KR100431277B1 - functional Chungkookjang and process for preparation thereof - Google Patents
functional Chungkookjang and process for preparation thereof Download PDFInfo
- Publication number
- KR100431277B1 KR100431277B1 KR10-2000-0049010A KR20000049010A KR100431277B1 KR 100431277 B1 KR100431277 B1 KR 100431277B1 KR 20000049010 A KR20000049010 A KR 20000049010A KR 100431277 B1 KR100431277 B1 KR 100431277B1
- Authority
- KR
- South Korea
- Prior art keywords
- cheonggukjang
- prepared
- soybeans
- fermentation
- minutes
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 78
- 230000008569 process Effects 0.000 title description 16
- 108010083364 chungkookjang Proteins 0.000 title description 8
- 238000002360 preparation method Methods 0.000 title description 4
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 84
- 244000068988 Glycine max Species 0.000 claims abstract description 83
- 150000003839 salts Chemical class 0.000 claims abstract description 44
- 240000002234 Allium sativum Species 0.000 claims abstract description 42
- 235000004611 garlic Nutrition 0.000 claims abstract description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000000843 powder Substances 0.000 claims abstract description 20
- 240000004160 Capsicum annuum Species 0.000 claims abstract description 19
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 claims abstract description 19
- 235000007862 Capsicum baccatum Nutrition 0.000 claims abstract description 19
- 239000001728 capsicum frutescens Substances 0.000 claims abstract description 19
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims description 63
- 230000004151 fermentation Effects 0.000 claims description 58
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 14
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 19
- 239000000796 flavoring agent Substances 0.000 abstract description 10
- 230000018044 dehydration Effects 0.000 abstract description 9
- 238000006297 dehydration reaction Methods 0.000 abstract description 9
- 235000019634 flavors Nutrition 0.000 abstract description 8
- 230000005070 ripening Effects 0.000 abstract description 7
- 210000004027 cell Anatomy 0.000 description 67
- 230000001093 anti-cancer Effects 0.000 description 55
- 235000002639 sodium chloride Nutrition 0.000 description 44
- 230000000694 effects Effects 0.000 description 34
- 230000005764 inhibitory process Effects 0.000 description 26
- 239000000401 methanolic extract Substances 0.000 description 24
- 230000035772 mutation Effects 0.000 description 23
- 239000002994 raw material Substances 0.000 description 22
- 239000000523 sample Substances 0.000 description 22
- 239000007858 starting material Substances 0.000 description 21
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 20
- 206010028980 Neoplasm Diseases 0.000 description 20
- 238000000338 in vitro Methods 0.000 description 20
- 239000010902 straw Substances 0.000 description 20
- 244000063299 Bacillus subtilis Species 0.000 description 19
- 235000014469 Bacillus subtilis Nutrition 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 18
- 208000005718 Stomach Neoplasms Diseases 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 17
- 206010017758 gastric cancer Diseases 0.000 description 17
- 201000011549 stomach cancer Diseases 0.000 description 17
- 241000209094 Oryza Species 0.000 description 16
- 235000007164 Oryza sativa Nutrition 0.000 description 16
- 235000009566 rice Nutrition 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 15
- 101000856500 Bacillus subtilis subsp. natto Glutathione hydrolase proenzyme Proteins 0.000 description 13
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 13
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
- 235000013557 nattō Nutrition 0.000 description 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- 230000004083 survival effect Effects 0.000 description 13
- 230000002401 inhibitory effect Effects 0.000 description 12
- 230000002790 anti-mutagenic effect Effects 0.000 description 11
- 230000001965 increasing effect Effects 0.000 description 11
- 238000011160 research Methods 0.000 description 11
- 210000004881 tumor cell Anatomy 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 238000010025 steaming Methods 0.000 description 10
- 241000194108 Bacillus licheniformis Species 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 230000002747 voluntary effect Effects 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000003471 mutagenic agent Substances 0.000 description 8
- 231100000707 mutagenic chemical Toxicity 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 8
- 238000010953 Ames test Methods 0.000 description 7
- 231100000039 Ames test Toxicity 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 7
- 231100000002 MTT assay Toxicity 0.000 description 7
- 238000000134 MTT assay Methods 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 102000005936 beta-Galactosidase Human genes 0.000 description 7
- 108010005774 beta-Galactosidase Proteins 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 230000003505 mutagenic effect Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 241000193830 Bacillus <bacterium> Species 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 6
- 208000006268 Sarcoma 180 Diseases 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 229960005322 streptomycin Drugs 0.000 description 6
- 235000019750 Crude protein Nutrition 0.000 description 5
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 description 5
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 description 5
- 230000027151 SOS response Effects 0.000 description 5
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 5
- 235000013339 cereals Nutrition 0.000 description 5
- 235000019784 crude fat Nutrition 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 235000021107 fermented food Nutrition 0.000 description 5
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- 210000004698 lymphocyte Anatomy 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108010020346 Polyglutamic Acid Proteins 0.000 description 4
- 239000002115 aflatoxin B1 Substances 0.000 description 4
- 229930020125 aflatoxin-B1 Natural products 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 229920000370 gamma-poly(glutamate) polymer Polymers 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 3
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000002019 anti-mutation Effects 0.000 description 3
- 230000005907 cancer growth Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000005760 tumorsuppression Effects 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 235000002566 Capsicum Nutrition 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101100150784 Escherichia coli (strain K12) sulA gene Proteins 0.000 description 2
- 229920002670 Fructan Polymers 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000006002 Pepper Substances 0.000 description 2
- 241000722363 Piper Species 0.000 description 2
- 235000016761 Piper aduncum Nutrition 0.000 description 2
- 235000017804 Piper guineense Nutrition 0.000 description 2
- 235000008184 Piper nigrum Nutrition 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 2
- VIYFPAMJCJLZKD-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate Chemical compound [Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 VIYFPAMJCJLZKD-UHFFFAOYSA-L 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000005965 immune activity Effects 0.000 description 2
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 2
- 101150066555 lacZ gene Proteins 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- AIHDCSAXVMAMJH-GFBKWZILSA-N levan Chemical group O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 231100000299 mutagenicity Toxicity 0.000 description 2
- 230000007886 mutagenicity Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 150000003071 polychlorinated biphenyls Chemical group 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- JNUMDLCHLVUHFS-QYZPTAICSA-M sodium;[[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-oxidophosphoryl] [(2r,3s,4r,5r)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound [Na+].NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 JNUMDLCHLVUHFS-QYZPTAICSA-M 0.000 description 2
- 235000013555 soy sauce Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 101150060445 uvrB gene Proteins 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 description 1
- CMXXUDSWGMGYLZ-XRIGFGBMSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride;hydrate Chemical compound O.Cl.OC(=O)[C@@H](N)CC1=CN=CN1 CMXXUDSWGMGYLZ-XRIGFGBMSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- LLIANSAISVOLHR-GBCQHVBFSA-N 5-[(3as,4s,6ar)-2-oxidanylidene-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LLIANSAISVOLHR-GBCQHVBFSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 101150082239 G gene Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
- 238000007696 Kjeldahl method Methods 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 241000405383 Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 Species 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical class [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000000944 Soxhlet extraction Methods 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- OGMVAUOXEUCOHJ-UHFFFAOYSA-N [Br-].CC=1N=CSC=1C.C1=CC=CC=C1C1=[NH+]N(C=2C=CC=CC=2)N=N1 Chemical compound [Br-].CC=1N=CSC=1C.C1=CC=CC=C1C1=[NH+]N(C=2C=CC=CC=2)N=N1 OGMVAUOXEUCOHJ-UHFFFAOYSA-N 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 150000004683 dihydrates Chemical class 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000006846 excision repair Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 229960004279 formaldehyde Drugs 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000003228 microsomal effect Effects 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- -1 monosodium salt Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000013379 physicochemical characterization Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 235000002949 phytic acid Nutrition 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 239000001967 plate count agar Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 235000013547 stew Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Classifications
-
- 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
- A23L23/00—Soups; Sauces; Preparation or treatment thereof
-
- 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
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
- A23L27/105—Natural spices, flavouring agents or condiments; Extracts thereof obtained from liliaceae, e.g. onions, garlic
-
- 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
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
- A23L27/14—Dried spices
-
- 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
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/40—Table salts; Dietetic salt substitutes
-
- 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
- A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention
Landscapes
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
본 발명은 기능성 청국장 및 그 제조방법에 관한 것으로 원료대두로 중소립콩을 정선 및 수세하고 1.5배의 15℃ 물에 12시간 침지한 후 탈수시간을 45분으로 하고 1.0∼1.5㎏/㎠ 의 오토클래브(Autoclave)를 사용하여 60분간 증자한 다음 전배양한 종균액을 접종하여 40℃에서 48시간 발효시킨 후 소금 7.9%, 고춧가루 2.0%, 마늘 0.2%를 첨가하여 혼합 및 마쇄하고 5℃에서 15일간 후숙하여 제조한 청국장은 풍미가 우수한 뛰어난 효과가 있고 또 중소립콩을 정선 및 수세하고 1.5배의 15℃ 물에 12시간 침지한 후 탈수시간을 45분으로 하고 오토클래브를 사용하여 60분 증자하고 전배양한 종균액을 접종하여 40℃에서 72시간 발효시킨 후 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%를 첨가하고 혼합 및 마쇄하여 제조한 청국장은 항암 기능성이 우수한 뛰어난 효과가 있다.The present invention relates to a functional Cheonggukjang and a method of manufacturing the same. Selected and washed small and medium soybeans as raw soybeans, soaked in 1.5 times of 15 ° C. for 12 hours, and then dehydrated to 45 minutes with an autoclave of 1.0 to 1.5 kg / cm 2. The mixture was incubated for 60 minutes using autoclave, inoculated with precultured seed solution, fermented at 40 ° C for 48 hours, mixed and ground by adding 7.9% salt, red pepper powder 2.0%, and garlic 0.2%. Cheonggukjang prepared after ripening for one day has excellent flavor and has excellent flavor, and small and medium soybeans are selected and washed, immersed in 1.5 times of 15 ℃ water for 12 hours, and dehydration time is 45 minutes and 60 minutes using autoclave. After inoculating the pre-cultivated spawn solution and fermented at 40 ° C. for 72 hours, salt 7.9%, red pepper powder 1.1%, and garlic 1.1% were added, mixed and crushed.
Description
본 발명은 기능성 청국장 및 그 제조방법에 관한 것이다. 더욱 상세하게는, 본 발명은 대두품종과 스타터 균주를 선정하여 증자 및 발효시킨 후 부재료를 혼합하여 제조한 풍미가 우수하고 항암 기능성이 있는 청국장 및 그 제조방법에 관한 것이다.The present invention relates to a functional Cheonggukjang and its manufacturing method. More specifically, the present invention relates to a Cheonggukjang with excellent flavor and anti-cancer function, prepared by mixing soybean varieties and starter strains, and then increasing and fermenting them, and a method for producing the same.
청국장은 콩을 삶아 볏짚을 군데군데 꽂고 따뜻한 아랫목에 덮어 두면 하룻밤 사이에 표면이 회백색이 되고 끈적끈적한 실이 나게 띄워진다. 여기에 소금이나 마늘, 고춧가루 등을 섞어 절구통에서 찧어 단지에 다져 넣고 두면서 찌개의 재료로 사용하여 온 것으로 우리의 전래 간장, 된장, 고추장과는 달리 전통장류 중 유일하게 소금을 첨가하지 않고 고온에서 속성으로 발효시킨 식품이기 때문에 전통적으로 콩을 수확한 뒤 늦가을부터 초봄 사이에 각 가정에서 손쉽게 제조 이용하여 왔다. 그 제조방법이 정형화 된 것은 없고 경험에 의해서 전래된 방법대로 제조하였다.Cheonggukjang boils soybeans and puts straw in several places and covers it with a warm lower neck to make the surface grayish and sticky threads overnight. It is mixed with salt, garlic, and red pepper powder, and then chopped in a mortar and chopped into a jar. It is used as an ingredient in stew. Since it is a fermented food, it has been traditionally harvested soybeans and then easily manufactured and used in each family from late autumn to early spring. The manufacturing method was not standardized and manufactured according to the method conventionally obtained by experience.
청국장은 삶은 콩을 볏짚에 붙어 있는 고초균(枯草菌)이라 부르는 바실러스 서브틸리스(Bacillus subtilis)를 이용하여 띄워 만든 것으로 발효과정 중에 고초균이 생산하는 효소에 의해서 그 특유의 맛과 냄새를 내는 동시에 원료대두의 당질과 단백질에서 유래된 레반 폼 프럭탄트(levan form fructan)와 폴리글루타메이트 (polyglutamate)의 중합물질인 끈적끈적한 점질물을 생성한다. 이와 같이 제조된 청국장은 콩을 원료로한 우리나라의 대표적 발효식품으로 간장, 된장, 고추장 등과 함께 오늘날까지 상용되어 온 전통장류 중의 하나로 된장보다 콩 단백질과 지방질 함량이 많고 소화 흡수율이 높으며, 칼슘과 비타민 A, B의 중요한 공급원, 청국장균의 정장효과, 섬유질의 변비예방효과, 발암물질과 콜레스테롤의 체외 배출효과, 점질물(mucin)의 알코올 흡수에 의한 해장효과, 사포닌의 혈관강화와 혈액순환 촉진과 젖산분해효과, 레시친의 뇌 노화와 치매, 고혈압과 동맥경화의 예방 등의 효과가 있다는 것이 많이 알려져 왔다(이서래, 한국의 발효식품, 1997., 이한창, 발효식품, 1996., 김상순 한국전통식품의 과학적 고찰, 1985., 김상순, 청국장, 1987., 이한창, 청국장론, 1991., 이한창, 청국장 소고, 1992., 주현규, 청국장은 기능성 건강식품, 1996.).Cheonggukjang is made by using Bacillus subtilis called Bacillus subtilis, which is attached to rice straw, and gives its unique taste and smell by enzymes produced by Bacillus subtilis during fermentation. It produces sticky slime, a polymer of levan form fructan and polyglutamate derived from soy sugar and protein. Cheonggukjang prepared in this way is one of Korea's representative fermented foods based on soybeans. Soy sauce, soybean paste, and red pepper paste are one of the traditional soybeans that have been commercialized to date. Important sources of A and B, intestinal effects of chungguk coli, fibrous constipation prevention effect, carcinogens and cholesterol in vitro excretion effect, haematological effect of alcohol absorption of mucin, stimulation of saponin and blood circulation and lactic acid It has been known that there are effects of degradation, lecithin, brain aging and dementia, prevention of hypertension and arteriosclerosis (Lee Seo-rae, Korean fermented food, 1997., Lee Han-chang, fermented food, 1996., Sang-Soon Kim REMARKS, 1985., Kim Sang-soon, Cheonggukjang, 1987., Lee Han-chang, Cheongguk Jang, 1991., Lee Han-chang, Cheonggukjang Sogo, 1992., Ju Hyun-kyu, Cheonggukjang is a functional health food, 1 996.).
