CN115873777A - Oriented domestication modified probiotics, microecological preparation containing probiotics and application of probiotics in gynecological inflammation - Google Patents

Abstract

The invention discloses a directional domestication modified probiotic, a microecological preparation containing the probiotic and application of the microecological preparation in preparation of a medicament for preventing and treating gynecological inflammation, and belongs to the technical field of synthetic biology.

Description

Oriented domestication modified probiotics, microecological preparation containing probiotics and application of probiotics in gynecological inflammation

Technical Field

The invention relates to the technical field of synthetic biology, in particular to a biosynthesis technology combining traditional Chinese medicine fermentation and domesticated probiotics.

Background

The treatment of gynecological inflammation mainly comprises drug treatment, physical treatment and operation treatment. Common gynecological inflammation such as vaginitis, cervicitis and adnexitis is treated by combining antibiotics with Chinese traditional medicines such as roxithromycin, amoxicillin, levofloxacin, metronidazole, tinidazole, norfloxacin, roxithromycin and the like, and combining gynecological Qianjin tablets, female capsules, fule tablets, golden chicken capsules and the like. Also used are vaginal topical drugs, such as metronidazole, baofukang suppository, erosion-eliminating suppository, etc. which have certain therapeutic effects. Because the female reproductive organs are positioned in the pelvic cavity, the medicine can hardly directly reach the focus, and the auxiliary treatment is carried out by using physical treatment methods such as laser, iontophoresis, microwave, red light and the like. For patients with severe gynecological inflammation, surgical methods are often used for treatment, for example, severe cervical erosion and cervical polyp need to be treated by surgery. The traditional Chinese patent medicines, antibiotic medicines and the like for treating gynecological inflammation have basically consistent principles, kill bacteria, kill beneficial bacteria of a human body while killing germs, cause micro-ecological imbalance of the human body, generate antibiotic drug resistance after long-term use, cause gynecological inflammation to be stubborn after repeated attack, destroy the micro-ecological environment of female genital tracts, cause anaphylactic reaction, need careful use by people with allergic constitution, cause the change of the local microenvironment of vagina, and cannot be used for pruritus caused by vulval white lesion and diabetes.

In the case of unmarried and postmenopausal women, the patients may experience stinging and hot flashes of the skin during the application of the drug, due to the immaturity of the vaginal mucosa or atrophy of the mucosa. Some patients may develop skin allergy such as burning, pain, rash, and skin pruritus.

The following hazards can arise from the long-term use of antibiotics: first, bacterial resistance is induced. Secondly, the organs of the human body are damaged. The antibiotic can also cause human body damage while sterilizing, influence liver and kidney functions and cause gastrointestinal reaction. Third, it causes a superinfection. Under normal conditions, all organs and parts of a human body are parasitized by bacteria, the parasitized flora and the parasitized flora maintain a balanced state under mutual antagonism, if broad-spectrum antibacterial drugs are used for a long time, sensitive bacteria can be killed, insensitive bacteria propagate by machine, and uninhibited bacteria, fungi and foreign bacteria can also enter by default to induce new infection. The recurrence rate of antibiotics is high, and the recurrence rate of three months is as high as 30%. Antibiotics have long been abused in China, and are therefore also once called "the world of infusion bottles". The world health organization, the resident representative department, the health system and the health safety group, namely Martin Taylor, receives the introduction of the authority data when the southern daily newspaper is specially visited in 2015, and in the last 10 years, more than half of outpatients in China obtain prescribed antibiotics which far exceed the limit (less than 30%) recommended by the world health organization. In the article of antibiotic abuse and countermeasure published in 2012 of inner Mongolia traditional Chinese medicine, 1995-2007 disease classification survey data shows that Chinese infectious diseases account for 49% of the total disease incidence, wherein bacterial infectivity accounts for 18% -21% of the total disease. That is, less than 20% of patients who actually need to use antibiotics are abused with antibiotics for more than 80%. On 7 d 4.2021, the national medical administration of health and welfare committee issued a "notice" on the work of further strengthening antimicrobial drug administration and suppressing drug resistance (hereinafter referred to as "notice"), and required to further fulfill the requirements of the "method for clinical administration and management of antimicrobial drugs", and to standardize the drug treatment of infectious diseases caused by pathogenic microorganisms such as bacteria, mycoplasma, chlamydia, rickettsia, spirochete, fungi, and the like. Among them, antibiotics are the main drugs of antimicrobial drugs, including penicillins, cephalosporins, aminoglycosides, macrolides, tetracyclines, chloramphenics, polypeptides, lincomycins, etc. Recent notice also means that antibiotic drugs will be further under scrutiny after a recent series of "resistance-limiting" s. This is not the first time the antimicrobial drug is called. In order to prevent the abuse of antibacterial drugs, the country of 2011 has started a national action for three years, and the reasonable use of antibiotics in second-level and above public hospitals is improved; the ministry of health published 2012 "antibacterial drugs clinical application management approach"; in the year 2015, 8 months, the "clinical application and management method for antibacterial drugs" were officially implemented (in the year 2020, 5 months, "antibacterial drugs" were named "antimicrobial drugs" by the world health organization, food and agricultural organizations of the united nations, and the world animal health organization).

