CN116004457B - Humanized lactobacillus plantarum and application thereof - Google Patents
Humanized lactobacillus plantarum and application thereof Download PDFInfo
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- CN116004457B CN116004457B CN202211698440.XA CN202211698440A CN116004457B CN 116004457 B CN116004457 B CN 116004457B CN 202211698440 A CN202211698440 A CN 202211698440A CN 116004457 B CN116004457 B CN 116004457B
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a human lactobacillus plantarum and application thereof, wherein the strain is lactobacillus plantarum (Lactobacillus plantarum) SQ1631, and the preservation number of the strain is CGMCCNO:23932. the lactobacillus plantarum SQ1631 has stronger acid and hydrogen peroxide production capacity; the strain can be normally planted in the genital tract environment; the composition has the functions of regulating vaginal flora, inhibiting the growth and colonization of vaginal pathogenic bacteria, promoting the reconstruction of vaginal ecology, recovering the original dominant vaginal flora and acidic environment, and can be used for preventing and treating bacterial vaginitis, vulvocandidiasis and other vaginal infection diseases, so that the traditional treatment concept of killing microorganisms is changed into a new treatment concept with the aim of increasing probiotics and recovering normal micro-ecological environment.
Description
Technical field:
the invention belongs to the technical field of microorganisms, and particularly relates to a humanized lactobacillus plantarum and application thereof.
The background technology is as follows:
The female genital tract microenvironment is a unique and complex dynamic system composed of multiple flora that plays a critical role in the health of the reproductive system. Vaginal dysbacteriosis is associated with many gynecological diseases, such as bacterial vaginosis, fungal infections such as candida, urinary tract infections, HIV, etc.
Bacterial Vaginitis (BV) is a common and frequently occurring gynecological disease associated with dysbacteriosis, which is accompanied by an increase in pathogenic bacteria and a decrease in the abundance of probiotic lactobacilli. Vulvocandidiasis (VVC) is a gynecological disease using candida albicans as a main infectious agent, and clinically related diseases such as VVC, BV and the like are mainly treated by antibiotics at present, however, the antibiotics are not easy to thoroughly treat, drug resistance is easy to generate, and vaginal dysbiosis is further aggravated, so that the disease is easy to repeat.
Studies have shown that several kinds of lactobacillus commonly found in female genital tracts can inhibit adhesion and proliferation of various vaginal pathogenic bacteria by producing various mechanisms such as bacteriostasis, antibacterial substances (acidogenesis, hydrogen peroxide, bacteriocin, etc.), competitive adhesion, etc., and therefore, the treatment of gynecological diseases such as BV, VVC, etc. by using the microbial therapy related to beneficial lactobacillus from healthy female genital tracts may have a better effect. At present, although the existing lactobacillus plantarum in the field has a bacteriostasis effect on common pathogenic bacteria of vagina, the effect is still to be further improved.
The invention comprises the following steps:
In order to make up the prior art, the invention aims to provide the humanized lactobacillus plantarum and the application thereof in medicaments for preventing and treating female genital tract infection, so as to at least achieve the effects of having good cell adhesion and generating antibacterial substances, thereby being capable of effectively inhibiting the growth of various vaginal pathogenic bacteria, and having the probiotic functions of improving the vaginal environment of a human body and preventing and treating vaginitis.
The purpose of the invention is implemented by the following technical scheme:
In a first aspect, the human lactobacillus plantarum is lactobacillus plantarum (Lactobacillus plantarum) SQ1631, and the preservation number of the strain is CGMCC NO:23932. the strain has a 16S rDNA sequence shown as a sequence 1 in a sequence table.
In a second aspect, the use of a strain as hereinbefore described for improving the vaginal environment of a human.
In particular to the application in the production of a composition for improving the vaginal environment of human bodies.
Further, the amount of Lactobacillus plantarum SQ1631 in the composition is not less than 1X 10 6 CFU/mL or not less than 1X 10 6 CFU/g.
Further, the composition comprises a microbial inoculum.
Further, the microbial inoculum comprises at least one of a live strain or an inactivated strain of the lactobacillus plantarum SQ1631 or a metabolite, a culture solution, a suspension, a fermentation liquor or an extraction liquor of the lactobacillus plantarum SQ1631, and the microbial inoculum further comprises a pharmaceutically acceptable carrier. The carrier is selected from one or a combination of a plurality of microcarriers, microcapsules, hydrogel or nanoscale materials. The carrier is beneficial to maintaining the biological activity of the strain or the strain product of the composition before use, prolonging the shelf life of the composition, and ensuring the use effect of the composition because the strain or the strain product can play a role more permanently when the composition is used.
