CN116200306A - Lactobacillus rhamnosus LRa16, and application and product thereof in preparation of medicines for treating genital tract infection - Google Patents
Lactobacillus rhamnosus LRa16, and application and product thereof in preparation of medicines for treating genital tract infection Download PDFInfo
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- CN116200306A CN116200306A CN202310104407.8A CN202310104407A CN116200306A CN 116200306 A CN116200306 A CN 116200306A CN 202310104407 A CN202310104407 A CN 202310104407A CN 116200306 A CN116200306 A CN 116200306A
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Abstract
The invention relates to a lactobacillus rhamnosus LRa16 and application and a product thereof in preparing a medicament for treating genital tract infection, belonging to the technical field of microorganisms. The deposit number of the lactobacillus rhamnosus LRa16 is GDMCC NO:62278. The invention also provides application of the lactobacillus rhamnosus LRa16 in bacteriostasis, and/or cell adhesion, and/or preparation of a traditional Chinese medicine compound preparation, and/or preparation of a medicine for treating genital tract infection, and a probiotic composition, a traditional Chinese medicine compound preparation and an in vitro bacteriostasis method based on the lactobacillus rhamnosus LRa 16. The lactobacillus rhamnosus LRa16 can remarkably treat colpitis, the total cure effective rate is up to 98%, the recurrence rate is 0 within 3 months, and meanwhile, pathogenic bacteria such as candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella and the like can be effectively inhibited in a broad spectrum.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a lactobacillus rhamnosus LRa16, and application and a product thereof in preparing medicines for treating genital tract infection.
Background
It is reported that more than 300 microorganisms are symbiotic in the vagina of a human body, and the microorganisms restrict each other and balance each other to form dynamic balance; the vaginal microecological environment imbalance is related to the occurrence of various vaginitis; vaginitis is a risk factor for histological chorionic inflammation, amniotic fluid infection, post-cesarean endometritis and other pregnancy failure and pregnancy complications.
At present, antibiotics such as metronidazole or clindamycin are generally adopted for clinically treating vaginal infection, the effect of the antibiotics is quick, but the recurrence rate is high, and according to the description in clinical study of probiotics for treating bacterial vaginitis and related mechanism, the recurrence rate after 3 months of traditional antibiotics is 40%; besides the use of antibiotics, the traditional Chinese medicine has the defects of slow effect, poor efficacy, easy recurrence and the like, especially candida vulvosa and recurrent candida vaginae, and the treatment course is long and easy to be repeated.
The treatment of the vaginal microecological imbalance comprises three steps of sterilization, mucous membrane repair and restoration of vaginal microecological balance. Sterilization is the first step in the treatment of vaginitis, including hyperproliferative aerobic and anaerobic bacteria, blastospores or hyphae, trichomonas, etc. After pathogenic microorganisms are prevented, immune repair of vaginal mucosa and recovery of dominant lactobacillus are the final targets for treating colpitis. If this recovery process is affected, the physical and chemical environment in the vagina is not restored to normal, and the suppressed pathogenic microorganisms or foreign microorganisms may again reproduce or even recur new vaginitis. And probiotics can rapidly occupy vaginal epithelial receptors in the vagina, so as to generate a protective effect on the vagina and promote the vagina to restore to normal microenvironment. Therefore, the probiotic microecological preparation is the most preferable mode in terms of the current technical means for treating the colpitis.
The research of more than ten years shows that the diversity of the lactobacillus in the vagina of the human body is very rich, different lactobacillus can act cooperatively in the micro-environment of the vagina, and the types of the lactobacillus in different regions and people are different. Among them, the most common dominant lactobacillus in the vagina of healthy women in China are Lactobacillus crispatus, lactobacillus gasseri, lactobacillus jensenii, lactobacillus rhamnosus, lactobacillus reuteri, lactobacillus johnsonii, lactobacillus acidophilus, lactobacillus inertia, etc. Therefore, the screening of products related to the development of probiotics microecological preparations of lactobacillus existing in female vagina has profound significance in preventing and treating diseases related to genital tract infection.
Although some probiotics strains (comprising lactobacillus rhamnosus) effective for preventing and treating vaginal infection or inhibiting bacteria of vaginal infection are reported in the prior art at present, the strains cannot simultaneously have strong antibacterial capability and good cell adhesion capability, cannot be compatible with antibacterial traditional Chinese medicines to form a preparation with curative effect on vaginal infection, and cannot completely solve the problem of short-term recurrence rate.
The field needs to develop new bacterial strains which have strong inhibition capability to vagina infection pathogens and high cell adhesion capability, are not influenced by inhibition of antibacterial traditional Chinese medicines, can be compatible with the traditional Chinese medicines, and can thoroughly stop short-term recurrence rate of vagina infection.
Disclosure of Invention
In order to solve the above-mentioned shortcomings and needs in the prior art, the invention separates lactobacillus rhamnosus which can effectively and continuously antagonize candida albicans, gardnerella vaginalis, escherichia coli and staphylococcus aureus and has high adhesion capability; the lactobacillus rhamnosus has the advantages of safety, reliability, less adverse reaction, good effect and the like, has good treatment effect on female genital tract health, particularly has prominent effect of reducing recurrence of vaginitis, fundamentally restores vaginal microecological homeostasis and solves female health trouble.
The technical scheme of the invention is as follows:
lactobacillus rhamnosus @ strainLacticaseibacillus rhamnosus) Strain LRa16, characterized by the deposit number GDMCC No. 62278.
Lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) The strain LRa16 is used for bacteriostasis, cell adhesion and/or preparation of a traditional Chinese medicine compound preparation and/or preparation of a medicine for treating genital tract infection.
The bacteriostasis refers to inhibiting pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
Preferably, the cell adhesion refers to adhesion of VK2/E6E7 cells;
preferably, the strain LRa16 has an adhesion rate to VK2/E6E7 cells of 65.47%;
preferably, the Chinese herbal compound preparation comprises Chinese herbal and strain LRa16;
preferably, the Chinese medicine is selected from the group consisting of radix Scutellariae, radix Sophorae Flavescentis, fructus Kochiae, cortex Phellodendri, and folium Artemisiae Argyi;
preferably, the Chinese herbal compound preparation comprises a Chinese herbal extract and a probiotic composition containing strain LRa16;
preferably, the probiotic composition comprises a strain LRa16 and lactobacillus plantarum N13 with a preservation number of CGMCC No. 20496;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
Preferably, the probiotic composition is prepared by mixing a fermented dilution of strain LRa16 and a fermented dilution of lactobacillus plantarum N13 in equal volume in a ratio of 1:1;
preferably, the treatment of genital tract infections refers to: treating colpitis;
preferably, the vaginitis is selected from the group consisting of bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis;
preferably, the treatment of colpitis refers to: treating vaginitis with a total effective rate of 98% and/or a short-term recurrence rate of 0%;
preferably, the short-term recurrence rate refers to a recurrence rate within 3 months.
A medicament for treating genital tract infection, comprising a pharmaceutically active ingredient; the pharmaceutically active ingredients include: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
The medicine for treating genital tract infection further comprises: pharmaceutical excipients.
A probiotic composition comprising: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16 and lactobacillus plantarum N13 with the preservation number of CGMCC No. 20496.