In 7 months of 2020, the provision of the Notification of the antibacterial clinical application management work of the national health Commission office on continuous well-being is stipulated in various places to strengthen the construction of infectious disease departments and improve the medical quality of infectious diseases; the medical institution optimizes the supply catalogue of the antibacterial drugs, improves the professional technical service level of pharmacy, strengthens key link management and the like. The 'notice' of this time further provides that the reasonable use condition of the antimicrobial drugs is brought into the work of hospital evaluation, public hospital performance assessment, reasonable medication assessment and the like, and the requirement of assessment weight is properly increased. The antibacterial drug called in the second method in the 84 th 'antibacterial drug clinical application management method' of the ministry of health of the people's republic of China' is a drug for treating infectious diseases caused by pathogenic microorganisms such as bacteria, mycoplasma, chlamydia, rickettsia, spirochete, fungi and the like, and does not include drugs for treating tuberculosis, parasitic diseases and infectious diseases caused by various viruses and traditional Chinese medicine preparations with antibacterial action.

The gynecological disease is mainly in the form of suppository and lotion. The suppository and lotion have the functions of diminishing inflammation and resisting bacteria, but have the defect that the suppository and lotion cannot achieve the purpose of durable treatment, in addition, the vagina of a woman has normal flora, the pH value of the vagina is suitable for the growth and the reproduction of the normal bacteria, the types of the lotion for treating gynecological inflammation are very large, and the misuse of the lotion can cause vaginal flora imbalance and destroy the acid-base balance of the vagina. The suppository is generally used at night, and if the suppository is used in the daytime, the medicine is easy to flow out through the vagina. Some Chinese patent medicine suppositories and washing liquid also have side effects such as residues after medicine application, residual toxicity and the like which are not good for female health.

The Chinese patent medicine lotion not only damages the micro-ecological environment of female genital tracts and causes anaphylactic reaction, but also can cause the change of the micro-ecological environment of local vagina when people with allergic constitution need to use with caution, such as white lesions of vulva and women after marriage and menopause, and the patients can have reactions of stabbing pain, hot skin and the like during the medication period because the vaginal mucosa is immature or the mucosa is atrophic. Some patients may develop skin allergy such as burning, pain, rash, and skin pruritus.

The novel treatment mode of gynecological diseases is a microecological preparation medicine, a microecological system is a unity of mutual restriction and interaction of normal microbiota, environment and host, and the microecological system comprises an open mucous membrane part and comprises: oral micro-ecosystem, digestive micro-ecosystem, skin micro-ecosystem, reproductive micro-ecosystem, other micro-ecosystem. More than 300 microorganisms are present in the vagina of women, 1) gram-positive aerobic (or facultative) bacteria, such as lactobacilli, non-hemolytic streptococci, enterococci, etc., and 2) gram-negative aerobic (or facultative) bacteria, such as escherichia coli, gardner bacteria, etc. 3) Anaerobic bacteria such as Clostridium, streptococcus digestns, bacteroides and Clostridium. 4) Others, such as mycoplasma and candida. The most abundant dominant bacteria in the normal vagina are lactobacilli, containing 10 per gram of vaginal secretions ⁷ ～10 ⁸ When the dominance of the lactobacillus CFU is changed, dysbacteriosis occurs, which causes vaginal inflammation. The normal pH value of the vagina is 3.8-4.5, and the change of the pH value of the vagina can directly reflect the change of microbial flora mainly comprising lactobacillus in the vagina.

Diseases related to genital tract microecological system imbalance are often called ABCDV for short, and the specific manifestations are as follows: bacterial Vaginosis (BV), aerobic Vaginitis (AV), candidiasis (VVC), trichomonas Vaginitis (TV), cytolytic Vaginosis (CV), senile vaginitis is also a dynamic balance system along with microecological imbalance of vagina, and 'physiological bacteria' and 'pathogenic bacteria' in the environment are mutually restricted and coordinated and change along with the change of factors such as ovarian function, hormone level, pregnancy, menstrual cycle and the like

The currently approved vaginal microecological preparation on the market in China has only two medicines, one is a capsule type medicine which is produced by inner Mongolia Shuangqi pharmaceutical industry Limited company and has the trade name of 'Dingjunsheng', the main component of the capsule type medicine is Lactobacillus delbrueckii, and the strain does not belong to the dominant bacterial strain of female vagina in China. One is a capsule medicine with "Yanhua" trade name, which is produced by Xian Zhenghao organism limited company, and the main component is an active Streptococcus enterobacteria (Streptococcus faecalis), which is not the dominant bacterial of vagina. Moreover, the two microecological preparation medicines are required to be stored at a low temperature, the application and use range is limited, the treatment cost is higher, and the market price of 1 granule is as high as 9 yuan. Meanwhile, the prescription is only prescribed in hospitals and medical institutions in towns, and the prescription cannot be popularized to vast remote rural areas at present.

However, about 40% of people in China still have rural areas, and the problem of gynecological diseases of rural women cannot be ignored.

Therefore, besides the education popularization of the gynecological health care and health knowledge, the safety, convenience, diversity and price bearing capacity of the gynecological inflammation are the problems to be solved in the field.