최근 많은 연구자들에 의해서 밝혀지고 있는 청국장의 생리활성 효과로는 청국장이 콩으로부터 유래된 이소플라본(isoflavons), 피트산(phytic acid), 사포닌(saponin), 트립신 저해제(trypsin inhibitor), 토코페롤(tocopherol), 불포화지방산, 식이섬유, 올리고당 등의 각종 생리활성 물질과 항산화물질 및 혈전용해 효소를 다량 함유하고 있는 기능성 식품으로서 고혈압방지 효과, 혈중 콜레스테롤 저하능, 항돌연변이성, 항암성, 항산화성, 혈전 용해능 등이 있다(김정상, 한국콩연구회지 1996., 성미경, 한국콩연구회지, 1996., 윤기도 외 4인, 1996., 정건섭외1인 산업미생물학회지, 1997., 길지은 외 2인, 1998., 건국대학교 식품개발연구소, 개교 50주년기념 심포지움 자료, 1996., 권익부 외 1인, 영남대학교 부설 장류 연구소, 1999., 윤기도 외 4인, 산업미생물학회지, 1996., 정건섭외 1인, 산업미생물학회지, 1997., 허석 외 2인, 한국농화학회지, 1998., 영양과 암, 한국콩연구회 소식, 1998.).Cheonggukjang's physiological effects, which have recently been identified by many researchers, include isoflavons, phytic acid, saponin, trypsin inhibitor, and tocopherol derived from soybeans. ), Functional foods containing various physiologically active substances such as unsaturated fatty acids, dietary fiber, oligosaccharides, antioxidants and thrombolytic enzymes, and antihypertensive effect, blood cholesterol lowering ability, antimutagenicity, anticancer activity, antioxidant activity, thrombus (Kim Jung-sang, Korea Soybean Research Journal 1996., Mi-Kyung Sung, Korea Soybean Research Journal, 1996., Yoon Doo et al., 4 people, 1996. 1998., Food Development Research Institute, Konkuk University, 50th Anniversary Symposium, 1996., Kwon, Ikbu et al., Yeungnam University affiliated research institute, 1999., Yoondo, et al., Journal of the Industrial Microbiology, 1996. Phosphorus, Journal of the Korean Society for Industrial Microbiology, 1997., Huh Seok and 2 others, The Korean Journal of Agricultural Chemistry, 1998., Nutrition and Cancer, News of the Korean Soybean Research Society, 1998.).
그러나 청국장은 각 지방 또는 가정마다 제조방법이 일정하지 않으며 그 품질 또한 볏짚에 부착된 고초균의 종류와 발효조건 등에 따라 크게 다르게 된다. 따라서 전통 장류 문화의 계승발전과 우수한 기능을 지닌 전통 청국장의 품질을 향상시키고 표준화하기 위해서는 제조방법을 확립하는 것이 절실히 요망되고 있다.However, Cheonggukjang does not have a uniform manufacturing method for each province or family, and its quality also varies greatly depending on the type of Bacillus subtilis attached to rice straw and fermentation conditions. Therefore, in order to improve and standardize the succession of traditional Korean culture and the quality of traditional Cheonggukjang, it is urgently needed to establish a manufacturing method.
지금까지 청국장 품질 향상과 산업화 기술에 관한 연구로는 전통 청국장 이용실태(최정숙 외 6인, 한국콩연구회지, 1996.)와 산업체 청국장 생산현황 조사 등에 대하여 설문을 통하여 고찰한 것과 청국장 발효우수균주 선발 및 대두 품종별 제조 청국장의 품질 특성 연구, 발효조건별 청국장 품질 특성 연구(한국식품개발연구원, 1998., 주현규, 한국식품과학회지 1971., 김경자 외 2인 1982., 김상순, 장유, 1997., 한만숙, 동국대학논총, 1985., 유진영, 식품과학기술지, 1997., 장창문, 제 1 회 장류심포지움 및 장류전시회, 1998., 권동진, 한국전통식품산업화연구회, 1999., 장창문, 생물공학적 기법에 의한 전통 장류의 제품화 연구 제 2 단계 결과 보고서, 1998., 장창문, 농촌생활과학, 1996., 고현숙, 건국대 석사학외논문, 1998.) 등이 있으나, 이는 전통 청국장을 계승하기보다는 일본 낫토(Natto)의 제조공정에 적용시킨 것이었다.So far, the research on the quality improvement and industrialization technology of Cheonggukjang was conducted through the questionnaire survey on the status of the use of traditional Cheonggukjang (6 people, Choi Sok Choi and Korean Soybean Research Journal, 1996.) Quality Characteristics of Cheonggukjang Prepared by Korean and Soybean Varieties, and Quality Characteristics of Cheonggukjang by Fermentation Conditions (Korea Food Research Institute, 1998., Hyun-Kyu Joo, Korea Food Science Review 1971., Kyung-Ja Kim and others 1982., Kim Sang-soon, Jang Yu, 1997., Man-sook Han, Journal of Dongguk University, 1985., Yoo Jin-young, Food Science and Technology Journal, 1997., Chang-Moon Chang, 1st Jang Symposium and Soy Sauce Exhibition, 1998., Kwon, Dong-Jin, Korean Traditional Food Industrialization Research Society, 1999., Chang-Chang Chang, Biotechnology The second stage result report on the research on the production of traditional Jangjang by technique, 1998., Chang-moon Chang, Rural Living Science, 1996., Koh Hyun-suk, Master's Thesis, Konkuk University, 1998.) That was applied to the production process of Japanese natto (Natto).
본 발명자들은 우리나라 청국장의 고유한 맛을 재현하면서 우수한 기능성을 갖는 고품질의 청국장을 제조하기 위하여 대두품종, 증자방법, 스타터(Starter), 발효기간, 부재료비를 달리하여 청국장을 제조하고 이화학적 실험을 행하여 청국장 제조 방법을 표준화하였다. 또한 상기 대두품종, 스타터(Starter), 발효기간, 부재료비를 달리하여 제조한 청국장의 Ames test와 SOS chromotest에 의한 항돌연변이 효과를 살펴보았고 MTT assay를 행하여시험관내(in vitro)에서 항암효과를 관찰하여 항암성이 우수한 항암 기능성 청국장 제조방법을 확립하였으며시험관내(in vitro)및생체내(in vivo)에서 상기 항암 기능성 청국장 제조방법에 의해 제조된 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장의 항암성을 조사하고 비교하여 본 발명 방법에 의해 제조된 항암 기능성 청국장이 가장 우수한 항암성을 나타냄을 확인하므로서 본 발명을 완성하였다.The present inventors produced Cheonggukjang by varying soybean varieties, steaming method, starter, fermentation period, and subsidiary materials to produce high-quality Cheonggukjang with excellent functionality while reproducing the unique taste of Cheonggukjang in Korea. To standardize the preparation of Chungkookjang. In addition, the effects of anti-mutation by Ames test and SOS chromotest of Cheonggukjang prepared by varying the soybean varieties, starter, fermentation period, and subsidiary material cost were examined, and the anticancer effect was observed in vitro by MTT assay. By establishing a method of producing anti-cancer functional cheonggukjang excellent in anti-cancer properties , and by improving the cheonggukjang, traditional methods and traditional methods manufactured by the method for producing anti-cancer functional cheonggukjang in vitro and in vivo (in vivo) The present invention was completed by investigating and comparing the anticancer properties of the prepared Cheonggukjang, confirming that the anticancer functional Cheonggukjang produced by the method of the present invention showed the best anticancer properties.
따라서, 본 발명의 목적은 대두품종과 스타터 균주를 선정하여 증자 및 발효시킨 후 부재료를 혼합하여 맛과 풍미가 우수한 표준화 청국장과 항암 기능성이 우수한 청국장을 제조하는 방법을 제공함에 있다.Accordingly, an object of the present invention is to select a soybean varieties and starter strains to increase the weight and fermentation, and then to mix the subsidiary materials to provide a standardized Cheonggukjang with excellent taste and flavor and a Cheonggukjang with excellent anticancer function.
본 발명의 다른 목적은 상기 제조한 풍미가 우수한 표준화 청국장과 항암 기능성 청국장을 제공함에 있다.Another object of the present invention is to provide a standardized Cheonggukjang and anti-cancer functional Cheonggukjang with excellent flavor.
본 발명의 상기 목적은 준저리, 만리콩, 황금콩 및 수입콩과 같은 4종의 대두를 다양한 방법으로 증자한 후 종균을 처리하여 발효시키므로 청국장을 제조하고 제조한 청국장의 수분, pH, 아미노태 질소, 암모니아태 질소, γ-글루타밀트랜스펩티다아제의 함량과 같은 이화학적 특성을 조사하여 풍미가 가장 우수한 청국장을 표준화하였다. 이어서, 상기 제조한 청국장들의 메탄올 추출물을 각각 Ames test와 SOS chromotest 및 MTT assay하여 항돌연변이성과 항암성이 가장 우수한 청국장을시험관내(in vitro)에서 조사하여 항암성이 우수한 청국장으로 표준화한 후 상기 표준화한 항암 기능성 청국장과 전통방법에 의해 제조된 청국장 및 전통방법을 개량한 방법에 의해 제조한 청국장을 시료로 사용하여시험관내(in vitro)에서 항돌연변이성과 항암성을 조사하고 또생체내(in vivo)에서 상기 청국장 시료들을 마우스에 투여하고 종양세포를 이식하여 세포독성효과, 고형암성장 저지효과 및 자연살해세포의 활성능을 측정하여 본 발명에서 표준화한 항암 기능성 청국장의 항암효과가 우수함을 재확인함으로서 달성하였다.The object of the present invention is to increase the four types of soybeans, such as Junjiri, Manri beans, golden beans and imported soybeans in various ways, and then fermented by treating the seed, so that the water, pH, amino nitrogen of Cheonggukjang prepared and manufactured Physicochemical properties such as nitrogen, ammonia nitrogen and γ-glutamyltranspeptidase were investigated to standardize the most flavorful Cheonggukjang. Subsequently, the methanol extracts of the cheonggukjang prepared above were subjected to Ames test, SOS chromotest, and MTT assay, respectively , to investigate the cheonggukjang with the best antimutagenicity and anticancer properties in vitro, and then standardized to the cheonggukjang with excellent anticancer properties. It investigated the anticancer functionality using a soybean prepared by soybean and methods improving the traditional method of producing by the soybean and the traditional method a sample in vitro mutagenesis and antitumor in (in vitro) and also in vivo (in vivo ) By reconfirming the superior anticancer effect of the anticancer functional Cheonggukjang standardized in the present invention by measuring the cytotoxic effect, the solid cancer growth inhibition effect and the natural killer cell activity by administering the samples of the Cheonggukjang to mice and transplanting tumor cells. It was.
이하, 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.
도 1은 다양한 대두품종을 40℃에서 3일동안 발효시켜 제조한 청국장의 아미노태 질소, 암모니아태 질소 및 γ-글루타밀트랜스펩티다아제(γ-glutamyltranspeptidase:γ-GTP)의 함량을 나타낸 그래프이다.1 is a graph showing the content of amino nitrogen, ammonia nitrogen and γ-glutamyltranspeptidase (γ-glutamyltranspeptidase: γ-GTP) of Cheonggukjang prepared by fermenting various soybean varieties at 40 ° C. for 3 days.
도 2는 다양한 증자방법에 의해 제조한 청국장의 아미노태 질소와 암모니아태 질소의 함량을 나타낸 그래프이다.Figure 2 is a graph showing the content of amino nitrogen and ammonia nitrogen of Cheonggukjang prepared by various steaming methods.
도 3은 다양한 종류의 스타터 균주를 사용하여 접종하거나 자연적으로 발효시켜 제조한 청국장의 아미노태 질소, 암모니아태 질소 및 γ-GTP의 함량을 나타낸 그래프이다.Figure 3 is a graph showing the contents of amino nitrogen, ammonia nitrogen and γ-GTP of Cheonggukjang prepared by inoculation or natural fermentation using various kinds of starter strains.
도 4는 바실러스 서브틸리스(Bacillus subtilis)를 접종하여 40℃에서 5일 동안 발효시켜 제조한 청국장의 아미노태 질소, 암모니아태 질소 및 γ-GTP의 함량을 나타낸 그래프이다.Figure 4 is a graph showing the contents of amino nitrogen, ammonia nitrogen and γ-GTP of Cheonggukjang prepared by fermenting Bacillus subtilis ( Bacillus subtilis ) for 5 days at 40 ℃.
도 5는 3일동안 40℃에서 배양한 후 다양한 부재료를 첨가하여 5℃에서 여러 기간동안 숙성시켜 제조한 청국장의 아미노태 질소, 암모니아태 질소 및 γ-GTP의 함량을 나타낸 그래프이다.5 is a graph showing the contents of amino nitrogen, ammonia nitrogen and γ-GTP of Cheonggukjang prepared by culturing at 40 ° C. for 3 days after incubation at 40 ° C. for 3 days.
도 6은 마우스 비장 세포의 자연살해 세포활성에 미치는 청국장의 영향을 나타낸 그래프이다.6 is a graph showing the effect of Chungkookjang on spontaneous killer cell activity of mouse spleen cells.
