CN116606781B - Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof - Google Patents
Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof Download PDFInfo
- Publication number
- CN116606781B CN116606781B CN202310861908.0A CN202310861908A CN116606781B CN 116606781 B CN116606781 B CN 116606781B CN 202310861908 A CN202310861908 A CN 202310861908A CN 116606781 B CN116606781 B CN 116606781B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- vhprobi
- helicobacter pylori
- strain
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 86
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 86
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 86
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 61
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 61
- 230000008485 antagonism Effects 0.000 title abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 3
- 238000000855 fermentation Methods 0.000 claims description 16
- 230000004151 fermentation Effects 0.000 claims description 16
- 238000011282 treatment Methods 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 9
- 208000007882 Gastritis Diseases 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000002207 metabolite Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 2
- 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 24
- 230000000694 effects Effects 0.000 abstract description 15
- 235000012000 cholesterol Nutrition 0.000 abstract description 12
- 238000012216 screening Methods 0.000 abstract description 12
- 230000012010 growth Effects 0.000 abstract description 7
- 230000003064 anti-oxidating effect Effects 0.000 abstract description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 56
- 230000001580 bacterial effect Effects 0.000 description 32
- 239000001963 growth medium Substances 0.000 description 31
- 239000007788 liquid Substances 0.000 description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- 230000005764 inhibitory process Effects 0.000 description 18
- 239000006228 supernatant Substances 0.000 description 18
- 239000003242 anti bacterial agent Substances 0.000 description 17
- 238000012360 testing method Methods 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 16
- 239000000725 suspension Substances 0.000 description 16
- 229940088710 antibiotic agent Drugs 0.000 description 15
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 12
- 238000009630 liquid culture Methods 0.000 description 12
- 238000012258 culturing Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 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 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 239000002253 acid Substances 0.000 description 9
- 230000002496 gastric effect Effects 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 230000003115 biocidal effect Effects 0.000 description 7
- 239000001569 carbon dioxide Substances 0.000 description 7
- 229910002092 carbon dioxide Inorganic materials 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 241000186660 Lactobacillus Species 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 210000004051 gastric juice Anatomy 0.000 description 6
- 229940039696 lactobacillus Drugs 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 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 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000004098 Tetracycline Substances 0.000 description 5
- 239000008186 active pharmaceutical agent Substances 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 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 5
- 230000015556 catabolic process Effects 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 229960003276 erythromycin Drugs 0.000 description 5
- 239000006041 probiotic Substances 0.000 description 5
- 235000018291 probiotics Nutrition 0.000 description 5
- 229960002180 tetracycline Drugs 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- 235000019364 tetracycline Nutrition 0.000 description 5
- 150000003522 tetracyclines Chemical class 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- -1 DPPH free radical Chemical class 0.000 description 4
- 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 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 239000012266 salt solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 229930182566 Gentamicin Natural products 0.000 description 3
- 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 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010083204 Proton Pumps Proteins 0.000 description 3
- 230000002292 Radical scavenging effect Effects 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 210000000981 epithelium Anatomy 0.000 description 3
- 239000000284 extract Substances 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
- 229960002518 gentamicin Drugs 0.000 description 3
- 230000009036 growth inhibition Effects 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000003978 infusion fluid Substances 0.000 description 3
- 230000003859 lipid peroxidation Effects 0.000 description 3
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 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
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 108010006464 Hemolysin Proteins Proteins 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
- 239000005662 Paraffin oil Substances 0.000 description 2
- 208000008469 Peptic Ulcer Diseases 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 102000006270 Proton Pumps Human genes 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 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
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 150000001622 bismuth compounds Chemical class 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent 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
- 239000006781 columbia blood agar Substances 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
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 239000000839 emulsion Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000008029 eradication Effects 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
- 201000005917 gastric ulcer Diseases 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 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
- 239000011777 magnesium Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 229960004025 sodium salicylate Drugs 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000012138 yeast extract Substances 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
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000004300 Atrophic Gastritis Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186000 Bifidobacterium 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
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000036495 Gastritis atrophic Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100032709 Potassium-transporting ATPase alpha chain 2 Human genes 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000016644 chronic atrophic gastritis Diseases 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 1
- 238000011970 concomitant therapy Methods 0.000 description 1
- 238000001816 cooling Methods 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
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000002949 hemolytic 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
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006166 lysate Substances 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
- 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
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000013586 microbial product Substances 0.000 description 1
- 238000005142 microbroth dilution method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 208000011906 peptic ulcer disease Diseases 0.000 description 1
- 230000008855 peristalsis Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008092 positive effect Effects 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
- 230000000529 probiotic effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 229960002181 saccharomyces boulardii Drugs 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000011450 sequencing therapy Methods 0.000 description 1
- 210000002966 serum Anatomy 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
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000012137 tryptone Substances 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
- 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
- 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
- C12R2001/25—Lactobacillus plantarum
-
- 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)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of functional microorganism screening and application, in particular to lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof. The strain has strong tolerance to gastrointestinal tract environment, can effectively degrade cholesterol, has remarkable antioxidation effect, and can obviously inhibit the growth and adhesion of helicobacter pylori. The strain is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china on 4 months and 10 days of 2023, and the preservation number is CCTCC NO: m2023501.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening and application, in particular to lactobacillus plantarum with antagonism to helicobacter pylori and application of the lactobacillus plantarum in medicines.
Background
Helicobacter pylori (Helicobacter pylori, hp) is present in the gastric epithelium and is able to cross the superficial mucus while also residing under the mucus on the surface of the gastric epithelium. The bacteria are mainly planted on the gastric, oral or intestinal mucosa and the like by flagella, are microaerophilic gram-negative bacillus, have great radical treatment difficulty after infection, are easy to repeatedly attack by patients, continuously develop peptic ulcer or atrophic gastritis in the course of disease, even develop gastric cancer, and cause great trouble to vast patients. Helicobacter pylori damages gastric mucosal epithelial cells by secreting catalase, lipase, proteolytic enzyme, toxins, etc. From 10 to 20% of people infected with helicobacter pylori may eventually develop peptic ulcers, eradicating helicobacter pylori, especially before cancerous changes, may be effective in reducing the risk of cancer occurrence.
