Lactobacillus, multi-lactobacillus complex, preparation and application thereof

Abstract

The present invention discloses lactobacillus, a multi-lactobacillus complex, a preparation and its application. The lactobacillus includes LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain; the LSA-858 strain is deposited with GDMCC No.62779; the LSA-946 strain is deposited with GDMCC No.62781; the LSA-1012 strain is deposited with GDMCC No.62782; the LSA-1066 strain is deposited with GDMCC No.62783. The multi-lactobacillus complex includes at least three of the LSA-858 strain, the LSA-946 strain, the LSA-1012 strain or the LSA-1066 strain. The multi-lactobacillus complex has a better VVC pathogen inhibition effect than any of the aforementioned lactobacillus single bacteria.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to multiple lactobacilli, a complex prepared by the multiple lactobacilli, a preparation and applications thereof. Background Art

[0002] Vulvovaginal candidiasis (VVC), also known as candidal vaginitis and vulvovaginal candidiasis, is one of the most common vaginal infections in women. Its pathogens are mainly Candida albicans (~90% of VVC patients), with Candida glabrata, Candida tropicalis, and Candida parapsilosis accounting for a small number. Common clinical symptoms include increased leucorrhea, burning pain and itching in the vagina and vulva. If not treated in time, it may lead to some serious complications of the upper reproductive tract, such as endometritis, salpingitis and pelvic inflammatory disease, which may further worsen and lead to tubal scarring, infertility or ectopic pregnancy.

[0003] Domestic and foreign studies have shown that approximately 70% to 75% of women experience VVC at least once in their lives, 50% of whom will experience multiple attacks of the disease in their lives, and 5% to 10% of whom may develop recurrent VVC (RVVC). At present, the main clinical treatment for vulvovaginal candidiasis is antibiotic treatment, which kills pathogens such as Candida albicans by oral or topical use of azole antifungal drugs to achieve the purpose of treatment. Commonly used antibiotics include fluconazole, miconazole, and clotrimazole. However, due to the irregular clinical diagnosis and treatment and the excessive use of antifungal drugs, the stubbornness of vulvovaginal candidiasis is further aggravated, breaking the balance of vaginal flora, thereby causing drug resistance and recurrence.

[0004] The National Collaborative Group of Obstetrics and Gynecology proposed that restoring vaginal microecology to normal is the ultimate goal of vaginitis treatment, and proposed the concept of microecological treatment, using vaginal microecological preparations to restore a weakly acidic environment dominated by functional lactobacilli. The vaginal flora of healthy women is mainly composed of lactobacilli, including Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. Lactobacillus can produce lactic acid, hydrogen peroxide, antimicrobial peptides, etc., which can resist the invasion of pathogens and inhibit the growth of pathogens. It is a potential new generation of drugs that can be used to treat vaginitis. However, there are only two types of vaginal microecological preparations on the market in China, a commercially available drug named "Yanhua" containing enterococci and a commercially available drug named "Dingjunsheng" containing Lactobacillus delbrueckii. Both are single strain preparations for the treatment of bacterial vaginitis, and there are no microecological preparations for the treatment of VVC.

[0005] VVC patients are prone to recurring clinical symptoms and are difficult to cure, which seriously affects the quality of life and physical and mental health of patients. This is a key problem that clinicians are currently facing and need to solve urgently. Therefore, it is urgent and necessary to develop a microecological preparation for the treatment of VVC. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a multi-linked lactobacillus complex and further provides the application of the above-mentioned strain.

[0007] In a first aspect of the present invention, a strain of Lactobacillus crispatus is provided. The Lactobacillus crispatus is named as LSA-858 strain, and the LSA-858 strain is Lactobacillus crispatus ( Lactobacillus crispatus ), deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No.62779 and the deposit date September 9, 2022. The LSA-858 strain has a 16S rDNA gene fragment with a sequence as shown in SEQ ID No:1.

[0008] In a second aspect of the present invention, a strain of Lactobacillus jensenii is provided. The Lactobacillus jensenii is named as strain LSA-946, and the LSA-946 strain is Lactobacillus jensenii ( Lactobacillus jensenii ), deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No.62781, and the deposit date is September 9, 2022. The LSA-946 strain has a 16S rDNA gene fragment with a sequence as shown in SEQ ID No:2.