Disclosure of Invention

It is an object of the present invention to provide a domesticated probiotic to solve the above problems.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a method for the directed domestication of modified probiotics, the modification method of the probiotics comprises the following steps:

(1) Weighing 5-8 parts of dendrobium, 1-3 parts of coptis chinensis, 1-3 parts of phellodendron, 2-4 parts of polygonum cuspidatum, 3-6 parts of fructus cnidii, 3-6 parts of radix sophorae flavescentis, 5-8 parts of elsholtzia and 1-2 parts of borneol, mixing, crushing and extracting to obtain an extracting solution and medicine residues, wherein the extracting solution is used as a fermentation substrate;

(2) Firstly, adding 13-18% of yeast and 5-8% of bacillus natto into the fermentation substrate obtained in the step (1) according to the total weight of the fermentation raw materials, carrying out aseptic aerobic constant-temperature fermentation for 24-30 h at 28-32 ℃, adding lactobacillus after stopping fermentation, carrying out anaerobic fermentation for 42-48 h, and carrying out constant-temperature enzymolysis for 2-4h after fermentation is completed to obtain a basic culture solution;

(3) Adding 2-4 times of purified water into the medicine residue obtained in the step (1), adding chicory inulin and lactic acid bacteria, fermenting at a constant temperature of 28-32 ℃, extracting, mixing an extracting solution with the basic culture solution obtained in the step (2), and performing pasteurization to obtain a composite culture medium;

(4) Domestication of strains: mixing Lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus delbrueckii subspecies lactis (Lactobacillus), lactobacillus crispatus and Lactobacillus jensenii with the composite culture medium obtained in the step (3) for strain acclimatization to obtain the Lactobacillus acidophilus/Lactobacillus delbrueckii composite culture medium.

Synthetic biology is an emerging cross-fusion subject developed in this century, and research and application of the synthetic biology mainly comprises the fields of biological elements, gene circuits, metabolic engineering, genome engineering and the like. The invention relates to a result of cross fusion in two different subject fields of traditional Chinese medicine fermentation and strain domestication modification, wherein the domestication of the strain is purposeful and directional domestication, namely, the domestication is metabolic engineering application in the field of synthetic biology, namely, highly complex medicine molecule biological fermentation production, and the domestication depends on the high understanding of strain discovery and engineering.

The synthetic biology is a novel cross-technology subject taking 'construction' as a core, deeply and cross-fuses biology with other subjects and paradigms, fully exerts the characteristics of quantification, design, engineering and the like, is a basic and tool-combining subject, and brings possibility for breakthrough of DNA synthesis, gene editing and high-throughput biological experiments. For example, tens of millions of huge research and development expenses and years of research time of hundreds of research and development personnel are consumed for developing or discovering a new strain with economic value, and the characteristic of synthetic bioengineering can introduce an intelligent manufacturing idea from industrial 2.0/3.0/4.0 to biological research and development, so that automation and high-throughput equipment can be used rapidly at low cost, large-scale and structured data are used as new production data, experimental design is optimized, research and development experience is precipitated as platform research and development capability, and further, the large-scale promotion of the product landing success rate, speed and quantity in parallel is realized.

The invention is based on directional/quantitative design and cultivation, breaks the boundary of the two fields of traditional Chinese medicine fermentation and micro-ecology, carries out deep fusion, directional design and quantitative transformation, and achieves the purposes of design and production.

The probiotics are more applied in livestock and agriculture at present, including fermentation application. In human health, the intestinal tract is the hottest at present, and nearly 70% of bacterial strains in the human body are in the intestinal tract, and the intestinal tract is also the main strain in the world and nationwide. The application is specially directed at the application of the reproductive system, namely the invention combines the directional modification of the well-known safe beneficial bacteria with the field of traditional Chinese medicine fermentation, breaks through the traditional mode, and obtains a microecological preparation product which can be safely and effectively applied to gynecological inflammation, has good temperature tolerance, has a proper pH value suitable for female genital tracts and has high viable bacteria content. The invention obviously improves the activity and temperature tolerance of the strains of the existing probiotics, prolongs the decay period of the strains and obviously improves the content of the strains.

The invention preferably selects dominant strains of Lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus delbrueckii subspecies lactis (Lactobacillus), lactobacillus crispatus and Lactobacillus jensenii in the female vaginal environment, adopts special Chinese herbal compound extracts to domesticate and transform the strains, retains the original structure and characteristics of beneficial bacteria, improves the activity of the strains, and solves the problems of low environmental temperature bearing capacity and short life cycle of the original beneficial bacteria. The preservation temperature of the general probiotic preparation is 2-6 ℃, the active lactobacillus needs to be preserved at the refrigerated temperature of 0-4 ℃, and the production, transportation and preservation of the probiotic require a cold chain, because the probiotic enters a fermentation state along with the rise of the temperature, and the activity is gradually reduced; the strain domesticated by the invention has stronger viability, improved active ingredients, delayed degradation period and obviously improved survival rate of the strain in the microecological preparation at normal temperature for a long time. The fat-soluble components in the traditional Chinese medicine have side effects of different degrees in the production process of the Chinese patent medicine, and the invention adopts the subcritical extraction method for degreasing treatment before the processing of the traditional Chinese medicine, which is beneficial to reducing the toxicity of the medicine.

Based on the idea of biosynthesis, the invention achieves the set target by a multi-connection fermentation mode: including a pH of 3.8-4.4 or 4.2-4.5, which is the pH of the normal female reproductive tract environment; the temperature resistance of the strain is improved, the attenuation period is prolonged, the number of beneficial bacteria applied to human body environment is increased, and the number of bacterial colonies is the basic guarantee of the treatment effect.

As a preferred technical scheme: the extraction method comprises subcritical low-temperature extraction: under the pressure of 0.88-5.15Mpa, the material is extracted by liquid ammonia in subcritical state, the solvent in the extracting solution is evaporated to separate the solvent from the extracted target component to obtain the fermentation substrate, and the evaporated solvent is compressed for heat exchange and then liquefied for recycling.

