CN108004187B - Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug - Google Patents
Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug Download PDFInfo
- Publication number
- CN108004187B CN108004187B CN201810026176.2A CN201810026176A CN108004187B CN 108004187 B CN108004187 B CN 108004187B CN 201810026176 A CN201810026176 A CN 201810026176A CN 108004187 B CN108004187 B CN 108004187B
- Authority
- CN
- China
- Prior art keywords
- vaginal
- lactobacillus gasseri
- lactobacillus
- bacterial
- strain
- 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.)
- Active
Links
- 241000186606 Lactobacillus gasseri Species 0.000 title claims abstract description 97
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 230000003385 bacteriostatic effect Effects 0.000 title claims abstract description 12
- 229940079593 drug Drugs 0.000 title claims abstract description 12
- 210000001215 vagina Anatomy 0.000 title description 21
- 230000001580 bacterial effect Effects 0.000 claims abstract description 26
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 15
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 208000013464 vaginal disease Diseases 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- 238000011282 treatment Methods 0.000 claims abstract description 5
- 241000186660 Lactobacillus Species 0.000 claims description 23
- 229940039696 lactobacillus Drugs 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 10
- 206010046914 Vaginal infection Diseases 0.000 claims description 8
- 241000207201 Gardnerella vaginalis Species 0.000 claims description 5
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 5
- 241000607142 Salmonella Species 0.000 claims description 5
- 241000222122 Candida albicans Species 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 241000191967 Staphylococcus aureus Species 0.000 claims description 4
- 229940095731 candida albicans Drugs 0.000 claims description 4
- 206010003693 Atrophic vulvovaginitis Diseases 0.000 claims description 2
- 201000010808 postmenopausal atrophic vaginitis Diseases 0.000 claims description 2
- 210000000981 epithelium Anatomy 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 244000000072 vaginal pathogen Species 0.000 claims 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 26
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 24
- 235000014655 lactic acid Nutrition 0.000 abstract description 13
- 239000004310 lactic acid Substances 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 10
- 210000002919 epithelial cell Anatomy 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 229940124350 antibacterial drug Drugs 0.000 abstract description 6
- 101001076687 Lactobacillus gasseri Inulosucrase Proteins 0.000 abstract description 4
- 230000021164 cell adhesion Effects 0.000 abstract description 4
- 238000009629 microbiological culture Methods 0.000 abstract description 4
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 35
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 20
- 239000001963 growth medium Substances 0.000 description 20
- 238000000034 method Methods 0.000 description 18
- 241000894006 Bacteria Species 0.000 description 15
- 210000003756 cervix mucus Anatomy 0.000 description 15
- 230000035945 sensitivity Effects 0.000 description 14
- 229920001817 Agar Polymers 0.000 description 12
- 239000008272 agar Substances 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 230000003749 cleanliness Effects 0.000 description 8
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 7
- 238000012408 PCR amplification Methods 0.000 description 7
- 208000037009 Vaginitis bacterial Diseases 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000002207 metabolite Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 231100000820 toxicity test Toxicity 0.000 description 5
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000009642 citrate test Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 229960003306 fleroxacin Drugs 0.000 description 4
- XBJBPGROQZJDOJ-UHFFFAOYSA-N fleroxacin Chemical compound C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN(CCF)C2=C1F XBJBPGROQZJDOJ-UHFFFAOYSA-N 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 229910052742 iron Inorganic materials 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- DWHGNUUWCJZQHO-ZVDZYBSKSA-M potassium;(2s,5r,6r)-6-[[(2r)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;(2r,3z,5r)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylate Chemical compound [K+].[O-]C(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21.C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 DWHGNUUWCJZQHO-ZVDZYBSKSA-M 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 241000207202 Gardnerella Species 0.000 description 3
- 238000003794 Gram staining Methods 0.000 description 3
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 3
- 241000282567 Macaca fascicularis Species 0.000 description 3
- 201000008100 Vaginitis Diseases 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PLXMOAALOJOTIY-FPTXNFDTSA-N Aesculin Natural products OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1Oc2cc3C=CC(=O)Oc3cc2O PLXMOAALOJOTIY-FPTXNFDTSA-N 0.000 description 2
- 108010001478 Bacitracin Proteins 0.000 description 2
- WNBCMONIPIJTSB-BGNCJLHMSA-N Cichoriin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1c(O)cc2c(OC(=O)C=C2)c1 WNBCMONIPIJTSB-BGNCJLHMSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 235000000177 Indigofera tinctoria Nutrition 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 108010059993 Vancomycin Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229960003022 amoxicillin Drugs 0.000 description 2
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000002829 antibacterial sensitivity test Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229960004099 azithromycin Drugs 0.000 description 2
- MQTOSJVFKKJCRP-BICOPXKESA-N azithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)N(C)C[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 MQTOSJVFKKJCRP-BICOPXKESA-N 0.000 description 2
- 229960003071 bacitracin Drugs 0.000 description 2
- 229930184125 bacitracin Natural products 0.000 description 2
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 229960004755 ceftriaxone Drugs 0.000 description 2
- VAAUVRVFOQPIGI-SPQHTLEESA-N ceftriaxone Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C(=O)NN1C VAAUVRVFOQPIGI-SPQHTLEESA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229960002227 clindamycin Drugs 0.000 description 2
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 229940093496 esculin Drugs 0.000 description 2
- XHCADAYNFIFUHF-TVKJYDDYSA-N esculin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(C(=C1)O)=CC2=C1OC(=O)C=C2 XHCADAYNFIFUHF-TVKJYDDYSA-N 0.000 description 2
- AWRMZKLXZLNBBK-UHFFFAOYSA-N esculin Natural products OC1OC(COc2cc3C=CC(=O)Oc3cc2O)C(O)C(O)C1O AWRMZKLXZLNBBK-UHFFFAOYSA-N 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 229940097275 indigo Drugs 0.000 description 2
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229960002260 meropenem Drugs 0.000 description 2
- DMJNNHOOLUXYBV-PQTSNVLCSA-N meropenem Chemical compound C=1([C@H](C)[C@@H]2[C@H](C(N2C=1C(O)=O)=O)[C@H](O)C)S[C@@H]1CN[C@H](C(=O)N(C)C)C1 DMJNNHOOLUXYBV-PQTSNVLCSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- 239000006872 mrs medium Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 2
- 229940056360 penicillin g Drugs 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 229960002292 piperacillin Drugs 0.000 description 2
- IVBHGBMCVLDMKU-GXNBUGAJSA-N piperacillin Chemical compound O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC=CC=1)C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 IVBHGBMCVLDMKU-GXNBUGAJSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 229960003165 vancomycin Drugs 0.000 description 2
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 2
