CN116426413A - Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products - Google Patents
Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products Download PDFInfo
- Publication number
- CN116426413A CN116426413A CN202310180378.3A CN202310180378A CN116426413A CN 116426413 A CN116426413 A CN 116426413A CN 202310180378 A CN202310180378 A CN 202310180378A CN 116426413 A CN116426413 A CN 116426413A
- Authority
- CN
- China
- Prior art keywords
- enterococcus faecium
- microbial inoculum
- preparation
- pathogenic bacteria
- fermentation
- 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
- 241000194031 Enterococcus faecium Species 0.000 title claims abstract description 126
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 244000052616 bacterial pathogen Species 0.000 title abstract description 21
- 230000005764 inhibitory process Effects 0.000 title abstract description 21
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 241000193403 Clostridium Species 0.000 claims abstract description 24
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 241001052560 Thallis Species 0.000 claims abstract description 8
- 230000003213 activating effect Effects 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 33
- 239000000725 suspension Substances 0.000 claims description 25
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 230000004913 activation Effects 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 230000002401 inhibitory effect Effects 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 235000015278 beef Nutrition 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 229940099596 manganese sulfate Drugs 0.000 claims description 9
- 239000011702 manganese sulphate Substances 0.000 claims description 9
- 235000007079 manganese sulphate Nutrition 0.000 claims description 9
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 9
- 229920000053 polysorbate 80 Polymers 0.000 claims description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 230000000813 microbial effect Effects 0.000 claims description 7
- 235000013305 food Nutrition 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000002552 dosage form Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 235000019730 animal feed additive Nutrition 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 3
- 239000006071 cream Substances 0.000 claims description 3
- 239000000839 emulsion Substances 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 239000006187 pill Substances 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 239000000829 suppository Substances 0.000 claims description 3
- 239000003826 tablet Substances 0.000 claims description 3
- 244000052769 pathogen Species 0.000 claims 2
- 230000001717 pathogenic effect Effects 0.000 claims 2
- 206010009944 Colon cancer Diseases 0.000 abstract description 32
- 208000029742 colonic neoplasm Diseases 0.000 abstract description 32
- 238000011282 treatment Methods 0.000 abstract description 19
- 206010028980 Neoplasm Diseases 0.000 abstract description 16
- 239000003833 bile salt Substances 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 239000002253 acid Substances 0.000 abstract description 6
- 239000002609 medium Substances 0.000 description 32
- 239000001963 growth medium Substances 0.000 description 30
- 238000012258 culturing Methods 0.000 description 21
- 239000000243 solution Substances 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 108010010803 Gelatin Proteins 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 11
- 239000008273 gelatin Substances 0.000 description 11
- 229920000159 gelatin Polymers 0.000 description 11
- 235000019322 gelatine Nutrition 0.000 description 11
- 235000011852 gelatine desserts Nutrition 0.000 description 11
- 238000012408 PCR amplification Methods 0.000 description 10
- 241000191967 Staphylococcus aureus Species 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 150000001412 amines Chemical class 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- 230000001954 sterilising effect Effects 0.000 description 8
- 230000000035 biogenic effect Effects 0.000 description 7
- 230000002949 hemolytic effect Effects 0.000 description 7
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 7
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 7
- 238000009630 liquid culture Methods 0.000 description 7
- 229960001756 oxaliplatin Drugs 0.000 description 7
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 6
- 210000004940 nucleus Anatomy 0.000 description 6
- 238000007400 DNA extraction Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 230000000968 intestinal effect Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000015784 hyperosmotic salinity response Effects 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 102100023990 60S ribosomal protein L17 Human genes 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 108010014095 Histidine decarboxylase Proteins 0.000 description 3
- 102100037095 Histidine decarboxylase Human genes 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 210000004051 gastric juice Anatomy 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000003305 oral gavage Methods 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 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
- 102000004031 Carboxy-Lyases Human genes 0.000 description 2
- 108090000489 Carboxy-Lyases Proteins 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 102100021079 Ornithine decarboxylase Human genes 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000009654 indole test Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 159000000003 magnesium salts Chemical class 0.000 description 2
- 150000002696 manganese Chemical class 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008223 sterile water 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
- 210000001519 tissue Anatomy 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 125000000030 D-alanine group Chemical group [H]N([H])[C@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241001413625 Enterococcus faecium Aus0004 Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000605986 Fusobacterium nucleatum Species 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 1
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 101710124951 Phospholipase C Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000011226 adjuvant chemotherapy Methods 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
- 108010066657 azoreductase Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000004611 cancer cell death Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000006781 columbia blood agar Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 101150069320 ddl gene Proteins 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 231100000405 induce cancer Toxicity 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 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
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- 229960001327 pyridoxal phosphate Drugs 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QTTDXDAWQMDLOF-UHFFFAOYSA-J tetrasodium 3-[[4-[[4-[(6-amino-1-hydroxy-3-sulfonatonaphthalen-2-yl)diazenyl]-6-sulfonatonaphthalen-1-yl]diazenyl]naphthalen-1-yl]diazenyl]naphthalene-1,5-disulfonate Chemical compound [Na+].[Na+].[Na+].[Na+].Nc1ccc2c(O)c(N=Nc3ccc(N=Nc4ccc(N=Nc5cc(c6cccc(c6c5)S([O-])(=O)=O)S([O-])(=O)=O)c5ccccc45)c4ccc(cc34)S([O-])(=O)=O)c(cc2c1)S([O-])(=O)=O QTTDXDAWQMDLOF-UHFFFAOYSA-J 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
-
- 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
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Mycology (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Communicable Diseases (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a medical biotechnology, and discloses enterococcus faecium, a microbial inoculum containing the enterococcus faecium and application of the enterococcus faecium in preparation of pathogenic bacteria inhibition products. The preservation number of the enterococcus faecium (Enterococcus faecium) is CCTCC NO: M20221902. The microbial inoculum provided by the invention contains the enterococcus faecium. The invention also provides a preparation method of the microbial inoculum, which comprises the following steps: activating the enterococcus faecium to obtain activated thalli; inoculating the activated thalli into a fermentation medium for fermentation to prepare the liquid microbial inoculum. The invention also provides application of the enterococcus faecium, the microbial inoculum and the microbial inoculum prepared by the preparation method in preparation of a pathogenic bacteria inhibition product, wherein the pathogenic bacteria are clostridium nucleatum. The enterococcus faecium can effectively antagonize and inhibit pathogenic bacteria clostridium nucleatum, has acid resistance, bile salt resistance and high biological safety, and has potential clinical auxiliary treatment effect on colon cancer tumor.