Current methods for eradication of helicobacter pylori are triple therapy, quadruple therapy with or without bismuth (concomitant therapy, sequential therapy and mixed therapy) and double therapy. The standard mode of treatment is still a proton pump plus two antibiotics. However, with the increase of the use of antibiotics, the problem of antibiotic resistance is attracting more and more attention, and the disorder of antibiotics is also being reduced or avoided as much as possible. In addition, the recurrence rate of antibiotic treatment in clinical treatment is high, and the disease of most patients is repeated, so that the treatment difficulty is increased due to repeated infection of helicobacter pylori. And adverse reactions such as diarrhea, abdominal distension, nausea and vomiting often occur after the administration of the medicine to patients, so that the removal of helicobacter pylori is more difficult. Although there have been studies from the development of new antibiotics to replace the conventional antibiotics or to add strong antibacterial agents (bismuth compounds) in therapy or to use new proton pumps to increase the inhibition rate of helicobacter pylori, other side effects (especially in the presence of bismuth compounds) have been added. But the introduction of new antibiotics has also created new concerns. Thus, methods of acting against helicobacter pylori are of great importance for the treatment of helicobacter pylori.
In recent years, the clinical recommendation of the auxiliary treatment of gastritis by using probiotics is that good treatment effects are achieved. Probiotics are emerging alternatives to the treatment of gastrointestinal disorders, which act by modifying the microbiota as antibacterial agents or immunomodulators. The report shows that the probiotics can effectively kill helicobacter pylori for the helicobacter pylori infected patients, improve the clinical treatment effect and strengthen the gastritis rehabilitation of the patients. Domestic researches report that certain strains of lactic acid bacteria have the effect of relieving the side effects of antibiotics, and meanwhile, the external application also reports that certain lactic acid bacteria can be used in combination with antibiotics and proton pumps (mainly lactobacillus, bifidobacterium and saccharomyces boulardii), so that the eradication rate can be improved, and the side effects caused by the antibiotics can be reduced.
At present, a plurality of microbial products capable of inhibiting helicobacter pylori exist on the market, but the disadvantage of poor gastric juice tolerance exists generally, and probiotic bacterial strains must meet certain survival and vitality requirements to successfully reach gastric epithelium in a digestive tract to exert antibacterial effect. Therefore, screening of probiotics having a strong acid resistance and a remarkable inhibitory effect on helicobacter pylori is still the current research focus.
Disclosure of Invention
The invention aims to provide a novel lactobacillus plantarum (Lactiplantibacillus plantarum) with the helicobacter pylori antagonism function. 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 applied to the field of medicines.
The invention provides a lactobacillus plantarum which is separated from acid dough and named as lactobacillus plantarum (Lactiplantibacillus plantarum) VHProbi S strain, and the lactobacillus plantarum is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China at 4 months and 10 days in 2023: m2023501.
The Lactobacillus plantarum VHProbi S strain has a 16s rDNA sequence of SEQ ID NO:1.
The RAPD fingerprint of the Lactobacillus plantarum VHProbi S strain is shown in figure 4, the rep-PCR fingerprint is shown in figure 5, and the MALDI-TOF-MS protein fingerprint is shown in figure 6.
The invention also provides application of the lactobacillus plantarum VHProbi S strain in preparation of medicines with the function of preventing or treating gastritis.
The invention also provides a helicobacter pylori inhibitor, which comprises at least one of the live bacteria, the inactivated bacteria, the fermentation metabolite or the intracellular extract of the lactobacillus plantarum.
The invention also provides application of the helicobacter pylori inhibitor in preparation of medicines.
The lactobacillus plantarum VHProbi S strain has strong acid resistance, and the logarithmic value of the viable bacteria after re-screening by an acid-resistant culture medium is as high as 8.01 Log CFU/mL; the plant can grow at 15 ℃ and 45 ℃, can grow at 1% -8% salt concentration, and has an optimal tolerance salt concentration of 5%. The degradation rate of the strain on cholesterol reaches 21.27%, and the degradation rate on salt-containing cholesterol also reaches 8.4%; the clearance rate of the bacterial suspension to DPPH free radical and HRS free radical reaches 23.73 percent and 41.41 percent respectively, and the inhibition rate of lipid peroxidation reaches 48.63 +/-0.41 percent; the clearance rate of the fermentation supernatant to the HRS free radical is more up to 90.46%, and the antioxidation effect is remarkable.
The Lactobacillus plantarum VHProbi S strain can effectively inhibit the growth of helicobacter pylori, and the diameter of a bacteriostasis circle reaches 17.0+/-0.165 mm; the addition amount of the cell-free fermentation supernatant is positively correlated with the inhibition rate of helicobacter pylori, and when the addition amount reaches 15%, the inhibition rate of helicobacter pylori is up to 33.39%, so that unexpected technical effects are achieved. In addition, the strain can obviously reduce the adhesion effect of helicobacter pylori on human gastric adenocarcinoma cells (BGC-823), the adhesion inhibition rate is up to 29.30%, and the strain can be combined with helicobacter pylori to form obvious agglutinate and has stronger coagglutination capability.
The lactobacillus plantarum VHProbi S does not produce hemolysin, can not dissolve blood cells, and has good safety. Is sensitive to common antibiotics such as erythromycin, ampicillin, tetracycline, clindamycin and the like, has good biological safety, can be used for preparing medicines for preventing or treating diseases such as gastritis, gastric ulcer and the like caused by helicobacter pylori, and has broad prospect.
