CN116694498A - Vaginal probiotic composition for relieving various vaginitis - Google Patents
Vaginal probiotic composition for relieving various vaginitis Download PDFInfo
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- CN116694498A CN116694498A CN202310046334.1A CN202310046334A CN116694498A CN 116694498 A CN116694498 A CN 116694498A CN 202310046334 A CN202310046334 A CN 202310046334A CN 116694498 A CN116694498 A CN 116694498A
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Abstract
The invention discloses a vaginal probiotics composition for relieving various vaginitis, and belongs to the technical field of microorganisms. Based on the growth characteristics of the strain by utilizing the substrate, the invention preferably selects the combination of the lactobacillus crispatus CCFM1298, the lactobacillus gasseri CCFM1201 and the fructo-oligosaccharide, realizes the synergistic symbiosis of the combined strains, and realizes the effect of multiple remission of colpitis. The probiotic composition can relieve colpitis tissue inflammation, regulate local vaginal immune system, and help relieve and improve female bacterial vaginitis and candidal vaginitis. In addition, as the probiotics are all derived from healthy female vagina, the probiotics are used for preparing products and sanitary articles for female vaginitis, are beneficial to relieving and improving female vaginitis, and have good application prospects.
Description
Technical Field
The invention relates to a vaginal probiotic composition for relieving various vaginitis, and belongs to the technical field of microorganisms.
Background
Unlike the high diversity of the gastrointestinal tract, the microbiome diversity of the female genital tract is low and changes dynamically during the female menstrual cycle. Healthy vaginal flora is mainly of the genus Lactobacillus and mainly comprises Lactobacillus crispatus, lactobacillus gasseri, lactobacillus jensenii and Lactobacillus inertia. When an exogenous pathogen interferes with or is endogenous to the disorder, infection such as vaginitis, cervicitis, pelvic inflammatory disease, etc. can occur. Among them bacterial vaginosis (bacterial vaginosis, BV) and vulvovaginal candidiasis (or candidal vaginitis, vulvovaginal candidiasis, VVC) are the most common vaginal infection diseases for women of childbearing age. Meanwhile, BV and/or VVC have many associated complications, including premature labor, pelvic inflammatory disease, infertility, ectopic pregnancy, pelvic abscess, spontaneous abortion, menstrual disorder, increased susceptibility to hiv infection, and other chronic health problems. In view of the prevalence of BV and VVC and related complications, BV and VVC have been identified as important global public health issues.
BV is characterized by increased microbiome diversity in the vagina, decreased Lactobacillus in the vagina, and increased BV-related anaerobic bacteria. BV-related bacteria mainly include Gardnerella genus and Pr Lei Wote genus. BV is clinically manifested by increased vaginal secretion, fishy smell, and increased taste after sexual intercourse, and can be accompanied by mild pruritus vulvae or burning sensation. The secretion is off-white, homogeneous and thin. The initial cure rate of BV is 70% -90%, the recurrence rate of 3 months after treatment can reach 40%, and the recurrence rate of 12 months can reach 60%. The most common species associated with VVC are candida species, with candida albicans, candida glabrata, candida tropicalis being the most common. Clinical symptoms of VVC are non-specific and can be associated with a variety of vaginosis, such as bacterial vaginosis. The most common clinical manifestations are pruritus vulvae and burning with vaginal soreness and irritation, resulting in dyspareunia and dysuria. Vulvar and vaginal erythema, oedema and chapping are also common. It is estimated that about 10-15% of asymptomatic women are infected with candida, 50% of primary infected women experience a second VVC event, and 5-10% of women develop recurrent candida vaginitis. At present, the clinical treatment means aiming at BV and VVC mainly adopts antibiotic treatment, and the antibiotic treatment kills pathogenic bacteria and non-pathogenic bacteria indiscriminately. Finally, dysbacteriosis is caused, so that the problems of poor treatment effect, high recurrence rate and the like are caused.
Probiotics are widely used to promote gastrointestinal health and they may also be beneficial in the prevention and treatment of gynaecological disorders. At present, the probiotics reported in China and suitable for women are mainly Lactobacillus delbrueckii DM8909 which is a single lactobacillus preparation screened from vaginal secretions of healthy women by the professor Kang Bai of Dalian medical university (J. Chinese microecology journal, 2001) of Lactobacillus delbrueckii DM8909 strain, mainly by vaginal external application, and the lactobacillus is not a common bacterium of the genital tract of healthy women. In addition, some of the patent publications relate to vaginal probiotic mixtures (CN 113384601A, CN111281896, CN109674826 a) mostly aimed at single vaginitis, the mode of use is mainly vaginal external, and the effect after oral administration is unknown. And the combination of probiotic mixtures does not take into account factors that might reduce the effect, such as antagonism between strains, competition of nutrition and adhesion sites in the gastrointestinal tract, etc., while it is unclear whether the host immune system can be stimulated in vivo to thereby alleviate and/or treat vaginitis. Thus, there is currently a lack of vaginal probiotic compositions that are fully evaluated and have a clear effect in a variety of vaginitis bodies after oral administration.
Disclosure of Invention
Aiming at the problems existing at present, the invention provides a vaginal probiotic composition capable of synergistically regulating and controlling bacterial vaginitis and candidal vaginitis, and the key point is that the composition can enhance the immune regulation effect of a single probiotic preparation.
The invention provides a Lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 which is deposited with the microorganism strain collection in Guangdong province at 12/9 of 2022 with the deposit number of GDMCC No:62275.
the lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 provided by the invention is derived from a healthy female vaginal swab in Jiangsu local area, the genome of the CCFM1298 is extracted, the 16S rDNA of the strain CCFM1298 is amplified and sequenced (the nucleotide sequence of the 16S rDNA obtained by amplifying the CCFM1298 is shown as SEQ ID NO.1 by the biological technology Co., jin Weizhi, suzhou) and the nucleotide sequence of the sequence is compared in NCBI, and the result shows that the strain is the lactobacillus crispatus and is named as the lactobacillus crispatus (Lactobacillus crispatus) CCFM1298.
The invention provides a vaginal probiotics composition, which comprises, based on total colony count, lactobacillus crispatus CCFM1298 parts, lactobacillus grignard CCFM1201 1 parts and added with prebiotic fructo-oligosaccharides.
The invention provides a composition, which contains probiotics and prebiotics, wherein the probiotics are lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201; the prebiotic is fructo-oligosaccharide.
The lactobacillus gasseri CCFM1201 is described in the chinese patent application publication No. CN 114250186A, with accession number GDMCC No. 62136.
In one embodiment of the invention, in the composition, lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201 are added in a ratio of 1:1 viable count; the probiotic is added to the composition in an amount of at least: 10 6 CFU/mL or 10 6 CFU/g。
In one embodiment of the invention, the fructooligosaccharides are purchased from: quantum Gao (Guangdong) biological Co., ltd.
In one embodiment of the present invention, the fructooligosaccharide is added to the composition in an amount of at least: 2g/Kg b.w.
In one embodiment of the present invention, the composition has a content of Lactobacillus crispatus and Lactobacillus gasseri of not less than 10 6 CFU/mL or 10 6 CFU/g。
The invention also provides a product, which contains the composition.
In one embodiment of the invention, the product is a food, pharmaceutical or sanitary product.
In one embodiment of the invention, the food comprises dietary supplements, plain foods, health products.
In one embodiment of the invention, the pharmaceutical product comprises the above composition and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier comprises one or more of fillers, binders, humectants, disintegrants, lubricants, flavoring agents commonly used in medicine.
In one embodiment of the invention, the dosage form of the drug comprises granules, capsules, tablets, pills, suppositories or oral liquids.
In one embodiment of the invention, the medicine comprises tablets, capsules and oral liquid which are orally coated with enteric coatings; pessaries, tablets, gelatin capsules, sprays, creams, gels.
In one embodiment of the present invention, the sanitary product comprises a sanitary wet tissue, a sanitary napkin, a panty liner, a tampon, a sanitary napkin, a vaginal lotion, a women's antibacterial/bacteriostatic lotion.
The invention also provides application of the composition in preparing products for relieving and/or treating bacterial and candidal vaginitis.
In one embodiment of the invention, the relief and/or treatment of bacterial vaginitis and candidal vaginitis; comprises relieving the inflammation of vaginal tissue, regulating and controlling the local immunity of vagina; the vaginal local immune system includes reduced expression of myeloperoxidase MPO, TNF-alpha.
In one embodiment of the invention, the product is a pharmaceutical or hygiene product.
In one embodiment of the invention, the pharmaceutical product comprises the above composition and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier comprises one or more of fillers, binders, humectants, disintegrants, lubricants, flavoring agents commonly used in medicine.
In one embodiment of the invention, the dosage form of the drug comprises granules, capsules, tablets, pills, suppositories or oral liquids.
In one embodiment of the invention, the medicine comprises tablets, capsules and oral liquid which are orally coated with enteric coatings; pessaries, tablets, gelatin capsules, sprays, creams, gels.
In one embodiment of the present invention, the sanitary product comprises a sanitary wet tissue, a sanitary napkin, a panty liner, a tampon, a sanitary napkin, a vaginal lotion, a women's antibacterial/bacteriostatic lotion.
The invention also provides application of the composition in preparation of products for relieving vaginal epithelial shedding, relieving vaginal tissue inflammation and regulating and controlling local immunity of vagina.
In one embodiment of the invention, the product is a pharmaceutical or hygiene product.
In one embodiment of the invention, the pharmaceutical product comprises the above composition and a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier comprises one or more of fillers, binders, humectants, disintegrants, lubricants, flavoring agents commonly used in medicine.
In one embodiment of the invention, the dosage form of the drug comprises granules, capsules, tablets, pills, suppositories or oral liquids.
In one embodiment of the invention, the medicine comprises tablets, capsules and oral liquid which are orally coated with enteric coatings; pessaries, tablets, gelatin capsules, sprays, creams, gels.
In one embodiment of the present invention, the sanitary product comprises a sanitary wet tissue, a sanitary napkin, a panty liner, a tampon, a sanitary napkin, a vaginal lotion, a women's antibacterial/bacteriostatic lotion.
Advantageous effects
(1) The invention provides a composition of lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 and lactobacillus grignard (Lactobacillus gasseri) CCFM1201, which ensures the synergistic symbiosis of the combined strains based on the growth characteristics of substrates, preferably the combination of the strains and prebiotics, of the lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 and lactobacillus grignard (Lactobacillus gasseri) CCFM1201, and really realizes the corresponding multiple effects of the combined strains.
(2) The invention provides a vaginal probiotic composition composed of lactobacillus crispatus CCFM1298, lactobacillus grignard CCFM1201 and prebiotics, which can effectively relieve the inflammation of vaginal tissues, regulate the local immune system of the vagina and be helpful for relieving and improving female bacterial vaginitis and candidal vaginitis.
Preservation of biological materials
Lactobacillus crispatus (Lactobacillus crispatus) CCFM1298, taxonomically designated: lactobacillus crispatus, deposited on month 9 of 2022 under the accession number GDMCC No:62275 the preservation address is building 5 of Guangdong national institute of microbiology, guangzhou Miao road 100, university 59.
Drawings
FIG. 1 shows the co-culture of CCFM1201 and CCFM1298 in MRS broth at different ratios.
FIG. 2 shows the generation of CCFM1201 and CCFM1298 using different carbon sources.
FIG. 3 shows CCFM1201 and CCFM1298 co-cultured in different ratios of MRS-FOS broth.
Fig. 4 is a flow chart of BV animal experimental design.
FIG. 5 is the effect of probiotics on myeloperoxidase MPO in vaginal tissue of BV mice; * : p <0.05,: p <0.01,: p <0.001.
FIG. 6 is the effect of probiotics on TNF- α in vaginal tissue of BV mice; * : p <0.05,: p <0.01.
Fig. 7 is a graph of probiotic versus BV mouse vaginal histopathological evaluation: (A): blank control group; (B): bacterial vaginitis model group; (C): lactobacillus gasseri CCFM1201 group; (D): lactobacillus crispatus CCFM1298 group; (E): group 1 probiotic compositions; (F): probiotic compositions 2 groups.
FIG. 8 is the effect of probiotics on sialidase concentration in vaginal secretions of BV mice; * : p <0.05,: p <0.01,: p <0.001.
Fig. 9 is a flow chart of a VVC animal experiment design.
FIG. 10 is the effect of probiotics on myeloperoxidase MPO in VVC mouse vaginal tissue; * *: p <0.01.
FIG. 11 is the effect of probiotics on TNF- α in VVC mouse vaginal tissue; * : p <0.05.
FIG. 12 is a graph of probiotic versus VVC mouse vaginal histopathological evaluation; wherein, (A): blank control group; (B): candidal vaginitis model group; (C): lactobacillus gasseri CCFM1201 group; (D): lactobacillus crispatus CCFM1298 group; (E): group 1 probiotic compositions; (F): probiotic compositions 2 groups.
Detailed Description
The Gardnerella vaginalis referred to in the following examples was purchased from the culture collection GDMCC of the institute of microbiology, guangdong, and the Candida albicans referred to in the following examples was isolated from vaginal secretions of patients with candidal vaginitis and deposited in the culture collection of the institute of food biotechnology, university in Jiangnan.
The following examples relate to the following media:
MRS liquid Medium (/ L): 10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 20.0g of glucose, 2.0g of anhydrous sodium acetate, 2.6g of dipotassium phosphate trihydrate, 2.0g of diammonium hydrogen citrate, 0.25g of manganese sulfate monohydrate and 0.5g of magnesium sulfate heptahydrate, adding 1mL of Tween 80, adding 1000mL of distilled water, adjusting the pH to 6.2-6.4, and sterilizing at 115 ℃ under high pressure for 20min.
MRS solid medium: adding 1.5% -2% agar into the prepared MRS liquid culture medium, and sterilizing at 115 ℃ for 20min.
Selective medium (/ L): 39g NaCl is added into the prepared MRS liquid culture medium, 1.5 to 2 percent of agar is added, and the temperature is 115 ℃ for high-pressure sterilization for 20 minutes.
BHI broth (/ L): 10.0g of pancreatic protein, 17.5g of bovine heart powder, 5.0g of sodium chloride, 2.5g of disodium hydrogen phosphate and 2.0g of glucose, and then 1% of yeast extract, 0.1% of maltose and 0.1% of glucose are additionally added, and the pH value is regulated to 7.4. And autoclaving at 115℃for 20min. Before use, 10% sterile fetal bovine serum was added to the medium.
YPD liquid medium (/ L): 20.0g of peptone, 20.0g of glucose and 10.0g of yeast powder. And autoclaving at 115℃for 20min.
Plain agar solid medium (/ L): 2% agar was added per 1L of water and autoclaved at 115℃for 20min.
The detection method involved in the following examples is as follows:
determination of TNF- α and MPO concentrations:
at the end of the experiment, vaginal tissue was removed after dislocation of cervical vertebrae, vaginal tissue samples (20 mg) were placed in 200. Mu.L of RIPA lysate containing 2% (v/v) protease inhibitor cocktail and 2% (v/v) phosphatase inhibitor cocktail, and disrupted with a high throughput tissue mill (65 Hz,45 s/time, 6 times) to give vaginal tissue homogenates. The vaginal tissue supernatant was centrifuged at 12000 Xg for 15min at 4℃and assayed for MPO and TNF-. Alpha.concentrations according to the kit instructions.
Detection of sialidase content
On day 18, mouse vaginal lavage was collected, centrifuged at 3600 Xg for 3min at 4℃to remove cell debris, and 10. Mu.L of vaginal lavage was extracted from vaginal supernatant for experiments. Determination of sialidase content Using enzyme-linked immunosorbent assay (ELISA) and OD 450 nm The values are compared to a standard curve. The specific operation is carried out according to the instruction of the kit.
Example 1: lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 separation and identification method
(1) Separation
Taking a healthy female vaginal swab from Jiangsu local area as a sample, pretreating the sample, storing the sample in 30% glycerol at-80 ℃ in a refrigerator, taking out the sample after thawing, uniformly mixing the sample, sucking 0.5mL of the sample into 4.5mL of 0.9% physiological saline, and carrying out ten-fold gradient dilution. And (3) selecting proper gradient diluent to be coated on an MRS solid culture medium, culturing for 48 hours at 37 ℃, picking typical colonies of lactobacillus crispatus, streaking and purifying the typical colonies on the MRS solid culture medium, picking single colonies to the MRS liquid culture medium for enrichment, and suspending the strains in 30% glycerol for preservation to obtain the strain CCFM1298. Wherein, the typical colony of the lactobacillus crispatus is smaller, white, round, full in the middle and dispersed around.
(2) Authentication
The genome of CCFM1298 is extracted, the 16S rDNA of the strain CCFM1298 is amplified and sequenced (the nucleotide sequence of the 16S rDNA obtained by amplifying the CCFM1298 is shown as SEQ ID NO.1 by Jin Weizhi biotechnology Co., ltd. In Suzhou), and the nucleotide sequence of the sequence is aligned in NCBI, so that the strain is shown to be Lactobacillus crispatus and named as Lactobacillus crispatus (Lactobacillus crispatus) CCFM1298.
Example 2: lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298 co-culture
In order to observe whether lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 strain can stably coexist when co-culturing, we co-culture lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 in different proportions, and observe the change of community structure, the specific steps are as follows:
(1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298
Both Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201 were stored in-80℃freezer with 30% glycerol prior to activation. Respectively dipping a small amount of lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201 by using a sterile inoculating loop, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48 hours; obtaining single colony, picking the single colony to be inoculated into an MRS liquid culture medium for culture, inoculating the single colony into the MRS liquid culture medium with an inoculum size of 2% (v/v), culturing for 24 hours at 37 ℃, and activating for 2 generations to respectively obtain lactobacillus crispatus CCFM1298 bacterial suspension and lactobacillus griseus CCFM1201 bacterial suspension, wherein the bacterial concentrations are as follows: 4.07×10 8 CFU/mL、1.84×10 8 CFU/mL。
(2) Inoculating the activated lactobacillus crispatus CCFM1298 obtained in the step (1) and lactobacillus gasseri CCFM1201 bacterial suspension in any proportion to 5mL of MRS culture medium, and placing the culture medium in a 37 ℃ constant temperature incubator for incubation for 24 hours to prepare cultures respectively; wherein, the combination of any proportion refers to: respectively mixing lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 bacterial suspension according to the volume ratio of 1:1, 1:9 and 9:1 to prepare cultures.
(3) The cultures obtained in step (2) were inoculated into MRS medium at an inoculum size of 2% (v/v), respectively, and cultured again at 37℃for 24 hours, and the transfer was repeated 2 times.
As described above, growth measurement was performed for 72 hours according to the above method, samples were collected every 24 hours, total bacterial count was counted in MRS medium, viable bacterial count of Lactobacillus gasseri CCFM1201 in the co-culture system was counted in selective medium, and changes in relative abundance were observed, and the results are shown in FIG. 1.
The results show that the lactobacillus gasseri CCFM1201 and the lactobacillus crispatus CCFM1298 are co-cultured in different proportions, and the lactobacillus gasseri CCFM1201 is dominant in a co-culture system, which indicates that the lactobacillus gasseri CCFM1201 has competitive advantages in the co-culture system, so that the growth is unbalanced, and the stable coexistence of the combined strains cannot be realized.
Example 3: determination of the antibacterial effect of Lactobacillus gasseri CCFM1201 on Lactobacillus crispatus CCFM1298
Interference competition between microorganisms in co-culture systems can exist, and this interaction is important in the composition and maintenance of the microbial community. Wherein the generation of antibacterial compounds plays an important role in interfering with competition. Thus, to verify whether lactobacillus gasseri CCFM1201 would inhibit the growth of lactobacillus crispatus CCFM1298 by producing a secondary metabolite, we determined the antibacterial effect of lactobacillus gasseri CCFM1201 on lactobacillus crispatus CCFM1298 by the following steps:
(1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298
Both Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201 were stored in-80℃freezer with 30% glycerol prior to activation. Dipping a small amount of lactobacillus bacteria liquid by using a sterile inoculating loop respectively, streaking on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single bacterial colonies; and (3) selecting single bacterial colony, inoculating the single bacterial colony into an MRS liquid culture medium for culture, inoculating the single bacterial colony into the MRS liquid culture medium with an inoculum size of 2% (v/v), culturing for 24 hours at 37 ℃, and activating for 3 generations to respectively prepare CCFM1298 bacterial suspension and lactobacillus gasseri CCFM1201 bacterial suspension.
(2) Preparation of antibacterial substances
And (3) centrifuging the activated lactobacillus gasseri CCFM1201 bacterial suspension obtained in the step (1) at 4 ℃ for 1min at 15000 Xg, collecting supernatant, and obtaining lactobacillus gasseri CCFM1201 cell-free supernatant after passing through a 0.22 mu m microporous filter membrane.
(3) Double-layer agar antibacterial test
Antibacterial substance determination was performed on the lactobacillus gasseri CCFM1201 bacterial suspension obtained in step (1) and the cell-free supernatant obtained in step (2) respectively using double-layer agar containing lactobacillus crispatus CCFM1298. The method comprises the following steps:
pouring MRS solid culture medium containing 2% of Lactobacillus crispatus CCFM1298 bacterial suspension into a dry plain agar plate, and drying at room temperature for 15min;
mu.L of each of the antibacterial substances obtained in step (2) was dropped on top of the above agar and dried to complete absorption, MRS liquid medium was selected as a negative control, and 640. Mu.g/mL of streptomycin sulfate was used as a positive control, and the results are shown in Table 1.
Table 1: determination of Lactobacillus gasseri CCFM1201 antibacterial substance
| Lactobacillus crispatus CCFM1298 | |
| MRS liquid culture medium | (-) |
| Streptomycin sulfate (640. Mu.g/mL) | (+) |
| Lactobacillus gasseri CCFM1201 bacterial suspension | (-) |
| Lactobacillus gasseri CCFM1201 supernatant | (-) |
Note that: (+): positive; negative of (-)
The results showed that lactobacillus gasseri CCFM1201 did not produce an antibacterial substance that inhibits lactobacillus crispatus CCFM1298.
Example 4: generation time of lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 under different carbon sources
According to the competition correlation hypothesis, the closer the relationship of two organisms is, the more likely it is for the two organisms to compete and reject each other due to the overlapping niches. Alpha-amylase in the genital tract breaks down glycogen into smaller carbohydrates (such as maltose, maltotriose, maltopentaose, and maltodextrin) to power the lactobacilli in the vagina. Both lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201 are from the vagina, and it is presumed that the capacity of the lactobacillus gasseri CCFM1201 to compete for carbohydrates is superior to that of lactobacillus crispatus CCFM1298, leading to the rapid proliferation of the lactobacillus gasseri CCFM1201 into dominant bacteria. Thus, we determined the generation of glucose for Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298.
The method comprises the following specific steps:
1. preparation of different carbon source culture media:
the culture medium is MRS liquid culture medium, and is characterized in that glucose is replaced by different carbon sources (inulin, resistant dextrin, galacto-oligosaccharide, fructo-oligosaccharide, xylo-oligosaccharide and mannooligosaccharide), and the concentrations are as follows: 20.0g/L, and the initial pH value is adjusted to 7.0.
2. Determination of time of day
(1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298
Both Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201 were stored in-80℃freezer with 30% glycerol prior to activation. Dipping a small amount of lactobacillus bacteria liquid by using a sterile inoculating loop respectively, streaking on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single bacterial colonies; and (3) picking single bacterial colony, inoculating the single bacterial colony into an MRS liquid culture medium for culture, inoculating the single bacterial colony into the MRS liquid culture medium with an inoculum size of 2% (v/v), culturing for 24 hours at 37 ℃, and activating for 3 generations to obtain a lactobacillus crispatus CCFM1298 bacterial suspension and a lactobacillus griseus CCFM1201 bacterial suspension.
(2) The activated lactobacillus gasseri CCFM1201 obtained in step (1) and lactobacillus crispatus CCFM1298 were inoculated separately in an amount of 2% (v/v) to the medium of different carbon sources obtained in step (1) (5 mL/tube), and incubated in 3 parts at 37 ℃ in a constant temperature incubator. Sampling and measuring OD of bacterial liquid every 2 hours 600 Values. Then log with time as abscissa 2 OD 600 The values are on the ordinate, the strain growth curve is drawn,when the generation is calculated, the result is shown in fig. 2.
The results show that lactobacillus gasseri CCFM1201 takes advantage of glucose for a time (2.03±0.16 h) that is less than lactobacillus crispatus CCFM1298 (2.43±0.10 h) and has significant differences (p < 0.01). Thus, glucose is preferentially utilized when lactobacillus gasseri CCFM1201 is co-cultured with lactobacillus crispatus CCFM1298, thereby causing lactobacillus gasseri CCFM1201 to become a dominant bacterium.
To alleviate the nutritional niche competition between lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298, we aimed at balancing the generation of lactobacillus gasseri CFFM1201 and lactobacillus crispatus CCFM1298. As shown in fig. 2, when glucose was replaced with fructooligosaccharides, the generation of lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 was shortened without significant difference.
Example 5: effect of fructooligosaccharides on Co-culture of Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298
To observe the effect of fructooligosaccharides on colony composition when co-cultured in any ratio of lactobacillus gasseri CCFM1201 to lactobacillus crispatus CCFM1298. The glucose in MRS culture medium is replaced by fructo-oligosaccharides by equal amount, and the specific steps are as follows:
(1) Activation of Lactobacillus gasseri CCFM1201 and Lactobacillus crispatus CCFM1298
Both Lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201 were stored in-80℃freezer with 30% glycerol prior to activation. Dipping a small amount of lactobacillus bacteria liquid by using a sterile inoculating loop respectively, streaking on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single bacterial colonies; selecting single bacterial colony, inoculating the single bacterial colony into an MRS liquid culture medium for culture, inoculating the single bacterial colony into the MRS liquid culture medium with an inoculum size of 2% (v/v), culturing for 24 hours at 37 ℃, and activating for 3 generations to respectively prepare lactobacillus crispatus CCFM1298 and lactobacillus griseus CCFM1201 bacterial suspensions, wherein the bacterial concentrations are respectively: 7.2X10 8 CFU/mL、3.9×10 8 CFU/mL。
(2) Inoculating the activated lactobacillus gasseri CCFM1201 obtained in the step (1) and lactobacillus crispatus CCFM1298 in any proportion to 5mL of MRS-FOS culture medium (glucose in MRS is replaced by fructo-oligosaccharides in equal quantity), placing the culture medium in a constant temperature incubator at 37 ℃ for incubation for 24 hours, and respectively preparing cultures, wherein the MRS liquid culture medium is used as a control. Wherein, the combination of any proportion refers to: respectively mixing lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298 bacterial suspension according to the volume ratio of 1:1, 1:9 and 9:1 to prepare cultures. .
(3) The cultures obtained in step (2) were inoculated into the medium described in step (2) in an inoculum size of 2% (v/v), respectively, and again cultured at 37℃for 24 hours, and the transfer was repeated 2 times.
As described above, growth measurement was performed for 72 hours according to the above method, samples were collected every 24 hours, total bacterial count was counted in MRS medium, viable bacterial count of Lactobacillus gasseri CCFM1201 was counted in selective medium during co-culture, and changes in relative abundance of the two strains were observed, and the results are shown in FIG. 3.
The results show that the fructo-oligosaccharide can effectively relieve the competition between the Lactobacillus gasseri CCFM1201 and the Lactobacillus crispatus CCFM1298 when co-cultured in different proportions, promote the growth of the Lactobacillus crispatus CCFM1298 and avoid the occurrence of unbalanced growth.
Example 6: application of vaginal probiotic composition in relieving bacterial vaginitis of mice
The specific implementation mode is as follows:
(1) Preparation of bacterial suspension and composition
Lactobacillus crispatus CCFM1298 bacterial suspension: lactobacillus crispatus CCFM1298 preserved with 30% glycerol in a refrigerator at-80℃was activated in MRS liquid medium (the method of activation is described in example 2), centrifuged at 8000g for 15min after three generations of activation, and the bacterial sludge of the Lactobacillus crispatus suspension after three generations of activation was collected. The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:100. Finally, a concentration of 1X 10 was obtained 10 CFU/mL of lactobacillus crispatus CCFM1298 bacterial suspension for intervention experiments.
Gardnerella vaginalis suspension: gardnerella vaginalis preserved in a refrigerator at-80 ℃ with 30% glycerol is activated in a BHI liquid culture medium, and the method comprises the following steps: inoculating into BHI culture medium with 2% (v/v), anaerobic culturing at 37deg.C for 48 hr for the first generation, and anaerobic culturing at 37deg.C for 24 hr for the 2 nd and 3 rd generation. After the activation of the three-generation type of the catalyst,centrifuging at 8000g for 15min, and collecting bacterial mud of Gardner vaginalis after three generations of activation. The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:10. Finally, a concentration of 1X 10 was obtained 10 CFU/mL gardnerella vaginalis suspension was used for infection experiments.
Lactobacillus gasseri CCFM1201 suspension: lactobacillus gasseri CCFM1201 preserved with 30% glycerol in a refrigerator at-80deg.C was activated in MRS liquid medium (the method of activation is described in example 2), and after three generations of activation, 8000g was centrifuged for 15min to collect bacterial sludge of the Lactobacillus crispatus suspension after three generations of activation. The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:100. Finally, a concentration of 1X 10 was obtained 10 CFU/mL of lactobacillus gasseri CCFM1201 bacterial suspension for intervention experiments.
Probiotic composition 1: compounding the obtained lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201 bacterial suspension with the viable count of 1:1 to finally obtain the viable count of 1 multiplied by 10 10 CFU/mL probiotic composition 1.
(2) Animal experiment
Experimental animals and experimental strains:
SPF-class 7 week female BALB/c mice, weighing 17-19g, were housed in IVC system for one week. Purchased from beijing velarihua laboratory animal technology limited (production license number SCXK 2012-0001). During the feeding period, each mouse was free to drink water and fed with SPF mouse feed (Co 60 And (5) disinfection). Feeding environment: and the illumination is carried out in each half cycle of day and night, the temperature is 22-24 ℃, and the humidity is 45-55%.
Experimental flow chart 4 shows experimental protocols and groupings: mice were randomly divided into 6 groups according to body weight, see table 2.
Table 2: grouping of mice
The experimental period was 22 days (days-3-18), and mice were in the adaptation phase for one week before the start of the experiment. 3 days before infection and on the day of infection, except for the blank group, the remaining mice were subcutaneously injected with 100 μl of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μl of sesame oil), and the mice were induced to develop oestrus, once daily in the morning, 7 days thereafter, until the end of the experiment.
Infection experiment (molding phase): 20 mu L of PBS buffer solution is inoculated into the vagina of a 0 th sky white control group mouse, and the number of viable bacteria of the other mice is 10 by 20 mu L of vaginal inoculation 10 CFU/mL gardnerella vaginalis suspension. After inoculation, the mice are inverted for 1-2 minutes to prevent bacteria from flowing out, and the process is continued for 7 days until the 6 th day is finished.
Intervention experiment: starting on day 7, the specific following is:
blank control group: 200 mu L of physiological saline is infused once a day;
model group mice: 200 mu L of physiological saline is infused once a day;
lactobacillus gasseri CCFM1201 group: mice were perfused with 200 μl, 10 a day 9 CFU lactobacillus gasseri CCFM1201 bacterial suspension;
lactobacillus crispatus CCFM1298 group: mice were perfused with 200 μl, 10 a day 9 CFU lactobacillus crispatus CCFM1298 bacterial suspension;
group 1 probiotic composition: 200 mu L and 10 mu L of the medicine are infused once a day 9 Complex bacterial suspension of CFU;
group 2 probiotic compositions: 200 mu L and 10 mu L of the medicine are infused once a day 9 CFU probiotic composition 1, while gavage 2g/Kg b.w. complex of fructooligosaccharides.
Each group of mice was gavaged once daily for 12 consecutive days.
At the end of infection (day 7) and at the end of intervention (day 18), mice were sampled for vaginal aspiration with 50 μl of PBS buffer solution each time by the gun head, and finally 300 μl of vaginal lavage fluid was collected for determination of sialidase content. At the same time, on day 19, all experimental mice were sacrificed and vaginal tissue was dissected for subsequent experimental histopathological analysis to determine secretion of myeloperoxidase MPO and inflammatory factor TNF- α.
(3) Experimental results:
1) Effect of vaginal probiotic composition on local immunity in bacterial vaginitis mice
At the end of the experiment, after the mice are killed by cervical dislocation, vaginal tissues are stripped, vaginal tissue supernatant is taken to measure the concentration of MPO and TNF-alpha according to the specification of the kit, the results are shown in figures 5-6, and the concentrations of MPO and pro-inflammatory factor TNF-alpha are shown in figures 5 and 6 respectively.
The results show that the blank group: MPO and TNF- α concentrations were 19.3ng/L, 115.8pg/mg, respectively.
Model group: after the gardnerella vaginalis infection, the organism is subjected to oxidative stress, and the MPO content of the myeloperoxidase is obviously increased to 23.15ng/L (p < 0.001);
after the Lactobacillus gasseri CCFM1201, lactobacillus crispatus CCFM1298 and the probiotic composition 1 are interfered for 12 days, the MPO content is reduced to 21.40ng/L (p < 0.05), 22.63ng/L and 21.78ng/L respectively, and the effect of reducing the MPO content (obviously reduced to 20.28ng/L (p < 0.01)) of the probiotic composition 2 is obviously better than that of the CCFM1201, the CCFM1298 and the probiotic composition 1.
Likewise, model set: after infection with gardnerella vaginalis, the host's immune system is stimulated and the expression of the pro-inflammatory factor TNF-alpha is significantly increased to 378.6pg/mg (p < 0.01).
After 12 days of intervention with lactobacillus gasseri CCFM1201, the expression of the pro-inflammatory factor TNF- α was reduced to 266.3pg/mg without significant differences. Compared with CCFM1201, CCFM1298 and the probiotic composition 1 can obviously reduce the expression of TNF-alpha (the expression amounts are 177.9pg/mg and 240.3pg/mg respectively), and the inhibition rates are 53% (p < 0.01) and 36.5% (p < 0.05) respectively. The effect of the probiotic composition 2 (expression amount: 204.1 pg/mg) and the effect of Lactobacillus crispatus CCFM1298 are not significantly different, but are superior to the effect of the Lactobacillus gasseri CCFM1201 and the probiotic composition 1 (p < 0.01)
2) Mouse vaginal histopathological analysis
The vaginal tissue of the mice was fixed and preserved with 4% paraformaldehyde solution, sectioned after paraffin embedding, sectioned 5mm thick, and stained with hematoxylin and eosin (H & E). The slice specimens were observed under a pathological section scanner at 30-fold magnification, and the results are shown in fig. 7, and the HE staining patterns of the blank, bacterial vaginitis model, CCFM1201, CCFM1298, and probiotic composition intervention groups are shown in fig. 7A, 7B, 7C, 7D, 7E, and 7F, respectively.
The results show that the blank group: the surface of the spinous layer of the vaginal tissue is smooth and continuous, necrosis and shedding are avoided, obvious inflammatory cell infiltration phenomenon is not found, the connection among cells of the spinous layer is tight, and an obvious gap bridge structure is not found.
Model group: compared with a blank control, the surface of the vaginal tissue acantha layer is seriously damaged, obvious necrosis and shedding phenomena occur, and a large amount of neutrophils infiltrate to form micro-abscesses. The acantha cells relax and a distinct gap bridge structure appears. The dermis layer is heavily infiltrated by inflammatory cells.
CCFM1201 and CCFM1298 groups: after 12 days of intervention of CCFM1201 and CCFM1298, inflammatory cell infiltration phenomenon is obviously improved, continuity is obviously improved, and the intercellular structure of the acantha layer is gradually compact compared with a model group.
Group 1 probiotic composition: the effect of the probiotic composition 1 on relieving inflammation is less obvious compared with lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298, and a small amount of inflammatory cell infiltration and a gap bridge structure still exist.
Group 2 probiotic compositions: the probiotic composition 2 is similar to lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298, the surface continuity of cells of the acanthosis layer is obviously improved, the inflammatory cell infiltration is obviously improved, and the effect is better than that of the probiotic composition 1.
3) Effect of vaginal probiotic composition on sialidase content in mice with bacterial vaginitis
Mouse vaginal lavage fluid was collected on day 18, sialidase content was determined by enzyme-linked immunosorbent assay (ELISA), and OD was measured 450 nm The values are compared to a standard curve. The specific procedure was carried out according to the kit instructions and the results are shown in FIG. 8.
The results showed that the sialidase content in the vaginal secretions of mice after gardnerella vaginalis infection in the model group was significantly increased to 59.7ng/L (p < 0.001).
Compared with the model group, after 12 days of intervention of the Lactobacillus gasseri CCFM1201 and the Lactobacillus crispatus CCFM1298, the sialidase content is obviously reduced (49.4 ng/L and 50.7ng/L respectively) by 17.3 percent and 15.1 percent (p < 0.01).
Compared with lactobacillus gasseri CCFM1201 and lactobacillus crispatus CCFM1298, the effect of the probiotic composition 1 on reducing the sialidase content is less pronounced (p < 0.05), as: 53.5ng/L.
The effect of the probiotic composition 2 is opposite to that of the probiotic composition 1, and the content of sialidase is obviously reduced, namely: 44.6ng/L, the inhibition rate was 25.3% (p < 0.001).
Example 7: application of vaginal probiotic composition in relieving candidal vaginitis of mice
The specific implementation mode is as follows:
(1) Preparation of bacterial suspension and composition
Lactobacillus crispatus CCFM1298 bacterial suspension: lactobacillus crispatus CCFM1298 preserved with 30% glycerol in a refrigerator at-80℃was activated in MRS liquid medium (the method of activation is described in example 2), and after three generations of activation, 8000g was centrifuged for 15min to collect bacterial sludge of the Lactobacillus crispatus suspension after three generations of activation. The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:100. Finally, a concentration of 1X 10 was obtained 10 CFU/mL of lactobacillus crispatus CCFM1298 bacterial suspension for intervention experiments.
Candida albicans suspension: candida albicans preserved in a refrigerator at-80 ℃ with 30% glycerol is activated in YPD liquid medium by the following method: inoculated in YPD medium at an inoculum size of 2% (v/v), cultured at 37℃for the first generation for 48 hours, and cultured at 37℃for the 2 nd and 3 rd generation for 24 hours. After the third generation of activation, 8000g of the mixture is centrifuged for 15min, and the bacterial sludge of candida albicans after the third generation of activation is collected. The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:10. Finally, a concentration of 1X 10 was obtained 8 CFU/mL candida albicans suspension was used for infection experiments.
Lactobacillus gasseri CCFM1201 suspension: lactobacillus gasseri CCFM1201 preserved with 30% glycerol in a refrigerator at-80deg.C was activated in MRS liquid medium (the method of activation is described in example 2), and after three generations of activation, 8000g was centrifuged for 15min to collect bacterial sludge of the Lactobacillus crispatus suspension after three generations of activation.The bacterial sludge was washed 2 times with PBS and resuspended with PBS 1:100. Finally, a concentration of 1X 10 was obtained 10 CFU/mL of lactobacillus gasseri CCFM1201 bacterial suspension for intervention experiments.
Probiotic composition 1: compounding the obtained lactobacillus crispatus CCFM1298 and lactobacillus gasseri CCFM1201 bacterial suspension with a viable count of 1:1 to finally obtain 1 multiplied by 10 10 CFU/mL probiotic composition 1.
(2) Animal experiment
Experimental animals and experimental strains:
SPF-class 7 week female BALB/c mice, weighing 17-19g, were housed in IVC system for one week. Purchased from beijing velarihua laboratory animal technology limited (production license number SCXK 2012-0001). During the feeding period, each mouse was free to drink water and fed with SPF mouse feed (Co 60 And (5) disinfection). Feeding environment: and the illumination is carried out in each half cycle of day and night, the temperature is 22-24 ℃, and the humidity is 45-55%.
Experimental flow chart 9 shows experimental protocols and groupings: mice were randomly divided into 6 groups according to body weight, see table 3.
TABLE 3 grouping of mice experiments
| Grouping | Inducing estrus | Molding stage | Probiotic intervention phase |
| Blank control group | Day-3-0 | Day 0-6 | Day 7-18 |
| Model group of candidal vaginitis | Day-3-0 | Day 0-6 | Day 7-18 |
| Lactobacillus gasseri CCFM1201 group | Day-3-0 | Day 0-6 | Day 7-18 |
| Lactobacillus crispatus CCFM1298 group | Day-3-0 | Day 0-6 | Day 7-18 |
| Group 1 (1:1) of probiotic compositions | Day-3-0 | Day 0-6 | Day 7-18 |
| Group 2 probiotic composition (1:1-FOS) | Day-3-0 | Day 0-6 | Day 7-18 |
The experimental period was 22 days (days-3-18), and mice were in the adaptation phase for one week before the start of the experiment. 3 days before infection and on the day of infection, except for the blank group, the remaining mice were subcutaneously injected with 100 μl of estradiol valerate (0.5 mg of estradiol valerate dissolved in 100 μl of sesame oil), and the mice were induced to develop oestrus, once daily in the morning, 7 days thereafter, until the end of the experiment.
Infection experiment (molding phase): blank pair on day 0Group mice were inoculated vaginally with 20. Mu.L of PBS buffer, and the remaining mice were inoculated vaginally with 20. Mu.L of viable bacteria count of 10 8 CFU/mL candida albicans suspension. After inoculation, the mice are inverted for 1-2 minutes to prevent bacterial flow out, and the inoculation is continued for 7 days until the candida albicans inoculation is finished on the 6 th day.
Intervention experiment: starting on day 7, the specific following is:
blank control group: 200 mu L of physiological saline is infused once a day;
model group mice: 200 mu L of physiological saline is infused once a day;
lactobacillus gasseri CCFM1201 group: mice were perfused with 200 μl, 10 a day 9 CFU lactobacillus gasseri CCFM1201 bacterial suspension;
lactobacillus crispatus CCFM1298 group: mice were perfused with 200 μl, 10 a day 9 CFU lactobacillus crispatus CCFM1298 bacterial suspension;
group 1 probiotic composition: 200 mu L and 10 mu L of the medicine are infused once a day 9 Complex bacterial suspension of CFU;
group 2 probiotic compositions: 200 mu L and 10 mu L of the medicine are infused once a day 9 CFU probiotic composition 1, while gavage 2g/Kg b.w. complex of fructooligosaccharides.
Each group of mice was gavaged once daily for 12 consecutive days.
On day 19, all experimental mice were sacrificed and vaginal tissue was dissected for subsequent experimental histopathological analysis to determine myeloperoxidase MPO and inflammatory factor TNF- α expression.
(3) Experimental results:
1) Effect of vaginal probiotic composition on local immunity of candidal vaginitis mice
At the end of the experiment, after the mice are killed by cervical dislocation, vaginal tissues are stripped, vaginal tissue supernatant is taken to measure the concentration of MPO and TNF-alpha according to the specification of the kit, the results are shown in figures 10-11, and the expression of myeloperoxidase MPO and pro-inflammatory factor TNF-alpha is shown in figures 10 and 11 respectively.
The results show that the model set: when candida albicans infects the vagina, MPO expression was significantly elevated compared to the blank (p < 0.01), as: 24.3ng/L.
After 12 days of intervention with probiotic composition 1, the expression of MPO was not significantly reduced in the CCFM1201 and CCFM1298 groups, respectively: 22.6ng/L, 24.2ng/L, 24.4ng/L. After 12 days of the probiotic composition 2 intervention, the MPO content was significantly reduced to 19.2ng/L (p < 0.01), the effect was significantly better than the other three groups.
The expression level of the pro-inflammatory factor TNF-alpha in the model group was increased 1.8-fold (p < 0.001) compared to the blank group (115.8 pg/mg), and was 211.9pg/mg.
After 12 days of intervention of lactobacillus gasseri CCFM1201 with probiotic composition 2, the expression level of TNF-alpha could be significantly reduced (136.9 pg/mg, 138.7pg/mg, respectively), but without significant differences.
Compared with lactobacillus gasseri CCFM1201 and probiotic composition 2, CCFM1298 and probiotic composition 1 have less obvious effect of regulating inflammatory factor expression (195.8 pg/mg and 211.1pg/mg respectively).
2) Mouse vaginal histopathological analysis
The vaginal tissue of the mice was fixed and preserved with 4% paraformaldehyde solution, sectioned after paraffin embedding, sectioned 5mm thick, and stained with hematoxylin and eosin (H & E). The slice specimens were observed under a pathological section scanner at 30-fold magnification, and the results are shown in fig. 12, and the HE staining patterns of the blank group, the candidal vaginitis model group, the CCFM1201 group, the CCFM1298 group, and the probiotic composition group are shown in fig. 12A, 12B, 12C, 12D, 12E, and 12F, respectively.
The results show that the blank group: the surface of the vaginal tissue acantha layer is smooth and continuous, necrosis and shedding are avoided, obvious inflammatory cell infiltration phenomenon is not found, the acantha layer cells are tightly connected, and an obvious gap bridge structure is not found.
Model group: compared with the blank control, the vaginal tissue has obvious edema of the acantha layer, the keratinocytes are denatured and destroyed into a net shape, a large number of eosinophils, neutrophils and lymphocytes are distributed in the vaginal tissue, the acantha layer is loose, and the number of cell bridges is increased.
CCFM1201 and CCFM1298 groups: after 12 days of intervention with CCFM1201 and CCFM1298, respectively, the continuity was significantly improved compared to the model group, with only a small inflammatory cell infiltration and a small number of gap bridge structures.
Group 1 probiotic composition: compared with CCFM1201 and CCFM1298, the improvement effect is less obvious, and more inflammatory cell infiltration still exists.
Group 2 probiotic compositions: compared with lactobacillus gasseri CCFM1201, lactobacillus crispatus CCFM1298 and probiotic composition 1, the probiotic composition 2 has obviously improved inflammatory cell infiltration and intercellular bridge structure phenomenon.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. Lactobacillus crispatus (Lactobacillus crispatus) CCFM1298 deposited at the microorganism strain collection in Guangdong province at 12/9 of 2022 under the accession number GDMCC No:62275.
2. a composition comprising a probiotic bacterium and a prebiotic, wherein the probiotic bacterium is lactobacillus crispatus CCFM1298, lactobacillus griseus CCFM1201 according to claim 1, and the prebiotic is fructo-oligosaccharide; lactobacillus crispatus CCFM1298 and Lactobacillus gasseri CCFM1201 are added in a ratio of 1:1 viable count.
3. The composition according to claim 2, wherein the probiotic is added in an amount of at least: 10 6 CFU/mL or 10 6 CFU/g。
4. A composition according to claim 3, wherein the fructooligosaccharides are added to the composition in an amount of at least: 2g/Kg b.w.
5. A product comprising the composition of any one of claims 2 to 4.
6. The product of claim 5, wherein the product is a food, pharmaceutical or sanitary product.
7. The product of claim 6, wherein the food comprises a dietary supplement, a plain food, a health product; the pharmaceutical product comprises a composition according to any one of claims 2 to 4 and a pharmaceutically acceptable carrier; the sanitary articles comprise sanitary wet tissues, sanitary napkins, sanitary pads, tampons, sanitary napkins, vaginal lotions, and antibacterial/bacteriostatic lotions for women.
8. Use of a composition according to any one of claims 2 to 4 for the preparation of a product for the alleviation and/or treatment of bacterial and candidal vaginitis.
9. The use according to claim 8, wherein the product is a pharmaceutical or hygiene product.
10. Use of a composition according to any one of claims 2 to 4 for the preparation of a product for reducing vaginal epithelial desquamation, alleviating inflammation of vaginal tissue, regulating local immunity in the vagina.
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