Description
Technical Field
The invention relates to a medical biotechnology, in particular to enterococcus faecium, a microbial inoculum containing the enterococcus faecium and application of the enterococcus faecium in preparation of pathogenic bacteria inhibition products.
Background
Colon cancer is one of the tumors with the highest morbidity and mortality in solid cancers, and the morbidity is steadily rising. Colon cancer can develop along the circulation of intestinal wall, up and down along the longitudinal diameter of intestinal canal or deeply infiltrate into the intestinal wall, besides lymphatic vessel, blood flow transfer and local invasion, can also be planted in abdominal cavity or spread and transferred along suture lines and incision surfaces, so that the colon cancer has extremely high malignancy degree, strong transferability, lower cure rate and poor prognosis, and has a trend of increasing younger and younger.
At present, main treatment methods of colon cancer clinically comprise operation treatment, radiotherapy and chemotherapy, targeted treatment, traditional Chinese medicine treatment and the like, but the modes have certain limitations, such as high recurrence rate of operation treatment and poor compliance; chemotherapy can induce cancer cell death by inducing DNA damage or initiating various signaling pathways, and for colon cancer patients with earlier stages, the adjuvant chemotherapy treatment is also gradually maturing, but still has strong side effects, and patients are prone to relapse. Therefore, there is an urgent need for highly effective low-toxicity antitumor drugs to target colon cancer treatment.
Recent researches find that the richness of the clostridium with obvious correlation with the occurrence of colon cancer, and the clostridium with the nucleus is used as pathogenic bacteria, is enriched in colon cancer tissues, can influence a plurality of stages in colon cancer progression, and promotes proliferation of colon cancer cells, tumor immune escape, recurrence, chemotherapy drug resistance risks and the like.
In view of the defects of the operation and chemotherapy modes, if targeted inhibition intervention can be carried out on pathogenic bacteria of colon cancer, the method has important significance for treating colon cancer.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide enterococcus faecium, a microbial inoculum containing the enterococcus faecium and application of the enterococcus faecium in preparation of pathogenic bacteria inhibition products.
In order to achieve the aim, the first aspect of the invention provides enterococcus faecium (Enterococcus faecium), wherein the preservation number of the enterococcus faecium is CCTCC NO: M20221902.
The second aspect of the invention provides a microbial inoculum comprising the enterococcus faecium described above.
Preferably, the microbial inoculum contains at least one of viable bacteria, dead bacteria and fermentation products of the enterococcus faecium; preferably living bacterial cells.
Preferably, the microbial inoculum is a liquid microbial inoculum and/or a solid microbial inoculum.
Preferably, the bacterial agent is the enterococcus faecium with a colony concentration of 10 6 -10 8 cfu/mL of liquid microbial inoculum.
The third aspect of the invention provides a preparation method of a microbial inoculum, comprising the following steps:
(1) Activating enterococcus faecium with a preservation number of CCTCC NO: M20221902 to obtain activated thalli;
(2) Inoculating the activated thalli obtained in the step (1) into a fermentation medium for fermentation to obtain the liquid microbial inoculum.
Preferably, the activation medium used for the activation comprises: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of tween 80, 0.2-0.8g/L of magnesium sulfate, 0.15-0.4g/L of manganese sulfate and 12-18g/L of agar; the fermentation medium contains: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of Tween 80, 0.2-0.8g/L of magnesium sulfate and 0.15-0.4g/L of manganese sulfate.
Preferably, the conditions of activation at least satisfy: the temperature is 30-45 ℃ and the time is 24-48h; the fermentation conditions at least meet: the temperature is 30-45 ℃, the rotating speed is 150-200rpm, and the time is 20-60h.
The fourth aspect of the invention provides the enterococcus faecium, the microbial inoculum and the application of the microbial inoculum prepared by the preparation method in preparation of a pathogenic bacteria inhibition product, wherein the pathogenic bacteria are clostridium nucleatum.
Preferably, the pathogen-suppressing product is selected from at least one of a drug, a food product, and an animal feed additive.
Preferably, the dosage form of the drug is selected from at least one of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.
Through the technical scheme, the invention has the beneficial effects that:
the enterococcus faecium provided by the invention has the characteristic of unique inhibition of the clostridium nucleatum, has a remarkable inhibition effect on pathogenic bacteria of colon cancer, can sensitize oxaliplatin to the anti-tumor effect of colon cancer, has an auxiliary effect on the treatment of colon cancer, and inhibits the progress of tumor diseases when being applied to the treatment of colon cancer.
The enterococcus faecium provided by the invention also has the characteristics of acid resistance, bile salt resistance and the like, does not generate harmful metabolites such as indole, amine and the like, has high biological safety, can be used for producing additives of medicines and foods, is added into the medicines and the foods to realize the auxiliary treatment, health care effect and the like of colon cancer patients, and especially has better auxiliary treatment effect on patients enriched with clostridium nucleatum in colon cancer tumor tissues. Furthermore, the enterococcus faecium provided by the invention can also be used in animal feed additives to assist in the treatment and health care of colon cancer tumors of animals.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Preservation of organisms
The strain provided by the invention is enterococcus faecium (Enterococcus faecium LMX) and is preserved in China center for type culture collection (address: wuhan university in Wuchang district of Wuhan, hubei province) (across from first auxiliary university of Wuhan), 12 th month 09 of 2022, wherein the China center for type culture collection 211 room, postal code: 430072, CCTCC for preservation unit is abbreviated as CCTCC, and the preservation number is CCTCC NO: M20221902.