Compared with other separation methods, the subcritical fluid extraction method has the advantages of no toxicity, harmlessness, environmental protection, no pollution, non-thermal processing, no damage and no oxidation of active products of the extract, high capacity, large-scale industrial production, large polar range of extractable target substances according to the polarities of different solvents, energy conservation, low operation cost, certain extraction selectivity, less energy consumption of evaporation of extract liquid, easy separation from products and more contribution to extraction of active ingredients of traditional Chinese medicines (including compound medicines) and separation of harmful fat-soluble ingredients.

As a preferable technical scheme: in the step (3), the concentration of the added chicory inulin is 180-220g/L, and the amount of the added lactic acid bacteria is 22-30% of the total mass of the fermentation raw materials; the fermentation temperature is 30-35 ℃, and the fermentation time is 20-30h; the extraction method comprises the following steps: extracting with liquid ammonia at 55-65 deg.C for at least two times, mixing extractive solutions, concentrating, and removing solvent.

As a preferred technical scheme: in the step (4), the domestication method comprises the following steps: mixing each fermentation strain with composite culture medium according to the ratio of 150-200 × 10 ⁸ Mixing and stirring the CFU fermentation strain/kg composite culture medium, and culturing at constant temperature of 35-38 deg.C for 10-15 hr to obtain subspecies of domesticated Lactobacillus jensenii, lactobacillus crispatus, lactobacillus acidophilus, and Lactobacillus delbrueckii.

As a further preferred technical scheme: adding 2-5% of domesticated lactobacillus jensenii, 3.5-5% of domesticated lactobacillus crispatus, 2.5-5% of domesticated lactobacillus acidophilus and 2-5% of domesticated lactobacillus delbrueckii subspecies according to the total mass of fermentation raw materials into a composite culture medium, and placing the mixture at the temperature of 40-42 ℃ for constant-temperature fermentation for 10-15h to respectively obtain four fermentation raw liquids of the domesticated lactobacillus jensenii fermentation raw liquid, the domesticated lactobacillus crispatus fermentation raw liquid, the domesticated lactobacillus acidophilus fermentation raw liquid and the domesticated lactobacillus delbrueckii subspecies fermentation raw liquid.

The live bacteria content of each 0.25g of the two micro-ecological preparation medicines 'Dingjunsheng' and 'Yanhua' on the market is 2.5 multiplied by 10 ⁵ CFU、6×10 ⁶ The bacterial strain delivery content of the invention preferably improved by multi-stage fermentation and domestication can be increased to more than billions of CFU, the content of colony number in the microecological preparation is obviously improved, the content of beneficial bacteria required by female reproductive environment is achieved, and the prevention and adjuvant treatment effects are effectively improved.

The second purpose of the invention is to provide a microecological preparation containing the domesticated probiotics, which adopts the technical scheme that: the preparation method of the microecological preparation comprises the following steps of respectively and uniformly mixing 3-5% of domesticated lactobacillus jensenii fermentation stock solution, 5-8% of domesticated lactobacillus crispus fermentation stock solution, 2-5% of domesticated lactobacillus acidophilus fermentation stock solution and 2-5% of domesticated lactobacillus delbrueckii subspecies fermentation stock solution with the composite culture medium according to the mass of the composite culture medium serving as a reference, adding bacillus natto with the mass of 3-6% of the composite culture medium, standing at the constant temperature of 30 ℃ for 2-4h, dissolving with purified water, sequentially adding 0.05-0.08% of polyhexamethylene biguanide, 1.8-2.4% of lactic acid and 3-5% of sodium hyaluronate according to the proportion of 0.15-0.3 per mill of the mass of the dissolved material, and uniformly mixing to obtain a semi-finished product;

and adding fructo-oligosaccharide with the percentage content of 2-7 percent based on the mass of the semi-finished product into the semi-finished product, uniformly mixing, and filling and packaging under aseptic conditions to obtain a finished product.

As a preferred technical scheme: the formulation is in a liquid or semi-solid or foam form.

As mentioned above, the existing two types of microecological preparation medicines are only capsule formulations, are limited to medical institutions and pharmacies, have high cost, need to put the capsule into the deep vagina by hands during use, have certain unsanitary potential safety hazards, and can not completely cover the fold parts in the deep genital tract of women, thereby affecting the treatment effect. The preparation can be developed into various dosage forms such as paste, semisolid, liquid, foam and the like, has various morphological expression forms, can survive and be stored in various base materials for a long time, is safe and sanitary to use and more convenient for the product formed after the process, can be developed into various types of products, can be used as daily supplementary nursing, protects and stabilizes the healthy micro-ecological environment of the female reproductive system for a long time, effectively reduces the production cost, is easy to realize industrial mass production, effectively reduces the treatment cost, and solves the problems of limited application range, higher cost and inconvenient use of the existing micro-ecological preparation medicines.

The third purpose of the invention is to provide the application of the microecological preparation in preparing the medicine for preventing and treating gynecological inflammation.

The invention also provides application of the microecological preparation in preparing an external sanitary health-care product for women, which is used for preventing gynecological inflammation.

As a preferred technical scheme: the gynecological inflammation is caused by any one or more pathogenic bacteria of staphylococcus aureus, escherichia coli and candida albicans.

The microecological preparation can be used for traditional medicine, but the use scene of the medicine is limited, so the microecological preparation can also be prepared into women external health care products and other forms, namely, civil characters such as 'Xiaoquan' and 'mechanical characters' can be applied and matched with 'national medicine standard characters' so as to expand the application range and the use scene and mode; as mentioned previously, such new applications, solve the limitation that the prescription must be made to the hospital, thus expanding the application scope and scene.

