CN117487685A - Lactobacillus crispatus and application thereof in preventing or treating female colpitis - Google Patents
Lactobacillus crispatus and application thereof in preventing or treating female colpitis Download PDFInfo
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- CN117487685A CN117487685A CN202311080352.8A CN202311080352A CN117487685A CN 117487685 A CN117487685 A CN 117487685A CN 202311080352 A CN202311080352 A CN 202311080352A CN 117487685 A CN117487685 A CN 117487685A
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- lactobacillus crispatus
- vaginal
- lactobacillus
- strain
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Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to a lactobacillus crispatus strain which is prepared from lactobacillus crispatusLactobacillus crispatus) And applications thereof. The lactobacillus crispatus is obtained by screening vaginal secretion of healthy women, belongs to normal vaginal strains, and can effectively inhibit various vaginal pathogenic bacteria such as gardnerella, candida albicans, staphylococcus aureus and the like. The Lactobacillus crispatus has been deposited at university of Wuhan, china at 2023, 4 and 24 daysThe preservation number of the China center for type culture collection is CCTCC NO: m2023608 can be widely applied to the prevention or auxiliary treatment of female bacterial vaginitis and mycotic vaginitis.
Description
Technical Field
The invention relates to the technical field of screening and application of probiotics, in particular to a lactobacillus crispatus which has the effect of preventing or treating female colpitis.
Background
The vaginal flora plays an irreplaceable role in maintaining the stability and regulation of the intravaginal environment. The vaginal flora is very complex and comprises, in addition to protozoa and fungi, a number of aerobic and anaerobic bacteria, such as streptococcus lactis, enterobacteria, proteus, garfujicoccus, wei Yong cocci, which are classified as colonic and pathological, all grown in this common environment, and there is a potential for antagonism between the microorganisms. At pH 3.8-4.2, the culture of co-perching probiotics, especially lactobacillus, is favorable to the reproduction of the main probiotics in healthy vagina, and the density in the vaginal fluid can reach 10 5 ～10 8 mL, when the vagina is infected by pathogenic microorganism, if lactobacillus is dominant, weak acid environment with pH of 3.8-4.2 in vagina can be maintained, and some lactobacillus can also produce H 2 0 2 Has toxic effect on other microorganisms to inhibit its proliferation, and can prevent vagina diseases. Once the fertility of pathogenic microorganisms exceeds that of probiotics, the growth environment of lactic acid bacteria is destroyed, and the environment in the vagina is further destroyed, and finally gynecological diseases are caused. After the gynecological diseases occur, the treatment is often not very effective, and intractable inflammation repeatedly attacks, which seriously affects female reproductive health. Wherein bacterial vaginitis (Ba)cterial vaginosis, BV) is one of the diseases caused by dysregulation of the vaginal flora and is characterized by a decrease in Lactobacillus and an increase in anaerobic bacteria, including Gardnerella vaginalis and Altobossis vaginalis. BV incidence is between 5% and 50%, itching, peculiar smell and leucorrhea abnormality can be caused, and the BV incidence becomes an important problem affecting the daily life, the quality of sexual life and public health safety of women in the global scope. Currently, bacterial vaginitis is mainly treated by antibiotics (including metronidazole and clindamycin), but the antibiotic therapy has low cure rate and high recurrence rate, and is difficult to reach the expectations of patients. Thus, there is a great need for a safe and effective treatment to alleviate the physiological and psychological burden of patients with bacterial vaginitis.
Probiotics were first discovered by russian microbiologists at the beginning of the 20 th century and developed for over a century. During this century, the concept and efficacy of probiotics has been recognized by scientists and consumers. The probiotics not only can form dominant flora on human intestinal tracts or skin, but also can be tightly combined on intestinal mucosa or skin to form a natural biological barrier, so that the whole human intestinal tracts or skin is in a healthy state. Probiotics regulate intestinal health and physical health mainly through antagonism of bacteria or production of antibacterial peptides. The types of lactobacillus in vagina are complex, and about 25 types of lactobacillus vaginalis reported so far are mainly Lactobacillus crispatus, lactobacillus grignard, lactobacillus inerticus and Lactobacillus jensenii. In vitro studies have shown that lactobacillus isolated from the vagina of healthy women can inhibit the adhesion of gardnerella vaginalis and even replace gardnerella vaginalis that has adhered to the vaginal epithelium. KremLeva et al found that H was produced 2 O 2 The lactobacillus can stimulate vaginal epithelial cells to secrete antibacterial substances, and increase the activity of synthesized protective factors, such as lysozyme, lactoferrin and the like. Such a severe deficiency of lactobacillus is found in women with bacterial vaginosis, particularly pregnant women. Therefore, the preparation of medicament or sanitary article by utilizing the vaginal dominant strain screened from the vaginal secretion of healthy women is a safe and effective method for preventing and treating bacterial vaginitisA method of manufacturing the same.
Disclosure of Invention
The invention aims to provide a Lactobacillus crispatus strainLactobacillus crispatus) And applications thereof. The lactobacillus crispatus is obtained by screening vaginal secretion of healthy women, belongs to normal vaginal strains, can effectively inhibit various vaginal pathogenic bacteria such as gardnerella, candida albicans, staphylococcus aureus and the like, and can be widely applied to prevention or auxiliary treatment of female bacterial vaginitis and mycotic vaginitis.
The invention firstly provides a strain of lactobacillus crispatusLactobacillus crispatus) VHProbi E04 strain, which was preserved in the China center for type culture collection, university of martial arts, in China, with a preservation number of cctccc NO: m2023608.
The 16s rDNA sequence of the Lactobacillus crispatus VHProbi E04 strain is SEQ ID NO. 1.
The MALDI-TOF-MS protein fingerprint of the Lactobacillus crispatus VHProbi E04 strain is shown in figure 3; the rep-PCR fingerprint is shown in FIG. 4; the RAPD finger print is shown in figure 5.
The invention also provides application of the Lactobacillus crispatus VHProbi E04 strain in preparation of a bacteriostatic agent.
The invention also provides application of the Lactobacillus crispatus VHProbi E04 strain in preparing medicines for preventing or treating bacterial or fungal induced vaginitis.
The bacteria are gardnerella or staphylococcus aureus.
The mould is candida albicans.
The invention also provides a composition for treating colpitis, which comprises viable bacteria, inactivated bacteria or fermentation products of the lactobacillus crispatus E04 strain.
The composition is a non-therapeutic vaginal health product, including vaginal health products, vaginal cleaning products, vaginal care products, vaginal cosmetics, and vaginal hygiene products.
The composition is a therapeutic vaginal health product, including vaginal medicines.
The composition is a medical device for vagina.
The Lactobacillus crispatus VHProbi E04 provided by the invention can produce antibacterial substances H 2 O 2 The growth of pathogenic bacteria Gardnerella vaginalis and staphylococcus aureus of vagina can be effectively inhibited, and the diameters of the inhibition zones respectively reach 1.38+/-0.05 cm and 2.15+/-0.21 cm; the strain can inhibit the growth of gardnerella and candida albicans with high efficiency under the condition of mixed culture, the inhibition rates respectively reach 94 percent and 100 percent, and the strain has certain adhesiveness to human vaginal epithelial cells and can be planted in vagina, so that the strain can be widely applied to the prevention and auxiliary treatment of bacterial vaginitis and colpitis mycotica.
The Lactobacillus crispatus VHProbi E04 provided by the invention is sensitive to common antibiotics and has good biological safety; can tolerate higher salinity, and the maximum tolerant salt concentration is 3%; can grow at 45 ℃ and has certain heat resistance. The strain has stronger antioxidation function, wherein the clearance rate of DPPH and HRS free radicals is 24.28+/-1.03% and 24.19+/-4.63% respectively; the anti-lipid peroxidation inhibition rates of the supernatant, the thalli and the intracellular extract are 39.18% +/-0.17%, 10.71% +/-0.19% and 15.40% +/-0.19%, respectively, and the effect is remarkable.
The Lactobacillus crispatus VHProbi E04 provided by the invention can be prepared into a bacteriostatic agent, a drug or a female sanitary product, and can be used for relieving the uncomfortable symptoms of bacterial vaginitis and mycotic vagina and keeping the health of female vagina.
Drawings
FIG. 1 is a diagram of the hydrogen peroxide production of Lactobacillus crispatus VHProbi E04;
FIG. 2 is a colony morphology of Lactobacillus crispatus VHProbi E04;
FIG. 3 shows a MALDI-TOF-MS protein fingerprint of Lactobacillus crispatus VHProbi E04;
FIG. 4 is a rep-PCR fingerprint of Lactobacillus crispatus VHProbi E04;
FIG. 5 is a RAPD fingerprint of Lactobacillus crispatus VHProbi E04;
FIG. 6 is a graph showing adhesion of Lactobacillus crispatus VHProbi E04 to human vaginal epithelial cells.
Detailed Description
The examples are only illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
EXAMPLE 1 isolation screening of Lactobacillus crispatus VHProbi E04
1. Primary screening of lactic acid bacteria
According to 2019 edition of human genetic resource coulomb theory specification, after signing project commitment and informed consent with a sample provider, collecting healthy female vaginal secretion which does not eat probiotic preparations within half a year according to biological sample library standard operation specifications; serial dilution of the secretion was carried out to obtain 10 -1 、10 -2 、10 -3 100 mu L of each of the three dilution gradients is respectively coated on MRS selective culture medium and anaerobically cultured for 48 hours at 37 ℃; after the single colony of the flat plate grows, lactobacillus with the shape of bacillus is selected through microscopic examination, 18 strains are totally named as E01, E02, … … and E18 respectively.
2. Anti-gardnerella vaginalis strain and hydrogen peroxide producing strain rescreening
(1) Preparation of Columbia blood agar plates
The Columbia blood agar plates were prepared in advance and dried.
(2) Preparation of gardnerella vaginalis bacterial liquid
Streaking and activating the gardnerella vaginalis BNCC337545 and BNCC354890 on a Columbia blood agar plate respectively, then picking single colonies into a modified BHI broth culture medium (BHI plus 5% serum), aerobically culturing for 24 hours at 37 ℃, then transferring the single colonies into a new modified BHI broth culture medium according to the proportion of 1%, aerobically culturing for 24 hours at 37 ℃ to obtain fresh bacterial liquid, wherein the fresh bacterial liquid is prepared according to the following steps of 1:1, uniformly mixing the mixture in equal volume to obtain gardnerella vaginalis bacterial liquid.
The gardnerella vaginalis bacterial solutions described in the following examples are the same as in this example.
Two different gardnerella vaginalis strains are used as indication strains, so that the antibacterial capability of the screened lactobacillus to gardnerella vaginalis can be reflected.
(3) Oxford cup bacteriostasis experiment
Preparing Columbia blood plates in advance, then coating 50 mu L of vaginal Lactobacillus gasseri liquid on the blood plates, placing oxford cups, respectively adding 100 mu L of fermentation broth of lactobacillus as a primary sieve into the holes, culturing at 37 ℃ for 48 hours, and observing whether a bacteriostasis ring exists or not.
The results show that the E01, E04 and E11 strains in 18 lactobacillus strains obtained by primary screening have obvious inhibition effect on gardnerella, and the diameters of inhibition zones generated by fermentation liquor are respectively 1.23+/-0.03 cm, 1.38+/-0.05 cm and 1.28+/-0.02 cm.
3. Determination of Hydrogen peroxide production Capacity
The tetramethylaniline is prepared into 0.1g/100mL by ethanol, and then filtered by a 0.22 mu m filter membrane; horseradish peroxidase was dissolved in water to prepare 0.01g/100mL and filtered through a 0.22 μm filter.
Glucose (1% (w/v) is added into MRS culture medium, then the culture medium is autoclaved at 121 ℃, and when the temperature is reduced to 50 ℃, tetramethylaniline and horseradish peroxidase are added, so that a solid culture medium with the final concentration of tetramethylaniline of 25mg/100mL and the final concentration of horseradish peroxidase of 1mg/100mL is obtained.
Then, the fresh bacterial solutions of E01, E04 and E11 were diluted, 50. Mu.L of each was spread on the above solid medium plate, and cultured at 37℃for 48 hours, and then, whether or not blue colonies were produced was observed.
As a result, as shown in FIG. 1, blue colonies were produced around E04 colony, namely hydrogen peroxide was produced, and the other two strains did not produce blue colonies. Thus, it was demonstrated that E04 strain is capable of producing hydrogen peroxide.
EXAMPLE 2 identification of Lactobacillus crispatus VHProbi E04
2.1 Colony morphology identification
The colony morphology of E04 strain is shown in figure 2, single colony is semitransparent, matt, irregular and uneven in edge, colony diameter is 2-3mm, and thallus is rod-shaped under microscope.
2.2 Identification of physiological and biochemical characteristics
The inoculum preparation in this example was as follows: under aseptic condition, taking a proper amount of fresh bacterial liquid, centrifuging for 5min at 5000rpm/min, washing for 2 times by using PBS buffer solution, and diluting for 50 times after re-suspending by using the PBS buffer solution with the same volume to serve as an inoculation liquid.
1. Temperature growth Range experiment
Under aseptic conditions, the inoculation liquid is inoculated into 10mL of MRS liquid culture medium according to the inoculation amount of 10%, 10mL of MRS liquid culture medium without bacteria is used as a control, and the culture liquid is respectively placed in a 15 ℃ constant temperature shaking incubator for 7 days and a 45 ℃ constant temperature shaking incubator for 2 days, and whether the culture liquid becomes turbid is observed.
The results show that: after the culture is carried out for 7 days at the constant temperature of 15 ℃, the culture medium is still clear; after incubation at 45℃for 2 days, the medium became turbid. Thus, it was demonstrated that E04 strain was unable to grow at 15℃and grew normally at 45 ℃.
2. Experiment of carbon Source metabolism
The metabolic effect of E04 strain on 49 carbon sources was determined using the API 50CHL reagent strip.
Under aseptic condition, taking a proper amount of fresh bacterial liquid of E04 bacterial strain, centrifuging at 5000rpm for 5min, washing with pH7.0 phosphate buffer solution for 2 times, and re-suspending with the same volume of buffer solution to obtain bacterial suspension. Adding the bacterial suspension into the culture medium of the API kit according to the addition amount of 10%, adding the culture medium into the holes of the test paper strips according to the operation of the kit, sealing the paraffin, putting the test paper strips into a box, covering a cover with about 10mL of sterile deionized water on the substrate of the box, placing the box into a 37 ℃ incubator for culturing for 24-48 hours, observing color change, changing the color from blue to yellow if the bacteria grow, and keeping the color unchanged if the bacteria do not grow. And recording test results, and uploading the test results to an authentication software API web.
The results show that E04 strain can utilize galactose, glucose, fructose, mannose, N-acetylglucosamine, phellodendrine, esculin, salicin, cellobiose, maltose, sucrose, raffinose, starch; glycerol, mannitol, sorbitol, trehalose and D-arabitol cannot be utilized; erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, ribose, D-adonitol, beta-methyl-D-xyloside, sorbose, alpha-methyl-D-glucoside, dulcitol, amygdalin, rhamnose, inositol, alpha-methyl-mannoside, lactose, melibiose, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, inulin, melezitose, gentiobiose, turinose, D-tagatose, gluconate, L-arabinitol, 2-ketogluconate and 5-ketogluconate.
API authentication results: the E04 strain was lactobacillus crispatus, id=99.6%, T value=0.87.
3. Glucose acidogenesis and gas production test
The medium formulation used in this example is as follows:
peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; salt solution a 0.5mL; salt solution B0.5 mL; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100mL; the pH is 6.8-7.0.
The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.
Salt solution A: KH (KH) 2 PO 4 10g、K 2 HPO 4 1.0g, dissolved in distilled water, was fixed to a volume of 100mL.
Salt solution B: mgSO (MgSO) 4 ·7H 2 O 11.5g、MnSO 4 ·2H 2 O 2.4g、FeSO 4 ·7H 2 O0.68 g, dissolved in distilled water, was fixed to a volume of 100mL.
Under aseptic conditions, the bacterial suspension is inoculated with a culture medium in an inoculum size of 10%, the culture medium without bacteria is used as a control, then the top is sealed by 2mL of sterile liquid paraffin, and the culture is carried out at 37 ℃. The culture was continued for 6 days, and the presence or absence of change in the color of the medium was observed every day.
The results show that: after 6d of culture at 37 ℃, the culture medium turns yellow from green, and no gas exists in the small inverted tube, which indicates that the E04 strain ferments glucose to produce acid and does not produce gas.
In summary, the physiological and biochemical identification results of the E04 strain are as follows: can not grow under the condition of 15 ℃ and can normally grow under the condition of 45 ℃; fermenting glucose to produce acid without producing gas; the carbon source metabolism was identified as lactobacillus crispatus.
2.3 Molecular biological identification
Single colonies of E04 strain on the plate were picked up and cultured in MRS broth medium at 37℃for 24 hours, then 500. Mu.L of the fermentation broth was taken, and the genome of the strain was obtained by referring to the Tiangen bacterium genome DNA extraction kit (catalog number: DP 302) and used for the subsequent molecular biological identification.
2.3.1 Mass Spectrometry identification of proteins
The single colony of E04 strain on the flat plate is dipped on a protein mass spectrum plate by a toothpick, then bacterial mud is uniformly coated in a disc on the mass spectrum plate by the toothpick, the coated bacterial mud is not required to be too thick, then 1 mu L of matrix solution in a mass spectrum sample pretreatment box is added to cover the sample according to the specification of the protein mass spectrum kit, and the sample is naturally dried at room temperature. And (5) placing the dried mass spectrum plate on a motitop protein mass spectrometer for identification.
MALDI-TOF-MS protein fingerprint is shown in FIG. 3, and the E04 strain is identified as Lactobacillus crispatus.
2.3.1 Identification of the 16s rDNA Gene sequence
(1) 16s rDNA Gene amplification
Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
TABLE 1 16s rDNA PCR amplification System (50. Mu.L)
| Composition of the components | Reaction volume |
| 10×PCR buffer | 5μL |
| dNTPs | 4μL |
| 27F | 2μL |
| 1492R | 2μL |
| DNA | 2.5μL |
| rTaq | 0.5μL |
| ddH2O | 34μL |
(2) Electrophoresis verifies that the size of the PCR amplification product is about 1500bp, which meets the requirements.
(3) Sequencing of PCR products
Sequencing results show that the 16s rDNA sequence of the E04 strain is SEQ ID NO. 1. The sequence was subjected to BALST alignment in NCBI database against Lactobacillus crispatus @Lactobacillus crispatus) Is the highest in similarity. Thus, E04 strain was preliminarily determined to be Lactobacillus crispatusLactobacillus crispatus）。
SEQ ID NO. 1 is shown below:
GACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTAAAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACCGAAGTAAATCTGTTAGTTCCGCTCGCTCGACTGC。
2.3.3 RAPD fingerprint identification
1. RAPD fingerprint identification
1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3').
2) RAPD reaction system
TABLE 2 RAPD reaction System
| Composition of the components | Reaction volume |
| TaqDNA polymerase (5U/. Mu.L) | 0.2 μL |
| 10 XBuffer (containing Mg) 2+ ） | 2 μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 0.8 μL |
| DNA template | 2 μL |
| Sterile double distilled water | 14 μL |
| Total volume of | 20 μL |
3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electrophoresis pattern was detected by using a gel imaging system, and the RAPD fingerprint pattern of E04 strain was shown in FIG. 4.
By comparison, no RAPD fingerprint matching FIG. 4 was found in the prior published reports, thus indicating that E04 strain is a novel Lactobacillus crispatus strain.
2.3.4 rep-PCR fingerprint identification
1) rep-PCR primer
GTGGTGGTGGTGGTG。
2) reaction system of rep-PCR
TABLE 3 rep-PCR reaction System
| Reaction components | Volume of |
| r TaqDNA polymerase | 0.2μL |
| 10X Ex Taq DNA Buffer (containing Mg) 2+ ） | 2μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 2μL |
| DNA template | 2μL |
| Sterile double distilled water | 12.8 μL |
3) Electrophoresis
DL2000 DNA Marker served as a result control. The voltage is 100V, the electrophoresis time is 80min, and the detection and amplification result is shown in FIG. 5.
By comparison, no rep-PCR fingerprint matching with FIG. 5 was found in the prior published report, thus demonstrating that the E04 strain screened by the invention is a novel Lactobacillus crispatus strain.
2.3.5 Whole genome sequencing
The E04 strain was inoculated into 500 mM RS broth at a volume ratio of 1%, cultured at 37℃for 22 hours, and centrifuged at 8000rpm for 10 minutes to collect the cells. The thallus is sent to a sequencing center to obtain the complete gene sequence of the bacterium, the gene sequence is uploaded to NCBI gene database, the accession number is CP128800, the whole genome is 2133556bp, the GC content is 36.59%, and the total number of genes is 2055.
Comparing colony morphology and physiological and biochemical characteristic results of E04 strain, and combining molecular biology identification results to determine E04 strain as a novel Lactobacillus crispatus, and named Lactobacillus crispatusLactobacillus crispatus）VHProbi E04。
EXAMPLE 3 Lactobacillus crispatus VHProbi E04 salinity tolerance test
Under aseptic conditions, the inoculation liquid is inoculated into 5mL MRS liquid culture media with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% respectively according to 10% inoculation amount, 5mL MRS liquid culture media without bacteria is used as a control, and the culture media are subjected to constant temperature shaking culture at 37 ℃ to observe whether the culture media become turbid.
The results show that: lactobacillus crispatus VHProbi E04 grows at 1% -3% salt concentration and does not grow at 4% -8% salt concentration, and the optimal tolerance salt concentration of Lactobacillus crispatus VHProbi E04 is 3%.
EXAMPLE 4 antibiotic resistance test of Lactobacillus crispatus VHProbi E04
1. Preparing antibiotics: ampicillin, gentamicin, clindamycin, erythromycin, streptomycin and tetracycline are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using MRS liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
2. Preparing an inoculation liquid: preparation of the inoculum: taking a proper amount of fresh bacterial liquid (24-48 h, culturing at 40 ℃) and centrifuging at 5000rpm for 5min, washing once with sterile physiological saline, and diluting 50 times after re-suspending with the same volume of physiological saline to obtain an inoculation liquid.
3. Minimal inhibitory concentration MIC of antibiotics against lactobacillus crispatus VHProbi E04 was determined by a mini broth dilution method.
(1) The MRS liquid culture medium without antibiotics is added to the 96-well plate in column 1 for the time, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 96-well plate in columns 2 to 12 as a negative control, 10 mu L of the inoculation liquid is respectively inoculated, 3 parallel wells are made, and 1 well of the inoculation liquid without bacteria is used as a blank.
(2) 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
(3) Shake culturing 96-well plate at 40deg.C for 48 hr, taking out, and measuring OD 600 Values, MIC values of antibiotics against strains were counted with 48h results, which are shown in Table 5.
TABLE 4 antibiotic MIC values for Lactobacillus crispatus VHProbi E04
Note that: MIC units μg/mL.
From the results shown in Table 4, the Lactobacillus crispatus VHProbi E04 provided by the invention is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline and clindamycin, is slightly sensitive to streptomycin and gentamicin, and has good overall biosafety.
Example 5 hydrophobic cell surface test of Lactobacillus crispatus VHProbi E04
1. Preparing a bacterial liquid to be tested: the purified Lactobacillus crispatus VHProbi E04 colony is picked and inoculated in a newly prepared MRS liquid culture medium, and is cultured for 24 to 48 hours at 40 ℃ in a shaking way. Inoculating 1% (V/V) of the strain into MRS liquid culture medium, shaking culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1M KNO 3 The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.
2. Surface hydrophobicity determination: mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO 3 And record OD 600 Is A 0 1.5mL of the bacterial suspension was mixed with 500. Mu.L of xylene, and the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase (not to the organic phase) was carefully aspirated and absorbance A was measured at 600nm 1 . Cell hydrophobicity as formula hydrophobicity= (a) 0 -A 1 )/A 1 The x 100% calculation was performed and the average of three experiments was taken.
The results show that: the hydrophobicity of the surface of the Lactobacillus crispatus VHProbi E04 cell provided by the invention is 50.61%.
EXAMPLE 6 measurement of antioxidant function of Lactobacillus crispatus VHProbi E04
1. Determination of the ability of the Strain to scavenge DPPH and Hydroxyl Radical (HRS)
1) Preparation of PBS bacterial suspension
Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, cultured for 18-20h at 37 ℃, the culture solution is taken as an inoculating solution, inoculated into 50mL of MRS liquid culture medium according to 2% of inoculating amount, and subjected to static culture for 18h, thus obtaining the culture solution of the strain. After 1mL of bacterial liquid is sucked and the bacterial cells are collected, the bacterial cells are washed by 1mL of PBS buffer solution for 2 times, and then 2mL of PBS solution is added to resuspend the bacterial cells for standby.
2) Determination of DPPH free radical scavenging ability of Strain
Taking 1mL of PBS bacterial suspension of the strain to be detected, adding 1mL of 0.4 mM of the freshly prepared DPPH free radical solution, uniformly mixing, then placing the mixture at room temperature for shading reaction for 30min, and then measuring the absorbance A of the sample at the wavelength of 517nm Sample of 3 replicates were measured. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution.
The clearance is calculated according to the following formula: clearance = [1- (A) Sample of -A Blank space ）/A Control ]×100%。
The results show that: the clearance rate of the Lactobacillus crispatus VHProbi E04 to DPPH free radical is up to 24.28% +/-1.03%.
3) Determination of the ability of the Strain to scavenge hydroxyl radical HRS
mu.L of 5mM sodium salicylate-ethanol solution, 100. Mu.L of 5mM ferrous sulfate, 500. Mu.L of deionized water and 200. Mu.L of Lactobacillus crispatus VHProbi E04 bacterial suspension were mixed uniformly, 100. Mu.L of hydrogen peroxide solution (3 mM) was added thereto, and after 15min in a 37℃water bath, the absorbance of the sample was measured at a wavelength of 510 nm.
The hydroxyl radical scavenging rate was calculated according to the following formula: clearance= (a) Sample of -A Control of ）/（A Blank space -A Control of ）×100%。
Wherein: a is that Control of For deionized water to replace the sample, A Blank space Substitution of deionized water for sample and H 2 O 2 。
The results show that: the clearance rate of the Lactobacillus crispatus VHProbi E04 provided by the invention on the HRS free radical is up to 24.19% +/-4.63%.
2. Determination of lipid peroxidation resistance of Strain
1) Preparation of strain culture and fermentation supernatant, thallus and intracellular extract:
culturing the strain in MRS liquid culture medium at 37deg.C for 24 hr, transferring for 3 generations, centrifuging at 4deg.C for 10min at 6000 r/min, and collecting supernatant to obtain fermentation supernatant. PBS buffer solution for collected thallipH 7.4) was centrifuged at 6000 r/min for 10min and washed 3 times. The bacterial cells were resuspended in PBS buffer to a bacterial cell concentration of 1.0X10 9 cells/mL to obtain a bacterial suspension. The bacterial suspension is subjected to ultrasonic crushing for 20 minutes by an ultrasonic crusher to obtain intracellular extracts.
2) Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
3) 0.5mL of PBS solution (pH 7.4) was added with 1mL of emulsion of linoleic acid (mL), 1mL of LFASO 4 (1%) and 0.5mL of sample were added, the mixture was added with 1.5. 1.5 h in a 37℃water bath, 0.2mL of TCA (4%), 2mL of TBA (0.8%), the mixture was cooled rapidly in a 100℃water bath for 30min, centrifuged at 4000r/min for 15min, and the supernatant was collected at OD 532 The absorbance measured at nm is A; the control group was A with 0.5. 0.5mL distilled water instead of the sample 0 。
Inhibition ratio = (a 0 －A)/ A 0 ×100%。
The results show that: the supernatant anti-lipid peroxidation inhibition rate of the Lactobacillus crispatus VHProbi E04 is 39.18% +/-0.17%; the inhibition rate of the bacterial body to lipid peroxidation is 10.71% +/-0.19%; the lipid peroxidation inhibition rate of the intracellular extract was 15.40% ± 0.19%.
Example 8 inhibitory Effect of Lactobacillus crispatus VHProbi E04 on Gardnerella
100. Mu.L of Lactobacillus crispatus bacterial liquid (10) 9 CFU/mL) and 100. Mu.L of gardnerella liquid (10) 9 CFU/mL) was inoculated into a test tube containing 5mL of the modified BHI liquid medium, shaken well, and the test tube was incubated in an incubator at 37℃for 24 hours to obtain an experimental group broth.
100 mu L of MRS liquid culture medium and 100 mu L of gardnerella bacteria liquid are inoculated into a test tube filled with 5mL of modified BHI culture medium, the test tube is evenly vibrated, and the test tube is cultured in a 37 ℃ incubator for 24 hours, so as to obtain a control group culture solution.
After each group of culture solutions was diluted with sterilized PBS buffer solution in a gradient manner, 100. Mu.L of the culture solution was pipetted and spread on Columbia blood agar solid medium, and after culturing in an incubator at 37℃for 48 hours, plate counting was performed to obtain the colony count of Gardnerella in each group of culture solutions.
The results showed that the gardnerella concentration in the culture solution of the control group was 2.15X10 9 CFU/mL, and gardnerella concentration in the Lactobacillus crispatus group culture solution was 3.45X10 7 CFU/mL shows that the Lactobacillus crispatus VHProbi E04 can obviously inhibit the growth of gardnerella under the co-culture condition, and the inhibition rate reaches 98%.
Example 9 inhibitory Effect of Lactobacillus crispatus VHProbi E04 on Candida albicans
50. Mu.L of Lactobacillus crispatus bacterial liquid (10) 8 CFU/mL) and 50. Mu.L of Candida albicans liquid (10 7 CFU/mL) is inoculated in a test tube filled with 5mL MRS liquid culture medium, and the culture medium is evenly vibrated to be used as an experimental group culture solution;
mu.L of MRS liquid medium and 50. Mu.L of Candida albicans liquid were inoculated into a test tube containing 5mL of MRS liquid medium as a control group culture liquid.
Culturing the culture solutions of the control group and the experimental group in a culture box at 37 ℃ for 24 hours; after the culture medium is subjected to gradient dilution by using sterilized PBS buffer solution, 100 mu L of the culture medium is sucked by a liquid transfer device and coated on a glucose solid culture medium, and after the culture medium is subjected to inversion culture for 48 hours in a 37 ℃ incubator, the plate count is carried out, so that the colony number of candida albicans in each group of culture solution is obtained.
The results showed that the concentration of Candida albicans in the culture broth of the control group was 5.05X10 6 CFU/mL, while Candida albicans concentration in Lactobacillus crispatus culture broth was only 430 CFU/mL. The lactobacillus crispatus VHProbi E04 has strong inhibiting effect on candida albicans, and the inhibiting rate is almost 100%.
EXAMPLE 10 antibacterial Effect of Lactobacillus crispatus VHProbi E04 on Staphylococcus aureus
And taking out a glycerin tube of staphylococcus aureus, drawing a small amount of bacterial liquid on a nutrient agar plate by using an inoculating loop, and after bacterial colonies grow out, picking single bacterial colonies to culture in nutrient broth for 24 hours, wherein the bacterial liquid is reserved.
Referring to the oxford cup bacteriostasis test method described in example 1, the inhibitory effect of lactobacillus crispatus VHProbi E04 broth on staphylococcus aureus causing bacterial vaginitis was examined.
The result shows that the diameter of the inhibition zone of the strain on staphylococcus aureus reaches 2.15+/-0.21 cm. Thus, it was demonstrated that Lactobacillus crispatus VHProbi E04 is effective against Staphylococcus aureus.
EXAMPLE 11 adhesion of Lactobacillus crispatus VHProbi E04 to human vaginal epithelial cells
1. Cell preculture
Resuscitating human vaginal epithelial cells in liquid nitrogen, culturing to desired amount, and digesting with pancreatin to obtain single cell suspension when cell density is about 80%, counting with a blood cell counting plate, and counting with cell number of 5×10 5 cells/mL. Then, 500. Mu.L of the cell suspension was inoculated into a 24-well plate with a cell slide at a density of 2.5X10 5 cells/well were cultured overnight to complete adherence and the broth was discarded and rinsed 2 times with fresh broth for use.
2. Preparation of bacterial suspension
Washing fresh bacterial liquid of Lactobacillus crispatus VHProbi E04 with phosphate buffer solution of pH7.0 twice, then re-suspending with RPMI-1640 culture solution containing 10% calf serum in the same volume, adjusting absorbance to make OD 600 Between=0.8-1.0.
3. Cell culture
Adding 500 mu L of bacterial suspension into a prepared 24-well plate containing human vaginal epithelial cells, and co-culturing for 2h in a carbon dioxide incubator; the non-adherent bacteria were removed by washing 3 times with phosphate buffer pH 7.0.
4. Counting
The cell slide was fixed with methanol for 15min, then stained with Giemsa stain for 5min, washed clean with phosphate buffer at pH7.0, and then removed from the slide. And observed under a microscope.
As a result, as shown in FIG. 6, lactobacillus crispatus VHProbi E04 has a certain adhesiveness to human vaginal epithelial cells, and thus can be effectively transplanted in the vagina.
The Lactobacillus crispatus VHProbi E04 is selected from healthy female vaginal secretion, is one of normal vaginal strains, can be adhered to the surface of vaginal epithelial cells for field planting, has a wide antibacterial spectrum, can inhibit gardnerella and candida albicans and can also effectively inhibit staphylococcus aureus, so that the Lactobacillus crispatus VHProbi E04 can be prepared into a microbial inoculum, a drug or a female sanitary product, and is helpful for preventing or treating bacterial vaginitis and mycotic vaginitis, and keeping the health of female vagina.
Claims (10)
1. Lactobacillus crispatusLactobacillus crispatus) The method is characterized in that the lactobacillus crispatus is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china at 2023, 4 months and 24 days: m2023608.
2. The lactobacillus crispatus of claim 1, wherein the 16s rDNA sequence of the lactobacillus crispatus is SEQ ID NO:1.
3. use of lactobacillus crispatus according to claim 1 in the preparation of a bacteriostatic agent.
4. Use of lactobacillus crispatus according to claim 1 for the manufacture of a medicament for the prevention or treatment of bacterial or fungal induced vaginitis.
5. The use according to claim 4, wherein the bacterium is gardnerella or staphylococcus aureus.
6. The use according to claim 4, wherein the mould is candida albicans.
7. A composition for use in the vagina comprising the viable bacteria, inactivated bacteria, or fermentation products thereof of lactobacillus crispatus of claim 1.
8. The composition of claim 7, wherein the composition is a non-therapeutic vaginal health product, including vaginal health products, vaginal cleaning products, vaginal care products, vaginal cosmetics, and vaginal hygiene products.
9. The composition of claim 7, wherein the composition is a therapeutic vaginal health product, including a vaginal drug.
10. The composition of claim 7, wherein the composition is a medical vaginal device.
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