Drawings
FIG. 1 shows the results of identification of LMX46 and LMX47 specific genes ddI and SodA of enterococcus faecium in example 1;
FIG. 2 is a graph showing the results of acid and bile salt tolerance tests of enterococcus faecium LMX46 and LMX47 in example 2, wherein A is a simulated gastrointestinal fluid tolerance test and B is a graph showing the bile salt tolerance test;
FIG. 3 is a safety test result of enterococcus faecium LMX46 and LMX47 in example 3, wherein A is a hemolytic activity test result, B is an azo reduction test result, C is a gelatin liquefaction test result, D is an indole test result, E is a biogenic amine production test result, ODC is ornithine decarboxylase, LDC is lysine decarboxylase, and HDC is histidine decarboxylase;
FIG. 4 is a graph showing the inhibition of Clostridium nucleatum by the culture supernatants, bacterial cells, and bacterial suspensions of enterococcus faecium LMX46 and LMX47 of example 4;
FIG. 5 is a graph of the therapeutic effect of enterococcus faecium LMX 47-assisted oxaliplatin in a mouse colon cancer model as described in example 5;
FIG. 6 is a graph showing the inhibitory effects of enterococcus faecium LMX47 and enterococcus faecium ATCC 51559 on Staphylococcus aureus in example 6, wherein a is enterococcus faecium LMX47 and b is enterococcus faecium ATCC 51559.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the invention provides enterococcus faecium (Enterococcus faecium), wherein the preservation number of the enterococcus faecium is CCTCC NO: M20221902.
The enterococcus faecium provided by the invention is separated from the faeces of lung cancer patients with better response to PD-1 treatment (acquired by the oncology department of Min Hospital in Jiangsu province, nanjing). The enterococcus faecium may be isolated using methods conventional in the art for isolation of new strains. Illustratively, the enterococcus faecium separation process provided by the invention specifically comprises the following steps: diluting and plating faeces of lung cancer patients with good PD-1 antibody treatment responsiveness on an MRS culture medium, a TSA culture medium and a Columbia blood plate respectively, culturing at 37 ℃ for 24-48 hours, picking single colony on each plate to a corresponding liquid culture medium for activation, and standing and culturing at 37 ℃ for 24-48 hours; then, quick identification is carried out on bacteria by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), two strains of enterococcus faecium are primarily screened out, genomic DNA of the two strains is extracted according to the operation on a bacterial genomic DNA extraction kit, PCR amplification sequencing is carried out, and molecular biological identification is carried out on the strains.
The inventor selects a strain which can obviously form a bacteriostasis ring for inhibiting clostridium with nucleuses from the screened strain, and performs DNA extraction and identification, and the identification result shows that the 16srDNA sequence of the strain is highly similar to enterococcus faecium (Enterococcus faecium), has specific genes dd I and SodA of enterococcus faecium, can determine that the strain is enterococcus faecium (Enterococcus faecium LMX 47), and is preserved in China Center for Type Culture Collection (CCTCC) No. M20221902 at 12 months 09 of 2022.
The enterococcus faecium provided by the invention can generate a large amount of viable bacteria and/or fermentation products of the enterococcus faecium after being cultured. The method of the present invention is not particularly limited as long as the enterococcus faecium can be proliferated in large amounts by the method of the present invention, and for example, a live cell of enterococcus faecium can be inoculated in a culture medium in an amount of not less than 0.5% by volume and cultured at a temperature of 30 to 45℃for not less than 12 hours to obtain a culture solution. The medium may be a medium conventionally used in the art, for example, an MRS liquid medium.
The method of separating the enterococcus faecium cells in the culture solution according to the present invention is not particularly limited, so long as the cells can be enriched from the culture solution, and the method may be, for example, centrifugation and/or filtration, and the conditions of the centrifugation and the filtration may be conventional conditions in the art, and are well known to those skilled in the art, and will not be described herein.
The second aspect of the invention provides a microbial inoculum comprising the enterococcus faecium described above.
In the present invention, the concentration of enterococcus faecium in the microbial inoculum is not particularly limited, and may be specifically selected according to the specific circumstances.
According to the present invention, the microbial inoculum preferably contains at least one of a viable cell, a dead cell and a fermentation product of the enterococcus faecium, and more preferably a viable cell. In the present invention, the term "fermentation product" refers to a metabolite (including intracellular and/or extracellular metabolites) produced by enterococcus faecium during fermentation or culture.
According to the present invention, the form of the microbial inoculum is not particularly limited, and may be prepared into various forms according to the intended use, and components such as corresponding excipients and the like are added, for example, the microbial inoculum may be a liquid microbial inoculum (for example, may be a bacterial liquid of enterococcus faecium) and/or a solid microbial inoculumThe ecological microbial inoculum (for example, the microbial inoculum can be powder prepared by drying enterococcus faecium or is prepared into a high-purity preparation by steps of separation, purification and the like). The addition of excipients to the bacterial agents in which the dosage forms are described herein is well known to those skilled in the art and will not be described in detail herein. Further preferably, the bacterial agent is the enterococcus faecium with a colony concentration of 10 6 -10 8 cfu/mL of liquid microbial inoculum.
The third aspect of the invention provides a preparation method of a microbial inoculum, comprising the following steps:
(1) Activating enterococcus faecium with a preservation number of CCTCC NO: M20221902 to obtain activated thalli;
(2) Inoculating the activated thalli obtained in the step (1) into a fermentation medium for fermentation to obtain the liquid microbial inoculum.
According to the invention, the medium used for the activation may be a medium common in the art, preferably the medium used for the activation comprises the following components: the carbon source, nitrogen source, potassium salt, magnesium salt, ammonium salt, sodium salt and manganese salt may specifically contain: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of tween 80, 0.2-0.8g/L of magnesium sulfate, 0.15-0.4g/L of manganese sulfate and 12-18g/L of agar.
According to the present invention, the fermentation medium may be a liquid medium as is common in the art, preferably the components of the fermentation medium comprise: the carbon source, nitrogen source, potassium salt, magnesium salt, ammonium salt, sodium salt and manganese salt may specifically contain: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of Tween 80, 0.2-0.8g/L of magnesium sulfate and 0.15-0.4g/L of manganese sulfate.