The invention can also solve the problems of adverse reaction and long-term use side effect of antibiotic medicines on human bodies, and also solve the problems of repeated side effect of inflammation caused by damage, allergy, incomplete absorption and residual toxicity of the traditional Chinese patent medicine on the micro-ecological environment of human bodies, sterilization of germs and sterilization of probiotics.

Compared with the prior art, the invention has the advantages that: the traditional Chinese medicine extract is used for multi-connection fermentation domestication and transformation of probiotics, the active pharmacological function of the traditional Chinese medicine essence is kept, and meanwhile, the product obtained by adding the high molecular polymer can effectively inhibit candida albicans (ATCC 10231), escherichia coli (8099) and staphylococcus aureus (ATCC 6538) in female vagina, regulate the balance of the flora of the female reproductive environment, and improve and enhance the immunity and resistance of the female reproductive tract; in addition, the temperature resistance of the strain is improved, the decay period is prolonged, and the pH value is reduced by a multi-connection fermentation mode.

Drawings

FIG. 1 is a flow chart showing the preparation of example 1 of the present invention;

FIG. 2 is a test chart of the adhesion of the domesticated strain and the genital mucosal cells in example 1 of the present invention;

FIG. 3 is a graph showing the effect of the domesticated strain of Candida albicans of the present invention in example 1.

Detailed Description

The invention will be further explained with reference to the drawings.

Example 1:

a microecological preparation containing domesticated probiotic bacteria is prepared by the method shown in figure 1, and comprises the following steps:

(1) Determining a traditional Chinese medicine formula: 60g of dendrobium, 38g of coptis chinensis, 45g of phellodendron, 55g of polygonum cuspidatum, 40g of fructus cnidii, 40g of radix sophorae flavescentis, 35g of elsholtzia and 15g of borneol, weighing the Chinese herbal medicines according to the formula, screening to remove impurities, crushing the Chinese herbal medicines into 200 meshes by using an ultrafine grinder, conveying the crushed Chinese herbal medicines into an extraction tank by air flow, pumping to directly dissolve the crushed Chinese herbal medicines, injecting subcritical low-temperature solvent liquid ammonia into the extraction tank for soaking, wherein the volume ratio of material dissolution is 1:1.4, extracting at 38 ℃ and under the working pressure of 1.5Mpa for 60min for 6 times, pumping the primary extracted extract into a separation tank to separate the solvent and the liquid medicine, extracting the supernatant, collecting the rest of the extract by countercurrent extraction to be used as the solvent of the next batch of materials, adjusting the pH value of the acid-base precipitate of the supernatant to 4.5-6.5, performing solid-liquid separation, and performing pasteurization to obtain a fermentation substrate; the gasified solvent gas can be recycled after being compressed, condensed and liquefied by a compressor;

(2) Putting the fermentation substrate obtained in the step (1) into a clean and sterilized fermentation tank, adding yeast and bacillus natto, wherein the addition amount of the yeast, the yeast and the bacillus natto is 78%, 10% and 12% in total by mass ratio, fermenting for 24 hours at constant temperature of 35 ℃ under aseptic and aerobic conditions, adding lactobacillus accounting for 3.5% of the total mass of the fermentation raw materials after fermentation is stopped, performing anaerobic fermentation for 48 hours, and standing for 2 hours at constant temperature of 30 ℃ after fermentation is completed to obtain a basic culture solution, wherein the pH value of the basic culture solution is 5.2;

(3) Adding chicory inulin into the dregs obtained in the step (1), adding purified water to enable the concentration of the chicory inulin to reach 200g/L, then adding lactic acid bacteria, wherein the addition amount of the lactic acid bacteria accounts for 3wt% of the material, fermenting at the constant temperature of 35 ℃ for 24h, conveying the material into an extraction tank, injecting liquid ammonia serving as a subcritical low-temperature solvent, and enabling the volume ratio of material to solution to be 1:1.4, setting the extraction temperature at 38 ℃, the extraction working pressure at 0.5Mpa for 40min, extracting for 5 times, pumping the extract liquor obtained in the primary extraction into a separation tank for separating a solvent from a liquid medicine, collecting the rest extract liquor through countercurrent extraction to be used as a solvent for the next batch of materials, performing solid-liquid separation, and performing pasteurization to obtain a composite culture medium, wherein the pH value of the composite culture medium is 4.5; the gasified solvent gas is compressed, condensed and liquefied by a compressor and then recycled;

(4) Domesticating the strain: lactobacillus acidophilus (160X 10) ⁸ CFU fermentation strain/kg composite culture medium), lactobacillus delbrueckii subspecies lactis (160X 10) ⁸ CFU zymocyte/kg composite culture medium), lactobacillus crispatus (180 × 10) ⁸ CFU zymocyte/kg composite culture medium), lactobacillus jensenii (160 multiplied by 10) ⁸ CFU zymocyte/kg composite culture medium) and the composite culture medium are fully mixed and stirred, and are placed at the constant temperature of 38 ℃ for 12 hours of culture, and strain domestication is carried out to respectively obtain domesticated lactobacillus jensenii, domesticated lactobacillus crispatus, domesticated lactobacillus acidophilus and domesticated lactobacillus delbrueckii subspecies;