본 발명은 준저리, 만리콩, 황금콩, 수입콩 각각을 다양한 증자방법으로 증자한 후 스타터로 다양한 종균을 각각 처리하여 발효시킨 다음 부재료로 소금, 고춧가루, 마늘 등을 첨가하여 혼합·마쇄하고 후숙시켜 청국장을 제조하는 단계; 상기 제조된 청국장에 함유된 수분, pH, 아미노태 질소, 암모니아태 질소, γ-글루타밀트랜스펩티다아제의 함량 및 총균수를 대두품종별, 증자방법별, 스타터 균주별로 측정하여 청국장의 이화학적 특성을 조사하므로써 가장 양호한 풍미를 갖는 청국장을 표준화하는 단계; 대두품종별, 스타터 균주별, 발효기간별, 첨가한 부재료 비율별로 제조한 각각의 청국장 메탄올 추출물을 Ames test 및 SOS chromotest하여 가장 우수한 항돌연변이성을 나타내는 대두품종, 종균, 부재료를 조사하고 발효기간을 조사하여 항돌연변이성이 가장 우수한 청국장 제조조건을시험관내(in vitro)에서 결정하는 단계; 암세포에 대두품종별, 스타터 균주별, 발효기간별, 첨가한 부재료 비율별로 제조한 각각의 청국장 메탄올 추출물을 첨가하고 배양한 후 MTT(3-(4,5-dimethyl-thiazol-2,5-diphenyltetrazolium bromide) 용액을 첨가하고 다시 한번 배양한 후 ELISA reader로 흡광도를 측정하여 세포독성을 조사하므로써 가장 항암 기능성이 우수한 청국장의 제조조건을시험관내(in vitro)에서 결정하여 표준화하는 단계; 상기 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장을 시료로 사용하여 항돌연변이성 및 항암성을 각각 조사하여 비교하는 단계; 다이익스클루젼(dye exclusion) 방법을 이용하여 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장 시료들의 세포독성을 조사하는 단계; 상기 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장 시료를 공급한 마우스의 서혜부에 sarcoma-180 종양세포를 이식하고 26일째 되는 날 치사시켜 각 장기의 중량을 측정한 후 생성된 고형암을 적출하고 그 무게를 측정하여생체내(in vivo)에서 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장의 항암성을 조사하여 비교하는 단계 및: 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장 시료를 공급한 마우스의 비장세포를 분리하여 이펙터 세포 (effecter cell)로 준비하고 Yac-1 마우스 임파구 세포를 타겟세포로 사용하여 자연살해능을 측정함으로서 본 발명에서 표준화한 항암 기능성 청국장과 전통방법 및 전통방법을 개량한 방법에 의해 제조된 청국장의 면역활성 증강효과를 조사하여 비교하는 단계로 구성된다.In the present invention, after adding each of the jejunri, Manri beans, golden beans, and imported beans by various methods of increasing the weight, each seed is treated with a starter and fermented, followed by mixing, grinding and ripening by adding salt, red pepper powder, garlic, etc. Preparing a cheongukjang; Moisture, pH, amino nitrogen, ammonia nitrogen, γ-glutamyltranspeptidase content and total number of bacteria contained in the prepared Cheonggukjang were measured by soybean varieties, by steaming method, and by starter strain to determine the physicochemical characteristics of Cheonggukjang. Standardizing Cheonggukjang having the best flavor by surveying; Investigate the soybean varieties, spawns, and subsidiary materials showing the best antimutagenicity by Ames test and SOS chromotest of methanol extracts prepared by soybean varieties, starter strains, fermentation periods, and added ingredient ratios. To determine in vitro the production conditions of the best anti-mutation Cheonggukjang; MTT (3- (4,5-dimethyl-thiazol-2,5-diphenyltetrazolium bromide) was added to the cancer cells by adding soybean varieties, starter strains, fermentation periods, and subsidiary ingredient ratios. ) After adding the solution and incubating once again, measuring the absorbance with an ELISA reader to examine cytotoxicity and determining and standardizing in vitro the preparation conditions of Cheonggukjang having the most anticancer function; Investigating and comparing antimutagenicity and anticancer activity using a cheonggukjang prepared by an anticancer functional cheonggukjang and an improved method of traditional and traditional methods, respectively; using a multi-exclusion method To examine the cytotoxicity of the cheonggukjang samples prepared by the anti-cancer functional cheonggukjang standardized in the present invention and the traditional method and the improved method In the present invention, sarcoma-180 tumor cells were implanted into the inguinal part of mice fed with cheonggukjang samples prepared by the anti-cancer functional cheonggukjang standardized in the present invention and the method improved by the traditional method and the traditional method. The anticancer properties of the cheonggukjang prepared by the anticancer functional Cheonggukjang and the traditional method and the method improved by the standardized method of the present invention in vivo by measuring the weight and extracting the solid cancer produced. Investigation and comparison step: The splenocytes of the mice supplied with the cheonggukjang samples prepared by the anticancer functional cheonggukjang standardized in the present invention and the traditional method and the improved method are isolated and prepared as effector cells. Anticancer functional cheongguk standardized in the present invention by measuring natural killer ability using Yac-1 mouse lymphocyte cells as target cells And it consists of the traditional method and comparing to examine the immune activity enhancing effect of the soybean produced by a method in improving the traditional methods.
이하, 본 발명의 구체적인 방법을 실시예와 실험예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예와 실험예에만 한정되는 것은 아니다.Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.
실시예 1: 청국장 제조방법의 표준화Example 1: Standardization of Cheonggukjang
제 1 공정: 청국장 제조1st Process: Cheonggukjang Manufacturing
본 실시예에서 사용한 원료대두는 소립종인 준저리(100립중 10g, 조단백질 41%, 조지방 19%), 중립종인 만리콩(100립중 20g, 조단백질 41%, 조지방 20%), 대립종인 황금콩(100립중 25g, 조단백질 41%, 조지방 20%), 수입콩인 미국산 황색 1호(100립중 16g, 조단백질 35%, 조지방 18%)은 각각 영남 농업 시험장과 수원의 작물시험장, 장류조합에서 분양 받아 청국장 제조 재료로 사용하였다. 공시균주는 바실러스 서브틸리스(Bacillus subtilisKCCM 11315)로 한국 미생물 보존센터에서 분양 받아 사용하였으며 메주에서 분리한 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115), 재래간장으로부터 분리한 바실러스 서브틸리스 CCKS-111(Bacillus subtilisCCKS-111), 전통 청국장으로부터 분리한 바실러스 서브틸리스 DC-2(Bacillus subtilisDC-2) 균주는 영남대 식품가공학과에서 분양받아 사용하였다. 균은 글루코스(glucose) 0.5%를 첨가한 NB배지(Difco, nutrient broth : beef extract 0.3%, peptone 0.5%)에 균을 각각 접종하여 37℃에서 24시간 배양한 후, 균체 농도를 조절(흡광도 600nm = 0.8)하여 종균액으로 사용하였다.The raw soybean used in this example is semi-fermented (10g in 100 grains, crude protein 41%, crude fat 19%), medium grain manali beans (20g in 100 grains, crude protein 41%, crude fat 20%), golden bean (all 100 grains) 25g, crude protein 41%, crude fat 20%) and imported yellow soybean No. 1 (16g of 100 grains, crude protein 35%, crude fat 18%) were sold by Yeongnam Agricultural Experiment Station, Suwon Crop Experiment Station, and Soybean Association, respectively. Used as. The test strain was Bacillus subtilis KCCM 11315, which was used by the Korea Microbial Conservation Center, and was used as Bacillus licheniformis CN-115 isolated from Meju, and Bacillus subtilis isolated from conventional soy sauce. Bacillus subtilis DC-2 ( Bacillus subtilis DC-2) strain isolated from traditional Cheonggukjang was used by the Department of Food Science and Technology of Yeungnam University. The bacteria were inoculated with NB medium (Difco, nutrient broth: beef extract 0.3%, peptone 0.5%) added with 0.5% glucose, incubated at 37 ° C for 24 hours, and then the cell concentration was adjusted (absorbance 600 nm). = 0.8) was used as a seed solution.
먼저 대두를 정선 및 수세하여 1.5배의 물(15℃)에 12시간 침지하고 물빼기하였다. 이를 어렌메이어 플라스크(Erlenmeyer flask)에 담아 고압 멸균기 (1.0∼1.5kg/cm2)에서 60분 동안 증자하여 50℃로 냉각하였다. 전배양한 종균액(흡광도 600nm=0.8)을 원료의 1%(v/w) 접종하고 40℃ 배양기(incubator)에서 72시간 발효시켰다. 발효가 끝난 청국장 메주에 소금을 7.6% 첨가하여 혼합·마쇄하였다. 또한 전통 청국장은 증자시 상압에서 4시간 삶고 냉각한 후 5 ∼ 7cm로 절제한 볏짚을 깐 상자에 3cm 두께로 대두를 깔고 그 위에 볏짚을 덮어 발효시켰다. 발효가 끝난후 소금 7.93%, 고춧가루1.1%, 마늘 1.1%을 첨가하여 혼합·마쇄하고 5℃에서 15일간 후숙시켰다.First, soybeans were selected and washed with water, soaked in 1.5 times of water (15 ° C) for 12 hours and drained. This was immersed in an Erlenmeyer flask and the mixture was increased in a high pressure sterilizer (1.0-1.5 kg / cm 2 ) for 60 minutes and cooled to 50 ° C. Precultured seed stock (absorbance 600nm = 0.8) was inoculated 1% (v / w) of the raw material and fermented for 72 hours in a 40 ℃ incubator (incubator). Fermented chungkukjang meju was added with 7.6% of salt to mix and mill. In addition, the traditional Cheonggukjang was boiled for 4 hours at normal pressure at the time of steaming, cooled, and then fermented with rice straw covered with 3 cm thick in a box of rice straw cut into 5-7 cm. After the fermentation was completed, 7.93% of salt, 1.1% of red pepper powder and 1.1% of garlic were mixed and ground, and then ripened at 5 ° C. for 15 days.
제 2 공정: 대두 품종별 청국장의 품질 특성Process 2: Quality Characteristics of Cheonggukjang by Soybean Varieties
우리나라의 청국장과 비슷한 일본 낫또(Natto)의 원료 대두인 준저리, 항돌연변이, 항암효과가 우수하였던 만리콩, 민간에 장류의 원료 대두로 널리 이용되는 황금콩 그리고 수입콩인 미국산 황색 1호를 사용하여 상기 제 1 공정에서 제조한 청국장 발효 후 수분함량과 pH, 아미노태 질소와 암모니아태 질소함량, γ-GTP 활성(activity)를 조사하였다. 청국장의 일반성분은 A.O.A.C.법(Association ofofficial analytical chemists)에 준하여, 수분은 105℃ 건조법, 조단백질은 Micro-Kjeldahl법, 조지방은 Soxhlet 추출법으로 분석하였다. pH는 시료를 10배 희석하여 pH meter (Corning 220, USA)로 측정하였다. 아미노태 질소(NH2-N)는 청국장 시료 2g을 취하여 formol법으로 측정하였다. 암모니아태 질소(NH3-N)는 증류수를 가하여 청국장을 5배 희석, 진탕추출 후 여과지로 여과한 여액을 AM505-K(아산제약)에 의한 인돌페놀(Indophenol)법으로 측정하였다. γ-글루타밀트랜스펩티다아제 (γ-glutamyltranspeptidase;γ-GTP) 역가는 시료 10g을 취하여 증류수로 5배 희석하여 진탕추출한 다음 10,000×g에서 30분간 원심분리하여 상등액을 조효소액으로 하였다. 이 조효소액을 5-ami nolicylic acid법을 이용하는 AM158-K(아산제약)를 이용하여 측정하였다. 총균수는 청국장 1g을 취하여 멸균생리식염수를 이용한 10배 희석법으로 희석하고 플레이트 카운트 아가(plate count agar)에 도말한 후 30℃에서 48시간 배양한 다음 나타난 콜로니(colony)를 계수하여 호기성균수(CFU/g)로 나타내었다.Similar to Korea's Cheonggukjang, we use the Japanese natto raw soybeans, quasi-edge, anti-mutation, anti-cancer effect, soybeans, golden soybeans widely used as commercial soybeans, and US yellow 1, imported soybeans. The water content and pH, amino nitrogen and ammonia nitrogen, and γ-GTP activity were investigated after the fermentation of Cheonggukjang prepared in the first step. The general components of Cheonggukjang were analyzed according to AOAC method (Association ofofficial analytical chemists), moisture was dried at 105 ℃, crude protein was micro-Kjeldahl method, crude fat was analyzed by Soxhlet extraction method. pH was measured by pH meter (Corning 220, USA) by diluting the sample 10 times. Amino nitrogen (NH 2 -N) was measured by the formol method taking 2g of Cheonggukjang samples. Ammonia-like nitrogen (NH 3 -N) was distilled water was added to the distilled water 5 times dilution, shake extract and the filtrate filtered with filter paper was measured by the indophenol (Indophenol) method by AM505-K (Asan Pharmaceuticals). The γ-glutamyltranspeptidase (γ-GTP) titer was taken from 10 g of the sample, diluted five-fold with distilled water, shaken out, and centrifuged at 10,000 × g for 30 minutes to prepare the supernatant as a coenzyme solution. This coenzyme solution was measured using AM158-K (Asan Pharmaceutical Co., Ltd.) using the 5-ami nolicylic acid method. The total bacterial count was diluted by 10-fold dilution method using sterile physiological saline, 1 g of Cheonggukjang, and plated on plate count agar, incubated at 30 ° C for 48 hours, and then counted colonies (CFU). / g).
실험결과, 표 1에서 보는 바와 같이 황금콩 청국장의 수분함량이 가장 높고 준저리가 가장 낮았다. 한편 최종 청국장 제품의 pH는 준저리가 가장 높고 수입콩이 가장 낮았다. 이는 증자 대두 발효식품의 발효기간이 경과되면 탈아미노화 현상이 일어나 이취성분의 주체인 암모니아가 생성되기 때문에 아미노태 질소와 pH가 증가되는 것으로 볼 수 있다. 국산콩이 수입콩에 비하여 발효성적이 우수한 원인은 국산콩이 수입콩에 비해 상대적으로 단백질 함량이 높아 청국장균이 자라는 배지로서의 조건이 우수하며 수입콩은 수송과정에서 발생되는 부패와 해충 방제를 위한 약제 처리 등으로 인하여 청국장균이 자라는 조건을 저하시켰다고 생각된다. 도 1에서는 청국장 제품의 NH2-N, NH3-N, γ-GTP 활성을 보여주고 있다. 청국장의 구수한 맛을 좌우하는 아미노산 성분인 아미노태 질소는 만리콩이 비교적 높은 함량을 보였고 수입콩이 제일 낮았다. 청국장 불쾌취의 원인인 암모니아태 질소함량은 만리콩이 가장 낮았고 수입콩이 가장 높았다. 청국장의 점질물 구성성분은 프럭토스(fructose)와 글루타민산(glutamic acid)이 중합된 레반태프럭탄(levan form fructan)과 폴리글루타메이트(polyglutamate;PGA)의 혼합물질이며 PGA는 γ-GTP에 의해 생성되는 것으로 γ-GTP역가는 준저리가 가장 높고 수입콩이 가장 낮았다. 이로서 청국장 원료대두로는 수입콩과 대립종인 황금콩보다는 소립종 및 중립종이 우수하였는데 특히 중립종인 만리콩이 NH2-N 생성량이 높고 NH3-N 생성량은 낮으며 점질물 생성능이 적당하였다.As a result of the experiment, as shown in Table 1, the highest water content and lowest quasi-drangement of the golden soybeans Cheonggukjang. On the other hand, the pH of the final Cheonggukjang products was highest in quasi-edged and lowest in soybeans. This is because amino nitrogen and pH are increased because deamination occurs after fermentation period of steamed soybean fermented food and ammonia, a main ingredient of off-flavor, is produced. The reason that domestic soybeans have better fermentation performance than imported soybeans is that the domestic soybeans have a higher protein content than imported soybeans, so the conditions for growing soybean coli are excellent. It is thought that the conditions for the growth of Chungguk coli have been reduced due to drug treatment. Figure 1 shows the NH 2 -N, NH 3 -N, γ-GTP activity of the Cheonggukjang products. Amino-type nitrogen, an amino acid component that determines the taste of Cheonggukjang, has a relatively high content of soybeans and the lowest of soybeans. Ammonia-containing nitrogen, the cause of the odor of Cheonggukjang, was the lowest in Manali and the highest in imported soybean. Cheongukjang's viscous component is a mixture of levan form fructan and polyglutamate (PGA) polymerized with fructose and glutamic acid, and PGA is produced by γ-GTP. γ-GTP titers had the highest quasi-edge and lowest imported soybeans. As a result, raw soybeans were superior to imported soybeans and alleles such as golden soybeans and small and neutral soybeans. Especially, the neutral soybeans had high NH 2 -N production, low NH 3 -N production, and moderate viscous product ability.
