CN118389377A - Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health - Google Patents
Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health Download PDFInfo
- Publication number
- CN118389377A CN118389377A CN202410853045.7A CN202410853045A CN118389377A CN 118389377 A CN118389377 A CN 118389377A CN 202410853045 A CN202410853045 A CN 202410853045A CN 118389377 A CN118389377 A CN 118389377A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus gasseri
- nhnk
- improving
- vaginal
- candida albicans
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000186606 Lactobacillus gasseri Species 0.000 title claims abstract description 64
- 210000005000 reproductive tract Anatomy 0.000 title claims abstract description 19
- 230000036541 health Effects 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 230000001018 virulence Effects 0.000 claims abstract description 18
- 230000012010 growth Effects 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 230000004523 agglutinating effect Effects 0.000 claims abstract description 4
- 229940095731 candida albicans Drugs 0.000 claims description 68
- 241000222122 Candida albicans Species 0.000 claims description 67
- 244000052616 bacterial pathogen Species 0.000 claims description 46
- 238000000855 fermentation Methods 0.000 claims description 30
- 230000004151 fermentation Effects 0.000 claims description 30
- 230000035755 proliferation Effects 0.000 claims description 18
- 241000207201 Gardnerella vaginalis Species 0.000 claims description 17
- 241001148134 Veillonella Species 0.000 claims description 16
- 230000001737 promoting effect Effects 0.000 claims description 13
- 230000001717 pathogenic effect Effects 0.000 claims description 11
- 230000009286 beneficial effect Effects 0.000 claims description 10
- 230000005764 inhibitory process Effects 0.000 claims description 10
- 241000218492 Lactobacillus crispatus Species 0.000 claims description 8
- 230000002222 downregulating effect Effects 0.000 claims description 7
- 241001561398 Lactobacillus jensenii Species 0.000 claims description 6
- 101150057219 HWP1 gene Proteins 0.000 claims description 4
- 230000002538 fungal effect Effects 0.000 claims description 4
- 244000000072 vaginal pathogen Species 0.000 claims description 4
- 230000006872 improvement Effects 0.000 claims description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 230000028327 secretion Effects 0.000 claims description 2
- 244000053095 fungal pathogen Species 0.000 claims 2
- 238000002474 experimental method Methods 0.000 abstract description 19
- 230000002401 inhibitory effect Effects 0.000 abstract description 15
- 239000002131 composite material Substances 0.000 abstract description 7
- 230000001105 regulatory effect Effects 0.000 abstract description 7
- 239000012528 membrane Substances 0.000 abstract description 6
- 244000005700 microbiome Species 0.000 abstract description 6
- 230000006870 function Effects 0.000 abstract description 5
- 241000233866 Fungi Species 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 206010046914 Vaginal infection Diseases 0.000 abstract 6
- 239000000047 product Substances 0.000 description 40
- 239000007788 liquid Substances 0.000 description 33
- 230000001580 bacterial effect Effects 0.000 description 18
- 239000001963 growth medium Substances 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 11
- 238000005119 centrifugation Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000004364 calculation method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000009630 liquid culture Methods 0.000 description 8
- 241000965254 Apostichopus japonicus Species 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 230000004520 agglutination Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 244000052769 pathogen Species 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 238000012404 In vitro experiment Methods 0.000 description 6
- 241000186660 Lactobacillus Species 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 238000004220 aggregation Methods 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000005189 flocculation Methods 0.000 description 5
- 230000016615 flocculation Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 210000001215 vagina Anatomy 0.000 description 5
- 230000007923 virulence factor Effects 0.000 description 5
- 239000000304 virulence factor Substances 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 230000007918 pathogenicity Effects 0.000 description 4
- 239000004576 sand Substances 0.000 description 4
- 230000003068 static effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 101150094738 ALS3 gene Proteins 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000002247 constant time method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 239000013049 sediment Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000251511 Holothuroidea Species 0.000 description 2
- 101000664887 Homo sapiens Superoxide dismutase [Cu-Zn] Proteins 0.000 description 2
- 241001518729 Monilinia Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241001533207 Veillonella atypica Species 0.000 description 2
- 241001116698 Veillonella montpellierensis Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000032770 biofilm formation Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000004877 mucosa Anatomy 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010006533 ATP-Binding Cassette Transporters Proteins 0.000 description 1
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 241001448631 Candidula Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241000207202 Gardnerella Species 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001116693 Megasphaera micronuciformis Species 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 208000037009 Vaginitis bacterial Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000007366 host health Effects 0.000 description 1
- AKPUJVVHYUHGKY-UHFFFAOYSA-N hydron;propan-2-ol;chloride Chemical compound Cl.CC(C)O AKPUJVVHYUHGKY-UHFFFAOYSA-N 0.000 description 1
- 230000008004 immune attack Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- -1 typical Species 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- 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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Communicable Diseases (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Oncology (AREA)
- Gynecology & Obstetrics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses lactobacillus gasseri and application thereof in preparing a product for improving genital tract health, and relates to the technical field of microorganisms. The invention discloses a lactobacillus gasseri, which is lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024420 in 3 months and 7 days of 2024. Experiments show that NHNK-607 has the functions of inhibiting the growth of colpitis bacteria, regulating virulence genes of colpitis bacteria, inhibiting the composite biological membrane of colpitis bacteria-fungi, competing for combining colpitis fungus hypha and agglutinating colpitis bacteria, and can be used for preparing medicines or products for improving colpitis, etc.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus gasseri and application thereof in preparing a product for improving genital tract health.