According to the invention, the conditions of activation at least satisfy: the temperature is 30-45deg.C, specifically 30deg.C, 33deg.C, 36deg.C, 39deg.C, 42deg.C, 45deg.C, or any value between the above two values; the time is 24-48h, and can be specifically 24h, 30h, 36h, 42h, 48h, or any value between the two values; the fermentation conditions at least meet: the temperature is 30-45deg.C, specifically 30deg.C, 33deg.C, 36deg.C, 39deg.C, 42deg.C, 45deg.C, or any value between the above two values; the rotation speed is 150-200rpm, and can be 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the two values; the time is 20-60h, and can be specifically 20h, 30h, 40h, 50h, 60h, or any value between the two values.
The enterococcus faecium provided by the invention not only can inhibit staphylococcus aureus, but also has the characteristic of unique inhibition of clostridium nucleatum, and when the enterococcus faecium is applied to the treatment of colon cancer, the enterococcus faecium not only has a remarkable inhibition effect on pathogenic bacteria of colon cancer, but also can sensitize the anti-tumor effect of oxaliplatin on colon cancer, has an auxiliary effect on the treatment of colon cancer and inhibits the progress of tumor diseases. Based on this, the fourth aspect of the present invention provides the enterococcus faecium, the microbial inoculum, and the application of the microbial inoculum prepared by the preparation method in preparing a pathogenic bacteria inhibition product, wherein the pathogenic bacteria are clostridium nucleatum.
Preferably, according to the present invention, the pathogen-suppressing product is selected from at least one of a pharmaceutical, a food and an animal feed additive. Wherein, the dosage form of the pathogenic bacteria inhibiting drug is preferably at least one selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nano-particles, capsules, suppositories, injections, sprays and injections; the food may be a health product. The pathogenic bacteria inhibiting drugs in the above various dosage forms can be prepared according to the conventional methods in the art, and can also comprise pharmaceutically acceptable carriers.
According to the invention, the content of enterococcus faecium in the pathogenic bacteria inhibiting product can be designed and adjusted according to the dosage.
The present invention will be described in detail by examples.
In the following examples, matrix assisted laser Desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was manufactured by Beijing Naohos technologies Co., ltd., model 5301455816; colon cancer CT-26 cells were purchased from american type culture collection ATCC, no. ATCC-9118, bacterial genomic DNA extraction kit was purchased from the institute of biotechnology (shanghai) corporation, no. B518225-0100, columbia blood plates were purchased from the south kyo holotype biotechnology company, and clostridium with nuclei (Fusobacterium nucleatum) was supplied by the mouth hospital in south kyo city, staphylococcus aureus (Staphylococcus aureus) was purchased from the american type culture collection ATCC, no. ATCC 29213, escherichia coli (Escherichia coli) was purchased from the american type culture collection ATCC, no. ATCC 25922, and all experimental materials used were purchased from conventional biochemical reagent stores, unless otherwise specified.
In the following examples, MRS solid medium was used as the activation medium, and the formulation was: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium citrate, 5g/L of sodium acetate, 20g/L of glucose, 1g/L of tween 80, 0.5g/L of magnesium sulfate, 0.25g/L of manganese sulfate and 15g/L of agar, adjusting the pH to 6.2-6.4, and sterilizing under high pressure for 15min for later use;
the fermentation medium adopts MRS liquid medium, and the formula is as follows: 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium citrate, 5g/L of sodium acetate, 20g/L of glucose, 1g/L of tween 80, 0.5g/L of magnesium sulfate and 0.25g/L of manganese sulfate, adjusting the pH value to 6.2-6.4, and sterilizing under high pressure for 15min for later use;
the formula of the TSA culture medium is as follows: 15g/L of tryptone, 5g/L of soybean papain hydrolysate, 5g/L of sodium chloride and 15g/L of agar, adjusting the pH to 7.1-7.5, and sterilizing for 15min under high pressure for later use.
The formula of the TSB culture medium is as follows: 15g/L of tryptone, 5g/L of soybean peptone, 5g/L of sodium chloride and 15g/L of agar, regulating the pH to 7.0-7.4, and sterilizing for 15min under high pressure for later use.
Example 1
1. Isolation of strains
Taking the feces of a lung cancer patient with good PD-1 antibody treatment responsiveness (acquired places are Jiangsu people's hospital oncology, nanjing), respectively diluting and plating on MRS solid culture medium, TSA culture medium and Columbia blood plates, culturing at 37 ℃ for 24-48 hours, picking single colony on each plate to a corresponding liquid culture medium for activation, standing at 37 ℃ for 24-48 hours, then rapidly identifying bacteria by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and primarily screening two strains of enterococcus faecium with the serial numbers of LMX46 and LMX47;
2. molecular biological identification of strains
Extracting genome DNA of two strains of enterococcus faecium LMX46 and LMX47 respectively according to the operation on a bacterial genome DNA extraction kit, and performing PCR amplification sequencing;
wherein, the bacterial genome DNA extraction steps are as follows:
inoculating the single colony of the two screened enterococcus faecium streaks to MRS liquid culture medium, culturing at 37 ℃ for 24 hours, taking 1.5mL of bacterial liquid, centrifuging at room temperature and 8000rmp for 1min, collecting bacterial cells, adding 180 mu L of lysozyme solution (20 mg/mL) to resuspend the bacterial liquid, carrying out water bath at 37 ℃ for 30min-60min (reversing and mixing every 10min during the water bath), adding 400 mu L Buffer Digestion, and shaking and mixing uniformly; then adding 20 mu L of RNaseA (10 mg/mL) after completely lysing cells in a water bath at 65 ℃ for 1h, standing at room temperature for 2-5min to obtain RNA-free DNA, adding 200 mu L of Buffer PB into the DNA, fully reversing and uniformly mixing, standing at-20 ℃ for 5min, centrifuging at room temperature for 5min at 1000rmp, and sucking 500 mu L of supernatant and transferring into a new centrifuge tube; adding isopropanol with equal volume, reversing for 5-8 times to fully mix, standing at room temperature for 2-3min, centrifuging at room temperature 10000rmp for 5min, discarding supernatant, adding 1mL 75% ethanol, reversing for 1-3min, centrifuging at 10000rmp for 2min, discarding supernatant, and repeating the above steps twice; inverting the cover at room temperature for 5-10min until ethanol is completely volatilized, dissolving the obtained DNA with 50 mu L TE Buffer to obtain genomic DNA, and preserving at-20deg.C for use;
the genome DNA extracted from two strains of enterococcus faecium LMX46 and LMX47 is used as a template, a bacterial universal primer pair (the nucleotide sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2) is adopted for PCR amplification, and a 20 mu L system is adopted for PCR amplification: 1. Mu.L of forward primer, 1. Mu.L of reverse primer, 10. Mu.L of Prime STAR, 1. Mu.L of DNA template and ddH 2 O7 μl; the PCR amplification reaction conditions were: pre-denaturing at 98 ℃ for 3min, denaturing at 98 ℃ for 10s, annealing at 56 ℃ for 20s, extending at 72 ℃ for 1min, circulating for 36 times, extending at 72 ℃ for 10min, and preserving at 4 ℃;
forward primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1), reverse primer 1492R:5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2);
the fragment size of the PCR product was checked by 1.5% agarose gel electrophoresis to determine if it was correct, and the PCR product was sequenced by the Protocolysis of the family of the Biotech Co., ltd. And the sequencing result was compared by NCBI database Blast and highly similar to enterococcus faecium (Enterococcus faecium).