The probiotic composition is prepared by mixing fermented dilutions of a strain LRa16 and lactobacillus plantarum N13 in an equal volume ratio of 1:1;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL。
A Chinese medicinal compound preparation is characterized by comprising the following components: chinese medicine and Lactobacillus rhamnosus with deposit number of GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
The Chinese medicine is selected from the group consisting of radix scutellariae, radix sophorae flavescentis, fructus kochiae, cortex phellodendri and folium artemisiae argyi;
preferably, the Chinese herbal compound preparation comprises a Chinese herbal extract and a probiotic composition containing strain LRa 16;
preferably, the mass ratio of the traditional Chinese medicine extract to the probiotic composition in the traditional Chinese medicine compound preparation is 1:1;
Preferably, the probiotic composition is prepared by mixing a fermented dilution of strain LRa16 and a fermented dilution of lactobacillus plantarum N13 in equal volume in a ratio of 1:1;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
Preferably, the Chinese medicine extract is prepared by mixing filtrates obtained by decocting Chinese medicines with water twice, concentrating and centrifuging;
preferably, the traditional Chinese medicine comprises the following raw materials in parts by weight: 30 parts of radix scutellariae, 30 parts of radix sophorae flavescentis, 8 parts of fructus kochiae, 8 parts of phellodendron, and 10 parts of folium artemisiae argyi;
preferably, the dosage ratio of the Chinese medicine to the water for the first time is 1:10; the dosage ratio of the Chinese medicine dregs and water in the second decoction is 1:5;
preferably, the concentration refers to concentrating the filtrate into a traditional Chinese medicine liquid with the density of 1.0mg/mL by using a rotary evaporator;
preferably, the Chinese medicinal liquid is centrifugated after standing overnight;
preferably, the centrifugation means centrifugation at 5000 rpm for 10min;
preferably, the supernatant obtained by the centrifugation is the traditional Chinese medicine extract.
An in vitro bacteriostasis method is characterized in that a strain of Lactobacillus rhamnosus with the preservation number of GDMCC NO: 62278 is usedLacticaseibacillus rhamnosus) Bacterial strain LRa16, and/or the probiotic composition, and/or the Chinese herbal compound preparation are used for inhibiting bacteria;
Preferably, the bacteria are pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
preferably, the inhibition zone of the strain LRa16 on candida albicans is 18mm, and the inhibition rate is 73.84% -99%;
preferably, the inhibition zone of the strain LRa16 on gardnerella vaginalis is 39mm, and the inhibition rate is 98.38%;
preferably, the bacterial strain LRa16 has a bacteriostasis zone of 31mm for Escherichia coli, and the number of viable bacteria for Escherichia coli is reduced by 5 orders of magnitude;
preferably, the bacterial inhibition zone of the strain LRa16 on staphylococcus aureus is 25mm, and the number of viable bacteria on staphylococcus aureus is reduced by 5 orders of magnitude;
preferably, the bacteria inhibition zone of the strain LRa16 on salmonella is 22mm;
preferably, the probiotic composition has a zone of inhibition of 24mm for candida albicans;
preferably, the zone of inhibition of the probiotic composition against gardnerella vaginalis is 39.5mm;
preferably, the zone of inhibition of the probiotic composition against escherichia coli is 38mm;
preferably, the probiotic composition has a zone of inhibition of 24.5mm against staphylococcus aureus;
preferably, the probiotic composition has a zone of inhibition of 23mm for salmonella;
Preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on candida albicans is 24.5mm;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on gardnerella vaginalis is 40mm;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on escherichia coli is 39mm;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on staphylococcus aureus is 25.5mm;
preferably, the antibacterial circle of the Chinese herbal compound preparation on salmonella is 23mm.
The lactobacillus rhamnosus LRa16 is separated and preserved by the Wuhan micro Kang Yisheng bacteria limited company; the preservation units are as follows: the collection address of the Guangdong province microorganism strain collection center is: the collection number of the building 5 of the Guangzhou city first Zhonglu No. 100 college No. 59 is: GDMCC NO. 62278, the preservation date is: 2022 yearsOn 12 months 09, the classification was designated as Lactobacillus rhamnosusLacticaseibacillus rhamnosus。
The lactobacillus rhamnosus LRa16 is separated from healthy adult feces, and is verified to be safe and reliable through a mouse acute toxicity test and a genital tract mucous membrane irritation test, and has no adverse reaction phenomenon.
Compared with the prior art, the lactobacillus rhamnosus LRa16 has the advantages of high growth speed, strong acid production capability, better capability of antagonizing candida albicans, gardnerella vaginalis, escherichia coli and staphylococcus aureus, capability of adhering to vaginal epithelial cells and capability of inhibiting the maturation stage of candida albicans biological membranes than commercial strains GR-1 and RC-14.
Clinical trial experiments prove that the lactobacillus rhamnosus LRa16 is safe and effective, has high cure rate on various colpitis, and compared with the treatment of common antibiotics on the market, the cure rate is obviously improved, and the recurrence rate is 0 within 3 months.
The invention also aims to provide application of the lactobacillus rhamnosus LRa16 and/or the microbial inoculum and the traditional Chinese medicine compound in preparing products for treating gynecological inflammation.
The traditional Chinese medicine probiotic composition comprises the lactobacillus rhamnosus LRa16 fermentation culture and lactobacillus plantarum N13 fermentation culture with the preservation number of CGMCC No. 20496.
The number of viable bacteria in the lactobacillus rhamnosus LRa16 fermentation culture is 1.0X10 8 CFU/mL-5.0x10 8 CFU/mL; the viable count of lactobacillus plantarum N13 fermentation culture with the preservation number of CGMCC No.20496 is 2.0X10 8 CFU/mL-10.0x10 8 CFU/mL; the two are mixed in a volume ratio of 1:1.
The traditional Chinese medicine probiotic preparation comprises the following components in parts by weight: 40-50 parts of probiotic metabolism composition and 50-60 parts of traditional Chinese medicine extract.
The traditional Chinese medicine extract is prepared from the following raw materials in parts by weight: 30 parts of baical skullcap root, 30 parts of lightyellow sophora root and broom cypress fruit
8 parts of seeds, 8 parts of phellodendron, and 10 parts of mugwort leaves.
Furthermore, the traditional Chinese medicine probiotic composition can be combined with pharmaceutically acceptable auxiliary materials to prepare various types of products, including: health products, disinfectants, and medicines (e.g., lotions, gels, suppositories, etc.).
The invention has the following advantages and positive effects:
the strain of lactobacillus rhamnosus LRa16 is obtained by separating and screening from healthy adult feces, and has high growth speed and high cell density; the acid production capacity is strong, the pH value of the genital tract can be effectively reduced, and the growth of pathogenic bacteria can be inhibited; when co-culturing with pathogenic bacteria such as candida albicans, the growth of the pathogenic bacteria can be effectively and continuously inhibited; meanwhile, the adhesive capacity to the vaginal epithelial cells is strong, so that the vaginal epithelial cells can be successfully planted in the vagina, and pathogenic bacteria, especially candida albicans, and the formation and maturation of biological films are inhibited.
Animal experiments prove that the lactobacillus rhamnosus LRa16 is safe and effective, has no adverse reaction phenomenon, is beneficial to lactobacillus proliferation and vaginal mucosa repair, and prevents the recurrence of colpitis. In clinical experiments, the lactobacillus rhamnosus LRa16 can remarkably treat colpitis, the total cure effective rate is up to 98%, the recurrence rate is 0 within 3 months, and the total cure effective rate is remarkably stronger than that of an antibiotic treatment group. The lactobacillus rhamnosus LRa16 has the outstanding capability of repairing the microecological balance of the vagina and preventing recurrence while effectively improving the cure rate of various vaginitis, and fundamentally solves the health trouble of female genital tracts.