Background
Sea cucumber is an aquatic species with extremely high nutrition and economic value, and the rapid development of the sea cucumber culture industry has become an important component part of the sea water culture industry. Edible species are typically rich in symbiotic bacteria that are involved in the nutrient absorption, energy metabolism and growth and health of the host. Researches show that the richness of the intestinal bacterial community of apostichopus japonicus (Apostichopus japonicus) in Shandong Jib, shandong Qingdao and Liaoning Dalianxi coast is highest. Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes, with marine microorganisms being more likely than terrestrial microorganisms to synthesize structurally unique enzymes and secondary metabolites.
Virulence (virulence) refers to the severity of a disease caused by a pathogen, and is also known as virulence as the pathogenicity of the pathogen. Virulence factors (virulent factor) refer to the structural and physiological characteristics of the pathogen that cause the disease, and generally refer to a class of effector molecules, such as proteins, lipid molecules, and functional units of combinations thereof, that play an important role in the pathogenicity of the pathogen. Accurate detection of Virulence Factor Genes (VFGs) is critical to accurate treatment and prognostic management of infections by highly virulent pathogens. Pathogenic bacteria are also one of normal symbiotic flora of vagina, and only in specific environments, the pathogenic bacteria are shown, and virulence factors become important indexes for medical detection of toxicity of pathogenic bacteria.
Under normal conditions, lactobacillus is a dominant bacterium of vaginal flora, including lactobacillus jensenii (Lactobacillus jensenii), lactobacillus crispatus (Lactobacillus crispatus) and the like, and the lactobacillus can effectively inhibit the growth of pathogenic bacteria and maintain the acidic and healthy environment of vagina by competitively adhering to vaginal epithelial cells, metabolizing and secreting substances such as lactic acid, hydrogen peroxide (H 2O2) and bacteriocin.
Bacterial vaginosis is a common disease of women in women and severely threatens the reproductive health of the women. The common harmful bacteria are represented by gardnerella vaginalis (GARDNERELLA VAGINALIS), can secrete various virulence factors, can form a biological film to be adhered to vaginal epithelial cells, can avoid immune attack, and can increase the capability of resisting an organism immune system.
Three bacteria were found to have deleterious effects on the cervical microenvironment, atypical veillonella (Veillonella atypica), montrea (Veillonella montpellierensis) and megasphaerella (MEGASPHAERA MICRONUCIFORMIS), respectively. Atypical veillonella and monpeteri reduce lactic acid in the vagina, providing conditions for pathogen infection. Megasphaerella promotes cell death by increasing inflammation and by producing certain fat molecules, thereby further promoting disease progression. When the environment in the vagina of a female is changed or the immunity is low, candida albicans is easy to be infected, the pathogenicity of the Candida albicans is related to a plurality of virulence factors, and hyphae formed by budding in the morbidity process are attached to vaginal mucosa epithelial cells, so that the damage to the mucosa cells is increased. The vaginal pathogenic bacteria can be attached to any position of candida albicans, including candida albicans itself, and even polysaccharides on the surface of thalli. In addition to the wide range of movement, this intricate cell pellet is also very flexible, and studies have also demonstrated that bacterial-fungal cooperation produces a greater number of complex biofilms than those produced by a single species.
Marine microorganisms are one of the most well known sources of marine bioactive substances, including algae, bacteria, fungi, protozoa, and the like. The bioactive substances have important application values in various fields such as medicines, foods, cosmetics and the like. Therefore, the marine industry related product developed by utilizing the microbiological technology is provided, and the marine industry related product has important practical significance.
Disclosure of Invention
In view of the above, the invention provides lactobacillus gasseri and application thereof.
The invention provides a lactobacillus gasseri (Lactobacillus gasseri), which is lactobacillus gasseri (Lactobacillus gasser) NHNK-607 and is preserved in China center for type culture collection (CCTCC for short, address: eight channel 299 No. in Wuchang district of Wuhan, university of Wuhan, post code 430072) for 3 months and 7 days in 2024, and the preservation number is lactobacillus gasseri with CCTCC NO: M2024420.
The invention provides application of the lactobacillus gasseri in preparing a product for improving genital tract health, wherein the improvement of genital tract health comprises inhibition of vaginal pathogenic bacteria proliferation, and the inhibition of vaginal pathogenic bacteria proliferation comprises inhibition of atypical veillonella and Montrea growth.
Further, the improving genital tract health includes modulating a vaginal pathogenic virulence gene including at least one of downregulating candida albicans virulence genes HWP1, ALS3 and downregulating gardnerella vaginalis virulence gene vly, sld, pat, gtf, atm.
Further, the improving genital tract health includes competing for binding to a fungal hypha of a vaginal pathogen, the competing for binding to a fungal hypha of a vaginal pathogen being a candida albicans hypha.
Further, the improvement of genital tract health includes agglutinating vaginal pathogens including candida agglutinates, atypical veillonella, gardnerella vaginalis.
Further, the improving genital tract health comprises promoting vaginal beneficial proliferation, including promoting lactobacillus jensenii proliferation, lactobacillus crispatus proliferation.
Further, the Lactobacillus gasseri in the genital tract health improving product comprises one or two of the following (1) or (2):
(1) The strain and/or inactivated strain of lactobacillus gasseri described above;
(2) Fermentation and/or secretion products of the above-mentioned lactobacillus gasseri.
The invention discloses Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607, the preservation number of which is CCTCC NO: M2024420. Experiments show that NHNK-607 has the functions of inhibiting the proliferation of vaginal pathogenic bacteria, regulating the virulence genes of the vaginal pathogenic bacteria, inhibiting the complex biological membrane of the vaginal pathogenic bacteria and fungi, competing for combining with the fungus hyphae of the vaginal pathogenic bacteria, agglomerating the vaginal pathogenic bacteria and promoting the proliferation of beneficial bacteria of the vagina, and can be used for preparing products for improving the health of genital tracts, etc.
