Lactobacillus casei, probiotic preparation for relieving vaginitis, and preparation method and application thereof

Abstract

The present invention relates to the screening of functional probiotics and its application, and specifically provides a strain of Lactobacillus casei RH99 that produces high extracellular polysaccharides and relieves vaginitis, and its preservation number is CGMCC No.32108. The Lactobacillus casei can inhibit the growth of Gardnerella, Candida albicans, and Peptostreptococcus and their biofilm generation ability; the extracellular polysaccharides produced by it have a significant promoting effect on the biofilm formation of Lactobacillus casei itself. RH99 is inoculated into the vagina, and its strain, the metabolites (exocellular polysaccharides) produced, and the core monosaccharide (D-mannitol) can all inhibit the colonization of pathogenic bacteria and promote the colonization of lactic acid bacteria; and RH99 can also relieve vaginitis by inhibiting inflammatory factors. It can be seen that RH99 can inhibit the growth of pathogenic bacteria + inhibit biofilm formation + inhibit pathogenic bacteria adhesion + inhibit the level of inflammatory factors through key core metabolites, and realize the all-round inhibition of four-in-one.

Description

Technical Field

[0001] The invention relates to the field of probiotics, in particular to Lactobacillus casei, a probiotic preparation comprising Lactobacillus casei, and a preparation method and application thereof. Background Art

[0002] The most common infectious disease in women is vaginitis, and the most common types of vaginitis are bacterial vaginitis (BV), vulvovaginal candidiasis, aerobic bacterial vaginitis, and Trichomonas vaginitis. Vaginitis is mainly caused by an imbalance in the vaginal microbial ecology, and about one-third of women worldwide suffer from vaginitis. According to the Amsel criteria, the diagnosis of BV mainly has the following characteristics: increased vaginal secretions, changes in the texture and color of the secretions, the secretions may be accompanied by a fishy odor, the pH of the vaginal environment is greater than 4.5, clue cells are present in the vaginal secretions, the secretions produce a large amount of amines, an increase in pathogenic bacteria, and a decrease in lactobacilli.

[0003] Pathogenic bacteria associated with vaginitis include Gardnerella, Candida albicans, Atopobium, Prevotella, and Peptostreptococcus, which damage the vaginal mucosa and epithelial barrier by releasing cytotoxins such as vaginal hemolysin and sialidase. BV-related vaginal metabolites may promote vaginal inflammation by increasing the production of proinflammatory factors (tumor necrosis factor TNF-α, IL-8, etc.). The formation of pathogenic biofilms can also damage the integrity of the vaginal epithelial barrier, especially the initial adhesion of vaginal Gardnerella to vaginal epithelial cells, which is a key link in the formation of pathogenic biofilms and BV. Biogenic amines (tyrosine, cadaverine) are associated with increased pH in the vaginal environment, vaginal odor, and abnormal secretions, while metabolites produced by related lactic acid bacteria (lactic acid, glutathione, phenylalanine, etc.) can protect the vaginal environment. Pathogenic bacteria can also reduce the redox potential in the vaginal microbial environment, leading to a significant decrease in Lactobacillus and an increase in other BV-related bacteria such as Prevotella and Atopobium, causing vaginal microecological disorders.

[0004] There are two main methods for treating bacterial vaginitis: one is mainly based on antibacterial drugs, and the other is mainly based on microbial intervention. Antibacterial drugs are mainly antibiotics such as metronidazole, clindamycin or tinidazole, but antibiotic therapy is difficult to rebuild the already disordered vaginal environment, and may even cause digestive tract reactions such as nausea, vomiting or gastrointestinal discomfort. In addition, antibiotics are not suitable for pregnant and lactating women. Microecological therapy can not only destroy the virulence of pathogens, but also regulate the immune system and improve the vaginal environment. Probiotics play an important role in alleviating bacterial vaginitis, such as inhibiting the growth and colonization of vaginitis-related pathogens, inhibiting the formation of pathogenic bacterial biofilms, and regulating local inflammatory immune responses in the vagina. Lactobacillus can effectively relieve BV symptoms. The specific mechanisms are as follows: Lactobacillus can inhibit the growth of pathogenic bacteria by producing antibacterial substances such as organic acids, hydrogen peroxide (H2O2), and bacteriocins; Lactobacillus competes with pathogenic bacteria for the adhesion sites of epithelial cells, forming steric hindrance to prevent the adhesion and colonization of pathogenic bacteria; Lactobacillus inhibits the expression of known virulence factors of pathogenic bacteria (such as vaginal lysin and sialidase), slows down the shedding of vaginal epithelial cells, improves damaged vaginal mucosa, and restores the vaginal mucosal barrier. Although vaginal administration directly acts on the affected area, the bacteria metabolize rapidly and have poor colonization ability, resulting in short-term efficacy and repeated infections, and most drugs will destroy the balance of vaginal flora.

