KR20220125785A - Composition for verification of human-derived enzyme degradation and immune function of Lactic acid bacteria from Kimchi containing Lipoteichoic acid as an indicator - Google Patents
Composition for verification of human-derived enzyme degradation and immune function of Lactic acid bacteria from Kimchi containing Lipoteichoic acid as an indicator Download PDFInfo
- Publication number
- KR20220125785A KR20220125785A KR1020220027911A KR20220027911A KR20220125785A KR 20220125785 A KR20220125785 A KR 20220125785A KR 1020220027911 A KR1020220027911 A KR 1020220027911A KR 20220027911 A KR20220027911 A KR 20220027911A KR 20220125785 A KR20220125785 A KR 20220125785A
- Authority
- KR
- South Korea
- Prior art keywords
- lactobacillus casei
- dkgf7
- kctc
- dead cells
- function
- Prior art date
Links
- 241000894006 Bacteria Species 0.000 title claims description 30
- 230000036737 immune function Effects 0.000 title claims description 3
- 239000000203 mixture Substances 0.000 title claims description 3
- 239000002253 acid Substances 0.000 title abstract description 23
- 230000015556 catabolic process Effects 0.000 title abstract description 8
- 238000006731 degradation reaction Methods 0.000 title abstract description 8
- 102000004190 Enzymes Human genes 0.000 title abstract description 7
- 108090000790 Enzymes Proteins 0.000 title abstract description 7
- 235000021109 kimchi Nutrition 0.000 title abstract description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title description 34
- 239000004310 lactic acid Substances 0.000 title description 17
- 235000014655 lactic acid Nutrition 0.000 title description 17
- 238000012795 verification Methods 0.000 title description 3
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 64
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 60
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 60
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 12
- 230000000968 intestinal effect Effects 0.000 claims abstract description 11
- 208000024891 symptom Diseases 0.000 claims abstract description 9
- 235000013373 food additive Nutrition 0.000 claims abstract description 6
- 239000002778 food additive Substances 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims description 30
- 230000002708 enhancing effect Effects 0.000 claims description 16
- 238000000527 sonication Methods 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 230000006870 function Effects 0.000 claims description 13
- 208000002551 irritable bowel syndrome Diseases 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 230000003871 intestinal function Effects 0.000 claims description 11
- 239000004480 active ingredient Substances 0.000 claims description 10
- 239000006872 mrs medium Substances 0.000 claims description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims description 9
- 235000013361 beverage Nutrition 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000003674 animal food additive Substances 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 13
- 241001465754 Metazoa Species 0.000 abstract description 12
- 241000186660 Lactobacillus Species 0.000 abstract description 9
- 229940039696 lactobacillus Drugs 0.000 abstract description 9
- 238000012790 confirmation Methods 0.000 abstract description 7
- 230000008944 intestinal immunity Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 110
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 28
- 102000004127 Cytokines Human genes 0.000 description 20
- 108090000695 Cytokines Proteins 0.000 description 20
- 238000011282 treatment Methods 0.000 description 19
- 239000006041 probiotic Substances 0.000 description 17
- 235000018291 probiotics Nutrition 0.000 description 17
- 102100026238 Lymphotoxin-alpha Human genes 0.000 description 14
- 238000005259 measurement Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 9
- 102100040247 Tumor necrosis factor Human genes 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 230000002757 inflammatory effect Effects 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 8
- 238000000354 decomposition reaction Methods 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 230000000529 probiotic effect Effects 0.000 description 8
- 241000700159 Rattus Species 0.000 description 7
- 102000004139 alpha-Amylases Human genes 0.000 description 7
- 108090000637 alpha-Amylases Proteins 0.000 description 7
- 229940024171 alpha-amylase Drugs 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- -1 IL-1α Proteins 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 102000057297 Pepsin A Human genes 0.000 description 6
- 108090000284 Pepsin A Proteins 0.000 description 6
- 102000044820 Zonula Occludens-1 Human genes 0.000 description 6
- 108700007340 Zonula Occludens-1 Proteins 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 210000001072 colon Anatomy 0.000 description 6
- 230000002550 fecal effect Effects 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000003364 immunohistochemistry Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 229940111202 pepsin Drugs 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000028327 secretion Effects 0.000 description 6
- 239000003270 steroid hormone Substances 0.000 description 6
- 241000186612 Lactobacillus sakei Species 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 102000004162 Claudin-1 Human genes 0.000 description 4
- 108090000600 Claudin-1 Proteins 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 230000035939 shock Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 102000000591 Tight Junction Proteins Human genes 0.000 description 3
- 108010002321 Tight Junction Proteins Proteins 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 102000038379 digestive enzymes Human genes 0.000 description 3
- 108091007734 digestive enzymes Proteins 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000007515 enzymatic degradation Effects 0.000 description 3
- 230000006862 enzymatic digestion Effects 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 244000005709 gut microbiome Species 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 208000007882 Gastritis Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241000186605 Lactobacillus paracasei Species 0.000 description 2
- 229920002774 Maltodextrin Polymers 0.000 description 2
- 239000005913 Maltodextrin Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 229940035034 maltodextrin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- JQMFQLVAJGZSQS-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)NC1=CC2=C(NC(O2)=O)C=C1 JQMFQLVAJGZSQS-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- YLZOPXRUQYQQID-UHFFFAOYSA-N 3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]propan-1-one Chemical compound N1N=NC=2CN(CCC=21)CCC(=O)N1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F YLZOPXRUQYQQID-UHFFFAOYSA-N 0.000 description 1
- CONKBQPVFMXDOV-QHCPKHFHSA-N 6-[(5S)-5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-2-oxo-1,3-oxazolidin-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C[C@H]1CN(C(O1)=O)C1=CC2=C(NC(O2)=O)C=C1 CONKBQPVFMXDOV-QHCPKHFHSA-N 0.000 description 1
- WTFUTSCZYYCBAY-SXBRIOAWSA-N 6-[(E)-C-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-N-hydroxycarbonimidoyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C/C(=N/O)/C1=CC2=C(NC(O2)=O)C=C1 WTFUTSCZYYCBAY-SXBRIOAWSA-N 0.000 description 1
- DFGKGUXTPFWHIX-UHFFFAOYSA-N 6-[2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]acetyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)C1=CC2=C(NC(O2)=O)C=C1 DFGKGUXTPFWHIX-UHFFFAOYSA-N 0.000 description 1
- LLQHSBBZNDXTIV-UHFFFAOYSA-N 6-[5-[[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]methyl]-4,5-dihydro-1,2-oxazol-3-yl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC1CC(=NO1)C1=CC2=C(NC(O2)=O)C=C1 LLQHSBBZNDXTIV-UHFFFAOYSA-N 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 208000031873 Animal Disease Models Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 241000723346 Cinnamomum camphora Species 0.000 description 1
- 102000002029 Claudin Human genes 0.000 description 1
- 108050009302 Claudin Proteins 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 241000087799 Koma Species 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 101001002629 Mus musculus Interleukin-1 alpha Proteins 0.000 description 1
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 description 1
- 101000648740 Mus musculus Tumor necrosis factor Proteins 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 238000011558 animal model by disease Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229960000846 camphor Drugs 0.000 description 1
- 229930008380 camphor Natural products 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000004953 colonic tissue Anatomy 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000001200 fecal consistency Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 230000007366 host health Effects 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000008975 immunomodulatory function Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003870 intestinal permeability Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000002438 stress hormone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 210000001578 tight junction Anatomy 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000006433 tumor necrosis factor production Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Images
Classifications
-
- 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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
-
- 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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
-
- 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
- A23V2300/00—Processes
- A23V2300/24—Heat, thermal treatment
-
- 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
- A23V2300/00—Processes
- A23V2300/48—Ultrasonic treatment
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
-
- A23Y2220/17—
-
- 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
- C12R2001/245—Lactobacillus casei
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
본 발명은 김치로부터 분리된 락토바실러스 카제이 DKGF7(Lactobacillus casei, 기탁기관 : 한국생명공학연구원 KCTC에 2020년 7월 20일자로 기탁됨, 수탁번호 : KCTC 16246BP) 유산균의 열처리한 사균체의 리포테이코산(Lipoteichoic acid)를 지표물질로 선정하여 체내에서의 사균체 안정성을 검증하기 위한 인체 유래 효소분해능 및 LTA 함량 변화를 확인하여 사균체의 인체 적용 가능성 확인에 관한 것이다. 또한, 동물실험을 통하여 사균체의 장증상 및 장면역 개선 효과 확인하여, 상기 사균체를 유효성분으로 포함하는 장면역 기능 증진용 약학적 조성물, 식품 첨가제, 음료수 정장제, 사료 첨가제 및 상기 사균체의 제조 방법에 관한 것이다.The present invention is Lactobacillus casei DKGF7 ( Lactobacillus casei , deposited from KCTC, Korea Research Institute of Bioscience and Biotechnology on July 20, 2020, accession number: KCTC 16246BP) lipotay of heat-treated dead cells of lactic acid bacteria isolated from kimchi It relates to confirming the applicability of dead cells to the human body by checking the enzyme decomposition ability and LTA content change derived from the human body to verify the stability of dead cells in the body by selecting Lipoteichoic acid as an indicator material. In addition, by confirming the effect of improving intestinal symptoms and intestinal function of dead cells through animal experiments, a pharmaceutical composition for enhancing intestinal function containing the dead cells as an active ingredient, food additives, beverage preparations, feed additives, and the dead cells It relates to a manufacturing method.
사람이나 동물에게 건조세포나 발효산물의 형태로 투여하여 숙주의 장내 균총을 개선하여 좋은 영향을 주는, 단일 혹은 복합 형태의 살아있는 균을 뜻하는 프로바이오틱스에 대해 2001년 FAO와 WHO는 “살아있는 미생물로써 투여량이 적정하면 숙주의 건강에 이로우며 생리적 이익을 주기 위해서는 살아있는 상태를 유지해야 함”으로 정의하였다. 그러나 다수의 연구논문을 통해 “숙주에 유익한 작용을 하는 미생물 또는 미생물의 성분”으로 사균체를 포함한 프로바이오틱스의 개념이 주장된다.Regarding probiotics, which are single or complex types of live bacteria, which are administered to humans or animals in the form of dried cells or fermented products to improve the intestinal flora of the host and have a positive effect, in 2001, FAO and WHO, “administered as a living microorganism If the dose is appropriate, it is beneficial to the health of the host, and in order to provide physiological benefits, it must be maintained in a living state.” However, through a number of research papers, the concept of probiotics including dead cells is claimed as “a microorganism or a component of a microorganism that has a beneficial action on the host”.
유산균의 세포벽과 핵산이 우리 몸의 면역을 조절하는 대식세포와 수지상세포를 자극하여 면역세포의 활성을 강화시키는데 이 유산균의 세포벽과 핵산은 살아있거나 죽어있을 때 모두 동일한 역할을 한다.The cell wall and nucleic acid of lactic acid bacteria stimulate the macrophages and dendritic cells that control the immune system of our body to enhance the activity of immune cells.