Description of biological preservation
Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 was deposited at China center for type culture collection (CCTCC, address: eight-channel 299 No. of Wuchang district of Wuhan, university of Wuhan, post code 430072) at 3 and 7 days 2024, and the preservation number was CCTCC NO: M2024420.
Drawings
FIG. 1 is a colony chart and gram stain chart of MRS plates of Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 of the present invention;
FIG. 2 shows the coagglutination of the inactivated bacteria and the pathogenic bacteria of Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 of the present invention;
FIG. 3 shows the rapid coagglutination of live Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 and Candida albicans in accordance with the present invention;
FIG. 4 shows the combination of Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 inactivated bacteria and Candida albicans mycelium according to the present invention;
FIG. 5 is a gram stain of the combination of Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 inactivated bacteria and Candida albicans mycelium according to the present invention.
The invention will be further described in the following detailed description in conjunction with the above-described figures.
Detailed Description
The invention provides lactobacillus gasseri and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The lactobacillus gasseri NHNK-607 of the invention is derived from the intestinal tract of apostichopus japonicus and is identified as lactobacillus gasseri (Lactobacillus gasseri) by 16S rDNA. The strain is gram-positive, rod-shaped and slightly bent under a microscope; is suitable for standing growth in MRS liquid culture medium at 37 ℃, and the thalli presents white precipitation. The MRS plate grows to form flat, peripheral diffuse and edge regular colonies.
Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607, accession number: china center for type culture Collection, address: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: and 2024, 3 and 7 days, wherein the preservation number is CCTCC NO: M2024420.
Further, the present invention provides lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 for use in the described invention, either in live or inactivated form, or in fermentation product (i.e. supernatant) form, said derivative form preferably being selected from the group consisting of: metabolites, metabolic biological products, probiotics, cell walls and components thereof, extracellular polysaccharides and compounds containing immunogenic components, preferably selected from the group consisting of: fermentation product, living bacteria and inactivated bacteria.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 has the effect of inhibiting the growth of atypical veillonella and Montrea of vaginal pathogenic bacteria, and the inhibition rate reaches 27.27% -78.39%.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 has the function of down regulating the expression of the gardnerella vaginalis lysozyme gene vly, sialidase gene sld, vaginal epithelial cell adhesion related TadE-like protein gene pat, glycosyltransferase gene gtf and self metabolism related ABC transporter permease gene atm, and the relative expression of the genes is down regulated to 0.04-0.81 times.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 has the effect of inhibiting the expression of candida albicans related cell surface glycoprotein genes ALS1/ALS3 and hypha cell wall protein gene HWP1, and the relative expression quantity of the genes is reduced to 0.06-0.91 times.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 has the effects of inhibiting the formation of gardnerella vaginalis, monilinia mongolica, atypical veillonella, candida albicans biomembrane and candida albicans vaginalis, monilinia candidula and atypical veillonella-candida albicans composite biomembrane, and the inhibition rate is 31.41% -94.41%.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 has the functions of agglutinating vaginal pathogenic bacteria candida albicans, atypical veillonella and gardnerella vaginalis, and the agglutination rate can reach 23.98% -43.66%.
In vitro experiments show that the lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 fermentation product has the effect of promoting the proliferation of beneficial vaginal bacteria lactobacillus janus and lactobacillus crispatus, and the proliferation promoting rate can reach 13.92% -44.19%.
The reagent consumables adopted by the invention are all common commercial products, and the invention is further described below by combining examples:
examples 1 NHNK-607 isolation
Taking fresh Apostichopus japonicus in Qingdao market, wiping body surface of Stichopus japonicus with cotton ball stained with sterile physiological saline, taking out intestinal canal of Stichopus japonicus, cutting with sterile surgical scissors and surgical knife, washing with 0.9% sterile physiological saline for 2-3 times, placing washed and cut intestinal canal of Stichopus japonicus together into triangular flask containing MRS liquid culture medium of small glass beads for enrichment culture, culturing at 37deg.C at 160r/min, and shake culturing for 24 hr. And (3) coating a proper amount of culture bacterial liquid on an MRS solid culture medium, placing the culture bacterial liquid in an anaerobic bag, culturing the culture bacterial liquid at the constant temperature of 37 ℃ for 48 hours, and then picking white bacterial colonies, repeatedly marking and screening until uniform single bacterial colonies are obtained, and naming the single bacterial colonies as NHNK-607.
Gram staining microscopy: strains NHNK-607 are gram-positive colonies, are rod-shaped and slightly bent under a microscope; the white semitransparent circular colony with smooth and round surface can be formed by growing on an MRS flat plate, and the edge is neat; can grow in uniform turbidity in MRS liquid culture medium, and the thalli are white precipitate after long-term storage. As shown in fig. 1.
Nucleic acid identification of examples 2NHNK-607
1. 16S rDNA gene sequence analysis:
Single colonies were picked up in MRS liquid medium, cultured overnight at 37℃and centrifuged at 8000 rpm for 1min to collect the colonies, which were then subjected to the procedure according to the instructions of the gram-positive DNA extraction kit. The primers were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20. Mu.L. The PCR amplification procedure was 95℃for 5min,94℃for 15s,58℃for 15s,72℃for 1min,35 cycles; extending at 72℃for 10min.
2. Results
The PCR products were sequenced and compared for homology (BLASTN) with published standard sequences in the GenBank database to give strain NHNK-607 as Lactobacillus gasseri (Lactobacillus gasseri).
