WO2021027740A1 - 多联乳杆菌组合物及其在女性阴道健康中的应用 - Google Patents

多联乳杆菌组合物及其在女性阴道健康中的应用 Download PDF

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WO2021027740A1
WO2021027740A1 PCT/CN2020/107915 CN2020107915W WO2021027740A1 WO 2021027740 A1 WO2021027740 A1 WO 2021027740A1 CN 2020107915 W CN2020107915 W CN 2020107915W WO 2021027740 A1 WO2021027740 A1 WO 2021027740A1
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lactobacillus
bacteria
bacterial
composition
preparation
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PCT/CN2020/107915
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French (fr)
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承磊
刘瑶
王琼
曾婉秋
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四川厌氧生物科技有限责任公司
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Priority to KR1020217029194A priority Critical patent/KR20210128429A/ko
Priority to JP2021553809A priority patent/JP7354274B2/ja
Priority to EP20853382.8A priority patent/EP3926040A4/en
Priority to SG11202112633PA priority patent/SG11202112633PA/en
Priority to US17/435,670 priority patent/US20220133819A1/en
Publication of WO2021027740A1 publication Critical patent/WO2021027740A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/36—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of microorganisms, in particular to a Lactobacillus polycondensate composition and its application in female vagina health.
  • the vagina of healthy women of childbearing age is a microenvironment with Lactobacillus as the dominant flora. They can use the glycogen in the vaginal epithelial cells to grow and produce H 2 O 2 , lactic acid, bacteriocin, etc., and can also competitively adhere to the vagina
  • the epithelium occupies the binding site and consumes nutrients in the vagina, thereby occupying a dominant position in the vagina and inhibiting the excessive proliferation of other pathogenic bacteria and anaerobic bacteria.
  • the ecological flora of the vagina of North American women can be divided into 5 types, 4 of which are Lactobacillus crispatus and Lactobacillus iners. , Lactobacillus jensenii and Lactobacillus gasseri are the dominant bacteria.
  • the fifth type is other lactic acid bacteria and anaerobic bacteria.
  • the most common dominant lactobacilli in the vagina of healthy women in China are Lactobacillus crispatus and Lactobacillus gasseri. Lactobacillus, Lactobacillus jannaschii, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus inert, etc.
  • Bacterial vaginosis bacterial vaginosis, hereinafter referred to as "BV"
  • BV Bacterial vaginosis
  • Bacillus a vaginal infectious disease that causes the clinical symptoms of the vaginal flora to be imbalanced. According to data, BV is the cause of tissue choriodritis, amniotic fluid infection, post-cesarean endometritis, and other poor pregnancy and pregnancy complications Risk factors for disease.
  • the clinical treatment method is to use metronidazole or clindamycin.
  • Metronidazole is a prodrug.
  • bacterial intracellular enzymatic reduction reduces the nitro group of metronidazole to an amino group.
  • Clindamycin can interact with the 50S ribosome on the bacterial ribosome The combination of subunits prevents the extension of the peptide chain, thereby inhibiting the protein synthesis of bacterial cells, leading to the death of bacteria.
  • antibiotic treatment is effective, it also has great shortcomings. It has the following two aspects: (1) It has an inhibitory effect on all antibiotic-sensitive microorganisms in the vaginal microenvironment. Therefore, after treatment, the vaginal microecology has not recovered to a healthy, A balanced state that can resist the invasion of pathogenic bacteria. The inhibited or killed pathogenic microorganisms or foreign pathogenic microorganisms will reproduce or even cause disease and relapse or new vaginal inflammation; (2) The microorganisms develop drug resistance and antibiotics The inability to balance the vaginal microecological environment results in refractory BV. Therefore, although antibiotic treatment is effective, the recurrence rate is high, and the recurrence rate is as high as 30% within 3 months.
  • the treatment of vaginal microecological imbalance includes three steps: sterilization, mucosal repair, and restoration of vaginal microecological balance.
  • Sterilization is the first step in the treatment of vaginal inflammation. It inhibits or kills pathogenic microorganisms, including over-proliferation of aerobic and anaerobes, spores or hyphae, trichomonas, etc. After the pathogenic microorganisms are inhibited or killed, the immune repair of the vaginal mucosa and the recovery of dominant lactobacilli are the ultimate goals of treating vaginal inflammation.
  • vaginal microecological preparations there are only two types of vaginal microecological preparations on the market in China.
  • One is a commercial drug produced by Xi’an Zhenghao Biopharmaceutical Co., Ltd. under the trade name "Yanhua”, which contains a type of Streptococcus faecalis. It is not a vaginal dominant strain, and the bacteria under this species are conditionally pathogenic; the other is a commercial drug produced by Inner Mongolia Shuangqi Pharmaceutical Co., Ltd. under the trade name "Wanze Shuangqi", which contains 1 milk Bacillus-Lactobacillus delbrueckii (Lactobacillus delbrueckii), this strain is not a dominant strain of Chinese women's vagina. Therefore, the use of dominant Lactobacillus strains to prepare vaginal microecological preparations for the treatment of bacterial vaginitis has more therapeutic advantages.
  • Chinese patent document CN102851248 A discloses a Lactobacillus jannaschii used to place bacterial vaginosis, which is a dominant Lactobacillus isolated from the vagina of healthy Chinese women.
  • Chinese patent document CN107794236 A discloses a Lactobacillus crispatus and its application. It is a dominant Lactobacillus isolated from the vagina of healthy Chinese women and is used to treat bacterial vaginitis.
  • Chinese patent document CN 108004187 A discloses a Lactobacillus gasseri and its application in the preparation of vaginal antibacterial drugs. Lactobacillus gasseri is a dominant bacterium isolated from the vagina of healthy Chinese women.
  • lactobacilli Due to the rich diversity of lactobacilli in the human vagina, different lactobacilli exhibit different probiotic abilities, and they act synergistically in the vaginal microenvironment, and there are individual differences.
  • the dominant strains in the vagina of different women are slightly different, so when choosing the corresponding vaginal use
  • Lactobacillus probiotics it is necessary to comprehensively consider the types of Lactobacillus and the probiotic ability of different strains. This also shows that the coverage of a single strain in the treatment of bacterial vaginosis is not wide, and it is necessary to adopt a combination of dominant Lactobacillus strains.
  • the method for preparing vaginal microecological preparation is used to treat bacterial vaginosis.
  • the purpose of the present invention is to provide a Lactobacillus polymorphic composition of dominant strains belonging to the vagina of Chinese women in view of the problems in the prior art.
  • the strains mutually beneficial symbiosis, does not produce antagonism, and has a synergistic effect.
  • Single-dominant strains have stronger probiotic ability, and because they are Lactobacillus polyadhas, they have a wider coverage in the treatment of bacterial vaginosis.
  • the Lactobacillus polycondensate composition of the present invention is based on the dominant strains isolated and screened from the vagina of healthy Chinese women. Specifically, the Lactobacillus polycondensate composition includes at least the following three active ingredients:
  • Lactobacillus johnsonii (Lactobacillus johnsonii), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus jensenii.
  • Lactobacillus johnsonii is Lactobacillus johnsonii Ljohn-1 (deposit number: CCTCC No. 2019426); the above-mentioned Lactobacillus gasseri is Lactobacillus gasseri Lgass-17 (deposit number: CCTCC No. 2019426). 2019430); the above-mentioned Lactobacillus rhamnosus is Lactobacillus rhamnosus Lrham-6 (preservation number: CCTCC No. 2019428); the above-mentioned Lactobacillus crispatus is Lactobacillus crispatus Lcris-2 (preservation number: CCTCC No.
  • Lactobacillus jannaschii is Lactobacillus jannaschii Ljen-10 (preservation number: CCTCC No. 2019429).
  • the above-mentioned Lactobacilli are strains isolated from the vaginal screening of healthy Chinese women by the inventor of the present application and deposited to the China Center for Type Culture Collection (CCTCC) on June 4.
  • the above-described multi-viable count of lactobacilli composition is 10 5 ⁇ 10 11 cfu / g , each single strain is not less than 10 5 cfu / g.
  • an inoculum containing the above-mentioned Lactobacillus polycondensate composition as an active ingredient.
  • the inoculum can be a suspension or a freeze-dried bacterial powder.
  • the above-mentioned polylactic acid composition is used in the preparation of medicines or health products for the prevention and treatment of pathogenic bacteria of bacterial vaginitis.