Drawings
FIG. 1 is a colony chart of strain S26;
FIG. 2 is a gram of strain S26;
FIG. 3 is a graph of API test results for strain S26;
FIG. 4 is a RAPD fingerprint of strain S26;
FIG. 5 is a rep-PCR fingerprint of strain S26;
FIG. 6 shows MALDI-TOF-MS fingerprint of strain S26;
FIG. 7 is a diagram showing the zone of inhibition of helicobacter pylori by strain S26;
FIG. 8 is a graph showing the inhibitory effect of the strain S26 on H.pylori adhesion to human gastric adenocarcinoma cells;
FIG. 9 is a coagglutination pattern of strain S26 against H.pylori.
Detailed Description
The lactobacillus plantarum VHProbi S provided by the invention meets the requirement of regulations, and the lactobacillus plantarum VHProbi S is a newly discovered strain through multiphase taxonomy identification. The lactobacillus plantarum VHProbi S provided by the invention has the application of inhibiting the growth of helicobacter pylori, and has important application value for preventing or treating gastritis, gastric ulcer and other 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 plantarum VHProbi S26
MRS liquid medium: 1000mL 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, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ of 15 min.
MRS solid medium: agar 15g was added to the liquid medium for sterilization.
1.1 Lactobacillus primary screening
Taking 1g of sour dough, flushing the sour dough by using sterile normal saline, putting the sour dough into a sterile sample bag, and beating and uniformly mixing the sour dough by using a homogenizer; and (3) taking 100 mu L of mixed solution, carrying out gradient dilution, coating the mixed solution on an MRS solid plate culture 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 out 50 potential lactobacillus strains, which are named S1, S2, … … and S50 respectively.
1.2 Lactobacillus re-screening
Taking 1L of MRS liquid culture medium, adding 3.2g of porcine mucosa pepsin, shaking to dissolve, and placing in a 37 ℃ water bath shaking table for water bath for 1h to prepare the acid-resistant culture medium. 50 strains of lactobacillus S1, S2, … … and S50 obtained by screening are respectively inoculated into the acid-resistant culture medium according to the inoculum size of 6 percent, and are subjected to stationary culture for 72 hours at 37 ℃, and fermentation liquor is taken for bacterial count.
The result shows that the S26 strain in the 50 lactobacillus fermentation liquid has the maximum viable count after being rescreened by the acid-resistant culture medium, and the logarithmic value is as high as 8.01 Log CFU/mL.
Example 2 identification of strains
The inoculum preparation in this example was as follows: under the aseptic condition, a proper amount of fresh S26 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are resuspended by the same volume of PBS buffer to be used as inoculation liquid.
2.1 Colony morphology identification
The S26 strain was inoculated on MRS agar medium and cultured at 37℃for 48 hours. As shown in FIG. 1, the colony pattern of the S26 strain is milky white, the colony diameter is about 1.5-2mm, and the surface is wet.
As shown in FIG. 2, the S26 strain has positive gram staining, short bar shape under microscope, and round two ends.
2.2 Physiological Biochemical identification
2.2.1 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, and observing whether the culture medium becomes turbid.
The results show that the S26 strain can grow at a salt concentration of 1% -8%, and the optimal tolerance salt concentration is 5%.
2.2.2 Temperature tolerance test
Inoculating the inoculation liquid into 10mL MRS liquid culture medium according to 10% inoculation amount, taking 5mLMRS liquid culture medium without inoculating bacteria as control, respectively placing into 15 ℃ constant temperature incubator for 7 days, 45 ℃ constant temperature incubator for 2 days, and observing whether the bacterial liquid becomes turbid.
The results showed that S26 strain can reproduce at 15℃and 45 ℃.
2.2.3 Catalase experiment
The fresh bacterial liquid is taken, dropped on a clean glass slide, and then a drop of 3% hydrogen peroxide solution is dropped on the glass slide, and the S26 strain is observed to generate no bubbles and is a negative reaction.
2.2.4 Carbon source metabolism test
The carbon source metabolism experiment of strain S26 was validated using API 50CHL reagent. The API 50CHL reagent can be used to identify differences in the strain at the genus or species level. The experimental method and the result analysis are specifically described in the API 50CHL kit instruction.
The API test results are shown in FIG. 3, and the ID value of the S26 strain and the lactobacillus plantarum is 99.9%, which is an excellent identification result. The bacterium can be primarily identified as lactobacillus plantarum (Lactiplantibacillus plantarum) according to the carbon source metabolism result.
2.2.5 Glucose acidogenesis 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; salt solution a 0.5mL; 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 A: KH 2PO4 10g、K2HPO4 1.0.0 g was dissolved in distilled water to a volume of 100mL.
Salt solution B: mgSO 4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2 O0.68 g was dissolved in distilled water and the volume was set to 100mL.
Under aseptic conditions, inoculating the culture medium with 10% of inoculation amount, taking the culture medium without inoculating bacteria as a control, sealing the top with 2mL of aseptic liquid paraffin, culturing at 37 ℃ for 24 hours, 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 S26 strain produces acid and does not produce gas.
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 sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
(2) Reaction system (50. Mu.L)
TABLE 116 s rDNA PCR amplification System
| Composition of components | Reaction volume |
| 10×PCR buffer | 5μL |
| dNTPs | 4μL |
| 27F | 2μL |
| 1492R | 2μL |
| DNA | 2.5μL |
| rTaq | 0.5μL |
| ddH2O | 34μL |
(3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
(4) Sequencing PCR products: the 16S rDNA sequence of the S26 strain was obtained by sequencing and the sequences were aligned in NCBI database to preliminarily determine that the S26 strain was Lactobacillus plantarum (Lactiplantibacillus plantarum). SEQ ID NO: the sequence 1 is specifically as follows:
CCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTCAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTGC.
