CN107299065B - Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs - Google Patents
Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs Download PDFInfo
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Abstract
The invention provides a lactobacillus plantarum and application thereof in preparing vagina bacteriostatic drugs, wherein the lactobacillus plantarum is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14111. The research on the metabolic performance of the lactobacillus plantarum shows that compared with the common lactobacillus plantarum, the lactobacillus plantarum has the advantages of strong lactic acid production capacity, strong hydrogen peroxide production capacity, strong bacteriostatic capacity on pathogenic bacteria and outstanding vaginal epithelial cell adhesion capacity. Based on the new properties discovered above, the invention determines the new application of the vaginal antibacterial drug prepared by the vaginal antibacterial drug, and can realize the treatment of various bacterial vaginal diseases. The bacterial agent has obvious effect on bacterial vaginal diseases, is safe, nontoxic and good in stability, can be stored for a long time, and also relates to application of the bacterial agent in medicines for preventing and/or treating gynecological diseases.
Description
Technical Field
The invention relates to the technical field of microorganisms, further relates to adjustment of vaginal micro-ecological environment and treatment of bacterial vaginal diseases, and particularly relates to lactobacillus plantarum and application thereof in preparation of vaginal antibacterial drugs.
Background
A plurality of microorganisms exist in the vagina of healthy women, and the microorganisms, a host and the environment form a vaginal microecosystem which is mutually restricted, coordinated and dynamically balanced. The vaginal flora of healthy women is mainly composed of lactobacillus, including lactobacillus plantarum, lactobacillus jensenii, lactobacillus gasseri, lactobacillus crispatus, lactobacillus vaginalis, lactobacillus rhamnosus and the like. Under normal conditions, the lactobacillus can protect the vagina, and when the lactobacillus disorder of the microecology of the vagina can cause the invasion of pathogenic bacteria to generate vaginitis.
Bacterial Vaginosis (BV) occurs due to a deregulation of the vaginal flora and a reduction of the host's own lactobacilli, which leads to a massive proliferation of other conditionally pathogenic microorganisms such as Gardnerella, various anaerobes, Vibrio flexuosus, etc., usually BV is actually a mixed infection mainly of Gardnerella. By using antibiotic therapy, the symptoms of BV can be temporarily relieved, but the reduced lactobacilli are further reduced, and vaginal microecological imbalance is aggravated, so that BV can relapse repeatedly. How to control relapse and radically cure bacterial vaginosis is a delicate problem which needs to be solved urgently by gynecologists.
The research shows that: produce H2O2And lactobacillus is the dominant bacterium in the vagina of healthy women, is an important factor for protecting the vagina of women from being infected by pathogens, and in addition, acid produced by the metabolism of lactobacillus and some antimicrobial factors can also effectively inhibit the growth and the propagation of other bacteria.
Various lactobacilli exist in the vagina of healthy women, the individual difference is achieved, and the difference of pathogenic bacteria resistance among lactobacillus strains is obvious. When selecting lactobacillus probiotics, the species of lactobacillus, acid production and H production need to be comprehensively considered2O2The ability, and the ability to adhere to vaginal epithelial cells, wherein whether lactobacillus can successfully colonize the vagina or not, is the basis of the sustained action of lactobacillus and is also a key factor for the curative effect of lactobacillus. Lactobacillus plantarum (Lactobacillus sp)lantarum), a genus of lactobacillus, is one of the normal flora of the human body and is widely distributed in the human intestinal tract, as well as in the female vagina. For conventional strains, they do not have specific bacteriostatic effects in the cognitive domain of the prior art.
The existing product, namely lactobacillus delbrueckii, in the market at present is actually not the dominant bacterial flora in the vagina of women in China, has poor planting capability, cannot maintain stable viable bacteria content, and cannot meet the clinical requirement of gynecology.
Disclosure of Invention
The invention aims to provide a specific lactobacillus plantarum to solve the technical problem that conventional lactobacillus plantarum does not have the capability of inhibiting pathogenic bacteria in vagina in the prior art.
Another technical problem to be solved by the invention is that the conventional Lactobacillus plantarum has a low hydrogen peroxide production capacity.
The invention also aims to solve the technical problem that the conventional lactobacillus plantarum has low lactic acid yield.
The invention also aims to solve the technical problem that the conventional lactobacillus plantarum in the prior art has poor colonization and survival effects in the vagina.
The invention also aims to solve the technical problem that the treatment effect of the bacteriostatic medicaments of the microbial inoculum type aiming at the vaginal pathogenic bacteria in the prior art is poor.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the invention relates to a separated Lactobacillus plantarum (Lactobacillus plantarum), which is disclosed as RD-0025 (Lactobacillus plantarum) and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14111.
The Lactobacillus plantarum RD-0025 is selected from vaginal secretion of Chinese healthy women of child bearing age, and has been preserved in China general microbiological culture collection center (CGMCC) in 5-10.2017, wherein the preservation unit address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, China academy of sciences microbiological research institute, the collection registration number is CGMCC No.14111, and the strain is classified and named as Lactobacillus plantarum.
The separated lactobacillus plantarum RD-0025 adopts 16SrDNA for sequencing, and the highest homology score with the lactobacillus plantarum base sequence in a GenBank database is more than 98 percent.
On the basis of the technical scheme, the invention further provides application of the lactobacillus plantarum in preparation of bacteriostatic drugs for vagina pathogenic bacteria.
Preferably, the drug is a bacterial agent that colonizes and survives vaginal epithelial cells in the vaginal environment.
Preferably, the drug is a bacterial agent which is metabolized to produce H in the vaginal environment2O2。
Preferably, the pathogenic bacteria is gardnerella vaginalis.
Preferably, the pathogenic bacteria is atrophaerella.
Preferably, the pathogenic bacteria are candida albicans, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa or salmonella.
Meanwhile, the invention further provides application of the lactobacillus plantarum in preparing a medicine for preventing or treating vaginal diseases.
Preferably, the vaginal disease is vulvovaginal candidiasis, trichomonas vaginitis, senile vaginitis, non-specific vaginal infection or mixed vaginal infection.
According to the invention, a lactobacillus plantarum strain is obtained by separation at first, and research on metabolic performance of the lactobacillus plantarum strain shows that the lactobacillus plantarum strain has excellent lactic acid production capacity and hydrogen peroxide production capacity, has strong bacteriostatic ability on pathogenic bacteria, and has outstanding vaginal epithelial cell adhesion capacity. Based on the new properties discovered above, the invention determines the new application of the vaginal antibacterial drug prepared by the vaginal antibacterial drug, and realizes the treatment of various bacterial vaginal diseases.
The beneficial effect that adopts above-mentioned technical scheme to produce lies in: (1) the lactobacillus plantarum RD-0025 strain disclosed by the invention can be preserved for a long time and resists bacterial vaginosis and various vaginal infections, including candida albicans vaginitis, gonorrhea, viral vaginitis, urinary tract infection and the like. (2) The strain is directly collected from a healthy human body, has active and stable biological characteristics, does not need domestication and rejuvenation processes, and can be directly prepared for use. (3) The strain has the effect of inhibiting pathogenic bacteria such as gardnerella vaginalis and the like, has superior vaginal epithelial cell adhesion compared with the commercial contrast bacteria, and has superior capability of permanent planting in the vagina of primates.
Drawings
FIG. 1 is a photograph showing the colony morphology of Lactobacillus plantarum RD-0025 according to the present invention;
FIG. 2 is a gram-stained microscopic photograph of Lactobacillus plantarum RD-0025 according to the present invention;
FIG. 3 is an electrophoretogram of PCR amplification product of 16SrDNA gene of Lactobacillus plantarum RD-0025 according to the present invention;
FIG. 4 is a lactic acid detection spectrum of Lactobacillus plantarum RD-0025 according to the present invention;
FIG. 5 is a 5min and 10min picture of the coloration of Lactobacillus plantarum RD-0025 hydrogen peroxide in accordance with the present invention;
FIG. 6 is a colony chart of the antibiotic susceptibility test of Lactobacillus plantarum RD-0025 of the present invention to vancomycin, gentamicin and erythromycin;
FIG. 7 is a photograph showing the bacteriostatic effects of Lactobacillus plantarum RD-0025 (left) and Lactobacillus delbrueckii (right) on Gardnerella vaginalis according to the present invention;
FIG. 8 is a photograph showing the bacteriostatic effect of Lactobacillus plantarum RD-0025 (left) and Lactobacillus delbrueckii (right) on Escherichia coli in accordance with the present invention;
FIG. 9 is a photograph showing the bacteriostatic effects of Lactobacillus plantarum RD-0025 (left) and Lactobacillus delbrueckii (right) on Staphylococcus aureus in accordance with the present invention;
FIG. 10 is an electrophoresis picture of PCR amplification products of a partial bacterial strain 16SrDNA fragment in a vaginal microbial area of a cynomolgus monkey after lactobacillus plantarum RD-0025 is fixedly planted in the vagina of the cynomolgus monkey.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified; in the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged; in the following examples,% is by mass unless otherwise specified.