Further, the composition is a pharmaceutical or disposable hygienic product. .
Further, the method is particularly applied to the production of disposable hygienic products for inhibiting/killing vaginal pathogenic bacteria. The disposable sanitary article is a disposable daily life article which is only in direct contact with a human body and aims at achieving the purposes of physiological sanitation, antibiosis or bacteriostasis of the human body, and mainly comprises the following components: menstrual hygiene products for women such as: sanitary pads, sanitary napkins, panty liners, tampons and the like; faecal hygiene products such as: diaper pants, diaper pads, diaper paper, urine insulation pads, and the like; other sanitary products such as tissues, wet tissues, sanitary wet tissues, antibacterial agents, bacteriostats, tissues, cosmetic cottons, disposable underpants, disposable gloves or finger-stalls (excluding medical gloves or finger stalls), condoms, and the like.
Further, the application in improving the change of human vagina environment caused by vagina pathogenic bacteria.
Further, the vaginal pathogenic bacteria include at least one of candida albicans, escherichia coli and staphylococcus aureus.
In a third aspect, the use of a strain as hereinbefore described in the manufacture of a medicament for the treatment of female genital tract infections. For example, a medicament for preventing and treating genital tract infection similar to a lactobacillus live bacteria capsule for vagina and the like.
Biological preservation information:
Classification naming: lactobacillus plantarum (Lactobacillus plantarum);
Preservation unit: china general microbiological culture Collection center (CGMCC);
preservation number: CGMCC NO:23932;
Preservation date: 2021, 11, 18;
preservation address: north Star, chaoyang district, beijing, no. 1, no. 3.
The invention has the advantages that:
(1) The lactobacillus plantarum SQ1631 has stronger acid and hydrogen peroxide production capability.
(2) The lactobacillus plantarum SQ1631 has strong acid resistance, can adapt to the acidic environment of the vagina of a healthy woman, and has the advantage of adhesion to vaginal epithelial cells, so that the strain can be normally planted in the genital tract environment.
(3) The lactobacillus plantarum SQ1631 has better inhibition effect on common vaginal pathogenic bacteria such as escherichia coli, staphylococcus aureus and candida albicans, and particularly has more advantages on the inhibition effect on candida albicans, and under the same experimental condition, the inhibition zone of the lactobacillus plantarum SQ1631 on candida albicans is 1.6 times of that of lactobacillus delbrueckii.
Therefore, the novel vaginal antibacterial agent has the functions of regulating vaginal flora, inhibiting growth and field planting of vaginal pathogenic bacteria, promoting reconstruction of vaginal ecology, recovering original dominant vaginal flora and acidic environment, and can be used for preventing and treating bacterial vaginitis, so that the traditional treatment concept of killing microorganisms is changed into a novel treatment concept with the aim of increasing probiotics and recovering normal micro-ecological environment.
Description of the drawings:
in order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph showing colony morphology of Lactobacillus plantarum SQ1631 of the present invention on MRS plates;
FIG. 2 is a diagram showing the shape of the fungus of Lactobacillus plantarum SQ1631 according to the invention under a microscope;
FIG. 3 is a post-acidogenesis plate of Lactobacillus plantarum SQ1631 of the invention;
FIG. 4 is a graph showing the acid producing capacity of Lactobacillus plantarum SQ1631 according to the invention;
FIG. 5 is a photograph showing the result of an experiment of the hydrogen peroxide producing ability of Lactobacillus plantarum SQ1631 according to the present invention;
FIG. 6 is a bar graph of the acid resistance test of Lactobacillus plantarum SQ1631 of the present invention;
FIG. 7 is a graph showing the effect of Lactobacillus plantarum SQ1631 strain of the present invention before rinsing due to adhesion to vaginal epithelial cells;
FIG. 8 is a graph showing the effect of the adhesion of Lactobacillus plantarum SQ1631 strain of the present invention to vaginal epithelial cells after rinsing;
FIG. 9 is a graph showing the results of quantitative antibacterial tests of the biofilm formation capacity of Lactobacillus plantarum SQ1631 according to the invention.
The specific embodiment is as follows:
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents in the following examples, unless otherwise specified, are all conventional biochemical reagents; the quantitative tests are all average results of three repeated experiments; the medium used was as follows:
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 2g of K 2HPO4, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 1mL of Tween 80, 0.25g of MgSO 4.7H2O 0.58g,MnSO44H2 O and 1000ml of distilled water.
MRS solid medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 2g of K 2HPO4 g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 1mL of Tween 80, 0.25g of MgSO 4.7H2O 0.58g,MnSO44H2 O and 18g of agar.
TMB-HRP solid Medium: 25.0mg of 3,3', 5' -Tetramethylbenzidine (TMB) was dissolved in a small amount of absolute ethanol, and then added to 100ml of MRS solid medium having a pH of 7.0, followed by sterilization. Then 0.5mL 10mg/mL horseradish peroxidase (HRP) (0.22 μm filter degerming) is added into the MRS solid culture medium which is sterilized and cooled to about 50 ℃, and after uniform mixing, the mixture is immediately poured into a flat plate and stored under the refrigerating condition after solidification for standby.
The strains used in the following examples: lactobacillus delbrueckii DM8909, lactobacillus plantarum SQ1662, lactobacillus plantarum SQ1651, lactobacillus plantarum SQ1685 and Lactobacillus plantarum SQ1686 are all self-owned strains of the inner Mongolian Biqi pharmaceutical industry Co., ltd. In this example, candida albicans CMCC (F) 98001, escherichia coli CMCC (B) 44102 and Staphylococcus aureus CMCC (B) 26003 were purchased from standard strains of the national institute for biological product testing.
Example 1 screening of target strains
The aseptic cotton swab is used for taking the secretion from the vaginal post-fornix of the female of the middle and young women, the secretion is put into a sterilizing test tube, and the sterilizing test tube is put into an ice box for rapid transportation to a laboratory. Under the aseptic condition, the taken samples are diluted to 10 -3 times by ten times by using phosphate buffer PBS, 100 microliters of different gradient dilutions are taken and coated with an MRS solid plate containing bromocresol purple for strain separation. After the flat plate is subjected to anaerobic culture for 48 hours at 37 ℃, bacterial colony with strong acid production capability to make the purple flat plate obviously yellow (see figure 3) is selected for bacterial strain purification, the bacterial colony is selected and purified for a plurality of times until the separated bacterial strain is a pure culture, the obtained pure bacteria are subjected to gram-staining microscopic examination and hydrogen peroxide production analysis, gram-positive and hydrogen peroxide-producing bacillus is reserved, the primarily prepared glycerol strain is preserved at a low temperature for standby, and the cultured bacterial colony is smooth and full, has clean edges and white and has a size of about 2.5mm; the cells were observed under a microscope as short rods with rounded ends, arranged singly or in a chain, see in particular figures 1-2.
Example 2 identification of strains
The bacterial 16S rRNA amplification universal primer 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3') are used for carrying out PCR amplification on the selected strain, amplified products are sent to a sequencing company for sequencing, the obtained sequencing result is compared with sequences in an NCBI database through BLAST and is divided into a phylogenetic tree, and the sequencing result is shown in a sequence table.
The bacterial strain separated by the invention has the advantages of rod shape, gram positive and strong acid production, and the acid production effect is shown in figures 3-4, and is facultative anaerobic. The 16S rRNA sequencing result shows that the screened strain has high sequence similarity with lactobacillus plantarum, the similarity is up to 100%, and the selected strain belongs to lactobacillus plantarum and is named as SQ1631.
EXAMPLE 3 carbohydrate utilization of Lactobacillus plantarum SQ1631 Strain
After the lactobacillus plantarum SQ1631 strain is subjected to plate activation culture, a single colony is selected and penetrated into an MRS solid culture medium containing carbohydrates as shown in table 1, the lactobacillus plantarum SQ1631 strain is subjected to aerobic culture at 38 ℃, and after 3 days, the change condition of bromocresol purple color in the culture medium is observed and recorded, and the result is shown in table 1.
Table 1: results of experiments on carbohydrate utilization of Lactobacillus plantarum SQ1631
Note that: + indicates positive; -negative representation
Lactobacillus plantarum SQ1631 of the invention, except rhamnose and melezitose, the other carbohydrates mentioned above are available.
Example 4 acid production ability of Lactobacillus plantarum SQ1631 Strain
The lactobacillus plantarum SQ1631 glycerol strain is dipped in a loop by an inoculating loop, is inoculated into a test tube filled with 10mLMRS liquid culture medium, is subjected to anaerobic culture at 37 ℃ for 12hrs, then the cultured bacterial liquid is inoculated into 80mL MRS liquid culture medium with an inoculating amount of 4%, is subjected to anaerobic culture at 37 ℃ for 8hrs, and the pH of the fermentation liquid is measured at 2,3, 4, 5, 6, 7 and 8 hours respectively, and the result is shown in figure 4, and the result shows that the bacterial has strong acid producing capability.
EXAMPLE 5 Hydrogen peroxide production ability of Lactobacillus plantarum SQ1631 Strain
The lactobacillus plantarum SQ1631, lactobacillus delbrueckii DM8909, lactobacillus plantarum SQ1662 and lactobacillus plantarum SQ1651, lactobacillus plantarum SQ1685 and lactobacillus plantarum SQ1686 are respectively activated, then are divided into areas and streaked on a TMB-HRP plate, are subjected to anaerobic culture at 37 ℃ for 24 hours and then are taken out, are exposed to an aerobic air environment, are observed to change color after 20 minutes, and are judged to be strong and weak in hydrogen peroxide production and capability according to the shade of blue, the result is shown in FIG. 5, 6 different strains corresponding to 6 areas in the graph respectively, wherein reference numeral 104-1 corresponds to the lactobacillus plantarum SQ1631 color development result, 106-1 corresponds to lactobacillus delbrueckii DM8909 color development result, 114-1 corresponds to lactobacillus plantarum SQ1662 color development result, 113-1 corresponds to lactobacillus plantarum SQ1651 color development result, 110-1 corresponds to lactobacillus plantarum SQ1685 color development result, and 108-2 corresponds to lactobacillus plantarum SQ1686 color development result. From this, the lactobacillus plantarum SQ1631 of the present invention has the strongest hydrogen peroxide production capacity.
Example 6 liquid fermentation and lyophilization of Lactobacillus plantarum SQ1631 Strain
Inoculating lactobacillus plantarum SQ1631 strain half branch into 80mL of MRS liquid culture medium, performing anaerobic culture at 37 ℃ for 12hrs, inoculating the cultured bacterial liquid into 200mL of MRS liquid culture medium with an inoculum size of 4%, performing anaerobic culture at 37 ℃ for 8hrs, continuously transferring into 400mlMRS liquid culture medium, performing anaerobic culture at 37 ℃ to a fermentation end point, and obtaining lactobacillus plantarum SQ1631 fermentation broth, and determining the viable count of the lactobacillus plantarum SQ1631 fermentation broth to be 20 hundred million CFU/mL. And centrifuging the end fermentation liquid, removing the supernatant, adding a protective agent, and freeze-drying to obtain the bacterial powder, wherein the viable count of the bacterial powder is 600 hundred million.
EXAMPLE 7 acid resistance of Lactobacillus plantarum SQ1631 Strain
The fermentation broth of Lactobacillus plantarum SQ1631 in example 6 was collected by centrifugation, 1.0g of the pellet was weighed and resuspended in 9.0mL of sterile PBS having pH values of 3.7, 4.5 and 5.0, respectively, and viable counts were obtained after treatments of 0, 1,2 and 3hrs at 37℃as shown in Table 2, and the results of the viable counts were plotted and analyzed, as shown in FIG. 6.
Table 2: acid resistance test of the Strain viable count results
The result shows that the viable count of the strain is not obviously reduced after the strain is treated for 3 hours under the condition that the pH value is 3.7-5, which proves that the strain has stronger acid resistance; the pH value of vagina of healthy women is about 3.8-4.4, so the strain can be normally planted in the environment of the genital tract.
Example 8 adhesion ability of Lactobacillus plantarum SQ1631 Strain
Subculture of cells:
(1) Placing the purchased fresh cell culture fluid finished product in a 37 ℃ incubator for temperature return, spraying 70% alcohol after temperature return, wiping, and transferring into a sterile operation table.
(2) Taking out the freezing tube, immediately placing into a water bath kettle at 37 ℃, quickly thawing, wiping the outside of the preservation tube with 70% alcohol, and transferring into a sterile operation table.
(3) The thawed human vaginal epithelial cell suspension was removed, placed in a sterile, enzyme-free 1.5mL centrifuge tube, centrifuged at 1500rpm for 5 minutes, and the supernatant removed.
(4) The washing was performed three times using DPBS as a phosphate buffered saline solution.
(5) Removing supernatant, adding the above cell culture solution, mixing, transferring into cell culture bottle (25 cm 2), and culturing in CO 2 incubator.
(6) After the cells adhere to the wall, the liquid is changed every day so as to keep the fresh of the human epithelial cell culture liquid.
(7) When the cells are attached to 80%, the cells are digested and passaged into six-hole plates by using pancreatin with the mass-volume ratio of 0.25% and disodium ethylenediamine tetraacetate EDTA with the mass-volume ratio of 0.53nm, and after passaging, the cells grow to the usable concentration, and the culture is stopped.
Adhesion of thalli to human vaginal epithelial cells
(1) DPBS and fresh broth were incubated at 37℃for 1h for further use.
(2) The cultured human vaginal epithelial cells are washed three times by using DPBS buffer solution without antibiotics for standby.
(3) And (3) selecting the activated lactobacillus plantarum SQ1631, marking a flat plate, and directly scraping a proper amount of thalli into the fresh cell culture solution without resistance by using a sterile inoculating loop after the thalli grow to be dense in the flat plate, and ensuring the concentration of the thalli to be 10 9/mL for later use.
(4) Directly adding the bacterial liquid with the adjusted concentration into the six-hole plate of the cultured cells, sucking out the culture liquid in the six-hole plate after 1h of treatment, adding the DPBS buffer solution, and washing the decolorizing shaker for 20min at the rotating speed of 65 rmp.
(5) Switzerland-Giemsa staining, rinsing, and microscopic observation.
The experimental results are shown in fig. 7-8, and the bacteria colonization ability is seen to be advantageous from fig. 7-8.
EXAMPLE 9 antibacterial Properties of Lactobacillus plantarum SQ1631 Strain
Firstly, pouring a sterilized MRS solid culture medium into a flat plate until the bottom of the flat plate is just fully paved, standing and uniformly placing 3 oxford cups into each flat plate after solidification.
Secondly, culturing the indicator bacteria liquid overnight according to a certain proportion: coli CMCC (B) 44102, staphylococcus aureus CMCC (B) 26003, candida albicans CMCC (F) 98001 were mixed with MRS solid medium cooled to a suitable temperature, respectively, to give pathogen concentrations on the order of 10 5 CFU/ml. Respectively pouring the materials into a flat plate with the oxford cup put in advance until the materials are 2mm away from the upper edge of the oxford cup. Standing for solidification, and taking out the oxford cup gently.
The experimental group 1 is a blank control, the experimental group 2 is lactobacillus delbrueckii DM8909 bacterial powder suspension (10 8 CFU/ml order), and the experimental group 3 is lactobacillus plantarum SQ1631 bacterial powder suspension (10 8 CFU/ml order).
Finally, 200 microliters of each experimental group suspension was added to each well, each group of plates was placed in an anaerobic box at 37 ℃ for culturing for 24hrs, photographed, and the diameter of the inhibition zone was measured with a ruler, and the test results are shown in tables 3-5 and fig. 9.
Table 3: candida albicans inhibiting CMCC (F) 98001 detection result
Table 4: detection result of staphylococcus aureus inhibiting CMCC (B) 26003
Table 5: detection result of colibacillus inhibiting CMCC (B) 44102
The result shows that the lactobacillus plantarum SQ1631 has better inhibiting effect on the escherichia coli CMCC (B) 44102, the staphylococcus aureus CMCC (B) 26003 and the candida albicans CMCC (F) 98001 than the lactobacillus delbrueckii DM8909, and particularly has obviously improved inhibiting effect on the candida albicans CMCC (F) 98001 than the lactobacillus delbrueckii DM 8909.
Example 10 animal experiments with Lactobacillus plantarum SQ1631 Strain inhibiting vaginal bacterial infection in rats
Step 1, selecting 40 normal female rats, and randomly dividing the normal female rats into 4 groups: normal, model, natural recovery and lactobacillus plantarum treated groups of 10 animals each. The specific arrangement is shown in Table 6.
Table 6: animal experiment arrangement for inhibiting vaginal bacterial infection of rats by lactobacillus plantarum SQ1631 strain
Note that: the mixed bacterial liquid contains 10 8 cfu/ml of escherichia coli CMCC (B) 44102 and staphylococcus aureus CMCC (B) 26003, and 10 9 cfu/ml of lactobacillus plantarum SQ1631 bacterial liquid
Step 2. Determination and analysis of vaginal flora of rats
The vagina of the rat was repeatedly rinsed with 0.2mL of physiological saline for 8-10 times by a microsampler, 50. Mu.L of vaginal rinse solution was taken to count live bacteria of E.coli CMCC (B) 44102 and staphylococcus aureus CMCC (B) 26003, respectively, and the count results are shown in Table 7.
Table 7: detection result of rat vaginal flushing fluid flora
As can be seen from Table 7, both the Lactobacillus plantarum treated group and the natural recovery group had a lower number of colonisation than E.coli CMCC (B) 44102 and S.aureus CMCC (B) 26003 in the rat vagina in the model group, and in particular the treated group had substantially normal levels of E.coli CMCC (B) 44102 and S.aureus CMCC (B) 26003 in the rat vagina.
The results show that the lactobacillus plantarum SQ1631 has the functions of regulating vaginal flora, inhibiting the growth and colonization of vaginal pathogenic bacteria, promoting the reconstruction of vaginal ecology, recovering the original dominant vaginal flora and acidic environment, and can be used for preventing and treating colpitis diseases such as bacterial vaginitis, vulvocandidiasis and the like, so that the traditional treatment concept of killing microorganisms is changed into a new treatment concept with the purposes of increasing probiotics and recovering normal microecological environment.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. The human lactobacillus plantarum (Lactobacillus plantarum) SQ1631 is characterized in that the preservation number of the lactobacillus plantarum (Lactobacillus plantarum) SQ1631 is CGMCC NO:23932.
2. A microbial agent comprising a live strain of lactobacillus plantarum SQ1631 as claimed in claim 1.
3. The microbial agent of claim 2, further comprising a pharmaceutically acceptable carrier.
4. Use of the strain according to claim 1 for the preparation of a medicament for improving the vaginal environment of humans.
5. Use of the microbial agent of claim 2 or 3 for the preparation of a medicament for improving the vaginal environment of a human.
6. The use according to claim 4, wherein the number of lactobacillus plantarum SQ1631 in the medicament is equal to or greater than 1 x 10 6 CFU/mL or equal to or greater than 1 x 10 6 CFU/g.
7. The use according to claim 5, wherein the number of lactobacillus plantarum SQ1631 in the medicament is ≡1×10 6 CFU/mL or ≡1×10 6 CFU/g.
8. Use of the strain of claim 1 for the preparation of a disposable hygienic product for the inhibition/killing of vaginal pathogenic bacteria; the vaginal pathogenic bacteria are escherichia coli, staphylococcus aureus or candida albicans.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888051A (en) * | 2005-06-28 | 2007-01-03 | 北京天佑达生物工程科技有限公司 | Plant lactobacillus strain and its application |
| CN107299065A (en) * | 2017-06-20 | 2017-10-27 | 广东强基药业有限公司 | A kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines |
| CN107794236A (en) * | 2017-10-10 | 2018-03-13 | 内蒙古双奇药业股份有限公司 | A kind of Lactobacillus crispatus and its application |
| CN113699071A (en) * | 2021-08-30 | 2021-11-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicine for treating infectious vaginosis |
| CN114869917A (en) * | 2022-07-07 | 2022-08-09 | 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 |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888051A (en) * | 2005-06-28 | 2007-01-03 | 北京天佑达生物工程科技有限公司 | Plant lactobacillus strain and its application |
| CN107299065A (en) * | 2017-06-20 | 2017-10-27 | 广东强基药业有限公司 | A kind of Lactobacillus plantarum and its application for preparing vagina antibacterial medicines |
| CN107794236A (en) * | 2017-10-10 | 2018-03-13 | 内蒙古双奇药业股份有限公司 | A kind of Lactobacillus crispatus and its application |
| CN113699071A (en) * | 2021-08-30 | 2021-11-26 | 天津科技大学 | Lactobacillus plantarum and application thereof in preparation of medicine for treating infectious vaginosis |
| CN114869917A (en) * | 2022-07-07 | 2022-08-09 | 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 |
Non-Patent Citations (1)
| Title |
|---|
| Hydrogen peroxide-producing Lactobacillus species in healthy women and in women with bacterial vaginosis;E Shopova;Akusherstvo i ginekologiia;20031231;参见全文 * |
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