[0009] In a third aspect of the present invention, a strain of Lactobacillus johnsonii is provided. The Lactobacillus johnsonii is named as LSA-1012 strain, and the LSA-1012 strain is Lactobacillus johnsonii ( Lactobacillus johnsonii ), deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No.62782 and a deposit date of September 9, 2022. The LSA-1012 strain has a 16S rDNA gene fragment with a sequence as shown in SEQ ID No:3.

[0010] In a fourth aspect of the present invention, a strain of Lactobacillus reuteri is provided. The Lactobacillus reuteri strain is named as LSA-1066 strain, and the LSA-1066 strain is Lactobacillus reuteri ( Limosilactobacillus reuteri ), deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC No.62783, and the deposit date is September 9, 2022. The LSA-1066 strain has a 16S rDNA gene fragment with a sequence as shown in SEQ ID No:4.

[0011] The above four strains of lactobacillus all have high acid production and hydrogen peroxide production capabilities, and have excellent antibacterial effects on a variety of female vaginitis pathogens.

[0012] In a fifth aspect of the present invention, a multi-lactobacillus complex is provided. The multi-lactobacillus complex comprises at least three of the LSA-858 strain, the LSA-946 strain, the LSA-1012 strain or the LSA-1066 strain. During the experiment, it was found that the multi-lactobacillus complex has a better VCC pathogen inhibition effect than any of the above single bacteria.

[0013] In a sixth aspect, the present invention provides a preparation comprising at least one of the following (1) to (5):

[0014] (1) containing the multi-lactobacillus complex, wherein the bacteria in the multi-lactobacillus complex may be active freeze-dried bacterial powder or inactivated freeze-dried bacterial powder;

[0015] (2) a live bacterial liquid containing the polylactobacillus complex;

[0016] (3) a dead bacteria solution containing the polylactobacillus complex;

[0017] (4) metabolites obtained from the metabolic pathway of the polylactobacillus complex;

[0018] (5) An extract obtained from the polylactobacillus complex.

[0019] In a seventh aspect, the present invention provides the use of the above-mentioned multi-lactobacillus complex or preparation in the preparation of medicines or sanitary products for preventing or treating gynecological diseases.

[0020] In some embodiments of the present invention, the gynecological disease includes at least one of vaginal microecological flora imbalance, vulvovaginal candidiasis, bacterial vaginosis, HPV or urinary tract infection.

[0021] In some embodiments of the present invention, the dosage form of the drug is one of powder, ointment, gel, lozenge, granule, capsule, spray, tablet, pill or oral solution.

[0022] In some embodiments of the present invention, the drug further comprises pharmaceutical excipients, which include at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sweeteners and flavoring agents.

[0023] In some embodiments of the present invention, the sanitary product is one of a tampon, a sanitary napkin, a menstrual pad, a diaper, a soap and a condom.

[0024] In an eighth aspect, the present invention provides use of the above-mentioned multi-lactobacillus complex or preparation in the preparation of an antibacterial product.

[0025] In some embodiments of the present invention, the pathogenic bacteria inhibited by the antibacterial product include at least one of Candida albicans, Candida glabrata, Candida tropicalis, Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa.

[0026] In a ninth aspect, the present invention provides use of the above-mentioned multi-lactobacillus complex or preparation in the preparation of a drug delivery carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 1 is a diagram of the colony morphology of Lactobacillus, wherein A is the LSA-858 strain, B is the LSA-946 strain, C is the LSA-1012 strain, and D is the LSA-1066 strain;

[0028] Figure 2 The Gram staining microscopic examination images of lactobacilli, A is the LSA-858 strain, B is the LSA-946 strain, C is the LSA-1012 strain, and D is the LSA-1066 strain;

[0029] Figure 3 This is a diagram showing the detection results of the antagonism and symbiotic effects between strains when the multi-lactobacillus complex is co-cultured;

[0030] Figure 4 This is a diagram showing the inhibitory effect of the poly-lactobacillus complex on Candida albicans;

[0031] Figure 5 This is a diagram showing the inhibitory effect of the poly-lactobacillus complex on Candida glabrata;

[0032] Figure 6 It is a diagram of hemolysis experiment of Lactobacillus strains, A is the LSA-858 strain, B is the LSA-946 strain, C is the LSA-1012 strain, and D is the LSA-1066 strain;

[0033] Figure 7 This is the detection of Candida albicans and Lactobacillus counts in the rat vagina in Example 7 of the present invention. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0035] If no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the field or the product instructions are used. All reagents or instruments without manufacturer specified are conventional products that can be purchased commercially; the quantitative tests in the following examples were repeated three times, and the results were averaged.

[0036] The culture medium preparation and PCR method involved in the embodiment are as follows:

[0037] MRS broth / agar medium: weigh 24.15 g of MRS broth powder, add 500 mL of deionized water, stir to dissolve and adjust the pH to 6.2±0.2; for the preparation of agar medium, add 1.5~2% (W / V) agar content and sterilize at 121°C for 15 min.

[0038] YPD liquid culture medium: Weigh 17.50 g of YPD powder, add 500 mL of deionized water, stir to dissolve, and sterilize by high pressure at 121 °C for 20 min.

[0039] Blood agar plates: purchased from Beekman Biotechnology.

[0040] The sources or preparation methods of the control microorganisms and pathogenic microorganisms involved in the examples are as follows:

[0041] Lactobacillus delbrueckii DM8909: Purchase the "Dingjunsheng" drug from a very large pharmacy store, dip the bacterial powder into the MRS broth medium with an inoculation loop, and place it in a 37 ℃ incubator for anaerobically cultured for 24 hours. The bacterial liquid was streaked and purified, and cultured anaerobically at 37 ℃ for 48 hours. A single colony was picked for liquid enrichment culture, and the bacterial liquid was obtained for 16S rDNA sequence identification, which was identified as Lactobacillus delbrueckii (sequence as SEQ ID No. 5), and the bacterial liquid was preserved and frozen in a -80 ℃ refrigerator.

[0042] Candida albicans a: purchased from China General Microbiological Culture Collection Center, numbered CGMCC 2.4159.

[0043] Candida albicans b: isolated from vaginal secretions of VVC patients (provided by the First Affiliated Hospital of Jinan University), numbered LSJ-142 (ITS sequence as SEQ ID No. 6).

[0044] Candida albicans c: isolated from vaginal secretions of VVC patients (provided by the First Affiliated Hospital of Jinan University), numbered LSJ-143 (ITS sequence as SEQ ID No. 7).

[0045] Candida glabrata: isolated from a VVC patient and provided by the laboratory doctor of the First Affiliated Hospital of Jinan University in Guangzhou, numbered LSJ-160 (ITS sequence as SEQ ID No. 8).

[0046] Example 1: Isolation and identification of lactobacilli

[0047] 1. Sample Collection and Separation

[0048] The isolated samples were all from the vaginal secretions of healthy women. Vaginal secretion samples of women of childbearing age who had passed the health examination were collected with vaginal swabs and placed in ESwab transport preservation solution (Copan Company) and fully shaken and mixed. The samples were diluted in gradients, spread on MRS agar medium, and placed in a 37 ℃ incubator for anaerobically cultured for 48 h. After obvious colonies grew, strains with typical characteristics of lactic acid bacteria such as white round edges, neat edges, and shiny surfaces were selected, and streaked and cultured on MRS plates one by one to obtain pure culture strains. Finally, four strains with high lactic acid and hydrogen peroxide production capabilities were isolated, and their naming numbers were LSA-858, LSA-946, LSA-1012, and LSA-1066.

[0049] 2. Strain preservation and identification

[0050] The isolated strain was cultured in liquid MRS medium for 24 h, and the bacterial solution was transferred to a cryovial containing pure glycerol with a final glycerol concentration of 20-30%, and stored at -80°C.

[0051] 1 mL of bacterial solution was used to extract DNA, and the DNA was used as a template to amplify the conserved region of the 16S rDNA gene of the strain using primers 27F and 1492R. The 16S rDNA amplification product was obtained and then subjected to electrophoresis detection, purification and sequencing. By sequence comparison, the strain LSA-858 was identified as Lactobacillus crispatus ( Lactobacillus crispatus ), the homology score is 100.00%, and its 16S rDNA sequence is shown in SEQ ID No.1; LSA-946 strain is Lactobacillus jensenii ( Lactobacillus jensenii ), the homology score is 100.00%, and its 16S rDNA sequence is shown in SEQ ID No.2; LSA-1012 strain is Lactobacillus johnsonii ( Lactobacillus johnsonii ), the homology score is 100.00%, and its 16S rDNA sequence is shown in SEQ ID No.3; LSA-1066 strain is Lactobacillus reuteri ( Limosilactobacillus reuteri ; formerly known as Lactobacillus reuteri ), the homology score is 100.00%, and its 16S rDNA sequence is shown in SEQ ID No.4.

[0052] The PCR program and primers involved in the embodiment are as follows:

[0053] PCR system (25 μL): 12.5 μL Taq PCR Master Mix buffer (including Taq DNA polymerase, dNTP, MgCl2, PCR buffer, PCR reaction stabilizer, loading buffer and bromophenol blue dye), 1 μL of 10 μmol / L upstream and downstream primers, 1 μL DNA template, and add nuclease-free water to 25 μL. PCR conditions: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 90 s, a total of 35 cycles; 72 ℃ extension for 10 min.

[0054] Bacterial universal primers:

[0055] Upstream primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0056] Downstream primer 1492R: 5′-TACGGTTACCTTGTTACGACTT-3′.

[0057] Primers specific for Lactobacillus crispatus:

[0058] Upstream primer Lcris-F2: 5′-GATTTACTTCGGTAATGACGTTAGGA-3′;

[0059] Downstream primer Lcris-R2: 5′-AGCTGATCATGCGATCTGCTTTC-3′.

[0060] Lactobacillus jensenii specific primers:

[0061] Upstream primer Ljens-F: 5′-AAGTCGAGCGAGCTTGCCTATAGA-3′;

[0062] Downstream primer Ljens-R: 5′-CTTCTTTCATGCGAAAGTAGC-3′.

[0063] Lactobacillus johnsonii specific primers:

[0064] Upstream primer Ljohn-F: 5′-TCGAGCGAGCTTGCCTAGATGA-3′;

[0065] Downstream primer Ljohn-R: 5′-TCCGGACAACGCTTGCCACC-3′.

[0066] Lactobacillus reuteri specific primers:

[0067] Upstream primer Lreute-F2: 5′-CCCTTTCTTTCAGATCCACAGG-3′;

[0068] Downstream primer Lreute-R2: 5′-TGGTTGTAGAGAAGGGGACG-3′.

[0069] Example 2: Strain culture characteristics

[0070] 1. Morphological characteristics of each single bacteria

[0071] The activated LSA-858, LSA-946, LSA-1012 and LSA-1066 strains were streaked onto MRS plates and cultured anaerobically at 37 °C for 48 h before observing the colony morphology. Single colonies on the plates were picked for Gram staining and the strain characteristics were observed under a microscope.

[0072] Colony morphology and strain characteristics of LSA-858 strain Figure 1 A and Figure 2 As shown in A: The colonies appear as grayish-white round colonies with a diameter of about 1-2 mm, opaque, and a protrusion in the middle. The colonies have neat edges, are relatively moist, and are stable in size and shape. The strain is stained as a Gram-positive bacterium, and the bacteria are long rod-shaped.

[0073] Colony morphology and strain characteristics of LSA-946 strain Figure 1 B and Figure 2 As shown in B: The colonies appear as milky white round colonies with a diameter of about 0.5-1.5 mm, opaque, and a protrusion in the middle. The colonies have neat edges, are relatively moist, and are stable in size and shape. The strain is stained as a Gram-positive bacterium, and the bacteria are long rod-shaped.

[0074] Colony morphology and strain characteristics of LSA-1012 strain Figure 1 C and Figure 2 As shown in C: The colonies appear as milky white round colonies with a diameter of about 1-2 mm, opaque, and a protrusion in the middle. The colony edges are neat and moist, and the size and shape are stable. The strain is stained as Gram-positive bacteria, and the bacteria are long rod-shaped.

[0075] Colony morphology and strain characteristics of LSA-1066 Figure 1 D and Figure 2 As shown in D: The colonies appear as milky white round colonies with a diameter of about 1-2 mm, opaque, and a protrusion in the middle. The edges of the colonies are neat and moist, and the size and shape are stable. The strain is stained as Gram-positive bacteria, and the bacteria are long rod-shaped.

[0076] Example 3: Comparison of lactic acid production capacity, hydrogen peroxide capacity, fermentation capacity and adhesion capacity

[0077] 1. Comparative determination of lactic acid (D-lactic acid & L-lactic acid) production capacity

[0078] Single bacteria: Lactobacillus Germani DM8909 was used as the control group. Each lactobacillus strain was activated twice, centrifuged, the supernatant was discarded, the bacteria were resuspended and the turbidity of the liquid was adjusted to an appropriate turbidity (~10 8 CFU / mL), the bacterial suspension was inoculated into MRS liquid culture medium (inoculation volume was 6% v / v).

[0079] Lactobacillus multifilament complex: centrifuge the activated LSA-858, LSA-946, LSA-1012 and LSA-1066 strains, discard the supernatant, and resuspend the cells in PBS solution until the turbidity is ~10 8 CFU / mL), three or four strains are selected for equal ratio inoculation into MRS liquid culture medium (total inoculation volume is 6% v / v), and the mixture is obtained after anaerobic culture at 37 °C for 24 h. The multi-linked lactobacillus complex has 5 combinations, namely, group A consisting of LSA-858 strain, LSA-946 strain and LSA-1012 strain, group B consisting of LSA-858 strain, LSA-946 strain and LSA-1066 strain, group C consisting of LSA-858 strain, LSA-1012 strain and LSA-1066 strain, group D consisting of LSA-946 strain, LSA-1012 strain and LSA-1066 strain, and group E consisting of LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain.

[0080] After anaerobic culture at 37 °C for 24 h, the bacterial solution was centrifuged and the supernatant was taken. The content of D-lactic acid and L-lactic acid in the supernatant was detected by high performance liquid chromatography. The specific chromatographic conditions are shown in Table 1.

[0081] Table 1 Chromatographic conditions for determination of lactic acid content in Lactobacillus fermentation broth by HPLC

[0082]

[0083] 2. Comparison of H2O2 production capacity

[0084] After the activated single bacteria and multi-linked lactobacillus complex were cultured anaerobically at 37°C for 24 h, the bacterial solution was centrifuged, and the supernatant was taken. The H2O2 content in the supernatant was detected by high performance liquid chromatography. The specific chromatographic conditions are shown in Table 2:

[0085] Table 2 Chromatographic conditions for determination of H2O2 content in lactobacillus fermentation broth by HPLC

[0086]

[0087] 3. Comparison of fermentation capacity

[0088] After anaerobic culture at 37 °C for 24 h, the bacterial solution was centrifuged and the pH of the supernatant was measured with a pH meter.

[0089] 4. Comparison of Adhesion Ability to Hela Cells

[0090] Hela cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in T75 cell culture flasks at a concentration of 6 × 10 4 2 mL was inoculated in a 6-well plate at a density of cells / mL, and a single cell layer was formed after 48 h of culture. Single bacteria and multi-lactobacillus complex were used as experimental groups, and Lactobacillus Germani DM8909 was used as the reference group. After centrifugation of each group of culture fluid to remove the supernatant, PBS was resuspended, the turbidity of the bacterial solution was adjusted to 1.0 MCF, the supernatant was removed by centrifugation, and the bacteria were resuspended with an equal volume of DMEM high-glucose medium. The bacterial suspension was diluted and plated on MRS agar. After anaerobic culture for 48 h, the number of clones on each plate was counted.

[0091] Before the adhesion experiment, the cell culture medium was aspirated, and each well was washed with PBS solution, and then 2 mL of bacterial suspension was added. The cell culture plate and the remaining bacterial suspension were placed in a 37 °C, 5% CO2 incubator for adhesion for 2 h. After the adhesion was completed, the culture medium in the cell culture plate was aspirated, and each well was washed with PBS solution, and then trypsin-EDTA solution was added to digest for 150 s, and the digestion solution was aspirated. Then PBS solution was added to blow the cells to detach them and TritonX-100 was added to lyse the cells to make a bacterial suspension, which was graded diluted, and 100 μL of bacterial solution was spread on MRS agar. After anaerobic culture for 48 h, the number of clones on each plate was counted.

[0092] After the comparative experiments of the above embodiments, the comparative results are shown in Table 3 below:

[0093] Table 3 Comparison of the probiotic ability of each single bacteria and multi-lactobacillus complex:

[0094]

[0095] Note: “ND” means not produced.

[0096] Example 4: Detection of antagonistic and symbiotic effects between strains in a polyvalent lactobacillus complex

[0097] The LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain activated twice were discarded, and the supernatant was resuspended in PBS solution to an appropriate turbidity (~10 8CFU / mL) for later use, three or more strains were mixed and evenly inoculated into MRS liquid medium (total inoculation volume was 6% v / v) in equal proportions, and the strains composed of LSA-858, LSA-946 and LSA-1012 were recorded as group A, the strains composed of LSA-858, LSA-946 and LSA-1066 were recorded as group B, the strains composed of LSA-858, LSA-1012 and LSA-1066 were recorded as group C, the strains composed of LSA-946, LSA-1012 and LSA-1066 were recorded as group D, and the strains composed of LSA-858, LSA-946, LSA-1012 and LSA-1066 were recorded as group E. The culture was carried out at 37 ℃ for 24 h, and 1 mL of bacterial solution was taken from each group to extract DNA. The qPCR method was used to quantitatively detect the gene copy number of a single bacterium in each group of multi-lactobacillus complexes to determine whether there was antagonism between lactobacillus strains.

[0098] The fluorescence quantitative PCR system is shown in Table 4:

[0099] Table 4: Fluorescence quantitative PCR method

[0100]

[0101] The results are as follows Figure 3 As shown: Overall, the growth of each single bacteria in each group of multi-lactobacillus complexes is close, there is no obvious inhibition between each other, they can coexist with each other, and there is no mutual antagonism; among them, the growth of LSA-858 strain and LSA-946 strain is relatively dominant.

[0102] Example 5: In vitro inhibition of vulvovaginal candidiasis pathogen detection

[0103] The strains LSA-858, LSA-946, LSA-1012, LSA-1066 and Lactobacillus polysaccharide complex A to E were used as experimental groups; Lactobacillus germanium DM8909 was used as the control group. Each experimental group was cultured anaerobically at 37°C for 24 h, and then the bacterial solution was centrifuged. The supernatant was filtered through a 0.22 μm filter membrane and 100 μL was taken out and placed in a 96-well plate for later use.

[0104] The activated pathogens (Candida albicans, Candida glabrata) were centrifuged, the supernatant was discarded, and the cells were resuspended in PBS solution until the turbidity (~10 8CFU / mL), inoculated into YPD liquid medium (6% v / v), where the mixed Candida albicans sample was inoculated into YPD liquid medium (total inoculation volume was 6% v / v) by mixing a variety of Candida albicans (Candida albicans CGMCC2.4159, Candida albicans LSJ-142, Candida albicans LSJ-142) in equal proportions, and then 100 μL of YPD medium containing pathogens was added to 96-well plates containing 100 μL of supernatant from each experimental group and control group, and another 100 μL of YPD medium containing pathogens was added to a 96-well plate containing 100 μL of pure MRS medium as a positive control, and 100 μL of pure YPD medium without pathogens was added to a 96-well plate containing 100 μL of pure MRS medium as a negative control. After adding, the 96-well plate was placed in an incubator and cultured for 48 h. The growth of the pathogen was determined by measuring the OD values ​​of the control group and each experimental group after 0 h and 48 h of culture, and then the ability of single bacteria and composite bacteria to inhibit VVC pathogens was determined.

[0105] Experimental results: After 48 h of culture, the OD value of the white mixed Candida albicans in the control group can reach about 1.50. Figure 4 As shown; after culturing Candida glabrata for 48 h, the OD value can reach about 1.45. Figure 5 shown.

[0106] Single bacteria antibacterial ability: After the supernatant of Lactobacillus delbrueckii DM8909 was co-cultured with mixed Candida albicans for 48 hours, Candida albicans could still grow, and the OD value could be stabilized to about 0.8, and the inhibitory ability was not obvious. After co-cultured with Candida glabrata for 48 hours, Candida glabrata could still grow, and the OD value could be stabilized to about 1.0, showing only slight inhibitory ability. After each single bacteria was co-cultured with mixed Candida albicans for 48 hours, Candida albicans in the LSA-946 strain and LSA-1012 strain groups almost did not grow, showing strong antibacterial activity; the OD value of Candida albicans in the LSA-858 strain group was stabilized to about 0.5; the OD value of Candida albicans in the LSA-1066 group was stabilized to about 0.6, showing certain antibacterial activity. After each single bacteria was co-cultured with Candida glabrata for 48 hours, the OD value of Candida glabrata was between 0.8 and 1.0, showing only slight antibacterial ability, and there was no significant difference with Lactobacillus delbrueckii.

[0107] Antibacterial ability of the poly-lactobacillus complex: After each poly-lactobacillus complex was co-cultured with Candida albicans, Candida albicans basically had no growth, and had a strong inhibitory ability against Candida albicans; after the supernatant was co-cultured with Candida glabrata for 48 h, the OD value of Candida glabrata was between 0.4 and 0.5, which had a significant inhibitory effect, significantly stronger than the strain of Dingjunsheng microecological preparation. Unexpectedly, combined with the growth data analysis of Example 4, when the growth of LSA-858 strain and LSA-946 strain was relatively dominant, the inhibitory effect of each group of poly-lactobacillus complex on Candida glabrata was improved, which was better than any single bacteria.

[0108] Example 6: Lactobacillus in vitro safety testing

[0109] 1. Hemolyticity test of strains

[0110] Staphylococcus aureus CMCC(B)26003 (purchased from Guangdong Huankai Microbiology Co., Ltd.) was used as the positive control group. The control group and each lactobacillus were inoculated on blood agar plates and cultured anaerobically at 37°C for 48 h. The hemolysis of the colonies was observed.

[0111] The experimental results are as follows Figure 5 As shown: Staphylococcus aureus CMCC (B) 26003 has a very obvious hemolytic ring, while the lactobacillus strains in the present invention have no hemolytic ring, are non-pathogenic, and are safe and reliable.

[0112] Example 7: Therapeutic ability against vulvovaginal candidiasis in rats infected with Candida albicans

[0113] The rat model of infection was established using SD female rats, 7 weeks old, weighing 180-200 g. The experimental animals were divided into 9 groups, namely, healthy group, model group, clotrimazole group, Lactobacillus delbrueckii group, and complex A-E groups. Each multi-lactobacillus complex group was prepared by mixing each single bacteria in equal proportion (v / v), with a turbidity of 1.0×10 8 CFU / mL, the Lactobacillus delbrueckii group was made from DM8909 bacterial suspension, and the turbidity was 1.0×10 8 CFU / mL, 8 mice in each group, a total of 72 mice, all fed with standard diet.

[0114] The experiment was divided into an adaptation period, a modeling period, an intervention period, and an observation period. During the adaptation period, all animals were adaptively raised in the animal laboratory for one week. The ovaries of female animals were removed in advance to artificially exclude the influence of endogenous estrogen on the experiment. Seven days after the ovariectomy, 0.5 mg of estradiol benzoate was subcutaneously injected every other day to make the animals in a pseudo-estrus. Before modeling, the phenotypic characteristics of the rats' vagina, such as redness, swelling, and discharge, were observed and recorded.

[0115] Modeling stage: Candida albicans turbidity was adjusted to 1.0×108 CFU / mL, 1 mL was taken to flush the rat vagina, and the healthy group was flushed with normal saline; once a day for 3 consecutive days, and the rat vagina was flushed and sampled with sterile PBS on the 4th day. The yeast hyphae observed under the microscope, Gram staining of vaginal secretions, and appearance inspection of redness, swelling and inflammation were used to confirm whether the infection of Candida albicans was successful. Intervention period: a total of 7 days, the rats in the healthy group and the model group were not treated; 1 mL of clotrimazole solution (8 μg / mL) was injected into the vagina of the rats in the clotrimazole group; 1 mL of Lactobacillus solution of each group was injected into the vagina of the rats in each probiotic group, once a day, for 7 consecutive days. Observation period: during the intervention period and 3 days after the end of treatment, the phenotypic characteristics of the rat vagina, such as redness, swelling and discharge, were observed and recorded every day. While observing the phenotype of the rats each time, the vaginal washing fluid of the rats was taken to detect the colony counts of Candida albicans and Lactobacillus, respectively.

[0116] Table 5 Observation and statistics of rat vulvar swelling and secretions 3 days after treatment

[0117]

[0118] The experimental results are shown in Table 5 and Figure 7 As shown in the results, after the rats infected with Candida albicans were treated with the multi-lactobacillus complex, the number of Candida albicans in the body decreased significantly, approaching the level of the healthy group, and the treatment effect was equivalent to that of clotrimazole. After treatment, the vulva and secretions of the rats were normal, with no adverse reactions, while the treatment effect of the Lactobacillus delbrueckii group was not obvious. In the clotrimazole treatment group, while killing Candida albicans, the abundance of Lactobacillus in the rat vagina was also reduced accordingly, and the normal microecology in the rat vagina could not be restored, which was easy to cause VVC recurrence or infection by other pathogens; in the multi-lactobacillus complex group, the number of Lactobacillus in the rat vagina returned to normal and had an increasing trend, and it could be well colonized in the vagina, reducing the recurrence of VVC.

[0119] In summary, strains LSA-858, LSA-946, LSA-1012, and LSA-1066 have the ability to produce beneficial secretions such as lactic acid and hydrogen peroxide; can tolerate the acidic environment of the vagina well; have a certain adhesion ability to vaginal cervical cells; are non-hemolytic and have good safety; and have a certain inhibitory effect on common pathogens of VVC. The multi-lactobacillus complex composed of any three combinations has stronger probiotic properties than single bacteria; there is no antagonistic effect between the strains and they grow well; the therapeutic effect on the VVC animal disease model is obvious, and it can be used as a potential development drug for the treatment of vulvovaginal candidiasis.

[0120] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A strain of Lactobacillus crispatus, characterized in that: The Lactobacillus crispatus is named as LSA-858 strain, and the LSA-858 strain is Lactobacillus crispatus ( Lactobacillus crispatus ), deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No.62779 and the deposit date being September 9, 2022.

2. A strain of Lactobacillus jensenii, characterized in that The Lactobacillus jensenii is named as LSA-946 strain, and the LSA-946 strain is Lactobacillus jensenii ( Lactobacillus jensenii ), deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No.62781 and the deposit date being September 9, 2022.

3. A strain of Lactobacillus johnsonii, characterized in that The Lactobacillus johnsonii is named as LSA-1012 strain, and the LSA-1012 strain is Lactobacillus johnsonii ( Lactobacillus johnsonii ), deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No.62782 and the deposit date being September 9, 2022.

4. A strain of Lactobacillus reuteri, characterized in that The Lactobacillus reuteri strain is named as LSA-1066 strain, and the LSA-1066 strain is Lactobacillus reuteri strain ( Limosilactobacillus reuteri ), deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No.62783 and the deposit date of September 9, 2022.

5. A multi-lactobacillus complex, characterized in that: The method comprises at least three of the Lactobacillus crispatus LSA-858 strain as claimed in claim 1, the Lactobacillus jensenii LSA-946 strain as claimed in claim 2, the Lactobacillus johnsonii LSA-1012 strain as claimed in claim 3, or the Lactobacillus reuteri LSA-1066 strain as claimed in claim 4.

6. A preparation, characterized in that Include at least one of the following (1) to (3): (1) containing the multi-lactobacillus complex according to claim 5; (2) A live bacterial liquid containing the multi-lactobacillus complex of claim 5; (3) A dead bacteria solution containing the multi-lactobacillus complex according to claim 5.

7. Use of the multi-lactobacillus complex according to claim 5 or the preparation according to claim 6 in the preparation of medicines or sanitary products for preventing or treating gynecological diseases.

8. The use according to claim 7, characterized in that: The gynecological disease includes at least one of vaginal microecological flora imbalance, vulvovaginal candidiasis or bacterial vaginosis.

9. Use of the multi-lactobacillus complex according to claim 5 or the preparation according to claim 6 in the preparation of an antibacterial product.

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