CN116875480A - Lactobacillus rhamnosus with antagonism to helicobacter pylori - Google Patents
Lactobacillus rhamnosus with antagonism to helicobacter pylori Download PDFInfo
- Publication number
- CN116875480A CN116875480A CN202310580586.2A CN202310580586A CN116875480A CN 116875480 A CN116875480 A CN 116875480A CN 202310580586 A CN202310580586 A CN 202310580586A CN 116875480 A CN116875480 A CN 116875480A
- Authority
- CN
- China
- Prior art keywords
- lactobacillus rhamnosus
- strain
- vhprobi
- helicobacter pylori
- rhamnosus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000218588 Lactobacillus rhamnosus Species 0.000 title claims abstract description 91
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 53
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 52
- 230000008485 antagonism Effects 0.000 title abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 10
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims description 31
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- 239000000284 extract Substances 0.000 claims description 9
- 230000003834 intracellular effect Effects 0.000 claims description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 6
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 208000007882 Gastritis Diseases 0.000 claims description 5
- 235000013305 food Nutrition 0.000 claims description 5
- 230000036541 health Effects 0.000 claims description 5
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 3
- 239000002207 metabolite Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims 3
- 229940127557 pharmaceutical product Drugs 0.000 claims 3
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 31
- 230000000694 effects Effects 0.000 abstract description 20
- 235000012000 cholesterol Nutrition 0.000 abstract description 15
- 238000012216 screening Methods 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000012010 growth Effects 0.000 abstract description 6
- 235000013376 functional food Nutrition 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 241000589989 Helicobacter Species 0.000 abstract description 2
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 60
- 239000001963 growth medium Substances 0.000 description 29
- 241000894006 Bacteria Species 0.000 description 22
- 239000000243 solution Substances 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 18
- 239000006228 supernatant Substances 0.000 description 18
- 239000000725 suspension Substances 0.000 description 18
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 17
- 239000006041 probiotic Substances 0.000 description 17
- 235000018291 probiotics Nutrition 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 16
- 239000007788 liquid Substances 0.000 description 16
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 14
- 230000002496 gastric effect Effects 0.000 description 14
- 238000012258 culturing Methods 0.000 description 13
- 238000009630 liquid culture Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 230000000529 probiotic effect Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 239000003242 anti bacterial agent Substances 0.000 description 10
- 229940088710 antibiotic agent Drugs 0.000 description 10
- 230000029087 digestion Effects 0.000 description 10
- 239000012530 fluid Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 8
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 7
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 7
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 7
- 241000186660 Lactobacillus Species 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 239000001569 carbon dioxide Substances 0.000 description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- 229940039696 lactobacillus Drugs 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 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 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 108010019160 Pancreatin Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 210000000601 blood cell Anatomy 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- -1 citric acid diamine Chemical class 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 5
- 210000004051 gastric juice Anatomy 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229940055695 pancreatin Drugs 0.000 description 5
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 235000011511 Diospyros Nutrition 0.000 description 4
- 244000236655 Diospyros kaki Species 0.000 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 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000008186 active pharmaceutical agent Substances 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 235000013351 cheese Nutrition 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 210000001156 gastric mucosa Anatomy 0.000 description 4
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000005502 peroxidation Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000003794 Gram staining Methods 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- 108010006464 Hemolysin Proteins Proteins 0.000 description 3
- 244000199866 Lactobacillus casei Species 0.000 description 3
- 235000013958 Lactobacillus casei Nutrition 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 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 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000009194 climbing Effects 0.000 description 3
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 3
- 239000011790 ferrous sulphate Substances 0.000 description 3
- 235000003891 ferrous sulphate Nutrition 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 230000009036 growth inhibition Effects 0.000 description 3
- 239000003228 hemolysin Substances 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 239000003978 infusion fluid Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 3
- 229940017800 lactobacillus casei Drugs 0.000 description 3
- 230000003859 lipid peroxidation Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 238000007747 plating Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000012266 salt solution Substances 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000272203 Columba Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 208000018522 Gastrointestinal disease Diseases 0.000 description 2
- 240000001046 Lactobacillus acidophilus Species 0.000 description 2
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000005662 Paraffin oil Substances 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 2
- 208000007107 Stomach Ulcer Diseases 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000006161 blood agar Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Natural products OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 201000005917 gastric ulcer Diseases 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229960004025 sodium salicylate Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241001608472 Bifidobacterium longum Species 0.000 description 1
- 241000186015 Bifidobacterium longum subsp. infantis Species 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000036649 Dysbacteriosis Diseases 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 241000194032 Enterococcus faecalis Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 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 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
- 240000002605 Lactobacillus helveticus Species 0.000 description 1
- 235000013967 Lactobacillus helveticus Nutrition 0.000 description 1
- 241001468157 Lactobacillus johnsonii Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000194046 Streptococcus intermedius Species 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 229940004120 bifidobacterium infantis Drugs 0.000 description 1
- 229940009291 bifidobacterium longum Drugs 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 239000006781 columbia blood agar Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 244000000075 gastric pathogen Species 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 230000007160 gastrointestinal dysfunction Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229940054346 lactobacillus helveticus Drugs 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000004682 mucosal barrier function Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 208000011906 peptic ulcer disease Diseases 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- OSWPSODKJQKKLC-UHFFFAOYSA-M sodium ethanol 2-hydroxybenzoate Chemical compound [Na+].CCO.OC1=CC=CC=C1C([O-])=O OSWPSODKJQKKLC-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oncology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Toxicology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to a strain with antagonism to helicobacter pyloriLactobacillus rhamnosus usedLacticaseibacillus rhamnosus). The strain has strong tolerance to gastrointestinal tract environment, can effectively degrade cholesterol, has remarkable antioxidation effect, can obviously inhibit the growth and adhesion of helicobacter pylori, and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China 4 months and 10 days in 2023: m2023500. The lactobacillus rhamnosus can be widely applied to functional foods, health-care products or medicines.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening and application, in particular to a lactobacillus rhamnosus strain with antagonism to helicobacter pylori.
Background
The infection rate of helicobacter pylori (Helicobacter pylori, hp) in the global natural population is over 50 percent, the infection rate is about 30 percent in developed countries, the infection rate can reach 80 percent in developing countries, the Hp infection rate in China is 40 to 90 percent, and the average of the infection rate is 59 percent. Helicobacter pylori is a spiral curved shape, has flagella and gram-negative bacteria accompanied with power, is mainly planted on gastric mucosa through flagella, is occasionally planted on the parts of ear, nose and throat, oral cavity, intestine, gall bladder and the like, is considered as main pathogenic bacteria of peptic ulcer and chronic gastritis, is proved to be a high risk factor of gastric adenocarcinoma and gastric mucosa-related lymphoid tissue lymphoma, is also related to gastroesophageal reflux disease, functional dyspepsia and other gastrointestinal diseases, and is classified as a class I cancerogenic substance by WHO and International anticancer alliance. At present, the diseases caused by Hp are mainly treated by two methods of a standard triple method and a standard quadruple method, and the common method is mixed treatment of PPI and two other antibiotics. Although the cure rate is improved, the treatment scheme can cause the drug resistance of organism antibiotics, and the long-term unreasonable application of antibiotics can cause adverse reactions such as gastrointestinal dysfunction and gastrointestinal dysbacteriosis, so that another reasonable and effective treatment scheme is suggested clinically. .
Probiotics are living microorganisms that encompass synthetic antimicrobial substances such as bacteriocins, hydrogen peroxide, and the like, and attachment points for microorganisms that regulate the immune response of the host and compete for pathogens. Common probiotics include: (1) lactobacillus genus: lactobacillus helveticus, lactobacillus acidophilus, lactobacillus brevis, lactobacillus casei, lactobacillus johnsonii, lactobacillus rhamnosus, and the like; (2) bifidobacterium genus: bifidobacterium longum, bifidobacterium adolescentis, bifidobacterium infantis, bifidobacterium breve and the like; (3) streptococcus genus: streptococcus thermophilus, streptococcus faecalis, lactococcus, streptococcus intermedius, etc.; (4) saccharomyces, and the like. The action mechanism of probiotics on Hp may comprise the functions of regulating intestinal flora, forming mechanical and chemical biological barriers, forming anti-inflammatory factors and regulating immune mechanisms, so that the growth and colonization of Hp on gastric mucosal epithelium are effectively inhibited, the immune response and inflammation after Hp infection are inhibited, and the gastric mucosal barrier is stabilized, so that gastric mucosa of a patient is protected. There are many studies and documents on the application of probiotics in Hp treatment at present, and the studies prove that the probiotic preparation combined with the antibacterial drug can effectively relieve clinical symptoms of patients suffering from Hp infection, improve the radical treatment effect of Hp and reduce the incidence rate of adverse reactions of the drug. It is considered that fermentation such as probiotic preparation can raise the Hp eradication rate by 5% -15%. The probiotic preparation commonly used in clinic comprises lactobacillus acidophilus, bifidobacterium triple viable bacteria, bacillus subtilis double viable bacteria, bacillus licheniformis and the like.
Various probiotic preparations such as Lizhuchang le, pefeikang, mei Chang An, lefukang and the like in the current market are widely applied to clinic, and the clinic effect has a great relationship with the used strain. The existing products have the following defects: the survival rate of probiotics in gastrointestinal fluid is low, only a small part of probiotics can live to reach focus, and only weak effect can be exerted; secondly, the problem of poor helicobacter pylori inhibition by probiotics is also in need of solving. Therefore, it is the direction of this patent whether new probiotic candidates can be provided, and their ability to combat gastric pathogens such as Hp is enhanced. The lactobacillus rhamnosus strain with remarkable inhibiting effect on helicobacter pylori is screened in the research, has strong survival ability in gastric acid and intestinal juice, has good adhesion ability on gastric epithelial cells and intestinal cells, provides a solid foundation for exerting the probiotics effect, and has very important application value on gastrointestinal diseases caused by Hp.
Disclosure of Invention
The invention provides a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) with antagonism to helicobacter pylori for solving the problems in the prior art. The strain has strong tolerance to gastrointestinal tract environment, can effectively degrade cholesterol, has remarkable antioxidation effect, can obviously inhibit the growth and adhesion of helicobacter pylori, and can be widely applied to functional foods, health-care products or medicines.
One aspect of the invention relates to a lactobacillus rhamnosus, which is a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) VHProbi V77 strain, which has been preserved in the China center for type culture collection of university of martial arts in China, with a preservation number of CCTCC NO: m2023500.
The 16s rDNA sequence of the lactobacillus rhamnosus is SEQ ID NO. 1.
The RAPD fingerprint of the lactobacillus rhamnosus is shown in figure 3; the rep-PCR fingerprint is shown in FIG. 4; the MALDI-TOF-MS protein fingerprint is shown in FIG. 5.
One aspect of the invention relates to the use of said Lactobacillus rhamnosus for the preparation of a helicobacter pylori inhibitor.
One aspect of the invention relates to the application of the lactobacillus rhamnosus in preparing food or health care products.
One aspect of the invention relates to the use of said lactobacillus rhamnosus for the preparation of a medicament.
The medicine has the function of preventing or treating digestive tract diseases caused by helicobacter pylori.
The invention also relates to a probiotic preparation comprising at least one of a live thallus, an inactivated thallus, a fermentation metabolite or an intracellular extract of said lactobacillus rhamnosus.
The invention also relates to application of the probiotic preparation in preparing foods or health care products.
The invention also relates to application of the probiotic preparation in preparing medicines with the function of preventing or treating gastritis.
The lactobacillus rhamnosus VHProbi V77 strain provided by the invention has stronger anti-gastrointestinal fluid digestion capability, and the bacterial load is less reduced after gastric fluid is digested for 2 hours and intestinal fluid is digested for 3 hours; can be propagated in large quantities at 15 ℃ and 45 ℃; can grow under the salt concentration of 1% -8%, and the maximum tolerant salt concentration is 6%; does not produce hemolysin, can not dissolve blood cells, has no toxic or harmful effect on organisms, is sensitive to common antibiotics such as erythromycin, tetracycline, ampicillin and the like, and has good safety.
The rhamnose cheese bacillus VHProbi V77 has remarkable inhibition effect on two different helicobacter pylori strains, and the diameter of a bacteriostasis circle of a fermentation liquor containing living bacteria on the mixed helicobacter pylori reaches 16.50+/-0.35 mm, so that the effect is remarkable; the inhibiting effect of the fermentation supernatant on helicobacter pylori is positively correlated with the addition amount, and when the addition amount is 15%, the inhibiting rate on helicobacter pylori is as high as 33.39%. The strain has strong coagglutination ability with helicobacter pylori, and provides necessary conditions for coagglutination with helicobacter pylori and discharge of the helicobacter pylori in gastrointestinal environment. The strain can also obviously inhibit the adhesion of helicobacter pylori to human gastric adenocarcinoma cells, the adhesion inhibition rate is up to 49.30 percent, and unexpected technical effects are achieved.
The lactobacillus rhamnosus VHProbi V77 also has strong adhesion effect on human gastric adenocarcinoma cells (BGC-823) and intestinal epithelial cells (Caco-2), and provides favorable conditions for adhesion and colonization in gastric and intestinal mucosa.
The degradation rate of the rhamnose cheese bacillus VHProbi V77 strain on cholesterol reaches 21.7%, and the rhamnose cheese bacillus VHProbi V77 strain has the probiotic property of reducing serum cholesterol. The strain also has strong antioxidant capacity, the clearance rate of the strain on DPPH free radicals and HRS free radicals reaches 18.66% and 52.36%, and the clearance rate of the supernatant fluid on the HRS free radicals reaches 92.31%; the inhibition rate of lipid peroxidation of fermentation supernatant, thallus and intracellular extract is 43.41+ -0.09%, 40.01+ -0.21% and 19.06+ -0.41%.
The lactobacillus rhamnosus VHProbi V77 strain provided by the invention can be widely used for producing functional foods or health products, and can also be used for preparing medicines for preventing or treating diseases such as gastritis, gastric ulcer and the like caused by helicobacter pylori; the preparation can also be used for preparing a probiotic preparation, and the preparation comprises at least one of live bacteria, dead bacteria, metabolites or intracellular extracts of the Lactobacillus rhamnosus VHProbi V77, and can be used for preparing functional foods, health-care products or medicines, and has wide prospects.
Drawings
FIG. 1 is a colony map and gram stain map of strain V77; wherein A is a colony chart and B is a gram staining chart;
FIG. 2 shows the API test results of strain V77;
FIG. 3 is a RAPD fingerprint of strain V77;
FIG. 4 is a rep-PCR fingerprint of strain V77;
FIG. 5 shows MALDI-TOF-MS fingerprint of strain V77;
FIG. 6 is a diagram showing the zone of inhibition of helicobacter pylori by strain V77;
FIG. 7 is a graph showing the effect of strain V77 on adhesion to human gastric adenocarcinoma cells;
FIG. 8 is a coagglutination diagram of strain V77 against H.pylori.
Detailed Description
The lactobacillus rhamnosus VHProbi V77 provided by the invention meets the requirement of regulation, and is a newly discovered strain through multiphase taxonomy identification. The rhamnose cheese bacillus VHProbi V77 provided by the invention has strong acid resistance, can obviously inhibit the growth of helicobacter pylori, and has important application value for preventing and treating digestive tract diseases caused by helicobacter pylori.
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The invention will be further illustrated with reference to specific examples.
Example 1 isolation screening of Lactobacillus rhamnosus VHProbi V77
1.1 Lactobacillus Primary screening
Taking 1g of dried persimmon, flushing the dried persimmon by using sterile normal saline, putting the dried persimmon into a sterile sample bag, and beating and uniformly mixing the dried persimmon with a homogenizer; and (3) taking 100 mu L of the mixed solution, carrying out gradient dilution, coating the mixed solution on an MRS agar medium, and then culturing at 37 ℃ for 48 hours, and carrying out microscopic examination on a single colony after the plate grows. According to the microscopic examination result, the applicant screens 78 potential lactobacillus strains, which are respectively named as V1, V2, … … and V78.
1.2 Lactobacillus re-screening
Preparing 1L of MRS liquid culture medium (1000 mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, adjusting pH to 6.2-6.5), sterilizing at 115 ℃ for 30min, adding 3.2g of porcine mucosa pepsin after the culture medium is cooled, shaking uniformly for dissolving, and placing in a water bath shaker at 37 ℃ for 1h to prepare the acid-resistant culture medium. Inoculating 78 strains of lactobacillus obtained by primary screening into the acid-resistant culture medium according to the inoculation amount of 6%, standing and culturing for 2 hours at 37 ℃, and counting the bacterial amount of the fermentation liquor.
The results show that the lactobacillus obtained by the primary screening of the invention has the strongest acid resistance, the biggest viable bacteria amount after being re-screened by the acid-resistant culture medium, and the logarithmic value reaches 7.78Log CFU/mL, thus obtaining unexpected technical effects.
EXAMPLE 2 identification of the V77 Strain
The inoculum preparation in this example was as follows: taking a proper amount of fresh V77 bacterial liquid, centrifuging at 5000rpm/min for 5min, washing with PBS buffer for 2 times, and then diluting the bacterial cells with the same volume of PBS buffer for 50 times later as an inoculation liquid.
2.1 identification of colony and fungus shape
The V77 strain is inoculated on MRS agar culture medium, and after culturing for 48h at 37 ℃, the single colony of the V77 strain is milky white, the diameter of the colony is about 1.5-2mm, and the surface is moist. The V77 colony image is shown in FIG. 1 (A).
Colony smears of the V77 strain are shown in fig. 1 (B): gram staining is positive, short bar-shaped under a microscope, and round at two ends.
2.2 physiological Biochemical identification
2.2.1 carbon Source metabolism test
The carbon source metabolism experiment of strain V77 was validated using API 50CHL reagent. The experimental method and the result analysis are specifically described in the API 50CHL kit instruction. The results showed that strain V77 had an ID value of 99.9% with Lactobacillus rhamnosus and the API test results are shown in FIG. 2. The results can be used to preliminarily identify the bacterium as Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus).
2.2.2 glucose acid and gas production test
The formula of the culture medium is as follows: peptone 0.5g; 0.3g of yeast extract; tween 80.1 ml; 80.5mL of saline solution A; salt solution B0.5 mL; 0.5g of sodium acetate; glucose 2.5g; 0.05mL of 2% bromocresol green (w/v); distilled water 100mL;
the pH is 6.8-7.0. The prepared culture medium was dispensed into large tubes containing inverted small tubes, 3 mL/tube, and autoclaved at 121℃for 15min.
Salt solution A9 composition: KH (KH) 2 PO 4 10g、K 2 HPO 4 1.0g, dissolved in distilled water, was fixed to a volume of 100mL.
Salt solution B: mgSO (MgSO) 4 ·7H 2 O 11.5g、MnSO 4 ·2H 2 O 2.4g、FeSO 4 ·7H 2 O0.68 g, dissolved in distilled water, was fixed to a volume of 100mL.
Under aseptic condition, inoculating the inoculating solution with 10% inoculating amount, inoculating the culture medium without inoculating bacteria as control, sealing the top with 2mL sterile liquid paraffin, culturing at 37deg.C for 24 hr, and observing whether the color of the culture medium changes.
The results show that: after 24h of culture at 37 ℃, the culture medium turns from green to yellow, and no gas exists in the small inverted tube, which indicates that the V77 strain ferments glucose to produce acid and does not produce gas.
2.2.3 salinity tolerance test
Under aseptic conditions, 190. Mu.L of MRS liquid culture medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, 3 replicates of each salt concentration, and then 10. Mu.L of inoculation liquid was added, and wells without bacteria were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C for 36h, and observing whether the culture medium becomes turbid. The results show that the V77 strain can grow at a salt concentration of 1% -8%, and the maximum tolerant salt concentration is 6%.
2.2.4 catalase experiments
The fresh bacterial liquid was taken and dropped onto a clean glass slide, and then a drop of 3% hydrogen peroxide solution was dropped thereon, and no bubbles were observed to be generated by the V77 strain, which was a negative reaction.
2.2.5 temperature tolerance test
The inoculation liquid is inoculated into 10mL MRS liquid culture medium according to 10% inoculation amount, 5mL MRS liquid culture medium without bacteria is used as a control, and is respectively placed in a constant temperature incubator at 15 ℃ for 7 days, a constant temperature incubator at 45 ℃ for 2 days, and whether bacteria liquid becomes turbid is observed. The results showed that the V77 strain can be propagated in large amounts at 15℃and 45 ℃.
2.3 molecular biological identification
2.3.1 16s rDNA Gene sequence analysis
1. Genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
2. 16s rDNA Gene amplification
(1) Primer sequences
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
(2) Reaction system (50. Mu.L)
TABLE 1.16s rDNA PCR amplification System
(3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
(4) Sequencing of PCR products
Sequencing results show that the 16s rDNA sequence of the V77 strain is SEQ ID NO. 1. The sequences were aligned in the NCBI database and the V77 strain was initially determined to be Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus). SEQ ID NO. 1 sequence is as follows:
CGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGG
GAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGC
GAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGT
CTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA
TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCA
ACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCT
CACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAAC
CTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGA
ATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTT
GCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAA
ACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTT
CGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACT
GGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTG
CACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAG
ACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCT
ACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCAC
GCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAA
GCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTA
CTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCT
CAGTTCGGCTACGTATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCG
CGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTG
GATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCA
CGTGTTACTCACCCGTCCGCCACTCGTTCAAAATTAAATCAAGATGCAAGCACCTTTC。
3.2RAPD and rep-PCR fingerprint identification
1. RAPD fingerprint identification
(1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
(2) RAPD reaction system
TABLE 2RAPD reaction System
(3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger-prints of the V77 strain are shown in FIG. 3.
2. rep-PCR fingerprint
(1) rep-PCR primer
CTACGGCAAGGCGACGCTGACG。
(2) reaction system of rep-PCR
TABLE 3 reaction System for rep-PCR
(3) Electrophoresis
DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of the V77 strain is shown in FIG. 4.
Detection of ribosomal protein expression in strains by 2.3.3MALDI-TOF-MS
Inoculating the V77 strain into a fresh MRS liquid culture medium, and culturing at 37 ℃ for 24 hours to obtain a V77 fresh bacterial liquid; the fresh bacterial liquid is washed 3 times by sterile normal saline, and the surface moisture is dried. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. The main ion peaks of strain V77 are: m/z 4688.509, 5349.062, 5887.118, 7573.921, 9374.886, etc., and the results of the identification are shown in FIG. 5.
In summary, by combining the colony morphology, the physiological and biochemical characteristic results and the molecular biological identification results of the V77 strain, it can be concluded that the V77 strain is a novel strain of Lactobacillus rhamnosus, which is named as Lactobacillus rhamnosus VHProbi V77 (Lacticaseibacillus rhamnosus VHProbi V).
Example 3 analysis of resistance of Lactobacillus rhamnosus VHProbi V77 to gastrointestinal fluids
3.1 Artificial gastric juice digestion
9ml of artificial gastric juice was placed on a 37℃water bath shaker for 1h to simulate the human body temperature. 1mL of fresh bacterial liquid of Lactobacillus rhamnosus VHProbi V77 (10) 8 cfu/mL or so), added to 9mL of artificial gastric juice, and placed on a 37℃water bath shaker (200 rpm) for 2h. 1ml of the sample was sampled at 0h and 2h after inoculation to examine the viable bacteria amount.
3.2 Artificial intestinal juice digestion
24ml of the artificial intestinal juice was placed on a 37℃water bath shaker for 1h to simulate the temperature of the human body. 1mL of the above-mentioned artificial gastric juice after 2 hours of digestion was taken and added to 24mL of artificial intestinal juice, and the mixture was placed on a 37℃water bath shaker (200 rpm) for 3 hours. 1ml of the sample was sampled at 3 hours to measure the amount of viable bacteria. The gastrointestinal fluid digestion resistance of the Lactobacillus rhamnosus VHProbi V77 was estimated from the viable counts of the viable bacteria of the gastrointestinal fluid before and after the measurement.
The results show that the bacterial load of the Lactobacillus rhamnosus VHProbi V77 is reduced by 0.07Log CFU/mL after 2 hours of gastric juice digestion and reduced by 2.12Log CFU/mL after 3 hours of intestinal juice digestion. Therefore, the lactobacillus rhamnosus VHProbi V77 provided by the invention has strong tolerance to gastrointestinal fluids.
EXAMPLE 4 Lactobacillus rhamnosus VHProbi V77 hemolysis and antibiotic resistance experiments
Preparation of a lactobacillus rhamnosus VHProbi V77 bacterial suspension:
picking up purified rhamnose cheeseBacterial VHProbi V77 colony is inoculated in fresh MRS liquid culture medium and cultured for 24 hours at 37 ℃; inoculating the strain into MRS liquid culture medium according to the inoculum size of 1% (V/V), and continuously culturing at 37 ℃ for 24-48 h; taking 1ml of fresh bacterial liquid of the Lactobacillus rhamnosus VHProbi V77, centrifuging at 5000rpm for 5min, and respectively collecting fermentation supernatant and thalli; washing thallus with PBS buffer solution of pH7.4 for 2 times, and regulating bacterial concentration to 5×10 with PBS buffer solution 7 CFU/mL (OD 600 absorbance value about 0.4) to give a bacterial suspension.
TBS basal medium: tryptone 17.0g, soyase 3.0g, sodium chloride 5.0g, potassium dihydrogen phosphate (anhydrous) 2.5g, glucose 2.5g, and distilled water 1000.0ml.
4.1 hemolysis experiments
Weighing the various components of TBS basic culture medium, dissolving, autoclaving at 121deg.C for 15min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, and plating. And streaking the test strain, inoculating the streaked strain to a prepared blood cell plate, culturing the strain in a 37 ℃ incubator, and observing whether the test strain has a hemolysis phenomenon or not in 24-48 hours.
The results show that: the lactobacillus rhamnosus VHProbi V77 was able to grow and the blood cell plates were unchanged, indicating that the lactobacillus rhamnosus VHProbi V77 did not produce hemolysin and was unable to lyse blood cells.
4.2 antibiotic resistance experiments
Preparing antibiotics: ampicillin, erythromycin, gentamicin, streptomycin and tetracycline are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using MRS liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
Minimal inhibitory concentration MIC values of antibiotics against lactobacillus rhamnosus VHProbi V77 were determined by a mini-broth dilution method.
(1) The MRS liquid culture medium without antibiotics is added to the 96-well plate in column 1 for the time, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the columns 2 to 12 as a negative control, 10 mu L of the bacterial suspension is inoculated respectively, 3 parallel wells are made, and 1 well without bacterial solution is used as a blank.
(2) 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
(3) The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 4.
Table 4 antibiotic MIC values of Lactobacillus rhamnosus VHProbi V77
MIC units μg/mL.
From the results shown in Table 5, the Lactobacillus rhamnosus VHProbi V77 provided by the invention is sensitive to common antibiotics such as tetracycline and ampicillin, and has good biological safety.
EXAMPLE 5 antibacterial Effect of Lactobacillus rhamnosus VHProbi V77 on helicobacter pylori
5.1 cultivation of pathogenic bacteria:
collecting frozen glycerol tube (helicobacter pylori ATCC353909/ATCC 354364), spreading on Columba blood agar medium (Columba blood agar is autoclaved, cooled to about 50deg.C, 10% sterilized defibrinated sheep blood is added, cooled to 45-50deg.C, poured into a sterile plate, sealed at 4deg.C), and placed at 37deg.C, 10% CO 2 Culturing for 72h under the condition; scraping bacterial mud into brain heart infusion liquid culture medium (brain heart infusion broth, sterilizing at 121deg.C for 15min, cooling to about 50deg.C, adding 10% fetal bovine serum, mixing, cooling to room temperature), culturing under micro-oxygen condition (oxygen concentration 5%, carbon dioxide concentration 10%, nitrogen concentration 85% environment, and the% is volume%) at 37deg.C for 24 hr, collecting 5mL bacterial solution, centrifuging at 5000r/min for 5min, re-suspending bacterial cells with physiological saline (0.8%, w/v), and adjusting bacterial solution concentration to about 10 6 cfu/mL。
5.2 inhibition zone test:
the method is carried out by adopting an oxford cup double-layer flat plate method.
In a clean bench, sterile plates were taken, sterilized with nutrient agar, poured into the plates, and the plates were confluent as the lower medium. After the agar solidified, 10ml of Columbia blood agar medium was spread on the upper layer uniformly as an upper layer medium. After the upper layer culture medium is solidified, 0.2mL of helicobacter pylori bacteria liquid is absorbed on a solid culture medium plate, and the coating is uniform. Standing for 1 hr, fixing the bacterial liquid on the surface of the plate, perforating to obtain a hole with diameter of 8mm and depth of 3mm, and collecting V77 (10 8 cfu/mL or so) was added to 0.1mL of sterile water, and then the mixture was poured into a hole, and incubated at 37℃for 8 hours for observation, and the diameter of the zone of inhibition was measured.
The results are shown in FIG. 6, and the measurement shows that the diameter of the inhibition zone of the Lactobacillus rhamnosus VHProbi V77 on helicobacter pylori reaches 16.50+/-0.30 mm, so that the strain has remarkable inhibition effect on helicobacter pylori and unexpected technical effect is achieved.
5.3 growth inhibition experiments:
(1) Experimental group: inoculating helicobacter pylori fresh bacterial liquid into brain heart infusion liquid culture medium added with 2.5% (V/V), 5% (V/V) and 10% (V/V) of lactobacillus rhamnosus VHProbi V77 fermentation supernatant described in example 4 according to a volume ratio of 1%;
(2) Control group: the fresh bacterial liquid of helicobacter pylori is inoculated into brain heart infusion liquid culture medium according to the volume ratio of 1 percent.
37℃、10％CO 2 Culturing for 24h under the condition; OD values of the culture solutions were measured at 600nm, respectively. OD of culture solution of control group 600 The values were calculated to be 100%, and the growth inhibition ratio of Lactobacillus rhamnosus VHProbi V77 cell-free fermentation supernatant to helicobacter pylori was calculated. The specific results are shown in Table 5.
Growth inhibition (%) = (control OD) 600 Experimental group OD 600 ) Control group OD 600 ×100％。
TABLE 5 inhibition of helicobacter pylori by Lactobacillus rhamnosus VHProbi V77 fermentation supernatant
From the data in Table 5, it is clear that the cell-free fermentation supernatant of Lactobacillus rhamnosus VHProbi V77 can significantly inhibit the growth of helicobacter pylori, and the effect of inhibiting helicobacter pylori is continuously enhanced with the increase of the addition amount of the fermentation supernatant, and when the addition amount is 15%, the inhibition rate of helicobacter pylori is as high as 33.39%, and unexpected technical effects are obtained.
Example 6 Lactobacillus rhamnosus VHProbi V77 cytotoxicity test
The suspension of the Lactobacillus rhamnosus VHProbi V77 strain described in example 4 was inactivated in a water bath at 70℃for 20 minutes for use.
Culture of human gastric adenocarcinoma cells (BGC-823) cells: human gastric adenocarcinoma cells (BGC-823) are taken out from the liquid nitrogen tank, resuscitated and subcultured, and the cells are diluted. Human gastric adenocarcinoma cells (BGC-823) are inoculated into a six-hole culture plate with built-in cell climbing plates and 10% fetal bovine serum (DMEM) culture solution, and the number of cell plates of each hole is about 2 multiplied by 10 6 cells, six well plates were placed in a carbon dioxide incubator for 24 hours.
Cytotoxicity test: resuscitates human gastric adenocarcinoma cells BGC-823, inoculates the cells into a 24-hole culture plate containing 10% calf serum cell culture solution, and cultures the cells for 24 hours, wherein the inoculation density is 2×105 cells/hole. The inactivated Lactobacillus rhamnosus VHProbi V77 was added to the cells at a ratio of MOI (Multiplicity of Infection ) of 10, and a blank control group without bacteria was set up for continuous culture in an incubator for 24 hours. MTT solution was added to each cell culture well to be tested at a final concentration of 0.3mg/ml and incubated for 3h in a carbon dioxide incubator. The supernatant was carefully discarded, and 500ul of DMSO was added to each 24-well plate cell culture well and incubated at 37℃for 30min to allow the purple crystals to dissolve well. Absorbance at 490nm was measured.
The results show that the lactobacillus rhamnosus VHProbi V77 has no obvious effect on the proliferation activity of human gastric adenocarcinoma cells, no cytotoxicity and good safety.
Example 7 adhesion experiments of Lactobacillus rhamnosus VHProbi V77 on human gastric adenocarcinoma cells
The adherent human gastric adenocarcinoma cell (BGC-823) single cell layer in the six-hole plate is washed 3 times by using PBS buffer solution, added with the suspension of the Lactobacillus rhamnosus VHProbi V77 strain described in the example 4, and placed in a carbon dioxide incubator for culturing for 2 hours. The cell slide was repeatedly washed 3 times with PBS buffer to remove non-adherent bacteria. The PBS was repeatedly washed 5 times to remove non-adhering bacteria, and the PBS was slowly added along the walls to avoid the cell layers from being washed up. Digestion was stopped by adding 500ul of pancreatin for 3 minutes, followed by 1.5ml of cell culture broth, repeated pipetting, and the resulting solution was collected in sterile EP tubes and the collected solution was subjected to gradient dilution and plating counting. Cells from the blank group were counted simultaneously. The adhesion ability of the test strain was calculated according to the following formula:
adhesion capacity (CFU/cells) =total number of bacteria adhered per culture well/total number of cells per culture well.
The statistical analysis result shows that the adhesion amount of the lactobacillus rhamnosus VHProbi V77 to human gastric adenocarcinoma cells is 1.28+/-0.55 CFU/cell, which indicates that the strain can be effectively adhered to the surface of human gastric adenocarcinoma cells (BGC-823).
Example 8 adhesion inhibition experiment of Lactobacillus rhamnosus VHProbi V77 against helicobacter pylori
Sterile cell climbing sheets were placed in six well plates, washed 3 times with PBS buffer, the adherent human gastric adenocarcinoma cell BGC-823 single cell layer was retained, washed 3 times with PBS buffer, 1mL of the Lactobacillus rhamnosus VHProbi V77 bacterial suspension and helicobacter pylori bacterial suspension described in example 4 were added respectively (10 6 cfu/mL), cells without strain were used as a blank, and cultured in a carbon dioxide incubator for 2 hours. The cell slide was repeatedly washed 3 times with PBS buffer to remove non-adherent bacteria. Fixing with anhydrous methanol for 20 min, taking out the cell climbing sheet, air drying, gram staining, observing 20 random fields under a 100 times oil microscope, and calculating the number of helicobacter pylori adhered on each cell. Comparison of the Presence of Lactobacillus rhamnosus VHProbi V77 andin the absence, the adhesion inhibition rate of helicobacter pylori VHProbi V77 was calculated on the basis of the adhesion rate of no lactobacillus added as 100% by changing the adhesion amount of helicobacter pylori on human gastric adenocarcinoma cells.
Adhesion inhibition (%) = (number of adhesion of control group helicobacter pylori-number of adhesion of experimental group helicobacter pylori)/number of adhesion of control group helicobacter pylori x 100%.
The results show that the Lactobacillus rhamnosus VHProbi V77 can remarkably inhibit the adhesion of helicobacter pylori to human gastric adenocarcinoma cells, and the adhesion inhibition rate is as high as 49.30%. It is also apparent from FIG. 7 that the experimental group significantly reduced the number of H.pylori adhering to human gastric adenocarcinoma cells in the presence of Lactobacillus rhamnosus VHProbi V77 and the effect was very pronounced compared to the control group.
Example 9 Lactobacillus rhamnosus VHProbi V77 agglutination adsorption assay
300. Mu.L of the Lactobacillus rhamnosus VHProbi V77 strain suspension described in example 4 was added to a 24-well plate, followed by 300. Mu.L of the helicobacter pylori strain suspension (10 6 cfu/mL) as experimental group; equal amounts of the suspension of the Lactobacillus rhamnosus VHProbi V77 strain and the buffer were mixed as control groups, and 2 groups of the control groups and the experimental groups were arranged in parallel. The 24-well plate was placed in a microplate thermostatted shaker at 400rpm, room temperature and incubated with shaking. The initial well plate state and the well plate state at different times are observed microscopically and photographed to observe whether agglutination phenomenon occurs.
As a result, as shown in FIG. 8, the binding of Lactobacillus rhamnosus VHProbi V77 to helicobacter pylori showed a distinct agglutinate, whereas there was no agglutination in the control group.
EXAMPLE 10 adhesion experiment of Lactobacillus rhamnosus VHProbi V77 to human intestinal epithelial cells (CaCO-2)
1. Culture of human intestinal epithelial cells (CaCO-2) cells:
and taking out Caco-2 cells from the liquid nitrogen tank, resuscitating, subculturing, and amplifying the number of the cultured cells to the required dosage. Subsequent experiments can be performed when the cell growth confluence is observed to be close to 80% under an inverted microscope. Discarding the original mediumThe PBS is rinsed twice, and a proper amount of pancreatin is added. After pancreatin is added, the cells are put back into an incubator, after complete shedding of the cells is observed by naked eyes, 2-3 times of pancreatin volume of culture solution is added to stop digestion, the cells are repeatedly blown for about ten times, and the cells are observed under a mirror to be in a single cell state as much as possible. The single cell suspension is sucked into a 15ml or 50ml centrifuge tube, centrifuged for 5 minutes at 1000 revolutions, the supernatant is discarded, the cell sediment is scattered slightly, and a proper amount of new culture medium is added for blowing and resuspension. The cell counting plate is used for cell counting, and a proper amount of PBS is used for diluting the cell suspension, and the cell dilution is recommended to be 20-50 cells per big cell. The number of plates per well cell in the six well plate was 1.5 x 10 6 The amount of the culture medium added per well was 2ml. After placing the six-well plate in a carbon dioxide incubator for 24 hours, a subsequent cell adhesion experiment can be performed.
2. Adhesion test:
washing the adhered Caco-2 single cell layer in the six-hole plate with PBS for 2 times; respectively adding 1mL of an antibiotic-free cell culture solution into each of the experimental groups, and placing 1mL of the lactobacillus rhamnosus VHProbi V77 bacterial suspension described in the example 4 into a carbon dioxide incubator for culturing for 2 hours; repeatedly washing with PBS for 5 times to remove non-adhering bacteria; digestion was stopped by adding 500ul pancreatin for 3 min, then 1.5ml cell culture medium was added, repeated pipetting was performed, and the resulting solution was collected into sterile EP tubes and the collected solution was subjected to 10-fold, 100-fold, 1000-fold, 10000-fold gradient dilution, plating to count the bacterial load.
Meanwhile, lactobacillus rhamnosus LGG strain having strong cell adhesion was used as a control group, and the procedure was performed with reference to the above steps.
The adhesion ability of lactobacillus rhamnosus VHProbi V77 was calculated according to the following formula:
adhesion capacity (CFU/cells) =total number of bacteria adhered per culture well/total number of cells per culture well.
The results show that: the adhesion capability of the rhamnose lactobacillus casei VHProbi V77 provided by the invention to human intestinal epithelial cells is 34.6 times that of the control lactobacillus rhamnosus LGG, so that the rhamnose lactobacillus casei VHProbi V77 can be more effectively adhered in human intestinal tracts, and a foundation is provided for the fixation and the probiotic effect in the intestinal tracts.
Example 11 Lactobacillus rhamnosus VHProbi V77 in vitro cholesterol degradation experiments
Preparing a cholesterol micelle solution: 1g of cholesterol was accurately weighed, dissolved in absolute ethanol, and fixed to 100mL, and sterilized by filtration through a 0.22 μm microporous filter under aseptic conditions.
10.0g of peptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of diammonium hydrogen citrate, 20.0g of glucose, 1.0mL of tween 80, 5.0g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate and 1g of bile salt are weighed, 1000mL of distilled water is used for adjusting the pH value to 7.3, sterilization is carried out for 30min at 115 ℃, and then a cholesterol solution is added to ensure that the final concentration of cholesterol is 0.1 percent.
Inoculating fresh strain of Lactobacillus rhamnosus VHProbi V77 according to 0.1% inoculum size, standing at 37deg.C for 48 hr, collecting 0.2mL strain, adding 1.8mL absolute ethanol, mixing, standing for 10min, centrifuging at 3000 rpm for 5min, and collecting supernatant for measuring cholesterol content. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >. The specific results are shown in Table 7.
TABLE 7 degradation effect of Lactobacillus rhamnosus VHProbi V77 on cholesterol
From the results in Table 7, the degradation rate of the lactobacillus rhamnosus VHProbi V77 provided by the invention on cholesterol reaches 21.7%, and the degradation rate on salt-containing cholesterol also reaches 8.4%, so that the effect is remarkable.
Example 12 determination of antioxidant Capacity of Lactobacillus rhamnosus VHProbi V77
12.1 determination of the ability of Strain to scavenge Hydroxyl Radicals (HRS)
200ul of the Lactobacillus rhamnosus VHProbi V77 strain suspension described in example 4, 100ul of 5mM sodium salicylate-ethanol solution, 100ul of 5mM ferrous sulfate, 500ul of deionized water were mixed uniformly, 100ul of hydrogen peroxide solution (3 mM) was added, absorbance of the strain suspension was measured at a wavelength of 510nm after 15min in a 37℃water bath, and the rate of elimination of HRS by the strain suspension was calculated.
In addition, experiments were performed with equal doses of the lactobacillus rhamnosus VHProbi V77 fermentation supernatant described in example 4 instead of the bacterial suspension to determine the clearance of HRS by the supernatant. The specific results are shown in Table 8.
The hydroxyl radical scavenging rate was calculated according to the following formula.
Clearance (%) = (a) Sample of -A Control of )/(A Blank space -A Control of )×100％。
Wherein: a is that Control of Is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate, A Blank space Is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate.
TABLE 8 scavenging effect of Lactobacillus rhamnosus VHProbi V77 on HRS free radicals
12.2 identification of Strain anti-lipid peroxidation experiments
The anti-lipid peroxidation inhibition rates were measured using the Lactobacillus rhamnosus VHProbi V77 bacterial suspension, fermentation supernatant and intracellular extract described in example 4 as samples, respectively. Wherein the preparation method of the intracellular extract comprises the following steps:
taking the suspension of the Lactobacillus rhamnosus VHProbi V77 strain in the embodiment 4, carrying out ice bath ultrasonic disruption on cells (with the output power of 300W for every 2s of intermittent 1 s) for 10min, centrifuging at 4 ℃ and 10000r/min for 30min, and collecting supernatant, namely the intracellular extract of the Lactobacillus rhamnosus VHProbi V77 strain.
The anti-lipid peroxidation experiment comprises the following specific steps:
preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
Adding 1mL of linoleic acid emulsion, 1mL of LFASO 4 (1%) into 0.5mL of PBS buffer solution (pH 7.4), adding 0.5mL of sample, carrying out water bath at 37 ℃ for 1.5h, adding 0.2mL of TCA (4%), 2mL of TBA (0.8%), carrying out water bath at 100 ℃ for 30min, rapidly cooling, centrifuging at 4000rpm/min for 15min, and collecting supernatant, wherein absorbance is measured at 532nm to obtain A; the control group replaced the sample, A0, with 0.5mL distilled water.
The anti-lipid peroxidation inhibition was calculated according to the following formula.
Inhibition (%) = (A0-a)/a0×100%.
Wherein: a is absorbance of a sample group; a0 is absorbance of the control group.
The specific results are shown in Table 9.
TABLE 9 inhibitory effect of Lactobacillus rhamnosus VHProbi V77 on lipid peroxidation
12.3 determination of the ability of the Strain to clear DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine)
1mL of the Lactobacillus rhamnosus VHProbi V77 strain suspension described in example 4 was taken, 1mL of 0.4mM DPPH free radical solution was added, the mixture was uniformly mixed, then the mixture was subjected to shading reaction at room temperature for 30min, then the absorbance A sample of the sample at 517nm was measured, and the sample was measured in 3 times of parallelism. Control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS buffer and ethanol mixed solution.
The clearance is calculated according to the following formula:
clearance% = [1- (a sample-a blank)/a control ] ×100%.
The specific results are shown in Table 10.
TABLE 10 scavenging effect of Lactobacillus rhamnosus VHProbi V77 on DPPH free radicals
In conclusion, the lactobacillus rhamnosus (Lacticaseibacillus paracasei) VHProbi V77 obtained by separation has strong tolerance to gastrointestinal fluid, does not generate hemolysin, does not dissolve blood cells, and has good biological safety; can effectively degrade cholesterol in serum, remove DPPH free radical and HRS free radical, inhibit lipid peroxidation, and have strong antioxidant activity. In addition, both thalli and supernatant of the strain can effectively inhibit the growth of helicobacter pylori, realize co-aggregation with the helicobacter pylori, can obviously inhibit the adhesion of the helicobacter pylori on the surface of gastrointestinal cells, can be widely used for preparing foods, health care products and medicines for preventing or treating diseases such as gastritis, gastric ulcer and the like caused by the helicobacter pylori, and has wide prospect.
Claims (8)
1. The lactobacillus rhamnosus is characterized in that the preservation number of the lactobacillus rhamnosus is CCTCC NO: m2023500.
2. The lactobacillus rhamnosus of claim 1 wherein the 16s rDNA sequence of lactobacillus rhamnosus is SEQ ID No. 1.
3. The lactobacillus rhamnosus of claim 1 wherein the RAPD fingerprint of the lactobacillus rhamnosus is shown in figure 3; the rep-PCR fingerprint is shown in FIG. 4; the MALDI-TOF-MS protein fingerprint is shown in FIG. 5.
4. Use of lactobacillus rhamnosus as claimed in claim 1 in the preparation of a helicobacter pylori inhibitor.
5. The use of lactobacillus rhamnosus as claimed in claim 1 in the preparation of a food or a health product.
6. The use of lactobacillus rhamnosus as claimed in claim 1 in the manufacture of a medicament.
7. The use according to claim 6, wherein the pharmaceutical product has a function of preventing or treating a digestive tract disease caused by helicobacter pylori.
8. A pharmaceutical product for preventing or treating gastritis, characterized in that the pharmaceutical product comprises at least one of living bacterial cells, inactivated bacterial cells, fermentation metabolites or intracellular extracts of lactobacillus rhamnosus according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310580586.2A CN116875480A (en) | 2023-05-23 | 2023-05-23 | Lactobacillus rhamnosus with antagonism to helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310580586.2A CN116875480A (en) | 2023-05-23 | 2023-05-23 | Lactobacillus rhamnosus with antagonism to helicobacter pylori |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116875480A true CN116875480A (en) | 2023-10-13 |
Family
ID=88263301
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310580586.2A Pending CN116875480A (en) | 2023-05-23 | 2023-05-23 | Lactobacillus rhamnosus with antagonism to helicobacter pylori |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116875480A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467586A (en) * | 2023-12-28 | 2024-01-30 | 潍坊康地恩生物科技有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
-
2023
- 2023-05-23 CN CN202310580586.2A patent/CN116875480A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467586A (en) * | 2023-12-28 | 2024-01-30 | 潍坊康地恩生物科技有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
| CN117467586B (en) * | 2023-12-28 | 2024-05-10 | 青岛蔚蓝生物集团有限公司 | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114717129B (en) | Lactobacillus rhamnosus and application thereof in preventing and relieving constipation symptoms | |
| CN114703108B (en) | Fermented lactobacillus mucilaginosus and application thereof in improving facial redness and type I rose acnes | |
| CN115044504B (en) | Enterococcus faecalis YZ-1 and probiotic application thereof | |
| CN112625979A (en) | Lactobacillus casei for resisting helicobacter pylori and application thereof | |
| CN114672443A (en) | Lactobacillus plantarum with function of preventing or improving facial redness and type I rosacea | |
| CN114717157A (en) | Lactobacillus paracasei for preventing streptococcus infection of infants and application thereof | |
| CN114774315B (en) | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product | |
| CN114752529A (en) | Lactobacillus plantarum HOM3201 strain, viable bacteria preparation thereof, preparation method and application | |
| CN116875480A (en) | Lactobacillus rhamnosus with antagonism to helicobacter pylori | |
| CN116515666A (en) | Lactobacillus helveticus with acne treatment effect and application thereof | |
| CN116445356A (en) | Bifidobacterium animalis subspecies BA67 for regulating intestinal flora and enhancing immunity and application thereof | |
| CN111718903A (en) | Method for screening medicine capable of preventing and/or treating osteoporosis | |
| CN117448243A (en) | Acremonium muciniphilum Akk007 with probiotic function and immunity enhancing function, application thereof and health care product | |
| CN116925980A (en) | Lactobacillus gasseri strain for relieving salmonella typhimurium infection and application thereof | |
| CN114657106B (en) | Lactobacillus plantarum and application thereof in preventing and treating acne | |
| CN114703107B (en) | Lactobacillus paracasei and application thereof in preventing streptococcus infection of infants | |
| CN114806953A (en) | Lactobacillus gasseri with characteristic of improving type 1 diabetes | |
| CN116640704B (en) | Pediococcus pentosaceus for inhibiting helicobacter pylori and application thereof | |
| CN117187101A (en) | Lactobacillus plantarum strain and application thereof in inhibiting helicobacter pylori | |
| CN116606781B (en) | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof | |
| CN116731911A (en) | Lactobacillus reuteri for inhibiting helicobacter pylori and application thereof | |
| CN114657107B (en) | Lactobacillus plantarum with acne treatment effect and application thereof | |
| CN117467586B (en) | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process | |
| WO2023093566A1 (en) | Lactobacillus helveticus having effect of treating acne and use thereof | |
| CN114574405B (en) | Lactobacillus plantarum WKA86, application thereof in preparation of halitosis preventing and treating product and halitosis preventing and treating product |
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 |