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 2
- LVSQXDHWDCMMRJ-UHFFFAOYSA-N 4-hydroxybutan-2-one Chemical compound CC(=O)CCO LVSQXDHWDCMMRJ-UHFFFAOYSA-N 0.000 description 1
- KKADPXVIOXHVKN-UHFFFAOYSA-N 4-hydroxyphenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=C(O)C=C1 KKADPXVIOXHVKN-UHFFFAOYSA-N 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 239000005730 Azoxystrobin Substances 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229930182843 D-Lactic acid Natural products 0.000 description 1
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 241000218492 Lactobacillus crispatus Species 0.000 description 1
- 241001561398 Lactobacillus jensenii Species 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 241000186783 Lactobacillus vaginalis Species 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 208000007074 Trichomonas Vaginitis Diseases 0.000 description 1
- 208000025206 Trichomonas vaginitis urogenital infection Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 201000007096 Vulvovaginal Candidiasis Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- -1 acetyl methyl Chemical group 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- WFDXOXNFNRHQEC-GHRIWEEISA-N azoxystrobin Chemical compound CO\C=C(\C(=O)OC)C1=CC=CC=C1OC1=CC(OC=2C(=CC=CC=2)C#N)=NC=N1 WFDXOXNFNRHQEC-GHRIWEEISA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940022769 d- lactic acid Drugs 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 208000001786 gonorrhea Diseases 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- 229960001019 oxacillin Drugs 0.000 description 1
- UWYHMGVUTGAWSP-JKIFEVAISA-N oxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=CC=CC=C1 UWYHMGVUTGAWSP-JKIFEVAISA-N 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- PRWXGRGLHYDWPS-UHFFFAOYSA-L sodium malonate Chemical compound [Na+].[Na+].[O-]C(=O)CC([O-])=O PRWXGRGLHYDWPS-UHFFFAOYSA-L 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
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
-
- 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
-
- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a Lactobacillus gasseri and application thereof in preparing vaginal antibacterial drugs, the Lactobacillus gasseri is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 14109. The research on the metabolic performance of the strain shows that compared with the common Lactobacillus gasseri, the strain has stronger production capacity of lactic acid and hydrogen peroxide, has very strong bacteriostatic ability on pathogenic bacteria, and has outstanding vaginal epithelial cell adhesion capacity. Based on the new properties discovered above, the invention determines the new application of the vaginal antibacterial drug prepared by the vaginal antibacterial drug, and can realize the treatment of various bacterial vaginal diseases. The bacterial agent has obvious effect on bacterial vaginal diseases, is safe, nontoxic and good in stability, can be stored for a long time, and also relates to application of the bacterial agent in medicines for preventing and/or treating gynecological diseases.
Description
Technical Field
The invention relates to the technical field of microorganisms, further relates to adjustment of vaginal micro-ecological environment and treatment of bacterial vaginal diseases, and particularly relates to lactobacillus gasseri and application thereof in preparation of vaginal antibacterial drugs.
Background
A plurality of microorganisms exist in the vagina of healthy women, and the microorganisms, a host and the environment form a vaginal microecosystem which is mutually restricted, coordinated and dynamically balanced. The vaginal flora of healthy women is mainly composed of lactobacillus, including lactobacillus plantarum, lactobacillus jensenii, lactobacillus gasseri, lactobacillus crispatus, lactobacillus vaginalis, lactobacillus gasseri and the like. Under normal conditions, the lactobacillus can protect the vagina, and when the lactobacillus disorder of the microecology of the vagina can cause the invasion of pathogenic bacteria to generate vaginitis.
Bacterial Vaginosis (BV) occurs due to a deregulation of the vaginal flora and a reduction of the host's own lactobacilli, which leads to a massive proliferation of other conditionally pathogenic microorganisms such as Gardnerella, various anaerobes, Vibrio flexuosus, etc., usually BV is actually a mixed infection mainly of Gardnerella. By using antibiotic therapy, the symptoms of BV can be temporarily relieved, but the reduced lactobacilli are further reduced, and vaginal microecological imbalance is aggravated, so that BV can relapse repeatedly. How to control relapse and radically cure bacterial vaginosis is a delicate problem which needs to be solved urgently by gynecologists.
The research shows that: produce H2O2Lactobacillus which is lactic acid is the dominant bacterium in the vagina of healthy women, is an important factor for protecting the vagina of women from pathogen infection, and in addition, acid produced by the metabolism of lactobacillus and some antimicrobial factors can also be usedEffectively inhibit the growth and the propagation of other bacteria.
Various lactobacilli exist in the vagina of healthy women, the individual difference is achieved, and the difference of pathogenic bacteria resistance among lactobacillus strains is obvious. When selecting lactobacillus probiotics, the species of lactobacillus, acid production and H production need to be comprehensively considered2O2The ability, and the ability to adhere to vaginal epithelial cells, wherein whether lactobacillus can successfully colonize the vagina or not, is the basis of the sustained action of lactobacillus and is also a key factor for the curative effect of lactobacillus. The existing product, namely lactobacillus delbrueckii, in the market at present is actually not the dominant bacterial flora in the vagina of women in China, has poor planting capability, cannot maintain stable viable bacteria content, and cannot meet the clinical requirement of gynecology.
Lactobacillus gasseri (Lactobacillus gasseri), which is a Lactobacillus species, is one of the normal flora of human body, and is widely distributed in the intestinal tract of human body and also in the vagina of women. In the prior art, lactobacillus gasseri is mainly used for stimulating immune cells to secrete anti-allergy related cell hormones; in addition, the characteristics of the intestinal flora can be improved by the self flora dominance, so that the conventional strains have no specific bacteriostatic action on the Lactobacillus gasseri in the cognitive category of the prior art.
Disclosure of Invention
The invention aims to provide a specific lactobacillus gasseri to solve the technical problem that the conventional lactobacillus gasseri in the prior art does not have the capability of inhibiting pathogenic bacteria in vagina.
Another technical problem to be solved by the invention is that the conventional Lactobacillus gasseri has low hydrogen peroxide production capacity.
The invention also aims to solve the technical problem that the conventional lactobacillus gasseri has low lactic acid yield.
The invention also aims to solve the technical problem that the conventional lactobacillus gasseri has poor colonization and survival effects in the vagina in the prior art.
The invention also aims to solve the technical problem that the treatment effect of the bacteriostatic medicaments of the microbial inoculum type aiming at the vaginal pathogenic bacteria in the prior art is poor.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the separated Lactobacillus gasseri (Lactobacillus gasseri) is disclosed as RD-0046 (Lactobacillus gasseri) and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14109.
The Lactobacillus gasseri RD-0046 is selected from vaginal secretion of healthy women of childbearing age in China, and has been preserved in China general microbiological culture Collection center (CGMCC) in 5-10 th of 2017, wherein the preservation unit address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, the institute of microbiology of Chinese academy of sciences, the registration number of the collection is CGMCC No.14109, and the classification name of the strain is Lactobacillus gasseri.
The separated lactobacillus gasseri RD-0046 adopts 16SrDNA for sequencing, and the homology score of the lactobacillus gasseri base sequence in a GenBank database is more than 98 percent.
On the basis of the technical scheme, the invention further provides application of the lactobacillus gasseri in preparing bacteriostatic drugs for vaginal pathogenic bacteria.
Preferably, the drug is a bacterial agent that colonizes and survives vaginal epithelial cells in the vaginal environment.
Preferably, the drug is a bacterial agent which is metabolized to produce H in the vaginal environment2O2。
Preferably, the pathogenic bacteria is gardnerella vaginalis.
Preferably, the pathogenic bacteria is atrophaerella.
Preferably, the pathogenic bacteria are Candida albicans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella, or the like.
Meanwhile, the invention further provides the application of the lactobacillus gasseri in preparing a medicament for preventing or treating vaginal diseases.
Preferably, the vaginal disease is vulvovaginal candidiasis, trichomonas vaginitis, senile vaginitis, non-specific vaginal infection or mixed vaginal infection.
According to the invention, a Lactobacillus gasseri strain is obtained by separation, and the metabolic performance of the Lactobacillus gasseri strain is researched, so that the Lactobacillus gasseri strain has excellent lactic acid production capacity and hydrogen peroxide production capacity, has strong bacteriostatic capacity on pathogenic bacteria, and has outstanding vaginal epithelial cell adhesion capacity. Based on the new properties discovered above, the invention determines the new application of the vaginal antibacterial drug prepared by the vaginal antibacterial drug, and realizes the treatment of various bacterial vaginal diseases.
The beneficial effect that adopts above-mentioned technical scheme to produce lies in: (1) the Lactobacillus gasseri RD-0046 strain of the invention can be preserved for a long time and can resist bacterial vaginosis and various vaginal infections, including Candida albicans vaginitis, gonorrhea, viral vaginitis, urinary tract infection and the like. (2) The strain is directly collected from a healthy human body, has active and stable biological characteristics, does not need domestication and rejuvenation processes, and can be directly prepared by a preparation process. (3) The strain has the effects of inhibiting Gardnerella and Candida albicans, has superior vaginal epithelial cell adhesion compared with a commercially available control bacterium, and has superior capability of permanent planting in the vagina of a primate.
Drawings
FIG. 1 is a photograph showing the morphology of a colony of Lactobacillus gasseri RD-0046 according to an embodiment of the present invention;
FIG. 2 is a gram-stained microscopic photograph of Lactobacillus gasseri RD-0046 according to an embodiment of the present invention;
FIG. 3 is an electrophoretogram of PCR amplification product of 16SrDNA gene of Lactobacillus gasseri RD-0046 in accordance with an embodiment of the present invention;
FIG. 4 is a lactic acid detection map of Lactobacillus gasseri RD-0046 in accordance with an embodiment of the present invention;
FIG. 5 is a 5min and 10min image of the coloration of Lactobacillus gasseri RD-046 with hydrogen peroxide in accordance with an embodiment of the present invention;
FIG. 6 is a plot of antibiotic susceptibility test colonies of Lactobacillus gasseri RD-0046 against kanamycin, fleroxacin and amoxicillin/clavulanic acid in an embodiment of the present invention;
FIG. 7 is a photograph showing the bacteriostatic effects of Lactobacillus gasseri RD-0046 (left) and Lactobacillus delbrueckii (right) on Gardner vaginalis in an embodiment of the present invention;
FIG. 8 is a photograph showing the bacteriostatic effects of Lactobacillus gasseri RD-0046 (left) and Lactobacillus delbrueckii (right) on Salmonella bacteria in accordance with an embodiment of the present invention;
FIG. 9 is a photograph showing the bacteriostatic effects of Lactobacillus gasseri RD-0046 (left) and Lactobacillus delbrueckii (right) on Pseudomonas aeruginosa according to the embodiment of the present invention;
FIG. 10 is an electrophoresis diagram of PCR amplification products of a partial strain 16SrDNA fragment in a vaginal microbial area of a cynomolgus monkey after lactobacillus gasseri RD-0046 is fixedly planted in the vagina of the cynomolgus monkey in the embodiment of the invention;
FIG. 11 is a scanning electron micrograph of Lactobacillus gasseri RD-0046 of the present invention.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The test reagent consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified; in the quantitative tests in the following examples, three repeated experiments are set, and the results are averaged; in the following examples,% is by mass unless otherwise specified.
The bacterial culture medium components and preparation methods used in the following examples were as follows:
1. broth solid Medium (MRS) preparation:
(1) preparing agar powder into a solution, and adding 1.5g/100ml deionized water;
(2) adding 17.91g/100ml agar solution of MRS culture medium, and mixing;
(3) placing into a sterilizing pot, and sterilizing for 20 minutes by 1.0MPa steam;
(4) pouring the culture medium into a culture dish after the temperature of the culture medium is reduced to room temperature, wherein the culture medium is about 10 ml/piece or 20 ml/piece according to the size of the culture dish;
(5) operating in a clean bench. Cooling to become agar, marking the name of the culture medium and the preparation date, and placing in a refrigerator at 4 ℃ for later use.
2. Broth liquid Medium (MRS) preparation:
(1) adding the MRS culture medium into deionized water in a ratio of 17.9l g/100 ml;
(2) placing into a sterilizing pot, and sterilizing for 20 minutes by 1.0MPa steam;
(3) taking out the sterilized pan when the pressure of the sterilized pan is not high, and subpackaging the sterilized pan into EP tubes with each 1.0 ml. Marking the name and preparation date of the culture medium, and placing the culture medium in a refrigerator at 4 ℃ for later use.
3. Hydrogen peroxide (H)2O2) Preparing an identification culture medium:
(1) preparing steps (1) to (4) with a broth solid Medium (MRS);
(2) taking out the autoclave without pressure, slightly cooling, adding TMB (final concentration of 0.25mg/m1) and HRP (final concentration of 0.01mg/m1) in a super clean bench when the autoclave is still in a liquid state, and mixing;
(3) and (3) after the temperature of the culture medium is reduced to about 45 ℃, pouring the culture medium into a culture dish, cooling the culture medium into an agar shape, marking the name and preparation date of the culture medium, and placing the culture medium in a refrigerator at 4 ℃ for later use.
Example 1 isolation and inoculation of Lactobacillus gasseri RD-0046 flora, purification, enrichment culture
1. Isolation and inoculation of the Lactobacillus gasseri RD-0046 flora: collecting secretion on vaginal side wall of subject with two sterile cotton swabs, inoculating in culture dish containing prepared MRS culture medium at different concentrations within 24 hr, marking information, placing the culture dish in anaerobic jar, and adding CO2And (5) generating air bags, placing in an incubator at 37 ℃, and incubating for more than 48 h.
2. Purifying and enrichment culturing of the Lactobacillus gasseri strain RD-0046: counting according to different forms (surfaces, edges and the like) and sizes of colonies, respectively, recording as one form with the same form and the same size, picking a few bacteria in a single colony by an inoculating loop, and inoculating to an MRS solid culture medium according to a diagonal method to obtain a separated and purified single colony; the bacteria toothpick picks a little bacteria of single colony on MRS solid culture medium, inoculates to MRS liquid culture medium, puts in 37 degrees C incubator, anaerobic culture 24h-48h, screens out the new bacterial strain.
Example 2 (identification and preservation of Lactobacillus gasseri RD-0046 Strain)
1. Culture characteristics, staining microscopy and morphological characteristics: the colony obtained after culture is as shown in figure 1, and is round, neat in edge, smooth in surface, flat and semitransparent; gram staining is carried out on the smear of the pure culture of the bacteria, the result is shown in figure 2, the gram staining is positive, the smear is in a thin rod shape, the end is blunt, and the smear is arranged in a chain shape or a thin wire shape, and the result shows that: the isolated strain was initially judged to be of the genus Lactobacillus.
2. 16SrDNA gene sequence identification: extracting DNA with bacterial genome DNA extraction kit, performing PCR amplification with primer pair 27F (5 '-AGAGTTTGATCMTGGCTCAG-3'), 1492R (5 '-TACGGYTACCTTGTTACGACTT-3'), performing gel electrophoresis to obtain 16SrDNA gene fragment, and obtaining single clear PCR product band at about 1500bp, as shown in figure 3. And (3) purifying the PCR product and determining a DNA sequence, wherein a Sanger sequencing method is adopted, a sequencing primer pair is 27F/1492R, a sequencing instrument ABI3730XL is adopted, and the sequencing result is compared with a GenBank database, so that the homology similarity of the PCR product and the Lactobacillus gasseri is more than 99%. The finally identified genus species is lactobacillus gasseri. The sequence of the variable region of the 16SrDNA is shown in a sequence table SEQID NO. 1.
3. Physiological and biochemical characteristics: the physiological and biochemical reactions of the strains were measured by an esculin hydrolysis test, a methyl red test (MR test), an acetyl methyl alcohol test (VP test), an indigo matrix test, a trisaccharide iron test, a crimson disaccharide iron test, a urease test, a phenylalanine deaminase test, an amino acid decarboxylase test, a gelatin liquefaction test, a sodium malonate test, a citrate test (citrate test), a nitrate reduction test, a litmus milk test, a bacterial motility test, and the results are shown in table 1:
TABLE 1 physiological and biochemical characteristics test results of Lactobacillus gasseri RD0046
| Physiological and biochemical project | Results |
| Esculin hydrolysis test | + |
| Methyl Red test (MR test) | + |
| Acetylmethylmethanol test (VP test) | - |
| Indigo substrate test | - |
| Trisaccharide iron test | - |
| Iron keshi disaccharide test | - |
| Urease test | - |
| Phenylalanine deaminase assay | - |
| Amino acid decarboxylase test | - |
| Liquefaction test of gelatin | - |
| Sodium malonate test | - |
| Citrate test (citrate test) | - |
| Nitrate reduction test | - |
| Litmus milk test | Acid production by solidification |
| Bacterial motility test | - |
+: indicates positive; -: indicates negativity
The strain was biochemically identified by API 50CHL Lactobacillus identification system produced by Meiriee, France, and statistics of the identification results are shown in Table 2
TABLE 2 Lactobacillus gasseri RD-0046API 50CH test strip reaction results
+: indicates positive; -: indicating a negative; d: indicating weak positivity
The biochemical characteristics of the strain are judged to be in accordance with the biochemical characteristics of the Lactobacillus gasseri by the biochemical map.
3. Preservation of the Strain
The Lactobacillus gasseri RD-0046 is screened from vaginal secretion of Chinese healthy women of child bearing age, and is preserved in China general microbiological culture Collection center (CGMCC for short) in 5 months and 10 days in 2017, and the preservation unit address is as follows: no. 3 of Xilu No.1 of Beijing, Chaoyang, the institute of microbiology of Chinese academy of sciences, the registration number of the collection is CGMCC No.14109, and the classification name of the strain is Lactobacillus gasseri.
Example 3 (Lactobacillus gasseri RD-0046 metabolite assay)
1. And (3) determining the content of lactic acid in the metabolite of the Lactobacillus gasseri RD-0046: the lactic acid in the fermentation liquor of the strain cultured for 24-48h is detected by a high performance liquid phase method (0.005M sulfuric acid aqueous solution (0.28mL sulfuric acid (98%)) -1000mL water, pH is about 2.1), and the result shows that the lactic acid (L/D lactic acid sum) is about 35mg/mL and is far higher than the lactic acid content of the commercial Lactobacillus delbrueckii (about 15mg/mL) and the conventional Lactobacillus gasseri, and the lactic acid detection pattern of the invention is shown in figure 4
2. Determination of hydrogen peroxide content in the metabolite of Lactobacillus gasseri RD-0046: hydrogen peroxide semiquantitative determination was carried out by the peroxidase method of Mcgrooarty et al, and isolated and identified Lactobacillus gasseri RD-0046 was inoculated into H2O2And (3) identifying the MRS-TMB plate, carrying out anaerobic culture at 37 ℃ for 24h, taking out the plate, and exposing the thallus in the air. Produce H2O2The lactobacillus colonies will turn blue without producing H2O2The colony does not change color, and the produced H is subjected to color change according to the color change time2O2The results of semiquantitative analysis are shown in FIG. 5, in which the blue color of the colony appears at 5min and a large amount of blue color appears at 10min, which shows that the bacterium is easy to produce hydrogen peroxide and has strong hydrogen peroxide producing ability, and the results show that the Lactobacillus gasseri RD-0046 can produce lactic acid and hydrogen peroxide and is helpful for maintaining the vaginal microecological balance.
Example 4 (antibiotic sensitivity test)
According to the requirement of an antibiotic susceptibility test in the third general theory of microecological live bacteria products of the pharmacopoeia of 2010 edition, the sensitivity of the strain to the antibiotic is determined by adopting an agar diffusion paper sheet method, the sensitivity of the lactobacillus gasseri RD-0046 of 0 generation and 30 generation passage to each antibiotic is examined, the level of the sensitivity of the strain to the antibiotic is judged according to the size of an inhibition zone, and the determination result is shown in Table 3:
TABLE 3 antibiotic susceptibility test results of Lactobacillus gasseri RD0046
The inhibition zone is less than 10mm, the mild sensitivity is judged, the moderate sensitivity is judged at 10-20mm, and the sensitivity is judged at more than 20 mm.
Experimental data show that the bacillus subtilis is slightly sensitive to bacitracin and fleroxacin, moderately sensitive to oxacillin, kanamycin clindamycin and gentamycin, and sensitive to ampicillin, penicillin G, tetracycline, erythromycin, piperacillin, ceftriaxone, vancomycin, amoxicillin/clavulanic acid, azithromycin, amoxicillin and meropenem.
Wherein the antibiotic sensitivity to kanamycin, fleroxacin and amoxicillin/clavulanic acid is shown in figure 6.
Example 5 (toxicity test)
5 SPF-level Kunming mice were injected with 0.3ml of fresh Lactobacillus gasseri RD-0046 suspension (more than 1 × 10) per mouse9CFU/mouse). The body weight of each mouse was measured daily according to the requirements of chinese pharmacopoeia 2015, and the behavior, physiology, etc. of each mouse before and after injection were observed and recorded. The results show that the weight average of all animals is increased within 7 days, no obvious poisoning symptom is seen, the activity behaviors are not abnormal, no animal is dead, and the strain is considered to belong to the nontoxic strain.
Example 6 (Lactobacillus gasseri RD-0046 passage stability test)
This example carried out a stability study of this Lactobacillus gasseri strain RD-0046 for 30 passages (C30) in terms of growth characteristics, morphology, biochemical characteristics, metabolite composition, antibiotic sensitivity characteristics, genetic characteristics, and toxicity test.
1. The method for separating and purifying the lactobacillus gasseri RD-0046, observing colony morphology, performing stainboscopy and detecting biochemical characteristics is the same as the first part of the example 1 and the example 2. The results show that: after passage, the colony morphology is round, the edge is neat, the surface is smooth, flat and semitransparent, and the passage is stable; gram staining appeared as gram positive bacilli, with no change from the stained microscopic photograph to passage 0.
2. Analysis of genetic characteristics: the procedure is as in the second part of example 2. 16SrDNA fragment PCR amplification is respectively carried out on strains of the 0 th generation (C0) and the 30 th generation (C30) of the Lactobacillus gasseri RD-0046, electrophoresis analysis is carried out on PCR amplification products, target bands are clear and single, the size is about 1500bp, the amplification is correct, the results of the two PCR amplifications of C0 and C30 are consistent, and the determined sequences are compared and analyzed with known sequences in a GenBank database by using a BLAST tool in NCBI, so that the homology similarity of the Lactobacillus gasseri RD-0046 is 100%.
3. And (3) metabolite determination: the method is the same as example 3, the content of the lactic acid is about 35mg/mL, hydrogen peroxide experiments show that each generation of bacterial colony has blue color at 5min, and a large amount of blue color is obvious at 10min, which proves that the capability of the bacterial strain to generate hydrogen peroxide by metabolism and the capability of generating lactic acid are stable.
4. Antibiotic sensitivity test: the method is the same as example 4, the sensitivity of the strain to antibiotics is determined by adopting an agar diffusion paper sheet method, and the sensitivity of the lactobacillus gasseri strain to bacitracin and fleroxacin is determined according to the bacteriostasis range interpretation standard of a drug sensitivity test paper sheet method, and the sensitivity of the lactobacillus gasseri strain to azoxystrobin, kanamycin clindamycin and gentamicin is determined to be moderate, and the sensitivity of the lactobacillus gasseri strain to ampicillin, penicillin G, tetracycline, erythromycin, piperacillin, ceftriaxone, vancomycin, amoxicillin/clavulanic acid, azithromycin, amoxicillin and meropenem is determined.
5. Toxicity test: the procedure is as in example 5, and toxicity tests were carried out on the strains C0, C30 and generations of Lactobacillus Benger RD-0046 by intraperitoneal injection in mice, at concentrations > 109CFU/mouse. The results were: all the tested mice have no toxic symptoms within 7 days, the weight is increased, and no animal is dead. According to the above results, the strain belongs to the nontoxic strain according to the supplement instruction of the research technical requirements of pharmacology and toxicology of new drugs.
In this example, the Lactobacillus gasseri RD-0046 is cultured in MRS medium for multiple passages, and the influence of passage propagation on Lactobacillus gasseri is discussed in the aspects of morphology, biochemistry, metabolite characteristics, genetic characteristics, drug sensitivity characteristics, toxicity test and the like. The results show that: the morphological, biochemical, genetic, metabolite and drug sensitive characteristics of MRS cultured and passaged within 30 generations are consistent with those of the initially isolated strain.
Example 7 (pharmacodynamic experiment of Lactobacillus gasseri RD-0046 strain)
In vitro bacteriostasis experiment of Lactobacillus gasseri RD-0046 strain
(1) Experiment of inhibiting Gardnerella vaginalis in vitro by Lactobacillus gasseri RD-0046 and Lactobacillus delbrueckii: preparing MRS agar cake of Lactobacillus gasseri RD-0046 according to the inoculation concentration of 3%, anaerobically culturing at 37 deg.C for 24h, and preparing commercially available Lactobacillus delbrueckii cake by the same method; taking 100 mu L of Gardnerella vaginalis, inoculating the Gardnerella vaginalis 100 mu L into 10mL of BHI solid culture medium, adding Lactobacillus gasseri RD-0046 and Lactobacillus delbrueckii cake, and carrying out anaerobic culture at 37 ℃ for 48 h; obvious inhibition zones can appear around the lactobacillus. The results are shown in FIG. 7, wherein the left graph shows the inhibition zone effect of Lactobacillus gasseri RD-0046, the diameter of the inhibition zone measured by a vernier caliper is 30.7mm, the right graph shows the inhibition zone effect of Lactobacillus delbrueckii, and the inhibition diameter is 24.6mm, and the conclusion is that the inhibition effect of Lactobacillus gasseri RD-0046 on Gardner's bacteria in vagina is stronger than that of Lactobacillus delbrueckii.