The bacterial culture medium components and preparation methods used in the following examples were as follows:
1. broth solid Medium (MRS) preparation:
(1) preparing agar powder into a solution, and adding 1.5g/100ml deionized water;
(2) adding 17.91g/100ml agar solution of MRS culture medium, and mixing;
(3) placing into a sterilizing pot, and sterilizing for 20 minutes by 1.0MPa steam;
(4) pouring the culture medium into a culture dish after the temperature of the culture medium is reduced to room temperature, wherein the culture medium is about 10 ml/piece or 20 ml/piece according to the size of the culture dish;
(5) operating in a clean bench. Cooling to become agar, marking the name of the culture medium and the preparation date, and placing in a refrigerator at 4 ℃ for later use.
2. Broth liquid Medium (MRS) preparation:
(1) adding the MRS culture medium into deionized water in a ratio of 17.9l g/100 ml;
(2) placing into a sterilizing pot, and sterilizing for 20 minutes by 1.0MPa steam;
(3) taking out the sterilized pan when the pressure of the sterilized pan is not high, and subpackaging the sterilized pan into EP tubes with each 1.0 ml. Marking the name and preparation date of the culture medium, and placing the culture medium in a refrigerator at 4 ℃ for later use.
3. Hydrogen peroxide (H)2O2) Preparing an identification culture medium:
(1) preparing steps (1) to (4) with a broth solid Medium (MRS);
(2) taking out the autoclave without pressure, slightly cooling, adding TMB (final concentration of 0.25mg/m1) and HRP (final concentration of 0.01mg/m1) in a super clean bench when the autoclave is still in a liquid state, and mixing;
(3) and (3) after the temperature of the culture medium is reduced to about 45 ℃, pouring the culture medium into a culture dish, cooling the culture medium into an agar shape, marking the name and preparation date of the culture medium, and placing the culture medium in a refrigerator at 4 ℃ for later use.
Example 1 isolation and inoculation of Lactobacillus plantarum RD-0025 flora, purification, enrichment culture)
1. Isolation and inoculation of the Lactobacillus plantarum RD-0025 flora: collecting vaginal secretion of a subject with two sterile cotton swabs, inoculating the vaginal secretion to a culture dish containing prepared MRS culture medium at different concentrations, marking information, placing the culture dish in an anaerobic jar, and adding CO2And (5) generating air bags, placing in an incubator at 37 ℃, and incubating for more than 48 h.
2. Purifying and culturing lactobacillus plantarum strain RD-0025: counting according to different forms (surfaces, edges and the like) and sizes of colonies, respectively, recording as one form with the same form and the same size, picking a few bacteria in a single colony by an inoculating loop, and inoculating to an MRS solid culture medium according to a diagonal method to obtain a separated and purified single colony; a small amount of bacteria in a single colony on an MRS solid culture medium are picked by the aseptic toothpick, inoculated to an MRS liquid culture medium, placed in an incubator at 37 ℃, subjected to anaerobic culture for 24-48h, and screened out a new strain.
Example 2 identification and preservation of Lactobacillus plantarum RD-0025 Strain
1. Culture characteristics, staining microscopy and morphological characteristics: the colony obtained after culture is as shown in figure 1, and is generally white round, with a diameter of about 3mm, a convex middle part, and smooth and fine surface; gram staining is carried out on the smear of the pure culture of the bacteria, the result is shown in figure 2, the smear is gram positive, the smear is straight and round-ended, and the smear is distributed in a single, paired or short-chain shape, and the result shows that: the isolated strain was initially judged to be of the genus Lactobacillus.
2. 16SrDNA gene sequence identification: extracting DNA with bacterial genome DNA extraction kit, performing PCR amplification with primer pair 27F (5 '-AGAGTTTGATCMTGGCTCAG-3'), 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'), performing gel electrophoresis to obtain 16SrDNA gene fragment, and obtaining single clear PCR product band at about 1500bp, as shown in figure 3. And (3) purifying the PCR product and determining a DNA sequence, wherein a Sanger sequencing method is adopted, a sequencing primer pair is 27F/1492R, a sequencing instrument ABI3730XL is adopted, the sequencing result is compared with a GenBank database, and the homology similarity of the PCR product and the Lactobacillus plantarum is more than 99%. The finally identified genus species is lactobacillus plantarum. The sequence of the variable region of the 16SrDNA is shown in a sequence table SEQID NO. 1.
3. Physiological and biochemical characteristics: the physiological and biochemical reactions of the strains were measured by an esculin hydrolysis test, a methyl red test (MR test), an acetyl methyl alcohol test (VP test), an indigo matrix test, a trisaccharide iron test, a crimson disaccharide iron test, a urease test, a phenylalanine deaminase test, an amino acid decarboxylase test, a gelatin liquefaction test, a sodium malonate test, a citrate test (citrate test), a nitrate reduction test, a litmus milk test, a bacterial motility test, and the results are shown in table 1:
TABLE 1 results of physiological and biochemical characteristics experiment of Lactobacillus plantarum RD0025
| Physiological and biochemical project | Results |
| Esculin hydrolysis test | + |
| Methyl Red test (MR test) | + |
| Acetylmethylmethanol test (VP test) | - |
| Indigo substrate test | - |
| Trisaccharide iron test | - |
| Iron keshi disaccharide test | - |
| Urease test | - |
| Phenylalanine deaminase assay | - |
| Amino acid decarboxylase test | - |
| Liquefaction test of gelatin | - |
| Sodium malonate test | - |
| Citrate test (citrate test) | - |
| Nitrate reduction test | - |
| Litmus milk test | - |
| Bacterial motility test | - |
+: indicates positive; -: indicates negativity
The strain was biochemically identified by API 50CHL Lactobacillus identification system produced by Meiriee, France, and statistics of the identification results are shown in Table 2
TABLE 2 Lactobacillus plantarum RD0025API 50CH test strip reaction results
+: indicates positive; -: indicates negativity
The biochemical characteristics of the strain are judged to be in accordance with the biochemical characteristics of the lactobacillus plantarum by the biochemical map.
3. Preservation of the Strain
The Lactobacillus plantarum RD-0025 is screened from vaginal secretion of Chinese healthy women of child bearing age, and is preserved in China general microbiological culture collection center (CGMCC for short) in 5 months and 10 days in 2017, and the preservation unit address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, China academy of sciences microbiological research institute, the collection registration number is CGMCC No.14111, and the strain is classified and named as Lactobacillus plantarum.
Example 3 (Lactobacillus plantarum RD-0025 metabolite determination)
1. And (3) measuring the content of lactic acid in the metabolite of the lactobacillus plantarum RD-0025: the lactic acid in the fermentation liquor of the strain cultured for 24-48h is detected by a high performance liquid phase method (0.005M sulfuric acid aqueous solution (0.28mL sulfuric acid (98%)) -1000mL water, pH is about 2.1), and the result shows that the lactic acid (total L/D lactic acid) is about 30mg/mL and is far higher than the lactic acid content (about 15mg/mL) of the lactobacillus delbrueckii (commercially available), and the lactic acid detection spectrum of the invention is shown in figure 4
2. Determination of hydrogen peroxide content in the metabolite of lactobacillus plantarum RD-0025: hydrogen peroxide semiquantitative determination was carried out by the peroxidase method of Mcgrooarty et al, and isolated and identified Lactobacillus plantarum RD-0025 was inoculated to H2O2Identification of MRS-TMB plates, anaerobic reaction at 37 ℃After 24h of oxygen culture, the plates were removed and the cells were exposed to air. Produce H2O2The lactobacillus colonies will turn blue without producing H2O2The colony does not change color, and the produced H is subjected to color change according to the color change time2O2The results of semi-quantitative determination are shown in figure 5, the colony is obviously blue at 5min and a large amount of blue is obviously appeared at 10min, the bacterium is obviously easy to generate hydrogen peroxide and has strong hydrogen peroxide generation capacity, and the results show that the lactobacillus plantarum RD-0025 can obviously generate lactic acid and hydrogen peroxide and is beneficial to maintaining vaginal microecological balance.
Example 4 (antibiotic sensitivity test)
According to the requirements of antibiotic susceptibility tests in the general theory of live microbial products of the third portion of the pharmacopoeia of the 2015 edition, the sensitivity of the strain to the antibiotics is determined by adopting an agar diffusion paper sheet method, the sensitivity of lactobacillus plantarum RD-0025 of 0 generation (C0) and 30 generations (C30) to each antibiotic is examined, the sensitivity level of the strain to the antibiotics is judged according to the size of the inhibition zone, and the determination results are shown in the following table 3:
TABLE 3 antibiotic sensitivity test results of Lactobacillus plantarum RD0025
The inhibition zone is less than 10mm, the mild sensitivity is judged, the moderate sensitivity is judged at 10-20mm, and the sensitivity is judged at more than 20 mm.
Experimental data show that the bacillus is slightly sensitive to erythromycin and fleroxacin, moderately sensitive to meropenem and gentamycin, and sensitive to ampicillin, oxacillin, penicillin G, kanamycin, tetracycline, clindamycin, erythromycin, piperacillin, ceftriaxone, fleroxacin, vancomycin, amoxicillin/clavulanic acid, azithromycin, amoxicillin and bacitracin.
The antibiotic sensitivity to vancomycin, gentamicin and erythromycin is shown in figure 6.
Example 5 (toxicity test)
5 SPF-level Kunming mice are inoculated with 0.5ml of lactobacillus plantarum RD-0025 suspension liquid (more than 1 x 10) into the vagina of each mouse9CFU/mouse). The body weight of each mouse was measured daily according to the requirements of chinese pharmacopoeia 2015, and the behavior, physiology, etc. of each mouse before and after injection were observed and recorded. The results show that the weight average of all animals is increased within 7 days, no obvious poisoning symptom is seen, the activity behaviors are not abnormal, no animal is dead, and the strain is considered to belong to the nontoxic strain.
Example 6 (Lactobacillus plantarum RD-0025 passage stability test)
In this example, the lactobacillus plantarum strain RD-0025 was investigated for stability for 30 passages (C30) in terms of growth characteristics, morphology, biochemical characteristics, metabolite composition, antibiotic sensitivity, genetic characteristics, and toxicity test.
1. The method for separating and purifying lactobacillus plantarum RD-0025, observing colony morphology, performing stainboscopy and detecting biochemical characteristics are the same as the first part of example 1 and example 2. The results show that: after passage, the colony morphology is a white round colony with the size of about 3mm, the middle part is convex, the surface is smooth, no obvious change occurs, and the passage is stable; gram staining appeared as gram positive bacilli, with no change from the stained microscopic photograph to passage 0.
2. Analysis of genetic characteristics: the procedure is as in the second part of example 2. 16SrDNA fragment PCR amplification is respectively carried out on strains of the 0 th generation (C0) and the 30 th generation (C30) of the lactobacillus plantarum RD-0025, electrophoresis analysis is carried out on PCR amplification products, target bands are clear and single, the size is about 1500bp, the amplification is correct, the results of the two PCR amplifications of C0 and C30 are consistent, the determined sequences are compared and analyzed with known sequences in a GenBank database by using a BLAST tool in NCBI, and the homology is 100 percent for the lactobacillus plantarum.
3. And (3) metabolite determination: the method is the same as example 3, the content of the lactic acid is about 30mg/mL, hydrogen peroxide experiments show that each generation of bacterial colony has blue color at 5min, and a large amount of blue color is obvious at 10min, which proves that the capability of the bacterial strain to generate hydrogen peroxide by metabolism and the capability of generating lactic acid are stable.
4. Antibiotic sensitivity test: the method is the same as example 4, the sensitivity of the strain to antibiotics is determined by adopting an agar diffusion paper sheet method, the lactobacillus plantarum strain is judged to be slightly sensitive to erythromycin and fleroxacin, moderately sensitive to meropenem and gentamicin, and sensitive to ampicillin, oxacillin, penicillin G, kanamycin, tetracycline, clindamycin, erythromycin, piperacillin, ceftriaxone, fleroxacin, vancomycin, amoxicillin/clavulanic acid, azithromycin, amoxicillin and bacitracin according to the bacteriostasis range interpretation standard of a drug sensitive test paper sheet method.
5. Toxicity test: the procedure is as in example 5, and toxicity tests are carried out using the mouse vaginal infusion of Lactobacillus plantarum RD-0025 for passage 30(C30), at concentrations > 109CFU/mouse. The results were: all the tested mice have no toxic symptoms within 7 days, the weight is increased, and no animal is dead. According to the above results, the strain belongs to the nontoxic strain according to the supplement instruction of the research technical requirements of pharmacology and toxicology of new drugs.
In this example, lactobacillus plantarum RD-0025 was cultured in MRS medium for multiple passages, and the influence of the passage propagation on lactobacillus plantarum was studied in terms of morphology, biochemistry, metabolite characteristics, genetic characteristics, drug sensitivity characteristics, toxicity test, and the like. The results show that: the morphological, biochemical, genetic, metabolite and drug sensitive characteristics of MRS cultured and passaged within 30 generations are consistent with those of the initially isolated strain.
Example 7 (pharmacodynamic experiment of Lactobacillus plantarum RD-0025 Strain)
In-vitro bacteriostatic experiment of lactobacillus plantarum RD-0025 strain
(1) Experiment of Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii inhibiting Gardnerella vaginalis in vitro: preparing MRS agar cake of Lactobacillus plantarum RD-0025 according to 3% inoculation concentration, anaerobically culturing at 37 deg.C for 24h, and preparing commercially available Lactobacillus delbrueckii cake by the same method; taking 100 mu L of Gardnerella vaginalis, inoculating the Gardnerella vaginalis into 10mL of BHI solid culture medium, adding Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii cake, and carrying out anaerobic culture at 37 ℃ for 48 h; obvious inhibition zones can appear around the lactobacillus. The results are shown in FIG. 7, wherein the left graph shows the inhibition zone effect of Lactobacillus plantarum RD-0025, the diameter of the inhibition zone measured by a vernier caliper is 32.3mm, the right graph shows the inhibition zone effect of Lactobacillus delbrueckii, and the inhibition diameter is 21.5mm, and the conclusion is that the inhibition effect of Lactobacillus plantarum RD-0025 on Gardnerella vaginalis is stronger than that of Lactobacillus delbrueckii.
(2) Experiment of Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii in vitro inhibition of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella: preparing MRS agar cake of Lactobacillus plantarum RD-0025 according to 3% inoculation concentration, anaerobically culturing at 37 deg.C for 24h, and preparing commercially available Lactobacillus delbrueckii cake by the same method; staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella were inoculated into trypticase Soy peptone liquid (TSB) agar medium, and Lactobacillus plantarum RD-0025 and Lactobacillus delbrueckii cake were added. Culturing at 33 deg.C for 18-24h, observing the inhibition zone, measuring RD-0025 with vernier caliper to obtain inhibition zone diameter of 22mm, and determining Lactobacillus delbrueckii inhibition zone effect and inhibition diameter of 12mm, to obtain Lactobacillus plantarum RD-0025 with stronger inhibition effect on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella than Lactobacillus delbrueckii, and representative figures shown in figures 8-9
II, cell adhesion experiment: according to the number of the lactobacillus adhered to the vaginal epithelial cell monolayer, the adhesion performance of different lactobacillus is determined. The method comprises the following steps: taking human vaginal epithelial cells Vk2/E6E7 and human cervical cancer epithelial cells Hela, inoculating the cells into a 12-well plate at the density of 50 ten thousand per well, and forming a single cell layer by VK2/E6E7 after 48 hours; adding commercially available lactobacillus (Lactobacillus delbrueckii) and lactobacillus plantarum RD-0025 into each hole according to different amounts of CFU respectively, adhering for 4 hours, slightly oscillating on a shaking table in the adhering process, and setting two parallel experiments for each group; after the adhesion is finished, 1ml of 0.05% triton X-100 is used for cell lysis to prepare suspension bacterial liquid, the suspension bacterial liquid is diluted, and 100ul of bacterial liquid is respectively and uniformly inoculated on an MRS agar medium plate; after 48 hours of anaerobic culture, the number of clones per plate was counted.
The results show that: the 4h adhesion rate of the lactobacillus plantarum RD-0025 is 35.2 percent and 45.3 percent respectively, the 4h adhesion rate of the similar commercially available lactobacillus delbrueckii strain is 23.6 percent and 20.7 percent respectively, and the adhesion rate of the lactobacillus plantarum RD-0025 is higher than that of the similar commercially available lactobacillus delbrueckii strain.
Third, domestic rabbit vagina field planting experiment
1. Experimental methods
10 healthy animals were selected for stratified random grouping by weight into 2 groups, 5 animals in the positive control group, 5 animals in the experimental group:
preparing lactobacillus plantarum RD-0025 for field planting: weighing freeze-dried powder of Lactobacillus plantarum RD-0025 with field planting amount of 106. In the control group, a commercial product (Lactobacillus delbrueckii capsule) was used, 0.5mL of MRS liquid medium was added to each control group under aseptic conditions, the mixture was mixed well, and the mixture was completely sucked by a vaginal applicator and then implanted into the vagina.
And (3) field planting molding and sampling: after the monkey menstruation observed in the normal menstrual cycle, the molding bacteria were implanted for 7 consecutive days. The vagina of the animal is observed once a week, the color, the character and the secretion amount of the vaginal secretion are checked, the pH value of the vaginal secretion is measured, 2 sterile swabs are sampled, one swab is used for microscopic examination of the cleanness of the vaginal secretion, and the other swab is used for flora analysis.
Separation and purification culture of vaginal bacteria: the collected vaginal secretion is shaken in 2mL of D-Hanks buffer solution, and is subjected to gradient dilution by phosphate buffer solution, and the obtained product is respectively coated on MRS agar plates and cultured for 24-48h at 37 ℃ under an anaerobic condition. Single colony morphology and the like are recorded, and the MRS agar plate is restreaked to obtain purified colonies and subjected to biochemical and molecular identification.
Molecular biological method identification (16SrDNA gene sequence analysis): and (3) carrying out 16SrDNA sequence amplification, sequencing and analysis on the separated and purified strain, and carrying out sequencing after a PCR amplification product identified as a 16SrDNA fragment is purified on a target fragment by using a gel cutting recovery method. The determined 16S rDNA gene sequence was analyzed by alignment with known sequences in GenBank database using BLAST tool in NCBI, and the homology was 99%, and identified as the same species.
2. Results and analysis of the experiments
General observations of vaginal mucosa and secretions: after implantation, the vaginal mucosal secretion was observed once a week, and as a result, no obvious abnormality was found in all the test animals.
Measuring the pH value of vaginal secretion: after the lactobacillus plantarum RD-0025 is implanted, the pH value of vaginal secretion of most experimental animals is obviously lower than that of a control group of a marketed product, and is obviously reduced relative to that before the implantation, and the pH value measurement result is shown in Table 4.
TABLE 4 influence of Lactobacillus plantarum RD0025 on the pH of vaginal secretions of experimental animals
Cleanliness of vaginal secretions: compared with the control group before and after implantation, the quantity of the vaginal mixed bacteria in the control group is obviously increased, and the cleanliness is reduced; after the lactobacillus plantarum RD-0025 is implanted, the cleanliness of vaginal secretion of the experimental group is obviously better than that of the control group, gram-positive vaginal bacilli with different quantities can be seen, and the cleanliness is obviously higher than that of the control group. The results of the vaginal secretion cleanliness determinations are shown in table 5.
TABLE 5 influence of Lactobacillus plantarum RD0025 on the cleanliness of vaginal secretion of experimental animals
I to III represent the cleanliness, and III represents the lowest cleanliness.
Amplifying, separating and purifying the secretion of the experimental group, and analyzing the vaginal flora to find the test lactobacillus plantarum RD-0025 in all the vaginal secretions of 3 animals in the experimental group, wherein the information of the separated test lactobacillus plantarum RD-0025 is shown in figure 10. As can be seen from the figure, the tested lactobacillus plantarum RD-0025 is found in vaginal secretions of tested animals, so that the lactobacillus plantarum RD-0025 can be effectively planted in the vagina of the cynomolgus monkey.
Example 8 (Lactobacillus plantarum RD-0025 Freeze-drying preservation and stability of the Freeze-dried powder)
To test the survival rate of Lactobacillus plantarum RD-0025 in fermentation and freeze-drying conditionsLactobacillus plantarum RD-0025 was grown in modified MRS medium pH 6.5 and fermented using a 100 liter scale fermentor. Collecting thallus in early stage of plateau stage, and the viable count reaches about 3 × 109CFU/ml. The cells were collected by centrifugation, washed with phosphate buffer and mixed with lyoprotectants (including skimmed milk powder and sucrose). The mixture was then freeze-dried in a freeze-dryer. The samples were frozen at-40 ℃ for about 2 hours, then vacuum dried at-20 ℃ for 20-30 hours, and then dried at 30 ℃ for 3-5 hours. The dry powder was packaged in foil bags and stored at 4 ℃ and room temperature (25 ℃). Viable counts were determined by plate counting at months 0, 1, 3, 6, 9, and 12.
The starting Lactobacillus plantarum RD-0025 contains up to 500 billion viable bacteria per gram of dry powder (5X 10)10cfu/g) with optimum storage stability at 4 ℃. After 6 months of storage at 4 ℃, 80% of the initial viable count was retained, see table 6.
TABLE 6 stability test results of Lactobacillus plantarum RD0025 lyophilized powder agent
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Qiangji pharmaceutical Co., Ltd; guangdong Longchuang base pharmaceutical Co., Ltd; zhao Xiang Jiang
<120> Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs
<160>1
<210>1
<211>1471
<212>DNA
<213> Artificial sequence
<400>1
aatctctggtccacttaggcggctggttctaaaaggttac 40
cccaccgactttgggtgttacaaactctcatggtgtgacg 80
GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCA 120
TGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGG 160
AGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCA 240
TCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCAT 280
GATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCAC 320
CGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACT 360
TCTCACGACACGAGCTGACGACAACCATGCACCACCTGTA 440
TCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGC 480
ATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTC 520
GAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT 560
GCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGG 640
AAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGA 680
CTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCG 720
AGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCC 760
ACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACA 800
CATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCA 840
GTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCAC 880
ATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAA 920
TAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 960
GTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTT 1040
AACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCAC 1080
GCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGAT 1120
TCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTC 1160
AGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGT 1200
ATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAA 1240
TACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATC 1280
TTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTA 1320
TTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCA 1360
GGTTTCCCACGTGTTACTCACCAGTTCGCCActcactcaa 1400
atgtaaatcatgatgcaagcaccaatcaataccagagttc 1440
gttcgactgcatgtatagcacgccgcacctc1471
Claims (8)
1. Lactobacillus plantarum has a preservation number of CGMCC 14111.
2. Use of a lactobacillus plantarum as claimed in claim 1 for the preparation of a bacteriostatic medicament for bacterial vaginal pathogenic bacteria.
3. Use according to claim 2, characterized in that the drug is a bacterial agent which colonizes and survives the vaginal epithelium in the vaginal environment.
4. Use according to claim 2, characterized in that said drug is a bacterial agent which is metabolized to produce H in the vaginal environment2O2。
5. Use according to claim 2, characterized in that the bacterial vaginal pathogenic bacterium is gardnerella vaginalis.
6. Use according to claim 2, characterized in that the bacterial vaginal pathogenic bacterium is candida albicans, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa or salmonella.
7. Use of the lactobacillus plantarum described in claim 1 for the preparation of a medicament for the prophylaxis or treatment of bacterial vaginal diseases.
8. Use according to claim 7, characterized in that the bacterial vaginal disease is senile vaginitis, non-specific vaginal infections or mixed vaginal infections.
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| CN110339216B (en) * | 2018-04-02 | 2022-08-26 | 景岳生物科技股份有限公司 | Lactic acid bacteria composition for preventing and treating bacterial vaginitis |
| CN110540945B (en) * | 2018-05-29 | 2023-04-25 | 广东强基药业有限公司 | Lactobacillus jensenii and application thereof in preparing vaginal antibacterial drugs |
| KR101930438B1 (en) * | 2018-10-12 | 2018-12-18 | (주) 에이투젠 | Novel Lactobacillus plantarum strain ATG-K2, ATG-K6 or ATG-K8, and composition comprising thereof for preventing or treating vaginosis |
| CN109439592A (en) * | 2018-12-06 | 2019-03-08 | 青岛东海药业有限公司 | Lactobacillus plantarum preparation and its application |
| CN110016442B (en) * | 2019-02-21 | 2021-03-23 | 河北一然生物科技有限公司 | Application of lactobacillus rhamnosus and compound bacterium powder thereof in product for preventing and treating vaginitis |
| CN110777087B (en) * | 2019-08-09 | 2020-08-07 | 四川厌氧生物科技有限责任公司 | Lactobacillus johnsonii and application thereof |
| CN112342154B (en) * | 2020-09-03 | 2022-08-05 | 上海上药信谊药厂有限公司 | Probiotics for preventing and treating female genital tract inflammation |
| CN113699071B (en) * | 2021-08-30 | 2023-05-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicines for treating infectious vaginosis |
| CN114869917B (en) * | 2022-07-07 | 2022-10-28 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus plantarum N13 in preparation of medicine for preventing and treating vaginitis or inhibiting pathogenic bacteria |
| WO2024032452A1 (en) * | 2022-08-09 | 2024-02-15 | 成都倍特生物制药有限公司 | Lactiplantibacillus plantarum strain and use thereof |
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