The housekeeping genes of enterococcus faecium have high conservation and variability, and have higher resolution than 16S rRNA gene sequencing, so that the species of the near bacteria can be identified more easily; therefore, two species-specific genes ddl and SodA of enterococcus faecium are selected for PCR amplification, and LMX46 and LMX47 are further identified and confirmed, wherein the ddl gene is d-alanine polylinker gene, and the sequence size is about 550bp; the SodA gene is a manganese superoxide dismutase gene of enterococcus faecium, is an important antioxidant active gene required by complete virulence of enterococcus faecium AUS0004 strain, and has a sequence size of about 216 bp; the PCR amplification adopts a 20 mu L system: 1. Mu.L of DNA template, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 10. Mu.L of Prime STAR, ddH 2 O7 μl; the PCR amplification reaction conditions were: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1min, circulation for 30 times, extension at 72 ℃ for 7min, and preservation at 4 ℃;
the nucleotide sequences of primer pairs for PCR amplification of ddl fragments are shown as SEQ ID NO.3 and SEQ ID NO.4,
ddl forward primer 27F:5'-GCAAGGCTTCTTAGAGA-3' (SEQ ID NO. 3), ddl reverse primer 1492R:5'-CATCGTGTAAGCTAACTTC-3' (SEQ ID NO. 4),
the nucleotide sequences of primer pairs for amplifying the SodA fragment by PCR are shown as SEQ ID NO.5 and SEQ ID NO.6,
SodA forward primer 27F:5'-GAAAAAACAATAGAAGAATTAT-3' (SEQ ID NO. 5),
sodA reverse primer 1492R:5'-TGCTTTTTTGAATTCTTCTTTA-3' (SEQ ID NO. 6);
detecting whether the fragments of the PCR products are consistent with expected results by using 1.5% agarose gel electrophoresis, wherein the results are shown in figure 1, and the PCR amplification results of specific genes ddl and sodA of two strains LMX46 and LMX47 are consistent with the expected fragments, so that the two strains are proved to be determined to be enterococcus faecium; two strains of enterococcus faecium were designated enterococcus faecium LMX46 (Enterococcus faecium LMX) and enterococcus faecium LMX47 (Enterococcus faecium LMX) respectively.
Example 2
Acid and bile salt resistant experimental researches are carried out on enterococcus faecium LMX46 and enterococcus faecium LMX 47.
The simulated gastrointestinal fluid tolerance experiment process comprises the following steps:
a. preparation of simulated gastric fluid: sucking 1.64mL of dilute hydrochloric acid, adding 80mL of water and 1g of pepsin, shaking uniformly, adding water to dilute to 100mL, adjusting the pH to 3.0, and placing into an ultra-clean workbench to sterilize for half an hour for later use;
b. preparation of simulated intestinal juice: weighing 0.68g of monopotassium phosphate, adding 50mL of water for dissolution, and adjusting the pH to 6.8 by 0.1 mol/LNaOH; dissolving 1g of trypsin in water, mixing, diluting to 100ml with water, and sterilizing in an ultra-clean bench for half an hour;
c. inoculating enterococcus faecium LMX46 and LMX47 onto MRS solid state culture medium plate for activation, inoculating single colony into MRS liquid culture medium, culturing at 37deg.C for 24 hr to obtain culture solution, inoculating 10% (v/v) of culture solution into artificial gastric juice, and standing at 37deg.C for culturing; inoculating culture solution for 0h, 1h, 2h, and 3h, and continuously diluting with sterile water 10 times to 10 5 Multiple of 10 6 Doubling, coating 100 mu L on an MRS plate, observing growth conditions and counting colonies; then taking the artificial gastric juice culture solution after 3 hours, inoculating the artificial gastric juice culture solution into the artificial intestinal juice with 10 percent of inoculation amount, standing and culturing at 37 ℃, taking the culture solution inoculated for 8 hours, 13 hours and 19 hours, and continuously diluting the culture solution with sterile water for 10 times until the culture solution is 10 times 5 Multiple of 10 6 100. Mu.L of the solution was applied to MRS plates, and the growth was observed and colony counts were performed, as shown in FIG. 2.
Bile salt tolerance test
After enterococcus faecium LMX46 and LMX47 are inoculated onto an MRS solid medium plate for activation, single colonies are selected and inoculated into an MRS liquid medium, the bacteria suspension is obtained by culturing at 37 ℃ for 24 hours, the bacteria suspension is respectively inoculated into the MRS liquid medium with the bile salt mass concentration of 0.1 percent, 0.3 percent and 0.5 percent by weight according to the inoculum concentration of 2 percent (v/v), the bacteria suspension is subjected to stationary culture at 37 ℃ for 3 hours, the number of bacteria in a sample is measured by adopting an MRS plate colony counting method, and the result is shown in figure 2.
As shown in FIG. 2, both LMX46 and LMX47 enterococcus faecium have stable tolerance to gastric acid environment with pH of 3.0, and the survival rate is maintained at 100% over 3 h. When the concentration of bile salt is 0.1 weight percent and 0.3 weight percent, compared with LMX46, LMX47 has better bile salt tolerance, and after the culture of the bile salt culture medium for 24 hours, the survival rate of LMX47 is still more than 90 percent, which indicates that enterococcus faecium is used as probiotics for oral administration and has better acid resistance and bile salt resistance.
Example 3
A series of tests are carried out on the safety of the two strains of enterococcus faecium LMX46 and LMX47, including a hemolytic activity test, an azo reduction test, a gelatin liquefaction test, an indole test and a biogenic amine production test.
1. Analysis of hemolytic Activity
After enterococcus faecium LMX46 and LMX47 are inoculated on a MRS solid medium plate for activation, single colony is selected and inoculated in MRS liquid medium, bacterial suspension is obtained by culturing for 24 hours at 37 ℃, streaking is inoculated on a Columbia blood agar plate, inverted culturing is carried out for 24-48 hours, whether a hemolytic transparent ring exists around the colony is observed and recorded, and staphylococcus aureus (S.aureus, ATCC 29213) with strong hemolytic property is used as a positive control; if grass green ring is formed around the colony due to incomplete rupture of red blood cells, alpha hemolysis is performed; beta hemolysis occurs if a well-defined, completely transparent hemolytic ring is formed around the colony due to complete rupture of the red blood cells; if the medium surrounding the colonies was not changed, it was gamma-hemolyzed, i.e., not hemolyzed, and the results are shown in FIG. 3.
2. Azo reduction experiment
After enterococcus faecium LMX46 and LMX47 are inoculated on a MRS solid medium plate for activation, single colonies are selected and inoculated in MRS liquid medium, bacterial suspension is obtained by culturing at 37 ℃ for 24 hours, the bacterial suspension is streaked and inoculated on the MRS solid medium plate containing direct blue 71 (with the final mass concentration of 5 mg/L), culturing is carried out at 37 ℃ for 72 hours, whether hydrolysis rings are generated or not is observed, the positive hydrolysis rings appear, and staphylococcus aureus (S. Aureus, ATCC 29213) is used as a positive control, and the result is shown in figure 3.
3. Gelatin liquefaction experiment
Preparation of gelatin medium: 0.5g of sodium chloride, 1g of peptone, 0.3g of beef extract, 12g of gelatin and 100mL of distilled water, adjusting the pH to be 7.2-7.4, and sterilizing at 115 ℃ for 30min;
after enterococcus faecium LMX46 and LMX47 are inoculated onto a MRS solid medium flat plate for activation, single colonies are selected and inoculated into MRS liquid medium, bacterial suspension is obtained by culturing for 24 hours at 37 ℃, puncture inoculation is carried out on 5mL of gelatin medium, after culturing for 24-48 hours at 37 ℃, the enterococcus faecium is taken out and placed in a refrigerator at 4 ℃ for 2 hours to completely coagulate residual gelatin, meanwhile, a negative control group BC (sterile normal saline is used for replacing experimental bacteria) and a positive control group BC (staphylococcus aureus S.aureus is used for replacing experimental bacteria) are arranged, the melting condition in a tube is observed, a test tube with gelatin melting phenomenon is judged to be a positive reaction of gelatin, and the result is shown in figure 3.
4. Indole experiments
The formula of the peptone water culture medium comprises: tryptone 20g, sodium chloride 5g, distilled water 1000mL, pH value of 7.4 plus or minus 0.2, sterilizing at 121 ℃ for 15min;
the indole reagent formula is as follows: 0.1g of p-dimethylaminobenzaldehyde is dissolved by 5mL of ethanol solution with the volume fraction of 95%, and then 2mL of concentrated hydrochloric acid is slowly added, so that the p-dimethylaminobenzaldehyde is prepared for use and stored in a dark place;
inoculating enterococcus faecium LMX46 and LMX47 to an MRS solid culture medium plate for activation, then inoculating single colony to an MRS liquid culture medium, culturing at 37 ℃ for 24 hours to obtain bacterial suspension, inoculating 2% (v/v) of the bacterial suspension to a peptone water culture medium for 24 hours at 37 ℃, dropwise adding an indole reagent, wherein the liquid level shows rose, and the result is positive, otherwise the result is negative; the results of the experiment using E.coli (E.coli) as a positive control and peptone water medium without bacterial liquid inoculation as a Blank (BC) are shown in FIG. 3.
5. Biogenic amine production experiments
The formula of the biogenic amine detection culture medium is as follows: 5g of peptone, 5g of yeast powder, 5g of beef extract, 2.5g of sodium chloride, 0.5g of glucose, 1mL of Tween-80, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 0.04g of ferric sulfate, 2g of ammonium citrate and 2g of dibasic phosphate2g of potassium, 0.1g of calcium carbonate and VB 1 0.01g of pyridoxal-5-phosphate, 0.05g of bromocresol purple, 0.06g of distilled water, 1000mL; respectively adding 10g/L of corresponding precursor amino acids (histidine, ornithine and lysine) into biogenic amine detection culture medium to prepare improved amino decarboxylase detection culture medium, adjusting pH value to 5.3-5.5, and sterilizing at 115 ℃ for 30min;
inoculating enterococcus faecium LMX46 and LMX47 to an MRS solid state culture medium plate for activation, then inoculating single colony to an MRS liquid culture medium, culturing at 37 ℃ for 24 hours to obtain bacterial suspension, marking lines on an improved amino decarboxylase detection plate added with precursor amino acids (histidine HDC, ornithine ODC and lysine LDC) and a detection plate not added with the precursor amino acids respectively, culturing at 37 ℃ and observing color change, wherein the color change is purple, the color is a positive result, and the color is not changed to a negative result; the experiment was performed with E.coli (E.coli, ATCC 25922) as positive control and without the addition of the precursor amino acid as negative control, the results being shown in FIG. 3.
As shown in the results of FIG. 3, the hemolytic activity experiments show that the two strains of enterococcus faecium LMX46 and LMX47 do not produce beta hemolysin (see FIG. 3A); gelatin liquefaction experiments show that neither LMX46 nor LMX47 enterococcus faecium produces gelatinase, and gelatin liquefaction phenomenon does not occur (see figure 3C); in addition, biogenic amine experiments, azo reduction experiments and indole experiments show that LMX46 and LMX47 do not produce biogenic amine and azo reductase (see fig. 3B and 3E), and that LMX47 strain does not metabolize tryptophan to produce the harmful substance indole (see fig. 3D) in comparison to LMX46 strain, further demonstrating the biosafety of LMX 47.
Example 4
The antibacterial activity of the two strains of enterococcus faecium suspension and thallus suspension and supernatant on the clostridium nucleatum is measured by an oxford cup method. The specific operation is as follows:
preparation and treatment of enterococcus faecium bacterial suspension, thallus suspension and supernatant: enterococcus faecium is inoculated in MRS liquid culture medium with an inoculum size of 1% (v/v), and is subjected to stationary culture at 37 ℃ for 24 hours; inoculating the clostridium with 2% (v/v) inoculum size into TSB culture medium containing 5% of aseptic defibrinated sheep blood, standing and culturing at 37 ℃ for 48 hours in an anaerobic incubator, and balancing OD values of activated enterococcus faecium and clostridium with fungus liquid to about 0.8 to obtain activated clostridium with fungus suspension and fungus suspension of two strains of enterococcus faecium; taking 1mL of bacterial suspension of two strains of enterococcus faecium LMX46 and LMX47 after balancing OD value, centrifuging at 5000rmp for 5min, taking supernatant and bacterial precipitate respectively, transferring the supernatant into a new centrifuge tube again to be used as supernatant, re-suspending the bacterial precipitate with 1mL of PBS buffer to be used as bacterial suspension, and placing the prepared sample at 4 ℃ for standby.
The preparation method of the oxford cup double-layer flat plate method comprises the steps of pouring 20-25mL of TSB culture medium containing agar into a culture dish, horizontally placing until solidification is achieved, taking the culture medium as a bottom layer, and placing the oxford cup on the surface; mixing 200 μl of activated suspension of Clostridium nucleatum with 25mL of semi-solid TSB culture medium containing 5% sterilized defibrinated sheep blood at 50-55deg.C, shaking thoroughly, pouring the mixture into a plate with oxford cup, standing in a sterile operation table, and taking out oxford cup after the culture medium is solidified; then, 140. Mu.L of enterococcus faecium suspension, thallus suspension and supernatant were added to the wells, and the mixture was spread at 4℃for 3 hours, cultured at 37℃for 48 hours under anaerobic conditions, and the diameter of the inhibition zone was measured, the results of which are shown in FIG. 4.
The results shown in fig. 4 show that the inhibition zone of LMX47 on the clostridium with nucleuses is obviously larger than that of LMX46, and the LMX has obvious inhibition effect on the clostridium with nucleuses of pathogenic bacteria of colon cancer.
The experimental results combined with the embodiment 1 to the embodiment 4 show that the enterococcus faecium LMX47 not only has the unique characteristic of inhibiting the colon cancer pathogenic bacteria clostridium nucleatum, but also has the characteristics of acid resistance, bile salt resistance and the like, does not generate harmful metabolites such as indole, amine and the like, and has high biological safety; the strain is preserved in China center for type culture collection (address: wuhan university of Wuchang district of Wuhan, hubei province, across from the first affiliated primary school of Wuhan), china center for type culture collection 211, postal code: 430072, abbreviated as CCTCC of preservation unit), and the preservation number is CCTCC NO: M20221902.
Example 5
Experiment for evaluating anti-tumor drug effect of enterococcus faecium LMX47 by using colon cancer and clostridium nucleatum co-modeling mouse model. In vitro culturing colon cancer CT-26 cells, inoculating the cells into a cell culture flask, adding DMEM medium, and culturing in 5% CO 2 Culturing in a cell culture incubator at 37 ℃, wherein the complete culture medium comprises 10% of fetal bovine serum, 1% of double antibody (100 IU/ml penicillin and 100IU/ml streptomycin) and RPMI 1640 culture medium; when the cells grow to 70-90% of proper density, adding trypsin for digestion, stopping digestion after 3min, collecting cells, adding PBS, and re-suspending to a concentration of 10 6 /mL; CT26 cell suspension and Clostridium sclerotium bacteria solution (concentration 10) 3 CFU/mL), 0.5mL was inoculated to the subcutaneous area of each mouse, respectively, until the tumor size was 100-150mm 3 The administration was grouped as follows: 1) PBS group, 2) LMX47 group, 3) LMX47+oxa group, 4) Oxa group; wherein the PBS group refers to 0.5mL of PBS buffer solution for oral gavage daily, and the LMX47 group refers to 0.5mL of LMX47 bacterial solution for oral gavage daily (bacterial solution concentration is 10) 8 CFU/mL), LMX47+oxa group refers to oral gavage of 0.5mL LMX47 bacterial liquid (bacterial liquid concentration 10) 8 CFU/mL), and oxaliplatin (oxa) was intravenously injected every four days in an amount of 0.1mL (dose is 15mg/kg in terms of the weight of the mouse), the oxa group was oxaliplatin every four days in an amount of 0.1mL (dose is 15mg/kg in terms of the weight of the mouse), tumors of the mice were dissected and weighed on day 20, and the tumor inhibition rate was calculated, and the result is shown in fig. 5.
As shown in the results of FIG. 5, when enterococcus faecium LMX47 is singly orally administered, the inhibition rate of colon cancer tumor is about 18%, and the inhibition rate is mainly due to that the enterococcus faecium LMX47 inhibits the activity of clostridium with nucleuses so as to slow down the proliferation of tumor; in addition, enterococcus faecium LMX47 can remarkably improve the tumor inhibiting effect of the positive drug oxaliplatin, and the tumor inhibiting rate is improved from 65% to 80% after the combined administration. In conclusion, experiments show that enterococcus faecium LMX47 can sensitize oxaliplatin to the anti-tumor effect, and can be used as a potential auxiliary therapeutic drug for colon cancer.
Example 6
The inhibition zone of enterococcus faecium LMX47 and enterococcus faecium ATCC 51559 (purchased from American type culture Collection ATCC, accession No. ATCC 51559) against Staphylococcus aureus (S.aureus, ATCC 29213) was determined by the oxford cup method described in example 4, and the results are shown in FIG. 6. The results show that enterococcus faecium LMX47 also has a significant inhibitory effect on Staphylococcus aureus, while enterococcus faecium ATCC 51559 has no inhibitory effect on Staphylococcus aureus.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. Enterococcus faecium (Enterococcus faecium) is characterized in that the preservation number of the enterococcus faecium is CCTCC NO: M20221902.
2. A microbial inoculum comprising the enterococcus faecium according to claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent contains at least one of viable bacteria, dead bacteria, and fermentation products of the enterococcus faecium; preferably living bacterial cells.
4. A microbial agent according to claim 2 or 3, wherein the microbial agent is a liquid microbial agent and/or a solid microbial agent.
5. The microbial inoculum of claim 4, wherein the microbial inoculum is a colony concentration of the enterococcus faecium of 10 6 -10 8 cfu/mL of liquid microbial inoculum.
6. The preparation method of the microbial inoculum is characterized by comprising the following steps:
(1) Activating enterococcus faecium with a preservation number of CCTCC NO: M20221902 to obtain activated thalli;
(2) Inoculating the activated thalli obtained in the step (1) into a fermentation medium for fermentation to obtain the liquid microbial inoculum.
7. The method according to claim 6, wherein the activating medium used for the activation comprises: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of tween 80, 0.2-0.8g/L of magnesium sulfate, 0.15-0.4g/L of manganese sulfate and 12-18g/L of agar;
the fermentation medium contains: 8-12g/L of peptone, 8-12g/L of beef extract, 3-8g/L of yeast extract, 1-3g/L of dipotassium hydrogen phosphate, 1-3g/L of diammonium citrate, 3-8g/L of sodium acetate, 15-25g/L of glucose, 0.5-1.5g/L of Tween 80, 0.2-0.8g/L of magnesium sulfate and 0.15-0.4g/L of manganese sulfate.
8. The preparation method according to claim 6 or 7, characterized in that the conditions of the activation at least satisfy: the temperature is 30-45 ℃ and the time is 24-48h;
the fermentation conditions at least meet: the temperature is 30-45 ℃, the rotating speed is 150-200rpm, and the time is 20-60h.
9. Use of the enterococcus faecium according to claim 1, the microbial inoculum according to any one of claims 2 to 5, and the microbial inoculum prepared by the preparation method according to any one of claims 6 to 8 for the preparation of a pathogen-inhibiting product, said pathogen being clostridium nucleatum.
10. The use of claim 9, wherein the pathogen-suppressing product is selected from at least one of a drug, a food, and an animal feed additive;
preferably, the dosage form of the drug is selected from at least one of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310180378.3A CN116426413A (en) | 2023-02-24 | 2023-02-24 | Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310180378.3A CN116426413A (en) | 2023-02-24 | 2023-02-24 | Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116426413A true CN116426413A (en) | 2023-07-14 |
Family
ID=87080413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310180378.3A Pending CN116426413A (en) | 2023-02-24 | 2023-02-24 | Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116426413A (en) |
-
2023
- 2023-02-24 CN CN202310180378.3A patent/CN116426413A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2023072305A1 (en) | Akkermansia muciniphila and application thereof in preparation of antitumor drugs | |
| CN107619811A (en) | Lactobacillus plantarum CCFM200 bacterial strains and application | |
| WO2017020784A1 (en) | Bacteroides fragilis and application thereof | |
| CN113604384A (en) | Lactobacillus rhamnosus and application thereof | |
| CN110564638A (en) | Lactobacillus reuteri with probiotic characteristics and application thereof | |
| CN115093999B (en) | Clostridium praecox capable of improving blood lipid disorders and application thereof | |
| CN115969888A (en) | Saliva combined lactobacillus and application thereof in preparation of medicine for treating cancer | |
| WO2012103785A1 (en) | Lactobacillus salivarius and method for preparing metabolite thereof, composition of lactobacillus salivarius and metabolite thereof and use of the composition | |
| CN112940970A (en) | Intestinal probiotics and application thereof in treating tumors and replacing antibiotics | |
| CN114107134B (en) | Brevibacillus laterosporus and application thereof | |
| CN115103901A (en) | Methods and compositions for culturing hemoglobin-dependent bacteria | |
| CN114107088B (en) | Lactobacillus reuteri LRSY523 and application thereof | |
| JP4540664B2 (en) | Novel Bifidobacterium strain having glutamine-producing ability | |
| Xu et al. | Lactobacillus casei JY300-8 generated by 12C6+ beams mutagenesis inhibits tumor progression by modulating the gut microbiota in mice | |
| CN117327632A (en) | Bifidobacterium animalis and application thereof | |
| CN116426406B (en) | Saliva combined lactobacillus DY802 and application thereof | |
| CN117264814A (en) | Lactobacillus rhamnosus with effects of preventing and treating digestive tract diseases | |
| CN116769658A (en) | Bacillus coagulans AP15 and application thereof in intestinal tract regulation and uric acid and cholesterol metabolism | |
| CN116426413A (en) | Enterococcus faecium, microbial inoculum containing enterococcus faecium and application of microbial inoculum in preparation of pathogenic bacteria inhibition products | |
| TWI810852B (en) | Use of Bacillus coagulans BC198 or its metabolites for the prevention or adjuvant treatment of intestinal damage-related lesions or flora imbalance caused by chemotherapy | |
| CN114164148B (en) | Lactobacillus equi-like bacterium, microbial inoculum and application thereof | |
| CN117286078B (en) | Lactobacillus plantarum for improving gastrointestinal health and application thereof | |
| CN117402794B (en) | Lactobacillus gasseri and application thereof | |
| CN116790402B (en) | Bacteroides simplex strain with anti-inflammatory property, culture method and application | |
| CN117305187B (en) | Pediococcus acidilactici for improving intestinal health condition 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 |