CN116083269A - Lactobacillus delbrueckii subspecies indicum and culture method and application thereof - Google Patents
Lactobacillus delbrueckii subspecies indicum and culture method and application thereof Download PDFInfo
- Publication number
- CN116083269A CN116083269A CN202210966253.9A CN202210966253A CN116083269A CN 116083269 A CN116083269 A CN 116083269A CN 202210966253 A CN202210966253 A CN 202210966253A CN 116083269 A CN116083269 A CN 116083269A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus delbrueckii
- delbrueckii subspecies
- indicum
- subspecies
- glucose
- 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.)
- Granted
Links
- 241000186673 Lactobacillus delbrueckii Species 0.000 title claims abstract description 65
- 238000012136 culture method Methods 0.000 title abstract description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 18
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 16
- 210000004369 blood Anatomy 0.000 claims abstract description 12
- 239000008280 blood Substances 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 11
- 230000001580 bacterial effect Effects 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 8
- 208000013464 vaginal disease Diseases 0.000 claims abstract description 8
- 229940124530 sulfonamide Drugs 0.000 claims abstract description 7
- 150000003456 sulfonamides Chemical class 0.000 claims abstract description 7
- GUBGYTABKSRVRQ-UHFFFAOYSA-N D-Cellobiose Natural products OCC1OC(OC2C(O)C(O)C(O)OC2CO)C(O)C(O)C1O GUBGYTABKSRVRQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims abstract description 5
- 229930006000 Sucrose Natural products 0.000 claims abstract description 5
- 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 claims abstract description 5
- 239000005720 sucrose Substances 0.000 claims abstract description 5
- 239000000843 powder Substances 0.000 claims description 21
- 239000003814 drug Substances 0.000 claims description 12
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 8
- 241001494479 Pecora Species 0.000 claims description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- 210000003743 erythrocyte Anatomy 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 7
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012154 double-distilled water Substances 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 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 claims description 4
- 235000004279 alanine Nutrition 0.000 claims description 4
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 4
- 239000012091 fetal bovine serum Substances 0.000 claims description 4
- 235000000346 sugar Nutrition 0.000 claims description 4
- LKDRXBCSQODPBY-OEXCPVAWSA-N D-tagatose Chemical compound OCC1(O)OC[C@@H](O)[C@H](O)[C@@H]1O LKDRXBCSQODPBY-OEXCPVAWSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims description 3
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 3
- DKXNBNKWCZZMJT-UHFFFAOYSA-N O4-alpha-D-Mannopyranosyl-D-mannose Natural products O=CC(O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O DKXNBNKWCZZMJT-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229960001031 glucose Drugs 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229960002160 maltose Drugs 0.000 claims description 3
- 229940099596 manganese sulfate Drugs 0.000 claims description 3
- 239000011702 manganese sulphate Substances 0.000 claims description 3
- 235000007079 manganese sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 3
- 239000001393 triammonium citrate Substances 0.000 claims description 3
- 235000011046 triammonium citrate Nutrition 0.000 claims description 3
- XUBOMFCQGDBHNK-JTQLQIEISA-N (S)-gatifloxacin Chemical compound FC1=CC(C(C(C(O)=O)=CN2C3CC3)=O)=C2C(OC)=C1N1CCN[C@@H](C)C1 XUBOMFCQGDBHNK-JTQLQIEISA-N 0.000 claims 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 claims description 2
- 229930182566 Gentamicin Natural products 0.000 claims description 2
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 claims description 2
- 229930182555 Penicillin Natural products 0.000 claims description 2
- 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 claims description 2
- 239000004098 Tetracycline Substances 0.000 claims description 2
- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 claims description 2
- 108010059993 Vancomycin Proteins 0.000 claims description 2
- 229960004821 amikacin Drugs 0.000 claims description 2
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 claims description 2
- 229960000723 ampicillin Drugs 0.000 claims 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 claims description 2
- 229960002100 cefepime Drugs 0.000 claims description 2
- HVFLCNVBZFFHBT-ZKDACBOMSA-N cefepime 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=1C[N+]1(C)CCCC1 HVFLCNVBZFFHBT-ZKDACBOMSA-N 0.000 claims description 2
- 229960004261 cefotaxime Drugs 0.000 claims description 2
- GPRBEKHLDVQUJE-VINNURBNSA-N cefotaxime Chemical compound N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C(O)=O)=O)C(=O)/C(=N/OC)C1=CSC(N)=N1 GPRBEKHLDVQUJE-VINNURBNSA-N 0.000 claims description 2
- 229960004755 ceftriaxone Drugs 0.000 claims 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 claims description 2
- 229960005091 chloramphenicol Drugs 0.000 claims description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 2
- 229960003405 ciprofloxacin Drugs 0.000 claims description 2
- 229960002227 clindamycin Drugs 0.000 claims 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 claims description 2
- 229960003276 erythromycin Drugs 0.000 claims description 2
- 229960003923 gatifloxacin Drugs 0.000 claims description 2
- 229960002518 gentamicin Drugs 0.000 claims description 2
- 229960002182 imipenem Drugs 0.000 claims description 2
- ZSKVGTPCRGIANV-ZXFLCMHBSA-N imipenem Chemical compound C1C(SCC\N=C\N)=C(C(O)=O)N2C(=O)[C@H]([C@H](O)C)[C@H]21 ZSKVGTPCRGIANV-ZXFLCMHBSA-N 0.000 claims description 2
- 229960003376 levofloxacin Drugs 0.000 claims description 2
- 229960003907 linezolid Drugs 0.000 claims description 2
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 claims description 2
- 229960002260 meropenem Drugs 0.000 claims 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 claims description 2
- 229940049954 penicillin Drugs 0.000 claims description 2
- 229960001225 rifampicin Drugs 0.000 claims description 2
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 2
- 229960002180 tetracycline Drugs 0.000 claims description 2
- 229930101283 tetracycline Natural products 0.000 claims description 2
- 235000019364 tetracycline Nutrition 0.000 claims description 2
- 150000003522 tetracyclines Chemical class 0.000 claims description 2
- 229960003165 vancomycin Drugs 0.000 claims 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 claims 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 claims description 2
- 238000011321 prophylaxis Methods 0.000 claims 2
- 150000008163 sugars Chemical class 0.000 claims 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims 1
- WQZGKKKJIJFFOK-CBPJZXOFSA-N D-Gulose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O WQZGKKKJIJFFOK-CBPJZXOFSA-N 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 14
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 14
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 11
- 241000588724 Escherichia coli Species 0.000 abstract description 7
- 239000004310 lactic acid Substances 0.000 abstract description 7
- 235000014655 lactic acid Nutrition 0.000 abstract description 7
- 241000193985 Streptococcus agalactiae Species 0.000 abstract description 6
- 244000052616 bacterial pathogen Species 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000002949 hemolytic effect Effects 0.000 abstract description 3
- 229920002527 Glycogen Polymers 0.000 abstract description 2
- 229940124350 antibacterial drug Drugs 0.000 abstract description 2
- 210000002919 epithelial cell Anatomy 0.000 abstract description 2
- 229940096919 glycogen Drugs 0.000 abstract description 2
- 230000003385 bacteriostatic effect Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 11
- 229940079593 drug Drugs 0.000 description 9
- 239000003242 anti bacterial agent Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 210000001215 vagina Anatomy 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 210000003756 cervix mucus Anatomy 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000035935 pregnancy Effects 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 206010018910 Haemolysis Diseases 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- 241001060359 Lactobacillus delbrueckii subsp. indicus Species 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 230000004544 DNA amplification Effects 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 238000005842 biochemical reaction Methods 0.000 description 3
- 238000009640 blood culture Methods 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000013365 dairy product Nutrition 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 3
- 229940093181 glucose injection Drugs 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108700023418 Amidases Proteins 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 206010046914 Vaginal infection Diseases 0.000 description 2
- 102000005922 amidase Human genes 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- WZRJTRPJURQBRM-UHFFFAOYSA-N 4-amino-n-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide;5-[(3,4,5-trimethoxyphenyl)methyl]pyrimidine-2,4-diamine Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1.COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 WZRJTRPJURQBRM-UHFFFAOYSA-N 0.000 description 1
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 1
- 101000894568 Catharanthus roseus Catharanthine synthase Proteins 0.000 description 1
- 101000658635 Catharanthus roseus Tabersonine synthase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229940047766 co-trimoxazole Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019985 fermented beverage Nutrition 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 235000021109 kimchi Nutrition 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000013618 yogurt Nutrition 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
-
- 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
-
- 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
-
- 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
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Gynecology & Obstetrics (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a Lactobacillus delbrueckii subspecies Indici, a culture method and application thereof, wherein the Lactobacillus delbrueckii subspecies Indici is preserved in China center for type culture collection (CCTCCNO. M20221249). The lactobacillus delbrueckii subspecies indicum hydrogen peroxide test of the invention is negative, and can decompose D-glucose, sucrose and D-cellobiose; can generate an a-hemolytic ring and a bacteriostatic ring, can inhibit the growth phenomenon of staphylococcus aureus and escherichia coli visually when being cultured on a blood plate for 48 hours, and can generate a regular concentric circular hemolytic ring with the staphylococcus aureus; acid production on the culture medium can inhibit the growth of streptococcus agalactiae; can decompose glycogen in vaginal epithelial cells to generate lactic acid, reduce pH value, inhibit the growth of pathogenic bacteria and the excessive reproduction of conditional pathogenic bacteria; is sensitive to 19 common antibacterial drugs, is only resistant to sulfonamides, and is suitable for preventing or treating bacterial vaginal diseases, especially bacterial vaginal diseases of pregnant women.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus delbrueckii subspecies indicum and a culture method and application thereof.
Background
Lactobacillus delbrueckii includes six subspecies of delbrueckii, subspecies lactis, subspecies bulgaricus, subspecies indiae, subspecies sunkii and subspecies jacobson. The six subspecies were derived from: de's subspecies are mainly derived from fermented potato, cereal and vegetable ferments, bulgaria subspecies are mainly derived from Bao's calix and yoghurt etc., lactic subspecies are mainly derived from milk, cheese, compressed yeast and cereal wort etc., indian subspecies are mainly derived from dairy products, sunkii subspecies are mainly derived from river water and Japanese kimchi etc., and jacob subspecies are found in an alcoholic fermented beverage. Lactobacillus dellaginella subspecies indicum was originally found in indian dairy at university of verona study in 2005.
There are few reports in the literature on the lactobacillus delbrueckii subspecies india at home and abroad, and the research specimens on the 16s rRNA sequence of the lactobacillus delbrueckii subspecies india in the NCBI genome database are all from the external environment such as dairy products, and no vaginal secretion from pregnant women is found. Moreover, lactobacillus delbrueckii is the most studied in the food industry and the livestock breeding industry, and has little research in the aspects of medical care and disease prevention and treatment.
Disclosure of Invention
The invention aims to provide lactobacillus delbrueckii subspecies indicus (Lactobacillus delbrueckii subsp. Indicus) for solving the problems existing in the prior art.
The embodiment of the invention is realized by that a Lactobacillus delbrueckii subspecies Indici (Lactobacillus delbrueckii subsp. Indicus) named WuYI2021-2 is preserved in China center for type culture collection (China Center for Type Culture Collection, CCTCC) on 8 months and 5 days in 2022, the preservation address is university of Wuhan in Wuhan, hubei province, china, and the preservation number is CCTCC NO. M20221249.
Still further, the Lactobacillus delbrueckii subspecies Indicus can decompose saccharides including D-glucose, sucrose and D-cellobiose, and the non-decomposable saccharides include D-glucose, N-acetyl-D-glucosamine, D-maltose, ancient sugar, D-tagatose, D-trehalose, alpha-glucose, D-trehalose, D-galactose, D-ribose, lactose and D-xylose; can decompose arginine biohydrolase 1, arginine biohydrolase 2, alanine arylamidase and alanine-phenylalanine-proline arylamidase; the olprixin toleration test was positive and the hydrogen peroxide test was negative.
Still further, the lactobacillus delbrueckii subspecies indicum are sensitive to linezolid, ciprofloxacin, levofloxacin, gatifloxacin, tetracycline, rifampin, chloramphenicol, ampicillin, penicillin, vancomycin, gentamicin, amikacin, clindamycin, ceftriaxone, cefepime, cefotaxime, erythromycin, imipenem, meropenem; resistant to sulfonamides such as trimethoprim/sulfamethoxazole.
The embodiment of the invention also provides a culture method of the Lactobacillus delbrueckii subspecies indicum, wherein the separating culture adopts MRS blood plates, and the components comprise: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium acetate, 5g of beef extract powder, 4g of yeast extract powder, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 1ml of Tween 80, 50ml of 5% sterile defibrinated sheep erythrocytes, 750 ml of deionized double distilled water and citric acid-sodium citrate buffer solution with pH=4.0.
Further, the pure culture adopts modified acid blood plates, and the components comprise: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium chloride, 5g of beef extract powder, 5g of yeast extract powder, 5g of beef heart extract powder, 50ml of 5% sterile defibrinated sheep red blood cells, 50ml of fetal bovine serum, 900ml of deionized double distilled water and a citric acid-sodium citrate buffer solution with the pH value of 4.0.
The embodiment of the invention also provides the application of the lactobacillus delbrueckii subspecies indicum in preparing medicines for preventing or treating bacterial vaginal diseases.
Further, the application of the medicine for preventing or treating bacterial vaginal diseases of pregnant women is provided. The pregnant woman is successfully separated and cultured in vaginal secretion of pregnant women for the first time. The invention discovers that the structure of the lactic acid bacteria flora in pregnant women is different from the structure of the bacteria flora of women in general childbearing period and women with asexual life history, and the types of the lactic acid bacteria in the vagina are obviously reduced, which possibly relates to the hormone level in the pregnant women, and inhibit the growth and reproduction of other lactic acid bacteria, so that the value of the Lactobacillus delbrueckii existing in the vagina of the pregnant women is very important, which possibly relates to the self-cleaning effect of the vagina and the micro-ecological balance of the vaginal bacteria flora, and plays an important role in inhibiting the overgrowth and reproduction of pathogenic bacteria and miscellaneous bacteria. The Lactobacillus delbrueckii subspecies indicum drug sensitivity test finds that the Lactobacillus delbrueckii subspecies indicum drug sensitivity test is sensitive to most antibacterial drugs and only resistant to sulfonamides, which suggests that clinically various antibiotics can influence the growth and propagation of Lactobacillus delbrueckii flora, so that the content of Lactobacillus delbrueckii is reduced, the vaginal microecological balance is indirectly influenced, and the occurrence and development of related diseases are promoted. It is reflected that the pregnant woman should not use antibiotics during gestation period to avoid inhibiting the growth and reproduction of lactobacillus, breaking the microecological balance of vagina, causing flora disorder to induce colpitis and causing fetal development to be affected. Thus, the microecological preparation of Lactobacillus delbrueckii subspecies indicum may have a very good effect on the treatment of bacterial vaginitis in pregnant women during gestation.
Compared with the prior art, the invention has positive oltuin tolerance test of the lactobacillus delbrueckii subspecies indicum, negative hydrogen peroxide test, and can decompose D-glucose, sucrose, D-cellobiose, arginine double hydrolase 1, arginine double hydrolase 2, alanine aromatic amidase and alanine-phenylalanine-proline aromatic amidase, generate a-hemolytic ring and antibacterial ring, and can inhibit the growth phenomenon of staphylococcus aureus and escherichia coli visible to naked eyes and generate regular concentric circular hemolytic ring with the staphylococcus aureus after being cultured on a blood plate for 48 hours. Meanwhile, the streptococcus agalactiae can inhibit the growth of the lactobacillus delbrueckii subspecies indicum in an external culture medium for a short time, and can inhibit the growth of the streptococcus agalactiae after the lactobacillus delbrueckii subspecies indicum of the invention produces a large amount of lactic acid; can decompose glycogen in vaginal epithelial cells to generate lactic acid, reduce pH value, inhibit the growth of pathogenic bacteria and the excessive reproduction of conditional pathogenic bacteria; although hydrogen peroxide is not generated, the pH value of the culture medium can be obviously reduced, which indicates that the acid generating capacity is strong; is suitable for preventing or treating bacterial vaginal diseases, especially bacterial vaginal diseases of pregnant women.
Drawings
FIG. 1 is a phylogenetic tree of the Lactobacillus delbrueckii subspecies india of the present invention;
FIG. 2 is a graph of partial sequencing peaks of the Lactobacillus delbrueckii subspecies Indici of the present invention;
FIG. 3 is a Lactobacillus delbrueckii subspecies Indici of the present invention;
FIG. 4 is a gram positive plot of Lactobacillus delbrueckii subspecies Indicum of the present invention;
FIG. 5 is a graph showing the inhibition of Staphylococcus aureus growth by Lactobacillus delbrueckii subspecies Indici of the present invention;
FIG. 6 shows the inhibition of E.coli growth by Lactobacillus delbrueckii subspecies Indici of the present invention;
FIG. 7 is a graph showing that Lactobacillus delbrueckii subspecies Indici and Staphylococcus aureus of the present invention produce regular concentric circular hemolytic rings.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
At present, some Lactobacillus delbrueckii subspecies are internationally found and are separated from the external environment, no human body has successfully separated and cultured the Lactobacillus delbrueckii subspecies in the human body, and the invention truly separates and cultures the Lactobacillus delbrueckii subspecies from vaginal secretion of pregnant women for the first time. Separated from human body, and then applied to disease prevention and treatment of human body, the safety coefficient is relatively high, and the method can also be applied to food industry.
Example 1
Isolated culture and identification of lactobacillus delbrueckii subspecies indicum (Lactobacillus delbrueckii subsp. Indicus) WuYi2021-2, comprising the steps of:
(1) And (5) collecting and separating the sample and culturing the sample. The vaginal secretion of pregnant women is collected by using a sterile sampling cotton swab in strict sterile operation. All collected samples were examined in time and inoculated in MRS platelets by partition streaking, and 5% CO was added 2 The incubator is used for isolated culture for 48 hours, and suspicious colonies are selected for further pure culture on the modified acid blood plates.
(2) The mass spectrometer identifies the strain and observes the colony growth and staining effect. After pure culture is separated, a mass spectrometer on a single colony is selected for strain identification, and specific operations are carried out according to the instruction of instrument operation. Colony growth was also observed and gram stained.
(3) Biochemical reaction, acid production test and bacteriostasis test. And carrying out biochemical reaction on the lactobacillus delbrueckii initially identified by the mass spectrometer, and respectively inoculating the lactobacillus delbrueckii to a GP gram positive bacteria identification card and a GN gram negative bacteria identification card. Lactobacillus delbrueckii is prepared into 0.5 McUth bacteria liquid, 1ml of 5% medical glucose injection and 0.1ml of bacteria liquid are added into 5ml of nutrient broth culture medium for acid production test; simultaneously, a staphylococcus aureus colony (standard strain ATCC 25923) or an escherichia coli standard strain colony (standard strain ATCC 25922) is picked by an inoculating needle and inoculated in the center of a closed streak, and a staphylococcus aureus colony (standard strain ATCC 25923) or an escherichia coli standard strain colony (standard strain ATCC 25922) is also inoculated on the other side of the blood plate for a control experiment, and after culturing for 48 hours, the acid production condition and the antibacterial effect are observed.
(4) And (5) extracting sample DNA. Firstly, taking a 1.5ml centrifuge tube, adding 200ul of pretreatment liquid and 3 glass beads, then adding a proper amount of bacteria sample, and fully grinding in a grinder. Then 20ul of protease K solution is added, and the mixture is placed for 30 to 60 minutes at 37 ℃ after uniform mixing. Then 200ul of lysate is added, the mixture is fully and reversely mixed, and the mixture is placed at 70 ℃ for 10min. Immediately add 200ul of absolute ethanolFully reversing and uniformly mixing, removing liquid on the inner wall of the pipe cover by short-time centrifugation, passing through an adsorption column, washing the washing liquid for 1 time, and washing the washing liquid for 2 times. And placing the adsorption column at room temperature for 5-10 min to thoroughly dry residual rinse liquid in the adsorption material. Finally, transferring the adsorption column into a new centrifuge tube, and suspending and dripping 50-100 ul ddH into the central position of the adsorption film 2 O, standing at room temperature for 5-10 min, centrifuging at 12000rpm for 2min, and placing the sample DNA template solution extracted from the centrifuge tube in a refrigerator at-20 ℃ for standby.
(5) 16s rRNA Gene amplification the 16s rRNA gene amplification 27F primer sequence was AGAGAGTTTGATCCTGGCTCAG, the 1492R primer sequence was TACGGCTACCTTGTTACGACTT, and the final product fragment size was about 1500bp. Into a 0.5mL PCR reaction tube was added 15. Mu.L of the Super Mix enzyme mixture, 1. Mu.L each of the F primer, R primer and DNA template, and ddH was further added 2 O to 30 mu L, and performing amplification on the sample after 5s of instantaneous high-speed centrifugation. The PCR amplification procedure was: firstly, the temperature is 96 ℃ for 5min; then, the temperature is 96 ℃ for 20s, 62 ℃ for 20s and 72 ℃ for 30s for 35 cycles; finally, the amplification procedure was ended at 72℃for 10min and 16 ℃.
(6) PCR product detection, purification and sequencing. 3ul of PCR products were added to 1.0% agarose gel and electrophoresed, and the presence or absence of product bands and brightness were observed. And (3) purifying the PCR product by using magnetic beads according to the instruction of the kit, and sequencing the purified PCR product on a machine.
(7) And (5) data analysis. BLAST comparison analysis is carried out on the sequencing result and the database on the NCBI website to identify strains. BLAST comparison of the sequencing result of the 16S rRNA gene amplification product with a database on NCBI website shows that the sequencing result has the closest parent relationship with Lactobacillus delbrueckii subsp.indicus, the evolutionary tree is shown in figure 1 and named as WuYI2021-2, the variable region sequence of the 16S rRNA is shown in a sequence table SEQ ID NO:1, and the sequencing result is shown in figure 2.
Example 2
Preparation of Lactobacillus delbrueckii Indian subspecies WuYi2021-2 isolation Medium
1. Preparation of Lactobacillus delbrueckii isolation Medium
Preparing MRS blood plate according to the formula, sterilizing at 121deg.C for 15 min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep red blood cells, mixing, rapidly pouring 15-20ml into a 90cm sterilized plate, cooling to room temperature, solidifying, and quality testing. The prepared culture medium has about ph=6.0, 3ml of citric acid-sodium citrate buffer solution (ph=4.0, room temperature 25 ℃) is added on the surface of the MRS blood culture medium before use, and the mixture is placed in an incubator for 15-20 minutes, so that the citric acid-sodium citrate buffer solution is automatically absorbed into the culture medium.
MRS blood plate composition (g/L) includes: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium acetate, 5g of beef extract powder, 4g of yeast extract powder, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 1ml of tween 80, 50ml of 5% sterile defibrinated sheep erythrocytes, 950ml of deionized double distilled water and a citric acid-sodium citrate buffer (pH=4.0, room temperature 25 ℃).
2. Preparing pure culture medium of Lactobacillus delbrueckii
Preparing an improved acidic blood plate according to a formula, sterilizing at 121 ℃ under high pressure for 15 minutes, cooling to about 50 ℃, adding 5% of aseptic defibrinated sheep red blood cells and aseptic fetal bovine serum, uniformly mixing, rapidly pouring 15-20ml into a 90cm aseptic plate, cooling to room temperature, solidifying, and checking quality to be qualified for later use. The prepared culture medium has about ph=7.0, and 3ml of citric acid-sodium citrate buffer solution (ph=4.0, room temperature 25 ℃) is added on the surface of the blood culture medium before use, and the blood culture medium is placed in an incubator for 15-20 minutes, so that the citric acid-sodium citrate buffer solution is automatically absorbed into the culture medium.
The modified acid platelet component (g/L) comprises: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium chloride, 5g of beef extract powder, 5g of yeast extract powder, 5g of beef extract powder, 50ml of 5% sterile defibrinated sheep red blood cells, 50ml of fetal bovine serum, 900ml of deionized double distilled water and citric acid-sodium citrate buffer (pH=4.0, room temperature 25 ℃).
Example 3
The isolated and cultured Lactobacillus delbrueckii subspecies WuYi2021-2 of example 1 was tested for physiological and biochemical characteristics.
1. Colony morphological characteristics and staining conditions
Lactobacillus delbrueckii subspecies WuYi2021-2 has irregular colony edges and rough colony on the blood plate, and can generate a-hemolysis ring; in the form of a gram-positive long rod, as shown in figures 3 and 4.
2. Results of biochemical reactions on GP and GN identification cards
The saccharide decomposed by the Lactobacillus delbrueckii subspecies WuYi2021-2 mainly comprises D-glucose, sucrose and D-cellobiose, and can not decompose D-glucose, N-acetyl-D-glucosamine, D-maltose, old sugar, D-tagatose, D-trehalose, alpha-glucose, D-trehalose, D-galactose, D-ribose, lactose and D-xylose; can decompose arginine biohydrolase 1, arginine biohydrolase 2, alanine arylamidase and alanine-phenylalanine-proline arylamidase; the olprixin tolerability test was positive and the hydrogen peroxide test was negative, as shown in tables 1 and 2.
TABLE 1 identification of card Positive results for GP gram-Positive bacteria by Lactobacillus delbrueckii subspecies Indici WuYi2021-2
TABLE 2 identification of card-positive results for GN gram-negative bacteria by Lactobacillus delbrueckii subspecies Indici WuYI2021-2
3. Acid production condition and antibacterial effect
The Lactobacillus delbrueckii subspecies WuYi2021-2 can decompose 5% medical glucose injection to produce acid, so that the nutrient broth culture medium containing 5% medical glucose injection is reduced from pH 7.8 to about pH 4, and the glucose concentration is reduced from 277mmol/L to about 140 mmol/L; the growth of staphylococcus aureus and escherichia coli can be inhibited visually after the culture on a blood plate for 48 hours, and a bacteriostasis ring and a hemolysis ring can be generated as shown in fig. 5 and 6, and fig. 7 is a very regular round semitransparent hemolysis ring generated when staphylococcus aureus and lactobacillus delbrueckii subspecies india are simultaneously cultured.
4. Drug sensitivity test
The method comprises the following steps: the Lactobacillus delbrueckii subspecies WuYi2021-21 strain is separated from vaginal secretion of 20 women with normal pregnancy more than 28 weeks in the year 2020, 7 months and 12 months 2020. 20 drug susceptibility tests were performed on Lactobacillus delbrueckii subspecies Indici by K-B diffusion and statistical analysis was performed on the drug susceptibility results of the strains, as shown in Table 3, it was found that Lactobacillus delbrueckii subspecies Indici were susceptible to the other drugs except for the sulfonamide drug resistance.
TABLE 3 analysis of the results of the De Lactobacillus subspecies Indici drug sensitivity test
5. Application prospect
The lactobacillus delbrueckii subspecies india WuYi2021-2 hydrogen peroxide test of the present invention is negative and does not produce hydrogen peroxide; meanwhile, the streptococcus agalactiae can inhibit the growth of the lactobacillus delbrueckii subspecies WuYi2021-2 in an external culture medium for a short period, and can inhibit the growth of the streptococcus agalactiae after the lactobacillus delbrueckii subspecies WuYi2021-2 produces a large amount of lactic acid (namely, the acidogenesis can inhibit the growth of the streptococcus agalactiae); meanwhile, the phenomena that lactobacillus delbrueckii subspecies india WuYi2021-2 can generate a bacteriostasis ring, a hemolysis ring and the like with staphylococcus aureus are also observed; can also inhibit the growth of Escherichia coli.
For pregnant females, infectious diseases may often occur during pregnancy, and if antibiotics are applied at this time, the level of lactobacillus delbrueckii may be affected, further increasing the risk of occurrence of related diseases, and ultimately affecting the life health and safety of the fetus. The study of the invention shows that Lactobacillus delbrueckii subspecies WuYi2021-2 is resistant to sulfonamides, but is sensitive to other antibacterial agents. The method suggests that various antibiotics in clinic can influence the activity of lactobacillus delbrueckii, promote the reduction of the content of lactobacillus delbrueckii, indirectly influence the microecological balance of vagina, and promote the occurrence and development of related diseases. It is reflected that the pregnant woman should not use antibiotics during gestation period to avoid inhibiting the growth and reproduction of lactobacillus, breaking the microecological balance of vagina, causing flora disorder to induce colpitis and causing the fetus to be affected. The Lactobacillus delbrueckii subspecies indicum has obvious drug resistance to sulfonamides, is sensitive to other drugs to different degrees, provides guidance for clinical pregnant women, and fully illustrates that the pregnant women should avoid the application of antibiotics as much as possible so as to ensure the life health and safety of the women and fetuses.
Claims (7)
1. A lactobacillus delbrueckii subspecies indicum, which is preserved in the China center for type culture collection (cctccc) No. m20221249.
2. Lactobacillus delbrueckii subspecies indicum according to claim 1, wherein the decomposable sugars comprise D-glucose, sucrose and D-cellobiose, and the non-decomposable sugars comprise D-glucose, N-acetyl-D-glucosamine, D-maltose, gulose, D-tagatose, D-trehalose, a-glucose, D-trehalose, D-galactose, D-ribose, lactose and D-xylose; can decompose arginine biohydrolase 1, arginine biohydrolase 2, alanine arylamidase and alanine-phenylalanine-proline arylamidase; the olprixin toleration test was positive and the hydrogen peroxide test was negative.
3. Lactobacillus delbrueckii subspecies indicum according to claim 1, characterized in that it is sensitive to linezolid sheets, ciprofloxacin, levofloxacin, gatifloxacin, tetracycline, rifampin, chloramphenicol, ampicillin, penicillin, vancomycin, gentamicin, amikacin, clindamycin, ceftriaxone, cefepime, cefotaxime, erythromycin, imipenem, meropenem; is resistant to sulfonamides.
4. The method according to claim 1, wherein the isolated culture is performed using MRS blood plates comprising the following components: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium acetate, 5g of beef extract powder, 4g of yeast extract powder, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 2g of triammonium citrate, 1ml of Tween 80, 50ml of 5% sterile defibrinated sheep erythrocytes, 750 ml of deionized double distilled water and citric acid-sodium citrate buffer solution with pH=4.0.
5. The method according to claim 4, wherein the pure culture is performed using modified acid platelets, comprising the following components: 10g of peptone, 20g of glucose, 15g of agar powder, 5g of sodium chloride, 5g of beef extract powder, 5g of yeast extract powder, 5g of beef heart extract powder, 50ml of 5% sterile defibrinated sheep red blood cells, 50ml of fetal bovine serum, 900ml of deionized double distilled water and a citric acid-sodium citrate buffer solution with the pH value of 4.0.
6. Use of lactobacillus delbrueckii subspecies indicum according to claim 1, for the preparation of a medicament for the prophylaxis or treatment of bacterial vaginal disease.
7. The use according to claim 6, for the preparation of a medicament for the prophylaxis or treatment of bacterial vaginal disease in pregnant women.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210966253.9A CN116083269B (en) | 2022-08-12 | 2022-08-12 | Lactobacillus delbrueckii subspecies indicum and culture method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210966253.9A CN116083269B (en) | 2022-08-12 | 2022-08-12 | Lactobacillus delbrueckii subspecies indicum and culture method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116083269A true CN116083269A (en) | 2023-05-09 |
| CN116083269B CN116083269B (en) | 2024-09-20 |
Family
ID=86199712
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210966253.9A Active CN116083269B (en) | 2022-08-12 | 2022-08-12 | Lactobacillus delbrueckii subspecies indicum and culture method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116083269B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101543514A (en) * | 2009-05-06 | 2009-09-30 | 大连润生堂生物科技工程有限公司 | Compound lactobacillus vagina cleaning hygiene body lotion for preventing or treating vaginitis and technology thereof |
| EP3172973A1 (en) * | 2015-11-27 | 2017-05-31 | DAFLORN Ltd. | Probiotic formula, process of its preparation and use |
| CN110607255A (en) * | 2019-08-28 | 2019-12-24 | 华南理工大学 | Preparation method and application of lactobacillus delbrueckii and direct vat set lactobacillus delbrueckii starter |
-
2022
- 2022-08-12 CN CN202210966253.9A patent/CN116083269B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101543514A (en) * | 2009-05-06 | 2009-09-30 | 大连润生堂生物科技工程有限公司 | Compound lactobacillus vagina cleaning hygiene body lotion for preventing or treating vaginitis and technology thereof |
| EP3172973A1 (en) * | 2015-11-27 | 2017-05-31 | DAFLORN Ltd. | Probiotic formula, process of its preparation and use |
| CN110607255A (en) * | 2019-08-28 | 2019-12-24 | 华南理工大学 | Preparation method and application of lactobacillus delbrueckii and direct vat set lactobacillus delbrueckii starter |
Non-Patent Citations (2)
| Title |
|---|
| DELLAGLIO FRANCO 等: "Lactobacillus delbrueckii subsp. indicus subsp. nov., isolated from Indian dairy products", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 55, no. 1, 21 January 2005 (2005-01-21), pages 401 - 404 * |
| 李佳凤 等: "治疗细菌性阴道炎的药物——德氏乳杆菌 DM 8909活菌制剂", 海峡药学, vol. 15, no. 3, 31 March 2003 (2003-03-31), pages 91 - 92 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116083269B (en) | 2024-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110656060B (en) | Multi-linked lactobacillus composition and application thereof in female vaginal health | |
| CN108102959B (en) | Humanized lactobacillus plantarum ZY08 for reducing cholesterol and application thereof | |
| Thomsen et al. | Leukocyte counts and microbiologic cultivation in the diagnosis of puerperal mastitis | |
| Lotfollahi et al. | Prevalence and antimicrobial resistance profiles of Listeria monocytogenes in spontaneous abortions in humans | |
| JP2017501741A (en) | Lactobacillus crispatus and its applications | |
| Goossens et al. | A new selective medium for the isolation of Campylobacter jejuni from human faeces | |
| CN112143680B (en) | Lactobacillus paracasei ZJUIDS05 with antioxidant effect and application thereof | |
| CN113462613A (en) | Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof | |
| Riegel et al. | Isolations of Corynebacterium kroppenstedtii from a breast abscess | |
| CN109402002A (en) | Lactobacillus gasseri and application thereof in preparation of premature delivery prevention medicine | |
| CN117327608A (en) | Lactobacillus rhamnosus strain and application thereof | |
| CN112442464A (en) | Bifidobacterium breve grx201 resistant to oxidation stress and application thereof | |
| CN116162562A (en) | Lactobacillus delbrueckii subspecies and culture method and application thereof | |
| CN114561318B (en) | Lactobacillus murinus and application thereof in treatment of type II diabetes | |
| CN106222240A (en) | Lactobacillus fermenti special media and application thereof | |
| Bahar et al. | Mobiluncus species in gynaecological and obstetric infections: antimicrobial resistance and prevalence in a Turkish population | |
| CN113528383B (en) | Hypoglycemic lactobacillus ZJUIDS09 and application thereof | |
| CN116083269B (en) | Lactobacillus delbrueckii subspecies indicum and culture method and application thereof | |
| Sokolova et al. | Characteristics of species composition, biochemical and pathogenic nature of the microbiota of mammary gland and the reproductive tract in dairy cows | |
| CN116769655A (en) | Streptococcus salivarius thermophilus JIAN+ and application thereof | |
| Önat et al. | Frequency and antibiotic susceptibility of Pasteurella multocida and Mannheimia haemolytica isolates from nasal cavities of cattle | |
| CN114717150B (en) | Lactobacillus plantarum CRS33 and application thereof | |
| CN114317366B (en) | Bacterial strain and application thereof | |
| Mosabi et al. | Isolation and characterization of group B streptococci from human and bovine sources within and around Nairobi | |
| Tompkins et al. | Use of selective broth enrichment to determine the prevalence of multiply resistant JK corynebacteria on skin |
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 |