[0005] The Chinese invention application with the publication number CN 117778221 A discloses a strain of Lactobacillus rhamnosus that has the effect of preventing and treating female vaginitis. This invention screened a strain of Lactobacillus rhamnosus from infant feces, which can significantly inhibit the growth of pathogens such as vaginal Gardnerella, Candida albicans, and Staphylococcus aureus. It is sensitive to common antibiotics, has a maximum salt tolerance of 8%, can grow at 45°C, has a certain heat resistance, and has an adhesion effect on human vaginal epithelial cells and can colonize in the vagina. The application only discusses the intervention effect on vaginitis from the aspects of the ability to inhibit pathogenic bacteria, adhesion to reproductive tract cells, and the probiotic properties of the strain itself (resistance to gastric acid, resistance to bile salts, sensitivity to antibiotics, and adhesion to human vaginal epithelial cells), and no animal experiment efficacy verification has been conducted.

[0006] The Chinese invention application with publication number CN 114350560 B discloses a strain of Lactobacillus paragaseri that inhibits the growth of vaginal Gardnerella and biofilm and produces hydrogen peroxide in high yield. The Lactobacillus paragaseri of the present invention has significantly improved the production capacity of hydrogen peroxide, the growth of pathogenic bacteria (Gardnerella) and the inhibition of biofilm, and has a strong adhesion ability to the vagina. In addition, since the Lactobacillus paragaseri is derived from the vagina of a healthy woman, it has little side effects on the body and is a probiotic strain suitable for human vaginitis. It is used in the preparation of products and sanitary products for female vaginitis, which helps to alleviate and improve female bacterial vaginitis. The application only compares in vitro antibacterial, hydrogen peroxide production and adhesion ability, and no animal experiment verification is carried out, nor is the reason for the strong antibacterial ability / strong adhesion ability analyzed from a mechanism perspective. Summary of the invention

[0007] Therefore, in order to solve the above problems, the present invention screens out lactic acid bacteria that can inhibit vaginitis, improve the treatment effect of vaginitis, reduce the recurrence rate, improve the health of vaginal mucosa, and provide comprehensive and lasting protection for women's health.

[0008] The present invention is achieved through the following technical solutions.

[0009] A Lacticaseibacillus casei RH99, whose preservation number is CGMCC No. 32108. A fermentation product obtained by fermenting the Lacticaseibacillus casei RH99.

[0010] A probiotic preparation of Lactobacillus casei RH99 comprises the steps of streaking the probiotics and then picking out a single bacterium into a liquid MRS culture medium, culturing at 37° C., performing two rounds of activation, and centrifuging to obtain a probiotic mud.

[0011] The culture medium comprises the following components by weight percentage: 0.5-1.0% yeast peptone, 0.5-1.0% soy peptone, 0.4-0.5% yeast extract powder, 1.5-2.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate, 0.4-0.6% sodium acetate, 0.02-0.03% magnesium sulfate, 0.002-0.004% manganese sulfate monohydrate, and the balance distilled water.

[0012] The probiotic preparation is used for preparing medicine for inhibiting the growth of Gardnerella, Candida albicans and / or Peptostreptococcus.

[0013] The probiotic preparation is used for preparing medicine for inhibiting the biofilm formation of Gardnerella, Candida albicans and / or Peptostreptococcus.

[0014] The probiotic preparation is used to prepare medicines for inhibiting inflammatory factors.

[0015] The inflammatory factors include NF-α and IL-1β.

[0016] An application of Lactobacillus casei RH99 in preparing a microbial preparation; or

[0017] For use in addition to sanitary products; or

[0018] Used in the preparation of daily necessities; or

[0019] The sanitary products include menstrual hygiene products, excrement hygiene products, disposable underwear, and condoms;

[0020] The daily necessities include vaginal washes, gels, ointments, sprays or creams.

[0021] Biological deposit description Biological material: Lactobacillus casei RH99, classification name: Lactobacillus casei, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on September 29, 2024, the address of the deposit center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101; the deposit number is CGMCC No. 32108. The bacteria are alive when deposited.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides that Lactobacillus casei has the effect of high production of extracellular polysaccharides (especially containing D-mannose);

[0024] The present invention provides Lactobacillus casei RH99 which has the effect of alleviating vaginitis;

[0025] The extracellular polysaccharide produced by the Lactobacillus casei RH99 provided by the invention has a promoting effect on its own biofilm and an inhibitory effect on the biofilm of pathogenic bacteria.

[0026] The Lactobacillus casei RH99 provided by the present invention can compete with Gardnerella vaginalis for colonization sites, inhibit pathogenic bacteria from adhering to HeLa cells by means of competition, exclusion, and replacement of the occupied sites, thereby inhibiting the formation of biofilms of pathogenic bacteria and inhibiting the colonization of pathogenic bacteria in the vagina. The Lactobacillus casei RH99 provided by the present invention can also relieve vaginitis by inhibiting inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 The adhesion ability evaluation of dominant bacteria and pathogenic bacteria is shown;

[0029] Figure 2 The monosaccharide composition of Lactobacillus casei RH99 exopolysaccharide is shown;

[0030] Figure 3 The effects of exopolysaccharides or D-mannose on Lactobacillus casei RH99 are shown;

[0031] Figure 4 The effects of exopolysaccharides or D-mannose on Candida albicans are shown;

[0032] Figure 5 The effects of exopolysaccharides or D-mannose on Gardnerella are shown;

[0033] Figure 6 The effects of exopolysaccharides or D-mannose on Peptostreptococcus are shown;

[0034] Figure 7 Colonization assay of Gardnerella vaginalis in mouse vagina is shown;

[0035] Figure 8 A colonization assay of Lactobacillus casei in the mouse vagina is shown;

[0036] Fig. 9 Cytokine levels in mouse serum are shown. DETAILED DESCRIPTION

[0037] The present invention provides a Lactobacillus casei RH99, which is screened from the saliva of children. Specifically, the Lactobacillus casei RH99 provided by the present invention is screened out by the following method.

[0038] First, the present invention selects 200 edible lactic acid bacteria from children's saliva samples, breast milk samples, infant feces and other samples, and screens out strains that have an inhibitory effect on vaginitis pathogens (Gardnerella, Peptostreptococcus, Candida albicans) through antibacterial experiments.

[0039] Example 1

[0040] Antibacterial experiment

[0041] Take 200 μL of the bacterial solution of vaginitis pathogens (Gardnerella, Streptococcus, Candida albicans), and spread it evenly on blood agar medium, blood agar medium (containing 5% sterile defibrinated sheep whole blood) and Sabouraud medium plates, respectively. Leave it for 30 minutes. After the bacterial solution on the surface is fixed, use an Oxford cup to punch holes on the surface of the plate, take 200 μL of lactic acid bacteria solution (OD600 is about 1.0) and inject it into the holes. Use physiological saline as a control. After culturing at 37°C for 24 hours, observe the results and measure the diameter of the inhibition zone.

[0042] The present invention screened out 20 strains of lactic acid bacteria from 200 strains through antibacterial experiments, and found that 20 strains had significant inhibitory effects on pathogenic bacteria. The specific results are shown in Table 1 below:

[0043] Table 1 Effects of the initial screening strains on the diameters of the inhibition zones of three pathogenic bacteria

[0044]

[0045] As can be seen from Table 1, the top 5 strains with antibacterial effect on Candida albicans are: RH99, 24-34M4, 31MC4, 33M1, TY10-6; the top 5 strains with antibacterial effect on Gardnerella are: 24-34M4, RH99, TY21-1, TY19-7, 24-10M1; the top 5 strains with antibacterial effect on Peptostreptococcus are 3Y2, TY1-18, 13M4, RH99, 33M4. Then, the present invention further tests the inhibitory effect of 20 probiotics that have significant inhibitory effect on pathogenic bacteria on biofilm of pathogenic bacteria through experiments. As shown in Example 2.

[0046] Example 2

[0047] Pathogenic bacteria biofilm inhibition ability

[0048] Adjust the 24h culture of pathogenic bacteria to 10 8 CFU / mL, add 100μL of pathogenic bacteria suspension to each well of a 96-well plate. At the same time, add the supernatant of all lactic acid bacteria to the 96-well plate, 100μL per well, and use MRS as a blank control. Incubate the 96-well plate at 37°C for 24h. After 24h biofilm formation, wash 3 times with PBS to carefully remove planktonic cells, and add 100μL of methanol to each well for fixation for 30 minutes. After discarding the methanol, the biofilm was stained with 0.1% crystal violet for 5 minutes. Discard the crystal violet, wash 3 times with PBS, add 200μL of 36% glacial acetic acid to each well, and measure the absorbance of each well at 590nm using an enzyme reader.

[0049] Table 2 Effect of initial screening of lactic acid bacteria on the inhibition rate of pathogenic bacteria biofilm

[0050]

[0051] As can be seen from Table 2, the top five bacterial strains with higher inhibition rates on Candida albicans biofilm are: RH99, TY19-7, 24-34M4, TY20-8, and 3T4; the top five bacterial strains with higher inhibition rates on Gardnerella biofilm are: RH99, TY10-6, TY20-8, TY21-1, and 24-32M2; the top five bacterial strains with higher inhibition rates on Streptococcus biofilm are: RH99, 24-32M2, TY21-1, 3Y2, and 3T4. The results showed that there was no obvious correlation between the antibacterial ability and biofilm inhibition ability of lactic acid bacteria. This may be because the way in which lactic acid bacteria inhibit biofilm not only depends on antibacterial activity, but is also related to downregulating the expression of biofilm-related genes and inhibiting quorum sensing.

[0052] Based on the antibacterial ability against pathogenic bacteria and biofilm inhibition ability, it can be seen that RH99 has a strong comprehensive antibacterial effect, and this strain of bacteria was used for subsequent experiments.

[0053] Then, the probiotics with strain number RH99 of the present invention were identified. The specific identification process and results are shown in Example 3.