제 3 공정: 증자방법을 달리하여 제조한 청국장의 품질 특성Third Process: Quality Characteristics of Cheonggukjang Prepared by Different Capital Increase
상기 제 1 공정에서 제조한 청국장은 증자시에 침수 후 탈수 시간은 0분, 45분, 60분으로 한 후 증자방법은 전통적인 방법인 삶기(충분한 수분공급으로 상압에서 4시간)와 찌기(400cc의 수분 공급으로 0.9kg/cm2의 압력밥솥에서 15분 증자), 개량적인 방법으로 이용되고 있는 오토클래브(autoclave;1.0∼1.5kg/cm2, 60분간)를 이용하여 청국장을 제조하여 품질특성을 비교하였다. 품질특성 비교를 위한 이화학적 특성 조사는 상기 제 2 공정에서 실시한 수분함량과 pH, 아미노태 질소와 암모니아태 질소함량, γ-GTP 활성(activity)의 측정방법과 동일하게 실시하였다.Cheonggukjang prepared in the first process, the dehydration time after immersion at the time of the increase of the steam dehydration time is 0 minutes, 45 minutes, 60 minutes and then the steaming method is the traditional method of boiling (4 hours at normal pressure with sufficient water supply) and steaming (400cc of Cheonggukjang is made by using autoclave (1.0-1.5kg / cm 2 , 60 minutes), which is used as an improved method, in a pressure cooker of 0.9kg / cm 2 . Was compared. The physicochemical characterization for comparing quality characteristics was carried out in the same manner as the method for measuring the water content and pH, amino nitrogen and ammonia nitrogen, and γ-GTP activity performed in the second step.
실험결과, 침수 후 탈수 시간을 0분, 45분, 60분으로 달리하고 증자 방법을 달리하여 발효시켜 제조한 청국장의 품질 특성은 표 2와 도 2에 나타난 바와 같았다. 탈수시간을 0분으로 하여 오토클래브(autoclave)시켜 제조한 청국장과 삶기와 찌기를 이용한 청국장은 수분함량이 각각 67.6%, 67.9%, 61.7%로 전통 식품 표준 규격(전통식품표준 규격집, 한국식품개발연구원, 1996.)의 55%이하에 부적합하였으며 탈수 시간을 45분과 60분으로 하여 오토클래브(autoclave)시켜 제조한 청국장은 수분함량이 각각 54.3%와 52.1%였다. pH와 청국장의 구수한 맛을 좌우하는 아미노산 성분인 아미노태 질소가 탈수 시간이 0분인 경우와 삶기의 경우 각각 7.95, 550mg%와 8.04, 497mg%로 높았다. 청국장 불쾌취의 원인인 암모니아태 질소 함량은 탈수 45분으로 하여 오토클래브를 이용한 경우 363㎍/dl로 가장 낮았다. 따라서 청국장 제조시 증자방법은 45분 탈수하고 고압살균하여 증자한 청국장이 수분함량,pH, 아미노태 질소, 암모니아태 질소함량 등을 비교할때 가장 우수하였다.As a result, the quality characteristics of Cheonggukjang prepared by fermentation by changing the dehydration time to 0 minutes, 45 minutes, and 60 minutes after different immersion methods and increasing the steaming method were shown in Table 2 and FIG. 2. Cheonggukjang made by autoclave with dehydration time of 0 minutes and Cheonggukjang made with boil and steaming have 67.6%, 67.9%, and 61.7% moisture content, respectively. Cheonggukjang prepared by autoclave with dehydration time of 45 minutes and 60 minutes was 54.3% and 52.1%, respectively. The amino acid nitrogen, the amino acid component that influenced the pH and the taste of Cheonggukjang, was high at 7.95, 550mg%, 8.04, and 497mg% for dehydration time of 0 min and boil, respectively. The ammonia nitrogen content, the cause of the odor of Chungkookjang, was 45 minutes for dehydration, which was the lowest at 363㎍ / dl using autoclave. Therefore, the cooking method of Cheonggukjang was the best when the water content, pH, amino nitrogen, ammonia nitrogen content, etc. were increased by 45 minutes of dehydration and autoclaving.
제 4 공정: 스타터를 달리하여 제조한 청국장의 품질 특성Fourth Process: Quality Characteristics of Cheonggukjang Prepared with Different Starters
제 1 공정에서 제조한 청국장은 발효시에 증자·냉각된 대두를 전통적인 방법인 볏짚을 이용하여 발효시키거나 그대로 40℃ 항온기에서 3일간 발효 또는 균종을 달리하여 전배양한 종균액을 원료의 1%(v/w) 접종하여 동일조건으로 발효시켰다. 품질특성은 제 2 공정에서 실시한 수분함량과 pH, 아미노태 질소와 암모니아태 질소함량, γ-GTP 활성(activity)의 측정방법과 동일하게 실시하여 조사하였다.Cheonggukjang prepared in the first process ferments the cooked soybeans cooked with steamed or cooled soybeans using rice straw, which is a traditional method, or is pre-cultivated for 3 days at 40 ° C in a constant temperature or different species. (v / w) inoculated and fermented under the same conditions. Quality characteristics were investigated in the same manner as in the measurement of water content and pH, amino nitrogen and ammonia nitrogen, and γ-GTP activity in the second process.
실험결과, 볏짚을 이용하거나 무접종의 재래식 방법과 균을 접종하는 개량식 방법으로 제조한 청국장의 품질특성을 비교한 결과를 표 3과 도 3에 나타냈다. 수분함량은 모든 군에서 전통표준규격에 적합하였고 대두를 그대로 자연 발효시킨 청국장은 pH와 아미노태 질소함량이 낮아 전반적으로 발효가 되지 않았다. 전통적인 방법인 볏짚을 이용한 청국장은 아미노태 질소함량은 가장 많고 암모니아태 질소함량은 가장 적었는데 이는 볏짚에 부착된 고초균에 의한 것으로 보이며 특히 볏짚이 발효시 발생되는 이취를 흡수해 주는 것으로 보인다. 균종을 달리하여 발효시킨 청국장에서 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)을 종균으로 제조한 청국장의 점질능이 두드러지게 높았다. 따라서 청국장 제조시 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)을 종균으로 하였을 때 수분함량, pH, 아미노태질소, 암모니아태질소함량, γ-GTP acitivity 등 전체를 비교할때 가장 우수하였다.As a result of the experiment, the results of comparing the quality characteristics of Cheonggukjang prepared by using the straw or the conventional method of inoculation with bacteria and inoculated bacteria are shown in Table 3 and FIG. 3. Moisture content was in compliance with the traditional standard in all groups, and the fermented soybeans were naturally fermented so that the pH and amino nitrogen content were low. Cheonggukjang using rice straw, which is a traditional method, has the highest amino nitrogen content and the lowest ammonia nitrogen content, which appears to be caused by Bacillus subtilis attached to rice straw. In particular, rice straw absorbs the off-flavor generated during fermentation. Cheonggukjang prepared with Bacillus licheniformis CN-115 as a seed in Cheonggukjang fermented with different strains was significantly higher. Therefore, when Bacillus licheniformis CN-115 was used as a seed during the production of Cheonggukjang, it was the best when comparing the total water content, pH, amino nitrogen, ammonia nitrogen content, γ-GTP acitivity, etc.
제 5 공정: 발효기간을 달리하여 제조한 청국장의 품질 특성Process 5: Quality Characteristics of Cheonggukjang Prepared with Different Fermentation Periods
제 1 공정에서 제조한 청국장을 발효시에 40℃에서 0 ∼ 5일간 발효하면서 pH, 아미노태 질소(NH2-N) 함량, 암모니아태 질소(NH3-N) 함량, γ-GTP 활성(activity), 총균수를 측정하였다. 방법은 제 2 공정과 동일하게 실시하였다.The fermented Cheonggukjang prepared in the first step was fermented at 40 ° C. for 0-5 days while pH, amino nitrogen (NH 2 -N) content, ammonia nitrogen (NH 3 -N) content, γ-GTP activity ), The total number of bacteria was measured. The method was carried out in the same manner as in the second step.
실험결과, 표 4와 도 4에 나타낸 바와 같이 pH는 발효기간 중 계속적으로 상승하였으며 아미노태 질소는 2일째 급속하게 증가하였다. 암모니아태 질소는 4일이후 급속히 증가하였으며 점질능은 발효 2일째 최고치를 기록한 후 서서히 감소하였다. 따라서, 품질 특성을 고려하였을 때 발효 2일째가 청국장의 최적발효기였다.As a result, as shown in Table 4 and Figure 4, the pH continuously increased during the fermentation period, and amino nitrogen increased rapidly on the second day. The ammonia nitrogen increased rapidly after 4 days and the viscous capacity decreased slowly after recording the highest value at 2 days of fermentation. Therefore, considering the quality characteristics, the second day of fermentation was the optimum fermentation period of Cheonggukjang.
제 6 공정: 부재료 첨가량에 따른 청국장 품질 특성6th Process: Cheonggukjang Quality Characteristics According to Substance Addition
청국장의 부재료 첨가비 표준화를 위하여 소금, 고춧가루, 마늘의 첨가농도를 각각 달리하여 적절한 배합비를 결정하였다. 청국장 100에 대하여 소금의 농도는 7.9%로 하고 고춧가루는 0.2%, 1.1%, 2.0%로 첨가하였고 마늘은 0.2%, 1.1%,2.0%로 조절하였다. 품질특성은 제 2 공정에서 실시한 방법과 동일하게 실시하였다.In order to standardize the ratio of the additives in Cheonggukjang, the appropriate concentration ratio was determined by varying the concentrations of salt, pepper powder and garlic. The concentration of salt was 7.9% and the red pepper powder was added at 0.2%, 1.1%, 2.0% and the garlic was adjusted to 0.2%, 1.1%, 2.0%. Quality characteristics were carried out in the same manner as in the second process.
실험결과, 표 5와 도 5에 나타낸 바와 같았다. 총균수는 숙성 0일 이후 청국장의 마늘첨가 농도순으로 감소하다가 일정한 수준을 유지하였다. 아미노태 질소의 함량은 5일째까지 급속히 증가하다 다시 서서히 증가하였다. 암모니아태 질소 함량은 고춧가루 2.0%+마늘 0.2% 청국장이 숙성 5일 후 꾸준히 감소하였다. 점질능은 부재료 첨가후 감소하였으며 고춧가루 2.0%+마늘 0.2% 청국장이 숙성 10일후 증가하였다. 따라서 발효 후 부재료를 첨가하여 숙성시 아미노태 질소와 암모니아태 질소함량, 점질능을 고려했을 때 고춧가루 2.0%+마늘 0.2% 청국장이 가장 우수하였다.Experimental results were as shown in Table 5 and FIG. The total number of bacteria decreased after 0 days of fermentation in the order of garlic addition concentration in Cheonggukjang and then maintained at a constant level. The content of amino nitrogen increased rapidly until day 5 and then increased slowly. Ammonia nitrogen content decreased steadily after 5 days of fermentation of red pepper powder 2.0% + garlic 0.2%. The viscous capacity was decreased after the addition of subsidiary materials, and pepper powder 2.0% + garlic 0.2% was added after 10 days of fermentation. Therefore, red pepper powder 2.0% + garlic 0.2% Cheonggukjang was the best when considering the amino nitrogen, ammonia nitrogen content and viscosity when fermentation was added after fermentation.
상기 제 1 ∼ 6 공정의 결과를 종합하면, 원료대두로 만리콩을 사용하고 이를 정선 및 수세하여 1.5배의 물(15℃)에 12시간 침지한 후 이를 45분간 탈수하여 오토클래브(Autoclave;1.0 ∼ 1.5kg/cm2)를 사용하여 60분간 증자하고 50℃로 냉각한 다음, 전배양한 종균액 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)을 접종하여 40℃, 48시간 발효시킨 후 소금 7.9%, 고춧가루 2.0%, 마늘 0.2%를 첨가하여 혼합 및 마쇄하고 5℃에서 15일간 후숙한 청국장이 풍미가 가장 좋은 청국장으로 표준화하였다.To sum up the results of the first to sixth step, using the soybeans as raw material soybeans were selected and washed with water, soaked in 1.5 times of water (15 ℃) for 12 hours and then dehydrated for 45 minutes to autoclave (Autoclave; 1.0 to 1.5kg / cm 2 ) was used for 60 minutes, cooled to 50 ° C., and then inoculated with Bacillus licheniformis CN-115, pre-cultured, and fermented at 40 ° C. for 48 hours. After salting, 7.9%, red pepper powder 2.0%, and garlic 0.2% were added and mixed, and ground. Cheonggukjang ripened at 5 ° C. for 15 days was standardized as the best flavor.
실시예 2:Example 2: 시험관내(in vitro)In vitro 에서 청국장의 항돌연변이성Mutagenicity of Cheonggukjang in Korea
본 실시예에서는 상기 실시예 1에서 대두품종별, 스타터 균주별, 발효기간별, 부재료별로 제조하여 품질 특성을 조사한 청국장을 시료로 사용하였다.In this example, Cheonggukjang prepared by soybean varieties, starter strains, fermentation periods, and subsidiary materials in Example 1 was used as a sample.