EXAMPLES 3NHNK-607 experiments to inhibit the growth of vaginal pathogenic bacteria
1. NHNK-607 preparation of fermentation products
Single colony of lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 is selected and placed in MRS liquid culture medium, and after standing culture for 48 hours at 37 ℃, the dilution of MRS is adjusted to be OD 600 =0.2, centrifugation is carried out at 5000rpm for 10min, the supernatant is taken and filtered by 0.22 mu M, and the supernatant is filtered by a 0.22 mu M filter membrane, thus obtaining cell-free fermentation products.
2. Preparation of pathogenic bacteria liquid
Vaginal pathogenic bacteria: atypical veillonella (Veillonella Atypica) DSM 20739, montrea (Veillonella montpellierensis) DSM 17217 single colonies were picked and inoculated with Yu Weirong g liquid medium and PYG liquid medium, respectively, and incubated overnight at 37 ℃ to stationary phase.
3. Experiment for inhibiting growth of pathogenic bacteria
Atypical veillonella and Montrea are respectively inoculated with pathogenic bacteria liquid according to the proportion of 2%, the fermentation products are respectively added into the pathogenic bacteria liquid according to the proportion of 5% (V/V), an equal volume of MRS liquid culture medium is added as a reference, the culture is carried out for 24 hours at 37 ℃, the concentration (OD 600) of the bacterial liquid is detected, and the pathogenic bacteria inhibition rate is calculated. The calculation formula is as follows: inhibition = (1-absorbance of experimental group/absorbance of control group) ×100%
The results are shown in Table 1 below:
TABLE 1
The results show that NHNK-607 cell-free fermentation product can effectively inhibit the growth of atypical veillonella and Montrea vaginalis pathogenic bacteria.
EXAMPLES 4 NHNK-607 Regulation of the Gardnerella vaginalis virulence Gene expression experiments
1. NHNK-607 fermentation product and preparation of inactivated thallus
Selecting single colony of Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607 in MRS liquid culture medium, standing at 37deg.C for 48 hr, regulating bacterial liquid with MRS to OD 600 =1.0, centrifuging at 5000rpm for 10min to obtain supernatant, and filtering with 0.22 μm filter membrane to obtain aseptic fermentation product; the cells were then conditioned with PBS to OD 600 =1.0, washed twice with PBS solution and resuspended to OD 600 =1.0. Sterilizing at 121deg.C for 15min to obtain inactivated bacteria.
2. Experiment for regulating gardnerella vaginalis virulence gene expression
The vaginal gardnerella (GARDNERELLA VAGINALIS) BNCC354890 frozen tube obtained by separating vaginal secretion was inoculated into fresh Columbia liquid basal medium at a ratio of 2%, and anaerobic culture was performed at 37℃for 48 h. Taking 3mL of cultured gardnerella vaginalis bacterial liquid, adding 1mL NHNK-607 fermentation product or inactivated bacteria (equal volume MRS or PBS is added into a control group), and carrying out anaerobic culture for 2h at 37 ℃ by 1mL of fresh Columbia liquid basal culture medium. After the culture is finished, centrifuging at 8000rpm for 1min to obtain thalli, extracting total RNA according to the instruction of a kit, detecting the concentration and purity of the RNA, reversely transcribing the RNA into cDNA, taking 16S as an internal reference gene, and detecting the expression of vly, sld, pat, gtf, atm genes by adopting a qPCR fluorescent quantitative technology. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
The formula: f=2 -ΔΔCT, wherein:
△CT Experiment =CT Experiment -CT Internal reference （ Experiment ）;
△CT Control =CT Control -CT Internal reference （ Control ）;
△△CT=△CT Experiment -△CT Control .
the results are shown in tables 2 and 3 below:
TABLE 2
TABLE 3 Table 3
The results show that NHNK-607 has the effect of down regulating the expression of gardnerella vaginalis virulence genes.
EXAMPLES 5 NHNK-607 experiments on the modulation of the expression of the virulence Gene of Candida albicans
1. NHNK-607 fermentation product and preparation of inactivated thallus
The preparation is described in example 4.
2. Experiment for regulating virulence gene expression of candida albicans
The Candida albicans (Candida albicans) CGMCC 2.4550 freeze-storage tube is inoculated into fresh sand culture medium according to the proportion of 2 percent, and the culture is kept stand for 24 hours at 37 ℃. Adding 3 mL cultured candida albicans liquid into 1mL NHNK-607 fermentation product or inactivated bacteria (equal volume of MRS or PBS is added into a control group), and 1mL of fresh sand culture medium, and standing and culturing at 37 ℃ for 24 hours. After the culture is finished, centrifugation is carried out at 8000rpm for 1min to obtain thalli, total RNA is extracted according to the instruction of a kit, the concentration and purity of the RNA are detected, the RNA is reversely transcribed into cDNA, TDH1 is used as an internal reference gene, and qPCR is adopted to detect the expression of HWP1, ALS1 and ALS3 genes. The relative expression fold f=1 for the control group gene was calculated for each sample F value using the method of 2 -ΔΔCT, and the calculation method was referred to in example 3.
The results are shown in tables 4 and 5 below:
TABLE 4 Table 4
TABLE 5
The results show that NHNK-607 fermentation products and inactivated bacteria have the function of down regulating the expression of candida albicans virulence genes.
Examples 6 NHNK-607 experiments to inhibit the biofilm formation of pathogenic bacteria
1. NHNK-607 fermentation product and preparation of inactivated thallus
The preparation is described in example 4.
2. Preparation of pathogenic bacteria liquid
Pathogenic bacteria are prepared by the following steps: the single colonies of gardnerella vaginalis BNCC354890, atypical veillonella DSM 20739 and Montrea angustifolia DSM 17217 are respectively picked up by candida albicans CGMCC 2.4550 and respectively inoculated into a liquid basal medium of Columbia, a liquid medium of veillonella and a liquid medium of Serratia, cultured overnight at 37 ℃, and after the culture is finished, OD 600 =0.3 is respectively regulated by using the respective liquid medium.