(5) Adding 3% of domesticated lactobacillus jensenii, 5% of domesticated lactobacillus crispatus, 3% of domesticated lactobacillus acidophilus and 3% of domesticated lactobacillus delbrueckii subspecies lactobacillus to the composite culture medium obtained in the step (3) by taking the composite culture medium as a mass standard, uniformly mixing, and then placing at 43 ℃ for constant-temperature fermentation for 12 hours to respectively obtain four fermentation raw liquids of domesticated lactobacillus jensenii fermentation raw liquid (pH value of 4.1), domesticated lactobacillus crispatus fermentation raw liquid (pH value of 3.9), domesticated lactobacillus acidophilus fermentation raw liquid (pH value of 4.3) and domesticated lactobacillus delbrueckii subspecies fermentation raw liquid (pH value of 4.5);

(6) Adding 3% of domesticated lactobacillus jensenii fermentation stock solution, 6% of domesticated lactobacillus crispus fermentation stock solution, 3% of domesticated lactobacillus acidophilus fermentation stock solution and 3% of domesticated lactobacillus delbrueckii subspecies fermentation stock solution obtained in the step (5) into the composite culture medium obtained in the step (3) by taking the composite culture medium as a mass standard, uniformly mixing, adding bacillus natto with the mass of 2% of the composite culture medium, placing into a sterilized sealed tank for standing for 3 hours, dissolving with purified water, adding polyhexamethylene biguanide according to the mass proportion of 0.0.4 per mill of the total dissolved materials, and uniformly mixing all the materials to obtain a semi-finished product;

(7) And (4) adding fructo-oligosaccharide with the mass percentage of 5% into the semi-finished product obtained in the step (6) as a prebiotics component, uniformly mixing, and filling and packaging under aseptic conditions to obtain a finished product.

The obtained product is measured by colony counter according to GB4789.2-94 determination of colony count for national Standard of food sanitation and microbiology inspection of the people's republic of China, and the viable bacteria content in 0.25g product is 1.15 × 10 ¹⁰ CFU, and the same quality is the same conditionThe living bacteria contents of the products of 'Dingjunsheng', 'Yanhua' on the market measured by the same method are respectively 2.5 multiplied by 10 ⁵ CFU、6×10 ⁶ CFU。

Comparative example 1

This comparative example was the same as example 1 except that the extract of the Chinese medicinal herbs was not used but a minimal medium (1L of distilled water, 0.5% of glucose, 1.0g/L of ammonium nitrate, 0.5g/L of potassium dihydrogenphosphate, 1.5g/L of disodium hydrogenphosphate, 1.0g/L of sodium chloride, 0.2g/L of magnesium sulfate heptahydrate adjusted to pH 7.2) was used instead of the extract of the Chinese medicinal herbs.

Comparative example 2

This comparative example was compared with example 1, eliminating step (5), and the rest was the same as example 1.

Example 2

The finished products obtained in example 1, comparative example 1 and comparative example 2 were subjected to a relevant performance test:

(1) pH value: the pH of the finished product of example 1 was determined to be 4.2-4.5 (pH is the value required for the product to meet the human environment; two criteria are found for normal women, 3.8-4.4, 4.2-4.5).

While the pH of comparative example 1 was 5.7 and the pH of comparative example 2 was 6.3;

(2) Lactic acid content and stability:

detecting equipment: 1) BS224S of an electronic analytical balance, 2) ME155DU of the electronic analytical balance, 3) BSC-250 constant temperature and humidity chamber, 4) high performance liquid chromatograph U-3000, the lactic acid content of the combined prepared sample is 0.982%, after being stored for 90 days under the stability condition (37-40 ℃, 75% of relative humidity), the lactic acid content is 0.966%, and the reduction rate is 1.63%. The basis of the inspection is as follows: GB5009.157-2016, meets the Disinfection technical Specification 2002 edition, and the storage period of the sample obtained in example 1 is 2 years according to the relevant requirements of the enterprise standard Q/CDSF 02-2022;

while comparative example 1 had a storage life of 2 years and comparative example 2 had a storage life of 1.5 years.

(3) And (3) detecting lead and mercury: using a detection device: 1) an electronic analytical balance BS224S, 2) an atomic absorption spectrophotometer 990AFG, 3) an atomic fluorescence spectrophotometer AFS-8820, wherein the lead content of the preparation is less than 1.5mg/kg, the Kun content is less than 0.010mg/kg, and the mercury content is less than 0.002mg/kg through determination, so that the preparation meets the relevant requirements of the cosmetic safety technical specification 2015 edition.

(4) And (3) microbiological index detection:

detecting equipment: 1) STX1202ZH electronic balance, 2) common nutrient agar culture medium, sabouraud's agar culture medium, SCDLP liquid culture medium, lactose bile salt fermentation culture medium, blood agar plate, provided by Beijing land bridge technology, inc.; 3) HPX-9272MBE electrothermal constant temperature incubator, 4) carrier: filter paper (2.0cm x 3.0cm), sterile scale suction tube, sterile plate, sterile test tube, scissors, tweezers and alcohol lamp,

test strains: escherichia coli (8099), candida albicans (ATCC 10231) and Staphylococcus aureus (ATCC 6538) for 5 generations and Staphylococcus aureus for 4 generations, all provided by China center for culture Collection.

0.03mol/L Phosphate Buffer Solution (PBS)

The inspection basis is as follows: the test is directly carried out according to appendix B of GB15979-2002 sanitary Standard for Disposable sanitary articles and 2002 edition of Disinfection technical Specification.

The bacteriostatic test sample is a suspension test, the test temperature is 20 ℃, and the test is repeated three times. The preparation is applied for 20 minutes as it is, and the average bacteriostatic rates of the preparation on different (min) actions of Escherichia coli (8099), candida albicans (ATCC 10231) and Staphylococcus aureus (ATCC 6538) are respectively shown in the following table.

Example 1 bacteriostatic effect of samples on e.coli:

and the sample of comparative example 1 has bacteriostatic effect on Escherichia coli

Comparative example 2 bacteriostatic effect of sample on escherichia coli

Note: positive control group (Staphylococcus aureus broth culture/common agar), average number of growing colonies was 2.21X 10 ⁴ cfu/ml，

The negative control group (common broth) grows aseptically, and the test result has obvious bacteriostatic action.

Example 1 bacteriostatic effect of sample on staphylococcus aureus:

note: the average number of growing colonies in the positive control group was 2.36X 10 ⁴ cfu/ml。

The negative control group grows aseptically, and the test result has obvious bacteriostatic action.

And the sample of comparative example 1 has bacteriostatic effect on staphylococcus aureus

Comparative example 2 bacteriostatic effect of sample on staphylococcus aureus

Bacteriostatic effect of sample on candida albicans

Note: the average number of growing colonies in the positive control group was 2.51X 10 ⁴ cfu/ml；

The negative control group grows aseptically, and the test result has obvious bacteriostatic action.

And the bacteriostatic effect of the sample of comparative example 1 on the candida albicans

Comparative example 2 bacteriostatic effect of sample on Candida albicans

In addition, step (5) of example 1 acclimatized and modified Lactobacillus acidophilus

The effect of Lactobacillus acidophilus, lactobacillus delbrueckii subspecies Lactobacillus, lactobacillus crispatus and Lactobacillus jensenii on gathering Candida albicans is shown in figure 3, and as can be seen from figure 3, the combination of the Lactobacillus acidophilus, the Lactobacillus delbrueckii and the Lactobacillus jensenii forms an aggregation effect, and antibacterial substances are generated after the pathogenic bacteria are surrounded, so that the adsorption of the pathogenic bacteria is interfered.

(5) And (3) vagina mucosa stimulation test:

animal experiments: shanxi Jun row biotechnology limited 1) provides 6 New Zealand rabbits, female, with the weight of 2.0-2.5kg, license number: SYXK (Shaan) No. 2018-007, the feed source is the same as above.

The test was carried out according to the method 2.3.5 in the "Disinfection technical Specification" 2002 edition.

Dose of test substance: 2 ml/tube.

The test method comprises the following steps: the 6 non-estrous new zealand rabbits were randomly divided into 2 groups, namely a test subject group and a control group, and 3 rabbits were used in each group. During the test, the animal is fixed in the supine position, 2 ml/test object is injected into the test object group through the vagina, the same volume of normal saline is given to the control object, and the animal is fixed in the supine position for 4 hours, so that the test object is fully contacted with the vaginal mucosa. After the test is finished, the animals are returned to the cages for feeding, the animals are sacrificed after 24h, the vaginal tissues are taken out, the phenomena of blood vessel congestion, edema and the like are carefully observed by naked eyes, and if the phenomena are changed, the pathological histology examination is carried out.

And (3) test results: the observed shape of the vaginal mucosa of the rabbits in the test object group is normal without congestion and swelling. The test sample has an average score of 0 for the stimulation response to the vaginal mucosa of the rabbits. According to the classification standard of the vaginal mucosa irritation reaction in 2002 edition of the Ministry of health, the original sample has no irritation to the rabbit vaginal mucosa irritation reaction, and meets the relevant requirements of 2002 edition of the Ministry of health.

In addition, the domesticated and transformed strain in the step (5) is subjected to adhesion test with genital mucosal cells:

the following experimental materials were prepared:

fibronectin and Laminin;2.0.2% BSA (in PBS); 3.MTT;4, PBS; DMEM;6. a culture medium; 7. an enzyme-labeling instrument; 8. pipettors and tips;

the experimental procedure was as follows:

1) Adding fibrinectin (10. Mu.g/ml) or Laminin (10. Mu.g/ml) diluted with PBS to the enzyme label strip, and incubating overnight at 4 ℃;

2) After absorbing fibrinectin or lamin, washing with PBS for three times, blocking with 0.2% BSA at room temperature for 2 hours, and then washing with DMEM for three times;

3) Pancreatin digestion of cells, counting, dilution to 2.5X 105/ml cell suspension, adding 100. Mu.l cell dilution per well, and culturing at 37 ℃ for 30 minutes (the specific time is determined according to different cells); adding 100 mul DMEM hole as blank reference;

4) DMEM was washed three times to wash out non-adherent cells, and 200. Mu.l of DMEM was added.

5) The number of cells in the enzyme label was measured by MTT assay (see section "MTT assay for cell proliferation").

6) The percentage of adherent cells (%) = (OD assay well-OD control well)/OD 100 × 100%.

The results are shown in FIG. 2, indicating that: the domesticated and modified probiotics have good adhesion and colonization performance, can continuously exert the functions of maintaining the flora balance of the reproductive tract and reducing the probability of infection and recurrence of reproductive system inflammation.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. The directional domestication modified probiotic bacteria are characterized in that the modification method of the probiotic bacteria comprises the following steps:

(1) Weighing 5-8 parts of dendrobium, 1-3 parts of coptis chinensis, 1-3 parts of phellodendron, 2-4 parts of polygonum cuspidatum, 3-6 parts of fructus cnidii, 3-6 parts of radix sophorae flavescentis, 5-8 parts of elsholtzia and 1-2 parts of borneol, mixing, crushing and extracting to obtain an extracting solution and medicine residues, wherein the extracting solution is used as a fermentation substrate;

(2) Firstly, adding 13-18% of yeast and 5-8% of bacillus natto into the fermentation substrate obtained in the step (1) according to the total weight of the fermentation raw materials, carrying out aseptic aerobic constant-temperature fermentation for 24-30 h at 28-32 ℃, adding lactobacillus after stopping fermentation, carrying out anaerobic fermentation for 42-48 h, and carrying out constant-temperature enzymolysis for 2-4h after fermentation is completed to obtain a basic culture solution;

(3) Adding 2-4 times of purified water into the medicine residue obtained in the step (1), adding chicory inulin and lactic acid bacteria, fermenting at a constant temperature of 28-32 ℃, extracting, mixing an extracting solution with the basic culture solution obtained in the step (2), and performing pasteurization to obtain a composite culture medium;

(4) Domesticating the strain: mixing Lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus delbrueckii subspecies lactis (Lactobacillus), lactobacillus crispatus and Lactobacillus jensenii with the composite culture medium obtained in the step (3) for strain acclimatization to obtain the Lactobacillus acidophilus/Lactobacillus delbrueckii composite culture medium.

2. The targeted acclimatized, modified probiotic bacteria according to claim 1, characterized in that: in the step (1), the extraction method is subcritical low-temperature extraction: under the pressure of 0.88-5.15Mpa, performing countercurrent extraction on the material with liquid ammonia in subcritical state, evaporating the solvent in the extractive solution to separate the extracted target component from the solvent by vaporization, and obtaining the fermentation substrate.

3. The targeted acclimatized, modified probiotic bacteria according to claim 1, characterized in that: in the step (3), the concentration of the added chicory inulin is 180-220g/L, and the amount of the added lactic acid bacteria is 30-35% of the total mass of the fermentation raw materials; the fermentation temperature is 30-35 ℃, and the fermentation time is 20-30h; the extraction method comprises the following steps: extracting with liquid ammonia at 55-65 deg.C for at least two times, mixing extractive solutions, concentrating, and removing solvent.

4. According to claim 1The directional domestication modified probiotics is characterized in that: in the step (4), the domestication method comprises the following steps: mixing each fermentation strain with composite culture medium according to the ratio of 150-200 × 10 ⁸ Mixing and stirring the CFU fermentation strain/kg composite culture medium, and culturing at constant temperature of 35-38 deg.C for 10-15 hr to obtain subspecies of domesticated Lactobacillus jensenii, lactobacillus crispatus, lactobacillus acidophilus, and Lactobacillus delbrueckii.

5. The targeted acclimatized, modified probiotic bacteria according to claim 4, characterized in that: adding 2-5% of the domesticated lactobacillus jensenii, 3.5-5% of the domesticated lactobacillus crispatus, 2.5-5% of the domesticated lactobacillus acidophilus and 2-5% of the domesticated lactobacillus delbrueckii subspecies into a composite culture medium according to the total mass of fermentation raw materials, placing the mixture at the temperature of 40-42 ℃ for constant-temperature fermentation for 10-15h, and obtaining four kinds of fermentation raw solutions of the domesticated lactobacillus jensenii fermentation raw solution, the domesticated lactobacillus crispatus fermentation raw solution, the domesticated lactobacillus acidophilus fermentation raw solution and the domesticated lactobacillus delbrueckii subspecies fermentation raw solution respectively.

6. A microecological formulation comprising the probiotic of any one of claims 1 to 5, characterized in that: the preparation method of the microecological preparation comprises the following steps of respectively and uniformly mixing 3-5% of domesticated lactobacillus jensenii fermentation stock solution, 5-8% of domesticated lactobacillus crispus fermentation stock solution, 2-5% of domesticated lactobacillus acidophilus fermentation stock solution and 2-5% of domesticated lactobacillus delbrueckii subspecies fermentation stock solution with the composite culture medium according to the mass of the composite culture medium serving as a reference, adding bacillus natto with the mass of 3-6% of the composite culture medium, standing at the constant temperature of 30 ℃ for 2-4h, dissolving with purified water, sequentially adding 0.05-0.08% of polyhexamethylene biguanide, 1.8-2.4% of lactic acid and 3-5% of sodium hyaluronate according to the proportion of 0.15-0.3 per mill of the mass of the dissolved material, and uniformly mixing to obtain a semi-finished product;

and adding fructo-oligosaccharide with the percentage content of 2-7 percent based on the mass of the semi-finished product into the semi-finished product, uniformly mixing, and filling and packaging under aseptic condition to obtain the finished product.

7. The microecological formulation according to claim 6, wherein: the formulation is in a liquid or semi-solid or foam form.

8. The use of the probiotic of claim 6 for the preparation of a medicament for the prevention and treatment and for the adjuvant treatment of gynecological inflammation.

9. Use of the probiotic of claim 6 for the preparation of a feminine hygiene product for external use, for external genital hygiene products, for the prevention of gynecological inflammations, for the preparation of a medicament or a hygiene product for regulating the vaginal flora balance.

10. Use according to claim 8 or 9, characterized in that: the gynecological inflammation is caused by any one or more pathogenic bacteria of staphylococcus aureus, escherichia coli and candida albicans.

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