열처리한 유산균 유래 사균체는 소장에서 흡수되어 TNF-α 생산, 산화질소 분비와 macrophage의 phagocytic 작용을 활성화하여 면역조절 기능을 하는데 최근 미국, 일본 및 국내의 식의약 제품에 사균체의 활용이 증가하고 있다. 이는 사균체가 인체 생리활성 적용 분야에서 생균과 유사한 기능을 나타낼 수 있으면서, 인체내 소화흡수 과정에서의 안정성이 높고, 제품 가공 및 유통 과정에서 생균에 비해 활용 범위가 넓으며 일정한 사균체 함량을 정량적으로 조절할 수 있다는 장점이 인정된 것이다.The heat-treated lactic acid bacteria-derived dead cells are absorbed in the small intestine and function as an immunomodulatory function by activating TNF-α production, nitric oxide secretion, and phagocytic action of macrophages. have. This means that dead cells can exhibit similar functions to live cells in the field of human physiological activity application, have high stability in the digestive and absorption process in the human body, have a wider range of applications than live cells in product processing and distribution, and quantitatively measure a certain amount of dead cells. The advantage of being able to control it has been recognized.
유산균을 가열처리한 사균체는 면역부활에 의한 발암 예방까지 폭넓게 사람의 건강기능에 작용하는 것으로 알려져 있고 최근까지 연구결과에 따르면 생균 형태의 probiotics와 더불어 열처리 등에 의해 생산된 사균체에 의한 다양한 면역조절 기능이 보고되고 있고 유산균들의 장점이 유산균이 사멸되어도 유지된다고 알려진다.It is known that the dead cells treated with lactic acid bacteria have a wide range of effects on human health from cancer prevention by immune revitalization. According to research results until recently, various immune regulation by dead cells produced by heat treatment along with live probiotics. It is known that the function is reported and the advantages of the lactic acid bacteria are maintained even if the lactic acid bacteria are killed.
현재 가장 대표적으로 알려진 Enterococus faecalis를 열처리하여 생산된 사균체를 가지고 동물실험을 실시한 결과 면역력, 장질환, 급발성 위염 및 근위축증 개선에 효과적인 것으로 보고되었으며 이는 균체 유래 핵산과 리포테이코산(Lipoteichonic acid)에 의한 것으로 보고되고 있다. 리포테이코산은 그람 양성 박테리아의 세포벽 성분이며 양쪽 친매성 물질로 최근 많은 연구가 이루어지고 있으나 주로 병원성 세균의 리포테이코산에 대한 연구만 이루어져 있고, 병원균 및 유산균 자체의 리포테이코산을 분석 진행한 연구는 많이 보고되어왔지만 사균체의 리포테이코산 분석을 진행하여 그 함량을 생균제와 비교한 연구는 보고된 바 없다.As a result of animal experiments with dead cells produced by heat treatment of Enterococus faecalis , the most representative currently known, it was reported to be effective in improving immunity, intestinal disease, acute gastritis and muscular atrophy. It is reported to be caused by Lipoteichoic acid is a cell wall component of Gram-positive bacteria and is an amphiphilic substance, and many studies have been conducted recently. has been reported a lot, but there has been no report of a study comparing the content of lipoteichoic acid with a probiotic by analyzing the lipoteichoic acid of dead cells.
장내 미생물이 장 질환과 인간 건강에 중요한 역할을 한다는 많은 증거가 나오고 있다. 과민성 대장 증후군(IBS)은 최소 6개월 동안 배변 습관 변화를 수반하는, 즉, 반복적인 복통이나 불편함을 동반하는 반선 기능성 장 질환이다. 병리 생리학에서는 거의 알려지지 않았지만 최근 발전은 장내 미생물과 장점막, 복합 장뇌 장애, 낮은 정도의 위점막염증, 면역 반응 및 변화된 장 투과성 사이의 명확한 연관성을 나타낸다. 이는 장내 미생물과 숙주 면역 반응 사이의 균형을 유지하려는 치료의 기초이며, 그중 하나는 프로바이오틱스의 투여이다. 박테리아의 생존력은 건강상의 이점을 증진하는데 필수적이진 않다. 죽은 박테리아는 세포 조각을 포함하고 살아있는 박테리아와 유사한 숙주에서 면역 반응을 유도할 수 있는 생물학적 활성을 유지한다고 알려져있고 안전성, 긴 보관기간 및 운송 편의성을 포함하지만 사균체의 프로바이오틱 효과에 대한 증거는 거의 알려져 있지 않다.There is a lot of evidence that the gut microbiome plays an important role in gut disease and human health. Irritable Bowel Syndrome (IBS) is an anti-inflammatory bowel disease that is accompanied by changes in bowel habits for at least 6 months, i.e., recurrent abdominal pain or discomfort. Although little known in pathophysiology, recent advances indicate a clear link between the gut microbiota and intestinal mucosa, complex camphor disorders, low-grade gastric mucositis, immune responses and altered intestinal permeability. It is the basis of treatments aimed at maintaining a balance between the gut microbiota and the host immune response, one of which is the administration of probiotics. Bacterial viability is not essential to promote health benefits. Dead bacteria contain cell fragments and are known to retain biological activity capable of inducing an immune response in a host similar to live bacteria, including safety, long shelf life, and transport convenience, but evidence for probiotic effects of dead cells is lacking. Little is known about it.
유산균 사균체가 생리활성이 있으며 숙주, 즉 유산균을 섭취한 대상에 대해서 생균보다 사균체가 안전성이 높다고 평가되고 있음에도 불구하고 이에 대한 사균체 섭취 시, 생균제와 비교하였을 때 인체 내에서의 사균체의 안정성 (인체 유래 효소분해율) 확인 및 사균체 제조 시 LTA의 함량 변화에 대한 연구는 보고된 바가 없어 본 발명자들은 유산균 사균체의 인체 유래 효소분해능 및 LTA 함량 변화를 확인하여 사균체 섭취 시 인체 적용 가능성 확인하였으며, 이를 통해 사균화 처리를 통해 면역 조절 효능 확인 및 LTA 함량의 유의미한 증가를 발견하였기에 본 발명을 완성하게 되었다. 또한 동물 질병 모델에서 IBS 증상 개선에 대한 사균체의 유익한 효과를 평가하고 질병 자체에 대한 사균체의 작용 메커니즘을 조사하기 위하여 본 발명을 완성하게 되었다.Despite the fact that the dead cells of lactic acid bacteria are physiologically active and that the safety of the dead cells is higher than that of the live cells for the host, that is, the target ingesting the lactic acid bacteria, when the dead cells are ingested, compared to the live bacteria, There have been no reports of studies on the stability (enzyme decomposition rate derived from the human body) and the change in the LTA content during the production of dead cells, so the present inventors confirmed the human-derived enzyme decomposition ability and LTA content change of the dead cells of lactic acid bacteria. Possibility of human application when ingesting dead cells It was confirmed, and through this, the present invention was completed because it was found that the immunomodulatory efficacy was confirmed and a significant increase in the LTA content was confirmed through the bactericidal treatment. In addition, the present invention was completed to evaluate the beneficial effect of dead cells on the improvement of IBS symptoms in an animal disease model and to investigate the mechanism of action of dead cells on the disease itself.
종래의 사균체는 틴딜화 또는 열처리하여 사균체 제조하는 방법에 국한되어있고, 유산균에 의한 사이토카인 또는 면역반응 매개물질의 발현 증가에 대한 연구가 주로 진행되었다.Conventional dead cells are limited to methods for producing dead cells by tindylation or heat treatment, and studies on the increase in the expression of cytokines or immune response mediators by lactic acid bacteria have been mainly conducted.
또한 동물실험의 경우, 마우스 또는 쥐의 비장세포를 적출하여 비장세포 증식능 측정 및 사이토카인 분비 측정에 대한 내용이 주로 진행되었고, 살아있는 생균제의 면역학적 효과 확인을 위한 동물실험이 주를 이루고 있다.In addition, in the case of animal experiments, splenocytes were extracted from mice or mice to measure splenocyte proliferation and cytokine secretion, and animal experiments were mainly conducted to confirm the immunological effect of live probiotics.
본 발명은 상술한 바와 같은 문제점을 해결하고 필요한 기술을 제공하기 위하여 안출된 것으로서,The present invention has been devised to solve the problems as described above and provide the necessary technology,
본 발명은 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)에 pH 7 내지 8 의 1X PBS 를 첨가하여 열 처리 또는 초음파 처리하여 제조된 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체를 유효성분으로 포함하는 장면역 기능 증진용 약학적 조성물을 제공함에 그 목적이 있다.The present invention is Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that the addition of 1X PBS of
또한, 상기 사균체를 유효성분으로 포함하는 식품첨가제나, 음료수 정장제, 사료첨가제를 제공하거나, 상기 사균체의 제조방법을 제공함에 다른 목적이 있다.In addition, it is another object to provide a food additive, beverage regulating agent, feed additive containing the dead cells as an active ingredient, or to provide a method for producing the dead cells.
본 발명은 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)에 pH 7 내지 8 의 1X PBS 를 첨가하여 열 처리 또는 초음파 처리하여 제조된 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체를 유효성분으로 포함하는 장면역 기능 증진용 약학적 조성물을 제공한다.The present invention is Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that the addition of 1X PBS of
본 발명을 통해 얻어지는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 유산균의 열처리한 사균체의 리포테이코산(Lipoteichoic acid)을 지표물질로 선정하여 인체 유래 효소분해능 및 LTA 함량 변화를 확인하여 사균체 섭취 시 인체 적용 가능성 확인에 관한 것이다. 특히, 본 발명을 통해 개발된 락토바실러스 카제이 DKGF7 (Lactobacillus casei)는 김치로부터 분리된 타 균주에 비해 체내에서의 면역조절 효능이 우수하며, 사균화 시키는 과정에서 LTA가 발현율이 높아져 생균제보다 LTA 함유량이 높음을 확인하였다. 동물실험을 통하여 DKGF7의 장증상 및 장면역적 효과를 확인해 본 결과 DKGF7 (Lactobacillus casei)의 투여로 인한 스테로이드 호르몬 분비의 감소, 쥐의 결장 조직에서의 낮은 염증성 사이토카인의 수치 확인 및 tight junction에 관여하는 단백질 발현정도를 증가시킴으로써 사균체가 장증상 개선 및 장면역을 증가시킴을 확인하였다. 또한 본 발명을 통해 검증된 사균체는 약품 조성물, 음료수 정장제, 사균용 조성물, 식품첨가제 등에 다양하게 이용될 수 있다.Lactobacillus casei obtained through the present invention ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) Lipoteichoic acid of the heat-treated dead cells of the lactic acid bacteria was selected as an indicator material, It is about confirming the applicability of the human body when ingesting the cells. In particular, Lactobacillus casei DKGF7 ( Lactobacillus casei ) developed through the present invention has superior immunomodulatory efficacy in the body compared to other strains isolated from kimchi, and the LTA expression rate is increased during the killing process, so that the LTA content is higher than that of the probiotic. This high was confirmed. As a result of confirming the intestinal symptoms and intestinal effects of DKGF7 through animal experiments, the decrease in steroid hormone secretion due to the administration of DKGF7 ( Lactobacillus casei ), low levels of inflammatory cytokines in the colon tissue of mice, and By increasing the protein expression level, it was confirmed that the dead cells improved intestinal symptoms and increased the intestinal area. In addition, the dead cells verified through the present invention can be used in various ways, such as pharmaceutical compositions, beverage preparations, compositions for killing bacteria, food additives, and the like.
도 1은 60 ℃, 5, 15, 30, 60분동안 열처리한 락토바실러스 균주의 생균수를 측정한 결과이다.
도 2는 100 ℃, 5, 10분동안 열처리한 락토바실러스 균주의 생균수를 측정한 결과이다.
도 3은 400 W, 40 KHz에서 30, 60, 90분동안 음파처리한 락토바실러스 균주의 생균수를 측정한 결과이다.
도 4는 열처리 및 음파처리한 락토바실러스 균주의 리포테이코산 발현정도를 측정한 결과이다.
도 5는 열처리 및 음파처리한 락토바실러스 균주의 단백질 정량을 통한 단백질 함량을 측정한 결과이다.
도 6은 생균제의 세포생존률을 측정한 결과이다.
도 7은 사균체의 세포생존률을 측정한 결과이다.
도 8은 생균제의 NO 생성 억제 효과를 확인한 결과이다.
도 9는 사균체의 NO 생성 억제 효과를 확인한 결과이다.
도 10은 생균제를 이용한 사이토카인 (TNF-α, IL-1α, IL-6) 측정 결과이다.
도 11은 사균체를 이용한 사이토카인 (TNF-α, IL-1α, IL-6) 측정 결과이다.
도 12는 상용화된 사균체인 L.sakei를 효소처리 (α-Amylase, pepsin, 0.3% oxgall)한 후, SDS-PAGE를 통한 효소분해율을 확인한 결과이다.
도 13은 열처리한 DKGF1을 효소처리 (α-Amylase, pepsin, 0.3% oxgall)한 후, SDS-PAGE를 통한 효소분해율을 확인한 결과이다.
도 14는 열처리한 DKGF7을 효소처리 (α-Amylase, pepsin, 0.3% oxgall)한 후, SDS-PAGE를 통한 효소분해율을 확인한 결과이다.
도 15는 리포테이코산 standard를 이용하여 농도별로 HPLC 분석을 통해 확보한 직선성 결과이다.
도 16은 상용회된 사균체 L.sakei의 리포테이코산을 분석한 결과이다.
도 17은 상용회된 사균체 L.sakei를 효소분해하여 리포테이코산을 분석한 결과이다.
도 18은 사균체 DKGF1의 리포테이코산을 분석한 결과이다.
도 19는 사균체 DKGF1을 효소분해하여 리포테이코산을 분석한 결과이다.
도 20은 사균체 DKGF7의 리포테이코산을 분석한 결과이다.
도 21은 사균체 DKGF7을 효소분해하여 리포테이코산을 분석한 결과이다.
도 22는 실험 동물에 대한 연구계획을 나타낸 도면이다.
도 23은 시간의 경과에 따른 쥐의 몸무게 변화를 나타낸 도면이다.
도 24는 시간의 경과에 따른 실험군과 대조군의 분변의 굳기 변화를 나타낸 도면이다.
도 25는 시간의 경과에 따른 혈청 스테로이드 호르몬 수치 변화를 나타낸 도면이다.
도 26은 사균체 투여에 따른 염증성 사이토카인 측정 결과를 나타낸 도면이다.
도 27은 투여군과 대조군의 Claudin-1 및 ZO-1 의 발현 비교를 나타낸 도면이다.1 is a result of measuring the number of viable Lactobacillus strains subjected to heat treatment at 60° C. for 5, 15, 30, and 60 minutes.
Figure 2 is a result of measuring the number of viable cells of the Lactobacillus strain heat-treated at 100 ℃, 5, 10 minutes.
3 is a result of measuring the number of viable Lactobacillus strains subjected to sonic treatment at 400 W and 40 KHz for 30, 60, and 90 minutes.
4 is a result of measuring the expression level of lipoteichoic acid in Lactobacillus strains subjected to heat treatment and sonication.
5 is a result of measuring protein content through protein quantification of Lactobacillus strains subjected to heat treatment and sonication.
6 is a result of measuring the cell viability of a probiotic.
7 is a result of measuring the cell viability of dead cells.
8 is a result confirming the NO production inhibitory effect of the probiotic.
9 is a result confirming the NO production inhibitory effect of dead cells.
10 is a measurement result of cytokines (TNF-α, IL-1α, IL-6) using a probiotic.
11 is a measurement result of cytokines (TNF-α, IL-1α, IL-6) using dead cells.
12 is a result of confirming the enzymatic degradation rate through SDS-PAGE after enzymatic treatment (α-Amylase, pepsin, 0.3% oxgall) of commercialized dead cell L.sakei.
13 is a result of confirming the enzymatic degradation rate through SDS-PAGE after enzymatic treatment (α-Amylase, pepsin, 0.3% oxgall) of heat-treated DKGF1.
14 is a result of confirming the enzymatic degradation rate through SDS-PAGE after enzymatic treatment (α-Amylase, pepsin, 0.3% oxgall) of heat-treated DKGF7.
15 is a linearity result obtained through HPLC analysis for each concentration using a lipoteichoic acid standard.
16 is a result of analysis of lipoteichoic acid of L.sakei, a dead cell that has been commercialized.
17 is a result of analysis of lipoteichoic acid by enzymatic digestion of the commercialized dead cell L.sakei.
18 is a result of analyzing lipoteichoic acid of dead cell DKGF1.
19 is a result of analyzing lipoteichoic acid by enzymatic digestion of dead cell DKGF1.
20 is a result of analyzing lipoteichoic acid of dead cell DKGF7.
21 is a result of analyzing lipoteichoic acid by enzymatic digestion of dead cell DKGF7.
22 is a view showing a research plan for an experimental animal.
23 is a view showing the change in the weight of the rat over time.
24 is a view showing changes in the hardness of feces of the experimental group and the control group with the lapse of time.
25 is a diagram showing changes in serum steroid hormone levels over time.
26 is a view showing the measurement results of inflammatory cytokines according to the administration of dead cells.
27 is a view showing a comparison of the expression of Claudin-1 and ZO-1 in the administration group and the control group.
이하, 본 발명에 대해 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면에 따르면, 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)에 pH 7 내지 8 의 1X PBS 를 첨가하여 열 처리 또는 초음파 처리하여 제조된 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체를 유효성분으로 포함하는 장면역 기능 증진용 약학적 조성물을 제공한다.According to one aspect of the present invention, Lactobacillus casei ( Lactobacillus casei ) Lactobacillus casei ( Lactobacillus casei ), characterized in that prepared by heat treatment or sonication by adding 1X PBS of
구체적으로는, 상기 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)는, 10 mL MRS 배지에서 37 ℃ 온도 조건으로 18시간 배양한 뒤, 100mL MRS 배지에서 37 ℃ 온도 조건으로 18시간동안 배양된 것일 수 있다.Specifically, the Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) was cultured in 10 mL MRS medium at 37 ° C for 18 hours, and then in 100 mL MRS medium at 37 ° C for 18 hours. it could be
한편, 상기 열 처리는, 60 ℃ 이상의 조건에서 30분 이상 진행되는 것일 수 있으며, 바람직하게는 60 ℃ 이상의 온도에서 30분 내지 60분동안 진행되는 것일 수 있으며, 보다 바람직하게는, 60 ℃ 의 온도에서 30분 동안 진행되는 것일 수 있다.On the other hand, the heat treatment may be carried out at a temperature of 60 ° C or higher for 30 minutes or more, preferably at a temperature of 60 ° C or higher for 30 to 60 minutes, more preferably, at a temperature of 60 ° C. may be carried out for 30 minutes.
아울러, 상기 초음파 처리는, 초음파 출력 400 W 이상, 주파수 40kHz 에서 90분 이상 진행되는 것일 수 있으며, 보다 바람직하게는, 400 W, 주파수 40kHZ 에서 90분 동안 진행되는 것일 수 있다.In addition, the ultrasonic treatment may be performed for 90 minutes or more at an ultrasonic output of 400 W or more and a frequency of 40 kHz, and more preferably, may be performed for 90 minutes at 400 W and a frequency of 40 kHz.
이러한 장면역 기능 증진용 약학적 조성물은, 구체적으로는 장증상의 개선이나 장면역 개선 효과를 나타내는 것일 수 있으며, 보다 구체적으로는 과민성 대장 증후군(IBS)에 대한 개선 효과를 나타내는 것일 수 있다.The pharmaceutical composition for enhancing intestinal function may specifically exhibit an effect of improving intestinal symptoms or improving intestinal function, and more specifically, may exhibit an improving effect on irritable bowel syndrome (IBS).
본 발명의 또 다른 실시예에 따르면, 상기 장면역 기능 증진용 약학적 조성물 이외에도, 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)에 pH 7 내지 8 의 1X PBS 를 첨가하여 열 처리 또는 초음파 처리하여 제조된 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체를 유효성분으로 포함하는 장면역 기능 증진용 식품 첨가제를 제공할 수 있으며, 음료수 정장제, 사료 첨가제 등으로도 제공할 수 있다.According to another embodiment of the present invention, in addition to the pharmaceutical composition for enhancing intestinal function, Lactobacillus casei DKGF7 (KCTC 16246BP) by adding 1X PBS of
한편, 본 발명의 또 다른 실시예에 따르면,On the other hand, according to another embodiment of the present invention,
a) 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)를 10 mL MRS 배지에서 37 ℃ 온도 조건으로 18시간 배양한 뒤, 100mL MRS 배지에서 37 ℃ 온도 조건으로 18시간동안 배양하는 단계;a) Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) in 10 mL MRS medium at 37 ° C. for 18 hours, followed by culturing for 18 hours at 37 ° C. in 100 mL MRS medium;
b) 상기 배양액을 원심분리하여 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 균체를 회수하고, pH 7 내지 8 의 1X PBS 를 첨가하는 단계; 및b) centrifuging the culture solution to recover Lactobacillus casei DKGF7 (KCTC 16246BP) cells, and adding 1X PBS of
c) 열 처리 또는 초음파 처리하는 단계;c) heat treatment or sonication;
를 포함하는 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체의 제조 방법을 제공한다.Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) characterized in that it comprises a method for producing dead cells.
상기 열 처리 온도는 앞서 기술한 바와 같이, 60 ℃ 이상의 조건에서 30분 이상 열 처리하는 것일 수 있으며, 상기 초음파 처리는 초음파 출력 400 W, 주파수 40kHz 에서 90 분 이상 초음파 처리하는 것일 수 있다.As described above, the heat treatment temperature may be heat treatment at 60° C. or higher for 30 minutes or longer, and the ultrasonic treatment may be ultrasonic treatment at an ultrasonic power output of 400 W and a frequency of 40 kHz for 90 minutes or longer.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예 들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리 범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, in the description with reference to the accompanying drawings, the same components are assigned the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In the description of the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.
또한, 실시예의 구성 요소를 설명하는 데 있어서, 제 1, 제 2, A, B, (a), (b) 등의 용어를 사용할 수 있다. 이러한 용어는 그 구성 요소를 다른 구성 요소와 구별하기 위한 것일 뿐, 그 용어에 의해 해당 구성 요소의 본질이나 차례 또는 순서 등이 한정되지 않는다.In addition, in describing the components of the embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms.
어느 하나의 실시예에 포함된 구성요소와, 공통적인 기능을 포함하는 구성요소는, 다른 실시예에서 동일한 명칭을 사용하여 설명하기로 한다. 반대되는 기재가 없는 이상, 어느 하나의 실시예에 기재한 설명은 다른 실시 예에도 적용될 수 있으며, 중복되는 범위에서 구체적인 설명은 생략하기로 한다.Components included in one embodiment and components having a common function will be described using the same names in other embodiments. Unless otherwise stated, descriptions described in one embodiment may be applied to other embodiments as well, and detailed descriptions within the overlapping range will be omitted.
실시예 1 : 사균체 개발을 위한 락토바실러스 균주 전처리공정 시스템 구축Example 1: Establishment of Lactobacillus strain pretreatment process system for dead cell development
실시예 1-1 : 락토바실러스 균주의 사균체 개발을 위한 전처리 공정 확립Example 1-1: Establishment of pretreatment process for the development of dead cells of Lactobacillus strains
락토바실러스 파라카제이 DKGF1 및 락토바실러스 카제이 DKGF7의 사균화를 위하여 10 mL MRS 배지에서 37 ℃ 온도 조건으로 18시간 배양한 뒤, 100mL MRS 배지에서 37 ℃ 온도 조건으로 18시간동안 배양하였다.In order to kill Lactobacillus paracasei DKGF1 and Lactobacillus casei DKGF7, the cells were cultured for 18 hours in 10 mL MRS medium at 37 ° C., and then incubated in 100 mL MRS medium at 37 ° C. for 18 hours.
이후 원심분리하여 균체 회수 후 1X PBS (pH 7.4)를 첨가한 뒤, 열 처리 및 초음파 처리하여 처리 시간에 따른 생균수를 확인하였다. Then, after centrifugation to recover the cells, 1X PBS (pH 7.4) was added, and the number of viable cells according to the treatment time was confirmed by heat treatment and sonication.
그 결과, 도 1, 2 및 3 에서와 같이 60 ℃에서는 30분 이상, 100 ℃에서는 5분 이상의 조건에서 균이 사멸됨을 확인하였다. 또한 초음파 출력 400 W, 주파수 40 kHz에서 sonication 처리하여 생균수 측정한 결과 90분 이상 열처리 진행 후 균이 사멸됨을 확인하였다.As a result, as shown in FIGS. 1, 2 and 3, it was confirmed that the bacteria were killed at 60 °C for 30 minutes or longer and at 100 °C for 5 minutes or longer. In addition, it was confirmed that the results of the measurement of the number of viable cells by the ultrasonic treatment at 400 W, the frequency at 40 kHz and the frequency at 40 kHz kills the bacteria after heat treatment for more than 90 minutes.
실시예 1-2 : 사균체 지표물질인 세포막 당단백질 Lipoteichoic acid(리포테이코산) 분석Example 1-2: Analysis of cell membrane glycoprotein lipoteichoic acid (lipoteichoic acid) as a dead cell indicator material
General lipoteichoic acid ELISA kit (Mybiosource, USA)를 사용하여 lipoteichoic acid(리포테이코산) 분석을 진행하였다.The analysis of lipoteichoic acid (lipoteichoic acid) was performed using the General lipoteichoic acid ELISA kit (Mybiosource, USA).
락토바실러스 균주를 heat shock (60 ℃ 5, 15, 30, 60 min, 100 ℃ 5, 10 min) 및 sonication (400 W, 40 kHz 30, 60, 90 min) 처리하여 13,000 rpm, 10 min, 4 ℃에서 원심 분리하여 상등액을 취한 뒤 sample로 사용하였다.Lactobacillus strains were treated with heat shock (60
Sample과 detection reagent A를 각 well에 50 uL 분주한 뒤 37 ℃에 1시간 배양한 뒤 wash buffer로 총 3회 세척하고 Detection reagent B 100 ㎕ 분주하여 37 ℃에 45분 배양 후 wash buffer로 총 5회 세척하였다.Dispense 50 uL of Sample and detection reagent A into each well, incubate at 37 °C for 1 hour, wash 3 times with wash buffer, dispense 100 µl of Detection reagent B, incubate at 37 °C for 45 minutes, and use wash buffer for a total of 5 times washed.
세척 후 substrate (TMB 기질 용액) 90 ㎕ 분주하여 효소-기질 반응 시작을 위해 37 ℃에 10-20분 배양 후, stop solution(황산 용액) 50 ㎕ 첨가하여 효소-기질 반응 종료하고 micro plate reader기로 450 nm 에서 흡광도를 측정하였다.After washing, dispense 90 μl of substrate (TMB substrate solution) and incubate at 37°C for 10-20 minutes to start the enzyme-substrate reaction, then add 50 μl of stop solution (sulfuric acid solution) to terminate the enzyme-substrate reaction and 450 with a micro plate reader. The absorbance was measured in nm.
그 결과는 도 4 에 나타난 바와 같이 각 온도에서의 열 처리 및 초음파 처리 시간이 길어진수록 LTA 발현율이 높아짐을 확인하였다.As a result, as shown in FIG. 4 , it was confirmed that the LTA expression rate increased as the heat treatment and sonication times at each temperature increased.
초음파의 경우 400 W, 40 kHz에서 약 90분 이상 처리 시 균이 사멸됨을 확인하였는데, 음파처리를 통한 처리는 열처리와 비교하였을 때 균의 사멸을 위한 처리 시간이 길어 pilot scale로 사균체 제작 시 경제성이 떨어져 사균체 전처리 공정은 heat shock (열처리)으로 최종 선발하였다.In the case of ultrasound, it was confirmed that the bacteria were killed when treated at 400 W and 40 kHz for more than about 90 minutes. Compared with heat treatment, the treatment with sonication takes a long time to kill the bacteria, so it is economical to produce dead cells on a pilot scale. Due to this, the dead cell pretreatment process was finally selected by heat shock (heat treatment).
실시예 1-3 : 사균체 열처리 조건 확립을 위한 단백질 함량 측정Example 1-3: Protein content measurement for establishing heat treatment conditions for dead cells
사균체의 온도 및 처리 시간별 단백질 함량을 확인하기 위하여 Total protein assay kit (Abnova, Taiwan)를 사용하여 단백질 정량을 진행하였다.Protein quantification was performed using a total protein assay kit (Abnova, Taiwan) to check the protein content by temperature and treatment time of dead cells.
Heat shock (60 ℃ 5, 15, 30, 60 min / 100 ℃ 5, 10 min) 처리 후 13,000 rpm, 10 min, 4 ℃에서 원심 분리하여 상등액을 취한 뒤 sample로 사용하였다.After heat shock (60
96-well culture plate에 Sample 및 distilled water를 각각 5 ㎕ 분주한 뒤 reagent 200 ㎕ 혼합하여 상온에서 10분동안 반응 후 micro plate reader기로 600 nm에서 흡광도를 측정하였다.After dispensing 5 μl of each sample and distilled water in a 96-well culture plate, 200 μl of the reagent was mixed and reacted at room temperature for 10 minutes, and then absorbance was measured at 600 nm with a micro plate reader.
단백질 함량 측정 결과는 도 5와 같다. Heat shock (60 ℃ 15, 30, 60 min / 100 ℃ 5, 10 min) 처리하여 단백질 정량한 결과, lipoteichoic acid 발현율이 높았던 100 ℃ 에서 열처리한 사균체의 단백질 함량이 60 ℃ 에서보다 낮음을 확인하였다.The protein content measurement result is shown in FIG. 5 . As a result of protein quantification by heat shock (60
이는 100 ℃ 에서 단백질이 degradation되어 단백질 함량이 줄었음을 의미하기에 단백질 함량이 가장 높은 60 ℃, 60 min을 사균체 열처리 조건으로 최종 선발하였고, 도 4 와 도 5 에서 LTA 발현율 및 단백질 함량이 높은 DKGF1 및 DKGF7을 사균체 균주로 최종 선발하였다.This means that the protein was degraded at 100 ℃ and the protein content was reduced, so 60 ℃, 60 min, which had the highest protein content, was finally selected as the dead cell heat treatment condition, and LTA expression rate and protein content were high in FIGS. DKGF1 and DKGF7 were finally selected as dead cell strains.
실시예 2 : 선발 사균체의 면역효과 확인Example 2: Confirmation of immune effect of selected dead cells
실시예 2-1 : MTT assay를 통한 세포생존률 확인Example 2-1: Confirmation of cell viability through MTT assay
세포생존률 확인을 위한 실험에는 단국대학교에서 분양받은 RAW 264.7 macrophage cell (Cheonan, Korea)을 이용하였으며 10% heat-inactivated fetal bovine serum (FBS)이 혼합된 Dulbecco's Modified Eagle Medium (DMEM) 배지에서 배양하여 실험에 사용하였다.For the experiment to check the cell viability, RAW 264.7 macrophage cells (Cheonan, Korea) purchased from Dankook University were used and cultured in Dulbecco's Modified Eagle Medium (DMEM) medium mixed with 10% heat-inactivated fetal bovine serum (FBS). was used for
75 T cell culture flask에 배양된 RAW 264.7 cell을 1,000 rpm, 5 min의 조건에서 원심분리 후 상등액 제거하여 동량의 10% FBS+DMEM 배지 첨가 후 96-well plate에 5 × 104 수준의 cell 접종하여 37 ℃, 5% CO2 incubator에 16-18 hr 배양하였다.After centrifuging the RAW 264.7 cells cultured in a 75 T cell culture flask at 1,000 rpm and 5 min, the supernatant was removed, the same amount of 10% FBS+DMEM medium was added, and then 5 × 10 4 cells were inoculated into a 96-well plate. 37° C., 5% CO 2 Incubated for 16-18 hr in an incubator.
배지 제거 후, 유산균 sample을 생균 (1010-105 CFU/g), 사균 (1010-105 CFU/g)을 농도별로 준비하여 100 ㎕ 분주 후 20-22 h 배양한 뒤, 상등액을 제거하여 5 mg/mL 농도의 MTT solution을 분주하였다.After removing the medium, prepare lactic acid bacteria samples for each concentration of live (10 10 -10 5 CFU/g) and dead cells (10 10 -10 5 CFU/g), dispense 100 μl, incubate for 20-22 h, and remove the supernatant. Thus, a 5 mg/mL MTT solution was dispensed.
37 ℃, 5% CO2 incubator에 3 hr 배양 후 상등액을 제거한 뒤 DMSO 100 ㎕ 첨가하여 상온에서 5분 반응하여 micro plate reader기로 540 nm에서 흡광도를 측정하였다.After 3 hr incubation at 37 ° C., 5% CO 2 in an incubator, the supernatant was removed, followed by addition of 100 μl of DMSO, followed by reaction at room temperature for 5 minutes, and absorbance was measured at 540 nm with a micro plate reader.
Tetrazolium-based colorimetric(MTT) 시험법은 많은 시료를 쉽게 빠르게 판독할 수 있어 배양된 세포에서 세포 독성에 관한 연구에 주로 사용되는 방법이다.Tetrazolium-based colorimetric (MTT) test method is mainly used for cytotoxicity studies in cultured cells because it can read many samples easily and quickly.
이 방법은 대사 과정이 온전한 세포의 미토콘드리아 내의 탈수소효소가 노란색 수용성 MTT를 비수용성의 짙은 자주색 MTT formazan 결정으로 환원시키는 원리를 이용함으로써 적절한 파장인 540 nm에서 흡광도를 측정하여 세포독성을 평가할 수 있다.This method uses the principle that the dehydrogenase in the mitochondria of the metabolically intact cell reduces yellow soluble MTT to insoluble dark purple MTT formazan crystals.
생균제 및 사균체의 MTT 분석을 통한 세포 독성 시험 결과, 도 6, 7 에 나타낸 바와 같이, 생균제는 109 cfu/mL(도 6), 사균체는 1010 cfu/mL(도 7)의 농도까지 80% 이상의 생존률 보임으로써 선발 사균체의 세포독성이 거의 없는 것을 확인하였다.As a result of cytotoxicity test through MTT analysis of probiotics and dead cells, as shown in FIGS. 6 and 7 , probiotics up to a concentration of 10 9 cfu/mL (FIG. 6), and dead cells up to a concentration of 10 10 cfu/mL (FIG. 7) By showing a survival rate of 80% or more, it was confirmed that there was almost no cytotoxicity of the selected dead cells.
(DKGF1 = L.casei DKGF1; DKGF7 = L.casei DKGF7; DKGF8 = L.casei DKGF8; DK211 = L.casei DK211)(DKGF1 = L.casei DKGF1; DKGF7 = L.casei DKGF7; DKGF8 = L.casei DKGF8; DK211 = L.casei DK211)
실시예 2-2 : NO assay를 통한 산화질소 생성 억제 효과 확인Example 2-2: Confirmation of nitric oxide production inhibitory effect through NO assay
세포생존률 확인을 위한 실험에는 단국대학교에서 분양받은 RAW 264.7 macrophage cell (Cheonan, Korea)을 이용하였으며 10% heat-inactivated fetal bovine serum (FBS)이 혼합된 Dulbecco's Modified Eagle Medium (DMEM) 배지에서 배양하여 실험에 사용하였다.For the experiment to check the cell viability, RAW 264.7 macrophage cells (Cheonan, Korea) purchased from Dankook University were used and cultured in Dulbecco's Modified Eagle Medium (DMEM) medium mixed with 10% heat-inactivated fetal bovine serum (FBS). was used for
75 T cell culture flask에 배양된 RAW 264.7 cell을 1,000 rpm, 5 min의 조건에서 원심분리 후 상등액을 제거하여 동량의 10% FBS+DMEM 배지 첨가 후 96-well plate에 5 × 104 수준의 cell 접종하여 37 ℃, 5% CO2 incubator에 16-18 hr 배양하였다.After centrifuging the RAW 264.7 cells cultured in a 75 T cell culture flask at 1,000 rpm and 5 min, the supernatant was removed, the same amount of 10% FBS+DMEM medium was added, and then inoculated with 5 × 10 4 cells in a 96-well plate. and incubated for 16-18 hr in a 37 ℃, 5% CO 2 incubator.
배지 제거 후, 유산균 sample을 생균 (1010-105 CFU/g), 사균 (1010-105 CFU/g)을 농도별로 준비하여 100 ㎕ 분주 후 37 ℃, 5% CO2 incubator에 2-3 hr 배양하였다.After removing the medium, prepare lactic acid bacteria samples for each concentration of live cells (10 10 -10 5 CFU/g) and dead cells (10 10 -10 5 CFU/g), dispense 100 μl, and place 2- Incubated for 3 hr.
최종농도 100 ng/mL의 LPS를 제조하여 100 ㎕ 분주 후 37 ℃, 5% CO2 incubator에 20-22 hr 배양한 뒤, NO 시약(Griess reagent) 100 ㎕ 분주 후 상온에서 15분간 반응하여 micro plate reader기로 540 nm 에서 흡광도를 측정하였다.After preparing LPS with a final concentration of 100 ng/mL, dispensing 100 μl, incubating for 20-22 hr in an incubator at 37° C., 5% CO 2 , dispensing 100 μl of NO reagent (Griess reagent), and reacting for 15 minutes at room temperature to micro plate Absorbance was measured at 540 nm with a reader.
NO (Nitric oxide)는 혈액응고 및 혈압조절 가능, 암세포에 대한 면역 기능 등에 중요한 역할을 하지만 산화되어 활성 NO로 변화되며, 단백질 및 지질의 과산화를 유도하고 세포독성을 야기하는 강력한 산화제인 peroxynitrite를 생산한다.NO (Nitric oxide) plays an important role in blood coagulation, blood pressure regulation, and immune function against cancer cells, but it is oxidized and converted to active NO, and produces peroxynitrite, a powerful oxidizing agent that induces peroxidation of proteins and lipids and causes cytotoxicity. do.
염증성 매개 물질에 노출된 세포에 의해서 생성된 NO의 증가는 NO가 생체 방어와 자유기에 의해 유발된 조직 손상에 기여한다고 알려져 있으며 다양한 염증성 질환 시 증가된다.The increase in NO produced by cells exposed to inflammatory mediators is known to contribute to biodefense and tissue damage induced by free radicals, and it is increased in various inflammatory diseases.
생균제와 사균체의 RAW 264.7 cell로부터 생성된 NO의 양 측정 결과, 도 8, 9 에 나타낸 바와 같이 생균제는 105-109 cfu/mL에서(도 8), 사균체는 모든 농도(도 9)에서 NO 생성 억제 효과를 보임으로써 생균제와 비교하였을 때 사균체가 NO 생성이 억제되는 것을 확인할 수 있었다.As a result of measuring the amount of NO produced from RAW 264.7 cells of probiotics and dead cells, as shown in FIGS. 8 and 9, probiotics were at 10 5 -10 9 cfu/mL (FIG. 8), and dead cells were at all concentrations (FIG. 9) By showing the NO production inhibitory effect, it was confirmed that the dead cells suppressed NO production when compared with the probiotic.
실시예 2-3 : 사이토카인 분비 억제 확인 (TNF-α, IL-1α, IL-6)Example 2-3: Cytokine secretion inhibition confirmation (TNF-α, IL-1α, IL-6)
세포생존률 및 NO 생성 억제 효과 측정을 통해 생균제는 109 cfu/mL, 사균체는 1010 cfu/mL의 농도로 사이토카인 측정 진행하였다. 사이토카인 측정 (TNF-α, IL-1α, IL-6)은 Mouse IL-1α, Mouse IL-6, Mouse TNF-α ELISA kit (KOMA biotech, Korea)를 사용하여 진행하였다.Cytokines were measured at a concentration of 10 9 cfu/mL for probiotics and 10 10 cfu/mL for dead cells by measuring the cell viability and NO production inhibitory effect. Cytokine measurements (TNF-α, IL-1α, IL-6) were performed using Mouse IL-1α, Mouse IL-6, and Mouse TNF-α ELISA kit (KOMA biotech, Korea).
세포생존률 확인을 위한 실험에는 단국대학교에서 분양받은 RAW 264.7 macrophage cell (Cheonan, Korea)을 이용하였으며 10% heat-inactivated fetal bovine serum (FBS)이 혼합된 Dulbecco's Modified Eagle Medium (DMEM) 배지에서 배양하여 실험에 사용하였다.For the experiment to check the cell viability, RAW 264.7 macrophage cells (Cheonan, Korea) purchased from Dankook University were used and cultured in Dulbecco's Modified Eagle Medium (DMEM) medium mixed with 10% heat-inactivated fetal bovine serum (FBS). was used for
75 T cell culture flask에 배양된 RAW 264.7 cell을 1,000 rpm, 5 min의 조건에서 원심분리 후 상등액을 제거하여 동량의 10% FBS+DMEM 배지 첨가 후 96-well plate에 5 × 104 수준의 cell 접종하여 37 ℃, 5% CO2 incubator에 24 hr 배양하였다.After centrifuging the RAW 264.7 cells cultured in a 75 T cell culture flask at 1,000 rpm and 5 min, the supernatant was removed, the same amount of 10% FBS+DMEM medium was added, and then inoculated with 5 × 10 4 cells in a 96-well plate. and incubated for 24 hr in a 37 °C, 5% CO 2 incubator.
항체가 코팅되어있는 96-well ELISA microplate에 300 ㎕의 washing solution을 첨가한 뒤 세척하는 과정을 3회 진행하였다. Standard 및 sample을 각 well에 100 ㎕ 첨가한 뒤 상온에서 2시간 반응한 뒤 washing solution으로 세척 4회 진행하였다.After adding 300 μl of washing solution to the antibody-coated 96-well ELISA microplate, the washing process was performed 3 times. After adding 100 μl of standard and sample to each well, reaction was performed at room temperature for 2 hours, followed by washing with
Detection antibody를 각 well에 100 ㎕ 첨가한 뒤 상온에서 2시간 반응 후, washing solution으로 세척 4회 진행하였다. Streptavidin-HRP를 각 well에 100 ㎕ 첨가한 뒤 상온에서 30분 반응한 뒤 washing solution으로 세척 4회 진행하였다.100 μl of detection antibody was added to each well, and after 2 hours of reaction at room temperature, washing was performed 4 times with washing solution. After adding 100 μl of Streptavidin-HRP to each well, the reaction was performed at room temperature for 30 minutes, followed by washing with
TMB solution을 각 well에 100 ㎕ 첨가하여 상온에서 발색 반응 확인 후 발색이 충분히 진행되면 stop solution을 각 well에 100 ㎕ 첨가하여 발색 반응 종료시킨 뒤, micro plate reader기를 이용하여 450 nm 에서 흡광도를 측정하였다.100 μl of TMB solution was added to each well to confirm color development at room temperature, and when color development was sufficiently progressed, 100 μl of stop solution was added to each well to terminate the color reaction, and absorbance was measured at 450 nm using a micro plate reader. .
면역 균형을 유지하기 위해선 면역 세포의 직접 또는 간접적 상호작용이 있어야한다. 사이토카인은 다양한 면역 세포의 증식, 분화 및 기능과 활동의 변화를 유도할 수 있다. 질병은 대부분 염증과 관련이 있으며, 염증세포는 염증을 유발하는 염증성 사이토카인을 분비한다.To maintain the immune balance, there must be a direct or indirect interaction of immune cells. Cytokines can induce proliferation, differentiation, and changes in function and activity of various immune cells. Most diseases are related to inflammation, and inflammatory cells secrete inflammatory cytokines that cause inflammation.
ELISA를 이용하여 사이토카인 (TNF-α, IL-1α, IL-6) 측정 결과는 도 10, 11과 같다. 세가지 사이토카인 (TNF-α, IL-1α, IL-6) 생산이 LPS(+)군보다 유의하게 낮음을 확인할 수 있었다. 또한 생균제(도 10)와 비교하였을 때 사균체(도 11)의 사이토카인 분비가 억제됨을 확인할 수 있었다. 이 결과를 바탕으로 사균체에서 항염증 효과의 가능성을 확인할 수 있었다.Cytokines (TNF-α, IL-1α, IL-6) measurement results using ELISA are shown in FIGS. 10 and 11 . It was confirmed that the production of three cytokines (TNF-α, IL-1α, IL-6) was significantly lower than that of the LPS(+) group. In addition, it was confirmed that the cytokine secretion of dead cells (FIG. 11) was inhibited when compared with the probiotic (FIG. 10). Based on this result, it was possible to confirm the possibility of anti-inflammatory effect in dead cells.
실시예 3 : 사균체의 상용화 적용성 확인을 위한 인체 유래 효소분해율 검증Example 3: Verification of human-derived enzyme degradation rate for confirming commercial applicability of dead cells
사균체 sample을 동결건조하여 최종 농도 1 Х 1011으로 설정하여 인체 유래 효소분해율 검증을 위해 체내 소화관 조건과 유사한 환경에서 진행하였다.The dead cell sample was freeze-dried and set to a final concentration of 1
동결건조된 sample 4 g과 D.W 36 mL 혼합한 뒤, 10%의 α-Amylase (Novozymes, Denmark) 첨가하여 37 ℃에서 5, 15, 30 min 반응 후 SDS-PAGE를 이용한 분해율을 측정하였다.After mixing 4 g of lyophilized sample with 36 mL of D.W, 10% of α-Amylase (Novozymes, Denmark) was added, and reaction was performed at 37 ° C. for 5, 15, 30 min. The degradation rate was measured using SDS-PAGE.
α-Amylase를 첨가한 sample 조건별 처리 후, HCl로 보정하여 만든 pH 1.5, 3.0의 pepsin (1000 unit/mL) 첨가하여 37 ℃에서 조건별 (30, 60, 120 min) 반응 후 SDS-PAGE를 이용한 분해율을 측정하였다.After treatment for each condition in which α-Amylase was added, pepsin (1000 unit/mL) at pH 1.5 and 3.0 prepared by correction with HCl was added, and reaction was performed at 37 ° C for each condition (30, 60, 120 min), followed by SDS-PAGE. The decomposition rate used was measured.
Pepsin 첨가한 인공위액을 조건별 처리한 뒤, 원심분리하여 상등액 제거 후 0.3% Oxgall 첨가하여 37 ℃ 6, 12, 24 hrs 반응 후 SDS-PAGE를 이용한 분해율을 측정하였다.Pepsin-added artificial gastric juice was treated for each condition, centrifuged to remove the supernatant, 0.3% Oxgall was added, and the degradation rate was measured using SDS-PAGE after reaction at 37°C for 6, 12, and 24 hrs.
그 결과는 도 12, 13, 14에 나타난바와 같다.The results are as shown in FIGS. 12, 13 and 14 .
10% α-Amylase 처리 후, 조건별(5, 15, 30 min)로 SDS-PAGE를 통한 분해율 측정 결과 모든 조건에서 소화효소에 의해 사균체 분해가 없는 것으로 확인하였으며, 사람의 소화 과정 중 저작이 발생하는 시간은 5분 내외이므로 α-Amylase 처리 시간을 5 min으로 설정하였다.After 10% α-Amylase treatment, as a result of measuring the degradation rate through SDS-PAGE for each condition (5, 15, 30 min), it was confirmed that there was no decomposition of dead cells by digestive enzymes under all conditions. Since the generation time is about 5 minutes, the α-Amylase treatment time was set to 5 min.
Pepsin (pH 1.5, 3.0) 처리 후 조건별 (30, 60, 120 min)로 SDS-PAGE를 이용한 분해율 측정 결과 소화효소에 의해 pH 3.0, 30 min에서 사균체 분해가 없는 것으로 확인하였으며, 사람의 소화 과정 중 위를 통과하는 시간은 30분 내외이므로 pepsin 처리 시간을 30 min으로 설정하였다.After treatment with Pepsin (pH 1.5, 3.0), the degradation rate was measured using SDS-PAGE for each condition (30, 60, 120 min). As a result, it was confirmed that there was no decomposition of dead cells at pH 3.0, 30 min by digestive enzymes. The time to pass through the stomach during the process was about 30 minutes, so the pepsin treatment time was set to 30 min.
0.3% oxgall 처리 후 조건별 (6, 12, 24 hrs)로 SDS-PAGE를 이용한 분해율 측정 결과 모든 조건에서 소화효소에 의한 사균체 분해가 없는 것으로 확인하였으며, 사람의 소화 과정 중 췌장을 통과하는 시간은 6시간 내외이므로 oxgall 처리시간을 6시간으로 설정하였다.As a result of measuring the degradation rate using SDS-PAGE for each condition (6, 12, 24 hrs) after 0.3% oxgall treatment, it was confirmed that there was no decomposition of dead cells by digestive enzymes under all conditions, and the time passing through the pancreas during human digestion is about 6 hours, so the oxgall treatment time was set to 6 hours.
실시예 4 : 선발 사균체의 Lipoteichoic acid 분석을 위한 HPLC (High Performance Liquid Chromatography) 측정Example 4: High Performance Liquid Chromatography (HPLC) measurement for lipoteichoic acid analysis of selected dead cells
사균체의 Lipoteichoic acid 추출을 위하여 1011수준으로 농축된 사균체 20 mL를 0.1 M sodium citrate (pH 4.7) (Sigma, USA)로 세척한 뒤, 2 mL의 0.1 M sodium citrate (pH 4.7)로 재현탁한 뒤 400 W, 5 min 조건으로 sonication 진행하였다.For lipoteichoic acid extraction of dead cells, 20 mL of dead cells concentrated to the level of 10 11 were washed with 0.1 M sodium citrate (pH 4.7) (Sigma, USA), and then reproduced with 2 mL of 0.1 M sodium citrate (pH 4.7). After turbidity, sonication was performed under 400 W, 5 min conditions.
현탁액을 17,000 g, 20 min의 조건으로 원심분리한 뒤, 상청액을 제거하여 0.1 M sodium citrate 1 mL과 1-Butanol(Sigma, USA) 1 mL에 현탁하여 현탁액을 실온에서 30분간 shaking 후, 원심분리하여 상등액을 sample로 사용하였다.The suspension was centrifuged at 17,000 g, 20 min, and the supernatant was removed, suspended in 1 mL of 0.1 M sodium citrate and 1 mL of 1-Butanol (Sigma, USA), the suspension was shaken at room temperature for 30 minutes, and centrifuged. Thus, the supernatant was used as a sample.
Sample을 rotary evaporator로 증발시킨 뒤, 10 mL의 distilled water와 혼합하여 동결건조 진행하고 동결건조된 LTA sample을 1 mL의 0.1 M Ammonium acetate (pH 4.7) : 1-Propanol (Sigma, USA) = 5.5 : 4.5 (v/v)과 혼합하여 Symmetry® C8 column상에서 HPLC 분석하였다. LTA 분석을 위한 HPLC 조건은 표 1 에 나타내었다.After evaporating the sample with a rotary evaporator, it was mixed with 10 mL of distilled water and freeze-dried. The lyophilized LTA sample was subjected to 1 mL of 0.1 M Ammonium acetate (pH 4.7): 1-Propanol (Sigma, USA) = 5.5: Mixed with 4.5 (v/v) and analyzed by HPLC on a Symmetry® C8 column. HPLC conditions for LTA analysis are shown in Table 1.
결과는 도 15, 16, 17, 18, 19, 20, 21에 나타난 바와 같이 사균체의 LTA 추출하여 HPLC 분석하였을때 R2 = 0.998을 통하여 직선성을 확보하였다.As shown in FIGS. 15, 16, 17, 18, 19, 20, and 21, when LTA extraction of dead cells was analyzed by HPLC, linearity was secured through R 2 = 0.998.
LTA 분석을 위한 HPLC 분석 결과, L.sakei - 2920 ppm, DKGF1 - 3587 ppm, DKGF7 - 4711 ppm으로 DKGF7의 LTA 함유량이 가장 많음을 확인하였다.As a result of HPLC analysis for LTA analysis, L.sakei - 2920 ppm, DKGF1 - 3587 ppm, DKGF7 - 4711 ppm, confirming that DKGF7 had the highest LTA content.
또한 효소분해한 L.sakei - 2346 ppm, DKGF1 - 3305 ppm, DKGF7 - 4212 ppm으로 DKGF7의 LTA 함유량이 가장 많음을 확인하였다.In addition, it was confirmed that the LTA content of DKGF7 was the highest with enzyme-decomposed L.sakei - 2346 ppm, DKGF1 - 3305 ppm, and DKGF7 - 4212 ppm.
실시예 5 : 과민성대장증후군 동물모델을 이용한 사균체 효능평가Example 5: Efficacy evaluation of dead cells using an animal model for irritable bowel syndrome
실시예 5-1 : 실험동물 사육, 실험군 설정 및 처리Example 5-1: breeding of experimental animals, experimental group setting and treatment
본 연구에 사용된 동물은 8주령된 수컷 (체중 - 304.7 ± 1.4 g) 쥐를 ㈜오리엔탈 바이오로부터 분양 받아 일정한 온도와 명암주기 (light and dark cycle)는 12시간 단위로 조절하여 동일한 사료와 물을 자유로이 공급하며 7일동안 무균환경에서 성장시켰다. 쥐를 임의 배치법에 의해 대조군과 투여군으로 나눴으며, 실험군마다 7마리씩 사용하였다. 대조군 및 투여군은 모두 말토덱스트린을 경구 투여하였고, 투여군에는 단국대학교에서 자체 개발한 김치 유래 유산균을 ㈜지에프퍼멘텍에서 고농도 배양 및 동결건조 진행하여 공급받은 락토바실러스 파라카제이 DKGF1 및 락토바실러스 카제이 DKGF7의 사균체 분말을 각각 1 x 1011 cfu 수준의 500 ㎕로 28일간 경구투여하였다. 모든 쥐는 실험 4주동안 매일 2시간 몸에 밀착된 플라스틱 케이지를 이용하여 고정되었다. 연구 설계에 대한 계획은 도 22 에 나타낸 바와 같다.The animals used in this study were 8-week-old male (weight - 304.7 ± 1.4 g) rats from Oriental Bio Co., Ltd. It was supplied freely and grown in an aseptic environment for 7 days. The rats were divided into a control group and an administration group by random placement method, and 7 mice were used in each experimental group. Both the control group and the administration group were orally administered maltodextrin, and Lactobacillus paracasei DKGF1 and Lactobacillus casei DKGF7 were supplied through high-concentration culture and freeze-drying of kimchi-derived lactic acid bacteria developed by Dankook University at GF Fermentec. of the dead cell powder was orally administered for 28 days at a level of 1 x 10 11 cfu of 500 μl. All rats were immobilized using plastic cages attached to the body for 2 hours every day for 4 weeks of the experiment. The plan for the study design is shown in FIG. 22 .
본 연구는 삼성 생명 과학 연구소의 동물실험 윤리 위원회 (IACUC)로부터 과학성과 윤리성에 대한 심사를 거쳐 승인 (승인번호 : 20200219001)을 받아 수행되었다. 또한 국제 실험동물 관리 평가인증 협회 (AAALAC)의 공인된 시설로써 ILAR (Institute of laboratory animal resources) 지침을 준수하였다.This study was carried out after being approved (approval number: 20200219001) by the Animal Experiment Ethics Committee (IACUC) of the Samsung Life Sciences Research Institute after reviewing the scientific and ethical aspects. In addition, as an accredited facility of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), it complied with ILAR (Institute of laboratory animal resources) guidelines.
실시예 5-2 : 유산균 사균체 락토바실러스 카제이 DKGF7의 동물실험용 샘플 제조Example 5-2: Preparation of animal test samples of lactic acid bacteria dead cells Lactobacillus casei DKGF7
본 연구에 사용된 유산균 사균체는 기능성 검증에서 가장 우수한 락토바실러스 카제이 DKGF7로서 2% maltose가 보충된 10 mL의 MRS broth에서 30 ℃, 24시간동안 배양되었다.The lactic acid bacteria dead cells used in this study were Lactobacillus casei DKGF7, the best in functional verification, and were cultured for 24 hours at 30°C in 10 mL of MRS broth supplemented with 2% maltose.
2-3회 반복 배양 후 5,000 rpm에서 10분동안 원심분리 후 상등액 제거하여 수분함량이 90%가 되도록 증류수를 첨가한 뒤, 121 ℃에서 15분동안 고압 열처리하여 불활성화된 균체를 -40 ℃이하 온도의 진공조건에서 24시간 이상 동결건조하였다. 분말화된 균을 샘플로 사용하기 위하여 부형제로 과민성 대장 증후군 (IBS; Irritable bowel syndrome)에 거의 영향을 미치지 않는 말토덱스트린을 선정하여 혼합하였다. 투여군의 쥐에게 매일 500 ㎕의 락토바실러스 카제이 DKGF7 (1 x 1011 CFU)을 1회 투여하였다.After repeated incubation 2-3 times, centrifuge at 5,000 rpm for 10 minutes, remove the supernatant, add distilled water so that the water content becomes 90%, and heat treatment at 121 ° C for 15 minutes under high pressure to reduce the inactivated cells to -40 ° C or less It was freeze-dried for more than 24 hours under vacuum conditions of temperature. To use the powdered bacteria as a sample, maltodextrin, which has little effect on Irritable Bowel Syndrome (IBS), was selected and mixed as an excipient. 500 μl of Lactobacillus casei DKGF7 (1×10 11 CFU) was administered to the mice in the administration group once daily.
실시예 5-3 : 사균체의 투여로 인한 몸무게 및 분변 굳기 측정Example 5-3: Measurement of body weight and fecal hardness due to administration of dead cells
체중은 매일 측정되었고, 4주에 걸쳐 7일마다 분변의 굳기를 측정하였다. 분변 굳기의 일관성을 측정하기 위해 3등급 나누어 점수를 측정 (0; 정상, 2; 살짝 무름, 4; 설사) 하였고, 실험이 끝난 뒤 결장 조직을 수집하여 염증성 사이토카인과 tight junction 단백질 발현정도를 측정하였다. Body weight was measured daily, and fecal hardness was measured every 7 days over 4 weeks. To measure the consistency of fecal hardness, the score was divided into three grades (0; normal, 2; mildly soft, 4; diarrhea). After the experiment, colon tissues were collected to measure the expression levels of inflammatory cytokines and tight junction proteins. did.
그 결과, 쥐의 몸무게는 도 23 에 나타난 바와 같이 대조군과 투여군 모두 실험기간 동안 점진적으로 증가하였다. 투여 시작 4주 후, 모든 쥐에서 약 1.25%의 체중 증가가 있었다 (대조군; 378 ± 7.9 g, 투여군; 380 ±9.8 g). As a result, as shown in FIG. 23 , the body weight of the mice gradually increased during the experimental period in both the control group and the administration group. After 4 weeks of initiation of dosing, there was a weight gain of about 1.25% in all rats (control group; 378 ± 7.9 g, administration group; 380 ± 9.8 g).
분변의 굳기 측정 결과는 도 24 에 나타난 바와 같이 유의적인 분변의 굳기 차이는 연구 1주차부터 확인이 가능하였다. 투여기간동안 투여군의 분변 굳기값은 모두 1미만으로 정상에 가까운 수준이었다. 대조군의 분변 굳기값은 지속적으로 무른 변을 배설하였으며 정상으로 회복되지 않았다. 이를 통해 사균체 투여 4주차에 투여군이 대조군에 비해 분변 굳기의 일관성에 대한 점수가 유의하게 낮음을 확인하였다 (대조군; 1.9, 투여군; 0.4, p 〈0.05). As shown in FIG. 24 , a significant difference in fecal hardness was confirmed from the first week of the study. During the administration period, all fecal hardness values of the administration group were less than 1, which was close to normal. The fecal hardness value of the control group continued to excrete soft feces and did not return to normal. Through this, it was confirmed that at the 4th week of administration of dead cells, the administration group had a significantly lower score for fecal consistency than the control group (control group; 1.9, administration group; 0.4, p <0.05).
실시예 5-4 : 사균체의 투여로 인한 스테로이드 호르몬 측정Example 5-4: Steroid hormone measurement due to administration of dead cells
스테로이드 호르몬의 혈청 수준을 측정하기 위해 모든 쥐의 꼬리 정맥에서 매주 0.5 mL의 전혈을 수득하였다. 혈청은 전혈에서 분리되어 분석 전에 -80 ℃에서 보관되었다. 스테로이드 호르몬 농도는 corticosterone ELISA kit (Arigo, Hsinchu, Taiwan, China)를 사용하여 정량화하고 흡광도는 ELISA microplate reader (Thermo scientific, Waltham, MA, USA)를 이용하여 450 nm 에서 측정하였다. 측정 가능한 최소 농도는 6.1 ng/mL이고 모든 실험은 중복하여 수행하였다.0.5 mL of whole blood was obtained weekly from the tail vein of all rats to measure the serum levels of steroid hormones. Serum was isolated from whole blood and stored at -80 °C prior to analysis. The steroid hormone concentration was quantified using a corticosterone ELISA kit (Arigo, Hsinchu, Taiwan, China) and the absorbance was measured at 450 nm using an ELISA microplate reader (Thermo scientific, Waltham, MA, USA). The minimum measurable concentration was 6.1 ng/mL, and all experiments were performed in duplicate.
그 결과는 도 25 에 나타난 바와 같이 혈청 스테로이드 호르몬 수치는 연구 기간 내내 투여군이 대조군보다 유의하게 낮음을 확인하였다. 사균체의 투여가 억제 스트레스로 인한 혈장 내 스트레스 호르몬 분비를 감소시키는 것으로 보아 과민성 대장 증후군 (IBS) 증상 개선의 영향을 줄 것으로 판단된다.As a result, as shown in FIG. 25 , it was confirmed that the serum steroid hormone level was significantly lower in the treated group than in the control group throughout the study period. Since the administration of dead cells reduces the secretion of stress hormones in plasma due to inhibitory stress, it is judged to have an effect on improving the symptoms of irritable bowel syndrome (IBS).
실시예 5-5 : 사균체의 투여로 인한 쥐의 결장 조직에서의 염증성 사이토카인 측정Example 5-5: Measurement of inflammatory cytokines in colon tissues of mice due to the administration of dead cells
연구 종료 전, 쥐의 결장 조직을 적출하여 Milliplex map rat cytokine/chemokine magnetic bead kit (Millipore Sigma, Burlington, MA, USA)를 사용하여 인터루킨 IL-1β, IL-12p70, IL-17A 및 종양 괴사 인자 TNF-α를 포함한 염증성 사이토카인을 측정하였다. Masterplex QT2010 소프트웨어 (Luminex, Austin, TX, USA)를 사용하여 Luminex 100/200 reader기로 플레이트를 분석하였다. 모든 샘플은 3반복하여 실험 진행하였다.Before the end of the study, the colon tissues of mice were removed and interleukins IL-1β, IL-12p70, IL-17A and tumor necrosis factor TNF using Milliplex map rat cytokine/chemokine magnetic bead kit (Millipore Sigma, Burlington, MA, USA) Inflammatory cytokines including -α were measured. Plates were analyzed with a
그 결과는 도 26 에 나타난 바와 같이 투여군은 IL-1β, IL-12p70 및 TNF-α를 포함한 결장 조직에서 낮은 수준의 염증성 사이토카인을 보였다. IL-1β 및 TNF-α는 대조군보다 투여군에서 더 낮은 수치의 염증성 사이토카인을 보였다 (p <0.05). IL-12p70의 수치는 투여군에서 최소 검출 한계에 가까웠으며 대조군과 비교하였을 때 유의한 차이를 보였다 (대조군; 4.21 ±1.43 pg/mL, 투여군; 0 pg/mL, p <0.01).As a result, as shown in FIG. 26 , the administration group showed low levels of inflammatory cytokines in colon tissues including IL-1β, IL-12p70 and TNF-α. IL-1β and TNF-α showed lower levels of inflammatory cytokines in the administration group than in the control group (p <0.05). The level of IL-12p70 was close to the minimum detection limit in the administration group and showed a significant difference compared to the control group (control group; 4.21 ±1.43 pg/mL, administration group; 0 pg/mL, p <0.01).
실시예 5-6 : 면역조직화학 (IHC; Immunohistochemistry)을 이용한 tight junction 단백질의 발현 측정Example 5-6: Measurement of expression of tight junction protein using immunohistochemistry (IHC; Immunohistochemistry)
Claudin-1 및 zona occludens-1 (ZO-1)을 포함하는 tight junction 단백질은 면역 조직 화학 (IHC)을 사용하여 결장 조직에서 측정되었다. 4 ㎛ 파라핀이 있는 조직 절편을 자일렌으로 탈파리핀화한 뒤, 내인성 페록시다아제 (peroxidase) 활성을 PBS에서 3% 과산화수소로 차단하였다. 차단된 조직 절편을 1차 항체로 처리하여 4 ℃에서 밤새 배양한 뒤, horseradish peroxidase P-labeled 폴리머-접합 항체와 함께 배양하였다. 모든 IHC 슬라이드는 Aperio scanscope XT slide scanner (Leica, Wetzlar, Germany)를 사용하여 스캔되었으며, 정량 분석은 ImageJ software를 사용하여 수행되었다.Tight junction proteins, including claudin-1 and zona occludens-1 (ZO-1), were measured in colonic tissues using immunohistochemistry (IHC). After deparaffinizing the tissue sections with 4 μm paraffin with xylene, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS. The blocked tissue sections were treated with a primary antibody and incubated overnight at 4°C, followed by incubation with a horseradish peroxidase P-labeled polymer-conjugated antibody. All IHC slides were scanned using an Aperio scanscope XT slide scanner (Leica, Wetzlar, Germany), and quantitative analysis was performed using ImageJ software.
그 결과는 도 27 에 나타난 바와 같이 투여군은 Claudin-1과 ZO-1의 발현이 유의하게 높음을 확인할 수 있다. 투여군은 결장 상피에서 더 강한 강도와 넓은 갈색으로 염색된 영역을 확인할 수 있었다. ImageJ를 사용하여 슬라이드를 스캔한 결과, 전체 스캔된 표본의 양성으로 염색된 영역의 비율로 표현되는 Claudin-1의 염색 강도는 대조군보다 투여군에서 더 높았다 (대조군; 3.53% 양성, 투여군; 8.38% 양성, p <0.001). 또한 ZO-1에서도 투여군에서 염색 강도가 더 높음을 확인할 수 있었다 (대조군; 5.51% 양성, 투여군; 9.43% 양성, p <0.01). 따라서 Claudin, ZO-1과 같은 tight junction에 관여하는 단백질을 증가시키는 것으로 보아 사균체의 장점막 보호 효과를 기대할 수 있다고 생각된다.As a result, it can be confirmed that the expression of Claudin-1 and ZO-1 is significantly higher in the administration group as shown in FIG. 27 . In the treated group, stronger intensity and wider brown-stained areas were observed in the colon epithelium. As a result of scanning the slides using ImageJ, the staining intensity of Claudin-1 expressed as a percentage of the positively stained area of the entire scanned sample was higher in the treated group than in the control group (control group; 3.53% positive, dose group; 8.38% positive). , p < 0.001). In addition, it was confirmed that the staining intensity was higher in the ZO-1 group (control group; 5.51% positive, administration group; 9.43% positive, p <0.01). Therefore, it is thought that the intestinal membrane protection effect of dead cells can be expected by increasing the proteins involved in tight junctions such as Claudin and ZO-1.
Claims (12)
Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that produced by heat treatment or sonication by adding 1X PBS of pH 7 to 8 to Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) company A pharmaceutical composition for enhancing the function of the scene area comprising the cells as an active ingredient.
상기 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP)는, 10 mL MRS 배지에서 37 ℃ 온도 조건으로 18시간 배양한 뒤, 100mL MRS 배지에서 37 ℃ 온도 조건으로 18시간동안 배양된 것을 특징으로 하는, 장면역 기능 증진용 약학적 조성물.
According to claim 1,
The Lactobacillus casei DKGF7 (KCTC 16246BP) is cultured for 18 hours in 10 mL MRS medium at 37 ° C. temperature condition, and then in 100 mL MRS medium at 37 ° C. temperature condition for 18 hours , A pharmaceutical composition for enhancing the function of the scene area.
상기 열 처리는, 60 ℃ 이상의 조건에서 30분 이상 진행되는 것인, 장면역 기능 증진용 약학적 조성물.
According to claim 1,
The heat treatment, which will be carried out for 30 minutes or more under conditions of 60 ° C. or higher, a pharmaceutical composition for enhancing the function of the scene area.
상기 초음파 처리는, 초음파 출력 400 W, 주파수 40kHz 에서 90 분 이상 진행되는 것인, 장면역 기능 증진용 약학적 조성물.
According to claim 1,
The ultrasonic treatment, the ultrasonic output 400 W, the frequency of 40 kHz will proceed for 90 minutes or more, the pharmaceutical composition for enhancing the scene area function.
상기 면역 기능 증진용 조성물은 장증상 및 장면역 개선 효과를 나타내는 것인, 장면역 기능 증진용 약학적 조성물.
According to claim 1,
The composition for enhancing immune function is to exhibit the effect of improving intestinal symptoms and intestinal function, a pharmaceutical composition for enhancing intestinal function.
상기 장증상은 과민성 대장 증후군(IBS)인 것을 특징으로 하는, 장면역 기능 증진용 약학적 조성물.
6. The method of claim 5,
The intestinal symptoms are irritable bowel syndrome (IBS), characterized in that, a pharmaceutical composition for improving intestinal function.
Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that produced by heat treatment or sonication by adding 1X PBS of pH 7 to 8 to Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) company A food additive for enhancing the function of the scene area that contains bacteria as an active ingredient.
Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that produced by heat treatment or sonication by adding 1X PBS of pH 7 to 8 to Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) company Beverage bowel preparation for enhancing the function of the scene area containing bacteria as an active ingredient.
Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP), characterized in that produced by heat treatment or sonication by adding 1X PBS of pH 7 to 8 to Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) company A feed additive for enhancing the function of the scene area that contains bacteria as an active ingredient.
b) 상기 배양액을 원심분리하여 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 균체를 회수하고, pH 7 내지 8 의 1X PBS 를 첨가하는 단계; 및
c) 열 처리 또는 초음파 처리하는 단계;
를 포함하는 것을 특징으로 하는 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체의 제조 방법.
a) Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) in 10 mL MRS medium at 37 ° C. for 18 hours, followed by culturing for 18 hours at 37 ° C. in 100 mL MRS medium;
b) centrifuging the culture solution to recover Lactobacillus casei DKGF7 (KCTC 16246BP) cells, and adding 1X PBS of pH 7 to 8; and
c) heat treatment or sonication;
Lactobacillus casei, characterized in that it comprises a ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) production method of dead cells.
상기 열 처리는, 60 ℃ 이상의 조건에서 30분 이상 열 처리하는 것을 특징으로 하는, 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체의 제조 방법.
11. The method of claim 10,
The heat treatment, Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) production method of dead cells, characterized in that the heat treatment at 60 ℃ or more conditions for 30 minutes or more.
상기 초음파 처리는, 초음파 출력 400 W, 주파수 40kHz 에서 90 분 이상 초음파 처리하는 것을 특징으로 하는, 락토바실러스 카제이 (Lactobacillus casei) DKGF7 (KCTC 16246BP) 사균체의 제조 방법.
11. The method of claim 10,
The ultrasonic treatment, characterized in that the ultrasonic treatment for more than 90 minutes at an ultrasonic power of 400 W, a frequency of 40 kHz, Lactobacillus casei ( Lactobacillus casei ) DKGF7 (KCTC 16246BP) production method of dead cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020210029676 | 2021-03-05 | ||
| KR20210029676 | 2021-03-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20220125785A true KR20220125785A (en) | 2022-09-14 |
Family
ID=83279234
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020220027911A KR20220125785A (en) | 2021-03-05 | 2022-03-04 | Composition for verification of human-derived enzyme degradation and immune function of Lactic acid bacteria from Kimchi containing Lipoteichoic acid as an indicator |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR20220125785A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116751732A (en) * | 2023-07-11 | 2023-09-15 | 内蒙古农业大学 | Culture method for increasing number of living cells of cheese bacillus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101729478B1 (en) | 2014-11-28 | 2017-04-25 | 주식회사농심 | Immune regulating tyndalized lactic acid bacteria and a method for manufacturing the same |
| KR101919938B1 (en) | 2017-06-16 | 2019-02-08 | 주식회사 락토메이슨 | Composition comprising heat-killed lactobacillus plantarum lm1004 having immunostimulating activity |
| KR102106737B1 (en) | 2019-09-27 | 2020-05-04 | (주)바이오일레븐 | Immunopotentiating composition comprising heat-killed Lactobacillus fermentum BioE LF11 having immunopotentiating activity |
| KR102122194B1 (en) | 2019-01-16 | 2020-06-15 | 에스피씨 주식회사 | Immune enhanced dead cell of lactic acid bacteria and a method for manufacturing thereof |
-
2022
- 2022-03-04 KR KR1020220027911A patent/KR20220125785A/en unknown
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101729478B1 (en) | 2014-11-28 | 2017-04-25 | 주식회사농심 | Immune regulating tyndalized lactic acid bacteria and a method for manufacturing the same |
| KR101919938B1 (en) | 2017-06-16 | 2019-02-08 | 주식회사 락토메이슨 | Composition comprising heat-killed lactobacillus plantarum lm1004 having immunostimulating activity |
| KR102122194B1 (en) | 2019-01-16 | 2020-06-15 | 에스피씨 주식회사 | Immune enhanced dead cell of lactic acid bacteria and a method for manufacturing thereof |
| KR102106737B1 (en) | 2019-09-27 | 2020-05-04 | (주)바이오일레븐 | Immunopotentiating composition comprising heat-killed Lactobacillus fermentum BioE LF11 having immunopotentiating activity |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116751732A (en) * | 2023-07-11 | 2023-09-15 | 内蒙古农业大学 | Culture method for increasing number of living cells of cheese bacillus |
| CN116751732B (en) * | 2023-07-11 | 2024-05-24 | 内蒙古农业大学 | Culture method for increasing number of living cells of cheese bacillus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6954563B2 (en) | Use of pasteurized Akkermansia to treat metabolic disorders | |
| Su et al. | Flammulina velutipes polysaccharides improve scopolamine-induced learning and memory impairment in mice by modulating gut microbiota composition | |
| Liu et al. | Antimutagenic and antioxidant properties of milk− kefir and soymilk− kefir | |
| CN101983237B (en) | Microorganisms for improving the health of individuals with disorders related to gluten ingestion | |
| US9636366B2 (en) | Bacteroides CECT 7771 and the use thereof in the prevention and treatment of excess weight, obesity and metabolic and immunological alterations | |
| TW201124089A (en) | A nutritional composition comprising bifidobacterium longum strains and reducing food allergy symptoms, especially in infants and children | |
| KR20080081186A (en) | Novel lactobacillus strains and their use against helicobacter pylori | |
| RU2671209C2 (en) | Lactobacillus rhamnosus rht-3201 conjugated with polysaccharide polymer binder, and use thereof for prevention or treatment of atopic diseases | |
| TW201408311A (en) | New use of Lactobacillus plantarum CMU995 strain | |
| Dai et al. | Anti-inflammatory effects of newly synthesized α-galacto-oligosaccharides on dextran sulfate sodium-induced colitis in C57BL/6J mice | |
| KR102181606B1 (en) | Probiotic strains for the treatment and/or prevention of diarrhea | |
| JP7414328B2 (en) | Lactobacillus acidophilus KBL409 strain and its uses | |
| JP2020074751A (en) | Bifidobacterium lactis gkk2, composition containing the same, and uses thereof for amelioration of allergic asthma | |
| Fabersani et al. | Lactic acid bacteria strains differently modulate gut microbiota and metabolic and immunological parameters in high-fat diet-fed mice | |
| Lee et al. | Anti-obesity effect of vegetable juice fermented with lactic acid bacteria isolated from kimchi in C57BL/6J mice and human mesenchymal stem cells | |
| KR20220125785A (en) | Composition for verification of human-derived enzyme degradation and immune function of Lactic acid bacteria from Kimchi containing Lipoteichoic acid as an indicator | |
| JP5531321B2 (en) | Immunostimulatory composition having IL-10 production promoting action | |
| Dong et al. | Immunomodulatory effects of mixed Lactobacillus plantarum on lipopolysaccharide-induced intestinal injury in mice | |
| JP5525511B2 (en) | Drugs for non-alcoholic fatty liver disease and / or non-alcoholic steatohepatitis | |
| Fujiwara et al. | A double-blind trial of Lactobacillus paracasei strain KW3110 administration for immunomodulation in patients with pollen allergy | |
| CN114921383A (en) | Probiotic preparation with cholesterol removing function and preparation method thereof | |
| JP2024510374A (en) | Composition containing polysaccharide or extract derived from Lactobacillus plantarum | |
| KR20180110848A (en) | A novel Lactobacillus reuteri BM36301 and a Probiotic Benefits of the same | |
| Shin et al. | Enhancement of immunological activity in mice with oral administration of cell wall components of Bifidobacterium bifidum | |
| KR102670126B1 (en) | Composition of synbiotics for improving Irritable Bowel Syndrome comprising Opuntia humifusa extract and lactobacillus |