3. Biological film experiment for inhibiting pathogenic bacteria
100 Mu L of pathogenic bacteria liquid is added into a 96-well plate, 100 mu L NHNK-607 fermentation products or inactivated bacteria are added into an experimental group, an equal volume of MRS culture medium or PBS solution is added into a control group, 3 groups are arranged in parallel, and the culture is carried out for 24 hours at 37 ℃. After the incubation was completed, the supernatant was discarded, and 100. Mu.L of sterile PBS was added to each well and washed twice, followed by adding 100. Mu.L of 4% paraformaldehyde fixing solution to each well, and fixing at room temperature for 30min. The fixative was discarded, 100. Mu.L of crystal violet was added to each well and stained at room temperature for 30min. After the dyeing is finished, the substrate is washed twice by using sterile PBS and dried, 100 mu L of absolute ethyl alcohol is added into each hole, the substrate is kept stand for 1min, the light absorption value at 600nm is measured, and the inhibition rate is calculated according to a formula.
The calculation formula and the results are shown in the following tables 6 and 7:
Table 6: NHNK-607 fermentation products inhibit pathogenic bacteria biofilm formation
Table 7: NHNK-607 inactivated bacteria for inhibiting formation of pathogenic bacteria biofilm
The results show that NHNK-607 fermentation products and inactivated bacteria have the effect of inhibiting the formation of pathogenic bacteria biological films.
Examples 7 NHNK-607 experiments to inhibit the formation of a pathogenic bacterial-fungal composite biofilm
1. NHNK-607 fermentation product and preparation of inactivated thallus
The preparation is described in example 4.
2. Preparation of pathogenic bacteria liquid
The preparation is described in example 6.
3. Experiment for inhibiting pathogenic bacteria-fungi composite biological film
100 Mu L of candida albicans liquid is mixed with 100 mu L of other bacterial pathogenic bacteria liquid and added into a 96-well plate respectively, 100 mu L NHNK-607 fermentation products or inactivated bacteria are added into an experimental group, an equal volume of MRS culture medium or PBS solution is added into a control group, 3 groups are parallel, and the culture is carried out for 24 hours at 37 ℃. The method of fixing, staining and measuring 96-well plates is described in example 5.
The calculation formula and the results are shown in the following tables 8 and 9:
table 8: NHNK-607 fermentation products inhibit formation of bacterial-fungal composite biofilm
Table 9: NHNK-607 inactivated bacteria for inhibiting formation of bacteria-fungi composite biological film
The results show that NHNK-607 fermentation products/inactivated bacteria inhibit the formation of pathogenic bacteria-fungi composite biological film.
EXAMPLES 8 NHNK-607 experiments on agglutination of pathogenic bacteria
1. Preparation of NHNK-607 inactivated thallus
The preparation is described in example 4.
2. Preparation of pathogenic bacteria liquid
The method for culturing candida albicans, atypical veillonella and gardnerella vaginalis is described in example 6. After the incubation was completed, the supernatant was discarded by centrifugation at 5000rpm for 10min, followed by washing twice with sterile PBS buffer, and then resuspended in sterile PBS buffer to adjust OD 600 = 1.0.
3. Agglutination
Mixing pathogenic bacteria liquid and NHNK-607 inactivated bacteria liquid according to the volume ratio of 1:1, using pathogenic bacteria and PBS as control, standing for 30min to observe flocculation condition.
4. Index measurement
The reaction was carried out for 30 minutes by sampling the reaction mixture of NHNK-607, the pathogenic bacteria and NHNK-607 and the pathogenic bacteria in a range of 50. Mu.L at the uppermost layer of the liquid surface, pipetting the reaction mixture, transferring the reaction mixture to a 96-well plate, and measuring the absorbance at OD=600 nm. Meanwhile, the aggregated precipitate was stained with a gram-type dye and then observed for the state of bacterial aggregation. As shown in fig. 2.
The calculation formula is as follows:
aggregation ratio (%) = [ (ax+ay) -2Amix ]/(ax+ay) ×100%;
Note that: ax, OD 600 values were measured at reaction time for NHNK-607 alone; ay, OD 600 value is measured by single pathogenic bacteria in the reaction time; amix OD 600 values were measured at the reaction time after mixing NHNK-607 with the pathogen.
Table 10
The result shows that NHNK-607 inactivated bacteria can agglutinate and catch candida albicans, atypical veillonella and gardnerella vaginalis, and further realize physical contact with pathogenic bacteria, thereby playing the roles of NHNK-607 in down-regulating virulence genes of the pathogenic bacteria, reducing pathogenicity, inhibiting formation of biological films of the pathogenic bacteria and reducing the colonization capability of the pathogenic bacteria.
EXAMPLES 9NHNK-607 live bacteria quick-setting Candida albicans
1. NHNK-607 activation and preparation of bacterial suspension
NHNK-607 glycerol bacteria are inoculated into fresh MRS culture medium according to the proportion of 2 percent, and are subjected to static culture for 24 hours at 37 ℃. After the end of the incubation, viable cells were obtained by centrifugation at 5000rpm for 10min, washed twice with sterile PBS, and then OD 600 = 1.0 was adjusted with PBS.
2. Preparation of candida albicans liquid
The glycerol bacteria of candida albicans are inoculated into a fresh sand culture medium according to the proportion of 2 percent, and the static culture is carried out for 24 hours at 37 ℃. After the incubation was completed, the cells were obtained by centrifugation at 5000rpm for 10min, washed twice with sterile PBS, and then OD 600 =1.0 was adjusted with PBS for use.