(2) Experiment of inhibiting aerobic pathogenic bacteria in vitro by Lactobacillus gasseri RD-0046 and Lactobacillus delbrueckii: preparing MRS agar cake of Lactobacillus gasseri RD-0046 according to the inoculation concentration of 3%, anaerobically culturing at 37 deg.C for 24h, and preparing commercially available Lactobacillus delbrueckii cake by the same method; staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella were inoculated into trypticase Soy peptone liquid (TSB) agar medium, and Lactobacillus gasseri RD-0046 and Lactobacillus delbrueckii cake were added. Culturing at 33 deg.C for 18-24h, observing the inhibition zone, measuring RD-0046 with vernier caliper with diameter of 235mm, and determining the inhibition zone effect of Lactobacillus delbrueckii with diameter of 15.3mm, to obtain the result that the inhibition effect of Lactobacillus delbrueckii RD-0046 on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella is better than that of Lactobacillus delbrueckii, and the representative graph is shown in figures 8-9
II, cell adhesion experiment: according to the number of the lactobacillus adhered to the vaginal epithelial cell monolayer, the adhesion performance of different lactobacillus is determined. The method comprises the following steps: taking human vaginal epithelial cells Vk2/E6E7 and human cervical cancer epithelial cells Hela, inoculating the cells into a 12-well plate at the density of 50 ten thousand per well, and forming a single cell layer by VK2/E6E7 after 48 hours; adding commercially available lactobacillus (Lactobacillus delbrueckii) and Lactobacillus gasseri RD-0046 into each hole according to different amounts of CFU, adhering for 4 hours, slightly oscillating on a shaking table in the adhering process, and setting two parallel experiments for each group; after the adhesion is finished, 1ml of 0.05% triton X-100 is used for cell lysis to prepare suspension bacterial liquid, the suspension bacterial liquid is diluted, and 100ul of bacterial liquid is respectively and uniformly inoculated on an MRS agar medium plate; after 48 hours of anaerobic culture, the number of clones per plate was counted.
The results show that: the 4h adhesion rate of the Lactobacillus gasseri RD-0046 is 42.4 percent and 48.2 percent respectively, the 4h adhesion rate of the similar commercially available Lactobacillus delbrueckii strain is 11.8 percent and 17.3 percent respectively, and the adhesion rate of the Lactobacillus gasseri RD-0046 is higher than that of the similar commercially available Lactobacillus delbrueckii strain.
Third, domestic rabbit vagina field planting experiment
1. Experimental methods
4 healthy animals were selected for stratified random grouping by weight into 2 groups, 2 animals in the positive control group, 2 animals in the experimental group:
the planting is prepared by using lactobacillus gasseri RD-0046: weighing freeze-dried powder of Lactobacillus gasseri RD-0046 to make the planting amount 106. In the control group, a commercial product (Lactobacillus delbrueckii capsule) was used, 0.5mL of MRS liquid medium was added to each control group under aseptic conditions, the mixture was mixed well, and the mixture was completely sucked by a vaginal applicator and then implanted into the vagina.
And (3) field planting molding and sampling: after the monkey menstruation observed in the normal menstrual cycle, the molding bacteria were implanted for 7 consecutive days. The vagina of the animal is observed once a week, the color, the character and the secretion amount of the vaginal secretion are checked, the pH value of the vaginal secretion is measured, 2 sterile swabs are sampled, one swab is used for microscopic examination of the cleanness of the vaginal secretion, and the other swab is used for flora analysis.
Separation and purification culture of vaginal bacteria: the collected vaginal secretion is shaken in 2mL of D-Hanks buffer solution, and is subjected to gradient dilution by phosphate buffer solution, and the obtained product is respectively coated on MRS agar plates and cultured for 24-48h at 37 ℃ under an anaerobic condition. Single colony morphology and the like are recorded, and the MRS agar plate is restreaked to obtain purified colonies and subjected to biochemical and molecular identification.
Molecular biological method identification (16SrDNA gene sequence analysis): and (3) carrying out 16SrDNA sequence amplification, sequencing and analysis on the separated and purified strain, and carrying out sequencing after a PCR amplification product identified as a 16SrDNA fragment is purified on a target fragment by using a gel cutting recovery method. The determined 16SrDNA gene sequence is aligned with known sequences in GenBank databases using the BLAST tool in NCBI, and the same species is identified when the alignment homology is greater than or equal to 99%.
2. Results and analysis of the experiments
General observations of vaginal mucosa and secretions: after implantation, the vaginal mucosal secretion was observed once a week, and as a result, no obvious abnormality was found in all the test animals.
Measuring the pH value of vaginal secretion: after the lactobacillus gasseri RD-0046 is implanted, the pH value of vaginal secretion of most experimental animals is obviously lower than that of a marketed control group, and is obviously reduced relative to that before the implantation, and the measurement result of the pH value is shown in a table 4.
TABLE 4 influence of Lactobacillus gasseri RD-0046 on pH of vaginal secretion of experimental animals
Cleanliness of vaginal secretions: compared with the control group before and after implantation, the quantity of the vaginal mixed bacteria in the control group is obviously increased, and the cleanliness is reduced; after the lactobacillus gasseri RD-0046 is implanted in the experimental group, the cleanliness of the vaginal secretion is obviously better than that of the control group, so that gram-positive vaginal bacilli with different quantities can be seen, and the cleanliness is obviously higher than that of the control group. The results of the vaginal secretion cleanliness determinations are shown in table 5.
TABLE 5 influence of Lactobacillus gasseri RD-0046 on the cleanliness of vaginal secretion of experimental animals
I to III represent the cleanliness, and III represents the lowest cleanliness.
The secretion of the experimental group is amplified, separated and purified, and after vaginal flora analysis, the tested lactobacillus gasseri RD-0046 is found in all the vaginal secretion of 3 animals in the experimental group, and the information of the separated tested lactobacillus gasseri RD-0046 is shown in FIG. 10. As can be seen from the figure, the tested Lactobacillus gasseri RD-0046 is found in vaginal secretion of tested animals, so that the Lactobacillus gasseri RD-0046 can be effectively colonized in the vagina of the cynomolgus monkey.