The probiotic metabolism composition is formed by compounding lactobacillus rhamnosus LRa16 and lactobacillus plantarum with the preservation number of CGMCC No.20496, and the lactobacillus rhamnosus LRa16 is combined, so that the lactobacillus and lactobacillus rhamnosus LRa16 are combined, and the two strains are found to have remarkable synergistic effect when being matched for use, and particularly have more remarkable antibacterial effect on candida albicans, gardnerella vaginalis and escherichia coli causing vaginitis.
The traditional Chinese medicine probiotic preparation is prepared by scientifically matching a probiotic metabolism composition with a traditional Chinese medicine extract, and in-vitro antibacterial experiments prove that compared with a pure probiotic product or a pure traditional Chinese medicine product, the traditional Chinese medicine probiotic preparation has the effects of broad antibacterial spectrum, clearing heat, drying dampness, expelling toxin, relieving itching, obvious endocrine regulating effect and the like.
The deposit information of lactobacillus rhamnosus LRa16 of the present invention is as follows:
preservation number: GDMCC NO 62278;
classification naming:Lacticaseibacillus rhamnosus；
preservation date: 2022, 12, 09;
preservation unit: the collection of microorganism strains in Guangdong province;
preservation address: guangzhou city first middle road No. 100 college No. 59 building 5.
Drawings
FIG. 1 is a graph showing growth rates of Lactobacillus rhamnosus LRa16, commercial strains RC-14, GR-1, and LGG according to Experimental example 1 of the present invention.
FIG. 2 is a graph showing the results of the acid production capacity test of Lactobacillus rhamnosus LRa16 according to Experimental example 1 of the present invention at different times.
FIG. 3 is a line graph showing the effect of co-cultivation of Lactobacillus rhamnosus LRa16 with Candida albicans on Candida albicans viability according to experimental example 1 of the present invention.
FIG. 4 is a graph showing the ability of Lactobacillus rhamnosus LRa16, commercial strains RC-14 and GR-1, respectively, of experimental example 1 of the present invention to reduce the number of viable candida albicans by co-cultivation with candida albicans.
FIG. 5 is a bar graph of the inhibition of Candida albicans biofilm by Lactobacillus rhamnosus LRa16, commercial strains RC-14 and GR-1 of Experimental example 2 of the present invention at various stages.
FIG. 6 is a graph showing the effect of co-culture of Lactobacillus rhamnosus LRa16, commercial strain RC-14 and GR-1 with GV/EC/SA on GV/EC/SA viability according to Experimental example 3 of the present invention; wherein, A is a histogram of inhibition ratio to GV, B is a comparison graph for reducing the number of EC viable bacteria, and C is a comparison graph for reducing the number of SA viable bacteria.
FIG. 7 is a bar graph of the adhesion capacity of Lactobacillus rhamnosus LRa16, commercial strain RC-14 and GR-1 of Experimental example 4 to VK2/E6E7 cells according to the present invention.
Description of the embodiments
In order to further illustrate the technical means adopted by the present invention and the effects thereof, the following is a preferred experimental example of the present invention, but the present invention is not limited to the experimental example.
Sources of biological materials
The VK2/E6E7 cells used in Experimental example 4 are commercially available.
Experimental example 6 the experimental animals used were normal adult healthy female laboratory rabbits, kunming mice, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd.
The candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus used in experimental examples 1, 2, 3, 10 below were purchased from the guangdong collection of microorganism strains.
Commercial strains (RC-14, GR-1, LGG) used in experimental examples 1, 2, 3 were commercially available.
The genital tract infected volunteers referred to in experimental example 7 were from socially recruited volunteers, each signed an informed consent form relevant to the experiment.
The Lactobacillus plantarum N13 with the preservation number of CGMCC No.20496 used in experimental examples 8 and 10 is Lactobacillus plantarum N13 described in Chinese patent No. ZL202111137085.4, and is preserved in the laboratory of the applicant. The applicant promises to deliver to the public within 20 years from the date of filing of the present invention the effect of the present invention for verification.
The baikal skullcap root, kuh-seng, broom cypress fruit, phellodendron bark and mugwort leaf used in experimental example 9 are all traditional Chinese medicine raw materials which are commercially available from pharmacies.
Experimental reagent and consumable
BHI medium, eosin blue medium, and BG medium were purchased from Qingdao sea Bo biotechnology Co., ltd; DMEM medium was purchased from gibco;
The following experimental examples relate to the following media:
MRS liquid medium: glucose 20g/L, yeast peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, anhydrous sodium acetate 5g/L, tween-80 1g/L, diammonium citrate 2g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate heptahydrate
0.58g/L, 0.19g/L manganese sulfate monohydrate, 1000mL of water, ph6.8.
YPD liquid medium: glucose 10g/L, peptone 10g/L, yeast extract 5g/L, water 1000mL, ph7.0.
LB liquid medium: 10g/L tryptone, 5g/L yeast, 5g/L NaCl, 1000mL water and ph7.2.
1.5% of agar powder is added into the solid culture medium.
BHIs liquid medium: to commercial BHI medium was added 1% yeast extract, 0.1% maltose, 0.1% glucose and 10% horse serum.
The embodiment provides a strain of Lactobacillus rhamnosus @Lacticaseibacillus rhamnosus) Strain LRa16, deposited under accession number GDMCC No. 62278.
Any one cultivates, propagates, ferments, enriches, produces, prepares, uses, inoculates, amplifies, transforms, modifies, reforms, utilizes, sells, offers to sell the Lactobacillus rhamnosus with the deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Behavior of Strain LRa16 Lactobacillus rhamnosus with accession number GDMCC NO. 62278 Lacticaseibacillus rhamnosus) The strain LRa16 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 a traditional Chinese medicine compound preparation, and/or be prepared into a probiotic composition, and/or be prepared into a microbial inoculum, which all fall into the protection scope of the invention.
In a specific embodiment, the lactobacillus rhamnosus @ isLacticaseibacillus rhamnosus) The pH after 13h incubation of strain LRa16 can be as low as 3.32.
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.
This example provides Lactobacillus rhamnosus with accession number GDMCC NO. 62278Lacticaseibacillus rhamnosus) The strain LRa16 is used for bacteriostasis, cell adhesion and/or preparation of a traditional Chinese medicine compound preparation and/or preparation of a medicine for treating genital tract infection.
In some embodiments, the bacteriostatic means inhibiting pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
preferably, the cell adhesion refers to adhesion of VK2/E6E7 cells;
preferably, the strain LRa16 has an adhesion rate to VK2/E6E7 cells of 65.47%;
preferably, the Chinese herbal compound preparation comprises Chinese herbal and strain LRa16;
preferably, the Chinese medicine is selected from the group consisting of radix Scutellariae, radix Sophorae Flavescentis, fructus Kochiae, cortex Phellodendri, and folium Artemisiae Argyi;
in a specific embodiment, the traditional Chinese medicine comprises: radix Scutellariae, radix Sophorae Flavescentis, fructus Kochiae, cortex Phellodendri, and folium Artemisiae Argyi.
In a more specific embodiment, the traditional Chinese medicine comprises the following raw materials in parts by weight: 30 parts of radix scutellariae, 30 parts of radix sophorae flavescentis, 8 parts of fructus kochiae, 8 parts of phellodendron, and 10 parts of folium artemisiae argyi.
Preferably, the Chinese herbal compound preparation comprises a Chinese herbal extract and a probiotic composition containing strain LRa16;
preferably, the probiotic composition comprises a strain LRa16 and lactobacillus plantarum N13 with a preservation number of CGMCC No. 20496;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
Preferably, the probiotic composition is prepared by mixing a fermented dilution of strain LRa16 and a fermented dilution of lactobacillus plantarum N13 in equal volume in a ratio of 1:1;
preferably, the treatment of genital tract infections refers to: treating colpitis;
preferably, the vaginitis is selected from the group consisting of bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis;
preferably, the treatment of colpitis refers to: treating vaginitis with a total effective rate of 98% and/or a short-term recurrence rate of 0%;
preferably, the short-term recurrence rate refers to a recurrence rate within 3 months.
In a further embodiment, the compound traditional Chinese medicine preparation further comprises: auxiliary materials.
In still further embodiments, the medicament for treating genital tract infections further comprises: auxiliary materials.
In yet further embodiments, the probiotic composition further comprises: auxiliary materials.
In particular embodiments, the adjunct is selected from: 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-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
According to the invention, in view of different demands 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 the auxiliary materials and store the rhamnose cheese bacillus with the preservation number of GDMCC NO: 62278Lacticaseibacillus rhamnosus) The strain LRa16 and lactobacillus plantarum N13 with the preservation number of CGMCC No.20496 are prepared into different dosage forms, such as powder, tablets, injection, oral liquid, capsules, granules, spray, gel, ointment and the like.
The present group of implementationAn example provides a medicament for treating genital tract infection. All embodiments of this group share the following common features: the medicine for treating genital tract infection comprises active ingredients with efficacy; the pharmaceutically active ingredients include: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
In a further embodiment, the medicament for treating genital tract infection further comprises: pharmaceutical excipients.
In specific embodiments, the pharmaceutical excipients are selected from the group consisting of 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-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
According to the invention, the technical means (such as encyclopedia of preparation technology, pharmaceutical preparation technology and the like) in the field of medicine preparation are combined for different demands in practical production application, and the technical staff in the field can select and blend the medicinal auxiliary materials and store the rhamnose cheese bacillus with the preservation number of GDMCC NO: 62278Lacticaseibacillus rhamnosus) The strain LRa16 can be made into various dosage forms, such as powder, tablet, injection, oral liquid, capsule, granule, spray, gel, paste, etc.
Group 4 examples, probiotic compositions of the invention
The present set of examples provides a probiotic composition. All embodiments of this group share the following common features: the probiotic composition comprises: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16 and lactobacillus plantarum N13 with the preservation number of CGMCC No. 20496.
In specific embodiments, the probiotic composition is prepared by mixing a fermented dilution of strain LRa16 and a fermented dilution of lactobacillus plantarum N13 in equal volume in a ratio of 1:1;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL。
In some specific embodiments, the probiotic composition may be a microbial agent.
In other embodiments, the probiotic composition further comprises adjuvants useful for the microbial agent.
In specific embodiments, the bacterial agent may be used as an adjuvant selected from the group consisting of solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
According to the invention, the technical means (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) conventional in the field of preparation of the microbial agent are combined for different requirements in practical production application, and a person skilled in the art can select and blend auxiliary materials available for the microbial agent and prepare the probiotic composition into different dosage forms, such as powder, tablets, injection, oral liquid, capsules, granules, spray, gel, ointment and the like.
Group 5 examples, the Chinese herbal Compound preparation of the invention
The embodiment of the group provides a traditional Chinese medicine compound preparation. All embodiments of this group share the following common features: the traditional Chinese medicine compound preparation comprises the following components: chinese medicine and Lactobacillus rhamnosus with deposit number of GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
In a specific embodiment, the traditional Chinese medicine is selected from the group consisting of radix scutellariae, radix sophorae flavescentis, fructus kochiae, cortex phellodendri and folium artemisiae argyi;
preferably, the Chinese herbal compound preparation comprises a Chinese herbal extract and a probiotic composition containing strain LRa 16;
preferably, the mass ratio of the traditional Chinese medicine extract to the probiotic composition in the traditional Chinese medicine compound preparation is 1:1;
preferably, the probiotic composition is prepared by mixing a fermented dilution of strain LRa16 and a fermented dilution of lactobacillus plantarum N13 in equal volume in a ratio of 1:1;
preferably, the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
Preferably, the Chinese medicine extract is prepared by mixing filtrates obtained by decocting Chinese medicines with water twice, concentrating and centrifuging;
preferably, the traditional Chinese medicine comprises the following raw materials in parts by weight: 30 parts of radix scutellariae, 30 parts of radix sophorae flavescentis, 8 parts of fructus kochiae, 8 parts of phellodendron, and 10 parts of folium artemisiae argyi;
Preferably, the dosage ratio of the Chinese medicine to the water for the first time is 1:10; the dosage ratio of the Chinese medicine dregs and water in the second decoction is 1:5;
preferably, the concentration refers to concentrating the filtrate into a traditional Chinese medicine liquid with the density of 1.0mg/mL by using a rotary evaporator;
preferably, the Chinese medicinal liquid is centrifugated after standing overnight;
preferably, the centrifugation means centrifugation at 5000 rpm for 10min;
preferably, the supernatant obtained by the centrifugation is the traditional Chinese medicine extract.
In a further embodiment, the compound traditional Chinese medicine preparation further comprises: pharmaceutically acceptable auxiliary materials.
In specific embodiments, the pharmaceutically acceptable excipients are selected from the group consisting of: 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-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders.
According to the present invention, in view of different requirements in practical production applications, in combination with conventional technical means in the field of pharmaceutical preparation (for example, encyclopedia of formulation technology, pharmaceutical preparation technology, etc.), a person skilled in the art may select and formulate the above pharmaceutically acceptable auxiliary materials, and make a mixture of the traditional Chinese medicine extract and the probiotic composition into different dosage forms, such as powder, tablet, injection, oral liquid, capsule, granule, spray, gel, paste, patch, etc.
The present set of embodiments provides a method of inhibiting bacteria in vitro. All embodiments of this group share the following common features: using a strain of Lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Bacterial strain LRa16, and/or the probiotic composition provided by any one of the embodiments of group 4, and/or the Chinese herbal compound preparation provided by any one of the embodiments of group 5.
Preferably, the bacteria are pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
Preferably, the inhibition zone of the strain LRa16 on candida albicans is 18mm, and the inhibition rate is 73.84% -99%;
preferably, the inhibition zone of the strain LRa16 on gardnerella vaginalis is 39mm, and the inhibition rate is 98.38%;
preferably, the bacterial strain LRa16 has a bacteriostasis zone of 31mm for Escherichia coli, and the number of viable bacteria for Escherichia coli is reduced by 5 orders of magnitude;
preferably, the bacterial inhibition zone of the strain LRa16 on staphylococcus aureus is 25mm, and the number of viable bacteria on staphylococcus aureus is reduced by 5 orders of magnitude;
preferably, the bacteria inhibition zone of the strain LRa16 on salmonella is 22mm;
preferably, the probiotic composition has a zone of inhibition of 24mm for candida albicans;
preferably, the zone of inhibition of the probiotic composition against gardnerella vaginalis is 39.5mm;
preferably, the zone of inhibition of the probiotic composition against escherichia coli is 38mm;
preferably, the probiotic composition has a zone of inhibition of 24.5mm against staphylococcus aureus;
preferably, the probiotic composition has a zone of inhibition of 23mm for salmonella;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on candida albicans is 24.5mm;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on gardnerella vaginalis is 40mm;
preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on escherichia coli is 39mm;
Preferably, the antibacterial circle of the traditional Chinese medicine compound preparation on staphylococcus aureus is 25.5mm;
preferably, the antibacterial circle of the Chinese herbal compound preparation on salmonella is 23mm.
Experimental example 1 isolation screening of Lactobacillus rhamnosus LRa16
The method for separating the lactobacillus rhamnosus comprises the following steps of:
collecting a healthy adult excrement sample, performing 10-time gradient dilution by using physiological saline with the mass concentration of 0.85%, diluting for 5 times, coating on an MRS solid culture medium, culturing at 37 ℃ for 48 hours, picking a typical colony, continuously performing proper gradient dilution in the physiological saline with the mass concentration of 0.85%, coating on the surface of the MRS solid culture medium again, purifying and culturing at 37 ℃ for 48 hours, determining a pure culture system by colony morphology observation and thallus morphology microscopic examination, picking single colonies, performing expansion culture at 37 ℃ by using the MRS liquid culture medium, and preserving by using glycerol with the mass concentration of 30%.
The lactobacillus rhamnosus with the best performance is obtained after the 300 strains of lactobacillus preserved are subjected to a primary screening test with high efficiency and low pH value and a co-culture test with candida albicans, and is named as LRa16.
The screening method of the lactobacillus rhamnosus comprises the following steps:
1. High efficiency and low pH.
After the preserved lactobacillus is subjected to overnight activation culture at 37 ℃ in MRS liquid culture medium, the lactobacillus is transferred to 2-generation activation culture for 8 hours at 5% of inoculum size, and then transferred to 3-generation at 2% of inoculum size, and OD (OD) of 3h,5h,7h,9h,11h,12h,13h,15h,18h,24h and the like is measured 600 And a pH value. By using commercial strains (RC-14 and GR-1) as controls, strains from different samples were screened for rapid proliferation and acid production (lower pH).
Co-culture screening of candida albicans
Strain activation: inoculating lactobacillus strain into MRS liquid culture medium respectively for two-generation activation, inoculating after 8 hr of second-generation activation (the number of activated lactobacillus is 10 9 CFU/mL); shaking culture of 50ul Candida albicans liquid in No. 13 liquid culture medium at 37deg.C for 16 hr (activated Candida albicans viable count is 10) 8 CFU/mL）。
Co-cultivation: inoculating lactobacillus 200ul and candida albicans 200ul simultaneously into 10mLMRS liquid culture
Culture medium is cultured for 12h,16h,20h,24h,36h,40h,44h and 48h respectively, and the non-connected lactobacillus is used as a blank control. Candida albicans at each time point was counted with YPD solid medium to determine the optimal time point for co-culture with candida albicans. The best time point lactobacillus and candida albicans co-culture experiments were repeated to ensure the candida albicans inhibition effect.
Experimental results: as can be seen from the growth curve of FIG. 1, the screened Lactobacillus rhamnosus LRa16 grows slowly in 0-4h, grows quickly in 4h-12h, is in the logarithmic growth phase, and is stable in 12h-14h, the light absorption value in the later phase (11 h) of logarithmic growth is between 12.0-12.5, and compared with commercial strains LGG, GR-1 and RC-14, the Lactobacillus rhamnosus LRa16 has stronger reproductive capacity and potential for industrial production.
As can be seen from the acid production curve of FIG. 2, the pH of the screened Lactobacillus rhamnosus LRa16 and commercial strains (LGG, GR-1, RC-14) decreased rapidly at 0-13h, indicating that the acid production rate was rapid during this period, and the pH change was small or remained substantially unchanged after 15 h. The pH of the Lactobacillus rhamnosus LRa16 after 13h of culture was less than 4 (pH 3.32), whereas the commercial strains were all above 4. The result shows that the lactobacillus rhamnosus LRa16 obtained by screening has stronger acid-producing capability than commercial strains GR-1 and RC-14, and has important effect on maintaining the acidic environment of vagina.
As can be seen from the co-culture of fig. 3 and candida albicans, the inhibition rate of the screened lactobacillus rhamnosus LRa16 on candida albicans reaches the minimum value in 24 hours; and continuously inhibiting the activity of candida albicans in a low concentration state in the subsequent 36 hours and 48 hours; the lactobacillus rhamnosus LRa16 continuously produces acid through fermentation, has a continuous inhibition effect on vaginal pathogenic bacteria, so as to improve vaginal microecological balance, and restore microbial homeostasis, so that the defect of curative effect of antibiotics on treating vaginal microbial infection is overcome.
As can be seen from the repeated verification and comparison experiments of FIG. 4, the Lactobacillus rhamnosus LRa16 has a candida albicans concentration of 1.71×10 compared with the number of live candida albicans in pure culture 7 CFU/mL was reduced to 5.82×10 4 CFU/mL inhibited Candida albicans viability by nearly 3 concentration gradients, significantly stronger than commercial strains GR-1 and RC-14.
The lactobacillus rhamnosus LRa16 is sent to a preservation, and the preservation information is as follows:
preservation number: GDMCC NO 62278;
classification naming:Lacticaseibacillus rhamnosus；
preservation date: 2022, 12, 09;
preservation unit: the collection of microorganism strains in Guangdong province;
preservation address: guangzhou city first middle road No. 100 college No. 59 building 5.
Experimental example 2 evaluation of ability to inhibit Candida albicans biofilm adhesion, formation and maturation stage
Reagent: XTT: sodium 3,3' - [1- (anilinoyl) -3, 4-tetrazole ] -bis (4-methoxy-6-nitro) benzoate (XTT) (sigma aldrich, usa); menaquinone (sigma aldrich, usa);
preparation of 1mg/ml XTT: the XTT is prepared into 1mg/mL solution by PBS solution, filtered by a 0.22um filter membrane, and stored in a dark place at-70 ℃ after sub-packaging.
Preparation of 0.4mM menadione solution: is prepared by absolute ethyl alcohol and is ready to be used.
T1: evaluation of ability to inhibit Candida albicans biofilm adhesion stage: 100ul of candida albicans liquid (10) 7 CFU/ml standard suspension) was inoculated in 96-well plates with the addition of lactobacillus rhamnosus CFS (cell-free supernatant), each group: experimental group: 100ul of candida albicans standard suspension is added into 100ul of lactobacillus rhamnosus CFS; blank control group: 100ul YPD was added to 100ul Lactobacillus rhamnosus CFS, negative control: 100ul of MRS was added to 100ul of Candida albicans standard suspension.
T2: evaluation of ability to inhibit Candida albicans biofilm formation: 100ul of candida albicans liquid (10) 7 CFU/ml standard suspension) was inoculated into 96-well plates, the 96-well plates were incubated at 37 ℃ for 1.5 hours at 75rpm/min, the suspensions were discarded, and after 3 washes with PBS, each group was: experimental group: 200ul of Lactobacillus rhamnosus CFS was added to the wells with Candida albicans adhered thereto; blank control group: 200ul of Lactobacillus rhamnosus CFS was added to the sterile 96 wells; negative control group: 200ul MRS was added to the wells with Candida albicans adhered.
T3: evaluation of ability to inhibit the maturation stage of candida albicans biofilm: taking 100ul candida albicans liquid
（10 7 CFU/ml standard suspension) was inoculated into 96-well plates, the 96-well plates were incubated at 37 ℃ for 1.5h at 75rpm/min, the suspensions were discarded, after washing 3 times with PBS, 200ul ypd was added, incubation was continued at 37 ℃ for 24h at 75rpm/min, and the upper suspension was carefully aspirated, and the following groups were used: 200ul of Lactobacillus rhamnosus CFS was added to the wells formed by Candida albicans biofilm; blank control group: 200ul of Lactobacillus rhamnosus CFS was added to the sterile 96 wells; negative control group: 200ul MRS was added to the wells formed by Candida albicans biofilm.
After incubating the 96-well plates in T1, T2, T3 at 37℃for 24h at 75rpm/min, the suspension was discarded, after 3 washes with PBS, 79ul of PBS, 20ul of XTT (1 mg/mL) and 1ul of PMS (0.4 mM menaquinone solution) were added to the 96-well platesAfter 2h of light-shielding culture, the culture was performed on OD (optical density) by using an enzyme-labeled instrument 492nm Absorbance values were measured for each well of the 96-well plates and the inhibition rates of lactobacillus rhamnosus CFS on candida albicans biofilm adhesion, formation and maturation stages were calculated separately. The calculation formula is as follows: y= [1- (a experimental group-a blank control group)/(a negative control group-a blank control group)]*100%
The formula: the larger the Y value, the stronger the antibacterial intensity. Setting candida albicans-free blank control group; the cheese bacillus without rhamnose was used as a negative control group.
As can be seen from the experimental results in fig. 5: compared with commercial strains GR-1 and RC-14, the CFS of the Lactobacillus rhamnosus LRa16 has no significant difference in the formation stage of candida albicans biofilm (both 98-99%), but has a higher inhibition rate in the adhesion stage of candida albicans (73.84%) and a higher inhibition rate in the maturation stage of the biofilm (85.62%), which is significantly higher than that of commercial strains GR-1 and RC-14.
Experimental example 3, pathogenic bacteria inhibition ability analysis
Activation of lactobacillus rhamnosus LRa 16: lactobacillus rhamnosus LRa16 was thawed, activated for 3 passages in modified MRS broth, facultative anaerobic cultured for 24h at 37 ℃ and supernatant was prepared.
Activation of pathogenic bacteria: gardnerella vaginalis is cultured in BHIS culture medium at 37deg.C under anaerobic condition for 48 hr, and Escherichia coli and Staphylococcus aureus are respectively activated in LB liquid culture medium for 16 hr for subsequent experiment.
Co-culture antagonistic pathogen assay:
1) Lactobacillus rhamnosus LRa16 CFS co-cultured with gardnerella vaginalis:
experimental group: to 2.3ml of BHIS medium was added 2.5ml of Lactobacillus rhamnosus cell-free supernatant (CFS) and 200ul of Gardnerella vaginalis liquid (5 ml system). Control group 2.3ml BHIs medium was added with 2.5ml modified MRS and 200ul gardnerella vaginalis broth.
The culture was subjected to facultative anaerobic culture at 37℃for 48 hours. The cultured liquid was added to a sterile 96-well plate. Enzyme-labeled instrument for measuring OD 600 Values, average of 3 replicates.
2) Co-culture of lactobacillus rhamnosus LRa16 with escherichia coli, staphylococcus aureus: inoculating activated escherichia coli, staphylococcus aureus and lactobacillus rhamnosus in an improved MRS liquid culture medium (10 mL culture system/15 mL centrifuge tube) according to a bacterial inoculation amount of 2 percent and a bacterial inoculation ratio of 1:1, and performing anaerobic culture for 24 hours at 37 ℃, wherein the non-inoculated lactobacillus is used as a blank control. Coli and staphylococcus aureus were counted in LB solid medium and the experiment was repeated 2 times.
As can be seen from the experimental results in fig. 6: the inhibition rate of the lactobacillus rhamnosus LRa16 on the gardnerella vaginalis is as high as 98.38 percent, which is obviously higher than that of commercial strains GR-1 (83.03 percent) and RC-14 (72.71 percent); co-culture of Lactobacillus rhamnosus LRa16 with pathogenic bacteria inhibited E.coli, staphylococcus aureus, 5 concentration gradients, significantly higher than commercial strains GR-1 and RC-14, respectively, compared to pure cultured EC/SA.
Experimental example 4 adhesion to VK2/E6E7 cells
1) 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 bacterial 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, the cultured target strain bacterial liquid is taken, and the bacterial liquid is collected by centrifugation at 10000 r/min at room temperature for 1 min, and washed twice with sterile PBS, and finally resuspended in DMEM medium (the number of live lactobacillus bacteria is adjusted to be 1X 10) 8 CFU/mL）；
Co-cultivation: sucking the culture medium from single cell layer of prepared VK2/E6E7 cells, adding PBS buffer solution, rinsing for 2 times, sucking the buffer solution, adding prepared bacterial suspension 1 mL/hole, mixing, and adding 5% CO 2 Co-incubating at 37 ℃ for 2 h; careful attention was paid toThe culture supernatant was 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;
the collected samples were subjected to gradient dilution and viable counts. The adhesion capacity is calculated as follows:
adhesion ability (CFU/cell) =number of cells in Cell (CFU)/number of cells adhered to cells
Adhesion rate (%) = number of cells adhered to Cells (CFU)/total number of cells added to the well x100%
TABLE 1 capacity of Lactobacillus rhamnosus LRa16 to adhere to VK2/E6E7 cells and adhesion Rate
As can be seen from the experimental results in Table 1 and FIG. 7, the capacity of the Lactobacillus rhamnosus Lra16 to adhere to VK2/E6E7 cells is as high as 70.78CFU/cell, the adhesion rate is as high as 65.47%, and the adhesion rate is significantly higher than that of commercial strains GR-1 (3.88 CFU/cell, adhesion rate is 0.95%) and RC-14 (30.56 CFU/cell, adhesion rate is 11.25%). It is fully demonstrated that Lactobacillus rhamnosus Lra16 has a strong adhesion to vaginal epithelial cells. 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.
Experimental example 5 preparation of Lactobacillus rhamnosus Lra16 lyophilized powder
Preparation of lactobacillus rhamnosus Lra16 bacterial suspension: taking out the lactobacillus rhamnosus Lra16 strain preservation tube from the micro Kang Yisheng strain library, carrying out 2-generation activation culture on the lactobacillus rhamnosus Lra16 by using an MRS (media stop) culture medium for 8 hours, inoculating the seeds into the MRS culture medium by using 2% of inoculation amount, and carrying out fermentation culture until the concentration of the lactobacillus rhamnosus Lra16 reaches 30.0-50.0 hundred million CFU/mL, thus obtaining the lactobacillus rhamnosus Lra16 strain suspension.
Physiological use of Lactobacillus rhamnosus Lra16 fermentation culturesDiluting the brine to 1X 10 8 And (3) centrifuging after CFU/mL to obtain thalli, mixing thalli with a protective agent solution (comprising 4.5% of skimmed milk powder, 5.5% 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 thalli is 2:1, obtaining a heavy suspension, and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the lactobacillus rhamnosus Lra16 probiotic freeze-dried powder.
Experimental example 6, safety test of Lactobacillus rhamnosus Lra16
(1) Toxicity test
10 SPF-grade Kunming mice were intraperitoneally injected with 0.3ml of the Lactobacillus rhamnosus LRa16 strain suspension (greater than 1X 10) prepared in Experimental example 5 9 CFU/mouse), the body weight of each mouse was measured daily according to the requirements of the chinese pharmacopoeia of 2015 edition, and the behavior and physiological changes before and after injection of each mouse were observed and recorded.
The results show that all animals have increased weight within 7 days, no obvious poisoning symptoms are seen, no abnormal activity and no animal death occur, and the strain is considered to be safe and nontoxic.
(2) Genital tract mucosa irritation test
According to the vaginal mucosa stimulation experimental standard in the national ministry of health, female New Zealand rabbits which are healthy as early as possible and in the non-estrus period are selected, and whether secretion, congestion, edema and other damage conditions of the vaginal orifice of the animal are detected before the experiment. 3 female New Zealand rabbits are randomly selected to be infected by using the lactobacillus rhamnosus LRa16 as a test solution, and 2mL of the test solution is injected each time. Animals in the control group were treated in the same manner with physiological saline. Each time interval is 24 hours, and the time is 5 days. The animals are sacrificed by an air embolism method 24 hours after the last contamination, the vagina is cut off completely, the vagina is cut longitudinally, the appearance of edema, congestion and the like of mucous membrane is observed visually, and then the mucous membrane is fixed in 10% formaldehyde solution for histopathological examination.
According to the grading standard and the grading of the irritation intensity of the vaginal mucosa side mark in the disinfection technical specification, the vaginal mucosa irritation index of the traditional Chinese medicine microecological preparation is 0.78<1, and the traditional Chinese medicine microecological preparation is judged to have no irritation to the vaginal mucosa. Can be used as a biological agent for directly applying to vaginal microecology intervention.
Experimental example 7 clinical trials of Lactobacillus rhamnosus LRa16 gel for treating volunteers with genital tract infection
(1) Material
Lactobacillus rhamnosus LRa16 gel: weighing 800mL of the freeze-dried powder suspension of lactobacillus rhamnosus LRa16 probiotics prepared in experimental example 5, weighing 942.5g of carbomer, adding the carbomer into a container containing the carbomer, overnight, stirring uniformly until the carbomer is fine and uniform and has no particles, regulating the pH value to 5.0 by using sodium hydroxide with the mass concentration of 10%, adding 5g of glycerol and 0.2g of peppermint oil, and adding water to 1000g and stirring uniformly to obtain the lactobacillus rhamnosus LRa probiotic freeze-dried powder. The recommended dosage is 3 g/person/day, the injection is carried out, and 6 days is a small treatment course.
(2) Group of people to be tested
Subject inclusion criteria: 200 cases of vaginitis (bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis) are all in a sexual life history with ages of 18-65 years. A non-menstrual period; the clinical symptoms are compounded with clinical diagnosis standards of colpitis, patients with serious mental diseases are excluded from patients, and sexual life history is forbidden within 1 week; no other medications and vaginal cleaners were used or were ineffective with other medications within 1 week prior to the test.
(3) Therapeutic method
Control group: 400mg of metronidazole was taken for 100 patients. 2 times daily, 7 days of oral administration, experimental group: 100 patients use the Lactobacillus rhamnosus LRa16 gel 1 time a day, 3g each time, and two groups of patients observe clinical effects after continuous administration for 1 week.
(4) Observation index
The curative effect is divided into three indexes: and (A) curing: the clinical symptoms of the patient disappear, and the detection result is negative; b is effective: the clinical symptoms of the patient are basically improved, and the detection result is negative; c is invalid: the clinical symptoms of the patients are not improved or the disease is aggravated, and the patients have recurrence after 3 months of treatment. Total effective rate = (cure+effective)/total case x 100%
TABLE 2 comprehensive comparison of the therapeutic effects of the two groups of patients
Note that: the two groups of effective rates were statistically compared. P <0.05, the difference is statistically significant.
TABLE 3 comparison of recurrence rates for two groups of patients
The experimental results show that: after 100 patients in the experimental group are treated, the total effective rate of treatment is obviously higher than that of the control group, and the treatment effect of the experimental group is obviously higher than that of the control group (antibiotic group). After 3 months, the clinical recurrence rates of the experimental groups are 0, and the recurrence rate of the control group is obviously higher. Demonstrating that lactobacillus rhamnosus LRa16 itself has a prominent capacity to reduce vaginitis recurrence.
Experimental example 8 preparation of Lactobacillus rhamnosus LRa16 probiotic preparation
Preparation of probiotic metabolizing composition: after 2 generations of activated culture is carried out on lactobacillus plantarum N13 with the preservation number of CGMCC No.20496 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, and when the concentration of lactobacillus plantarum fermentation culture thallus reaches more than 30 hundred million CFU/mL. Diluting the fermentation culture with physiological saline to 1×10 8 After CFU/mL, the fermented dilution of Lactobacillus rhamnosus LRa16 (the viable count of Lactobacillus rhamnosus LRa16 is 1×10) was obtained in experimental example 5 8 CFU/mL) were mixed in equal volume at a ratio of 1:1 for use.
Preparation of probiotic preparation: and (3) centrifuging the diluted fermentation culture to obtain thalli, mixing the thalli with a protective agent solution (comprising 4.5% of skimmed milk powder, 5.5% 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 2:1, obtaining a heavy suspension, and freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the lactobacillus rhamnosus LRa16 probiotic freeze-dried powder. Mixing the freeze-dried powder with lactose and inulin to obtain a lactobacillus rhamnosus LRa16 probiotic preparation; the mass ratio of the freeze-dried powder to lactose to inulin is 2:1:1.
Experimental example 9 preparation of probiotic preparation with Chinese herbal Compound
Preparation of traditional Chinese medicine extract: mixing 30 parts of radix scutellariae, 30 parts of radix sophorae flavescentis, 8 parts of fructus kochiae, 8 parts of cortex phellodendri and 10 parts of folium artemisiae argyi, soaking the mixture in 10 times of water for 60 minutes, decocting for 60 minutes after the water is boiled, and filtering to obtain a first filtrate; decocting the residue with 5 times of water for 30 min, filtering to obtain second filtrate, mixing with the first filtrate, concentrating the Chinese medicinal liquid by rotary evaporator until the density of the Chinese medicinal liquid is about 1.0mg/mL, standing overnight, centrifuging at 5000 rpm for 10min, and collecting supernatant.
Preparation of a traditional Chinese medicine probiotic preparation: and uniformly mixing the probiotic composition of experimental example 8 and the traditional Chinese medicine extract in a ratio of 1:1 to prepare the traditional Chinese medicine probiotic preparation.
Experimental example 10, lactobacillus rhamnosus LRa16 and in vitro bacteriostasis experiment of probiotic preparation thereof
Test material: lactobacillus rhamnosus LRa16, a probiotic metabolism composition, a traditional Chinese medicine extract and a traditional Chinese medicine probiotic preparation.
Indicator bacteria: candida albicans, gardnerella vaginalis, escherichia coli, staphylococcus aureus, salmonella, lactobacillus plantarum N13, lactobacillus rhamnosus LRa16.
Oxford cup method: cooling YPD culture medium, BHI culture medium (5% defibrinated sheep blood is added), eosin blue agar culture medium, BG agar culture medium, LB agar culture medium, MRS culture medium to about 55deg.C, sequentially mixing with Candida albicans, gardnerella vaginalis, escherichia coli, salmonella, staphylococcus aureus, lactobacillus plantarum N13, and Lactobacillus rhamnosus LRa16 bacterial suspension at a certain ratio to obtain viable count of indicator bacteria of 10 6 On the order of CFU/mL, then rapidly poured into a plate in which an oxford cup is placed in advance, after the culture medium is cooled and solidified, the oxford cup is taken out, 200uL of Lactobacillus rhamnosus LRa16 is respectively injected into each hole, and the probiotic metabolic composition (viable count is 10 8 CFU/mL), traditional Chinese medicine extract, traditional Chinese medicine probiotic preparation, and measuring the diameter of the inhibition zone after overnight culture at 37 ℃.
TABLE 4 in vitro antibacterial test results of traditional Chinese medicine probiotic preparation
The test results are shown in table 4: the lactobacillus rhamnosus LRa16 and lactobacillus plantarum N13 are compounded to form a probiotic fermentation liquor, and the probiotic preparation is compounded with a traditional Chinese medicine extract, has strong antibacterial activity on candida albicans, gardnerella vaginalis, escherichia coli, staphylococcus aureus and other indicator bacteria, but has no antibacterial activity on lactobacillus, so that the probiotic preparation has cooperativity with the traditional Chinese medicine extract, and the antibacterial spectrum is enlarged, and the antibacterial effect is stronger.
Claims (10)
1. Lactobacillus rhamnosus @ strainLacticaseibacillus rhamnosus) Strain LRa16, characterized by the deposit number GDMCC No. 62278.
2. Lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) The strain LRa16 is used for bacteriostasis, cell adhesion and/or preparation of a traditional Chinese medicine compound preparation and/or preparation of a medicine for treating genital tract infection.
3. The Lactobacillus rhamnosus strain with accession number GDMCC NO. 62278 as claimed in claim 2Lacticaseibacillus rhamnosus) The application of the strain LRa16 in bacteriostasis, and/or cell adhesion, and/or preparation of a traditional Chinese medicine compound preparation, and/or preparation of a medicine for treating genital tract infection is characterized in that the bacteriostasis refers to inhibition of pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
And/or, the cell adhesion refers to adhering VK2/E6E7 cells;
and/or, the adhesion rate of the strain LRa16 to the VK2/E6E7 cells is 65.47%;
and/or, the traditional Chinese medicine compound preparation comprises traditional Chinese medicines and bacterial strain LRa16;
and/or the traditional Chinese medicine is selected from the group consisting of radix scutellariae, radix sophorae flavescentis, fructus kochiae, cortex phellodendri and folium artemisiae argyi;
and/or the traditional Chinese medicine compound preparation comprises a traditional Chinese medicine extract and a probiotic composition containing strain LRa16;
and/or the probiotic composition comprises a bacterial strain LRa16 and lactobacillus plantarum N13 with a preservation number of CGMCC No. 20496;
and/or the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
And/or, the probiotic composition is prepared by mixing fermented dilutions of the strain LRa16 and lactobacillus plantarum N13 in equal volumes in a ratio of 1:1;
and/or, the treatment of genital tract infections refers to: treating colpitis;
and/or, the vaginitis is selected from the group consisting of bacterial vaginitis, mycotic vaginitis, and trichomonas vaginitis;
and/or, the treatment of colpitis refers to: treating vaginitis with a total effective rate of 98% and/or a short-term recurrence rate of 0%;
and/or, the short-term recurrence rate refers to a recurrence rate within 3 months.
4. A medicament for treating genital tract infection, comprising a pharmaceutically active ingredient; the pharmaceutical active ingredients are characterized by comprising: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
5. A medicament for treating genital tract infections according to claim 4, further comprising: pharmaceutical excipients.
6. A probiotic composition comprising: lactobacillus rhamnosus with deposit number GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16 and lactobacillus plantarum N13 with the preservation number of CGMCC No. 20496.
7. A probiotic composition according to claim 6, characterized in that it is prepared by mixing the fermented dilution of strain LRa16 and the fermented dilution of lactobacillus plantarum N13 in equal volumes in a ratio of 1:1;
and/or the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL。
8. A Chinese medicinal compound preparation is characterized by comprising the following components: chinese medicine and Lactobacillus rhamnosus with deposit number of GDMCC NO. 62278Lacticaseibacillus rhamnosus) Strain LRa16.
9. The compound Chinese medicinal preparation according to claim 8, wherein the Chinese medicinal materials are selected from the group consisting of baikal skullcap root, kuh-seng, broom cypress fruit, phellodendron bark and mugwort leaf;
And/or the traditional Chinese medicine compound preparation comprises a traditional Chinese medicine extract and a probiotic composition containing strain LRa 16;
and/or the mass ratio of the traditional Chinese medicine extract to the probiotic composition in the traditional Chinese medicine compound preparation is 1:1;
and/or, the probiotic composition is prepared by mixing fermented dilutions of the strain LRa16 and lactobacillus plantarum N13 in equal volumes in a ratio of 1:1;
and/or the live bacterial numbers of the strain LRa16 and the lactobacillus plantarum N13 in the probiotic composition are respectively 1 multiplied by 10 8 CFU/mL；
And/or the traditional Chinese medicine extract is prepared by mixing filtrate obtained by decocting the traditional Chinese medicine twice with water, concentrating and centrifuging;
and/or the traditional Chinese medicine comprises the following raw material medicines in parts by weight: 30 parts of radix scutellariae, 30 parts of radix sophorae flavescentis, 8 parts of fructus kochiae, 8 parts of phellodendron, and 10 parts of folium artemisiae argyi;
and/or the dosage ratio of the Chinese medicine decocted for the first time to the water is 1:10; the dosage ratio of the Chinese medicine dregs and water in the second decoction is 1:5;
and/or concentrating the filtrate into a Chinese medicinal liquid with the density of 1.0mg/mL by using a rotary evaporator;
and/or, standing the traditional Chinese medicine liquid overnight and centrifuging;
and/or, the centrifugation means 5000-rotation centrifugation for 10min;
and/or, the supernatant obtained by the centrifugation is the traditional Chinese medicine extract.
10. An in vitro bacteriostasis method is characterized in that a strain of Lactobacillus rhamnosus with the preservation number of GDMCC NO: 62278 is usedLacticaseibacillus rhamnosus) Bacterial strain LRa16, and/or the probiotic composition of claim 6 or 7, and/or the Chinese herbal compound preparation of claim 8 or 9;
and/or, the bacteria are pathogenic bacteria; the pathogenic bacteria are selected from the group consisting of candida albicans, escherichia coli, staphylococcus aureus, gardnerella vaginalis, salmonella;
and/or the inhibition zone of the strain LRa16 on candida albicans is 18mm, and the inhibition rate is 73.84% -99%;
and/or the inhibition zone of the strain LRa16 on gardnerella vaginalis is 39mm, and the inhibition rate is 98.38%;
and/or the bacterial strain LRa16 has a bacteriostasis zone of 31mm for Escherichia coli, and the number of viable bacteria for Escherichia coli is reduced by 5 orders of magnitude;
and/or the bacteria inhibition zone of the strain LRa16 to staphylococcus aureus is 25mm, and the number of viable bacteria to staphylococcus aureus is reduced by 5 orders of magnitude;
and/or the bacteria inhibition zone of the strain LRa16 on salmonella is 22mm;
and/or the zone of inhibition of the probiotic composition against candida albicans is 24mm;
and/or the zone of inhibition of the probiotic composition against gardnerella vaginalis is 39.5mm;
And/or the zone of inhibition of the probiotic composition against escherichia coli is 38mm;
and/or the zone of inhibition of the probiotic composition against staphylococcus aureus is 24.5mm;
and/or the zone of inhibition of the probiotic composition against salmonella is 23mm;
and/or the antibacterial circle of the traditional Chinese medicine compound preparation on candida albicans is 24.5mm;
and/or the antibacterial circle of the traditional Chinese medicine compound preparation on gardnerella vaginalis is 40mm;
and/or the antibacterial circle of the traditional Chinese medicine compound preparation on escherichia coli is 39mm;
and/or the antibacterial circle of the traditional Chinese medicine compound preparation on staphylococcus aureus is 25.5mm;
and/or the antibacterial circle of the Chinese herbal compound preparation on salmonella is 23mm.
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