3. NHNK-607 determination of the time curve of the agglutination of Candida albicans
Co-aggregation: NHNK-607 with OD 600 =1.0 and candida albicans suspension were mixed uniformly in a ratio of 1:1 (v/v), candida albicans was added with equal volume of PBS as a control, and the flocculation was observed after standing for 5min.
And (3) measuring indexes: mixing at 10s, 30s, 1min, 5min, and 10min, collecting upper liquid of NHNK-607, candida albicans, NHNK-607, and mixing with candida albicans at 50 μl, and measuring absorbance at 600 nm. Finally, the aggregated precipitate is stained with a gram-type dye, and the bacterial aggregation state is observed. As shown in fig. 3.
The calculation formula is as follows:
Percent of agglutination (%) = [ (ax+ay) -2Amix ]/(ax+ay) ×100%;
note that: ax is a number measured at a reaction time of 600nm from NHNK to 607 alone; ay, measured number of candida albicans alone at the reaction time of 600 nm; amix A value was measured at a reaction time of 600nm after mixing NHNK-607 with Candida albicans.
NHNK-607 the flocculation rate of candida albicans at different times is shown in table 11 below:
TABLE 11
The results show that NHNK-607 viable cells mainly rapidly coagulate candida albicans within 30s-1min, and gradually enter the plateau phase about 1min-5 min.
EXAMPLE 10 different proportions NHNK-607 live bacteria agglutinate Candida albicans
1. NHNK-607 activation and preparation of bacterial suspension
NHNK-607 glycerol bacteria are inoculated into fresh MRS culture medium according to the proportion of 2 percent, and are subjected to static culture for 24 hours at 37 ℃. After the incubation was completed, the cells were obtained by centrifugation at 5000rpm for 10min, washed twice with sterile PBS, and then OD 600 =1.0 was adjusted with PBS for use.
2. Preparation of candida albicans liquid
The glycerol bacteria of candida albicans are inoculated into a fresh sand culture medium according to the proportion of 2 percent, and the static culture is carried out for 24 hours at 37 ℃. After the incubation was completed, the cells were obtained by centrifugation at 5000rpm for 10min, washed twice with sterile PBS, and then OD 600 =1.0 was adjusted with PBS for use.
3. NHNK-607 test on agglutination of Candida albicans
Co-aggregation: when the ratio of NHNK-607 to candida albicans is 1:1, NHNK-607 with OD 600 =1.0 and candida albicans suspension with OD 600 =1.0 are respectively added into a centrifuge tube according to the ratio of 1:1 (v/v), the total volume is supplemented to 1ml of bacterial suspension by PBS after being added into the centrifuge tube, the candida albicans is uniformly mixed, the equal volume PBS is added as a control, and the flocculation state is observed after standing for 1 min. NHNK-607 with OD 600 =1.0 and candida albicans suspension with OD being equal to 1:2 (v/v) when NHNK-607 and candida albicans are 1:2, NHNK-607 is 0.25 ml and candida albicans is 0.5 ml are added into a centrifuge tube, sterile PBS is added to 1ml of total volume, then bacterial suspension is uniformly mixed, candida albicans is added into equal volume of PBS as a control, and flocculation is observed after standing for 1 min. As shown in fig. 4.
And (3) measuring indexes: at the time of 1min of reaction, 50. Mu.L of the supernatant of the mixture of NHNK-607, candida albicans and NHNK-607 with Candida albicans was taken and the absorbance at 600nm was measured, and the calculation formula was as described in example 9.
The experimental results are shown in table 12 below:
table 12
Experimental results show that NHNK-607 candida albicans has similar agglutination rates of 59.32% -64.45% in 1:1 and 1:2, and NHNK-607 can rapidly agglutinate twice the candida albicans per se in one minute, as shown in figure 4.
Examples 9NHNK-607 experiments to inhibit the formation of candida albicans hyphae
1. NHNK-607 fermentation product and preparation of inactivated thallus
The preparation is described in example 4.
2. Candida albicans mycelium morphology induction
Reference example 5 refers to the Candida albicans culture method. After the completion of the culture, the cells were obtained by centrifugation at 5000rpm for 10 minutes. After washing the cells twice with PBS, the cells were centrifuged, resuspended with Fetal Bovine Serum (FBS), and OD 600 =0.15 was adjusted.
3. NHNK-607 test for inhibiting the formation of candida albicans hyphae
The experimental group is prepared by adding 100 mu L of FBS resuspended candida albicans liquid into a 96-well plate, adding 100 mu L NHNK-607 fermentation products, adding the same amount of MRS liquid culture medium into a control group, and culturing for 2 hours at 37 ℃ in parallel. After the completion of the culture, the medium was discarded, washed 1 time with 70% alcohol, 1 time with 0.25% SDS solution, 3 times with sterile water, and then stained with 0.1% crystal violet solution for 30min. After the dyeing is finished, the membrane is washed by 0.25% SDS solution for 1 time and sterile water for 3 times, and the biological membrane at the bottom is observed after the pore plate is naturally dried. 200. Mu.L of 40mmol/L HCl-isopropanol solution and 50. Mu.L of 0.25% SDS solution were added to each well, and the absorbance at OD=600 nm was measured after standing at room temperature for 1 min. The calculation formula and the results are shown in the following table 13:
The results show that NHNK-607 fermentation product and inactivated bacteria can effectively inhibit candida albicans hypha formation.
EXAMPLES 10 NHNK-607 experiments on the combination of inactivated bacteria with Candida albicans hyphae
1. Preparation of NHNK-607 inactivated thallus
The preparation is described in example 3.