  • the above-mentioned polylactic acid composition can be used in the preparation of external genital sanitary products, such as sanitary napkins, tampons or sanitary care solutions for external genitals.
  • the above-mentioned Lactobacillus polycondensate composition can be applied and can also be used in the preparation of medicines or health care products for regulating the balance of vaginal flora.
  • Lactobacillus polycondensate composition can be used in the preparation of drugs or health care products with vaginal epithelial cell adhesion function.
  • the above-mentioned Lactobacillus polycondensate composition can be used in the preparation of medicines, health products, and food additives with the prevention and treatment of pathogenic bacteria.
  • the pathogenic bacteria include but are not limited to Gardnerella vaginalis and Staphylococcus aureus. , Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B, and Shigella dysenteriae.
  • the Lactobacillus polymorphic composition can also be used in the preparation of external care products for infants delivered by caesarean section. Because babies born by caesarean section do not pass through the female vagina, no exogenous probiotics are obtained during the production process. Therefore, the probiotics screened in the female vagina can be made into external care products for the baby's body to smear or bathe.
  • the present invention has the following beneficial effects:
  • the Lactobacillus colonized in the vagina of healthy women has a diversity of bacterial species. Different bacterial species or different strains of the same bacterial species have different probiotic abilities. Therefore, when choosing a vaginal Lactobacillus probiotic, it is necessary to comprehensively consider the type of Lactobacillus.
  • the inventor will select suitable strains from the various strains isolated and screened by healthy women in China for reasonable compatibility.
  • Lactobacillus uses a combination of three or more Lactobacillus formulas to prevent and treat bacterial vaginitis. Due to the experimental verification, the polylactic acid Lactobacillus composition produces better probiotics and belongs to the dominant strain of Chinese female vagina. Therefore, it can be expected that the treatment and prevention of vaginal diseases in Chinese women will produce better results and cover a wide range.
  • Figure 1 is a comparison diagram of the hydrogen peroxide production scores of single bacteria and combined bacteria.
  • Figure 2 is a comparison diagram of lactic acid production capacity of single bacteria and combined bacteria.
  • Figure 3 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against Gardnerella vaginalis.
  • Figure 4 is a comparison diagram of the adhesion ability of single bacteria and combined bacteria to hela cells.
  • Figure 5 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against Staphylococcus aureus.
  • Figure 6 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against E. coli.
  • Figure 7 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against Pseudomonas aeruginosa.
  • Figure 8 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against Shigella dysentery.
  • Figure 9 is a comparison diagram of the inhibitory ability of single bacteria and combined bacteria against Salmonella paratyphi B.
  • Figure 10 is the solid symbiosis effect test diagram 1 between the individual bacteria in Lactobacillus polycondens.
  • Fig. 11 is a graph 2 of the broth symbiosis effect test among the individual bacteria in Lactobacillus polysporum.
  • Lactobacillus johnsonii Ljohn-1 Lactobacillus gasseri Lgass-17
  • Lactobacillus crispatus Lcris-2 Lactobacillus jannaschii Ljen-10
  • Lactobacillus rhamnosus Lrham-6 The methods for screening and separating Lactobacillus johnsonii Ljohn-1, Lactobacillus gasseri Lgass-17, Lactobacillus crispatus Lcris-2, Lactobacillus jannaschii Ljen-10, and Lactobacillus rhamnosus Lrham-6 are as follows:
  • vaginal cotton swabs to collect samples of vaginal secretions from Chinese women aged 20-40 who have passed the health examination; take 2 mL of sterile, oxygen-free PBS buffer in an anaerobic tube containing the cotton swabs, shake and mix thoroughly, And use it as the original solution for continuous 10-fold gradient dilution; take 100 ⁇ L of the 10,000-fold diluted liquid and spread it on the MRS solid medium and place it in an anaerobic incubator at 37°C.
  • the method for screening Lactobacillus of the present invention includes the following steps:
  • 16S rRNA was performed on the lactobacilli selected by the low pH culture vigor comparison Genotype screening.
  • the Lactobacillus was transferred to the MRS broth medium, and the two were cultured in parallel at 37°C for 48 hours.
  • the pH value of the Lactobacillus broth cultured for 48 hours was measured and recorded with a pH 0.5-5.0 test paper.
  • Two conditions are used to select strains for liquid chromatography: condition one, the strain with pH value of 2.5 is subjected to liquid chromatography; condition two, the same species of Lactobacillus, select the strain with lower pH value for liquid chromatography; confirm the liquid chromatography After the sample is taken, the supernatant is diluted 5 times, concentrated sulfuric acid is added for pretreatment, and the sample is filtered with a 0.22um syringe filter.
  • the relevant parameters of liquid chromatography are as follows:
  • Detector and detection wavelength DAD, 207nm; RID, refractive index signal
  • Injection volume 20 ⁇ L.
  • Lactobacillus delbrueckii Take Lactobacillus delbrueckii as the positive control, the blue depth produced by Lactobacillus bideti is counted as 4 points.
  • the blue color produced by Lactobacillus is equivalent to 3 points, the light blue color produced by Lactobacillus Biederi is 2 points, the blue color is very weak, 1 point (slight color reaction), and 0 points are not changed.
  • the cells are washed twice by centrifugation, resuspended in PBS, and 100 ⁇ L of Lactobacillus suspension is sucked into a 96-well cell culture plate containing Hela cells, incubated at 37°C for 30 minutes, and washed twice with sterile PBS to wash Remove non-adherent Lactobacillus, then add 25 ⁇ L of pancreatin solution to each well, and place the cells in a 37°C incubator to digest the cells. After Hela cells are digested and become spherical, add 75 ⁇ L of complete medium to each well, pipette repeatedly, and pipette after complete digestion. 20 microliters of bacterial suspension, diluted with sterile PBS 10-fold gradient, select an appropriate dilution gradient for counting experiment by pouring method, and count after 48 hours incubation at 37°C.
  • the bacterial culture medium components and preparation methods used above and below are as follows:
  • MRS broth Preparation of MRS broth: Weigh 52.0g of MRS product culture medium powder and dissolve it in 1L of distilled water; heat to boil, cool to room temperature and add 0.55g of cysteine hydrochloride, stir to dissolve and adjust the pH to 6.5; load quantitatively Dispense the dispenser with N 2 , heat it to boiling, boil for 20 minutes in a slightly boiling state, and dispense into 10 mL anaerobic tubes after cooling, sterilize at 118°C with high temperature and humidity for 20 minutes, store in the shade, avoid light, and reserve.
  • MRS solid medium weigh 52.0g of MRS finished medium powder and 15.0g of agar powder, dissolve in 1L of distilled water, heat to boil, add 0.55g of cysteine hydrochloride after boiling, adjust the pH to 6.5,118 Sterilize at high temperature, humidity and heat for 20 minutes, store in a cool, dark place for later use.
  • Hydrogen peroxide semi-quantitative medium preparation Weigh 52.0g of MRS finished medium powder and 15.0g of agar powder, dissolve in 1L distilled water, adjust pH to 6.5, sterilize at 118°C high temperature and humidity for 20min, put it in after sterilization Incubate the 50°C water bath for 30 minutes, add TMB (to make the final concentration of TMB be 0.25mg/mL) and HRP (to make the final concentration of HRP be 0.01mg/mL) and mix well; after cooling and solidification, mark the name of the medium and the date of preparation, and place it in Refrigerator at 4°C for use.
  • TMB to make the final concentration of TMB be 0.25mg/mL
  • HRP to make the final concentration of HRP be 0.01mg/mL
  • anaerobic PBS weigh 0.27g potassium dihydrogen phosphate, 1.42g disodium hydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, dissolve in 1L of distilled water, heat to boil, cool to room temperature and add 0.55g half Cystine hydrochloride, stir to dissolve, adjust the pH value to 6.5, install a quantitative dispenser and pass N 2 , heat to boiling, boil for 30 minutes at a slight boiling state, and then dispensed into a 10 mL anaerobic tube after cooling. Sterilize at 121°C for 30min under high temperature, humidity and heat. Store in a cool, dark place for later use.
  • Anaerobic BHI liquid medium preparation Weigh 37.0g of BHI finished medium powder, dissolve it in 1L of distilled water, heat to boil, cool to room temperature, add 0.55g of cysteine hydrochloride, stir to dissolve and adjust the pH to 6.5, Install a quantitative dispenser and pass N 2 , heat to boiling, boil for 20 minutes in a slightly boiling state, pass in N 2 and CO 2 (1:1 ratio) during cooling and dispensing, and dispense to 10 mL anaerobic tubes Sterilize at high temperature and humidity at 118°C for 20 minutes, store in a cool, dark place for later use.
  • Anaerobic BHI semi-solid medium preparation weigh 37.0g of BHI finished medium powder and dissolve in 1L distilled water; heat to boil, cool to room temperature, add 6g agar powder and 0.55g cysteine hydrochloride, stir to dissolve and adjust When the pH value reaches 6.5, install a quantitative dispenser and pass N 2 , heat to boiling, boil for 20 minutes, cool slightly, and pass in N 2 and CO 2 (1:1 ratio) during the cooling and dispensing process , Timely aliquot into 10mL anaerobic tubes, sterilize at 118°C high temperature and humidity for 20min, store in a cool, dark place.
  • Preparation of nutrient broth liquid medium weigh 10g peptone, 3g beef powder, 5g sodium chloride and dissolve into 1L distilled water, adjust the pH to 7.2, heat and boil, cool to room temperature and then aliquot, each 10mL; 121°C high temperature and humidity Sterilize for 15 minutes, store in a cool, dark place for later use.
  • Preparation of nutrient broth solid medium weigh 10g peptone, 3g beef powder, 5g sodium chloride, 6g agar powder and dissolve it in 1L distilled water, adjust the pH to 7.2, cool it slightly, and timely aliquot it into a 10mL anaerobic tube; Sterilize at high temperature, humidity and heat for 15 minutes, store in a cool, dark place for later use.
  • Lactobacillus johnsonii Ljohn-1 (deposit number: CCTCC No. 2019426) is hereinafter referred to as "A”
  • Lactobacillus crispatus Lcris-2 (deposit number: CCTCC No. 2019427) is hereinafter referred to as "B”.
  • Lactobacillus Leesose Lrham-6 (deposit number: CCTCC No. 2019428) hereinafter referred to as "C”
  • Lactobacillus jannaschii Ljen-10 (deposit number: CCTCC No. 2019429) hereinafter referred to as "D”
  • Lactobacillus gasseri Lgass- 17 (Deposit No.: CCTCC No. 2019430) hereinafter referred to as "E”.
  • Example 1 Comparison of lactic acid production capacity of single bacteria and combined lactobacilli.
  • Combination bacteria After activation and transfer of Lactobacillus, select 3 or more strains to mix and culture, 3 parallel, cultivate 48h at 37°C, centrifuge, dilute the supernatant 5 times, add concentrated sulfuric acid for pretreatment, load the sample Use a 0.22um syringe filter before filtering.
  • the relevant parameters of liquid chromatography are as follows:
  • Detector and detection wavelength DAD, 207nm; RID, refractive index signal
  • Injection volume 20 ⁇ L.
  • the growth of microbial biomass and lactic acid production mainly depend on the type and concentration of carbon source in the medium, the pathway of microbial metabolism, and the tolerance of the strain to organic acids such as lactic acid.
  • the amount of inoculum generally only affects the delay period of microbial growth, but has little effect on the growth of microbial cells and the production of lactic acid.
  • the test details that need to be paid attention to here are that different inoculum amounts will bring different background organic content. Then in the new medium, the organic matter content of the high inoculum group is higher, resulting in different growth and lactic acid production for different inoculum levels. If this part of the details is ignored in the test process, it will cause wrong judgments.
  • the selected inoculation amount was the same, and the lactobacillus inoculation amount in the above lactic acid production experiment was 2%.
  • the lactic acid production of Lactobacillus in the logarithmic growth phase is positively correlated with the growth curve of the microorganism.
  • the lactic acid production will slightly increase, which is consistent with literature reports.
  • the time for Lactobacillus to reach a stable growth period generally does not exceed 24h, but from 24h to 48h, the lactic acid production of some Lactobacilli will increase slightly. Therefore, the 48h time point is selected to analyze the lactic acid production.
  • Example 2 Comparison of hydrogen peroxide production ability between single bacteria and combined lactobacilli.
  • Combination bacteria After the activation and transfer of Lactobacillus, select 3 or more strains to mix, use a pipette to suck 2 ⁇ L of bacterial liquid and spot in MRS agar containing TMB and HRP, cultivate at 37°C, and after the corresponding time of cultivation, Take out the corresponding plates, expose them to the air, observe the color reaction after 30 minutes and take pictures to record: Take Lactobacillus delbrueckii as the positive control, the blue depth produced by Lactobacillus biderlii is counted as 4 points. Blue is equivalent to 3 points, blue light produced by Lactobacillus bideti is counted as 2 points, very weak blue is counted as 1 point (slight color reaction), and no color is counted as 0 points.
  • Lactobacillus converts pyruvate and or lactate and oxygen into hydrogen peroxide through lactate oxidase and/or pyruvate oxidase. Therefore, the hydrogen peroxide production of Lactobacillus is affected by many factors (metabolic products and oxygen content, bacterial growth stage and enzyme activity, etc.). In this type of experiment, it is not meaningful to only quantify the inoculation amount of Lactobacillus. Big. Therefore, we adopted a semi-quantitative method in this experiment. The inoculum of all strains was 2 microliters, and they were spotted on solid medium for growth.
  • Example 3 Comparison of the ability of single bacteria and combined lactobacilli to inhibit Gardnerella vaginalis.
  • Lactobacillus After Lactobacillus is activated, take 0.1 mL of bacteria liquid and MRS solid medium and mix it, pour it into a 6cm petri dish, incubate at 37°C for 48h after complete solidification, take out the petri dish, and use a hole punch with an inner diameter of 6mm in the agar medium Punch a hole on the top to obtain a bacterial cake; after the Gardnerella vaginalis is activated and transferred, the Gardnerella bacterial solution is diluted to 100 times with a 10-fold gradient with anaerobic sterile PBS buffer, and 10 -1 and 10 -1 are taken respectively.
  • Combination bacteria After the activation and transfer of Lactobacillus, select 3 or more strains to mix, take 0.1mL of bacterial liquid and MRS solid medium and mix it, pour it into a 6cm plate, culture it at 37°C for 48h after complete solidification, and remove the plate.
  • the lactobacillus was prepared by pouring method with 1% inoculum.
  • the bacterial content of the bacterial cake after 48 hours of culture was similar, and the color on the bacterial cake was similar, indicating that the bacterial cakes of different strains had considerable homogeneity .
  • Example 4 Comparison of the ability of single bacteria and combined bacteria to adhere to Hela cells.
  • Single bacteria After the activation of Lactobacillus, wash the bacteria by centrifugation twice, resuspend in PBS, draw 100 ⁇ L of Lactobacillus suspension in a 96-well cell culture plate containing Hela cells, incubate at 37°C for 30min, then use sterile PBS after 30min Wash twice to remove the adherent Lactobacillus. Add 25 ⁇ L of trypsin solution to each well of a 96-well cell culture plate containing Hela cells, and place it in a 37°C incubator to digest the cells.
  • Hela cells are digested and become spherical, add to each well 75 ⁇ L complete medium, pipet repeatedly and evenly, after complete digestion, draw 20 ⁇ l of bacterial suspension, dilute with sterile PBS 10-fold gradient, select appropriate dilution gradient for counting experiment by pouring method, culture at 37°C for 48h and count.
  • Combination bacteria After the activation and transfer of the Lactobacillus, wash the bacteria by centrifugation twice, resuspend in PBS, select 3 or more strains and mix them evenly, draw 100 ⁇ L of Lactobacillus suspension on a 96-well cell culture plate containing Hela cells Incubate at 37°C for 30 min. After 30 min, wash twice with sterile PBS to remove the adherent Lactobacillus; after incubating for the corresponding time, add 25 ⁇ L of trypsin solution to each well of a 96-well cell culture plate containing Hela cells, and place Digest the cells in a 37°C incubator.
  • Hela cells are digested into a spherical shape, add 75 ⁇ L of complete medium to each well, repeatedly pipette evenly. After complete digestion, draw 20 microliters of bacterial suspension and dilute it with sterile PBS 10 times. A 10 -4 dilution gradient was used for counting experiments by pouring method, and counting was carried out after 48 hours incubation at 37°C.
  • the adhesion time of single bacteria to Hela cells was only set at a 30-minute observation time point. After determining the preferred strains, 16 formulas were combined, so an observation time point of 4h was added. The test results of 4h and 30min showed that as the adhesion time increased, the adhesion effect of the combined bacteria on Hela cells increased.
  • the figures and tables in the specification are 30min data.
  • Example 5 Comparison of the ability of single bacteria and combined bacteria to inhibit Staphylococcus aureus, inhibit Pseudomonas aeruginosa, inhibit E. coli, inhibit Salmonella paratyphi B, and inhibit Shigella dysenteriae.
  • Lactobacillus After Lactobacillus is activated, take 0.1 mL of bacteria liquid and MRS solid medium and mix it, pour it into a 6cm petri dish, incubate at 37°C for 48h after complete solidification, take out the petri dish, and use a hole punch with an inner diameter of 6mm in the agar medium Punch holes on the top to obtain a bacterial cake; Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella paratyphi B and Shigella dysentery are activated and transferred, and 0.9% saline is used in a 10-fold gradient.
  • Combination bacteria ability to inhibit Salmonella paratyphi B and inhibit Shigella dysentery
  • Lactobacillus bacteria cakes are placed on the surface of nutrient agar lightly, 4 bacteria cakes are symmetrically placed in each dish, 2 of each strain are parallel, placed in a sealed tank, the dish is placed upright for 24 hours, and the size of the inhibition zone is measured with a vernier caliper.
  • the lactobacillus was prepared by pouring method with 1% inoculum.
  • the bacterial content of the bacterial cake after 48 hours of culture was similar, and the color on the bacterial cake was similar, indicating that the bacterial cakes of different strains had considerable homogeneity .
  • the lactobacillus was prepared by pouring method with 1% inoculum.
  • the bacterial content of the bacterial cake after 48 hours of culture was similar, and the color on the bacterial cake was similar, indicating that the bacterial cakes of different strains had considerable homogeneity .
  • Example 6 Test of antagonistic effect and symbiotic effect among various strains of Lactobacillus polysporum
  • the single strain before inoculation is diluted to a suitable gradient by 10-fold, and the concentration of the bacterial solution before inoculation is counted.
  • Each gradient is 3 parallel, and the plate is placed in anaerobic In a sealed tank, add an anaerobic gas bag and incubate for 48 hours at 37°C; then mix the strains to be tested and inoculate it in MRS broth.
  • the inoculation concentration of each strain in the mixed strain is the same as that of a single strain.
  • the amplification multiples of single bacteria and mixed culture are calculated according to the average number, and single bacteria are inoculated once. Therefore, the amplification multiple calculated by single bacteria culture has only one set of data, considering the uniformity of mixed bacteria inoculation, so mixed The culture is inoculated twice at the same time, so there are two sets of parallel data for the amplification factor calculated by the mixed culture.
  • the present invention can be well realized. It is worth noting that, based on the above-mentioned structural design, in order to solve the same technical problem, even if some insubstantial changes or polishes are made in the present invention, the essence of the technical solution adopted is still the same as that of the present invention. Therefore, it should also fall within the protection scope of the present invention.

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Abstract

一种多联乳杆菌组合物，所述多联乳杆菌组合物是基于中国健康女性阴道内分离筛选出的优势菌株。多联乳杆菌组合物至少包括以下三种活性成分：约氏乳杆菌(Lactobacillus johnsonii)、格氏乳杆菌(Lactobacillus gasseri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、卷曲乳杆菌(Lactobacillus crispatus)、詹氏乳杆菌(Lactobacillus jensenii)。

Description

多联乳杆菌组合物及其在女性阴道健康中的应用 技术领域
本发明涉及微生物领域，具体涉及多联乳杆菌组合物及其在女性阴道健康中的应用。
背景技术
从期刊实用妇科杂志公开的文献《阴道微生态的研究进展及临床意义》、期刊中国微生态学杂志公开的文献《乳杆菌在阴道微生态中生存状态的研究进展》等各方研究报告可知，人体阴道内有300多种微生物共生，它们彼此制约、相互制衡，形成动态平衡，多种阴道炎的发生和阴道微生态环境失衡有关。
健康的育龄女性阴道是一个以乳杆菌为优势菌群的微环境，它们可以利用阴道上皮细胞内的糖原生长，并产H 2O 2、乳酸、细菌素等，还可以竞争性黏附于阴道上皮，占据结合位并消耗阴道内营养，从而在阴道内占据优势地位，抑制其他致病菌和厌氧菌的过度增殖。最近十多年的研究发现，人体阴道内乳杆菌多样性非常丰富，不同乳杆菌在阴道微环境协同发挥作用，而且不同地域和人群中的乳杆菌类型不同，从宏观上看，不同的种族，健康女性阴道内的优势菌有明显差异(据研究，80％-90％的亚洲人和白人优势菌为乳杆菌，而不到60％的黑人和西班牙人为乳杆菌)，从微观上看，同种族，不同个体之间优势乳杆菌存在个体差异。目前，从阴道微环境分离出的乳杆菌已超过20种，北美女性阴道生态菌群主要可分为5种类型，有4种分别以卷曲乳杆菌(Lactobacillus crispatus)、惰性乳杆菌(Lactobacillus iners)、詹氏乳杆菌(Lactobacillus jensenii)和格氏乳杆菌(Lactobacillus gasseri)为优势菌，第5种是其他乳酸菌和厌氧菌，中国健康女性阴道最常见的优势乳杆菌有卷曲乳杆菌、格氏乳杆菌、詹氏乳杆菌、鼠李糖乳杆菌、罗伊氏乳杆菌、约氏乳杆菌、嗜酸乳杆菌、惰性乳杆菌等。
当环境因素或者外界干预打破了阴道微生态平衡，会使阴道微生态环境进入一个脆弱的状态，不容易抵御致病菌的繁殖、侵犯，出现各种阴道炎症。细菌性阴道炎(bacterial vaginosis，以下简称“BV”)，是一种常见的妇科疾病，感染率在15％-52％，是以阴道加德纳菌和其他厌氧菌为主过度繁殖取代乳杆菌，造成阴道内菌群失调而出现的临床症候群的一种阴道感染性疾病，据资料报道，BV是导致组织性绒毛膜炎、羊水感染、剖腹产后子宫内膜炎及其他妊娠不良和妊娠并发症的危险因素。
针对BV，临床治疗方法是采用甲硝唑或克林霉素，甲硝唑是一种前体药，在无氧环境中，细菌胞内酶促还原将甲硝唑的硝基还原成氨基，从而将抗生素转化为活性形式，然后通过与DNA共价结合破坏其螺旋结构并导致单链和双链断裂，进而使DNA降解和病原体死亡；克林霉素能够与细菌核糖体上的50S核糖体亚基结合，阻止肽链的延长，从而抑制细菌 细胞的蛋白质合成，导致细菌死亡。采用抗生素治疗虽然见效快，但也存在很大缺陷，有以下两方面：(1)对阴道微环境中所有抗生素敏感微生物均有抑制作用，因此，治疗后阴道微生态没有恢复到一个健康的、能抵御致病菌侵袭的平衡状态，被抑制或杀灭的病原微生物或者外来的病原微生物还会再度繁殖甚至致病而出现复发或新的阴道炎症；(2)微生物产生耐药性、且抗生素无法使得阴道微生态环境平衡，导致了难治性BV。因而，抗生素治疗虽然见效快，但复发率高，3月内复发率高达30％。
阴道微生态失衡的治疗包括杀菌、黏膜修复、恢复阴道微生态平衡三步。杀菌是治疗阴道炎症的第一步，抑制或灭杀病原微生物，包括过度增殖的需氧菌和厌氧菌、芽生孢子或者菌丝、滴虫等。病原微生物被抑制或灭杀后，阴道黏膜的免疫修复和优势乳杆菌的恢复才是治疗阴道炎症的最终目标。在这期间，如果阴道黏膜的修复、乳杆菌的恢复过程被影响、阴道内理化环境未恢复至正常，被抑制的病原微生物或者外来的病原微生物还会再度繁殖甚至致病而出现复发或新的阴道炎症。而益生菌在阴道内可以迅速占据阴道上皮的受体，产生对阴道的保护作用，从而促使阴道恢复至正常微环境，减少阴道炎症的复发。因此，采用益生菌微生态制剂治疗BV是以现在的技术手段看来，最为优选的一种方式。
目前国内上市的阴道微生态制剂仅有2种，一种是西安正浩生物制药有限公司生产的商品名为“延华”的市售药品，包含一种肠链球菌(Streptococcus faecalis)，该菌种不是阴道优势菌种，而且这个物种下的细菌具有条件致病性；另一种是内蒙古双奇药业股份有限公司生产的商品名为“万泽双奇”的市售药品，包含1种乳杆菌——德氏乳杆菌(Lactobacillus delbrueckii)，该菌种不属于中国妇女阴道优势菌种。因此，采用优势乳杆菌菌种制备阴道微生态制剂用于治疗细菌性阴道炎更具有治疗优势。
中国专利文件CN102851248 A公开了一种用于放置细菌性阴道病的詹氏乳杆菌，其是从中国健康女性体阴道内分离的优势乳杆菌。中国专利文件CN 107794236 A公开的一种卷曲乳杆菌及其应用，其是从中国健康女性阴道内分离的优势乳杆菌，用于治疗细菌性阴道炎。中国专利文件CN 108004187 A公开了一种格氏乳杆菌及其用于制备阴道抑制菌药物中的应用，其格氏乳杆菌是从中国健康女性阴道内分离的优势杆菌。而除上述列举的三份中国专利文件外，还有很多类似的从中国健康女性阴道内分离的优势乳杆菌，且其用于治疗细菌性阴道炎的相关专利文件，他们都是采用从中国健康女性阴道筛选的单一乳杆菌去治疗细菌性阴道炎。
由于人体阴道内乳杆菌多样性丰富，不同乳杆菌表现出不同的益生能力，在阴道微环境协同发挥作用，且存在个体差异，不同女性阴道内优势菌株略有区别，因此在选择对应的阴道用乳杆菌益生菌时，需要综合考虑乳杆菌的种类及不同菌种的益生能力，这也说明了单一 菌株在细菌性阴道病治疗中的应用覆盖面不广，需要对优势乳杆菌菌种采用复配的方法制备阴道微生态制剂用于治疗细菌性阴道病。
发明内容
本发明的目的在于针对现有技术存在的问题，提供一种属于中国妇女阴道的优势菌株的多联乳杆菌组合物，菌株之间互利共生、不产生拮抗作用，且具有协同效应，产生了比单一优势菌株更强的益生能力，且由于是多联乳杆菌，其在细菌性阴道病的治疗中覆盖面更广。
本发明所述多联乳杆菌组合物是基于中国健康女性阴道内分离筛选出的优势菌株。具体的说，多联乳杆菌组合物至少包括以下三种活性成分：
约氏乳杆菌(Lactobacillus johnsonii)、格氏乳杆菌(Lactobacillus gasseri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、卷曲乳杆菌(Lactobacillus crispatus)、詹氏乳杆菌(Lactobacillus jensenii)。
值得也别强调的是，上述约氏乳杆菌为约氏乳杆菌Ljohn-1(保藏编号：CCTCC No.2019426)；上述格氏乳杆菌为格氏乳杆菌Lgass-17(保藏编号：CCTCC No.2019430)；上述鼠李糖乳杆菌为鼠李糖乳杆菌Lrham-6(保藏编号：CCTCC No.2019428)；上述卷曲乳杆菌为卷曲乳杆菌Lcris-2(保藏编号：CCTCC No.2019427)、上述詹氏乳杆菌为詹氏乳杆菌Ljen-10(保藏编号：CCTCC No.2019429)。上述各乳杆菌是本申请的发明人从中国健康女性阴道筛选分离出，并在6月4日保藏至中国典型培养物保藏中心(CCTCC)的菌株。
鼠李糖乳杆菌Lrham-6的全基因组序列上传到EzBiocloud后，与其能链接到所有的同种的全基因组序列比较，得到的平均核苷酸一致性(average nucleotide identity，ANI)比值如表1所示，证明其为新的乳杆菌株。
表1：Lrham-6与不同鼠李糖乳杆菌全基因组ANI比较
Figure PCTCN2020107915-appb-000001
Figure PCTCN2020107915-appb-000002
将格氏乳杆菌Lgass-17的全基因组序列上传到EzBiocloud后，与其能链接到所有的同种的全基因组序列比较，得到的平均核苷酸一致性(average nucleotide identity，ANI)比值如表2所示，证明其为新的乳杆菌株。
表2：Lgass-17与不同格氏乳杆菌全基因组ANI比较
Figure PCTCN2020107915-appb-000003
将约氏乳杆菌Ljohn-1的全基因组序列上传到EzBiocloud后，与其能链接到所有的同种的全基因组序列比较，得到的平均核苷酸一致性(average nucleotide identity，ANI)比值如表3所示，证明其为新的乳杆菌株。
表3：Ljohn-1与不同约氏乳杆菌全基因组ANI比较
Figure PCTCN2020107915-appb-000004
将詹氏乳杆菌Ljen-10的全基因组序列上传到EzBiocloud后，与其能链接到所有的同种的全基因组序列比较，得到的平均核苷酸一致性(average nucleotide identity，ANI)比值如表4所示，证明其为新的乳杆菌株。
表4：Ljen-10与不同詹氏乳杆菌全基因组ANI比较
Figure PCTCN2020107915-appb-000005
Figure PCTCN2020107915-appb-000006
2.曲乳杆菌Lcris-2的全基因组序列上传到EzBiocloud后，与其能链接到所有的同种的全基因组序列比较，得到的平均核苷酸一致性(average nucleotide identity，ANI)比值如表5所示，证明其为新的乳杆菌株。
表5：Lcris-2与不同卷曲乳杆菌全基因组ANI比较
Figure PCTCN2020107915-appb-000007
Figure PCTCN2020107915-appb-000008
上述多联乳杆菌组合物活菌数为10 5～10 11cfu/g，每种单菌含量不低于10 5cfu/g。
本发明中，提供以上述多联乳杆菌组合物为活性成分的菌剂，菌剂可为悬浮液或冻干菌粉。
本发明中，上述多联乳杆菌组合物在在制备用于预防、治疗细菌性阴道炎致病菌药品或保健品中的应用。
本发明中，上述多联乳杆菌组合物能应用在制备外生殖器卫生用品中，比如卫生巾、卫生棉条或用于外生殖器的卫生护理液。
本发明中，上述多联乳杆菌组合物能应用还能够应用在制备调节阴道菌群平衡的药品或卫生保健品中。
本发明中，上述多联乳杆菌组合物能应用在制备具有阴道上皮细胞粘附功能的药品或卫生保健品中。
本发明中，上述多联乳杆菌组合物能应用在制备具有预防、治疗致病菌的药品、保健品、食品添加剂的用途，致病菌包括但不限于阴道加德纳菌、金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌中的任意一种或多种。
多联乳杆菌组合物还能够应用在制备剖腹产婴儿外用护理品中的应用。由于剖腹产的婴儿未经过女性阴道，因此，在其生产过程中没有获得外源的益生菌，因此，可以将妇女阴道中筛选得到的益生菌做成外用护理品给婴儿身体涂抹或洗澡。
本发明与现有技术相比，具有以下有益效果：
健康女性阴道内定植的乳杆菌具有菌种多样性，不同菌种或相同菌种的不同菌株在不同益生能力存在差异，因此在选择阴道用乳杆菌益生菌时，需要综合考虑乳杆菌的种类，产乳酸、产过氧化氢能力、对致病菌的抑制能力、阴道细胞粘附的能力。为了尽可能覆盖阴道菌群失调的女性，同时为了增强抑制不同致病菌的能力，发明人将在中国健康女性分离筛选得到的多种菌株中选出合适菌株进行合理配伍，将分离筛选得到的乳杆菌，采用三种或以上乳杆菌组合配方的方式，来预防和治疗细菌性阴道炎，由于通过实验验证，多联乳杆菌组合物产生了更好益生能力，且属于中国女性阴道优势菌株，因此，可期的，其对中国女性阴道疾病的治疗和预防会产生更好的效果，且覆盖范围广。
附图说明
图1为单菌及组合菌产过氧化氢评分比较图。
图2为单菌及组合菌产乳酸能力比较图。
图3为单菌及组合菌对阴道加德纳菌抑制能力比较图。
图4为单菌及组合菌对hela细胞粘附能力比较图。
图5为单菌及组合菌对金黄色葡萄球菌抑制能力比较图。
图6为单菌及组合菌对大肠杆菌抑制能力比较图。
图7为单菌及组合菌对绿脓假单胞菌抑制能力比较图。
图8为单菌及组合菌对痢疾志贺菌抑制能力比较图。
图9为单菌及组合菌对乙型副伤寒沙门氏菌抑制能力比较图。
图10为多联乳杆菌中各单菌之间的固体共生效应试验图1。
图11为多联乳杆菌中各单菌之间的肉汤共生效应试验图2。
具体实施方式
以下将对本发明的具体实施方式进行详细描述，本发明的实施方式包括但不限于下列实施例。为了避免过多不必要的细节，在以下实施例中对属于公知的结构或功能将不进行详细描述。除有定义外，以下实施例中所用的技术和科学术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试剂耗材，如无特殊说明，均为常规生化试剂。
约氏乳杆菌Ljohn-1、格氏乳杆菌Lgass-17、卷曲乳杆菌Lcris-2、詹氏乳杆菌Ljen-10、鼠李糖乳杆菌Lrham-6的筛选分离方法如下：
用阴道棉拭子采集20-40岁通过健康体检的中国女性的阴道分泌物样品；取2mL无菌无氧的PBS缓冲液于装有上述棉拭子的厌氧管中，充分震荡混匀，并以其为原液连续十倍梯度稀释；取100μL稀释10000倍的液体涂布于MRS固体培养基，置于厌氧培养箱中37℃培养，培养48h后，挑取疑似乳杆菌单菌落在MRS肉汤培养基中培养24h，并将培养后得到菌液的一部分转接继续培养，菌液的另一部分进行细菌DNA提取；通过细菌16S rRNA基因扩增及测序，并将测序结果BLAST对比，根据对比结果分析菌种并分别保藏，得到共计1336株乳杆菌。通过对上述保藏的1336株乳杆菌进行低pH生长耐受试验、16S rRNA基因型筛选、抑制阴道加德纳菌试验、产乳酸试验、产过氧化氢试验、粘附Hela细胞试验筛选后，筛选出对应的上述乳杆菌。
本发明所述的乳杆菌的筛选方法包括以下步骤：
1.低pH生长耐受试验
1.1活化：保藏乳杆菌在pH值6.5的MRS肉汤活化，37℃过夜培养；
1.2转接：将活化菌液转接至pH值4-5的MRS肉汤培养中，每2-3h测量一次OD 600值；
1.3比较OD 600值，筛选能够快速增殖或OD 600值较高的来自不同样品的菌株。
2. 16S rRNA基因型筛选
分离自同一样品的相同种属的乳杆菌，若16S rRNA序列不同，表明其基因型不同，则其生理特性也可能有差异，因此对经过低pH培养活力比对筛选出的乳杆菌进行16S rRNA基因型筛选。
3.抑制阴道加德纳菌试验
乳杆菌经活化后，取0.1mL菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后37℃培养48h，取出平皿，用内径6mm的打孔器在琼脂培养基上打孔，获得菌饼；阴道加德纳菌经活化转接后，用无氧无菌PBS缓冲液以10倍梯度，将加德纳菌菌液稀释至100倍，分别取10 -1和10 -2稀释液0.5mL与5.25mL含5％马血清的BHI固体培养基混匀，倾注入9cm平皿内,完全凝固后，将乳酸菌菌饼轻放在BHI琼脂表面，每皿对称放4个菌饼，每株菌2个平行，放入厌氧的密封罐中，加厌氧产气袋，平皿正置培养48h，用游标卡尺测量抑菌圈大小。
4.产乳酸试验
乳杆菌经活化后，转接至MRS肉汤培养基中，2个平行，37℃条件下培养48h，用pH0.5-5.0试纸测定并记录培养48h的乳杆菌菌液pH值，按以下两个条件选择菌株进行液相色谱：条件一，pH值为2.5的菌株进行液相色谱；条件二，同种属的乳杆菌，挑取pH值较低的菌株进行液相色谱；确定液相色谱样品后，将上清液稀释5倍，加入浓硫酸进行前处理，上样前用0.22um针头滤器过滤。液相色谱相关参数如下：
仪器型号：Agilent，分析型液相色谱1200
色谱柱型号：伯乐，Aminex HPX-87H
流动相：0.005M H 2SO 4，速度0.6mL/min
检测器及检测波长：DAD，207nm；RID,示差折光信号
进样量：20μL。
5.产过氧化氢能力
乳杆菌经活化后，用移液器吸取2μL菌液点种于含0.25mg/mL 3,3',5,5'-四甲基联苯胺溶液及0.01mg/mL辣根过氧化物酶的MRS琼脂中，在24h、48h和72h观察时间点设置2个平行并将相同观察时间点的平皿置于相同的厌氧密封罐中，加入厌氧产气袋，37℃条件 下培养，培养满相应时间后，取出相应平皿，暴露于空气中，30min后观察显色反应并拍照记录：以德氏乳杆菌为阳性对照，比德氏乳杆菌产生的蓝色深计为4分，与德氏乳杆菌产生的蓝色相当计为3分，比德氏乳杆菌产生的蓝色浅计为2分，显蓝色非常弱计为1分(轻微显色反应)，不变色的计0分。
6.粘附Hela细胞试验
乳杆菌经活化后，离心洗涤菌体2次，PBS重悬，吸取100μL乳杆菌悬浮液于含Hela细胞的96孔细胞培养板，37℃静置孵育30min，用无菌PBS洗涤2次以洗去未粘附乳杆菌，然后每孔加入25μL胰酶溶液，置于37℃恒温箱消化细胞，待Hela细胞消化变成球状后，每孔加入75μL完全培养基，反复吹打均匀，完全消化后吸取20微升菌悬液，用无菌PBS 10倍梯度稀释，选择适当稀释梯度进行倾注法计数实验，37℃培养48h后计数。
上述及下述所用到的细菌培养基成分及配制方法如下：
MRS肉汤制备：称量MRS成品培养基粉末52.0g，溶解至1L蒸馏水中；加热煮沸，凉至室温加入0.55g半胱氨酸盐酸盐，搅拌溶解后调节pH值至6.5；装上定量分液器并通N 2，加热至沸腾，在微沸状态下煮20min，冷却后分装至10mL厌氧管中，118℃高温湿热灭菌20min，荫凉、避光存放、备用。
MRS固体培养基制备：称量MRS成品培养基粉末52.0g，琼脂粉15.0g，溶解至1L蒸馏水中，加热煮沸，沸腾后加入0.55g半胱氨酸盐酸盐，调节pH值至6.5，118℃高温湿热灭菌20min，荫凉、避光存放、备用。
过氧化氢半定量培养基制备：称量MRS成品培养基粉末52.0g，琼脂粉15.0g，溶解至1L蒸馏水中，调节pH值至6.5，118℃高温湿热灭菌20min，灭菌结束后放入50℃水浴锅保温30min，加入TMB(使TMB终浓度为0.25mg/mL)和HRP(使HRP终浓度为0.01mg/mL)混匀；冷却凝固后，标记培养基名称及配制日期，放于4℃冰箱待用。
无氧PBS的配制：称量磷酸二氢钾0.27g，磷酸氢二钠1.42g，氯化钠8g，氯化钾0.2g，溶解至1L的蒸馏水中，加热煮沸，凉至室温加入0.55g半胱氨酸盐酸盐，搅拌溶解后调节pH值至6.5，装上定量分液器并通N 2，加热至沸腾，在微沸状态下煮30min，冷却后分装至10mL厌氧管中，121℃高温湿热灭菌30min，荫凉、避光存放、备用。
无氧BHI液体培养基配制：称量BHI成品培养基粉末37.0g，溶解至1L蒸馏水中，加热煮沸，凉至室温加入0.55g半胱氨酸盐酸盐，搅拌溶解后调节pH值至6.5，装上定量分液器并通N 2，加热至沸腾，在微沸状态下煮20min，冷却和分装过程中通入N 2和CO 2(1:1比例)，分装至10mL厌氧管中，118℃高温湿热灭菌20min，荫凉、避光存放、备用。
无氧BHI半固体培养基配制：称量BHI成品培养基粉末37.0g，溶解至1L蒸馏水中；加热煮沸，凉至室温加入6g琼脂粉，0.55g半胱氨酸盐酸盐，搅拌溶解后调节pH值至6.5，装上定量分液器并通N 2，加热至沸腾，在微沸状态下煮20min，稍稍冷却，冷却和分装过程中通入N 2和CO 2(1:1比例)，及时分装至10mL厌氧管中，118℃高温湿热灭菌20min，荫凉、避光存放。
营养肉汤液体培养基配制：称量蛋白胨10g，牛肉粉3g，氯化钠5g溶解至1L蒸馏水中，调节pH至7.2，加热煮沸，凉至室温后分装，每支10mL；121℃高温湿热灭菌15min，荫凉、避光存放、备用。
营养肉汤固体培养基配制：称量蛋白胨10g，牛肉粉3g，氯化钠5g，琼脂粉6g溶解至1L蒸馏水中，调节pH至7.2，稍稍冷却，及时分装至10mL厌氧管中；121℃高温湿热灭菌15min，荫凉、避光存放、备用。
为描述方便，下述约氏乳杆菌Ljohn-1(保藏编号：CCTCC No.2019426)以下简称“A”,卷曲乳杆菌Lcris-2(保藏编号：CCTCC No.2019427)以下简称“B”,鼠李糖乳杆菌Lrham-6(保藏编号：CCTCC No.2019428)以下简称“C”，詹氏乳杆菌Ljen-10(保藏编号：CCTCC No.2019429)以下简称“D”，格氏乳杆菌Lgass-17(保藏编号：CCTCC No.2019430)以下简称“E”。
实施例1：单菌和组合乳杆菌产乳酸能力对比。
单一菌：乳杆菌经活化后，转接至MRS肉汤培养基中，2个平行，37℃条件下培养48h，用pH 0.5-5.0试纸测定并记录培养48h的乳杆菌菌液pH值，让菌株进行液相色谱，将上清液稀释5倍，加入浓硫酸进行前处理，上样前用0.22um针头滤器过滤。
组合菌：乳杆菌经活化转接后，选择3种及以上的菌株混合培养，3个平行，37℃条件下培养48h，离心，上清液稀释5倍，加入浓硫酸进行前处理，上样前用0.22um针头滤器过滤。液相色谱相关参数如下：
仪器型号：Agilent，分析型液相色谱1200
色谱柱型号：伯乐，Aminex HPX-87H
流动相：0.005M H 2SO 4，速度0.6mL/min
检测器及检测波长：DAD，207nm；RID,示差折光信号
进样量：20μL。
需要说明的是，值得说明的是，微生物生长长的菌体生物量和乳酸产量主要取决于培养基中的碳源类型和浓度、微生物代谢的途径、菌株对乳酸等有机酸的耐受度，接种量 一般只会影响到微生物生长的延迟期，而对微生物菌体的生长量和乳酸产量的影响较小。但是在这里需要注意的试验细节是，不同的接种量中，带入的背景有机物含量也会不同。那么在新的培养基中，高接种量组的有机物含量更高，导致不同接种量其生长量和产乳酸量不同，如果在试验过程忽略这部分细节，会造成错误的判断。
本实施例在同批次试验中，所选择的接种量都是一致的，上述产乳酸试验的乳杆菌接种量都是2％。
本实施例中，通过预实验发现乳杆菌在对数生长期的乳酸产量和微生物的生长曲线成正相关，在乳杆菌生长进入稳定期后，乳酸产量会有微弱的增加，与文献报道一致。乳杆菌达到生长稳定期的时间一般不超过24h，但是从24h到48h，部分乳杆菌的乳酸产量还会微弱增加，因此，选择了48h的时间点来分析乳酸产量。
实施例2:单菌和组合乳杆菌产过氧化氢能力对比。
单一菌：乳杆菌经活化后，用移液器吸取2μL菌液，点种于含0.25mg/mL的3,3',5,5'-四甲基联苯胺溶液及0.01mg/mL的辣根过氧化物酶的MRS琼脂中，在24h、48h和72h观察时间点设置2个平行并将相同观察时间点的平皿置于相同的厌氧密封罐中(以下表格及附图中提供的是48h的数据)，37℃培养，培养满相应时间后，取出相应平皿，暴露于空气中，30min后观察显色反应并拍照记录：以德氏乳杆菌为阳性对照，比德氏乳杆菌产生的蓝色深计为4分，与德氏乳杆菌产生的蓝色相当计为3分，比德氏乳杆菌产生的蓝色浅计为2分，显蓝色非常弱计为1分(轻微显色反应)，不变色的计0分。
组合菌：乳杆菌经活化转接后，选择3种及以上的菌株混合，用移液器吸取2μL菌液点种于含TMB及HRP的MRS琼脂中，37℃培养，培养满相应时间后，取出相应平皿，暴露于空气中，30min后观察显色反应并拍照记录：以德氏乳杆菌为阳性对照，比德氏乳杆菌产生的蓝色深计为4分，与德氏乳杆菌产生的蓝色相当计为3分，比德氏乳杆菌产生的蓝色浅计为2分，显蓝色非常弱计为1分(轻微显色反应)，不变色的计0分。
值得说明的是，乳杆菌通过乳酸氧化酶和/或丙酮酸氧化酶，将丙酮酸盐和或乳酸盐和氧气转化成过氧化氢。因此，乳杆菌的过氧化氢的产量受多种因素影响(代谢产物和氧气含量、菌体生长阶段和酶活性等有关)，在此类试验中，只去绝对定量乳杆菌的接种量意义不大。因此，我们在本试验中采取了半定量方法，所有菌株的接种量均为2微升，并点种到固体培养基上生长。设置了24h、48h和72h三个不同的时间点观察，并以参比菌株德氏乳杆菌作为参比，比较分离自健康妇女的乳杆菌与参比菌株的产过氧化氢能力，并发现过氧化氢显示度会随时间变化而变化。在单菌过氧化氢显示中，我们筛选的菌株除单菌D(詹氏 乳杆菌Ljen-10)和参比菌株的显示相当，其它菌株都不及德氏乳杆菌。但是，当我们组成配方菌后以后，大部分配方菌的过氧化氢显色强度超过或与德氏乳杆菌相当。另外，我们的前期研究发现用于配方的本申请所述的5个乳杆菌，共同生长时不会相互抑制，这些经过筛选评价的菌株组成的配方菌，以群落的形式共同生长繁殖，其微生态效应显著高于单菌。
实施例3单菌和组合乳杆菌抑制阴道加德纳菌能力对比。
单菌：乳杆菌经活化后，取0.1mL菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后37℃培养48h，取出平皿，用内径6mm的打孔器在琼脂培养基上打孔，获得菌饼；阴道加德纳菌经活化转接后，用无氧无菌PBS缓冲液以10倍梯度，将加德纳菌菌液稀释至100倍，分别取10 -1和10 -2稀释液0.5mL与5.25mL含5％马血清的BHI固体培养基混匀，倾注入9cm平皿内,完全凝固后，将乳杆菌菌饼轻放在BHI琼脂表面，每皿对称放4个菌饼，每株菌2个平行，放入厌氧的密封罐中，加厌氧产气袋，平皿正置培养48h，用游标卡尺测量抑菌圈大小。
组合菌：乳杆菌经活化转接后，选择3种及以上的菌株混合，取0.1mL菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后37℃培养48h，取出平皿，用内径6mm的打孔器在琼脂培养基上打孔，获得菌饼；阴道加德纳菌经活化转接后，用无氧无菌PBS缓冲液以10倍梯度，将加德纳菌菌液稀释至100倍，分别取10 -1和10 -2稀释液0.5mL与5.25mL含5％马血清的BHI固体培养基混匀(以下表格和附图显示的是10 -1的数据)，倾注入9cm平皿内，完全凝固后，将乳杆菌菌饼轻放在BHI琼脂表面，每皿对称放4个菌饼，每株菌2个平行，放入厌氧的密封罐中，加厌氧产气袋，平皿正置培养48h，用游标卡尺测量抑菌圈大小。
本实施例中，乳杆菌以1％接种量采用倾注法制备菌饼，培养48h后的菌饼的菌含量相当，菌饼上的色度类似，表明不同菌株的菌饼具有相当的均质性。
本实施例中，阴道加德纳菌活化以后，分别以1％和0.1％接种量，倾注于平板中(表格及附图显示的是1％的数据)。
实施例4：单菌和组合菌粘附Hela细胞能力对比。
单菌：乳杆菌经活化后，离心洗涤菌体2次，PBS重悬，吸取100μL乳杆菌悬浮液于含Hela细胞的96孔细胞培养板，37℃静置孵育30min，30min后用无菌PBS洗涤2次以未洗去粘附乳杆菌，向含Hela细胞96孔细胞培养板每孔加入25μL胰酶溶液，置于37℃恒温箱消化细胞，待Hela细胞消化变成球状后，每孔加入75μL完全培养基，反复吹打均匀，完全消化后吸取20微升菌悬液，用无菌PBS 10倍梯度稀释，选择适当稀释梯度进行倾 注法计数实验，37℃培养48h后计数。
组合菌：乳杆菌经活化转接后，离心洗涤菌体2次，PBS重悬，选择3种及以上的菌株菌液混合均匀，吸取100μL乳杆菌悬浮液于含Hela细胞的96孔细胞培养板，37℃静置孵育30min，30min后用无菌PBS洗涤2次以未洗去粘附乳杆菌；孵育满相应时间后，向含Hela细胞96孔细胞培养板每孔加入25μL胰酶溶液，置于37℃恒温箱消化细胞，待Hela细胞消化变成球状后，每孔加入75μL完全培养基，反复吹打均匀，完全消化后吸取20微升菌悬液，用无菌PBS 10倍梯度稀释，选择10 -4稀释梯度进行倾注法计数实验，37℃培养48h后计数。
本实施例中，们采用台盼蓝染色显微镜计数法，随机选择96孔细胞培养板上的其中4孔的菌悬液，进行Hela细胞计数，计算出平均每孔的Hela细胞数量；再取96孔板上的菌悬液，梯度稀释后倾注到MRS固体培养基中(称为“倾注法”)，培养后计算培养菌悬液的乳杆菌数量，计算出每孔粘附的乳杆菌数量。从而算出相应时间条件下单个Hela细胞粘附的乳杆菌数量，
本实施例中，考虑到益生筛选时的单菌数量较多，为节省实验时间，单菌对Hela细胞的粘附时间只设置了30min观察时间点。在确定优选菌株后，并组合出了16个配方，故而增加了一个4h的观察时间点。4h和30min的试验结果显示，随着粘附时间增长，组合菌对Hela细胞的粘附效果增加。说明书附图及表中为30min的数据。
实施例5：单菌和组合菌抑制金黄色葡萄球菌能力、抑制绿脓假单胞菌能力、抑制大肠杆菌能力、抑制乙型副伤寒沙门氏菌能力和抑制痢疾志贺菌能力对比。
单菌：乳杆菌经活化后，取0.1mL菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后37℃培养48h，取出平皿，用内径6mm的打孔器在琼脂培养基上打孔，获得菌饼；金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌经活化转接后，用0.9％生理盐水以10倍梯度，将病原菌菌液稀释至100倍，分别取10 -1和10 -2稀释液0.5mL与5mL营养琼脂固体培养基混匀(以下表格和附图显示的是10 -1的数据)，倾注入9cm平皿内,完全凝固后，将乳杆菌菌饼轻放在营养琼脂表面，每皿对称放4个菌饼，每株菌2个平行，放入密封罐中，平皿正置培养24h，用游标卡尺测量抑菌圈大小。
组合菌：抑制乙型副伤寒沙门氏菌能力和抑制痢疾志贺菌能力
乳杆菌经活化转接后，选择3种及以上的菌株混合，取0.1mL菌液与MRS固体培养基混匀，倾注入6cm平皿内，完全凝固后37℃培养48h，取出平皿，用内径6mm的打孔器在琼脂培养基上打孔，获得菌饼；金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌经活化转接后，用0.9％生理盐水以10倍梯度，将病原菌菌液稀释至 100倍，分别取10 -1和10 -2稀释液0.5mL与5mL营养琼脂固体培养基混匀，倾注入9cm平皿内,完全凝固后，将乳杆菌菌饼轻放在营养琼脂表面，每皿对称放4个菌饼，每株菌2个平行，放入密封罐中，平皿正置培养24h，用游标卡尺测量抑菌圈大小。
本实施例中，乳杆菌以1％接种量采用倾注法制备菌饼，培养48h后的菌饼的菌含量相当，菌饼上的色度类似，表明不同菌株的菌饼具有相当的均质性。
本实施例中，乳杆菌以1％接种量采用倾注法制备菌饼，培养48h后的菌饼的菌含量相当，菌饼上的色度类似，表明不同菌株的菌饼具有相当的均质性。
本实施例中，本实施例所述致病菌活化以后，分别以1％和0.1％接种量，倾注于平板中(表格及附图显示的是1％的数据)。
经过上述实施例1～实施例5对比实验，对比结果如下表6：
表6同一批次单菌及组合菌各益生能力比较
Figure PCTCN2020107915-appb-000009
Figure PCTCN2020107915-appb-000010
实施例6：多联乳杆菌中各菌株之间的拮抗效应和共生效应试验
将待测定的菌株之一在培养基平板划直线，再将待测定菌株之二横向接种在待测菌株之一的两侧，成十字交叉培养18～24h，测定交叉点抑菌宽度。
经过试验，各菌株之间的拮抗效应和共生效应(抑菌半径mm)结果如下表7：
表7各单菌在固体平皿上共生的抑菌圈直径(mm)
  A B C D E
A 0.00 0.00 0.00 0.00 0.00
B 0.00 0.00 0.00 0.00 0.00
C 0.00 0.00 0.00 0.00 0.00
D 0.00 0.00 0.00 0.00 0.00
E 0.00 0.00 0.00 0.00 0.00
将待测定的单一菌株接种于MRS肉汤，37℃培养约8h，同时对接种前单一菌株10倍梯度稀释至合适梯度，计数接种前菌液浓度，每梯度3平行，平皿放入厌氧的密封罐中，加厌氧产气袋，37℃条件下培养48h；然后将待测定的菌株混合后接种于MRS肉汤，混合菌中各菌接种浓度与单一菌株接种浓度相同，37℃培养约8h；单菌及混合菌MRS肉汤培养约8h后，10倍梯度稀释至合适梯度，涂布计数，每梯度三个平行，平皿放入厌氧的 密封罐中，加厌氧产气袋，37℃条件下培养48h；平皿厌氧培养48h后，对单菌平皿计算接种前后菌落数，分析单菌扩增倍数，同时对混合菌平皿上的菌落进行质谱鉴定，通过质谱判定菌种，进而分析混合菌中各单菌扩增倍数，经过试验，各菌株之间无抑制，能共同生长，肉汤共培养扩增数据见表8。
表8各单菌在MRS肉汤上共生的扩增倍数
Figure PCTCN2020107915-appb-000011
本实施例中，单菌和混合培养的扩增倍数均按照平均数计算，单菌做一次接种，因此单菌培养计算的扩增倍数只有一组数据，考虑混合菌接种的均一性，所以混合培养同时做两次接种，因而混合培养计算的扩增倍数有两组平行数据。
按照上述实施例，便可很好地实现本发明。值得说明的是，基于上述结构设计的前提下，为解决同样的技术问题，即使在本发明上做出的一些无实质性的改动或润色，所采用的技术方案的实质仍然与本发明一样，故其也应当在本发明的保护范围内。

Claims (8)

  1. 多联乳杆菌组合物，其特征在于，多联乳杆菌组合物至少包括以下三种活性成分：
    约氏乳杆菌(Lactobacillus johnsonii)、格氏乳杆菌(Lactobacillus gasseri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、卷曲乳杆菌(Lactobacillus crispatus)、詹氏乳杆菌(Lactobacillus jensenii)；
    约氏乳杆菌为约氏乳杆菌CCTCC No.2019426；格氏乳杆菌为格氏乳杆菌CCTCC No.2019430；鼠李糖乳杆菌为鼠李糖乳杆菌CCTCC No.2019428；卷曲乳杆菌为卷曲乳杆菌CCTCC No.2019427；詹氏乳杆菌为詹氏乳杆菌CCTCC No.2019429。
  2. 根据权利要求2所述的多联乳杆菌组合物，其特征在于，组合物活菌数为105～1011CFU/g，每种单菌含量不低于105CFU/g。
  3. 根据权利要求2所述的多联乳杆菌组合物的发酵液。
  4. 权利要求1～2任一项所述的多联乳杆菌组合物或权利要求3所述的发酵液在制备预防、治疗细菌性阴道炎致病菌药品或卫生保健品中的应用。
  5. 权利要求1～2任一项所述的多联乳杆菌组合物或权利要求3所述的发酵液在制备外生殖器卫生用品中的应用。
  6. 权利要求1～2任一项所述的多联乳杆菌组合物或权利要求3所述的发酵液在制备调节阴道菌群平衡的药品或卫生保健品中的应用。
  7. 权利要求1～2任一项所述的多联乳杆菌组合物或权利要求3所述的发酵液在制备具有预防、治疗致病菌的药品、保健品中的用途，致病菌包括但不限于阴道加德纳菌、金黄色葡萄球菌、绿脓假单胞菌、大肠杆菌、乙型副伤寒沙门氏菌和痢疾志贺菌中的任意一种或多种。
  8. 权利要求1～2任一项所述的多联乳杆菌组合物或权利要求3所述的发酵液在制备剖腹产婴儿外用护理品中的应用。
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