CN116218708A

CN116218708A - Lactobacillus, multi-lactobacillus complex, preparation and application thereof

Info

Publication number: CN116218708A
Application number: CN202211606071.7A
Authority: CN
Inventors: 王梅; 崔鸿亮; 宾金梅; 梁姝婕; 欧景森
Original assignee: Guangdong Junwei Biotechnology Co ltd
Current assignee: Guangdong Junwei Biotechnology Co ltd
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-06-06

Abstract

The invention discloses lactobacillus, a lactobacillus polynicotinus compound, a preparation and application thereof. The lactobacillus comprises LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain; the LSA-858 strain deposit number is GDMCC No.62779; the LSA-946 strain is deposited with the GDMCCNo.62781; the LSA-1012 strain is deposited with the number GDMCC No.62782; the LSA-1066 strain is deposited with the GDMCC No.62783. The Lactobacillus reuteri complex includes at least three of the strain LSA-858, the strain LSA-946, the strain LSA-1012, or the strain LSA-1066. The Lactobacillus deltoidea complex has a better VVC pathogen inhibition effect than any of the Lactobacillus deltoidea single bacteria.

Description

Lactobacillus, multi-lactobacillus complex, preparation and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a plurality of lactobacillus, a compound prepared from the lactobacillus, a preparation and application thereof.

Background

Vulvovaginal candidiasis (vulvovaginal candidiasis, VVC), also known as colpitis mycotica, vulvovaginal candidiasis, and the like, is one of the most common vaginal infection diseases of women, and has pathogenic bacteria mainly including candida albicans (90% of VVC patients), and a minority of candida glabrata, candida tropicalis, candida parapsilosis, and the like. Clinically common symptoms include increased leucorrhea, burning pain and itching of the vagina and vulva, and if not treated in time, serious complications of upper genital tracts, such as endometritis, salpingitis and pelvic inflammatory disease, can be caused, and further worsening can lead to salpingemphraxis, infertility or ectopic pregnancy.

Studies at home and abroad show that about 70% -75% of women experience VVC at least once in their lifetime, 50% of women will experience the attack of the disease many times in their lifetime, and 5% -10% of women are likely to develop recurrent VVC (Recurrent Vulvovaginal Candidiasis, RVVC). At present, the main clinical treatment means for vulvovaginal candida diseases is antibiotic treatment, and pathogenic bacteria such as candida albicans are killed by oral administration or local use of azole antifungal medicaments, so as to achieve the treatment purpose. The common antibiotics are fluconazole, miconazole, clotrimazole, etc. However, due to the irregular clinical diagnosis and excessive use of antifungal drugs, the intractable nature of vulvovaginal candidiasis is further exacerbated, and the vaginal flora balance is broken, thereby causing drug resistance and recurrence.

The national gynaecology and obstetrics cooperative group proposes that the vaginal microecology is recovered to be the ultimate target of colpitis treatment, and proposes the microecology treatment concept, and the application of the vaginal microecology preparation recovers the weak acidic environment mainly containing functional lactobacillus. The vaginal flora of healthy women is mainly composed of lactobacilli, including lactobacillus crispatus, lactobacillus grignard, lactobacillus jensenii, etc. The lactobacillus can produce lactic acid, hydrogen peroxide, antibacterial peptide and the like, can resist invasion of pathogenic bacteria and inhibit growth of the pathogenic bacteria, and is a potential new-generation medicament for treating colpitis. However, there are only 2 vaginal microecologics currently marketed in China, namely, a commercial medicine named "Yanhua" containing streptococcus enteriae and a commercial medicine named "dingjunsheng" containing lactobacillus delbrueckii, and the preparations are single strain preparations for treating bacterial vaginitis, and no microecologics for treating VVC exist temporarily.

Clinical symptoms of VVC patients are easy to repeatedly attack, difficult to radically cure, and seriously affect the life quality and physical and mental health of the patients, and are key problems which are urgently needed to be solved by clinicians at present. Therefore, development of a microecological preparation for VVC therapy is needed and necessary.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. For this purpose, the invention provides a lactobacillus polymyxa complex and further provides the application of the strain.

In a first aspect of the invention there is provided a strain of Lactobacillus crispatus. The Lactobacillus crispatus is named as LSA-858 strain, the LSA-858 strain is Lactobacillus crispatus (Lactobacillus crispatus) and is deposited with the Guangdong province microorganism strain collection center with the deposit number of GDMCC No.62779 and the deposit date of 2022, 9 months and 9 days. The LSA-858 strain has a 16S rDNA gene fragment with a sequence shown as SEQ ID No. 1.

In a second aspect the invention provides a strain of lactobacillus jannaschii. The Lactobacillus jensenii is named as LSA-946 strain, the LSA-946 strain is Lactobacillus jensenii (Lactobacillus jensenii) and is preserved in the Guangdong province microorganism strain collection, the preservation number is GDMCC No.62781, and the preservation date is 2022, 9 and 9. The LSA-946 strain has a 16S rDNA gene fragment with a sequence shown as SEQ ID No. 2.

In a third aspect of the invention there is provided a strain of Lactobacillus johnsonii. The Lactobacillus johnsonii is named as LSA-1012 strain, the LSA-1012 strain is Lactobacillus johnsonii (Lactobacillus johnsonii) and is deposited with the Guangdong province microorganism strain collection, the deposition number is GDMCC No.62782, and the deposition date is 2022, 9 months and 9 days. The LSA-1012 strain has a 16S rDNA gene fragment with a sequence shown as SEQ ID No. 3.

In a fourth aspect of the invention there is provided a strain of lactobacillus reuteri. The Lactobacillus reuteri is named as LSA-1066 strain, the LSA-1066 strain is Lactobacillus reuteri (Limosilactobacillus reuteri) and is deposited in the Guangdong province microorganism strain collection center, the deposition number is GDMCC No.62783, and the deposition date is 2022, 9 months and 9 days. The LSA-1066 strain has a 16S rDNA gene fragment with a sequence shown as SEQ ID No. 4.

The four lactobacillus strains have higher acid production capacity and hydrogen peroxide production capacity, and have excellent antibacterial effect on various female colpitis pathogenic bacteria.

In a fifth aspect of the invention there is provided a Lactobacillus multiplex complex. The Lactobacillus reuteri complex comprises at least three of strain LSA-858, strain LSA-946, strain LSA-1012 or strain LSA-1066. During the test, the lactobacillus deltoidea compound is found to have better VCC pathogenic bacteria inhibition effect compared with any single bacteria.

In a sixth aspect of the present invention, there is provided a formulation comprising at least one of the following (1) to (5):

(1) The lactobacillus polymyxa compound is contained, and thalli in the lactobacillus polymyxa compound can be active freeze-dried bacteria powder or inactive freeze-dried bacteria powder;

(2) A live bacterial liquid containing the Lactobacillus deltoidea complex;

(3) A dead bacterial liquid containing the lactobacillus polymyxa complex;

(4) A metabolite obtained from the metabolic pathway of the lactobacillus multiplex complex;

(5) An extract obtained by extraction from the Lactobacillus deltoidea complex.

In a seventh aspect, the invention provides the use of a lactobacillus reuteri complex or formulation as described above in the manufacture of a medicament, food or sanitary product for the prophylaxis or treatment of gynaecological disorders.

In some embodiments of the invention, the gynecological disease comprises at least one of vaginal dysbiosis, vulvovaginal candidiasis, bacterial vaginosis, HPV, or urinary tract infection.

In some embodiments of the invention, the pharmaceutical formulation is one of a powder, an ointment, a gel, a lozenge, a granule, a capsule, a spray, a tablet, a pill, or an oral liquid.

In some embodiments of the invention, the pharmaceutical product further comprises a pharmaceutical adjuvant comprising at least one of a diluent, excipient, filler, binder, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, sweetener, and flavoring agent.

In some embodiments of the invention, the sanitary product is one of a tampon, a sanitary towel, a catamenial pad, a diaper, a soap, and a condom.

In an eighth aspect of the invention there is provided the use of a lactobacillus reuteri complex or formulation as described above in the preparation of a bacteriostatic product.

In some embodiments of the invention, the pathogenic bacteria inhibited by the bacteriostatic product comprises at least one of candida albicans, candida glabrata, candida tropicalis, gardnerella vaginalis, escherichia coli, staphylococcus aureus, or pseudomonas aeruginosa.

In a ninth aspect, the invention provides the use of a lactobacillus reuteri complex or formulation as described above in the preparation of a delivery vehicle.

Drawings

FIG. 1 is a diagram of the colony morphology of Lactobacillus, A being the strain LSA-858, B being the strain LSA-946, C being the strain LSA-1012, D being the strain LSA-1066;

FIG. 2 is a gram of Lactobacillus with A being the strain LSA-858, B being the strain LSA-946, C being the strain LSA-1012, and D being the strain LSA-1066;

FIG. 3 is a graph showing the results of detection of antagonism and symbiotic effects between strains during co-cultivation of the Lactobacillus deltoides complex;

FIG. 4 is a graph showing the inhibitory effect of the Lactobacillus delbrueckii complex on Candida albicans;

FIG. 5 is a graph showing the inhibitory effect of the Lactobacillus deltoidea complex on Candida parapsilosis;

FIG. 6 is a graph of a hemolysis experiment of a Lactobacillus strain, 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;

FIG. 7 shows the count of candida albicans and lactobacillus in the vagina of a rat in example 7 of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications. All reagents or instruments are commercially available conventional products, with no manufacturer noted; the quantitative tests in the following examples were all set up in triplicate and the results averaged.

The preparation of the medium and the PCR method involved in the examples are as follows:

MRS broth/agar medium: weighing 24.15g of MRS broth powder, adding 500mL of deionized water, stirring and dissolving, and regulating the pH to 6.2+/-0.2; the agar culture medium is prepared by adding 1.5-2% (W/V) agar content, and sterilizing at 121deg.C for 15 min.

YPD liquid medium: 17.50g of YPD powder is weighed, 500mL of deionized water is added, and the mixture is stirred and dissolved and then autoclaved at 121 ℃ for 20min, thus obtaining the YPD powder.

Blood agar plates: purchased from bikmann biosystems.

The sources or methods of preparation of the control microorganisms and pathogenic microorganisms referred to in the examples are as follows:

lactobacillus delbrueckii DM8909: the "Dingjunsheng" medicine was purchased from a very large pharmacy store, dipped with fungus powder in MRS broth medium with an inoculating loop, and placed in an incubator at 37℃for anaerobic cultivation for 24 hours. And (3) streaking and purifying bacterial liquid, carrying out anaerobic culture for 48 hours at 37 ℃, picking single bacterial colony, carrying out liquid enrichment culture, obtaining bacterial liquid, carrying out 16S rDNA sequence identification, obtaining lactobacillus delbrueckii (with the sequence shown as SEQ ID No. 5) through identification, preserving the bacterial liquid, and freezing the bacterial liquid at-80 ℃ in a refrigerator.

Candida albicans a: the strain is purchased from China general microbiological culture collection center (CGMCC) 2.4159.

Candida albicans b: vaginal secretion isolated from VVC patients (supplied by the first affiliated hospital of university of south and south China) was numbered LSJ-142 (ITS sequence shown in SEQ ID No. 6).

Candida albicans c: vaginal secretion isolated from VVC patients (supplied by the first affiliated hospital of university of south and south China) was numbered LSJ-143 (ITS sequence shown in SEQ ID No. 7).

Candida glabrata: isolated from VVC patients, provided by the first affiliated hospital clinical laboratory doctor of Guangzhou and south university under the designation LSJ-160 (ITS sequence shown in SEQ ID No. 8).

Example 1: isolation and identification of Lactobacillus

1. Sample collection and separation

The isolated samples are all from vaginal secretions of healthy females, vaginal swabs are used for collecting vaginal secretion samples of women in the childbearing period passing through the physical examination, and the vaginal secretion samples are placed in ESwab transportation preservation solution (Copan company) for full shaking and uniform mixing. Sampling, gradient diluting, coating on MRS agar culture medium, and anaerobic culturing in 37 deg.C incubator for 48 hr. After obvious colonies grow, strains with the typical characteristics of lactic acid bacteria such as white round edges, regular edges, glossy surfaces and the like in the form of growing colonies are selected, and streak culture separation is carried out on an MRS flat plate one by one to obtain pure culture strains. Finally, four strains with high lactic acid and hydrogen peroxide production capacity are separated, and the serial numbers of the strains are respectively LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain.

2. Strain preservation and identification

Culturing the obtained isolated strain in liquid MRS culture medium for 24 hr, collecting bacterial liquid, adding into freezing tube containing pure glycerol with final glycerol concentration of 20-30%, and storing at-80deg.C.

1mL of bacterial liquid is taken to extract DNA, and 27F and 1492R primers are adopted to amplify a 16S rDNA gene conservation region of the strain to obtain a 16S rDNA amplified product, and electrophoresis detection, purification and sequencing are carried out. By sequence alignment, the LSA-858 strain is determined to be lactobacillus crispatus (Lactobacillus crispatus), the homology score is 100.00%, and the 16S rDNA sequence is shown as SEQ ID No. 1; the LSA-946 strain is Lactobacillus jensenii (Lactobacillus jensenii), the homology score is 100.00%, and the 16S rDNA sequence is shown as SEQ ID No. 2; the LSA-1012 strain is Lactobacillus johnsonii (Lactobacillus johnsonii), the homology score is 100.00%, and the 16S rDNA sequence is shown as SEQ ID No. 3; the LSA-1066 strain is Lactobacillus reuteri (Limosilactobacillus reuteri; zehnder name is Lactobacillus reuteri), the homology score is 100.00%, and the 16S rDNA sequence is shown as SEQ ID No. 4.

The PCR procedure and primers involved in the examples are as follows:

PCR System (25. Mu.L): 12.5 mu L Taq PCR Master Mix buffer (containing Taq DNA polymerase, dNTP, mgCl) ₂ PCR buffer, PCR reaction stabilizer, loading buffer and bromophenol blue dye), 1. Mu.L each of 10. Mu. Mol/L upstream and downstream primers, 1. Mu.L DNA template, and 25. Mu.L supplemented with nuclease-free water. PCR conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 90s for 35 cycles; extending at 72℃for 10min.

Bacterial universal primers:

upstream primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3';

downstream primer 1492R:5'-TACGGTTACCTTGTTACGACTT-3'.

Lactobacillus crispatus specific primers:

the upstream primer Lcris-F2:5'-GATTTACTTCGGTAATGACGTTAGGA-3';

the downstream primer Lcris-R2:5'-AGCTGATCATGCGATCTGCTTTC-3'.

Lactobacillus jensenii specific primers:

the upstream primer Ljens-F:5'-AAGTCGAGCGAGCTTGCCTATAGA-3';

downstream primer Ljens-R:5'-CTTCTTTCATGCGAAAGTAGC-3'.

Lactobacillus johnsonii specific primers:

the upstream primer Ljohn-F:5'-TCGAGCGAGCTTGCCTAGATGA-3';

the downstream primer Ljohn-R:5'-TCCGGACAACGCTTGCCACC-3'.

Lactobacillus reuteri specific primers:

the upstream primer Lroute-F2: 5'-CCCTTTCTTTCAGATCCACAGG-3';

the downstream primer Lroute-R2: 5'-TGGTTGTAGAGAAGGGGACG-3'.

Example 2: strain culture characterization

1. Characteristics of the form of each single bacterium

Activated strain LSA-858, strain LSA-946, strain LSA-1012 and strain LSA-1066 were streaked onto MRS plates and colony morphology was observed after anaerobic culture at 37℃for 48h. Single colonies on the plates were picked for gram staining and the strain characteristics were observed with a microscope.

The colony morphology and strain characteristics of LSA-858 strain are shown in fig. 1A and 2A: the bacterial colony is an off-white circular bacterial colony with the diameter of about 1-2mm, opacity and middle protuberance, and the edge of the bacterial colony is neat, moist and stable in size and shape; the strain is dyed into gram positive bacteria, and the thallus is in a long rod shape.

The colony morphology and strain characteristics of LSA-946 strain are shown in fig. 1B and 2B: the bacterial colony is a milky white circular bacterial colony with the diameter of about 0.5-1.5mm, opacity and middle protuberance, the edge of the bacterial colony is neat, moist and stable in size and shape; the strain is dyed into gram positive bacteria, and the thallus is in a long rod shape.

Colony morphology and strain characteristics of LSA-1012 strain are shown in fig. 1C and fig. 2C: the bacterial colony is a milky white circular bacterial colony with the diameter of about 1-2mm, opacity and middle protuberance, and the edge of the bacterial colony is neat and moist, and the size and shape are stable; the strain is dyed into gram positive bacteria, and the thallus is in a long rod shape.

Colony morphology and strain characteristics of LSA-1066 are shown in fig. 1D and fig. 2D: the bacterial colony is a milky white circular bacterial colony with the diameter of about 1-2mm, opacity and middle protuberance, and the edge of the bacterial colony is neat and moist, and the size and shape are stable; the strain is dyed into gram positive bacteria, and the thallus is in a long rod shape.

Example 3: lactic acid production ability, hydrogen peroxide ability, fermentation ability and adhesion ability are compared

1. Comparative measurement of lactic acid production (D-lactic acid & L-lactic acid)

Single bacteria: lactobacillus delbrueckii DM8909 was used as a control, each Lactobacillus strain was activated 2 times, centrifuged, the supernatant was discarded, the cells resuspended and the turbidity of the liquid was adjusted to the appropriate turbidity (-10) ⁸ CFU/mL), inoculating the bacterial suspension into MRS liquid culture medium (inoculum size is 6%v/v)。

Lactobacillus polynucedanum complex: the activated strain LSA-858, strain LSA-946, strain LSA-1012 and strain LSA-1066 were centrifuged and the supernatant was discarded and the bacterial body was resuspended to turbidity (. About.10) with PBS solution ⁸ CFU/mL), three or four strains are selected to be inoculated into an MRS liquid culture medium in an equal ratio (the total inoculum size is 6% v/v), and the strain is obtained after anaerobic culture for 24 hours at 37 ℃. The Lactobacillus multiplex complex has 5 combination modes, namely an A group consisting of an LSA-858 strain, an LSA-946 strain and an LSA-1012 strain, a B group consisting of an LSA-858 strain, an LSA-946 strain and an LSA-1066 strain, a C group consisting of an LSA-858 strain, an LSA-1012 strain and an LSA-1066 strain, a D group consisting of an LSA-946 strain, an LSA-1012 strain and an LSA-1066 strain, and an E group consisting of an LSA-858 strain, an LSA-946 strain, an LSA-1012 strain and an LSA-1066 strain.

After anaerobic culture at 37deg.C for 24 hr, the bacterial liquid was centrifuged, and the supernatant was subjected to high performance liquid chromatography to determine the content of D-lactic acid and L-lactic acid in the supernatant, and the specific chromatographic conditions are shown in Table 1.

TABLE 1 chromatographic conditions for measuring lactic acid content of lactobacillus fermentation broth by high performance liquid chromatography

2. H production ₂ O ₂ Comparison of competence

The activated single bacteria and multi-lactobacillus complex is subjected to anaerobic culture at 37 ℃ for 24 hours, bacterial liquid is centrifuged, supernatant is taken, and H in the supernatant is detected by a high performance liquid phase method ₂ O ₂ The content and specific chromatographic conditions are shown in Table 2:

TABLE 2 high performance liquid phase method for detecting lactobacillus fermentation broth H ₂ O ₂ Chromatographic conditions for content determination

3. Comparative fermentation Capacity

After anaerobic culture at 37℃for 24 hours, the bacterial solution was centrifuged, and the pH of the supernatant was measured by a pH meter.

4. Comparison of Hela cell adhesion Capacity

HeLa cells (purchased from the national academy of sciences cell Bank) were cultured in T75 cell culture flasks at 6X 10 ⁴ cells/mL were seeded at a density of 2mL in 6-well plates and a single cell layer was formed after 48h of incubation. The Lactobacillus delbrueckii DM8909 was used as reference group, with the single and multiple Lactobacillus complexes as experimental groups. After the supernatant is removed by centrifugation of each group of culture solutions, PBS is used for resuspension, the turbidity of the bacterial solution is adjusted to be 1.0MCF, the supernatant is removed by centrifugation, the bacterial cells are resuspended by using an equal volume of DMEM high-sugar culture medium, bacterial suspension is diluted and coated on MRS agar, and after anaerobic culture is performed for 48 hours, the clone number of each plate is counted.

Before the adhesion experiment, the cell culture solution was aspirated, each well was washed with PBS solution and then 2mL of the bacterial suspension was added, and the cell culture plate and the remaining bacterial suspension were placed at 37℃with 5% CO ₂ Adhering in an incubator for 2 hours. After the adhesion is finished, the culture solution in the cell culture plate is sucked out, PBS solution is added into each hole for washing, trypsin-EDTA solution is added for digestion for 150s, the digestion solution is sucked out, PBS solution is added for blowing the cells to separate the cells, triton X-100 is added for lysing the cells to prepare bacterial suspension, the bacterial suspension is diluted in a gradient manner, 100 mu L of bacterial solution is coated on MRS agar, and after anaerobic culture is carried out for 48h, the clone number of each plate is counted.

Through the comparative experiments of the above examples, the comparative results are shown in the following table 3:

table 3 probiotic capacity of each of the single and multiple lactobacillus complexes was compared:

note that: "ND" means non-productive.

Example 4: antagonism and symbiotic effect detection among various strains in multi-linked lactobacillus complex

The LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain, which were activated 2 times, were discarded and the supernatant was resuspended in PBS to an appropriate turbidity (. About.10) ⁸ CFU/mL), mixing 3 or more strains according to an equal ratioThe cells were evenly inoculated into MRS liquid medium (total inoculum size: 6% v/v), designated A group consisting of LSA-858 strain, LSA-946 strain and LSA-1012 strain, designated B group consisting of LSA-858 strain, LSA-946 strain and LSA-1066 strain, designated C group consisting of LSA-858 strain, LSA-1012 strain and LSA-1066 strain, designated D group consisting of LSA-946 strain, LSA-1012 strain and LSA-1066 strain, and designated E group consisting of LSA-858 strain, LSA-946 strain, LSA-1012 strain and LSA-1066 strain. Anaerobic culture was carried out at 37℃for 24 hours, and 1mL of the bacterial liquid was used for each group to extract DNA. Quantitative detection of gene copy number of single bacteria in each group of multi-linked lactobacillus complex is carried out by qPCR method, so as to judge whether antagonism exists between lactobacillus strains.

The fluorescent quantitative PCR system is shown in table 4:

table 4: fluorescent quantitative PCR method

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The results are shown in FIG. 3: in general, the growth amounts of the single bacteria in each group of the multi-linked lactobacillus compound are close, the phenomenon of inhibition is not obvious, the single bacteria and the compound can coexist, and the antagonism is not generated; wherein the LSA-858 strain and the LSA-946 strain grow relatively predominantly.

Example 5: detection of pathogenic bacteria of in vitro inhibition vulvovaginal candida diseases

Taking LSA-858 strain, LSA-946 strain, LSA-1012 strain, LSA-1066 strain and Lactobacillus deluxe complexes A-E as experimental groups; lactobacillus delbrueckii DM8909 was used as a control group. Anaerobic culture is carried out for 24 hours at 37 ℃ on each experimental group, bacterial liquid is taken out, centrifugation is carried out, supernatant fluid is taken out, and 100 mu L to 96 pore plates are respectively taken out for standby after passing through a 0.22 mu m filter membrane.

Centrifuging the activated pathogenic bacteria (Candida albicans, candida glabrata), discarding the supernatant, and adjusting the bacterial weight to turbidity (-10) with PBS solution ⁸ CFU/mL), inoculating into YPD liquid medium (6% v/v), wherein the mixed candida albicans sample is prepared from multiple candida albicans (candida albicans CGMCC2.4159, candida albicans LSJ-142) by equal ratioMixed inoculated into YPD liquid medium (total inoculum size is 6% v/v), 100 μl of YPD medium containing pathogenic bacteria was added to 96-well plates containing 100 μl of supernatants of each of the experimental group and the control group, and another 100 μl of YPD medium containing pathogenic bacteria was added to 96-well plates containing 100 μl of pure MRS medium as positive control, and 100 μl of pure YPD medium containing no pathogenic bacteria was added to 96-well plates containing 100 μl of pure MRS medium as negative control. After the addition, placing the 96-well plate into an incubator for culturing for 48 hours, and judging the growth condition of pathogenic bacteria by measuring OD values after culturing for 0 hour and 48 hours of a control group and each experimental group, thereby judging the capacity of inhibiting VVC pathogenic bacteria of single bacteria and composite bacteria.

Experimental results: after the control group white mixed candida albicans is cultured for 48 hours, the OD value can reach about 1.50, as shown in figure 4; after the candida glabrata is cultured for 48 hours, the OD value can reach about 1.45, as shown in figure 5.

Single bacteria bacteriostasis ability: the candida albicans can still grow after the supernatant of the reference strain lactobacillus delbrueckii DM8909 is co-cultured with the mixed candida albicans for 48 hours, the OD value can be stabilized to about 0.8, the inhibition capacity is not obvious, the candida glabrata can still grow after the reference strain lactobacillus delbrueckii DM8909 is co-cultured with the candida glabrata for 48 hours, and the OD value can be stabilized to about 1.0, so that only slight inhibition capacity is shown. After the single bacteria and the mixed candida albicans are co-cultured for 48 hours, the candida albicans of the LSA-946 strain group and the LSA-1012 strain group hardly grow, and has strong antibacterial property; the OD value of candida albicans in the LSA-858 strain group is stabilized to about 0.5; the candida albicans OD value of the LSA-1066 group is stabilized to about 0.6, and the candida albicans OD value has certain antibacterial property. After the single bacteria and the candida glabrata are co-cultured for 48 hours, the OD value of the candida glabrata is between 0.8 and 1.0, and the candida glabrata has only slight antibacterial capability and has no obvious difference with lactobacillus delbrueckii.

Bacteriostasis ability of the Lactobacillus deltoid complex: after co-culturing the lactobacillus polymyxa complexes and candida albicans, the candida albicans basically does not grow, and has strong inhibition capability on the candida albicans; after the supernatant and candida glabrata are co-cultured for 48 hours, the OD value of the candida glabrata is between 0.4 and 0.5, and the strain has obvious inhibition effect and is remarkably strong in Yu Dingjun raw microecological preparation. Surprisingly, in combination with the growth data analysis of example 4, the inhibition effect of each group of Lactobacillus delbrueckii complexes on candida slidingica was improved over either single strain, with the relative predominance of the growth of the LSA-858 strain and the LSA-946 strain. Example 6: in vitro safety detection of lactobacillus

1. Strain hemolytic assay

Staphylococcus aureus CMCC (B) 26003 (purchased from the company of microorganisms, inc., guangdong) was used as a positive control group, and the control group and each lactobacillus were inoculated onto blood agar plates, and anaerobic culture was performed at 37 ℃ for 48 hours to observe the hemolysis of colonies.

The experimental results are shown in fig. 5: the staphylococcus aureus CMCC (B) 26003 has obvious hemolytic rings, and each lactobacillus strain has no hemolytic rings, no pathogenicity, safety and reliability.

Example 7: therapeutic capacity on candida albicans infected vulvovaginal candidiasis rats

An SD female rat model is adopted to construct an infected rat model, the age of 7 weeks and the weight of 180-200 g are divided into 9 groups, namely a healthy group, a model group, a clotrimazole group, a Lactobacillus delbrueckii group and a compound A-E group respectively. Each Lactobacillus multiplex complex group is prepared by mixing each single bacterium with equal ratio (v/v), and has turbidity of 1.0X10 ⁸ CFU/mL of Lactobacillus delbrueckii group was prepared from DM8909 bacterial suspension, turbidity was 1.0X10 ⁸ CFU/mL, 8 in each group, 72 total, were fed on a standard diet.

The experiments were divided into adaptation phase, modeling phase, dry expectation and observation phase. The adaptation period is as follows: all animals are adaptively raised in an animal laboratory for 1 week, ovaries of female animals are removed in advance, the influence of endogenous estrogens on experiments is manually eliminated, 0.5mg of estradiol benzoate is injected subcutaneously every 1 day after 7 days of ovariectomy operation, so that the animals are in wig estrus, and the phenotypic characteristics such as whether the vagina of a rat has red swelling, spilled liquid and the like are observed and recorded before modeling. Modeling period: the turbidity of candida albicans is regulated to be 1.0 multiplied by 10 ⁸ CFU/mL, 1mL was taken to rinse the rat vagina, and healthy group was rinsed with normal saline; once daily, rinsing continuously for 3 days, rinsing and sampling rat vagina with sterile PBS on day 4, observing with microscopeThe resultant yeast mycelium was gram stained, and appearance checked for inflammation and redness to confirm success of candida albicans infection. Dry expectation: for 7 days, rats in healthy group and model group were not treated; the clotrimazole group rats were vaginally injected with 1mL of clotrimazole solution (8. Mu.g/mL); 1mL of each group of lactobacillus bacteria liquid is injected into the vagina of each probiotic group of rats, and the infusion is carried out for 7 days continuously, wherein the times are 1 day. Observation period: phenotypic characteristics such as whether the vagina of the rat is red and swollen or not and the exudates are observed and recorded every day during the intervention period and 3 days after the treatment. While observing the phenotype of the rat, taking vaginal rinse liquid of the rat, and respectively detecting colony counts of candida albicans and lactobacillus.

TABLE 5 observation and statistics of vulva redness and swelling and secretion of rats 3 days after the end of treatment

Viewing items	Red and swollen vulva	Secretion of	Vaginal congestion
				Health group	Whether or not	Normal state	Whether or not
Model group	Is that	Bean curd residue shape	Is that
				Clotrimazole group	Whether or not	Normal state	Whether or not
Lactobacillus delbrueckii group	However, there is a slight improvement	Abnormal, bean curd residue shape	However, there is a slight improvement
				Group A	Whether or not	Normal state	Whether or not
Group B	Whether or not	Normal state	Whether or not
				Group C	Whether or not	Normal state	Whether or not
Group D	Whether or not	Normal state	Whether or not
				Group E	Whether or not	Normal state	Whether or not

The experimental results are shown in table 5 and fig. 7: after the rats infected by candida albicans are treated by the multi-linked lactobacillus compound, the number of candida albicans in the body is obviously reduced, the candida albicans approaches to the level of a healthy group, the treatment effect is equivalent to the clotrimazole treatment effect, the external appearance of the vulva and secretion of the rats is normal after the treatment, no adverse reaction exists, and the treatment effect of the lactobacillus delbrueckii group is not obvious. The clotrimazole treatment group can kill candida albicans and correspondingly reduce the abundance of lactobacillus in the vagina of the rat, so that the normal microecology in the vagina of the rat can not be recovered, and the recurrence of VVC or the infection of other pathogenic bacteria can be easily caused; the multi-lactobacillus compound group has the advantages that the number of the lactobacillus in the vagina of the rat is recovered to be normal and has an increasing trend, and the multi-lactobacillus compound group can be well planted in the vagina to reduce the recurrence of VVC.

To sum up: the LSA-858 strain, the LSA-946 strain, the LSA-1012 strain and the LSA-1066 strain have the capability of producing beneficial secretion such as lactic acid, hydrogen peroxide and the like; can well withstand the acidic environment of the vagina; has certain adhesion capability to vaginal cervical cells; the medicine has no hemolysis and good safety; has certain inhibiting effect on common pathogenic bacteria of VVC. The multi-lactobacillus compound formed by combining any 3 kinds of the compounds has the advantage of stronger probiotics than single bacteria; the strains have no antagonism effect and good growth; has obvious treatment effect on VVC animal disease models, and can be used as potential development medicine for treating vulvovaginal candida diseases.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. A strain of lactobacillus crispatus, wherein the strain is designated as LSA-858, the strain LSA-858 is lactobacillus crispatus (Lactobacillus crispatus) deposited with the cantonese collection of microorganism strains under accession number GDMCC No.62779 and a date of deposit of 2022, 9 and 9.

2. A lactobacillus jannaschii, which is named as LSA-946 strain, wherein the LSA-946 strain is lactobacillus jannaschii (Lactobacillus jensenii) and is deposited with the cantonese province microorganism strain collection, wherein the deposit number is GDMCC No.62781, and the deposit date is 2022, 9 and 9.

3. A lactobacillus johnsonii, wherein the lactobacillus johnsonii is designated as LSA-1012 strain, the LSA-1012 strain is lactobacillus johnsonii (Lactobacillus johnsonii) and is deposited with the cantonese microorganism strain collection under the accession number GDMCC No.62782 and the date of deposition is 2022, 9 and 9.

4. Lactobacillus reuteri, characterized in that said lactobacillus reuteri is designated as LSA-1066 strain, said LSA-1066 strain is lactobacillus reuteri (limosilactobacilli), deposited with the cantonese province microorganism strain collection under accession number GDMCC No.62783, and the date of deposit is 2022, 9.

5. A lactobacillus reuteri complex comprising at least three of strain LSA-858, strain LSA-946, strain LSA-1012, or strain LSA-1066.

6. A formulation characterized by comprising at least one of the following (1) to (5):

(1) Comprising the Lactobacillus multiplex complex according to claim 5;

(2) A live bacterial liquid comprising the lactobacillus polymorphus complex of claim 5;

(3) A dead bacterial liquid comprising the lactobacillus polymyxa complex of claim 5;

(4) A metabolite obtained from the metabolic pathway of the lactobacillus polymyxa complex of claim 5;

(5) An extract obtained by extraction from the Lactobacillus deltoidea complex of claim 5.

7. Use of a lactobacillus reuteri complex according to claim 5 or a formulation according to claim 6 for the manufacture of a medicament, food or sanitary product for the prevention or treatment of gynaecological disorders.

8. The use according to claim 7, wherein the gynaecological disease comprises at least one of vaginal dysbiosis, vulvovaginal candidiasis, bacterial vaginosis, HPV or urinary tract infection.

9. Use of a lactobacillus reuteri complex according to claim 5 or a formulation according to claim 6 for the preparation of a bacteriostatic product.

10. Use of the lactobacillus reuteri complex of claim 5 or the formulation of claim 6 for the preparation of a delivery vehicle.