liquid was freeze-dried to obtain a fermentation product;

[0097] S5. Preparation of acid-soluble bacteria-loaded microcapsules: 13.5 parts by weight of carboxymethyl chitosan and 8.5 parts by weight of gelatin were added to 230 parts by weight of water, heated to 55 ° C, stirred to dissolve, 5 parts by weight of tannic acid, 0.7 parts by weight of Tween-80 and 22 parts by weight of the bacterial sludge obtained in step S4 were added, stirred and mixed for 20 min, added dropwise to 500 parts by weight of fish oil, emulsified at 10000 r / min for 10 min, the pH value of the solution was adjusted to 8.5, stirred for 45 min, and freeze-dried to obtain acid-soluble bacteria-loaded microcapsules;

[0098] S6. Preparation of a probiotic composition for treating vaginitis: add 17 parts by weight of sodium alginate, 4 parts by weight of inulin, and 2.5 parts by weight of arabinose to 200 parts by weight of water, add 3.5 parts by weight of citric acid, 5 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5 parts by weight of N-hydroxysuccinimide, stir and react at 2°C for 30 minutes, add 7.5 parts by weight of silk fibroin peptide, heat to 75°C, stir and react for 4.5 hours, then add 5 parts by weight of the extract prepared in step S1, 6 parts by weight of the fermentation product prepared in step S4, 2.5 parts by weight of the antimicrobial peptide prepared in step S4, and 8.5 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S5, stir and mix for 20 minutes, and swell for 13 hours to prepare a probiotic composition for treating vaginitis.

[0099] Comparative Example 6

[0100] Compared with the embodiment 3, the difference is that step S6 is not performed.

[0101] The details are as follows:

[0102] S1. Chinese medicine extraction: 6 parts by weight of Phellodendron, 1.5 parts by weight of honeysuckle, 5 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 4h, filtered to obtain Chinese medicine residue, and the filtrate was dried to obtain an extract;

[0103] S2. Preparation of medium: 17 parts by weight of the Chinese medicine residue obtained in step S1, 6 parts by weight of desalted whey powder, 11 parts by weight of glucose, 4 parts by weight of yeast extract powder were added to 250 parts by weight of water and sterilized to obtain a medium;

[0104] S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium respectively, and activated and cultured for 21 hours at 37°C and 150 r / min under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0105] S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1.5 v / v%, 3 v / v%, 2.5 v / v% and 2.5 v / v%, respectively, under anaerobic conditions, 37 ° C, 150 r / min, fermented and cultured for 53 h, filtered, the bacterial mud was collected, the filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10 KDa, freeze-dried, and antimicrobial peptides were obtained, ethanol was added to the remaining liquid to a system ethanol content of 82 wt%, precipitated for 1.5 h, filtered, washed, dried, and a complex polysaccharide was obtained, and the filtrate was recovered with ethanol, freeze-dried, and a fermentation product was obtained;

[0106] S5. Preparation of antimicrobial peptide-polysaccharide complex: 11 parts by weight of the antimicrobial peptide prepared in step S4 were dissolved in 200 parts by weight of water, 5 parts by weight of N-hydroxysuccinimide and 6 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated. 13 parts by weight of the complex polysaccharide prepared in step S4 were added, and the mixture was heated to 55° C., stirred for reaction for 5 h, dialyzed, and dried to obtain an antimicrobial peptide-polysaccharide complex.

[0107] S6. Preparation of a probiotic composition for treating vaginitis: add 17 parts by weight of sodium alginate, 4 parts by weight of inulin, and 2.5 parts by weight of arabinose to 200 parts by weight of water, add 3.5 parts by weight of citric acid, 5 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5 parts by weight of N-hydroxysuccinimide, stir and react at 2°C for 30 minutes, add 7.5 parts by weight of silk fibroin peptide, heat to 75°C, stir and react for 4.5 hours, then add 5 parts by weight of the extract prepared in step S1, 6 parts by weight of the fermentation product prepared in step S4, 2.5 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 8.5 parts by weight of the bacterial mud prepared in step S4, stir and mix for 20 minutes, and swell for 13 hours to prepare a probiotic composition for treating vaginitis.

[0108] Comparative Example 7

[0109] Compared with Example 3, the difference is that in step S6, the microcapsules are embedded with ordinary sodium alginate.

[0110] The details are as follows:

[0111] S6. Preparation of microcapsules: Add 22 parts by weight of sodium alginate to 230 parts by weight of water, heat to 55°C, stir to dissolve, add 0.7 parts by weight of Tween-80 and 22 parts by weight of the bacterial sludge obtained in step S4, stir and mix for 20 minutes, add dropwise to 500 parts by weight of fish oil, emulsify at 10000 r / min for 10 minutes, add dropwise 10 parts by weight of 2wt% calcium chloride solution, stir to react for 45 minutes, and freeze-dry to obtain microcapsules.

[0112] Comparative Example 8

[0113] Compared with Example 3, the difference is that inulin and gum arabic are not added in step S7.

[0114] The details are as follows:

[0115] S7. Preparation of a probiotic composition for treating vaginitis: add 23.5 parts by weight of sodium alginate to 200 parts by weight of water, add 3.5 parts by weight of citric acid, 5 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5 parts by weight of N-hydroxysuccinimide, stir and react at 2°C for 30 minutes, add 7.5 parts by weight of silk fibroin peptide, heat to 75°C, stir and react for 4.5 hours, then add 5 parts by weight of the extract prepared in step S1, 6 parts by weight of the fermentation product prepared in step S4, 2.5 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 8.5 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix for 20 minutes, and swell for 13 hours to prepare a probiotic composition for treating vaginitis.

[0116] Test Example 1

[0117] The mice were randomly divided into a blank group, a model group, a levofloxacin group, and the Example 1-3 and Comparative Example 1-8 groups, with 10 mice in each group. The model group, the levofloxacin group, the Example 1-3 and Comparative Example 1-8 groups were subjected to bacterial vaginosis modeling, and a mixed bacterial solution of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa (1:1:1) (containing 10 7 -10 8 cfu / mL) was injected into the vagina of mice once a day. After 7 consecutive days of injection, the vaginal opening of the mice was obviously swollen and purulent. Take a swab of vaginal secretions from the mice and place it in a small test tube containing sample diluent. After rinsing thoroughly, discard the swab. Pipette the sample liquid in the test tube, add 1 drop to the sample hole of the test card, and then add 1 drop of color development liquid. After incubation at 37℃ for 10 minutes, observe the color development. A positive result indicates that the model is successfully established.

[0118] After the film was successfully formed, the levofloxacin group and the Example 1-3 and Comparative Example 1-8 groups were treated with vaginal local administration. The levofloxacin group was treated with 0.2 mL of levofloxacin solution (0.1 mg / mL) for external use, once a day, for 7 consecutive days. The Example 1-3 and Comparative Example 1-8 groups were gavaged with 1 g / kg of the corresponding probiotic composition for treating vaginitis, once a day, for 7 consecutive days. After 7 days, the blank group, model group, levofloxacin group and the Example 1-3 and Comparative Example 1-8 groups of mice vaginal secretion specimens were taken for smear microscopy, and the vaginal condition was evaluated using Nugent scoring: 0 points for the best vaginal environment, 10 points for the worst vaginal environment, >4 points for the beginning of vaginal inflammation, and the higher the score, the higher the degree of inflammation. The mouse vaginal secretion sialidase method was used for qualitative detection to determine whether it turned negative, and the negative conversion rate of each group of mice was obtained. The results are shown in Table 1.

[0119] Table 1

[0120]

[0121] Note: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

[0122] It can be seen from the above table that the probiotic composition for treating vaginitis prepared in Examples 1-3 of the present invention can significantly improve the symptoms of vaginitis caused by bacteria, and has a high negative conversion rate.

[0123] After scoring, orbital venous blood was collected from each group of mice in a tube, part of the blood was taken in a tube with anticoagulant to calculate WBC (white blood cell count), the rest was centrifuged, and the upper serum was taken. The concentrations of CRP (C-reactive protein), IL-6 (interleukin-6), and IL-10 (interleukin-10) in mouse serum were detected according to the enzyme-linked immunosorbent assay detection method of the kit. The results are shown in Table 2.

[0124] Table 2

[0125]

[0126]

[0127] Note: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05.

[0128] It can be seen from the above table that the probiotic composition for treating vaginitis prepared in Examples 1-3 of the present invention can significantly reduce the inflammatory indexes in serum.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a probiotic composition for treating vaginitis, characterized in that: Extracts are obtained by water extraction of Phellodendron amurense, honeysuckle and Platycladus orientalis leaves, the residues are inoculated with probiotics for fermentation, antimicrobial peptides, bacterial mud and fermentation products are separated, antimicrobial peptides are used to prepare antimicrobial peptide-polysaccharide complexes, the bacterial mud is embedded in an acid-soluble shell layer to prepare acid-soluble bacteria-loaded microcapsules, the antimicrobial peptide-polysaccharide complexes, extracts and prebiotics are mixed with the microcapsules and added to a hydrogel to prepare a probiotic composition for treating vaginitis.

2. The preparation method according to claim 1, characterized in that: The following steps are involved: S1. Extraction of Chinese medicine: Wash Phellodendron chinense, honeysuckle and Platycladus orientalis leaves, add them to water, heat and boil to extract, filter to obtain Chinese medicine residue, dry the filtrate to obtain an extract; S2. Preparation of culture medium: The Chinese medicine residue obtained in step S1, desalted whey powder, glucose, yeast extract powder were added to water and sterilized to obtain a culture medium; S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, activated and cultured to obtain bacterial seed liquid; S4. Fermentation: The seed solution of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051 and Lactobacillus gasseri LG019 obtained in step S3 was inoculated into the medium obtained in step S2, and the culture was fermented, filtered, and the bacterial sludge was collected. The filtrate was ultrafiltrated to collect the solution with a molecular weight of 2-10KDa, and freeze-dried to obtain antimicrobial peptides. The remaining liquid was added with ethanol for precipitation, filtered, washed, and dried to obtain a complex polysaccharide. The filtrate was recovered with ethanol, and freeze-dried to obtain a fermentation product. S5. Preparation of antimicrobial peptide-polysaccharide complex: dissolving the antimicrobial peptide obtained in step S4 in water, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activating the reaction, adding the complex polysaccharide obtained in step S4, heating and stirring the reaction, dialyzing, and drying to obtain an antimicrobial peptide-polysaccharide complex; S6. Preparation of acid-soluble bacteria-carrying microcapsules: carboxymethyl chitosan and gelatin are added to water, heated to dissolve, tannic acid, lecithin and the bacterial sludge obtained in step S4 are added, dispersed evenly, added dropwise to fish oil, emulsified, the pH value of the solution is adjusted, the reaction is stirred, and freeze-dried to obtain acid-soluble bacteria-carrying microcapsules; S7. Preparation of a probiotic composition for treating vaginitis: add sodium alginate, inulin and arabinose into water, add citric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir to react, add silk fibroin peptide, heat and stir to react, then add the extract prepared in step S1, the fermentation product prepared in step S4, the antimicrobial peptide-polysaccharide complex prepared in step S5, and the acid-soluble bacteria-loaded microcapsule prepared in step S6, stir and mix evenly to prepare a probiotic composition for treating vaginitis.

3. The preparation method according to claim 2, characterized in that: The mass ratio of Phellodendron chinense, honeysuckle and Platycladus orientalis leaves in step S1 is 5-7:1-2:4-6, and the heating and boiling extraction time is 3-5h; the mass ratio of Chinese medicinal residues, desalted whey powder, glucose, yeast extract powder and water in step S2 is 15-20:5-7:10-12:3-5:200-300.

4. The preparation method according to claim 2, characterized in that: The activation culture conditions in step S3 are anaerobic conditions, 36-38°C, 100-200r / min, culture for 18-24h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

5. The preparation method according to claim 2, characterized in that: The inoculation amounts of the seed solutions of Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 in step S4 are 1-2v / v%, 2-4v / v%, 2-3v / v%, and 2-3v / v%, respectively. The fermentation and culture conditions are anaerobic conditions, 36-38°C, 100-200r / min, and the fermentation and culture is 50-56h.

6. The preparation method according to claim 2, characterized in that: In step S5, the mass ratio of the antimicrobial peptide, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and the complex polysaccharide is 10-12:4-7:5-8:12-15, the temperature of the activation reaction is 0-4°C, the time is 30-40min, and the temperature of the heating and stirring reaction is 50-60°C, and the time is 4-6h.

7. The preparation method according to claim 2, characterized in that: In step S6, the mass ratio of carboxymethyl chitosan, gelatin, tannic acid, lecithin, bacterial mud, water and fish oil is 12-15:7-10:4-6:0.5-1:20-25:200-250:500, the pH value of the adjusted solution is 8.4-8.6, and the stirring reaction time is 30-60 min.

8. The preparation method according to claim 2, characterized in that: The mass ratio of sodium alginate, inulin, arabinose, citric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, silk fibroin peptide, extract, fermentation product, antimicrobial peptide-polysaccharide complex, and acid-soluble bacteria-loaded microcapsule in step S7 is 15-20:3-5:2-3:3-4:4-6:4-6:6-9:4-6:5-7:2-3:7-10, and the temperature of the heated and stirred reaction is 70-80°C, and the time is 4-5h.

9. The preparation method according to claim 2, characterized in that: The specific steps include: S1. Chinese medicine extraction: 5-7 parts by weight of Phellodendron, 1-2 parts by weight of honeysuckle, 4-6 parts by weight of Platycladus orientalis leaves were washed and added to 100 parts by weight of water, heated to boiling and extracted for 3-5h, filtered to obtain Chinese medicine residues, and the filtrate was dried to obtain an extract; S2. Preparation of medium: 15-20 parts by weight of the Chinese medicine residue obtained in step S1, 5-7 parts by weight of desalted whey powder, 10-12 parts by weight of glucose, 3-5 parts by weight of yeast extract powder are added to 200-300 parts by weight of water and sterilized to obtain a medium; S3. Activation of probiotics: Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 were inoculated into MRS broth medium, and activated and cultured at 36-38°C for 18-24h under anaerobic conditions to obtain a probiotic with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution; S4. Fermentation: The Lactobacillus rhamnosus LRYZU021, Lactobacillus reuteri FPHC2951, Lactobacillus crispatus LCP051, and Lactobacillus gasseri LG019 obtained in step S3 were inoculated into the culture medium obtained in step S2, and the inoculation amounts were 1-2v / v%, 2-4v / v%, 2-3v / v%, and 2-3v / v%, respectively, under anaerobic conditions, 36-38°C, 100-200r / min, and fermented for 50-56h, filtered, and the sludge was collected. The filtrate was ultrafiltrated to collect a solution with a molecular weight of 2-10KDa, and freeze-dried to obtain an antimicrobial peptide, and ethanol was added to the remaining liquid to a system ethanol content of 80-85wt%, precipitated for 1-2h, filtered, washed, and dried to obtain a complex polysaccharide, and the filtrate was recovered with ethanol, freeze-dried, and a fermentation product was obtained; S5. Preparation of antimicrobial peptide-polysaccharide complex: dissolve 10-12 parts by weight of the antimicrobial peptide prepared in step S4 in 200 parts by weight of water, add 4-7 parts by weight of N-hydroxysuccinimide and 5-8 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, activate the reaction, add 12-15 parts by weight of the complex polysaccharide prepared in step S4, heat to 50-60° C., stir to react for 4-6 hours, dialyze, and dry to obtain an antimicrobial peptide-polysaccharide complex; S6. Preparation of acid-soluble bacteria-loaded microcapsules: 12-15 parts by weight of carboxymethyl chitosan, 7-10 parts by weight of gelatin are added to 200-250 parts by weight of water, heated to 50-60 ° C, stirred to dissolve, 4-6 parts by weight of tannic acid, 0.5-1 parts by weight of lecithin and 20-25 parts by weight of the bacterial mud obtained in step S4 are added, uniformly dispersed, added dropwise to 500 parts by weight of fish oil, emulsified, the pH value of the solution is adjusted to 8.4-8.6, stirred for 30-60 min, freeze-dried, and acid-soluble bacteria-loaded microcapsules are obtained; S7. Preparation of a probiotic composition for treating vaginitis: add 15-20 parts by weight of sodium alginate, 3-5 parts by weight of inulin, and 2-3 parts by weight of arabinose to 200 parts by weight of water, add 3-4 parts by weight of citric acid, 4-6 parts by weight of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4-6 parts by weight of N-hydroxysuccinimide, stir and react at 0-4°C for 20-40 minutes, add 6-9 parts by weight of silk fibroin peptide, heat to 70-80°C, stir and react for 4-5 hours, then add 4-6 parts by weight of the extract prepared in step S1, 5-7 parts by weight of the fermentation product prepared in step S4, 2-3 parts by weight of the antimicrobial peptide-polysaccharide complex prepared in step S5, and 7-10 parts by weight of the acid-soluble bacteria-loaded microcapsules prepared in step S6, stir and mix evenly, and freeze-dry to prepare a probiotic composition for treating vaginitis.

10. A probiotic composition for treating vaginitis obtained by the preparation method according to any one of claims 1 to 9.

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