2.3.2 RAPD and rep-PCR fingerprint identification
1. RAPD fingerprint identification
(1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
(2) RAPD reaction system
TABLE 2 RAPD reaction System
| Reaction components | Volume of |
| TaqDNA polymerase (5U/. Mu.L) | 0.2 μL |
| 10 XBuffer (with Mg 2+) | 2 μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 0.8 μL |
| DNA template | 2 μL |
| Sterile double distilled water | 14 μL |
| Total volume of | 20 μL |
(3) Electrophoresis: a1.5% agarose gel plate was prepared, DL2000 DNA MARKER was used as a result control, 100V was stabilized for 80min, and the electropherograms were detected using a gel imaging system. RAPD finger print of S26 strain is shown in FIG. 4.
2. Rep-PCR fingerprint
(1) Rep-PCR primer: CTACGGCAAGGCGACGCTGACG.
(2) Reaction system of rep-PCR
TABLE 3 reaction System for rep-PCR
| Reaction components | Volume of |
| R TaqDNA polymerase | 0.2μL |
| 10X Ex Taq DNA Buffer (with Mg 2+) | 2μL |
| Primer (10 uM) | 1 μL |
| dNTPs（2.5 mM） | 2μL |
| DNA template | 2μL |
| Sterile double distilled water | 12.8 μL |
(3) Electrophoresis: the voltage is 100V, and the electrophoresis time is 80min to detect the amplification result. DL2000 DNA MARKER served as a result control. The rep-PCR fingerprint of S26 strain is shown in FIG. 5.
2.3.3 MALDI-TOF-MS detection of strain ribosomal protein expression
S26, centrifuging the fresh bacterial liquid at 5000rpm/min for 5min, washing with sterile water for 4 times, and airing surface moisture. 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. Protein fingerprint is obtained by Autofms 1000 analysis software Autof Analyzer v 1.0.0, and main ion peaks of the strain S26 are as follows: m/z 2594.389, 3932.785, 5192.135, 7865.555, 9500.578, etc., and the results of the identification are shown in FIG. 6.
In summary, in combination with the colony morphology, the physiological and biochemical characteristic results and the molecular biological identification results of the S26 strain, it can be concluded that the S26 strain is a new lactobacillus plantarum, which is named lactobacillus plantarum VHProbi S (Lactiplantibacillus plantarum VHProbi S).
EXAMPLE 3 test of haemolytic and antibiotic resistance of Lactobacillus plantarum VHProbi S26
Preparation of lactobacillus plantarum VHProbi S bacterial suspension:
Selecting purified lactobacillus plantarum VHProbi S colony, inoculating the colony into fresh MRS liquid culture medium, and culturing at 37 ℃ for 24 hours; 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 lactobacillus plantarum VHProbi S fresh bacterial liquid, centrifuging at 5000rpm for 5 min, and respectively collecting fermentation supernatant and thalli; the cells were washed 2 times with PBS buffer at pH7.4, and the concentration of the cells was adjusted to 5X 10 7 CFU/mL (OD 600 absorbance value: about 0.4) with PBS buffer to obtain a bacterial suspension.
3.1 Hemolysis test
The components of TBS basal medium (tryptone 17.0 g, soytone 3.0 g, sodium chloride 5.0 g, potassium dihydrogen phosphate (anhydrous) 2.5 g, glucose 2.5 g, distilled water 1000.0 ml) were weighed and dissolved, and then autoclaved at 121℃for 15 min, and 5% of sterilized defibrinated sheep blood was added and mixed evenly and poured into a plate when the medium was cooled to 50 ℃. And (3) streaking and inoculating the test strain to a prepared blood cell plate, culturing in an incubator at 37 ℃ for 24-48 hours, and observing whether the test strain has a hemolysis phenomenon.
The results show that: the lactobacillus plantarum VHProbi S can grow, and the blood cell plate is unchanged, which indicates that the lactobacillus plantarum VHProbi S does not produce hemolysin and can not dissolve blood cells.
3.2 Antibiotic resistance test
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 storage liquid is subjected to 2-time serial gradient dilution by using the MRS liquid culture medium to form a use liquid, wherein the gradient dilution concentration is 1-1024 mu g/mL and 11 gradients are total.
The minimum inhibitory concentration MIC of the antibiotic for Lactobacillus plantarum VHProbi S was determined by a micro broth dilution method.
Sequentially adding MRS liquid culture medium without antibiotics into the 1 st column of the 96-well plate as a negative control, sequentially adding 190 mu L of MRS liquid culture medium with antibiotics with different concentrations into the 2 nd-12 nd column, then respectively inoculating 10 mu L of the inoculation liquid, making 3 parallel wells, and taking 1 well of the non-added bacteria liquid 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, OD 600 was measured, and the MIC value of the antibiotic for the strain was counted using the 24-hour result, and the result is shown in Table 4.
TABLE 4 antibiotic MIC values for Lactobacillus plantarum VHProbi S26
| Erythromycin MIC | Erythromycin R/S | Gentamicin MIC | Gentamicin R/S | Streptomycin MIC | Streptomycin R/S | Ampicillin MIC | Ampicillin R/S | Tetracycline MIC | Tetracycline R/S |
| 4 | / | 32 | / | 64 | / | 2 | / | 2 | / |
MIC units μg/mL.
From Table 4, the lactobacillus plantarum VHProbi S provided by the invention is sensitive to common antibiotics such as erythromycin, tetracycline, ampicillin and the like, and has good biological safety.
Example 4 gastric juice tolerance test of Lactobacillus plantarum VHProbi S26
9Ml of artificial gastric juice was placed on a 37℃water bath shaker for 1h to simulate the human body temperature. 1mL of Lactobacillus plantarum VHProbi S fresh bacterial liquid (about 10 8 cfu/mL) is added into 9mL of artificial gastric juice, and placed in 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.
The results show that the lactobacillus plantarum VHProbi S is digested by gastric juice for 2 hours, and the bacterial load is only reduced by 0.07 Log CFU/mL, thereby indicating that the lactobacillus plantarum VHProbi S has strong tolerance to gastric juice.
EXAMPLE 5 inhibitory Effect of Lactobacillus plantarum VHProbi S on helicobacter pylori
5.1 And (3) culturing pathogenic bacteria:
The frozen and preserved glycerin helicobacter pylori (ATCC 353909/ATCC 354364) is coated on Columbia blood agar medium, and is placed under the condition of 37 ℃ and 10% CO 2 for culturing for 72 hours; scraping bacterial mud into brain heart infusion liquid culture medium under aseptic condition, and culturing at 37deg.C for 24 hr under micro-oxygen condition (oxygen concentration 5%, carbon dioxide concentration 10%, nitrogen concentration 85%, which is volume%); 5mL of the bacterial liquid was centrifuged at 5000r/min for 5min, and the bacterial cells were resuspended in phosphate buffer (PBS, pH 7.4) to adjust the concentration of the helicobacter pylori bacterial liquid to about 10 6 cfu/mL.
5.2 And (3) bacteriostasis circle test:
The method is carried out by adopting an oxford cup double-layer flat plate method.
In a clean workbench, taking a sterile flat plate, sterilizing nutrient agar, pouring the sterilized nutrient agar into the flat plate, and spreading the flat plate to serve as a lower culture medium; after the agar is solidified, uniformly spreading 10ml of Columbia blood agar culture medium on the upper layer to serve as an upper layer culture medium; after the upper layer culture medium is solidified, sucking 0.2mL of helicobacter pylori bacterial liquid on a solid culture medium plate, and uniformly coating; placing for 1h, and punching after bacterial liquid on the surface is fixed on the surface of the flat plate, wherein the diameter of the hole is 8mm, and the depth of the hole is 3mm; 0.1mL of the Lactobacillus plantarum VHProbi S strain suspension described in example 3 was injected into the well, and after 8h incubation at 37℃the results were observed and the diameter of the inhibition zone was determined.
As shown in FIG. 7, the diameter of the inhibition zone of the Lactobacillus plantarum VHProbi S on helicobacter pylori reaches 17+ -0.16 mm, which indicates that the strain has remarkable inhibition effect on helicobacter pylori.
EXAMPLE 6 inhibitory Effect of Lactobacillus plantarum VHProbi S fermentation supernatant on helicobacter pylori growth
(1) Experimental group:
The fresh bacterial solutions of helicobacter pylori were inoculated into brain heart infusion liquid culture media to which 2.5% (v/v), 5% (v/v), 10% (v/v) of the fermentation supernatant of lactobacillus plantarum VHProbi S described in example 3 was added, respectively, at a volume ratio of 1%.
(2) Control group:
inoculating fresh helicobacter pylori bacterial liquid into brain heart infusion liquid culture medium according to a volume ratio of 1%; culturing for 24h at 37 ℃ under the condition of 10% CO 2; OD values of the culture solutions were measured at 600 nm wavelength, respectively. The growth inhibition ratio of Lactobacillus plantarum VHProbi S cell-free fermentation supernatant to helicobacter pylori was calculated as 100% of the OD600 value of the control culture broth. The specific results are shown in Table 5.
Growth inhibition (%) = (control OD 600-experimental OD 600)/control OD600 x 100%.
TABLE 5 growth inhibition of Lactobacillus plantarum VHProbi S fermentation supernatant on helicobacter pylori
| Supernatant inoculum size | Inhibition rate | Standard deviation of |
| 2.5% | 4.31% | 0.19% |
| 5% | 12.10% | 0.31% |
| 10% | 23.51% | 0.71% |
| 15% | 33.39% | 0.54% |
From the results shown in Table 5, the addition of the cell-free fermentation supernatant of Lactobacillus plantarum VHProbi S significantly inhibited the growth of the pathogenic helicobacter pylori, and the inhibition rate of helicobacter pylori was continuously increased as the addition of the fermentation supernatant of Lactobacillus plantarum VHProbi S increased; when the addition amount reaches 15%, the inhibition rate of helicobacter pylori reaches 33.39%, and unexpected technical effects are achieved.
EXAMPLE 7 adhesion inhibition experiment of Lactobacillus plantarum VHProbi S on helicobacter pylori
(1) Culture of human gastric adenocarcinoma cells (BGC-823): 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) were inoculated into six-well plates containing 10% fetal bovine serum cells DMEM medium with built-in cell slide plates, the number of cell plates per well was about 2X 10 6 cells, and the six-well plates were placed in a carbon dioxide incubator for 24 hours.
(2) Adhesion inhibition test: the BGC-823 single cell layer adhered to the six-hole plate is washed 3 times by using PBS buffer solution, 1mL of lactobacillus plantarum VHProbi S and helicobacter pylori suspension described in the example 3 are respectively added, cells without the strain are used as blank control, and the blank control is placed into 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. Fixing with anhydrous methanol for 20min, 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. The ability of Lactobacillus plantarum VHProbi S to inhibit adhesion of helicobacter pylori to BGC-823 cells was evaluated by comparing the change in the adhesion number of helicobacter pylori in the presence and absence of Lactobacillus plantarum VHProbi S and examining the decrease in the adhesion rate of helicobacter pylori in the presence of Lactobacillus plantarum VHProbi S with the adhesion rate of Lactobacillus plantarum VHProbi S being 100% without adding Lactobacillus plantarum 3926.
As a result, as shown in FIG. 8, when treated with Lactobacillus plantarum VHProbi S, the adhesion rate of H.pylori to human gastric adenocarcinoma cells was 1, and the adhesion inhibition rate of Lactobacillus plantarum VHProbi S to H.pylori was 29.30%. As is evident from the figure, the number of H.pylori adhering to the human gastric adenocarcinoma cells of the experimental group is significantly smaller than that of the control group. Thus, it was demonstrated that Lactobacillus plantarum VHProbi S was able to significantly inhibit the adhesion of helicobacter pylori to human gastric adenocarcinoma cells.
EXAMPLE 8 Lactobacillus plantarum VHProbi S agglutination adsorption test
300. Mu.L of the Lactobacillus plantarum VHProbi S strain suspension described in example 3 was added to a 24-well plate, and 300. Mu.L of helicobacter pylori strain suspension (10 6 cfu/mL) was added as an experimental group; equal amounts of lactobacillus plantarum VHProbi S bacterial suspension and buffer were mixed as control groups, 2 replicates per control and experimental group. The 24-well plate was placed in a microplate thermostatted shaker at 400rpm/min, room temperature and incubated with shaking. And observing and photographing by a microscope, recording the initial orifice plate state and the orifice plate states at different times, and observing whether agglutination phenomenon occurs.
As a result, as shown in FIG. 9, the combination of Lactobacillus plantarum VHProbi S and helicobacter pylori showed a significant aggregate.
Example 9 adhesion experiments of human intestinal epithelial cells (Caco-2)
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. The original medium was discarded, rinsed twice with PBS buffer, and a suitable amount of pancreatin was added. And (3) placing the cells back into an incubator after adding pancreatin, stopping digestion after visually observing complete cell shedding, adding a culture solution with the volume of 2-3 times of pancreatin, repeatedly blowing for about ten times, and observing under a mirror to obtain 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 counting cells, 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 plated cells per well in the six-well plate was 1.5X10 6 cells, and the amount of culture medium added per well was 2ml. The six-hole plate is placed in a carbon dioxide incubator for 24 hours, and 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; 1mL of the cell culture solution without resistance and 1mL of the lactobacillus plantarum VHProbi S bacterial suspension described in the example 3 are respectively added into the experiment, and the experiment is put into a carbon dioxide incubator for 2 hours of culture; 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 plantarum VHProbi S 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.
TABLE 6 adhesion of Lactobacillus plantarum VHProbi S26.38326 to human intestinal epithelial cells (Caco-2)
| Strain | Adhesion capability | Standard deviation of |
| Lactobacillus plantarum VHProbi S26 | 2.08 | 0.1% |
| Lactobacillus rhamnosus LGG | 0.06 | 0.01% |
The adhesion test results are shown in Table 6, and the adhesion with lactobacillus rhamnosus LGG strain is used as a positive control, and the adhesion capability of lactobacillus plantarum VHProbi S to human intestinal epithelial cells is 34.6 times that of lactobacillus rhamnosus LGG, which indicates that the adhesion capability of lactobacillus plantarum VHProbi S to cells is stronger, and provides a foundation for the positive effect of lactobacillus plantarum VHProbi S in intestinal tracts.
EXAMPLE 10 cytotoxicity test of Lactobacillus plantarum VHProbi S26
The lactobacillus plantarum VHProbi S strain suspension described in example 3 was inactivated in a water bath at 70 ℃ for 20 minutes for later use.
Culture of human gastric adenocarcinoma cells (BGC-823): 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) were inoculated into six-well plates containing 10% fetal bovine serum cells DMEM medium with built-in cell slide plates, the number of cell plates per well was about 2X 10 6 cells, and the 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 2×10 5 cells/hole. The inactivated lactobacillus plantarum VHProbi S is added into cells according to the ratio of MOI (multiplicity of infection) value of 10, and a blank control group without bacteria is arranged for continuous culture 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 detection result shows that the lactobacillus plantarum VHProbi S has no obvious influence on the proliferation activity of human gastric epithelial cell BGC-823, has no cytotoxicity on human gastric cells, and has good safety.
Example 11 measurement of antioxidant Capacity of Lactobacillus plantarum VHProbi S26.3835
11.1 DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) scavenging ability determination
1ML of the Lactobacillus plantarum VHProbi S strain suspension described in example 3 was taken, 1mL of a 0.4 mM freshly prepared DPPH radical solution was added, the mixture was mixed uniformly, then the mixture was subjected to shading reaction at room temperature for 30 min, and then the absorbance A sample of the sample at a wavelength of 517nm was measured for 3 times. The control group sample is equal volume PBS solution and DPPH-ethanol mixed solution, and blank zeroing is carried out by equal volume bacterial suspension 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 7.
TABLE 7 DPPH radical scavenging
| Strain | Clearance rate% | Standard deviation of |
| Lactobacillus plantarum VHProbi S26 | 23.73% | 3.27% |
11.2 Determination of Hydroxy Radical (HRS) scavenging Capacity
200Ul of the bacterial suspension, 100ul of 5mM sodium salicylate-ethanol solution, 100ul of 5mM ferrous sulfate and 500ul of deionized water are uniformly mixed, 100ul of hydrogen peroxide solution (3 mM) is added, the absorbance of the bacterial suspension is measured at the wavelength of 510nm after 15min in a water bath at 37 ℃, and the clearance rate of the bacterial suspension to HRS is calculated. In addition, experiments were performed with equal doses of fermentation supernatant instead of bacterial suspension, and the clearance of the supernatant to HRS was determined. 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%, where a control of is absorbance of a mixed solution of ferrous sulfate, hydrogen peroxide, and sodium salicylate, and a Blank space is absorbance of a mixed solution of ferrous sulfate and sodium salicylate. The specific results are shown in Table 8.
Table 8 Lactobacillus plantarum VHProbi S for HRS radical scavenging
| Lactobacillus plantarum VHProbi S26 | Clearance rate% | Standard deviation of |
| Thallus | 41.41% | 10.16% |
| Fermentation supernatant | 90.46% | 0.10% |
11.3 Measurement of lipid peroxidation resistance
Preparation of linoleic acid emulsion: 0.1mL linoleic acid, 0.2mL Tween 20, 19.7mL deionized water.