Example 8 (Lactobacillus gasseri RD-0046 freeze-dried preservation and stability of the freeze-dried powder)
To examine the survival rate of Lactobacillus gasseri RD-0046 in the case of fermentation and lyophilization, Lactobacillus gasseri RD-0046 was grown in modified MRS medium at pH 6.5 and fermented using a 100 liter-scale fermenter. Collecting thallus in early stage of plateau stage, and the viable count reaches about 3 × 109CFU/ml. The cells were collected by centrifugation, washed with phosphate buffer and mixed with lyoprotectants (including skimmed milk powder and sucrose). The mixture was then freeze-dried in a freeze-dryer. The samples were frozen at-40 ℃ for about 2 hours, then vacuum dried at-20 ℃ for 20-30 hours, and then dried at 30 ℃ for 3-5 hours. The dry powder was packaged in aluminum foil bags with desiccant and stored at 4 ℃ and room temperature (25 ℃). Viable counts were determined by plate counting at months 0, 1, 2, 3, 6, 12.
The starting Lactobacillus gasseri RD-0046 contains up to 750 billion viable bacteria per gram of dry powder (7.5X 10)10cfu/g) has optimum storage stability at 2-8 ℃. After 6 months of storage at 2-8 ℃, 77.3% of the initial viable count was retained, see table 6.
TABLE 6 stability test results of Lactobacillus gasseri RD-0046 lyophilized powder agent
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Zhao Xiang Jiang
<120> lactobacillus rhamnosus and application thereof in preparing vagina bacteriostatic drugs
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1506
<212>DNA
<213> Lactobacillus rhamnosus (Lactobacillus rhamnosus)
<400>1
aatttttgtc caccttagac ggctcgctcc ctaaaagggt tacgccaccg gcttcgggtg 60
ttacaaactc tcatggtgtg acgggcggtg tgtacaaggcccgggaacgt attcaccgcg 120
gcgtgctgat ccgcgattac tagcgattcc gacttcgtgt aggcgagttg cagcctacag 180
tccgaactga gaatggcttt aagagattag cttgacctcg cggtctcgca actcgttgta 240
ccatccattg tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc 300
cccaccttcc tccggtttgt caccggcagt cttactagag tgcccaacta aatgctggca 360
actagtcata agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 420
acgacaacca tgcaccacct gtcattttgc ccccgaaggg gaaacctgat ctctcaggtg 480
atcaaaagat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct 540
ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg tcgtactccc 600
caggcggaat gcttaatgcg ttagctgcgg cactgaaggg cggaaaccct ccaacaccta 660
gcattcatcg tttacggcat ggactaccag ggtatctaat cctgttcgct acccatgctt 720
tcgagcctca gcgtcagtta cagaccagac agccgccttc gccactggtg ttcttccata 780
tatctacgca tttcaccgct acacatggag ttccactgtc ctcttctgca ctcaagtttc 840
ccagtttccg atgcacttcc tcggttaagc cgagggcttt cacatcagac ttaaaaaacc 900
gcctgcgctc gctttacgcc caataaatcc ggataacgct tgccacctac gtattaccgc 960
ggctgctggc acgtagttag ccgtggcttt ctggttggat accgtcacgc cgacaacagt 1020
tactctgccg accattcttc tccaacaaca gagttttacg acccgaaagc cttcttcact 1080
cacgcggcgt tgctccatca gacttgcgtc cattgtggaa gattccctac tgctgcctcc 1140
cgtaggagtt tgggccgtgt ctcagtccca atgtggccga tcaacctctc agttcggcta 1200
cgtatcattg ccttggtgag ccgttacctc accaactagc taatacgccg cgggtccatc 1260
caaaagcgat agcttacgcc atctttcagc caagaaccat gcggttcttg gatttatgcg 1320
gtattagcat ctgtttccaa atgttatccc ccacttaagg gcaggttacc cacgtgttac 1380
tcacccgtcc gccactcgtt caaaattaaa tcaagatgca agcacctttc aataatcaga 1440
actcgttcga cttgcatgta ttaggcacgc cgccagcgtt catcctgacc agaaaaaaaa 1500
actcat 1506
Claims (8)
1. Lactobacillus gasseriLactobacillus gasseriThe preservation number is CGMCC number 14109.
2. Use of lactobacillus gasseri according to claim 1 for the preparation of a bacteriostatic medicament for bacterial vaginal pathogens.
3. Use according to claim 2, characterized in that the drug is a bacterial agent which colonizes and survives the vaginal epithelium in the vaginal environment.
4. Use according to claim 2, characterized in that said drug is a bacterial agent which is metabolized to produce H in the vaginal environment2O2。
5. Use according to claim 2, characterized in that the bacterial vaginal pathogenic bacterium is gardnerella vaginalis.
6. Use according to claim 2, characterized in that the bacterial vaginal pathogenic bacterium is candida albicans, staphylococcus aureus, escherichia coli, pseudomonas aeruginosa or salmonella.
7. Use of the lactobacillus gasseri as defined in claim 1 for the preparation of a medicament for the prophylaxis or treatment of bacterial vaginal diseases.
8. Use according to claim 7, characterized in that the bacterial vaginal disease is senile vaginitis, non-specific vaginal infections or mixed vaginal infections.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810026176.2A CN108004187B (en) | 2018-01-11 | 2018-01-11 | Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810026176.2A CN108004187B (en) | 2018-01-11 | 2018-01-11 | Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108004187A CN108004187A (en) | 2018-05-08 |
| CN108004187B true CN108004187B (en) | 2020-03-10 |
Family
ID=62049985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810026176.2A Active CN108004187B (en) | 2018-01-11 | 2018-01-11 | Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108004187B (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110538201A (en) * | 2018-05-28 | 2019-12-06 | 赵湘江 | Lactobacillus composition and application thereof |
| CN110540945B (en) * | 2018-05-29 | 2023-04-25 | 广东强基药业有限公司 | Lactobacillus jensenii and application thereof in preparing vaginal antibacterial drugs |
| CN111088178B (en) * | 2018-10-23 | 2022-11-22 | 上海上药信谊药厂有限公司 | Produce lactic acid and H 2 O 2 Lactobacillus and application thereof |
| CN109402002B (en) * | 2018-11-01 | 2021-10-08 | 南京工业大学 | Lactobacillus gasseri and application thereof in preparation of premature delivery prevention medicine |
| CN110016442B (en) * | 2019-02-21 | 2021-03-23 | 河北一然生物科技有限公司 | Application of lactobacillus rhamnosus and compound bacterium powder thereof in product for preventing and treating vaginitis |
| CN110656060B (en) | 2019-08-09 | 2020-08-07 | 四川厌氧生物科技有限责任公司 | Multi-linked lactobacillus composition and application thereof in female vaginal health |
| CN111471623A (en) * | 2020-04-21 | 2020-07-31 | 广东龙创基药业有限公司 | Composition of three lactobacilli and application thereof |
| CN112458006A (en) * | 2020-11-10 | 2021-03-09 | 深圳华大生命科学研究院 | Lactobacillus gasseri for the prevention and/or treatment of diseases associated with disturbances of the genital flora |
| CN113862188B (en) * | 2021-10-15 | 2023-06-16 | 广东一元兰欣生物科技有限公司 | Lactobacillus gasseri LS03 and application thereof |
| CN114350560B (en) * | 2022-01-05 | 2023-07-04 | 江南大学 | Lactobacillus paracasei capable of inhibiting growth and biomembrane of gardnerella vaginalis and producing hydrogen peroxide |
| CN114250186B (en) * | 2022-01-05 | 2023-06-13 | 江南大学 | Lactobacillus gasseri strain for relieving bacterial vaginitis and application thereof |
| CN114214256B (en) * | 2022-01-12 | 2022-06-28 | 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 |
| CN116948922B (en) * | 2023-09-20 | 2024-03-01 | 杭州微致生物科技有限公司 | Lactobacillus gasseri VB247 and application thereof |
| CN117327628B (en) * | 2023-11-16 | 2024-03-22 | 江苏科荣生物医药有限公司 | Lactobacillus gasseri Mia capable of fermenting Chinese herbal medicine and resisting HPV virus 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2364712A1 (en) * | 2010-03-11 | 2011-09-14 | Kloarys Developpement | Topical cosmetic or pharmaceutical composition comprising probiotic lactobacillus strains and use of same |
| CN103911309A (en) * | 2013-01-05 | 2014-07-09 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri strain and its uses |
| CN103911306A (en) * | 2013-01-05 | 2014-07-09 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri strain and uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL210465B1 (en) * | 2007-06-04 | 2012-01-31 | Inst Biotechnologii Surowic I Szczepionek Biomed Społka Akcyjna | Composition of strains type Lactobacillus and application of the composition of strains type Lactobacillus |
-
2018
- 2018-01-11 CN CN201810026176.2A patent/CN108004187B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2364712A1 (en) * | 2010-03-11 | 2011-09-14 | Kloarys Developpement | Topical cosmetic or pharmaceutical composition comprising probiotic lactobacillus strains and use of same |
| CN103911309A (en) * | 2013-01-05 | 2014-07-09 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri strain and its uses |
| CN103911306A (en) * | 2013-01-05 | 2014-07-09 | 欣力菲生物医药技术（天津）有限公司 | Lactobacillus gasseri strain and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| Identification and H2O2 Production of Vaginal Lactobacilli from Pregnant Women at High Risk of Preterm Birth and Relation with Outcome;Mark Wilks等;《JOURNAL OF CLINICAL MICROBIOLOGY》;20040228;第42卷(第2期);第713-717页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108004187A (en) | 2018-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108004187B (en) | Lactobacillus gasseri and application thereof in preparing vagina bacteriostatic drug | |
| CN107299065B (en) | Lactobacillus plantarum and application thereof in preparation of vagina bacteriostatic drugs | |
| CN108004188B (en) | Lactobacillus rhamnosus and application thereof in preparing vagina bacteriostatic medicine | |
| CN107267415B (en) | Lactobacillus reuteri and application thereof in preparing vagina bacteriostatic drug | |
| US9937214B2 (en) | Lactobacillus crispatus and application thereof | |
| WO2021027740A1 (en) | Multi-lactobacillus composition and application thereof to vaginal health of females | |
| CN111088178B (en) | Produce lactic acid and H 2 O 2 Lactobacillus and application thereof | |
| CN110777087B (en) | Lactobacillus johnsonii and application thereof | |
| CN110540945B (en) | Lactobacillus jensenii and application thereof in preparing vaginal antibacterial drugs | |
| WO2022100631A1 (en) | Lactobacillus crispatus for preventing and/or treating genital tract flora disorder related diseases | |
| CN110982726B (en) | Lactobacillus crispatus and application thereof | |
| CN114561330A (en) | Composite microbial inoculum for preventing and treating genital tract infection | |
| CN111471623A (en) | Composition of three lactobacilli and application thereof | |
| CN110538201A (en) | Lactobacillus composition and application thereof | |
| CN117511826B (en) | Lactobacillus mucilaginosus and application thereof | |
| CN117535206B (en) | Lactobacillus salivarius and application thereof | |
| CN116769676B (en) | Lactobacillus crispatus and application thereof | |
| CN117535208B (en) | Lactobacillus crispatus and application thereof in female genital tract health | |
| CN117327628B (en) | Lactobacillus gasseri Mia capable of fermenting Chinese herbal medicine and resisting HPV virus and application thereof | |
| WO2024032452A1 (en) | Lactiplantibacillus plantarum strain and use thereof | |
| CN117363511A (en) | Webster-fused-type medicine capable of improving colpitis diseases and application thereof | |
| CN117535207A (en) | Lactobacillus gasseri and application thereof | |
| CN117683691A (en) | Lactobacillus reuteri and application thereof in preparation of medicines for preventing and treating vaginitis |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: 521000 self built R & D Building 1 / F and 2 / F, plot jn02-15-1, shenchao Road, Jingnan sub Park, Zhongshan (Chaozhou) industrial transfer industrial park, Chaozhou City, Guangdong Province Patentee after: GUANGDONG LONGCHUANGJI PHARMACEUTICAL Co.,Ltd. Patentee after: Zhao Xiangjiang Address before: 521000 Chaozhou Avenue, Chaozhou City, Guangdong Province, North End of Chaozhou Avenue, East Chaozhou Economic Development Zone Management Committee Office Building, Fifth Floor, 535 Rooms Patentee before: GUANGDONG LONGCHUANGJI PHARMACEUTICAL Co.,Ltd. Patentee before: Zhao Xiangjiang |
|
| CP02 | Change in the address of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240319 Address after: 521000 Factory Building on Plot JN02-15-1, Shenchao Road, Jingnan Branch, Zhongshan (Chaozhou) Industrial Transfer Industrial Park, Fengquanhu High tech Zone, Chaozhou City, Guangdong Province Patentee after: GUANGDONG LONGCHUANGJI PHARMACEUTICAL CO.,LTD. Country or region after: Zhong Guo Address before: 521000 self built R & D Building 1 / F and 2 / F, plot jn02-15-1, shenchao Road, Jingnan sub Park, Zhongshan (Chaozhou) industrial transfer industrial park, Chaozhou City, Guangdong Province Patentee before: GUANGDONG LONGCHUANGJI PHARMACEUTICAL CO.,LTD. Country or region before: Zhong Guo Patentee before: Zhao Xiangjiang |
|
| TR01 | Transfer of patent right |