[0054] Example 3

[0055] 3.16S rDNA identification

[0056] The DNA of RH99 and 24-15M6 strains was extracted using a bacterial genomic DNA extraction kit, and the extracted genomic DNA of the strains was used as a template for PCR amplification. The lactic acid bacteria primers were universal primers:

[0057] 27F (SEQ ID No. 1): AGAGTTTGATCCTGGCTCAG;

[0058] 1492R (SEQ ID No. 2): GGTTACCTTGTTACGACTT;

[0059] After electrophoresis detection of the amplified product, it was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing peak graph was integrated to cut off the 800 bp region of the sequencing result; the bacteria were compared with the GenBank gene library through the BLAST program and it was concluded that the bacteria was Lactobacillus casei.

[0060] Specifically, the 16s rDNA sequence of Lactobacillus casei RH99 is as follows:

[0061] GGCCGCTACAGTAGAGAGTGTTCGAGCGCTCGAGTTCTCGTTGATGATCGGCGCTTG

[0062] CACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTG

[0063] CCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAAC

[0064] CGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGC

[0065] GTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGA

[0066] GAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAG

[0067] CAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGA

[0068] AGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACT

[0069] GTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCC

[0070] GCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCA

[0071] GGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAA

[0072] ACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATG

[0073] CGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACG

[0074] CTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCG

[0075] TAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCA

[0076] TTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGG

[0077] GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA

[0078] CCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAA

[0079] TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

[0080] CAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGAC

[0081] TGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATG

[0082] ACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGT

[0083] CAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACAC

[0084] GAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGG

[0085] CCTGGTACACACCGCCCGTCACACCATCAAACTAGTCACACATAGAGTCGATCGTGTC

[0086] TCAGTAAGTCGAACAACGCAACCATAGGAGTT

[0087] The present invention evaluates the acid production capacity, hydrogen peroxide production capacity, and exopolysaccharide production capacity of Lactobacillus casei RH99, as shown in Example 4.

[0088] Example 4

[0089] Determination of the probiotic properties of dominant bacteria

[0090] Evaluation of the acid production, hydrogen peroxide production and exopolysaccharide production capacity of dominant bacteria

[0091] Because lactobacilli colonizing the vagina produce organic acids, the pH value of the vaginal environment of healthy women is between 3.8 and 4.5, and the acidic environment of the vagina can inhibit the colonization and growth of pathogens. Among them, lactobacilli use glucose in vaginal epithelial cells to produce a large amount of organic acids. Among them, the fat-soluble carboxylic acids produced can pass through the cell membrane of acid-intolerant pathogens in a non-dissociated form, dissociate in the cytoplasm, lower the intracellular pH value, inhibit important cell functions, and lead to the death of pathogens. Therefore, when evaluating the probiotic properties of dominant bacteria, it is necessary to evaluate their acid production ability.

[0092] H2O2 is an active substance produced by lactobacilli that can hinder the colonization and growth of pathogens associated with bacterial vaginitis. A large number of in vitro experiments have shown that lactobacilli that produce H2O2 can kill vaginal pathogens such as Gardnerella vaginitis, Escherichia coli, Staphylococcus aureus, and Peptostreptococcus. Therefore, the ability of lactic acid bacteria to produce H2O2 is one of the indicators for evaluating dominant bacteria.

[0093] Extracellular polysaccharides are secondary metabolites produced during the growth and metabolism of lactic acid bacteria. They can be loosely attached to the cell surface or directly secreted into the extracellular environment. Polysaccharides attached to the cell wall form capsules, called capsular polysaccharides (c-EPS); those that enter the culture medium to form mucus are called mucus polysaccharides (s-EPS). c-EPS and s-EPS are collectively called EPS, which are the products of microbial adaptation to environmental changes. As the concentration of EPS increases, its antibacterial ability gradually increases. EPS can inhibit the growth of pathogens in the host through competitive inhibition. Therefore, the exopolysaccharide index is also one of the evaluation indicators.

[0094] Index detection:

[0095] ① Evaluation of acid production capacity: Two commercially available dominant strains (Lactobacillus rhamnosus of Lactobacillus reuteri RC14) and Lactobacillus casei RH99 were inoculated into MRS liquid culture medium, cultured at 37°C for 24 h. After activation for three generations, the fermentation supernatant of each strain was collected and the pH of each fermentation supernatant was tested.

[0096] ② The determination of hydrogen peroxide shall refer to GB 5009.226-2016 National Food Safety Standard Determination of Residual Hydrogen Peroxide in Food.

[0097] ③ Determination of extracellular polysaccharide content: refer to DB15 / T 2033-2020 Technical procedures for separation, purification and identification of extracellular polysaccharides of lactic acid bacteria.

[0098] Table 4 Probiotic functions of dominant bacteria

[0099]

[0100] Compared with the two commercially available lactic acid bacteria, Lactobacillus casei RH99 had no significant difference in acid and hydrogen peroxide production, while the extracellular polysaccharide content of RH99 was significantly higher than that of the control strain (p < 0.05). For the comprehensive antibacterial ability of RH99, its extracellular polysaccharide production may be a key indicator. The possible mechanism is that EPS is a foreign substance to other organisms and cannot enter the cell body. It can only bind to signal molecules or glycocalyx receptors related to the biofilm on the surface of pathogenic bacteria, destroying cell communication, thereby destroying the integrity and flow proteins of the cell membrane and affecting the formation of the cell membrane. In addition, the antibacterial effect of EPS may be exerted by inhibiting the colonization and reproduction of harmful bacteria in the vagina.

[0101] Evaluation of adhesion ability of dominant bacteria

[0102] Pathogen adhesion to vaginal epithelial cells is the first step in the development of vaginitis, which helps pathogen colonization and establishment of infection. Adhesion is the basis of the interaction between lactobacilli and host cells. In order to ensure the growth and colonization of lactobacilli in the body and to play a beneficial role in the host, good adhesion is essential. Related studies have shown that the adhesion ability of different lactobacilli to epithelial cells and the ability to colonize in mucosal tissues are the main factors that enable them to compete with pathogens in vaginal tissues and eliminate pathogens from vaginal tissues.

[0103] Adhesion index determination: Human cervical cancer cell Hela cell line was purchased from Jin Shaoyuan (Shanghai) Biotechnology Co., Ltd. After the HeLa cells that grew to 80% fusion were digested, the cell density was adjusted with complete cell culture medium. 2 mL of 1×10 cells were added to a 6-well culture plate with a sterile cover glass. 5 The cells were cultured in a 5% CO2 incubator at 37°C. After the cells adhered to the wall, the culture medium was discarded and 2 mL of 1×10 8CFU / mL of lactobacillus / pathogenic bacteria suspension. After culturing for 2 hours, the culture medium was discarded, and the excess non-adherent lactobacilli were washed with PBS buffer, fixed with methanol for 20 minutes, and then Gram-stained and observed under a microscope. Twenty fields of view were randomly selected for evaluation, and the number of bacteria adhered to 100 cells was recorded and expressed as the number of bacteria adhered to each Hela cell, which is the adhesion index.

[0104] As for the adhesion ability of Hela cells, the adhesion index of RH99 was 25.34%, which was higher than that of the control commercial strains RC-14 and GR-1, and the adhesion ability of lactic acid bacteria was higher than that of pathogenic bacteria.

[0105] The invention also characterizes the pathogenic bacteria inhibition mechanism of Lactobacillus casei RH99.

[0106] Example 5

[0107] Study on the mechanism of antibacterial ability against pathogenic bacteria (in vitro experiment)

[0108] In order to analyze the antibacterial properties of dominant strains and understand the antibacterial ability of extracellular polysaccharides on pathogenic bacteria and the inhibitory ability of pathogenic biofilm, it is necessary to explore its internal mechanism.

[0109] Determination of Monosaccharide Composition of Extracellular Polysaccharides of Dominant Bacteria

[0110] The monosaccharide composition was extracted and tested using the DB15 / T 2033-2020 method, mainly providing D-mannose, D-glucosamine, D-galactosamine, D-rhamnose, D-glucose, D-galactose, D-xylose and D-fucose. The results were as follows: Figure 2 As shown. Figure 2 It can be seen that the monosaccharide composition of the extracellular polysaccharide of Lactobacillus casei RH99 is mainly D-mannose, with a relative content of 50.12%, and D-glucose accounts for 14.08%. It can be inferred that D-mannose may be a key substance.

[0111] Exopolysaccharides stimulate biofilm formation of lactic acid bacteria / inhibit biofilm formation of pathogenic bacteria

[0112] Adjust the 24h culture of lactic acid bacteria (Lactobacillus casei) and pathogenic bacteria (Gardnerella, Peptostreptococcus, Candida albicans) to 10 8CFU / mL, add 100 μL of pathogenic bacteria / lactic acid bacteria suspension to each well of a 96-well plate. At the same time, the freeze-dried exopolysaccharide or purchased D-mannose was dissolved in sterile distilled water at a concentration of 1 and 2 mg / mL, and different concentrations of exopolysaccharide / mannose were added to the 96-well plate, 100 μL per well, so that the concentration of exopolysaccharide / mannose was 0.5 and 1 mg / mL, and MRS, BHI, and distilled water were used as blank controls. The 96-well plate was incubated at 37°C for 24 hours with culture medium and exopolysaccharide / D-mannose solution as sterile control wells. After 24 hours of biofilm formation, the floating cells were carefully removed by washing 3 times with PBS, and 100 μL of methanol was added to each well for fixation for 30 minutes. After discarding the methanol, the biofilm was stained with 0.1% crystal violet for 5 minutes. The crystal violet was discarded, the wells were washed three times with PBS, 200 μL of 36% glacial acetic acid was added to each well, and the absorbance of each well at 590 nm was measured using an enzyme-labeled instrument.

[0113] EPS is an important component of microbial biofilm. Therefore, this example explores the effect of extracellular polysaccharides or D-mannose of Lactobacillus casei RH99 on biofilm of lactic acid bacteria and pathogenic bacteria. Figure 3 It can be seen that 1 mg / mL of extracellular polysaccharide can significantly promote the formation of Lactobacillus casei RH99 biofilm, followed by 1 mg / mL D-mannose.

[0114] Depend on Figure 4 and Figure 5 It can be seen that 1 mg / mL of exopolysaccharide can significantly inhibit the formation of biofilm of pathogenic bacteria, and 1 mg / mL of D-mannose has the second best inhibitory ability. In summary, EPS produced by the same species is more likely to stimulate the formation of biofilm of the species, that is, the EPS produced by Lactobacillus casei RH99 has a significant promoting effect on its own biofilm formation and a significant inhibitory effect on pathogenic bacteria. The inhibitory effect of D-mannose is second only to the extracted exopolysaccharide.

[0115] Exopolysaccharide / D-mannose from RH99 inhibits pathogenic bacteria from adhering to HeLa cells

[0116] In complex microbial communities, there is competition for adhesion sites among bacteria. Bacteria with high EPS content may have an advantage in the competition. When multiple bacteria coexist near a surface, EPS can help bacteria quickly occupy favorable adhesion sites. Therefore, it is necessary to explore the effects of exopolysaccharides or D-mannose on the adhesion of Hela cells.

[0117] After the HeLa cells that have grown to 80% fusion are digested, the cell density is adjusted with complete cell culture medium. Add 2 mL of 5×10 cells to a 6-well culture plate with a sterile coverslip. 5The cells were cultured in a complete cell culture suspension of 1 mg / mL. The cells were cultured in a 5% CO2 incubator at 37°C. After the cells adhered to the wall, the culture medium was discarded. The inhibitory effect of the extracellular polysaccharide / D-mannose suspension containing 1 mg / mL on the adhesion of the pathogenic bacteria Gardnerella was investigated by competition, exclusion, and replacement. The details are as follows:

[0118] 1) Control group: 2 mL of Gardnerella suspension was added to the well plate and incubated with HeLa cells for 4 h;

[0119] 2) Competition group: 1 mg / mL exopolysaccharide / D-mannose suspension and Gardnerella suspension were incubated with HeLa cells for 4 h;

[0120] 3) Rejection group: First, incubate HeLa cells with 1 mg / mL of exopolysaccharide / D-mannose for 2 h, then add 2 ml of Gardnerella suspension and continue to incubate in the incubator for 2 h;

[0121] 4) Replacement group: Gardnerella was first incubated with HeLa cells for 2 h, then 1 mg / mL of exopolysaccharide / D-mannose was added and the cells were incubated in the incubator for another 2 h;

[0122] After the cell incubation is completed, the upper cell culture medium is removed and washed three times with sterile PBS buffer. First, 200 μL of 0.25% trypsin is added to each well for digestion for 2 minutes, and then 800 μL of DMEM medium without antibiotics is added to terminate the reaction, and the cells are fully blown to completely detach. The mixed solution is transferred to a centrifuge tube, graded diluted, and spread on a blood agar plate for live bacteria count. The experiment is repeated three times.

[0123] Table 5 Inhibitory effect of extracellular polysaccharide / D-mannose on the adhesion of Gardnerella to HeLa cells

[0124]

[0125] In the competition experiment, 1 mg / mL extracellular polysaccharide had a significant inhibitory effect on the adhesion of Gardnerella to HeLa cells; in the exclusion experiment, 1 mg / mL extracellular polysaccharide / D-mannose could significantly reduce the number of Gardnerella adhering to HeLa cells; in the replacement experiment, 1 mg / mL extracellular polysaccharide had the most significant effect in antagonizing the adhesion of Gardnerella to HeLa cells.

[0126] In summary, the antibacterial ability of Lactobacillus casei RH99 is mainly achieved by inhibiting the formation of biofilms of pathogenic bacteria. In particular, the metabolites of RH99, exopolysaccharides (especially the monosaccharide component D-mannitol), can promote the formation of lactic acid bacteria biofilms and inhibit the biofilms of pathogenic bacteria, and can inhibit the adhesion of pathogenic bacteria to HeLa cells by competition, exclusion, and replacement of occupancy.

[0127] Finally, the present invention also provides animal experiments on RH99.

[0128] Example 6

[0129] Study on the antagonistic effect of RH99 and its extracellular polysaccharide on mice infected with Gardnerella

[0130] Forty 7-week-old female BALB / c mice were placed in an environment with an alternating cycle of 20-22℃, 50±10% humidity, and a light / dark cycle of 12h. Before the experiment, the mice were in a one-week adaptation phase. The mice were randomly divided into 4 groups, including: control group (Control), Gardnerella infection group (GV), intervention group A (GV+1mg / mL EPS), intervention group B (GV+1mg / mL D-mannose), intervention group C (RH99 group), and intervention group D (metronidazole 1mg / 20g). Three days before infection (day -3) and on the day of infection (day 0), except for the control group, all other mice were subcutaneously injected with 100μL estradiol valerate (0.5mg dissolved in 100μL sesame oil) to induce estrus in mice. On day 0, the number of live bacteria in the vagina was 20μL of 108CFU / mL vaginal Gardnerella suspension, which was continued for 5 days until the vaginal Gardnerella inoculation ended on day 4. The specific operation is to insert the tip of the pipette into the vagina about 5mm deep for vulvar inoculation. After inoculation, the mouse is inverted for 1-2 minutes to prevent the bacteria from flowing out. Starting from the 5th day, the experimental mice were inoculated with 10 9 CFU / mL lactobacillus 20μL (containing 1mg / mL EPS), 1mg / mL EPS, 1mg / mL D-mannose, once a day for 12 days. The control group of mice was inoculated with 20μL PBS buffer intravaginally once a day. On the 17th day, all experimental mice were killed by cervical dislocation. Index determination:

[0131] ① Determination of vaginal colonization of Gardnerella vaginalis and Lactobacillus in mice

[0132] To assess the load of vaginal Gardnerella and Lactobacillus, vaginal wash fluid was collected by flushing the mouse vagina with 300 μL sterile PBS buffer on days 5 and 17. DNA from vaginal wash fluid was extracted using the Fast DNA Spin Kit for soil and the QIAickGel extraction kit according to the manufacturer's instructions. Subsequently, the number of vaginal Gardnerella and Lactobacillus casei was quantified using real-time fluorescence quantitative PCR (qPCR).

[0133] ②ELISA to detect the content of cytokines in mouse serum

[0134] ELISA was used to determine the content of mouse serum cytokines: refer to the instructions of the mouse TNF-α and IL-1β ELISA kit. To analyze the colonization of lactic acid bacteria in the mouse vagina and its effect on the growth of vaginal Gardnerella in vivo, we detected the load of vaginal Gardnerella and Lactobacillus in the vagina on the 5th and 17th days by real-time fluorescence quantitative method.

[0135] This experiment evaluated the colonization of pathogenic bacteria and Lactobacillus during the intervention period, e.g. Figure 5 , 6 As shown. Except for the blank control group, about 7lg copies / μL of vaginal Gardnerella were detected in all groups of mice on the 5th day, indicating that vaginal Gardnerella colonization was successful and modeling was successful. Figure 5 As shown in the figure, vaginal Gardnerella was not detected in the blank control group during the entire experiment. After probiotic intervention, all intervention groups were able to significantly inhibit the colonization of vaginal Gardnerella compared with the model group, but intervention group D reduced the content of Lactobacillus. It can be seen that although metronidazole has the ability to inhibit pathogenic bacteria, it can also simultaneously inhibit the colonization of lactic acid bacteria. The ability of intervention group C (RH99 probiotic group) to inhibit pathogenic bacteria was more significant, reduced to 5.85lg copies / μL, and the colonization rate of pathogenic bacteria was reduced by 28.75% compared with the positive control group (8.21lg copies / μL). The inhibitory ability of exopolysaccharides and D-mannitol is comparable, which shows that exopolysaccharides and D-mannitol are key components for inhibiting the colonization of pathogenic bacteria.

[0136] Figure 7 Shown are the results of a colonization assay of Gardnerella vaginalis in the mouse vagina.

[0137] At the same time, this experiment also evaluated the colonization of Lactobacillus casei during the intervention phase. Lactobacillus casei was not detected in the vagina of mice before intervention. On the 17th day, Lactobacillus casei colonization occurred in all intervention groups, and the colonization amount in intervention group C was the most obvious, with a copy number of 7.35lg / μL. It can be seen that Lactobacillus casei RH99 can compete with vaginal Gardnerella for colonization sites and show good antagonism to the growth of pathogenic bacteria. Secondly, the colonization of Lactobacillus casei in intervention groups A and B was second, and both could significantly reduce the infection of Gardnerella. It may be possible to inhibit the adhesion of pathogenic bacteria to HeLa cells by competition, exclusion, and replacement of occupancy, thereby inhibiting the formation of pathogenic biofilm and then inhibiting the colonization of pathogenic bacteria.

[0138] Figure 8 Shown is a colonization assay of Lactobacillus casei in the mouse vagina.

[0139] Fig. 9 Cytokine levels in mouse serum are shown.

[0140] After Gardnerella invades the body, it will cause different degrees of inflammatory response. The ELISA kit method was used to detect the level of inflammatory factors in mouse serum. The results are as follows Figure 6 After infection with Gardnerella, the levels of TNF-α and IL-1β in mouse serum showed an upward trend. After intervention, the anti-inflammatory effect of intervention group C was the most obvious, which could significantly reduce the levels of TNF-α and IL-1β. It can be seen that RH99 can also relieve vaginitis by inhibiting inflammation.

[0141] It can be seen that the Lactobacillus casei RH99 provided by the present invention can significantly alleviate vaginitis. First, the Lactobacillus casei RH99 has the ability of high-yield extracellular polysaccharides of D-mannose monomers, and the content of D-mannose monomers is more than 50%. Secondly, Lactobacillus casei RH99, the extracellular polysaccharides produced by it have a significant promoting effect on the biofilm formation of lactic acid bacteria itself, and have a significant inhibitory effect on the biofilm of vaginal pathogens; moreover, RH99 can compete with vaginal Gardnerella for colonization sites, and can inhibit pathogenic bacteria from adhering to HeLa cells by means of competition, repulsion, and replacement of occupancy, thereby inhibiting the formation of pathogenic biofilm and inhibiting the colonization of pathogens in the vagina. Therefore, RH99 is inoculated into the vagina, and its strain, the metabolites produced (exopolysaccharides), and the core monosaccharide (D-mannitol) may all inhibit the colonization of pathogens and promote the colonization of lactic acid bacteria.

[0142] In short: RH99 can inhibit the growth of pathogenic bacteria + inhibit biofilm formation + inhibit the adhesion of pathogenic bacteria + inhibit the level of inflammatory factors through key core metabolites (extracellular polysaccharides containing more than 50% D-mannose), achieving a four-in-one all-round inhibition and comprehensive relief of vaginitis.

[0143] Example 7 Probiotic Condom Preparation Method and Verification:

[0144] ① Probiotic preparation of Lactobacillus casei RH99:

[0145] The method comprises the steps of streaking the probiotics, picking out single bacteria and putting them into a liquid MRS culture medium, culturing at 37°C, performing two rounds of activation, and centrifuging to obtain probiotic bacterial slurry.

[0146] The culture medium comprises the following components by weight percentage: 0.5-1.0% yeast peptone, 0.5-1.0% soy peptone, 0.4-0.5% yeast extract powder, 1.5-2.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate, 0.4-0.6% sodium acetate, 0.02-0.03% magnesium sulfate, 0.002-0.004% manganese sulfate monohydrate, and the balance distilled water.

[0147] After centrifugation, the bacterial sludge was freeze-dried in a freeze dryer, and the freeze-dried powder was collected for later use.

[0148] ② Condoms Condom preparation: Condoms were prepared according to existing condom preparation technology, and the probiotic preparation and medium-chain triglycerides were mixed in a ratio of 1:10, wherein the number of live bacteria in the probiotic preparation was 1 billion, and added to the front end chamber of the condom containing dimethyl silicone oil, and a storage stability experiment and an antibacterial experiment were performed with reference to Example 2.

[0149] Table 6 Storage stability and antibacterial properties of condoms containing probiotics

[0150]

[0151] As shown in Table 6, the probiotic condoms also have antibacterial properties, and at 37°C, the viable bacterial survival rate after one month is 84.30%.

Claims

1. A Lactobacillus casei RH99, characterized in that: Its deposit number is CGMCC No.32108.

2. A fermentation product, characterized in that: The method is obtained by fermenting the Lactobacillus casei RH99 as claimed in claim 1.

3. A probiotic preparation comprising Lactobacillus casei RH99 as claimed in claim 1, characterized in that: The method comprises the steps of streaking the probiotics, picking out single bacteria and putting them into a liquid MRS culture medium, culturing at 37°C, performing two rounds of activation, and centrifuging to obtain probiotic bacterial slurry.

4. The method for preparing the probiotic preparation according to claim 3, characterized in that: The culture medium comprises the following components by weight percentage: 0.5-1.0% yeast peptone, 0.5-1.0% soy peptone, 0.4-0.5% yeast extract powder, 1.5-2.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate, 0.4-0.6% sodium acetate, 0.02-0.03% magnesium sulfate, 0.002-0.004% manganese sulfate monohydrate, and the balance distilled water.

5. The use of the probiotic preparation according to claim 2, characterized in that: The invention is used for preparing a drug for inhibiting the growth of Gardnerella, Candida albicans and / or Peptostreptococcus.

6. The use of the probiotic preparation according to claim 2, characterized in that: The invention is used for preparing drugs for inhibiting biofilm formation of Gardnerella, Candida albicans and / or Peptostreptococcus.

7. The use of the probiotic preparation according to claim 2, characterized in that: Used in preparing drugs for inhibiting inflammatory factors.

8. The use of the probiotic preparation according to claim 7, characterized in that: The inflammatory factors include NF-α and IL-1β.

9. An application of Lactobacillus casei RH99 as claimed in claim 1, characterized in that: for use in the preparation of microbial preparations; or For use in addition to sanitary products; or Used in the preparation of daily necessities; or The sanitary products include menstrual hygiene products, excrement hygiene products, disposable underwear, and condoms; The daily necessities include vaginal washes, gels, ointments, sprays or creams.

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