본 실시예에서 돌연변이원/발암원으로 간접돌연변이원인 아플라톡신 B1(Aflatoxin B1; AFB1)은 Sigma사에서 구입하여 디메틸 설폭사이드(dimethyl sufoxide;DMSO)에 녹여서 사용하였고 직접돌연변이원인 N-메틸-N'-니트로-N-니트로소구아니딘(N-methyl-N'-nitro-N-nitrosoguanidine;MNNG)는 Aldrich Chemical Co.(USA)에서 구입하여 증류수에 녹여서 사용하였다.Mutagen / Carcinogens in indirect mutation causes aflatoxin B 1 in this embodiment (Aflatoxin B 1; AFB 1) was purchased from Sigma, dimethylsulfoxide; was used to dissolve (DMSO dimethyl sufoxide) direct mutation causes N- methyl- N'-nitro-N-nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine; MNNG) was purchased from Aldrich Chemical Co. (USA) and dissolved in distilled water.
본 실시예에서 Ames test시에 사용한 시약 D-바이오틴(D-Biotin), L-히스티딘·HCl(모노하이드레이트){L-histidine·HCl(monohydrate)}, D-글루코스-6-포스페이트(모노 소듐염){D-glucose-6-phosphate(mono sodium salt)}, NADP(sodium salt){NADP(소듐염)}는 Sigma Chemical Co.(USA)에서 구입하였으며, 박토뉴트리언트 브로쓰(디하이드레이트){Bacto nutrient broth(dehydrated)}와 비테크 아가(Bitek agar)는 Difco Laboratories (USA)로부터 구입하였다.Reagents D-Biotin (D-Biotin), L-histidine-HCl (monohydrate) {L-histidine-HCl (monohydrate)}, D-glucose-6-phosphate (monosodium salt) used in the Ames test in this Example ) {D-glucose-6-phosphate (mono sodium salt)}, NADP (sodium salt) {NADP (sodium salt)} was purchased from Sigma Chemical Co. (USA), and bactonutrient broth (dihydrate) { Bacto nutrient broth (dehydrated)} and Bitek agar were purchased from Difco Laboratories (USA).
본 실시예에서 Ames test 시에 사용한 균주는 살모넬라 타이피무리움 LT-2 히스티딘(Salmonella typhimuriumLT-2 histidine) 영양요구성인 살모넬라 타이피무리움 TA100(Salmonella typhimuriumTA100)은 미국 캘리포니아 대학의 Ames B.N.박사로부터 제공받아 실험에 사용하였다. 이들 균주는 모균의 히스티딘 오페론(histidine operon)에서 돌연변이된 것으로 돌연변이 물질을 보다 효과적으로 검출하기 위해 세포벽의 리포폴리사카라이드(lipopolysaccharide)가 부분적으로 손실된rfa(deep rough) 돌연변이와, DNA 절단 리페어 시스템(DNA excision repair system)에 대한 유전자 암호(gene coding)가 부분적으로 결여된uvrB 결손 돌연변이, 그리고 암피실린(ampicillin)에 대해 내성을 가지는 플라스미드 pKM101(plasmid pKM101)을 가지는 균주이다. 살모넬라 타이피무리움 TA100(Salmonella typhimuriumTA100)은 히스티딘(histidine) 생합성의 첫번째 효소(enzyme)를 암호화(coding)하는 his G gene에 돌연변이를 일으켜 wide type(leucine)이(proline)으로 치환되어 있다. 그러므로 살모넬라 타이피무리움 TA100(Salmonella typhimuriumTA100)은 주로 이들 G-C 쌍들의 하나에서 염기-쌍 치환(base-pair substitution)을 일으키는 돌연변이 물질을 검출하기 위해 쓰인다. 그리고 이들 실험균주들은 새로운 동결보존(frozen permanent)이 준비되었을 때나 매 실험 직전 히스티딘(histidine) 요구성,rfa(deep rough) 돌연변이, uvrB 돌연변이, R factor 등의 유전형질을 Maron과 Ames 등의 방법으로 확인하여 사용하였다.The strain used in the Ames test in this embodiment is Salmonella typhimurium LT-2 histidine nutritional component Salmonella typhimurium TA100 (Dr. Salmonella typhimurium TA100) is Dr. Ames BN of the University of California, USA It was provided from and used for the experiment. These strains are mutated from the histidine operon of the parent strain, and in order to more effectively detect the mutant, a deep rough mutation (rfa ) with a partial loss of lipopolysaccharide in the cell wall, and a DNA cleavage repair system ( It is a strain having a vrmid pKM101 (plasmid pKM101) resistant to ampicillin, and uvr B deletion mutant partially lacking the gene coding for DNA excision repair system. Salmonella typhimurium TA100 mutated his G gene, which encodes the first enzyme of histidine biosynthesis. (leucine) replaced with (proline). Therefore Salmonella typhimurium TA100 is mainly used to detect mutants that cause base-pair substitution in one of these GC pairs. These strains were then analyzed for genotypes such as histidine requirements, rfa ( deep rough) mutations, uvrB mutations, and R factor, either when new frozen permanents were prepared or before each experiment. It was confirmed and used.
본 실시예에서 SOS chromotest에서 사용한 실험균주는 에스케리키아 콜리 GC4436(Escherichia coliGC4436)으로부터 유래된 에스케리키아 콜리 PQ37/플라스미드 pKM101(PQ37){Escherichia coliPQ37/plasmid pKM101(PQ37)}로써lacZgene을sfiAgene에 삽입하고 정상적인lacZgene은 제거되어 β-갈락토시다아제(β-galactosidase;β-G)의 활성은sfiA의 발현에 의해서만 나타날 수 있다. 또한 Ames test의 균주에서 처럼 uvrB 돌연변이(mutation)과rfa돌연변이(mutation)이 되어 있어 SOS 반응이 증폭되고 돌연변이원이 용이하게 균체내로 침투할 수 있다.Experimental strains used in the SOS chromotest in the present example is lacZ gene as Escherichia coli PQ37 / plasmid pKM101 (PQ37) derived from Escherichia coli GC4436 ( Escherichia coli GC4436) { Escherichia coli PQ37 / plasmid pKM101 (PQ37)} Insertion into the sfiA gene and normal lacZ gene is eliminated so that the activity of β-galactosidase (β-G) can only be expressed by the expression of sfiA . In addition, as in the strain of Ames test, uvrB mutations and rfa mutations are used to amplify the SOS response and mutagen can easily penetrate into cells.
이하, 청국장의 항돌연변이성 실험은 하기 공정과 같이 실시하였다.Hereinafter, the antimutagenicity test of Cheonggukjang was performed as follows.
제 1 공정: 청국장 시료 조제 및 추출Process 1: Preparation and Extraction of Cheonggukjang Samples
상기 제 1 실시예에서 제조하여 동결건조시킨 시료를 마쇄하여 시료에 20배(w/v)의 메탄올을 첨가하여 12시간 교반을 3회 반복한 후 여과하여 회전식 진공 농축기(EYELA, Tokyo Rikakikai Co., Japen)로 농축하여 메탄올 추출물(methanol extract)을 얻었다. 이들 추출물들은 증류수에 희석하여 실험에 사용하였다.The sample prepared in Example 1 was ground and lyophilized, and 20 times (w / v) of methanol was added to the sample, followed by repeated stirring for three hours for 12 hours, followed by filtration and a rotary vacuum concentrator (EYELA, Tokyo Rikakikai Co., Ltd.). , Japen) to give a methanol extract (methanol extract). These extracts were diluted in distilled water and used for the experiment.
제 2 공정: Ames test2nd process: Ames test
본 공정에서는 먼저 간접적 돌연변이원(Indirect mutagen)을 활성화시키기 위하여 Maron과 Ames의 방법에 따라 간의 마이크로좀 효소 혼합물(microsomal enzyme mixture)인 S9 혼합물(mixture)을 조제하였다. 약 200g의 웅성 Sprague-Dawley rat의 간 효소의 도입(induction)을 위하여 폴리크로리네이트 바이페닐 (polychlorinated biphenyl;PCB) 혼합물(mixture)인 Aroclor 1254를 옥수수 오일(corn oil) 1mL당 200mg의 농도로 희석하여 1회 복강 주사하고(500mg/kg) 5일 후에 간을 적출하였다. 4℃ 무균 상태에서 적출한 간을 0.15M KCl로 수회 세척하고 간 무게의 3배량의 0.15M KCl 용액을 가하여 균질기(homogenizer;Potter-Elvehiem apparatus, USA)로 균질화하였다. 이것을 9000×g에서 10분간 원심분리하여 상등액인 S9 분획(fraction)을 얻었으며 Cryo tube에 1∼2mL씩 분주하여 드라이 아이스(dry ice)에서 급속동결한 후 -80℃ 급속 동결(deep freezer)하여 보관하면서 실험에 사용하였다. 상기 S9 분획(fraction;10%)을 MgCl-KCl 염(salts;2%), 1M 글루코스-6-포스페이트(glucose-6-phosphate;0.5%), 1M NADP(4%), 0.2M 포스페이트 버퍼(phosphate buffer;pH 7.4) 및 멸균수를 역순으로 혼합하여 S9 혼합물을 조제하였다. 이어서, S9 혼합물 0.5mL(간접돌연변이인 경우), 하룻밤 배양된 균주(1 ∼2×109cells/mL) 0.1mL, 희석된 시료(50㎕)와 돌연변이 유발물질(50㎕)을 아이스 배스(ice bath)에 담긴 캡 튜브(cap tube)에 넣고 가볍게 볼텍스(vortex)한 후 37℃에서 30분간 예비배양하였다. 45℃의 상층 아가(top agar) 2mL씩을 각 튜브(tube)에 붓고 3초간 볼텍스(vortex)하여 최소 글루코스 아가 플레이트(minimal glucose agar plate)에 도말하고 37℃에서 48시간 배양한 후 복귀돌연변이 숫자를 계수하였다. 한편 실험에 사용된 시료와 돌연변이 유발물질의 농도는 예비실험(dose response 및 독성실험)을 통하여 결정하였다. 돌연변이 억제효과의 정도(inhibition rate)는 하기 식에 의해 계산하였다.In this process, S9 mixture, which is a microsomal enzyme mixture of liver, was prepared according to the method of Maron and Ames in order to activate indirect mutagen. Aroclor 1254, a polychlorinated biphenyl (PCB) mixture, was introduced at a concentration of 200 mg per 1 mL of corn oil for the introduction of about 200 g of male Sprague-Dawley rat liver enzymes. Diluted once intraperitoneally (500 mg / kg) and liver removed 5 days later. Liver harvested at 4 ° C. aseptic state was washed several times with 0.15M KCl, and homogenized with a homogenizer (Potter-Elvehiem apparatus, USA) by adding 0.15M KCl solution three times the liver weight. This was centrifuged at 9000 × g for 10 minutes to obtain S9 fraction as a supernatant. Dispense 1-2 mL in Cryo tube and rapidly freeze in dry ice and then deep freezer at -80 ℃. It was used for the experiment while storing. The S9 fraction (10%) was added with MgCl-KCl salts (2%), 1M glucose-6-phosphate (0.5%), 1M NADP (4%), 0.2M phosphate buffer ( phosphate buffer; pH 7.4) and sterile water were mixed in the reverse order to prepare an S9 mixture. Subsequently, 0.5 mL of S9 mixture (if indirect mutation), 0.1 mL of overnight cultured strain (1-2 × 10 9 cells / mL), diluted sample (50 μL) and mutagen (50 μL) were added to ice bath ( In a cap tube (ice bath) contained in an ice bath) was lightly vortex (vortex) and pre-incubated for 30 minutes at 37 ℃. Pour 2 mL of top agar at 45 ° C into each tube, vortex for 3 seconds, spread on minimal glucose agar plate, incubate at 37 ° C for 48 hours, and return mutations. Counted. Meanwhile, the concentrations of samples and mutagens used in the experiments were determined through preliminary experiments (dose response and toxicity experiments). Inhibition rate (mutation rate) was calculated by the following formula.
억제정도(%) = 100×[(ab)/(ac)]Suppression degree (%) = 100 × [(ab) / (ac)]
여기서 a는 돌연변이원에 의해 유도된 복귀돌연변이수, b는 시료를 처리하였을 때의 복귀돌연변이의 수이며, c는 돌연변이원과 시료가 없을 경우의 자연 복귀돌연변이의 수이다.Where a is the number of back mutations induced by the mutagen, b is the number of back mutations when the sample is processed, and c is the number of back mutations in the absence of the mutagen and the sample.
실험결과, 대두의 품종을 달리하였을 때에는 표 6에 나타낸 바와 같이 항돌연변이 효과는 큰 차이를 보이지 않았으나, 만리콩으로 제조한 청국장이 54%의 높은 항돌연변이 효과를 보였다. 스타터 균주를 달리하였을 때에는 표 7에 나타낸 바와 같으며 모든 청국장의 메탄올 추출물은 살모넬라 타이피무리움 TA100 (Salmonella typhimuriumTA100) 균주에서 AFB1에 대한 돌연변이 유발을 50%이상 억제하였으며, 균종을 달리하였을 때의 항돌연변이 효과는 큰 차이를 보이지 않았으나, 점질능이 우수하였던 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)을 사용하여 제조한 청국장의 항돌연변이 효과가 가장 뛰어나 63%의 돌연변이 유발억제효과를 나타내었다. 발효기간을 달리하여 제조한 청국장의 메탄올 추출물은 표 8에 나타낸 바와 같이 3일 발효시킨 청국장의 항돌연변이 효과가 가장 뛰어나 54%의 돌연변이 유발억제효과를 나타내었으며, 5일 발효시킨 청국장과 3일 발효 후 15일 후숙한 청국장은 항돌연변이 효과를 나타내지 못했다. 부재료를 달리하여 제조한 청국장의 메탄올 추출물은 표 9에 나타낸 바와 같이 항돌연변이 효과는 큰 차이를 보이지 않았으나 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%를 첨가하여 제조한 청국장이 44%의 돌연변이 유발억제효과를 나타내었다.As a result, when the varieties of soybeans were different, the antimutagenic effect did not show a big difference as shown in Table 6, but the Cheonggukjang made with the soybean showed a high antimutagenic effect of 54%. When different starter strains are shown in Table 7, methanol extracts of all Cheonggukjang inhibited more than 50% of the mutagenesis of AFB 1 in Salmonella typhimurium TA100 strains. The antimutagenic effect of did not show a significant difference, but the antimutagenic effect of Cheonggukjang prepared using Bacillus licheniformis CN-115, which had excellent viscosity, showed the highest antimutagenic effect of 63%. Indicated. Methanol extract of Cheonggukjang prepared with different fermentation periods showed the highest antimutagenic effect of Cheonggukjang fermented for 3 days as shown in Table 8, showing 54% mutagenesis-inhibiting effect. After 15 days, Cheonggukjang did not show antimutagenic effects. Methanol extract of Cheonggukjang prepared with different subsidiary ingredients showed no significant difference in antimutagenic effect as shown in Table 9, but 44% of Mookkukjang prepared by adding salt 7.9%, red pepper powder 1.1%, and garlic 1.1% The effect was shown.
제 3 공정: SOS chromotest3rd process: SOS chromotest
SOS chromotest는 구조 이동 돌연변이(Frame shift mutation)와 점 돌연변이(point mutation)를 동시에 측정할 수 있다. 냉동 보관된 균주 50㎕를 5mL의 L medium에 접종하고 37℃에서 12 ∼ 18시간 진탕배양시켰다. 이를 다시 5mL의 L medium에 접종하고 37℃에서 흡광도 660nm에서의 측정치가 0.3 ∼ 0.4에 이를 때까지 2시간동안 진탕배양시켰다. 여기서 얻은 균주를 L medium에 1/10로 희석하여 각 농도별로 준비된 시료와 돌연변이 유발물질에 혼합된 것을 20㎕를 미리 분주해 둔 96well 플레이트(plate)의 각 well에 100㎕씩 분주하고 90분간 37℃에서 진탕후 SOS 반응을 유도하고 한쪽에는 β-갈락토시다아제(β-galactosidase;β-G)의 활성측정을 위해 ONPG(o-nitrophenyl-β-D-galactopyranoside) 100㎕, 다른쪽에서는 알카린 포스파타아제(alkaline phosphatase;A-P)의 활성 측정을 위해 PNPP (p-nitrophenyl phosphate disodium) 100㎕를 첨가하였다. 발색시간은 30분으로 하였으며 β-갈락토시다아제는 1.5M Na2CO3100㎕로 알카린 포스파타아제(alkaline phosphatase)는 1M HCl 50㎕로서 효소에 의한 발색반응을 정지시키고 5분 후 알카린 포스페이트(alkaline phosphate)쪽에 50㎕의 2M 트리스 버퍼(Tris buffer)를 첨가하여 HCl을 중화하고 분광광도계(spectrophotometer)로 420nm에서 흡광도를 측정하였다. 측정된 O.D.값은 Miller의 공식에 의해 효소 단위(enzyme unit;Eu) 값을 구하였다.SOS chromotest can simultaneously measure frame shift mutations and point mutations. 50 μl of the frozen strain was inoculated in 5 mL of L medium and shaken at 37 ° C. for 12-18 hours. This was again inoculated in 5mL L medium and shaken for 2 hours until the measured value at absorbance at 660nm at 0.3 ℃ reached 0.3 ~ 0.4. Dilute the strain obtained here by 1/10 in L medium, and mix 100 μl of each well of the 96 well plate with 20 μl of the sample prepared at each concentration and mixed with the mutagen. Induces SOS response after shaking at ℃ and 100 μl of ONPG (o-nitrophenyl-β-D-galactopyranoside) to measure activity of β-galactosidase (β-G) on one side and eggs on the other 100 μl of PNPP (p-nitrophenyl phosphate disodium) was added to determine the activity of alkaline phosphatase (AP). The color development time was 30 minutes, β-galactosidase was 100M of 1.5M Na 2 CO 3 and alkaline phosphatase was 50M of 1M HCl. 50 μl of 2M Tris buffer was added to the alkaline phosphate to neutralize HCl, and the absorbance was measured at 420 nm with a spectrophotometer. The measured OD value was obtained by enzyme unit (Eu) value by Miller's formula.
Eu = (1000×A420)/t(min)Eu = (1000 × A 420 ) / t (min)
실험결과, MNNG에 대한 대두 4종류의 청국장의 SOS 반응 억제효과는 표 10에 나타낸 바와 같이 SOS chromotest에서도 Ames 실험계에서와 유사한 효과를 보였다. 만리콩으로 제조한 청국장이 높은 SOS 반응 억제효과를 보였고, 또한 준저리로 제조한 청국장도 높은 저해효과를 보였다(p＜0.05). MNNG에 대한 스타터 균주 4종류의 청국장 SOS 반응 억제효과는 표 11에 나타낸 바와 같이 균종을 달리하였을 때 큰 차이를 보이지 않았다. MNNG에 대한 발효기간별 청국장의 SOS 반응 억제효과는 표 12에 나타낸 바와 같이 3일 발효 후 SOS 반응 억제효과는 큰 차이를 보이지 않았다. MNNG에 대한 부재료의 양을 달리한 4종류의 청국장의 SOS 반응 억제효과는 표 13에 나타낸 바와 같이 Ames 실험계에서와 유사한 효과를 보였으며 부재료의 종류를 달리하였을 때의 SOS 반응 억제효과는 큰 차이를 보이지 않았다.As a result, SOS reaction inhibition effect of four types of Chungkookjang on MNNG showed similar effects to Ames experimental system in SOS chromotest as shown in Table 10. Cheonggukjang made with manly beans showed a high inhibitory effect on SOS reactions, while cheonggukjang made with quasi-edge foods showed a high inhibitory effect (p <0.05). Inhibition effect of the four kinds of starter strains SOS response to MNNG did not show a big difference when different strains as shown in Table 11. Inhibition effect of SOS response of Cheonggukjang by fermentation period for MNNG showed no significant difference in SOS response inhibition after 3 days fermentation. As shown in Table 13, the SOS reaction inhibition effect of four types of Chungkookjang with different amounts of subsidiary materials to MNNG showed similar effects as in the Ames experimental system. I didn't see it.
실시예 3:Example 3: 시험관내(in vitro)In vitro 에서 청국장의 항암성Cheonggukjang's anticancer activity
본 실시예에서는 상기 실시예 2와 같이 대두품종별, 스타터 균주별, 발효기간별, 부재료별로 제조하여 품질 특성을 조사한 청국장을 시료로 사용하였다.In this example, Cheonggukjang prepared by soybean varieties, starter strains, fermentation periods, and subsidiary materials as in Example 2 was used as a sample.
본 실시예에서 사용한 사용시약 및 기기는 세포배양을 위해 DMEM(Dulbeco's modified Eagle's medium), FBS(Fetal bovine serum), 0.05% 트립신(trypsin)-0.02% EDTA 그리고 100units/mL 페니실린-스트렙토마이신(Penicillin- Streptomycin)을 GIBCO사(USA)로 부터 구입하여 사용하였다. 세포배양은 CO2배양기(incubator;Sanyo, model MCO96, Japan)를 사용하였으며 암세포형태의 조직학적 관찰은 Olympus사의 inverted microscope(Olympus, model PM-10AK, Japan)를 이용하여 실험하였다.The reagents and devices used in this Example were Dulbeco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS), 0.05% trypsin-0.02% EDTA, and 100 units / mL penicillin-streptomycin (Penicillin-) for cell culture. Streptomycin was purchased from GIBCO (USA) and used. Cell culture was performed using a CO 2 incubator (Sanyo, model MCO96, Japan) and histological observations of cancer cell types were performed using an inverted microscope (Olympus, model PM-10AK, Japan) of Olympus.
본 실시예에서 실험에 사용된 암세포인 인체 위암세포(AGS, human gastric adenocarcinoma cell)는 한국 세포주 은행(서울의대)으로부터 분양받아 배양하면서 실험에 사용하였다. 암세포 배양 AGS 위암세포는 100units/mL의 페니실린-스트렙토마이신(Penicilin-Streptomycin)과 10%의 FBS가 함유된 DMEM을 사용하여 37℃, 5% CO2배양기에서 배양하였다. 배양된 각각의 암세포는 일주일에 2∼3회 재급여(refeeding)하고 6∼7일 만에 PBS로 세척한 후 0.05% 트립신(trypsin)-0.02% EDTA로 부착된 세포를 분리하여 원심분리한 후 집적된 암세포에 배지를 넣고 피펫으로 암세포가 골고루 분산되도록 잘 혼합하여 75mL 세포 배양 플라스크(cell culture flask)에 5mL씩 일정수 분할하여 주입하고 계속 6∼7일 마다 계대 배양하면서 실험에 사용하였다. 상기 배양된 암세포는 96 well plate에 well당 1×104mL이 되게 180㎕씩 분주하고 시료를 일정농도로 제조하여 20㎕ 첨가하여 37℃, 5% CO2배양기에서 72시간 배양하였다. 여기에 인산생리식염수에 5mg/mL의 농도로 제조한 MTT (3-(4,5-dimethyl-thiazol)-2,5-diphenyltetrazolium bromide) 용액 20㎕를 첨가하여 동 배양조건에서 4시간 더 배양하였다. 이를 550×g에서 10분간 원심분리하여 상징액을 제거하고, 각 well 당 DMSO 150㎕를 가하여 30분간 교반한 후 ELISA reader로 550nm에서 흡광도를 측정하였다. 세포독성은 하기의 식으로 구하였다.Human gastric adenocarcinoma cells (AGS), which are cancer cells used in the experiments in this example, were used for the experiment while being cultured by being fed from the Bank of Korea Cell Line (Seoul National University of Medicine). Cancer Cell Culture AGS gastric cancer cells were cultured in a 37 ° C., 5% CO 2 incubator using 100units / mL of Penicillin-Streptomycin and DMEM containing 10% FBS. Each cultured cancer cell was refeeded 2-3 times a week, washed with PBS in 6-7 days, centrifuged by detaching cells attached with 0.05% trypsin-0.02% EDTA. The medium was added to the integrated cancer cells, and the pipettes were mixed well so that the cancer cells were evenly distributed. The cells were injected into a 75 mL cell culture flask by dividing a predetermined number of 5 mL, and then used in experiments while subcultured every 6 to 7 days. The cultured cancer cells were divided into 180 μl so that 1 × 10 4 mL per well was put into a 96 well plate, and a sample was prepared at a predetermined concentration, and 20 μl was added thereto and incubated in 37 ° C. and 5% CO 2 incubator for 72 hours. To this was added 20 μl of a solution of MTT (3- (4,5-dimethyl-thiazol) -2,5-diphenyltetrazolium bromide) prepared at a concentration of 5 mg / mL to physiological phosphate saline, followed by further incubation for 4 hours under the same culture conditions. . The supernatant was removed by centrifugation at 550 × g for 10 minutes, and 150 μl of DMSO was added to each well, followed by stirring for 30 minutes. The absorbance was measured at 550 nm using an ELISA reader. Cytotoxicity was calculated by the following formula.
실험결과, AGS 인체 위암세포를 이용하여 대두 품종을 달리하여 제조한 청국장의시험관내(in vitro)항암효과는 표 14에 나타낸 바와 같이 만리콩으로 제조한 청국장이 MTT assay에서 AGS 인체 위암세포의 생존을 가장 크게 억제하여 각각 61%, 76%의 생존 저해효과를 나타내었다. 또한 준저리로 제조한 청국장도 AGS 인체 위암세포의 생존 저해효과가 다소 높아 50㎕/assay와 100㎕/assay에서 각각 51%, 64%의 저해효과를 보였다. 이상의 결과로부터 기능성의 측면에서 가장 우수한 청국장의 원료 대두로는 대립종보다는 소립 및 중립종이 우수하였는데 그중 만리콩이 가장 효과가 좋았다. AGS 인체 위암세포를 이용하여 스타터(Starter)의 종류를 달리하여 제조한 청국장의시험관내(in vitro)항암효과는 표 15에 나타낸 바와 같이 볏짚을 사용하여 제조한 청국장이 MTT assay에서 AGS 인체 위암세포의 생존을 가장 크게 억제하여 각각 74%, 79%의 생존 저해효과를 나타나 자연발효시는 볏짚사용이 우수하였다. 또한 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)로 제조한 청국장도 AGS 인체 위암세포의 생존 저해효과가 다소 높아 50㎕/assay와 100㎕/assay에서 각각 63%, 72%의 저해효과를 보였다. 이상의 결과로부터 기능성의 측면에서 가정 적절한 청국장의 종균으로 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)이 우수함을 알 수 있었다. AGS 인체 위암세포를 이용하여 발효기간을 달리하여 제조한 청국장의시험관내(in vitro)항암효과는 표 16에 나타내었다. 3일 발효 이후의 청국장 메탄올 추출물의 처리농도가 50㎕/assay일 때는 대개 AGS 인체 위암세포의 생존을 50%정도 억제하였으며, 100㎕/assay의 처리농도에서 60%이상의 생존 저해효과를 보였다. 특히 5일 발효한 청국장이 MTT assay에서 AGS 인체 위암세포의 생존을 가장 크게 억제하여 각각 55%, 79%의 생존 저해효과를 나타내었다. 한편 발효 이후 후숙과정에서 암세포의 생존 저해효과는 큰 차이를 보이지 않았다. 이상의 결과로부터 기능성의 측면에서 가장 적절한 청국장의 발효기간은 3일이 가장 적절함을 알 수 있었다. AGS 인체 위암세포를 이용하여 부재료를 달리하여 제조한 청국장의시험관내(in vitro)항암효과는 표 17에 나타내었다. 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%를 첨가하여 제조한 청국장이 MTT assay에서 AGS 인체 위암세포의 생존을 가장 크게 억제하여 각각 59%, 64%의 생존 저해효과를 나타내었고 부재료 첨가를 달리하여 제조한 청국장의 암세포의 생존 저해효과는 차이를 보이지 않았다. 이상의 결과로부터 기능성의 측면에서 청국장의 부재료 첨가 비율은 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%가 가장 우수함을 알 수 있었다.As a result, the in vitro anticancer effect of Cheonggukjang prepared by using different soybean varieties using AGS human gastric cancer cells was shown in Table 14. The greatest inhibition was found to be 61% and 76%, respectively. Cheonggukjang prepared by quasi-education also showed a slightly higher inhibitory effect on the survival of AGS human gastric cancer cells, showing 51% and 64% inhibition at 50µl / assay and 100µl / assay, respectively. From the above results, the most advanced soybeans in terms of functionality were small and neutral soybeans rather than alleles. Among them, the soybean was the most effective. In vitro anticancer effect of Cheonggukjang prepared by using different types of starter using AGS human gastric cancer cells was shown in Table 15. AGS human gastric cancer cells in MTT assay by Cheonggukjang prepared with rice straw The most inhibited survival was 74% and 79%, respectively, and the use of rice straw was excellent during natural fermentation. In addition, Cheonggukjang prepared with Bacillus licheniformis CN-115 has a slightly higher inhibitory effect on the survival of AGS human gastric cancer cells, which is 63% and 72% at 50µl / assay and 100µl / assay, respectively. Showed. From the above results, it was found that Bacillus licheniformis CN-115 was excellent as a seed of suitable Chungkookjang in terms of functionality. In vitro anticancer effects of Cheonggukjang prepared with different fermentation periods using AGS human gastric cancer cells are shown in Table 16. When the concentration of methanol extract of Chunggukjang after 50 days fermentation was 50µl / assay, AGS human gastric cancer cells generally inhibited about 50% survival and showed a 60% survival inhibition at 100µl / assay concentration. In particular, Cheonggukjang fermented for 5 days inhibited the survival of AGS human gastric cancer cells in the MTT assay, showing 55% and 79% survival inhibition, respectively. On the other hand, there was no significant difference in the inhibition of survival of cancer cells during the ripening process after fermentation. From the above results, it was found that the most appropriate period of fermentation of Chonggukjang was 3 days. In vitro anticancer effects of Cheonggukjang prepared with different substances using AGS human gastric cancer cells are shown in Table 17. Cheonggukjang prepared by adding salt 7.9%, red pepper powder 1.1% and garlic 1.1% showed the greatest inhibition of survival of AGS human gastric cancer cells in MTT assay, showing 59% and 64% survival inhibition effects, respectively. Survival inhibitory effect of cancer cells of the prepared Cheonggukjang did not show a difference. From the above results, it was found that the content of the additives in Cheonggukjang was 7.9%, red pepper powder 1.1% and garlic 1.1%.
따라서, 상기 실시예 2와 실시예 3의 결과에 따라 본 발명 항암 기능성 청국장은 만리콩을 정선 및 수세하여 1.5배의 물에 12시간 침지한 후 45분간 물빼기를 하고 이를 오토클래브(Autoclave)를 이용하여 60분 증자하고 50℃에서 냉각한 다음 전 배양한 종균액 바실러스 리체니포미스 CN-115(Bacillus licheniformisCN-115)를 접종하여 40℃에서 72시간 발효하여 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%를 첨가하여 혼합 및 마쇄한 청국장을 항암 기능성이 가장 우수한 청국장으로 표준화하였다.Therefore, according to the results of Examples 2 and 3, the anticancer functional cheonggukjang of the present invention was selected and washed with soybeans for 12 hours in 1.5 times of water, followed by 45 minutes of drainage, and the autoclave. After 60 minutes of steaming and cooling at 50 ° C, inoculated with Bacillus licheniformis CN-115, pre-cultivated seed solution Bacillus licheniformis CN-115, fermented at 40 ° C for 72 hours, salt 7.9%, red pepper powder 1.1%, Cheonggukjang mixed and ground by adding 1.1% garlic was normalized to the best cheonggukjang.
실시예 4:Example 4: 시험관내(in vitro)In vitro 에서 청국장의 항암효과Anticancer Effect of Cheonggukjang in Korea
본 실시예에서는 상기 실시예 3에서 항돌연변이성과 항암성이 있는 것으로 확인되어 표준화한 본 발명 항암 기능성 청국장과 전통방식으로 제조된 청국장, 개량된 방식으로 제조된 청국장 및 낫또(Natto)의 항돌연변이효과와 항암효과를시험관내(in vitro)에서 조사하여 비교하였다. 이때, 전통적 방법은 상압에서 시루에 물을 넉넉히 하여 4시간 푹 삶고 이를 50℃로 식혀 볏짚을 깐 시루에 넣고 볏짚을 덮어서 축축한 면보자기나 담요를 쒸워 따뜻한 방아랫목(습도 80∼90%, 40℃)에서 3일 발효시키고 발효가 끝난 청국장 메주는 소금 7.9%, 마늘 1.1%, 고춧가루 1.1%를 첨가하여 절구에 굵고 균일하게 찧어 항아리에 담아 서늘한 곳(5∼10℃)에서 15일간 후숙시켰다. 개량적 방법은 stainless steel용기에 1.0∼1.5 kg/cm2에서 60분 증자한 뒤 50℃로 식혀Bacillus subtilis종균을 원료무게의 1% 접종하여 40℃에서 72시간 발효시킨다. 발효가 끝나면 소금만을 첨가하여 혼합·마쇄한 후 숙성과정을 생략하였다.In this embodiment, the antimutagenicity and anti-mutagenic effects of the anti-cancer functional cheonggukjang prepared in the traditional embodiment and the cheonggukjang prepared in a traditional manner, the cheonggukjang prepared in an improved manner and natto (Natto) And anticancer effects were investigated in vitro . At this time, the traditional method is to boil water for 4 hours in normal pressure at normal pressure, cool it to 50 ℃, put it in rice straw covered with rice straw, and cover it with rice straw cloth or steamed cotton cloth or blanket. ) Fermented for 3 days and fermented chungkukjang meju was added 7.9% of salt, 1.1% of garlic, and 1.1% of red pepper powder, and coarsely and steamed in mortar and put in a jar for 15 days in a cool place (5 ~ 10 ℃). An improved method is to add 60 minutes at 1.0∼1.5 kg / cm 2 in stainless steel container, cool to 50 ℃ and inoculate Bacillus subtilis spawn with 1% of raw material weight and ferment at 40 ℃ for 72 hours. After fermentation, only salt was added and mixed and crushed, and the aging process was omitted.
상기와 같이 제조방법에 따른 청국장의 항돌연변이성 조사는 실시예 2와 동일하게 실시하였고 항암성은 실시예 3의 방법과 동일하게 실시하였다.As described above, the antimutagenicity of Cheonggukjang was conducted in the same manner as in Example 2, and the anticancer activity was performed in the same manner as in Example 3.
실험결과, 표 18에 나타낸 바와 같이 상기 실시예 2에서 항돌연변이성이 확인된 기능성 청국장 메탄올 추출물이 첨가농도 2.5mg/plate에서 31% 정도로 AFB1의 돌연변이성을 저해하였고 첨가농도 5mg/plate에서는 63% 정도로 유사한 항돌연변이 효과를 관찰할 수 있었다. AGS 인체 위암세포를 이용하여 기능성 청국장, 전통식과 개량식으로 제조된 청국장 및 낫또(Natto)의 메탄올 추출물의시험관내(in vitro)항암효과는 표 19에 나타낸 바와 같이 기능성 청국장이 MTT assay에서 AGS 인체 위암세포의 생존을 억제하여 각각 44%, 67%의 생존 저해효과를 나타내었다.As a result, as shown in Table 18, the functional Cheongguk methanol extract confirmed antimutagenicity in Example 2 inhibited the mutagenicity of AFB 1 to about 31% at 2.5 mg / plate concentration and 63 at 5 mg / plate concentration. Similar antimutagenic effects could be observed. In vitro anticancer effect of functional Cheonggukjang, Chunggukjang and Natto's methanol extracts prepared using AGS human gastric cancer cells in the AGS human stomach in MTT assay Inhibiting the survival of cancer cells showed 44%, 67% survival inhibition effect, respectively.
따라서 본 실시예의시험관내(in vitro)실험결과로부터 표준화한 본 발명 항암 기능성 청국장이 전통식 또는 개량식으로 제조된 청국장 및 일본의 낫또(Natto)보다 항돌연변이 및 항암효과가 우수함을 알 수 있었다.Therefore, from the in vitro test results of this embodiment, the anticancer functional cheonggukjang of the present invention was found to have better antimutagenic and anticancer effects than Cheonggukjang prepared in a traditional or improved manner and natto of Japan.
실시예 5:Example 5: 생체내(in vivo)In vivo 에서 청국장의 항암효과Anticancer Effect of Cheonggukjang in Korea
본 실시예에서는 상기 실시예 4에 의해시험관내(in vitro)에서 확인된 본 발명에서 표준화한 항암 기능성 청국장 제조방법에 의해 제조된 본 발명 항암 기능성 청국장의 항암효과를생체내(in vivo)에서 조사하기 위해 먼저 청국장의 세포독성실험을 행한 후 고형암 성장저지실험과 자연살해세포 활성조사 실험을 차례로 실시하였다. 실험동물은 웅성 Balb/c mouse(한국화학연구소, 대전)로, 체중이 25g 전후의 것을 사용하였으며, 사료는 표준사료로 사육하였다. 사육시 물과 사료는 충분한 양을 공급하였고, 동물실험실은 온도 22±1℃, 습도 55±5%를 유지하였으며, 12시간 간격으로 명암주기(light-dark cycle)를 유지하였다.In this embodiment, the anticancer effect of the anticancer functional Cheonggukjang of the present invention prepared by the anticancer functional Cheonggukjang production standardized in the present invention confirmed in vitro by Example 4 was investigated in vivo. To do this, first, the cytotoxicity test of Cheonggukjang was conducted, followed by solid cancer growth inhibition test and natural killer cell activity test. The experimental animals were male Balb / c mouse (Korea Chemistry Research Institute, Daejeon), and weighed about 25g. The feed was raised as standard feed. During breeding, water and feed were supplied in sufficient quantities, and the animal laboratory maintained a temperature of 22 ± 1 ° C and a humidity of 55 ± 5%, and maintained a light-dark cycle at 12 hour intervals.
종양세포는 Balb/c mouse의 복강내에 1주일 간격으로 계대배양하여 보존하고 있는 sarcoma-180 세포를 실험용 종양세포로 사용하였다. 즉 실험동물의 복강내에서 1주일간 배양된 sarcoma-180세포를 복수와 함께 취하고 포스페이트 버퍼 사린(phosphate buffered saline;PBS)과 함께 원심분리(1,200 rpm, 10min.)하여 종양세포를 분리하였다. 분리된 세포를 다시 PBS에 부유시켜 재차 원심분리하여 상등액을 제거한 후 1.0×106cells/mL가 되도록 종양세포 부유액을 만들어 1mL씩을 복강주사하여 이식 보존하면서 실험에 사용하였다. 사용한 청국장 시료는 멸균된 PBS를 사용하여 조제하였으며 투여량은 마우스 ㎏당, 1㎎으로 하고, 대조군은 멸균 PBS만 투여하였으며, 투여하지 않을 때는 냉장고에 보관하면서 사용하였다.Tumor cells were subcultured at weekly intervals in the abdominal cavity of Balb / c mice, and sarcoma-180 cells were used as experimental tumor cells. That is, sarcoma-180 cells cultured for one week in the abdominal cavity of experimental animals were taken with ascites and centrifuged (1,200 rpm, 10 min.) With phosphate buffered saline (PBS) to separate tumor cells. The separated cells were suspended in PBS again, centrifuged again to remove the supernatant, and tumor suspensions were made to 1.0 × 10 6 cells / mL. The used Cheonggukjang samples were prepared using sterile PBS, and the dosage was 1 mg / kg mice, and the control group was administered only sterile PBS.
제 1 공정: 세포독성실험Process 1: Cytotoxicity Test
항암효과를 나타낸 시료의 직접적인 세포 독성작용의 유무를 알아보기 위해서 다이 익스클루젼법(dye exclusion method)를 이용하여시험관내(in vitro)에서 생존시험(viability test)을 행하였다. 1회용 24well plate에 조제한 종양세포 부유액 1mL(2.0 ×105cells)과 최종 농도 20% HFCS(heat inactivated fetal calf serum: Gibco Lab., USA)의 RPMI 배지 1mL를 가하였다. 여기에 100㎕의 시료를 넣어서 37℃, 5% CO2배양기(incubator)에서 24시간 배양하였다. 배양 후 세포 50㎕를 0.2% 트립신 블루(trypan blue) 용액 50㎕와 잘 섞어 헤모사이토메터 (hemocytometer)를 사용하여 전체의 세포수와 염색되어진 세포(non-viable cell) 및 염색되지 않은 세포(viable cell)의 수를 측정한 다음, 시료를 넣지 않은 대조 세포군과 비교하여 생존(viability) 비율을 계산하였다.In order to examine the direct cytotoxicity of the anti-cancer sample, viability test was performed in vitro using the die exclusion method. 1 mL (2.0 × 10 5 cells) of tumor cell suspension prepared in a disposable 24well plate and 1 mL of RPMI medium of 20% HFCS (heat inactivated fetal calf serum: Gibco Lab., USA) were added. 100 μl of sample was added thereto and incubated at 37 ° C. in a 5% CO 2 incubator for 24 hours. After incubation, 50 μl of cells were mixed well with 50 μl of 0.2% trypan blue solution, and the total number of cells, non-viable and non-stained cells (viable) using a hemocytometer. The number of cells) was measured and then the viability ratio was calculated compared to the control cell group without the sample.
실험결과, 표 20에 나타낸 바와 같이 모든 시료가 1.0mg/mL까지 독성이 없었으므로 1.0mg/kg의 시료를 마우스 복강에 투여하여 고형암성장저지 효과를 조사할 수 있음을 알 수 있었다.As a result of the experiment, as shown in Table 20, all the samples were not toxic to 1.0 mg / mL, so it can be seen that the administration of the 1.0 mg / kg sample to the abdominal cavity of the mouse was investigated the effect of inhibiting solid cancer growth.
제 2 공정: 고형암 성장저지 실험Second Process: Solid Rock Growth Prevention Experiment
실험동물을 각 군당 6마리씩으로 하여 실험실에서 1주일 간격으로 계대 보관 중인 종양세포부유액 0.2㎖(6×106cells/mouse)씩을 실험동물의 왼쪽 서혜부(left groin)에 피하 이식한 후 24시간 후부터 20일간 매일 1회씩 시료 용액를 복강으로 투여하였다. 종양세포 이식 26일째 되는 날 치사시켜 간, 비장 등의 장기의 중량변화를 조사하고 생성된 고형암을 적출하여 그 무게를 측정한 후 하기 식에 따라 종양 성장 저지 백분율( tumor growth inhibition ratio, I.R.: %)을 계산하였다.Six experiment animals were used in each group, and 0.2 ml (6 × 10 6 cells / mouse) of tumor cell suspension, which was kept in the laboratory at weekly intervals, was subcutaneously implanted into the left groin of the experimental animals 24 hours later. The sample solution was administered intraperitoneally once daily for 20 days. On the 26th day after tumor cell transplantation, the weight change of organs such as liver and spleen were examined, and the resulting solid cancer was extracted and weighed. Tumor growth inhibition ratio (IR:%) was calculated according to the following formula. ) Was calculated.
Cw : 대조군의 평균 종양무게Cw: mean tumor weight of control group
Tw : 처리군의 평균 종양무게Tw: mean tumor weight of treatment group
실험결과, 표 21에 나타낸 바와 같이 종양세포만 이식한 대조군은 정상마우스보다 비장, 간의 중량비가 크게 증가하였고 청국장 시료를 투여한 군에서 이들의 중량비가 감소하였다. 종양이 무게는 표 22에 나타낸 바와 같이 종양세포만 이식한 대조군은 종양의 무게가 7.1g이었으나 전통식 청국장 투여군은 종양의 무게가 5.0g으로 감소하여 30%의 종양생성억제효과를 나타내었다. 개량식청국장 투여군은 전통식 청국장보다 높은 35%의 종양형성억제효과를 보였고, 표준화한 본발명 항암 기능성 청국장 투여군은 종양의 무게가 3.1g으로 56%의 종양형성 억제효과를 보여 청국장 중에서는 가장 높은 고형암 성장저지효과를 나타내었다.As a result, as shown in Table 21, the control group transplanted with tumor cells only showed a greater increase in the weight ratio of the spleen and liver than normal mice, and their weight ratio decreased in the group administered with Cheonggukjang samples. As shown in Table 22, the weight of the tumor was 7.1g in the control group transplanted with only the tumor cells, but the weight of the conventional Cheonggukjang group was reduced to 5.0g, resulting in 30% tumor suppression effect. The improved Chungcheongjang-administered group showed 35% higher tumor suppression effect than the traditional Cheonggukjang, and the standardized anti-cancer functional Cheonggukjang-administered group showed 3.1% tumor weight and 56% tumor suppression effect. It has a blocking effect.
제 3 공정: 면역활성 증강 실험Third Step: Immune Activity Enhancement Experiment
표준화한 본 발명 항암 기능성 청국장의 면역활성 증강실험을 실시하기 위해먼저 마우스 비장 림프구의 분리하여 이펙터 세포(Effector cell)를 제조하였다. 즉, Balb/c 마우스의 비장을 clean bench 내에서 무균적으로 적출하여 penicillin(100 U/mL), 스트렙토마이신(streptomycine;100㎍/mL)를 함유한 5mL의 RPMI 1640 배지로 3회 세척한 후 곱게 마쇄하였다. 이 세포부유액을 70㎛ nylon mesh로 여과하고 원심분리하여 림프구를 모은 다음 배지에 부유시킨 후 histopaque-1077을 이용하여 원심분리(500×g, 30 min, 18℃)하여 림프구를 분리하였다. 준비된 세포를 10% FCS, 2mM L-glutamine, penicillin(100 U/㎖), streptomycin(100㎍/㎖)를 함유한 RPMI 1640 배지에 재현탁시키고, 이것을 37℃, CO2incubator에서 1∼2시간 배양시켜서 세포가 배양 플라스크(60㎜ dish)에 부착되도록 하였다. 비부착성 자연살해(natural killer;NK) 세포를 원심분리를 통해서 수집하여 배양배지에 재현탁시킨 다음 적정 세포수를 계수하여 사용하였다. 이어서, 자연살해세포의 활성을 측정하였다. Yac-1 마우스 임파구 세포(mouse lymphoma cell)를 타겟세포(target cell)로 사용하였는데, Yac-1 세포는 10% FCS, 2mM L-glutamin, penicillin(100 U/mL), streptomycin(100㎍/㎖)를 함유한 RPMI 1640 배지에 5×104cells/mL의 밀도가 되도록 희석한 후, 각 well에 50㎕씩 첨가하였다. NK cell은 1×107cells/mL의 밀도로 현탁시켜 50㎕씩을 96well plate-bottomed microtire plate에 첨가하였다. 이것을 37℃, CO2incubator 안에서 3일간 배양시킨 후 MTT(5㎎/㎖)용액을 10㎕씩 각 well에 가한 다음 37℃에서 4시간 배양하였다. 여기에 SDS(10% in 0.02N HCl)을 25㎕ 첨가하여 실온에서 30분간 발색시킨 후에 540nm에서 OD를 측정하였다. 세포독성 백분율(Cell cytolysis percentage)는 하기 식에 의해서 계산하였다.In order to carry out a standardized immunological enhancement of the anticancer functional cheongukjang of the present invention, mouse spleen lymphocytes were first isolated to prepare effector cells. In other words, spleens of Balb / c mice were removed aseptically on a clean bench and washed three times with 5 mL of RPMI 1640 medium containing penicillin (100 U / mL) and streptomycin (100 μg / mL). Finely ground. The cell suspension was filtered through a 70 μm nylon mesh, centrifuged to collect lymphocytes, suspended in media, and then centrifuged using histopaque-1077 (500 × g, 30 min, 18 ° C.) to separate lymphocytes. The prepared cells were resuspended in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, penicillin (100 U / ml), streptomycin (100 µg / ml), and these were reconstituted in 37 ° C., CO 2 incubator for 1-2 hours. The cells were allowed to adhere to the culture flask (60 mm dish). Non-adherent natural killer (NK) cells were collected by centrifugation, resuspended in culture medium, and the appropriate number of cells was counted. Subsequently, the activity of natural killer cells was measured. Yac-1 mouse lymphocyte cells were used as target cells. Yac-1 cells were 10% FCS, 2 mM L-glutamin, penicillin (100 U / mL), streptomycin (100 µg / mL). ) Was diluted to a density of 5 × 10 4 cells / mL in RPMI 1640 medium containing 50 μl to each well. NK cells were suspended at a density of 1 × 10 7 cells / mL and 50 μl each was added to 96well plate-bottomed microtire plates. This was incubated for 3 days in 37 ℃, CO 2 incubator, Ml (5mg / ㎖) solution was added to each well 10μl each incubated at 37 ℃ 4 hours. 25 μl of SDS (10% in 0.02N HCl) was added thereto, followed by color development at room temperature for 30 minutes, and then OD was measured at 540 nm. Cell cytolysis percentage was calculated by the following formula.
실험결과, 도 6에 나타낸 바와 같이 Yac-1 세포에 대한 세포파괴 활성이 전통방법으로 제조한 청국장 투여군은 40%, 전통방법을 개량한 방법으로 제조한 청국장과 표준화한 본 발명 항암 기능성 청국장을 투여한 군은 각각 44%와 64%로 기능성 청국장 투여군이 청국장 투여군 중에서는 NK cell의 면역활성이 가장 높았다. 또한 청국장 투여군들은 모두 낫또(37%)보다는 높은 세포파괴활성을 보였다.As a result, as shown in FIG. 6, 40% of the cell-destructive activity of Yac-1 cells prepared by the traditional method was administered 40%, and the standardized anticancer functional cheonggukjang of the present invention was prepared by the conventional method In one group, 44% and 64%, respectively, were the highest in NK cells. In addition, all of the cheongguk administration group showed higher cell destruction activity than natto (37%).
이상, 상기 실시예와 실험예를 통하여 설명한 바와 같이, 원료대두로 중소립콩을 정선 및 수세하고 1.5배의 15℃ 물에 12시간 침지한 후 탈수시간을 45분으로 하고 1.0∼1.5㎏/㎠ 오토클래브(Autoclave)를 사용하여 60분간 증자하고 전배양한 종균액을 접종하여 40℃에서 48시간 발효시킨 후 소금 7.9%, 고춧가루 2.0%, 마늘 0.2%를 첨가하여 혼합 및 마쇄하고 5℃에서 15일간 후숙하여 제조한 청국장은 풍미가 우수한 뛰어난 효과가 있고 또 중소립콩을 위와 같은 방법으로 처리하고 오토클래브를 사용하여 60분 증자하고 전배양한 종균액을 접종하여 40℃에서 72시간 발효시킨 후 소금 7.9%, 고춧가루 1.1%, 마늘 1.1%를 첨가하고 혼합 및 마쇄하여 바로 제조한 청국장은 항암 기능성이 우수한 항암 기능성 청국장으로 뛰어난 효과가 있으므로 식품산업상 매우 유용한 발명인 것이다.As described above, the small and medium soybeans were selected and washed with raw soybeans and immersed in 1.5 times of 15 ° C. water for 12 hours as described in the above Examples and Experimental Examples. Incubate with autoclave for 60 minutes, inoculate precultured seed stock, ferment for 48 hours at 40 ℃, mix and grind with salt 7.9%, red pepper powder 2.0%, garlic 0.2% and mix and grind 15 at 5 ℃. Cheonggukjang prepared after ripening for one day has excellent flavor, and it is processed with small and medium soybeans in the same way as above. Cheonggukjang prepared directly by adding, mixing, and grinding salt 7.9%, red pepper powder 1.1%, and garlic 1.1% is an anti-cancer functional Cheonggukjang with excellent anticancer function. will be.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2000-0049010A KR100431277B1 (en) | 2000-08-23 | 2000-08-23 | functional Chungkookjang and process for preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2000-0049010A KR100431277B1 (en) | 2000-08-23 | 2000-08-23 | functional Chungkookjang and process for preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20020021178A KR20020021178A (en) | 2002-03-20 |
| KR100431277B1 true KR100431277B1 (en) | 2004-05-12 |
Family
ID=19684755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR10-2000-0049010A KR100431277B1 (en) | 2000-08-23 | 2000-08-23 | functional Chungkookjang and process for preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100431277B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040046570A (en) * | 2002-11-27 | 2004-06-05 | 박정이 | Preparation of Powdered Cheonggukjang Using Onion and Garlic |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100903186B1 (en) * | 2006-09-12 | 2009-06-17 | 최청 | A preparation method of chungkukjang using Bacilluse licheniformis |
| KR100823896B1 (en) * | 2007-01-12 | 2008-04-21 | 전북대학교산학협력단 | Enhancement of isoflavone contents in soybean by pretreatment and method for chungukukjang with improved functionality |
| KR100886154B1 (en) * | 2007-02-01 | 2009-02-27 | 황민제 | The hot taste is removed and the garlic exchange where the bamboo salt which is kneaded in garlic pollination and position misfortune and Chung director of a bureau are mixed and the manufacturing method |
| KR101249516B1 (en) * | 2010-11-16 | 2013-04-01 | 민 환 오 | A fermented soybeans and manufacturing methodof that |
| KR101870419B1 (en) | 2016-11-16 | 2018-06-27 | 농업회사법인주식회사니파바이오 | Method for preparing chungkookchang comprising nipa fruticans wurmb and chungkookchang by the method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR910007446A (en) * | 1989-10-17 | 1991-05-30 | 황주석 | Manufacturing method of unsalted fortified food which improved Chunggukjang |
| KR100324132B1 (en) * | 1999-08-17 | 2002-02-20 | 김근실 | Chonggugjang Made of Sword Bean and Process for Preparing the Same |
-
2000
- 2000-08-23 KR KR10-2000-0049010A patent/KR100431277B1/en active IP Right Grant
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR910007446A (en) * | 1989-10-17 | 1991-05-30 | 황주석 | Manufacturing method of unsalted fortified food which improved Chunggukjang |
| KR100324132B1 (en) * | 1999-08-17 | 2002-02-20 | 김근실 | Chonggugjang Made of Sword Bean and Process for Preparing the Same |
Non-Patent Citations (3)
| Title |
|---|
| Paper 1997. 논문 1997 * |
| 한국농화학회지, 42권 2호, 91~98쪽 * |
| 한국식품과학회, 창립 30주년 기념 심포지움, 제 61차 학술발표회 및 정기총회, 468쪽 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040046570A (en) * | 2002-11-27 | 2004-06-05 | 박정이 | Preparation of Powdered Cheonggukjang Using Onion and Garlic |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20020021178A (en) | 2002-03-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102134209B1 (en) | Novel strains derived from fermented food and having with excellent enzyme activity and method for producing grains-fermented food using the same | |
| KR101408568B1 (en) | Process of fermented food containing enzyme by using mixed Aspergillus oryzae and lactic acid bacteria | |
| KR101761379B1 (en) | Novel Lactobacillus sakei K040706 for multi function and culture method thereof | |
| US11044928B2 (en) | Method for preparing rice hot pepper paste and rice hot pepper paste prepared thereby | |
| KR100445685B1 (en) | Medium composition for lactobacillus comprising mushroom powder or mushroom extracts, composition of mushroom lactic acid fermentation using the medium composition, and method for manufacturing the composition of mushroom lactic acid fermentation | |
| KR100431277B1 (en) | functional Chungkookjang and process for preparation thereof | |
| JP2023000116A (en) | Production method of honey fermentation product | |
| KR100987990B1 (en) | Mixed Culturing method of Aspergillus oryzae and Bacillus subtilis in Rice bran, and it's processing methods | |
| KR102536627B1 (en) | Composition for preventing or improving alcoholic liver injury comprising fermented kiwi | |
| WO2020146650A1 (en) | Food and beverage products comprising ascomycetes | |
| KR100503570B1 (en) | Novel Pediococcus pentosaceus EROM101 having immune enhancement, anticancer and antibacterial activities | |
| KR100787756B1 (en) | Culturing method of aspergillus oryzae and bacillus subtilis in the mixture of rice bran and soybean, and it's processing method | |
| KR100242830B1 (en) | New strain bacillus sk-31, fermentative bean milk using the same and processes for the preparation thereof | |
| KR102282725B1 (en) | Weissella cibaria strain having rice cake anti-retrogradation activity and use thereof | |
| KR100311256B1 (en) | Nato products using black-soybean and process of preparation thereof | |
| KR20180007633A (en) | Dough fermentation method for producing an anti-obesity effect | |
| KR102007569B1 (en) | Fermentation product of seomae mugwort with high palatability and functionality, and method for preparing the same | |
| KR100385658B1 (en) | A grain culture composition of inonotus obliquus, a method of preparing the antitumor and immuno-activity polysaccharide from it and their use | |
| CN106795541A (en) | The autofrettage of the culture containing menadione 7 and menadione 7 | |
| KR101871904B1 (en) | Leuconostoc mesenteroides YSM1219 and its use | |
| KR101097838B1 (en) | A method for preparing fermented soymilk having improved good sensory property and Lipid lowering ability by Bacillus subtilis and Corni fructus, and fermented soymilk for prevention atherosclerosis obtained from this method | |
| KR100368183B1 (en) | New Strain Bacillus licheniformis B1 and Usages Thereof | |
| KR20180088947A (en) | Manufacturing method of lotus leaf fermented extract, meat producing stuff having anti-obesity functional using the fermented extract anc meat product thereby | |
| KR102577648B1 (en) | Composition for preventing, ameliorating or treating obesity comprising Bacillus subtilis SB051 strain or fermented soybean using the same as effective component | |
| KR102586000B1 (en) | Lactobacillus fermentum HDB1098 that selectively degrades acetaldehyde and composition for removing hangover containing the same as an active ingredient |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| N231 | Notification of change of applicant | ||
| GRNT | Written decision to grant | ||
| FPAY | Annual fee payment |
Payment date: 20130415 Year of fee payment: 10 |
|
| FPAY | Annual fee payment |
Payment date: 20140502 Year of fee payment: 11 |
|
| FPAY | Annual fee payment |
Payment date: 20150421 Year of fee payment: 12 |
|
| FPAY | Annual fee payment |
Payment date: 20160503 Year of fee payment: 13 |
|
| FPAY | Annual fee payment |
Payment date: 20170403 Year of fee payment: 14 |
|
| FPAY | Annual fee payment |
Payment date: 20190715 Year of fee payment: 16 |