2. Preparation of candida albicans mycelium
Reference example 6 refers to the Candida albicans culture method. After the completion of the culture, the cells were obtained by centrifugation at 5000rpm for 10 minutes. The mycelia were resuspended in Fetal Bovine Serum (FBS) and adjusted to od=0.1 and cultured at 37 ℃ for 3 hours to obtain mycelia.
3. Experiment of combining inactivated bacteria with candida albicans mycelium
0.7Ml of candida albicans mycelium and 0.7ml of NHNK-607 inactivated cells are added into a test tube, the mixture is left stand after being fully vortexed, white sediment appears in the test tube after 1h, the sediment is gently sucked out and is dyed by using a gram stain, and the sediment is observed and photographed by a microscope with a 1000-time oil microscope. As shown in fig. 5.
NHNK-607 inactivated bacteria and candida albicans mycelium have obvious size difference, and NHNK-607 inactivated bacteria and candida albicans mycelium can be distinguished by observation. The results show that NHNK-607 inactivated bacteria can obviously bind to the hypha part of candida albicans mycelium, and has competitive inhibition effect on other pathogenic bacteria binding to candida albicans mycelium.
Examples 11 NHNK-607 experiments to promote vaginal beneficial proliferation
1. NHNK-607 preparation of fermentation products
The preparation is described in example 3.
2. Acquisition and culture of vaginal beneficial bacteria
Single colonies of Lactobacillus jensenii (Lactobacillus jensenii) ATCC 25258 and Lactobacillus crispatus (Lactobacillus crispatus) CICC 24879 were picked up and inoculated into MRS broth, and subjected to anaerobic stationary culture at 37℃for 48 h.
3. NHNK-607 fermentation product for promoting vaginal beneficial bacteria proliferation experiment
The experimental group took 4 ml MRS and 1 ml NHNK-607 fermented product to add into centrifuge tube, the control group added with equal volume MRS liquid culture medium, inoculated with vaginal beneficial bacteria liquid according to 1% inoculum size, cultured for 24 hours at 37 ℃ and measured absorbance at OD=600 nm, the results are shown in Table 14 below:
Table 14: NHNK-607 fermentation product for promoting lactobacillus proliferation
The result shows that NHNK-607 fermentation product has the effect of promoting the proliferation of beneficial vaginal bacteria Lactobacillus janus and Lactobacillus crispatus, and the proliferation promoting rate reaches 27.09% -30.87%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A Lactobacillus gasseri (Lactobacillus gasseri) is Lactobacillus gasseri (Lactobacillus gasseri) NHNK-607, which has been preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of M2024420 at 3 and 7 of 2024.
2. Use of lactobacillus gasseri according to claim 1 for the preparation of a product for improving genital tract health, wherein the improvement of genital tract health comprises inhibition of vaginal pathogenic bacteria proliferation, which comprises inhibition of atypical veillonella and monthlies growth.
3. The use of claim 2, wherein the improving genital tract health comprises modulating a vaginal pathogenic virulence gene comprising down-regulating at least one of candida albicans virulence genes HWP1, ALS3, and gardnerella vaginalis virulence gene vly, sld, pat, gtf, atm.
4. The use according to claim 2, wherein said improving genital tract health comprises competing for binding to a fungal hypha of a vaginal pathogenic fungus, said competing for binding to a fungal hypha of a vaginal pathogenic fungus being a candida albicans hypha.
5. The use according to claim 2, wherein said improving genital tract health comprises agglutinating vaginal pathogens including candida agglutinates, atypical veillonella, gardnerella vaginalis.
6. The use of claim 2, wherein said improving genital tract health comprises promoting vaginal beneficial proliferation, said promoting vaginal beneficial proliferation comprising promoting proliferation of lactobacillus jensenii, lactobacillus crispatus.
7. The use according to any one of claims 2 to 6, wherein the lactobacillus gasseri in the genital tract improving product comprises one or both of the following (1) or (2):
(1) The strain and/or inactivated bacterium of lactobacillus gasseri according to claim 1;
(2) Fermentation and/or secretion product of lactobacillus gasseri as claimed in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410853045.7A CN118389377A (en) | 2024-06-28 | 2024-06-28 | Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410853045.7A CN118389377A (en) | 2024-06-28 | 2024-06-28 | Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118389377A true CN118389377A (en) | 2024-07-26 |
Family
ID=92006382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410853045.7A Pending CN118389377A (en) | 2024-06-28 | 2024-06-28 | Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118389377A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016020081A1 (en) * | 2014-08-05 | 2016-02-11 | Alfa Wassermann S.P.A. | Identification of vaginal bacteria |
| WO2016167489A1 (en) * | 2015-04-16 | 2016-10-20 | 주식회사 고바이오랩 | Lactobacillus sp. strain having ability to inhibit proliferation of virginal pathogenic microorganisms |
| CN107949393A (en) * | 2015-03-12 | 2018-04-20 | 不列颠哥伦比亚大学 | Bacteria composition and its application process |
| WO2018223146A1 (en) * | 2017-06-02 | 2018-12-06 | uBiome, Inc. | Method and system for characterizing microorganism-associated sleep-related conditions |
| CN114214256A (en) * | 2022-01-12 | 2022-03-22 | 哈尔滨美华生物技术股份有限公司 | Lactobacillus gasseri for preventing and treating urogenital infection and application thereof |
| CN115418338A (en) * | 2022-11-03 | 2022-12-02 | 山东锦鲤生物工程有限公司 | Lactobacillus paracasei and application thereof |
| CN117487687A (en) * | 2023-09-01 | 2024-02-02 | 青岛蔚蓝生物集团有限公司 | Lactobacillus gasseri with effect of preventing and treating female colpitis and application thereof |
| CN117535207A (en) * | 2024-01-04 | 2024-02-09 | 四川厌氧生物科技有限责任公司 | Lactobacillus gasseri and application thereof |
| CN118516281A (en) * | 2024-06-28 | 2024-08-20 | 青岛诺和诺康生物科技有限公司 | Lactobacillus crispatus and application thereof in preparation of products for improving genital tract flora |
-
2024
- 2024-06-28 CN CN202410853045.7A patent/CN118389377A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016020081A1 (en) * | 2014-08-05 | 2016-02-11 | Alfa Wassermann S.P.A. | Identification of vaginal bacteria |
| CN107949393A (en) * | 2015-03-12 | 2018-04-20 | 不列颠哥伦比亚大学 | Bacteria composition and its application process |
| WO2016167489A1 (en) * | 2015-04-16 | 2016-10-20 | 주식회사 고바이오랩 | Lactobacillus sp. strain having ability to inhibit proliferation of virginal pathogenic microorganisms |
| WO2018223146A1 (en) * | 2017-06-02 | 2018-12-06 | uBiome, Inc. | Method and system for characterizing microorganism-associated sleep-related conditions |
| CN114214256A (en) * | 2022-01-12 | 2022-03-22 | 哈尔滨美华生物技术股份有限公司 | Lactobacillus gasseri for preventing and treating urogenital infection and application thereof |
| CN115418338A (en) * | 2022-11-03 | 2022-12-02 | 山东锦鲤生物工程有限公司 | Lactobacillus paracasei and application thereof |
| CN117487687A (en) * | 2023-09-01 | 2024-02-02 | 青岛蔚蓝生物集团有限公司 | Lactobacillus gasseri with effect of preventing and treating female colpitis and application thereof |
| CN117535207A (en) * | 2024-01-04 | 2024-02-09 | 四川厌氧生物科技有限责任公司 | Lactobacillus gasseri and application thereof |
| CN118516281A (en) * | 2024-06-28 | 2024-08-20 | 青岛诺和诺康生物科技有限公司 | Lactobacillus crispatus and application thereof in preparation of products for improving genital tract flora |
Non-Patent Citations (4)
| Title |
|---|
| SALLISS M 等: "NPJ Biofilms Microbiomes", 《NPJ BIOFILMS MICROBIOMES》, vol. 7, no. 1, 6 July 2021 (2021-07-06), pages 1 - 11 * |
| VASUNDHARA D 等: "Vaginal & gut microbiota diversity in pregnant women with bacterial vaginosis & effect of oral probiotics: An exploratory study", 《INDIAN J MED RES》, vol. 153, no. 4, 30 April 2021 (2021-04-30), pages 492 - 502 * |
| 肖冰冰 等: "利用DNA指纹图谱技术对健康女性阴道菌群多样性的分析", 《北京大学学报(医学版)》, no. 2, 18 April 2012 (2012-04-18), pages 123 - 129 * |
| 董勇宏: "女性生殖道微生物对宫颈癌发生发展的影响", 《中国优秀硕士学位论文全文数据库(电子期刊)医药卫生科技辑》, no. 3, 15 March 2023 (2023-03-15), pages 068 - 35 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108728382B (en) | Lactobacillus plantarum capable of reducing cholesterol and promoting intestinal tract short-chain fatty acid production and application thereof | |
| CN110577912B (en) | Lactobacillus gasseri and application thereof in preparing fermented milk | |
| CN118516281A (en) | Lactobacillus crispatus and application thereof in preparation of products for improving genital tract flora | |
| CN107988115B (en) | Lactobacillus plantarum and composite probiotic fermentation liquor and preparation method thereof | |
| CN110157645B (en) | Lactobacillus salivarius Y4 and application thereof | |
| CN111944713B (en) | Pediococcus acidilactici with excellent alcohol stress resistance and application thereof | |
| CN114642686B (en) | Composite probiotics and its functions of delaying senility and resisting oxidation | |
| CN111944712B (en) | Lactobacillus plantarum with excellent alcohol tolerance and application thereof | |
| CN116555115B (en) | Lactobacillus plantarum NXU0011 and application thereof | |
| CN118345015B (en) | Fermented lactobacillus mucilaginosus and application thereof in preparation of oral health improving products | |
| CN118421540B (en) | Lactobacillus paracasei, product and application thereof in improvement of infectious gastritis | |
| CN111454863A (en) | Lactobacillus fermentum and application thereof in preparation of lactobacillus fermentation liquor with anti-aging function | |
| CN115261262A (en) | Lactobacillus plantarum HSF-LAB-1303 and application thereof | |
| CN116694535B (en) | Lactobacillus pentosus W19 and application thereof | |
| CN116622568A (en) | Lactobacillus harbini P1-1 and application thereof | |
| CN118389377A (en) | Lactobacillus gasseri and application thereof in preparation of products for improving genital tract health | |
| CN109266581A (en) | One plant of streptococcus thermophilus and its application | |
| CN111621461B (en) | Method for improving activity and tolerance of bacillus coagulans BC99 and application thereof | |
| CN112708577B (en) | Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof | |
| CN116254187A (en) | Lactobacillus plantarum and application thereof in resisting streptococcus pneumoniae respiratory tract infection | |
| CN113621572A (en) | Application of lactobacillus rhamnosus in adhering colon cells | |
| CN118374413B (en) | Saliva combined lactobacillus NHNK-612 for reducing pathogenicity of oral pathogenic bacteria, and product and application thereof | |
| CN111154677B (en) | Lactobacillus acidophilus and application thereof | |
| CN114395514B (en) | Lactobacillus acidophilus, microbial inoculum and application thereof | |
| CN118126850A (en) | Kluyveromyces marxianus LSY9-3 with probiotics and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |