CN116622570B - Lactobacillus reuteri LR08, and application, product and method thereof in preparation of medicines for preventing and treating vaginitis - Google Patents
Lactobacillus reuteri LR08, and application, product and method thereof in preparation of medicines for preventing and treating vaginitis Download PDFInfo
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- CN116622570B CN116622570B CN202310575185.8A CN202310575185A CN116622570B CN 116622570 B CN116622570 B CN 116622570B CN 202310575185 A CN202310575185 A CN 202310575185A CN 116622570 B CN116622570 B CN 116622570B
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Abstract
The invention relates to lactobacillus reuteri LR08, and application, a product and a method for preparing a medicine for preventing and treating colpitis, belonging to the technical field of microorganisms. The Lactobacillus reuteri isLactobacillus reuteri) The preservation number of the strain LR08 is CGMCC NO.1.12733. The invention also provides application of the strain LR08 in-vitro bacteriostasis and/or preparation of products for adhering to vaginal epithelial cells and/or preparation of products for bacteriostasis and/or preparation of medicines for preventing and treating colpitis, and provides products for bacteriostasis, medicines for preventing and treating colpitis and products for adhering to vaginal epithelial cells based on the strain LR08. The strain LR08 has wide antibacterial spectrum and strong adhesion performance to vaginal epithelial cells, and can effectively prevent and treat colpitis, reduce colpitis score and cleanliness of colpitis model mice and regulate the level of each inflammatory factor.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus reuteri LR08, and application, a product and a method for preparing a medicine for preventing and treating colpitis.
Background
The vagina of female is a complex micro-ecological system, which consists of the anatomy of vagina, micro-ecological flora, local immunity and endocrine regulation function of organism, and the disease is governed by 3 factors of etiology, environment and host, but the core is the interrelation of normal microbiota and host. From a clinical perspective, the onset of various vaginitis is mostly associated with vaginal microecological imbalance, which can cause various female vulvovaginal discomfort.
At present, medicines such as antibiotics and the like are mainly used for clinic treatment; the drug treatment often causes the immunity of the patient to be reduced, so that the vaginal fungus phase of the patient is changed, the infection is further repeated, even pathogenic bacteria generate drug resistance to cause treatment barriers, in addition, the risk of potential drug side effects exists, the problem of no side effect and drug resistance is found, and the drug has higher curative effect and can avoid the replacement drugs or treatment schemes of recurrence, thus being an important issue related to recent colpitis research.
In addition, in the index for treating colpitis, besides the change of pathogenic bacteria and probiotics, symptoms such as inflammation of affected parts are involved in activation of immune response, so that molecular expression quantity related to immune regulation is also a reference basis, wherein IL-10 and IL-12 are immune regulation molecules which are produced in large quantity when invaded by pathogenic bacteria, the two are in synergistic expression, immune defense of a host is promoted, besides IL-10 and IL-12, IL-1 beta and IL-6 are used as pro-inflammatory cytokines when infected by bacteria, the occurrence of inflammatory response is regulated (IL-1 beta can promote phagocytosis of bacteria by immune cells of the host, IL-6 can assist neutrophils to remove bacteria in a non-phagocytic mode), and the expression quantity of the anti-inflammatory molecules can represent alleviation of the inflammatory response of the affected parts, so that the immune regulation factors can be used as a curative reference index for treating vaginal infection.
In view of the high threat and high infection rate caused by colpitis to women in recent years and the possible cross infection caused by different pathogenic bacteria, reports of inhibiting pathogenic bacteria and treating colpitis by adopting probiotics appear in the prior art, but the types of the strains reported at present are limited, the bacteriostasis spectrum is narrow, and the effect of reducing inflammatory factors is poor.
The development of new bacterial strains with strong adhesion and colonization capability and simultaneously inhibiting different pathogenic bacteria can improve the vaginal cleanliness and relieve inflammatory reaction, thereby effectively improving the prevention and treatment effects of clinical vaginitis and being an innovative requirement in the field.
Therefore, there is a need in the art to develop more new strains for better clinical treatment of vaginitis, greater ability to reduce inflammatory factors, better performance in all aspects, and broader antimicrobial spectrum.
Disclosure of Invention
Aiming at solving the problems of narrow bacteriostasis spectrum, poor effect of reducing inflammatory factors and the like in the prior art, the invention provides a novel screened lactobacillus reuteriLactobacillus reuteri) Strain LR08 and its use, products and methods for the preparation of products for adhesion to vaginal epithelial cells and/or bacteriostatic products.
The technical scheme of the invention is as follows:
lactobacillus reuteri @Lactobacillus reuteri) Strain LR08, characterized in that its preservation number is CGMCC No.1.12733.
Lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) The application of the strain LR08 in-vitro bacteriostasis and/or preparation of products for adhering vaginal epithelial cells and/or preparation of bacteriostasis products and/or preparation of medicines for preventing and treating colpitis is characterized in that the bacteriostasis refers to: inhibit one or more of Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, and Prevotella intermedia.
The vaginal epithelial cells refer to VK2/E6E7 cells;
preferably, the prevention and treatment of colpitis means reducing inflammation score, and/or improving cleanliness, and/or regulating inflammatory factor content;
preferably, the inflammatory factor comprises: TNF-alpha, IL-1 beta, IL-6, IL-10;
preferably, said modulating the inflammatory factor content comprises: lowering levels of TNF- α and/or IL-1β and/or IL-6, and/or increasing levels of IL-10.
A bacteriostatic product, which is prepared from the following components,comprises antibacterial active ingredients; the antibacterial active ingredients comprise: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
The antibacterial product also comprises: auxiliary materials.
A medicine for preventing and treating colpitis, which comprises active ingredients with medicinal effects; the pharmaceutically active ingredients include: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
The medicine for preventing and treating colpitis further comprises: pharmaceutically acceptable auxiliary materials.
A product for adhering to vaginal epithelial cells, comprising: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
An in vitro antibacterial method adopts Lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Bacterial strain LR08 inhibits bacteria.
The bacteriostasis refers to: inhibit one or more of Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, and Prevotella intermedia.
The invention provides a Lactobacillus reuteri on the one handLactobacillus reuteri) LR08. The lactobacillus reuteri LR08 is self-produced bacterial powder, which takes antibacterial performance as an index to screen a self-owned bacterial resource library of the applicant, and has broad-spectrum antibacterial characteristics to vaginal pathogenic bacteria, which are not possessed by other bacterial strains. Lactobacillus reuteri has been deposited in the China general microbiological culture Collection center, designated by the classification: lactobacillus reuteri @Lactobacillus reuteri) The preservation number is CGMCC NO.1.12733, the preservation date is 2020, 07 and 20, and the preservation unit address is: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.
The lactobacillus reuteri LR08 of the invention can produce various metabolites including teichoic acid, peptidoglycan, acidic substances, lactic acid bacteria, reuterin and H 2 O 2 Etc. Wherein lipoteichoic acid and peptidoglycan have adhesion effect on epithelial cells, so as to form space occupation to achieve the purpose of preventing pathogenic bacteria from adhering to vaginal epithelial cells; the metabolites such as acidic substances can consume a large amount of cell energy and influence the stability of cell membranes, thereby achieving the purpose of inhibiting the proliferation of pathogenic bacteria.
Another aspect of the invention provides the above Lactobacillus reuteriLactobacillus reuteri) Application of LR08 in-vitro bacteriostasis and/or preparation of products adhered to vaginal epithelial cells and/or preparation of bacteriostasis products and/or preparation of medicines for preventing and treating colpitis. The invention detects the average level of cell inflammatory factors TNF-alpha, IL-1 beta, IL-6 and IL-10, researches the effect of lactobacillus reuteri in cell inflammatory factor expression, and researches the action mechanism of lactobacillus reuteri in vaginal bacteriostasis, thus providing basis for developing microecological preparations.
The lactobacillus reuteri of the invention has stronger capability of antagonizing candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, prasugrel intermedium and other vaginal pathogenic bacteria,
the lactobacillus reuteri has strong adhesiveness, the capability of adhering to VK2/E6E7 cells is as high as 116.10CFU/cell, the adhesiveness rate is 71.47 percent, and the lactobacillus reuteri is obviously higher than that of commercial strains lactobacillus rhamnosus GR-1 and lactobacillus reuteri RC-14.
The results of the study compared with the adhesion force of gardnerella vaginalis, escherichia coli, staphylococcus and candida albicans on vaginal epithelial cells show that the adhesion capacity of lactobacillus reuteri LR08 on vaginal epithelial cells VK2/E6E7 is obviously higher than that of the escherichia coli, the staphylococcus and the candida albicans; lactobacillus reuteri can strongly inhibit adhesion of gardnerella vaginalis, escherichia coli, staphylococcus and candida albicans to vaginal epithelial cells. The space occupation formed by the adhesion of lactobacillus reuteri is an important protective mechanism for preventing the adhesion of other bacteria to vaginal tissue.
The lactobacillus reuteri has excellent acid resistance, and the survival rate of the lactobacillus reuteri reaches 98.9% after the lactobacillus reuteri is incubated for 3 hours in a pH4 environment. The strain can be planted and survived in normal vagina environment, and has biological characteristics for preparing vagina health cleaning products.
The lactobacillus reuteri is proved to be sensitive to common antibiotics, has no drug resistance, and is safe and has no side effect through in vitro experiments.
In a mouse colpitis model, metronidazole is used as a control, and after intervention treatment by using lactobacillus reuteri, the result shows that the cure rate of the lactobacillus reuteri on the mouse mixed bacteria colpitis is 90%, the inflammatory response of uterus is obviously improved, the phenomena of tissue congestion and edema are reduced, the expression of pro-inflammatory factors IL-1 beta, TNF-alpha and IL-6 in mouse serum is reduced by the lactobacillus reuteri, and the expression of anti-inflammatory factors IL-10 is improved, so that the lactobacillus reuteri has positive effects on recovery of the mouse colpitis.
The lactobacillus reuteri has the characteristic of high viable count, and the viable count of the lactobacillus reuteri reaches 20 hundred million CFU/mL after the lactobacillus reuteri is subjected to small-scale fermentation culture, so that the lactobacillus reuteri has the potential of realizing industrial production.
The invention screens and obtains the lactobacillus reuteri which has wide antibacterial spectrum and strong adhesion performance to vaginal epithelial cells by taking antibacterial performance as a targetLactobacillus reuteri) The strain LR08 is proved to be a strain which is suitable for the vaginal environment, is sensitive to antibiotics and is safe to human body by experimental verification of acid resistance, antibiotic sensitivity and the like. Meanwhile, the mouse experiment further proves that the strain can effectively prevent and treat colpitis, and the colpitis score, cleanliness and inflammatory factor level of a colpitis model mouse are reduced. The invention provides lactobacillus reuteriLactobacillus reuteri) The strain LR08 can be used for preparing products adhered to vaginal epithelial cells, antibacterial products and medicines for preventing and treating colpitis, and has higher industrial production value.
The invention relates to lactobacillus reuteriLactobacillus reuteri) The deposit information for strain LR08 is as follows:
strain name: LR08;
deposit number: CGMCC No. 1.12733;
classification naming: lactobacillus reuteriLactobacillus reuteri；
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
deposit unit address: beijing, chaoyang area, north Chenxi Lu No.1, 3;
preservation date: 2020, 07, 20.
Drawings
FIG. 1 is an analysis of the ability of L.reuteri LR08 of experimental example 2 of the present invention to inhibit adhesion of pathogenic bacteria to VK2/E6E7 cells.
FIG. 2 is a graph showing the cleanliness ratings of each experimental group of experimental example 6 according to the present invention.
FIG. 3 shows the expression level of inflammatory factor TNF-. Alpha.in each experimental group of Experimental example 6 according to the present invention.
FIG. 4 shows the expression level of inflammatory factor IL-1. Beta. In each experimental group of Experimental example 6 according to the present invention.
FIG. 5 shows the expression level of inflammatory factor IL-6 in each experimental group of Experimental example 6 according to the present invention.
FIG. 6 shows the expression level of inflammatory factor IL-10 in each experimental group of Experimental example 6 according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples, experimental examples and drawings, but the scope of the present invention is not limited thereto.
Sources of biological materials
1. Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus used in experimental example 1 and experimental example 3 of the present invention were purchased from the cantonese microorganism strain collection, and prasugrel intermedia was obtained from ATCC.
2. The VK2/E6E7 cells used in Experimental example 2 and Experimental example 3 of the present invention are commercially available.
3. Commercial strains GR-1 and RC-14 used in Experimental example 2 of the present invention are commercially available.
4. The mice used in experimental example 6 of the present invention are commercially available.
Lactobacillus reuteri strain LR08, lactobacillus reuteri LR08, strain LR08, all refer to LR08 herein: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
The terms "above" and "below" as used herein include the present numbers.
Group 1 example, strain LR08 of the invention
The embodiment of the group provides lactobacillus reuteri @Lactobacillus reuteri) Strain LR08. All embodiments of this group share the following common features: lactobacillus reuteri @Lactobacillus reuteri) The preservation number of the strain LR08 is CGMCC NO.1.12733.
Any one cultivates, propagates, ferments, enriches, produces, prepares, uses, inoculates, amplifies, transforms, modifies, reforms, utilizes, sells, offers to sell, imports, exports and reserves the number CGMCC NO.1.12733 lactobacillus reuteri @Lactobacillus reuteri) Behavior of strain LR08 using Lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) The strain LR08 can prevent/treat genital tract infection, and/or prepare medicines for preventing and treating genital tract infection, and/or inhibit bacteria, and/or be compatible with traditional Chinese medicines, and/or be prepared into traditional Chinese medicine compound preparations, and/or be prepared into probiotic compositions, and/or be prepared into microbial agents, which fall into the protection scope of the invention.
According to the actual production requirement, the person skilled in the art can combine the conventional technical means or the common general knowledge of the production process in the pharmaceutical field (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) to perform conventional selection or adjustment on the pharmaceutical auxiliary materials, so as to prepare the medicaments with different dosage forms, different storage conditions and different shelf lives, which is free from technical barriers and can be easily achieved for the person skilled in the art.
Group 2 examples, use of strain LR08 of the invention
The embodiment provides lactobacillus reuteri with the preservation number of CGMCC No.1.12733Lactobacillus reuteri) The application of the strain LR08 in-vitro bacteriostasis and/or preparation of products for adhering to vaginal epithelial cells and/or preparation of bacteriostasis products and/or preparation of medicines for preventing and treating colpitis.
In a specific embodiment, the bacteriostasis means: inhibit one or more of Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, and Prevotella intermedia.
In some embodiments, the vaginal epithelial cells refer to VK2/E6E7 cells;
preferably, the prevention and treatment of colpitis refers to reducing the inflammation score, and/or, cleanliness, and/or, inflammatory factor content;
preferably, the inflammatory factor comprises: TNF-alpha, IL-1 beta, IL-6, IL-10.
According to the invention, in view of different requirements in practical production and application, in combination with conventional technical means in the field of medicine preparation (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like), a person skilled in the art can select and blend common auxiliary materials and store lactobacillus reuteri with the preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08 is formulated into various dosage forms such as powder, tablet, injection, oral liquid, capsule, granule, spray, gel, paste, suppository, lotion, etc.
Group 3 examples, bacteriostatic products of the invention
The present set of embodiments provides a bacteriostatic product. All embodiments of this group share the following common features: the antibacterial product comprises antibacterial active ingredients; the antibacterial active ingredients comprise: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
In a further embodiment, the bacteriostatic product further comprises: auxiliary materials.
In particular embodiments, the adjuvants include, but are not limited to: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, protectants, additives, and the like.
In some embodiments, the adjunct is a protectant.
Preferably, the protective agent is selected from: skimmed milk powder and/or soybean milk powder.
In other embodiments, the adjunct is an additive.
The additive comprises any one or a combination of at least two of rose essential oil, propolis, sodium hyaluronate, glycerol or collagen.
According to the invention, the person skilled in the art can select and blend the auxiliary materials and prepare lactobacillus reuteri (Lactobacillus reuteri) strain LR08 with the preservation number of CGMCC No.1.12733 into different dosage forms, such as powder, tablets, injections, oral liquids, capsules, granules, sprays, gels, ointments, suppositories, lotions and the like, according to different requirements in practical production and application and in combination with conventional technical means in the field of medicament preparation (for example, encyclopedia of formulation technology, pharmaceutical preparation technology and the like).
Group 4 example, the medicine for preventing and treating colpitis of the invention
The embodiment provides a medicine for preventing and treating colpitis. All embodiments of this group share the following common features: the medicine for preventing and treating colpitis comprises active ingredients with medicinal effects; the pharmaceutically active ingredients include: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
In a further embodiment, the medicine for preventing and treating colpitis further comprises: pharmaceutically acceptable auxiliary materials.
In specific embodiments, the pharmaceutically acceptable excipients are selected from one or more of the following: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, protectants, additives, and the like.
In some embodiments, the pharmaceutically acceptable adjuvant is a protective agent.
Preferably, the protective agent is selected from: skimmed milk powder and/or soybean milk powder.
More preferably, the protective agent is skimmed milk powder and soybean milk powder with the mass ratio of (1-3) to (1-3).
Preferably, in the medicament, the mass ratio of the protective agent to the lactobacillus reuteri LR08 is (1-10) to 1.
In other embodiments, the pharmaceutically acceptable adjuvant is an additive.
The additive comprises any one or a combination of at least two of rose essential oil, propolis, sodium hyaluronate, glycerol or collagen.
In a specific embodiment, the medicine for preventing and treating colpitis can be prepared into an external product or an oral product for maintaining the vaginal health of women.
The external product comprises at least one of cleaning products (such as washing liquid, bath lotion, cleaning soap and the like), nursing products (nursing liquid, nursing cream and the like), disposable sanitary products (such as sanitary napkins, wet tissues, tampons and the like), external medicines (dosage forms including but not limited to lotion, tincture, emulsion, ointment, plaster and the like) or external medical devices (such as colposcope or hysteroscope and the like).
The oral products include oral medications (including, but not limited to, solutions, extracts, powders, pills, granules/powders, capsules, tablets, pills, etc. by dosage form classification).
Preferably, in the external product or oral product, the viable count of the lactobacillus reuteri LR08 strain is not less than 1×10 8 CFU/mL。
The cleaning product for maintaining female vaginal health comprises lactobacillus reuteri LR08 or a microbial agent and an additive thereof, wherein the lactobacillus reuteri LR08 has strong antibacterial capability and adhesion capability.
Group 5 example, vaginal epithelial cell adhesion product of the invention
This set of examples provides a product that adheres to vaginal epithelial cells. All embodiments of this group share the following common features: the product for adhering to vaginal epithelial cells comprises: lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08.
In a specific embodiment, the vaginal epithelial cells are VK2/E6E7 cells.
In a further embodiment, the product for adhering to vaginal epithelial cells further comprises: auxiliary materials.
In particular embodiments, the adjuvants include, but are not limited to: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, protectants, additives, and the like.
In some embodiments, the adjunct is a protectant.
Preferably, the protective agent is selected from: skimmed milk powder and/or soybean milk powder.
In other embodiments, the adjunct is an additive.
The additive comprises any one or a combination of at least two of rose essential oil, propolis, sodium hyaluronate, glycerol or collagen.
According to the invention, the above auxiliary materials can be selected and adjusted by those skilled in the art according to different requirements in practical production and application, in combination with conventional technical means in the field of medicine preparation (e.g. encyclopedia of preparation technology, pharmaceutical preparation technology, etc.)Preparing lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) Strain LR08 is formulated into various dosage forms such as powder, tablet, injection, oral liquid, capsule, granule, spray, gel, paste, suppository, lotion, patch, etc.
Group 6 example, in vitro method of inhibiting bacteria of the invention
The present set of embodiments provides an in vitro bacteriostasis method. All embodiments of this group share the following common features: adopts lactobacillus reuteri with the preservation number of CGMCC No.1.12733Lactobacillus reuteri) Bacterial strain LR08 inhibits bacteria.
In some embodiments, the bacteriostatic means: inhibit one or more of Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, and Prevotella intermedia.
Experimental example 1, antibacterial screening
The screened lactobacillus is derived from a micro Kang Yisheng strain resource library, and is transferred to a 2-generation (10 ml/15ml centrifuge tube) with an inoculum size of 2% after a first-generation activation culture is carried out from a strain library glycerol tube, and is used for subsequent experiments after 16h of culture.
Preparation of indicator bacteria suspension: activating Gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus and Prevotella intermedia on plate culture medium, selecting thallus Porphyrae to normal saline to prepare bacterial suspension, and regulating bacterial concentration to 10 8 CFU/mL; candida albicans is subjected to shake flask YPD liquid activation culture for 16 hours, and the concentration of bacterial liquid is adjusted to 10 8 CFU/mL。
Bacteriostasis experiment: cooling YPD culture medium, 5% defibrinated sheep blood-containing BHI culture medium, eosin blue agar culture medium, BG agar culture medium, LB agar culture medium to 55deg.C, sequentially mixing with Candida albicans, gardnerella vaginalis, prevotella intermedia, escherichia coli, salmonella, and Staphylococcus aureus suspension at a certain ratio to obtain viable count of indicator bacteria at 10 6 On the order of CFU/mL, then rapidly poured into a plate pre-placed in an oxford cup, and the medium cooled to condenseAfter fixation, the oxford cup was removed, and 200uL of lactic acid bacteria fermentation broth (viable count 10) was injected into each well 8 CFU), after overnight incubation at 37 ℃, the diameter of the zone of inhibition was measured.
As shown in Table 1, a strain having different degrees of inhibition on Candida albicans, gardnerella vaginalis, escherichia coli, staphylococcus aureus, salmonella, prevotella intermedia was selected and identified as Lactobacillus reuteriLactobacillus reuteri) This was designated LR08.
TABLE 1 inhibition of pathogenic bacteria by Lactobacillus reuteri LR08
The deposit information for strain LR08 is as follows:
strain name: LR08;
deposit number: CGMCC No. 1.12733;
classification naming: lactobacillus reuteriLactobacillus reuteri；
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection);
deposit unit address: beijing, chaoyang area, north Chenxi Lu No.1, 3;
preservation date: 2020, 07, 20.
Experimental example 2, analysis of ability to adhere VK2/E6E7 cells
Liquid culture of experimental strains: the culture was allowed to stand at 37℃for 15 hours using a 2-generation activated bacteria solution, 2% inoculum size, 9mL medium/15 mL centrifuge tube.
Preparation of a single cell layer: inoculating resuscitated VK2/E6E7 cells into DMEM medium supplemented with 10% (v/v) fetal bovine serum, transferring to 12-well cell culture plate, and adding 1.5X10 cells per well 1mL 5 cell/ml, 5% CO 2 Culturing at 37 ℃ for 69 hours to obtain a single cell layer for later use (culturing for 24 hours, changing liquid, culturing for 48 hours, continuously changing liquid, and culturing for 24 hours again);
preparation of bacterial suspension: at the same time as the time of this,collecting the cultured target strain bacterial liquid, centrifuging at 10000 r/min at room temperature for 1 min to collect bacterial cells, washing twice with sterile PBS, and re-suspending in DMEM culture medium (adjusting live lactobacillus bacterial count to 1×10) 8 CFU/mL）；
Co-cultivation: sucking the culture medium from the single cell layer of the prepared VK2/E6E7 cells, adding PBS buffer solution for rinsing for 2 times, sucking the buffer solution, adding the prepared bacterial suspension 1 mL/hole, uniformly mixing, and respectively incubating at 5% CO2 and 37 ℃ for 2 hours and 4 hours; carefully remove the culture supernatant, add sterile PBS and rinse 3 times to remove non-adherent bacteria;
adding pancreatin cell digestive juice 0.2 mL/hole, digesting for 5min to separate cells from cell culture plate hole wall, collecting solution as sample;
the collected samples were subjected to gradient dilution and viable counts. The adhesion capacity is calculated as follows:
adhesion capacity (CFU/cell) =number of cells in Cell (CFU)/number of cells adhered to the cells.
Adhesion (%) = number of cells adhered to Cells (CFU)/total number of cells added to the well x100%
As shown in the experimental results of Table 2, the capacity of the Lactobacillus reuteri LR08 to adhere to VK2/E6E7 cells for 4 hours is as high as 121.23 CFU/cell, the adhesion rate is as high as 76.12%, and the adhesion rate is remarkably higher than that of commercial strains GR-1 (4.98 CFU/cell, adhesion rate of 2.54%) and RC-14 (41.47 CFU/cell, adhesion rate of 24.35%). It is well demonstrated that lactobacillus reuteri LR08 has a strong vaginal epithelial cell adhesion capacity. The stronger the adhesion capability is, the stronger the strain implantation capability is proved, and the strain is easier to be remained in the vaginal environment for reproduction, thereby playing the roles of inhibiting pathogenic bacteria, protecting vaginal mucosa and recovering vaginal flora, and achieving the curative effect of treating or preventing vaginal infection.
TABLE 2 Lactobacillus reuteri LR08 ability to adhere to VK2/E6E7 cells and adhesion Rate
Experimental example 3 Lactobacillus reuteri inhibition of pathogenic bacteria from adhering to vaginal epithelial cells VK2/E6E7 analysis
Preparation of VK2/E6E7 single cell layer: inoculating resuscitated VK2/E6E7 cells into DMEM medium supplemented with 10% (v/v) fetal bovine serum, transferring to 12-well cell culture plate, and adding 1.5X10 cells per well 1mL 5 cell/ml, 5% CO 2 Culturing at 37 ℃ for 69 hours to obtain a single cell layer for later use (culturing for 24 hours, changing liquid, culturing for 48 hours, continuously changing liquid, and culturing for 24 hours again);
preparation of lactobacillus reuteri LR08 suspension: after the lactobacillus is activated and cultured in MRS culture medium, the lactobacillus is centrifuged, supernatant is removed, PBS is washed 3 times, RPMI1640 culture medium is resuspended, and the final concentration is adjusted to 10 8 CFU/mL (colony count and OD) 600 Is measured).
Preparation of pathogenic bacteria suspension: the pathogenic bacteria are respectively activated and cultured on YPD/BHI agar, single colony is selected and inoculated into DMEM culture medium, and the final concentration is adjusted to 10 7 CFU/mL (blood cell count plate count).
Lactobacillus reuteri cultured overnight was used to obtain lactobacillus reuteri LR08: the effect of Lactobacillus reuteri on pathogen adhesion was evaluated at a VK2/E6E7 ratio of 1000:1. A. Adhesion repellency test: 1*10 8 Lactobacillus reuteri and vaginal epithelial cells at 37℃with 5% CO 2 Co-cultivation under incubator 1 h. Then add 1 x10 7 Pathogenic bacterial cells, incubating for 1h, pH4.5.B. Competitive adhesion test: 1*10 8 Lactobacillus reuteri and 1 x10 7 Pathogenic bacteria are added to the VK2/E6E7 cell layer in a mixture, and then at 37 ℃ 5% CO 2 Incubating under the incubator for 2 hours at pH4.5. C, adhesion shift experiment: 1*10 7 Pathogenic bacteria were incubated with VK2/E6E7 cells at 37℃for 1.cndot. 1h, 1.cndot.10 was added 8 After further incubation for 1h (pH 4.5) after lactobacillus reuteri, the culture supernatant was carefully removed and rinsed 3 times with sterile PBS to remove non-adherent bacteria.
Adding pancreatin cell digestive juice 0.2 mL/hole, digesting for 5min to separate cells from cell culture plate hole wall, collecting solution as sample; samples were diluted in appropriate multiples, lactobacillus reuteri was counted in MRS medium, candida albicans was counted in YPD medium, bacteria such as escherichia coli were counted in LB medium, and the results of the above three experiments were used as a control group to calculate pathogen adhesion inhibition rate by using the average number of pathogen adhering to VK2/E6E7 cells without lactobacillus adhesion. Adhesion experiments were performed 3 times, with at least 3 replicates per group.
Adhesion inhibition = 1-number of viable bacteria of pathogenic bacteria in test wells of added amount of probiotics/number of viable bacteria of pathogenic bacteria in control wells x100%
As shown by the experimental result of experimental example 2, the lactobacillus reuteri LR08 has strong capability of adhering to the VK2/E6E7 cells, so that the pathogenic bacteria can be effectively inhibited from adhering to the VK2/E6E7 cells. As shown in the experimental results of Table 3, in the rejection, competition and replacement experiments, the adhesion capacity of the Lactobacillus reuteri LR08 to the vaginal epithelial cells VK2/E6E7 is obviously higher than that of Candida albicans, escherichia coli and Staphylococcus aureus; as can be seen from the experimental results in FIG. 1, in the rejection experiment, the adhesion inhibition rates of the Lactobacillus reuteri LR08 on Escherichia coli, staphylococcus aureus and Candida albicans were as high as 37.27%,24.95% and 44.42%, respectively. In competition experiments, the adhesion inhibition rate of lactobacillus reuteri LR08 on escherichia coli, staphylococcus aureus and candida albicans is up to 47.52%,27.96% and 54.66% respectively; in the substitution experiments, the adhesion inhibition rate of lactobacillus reuteri LR08 on escherichia coli, staphylococcus aureus and candida albicans is up to 48.60%,24.26% and 46.50%, respectively.
TABLE 3 number of bacteria adhering to vaginal epithelial cells VK2/E6E7
Experimental example 4 evaluation of acid resistance of Lactobacillus reuteri LR08
Taking out strain preserved at-80deg.C, inoculating into centrifuge tube containing 10 mM RS liquid medium at 2%, culturing at 37deg.C for 20 hr, centrifuging at 10000rpm for min, removing supernatant, and washing with PBS for 2 times to obtain suspension with concentration of (10) 9 CFU/mL). Taking 100uL heavy suspension to 900uL of sterile PBS with different pH values (pH 2.0, pH2.5, pH3.0 and pH 4.0), performing anaerobic stationary culture at 37deg.C, and respectively beginning [ ] at the beginning0h) And sampling after the treatment (3 h), measuring the number of viable bacteria by a pour culture method and calculating the survival rate thereof, wherein the formula is as follows: survival (%) =n 1 /N 0 *100%, wherein N1: viable count after 3h of treatment with sterile PBS of different pH; n0: viable count of 0 h. The measurement results are shown in Table 4.
As can be seen from Table 4, the Lactobacillus reuteri LR08 has strong acid resistance, and the survival rate is up to more than 95.4% after incubation for 3 hours at pH 2.5; at pH3.0 and 4.0, the survival rate is close to 100%. The pH value of the vagina of a normal healthy female is about 4.0, so that lactobacillus reuteri has excellent acid resistance, can be planted in the vagina normally, and also provides conditions for preparing a cleaning product for maintaining the healthy environment of the vagina.
TABLE 4 evaluation of acid resistance of Lactobacillus reuteri LR08
Experimental example 5, evaluation of safety of Lactobacillus reuteri LR08
(1) Evaluation of susceptibility to common antibiotics
Marking and activating Lactobacillus reuteri LR08 on MRS solid plate, picking up thallus Porphyrae, adding into physiological saline to prepare bacterial suspension, and adjusting concentration of bacterial suspension to 10 8 CFU/mL, taking 100 mu L of bacterial suspension, uniformly coating the bacterial suspension on an MRS solid flat plate by using a sterile cotton swab, orderly placing antibiotic drug sensitive test paper sheets on the surface of the flat plate, placing the flat plate under the facultative anaerobic condition, culturing at 37 ℃ for 24-36h, and measuring the diameter of a bacteriostasis ring by using a vernier caliper.
According to the evaluation standard of the American clinical and experimental standards institute CLSI, the drug resistance of the antibiotics to the lactobacillus reuteri LR08 is judged, the result is shown in Table 5, and the lactobacillus reuteri LR08 is sensitive to most antibiotics, which indicates that the strain is safe and can be potentially used for biological products.
TABLE 5 sensitivity of Lactobacillus reuteri LR08 to antibiotics
Note that: r-insensitivity; i-intermediation; s-sensitivity
(2) Hemolysis test
The strain was subjected to a hemolysis experiment using Streptococcus pyogenes as a control. Transparent hemolysis rings appear around colonies after the streptococcus pyogenes is inoculated on a Columbia agar plate for culture, and the bacteria are B type hemolysis (beta hemolysis); after lactobacillus reuteri LR08 is inoculated in the culture medium, a hemolytic ring does not appear around a colony, so that the strain is judged to have no hemolysis and does not generate hemolytic hazard.
Experimental example 6, mouse vaginitis model
1. Establishment of mouse colpitis model:
taking 40C 57CL/6J mice which are female, 8 weeks old and have a weight of 20-25g, and dividing the mice into 4 groups, wherein each group is a healthy control group (CTL group), a model group, a metronidazole treatment group (metronidazole group) and a lactobacillus reuteri LR08 group (LR 08 group), and 10 mice in each group are treated by the model group;
modeling of bacterial infected mice: mice in both model and treatment groups were subcutaneously injected with 100ul estradiol valerate to induce oestrus in the mice 2 times per week. Mice in the postestrus model group and the treatment group were inoculated with 10ul of bacteria at a concentration of 1X 10 in the vagina 7 CFU/mL gardnerella vaginalis and 10ul bacteria concentration of 1X 10 7 CFU/mL staphylococcus aureus, once daily, was inoculated continuously for 3 days until the molding was completed.
Treatment of infected mice: healthy control mice were not treated; mice in the model group were injected intravaginally with 20ul of physiological saline; the administration dosage of the metronidazole treatment group is 8.1mg/200g according to the purchased metronidazole drug specification and the equivalent dosage conversion; 40.5mg of metronidazole was dissolved in 250ul of sterile PBS to prepare a 0.16g/mL solution, giving a dose of 8.1mg/200g per mouse. Lactobacillus reuteri LR08 mice were inoculated intravaginally at a 20ul concentration of 1X 10 7 CFU/mL of Lactobacillus reuteri LR08 was inoculated 1 time per day for 10 consecutive days.
Experimental mice were sacrificed for cervical dislocation. Dissecting and taking the uterine tissue of the mouse, partially placing the uterine tissue in a 4% paraformaldehyde solution for preservation, and directly placing the uterine tissue of the mouse in a refrigerator at the temperature of minus 80 ℃ for later use.
2. Detection of various indexes after treatment of animal experiment colpitis model
(1) Classification of vaginal cleanliness: vaginal secretions are smeared on a glass slide with 1-2 drops of physiological saline, examined under a microscope and classified according to Table 6
TABLE 6 vaginal cleanliness grading Table
(2) Degree of inflammation of vaginal lesions and cure rate
The mouse anatomy was sacrificed on day 7 of drug withdrawal and vaginal specimens were taken for histopathological examination. The vaginal specimen is scored according to 4 basic indexes of congestion, edema, bleeding and infiltration, the sum of the inflammation scoring values of each index is the total inflammation score value, the score of the model group is 100% of the inflammation degree, the inflammation degree of the administration group can be obtained by comparing the administration group with the model group, and the inflammation degree of the administration group subtracted by 100% is the cure degree of colpitis (100% -drug group inflammation degree%)
TABLE 7 mice vaginal mucosal inflammatory stimulus scoring criteria
(3) Measurement of cytokines TNF-alpha, IL-1 beta, IL-6 and IL-10.
The mice are killed by taking about 3mL of blood through cardiac puncture, blood is collected by blood collection tubes of corresponding groups, after standing for 1h at room temperature, the blood is coagulated, serum is separated out, a pipette is used for transferring the upper serum, the serum is sucked into a new centrifuge tube, the centrifuge tube containing the serum is placed into a high-speed centrifuge with the temperature of 4 ℃ and 3000rmp, the centrifuge is carried out for 15min, and the centrifuge tube is immediately placed into a refrigerator with the temperature of minus 80 ℃ for freezing for standby. The average levels of cytokines TNF-alpha, IL-1 beta, IL-6 and IL-10 in the serum of each group of mice were examined by strictly following the instructions of ELISA kit.
3. Results and analysis
(1) From the results of the vaginal cleanliness test shown in fig. 2, the vaginal cleanliness of mice in the model group is mainly concentrated in level 3 and level 4, and the level of the vaginal cleanliness induced by the mixed bacteria is remarkably improved compared with that of the CTL group, which indicates that modeling of the vaginitis animal model combined with bacterial infection is successful. Compared with the model group, the LR08 group and the metronidazole group have obviously reduced 4-level cases, obviously increased 2-level cases and obviously improved vagina cleanliness of mice, and meanwhile, the 4-level cases of the LR08 group are less than those of the metronidazole group, which indicates that the treatment effect of the LR08 group is optimal and is not inferior to that of the metronidazole group.
(2) As shown in Table 8, the cure rates of the LR08 group and the metronidazole group on the mixed bacterial vaginosis of mice are 90% and 86.67%, and the cure rates are significantly different (P < 0.05) compared with the model control group.
TABLE 8 degree of inflammation of vaginal lesions and cure rate
(3) Cytokine TNF-alpha, IL-1 beta, IL-6, IL-10 expression
From the results shown in FIGS. 3-6, it can be seen that the mice in the vaginitis model group have much higher levels of pro-inflammatory factors TNF-alpha, IL-1β, IL-6 than the healthy control group, and the anti-inflammatory factor IL-10 is much lower than the healthy control group; the modeling is shown to raise the content of TNF-alpha, IL-1 beta and IL-6 in serum of mice in the model group, reduce the content of IL-10, and effectively reduce the content of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum after being treated by metronidazole or lactobacillus reuteri LR08, and obviously raise the content of IL-10. Wherein the inflammatory factors TNF-alpha, IL-1 beta, IL-6 and IL-10 content of the metronidazole group are not significantly different from that of the lactobacillus reuteri LR08 group. The lactobacillus reuteri LR08 strain can obviously reduce the content of TNF-alpha, IL-1 beta and IL-6 in vaginal tissues and improve the content of IL-10; the effect of relieving the colpitis of mice infected by the combined bacteria is not inferior to that of metronidazole; meanwhile, the lactobacillus has the effects of adjusting the vaginal microecology and preventing the recurrence of colpitis, which is incomparable with antibiotics. Therefore, the lactobacillus reuteri LR08 has wide application prospect as a microecological preparation for preventing or treating colpitis and regulating the vaginal immunity.
Experimental example 7 preparation of cleaning products containing Lactobacillus reuteri LR08
The experimental example provides a cleaning product of a liquid preparation of lactobacillus reuteri LR08 for maintaining vaginal health, the specification is 2g multiplied by 30 pieces/box, and the number of viable bacteria in each piece (2 g) is about 2 multiplied by 10 8 The CFU is clean and antibacterial, has good effect of maintaining vagina environment, is convenient to carry and use, and is prepared by the following steps:
preparation of lactobacillus reuteri LR08 lyophilized powder: and taking out the lactobacillus reuteri LR08 strain preservation tube from the micro Kang Yisheng strain library, carrying out 2-generation activation culture on the lactobacillus reuteri LR08 by using an MRS culture medium for 8 hours, inoculating 2% of strain inoculating amount of seeds into the MRS culture medium for fermentation culture for 11 hours, and taking the fermentation culture for viable count, wherein the concentration of the lactobacillus reuteri LR08 in the fermentation culture can reach 20 hundred million CFU/mL, which indicates that the viable count of the lactobacillus reuteri is high, and the method has the potential of realizing industrial production. Continuously fermenting and culturing the lactobacillus reuteri LR08 to a stable stage to obtain lactobacillus reuteri LR08 bacterial suspension; and (3) centrifuging lactobacillus reuteri LR08 zymophyte suspension to obtain thalli, mixing thalli with a protective agent solution (comprising 5% of skimmed milk powder, 5% of soybean milk powder and the balance of water in percentage by weight), wherein the mass ratio of the total mass of the skimmed milk powder and the soybean milk powder to the mass of the thalli is 1:1, obtaining heavy suspension, and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain lactobacillus reuteri LR08 probiotic freeze-dried powder. The defatted milk powder is obtained from Heng natural materials, and the soybean milk powder is obtained from Dragon king.
Respectively adding 15g of lactobacillus reuteri freeze-dried powder, 36g of rose essential oil, 24g of propolis, 3g of sodium hyaluronate, 36g of glycerol, 15g of collagen and 24g of purified water into a blending tank in sequence, shearing, circulating and stirring for 15min (stirring condition 35 Hz) until the color and the state of the mixture are uniform, and fixing the volume.
Degassing the obtained solution (vacuum degree 0.04 Mpa), homogenizing (19 Mpa), packaging, and packaging in sterile environment at normal temperature.
Claims (5)
1. Lactobacillus reuteri with preservation number of CGMCC No.1.12733Lactobacillus reuteri) The application of the strain LR08 in-vitro bacteriostasis and/or preparation of products for adhering vaginal epithelial cells and/or preparation of bacteriostasis products and/or preparation of medicines for preventing and treating bacterial vaginitis is characterized in that the bacteriostasis refers to: while inhibiting candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus and prasugrel intermedia.
2. The lactobacillus reuteri with the preservation number of CGMCC No.1.12733 as claimed in claim 1Lactobacillus reuteri) The application of the strain LR08 in-vitro bacteriostasis and/or preparation of products for adhering to vaginal epithelial cells and/or preparation of bacteriostasis products and/or preparation of medicines for preventing and treating bacterial vaginitis is characterized in that the vaginal epithelial cells refer to VK2/E6E7 cells;
and/or, the prevention and treatment of bacterial vaginitis refers to reducing the inflammation score, and/or, improving the cleanliness, and/or, regulating the inflammatory factor content.
3. The lactobacillus reuteri with the preservation number of CGMCC No.1.12733 as claimed in claim 2Lactobacillus reuteri) Use of strain LR08 for in vitro bacteriostasis and/or for the preparation of products adhering to vaginal epithelial cells and/or for the preparation of products for bacteriostasis and/or for the preparation of a medicament for the prevention and treatment of bacterial vaginitis, characterized in that said inflammatory factor comprises: TNF-alpha, IL-1 beta, IL-6, IL-10.
4. The lactobacillus reuteri with the preservation number of CGMCC No.1.12733 as claimed in claim 2Lactobacillus reuteri) Use of strain LR08 for in vitro bacteriostasis and/or for the preparation of products adhering to vaginal epithelial cells and/or for the preparation of products for bacteriostasis and/or for the preparation of a medicament for the prevention and treatment of bacterial vaginitis, characterized in that said modulation of the inflammatory factor content comprises: lowering levels of TNF- α and/or IL-1β and/or IL-6, and/or increasing levels of IL-10.
5. An in vitro bacteriostasis method is characterized in that Lactobacillus reuteri with the preservation number of CGMCC No.1.12733 is adoptedLactobacillus reuteri) Bacterial strain LR08 inhibits bacteria; the bacteriostasis refers to: while inhibiting candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus and prasugrel intermedia.
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