0.5 Adding 1 mL linoleic acid emulsion, 1 mLFeSO 4 (1%), 0.5 mL sample and 37 ℃ water bath 1.5 h into the PBS solution (pH 7.4), adding 0.2 mL TCA (4%), 2mL TBA (0.8%), 100 ℃ water bath 30 min, rapidly cooling, 4000 rpm/min centrifuging 15 min, collecting supernatant, and measuring absorbance at 532 nm to obtain A; the control group replaced the sample, A0, with 0.5 mL distilled water. Inhibition rate/% = (A0-a)/a0×100%.
Note that: a is absorbance of a sample group; a0 is absorbance of the control group. The specific results are shown in Table 9.
TABLE 9 anti-lipid peroxidation inhibition rate tables
| Lactobacillus plantarum VHProbi S26 | Inhibition rate | Standard deviation of |
| Thallus | 48.63% | 0.41% |
| Fermentation supernatant | 14.69% | 0.65% |
| Intracellular extracts | 5.39% | 0.06% |
The results show that the lactobacillus plantarum VHProbi S provided by the invention has strong antioxidation capability, can effectively remove DPPH and HRS free radicals, and has remarkable lipid peroxidation resistance.
Example 12 in vitro cholesterol degradation experiment of Lactobacillus plantarum VHProbi S26
1G of cholesterol is accurately weighed, dissolved in absolute ethyl alcohol, and is fixed to a volume of 100 mL, and is subjected to filtration sterilization by a microporous filter membrane of 0.22 mu m under the aseptic condition. Cholesterol measurement method according to GB/T5009.128-2003 < measurement of cholesterol in food >. Inoculating fresh bacterial liquid according to 0.1% of inoculation amount, standing at 37 ℃ for 48 hours, taking 0.2mL of bacterial liquid, adding 1.8mL of absolute ethyl alcohol, uniformly mixing, standing for 10 minutes, centrifuging at 3000r for 5 minutes, and taking supernatant for measuring cholesterol content.
The results show that: the degradation rate of the lactobacillus plantarum VHProbi S provided by the invention on cholesterol reaches 21.27%, and the degradation rate on salt-containing cholesterol also reaches 8.4%.
In conclusion, the lactobacillus plantarum (Lacticaseibacillus paracasei) VHProbi S strain obtained by separation has no hemolysis, is sensitive to antibiotics, has strong acid resistance, can effectively inhibit the growth of helicobacter pylori, degrades cholesterol, and has remarkable antioxidation effect. In addition, the strain has certain adhesion capability to gastric cells and intestinal cells, can be aggregated with helicobacter pylori, increases the possibility that the helicobacter pylori is discharged out of the body along with gastrointestinal peristalsis, and can be applied to the field of medicines. The strain is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan, china 4 months 10 years 2023, and the preservation number is CCTCC NO: m2023501.
Claims (4)
1. Lactobacillus plantarum (Lactiplantibacillus plantarum) strain VHProbi S, characterized in that lactobacillus plantarum strain VHProbi S has a preservation number of CCTCC NO: m2023501.
2. Use of lactobacillus plantarum strain VHProbi S according to claim 1 for the manufacture of a medicament for the prevention or treatment of gastritis.
3. A helicobacter pylori inhibitor, characterized in that the inhibitor comprises at least one of viable cells or fermentation metabolites of lactobacillus plantarum strain VHProbi S of claim 1.
4. Use of the helicobacter pylori inhibitor according to claim 3 for the preparation of a medicament for the prevention or treatment of gastritis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310861908.0A CN116606781B (en) | 2023-07-14 | 2023-07-14 | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310861908.0A CN116606781B (en) | 2023-07-14 | 2023-07-14 | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116606781A CN116606781A (en) | 2023-08-18 |
| CN116606781B true CN116606781B (en) | 2024-05-14 |
Family
ID=87675008
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310861908.0A Active CN116606781B (en) | 2023-07-14 | 2023-07-14 | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116606781B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117778269A (en) * | 2024-01-08 | 2024-03-29 | 宁夏大学 | Lactobacillus plantarum NXU0014 and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779343A (en) * | 2016-03-28 | 2016-07-20 | 济南康多宝生物技术有限公司 | Lactobacillus plantarum capable of effectively removing cholesterol and free radicals and application thereof |
| KR20170071214A (en) * | 2015-12-15 | 2017-06-23 | 조선대학교산학협력단 | ANTI-HELICOBACTER COMPOSITION CONTAINING LactIC ACID BACTERIA SEPARATED FROM KIMCHI |
| CN112080445A (en) * | 2020-08-20 | 2020-12-15 | 浙江工商大学 | Lactobacillus plantarum ZJ316 and application thereof in inhibiting helicobacter pylori |
| CN113980878A (en) * | 2021-12-29 | 2022-01-28 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum for resisting helicobacter pylori infection and application thereof |
| CN114350578A (en) * | 2022-03-17 | 2022-04-15 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactobacillus plantarum LP1Z for producing lysozyme and efficiently antagonizing multidrug-resistant helicobacter pylori and application thereof |
| CN114774326A (en) * | 2022-05-11 | 2022-07-22 | 广西爱生生命科技有限公司 | Lactobacillus plantarum and application thereof |
| CN114891675A (en) * | 2022-05-11 | 2022-08-12 | 广州维生君生物科技有限公司 | Stomach-derived lactobacillus plantarum LPF-01 and application thereof |
| CN114908020A (en) * | 2022-05-31 | 2022-08-16 | 仁和全域(上海)大健康研究院有限公司 | Lactobacillus plantarum for resisting helicobacter pylori infection and application of lactobacillus plantarum in edible herbal enzyme product |
-
2023
- 2023-07-14 CN CN202310861908.0A patent/CN116606781B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170071214A (en) * | 2015-12-15 | 2017-06-23 | 조선대학교산학협력단 | ANTI-HELICOBACTER COMPOSITION CONTAINING LactIC ACID BACTERIA SEPARATED FROM KIMCHI |
| CN105779343A (en) * | 2016-03-28 | 2016-07-20 | 济南康多宝生物技术有限公司 | Lactobacillus plantarum capable of effectively removing cholesterol and free radicals and application thereof |
| CN112080445A (en) * | 2020-08-20 | 2020-12-15 | 浙江工商大学 | Lactobacillus plantarum ZJ316 and application thereof in inhibiting helicobacter pylori |
| CN113980878A (en) * | 2021-12-29 | 2022-01-28 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum for resisting helicobacter pylori infection and application thereof |
| WO2023124018A1 (en) * | 2021-12-29 | 2023-07-06 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum against helicobacter pylori infection, and use thereof |
| CN114350578A (en) * | 2022-03-17 | 2022-04-15 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactobacillus plantarum LP1Z for producing lysozyme and efficiently antagonizing multidrug-resistant helicobacter pylori and application thereof |
| CN114774326A (en) * | 2022-05-11 | 2022-07-22 | 广西爱生生命科技有限公司 | Lactobacillus plantarum and application thereof |
| CN114891675A (en) * | 2022-05-11 | 2022-08-12 | 广州维生君生物科技有限公司 | Stomach-derived lactobacillus plantarum LPF-01 and application thereof |
| CN114908020A (en) * | 2022-05-31 | 2022-08-16 | 仁和全域(上海)大健康研究院有限公司 | Lactobacillus plantarum for resisting helicobacter pylori infection and application of lactobacillus plantarum in edible herbal enzyme product |
Non-Patent Citations (5)
| Title |
|---|
| Effects of Lactobacillus on the inhibition of Helicobacter pylori growth;Liyuan Sun等;《Biotechnology & Biotechnological Equipment》;第32卷(第06期);第1533-1540页 * |
| Isolation and characteristics of lactic acid bacteria with antibacterial activity against Helicobacter pylori;Hui Huang等;《Food Bioscience》;第44卷;第1-9页 * |
| 两株潜在益生菌拮抗幽门螺杆菌的作用机制研究及其乳品工艺初探;郭怡麟;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》(2018年第12期);第B024-109页 * |
| 吴兴壮等.《乳酸菌及其发酵食品》.中国轻工业出版社,2021,(第1版),第92、103页. * |
| 防治幽门螺杆菌感染的益生菌研究进展;陈晓华等;《食品工业科技》;第33卷(第02期);第195-198页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116606781A (en) | 2023-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114990011B (en) | Lactobacillus reuteri capable of reducing cholesterol and inhibiting gardnerella and application | |
| CN114081901B (en) | Probiotic composition, preparation method and application thereof | |
| CN113862188B (en) | Lactobacillus gasseri LS03 and application thereof | |
| AU2020103929A4 (en) | Bacillus coagulans strain BACO-17 with high germination rate in the intestines and its uses for promoting gastrointestinal health | |
| CN116606781B (en) | Lactobacillus plantarum with helicobacter pylori antagonism capability and application thereof | |
| CN114642686B (en) | Composite probiotics and its functions of delaying senility and resisting oxidation | |
| CN115992073B (en) | Lactobacillus plantarum capable of converting ellagitannin to regulate mitochondrial autophagy and application thereof | |
| CN112625979A (en) | Lactobacillus casei for resisting helicobacter pylori and application thereof | |
| CN114752529B (en) | Lactobacillus plantarum HOM3201 strain and viable bacteria preparation, preparation method and application thereof | |
| CN117327608A (en) | Lactobacillus rhamnosus strain and application thereof | |
| WO2018112740A1 (en) | Lactobacillus gasseri, culture method therefor and application thereof | |
| CN116875480A (en) | Lactobacillus rhamnosus with antagonism to helicobacter pylori | |
| CN117431173A (en) | Antibacterial altitude pediococcus pentosaceus TR-37, cell-free extract thereof and application thereof | |
| CN115992059B (en) | Lactobacillus johnsonii for producing feruloyl esterase and application thereof in relieving ulcerative colitis | |
| CN114657106B (en) | Lactobacillus plantarum and application thereof in preventing and treating acne | |
| KR101512482B1 (en) | Novel Strains of Lactobacillus plantarum Q180 with Anti-Obesity Ability | |
| CN116254187A (en) | Lactobacillus plantarum and application thereof in resisting streptococcus pneumoniae respiratory tract infection | |
| 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 | |
| CN117467586B (en) | Lactobacillus rhamnosus with effect of delaying chronic nephrosis process | |
| CN117487685A (en) | Lactobacillus crispatus and application thereof in preventing or treating female colpitis | |
| CN116731911A (en) | Lactobacillus reuteri for inhibiting helicobacter pylori and application thereof | |
| CN114574405B (en) | Lactobacillus plantarum WKA86, application thereof in preparation of halitosis preventing and treating product and halitosis preventing and treating product | |
| CN114657107B (en) | Lactobacillus plantarum with acne treatment effect and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240410 Address after: 266000 596-1 Industrial Park, Jiushui East Road, Laoshan District, Qingdao, Shandong Applicant after: QINGDAO VLAND BIOTECH GROUP Co.,Ltd. Country or region after: China Address before: 261500 south of Xianghe Road, east of Yi'an Avenue, Gaomi City, Weifang City, Shandong Province (in Xiazhuang Industrial Park) Applicant before: WEIFANG KDN BIOTECH CO.,LTD. Country or region before: China Applicant before: QINGDAO VLAND BIOTECH GROUP Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |